COMPOSITIONS AND METHODS FOR TREATING NEURODEGENERATIVE DISEASES

Abstract

The invention relates to inhibitory nucleic acids targeting the ataxin-2 gene (ATXN2), and expression cassettes and vectors comprising the same. Also provided herein are methods of treating neurodegenerative diseases, e.g., Amyotrophic Lateral Sclerosis and Spinocerebellar Ataxia-2.

Description

STATEMENT REGARDING SEQUENCE LISTING

The Sequence Listing associated with this application is provided in text format in lieu of a paper copy, and is hereby incorporated by reference into the specification. The name of the text file containing the Sequence Listing is 630264_401USPC_SEQUENCE_LISTING.txt. The text file is 666,463 bytes, was created on Jan. 6, 2023, and is being submitted electronically via EFS-Web.

BACKGROUND

Ataxin-2 (ATXN2) protein is a cytoplasmic protein that is a component of stress granules. Stress granules are thought to be transient subcellular compartments induced by arrest of protein translation, and include a number of proteins known to be mutated in subjects with neurodegenerative disease (Brown and Al-Chalabi, N Engl J Med (2017) 377:162-172). Ataxin-2 contains a sequence of glutamine residues, known as a polyglutamine repeat, that in normal individuals is ˜22 amino acids in length. Expansions of this polyglutamine repeat to a length of 34 or longer is found in individuals with a neurodegenerative disease Spinocerebellar Ataxia-2 (SCA2). This disease is characterized by progressive death of Purkinje neurons in the cerebellum and other neuronal cell types. Patients with Spinocerebellar Ataxia-2 develop ataxia, sensory problems, and other clinical features, which worsen over time. Moderate expansion of Ataxin-2 polyglutamine repeat, which are longer than that observed in most individuals but that are shorter than those typically observed in subjects with Spinocerebellar Ataxia-2 (e.g., between 27 and 33 glutamine residues), have been reported at a substantially elevated frequency in individuals with the motor neuron disease amyotrophic lateral sclerosis (ALS) as compared to normal subjects (Elden et al., Nature (2010) 466:7310). This suggests that these polyglutamine repeats of intermediate length, i.e., between those found in normal individuals and those found in spinocerebellar ataxia-2 patients, increase risk for ALS. Currently, treatment options for SCA2 and ALS are limited.

BRIEF SUMMARY

Aspects of the disclosure relate to compositions and methods for modulating expression of genes associated with spinocerebellar ataxia-2 (SCA2), amyotrophic lateral sclerosis (ALS), and conditions associated with TDP-43 proteinopathies. In particular, inhibitory nucleic acids are provided that are useful for inhibiting expression or activity of ataxin 2 (ATXN2). For example, inhibitory nucleic acids are provided that target one or more isoforms of ATXN2, e.g., a subset of ATXN2 isoforms, or all ATXN2 isoforms.

In one aspect, the disclosure provides an isolated nucleic acid molecule comprising an expression construct encoding an inhibitory nucleic acid that inhibits expression or activity of ATXN2, wherein the inhibitory nucleic acid comprises a guide strand sequence comprising the nucleic acid sequence set forth in any one of SEQ ID NOS: 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 124, 126, 128, 130, 132, 134, 136, 138, 140, 142, 144, 146, 148, 150, 152, 154, 156, 158, 160, 162, 164, 166, 168, 170, 172, 174, 176, 178, 180, 182, 184, 186, 188, 190, 192, 194, 196, 198, 200, 202, 204, 206, 208, 210, 212, 214, 216, 218, 220, 222, 224, 226, 228, 230, 232, 234, 236, 238, 240, 242, 244, 246, 248, 250, 252, 254, 256, 258, 260, 262, 264, 266, 268, 270, 272, 274, 276, 278, 280, 282, 284, 286, 288, 290, 292, 294, 296, 298, 300, 302, 304, 306, 308, 310, 312, 314, 316, 318, 320, 324, 326, 328, 330, 332, 334, 336, 338, 340, 342, 344, 346, 348, 350, 352, 354, 356, 358, 360, 362, 364, 366, 368, 370, 372, 374, 376, 378, 380, 382, 384, 386, 388, 390, 392, 394, 396, 398, 400, 402, 404, 406, 408, 410, 412, 414, 416, 418, 420, 422, 424, 426, 428, 430, 432, 434, 436, 1176-1288, 1811-1827, 2015, 2065, 2083, 2152, 2203, and 2209.

In some embodiments, the inhibitory nucleic acid is a siRNA duplex, shRNA, miRNA, or dsRNA.

In some embodiments, the inhibitory nucleic acid further comprises a passenger strand sequence, optionally wherein the passenger strand sequence is selected from Tables 1, 19, 23, and 24, or a passenger strand sequence selected from Tables 1, 19, 23, and 24, and having 1-10 insertions, deletions, substitutions, mismatches, wobbles, or any combination thereof.

In some embodiments, the inhibitory nucleic acid is an artificial miRNA wherein the guide strand sequence is contained within a miRNA backbone sequence.

In some embodiments, the guide strand sequence and passenger strand sequence of the artificial miRNA are contained within a miRNA backbone sequence. In some embodiments, the miRNA backbone sequence is a miR-155 backbone sequence, a miR-155E backbone sequence, a miR-155M backbone sequence, miR1-1 backbone sequence, a miR-1-1_M backbone sequence, a miR-100 backbone sequence, a miR-100_M backbone sequence, a miR-190a backbone sequence, a miR-190a_M backbone sequence, a miR-124 backbone sequence, a miR-124_M backbone sequence, a miR-132 backbone sequence, a miR-9 backbone sequence, a miR-138-2 backbone sequence, a miR-122 backbone sequence, a miR-122_M backbone sequence, a miR-130a backbone sequence, a miR-16-2 backbone sequence, a miR-128 backbone sequence, a miR-144 backbone sequence, a miR-451a backbone sequence, or a miR-223 backbone sequence.

In some embodiments, the inhibitory nucleic acid is a miRNA comprising the nucleic acid sequence set forth in any one of SEQ ID NOS: 443-490, 1109-1111, 1114, 1121-1168, 1405-1520, 1908-2007, 2011, 2017, 2021, 2025, 2027, 2031, 2035, 2039, 2041, 2045, 2049, 2053, 2057, 2061, 2067, 2071, 2075, 2079, 2085, 2089, 2093, 2097, 2101, 2105, 2109, 2113, 2117, 2120, 2124, 2128, 2132, 2136, 2140, 2144, 2148, 2154, 2158, 2162, 2166, 2170, 2174, 2176, 2180, 2182, 2184, 2187, 2189, 2191, 2193, 2195, 2197, 2199, 2205, 2211, 2261, 2263, 2265, and 2267.

In some embodiments, the nucleic acid sequence encoding the inhibitory nucleic acid is located in an untranslated region of the expression construct. In some embodiments, the untranslated region is an intron, a 5′ untranslated region (5′UTR), or a 3′ untranslated region (3′UTR).

In some embodiments, the isolated nucleic acid comprising an expression construct encoding an inhibitory nucleic acid furthers comprises a promoter. In some embodiments, the promoter is a RNA pol III promoter (e.g., U6, H1, etc.), a chicken-beta actin (CBA) promoter, a CAG promoter, a H1 promoter, a CD68 promoter, a human synapsin promoter, or a JeT promoter. In some embodiments, the promoter is an H1 promoter comprising nucleotides 113-203 of SEQ ID NO:1522, nucleotides 1798-1888 of SEQ ID NO:1521, nucleotides 113-343 of SEQ ID NO:2257, or nucleotides 244-343 of SEQ ID NO:2257.

In some embodiments, the expression construct is flanked by a 5′ adeno-associated virus (AAV) inverted terminal repeat (ITR) sequence and a 3′ AAV ITR sequence, or variants thereof. In some embodiments, one of the ITR sequences lacks a functional terminal resolution site. In some embodiments, the ITRs are derived from an AAV serotype selected from the group consisting of: AAV1, AAV2, AAV5, AAV6, AAV6.2, AAV7, AAV8, AAV9, AAVRh10, AAV11, and variants thereof. In some embodiments, the 5′ ITR comprises nucleotides 1-106 of SEQ ID NO:2257 and the 3′ ITR comprises nucleotides 2192-2358 of SEQ ID NO:2257.

In another aspect, the disclosure provides a vector comprising the isolated nucleic acid as provided in the present disclosure. In some embodiments, the vector is a plasmid or viral vector. In some embodiments, the viral vector is a recombinant adeno-associated virus (rAAV) vector or a Baculovirus vector. In some embodiments, the vector is a self-complementary rAAV vector. In some embodiments, the vector (e.g., rAAV vector) further comprises a stuffer sequence. In some embodiments, the stuffer sequence comprises nucleotides 348-2228 of SEQ ID NO:1522 or nucleotides 489-2185 of SEQ ID NO:2257. In some embodiments, the vector (e.g., rAAV vector) comprises the nucleotide sequence of any one of SEQ ID NOS:2257-2260.

In another aspect, the disclosure provides a recombinant adeno-associated (rAAV) particle comprising the isolated nucleic acid molecule or rAAV vector as provided in the present disclosure. In some embodiments, the rAAV particle comprises a capsid protein. In some embodiments, the capsid protein is capable of crossing the blood-brain barrier. In some embodiments, the capsid protein is an AAV9 capsid protein or AAVrh.10 capsid protein. In some embodiments, the rAAV particle transduces neuronal cells and/or non-neuronal cells of the central nervous system (CNS).

In another aspect, the disclosure provides a pharmaceutical composition comprising the isolated nucleic acid as provided in the present disclosure, the vector as provided in the present disclosure, or the rAAV particle as provided in the present disclosure, and optionally a pharmaceutically acceptable carrier.

In another aspect, the disclosure provides a host cell comprising the isolated nucleic acid as provided in the present disclosure, the vector as provided in the present disclosure, or the rAAV particle as provided in the present disclosure.

In another aspect, the disclosure provides method for treating a subject having or suspected of having a neurodegenerative disease, the method comprising administering to the subject the isolated nucleic acid molecule as provided in the present disclosure, the vector as provided in the present disclosure, the rAAV particle as provided in the present disclosure, or the pharmaceutical composition as provided in the present disclosure. In some embodiments, the administration comprises direct injection to the CNS of the subject. In some embodiments, the direct injection is intracerebral injection, intraparenchymal injection, intrathecal injection, intrastriatal injection subpial injection, or any combination thereof. In some embodiments, the direct injection is direct injection to the cerebrospinal fluid (CSF) of the subject, optionally wherein the direct injection is intracistemal injection, intraventricular injection, and/or intralumbar injection. In some embodiments, the subject is characterized as having an ATXN2 allele having at least 22 CAG trinucleotide repeats, optionally wherein the ATXN2 allele has at least 24 CAG trinucleotide repeats, at least 27 CAG trinucleotide repeats, at least 30 CAG trinucleotide repeats, or at least 33 or more CAG trinucleotide repeats. In some embodiments, the neurodegenerative disease is spinocerebellar ataxia-2, amyotrophic lateral sclerosis, frontotemporal dementia, primary lateral sclerosis, progressive muscular atrophy, limbic-predominant age-related TDP-43 encephalopathy, chronic traumatic encephalopathy, dementia with Lewy bodies, corticobasal degeneration, progressive supranuclear palsy (PSP), dementia Parkinsonism ALS complex of guam (G-PDC), Pick's disease, hippocampal sclerosis, Huntington's disease, Parkinson's disease, or Alzheimer's disease.

In another aspect, the disclosure provides a method of inhibiting ATXN2 expression in a cell, the method comprising delivering to the cell the isolated nucleic acid molecule as provided in the present disclosure, the vector as provided in the present disclosure, the rAAV particle as provided in the present disclosure, or the pharmaceutical composition as provided in the present disclosure. In some embodiments, the cell has an ATXN2 allele having at least 22 CAG trinucleotide repeats, optionally wherein the ATXN2 allele has at least 24 CAG trinucleotide repeats, at least 27 CAG trinucleotide repeats, at least 30 CAG trinucleotide repeats, or at least 33 or more CAG trinucleotide repeats. In some embodiments, the cell is a cell in the CNS, optionally a neuron, glial cell, astrocyte, or microglial cell. In some embodiments, the cell is in vitro. In some embodiments, the cell is from a subject having one or more symptoms of a neurodegenerative disease. In some embodiments, the cell is from a subject having or suspected of having a neurodegenerative disease. In some embodiments, the neurodegenerative disease is spinocerebellar ataxia-2, amyotrophic lateral sclerosis, frontotemporal dementia, primary lateral sclerosis, progressive muscular atrophy, limbic-predominant age-related TDP-43 encephalopathy, chronic traumatic encephalopathy, dementia with Lewy bodies, corticobasal degeneration, progressive supranuclear palsy (PSP), dementia Parkinsonism ALS complex of guam (G-PDC), Pick's disease, hippocampal sclerosis, Huntington's disease, Parkinson's disease, or Alzheimer's disease.

In another aspect the present disclosure provides a method of inhibiting ATXN2 expression in the central nervous system of a subject, the method comprising administering to the subject the isolated nucleic acid molecule as provided in the present disclosure, the vector as provided in the present disclosure, the rAAV particle as provided in the present disclosure, or the pharmaceutical composition as provided in the present disclosure. In some embodiments, the administration comprises direct injection to the CNS of the subject. In some embodiments, the direct injection is intracerebral injection, intraparenchymal injection, intrathecal injection, intrastriatal injection, subpial injection, or any combination thereof. In some embodiments, the direct injection is injection to the cerebrospinal fluid (CSF) of the subject, optionally wherein the direct injection is intracistemal injection, intraventricular injection, and/or intralumbar injection. In some embodiments, the subject has an ATXN2 allele having at least 24 CAG trinucleotide repeats, at least 27 CAG trinucleotide repeats, at least 30 CAG trinucleotide repeats, or at least 33 or more CAG trinucleotide repeats.

In another aspect, the present disclosure provides an artificial miRNA comprising a guide strand sequence and a passenger strand sequence, wherein the guide strand sequence comprises any one of SEQ ID NOS: 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 124, 126, 128, 130, 132, 134, 136, 138, 140, 142, 144, 146, 148, 150, 152, 154, 156, 158, 160, 162, 164, 166, 168, 170, 172, 174, 176, 178, 180, 182, 184, 186, 188, 190, 192, 194, 196, 198, 200, 202, 204, 206, 208, 210, 212, 214, 216, 218, 220, 222, 224, 226, 228, 230, 232, 234, 236, 238, 240, 242, 244, 246, 248, 250, 252, 254, 256, 258, 260, 262, 264, 266, 268, 270, 272, 274, 276, 278, 280, 282, 284, 286, 288, 290, 292, 294, 296, 298, 300, 302, 304, 306, 308, 310, 312, 314, 316, 318, 320, 324, 326, 328, 330, 332, 334, 336, 338, 340, 342, 344, 346, 348, 350, 352, 354, 356, 358, 360, 362, 364, 366, 368, 370, 372, 374, 376, 378, 380, 382, 384, 386, 388, 390, 392, 394, 396, 398, 400, 402, 404, 406, 408, 410, 412, 414, 416, 418, 420, 422, 424, 426, 428, 430, 432, 434, 436, 1176-1288, 1811-1827, 2015, 2065, 2083, 2152, 2203, and 2209.

In some embodiments, the guide strand sequence and passenger strand sequence are contained within a miR backbone sequence. In some embodiments, the miR backbone sequence is a miR-155 backbone sequence, a miR-155E backbone sequence, a miR-155M backbone sequence, miR1-1 backbone sequence, a miR-1-1_M backbone sequence, a miR-16-2 backbone sequence, a miR-100 backbone sequence, a miR-100_M backbone sequence, a miR-190a backbone sequence, a miR-190a_M backbone sequence, a miR-124 backbone sequence, a miR-124_M backbone sequence, a miR-132 backbone sequence, a miR-9 backbone sequence, a miR-138-2 backbone sequence, a miR-122 backbone sequence, a miR-122_M backbone sequence, a miR-130a backbone sequence, a miR-128 backbone sequence, a miR-144 backbone sequence, a miR-451a backbone sequence, or a miR-223 backbone sequence.

In some embodiments, the artificial miRNA comprises a sequence as set forth in any one of SEQ ID NOS: 443-490, 1109-1111, 1114, 1121-1168, 1405-1520, 1908-2007, 2011, 2017, 2021, 2025, 2027, 2031, 2035, 2039, 2041, 2045, 2049, 2053, 2057, 2061, 2067, 2071, 2075, 2079, 2085, 2089, 2093, 2097, 2101, 2105, 2109, 2113, 2117, 2120, 2124, 2128, 2132, 2136, 2140, 2144, 2148, 2154, 2158, 2162, 2166, 2170, 2174, 2176, 2180, 2182, 2184, 2187, 2189, 2191, 2193, 2195, 2197, 2199, 2205, 2211, 2261, 2263, 2265, and 2267.

In another aspect, the present disclosure provides an isolated RNA duplex comprising a guide strand sequence and a passenger strand sequence, wherein the guide strand sequence comprises the nucleic acid sequence set forth in any one of SEQ ID NOS: 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 124, 126, 128, 130, 132, 134, 136, 138, 140, 142, 144, 146, 148, 150, 152, 154, 156, 158, 160, 162, 164, 166, 168, 170, 172, 174, 176, 178, 180, 182, 184, 186, 188, 190, 192, 194, 196, 198, 200, 202, 204, 206, 208, 210, 212, 214, 216, 218, 220, 222, 224, 226, 228, 230, 232, 234, 236, 238, 240, 242, 244, 246, 248, 250, 252, 254, 256, 258, 260, 262, 264, 266, 268, 270, 272, 274, 276, 278, 280, 282, 284, 286, 288, 290, 292, 294, 296, 298, 300, 302, 304, 306, 308, 310, 312, 314, 316, 318, 320, 324, 326, 328, 330, 332, 334, 336, 338, 340, 342, 344, 346, 348, 350, 352, 354, 356, 358, 360, 362, 364, 366, 368, 370, 372, 374, 376, 378, 380, 382, 384, 386, 388, 390, 392, 394, 396, 398, 400, 402, 404, 406, 408, 410, 412, 414, 416, 418, 420, 422, 424, 426, 428, 430, 432, 434, 436, and 1176-1288, 1811-1827, 2015, 2065, 2083, 2152, 2203, and 2209, optionally wherein the guide strand sequence and passenger strand sequence are linked by a loop region to form a hairpin structure comprising a duplex structure and a loop region. In some embodiments, the loop structure comprises from 6 to 25 nucleotides.

In another aspect, the disclosure provides a kit comprising a container housing a composition as described by the present disclosure.

BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS

FIG. 1 shows tuning mean squared error for mir-30 dataset (Pelossof et al., Nature Biotechnology (2017) 35:350-353). Data showing the mean squared error (MSE) for prediction performance of the shRNA prediction algorithm on a set of shRNAs targeting Kras, held out from a data set of shRNAs used to train the support vector machine model to predict shRNA performance. Mean squared error is calculated as the square of the difference between the score of the support vector machine (SVM) predictor and the label 1 or −1, corresponding to shRNAs empirically determined to yield good knockdown or poor knockdown. These squared differences are averaged across shRNAs tested. The hyperparameter c was varied and the mean squared errors calculated for each value c.

FIG. 2 shows a plot of precision vs recall for SVM model applied to held-out shRNAs targeting Trp53 gene, after training on the shRNAs targeting the other genes in the TILE dataset (Pelossof et al, Nature Biotechnology (2017) 35:350-353). Horizontal line at approximately 0.19 represents the fraction of shRNAs that are positive, i.e., yielding good knockdown, out of the total number of shRNAs, in the set of all shRNAs targeting Trp53. The precision-recall line represents, varying across values of the SVM score, the fraction of true positives that are included in the dataset (‘recall’), versus the fraction of true positives relative to false positives (‘precision’), at a given SVM score cutoff. Thus, at the least stringent SVM score, all true positives are included (recall=1), but precision is low because many negative shRNAs are included.

FIG. 3 shows two curves are plotted against SVM score. In one, the cumulative fraction of positive shRNAs that are expected to be lost as the classifier score is increased is shown. This is denoted by the bold line. In the other, the percent improvement in rejection of low-performing shRNAs is shown. This is denoted by the lighter line. Vertical dashed lines, from left to right, represent the 25^th percentile (light dashed) and 50^th percentile (bold dashed) of SVM scores in the dataset, the shRNAs targeting Trp53.

FIG. 4 shows jitter plots of the distribution of SVM score predictions as a function of the first base of the guide sequence of the shRNA sequences targeting ATXN2. All data points are shown; the horizontal width of the ‘violin’ is proportional to the number of points at each SVM score, which is plotted on the y axis. On the left, the score is calculated for guide sequences that are perfectly complementary to the ATXN2 sequence (guide sequence base at position 1 is A, U, C, or G). On the right, the score is calculated if the first base is converted to U (edit guide sequence base at position 1 to U if guide at position 1 does not natively begin with U). Note that guide sequences which originally begin with U will have the same score in the right plot, whereas sequences which begin with A, G, or C will have different scores. In general, the SVM score increases if the first base is U.

FIG. 5 shows a plot of ATXN2 quantigene assay values across a panel of commonly used cell lines. Signal is reported with 30 μL (left bar) or 10 μL (right bar) of lysate. “−” represents negative control with no cellular material. Y-axis is the assay signal. Additional horizontal line represents the minimal signal selection criterion.

FIGS. 6A-6B show a ‘Sashimi’ plot of the alternative splicing of Ataxin-2 transcript from human brain or from HepG2.5 cells. FIG. 6A: For brain, representative plots from two different individuals are shown. The height of the bars in the plot represents the number of reads aligning to the position in Ataxin-2, according to the diagram underneath the plot. Numbers on curved arcs represent the count of reads aligning across exon-exon junctions. Injunctions where the arcs are on both top and bottom, this indicates potential alternative splicing of the transcript. Arrows point out exons subject to substantial alternative splicing, such that these exons do not appear in a substantial number of ATXN2 transcripts in human brain. The diagram at the bottom of the plot represents the structure of the transcript ENST00000377617.7, with exons as solid rectangles. The transcript is oriented from right to left, with exon 1 on the right. FIG. 6B: Similar data from HepG2 cells is shown. The alignment to the transcript is not to scale.

FIG. 7 shows ATXN2 mRNA values across tested siRNAs, at 20 nM, 1 nM, and 200 pM doses. The x-axis shows the position of the ATXN2 sequence (SEQ ID NO: 2) that the corresponding siRNA is complementary to. ATXN2 mRNA values represents the ratio of ATXN2 to GAPDH signal from quantigene assay, normalized to mock control. 3′ UTR on the X-axis shows the general position of the 3′ untranslated region of the ATXN2 transcript.

FIG. 8. Correlation plot of the ATXN2 mRNA knockdown (ratio of ATXN2 to GAPDH signal, normalized to mock transfected controls), versus the SVM score. The expected correlation is observed, indicating that high SVM scores predict good knockdown performance.

FIG. 9. Plot of ATXN2 signal from ATXN2 siRNA treated U2OS cells, derived from indirect immunofluorescence, for the indicated conditions. XD-ID Nos represent treatment with different siRNAs corresponding to Table 1, at the indicated dose (20 nM (top) or 1 nM (bottom)). Other treatments are indicated as follows: “no_primary_secondary”=during antibody staining, the Ataxin-2 primary antibody was omitted, secondary antibody was included; “no_primary_no secondary”=during antibody staining, both the Ataxin-2 primary antibody and the secondary fluorescent antibody against the Ataxin-2 antibody were omitted; “primary_no secondary”=during antibody staining, the Ataxin-2 primary antibody was included but the secondary fluorescent antibody against the Ataxin-2 antibody was omitted; “SMP”=a pool of 4 siRNAs targeting ATXN2, with chemically modified nucleotides, obtained from Dharmacon; “primary_secondary”=untreated cells stained with primary and secondary antibody; “NTC”=cells treated with a ‘non-targeting control’ siRNA, not expected to target any human transcripts, with chemically modified nucleotides, obtained from Dharmacon; “XD-LucControl”=an siRNA, comprised only of RNA bases as in the ATXN2 targeting siRNAs, expected to target the luciferase gene but not to target ATXN2. In the plot, each point represents signal averaged across all cells in a well. Outliers, which were excluded from calculation of mean knockdown across wells in Tables 6 and 7, are shown as lighter colored points.

FIGS. 10A-10B show representative images of ATXN2 siRNA treated U2OS cells as described in FIG. 9. FIG. 10A: Representative images of siRNA (20 nM) treated U2OS cells. Top panels, Hoechst staining demarcates cell nuclei. Bottom panels, ATXN2 indirect immunofluorescence. Treatment/staining procedure is shown below image panels. FIG. 10B: As in FIG. 11A, but for U2OS samples treated with siRNAs at 1 nM.

FIG. 11 shows a plot of normalized ATXN2 indirect immunofluorescence signal, as a function of position along ATXN2 transcript (SEQ ID NO: 2). The x-axis is restricted to the positions along the ATXN2 transcript spanning the binding sites of the tested siRNAs.

FIGS. 12A-12C show dose response of various siRNAs tested. FIG. 12A (top) shows a plot of log IC50 across siRNA IDs tested in Group 1. Bars represent span of 95% confidence interval for IC50 values. FIG. 12A (bottom) shows representative dose response curves for siRNAs. Y-axis represents ratio of ATXN2 to GAPDH signal from quantigene assay of mRNA levels, from lysates of HepG2 cells dosed with indicated concentration of siRNA. Fits represents 3-parameter logistic regression fits, with Hill slope set constant at 1. Outliers were automatically identified, excluded from curve fitting and IC50 estimation. FIG. 12B shows a plot of log IC50 across siRNA IDs tested in Group 2. Bars represent span of 95% confidence interval for IC50 values. FIG. 12C shows a representative dose response curves for siRNAs. Y-axis represents ratio of ATXN2 to GAPDH signal from quantigene assay of mRNA levels, from lysates of HepG2 cells dosed with indicated concentration of siRNA. Fits represents 3-parameter logistic regression fits, with Hill slope set constant at 1. Outliers were automatically identified, excluded from curve fitting and IC50 estimation, and are indicated on graph.

FIG. 13 shows predicted folding patterns of guide sequences embedded in miRNA backbones, as created using the web-based server mfold. Multiple fold predictions are obtained; a representative fold is shown. Note the unpaired, ‘bulged’ nucleotides at several positions in each miRNA in the vicinity of the guide sequence, except in the ‘sealed’ variant.

FIG. 14 shows fluorescence automated cell sorting data demonstrating reduction in signal intensity for a GFP (stop)-ATXN2 reporter construct-expressing U2OS cell line by artificial miRNAs. Cells were transfected with vectors containing inserts either including the guide sequence of XD-14792 (SEQ ID NO:112), or control guide sequences, embedded in miRNA backbones. Y-axis plots the median fluorescence intensity of cells within each replicate. Replicates derive from wells of a 96-well plate containing cells that were transfected with vectors. The cells were dissociated with trypsin prior to FACS analysis.

FIG. 15 shows thresholding procedure to distinguish transduced from untransduced cells in imaging experiments using lentivirally packaged ATXN2-specific artificial miRNAs. Lentiviral vectors (similar to pLVX-EF1A_mCherry-miR-1-1-XD_14890-WPRE_CMV (SEQ ID NO:546)) express mCherry and so identification of mCherry expression distinguishes transduced from untransduced cells. Left panel shows histogram of signal in the fluorescence channel used to detect mCherry signal (including indirect immunofluorescence from an anti-mCherry antibody and fluorescent secondary antibody). Right panel shows histogram of signal from cells transduced with mCherry-encoding vector, with a clear bimodal distribution of signals representing untransduced cells (low signal) and transduced cells (high signal). Vertical line shows threshold used to separate mCherry positive from mCherry negative cells, placed such that no untransduced cells exceed this signal threshold and such that the large majority of the right peak of the bimodal histogram of mCherry signal in transduced cells exceeds this threshold.

FIG. 16 shows ATXN2 signal normalization procedure for artificial miRNA high content imaging assay. Each point represents signal in the channel used to detect indirect immunofluorescence for ATXN2, average across cells in the well. ATXN2 knockout cells were used to determine the background levels of indirect immunofluorescence for the ATXN2 antibody. The different cell types and staining conditions are shown, with the y-axis normalized with 100% set to the signal from wild-type, untransduced cells and 0% set to the signal from untransduced ATXN2 knockout cells. The signal in the ATXN2-antibody stained ATXN2 knockout cells somewhat exceeds signal from cells not stained with antibody, indicating that there is some background associated with the antibody and that using the ATXN2 knockout can help correct for this background to improve accuracy in measuring ATXN2 protein signal.

FIGS. 17A-17B show plots of ATXN2 signal from wells transduced with lentiviral vectors expressing guide sequences (shown on x-axis) embedded in miRNA backbones (miR-155E—FIG. 17A; miR1-1—FIG. 17B). Guide sequences and miRNA context sequences are listed in Table 11.

FIG. 18A-18B show representative images of Hoechst 33342 stain (top row), mCherry signal (middle row), and ATXN2 indirect immunofluorescence signal (bottom row) from cells as quantified in FIG. 17. FIG. 18A shows data for guide sequences embedded in miR-155E backbone; FIG. 18B shows data for guide sequences embedded in miR1-1 backbone.

FIG. 19 shows a plot of ATXN2 protein signal from miRNA-embedded anti-ATXN2 guide sequences versus ATXN2 mRNA signal from anti-ATXN2 siRNA treatment. There is correlation between the mRNA and protein knockdown across conditions tested.

FIGS. 20A-20C show validation of CRISPR guide RNAs in disrupting Ataxin-2 gene and knocking out Ataxin-2 protein in U2OS cells. FIG. 20A shows western blot analysis of U2OS cells nucleofected with ATXN2-targeting CRISPR gRNAs, complexed with Cas9 protein. Treatments include no nucleofection controls, control guide RNAs targeting CD81 or expected to be non-targeting, and five unique ATXN2 targeting guides. Immunoblots against Ataxin-2 protein and alpha-tubulin loading control are shown. FIG. 20B shows representative histograms and FIG. 20C shows median fluorescence intensity within treated wells of Ataxin-2 indirect immunofluorescence signal for cells nucleofected with indicated treatments, as in FIG. 20A.

FIGS. 21A-21B show U2OS ATXN2 knockout clones generated for assay calibration. FIG. 21A shows ATXN2 U2OS knockout cell line generation scheme. FIG. 21B shows western blot analysis from clonal lines generated after nucleofection with indicated ATXN2 targeting gRNA. The lane containing protein from lysed material from the clone (clone 43) selected for use is indicated by the arrow.

FIG. 22 show knockdown of Ataxin-2 protein in vivo after AAV vectorized amiRNA delivery. AAV encoding miRNAs XD-14792 or XD-14887, embedded in the miR-1-1 backbone, or a control construct lacking a miRNA, was delivered intravenously to adult wild-type mice by tail vein injection. 15 days after injection, animals were euthanized and livers were harvested and snap-frozen. GFP fluorescence, resulting from vector encoded GFP, was detected in the liver upon blue light illumination. FIG. 22 (left): Liver lysate was immunoblotted for Ataxin-2, beta-actin, and GFP (not shown). Each lane is derived from a different animal. FIG. 22 (right): Ataxin-2 signal was normalized to beta-actin signal. All miRNA-dosed animals had lower Ataxin-2 signal than animals dosed with control AAV vector. Each point represents ratio of Atxn2 to Beta actin signal from an individual animal.

FIGS. 23A-23B show quality metrics of pooled library screen of Atxn2-targeting miRNAs (“Deep Screen 1”). FIG. 23A shows a scatter plot comparing ratios of high- and low-sorted samples in two replicates, showing tight correlation. FIG. 23B shows correlation matrix between all samples tested. Spearman correlation was calculated between guide sequence count vectors between all samples.

FIG. 24 shows ratio baseline subtraction procedure. Raw count ratios (log-base 2 transformed) are shown on x-axis, for top, ATXN2-targeting miRNAs, and bottom, scrambled miRNAs. For subsequent calculations, the median of the ratio for the scrambled miRNAs was subtracted.

FIG. 25 shows a plot of ATXN2 signal depletion versus cell depletion. Each point represents a library element, containing a miRNA targeting either the ATXN2 transcript; a scrambled sequence; or a sequence targeting an essential gene and expected to reduce cell proliferation and/or viability. The x-axis is the average across replicates of the ratio of sequence counts derived from cells in the high- and low-ATXN2 FACS gate populations. The y-axis is the average across replicates of the ratio of sequence counts derived from HeLa cells after initial transduction and after 16 days. Points falling toward the bottom of the axis represents elements that were depleted from the 16 day timepoint relative to the initial transduction timepoint.

FIG. 26 shows a plot of ATXN2 signal depletion versus position on ATXN2 transcript of complementarity of guide sequence. Points toward the bottom represent guide sequences with greater knockdown of ATXN2; points toward the top of the y-axis represent guide sequences with less knockdown of ATXN2.

FIG. 27 shows a similar plot as in FIG. 26, but zoomed-in on the 3′ end of the ATXN2 transcript. In black are sequences deemed part of ‘hotspots’ in the 3′ UTR of the ATXN2 transcript.

FIG. 28 shows the percent of reads, averaged across scrambled guide sequences, that match to a guide sequence excised from the pri-miRNA at the indicated position. The diagram above shows an example sequence, where the bold text to the left is miR backbone sequence and the regular text is the guide sequence. Arrows and numbers indicated cleavage position (for the tiled screened described here, in the miR 16-2 backbone, Drosha is the expected enzyme for this cleavage event). The seed sequence for a guide sequence cut at the expected position is shown. The position of this seed sequence will shift if the guide position is cut out of the pri-miRNA at a different position from the expected position.

FIG. 29 shows representative images used in assessing the production of motor neurons in the stem cell differentiation protocols. Upper left image shows overlay of indirect immunofluorescence signal from anti-HB9 and anti-Beta 3 tubulin (TUJ1) antibodies. Upper right shows overlay of signal from anti-ISLET1 and TUJ1 signal. Lower left shows overlay of HB9, ISLET1, and TUJ1 signal. Bottom right shows overlay of HB9, ISLET1, TUJ1, and nuclear DAPI stain. In the images, neuronal processes are clearly seen as labeled by TUJ1 antibody. Neuronal nuclei are labeled by the motor neuron markers HB9 and Islet1, with 25-35% of neurons labeled with HB9, 50-60% labeled by Islet1, and 70-80% of cells positive for TUJ1 signal.

FIG. 30A-30C show data from an experiment testing knockdown of ATXN2 mRNA and protein after transduction of ATXN2-targeting amiRNAs in lentiviral format in stem-cell derived motor neurons. FIG. 30A is a schematic of the cassette packaged in lentiviral vectors, with an H1 promoter driving the artificial miRNA, followed by a Pol III termination signal (6T). After this miR expression cassette, a CMV Pol II promoter drives expression of the fluorescent reporter GFP, and is followed by a WPRE element to stabilize the GFP transcript. FIG. 30B shows data from qPCR against ATXN2 mRNA. Each dot represents a biological replicate derived from a distinct tissue culture well of motor neurons. Data represent average signal calculated from change in qPCR threshold (CT) for ATXN2 versus either GUSB or B2M. Bars are mean of replicates, and error bars are standard deviation across replicates. ATXN2 signal is normalized to levels measured from motor neurons growing in wells not treated with vector. Data from wells treated with a control lentiviral vector with the multiple cloning site (MCS) in place of the amiRNA is shown as “MCS.” Two amiRNAs were tested, with amiRNAs targeting indicated position in ATXN2 transcript (1784 or 4402) indicated; amiRs were embedded in the miR16-2 backbone. The guide sequence targeting ATXN2 position 1784 is also referred to as XD-14792. Lentiviral vectors were dosed at two concentrations. The viral dose to achieve a multiplicity of infection (MOI) of 2.5 or 4.5 was calculated based on titration in U2OS cells (FACS analysis of GFP signal, calculating % cells positive for GFP). Using these values and the number of neurons plated per well, the corresponding dose of vector to achieve MOI of 2.5 or 4.5 in the motor neuron cultures (calculated based on the U2OS infectivity) was used. Observation of GFP fluorescence in cultures confirmed that transduction was near complete, as expected if the U2OS MOI was similar to the motor neuron MOI. FIG. 30C shows assessment of ATXN2 protein assessment from cultures treated the same as in FIG. 30B. The top panel shows the Western blot, with clear evidence of reduction in signal in lanes with protein from wells treated with amiRNAs targeting ATXN2 versus untreated wells or wells treated with the control MCS vector. Bottom panel quantifies ATXN2 immunoblot signal, with each point representing a biological replicate, the bars representing mean across replicates and the error bars standard deviation.

FIG. 31. Data is presented from an experiment performed similarly to that shown in FIG. 30. In this experiment, the MOI (as calculated by infectivity in U2OS cells) was 3.5. Knockdown in motor neurons treated with lentiviral vectors with miR 16-2 backbone-embedded amiRNAs targeting indicated ATXN2 transcript position is shown. Horizontal dashed line represents the threshold of 80% knockdown. In this experiment, it is apparent that the amiRNAs targeting the ATXN2 transcript in the 3′ UTR do not yield the same level of knockdown as amiRNAs targeting the ATXN2 coding sequence. Bars show mean knockdown, normalized to wells not treated with lentiviral vector; each point is a biological replicate (neurons from an individual well), and error bars are standard deviations across replicates. As above, MCS represents a lentiviral vector with a control multiple cloning site in place of a miR cassette.

FIG. 32. 2% agarose TAE gel demonstrating truncations in miR16-2 backbone-embedded amiRNAs packaged in AAV9. AAV genomic DNA was column purified and concentration quantified by Qubit fluorometer. Equal amounts of vector genome DNA, by Qubit measurement, were loaded into gel and subject to electrophoresis. Note that the gel image shown was spliced together for clarity. Leftmost lane is a DNA size ladder, with indicated DNA sizes in kilobases shown. From left to right, samples are (all DNA derived from purified AAV vector genomes): (1) H1 promoter driving miR1-1 XD-14792 (1784), followed by CBh promoter driving GFP; (2) H1 promoter followed by a non-miR multiple cloning site, followed by stuffer sequence “AMELY_V1”; (3-11) From left to right, AAV with amiRNAs targeting ATXN2 at positions 1784, 1479, 1755, 3330, 4402, 4405, 4406, 4409, and 4502. Each lane has an amiRNA targeting ATXN2, in the same vector genome format as lane 2 replacing the MCS with the indicated miR cassette, with miR16-2 backbone. Note in all of the material from AAV genomes with miR16-2 backbone miR cassettes the presence of both an upper band, running at the intended size, as well as a faster migrating lower band.

FIG. 33A-33B. Data from Deep Screen 2 showing replicate to replicate consistency (FIG. 33A) and performance across miR backbones (FIG. 33B). In FIG. 33A, each point represents the relative abundance of a library element, with position on the x-axis representing the log 2 fold change in abundance between the 10^th percentile ATXN2 sort and unsorted cells from the first screen replicate, and the y-axis the corresponding log 2 fold change from the second screen replicate. Points on the far right of the graph represent data where the denominator in the ratio of sequence counts for sorted and unsorted cells is 0, and hence undefined when log-transformed. There is good correspondence between the replicates for elements exhibiting substantial knockdown (log 2 fold change <−1), but for inactive controls (including essential gene targeting amiRNAs, 911 controls, and scramble controls), there is more variability from replicate to replicate in this screen compared to Deep Screen 1. As a result there is some deviation from screen replicate to replicate in the negative control medians. No baseline subtraction was done because of the agreement in log 2 fold change values for active amiRNAs. In FIG. 33B, boxplots represent the ATXN2 knockdown performance across amiRNAs embedded in various miR backbones. In each boxplot, the center line is the median, the upper and lower edges of the box represent the 75^th and 25^th percentiles, and the line extends beyond the box edges to either the maxima/minima or 1.5 times the interquartile range (difference between 25^th and 75^th percentiles), whichever is closer to the median. Overlaying points (very faint, transparent) represent the ATXN2 knockdown signal from individual miRNAs. The y-axis represents the mean log 2 fold-change between the abundance of sequencing reads of elements detected in the 10^th percentile of ATXN2 signal relative to the abundance of the guide in unsorted cells. In this screen, the theoretical maximum fold-change is 10-fold between the 10^th percentile sort and un-sorted cells.

FIG. 34. Depletion of essential-gene targeting amiRNAs in various miR backbones at a late timepoint T₁ (18 days after transduction) versus an early timepoint T₀ (1 day after transduction). The y-axis represents the log₂ fold change in abundance between the two timepoints, and was not baseline subtracted. A similar ranking between the ‘performance’ of each miR backbone in inducing guide depletion over time, when expressing essential gene-targeting amiRNAs as in this figure, versus performance of miR backbones in ATXN2 knockdown when expressing ATXN2-targeting amiRNAs, as in FIG. 33, can be seen.

FIG. 35. Agarose gel with purified AAV vector genomes with various miR backbones, with amiRNA targeting Atxn2 at position 4402 (first 10) embedded, or targeting position 1784 (last 2; 1784 guide sequence is same as XD-14792). Note that image is spliced for clarity (to place lane including DNA size ladder immediately adjacent to relevant lanes). Some lanes have bands that both migrate differently than others (miR122, miR1-1-4402, miR-1-1XD14792), this is likely due to differences in loading or dye binding and not true migration differences. More importantly, across miR backbones there are differences in the relative intensity of the second most intense band, migrating farther than the most intense upper band which is the presumed intended vector genome. AAV vector genomes with miR100 and miR128 backbones in particular have a less intense faster migrating band than others.

FIG. 36. Agarose gel with AAV vector genomes derived from pools of cis plasmids. Each pool includes elements generated by PCR amplification from an oligonucleotide pool containing a mixture of amiRs embedded in multiple miR backbones, and the PCR primers used do not distinguish between parent and “_M” form miR backbones. Thus, the pool labeled miR-1-1 will include amiRs in backbones miR-1-1 and miR-1-1_M; the pool labeled miR-100 will contain miR-100 and miR-100_M backbones; the pool labeled miR-190a will contain miR-190a and miR-190a_M backbones; the pool miR-124 will contain miR-124 and miR-124_M backbones; the pool miR-138-2 will contain miR-138-2 and miR-138-2_M backbones. miR-155M and miR-155E, though not related to each other by the “_M” modification rules, also have high sequence similarity and therefore the pool labeled “miR-155M” likely contains a mix of miR-155M and miR-155E backbones. Each lane contains purified vector genome DNA from AAV generated with indicated plasmid pool. The last lane is derived from a mixture of the 5 micropools shown in the gel as well as micropools with miR backbones miR-124, miR-128, miR-138-2, miR-144, and miR-155M. As in FIG. 35, the AAV pool with the miR-100 backbone (dashed box) has a less intense faster migrating band than the other AAV pools.

FIG. 37. Data from Deep Screen 2, only including elements with miR-100 or miR-100_M backbones. As in FIG. 33A, each point represents the relative abundance of a library element, with position on the x-axis representing the log 2 fold change in abundance between the 10^th percentile ATXN2 sort and unsorted cells from the first screen replicate, and the y-axis the corresponding log 2 fold change from the second screen replicate.

FIG. 38. RT-ddPCR data demonstrating knockdown of ATXN2 mRNA in stem-cell derived motor neurons 7 days after treatment with scAAV-DJ vectors expressing ATXN2-targeting amiRNAs. Each point represents a biological replicate (a well of neurons treated with AAV at indicated dose of vector genomes per cell). Indicated amiRNAs, denoted as miR backbone—Atxn2 targeting position, mark x-axis. The amiRNAs were embedded in a self-complementary vector genome, with an H1 promoter driving the amiR, and a stuffer sequence modified from PSG11, “PSG11_V5” (nucleotides 489-2185 of SEQ ID NO:2257) 3′ of the miR cassette up to the wild-type ITR. The y-x is represents RT-ddPCR signal, with copies of each transcript per unit microliter derived from percentage of positive to negative droplets for primer/probesets specific to ATXN2, GUSB, or B2M. The points represent averages of ratios of ATXN2/GUSB and ATXN2/B2M ratios.

FIG. 39. This graph shows a RT-ddPCR experiment similar to that in FIG. 38, except spanning a broader range of indicated doses. Because of constraints on the number of available cells, not all amiRNAs were treated with all doses. In this experiment, the ATXN2 mRNA level is calculated by ATXN2/B2M RT-ddPCR ratios.

FIG. 40. Images of stem-cell derived motor neurons treated with scAAV-DJ vector as in FIGS. 38 and 39. Cells were treated with a dose of 1E4 vector genomes per cell. Representative images of DAPI stain (to label cell nuclei), indirect immunofluorescence signal for anti-ISL1 antibody (to label motor neurons), and TUJ1 signal, to label neuronal processes. No obvious differences were seen in neuronal processes between neurons treated with an active ATXN2-targeting amiRNA (1755) and an inactive (1755_911) amiRNA in scAAV-DJ. Panels at right (top) quantify total number of cells, defined by DAPI staining, and (bottom) quantify fraction of cells that are positive for ISL1. Each point represents average quantification across fields for a given well. Asterisks indicate significant (p<0.05) difference versus vehicle (PBS+0.001% PF-68) control, calculated by one-way ANOVA followed by Dunnett's multiple comparisons test. Vectors encode amiRNAs targeting indicated ATXN2 transcript position in miR100 or miR100_M backbone (FIGS. 38 and 39 show which amiRNA is in miR100 and which is in miR100_M backbones). “PBS” represents wells of motor neurons treated with vehicle (PBS+0.001% PF-68); GFP represents the amiRNA and GFP expressing vector H1-miR1-1.XD-14792-CBh-GFP packaged in scAAV-DJ.

FIGS. 41A-41C. Similar to FIG. 40, FIG. 41A shows representative images of neuronal morphology across stem-cell derived motor neuron treated with indicated scAAV-DJ vector encoding specified amiRNA, embedded in miR100 or miR100_M backbone vector. There is no readily apparent alteration in neuronal morphology for any treatment compared to vehicle. Total number of Hoechst+ nuclei (FIG. 41B) and the % of total nuclei that are Isl1+ (FIG. 41C) in AAV treated stem-cell derived motor neurons was quantified.

FIG. 42. Shows ‘volcano plots’ of RNAseq data, comparing gene expression in neurons treated with active amiRNA versus their inactive, ‘9-11’ control counterparts. The 911 controls do not reduce ATXN2 levels, but differ only by 3 nucleotides (bases 9, 10 and 11) from the active amiRNAs. Off-target effects of the amiRNAs not involving bases 9, 10 and 11 may therefore be conserved with the cognate non-911 control amiRNA, and the comparison can be considered to enrich the ‘on-target’ transcriptional impact of lowering Atxn2 levels. By far the most robust transcriptional effect observed in comparisons of miR100_1755 and miR100_2945 versus their 911 controls is ATXN2. In the plots, each point represents a gene (counts for different transcripts are collapsed gene-wise); the y-axis represents the nominal p value; the x-axis the log₂ fold change for gene expression between conditions. Data is derived from n=5-6 biological replicates per treatment. Neurons were treated with a dose of 1E4 vector genomes/cell, and RNA collected for RNAseq (quantseq) 7 days later.

FIG. 43. Panel of ‘volcano plots’ comparing each indicated amiR AAV treatment, with the same treatment conditions described as in FIG. 42, to all other amiRNA treatments shown (n=6 replicates/condition). Axes are as in FIG. 42; horizontal dashed line represents the false discovery rate threshold of 10%. Here, what are plotted are predicted off-target transcripts (with detectable expression levels in this system) for each amiR, that is transcripts with complementarity to bases 2-18 of the guide sequence with 2 or fewer mismatches. For most of the amiRNAs, none or only very few of the predicted off-targets are downregulated relative to the set of other amiRNAs, and exceed the 10% false discovery rate threshold.

FIG. 44. Plot of Atxn2 mRNA versus biodistribution of ATXN2 amiRNA expressing vectors (miR1-1-1784 (left) and miR100-3330 (right)) from mice dosed intrastriatally with vectors expressing indicated amiRNA AAV construct. Each point represents RT-ddPCR mRNA and vector distribution data from RNA and DNA isolated from an individual striatal biopsy, taking the average of Atxn2/Gusb and Atxn2/Tbp droplet ratios, normalized to vehicle treated animals. Multiple distinct vector formats are included, all with one version of the H1 promoter and various stuffer sequences.

FIGS. 45A-45B. Plot of Taqman qPCR data from striatal biopsies of animals dosed with indicated amiRNA AAV constructs (miR1784—FIG. 45A; miR3330—FIG. 45B). For each striatal biopsy assessed, two data points are shown: the y-axis plots the CT threshold difference between amplification of cDNA from an exogenous amiR and an endogenous miR, miR124; or the difference between amplification of two endogenous miRs. The x-axis shows the (log-base-2 transform of) vector distribution data, as in FIG. 44. Dashed lines are linear fits. Note that the relationship between CT and expression is of a form similar to expression ˜2{circumflex over ( )}^CT, consistent with the apparent linear relationship between CT difference and log₂ (vector genomes/diploid genome).

FIG. 46. qPCR data (a subset of the data shown in FIG. 45) is plotted against small RNAseq quantification of exogenous amiR expression/total miR expression, for RNA deriving from the same striatal punch biopsies. The relationship between the delta CT of exogenous amiR versus endogenous miR and small RNAseq quantification is separately fit to a linear model (linear regression) for each of the indicated amiRs. The slope of fits for the qPCR versus small RNAseq for the two amiRs are similar, and the fits are good as quantified by residuals, R².

FIG. 47. This graph shows use of the linear model in FIG. 46 to derive a predicted absolute amiR expression level, as a function of total miR expression, for the remaining samples that only had amiR expression measured by qPCR. This predicted amiR expression level is plotted on the x-axis. Each point represents an individual striatal punch biopsy. The y-axis represents the RT-ddPCR quantified Atxn2 mRNA level for that biopsy, same as in FIG. 44. A loess fit is used to separately fit a curve to data from biopsies from animals dosed with miR1-1.1784 expressing AAVs (black filled circles, dashed line); or miR100.3330 expressing AAVs (open diamonds; dotted line).

FIGS. 48A-48B. Liver enzyme data, alanine transaminase (ALT) (FIG. 48A) and aspartate aminotransferase (AST) (FIG. 48B) from blood collected from the submandibular vein, at 2 or 3 weeks after intravenous dosing of AAVs expressing indicated amiRs. Naïve animals were monitored in parallel.

FIG. 49. Plot of Atxn2 mRNA knockdown and vector distribution, as in FIG. 44, in striatal biopsies from animals dosed with AAVs expressing indicated amiRNAs. Lines represent loess (locally estimated scatterplot smoothing) fits for each series, implemented in R (stats::loess).

FIGS. 50A-50B. Expression of amiRNAs in tissue from animals dosed with AAVs expressing indicated amiRNAs. Liver tissue was analyzed from animals dosed intravenously (FIG. 50A); striatal tissue was analyzed from animals dosed via intrastriatal injection (FIG. 50B). amiRNA expression is plotted as normalized to total miRNA expression.

FIG. 51. Plot showing 5′ end homogeneity of processed miRNAs in striatal tissue in animals dosed intrastriatally. The y-axis (log₁₀ scale) plots cumulative sequencing reads, across all samples (n=4/AAV), for mature amiRNAs initiating at the ‘expected’ position 0, 5′ of the expected start site (negative numbers) or 3′ of the expected start site (positive numbers). For all of these amiRs, the vast majority of mature processed amiRNAs initiate at the expected start site.

FIGS. 52A-52D. (Top) Diagrams of representative predicted folding structures (mfold) of amiRNAs miR100_1755 (FIG. 52A), miR100_2586 (FIG. 52B), miR100_2945 (FIG. 52C), and miR100_3330 (FIG. 52D), embedded in miR100 backbone. Arrow indicates typical start position of processed miRNA guide strand. (Bottom) Observed small RNAseq sequencing reads. On the left are observed sequences, on the right the number of observations across all samples (n=3-4 liver, n=6 striatal biopsy). Note that the sequence reads are DNA, and in the corresponding miRNA the sequence would be generated by substituting “U” bases for “T” in the reads. A small number of sequences were fusions between the amiR and endogenous miRs, but these are considered to be artifacts of the ligation reaction during the small RNAseq procedure and were excluded. By comparing the observed sequences to the pri-miRNA sequence on top, it. An be noted that in some cases 3′ modifications are occurring, such as addition of ‘A’ or ‘U’ bases (‘T’ in the DNA sequencing reads) at the 3′ terminus of the amiRNA.

FIGS. 53A-53C show knockdown of Ataxin-2 protein in vivo after AAV9 vectorized miRNA delivery into cerebrospinal fluid. As in FIG. 22, AAVs encoding miRNAs XD-14792 or XD-14887, embedded in the miR-1-1 backbone, or a control construct lacking a miRNA, were dosed, in this case injected bilaterally intracerebroventricularly (ICV) in postnatal day 0 mice, 3 microliters per hemisphere. amiRNAs were expressed either under the control of the neuron-specific Synapsin promoter (as in nucleotides 1128-1575 of SEQ ID NO:622 or nucleotides 1128-1575 of SEQ ID NO:623), or the ubiquitous CAG promoter. Brain tissue (cortex) was harvested at indicated timepoint after injection. (FIG. 53A) Diagrams are shown of the expression cassettes used. (FIG. 53B) Representative immunoblot from Western analysis, similar to FIG. 22. Immunoblotting was performed against Ataxin-2, Beta-actin and GFP. For each treatment dose administered per hemisphere is listed, calculated by qPCR titering against the GFP region in the vector genome. In FIG. 53C, quantification of signal intensity of Atxn2 protein or GFP protein, normalized to total protein signal intensity (Revert 700, Licor), are shown. Atxn2 signal is scaled to the average of CAG-MCS and SYN-MCS controls at the indicated times, and GFP signal is scaled to the GFP maximum for the 4 week timepoint or to the average GFP signal of multiple CAG-MCS vector IV dosed liver samples that were loaded onto each Western blot for the 8 week timepoint. Each point represents data from an individual cortex (from a single animal), averaging across technical replicates. Error bars show standard deviation across technical replicates. A reduction in Atxn2 levels relative to control AAV vectors (MCS) is apparent for CAG vectors expressing the XD-14792 miR at 4 and 8 week timepoints, and for the 8 week timepoint for vectors with the Synapsin promoter.

FIGS. 54A-54B show representative immunofluorescence micrographs of tissue sections of cortex and cerebellum from animals dosed i.c.v. with AAV9 control or amiRNA vectors expressing (XD-14792 in miR-1-1 backbone, SEQ ID NO:1133), as in FIG. 53. Red corresponds to indirect immunofluorescence signal for anti-Atxn2 antibodies; Green to anti-GFP signal; and blue are nuclei (Dapi stained). In FIG. 54A, presumptive layer 5 cortical pyramidal neurons are seen, with apical dendrites projecting up in the image. Intensity from the GFP reporter is present in neurons, which are likely transduced with the AAV. On the left, GFP-expressing neurons in the animal transduced with the control amiRNA also have strong Atxn2 (red) signal, and neurons can be clearly seen with both GFP and Atxn2 signal. On the right, which corresponds to an image of tissue from an animal dosed with an ATXN2 amiRNA (XD-14792 in miR-1-1 backbone, SEQ ID NO:1133) expressing vector, by contrast, neurons with strong GFP intensity do not also have strong Atxn2 intensity, and overall the number of neurons with strong Atxn2 signal appears to be reduced. FIG. 54B shows similar results as FIG. 54A, but captures Purkinje cells in the cerebellum. On the right, the image shows Cerebellar tissue from an animal injected with Atxn2 amiRNA (XD-14792 in miR-1-1 backbone, SEQ ID NO:1133) expressing vector. GFP labeled, AAV transduced Purkinje cells do not have strong Atxn2 signal, whereas Purkinje cells lacking GFP transduction have strong Atxn2 expression. By contrast, on the left, which corresponds to an image from an animal dosed with control vector, cells with GFP signal also have Atxn2 signal.

DETAILED DESCRIPTION

Expansions of ATXN2 polyglutamine repeat to a length of 34 or longer causes spinocerebellar ataxia type 2 (SCA2). Moreover, intermediate length polyglutamine expansions in ATXN2 increase risk of ALS. Reduction of ATXN2 levels has been demonstrated to have therapeutic benefit in animal models of spinocerebellar ataxia-2 and ALS. Knocking down the ATXN2 protein using nucleic acid based therapies alleviates the progressive neurodegeneration that occurs in animal models expressing a variant of the human ATXN2 containing an expanded polyglutamine repeat. In an animal model of ALS, which overexpresses the TDP-43 protein, a component of the most common neuropathology found in patients with ALS, animals normally develop a progressive death of motor neurons. However, breeding these animals with ATXN2 knock out mice dramatically increased survival time (Elden et al., Nature (2010) 466:7310). Similarly, reducing ATXN2 protein levels by introducing antisense oligonucleotide nucleic acids also increased survival of TDP-43 transgenic mice. Lowering ATXN2 levels markedly increased lifespan and improved motor function in TDP-43 transgenic mice and decreased the burden of TDP-43 inclusions. AXTN2 may modulate toxicity by affecting the aggregation propensity of TDP-43. TDP-43 proteinopathy has also been observed in a number of neurodegenerative diseases, including ALS, FTD, primary lateral sclerosis, progressive muscular atrophy, limbic-predominant age-related TDP-43 encephalopathy, chronic traumatic encephalopathy, dementia with Lewy bodies, corticobasal degeneration, progressive supranuclear palsy (PSP), dementia Parkinsonism ALS complex of guam (G-PDC), Pick's disease, hippocampal sclerosis, Huntington's disease, Parkinson's disease, and Alzheimer's disease. Thus, reducing ATXN2 levels may be useful for treating neurodegenerative diseases where ATXN2 is a causative agent (e.g., SCA2), as well as neurodegenerative diseases where ATXN2 is not the causative agent but modifies TDP-43 pathological aggregation.

Aspects of the invention relate to inhibitory nucleic acids (e.g., siRNAs, shRNAs, miRNAs, including artificial miRNAs) that when administered to a subject reduce the expression or activity of Ataxin-2 in the subject. Accordingly, compositions and methods provided in the present disclosure are useful for the treatment of neurodegenerative diseases, including spinocerebellar ataxia type 2 (SCA2), amyotrophic lateral sclerosis (ALS), Alzheimer's frontotemporal dementia (FTD), parkinsonism, and conditions associated with TDP-43 proteinopathies.

Prior to setting forth this disclosure in more detail, it may be helpful to an understanding thereof to provide definitions of certain terms to be used herein. Additional definitions are set forth throughout this disclosure.

In the present description, any concentration range, percentage range, ratio range, or integer range is to be understood to include the value of any integer within the recited range and, when appropriate, fractions thereof (such as one tenth and one hundredth of an integer), unless otherwise indicated. Also, any number range recited herein relating to any physical feature, such as polymer subunits, size or thickness, are to be understood to include any integer within the recited range, unless otherwise indicated. As used herein, the term “about” means±20% of the indicated range, value, or structure, unless otherwise indicated. It should be understood that the terms “a” and “an” as used herein refer to “one or more” of the enumerated components. The use of the alternative (e.g., “or”) should be understood to mean either one, both, or any combination thereof of the alternatives. As used herein, the terms “include,” “have” and “comprise” are used synonymously, which terms and variants thereof are intended to be construed as non-limiting.

As used herein, the term “nucleic acid” or “polynucleotide” refer to any nucleic acid polymer composed of covalently linked nucleotide subunits, such as polydeoxyribonucleotides or polyribonucleotides. Examples of nucleic acids include RNA and DNA.

As used herein, “RNA” refers to a molecule comprising one or more ribonucleotides and includes double-stranded RNA, single-stranded RNA, isolated RNA, synthetic RNA, recombinant RNA, as well as modified RNA that differs from naturally-occurring RNA by the addition, deletion, substitution, and/or alternation of one or more nucleotides. Nucleotides of RNA molecules may comprise standard nucleotides or non-standard nucleotides, such as non-naturally occurring nucleotides or chemically synthesized nucleotides.

As used herein, “DNA” refers to a molecule comprising one or more deoxyribonucleotides and includes double-stranded DNA, single-stranded DNA, isolated DNA, synthetic DNA, recombinant DNA, as well as modified DNA that differs from naturally-occurring DNA by the addition, deletion, substitution, and/or alteration of one or more nucleotides. Nucleotides of DNA molecules may comprise standard nucleotides or non-standard nucleotides, such as non-naturally occurring nucleotides or chemically synthesized nucleotides.

“Isolated” refers to a substance that has been isolated from its natural environment or artificially produced. As used herein with respect to a cell, “isolated” refers to a cell that has been isolated from its natural environment (e.g., from a subject, organ, tissue, or bodily fluid). As used herein with respect to a nucleic acid, “isolated” refers to a nucleic acid that has been isolated or purified from its natural environment (e.g., from a cell, cell organelle, or cytoplasm), recombinantly produced, amplified, or synthesized. In embodiments, an isolated nucleic acid includes a nucleic acid contained within a vector.

As used herein, the term “wild-type” or “non-mutant” form of a gene refers to a nucleic acid that encodes a protein associated with normal or non-pathogenic activity (e.g., a protein lacking a mutation, such as a repeat region expansion that results in higher risk of developing, onset, or progression of a neurodegenerative disease).

As used herein, the term “mutation” refers to any change in the structure of a gene, e.g., gene sequence, resulting in an altered form of the gene, which may be passed onto subsequent generations (hereditary mutation) or not (somatic mutation). Gene mutations include the substitution, insertion, or deletion of a single base in DNA or the substitution, insertion, deletion, or rearrangement of multiple bases or larger sections of genes or chromosomes, including repeat expansions.

As used herein, the term “Ataxin 2” or “ATNX2” refers to a protein encoded by the ATXN2 gene, which contains a polyglutamine (polyQ, CAG repeat) tract. ATXN2 gene or transcript may refer to normal alleles of ATXN2, which usually have 22 or 23 repeats, or mutated alleles having intermediate (˜24-32 repeats) or longer repeat expansions (˜33 to >100 repeats). In some embodiments, ATXN2 refers to mammalian ATNX2, including human ATXN2. In some embodiments, wild-type ATXN2 refers to a protein sequence of Q99700.2 as set forth in SEQ ID NO:1 or naturally occurring variants thereof. In some embodiments, wild-type ATXN2 nucleic acid refers to a nucleic acid sequence of NM_002973.3 (SEQ ID NO:2), ENST00000377617.7, ENST00000550104.5, ENST00000608853.5, or ENST00000616825.4, or naturally occurring variants thereof.

As used herein, the term “inhibitory nucleic acid” refers to a nucleic acid that comprises a guide strand sequence that hybridizes to at least a portion of a target nucleic acid, e.g., ATXN2 RNA, mRNA, pre-mRNA, or mature mRNA, and inhibits its expression or activity. An inhibitory nucleic acid may target a protein coding region (e.g., exon) or non-coding region (e.g., 5′UTR, 3′UTR, intron, etc.) of a target nucleic acid. In some embodiments, an inhibitory nucleic acid is a single stranded or double stranded molecule. An inhibitory nucleic acid may further comprise a passenger strand sequence on a separate strand (e.g., double stranded duplex) or in the same strand (e.g., single stranded, self-annealing duplex structure). In some embodiments, an inhibitory nucleic acid is an RNA molecule, such as a siRNA, shRNA, miRNA, or dsRNA.

As used herein, a “microRNA” or “miRNA” refers to a small non-coding RNA molecule capable of mediating silencing of a target gene by cleavage of the target mRNA, translational repression of the target mRNA, target mRNA degradation, or a combination thereof. Typically, miRNA is transcribed as a hairpin or stem-loop (e.g., having a self-complementary, single-stranded backbone) duplex structure, referred to as a primary miRNA (pri-miRNA), which is enzymatically processed (e.g., by Drosha, DGCR8, Pasha, etc.) into a pre-miRNA. Pre-miRNA is exported into the cytoplasm, where it is enzymatically processed by Dicer to produce a miRNA duplex with the passenger strand and then a single-stranded mature miRNA molecule, which is subsequently loaded into the RNA-induced silencing complex (RISC). Reference to a miRNA may include synthetic or artificial miRNAs.

As used herein, a “synthetic miRNA” or “artificial miRNA” or “amiRNA” refers to an endogenous, modified, or synthetic pri-miRNA or pre-miRNA (e.g., miRNA backbone or scaffold) in which the endogenous miRNA guide sequence and passenger sequence within the stem sequence have been replaced with a miRNA guide sequence and a miRNA passenger sequence that direct highly efficient RNA silencing of the targeted gene (see, e.g., Eamens et al. (2014), Methods Mol. Biol. 1062:211-224). In some embodiments, the nature of the complementarity of the guide and passenger sequences (e.g., number of bases, position of mismatches, types of bulges, etc.) can be similar or different from the nature of complementarity of the guide and passenger sequences in the endogenous miRNA backbone upon which the synthetic miRNA is constructed.

As used herein, the term “microRNA backbone,” “miR backbone,” “microRNA scaffold,” or “miR scaffold” refers to a pri-miRNA or pre-miRNA scaffold, with the stem sequence replaced by a miRNA of interest, and is capable of producing a functional, mature miRNA that directs RNA silencing at the gene targeted by the miRNA of interest. A miR backbone comprises a 5′ flanking region (also referred to 5′ miR context, ≥9 nucleotides), a stem region comprising the miRNA duplex (guide strand sequence and passenger strand sequence) and basal stem (5′ and 3′, each about 4-13 nucleotides), at least one loop motif region including the terminal loop (≥10 nucleotides for terminal loop), a 3′ flanking region (also referred to 3′ miR context, ≥9 nucleotides), and optionally one or more bulges in the stem. A miR backbone may be derived completely or partially from a wild type miRNA scaffold or be a completely artificial sequence.

As used herein, the term “antisense strand sequence” or “guide strand sequence” of an inhibitory nucleic acid refers to a sequence that is substantially complementary (e.g., at least 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% complementary) to a region of about 10-50 nucleotides (e.g., about 15-30, 16-25, 18-23, or 19-22 nucleotides) of the mRNA of the gene targeted for silencing. The antisense sequence is sufficiently complementary to the target mRNA sequence to direct target-specific silencing, e.g., to trigger the destruction of the target mRNA by the RNAi machinery or process. In some embodiments, the antisense sequence or guide strand sequence refers to the mature sequence remaining following cleavage by Dicer.

As used herein, the term “sense sequence” or “passenger strand sequence” of an inhibitory nucleic acid refers to a sequence that is homologous to the target mRNA and partially or completely complementary to the antisense strand sequence or guide strand sequence of an inhibitory nucleic acid. The antisense strand sequence and sense strand sequence of an inhibitory nucleic acid are hybridized to form a duplex structure (e.g., forming a double-stranded duplex or single-stranded self-annealing duplex structure). In some embodiments, the sense sequence or passenger strand sequence refers to the mature sequence remaining following cleavage by Dicer.

As used herein, a “duplex,” when used in reference to an inhibitory nucleic acid, refers to two nucleic acid strands (e.g., a guide strand and passenger strand) hybridizing together to form a duplex structure. A duplex may be formed by two separate nucleic acid strands or by a single nucleic acid strand having a region of self-complementarity (e.g., hairpin or stem-loop).

As used herein, the term “complementary” refers to the ability of polynucleotides to form base pairs with each other. Base pairs are typically formed by hydrogen bonds between nucleotide subunits in antiparallel polynucleotide strands or a single, self-annealing polynucleotide strand. Complementary polynucleotide strands can form base pairs in the Watson-Crick manner (e.g., A to T, A to U, C to G), or in any other manner that allows for the formation of duplexes. As apparent to skilled persons in the art, when using RNA as opposed to DNA, uracil rather than thymine is the base that is considered to be complementary to adenosine. Furthermore, when a “U” is denoted in the context of the present invention, the ability to substitute a “T” is understood, unless otherwise stated. Complementarity also encompasses Watson-Crick base pairing between non-modified and modified nucleobases (e.g., 5-methyl cytosine substituted for cytosine). Full complementarity, perfect complementarity or 100% complementarity between two polynucleotide strands is where each nucleotide of one polynucleotide strand can form hydrogen bond with a nucleotide unit of a second polynucleotide strand. % complementarity refers to the number of nucleotides of a contiguous nucleotide sequence in a nucleic acid molecule that are complementary to an aligned reference sequence (e.g., a target mRNA, passenger strand), divided by the total number of nucleotides and multiplying by 100. In such an alignment, a nucleobase/nucleotide which does not form a base pair is called a mismatch. Insertions and deletions are not permitted in calculating % complementarity of a contiguous nucleotide sequence. It is understood by skilled persons in the art that in calculating complementarity, chemical modifications to nucleobases are not considered as long as the Watson-Crick base pairing capacity of the nucleobase is retained (e.g., 5-methyl cytosine is considered the same as cytosine for the purpose of calculating % complementarity).

The “percent identity” between two or more nucleic acid sequences refers to the proportion nucleotides of a contiguous nucleotide sequence in a nucleic acid molecule that are shared by a reference sequence (i.e., % identity=number of identical nucleotides/total number of nucleotides in the aligned region (e.g., the contiguous nucleotide sequence)×100). Insertions and deletions are not permitted in the calculation of % identity of a contiguous nucleotide sequence. It is understood by skilled persons in the art that in calculating identity, chemical modifications to nucleobases are not considered as long as the Watson-Crick base pairing capacity of the nucleobase is retained (e.g., 5-methyl cytosine is considered the same as cytosine for the purpose of calculating % identity).

As used herein, the term “hybridizing” or “hybridizes” refers to two nucleic acids strands forming hydrogen bonds between base pairs on antiparallel strands, thereby forming a duplex. The strength of hybridization between two nucleic acid strands may be described by the melting temperature (T_m), defined as at a given ionic strength and pH, the temperature at which 50% of a target sequence hybridizes to a complementary polynucleotide.

As used herein, “expression construct” refers to any type of genetic construct containing a nucleic acid (e.g., transgene) in which part or all of the nucleic acid encoding sequence is capable of being transcribed. In some embodiments, expression includes transcription of the nucleic acid, for example, to generate a biologically-active polypeptide product or inhibitory RNA (e.g., siRNA, shRNA, miRNA) from a transcribed gene. In some embodiments, the transgene is operably linked to expression control sequences.

As used herein, the term “transgene” refers to an exogenous nucleic acid that has been transferred naturally or by genetic engineering means into another cell and is capable of being transcribed, and optionally translated.

As used herein, the term “gene expression” refers to the process by which a nucleic acid is transcribed from a nucleic acid molecule, and often, translated into a peptide or protein. The process can include transcription, post-transcriptional control, post-transcriptional modification, translation, post-translational control, post translational modification, or any combination thereof. Reference to a measurement of “gene expression” may refer to measurement of the product of transcription (e.g., RNA or mRNA), the product of translation (e.g., peptides or proteins).

As used herein, the term “inhibit expression of a gene” means to reduce, down-regulate, suppress, block, lower, or stop expression of the gene. The expression product of a gene can be a RNA molecule transcribed from the gene (e.g., an mRNA) or a polypeptide translated from an mRNA transcribed from the gene. Typically a reduction in the level of an mRNA results in a reduction in the level of a polypeptide translated therefrom. The level of expression may be determined using standard techniques for measuring mRNA or protein.

As used herein, “vector” refers to a genetic construct that is capable of transporting a nucleic acid molecule (e.g., transgene encoding inhibitory nucleic acid) between cells and effecting expression of the nucleic acid molecule when operably-linked to suitable expression control sequences. Expression control sequences may include transcription initiation, termination, promoter and enhancer sequences; efficient RNA processing signals such as splicing and polyadenylation (polyA) signals; sequences that stabilize cytoplasmic mRNA; sequences that enhance translation efficiency (i.e., Kozak consensus sequence); sequences that enhance protein stability; and when desired, sequences that enhance secretion of the encoded product. The vector may be a plasmid, phage particle, transposon, cosmid, phagemid, chromosome, artificial chromosome, virus, virion, etc. Once transformed into a suitable host cell, the vector may replicate and function independently of the host genome, or may, in some instances, integrate into the genome itself.

As used herein, “host cell” refers to any cell that contains, or is capable of containing a composition of interest, e.g., an inhibitory nucleic acid. In embodiments, a host cell is a mammalian cell, such as a rodent cell, (mouse or rat) or primate cell (monkey, chimpanzee, or human). In embodiments, a host cell may be in vitro or in vivo. In embodiments, a host cell may be from an established cell line or primary cells. In embodiments, a host cell is a cell of the CNS, such as a neuron, glial cell, astrocyte, and microglial cell.

As used herein, “neurodegenerative disease” or “neurodegenerative disorder” refers to diseases or disorders that exhibit neural cell death as a pathological state. A neurodegenerative disease may exhibit chronic neurodegeneration, e.g., slow, progressive neural cell death over a period of several years, or acute neurodegeneration, e.g., sudden onset or neural cell death. Examples of chronic, neurodegenerative diseases include Alzheimer's disease, Parkinson's disease, Huntington's disease, spinocerebellar ataxia type 2 (SCA2), frontotemporal dementia (FTD), and amyotrophic lateral schlerosis (ALS). Chronic neurodegenerative diseases include diseases that feature TDP-43 proteinopathy, which is characterized by nucleus to cytoplasmic mislocalization, deposition of ubiquitinated and hyper-phosphorylated TDP-43 into inclusion bodies, protein truncation leading to formation of toxic C-terminal TDP-43 fragments, and protein aggregation. TDP-43 proteinopathy diseases include ALS, FTD, primary lateral sclerosis, progressive muscular atrophy, limbic-predominant age-related TDP-43 encephalopathy, chronic traumatic encephalopathy, dementia with Lewy bodies, corticobasal degeneration, progressive supranuclear palsy (PSP), dementia Parkinsonism ALS complex of guam (G-PDC), Pick's disease, hippocampal sclerosis, Huntington's disease, Parkinson's disease, and Alzheimer's disease. Acute neurodegeneration may be caused by ischemia (e.g., stroke, traumatic brain injury), axonal transection by demyelination or trauma (e.g., spinal cord injury or multiple sclerosis). A neurodegenerative disease may exhibit death of mainly one type of neuron or of multiple types of neurons.

As used herein, “subject,” “patient,” and “individual” are used interchangeably herein and refer to living organisms (e.g., mammals) selected for treatment or therapy. Examples of subjects include human and non-human mammals, such as primates (monkey, chimpanzee), cows, horses, sheep, dogs, cats, rats, mice, guinea pigs, pigs, and transgenic species thereof.

Inhibitory Nucleic Acids

In one aspect, the disclosure provides isolated inhibitory nucleic acids that inhibit expression or activity of Ataxin 2 (ATXN2). The inhibitory nucleic acid is a nucleic acid that specifically binds (e.g., hybridizes to) at least a portion of the ATXN2 nucleic acid, such as an ATXN2 RNA, pre-mRNA, mRNA, and inhibits its expression or activity. In some embodiments, the inhibitory nucleic acid is complementary to a protein coding region or non-coding region (e.g., 5′UTR, 3′UTR, intron, etc.) of ATXN2. In some embodiments, the inhibitory nucleic acid is complementary to a wild type ATXN2 nucleic acid or a naturally occurring variant thereof. In some embodiments, the ATXN2 gene encodes a polypeptide identified by NCBI Reference Sequence NP_002964.4 or NP_002964.3. In some embodiments, an ATXN2 transcript comprises the sequence set forth in SEQ ID NO:2 or encodes an amino acid sequence set forth in SEQ ID NO:1. In some embodiments, the ATXN2 allele contains approximately 22 CAG trinucleotide repeats. In some embodiments, the ATXN2 allele has at least 22 CAG trinucleotide repeats, at least 24 CAG trinucleotide repeats, at least 27 CAG trinucleotide repeats, at least 30 CAG trinucleotide repeats, or at least 33 or more CAG trinucleotide repeats. In some embodiments, the inhibitory nucleic acid is single stranded or double-stranded. In some embodiments, the inhibitory nucleic acid is a siRNA, shRNA, miRNA, or dsRNA.

In some embodiments, the inhibitory nucleic acid is capable of inhibiting expression or activity of ATXN2 by at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90% at least 95% or more in a cell compared to the expression level of ATXN2 in a cell that has not been contacted with the inhibitory nucleic acid. In some embodiments, the inhibitory nucleic acid is capable of inhibiting expression or activity of ATXN2 by 10-20%, 10-30%, 10-40%, 10-50%, 10-60%, 10-70%, 10-80%, 10-90%, 10-95%, 20-30%, 20-40%, 20-50%, 20-60%, 20-70%, 20-80%, 20-90%, 20-95%, 20-100%, 30-40%, 30-50%, 30-60%, 30-70%, 30-80%, 30-90%, 30-95%, 30-100%, 40-50%, 40-60%, 40-70%, 40-80%, 40-90%, 40-95%, 40-100%, 50-60%, 50-70%, 50-80%, 50-90%, 50-95%, 50-100%, 60-70%, 60-80%, 60-90%, 60-95%, 60-100%, 70-80%, 70-90%, 70-95%, 70-100%, 80-90%, 80-95%, 80-100%, 90-95%, 90-100% compared to the expression level of ATXN2 in a cell that has not been contacted with the inhibitory nucleic acid. Methods of measuring ATXN2 expression, e.g., levels of RNA, mRNA polypeptides, are known in the art including those described herein.

In some embodiments, the inhibitory nucleic acid comprises a guide strand sequence comprising or consisting of the nucleic acid sequence set forth in any one of the guide sequences in Tables 1, 3, 9, 11, 12, 13, 19, 23, 24, and 25. In some embodiments, the inhibitory nucleic acid comprises a guide strand sequence comprising or consisting of the nucleic acid sequence set forth in any one of SEQ ID NOS: 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 124, 126, 128, 130, 132, 134, 136, 138, 140, 142, 144, 146, 148, 150, 152, 154, 156, 158, 160, 162, 164, 166, 168, 170, 172, 174, 176, 178, 180, 182, 184, 186, 188, 190, 192, 194, 196, 198, 200, 202, 204, 206, 208, 210, 212, 214, 216, 218, 220, 222, 224, 226, 228, 230, 232, 234, 236, 238, 240, 242, 244, 246, 248, 250, 252, 254, 256, 258, 260, 262, 264, 266, 268, 270, 272, 274, 276, 278, 280, 282, 284, 286, 288, 290, 292, 294, 296, 298, 300, 302, 304, 306, 308, 310, 312, 314, 316, 318, 320, 324, 326, 328, 330, 332, 334, 336, 338, 340, 342, 344, 346, 348, 350, 352, 354, 356, 358, 360, 362, 364, 366, 368, 370, 372, 374, 376, 378, 380, 382, 384, 386, 388, 390, 392, 394, 396, 398, 400, 402, 404, 406, 408, 410, 412, 414, 416, 418, 420, 422, 424, 426, 428, 430, 432, 434, 436, 1176-1288, 1811-1827, 2015, 2065, 2083, 2152, 2203, and 2209.

In some embodiments, the inhibitory nucleic acid comprises a guide strand sequence comprising or consisting of the nucleic acid sequence set forth in any one of the guide sequences in Tables 1, 3, 9, 11, 12, 13, 19, 23, 24, and 25, e.g., any one of SEQ ID NOS: 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 124, 126, 128, 130, 132, 134, 136, 138, 140, 142, 144, 146, 148, 150, 152, 154, 156, 158, 160, 162, 164, 166, 168, 170, 172, 174, 176, 178, 180, 182, 184, 186, 188, 190, 192, 194, 196, 198, 200, 202, 204, 206, 208, 210, 212, 214, 216, 218, 220, 222, 224, 226, 228, 230, 232, 234, 236, 238, 240, 242, 244, 246, 248, 250, 252, 254, 256, 258, 260, 262, 264, 266, 268, 270, 272, 274, 276, 278, 280, 282, 284, 286, 288, 290, 292, 294, 296, 298, 300, 302, 304, 306, 308, 310, 312, 314, 316, 318, 320, 324, 326, 328, 330, 332, 334, 336, 338, 340, 342, 344, 346, 348, 350, 352, 354, 356, 358, 360, 362, 364, 366, 368, 370, 372, 374, 376, 378, 380, 382, 384, 386, 388, 390, 392, 394, 396, 398, 400, 402, 404, 406, 408, 410, 412, 414, 416, 418, 420, 422, 424, 426, 428, 430, 432, 434, 436, 1176-1288, 1811-1827, 2015, 2065, 2083, 2152, 2203, and 2209, with at least 1, 2, 3, 4, or 5 mismatches to the target ATXN2 mRNA sequence.

In some embodiments, the inhibitory nucleic acid comprises a guide strand sequence comprising or consisting of a nucleic acid sequence that is at least 60%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 99%, or 100% identical to any one of the guide sequences in Tables 1, 3, 9, 11, 12, 13, 19, 23, 24, and 25, e.g., any one of SEQ ID NOS: 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 124, 126, 128, 130, 132, 134, 136, 138, 140, 142, 144, 146, 148, 150, 152, 154, 156, 158, 160, 162, 164, 166, 168, 170, 172, 174, 176, 178, 180, 182, 184, 186, 188, 190, 192, 194, 196, 198, 200, 202, 204, 206, 208, 210, 212, 214, 216, 218, 220, 222, 224, 226, 228, 230, 232, 234, 236, 238, 240, 242, 244, 246, 248, 250, 252, 254, 256, 258, 260, 262, 264, 266, 268, 270, 272, 274, 276, 278, 280, 282, 284, 286, 288, 290, 292, 294, 296, 298, 300, 302, 304, 306, 308, 310, 312, 314, 316, 318, 320, 324, 326, 328, 330, 332, 334, 336, 338, 340, 342, 344, 346, 348, 350, 352, 354, 356, 358, 360, 362, 364, 366, 368, 370, 372, 374, 376, 378, 380, 382, 384, 386, 388, 390, 392, 394, 396, 398, 400, 402, 404, 406, 408, 410, 412, 414, 416, 418, 420, 422, 424, 426, 428, 430, 432, 434, 436, 1176-1288, 1811-1827, 2015, 2065, 2083, 2152, 2203, and 2209.

In some embodiments, the inhibitory nucleic acid comprises a guide strand sequence comprising or consisting of at least 15, 16, 17, 18, 19, 20, 21, or 22 contiguous nucleotides of a sequence of any one of the guide sequences in Tables 1, 3, 9, 11, 12, 13, 19, 23, 24, and 25, e.g., any one of SEQ ID NOS: 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 124, 126, 128, 130, 132, 134, 136, 138, 140, 142, 144, 146, 148, 150, 152, 154, 156, 158, 160, 162, 164, 166, 168, 170, 172, 174, 176, 178, 180, 182, 184, 186, 188, 190, 192, 194, 196, 198, 200, 202, 204, 206, 208, 210, 212, 214, 216, 218, 220, 222, 224, 226, 228, 230, 232, 234, 236, 238, 240, 242, 244, 246, 248, 250, 252, 254, 256, 258, 260, 262, 264, 266, 268, 270, 272, 274, 276, 278, 280, 282, 284, 286, 288, 290, 292, 294, 296, 298, 300, 302, 304, 306, 308, 310, 312, 314, 316, 318, 320, 324, 326, 328, 330, 332, 334, 336, 338, 340, 342, 344, 346, 348, 350, 352, 354, 356, 358, 360, 362, 364, 366, 368, 370, 372, 374, 376, 378, 380, 382, 384, 386, 388, 390, 392, 394, 396, 398, 400, 402, 404, 406, 408, 410, 412, 414, 416, 418, 420, 422, 424, 426, 428, 430, 432, 434, 436, 1176-1288, 1811-1827, 2015, 2065, 2083, 2152, 2203, and 2209, preferably wherein the guide strand sequence retains positions 2-7 (“seed sequence”) of the selected SEQ ID NO.

In some embodiments, the inhibitory nucleic acid comprises a guide strand sequence comprising or consisting of a sequence of any one of the guide sequences of Tables 1, 3, 9, 11, 12, 13, 19, 23, 24, and 25, e.g., any one of SEQ ID NOS: 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 124, 126, 128, 130, 132, 134, 136, 138, 140, 142, 144, 146, 148, 150, 152, 154, 156, 158, 160, 162, 164, 166, 168, 170, 172, 174, 176, 178, 180, 182, 184, 186, 188, 190, 192, 194, 196, 198, 200, 202, 204, 206, 208, 210, 212, 214, 216, 218, 220, 222, 224, 226, 228, 230, 232, 234, 236, 238, 240, 242, 244, 246, 248, 250, 252, 254, 256, 258, 260, 262, 264, 266, 268, 270, 272, 274, 276, 278, 280, 282, 284, 286, 288, 290, 292, 294, 296, 298, 300, 302, 304, 306, 308, 310, 312, 314, 316, 318, 320, 324, 326, 328, 330, 332, 334, 336, 338, 340, 342, 344, 346, 348, 350, 352, 354, 356, 358, 360, 362, 364, 366, 368, 370, 372, 374, 376, 378, 380, 382, 384, 386, 388, 390, 392, 394, 396, 398, 400, 402, 404, 406, 408, 410, 412, 414, 416, 418, 420, 422, 424, 426, 428, 430, 432, 434, 436, 1176-1288, 1811-1827, 2015, 2065, 2083, 2152, 2203, and 2209, wherein 1, 2, 3, or 4 nucleotides at positions 19-22 differ from the selected SEQ ID NO (variant nucleotide(s)) such that the guide strand sequence is no longer complementary to the ATXN2 target sequence at the variant nucleotide(s).

In some embodiments, the inhibitory nucleic acid comprises a guide strand sequence of Table 12. In some embodiments, the inhibitory nucleic acid comprises a guide strand sequence comprising or consisting of the nucleic acid sequence set forth in any one of SEQ ID NOS: 12, 14, 40, 60, 100, 104, 108, 112, 124, 126, 128, 166, 198, 220, 242, 302, 306, 308, 330, 336, and 362. In some embodiments, the inhibitory nucleic acid comprises a guide strand sequence comprising or consisting of the nucleic acid sequence set forth in any one of SEQ ID NOS: 12, 14, 40, 60, 100, 104, 108, 112, 124, 126, 128, 166, 198, 220, 242, 302, 306, 308, 330, 336, and 362 with at least 1, 2, 3, 4, or 5 mismatches to the target ATXN2 mRNA sequence. In some embodiments, the inhibitory nucleic acid comprises a guide strand sequence comprising or consisting of a nucleic acid sequence that is at least 60%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 99%, or 100% identical to any one of SEQ ID NOS: 12, 14, 40, 60, 100, 104, 108, 112, 124, 126, 128, 166, 198, 220, 242, 302, 306, 308, 330, 336, and 362. In some embodiments, the inhibitory nucleic acid comprises a guide strand sequence comprising or consisting of at least 15, 16, 17, 18, 19, 20, 21, or 22 contiguous nucleotides of a sequence of any one of SEQ ID NOS: 12, 14, 40, 60, 100, 104, 108, 112, 124, 126, 128, 166, 198, 220, 242, 302, 306, 308, 330, 336, and 362, preferably wherein the guide strand sequence retains positions 2-7 (“seed sequence”) of the selected SEQ ID NO. In some embodiments, the inhibitory nucleic acid comprises a guide strand sequence comprising or consisting of a sequence of any one of SEQ ID NOS: 12, 14, 40, 60, 100, 104, 108, 112, 124, 126, 128, 166, 198, 220, 242, 302, 306, 308, 330, 336, and 362, wherein 1, 2, 3, or 4 nucleotides at positions 19-22 differ from the selected SEQ ID NO (variant nucleotide(s)) such that the guide strand sequence is no longer complementary to the ATXN2 target sequence at the variant nucleotide(s).

In some embodiments, the inhibitory nucleic acid comprises a guide strand sequence of Table 13. In some embodiments, the inhibitory nucleic acid comprises a guide strand sequence comprising or consisting of the nucleic acid sequence set forth in any one of SEQ ID NOS: 14, 40, 100, 108, 112, 128, 166, 198, 242, 308, 336, and 362. In some embodiments, the inhibitory nucleic acid comprises a guide strand sequence comprising or consisting of the nucleic acid sequence set forth in any one of SEQ ID NOS: 14, 40, 100, 108, 112, 128, 166, 198, 242, 308, 336, and 362 with at least 1, 2, 3, 4, or 5 mismatches to the target ATXN2 mRNA sequence. In some embodiments, the inhibitory nucleic acid comprises a guide strand sequence comprising or consisting of a nucleic acid sequence that is at least 60%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 99%, or 100% identical to any one of SEQ ID NOS: 14, 40, 100, 108, 112, 128, 166, 198, 242, 308, 336, and 362. In some embodiments, the inhibitory nucleic acid comprises a guide strand sequence comprising or consisting of at least 15, 16, 17, 18, 19, 20, 21, or 22 contiguous nucleotides of a sequence of any one of SEQ ID NOS: 14, 40, 100, 108, 112, 128, 166, 198, 242, 308, 336, and 362, preferably wherein the guide strand sequence retains positions 2-7 (“seed sequence”) of the selected SEQ ID NO. In some embodiments, the inhibitory nucleic acid comprises a guide strand sequence comprising or consisting of a sequence of any one of SEQ ID NOS: 14, 40, 100, 108, 112, 128, 166, 198, 242, 308, 336, and 362, wherein 1, 2, 3, or 4 nucleotides at positions 19-22 differ from the selected SEQ ID NO (variant nucleotide(s)) such that the guide strand sequence is no longer complementary to the ATXN2 target sequence at the variant nucleotide(s).

In some embodiments, the inhibitory nucleic acid comprises a guide strand sequence of Table 19. In some embodiments, the inhibitory nucleic acid comprises a guide strand sequence comprising or consisting of the nucleic acid sequence set forth in any one of SEQ ID NOS:1176-1288, 40, 108, and 166. In some embodiments, the inhibitory nucleic acid comprises a guide strand sequence comprising or consisting of the nucleic acid sequence set forth in any one of SEQ ID NOS:1176-1288, 40, 108, and 166, with at least 1, 2, 3, 4, or 5 mismatches to the target ATXN2 mRNA sequence. In some embodiments, the inhibitory nucleic acid comprises a guide strand sequence comprising or consisting of a nucleic acid sequence that is at least 60%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 99%, or 100% identical to any one of SEQ ID NOS:1176-1288, 40, 108, and 166. In some embodiments, the inhibitory nucleic acid comprises a guide strand sequence comprising or consisting of at least 15, 16, 17, 18, 19, 20, 21, or 22 contiguous nucleotides of a sequence of any one of SEQ ID NOS: 1176-1288, 40, 108, and 166, preferably wherein the guide strand sequence retains positions 2-7 (“seed sequence”) of the selected SEQ ID NO. In some embodiments, the inhibitory nucleic acid comprises a guide strand sequence comprising or consisting of a sequence of any one of SEQ ID NOS:1176-1288, 40, 108, and 166, wherein 1, 2, 3, or 4 nucleotides at positions 19-22 differ from the selected SEQ ID NO (variant nucleotide(s)) such that the guide strand sequence is no longer complementary to the ATXN2 target sequence at the variant nucleotide(s).

In some embodiments, the inhibitory nucleic acid comprises a guide strand sequence of Table 23. In some embodiments, the inhibitory nucleic acid comprises a guide strand sequence comprising or consisting of the nucleic acid sequence set forth in any one of SEQ ID NOS:1908-2007. In some embodiments, the inhibitory nucleic acid comprises a guide strand sequence comprising or consisting of the nucleic acid sequence set forth in any one of SEQ ID NOS:1908-2007, with at least 1, 2, 3, 4, or 5 mismatches to the target ATXN2 mRNA sequence. In some embodiments, the inhibitory nucleic acid comprises a guide strand sequence comprising or consisting of a nucleic acid sequence that is at least 60%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 99%, or 100% identical to any one of SEQ ID NOS:1908-2007. In some embodiments, the inhibitory nucleic acid comprises a guide strand sequence comprising or consisting of at least 15, 16, 17, 18, 19, 20, 21, or 22 contiguous nucleotides of a sequence of any one of SEQ ID NOS:1908-2007, preferably wherein the guide strand sequence retains positions 2-7 (“seed sequence”) of the selected SEQ ID NO. In some embodiments, the inhibitory nucleic acid comprises a guide strand sequence comprising or consisting of a sequence of any one of SEQ ID NOS:1908-2007, wherein 1, 2, 3, or 4 nucleotides at positions 19-22 differ from the selected SEQ ID NO (variant nucleotide(s)) such that the guide strand sequence is no longer complementary to the ATXN2 target sequence at the variant nucleotide(s).

In some embodiments, the inhibitory nucleic acid comprises a guide strand sequence of Table 24. In some embodiments, the inhibitory nucleic acid comprises a guide strand sequence comprising or consisting of the nucleic acid sequence set forth in any one of SEQ ID NOS:100, 112, 166, 202, 246, 306, 308, 314, 1180, 1185, 1196, 1200, 1211, 1213, 1215, 1216, 1224, 1811-1822, 1824-1827, 2015, 2065, 2083, 2152, 2203, and 2209. In some embodiments, the inhibitory nucleic acid comprises a guide strand sequence comprising or consisting of the nucleic acid sequence set forth in any one of SEQ ID NOS:100, 112, 166, 202, 246, 306, 308, 314, 1180, 1185, 1196, 1200, 1211, 1213, 1215, 1216, 1224, 1811-1822, 1824-1827, 2015, 2065, 2083, 2152, 2203, and 2209, with at least 1, 2, 3, 4, or 5 mismatches to the target ATXN2 mRNA sequence. In some embodiments, the inhibitory nucleic acid comprises a guide strand sequence comprising or consisting of a nucleic acid sequence that is at least 60%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 99%, or 100% identical to any one of SEQ ID NOS:100, 112, 166, 202, 246, 306, 308, 314, 1180, 1185, 1196, 1200, 1211, 1213, 1215, 1216, 1224, 1811-1822, 1824-1827, 2015, 2065, 2083, 2152, 2203, and 2209. In some embodiments, the inhibitory nucleic acid comprises a guide strand sequence comprising or consisting of at least 15, 16, 17, 18, 19, 20, 21, or 22 contiguous nucleotides of a sequence of any one of SEQ ID NOS:100, 112, 166, 202, 246, 306, 308, 314, 1180, 1185, 1196, 1200, 1211, 1213, 1215, 1216, 1224, 1811-1822, 1824-1827, 2015, 2065, 2083, 2152, 2203, and 2209, preferably wherein the guide strand sequence retains positions 2-7 (“seed sequence”) of the selected SEQ ID NO. In some embodiments, the inhibitory nucleic acid comprises a guide strand sequence comprising or consisting of a sequence of any one of SEQ ID NOS:100, 112, 166, 202, 246, 306, 308, 314, 1180, 1185, 1196, 1200, 1211, 1213, 1215, 1216, 1224, 1811-1822, 1824-1827, 2015, 2065, 2083, 2152, 2203, and 2209, wherein 1, 2, 3, or 4 nucleotides at positions 19-22 differ from the selected SEQ ID NO (variant nucleotide(s)) such that the guide strand sequence is no longer complementary to the ATXN2 target sequence at the variant nucleotide(s).

In some embodiments, the inhibitory nucleic acid comprises a guide strand sequence of Table 25. In some embodiments, the inhibitory nucleic acid comprises a guide strand sequence comprising or consisting of the nucleic acid sequence set forth in any one of SEQ ID NOS:1185, 1816, 1213, 1819, 2083, 1215, 1216, 1811, and 314. In some embodiments, the inhibitory nucleic acid comprises a guide strand sequence comprising or consisting of the nucleic acid sequence set forth in any one of SEQ ID NOS:1185, 1816, 1213, 1819, 2083, 1215, 1216, 1811, and 314, with at least 1, 2, 3, 4, or 5 mismatches to the target ATXN2 mRNA sequence. In some embodiments, the inhibitory nucleic acid comprises a guide strand sequence comprising or consisting of a nucleic acid sequence that is at least 60%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 99%, or 100% identical to any one of SEQ ID NOS:1185, 1816, 1213, 1819, 2083, 1215, 1216, 1811, and 314. In some embodiments, the inhibitory nucleic acid comprises a guide strand sequence comprising or consisting of at least 15, 16, 17, 18, 19, 20, 21, or 22 contiguous nucleotides of a sequence of any one of SEQ ID NOS:1185, 1816, 1213, 1819, 2083, 1215, 1216, 1811, and 314, preferably wherein the guide strand sequence retains positions 2-7 (“seed sequence”) of the selected SEQ ID NO. In some embodiments, the inhibitory nucleic acid comprises a guide strand sequence comprising or consisting of a sequence of any one of SEQ ID NOS:1185, 1816, 1213, 1819, 2083, 1215, 1216, 1811, and 314, wherein 1, 2, 3, or 4 nucleotides at positions 19-22 differ from the selected SEQ ID NO (variant nucleotide(s)) such that the guide strand sequence is no longer complementary to the ATXN2 target sequence at the variant nucleotide(s).

In some embodiments, the inhibitory nucleic acid comprises a guide strand sequence comprising or consisting of the nucleic acid sequence set forth in any one of SEQ ID NOS:1185, 1816, 1213, and 1811. In some embodiments, the inhibitory nucleic acid comprises a guide strand sequence comprising or consisting of the nucleic acid sequence set forth in any one of SEQ ID NOS:1185, 1816, 1213, and 1811, with at least 1, 2, 3, 4, or 5 mismatches to the target ATXN2 mRNA sequence. In some embodiments, the inhibitory nucleic acid comprises a guide strand sequence comprising or consisting of a nucleic acid sequence that is at least 60%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 99%, or 100% identical to any one of SEQ ID NOS:1185, 1816, 1213, and 1811. In some embodiments, the inhibitory nucleic acid comprises a guide strand sequence comprising or consisting of at least 15, 16, 17, 18, 19, 20, 21, or 22 contiguous nucleotides of a sequence of any one of SEQ ID NOS:1185, 1816, 1213, and 1811, preferably wherein the guide strand sequence retains positions 2-7 (“seed sequence”) of the selected SEQ ID NO. In some embodiments, the inhibitory nucleic acid comprises a guide strand sequence comprising or consisting of a sequence of any one of SEQ ID NOS:1185, 1816, 1213, and 1811, wherein 1, 2, 3, or 4 nucleotides at positions 19-22 differ from the selected SEQ ID NO (variant nucleotide(s)) such that the guide strand sequence is no longer complementary to the ATXN2 target sequence at the variant nucleotide(s).

In some embodiments, the inhibitory nucleic acid is an isolated siRNA duplex that targets ATXN2 mRNA to interfere with ATXN2 expression by mRNA degradation or translational inhibition. A siRNA duplex is a short, double stranded RNA comprising a guide strand, which is complementary to the target ATXN2 mRNA, and a passenger strand, which is homologous to the target ATNX2 mRNA. The guide strand and passenger strand hybridize together to form a duplex structure, and the guide strand has sufficient complementarity to the ATXN2 mRNA sequence to direct ATXN2-specific RNA interference. The guide strand of the siRNA duplex may be about 18 nucleotides, 19 nucleotides, 20 nucleotides, 21 nucleotides, 22 nucleotides, 23 nucleotides, 24 nucleotides, 25 nucleotides, 26 nucleotides, 27 nucleotides, 28 nucleotides, 29 nucleotides, or 30 nucleotides in length or 18-30, 18-29, 18-28, 18-27, 18-26, 18-25, 18-24, 18-23, 18-22, 18-20, 19-30, 19-29, 19-28, 19-27, 19-26, 19-25, 19-24, 19-23, 19-22, 19-21, 20-30, 20-29, 20-28, 20-27, 20-26, 20-25, 20-24, 20-23, 20-22, 21-30, 21-29, 21-28, 21-27, 21-26, 21-25, 21-24, 21-23, 22-30, 22-29, 22-28, 22-27, 22-26, 22-24, 23-30, 23-29, 23-28, 23-27, 23-26, 23-25, 24-30, 24-29, 24-28, 24-27, 24-26, 25-30, 25-29, 25-28, 25-27, 26-30, 26-29, 26-28, 27-30, 27-29, 28-30 nucleotides in length. The passenger strand of the siRNA duplex may be about 18 nucleotides, 19 nucleotides, 20 nucleotides, 21 nucleotides, 22 nucleotides, 23 nucleotides, 24 nucleotides, 25 nucleotides, 26 nucleotides, 27 nucleotides, 28 nucleotides, 29 nucleotides, or 30 nucleotides in length or 18-30, 18-29, 18-28, 18-27, 18-26, 18-25, 18-24, 18-23, 18-22, 18-20, 19-30, 19-29, 19-28, 19-27, 19-26, 19-25, 19-24, 19-23, 19-22, 19-21, 20-30, 20-29, 20-28, 20-27, 20-26, 20-25, 20-24, 20-23, 20-22, 21-30, 21-29, 21-28, 21-27, 21-26, 21-25, 21-24, 21-23, 22-30, 22-29, 22-28, 22-27, 22-26, 22-24, 23-30, 23-29, 23-28, 23-27, 23-26, 23-25, 24-30, 24-29, 24-28, 24-27, 24-26, 25-30, 25-29, 25-28, 25-27, 26-30, 26-29, 26-28, 27-30, 27-29, 28-30 nucleotides in length. In some embodiments, the siRNA duplex contains 2 or 3 nucleotide 3′ overhangs on each strand. In some embodiments, the 3′ overhangs are complementary to the ATXN2 transcript. In some embodiments, the guide strand and passenger strand of the siRNA duplex are at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, or 100% complementary to each other, not including any nucleotides in overhang(s).

In some embodiments, the siRNA duplex comprises a guide strand sequence comprising or consisting of the nucleic acid sequence set forth in any one of the guide sequences of Tables 1, 3, 9, 11, 12, 13, 19, 23, 24, and 25, e.g., any one of SEQ ID NOS: 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 124, 126, 128, 130, 132, 134, 136, 138, 140, 142, 144, 146, 148, 150, 152, 154, 156, 158, 160, 162, 164, 166, 168, 170, 172, 174, 176, 178, 180, 182, 184, 186, 188, 190, 192, 194, 196, 198, 200, 202, 204, 206, 208, 210, 212, 214, 216, 218, 220, 222, 224, 226, 228, 230, 232, 234, 236, 238, 240, 242, 244, 246, 248, 250, 252, 254, 256, 258, 260, 262, 264, 266, 268, 270, 272, 274, 276, 278, 280, 282, 284, 286, 288, 290, 292, 294, 296, 298, 300, 302, 304, 306, 308, 310, 312, 314, 316, 318, 320, 324, 326, 328, 330, 332, 334, 336, 338, 340, 342, 344, 346, 348, 350, 352, 354, 356, 358, 360, 362, 364, 366, 368, 370, 372, 374, 376, 378, 380, 382, 384, 386, 388, 390, 392, 394, 396, 398, 400, 402, 404, 406, 408, 410, 412, 414, 416, 418, 420, 422, 424, 426, 428, 430, 432, 434, 436, 1176-1288, 1811-1827, 2015, 2065, 2083, 2152, 2203, and 2209.

In some embodiments, the siRNA duplex comprises a guide strand sequence comprising or consisting of the nucleic acid sequence set forth in any one of the guide sequences of Tables 1, 3, 9, 11, 12, 13, 19, 23, 24, and 25, e.g., any one of SEQ ID NOS: 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 124, 126, 128, 130, 132, 134, 136, 138, 140, 142, 144, 146, 148, 150, 152, 154, 156, 158, 160, 162, 164, 166, 168, 170, 172, 174, 176, 178, 180, 182, 184, 186, 188, 190, 192, 194, 196, 198, 200, 202, 204, 206, 208, 210, 212, 214, 216, 218, 220, 222, 224, 226, 228, 230, 232, 234, 236, 238, 240, 242, 244, 246, 248, 250, 252, 254, 256, 258, 260, 262, 264, 266, 268, 270, 272, 274, 276, 278, 280, 282, 284, 286, 288, 290, 292, 294, 296, 298, 300, 302, 304, 306, 308, 310, 312, 314, 316, 318, 320, 324, 326, 328, 330, 332, 334, 336, 338, 340, 342, 344, 346, 348, 350, 352, 354, 356, 358, 360, 362, 364, 366, 368, 370, 372, 374, 376, 378, 380, 382, 384, 386, 388, 390, 392, 394, 396, 398, 400, 402, 404, 406, 408, 410, 412, 414, 416, 418, 420, 422, 424, 426, 428, 430, 432, 434, 436, 1176-1288, 1811-1827, 2015, 2065, 2083, 2152, 2203, and 2209, with at least 1, 2, 3, 4, or 5 mismatches to the target ATXN2 mRNA sequence.

In some embodiments, the siRNA duplex comprises a guide strand sequence comprising or consisting of a nucleic acid sequence that is at least 60%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 99%, or 100% identical to any one of the guide sequences of Tables 1, 3, 9, 11, 12, 13, 19, 23, 24, and 25, e.g., any one of SEQ ID NOS: 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 124, 126, 128, 130, 132, 134, 136, 138, 140, 142, 144, 146, 148, 150, 152, 154, 156, 158, 160, 162, 164, 166, 168, 170, 172, 174, 176, 178, 180, 182, 184, 186, 188, 190, 192, 194, 196, 198, 200, 202, 204, 206, 208, 210, 212, 214, 216, 218, 220, 222, 224, 226, 228, 230, 232, 234, 236, 238, 240, 242, 244, 246, 248, 250, 252, 254, 256, 258, 260, 262, 264, 266, 268, 270, 272, 274, 276, 278, 280, 282, 284, 286, 288, 290, 292, 294, 296, 298, 300, 302, 304, 306, 308, 310, 312, 314, 316, 318, 320, 324, 326, 328, 330, 332, 334, 336, 338, 340, 342, 344, 346, 348, 350, 352, 354, 356, 358, 360, 362, 364, 366, 368, 370, 372, 374, 376, 378, 380, 382, 384, 386, 388, 390, 392, 394, 396, 398, 400, 402, 404, 406, 408, 410, 412, 414, 416, 418, 420, 422, 424, 426, 428, 430, 432, 434, 436, 1176-1288, 1811-1827, 2015, 2065, 2083, 2152, 2203, and 2209.

In some embodiments, the siRNA duplex comprises a guide strand sequence comprising or consisting of at least 15, 16, 17, 18, 19, 20, 21, or 22 contiguous nucleotides of a sequence of any one of the guide sequences of Tables 1, 3, 9, 11, 12, 13, 19, 23, 24, and 25, e.g., any one of SEQ ID NOS: 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 124, 126, 128, 130, 132, 134, 136, 138, 140, 142, 144, 146, 148, 150, 152, 154, 156, 158, 160, 162, 164, 166, 168, 170, 172, 174, 176, 178, 180, 182, 184, 186, 188, 190, 192, 194, 196, 198, 200, 202, 204, 206, 208, 210, 212, 214, 216, 218, 220, 222, 224, 226, 228, 230, 232, 234, 236, 238, 240, 242, 244, 246, 248, 250, 252, 254, 256, 258, 260, 262, 264, 266, 268, 270, 272, 274, 276, 278, 280, 282, 284, 286, 288, 290, 292, 294, 296, 298, 300, 302, 304, 306, 308, 310, 312, 314, 316, 318, 320, 324, 326, 328, 330, 332, 334, 336, 338, 340, 342, 344, 346, 348, 350, 352, 354, 356, 358, 360, 362, 364, 366, 368, 370, 372, 374, 376, 378, 380, 382, 384, 386, 388, 390, 392, 394, 396, 398, 400, 402, 404, 406, 408, 410, 412, 414, 416, 418, 420, 422, 424, 426, 428, 430, 432, 434, 436, 1176-1288, 1811-1827, 2015, 2065, 2083, 2152, 2203, and 2209, preferably wherein the guide strand sequence retains positions 2-7 (“seed sequence”) of the selected SEQ ID NO.

In some embodiments, the siRNA duplex comprises a guide strand sequence comprising or consisting of a sequence of any one of the guide sequences of Tables 1, 3, 9, 11, 12, 13, 19, 23, 24, and 25, e.g., any one of SEQ ID NOS: 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 124, 126, 128, 130, 132, 134, 136, 138, 140, 142, 144, 146, 148, 150, 152, 154, 156, 158, 160, 162, 164, 166, 168, 170, 172, 174, 176, 178, 180, 182, 184, 186, 188, 190, 192, 194, 196, 198, 200, 202, 204, 206, 208, 210, 212, 214, 216, 218, 220, 222, 224, 226, 228, 230, 232, 234, 236, 238, 240, 242, 244, 246, 248, 250, 252, 254, 256, 258, 260, 262, 264, 266, 268, 270, 272, 274, 276, 278, 280, 282, 284, 286, 288, 290, 292, 294, 296, 298, 300, 302, 304, 306, 308, 310, 312, 314, 316, 318, 320, 324, 326, 328, 330, 332, 334, 336, 338, 340, 342, 344, 346, 348, 350, 352, 354, 356, 358, 360, 362, 364, 366, 368, 370, 372, 374, 376, 378, 380, 382, 384, 386, 388, 390, 392, 394, 396, 398, 400, 402, 404, 406, 408, 410, 412, 414, 416, 418, 420, 422, 424, 426, 428, 430, 432, 434, 436, 1176-1288, 1811-1827, 2015, 2065, 2083, 2152, 2203, and 2209, wherein 1, 2, 3, or 4 nucleotides at positions 19-22 differ from the selected SEQ ID NO (variant nucleotide(s)), such that the guide strand sequence is no longer complementary to the ATXN2 target sequence at the variant nucleotide(s).

In some embodiments, the siRNA duplex comprises a guide strand sequence of Table 12. In some embodiments, the siRNA duplex comprises a guide strand sequence comprising or consisting of the nucleic acid sequence set forth in any one of SEQ ID NOS: 12, 14, 40, 60, 100, 104, 108, 112, 124, 126, 128, 166, 198, 220, 242, 302, 306, 308, 330, 336, and 362. In some embodiments, the siRNA duplex comprises a guide strand sequence comprising or consisting of the nucleic acid sequence set forth in any one of SEQ ID NOS: 12, 14, 40, 60, 100, 104, 108, 112, 124, 126, 128, 166, 198, 220, 242, 302, 306, 308, 330, 336, and 362 with at least 1, 2, 3, 4, or 5 mismatches to the target ATXN2 mRNA sequence. In some embodiments, the siRNA duplex comprises a guide strand sequence comprising or consisting of a nucleic acid sequence that is at least 60%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 99%, or 100% identical to any one of SEQ ID NOS: 12, 14, 40, 60, 100, 104, 108, 112, 124, 126, 128, 166, 198, 220, 242, 302, 306, 308, 330, 336, and 362. In some embodiments, the siRNA duplex comprises a guide strand sequence comprising or consisting of at least 15, 16, 17, 18, 19, 20, 21, or 22 contiguous nucleotides of a sequence of any one of SEQ ID NOS: 12, 14, 40, 60, 100, 104, 108, 112, 124, 126, 128, 166, 198, 220, 242, 302, 306, 308, 330, 336, and 362, preferably wherein the guide strand sequence retains positions 2-7 (“seed sequence”) of the selected SEQ ID NO. In some embodiments, the siRNA duplex comprises a guide strand sequence comprising or consisting of a sequence of any one of SEQ ID NOS: 12, 14, 40, 60, 100, 104, 108, 112, 124, 126, 128, 166, 198, 220, 242, 302, 306, 308, 330, 336, and 362, wherein 1, 2, 3, or 4 nucleotides at positions 19-22 differ from the selected SEQ ID NO (variant nucleotide(s)), such that the guide strand sequence is no longer complementary to the ATXN2 target sequence at the variant nucleotide(s).

In some embodiments, the siRNA duplex comprises a guide strand sequence of Table 13. In some embodiments, the siRNA duplex comprises a guide strand sequence comprising or consisting of the nucleic acid sequence set forth in any one of SEQ ID NOS: 14, 40, 100, 108, 112, 128, 166, 198, 242, 308, 336, and 362. In some embodiments, the siRNA duplex comprises a guide strand sequence comprising or consisting of the nucleic acid sequence set forth in any one of SEQ ID NOS: 14, 40, 100, 108, 112, 128, 166, 198, 242, 308, 336, and 362 with at least 1, 2, 3, 4, or 5 mismatches to the target ATXN2 mRNA sequence. In some embodiments, the siRNA duplex comprises a guide strand sequence comprising or consisting of a nucleic acid sequence that is at least 60%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 99%, or 100% identical to any one of SEQ ID NOS: 14, 40, 100, 108, 112, 128, 166, 198, 242, 308, 336, and 362. In some embodiments, the siRNA duplex comprises a guide strand sequence comprising or consisting of at least 15, 16, 17, 18, 19, 20, 21, or 22 contiguous nucleotides of a sequence of any one of SEQ ID NOS: 14, 40, 100, 108, 112, 128, 166, 198, 242, 308, 336, and 362, preferably wherein the guide strand sequence retains positions 2-7 (“seed sequence”) of the selected SEQ ID NO. In some embodiments, the siRNA duplex comprises a guide strand sequence comprising or consisting of a sequence of any one of SEQ ID NOS: 14, 40, 100, 108, 112, 128, 166, 198, 242, 308, 336, and 362, wherein 1, 2, 3, or 4 nucleotides at positions 19-22 differ from the selected SEQ ID NO (variant nucleotide(s)), such that the guide strand sequence is no longer complementary to the ATXN2 target sequence at the variant nucleotide(s).

In some embodiments, the siRNA duplex comprises a guide strand sequence of Table 19. In some embodiments, the siRNA duplex comprises a guide strand sequence comprising or consisting of the nucleic acid sequence set forth in any one of SEQ ID NOS:1176-1288, 40, 108, and 166. In some embodiments, the siRNA duplex comprises a guide strand sequence comprising or consisting of the nucleic acid sequence set forth in any one of SEQ ID NOS:1176-1288, 40, 108, and 166, with at least 1, 2, 3, 4, or 5 mismatches to the target ATXN2 mRNA sequence. In some embodiments, the siRNA duplex comprises a guide strand sequence comprising or consisting of a nucleic acid sequence that is at least 60%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 99%, or 100% identical to any one of SEQ ID NOS:1176-1288, 40, 108, and 166. In some embodiments, the siRNA duplex comprises a guide strand sequence comprising or consisting of at least 15, 16, 17, 18, 19, 20, 21, or 22 contiguous nucleotides of a sequence of any one of SEQ ID NOS:1176-1288, 40, 108, and 166, preferably wherein the guide strand sequence retains positions 2-7 (“seed sequence”) of the selected SEQ ID NO. In some embodiments, the siRNA duplex comprises a guide strand sequence comprising or consisting of a sequence of any one of SEQ ID NOS:1176-1288, 40, 108, and 166, wherein 1, 2, 3, or 4 nucleotides at positions 19-22 differ from the selected SEQ ID NO (variant nucleotide(s)), such that the guide strand sequence is no longer complementary to the ATXN2 target sequence at the variant nucleotide(s).

In some embodiments, the siRNA duplex comprises a guide strand sequence of Table 23. In some embodiments, the siRNA duplex comprises a guide strand sequence comprising or consisting of the nucleic acid sequence set forth in any one of SEQ ID NOS:1908-2007. In some embodiments, the siRNA duplex comprises a guide strand sequence comprising or consisting of the nucleic acid sequence set forth in any one of SEQ ID NOS:1908-2007, with at least 1, 2, 3, 4, or 5 mismatches to the target ATXN2 mRNA sequence. In some embodiments, the siRNA duplex comprises a guide strand sequence comprising or consisting of a nucleic acid sequence that is at least 60%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 99%, or 100% identical to any one of SEQ ID NOS:1908-2007. In some embodiments, the siRNA duplex comprises a guide strand sequence comprising or consisting of at least 15, 16, 17, 18, 19, 20, 21, or 22 contiguous nucleotides of a sequence of any one of SEQ ID NOS:1908-2007, preferably wherein the guide strand sequence retains positions 2-7 (“seed sequence”) of the selected SEQ ID NO. In some embodiments, the siRNA duplex comprises a guide strand sequence comprising or consisting of a sequence of any one of SEQ ID NOS:1908-2007, wherein 1, 2, 3, or 4 nucleotides at positions 19-22 differ from the selected SEQ ID NO (variant nucleotide(s)), such that the guide strand sequence is no longer complementary to the ATXN2 target sequence at the variant nucleotide(s).

In some embodiments, the siRNA duplex comprises a guide strand sequence of Table 24. In some embodiments, the siRNA duplex comprises a guide strand sequence comprising or consisting of the nucleic acid sequence set forth in any one of SEQ ID NOS:100, 112, 166, 202, 246, 306, 308, 314, 1180, 1185, 1196, 1200, 1211, 1213, 1215, 1216, 1224, 1811-1822, 1824-1827, 2015, 2065, 2083, 2152, 2203, and 2209. In some embodiments, the siRNA duplex comprises a guide strand sequence comprising or consisting of the nucleic acid sequence set forth in any one of SEQ ID NOS:100, 112, 166, 202, 246, 306, 308, 314, 1180, 1185, 1196, 1200, 1211, 1213, 1215, 1216, 1224, 1811-1822, 1824-1827, 2015, 2065, 2083, 2152, 2203, and 2209, with at least 1, 2, 3, 4, or 5 mismatches to the target ATXN2 mRNA sequence. In some embodiments, the siRNA duplex comprises a guide strand sequence comprising or consisting of a nucleic acid sequence that is at least 60%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 99%, or 100% identical to any one of SEQ ID NOS:100, 112, 166, 202, 246, 306, 308, 314, 1180, 1185, 1196, 1200, 1211, 1213, 1215, 1216, 1224, 1811-1822, 1824-1827, 2015, 2065, 2083, 2152, 2203, and 2209. In some embodiments, the siRNA duplex comprises a guide strand sequence comprising or consisting of at least 15, 16, 17, 18, 19, 20, 21, or 22 contiguous nucleotides of a sequence of any one of SEQ ID NOS:100, 112, 166, 202, 246, 306, 308, 314, 1180, 1185, 1196, 1200, 1211, 1213, 1215, 1216, 1224, 1811-1822, 1824-1827, 2015, 2065, 2083, 2152, 2203, and 2209, preferably wherein the guide strand sequence retains positions 2-7 (“seed sequence”) of the selected SEQ ID NO. In some embodiments, the siRNA duplex comprises a guide strand sequence comprising or consisting of a sequence of any one of SEQ ID NOS:100, 112, 166, 202, 246, 306, 308, 314, 1180, 1185, 1196, 1200, 1211, 1213, 1215, 1216, 1224, 1811-1822, 1824-1827, 2015, 2065, 2083, 2152, 2203, and 2209, wherein 1, 2, 3, or 4 nucleotides at positions 19-22 differ from the selected SEQ ID NO (variant nucleotide(s)), such that the guide strand sequence is no longer complementary to the ATXN2 target sequence at the variant nucleotide(s).

In some embodiments, the siRNA duplex comprises a guide strand sequence of Table 25. In some embodiments, the siRNA duplex comprises a guide strand sequence comprising or consisting of the nucleic acid sequence set forth in any one of SEQ ID NOS:1185, 1816, 1213, 1819, 2083, 1215, 1216, 1811, and 314. In some embodiments, the siRNA duplex comprises a guide strand sequence comprising or consisting of the nucleic acid sequence set forth in any one of SEQ ID NOS:1185, 1816, 1213, 1819, 2083, 1215, 1216, 1811, and 314, with at least 1, 2, 3, 4, or 5 mismatches to the target ATXN2 mRNA sequence. In some embodiments, the siRNA duplex comprises a guide strand sequence comprising or consisting of a nucleic acid sequence that is at least 60%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 99%, or 100% identical to any one of SEQ ID NOS:1185, 1816, 1213, 1819, 2083, 1215, 1216, 1811, and 314. In some embodiments, the siRNA duplex comprises a guide strand sequence comprising or consisting of at least 15, 16, 17, 18, 19, 20, 21, or 22 contiguous nucleotides of a sequence of any one of SEQ ID NOS:1185, 1816, 1213, 1819, 2083, 1215, 1216, 1811, and 314, preferably wherein the guide strand sequence retains positions 2-7 (“seed sequence”) of the selected SEQ ID NO. In some embodiments, the siRNA duplex comprises a guide strand sequence comprising or consisting of a sequence of any one of SEQ ID NOS:1185, 1816, 1213, 1819, 2083, 1215, 1216, 1811, and 314, wherein 1, 2, 3, or 4 nucleotides at positions 19-22 differ from the selected SEQ ID NO (variant nucleotide(s)), such that the guide strand sequence is no longer complementary to the ATXN2 target sequence at the variant nucleotide(s).

In some embodiments, the siRNA duplex comprises a guide strand sequence comprising or consisting of the nucleic acid sequence set forth in any one of SEQ ID NOS:1185, 1816, 1213, and 1811. In some embodiments, the siRNA duplex comprises a guide strand sequence comprising or consisting of the nucleic acid sequence set forth in any one of SEQ ID NOS:1185, 1816, 1213, and 1811, with at least 1, 2, 3, 4, or 5 mismatches to the target ATXN2 mRNA sequence. In some embodiments, the siRNA duplex comprises a guide strand sequence comprising or consisting of a nucleic acid sequence that is at least 60%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 99%, or 100% identical to any one of SEQ ID NOS:1185, 1816, 1213, and 1811. In some embodiments, the siRNA duplex comprises a guide strand sequence comprising or consisting of at least 15, 16, 17, 18, 19, 20, 21, or 22 contiguous nucleotides of a sequence of any one of SEQ ID NOS:1185, 1816, 1213, and 1811, preferably wherein the guide strand sequence retains positions 2-7 (“seed sequence”) of the selected SEQ ID NO. In some embodiments, the siRNA duplex comprises a guide strand sequence comprising or consisting of a sequence of any one of SEQ ID NOS:1185, 1816, 1213, and 1811, wherein 1, 2, 3, or 4 nucleotides at positions 19-22 differ from the selected SEQ ID NO (variant nucleotide(s)), such that the guide strand sequence is no longer complementary to the ATXN2 target sequence at the variant nucleotide(s).

In some embodiments the siRNA duplex comprises a guide strand sequence and passenger strand sequence of any one of siRNA duplexes provided by Tables 1, 19, 23, and 24. In some embodiments, the siRNA duplex comprises a guide strand sequence and passenger strand sequence comprising any one of: SEQ ID NOS:12 and 11; SEQ ID NOS: 14 and 13; SEQ ID NOS: 40 and 39; SEQ ID NOS: 60 and 59; SEQ ID NOS: 100 and 99; SEQ ID NOS: 104 and 103; SEQ ID NOS: 108 and 107; SEQ ID NOS: 112 and 111; SEQ ID NOS: 124 and 123; SEQ ID NOS: 126 and 125; SEQ ID NOS: 128 and 127; SEQ ID NOS: 166 and 165; SEQ ID NOS: 198 and 197; SEQ ID NOS: 220 and 219; SEQ ID NOS: 242 and 241; SEQ ID NOS: 302 and 301; SEQ ID NOS: 306 and 305; SEQ ID NOS: 308 and 307; SEQ ID NOS: 330 and 320; SEQ ID NOS: 336 and 335; and SEQ ID NOS: 362 and 361. In some embodiments, the siRNA duplex comprises a guide strand sequence and passenger strand sequence comprising any one of: SEQ ID NOS: 14 and 13; SEQ ID NOS: 40 and 39; SEQ ID NOS: 100 and 99; SEQ ID NOS: 8 and 107: SEQ ID NOS: 2 and 11; SEQ ID NOS 128 and 127; SEQ ID NOS: 166 and 165; SEQ ID NOS: 198 and 197; SEQ ID NOS: 242 and 241; SEQ ID NOS: 308 and 307; SEQ ID NOS: 336 and 335; and SEQ ID NOS: 362 and 361.

TABLE 1
ATXN2 Specific siRNA Duplex Sequences
	sense sequence	antisense sequence
duplex ID	(passenger)	(guide)
XD-14738	CCUCCGCCUCAGACUGUUUUGG	AAAACAGUCUGAGGCGGAGGGA
	[SEQ ID NO: 3]	[SEQ ID NO: 4]
XD-14739	CUCCGCCUCAGACUGUUUUAGU	UAAAACAGUCUGAGGCGGAGGG
	[SEQ ID NO: 5]	[SEQ ID NO: 6]
XD-14740	CGGCGGCAGCGGCCUUCUAACG	UUAGAAGGCCGCUGCCGCCGGG
	[SEQ ID NO: 7]	[SEQ ID NO: 8]
XD-14741	GGACUGCCUCAGUCUACGAUUU	AUCGUAGACUGAGGCAGUCCUU
	[SEQ ID NO: 9]	[SEQ ID NO: 10]
XD-14742	CUGCCUCAGUCUACGAUUUAUU	UAAAUCGUAGACUGAGGCAGUC
	[SEQ ID NO: 11]	[SEQ ID NO: 12]
XD-14743	UGCCUCAGUCUACGAUUUCUUU	AGAAAUCGUAGACUGAGGCAGU
	[SEQ ID NO: 13]	[SEQ ID NO: 14]
XD-14744	CCUCAGUCUACGAUUUCUUUUG	AAAGAAAUCGUAGACUGAGGCA
	[SEQ ID NO: 15]	[SEQ ID NO: 16]
XD-14745	CAGUCUACGAUUUCUUUUGAUG	UCAAAAGAAAUCGUAGACUGAG
	[SEQ ID NO: 17]	[SEQ ID NO: 18]
XD-14746	GAGGAUGGUUCAUAUACUUACA	UAAGUAUAUGAACCAUCCUCAU
	[SEQ ID NO: 19]	[SEQ ID NO: 20]
XD-14747	AGGAUGGUUCAUAUACUUAAAU	UUAAGUAUAUGAACCAUCCUCA
	[SEQ ID NO: 21]	[SEQ ID NO: 22]
XD-14748	GGAUGGUUCAUAUACUUACAUC	UGUAAGUAUAUGAACCAUCCUC
	[SEQ ID NO: 23]	[SEQ ID NO: 24]
XD-14749	UUCAUAUACUUACAUCAGUUGU	AACUGAUGUAAGUAUAUGAACC
	[SEQ ID NO: 25]	[SEQ ID NO: 26]
XD-14750	AUGAGAAAAGUACAGAAUCAAG	UGAUUCUGUACUUUUCUCAUGU
	[SEQ ID NO: 27]	[SEQ ID NO: 28]
XD-14751	GAGAAAAGUACAGAAUCCAAUU	UUGGAUUCUGUACUUUUCUCAU
	[SEQ ID NO: 29]	[SEQ ID NO: 30]
XD-14752	AAAGUACAGAAUCCAGUUCAGG	UGAACUGGAUUCUGUACUUUUC
	[SEQ ID NO: 31]	[SEQ ID NO: 32]
XD-14753	AAGUACAGAAUCCAGUUCGAGG	UCGAACUGGAUUCUGUACUUUU
	[SEQ ID NO: 33]	[SEQ ID NO: 34]
XD-14754	GUACAGAAUCCAGUUCGGGACC	UCCCGAACUGGAUUCUGUACUU
	[SEQ ID NO: 35]	[SEQ ID NO: 36]
XD-14755	UACAGAAUCCAGUUCGGGGACG	UCCCCGAACUGGAUUCUGUACU
	[SEQ ID NO: 37]	[SEQ ID NO: 38]
XD-14756	UCAGACUUUGUUGUGGUACAGU	UGUACCACAACAAAGUCUGAAC
	[SEQ ID NO: 39]	[SEQ ID NO: 40]
XD-14757	UUUGUUGUGGUACAGUUUAAAG	UUAAACUGUACCACAACAAAGU
	[SEQ ID NO: 41]	[SEQ ID NO: 42]
XD-14758	UUGUUGUGGUACAGUUUAAAGA	UUUAAACUGUACCACAACAAAG
	[SEQ ID NO: 43]	[SEQ ID NO: 44]
XD-14759	UGUGGUACAGUUUAAAGAUAUG	UAUCUUUAAACUGUACCACAAC
	[SEQ ID NO: 45]	[SEQ ID NO: 46]
XD-14760	UUACUGACUCUGCUAUCAGUGC	ACUGAUAGCAGAGUCAGUAAAA
	[SEQ ID NO: 47]	[SEQ ID NO: 48]
XD-14761	CUGACUCUGCUAUCAGUGCUAA	AGCACUGAUAGCAGAGUCAGUA
	[SEQ ID NO: 49]	[SEQ ID NO: 50]
XD-14762	UGACUCUGCUAUCAGUGCUAAA	UAGCACUGAUAGCAGAGUCAGU
	[SEQ ID NO: 51]	[SEQ ID NO: 52]
XD-14763	CUAAAGUGAAUGGCGAACAAAA	UUGUUCGCCAUUCACUUUAGCA
	[SEQ ID NO: 53]	[SEQ ID NO: 54]
XD-14764	AAAGUGAAUGGCGAACACAAAG	UUGUGUUCGCCAUUCACUUUAG
	[SEQ ID NO: 55]	[SEQ ID NO: 56]
XD-14765	GUGAAUGGCGAACACAAAGAGA	UCUUUGUGUUCGCCAUUCACUU
	[SEQ ID NO: 57]	[SEQ ID NO: 58]
XD-14766	CUUUGGAAAAUGACGUAUCUAA	AGAUACGUCAUUUUCCAAAGCC
	[SEQ ID NO: 59]	[SEQ ID NO: 60]
XD-14767	UUGGAAAAUGACGUAUCUAAUG	UUAGAUACGUCAUUUUCCAAAG
	[SEQ ID NO: 61]	[SEQ ID NO: 62]
XD-14768	GGAAAAUGACGUAUCUAAUAGA	UAUUAGAUACGUCAUUUUCCAA
	[SEQ ID NO: 63]	[SEQ ID NO: 64]
XD-14769	AAAUGACGUAUCUAAUGGAUGG	AUCCAUUAGAUACGUCAUUUUC
	[SEQ ID NO: 65]	[SEQ ID NO: 66]
XD-14770	AUGACGUAUCUAAUGGAUGAGA	UCAUCCAUUAGAUACGUCAUUU
	[SEQ ID NO: 67]	[SEQ ID NO: 68]
XD-14771	UAAUGGAUGGGAUCCCAAUAAU	UAUUGGGAUCCCAUCCAUUAGA
	[SEQ ID NO: 69]	[SEQ ID NO: 70]
XD-14772	AAUGGAUGGGAUCCCAAUGAUA	UCAUUGGGAUCCCAUCCAUUAG
	[SEQ ID NO: 71]	[SEQ ID NO: 72]
XD-14773	AUAUGUUUCGAUAUAAUGAAGA	UUCAUUAUAUCGAAACAUAUCA
	[SEQ ID NO: 73]	[SEQ ID NO: 74]
XD-14774	AUGUUUCGAUAUAAUGAAGAAA	UCUUCAUUAUAUCGAAACAUAU
	[SEQ ID NO: 75]	[SEQ ID NO: 76]
XD-14775	GUCUACGUAUGAUAGCAGUUUA	AACUGCUAUCAUACGUAGACAC
	[SEQ ID NO: 77]	[SEQ ID NO: 78]
XD-14776	CGUAUACAGUGCCCUUAGAAAG	UUCUAAGGGCACUGUAUACGAA
	[SEQ ID NO: 79]	[SEQ ID NO: 80]
XD-14777	UAUACAGUGCCCUUAGAAAAAG	UUUUCUAAGGGCACUGUAUACG
	[SEQ ID NO: 81]	[SEQ ID NO: 82]
XD-14778	CAAGGGCAAACCAGUUAGCAGA	UGCUAACUGGUUUGCCCUUGCU
	[SEQ ID NO: 83]	[SEQ ID NO: 84]
XD-14779	AGGGCAAACCAGUUAGCAGAAG	UCUGCUAACUGGUUUGCCCUUG
	[SEQ ID NO: 85]	[SEQ ID NO: 86]
XD-14780	UGAGUCAAGUGCCCAGUACAAA	UGUACUGGGCACUUGACUCAAU
	[SEQ ID NO: 87]	[SEQ ID NO: 88]
XD-14781	GAGUCAAGUGCCCAGUACAAAG	UUGUACUGGGCACUUGACUCAA
	[SEO ID NO: 89]	[SEO ID NO: 90]
XD-14782	GCCCAGUACAAAGCUCGAGUGG	ACUCGAGCUUUGUACUGGGCAC
	[SEQ ID NO: 91]	[SEQ ID NO: 92]
XD-14783	AAGAAAAAUACACAGCAGUUCA	AACUGCUGUGUAUUUUUCUUCC
	[SEQ ID NO: 93]	[SEQ ID NO: 94]
XD-14784	GAGGGGCACAGCAUAAACAAUA	UUGUUUAUGCUGUGCCCCUCAC
	[SEQ ID NO: 95]	[SEQ ID NO: 96]
XD-14785	AGUCAUAUCCUGGGGAAGUAGG	UACUUCCCCAGGAUAUGACUUC
	[SEQ ID NO: 97]	[SEQ ID NO: 98]
XD-14786	GAGACAGAAUUCACCGCGUAUG	UACGCGGUGAAUUCUGUCUCCC
	[SEQ ID NO: 99]	[SEQ ID NO: 100]
XD-14787	AGACAGAAUUCACCGCGUAUGG	AUACGCGGUGAAUUCUGUCUCC
	[SEQ ID NO: 101]	[SEQ ID NO: 102]
XD-14788	GACAGAAUUCACCGCGUAUAGG	UAUACGCGGUGAAUUCUGUCUC
	[SEQ ID NO: 103]	[SEQ ID NO: 104]
XD-14789	ACAGAAUUCACCGCGUAUGAGC	UCAUACGCGGUGAAUUCUGUCU
	[SEQ ID NO: 105]	[SEQ ID NO: 106]
XD-14790	ACUUCAGAUUUCAACCCGAAUU	UUCGGGUUGAAAUCUGAAGUGU
	[SEQ ID NO: 107]	[SEQ ID NO: 108]
XD-14791	UCAGACCAAAGAGUAGUUAAUG	UUAACUACUCUUUGGUCUGAAC
	[SEQ ID NO: 109]	[SEQ ID NO: 110]
XD-14792	CAGACCAAAGAGUAGUUAAUGG	AUUAACUACUCUUUGGUCUGAA
	[SEQ ID NO: 111]	[SEQ ID NO: 112]
XD-14793	AGACCAAAGAGUAGUUAAUAGA	UAUUAACUACUCUUUGGUCUGA
	[SEQ ID NO: 113]	[SEQ ID NO: 114]
XD-14794	GACCAAAGAGUAGUUAAUGAAG	UCAUUAACUACUCUUUGGUCUG
	[SEQ ID NO: 115]	[SEQ ID NO: 116]
XD-14795	CCAAAGAGUAGUUAAUGGAAGU	UUCCAUUAACUACUCUUUGGUC
	[SEQ ID NO: 117]	[SEQ ID NO: 118]
XD-14796	UGUCCCCAAAGGCCCAGCGACA	UCGCUGGGCCUUUGGGGACAUC
	[SEQ ID NO: 119]	[SEQ ID NO: 120]
XD-14797	GCCCAGCGACAUCCUCGAAAUC	UUUCGAGGAUGUCGCUGGGCCU
	[SEQ ID NO: 121]	[SEQ ID NO: 122]
XD-14798	CCCAGCGACAUCCUCGAAAUCA	AUUUCGAGGAUGUCGCUGGGCC
	[SEQ ID NO: 123]	[SEQ ID NO: 124]
XD-14799	CCAGCGACAUCCUCGAAAUAAC	UAUUUCGAGGAUGUCGCUGGGC
	[SEQ ID NO: 125]	[SEQ ID NO: 126]
XD-14800	AGCGACAUCCUCGAAAUCAAAG	UUGAUUUCGAGGAUGUCGCUGG
	[SEQ ID NO: 127]	[SEQ ID NO: 128]
XD-14801	GCGACAUCCUCGAAAUCACAGA	UGUGAUUUCGAGGAUGUCGCUG
	[SEQ ID NO: 129]	[SEQ ID NO: 130]
XD-14802	UGCUGGGAGGGGUUCCAUAUCC	AUAUGGAACCCCUCCCAGCAGA
	[SEQ ID NO: 131]	[SEQ ID NO: 132]
XD-14803	GCUACUCCUCCAGUAGCAAAGA	UUUGCUACUGGAGGAGUAGCUG
	[SEQ ID NO: 133]	[SEQ ID NO: 134]
XD-14804	CCUCCAGUAGCAAGGACCAAUC	UUGGUCCUUGCUACUGGAGGAG
	[SEQ ID NO: 135]	[SEQ ID NO: 136]
XD-14805	GUGGUCAGUGGGGUUCCAAAAU	UUUGGAACCCCACUGACCACUG
	[SEQ ID NO: 137]	[SEQ ID NO: 138]
XD-14806	GUGGGGUUCCAAGAUUAUCACC	UGAUAAUCUUGGAACCCCACUG
	[SEQ ID NO: 139]	[SEQ ID NO: 140]
XD-14807	UCCAAGAUUAUCCCCUAAAACU	UUUUAGGGGAUAAUCUUGGAAC
	[SEQ ID NO: 141]	[SEQ ID NO: 142]
XD-14808	CCAAGAUUAUCCCCUAAAAAUC	UUUUUAGGGGAUAAUCUUGGAA
	[SEQ ID NO: 143]	[SEQ ID NO: 144]
XD-14809	CAAGAUUAUCCCCUAAAACUCA	AGUUUUAGGGGAUAAUCUUGGA
	[SEQ ID NO: 145]	[SEQ ID NO: 146]
XD-14810	AAGAUUAUCCCCUAAAACUAAU	UAGUUUUAGGGGAUAAUCUUGG
	[SEQ ID NO: 147]	[SEQ ID NO: 148]
XD-14811	AGAUUAUCCCCUAAAACUCAUA	UGAGUUUUAGGGGAUAAUCUUG
	[SEQ ID NO: 149]	[SEQ ID NO: 150]
XD-14812	GAUUAUCCCCUAAAACUCAUAG	AUGAGUUUUAGGGGAUAAUCUU
	[SEQ ID NO: 151]	[SEQ ID NO: 152]
XD-14813	AUUAUCCCCUAAAACUCAUAGA	UAUGAGUUUUAGGGGAUAAUCU
	[SEQ ID NO: 153]	[SEQ ID NO: 154]
XD-14814	UAUCCCCUAAAACUCAUAGACC	UCUAUGAGUUUUAGGGGAUAAU
	[SEQ ID NO: 155]	[SEQ ID NO: 156]
XD-14815	AACUCAUAGACCCAGGUCUACC	UAGACCUGGGUCUAUGAGUUUU
	[SEQ ID NO: 157]	[SEQ ID NO: 158]
XD-14816	UCCCCAAGCUGGUAUUAUUACA	UAAUAAUACCAGCUUGGGGAGA
	[SEQ ID NO: 159]	[SEQ ID NO: 160]
XD-14817	CCCCAAGCUGGUAUUAUUCAAA	UGAAUAAUACCAGCUUGGGGAG
	[SEQ ID NO: 161]	[SEQ ID NO: 162]
XD-14818	CAUCUCCUACGCCUGCUAGUCC	ACUAGCAGGCGUAGGAGAUGCA
	[SEQ ID NO: 163]	[SEQ ID NO: 164]
XD-14819	CCUGCUAGUCCUGCAUCGAACA	UUCGAUGCAGGACUAGCAGGCG
	[SEQ ID NO: 165]	[SEQ ID NO: 166]
XD-14820	UGCUAGUCCUGCAUCGAACAGA	UGUUCGAUGCAGGACUAGCAGG
	[SEQ ID NO: 167]	[SEQ ID NO: 168]
XD-14821	CUGCAUCGAACAGAGCUGUUAC	AACAGCUCUGUUCGAUGCAGGA
	[SEQ ID NO: 169]	[SEQ ID NO: 170]
XD-14822	GCAUCGAACAGAGCUGUUAACC	UUAACAGCUCUGUUCGAUGCAG
	[SEQ ID NO: 171]	[SEQ ID NO: 172]
XD-14823	ACCCCUUCUAGUGAGGCUAAAG	UUAGCCUCACUAGAAGGGGUAA
	[SEQ ID NO: 173]	[SEQ ID NO: 174]
XD-14824	CCCCUUCUAGUGAGGCUAAAGA	UUUAGCCUCACUAGAAGGGGUA
	[SEQ ID NO: 175]	[SEQ ID NO: 176]
XD-14825	CCUUCUAGUGAGGCUAAAGAUU	UCUUUAGCCUCACUAGAAGGGG
	[SEQ ID NO: 177]	[SEQ ID NO: 178]
XD-14826	UUCUAGUGAGGCUAAAGAUUCC	AAUCUUUAGCCUCACUAGAAGG
	[SEQ ID NO: 179]	[SEQ ID NO: 180]
XD-14827	AAUGAAACAUCACCUAGCUUCU	AAGCUAGGUGAUGUUUCAUUGG
	[SEQ ID NO: 181]	[SEQ ID NO: 182]
XD-14828	ACAUCACCUAGCUUCUCAAAAG	UUUGAGAAGCUAGGUGAUGUUU
	[SEQ ID NO: 183]	[SEQ ID NO: 184]
XD-14829	AUCACCUAGCUUCUCAAAAACU	UUUUUGAGAAGCUAGGUGAUGU
	[SEQ ID NO: 185]	[SEQ ID NO: 186]
XD-14830	CCUAGCUUCUCAAAAGCUGAAA	UCAGCUUUUGAGAAGCUAGGUG
	[SEQ ID NO: 187]	[SEQ ID NO: 188]
XD-14831	AAAACAAAGGUAUAUCACCAGU	UGGUGAUAUACCUUUGUUUUCA
	[SEQ ID NO: 189]	[SEQ ID NO: 190]
XD-14832	UAAGAAUGAUUUUAGGUUAAAG	UUAACCUAAAAUCAUUCUUAAA
	[SEQ ID NO: 191]	[SEQ ID NO: 192]
XD-14833	AAGAAUGAUUUUAGGUUACAGC	UGUAACCUAAAAUCAUUCUUAA
	[SEQ ID NO: 193]	[SEQ ID NO: 194]
XD-14834	ACUUCUGAAUCUAUGGAUCAAC	UGAUCCAUAGAUUCAGAAGUAG
	[SEQ ID NO: 195]	[SEQ ID NO: 196]
XD-14835	UGAAUCUAUGGAUCAACUAAUA	UUAGUUGAUCCAUAGAUUCAGA
	[SEQ ID NO: 197]	[SEQ ID NO: 198]
XD-14836	AUCUAUGGAUCAACUACUAAAC	UUAGUAGUUGAUCCAUAGAUUC
	[SEQ ID NO: 199]	[SEQ ID NO: 200]
XD-14837	UCUAUGGAUCAACUACUAAACA	UUUAGUAGUUGAUCCAUAGAUU
	[SEQ ID NO: 201]	[SEQ ID NO: 202]
XD-14838	UAUGGAUCAACUACUAAACAAA	UGUUUAGUAGUUGAUCCAUAGA
	[SEQ ID NO: 203]	[SEQ ID NO: 204]
XD-14839	AUGGAUCAACUACUAAACAAAA	UUGUUUAGUAGUUGAUCCAUAG
	[SEQ ID NO: 205]	[SEQ ID NO: 206]
XD-14840	UGGAUCAACUACUAAACAAAAA	UUUGUUUAGUAGUUGAUCCAUA
	[SEQ ID NO: 207]	[SEQ ID NO: 208]
XD-14841	GGAUCAACUACUAAACAAAAAU	UUUUGUUUAGUAGUUGAUCCAU
	[SEQ ID NO: 209]	[SEQ ID NO: 210]
XD-14842	GCCGAAUAGCCCCAGCAUUUCC	AAAUGCUGGGGCUAUUCGGCUU
	[SEQ ID NO: 211]	[SEQ ID NO: 212]
XD-14843	CAGCAUUUCCCCUUCAAUAAUU	UUAUUGAAGGGGAAAUGCUGGG
	[SEQ ID NO: 213]	[SEQ ID NO: 214]
XD-14844	UUUCCCCUUCAAUACUUAGUAA	ACUAAGUAUUGAAGGGGAAAUG
	[SEQ ID NO: 215]	[SEQ ID NO: 216]
XD-14845	UUCCCCUUCAAUACUUAGUAAC	UACUAAGUAUUGAAGGGGAAAU
	[SEQ ID NO: 217]	[SEQ ID NO: 218]
XD-14846	UCCCCUUCAAUACUUAGUAACA	UUACUAAGUAUUGAAGGGGAAA
	[SEQ ID NO: 219]	[SEQ ID NO: 220]
XD-14847	CCCCUUCAAUACUUAGUAAAAC	UUUACUAAGUAUUGAAGGGGAA
	[SEQ ID NO: 221]	[SEQ ID NO: 222]
XD-14848	UUCAAUACUUAGUAACACGAAG	UCGUGUUACUAAGUAUUGAAGG
	[SEQ ID NO: 223]	[SEQ ID NO: 224]
XD-14849	UCAAUACUUAGUAACACGGAGC	UCCGUGUUACUAAGUAUUGAAG
	[SEQ ID NO: 225]	[SEQ ID NO: 226]
XD-14850	AUACUUAGUAACACGGAGCACA	UGCUCCGUGUUACUAAGUAUUG
	[SEQ ID NO: 227]	[SEQ ID NO: 228]
XD-14851	GUCACUUCCCAAGGGGUUCAGA	UGAACCCCUUGGGAAGUGACCU
	[SEQ ID NO: 229]	[SEQ ID NO: 230]
XD-14852	CCAAGGGGUUCAGACUUCCAGC	UGGAAGUCUGAACCCCUUGGGA
	[SEQ ID NO: 231]	[SEQ ID NO: 232]
XD-14853	GACGCAGCUGAGCAAGUUAAGA	UUAACUUGCUCAGCUGCGUCUU
	[SEQ ID NO: 233]	[SEQ ID NO: 234]
XD-14854	CUGAGCAAGUUAGGAAAUCAAC	UGAUUUCCUAACUUGCUCAGCU
	[SEQ ID NO: 235]	[SEQ ID NO: 236]
XD-14855	GAGCAAGUUAGGAAAUCAAAAU	UUUGAUUUCCUAACUUGCUCAG
	[SEQ ID NO: 237]	[SEQ ID NO: 238]
XD-14856	CCAAUGCAAAGGAGUUCAAACC	UUUGAACUCCUUUGCAUUGGGA
	[SEQ ID NO: 239]	[SEQ ID NO: 240]
XD-14857	GGAGUUCAACCCACGUUCCUUC	AGGAACGUGGGUUGAACUCCUU
	[SEQ ID NO: 241]	[SEQ ID NO: 242]
XD-14858	UCUCAGCCAAAGCCUUCUAAUA	UUAGAAGGCUUUGGCUGAGAGA
	[SEQ ID NO: 243]	[SEQ ID NO: 244]
XD-14859	CAGCCAAAGCCUUCUACUAACC	UUAGUAGAAGGCUUUGGCUGAG
	[SEQ ID NO: 245]	[SEQ ID NO: 246]
XD-14860	AGCCAAAGCCUUCUACUACACC	UGUAGUAGAAGGCUUUGGCUGA
	[SEQ ID NO: 247]	[SEQ ID NO: 248]
XD-14861	GCCAAAGCCUUCUACUACCACA	UGGUAGUAGAAGGCUUUGGCUG
	[SEQ ID NO: 249]	[SEQ ID NO: 250]
XD-14862	ACCCCAACUUCACCUCGGCAUC	UGCCGAGGUGAAGUUGGGGUAG
	[SEQ ID NO: 251]	[SEQ ID NO: 252]
XD-14863	CCCCAACUUCACCUCGGCCUCA	AGGCCGAGGUGAAGUUGGGGUA
	[SEQ ID NO: 253]	[SEQ ID NO: 254]
XD-14864	CCCAACUUCACCUCGGCCUAAA	UAGGCCGAGGUGAAGUUGGGGU
	[SEQ ID NO: 255]	[SEQ ID NO: 256]
XD-14865	ACUUCACCUCGGCCUCAAGAAC	UCUUGAGGCCGAGGUGAAGUUG
	[SEQ ID NO: 257]	[SEQ ID NO: 258]
XD-14866	ACCUCGGCCUCAAGCACAAACU	UUUGUGCUUGAGGCCGAGGUGA
	[SEQ ID NO: 259]	[SEQ ID NO: 260]
XD-14867	CCUCGGCCUCAAGCACAACAUA	UGUUGUGCUUGAGGCCGAGGUG
	[SEQ ID NO: 261]	[SEQ ID NO: 262]
XD-14868	AGCACAACCUAGCCCAUCUAUG	UAGAUGGGCUAGGUUGUGCUUG
	[SEQ ID NO: 263]	[SEQ ID NO: 264]
XD-14869	GCACAACCUAGCCCAUCUAUGG	AUAGAUGGGCUAGGUUGUGCUU
	[SEQ ID NO: 265]	[SEQ ID NO: 266]
XD-14870	ACAACCUAGCCCAUCUAUGAUG	UCAUAGAUGGGCUAGGUUGUGC
	[SEQ ID NO: 267]	[SEQ ID NO: 268]
XD-14871	CAACCUAGCCCAUCUAUGGUGG	ACCAUAGAUGGGCUAGGUUGUG
	[SEQ ID NO: 269]	[SEQ ID NO: 270]
XD-14872	GCCCAUCUAUGGUGGGUCAUCA	AUGACCCACCAUAGAUGGGCUA
	[SEQ ID NO: 271]	[SEQ ID NO: 272]
XD-14873	CCCAUCUAUGGUGGGUCAUAAA	UAUGACCCACCAUAGAUGGGCU
	[SEQ ID NO: 273]	[SEQ ID NO: 274]
XD-14874	CCAUCUAUGGUGGGUCAUCAAC	UGAUGACCCACCAUAGAUGGGC
	[SEQ ID NO: 275]	[SEQ ID NO: 276]
XD-14875	CAUCUAUGGUGGGUCAUCAACA	UUGAUGACCCACCAUAGAUGGG
	[SEQ ID NO: 277]	[SEQ ID NO: 278]
XD-14876	GCCAACUCCAGUUUAUACUAAG	UAGUAUAAACUGGAGUUGGCUG
	[SEQ ID NO: 279]	[SEQ ID NO: 280]
XD-14877	CACCAAAUAUGAUGUAUCCAGU	UGGAUACAUCAUAUUUGGUGCA
	[SEQ ID NO: 281]	[SEQ ID NO: 282]
XD-14878	AGCCCAGGCGUGCAACCUUUAU	AAAGGUUGCACGCCUGGGCUCA
	[SEQ ID NO: 283]	[SEQ ID NO: 284]
XD-14879	UACCCAAUACCUAUGACGCACA	UGCGUCAUAGGUAUUGGGUAUA
	[SEQ ID NO: 285]	[SEQ ID NO: 286]
XD-14880	CAAUACCUAUGACGCCCAUACC	UAUGGGCGUCAUAGGUAUUGGG
	[SEQ ID NO: 287]	[SEQ ID NO: 288]
XD-14881	AUACCUAUGACGCCCAUGCAAG	UGCAUGGGCGUCAUAGGUAUUG
	[SEQ ID NO: 289]	[SEQ ID NO: 290]
XD-14882	GAAUCAAGCCAAGACAUAUAGA	UAUAUGUCUUGGCUUGAUUCAC
	[SEQ ID NO: 291]	[SEQ ID NO: 292]
XD-14883	AAUCAAGCCAAGACAUAUAAAG	UUAUAUGUCUUGGCUUGAUUCA
	[SEQ ID NO: 293]	[SEQ ID NO: 294]
XD-14884	AUCAAGCCAAGACAUAUAGAGC	UCUAUAUGUCUUGGCUUGAUUC
	[SEQ ID NO: 295]	[SEQ ID NO: 296]
XD-14885	AAGCCAAGACAUAUAGAGCAGU	UGCUCUAUAUGUCUUGGCUUGA
	[SEQ ID NO: 297]	[SEQ ID NO: 298]
XD-14886	CCAAAUAUGCCCCAACAGCAGC	UGCUGUUGGGGCAUAUUUGGUA
	[SEQ ID NO: 299]	[SEQ ID NO: 300]
XD-14887	CAAAUAUGCCCCAACAGCGACA	UCGCUGUUGGGGCAUAUUUGGU
	[SEQ ID NO: 301]	[SEQ ID NO: 302]
XD-14888	GCAGCGGGCCCACCGAUUGAAG	UCAAUCGGUGGGCCCGCUGCUG
	[SEQ ID NO: 303]	[SEQ ID NO: 304]
XD-14889	ACCAGCUUACUCCACGCAAUAU	AUUGCGUGGAGUAAGCUGGUGG
	[SEQ ID NO: 305]	[SEQ ID NO: 306]
XD-14890	CCAGCUUACUCCACGCAAUAUG	UAUUGCGUGGAGUAAGCUGGUG
	[SEQ ID NO: 307]	[SEQ ID NO: 308]
XD-14891	AGCUUACUCCACGCAAUAUAUU	UAUAUUGCGUGGAGUAAGCUGG
	[SEQ ID NO: 309]	[SEQ ID NO: 310]
XD-14892	UCCACGCAAUAUGUUGCCUACA	UAGGCAACAUAUUGCGUGGAGU
	[SEQ ID NO: 311]	[SEQ ID NO: 312]
XD-14893	AGUCUCAGCAUCCUCAUGUAUA	UACAUGAGGAUGCUGAGACUGA
	[SEQ ID NO: 313]	[SEQ ID NO: 314]
XD-14894	CAGCAUCCUCAUGUCUAUAAUC	UUAUAGACAUGAGGAUGCUGAG
	[SEQ ID NO: 315]	[SEQ ID NO: 316]
XD-14895	GUCCUGUAAUACAGGGUAAUGC	AUUACCCUGUAUUACAGGACUA
	[SEQ ID NO: 317]	[SEQ ID NO: 318]
XD-14896	ACAGGGUAAUGCUAGAAUGAUG	UCAUUCUAGCAUUACCCUGUAU
	[SEQ ID NO: 319]	[SEQ ID NO: 320]
XD-14897	ACUCAGUACGGGGCUCAUGAGC	UCAUGAGCCCCGUACUGAGUUG
	[SEQ ID NO: 321]	[SEQ ID NO: 322]
XD-14898	GAGCAGACGCAUGCGAUGUAUG	UACAUCGCAUGCGUCUGCUCAU
	[SEQ ID NO: 323]	[SEQ ID NO: 324]
XD-14899	AGCAGACGCAUGCGAUGUAUGC	AUACAUCGCAUGCGUCUGCUCA
	[SEQ ID NO: 325]	[SEQ ID NO: 326]
XD-14900	GCAGACGCAUGCGAUGUAUACA	UAUACAUCGCAUGCGUCUGCUC
	[SEQ ID NO: 327]	[SEQ ID NO: 328]
XD-14901	CUUGCUCAGCAGUAUGCGCACC	UGCGCAUACUGCUGAGCAAGGG
	[SEQ ID NO: 329]	[SEQ ID NO: 330]
XD-14902	CAGCAGUAUGCGCACCCUAACG	UUAGGGUGCGCAUACUGCUGAG
	[SEQ ID NO: 331]	[SEQ ID NO: 332]
XD-14903	CAGUAUGCGCACCCUAACGAUA	UCGUUAGGGUGCGCAUACUGCU
	[SEQ ID NO: 333]	[SEQ ID NO: 334]
XD-14904	AGUAUGCGCACCCUAACGCUAC	AGCGUUAGGGUGCGCAUACUGC
	[SEQ ID NO: 335]	[SEQ ID NO: 336]
XD-14905	GUAUGCGCACCCUAACGCUACC	UAGCGUUAGGGUGCGCAUACUG
	[SEQ ID NO: 337]	[SEQ ID NO: 338]
XD-14906	CAGCAGUCAGCCAUUUACCACG	UGGUAAAUGGCUGACUGCUGCU
	[SEQ ID NO: 339]	[SEQ ID NO: 340]
XD-14907	AGCCAUUUACCACGCGGGGAUU	UCCCCGCGUGGUAAAUGGCUGA
	[SEQ ID NO: 341]	[SEQ ID NO: 342]
XD-14908	UCCAACACGCAGUCGCCACAGA	UGUGGCGACUGCGUGUUGGAGG
	[SEQ ID NO: 343]	[SEQ ID NO: 344]
XD-14909	AACACGCAGUCGCCACAGAAUA	UUCUGUGGCGACUGCGUGUUGG
	[SEQ ID NO: 345]	[SEQ ID NO: 346]
XD-14910	ACGCAGUCGCCACAGAAUAAUU	UUAUUCUGUGGCGACUGCGUGU
	[SEQ ID NO: 347]	[SEQ ID NO: 348]
XD-14911	GCAGUCGCCACAGAAUAGUUUC	AACUAUUCUGUGGCGACUGCGU
	[SEQ ID NO: 349]	[SEQ ID NO: 350]
XD-14912	ACAGAAUAGUUUCCCAGCAACA	UUGCUGGGAAACUAUUCUGUGG
	[SEQ ID NO: 351]	[SEQ ID NO: 352]
XD-14913	AGUUUCCCAGCAGCACAACAGA	UGUUGUGCUGCUGGGAAACUAU
	[SEQ ID NO: 353]	[SEQ ID NO: 354]
XD-14914	ACGAUCCAUCCUUCUCACGUUC	ACGUGAGAAGGAUGGAUCGUAA
	[SEQ ID NO: 355]	[SEQ ID NO: 356]
XD-14915	CUCACGUUCAGCCGGCGUAUAC	AUACGCCGGCUGAACGUGAGAA
	[SEQ ID NO: 357]	[SEQ ID NO: 358]
XD-14916	UCACGUUCAGCCGGCGUAUACC	UAUACGCCGGCUGAACGUGAGA
	[SEQ ID NO: 359]	[SEQ ID NO: 360]
XD-14917	ACGUUCAGCCGGCGUAUACAAA	UGUAUACGCCGGCUGAACGUGA
	[SEQ ID NO: 361]	[SEQ ID NO: 362]
XD-14918	CGUUCAGCCGGCGUAUACCAAC	UGGUAUACGCCGGCUGAACGUG
	[SEQ ID NO: 363]	[SEQ ID NO: 364]
XD-14919	CCCACAUGGCCCACGUACCUCA	AGGUACGUGGGCCAUGUGGGGU
	[SEQ ID NO: 365]	[SEQ ID NO: 366]
XD-14920	CCACAUGGCCCACGUACCUAAG	UAGGUACGUGGGCCAUGUGGGG
	[SEQ ID NO: 367]	[SEQ ID NO: 368]
XD-14921	ACAUGGCCCACGUACCUCAAGC	UUGAGGUACGUGGGCCAUGUGG
	[SEQ ID NO: 369]	[SEQ ID NO: 370]
XD-14922	CCAACAGCAGUUGUAAGGCUGC	AGCCUUACAACUGCUGUUGGUG
	[SEQ ID NO: 371]	[SEQ ID NO: 372]
XD-14923	UCUUGUAACAUCCAAUAGGAAU	UCCUAUUGGAUGUUACAAGAAA
	[SEQ ID NO: 373]	[SEQ ID NO: 374]
XD-14924	GACCGAGUAGAGGCAUUUAAGA	UUAAAUGCCUCUACUCGGUCCA
	[SEQ ID NO: 375]	[SEQ ID NO: 376]
XD-14925	CGAGUAGAGGCAUUUAGGAACU	UUCCUAAAUGCCUCUACUCGGU
	[SEQ ID NO: 377]	[SEQ ID NO: 378]
XD-14926	GGAACUUGGGGGCUAUUCCAUA	UGGAAUAGCCCCCAAGUUCCUA
	[SEQ ID NO: 379]	[SEQ ID NO: 380]
XD-14927	ACUUGGGGGCUAUUCCAUAAUU	UUAUGGAAUAGCCCCCAAGUUC
	[SEQ ID NO: 381]	[SEQ ID NO: 382]
XD-14928	UUGGGGGCUAUUCCAUAAUUCC	AAUUAUGGAAUAGCCCCCAAGU
	[SEQ ID NO: 383]	[SEQ ID NO: 384]
XD-14929	GGGGGCUAUUCCAUAAUUCAAU	UGAAUUAUGGAAUAGCCCCCAA
	[SEQ ID NO: 385]	[SEQ ID NO: 386]
XD-14930	GGGGCUAUUCCAUAAUUCCAUA	UGGAAUUAUGGAAUAGCCCCCA
	[SEQ ID NO: 387]	[SEQ ID NO: 388]
XD-14931	CUCUGCUUGCCGAAACUGGAAG	UCCAGUUUCGGCAAGCAGAGCU
	[SEQ ID NO: 389]	[SEQ ID NO: 390]
XD-14932	UUGCCGAAACUGGAAGUUAUUU	AUAACUUCCAGUUUCGGCAAGC
	[SEQ ID NO: 391]	[SEQ ID NO: 392]
XD-14933	AUAACCCUUGAAAGUCAUGAAC	UCAUGACUUUCAAGGGUUAUUA
	[SEQ ID NO: 393]	[SEQ ID NO: 394]
XD-14934	UGAACACAUCAGCUAGCAAAAG	UUUGCUAGCUGAUGUGUUCAUG
	[SEQ ID NO: 395]	[SEQ ID NO: 396]
XD-14935	UGAUUCUUGCUGCUAUUACUGC	AGUAAUAGCAGCAAGAAUCACU
	[SEQ ID NO: 397]	[SEQ ID NO: 398]
XD-14936	UGGAACGCCCUUUUACUAAACU	UUUAGUAAAAGGGCGUUCCAAG
	[SEQ ID NO: 399]	[SEQ ID NO: 400]
XD-14937	GGAACGCCCUUUUACUAAAAUU	UUUUAGUAAAAGGGCGUUCCAA
	[SEQ ID NO: 401]	[SEQ ID NO: 402]
XD-14938	GAACGCCCUUUUACUAAACUUG	AGUUUAGUAAAAGGGCGUUCCA
	[SEQ ID NO: 403]	[SEQ ID NO: 404]
XD-14939	AACGCCCUUUUACUAAACUUGA	AAGUUUAGUAAAAGGGCGUUCC
	[SEQ ID NO: 405]	[SEQ ID NO: 406]
XD-14940	AGUAAAUUCUUACCGUCAAACU	UUUGACGGUAAGAAUUUACUGA
	[SEQ ID NO: 407]	[SEQ ID NO: 408]
XD-14941	GUAAAUUCUUACCGUCAAAAUG	UUUUGACGGUAAGAAUUUACUG
	[SEQ ID NO: 409]	[SEQ ID NO: 410]
XD-14942	UCUUACCGUCAAACUGACGAAU	UCGUCAGUUUGACGGUAAGAAU
	[SEQ ID NO: 411]	[SEQ ID NO: 412]
XD-14943	CUUACCGUCAAACUGACGGAUU	UCCGUCAGUUUGACGGUAAGAA
	[SEQ ID NO: 413]	[SEQ ID NO: 414]
XD-14944	UUACCGUCAAACUGACGGAUUA	AUCCGUCAGUUUGACGGUAAGA
	[SEQ ID NO: 415]	[SEQ ID NO: 416]
XD-14945	ACCGUCAAACUGACGGAUUAUU	UAAUCCGUCAGUUUGACGGUAA
	[SEQ ID NO: 417]	[SEQ ID NO: 418]
XD-14946	CCGUCAAACUGACGGAUUAUUA	AUAAUCCGUCAGUUUGACGGUA
	[SEQ ID NO: 419]	[SEQ ID NO: 420]
XD-14947	CGUCAAACUGACGGAUUAUUAU	AAUAAUCCGUCAGUUUGACGGU
	[SEQ ID NO: 421]	[SEQ ID NO: 422]
XD-14948	GUCAAACUGACGGAUUAUUAUU	UAAUAAUCCGUCAGUUUGACGG
	[SEQ ID NO: 423]	[SEQ ID NO: 424]
XD-14949	UCAAACUGACGGAUUAUUAUUU	AUAAUAAUCCGUCAGUUUGACG
	[SEQ ID NO: 425]	[SEQ ID NO: 426]
XD-14950	ACUGACGGAUUAUUAUUUAUAA	AUAAAUAAUAAUCCGUCAGUUU
	[SEQ ID NO: 427]	[SEQ ID NO: 428]
XD-14951	CUGACGGAUUAUUAUUUAUAAA	UAUAAAUAAUAAUCCGUCAGUU
	[SEQ ID NO: 429]	[SEQ ID NO: 430]
XD-14952	AUGAGGUGAUCACUGUCUAAAG	UUAGACAGUGAUCACCUCAUCA
	[SEQ ID NO: 431]	[SEQ ID NO: 432]
XD-14953	GAGGUGAUCACUGUCUACAAUG	UUGUAGACAGUGAUCACCUCAU
	[SEQ ID NO: 433]	[SEQ ID NO: 434]
XD-14954	CUGUCUACAGUGGUUCAACUUU	AGUUGAACCACUGUAGACAGUG
	[SEQ ID NO: 435]	[SEQ ID NO: 436]

In some embodiments, the isolated siRNA duplexes of the present disclosure, particularly when not delivered as an expression construct or within a vector, comprise at least one modified nucleotide, including a modified base, modified sugar, or modified backbone. siRNA having nucleotide modification(s) may have increased stability, increased specificity, reduced immunogenicity, or a combination thereof. Modified nucleotides may occur on either the guide strand, passenger strand, or both the guide strand and passenger strand.

Modified bases refer to nucleotide bases such as, for example, adenine, guanine, cytosine, thymine, uracil, xanthine, inosine, and queuosine that have been modified by the replacement or addition of one or more atoms or groups. Some examples of modifications on the nucleobase moieties include, but are not limited to, alkylated, halogenated, thiolated, aminated, amidated, or acetylated bases, individually or in combination. More specific examples include, for example, 5-propynyluridine, 5-propynylcytidine, 6-methyladenine, 6-methylguanine, N,N-dimethyladenine, 2-propyladenine, 2-propylguanine, 2-aminoadenine, 1-methylinosine, 3-methyluridine, 5-methylcytidine, 5-methyluridine and other nucleotides having a modification at the 5 position, 5-(2-amino)propyl uridine, 5-halocytidine, 5-halouridine, 4-acetylcytidine, 1-methyladenosine, 2-methyladenosine, 3-methylcytidine, 6-methyluridine, 2-methylguanosine, 7-methylguanosine, 2,2-dimethylguanosine, 5-methylaminoethyluridine, 5-methyloxyuridine, deazanucleotides such as 7-deaza-adenosine, 6-azouridine, 6-azocytidine, 6-azothymidine, 5-methyl-2-thiouridine, other thio bases such as 2-thiouridine and 4-thiouridine and 2-thiocytidine, dihydrouridine, pseudouridine, queuosine, archaeosine, naphthyl and substituted naphthyl groups, any O- and N-alkylated purines and pyrimidines such as N6-methyladenosine, 5-methylcarbonylmethyluridine, uridine 5-oxyacetic acid, pyridine-4-one, pyridine-2-one, phenyl and modified phenyl groups such as aminophenol or 2,4,6-trimethoxy benzene, modified cytosines that act as G-clamp nucleotides, 8-substituted adenines and guanines, 5-substituted uracils and thymines, azapyrimidines, carboxyhydroxyalkyl nucleotides, carboxyalkylaminoalkyl nucleotides, and alkylcarbonylalkylated nucleotides. Sugar modified nucleotides include, but are not limited to 2′-fluoro, 2′-amino and 2′-thio modified ribonucleotides, e.g., 2′-fluoro modified ribonucleotides.

Modified nucleotides may be modified on the sugar moiety, as well as be nucleotides having non-ribosyl sugars or analogs thereof. For example, the sugar moieties may be, or be based on, mannoses, arabinoses, glucopyranoses, galactopyranoses, 4′-thioribose, and other sugars, heterocycles, or carbocycles.

A normal “backbone,” as used herein, refers to the repeatingly alternating sugar-phosphate sequences in a DNA or RNA molecule. The deoxyribose/ribose sugars are joined at both the 3′-hydroxyl and 5′-hydroxyl groups to phosphate groups in ester links, also known as “phosphodiester” bonds or linkages. One or more, or all phosphodiester linkage(s) may be modified as phosphorothioate linkages, boranophosphate linkages, amide linkages, phosphorodithioate linkages, or triazole linkages.

In some embodiments, the inhibitory nucleic acid is a shRNA. In some embodiments, the shRNA is a stem-loop duplex molecule comprising a guide strand and passenger strand of a siRNA duplex as provided herein (e.g., siRNA duplexes of Tables 1 and 19), linked by a spacer sequence, i.e., loop. In some embodiments, loop sequence is 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 nucleotides in length or 4-25, 4-24, 4-23, 4-22, 4-21, 4-20, 4-19, 4-18, 4-17, 4-16, 4-15, 4-14, 4-11, 4-10, 4-9, 4-8, 4-7, 4-6, 5-25, 5-24, 5-23, 5-22, 5-21, 5-20, 5-19, 5-18, 5-17, 5-16, 5-15, 5-14, 5-13, 5-12, 5-11, 5-10, 5-9, 5-8, 5-7, 6-25, 6-24, 6-23, 6-22, 6-21, 6-20, 6-19, 6-18, 6-17, 6-16, 6-15, 6-14, 6-13, 6-12, 6-11, 6-10, 6-9, 6-8, 7-25, 7-24, 7-23, 7-22, 7-21, 7-20, 7-19, 7-18, 7-17, 7-16, 7-15, 7-14, 7-13, 7-12, 7-11, 7-10, 7-9, 8-25, 8-24, 8-23, 8-22, 8-21, 8-20, 8-19, 8-18, 8-11, 8-10, 9-25, 9-24, 9-23, 9-22, 9-21, 9-20, 9-19, 9-18, 9-17, 9-16, 9-15, 9-14, 9-13, 9-12, 9-11, 10-25, 10-24, 10-23, 10-22, 10-21, 10-20, 10-19, 10-18, 10-17, 10-16, 10-15, 10-14, 10-13, 10-12, 11-25, 11-24, 11-23, 11-22, 11-20, 11-19, 11-18, 11-17, 11-16, 11-15, 11-14, 11-13, 12-25, 12-24, 12-23, 12-22, 12-21, 12-20, 12-19, 12-18, 12-17, 12-16, 12-15, or 12-14 nucleotides in length.

In some embodiments, the inhibitory nucleic acid is an isolated miRNA. A miRNA may be a pri-mRNA, a pre-mRNA, mature miRNA, or artificial miRNA. In some embodiments, a miRNA is comprised of a guide strand and passenger strand. In some embodiments, the guide strand and passenger strand are within the same nucleic acid strand, where the guide strand and passenger strand hybridize together to form a self-annealing duplex structure. MiRNA is initially transcribed as a pri-mRNA, which is processed by nuclear nuclease (e.g., Drosha-DGCR8 complex) into pre-mRNA. A pri-mRNA is a single-stranded molecule having a stem-loop structure. In some embodiments, the pri-miRNA is about 100, 150, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, 2000, 2500, 3000 or more nucleotides in length or about 100-3000, 100-2500, 100-2000, 100-1900, 100-1800, 100-1700, 100-1600, 100-1500, 100-1400, 100-1300, 100-1200, 100-1100, 100-1000, 100-900, 100-800, 100-700, 100-600, 100-500, 100-400, 100-300, 100-200, 100-150, 150-3000, 150-2500, 150-2000, 150-1900, 150-1800, 150-1700, 150-1600, 150-1500, 150-1400, 150-1300, 150-1200, 150-1100, 150-1000, 150-900, 150-800, 150-700, 150-600, 150-500, 150-400, 150-300, 150-200, 200-3000, 200-2500, 200-2000, 200-1900, 200-1800, 200-1700, 200-1600, 200-1500, 200-1400, 200-1300, 200-1200, 200-1100, 200-1000, 200-900, 200-800, 200-700, 200-600, 200-500, 200-400, 200-300, 300-3000, 300-2500, 300-2000, 300-1900, 300-1800, 300-1700, 300-1600, 300-1500, 300-1400, 300-1300, 300-1200, 300-1100, 300-1000, 300-900, 300-800, 300-700, 300-600, 300-500, 300-400, 400-3000, 400-2500, 400-2000, 400-1900, 400-1800, 400-1700, 400-1600, 400-1500, 400-1400, 400-1300, 400-1200, 400-1100, 400-1000, 400-900, 400-800, 400-700, 400-600, 400-500, 500-3000, 500-2500, 500-2000, 500-1900, 500-1800, 500-1700, 500-1600, 500-1500, 500-1400, 500-1300, 500-1200, 500-1100, 500-1000, 500-900, 500-800, 500-700, or 500-600 nucleotides in length.

Pre-miRNA is also a single-stranded molecule having a stem-loop structure. In some embodiments, the pre-miRNA is about 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, or 500 nucleotides in length, or about 40-500, 40-400, 40-300, 40-200, 40-100, 40-90, 40-80, 40-70, 40-60, 40-50, 50-500, 50-400, 50-300, 50-200, 50-100, 50-90, 50-80, 50-70, 60-500, 60-400, 60-300, 60-200, 60-100, 60-90, 60-80, 70-500, 70-400, 70-300, 70-200, 70-100, 70-90, 80-500, 80-400, 80-300, 80-200, 80-100, 90-500, 90-400, 90-300, 90-200, 100-500, 100-400, 100-300, 100-200, 200-500, 200-400, 200-300, 300-500, 300-400, or 400-500 nucleotides in length.

The pre-miRNA is transported from the nucleus to the cytoplasm by exportin-5 and further processed by Dicer to produce a mature, double-stranded miRNA duplex comprising a guide strand and a passenger strand. The mature miRNA duplex is then incorporated into the RNA inducing silencing complex (RISC), mediated by TRBP (HIV transactivating response RNA-binding protein). The passenger strand is generally released and cleaved, while the guide strand remains in RISC and binds to the target mRNA and mediates silencing. In some embodiments, a mature miRNA refers to the guide strand of a mature miRNA duplex. In some embodiments, a mature miRNA is about 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides in length, or ranges from about 19-30 nucleotides, 19-29 nucleotides, 19-28 nucleotides, 19-27 nucleotides, 19-26 nucleotides, 19-25 nucleotides, 19-24 nucleotides, 19-23 nucleotides, 19-21 nucleotides, 20-30 nucleotides, 20-29 nucleotides, 20-28 nucleotides, 20-27 nucleotides, 20-26 nucleotides, 20-25 nucleotides, 20-24 nucleotides, 20-23 nucleotides, 20-22 nucleotides, 21-30 nucleotides, 21-29 nucleotides, 21-28 nucleotides, 21-27 nucleotides, 21-26 nucleotides, 21-25 nucleotides, 21-24 nucleotides, 21-23 nucleotides, 22-30 nucleotides, 22-29 nucleotides, 22-28 nucleotides, 22-27 nucleotides, 22-26 nucleotides, 22-25 nucleotides, 22-24 nucleotides, 23-30 nucleotides, 23-29 nucleotides, 23-28 nucleotides, 23-27 nucleotides, 23-26 nucleotides, 23-25 nucleotides, 24-30 nucleotides, 24-29 nucleotides, 24-28 nucleotides, 24-27 nucleotides, 24-26 nucleotides, 25-30 nucleotides, 25-29 nucleotides, 25-28 nucleotides, 25-27 nucleotides, 26-30 nucleotides, 26-29 nucleotides, 26-28 nucleotides, 27-30 nucleotides, 27-29 nucleotides, or 28-30 nucleotides in length.

Artificial miRNA refers to an endogenous, modified or synthetic pri-mRNA or pre-mRNA scaffold or backbone capable of producing a functional mature miRNA, where the guide strand sequence and passenger strand sequence of the miRNA duplex within the stem region have been replaced with a guide strand sequence and passenger strand sequence of interest that directs silencing of the target mRNA of interest. Artificial miRNA design is described in Eamens et al. (2014) Methods Mol Biol. 1062:211-24 (incorporated by reference in its entirety). Synthetic miRNA backbones are described in U.S. Patent Publication 2008/0313773 (incorporated by reference in its entirety). In some embodiments, the artificial miRNA is about 100-200 nucleotides, 100-175 nucleotides 100-150 nucleotides, 125-200 nucleotides 125-175 nucleotides, or 125-150 nucleotides in length. In some embodiments, the artificial miRNA is about 100 nucleotides, about 120 nucleotides, about 130 nucleotides, about 140 nucleotides, about 150 nucleotides, about 160 nucleotides, about 170 nucleotides, about 180 nucleotides, about 190 nucleotides, or about 200 nucleotides in length.

In some embodiments, the miRNA is a pri-miRNA, a pre-mRNA, an artificial miRNA, or a mature miRNA comprising a guide strand sequence comprising or consisting of the nucleic acid sequence set forth in any one of the guide sequences of Tables 1, 3, 9, 11, 12, 13, 19, 23, 24, and 25, e.g., any one of SEQ ID NOS: 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 124, 126, 128, 130, 132, 134, 136, 138, 140, 142, 144, 146, 148, 150, 152, 154, 156, 158, 160, 162, 164, 166, 168, 170, 172, 174, 176, 178, 180, 182, 184, 186, 188, 190, 192, 194, 196, 198, 200, 202, 204, 206, 208, 210, 212, 214, 216, 218, 220, 222, 224, 226, 228, 230, 232, 234, 236, 238, 240, 242, 244, 246, 248, 250, 252, 254, 256, 258, 260, 262, 264, 266, 268, 270, 272, 274, 276, 278, 280, 282, 284, 286, 288, 290, 292, 294, 296, 298, 300, 302, 304, 306, 308, 310, 312, 314, 316, 318, 320, 324, 326, 328, 330, 332, 334, 336, 338, 340, 342, 344, 346, 348, 350, 352, 354, 356, 358, 360, 362, 364, 366, 368, 370, 372, 374, 376, 378, 380, 382, 384, 386, 388, 390, 392, 394, 396, 398, 400, 402, 404, 406, 408, 410, 412, 414, 416, 418, 420, 422, 424, 426, 428, 430, 432, 434, 436, 1176-1288, 1811-1827, 2015, 2065, 2083, 2152, 2203, and 2209.

In some embodiments, the miRNA is a pri-miRNA, a pre-mRNA, an artificial miRNA, or a mature miRNA comprising a guide strand sequence comprising or consisting of the nucleic acid sequence set forth in any one of the guide sequences of Tables 1, 3, 9, 11, 12, 13, 19, 23, 24, and 25, e.g., any one of SEQ ID NOS: 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 124, 126, 128, 130, 132, 134, 136, 138, 140, 142, 144, 146, 148, 150, 152, 154, 156, 158, 160, 162, 164, 166, 168, 170, 172, 174, 176, 178, 180, 182, 184, 186, 188, 190, 192, 194, 196, 198, 200, 202, 204, 206, 208, 210, 212, 214, 216, 218, 220, 222, 224, 226, 228, 230, 232, 234, 236, 238, 240, 242, 244, 246, 248, 250, 252, 254, 256, 258, 260, 262, 264, 266, 268, 270, 272, 274, 276, 278, 280, 282, 284, 286, 288, 290, 292, 294, 296, 298, 300, 302, 304, 306, 308, 310, 312, 314, 316, 318, 320, 324, 326, 328, 330, 332, 334, 336, 338, 340, 342, 344, 346, 348, 350, 352, 354, 356, 358, 360, 362, 364, 366, 368, 370, 372, 374, 376, 378, 380, 382, 384, 386, 388, 390, 392, 394, 396, 398, 400, 402, 404, 406, 408, 410, 412, 414, 416, 418, 420, 422, 424, 426, 428, 430, 432, 434, 436, 1176-1288, 1811-1827, 2015, 2065, 2083, 2152, 2203, and 2209, with at least 1, 2, 3, 4, or 5 mismatches to the target ATXN2 mRNA sequence.

In some embodiments, the miRNA is a pri-miRNA, a pre-mRNA, an artificial miRNA, or a mature miRNA comprising a guide strand sequence comprising or consisting of a sequence that is at least 60%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 99%, or 100% identical to any one of the guide sequences of Tables 1, 3, 9, 11, 12, 13, 19, 23, 24, and 25, e.g., any one of SEQ ID NOS: 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 124, 126, 128, 130, 132, 134, 136, 138, 140, 142, 144, 146, 148, 150, 152, 154, 156, 158, 160, 162, 164, 166, 168, 170, 172, 174, 176, 178, 180, 182, 184, 186, 188, 190, 192, 194, 196, 198, 200, 202, 204, 206, 208, 210, 212, 214, 216, 218, 220, 222, 224, 226, 228, 230, 232, 234, 236, 238, 240, 242, 244, 246, 248, 250, 252, 254, 256, 258, 260, 262, 264, 266, 268, 270, 272, 274, 276, 278, 280, 282, 284, 286, 288, 290, 292, 294, 296, 298, 300, 302, 304, 306, 308, 310, 312, 314, 316, 318, 320, 324, 326, 328, 330, 332, 334, 336, 338, 340, 342, 344, 346, 348, 350, 352, 354, 356, 358, 360, 362, 364, 366, 368, 370, 372, 374, 376, 378, 380, 382, 384, 386, 388, 390, 392, 394, 396, 398, 400, 402, 404, 406, 408, 410, 412, 414, 416, 418, 420, 422, 424, 426, 428, 430, 432, 434, 436, 1176-1288, 1811-1827, 2015, 2065, 2083, 2152, 2203, and 2209.

In some embodiments, the miRNA is a pri-miRNA, a pre-mRNA, an artificial miRNA, or a mature miRNA comprising a guide strand sequence comprising or consisting of at least 15, 16, 17, 18, 19, 20, 21, or 22 contiguous nucleotides of a sequence of any one of the guide sequences of Tables 1, 3, 9, 11, 12, 13, 19, 23, 24, and 25, e.g., any one of SEQ ID NOS: 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 124, 126, 128, 130, 132, 134, 136, 138, 140, 142, 144, 146, 148, 150, 152, 154, 156, 158, 160, 162, 164, 166, 168, 170, 172, 174, 176, 178, 180, 182, 184, 186, 188, 190, 192, 194, 196, 198, 200, 202, 204, 206, 208, 210, 212, 214, 216, 218, 220, 222, 224, 226, 228, 230, 232, 234, 236, 238, 240, 242, 244, 246, 248, 250, 252, 254, 256, 258, 260, 262, 264, 266, 268, 270, 272, 274, 276, 278, 280, 282, 284, 286, 288, 290, 292, 294, 296, 298, 300, 302, 304, 306, 308, 310, 312, 314, 316, 318, 320, 324, 326, 328, 330, 332, 334, 336, 338, 340, 342, 344, 346, 348, 350, 352, 354, 356, 358, 360, 362, 364, 366, 368, 370, 372, 374, 376, 378, 380, 382, 384, 386, 388, 390, 392, 394, 396, 398, 400, 402, 404, 406, 408, 410, 412, 414, 416, 418, 420, 422, 424, 426, 428, 430, 432, 434, 436, 1176-1288, 1811-1827, 2015, 2065, 2083, 2152, 2203, and 2209, preferably wherein the guide strand sequence retains positions 2-7 (“seed sequence”) of the selected SEQ ID NO.

In some embodiments, the miRNA is a pri-miRNA, a pre-mRNA, an artificial miRNA, or a mature miRNA comprising a guide strand sequence comprising or consisting of a sequence of any one of the guide sequences of Tables 1, 3, 9, 11, 12, 13, 19, 23, 24, and 25, e.g., any one of SEQ ID NOS: 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 124, 126, 128, 130, 132, 134, 136, 138, 140, 142, 144, 146, 148, 150, 152, 154, 156, 158, 160, 162, 164, 166, 168, 170, 172, 174, 176, 178, 180, 182, 184, 186, 188, 190, 192, 194, 196, 198, 200, 202, 204, 206, 208, 210, 212, 214, 216, 218, 220, 222, 224, 226, 228, 230, 232, 234, 236, 238, 240, 242, 244, 246, 248, 250, 252, 254, 256, 258, 260, 262, 264, 266, 268, 270, 272, 274, 276, 278, 280, 282, 284, 286, 288, 290, 292, 294, 296, 298, 300, 302, 304, 306, 308, 310, 312, 314, 316, 318, 320, 324, 326, 328, 330, 332, 334, 336, 338, 340, 342, 344, 346, 348, 350, 352, 354, 356, 358, 360, 362, 364, 366, 368, 370, 372, 374, 376, 378, 380, 382, 384, 386, 388, 390, 392, 394, 396, 398, 400, 402, 404, 406, 408, 410, 412, 414, 416, 418, 420, 422, 424, 426, 428, 430, 432, 434, 436, 1176-1288, 1811-1827, 2015, 2065, 2083, 2152, 2203, and 2209, wherein 1, 2, 3, or 4 nucleotides at positions 19-22 differ from the selected SEQ ID NO (variant nucleotide(s)) such that the guide strand sequence is no longer complementary to the ATXN2 target sequence at the variant nucleotide(s).

In some embodiments, the miRNA is a pri-miRNA, a pre-mRNA, an artificial miRNA, or a mature miRNA comprising a guide strand sequence of Table 12. In some embodiments, the miRNA is a pri-miRNA, a pre-mRNA, an artificial miRNA, or a mature miRNA comprising a guide strand sequence comprising or consisting of the nucleic acid sequence set forth in any one of SEQ ID NOS: 12, 14, 40, 60, 100, 104, 108, 112, 124, 126, 128, 166, 198, 220, 242, 302, 306, 308, 330, 336, and 362. In some embodiments, the miRNA is a pri-miRNA, a pre-mRNA, an artificial miRNA, or a mature miRNA comprising a guide strand sequence comprising or consisting of the nucleic acid sequence set forth in any one of SEQ ID NOS: 12, 14, 40, 60, 100, 104, 108, 112, 124, 126, 128, 166, 198, 220, 242, 302, 306, 308, 330, 336, and 362, with at least 1, 2, 3, 4, or 5 mismatches to the target ATXN2 mRNA sequence. In some embodiments, the miRNA is a pri-miRNA, a pre-mRNA, an artificial miRNA, or a mature miRNA comprising a guide strand sequence comprising or consisting of a nucleic acid sequence that is at least 60%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 99%, or 100% identical to any one of SEQ ID NOS: 12, 14, 40, 60, 100, 104, 108, 112, 124, 126, 128, 166, 198, 220, 242, 302, 306, 308, 330, 336, and 362. In some embodiments, the miRNA is a pri-miRNA, a pre-mRNA, an artificial miRNA, or a mature miRNA comprising a guide strand sequence comprising or consisting of at least 15, 16, 17, 18, 19, 20, 21, or 22 contiguous nucleotides of a sequence of any one of SEQ ID NOS: 12, 14, 40, 60, 100, 104, 108, 112, 124, 126, 128, 166, 198, 220, 242, 302, 306, 308, 330, 336, and 362, preferably wherein the guide strand sequence retains positions 2-7 (“seed sequence”) of the selected SEQ ID NO. In some embodiments, the miRNA is a pri-miRNA, a pre-mRNA, an artificial miRNA, or a mature miRNA comprising a guide strand sequence comprising or consisting of a sequence of any one of SEQ ID NOS: 12, 14, 40, 60, 100, 104, 108, 112, 124, 126, 128, 166, 198, 220, 242, 302, 306, 308, 330, 336, and 362, wherein 1, 2, 3, or 4 nucleotides at positions 19-22 differ from the selected SEQ ID NO (variant nucleotide(s)) such that the guide strand sequence is no longer complementary to the ATXN2 target sequence at the variant nucleotide(s).

In some embodiments, the miRNA is a pri-miRNA, a pre-mRNA, an artificial miRNA, or a mature miRNA comprising a guide strand sequence of Table 13. In some embodiments, the miRNA is a pri-miRNA, a pre-mRNA, an artificial miRNA, or a mature miRNA comprising a guide strand sequence comprising or consisting of the nucleic acid sequence set forth in any one of SEQ ID NOS: 14, 40, 100, 108, 112, 128, 166, 198, 242, 308, 336, and 362. In some embodiments, the miRNA is a pri-miRNA, a pre-mRNA, an artificial miRNA, or a mature miRNA comprising a guide strand sequence comprising or consisting of the nucleic acid sequence set forth in any one of SEQ ID NOS: 14, 40, 100, 108, 112, 128, 166, 198, 242, 308, 336, and 362, with at least 1, 2, 3, 4, or 5 mismatches to the target ATXN2 mRNA sequence. In some embodiments, the miRNA is a pri-miRNA, a pre-mRNA, an artificial miRNA, or a mature miRNA comprising a guide strand sequence comprising or consisting of a nucleic acid sequence that is at least 60%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 99%, or 100% identical to any one of SEQ ID NOS: 14, 40, 100, 108, 112, 128, 166, 198, 242, 308, 336, and 362. In some embodiments, the miRNA is a pri-miRNA, a pre-mRNA, an artificial miRNA, or a mature miRNA comprising a guide strand sequence comprising or consisting of at least 15, 16, 17, 18, 19, 20, 21, or 22 contiguous nucleotides of a sequence of any one of SEQ ID NOS: 14, 40, 100, 108, 112, 128, 166, 198, 242, 308, 336, and 362, preferably wherein the guide strand sequence retains positions 2-7 (“seed sequence”) of the selected SEQ ID NO. In some embodiments, the miRNA is a pri-miRNA, a pre-mRNA, an artificial miRNA, or a mature miRNA comprising a guide strand sequence comprising or consisting of a sequence of any one of SEQ ID NOS: 14, 40, 100, 108, 112, 128, 166, 198, 242, 308, 336, and 362, wherein 1, 2, 3, or 4 nucleotides at positions 19-22 differ from the selected SEQ ID NO (variant nucleotide(s)) such that the guide strand sequence is no longer complementary to the ATXN2 target sequence at the variant nucleotide(s).

In some embodiments, the miRNA is a pri-miRNA, a pre-mRNA, an artificial miRNA, or a mature miRNA comprising a guide strand sequence of Table 19. In some embodiments, the miRNA is a pri-miRNA, a pre-mRNA, an artificial miRNA, or a mature miRNA comprising a guide strand sequence comprising or consisting of the nucleic acid sequence set forth in any one of SEQ ID NOS:1176-1288, 40, 108, and 166. In some embodiments, the miRNA is a pri-miRNA, a pre-mRNA, an artificial miRNA, or a mature miRNA comprising a guide strand sequence comprising or consisting of the nucleic acid sequence set forth in any one of SEQ ID NOS:1176-1288, 40, 108, and 166, with at least 1, 2, 3, 4, or 5 mismatches to the target ATXN2 mRNA sequence. In some embodiments, the miRNA is a pri-miRNA, a pre-mRNA, an artificial miRNA, or a mature miRNA comprising a guide strand sequence comprising or consisting of a nucleic acid sequence that is at least 60%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 99%, or 100% identical to any one of SEQ ID NOS:1176-1288, 40, 108, and 166. In some embodiments, the miRNA is a pri-miRNA, a pre-mRNA, an artificial miRNA, or a mature miRNA comprising a guide strand sequence comprising or consisting of at least 15, 16, 17, 18, 19, 20, 21, or 22 contiguous nucleotides of a sequence of any one of SEQ ID NOS: 1176-1288, 40, 108, and 166, preferably wherein the guide strand sequence retains positions 2-7 (“seed sequence”) of the selected SEQ ID NO. In some embodiments, the miRNA is a pri-miRNA, a pre-mRNA, an artificial miRNA, or a mature miRNA comprising a guide strand sequence comprising or consisting of a sequence of any one of SEQ ID NOS:1176-1288, 40, 108, and 166, wherein 1, 2, 3, or 4 nucleotides at positions 19-22 differ from the selected SEQ ID NO (variant nucleotide(s)) such that the guide strand sequence is no longer complementary to the ATXN2 target sequence at the variant nucleotide(s).

In some embodiments, the miRNA is a pri-miRNA, a pre-mRNA, an artificial miRNA, or a mature miRNA comprising a guide strand sequence of Table 23. In some embodiments, the miRNA is a pri-miRNA, a pre-mRNA, an artificial miRNA, or a mature miRNA comprising a guide strand sequence comprising or consisting of the nucleic acid sequence set forth in any one of SEQ ID NOS:1908-2007. In some embodiments, the miRNA is a pri-miRNA, a pre-mRNA, an artificial miRNA, or a mature miRNA comprising a guide strand sequence comprising or consisting of the nucleic acid sequence set forth in any one of SEQ ID NOS:1908-2007, with at least 1, 2, 3, 4, or 5 mismatches to the target ATXN2 mRNA sequence. In some embodiments, the miRNA is a pri-miRNA, a pre-mRNA, an artificial miRNA, or a mature miRNA comprising a guide strand sequence comprising or consisting of a nucleic acid sequence that is at least 60%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 99%, or 100% identical to any one of SEQ ID NOS:1908-2007. In some embodiments, the miRNA is a pri-miRNA, a pre-mRNA, an artificial miRNA, or a mature miRNA comprising a guide strand sequence comprising or consisting of at least 15, 16, 17, 18, 19, 20, 21, or 22 contiguous nucleotides of a sequence of any one of SEQ ID NOS:1908-2007, preferably wherein the guide strand sequence retains positions 2-7 (“seed sequence”) of the selected SEQ ID NO. In some embodiments, the miRNA is a pri-miRNA, a pre-mRNA, an artificial miRNA, or a mature miRNA comprising a guide strand sequence comprising or consisting of a sequence of any one of SEQ ID NOS:1908-2007, wherein 1, 2, 3, or 4 nucleotides at positions 19-22 differ from the selected SEQ ID NO (variant nucleotide(s)) such that the guide strand sequence is no longer complementary to the ATXN2 target sequence at the variant nucleotide(s).

In some embodiments, the miRNA is a pri-miRNA, a pre-mRNA, an artificial miRNA, or a mature miRNA comprising a guide strand sequence of Table 24. In some embodiments, the miRNA is a pri-miRNA, a pre-mRNA, an artificial miRNA, or a mature miRNA comprising a guide strand sequence comprising or consisting of the nucleic acid sequence set forth in any one of SEQ ID NOS:100, 112, 166, 202, 246, 306, 308, 314, 1180, 1185, 1196, 1200, 1211, 1213, 1215, 1216, 1224, 1811-1822, 1824-1827, 2015, 2065, 2083, 2152, 2203, and 2209. In some embodiments, the miRNA is a pri-miRNA, a pre-mRNA, an artificial miRNA, or a mature miRNA comprising a guide strand sequence comprising or consisting of the nucleic acid sequence set forth in any one of SEQ ID NOS:100, 112, 166, 202, 246, 306, 308, 314, 1180, 1185, 1196, 1200, 1211, 1213, 1215, 1216, 1224, 1811-1822, 1824-1827, 2015, 2065, 2083, 2152, 2203, and 2209, with at least 1, 2, 3, 4, or 5 mismatches to the target ATXN2 mRNA sequence. In some embodiments, the miRNA is a pri-miRNA, a pre-mRNA, an artificial miRNA, or a mature miRNA comprising a guide strand sequence comprising or consisting of a nucleic acid sequence that is at least 60%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 99%, or 100% identical to any one of SEQ ID NOS:100, 112, 166, 202, 246, 306, 308, 314, 1180, 1185, 1196, 1200, 1211, 1213, 1215, 1216, 1224, 1811-1822, 1824-1827, 2015, 2065, 2083, 2152, 2203, and 2209. In some embodiments, the miRNA is a pri-miRNA, a pre-mRNA, an artificial miRNA, or a mature miRNA comprising a guide strand sequence comprising or consisting of at least 15, 16, 17, 18, 19, 20, 21, or 22 contiguous nucleotides of a sequence of any one of SEQ ID NOS:100, 112, 166, 202, 246, 306, 308, 314, 1180, 1185, 1196, 1200, 1211, 1213, 1215, 1216, 1224, 1811-1822, 1824-1827, 2015, 2065, 2083, 2152, 2203, and 2209, preferably wherein the guide strand sequence retains positions 2-7 (“seed sequence”) of the selected SEQ ID NO. In some embodiments, the miRNA is a pri-miRNA, a pre-mRNA, an artificial miRNA, or a mature miRNA comprising a guide strand sequence comprising or consisting of a sequence of any one of SEQ ID NOS:100, 112, 166, 202, 246, 306, 308, 314, 1180, 1185, 1196, 1200, 1211, 1213, 1215, 1216, 1224, 1811-1822, 1824-1827, 2015, 2065, 2083, 2152, 2203, and 2209, wherein 1, 2, 3, or 4 nucleotides at positions 19-22 differ from the selected SEQ ID NO (variant nucleotide(s)) such that the guide strand sequence is no longer complementary to the ATXN2 target sequence at the variant nucleotide(s).

In some embodiments, the miRNA is a pri-miRNA, a pre-mRNA, an artificial miRNA, or a mature miRNA comprising a guide strand sequence of Table 25. In some embodiments, the miRNA is a pri-miRNA, a pre-mRNA, an artificial miRNA, or a mature miRNA comprising a guide strand sequence comprising or consisting of the nucleic acid sequence set forth in any one of SEQ ID NOS:1185, 1816, 1213, 1819, 2083, 1215, 1216, 1811, and 314. In some embodiments, the miRNA is a pri-miRNA, a pre-mRNA, an artificial miRNA, or a mature miRNA comprising a guide strand sequence comprising or consisting of the nucleic acid sequence set forth in any one of SEQ ID NOS:1185, 1816, 1213, 1819, 2083, 1215, 1216, 1811, and 314, with at least 1, 2, 3, 4, or 5 mismatches to the target ATXN2 mRNA sequence. In some embodiments, the miRNA is a pri-miRNA, a pre-mRNA, an artificial miRNA, or a mature miRNA comprising a guide strand sequence comprising or consisting of a nucleic acid sequence that is at least 60%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 99%, or 100% identical to any one of SEQ ID NOS:1185, 1816, 1213, 1819, 2083, 1215, 1216, 1811, and 314. In some embodiments, the miRNA is a pri-miRNA, a pre-mRNA, an artificial miRNA, or a mature miRNA comprising a guide strand sequence comprising or consisting of at least 15, 16, 17, 18, 19, 20, 21, or 22 contiguous nucleotides of a sequence of any one of SEQ ID NOS:1185, 1816, 1213, 1819, 2083, 1215, 1216, 1811, and 314, preferably wherein the guide strand sequence retains positions 2-7 (“seed sequence”) of the selected SEQ ID NO. In some embodiments, the miRNA is a pri-miRNA, a pre-mRNA, an artificial miRNA, or a mature miRNA comprising a guide strand sequence comprising or consisting of a sequence of any one of SEQ ID NOS:1185, 1816, 1213, 1819, 2083, 1215, 1216, 1811, and 314, wherein 1, 2, 3, or 4 nucleotides at positions 19-22 differ from the selected SEQ ID NO (variant nucleotide(s)) such that the guide strand sequence is no longer complementary to the ATXN2 target sequence at the variant nucleotide(s).

In some embodiments, the miRNA is a pri-miRNA, a pre-mRNA, an artificial miRNA, or a mature miRNA comprising a guide strand sequence comprising or consisting of the nucleic acid sequence set forth in any one of SEQ ID NOS:1185, 1816, 1213, and 1811. In some embodiments, the miRNA is a pri-miRNA, a pre-mRNA, an artificial miRNA, or a mature miRNA comprising a guide strand sequence comprising or consisting of the nucleic acid sequence set forth in any one of SEQ ID NOS:1185, 1816, 1213, and 1811, with at least 1, 2, 3, 4, or 5 mismatches to the target ATXN2 mRNA sequence. In some embodiments, the miRNA is a pri-miRNA, a pre-mRNA, an artificial miRNA, or a mature miRNA comprising a guide strand sequence comprising or consisting of a nucleic acid sequence that is at least 60%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 99%, or 100% identical to any one of SEQ ID NOS:1185, 1816, 1213, and 1811. In some embodiments, the miRNA is a pri-miRNA, a pre-mRNA, an artificial miRNA, or a mature miRNA comprising a guide strand sequence comprising or consisting of at least 15, 16, 17, 18, 19, 20, 21, or 22 contiguous nucleotides of a sequence of any one of SEQ ID NOS:1185, 1816, 1213, and 1811, preferably wherein the guide strand sequence retains positions 2-7 (“seed sequence”) of the selected SEQ ID NO. In some embodiments, the miRNA is a pri-miRNA, a pre-mRNA, an artificial miRNA, or a mature miRNA comprising a guide strand sequence comprising or consisting of a sequence of any one of SEQ ID NOS:1185, 1816, 1213, and 1811, wherein 1, 2, 3, or 4 nucleotides at positions 19-22 differ from the selected SEQ ID NO (variant nucleotide(s)) such that the guide strand sequence is no longer complementary to the ATXN2 target sequence at the variant nucleotide(s).

In some embodiments, an artificial miRNA comprises a guide strand sequence according to any of the embodiments described herein, contained within a miR backbone sequence. In some embodiments, the guide strand sequence and passenger strand sequence of the artificial miRNA are contained with a miRNA backbone sequence. In some embodiments, the miRNA backbone sequence is a miR-155 backbone sequence, a miR-155E backbone sequence, a miR-155M backbone sequence, a miR1-1 backbone sequence, a miR-1-1_M backbone sequence, a miR-100 backbone sequence, a miR-100_M backbone sequence, a miR-190A backbone sequence, a miR-124 backbone sequence, a miR-124_M backbone sequence, a miR-16-2 backbone sequence, a miR-132 backbone sequence, a miR-9 backbone sequence, a miR-138-2 backbone sequence, a miR-122 backbone sequence, a miR-122_M backbone sequence, a miR-130a backbone sequence, miR-128 backbone sequence, a miR-144 backbone sequence, a miR-451a backbone sequence, or a miR-223 backbone sequence.

In some embodiments, the miRNA backbone sequence is a miR-155E backbone sequence, a miR-155M backbone sequence, a miR1-1 backbone sequence, a miR-1-1_M backbone sequence, a miR-100 backbone sequence, a miR-100_M backbone sequence, a miR-190a backbone sequence, a miR-190a_M backbone sequence, a miR-124 backbone sequence, a miR-124_M backbone sequence, a miR-132 backbone sequence, a miR-138-2 backbone sequence, a miR-122 backbone sequence, a miR-122_M backbone sequence, a miR-130a backbone sequence, a miR-16-2 backbone sequence, a miR-128 backbone sequence, a miR-144 backbone sequence, a miR-451a backbone sequence, or a miR-223 backbone sequence.

In some embodiments, the miRNA backbone sequence is a miR1-1 backbone sequence, a miR-1-1_M backbone sequence, a miR-100 backbone sequence, a miR-100_M backbone sequence, a miR-122 backbone sequence, a miR-122_M backbone sequence, a miR-124 backbone sequence, a miR-130a backbone sequence, a miR-132 backbone sequence, a miR-138-2 backbone sequence, a miR-144 backbone sequence, a miR-155E backbone sequence, a miR-155M backbone sequence, a miR-190a_M backbone sequence, or a miR-190a_M backbone sequence.

In some embodiments, the miRNA backbone sequence is a miR-100 backbone sequence or miR-100_M backbone sequence.

Table 2 provides examples of DNA sequences representing segments in miR-1-1, miR-100, miR-122, miR-124, miR-128, miR-130a, miR-155E, miR-155-M, and miR-138-2 backbones. Table 21 provides examples of DNA sequences representing segments in miR-1-1, miR-1-1_M, miR-100, miR-100_M, miR-122, miR-122_M, miR-124, miR-124 M, miR-128, miR-130a, miR-155E, miR-155M, miR-138-2, miR-144, miR-190a, miR-190a_M, miR-132, miR-451a, miR-223, and miR-16-2 backbones. It is understood that RNA sequences of the miR backbone segments in Tables 2 and 21 may be obtained by converting the “T” nucleotides in the sequences of Tables 2 and 21 to “U” nucleotides. Artificial miRNAs may be designed to insert desired guide and passenger sequences of the present disclosure into the miRNA backbones as defined in Table 2 or 21, and optionally wherein the passenger sequence is designed according to the rules in Table 8. For example, an artificial miRNA with miR-100 backbone in DNA format (e.g., for insertion into a transfer plasmid) may be designed according to Table 21 comprising from 5′ to 3′:5′ miR context (flanking) sequence of SEQ ID NO:1529; 5′ basal stem sequence of SEQ ID NO:1530; desired guide sequence; loop sequence of SEQ ID NO:1531; desired passenger sequence designed according to the rules in Table 8; 3′ basal stem sequence of SEQ ID NO:1532; and 3′ miR context (flanking) sequence of SEQ ID NO:1533.

TABLE 2
Annotation of miR Backbone Sequences
	5′ miR context						3′ miR context
	(flanking					3′ basal	(flanking
miR	segment)	5′ basal stem	5p	Terminal loop	3p	stem	segment)
miR-1-1	catgcagactgcct	TGGG	pas-	TATGGACCTGCTA	guide	CTCA	ggccgggacctctc
	gct	[SEQ ID NO:	sen-	AGCTA		[SEQ ID NO:	tcgccgcactgagg
	[SEQ ID NO:	492]	ger	[SEQ ID NO:		494]	ggcactccacacca
	491]			493]			cgggggccg
							[SEQ ID NO:
							495]
miR-100	CCCAAAAGAGAGAA	CCTGTTGCCACA	guide	GTATTAGTCCG	pas-	TGTGTCTGTTA	CAATCTCACGGACC
	GATATTGAGG	[SEQ ID NO:		[SEQ ID NO:	sen-	GG	TGGGGCTTTGCTTA
	[SEQ ID NO:	497]		498]	ger	[SEQ ID NO:	TATGCC
	496]					499]	[SEQ ID NO:
							500]
miR-122	ggctacagagttt	CCTTAGCAGAGCT	guide	TGTCTAAACTAT	pas-	TAGCTACTGCT	aatccttccctcga
	[SEQ ID NO:	G		[SEQ ID NO:	sen-	AGGC	taaatgtcttggca
	501]	[SEQ ID NO:		503]	ger	[SEQ ID NO:	tcgtttgctttg
		502]				504]	[SEQ ID NO:
							505]
miR-124	TTCCTTCCTCAGGA	AGGCCTCTCTC	pas-	ATTTAAATGTCCA	guide	GAATGGGGCTC	GCTGAGCACCGTGG
	GAA	[SEQ ID NO:	sen-	TACAAT		[SEQ ID NO:	GTCGGCGAGGGCCC
	[SEQ ID NO:	507]	ger	[SEQ ID NO:		509]	GCCAagga
	506]			508]			[SEQ ID NO:
							510]
miR-128	ATTTtgcaataatt	TGAGCTGTTGGA	pas-	GAGGTTTACATTT	guide	TTCAGCTGCTT	ctggcttcttttta
	ggccttgttcc	[SEQ ID NO:	sen-	C		C	ctcaggtttccact
	[SEQ ID NO:	512]	ger	[SEQ ID NO:		[SEQ ID NO:	gct
	511]			513]		514]	[SEQ ID NO:
							515]
miR-130a	gcagggccggcatg	TGCTGCTGGCCA	pas-	CTGTCTGCACCTG	guide	TGGCCGTGTAG	Ctacccagcgctgg
	cctc	[SEQ ID NO:	sen-	TCACTAG		TG	ctgcctcctcagca
	[SEQ ID NO:	517]	ger	[SEQ ID NO:		[SEQ ID NO:	ttg
	516]			518]		519]	[SEQ ID NO:
							520]
miR-155E	CTGGAGGCTTGCTT	GGGCTGTATGCTG	guide	TTTTGGCCTCTGA	pas-	CAGGACAAGGC	TTTATCAGCACTCA
	T	[SEQ ID NO:		CTGA	sen-	CC	CATGGAACAAATGG
	[SEQ ID NO:	522]		[SEQ ID NO:	ger	[SEQ ID NO:	CCACCGTG
	521]			523]		524]	[SEQ ID NO:
							525]
miR-155-	CCTGGAGGCTTGCT	AGGCTGTATGCTG	guide	TTTTGGCCACTGA	pas-	CAGGACACAAG	TGTTACTAGCACTC
M	GA	[SEQ ID NO:		CTGA	sen-	GCC	ACATGGAACAAATG
	[SEQ ID NO:	527]		[SEQ ID NO:	ger	[SEQ ID NO:	GCCACC
	526]			528]		529]	[SEQ ID NO:
							530]
miR-138-	gccggcggagttct	CGTTGCTGC	guide	GACGAGCAGCGCA	pas-	GTTGCATCA	tacccatcctctcc
2	ggtat	[SEQ ID NO:		TCCTCTTACCC	sen-	[SEQ ID NO:	aggcgagcctcgtg
	[SEQ ID NO:	532]		[SEQ ID NO:	ger	534]	ggacc
	531]			533]			[SEQ ID NO:
							535]

In some embodiments, the terminal loop, stem, 5′ flanking segment, 3′ flanking segment, or any combination thereof of the miR-155 backbone sequence, miR1-1 backbone sequence, miR-100 backbone sequence, miR-190A backbone sequence, miR-124 backbone sequence, miR-16-2 backbone sequence, miR-132 backbone sequence, miR-9 backbone sequence, miR-138-2 backbone sequence, miR-122 backbone sequence, miR-130a backbone sequence, miR-128 backbone sequence, miR-144 backbone sequence, miR-451a backbone sequence, or miR-223 backbone sequence is modified (e.g., has nucleotide insertion, deletion, substitution, mismatch, wobble, or any combination thereof).

Sequence motifs that enable efficient processing of pri-miRNA backbones have previously been identified. These include an UG motif at the 5′ end of the pre-miRNA, a mismatched GHG motif in the stem, and a 3′ CNNC motif. In some embodiments, the miR backbone sequence has been modified to incorporate these motifs, including for example, miR-155E backbone sequence, miR-1-1_M backbone, miR-100_M backbone sequence, miR-124_M backbone sequence, and miR-122_M backbone sequence. Such modified miR backbones are labeled herein by the suffix “_M.”

In some embodiments, the miRNA (pri-miRNA, a pre-mRNA, an artificial miRNA, or a mature miRNA) comprises or consists of a guide strand sequence and corresponding passenger strand sequence of any one of the duplexe sequences set forth in Tables 1, 19, 23, and 24. In some embodiments, the passenger strand sequence of the miRNA comprises a sequence that is 100% complementary or perfectly complementary to the guide strand sequence. For example, a guide strand sequence may comprise or consist of a sequence of SEQ ID NO: 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 124, 126, 128, 130, 132, 134, 136, 138, 140, 142, 144, 146, 148, 150, 152, 154, 156, 158, 160, 162, 164, 166, 168, 170, 172, 174, 176, 178, 180, 182, 184, 186, 188, 190, 192, 194, 196, 198, 200, 202, 204, 206, 208, 210, 212, 214, 216, 218, 220, 222, 224, 226, 228, 230, 232, 234, 236, 238, 240, 242, 244, 246, 248, 250, 252, 254, 256, 258, 260, 262, 264, 266, 268, 270, 272, 274, 276, 278, 280, 282, 284, 286, 288, 290, 292, 294, 296, 298, 300, 302, 304, 306, 308, 310, 312, 314, 316, 318, 320, 324, 326, 328, 330, 332, 334, 336, 338, 340, 342, 344, 346, 348, 350, 352, 354, 356, 358, 360, 362, 364, 366, 368, 370, 372, 374, 376, 378, 380, 382, 384, 386, 388, 390, 392, 394, 396, 398, 400, 402, 404, 406, 408, 410, 412, 414, 416, 418, 420, 422, 424, 426, 428, 430, 432, 434, or 436 (guide sequences in Table 1), and the passenger strand sequence may comprise or consist of a sequence of SEQ ID NO: 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 119, 121, 123, 125, 127, 129, 131, 133, 135, 137, 139, 141, 143, 145, 147, 149, 151, 153, 155, 157, 159, 161, 163, 165, 167, 169, 171, 173, 175, 177, 179, 181, 183, 185, 187, 189, 191, 193, 195, 197, 199, 201, 203, 205, 207, 209, 211, 213, 215, 217, 219, 221, 223, 225, 227, 229, 231, 233, 235, 237, 239, 241, 243, 245, 247, 249, 251, 253, 255, 257, 259, 261, 263, 265, 267, 269, 271, 273, 275, 277, 279, 281, 283, 285, 287, 289, 291, 293, 295, 297, 299, 301, 303, 305, 307, 309, 311, 313, 315, 317, 319, 321, 323, 325, 327, 329, 331, 333, 335, 337, 339, 341, 343, 345, 347, 349, 351, 353, 355, 357, 359, 361, 363, 365, 367, 369, 371, 373, 375, 377, 379, 381, 383, 385, 387, 389, 391, 393, 395, 397, 399, 401, 403, 405, 407, 409, 411, 413, 415, 417, 419, 421, 423, 425, 427, 429, 431, 433, or 435 (passenger sequences in Table 1), respectively. In some embodiments, the passenger strand sequence of the miRNA is not 100% complementary or to the guide strand sequence. For example, a guide strand sequence may comprise or consist of a sequence of SEQ ID NO:1176 and the corresponding passenger strand sequence may comprise or consist of a sequence of SEQ ID NO:1289 (see, Table 19).

In some embodiments, the miRNA (pri-miRNA, a pre-mRNA, an artificial miRNA, or a mature miRNA) comprises a guide strand sequence comprising or consisting of any one of SEQ ID NOS: 12, 14, 40, 60, 100, 104, 108, 112, 124, 126, 128, 166, 198, 220, 242, 302, 306, 308, 330, 336, and 362, and a passenger strand sequence of comprising a sequence that is 100% complementary or perfectly complementary to the guide strand sequence. For example, a guide strand sequence may comprise or consist of a sequence of SEQ ID NO: 12, 14, 40, 60, 100, 104, 108, 112, 124, 126, 128, 166, 198, 220, 242, 302, 306, 308, 330, 336, or 362, and the passenger strand sequence may comprise or consist of a sequence of SEQ ID NO: 11, 13, 39, 59, 99, 103, 107, 111, 123, 125, 127, 165, 197, 219, 241, 301, 305, 307, 329, 335, or 361, respectively.

In some embodiments, the miRNA (pri-miRNA, a pre-mRNA, an artificial miRNA, or a mature miRNA) comprises a guide strand sequence comprising or consisting of any one of SEQ ID NOS: 14, 40, 100, 108, 112, 128, 166, 198, 242, 308, 336, and 362, and the passenger strand sequence of the miRNA comprises or consists of a sequence that is 100% complementary or perfectly complementary to the guide strand. For example, a guide strand sequence may comprise a sequence of SEQ ID NO: 14, 40, 100, 108, 112, 128, 166, 198, 242, 308, 336, or 362, and the passenger strand sequence may comprise a sequence of SEQ ID NO: 13, 39, 99, 107, 111, 127, 165, 197, 241, 307, 335, or 361, respectively.

In some embodiments, the miRNA (pri-miRNA, a pre-mRNA, an artificial miRNA, or a mature miRNA) comprises a guide strand sequence comprising or consisting of any one of the guide sequences of Tables 1, 19, 23, and 24 and the passenger strand sequence comprises or consists of a corresponding passenger sequence of Tables 1, 19, 23, and 24 that has 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more insertions, deletions, substitutions, mismatches, wobbles, or any combination thereof relative to the passenger strand sequence of Tables 1, 19, 23 and 24. In some embodiments, the miRNA (pri-miRNA, a pre-mRNA, an artificial miRNA, or a mature miRNA) comprises a guide strand sequence comprising or consisting of any one of SEQ ID NOs: 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 124, 126, 128, 130, 132, 134, 136, 138, 140, 142, 144, 146, 148, 150, 152, 154, 156, 158, 160, 162, 164, 166, 168, 170, 172, 174, 176, 178, 180, 182, 184, 186, 188, 190, 192, 194, 196, 198, 200, 202, 204, 206, 208, 210, 212, 214, 216, 218, 220, 222, 224, 226, 228, 230, 232, 234, 236, 238, 240, 242, 244, 246, 248, 250, 252, 254, 256, 258, 260, 262, 264, 266, 268, 270, 272, 274, 276, 278, 280, 282, 284, 286, 288, 290, 292, 294, 296, 298, 300, 302, 304, 306, 308, 310, 312, 314, 316, 318, 320, 324, 326, 328, 330, 332, 334, 336, 338, 340, 342, 344, 346, 348, 350, 352, 354, 356, 358, 360, 362, 364, 366, 368, 370, 372, 374, 376, 378, 380, 382, 384, 386, 388, 390, 392, 394, 396, 398, 400, 402, 404, 406, 408, 410, 412, 414, 416, 418, 420, 422, 424, 426, 428, 430, 432, 434, 436, and a passenger strand sequence comprising or consisting a sequence of SEQ ID NOS: 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 119, 121, 123, 125, 127, 129, 131, 133, 135, 137, 139, 141, 143, 145, 147, 149, 151, 153, 155, 157, 159, 161, 163, 165, 167, 169, 171, 173, 175, 177, 179, 181, 183, 185, 187, 189, 191, 193, 195, 197, 199, 201, 203, 205, 207, 209, 211, 213, 215, 217, 219, 221, 223, 225, 227, 229, 231, 233, 235, 237, 239, 241, 243, 245, 247, 249, 251, 253, 255, 257, 259, 261, 263, 265, 267, 269, 271, 273, 275, 277, 279, 281, 283, 285, 287, 289, 291, 293, 295, 297, 299, 301, 303, 305, 307, 309, 311, 313, 315, 317, 319, 321, 323, 325, 327, 329, 331, 333, 335, 337, 339, 341, 343, 345, 347, 349, 351, 353, 355, 357, 359, 361, 363, 365, 367, 369, 371, 373, 375, 377, 379, 381, 383, 385, 387, 389, 391, 393, 395, 397, 399, 401, 403, 405, 407, 409, 411, 413, 415, 417, 419, 421, 423, 425, 427, 429, 431, 433, 435, respectively, wherein the passenger strand sequence has 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more insertions, deletions, substitutions, mismatches, wobbles, or any combination thereof relative to the passenger strand sequence of SEQ ID NOS: 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 119, 121, 123, 125, 127, 129, 131, 133, 135, 137, 139, 141, 143, 145, 147, 149, 151, 153, 155, 157, 159, 161, 163, 165, 167, 169, 171, 173, 175, 177, 179, 181, 183, 185, 187, 189, 191, 193, 195, 197, 199, 201, 203, 205, 207, 209, 211, 213, 215, 217, 219, 221, 223, 225, 227, 229, 231, 233, 235, 237, 239, 241, 243, 245, 247, 249, 251, 253, 255, 257, 259, 261, 263, 265, 267, 269, 271, 273, 275, 277, 279, 281, 283, 285, 287, 289, 291, 293, 295, 297, 299, 301, 303, 305, 307, 309, 311, 313, 315, 317, 319, 321, 323, 325, 327, 329, 331, 333, 335, 337, 339, 341, 343, 345, 347, 349, 351, 353, 355, 357, 359, 361, 363, 365, 367, 369, 371, 373, 375, 377, 379, 381, 383, 385, 387, 389, 391, 393, 395, 397, 399, 401, 403, 405, 407, 409, 411, 413, 415, 417, 419, 421, 423, 425, 427, 429, 431, 433, 435, respectively. In some embodiments, a mismatch is a G→C, C→G, A→T, or T→A conversion in the passenger strand sequence. In some embodiments, a mismatch (to create a bulge with the guide strand) is a G→T, C→A, A→C, or T→G conversion in the passenger strand sequence. In some embodiments, a wobble is a G-U wobble, wherein a C is converted to a T in the passenger strand sequence. In some embodiments, the passenger strand sequence is modified according to the rules of Table 8.

In some embodiments, the miRNA (pri-miRNA, a pre-mRNA, an artificial miRNA, or a mature miRNA) comprises a guide strand sequence comprising or consisting of any one of SEQ ID NOS: 12, 14, 40, 60, 100, 104, 108, 112, 124, 126, 128, 166, 198, 220, 242, 302, 306, 308, 330, 336, and 362, and a passenger strand sequence comprising or consisting of a sequence that has 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more insertions, deletions, substitutions, mismatches, wobbles, or any combination thereof relative to the passenger strand sequence comprising or consisting of a sequence of SEQ ID NOS: 11, 13, 39, 59, 99, 103, 107, 11, 123, 125, 127, 165, 197, 219, 241, 301, 305, 307, 329, 335, and 361, respectively. In some embodiments, a mismatch is a G→C, C→G, A→T, or T→A conversion in the passenger strand sequence. In some embodiments, a mismatch (to create a bulge with the guide strand) is a G→T, C→A, A→C, or T→G conversion in the passenger strand sequence. In some embodiments, a wobble is a G-U wobble, wherein a C is converted to a T in the passenger strand sequence. In some embodiments, the passenger strand sequence is modified according to the rules of Table 8.

In some embodiments, the miRNA (pri-miRNA, a pre-mRNA, an artificial miRNA, or a mature miRNA) comprises a guide strand sequence comprising or consisting of any one of SEQ ID NOS: 14, 40, 100, 108, 112, 128, 166, 198, 242, 308, 336, and 362, and a passenger strand sequence comprising or consisting of a sequence that has 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more insertions, deletions, substitutions, mismatches, wobbles, or any combination thereof relative to the passenger strand sequence comprising or consisting of a sequence of SEQ ID NO: 13, 39, 99, 107, 111, 127, 165, 197, 241, 307, 335, or 361, respectively. In some embodiments, a mismatch is a G→C, C→G, A→T, or T→A conversion in the passenger strand sequence. In some embodiments, a mismatch (to create a bulge with the guide strand) is a G→T, C→A, A→C, or T→G conversion in the passenger strand sequence. In some embodiments, a wobble is a G-U wobble, wherein a C is converted to a T in the passenger strand sequence. In some embodiments, the passenger strand sequence is modified according to the rules of Table 8.

In some embodiments, the miRNA is an artificial miRNA comprising a guide strand sequence according to any of the embodiments described herein, contained within a miR-155 backbone sequence, miR1-1 backbone sequence, miR-100 backbone sequence, miR-124 backbone sequence, mIR-138-2 backbone sequence, miR-122 backbone sequence, miR-128 backbone sequence, miR-130a backbone sequence, or miR-16-2 backbone sequence, wherein the artificial miRNA comprises a passenger strand sequence that is modified according to Table 8. In some embodiments, the passenger strand sequence comprises a mismatch, wherein a mismatch is a G→C, C→G, A→T, or T→A conversion in the passenger strand sequence; a mismatch (to create a bulge with the guide strand) is a G→T, C→A, A→C, or T→G conversion in the passenger strand sequence; and a wobble is a G-U wobble, wherein a C is converted to a T in the passenger strand sequence.

In some embodiments, an artificial miRNA comprises or consists of a nucleic acid sequence set forth in any one of Tables 3, 9, 11, 19, 23, 24, and 25. In some embodiments, an artificial miRNA comprises or consists of a nucleic acid sequence of any one of SEQ ID NOS: 443-490, 1109-1111, 1114, 1121-1168, 1405-1520, 1908-2007, 2011, 2017, 2021, 2025, 2027, 2031, 2035, 2039, 2041, 2045, 2049, 2053, 2057, 2061, 2067, 2071, 2075, 2079, 2085, 2089, 2093, 2097, 2101, 2105, 2109, 2113, 2117, 2120, 2124, 2128, 2132, 2136, 2140, 2144, 2148, 2154, 2158, 2162, 2166, 2170, 2174, 2176, 2180, 2182, 2184, 2187, 2189, 2191, 2193, 2195, 2197, 2199, 2205, 2211, 2261, 2263, 2265, and 2267.

TABLE 3
ATXN2 Specific amiRNAs
	Parent	miR
Parent Guide Sequence	Duplex ID	Backbone	Category	amiRNA Sequence
AGGAACGUGGGUUGAACUCCUU	XD-14857	miR-1-1	911 Control	CAUGCAGACUGCCUGCUUGGGAUGGAGUUCAAGGGACGUCGCC
[SEQ ID NO: 242]				UUAUGGACCUGCUAAGCUAAGGAACGUCCCUUGAACUCCUUCU
				CAGGCCGGGACCUCUCUCGCCGCACUGAGGGGCACUCCACACC
				ACGGGGGCC
				[SEQ ID NO: 437]
AGGAACGUGGGUUGAACUCCUU	XD-14857	miR-155E	911 Control	CUGGAGGCUUGCUUUGGGCUGUAUGCUGAGGAACGUCCCUUGA
[SEQ ID NO: 242]				ACUCCUUUUUUGGCCUCUGACUGAAAGGAGUUAAGGACGUUCC
				UCAGGACAAGGCCCUUUAUCAGCACUCACAUGGAACAAAUGGC
				CACCGUG
				[SEQ ID NO: 438]
UUCGGGUUGAAAUCUGAAGUGU	XD-14790	miR-155E	911 Control	CUGGAGGCUUGCUUUGGGCUGUAUGCUGUUCGGGUUCUUAUCU
[SEQ ID NO: 108]				GAAGUGUUUUUGGCCUCUGACUGAACACUUCAAUAGAACCCGA
				ACAGGACAAGGCCCUUUAUCAGCACUCACAUGGAACAAAUGGC
				CACCGUG
				[SEQ ID NO: 439]
UUCGGGUUGAAAUCUGAAGUGU	XD-14790	miR-1-1	911 Control	CAUGCAGACUGCCUGCUUGGGAGACUUCAGAUAAGAACCGAGA
[SEQ ID NO: 108]				AUAUGGACCUGCUAAGCUAUUCGGGUUCUUAUCUGAAGUGUCU
				CAGGCCGGGACCUCUCUCGCCGCACUGAGGGGCACUCCACACC
				ACGGGGGCC
				[SEQ ID NO: 440]
UUGAUUUCGAGGAUGUCGCUGG	XD-14800	miR-155E	911 Control	CUGGAGGCUUGCUUUGGGCUGUAUGCUGUUGAUUUCCUCGAUG
[SEQ ID NO: 128]				UCGCUGGUUUUGGCCUCUGACUGACCAGCGACUCGGGAAAUCA
				ACAGGACAAGGCCCUUUAUCAGCACUCACAUGGAACAAAUGGC
				CACCGUG
				[SEQ ID NO: 441]
UUGAUUUCGAGGAUGUCGCUGG	XD-14800	miR-1-1	911 Control	CAUGCAGACUGCCUGCUUGGGCGAGCGACAUCGAGGAAACGCA
[SEQ ID NO: 128]				AUAUGGACCUGCUAAGCUAUUGAUUUCCUCGAUGUCGCUGGCU
				CAGGCCGGGACCUCUCUCGCCGCACUGAGGGGCACUCCACACC
				ACGGGGGCC
				[SEQ ID NO: 442]
AGAAAUCGUAGACUGAGGCAGU	XD-14743	miR-1-1	Atxn2	CAUGCAGACUGCCUGCUUGGGAGUGCCUCAGUCUACGAUCGUC
[SEQ ID NO: 14]			targeting	UUAUGGACCUGCUAAGCUAAGAAAUCGUAGACUGAGGCAGUCU
				CAGGCCGGGACCUCUCUCGCCGCACUGAGGGGCACUCCACACC
				ACGGGGGCC
				[SEQ ID NO: 443]
AGAAAUCGUAGACUGAGGCAGU	XD-14743	miR-155E	Atxn2	CUGGAGGCUUGCUUUGGGCUGUAUGCUGAGAAAUCGUAGACUG
[SEQ ID NO: 14]			targeting	AGGCAGUUUUUGGCCUCUGACUGAACUGCCUCGUCACGAUUUC
				UCAGGACAAGGCCCUUUAUCAGCACUCACAUGGAACAAAUGGC
				CACCGUG
				[SEQ ID NO: 444]
AGAUACGUCAUUUUCCAAAGCC	XD-14766	miR-1-1	Atxn2	CAUGCAGACUGCCUGCUUGGGGCCUUUGGAAAAUGACGUCCUC
[SEQ ID NO: 60]			targeting	UUAUGGACCUGCUAAGCUAAGAUACGUCAUUUUCCAAAGCCCU
				CAGGCCGGGACCUCUCUCGCCGCACUGAGGGGCACUCCACACC
				ACGGGGGCC
				[SEQ ID NO: 445]
AGAUACGUCAUUUUCCAAAGCC	XD-14766	miR-155E	Atxn2	CUGGAGGCUUGCUUUGGGCUGUAUGCUGAGAUACGUCAUUUUC
[SEQ ID NO: 60]			targeting	CAAAGCCUUUUGGCCUCUGACUGAGGCUUUGGAAAGACGUAUC
				UCAGGACAAGGCCCUUUAUCAGCACUCACAUGGAACAAAUGGC
				CACCGUG
				[SEQ ID NO: 446]
AGCGUUAGGGUGCGCAUACUGC	XD-14904	miR-155E	Atxn2	CUGGAGGCUUGCUUUGGGCUGUAUGCUGAGCGUUAGGGUGCGC
[SEQ ID NO : 336]			targeting	AUACUGCUUUUGGCCUCUGACUGAGCAGUAUGGCACCUAACGC
				UCAGGACAAGGCCCUUUAUCAGCACUCACAUGGAACAAAUGGC
				CACCGUG
				[SEQ ID NO: 447]
AGCGUUAGGGUGCGCAUACUGC	XD-14904	miR-1-1	Atxn2	CAUGCAGACUGCCUGCUUGGGGGAGUAUGCGCACCCUAAGAGC
[SEQ ID NO: 336]			targeting	UUAUGGACCUGCUAAGCUAAGCGUUAGGGUGCGCAUACUGCCU
				CAGGCCGGGACCUCUCUCGCCGCACUGAGGGGCACUCCACACC
				ACGGGGGCC
				[SEQ ID NO: 448]
AGGAACGUGGGUUGAACUCCUU	XD-14857	miR-155E	Atxn2	CUGGAGGCUUGCUUUGGGCUGUAUGCUGAGGAACGUGGGUUGA
[SEQ ID NO: 242]			targeting	ACUCCUUUUUUGGCCUCUGACUGAAAGGAGUUAACCACGUUCC
				UCAGGACAAGGCCCUUUAUCAGCACUCACAUGGAACAAAUGGC
				CACCGUG
				[SEQ ID NO: 449]
AGGAACGUGGGUUGAACUCCUU	XD-14857	miR-1-1	Atxn2	CAUGCAGACUGCCUGCUUGGGAUGGAGUUCAACCCACGUCGCC
[SEQ ID NO: 242]			targeting	UUAUGGACCUGCUAAGCUAAGGAACGUGGGUUGAACUCCUUCU
				CAGGCCGGGACCUCUCUCGCCGCACUGAGGGGCACUCCACACC
				ACGGGGGCC
				[SEQ ID NO: 450]
AUAAUAAUCCGUCAGUUUGACG	XD-14949	miR-155E	Atxn2	CUGGAGGCUUGCUUUGGGCUGUAUGCUGAUAAUAAUCCGUCAG
[SEQ ID NO: 426]			targeting	UUUGACGUUUUGGCCUCUGACUGACGUCAAACGACGAUUAUUA
				UCAGGACAAGGCCCUUUAUCAGCACUCACAUGGAACAAAUGGC
				CACCGUG
				[SEQ ID NO: 451]
AUAAUAAUCCGUCAGUUUGACG	XD-14949	miR-1-1	Atxn2	CAUGCAGACUGCCUGCUUGGGCCUCAAACUGACGGAUUACGUA
[SEQ ID NO: 426]			targeting	UUAUGGACCUGCUAAGCUAAUAAUAAUCCGUCAGUUUGACGCU
				CAGGCCGGGACCUCUCUCGCCGCACUGAGGGGCACUCCACACC
				ACGGGGGCC
				[SEQ ID NO: 452]
AUACGCGGUGAAUUCUGUCUCC	XD-14787	miR-155E	Atxn2	CUGGAGGCUUGCUUUGGGCUGUAUGCUGAUACGCGGUGAAUUC
[SEQ ID NO: 102]			targeting	UGUCUCCUUUUGGCCUCUGACUGAGGAGACAGAUUACCGCGUA
				UCAGGACAAGGCCCUUUAUCAGCACUCACAUGGAACAAAUGGC
				CACCGUG
				[SEQ ID NO: 453]
AUACGCGGUGAAUUCUGUCUCC	XD-14787	miR-1-1	Atxn2	CAUGCAGACUGCCUGCUUGGGGCAGACAGAAUUCACCGCCUUA
[SEQ ID NO: 102]			targeting	UUAUGGACCUGCUAAGCUAAUACGCGGUGAAUUCUGUCUCCCU
				CAGGCCGGGACCUCUCUCGCCGCACUGAGGGGCACUCCACACC
				ACGGGGGCC
				[SEQ ID NO: 454]
AUUAACUACUCUUUGGUCUGAA	XD-14792	miR-1-1	Atxn2	CAUGCAGACUGCCUGCUUGGGUACAGACCAAAGAGUAGUCGAA
[SEQ ID NO: 112]			targeting	UUAUGGACCUGCUAAGCUAAUUAACUACUCUUUGGUCUGAACU
				CAGGCCGGGACCUCUCUCGCCGCACUGAGGGGCACUCCACACC
				ACGGGGGCC
				[SEQ ID NO: 455]
AUUAACUACUCUUUGGUCUGAA	XD-14792	miR-155E	Atxn2	CUGGAGGCUUGCUUUGGGCUGUAUGCUGAUUAACUACUCUUUG
[SEQ ID NO: 112]			targeting	GUCUGAAUUUUGGCCUCUGACUGAUUCAGACCAAGGUAGUUAA
				UCAGGACAAGGCCCUUUAUCAGCACUCACAUGGAACAAAUGGC
				CACCGUG
				[SEQ ID NO: 456]
AUUGCGUGGAGUAAGCUGGUGG	XD-14889	miR-155E	Atxn2	CUGGAGGCUUGCUUUGGGCUGUAUGCUGAUUGCGUGGAGUAAG
[SEQ ID NO: 306]			targeting	CUGGUGGUUUUGGCCUCUGACUGACCACCAGCUACCCACGCAA
				UCAGGACAAGGCCCUUUAUCAGCACUCACAUGGAACAAAUGGC
				CACCGUG
				[SEQ ID NO: 457]
AUUGCGUGGAGUAAGCUGGUGG	XD-14889	miR-1-1	Atxn2	CAUGCAGACUGCCUGCUUGGGCGACCAGCUUACUCCACGGAAA
[SEQ ID NO: 306]			targeting	UUAUGGACCUGCUAAGCUAAUUGCGUGGAGUAAGCUGGUGGCU
				CAGGCCGGGACCUCUCUCGCCGCACUGAGGGGCACUCCACACC
				ACGGGGGCC
				[SEQ ID NO: 458]
AUUUCGAGGAUGUCGCUGGGCC	XD-14798	miR-155E	Atxn2	CUGGAGGCUUGCUUUGGGCUGUAUGCUGAUUUCGAGGAUGUCG
[SEQ ID NO: 124]			targeting	CUGGGCCUUUUGGCCUCUGACUGAGGCCCAGCACACCUCGAAA
				UCAGGACAAGGCCCUUUAUCAGCACUCACAUGGAACAAAUGGC
				CACCGUG
				[SEQ ID NO: 459]
AUUUCGAGGAUGUCGCUGGGCC	XD-14798	miR-1-1	Atxn2	CAUGCAGACUGCCUGCUUGGGGCCCCAGCGACAUCCUCGCCAA
[SEQ ID NO: 124]			targeting	UUAUGGACCUGCUAAGCUAAUUUCGAGGAUGUCGCUGGGCCCU
				CAGGCCGGGACCUCUCUCGCCGCACUGAGGGGCACUCCACACC
				ACGGGGGCC
				[SEQ ID NO: 460]
UAAAUCGUAGACUGAGGCAGUC	XD-14742	miR-1-1	Atxn2	CAUGCAGACUGCCUGCUUGGGGUCUGCCUCAGUCUACGACGUU
[SEQ ID NO: 12]			targeting	AUAUGGACCUGCUAAGCUAUAAAUCGUAGACUGAGGCAGUCCU
				CAGGCCGGGACCUCUCUCGCCGCACUGAGGGGCACUCCACACC
				ACGGGGGCC
				[SEQ ID NO: 461]
UAAAUCGUAGACUGAGGCAGUC	XD-14742	miR-155E	Atxn2	CUGGAGGCUUGCUUUGGGCUGUAUGCUGUAAAUCGUAGACUGA
[SEQ ID NO: 12]			targeting	GGCAGUCUUUUGGCCUCUGACUGAGACUGCCUAGUUACGAUUU
				ACAGGACAAGGCCCUUUAUCAGCACUCACAUGGAACAAAUGGC
				CACCGUG
				[SEQ ID NO: 462]
UACGCGGUGAAUUCUGUCUCCC	XD-14786	miR-1-1	Atxn2	CAUGCAGACUGCCUGCUUGGGGCGAGACAGAAUUCACCGGAGU
[SEQ ID NO: 100]			targeting	AUAUGGACCUGCUAAGCUAUACGCGGUGAAUUCUGUCUCCCCU
				CAGGCCGGGACCUCUCUCGCCGCACUGAGGGGCACUCCACACC
				ACGGGGGCC
				[SEQ ID NO: 463]
UACGCGGUGAAUUCUGUCUCCC	XD-14786	miR-155E	Atxn2	CUGGAGGCUUGCUUUGGGCUGUAUGCUGUACGCGGUGAAUUCU
[SEQ ID NO: 100]			targeting	GUCUCCCUUUUGGCCUCUGACUGAGGGAGACAAAUCACCGCGU
				ACAGGACAAGGCCCUUUAUCAGCACUCACAUGGAACAAAUGGC
				CACCGUG
				[SEQ ID NO: 464]
UAUACGCGGUGAAUUCUGUCUC	XD-14788	miR-1-1	Atxn2	CAUGCAGACUGCCUGCUUGGGGUGACAGAAUUCACCGCGCGAU
[SEQ ID NO: 104]			targeting	AUAUGGACCUGCUAAGCUAUAUACGCGGUGAAUUCUGUCUCCU
				CAGGCCGGGACCUCUCUCGCCGCACUGAGGGGCACUCCACACC
				ACGGGGGCC
				[SEQ ID NO: 465]
UAUACGCGGUGAAUUCUGUCUC	XD-14788	miR-155E	Atxn2	CUGGAGGCUUGCUUUGGGCUGUAUGCUGUAUACGCGGUGAAUU
[SEQ ID NO: 104]			targeting	CUGUCUCUUUUGGCCUCUGACUGAGAGACAGAUUCCCGCGUAU
				ACAGGACAAGGCCCUUUAUCAGCACUCACAUGGAACAAAUGGC
				CACCGUG
				[SEQ ID NO: 466]
UAUUGCGUGGAGUAAGCUGGUG	XD-14890	miR-1-1	Atxn2	CAUGCAGACUGCCUGCUUGGGCUCCAGCUUACUCCACGCCCAU
[SEQ ID NO: 308]			targeting	AUAUGGACCUGCUAAGCUAUAUUGCGUGGAGUAAGCUGGUGCU
				CAGGCCGGGACCUCUCUCGCCGCACUGAGGGGCACUCCACACC
				ACGGGGGCC
				[SEQ ID NO: 467]
UAUUGCGUGGAGUAAGCUGGUG	XD-14890	miR-155E	Atxn2	CUGGAGGCUUGCUUUGGGCUGUAUGCUGUAUUGCGUGGAGUAA
[SEQ ID NO: 308]			targeting	GCUGGUGUUUUGGCCUCUGACUGACACCAGCUACUCACGCAAU
				ACAGGACAAGGCCCUUUAUCAGCACUCACAUGGAACAAAUGGC
				CACCGUG
				[SEQ ID NO: 468]
UAUUUCGAGGAUGUCGCUGGGC	XD-14799	miR-155E	Atxn2	CUGGAGGCUUGCUUUGGGCUGUAUGCUGUAUUUCGAGGAUGUC
[SEQ ID NO: 126]			targeting	GCUGGGCUUUUGGCCUCUGACUGAGCCCAGCGCAUCUCGAAAU
				ACAGGACAAGGCCCUUUAUCAGCACUCACAUGGAACAAAUGGC
				CACCGUG
				[SEQ ID NO: 469]
UAUUUCGAGGAUGUCGCUGGGC	XD-14799	miR-1-1	Atxn2	CAUGCAGACUGCCUGCUUGGGGGCCAGCGACAUCCUCGACCAU
[SEQ ID NO: 126]			targeting	AUAUGGACCUGCUAAGCUAUAUUUCGAGGAUGUCGCUGGGCCU
				CAGGCCGGGACCUCUCUCGCCGCACUGAGGGGCACUCCACACC
				ACGGGGGCC
				[SEQ ID NO: 470]
UCGCUGUUGGGGCAUAUUUGGU	XD-14887	miR-1-1	Atxn2	CAUGCAGACUGCCUGCUUGGGAGCAAAUAUGCCCCAACACUCG
[SEQ ID NO: 302]			targeting	AUAUGGACCUGCUAAGCUAUCGCUGUUGGGGCAUAUUUGGUCU
				CAGGCCGGGACCUCUCUCGCCGCACUGAGGGGCACUCCACACC
				ACGGGGGCC
				[SEQ ID NO: 471]
UCGCUGUUGGGGCAUAUUUGGU	XD-14887	miR-155E	Atxn2	CUGGAGGCUUGCUUUGGGCUGUAUGCUGUCGCUGUUGGGGCAU
[SEQ ID NO: 302]			targeting	AUUUGGUUUUUGGCCUCUGACUGAACCAAAUAGCCCAACAGCG
				ACAGGACAAGGCCCUUUAUCAGCACUCACAUGGAACAAAUGGC
				CACCGUG
				[SEQ ID NO: 472]
UGCGCAUACUGCUGAGCAAGGG	XD-14901	miR-1-1	Atxn2	CAUGCAGACUGCCUGCUUGGGCGCUUGCUCAGCAGUAUGGAGC
[SEQ ID NO: 330]			targeting	AUAUGGACCUGCUAAGCUAUGCGCAUACUGCUGAGCAAGGGCU
				CAGGCCGGGACCUCUCUCGCCGCACUGAGGGGCACUCCACACC
				ACGGGGGCC
				[SEQ ID NO: 473]
UGCGCAUACUGCUGAGCAAGGG	XD-14901	miR-155E	Atxn2	CUGGAGGCUUGCUUUGGGCUGUAUGCUGUGCGCAUACUGCUGA
[SEQ ID NO: 330]			targeting	GCAAGGGUUUUGGCCUCUGACUGACCCUUGCUAGCGUAUGCGC
				ACAGGACAAGGCCCUUUAUCAGCACUCACAUGGAACAAAUGGC
				CACCGUG
				[SEQ ID NO: 474]
UGUACCACAACAAAGUCUGAAC	XD-14756	miR-1-1	Atxn2	CAUGCAGACUGCCUGCUUGGGGAUCAGACUUUGUUGUGGCGAC
[SEQ ID NO: 40]			targeting	AUAUGGACCUGCUAAGCUAUGUACCACAACAAAGUCUGAACCU
				CAGGCCGGGACCUCUCUCGCCGCACUGAGGGGCACUCCACACC
				ACGGGGGCC
				[SEQ ID NO: 475]
UGUACCACAACAAAGUCUGAAC	XD-14756	miR-155E	Atxn2	CUGGAGGCUUGCUUUGGGCUGUAUGCUGUGUACCACAACAAAG
[SEQ ID NO: 40]			targeting	UCUGAACUUUUGGCCUCUGACUGAGUUCAGACUUGUGUGGUAC
				ACAGGACAAGGCCCUUUAUCAGCACUCACAUGGAACAAAUGGC
				CACCGUG
				[SEQ ID NO: 476]
UGUAUACGCCGGCUGAACGUGA	XD-14917	miR-1-1	Atxn2	CAUGCAGACUGCCUGCUUGGGUGACGUUCAGCCGGCGUACGAC
[SEQ ID NO: 362]			targeting	AUAUGGACCUGCUAAGCUAUGUAUACGCCGGCUGAACGUGACU
				CAGGCCGGGACCUCUCUCGCCGCACUGAGGGGCACUCCACACC
				ACGGGGGCC
				[SEQ ID NO: 477]
UGUAUACGCCGGCUGAACGUGA	XD-14917	miR-155E	Atxn2	CUGGAGGCUUGCUUUGGGCUGUAUGCUGUGUAUACGCCGGCUG
[SEQ ID NO: 362]			targeting	AACGUGAUUUUGGCCUCUGACUGAUCACGUUCGCCGCGUAUAC
				ACAGGACAAGGCCCUUUAUCAGCACUCACAUGGAACAAAUGGC
				CACCGUG
				[SEQ ID NO: 478]
UUACUAAGUAUUGAAGGGGAAA	XD-14846	miR-155E	Atxn2	CUGGAGGCUUGCUUUGGGCUGUAUGCUGUUACUAAGUAUUGAA
[SEQ ID NO: 220]			targeting	GGGGAAAUUUUGGCCUCUGACUGAUUUCCCCUCAAACUUAGUA
				ACAGGACAAGGCCCUUUAUCAGCACUCACAUGGAACAAAUGGC
				CACCGUG
				[SEQ ID NO: 479]
UUACUAAGUAUUGAAGGGGAAA	XD-14846	miR-1-1	Atxn2	CAUGCAGACUGCCUGCUUGGGUAUCCCCUUCAAUACUUACUUA
[SEQ ID NO: 220]			targeting	AUAUGGACCUGCUAAGCUAUUACUAAGUAUUGAAGGGGAAACU
				CAGGCCGGGACCUCUCUCGCCGCACUGAGGGGCACUCCACACC
				ACGGGGGCC
				[SEQ ID NO: 480]
UUAGUUGAUCCAUAGAUUCAGA	XD-14835	miR-1-1	Atxn2	CAUGCAGACUGCCUGCUUGGGUGUGAAUCUAUGGAUCAAGAUA
[SEQ ID NO: 198]			targeting	AUAUGGACCUGCUAAGCUAUUAGUUGAUCCAUAGAUUCAGACU
				CAGGCCGGGACCUCUCUCGCCGCACUGAGGGGCACUCCACACC
				ACGGGGGCC
				[SEQ ID NO: 481]
UUAGUUGAUCCAUAGAUUCAGA	XD-14835	miR-155E	Atxn2	CUGGAGGCUUGCUUUGGGCUGUAUGCUGUUAGUUGAUCCAUAG
[SEQ ID NO: 198]			targeting	AUUCAGAUUUUGGCCUCUGACUGAUCUGAAUCAUGAUCAACUA
				ACAGGACAAGGCCCUUUAUCAGCACUCACAUGGAACAAAUGGC
				CACCGUG
				[SEQ ID NO: 482]
UUCGAUGCAGGACUAGCAGGCG	XD-14819	miR-155E	Atxn2	CUGGAGGCUUGCUUUGGGCUGUAUGCUGUUCGAUGCAGGACUA
[SEQ ID NO: 166]			targeting	GCAGGCGUUUUGGCCUCUGACUGACGCCUGCUGUCUGCAUCGA
				ACAGGACAAGGCCCUUUAUCAGCACUCACAUGGAACAAAUGGC
				CACCGUG
				[SEQ ID NO: 483]
UUCGAUGCAGGACUAGCAGGCG	XD-14819	miR-1-1	Atxn2	CAUGCAGACUGCCUGCUUGGGCCCCUGCUAGUCCUGCAUGAGA
[SEQ ID NO: 166]			targeting	AUAUGGACCUGCUAAGCUAUUCGAUGCAGGACUAGCAGGCGCU
				CAGGCCGGGACCUCUCUCGCCGCACUGAGGGGCACUCCACACC
				ACGGGGGCC
				[SEQ ID NO: 484]
UUCGGGUUGAAAUCUGAAGUGU	XD-14790	miR-155E	Atxn2	CUGGAGGCUUGCUUUGGGCUGUAUGCUGUUCGGGUUGAAAUCU
[SEQ ID NO: 108]			targeting	GAAGUGUUUUUGGCCUCUGACUGAACACUUCAAUUCAACCCGA
				ACAGGACAAGGCCCUUUAUCAGCACUCACAUGGAACAAAUGGC
				CACCGUG
				[SEQ ID NO: 485]
UUCGGGUUGAAAUCUGAAGUGU	XD-14790	miR-1-1	Atxn2	CAUGCAGACUGCCUGCUUGGGAGACUUCAGAUUUCAACCGAGA
[SEQ ID NO: 108]			targeting	AUAUGGACCUGCUAAGCUAUUCGGGUUGAAAUCUGAAGUGUCU
				CAGGCCGGGACCUCUCUCGCCGCACUGAGGGGCACUCCACACC
				ACGGGGGCC
				[SEQ ID NO: 486]
UUGAUUUCGAGGAUGUCGCUGG	XD-14800	miR-1-1	Atxn2	CAUGCAGACUGCCUGCUUGGGCGAGCGACAUCCUCGAAACGCA
[SEQ ID NO: 128]			targeting	AUAUGGACCUGCUAAGCUAUUGAUUUCGAGGAUGUCGCUGGCU
				CAGGCCGGGACCUCUCUCGCCGCACUGAGGGGCACUCCACACC
				ACGGGGGCC
				[SEQ ID NO: 487]
UUGAUUUCGAGGAUGUCGCUGG	XD-14800	miR-155E	Atxn2	CUGGAGGCUUGCUUUGGGCUGUAUGCUGUUGAUUUCGAGGAUG
[SEQ ID NO: 128]			targeting	UCGCUGGUUUUGGCCUCUGACUGACCAGCGACUCCCGAAAUCA
				ACAGGACAAGGCCCUUUAUCAGCACUCACAUGGAACAAAUGGC
				CACCGUG
				[SEQ ID NO: 488]
UUGUACUGGGCACUUGACUCAA	XD-14781	miR-155E	Atxn2	CUGGAGGCUUGCUUUGGGCUGUAUGCUGUUGUACUGGGCACUU
[SEQ ID NO: 90]			targeting	GACUCAAUUUUGGCCUCUGACUGAUUGAGUCAGUGCCAGUACA
				ACAGGACAAGGCCCUUUAUCAGCACUCACAUGGAACAAAUGGC
				CACCGUG
				[SEQ ID NO: 489]
UUGUACUGGGCACUUGACUCAA	XD-14781	miR-1-1	Atxn2	CAUGCAGACUGCCUGCUUGGGUAGAGUCAAGUGCCCAGUCCCA
[SEQ ID NO: 90]			targeting	AUAUGGACCUGCUAAGCUAUUGUACUGGGCACUUGACUCAACU
				CAGGCCGGGACCUCUCUCGCCGCACUGAGGGGCACUCCACACC
				ACGGGGGCC
				[SEQ ID NO: 490]

In some embodiments, an artificial miRNA comprises or consists of a nucleic acid sequence set forth in Table 3. In some embodiments, an artificial miRNA comprises or consists of a nucleic acid sequence of any one of SEQ ID NOS:443-490.

In some embodiments, an artificial miRNA comprises or consists of a nucleic acid sequence set forth in Table 9. In some embodiments, an artificial miRNA comprises or consists of a nucleic acid sequence of any one of SEQ ID NOS:1109-1111, and 1114.

In some embodiments, an artificial miRNA comprises or consists of a nucleic acid sequence set forth in Table 11. In some embodiments, an artificial miRNA comprises or consists of a nucleic acid sequence of any one of SEQ ID NOS:1121-1168.

In some embodiments, an artificial miRNA comprises or consists of a nucleic acid sequence set forth in Table 19. In some embodiments, an artificial miRNA comprises or consists of a nucleic acid sequence of any one of SEQ ID NOS:1405-1520.

In some embodiments, an artificial miRNA comprises or consists of a nucleic acid sequence set forth in Table 23. In some embodiments, an artificial miRNA comprises or consists of a nucleic acid sequence of any one of SEQ ID NOS:1908-2007.

In some embodiments, an artificial miRNA comprises or consists of a nucleic acid sequence set forth in Table 24. In some embodiments, an artificial miRNA comprises or consists of a nucleic acid sequence of any one of SEQ ID NOS:1908-1934, 1936-1977, 1979-1982, 1984-1994, 1997, 1998, 2000, 2001, 2005-2007, 2011, 2017, 2021, 2025, 2027, 2031, 2035, 2039, 2041, 2045, 2049, 2053, 2057, 2061, 2067, 2071, 2075, 2079, 2085, 2089, 2093, 2097, 2101, 2105, 2109, 2113, 2117, 2120, 2124, 2128, 2132, 2136, 2140, 2144, 2148, 2154, 2158, 2162, 2166, 2170, 2174, 2176, 2180, 2182, 2184, 2187, 2189, 2191, 2193, 2195, 2197, 2199, 2205, 2211, 2261, 2263, 2265, and 2267.

In some embodiments, an artificial miRNA comprises or consists of a nucleic acid sequence set forth in Table 25. In some embodiments, an artificial miRNA comprises or consists of a nucleic acid sequence of any one of SEQ ID NOS:1915, 1982, 1965, 1937, 1985, 1921, and 2021.

Expression Constructs

In another aspect, the present disclosure provides an isolated nucleic acid comprising an expression construct or expression cassette encoding any one of the inhibitory nucleic acids (e.g., siRNA, shRNA, dsRNA, miRNA, amiRNA, etc.) that inhibit the expression or activity of ATXN2 as described herein.

In some embodiments, the isolated nucleic acid comprises an expression construct encoding an inhibitory nucleic acid that inhibits the expression or activity of ATXN2, wherein the inhibitory nucleic acid comprises a guide strand sequence comprising or consisting of the nucleic acid sequence set forth in any one of the guide sequences of Tables 1, 3, 9, 11, 12, 13, 19, 23, 24, and 25, e.g., SEQ ID NOS: 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 124, 126, 128, 130, 132, 134, 136, 138, 140, 142, 144, 146, 148, 150, 152, 154, 156, 158, 160, 162, 164, 166, 168, 170, 172, 174, 176, 178, 180, 182, 184, 186, 188, 190, 192, 194, 196, 198, 200, 202, 204, 206, 208, 210, 212, 214, 216, 218, 220, 222, 224, 226, 228, 230, 232, 234, 236, 238, 240, 242, 244, 246, 248, 250, 252, 254, 256, 258, 260, 262, 264, 266, 268, 270, 272, 274, 276, 278, 280, 282, 284, 286, 288, 290, 292, 294, 296, 298, 300, 302, 304, 306, 308, 310, 312, 314, 316, 318, 320, 324, 326, 328, 330, 332, 334, 336, 338, 340, 342, 344, 346, 348, 350, 352, 354, 356, 358, 360, 362, 364, 366, 368, 370, 372, 374, 376, 378, 380, 382, 384, 386, 388, 390, 392, 394, 396, 398, 400, 402, 404, 406, 408, 410, 412, 414, 416, 418, 420, 422, 424, 426, 428, 430, 432, 434, 436, 1176-1288, 1811-1827, 2015, 2065, 2083, 2152, 2203, and 2209.

In some embodiments, the isolated nucleic acid comprises an expression construct encoding an inhibitory nucleic acid that inhibits the expression or activity of ATXN2, wherein the inhibitory nucleic acid comprises a guide strand sequence comprising or consisting of the nucleic acid sequence set forth in any one of the guide sequences of Tables 1, 3, 9, 11, 12, 13, 19, 23, 24, and 25, e.g., SEQ ID NOS: 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 124, 126, 128, 130, 132, 134, 136, 138, 140, 142, 144, 146, 148, 150, 152, 154, 156, 158, 160, 162, 164, 166, 168, 170, 172, 174, 176, 178, 180, 182, 184, 186, 188, 190, 192, 194, 196, 198, 200, 202, 204, 206, 208, 210, 212, 214, 216, 218, 220, 222, 224, 226, 228, 230, 232, 234, 236, 238, 240, 242, 244, 246, 248, 250, 252, 254, 256, 258, 260, 262, 264, 266, 268, 270, 272, 274, 276, 278, 280, 282, 284, 286, 288, 290, 292, 294, 296, 298, 300, 302, 304, 306, 308, 310, 312, 314, 316, 318, 320, 324, 326, 328, 330, 332, 334, 336, 338, 340, 342, 344, 346, 348, 350, 352, 354, 356, 358, 360, 362, 364, 366, 368, 370, 372, 374, 376, 378, 380, 382, 384, 386, 388, 390, 392, 394, 396, 398, 400, 402, 404, 406, 408, 410, 412, 414, 416, 418, 420, 422, 424, 426, 428, 430, 432, 434, 436, 1176-1288, 1811-1827, 2015, 2065, 2083, 2152, 2203, and 2209, with at least 1, 2, 3, 4, or 5 mismatches to the target ATXN2 mRNA sequence.

In some embodiments, the isolated nucleic acid comprises an expression construct encoding an inhibitory nucleic acid that inhibits the expression or activity of ATXN2, wherein the inhibitory nucleic acid comprises a guide strand sequence comprising or consisting of a sequence at least 60%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 99%, or 100% identical to any one of the guide sequences of Tables 1, 3, 9, 11, 12, 13, 19, 23, 24, and 25, e.g., SEQ ID NOS: 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 124, 126, 128, 130, 132, 134, 136, 138, 140, 142, 144, 146, 148, 150, 152, 154, 156, 158, 160, 162, 164, 166, 168, 170, 172, 174, 176, 178, 180, 182, 184, 186, 188, 190, 192, 194, 196, 198, 200, 202, 204, 206, 208, 210, 212, 214, 216, 218, 220, 222, 224, 226, 228, 230, 232, 234, 236, 238, 240, 242, 244, 246, 248, 250, 252, 254, 256, 258, 260, 262, 264, 266, 268, 270, 272, 274, 276, 278, 280, 282, 284, 286, 288, 290, 292, 294, 296, 298, 300, 302, 304, 306, 308, 310, 312, 314, 316, 318, 320, 324, 326, 328, 330, 332, 334, 336, 338, 340, 342, 344, 346, 348, 350, 352, 354, 356, 358, 360, 362, 364, 366, 368, 370, 372, 374, 376, 378, 380, 382, 384, 386, 388, 390, 392, 394, 396, 398, 400, 402, 404, 406, 408, 410, 412, 414, 416, 418, 420, 422, 424, 426, 428, 430, 432, 434, 436, 1176-1288, 1811-1827, 2015, 2065, 2083, 2152, 2203, and 2209.

In some embodiments, the isolated nucleic acid comprises an expression construct encoding an inhibitory nucleic acid that inhibits the expression or activity of ATXN2, wherein the inhibitory nucleic acid comprises a guide strand sequence comprising or consisting of at least 15, 16, 17, 18, 19, 20, 21, or 22 contiguous nucleotides of a sequence of any one of the guide sequences of Tables 1, 3, 9, 11, 12, 13, 19, 23, 24, and 25 e.g., SEQ ID NOS: 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 124, 126, 128, 130, 132, 134, 136, 138, 140, 142, 144, 146, 148, 150, 152, 154, 156, 158, 160, 162, 164, 166, 168, 170, 172, 174, 176, 178, 180, 182, 184, 186, 188, 190, 192, 194, 196, 198, 200, 202, 204, 206, 208, 210, 212, 214, 216, 218, 220, 222, 224, 226, 228, 230, 232, 234, 236, 238, 240, 242, 244, 246, 248, 250, 252, 254, 256, 258, 260, 262, 264, 266, 268, 270, 272, 274, 276, 278, 280, 282, 284, 286, 288, 290, 292, 294, 296, 298, 300, 302, 304, 306, 308, 310, 312, 314, 316, 318, 320, 324, 326, 328, 330, 332, 334, 336, 338, 340, 342, 344, 346, 348, 350, 352, 354, 356, 358, 360, 362, 364, 366, 368, 370, 372, 374, 376, 378, 380, 382, 384, 386, 388, 390, 392, 394, 396, 398, 400, 402, 404, 406, 408, 410, 412, 414, 416, 418, 420, 422, 424, 426, 428, 430, 432, 434, 436, 1176-1288, 1811-1827, 2015, 2065, 2083, 2152, 2203, and 2209, preferably wherein the guide strand sequence retains positions 2-7 (“seed sequence”) of the selected SEQ ID NO.

In some embodiments, the isolated nucleic acid comprises an expression construct encoding an inhibitory nucleic acid that inhibits the expression or activity of ATXN2, wherein the inhibitory nucleic acid comprises a guide strand sequence comprising or consisting of a sequence of any one of the guide sequences of Tables 1, 3, 9, 11, 12, 13, 19, 23, 24, and 25, e.g., SEQ ID NOS: 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 124, 126, 128, 130, 132, 134, 136, 138, 140, 142, 144, 146, 148, 150, 152, 154, 156, 158, 160, 162, 164, 166, 168, 170, 172, 174, 176, 178, 180, 182, 184, 186, 188, 190, 192, 194, 196, 198, 200, 202, 204, 206, 208, 210, 212, 214, 216, 218, 220, 222, 224, 226, 228, 230, 232, 234, 236, 238, 240, 242, 244, 246, 248, 250, 252, 254, 256, 258, 260, 262, 264, 266, 268, 270, 272, 274, 276, 278, 280, 282, 284, 286, 288, 290, 292, 294, 296, 298, 300, 302, 304, 306, 308, 310, 312, 314, 316, 318, 320, 324, 326, 328, 330, 332, 334, 336, 338, 340, 342, 344, 346, 348, 350, 352, 354, 356, 358, 360, 362, 364, 366, 368, 370, 372, 374, 376, 378, 380, 382, 384, 386, 388, 390, 392, 394, 396, 398, 400, 402, 404, 406, 408, 410, 412, 414, 416, 418, 420, 422, 424, 426, 428, 430, 432, 434, 436, 1176-1288, 1811-1827, 2015, 2065, 2083, 2152, 2203, and 2209, wherein 1, 2, 3, or 4 nucleotides at positions 19-22 differ from the selected SEQ ID NO (variant nucleotide(s)) such that the guide strand sequence is no longer complementary to the ATXN2 target sequence at the variant nucleotide(s).

In some embodiments, the isolated nucleic acid molecule comprises an expression construct encoding an inhibitory nucleic acid that inhibits the expression or activity of ATXN2, wherein the inhibitory nucleic acid comprises a guide strand sequence of Table 12. In some embodiments, the inhibitory nucleic acid comprises a guide strand sequence comprising or consisting of the nucleic acid sequence set forth in any one of SEQ ID NOS: 12, 14, 40, 60, 100, 104, 108, 112, 124, 126, 128, 166, 198, 220, 242, 302, 306, 308, 330, 336, and 362. In some embodiments, the isolated nucleic acid comprises an expression construct encoding an inhibitory nucleic acid that inhibits the expression or activity of ATXN2, wherein the inhibitory nucleic acid comprises a guide strand sequence comprising or consisting of the nucleic acid sequence set forth in any one of SEQ ID NOS: 12, 14, 40, 60, 100, 104, 108, 112, 124, 126, 128, 166, 198, 220, 242, 302, 306, 308, 330, 336, and 362 with at least 1, 2, 3, 4, or 5 mismatches to the target ATXN2 mRNA sequence. In some embodiments, the isolated nucleic acid comprises an expression construct encoding an inhibitory nucleic acid that inhibits the expression or activity of ATXN2, wherein the inhibitory nucleic acid comprises a guide strand sequence comprising or consisting of a nucleic acid sequence that is at least 60%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 99%, or 100% identical to any one of SEQ ID NOS: 12, 14, 40, 60, 100, 104, 108, 112, 124, 126, 128, 166, 198, 220, 242, 302, 306, 308, 330, 336, and 362. In some embodiments, the isolated nucleic acid comprises an expression construct encoding an inhibitory nucleic acid that inhibits the expression or activity of ATXN2, wherein the inhibitory nucleic acid comprises a guide strand sequence comprising or consisting of at least 15, 16, 17, 18, 19, 20, 21, or 22 contiguous nucleotides of a sequence of any one of SEQ ID NOS: 12, 14, 40, 60, 100, 104, 108, 112, 124, 126, 128, 166, 198, 220, 242, 302, 306, 308, 330, 336, and 362, preferably wherein the guide strand sequence retains positions 2-7 (“seed sequence”) of the selected SEQ ID NO. In some embodiments, the isolated nucleic acid comprises an expression construct encoding an inhibitory nucleic acid that inhibits the expression or activity of ATXN2, wherein the inhibitory nucleic acid comprises a guide strand sequence comprising or consisting of a sequence of any one of SEQ ID NOS: 12, 14, 40, 60, 100, 104, 108, 112, 124, 126, 128, 166, 198, 220, 242, 302, 306, 308, 330, 336, and 362, wherein 1, 2, 3, or 4 nucleotides at positions 19-22 differ from the selected SEQ ID NO (variant nucleotide(s)), such that the guide strand sequence is no longer complementary to the ATXN2 target sequence at the variant nucleotide(s).

In some embodiments, the isolated nucleic acid comprises an expression construct encoding an inhibitory nucleic acid that inhibits the expression or activity of ATXN2, wherein the inhibitory nucleic acid comprises a guide strand sequence of Table 13. In some embodiments, the inhibitory nucleic acid comprises a guide strand sequence comprising or consisting of the nucleic acid sequence set forth in any one of SEQ ID NOS: 14, 40, 100, 108, 112, 128, 166, 198, 242, 308, 336, and 362. In some embodiments, the isolated nucleic acid comprises an expression construct encoding an inhibitory nucleic acid that inhibits the expression or activity of ATXN2, wherein the inhibitory nucleic acid comprises a guide strand sequence comprising or consisting of the nucleic acid sequence set forth in any one of SEQ ID NOS: 14, 40, 100, 108, 112, 128, 166, 198, 242, 308, 336, and 362 with at least 1, 2, 3, 4, or 5 mismatches to the target ATXN2 mRNA sequence. In some embodiments, the isolated nucleic acid comprises an expression construct encoding an inhibitory nucleic acid that inhibits the expression or activity of ATXN2, wherein the inhibitory nucleic acid comprises a guide strand sequence comprising or consisting of a nucleic acid sequence that is at least 60%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 99%, or 100% identical to any one of SEQ ID NOS: 14, 40, 100, 108, 112, 128, 166, 198, 242, 308, 336, and 362. In some embodiments, the isolated nucleic acid comprises an expression construct encoding an inhibitory nucleic acid that inhibits the expression or activity of ATXN2, wherein the inhibitory nucleic acid comprises a guide strand sequence comprising or consisting of at least 15, 16, 17, 18, 19, 20, 21, or 22 contiguous nucleotides of a sequence of any one of SEQ ID NOS: 14, 40, 100, 108, 112, 128, 166, 198, 242, 308, 336, and 362, preferably wherein the guide strand sequence retains positions 2-7 (“seed sequence”) of the selected SEQ ID NO. In some embodiments, the isolated nucleic acid comprises an expression construct encoding an inhibitory nucleic acid that inhibits the expression or activity of ATXN2, wherein the inhibitory nucleic acid comprises a guide strand sequence comprising or consisting of a sequence of any one of SEQ ID NOS: 14, 40, 100, 108, 112, 128, 166, 198, 242, 308, 336, and 362, wherein 1, 2, 3, or 4 nucleotides at positions 19-22 differ from the selected SEQ ID NO (variant nucleotide(s)), such that the guide strand sequence is no longer complementary to the ATXN2 target sequence at the variant nucleotide(s).

In some embodiments, the isolated nucleic acid comprises an expression construct encoding an inhibitory nucleic acid that inhibits the expression or activity of ATXN2, wherein the inhibitory nucleic acid comprises a guide strand sequence of Table 19. In some embodiments, the inhibitory nucleic acid comprises a guide strand sequence comprising or consisting of the nucleic acid sequence set forth in any one of SEQ ID NOS: 1176-1288, 40, 108, and 166. In some embodiments, the isolated nucleic acid comprises an expression construct encoding an inhibitory nucleic acid that inhibits the expression or activity of ATXN2, wherein the inhibitory nucleic acid comprises a guide strand sequence comprising or consisting of the nucleic acid sequence set forth in any one of SEQ ID NOS: 1176-1288, 40, 108, and 166 with at least 1, 2, 3, 4, or 5 mismatches to the target ATXN2 mRNA sequence. In some embodiments, the isolated nucleic acid comprises an expression construct encoding an inhibitory nucleic acid that inhibits the expression or activity of ATXN2, wherein the inhibitory nucleic acid comprises a guide strand sequence comprising or consisting of a nucleic acid sequence that is at least 60%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 99%, or 100% identical to any one of SEQ ID NOS:1176-1288, 40, 108, and 166. In some embodiments, the isolated nucleic acid comprises an expression construct encoding an inhibitory nucleic acid that inhibits the expression or activity of ATXN2, wherein the inhibitory nucleic acid comprises a guide strand sequence comprising or consisting of at least 15, 16, 17, 18, 19, 20, 21, or 22 contiguous nucleotides of a sequence of any one of SEQ ID NOS:1176-1288, 40, 108, and 166, preferably wherein the guide strand sequence retains positions 2-7 (“seed sequence”) of the selected SEQ ID NO. In some embodiments, the isolated nucleic acid comprises an expression construct encoding an inhibitory nucleic acid that inhibits the expression or activity of ATXN2, wherein the inhibitory nucleic acid comprises a guide strand sequence comprising or consisting of a sequence of any one of SEQ ID NOS:1176-1288, 40, 108, and 166, wherein 1, 2, 3, or 4 nucleotides at positions 19-22 differ from the selected SEQ ID NO (variant nucleotide(s)), such that the guide strand sequence is no longer complementary to the ATXN2 target sequence at the variant nucleotide(s).

In some embodiments, the isolated nucleic acid comprises an expression construct encoding an inhibitory nucleic acid that inhibits the expression or activity of ATXN2, wherein the inhibitory nucleic acid comprises a guide strand sequence of Table 23. In some embodiments, the inhibitory nucleic acid comprises a guide strand sequence comprising or consisting of the nucleic acid sequence set forth in any one of SEQ ID NOS:1908-2007. In some embodiments, the isolated nucleic acid comprises an expression construct encoding an inhibitory nucleic acid that inhibits the expression or activity of ATXN2, wherein the inhibitory nucleic acid comprises a guide strand sequence comprising or consisting of the nucleic acid sequence set forth in any one of SEQ ID NOS:1908-2007 with at least 1, 2, 3, 4, or 5 mismatches to the target ATXN2 mRNA sequence. In some embodiments, the isolated nucleic acid comprises an expression construct encoding an inhibitory nucleic acid that inhibits the expression or activity of ATXN2, wherein the inhibitory nucleic acid comprises a guide strand sequence comprising or consisting of a nucleic acid sequence that is at least 60%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 99%, or 100% identical to any one of SEQ ID NOS:1908-2007. In some embodiments, the isolated nucleic acid comprises an expression construct encoding an inhibitory nucleic acid that inhibits the expression or activity of ATXN2, wherein the inhibitory nucleic acid comprises a guide strand sequence comprising or consisting of at least 15, 16, 17, 18, 19, 20, 21, or 22 contiguous nucleotides of a sequence of any one of SEQ ID NOS:1908-2007, preferably wherein the guide strand sequence retains positions 2-7 (“seed sequence”) of the selected SEQ ID NO. In some embodiments, the isolated nucleic acid comprises an expression construct encoding an inhibitory nucleic acid that inhibits the expression or activity of ATXN2, wherein the inhibitory nucleic acid comprises a guide strand sequence comprising or consisting of a sequence of any one of SEQ ID NOS: 1908-2007, wherein 1, 2, 3, or 4 nucleotides at positions 19-22 differ from the selected SEQ ID NO (variant nucleotide(s)), such that the guide strand sequence is no longer complementary to the ATXN2 target sequence at the variant nucleotide(s).

In some embodiments, the isolated nucleic acid comprises an expression construct encoding an inhibitory nucleic acid that inhibits the expression or activity of ATXN2, wherein the inhibitory nucleic acid comprises a guide strand sequence of Table 24. In some embodiments, the inhibitory nucleic acid comprises a guide strand sequence comprising or consisting of the nucleic acid sequence set forth in any one of SEQ ID NOS:100, 112, 166, 202, 246, 306, 308, 314, 1180, 1185, 1196, 1200, 1211, 1213, 1215, 1216, 1224, 1811-1822, 1824-1827, 2015, 2065, 2083, 2152, 2203, and 2209. In some embodiments, the isolated nucleic acid comprises an expression construct encoding an inhibitory nucleic acid that inhibits the expression or activity of ATXN2, wherein the inhibitory nucleic acid comprises a guide strand sequence comprising or consisting of the nucleic acid sequence set forth in any one of SEQ ID NOS:100, 112, 166, 202, 246, 306, 308, 314, 1180, 1185, 1196, 1200, 1211, 1213, 1215, 1216, 1224, 1811-1822, 1824-1827, 2015, 2065, 2083, 2152, 2203, and 2209 with at least 1, 2, 3, 4, or 5 mismatches to the target ATXN2 mRNA sequence. In some embodiments, the isolated nucleic acid comprises an expression construct encoding an inhibitory nucleic acid that inhibits the expression or activity of ATXN2, wherein the inhibitory nucleic acid comprises a guide strand sequence comprising or consisting of a nucleic acid sequence that is at least 60%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 99%, or 100% identical to any one of SEQ ID NOS:100, 112, 166, 202, 246, 306, 308, 314, 1180, 1185, 1196, 1200, 1211, 1213, 1215, 1216, 1224, 1811-1822, 1824-1827, 2015, 2065, 2083, 2152, 2203, and 2209. In some embodiments, the isolated nucleic acid comprises an expression construct encoding an inhibitory nucleic acid that inhibits the expression or activity of ATXN2, wherein the inhibitory nucleic acid comprises a guide strand sequence comprising or consisting of at least 15, 16, 17, 18, 19, 20, 21, or 22 contiguous nucleotides of a sequence of any one of SEQ ID NOS:100, 112, 166, 202, 246, 306, 308, 314, 1180, 1185, 1196, 1200, 1211, 1213, 1215, 1216, 1224, 1811-1822, 1824-1827, 2015, 2065, 2083, 2152, 2203, and 2209, preferably wherein the guide strand sequence retains positions 2-7 (“seed sequence”) of the selected SEQ ID NO. In some embodiments, the isolated nucleic acid comprises an expression construct encoding an inhibitory nucleic acid that inhibits the expression or activity of ATXN2, wherein the inhibitory nucleic acid comprises a guide strand sequence comprising or consisting of a sequence of any one of SEQ ID NOS:100, 112, 166, 202, 246, 306, 308, 314, 1180, 1185, 1196, 1200, 1211, 1213, 1215, 1216, 1224, 1811-1822, 1824-1827, 2015, 2065, 2083, 2152, 2203, and 2209, wherein 1, 2, 3, or 4 nucleotides at positions 19-22 differ from the selected SEQ ID NO (variant nucleotide(s)), such that the guide strand sequence is no longer complementary to the ATXN2 target sequence at the variant nucleotide(s).

In some embodiments, the isolated nucleic acid comprises an expression construct encoding an inhibitory nucleic acid that inhibits the expression or activity of ATXN2, wherein the inhibitory nucleic acid comprises a guide strand sequence of Table 25. In some embodiments, the inhibitory nucleic acid comprises a guide strand sequence comprising or consisting of the nucleic acid sequence set forth in any one of SEQ ID NOS:1185, 1816, 1213, 1819, 2083, 1215, 1216, 1811, and 314. In some embodiments, the isolated nucleic acid comprises an expression construct encoding an inhibitory nucleic acid that inhibits the expression or activity of ATXN2, wherein the inhibitory nucleic acid comprises a guide strand sequence comprising or consisting of the nucleic acid sequence set forth in any one of SEQ ID NOS:1185, 1816, 1213, 1819, 2083, 1215, 1216, 1811, and 314 with at least 1, 2, 3, 4, or 5 mismatches to the target ATXN2 mRNA sequence. In some embodiments, the isolated nucleic acid comprises an expression construct encoding an inhibitory nucleic acid that inhibits the expression or activity of ATXN2, wherein the inhibitory nucleic acid comprises a guide strand sequence comprising or consisting of a nucleic acid sequence that is at least 60%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 99%, or 100% identical to any one of SEQ ID NOS:1185, 1816, 1213, 1819, 2083, 1215, 1216, 1811, and 314. In some embodiments, the isolated nucleic acid comprises an expression construct encoding an inhibitory nucleic acid that inhibits the expression or activity of ATXN2, wherein the inhibitory nucleic acid comprises a guide strand sequence comprising or consisting of at least 15, 16, 17, 18, 19, 20, 21, or 22 contiguous nucleotides of a sequence of any one of SEQ ID NOS:1185, 1816, 1213, 1819, 2083, 1215, 1216, 1811, and 314, preferably wherein the guide strand sequence retains positions 2-7 (“seed sequence”) of the selected SEQ ID NO. In some embodiments, the isolated nucleic acid comprises an expression construct encoding an inhibitory nucleic acid that inhibits the expression or activity of ATXN2, wherein the inhibitory nucleic acid comprises a guide strand sequence comprising or consisting of a sequence of any one of SEQ ID NOS:1185, 1816, 1213, 1819, 2083, 1215, 1216, 1811, and 314, wherein 1, 2, 3, or 4 nucleotides at positions 19-22 differ from the selected SEQ ID NO (variant nucleotide(s)), such that the guide strand sequence is no longer complementary to the ATXN2 target sequence at the variant nucleotide(s).

In some embodiments, the isolated nucleic acid comprises an expression construct encoding an inhibitory nucleic acid that inhibits the expression or activity of ATXN2, wherein the inhibitory nucleic acid comprises a guide strand sequence comprising or consisting of the nucleic acid sequence set forth in any one of SEQ ID NOS:1185, 1816, 1213, and 1811. In some embodiments, the isolated nucleic acid comprises an expression construct encoding an inhibitory nucleic acid that inhibits the expression or activity of ATXN2, wherein the inhibitory nucleic acid comprises a guide strand sequence comprising or consisting of the nucleic acid sequence set forth in any one of SEQ ID NOS:1185, 1816, 1213, and 1811, with at least 1, 2, 3, 4, or 5 mismatches to the target ATXN2 mRNA sequence. In some embodiments, the isolated nucleic acid comprises an expression construct encoding an inhibitory nucleic acid that inhibits the expression or activity of ATXN2, wherein the inhibitory nucleic acid comprises a guide strand sequence comprising or consisting of a nucleic acid sequence that is at least 60%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 99%, or 100% identical to any one of SEQ ID NOS:1185, 1816, 1213, and 1811. In some embodiments, the isolated nucleic acid comprises an expression construct encoding an inhibitory nucleic acid that inhibits the expression or activity of ATXN2, wherein the inhibitory nucleic acid comprises a guide strand sequence comprising or consisting of at least 15, 16, 17, 18, 19, 20, 21, or 22 contiguous nucleotides of a sequence of any one of SEQ ID NOS:1185, 1816, 1213, and 1811, preferably wherein the guide strand sequence retains positions 2-7 (“seed sequence”) of the selected SEQ ID NO. In some embodiments, the isolated nucleic acid comprises an expression construct encoding an inhibitory nucleic acid that inhibits the expression or activity of ATXN2, wherein the inhibitory nucleic acid comprises a guide strand sequence comprising or consisting of a sequence of any one of SEQ ID NOS:1185, 1816, 1213, and 1811, wherein 1, 2, 3, or 4 nucleotides at positions 19-22 differ from the selected SEQ ID NO (variant nucleotide(s)), such that the guide strand sequence is no longer complementary to the ATXN2 target sequence at the variant nucleotide(s).

In some embodiments, the isolated nucleic acid comprises an expression construct encoding a siRNA duplex that targets ATXN2 mRNA to interfere with ATXN2 expression by mRNA degradation or translational inhibition. In some embodiments, the guide strand of the siRNA duplex may be about 18 nucleotides, 19 nucleotides, 20 nucleotides, 21 nucleotides, 22 nucleotides, 23 nucleotides, 24 nucleotides, 25 nucleotides, 26 nucleotides, 27 nucleotides, 28 nucleotides, 29 nucleotides, or 30 nucleotides in length or 18-30, 18-29, 18-28, 18-27, 18-26, 18-25, 18-24, 18-23, 18-22, 18-20, 19-30, 19-29, 19-28, 19-27, 19-26, 19-25, 19-24, 19-23, 19-22, 19-21, 20-30, 20-29, 20-28, 20-27, 20-26, 20-25, 20-24, 20-23, 20-22, 21-30, 21-29, 21-28, 21-27, 21-26, 21-25, 21-24, 21-23, 22-30, 22-29, 22-28, 22-27, 22-26, 22-24, 23-30, 23-29, 23-28, 23-27, 23-26, 23-25, 24-30, 24-29, 24-28, 24-27, 24-26, 25-30, 25-29, 25-28, 25-27, 26-30, 26-29, 26-28, 27-30, 27-29, 28-30 nucleotides in length. In some embodiments, the passenger strand of the siRNA duplex may be about 18 nucleotides, 19 nucleotides, 20 nucleotides, 21 nucleotides, 22 nucleotides, 23 nucleotides, 24 nucleotides, 25 nucleotides, 26 nucleotides, 27 nucleotides, 28 nucleotides, 29 nucleotides, or 30 nucleotides in length or 18-30, 18-29, 18-28, 18-27, 18-26, 18-25, 18-24, 18-23, 18-22, 18-20, 19-30, 19-29, 19-28, 19-27, 19-26, 19-25, 19-24, 19-23, 19-22, 19-21, 20-30, 20-29, 20-28, 20-27, 20-26, 20-25, 20-24, 20-23, 20-22, 21-30, 21-29, 21-28, 21-27, 21-26, 21-25, 21-24, 21-23, 22-30, 22-29, 22-28, 22-27, 22-26, 22-24, 23-30, 23-29, 23-28, 23-27, 23-26, 23-25, 24-30, 24-29, 24-28, 24-27, 24-26, 25-30, 25-29, 25-28, 25-27, 26-30, 26-29, 26-28, 27-30, 27-29, 28-30 nucleotides in length. In some embodiments, the siRNA duplex contains 2 or 3 nucleotide 3′ overhangs on each strand. In some embodiments, the 3′ overhangs are complementary to the ATXN2 transcript. In some embodiments, the guide strand and passenger strand of the siRNA duplex are at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, or 100% complementary to each other, not including any nucleotides in overhang(s).

In some embodiments, the isolated nucleic acid comprises an expression construct encoding a siRNA duplex that comprises a guide strand sequence comprising or consisting of the nucleic acid sequence set forth in any one of the guide sequences of Tables 1, 3, 9, 11, 12, 13, 19, 23, and 24, e.g., any one of SEQ ID NOS: 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 124, 126, 128, 130, 132, 134, 136, 138, 140, 142, 144, 146, 148, 150, 152, 154, 156, 158, 160, 162, 164, 166, 168, 170, 172, 174, 176, 178, 180, 182, 184, 186, 188, 190, 192, 194, 196, 198, 200, 202, 204, 206, 208, 210, 212, 214, 216, 218, 220, 222, 224, 226, 228, 230, 232, 234, 236, 238, 240, 242, 244, 246, 248, 250, 252, 254, 256, 258, 260, 262, 264, 266, 268, 270, 272, 274, 276, 278, 280, 282, 284, 286, 288, 290, 292, 294, 296, 298, 300, 302, 304, 306, 308, 310, 312, 314, 316, 318, 320, 324, 326, 328, 330, 332, 334, 336, 338, 340, 342, 344, 346, 348, 350, 352, 354, 356, 358, 360, 362, 364, 366, 368, 370, 372, 374, 376, 378, 380, 382, 384, 386, 388, 390, 392, 394, 396, 398, 400, 402, 404, 406, 408, 410, 412, 414, 416, 418, 420, 422, 424, 426, 428, 430, 432, 434, 436, 1176-1288, 1811-1827, 2015, 2065, 2083, 2152, 2203, and 2209.

In some embodiments, the isolated nucleic acid comprises an expression construct encoding a siRNA duplex that comprises a guide strand sequence comprising or consisting of the nucleic acid sequence set forth in any one of the guide sequences of Tables 1, 3, 9, 11, 12, 13, 19, 23, and 24, e.g., any one of SEQ ID NOS: 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 124, 126, 128, 130, 132, 134, 136, 138, 140, 142, 144, 146, 148, 150, 152, 154, 156, 158, 160, 162, 164, 166, 168, 170, 172, 174, 176, 178, 180, 182, 184, 186, 188, 190, 192, 194, 196, 198, 200, 202, 204, 206, 208, 210, 212, 214, 216, 218, 220, 222, 224, 226, 228, 230, 232, 234, 236, 238, 240, 242, 244, 246, 248, 250, 252, 254, 256, 258, 260, 262, 264, 266, 268, 270, 272, 274, 276, 278, 280, 282, 284, 286, 288, 290, 292, 294, 296, 298, 300, 302, 304, 306, 308, 310, 312, 314, 316, 318, 320, 324, 326, 328, 330, 332, 334, 336, 338, 340, 342, 344, 346, 348, 350, 352, 354, 356, 358, 360, 362, 364, 366, 368, 370, 372, 374, 376, 378, 380, 382, 384, 386, 388, 390, 392, 394, 396, 398, 400, 402, 404, 406, 408, 410, 412, 414, 416, 418, 420, 422, 424, 426, 428, 430, 432, 434, 436, 1176-1288, 1811-1827, 2015, 2065, 2083, 2152, 2203, and 2209, with at least 1, 2, 3, 4, or 5 mismatches to the target ATXN2 mRNA sequence.

In some embodiments, the isolated nucleic acid comprises an expression construct encoding a siRNA duplex that comprises a guide strand sequence sequence comprising of consisting of a sequence that at least 60%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 99%, or 100% identical to any one of the guide sequences of Tables 1, 3, 9, 11, 12, 13, 19, 23, and 24, e.g., any one of SEQ ID NOS: 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 124, 126, 128, 130, 132, 134, 136, 138, 140, 142, 144, 146, 148, 150, 152, 154, 156, 158, 160, 162, 164, 166, 168, 170, 172, 174, 176, 178, 180, 182, 184, 186, 188, 190, 192, 194, 196, 198, 200, 202, 204, 206, 208, 210, 212, 214, 216, 218, 220, 222, 224, 226, 228, 230, 232, 234, 236, 238, 240, 242, 244, 246, 248, 250, 252, 254, 256, 258, 260, 262, 264, 266, 268, 270, 272, 274, 276, 278, 280, 282, 284, 286, 288, 290, 292, 294, 296, 298, 300, 302, 304, 306, 308, 310, 312, 314, 316, 318, 320, 324, 326, 328, 330, 332, 334, 336, 338, 340, 342, 344, 346, 348, 350, 352, 354, 356, 358, 360, 362, 364, 366, 368, 370, 372, 374, 376, 378, 380, 382, 384, 386, 388, 390, 392, 394, 396, 398, 400, 402, 404, 406, 408, 410, 412, 414, 416, 418, 420, 422, 424, 426, 428, 430, 432, 434, 436, 1176-1288, 1811-1827, 2015, 2065, 2083, 2152, 2203, and 2209.

In some embodiments, the isolated nucleic acid comprises an expression construct encoding a siRNA duplex that comprises a guide strand sequence comprising or consisting of at least 15, 16, 17, 18, 19, 20, 21, or 22 contiguous nucleotides of a sequence of any one of the guide sequences of Tables 1, 3, 9, 11, 12, 13, 19, 23, and 24, e.g., any one of SEQ ID NOS: 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 124, 126, 128, 130, 132, 134, 136, 138, 140, 142, 144, 146, 148, 150, 152, 154, 156, 158, 160, 162, 164, 166, 168, 170, 172, 174, 176, 178, 180, 182, 184, 186, 188, 190, 192, 194, 196, 198, 200, 202, 204, 206, 208, 210, 212, 214, 216, 218, 220, 222, 224, 226, 228, 230, 232, 234, 236, 238, 240, 242, 244, 246, 248, 250, 252, 254, 256, 258, 260, 262, 264, 266, 268, 270, 272, 274, 276, 278, 280, 282, 284, 286, 288, 290, 292, 294, 296, 298, 300, 302, 304, 306, 308, 310, 312, 314, 316, 318, 320, 324, 326, 328, 330, 332, 334, 336, 338, 340, 342, 344, 346, 348, 350, 352, 354, 356, 358, 360, 362, 364, 366, 368, 370, 372, 374, 376, 378, 380, 382, 384, 386, 388, 390, 392, 394, 396, 398, 400, 402, 404, 406, 408, 410, 412, 414, 416, 418, 420, 422, 424, 426, 428, 430, 432, 434, 436, 1176-1288, 1811-1827, 2015, 2065, 2083, 2152, 2203, and 2209, preferably wherein the guide strand sequence retains positions 2-7 (“seed sequence”) of the selected SEQ ID NO.

In some embodiments, the isolated nucleic acid comprises an expression construct encoding a siRNA duplex that comprises a guide strand sequence comprising or consisting of any one of the guide sequences of Tables 1, 3, 9, 11, 12, 13, 19, 23, and 24, e.g., any one of SEQ ID NOS: 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 124, 126, 128, 130, 132, 134, 136, 138, 140, 142, 144, 146, 148, 150, 152, 154, 156, 158, 160, 162, 164, 166, 168, 170, 172, 174, 176, 178, 180, 182, 184, 186, 188, 190, 192, 194, 196, 198, 200, 202, 204, 206, 208, 210, 212, 214, 216, 218, 220, 222, 224, 226, 228, 230, 232, 234, 236, 238, 240, 242, 244, 246, 248, 250, 252, 254, 256, 258, 260, 262, 264, 266, 268, 270, 272, 274, 276, 278, 280, 282, 284, 286, 288, 290, 292, 294, 296, 298, 300, 302, 304, 306, 308, 310, 312, 314, 316, 318, 320, 324, 326, 328, 330, 332, 334, 336, 338, 340, 342, 344, 346, 348, 350, 352, 354, 356, 358, 360, 362, 364, 366, 368, 370, 372, 374, 376, 378, 380, 382, 384, 386, 388, 390, 392, 394, 396, 398, 400, 402, 404, 406, 408, 410, 412, 414, 416, 418, 420, 422, 424, 426, 428, 430, 432, 434, 436, 1176-1288, 1811-1827, 2015, 2065, 2083, 2152, 2203, and 2209, wherein 1, 2, 3, or 4 nucleotides at positions 19-22 differ from the selected SEQ ID NO (variant nucleotide(s)), such that the guide strand sequence is no longer complementary to the ATXN2 target sequence at the variant nucleotide(s).

In some embodiments, the isolated nucleic acid comprises an expression construct encoding a siRNA duplex that comprises a guide strand sequence of Table 12. In some embodiments, the isolated nucleic acid comprises an expression construct encoding a siRNA duplex that comprises a guide strand sequence comprising or consisting of the nucleic acid sequence set forth in any one of SEQ ID NOS: 12, 14, 40, 60, 100, 104, 108, 112, 124, 126, 128, 166, 198, 220, 242, 302, 306, 308, 330, 336, and 362. In some embodiments, the isolated nucleic acid comprises an expression construct encoding a siRNA duplex that comprises a guide strand sequence comprising or consisting of the nucleic acid sequence set forth in any one of SEQ ID NOS: 12, 14, 40, 60, 100, 104, 108, 112, 124, 126, 128, 166, 198, 220, 242, 302, 306, 308, 330, 336, and 362 with at least 1, 2, 3, 4, or 5 mismatches to the target ATXN2 mRNA sequence. In some embodiments, the siRNA duplex comprises a guide strand sequence comprising or consisting of a nucleic acid sequence that is at least 60%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 99%, or 100% identical to any one of SEQ ID NOS: 12, 14, 40, 60, 100, 104, 108, 112, 124, 126, 128, 166, 198, 220, 242, 302, 306, 308, 330, 336, and 362. In some embodiments, the isolated nucleic acid comprises an expression construct encoding a siRNA duplex that comprises a guide strand sequence comprising or consisting of at least 15, 16, 17, 18, 19, 20, 21, or 22 contiguous nucleotides of a sequence of any one of SEQ ID NOS: 12, 14, 40, 60, 100, 104, 108, 112, 124, 126, 128, 166, 198, 220, 242, 302, 306, 308, 330, 336, and 362, preferably wherein the guide strand sequence retains positions 2-7 (“seed sequence”) of the selected SEQ ID NO. In some embodiments, the isolated nucleic acid comprises an expression construct encoding a siRNA duplex that comprises a guide strand sequence comprising or consisting of a sequence of any one of SEQ ID NOS: 12, 14, 40, 60, 100, 104, 108, 112, 124, 126, 128, 166, 198, 220, 242, 302, 306, 308, 330, 336, and 362, wherein 1, 2, 3, or 4 nucleotides at positions 19-22 differ from the selected SEQ ID NO (variant nucleotide(s)), such that the guide strand sequence is no longer complementary to the ATXN2 target sequence at the variant nucleotide(s).

In some embodiments, the isolated nucleic acid comprises an expression construct encoding a siRNA duplex that comprises a guide strand sequence of Table 13. In some embodiments, the isolated nucleic acid comprises an expression construct encoding a siRNA duplex that comprises a guide strand sequence comprising or consisting of the nucleic acid sequence set forth in any one of SEQ ID NOS: 14, 40, 100, 108, 112, 128, 166, 198, 242, 308, 336, and 362. In some embodiments, the isolated nucleic acid comprises an expression construct encoding a siRNA duplex that comprises a guide strand sequence comprising or consisting of the nucleic acid sequence set forth in any one of SEQ ID NOS: 14, 40, 100, 108, 112, 128, 166, 198, 242, 308, 336, and 362 with at least 1, 2, 3, 4, or 5 mismatches to the target ATXN2 mRNA sequence. In some embodiments, the isolated nucleic acid comprises an expression construct encoding a siRNA duplex that comprises a guide strand sequence comprising or consisting of a nucleic acid sequence that is at least 60%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 99%, or 100% identical to any one of SEQ ID NOS: 14, 40, 100, 108, 112, 128, 166, 198, 242, 308, 336, and 362. In some embodiments, the isolated nucleic acid comprises an expression construct encoding a siRNA duplex that comprises a guide strand sequence comprising a guide strand sequence comprising or consisting of at least 15, 16, 17, 18, 19, 20, 21, or 22 contiguous nucleotides of a sequence of any one of SEQ ID NOS: 14, 40, 100, 108, 112, 128, 166, 198, 242, 308, 336, and 362, preferably wherein the guide strand sequence retains positions 2-7 (“seed sequence”) of the selected SEQ ID NO. In some embodiments, the isolated nucleic acid comprises an expression construct encoding a siRNA duplex that comprises a guide strand sequence comprising or consisting of a sequence of any one of SEQ ID NOS: 14, 40, 100, 108, 112, 128, 166, 198, 242, 308, 336, and 362, wherein 1, 2, 3, or 4 nucleotides at positions 19-22 differ from the selected SEQ ID NO (variant nucleotide(s)), such that the guide strand sequence is no longer complementary to the ATXN2 target sequence at the variant nucleotide(s).

In some embodiments, the isolated nucleic acid comprises an expression construct encoding a siRNA duplex that comprises a guide strand sequence of Table 19. In some embodiments, the isolated nucleic acid comprises an expression construct encoding a siRNA duplex that comprises a guide strand sequence comprising or consisting of the nucleic acid sequence set forth in any one of SEQ ID NOS:1176-1288, 40, 108, and 166. In some embodiments, the isolated nucleic acid comprises an expression construct encoding a siRNA duplex that comprises a guide strand sequence comprising or consisting of the nucleic acid sequence set forth in any one of SEQ ID NOS:1176-1288, 40, 108, and 166 with at least 1, 2, 3, 4, or 5 mismatches to the target ATXN2 mRNA sequence. In some embodiments, the isolated nucleic acid comprises an expression construct encoding a siRNA duplex that comprises a guide strand sequence comprising or consisting of a nucleic acid sequence that is at least 60%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 99%, or 100% identical to any one of SEQ ID NOS:1176-1288, 40, 108, and 166. In some embodiments, the isolated nucleic acid comprises an expression construct encoding a siRNA duplex that comprises a guide strand sequence comprising a guide strand sequence comprising or consisting of at least 15, 16, 17, 18, 19, 20, 21, or 22 contiguous nucleotides of a sequence of any one of SEQ ID NOS:1176-1288, 40, 108, and 166, preferably wherein the guide strand sequence retains positions 2-7 (“seed sequence”) of the selected SEQ ID NO. In some embodiments, the isolated nucleic acid comprises an expression construct encoding a siRNA duplex that comprises a guide strand sequence comprising or consisting of a sequence of any one of SEQ ID NOS:1176-1288, 40, 108, and 166, wherein 1, 2, 3, or 4 nucleotides at positions 19-22 differ from the selected SEQ ID NO (variant nucleotide(s)), such that the guide strand sequence is no longer complementary to the ATXN2 target sequence at the variant nucleotide(s).

In some embodiments, the isolated nucleic acid comprises an expression construct encoding a siRNA duplex that comprises a guide strand sequence of Table 23. In some embodiments, the isolated nucleic acid comprises an expression construct encoding a siRNA duplex that comprises a guide strand sequence comprising or consisting of the nucleic acid sequence set forth in any one of SEQ ID NOS:1908-2007. In some embodiments, the isolated nucleic acid comprises an expression construct encoding a siRNA duplex that comprises a guide strand sequence comprising or consisting of the nucleic acid sequence set forth in any one of SEQ ID NOS:1908-2007 with at least 1, 2, 3, 4, or 5 mismatches to the target ATXN2 mRNA sequence. In some embodiments, the isolated nucleic acid comprises an expression construct encoding a siRNA duplex that comprises a guide strand sequence comprising or consisting of a nucleic acid sequence that is at least 60%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 99%, or 100% identical to any one of SEQ ID NOS:1908-2007. In some embodiments, the isolated nucleic acid comprises an expression construct encoding a siRNA duplex that comprises a guide strand sequence comprising a guide strand sequence comprising or consisting of at least 15, 16, 17, 18, 19, 20, 21, or 22 contiguous nucleotides of a sequence of any one of SEQ ID NOS:1908-2007, preferably wherein the guide strand sequence retains positions 2-7 (“seed sequence”) of the selected SEQ ID NO. In some embodiments, the isolated nucleic acid comprises an expression construct encoding a siRNA duplex that comprises a guide strand sequence comprising or consisting of a sequence of any one of SEQ ID NOS:1908-2007, wherein 1, 2, 3, or 4 nucleotides at positions 19-22 differ from the selected SEQ ID NO (variant nucleotide(s)), such that the guide strand sequence is no longer complementary to the ATXN2 target sequence at the variant nucleotide(s).

In some embodiments, the isolated nucleic acid comprises an expression construct encoding a siRNA duplex that comprises a guide strand sequence of Table 24. In some embodiments, the isolated nucleic acid comprises an expression construct encoding a siRNA duplex that comprises a guide strand sequence comprising or consisting of the nucleic acid sequence set forth in any one of SEQ ID NOS:100, 112, 166, 202, 246, 306, 308, 314, 1180, 1185, 1196, 1200, 1211, 1213, 1215, 1216, 1224, 1811-1822, 1824-1827, 2015, 2065, 2083, 2152, 2203, and 2209. In some embodiments, the isolated nucleic acid comprises an expression construct encoding a siRNA duplex that comprises a guide strand sequence comprising or consisting of the nucleic acid sequence set forth in any one of SEQ ID NOS:100, 112, 166, 202, 246, 306, 308, 314, 1180, 1185, 1196, 1200, 1211, 1213, 1215, 1216, 1224, 1811-1822, 1824-1827, 2015, 2065, 2083, 2152, 2203, and 2209, with at least 1, 2, 3, 4, or 5 mismatches to the target ATXN2 mRNA sequence. In some embodiments, the isolated nucleic acid comprises an expression construct encoding a siRNA duplex that comprises a guide strand sequence comprising or consisting of a nucleic acid sequence that is at least 60%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 99%, or 100% identical to any one of SEQ ID NOS:100, 112, 166, 202, 246, 306, 308, 314, 1180, 1185, 1196, 1200, 1211, 1213, 1215, 1216, 1224, 1811-1822, 1824-1827, 2015, 2065, 2083, 2152, 2203, and 2209. In some embodiments, the isolated nucleic acid comprises an expression construct encoding a siRNA duplex that comprises a guide strand sequence comprising a guide strand sequence comprising or consisting of at least 15, 16, 17, 18, 19, 20, 21, or 22 contiguous nucleotides of a sequence of any one of SEQ ID NOS:100, 112, 166, 202, 246, 306, 308, 314, 1180, 1185, 1196, 1200, 1211, 1213, 1215, 1216, 1224, 1811-1822, 1824-1827, 2015, 2065, 2083, 2152, 2203, and 2209, preferably wherein the guide strand sequence retains positions 2-7 (“seed sequence”) of the selected SEQ ID NO. In some embodiments, the isolated nucleic acid comprises an expression construct encoding a siRNA duplex that comprises a guide strand sequence comprising or consisting of a sequence of any one of SEQ ID NOS:100, 112, 166, 202, 246, 306, 308, 314, 1180, 1185, 1196, 1200, 1211, 1213, 1215, 1216, 1224, 1811-1822, 1824-1827, 2015, 2065, 2083, 2152, 2203, and 2209, wherein 1, 2, 3, or 4 nucleotides at positions 19-22 differ from the selected SEQ ID NO (variant nucleotide(s)), such that the guide strand sequence is no longer complementary to the ATXN2 target sequence at the variant nucleotide(s).

In some embodiments, the isolated nucleic acid comprises an expression construct encoding a siRNA duplex that comprises a guide strand sequence of Table 25. In some embodiments, the isolated nucleic acid comprises an expression construct encoding a siRNA duplex that comprises a guide strand sequence comprising or consisting of the nucleic acid sequence set forth in any one of SEQ ID NOS:1185, 1816, 1213, 1819, 2083, 1215, 1216, 1811, and 314. In some embodiments, the isolated nucleic acid comprises an expression construct encoding a siRNA duplex that comprises a guide strand sequence comprising or consisting of the nucleic acid sequence set forth in any one of SEQ ID NOS:1185, 1816, 1213, 1819, 2083, 1215, 1216, 1811, and 314, with at least 1, 2, 3, 4, or 5 mismatches to the target ATXN2 mRNA sequence. In some embodiments, the isolated nucleic acid comprises an expression construct encoding a siRNA duplex that comprises a guide strand sequence comprising or consisting of a nucleic acid sequence that is at least 60%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 99%, or 100% identical to any one of SEQ ID NOS:1185, 1816, 1213, 1819, 2083, 1215, 1216, 1811, and 314. In some embodiments, the isolated nucleic acid comprises an expression construct encoding a siRNA duplex that comprises a guide strand sequence comprising a guide strand sequence comprising or consisting of at least 15, 16, 17, 18, 19, 20, 21, or 22 contiguous nucleotides of a sequence of any one of SEQ ID NOS:1185, 1816, 1213, 1819, 2083, 1215, 1216, 1811, and 314, preferably wherein the guide strand sequence retains positions 2-7 (“seed sequence”) of the selected SEQ ID NO. In some embodiments, the isolated nucleic acid comprises an expression construct encoding a siRNA duplex that comprises a guide strand sequence comprising or consisting of a sequence of any one of SEQ ID NOS:1185, 1816, 1213, 1819, 2083, 1215, 1216, 1811, and 314, wherein 1, 2, 3, or 4 nucleotides at positions 19-22 differ from the selected SEQ ID NO (variant nucleotide(s)), such that the guide strand sequence is no longer complementary to the ATXN2 target sequence at the variant nucleotide(s).

In some embodiments the isolated nucleic acid comprises an expression construct encoding a siRNA duplex comprising a guide strand sequence and passenger strand sequence of any one of siRNA duplexes provided in Tables 1, 19, 23, and 24. In some embodiments the isolated nucleic acid comprises an expression construct encoding a siRNA duplex comprising a guide strand sequence and passenger strand sequence, comprising or consisting of any one of: SEQ ID NOS:12 and 11; SEQ ID NOS: 14 and 13; SEQ ID NOS: 40 and 39; SEQ ID NOS: 60 and 59; SEQ ID NOS: 100 and 99; SEQ ID NOS: 104 and 103; SEQ ID NOS: 108 and 107; SEQ ID NOS: 112 and 111; SEQ ID NOS: 124 and 123; SEQ ID NOS: 126 and 125; SEQ ID NOS: 128 and 127; SEQ ID NOS: 166 and 165; SEQ ID NOS: 198 and 197; SEQ ID NOS: 220 and 219; SEQ ID NOS: 242 and 241; SEQ ID NOS: 302 and 301; SEQ ID NOS: 306 and 305; SEQ ID NOS: 308 and 307; SEQ ID NOS: 330 and 320; SEQ ID NOS: 336 and 335; and SEQ ID NOS: 362 and 361. In some embodiments the isolated nucleic acid comprises an expression construct encoding a siRNA duplex comprising a guide strand sequence and passenger strand sequence comprising or consisting of any one of: SEQ ID NOS:14 and 13; SEQ ID NOS: 40 and 39; SEQ ID NOS: 100 and 99; SEQ ID NOS: 108 and 107: SEQ ID NOS: 112 and 11; SEQ ID NOS: 128 and 127; SEQ ID NOS: 166 and 165; SEQ ID NOS: 198 and 197; SEQ ID NOS: 242 and 241; SEQ ID NOS: 308 and 307; SEQ ID NOS: 336 and 335; and SEQ ID NOS: 362 and 361.

In some embodiments, the isolated nucleic acid comprises an expression construct encoding a shRNA comprising a guide strand and passenger strand of a siRNA duplex as provided herein, linked by a short spacer sequence, i.e., loop. In some embodiments, loop sequence is 4, 5, 6, 7, 8, 9, or 10 nucleotides in length or 4-10, 4-9, 4-8, 4-7, 4-6, 5-10, 5-9, 5-8, 5-7, 6-9, 6-8, 7-10, 7-9, or 8-10 nucleotides in length.

In some embodiments, the isolated nucleic acid comprises an expression construct encoding a miRNA, such as a pri-miRNA, a pre-mRNA, an artificial miRNA, or a mature miRNA, wherein the miRNA comprises a guide strand sequence comprising or consisting of the nucleic acid sequence set forth in any one of the guide sequences of Tables 1, 3, 9, 11, 12, 13, 19, 23, 24, and 25, e.g., any one of SEQ ID NOS: 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 124, 126, 128, 130, 132, 134, 136, 138, 140, 142, 144, 146, 148, 150, 152, 154, 156, 158, 160, 162, 164, 166, 168, 170, 172, 174, 176, 178, 180, 182, 184, 186, 188, 190, 192, 194, 196, 198, 200, 202, 204, 206, 208, 210, 212, 214, 216, 218, 220, 222, 224, 226, 228, 230, 232, 234, 236, 238, 240, 242, 244, 246, 248, 250, 252, 254, 256, 258, 260, 262, 264, 266, 268, 270, 272, 274, 276, 278, 280, 282, 284, 286, 288, 290, 292, 294, 296, 298, 300, 302, 304, 306, 308, 310, 312, 314, 316, 318, 320, 324, 326, 328, 330, 332, 334, 336, 338, 340, 342, 344, 346, 348, 350, 352, 354, 356, 358, 360, 362, 364, 366, 368, 370, 372, 374, 376, 378, 380, 382, 384, 386, 388, 390, 392, 394, 396, 398, 400, 402, 404, 406, 408, 410, 412, 414, 416, 418, 420, 422, 424, 426, 428, 430, 432, 434, 436, 1176-1288, 1811-1827, 2015, 2065, 2083, 2152, 2203, and 2209.

In some embodiments, the isolated nucleic acid comprises an expression construct encoding a miRNA, such as a pri-miRNA, a pre-mRNA, an artificial miRNA, or a mature miRNA, wherein the miRNA comprises a guide strand sequence comprising or consisting of the nucleic acid sequence set forth in any one of the guide sequences of Tables 1, 3, 9, 11, 12, 13, 19, 24, and 25, e.g., any one of SEQ ID NOS: 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 124, 126, 128, 130, 132, 134, 136, 138, 140, 142, 144, 146, 148, 150, 152, 154, 156, 158, 160, 162, 164, 166, 168, 170, 172, 174, 176, 178, 180, 182, 184, 186, 188, 190, 192, 194, 196, 198, 200, 202, 204, 206, 208, 210, 212, 214, 216, 218, 220, 222, 224, 226, 228, 230, 232, 234, 236, 238, 240, 242, 244, 246, 248, 250, 252, 254, 256, 258, 260, 262, 264, 266, 268, 270, 272, 274, 276, 278, 280, 282, 284, 286, 288, 290, 292, 294, 296, 298, 300, 302, 304, 306, 308, 310, 312, 314, 316, 318, 320, 324, 326, 328, 330, 332, 334, 336, 338, 340, 342, 344, 346, 348, 350, 352, 354, 356, 358, 360, 362, 364, 366, 368, 370, 372, 374, 376, 378, 380, 382, 384, 386, 388, 390, 392, 394, 396, 398, 400, 402, 404, 406, 408, 410, 412, 414, 416, 418, 420, 422, 424, 426, 428, 430, 432, 434, 436, 1176-1288, 1811-1827, 2015, 2065, 2083, 2152, 2203, and 2209, with at least 1, 2, 3, 4, or 5 mismatches to the target ATXN2 mRNA sequence.

In some embodiments, the isolated nucleic acid comprises an expression construct encoding a miRNA, such as a pri-miRNA, a pre-mRNA, an artificial miRNA, or a mature miRNA, wherein the miRNA comprises a guide strand sequence comprising or consisting of a sequence that is at least 60%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 99%, or 100% identical to any one of the guide sequences of Tables 1, 3, 9, 11, 12, 13, 19, 23, 24, and 25, e.g., any one of SEQ ID NOS: 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 124, 126, 128, 130, 132, 134, 136, 138, 140, 142, 144, 146, 148, 150, 152, 154, 156, 158, 160, 162, 164, 166, 168, 170, 172, 174, 176, 178, 180, 182, 184, 186, 188, 190, 192, 194, 196, 198, 200, 202, 204, 206, 208, 210, 212, 214, 216, 218, 220, 222, 224, 226, 228, 230, 232, 234, 236, 238, 240, 242, 244, 246, 248, 250, 252, 254, 256, 258, 260, 262, 264, 266, 268, 270, 272, 274, 276, 278, 280, 282, 284, 286, 288, 290, 292, 294, 296, 298, 300, 302, 304, 306, 308, 310, 312, 314, 316, 318, 320, 324, 326, 328, 330, 332, 334, 336, 338, 340, 342, 344, 346, 348, 350, 352, 354, 356, 358, 360, 362, 364, 366, 368, 370, 372, 374, 376, 378, 380, 382, 384, 386, 388, 390, 392, 394, 396, 398, 400, 402, 404, 406, 408, 410, 412, 414, 416, 418, 420, 422, 424, 426, 428, 430, 432, 434, 436, 1176-1288, 1811-1827, 2015, 2065, 2083, 2152, 2203, and 2209.

In some embodiments, the isolated nucleic acid comprises an expression construct encoding a miRNA, such as a pri-miRNA, a pre-mRNA, an artificial miRNA, or a mature miRNA, wherein the miRNA comprises a guide strand sequence comprising or consisting of at least 15, 16, 17, 18, 19, 20, 21, or 22 contiguous nucleotides of a sequence of any one of the guide sequences of Tables 1, 3, 9, 11, 12, 13, 19, 23, 24, and 25, e.g., any one of SEQ ID NOS: 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 124, 126, 128, 130, 132, 134, 136, 138, 140, 142, 144, 146, 148, 150, 152, 154, 156, 158, 160, 162, 164, 166, 168, 170, 172, 174, 176, 178, 180, 182, 184, 186, 188, 190, 192, 194, 196, 198, 200, 202, 204, 206, 208, 210, 212, 214, 216, 218, 220, 222, 224, 226, 228, 230, 232, 234, 236, 238, 240, 242, 244, 246, 248, 250, 252, 254, 256, 258, 260, 262, 264, 266, 268, 270, 272, 274, 276, 278, 280, 282, 284, 286, 288, 290, 292, 294, 296, 298, 300, 302, 304, 306, 308, 310, 312, 314, 316, 318, 320, 324, 326, 328, 330, 332, 334, 336, 338, 340, 342, 344, 346, 348, 350, 352, 354, 356, 358, 360, 362, 364, 366, 368, 370, 372, 374, 376, 378, 380, 382, 384, 386, 388, 390, 392, 394, 396, 398, 400, 402, 404, 406, 408, 410, 412, 414, 416, 418, 420, 422, 424, 426, 428, 430, 432, 434, 436, 1176-1288, 1811-1827, 2015, 2065, 2083, 2152, 2203, and 2209, preferably wherein the guide strand sequence retains positions 2-7 (“seed sequence”) of the selected SEQ ID NO.

In some embodiments, the isolated nucleic acid comprises an expression construct encoding a miRNA, such as a pri-miRNA, a pre-mRNA, an artificial miRNA, or a mature miRNA, wherein the miRNA comprises a guide strand sequence comprising or consisting of a nucleic acid sequence of any one of the guide sequences of Tables 1, 3, 9, 11, 12, 13, 19, 23, 24, and 25, e.g., any one of SEQ ID NOS: 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 124, 126, 128, 130, 132, 134, 136, 138, 140, 142, 144, 146, 148, 150, 152, 154, 156, 158, 160, 162, 164, 166, 168, 170, 172, 174, 176, 178, 180, 182, 184, 186, 188, 190, 192, 194, 196, 198, 200, 202, 204, 206, 208, 210, 212, 214, 216, 218, 220, 222, 224, 226, 228, 230, 232, 234, 236, 238, 240, 242, 244, 246, 248, 250, 252, 254, 256, 258, 260, 262, 264, 266, 268, 270, 272, 274, 276, 278, 280, 282, 284, 286, 288, 290, 292, 294, 296, 298, 300, 302, 304, 306, 308, 310, 312, 314, 316, 318, 320, 324, 326, 328, 330, 332, 334, 336, 338, 340, 342, 344, 346, 348, 350, 352, 354, 356, 358, 360, 362, 364, 366, 368, 370, 372, 374, 376, 378, 380, 382, 384, 386, 388, 390, 392, 394, 396, 398, 400, 402, 404, 406, 408, 410, 412, 414, 416, 418, 420, 422, 424, 426, 428, 430, 432, 434, 436, 1176-1288, 1811-1827, 2015, 2065, 2083, 2152, 2203, and 2209, wherein 1, 2, 3, or 4 nucleotides at positions 19-22 differ from the selected SEQ ID NO (variant nucleotide(s)) such that the guide strand sequence is no longer complementary to the ATXN2 target sequence at the variant nucleotide(s).

In some embodiments, the isolated nucleic acid comprises an expression construct encoding a miRNA, such as a pri-miRNA, a pre-mRNA, an artificial miRNA, or a mature miRNA comprising a guide strand sequence of Table 12. In some embodiments, the isolated nucleic acid comprises an expression construct encoding a miRNA, such as a pri-miRNA, a pre-mRNA, an artificial miRNA, or a mature miRNA comprising a guide strand sequence comprising or consisting of the nucleic acid sequence set forth in any one of SEQ ID NOS: 12, 14, 40, 60, 100, 104, 108, 112, 124, 126, 128, 166, 198, 220, 242, 302, 306, 308, 330, 336, and 362. In some embodiments, the isolated nucleic acid comprises an expression construct encoding a miRNA, such as a pri-miRNA, a pre-mRNA, an artificial miRNA, or a mature miRNA, wherein the miRNA comprises a guide strand sequence comprising or consisting of the nucleic acid sequence set forth in any one of SEQ ID NOS: 12, 14, 40, 60, 100, 104, 108, 112, 124, 126, 128, 166, 198, 220, 242, 302, 306, 308, 330, 336, and 362, with at least 1, 2, 3, 4, or 5 mismatches to the target ATXN2 mRNA sequence. In some embodiments, the isolated nucleic acid comprises an expression construct encoding a miRNA, such as a pri-miRNA, a pre-mRNA, an artificial miRNA, or a mature miRNA, wherein the miRNA comprises a guide strand sequence comprising or consisting of a nucleic acid sequence that is at least 60%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 99%, or 100% identical to any one of SEQ ID NOS: 12, 14, 40, 60, 100, 104, 108, 112, 124, 126, 128, 166, 198, 220, 242, 302, 306, 308, 330, 336, and 362. In some embodiments, the isolated nucleic acid comprises an expression construct encoding a miRNA, such as a pri-miRNA, a pre-mRNA, an artificial miRNA, or a mature miRNA, wherein the miRNA comprises a guide strand sequence comprising or consisting of at least 15, 16, 17, 18, 19, 20, 21, or 22 contiguous nucleotides of a sequence of any one of SEQ ID NOS: 12, 14, 40, 60, 100, 104, 108, 112, 124, 126, 128, 166, 198, 220, 242, 302, 306, 308, 330, 336, and 362, preferably wherein the guide strand sequence retains positions 2-7 (“seed sequence”) of the selected SEQ ID NO. In some embodiments, the isolated nucleic acid comprises an expression construct encoding a miRNA, such as a pri-miRNA, a pre-mRNA, an artificial miRNA, or a mature miRNA, wherein the miRNA comprises a guide strand sequence comprising or consisting of a sequence of any one of SEQ ID NOS: 12, 14, 40, 60, 100, 104, 108, 112, 124, 126, 128, 166, 198, 220, 242, 302, 306, 308, 330, 336, and 362, wherein 1, 2, 3, or 4 nucleotides at positions 19-22 differ from the selected SEQ ID NO (variant nucleotide(s)) such that the guide strand sequence is no longer complementary to the ATXN2 target sequence at the variant nucleotide(s).

In some embodiments, the isolated nucleic acid comprises an expression construct encoding a miRNA, such as a pri-miRNA, a pre-mRNA, an artificial miRNA, or a mature miRNA comprising a guide strand sequence of Table 13. In some embodiments, the isolated nucleic acid comprises an expression construct encoding a miRNA, such as a pri-miRNA, a pre-mRNA, an artificial miRNA, or a mature miRNA, comprising a guide strand sequence comprising or consisting of the nucleic acid sequence set forth in any one of SEQ ID NOS: 14, 40, 100, 108, 112, 128, 166, 198, 242, 308, 336, and 362. In some embodiments, the isolated nucleic acid comprises an expression construct encoding a miRNA, such as a pri-miRNA, a pre-mRNA, an artificial miRNA, or a mature miRNA, wherein the miRNA comprises a guide strand sequence comprising or consisting of the nucleic acid sequence set forth in any one of SEQ ID NOS: 14, 40, 100, 108, 112, 128, 166, 198, 242, 308, 336, and 362, with at least 1, 2, 3, 4, or 5 mismatches to the target ATXN2 mRNA sequence. In some embodiments, the miRNA is a pri-miRNA, a pre-mRNA, an artificial miRNA, or a mature miRNA wherein the miRNA comprises a guide strand sequence comprising or consisting of a nucleic acid sequence that is at least 60%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 99%, or 100% identical to any one of SEQ ID NOS: 14, 40, 100, 108, 112, 128, 166, 198, 242, 308, 336, and 362. In some embodiments, the isolated nucleic acid comprises an expression construct encoding a miRNA, such as a pri-miRNA, a pre-mRNA, an artificial miRNA, or a mature miRNA, wherein the miRNA comprises a guide strand sequence comprising or consisting of at least 15, 16, 17, 18, 19, 20, 21, or 22 contiguous nucleotides of a sequence of any one of SEQ ID NOS: 14, 40, 100, 108, 112, 128, 166, 198, 242, 308, 336, and 362, preferably wherein the guide strand sequence retains positions 2-7 (“seed sequence”) of the selected SEQ ID NO. In some embodiments, the isolated nucleic acid comprises an expression construct encoding a miRNA, such as a pri-miRNA, a pre-mRNA, an artificial miRNA, or a mature miRNA, wherein the miRNA comprises a guide strand sequence comprising or consisting of a sequence of any one of SEQ ID NOS: 14, 40, 100, 108, 112, 128, 166, 198, 242, 308, 336, and 362, wherein 1, 2, 3, or 4 nucleotides at positions 19-22 differ from the selected SEQ ID NO (variant nucleotide(s)), such that the guide strand sequence is no longer complementary to the ATXN2 target sequence at the variant nucleotide(s).

In some embodiments, the isolated nucleic acid comprises an expression construct encoding a miRNA, such as a pri-miRNA, a pre-mRNA, an artificial miRNA, or a mature miRNA comprising a guide strand sequence of Table 19. In some embodiments, the isolated nucleic acid comprises an expression construct encoding a miRNA, such as a pri-miRNA, a pre-mRNA, an artificial miRNA, or a mature miRNA, comprising a guide strand sequence comprising or consisting of the nucleic acid sequence set forth in any one of SEQ ID NOS:1176-1288, 40, 108, and 166. In some embodiments, the isolated nucleic acid comprises an expression construct encoding a miRNA, such as a pri-miRNA, a pre-mRNA, an artificial miRNA, or a mature miRNA, wherein the miRNA comprises a guide strand sequence comprising or consisting of the nucleic acid sequence set forth in any one of SEQ ID NOS:1176-1288, 40, 108, and 166, with at least 1, 2, 3, 4, or 5 mismatches to the target ATXN2 mRNA sequence. In some embodiments, the miRNA is a pri-miRNA, a pre-mRNA, an artificial miRNA, or a mature miRNA wherein the miRNA comprises a guide strand sequence comprising or consisting of a nucleic acid sequence that is at least 60%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 99%, or 100% identical to any one of SEQ ID NOS:1176-1288, 40, 108, and 166. In some embodiments, the isolated nucleic acid comprises an expression construct encoding a miRNA, such as a pri-miRNA, a pre-mRNA, an artificial miRNA, or a mature miRNA, wherein the miRNA comprises a guide strand sequence comprising or consisting of at least 15, 16, 17, 18, 19, 20, 21, or 22 contiguous nucleotides of a sequence of any one of SEQ ID NOS:1176-1288, 40, 108, and 166, preferably wherein the guide strand sequence retains positions 2-7 (“seed sequence”) of the selected SEQ ID NO. In some embodiments, the isolated nucleic acid comprises an expression construct encoding a miRNA, such as a pri-miRNA, a pre-mRNA, an artificial miRNA, or a mature miRNA, wherein the miRNA comprises a guide strand sequence comprising or consisting of a sequence of any one of SEQ ID NOS:1176-1288, 40, 108, and 166, wherein 1, 2, 3, or 4 nucleotides at positions 19-22 differ from the selected SEQ ID NO (variant nucleotide(s)), such that the guide strand sequence is no longer complementary to the ATXN2 target sequence at the variant nucleotide(s).

In some embodiments, the isolated nucleic acid comprises an expression construct encoding a miRNA, such as a pri-miRNA, a pre-mRNA, an artificial miRNA, or a mature miRNA comprising a guide strand sequence of Table 23. In some embodiments, the isolated nucleic acid comprises an expression construct encoding a miRNA, such as a pri-miRNA, a pre-mRNA, an artificial miRNA, or a mature miRNA, comprising a guide strand sequence comprising or consisting of the nucleic acid sequence set forth in any one of SEQ ID NOS:1908-2007. In some embodiments, the isolated nucleic acid comprises an expression construct encoding a miRNA, such as a pri-miRNA, a pre-mRNA, an artificial miRNA, or a mature miRNA, wherein the miRNA comprises a guide strand sequence comprising or consisting of the nucleic acid sequence set forth in any one of SEQ ID NOS:1908-2007, with at least 1, 2, 3, 4, or 5 mismatches to the target ATXN2 mRNA sequence. In some embodiments, the miRNA is a pri-miRNA, a pre-mRNA, an artificial miRNA, or a mature miRNA wherein the miRNA comprises a guide strand sequence comprising or consisting of a nucleic acid sequence that is at least 60%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 99%, or 100% identical to any one of SEQ ID NOS:1908-2007. In some embodiments, the isolated nucleic acid comprises an expression construct encoding a miRNA, such as a pri-miRNA, a pre-mRNA, an artificial miRNA, or a mature miRNA, wherein the miRNA comprises a guide strand sequence comprising or consisting of at least 15, 16, 17, 18, 19, 20, 21, or 22 contiguous nucleotides of a sequence of any one of SEQ ID NOS: 1908-2007, preferably wherein the guide strand sequence retains positions 2-7 (“seed sequence”) of the selected SEQ ID NO. In some embodiments, the isolated nucleic acid comprises an expression construct encoding a miRNA, such as a pri-miRNA, a pre-mRNA, an artificial miRNA, or a mature miRNA, wherein the miRNA comprises a guide strand sequence comprising or consisting of a sequence of any one of SEQ ID NOS:1908-2007, wherein 1, 2, 3, or 4 nucleotides at positions 19-22 differ from the selected SEQ ID NO (variant nucleotide(s)), such that the guide strand sequence is no longer complementary to the ATXN2 target sequence at the variant nucleotide(s).

In some embodiments, the isolated nucleic acid comprises an expression construct encoding a miRNA, such as a pri-miRNA, a pre-mRNA, an artificial miRNA, or a mature miRNA comprising a guide strand sequence of Table 24. In some embodiments, the isolated nucleic acid comprises an expression construct encoding a miRNA, such as a pri-miRNA, a pre-mRNA, an artificial miRNA, or a mature miRNA, comprising a guide strand sequence comprising or consisting of the nucleic acid sequence set forth in any one of SEQ ID NOS:100, 112, 166, 202, 246, 306, 308, 314, 1180, 1185, 1196, 1200, 1211, 1213, 1215, 1216, 1224, 1811-1822, 1824-1827, 2015, 2065, 2083, 2152, 2203, and 2209. In some embodiments, the isolated nucleic acid comprises an expression construct encoding a miRNA, such as a pri-miRNA, a pre-mRNA, an artificial miRNA, or a mature miRNA, wherein the miRNA comprises a guide strand sequence comprising or consisting of the nucleic acid sequence set forth in any one of SEQ ID NOS:100, 112, 166, 202, 246, 306, 308, 314, 1180, 1185, 1196, 1200, 1211, 1213, 1215, 1216, 1224, 1811-1822, 1824-1827, 2015, 2065, 2083, 2152, 2203, and 2209, with at least 1, 2, 3, 4, or 5 mismatches to the target ATXN2 mRNA sequence. In some embodiments, the miRNA is a pri-miRNA, a pre-mRNA, an artificial miRNA, or a mature miRNA wherein the miRNA comprises a guide strand sequence comprising or consisting of a nucleic acid sequence that is at least 60%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 99%, or 100% identical to any one of SEQ ID NOS:100, 112, 166, 202, 246, 306, 308, 314, 1180, 1185, 1196, 1200, 1211, 1213, 1215, 1216, 1224, 1811-1822, 1824-1827, 2015, 2065, 2083, 2152, 2203, and 2209. In some embodiments, the isolated nucleic acid comprises an expression construct encoding a miRNA, such as a pri-miRNA, a pre-mRNA, an artificial miRNA, or a mature miRNA, wherein the miRNA comprises a guide strand sequence comprising or consisting of at least 15, 16, 17, 18, 19, 20, 21, or 22 contiguous nucleotides of a sequence of any one of SEQ ID NOS:100, 112, 166, 202, 246, 306, 308, 314, 1180, 1185, 1196, 1200, 1211, 1213, 1215, 1216, 1224, 1811-1822, 1824-1827, 2015, 2065, 2083, 2152, 2203, and 2209, preferably wherein the guide strand sequence retains positions 2-7 (“seed sequence”) of the selected SEQ ID NO. In some embodiments, the isolated nucleic acid comprises an expression construct encoding a miRNA, such as a pri-miRNA, a pre-mRNA, an artificial miRNA, or a mature miRNA, wherein the miRNA comprises a guide strand sequence comprising or consisting of a sequence of any one of SEQ ID NOS:100, 112, 166, 202, 246, 306, 308, 314, 1180, 1185, 1196, 1200, 1211, 1213, 1215, 1216, 1224, 1811-1822, 1824-1827, 2015, 2065, 2083, 2152, 2203, and 2209, wherein 1, 2, 3, or 4 nucleotides at positions 19-22 differ from the selected SEQ ID NO (variant nucleotide(s)), such that the guide strand sequence is no longer complementary to the ATXN2 target sequence at the variant nucleotide(s).

In some embodiments, the isolated nucleic acid comprises an expression construct encoding a miRNA, such as a pri-miRNA, a pre-mRNA, an artificial miRNA, or a mature miRNA comprising a guide strand sequence of Table 25. In some embodiments, the isolated nucleic acid comprises an expression construct encoding a miRNA, such as a pri-miRNA, a pre-mRNA, an artificial miRNA, or a mature miRNA, comprising a guide strand sequence comprising or consisting of the nucleic acid sequence set forth in any one of SEQ ID NOS:1185, 1816, 1213, 1819, 2083, 1215, 1216, 1811, and 314. In some embodiments, the isolated nucleic acid comprises an expression construct encoding a miRNA, such as a pri-miRNA, a pre-mRNA, an artificial miRNA, or a mature miRNA, wherein the miRNA comprises a guide strand sequence comprising or consisting of the nucleic acid sequence set forth in any one of SEQ ID NOS:1185, 1816, 1213, 1819, 2083, 1215, 1216, 1811, and 314, with at least 1, 2, 3, 4, or 5 mismatches to the target ATXN2 mRNA sequence. In some embodiments, the miRNA is a pri-miRNA, a pre-mRNA, an artificial miRNA, or a mature miRNA wherein the miRNA comprises a guide strand sequence comprising or consisting of a nucleic acid sequence that is at least 60%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 99%, or 100% identical to any one of SEQ ID NOS:1185, 1816, 1213, 1819, 2083, 1215, 1216, 1811, and 314. In some embodiments, the isolated nucleic acid comprises an expression construct encoding a miRNA, such as a pri-miRNA, a pre-mRNA, an artificial miRNA, or a mature miRNA, wherein the miRNA comprises a guide strand sequence comprising or consisting of at least 15, 16, 17, 18, 19, 20, 21, or 22 contiguous nucleotides of a sequence of any one of SEQ ID NOS:1185, 1816, 1213, 1819, 2083, 1215, 1216, 1811, and 314, preferably wherein the guide strand sequence retains positions 2-7 (“seed sequence”) of the selected SEQ ID NO. In some embodiments, the isolated nucleic acid comprises an expression construct encoding a miRNA, such as a pri-miRNA, a pre-mRNA, an artificial miRNA, or a mature miRNA, wherein the miRNA comprises a guide strand sequence comprising or consisting of a sequence of any one of SEQ ID NOS:1185, 1816, 1213, 1819, 2083, 1215, 1216, 1811, and 314, wherein 1, 2, 3, or 4 nucleotides at positions 19-22 differ from the selected SEQ ID NO (variant nucleotide(s)), such that the guide strand sequence is no longer complementary to the ATXN2 target sequence at the variant nucleotide(s).

In some embodiments, the isolated nucleic acid comprises an expression construct encoding a miRNA, such as a pri-miRNA, a pre-mRNA, an artificial miRNA, or a mature miRNA, comprising a guide strand sequence comprising or consisting of the nucleic acid sequence set forth in any one of SEQ ID NOS:1185, 1816, 1213, and 1811. In some embodiments, the isolated nucleic acid comprises an expression construct encoding a miRNA, such as a pri-miRNA, a pre-mRNA, an artificial miRNA, or a mature miRNA, wherein the miRNA comprises a guide strand sequence comprising or consisting of the nucleic acid sequence set forth in any one of SEQ ID NOS:1185, 1816, 1213, and 1811, with at least 1, 2, 3, 4, or 5 mismatches to the target ATXN2 mRNA sequence. In some embodiments, the miRNA is a pri-miRNA, a pre-mRNA, an artificial miRNA, or a mature miRNA wherein the miRNA comprises a guide strand sequence comprising or consisting of a nucleic acid sequence that is at least 60%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 99%, or 100% identical to any one of SEQ ID NOS: 1185, 1816, 1213, and 1811. In some embodiments, the isolated nucleic acid comprises an expression construct encoding a miRNA, such as a pri-miRNA, a pre-mRNA, an artificial miRNA, or a mature miRNA, wherein the miRNA comprises a guide strand sequence comprising or consisting of at least 15, 16, 17, 18, 19, 20, 21, or 22 contiguous nucleotides of a sequence of any one of SEQ ID NOS:1185, 1816, 1213, and 1811, preferably wherein the guide strand sequence retains positions 2-7 (“seed sequence”) of the selected SEQ ID NO. In some embodiments, the isolated nucleic acid comprises an expression construct encoding a miRNA, such as a pri-miRNA, a pre-mRNA, an artificial miRNA, or a mature miRNA, wherein the miRNA comprises a guide strand sequence comprising or consisting of a sequence of any one of SEQ ID NOS:1185, 1816, 1213, and 1811, wherein 1, 2, 3, or 4 nucleotides at positions 19-22 differ from the selected SEQ ID NO (variant nucleotide(s)), such that the guide strand sequence is no longer complementary to the ATXN2 target sequence at the variant nucleotide(s).

In some embodiments, the isolated nucleic acid comprises an expression construct encoding an artificial miRNA comprising a guide strand sequence according to any of the embodiments described herein, contained within a miR backbone sequence. In some embodiments, the guide strand sequence and passenger strand sequence of the artificial miRNA are contained with a miRNA backbone sequence. In some embodiments, the miRNA backbone sequence is contained within a miR-155 backbone sequence, a miR-155E backbone sequence, a miR-155M backbone sequence, a miR1-1 backbone sequence, a miR-1-1_M backbone sequence, a miR-100 backbone sequence, a miR-100_M backbone sequence, a miR-190A backbone sequence, a miR-124 backbone sequence, a miR-124_M backbone sequence, a miR-16-2 backbone sequence, a miR-132 backbone sequence, a miR-9 backbone sequence, a miR-138-2 backbone sequence, a miR-122 backbone sequence, a miR-122_M backbone sequence, a miR-130a backbone sequence, a miR-128 backbone sequence, a miR-144 backbone sequence, a miR-451a backbone sequence, or a miR-223 backbone sequence. In some embodiments, the terminal loop, stem, 5′ flanking segment, 3′ flanking segment, or any combination thereof of the miR-155 backbone sequence, miR1-1 backbone sequence, miR-100 backbone sequence, miR-190A backbone sequence, miR-124 backbone sequence, miR-16-2 backbone sequence, miR-132 backbone sequence, miR-9 backbone sequence, miR-138-2 backbone sequence, miR-122 backbone sequence, miR-130a backbone sequence, miR-128 backbone sequence, miR-144 backbone sequence, miR-451a backbone sequence, or miR-223 backbone sequence is modified (e.g., nucleotide insertion, deletion, substitution, mismatch, wobble, or any combination thereof).

In some embodiments, the isolated nucleic acid comprises an expression construct encoding a miRNA (pri-miRNA, a pre-mRNA, an artificial miRNA, or a mature miRNA) comprising a guide strand sequence and corresponding passenger strand sequence comprising or consisting of any one of the duplex sequences set forth in Tables 1, 19, 23, and 24. In some embodiments, the passenger strand sequence of the miRNA comprises a sequence that is 100% complementary or perfectly complementary to the guide strand sequence. For example, the encoded guide strand sequence may comprise of consist of a sequence of SEQ ID NO: 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 124, 126, 128, 130, 132, 134, 136, 138, 140, 142, 144, 146, 148, 150, 152, 154, 156, 158, 160, 162, 164, 166, 168, 170, 172, 174, 176, 178, 180, 182, 184, 186, 188, 190, 192, 194, 196, 198, 200, 202, 204, 206, 208, 210, 212, 214, 216, 218, 220, 222, 224, 226, 228, 230, 232, 234, 236, 238, 240, 242, 244, 246, 248, 250, 252, 254, 256, 258, 260, 262, 264, 266, 268, 270, 272, 274, 276, 278, 280, 282, 284, 286, 288, 290, 292, 294, 296, 298, 300, 302, 304, 306, 308, 310, 312, 314, 316, 318, 320, 324, 326, 328, 330, 332, 334, 336, 338, 340, 342, 344, 346, 348, 350, 352, 354, 356, 358, 360, 362, 364, 366, 368, 370, 372, 374, 376, 378, 380, 382, 384, 386, 388, 390, 392, 394, 396, 398, 400, 402, 404, 406, 408, 410, 412, 414, 416, 418, 420, 422, 424, 426, 428, 430, 432, 434, or 436 (guide sequences in Table 1), and the encoded passenger strand sequence may comprise or consist of a sequence of SEQ ID NO: 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 119, 121, 123, 125, 127, 129, 131, 133, 135, 137, 139, 141, 143, 145, 147, 149, 151, 153, 155, 157, 159, 161, 163, 165, 167, 169, 171, 173, 175, 177, 179, 181, 183, 185, 187, 189, 191, 193, 195, 197, 199, 201, 203, 205, 207, 209, 211, 213, 215, 217, 219, 221, 223, 225, 227, 229, 231, 233, 235, 237, 239, 241, 243, 245, 247, 249, 251, 253, 255, 257, 259, 261, 263, 265, 267, 269, 271, 273, 275, 277, 279, 281, 283, 285, 287, 289, 291, 293, 295, 297, 299, 301, 303, 305, 307, 309, 311, 313, 315, 317, 319, 321, 323, 325, 327, 329, 331, 333, 335, 337, 339, 341, 343, 345, 347, 349, 351, 353, 355, 357, 359, 361, 363, 365, 367, 369, 371, 373, 375, 377, 379, 381, 383, 385, 387, 389, 391, 393, 395, 397, 399, 401, 403, 405, 407, 409, 411, 413, 415, 417, 419, 421, 423, 425, 427, 429, 431, 433, or 435, respectively (passenger sequences in Table 1). In some embodiments, the passenger strand sequence of the miRNA is not 100% complementary or to the guide strand sequence. For example, a guide strand sequence may comprise or consist of a sequence of SEQ ID NO: 1176 and the corresponding passenger strand sequence may comprise or consist of a sequence of SEQ ID NO:1289 (see, Table 19).

In some embodiments, the isolated nucleic acid comprises an expression construct encoding a miRNA (pri-miRNA, a pre-mRNA, an artificial miRNA, or a mature miRNA) comprising a guide strand sequence comprising or consisting of any one of SEQ ID NOS: 12, 14, 40, 60, 100, 104, 108, 112, 124, 126, 128, 166, 198, 220, 242, 302, 306, 308, 330, 336, and 362, and a passenger strand sequence of comprising a sequence that is 100% complementary or perfectly complementary to the guide strand sequence. For example, the encoded guide strand sequence may comprise or consist of a sequence of SEQ ID NO: 12, 14, 40, 60, 100, 104, 108, 112, 124, 126, 128, 166, 198, 220, 242, 302, 306, 308, 330, 336, or 362, and the encoded passenger strand sequence may comprise or consist of a sequence of SEQ ID NO: 11, 13, 39, 59, 99, 103, 107, 111, 123, 125, 127, 165, 197, 219, 241, 301, 305, 307, 329, 335, or 361, respectively.

In some embodiments, the isolated nucleic acid comprises an expression construct encoding a miRNA (pri-miRNA, a pre-mRNA, an artificial miRNA, or a mature miRNA) wherein the miRNA comprises a guide strand sequence comprising or consisting of any one of SEQ ID NOS: 14, 40, 100, 108, 112, 128, 166, 198, 242, 308, 336, and 362, and a passenger strand sequence comprising a sequence that is 100% complementary or perfectly complementary to the guide strand. For example, the encoded guide strand sequence may comprise or consist of a sequence of SEQ ID NO: 14, 40, 100, 108, 112, 128, 166, 198, 242, 308, 336, or 362, and the encoded passenger strand sequence may comprise or consisting of a sequence of SEQ ID NO: 13, 39, 99, 107, 111, 127, 165, 197, 241, 307, 335, or 361, respectively.

In some embodiments, the isolated nucleic acid comprises an expression construct encoding a miRNA (pri-miRNA, a pre-mRNA, an artificial miRNA, or a mature miRNA), wherein the miRNA comprises a guide strand sequence comprising or consisting of any one of the guide sequences of Tables 1, 19, 23, and 24, and the passenger strand sequence comprises or consists of a corresponding passenger sequence of Tables 1, 19, 23, and 24 that has 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more insertions, deletions, substitutions, mismatches, wobbles, or any combination thereof relative to the passenger strand sequence of Tables 1, 19, 23, and 24. In some embodiments, the isolated nucleic acid comprises an expression construct encoding a miRNA (pri-miRNA, a pre-mRNA, an artificial miRNA, or a mature miRNA), wherein the miRNA comprises a guide strand sequence comprising or consisting of any one of SEQ ID NOs: 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 124, 126, 128, 130, 132, 134, 136, 138, 140, 142, 144, 146, 148, 150, 152, 154, 156, 158, 160, 162, 164, 166, 168, 170, 172, 174, 176, 178, 180, 182, 184, 186, 188, 190, 192, 194, 196, 198, 200, 202, 204, 206, 208, 210, 212, 214, 216, 218, 220, 222, 224, 226, 228, 230, 232, 234, 236, 238, 240, 242, 244, 246, 248, 250, 252, 254, 256, 258, 260, 262, 264, 266, 268, 270, 272, 274, 276, 278, 280, 282, 284, 286, 288, 290, 292, 294, 296, 298, 300, 302, 304, 306, 308, 310, 312, 314, 316, 318, 320, 324, 326, 328, 330, 332, 334, 336, 338, 340, 342, 344, 346, 348, 350, 352, 354, 356, 358, 360, 362, 364, 366, 368, 370, 372, 374, 376, 378, 380, 382, 384, 386, 388, 390, 392, 394, 396, 398, 400, 402, 404, 406, 408, 410, 412, 414, 416, 418, 420, 422, 424, 426, 428, 430, 432, 434, 436, and a passenger strand sequence comprising or consisting of a sequence that has 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more insertions, deletions, substitutions, mismatches, wobbles, or any combination thereof relative to the corresponding passenger strand sequence of SEQ ID NOS: 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 119, 121, 123, 125, 127, 129, 131, 133, 135, 137, 139, 141, 143, 145, 147, 149, 151, 153, 155, 157, 159, 161, 163, 165, 167, 169, 171, 173, 175, 177, 179, 181, 183, 185, 187, 189, 191, 193, 195, 197, 199, 201, 203, 205, 207, 209, 211, 213, 215, 217, 219, 221, 223, 225, 227, 229, 231, 233, 235, 237, 239, 241, 243, 245, 247, 249, 251, 253, 255, 257, 259, 261, 263, 265, 267, 269, 271, 273, 275, 277, 279, 281, 283, 285, 287, 289, 291, 293, 295, 297, 299, 301, 303, 305, 307, 309, 311, 313, 315, 317, 319, 321, 323, 325, 327, 329, 331, 333, 335, 337, 339, 341, 343, 345, 347, 349, 351, 353, 355, 357, 359, 361, 363, 365, 367, 369, 371, 373, 375, 377, 379, 381, 383, 385, 387, 389, 391, 393, 395, 397, 399, 401, 403, 405, 407, 409, 411, 413, 415, 417, 419, 421, 423, 425, 427, 429, 431, 433, 435 respectively. In some embodiments, a mismatch is a G→C, C→G, A→T, or T→A conversion in the encoded passenger strand sequence. In some embodiments, a mismatch (to create a bulge with the guide strand) is a G→T, C→A, A→C, or T→G conversion in the encoded passenger strand sequence. In some embodiments, a wobble is a G-U wobble, wherein a C is converted to a T in the encoded passenger strand sequence. In some embodiments, the passenger strand sequence is modified according to the rules of Table 8.

In some embodiments, the isolated nucleic acid comprises an expression construct encoding a miRNA (pri-miRNA, a pre-mRNA, an artificial miRNA, or a mature miRNA) wherein the miRNA comprises a guide strand sequence comprising or consisting of any one of SEQ ID NOS: 12, 14, 40, 60, 100, 104, 108, 112, 124, 126, 128, 166, 198, 220, 242, 302, 306, 308, 330, 336, and 362, and a passenger strand sequence comprising or consisting of a sequence that has 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more insertions, deletions, substitutions, mismatches, wobbles, or any combination thereof, relative to the passenger strand sequence comprising or consisting of SEQ ID NOS: 11, 13, 39, 59, 99, 103, 107, 11, 123, 125, 127, 165, 197, 219, 241, 301, 305, 307, 329, 335, and 361, respectively. In some embodiments, a mismatch is a G→C, C→G, A→T, or T→A conversion in the passenger strand sequence. In some embodiments, a mismatch (to create a bulge with the guide strand) is a G→T, C→A, A→C, or T→G conversion in the passenger strand sequence. In some embodiments, a wobble is a G-U wobble, wherein a C is converted to a T in the passenger strand sequence. In some embodiments, the passenger strand sequence is modified according to the rules of Table 8.

In some embodiments, the isolated nucleic acid comprises an expression construct encoding a miRNA (pri-miRNA, a pre-mRNA, an artificial miRNA, or a mature miRNA) wherein the miRNA comprises a guide strand sequence comprising or consisting of any one of SEQ ID NOS: 14, 40, 100, 108, 112, 128, 166, 198, 242, 308, 336, and 362, and a passenger strand sequence comprising a sequence that has 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more insertions, deletions, substitutions, mismatches, wobbles, or any combination thereof relative to the passenger strand sequence comprising or consisting of SEQ ID NOS: 13, 39, 99, 107, 111, 127, 165, 197, 241, 307, 335, and 361, respectively. In some embodiments, a mismatch is a G→C, C→G, A→T, or T→A conversion in the encoded passenger strand sequence. In some embodiments, a mismatch (to create a bulge with the guide strand) is a G→T, C→A, A→C, or T→G conversion in the encoded passenger strand sequence. In some embodiments, a wobble is a G-U wobble, wherein a C is converted to a T in the encoded passenger strand sequence. In some embodiments, the passenger strand sequence is modified according to the rules of Table 8.

In some embodiments, the isolated nucleic acid comprises an expression construct encoding an artificial miRNA comprising a guide strand sequence according to any of the embodiments described herein, contained within a miR-155M backbone sequence, miR-155E backbone sequence, miR1-1 backbone sequence, miR-100 backbone sequence, miR-124 backbone sequence, mIR-138-2 backbone sequence, miR-122 backbone sequence, miR-128 backbone sequence, miR-130a backbone sequence, or miR-16-2 backbone sequence, wherein the artificial miRNA comprises a passenger strand sequence that is modified according to Table 8. In some embodiments, the passenger strand sequence comprises a mismatch, wherein a mismatch is a G→C, C→G, A→T, or T→A conversion in the passenger strand sequence; a mismatch (to create a bulge with the guide strand) is a G→T, C→A, A→C, or T→G conversion in the passenger strand sequence; and a wobble is a G-U wobble, wherein a C is converted to a T in the passenger strand sequence.

In some embodiments, the isolated nucleic acid comprises an expression construct encoding an artificial miRNA comprising or consisting of a nucleic acid sequence set forth in any one of Tables 3, 9, 11, 1923, 24, and 25. In some embodiments, the isolated nucleic acid comprises an expression construct encoding an artificial miRNA comprising or consisting of any one of SEQ ID NOS: 443-490, 1109-1111, 1114, 1121-1168, 1405-1520, 1908-2007, 2011, 2017, 2021, 2025, 2027, 2031, 2035, 2039, 2041, 2045, 2049, 2053, 2057, 2061, 2067, 2071, 2075, 2079, 2085, 2089, 2093, 2097, 2101, 2105, 2109, 2113, 2117, 2120, 2124, 2128, 2132, 2136, 2140, 2144, 2148, 2154, 2158, 2162, 2166, 2170, 2174, 2176, 2180, 2182, 2184, 2187, 2189, 2191, 2193, 2195, 2197, 2199, 2205, 2211, 2261, 2263, 2265, and 2267.

In some embodiments, the isolated nucleic acid comprises an expression construct encoding an artificial miRNA that comprises or consists of a nucleic acid sequence set forth in Table 3. In some embodiments, the isolated nucleic acid comprises an expression construct encoding an artificial miRNA that comprises or consists of a nucleic acid sequence of any one of SEQ ID NOS:443-490.

In some embodiments, the isolated nucleic acid comprises an expression construct encoding an artificial miRNA that comprises or consists of a nucleic acid sequence set forth in Table 9. In some embodiments, the isolated nucleic acid comprises an expression construct encoding an artificial miRNA that comprises or consists of a nucleic acid sequence of any one of SEQ ID NOS:1109-1111, and 1114.

In some embodiments, the isolated nucleic acid comprises an expression construct encoding an artificial miRNA that comprises or consists of a nucleic acid sequence set forth in Table 11. In some embodiments, the isolated nucleic acid comprises an expression construct encoding an artificial miRNA that comprises or consists of a nucleic acid sequence of any one of SEQ ID NOS:1121-1168.

In some embodiments, the isolated nucleic acid comprises an expression construct encoding an artificial miRNA that comprises or consists of a nucleic acid sequence set forth in Table 19. In some embodiments, the isolated nucleic acid comprises an expression construct encoding an artificial miRNA that comprises or consists of a nucleic acid sequence of any one of SEQ ID NOS:1405-1520.

In some embodiments, the isolated nucleic acid comprises an expression construct encoding an artificial miRNA that comprises or consists of a nucleic acid sequence set forth in Table 23. In some embodiments, the isolated nucleic acid comprises an expression construct encoding an artificial miRNA that comprises or consists of a nucleic acid sequence of any one of SEQ ID NOS:1908-2007.

In some embodiments, the isolated nucleic acid comprises an expression construct encoding an artificial miRNA that comprises or consists of a nucleic acid sequence set forth in Table 24. In some embodiments, the isolated nucleic acid comprises an expression construct encoding an artificial miRNA that comprises or consists of a nucleic acid sequence of any one of SEQ ID NOS:1908-1934, 1936-1977, 1979-1982, 1984-1994, 1997, 1998, 2000, 2001, 2005-2007, 2011, 2017, 2021, 2025, 2027, 2031, 2035, 2039, 2041, 2045, 2049, 2053, 2057, 2061, 2067, 2071, 2075, 2079, 2085, 2089, 2093, 2097, 2101, 2105, 2109, 2113, 2117, 2120, 2124, 2128, 2132, 2136, 2140, 2144, 2148, 2154, 2158, 2162, 2166, 2170, 2174, 2176, 2180, 2182, 2184, 2187, 2189, 2191, 2193, 2195, 2197, 2199, 2205, 2211, 2261, 2263, 2265, and 2267.

In some embodiments, the isolated nucleic acid comprises an expression construct encoding an artificial miRNA that comprises or consists of a nucleic acid sequence set forth in Table 25. In some embodiments, the isolated nucleic acid comprises an expression construct encoding an artificial miRNA that comprises or consists of a nucleic acid sequence of any one of SEQ ID NOS:1915, 1982, 1965, 1937, 1985, 1921, and 2021.

In some embodiments, expression constructs encoding the inhibitory nucleic acids that target ATXN2 mRNA comprises or consists of any of the guide strand sequences or artificial miRNA sequences disclosed in DNA format. For example, Tables 9, 11, 23, and 24 provide amiRNA sequences in DNA format, which DNA sequence may be inserted into expression constructs. Alternatively, amiRNA sequences provided herein can be converted to DNA format by replacing each “U” nucleotide with a “T” nucleotide.

In some embodiments, the expression construct encodes two or more inhibitory nucleic acids that target an ATXN2 mRNA transcript described herein. In some embodiments, the expression construct encodes an inhibitory nucleic acid that targets ATXN2 transcript and an inhibitory nucleic acid that targets a second target transcript other than ATXN2. In some embodiments, the second target transcript is C9ORF72. Examples of inhibitory nucleic acids targeting C9ORF72 are described in US Patent Publication US2019/0316126 (incorporated by reference in its entirety). In some embodiments, the expression construct encodes an inhibitory nucleic acid that targets ATXN2 transcript and encodes a therapeutic polypeptide or protein.

In some embodiments, the expression construct is monocistronic. In some embodiments, the expression construct is polycistronic (e.g., expression construct encodes two or more peptides or polypeptides). In some embodiments, a nucleic acid sequence encoding a first gene product (e.g., inhibitory nucleic acid targeting ATXN2 mRNA) and a nucleic acid sequence encoding a second gene product within an expression construct are separated by an internal ribosome entry site (IRES), furin cleavage site, or viral 2A peptide. In some embodiments, a viral 2A peptide is a porcine teschovirus-1 (P2A), Thosea asigna virus (T2A), equine rhinitis A virus (E2A), foot-and-mouth disease virus (F2A), B. mori cytoplasmic polyhedrosis virus (BmCPV 2A), B. mori flacherie virus (BmIFV 2A), or variant thereof.

In some embodiments, the expression construct further comprises one or more expression control sequences (regulatory sequences) operably linked with the transgene (e.g., nucleic acid encoding an artificial miRNA). “Operably linked” sequences include expression control sequences that are contiguous with the transgene or act in trans or at a distance from the transgene to control its expression. Examples of expression control sequences include transcription initiation sequences, termination sequences, promoter sequences, enhancer sequences, repressor sequences, splice site sequences, polyadenylation (polyA) signal sequences, or any combination thereof.

In some embodiments, a promoter is an endogenous promoter, synthetic promoter, constitutive promoter, inducible promoter, tissue-specific promoter (e.g., CNS-specific), or cell-specific promoter (neurons, glial cells, or astrocytes). Examples of constitutive promoters include, Rous sarcoma virus (RSV) LTR promoter (optionally with the RSV enhancer), cytomegalovirus (CMV) promoter (optionally with the CMV enhancer), SV40 promoter, and dihydrofolate reductase promoter. Examples of inducible promoters include zinc-inducible sheep metallothionine (MT) promoter, dexamethasone (Dex)-inducible mouse mammary tumor virus (MMTV) promoter, T7 polymerase promoter system, the ecdysone insect promoter, tetracycline-repressible system, tetracycline-inducible system, RU486-inducible system, and the rapamycin-inducible system. Further examples of promoters that may be used include, for example, chicken beta-actin promoter (CBA promoter), a CAG promoter, a H1 promoter, a CD68 promoter, a JeT promoter, synapsin promoter, RNA pol II promoter, or a RNA pol III promoter (e.g., U6, H1, etc.). In some embodiments, the promoter is a tissue-specific RNA pol II promoter. In some embodiments, the tissue-specific RNA pol II promoter is derived from a gene that exhibits neuron-specific expression. In some embodiments, the neuron-specific promoter is a synapsin 1 promoter or synapsin 2 promoter.

In some embodiments, the promoter is an H1 promoter comprising or consisting of the sequence set forth in nucleotides 113-203 of SEQ ID NO:1522. In some embodiments, the promoter is an H1 promoter comprising or consisting of the sequence set forth in nucleotides 1798-1888 of SEQ ID NO:1521. In some embodiments, the promoter is an H1 promoter comprising or consisting of the sequence set forth in nucleotides 113-343 of any one of SEQ ID NOS:2257-2260. In some embodiments, the promoter is an H1 promoter comprising or consisting of the sequence set forth in nucleotides 244-343 of any one of SEQ ID NOS:2257-2260.

In some embodiments, the sequence encoding the inhibitory nucleic acid of the present disclosure is positioned in an untranslated region of an expression construct. In some embodiments, the sequence encoding the inhibitory nucleic acid of the present disclosure is positioned in an intron, a 5′ untranslated region (5′UTR), or a 3′ untranslated region (3′UTR) of the expression construct. In some embodiments, the sequence encoding the inhibitory nucleic acid of the present disclosure is positioned in an intron downstream of the promoter and upstream of an expressed gene.

In some embodiments, the isolated nucleic acid comprises an expression construct encoding an inhibitory nucleic, flanked by two AAV inverted terminal repeats (ITRs) (e.g., 5′ ITR and 3′ ITR). In some embodiments, each AAV ITR is a full length ITR (e.g., approximately 145 bp in length, and containing functional Rep binding site (RBS) and terminal resolution site (trs)). In some embodiments, one of the ITRs is truncated (e.g., shortened or not full-length). In some embodiments, a truncated ITR lacks a functional terminal resolution site (trs) and is used for production of self-complementary AAV vectors (scAAV vectors). In some embodiments, a truncated ITR is a truncated version of AAV2 ITR referred to as AITR (D-sequence and TRS are deleted). In some embodiments, the ITRs are selected from AAV serotypes of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV9.47, AAV9(hu14), AAV10, AAV11, AAV 12, AAVrh8, AAVrh10, AAV-DJ8, AAV-DJ, AAV-PUP.A, AAV-PHP.B, AAVPHP.B2, AAVPHP.B3, AAVPHP.N/PHP.B-DGT, AAVPHP.B-EST, AAVPHP.B-GGT, AAVPHP.B-ATP, AAVPHP.B-ATT-T, AAVPHP.B-DGT-T, AAVPHP.B-GGT-T, AAVPHP.B-SGS, AAVPHP.B-AQP, AAVPHP.B-QQP, AAVPHP.B-SNP(3), AAVPHP.B-SNP, AAVPHP.B-QGT, AAVPHP.B-NQT, AAVPHP.B-EGS, AAVPHP.B-SGN, AAVPHP.B-EGT, AAVPHP.B-DST, AAVPHP.B-DST, AAVPHP.B-STP, AAVPHP.B-PQP, AAVPHP.B-SQP, AAVPHP.B-QLP, AAVPHP.B-TMP, AAVPHP.B-TTP, AAVPHP.S/G2A12, AAVG2A 15/G2A3, AAVG2B4, AAVG2B5, and variants thereof.

In some embodiments, the isolated nucleic acid molecule comprising an expression construct encoding an inhibitory nucleic acid that inhibits expression or activity of ATXN2 comprises the nucleotide sequence set forth in any one of SEQ ID NOS:2257-2260. In some embodiments, the isolated nucleic acid molecule comprising an expression construct encoding an inhibitory nucleic acid that inhibits expression or activity of ATXN2 comprises the nucleotide sequence set forth in SEQ ID NO:2257. In some embodiments, the isolated nucleic acid molecule comprising an expression construct encoding an inhibitory nucleic acid that inhibits expression or activity of ATXN2 comprises the nucleotide sequence set forth in SEQ ID NO:2258. In some embodiments, the isolated nucleic acid molecule comprising an expression construct encoding an inhibitory nucleic acid that inhibits expression or activity of ATXN2 comprises the nucleotide sequence set forth in SEQ ID NO:2259. In some embodiments, the isolated nucleic acid molecule comprising an expression construct encoding an inhibitory nucleic acid that inhibits expression or activity of ATXN2 comprises the nucleotide sequence set forth in SEQ ID NO:2260.

Additional isolated nucleic acid molecules comprising an expression construct encoding an inhibitory nucleic acid that inhibits expression or activity of ATXN2 may be constructed using the nucleotide sequence set forth in any one of SEQ ID NOS:2257-2260, by substituting the desired inhibitory nucleic acid sequence (e.g., artificial miRNA cassette) of the present disclosure into nucleotide positions 344-481 of any one of SEQ ID NOS:2257-2260.

Vectors and Host Cells

Inhibitory nucleic acid molecules (siRNAs, shRNAs, miRNAs) described herein can be encoded by vectors. The use of vectors, e.g., AAV, for expressing inhibitory nucleic acids of the present disclosure may allow for continual or controlled expression of inhibitory nucleic acid in the subject, rather than multiple doses of isolated inhibitory nucleic acids to the subject. The present disclosure provides a vector comprising an isolated nucleic acid comprising an expression construct encoding an inhibitory nucleic described herein. A vector can be a plasmid, cosmid, phagemid, bacterial artificial chromosome (BAC) or viral vector. Examples of viral vectors include herpesvirus (HSV) vectors, retroviral vectors, adenoviral vectors, adeno-associated viral (AAV) vectors, lentiviral vectors, baculoviral vectors, and the like. In some embodiments, a retroviral vector is a mouse stem cell virus, murine leukemia virus (e.g. Moloney murine leukemia virus vector), feline leukemia virus, feline sarcoma virus, or avian reticuloendotheliosis virus vector. In some embodiments, a lentiviral vector is a HIV (human immunodeficiency virus, including HIV type 1 and HIV type 2, equine infectious anemia virus, feline immunodeficiency virus (FIV), bovine immune deficiency virus (BIV), and simian immunodeficiency virus (SIV), equine infectious anemia virus, or Maedi-Visna viral vector.

In some embodiments, the vector is an AAV (AAV) vector, such as a recombinant AAV (rAAV) vector, which is produced by recombinant methods. AAV is a single-stranded, non-enveloped DNA virus having a genome that encodes proteins for replication (rep) and the capsid (Cap), flanked by two ITRs, which serve as the origin of replication of the viral genome. AAV also contains a packaging sequence, allowing packaging of the viral genome into an AAV capsid. A recombinant AAV vector (rAAV) may be obtained from the wild type genome of AAV by using molecular methods to remove the all or part of the wild type genome (e.g., Rep, Cap) from the AAV, and replacing with a non-native nucleic acid, such as a heterologous nucleic acid sequence (e.g., a nucleic acid molecule encoding an inhibitory nucleic acid). Typically, for AAV one or both inverted terminal repeat (ITR) sequences are retained in the AAV vector. In some embodiments, the rAAV vector comprises an expression construct encoding an inhibitory nucleic acid of the present disclosure flanked by two cis-acting AAV ITRs (5′ ITR and 3′ ITR). Functional ITR sequences are necessary for the rescue, replication and packaging of the AAV viral particle. Thus, an AAV vector is defined herein to include at least those sequences required in cis for replication and packaging (e.g., functional ITRs) of the virus. In some embodiments, each AAV ITR is a full length ITR (e.g., approximately 145 bp in length, and containing functional Rep binding site (RBS) and terminal resolution site (trs)). In some embodiments, one or both of the ITRs is is modified, e.g., by insertion, deletion, or substitution, provided that the ITRs provide for functional rescue, replication, and packaging. In some embodiments, a modified ITR lacks a functional terminal resolution site (trs) and is used for production of self-complementary AAV vectors (scAAV vectors). In some embodiments, a modified ITR is a truncated version of AAV2 ITR referred to as AITR (D-sequence and TRS are deleted).

In some embodiments, the AAV vector comprises a 5′ ITR comprising or consisting of nucleotides 1-106 of any one of SEQ ID NOS:2257-2260. In some embodiments, the AAV vector comprises a 3′ ITR comprising or consisting of nucleotides 2192-2358 of any one of SEQ ID NOS:2257-2260. In some embodiments, the AAV vector comprises: a 5′ ITR comprising or consisting of nucleotides 1-106 of SEQ ID NO:2257 and a 3′ ITR comprising or consisting of nucleotides 2192-2358 of SEQ ID NO:2257; a 5′ ITR comprising or consisting of nucleotides 1-106 of SEQ ID NO:2258 and a 3′ ITR comprising or consisting of nucleotides 2192-2358 of SEQ ID NO:2258; a 5′ ITR comprising or consisting of nucleotides 1-106 of SEQ ID NO:2259 and a 3′ ITR comprising or consisting of nucleotides 2192-2358 of SEQ ID NO:2259; or a 5′ ITR comprising or consisting of nucleotides 1-106 of SEQ ID NO:2260 and a 3′ ITR comprising or consisting of nucleotides 2192-2358 of SEQ ID NO:2260.

In some embodiments, the rAAV vector is a mammalian serotype AAV vector (e.g., AAV genome and ITRs derived from mammalian serotype AAV), including a primate serotype AAV vector or human serotype AAV vector. In some embodiments, the AAV vector is a chimeric AAV vector. In some embodiments, the ITRs are selected from AAV serotypes of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV9.47, AAV9(hu14), AAV10, AAV11, AAV 12, AAVrh8, AAVrh10, AAV-DJ8, AAV-DJ, AAV-PUPA, AAV-PHP.B, AAVPHP.B2, AAVPHP.B3, AAVPHP.N/PHP.B-DGT, AAVPHP.B-EST, AAVPHP.B-GGT, AAVPHP.B-ATP, AAVPHP.B-ATT-T, AAVPHP.B-DGT-T, AAVPHP.B-GGT-T, AAVPHP.B-SGS, AAVPHP.B-AQP, AAVPHP.B-QQP, AAVPHP.B-SNP(3), AAVPHP.B-SNP, AAVPHP.B-QGT, AAVPHP.B-NQT, AAVPHP.B-EGS, AAVPHP.B-SGN, AAVPHP.B-EGT, AAVPHP.B-DST, AAVPHP.B-DST, AAVPHP.B-STP, AAVPHP.B-PQP, AAVPHP.B-SQP, AAVPHP.B-QLP, AAVPHP.B-TMP, AAVPHP.B-TTP, AAVPHP.S/G2A12, AAVG2A 15/G2A3, AAVG2B4, AAVG2B5, and variants thereof.

Other expression control sequences may be present in the rAAV vector operably linked to the inhibitory nucleic acid, including one or more of transcription initiation sequences, termination sequences, promoter sequences, enhancer sequences, repressor sequences, splice site sequences, polyadenylation (polyA) signal sequences, or any combination thereof.

AAV preferentially packages a full-length genome, i.e., one that is approximately the same size as the native genome, and is not too big or too small. However, expression cassettes encoding inhibitory nucleic acid sequences are rather small. To avoid packaging of fragmented genomes, a stuffer sequence may be linked to an expression construct encoding inhitory nucleic acids of the present disclosure and flanked by the 5′ ITR and 3′ ITR to expand the packagable genome, resulted in a genome whose size was near-normal in length between the ITRs. In some embodiments, the rAAV vector comprising a stuffer sequence and expression cassette encoding an inhibitory nucleic acid sequence of the present disclosure has a total length of about 4.7 kb between the 5′ ITR and 3′ ITR. In some embodiments, the rAAV vector is a self-complementary rAAV vector comprising a stuffer sequence and expression cassette encoding an inhibitory nucleic acid sequence of the present disclosure and has a total length of about 2.4 kb between the 5′ ITR and 3′ ITR. An exemplary stuffer sequence for use in the rAAV vectors of the present disclosure includes a sequence comprising or consisting of nucleotides 348-2228 of SEQ ID NO:1522 and a sequence comprising or consisting of nucleotides 489-2185 of any one of SEQ ID NOS:2257-2260.

rAAV vectors may have one or more AAV wild type genes deleted in whole or in part. In some embodiments the rAAV vector is replication defective. In some embodiments, the rAAV vector lacks a functional Rep protein and/or capsid protein. In some embodiments, the rAAV vector is a self-complementary AAV (scAAV) vector.

In some embodiments, the rAAV vector comprises from 5′ ITR to 3′ ITR the nucleotide sequence set forth in any one of SEQ ID NOS:2257-2260. In some embodiments, the rAAV vector comprises from 5′ ITR to 3′ ITR the nucleotide sequence set forth in SEQ ID NO:2257. In some embodiments, the rAAV vector comprises from 5′ ITR to 3′ ITR the nucleotide sequence set forth in SEQ ID NO:2258. In some embodiments, the rAAV vector comprises from 5′ ITR to 3′ ITR the nucleotide sequence set forth in SEQ ID NO:2259. In some embodiments, the rAAV vector comprises the nucleotide sequence set forth in SEQ ID NO:2260.

Recombinant AAV vectors of the present disclosure may be encapsidated by one or more AAV capsid proteins to form a rAAV particle. A “rAAV particle” or “rAAV virion” refers to an infectious, replication-defective virus including an AAV protein shell, encapsidating a rAAV vector comprising a transgene of interest, which is flanked on each side by a 5′ AAV ITR and 3′ AAV ITR. A rAAV particle is produced in a suitable host cell which has had sequences specifying a rAAV vector, AAV helper functions and accessory functions introduced therein to render the host cell capable of encoding AAV polypeptides that are required for packaging the rAAV vector (containing the transgene sequence of interest) into infectious rAAV particles for subsequent gene delivery.

Methods of packaging recombinant AAV vector into AAV capsid proteins using host cell culture are known in the art. In some embodiments, one or more of the required components for packaging the rAAV vector, (e.g., Rep sequence, cap sequence, and/or accessory functions) may be provided by a stable host cell that has been engineered to to contain the one or more required components (e.g., by a vector). Expression of the required components for AAV packaging may be under control of an inducible or constitutive promoter in the host packaging cell. AAV helper vectors are commonly used to provide transient expression of AAV rep and/or cap genes, which function in trans, to complement missing AAV functions that are necessary for AAV replication. In some embodiments, AAV helper vectors lack AAV ITRs and can neither replicate nor package themselves. AAV helper vectors can be in the form of a plasmid, phage, transposon, cosmid, virus, or virion.

In some embodiments, rAAV particles may be produced using the triple transfection method (see, e.g., U.S. Pat. No. 6,001,650, incorporated herein by reference in its entirety). In this approach, the rAAV particles are produced by transfecting a host cell with a rAAV vector (comprising a transgene) to be packaged into rAAV particles, an AAV helper vector, and an accessory function vector. In some embodiments, the AAV helper function vector supports efficient AAV vector production without generating any detectable wild-type AAV virions (e.g., AAV virions containing functional rep and cap genes). The accessory function vector encodes nucleotide sequences for non-AAV derived viral and/or cellular functions upon which AAV is dependent for replication (e.g., “accessory functions”). The accessory functions include those functions required for AAV replication, including, without limitation, those moieties involved in activation of AAV gene transcription, stage specific AAV mRNA splicing, AAV DNA replication, synthesis of cap expression products, and AAV capsid assembly. Viral-based accessory functions can be derived from any of the known helper viruses such as adenovirus, herpesvirus (other than herpes simplex virus type-1), and vaccinia virus. In some embodiments, a double transfection method, wherein the AAV helper function and accessory function are cloned on a single vector, which is used to generate rAAV particles.

The AAV capsid is an important element in determining these tissue-specificity of the rAAV particle. Thus, a rAAV particle having a capsid tissue specificity can be selected. In some embodiments, the rAAV particle comprises a capsid protein selected from a AAV serotype of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV9.47, AAV9(hu14), AAV10, AAV11, AAV 12, AAVrh8, AAVrh10, AAV-DJ8, AAV-DJ, AAV-PUPA, AAV-PHP.B, AAVPHP.B2, AAVPHP.B3, AAVPHP.N/PHP.B-DGT, AAVPHP.B-EST, AAVPHP.B-GGT, AAVPHP.B-ATP, AAVPHP.B-ATT-T, AAVPHP.B-DGT-T, AAVPHP.B-GGT-T, AAVPHP.B-SGS, AAVPHP.B-AQP, AAVPHP.B-QQP, AAVPHP.B-SNP(3), AAVPHP.B-SNP, AAVPHP.B-QGT, AAVPHP.B-NQT, AAVPHP.B-EGS, AAVPHP.B-SGN, AAVPHP.B-EGT, AAVPHP.B-DST, AAVPHP.B-DST, AAVPHP.B-STP, AAVPHP.B-PQP, AAVPHP.B-SQP, AAVPHP.B-QLP, AAVPHP.B-TMP, AAVPHP.B-TTP, AAVPHP.S/G2A12, AAVG2A 15/G2A3, AAVG2B4, AAVG2B5, and variants thereof. In some embodiments, the AAV capsid is selected from a serotype that is capable of crossing the blood-brain barrier, e.g., AAV9, AAVrh.10, AAV-PHP-B, or a variant thereof. In some embodiments, the AAV capsid is a chimeric AAV capsid. In some embodiments, the AAV particle is a pseudotyped AAV, having capsid and genome from different AAV serotypes.

In some embodiments, the rAAV particle is capable of transducing cells of the CNS. In some embodiments, the rAAV particle is capable of transducing non-neuronal cells or neuronal cells of the CNS. In some embodiments, the CNS cell is a neuron, glial cell, astrocyte, or microglial cell.

In another aspect, the present disclosure provides host cells transfected with the rAAV comprising the inhibitory nucleic acids or vectors described herein. In some embodiments, the host cell is a prokaryotic cell or a eukaryotic cell. In some embodiments, the host cell is a mammalian cell (e.g., HEK293T, COS cells, HeLa cells, KB cells), bacterial cell (E. coli), yeast cell, insect cell (Sf9, Sf21, Drosophila, mosquito), etc.

Pharmaceutical Compositions

In some aspects, the disclosure provides pharmaceutical compositions comprising an inhibitory nucleic acid, isolated nucleic acid comprising an expression construct, or vector as described herein and a pharmaceutically acceptable carrier. As used herein, the term “pharmaceutically acceptable” refers to those compounds, materials, compositions, and/or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with cells and/or tissues without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit/risk ratio.

As used herein, the term “pharmaceutically acceptable carrier” means a pharmaceutically acceptable material, composition or carrier, such as a liquid or solid filler, stabilizer, dispersing agent, suspending agent, diluent, excipient, thickening agent, solvent or encapsulating material, involved in carrying or transporting a compound useful within the invention within or to the patient such that it may perform its intended function. Each carrier must be “acceptable” in the sense of being compatible with the other ingredients of the formulation and not injurious to the cell or tissue being contacted. Additional ingredients that may be included in the pharmaceutical compositions used in the practice of the invention are known in the art and described, for example in Remington's Pharmaceutical Sciences (Genaro, Ed., Mack Publishing Co., 1985, Easton, Pa.), which is incorporated herein by reference.

As is well known in the medical arts, the dosage for any one patient depends upon many factors, including the patient's size, weight, body surface area, age, the level of expression of inhibitory RNA expression required to achieve a therapeutic effect, stability of the inhibitory nucleic acid, specific disease being treated, stage of disease, sex, time and route of administration, general health, and other drugs being administered concurrently. In some embodiments, a rAAV particle as described herein is administered to a subject in an amount of about 1×10⁶ VG (viral genomes) to about 1×10¹⁶ VG per subject, or about 1×10⁶, 2×10⁶, 3×10⁶, 4×10⁶, 5×10⁶, 6×10⁶, 7×10⁶, 8×10⁶, 9×10⁶, 1×10⁷, 2×10⁷, 3×10⁷, 4×10⁷, 5×10⁷, 6×10⁷, 7×10⁷, 8×10⁷, 9×10⁷, 1×10⁸, 2×10⁸, 3×10⁸, 4×10⁸, 5×10⁸, 6×10⁸, 7×10⁸, 8×10⁸, 9×10⁸, 1×10⁹, 2×10⁹, 3×10⁹, 4×10⁹, 5×10⁹, 6×10⁹, 7×10⁹, 8×10⁹, 9×10⁹, 1×10¹⁰, 2×10¹⁰, 3×10¹⁰, 4×10¹⁰, 5×10¹⁰, 6×10¹⁰, 7×10¹⁰, 8×10¹⁰, 9×10¹⁰, 1×10¹¹, 2×10¹¹, 2.1×10¹¹, 2.2×10¹¹, 2.3×10¹¹, 2.4×10¹¹, 2.5×10¹¹, 2.6×10¹¹, 2.7×10¹¹, 2.8×10¹¹, 2.9×10¹¹, 3×10¹¹, 4×10¹¹, 5×10¹¹, 6×10¹¹, 7×10¹¹, 7.1×10¹¹, 7.2×10¹¹, 7.3×10¹¹, 7.4×10¹¹, 7.5×10¹¹, 7.6×10¹¹, 7.7×10¹¹, 7.8×10¹¹, 7.9×10¹¹, 8×10¹¹, 9×10¹¹, 1×10¹², 1.1×10¹², 1.2×10¹², 1.3×10¹², 1.4×10¹², 1.5×10¹², 1.6×10¹², 1.7×10¹², 1.8×10¹², 1.9×10¹², 2×10¹², 3×10¹², 4×10¹², 4.1×10¹², 4.2×10¹², 4.3×10¹², 4.4×10¹², 4.5×10¹², 4.6×10¹², 4.7×10¹², 4.8×10¹², 4.9×10¹², 5×10¹², 6×10¹², 7×10¹², 8×10¹², 8.1×10¹², 8.2×10¹², 8.3×10¹², 8.4×10¹², 8.5×10¹², 8.6×10¹², 8.7×10¹², 8.8×10¹², 8.9×10¹², 9×10¹², 1×10¹³, 2×10¹³, 3×10¹³, 4×10¹³, 5×10¹³, 6×10¹³, 6.7×10¹³, 7×10¹³, 8×10¹³, 9×10¹³, 1×10¹⁴, 2×10¹⁴, 3×10¹⁴, 4×10¹⁴, 5×10¹⁴, 6×10¹⁴, 7×10¹⁴, 8×10¹⁴, 9×10¹⁴, 1×10¹⁵, 2×10¹⁵, 3×10¹⁵, 4×10¹⁵, 5×10¹⁵, 6×10¹⁵, 7×10¹⁵, 8×10¹⁵, 9×10¹⁵, or 1×10¹⁶ VG/subject. In some embodiments, a rAAV particle as described herein is administered to a subject in an amount of about 1×10⁶ VG/kg to about 1×10¹⁶ VG/kg, or about 1×10⁶, 2×10⁶, 3×10⁶, 4×10⁶, 5×10⁶, 6×10⁶, 7×10⁶, 8×10⁶, 9×10⁶, 1×10⁷, 2×10⁷, 3×10⁷, 4×10⁷, 5×10⁷, 6×10⁷, 7×10⁷, 8×10⁷, 9×10⁷, 1×10⁸, 2×10⁸, 3×10⁸, 4×10⁸, 5×10⁸, 6×10⁸, 7×10⁸, 8×10⁸, 9×10⁸, 1×10⁹, 2×10⁹, 3×10⁹, 4×10⁹, 5×10⁹, 6×10⁹, 7×10⁹, 8×10⁹, 9×10⁹, 1×10¹⁰, 2×10¹⁰, 3×10¹⁰, 4×10¹⁰, 5×10¹⁰, 6×10¹⁰, 7×10¹⁰, 8×10¹⁰, 9×10¹⁰, 1×10¹¹, 2×10¹¹, 2.1×10¹¹, 2.2×10¹¹, 2.3×10¹¹, 2.4×10¹¹, 2.5×10¹¹, 2.6×10¹¹, 2.7×10¹¹, 2.8×10¹¹, 2.9×10¹¹, 3×10¹¹, 4×10¹¹, 5×10¹¹, 6×10¹¹, 7×10¹¹, 7.1×10¹¹, 7.2×10¹¹, 7.3×10¹¹, 7.4×10¹¹, 7.5×10¹¹, 7.6×10¹¹, 7.7×10¹¹, 7.8×10¹¹, 7.9×10¹¹, 8×10¹¹, 9×10¹¹, 1×10¹², 1.1×10¹², 1.2×10¹², 1.3×10¹², 1.4×10¹², 1.5×10¹², 1.6×10¹², 1.7×10¹², 1.8×10¹², 1.9×10¹², 2×10¹², 3×10¹², 4×10¹², 4.1×10¹², 4.2×10¹², 4.3×10¹², 4.4×10¹², 4.5×10¹², 4.6×10¹², 4.7×10¹², 4.8×10¹², 4.9×10¹², 5×10¹², 6×10¹², 7×10¹², 8×10¹², 8.1×10¹², 8.2×10¹², 8.3×10¹², 8.4×10¹², 8.5×10¹², 8.6×10¹², 8.7×10¹², 8.8×10¹², 8.9×10¹², 9×10¹², 1×10¹³, 2×10¹³, 3×10¹³, 4×10¹³, 5×10¹³, 6×10¹³, 6.7×10¹³, 7×10¹³, 8×10¹³, 9×10¹³, 1×10¹⁴, 2×10¹⁴, 3×10¹⁴, 4×10¹⁴, 5×10¹⁴, 6×10¹⁴, 7×10¹⁴, 8×10¹⁴, 9×10¹⁴, 1×10¹⁵, 2×10¹⁵, 3×10¹⁵, 4×10¹⁵, 5×10¹⁵, 6×10¹⁵, 7×10¹⁵, 8×10¹⁵, 9×10¹⁵, or 1×10¹⁶VG/kg.

Pharmaceutical compositions may be administered in a manner appropriate to the disease or condition to be treated (or prevented) as determined by persons skilled in the medical art. An appropriate dose and a suitable duration and frequency of administration of the compositions will be determined by such factors as the health condition of the patient, size of the patient (i.e., weight, mass, or body area), the type and severity of the patient's disease, the particular form of the active ingredient, and the method of administration. In general, an appropriate dose and treatment regimen provide the composition(s) in an amount sufficient to provide therapeutic and/or prophylactic benefit (such as described herein, including an improved clinical outcome, such as more frequent complete or partial remissions, or longer disease-free and/or overall survival, or a lessening of symptom severity). For prophylactic use, a dose should be sufficient to prevent, delay the onset of, or diminish the severity of a disease associated with disease or disorder. Prophylactic benefit of the compositions administered according to the methods described herein can be determined by performing pre-clinical (including in vitro and in vivo animal studies) and clinical studies and analyzing data obtained therefrom by appropriate statistical, biological, and clinical methods and techniques, all of which can readily be practiced by a person skilled in the art.

Compositions (e.g., pharmaceutical compositions) may be administered by any route, including enteral (e.g., oral), parenteral, intravenous, intramuscular, intra-arterial, intramedullary, intrathecal, subpial, intraparenchymal, intrastriatal, intracranial, intracisternal, intra-cerebral, intracerebral ventricular, intraocular, intraventricular, intralumbar, subcutaneous, transdermal, interdermal, rectal, intravaginal, intraperitoneal, topical (as by powders, ointments, creams, and/or drops), mucosal, nasal, bucal, sublingual; by intratracheal instillation, bronchial instillation, and/or inhalation; and/or as an oral spray, nasal spray, and/or aerosol. In general, the most appropriate route of administration will depend upon a variety of factors including the nature of the agent (e.g., its stability in the environment of the gastrointestinal tract), and/or the condition of the subject. In some embodiments, compositions are directly injected into the CNS of the subject. In some embodiments, direct injection into the CNS is intracerebral injection, intraparenchymal injection, intrathecal injection, intrastriatal injection, subpial injection, or any combination thereof. In some embodiments, direct injection into the CNS is direct injection into the cerebrospinal fluid (CSF) of the subject, optionally wherein the direct injection is is intracisternal injection, intraventricular injection, and/or intralumbar injection.

In some embodiments, pharmaceutical compositions comprising rAAV particles are formulated to reduce aggregation of rAAV particles, particularly where high rAAV particle concentrations are present (e.g., ^˜10¹³ VG/ml or more). Methods for reducing aggregation of rAAV particles are well known in the art and, include, for example, addition of surfactants, pH adjustment, salt concentration adjustment, etc. (See, e.g., Wright F R, et al., Molecular Therapy (2005) 12:171-178, incorporated herein by reference in its entirety).

Kits

In some embodiments, the compositions provided herein may be assembled into pharmaceutical or research kits to facilitate their use in therapeutic or research use. A kit may include one or more containers comprising: (a) inhibitory nucleic acid, isolated nucleic acid comprising an expression construct, or vector as described herein; (b) instructions for use; and optionally (c) reagents for transducing the kit component (a) into a host cell. In some embodiments, the kit component (a) may be in a pharmaceutical formulation and dosage suitable for a particular use and mode of administration. For example, the kit component (a) may be presented in unit-dose or multi-dose containers, such as sealed ampoules or vials. The components of the kit may require mixing one or more components prior to use or may be prepared in a premixed state. The components of the kit may be in liquid or solid form, and may require addition of a solvent or further dilution. The components of the kit may be sterile. The instructions may be in written or electronic form and may be associated with the kit (e.g., written insert, CD, DVD) or provided via internet or web-based communication. The kit may be shipped and stored at a refrigerated or frozen temperature.

Methods of Treatment

In another aspect, the present disclosure provides methods for inhibiting the expression or activity of ATXN2 in a cell, comprising administering a composition of the present disclosure (e.g., inhibitory nucleic acid, isolated nucleic acid comprising an expression construct encoding an inhibitory nucleic acid, vector, rAAV particle, pharmaceutical composition) to a cell, thereby inhibiting the expression or activity of ATXN2 in the cell. In some embodiments, the cell is a CNS cell. In some embodiments, the cell is a non-neuronal cell or neuronal cell of the CNS. In some embodiments, the non-neuronal cell of the CNS is a glial cell, astrocyte, or microglial cell. In some embodiments, the cell is in vitro. In some embodiments, the cell is from a subject having one or more symptoms of a neurodegenerative disease or suspected of having a neurodegenerative disease. In some embodiments, the cell expresses an ATXN2 having at least 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34 or more CAG trinucleotide (polyglutamine) repeats. In some embodiments, the cell expresses an ATXN2 having about 22 or 23 repeats, 24-32 repeats, or 33-100 or more repeats.

In another aspect, the present disclosure provides methods for inhibiting the expression or activity of ATXN2 in the central nervous system of a subject, comprising administering a composition of the present disclosure (e.g., inhibitory nucleic acid, isolated nucleic acid comprising an expression construct encoding an inhibitory nucleic acid, vector, rAAV particle, pharmaceutical composition) to the subject, thereby inhibiting the expression or activity of ATXN2 in the subject.

In another aspect, the present disclosure provides methods for treating a subject having or suspected of having a neurodegenerative disease, comprising administering a composition of the present disclosure (e.g., inhibitory nucleic acid, isolated nucleic acid comprising an expression construct encoding an inhibitory nucleic acid, vector, rAAV particle, pharmaceutical composition) to the subject, thereby treating the subject. As used herein, the term “treat” refers to preventing or delaying onset of neurodegenerative disease (e.g., ALS/FTD, Alzheimer's disease, Parkinson's disease, etc.); reducing severity of neurodegenerative disease; reducing or preventing development of symptoms characteristic of neurodegenerative disease; preventing worsening of symptoms characteristic of neurodegenerative disease, or any combination thereof.

Neurodegenerative diseases that may be treated in a subject using the compositions of the present disclosure include neurodegenerative diseases where ATXN2 is a causative agent (e.g., SCA2), as well as neurodegenerative diseases where ATXN2 is not the causative agent but modifies TDP-43 pathological aggregation. Neurodegenerative diseases associated with TDP-43 proteinopathy include ALS, FTD, primary lateral sclerosis, progressive muscular atrophy, limbic-predominant age-related TDP-43 encephalopathy, chronic traumatic encephalopathy, dementia with Lewy bodies, corticobasal degeneration, progressive supranuclear palsy (PSP), dementia Parkinsonism ALS complex of guam (G-PDC), Pick's disease, hippocampal sclerosis, Huntington's disease, Parkinson's disease, and Alzheimer's disease.

In some embodiments, the subject is characterized as having an ATXN2 allele having at least 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34 or more CAG trinucleotide (polyglutamine) repeats. In some embodiments, the subject is characterized as having an ATXN2 allele having about 22 or 23 repeats, 24-32 repeats, or 33-100 or more repeats.

In some embodiments, the methods for treatment of the present disclosure reduces, prevents, or slows development or progression of one or more symptom characteristic of a neurodegenerative disease. Examples of symptoms characteristic of neurodegenerative disease include motor dysfunction, cognitive dysfunction, emotional/behavioral dysfunction, or any combination thereof. Paralysis, shaking, unsteadiness, rigidity, twitching, muscle weakness, muscle cramping, muscle stiffness, muscle atrophy, difficulty swallowing, difficulty breathing, speech and language difficulties (e.g., slurred speech), slowness of movement, difficulty with walking, dementia, depression, anxiety, or any combination thereof.

In some embodiments, the methods for treatment of the present disclosure of the present disclosure comprise administration as a monotherapy or in combination with one or more additional therapies for the treatment of the neurodegenerative disease. Combination therapy may mean administration of the compositions of the present disclosure (e.g., inhibitory nucleic acid, isolated nucleic acid comprising an expression construct encoding an inhibitory nucleic acid, vector, rAAV particle, pharmaceutical composition) to the subject concurrently, prior to, subsequent to one or more additional therapies. Concurrent administration of combination therapy may mean that the the compositions of the present disclosure (e.g., inhibitory nucleic acid, isolated nucleic acid comprising an expression construct encoding an inhibitory nucleic acid, vector, rAAV particle, pharmaceutical composition) and additional therapy are formulated for administration in the same dosage form or administered in separate dosage forms.

In some embodiments, the one or additional therapies that may be used in combination with the inhibitory nucleic acids of the present disclosure include: inhibitory nucleic acids or antisense oligonucleotides that target neurodegenerative disease related genes or transcripts (e.g., C9ORF72), gene editing agents (e.g., CRISPR, TALEN, ZFN based systems) that target neurodegenerative related genes (e.g., C9ORF72), agents that reduce oxidative stress, such as free radical scavengers (e.g., Radicava (edaravone), bromocriptine); antiglutamate agents (e.g., Riluzole, Topiramate, Lamotrigine, Dextromethorphan, Gabapentin and AMPA receptor antagonist (e.g., Talampanel)); Anti-apoptosis agents (e.g., Minocycline, Sodium phenylbutyrate and Arimoclomol); Anti-inflammatory agents (e.g., ganglioside, Celecoxib, Cyclosporine, Nimesulide, Azathioprine, Cyclophosphamide, Plasmapheresis, Glatiramer acetate and thalidomide); Beta-lactam antibiotics (penicillin and its derivatives, ceftriaxone, and cephalosporin); Dopamine agonists (Pramipexole, Dexpramipexole); and neurotrophic factors (e.g., IGF-1, GDNF, BDNF, CTNF, VEGF, Colivelin, Xaliproden, Thyrotrophin-releasing hormone and ADNF).

In some embodiments, an inhibitory nucleic acid of the present disclosure is administered in combination with an additional therapy targeting C9ORF72. In some embodiments, the additional therapy targeting C9ORF72 comprises an inhibitory nucleic acid targeting C9ORF72 transcript, a C9ORF72 specific antisense oligonucleotide, or a C9ORF72 specific gene editing agent. Examples of C9ORF72 specific therapies are described in U.S. Pat. No. 9,963,699 (antisense oligonucleotides); PCT Publication No. WO2019/032612 (antisense oligonucleotides); U.S. Pat. No. 10,221,414 (antisense oligonucleotides); U.S. Pat. No. 10,407,678 (antisense oligonucleotides); U.S. Pat. No. 9,963,699 (antisense oligonucleotides); US Patent Publication US2019/0316126 (inhibitory nucleic acids); US Patent Publication No. 2019/0167815 (gene editing); PCT Publication No. WO2017/109757 (gene editing), each of which is incorporated by reference in its entirety.

In some embodiments, a subject treated in any of the methods described herein is a mammal (e.g., mouse, rat), preferably a primate (e.g., monkey, chimpanzee), or human.

In any of the methods of treatment described herein, a composition of the present disclosure (e.g., inhibitory nucleic acid, isolated nucleic acid comprising an expression construct encoding an inhibitory nucleic acid, vector, rAAV particle, pharmaceutical composition) may be administered to the subject by intrathecal, subpial, intraparenchymal, intrastriatal, intracranial, intracisternal, intra-cerebral, intracerebral ventricular, intraocular, intraventricular, intralumbar administration, or any combination thereof.

In some embodiments, a composition of the present disclosure (e.g., inhibitory nucleic acid, isolated nucleic acid comprising an expression construct encoding an inhibitory nucleic acid, vector, rAAV particle, pharmaceutical composition) is directly injected into the CNS of the subject. In some embodiments, direct injection into the CNS is intracerebral injection, intraparenchymal injection, intrathecal injection, intrastriatal injection, subpial injection, or any combination thereof. In some embodiments, direct injection into the CNS is direct injection into the cerebrospinal fluid (CSF) of the subject, optionally wherein the direct injection is intracisternal injection, intraventricular injection, intralumbar injection, or any combination thereof.

In some embodiments, the methods of the present disclosure reduces ATXN2 expression or activity in a cell by at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90% at least 95% or more in a cell compared to the expression level of ATXN2 in a cell that has not been contacted with the inhibitory nucleic acid. In some embodiments, the methods of the present disclosure reduces ATXN2 expression or activity in a cell by 10-20%, 10-30%, 10-40%, 10-50%, 10-60%, 10-70%, 10-80%, 10-90%, 10-95%, 20-30%, 20-40%, 20-50%, 20-60%, 20-70%, 20-80%, 20-90%, 20-95%, 20-100%, 30-40%, 30-50%, 30-60%, 30-70%, 30-80%, 30-90%, 30-95%, 30-100%, 40-50%, 40-60%, 40-70%, 40-80%, 40-90%, 40-95%, 40-100%, 50-60%, 50-70%, 50-80%, 50-90%, 50-95%, 50-100%, 60-70%, 60-80%, 60-90%, 60-95%, 60-100%, 70-80%, 70-90%, 70-95%, 70-100%, 80-90%, 80-95%, 80-100%, 90-95%, 90-100% compared to the expression level of ATXN2 in a cell that has not been contacted with the inhibitory nucleic acid.

In some embodiments, the methods of the present disclosure reduces ATXN2 expression or activity in the CNS of a subject by at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90% at least 95% or more in the CNS compared to the expression level of ATXN2 in the CNS of an untreated subject. In some embodiments, the methods of the present disclosure reduces ATXN2 expression or activity in the CNS of a subject by 10-20%, 10-30%, 10-40%, 10-50%, 10-60%, 10-70%, 10-80%, 10-90%, 10-95%, 20-30%, 20-40%, 20-50%, 20-60%, 20-70%, 20-80%, 20-90%, 20-95%, 20-100%, 30-40%, 30-50%, 30-60%, 30-70%, 30-80%, 30-90%, 30-95%, 30-100%, 40-50%, 40-60%, 40-70%, 40-80%, 40-90%, 40-95%, 40-100%, 50-60%, 50-70%, 50-80%, 50-90%, 50-95%, 50-100%, 60-70%, 60-80%, 60-90%, 60-95%, 60-100%, 70-80%, 70-90%, 70-95%, 70-100%, 80-90%, 80-95%, 80-100%, 90-95%, 90-100% compared to the expression level of ATXN2 in the CNS of an untreated subject.

EXAMPLES

Example 1: Design and Testing of siRNA Sequences to Knock Down Human ATAXIN-2

A number of criteria were used to select and design siRNA sequences to knock down ATXN2. The potential siRNA sequences that were initially considered included all possible 22-nucleotide RNAs complementary to ENST00000377617.7 (ATXN2-201). Human transcripts encoding for human Ataxin-2 were first examined. Only sequences found in all five of ATXN2 transcripts, NM_002973.3 (SEQ ID NO:2), ENST00000377617.7, ENST00000550104.5( ), ENST00000608853.5( ), and ENST00000616825.4( ), were selected.

The set of sequences was then filtered by cross-reactivity to the orthologous ATXN2 gene in rhesus and cynomolgous monkey. This allows the sequences to be tested in these species if needed to establish the activity and safety of gene therapies containing these inhibitory nucleic acid sequences prior to therapeutic use in humans. Thus, the sequence was also required to be in rhesus (Macaca mulatta) ATXN2 (NCBI Reference Sequences: XM_015152804.1, XM_015152805.1, XM_015152806.1, XM_015152807.1, XM_015152809.1, XM_015152810.1, XM_015152811.1, XM_015152812.1, XM_015152814.1, (Ensemble ID:) ENSMMUT00000062319, and ENSMMUT00000074794) and cynomolgous monkeys (Macaca fascicularis) ATXN2 (NCBI Reference Sequences: XM_005572266.2, XM_005572267.2, XM_015431532.1, XM_015431533.1, XM_015431534.1, XM_015431535.1, XM_015431536.1, XM_015431537.1, XM_015431538.1, XM_015431539.1, XM_015431540.1, XM_015431541.1, XM_015431542.1, XM_015431543.1, XM_015431544.1, XM_015431546.1, XM_015431547.1, XM_015431548.1, XM_015431549.1, XM_015431550.1, ENSMFAT00000019903.1). The ATXN2 transcript XM_015152813.1 of rhesus was also examined. This transcript was observed to be lacking a component of exon 1 and exon 2 (by comparison to human ATXN2 sequence SEQ ID NO:2). As described above for rhesus sequences, the following Macaca ATXN2 transcripts were identified to lack upstream sequence in exon 1: XM_015431551.1 and ENSMFAT00000019905.1. For these sequences, the exon 1 sequence was added back from human (SEQ ID NO:2) so as not to filter out that sequence. The nucleotide sequence in the ATXN2 gene encoding for the poly-glutamine repeat contains elements likely found elsewhere in the genome in other poly-glutamine repeat sequences. It is possible that automated transcript assignment algorithms, relying on alignment of RNAseq data, would mis-align sequencing reads overlapping with the poly-glutamine-encoding stretch (CAG repeating sequence) elsewhere in the genome, undercounting this sequence. These sequences in the upstream part of ATXN2 were therefore not excluded, except due to non-conservation from human to primate sequences.

Based on an analysis of brain RNAseq, exon 12 skipping is about 3% frequency, so this was not filtered out despite some alternative splice isoforms not including this isoform.

After defining the sequences expected to be present in human ATXN2 and key toxicology species, siRNAs were further selected based on criteria to reduce likelihood of off-target effects and to improve likelihood of strong ATXN2 knockdown. The seed sequences of both the antisense and sense strands of siRNAs, that is, bases 2-7 of the sequences which are known to be key determinants of activity of endogenous microRNAs, were examined for conservation in endogenous miRNAs expressed in human, mouse and rat. Antisense sequences present in any human endogenous miRNA were excluded, as were all sequences that were conserved in both mouse and rat. Sense sequences were excluded if seed regions were conserved in endogenous miRNAs present in more than 2 species out of human, mouse and rat.

A predicted knockdown ranking was calculated by adapting a version of an algorithm published in Pelossof et al. (Nature Biotechnology (2017) 35:350-353). Essentially, a support vector machine was trained on tiled sequencing data, provided in the publication. To generate the points in the space in which the support vector machine attempts to separate training examples which are labeled positive and negative, for good and bad knockdown respectively, features were selected as a weighted degree kernel. Features input to the support vector machine classifier were essentially the same as in Pelossof et al. For the SVM model, the “LibSVM” function from the Shogun module (version 6.1.3, Python version 2.7) was used instead of “SVMlite.”

The training set included 18,421 shRNA sequences from the genes PCNA, Trp53, Hras, Rpa3, Mcl1, hMyc, Myc, Bcl2, and Kras, all from the ‘TILE’ data set included in Pelossof et al. The TILE dataset empirically tests the performance of unbiased libraries of shRNAs covering sequences in the 9 genes described. The cost function c was assessed across a range of values training the SVM classifier on all genes except one of the nine left out, and calculating mean squared error on predictions for performance on data from the held-out gene. An example with Kras as the held out gene is shown (FIG. 1). A value of c=4 was selected which minimized the mean square error among values of c tested.

To further assess the performance of the classifier, knockdown data from another gene in the data set (Trp53) was held out after training the classifier on the other 8 genes. FIG. 2 shows a precision-recall curve for the classifier, as trained on data not including the Trp53 shRNAs, predicting performance of shRNA knockdown in the Trp53 targeting shRNAs. That is, after filtering shRNAs by a given classifier score, the fraction of of true positives identified by the classifier (recall) is plotted as a function of the number of true positives versus false positives (precision) (FIG. 2). Additionally, the anticipated cumulative fraction of ‘positive’ shRNAs (high performing) shRNAs that are expected to be lost as the classifier score was increased in stringency was plotted (FIG. 3), alongside the percent improvement in rejection of low-performing shRNAs. A separation in the curves was noted between scores of approximately −1.5 to −0.8, going from roughly the 25^th to 50^th percentiles of scores for Trp53 targeting shRNAs.

Next, siRNA sequences were triaged by specificity considerations, then ranked by the score from the above classifier. In addition to conservation of the seed sequences with endogenous miRNAs, as described above, metrics of specificity were: (a) comparison of seed sequences (guide bases 2-7) to a published data set of transfected siRNA seed sequences versus cell proliferation (Gaoao et al. Nature Communications (2018) 9:4504), excluding sequences with a >70% reduction of cell proliferation in the published assay; (b) the number of transcripts complementary to the first 19 nucleotides of the guide sequence, with 2 or fewer mismatches, was required to be less than 15; and (c) other considerations such as an internal algorithm of specificity were also factored in but triaged fewer samples than the criteria of (a) and (b).

Following filtering by specificity, sequences in the most common ATXN2 transcript, were ranked by SVM score and top-ranked candidate sequences selected. In calculating the SVM classifier score for shRNAs, however, it was found that the classifier score significantly increased for shRNAs beginning with U (FIG. 4). This was consistent with prior prediction algorithms (e.g., Vert et al., BMC Bioinformatics (2006) BMC Bioinformatics 7:520) and literature suggesting that the argonaute 2 binding pocket interacts best with this base, although guide base 1 does not base pair with the target mRNA (Boland et al., EMBO Reports (2010) 11:522-527). Therefore, for shRNA design, if the base was a ‘G’ or ‘C,’ based on complementarity to the target mRNA sequence, that base was replaced with a ‘U’ and the corresponding performance score calculated. The top 93 sequences beginning with A or U (SVM score >−0.8) and 34 sequences edited from a shRNA beginning with G or C, with a more stringent filter (SVM score >0.4).

Additional sequences were included for testing based on other criteria, including: (a) cross-reactivity with ATXN2L. ATXN2L shares considerable amino acid sequence similarity with ATXN2. Homologous genes often execute similar functions in a cell, and it is possible that knockdown of ATXN2L may serve similar therapeutic functions as knocking down ATXN2. Sequences which match both ATXN2 and ATXN2L may therefore have additional therapeutic benefit, and thus, 10 sequences were selected with potential to target both ATXN2 and ATXN2L; (b) sequences meeting a stringent off-target match criteria, with 2 or fewer transcripts matching at 2 or fewer positions in the first 19 nucleotides of the siRNA guide sequence (10 siRNAs), but ignoring SVM-based efficacy prediction; (c) sequences with perfect match or single mismatch to mouse ATXN2 in the first 19 nucleotides of the guide sequence. ‘Single mismatch’ guide sequences were defined as those where only one mismatch occurs between bases 12 and 19 nts against the mouse sequence, and none in bases 1-11. For guide sequences perfect-matching or single-mismatching mouse, the specificity criteria were relaxed, with guide sequences accepted with fewer than 50 complementary transcripts with 2 or fewer mismatches.

Selection of Cell Line to Screen siRNA Candidates

Following selection of siRNAs for testing, an in vitro cell system was established to assess knockdown of ATXN2 by siRNAs. ATXN2 levels were assessed by quantigene assay (Thermo Fisher), across a panel of cell lines (FIG. 5). The cell lines HepG2, KB, HT-29, LNCAP, C4-2 and Panc-1 all showed robust ATXN2 expression. To see if the splice patterns of cells were similar to that of ATXN2 in relevant target tissues, including in neurodegenerative disease states, RNAseq of postmortem human brain (Mayo Clinic Alzheimer's Disease Genetics Studies**; accessed via the synapse.org platform) was examined for splice patterns of ATXN2 and compared to data from cell lines (National Cancer Institute GDC Legacy Archive). In FIG. 6A, alternatively spliced exons were identified by reads crossing genomic regions that skip over the alternatively spliced exons. Exons 10, 21, and 24 in brain are frequently alternatively spliced. Examining alternative splicing in cell lines, HepG2 were similar to human brain (FIG. 6B). This line was selected for ATXN2 siRNA studies because of the high level of ATXN2 expression relative to background and consistent alternative splice patterns.

With regard to the synapse.org platform, study data were provided by the following sources: The Mayo Clinic Alzheimer's Disease Genetic Studies, led by Dr. Nilüfer Ertekin-Taner and Dr. Steven G. Younkin, Mayo Clinic, Jacksonville, Fla. using samples from the Mayo Clinic Study of Aging, the Mayo Clinic Alzheimer's Disease Research Center, and the Mayo Clinic Brain Bank. Data collection was supported through funding by NIA grants P50 AG016574, R01 AG032990, U01 AG046139, R01 AG018023, U01 AG006576, U01 AG006786, R01 AG025711, R01 AG017216, R01 AG003949, NINDS grant R01 NS080820, CurePSP Foundation, and support from Mayo Foundation. The following publications are applicable:

• [1] Carrasquillo et. al., Nat Genet. (2009) 41:192-8.
• [2] Zou et. al. PLoS Genet. (2012) 8(6):e1002707. [3] Allen et al. Sci Data. (2016) 3:160089.Synthesis and Testing of siRNAs

siRNAs were synthesized as 22 nucleotide RNAs, with 20 bp of complementarity (complementarity from positions 1-20, of guide and passenger strands). Here, guide strand refers to the sequence complementary to, or antisense to, the ATXN2 target mRNA, and passenger strand refers to the strand complementary to guide strand. Guide and passenger strands, also referred to as antisense and sense strand RNAs, are shown in Table 1. Sequences were synthesized as guide and passenger strands. All but 6 of the sequences met the following criteria: single strands within 0.05% of calculated mass (by LC/MS). At least 85% of full-length oligonucleotide purity (by HPLC). After annealing guide and passenger strands, duplex purity of >90% by non-denaturing HPLC. Oligonucleotides not meeting these criteria are noted as “FAIL,” but data are included for completeness.

Annealed siRNAs were reverse transfected, adding 20,000 cells per well of a 96-well plate, on top of a solution of lipofectamine 2000 with siRNA to yield a final siRNA concentration in the diluted culture media as noted below, in a volume of 0.5 microliters of transfection solution per well. siRNAs were tested in quadruplicate wells and incubated for 24 hours. ATXN2 and GAPDH levels were assayed in cell lysates by Quantigene assay using ATXN2 and GAPDH probes (Thermo Fisher). The ratio of ATXN2 mRNA levels to levels of the housekeeping gene GAPDH was calculated, and values were normalized to ATXN2/GAPDH ratios obtained for cells mock-treated with lipofectamine not containing siRNA.

All siRNAs were tested at doses of 20 nM or 1 nM (final calculated concentration of siRNA in cell culture media) for level of ATXN2 following knockdown (Table 4). A significant correlation, as assessed by a linear model fit, was observed plotting the predicted SVM score classifier against the 20 nM siRNA knockdown data (FIG. 8) (p<10⁻⁸, R²=0.15). Subsequently, the top ranked 100 siRNAs, by ATXN2 knockdown from 1 nM siRNA dosing data, were rescreened at 200 pM (Table 5). FIG. 7 plots the knockdown of ATXN2 mRNA for siRNAs as a function for position along the ATXN2 transcript that they transcript.

TABLE 4
siRNA Single Point Testing Data 20 nM-knockdown of ATXN2 mRNA
siRNA Duplex ID	20 nM_mean	20 nM_SD	1 nM_mean	1 nM_SD
XD-14738	84.1	6.8	91.1	3.6
XD-14739	88.6	4.7	96.8	6.9
XD-14740	87.1	3.4	82.9	3.4
XD-14741	56.4	2.3	54.6	3.2
XD-14742	44.5	2.5	44.7	3.5
XD-14743	44.9	2	48.1	3.5
XD-14744	62	2.9	65.7	2.1
XD-14745	42.5	3.9	47.3	3.5
XD-14746	65.4	3.9	62.5	2
XD-14747	43.2	2.4	41.2	1.8
XD-14748	52.4	1.6	49.3	1.9
XD-14749	60.2	3	57.4	1.3
XD-14750	52.3	3.3	53.5	1.2
XD-14751	47.7	4	54.6	1.1
XD-14752	78.4	3.3	76.4	6.4
XD-14753	47.7	2.4	55	1.1
XD-14754	47.5	8.4	50.6	2.8
XD-14755	79	3.6	73.3	3.9
XD-14756	37.6	5.2	46.8	7.8
XD-14757	32.5	1	39.7	0.4
XD-14758	29.6	0.9	35.3	5.2
XD-14759	43.6	4.3	51.8	5.5
XD-14760	45	1.3	52.3	3.6
XD-14761	40.2	1.8	54.7	4.1
XD-14762	39.4	2.5	47.8	2.3
XD-14763	39.2	3	50.6	1.7
XD-14764	80.9	4.4	81.2	8.7
XD-14765	43.1	2.9	49.5	5.1
XD-14766	30.3	0.7	33.1	3.6
XD-14767	43.6	5.6	38.5	2.5
XD-14768	35.9	3.3	40.9	2
XD-14769	72	7.6	78.7	2
XD-14770	50.7	7.6	56.9	3.1
XD-14771	59.8	6.5	77.4	17.7
XD-14772	81.1	2.1	80.2	1.1
XD-14773	44.2	3.1	52.1	5.2
XD-14774	81	7.1	74.8	12.1
XD-14775	42.3	4.3	44.3	2.6
XD-14776	50.3	2.2	45.7	2.7
XD-14777	51	5.4	53.2	1.9
XD-14778	71.2	5.2	69.2	6.1
XD-14779	36.5	2.8	34.7	4.3
XD-14780	121.4	8.7	97.2	6
XD-14781	52.7	5.9	69.7	3.3
XD-14782	46.3	2	61.1	3.2
XD-14783	44.6	2.1	56.9	2.8
XD-14784	49.8	1.3	64.4	2.7
XD-14785	48.1	8.7	61.4	4.5
XD-14786	45.1	2.5	44.7	6.5
XD-14787	43.3	7.4	48.2	2.8
XD-14788	34.8	1.4	41.7	4.7
XD-14789	44.6	0.8	62.3	2.8
XD-14790	31.3	0.8	36.7	2.6
XD-14791	29.5	1.5	38.9	2.9
XD-14792	31.2	2.6	38	0.9
XD-14793	38.3	1.7	47.9	2.9
XD-14794	34.7	1.4	38.6	1.3
XD-14795	36.4	5.4	47.9	4.1
XD-14796	91.9	7	91.1	8.3
XD-14797	61.9	1.2	74.9	17.3
XD-14798	31.2	2.3	33.9	2
XD-14799	32.5	2.1	39.8	3.3
XD-14800	30.4	1.4	43.3	6
XD-14801	32.8	3.4	36.2	0.9
XD-14802	56.3	1	70.5	2.4
XD-14803	45.2	1.3	56.8	5.2
XD-14804	74.3	3.6	76.2	5
XD-14805	47.2	3.5	50.1	3.5
XD-14806	71.4	5.8	75	4.4
XD-14807	42.4	2.4	52.7	3.6
XD-14808	42.8	2.6	53.1	5
XD-14809	47.4	3.3	49	2.8
XD-14810	39.7	1.7	48	1.4
XD-14811	38.9	4.8	46.3	0.9
XD-14812	40.8	4.9	47.1	2.3
XD-14813	57.8	5.9	59.5	2.3
XD-14814	103.2	29.6	81.5	2.9
XD-14815	54.	4.4	48.6	7
XD-14816	35.9	2.8	40.8	3.3
XD-14817	64	11.2	66.3	1.4
XD-14818	49.4	2.8	49.5	1
XD-14819	41.9	4	40.8	1.5
XD-14820	42.8	4.3	46	1.3
XD-14821	47.9	3.4	63	4.3
XD-14822	37.3	3.1	45.5	2.9
XD-14823	51.7	4.6	66.4	0.4
XD-14824	38.6	1.3	45.5	2.3
XD-14825	35.9	2.7	41.3	0.9
XD-14826	39.2	1.5	46.1	3
XD-14827	54.3 5.	2.7	62.3	1.4
XD-14828	63.8	1.9	77.1	3.5
XD-14829	40.1	0.6	38.2	8.1
XD-14830	36.7	0.7	46	0.9
XD-14831	42.5	2.9	61.8	3.4
XD-14832	71.3	2	95.6	1.5
XD-14833	56.5	2.8	75.1	2.3
XD-14834	38.3	1.6	47	2.6
XD-14835	30.6	2.4	38.5	1.6
XD-14836	44.5	3.2	58	2.7
XD-14837	29.2	3.4	36.2	0.5
XD-14838	36.5	2	46.1	1.2
XD-14839	32	2.9	42.5	4
XD-14840	29.3	2.3	34.1	7.6
XD-14841	32.9	2.5	41.2	3.7
XD-14842	38.8	3.4	45.6	2.5
XD-14843	32.9	1.2	40	1.6
XD-14844	88.5	4.9	84.6	4.8
XD-14845	72.5	9.4	67.6	1.9
XD-14846	29	1.7	34.6	3.4
XD-14847	34.1	1.2	38.6	2.5
XD-14848	40.4	0.5	46.3	3
XD-14849	58.5	2.1	67	1.4
XD-14850	34.7	0.6	38.7	0.7
XD-14851	46.6	3.4	47	0.9
XD-14852	61.4	2.4	55.9	2.6
XD-14853	47	3.7	42	2.4
XD-14854	43.3	5.3	40.8	4.9
XD-14855	45.5	1.6	43.7	2.1
XD-14856	43.9	3.3	40.7	5
XD-14857	39.5	5.3	36.1	2.3
XD-14858	63.8	4.7	54.1	1.1
XD-14859	39	3.6	40.3	1
XD-14860	35.5	3.2	36.2	1.6
XD-14861	35.8	2.9	42.3	2.2
XD-14862	85.3	2.6	84.2	7
XD-14863	56.3	6.3	52.6	3.4
XD-14864	46.3	3.1	47.2	2.3
XD-14865	77.5	2.6	79.6	4
XD-14866	52.6	4.4	53.2	7.4
XD-14867	70.6	4.6	56.7	5.4
XD-14868	79.8	3.4	72.5	3.8
XD-14869	93.1	3	89.4	3.7
XD-14870	60.9	0.9	65.1	4.2
XD-14871	86.1	1.6	94	8.6
XD-14872	93.1	3.6	91.8	5.5
XD-14873	94.4	2.3	90.2	4.6
XD-14874	60.7	4.3	56.9	7.5
XD-14875	50	1.3	49.5	2.7
XD-14876	53.5	15.9	51.7	4.1
XD-14877	55	4	61	1.4
XD-14878	49.6	1.5	47.4	2.2
XD-14879	65	2.2	64.9	2.3
XD-14880	47	1	44.5	1.8
XD-14881	90.3	4.7	90.6	2
XD-14882	39.7	1.5	40.1	5.1
XD-14883	58	1.4	65.9	5
XD-14884	62.7	2.2	67.9	6.1
XD-14885	46.6	3.3	53.8	6.6
XD-14886	39.5	1.4	44.4	7.5
XD-14887	38.2	0.4	41.7	5.1
XD-14888	61.4	2.1	68.9	4.9
XD-14889	31.2	8.8	40.7	2.7
XD-14890	36.3	1.9	37.3	2.7
XD-14891	45.2	4	46.8	2.1
XD-14892	49.1	2.8	62.5	1.7
XD-14893	41.7	2.3	49.7	6
XD-14894	47	2.7	64.9	3
XD-14895	53.3	4	67.2	2.3
XD-14896	45.1	1.3	66.8	2.4
XD-14897	74.3	8.9	87.7	4.6
XD-14898	68.4	5.8	73	4.8
XD-14899	44.3	6.4	55	1.2
XD-14900	45.6	2.8	47.8	1.6
XD-14901	47.9	12.1	41.4	1.7
XD-14902	56.1	6.3	52.8	2.5
XD-14903	52.1	4.2	46.6	2.7
XD-14904	47	3.7	36.6	8.4
XD-14905	40.6	2	40.6	3.2
XD-14906	47.1	4.6	50.7	3.3
XD-14907	57	4.1	51.1	2.7
XD-14908	58.8	6.3	51.1	4
XD-14909	47.1	2.8	50	2.6
XD-14910	46.9	2.8	53.5	9.2
XD-14911	61.7	3.6	55	4.3
XD-14912	58	3.8	59.3	3.9
XD-14913	72.1	5.5	73.3	6.4
XD-14914	68.9	4.7	66.3	5.7
XD-14915	47.4	5.1	59.1	9.5
XD-14916	43.1	5.7	55.6	9.4
XD-14917	43.4	4.6	42.7	4.3
XD-14918	64.2	2.3	67.4	5.7
XD-14919	62.5	6.6	61.8	3.8
XD-14920	76.1	1.8	67.6	4.2
XD-14921	64.4	3.9	73.9	7
XD-14922	52.2	6.9	66.7	7.5
XD-14923	48.7	2.6	56.3	3.8
XD-14924	47.6	1	51.8	2.1
XD-14925	51.7	3.7	56.5	6.2
XD-14926	43	3.9	54.9	3.2
XD-14927	53.3	6.2	64.3	8.2
XD-14928	61.7	1.7	70.4	3.8
XD-14929	54.7	3.9	64.4	2.8
XD-14930	50.1	3	63.6	5.5
XD-14931	52.9	3.9	62.4	4.9
XD-14932	62.6	3.7	69	3.8
XD-14933	62.2	5	66.7	3.3
XD-14934	59.7	7.6	62	4.8
XD-14935	60.5	4.1	65.9	7.5
XD-14936	54.4	2.6	71.3	4.7
XD-14937	61.2	5.5	74.1	2.9
XD-14938	65.8	6.6	71.9	3.7
XD-14939	61.9	7.8	73.8	2.6
XD-14940	68.8	6.8	79.6	2.2
XD-14941	57.8	4.1	69.7	3.1
XD-14942	75.3	9.3	85.4	4.4
XD-14943	69.8	4.2	77.7	6.7
XD-14944	84.1	4.3	84.2	8.5
XD-14945	53.5	3	55.2	2
XD-14946	58	1.8	56.9	3.7
XD-14947	59.2	2.2	59.2	1.3
XD-14948	56.8	4.4	53.4	2.4
XD-14949	51.7	2.5	52.4	2.3
XD-14950	63.2	2.2	69.6	4.3
XD-14951	53.9	2.7	62.6	3.8
XD-14952	40.3	0.9	51.3	6.2
XD-14953	46.6	2.5	56.1	4.1
XD-14954	56.1	8.6	55.9	6.6

TABLE 5
siRNA Single Point Testing Data 200 pM
	siRNA Duplex ID	0.2 nM_mean	0.2 nM_SD
	XD-14742	55.2	5.4
	XD-14743	49.9	3.2
	XD-14745	68.9	2.5
	XD-14747	59.3	5.8
	XD-14748	63.9	3.8
	XD-14754	75.9	3.2
	XD-14756	52	8.9
	XD-14757	57.2	6.7
	XD-14758	60.8	3.3
	XD-14759	60.3	3.4
	XD-14760	65.8	4.2
	XD-14762	61.7	7.7
	XD-14763	58.1	5.1
	XD-14765	72.6	6
	XD-14766	59.5	4.1
	XD-14767	66.2	3.5
	XD-14768	56.5	3.7
	XD-14773	73.1	4.5
	XD-14775	69.1	5.1
	XD-14776	62.5	1.8
	XD-14779	58.2	1.3
	XD-14786	51.1	1.4
	XD-14787	61.7	1.5
	XD-14788	58.1	4.7
	XD-14790	49.7	1
	XD-14791	51.2	4.3
	XD-14792	41.9	4.6
	XD-14793	62.2	8.4
	XD-14794	60.6	5.3
	XD-14795	75.9	7.3
	XD-14798	53.1	2.4
	XD-14799	60.4	2.2
	XD-14800	54.1	4
	XD-14801	59.7	13.7
	XD-14805	56.7	2.4
	XD-14807	66.9	7
	XD-14809	48.4	2.9
	XD-14810	54.5	4.6
	XD-14811	51.1	3.5
	XD-14812	62.3	6.6
	XD-14815	76.1	2
	XD-14816	71.8	1.7
	XD-14818	75.9	1.9
	XD-14819	57.2	2.6
	XD-14820	68.9	2.7
	XD-14822	63.7	0.7
	XD-14824	69.8	3.6
	XD-14825	52.5	2.2
	XD-14826	60.6	3.6
	XD-14829	67.7	4.5
	XD-14830	59	5.7
	XD-14834	67.7	2.8
	XD-14835	51.7	12.4
	XD-14837	63.4	10.1
	XD-14838	73.2	4
	XD-14839	65.7	1.9
	XD-14840	60.7	2.7
	XD-14841	65.5	2.2
	XD-14842	67.7	2
	XD-14843	76.7	9.6
	XD-14846	54.9	6.5
	XD-14847	69.6	1.7
	XD-14848	77.5	1.1
	XD-14850	74.4	3.2
	XD-14851	89.7	2.4
	XD-14853	77	3.2
	XD-14854	73	3.6
	XD-14855	82.4	4.2
	XD-14856	72.7	4.7
	XD-14857	54.4	3.6
	XD-14859	63.8	3.2
	XD-14860	52	2.3
	XD-14861	63.2	3.5
	XD-14863	72.3	3.1
	XD-14864	63.9	2.2
	XD-14875	63.1	2.9
	XD-14876	62.9	2.5
	XD-14878	66.1	3.8
	XD-14880	64.5	4.5
	XD-14882	52.7	2.8
	XD-14886	67.8	4.4
	XD-14887	63.1	2.3
	XD-14889	51.5	3.5
	XD-14890	52.3	0.6
	XD-14891	67.7	5.1
	XD-14893	51.7	2.4
	XD-14900	66.2	2.8
	XD-14901	55.3	1
	XD-14902	72.5	2.1
	XD-14903	69.5	2.5
	XD-14904	62.2	2.2
	XD-14905	63.9	3.9
	XD-14906	76.6	4.8
	XD-14907	77.2	8.1
	XD-14908	76.5	6.7
	XD-14909	75.1	7.7
	XD-14917	59.8	7.9
	XD-14924	68	5.5
	XD-14949	75.9	7.4
	XD-14952	75.8	5.2

Overall, the siRNA treatment data shows successful ATXN2 mRNA knockdown.

Confirmation of ATXN2 Protein Level Reduction by siRNA Treatment

To assess whether ATXN2 protein levels were also reduced by the informatically predicted siRNAs, 56 siRNAs were resynthesized (44 top ranked siRNAs by knockdown at 200 pM; 2 additional siRNAs near the top ranked, but having ATXN2L cross-reactivity (XD-14776) or mouse cross-reactivity (XD-14887) as characteristics which merited their re-testing; additional 10 siRNAs selected by a joint assessment of the ranking by knockdown at 20 nM dosed siRNA (from the top 55 ranked by knockdown), and also taking into account an informatic prediction of off-target likelihood. These siRNAs were synthesized to a reported purity of 80-85% (Dharmacon). As before, siRNAs were synthesized as 22 nucleotide guide and passenger strands, with a 20 nucleotide complementary sequence between guide base 1-20 and passenger bases 1-20, with 2 nucleotide 3′ overhangs on each strand, and introduced by transient transfection. Three additional controls were included. A non-targeting control (NTC) (Dharmacon, ON-Target plus Control Non-Targeting siRNA #1, D-001810-01-05) and a sequence targeting luciferase controlled for any nonspecific effects of siRNA treatment, including transfection reagents, on ATXN2 signal. For the luciferase control, sense sequence: GGAATTATAATGCTTATCTATA (SEQ ID NO:536); antisense sequence: TAGATAAGCATTATAATTCCTA (SEQ ID NO:537). A ‘SMARTPool’ (SMP), a combination of 4 siRNAs targeting ATXN2 (Dharmacon; ON-TARGETplus Human ATXN2 siRNA SMARTPool, L-011772-00-0005) was used as a positive control for specific targeting of ATXN2. Both the NTC and SMARTPool siRNAs are chemically modified to limit off-target effects.

An imaging based assay used indirect immunofluorescence signal by antibodies against ATXN2 to quantify ATXN2 levels. For these experiments U2OS cells were selected because of their large and uniform cell bodies, which permit good visualization of Ataxin-2 levels in the cytoplasm. siRNAs were introduced by transient transfection, and then 3 days later cells were fixed in paraformaldehyde, and then blocked and immunostained for Ataxin-2 and counterstained with Hoechst dye 33342 to identify cell nuclei.

Images were segmented using custom pipelines developed in Cell Profiler. First, cell nuclei are identified and outlined based on Hoechst 33342 signal. Subsequently, the nuclei outline is expanded to generate a ring. Within this ring, for each cell, the signal from the indirect immunofluorescence channel corresponding to a fluorescent secondary antibody binding to anti-Ataxin-2 is quantified. To calculate the ATXN2 signal for a well, the mean across cells in the well (typically 1000-3500 cells imaged/well) of cellular ATXN2 signal was calculated. The upper quartile ATXN2 signal within the cytoplasmic region was used. By taking the upper quartile of signal, this avoids the influence of signal from segmented regions of the image that may inadvertently not contain cells.

Cells were dosed with 20 or 1 nM siRNA in 96-well format, across multiple plates with controls in each plate. Background was subtracted by, within each imaging plate, wells stained with secondary antibody but not primary antibody, and not transfected. This reflects background intensity due to nonspecific binding of the secondary antibody. Ataxin-2 intensity values were normalized to those from wells transfected with non-targeting control (‘NTC’). From this, normalized ATXN2 signal represents a proxy for degree of protein level knockdown. Importantly, ATXN2 signal was similar for wells treated with luciferase targeting siRNA as with cells treated with NTC control. Note that the ‘NTC’ control (Dharmacon) chemistry is modified to reduce off-target effects whereas all ATXN2-targeting and luciferase-targeting siRNAs tested were unmodified. FIG. 9 quantifies knockdown of ATXN2 signal for siRNAs at 20 and 1 nM dose levels. FIGS. 10A and 10B show representative images from the knockdown experiments, with evidence of clear reduction of Ataxin-2 intensity from the indicated siRNAs. FIG. 11 plots the siRNAs protein knockdown data, at either 20 or 1 nM siRNA, as a function of ATXN2 transcript position. Almost all of these top siRNAs yielded substantial knockdown of siRNA at the protein level. At 1 nM, all of these top siRNAs exceeded the knockdown performance of the SMARTPool siRNA. Tables 6 and 7 display the mean and standard deviation of ATXN2 signal across wells. Sequences of the siRNAs from Tables 6 and 7 are provided in Table 1. For mean and SD calculations, outliers were excluded (outliers defined as wells where value deviated from the median value across wells by more than 1.5 standard deviations and by greater than 1000 normalized ATXN2 signal). Outlier wells are highlighted in FIG. 9.

TABLE 6
ATXN2 protein knockdown, measured by high content
imaging, after SiRNA treatment at 20 nM
		Mean	Standard
Treatment/	Dose	ATXN2	Deviation
Immunostain	in nM	Signal	ATXN2 Signal	N
no_primary_no_	20	−0.7	2.2	23
secondary
no_primary_	20	0	1.3	24
secondary
NTC	20	100	6.1	64
primary_no_	20	1.7	1.9	24
secondary
primary_secondary	20	92.5	7.2	46
SMP	20	23.4	5.8	61
XD-14742	20	13.6	3.5	4
XD-14743	20	20.7	4.9	4
XD-14747	20	11.4	1.9	3
XD-14756	20	6.9	3	3
XD-14757	20	18.2	6.9	4
XD-14758	20	14.6	2.3	4
XD-14759	20	11.1	3.2	4
XD-14762	20	13.7	5.9	4
XD-14763	20	7.4	5.7	4
XD-14766	20	15.7	5	4
XD-14768	20	11.6	3.9	4
XD-14775	20	14.1	3.3	4
XD-14776	20	15.9	1.7	4
XD-14779	20	13.2	1.5	3
XD-14786	20	19.8	4.9	3
XD-14787	20	12.7	6.7	4
XD-14788	20	16.7	7.4	4
XD-14790	20	17.9	8.1	4
XD-14791	20	15.6	5.4	4
XD-14792	20	12.5	9.1	4
XD-14793	20	17.4	4.4	4
XD-14794	20	14.5	4.2	4
XD-14798	20	15.9	4.7	3
XD-14799	20	14.5	1.8	4
XD-14800	20	13.1	5.7	3
XD-14801	20	9	6.1	4
XD-14805	20	22.2	4.3	4
XD-14809	20	44.2	4.9	4
XD-14810	20	24.8	6.4	4
XD-14811	20	29	6.3	4
XD-14819	20	27.1	9.1	4
XD-14822	20	29.4	4.6	4
XD-14825	20	19.9	5.2	4
XD-14826	20	22.2	7.5	4
XD-14830	20	11.7	5.4	4
XD-14834	20	7.5	4.3	4
XD-14835	20	6.2	3.5	3
XD-14837	20	13.3	4.8	4
XD-14839	20	23.4	0.9	4
XD-14840	20	19.7	4.2	4
XD-14841	20	25.3	2.7	4
XD-14843	20	16.8	2.8	4
XD-14846	20	11.2	2	3
XD-14847	20	8.9	2.8	4
XD-14850	20	21.6	3.1	4
XD-14857	20	16.8	7.4	4
XD-14860	20	8.7	4	3
XD-14882	20	14.2	1.3	3
XD-14887	20	19.2	4.2	4
XD-14889	20	16.2	2.6	4
XD-14890	20	13.8	4.9	4
XD-14893	20	24.7	12.4	4
XD-14901	20	17.7	4	4
XD-14904	20	15.9	5.8	4
XD-14905	20	13.6	5.7	3
XD-14917	20	18.7	5.9	4
XD-LucControl	20	101	4.8	3

TABLE 7
ATXN2 protein knockdown, measured by high content imaging, after
siRNA treatment at 1 nM
Treatment/	Dose	Mean ATXN2	Standard Deviation
Immunostain	in nM	Signal	ATXN2 Signal	N
no_primary_no_	1	−1.3	2.8	29
secondary
no_primary_secondary	1	0	2.7	30
NTC	1	99	17.1	78
primary_no_secondary	1	1.4	2.8	28
primary_secondary	1	97.9	10	62
SMP	1	50.1	7.1	78
XD-14742	1	27.2	5.5	5
XD-14743	1	18.1	3.3	4
XD-14747	1	21.7	3.8	5
XD-14756	1	18.2	5.5	4
XD-14757	1	26.7	3.6	5
XD-14758	1	19.8	5.2	5
XD-14759	1	16	3.2	5
XD-14762	1	18.3	2.3	5
XD-14763	1	20.7	3.8	5
XD-14766	1	19.7	2.9	5
XD-14768	1	25.5	7.6	5
XD-14775	1	28	2.5	4
XD-14776	1	25.4	4.7	5
XD-14779	1	26.8	4.8	5
XD-14786	1	24.4	4	4
XD-14787	1	23.2	4.6	5
XD-14788	1	25.7	5.7	4
XD-14790	1	22.9	5.6	5
XD-14791	1	25.5	5.9	5
XD-14792	1	18.6	5.7	5
XD-14793	1	37.6	4	5
XD-14794	1	23.4	4.7	5
XD-14798	1	20.2	6.1	4
XD-14799	1	29.5	3.7	5
XD-14800	1	22.6	1.8	4
XD-14801	1	16.2	4.5	4
XD-14805	1	35.4	2.5	5
XD-14809	1	40	8.5	5
XD-14810	1	40.7	4.3	5
XD-14811	1	42	14.5	4
XD-14819	1	36.6	0.7	4
XD-14822	1	31.5	5.6	5
XD-14825	1	28.2	3.4	5
XD-14826	1	32.7	6.5	4
XD-14830	1	26.6	4.6	5
XD-14834	1	19.7	4.9	5
XD-14835	1	20.3	4.9	5
XD-14837	1	19	4.6	5
XD-14839	1	21.6	3.1	4
XD-14840	1	20.7	5.4	5
XD-14841	1	29.8	4.9	5
XD-14843	1	26.3	2.5	4
XD-14846	1	23.1	5.8	5
XD-14847	1	15.1	2.9	5
XD-14850	1	26.4	8.2	5
XD-14857	1	26.1	3.8	5
XD-14860	1	18.5	4	5
XD-14882	1	23.5	4.9	5
XD-14887	1	22.3	4.2	5
XD-14889	1	24.4	7	5
XD-14890	1	21.6	2.4	4
XD-14893	1	17.3	2	3
XD-14901	1	25.6	2.9	4
XD-14904	1	19.9	2.1	5
XD-14905	1	26.5	2.5	5
XD-14917	1	19.2	2.7	5
XD-LucControl	1	114.6	12.1	5

Remarkably, 53 out of 56 of the ATXN2-targeting sequences achieved greater than 60% ATXN2 signal knockdown by this assay. In this assay, nonspecific antibody signal was not corrected. In subsequent assays (see below), ATXN2 knockout cells were used as controls demonstrating that some ATXN2 antibody background is present. Therefore, the ATXN2 protein level knockdown values here may underestimate the amount of protein knockdown caused by the ATXN2-targeting siRNA treatments.

Selection of Top-Ranked Sequences for Evaluation in siRNA Dose Response and in miRNA Backbones

To assess the potency of guide sequences targeting ATXN2, dose-response profiling of siRNAs and testing of guide sequences in miRNA format of 22 top sequences was conducted. To select top sequences for this detailed profiling, rankings of RNA knockdown for siRNAs at 20 nM and 200 pM were first assessed. In addition to this ranking of RNA knockdown, a method for predicting the number of off-target transcripts that would be influenced by the guide sequence was used, generating a probability of off-targeting score (POTS). https://sispotr.icts.uiowa.edu/sispotr/tools/lookup/evaluate.html) (Boudreau et al., Nucleic Acids Research 2013 41(1):e9). This score considers the seed sequence of the siRNA, and as such is supplementary to the initial assessment of off-target prediction based on the number of transcripts with 2 or fewer mismatches to the first 19 nucleotides of the guide sequence. Going down the knockdown ranks of siRNAs, sequences with increasingly stringent POTS score were favored. Additional criteria evaluated were: proximity to the region of ATXN2 complementarity for other guide sequences; re-examination of the number of transcripts closely complementary to nucleotides 2-19 were taken into account and resulted in the exclusion of two other sequences. The specific predicted off-targets were not examined for the selection of sequences for these experiments.

In addition to top-ranked sequences, two low-performing siRNAs (XD-14781 and XD-14949) that had low mRNA knockdown when assessed as siRNAs at 20 nM or 1 nM, were included to confirm the range and sensitivity of downstream assays.

siRNA Dose Response Versus ATXN2 mRNA Knockdown Testing

Dose response profiling was performed by testing dilution series of siRNAs transfected into HepG2 cells (FIG. 12), as described above for single-dose experiments. As expected, sequences XD-14781 (guide SEQ ID NO:90; passenger SEQ ID NO:89) and XD-14949 (guide SEQ ID NO:426; passenger SEQ ID NO:425), which had poor performance when assessed at 20 and 1 nM, had low potency and reduced maximal knockdown when assessed in dose response. IC50s of all other top-ranked siRNAs separated from these values. Two batches of testing were performed. Top sequences from one of the batches were estimated to have concentrations achieving half-maximal knockdown of <10 pM, indicating that the top-ranked siRNAs are highly potent. Performances of siRNAs had some dissimilarities between the batches but this was not investigated further, and the sequences were advanced into further testing in miRNA format. This miRNA testing, discussed below, showed that the lowest performing sequences from each batch were separated from the highest performing sequences in efficacy of ATXN2 protein lowering, but that the performance of top siRNAs from the two batches were similar. The miRNA testing is therefore regarded as more relevant for precise ranking of sequences.

Design and Production of ATXN2-Targeting Sequences in miRNA Backbones

Following identification of active siRNA sequences, siRNAs were embedded in miRNAs for expression from DNA vectors. The miR-155 and miR-1-1 backbones were considered.

The miR-155 was originally identified as a promising scaffold for construction of RNA polymerase II-based miRNA vectors due to its location within a conserved non-coding RNA⁸. After initial identification and design of miR-155 shRNA, subsequent sequence improvements increased microprocessor cleavage³. Many groups took the miR-155 scaffold to preclinical use in mice^10,11, sheep¹² and non-human primates¹³, enabling gene therapy approaches in genetically-driven human disease.

Initial experiments were conducted using a version of the miR-155 scaffold that, in one previous report, was engineered into an artificial mRNA and used in a mouse in vivo proof of concept study to knockdown HTT¹⁰. Small RNA sequencing had demonstrated high strand bias by this miRNA backbone¹⁰. ATXN2 targeting guide sequences and controls were incorporated into this scaffold sequence, which was termed “mR-155M” and assayed for protein knockdown after transfection of U2OS cells.

To rationally improve miR-155, human genomic sequence was examined, and the span of flanking miR-155 sequence to be used was defined by the region surrounding miR-155 with high evolutionary conservation across similar species. That is, a plot of sequence conservation versus position was visualized, and the genomic position from the endogenous miR-155 at which this sequence conservation dropped off was used to determine how much flanking context around the miR-155 stem structure should be included. Next, the mIR-155 loop was examined for features which might impact the use of this miR in different expression systems. A homotetrameric UUUU in the miR-155 loop was noted. UUUU sequences have been reported to induce Polymerase III termination¹⁴, which would lead to aberrantly truncated miRNAs which do not undergo stem pairing. To interrupt this homotetrameric UUUU, an apical UGU motif within the miR-155 loop was added. This motif additionally has been reported to enhance miRNA processing.^1,2 In addition to previously engineered UG and CNNC motifs³, a basal stem mismatched GHG motif² was added to improve precise processing.

To expand the number of amiRNA scaffolds beyond the miR-155 backbones, backbones from endogenous miRNAs reported to have high processing precision were prioritized. The miR-1-1 backbone ranks among the highest in processing precision according to reference:¹⁵, has high strand bias by small RNAseq⁵, and the guide strand is on the 3 prime arm of the miRNA stem, which may improve processing accuracy compared to 5 prime-arm positioned guide strands¹⁶. Natively integrated favorable sequence motifs include a basal mismatched GHG motif and downstream CNNC motif. It also has a short context for sequencing and has been successfully engineered for artificial miRNA expression in Drosophila models¹⁷.

Additional miRNA scaffolds that may be considered for the amiRNAs of the present disclosure include:

• • miR-100 and miR-190a—high throughput screen identified high on-target/off-target ratio¹⁵.
  • miR-124 and miR-132—both motor-neuron expressed miRNAs do not change expression in an ALS rat model¹⁸. The cell-type specific expression and consistent levels throughout ALS disease course are favorable miRNA characteristics. Neuronal specificity has been confirmed in a sRNAseq cross-tissue expression database¹⁹ (https://ccb-web.cs.uni-saarland.de/tissueatlas/).
  • miR-9—neuron-specific expression²⁰.
  • miR-138-2, miR-122, miR-130a, and miR-128 were selected to be naturally asymmetric (either exclusively 5′ or 3′ strand is observed in small RNAseq datasets), highly homogeneous (i.e. high “5′ homogeneity score”¹⁵), not reported to undergo post-transcriptional regulation (e.g. which occurs for clustered miRNAs), are consensus miRNAs on miRBase, have flexible loop structure and simple duplex stem.

To further mimic the miRNA backbones, bulges and mismatches can be inserted into the guide:passenger strand duplex in a manner to replicate the bulge pattern observed in endogenous miRNAs, but applied to artificial miRNAs targeting ATXN2. The modifications that can be done to the passenger strand to introduce these native-miRNA mimicking structures are provided in Table 8.

TABLE 8
Design Rules for Exemplary miRNA formats
miR      Modifications to passenger sequence
miR-1-1  Mismatch base 2 | Bulge mismatch transversion base 19 |
         Insert a base between bases 18 and 19, either G or C but
         not complementary to the guide
miR155   Delete bases 9 and 13 of passenger
(M or E)
miR100   Bulge mismatch transversion base 11 | Bulge mismatch
         transition bases 7, 18 | add GU wobble base 17 | Mismatch
         base 22
miR124   Mismatch base 3 | Bulge mismatch transition bases 2, 17 |
         Bulge mismatch transversion 12, 16
miR138-2 Mismatch bases 5, 21 | Bulge mismatch bases 6 | Add GU
         wobble base 13 | delete bases 7, 22
miR122   Bulge mismatch transition base 12 | Bulge mismatch
         transversion bases 20, 21
miR-128  Truncate guide to 21 bp | Mismatch bases 1, 2, 12 | Bulge
         mismatch transversion bases 3, 11, 13 | Insert 2 bases
         between bases 20 and 21, not complementary to the guide |
         add GU wobbles bases 5, 7
miR130a  Mismatch base 2 | Add GU wobbles bases 18, 19 | bulge
         mismatch transversion base 11 | Bulge mismatch transition
         base 22
miR 16-2 Bulge mismatch transversion bases 11, 12 | Mismatch base
         22
Note:
For the above, ‘passenger’ sequence refers to a sequence complementary to the 22 nucleotides of the guide sequence. This is not the same as passenger sequences as used in describing siRNA duplexes. Mismatch refers to the following substitution rule: G → C, C → G, A → T, T → A. Bule mismatch transition refers to the rule: T → C, C → A, A → C, G → A. Bulge mismatch transversion refers to the rule: G → T, C → A, A → C, T → G. Add GU wobble refers to the rule: If base is C, then convert to T.

Initial Testing of ATXN2 Targeting Guide Sequences in miR155-M and miR1-1 Backbones

As an initial test of the ability of the Atxn2 targeting siRNAs to knock down Atxn2 when embedded in a miRNA context, the guide sequence of XD-14792 (SEQ ID NO:112), which had the highest ranked ATXN2 mRNA knockdown when dosed at 200 pM as an siRNA, was embedded in several miRNA contexts as shown in Table 9. The amiRNA DNA sequences are provided in Table 9 as SEQ ID NOS:538-543. The corresponding amiRNA RNA sequences are provided in Table 9 as SEQ ID NOS:1109-1114, respectively.

TABLE 9
XD-14792 sequences embedded in amiRNAs
	Target/	Guide		miR	Artificial miRNA	Artificial miRNA
Name	Type	Sequence	Variation	Backbone	(DNA Sequence)	(RNA Sequence)
XD-14792_miR1-1	ATXN2	AUUAACUACUCUU		1-1	CATGCAGACTGCCTGCTTGGGTA	CAUGCAGACUGCCUGCUUGGGU
		UGGUCUGAA			CAGACCAAAGAGTAGTCGAATTA	ACAGACCAAAGAGUAGUCGAAU
		[SEQ ID NO: 112]			TGGACCTGCTAAGCTAATTAACT	UAUGGACCUGCUAAGCUAAUUA
					ACTCTTTGGTCTGAACTCAGGCC	ACUACUCUUUGGUCUGAACUCA
					GGGACCTCTCTCGCCGCACTGAG	GGCCGGGACCUCUCUCGCCGCAC
					GGGCACTCCACACCACGGGGGCC	UGAGGGGCACUCCACACCACGG
					[SEQ ID NO: 538]	GGGCC
						[SEQ ID NO: 1109]
XD-14792_miR1-1	ATXN2	AUUAACUACUCUU	E	1-1	CATGCAGACTGCCTGCTTGGGTA	CAUGCAGACUGCCUGCUUGGGU
enhanced		UGGUCUGAA			CAGACCAAAGAGTAGTCGAATTA	ACAGACCAAAGAGUAGUCGAAU
		[SEQ ID NO: 112]			TGGACCTGCTAAGCTAATTAACT	UAUGGACCUGCUAAGCUAAUUA
					ACTCTTTGGTCTGAACTCAGGCC	ACUACUCUUUGGUCUGAACUCA
					GGGACCTCTTCCGCCGCACTGAG	GGCCGGGACCUCUUCCGCCGCAC
					GGGCACTCCACACCACGGGGGCC	UGAGGGGCACUCCACACCACGG
					[SEQ ID NO: 539]	GGGCC
						[SEQ ID NO: 1110]
XD-14792_miR155	ATXN2	AUUAACUACUCUU		155	CCTGGAGGCTTGCTGAAGGCTGT	CCUGGAGGCUUGCUGAAGGCUG
		UGGUCUGAA			ATGCTGATTAACTACTCTTTGGTC	UAUGCUGAUUAACUACUCUUUG
		[SEQ ID NO: 112]			TGAATTTTGGCCACTGACTGATTC	GUCUGAAUUUUGGCCACUGACU
					AGACCAAGGTAGTTAATCAGGAC	GAUUCAGACCAAGGUAGUUAAU
					ACAAGGCCTGTTACTAGCACTCA	CAGGACACAAGGCCUGUUACUA
					CATGGAACAAATGGCCACCGG	GCACUCACAUGGAACAAAUGGC
					[SEQ ID NO: 540]	CACCGG
						[SEQ ID NO: 1111]
XD14792_911_miR155	ATXN2	AUUAACUAGAGUU		155	CCTGGAGGCTTGCTGAAGGCTGT	CCUGGAGGCUUGCUGAAGGCUG
	911	UGGUCUGAA			ATGCTGATTAACTAGAGTTTGGT	UAUGCUGAUUAACUAGAGUUUG
	Control	[SEQ ID NO: 544]			CTGAATTTTGGCCACTGACT	GUCUGAAUUUUGGCCACUGACU
					GATTCAGACCAACCTAGTTAATC	GAUUCAGACCAACCUAGUUAAU
					AGGACACAAGGCCTGTTACTAGC	CAGGACACAAGGCCUGUUACUA
					ACTCACATGGAACAAATGGC	GCACUCACAUGGAACAAAUGGC
					CACCGG	CACCGG
					[SEQ ID NO: 541]	[SEQ ID NO: 1112]
XD-14792_SScr	ATXN2	AUUAACUAAGUAU		155	CCTGGAGGCTTGCTGAAGGCTGT	CCUGGAGGCUUGCUGAAGGCUG
miR155	SScr	CGGUCUCUU			ATGCTGATTAACTAAGTATCGGT	UAUGCUGAUUAACUAAGUAUCG
		[SEQ ID NO: 545]			CTCTTTTTTGGCCACTGACTGAAA	GUCUCUUUUUUGGCCACUGACU
					GAGACCATATTAGTTAATCAGGA	GAAAGAGACCAUAUUAGUUAAU
					CACAAGGCCTGTTACTAGCACTC	CAGGACACAAGGCCUGUUACUA
					ACATGGAACAAATGGCCACCGG	GCACUCACAUGGAACAAAUGGC
					[SEQ ID NO: 542]	CACCGG
						[SEQ ID NO: 1113]
XD-14792_miR155	ATXN2	AUUAACUACUCUU	S	155	CCTGGAGGCTTGCTGAAGGCTGT	CCUGGAGGCUUGCUGAAGGCUG
sealed		UGGUCUGAA			ATGCTGATTAACTACTCTTTGGTC	UAUGCUGAUUAACUACUCUUUG
		[SEQ ID NO: 112]			TGAATTTTGGCCACTGACTGATTC	GUCUGAAUUUUGGCCACUGACU
					AGACCAAAGAGTAGTTAATCAGG	GAUUCAGACCAAAGAGUAGUUA
					ACACAAGGCCTGTTACTAGCACT	AUCAGGACACAAGGCCUGUUAC
					CACATGGAACAAATGGCCACC	UAGCACUCACAUGGAACAAAUG
					[SEQ ID NO: 543]	GCCACC
						[SEQ ID NO: 1114]
In the variation column: “E” refers to “enhanced,” “S” refers to ‘sealed’

In Table 9, the guide sequences (including the guide sequence, any variants, as well as the parental guide sequence from which they are derived) are shown in RNA form, and the artificial miR sequence is provided in both RNA format, and for when embedded in the vector is shown in DNA form. The miR backbones used include: (a) miR155, preserving a bulge format reported in (Fowler et al., Nucleic Acids Res. (2015) 44:e48); (b) miR155, with no sequence bulges, yielding a perfectly complementary stem (“sealed”); (c) miR1-1, preserving a native bulge format as in the endogenous miRNA; and (d) miR1-1 with the “Enhanced” variation, including a modification in the 3′ arm that in other miRNAs was previously reported to enhance processing (Auyeung et al., Cell 2013). FIG. 13 shows one of the predicted RNA folds of the miRNA stems of several of the constructs, using the web server mfold. Bulges in the stem in the region including or apposed to the guide sequence are apparent, which are designed to mimic the native mismatches of the endogenous forms of the microRNAs from which derive the surrounding context for the guide sequence. As controls (“911 controls”), bases 9, 10, and 11 of XD-14792 guide sequence were modified to be the complementary bases (that is, substituting A->T, T->A, C->G, or G->C); or (“SScr”), in which all bases except bases 1-7 were scrambled. In both cases, any seed-mediated off-target activity (deriving from bases 1-7) should be preserved, whereas the on-target Atxn2 slicing activity should be blocked.

pLVX-EF1A_mCherry-miR-1-1-XD_14890-WPRE_CMV (SEQ ID NO:546) is a representative lentiviral vector that can be used for expressing these artificial microRNAs. Nucleotides 4275-4412 of SEQ ID NO:546 (XD-14890 guide sequence in a miR-1-1 backbone) can be substituted with another artificial miRNA of interest. In this lentiviral vector suitable for packaging into lentivirus, an EF1-alpha promoter drives expression of a mCherry protein. After a stop codon, the amiRNA stem is expressed downstream within a 3′ UTR. Downstream of that a WPRE element (Woodchuck Hepatitis Virus Posttranscriptional Regulatory Element) enhances the stability of the transcript. Adapters may be included upstream or downstream of the artificial miRNA construct to facilitate cloning and downstream detection of the sequences, but these adapters are not expected to influence the performance of the microRNA. A CMV promoter (as in sequence shown), or a PGK promoter (as in plasmids transfected for data shown FIG. 14), downstream, drives expression of the puromycin resistance protein for puromycin selection in mammalian cells. This is a similar design to the vector used in (Kampmann et al., PNAS 2015).

pcDNA3.1 NEGFP STOP ATXN2 3′UTR.gb (SEQ ID NO:547) represents a plasmid used to generate a GFP-ATXN2 reporter line. A CMV promoter was used to drive the expression of a transcript encoding enhanced green fluorescent protein (EGFP). A stop codon at the end of the EGFP open reading frame was followed by the ATXN2 sequence, but removing the initial ATG such that the sequence is expected to not be translated. A separate SV40 promoter downstream drives the expression of the NeoR/KanR protein product which enabled selection of U2OS cells stably integrating the plasmid by G418 selection. EGFP fluorescence was bright and diffuse, and not restricted to the cytoplasm as expected if the ATXN2 protein was translated and fused to the EGFP. Several lines were generated by single-cell cloning after G418 selection, and one line ultimately selected based on uniform fluorescent signal distribution by FACS as well as a larger differential between control-transfected (siNTC) and ATXN2 siRNA-transfected cells.

Constructs with the artificial miRNAs noted above were transfected into U2OS cells stably expressing the GFP-Atxn2 reporter by transient transfection (lipofectamine 3000). Four days later, GFP-ATXN2 levels were quantified by fluorescence automated cell sorting (FACS), gating cells by the expression of the mCherry encoded on the miRNA vector to isolate cells expressing the artificial miRNA construct. FIG. 14 shows median fluorescence intensity signal of GFP intensity. XD-14792 sequences embedded in artificial miRNA backbones miR-155 or miR-1-1 considerably reduced ATXN2 GFP reporter intensity relative to cells expressing control constructs (XD-14792 911 and XD-14792 Sscr, embedded in the miR-155 stem backbone). A ‘sealed’ XD-14792 construct in a miR-155 backbone, in which the stem is perfectly complementary (FIG. 14) did not reduce the ATXN2 GFP reporter signal as much as did XD-14792 when embedded in either miR-155 or miR-1-1 with bulged residues.

Expanded Screening of ATXN2 Targeting Sequences in Artificial microRNA Vectors in Lentiviral Format

Given the encouraging results with the knockdown of the ATXN2 GFP reporter, a set of ATXN2 targeting sequences was cloned into the artificial microRNA expressing vector described above (SEQ ID NO:546). The same set of ATXN2 targeting sequences as were tested in dose-response testing for mRNA knockdown were incorporated into plasmids to enable lentiviral packaging. Vectors were packaged into lentivirus (see methods below) and transduced into unmodified U2OS cells or U2OS cells deficient for ATXN2 (described below) in a 96-well format, across multiple plates. Each plate had controls to enable plate-wise signal normalization. 3.5 days after transduction, cells were fixed with paraformaldehyde, blocked and stained with anti-ATXN2 antibodies, anti-mCherry antibodies, and Hoechst dye (33342) to demarcate cellular nuclei, and ATXN2 signal was quantified by image segmentation and signal intensity measurement as described above. Transduced and untransduced cells were differentiated by anti-mCherry signal. FIG. 15 shows histograms of the expected mCherry signal for untransduced cells as well as wildtype transduced cells. The threshold was set such that the signal from untransduced wild-type cells did not exceed this threshold, but most of the cells in the right peak of the bimodally distributed signals (right panel, wild-type transduced cells) were considered positive.

ATXN2 signal was subtracted for background measured in U2OS cells with the ATXN2 gene disrupted by CRISPR and in which ATXN2 protein had been verified to be eliminated by Western analysis. FIGS. 20-21 show the data for the knockout generation process. FIG. 20 shows Western and FACS analysis of Ataxin-2 signal in cells nucleofected with Cas9-gRNA complexes targeting Ataxin-2 or control targets. Robust reduction of Ataxin-2 protein is seen with multiple guides, consistent with editing and disruption of the Ataxin-2 gene. FIG. 21A shows the workstream to generate clonal ATXN2 knockout cells, and FIG. 21B shows Western analysis of single-cell clones derived from Cas9-gRNA nucleofected cells, from which clone 43 was confirmed to be null for Ataxin-2 and selected for further use. The clone was sequenced by Sanger sequencing, and using the ICE tool (Synthego), a mixture of disrupting mutations consistent with disruption of the ATXN2 alleles was confirmed.

As shown in FIG. 16, the signal in ATXN2 deleted cells was slightly increased relative to wild-type cells that were treated with secondary antibody but not primary anti-Ataxin-2 antibody, suggesting some nonspecific, background binding of the ATXN2 antibody. These cells were not transduced with virus. After background subtraction, signal was normalized relative to ATXN2 signal in untransduced wild-type cells.

FIG. 17 shows well-level quantification of ATXN2 signal intensities across artificial microRNA constructs, with representative images shown in FIG. 18. Transduced cells were identified by anti-mCherry levels exceeding the threshold defined above. A median of 3355 cells per well were mCherry positive and included for ATXN2 signal calculation, with a range of 2469-4582 cells and standard deviation of 391 cells per well.

Table 10 shows mean and standard deviations of ATXN2 signals, normalized as above, for sequences, embedded either in the enhanced miR-155 backbone or the miR1-1 backbone (sequences provided in Table 11). In general, for most but not all sequences, ATXN2 knockdown performance was superior when the guide sequence was embedded in the miR1-1 backbone. None of the 911 controls, where the artificial microRNA was engineered such that guide bases 9, 10 and 11 were complemented (A->T, T->A, C->G, or G->C), exhibited knockdown, indicating that the reduction in ATXN2 signal is dependent on the direct RNA interference activity of the microRNAs on the endogenous ATXN2 transcript. Additionally, protein level knockdown across guide sequences, when examined in the miR-1-1 backbone, correlated with mRNA knockdown in HepG2 cells after 200 pM siRNA treatment. (linear model p<0.001; R²=0.5; FIG. 19).

TABLE 10
ATXN2 protein levels following amiRNA treatment
Duplex	miR	Mean normalized	Standard
ID/Category	Backbone	ATXN2 Signal	Deviation
XD-14790 911	miR-1-1	111.7	15.9
XD-14790 911	miR-155E	116.8	5.4
XD-14800 911	miR-1-1	113.3	10.1
XD-14800 911	miR-155E	111.1	8
XD-14857 911	miR-1-1	112.7	11.5
XD-14857 911	miR-155E	116.7	10.1 1
XD-14742	miR-1-1	54.4	1.3
XD-14742	miR-155E	89.8	10.7
XD-14743	miR-1-1	39.8	2
XD-14743	miR-155E	89.9	8.1
XD-14756	miR-1-1	44.9	5.4
XD-14756	miR-155E	73.2	7.3
XD-14766	miR-1-1	55.4	1.2
XD-14766	miR-155E	53.3	5.1
XD-14781	miR-1-1	79.2	10.3
XD-14781	miR-155E	100.8	14.4
XD-14786	miR-1-1	45	1.7
XD-14786	miR-155E	77	11.4
XD-14787	miR-1-1	83.8	6.9
XD-14787	miR-155E	103.4	12
XD-14788	miR-1-1	69.7	9.8
XD-14788	miR-155E	98	3.8
XD-14790	miR-1-1	43.2	7.6
XD-14790	miR-155E	56.3	7.6
XD-14792	miR-1-1	37.9	4.8
XD-14792	miR-155E	51.5	9.9
XD-14798	miR-1-1	60.8	2.6
XD-14798	miR-155E	66.8	3
XD-14799	miR-1-1	73.5	6.7
XD-14799	miR-155E	84.9	8.9
XD-14800	miR-1-1	41	6.4
XD-14800	miR-155E	78.1	5.7
XD-14819	miR-1-1	46.6	7.1
XD-14819	miR-155E	63	7.5
XD-14835	miR-1-1	41.3	7.4
XD-14835	miR-155E	67.3	21.5
XD-14846	miR-1-1	69.3	9.3
XD-14846	miR-155E	97.5	5.6
XD-14857	miR-1-1	41.2	6.3
XD-14857	miR-155E	65.3	8.9
XD-14887	miR-1-1	71.8	11.7
XD-14887	miR-155E	87.8	16.3
XD-14889	miR-1-1	62.5	8.8
XD-14889	miR-155E	62.6	3.7
XD-14890	miR-1-1	24.8	7.2
XD-14890	miR-155E	83.8	9.2
XD-14901	miR-1-1	61.8	8.3
XD-14901	miR-155E	84	4.7
XD-14904	miR-1-1	44.2	8.3
XD-14904	miR-155E	55.2	9.9
XD-14917	miR-1-1	38.5	8.1
XD-14917	miR-155E	67.2	3.8
XD-14949	miR-1-1	106.8	9.7
XD-14949	miR-155E	55.2	6.7

Table 11 provides the parent guide RNA sequences, amiRNA sequences, and amiRNA DNA sequences as embedded in microRNA backbone-expressing vectors of both active guide sequences as well as a small set of control sequences. The guide sequence anticipated to be produced in cells is described in RNA form, and the sequence encoding the guide sequence (embedded in miRNA) is provided in DNA form.

TABLE 11
amiRNA Sequences
		miR
Parent Guide	ID	Backbone	Category	amiRNA DNA Sequence	amiRNA RNA Sequence
AGGAACGUGGGUU	XD-14857	miR-1-1	911 Control	CATGCAGACTGCCTGCTTGGGATG	CAUGCAGACUGCCUGCUUGGGAU
GAACUCCUU [SEQ				GAGTTCAAGGGACGTCGCCTTATG	GGAGUUCAAGGGACGUCGCCUUA
ID NO: 242]				GACCTGCTAAGCTAAGGAACGTCC	UGGACCUGCUAAGCUAAGGAACG
				CTTGAACTCCTTCTCAGGCCGGGA	UCCCUUGAACUCCUUCUCAGGCCG
				CCTCTCTCGCCGCACTGAGGGGCA	GGACCUCUCUCGCCGCACUGAGG
				CTCCACACCACGGGGGCC	GGCACUCCACACCACGGGGGCC
				[SEQ ID NO: 548]	[SEQ ID NO: 1115]
AGGAACGUGGGUU	XD-14857	miR-155E	911 Control	CTGGAGGCTTGCTTTGGGCTGTAT	CUGGAGGCUUGCUUUGGGCUGUA
GAACUCCUU [SEQ				GCTGAGGAACGTCCCTTGAACTCC	UGCUGAGGAACGUCCCUUGAACU
ID NO: 242]				TTTTTTGGCCTCTGACTGAAAGGA	CCUUUUUUGGCCUCUGACUGAAA
				GTTAAGGACGTTCCTCAGGACAAG	GGAGUUAAGGACGUUCCUCAGGA
				GCCCTTTATCAGCACTCACATGGA	CAAGGCCCUUUAUCAGCACUCAC
				ACAAATGGCCACCGTG	AUGGAACAAAUGGCCACCGUG
				[SEQ ID NO: 549]	[SEQ ID NO: 1116]
UUCGGGUUGAAAU	XD-14790	miR-155E	911 Control	CTGGAGGCTTGCTTTGGGCTGTAT	CUGGAGGCUUGCUUUGGGCUGUA
CUGAAGUGU [SEQ				GCTGTTCGGGTTCTTATCTGAAGTG	UGCUGUUCGGGUUCUUAUCUGAA
ID NO: 108]				TTTTTGGCCTCTGACTGAACACTTC	GUGUUUUUGGCCUCUGACUGAAC
				AATAGAACCCGAACAGGACAAGG	ACUUCAAUAGAACCCGAACAGGA
				CCCTTTATCAGCACTCACATGGAA	CAAGGCCCUUUAUCAGCACUCAC
				CAAATGGCCACCGTG	AUGGAACAAAUGGCCACCGUG
				[SEQ ID NO: 550]	[SEQ ID NO: 1117]
UUCGGGUUGAAAU	XD-14790	miR-1-1	911 Control	CATGCAGACTGCCTGCTTGGGAGA	CAUGCAGACUGCCUGCUUGGGAG
CUGAAGUGU [SEQ				CTTCAGATAAGAACCGAGAATATG	ACUUCAGAUAAGAACCGAGAAUA
ID NO: 108]				GACCTGCTAAGCTATTCGGGTTCTT	UGGACCUGCUAAGCUAUUCGGGU
				ATCTGAAGTGTCTCAGGCCGGGAC	UCUUAUCUGAAGUGUCUCAGGCC
				CTCTCTCGCCGCACTGAGGGGCAC	GGGACCUCUCUCGCCGCACUGAG
				TCCACACCACGGGGGCC	GGGCACUCCACACCACGGGGGCC
				[SEQ ID NO: 551]	[SEQ ID NO: 1118]
UUGAUUUCGAGGA	XD-14800	miR-155E	911 Control	CTGGAGGCTTGCTTTGGGCTGTAT	CUGGAGGCUUGCUUUGGGCUGUA
UGUCGCUGG [SEQ				GCTGTTGATTTCCTCGATGTCGCTG	UGCUGUUGAUUUCCUCGAUGUCG
ID NO: 128]				GTTTTGGCCTCTGACTGACCAGCG	CUGGUUUUGGCCUCUGACUGACC
				ACTCGGGAAATCAACAGGACAAG	AGCGACUCGGGAAAUCAACAGGA
				GCCCTTTATCAGCACTCACATGGA	CAAGGCCCUUUAUCAGCACUCAC
				ACAAATGGCCACCGTG	AUGGAACAAAUGGCCACCGUG
				[SEQ ID NO: 552]	[SEQ ID NO: 1119]
UUGAUUUCGAGGA	XD-14800	miR-1-1	911 Control	CATGCAGACTGCCTGCTTGGGCGA	CAUGCAGACUGCCUGCUUGGGCG
UGUCGCUGG [SEQ				GCGACATCGAGGAAACGCAATATG	AGCGACAUCGAGGAAACGCAAUA
ID NO: 128]				GACCTGCTAAGCTATTGATTTCCTC	UGGACCUGCUAAGCUAUUGAUUU
				GATGTCGCTGGCTCAGGCCGGGAC	CCUCGAUGUCGCUGGCUCAGGCC
				CTCTCTCGCCGCACTGAGGGGCAC	GGGACCUCUCUCGCCGCACUGAG
				TCCACACCACGGGGGCC	GGGCACUCCACACCACGGGGGCC
				[SEQ ID NO: 553]	[SEQ ID NO: 1120]
AGAAAUCGUAGAC	XD-14743	miR-1-1	Atxn2	CATGCAGACTGCCTGCTTGGGAGT	CAUGCAGACUGCCUGCUUGGGAG
UGAGGCAGU [SEQ			targeting	GCCTCAGTCTACGATCGTCTTATGG	UGCCUCAGUCUACGAUCGUCUUA
ID NO: 14]				ACCTGCTAAGCTAAGAAATCGTAG	UGGACCUGCUAAGCUAAGAAAUC
				ACTGAGGCAGTCTCAGGCCGGGAC	GUAGACUGAGGCAGUCUCAGGCC
				CTCTCTCGCCGCACTGAGGGGCAC	GGGACCUCUCUCGCCGCACUGAG
				TCCACACCACGGGGGCC	GGGCACUCCACACCACGGGGGCC
				[SEQ ID NO: 554]	[SEQ ID NO: 1121]
AGAAAUCGUAGAC	XD-14743	miR-155E	Atxn2	CTGGAGGCTTGCTTTGGGCTGTAT	CUGGAGGCUUGCUUUGGGCUGUA
UGAGGCAGU [SEQ			targeting	GCTGAGAAATCGTAGACTGAGGCA	UGCUGAGAAAUCGUAGACUGAGG
ID NO: 14]				GTTTTTGGCCTCTGACTGAACTGCC	CAGUUUUUGGCCUCUGACUGAAC
				TCGTCACGATTTCTCAGGACAAGG	UGCCUCGUCACGAUUUCUCAGGA
				CCCTTTATCAGCACTCACATGGAA	CAAGGCCCUUUAUCAGCACUCAC
				CAAATGGCCACCGTG	AUGGAACAAAUGGCCACCGUG
				[SEQ ID NO: 555]	[SEQ ID NO: 1122]
AGAUACGUCAUUU	XD-14766	miR-1-1	Atxn2	CATGCAGACTGCCTGCTTGGGGCC	CAUGCAGACUGCCUGCUUGGGGC
UCCAAAGCC [SEQ			targeting	TTTGGAAAATGACGTCCTCTTATG	CUUUGGAAAAUGACGUCCUCUUA
ID NO: 60]				GACCTGCTAAGCTAAGATACGTCA	UGGACCUGCUAAGCUAAGAUACG
				TTTTCCAAAGCCCTCAGGCCGGGA	UCAUUUUCCAAAGCCCUCAGGCC
				CCTCTCTCGCCGCACTGAGGGGCA	GGGACCUCUCUCGCCGCACUGAG
				CTCCACACCACGGGGGCC	GGGCACUCCACACCACGGGGGCC
				[SEQ ID NO: 556]	[SEQ ID NO: 1123]
AGAUACGUCAUUU	XD-14766	miR-155E	Atxn2	CTGGAGGCTTGCTTTGGGCTGTAT	CUGGAGGCUUGCUUUGGGCUGUA
UCCAAAGCC			targeting	GCTGAGATACGTCATTTTCCAAAG	UGCUGAGAUACGUCAUUUUCCAA
[SEQ ID NO: 60]				CCTTTTGGCCTCTGACTGAGGCTTT	AGCCUUUUGGCCUCUGACUGAGG
				GGAAAGACGTATCTCAGGACAAGG	CUUUGGAAAGACGUAUCUCAGGA
				CCCTTTATCAGCACTCACATGGAA	CAAGGCCCUUUAUCAGCACUCAC
				CAAATGGCCACCGTG	AUGGAACAAAUGGCCACCGUG
				[SEQ ID NO: 557]	[SEQ ID NO: 1124]
AGCGUUAGGGUGC	XD-14904	miR-155E	Atxn2	CTGGAGGCTTGCTTTGGGCTGTAT	CUGGAGGCUUGCUUUGGGCUGUA
GCAUACUGC [SEQ			targeting	GCTGAGCGTTAGGGTGCGCATACT	UGCUGAGCGUUAGGGUGCGCAUA
ID NO: 336]				GCTTTTGGCCTCTGACTGAGCAGT	CUGCUUUUGGCCUCUGACUGAGC
				ATGGCACCTAACGCTCAGGACAAG	AGUAUGGCACCUAACGCUCAGGA
				GCCCTTTATCAGCACTCACATGGA	CAAGGCCCUUUAUCAGCACUCAC
				ACAAATGGCCACCGTG	AUGGAACAAAUGGCCACCGUG
				[SEQ ID NO: 558]	[SEQ ID NO: 1125]
AGCGUUAGGGUGC	XD-14904	miR-1-1	Atxn2	CATGCAGACTGCCTGCTTGGGGGA	CAUGCAGACUGCCUGCUUGGGGG
GCAUACUGC [SEQ			targeting	GTATGCGCACCCTAAGAGCTTATG	AGUAUGCGCACCCUAAGAGCUUA
ID NO: 336]				GACCTGCTAAGCTAAGCGTTAGGG	UGGACCUGCUAAGCUAAGCGUUA
				TGCGCATACTGCCTCAGGCCGGGA	GGGUGCGCAUACUGCCUCAGGCC
				CCTCTCTCGCCGCACTGAGGGGCA	GGGACCUCUCUCGCCGCACUGAG
				CTCCACACCACGGGGGCC	GGGCACUCCACACCACGGGGGCC
				[SEQ ID NO: 559]	[SEQ ID NO: 1126]
AGGAACGUGGGUU	XD-14857	miR-155E	Atxn2	CTGGAGGCTTGCTTTGGGCTGTAT	CUGGAGGCUUGCUUUGGGCUGUA
GAACUCCUU [SEQ			targeting	GCTGAGGAACGTGGGTTGAACTCC	UGCUGAGGAACGUGGGUUGAACU
ID NO: 242]				TTTTTTGGCCTCTGACTGAAAGGA	CCUUUUUUGGCCUCUGACUGAAA
				GTTAACCACGTTCCTCAGGACAAG	GGAGUUAACCACGUUCCUCAGGA
				GCCCTTTATCAGCACTCACATGGA	CAAGGCCCUUUAUCAGCACUCAC
				ACAAATGGCCACCGTG	AUGGAACAAAUGGCCACCGUG
				[SEQ ID NO: 560]	[SEQ ID NO: 1127]
AGGAACGUGGGUU	XD-14857	miR-1-1	Atxn2	CATGCAGACTGCCTGCTTGGGATG	CAUGCAGACUGCCUGCUUGGGAU
GAACUCCUU [SEQ			targeting	GAGTTCAACCCACGTCGCCTTATG	GGAGUUCAACCCACGUCGCCUUA
ID NO: 242]				GACCTGCTAAGCTAAGGAACGTGG	UGGACCUGCUAAGCUAAGGAACG
				GTTGAACTCCTTCTCAGGCCGGGA	UGGGUUGAACUCCUUCUCAGGCC
				CCTCTCTCGCCGCACTGAGGGGCA	GGGACCUCUCUCGCCGCACUGAG
				CTCCACACCACGGGGGCC	GGGCACUCCACACCACGGGGGCC
				[SEQ ID NO: 561]	[SEQ ID NO: 1128]
AUAAUAAUCCGUC	XD-14949	miR-155E	Atxn2	CTGGAGGCTTGCTTTGGGCTGTAT	CUGGAGGCUUGCUUUGGGCUGUA
AGUUUGACG [SEQ			targeting	GCTGATAATAATCCGTCAGTTTGA	UGCUGAUAAUAAUCCGUCAGUUU
ID NO: 426]				CGTTTTGGCCTCTGACTGACGTCAA	GACGUUUUGGCCUCUGACUGACG
				ACGACGATTATTATCAGGACAAGG	UCAAACGACGAUUAUUAUCAGGA
				CCCTTTATCAGCACTCACATGGAA	CAAGGCCCUUUAUCAGCACUCAC
				CAAATGGCCACCGTG	AUGGAACAAAUGGCCACCGUG
				[SEQ ID NO: 562]	[SEQ ID NO: 1129]
AUAAUAAUCCGUC	XD-14949	miR-1-1	Atxn2	CATGCAGACTGCCTGCTTGGGCCT	CAUGCAGACUGCCUGCUUGGGCC
AGUUUGACG [SEQ			targeting	CAAACTGACGGATTACGTATTATG	UCAAACUGACGGAUUACGUAUUA
ID NO: 426]				GACCTGCTAAGCTAATAATAATCC	UGGACCUGCUAAGCUAAUAAUAA
				GTCAGTTTGACGCTCAGGCCGGGA	UCCGUCAGUUUGACGCUCAGGCC
				CCTCTCTCGCCGCACTGAGGGGCA	GGGACCUCUCUCGCCGCACUGAG
				CTCCACACCACGGGGGCC	GGGCACUCCACACCACGGGGGCC
				[SEQ ID NO: 563]	[SEQ ID NO: 1130]
AUACGCGGUGAAU	XD-14787	miR-155E	Atxn2	CTGGAGGCTTGCTTTGGGCTGTAT	CUGGAGGCUUGCUUUGGGCUGUA
UCUGUCUCC [SEQ			targeting	GCTGATACGCGGTGAATTCTGTCT	UGCUGAUACGCGGUGAAUUCUGU
ID NO: 102]				CCTTTTGGCCTCTGACTGAGGAGA	CUCCUUUUGGCCUCUGACUGAGG
				CAGATTACCGCGTATCAGGACAAG	AGACAGAUUACCGCGUAUCAGGA
				GCCCTTTATCAGCACTCACATGGA	CAAGGCCCUUUAUCAGCACUCAC
				ACAAATGGCCACCGTG	AUGGAACAAAUGGCCACCGUG
				[SEQ ID NO: 564]	[SEQ ID NO: 1131]
AUACGCGGUGAAU	XD-14787	miR-1-1	Atxn2	CATGCAGACTGCCTGCTTGGGGCA	CAUGCAGACUGCCUGCUUGGGGC
UCUGUCUCC [SEQ			targeting	GACAGAATTCACCGCCTTATTATG	AGACAGAAUUCACCGCCUUAUUA
ID NO: 102]				GACCTGCTAAGCTAATACGCGGTG	UGGACCUGCUAAGCUAAUACGCG
				AATTCTGTCTCCCTCAGGCCGGGA	GUGAAUUCUGUCUCCCUCAGGCC
				CCTCTCTCGCCGCACTGAGGGGCA	GGGACCUCUCUCGCCGCACUGAG
				CTCCACACCACGGGGGCC	GGGCACUCCACACCACGGGGGCC
				[SEQ ID NO: 565]	[SEQ ID NO: 1132]
AUUAACUACUCUU	XD-14792	miR-1-1	Atxn2	CATGCAGACTGCCTGCTTGGGTAC	CAUGCAGACUGCCUGCUUGGGUA
UGGUCUGAA [SEQ			targeting	AGACCAAAGAGTAGTCGAATTATG	CAGACCAAAGAGUAGUCGAAUUA
ID NO: 112]				GACCTGCTAAGCTAATTAACTACT	UGGACCUGCUAAGCUAAUUAACU
				CTTTGGTCTGAACTCAGGCCGGGA	ACUCUUUGGUCUGAACUCAGGCC
				CCTCTCTCGCCGCACTGAGGGGCA	GGGACCUCUCUCGCCGCACUGAG
				CTCCACACCACGGGGGCC	GGGCACUCCACACCACGGGGGCC
				[SEQ ID NO: 538]	[SEQ ID NO: 1133]
AUUAACUACUCUU	XD-14792	miR-155E	Atxn2	CTGGAGGCTTGCTTTGGGCTGTAT	CUGGAGGCUUGCUUUGGGCUGUA
UGGUCUGAA [SEQ			targeting	GCTGATTAACTACTCTTTGGTCTGA	UGCUGAUUAACUACUCUUUGGUC
ID NO: 112]				ATTTTGGCCTCTGACTGATTCAGAC	UGAAUUUUGGCCUCUGACUGAUU
				CAAGGTAGTTAATCAGGACAAGGC	CAGACCAAGGUAGUUAAUCAGGA
				CCTTTATCAGCACTCACATGGAAC	CAAGGCCCUUUAUCAGCACUCAC
				AAATGGCCACCGTG	AUGGAACAAAUGGCCACCGUG
				[SEQ ID NO: 566]	[SEQ ID NO: 1134]
AUUGCGUGGAGUA	XD-14889	miR-155E	Atxn2	CTGGAGGCTTGCTTTGGGCTGTAT	CUGGAGGCUUGCUUUGGGCUGUA
AGCUGGUGG [SEQ			targeting	GCTGATTGCGTGGAGTAAGCTGGT	UGCUGAUUGCGUGGAGUAAGCUG
ID NO: 306]				GGTTTTGGCCTCTGACTGACCACC	GUGGUUUUGGCCUCUGACUGACC
				AGCTACCCACGCAATCAGGACAAG	ACCAGCUACCCACGCAAUCAGGAC
				GCCCTTTATCAGCACTCACATGGA	AAGGCCCUUUAUCAGCACUCACA
				ACAAATGGCCACCGTG	UGGAACAAAUGGCCACCGUG
				[SEQ ID NO: 567]	[SEQ ID NO: 1135]
AUUGCGUGGAGUA	XD-14889	miR-1-1	Atxn2	CATGCAGACTGCCTGCTTGGGCGA	CAUGCAGACUGCCUGCUUGGGCG
AGCUGGUGG [SEQ			targeting	CCAGCTTACTCCACGGAAATTATG	ACCAGCUUACUCCACGGAAAUUA
ID NO: 306]				GACCTGCTAAGCTAATTGCGTGGA	UGGACCUGCUAAGCUAAUUGCGU
				GTAAGCTGGTGGCTCAGGCCGGGA	GGAGUAAGCUGGUGGCUCAGGCC
				CCTCTCTCGCCGCACTGAGGGGCA	GGGACCUCUCUCGCCGCACUGAG
				CTCCACACCACGGGGGCC	GGGCACUCCACACCACGGGGGCC
				[SEQ ID NO: 568]	[SEQ ID NO: 1136]
AUUUCGAGGAUGU	XD-14798	miR-155E	Atxn2	CTGGAGGCTTGCTTTGGGCTGTAT	CUGGAGGCUUGCUUUGGGCUGUA
CGCUGGGCC [SEQ			targeting	GCTGATTTCGAGGATGTCGCTGGG	UGCUGAUUUCGAGGAUGUCGCUG
ID NO: 124]				CCTTTTGGCCTCTGACTGAGGCCCA	GGCCUUUUGGCCUCUGACUGAGG
				GCACACCTCGAAATCAGGACAAGG	CCCAGCACACCUCGAAAUCAGGAC
				CCCTTTATCAGCACTCACATGGAA	AAGGCCCUUUAUCAGCACUCACA
				CAAATGGCCACCGTG	UGGAACAAAUGGCCACCGUG
				[SEQ ID NO: 569]	[SEQ ID NO: 1137]
AUUUCGAGGAUGU	XD-14798	miR-1-1	Atxn2	CATGCAGACTGCCTGCTTGGGGCC	CAUGCAGACUGCCUGCUUGGGGC
CGCUGGGCC [SEQ			targeting	CCAGCGACATCCTCGCCAATTATG	CCCAGCGACAUCCUCGCCAAUUAU
ID NO: 124]				GACCTGCTAAGCTAATTTCGAGGA	GGACCUGCUAAGCUAAUUUCGAG
				TGTCGCTGGGCCCTCAGGCCGGGA	GAUGUCGCUGGGCCCUCAGGCCG
				CCTCTCTCGCCGCACTGAGGGGCA	GGACCUCUCUCGCCGCACUGAGG
				CTCCACACCACGGGGGCC	GGCACUCCACACCACGGGGGCC
				[SEQ ID NO: 570]	[SEQ ID NO: 1138]
UAAAUCGUAGACU	XD-14742	miR-1-1	Atxn2	CATGCAGACTGCCTGCTTGGGGTC	UAGACUGAGGCAGUCCUCAGGCC
GAGGCAGUC [SEQ			targeting	TGCCTCAGTCTACGACGTTATATG	CAUGCAGACUGCCUGCUUGGGGU
ID NO: 12]				GACCTGCTAAGCTATAAATCGTAG	CUGCCUCAGUCUACGACGUUAUA
				ACTGAGGCAGTCCTCAGGCCGGGA	UGGACCUGCUAAGCUAUAAAUCG
				CCTCTCTCGCCGCACTGAGGGGCA	GGGACCUCUCUCGCCGCACUGAG
				CTCCACACCACGGGGGCC	GGGCACUCCACACCACGGGGGCC
				[SEQ ID NO: 571]	[SEQ ID NO: 1139]
UAAAUCGUAGACU	XD-14742	miR-155E	Atxn2	CTGGAGGCTTGCTTTGGGCTGTAT	CUGGAGGCUUGCUUUGGGCUGUA
GAGGCAGUC [SEQ			targeting	GCTGTAAATCGTAGACTGAGGCAG	UGCUGUAAAUCGUAGACUGAGGC
ID NO: 12]				TCTTTTGGCCTCTGACTGAGACTGC	AGUCUUUUGGCCUCUGACUGAGA
				CTAGTTACGATTTACAGGACAAGG	CUGCCUAGUUACGAUUUACAGGA
				CCCTTTATCAGCACTCACATGGAA	CAAGGCCCUUUAUCAGCACUCAC
				CAAATGGCCACCGTG	AUGGAACAAAUGGCCACCGUG
				[SEQ ID NO: 572]	[SEQ ID NO: 1140]
UACGCGGUGAAUU	XD-14786	miR-1-1	Atxn2	CATGCAGACTGCCTGCTTGGGGCG	CAUGCAGACUGCCUGCUUGGGGC
CUGUCUCCC [SEQ			targeting	AGACAGAATTCACCGGAGTATATG	GAGACAGAAUUCACCGGAGUAUA
ID NO: 100]				GACCTGCTAAGCTATACGCGGTGA	UGGACCUGCUAAGCUAUACGCGG
				ATTCTGTCTCCCCTCAGGCCGGGA	UGAAUUCUGUCUCCCCUCAGGCC
				CCTCTCTCGCCGCACTGAGGGGCA	GGGACCUCUCUCGCCGCACUGAG
				CTCCACACCACGGGGGCC	GGGCACUCCACACCACGGGGGCC
				[SEQ ID NO: 573]	[SEQ ID NO: 1141]
UACGCGGUGAAUU	XD-14786	miR-155E	Atxn2	CTGGAGGCTTGCTTTGGGCTGTAT	CUGGAGGCUUGCUUUGGGCUGUA
CUGUCUCCC [SEQ			targeting	GCTGTACGCGGTGAATTCTGTCTCC	UGCUGUACGCGGUGAAUUCUGUC
ID NO: 100]				CTTTTGGCCTCTGACTGAGGGAGA	UCCCUUUUGGCCUCUGACUGAGG
				CAAATCACCGCGTACAGGACAAGG	GAGACAAAUCACCGCGUACAGGA
				CCCTTTATCAGCACTCACATGGAA	CAAGGCCCUUUAUCAGCACUCAC
				CAAATGGCCACCGTG	AUGGAACAAAUGGCCACCGUG
				[SEQ ID NO: 574]	[SEQ ID NO: 1142]
UAUACGCGGUGAA	XD-14788	miR-1-1	Atxn2	CATGCAGACTGCCTGCTTGGGGTG	CAUGCAGACUGCCUGCUUGGGGU
UUCUGUCUC [SEQ			targeting	ACAGAATTCACCGCGCGATATATG	GACAGAAUUCACCGCGCGAUAUA
ID NO: 104]				GACCTGCTAAGCTATATACGCGGT	UGGACCUGCUAAGCUAUAUACGC
				GAATTCTGTCTCCTCAGGCCGGGA	GGUGAAUUCUGUCUCCUCAGGCC
				CCTCTCTCGCCGCACTGAGGGGCA	GGGACCUCUCUCGCCGCACUGAG
				CTCCACACCACGGGGGCC	GGGCACUCCACACCACGGGGGCC
				[SEQ ID NO: 575]	[SEQ ID NO: 1143]
UAUACGCGGUGAA	XD-14788	miR-155E	Atxn2	CTGGAGGCTTGCTTTGGGCTGTAT	CUGGAGGCUUGCUUUGGGCUGUA
UUCUGUCUC [SEQ			targeting	GCTGTATACGCGGTGAATTCTGTCT	UGCUGUAUACGCGGUGAAUUCUG
ID NO: 104]				CTTTTGGCCTCTGACTGAGAGACA	UCUCUUUUGGCCUCUGACUGAGA
				GATTCCCGCGTATACAGGACAAGG	GACAGAUUCCCGCGUAUACAGGA
				CCCTTTATCAGCACTCACATGGAA	CAAGGCCCUUUAUCAGCACUCAC
				CAAATGGCCACCGTG	AUGGAACAAAUGGCCACCGUG
				[SEQ ID NO: 576]	[SEQ ID NO: 1144]
UAUUGCGUGGAGU	XD-14890	miR-1-1	Atxn2	CATGCAGACTGCCTGCTTGGGCTC	CAUGCAGACUGCCUGCUUGGGCU
AAGCUGGUG [SEQ			targeting	CAGCTTACTCCACGCCCATATATG	CCAGCUUACUCCACGCCCAUAUAU
ID NO: 308]				GACCTGCTAAGCTATATTGCGTGG	GGACCUGCUAAGCUAUAUUGCGU
				AGTAAGCTGGTGCTCAGGCCGGGA	GGAGUAAGCUGGUGCUCAGGCCG
				CCTCTCTCGCCGCACTGAGGGGCA	GGACCUCUCUCGCCGCACUGAGG
				CTCCACACCACGGGGGCC	GGCACUCCACACCACGGGGGCC
				[SEQ ID NO: 577]	[SEQ ID NO: 1145]
UAUUGCGUGGAGU	XD-14890	miR-155E	Atxn2	CTGGAGGCTTGCTTTGGGCTGTAT	CUGGAGGCUUGCUUUGGGCUGUA
AAGCUGGUG [SEQ			targeting	GCTGTATTGCGTGGAGTAAGCTGG	UGCUGUAUUGCGUGGAGUAAGCU
ID NO: 308]				TGTTTTGGCCTCTGACTGACACCAG	GGUGUUUUGGCCUCUGACUGACA
				CTACTCACGCAATACAGGACAAGG	CCAGCUACUCACGCAAUACAGGA
				CCCTTTATCAGCACTCACATGGAA	CAAGGCCCUUUAUCAGCACUCAC
				CAAATGGCCACCGTG	AUGGAACAAAUGGCCACCGUG
				[SEQ ID NO: 578]	[SEQ ID NO: 1146]
UAUUUCGAGGAUG	XD-14799	miR-155E	Atxn2	CTGGAGGCTTGCTTTGGGCTGTAT	CUGGAGGCUUGCUUUGGGCUGUA
UCGCUGGGC [SEQ			targeting	GCTGTATTTCGAGGATGTCGCTGG	UGCUGUAUUUCGAGGAUGUCGCU
ID NO: 126]				GCTTTTGGCCTCTGACTGAGCCCA	GGGCUUUUGGCCUCUGACUGAGC
				GCGCATCTCGAAATACAGGACAAG	CCAGCGCAUCUCGAAAUACAGGA
				GCCCTTTATCAGCACTCACATGGA	CAAGGCCCUUUAUCAGCACUCAC
				ACAAATGGCCACCGTG	AUGGAACAAAUGGCCACCGUG
				[SEQ ID NO: 579]	[SEQ ID NO: 1147]
UAUUUCGAGGAUG	XD-14799	miR-1-1	Atxn2	CATGCAGACTGCCTGCTTGGGGGC	CAUGCAGACUGCCUGCUUGGGGG
UCGCUGGGC [SEQ			targeting	CAGCGACATCCTCGACCATATATG	CCAGCGACAUCCUCGACCAUAUA
ID NO: 126]				GACCTGCTAAGCTATATTTCGAGG	UGGACCUGCUAAGCUAUAUUUCG
				ATGTCGCTGGGCCTCAGGCCGGGA	AGGAUGUCGCUGGGCCUCAGGCC
				CCTCTCTCGCCGCACTGAGGGGCA	GGGACCUCUCUCGCCGCACUGAG
				CTCCACACCACGGGGGCC	GGGCACUCCACACCACGGGGGCC
				[SEQ ID NO: 580]	[SEQ ID NO: 1148]
UCGCUGUUGGGGC	XD-14887	miR-1-1	Atxn2	CATGCAGACTGCCTGCTTGGGAGC	CAUGCAGACUGCCUGCUUGGGAG
AUAUUUGGU [SEQ			targeting	AAATATGCCCCAACACTCGATATG	CAAAUAUGCCCCAACACUCGAUA
ID NO: 302]				GACCTGCTAAGCTATCGCTGTTGG	UGGACCUGCUAAGCUAUCGCUGU
				GGCATATTTGGTCTCAGGCCGGGA	UGGGGCAUAUUUGGUCUCAGGCC
				CCTCTCTCGCCGCACTGAGGGGCA	GGGACCUCUCUCGCCGCACUGAG
				CTCCACACCACGGGGGCC	GGGCACUCCACACCACGGGGGCC
				[SEQ ID NO: 581]	[SEQ ID NO: 1149]
UCGCUGUUGGGGC	XD-14887	miR-155E	Atxn2	CTGGAGGCTTGCTTTGGGCTGTAT	CUGGAGGCUUGCUUUGGGCUGUA
AUAUUUGGU [SEQ			targeting	GCTGTCGCTGTTGGGGCATATTTG	UGCUGUCGCUGUUGGGGCAUAUU
ID NO: 302]				GTTTTTGGCCTCTGACTGAACCAA	UGGUUUUUGGCCUCUGACUGAAC
				ATAGCCCAACAGCGACAGGACAA	CAAAUAGCCCAACAGCGACAGGA
				GGCCCTTTATCAGCACTCACATGG	CAAGGCCCUUUAUCAGCACUCAC
				AACAAATGGCCACCGTG	AUGGAACAAAUGGCCACCGUG
				[SEQ ID NO: 582]	[SEQ ID NO: 1150]
UGCGCAUACUGCUG	XD-14901	miR-1-1	Atxn2	CATGCAGACTGCCTGCTTGGGCGC	CAUGCAGACUGCCUGCUUGGGCG
AGCAAGGG [SEQ ID			targeting	TTGCTCAGCAGTATGGAGCATATG	CUUGCUCAGCAGUAUGGAGCAUA
NO: 330]				GACCTGCTAAGCTATGCGCATACT	UGGACCUGCUAAGCUAUGCGCAU
				GCTGAGCAAGGGCTCAGGCCGGGA	ACUGCUGAGCAAGGGCUCAGGCC
				CCTCTCTCGCCGCACTGAGGGGCA	GGGACCUCUCUCGCCGCACUGAG
				CTCCACACCACGGGGGCC	GGGCACUCCACACCACGGGGGCC
				[SEQ ID NO: 583]	[SEQ ID NO: 1151]
UGCGCAUACUGCUG	XD-14901	miR-155E	Atxn2	CTGGAGGCTTGCTTTGGGCTGTAT	CUGGAGGCUUGCUUUGGGCUGUA
AGCAAGGG [SEQ ID			targeting	GCTGTGCGCATACTGCTGAGCAAG	UGCUGUGCGCAUACUGCUGAGCA
NO: 330]				GGTTTTGGCCTCTGACTGACCCTTG	AGGGUUUUGGCCUCUGACUGACC
				CTAGCGTATGCGCACAGGACAAGG	CUUGCUAGCGUAUGCGCACAGGA
				CCCTTTATCAGCACTCACATGGAA	CAAGGCCCUUUAUCAGCACUCAC
				CAAATGGCCACCGTG	AUGGAACAAAUGGCCACCGUG
				[SEQ ID NO: 584]	[SEQ ID NO: 1152]
UGUACCACAACAAA	XD-14756	miR-1-1	Atxn2	CATGCAGACTGCCTGCTTGGGGAT	CAUGCAGACUGCCUGCUUGGGGA
GUCUGAAC [SEQ ID			targeting	CAGACTTTGTTGTGGCGACATATG	UCAGACUUUGUUGUGGCGACAUA
NO: 40]				GACCTGCTAAGCTATGTACCACAA	UGGACCUGCUAAGCUAUGUACCA
				CAAAGTCTGAACCTCAGGCCGGGA	CAACAAAGUCUGAACCUCAGGCC
				CCTCTCTCGCCGCACTGAGGGGCA	GGGACCUCUCUCGCCGCACUGAG
				CTCCACACCACGGGGGCC	GGGCACUCCACACCACGGGGGCC
				[SEQ ID NO: 585]	[SEQ ID NO: 1153]
UGUACCACAACAAA	XD-14756	miR-155E	Atxn2	CTGGAGGCTTGCTTTGGGCTGTAT	CUGGAGGCUUGCUUUGGGCUGUA
GUCUGAAC [SEQ ID			targeting	GCTGTGTACCACAACAAAGTCTGA	UGCUGUGUACCACAACAAAGUCU
NO: 40]				ACTTTTGGCCTCTGACTGAGTTCAG	GAACUUUUGGCCUCUGACUGAGU
				ACTTGTGTGGTACACAGGACAAGG	UCAGACUUGUGUGGUACACAGGA
				CCCTTTATCAGCACTCACATGGAA	CAAGGCCCUUUAUCAGCACUCAC
				CAAATGGCCACCGTG	AUGGAACAAAUGGCCACCGUG
				[SEQ ID NO: 586]	[SEQ ID NO: 1154]
UGUAUACGCCGGCU	XD-14917	miR-1-1	Atxn2	CATGCAGACTGCCTGCTTGGGTGA	CAUGCAGACUGCCUGCUUGGGUG
GAACGUGA [SEQ ID			targeting	CGTTCAGCCGGCGTACGACATATG	ACGUUCAGCCGGCGUACGACAUA
NO: 362]				GACCTGCTAAGCTATGTATACGCC	UGGACCUGCUAAGCUAUGUAUAC
				GGCTGAACGTGACTCAGGCCGGGA	GCCGGCUGAACGUGACUCAGGCC
				CCTCTCTCGCCGCACTGAGGGGCA	GGGACCUCUCUCGCCGCACUGAG
				CTCCACACCACGGGGGCC	GGGCACUCCACACCACGGGGGCC
				[SEQ ID NO: 587]	[SEQ ID NO: 1155]
UGUAUACGCCGGCU	XD-14917	miR-155E	Atxn2	CTGGAGGCTTGCTTTGGGCTGTAT	CUGGAGGCUUGCUUUGGGCUGUA
GAACGUGA [SEQ ID			targeting	GCTGTGTATACGCCGGCTGAACGT	UGCUGUGUAUACGCCGGCUGAAC
NO: 362]				GATTTTGGCCTCTGACTGATCACGT	GUGAUUUUGGCCUCUGACUGAUC
				TCGCCGCGTATACACAGGACAAGG	ACGUUCGCCGCGUAUACACAGGA
				CCCTTTATCAGCACTCACATGGAA	CAAGGCCCUUUAUCAGCACUCAC
				CAAATGGCCACCGTG	AUGGAACAAAUGGCCACCGUG
				[SEQ ID NO: 588]	[SEQ ID NO: 1156]
UUACUAAGUAUUG	XD-14846	miR-155E	Atxn2	CTGGAGGCTTGCTTTGGGCTGTAT	CUGGAGGCUUGCUUUGGGCUGUA
AAGGGGAAA [SEQ			targeting	GCTGTTACTAAGTATTGAAGGGGA	UGCUGUUACUAAGUAUUGAAGGG
ID NO: 220]				AATTTTGGCCTCTGACTGATTTCCC	GAAAUUUUGGCCUCUGACUGAUU
				CTCAAACTTAGTAACAGGACAAGG	UCCCCUCAAACUUAGUAACAGGA
				CCCTTTATCAGCACTCACATGGAA	CAAGGCCCUUUAUCAGCACUCAC
				CAAATGGCCACCGTG	AUGGAACAAAUGGCCACCGUG
				[SEQ ID NO: 589]	[SEQ ID NO: 1157]
UUACUAAGUAUUG	XD-14846	miR-1-1	Atxn2	CATGCAGACTGCCTGCTTGGGTAT	CAUGCAGACUGCCUGCUUGGGUA
AAGGGGAAA [SEQ			targeting	CCCCTTCAATACTTACTTAATATGG	UCCCCUUCAAUACUUACUUAAUA
ID NO: 220]				ACCTGCTAAGCTATTACTAAGTATT	UGGACCUGCUAAGCUAUUACUAA
				GAAGGGGAAACTCAGGCCGGGAC	GUAUUGAAGGGGAAACUCAGGCC
				CTCTCTCGCCGCACTGAGGGGCAC	GGGACCUCUCUCGCCGCACUGAG
				TCCACACCACGGGGGCC	GGGCACUCCACACCACGGGGGCC
				[SEQ ID NO: 590]	[SEQ ID NO: 1158]
UUAGUUGAUCCAU	XD-14835	miR-1-1	Atxn2	CATGCAGACTGCCTGCTTGGGTGT	CAUGCAGACUGCCUGCUUGGGUG
AGAUUCAGA [SEQ			targeting	GAATCTATGGATCAAGATAATATG	UGAAUCUAUGGAUCAAGAUAAUA
ID NO: 198]				GACCTGCTAAGCTATTAGTTGATC	UGGACCUGCUAAGCUAUUAGUUG
				CATAGATTCAGACTCAGGCCGGGA	AUCCAUAGAUUCAGACUCAGGCC
				CCTCTCTCGCCGCACTGAGGGGCA	GGGACCUCUCUCGCCGCACUGAG
				CTCCACACCACGGGGGCC	GGGCACUCCACACCACGGGGGCC
				[SEQ ID NO: 591]	[SEQ ID NO: 1159]
UUAGUUGAUCCAU	XD-14835	miR-155E	Atxn2	CTGGAGGCTTGCTTTGGGCTGTAT	CUGGAGGCUUGCUUUGGGCUGUA
AGAUUCAGA [SEQ			targeting	GCTGTTAGTTGATCCATAGATTCA	UGCUGUUAGUUGAUCCAUAGAUU
ID NO: 198]				GATTTTGGCCTCTGACTGATCTGAA	CAGAUUUUGGCCUCUGACUGAUC
				TCATGATCAACTAACAGGACAAGG	UGAAUCAUGAUCAACUAACAGGA
				CCCTTTATCAGCACTCACATGGAA	CAAGGCCCUUUAUCAGCACUCAC
				CAAATGGCCACCGTG	AUGGAACAAAUGGCCACCGUG
				[SEQ ID NO: 592]	[SEQ ID NO: 1160]
UUCGAUGCAGGAC	XD-14819	miR-155E	Atxn2	CTGGAGGCTTGCTTTGGGCTGTAT	CUGGAGGCUUGCUUUGGGCUGUA
UAGCAGGCG [SEQ			targeting	GCTGTTCGATGCAGGACTAGCAGG	UGCUGUUCGAUGCAGGACUAGCA
ID NO: 166]				CGTTTTGGCCTCTGACTGACGCCTG	GGCGUUUUGGCCUCUGACUGACG
				CTGTCTGCATCGAACAGGACAAGG	CCUGCUGUCUGCAUCGAACAGGA
				CCCTTTATCAGCACTCACATGGAA	CAAGGCCCUUUAUCAGCACUCAC
				CAAATGGCCACCGTG	AUGGAACAAAUGGCCACCGUG
				[SEQ ID NO: 593]	[SEQ ID NO: 1161]
UUCGAUGCAGGAC	XD-14819	miR-1-1	Atxn2	CATGCAGACTGCCTGCTTGGGCCC	CAUGCAGACUGCCUGCUUGGGCC
UAGCAGGCG [SEQ			targeting	CTGCTAGTCCTGCATGAGAATATG	CCUGCUAGUCCUGCAUGAGAAUA
ID NO: 166]				GACCTGCTAAGCTATTCGATGCAG	UGGACCUGCUAAGCUAUUCGAUG
				GACTAGCAGGCGCTCAGGCCGGGA	CAGGACUAGCAGGCGCUCAGGCC
				CCTCTCTCGCCGCACTGAGGGGCA	GGGACCUCUCUCGCCGCACUGAG
				CTCCACACCACGGGGGCC	GGGCACUCCACACCACGGGGGCC
				[SEQ ID NO: 594]	[SEQ ID NO: 1162]
UUCGGGUUGAAAU	XD-14790	miR-155E	Atxn2	CTGGAGGCTTGCTTTGGGCTGTAT	CUGGAGGCUUGCUUUGGGCUGUA
CUGAAGUGU [SEQ			targeting	GCTGTTCGGGTTGAAATCTGAAGT	UGCUGUUCGGGUUGAAAUCUGAA
ID NO: 108]				GTTTTTGGCCTCTGACTGAACACTT	GUGUUUUUGGCCUCUGACUGAAC
				CAATTCAACCCGAACAGGACAAGG	ACUUCAAUUCAACCCGAACAGGA
				CCCTTTATCAGCACTCACATGGAA	CAAGGCCCUUUAUCAGCACUCAC
				CAAATGGCCACCGTG	AUGGAACAAAUGGCCACCGUG
				[SEQ ID NO: 595]	[SEQ ID NO: 1163]
UUCGGGUUGAAAU	XD-14790	miR-1-1	Atxn2	CATGCAGACTGCCTGCTTGGGAGA	CAUGCAGACUGCCUGCUUGGGAG
CUGAAGUGU [SEQ			targeting	CTTCAGATTTCAACCGAGAATATG	ACUUCAGAUUUCAACCGAGAAUA
ID NO: 108]				GACCTGCTAAGCTATTCGGGTTGA	UGGACCUGCUAAGCUAUUCGGGU
				AATCTGAAGTGTCTCAGGCCGGGA	UGAAAUCUGAAGUGUCUCAGGCC
				CCTCTCTCGCCGCACTGAGGGGCA	GGGACCUCUCUCGCCGCACUGAG
				CTCCACACCACGGGGGCC	GGGCACUCCACACCACGGGGGCC
				[SEQ ID NO: 596]	[SEQ ID NO: 1164]
UUGAUUUCGAGGA	XD-14800	miR-1-1	Atxn2	CATGCAGACTGCCTGCTTGGGCGA	CAUGCAGACUGCCUGCUUGGGCG
UGUCGCUGG [SEQ			targeting	GCGACATCCTCGAAACGCAATATG	AGCGACAUCCUCGAAACGCAAUA
ID NO: 128]				GACCTGCTAAGCTATTGATTTCGA	UGGACCUGCUAAGCUAUUGAUUU
				GGATGTCGCTGGCTCAGGCCGGGA	CGAGGAUGUCGCUGGCUCAGGCC
				CCTCTCTCGCCGCACTGAGGGGCA	GGGACCUCUCUCGCCGCACUGAG
				CTCCACACCACGGGGGCC	GGGCACUCCACACCACGGGGGCC
				[SEQ ID NO: 597]	[SEQ ID NO: 1165]
UUGAUUUCGAGGA	XD-14800	miR-155E	Atxn2	CTGGAGGCTTGCTTTGGGCTGTAT	CUGGAGGCUUGCUUUGGGCUGUA
UGUCGCUGG [SEQ			targeting	GCTGTTGATTTCGAGGATGTCGCT	UGCUGUUGAUUUCGAGGAUGUCG
ID NO: 128]				GGTTTTGGCCTCTGACTGACCAGC	CUGGUUUUGGCCUCUGACUGACC
				GACTCCCGAAATCAACAGGACAAG	AGCGACUCCCGAAAUCAACAGGA
				GCCCTTTATCAGCACTCACATGGA	CAAGGCCCUUUAUCAGCACUCAC
				ACAAATGGCCACCGTG	AUGGAACAAAUGGCCACCGUG
				[SEQ ID NO: 598]	[SEQ ID NO: 1166]
UUGUACUGGGCAC	XD-14781	miR-155E	Atxn2	CTGGAGGCTTGCTTTGGGCTGTAT	CUGGAGGCUUGCUUUGGGCUGUA
UUGACUCAA [SEQ			targeting	GCTGTTGTACTGGGCACTTGACTC	UGCUGUUGUACUGGGCACUUGAC
ID NO: 90]				AATTTTGGCCTCTGACTGATTGAGT	UCAAUUUUGGCCUCUGACUGAUU
				CAGTGCCAGTACAACAGGACAAGG	GAGUCAGUGCCAGUACAACAGGA
				CCCTTTATCAGCACTCACATGGAA	CAAGGCCCUUUAUCAGCACUCAC
				CAAATGGCCACCGTG	AUGGAACAAAUGGCCACCGUG
				[SEQ ID NO: 599]	[SEQ ID NO: 1167]
UUGUACUGGGCAC	XD-14781	miR-1-1	Atxn2	CATGCAGACTGCCTGCTTGGGTAG	CAUGCAGACUGCCUGCUUGGGUA
UUGACUCAA [SEQ			targeting	AGTCAAGTGCCCAGTCCCAATATG	GAGUCAAGUGCCCAGUCCCAAUA
ID NO: 90]				GACCTGCTAAGCTATTGTACTGGG	UGGACCUGCUAAGCUAUUGUACU
				CACTTGACTCAACTCAGGCCGGGA	GGGCACUUGACUCAACUCAGGCC
				CCTCTCTCGCCGCACTGAGGGGCA	GGGACCUCUCUCGCCGCACUGAG
				CTCCACACCACGGGGGCC	GGGCACUCCACACCACGGGGGCC
				[SEQ ID NO: 600]	[SEQ ID NO: 1168]

ATXN2-targeting miRNA guide sequences having at least 25% ATXN2 immunofluorescence signal knockdown are shown in Table 12 (both RNA and DNA versions). ATXN2-targeting miRNA guide sequences having at least 50% ATXN2 immunofluorescence signal knockdown are shown in Table 13 (both RNA and DNA versions).

TABLE 12
miRNA guide sequences with at least 25% knockdown of ATXN2
siRNA	Guide (antisense sequence)	Guide (antisense sequence)
duplex ID	(RNA)	(DNA)
XD-14742	UAAAUCGUAGACUGAGGCA	TAAATCGTAGACTGAGGCA
	GUC	GTC
	[SEQ ID NO: 12]	[SEQ ID NO: 601]
XD-14743	AGAAAUCGUAGACUGAGGC	AGAAATCGTAGACTGAGGC
	AGU	AGT
	[SEQ ID NO: 14]	[SEQ ID NO: 602]
XD-14756	UGUACCACAACAAAGUCUG	TGTACCACAACAAAGTCTGA
	AAC	AC
	[SEQ ID NO: 40]	[SEQ ID NO: 603]
XD-14766	AGAUACGUCAUUUUCCAAA	AGATACGTCATTTTCCAAAG
	GCC	CC
	[SEQ ID NO: 601	SEQ ID NO: 604]
XD-14786	UACGCGGUGAAUUCUGUCU	TACGCGGTGAATTCTGTCTC
	CCC	CC
	[SEQ ID NO: 100]	[SEQ ID NO: 605]
XD-14788	UAUACGCGGUGAAUUCUGU	TATACGCGGTGAATTCTGTC
	CUC	TC
	[SEQ ID NO: 104]	[SEQ ID NO: 606]
XD-14790	UUCGGGUUGAAAUCUGAAG	TTCGGGTTGAAATCTGAAGT
	UGU	GT
	[SEQ ID NO: 108]	[SEQ ID NO: 607]
XD-14792	AUUAACUACUCUUUGGUCU	ATTAACTACTCTTTGGTCTG
	GAA	AA
	[SEQ ID NO: 112]	[SEQ ID NO: 608]
XD-14798	AUUUCGAGGAUGUCGCUGG	ATTTCGAGGATGTCGCTGGG
	GCC	CC
	[SEQ ID NO: 124	[SEQ ID NO: 609]
XD-14799	UAUUUCGAGGAUGUCGCUG	TATTTCGAGGATGTCGCTGG
	GGC	GC
	[SEQ ID NO: 126]	[SEQ ID NO: 610]
XD-14800	UUGAUUUCGAGGAUGUCGC	TTGATTTCGAGGATGTCGCT
	UGG	GG
	[SEQ ID NO: 128]	[SEQ ID NO: 611]
XD-14819	UUCGAUGCAGGACUAGCAG	TTCGATGCAGGACTAGCAGG
	GCG	CG
	[SEQ ID NO: 166]	[SEQ ID NO: 612]
XD-14835	UUAGUUGAUCCAUAGAUUC	TTAGTTGATCCATAGATTCA
	AGA	GA
	[SEQ ID NO: 198]	[SEQ ID NO: 613]
XD-14846	UUACUAAGUAUUGAAGGGG	TTACTAAGTATTGAAGGGGA
	AAA	AA
	[SEQ ID NO: 220]	[SEQ ID NO: 614]
XD-14857	AGGAACGUGGGUUGAACUC	AGGAACGTGGGTTGAACTCC
	CUU	TT
	[SEQ ID NO: 242]	[SEQ ID NO: 615]
XD-14887	UCGCUGUUGGGGCAUAUUU	TCGCTGTTGGGGCATATTTG
	GGU	GT
	[SEQ ID NO: 302]	[SEQ ID NO: 616]
XD-14889	AUUGCGUGGAGUAAGCUGG	ATTGCGTGGAGTAAGCTGGT
	UGG	GG
	[SEQ ID NO: 306]	[SEQ ID NO: 617]
XD-14890	UAUUGCGUGGAGUAAGCUG	TATTGCGTGGAGTAAGCTGG
	GUG	TG
	[SEQ ID NO: 308]	[SEQ ID NO: 618]
XD-14901	UGCGCAUACUGCUGAGCAA	TGCGCATACTGCTGAGCAAG
	GGG	GG
	[SEQ ID NO: 330]	[SEQ ID NO: 619]
XD-14904	AGCGUUAGGGUGCGCAUAC	AGCGTTAGGGTGCGCATACT
	UGC	GC
	[SEQ ID NO: 336]	[SEQ ID NO: 620]
XD-14917	UGUAUACGCCGGCUGAACG	TGTATACGCCGGCTGAACGT
	UGA	GA
	[SEQ ID NO: 362]	[SEQ ID NO: 621]

TABLE 13
miRNA sequences with at least 50% knockdown of ATXN2
SiRNA	Guide (antisense sequence)	Guide (antisense sequence)
duplex ID	(RNA)	(DNA)
XD-14743	AGAAAUCGUAGACUGAGGC	AGAAATCGTAGACTGAGGC
	AGU	AGT
	[SEQ ID NO: 14]	[SEQ ID NO: 602]
XD-14756	UGUACCACAACAAAGUCUG	TGTACCACAACAAAGTCTGA
	AAC	AC
	[SEQ ID NO: 40]	[SEQ ID NO: 603]
XD-14786	UACGCGGUGAAUUCUGUCU	TACGCGGTGAATTCTGTCTC
	CCC	CC
	[SEQ ID NO: 100]	[SEQ ID NO: 605]
XD-14790	UUCGGGUUGAAAUCUGAAG	TTCGGGTTGAAATCTGAAGT
	UGU	GT
	[SEQ ID NO: 108]	[SEQ ID NO: 607]
XD-14792	AUUAACUACUCUUUGGUCU	ATTAACTACTCTTTGGTCTG
	GAA	AA
	[SEQ ID NO: 112]	[SEQ ID NO: 608]
XD-14800	UUGAUUUCGAGGAUGUCGC	TTGATTTCGAGGATGTCGCT
	UGG	GG
	[SEQ ID NO: 128]	[SEQ ID NO: 611]
XD-14819	UUCGAUGCAGGACUAGCAG	TTCGATGCAGGACTAGCAGG
	GCG	CG
	[SEQ ID NO: 166]	[SEQ ID NO: 612]
XD-14835	UUAGUUGAUCCAUAGAUUC	TTAGTTGATCCATAGATTCA
	AGA	GA
	[SEQ ID NO: 198]	[SEQ ID NO: 613]
XD-14857	AGGAACGUGGGUUGAACUC	AGGAACGTGGGTTGAACTCC
	CUU	TT
	[SEQ ID NO: 242]	[SEQ ID NO: 615]
XD-14890	UAUUGCGUGGAGUAAGCUG	TATTGCGTGGAGTAAGCTGG
	GUG	TG
	[SEQ ID NO: 308]	[SEQ ID NO: 618]
XD-14904	AGCGUUAGGGUGCGCAUAC	AGCGTTAGGGTGCGCATACT
	UGC	GC
	[SEQ ID NO: 336]	[SEQ ID NO: 620]
XD-14917	UGUAUACGCCGGCUGAACG	TGTATACGCCGGCTGAACGT
	UGA	GA
	[SEQ ID NO: 362]	[SEQ ID NO: 621]

Embedding of miRNAs in AAV Plasmids

miRNA sequences such as the above are envisioned to have a therapeutic benefit for patients with neurodegenerative disease when expressed from an AAV genome. Therefore, miRNA sequences were inserted into AAV cis-plasmids, flanked by AAV2 inverted terminal repeats (ITRs). miRNAs were inserted in an intron, then followed by an exon expressing green fluorescent protein (GFP). After a stop codon, a SV40 poly adenylation sequence was inserted to ensure robust polyadenylation. The miRNA-encoding transcript was inserted downstream of either a CAG or human Synapsin promoter, as Polymerase-II promoters. The sequence was also inserted into a vector downstream of an H1 promoter, with a CBh promoter controlling the expression of GFP downstream of the H1 miRNA insert. Sequences scAAV.Syn.miR1-1.XD14792.GFP.SV40 (SEQ ID NO:622), scAAV.Syn.miR1-1.XD-14887.GFP.SV40 (SEQ ID NO:623), ssAAV.CAG.miR1-1.XD-14792.GFP.SV40pA (SEQ ID NO:624), ssAAV.CAG.miR-1-1.XD-14887.GFP.SV40pA (SEQ ID NO:625) show representative cis-plasmids with miRNA XD-14792 or XD-147887 inserted. For clinical constructs, GFP sequence are replaced by inert sequence, derived from portions of the genome expected to have no effect if expressed. For Synapsin or H1-promoter containing vectors, the insert was flanked by one full-length ITR and one ITR with a truncated terminal resolution site.

AAV plasmids were generated by conventional large-scale DNA preparation and the integrity of ITRs verified by digestion with the restriction endonuclease SmaI, with the expected banding pattern observed. Plasmids were used to package genomes containing the miRNAs into AAV9-capsid encapsidated viruses (Vector Biolabs). AAVs were titered by qPCR with primers against GFP to calculate genome counts per mL.

AAV Tail Vein Injection

Guide sequences of XD-14792 (SEQ ID NO:112) and XD-14887 (SEQ ID NO:302) are complementary to the mouse ATXN2 transcript, with one base pair mismatch at base 22 of XD-14792. Wu et al. (PLoS One (2011) 6:e28580) and Ohnishi et al. (Biochem Biophys Res Commun (2005) 329:516-21) suggest that these 3′ mismatches do not impair knockdown.

In order to assess the ability of these viruses to knockdown ATXN2 in vivo, concentrated AAV was diluted to a concentration of 3*10¹¹ genome counts per 200 microliters in 0.001% Pluronic F-68 (Gibco #24040-032) in PBS (VWR #K812-500ML). 2 month old C57Bl/6 male mice were each injected intravenously (IV) into the tail vein with 200 microliters of virus (3*10¹¹ GC total injected for viruses with CAG promoters; 2*10¹¹ GC injected for viruses with Synapsin promoters). Fifteen days after injection, mouse tissue was processed for analysis. Following carbon dioxide-induced euthanasia and transcardial perfusion with PBS, tissues were immediately snap-frozen in liquid nitrogen. Samples were subsequently stored at −80° C.

Western Analysis of ATXN2 Levels:

Protein extraction was performed by cutting approximately 50 mg of right medial liver tissue samples on dry ice, placing each into 500 microliters RIPA buffer (TEKNOVA #50-843-016) supplemented with protease and phosphatase inhibitor tablet (Pierce #A32959), Halt protease inhibitor cocktail (Thermo #1861279) and PMSF (Cell Signaling Technology #8553S). Tissues were disrupted in a Precellys Evolution Homogenizer (tubes=0.5 mL CK14, protocol=3×45 s 5000 rpm, 15 s break, 0° C.). Samples were incubated on ice for 30 min, centrifuged for 15 min at 17,000×g at 4° C., and supernatant was transferred to a fresh tube and stored at −80° C. Protein lysates were quantitated (Pierce, 23225), resulting in approximately 8 μg/μl protein per sample.

The NuPage system (Thermo) was used for gel electrophoresis. 20 μg of each sample was loaded onto 4-12% Bis-Tris protein gels (Thermo, NP0321BOX) and run at constant 200V for 1 hr. Revert 700 (Licor, 926-11010) was used to assay for protein loading. Proteins were transferred onto PVDF membrane (EMD Millipore, IPFL00005) overnight at 4° C. using constant 30V and 90 mA. Membranes were blocked for 1 hr at RT (Rockland, MB-070). Primary antibody incubation was performed overnight rocking at 4° C., including anti-Atxn2 (1:1000, BD, 611378), anti-GFP (1:2000, CST, 2956) and beta-actin (1:2000, CST, 4970). Washing was performed 4×5 min with TBS+0.1% tween-20, and secondary antibodies were incubated for 1 hr rocking at RT (1:15,000 each of 800CW goat anti-mouse and 680RD donkey anti-rabbit, Licor). Membranes were washed again and imaging was performed on an Odyssey Fc Imaging system (Licor). Signal quantitation was by Licor image-studio lite.

FIG. 22 (left panel) shows Western analysis of tissues from animals dosed with CAG-promoter containing viruses. Liver tissue from animals dosed with viruses expressing miRNA XD-14792 miR1-1 (SEQ ID NO:1133) or XD-14887 miR1-1 (SEQ ID NO:1149) showed a substantial reduction in ATXN2 signal, as quantified by the ratio of ATXN2 immunoblot signal to Beta-actin signal, relative to a control virus lacking a miRNA (FIG. 22 (right panel)). As expected, since expression from the synapsin promoter is CNS-enriched, AAV with a synapsin promoter expressing the same miRNAs showed much less GFP expression, and did not reduce ATXN2 protein levels (data not shown). Therefore, AAV-mediated delivery of ATXN2 targeting miRNAs can modulate ATXN2 protein levels in vivo, consistent with the therapeutic objective.

To assess whether ATXN2-targeting amiRNAs expressed from AAV dosed into the cerebrospinal fluid could lower ATXN2 levels, neonatal mice were dosed via the intracerebroventricular route (i.c.v.) at postnatal day 0 with AAV-amiRNAs with either CAG or Synapsin promoters (FIG. 53A). AAV expressing XD-14792 in miR1-1 backbone (SEQ ID NO:1133) or XD-14887 in miR1-1 backbone (SEQ ID NO:1149) were used. As in the intravenous dosing experiment, the vectors also included GFP reporters to allow for identification of transduced cells. Cortex tissue was harvested after either 4 or 8 weeks, and ATXN2 protein levels assessed by Western along with GFP levels (FIGS. 53B-53C). Decreased levels of ATXN2 protein were observed relative to tissue from animals dosed with control, non-amiRNA vectors (MCS) at both 4 and 8 weeks with CAG vectors, for XD-14792 amiRNAs, and at 8 weeks with Synapsin promoter vectors.

To verify that the cell types that experienced knockdown included the therapeutically intended target cell types, i.e., neurons, fixed cortex from i.c.v. dosed animals was subject to immunofluorescence analysis with antibodies against Atxn2 and GFP. Clear evidence of reduced anti-ATXN2 immunofluorescence signal was seen in the brain of animals dosed with ATXN2 amiRNAs versus animals dosed with control vector. Within individual tissue sections, transduced and untransduced cells can be distinguished by the expression of the GFP reporter. FIG. 54A shows immunofluorescence of cortex; in tissue from animals dosed with ATXN2 amiRNA (XD-14792 in miR-1-1 backbone, SEQ ID NO:1133) expressing AAVs, comparing neurons expressing GFP with neurons without GFP shows a clear reduction in Axn2 signal in GFP expressing neurons, which will also express the active amiRNA, versus neurons without the GFP. In slices from animals treated with vector without an active amiRNA, there is not an apparent difference in Atxn2 expression level between GFP expressing and non-GFP expressing neurons. Similarly, FIG. 54B shows sections of the cerebellum from animals treated with Atxn2 miRNA (XD-14792 in miR-1-1 backbone, SEQ ID NO:1133) expressing AAV or control virus. In animals treated with Atxn2 miRNA (XD-14792 in miR-1-1 backbone, SEQ ID NO: 1133), GFP expressing neurons (which will also express the Atxn2 miRNA) have lower levels of Atxn2 signal.

Materials and Methods

ATXN2 siRNA Transfection for Immunostaining

U2OS cells (unmodified; wildtype) were seeded at 5,000 cells/well 1 day prior to siRNA transfection in 96-well Flat Clear Bottom Black Polystyrene TC-treated microplates (Corning, P/N 3094). After siRNAs were diluted from stock solutions into Opti-MEM I Reduced Serum Medium (Gibco, P/N 31985-062), transfection mixtures were generated using Lipofectamine RNAiMAX Transfection Reagent (Invitrogen P/N 56532). Transfection mixtures were then aliquoted onto U2OS cells using the Apricot S-PIPETTE S2 and placed into the tissue culture incubator at 37 C/5% CO₂/20% 0₂.

ATXN2 siRNA Immunostaining and Imaging Protocol

Three days after transfection, cells were fixed (4% paraformaldehyde/4% sucrose final), followed by washing (PBS), blocking and permeabilization (IF buffer: 0.5% BSA, 0.2% saponin, 5% goat serum). Primary antibody (BD 611378) was applied to the cells at 1:200 in IF buffer in an overnight incubation. Following PBS washing, cells were incubated in secondary antibody (Life Technologies, Alexa Fluor Plus 488) followed by a DNA stain (Hoechst 33342). After final PBS washing, cells were incubated overnight at 4 C followed by imaging the next day. Using the Thermo Scientific Invitrogen EVOS FL Auto 2 Imaging System with a 20× objective, images were captured by autofocusing on the nuclear DNA stain, capturing the DNA stain, then auto-repositioning to capture the ATXN2 signal with a total of 9 fields imaged per well.

Artificial miRNA Construct Development

Oligonucleotides (Twist) containing Atxn2 targeting shRNAs embedded within miR-1-1 and miR-155E backbones were PCR amplified using regions common to all oligonucleotides (Forward: TAAGCCTGCAGGAATTGCCTAG (SEQ ID NO:626), Reverse: CATGTCTCGACCTGGCTTACTAG (SEQ ID NO:627)). Following amplification, PCR products were verified for the correct sized product by gel electrophoresis. Diluted PCR products were then inserted into a Xba1 and EcoRI-digested pLVX EF1alpha>mCherry CMV>Puro construct, similar to SEQ ID NO:546 using NEB HiFi DNA Assembly Master Mix (NEB P/N M5520AA). A portion of the reaction mixture was then incubated with NEB Stable Competent E. coli cells (NEB P/N C3040H) on ice, heat shocked at 42° C., allowed to recovery on ice, followed by addition of S.O.C. media and incubated at 30° C. The bacterial culture was then applied to LB agar plates with the antibiotic Carbenicillin and grown overnight at 30° C. Individual bacterial colonies were sanger sequence verified (Primer: CATAGCGTAAAAGGAGCAACA (SEQ ID NO:628)). After verifying the correct insert based on the Sanger sequencing, bacterial cultures were grown and the plasmid DNA purified and quantified.

Virus Production

With sequence-verified constructs, lentivirus was produced using Lenti-X 293T cells (Takara) and the pc-Pack2 Plasmid Mix (Cellecta P/N CPCP-K2A). Using the Lipofectamine 3000 Transfection Kit (Invitrogen, P/N L3000-008), Lenti-X 293Ts were transfected with individual pLVX EF1a>mCherry miR insert CMV>Puro constructs and the pc-Pack2 Plasmid Mix. The transfection-containing media was aspirated and replaced with viral product media (VPM; 293T media+20 mM HEPES (gibco, P/N 15630-08)). VPM was collected 48 hours later and aliquoted into 96-well 2.0 mL Deepwell plates (Thermo, P/N 4222) and frozen at −80° C.

Viral Transduction

Prior to adding the VPM to cells, U2OS wildtype (unmodified) and ATXN2 knockout (C43) were seeded at 5,000 cells/well 8 hours prior in 96-well Flat Clear Bottom Black Polystyrene TC-treated microplates (Corning, P/N 3094). After adding polybrene (8 μg/ml final, Cellecta, P/N LTDR1), thawed VPM was added using Apricot S-PIPETTE S2. The cells were then placed into the tissue culture incubator at 37° C./5% CO₂/20% O₂. The media on the cells containing the VPM and polybrene was removed 12 hours later and replace with fresh media (U2OS media only) and placed into the tissue culture incubator at 37° C./5% CO₂/20% O₂.

ATXN2 pLVX EF1a>mCherry miR Insert CMV>Puro Immunostaining and Imaging Protocol

Three days after changing the media (3.5 days since after the VPM), cells were fixed (4% paraformaldehyde/4% sucrose final), followed by washing (PBS), blocking and permeabilization (IF buffer: 0.5% BSA, 0.2% saponin, 5% goat serum). Primary antibodies (Atxn2; BD 611378, 1:200 dilution, mCherry; ab205402, 1:1000 dilution) were applied to the cells in IF buffer in an overnight incubation. Following PBS washing, cells were incubated in secondary antibody (Life Technologies, Alexa Fluor Plus 488 and Alexa Fluor Plus 647) followed by a DNA stain (Hoechst 33342). After final PBS washing, cells were incubated overnight at 4° C. followed by imaging the next day. Using the PerkinElmer Operetta CLS High-Content Analysis System with a 20× objective, non-confocal images were captured by autofocusing the bottom of the plate, then capturing the DNA signal, the ATXN2 signal and the mCherry signal with a total of 9 fields imaged per well.

miR-155 and miR-1-1 Transfection and ATXN2 Western Blot

A version of the miR-155 scaffold was engineered into an artificial miRNA and used in a mouse in vivo proof of concept study to knockdown HTT¹⁰. ATXN2-targeting guide sequences and controls were incorporated into this scaffold sequence, which we term “miR-155M,” and assayed for protein knockdown after transfection of U2OS cells.

The “miR-1-1E,” where “E” signifies “enhanced,” is taking the human miR-1-1 scaffold and simply introducing a downstream CNNC motif.

To perform the transfection, U2OS cells were plated at 90,000 cells/well in a 12-well dish, 24 hours later, transfected 2 micrograms/well of the 8 EF1alpha>mCherry constructs (7 with inserts, 1 control) with Lipofectamine 3000 (ThermoFisher). Specifically, each transfection used 2 μL enhancer reagent, 1.5 μL lipofectamine reagent; diluted samples in water to uniform amounts).

Following day imaging with Evos, a good number of mCherry cells observed. Much higher expression level observed in the control vector without insert.

Wells were aspirated and replaced with 1 ml of media with 1 microgram/mL puromycin. Selection occurred over the weekend and then puromycin was removed for recovery.

Three days post-selection, many dead cells were observed. Imaging of mCherry indicated there remained a good number of bright, surviving cells, however. Aspirated media and replaced with prewarmed media containing 200 ng/mL puromycin (a 5-fold dilution).

Two days later (7 days post-transfection), cells were lysed in RIPA buffer with Pierce phosphatase and protease inhibitor tablet.

Western blot was performed and imaged on Odyssey Fc (Licor).

Quantitation of ATXN2 band and control α-tubulin signal intensity was performed with ImageStudio software (LiCor).

Generation of ATXN2 Knockout in U2OS Cells

ATXN2 knockout cells in U2OS cells was performed using a Cas9-gRNA RNP nucleofection approach. In brief, crRNA and tracrRNA (IDT) were duplexed at equimolar ratios and complexed with recombinant Cas9 (IDT v3) and nucleofected using SF buffer and CM130 program (Lonza 4D nucleofector).

CRISPR guide RNAs were selected from two CRISPR library sources. Three CRISPR guide RNAs (gATXN2_1, gATXN2_2, gATXN2_3) were chosen from the Cellecta CRISPR cutting library (one was not selected due to its upstream position before the 2^nd ATG). Two additional guides (gATXN2_4 & gATXN2_5) were chosen from the another CRISPR cutting library reported by Bassik et al.²⁶. Additionally, a non-targeting control guide was chosen from the Cellecta library. CRISPR guide RNA sequences as well as DNA format are provided in Table 14.

TABLE 14
CRISPR Guide RNA Sequences for Targeting ATXN2
Name	Guide DNA Sequence	Guide RNA Sequence
gATXN2_1	AAGTTACTCACCGTAGACTG	AAGUUACUCACCGUAGACUG
	[SEQ ID NO: 629]	[SEQ ID NO: 1169]
gATXN2_2	AATAGAGAAGTCATATCCTG	AAUAGAGAAGUCAUAUCCUG
	[SEQ ID NO: 630]	[SEQ ID NO: 1170]
gATXN2_3	GTATTGGAAATACCCCCAGT	GUAUUGGAAAUACCCCCAGU
	[SEQ ID NO: 631]	[SEQ ID NO: 1171]
gNTC	GTAGCGAACGTGTCCGGCGT	GUAGCGAACGUGUCCGGCGU
	[SEQ ID NO: 632]	[SEQ ID NO: 1172]
gATXN2_4	GTGAGTTCACCTGCATCCCA	GUGAGUUCACCUGCAUCCCA
	[SEQ ID NO: 633]	[SEQ ID NO: 1173]
gATXN2_5	GCTATCAGTGCTAAAGTGAA	GCUAUCAGUGCUAAAGUGAA
	[SEQ ID NO: 634]	[SEQ ID NO: 1174]
gCD81	GTTGGCTTCCTGGGCTGCTA	GUUGGCUUCCUGGGCUGCUA
(cutting	[SEQ ID NO: 635]	[SEQ ID NO: 1175]
control that
cuts the CD81
gene)

Post nucleofection, bulk population of cells were allowed to recover for 5 days and lysed for western blot analysis.

U2OS Clone Selection

The bulk population of cells were also single cell sorted into 96-well plates for clonal expansion. Because guides gATXN2_1 and gATXN2_5 had the most decrease in ATXN2 protein signal by western blot (˜90% reduction), we proceeded with these cells for single cell cloning. After trypsinization and single cell suspension, a SONY SH800S was used to gate for singlet cells and to sort directly into U2OS growth media. Cells were allowed to grow for ˜2-3 weeks and lysed for genomic DNA extraction for Sanger sequencing and protein extraction for western blotting (10 μg of protein used per lane in this setting)

ICE Confirmation of Clones

Genomic DNA was extracted using a Qiagen Blood and Tissue Kit. Genomic primers were designed to amplify the genomic region surrounding the guide RNA cut site with the goal of sequencing the cut site by Sanger sequencing and validating an out-of-frame indel pattern consistent with a single clone.

Primer Blast (https://www.ncbi.nlm.nih.gov/tools/primer-blast/) was used with the following settings: For guide 1, we turned off repeat filter and low complexity filter due to the repetitive nature of ATXN2, but otherwise kept the default settings. The import function of Snapgene was used to import “6311” from NCBI. 500 upstream and 500 downstream bases from the protospacer sequence was used to as input for primer blast. Product size was set for 400-1000 and 2 distinct primer pairs were selected (Table 15).

TABLE 15
ATXN2 PCR primers
Primer Name             Primer Sequence
Forward primer          AAGTTTCCTGAGGCCTCCCC [SEQ ID
(ICE_gATXN2_1_set1_Fwd) NO: 636]
Reverse primer          TCCAGGCAAGCAGTGCATAG [SEQ ID
(ICE_gATXN2_1_set1_Rev) NO: 637]
Product length 522
Forward primer          AGTTTCCTGAGGCCTCCCC [SEQ ID NO: 638]
(ICE_gATXN2_1_set2_Fwd)
Reverse primer          CCCTACCTGTTGTGGGTCTC [SEQ ID
(ICE_gATXN2_1_set2_Rev) NO: 639]
Product length 539

Furthermore, amplicon internal sequencing primers were designed for Sanger sequencing in both forward and reverse directions to read the cut site (Table 16). The primer(+) algorithm (http://www.biology.wustl.edu/gcg/prime.html) was used to design the sequencing primers on this web interface (https://www.eurofinsgenomics.eu/en/ecom/tools/sequencing-primer-design/).

TABLE 16
ATXN2 Sequencing Primers
Primer Name       Primer Sequence
ICE_gATXN2_1_Rseq AGAGCCATCCTTAGGTAGCC [SEQ ID NO: 640]
ICE_gATXN2_1_Fseq GAGGTTTAATTGACTCATGCTCTG [SEQ ID
                  NO: 641]

For guide 5, we turned off repeat filter but turned on the low complexity filter, but otherwise kept the default settings. 500 upstream and 500 downstream bases from the protospacer sequence was used to as input for primer blast. Product size was set for 400-1000 and 2 distinct primer pairs were selected (Table 17).

TABLE 17
ATXN2 Primer Sequences
Primer Name             Primer Sequence
Forward primer          AAAACACACCGGCATTTCCC [SEQ ID NO: 642]
(ICE_gATXN2_5_set1_Fwd)
Reverse primer          CCTGGGCAACAGAACGAGAC [SEQ ID
(ICE_gATXN2_5_set1_Rev) NO: 643]
Product length 853
Forward primer          AGCAAAACACACCGGCATTT [SEQ ID
(ICE_gATXN2_5_set2_Fwd) NO: 644]
Reverse primer          ATCACGCCACTGCATTCCA [SEQ ID NO: 645]
(ICE_gATXN2_5_set2_Rev)
Product length

Internal sequencing primers were designed by the primer(+) algorithm (Table 18).

TABLE 18
ATXN2 Sequencing Primers
Primer Name       Primer Sequence
ICE_gATXN2_5_Fseq TCATGAGCATCCACAAGAACAG [SEQ ID NO: 646]
ICE_gATXN2_5_Rseq ACGTGTGTGAGTGTAACTGATTGC [SEQ ID
                  NO: 647]

PCR was performed with NEBNext Ultra II Q5 Master Mix (NEB, M0544S) with gDNA and primer pairs indicated above. Amplified products were visualized by agarose gel and correctly sized amplicons were gel purified and submitted for Sanger sequencing with forward and reverse sequencing primers. Chromatogram (.abi files) results were uploaded to the Inference of CRISPR Editing (ICE) tool²⁷ https://ice.synthego.com/#/ for deconvolution of Sanger reads to identify indels.

Clone 43 from guide 5 nucleofection, which verified both by western and Sanger sequencing as a bona fide knock-out clone, was carried forth for further studies.

Ataxin-2 Western Blot

To prepare lysates, 1×RIPA (Teknova, Tris-HCl 50 mM, NaCl 150 mM, 1% Triton X-100, Sodium Deoxycholate 1%, SDS 0.1%, EDTA 2 mM, pH 7.5) was supplemented with Pierce protease/phosphatase tablet (Thermo, A32959) and incubated 15 min on ice, spun down at 17,000×g at 4° C. for 15 min.

Pierce BCA kit (Thermo Scientific, 23225) was used for protein quantitation and 20 μg of protein was loaded per lane in SDS-PAGE gel electrophoresis (NuPage Bis Tris, Thermo Scientific).

Samples were prepared with 10 or 20 micrograms of protein, 4×LDS loading buffer (NP0007), 10× sample reducing agent (NP0004), water to 20 μl. Samples were heated at 70° C. for 10 min.

Protein size ladders were Precision plus protein dual color standard (Bio-rad, 1610374) or Chameleon® Duo Pre-stained Protein Ladder (Licor, 928-60000).

Samples were loaded onto 1.0 mm×10 well 4-12% Bis-Tris protein gel (NP0301PK2) and gel electrophoresis was run with MOPS SDS running buffer (NP0001) for 1 hr 20 min at constant 200V to resolve higher molecular weight bands.

Tris/glycine transfer buffer was used (Bio-rad, 1610734) without methanol. All components including sponges, filter paper, gel, and membrane were equilibrated at least 15 min with transfer buffer. The PVDF membrane was dipped in methanol for 15 seconds prior to equilibration with transfer buffer. Wet transfer was performed in a Mini Trans-Blot Electrophoretic Transfer Cell (Bio-rad, 1703930) overnight at 4° C. at constant 30V, 90 mA.

After overnight transfer, membranes were air dried for 1 hr at RT. Membranes were rinsed with 1×TBS (no tween) and blocked in Odyssey blocking buffer (LI-COR) at room temperature rocking for 30 min-1 hr.

Membranes were incubated with primary antibodies overnight at 4° C. at 1:1000 dilutions in Odyssey blocking buffer with 0.1% Tween-20. The mouse ATXN2 antibody (BD, 611378) and Rabbit α-Tubulin antibody (CST, 21445) was used as a loading control.

Membranes were washed 4×5 min with TBS-0.1% Tween-20.

Membranes were treated with two secondary antibodies for 1 hr rocking at RT at 1:20,000 dilutions in Odyssey blocking buffer with 0.1% Tween-20 and 0.01% SDS.

The secondary antibodies were IRDye 800CW Goat anti-mouse IgG, (Li-cor, 926-32210) and IRDye 680RD Donkey anti-rabbit IgG (Li-cor, 926-68073). Membranes were washed 4×5 min with TBS-0.1% Tween-20 and rinsed with TBS (no Tween) before imaging on a LI-COR Odyssey scanner (Fc) with both 700 and 800 channels.

Ataxin-2 FACS

Cells were trypsinized, transferred to a 96-well v-bottom format, each treatment assayed in triplicate, and washed in wash buffer (PBS/0.5% BSA (no EDTA)) and fixed with ice-cold methanol dropwise, incubated on ice for 10 min, then 200 μl of PBS were added and cells were rocked overnight at 4° C.

Cells were spun down at 1000×g, 5 min cold and washed twice with cold FACS wash buffer (PBS/0.5% BSA/2 mM EDTA/0.2% saponin). Primary antibody (BD 611378) was applied at 1:100 and incubated for 1 hr, rocking in 4° C. The buffer was supplemented with 5% goat serum to reduce non-specific binding. Cells were washed twice in cold FACS wash buffer. Cells were incubated in 1:100 secondary antibody (PE/Cy7 Biolegend clone RMG1-1) with cells resuspended in cold FACS wash buffer with 5% goat serum and incubated for 1 hr on ice. Cells were washed twice and resuspended in cold FACS wash buffer and sampled on an Attune (Thermo Scientific).

Intracerebroventricular Injections

For intracerebroventricular injections, postnatal day 0 pups were cryo-anesthetized and injected at a depth of 2 mm using Hamilton syringes, delivering a maximum volume of 3 uL per each ventricle.

Immunofluorescence Analysis

Animals dosed i.c.v with rAAV were euthanized 4 weeks after dosing with rAAV, fixed overnight in 4% paraformaldehyde. Tissue was then cryopreserved in cold 30% sucrose, then embedded in OCT media and frozen. 5 micrometer frozen sections were prepared on a cryostat. For staining, sections were thawed and dried, washed twice in PBS, heated in 95 C antigen retrieval solution (citra antigen retrieval, pH 6.0, Vector Labs #H-3300-250) for 10 minutes, then cooled for 30 minutes at room temperature. Sections were then washed 5 minutes each in water, PBS, and PBS-0.25% Triton-X-100, and 10 minutes in PBS. Sections were then blocked with 5% goat serum in PBS for 30 minutes in humidified chambers. Sections were treated with primary antibody solution in PBS+1% BSA, including: Mouse anti-ATXN2 antibody (BD #611378), 1:50; Rabbit anti-GFP antibody (Cell Signaling Technologies #25555), 1:2000 overnight at 4 C. After 3× washes in PBS, sections were incubated with secondary antibody solutions in PBS+1% BSA, including: goat anti-mouse Alexa Fluor 555 (Thermo Scientific #A21424) 1:250, Goat anti-Rabbit Alexa Fluor 488 (Thermo Scientific #A11008), 1:250 for 30 minutes at room temperature. Sections were then washed, and mounted in VectaShield PLUS with Dapi (H-2000-10). Images were collected with a Revolve microscope (Discover Echo).

Example 2: Identification of High Performing AmiRNAs by Tiled Screen of ATXN2 Targeting miRNAs in Lentiviral Format

As an alternative approach to siRNA screening followed by embedding of the associated guide sequences in miRNA backbones and testing one-by-one, pooled screening of ATXN2-targeting miRNAs was conducted (“Deep Screen 1”).

ATXN2 Target Sequences

Homo sapiens ATXN2 mRNA (NM_002973, transcript variant 1, SEQ ID NO:2) was used to identify target sequences for the artificial miRNAs. All human and primate cross-reactive sequences were identified and 22-nt guide sequences were designed taking into consideration criteria for effective shRNA and miRNA sequences, including the preference for A or U at guide position 1. Therefore, taking into consideration the 22 nucleotide antisense sequences complementary to the Ataxin-2 construct, if the first guide base was G or C this was converted to a ‘U’, whereas sequences that began with A or U were not changed from the base complementary to the corresponding position on the ATXN2 transcript. As above, U bases are encoded as T in the lentiviral expression construct. In total 2,381 ATXN2-targeting sequences were introduced into a modified variant of the miR-16-2 backbone. Passenger sequences (the sequence on the opposite side of the miRNA stem from the guide sequence) were generated following the rules in Table 8 for this backbone.

Toxicity Controls

By examining the abundance of elements of the library in cells that had been allowed to grow for lengthy periods of time versus initially transduced cells, the pooled screen can identify elements that alter cellular proliferation or viability. To calibrate the dynamic range of the assay, additional toxic elements were added to the library. Ten essential genes were selected with ten shRNAs each (removing 2 sequences that had polyT sequences deemed problematic because they may serve as termination signals for PolIII). To identify the “essential” gene list, genetic dropout screens performed in parallel with shRNA and CRISPR guide RNAs in the K562 cancer cell line²¹ were examined. Across both screens, genes were rank ordered by shRNA lethality, specifically genes that scored highly in the K562 shRNA dropout by combined Castle score (negative is more lethal). Since toxicity screen was performed in Hela cells, the K562 top genes were intersected and identified the top 10 genes that also scored highly (bayesian factor >100) in a Hela CRISPR cutting dropout screen²². The essential genes selected were: COPB1, COPB2, DHX15, EIF3A, EIF4A3, NUP93, PRPF8, PSMB6, PSMD1, and SF3B2.

To select 10 shRNA targeting each gene, the 25 shRNA/gene in a previously published shRNA library were considered and rank ordered by their performance in the dropout screen¹⁵. Specifically, the shRNAs were rank-ordered by the dropout metric (read counts in replicate 1 and replicate 2 divided by plasmid reads), and the top performing shRNAs that had at least one count across all replicates were selected.

GFP Controls

GFP controls (n=50) were designed to target two different GFP reporter systems. The first system involved tagging endogenous ATXN2 with the 11^th beta strand of GFP (GFP₁₁) in conjunction with overexpression of GFP_1-10 to constitute a self-complementary GFP system²³, and the second is a GFP-stop-ATXN2 overexpression reporter. The 11^th beta strand of GFP was targeted by entirely tiling the transcript with 28 individual 21 nt shRNA, adding an A at guide position 1 to form 22 nt oligomer sequences. Additional shGFP (n=22) were selected to target GFP_1-10 using the Design portal of the Broad Institute Genetic Perturbation Platform (https://portals.broadinstitute.org/gpp/public/seq/search), using the GFP_1-10 sequence as input. Although the split GFP system was not ultimately used to read out ATXN2 levels, the 50 shGFP still target the GFP-stop-ATXN2 reporter.

ATXN2 Transcript Scrambled Controls

Neutral controls were designed that should not have any effect in both the efficacy and toxicity screens. These elements can be used for baseline normalization. The guide sequences targeting ATXN2 were scrambled and 974 of these scrambled guide sequences used to construct amiRNAs as before. After scrambling, the same rules for the first base as with targeting sequence were imposed. Following this correction step, the GC content was adjusted by converting one of the guide bases 2-22 that were A or T, randomly selected, to G or C, randomly chosen, such that overall this set of scrambled controls maintains similar GC content relative to the ATXN2-targeting sequences.

Promoter Selection

The H1 promoter, an RNA polymerase III promoter, was selected to drive artificial miRNA expression as many groups have used it to achieve robust target knockdown.

Pooled Library Cloning

The oligonucleotide pool was synthesized on chip (oligo length 172 bp, Agilent), PCR amplified, and cloned into the pRSICPH1 vector (Cellecta) by Bpl1 restriction digestion and T4 ligase ligation. Each individual miRNA cassette was expressed under the control of an H1 promoter and subsequently followed by a short constant region and 17 bp barcode sequence that uniquely tags each miRNA. The elements were designed to contain both miRNA and barcode tags to enable multiple ways to amplify and sequence the constructs to readout the pooled screens. For instance, if PCR amplification bias were to confound the representation of high GC content sequences²⁴, comparison of the abundance of amplicons containing the guide sequence versus the abundance of amplicons containing the FREE barcode would resolve any discrepancies. FREE barcodes were used as they are indel-correcting and robust to DNA synthesis and NGS errors²⁵. The library was checked by Sanger sequencing and next-generation sequence (Illumina) to verify lack of synthesis errors, >99% amiRNA and FREE barcode were correctly paired, and the fold-representation between the top and bottom amiRNAs were within four fold-change.

Viral Production

Lenti-X 293T (Takara) cells were used to produce lentivirus by transfection of 4^th generation packaging plasmids (Lenti-X Packaging single shots, Takara) followed by viral concentration with Lenti-X concentrator and resuspension in PBS. Virus was titered in U2OS and Hela cells by infection and antibiotic selection followed with estimation of viral units and multiplicity of infection (MOI) by Cell-Titer-Glo (Promega).

Cell Culture and Transfections

U2OS cells and the GFP-ATXN2 reporter cell line were cultured in RPMI-1640 supplemented with 10% fetal bovine serum (FBS) and penicillin/streptomycin/glutamine. Hela cells were cultured in DMEM supplemented with 10% fetal bovine serum (FBS) and penicillin/streptomycin/glutamine.

Efficacy Screen

Two pooled lentiviral miRNA screens for on-target efficacy were performed to identify miRNA that diminish ATXN2 protein signal, reading out ATXN2 levels by 1) an exogenous GFP-stop-ATXN2 reporter or 2) endogenous ataxin-2 antibody in a FACS assay. Cells were infected with the pooled lentiviral library at a multiplicity of infection (MOI) of 0.1 into (˜5×10⁷ cells) with polybrene (8 μg/ml, EMD Millipore) and distributed across four T225 flasks. Two days post-infection, U2OS cells were selected with puromycin at 2 μg/ml. The MOI was confirmed by cell-titer-glo at day 5 (3 days after selection) in a 96 well format. An unsorted fraction (7×10⁶ cells) was collected at day 7 as a reference control. The remaining cells were washed in wash buffer (PBS/0.5% BSA (no EDTA)) and fixed with ice-cold methanol dropwise while vortexing on day 7, at a ratio of 1 ml methanol/2×10⁶ cells, incubated on ice for 10 min, then 10× volumes of PBS were added and cells were rocked overnight at 4° C.

Cells were spun down at 1000×g, 5 min cold (Corning 500 ml centrifuge tubes, 431123) and resuspended in cold FACS wash buffer (PBS/0.5% BSA/2 mM EDTA/0.2% saponin). Cells were counted and resuspended in 2×10⁶/ml in cold FACS wash buffer.

Primary antibody (BD 611378) was applied at 1:200 and incubated for 1 hr, rocking in 4° C. The buffer was supplemented with 5% goat serum to reduce non-specific binding.

Cells were washed twice in cold FACS wash buffer. Cells were incubated in 1:200 secondary antibody (PE/Cy7 Biolegend clone RMG1-1) with cells resuspend in 2×10⁶/ml cold FACS wash buffer with 5% goat serum and incubated for 1 hr on ice. Cells were washed twice in and resuspended in cold FACS wash buffer at 4-5×10⁶/ml to achieve 1000-2000 events per second on the Sony SH800S (approximately the maximal stable cell velocity on the instrument). Samples were filtered through a cell strainer directly into FACS tubes (FALCON 352235). Sorted cells were collected in 3 mL PBS/10% FBS in 15 ml conicals.

Dropout Screen

A pooled lentiviral miRNA screen for off-target toxicity was additionally performed, by identifying miRNA dropout between an early and late timepoint. HeLa cells were infected with polybrene (8 μg/ml, EMD Millipore) at a multiplicity of infection of 0.1 at 1000× representation (that is, the number of cells was >10,000× the number of library elements). Two days post-infection, HeLa cells were selected with puromycin at 0.5 micrograms/mL. Cells were passaged for a total of 10 doublings (˜16 days). The screen was performed in triplicate (3 separate infections).

DNA Processing

Genomic DNA was extracted from each sample using the Machery Nagel Blood L kit (FACS collections; early and late collection timepoints). A two-step PCR was conducted. In a first PCR reaction, an amplicon spanning both the guide and passenger sequences, and downstream past the FREE barcode, was generated. In a second PCR reaction, a nested amplicon was generated spanning either the guide and passenger sequence, or the FREE barcode. The second PCR was designed to incorporate Illumina binding sequences (P5 and P7) and sample index barcodes to enable demultiplexing on Illumina sequencing platforms. Each distinct sample (that is, FACS collection, or timepoint) was given a distinct index. Specifically, the guide and passenger amplicon was single-indexed, with an i7 sequence included upstream of the 6 nt sample barcode and P7 sequence. In contrast, the FREE barcode amplicon was single-indexed on the P5 end and no i7 sequence was included on the P7 end. Samples were sequenced on an Illumina MiSeq such that guide and passenger sequences can be matched in paired reads, with read 1 using a custom primer reading the 22 nt guide sequence, and read 2 being the standard Illumina primer reading the passenger sequence. FREE barcodes were also separately amplified and sequenced, with read 1 being a custom primer reading the 17 nt FREE barcode, and read 2 being a custom primer reading the 6 nt sample barcode. In general, calculations of abundances were highly similar for FREE barcode derived amplicons and guide/passenger sequence containing amplicons (using a lookup table of the association between FREE barcodes and guide/passenger sequences). Analyses below focused on counts of the guide sequences.

Computational Analysis

Occurrences of each guide sequence were counted, without tolerating sequencing or other errors (that is, no mismatches to the library input guide sequences were tolerated), in read 1 sequences, which directly sequences amiRNA guide sequences. To estimate ATXN2 knockdown efficiency, the abundance of guide sequence counts in the ATXN2 high FACS collection was divided by the abundance of guide sequence counts in the ATXN2 low FACS collection. Sequences that effectively knock down ATXN2 are enriched in the ATXN2 low FACS collection.

To assess whether the guide sequence influences cytotoxicity or reduces proliferation, the ratio of counts of each guide sequence for a pool of cells collected 16 days after library transduction, versus the ratio of counts for the library collected 18 hours after library transduction, were measured.

Data was highly consistent across replicates. FIG. 23A plots the high/low count ratios for two independent replicates against one another. Most points fall along y=x, indicating good correlation. FIG. 23B plots the matrix of Spearman correlation coefficients for count values for each condition against all others. The replicates are hierarchically clustered, and clustered blocks represent similar conditions. Note the strong anticorrelation between low and high conditions, as expected given that guides that deplete ATXN2 are expected to be differentially present in the low and high conditions. Note also that conditions where ATXN2 signal was visualized by antibody staining against endogenous Ataxin-2 protein, and conditions where the signal was visualized by fluorescence of the ATXN2 GFP reporter, correlate.

Following the calculation of count ratios, a normalization procedure was taken to rank ATXN2 targeting sequences by their ability to deplete ATXN2 signal. In FIG. 24, histograms for the distribution of high and low condition guide sequence counts for ATXN2 targeting guides, top trellis, and scrambled sequences, bottom trellis, are shown. The ATXN2 scrambled sequences exhibit a sharp, unimodal distribution of ratios of counts in the high and low ATXN2 FACS conditions. The median ratio from this distribution was taken to be no-effect, and the ATXN2 depleting effect of ATXN2 targeting miRNAs was therefore calculated by subtracting this (log base 2-transformed) value.

The ability of guide sequences to knock down ATXN2 and the presence of any altered proliferation or cytotoxicity were examined. FIG. 25 shows a plots of three classes of guide sequences in this experiment: ATXN2 targeting sequences, ATXN2 scrambled sequences, and amiRNAs targeting essential genes (predicted to be toxic). As expected, the log base-2 ATXN2 signal depletion (the scramble-baseline-corrected ATXN2 depletion in counts from high to low ATXN2 FACS conditions) was centered around 0 (no effect). However, many of these sequences exhibited remarkable shifts in abundance at a late collection timepoint, 16 days after transduction, versus an early timepoint after transduction. This is consistent with the reported essentiality for these sequences and demonstrates that this system can elicit cellular toxicity or proliferation impairment.

ATXN2 targeting guide sequences fall along a much wider spectrum along the axis of ATXN2 signal depletion compared to amiRNAs targeting essential genes or scrambled sequences, with targeting sequences exceeding 5 logs (base 2), corresponding to approximately 32-fold depletion of cells expressing these amiRNAs in high ATXN2 FACS collections versus low ATXN2 FACS collections.

The near complete tiling of the ATXN2 transcript enables the detection of ‘hotspots’ of Ataxin-2 targeting guide sequences, defined by the proximity of their complementary regions of the Ataxin-2 transcript. FIG. 26 shows a plot of the knockdown efficacy, as measured by the depletion of counts for a given guide from the high ATXN2 FACS collection versus low ATXN2 FACS collection. Across the transcript, multiple regions where adjacent ATXN2 targeting guide sequences exhibit strong ATXN2 knockdown are noted. FIG. 27 shows a ‘zoom-in’ of regions within the 3′ UTR of ATXN2, and highlights guide sequences (as dark points) with unusually high ATXN2 lowering, as measured by the count reduction.

Small RNAseq Confirmation of Pri-miRNA Processing Precision in the Pooled Screen

Guide sequences are excised from a miRNA stem by successive Drosha and Dicer processing. Each enzyme cuts the RNA. In the case of the miR backbone used for this tiled screen of ATXN2, the guide sequence from the corresponding endogenous miRNA (miR 16-2) is excised from the upstream, 5 prime arm, and therefore the guide sequence is cleaved from the parent stem at the 5′ side by Drosha. Because the position of the 5′ cut site determines the composition of the seed sequence, bases 2-7 counting from the 5′ nucleotide, the cutting position is important in determining both on- and off-target activity of the resulting guide sequence. Therefore, small RNAseq was conducted to assess the position of this cut.

The tiling library, in packaged lentiviral form, was transduced at high multiplicity of infection into U2OS cells. After selection by puromycin to eliminate untransduced cells (the library vector contains a puromycin selection cassette), RNA was extracted by standard methods, and small RNA was purified and ligated with adapters to enable small RNA sequencing using the Nextflex small RNAseq kit v3. After PCR amplification, the resulting library was subject to next-generation sequencing on an Illumina MiSeq. A high proportion of reads had sequences of length 21, 22, and 23 nucleotides, with a peak at 22 nucleotides, consistent with the detection of processed miRNAs (guide and passenger sequences). To examine the precision of 5′ processing, the number of observations of 22-mer sequences matching several models of processed guide sequences were calculated. In one model, the guide sequence was assumed to be correctly processed. In other models, the guide sequence was assumed to be processed either upstream or downstream of the expected nucleotide. If the guide sequence is cut upstream of the intended nucleotide, then the expected upstream bases are incorporated from the miRNA backbone sequence. If the guide sequence is cut downstream of the intended nucleotide, then the first base of the resulting guide sequence is downstream of expected. Because the scrambled sequences in the library do not generally overlap from one another, for example, lowering the likelihood of ‘collisions’ where a guide sequence processed by excision from the stem at a nucleotide one downstream of the intended first nucleotide is the same as a guide sequence aligning to a position in the ATXN2 transcript one bp shifted, the processing position across all scrambling sequences was analyzed and averaged to estimate the most probable cutting position. FIG. 28 plots the percent of reads of the guide sequence with cut position at each nucleotide relative to the intended first nucleotide, and shows a very high proportion of reads begin at the intended position.

Additional ATXN2 Targeting Sequences from Pooled Screen

By examination of the knockdown efficacy against ATXN2 (as measured by depletion from the high versus low ATXN2 FACS collections) across the positions of complementarity to the ATXN2 transcript, several regions of interest were noted where clusters of high performing ATXN2-targeting guide sequences were observed. Table 19 lists these guide sequences, the targeting position of the guide sequences relative to the ATXN2 transcript (SEQ ID NO:2), the guide sequences inserted into the miRNA16-2 backbone (which are also the highest probability sequence that will be generated in the cell according to the above small RNAseq experiments), and the passenger sequences generated for the miR16-2 backbone. The guide sequences, miRNA16-2 formatted passenger sequences, and amiRNA sequences are provided in Table 19 in RNA format and DNA format (e.g., for insertion into a plasmid for AAV). Exemplary passenger RNA sequences (e.g., not modified for a specific miRNA backbone) are also provided in Table 19 in both RNA and DNA format. Efficacy of ATXN2 knockdown is represented by the signal depletion column. Altogether, sequences with high efficacy and low potential for dropout may represent good candidates to incorporate into therapeutic vectors targeting ATXN2.

TABLE 19
Guide sequences in ‘hot spots’ targeting ATXN2 from tiled screen and corresponding passenger and miRNA sequences
					Mean
					Atxn2
Guide	Guide	16_2_format_	16_2_format_	siRNA	Log2
Sequence	Sequence	passenger	passenger	Passenger	Signal	Atxn2	amiRNA Sequence	amiRNA
(DNA)	(RNA)	(DNA)	(RNA)	(RNA)	Depletion	Position	(DNA)	Sequence (RNA)
TACCACAACAAA	UACCACAACAAA	ATGTTCAGACCC	AUGUUCAGACCC	AUGUUCAGACUU	-2.75633	1157	ACCGGACATACTTGTT	ACCGGACAUACUUGUU
GTCTGAACAT	GUCUGAACAU	TGTTGTGGTT	UGUUGUGGUU	UGUUGUGG			CCACTCTACCACAACA	CCACUCUACCACAACA
[SEQ ID	[SEQ ID	[SEQ ID	[SEQ ID	[SEQ ID			AAGTCTGAACATTAGT	AAGUCUGAACAUUAGU
NO: 648]	NO: 1176]	NO: 761]	NO: 1289]	NO: 993]			GAAATATATATTAAAA	GAAAUAUAUAUUAAAA
							TGTTCAGACCCTGTTG	UGUUCAGACCCUGUUG
							TGGTTTAGTGTGACAG	UGGUUUAGUGUGACAG
							GGATACAGCAACTATT	GGAUACAGCAACUAUU
							TTATCAA	UUAUCAA
							[SEQ ID NO: 877]	[SEQ ID NO: 1405]
TTACCACAACAA	UUACCACAACAA	TGTTCAGACTCC	UGUUCAGACUCC	UGUUCAGACUUU	-2.02091	1158	ACCGGACATACTTGTT	ACCGGACAUACUUGUU
AGTCTGAACA	AGUCUGAACA	GTTGTGGTAT	GUUGUGGUAU	GUUGUGGU			CCACTCTTACCACAAC	CCACUCUUACCACAAC
[SEQ ID	[SEQ ID	[SEQ ID	[SEQ ID	[SEQ ID			AAAGTCTGAACATAGT	AAAGUCUGAACAUAGU
NO: 649]	NO: 1177]	NO: 762]	NO: 1290]	NO: 994]			GAAATATATATTAAAT	GAAAUAUAUAUUAAAU
							GTTCAGACTCCGTTGT	GUUCAGACUCCGUUGU
							GGTATTAGTGTGACAG	GGUAUUAGUGUGACAG
							GGATACAGCAACTATT	GGAUACAGCAACUAUU
							TTATCAA	UUAUCAA
							[SEQ ID NO: 878]	[SEQ ID NO: 1406]
TGTACCACAACA	UGUACCACAACA	GTTCAGACTTCT	GUUCAGACUUCU	GUUCAGACUUUG	-3.6909	1159	ACCGGACATACTTGTT	ACCGGACAUACUUGUU
AAGTCTGAAC	AAGUCUGAAC	TTGTGGTACT	UUGUGGUACU	UUGUGGUA			CCACTCTGTACCACAA	CCACUCUGUACCACAA
[SEQ ID	[SEQ ID	[SEQ ID	[SEQ ID	[SEQ ID			CAAAGTCTGAACTAGT	CAAAGUCUGAACUAGU
NO: 603]	NO: 40]	NO: 763]	NO: 1291]	NO: 995]			GAAATATATATTAAAG	GAAAUAUAUAUUAAAG
	(same as						TTCAGACTTCTTTGTG	UUCAGACUUCUUUGUG
	guide in XD-						GTACTTAGTGTGACAG	GUACUUAGUGUGACAG
	14756)						GGATACAGCAACTATT	GGAUACAGCAACUAUU
							TTATCAA	UUAUCAA
							[SEQ ID NO: 879]	[SEQ ID NO: 1407]
TTGTACCACAAC	UUGUACCACAAC	TTCAGACTTTTC	UUCAGACUUUUC	UUCAGACUUUGU	-1.52769	1160	ACCGGACATACTTGTT	ACCGGACAUACUUGUU
AAAGTCTGAA	AAAGUCUGAA	TGTGGTACAT	UGUGGUACAU	UGUGGUAC			CCACTCTTGTACCACA	CCACUCUUGUACCACA
[SEQ ID	[SEQ ID	[SEQ ID	[SEQ ID	[SEQ ID			ACAAAGTCTGAATAGT	ACAAAGUCUGAAUAGU
NO: 650]	NO: 1178]	NO: 764]	NO: 1292]	NO: 996]			GAAATATATATTAAAT	GAAAUAUAUAUUAAAU
							TCAGACTTTTCTGTGG	UCAGACUUUUCUGUGG
							TACATTAGTGTGACAG	UACAUUAGUGUGACAG
							GGATACAGCAACTATT	GGAUACAGCAACUAUU
							TTATCAA	UUAUCAA
							[SEQ ID NO: 880]	[SEQ ID NO: 1408]
ACTGTACCACAA	ACUGUACCACAA	TCAGACTTTGCC	UCAGACUUUGCC	UCAGACUUUGUU	-3.46564	1161	ACCGGACATACTTGTT	ACCGGACAUACUUGUU
CAAAGTCTGA	CAAAGUCUGA	GTGGTACAGA	GUGGUACAGA	GUGGUACA			CCACTCACTGTACCAC	CCACUCACUGUACCAC
[SEQ ID	[SEQ ID	[SEQ ID	[SEQ ID	[SEQ ID			AACAAAGTCTGATAGT	AACAAAGUCUGAUAGU
NO: 651]	NO: 1179]	NO: 765]	NO: 1293]	NO: 997]			GAAATATATATTAAAT	GAAAUAUAUAUUAAAU
							CAGACTTTGCCGTGGT	CAGACUUUGCCGUGGU
							ACAGATAGTGTGACAG	ACAGAUAGUGUGACAG
							GGATACAGCAACTATT	GGAUACAGCAACUAUU
							TTATCAA	UUAUCAA
							[SEQ ID NO: 881]	[SEQ ID NO: 1409]
AACTGTACCACA	AACUGUACCACA	CAGACTTTGTCT	CAGACUUUGUCU	CAGACUUUGUUG	-3.89476	1162	ACCGGACATACTTGTT	ACCGGACAUACUUGUU
ACAAAGTCTG	ACAAAGUCUG	TGGTACAGTA	UGGUACAGUA	UGGUACAG			CCACTCAACTGTACCA	CCACUCAACUGUACCA
[SEQ ID	[SEQ ID	[SEQ ID	[SEQ ID	[SEQ ID			CAACAAAGTCTGTAGT	CAACAAAGUCUGUAGU
NO: 652]	NO: 1180]	NO: 766]	NO: 1294]	NO: 998]			GAAATATATATTAAAC	GAAAUAUAUAUUAAAC
							AGACTTTGTCTTGGTA	AGACUUUGUCUUGGUA
							CAGTATAGTGTGACAG	CAGUAUAGUGUGACAG
							GGATACAGCAACTATT	GGAUACAGCAACUAUU
							TTATCAA	UUAUCAA
							[SEQ ID NO: 882]	[SEQ ID NO: 1410]
AAACTGTACCAC	AAACUGUACCAC	AGACTTTGTTTC	AGACUUUGUUUC	AGACUUUGUUGU	-2.62277	1163	ACCGGACATACTTGTT	ACCGGACAUACUUGUU
AACAAAGTCT	AACAAAGUCU	GGTACAGTTA	GGUACAGUUA	GGUACAGU			CCACTCAAACTGTACC	CCACUCAAACUGUACC
[SEQ ID	[SEQ ID	[SEQ ID	[SEQ ID	[SEQ ID			ACAACAAAGTCTTAGT	ACAACAAAGUCUUAGU
NO: 653]	NO: 1181]	NO: 767]	NO: 1295	NO: 999]			GAAATATATATTAAAA	GAAAUAUAUAUUAAAA
							GACTTTGTTTCGGTAC	GACUUUGUUUCGGUAC
							AGTTATAGTGTGACAG	AGUUAUAGUGUGACAG
							GGATACAGCAACTATT	GGAUACAGCAACUAUU
							TTATCAA	UUAUCAA
							[SEQ ID NO: 883]	[SEQ ID NO: 1411]
TTAAACTGTACC	UUAAACUGUACC	ACTTTGTTGTTT	ACUUUGUUGUUU	ACUUUGUUGUGG	-0.49686	1165	ACCGGACATACTTGTT	ACCGGACAUACUUGUU
ACAACAAAGT	ACAACAAAGU	TACAGTTTAT	UACAGUUUAU	UACAGUUU			CCACTCTTAAACTGTA	CCACUCUUAAACUGUA
[SEQ ID	[SEQ ID	[SEQ ID	[SEQ ID	[SEQ ID			CCACAACAAAGTTAGT	CCACAACAAAGUUAGU
NO: 654]	NO: 1182]	NO: 768]	NO: 1296]	NO: 1000]			GAAATATATATTAAAA	GAAAUAUAUAUUAAAA
	(same as						CTTTGTTGTTTTACAG	CUUUGUUGUUUUACAG
	guide in XD-						TTTATTAGTGTGACAG	UUUAUUAGUGUGACAG
	14757)						GGATACAGCAACTATT	GGAUACAGCAACUAUU
							TTATCAA	UUAUCAA
							[SEQ ID NO: 884]	[SEQ ID NO: 1412]
TTGCTAACTGGT	UUGCUAACUGGU	GCAAGGGCAAGA	GCAAGGGCAAGA	GCAAGGGCAAAC	-3.48781	1479	ACCGGACATACTTGTT	ACCGGACAUACUUGUU
TTGCCCTTGC	UUGCCCUUGC	CAGTTAGCAT	CAGUUAGCAU	CAGUUAGC			CCACTCTTGCTAACTG	CCACUCUUGCUAACUG
[SEQ ID	[SEQ ID	[SEQ ID	[SEQ ID	[SEQ ID			GTTTGCCCTTGCTAGT	GUUUGCCCUUGCUAGU
NO: 655]	NO: 1183]	NO: 769]	NO: 1297]	NO: 1001]			GAAATATATATTAAAG	GAAAUAUAUAUUAAAG
							CAAGGGCAAGACAGTT	CAAGGGCAAGACAGUU
							AGCATTAGTGTGACAG	AGCAUUAGUGUGACAG
							GGATACAGCAACTATT	GGAUACAGCAACUAUU
							TTATCAA	UUAUCAA
							[SEQ ID NO: 885]	[SEQ ID NO: 1413]
TGGGTTGAAATC	UGGGUUGAAAUC	TCACACTTCATG	UCACACUUCAUG	UCACACUUCAGA	-1.9344	1754	ACCGGACATACTTGTT	ACCGGACAUACUUGUU
TGAAGTGTGA	UGAAGUGUGA	TTTCAACCCT	UUUCAACCCU	UUUCAACC			CCACTCTGGGTTGAAA	CCACUCUGGGUUGAAA
[SEQ ID	[SEQ ID	[SEQ ID	[SEQ ID	[SEQ ID			TCTGAAGTGTGATAGT	UCUGAAGUGUGAUAGU
NO: 656]	NO: 1184]	NO: 770]	NO: 1298]	NO: 1002]			GAAATATATATTAAAT	GAAAUAUAUAUUAAAU
							CACACTTCATGTTTCA	CACACUUCAUGUUUCA
							ACCCTTAGTGTGACAG	ACCCUUAGUGUGACAG
							GGATACAGCAACTATT	GGAUACAGCAACUAUU
							TTATCAA	UUAUCAA
							[SEQ ID NO: 886]	[SEQ ID NO: 1414]
TCGGGTTGAAAT	UCGGGUUGAAAU	CACACTTCAGGC	CACACUUCAGGC	CACACUUCAGAU	-4.71279	1755	ACCGGACATACTTGTT	ACCGGACAUACUUGUU
CTGAAGTGTG	CUGAAGUGUG	TTCAACCCGT	UUCAACCCGU	UUCAACCC			CCACTCTCGGGTTGAA	CCACUCUCGGGUUGAA
[SEQ ID	[SEQ ID	[SEQ ID	[SEQ ID	[SEQ ID			ATCTGAAGTGTGTAGT	AUCUGAAGUGUGUAGU
NO: 657]	NO: 1185]	NO: 771]	NO: 1299]	NO: 1003]			GAAATATATATTAAAC	GAAAUAUAUAUUAAAC
							ACACTTCAGGCTTCAA	ACACUUCAGGCUUCAA
							CCCGTTAGTGTGACAG	CCCGUUAGUGUGACAG
							GGATACAGCAACTATT	GGAUACAGCAACUAUU
							TTATCAA	UUAUCAA
							[SEQ ID NO: 887]	[SEQ ID NO: 1415]
TTCGGGTTGAAA	UUCGGGUUGAAA	ACACTTCAGACC	ACACUUCAGACC	ACACUUCAGAUU	-3.7055	1756	ACCGGACATACTTGTT	ACCGGACAUACUUGUU
TCTGAAGTGT	UCUGAAGUGU	TCAACCCGAT	UCAACCCGAU	UCAACCCG			CCACTCTTCGGGTTGA	CCACUCUUCGGGUUGA
[SEQ ID	[SEQ ID	[SEQ ID	[SEQ ID	[SEQ ID			AATCTGAAGTGTTAGT	AAUCUGAAGUGUUAGU
NO: 607]	NO: 108]	NO: 772]	NO: 1300]	NO: 1004]			GAAATATATATTAAAA	GAAAUAUAUAUUAAAA
	(same as						CACTTCAGACCTCAAC	CACUUCAGACCUCAAC
	guide in XD-						CCGATTAGTGTGACAG	CCGAUUAGUGUGACAG
	14790)						GGATACAGCAACTATT	GGAUACAGCAACUAUU
							TTATCAA	UUAUCAA
							[SEQ ID NO: 888]	[SEQ ID NO: 1416]
TGATGCAGGACT	UGAUGCAGGACU	TACGCCTGCTGT	UACGCCUGCUGU	UACGCCUGCUAG	-1.91676	2351	ACCGGACATACTTGTT	ACCGGACAUACUUGUU
AGCAGGCGTA	AGCAGGCGUA	TCCTGCATCT	UCCUGCAUCU	UCCUGCAU			CCACTCTGATGCAGGA	CCACUCUGAUGCAGGA
[SEQ ID	[SEQ ID	[SEQ ID	[SEQ ID	[SEQ ID			CTAGCAGGCGTATAGT	CUAGCAGGCGUAUAGU
NO: 658]	NO: 1186]	NO: 773]	NO: 1301]	NO: 1005]			GAAATATATATTAAAT	GAAAUAUAUAUUAAAU
							ACGCCTGCTGTTCCTG	ACGCCUGCUGUUCCUG
							CATCTTAGTGTGACAG	CAUCUUAGUGUGACAG
							GGATACAGCAACTATT	GGAUACAGCAACUAUU
							TTATCAA	UUAUCAA
							[SEQ ID NO: 889]	[SEQ ID NO: 1417]
TCGATGCAGGAC	UCGAUGCAGGAC	ACGCCTGCTATC	ACGCCUGCUAUC	ACGCCUGCUAGU	-2.27165	2352	ACCGGACATACTTGTT	ACCGGACAUACUUGUU
TAGCAGGCGT	UAGCAGGCGU	CCTGCATCGT	CCUGCAUCGU	CCUGCAUC			CCACTCTCGATGCAGG	CCACUCUCGAUGCAGG
[SEQ ID	[SEQ ID	[SEQ ID	[SEQ ID	[SEQ ID			ACTAGCAGGCGTTAGT	ACUAGCAGGCGUUAGU
NO: 659]	NO: 1187]	NO: 774]	NO: 1302]	NO: 1006]			GAAATATATATTAAAA	GAAAUAUAUAUUAAAA
							CGCCTGCTATCCCTGC	CGCCUGCUAUCCCUGC
							ATCGTTAGTGTGACAG	AUCGUUAGUGUGACAG
							GGATACAGCAACTATT	GGAUACAGCAACUAUU
							TTATCAA	UUAUCAA
							[SEQ ID NO: 890]	[SEQ ID NO: 1418]
TTCGATGCAGGA	UUCGAUGCAGGA	CGCCTGCTAGCA	CGCCUGCUAGCA	CGCCUGCUAGUC	-4.78943	2353	ACCGGACATACTTGTT	ACCGGACAUACUUGUU
CTAGCAGGCG	CUAGCAGGCG	CTGCATCGAT	CUGCAUCGAU	CUGCAUCG			CCACTCTTCGATGCAG	CCACUCUUCGAUGCAG
[SEQ ID	[SEQ ID	[SEQ ID	[SEQ ID	[SEQ ID			GACTAGCAGGCGTAGT	GACUAGCAGGCGUAGU
NO: 612]	NO: 166]	NO: 775]	NO: 1303]	NO: 1007]			GAAATATATATTAAAC	GAAAUAUAUAUUAAAC
	(same as						GCCTGCTAGCACTGCA	GCCUGCUAGCACUGCA
	guide in XD-						TCGATTAGTGTGACAG	UCGAUUAGUGUGACAG
	14819)						GGATACAGCAACTATT	GGAUACAGCAACUAUU
							TTATCAA	UUAUCAA
							[SEQ ID NO: 891]	[SEQ ID NO: 1419]
TTTCGATGCAGG	UUUCGAUGCAGG	GCCTGCTAGTAA	GCCUGCUAGUAA	GCCUGCUAGUCC	-3.98959	2354	ACCGGACATACTTGTT	ACCGGACAUACUUGUU
ACTAGCAGGC	ACUAGCAGGC	TGCATCGAAT	UGCAUCGAAU	UGCAUCGA			CCACTCTTTCGATGCA	CCACUCUUUCGAUGCA
[SEQ ID	[SEQ ID	[SEQ ID	[SEQ ID	[SEQ ID			GGACTAGCAGGCTAGT	GGACUAGCAGGCUAGU
NO: 660]	NO: 1188]	NO: 776]	NO: 1304]	NO: 1008]			GAAATATATATTAAAG	GAAAUAUAUAUUAAAG
							CCTGCTAGTAATGCAT	CCUGCUAGUAAUGCAU
							CGAATTAGTGTGACAG	CGAAUUAGUGUGACAG
							GGATACAGCAACTATT	GGAUACAGCAACUAUU
							TTATCAA	UUAUCAA
							[SEQ ID NO: 892]	[SEQ ID NO: 1420]
TGTTCGATGCAG	UGUUCGAUGCAG	CCTGCTAGTCAC	CCUGCUAGUCAC	CCUGCUAGUCCU	-3.78415	2355	ACCGGACATACTTGTT	ACCGGACAUACUUGUU
GACTAGCAGG	GACUAGCAGG	GCATCGAACT	GCAUCGAACU	GCAUCGAA			CCACTCTGTTCGATGC	CCACUCUGUUCGAUGC
[SEQ ID	[SEQ ID	[SEQ ID	[SEQ ID	[SEQ ID			AGGACTAGCAGGTAGT	AGGACUAGCAGGUAGU
NO: 661]	NO: 1189]	NO: 777]	NO: 1305]	NO: 1009]			GAAATATATATTAAAC	GAAAUAUAUAUUAAAC
	(same as						CTGCTAGTCACGCATC	CUGCUAGUCACGCAUC
	guide in XD-						GAACTTAGTGTGACAG	GAACUUAGUGUGACAG
	14820)						GGATACAGCAACTATT	GGAUACAGCAACUAUU
							TTATCAA	UUAUCAA
							[SEQ ID NO: 893]	[SEQ ID NO: 1421]
TTGTTCGATGCA	UUGUUCGAUGCA	CTGCTAGTCCCT	CUGCUAGUCCCU	CUGCUAGUCCUG	-2.10173	2356	ACCGGACATACTTGTT	ACCGGACAUACUUGUU
GGACTAGCAG	GGACUAGCAG	CATCGAACAT	CAUCGAACAU	CAUCGAAC			CCACTCTTGTTCGATG	CCACUCUUGUUCGAUG
[SEQ ID	[SEQ ID	[SEQ ID	[SEQ ID	[SEQ ID			CAGGACTAGCAGTAGT	CAGGACUAGCAGUAGU
NO: 662]	NO: 1190]	NO: 778]	NO: 1306]	NO: 1010]			GAAATATATATTAAAC	GAAAUAUAUAUUAAAC
							TGCTAGTCCCTCATCG	UGCUAGUCCCUCAUCG
							AACATTAGTGTGACAG	AACAUUAGUGUGACAG
							GGATACAGCAACTATT	GGAUACAGCAACUAUU
							TTATCAA	UUAUCAA
							[SEQ ID NO: 894]	[SEQ ID NO: 1422]
TCTGTTCGATGC	UCUGUUCGAUGC	TGCTAGTCCTTA	UGCUAGUCCUUA	UGCUAGUCCUGC	-1.98657	2357	ACCGGACATACTTGTT	ACCGGACAUACUUGUU
AGGACTAGCA	AGGACUAGCA	ATCGAACAGT	AUCGAACAGU	AUCGAACA			CCACTCTCTGTTCGAT	CCACUCUCUGUUCGAU
[SEQ ID	[SEQ ID	[SEQ ID	[SEQ ID	[SEQ ID			GCAGGACTAGCATAGT	GCAGGACUAGCAUAGU
NO: 663]	NO: 1191]	NO: 779]	NO: 1307]	NO: 1011]			GAAATATATATTAAAT	GAAAUAUAUAUUAAAU
							GCTAGTCCTTAATCGA	GCUAGUCCUUAAUCGA
							ACAGTTAGTGTGACAG	ACAGUUAGUGUGACAG
							GGATACAGCAACTATT	GGAUACAGCAACUAUU
							TTATCAA	UUAUCAA
							[SEQ ID NO: 895]	[SEQ ID NO: 1423]
TTCTGTTCGATG	UUCUGUUCGAUG	GCTAGTCCTGAG	GCUAGUCCUGAG	GCUAGUCCUGCA	-4.1672	2358	ACCGGACATACTTGTT	ACCGGACAUACUUGUU
CAGGACTAGC	CAGGACUAGC	TCGAACAGAT	UCGAACAGAU	UCGAACAG			CCACTCTTCTGTTCGA	CCACUCUUCUGUUCGA
[SEQ ID	[SEQ ID	[SEQ ID	[SEQ ID	[SEQ ID			TGCAGGACTAGCTAGT	UGCAGGACUAGCUAGU
NO: 664]	NO: 1192]	NO: 780]	NO: 1308]	NO: 1012]			GAAATATATATTAAAG	GAAAUAUAUAUUAAAG
							CTAGTCCTGAGTCGAA	CUAGUCCUGAGUCGAA
							CAGATTAGTGTGACAG	CAGAUUAGUGUGACAG
							GGATACAGCAACTATT	GGAUACAGCAACUAUU
							TTATCAA	UUAUCAA
							[SEQ ID NO: 896]	[SEQ ID NO: 1424]
TCTCTGTTCGAT	UCUCUGUUCGAU	CTAGTCCTGCGC	CUAGUCCUGCGC	CUAGUCCUGCAU	-1.7173	2359	ACCGGACATACTTGTT	ACCGGACAUACUUGUU
GCAGGACTAG	GCAGGACUAG	CGAACAGAGT	CGAACAGAGU	CGAACAGA			CCACTCTCTCTGTTCG	CCACUCUCUCUGUUCG
[SEQ ID	[SEQ ID	[SEQ ID	[SEQ ID	[SEQ ID			ATGCAGGACTAGTAGT	AUGCAGGACUAGUAGU
NO: 665]	NO: 1193]	NO: 781]	NO: 1309]	NO: 1013]			GAAATATATATTAAAC	GAAAUAUAUAUUAAAC
							TAGTCCTGCGCCGAAC	UAGUCCUGCGCCGAAC
							AGAGTTAGTGTGACAG	AGAGUUAGUGUGACAG
							GGATACAGCAACTATT	GGAUACAGCAACUAUU
							TTATCAA	UUAUCAA
							[SEQ ID NO: 897]	[SEQ ID NO: 1425]
TGAGAGAAGGAA	UGAGAGAAGGAA	TCAACCCACGCC	UCAACCCACGCC	UCAACCCACGUU	-0.42433	2926	ACCGGACATACTTGTT	ACCGGACAUACUUGUU
CGTGGGTTGA	CGUGGGUUGA	CCTTCTCTCT	CCUUCUCUCU	CCUUCUCU			CCACTCTGAGAGAAGG	CCACUCUGAGAGAAGG
[SEQ ID	[SEQ ID	[SEQ ID	[ SEQ ID	[SEQ ID			AACGTGGGTTGATAGT	AACGUGGGUUGAUAGU
NO: 666]	NO: 1194]	NO: 782]	NO: 1310]	NO: 1014]			GAAATATATATTAAAT	GAAAUAUAUAUUAAAU
							CAACCCACGCCCCTTC	CAACCCACGCCCCUUC
							TCTCTTAGTGTGACAG	UCUCUUAGUGUGACAG
							GGATACAGCAACTATT	GGAUACAGCAACUAUU
							TTATCAA	UUAUCAA
							[SEQ ID NO: 898]	[SEQ ID NO: 1426]
TTGAGAGAAGGA	UUGAGAGAAGGA	CAACCCACGTCA	CAACCCACGUCA	CAACCCACGUUC	-3.38604	2927	ACCGGACATACTTGTT	ACCGGACAUACUUGUU
ACGTGGGTTG	ACGUGGGUUG	CTTCTCTCAT	CUUCUCUCAU	CUUCUCUC			CCACTCTTGAGAGAAG	CCACUCUUGAGAGAAG
[SEQ ID	[SEQ ID	[SEQ ID	[SEQ ID	[SEQ ID			GAACGTGGGTTGTAGT	GAACGUGGGUUGUAGU
NO: 667]	NO: 1195]	NO: 783]	NO: 1311]	NO: 1015]			GAAATATATATTAAAC	GAAAUAUAUAUUAAAC
							AACCCACGTCACTTCT	AACCCACGUCACUUCU
							CTCATTAGTGTGACAG	CUCAUUAGUGUGACAG
							GGATACAGCAACTATT	GGAUACAGCAACUAUU
							TTATCAA	UUAUCAA
							[SEQ ID NO: 899]	[SEQ ID NO: 1427]
TCTGAGAGAAGG	UCUGAGAGAAGG	AACCCACGTTAA	AACCCACGUUAA	AACCCACGUUCC	-3.5546	2928	ACCGGACATACTTGTT	ACCGGACAUACUUGUU
AACGTGGGTT	AACGUGGGUU	TTCTCTCAGT	UUCUCUCAGU	UUCUCUCA			CCACTCTCTGAGAGAA	CCACUCUCUGAGAGAA
[SEQ ID	[SEQ ID	[SEQ ID	[SEQ ID	[SEQ ID			GGAACGTGGGTTTAGT	GGAACGUGGGUUUAGU
NO: 668]	NO: 1196]	NO: 784]	NO: 1312]	NO: 1016]			GAAATATATATTAAAA	GAAAUAUAUAUUAAAA
							ACCCACGTTAATTCTC	ACCCACGUUAAUUCUC
							TCAGTTAGTGTGACAG	UCAGUUAGUGUGACAG
							GGATACAGCAACTATT	GGAUACAGCAACUAUU
							TTATCAA	UUAUCAA
							[SEQ ID NO: 900]	[SEQ ID NO: 1428]
TGCTGAGAGAAG	UGCUGAGAGAAG	ACCCACGTTCAC	ACCCACGUUCAC	ACCCACGUUCCU	-2.96935	2929	ACCGGACATACTTGTT	ACCGGACAUACUUGUU
GAACGTGGGT	GAACGUGGGU	TCTCTCAGCT	UCUCUCAGCU	UCUCUCAG			CCACTCTGCTGAGAGA	CCACUCUGCUGAGAGA
[SEQ ID	[SEQ ID	[SEQ ID	[SEQ ID	[SEQ ID			AGGAACGTGGGTTAGT	AGGAACGUGGGUUAGU
NO: 669]	NO: 1197]	NO: 785]	NO: 1313]	NO: 1017]			GAAATATATATTAAAA	GAAAUAUAUAUUAAAA
							CCCACGTTCACTCTCT	CCCACGUUCACUCUCU
							CAGCTTAGTGTGACAG	CAGCUUAGUGUGACAG
							GGATACAGCAACTATT	GGAUACAGCAACUAUU
							TTATCAA	UUAUCAA
							[SEQ ID NO: 901]	[SEQ ID NO: 1429]
TGGCTGAGAGAA	UGGCUGAGAGAA	CCCACGTTCCCC	CCCACGUUCCCC	CCCACGUUCCUU	-1.84629	2930	ACCGGACATACTTGTT	ACCGGACAUACUUGUU
GGAACGTGGG	GGAACGUGGG	CTCTCAGCCT	CUCUCAGCCU	CUCUCAGC			CCACTCTGGCTGAGAG	CCACUCUGGCUGAGAG
[SEQ ID	[SEQ ID	[SEQ ID	[SEQ ID	[SEQ ID			AAGGAACGTGGGTAGT	AAGGAACGUGGGUAGU
NO: 670]	NO: 1198]	NO: 786]	NO: 1314]	NO: 1018]			GAAATATATATTAAAC	GAAAUAUAUAUUAAAC
							CCACGTTCCCCCTCTC	CCACGUUCCCCCUCUC
							AGCCTTAGTGTGACAG	AGCCUUAGUGUGACAG
							GGATACAGCAACTATT	GGAUACAGCAACUAUU
							TTATCAA	UUAUCAA
							[SEQ ID NO: 902]	[SEQ ID NO: 1430]
TTGGCTGAGAGA	UUGGCUGAGAGA	CCACGTTCCTCA	CCACGUUCCUCA	CCACGUUCCUUC	-4.19621	2931	ACCGGACATACTTGTT	ACCGGACAUACUUGUU
AGGAACGTGG	AGGAACGUGG	TCTCAGCCAT	UCUCAGCCAU	UCUCAGCC			CCACTCTTGGCTGAGA	CCACUCUUGGCUGAGA
[SEQ ID	[SEQ ID	[SEQ ID	[SEQ ID	[SEQ ID			GAAGGAACGTGGTAGT	GAAGGAACGUGGUAGU
NO: 671]	NO: 1199]	NO: 787]	NO: 1315]	NO: 1019]			GAAATATATATTAAAC	GAAAUAUAUAUUAAAC
							CACGTTCCTCATCTCA	CACGUUCCUCAUCUCA
							GCCATTAGTGTGACAG	GCCAUUAGUGUGACAG
							GGATACAGCAACTATT	GGAUACAGCAACUAUU
							TTATCAA	UUAUCAA
							[SEQ ID NO: 903]	[SEQ ID NO: 1431]
TTTGGCTGAGAG	UUUGGCUGAGAG	CACGTTCCTTAC	CACGUUCCUUAC	CACGUUCCUUCU	-3.26413	2932	ACCGGACATACTTGTT	ACCGGACAUACUUGUU
AAGGAACGTG	AAGGAACGUG	CTCAGCCAAT	CUCAGCCAAU	CUCAGCCA			CCACTCTTTGGCTGAG	CCACUCUUUGGCUGAG
[SEQ ID	[SEQ ID	[SEQ ID	[SEQ ID	[SEQ ID			AGAAGGAACGTGTAGT	AGAAGGAACGUGUAGU
NO: 672]	NO: 1200]	NO: 788]	NO: 1316]	NO: 1020]			GAAATATATATTAAAC	GAAAUAUAUAUUAAAC
							ACGTTCCTTACCTCAG	ACGUUCCUUACCUCAG
							CCAATTAGTGTGACAG	CCAAUUAGUGUGACAG
							GGATACAGCAACTATT	GGAUACAGCAACUAUU
							TTATCAA	UUAUCAA
							[SEQ ID NO: 904]	[SEQ ID NO: 1432]
TTTTGGCTGAGA	UUUUGGCUGAGA	ACGTTCCTTCCA	ACGUUCCUUCCA	ACGUUCCUUCUC	-0.25972	2933	ACCGGACATACTTGTT	ACCGGACAUACUUGUU
GAAGGAACGT	GAAGGAACGU	TCAGCCAAAT	UCAGCCAAAU	UCAGCCAA			CCACTCTTTTGGCTGA	CCACUCUUUUGGCUGA
[SEQ ID	[SEQ ID	[SEQ ID	[SEQ ID	[SEQ ID			GAGAAGGAACGTTAGT	GAGAAGGAACGUUAGU
NO: 673]	NO: 1201]	NO: 789]	NO: 1317]	NO: 1021]			GAAATATATATTAAAA	GAAAUAUAUAUUAAAA
							CGTTCCTTCCATCAGC	CGUUCCUUCCAUCAGC
							CAAATTAGTGTGACAG	CAAAUUAGUGUGACAG
							GGATACAGCAACTATT	GGAUACAGCAACUAUU
							TTATCAA	UUAUCAA
							[SEQ ID NO: 905]	[SEQ ID NO: 1433]
TCTTTGGCTGAG	UCUUUGGCUGAG	CGTTCCTTCTAC	CGUUCCUUCUAC	CGUUCCUUCUCU	-0.75797	2934	ACCGGACATACTTGTT	ACCGGACAUACUUGUU
AGAAGGAACG	AGAAGGAACG	CAGCCAAAGT	CAGCCAAAGU	CAGCCAAA			CCACTCTCTTTGGCTG	CCACUCUCUUUGGCUG
[SEQ ID	[SEQ ID	[SEQ ID	[SEQ ID	[SEQ ID			AGAGAAGGAACGTAGT	AGAGAAGGAACGUAGU
NO: 674]	NO: 1202]	NO: 790]	NO: 1318]	NO: 1022]			GAAATATATATTAAAC	GAAAUAUAUAUUAAAC
							GTTCCTTCTACCAGCC	GUUCCUUCUACCAGCC
							AAAGTTAGTGTGACAG	AAAGUUAGUGUGACAG
							GGATACAGCAACTATT	GGAUACAGCAACUAUU
							TTATCAA	UUAUCAA
							[SEQ ID NO: 906]	[SEQ ID NO: 1434]
TGCTTTGGCTGA	UGCUUUGGCUGA	GTTCCTTCTCCA	GUUCCUUCUCCA	GUUCCUUCUCUC	-1.45481	2935	ACCGGACATACTTGTT	ACCGGACAUACUUGUU
GAGAAGGAAC	GAGAAGGAAC	AGCCAAAGCT	AGCCAAAGCU	AGCCAAAG			CCACTCTGCTTTGGCT	CCACUCUGCUUUGGCU
[SEQ ID	[SEQ ID	[SEQ ID	[SEQ ID	[SEQ ID			GAGAGAAGGAACTAGT	GAGAGAAGGAACUAGU
NO: 675]	NO: 1203]	NO: 791]	NO: 1319]	NO: 1023]			GAAATATATATTAAAG	GAAAUAUAUAUUAAAG
							TTCCTTCTCCAAGCCA	UUCCUUCUCCAAGCCA
							AAGCTTAGTGTGACAG	AAGCUUAGUGUGACAG
							GGATACAGCAACTATT	GGAUACAGCAACUAUU
							TTATCAA	UUAUCAA
							[SEQ ID NO: 907]	[SEQ ID NO: 1435]
AGGCTTTGGCTG	AGGCUUUGGCUG	TTCCTTCTCTAG	UUCCUUCUCUAG	UUCCUUCUCUCA	-0.85197	2936	ACCGGACATACTTGTT	ACCGGACAUACUUGUU
AGAGAAGGAA	AGAGAAGGAA	GCCAAAGCCA	GCCAAAGCCA	GCCAAAGC			CCACTCAGGCTTTGGC	CCACUCAGGCUUUGGC
[SEQ ID	[SEQ ID	[SEQ ID	[SEQ ID	[SEQ ID			TGAGAGAAGGAATAGT	UGAGAGAAGGAAUAGU
NO: 676]	NO: 1204]	NO: 792]	NO: 1320]	NO: 1024]			GAAATATATATTAAAT	GAAAUAUAUAUUAAAU
							TCCTTCTCTAGGCCAA	UCCUUCUCUAGGCCAA
							AGCCATAGTGTGACAG	AGCCAUAGUGUGACAG
							GGATACAGCAACTATT	GGAUACAGCAACUAUU
							TTATCAA	UUAUCAA
							[SEQ ID NO: 908]	[SEQ ID NO: 1436]
AAGGCTTTGGCT	AAGGCUUUGGCU	TCCTTCTCTCGT	UCCUUCUCUCGU	UCCUUCUCUCAG	-3.87114	2937	ACCGGACATACTTGTT	ACCGGACAUACUUGUU
GAGAGAAGGA	GAGAGAAGGA	CCAAAGCCTA	CCAAAGCCUA	CCAAAGCC			CCACTCAAGGCTTTGG	CCACUCAAGGCUUUGG
[SEQ ID	[SEQ ID	[SEQ ID	[SEQ ID	[SEQ ID			CTGAGAGAAGGATAGT	CUGAGAGAAGGAUAGU
NO: 677]	NO: 1205]	NO: 793]	NO: 1321]	NO: 1025]			GAAATATATATTAAAT	GAAAUAUAUAUUAAAU
							CCTTCTCTCGTCCAAA	CCUUCUCUCGUCCAAA
							GCCTATAGTGTGACAG	GCCUAUAGUGUGACAG
							GGATACAGCAACTATT	GGAUACAGCAACUAUU
							TTATCAA	UUAUCAA
							[SEQ ID NO: 909]	[SEQ ID NO: 1437]
TAAGGCTTTGGC	UAAGGCUUUGGC	CCTTCTCTCATA	CCUUCUCUCAUA	CCUUCUCUCAGC	-0.34984	2938	ACCGGACATACTTGTT	ACCGGACAUACUUGUU
TGAGAGAAGG	UGAGAGAAGG	CAAAGCCTTT	CAAAGCCUUU	CAAAGCCU			CCACTCTAAGGCTTTG	CCACUCUAAGGCUUUG
[SEQ ID	[SEQ ID	[SEQ ID	[SEQ ID	[SEQ ID			GCTGAGAGAAGGTAGT	GCUGAGAGAAGGUAGU
NO: 678]	NO: 1206]	NO: 794]	NO: 1322]	NO: 1026]			GAAATATATATTAAAC	GAAAUAUAUAUUAAAC
							CTTCTCTCATACAAAG	CUUCUCUCAUACAAAG
							CCTTTTAGTGTGACAG	CCUUUUAGUGUGACAG
							GGATACAGCAACTATT	GGAUACAGCAACUAUU
							TTATCAA	UUAUCAA
							[SEQ ID NO: 910]	[SEQ ID NO: 1438]
AGAAGGCTTTGG	AGAAGGCUUUGG	CTTCTCTCAGAA	CUUCUCUCAGAA	CUUCUCUCAGCC	-2.37082	2939	ACCGGACATACTTGTT	ACCGGACAUACUUGUU
CTGAGAGAAG	CUGAGAGAAG	AAAGCCTTCA	AAAGCCUUCA	AAAGCCUU			CCACTCAGAAGGCTTT	CCACUCAGAAGGCUUU
[SEQ ID	[SEQ ID	[SEQ ID	[SEQ ID	[SEQ ID			GGCTGAGAGAAGTAGT	GGCUGAGAGAAGUAGU
NO: 679]	NO: 1207]	NO: 795]	NO: 1323]	NO: 1027]			GAAATATATATTAAAC	GAAAUAUAUAUUAAAC
							TTCTCTCAGAAAAAGC	UUCUCUCAGAAAAAGC
							CTTCATAGTGTGACAG	CUUCAUAGUGUGACAG
							GGATACAGCAACTATT	GGAUACAGCAACUAUU
							TTATCAA	UUAUCAA
							[SEQ ID NO: 911]	[SEQ ID NO: 1439]
TAGAAGGCTTTG	UAGAAGGCUUUG	TTCTCTCAGCAG	UUCUCUCAGCAG	UUCUCUCAGCCA	-0.15876	2940	ACCGGACATACTTGTT	ACCGGACAUACUUGUU
GCTGAGAGAA	GCUGAGAGAA	AAGCCTTCTT	AAGCCUUCUU	AAGCCUUC			CCACTCTAGAAGGCTT	CCACUCUAGAAGGCUU
[SEQ ID	[SEQ ID	[SEQ ID	[SEQ ID	[SEQ ID			TGGCTGAGAGAATAGT	UGGCUGAGAGAAUAGU
NO: 680]	NO: 1208]	NO: 796]	NO: 1324]	NO: 1028]			GAAATATATATTAAAT	GAAAUAUAUAUUAAAU
							TCTCTCAGCAGAAGCC	UCUCUCAGCAGAAGCC
							TTCTTTAGTGTGACAG	UUCUUUAGUGUGACAG
							GGATACAGCAACTATT	GGAUACAGCAACUAUU
							TTATCAA	UUAUCAA
							[SEQ ID NO: 912]	[SEQ ID NO: 1440]
TTAGAAGGCTTT	UUAGAAGGCUUU	TCTCTCAGCCGG	UCUCUCAGCCGG	UCUCUCAGCCAA	-0.48849	2941	ACCGGACATACTTGTT	ACCGGACAUACUUGUU
GGCTGAGAGA	GGCUGAGAGA	AGCCTTCTAT	AGCCUUCUAU	AGCCUUCU			CCACTCTTAGAAGGCT	CCACUCUUAGAAGGCU
[SEQ ID	[SEQ ID	[SEQ ID	[SEQ ID	[SEQ ID			TTGGCTGAGAGATAGT	UUGGCUGAGAGAUAGU
NO: 681]	NO: 1209]	NO: 797]	NO: 1325]	NO: 1029]			GAAATATATATTAAAT	GAAAUAUAUAUUAAAU
	(same as						CTCTCAGCCGGAGCCT	CUCUCAGCCGGAGCCU
	guide in XD-						TCTATTAGTGTGACAG	UCUAUUAGUGUGACAG
	14858)						GGATACAGCAACTATT	GGAUACAGCAACUAUU
							TTATCAA	UUAUCAA
							[SEQ ID NO: 913]	[SEQ ID NO: 1441]
AGTAGAAGGCTT	AGUAGAAGGCUU	CTCTCAGCCAGG	CUCUCAGCCAGG	CUCUCAGCCAAA	-2.61597	2942	ACCGGACATACTTGTT	ACCGGACAUACUUGUU
TGGCTGAGAG	UGGCUGAGAG	GCCTTCTACA	GCCUUCUACA	GCCUUCUA			CCACTCAGTAGAAGGC	CCACUCAGUAGAAGGC
[SEQ ID	[SEQ ID	[SEQ ID	[SEQ ID	[SEQ ID			TTTGGCTGAGAGTAGT	UUUGGCUGAGAGUAGU
NO: 682]	NO: 1210]	NO: 798]	NO: 1326]	NO: 1030]			GAAATATATATTAAAC	GAAAUAUAUAUUAAAC
							TCTCAGCCAGGGCCTT	UCUCAGCCAGGGCCUU
							CTACATAGTGTGACAG	CUACAUAGUGUGACAG
							GGATACAGCAACTATT	GGAUACAGCAACUAUU
							TTATCAA	UUAUCAA
							[SEQ ID NO: 914]	[SEQ ID NO: 1442]
TAGTAGAAGGCT	UAGUAGAAGGCU	TCTCAGCCAAGT	UCUCAGCCAAGU	UCUCAGCCAAAG	-2.42796	2943	ACCGGACATACTTGTT	ACCGGACAUACUUGUU
TTGGCTGAGA	UUGGCUGAGA	CCTTCTACTT	CCUUCUACUU	CCUUCUAC			CCACTCTAGTAGAAGG	CCACUCUAGUAGAAGG
[SEQ ID	[SEQ ID	[SEQ ID	[SEQ ID	[SEQ ID			CTTTGGCTGAGATAGT	CUUUGGCUGAGAUAGU
NO: 683]	NO: 1211]	NO: 799]	NO: 1327]	NO: 1031]			GAAATATATATTAAAT	GAAAUAUAUAUUAAAU
							CTCAGCCAAGTCCTTC	CUCAGCCAAGUCCUUC
							TACTTTAGTGTGACAG	UACUUUAGUGUGACAG
							GGATACAGCAACTATT	GGAUACAGCAACUAUU
							TTATCAA	UUAUCAA
							[SEQ ID NO: 915]	[SEQ ID NO: 1443]
TTAGTAGAAGGC	UUAGUAGAAGGC	CTCAGCCAAATA	CUCAGCCAAAUA	CUCAGCCAAAGC	-1.95956	2944	ACCGGACATACTTGTT	ACCGGACAUACUUGUU
TTTGGCTGAG	UUUGGCUGAG	CTTCTACTAT	CUUCUACUAU	CUUCUACU			CCACTCTTAGTAGAAG	CCACUCUUAGUAGAAG
[SEQ ID	[SEQ ID	[SEQ ID	[SEQ ID	[SEQ ID			GCTTTGGCTGAGTAGT	GCUUUGGCUGAGUAGU
NO: 684]	NO: 1212]	NO: 800]	NO: 1328]	NO: 1032]			GAAATATATATTAAAC	GAAAUAUAUAUUAAAC
	(same as						TCAGCCAAATACTTCT	UCAGCCAAAUACUUCU
	guide in XD-						ACTATTAGTGTGACAG	ACUAUUAGUGUGACAG
	14859)						GGATACAGCAACTATT	GGAUACAGCAACUAUU
							TTATCAA	UUAUCAA
							[SEQ ID NO: 916]	[SEQ ID NO: 1444]
TGTAGTAGAAGG	UGUAGUAGAAGG	TCAGCCAAAGAA	UCAGCCAAAGAA	UCAGCCAAAGCC	-4.21076	2945	ACCGGACATACTTGTT	ACCGGACAUACUUGUU
CTTTGGCTGA	CUUUGGCUGA	TTCTACTACT	UUCUACUACU	UUCUACUA			CCACTCTGTAGTAGAA	CCACUCUGUAGUAGAA
[SEQ ID	[SEQ ID	[SEQ ID	[SEQ ID	[SEQ ID			GGCTTTGGCTGATAGT	GGCUUUGGCUGAUAGU
NO: 685]	NO: 1213]	NO: 801]	NO: 1329]	NO: 1033]			GAAATATATATTAAAT	GAAAUAUAUAUUAAAU
	(same as						CAGCCAAAGAATTCTA	CAGCCAAAGAAUUCUA
	guide in XD-						CTACTTAGTGTGACAG	CUACUUAGUGUGACAG
	14860)						GGATACAGCAACTATT	GGAUACAGCAACUAUU
							TTATCAA	UUAUCAA
							[SEQ ID NO: 917]	[SEQ ID NO: 1445]
TGGTAGTAGAAG	UGGUAGUAGAAG	CAGCCAAAGCAC	CAGCCAAAGCAC	CAGCCAAAGCCU	-2.92638	2946	ACCGGACATACTTGTT	ACCGGACAUACUUGUU
GCTTTGGCTG	GCUUUGGCUG	TCTACTACCT	UCUACUACCU	UCUACUAC			CCACTCTGGTAGTAGA	CCACUCUGGUAGUAGA
[SEQ ID	[SEQ ID	[SEQ ID	[SEQ ID	[SEQ ID			AGGCTTTGGCTGTAGT	AGGCUUUGGCUGUAGU
NO: 686]	NO: 1214]	NO: 802]	NO: 1330]	NO: 1034]			GAAATATATATTAAAC	GAAAUAUAUAUUAAAC
	(same as						AGCCAAAGCACTCTAC	AGCCAAAGCACUCUAC
	guide in XD-						TACCTTAGTGTGACAG	UACCUUAGUGUGACAG
	14861)						GGATACAGCAACTATT	GGAUACAGCAACUAUU
							TTATCAA	UUAUCAA
							[SEQ ID NO: 918]	[SEQ ID NO: 1446]
TGAACAAGGGGC	UGAACAAGGGGC	TCCCAAATCATA	UCCCAAAUCAUA	UCCCAAAUCAGC	-4.19616	3301	ACCGGACATACTTGTT	ACCGGACAUACUUGUU
TGATTTGGGA	UGAUUUGGGA	CCCTTGTTCT	CCCUUGUUCU	CCCUUGUU			CCACTCTGAACAAGGG	CCACUCUGAACAAGGG
[SEQ ID	[SEQ ID	[SEQ ID	[SEQ ID	[SEQ ID			GCTGATTTGGGATAGT	GCUGAUUUGGGAUAGU
NO: 687]	NO: 1215]	NO: 803]	NO: 1331]	NO: 1035]			GAAATATATATTAAAT	GAAAUAUAUAUUAAAU
							CCCAAATCATACCCTT	CCCAAAUCAUACCCUU
							GTTCTTAGTGTGACAG	GUUCUUAGUGUGACAG
							GGATACAGCAACTATT	GGAUACAGCAACUAUU
							TTATCAA	UUAUCAA
							[SEQ ID NO: 919]	[SEQ ID NO: 1447]
TTGAACAAGGGG	UUGAACAAGGGG	CCCAAATCAGAA	CCCAAAUCAGAA	CCCAAAUCAGCC	-4.85857	3302	ACCGGACATACTTGTT	ACCGGACAUACUUGUU
CTGATTTGGG	CUGAUUUGGG	CCTTGTTCAT	CCUUGUUCAU	CCUUGUUC			CCACTCTTGAACAAGG	CCACUCUUGAACAAGG
[SEQ ID	[SEQ ID	[SEQ ID	[SEQ ID	[SEQ ID			GGCTGATTTGGGTAGT	GGCUGAUUUGGGUAGU
NO: 688]	NO: 1216]	NO: 804]	NO: 1332]	NO: 1036]			GAAATATATATTAAAC	GAAAUAUAUAUUAAAC
							CCAAATCAGAACCTTG	CCAAAUCAGAACCUUG
							TTCATTAGTGTGACAG	UUCAUUAGUGUGACAG
							GGATACAGCAACTATT	GGAUACAGCAACUAUU
							TTATCAA	UUAUCAA
							[SEQ ID NO: 920]	[SEQ ID NO: 1448]
TCTGAACAAGGG	UCUGAACAAGGG	CCAAATCAGCAA	CCAAAUCAGCAA	CCAAAUCAGCCC	-2.36325	3303	ACCGGACATACTTGTT	ACCGGACAUACUUGUU
GCTGATTTGG	GCUGAUUUGG	CTTGTTCAGT	CUUGUUCAGU	CUUGUUCA			CCACTCTCTGAACAAG	CCACUCUCUGAACAAG
[SEQ ID	[SEQ ID	[SEQ ID	[SEQ ID	[SEQ ID			GGGCTGATTTGGTAGT	GGGCUGAUUUGGUAGU
NO: 689]	NO: 1217]	NO: 805]	NO: 1333]	NO: 1037]			GAAATATATATTAAAC	GAAAUAUAUAUUAAAC
							CAAATCAGCAACTTGT	CAAAUCAGCAACUUGU
							TCAGTTAGTGTGACAG	UCAGUUAGUGUGACAG
							GGATACAGCAACTATT	GGAUACAGCAACUAUU
							TTATCAA	UUAUCAA
							[SEQ ID NO: 921]	[SEQ ID NO: 1449]
TGTGGCACATGC	UGUGGCACAUGC	CCCTTGTTCATA	CCCUUGUUCAUA	CCCUUGUUCAGC	-2.10246	3313	ACCGGACATACTTGTT	ACCGGACAUACUUGUU
TGAACAAGGG	UGAACAAGGG	ATGTGCCACT	AUGUGCCACU	AUGUGCCA			CCACTCTGTGGCACAT	CCACUCUGUGGCACAU
[SEQ ID	[SEQ ID	[SEQ ID	[SEQ ID	[SEQ ID			GCTGAACAAGGGTAGT	GCUGAACAAGGGUAGU
NO: 690]	NO: 1218]	NO: 806]	NO: 1334]	NO: 1038]			GAAATATATATTAAAC	GAAAUAUAUAUUAAAC
							CCTTGTTCATAATGTG	CCUUGUUCAUAAUGUG
							CCACTTAGTGTGACAG	CCACUUAGUGUGACAG
							GGATACAGCAACTATT	GGAUACAGCAACUAUU
							TTATCAA	UUAUCAA
							[SEQ ID NO: 922]	[SEQ ID NO: 1450]
TTGCCATCATTC	UUGCCAUCAUUC	GGTAATGCTATG	GGUAAUGCUAUG	GGUAAUGCUAGA	-0.43513	3378	ACCGGACATACTTGTT	ACCGGACAUACUUGUU
TAGCATTACC	UAGCAUUACC	ATGATGGCAT	AUGAUGGCAU	AUGAUGGC			CCACTCTTGCCATCAT	CCACUCUUGCCAUCAU
[SEQ ID	[SEQ ID	[SEQ ID	[SEQ ID	[SEQ ID			TCTAGCATTACCTAGT	UCUAGCAUUACCUAGU
NO: 691]	NO: 1219]	NO: 807]	NO: 1335]	NO: 1039]			GAAATATATATTAAAG	GAAAUAUAUAUUAAAG
							GTAATGCTATGATGAT	GUAAUGCUAUGAUGAU
							GGCATTAGTGTGACAG	GGCAUUAGUGUGACAG
							GGATACAGCAACTATT	GGAUACAGCAACUAUU
							TTATCAA	UUAUCAA
							[SEQ ID NO: 923]	[SEQ ID NO: 1451]
TTGCTGGGAAAC	UUGCUGGGAAAC	CCACAGAATATC	CCACAGAAUAUC	CCACAGAAUAGU	-2.70652	3804	ACCGGACATACTTGTT	ACCGGACAUACUUGUU
TATTCTGTGG	UAUUCUGUGG	TTCCCAGCAT	UUCCCAGCAU	UUCCCAGC			CCACTCTTGCTGGGAA	CCACUCUUGCUGGGAA
[SEQ ID	[SEQ ID	[SEQ ID	[SEQ ID	[SEQ ID			ACTATTCTGTGGTAGT	ACUAUUCUGUGGUAGU
NO: 692]	NO: 1220]	NO: 808]	NO: 1336]	NO: 1040]			GAAATATATATTAAAC	GAAAUAUAUAUUAAAC
	(same as						CACAGAATATCTTCCC	CACAGAAUAUCUUCCC
	guide in XD-						AGCATTAGTGTGACAG	AGCAUUAGUGUGACAG
	14861)						GGATACAGCAACTATT	GGAUACAGCAACUAUU
							TTATCAA	UUAUCAA
							[SEQ ID NO: 924]	[SEQ ID NO: 1452]
TCTGCTGGGAAA	UCUGCUGGGAAA	CACAGAATAGCC	CACAGAAUAGCC	CACAGAAUAGUU	-4.91212	3805	ACCGGACATACTTGTT	ACCGGACAUACUUGUU
CTATTCTGTG	CUAUUCUGUG	TCCCAGCAGT	UCCCAGCAGU	UCCCAGCA			CCACTCTCTGCTGGGA	CCACUCUCUGCUGGGA
[SEQ ID	[SEQ ID	[SEQ ID	[SEQ ID	[SEQ ID			AACTATTCTGTGTAGT	AACUAUUCUGUGUAGU
NO: 693]	NO: 1221]	NO: 809]	NO: 1337]	NO: 1041]			GAAATATATATTAAAC	GAAAUAUAUAUUAAAC
							ACAGAATAGCCTCCCA	ACAGAAUAGCCUCCCA
							GCAGTTAGTGTGACAG	GCAGUUAGUGUGACAG
							GGATACAGCAACTATT	GGAUACAGCAACUAUU
							TTATCAA	UUAUCAA
							[SEQ ID NO: 925]	[SEQ ID NO: 1453]
TGCTGCTGGGAA	UGCUGCUGGGAA	ACAGAATAGTCC	ACAGAAUAGUCC	ACAGAAUAGUUU	-3.54976	3806	ACCGGACATACTTGTT	ACCGGACAUACUUGUU
ACTATTCTGT	ACUAUUCUGU	CCCAGCAGCT	CCCAGCAGCU	CCCAGCAG			CCACTCTGCTGCTGGG	CCACUCUGCUGCUGGG
[SEQ ID	[SEQ ID	[SEQ ID	[SEQ ID	[SEQ ID			AAACTATTCTGTTAGT	AAACUAUUCUGUUAGU
NO: 694]	NO: 1222]	NO: 810]	NO: 1338]	NO: 1042]			GAAATATATATTAAAA	GAAAUAUAUAUUAAAA
							CAGAATAGTCCCCCAG	CAGAAUAGUCCCCCAG
							CAGCTTAGTGTGACAG	CAGCUUAGUGUGACAG
							GGATACAGCAACTATT	GGAUACAGCAACUAUU
							TTATCAA	UUAUCAA
							[SEQ ID NO: 926]	[SEQ ID NO: 1454]
TGAACGTGAGAA	UGAACGUGAGAA	CGATCCATCCCC	CGAUCCAUCCCC	CGAUCCAUCCUU	-3.0586	3844	ACCGGACATACTTGTT	ACCGGACAUACUUGUU
GGATGGATCG	GGAUGGAUCG	CTCACGTTCT	CUCACGUUCU	CUCACGUU			CCACTCTGAACGTGAG	CCACUCUGAACGUGAG
[SEQ ID	[SEQ ID	[SEQ ID	[SEQ ID	[SEQ ID			AAGGATGGATCGTAGT	AAGGAUGGAUCGUAGU
NO: 695]	NO: 1223]	NO: 811]	NO: 1339]	NO: 1043]			GAAATATATATTAAAC	GAAAUAUAUAUUAAAC
							GATCCATCCCCCTCAC	GAUCCAUCCCCCUCAC
							GTTCTTAGTGTGACAG	GUUCUUAGUGUGACAG
							GGATACAGCAACTATT	GGAUACAGCAACUAUU
							TTATCAA	UUAUCAA
							[SEQ ID NO: 927]	[SEQ ID NO: 1455]
TTGAACGTGAGA	UUGAACGUGAGA	GATCCATCCTCA	GAUCCAUCCUCA	GAUCCAUCCUUC	-4.40112	3845	ACCGGACATACTTGTT	ACCGGACAUACUUGUU
AGGATGGATC	AGGAUGGAUC	TCACGTTCAT	UCACGUUCAU	UCACGUUC			CCACTCTTGAACGTGA	CCACUCUUGAACGUGA
[SEQ ID	[SEQ ID	[SEQ ID	[SEQ ID	[SEQ ID			GAAGGATGGATCTAGT	GAAGGAUGGAUCUAGU
NO: 696]	NO: 1224]	NO: 812]	NO: 1340]	NO: 1044]			GAAATATATATTAAAG	GAAAUAUAUAUUAAAG
							ATCCATCCTCATCACG	AUCCAUCCUCAUCACG
							TTCATTAGTGTGACAG	UUCAUUAGUGUGACAG
							GGATACAGCAACTATT	GGAUACAGCAACUAUU
							TTATCAA	UUAUCAA
							[SEQ ID NO: 928]	[SEQ ID NO: 1456]
TCTGAACGTGAG	UCUGAACGUGAG	ATCCATCCTTAC	AUCCAUCCUUAC	AUCCAUCCUUCU	-2.54571	3846	ACCGGACATACTTGTT	ACCGGACAUACUUGUU
AAGGATGGAT	AAGGAUGGAU	CACGTTCAGT	CACGUUCAGU	CACGUUCA			CCACTCTCTGAACGTG	CCACUCUCUGAACGUG
[SEQ ID	[SEQ ID	[SEQ ID	[SEQ ID	[SEQ ID			AGAAGGATGGATTAGT	AGAAGGAUGGAUUAGU
NO: 697]	NO: 1225]	NO: 813]	NO: 1341]	NO: 1045]			GAAATATATATTAAAA	GAAAUAUAUAUUAAAA
							TCCATCCTTACCACGT	UCCAUCCUUACCACGU
							TCAGTTAGTGTGACAG	UCAGUUAGUGUGACAG
							GGATACAGCAACTATT	GGAUACAGCAACUAUU
							TTATCAA	UUAUCAA
							[SEQ ID NO: 929]	[SEQ ID NO: 1457]
AACTGTTAGCAT	AACUGUUAGCAU	TCCAATAGGAGC	UCCAAUAGGAGC	UCCAAUAGGAAU	-3.8378	4235	ACCGGACATACTTGTT	ACCGGACAUACUUGUU
TCCTATTGGA	UCCUAUUGGA	GCTAACAGTA	GCUAACAGUA	GCUAACAG			CCACTCAACTGTTAGC	CCACUCAACUGUUAGC
[SEQ ID	[SEQ ID	[SEQ ID	[SEQ ID	[SEQ ID			ATTCCTATTGGATAGT	AUUCCUAUUGGAUAGU
NO: 698]	NO: 1226]	NO: 814]	NO: 1342]	NO: 1046]			GAAATATATATTAAAT	GAAAUAUAUAUUAAAU
							CCAATAGGAGCGCTAA	CCAAUAGGAGCGCUAA
							CAGTATAGTGTGACAG	CAGUAUAGUGUGACAG
							GGATACAGCAACTATT	GGAUACAGCAACUAUU
							TTATCAA	UUAUCAA
							[SEQ ID NO: 930]	[SEQ ID NO: 1458]
TAACTGTTAGCA	UAACUGUUAGCA	CCAATAGGAACT	CCAAUAGGAACU	CCAAUAGGAAUG	-2.52035	4236	ACCGGACATACTTGTT	ACCGGACAUACUUGUU
TTCCTATTGG	UUCCUAUUGG	CTAACAGTTT	CUAACAGUUU	CUAACAGU			CCACTCTAACTGTTAG	CCACUCUAACUGUUAG
[SEQ ID	[SEQ ID	[SEQ ID	[SEQ ID	[SEQ ID			CATTCCTATTGGTAGT	CAUUCCUAUUGGUAGU
NO: 699]	NO: 1227]	NO: 815]	NO: 1343]	NO: 1047]			GAAATATATATTAAAC	GAAAUAUAUAUUAAAC
							CAATAGGAACTCTAAC	CAAUAGGAACUCUAAC
							AGTTTTAGTGTGACAG	AGUUUUAGUGUGACAG
							GGATACAGCAACTATT	GGAUACAGCAACUAUU
							TTATCAA	UUAUCAA
							[SEQ ID NO: 931]	[SEQ ID NO: 1459]
TGAACTGTTAGC	UGAACUGUUAGC	CAATAGGAATTA	CAAUAGGAAUUA	CAAUAGGAAUGC	-4.90789	4237	ACCGGACATACTTGTT	ACCGGACAUACUUGUU
ATTCCTATTG	AUUCCUAUUG	TAACAGTTCT	UAACAGUUCU	UAACAGUU			CCACTCTGAACTGTTA	CCACUCUGAACUGUUA
[SEQ ID	[SEQ ID	[SEQ ID	[ SEQ ID	[SEQ ID			GCATTCCTATTGTAGT	GCAUUCCUAUUGUAGU
NO: 700]	NO: 1228]	NO: 816]	NO: 1344]	NO: 1048]			GAAATATATATTAAAC	GAAAUAUAUAUUAAAC
							AATAGGAATTATAACA	AAUAGGAAUUAUAACA
							GTTCTTAGTGTGACAG	GUUCUUAGUGUGACAG
							GGATACAGCAACTATT	GGAUACAGCAACUAUU
							TTATCAA	UUAUCAA
							[SEQ ID NO: 932]	[SEQ ID NO: 1460]
TTGAACTGTTAG	UUGAACUGUUAG	AATAGGAATGAC	AAUAGGAAUGAC	AAUAGGAAUGCU	-3.95764	4238	ACCGGACATACTTGTT	ACCGGACAUACUUGUU
CATTCCTATT	CAUUCCUAUU	AACAGTTCAT	AACAGUUCAU	AACAGUUC			CCACTCTTGAACTGTT	CCACUCUUGAACUGUU
[SEQ ID	[ SEQ ID	[SEQ ID	[SEQ ID	[SEQ ID			AGCATTCCTATTTAGT	AGCAUUCCUAUUUAGU
NO: 701]	NO: 1229]	NO: 817]	NO: 1345]	NO: 1049]			GAAATATATATTAAAA	GAAAUAUAUAUUAAAA
							ATAGGAATGACAACAG	AUAGGAAUGACAACAG
							TTCATTAGTGTGACAG	UUCAUUAGUGUGACAG
							GGATACAGCAACTATT	GGAUACAGCAACUAUU
							TTATCAA	UUAUCAA
							[SEQ ID NO: 933]	[SEQ ID NO: 1461]
AGTGAACTGTTA	AGUGAACUGUUA	ATAGGAATGCCG	AUAGGAAUGCCG	AUAGGAAUGCUA	-4.39189	4239	ACCGGACATACTTGTT	ACCGGACAUACUUGUU
GCATTCCTAT	GCAUUCCUAU	ACAGTTCACA	ACAGUUCACA	ACAGUUCA			CCACTCAGTGAACTGT	CCACUCAGUGAACUGU
[SEQ ID	[SEQ ID	[SEQ ID	[SEQ ID	[SEQ ID			TAGCATTCCTATTAGT	UAGCAUUCCUAUUAGU
NO: 702]	NO: 1230]	NO: 818]	NO: 1346]	NO: 1050]			GAAATATATATTAAAA	GAAAUAUAUAUUAAAA
							TAGGAATGCCGACAGT	UAGGAAUGCCGACAGU
							TCACATAGTGTGACAG	UCACAUAGUGUGACAG
							GGATACAGCAACTATT	GGAUACAGCAACUAUU
							TTATCAA	UUAUCAA
							[SEQ ID NO: 934]	[SEQ ID NO: 1462]
AAGTGAACTGTT	AAGUGAACUGUU	TAGGAATGCTGG	UAGGAAUGCUGG	UAGGAAUGCUAA	-4.80102	4240	ACCGGACATACTTGTT	ACCGGACAUACUUGUU
AGCATTCCTA	AGCAUUCCUA	CAGTTCACTA	CAGUUCACUA	CAGUUCAC			CCACTCAAGTGAACTG	CCACUCAAGUGAACUG
[SEQ ID	[ SEQ ID	[SEQ ID	[SEQ ID	[SEQ ID			TTAGCATTCCTATAGT	UUAGCAUUCCUAUAGU
NO: 703]	NO: 1231]	NO: 819]	NO: 1347]	NO: 1051]			GAAATATATATTAAAT	GAAAUAUAUAUUAAAU
							AGGAATGCTGGCAGTT	AGGAAUGCUGGCAGUU
							CACTATAGTGTGACAG	CACUAUAGUGUGACAG
							GGATACAGCAACTATT	GGAUACAGCAACUAUU
							TTATCAA	UUAUCAA
							[SEQ ID NO: 935]	[SEQ ID NO: 1463]
TAAGTGAACTGT	UAAGUGAACUGU	AGGAATGCTAGA	AGGAAUGCUAGA	AGGAAUGCUAAC	-2.45702	4241	ACCGGACATACTTGTT	ACCGGACAUACUUGUU
TAGCATTCCT	UAGCAUUCCU	AGTTCACTTT	AGUUCACUUU	AGUUCACU			CCACTCTAAGTGAACT	CCACUCUAAGUGAACU
[SEQ ID	[SEQ ID	[SEQ ID	[SEQ ID	[SEQ ID			GTTAGCATTCCTTAGT	GUUAGCAUUCCUUAGU
NO: 704]	NO: 1232]	NO: 820]	NO: 1348]	NO: 1052]			GAAATATATATTAAAA	GAAAUAUAUAUUAAAA
							GGAATGCTAGAAGTTC	GGAAUGCUAGAAGUUC
							ACTTTTAGTGTGACAG	ACUUUUAGUGUGACAG
							GGATACAGCAACTATT	GGAUACAGCAACUAUU
							TTATCAA	UUAUCAA
							[SEQ ID NO: 936]	[SEQ ID NO: 1464]
TCAAGTGAACTG	UCAAGUGAACUG	GGAATGCTAAAG	GGAAUGCUAAAG	GGAAUGCUAACA	-4.855	4242	ACCGGACATACTTGTT	ACCGGACAUACUUGUU
TTAGCATTCC	UUAGCAUUCC	GTTCACTTGT	GUUCACUUGU	GUUCACUU			CCACTCTCAAGTGAAC	CCACUCUCAAGUGAAC
[SEQ ID	[SEQ ID	[SEQ ID	[SEQ ID	[SEQ ID			TGTTAGCATTCCTAGT	UGUUAGCAUUCCUAGU
NO: 705]	NO: 1233]	NO: 821]	NO: 1349]	NO: 1053]			GAAATATATATTAAAG	GAAAUAUAUAUUAAAG
							GAATGCTAAAGGTTCA	GAAUGCUAAAGGUUCA
							CTTGTTAGTGTGACAG	CUUGUUAGUGUGACAG
							GGATACAGCAACTATT	GGAUACAGCAACUAUU
							TTATCAA	UUAUCAA
							[SEQ ID NO: 937]	[SEQ ID NO: 1465]
TGCAAGTGAACT	UGCAAGUGAACU	GAATGCTAACGT	GAAUGCUAACGU	GAAUGCUAACAG	-4.05556	4243	ACCGGACATACTTGTT	ACCGGACAUACUUGUU
GTTAGCATTC	GUUAGCAUUC	TTCACTTGCT	UUCACUUGCU	UUCACUUG			CCACTCTGCAAGTGAA	CCACUCUGCAAGUGAA
[SEQ ID	[SEQ ID	[SEQ ID	[SEQ ID	[SEQ ID			CTGTTAGCATTCTAGT	CUGUUAGCAUUCUAGU
NO: 706]	NO: 1234]	NO: 822]	NO: 1350]	NO: 1054]			GAAATATATATTAAAG	GAAAUAUAUAUUAAAG
							AATGCTAACGTTTCAC	AAUGCUAACGUUUCAC
							TTGCTTAGTGTGACAG	UUGCUUAGUGUGACAG
							GGATACAGCAACTATT	GGAUACAGCAACUAUU
							TTATCAA	UUAUCAA
							[SEQ ID NO: 938]	[SEQ ID NO: 1466]
TTGCAAGTGAAC	UUGCAAGUGAAC	AATGCTAACATC	AAUGCUAACAUC	AAUGCUAACAGU	-3.92834	4244	ACCGGACATACTTGTT	ACCGGACAUACUUGUU
TGTTAGCATT	UGUUAGCAUU	TCACTTGCAT	UCACUUGCAU	UCACUUGC			CCACTCTTGCAAGTGA	CCACUCUUGCAAGUGA
[SEQ ID	[SEQ ID	[SEQ ID	[SEQ ID	[SEQ ID			ACTGTTAGCATTTAGT	ACUGUUAGCAUUUAGU
NO: 707]	NO: 1235]	NO: 823]	NO: 1351]	NO: 1055]			GAAATATATATTAAAA	GAAAUAUAUAUUAAAA
							ATGCTAACATCTCACT	AUGCUAACAUCUCACU
							TGCATTAGTGTGACAG	UGCAUUAGUGUGACAG
							GGATACAGCAACTATT	GGAUACAGCAACUAUU
							TTATCAA	UUAUCAA
							[SEQ ID NO: 939]	[SEQ ID NO: 1467]
ACTGCAAGTGAA	ACUGCAAGUGAA	ATGCTAACAGCC	AUGCUAACAGCC	AUGCUAACAGUU	-4.32689	4245	ACCGGACATACTTGTT	ACCGGACAUACUUGUU
CTGTTAGCAT	CUGUUAGCAU	CACTTGCAGA	CACUUGCAGA	CACUUGCA			CCACTCACTGCAAGTG	CCACUCACUGCAAGUG
[SEQ ID	[SEQ ID	[SEQ ID	[SEQ ID	[SEQ ID			AACTGTTAGCATTAGT	AACUGUUAGCAUUAGU
NO: 708]	NO: 1236]	NO: 824]	NO: 1352]	NO: 1056]			GAAATATATATTAAAA	GAAAUAUAUAUUAAAA
							TGCTAACAGCCCACTT	UGCUAACAGCCCACUU
							GCAGATAGTGTGACAG	GCAGAUAGUGUGACAG
							GGATACAGCAACTATT	GGAUACAGCAACUAUU
							TTATCAA	UUAUCAA
							[SEQ ID NO: 940]	[SEQ ID NO: 1468]
TACTGCAAGTGA	UACUGCAAGUGA	TGCTAACAGTCA	UGCUAACAGUCA	UGCUAACAGUUC	-4.77129	4246	ACCGGACATACTTGTT	ACCGGACAUACUUGUU
ACTGTTAGCA	ACUGUUAGCA	ACTTGCAGTT	ACUUGCAGUU	ACUUGCAG			CCACTCTACTGCAAGT	CCACUCUACUGCAAGU
[SEQ ID	[SEQ ID	[SEQ ID	[SEQ ID	[SEQ ID			GAACTGTTAGCATAGT	GAACUGUUAGCAUAGU
NO: 709]	NO: 1237]	NO: 825]	NO: 1353]	NO: 1057]			GAAATATATATTAAAT	GAAAUAUAUAUUAAAU
							GCTAACAGTCAACTTG	GCUAACAGUCAACUUG
							CAGTTTAGTGTGACAG	CAGUUUAGUGUGACAG
							GGATACAGCAACTATT	GGAUACAGCAACUAUU
							TTATCAA	UUAUCAA
							[SEQ ID NO: 941]	[SEQ ID NO: 1469]
TCACTGCAAGTG	UCACUGCAAGUG	GCTAACAGTTAG	GCUAACAGUUAG	GCUAACAGUUCA	-3.83727	4247	ACCGGACATACTTGTT	ACCGGACAUACUUGUU
AACTGTTAGC	AACUGUUAGC	CTTGCAGTGT	CUUGCAGUGU	CUUGCAGU			CCACTCTCACTGCAAG	CCACUCUCACUGCAAG
[SEQ ID	[SEQ ID	[SEQ ID	[SEQ ID	[SEQ ID			TGAACTGTTAGCTAGT	UGAACUGUUAGCUAGU
NO: 710]	NO: 1238]	NO: 826]	NO: 1354]	NO: 1058]			GAAATATATATTAAAG	GAAAUAUAUAUUAAAG
							CTAACAGTTAGCTTGC	CUAACAGUUAGCUUGC
							AGTGTTAGTGTGACAG	AGUGUUAGUGUGACAG
							GGATACAGCAACTATT	GGAUACAGCAACUAUU
							TTATCAA	UUAUCAA
							[SEQ ID NO: 942	[SEQ ID NO: 1470]
TCCACTGCAAGT	UCCACUGCAAGU	CTAACAGTTCGA	CUAACAGUUCGA	CUAACAGUUCAC	-4.9039	4248	ACCGGACATACTTGTT	ACCGGACAUACUUGUU
GAACTGTTAG	GAACUGUUAG	TTGCAGTGGT	UUGCAGUGGU	UUGCAGUG			CCACTCTCCACTGCAA	CCACUCUCCACUGCAA
[SEQ ID	[SEQ ID	[SEQ ID	[SEQ ID	[SEQ ID			GTGAACTGTTAGTAGT	GUGAACUGUUAGUAGU
NO: 711]	NO: 1239]	NO: 827]	NO: 1355]	NO: 1059]			GAAATATATATTAAAC	GAAAUAUAUAUUAAAC
							TAACAGTTCGATTGCA	UAACAGUUCGAUUGCA
							GTGGTTAGTGTGACAG	GUGGUUAGUGUGACAG
							GGATACAGCAACTATT	GGAUACAGCAACUAUU
							TTATCAA	UUAUCAA
							[SEQ ID NO: 943]	[SEQ ID NO: 1471]
TTCCACTGCAAG	UUCCACUGCAAG	TAACAGTTCAAC	UAACAGUUCAAC	UAACAGUUCACU	-3.84653	4249	ACCGGACATACTTGTT	ACCGGACAUACUUGUU
TGAACTGTTA	UGAACUGUUA	TGCAGTGGAT	UGCAGUGGAU	UGCAGUGG			CCACTCTTCCACTGCA	CCACUCUUCCACUGCA
[SEQ ID	[SEQ ID	[SEQ ID	[SEQ ID	[SEQ ID			AGTGAACTGTTATAGT	AGUGAACUGUUAUAGU
NO: 712]	NO: 1240]	NO: 828]	NO: 1356]	NO: 1060]			GAAATATATATTAAAT	GAAAUAUAUAUUAAAU
							AACAGTTCAACTGCAG	AACAGUUCAACUGCAG
							TGGATTAGTGTGACAG	UGGAUUAGUGUGACAG
							GGATACAGCAACTATT	GGAUACAGCAACUAUU
							TTATCAA	UUAUCAA
							[SEQ ID NO: 944]	[SEQ ID NO: 1472]
TTTCCACTGCAA	UUUCCACUGCAA	AACAGTTCACCC	AACAGUUCACCC	AACAGUUCACUU	-3.95941	4250	ACCGGACATACTTGTT	ACCGGACAUACUUGUU
GTGAACTGTT	GUGAACUGUU	GCAGTGGAAT	GCAGUGGAAU	GCAGUGGA			CCACTCTTTCCACTGC	CCACUCUUUCCACUGC
[SEQ ID	[SEQ ID	[SEQ ID	[SEQ ID	[SEQ ID			AAGTGAACTGTTTAGT	AAGUGAACUGUUUAGU
NO: 713]	NO: 1241]	NO: 829]	NO: 1357]	NO: 1061]			GAAATATATATTAAAA	GAAAUAUAUAUUAAAA
							ACAGTTCACCCGCAGT	ACAGUUCACCCGCAGU
							GGAATTAGTGTGACAG	GGAAUUAGUGUGACAG
							GGATACAGCAACTATT	GGAUACAGCAACUAUU
							TTATCAA	UUAUCAA
							[SEQ ID NO: 945]	[SEQ ID NO: 1473]
TCTTCCACTGCA	UCUUCCACUGCA	ACAGTTCACTCT	ACAGUUCACUCU	ACAGUUCACUUG	-4.68439	4251	ACCGGACATACTTGTT	ACCGGACAUACUUGUU
AGTGAACTGT	AGUGAACUGU	CAGTGGAAGT	CAGUGGAAGU	CAGUGGAA			CCACTCTCTTCCACTG	CCACUCUCUUCCACUG
[SEQ ID	[SEQ ID	[SEQ ID	[SEQ ID	[SEQ ID			CAAGTGAACTGTTAGT	CAAGUGAACUGUUAGU
NO: 714]	NO: 1242]	NO: 830]	NO: 1358]	NO: 1062]			GAAATATATATTAAAA	GAAAUAUAUAUUAAAA
							CAGTTCACTCTCAGTG	CAGUUCACUCUCAGUG
							GAAGTTAGTGTGACAG	GAAGUUAGUGUGACAG
							GGATACAGCAACTATT	GGAUACAGCAACUAUU
							TTATCAA	UUAUCAA
							[SEQ ID NO: 946]	[SEQ ID NO: 1474]
ATCTTCCACTGC	AUCUUCCACUGC	CAGTTCACTTTA	CAGUUCACUUUA	CAGUUCACUUGC	-2.98094	4252	ACCGGACATACTTGTT	ACCGGACAUACUUGUU
AAGTGAACTG	AAGUGAACUG	AGTGGAAGAA	AGUGGAAGAA	AGUGGAAG			CCACTCATCTTCCACT	CCACUCAUCUUCCACU
[SEQ ID	[SEQ ID	[SEQ ID	[SEQ ID	[SEQ ID			GCAAGTGAACTGTAGT	GCAAGUGAACUGUAGU
NO: 715]	NO: 1243]	NO: 831]	NO: 1359]	NO: 1063]			GAAATATATATTAAAC	GAAAUAUAUAUUAAAC
							AGTTCACTTTAAGTGG	AGUUCACUUUAAGUGG
							AAGAATAGTGTGACAG	AAGAAUAGUGUGACAG
							GGATACAGCAACTATT	GGAUACAGCAACUAUU
							TTATCAA	UUAUCAA
							[SEQ ID NO: 947]	[SEQ ID NO: 1475]
TATCTTCCACTG	UAUCUUCCACUG	AGTTCACTTGAG	AGUUCACUUGAG	AGUUCACUUGCA	-3.46197	4253	ACCGGACATACTTGTT	ACCGGACAUACUUGUU
CAAGTGAACT	CAAGUGAACU	GTGGAAGATT	GUGGAAGAUU	GUGGAAGA			CCACTCTATCTTCCAC	CCACUCUAUCUUCCAC
[SEQ ID	[SEQ ID	[SEQ ID	[SEQ ID	[SEQ ID			TGCAAGTGAACTTAGT	UGCAAGUGAACUUAGU
NO: 716]	NO: 1244]	NO: 832]	NO: 1360]	NO: 1064]			GAAATATATATTAAAA	GAAAUAUAUAUUAAAA
							GTTCACTTGAGGTGGA	GUUCACUUGAGGUGGA
							AGATTTAGTGTGACAG	AGAUUUAGUGUGACAG
							GGATACAGCAACTATT	GGAUACAGCAACUAUU
							TTATCAA	UUAUCAA
							[SEQ ID NO: 948]	[SEQ ID NO: 1476]
TGGCAAGCAGAG	UGGCAAGCAGAG	GGTACCCCAGAC	GGUACCCCAGAC	GGUACCCCAGCU	-3.53049	4348	ACCGGACATACTTGTT	ACCGGACAUACUUGUU
CTGGGGTACC	CUGGGGUACC	CTGCTTGCCT	CUGCUUGCCU	CUGCUUGC			CCACTCTGGCAAGCAG	CCACUCUGGCAAGCAG
[SEQ ID	[SEQ ID	[SEQ ID	[SEQ ID	[SEQ ID			AGCTGGGGTACCTAGT	AGCUGGGGUACCUAGU
NO: 717]	NO: 1245]	NO: 833]	NO: 1361]	NO: 1065]			GAAATATATATTAAAG	GAAAUAUAUAUUAAAG
							GTACCCCAGACCTGCT	GUACCCCAGACCUGCU
							TGCCTTAGTGTGACAG	UGCCUUAGUGUGACAG
							GGATACAGCAACTATT	GGAUACAGCAACUAUU
							TTATCAA	UUAUCAA
							[SEQ ID NO: 949]	[SEQ ID NO: 1477]
TCGGCAAGCAGA	UCGGCAAGCAGA	GTACCCCAGCCA	GUACCCCAGCCA	GUACCCCAGCUC	-4.36361	4349	ACCGGACATACTTGTT	ACCGGACAUACUUGUU
GCTGGGGTAC	GCUGGGGUAC	TGCTTGCCGT	UGCUUGCCGU	UGCUUGCC			CCACTCTCGGCAAGCA	CCACUCUCGGCAAGCA
[SEQ ID	[SEQ ID	[SEQ ID	[SEQ ID	[SEQ ID			GAGCTGGGGTACTAGT	GAGCUGGGGUACUAGU
NO: 718]	NO: 1246]	NO: 834]	NO: 1362]	NO: 1066]			GAAATATATATTAAAG	GAAAUAUAUAUUAAAG
							TACCCCAGCCATGCTT	UACCCCAGCCAUGCUU
							GCCGTTAGTGTGACAG	GCCGUUAGUGUGACAG
							GGATACAGCAACTATT	GGAUACAGCAACUAUU
							TTATCAA	UUAUCAA
							[SEQ ID NO: 950]	[SEQ ID NO: 1478]
TTCGGCAAGCAG	UUCGGCAAGCAG	TACCCCAGCTAC	UACCCCAGCUAC	UACCCCAGCUCU	-4.40568	4350	ACCGGACATACTTGTT	ACCGGACAUACUUGUU
AGCTGGGGTA	AGCUGGGGUA	GCTTGCCGAT	GCUUGCCGAU	GCUUGCCG			CCACTCTTCGGCAAGC	CCACUCUUCGGCAAGC
[SEQ ID	[SEQ ID	[SEQ ID	[SEQ ID	[SEQ ID			AGAGCTGGGGTATAGT	AGAGCUGGGGUAUAGU
NO: 719]	NO: 1247]	NO: 835]	NO: 1363]	NO: 1067]			GAAATATATATTAAAT	GAAAUAUAUAUUAAAU
							ACCCCAGCTACGCTTG	ACCCCAGCUACGCUUG
							CCGATTAGTGTGACAG	CCGAUUAGUGUGACAG
							GGATACAGCAACTATT	GGAUACAGCAACUAUU
							TTATCAA	UUAUCAA
							[SEQ ID NO: 951]	[SEQ ID NO: 1479]
TTTCGGCAAGCA	UUUCGGCAAGCA	ACCCCAGCTCCT	ACCCCAGCUCCU	ACCCCAGCUCUG	-3.51989	4351	ACCGGACATACTTGTT	ACCGGACAUACUUGUU
GAGCTGGGGT	GAGCUGGGGU	CTTGCCGAAT	CUUGCCGAAU	CUUGCCGA			CCACTCTTTCGGCAAG	CCACUCUUUCGGCAAG
[SEQ ID	[SEQ ID	[SEQ ID	[SEQ ID	[SEQ ID			CAGAGCTGGGGTTAGT	CAGAGCUGGGGUUAGU
NO: 720]	NO: 1248]	NO: 836]	NO: 1364]	NO: 1068]			GAAATATATATTAAAA	GAAAUAUAUAUUAAAA
							CCCCAGCTCCTCTTGC	CCCCAGCUCCUCUUGC
							CGAATTAGTGTGACAG	CGAAUUAGUGUGACAG
							GGATACAGCAACTATT	GGAUACAGCAACUAUU
							TTATCAA	UUAUCAA
							[SEQ ID NO: 952]	[SEQ ID NO: 1480]
TTTTCGGCAAGC	UUUUCGGCAAGC	CCCCAGCTCTTA	CCCCAGCUCUUA	CCCCAGCUCUGC	-2.43883	4352	ACCGGACATACTTGTT	ACCGGACAUACUUGUU
AGAGCTGGGG	AGAGCUGGGG	TTGCCGAAAT	UUGCCGAAAU	UUGCCGAA			CCACTCTTTTCGGCAA	CCACUCUUUUCGGCAA
[SEQ ID	[SEQ ID	[SEQ ID	[SEQ ID	[SEQ ID			GCAGAGCTGGGGTAGT	GCAGAGCUGGGGUAGU
NO: 721]	NO: 1249]	NO: 837]	NO: 1365]	NO: 1069]			GAAATATATATTAAAC	GAAAUAUAUAUUAAAC
							CCCAGCTCTTATTGCC	CCCAGCUCUUAUUGCC
							GAAATTAGTGTGACAG	GAAAUUAGUGUGACAG
							GGATACAGCAACTATT	GGAUACAGCAACUAUU
							TTATCAA	UUAUCAA
							[SEQ ID NO: 953]	[SEQ ID NO: 1481]
AGTTTCGGCAAG	AGUUUCGGCAAG	CCCAGCTCTGAC	CCCAGCUCUGAC	CCCAGCUCUGCU	-3.70458	4353	ACCGGACATACTTGTT	ACCGGACAUACUUGUU
CAGAGCTGGG	CAGAGCUGGG	TGCCGAAACA	UGCCGAAACA	UGCCGAAA			CCACTCAGTTTCGGCA	CCACUCAGUUUCGGCA
[SEQ ID	[SEQ ID	[SEQ ID	[SEQ ID	[SEQ ID			AGCAGAGCTGGGTAGT	AGCAGAGCUGGGUAGU
NO: 722]	NO: 1250]	NO: 838]	NO: 1366]	NO: 1070]			GAAATATATATTAAAC	GAAAUAUAUAUUAAAC
							CCAGCTCTGACTGCCG	CCAGCUCUGACUGCCG
							AAACATAGTGTGACAG	AAACAUAGUGUGACAG
							GGATACAGCAACTATT	GGAUACAGCAACUAUU
							TTATCAA	UUAUCAA
							[SEQ ID NO: 954]	[SEQ ID NO: 1482]
TAGTTTCGGCAA	UAGUUUCGGCAA	CCAGCTCTGCCC	CCAGCUCUGCCC	CCAGCUCUGCUU	-2.834	4354	ACCGGACATACTTGTT	ACCGGACAUACUUGUU
GCAGAGCTGG	GCAGAGCUGG	GCCGAAACTT	GCCGAAACUU	GCCGAAAC			CCACTCTAGTTTCGGC	CCACUCUAGUUUCGGC
[SEQ ID	[SEQ ID	[SEQ ID	[SEQ ID	[SEQ ID			AAGCAGAGCTGGTAGT	AAGCAGAGCUGGUAGU
NO: 723]	NO: 1251]	NO: 839]	NO: 1367]	NO: 1071]			GAAATATATATTAAAC	GAAAUAUAUAUUAAAC
							CAGCTCTGCCCGCCGA	CAGCUCUGCCCGCCGA
							AACTTTAGTGTGACAG	AACUUUAGUGUGACAG
							GGATACAGCAACTATT	GGAUACAGCAACUAUU
							TTATCAA	UUAUCAA
							[SEQ ID NO: 955]	[SEQ ID NO: 1483]
TCAGTTTCGGCA	UCAGUUUCGGCA	CAGCTCTGCTCT	CAGCUCUGCUCU	CAGCUCUGCUUG	-4.92603	4355	ACCGGACATACTTGTT	ACCGGACAUACUUGUU
AGCAGAGCTG	AGCAGAGCUG	CCGAAACTGT	CCGAAACUGU	CCGAAACU			CCACTCTCAGTTTCGG	CCACUCUCAGUUUCGG
[SEQ ID	[SEQ ID	[SEQ ID	[SEQ ID	[SEQ ID			CAAGCAGAGCTGTAGT	CAAGCAGAGCUGUAGU
NO: 724]	NO: 1252]	NO: 840]	NO: 1368]	NO: 1072]			GAAATATATATTAAAC	GAAAUAUAUAUUAAAC
							AGCTCTGCTCTCCGAA	AGCUCUGCUCUCCGAA
							ACTGTTAGTGTGACAG	ACUGUUAGUGUGACAG
							GGATACAGCAACTATT	GGAUACAGCAACUAUU
							TTATCAA	UUAUCAA
							[SEQ ID NO: 956]	[SEQ ID NO: 1484]
TCCAGTTTCGGC	UCCAGUUUCGGC	AGCTCTGCTTTA	AGCUCUGCUUUA	AGCUCUGCUUGC	-4.91921	4356	ACCGGACATACTTGTT	ACCGGACAUACUUGUU
AAGCAGAGCT	AAGCAGAGCU	CGAAACTGGT	CGAAACUGGU	CGAAACUG			CCACTCTCCAGTTTCG	CCACUCUCCAGUUUCG
[SEQ ID	[SEQ ID	[SEQ ID	[SEQ ID	[SEQ ID			GCAAGCAGAGCTTAGT	GCAAGCAGAGCUUAGU
NO: 725]	NO: 1253]	NO: 841]	NO: 1369]	NO: 1073]			GAAATATATATTAAAA	GAAAUAUAUAUUAAAA
	(same as						GCTCTGCTTTACGAAA	GCUCUGCUUUACGAAA
	guide in XD-						CTGGTTAGTGTGACAG	CUGGUUAGUGUGACAG
	14861)						GGATACAGCAACTATT	GGAUACAGCAACUAUU
							TTATCAA	UUAUCAA
							[SEQ ID NO: 957]	[SEQ ID NO: 1485]
TTCCAGTTTCGG	UUCCAGUUUCGG	GCTCTGCTTGAA	GCUCUGCUUGAA	GCUCUGCUUGCC	-4.12374	4357	ACCGGACATACTTGTT	ACCGGACAUACUUGUU
CAAGCAGAGC	CAAGCAGAGC	GAAACTGGAT	GAAACUGGAU	GAAACUGG			CCACTCTTCCAGTTTC	CCACUCUUCCAGUUUC
[SEQ ID	[SEQ ID	[SEQ ID	[SEQ ID	[SEQ ID			GGCAAGCAGAGCTAGT	GGCAAGCAGAGCUAGU
NO: 726]	NO: 1254]	NO: 842]	NO: 1370]	NO: 1074]			GAAATATATATTAAAG	GAAAUAUAUAUUAAAG
							CTCTGCTTGAAGAAAC	CUCUGCUUGAAGAAAC
							TGGATTAGTGTGACAG	UGGAUUAGUGUGACAG
							GGATACAGCAACTATT	GGAUACAGCAACUAUU
							TTATCAA	UUAUCAA
							[SEQ ID NO: 958]	[SEQ ID NO: 1486]
TTTCCAGTTTCG	UUUCCAGUUUCG	CTCTGCTTGCAT	CUCUGCUUGCAU	CUCUGCUUGCCG	-4.13551	4358	ACCGGACATACTTGTT	ACCGGACAUACUUGUU
GCAAGCAGAG	GCAAGCAGAG	AAACTGGAAT	AAACUGGAAU	AAACUGGA			CCACTCTTTCCAGTTT	CCACUCUUUCCAGUUU
[SEQ ID	[SEQ ID	[SEQ ID	[SEQ ID	[SEQ ID			CGGCAAGCAGAGTAGT	CGGCAAGCAGAGUAGU
NO: 727]	NO: 1255]	NO: 843]	NO: 1371]	NO: 1075]			GAAATATATATTAAAC	GAAAUAUAUAUUAAAC
							TCTGCTTGCATAAACT	UCUGCUUGCAUAAACU
							GGAATTAGTGTGACAG	GGAAUUAGUGUGACAG
							GGATACAGCAACTATT	GGAUACAGCAACUAUU
							TTATCAA	UUAUCAA
							[SEQ ID NO: 959]	[SEQ ID NO: 1487]
ACTTCCAGTTTC	ACUUCCAGUUUC	TCTGCTTGCCTG	UCUGCUUGCCUG	UCUGCUUGCCGA	-4.43988	4359	ACCGGACATACTTGTT	ACCGGACAUACUUGUU
GGCAAGCAGA	GGCAAGCAGA	AACTGGAAGA	AACUGGAAGA	AACUGGAA			CCACTCACTTCCAGTT	CCACUCACUUCCAGUU
[SEQ ID	[SEQ ID	[SEQ ID	[SEQ ID	[SEQ ID			TCGGCAAGCAGATAGT	UCGGCAAGCAGAUAGU
NO: 728]	NO: 1256]	NO: 844]	NO: 1372]	NO: 1076]			GAAATATATATTAAAT	GAAAUAUAUAUUAAAU
							CTGCTTGCCTGAACTG	CUGCUUGCCUGAACUG
							GAAGATAGTGTGACAG	GAAGAUAGUGUGACAG
							GGATACAGCAACTATT	GGAUACAGCAACUAUU
							TTATCAA	UUAUCAA
							[SEQ ID NO: 960]	[SEQ ID NO: 1488]
AACTTCCAGTTT	AACUUCCAGUUU	CTGCTTGCCGGG	CUGCUUGCCGGG	CUGCUUGCCGAA	-3.57411	4360	ACCGGACATACTTGTT	ACCGGACAUACUUGUU
CGGCAAGCAG	CGGCAAGCAG	ACTGGAAGTA	ACUGGAAGUA	ACUGGAAG			CCACTCAACTTCCAGT	CCACUCAACUUCCAGU
[SEQ ID	[SEQ ID	[SEQ ID	[SEQ ID	[SEQ ID			TTCGGCAAGCAGTAGT	UUCGGCAAGCAGUAGU
NO: 729]	NO: 1257]	NO: 845]	NO: 1373]	NO: 1077]			GAAATATATATTAAAC	GAAAUAUAUAUUAAAC
							TGCTTGCCGGGACTGG	UGCUUGCCGGGACUGG
							AAGTATAGTGTGACAG	AAGUAUAGUGUGACAG
							GGATACAGCAACTATT	GGAUACAGCAACUAUU
							TTATCAA	UUAUCAA
							[SEQ ID NO: 961]	[SEQ ID NO: 1489]
TAACTTCCAGTT	UAACUUCCAGUU	TGCTTGCCGAGG	UGCUUGCCGAGG	UGCUUGCCGAAA	-3.33142	4361	ACCGGACATACTTGTT	ACCGGACAUACUUGUU
TCGGCAAGCA	UCGGCAAGCA	CTGGAAGTTT	CUGGAAGUUU	CUGGAAGU			CCACTCTAACTTCCAG	CCACUCUAACUUCCAG
[SEQ ID	[SEQ ID	[SEQ ID	[SEQ ID	[SEQ ID			TTTCGGCAAGCATAGT	UUUCGGCAAGCAUAGU
NO: 730]	NO: 1258]	NO: 846]	NO: 1374]	NO: 1078]			GAAATATATATTAAAT	GAAAUAUAUAUUAAAU
							GCTTGCCGAGGCTGGA	GCUUGCCGAGGCUGGA
							AGTTTTAGTGTGACAG	AGUUUUAGUGUGACAG
							GGATACAGCAACTATT	GGAUACAGCAACUAUU
							TTATCAA	UUAUCAA
							[SEQ ID NO: 962]	[SEQ ID NO: 1490]
ATAACTTCCAGT	AUAACUUCCAGU	GCTTGCCGAAGA	GCUUGCCGAAGA	GCUUGCCGAAAC	-3.93535	4362	ACCGGACATACTTGTT	ACCGGACAUACUUGUU
TTCGGCAAGC	UUCGGCAAGC	TGGAAGTTAA	UGGAAGUUAA	UGGAAGUU			CCACTCATAACTTCCA	CCACUCAUAACUUCCA
[SEQ ID	[SEQ ID	[SEQ ID	[SEQ ID	[SEQ ID			GTTTCGGCAAGCTAGT	GUUUCGGCAAGCUAGU
NO: 731]	NO: 1259]	NO: 847]	NO: 1375]	NO: 1079]			GAAATATATATTAAAG	GAAAUAUAUAUUAAAG
	(same as						CTTGCCGAAGATGGAA	CUUGCCGAAGAUGGAA
	guide in XD-						GTTAATAGTGTGACAG	GUUAAUAGUGUGACAG
	14932)						GGATACAGCAACTATT	GGAUACAGCAACUAUU
							TTATCAA	UUAUCAA
							[SEQ ID NO: 963]	[SEQ ID NO: 1491]
AATAACTTCCAG	AAUAACUUCCAG	CTTGCCGAAAAC	CUUGCCGAAAAC	CUUGCCGAAACU	-3.56666	4363	ACCGGACATACTTGTT	ACCGGACAUACUUGUU
TTTCGGCAAG	UUUCGGCAAG	GGAAGTTATA	GGAAGUUAUA	GGAAGUUA			CCACTCAATAACTTCC	CCACUCAAUAACUUCC
[SEQ ID	[SEQ ID	[SEQ ID	[SEQ ID	[SEQ ID			AGTTTCGGCAAGTAGT	AGUUUCGGCAAGUAGU
NO: 732]	NO: 1260]	NO: 848]	NO: 1376]	NO: 1080]			GAAATATATATTAAAC	GAAAUAUAUAUUAAAC
							TTGCCGAAAACGGAAG	UUGCCGAAAACGGAAG
							TTATATAGTGTGACAG	UUAUAUAGUGUGACAG
							GGATACAGCAACTATT	GGAUACAGCAACUAUU
							TTATCAA	UUAUCAA
							[SEQ ID NO: 964]	[SEQ ID NO: 1492]
AAATAACTTCCA	AAAUAACUUCCA	TTGCCGAAACCT	UUGCCGAAACCU	UUGCCGAAACUG	-2.9553	4364	ACCGGACATACTTGTT	ACCGGACAUACUUGUU
GTTTCGGCAA	GUUUCGGCAA	GAAGTTATTA	GAAGUUAUUA	GAAGUUAU			CCACTCAAATAACTTC	CCACUCAAAUAACUUC
[SEQ ID	[SEQ ID	[SEQ ID	[SEQ ID	[SEQ ID			CAGTTTCGGCAATAGT	CAGUUUCGGCAAUAGU
NO: 733]	NO: 1261]	NO: 849]	NO: 1377]	NO: 1081]			GAAATATATATTAAAT	GAAAUAUAUAUUAAAU
							TGCCGAAACCTGAAGT	UGCCGAAACCUGAAGU
							TATTATAGTGTGACAG	UAUUAUAGUGUGACAG
							GGATACAGCAACTATT	GGAUACAGCAACUAUU
							TTATCAA	UUAUCAA
							[SEQ ID NO: 965]	[SEQ ID NO: 1493]
ATAAATAACTTC	AUAAAUAACUUC	GCCGAAACTGTG	GCCGAAACUGUG	GCCGAAACUGGA	-3.3862	4366	ACCGGACATACTTGTT	ACCGGACAUACUUGUU
CAGTTTCGGC	CAGUUUCGGC	AGTTATTTAA	AGUUAUUUAA	AGUUAUUU			CCACTCATAAATAACT	CCACUCAUAAAUAACU
[SEQ ID	[SEQ ID	[SEQ ID	[SEQ ID	[SEQ ID			TCCAGTTTCGGCTAGT	UCCAGUUUCGGCUAGU
NO: 734]	NO: 1262]	NO: 850]	NO: 1378]	NO: 1082]			GAAATATATATTAAAG	GAAAUAUAUAUUAAAG
							CCGAAACTGTGAGTTA	CCGAAACUGUGAGUUA
							TTTAATAGTGTGACAG	UUUAAUAGUGUGACAG
							GGATACAGCAACTATT	GGAUACAGCAACUAUU
							TTATCAA	UUAUCAA
							[SEQ ID NO: 966]	[SEQ ID NO: 1494]
AATAAATAACTT	AAUAAAUAACUU	CCGAAACTGGGG	CCGAAACUGGGG	CCGAAACUGGAA	-3.45626	4367	ACCGGACATACTTGTT	ACCGGACAUACUUGUU
CCAGTTTCGG	CCAGUUUCGG	GTTATTTATA	GUUAUUUAUA	GUUAUUUA			CCACTCAATAAATAAC	CCACUCAAUAAAUAAC
[SEQ ID	[SEQ ID	[SEQ ID	[SEQ ID	[SEQ ID			TTCCAGTTTCGGTAGT	UUCCAGUUUCGGUAGU
NO: 735]	NO: 1263]	NO: 851]	NO: 1379]	NO: 1083]			GAAATATATATTAAAC	GAAAUAUAUAUUAAAC
							CGAAACTGGGGGTTAT	CGAAACUGGGGGUUAU
							TTATATAGTGTGACAG	UUAUAUAGUGUGACAG
							GGATACAGCAACTATT	GGAUACAGCAACUAUU
							TTATCAA	UUAUCAA
							[SEQ ID NO: 967]	[SEQ ID NO: 1495]
AAATAAATAACT	AAAUAAAUAACU	CGAAACTGGAGT	CGAAACUGGAGU	CGAAACUGGAAG	-1.76663	4368	ACCGGACATACTTGTT	ACCGGACAUACUUGUU
TCCAGTTTCG	UCCAGUUUCG	TTATTTATTA	UUAUUUAUUA	UUAUUUAU			CCACTCAAATAAATAA	CCACUCAAAUAAAUAA
[SEQ ID	[SEQ ID	[SEQ ID	[SEQ ID	[SEQ ID			CTTCCAGTTTCGTAGT	CUUCCAGUUUCGUAGU
NO: 736]	NO: 1264]	NO: 852]	NO: 1380]	NO: 1084]			GAAATATATATTAAAC	GAAAUAUAUAUUAAAC
							GAAACTGGAGTTTATT	GAAACUGGAGUUUAUU
							TATTATAGTGTGACAG	UAUUAUAGUGUGACAG
							GGATACAGCAACTATT	GGAUACAGCAACUAUU
							TTATCAA	UUAUCAA
							[SEQ ID NO: 968]	[SEQ ID NO: 1496]
AAAATAAATAAC	AAAAUAAAUAAC	GAAACTGGAATC	GAAACUGGAAUC	GAAACUGGAAGU	-0.22922	4369	ACCGGACATACTTGTT	ACCGGACAUACUUGUU
TTCCAGTTTC	UUCCAGUUUC	TATTTATTTA	UAUUUAUUUA	UAUUUAUU			CCACTCAAAATAAATA	CCACUCAAAAUAAAUA
[SEQ ID	[SEQ ID	[SEQ ID	[SEQ ID	[SEQ ID			ACTTCCAGTTTCTAGT	ACUUCCAGUUUCUAGU
NO: 737]	NO: 1265]	NO: 853]	NO: 1381]	NO: 1085]			GAAATATATATTAAAG	GAAAUAUAUAUUAAAG
							AAACTGGAATCTATTT	AAACUGGAAUCUAUUU
							ATTTATAGTGTGACAG	AUUUAUAGUGUGACAG
							GGATACAGCAACTATT	GGAUACAGCAACUAUU
							TTATCAA	UUAUCAA
							[SEQ ID NO: 969]	[SEQ ID NO: 1497]
AAAAATAAATAA	AAAAAUAAAUAA	AAACTGGAAGCC	AAACUGGAAGCC	AAACUGGAAGUU	-0.00312	4370	ACCGGACATACTTGTT	ACCGGACAUACUUGUU
CTTCCAGTTT	CUUCCAGUUU	ATTTATTTTA	AUUUAUUUUA	AUUUAUUU			CCACTCAAAAATAAAT	CCACUCAAAAAUAAAU
[SEQ ID	[SEQ ID	[SEQ ID	[SEQ ID	[SEQ ID			AACTTCCAGTTTTAGT	AACUUCCAGUUUUAGU
NO: 738]	NO: 1266]	NO: 854]	NO: 1382]	NO: 1086]			GAAATATATATTAAAA	GAAAUAUAUAUUAAAA
							AACTGGAAGCCATTTA	AACUGGAAGCCAUUUA
							TTTTATAGTGTGACAG	UUUUAUAGUGUGACAG
							GGATACAGCAACTATT	GGAUACAGCAACUAUU
							TTATCAA	UUAUCAA
							[SEQ ID NO: 970]	[SEQ ID NO: 1498]
ATGACTTTCAAG	AUGACUUUCAAG	TTTAATAACCAC	UUUAAUAACCAC	UUUAAUAACCCU	-2.77085	4390	ACCGGACATACTTGTT	ACCGGACAUACUUGUU
GGTTATTAAA	GGUUAUUAAA	TGAAAGTCAA	UGAAAGUCAA	UGAAAGUC			CCACTCATGACTTTCA	CCACUCAUGACUUUCA
[SEQ ID	[SEQ ID	[SEQ ID	[SEQ ID	[SEQ ID			AGGGTTATTAAATAGT	AGGGUUAUUAAAUAGU
NO: 739]	NO: 1267]	NO: 855]	NO: 1383]	NO: 1087]			GAAATATATATTAAAT	GAAAUAUAUAUUAAAU
							TTAATAACCACTGAAA	UUAAUAACCACUGAAA
							GTCAATAGTGTGACAG	GUCAAUAGUGUGACAG
							GGATACAGCAACTATT	GGAUACAGCAACUAUU
							TTATCAA	UUAUCAA
							[SEQ ID NO: 971]	[SEQ ID NO: 1499]
TATGACTTTCAA	UAUGACUUUCAA	TTAATAACCCCC	UUAAUAACCCCC	UUAAUAACCCUU	-3.75832	4391	ACCGGACATACTTGTT	ACCGGACAUACUUGUU
GGGTTATTAA	GGGUUAUUAA	GAAAGTCATT	GAAAGUCAUU	GAAAGUCA			CCACTCTATGACTTTC	CCACUCUAUGACUUUC
[SEQ ID	[SEQ ID	[SEQ ID	[SEQ ID	[SEQ ID			AAGGGTTATTAATAGT	AAGGGUUAUUAAUAGU
NO: 740]	NO: 1268]	NO: 856]	NO: 1384]	NO: 1088]			GAAATATATATTAAAT	GAAAUAUAUAUUAAAU
							TAATAACCCCCGAAAG	UAAUAACCCCCGAAAG
							TCATTTAGTGTGACAG	UCAUUUAGUGUGACAG
							GGATACAGCAACTATT	GGAUACAGCAACUAUU
							TTATCAA	UUAUCAA
							[SEQ ID NO: 972]	[SEQ ID NO: 1500]
TCATGACTTTCA	UCAUGACUUUCA	TAATAACCCTCT	UAAUAACCCUCU	UAAUAACCCUUG	-4.61593	4392	ACCGGACATACTTGTT	ACCGGACAUACUUGUU
AGGGTTATTA	AGGGUUAUUA	AAAGTCATGT	AAAGUCAUGU	AAAGUCAU			CCACTCTCATGACTTT	CCACUCUCAUGACUUU
[SEQ ID	[SEQ ID	[SEQ ID	[SEQ ID	[SEQ ID			CAAGGGTTATTATAGT	CAAGGGUUAUUAUAGU
NO: 741]	NO: 1269]	NO: 857]	NO: 1385]	NO: 1089]			GAAATATATATTAAAT	GAAAUAUAUAUUAAAU
	(same as						AATAACCCTCTAAAGT	AAUAACCCUCUAAAGU
	guide in XD-						CATGTTAGTGTGACAG	CAUGUUAGUGUGACAG
	14933)						GGATACAGCAACTATT	GGAUACAGCAACUAUU
							TTATCAA	UUAUCAA
							[SEQ ID NO: 973]	[SEQ ID NO: 1501]
TTCATGACTTTC	UUCAUGACUUUC	AATAACCCTTTG	AAUAACCCUUUG	AAUAACCCUUGA	-3.88921	4393	ACCGGACATACTTGTT	ACCGGACAUACUUGUU
AAGGGTTATT	AAGGGUUAUU	AAGTCATGAT	AAGUCAUGAU	AAGUCAUG			CCACTCTTCATGACTT	CCACUCUUCAUGACUU
[SEQ ID	[SEQ ID	[SEQ ID	[SEQ ID	[SEQ ID			TCAAGGGTTATTTAGT	UCAAGGGUUAUUUAGU
NO: 742]	NO: 1270]	NO: 858]	NO: 1386]	NO: 1090]			GAAATATATATTAAAA	GAAAUAUAUAUUAAAA
							ATAACCCTTTGAAGTC	AUAACCCUUUGAAGUC
							ATGATTAGTGTGACAG	AUGAUUAGUGUGACAG
							GGATACAGCAACTATT	GGAUACAGCAACUAUU
							TTATCAA	UUAUCAA
							[SEQ ID NO: 974]	[SEQ ID NO: 1502]
TTTCATGACTTT	UUUCAUGACUUU	ATAACCCTTGGG	AUAACCCUUGGG	AUAACCCUUGAA	-3.57278	4394	ACCGGACATACTTGTT	ACCGGACAUACUUGUU
CAAGGGTTAT	CAAGGGUUAU	AGTCATGAAT	AGUCAUGAAU	AGUCAUGA			CCACTCTTTCATGACT	CCACUCUUUCAUGACU
[SEQ ID	[SEQ ID	[SEQ ID	[SEQ ID	[SEQ ID			TTCAAGGGTTATTAGT	UUCAAGGGUUAUUAGU
NO: 743]	NO: 1271]	NO: 859]	NO: 1387]	NO: 1091]			GAAATATATATTAAAA	GAAAUAUAUAUUAAAA
							TAACCCTTGGGAGTCA	UAACCCUUGGGAGUCA
							TGAATTAGTGTGACAG	UGAAUUAGUGUGACAG
							GGATACAGCAACTATT	GGAUACAGCAACUAUU
							TTATCAA	UUAUCAA
							[SEQ ID NO: 975]	[SEQ ID NO: 1503]
TGTTCATGACTT	UGUUCAUGACUU	TAACCCTTGAGG	UAACCCUUGAGG	UAACCCUUGAAA	-4.31654	4395	ACCGGACATACTTGTT	ACCGGACAUACUUGUU
TCAAGGGTTA	UCAAGGGUUA	GTCATGAACT	GUCAUGAACU	GUCAUGAA			CCACTCTGTTCATGAC	CCACUCUGUUCAUGAC
[SEQ ID	[SEQ ID	[SEQ ID	[SEQ ID	[SEQ ID			TTTCAAGGGTTATAGT	UUUCAAGGGUUAUAGU
NO: 744]	NO: 1272]	NO: 860]	NO: 1388]	NO: 1092]			GAAATATATATTAAAT	GAAAUAUAUAUUAAAU
							AACCCTTGAGGGTCAT	AACCCUUGAGGGUCAU
							GAACTTAGTGTGACAG	GAACUUAGUGUGACAG
							GGATACAGCAACTATT	GGAUACAGCAACUAUU
							TTATCAA	UUAUCAA
							[SEQ ID NO: 976]	[SEQ ID NO: 1504]
TTGTTCATGACT	UUGUUCAUGACU	AACCCTTGAAGT	AACCCUUGAAGU	AACCCUUGAAAG	-3.76092	4396	ACCGGACATACTTGTT	ACCGGACAUACUUGUU
TTCAAGGGTT	UUCAAGGGUU	TCATGAACAT	UCAUGAACAU	UCAUGAAC			CCACTCTTGTTCATGA	CCACUCUUGUUCAUGA
[SEQ ID	[SEQ ID	[SEQ ID	[SEQ ID	[SEQ ID			CTTTCAAGGGTTTAGT	CUUUCAAGGGUUUAGU
NO: 745]	NO: 1273]	NO: 861]	NO: 1389]	NO: 1093]			GAAATATATATTAAAA	GAAAUAUAUAUUAAAA
							ACCCTTGAAGTTCATG	ACCCUUGAAGUUCAUG
							AACATTAGTGTGACAG	AACAUUAGUGUGACAG
							GGATACAGCAACTATT	GGAUACAGCAACUAUU
							TTATCAA	UUAUCAA
							[SEQ ID NO: 977]	[SEQ ID NO: 1505]
TGTGTTCATGAC	UGUGUUCAUGAC	ACCCTTGAAATC	ACCCUUGAAAUC	ACCCUUGAAAGU	-4.07971	4397	ACCGGACATACTTGTT	ACCGGACAUACUUGUU
TTTCAAGGGT	UUUCAAGGGU	CATGAACACT	CAUGAACACU	CAUGAACA			CCACTCTGTGTTCATG	CCACUCUGUGUUCAUG
[SEQ ID	[SEQ ID	[SEQ ID	[SEQ ID	[SEQ ID			ACTTTCAAGGGTTAGT	ACUUUCAAGGGUUAGU
NO: 746]	NO: 1274]	NO: 862]	NO: 1390]	NO: 1094]			GAAATATATATTAAAA	GAAAUAUAUAUUAAAA
							CCCTTGAAATCCATGA	CCCUUGAAAUCCAUGA
							ACACTTAGTGTGACAG	ACACUUAGUGUGACAG
							GGATACAGCAACTATT	GGAUACAGCAACUAUU
							TTATCAA	UUAUCAA
							[SEQ ID NO: 978]	[SEQ ID NO: 1506]
ATGTGTTCATGA	AUGUGUUCAUGA	CCCTTGAAAGCA	CCCUUGAAAGCA	CCCUUGAAAGUC	-4.76135	4398	ACCGGACATACTTGTT	ACCGGACAUACUUGUU
CTTTCAAGGG	CUUUCAAGGG	ATGAACACAA	AUGAACACAA	AUGAACAC			CCACTCATGTGTTCAT	CCACUCAUGUGUUCAU
[SEQ ID	[SEQ ID	[SEQ ID	[SEQ ID	[SEQ ID			GACTTTCAAGGGTAGT	GACUUUCAAGGGUAGU
NO: 747]	NO: 1275]	NO: 863]	NO: 1391]	NO: 1095]			GAAATATATATTAAAC	GAAAUAUAUAUUAAAC
							CCTTGAAAGCAATGAA	CCUUGAAAGCAAUGAA
							CACAATAGTGTGACAG	CACAAUAGUGUGACAG
							GGATACAGCAACTATT	GGAUACAGCAACUAUU
							TTATCAA	UUAUCAA
							[SEQ ID NO: 979]	[SEQ ID NO: 1507]
TATGTGTTCATG	UAUGUGUUCAUG	CCTTGAAAGTAG	CCUUGAAAGUAG	CCUUGAAAGUCA	-4.33797	4399	ACCGGACATACTTGTT	ACCGGACAUACUUGUU
ACTTTCAAGG	ACUUUCAAGG	TGAACACATT	UGAACACAUU	UGAACACA			CCACTCTATGTGTTCA	CCACUCUAUGUGUUCA
[SEQ ID	[SEQ ID	[SEQ ID	[SEQ ID	[SEQ ID			TGACTTTCAAGGTAGT	UGACUUUCAAGGUAGU
NO: 748]	NO: 1276]	NO: 864]	NO: 1392]	NO: 1096]			GAAATATATATTAAAC	GAAAUAUAUAUUAAAC
							CTTGAAAGTAGTGAAC	CUUGAAAGUAGUGAAC
							ACATTTAGTGTGACAG	ACAUUUAGUGUGACAG
							GGATACAGCAACTATT	GGAUACAGCAACUAUU
							TTATCAA	UUAUCAA
							[SEQ ID NO: 980]	[SEQ ID NO: 1508]
TGATGTGTTCAT	UGAUGUGUUCAU	CTTGAAAGTCGC	CUUGAAAGUCGC	CUUGAAAGUCAU	-4.13084	4400	ACCGGACATACTTGTT	ACCGGACAUACUUGUU
GACTTTCAAG	GACUUUCAAG	GAACACATCT	GAACACAUCU	GAACACAU			CCACTCTGATGTGTTC	CCACUCUGAUGUGUUC
[SEQ ID	[SEQ ID	[SEQ ID	[SEQ ID	[SEQ ID			ATGACTTTCAAGTAGT	AUGACUUUCAAGUAGU
NO: 749]	NO: 1277]	NO: 865]	NO: 1393]	NO: 1097]			GAAATATATATTAAAC	GAAAUAUAUAUUAAAC
							TTGAAAGTCGCGAACA	UUGAAAGUCGCGAACA
							CATCTTAGTGTGACAG	CAUCUUAGUGUGACAG
							GGATACAGCAACTATT	GGAUACAGCAACUAUU
							TTATCAA	UUAUCAA
							[SEQ ID NO: 981]	[SEQ ID NO: 1509]
TTGATGTGTTCA	UUGAUGUGUUCA	TTGAAAGTCACT	UUGAAAGUCACU	UUGAAAGUCAUG	-4.24964	4401	ACCGGACATACTTGTT	ACCGGACAUACUUGUU
TGACTTTCAA	UGACUUUCAA	AACACATCAT	AACACAUCAU	AACACAUC			CCACTCTTGATGTGTT	CCACUCUUGAUGUGUU
[SEQ ID	[SEQ ID	[SEQ ID	[SEQ ID	[SEQ ID			CATGACTTTCAATAGT	CAUGACUUUCAAUAGU
NO: 750]	NO: 1278]	NO: 866]	NO: 1394]	NO: 1098]			GAAATATATATTAAAT	GAAAUAUAUAUUAAAU
							TGAAAGTCACTAACAC	UGAAAGUCACUAACAC
							ATCATTAGTGTGACAG	AUCAUUAGUGUGACAG
							GGATACAGCAACTATT	GGAUACAGCAACUAUU
							TTATCAA	UUAUCAA
							[SEQ ID NO: 982]	[SEQ ID NO: 1510]
TCTGATGTGTTC	UCUGAUGUGUUC	TGAAAGTCATTG	UGAAAGUCAUUG	UGAAAGUCAUGA	-4.95673	4402	ACCGGACATACTTGTT	ACCGGACAUACUUGUU
ATGACTTTCA	AUGACUUUCA	ACACATCAGT	ACACAUCAGU	ACACAUCA			CCACTCTCTGATGTGT	CCACUCUCUGAUGUGU
[SEQ ID	[SEQ ID	[SEQ ID	[SEQ ID	[SEQ ID			TCATGACTTTCATAGT	UCAUGACUUUCAUAGU
NO: 751]	NO: 1279]	NO: 867]	NO: 1395]	NO: 1099]			GAAATATATATTAAAT	GAAAUAUAUAUUAAAU
							GAAAGTCATTGACACA	GAAAGUCAUUGACACA
							TCAGTTAGTGTGACAG	UCAGUUAGUGUGACAG
							GGATACAGCAACTATT	GGAUACAGCAACUAUU
							TTATCAA	UUAUCAA
							[SEQ ID NO: 983]	[SEQ ID NO: 1511]
AGCTGATGTGTT	AGCUGAUGUGUU	GAAAGTCATGGG	GAAAGUCAUGGG	GAAAGUCAUGAA	-3.87865	4403	ACCGGACATACTTGTT	ACCGGACAUACUUGUU
CATGACTTTC	CAUGACUUUC	CACATCAGCA	CACAUCAGCA	CACAUCAG			CCACTCAGCTGATGTG	CCACUCAGCUGAUGUG
[SEQ ID	[SEQ ID	[SEQ ID	[SEQ ID	[SEQ ID			TTCATGACTTTCTAGT	UUCAUGACUUUCUAGU
NO: 752]	NO: 1280]	NO: 868]	NO: 1396]	NO: 1100]			GAAATATATATTAAAG	GAAAUAUAUAUUAAAG
							AAAGTCATGGGCACAT	AAAGUCAUGGGCACAU
							CAGCATAGTGTGACAG	CAGCAUAGUGUGACAG
							GGATACAGCAACTATT	GGAUACAGCAACUAUU
							TTATCAA	UUAUCAA
							[SEQ ID NO: 984]	[SEQ ID NO: 1512]
TAGCTGATGTGT	UAGCUGAUGUGU	AAAGTCATGAGA	AAAGUCAUGAGA	AAAGUCAUGAAC	-3.03908	4404	ACCGGACATACTTGTT	ACCGGACAUACUUGUU
TCATGACTTT	UCAUGACUUU	ACATCAGCTT	ACAUCAGCUU	ACAUCAGC			CCACTCTAGCTGATGT	CCACUCUAGCUGAUGU
[SEQ ID	[SEQ ID	[SEQ ID	[SEQ ID	[SEQ ID			GTTCATGACTTTTAGT	GUUCAUGACUUUUAGU
NO: 753]	NO: 1281]	NO: 869]	NO: 1397]	NO: 1101]			GAAATATATATTAAAA	GAAAUAUAUAUUAAAA
							AAGTCATGAGAACATC	AAGUCAUGAGAACAUC
							AGCTTTAGTGTGACAG	AGCUUUAGUGUGACAG
							GGATACAGCAACTATT	GGAUACAGCAACUAUU
							TTATCAA	UUAUCAA
							[SEQ ID NO: 985]	[SEQ ID NO: 1513]
TTAGCTGATGTG	UUAGCUGAUGUG	AAGTCATGAAAG	AAGUCAUGAAAG	AAGUCAUGAACA	-4. 75357	4405	ACCGGACATACTTGTT	ACCGGACAUACUUGUU
TTCATGACTT	UUCAUGACUU	CATCAGCTAT	CAUCAGCUAU	CAUCAGCU			CCACTCTTAGCTGATG	CCACUCUUAGCUGAUG
[SEQ ID	[SEQ ID	[SEQ ID	[SEQ ID	[SEQ ID			TGTTCATGACTTTAGT	UGUUCAUGACUUUAGU
NO: 754]	NO: 1282]	NO: 870]	NO: 1398]	NO: 1102]			GAAATATATATTAAAA	GAAAUAUAUAUUAAAA
							AGTCATGAAAGCATCA	AGUCAUGAAAGCAUCA
							GCTATTAGTGTGACAG	GCUAUUAGUGUGACAG
							GGATACAGCAACTATT	GGAUACAGCAACUAUU
							TTATCAA	UUAUCAA
							[SEQ ID NO: 986]	[SEQ ID NO: 1514]
TCTAGCTGATGT	UCUAGCUGAUGU	AGTCATGAACGA	AGUCAUGAACGA	AGUCAUGAACAC	-5.05901	4406	ACCGGACATACTTGTT	ACCGGACAUACUUGUU
GTTCATGACT	GUUCAUGACU	ATCAGCTAGT	AUCAGCUAGU	AUCAGCUA			CCACTCTCTAGCTGAT	CCACUCUCUAGCUGAU
[SEQ ID	[SEQ ID	[SEQ ID	[SEQ ID	[SEQ ID			GTGTTCATGACTTAGT	GUGUUCAUGACUUAGU
NO: 755]	NO: 1283]	NO: 871]	NO: 1399]	NO: 1103]			GAAATATATATTAAAA	GAAAUAUAUAUUAAAA
							GTCATGAACGAATCAG	GUCAUGAACGAAUCAG
							CTAGTTAGTGTGACAG	CUAGUUAGUGUGACAG
							GGATACAGCAACTATT	GGAUACAGCAACUAUU
							TTATCAA	UUAUCAA
							[SEQ ID NO: 987]	[SEQ ID NO: 1515]
TGCTAGCTGATG	UGCUAGCUGAUG	GTCATGAACAAG	GUCAUGAACAAG	GUCAUGAACACA	-4.47567	4407	ACCGGACATACTTGTT	ACCGGACAUACUUGUU
TGTTCATGAC	UGUUCAUGAC	TCAGCTAGCT	UCAGCUAGCU	UCAGCUAG			CCACTCTGCTAGCTGA	CCACUCUGCUAGCUGA
[SEQ ID	[SEQ ID	[SEQ ID	[SEQ ID	[SEQ ID			TGTGTTCATGACTAGT	UGUGUUCAUGACUAGU
NO: 756]	NO: 1284]	NO: 872]	NO: 1400]	NO: 1104]			GAAATATATATTAAAG	GAAAUAUAUAUUAAAG
							TCATGAACAAGTCAGC	UCAUGAACAAGUCAGC
							TAGCTTAGTGTGACAG	UAGCUUAGUGUGACAG
							GGATACAGCAACTATT	GGAUACAGCAACUAUU
							TTATCAA	UUAUCAA
							[SEQ ID NO: 988]	[SEQ ID NO: 1516]
TTGCTAGCTGAT	UUGCUAGCUGAU	TCATGAACACGC	UCAUGAACACGC	UCAUGAACACAU	-4.42302	4408	ACCGGACATACTTGTT	ACCGGACAUACUUGUU
GTGTTCATGA	GUGUUCAUGA	CAGCTAGCAT	CAGCUAGCAU	CAGCUAGC			CCACTCTTGCTAGCTG	CCACUCUUGCUAGCUG
[SEQ ID	[SEQ ID	[SEQ ID	[SEQ ID	[SEQ ID			ATGTGTTCATGATAGT	AUGUGUUCAUGAUAGU
NO: 757]	NO: 1285]	NO: 873]	NO: 1401]	NO: 1105]			GAAATATATATTAAAT	GAAAUAUAUAUUAAAU
							CATGAACACGCCAGCT	CAUGAACACGCCAGCU
							AGCATTAGTGTGACAG	AGCAUUAGUGUGACAG
							GGATACAGCAACTATT	GGAUACAGCAACUAUU
							TTATCAA	UUAUCAA
							[SEQ ID NO: 989]	[SEQ ID NO: 1517]
TTTGCTAGCTGA	UUUGCUAGCUGA	CATGAACACACA	CAUGAACACACA	CAUGAACACAUC	-5.35102	4409	ACCGGACATACTTGTT	ACCGGACAUACUUGUU
TGTGTTCATG	UGUGUUCAUG	AGCTAGCAAT	AGCUAGCAAU	AGCUAGCA			CCACTCTTTGCTAGCT	CCACUCUUUGCUAGCU
[SEQ ID	[SEQ ID	[SEQ ID	[SEQ ID	[SEQ ID			GATGTGTTCATGTAGT	GAUGUGUUCAUGUAGU
NO: 758]	NO: 1286]	NO: 874 ]	NO: 1402]	NO: 1106]			GAAATATATATTAAAC	GAAAUAUAUAUUAAAC
	(same as						ATGAACACACAAGCTA	AUGAACACACAAGCUA
	guide in XD-						GCAATTAGTGTGACAG	GCAAUUAGUGUGACAG
	14934)						GGATACAGCAACTATT	GGAUACAGCAACUAUU
							TTATCAA	UUAUCAA
							[SEQ ID NO: 990]	[SEQ ID NO: 1518]
TTTTGCTAGCTG	UUUUGCUAGCUG	ATGAACACATAG	AUGAACACAUAG	AUGAACACAUCA	-0.73644	4410	ACCGGACATACTTGTT	ACCGGACAUACUUGUU
ATGTGTTCAT	AUGUGUUCAU	GCTAGCAAAT	GCUAGCAAAU	GCUAGCAA			CCACTCTTTTGCTAGC	CCACUCUUUUGCUAGC
[SEQ ID	[SEQ ID	[SEQ ID	[SEQ ID	[SEQ ID			TGATGTGTTCATTAGT	UGAUGUGUUCAUUAGU
NO: 759]	NO: 1287]	NO: 875]	NO: 1403]	NO: 1107]			GAAATATATATTAAAA	GAAAUAUAUAUUAAAA
							TGAACACATAGGCTAG	UGAACACAUAGGCUAG
							CAAATTAGTGTGACAG	CAAAUUAGUGUGACAG
							GGATACAGCAACTATT	GGAUACAGCAACUAUU
							TTATCAA	UUAUCAA
							[SEQ ID NO: 991]	[SEQ ID NO: 1519]
TCTTTTGCTAGC	UCUUUUGCUAGC	GAACACATCATA	GAACACAUCAUA	GAACACAUCAGC	-1.29476	4412	ACCGGACATACTTGTT	ACCGGACAUACUUGUU
TGATGTGTTC	UGAUGUGUUC	TAGCAAAAGT	UAGCAAAAGU	UAGCAAAA			CCACTCTCTTTTGCTA	CCACUCUCUUUUGCUA
[SEQ ID	[SEQ ID	[SEQ ID	[SEQ ID	[SEQ ID			GCTGATGTGTTCTAGT	GCUGAUGUGUUCUAGU
NO: 760]	NO: 1288]	NO: 876]	NO: 1404]	NO: 1108]			GAAATATATATTAAAG	GAAAUAUAUAUUAAAG
							AACACATCATATAGCA	AACACAUCAUAUAGCA
							AAAGTTAGTGTGACAG	AAAGUUAGUGUGACAG
							GGATACAGCAACTATT	GGAUACAGCAACUAUU
							TTATCAA	UUAUCAA
							[SEQ ID NO: 992]	[SEQ ID NO: 1520]
TGTCAAGTTTAG		CGCCCTTTTAAC		CGCCCUUUUACU	-4.72	4502	ACCGGACATACTTGTT
TAAAAGGGCG		AAACTTGACT		AAACUUGA			CCACTCTGTCAAGTTT
[SEQ ID		[SEQ ID		[SEQ ID			AGTAAAAGGGCGTAGT
NO: 2269]		NO: 2270]		NO: 2271]			GAAATATATATTAAAC
							GCCCTTTTAACAAACT
							TGACTTAGTGTGACAG
							GGATACAGCAACTATT
							TTATCAA
							[SEQ ID NO: 2272]

Example 3: Testing of Top Hits from Pooled Screen in Lentiviral Transduction of Human Neurons

Several top hits from pooled Deep Screen 1 (Example 2) were cloned into lentiviral vectors, packaged, and tested in stem-cell derived motor neuron cultures for knockdown of ATXN2 mRNA and protein. An example lentiviral vector is given in H1-miR-16-2_1755-AMELY_V1_CMV_GFP_lenti (SEQ ID NO:1521) which contains a amiRNA targeting position 1755 of ATXN2 transcript embedded in a miR-16-2 backbone, or the other vectors described here. The amiRNA sequence in the vector (e.g., nucleotides 1889-2020 of SEQ ID NO:1521) may replaced with the corresponding amiR or control non-miRNA sequence (MCS) but the rest of the vector is left unchanged.) Characterization of motor neurons (FIG. 29) shows that cultures (differentiation protocol described in below methods) generated cultures enriched for motor neurons, with elaborated neuronal processes. amiRNAs were embedded in lentiviral vectors (FIG. 30A) with an H1 promoter as well as a GFP expression cassette. In a first experiment, two amiRNAs, targeting ATXN2 at position 1784 (guide sequence SEQ ID NO:112) in the coding sequence or ATXN2 at position 4402 (guide sequence SEQ ID NO:1279) having miR-16-2 backbones were tested at two different doses. Strong knockdown of ATXN2 mRNA and protein was detected by qPCR analysis of mRNA and Western analysis of protein, respectively (FIGS. 30B-30C). Protein levels as measured in this assay showed a greater fractional reduction of protein levels than mRNA levels, indicating that measurements of mRNA may represent at least the amount of ATXN2 protein reduced by a given amiRNA. Surprisingly, the amiRNA targeting the ATXN2 coding sequence (1784) yielded greater knockdown than the amiRNA targeting the 3′ UTR (4402), which is different than the relative performance of those amiRNAs in Deep Screen 1.

As a further investigation of amiRNA targeting the coding region versus the 3′ UTR, a second experiment was done (FIG. 31). In this case, all neurons were treated at a dose intermediate between the two levels tested in the first human neuron lentiviral dosing experiment. As before, amiRNAs targeting the coding sequence (1755 (guide sequence SEQ ID NO:1185), 1784 (guide sequence SEQ ID NO:112), 3302 (guide sequence SEQ ID NO:1216), 3330 (guide sequence SEQ ID NO:1811), and 3805 (guide sequence SEQ ID NO:1221) yielded stronger knockdown than amiRNAs targeting the 3′ UTR (4402 (guide sequence SEQ ID NO:1279), 4242 (guide sequence SEQ ID NO:1233), and 4502) in these neuronal cultures. The amount of mRNA reduction exceeded 75% for some amiRNAs, such as 1755 (guide sequence SEQ ID NO:1185), 1784 (guide sequence SEQ ID NO:112) and 3330 (guide sequence SEQ ID NO:1811).

Methods

Motor Neuron Production

Induced pluripotent stem cells (GM25256, Coriell Institute) were cultured in feeder-free conditions, in mTeSR1 media on Matrigel coated plates, according to standard procedures. To begin differentiation, iPSC colonies grown in 6-well dishes were dissociated with 500 uL ReLeSR, incubating 3 minutes at 37 C, and gently agitated. 1 mL of complete mTeSR1 media is added to stop dissociation. Cell suspension was collected, ReLeSR removed and cells resuspended in N2B27 differentiation media: 50 mL of 50% mTeSR1 and 50% NB27 differentiation media (50% DMEM-F12, 50% Neurobasal medium, 1×N-2 supplement, 1×B-27 supplement, XenoFree, 0.5× penicillin-streptomycin, 1×2-mercaptoethanol, 20 uM L-ascorbic acid). Rock Inhibitor Y-27632 (5 micromolar), LDN (200 nM), SB 431542 (40 micromolar), and Chir 99021 (3 micromolar) were added. Cell suspension was then transferred to a 75 cm² ultra low attachment U-flask for 24 hours. Cells then aggregated into small spheroids.

Media changes were then performed on days 2, 4, 6, 9, and 12. Media included (all based in N2B27 differentiation media): Day 2: Retinoic acid (1 micromolar), SAG (1 micromolar), LDN-193189 (0.2 micromolar), SB 431542 hydrate (40 micromolar), CHIR 99021 (3 micromolar). Day 4: Retinoic acid (1 micromolar), SAG (1 micromolar), LDN-193189 (0.2 micromolar). Day 6: Retinoic acid (1 micromolar); SAG (1 micromolar). Day 9: Retinoic acid (1 micromolar), SAG (1 micromolar), DAPT (10 micromolar). Day 12: DAPT (10 micromolar). By day 14, neuronal spheroids were present and were dissociated to plate motor neurons.

Neuronal spheroids were then dissociated with a papain:DNAse solution and triturated 4-5×. Cell suspensions were then divided into wells of 6-well plates; and after a 15 minute incubation, further triturated. Following this dissociation, enzyme was inactivated with a DMEM and knockout serum replacement (KOSR) mix, centrifuged, washed again in 90% DMEM/10% KOSR, centrifuged, and resuspended in complete neurobasal media: Neurobasal medium, 1×N-2 supplement, 1×B-27 supplement, XenoFree, 0.5× penicillin-streptomycin, 20 uM L-ascorbic acid, 1% KOSR, Rock Inhibitor Y-27632 (5 micromolar), GDNF (10 ng/mL), BDNF (20 ng/mL), CNTF (10 ng/mL), DAPT (5 micromolar). Cells were then centrifuged again, resuspended in complete neurobasal media, passed through a 40 micron cell strainer, counted via trypan blue staining and a hemocytometer, then diluted to 20K/well (96-well format) or 200K/well (24-well format) for plating in PDL/Laminin coated plates. Cells were cultured in a volume of neurobasal media: 200 uL/well (96-well format) or 1 mL/well (24-well format).

The PDL/Laminin coating was done by treating plates with a 100 microgram/mL solution of poly-D-lysine in PBS overnight at 4 C; washing 3 times with PBS; then treating plates overnight at 4 C with a 50 microgram/mL solution of laminin in PBS.

48 hours after plating, 50% of media was replaced with neuron maintenance media (Neurobasal, with 1×N-2 supplement, 1×Xeno-Free B-27 supplement, 0.5× penicillin-streptomycin, 20 micromolar L-ascorbic acid, with 10 ng/mL GDNF, 10 ng/mL BDNF, 10 ng/mL CNTF), including DAPT (5 micromolar). Thereafter, 50% of media was replaced 3 times per week, not including DAPT.

References relevant to the above protocol include: (Highly efficient neural conversion of human ES and iPS cells by dual inhibition of SMAD signaling (Chambers et al., Nat Biotechnol (2009) 27:275-280) and (Maury et al., Nat Biotechnol (2014) 33:89-96).

Reagents and Equipment for iPSC Embryoid Body Formation

Reagent	Vendor	Cat. No.
DMEM/F-12	ThermoFisher	11320-033
Neurobasal Medium		21103049
N-2 Supplement (100X)		17502-048
B-27 Supplement (50X), XenoFree		A1486701
Phosphate-Buffered Saline (PBS) pH 7.4		10010023
Penicillin-Streptomycin 100x		15140122
2-Mercaptoethanol 1000x		21985023
L-Ascorbic Acid	Millipore Sigma	A4403-
		100MG
Rock Inhibitor (Y-27632), 5MG		Y0503-5MG
SB 431542 hydrate		S4317-5MG
Retinoic Acid		R2625-50MG
DAPT		D5942-5MG
SAG, SHH agonist		566660-5MG
MTeSR1 cGMP, feeder-free maintenance	StemCell	85850
medium kit	Technologies,
ReLeSR	Inc	05872
LDN-193189 in solution	Fisher Scientific	NC0689818
CHIR 99021 10MG	Tocris	4423

Equipment/Supply	Vendor	Cat. No.
Ultra-Low Attachment 75 cm2 U-Flask	Corning	3814
6-well plate	Corning	353046
Nalgene Rapid-Flow 500 mL Filter Units	VWR (Nalgene)	73520-984

Reagents and Embryoid Body Dissociation and Motor Neuron Culture:

Reagent	Vendor	Cat. No.
Neurobasal Medium	ThermoFisher	21103049
DMEM/F-12		11320-033
N-2 Supplement (100X)		17502-048
B-27 Supplement (50X), XenoFree		A1486701
2-Mercaptoethanol 1000x		21985023
Phosphate-Buffered Saline (PBS) pH 7.4		10010-049
Penicillin-Streptomycin 100x		15140122
KnockOut Serum Replacement		10828010
Laminin Mouse Protein, 1 mg in		23017015
solution
Rock Inhibitor (Y-27632), 5 mg	Millipore Sigma	Y0503-5MG
DAPT		D5942-5MG
L-Ascorbic Acid		A4403-
		100MG
Poly-D-Lysine solution, 1 mg/mL		A-003-E
Recombinant Human GDNF, 50 μg	R&D Systems	212-GD-050
Recombinant Human BDNF, 50 μg		248-BDB-050
Recombinant Human CNTF, 50 μg		257-NT-050
Papain, ≥100 units per vial	Worthington	LK003178
DNase, ≥1,000 Kunitz units per vial		LK003172

Equipment for Embryoid Body Dissociation and Motor Neuron Culture

Equipment/Supply	Vendor	Cat. No.
6-well Clear TC-treated Plates	Corning	353046
12-well Clear TC-treated Plates	Corning	353043
24-well Clear TC-treated Plates	Corning	353047
96-well optic clear bottom, black wall	PerkinElmer	6005550
microplate
Nalgene Rapid-Flow 500 mL Filter Units	VWR (Nalgene)	73520-984

Lentiviral Production

To test the efficacy of miR16-2 embedded guides in stem-cell derived motor neurons, amiRNAs were expressed from an H1 promoter embedded within a lentiviral construct as described above. Lentivirus was generated with Lenti-X 293T (Takara, 632180) cells transfected with psPAX2 (Cellecta, P/N CPCP-PAX2) and pMD2.2 (Cellecta, CPCP-PM2G) using Lipofectamine LTX and PLUS Reagent (Thermo, P/N 15338-100). The following day after transfection media was changed to include ViralBoost Reagent (Alstem, P/N VB100) and then 2 days later the viral production media was filtered and concentrated using Lenti-X Concentrator (Takara, P/N 631232) and resuspended in N2B27 media.

qPCR Analysis

Stem-cell derived motor neurons were transduced, and 7 days post-transduction, media was removed, washed with PBS and cells lysed with Buffer RLT supplemented with beta-Mercaptoethanol. RNA was purified using Qiagen RNeasy Plus Mini Kit (Qiagen, P/N 74134) and reverse-transcribed using SuperScript VILO cDNA Synthesis Kit (Thermo, P/N 11754250). Using TaqMan Fast Advanced Master Mix (Thermo, P/N 4444556) and QuantStudio 6 Flex Real-Time PCR System (Thermo), Ct values were calculated using primer/probe sets to ATXN2 (Thermo, Hs01002847_m1), GUSB (Thermo, Hs00939627_m1), and B2M (Thermo, Hs00187842_m1). The average Ct across 4 replicates was calculated, and using the delta-delta Ct method, the delta Ct was calculated for ATXN2 to each internal control, then the delta-delta Ct was calculated to the average of the untreated conditions. The mean of the normalized values to untreated conditions were calculated and graphed as shown.

Western Analysis of ATXN2 Levels from Neurons Treated with ATXN2 amiRNA Expressing Lentiviruses

Protein extraction was performed by placing plates on ice, aspirating media, and adding 50-100 microliters cold RIPA buffer (TEKNOVA #50-843-016) supplemented with protease and phosphatase inhibitor tablet (Pierce #A32959), Halt protease inhibitor cocktail (Thermo #1861279) and PMSF (Cell Signaling Technology #8553S). Individual cell lifters were used to scrape each well thoroughly, plates were tilted and lysates were harvested and incubated on ice for an additional 30 min. Samples were centrifuged for 15 min at 17,000×g at 4° C., and supernatant was transferred to a fresh tube and stored at −80° C. Protein lysates were quantitated (Pierce, 23225), resulting in approximately 40 μg total protein per sample.

The NuPage system (Thermo) was used for gel electrophoresis. Five μg of each sample was loaded onto 4-12% Bis-Tris protein gels (Thermo, NP0321BOX) and run at constant 200V for 1 hr. Revert 700 (Licor, 926-11010) was used to assay for protein loading. Proteins were transferred onto PVDF membrane (EMD Millipore, IPFL00005) overnight at 4° C. using constant 30V and 90 mA. Membranes were blocked for 1 hr at RT (Rockland, MB-070). Primary antibody incubation was performed overnight rocking at 4° C., including anti-Atxn2 (1:1000, BD, 611378), anti-GFP (1:2000, CST, 2956) and beta-actin (1:2000, CST, 4970). Washing was performed 4×5 min with TBS+0.1% tween-20, and secondary antibodies were incubated for 1 hr rocking at RT (1:15,000 each of 800CW goat anti-mouse and 680RD donkey anti-rabbit, Licor). Membranes were washed again and imaging was performed on an Odyssey Fc Imaging system (Licor). Signal quantitation was by Licor image-studio lite.

Example 4: Embedding of Top Hits from Pooled Screen in AAV Cis-Plasmids and AAV Production

To test the ability of top performing amiRNAs identified from the pooled screen to knock down ATXN2 when embedded in AAV, 10 top miRNAs were cloned downstream of a H1 promoter (nucleotides 113-203 of SEQ ID NO:1522) in a cis plasmid (transfer plasmid) for AAV production. An example of a plasmid sequence (5′ ITR to 3′ ITR) (scAAV_AMELY_V1_H1_micropool_ITR_to_ITR) comprises the nucleotide sequence of SEQ ID NO:1522; where the desired amiRNA embedded in a miRNA backbone is inserted in nucleotides 204-341 of SEQ ID NO:1522. After AAV9 production by triple transfection of HEK293T cells with the cis-plasmid and helper plasmids and harvest of encapsidated AAV, vector genome DNA was extracted with Quick-DNA Viral Kit (Zymo, P/N D3015) to assess vector integrity. Purified vector was quantified using Qubit dsDNA HS Assay Kit (Thermo, P/N Q32854) and vector genome size was assessed by agarose gel electrophoresis and stained SyberSafe for visualization. Vector genome size was assessed by agarose gel electrophoresis (FIG. 32). Surprisingly, two bands were observed. The upper band migrated at the expected size 2284 bp, whereas the lower band migrated farther than the calculated vector size, or smaller in length than the full-length vector. Extraction of the band representing the full-length vector and subsequent Sanger sequencing with a primer amplifying towards the embedded aimRNA resulted in successful sequencing of the expected amiRNA. Whereas extraction of the smaller DNA product and sequencing failed to sequence through the embedded aimRNA, suggesting that the lower band might represent a vector truncation centered around the artificial miRNA, as noted in (Xie et al., Molecular Therapy (2013) 28:422-430). Calculation of predicted DNA secondary structure for miRs in the miR16-2 backbone using mfold (Zuker Nucleic Acids Research (2003) 31:3406-15) showed this sequence to form strong secondary structure, with Gibbs free binding energy of −26.78.

Using ImageJ, the individual vector genome lanes of an image gathered with the SyberSafe stained DNA gel were selected, the intensity of the lane plotted, and peaks quantified. Using the calculated lengths of the full-length and miR-centered truncated vector genomes of 2284 and 2077 bp respectively, the relative staining-intensity-derived molarity of each was calculated. With these values, the percentage full-length vector was calculated as the percentage of full-length divided by the combined amount of full-length and miR-centered truncated vector genomes (Table 20)

TABLE 20
Percentage of full-length vector genome
H1 AMELY         percentage full-length
V1 guide         vector genome
miR16-2-XD-14792 68.3
miR16-2-1479     56.7
miR16-2-1755     70.3
miR16-2-3330     62.5
miR16-2-4402     64.1
miR16-2-4405     67.8
miR16-2-4406     71.0
miR16-2-4409     67.2
miR16-2-4502     64.9

Example 5: Second Pooled miRNA Screen

Given the truncation observed in AAV vectors expressing miR16-2 embedded amiRNAs, a second pooled amiRNA screen was devised to embed the guide sequences from the top ATXN2 miRNA hits from the first pooled screen into a diverse set of 20 miRNA backbones.

ATXN2 Targeting Sequence Selection for DS2

ATXN2 targeting sequences presumed to be efficacious and safe were selected from Deep Screen 1 to enter “Deep Screen 2.” Sequences that were enriched in the low ATXN2 signal FACS bin and demonstrated low dropout (minimal change in representation comparing an early to a late timepoint) were prioritized. To calibrate the dynamic range of the assay, some sequences with high dropout were additionally included. Since there may be biological variability in the processing precision of the mature guide strand, guides bracketing efficacious guides (by position along the ATXN2 transcript) were additionally entered into Deep Screen 2.

Essential Gene Control miRNA Selection

A subset of the essential gene targeting amiRNAs with either ‘high’ or ‘medium’ dropout, with respect to other essential-gene targeting amiRNAs, were selected for Deep Screen 2 based on performance in Deep Screen 1.

911 Controls

A subset of sequences targeting ATXN2 were paired with their cognate 911 controls. In a 911 control, bases 9, 10, and 11 of the guide strand are complemented, along with corresponding change in the passenger strand, such that the resulting mature miRNA does not slice the target mRNA of the original guide. Because many aspects of amiRNA ‘off-target’ activity are presumed to occur through binding interactions with the seed region (bases 2-8), these 911 controls should in principle display a similar off-target profile as the original miRNA and should help distinguish on- and off-target activity.

ATXN2 Scramble Controls

A subset of the miRNA scramble controls from Deep Screen 1 was carried over into Deep Screen 2. These were considered for mean centering the data.

ATXN2 Backbone Selection, Processing Enhancement Motifs, and Passenger Variations

MicroRNA backbones were selected for naturally exhibiting high processing precision, high guide to passenger ratio, and efficient target knockdown as an artificial miRNA. Both miRNA performance in functional screens and 5′ guide processing homogeneity were considered^1-4.

Primary miRNA transcript sequence was identified in miRbase. The extended sequence contexts around the miRNAs were ascertained in EntrezGene. Surrounding 5′ and 3′ sequence with high mammalian conservation were used to define final 138 nt miRNA-embedded fragments that would be inserted into the pooled library.

Mfold and RNAfold were used to examine folding patterns and to consider Gibbs free energy, as there is evidence that high Gibbs free energy derived from extensive secondary structure in the miRNA may produce miR-centered truncations when later cloned and produced into AAV.

The basal stem, loop, and guide and passenger sequences were defined by stem loop folding predictions on miRbase and Mfold. The rules for passenger variations such as bulges and other asymmetries were chosen to mimic non-complementary base pairing in the endogenous hairpin stem and incorporated into the library construction algorithms.

Sequence motifs that enable efficient processing of pri-miRNA backbones have previously been identified. These include an UG motif at the 5′ end of the pre-miRNA, a mismatched GHG motif in the stem, and a 3′ CNNC motif Many of the primary miRNA transcripts selected naturally contain these motifs. Some of these motifs were artificially incorporated into five backbones, and these resulting miRNA backbones are denoted by “_M” (e.g., “miR-1-1_M”). Table 21 provides miRNA backbone sequences (in DNA format) used in Deep Screen 2. The RNA sequences of the miRNA backbone are provided by converting the “T” nucleotides in the sequences of Table 21 to “U” nucleotides.

TABLE 21
miRNA backbone sequences used in Deep Screen 2
miR_with	5′ miR context						3′ miR context
suffix	(flanking)	5′ basal stem	5p	loop	3p	3′ basal stem	(flanking)
miR-1-1	catgcagactgcctgct	TGGG	passenger	TATGGACCTGCTAA	guide	CTCA	ggccgggacctctctegccg
	[SEQ ID NO: 1523]			GCTA			cactgaggggcactccaca
				[SEQ ID NO: 1524]			ccacgggggccg
							[SEQ ID NO: 1525]
miR-1-1_M	catgcagactgcctgct	TGGG	passenger	TATGGACCTGCTAA	guide	CTCA	ggccgggacctcttccgccg
	[SEQ ID NO: 1526]			GCTA			cactgaggggcactccaca
				[SEQ ID NO: 1527]			ccacgggggccg
							[SEQ ID NO: 1528]
miR-100	CCCAAAAGAGA	CCTGTTGCCAC	guide	GTATTAGTCCG	passenger	TGTGTCTGTTA	CAATCTCACGGA
	GAAGATATTGA	A		[SEQ ID NO: 1531]		GG	CCTGGGGCTTTGC
	GG	[SEQ ID				[SEQ ID NO: 1532]	TTATATGCC
	[SEQ ID NO: 1529]	NO: 1530]					[SEQ ID NO: 1533]
miR-100_M	CCCAAAAGAGA	CCTGTTGCCAC	guide	GTATTAGTCCG	passenger	TGTGTCTGTTA	CtATtcCACGGACC
	GAAGATATTGAt	A		[SEQ ID NO: 1536]		GG	TGGGGCTTTGCTT
	G	[SEQ ID				[SEQ ID NO: 1537]	ATATGCC
	[SEQ ID NO: 1534]	NO: 1535]					[SEQ ID NO: 1538]
miR-122	ggctacagagttt	CCTTAGCAGAG	guide	TGTCTAAACTAT	passenger	TAGCTACTGCT	aatccttccctcgataaatgtc
	[SEQ ID NO: 1539]	CTG		[SEQ ID NO: 1541]		AGGC	ttggcatcgtttgctttg
		[SEQ ID				[SEQ ID NO: 1542]	[SEQ ID NO: 1543]
		NO: 1540]
miR-122 M	ggctacagagttt	GCTTAGCAGAG	guide	TGTCTAAACTAT	passenger	TAGCTACTGCT	catccttccctcgataaatgtc
	[SEQ ID NO: 1544]	CTG		[SEQ ID NO: 1546]		AGGC	ttggcatcgtttgctttg
		[SEQ ID				[SEQ ID NO: 1547]	[SEQ ID NO: 1548]
		NO: 1545]
miR-124	TTCCTTCCTCAG	AGGCCTCTCTC	passenger	ATTTAAATGTCCAT	guide	GAATGGGGCTG	GCTGAGCACCGT
	GAGAA	[SEQ ID		ACAAT		[SEQ ID NO: 1552]	GGGTCGGCGAGG
	[SEQ ID NO: 1549]	NO: 1550]		[SEQ ID NO: 1551]			GCCCGCCAagga
							[SEQ ID NO: 1553]
miR-124 M	TTCCTTCCTCAG	tGGCCTCTCTC	passenger	ATTTAAATGTCCAT	guide	GAATGGGGCTt	aCTGccgcaCGTGG
	GAGAA	[SEQ ID		ACAAT		[SEQ ID NO: 1557]	GTCGGCGAGGGC
	[SEQ ID NO: 1554]	NO: 1555]		[SEQ ID NO: 1556]			CCGCCAagga
							[SEQ ID NO: 1558]
miR-128	ATTTtgcaataattggc	TGAGCTGTTGG	passenger	GAGGTTTACATTTC	guide	TTCAGCTGCTTC	ctggcttctttttactcaggttt
	cttgttcc	A		[SEQ ID NO: 1561]		[SEQ ID NO: 1562]	ccactgct
	[SEQ ID NO: 1559]	[SEQ ID					[SEQ ID NO: 1563]
		NO: 1560]
miR-130a	gcagggccggcatgcct	TGCTGCTGGCC	passenger	CTGTCTGCACCTGTC	guide	TGGCCGTGTAG	ctacccagcgctggctgcct
	c	A		ACTAG		TG	cctcagcattg
	[SEQ ID NO: 1564]	[SEQ ID		[SEQ ID NO: 1566]		[SEQ ID NO: 1567]	[SEQ ID NO: 1568]
		NO: 1565]
miR-155E	CTGGAGGCTTG	GGGCTGTATGC	guide	TTTTGGCCTCTGACT	passenger	CAGGACAAGGC	TTTATCAGCACTC
	CTTT	TG		GA		CC	ACATGGAACAAA
	[SEQ ID NO: 1569]	[SEQ ID		[SEQ ID NO: 1571]		[SEQ ID NO: 1572]	TGGCCACCGTG
		NO: 1570]					[SEQ ID NO: 1573]
miR-155M	CCTGGAGGCTT	AGGCTGTATGC	guide	TTTTGGCCACTGACT	passenger	CAGGACACAAG	TGTTACTAGCACT
	GCTGA	TG		GA		GCC	CACATGGAACAA
	[SEQ ID NO: 1574]	[SEQ ID		[SEQ ID NO: 1576]		[SEQ ID NO: 1577]	ATGGCCACC
		NO: 15751					[SEQ ID NO: 1578]
miR-138-2	gccggcggagttctggta	CGTTGCTGC	guide	GACGAGCAGCGCAT	passenger	GTTGCATCA	tacccatcctctccaggcga
	t			CCTCTTACCC			gcctcgtgggaccGG
	[SEQ ID NO: 1579]			[SEQ ID NO: 1580]			[SEQ ID NO: 1581]
miR-144	TCAAGCCATGC	TGGGGCCCTGG	passenger	AGTTTGCGATGAGA	guide	AGTCCGGGCAC	AGCTCTGGAGCC
	TTCCTGTGCCCC	CT		CAC		CCCC	TGACAAGGAggaca
	CAG	[SEQ ID		[SEQ ID NO: 1584]		[SEQ ID NO: 1585]	[SEQ ID NO: 1586]
	[SEQ ID NO: 1582]	NO: 1583]
miR-190a	GAGCTCAGTCA	TGCAGGCCTCT	guide	TGTTATTTAATCCA	passenger	CTACAGTGTCT	CTGTCTCCGGGG
	AACCTGGATGC	GTG		[SEQ ID NO: 1589]		TGCC	GTTCCTAATAAA
	CTTTTC	[SEQ ID				[SEQ ID NO: 1590]	G
	[SEQ ID NO: 1587]	NO: 1588]					[SEQ ID NO: 1591]
miR-190a_	GAGCTCAGTCA	TGCAGGCgTCT	guide	TGTTATTTAATCCA	passenger	CTACAGTCTCTT	CTGTCTCCGGGG
M	AACCTGGATGC	GTG		[SEQ ID NO: 1594]		GCC	GTTCCTAATAAA
	CTTTTC	[SEQ ID				[SEQ ID NO: 1595]	G
	[SEQ ID NO: 1592]	NO: 15931					SEQ ID NO: 1596]
miR-132	GCCGTCCGCGC	CCGCCCCCGCG	Passenger	CTGTGGGAACTGGA	guide	CCCCGCAGCAC	CGCGCCACGCCG
	GCC	TCTCCAGGG		GG		GCCCACGCGC	CGCCCCGAGCC
	[SEQ ID NO: 1597]	[SEQ ID		[SEQ ID NO: 1599]		[SEQ ID NO: 1600]	[SEQ ID NO: 1601]
		NO: 1598]
miR-451a	GCTCTCTGCTC	CTTGGGAATGG	guide	None	passenger	TCTTGCTATACC	AAACGTGCCAGG
	AGCCTGTCACA	CAAGG				CAGA	AAGAGAACTCAG
	ACCTACTGACT	[SEQ ID				[SEQ ID NO: 1604]	[SEQ ID NO: 1605]
	GCCAGGGCA	NO: 1603]
	[SEQ ID NO: 1602]
miR-223	TCCCCACAGAA	CCTGGCCTCCT	passenger	CTCCATGTGGTAGA	guide	AGTGCGGCACA	CTCTAGGCC
	GCTCTTGG	GCAGTGCCACG		G		TGCTTACCAG
	[SEQ ID NO: 1606]	CT		[SEQ ID NO: 1608]		[SEQ ID NO: 1609]
		[SEQ ID
		NO: 1607]
miR-16-2	TTATGTTTGGAT	GTTCCACTC	guide	TAGTGAAATATATA	passenger	TAGTGTGAC	AGGGATACAGCA
	GAACTGACATA			TTAAA			ACTATTTTATCAA
	CTT			[SEQ ID NO: 1611]			TTGTTT
	[SEQ ID NO: 1610]						[SEQ ID NO: 1612]

Oligonucleotides were designed that embedded the guide sequences described in Table 19 into miRNA backbones, using flanking sequences as defined in Table 21, and with passenger sequences defined by the rules in Table 8. For example, an artificial miRNA with miR-100 backbone in DNA format for insertion into a transfer plasmid may be designed comprising from 5′ to 3′:5′ miR context (flanking) sequence of SEQ ID NO:1529; 5′ basal stem sequence of SEQ ID NO:1530; desired guide sequence; loop sequence of SEQ ID NO:1531; desired passenger sequence designed according to the rules in Table 8; 3′ basal stem sequence of SEQ ID NO:1532; and 3′ miR context (flanking) sequence of SEQ ID NO:1533. The artificial miRNA in RNA format may be obtained by converting the “T” nucleotides in these sequences to “U” nucleotides. The pooled library oligonucleotides were cloned into a lentiviral plasmid pLVX-EF1A-miR-CMV-Puro (5′ LTR to 3′ LTR sequence comprises the nucleotide sequence of SEQ ID:1613) with an EF1alpha promoter to express the amiR, and a CMV promoter to express a PuroR selection marker. The artificial miRNA oligonucleotide may be inserted at nucleotides 3126-3263 of SEQ ID NO:1613. After packaging the library in the plasmid, library composition was assessed by sequencing, and it was noted that the abundance of miRs embedded in the miR-16-2 backbone was in general substantially less than other backbones. One potential explanation would be that during library amplification—when all library elements undergo PCR amplification—elements including the miR-16-2 backbone are amplified less efficiently than other backbones. This could perhaps be because of the strong DNA hairpin that forms with the miR-16-2 backbone. Due to the low number of miR-16-2 backbone elements remaining in the library, counts of miR-16-2 containing guides were low and therefore noisy, and not included in further analyses.

After cloning, packaging, and execution of screen (see methods), sequencing data were analyzed essentially as for Deep Screen 1. Abundance of library elements were calculated by number of sequencing reads exactly matching input library elements. In this screen no baseline subtraction was done for either ATXN2 levels or for dropout. FIG. 33A shows a scatterplot plotting the correspondence in the ATXN2 knockdown metric for two screen replicates against each other. In this case what is plotted is the ratio of abundance of sequence reads for guide elements in the 10% low-ATXN2 signal sort bin versus an unsorted sort bin. There is good correspondence for elements that have low ratios for unsorted/10% low-ATXN2 signal—that is, elements that induce ATXN2 depletion—but there is less correspondence for elements with similar abundance in the unsorted and 10% unsorted bin.

FIG. 33B shows boxplots of knockdown performance of miRs embedded in the shown backbones; Table 22 shows the median and 95^th percentiles of performance. By this metric, some of the top performing miRs, as measured by median performing miRNA, were miR-1-1_M, miR1-1, miR-130a, miR-100, and miR-100_M. It is noted, however, that there were top miRNAs in each of these backbones that, as measured by this assay (ratio of counts of guides in a low-Atxn2 sorted pool versus guides in unsorted cells), performed similarly across miR backbones. Therefore, this assay made available multiple miR backbones with strong performance. This was likely due to good processing of the artificial pri-miRNA by the microprocessor and dicer complexes.

TABLE 22
Performance of miRNAs Across miRNA Backbones
	miR_with_suffix	median	ninety_fifth
1	miR-1-1_M	−0.6772802	−2.2788937
2	miR-1-1	−0.5968477	−2.1875722
3	miR-130a	−0.5111669	−2.157525
4	miR-100	−0.2912209	−2.0887075
5	miR-100_M	−0.2094112	−1.9758731
6	miR-155E	−0.1742495	−1.8818528
7	miR-132	−0.1144013	−2.0204587
8	miR-190a	−0.030798	−2.0813007
9	miR-190a_M	0.04198442	−2.1250904
10	miR-122	0.05806267	−1.9039473
11	miR-122_M	0.17311752	−1.851009
12	miR-155M	0.50609504	−1.11192
13	miR-124_M	0.55091372	−0.9016042
14	miR-124	0.57242321	−0.95078
15	miR-144	0.71869783	−1.3372836
16	miR-138-2	0.77876566	−0.8666664
17	miR-223	1.04743176	0.46242634

The depletion of elements targeting essential genes was also used as an orthogonal evaluation of miR backbone performance. FIG. 34 shows boxplots of the depletion of elements from the 18-day timepoint versus the 1-day post transduction timepoint. There is a similar ranking of ‘performance’ of the various miR backbones by this metric compared to the ATXN2 knockdown metric. This may be because of the ranking of miR backbones in processing to yield mature amiRNAs.

Table 23 lists the top 100 amiRNAs, ranked by mean enrichment in the ATXN2 low signal sorted cells. The miR backbone, guide sequence, targeting position within the complementary ATXN2 transcript sequence, passenger sequence, and the amiRNA sequence (including the miR backbone, loop, ATXN2 targeting guide and passenger), are provided in both RNA and DNA format. The ‘passenger’ sequence refers to sequence complementary to the guide sequence, but including bulges and mismatches designed according to the rules set forth in Table 8 to mimic endogenous miRNA structure. Note that after processing of the pri-miRNA, the passenger strand will likely initiate 1-3 nt downstream of the nucleotide shown in the table, and include 1-3 nt beyond the last nucleotide listed, derived from the miR cassette. Table 24 lists the top 10 amiRNAs for each miR backbone, excluding low performing backbones. Top amiRNAs were ranked by mean enrichment of sequence counts of the given amiR constructs in the ATXN2 low signal sorted cells. The miR backbone, guide sequence, targeting position within the complementary ATXN2 sequence, passenger sequence, and the amiRNA sequence are provided in RNA and DNA format.

TABLE 23
Top 100 amiRNAs
Atxn2		Atxn2		Guide	Passenger		Guide	Passenger
Targeting	miR	low/unsort	T1/T0	Sequence	Sequence	miR Cassette	Sequence	Sequence	miR Cassette
Position	Backbone	log2 FC	log2 FC	(DNA)	(DNA)	(DNA)	(RNA)	(RNA)	(RNA)
2945	miR-1-1_M	-2.89804	0.152222	TGTAGTAG	TGAGCCAA	CATGCAGACTGC	UGUAGUAG	UGAGCCAA	CAUGCAGACUG
				AAGGCTTTG	AGCCTTCTA	CTGCTTGGGTGA	AAGGCUUU	AGCCUUCU	CCUGCUUGGGU
				GCTGA	CCGACA	GCCAAAGCCTTC	GGCUGA	ACCGACA	GAGCCAAAGCC
				[SEQ ID	[SEQ ID	TACCGACATATG	[SEQ ID	[SEQ ID	UUCUACCGACA
				NO: 685]	NO: 1633]	GACCTGCTAAGC	NO: 1213]	NO: 1828]	UAUGGACCUGC
						TATGTAGTAGAA	(Same guide as		UAAGCUAUGUA
						GGCTTTGGCTGA	XD-14860)		GUAGAAGGCUU
						CTCAGGCCGGG			UGGCUGACUCA
						ACCTCTTCCGCC			GGCCGGGACCU
						GCACTGAGGGG			CUUCCGCCGCA
						CACTCCACACCA			CUGAGGGGCAC
						CGGGGGCC			UCCACACCACG
						[SEQ ID NO: 1711]			GGGGCC
									[SEQ ID NO: 1908]
3330	miR-190a	-2.86183	0.46905	TATGCTGAG	CCATTATCA	GAGCTCAGTCA	UAUGCUGA	CCAUUAUC	GAGCUCAGUCA
				ACTGATAAT	GTCTCAGCA	AACCTGGATGCC	GACUGAUA	AGUCUCAG	AACCUGGAUGC
				GTGG	CC	TTTTCTGCAGGC	AUGUGG	CACC	CUUUUCUGCAG
				[SEQ ID	[SEQ ID	CTCTGTGTATGC	[SEQ ID	SEQ ID	GCCUCUGUGUA
				NO: 1614]	NO: 1634]	TGAGACTGATA	NO: 1811]	NO: 1829]	UGCUGAGACUG
						ATGTGGTGTTAT			AUAAUGUGGUG
						TTAATCCACCAT			UUAUUUAAUCC
						TATCAGTCTCAG			ACCAUUAUCAG
						CACCCTACAGTG			UCUCAGCACCC
						TCTTGCCCTGTC			UACAGUGUCUU
						TCCGGGGGTTCC			GCCCUGUCUCC
						TAATAAAG			GGGGGUUCCUA
						[SEQ ID NO: 1712]			AUAAAG
									[SEQ ID NO: 1909]
3043	miR-144	-2.80802	0.325474	TTTGGTGCA	CCGGTTTGT	TCAAGCCATGCT	UUUGGUGC	CCGGUUUG	UCAAGCCAUGC
				AAACAAAC	TTATGCACC	TCCTGTGCCCCC	AAAACAAA	UUUAUGCA	UUCCUGUGCCC
				AGGCT	AAA	AGTGGGGCCCT	CAGGCU	CCAAA	CCAGUGGGGCC
				[SEQ ID	[SEQ ID	GGCTCCGGTTTG	[SEQ ID	[SEQ ID	CUGGCUCCGGU
				NO: 1615]	NO: 1635]	TTTATGCACCAA	NO: 1812]	NO: 1830]	UUGUUUAUGCA
						AAGTTTGCGATG			CCAAAAGUUUG
						AGACACTTTGGT			CGAUGAGACAC
						GCAAAACAAAC			UUUGGUGCAAA
						AGGAGTCCGGG			ACAAACAGGAG
						CACCCCCAGCTC			UCCGGGCACCC
						TGGAGCCTGAC			CCAGCUCUGGA
						AAGGAGGACA			GCCUGACAAGG
						[SEQ ID NO: 1713]			AGGACA
									[SEQ ID NO: 1910]
2602	miR-144	-2.76068	-0.25422	TTTAGTAGT	TCGATGGAT	TCAAGCCATGCT	UUUAGUAG	UCGAUGGA	UCAAGCCAUGC
				TGATCCATA	CATACTACT	TCCTGTGCCCCC	UUGAUCCA	UCAUACUA	UUCCUGUGCCC
				GATT	AAA	AGTGGGGCCCT	UAGAUU	CUAAA	CCAGUGGGGCC
				[SEQ ID	[SEQ ID	GGCTTCGATGGA	[SEQ ID	[SEQ ID	CUGGCUUCGAU
				NO: 1616]	NO: 1636]	TCATACTACTAA	NO: 202]	NO: 1831]	GGAUCAUACUA
						AAGTTTGCGATG	(Same guide as		CUAAAAGUUUG
						AGACACTTTAGT	XD-14837)		CGAUGAGACAC
						AGTTGATCCATA			UUUAGUAGUUG
						GAAGTCCGGGC			AUCCAUAGAAG
						ACCCCCAGCTCT			UCCGGGCACCC
						GGAGCCTGACA			CCAGCUCUGGA
						AGGAGGACA			GCCUGACAAGG
						[SEQ ID NO: 1714]			AGGACA
									[SEQ ID NO: 1911]
3341	miR-1-1_M	-2.70967	0.193529	ATAGACAT	GACTCAGC	CATGCAGACTGC	AUAGACAU	GACUCAGC	CAUGCAGACUG
				GAGGATGC	ATCCTCATG	CTGCTTGGGGAC	GAGGAUGC	AUCCUCAU	CCUGCUUGGGG
				TGAGAC	TGATAT	TCAGCATCCTCA	UGAGAC	GUGAUAU	ACUCAGCAUCC
				[SEQ ID	SEQ ID	TGTGATATTATG	[SEQ ID	[SEQ ID	UCAUGUGAUAU
				NO: 1617]	NO: 1637]	GACCTGCTAAGC	NO: 1813]	NO: 1832]	UAUGGACCUGC
						TAATAGACATG			UAAGCUAAUAG
						AGGATGCTGAG			ACAUGAGGAUG
						ACCTCAGGCCG			CUGAGACCUCA
						GGACCTCTTCCG			GGCCGGGACCU
						CCGCACTGAGG			CUUCCGCCGCA
						GGCACTCCACAC			CUGAGGGGCAC
						CACGGGGGCC			UCCACACCACG
						[SEQ ID NO: 1715]			GGGGCC
									[SEQ ID NO: 1912]
3302	miR-100	-2.68521	-0.3818	TTGAACAA	CCCAAACC	CCCAAAAGAGA	UUGAACAA	CCCAAACC	CCCAAAAGAGA
				GGGGCTGA	AGACCCTTG	GAAGATATTGA	GGGGCUGA	AGACCCUU	GAAGAUAUUGA
				TTTGGG	CTCAT	GGCCTGTTGCCA	UUUGGG	GCUCAU	GGCCUGUUGCC
				[SEQ ID	[SEQ ID	CATTGAACAAG	[SEQ ID	[SEQ ID	ACAUUGAACAA
				NO: 688]	NO: 1638]	GGGCTGATTTGG	NO: 1216]	NO: 1833]	GGGGCUGAUUU
						GGTATTAGTCCG			GGGGUAUUAGU
						CCCAAACCAGA			CCGCCCAAACC
						CCCTTGCTCATT			AGACCCUUGCU
						GTGTCTGTTAGG			CAUUGUGUCUG
						CAATCTCACGGA			UUAGGCAAUCU
						CCTGGGGCTTTG			CACGGACCUGG
						CTTATATGCC			GGCUUUGCUUA
						[SEQ ID NO: 1716]			UAUGCC
									[SEQ ID NO: 1913]
3043	miR-100	-2.67985	-0.0584	TTTGGTGCA	AGCCTGCTT	CCCAAAAGAGA	UUUGGUGC	AGCCUGCU	CCCAAAAGAGA
				AAACAAAC	GGTTTGCAA	GAAGATATTGA	AAAACAAA	UGGUUUGC	GAAGAUAUUGA
				AGGCT	CAAT	GGCCTGTTGCCA	CAGGCU	AACAAU	GGCCUGUUGCC
				[SEQ ID	[SEQ ID	CATTTGGTGCAA	[SEQ ID	[SEQ ID	ACAUUUGGUGC
				NO: 1615]	NO: 1639]	AACAAACAGGC	NO: 1812]	NO: 1834]	AAAACAAACAG
						TGTATTAGTCCG			GCUGUAUUAGU
						AGCCTGCTTGGT			CCGAGCCUGCU
						TTGCAACAATTG			UGGUUUGCAAC
						TGTCTGTTAGGC			AAUUGUGUCUG
						AATCTCACGGAC			UUAGGCAAUCU
						CTGGGGCTTTGC			CACGGACCUGG
						TTATATGCC			GGCUUUGCUUA
						[SEQ ID NO: 1717]			UAUGCC
									[SEQ ID NO: 1914]
1755	miR-100	-2.66915	-0.04161	TCGGGTTGA	CACACTCCA	CCCAAAAGAGA	UCGGGUUG	CACACUCC	CCCAAAAGAGA
				AATCTGAA	GCTTTCAAA	GAAGATATTGA	AAAUCUGA	AGCUUUCA	GAAGAUAUUGA
				GTGTG	CCGT	GGCCTGTTGCCA	AGUGUG	AACCGU	GGCCUGUUGCC
				[SEQ ID	[SEQ ID	CATCGGGTTGAA	[SEQ ID	[SEQ ID	ACAUCGGGUUG
				NO: 657]	NO: 1640]	ATCTGAAGTGTG	NO: 1185]	NO: 1835]	AAAUCUGAAGU
						GTATTAGTCCGC			GUGGUAUUAGU
						ACACTCCAGCTT			CCGCACACUCC
						TCAAACCGTTGT			AGCUUUCAAAC
						GTCTGTTAGGCA			CGUUGUGUCUG
						ATCTCACGGACC			UUAGGCAAUCU
						TGGGGCTTTGCT			CACGGACCUGG
						TATATGCC
						[SEQ ID NO: 1718]			GGCUUUGCUUA
									UAUGCC
									[SEQ ID NO: 1915]
3272	miR-100	-2.65898	-0.1606	AGGACTGT	GCAATACG	CCCAAAAGAGA	AGGACUGU	GCAAUACG	CCCAAAAGAGA
				AGGCAACA	TTTCCTACA	GAAGATATTGA	AGGCAACA	UUUCCUAC	GAAGAUAUUGA
				TATTGC	ATCCA	GGCCTGTTGCCA	UAUUGC	AAUCCA	GGCCUGUUGCC
				[SEQ ID	[SEQ ID	CAAGGACTGTA	[SEQ ID	[SEQ ID	ACAAGGACUGU
				NO: 1618]	NO: 1641]	GGCAACATATTG	NO: 1814]	NO: 1836]	AGGCAACAUAU
						CGTATTAGTCCG			UGCGUAUUAGU
						GCAATACGTTTC			CCGGCAAUACG
						CTACAATCCATG			UUUCCUACAAU
						TGTCTGTTAGGC			CCAUGUGUCUG
						AATCTCACGGAC			UUAGGCAAUCU
						CTGGGGCTTTGC			CACGGACCUGG
						TTATATGCC			GGCUUUGCUUA
						[SEQ ID NO: 1719]			UAUGCC
									[SEQ ID NO: 1916]
967	miR-	-2.64307	0.113083	ACTGATGTA	TGGCATATA	GAGCTCAGTCA	ACUGAUGU	UGGCAUAU	GAGCUCAGUCA
	190a_M			AGTATATG	CTTACATCA	AACCTGGATGCC	AAGUAUAU	ACUUACAU	AACCUGGAUGC
				AACCA	AG	TTTTCTGCAGGC	GAACCA	CAAG	CUUUUCUGCAG
				[SEQ ID	[SEQ ID	GTCTGTGACTGA	[SEQ ID	[SEQ ID	GCGUCUGUGAC
				NO: 1619]	NO: 1642]	TGTAAGTATATG	NO: 1815]	NO: 1837]	UGAUGUAAGUA
						AACCATGTTATT			UAUGAACCAUG
						TAATCCATGGCA			UUAUUUAAUCC
						TATACTTACATC			AUGGCAUAUAC
						AAGCTACAGTCT			UUACAUCAAGC
						CTTGCCCTGTCT			UACAGUCUCUU
						CCGGGGGTTCCT			GCCCUGUCUCC
						AATAAAG			GGGGGUUCCUA
						[SEQ ID NO: 1720]			AUAAAG
									[SEQ ID NO: 1917]
3302	miR-190a	-2.64186	0.124524	TTGAACAA	CCCATCAGC	GAGCTCAGTCA	UUGAACAA	CCCAUCAG	GAGCUCAGUCA
				GGGGCTGA	CCCTTGTTC	AACCTGGATGCC	GGGGCUGA	CCCCUUGU	AACCUGGAUGC
				TTTGGG	CC	TTTTCTGCAGGC	UUUGGG	UCCC	CUUUUCUGCAG
				[SEQ ID	[SEQ ID	CTCTGTGTTGAA	[SEQ ID	[SEQ ID	GCCUCUGUGUU
				NO: 688]	NO: 1643]	CAAGGGGCTGA	NO: 1216]	NO: 1838]	GAACAAGGGGC
						TTTGGGTGTTAT			UGAUUUGGGUG
						TTAATCCACCCA			UUAUUUAAUCC
						TCAGCCCCTTGT			ACCCAUCAGCC
						TCCCCTACAGTG			CCUUGUUCCCC
						TCTTGCCCTGTC			UACAGUGUCUU
						TCCGGGGGTTCC			GCCCUGUCUCC
						TAATAAAG			GGGGGUUCCUA
						[SEQ ID NO: 1721]			AUAAAG
									[SEQ ID NO: 1918]
3302	miR-130a	-2.62809	0.136636	TTGAACAA	CGCAAATC	GCAGGGCCGGC	UUGAACAA	CGCAAAUC	GCAGGGCCGGC
				GGGGCTGA	AGACCCTTG	ATGCCTCTGCTG	GGGGCUGA	AGACCCUU	AUGCCUCUGCU
				TTTGGG	TTCAC	CTGGCCACGCA	UUUGGG	GUUCAC	GCUGGCCACGC
				[SEQ ID	[SEQ ID	AATCAGACCCTT	[SEQ ID	[SEQ ID	AAAUCAGACCC
				NO: 688]	NO: 1644]	GTTCACCTGTCT	NO: 1216]	NO: 1839]	UUGUUCACCUG
						GCACCTGTCACT			UCUGCACCUGU
						AGTTGAACAAG			CACUAGUUGAA
						GGGCTGATTTGG			CAAGGGGCUGA
						GTGGCCGTGTAG			UUUGGGUGGCC
						TGCTACCCAGCG			GUGUAGUGCUA
						CTGGCTGCCTCC			CCCAGCGCUGG
						TCAGCATTG			CUGCCUCCUCA
						[SEQ ID NO: 1722]			GCAUUG
									[SEQ ID NO: 1919]
1755	miR-1-1_M	-2.62482	0.169485	TCGGGTTGA	CTCACTTCA	CATGCAGACTGC	UCGGGUUG	CUCACUUC	CAUGCAGACUG
				AATCTGAA	GATTTCAAC	CTGCTTGGGCTC	AAAUCUGA	AGAUUUCA	CCUGCUUGGGC
				GTGTG	GACGA	ACTTCAGATTTC	AGUGUG	ACGACGA	UCACUUCAGAU
				[SEQ ID	[SEQ ID	AACGACGATAT	[SEQ ID	[SEQ ID	UUCAACGACGA
				NO: 657]	NO: 1645]	GGACCTGCTAA	NO: 1185]	NO: 1840]	UAUGGACCUGC
						GCTATCGGGTTG			UAAGCUAUCGG
						AAATCTGAAGT			GUUGAAAUCUG
						GTGCTCAGGCCG			AAGUGUGCUCA
						GGACCTCTTCCG			GGCCGGGACCU
						CCGCACTGAGG			CUUCCGCCGCA
						GGCACTCCACAC			CUGAGGGGCAC
						CACGGGGGCC			UCCACACCACG
						[SEQ ID NO: 1723]			GGGGCC
									[SEQ ID NO: 1920]
3302	miR-100_M	-2.62238	-0.19177	TTGAACAA	CCCAAACC	CCCAAAAGAGA	UUGAACAA	CCCAAACC	CCCAAAAGAGA
				GGGGCTGA	AGACCCTTG	GAAGATATTGAT	GGGGCUGA	AGACCCUU	GAAGAUAUUGA
				TTTGGG	CTCAT	GCCTGTTGCCAC	UUUGGG	GCUCAU	UGCCUGUUGCC
				[SEQ ID	[SEQ ID	ATTGAACAAGG	[SEQ ID	[SEQ ID	ACAUUGAACAA
				NO: 688]	NO: 1638]	GGCTGATTTGGG	NO: 1216]	NO: 1833]	GGGGCUGAUUU
						GTATTAGTCCGC			GGGGUAUUAGU
						CCAAACCAGAC			CCGCCCAAACC
						CCTTGCTCATTG			AGACCCUUGCU
						TGTCTGTTAGGC			CAUUGUGUCUG
						TATTCCACGGAC			UUAGGCUAUUC
						CTGGGGCTTTGC			CACGGACCUGG
						TTATATGCC			GGCUUUGCUUA
						[SEQ ID NO: 1724]			UAUGCC
									[SEQ ID NO: 1921]
1755	miR-122_M	-2.60043	-0.03541	TCGGGTTGA	CACACTTCA	GGCTACAGAGTT	UCGGGUUG	CACACUUC	GGCUACAGAGU
				AATCTGAA	GACTTCAAC	TGCTTAGCAGAG	AAAUCUGA	AGACUUCA	UUGCUUAGCAG
				GTGTG	CATA	CTGTCGGGTTGA	AGUGUG	ACCAUA	AGCUGUCGGGU
				[SEQ ID	[SEQ ID	AATCTGAAGTGT	[SEQ ID	[SEQ ID	UGAAAUCUGAA
				NO: 657]	NO: 1646]	GTGTCTAAACTA	NO: 1185]	NO: 1841]	GUGUGUGUCUA
						TCACACTTCAGA			AACUAUCACAC
						CTTCAACCATAT			UUCAGACUUCA
						AGCTACTGCTAG			ACCAUAUAGCU
						GCCATCCTTCCC			ACUGCUAGGCC
						TCGATAAATGTC			AUCCUUCCCUC
						TTGGCATCGTTT			GAUAAAUGUCU
						GCTTTG			UGGCAUCGUUU
						[SEQ ID NO: 1725]			GCUUUG
									[SEQ ID NO: 1922]
1755	miR-1-1	-2.5734	-0.10352	TCGGGTTGA	CTCACTTCA	CATGCAGACTGC	UCGGGUUG	CUCACUUC	CAUGCAGACUG
				AATCTGAA	GATTTCAAC	CTGCTTGGGCTC	AAAUCUGA	AGAUUUCA	CCUGCUUGGGC
				GTGTG	GACGA	ACTTCAGATTTC	AGUGUG	ACGACGA	UCACUUCAGAU
				[SEQ ID	[SEQ ID	AACGACGATAT	[SEQ ID	[SEQ ID	UUCAACGACGA
				NO: 657]	NO: 1645]	GGACCTGCTAA	NO: 1185]	NO: 1840]	UAUGGACCUGC
						GCTATCGGGTTG			UAAGCUAUCGG
						AAATCTGAAGT			GUUGAAAUCUG
						GTGCTCAGGCCG			AAGUGUGCUCA
						GGACCTCTCTCG			GGCCGGGACCU
						CCGCACTGAGG			CUCUCGCCGCA
						GGCACTCCACAC			CUGAGGGGCAC
						CACGGGGGCC			UCCACACCACG
						[SEQ ID NO: 1726]			GGGGCC
									[SEQ ID NO: 1923]
3302	miR-1-1_M	-2.57066	0.05742	TTGAACAA	CGCAAATC	CATGCAGACTGC	UUGAACAA	CGCAAAUC	CAUGCAGACUG
				GGGGCTGA	AGCCCCTTG	CTGCTTGGGCGC	GGGGCUGA	AGCCCCUU	CCUGCUUGGGC
				TTTGGG	TCGCAA	AAATCAGCCCCT	UUUGGG	GUCGCAA	GCAAAUCAGCC
				[SEQ ID	[SEQ ID	TGTCGCAATATG	[SEQ ID	[SEQ ID	CCUUGUCGCAA
				NO: 688]	NO: 1647]	GACCTGCTAAGC	NO: 1216]	NO: 1842]	UAUGGACCUGC
						TATTGAACAAG			UAAGCUAUUGA
						GGGCTGATTTGG			ACAAGGGGCUG
						GCTCAGGCCGG			AUUUGGGCUCA
						GACCTCTTCCGC			GGCCGGGACCU
						CGCACTGAGGG			CUUCCGCCGCA
						GCACTCCACACC			CUGAGGGGCAC
						ACGGGGGCC			UCCACACCACG
						[SEQ ID NO: 1727]			GGGGCC
									[SEQ ID NO: 1924]
1755	miR-130a	-2.56989	0.011195	TCGGGTTGA	CTCACTTCA	GCAGGGCCGGC	UCGGGUUG	CUCACUUC	GCAGGGCCGGC
				AATCTGAA	GCTTTCAAT	ATGCCTCTGCTG	AAAUCUGA	AGCUUUCA	AUGCCUCUGCU
				GTGTG	TCGC	CTGGCCACTCAC	AGUGUG	AUUCGC	GCUGGCCACUC
				[SEQ ID	[SEQ ID	TTCAGCTTTCAA	[SEQ ID	[SEQ ID	ACUUCAGCUUU
				NO: 657]	NO: 1648]	TTCGCCTGTCTG	NO: 1185]	NO: 1843]	CAAUUCGCCUG
						CACCTGTCACTA			UCUGCACCUGU
						GTCGGGTTGAA			CACUAGUCGGG
						ATCTGAAGTGTG			UUGAAAUCUGA
						TGGCCGTGTAGT			AGUGUGUGGCC
						GCTACCCAGCGC			GUGUAGUGCUA
						TGGCTGCCTCCT			CCCAGCGCUGG
						CAGCATTG			CUGCCUCCUCA
						[SEQ ID NO: 1728]			GCAUUG
									[SEQ ID NO: 1925]
3272	miR-100_M	-2.56927	-0.01914	AGGACTGT	GCAATACG	CCCAAAAGAGA	AGGACUGU	GCAAUACG	CCCAAAAGAGA
				AGGCAACA	TTTCCTACA	GAAGATATTGAT	AGGCAACA	UUUCCUAC	GAAGAUAUUGA
				TATTGC	ATCCA	GCCTGTTGCCAC	UAUUGC	AAUCCA	UGCCUGUUGCC
				[SEQ ID	[SEQ ID	AAGGACTGTAG	[SEQ ID	[SEQ ID	ACAAGGACUGU
				NO: 1618]	NO: 1641]	GCAACATATTGC	NO: 1814]	NO: 1836]	AGGCAACAUAU
						GTATTAGTCCGG			UGCGUAUUAGU
						CAATACGTTTCC			CCGGCAAUACG
						TACAATCCATGT			UUUCCUACAAU
						GTCTGTTAGGCT			CCAUGUGUCUG
						ATTCCACGGACC			UUAGGCUAUUC
						TGGGGCTTTGCT			CACGGACCUGG
						TATATGCC			GGCUUUGCUUA
						[SEQ ID NO: 1729]			UAUGCC
									[SEQ ID NO: 1926]
3301	miR-130a	-2.55451	-0.07529	TGAACAAG	TGCCAAATC	GCAGGGCCGGC	UGAACAAG	UGCCAAAU	GCAGGGCCGGC
				GGGCTGATT	ATCCCCTTG	ATGCCTCTGCTG	GGGCUGAU	CAUCCCCU	AUGCCUCUGCU
				TGGGA	TTCC	CTGGCCATGCCA	UUGGGA	UGUUCC	GCUGGCCAUGC
				[SEQ ID	[SEQ ID	AATCATCCCCTT	[SEQ ID	[SEQ ID	CAAAUCAUCCC
				NO: 687]	NO: 1649]	GTTCCCTGTCTG	NO: 1215]	NO: 1844]	CUUGUUCCCUG
						CACCTGTCACTA			UCUGCACCUGU
						GTGAACAAGGG			CACUAGUGAAC
						GCTGATTTGGGA			AAGGGGCUGAU
						TGGCCGTGTAGT			UUGGGAUGGCC
						GCTACCCAGCGC			GUGUAGUGCUA
						TGGCTGCCTCCT			CCCAGCGCUGG
						CAGCATTG			CUGCCUCCUCA
						[SEQ ID NO: 1730]			GCAUUG
									[SEQ ID NO: 1927]
2943	miR-1-1	-2.54936	0.148183	TAGTAGAA	TGTCAGCCA	CATGCAGACTGC	UAGUAGAA	UGUCAGCC	CAUGCAGACUG
				GGCTTTGGC	AAGCCTTCT	CTGCTTGGGTGT	GGCUUUGG	AAAGCCUU	CCUGCUUGGGU
				TGAGA	CCCTA	CAGCCAAAGCC	CUGAGA	CUCCCUA	GUCAGCCAAAG
				[SEQ ID	[SEQ ID	TTCTCCCTATAT	[SEQ ID	[SEQ ID	CCUUCUCCCUA
				NO: 683]	NO: 1650]	GGACCTGCTAA	NO: 1211]	NO: 1845]	UAUGGACCUGC
						GCTATAGTAGA			UAAGCUAUAGU
						AGGCTTTGGCTG			AGAAGGCUUUG
						AGACTCAGGCC			GCUGAGACUCA
						GGGACCTCTCTC			GGCCGGGACCU
						GCCGCACTGAG			CUCUCGCCGCA
						GGGCACTCCAC			CUGAGGGGCAC
						ACCACGGGGGC			UCCACACCACG
						C			GGGGCC
						[SEQ ID NO: 1731]			[SEQ ID NO: 1928]
3338	miR-1-1	-2.52716	-0.2585	TACATGAG	TGAGTCTCA	CATGCAGACTGC	UACAUGAG	UGAGUCUC	CAUGCAGACUG
				GATGCTGA	GCATCCTCA	CTGCTTGGGTGA	GAUGCUGA	AGCAUCCU	CCUGCUUGGGU
				GACTGA	CGGTA	GTCTCAGCATCC	GACUGA	CACGGUA	GAGUCUCAGCA
				[SEQ ID	[SEQ ID	TCACGGTATATG	[SEQ ID	[SEQ ID	UCCUCACGGUA
				NO: 1620]	NO: 1651]	GACCTGCTAAGC	NO: 314]	NO: 1846]	UAUGGACCUGC
						TATACATGAGG	(Same guide as		UAAGCUAUACA
						ATGCTGAGACTG	XD-14893)		UGAGGAUGCUG
						ACTCAGGCCGG			AGACUGACUCA
						GACCTCTCTCGC			GGCCGGGACCU
						CGCACTGAGGG			CUCUCGCCGCA
						GCACTCCACACC			CUGAGGGGCAC
						ACGGGGGCC			UCCACACCACG
						[SEQ ID NO: 1732]			GGGGCC
									[SEQ ID NO: 1929]
3302	miR-	-2.52395	0.249656	TTGAACAA	CCCATCAGC	GAGCTCAGTCA	UUGAACAA	CCCAUCAG	GAGCUCAGUCA
	190a_M			GGGGCTGA	CCCTTGTTC	AACCTGGATGCC	GGGGCUGA	CCCCUUGU	AACCUGGAUGC
				TTTGGG	CC	TTTTCTGCAGGC	UUUGGG	UCCC	CUUUUCUGCAG
				[SEQ ID	[SEQ ID	GTCTGTGTTGAA	[SEQ ID	[SEQ ID	GCGUCUGUGUU
				NO: 688]	NO: 1643]	CAAGGGGCTGA	NO: 1216]	NO: 1838]	GAACAAGGGGC
						TTTGGGTGTTAT			UGAUUUGGGUG
						TTAATCCACCCA			UUAUUUAAUCC
						TCAGCCCCTTGT			ACCCAUCAGCC
						TCCCCTACAGTC			CCUUGUUCCCC
						TCTTGCCCTGTC			UACAGUCUCUU
						TCCGGGGGTTCC			GCCCUGUCUCC
						TAATAAAG			GGGGGUUCCUA
						[SEQ ID NO: 1733]			AUAAAG
									[SEQ ID NO: 1930]
3302	miR-155E	-2.52291	0.018122	TTGAACAA	CCCAAATC	CTGGAGGCTTGC	UUGAACAA	CCCAAAUC	CUGGAGGCUUG
				GGGGCTGA	GCCCTTGTT	TTTGGGCTGTAT	GGGGCUGA	GCCCUUGU	CUUUGGGCUGU
				TTTGGG	CAA	GCTGTTGAACAA	UUUGGG	UCAA	AUGCUGUUGAA
				[SEQ ID	[SEQ ID	GGGGCTGATTTG	[SEQ ID	[SEQ ID	CAAGGGGCUGA
				NO: 688]	NO: 1652]	GGTTTTGGCCTC	NO: 1216]	NO: 1847]	UUUGGGUUUUG
						TGACTGACCCAA			GCCUCUGACUG
						ATCGCCCTTGTT			ACCCAAAUCGC
						CAACAGGACAA			CCUUGUUCAAC
						GGCCCTTTATCA			AGGACAAGGCC
						GCACTCACATGG			CUUUAUCAGCA
						AACAAATGGCC			CUCACAUGGAA
						ACCGTGGG			CAAAUGGCCAC
						[SEQ ID NO: 1734]			CGUGGG
									[SEQ ID NO: 1931]
3302	miR-1-1	-2.51939	-0.06675	TTGAACAA	CGCAAATC	CATGCAGACTGC	UUGAACAA	CGCAAAUC	CAUGCAGACUG
				GGGGCTGA	AGCCCCTTG	CTGCTTGGGCGC	GGGGCUGA	AGCCCCUU	CCUGCUUGGGC
				TTTGGG	TCGCAA	AAATCAGCCCCT	UUUGGG	GUCGCAA	GCAAAUCAGCC
				[SEQ ID	[SEQ ID	TGTCGCAATATG	[SEQ ID	[SEQ ID	CCUUGUCGCAA
				NO: 688]	NO: 1647]	GACCTGCTAAGC	NO: 1216]	NO: 1842]	UAUGGACCUGC
						TATTGAACAAG			UAAGCUAUUGA
						GGGCTGATTTGG			ACAAGGGGCUG
						GCTCAGGCCGG			AUUUGGGCUCA
						GACCTCTCTCGC			GGCCGGGACCU
						CGCACTGAGGG			CUCUCGCCGCA
						GCACTCCACACC			CUGAGGGGCAC
						ACGGGGGCC			UCCACACCACG
						[SEQ ID NO: 1735]			GGGGCC
									[SEQ ID NO: 1932]
2586	miR-155E	-2.5179	-0.10172	TAGATTCAG	CCAAGTTCA	CTGGAGGCTTGC	UAGAUUCA	CCAAGUUC	CUGGAGGCUUG
				AAGTAGAA	CTCTGAATC	TTTGGGCTGTAT	GAAGUAGA	ACUCUGAA	CUUUGGGCUGU
				CTTGG	TA	GCTGTAGATTCA	ACUUGG	UCUA	AUGCUGUAGAU
				[SEQ ID	[SEQ ID	GAAGTAGAACT	[SEQ ID	[SEQ ID	UCAGAAGUAGA
				NO: 1621]	NO: 1653]	TGGTTTTGGCCT	NO: 1816]	NO: 1848]	ACUUGGUUUUG
						CTGACTGACCAA			GCCUCUGACUG
						GTTCACTCTGAA			ACCAAGUUCAC
						TCTACAGGACA			UCUGAAUCUAC
						AGGCCCTTTATC			AGGACAAGGCC
						AGCACTCACATG			CUUUAUCAGCA
						GAACAAATGGC			CUCACAUGGAA
						CACCGTGGG			CAAAUGGCCAC
						[SEQ ID NO: 1736]			CGUGGG
									[SEQ ID NO: 1933]
3341	miR-1-1	-2.50291	-0.1115	ATAGACAT	GACTCAGC	CATGCAGACTGC	AUAGACAU	GACUCAGC	CAUGCAGACUG
				GAGGATGC	ATCCTCATG	CTGCTTGGGGAC	GAGGAUGC	AUCCUCAU	CCUGCUUGGGG
				TGAGAC	TGATAT	TCAGCATCCTCA	UGAGAC	GUGAUAU	ACUCAGCAUCC
				[SEQ ID	[SEQ ID	TGTGATATTATG	[SEQ ID	[SEQ ID	UCAUGUGAUAU
				NO: 1617]	NO: 1637]	GACCTGCTAAGC	NO: 1813]	NO: 1832]	UAUGGACCUGC
						TAATAGACATG			UAAGCUAAUAG
						AGGATGCTGAG			ACAUGAGGAUG
						ACCTCAGGCCG			CUGAGACCUCA
						GGACCTCTCTCG			GGCCGGGACCU
						CCGCACTGAGG			CUCUCGCCGCA
						GGCACTCCACAC			CUGAGGGGCAC
						CACGGGGGCC			UCCACACCACG
						[SEQ ID NO: 1737]			GGGGCC
									[SEQ ID NO: 1934]
1580	miR-190a	-2.50168	-0.06321	ACTGGAATT	AGCTTCAG	GAGCTCAGTCA	ACUGGAAU	AGCUUCAG	GAGCUCAGUCA
				TCTCTGAAC	AGAAATTC	AACCTGGATGCC	UUCUCUGA	AGAAAUUC	AACCUGGAUGC
				TGCT	CAAG	TTTTCTGCAGGC	ACUGCU	CAAG	CUUUUCUGCAG
				[SEQ ID	[SEQ ID	CTCTGTGACTGG	[SEQ ID	[SEQ ID	GCCUCUGUGAC
				NO: 1622]	NO: 1654]	AATTTCTCTGAA	NO: 1817]	NO: 1849]	UGGAAUUUCUC
						CTGCTTGTTATT			UGAACUGCUUG
						TAATCCAAGCTT			UUAUUUAAUCC
						CAGAGAAATTC			AAGCUUCAGAG
						CAAGCTACAGT			AAAUUCCAAGC
						GTCTTGCCCTGT			UACAGUGUCUU
						CTCCGGGGGTTC			GCCCUGUCUCC
						CTAATAAAG			GGGGGUUCCUA
						[SEQ ID NO: 1738]			AUAAAG
									[SEQ ID NO: 1935]
3332	miR-122	-2.50104	-0.209	AGGATGCT	ACATTATCA	GGCTACAGAGTT	AGGAUGCU	ACAUUAUC	GGCUACAGAGU
				GAGACTGA	GTATCAGC	TCCTTAGCAGAG	GAGACUGA	AGUAUCAG	UUCCUUAGCAG
				TAATGT	ATAAT	CTGAGGATGCTG	UAAUGU	CAUAAU	AGCUGAGGAUG
				[SEQ ID	[SEQ ID	AGACTGATAAT	[SEQ ID	[SEQ ID	CUGAGACUGAU
				NO: 1623]	NO: 1655]	GTTGTCTAAACT	NO: 1818]	NO: 1850]	AAUGUUGUCUA
						ATACATTATCAG			AACUAUACAUU
						TATCAGCATAAT			AUCAGUAUCAG
						TAGCTACTGCTA			CAUAAUUAGCU
						GGCAATCCTTCC			ACUGCUAGGCA
						CTCGATAAATGT			AUCCUUCCCUC
						CTTGGCATCGTT			GAUAAAUGUCU
						TGCTTTG			UGGCAUCGUUU
						[SEQ ID NO: 1739]			GCUUUG
									[SEQ ID NO: 1936]
3133	miR-100_M	-2.49525	0.238806	TATGTCTTG	CAGTGACTC	CCCAAAAGAGA	UAUGUCUU	CAGUGACU	CCCAAAAGAGA
				GCTTGATTC	ACGCCAAG	GAAGATATTGAT	GGCUUGAU	CACGCCAA	GAAGAUAUUGA
				ACTG	CCATT	GCCTGTTGCCAC	UCACUG	GCCAUU	UGCCUGUUGCC
				[SEQ ID	[SEQ ID	ATATGTCTTGGC	[SEQ ID	[SEQ ID	ACAUAUGUCUU
				NO: 1624]	NO: 1656]	TTGATTCACTGG	NO: 1819]	NO: 1851]	GGCUUGAUUCA
						TATTAGTCCGCA			CUGGUAUUAGU
						GTGACTCACGCC			CCGCAGUGACU
						AAGCCATTTGTG			CACGCCAAGCC
						TCTGTTAGGCTA			AUUUGUGUCUG
						TTCCACGGACCT			UUAGGCUAUUC
						GGGGCTTTGCTT			CACGGACCUGG
						ATATGCC			GGCUUUGCUUA
						[SEQ ID NO: 1740]			UAUGCC
									[SEQ ID NO: 1937]
3341	miR-122	-2.4857	0.187025	ATAGACAT	GTCTCAGCA	GGCTACAGAGTT	AUAGACAU	GUCUCAGC	GGCUACAGAGU
				GAGGATGC	TCATCATGT	TCCTTAGCAGAG	GAGGAUGC	AUCAUCAU	UUCCUUAGCAG
				TGAGAC	CGCT	CTGATAGACATG	UGAGAC	GUCGCU	AGCUGAUAGAC
				[SEQ ID	[SEQ ID	AGGATGCTGAG	[SEQ ID	[SEQ ID	AUGAGGAUGCU
				NO: 1617]	NO: 1657]	ACTGTCTAAACT	NO: 1813]	NO: 1852]	GAGACUGUCUA
						ATGTCTCAGCAT			AACUAUGUCUC
						CATCATGTCGCT			AGCAUCAUCAU
						TAGCTACTGCTA			GUCGCUUAGCU
						GGCAATCCTTCC			ACUGCUAGGCA
						CTCGATAAATGT			AUCCUUCCCUC
						CTTGGCATCGTT			GAUAAAUGUCU
						TGCTTTG			UGGCAUCGUUU
						[SEQ ID NO: 1741]			GCUUUG
									[SEQ ID NO: 1938]
3341	miR-132	-2.48196	0.14613	ATAGACAT	GCCTCAGC	GCCGTCCGCGCG	AUAGACAU	GCCUCAGC	GCCGUCCGCGC
				GAGGATGC	ATCATAATG	CCCCGCCCCCGC	GAGGAUGC	AUCAUAAU	GCCCCGCCCCC
				TGAGAC	TCTAT	GTCTCCAGGGGC	UGAGAC	GUCUAU	GCGUCUCCAGG
				[SEQ ID	[SEQ ID	CTCAGCATCATA	[SEQ ID	[SEQ ID	GGCCUCAGCAU
				NO: 1617]	NO: 1658]	ATGTCTATCTGT	NO: 1813]	NO: 1853]	CAUAAUGUCUA
						GGGAACTGGAG			UCUGUGGGAAC
						GATAGACATGA			UGGAGGAUAGA
						GGATGCTGAGA			CAUGAGGAUGC
						CCCCCGCAGCAC			UGAGACCCCCG
						GCCCACGCGCC			CAGCACGCCCA
						GCGCCACGCCG			CGCGCCGCGCC
						CGCCCCGAGCC			ACGCCGCGCCC
						[SEQ ID NO: 1742]			CGAGCC
									[SEQ ID NO: 1939]
3330	miR-1-1_M	-2.47751	0.392579	TATGCTGAG	CGACATTAT	CATGCAGACTGC	UAUGCUGA	CGACAUUA	CAUGCAGACUG
				ACTGATAAT	CAGTCTCAG	CTGCTTGGGCGA	GACUGAUA	UCAGUCUC	CCUGCUUGGGC
				GTGG	GAATA	CATTATCAGTCT	AUGUGG	AGGAAUA	GACAUUAUCAG
				[SEQ ID	[SEQ ID	CAGGAATATAT	[SEQ ID	[SEQ ID	UCUCAGGAAUA
				NO: 1614]	NO: 1659]	GGACCTGCTAA	NO: 1811]	NO: 1854]	UAUGGACCUGC
						GCTATATGCTGA			UAAGCUAUAUG
						GACTGATAATGT			CUGAGACUGAU
						GGCTCAGGCCG			AAUGUGGCUCA
						GGACCTCTTCCG			GGCCGGGACCU
						CCGCACTGAGG			CUUCCGCCGCA
						GGCACTCCACAC			CUGAGGGGCAC
						CACGGGGGCC			UCCACACCACG
						[SEQ ID NO: 1743]			GGGGCC
									[SEQ ID NO: 1940]
3255	miR-144	-2.47679	-0.08595	ATTGCGTGG	ACAAGCTT	TCAAGCCATGCT	AUUGCGUG	ACAAGCUU	UCAAGCCAUGC
				AGTAAGCT	ACTGCCAC	TCCTGTGCCCCC	GAGUAAGC	ACUGCCAC	UUCCUGUGCCC
				GGTGG	GCAAT	AGTGGGGCCCT	UGGUGG	GCAAU	CCAGUGGGGCC
				[SEQ ID	[SEQ ID	GGCTACAAGCTT	[SEQ ID	[SEQ ID	CUGGCUACAAG
				NO: 617]	NO: 1660]	ACTGCCACGCA	NO: 306]	NO: 1855]	CUUACUGCCAC
						ATAGTTTGCGAT	(Same guide as		GCAAUAGUUUG
						GAGACACATTG	XD-14889)		CGAUGAGACAC
						CGTGGAGTAAG			AUUGCGUGGAG
						CTGGTAGTCCGG			UAAGCUGGUAG
						GCACCCCCAGCT			UCCGGGCACCC
						CTGGAGCCTGAC			CCAGCUCUGGA
						AAGGAGGACA			GCCUGACAAGG
						[SEQ ID NO: 1744]			AGGACA
									[SEQ ID NO: 1941]
1755	miR-100 M	-2.4723	-0.07055	TCGGGTTGA	CACACTCCA	CCCAAAAGAGA	UCGGGUUG	CACACUCC	CCCAAAAGAGA
				AATCTGAA	GCTTTCAAA	GAAGATATTGAT	AAAUCUGA	AGCUUUCA	GAAGAUAUUGA
				GTGTG	CCGT	GCCTGTTGCCAC	AGUGUG	AACCGU	UGCCUGUUGCC
				SEQ ID	[SEQ ID	ATCGGGTTGAA	[SEQ ID	[SEQ ID	ACAUCGGGUUG
				NO: 657]	NO: 1640]	ATCTGAAGTGTG	NO: 1185]	NO: 1835]	AAAUCUGAAGU
						GTATTAGTCCGC			GUGGUAUUAGU
						ACACTCCAGCTT			CCGCACACUCC
						TCAAACCGTTGT			AGCUUUCAAAC
						GTCTGTTAGGCT			CGUUGUGUCUG
						ATTCCACGGACC			UUAGGCUAUUC
						TGGGGCTTTGCT			CACGGACCUGG
						TATATGCC			GGCUUUGCUUA
						[SEQ ID NO: 1745]			UAUGCC
									[SEQ ID NO: 1942]
2586	miR-	-2.46486	0.014777	TAGATTCAG	CCATTCTAC	GAGCTCAGTCA	UAGAUUCA	CCAUUCUA	GAGCUCAGUCA
	190a_M			AAGTAGAA	TTCTGAATC	AACCTGGATGCC	GAAGUAGA	CUUCUGAA	AACCUGGAUGC
				CTTGG	CC	TTTTCTGCAGGC	ACUUGG	UCCC	CUUUUCUGCAG
				SEQ ID	[SEQ ID	GTCTGTGTAGAT	[SEQ ID	[SEQ ID	GCGUCUGUGUA
				NO: 1621]	NO: 1661]	TCAGAAGTAGA	NO: 1816]	NO: 1856]	GAUUCAGAAGU
						ACTTGGTGTTAT			AGAACUUGGUG
						TTAATCCACCAT			UUAUUUAAUCC
						TCTACTTCTGAA			ACCAUUCUACU
						TCCCCTACAGTC			UCUGAAUCCCC
						TCTTGCCCTGTC			UACAGUCUCUU
						TCCGGGGGTTCC			GCCCUGUCUCC
						TAATAAAG			GGGGGUUCCUA
						[SEQ ID NO: 1746]			AUAAAG
									[SEQ ID NO: 1943]
2586	miR-1-1_M	-2.46317	0.179187	TAGATTCAG	CGAAGTTCT	CATGCAGACTGC	UAGAUUCA	CGAAGUUC	CAUGCAGACUG
				AAGTAGAA	ACTTCTGAA	CTGCTTGGGCGA	GAAGUAGA	UACUUCUG	CCUGCUUGGGC
				CTTGG	CGCTA	AGTTCTACTTCT	ACUUGG	AACGCUA	GAAGUUCUACU
				[SEQ ID	[SEQ ID	GAACGCTATATG	SEQ ID	[SEQ ID	UCUGAACGCUA
				NO: 1621]	NO: 1662]	GACCTGCTAAGC	NO: 1816]	NO: 1857]	UAUGGACCUGC
						TATAGATTCAGA			UAAGCUAUAGA
						AGTAGAACTTG			UUCAGAAGUAG
						GCTCAGGCCGG			AACUUGGCUCA
						GACCTCTTCCGC			GGCCGGGACCU
						CGCACTGAGGG			CUUCCGCCGCA
						GCACTCCACACC			CUGAGGGGCAC
						ACGGGGGCC			UCCACACCACG
						[SEQ ID NO: 1747]			GGGGCC
									[SEQ ID NO: 1944]
3272	miR-1-1_M	-2.45517	0.050153	AGGACTGT	GGAATATG	CATGCAGACTGC	AGGACUGU	GGAAUAUG	CAUGCAGACUG
				AGGCAACA	TTGCCTACA	CTGCTTGGGGGA	AGGCAACA	UUGCCUAC	CCUGCUUGGGG
				TATTGC	GCGCCT	ATATGTTGCCTA	UAUUGC	AGCGCCU	GAAUAUGUUGC
				SEQ ID	SEQ ID	CAGCGCCTTATG	[SEQ ID	[SEQ ID	CUACAGCGCCU
				NO: 1618]	NO: 1663]	GACCTGCTAAGC	NO: 1814]	NO: 1858]	UAUGGACCUGC
						TAAGGACTGTA			UAAGCUAAGGA
						GGCAACATATTG			CUGUAGGCAAC
						CCTCAGGCCGG			AUAUUGCCUCA
						GACCTCTTCCGC			GGCCGGGACCU
						CGCACTGAGGG			CUUCCGCCGCA
						GCACTCCACACC			CUGAGGGGCAC
						ACGGGGGCC			UCCACACCACG
						[SEQ ID NO: 1748]			GGGGCC
									[SEQ ID NO: 1945]
2943	miR-1-1_M	-2.45216	0.338577	TAGTAGAA	TGTCAGCCA	CATGCAGACTGC	UAGUAGAA	UGUCAGCC	CAUGCAGACUG
				GGCTTTGGC	AAGCCTTCT	CTGCTTGGGTGT	GGCUUUGG	AAAGCCUU	CCUGCUUGGGU
				TGAGA	CCCTA	CAGCCAAAGCC	CUGAGA	CUCCCUA	GUCAGCCAAAG
				SEQ ID	[SEQ ID	TTCTCCCTATAT	[SEQ ID	[SEQ ID	CCUUCUCCCUA
				NO: 683]	NO: 1650]	GGACCTGCTAA	NO: 1211]	NO: 1845]	UAUGGACCUGC
						GCTATAGTAGA			UAAGCUAUAGU
						AGGCTTTGGCTG			AGAAGGCUUUG
						AGACTCAGGCC			GCUGAGACUCA
						GGGACCTCTTCC			GGCCGGGACCU
						GCCGCACTGAG			CUUCCGCCGCA
						GGGCACTCCAC			CUGAGGGGCAC
						ACCACGGGGGC			UCCACACCACG
						C			GGGGCC
						[SEQ ID NO: 1749]			[SEQ ID NO: 1946]
3043	miR-	-2.44458	0.289334	TTTGGTGCA	AGCGTTTGT	GAGCTCAGTCA	UUUGGUGC	AGCGUUUG	GAGCUCAGUCA
	190a M			AAACAAAC	TTTGCACCA	AACCTGGATGCC	AAAACAAA	UUUUGCAC	AACCUGGAUGC
				AGGCT	CC	TTTTCTGCAGGC	CAGGCU	CACC	CUUUUCUGCAG
				[SEQ ID	[SEQ ID	GTCTGTGTTTGG	[SEQ ID	[SEQ ID	GCGUCUGUGUU
				NO: 1615]	NO: 1664]	TGCAAAACAAA	NO: 1812]	NO: 1859]	UGGUGCAAAAC
						CAGGCTTGTTAT			AAACAGGCUUG
						TTAATCCAAGCG			UUAUUUAAUCC
						TTTGTTTTGCAC			AAGCGUUUGUU
						CACCCTACAGTC			UUGCACCACCC
						TCTTGCCCTGTC			UACAGUCUCUU
						TCCGGGGGTTCC			GCCCUGUCUCC
						TAATAAAG			GGGGGUUCCUA
						[SEQ ID NO: 1750]			AUAAAG
									[SEQ ID NO: 1947]
1578	miR-100	-2.43871	-0.43493	TGGAATTTC	ACAGCAAT	CCCAAAAGAGA	UGGAAUUU	ACAGCAAU	CCCAAAAGAGA
				TCTGAACTG	TCCGAGAA	GAAGATATTGA	CUCUGAAC	UCCGAGAA	GAAGAUAUUGA
				CTGT	ACTCCT	GGCCTGTTGCCA	UGCUGU	ACUCCU	GGCCUGUUGCC
				[SEQ ID	[SEQ ID	CATGGAATTTCT	[SEQ ID	[SEQ ID	ACAUGGAAUUU
				NO: 1626]	NO: 1665]	CTGAACTGCTGT	NO: 1820]	NO: 1860]	CUCUGAACUGC
						GTATTAGTCCGA			UGUGUAUUAGU
						CAGCAATTCCGA			CCGACAGCAAU
						GAAACTCCTTGT			UCCGAGAAACU
						GTCTGTTAGGCA			CCUUGUGUCUG
						ATCTCACGGACC			UUAGGCAAUCU
						TGGGGCTTTGCT			CACGGACCUGG
						TATATGCC			GGCUUUGCUUA
						[SEQ ID NO: 1751]			UAUGCC
									[SEQ ID NO: 1948]
3133	miR-122_M	-2.43848	0.197681	TATGTCTTG	CAGTGAAT	GGCTACAGAGTT	UAUGUCUU	CAGUGAAU	GGCUACAGAGU
				GCTTGATTC	CAAACCAA	TGCTTAGCAGAG	GGCUUGAU	CAAACCAA	UUGCUUAGCAG
				ACTG	GACCGA	CTGTATGTCTTG	UCACUG	GACCGA	AGCUGUAUGUC
				[SEQ ID	[SEQ ID	GCTTGATTCACT	[SEQ ID	[SEQ ID	UUGGCUUGAUU
				NO: 1624]	NO: 1666]	GTGTCTAAACTA	NO: 1819]	NO: 1861]	CACUGUGUCUA
						TCAGTGAATCAA			AACUAUCAGUG
						ACCAAGACCGA			AAUCAAACCAA
						TAGCTACTGCTA			GACCGAUAGCU
						GGCCATCCTTCC			ACUGCUAGGCC
						CTCGATAAATGT			AUCCUUCCCUC
						CTTGGCATCGTT			GAUAAAUGUCU
						TGCTTTG			UGGCAUCGUUU
						[SEQ ID NO: 1752]			GCUUUG
									[SEQ ID NO: 1949]
3341	miR-130a	-2.43802	0.12379	ATAGACAT	GACTCAGC	GCAGGGCCGGC	AUAGACAU	GACUCAGC	GCAGGGCCGGC
				GAGGATGC	ATACTCATG	ATGCCTCTGCTG	GAGGAUGC	AUACUCAU	AUGCCUCUGCU
				TGAGAC	TTTAC	CTGGCCAGACTC	UGAGAC	GUUUAC	GCUGGCCAGAC
				[SEQ ID	[SEQ ID	AGCATACTCATG	[SEQ ID	[SEQ ID	UCAGCAUACUC
				NO: 1617]	NO: 1667]	TTTACCTGTCTG	NO: 1813]	NO: 1862]	AUGUUUACCUG
						CACCTGTCACTA			UCUGCACCUGU
						GATAGACATGA			CACUAGAUAGA
						GGATGCTGAGA			CAUGAGGAUGC
						CTGGCCGTGTAG			UGAGACUGGCC
						TGCTACCCAGCG			GUGUAGUGCUA
						CTGGCTGCCTCC			CCCAGCGCUGG
						TCAGCATTG			CUGCCUCCUCA
						[SEQ ID NO: 1753]			GCAUUG
									[SEQ ID NO: 1950]
3330	miR-130a	-2.43606	0.221874	TATGCTGAG	CGACATTAT	GCAGGGCCGGC	UAUGCUGA	CGACAUUA	GCAGGGCCGGC
				ACTGATAAT	CCGTCTCAG	ATGCCTCTGCTG	GACUGAUA	UCCGUCUC	AUGCCUCUGCU
				GTGG	TATC	CTGGCCACGAC	AUGUGG	AGUAUC	GCUGGCCACGA
				[SEQ ID	[SEQ ID	ATTATCCGTCTC	[SEQ ID	[SEQ ID	CAUUAUCCGUC
				NO: 1614]	NO: 1668]	AGTATCCTGTCT	NO: 1811]	NO: 1863]	UCAGUAUCCUG
						GCACCTGTCACT			UCUGCACCUGU
						AGTATGCTGAG			CACUAGUAUGC
						ACTGATAATGTG			UGAGACUGAUA
						GTGGCCGTGTAG			AUGUGGUGGCC
						TGCTACCCAGCG			GUGUAGUGCUA
						CTGGCTGCCTCC			CCCAGCGCUGG
						TCAGCATTG			CUGCCUCCUCA
						[SEQ ID NO: 1754]			GCAUUG
									[SEQ ID NO: 1951]
3255	miR-130a	-2.43262	0.01754	ATTGCGTGG	CGACCAGC	GCAGGGCCGGC	AUUGCGUG	CGACCAGC	GCAGGGCCGGC
				AGTAAGCT	TTCCTCCAC	ATGCCTCTGCTG	GAGUAAGC	UUCCUCCA	AUGCCUCUGCU
				GGTGG	GTAAC	CTGGCCACGACC	UGGUGG	CGUAAC	GCUGGCCACGA
				[SEQ ID	[SEQ ID	AGCTTCCTCCAC	[SEQ ID	[SEQ ID	CCAGCUUCCUC
				NO: 617]	NO: 1669]	GTAACCTGTCTG	NO: 306]	NO: 1864]	CACGUAACCUG
						CACCTGTCACTA	(Same guide as		UCUGCACCUGU
						GATTGCGTGGA	XD-14889)		CACUAGAUUGC
						GTAAGCTGGTG			GUGGAGUAAGC
						GTGGCCGTGTAG			UGGUGGUGGCC
						TGCTACCCAGCG			GUGUAGUGCUA
						CTGGCTGCCTCC			CCCAGCGCUGG
						TCAGCATTG			CUGCCUCCUCA
						[SEQ ID NO: 1755]			GCAUUG
									[SEQ ID NO: 1952]
3302	miR-132	-2.43028	0.018802	TTGAACAA	CACAAATC	GCCGTCCGCGCG	UUGAACAA	CACAAAUC	GCCGUCCGCGC
				GGGGCTGA	AGCACATT	CCCCGCCCCCGC	GGGGCUGA	AGCACAUU	GCCCCGCCCCC
				TTTGGG	GTTCAA	GTCTCCAGGGCA	UUUGGG	GUUCAA	GCGUCUCCAGG
				[SEQ ID	[SEQ ID	CAAATCAGCAC	[SEQ ID	[SEQ ID	GCACAAAUCAG
				NO: 688]	NO: 1670]	ATTGTTCAACTG	NO: 1216]	NO: 1865]	CACAUUGUUCA
						TGGGAACTGGA			ACUGUGGGAAC
						GGTTGAACAAG			UGGAGGUUGAA
						GGGCTGATTTGG			CAAGGGGCUGA
						GCCCCGCAGCA			UUUGGGCCCCG
						CGCCCACGCGCC			CAGCACGCCCA
						GCGCCACGCCG			CGCGCCGCGCC
						CGCCCCGAGCC			ACGCCGCGCCC
						[SEQ ID NO: 1756]			CGAGCC
									[SEQ ID NO: 1953]
3133	miR-130a	-2.42664	0.412413	TATGTCTTG	CTGTGAATC	GCAGGGCCGGC	UAUGUCUU	CUGUGAAU	GCAGGGCCGGC
				GCTTGATTC	ACGCCAAG	ATGCCTCTGCTG	GGCUUGAU	CACGCCAA	AUGCCUCUGCU
				ACTG	ATATC	CTGGCCACTGTG	UCACUG	GAUAUC	GCUGGCCACUG
				[SEQ ID	[SEQ ID	AATCACGCCAA	[SEQ ID	[SEQ ID	UGAAUCACGCC
				NO: 1624]	NO: 1671]	GATATCCTGTCT	NO: 1819]	NO: 1866]	AAGAUAUCCUG
						GCACCTGTCACT			UCUGCACCUGU
						AGTATGTCTTGG			CACUAGUAUGU
						CTTGATTCACTG			CUUGGCUUGAU
						TGGCCGTGTAGT			UCACUGUGGCC
						GCTACCCAGCGC			GUGUAGUGCUA
						TGGCTGCCTCCT			CCCAGCGCUGG
						CAGCATTG			CUGCCUCCUCA
						[SEQ ID NO: 1757]			GCAUUG
									[SEQ ID NO: 1954]
3302	miR 155-M	-2.42428	0.285147	TTGAACAA	CCCAAATC	CCTGGAGGCTTG	UUGAACAA	CCCAAAUC	CCUGGAGGCUU
				GGGGCTGA	GCCCTTGTT	CTGAAGGCTGTA	GGGGCUGA	GCCCUUGU	GCUGAAGGCUG
				TTTGGG	CAA	TGCTGTTGAACA	UUUGGG	UCAA	UAUGCUGUUGA
				[SEQ ID	[SEQ ID	AGGGGCTGATTT	[SEQ ID	[SEQ ID	ACAAGGGGCUG
				NO: 688]	NO: 1652]	GGGTTTTGGCCA	NO: 1216]	NO: 1847]	AUUUGGGUUUU
						CTGACTGACCCA			GGCCACUGACU
						AATCGCCCTTGT			GACCCAAAUCG
						TCAACAGGACA			CCCUUGUUCAA
						CAAGGCCTGTTA			CAGGACACAAG
						CTAGCACTCACA			GCCUGUUACUA
						TGGAACAAATG			GCACUCACAUG
						GCCACCGG			GAACAAAUGGC
						[SEQ ID NO: 1758]			CACCGG
									[SEQ ID NO: 1955]
2586	miR 155-M	-2.4228	-0.13209	TAGATTCAG	CCAAGTTCA	CCTGGAGGCTTG	UAGAUUCA	CCAAGUUC	CCUGGAGGCUU
				AAGTAGAA	CTCTGAATC	CTGAAGGCTGTA	GAAGUAGA	ACUCUGAA	GCUGAAGGCUG
				CTTGG	TA	TGCTGTAGATTC	ACUUGG	UCUA	UAUGCUGUAGA
				[SEQ ID	[SEQ ID	AGAAGTAGAAC	[SEQ ID	[SEQ ID	UUCAGAAGUAG
				NO: 1621]	NO: 1653]	TTGGTTTTGGCC	NO: 1816]	NO: 1848]	AACUUGGUUUU
						ACTGACTGACCA			GGCCACUGACU
						AGTTCACTCTGA			GACCAAGUUCA
						ATCTACAGGAC			CUCUGAAUCUA
						ACAAGGCCTGTT			CAGGACACAAG
						ACTAGCACTCAC			GCCUGUUACUA
						ATGGAACAAAT			GCACUCACAUG
						GGCCACCGG			GAACAAAUGGC
						[SEQ ID NO: 1759]			CACCGG
									[SEQ ID NO: 1956]
1755	miR-122	-2.42198	0.034447	TCGGGTTGA	CACACTTCA	GGCTACAGAGTT	UCGGGUUG	CACACUUC	GGCUACAGAGU
				AATCTGAA	GACTTCAAC	TCCTTAGCAGAG	AAAUCUGA	AGACUUCA	UUCCUUAGCAG
				GTGTG	CATA	CTGTCGGGTTGA	AGUGUG	ACCAUA	AGCUGUCGGGU
				[SEQ ID	[SEQ ID	AATCTGAAGTGT	[SEQ ID	[SEQ ID	UGAAAUCUGAA
				NO: 657]	NO: 1646]	GTGTCTAAACTA	NO: 1185]	NO: 1841]	GUGUGUGUCUA
						TCACACTTCAGA			AACUAUCACAC
						CTTCAACCATAT			UUCAGACUUCA
						AGCTACTGCTAG			ACCAUAUAGCU
						GCAATCCTTCCC			ACUGCUAGGCA
						TCGATAAATGTC			AUCCUUCCCUC
						TTGGCATCGTTT			GAUAAAUGUCU
						GCTTTG			UGGCAUCGUUU
						[SEQ ID NO: 1760]			GCUUUG
									[SEQ ID NO: 1957]
2945	miR-1-1	-2.42094	-0.06694	TGTAGTAG	TGAGCCAA	CATGCAGACTGC	UGUAGUAG	UGAGCCAA	CAUGCAGACUG
				AAGGCTTTG	AGCCTTCTA	CTGCTTGGGTGA	AAGGCUUU	AGCCUUCU	CCUGCUUGGGU
				GCTGA	CCGACA	GCCAAAGCCTTC	GGCUGA	ACCGACA	GAGCCAAAGCC
				[SEQ ID	[SEQ ID	TACCGACATATG	[SEQ ID	[SEQ ID	UUCUACCGACA
				NO: 685]	NO: 1633]	GACCTGCTAAGC	NO: 1213]	NO: 1828]	UAUGGACCUGC
						TATGTAGTAGAA	(Same guide as		UAAGCUAUGUA
						GGCTTTGGCTGA	XD-14860)		GUAGAAGGCUU
						CTCAGGCCGGG			UGGCUGACUCA
						ACCTCTCTCGCC			GGCCGGGACCU
						GCACTGAGGGG			CUCUCGCCGCA
						CACTCCACACCA			CUGAGGGGCAC
						CGGGGGCC			UCCACACCACG
						[SEQ ID NO: 1761]			GGGGCC
									[SEQ ID NO: 1958
1755	miR-132	-2.41844	0.196175	TCGGGTTGA	CCCACTTCA	GCCGTCCGCGCG	UCGGGUUG	CCCACUUC	GCCGUCCGCGC
				AATCTGAA	GAGTGCAA	CCCCGCCCCCGC	AAAUCUGA	AGAGUGCA	GCCCCGCCCCC
				GTGTG	CCCGA	GTCTCCAGGGCC	AGUGUG	ACCCGA	GCGUCUCCAGG
				TSEQ ID	[SEQ ID	CACTTCAGAGTG	[SEQ ID	[SEQ ID	GCCCACUUCAG
				NO: 657]	NO: 1672]	CAACCCGACTGT	NO: 1185]	NO: 1867]	AGUGCAACCCG
						GGGAACTGGAG			ACUGUGGGAAC
						GTCGGGTTGAA			UGGAGGUCGGG
						ATCTGAAGTGTG			UUGAAAUCUGA
						CCCCGCAGCAC			AGUGUGCCCCG
						GCCCACGCGCC			CAGCACGCCCA
						GCGCCACGCCG			CGCGCCGCGCC
						CGCCCCGAGCC			ACGCCGCGCCC
						[SEQ ID NO: 1762]			CGAGCC
									[SEQ ID NO: 1959]
3301	miR-1-1	-2.41082	0.051034	TGAACAAG	TGCCAAATC	CATGCAGACTGC	UGAACAAG	UGCCAAAU	CAUGCAGACUG
				GGGCTGATT	AGCCCCTTG	CTGCTTGGGTGC	GGGCUGAU	CAGCCCCU	CCUGCUUGGGU
				TGGGA	CGTCA	CAAATCAGCCCC	UUGGGA	UGCGUCA	GCCAAAUCAGC
				[SEQ ID	[SEQ ID	TTGCGTCATATG	[SEQ ID	[SEQ ID	CCCUUGCGUCA
				NO: 687]	NO: 1673]	GACCTGCTAAGC	NO: 1215]	NO: 1868]	UAUGGACCUGC
						TATGAACAAGG			UAAGCUAUGAA
						GGCTGATTTGGG			CAAGGGGCUGA
						ACTCAGGCCGG			UUUGGGACUCA
						GACCTCTCTCGC			GGCCGGGACCU
						CGCACTGAGGG			CUCUCGCCGCA
						GCACTCCACACC			CUGAGGGGCAC
						ACGGGGGCC			UCCACACCACG
						[SEQ ID NO: 1763]			GGGGCC
									[SEQ ID NO: 1960]
2602	miR-132	-2.40987	-0.18915	TTTAGTAGT	ACTCTATGG	GCCGTCCGCGCG	UUUAGUAG	ACUCUAUG	GCCGUCCGCGC
				TGATCCATA	ATAACCTAC	CCCCGCCCCCGC	UUGAUCCA	GAUAACCU	GCCCCGCCCCC
				GATT	TAAA	GTCTCCAGGGAC	UAGAUU	ACUAAA	GCGUCUCCAGG
				SEQ ID	SEQ ID	TCTATGGATAAC	ISEQ ID	[SEQ ID	GACUCUAUGGA
				NO: 1616]	NO: 1674]	CTACTAAACTGT	NO: 202]	NO: 1869]	UAACCUACUAA
						GGGAACTGGAG	(Same guide as		ACUGUGGGAAC
						GTTTAGTAGTTG	XD-14837)		UGGAGGUUUAG
						ATCCATAGATTC			UAGUUGAUCCA
						CCCGCAGCACG			UAGAUUCCCCG
						CCCACGCGCCGC			CAGCACGCCCA
						GCCACGCCGCG			CGCGCCGCGCC
						CCCCGAGCC			ACGCCGCGCCC
						[SEQ ID NO: 1764]			CGAGCC
									[SEQ ID NO: 1961]
3302	miR-122 M	-2.40308	-0.14782	TTGAACAA	CCCAAATC	GGCTACAGAGTT	UUGAACAA	CCCAAAUC	GGCUACAGAGU
				GGGGCTGA	AGCACCTTG	TGCTTAGCAGAG	GGGGCUGA	AGCACCUU	UUGCUUAGCAG
				TTTGGG	TTACA	CTGTTGAACAAG	UUUGGG	GUUACA	AGCUGUUGAAC
				SEQ ID	[SEQ ID	GGGCTGATTTGG	[SEQ ID	SEQ ID	AAGGGGCUGAU
				NO: 688]	NO: 1675]	GTGTCTAAACTA	NO: 1216]	NO: 1870]	UUGGGUGUCUA
						TCCCAAATCAGC			AACUAUCCCAA
						ACCTTGTTACAT			AUCAGCACCUU
						AGCTACTGCTAG			GUUACAUAGCU
						GCCATCCTTCCC			ACUGCUAGGCC
						TCGATAAATGTC			AUCCUUCCCUC
						TTGGCATCGTTT			GAUAAAUGUCU
						GCTTTG			UGGCAUCGUUU
						[SEQ ID NO: 1765]			GCUUUG
									[SEQ ID NO: 1962]
1755	miR-155E	-2.39604	-0.03915	TCGGGTTGA	CACACTTCG	CTGGAGGCTTGC	UCGGGUUG	CACACUUC	CUGGAGGCUUG
				AATCTGAA	ATTCAACCC	TTTGGGCTGTAT	AAAUCUGA	GAUUCAAC	CUUUGGGCUGU
				GTGTG	GA	GCTGTCGGGTTG	AGUGUG	CCGA	AUGCUGUCGGG
				[SEQ ID	[SEQ ID	AAATCTGAAGT	[SEQ ID	[SEQ ID	UUGAAAUCUGA
				NO: 657]	NO: 1676]	GTGTTTTGGCCT	NO: 1185]	NO: 1871]	AGUGUGUUUUG
						CTGACTGACACA			GCCUCUGACUG
						CTTCGATTCAAC			ACACACUUCGA
						CCGACAGGACA			UUCAACCCGAC
						AGGCCCTTTATC			AGGACAAGGCC
						AGCACTCACATG			CUUUAUCAGCA
						GAACAAATGGC			CUCACAUGGAA
						CACCGTGGG			CAAAUGGCCAC
						[SEQ ID NO: 1766]			CGUGGG
									[SEQ ID NO: 1963]
3842	miR-130a	-2.39495	-0.55009	AACGTGAG	TTCGATCCA	GCAGGGCCGGC	AACGUGAG	UUCGAUCC	GCAGGGCCGGC
				AAGGATGG	TACTTCTCA	ATGCCTCTGCTG	AAGGAUGG	AUACUUCU	AUGCCUCUGCU
				ATCGTA	TGTC	CTGGCCATTCGA	AUCGUA	CAUGUC	GCUGGCCAUUC
				[SEQ ID	[SEQ ID	TCCATACTTCTC	[SEQ ID	[SEQ ID	GAUCCAUACUU
				NO: 1625]	NO: 1677]	ATGTCCTGTCTG	NO: 1824]	NO: 1872]	CUCAUGUCCUG
						CACCTGTCACTA			UCUGCACCUGU
						GAACGTGAGAA			CACUAGAACGU
						GGATGGATCGT			GAGAAGGAUGG
						ATGGCCGTGTAG			AUCGUAUGGCC
						TGCTACCCAGCG			GUGUAGUGCUA
						CTGGCTGCCTCC			CCCAGCGCUGG
						TCAGCATTG			CUGCCUCCUCA
						[SEQ ID NO: 1767]			GCAUUG
									[SEQ ID NO: 1964
2945	miR-100	-2.38747	-0.10088	TGTAGTAG	TCAGCCCA	CCCAAAAGAGA	UGUAGUAG	UCAGCCCA	CCCAAAAGAGA
				AAGGCTTTG	AGACTTCTA	GAAGATATTGA	AAGGCUUU	AGACUUCU	GAAGAUAUUGA
				GCTGA	ATACT	GGCCTGTTGCCA	GGCUGA	AAUACU	GGCCUGUUGCC
				SEQ ID	[SEQ ID	CATGTAGTAGA	[SEQ ID	[SEQ ID	ACAUGUAGUAG
				NO: 685]	NO: 1678]	AGGCTTTGGCTG	NO: 1213]	NO: 1873]	AAGGCUUUGGC
						AGTATTAGTCCG	(Same guide as		UGAGUAUUAGU
						TCAGCCCAAGA	XD-14860)		CCGUCAGCCCA
						CTTCTAATACTT			AGACUUCUAAU
						GTGTCTGTTAGG			ACUUGUGUCUG
						CAATCTCACGGA			UUAGGCAAUCU
						CCTGGGGCTTTG			CACGGACCUGG
						CTTATATGCC			GGCUUUGCUUA
						[SEQ ID NO: 1768]			UAUGCC
									[SEQ ID NO: 1965]
2586	miR-130a	-2.38521	0.042149	TAGATTCAG	CGAAGTTCT	GCAGGGCCGGC	UAGAUUCA	CGAAGUUC	GCAGGGCCGGC
				AAGTAGAA	AATTCTGAA	ATGCCTCTGCTG	GAAGUAGA	UAAUUCUG	AUGCCUCUGCU
				CTTGG	TCTC	CTGGCCACGAA	ACUUGG	AAUCUC	GCUGGCCACGA
				[SEQ ID	[SEQ ID	GTTCTAATTCTG	[SEQ ID	[SEQ ID	AGUUCUAAUUC
				NO: 1621]	NO: 1679]	AATCTCCTGTCT	NO: 1816]	NO: 1874]	UGAAUCUCCUG
						GCACCTGTCACT			UCUGCACCUGU
						AGTAGATTCAG			CACUAGUAGAU
						AAGTAGAACTT			UCAGAAGUAGA
						GGTGGCCGTGTA			ACUUGGUGGCC
						GTGCTACCCAGC			GUGUAGUGCUA
						GCTGGCTGCCTC			CCCAGCGCUGG
						CTCAGCATTG			CUGCCUCCUCA
						[SEQ ID NO: 1769]			GCAUUG
									[SEQ ID NO: 1966]
3301	miR-1-1_M	-2.38494	0.327128	TGAACAAG	TGCCAAATC	CATGCAGACTGC	UGAACAAG	UGCCAAAU	CAUGCAGACUG
				GGGCTGATT	AGCCCCTTG	CTGCTTGGGTGC	GGGCUGAU	CAGCCCCU	CCUGCUUGGGU
				TGGGA	CGTCA	CAAATCAGCCCC	UUGGGA	UGCGUCA	GCCAAAUCAGC
				[SEQ ID	[SEQ ID	TTGCGTCATATG	[SEQ ID	[SEQ ID	CCCUUGCGUCA
				NO: 687]	NO: 1673]	GACCTGCTAAGC	NO: 1215]	NO: 1868]	UAUGGACCUGC
						TATGAACAAGG			UAAGCUAUGAA
						GGCTGATTTGGG			CAAGGGGCUGA
						ACTCAGGCCGG			UUUGGGACUCA
						GACCTCTTCCGC			GGCCGGGACCU
						CGCACTGAGGG			CUUCCGCCGCA
						GCACTCCACACC			CUGAGGGGCAC
						ACGGGGGCC			UCCACACCACG
						[SEQ ID NO: 1770]			GGGGCC
									[SEQ ID NO: 1967]
1755	miR-190a	-2.38113	0.329455	TCGGGTTGA	CACTTCAGA	GAGCTCAGTCA	UCGGGUUG	CACUUCAG	GAGCUCAGUCA
				AATCTGAA	TTTCAACCC	AACCTGGATGCC	AAAUCUGA	AUUUCAAC	AACCUGGAUGC
				GTGTG	AC	TTTTCTGCAGGC	AGUGUG	CCAC	CUUUUCUGCAG
				[SEQ ID	[SEQ ID	CTCTGTGTCGGG	[SEQ ID	[SEQ ID	GCCUCUGUGUC
				NO: 657]	NO: 1680]	TTGAAATCTGAA	NO: 1185]	NO: 1875]	GGGUUGAAAUC
						GTGTGTGTTATT			UGAAGUGUGUG
						TAATCCACACTT			UUAUUUAAUCC
						CAGATTTCAACC			ACACUUCAGAU
						CACCTACAGTGT			UUCAACCCACC
						CTTGCCCTGTCT			UACAGUGUCUU
						CCGGGGGTTCCT			GCCCUGUCUCC
						AATAAAG			GGGGGUUCCUA
						[SEQ ID NO: 1771]			AUAAAG
									[SEQ ID NO: 1968]
2602	miR-1-1_M	-2.37919	0.040602	TTTAGTAGT	ATTCTATGG	CATGCAGACTGC	UUUAGUAG	AUUCUAUG	CAUGCAGACUG
				TGATCCATA	ATCAACTAC	CTGCTTGGGATT	UUGAUCCA	GAUCAACU	CCUGCUUGGGA
				GATT	CGAAA	CTATGGATCAAC	UAGAUU	ACCGAAA	UUCUAUGGAUC
				[SEQ ID	[SEQ ID	TACCGAAATATG	[SEQ ID	[SEQ ID	AACUACCGAAA
				NO: 1616]	NO: 1681]	GACCTGCTAAGC	NO: 202]	NO: 1876]	UAUGGACCUGC
						TATTTAGTAGTT	(Same guide as		UAAGCUAUUUA
						GATCCATAGATT	XD-14837)		GUAGUUGAUCC
						CTCAGGCCGGG			AUAGAUUCUCA
						ACCTCTTCCGCC			GGCCGGGACCU
						GCACTGAGGGG			CUUCCGCCGCA
						CACTCCACACCA			CUGAGGGGCAC
						CGGGGGCC			UCCACACCACG
						[SEQ ID NO: 1772]			GGGGCC
									[SEQ ID NO: 1969]
1231	miR-100_M	-2.37228	-0.26919	TTCACTTTA	CTGCTACCA	CCCAAAAGAGA	UUCACUUU	CUGCUACC	CCCAAAAGAGA
				GCACTGAT	GGGCTAAA	GAAGATATTGAT	AGCACUGA	AGGGCUAA	GAAGAUAUUGA
				AGCAG	ATGAT	GCCTGTTGCCAC	UAGCAG	AAUGAU	UGCCUGUUGCC
				[SEQ ID	[SEQ ID	ATTCACTTTAGC	[SEQ ID	[SEQ ID	ACAUUCACUUU
				NO: 1627]	NO: 1682]	ACTGATAGCAG	NO: 1825]	NO: 1877]	AGCACUGAUAG
						GTATTAGTCCGC			CAGGUAUUAGU
						TGCTACCAGGGC			CCGCUGCUACC
						TAAAATGATTGT			AGGGCUAAAAU
						GTCTGTTAGGCT			GAUUGUGUCUG
						ATTCCACGGACC			UUAGGCUAUUC
						TGGGGCTTTGCT			CACGGACCUGG
						TATATGCC			GGCUUUGCUUA
						[SEQ ID NO: 1773]			UAUGCC
									[SEQ ID NO: 1970]
3273	miR-155E	-2.36984	-0.0801	TAGGACTGT	CAATATGTG	CTGGAGGCTTGC	UAGGACUG	CAAUAUGU	CUGGAGGCUUG
				AGGCAACA	CCACAGTCC	TTTGGGCTGTAT	UAGGCAAC	GCCACAGU	CUUUGGGCUGU
				TATTG	TA	GCTGTAGGACTG	AUAUUG	CCUA	AUGCUGUAGGA
				[SEQ ID	[SEQ ID	TAGGCAACATAT	[SEQ ID	[SEQ ID	CUGUAGGCAAC
				NO: 1628]	NO: 1683]	TGTTTTGGCCTC	NO: 1821]	NO: 1878]	AUAUUGUUUUG
						TGACTGACAATA			GCCUCUGACUG
						TGTGCCACAGTC			ACAAUAUGUGC
						CTACAGGACAA			CACAGUCCUAC
						GGCCCTTTATCA			AGGACAAGGCC
						GCACTCACATGG			CUUUAUCAGCA
						AACAAATGGCC			CUCACAUGGAA
						ACCGTGGG			CAAAUGGCCAC
						[SEQ ID NO: 1774]			CGUGGG
									[SEQ ID NO: 1971]
3301	miR-190a	-2.36785	0.195332	TGAACAAG	TCCAATCAG	GAGCTCAGTCA	UGAACAAG	UCCAAUCA	GAGCUCAGUCA
				GGGCTGATT	CCCCTTGTT	AACCTGGATGCC	GGGCUGAU	GCCCCUUG	AACCUGGAUGC
				TGGGA	AC	TTTTCTGCAGGC	UUGGGA	UUAC	CUUUUCUGCAG
				[SEQ ID	[SEQ ID	CTCTGTGTGAAC	[SEQ ID	[SEQ ID	GCCUCUGUGUG
				NO: 687]	NO: 1684]	AAGGGGCTGAT	NO: 1215]	NO: 1879]	AACAAGGGGCU
						TTGGGATGTTAT			GAUUUGGGAUG
						TTAATCCATCCA			UUAUUUAAUCC
						ATCAGCCCCTTG			AUCCAAUCAGC
						TTACCTACAGTG			CCCUUGUUACC
						TCTTGCCCTGTC			UACAGUGUCUU
						TCCGGGGGTTCC			GCCCUGUCUCC
						TAATAAAG			GGGGGUUCCUA
						[SEQ ID NO: 1775]			AUAAAG
									[SEQ ID NO: 1972]
3043	miR-100_M	-2.35911	0.045827	TTTGGTGCA	AGCCTGCTT	CCCAAAAGAGA	UUUGGUGC	AGCCUGCU	CCCAAAAGAGA
				AAACAAAC	GGTTTGCAA	GAAGATATTGAT	AAAACAAA	UGGUUUGC	GAAGAUAUUGA
				AGGCT	CAAT	GCCTGTTGCCAC	CAGGCU	AACAAU	UGCCUGUUGCC
				[SEQ ID	[SEQ ID	ATTTGGTGCAAA	[SEQ ID	[SEQ ID	ACAUUUGGUGC
				NO: 1615]	NO: 1639]	ACAAACAGGCT	NO: 1812]	NO: 1834]	AAAACAAACAG
						GTATTAGTCCGA			GCUGUAUUAGU
						GCCTGCTTGGTT			CCGAGCCUGCU
						TGCAACAATTGT			UGGUUUGCAAC
						GTCTGTTAGGCT			AAUUGUGUCUG
						ATTCCACGGACC			UUAGGCUAUUC
						TGGGGCTTTGCT			CACGGACCUGG
						TATATGCC			GGCUUUGCUUA
						[SEQ ID NO: 1776]			UAUGCC
									[SEQ ID NO: 1973]
2586	miR-122	-2.35707	0.153236	TAGATTCAG	CCAAGTTCT	GGCTACAGAGTT	UAGAUUCA	CCAAGUUC	GGCUACAGAGU
				AAGTAGAA	ACCTCTGAA	TCCTTAGCAGAG	GAAGUAGA	UACCUCUG	UUCCUUAGCAG
				CTTGG	TAGA	CTGTAGATTCAG	ACUUGG	AAUAGA	AGCUGUAGAUU
				[SEQ ID	[SEQ ID	AAGTAGAACTT	[SEQ ID	[SEQ ID	CAGAAGUAGAA
				NO: 1621]	NO: 1685]	GGTGTCTAAACT	NO: 1816]	NO: 1880]	CUUGGUGUCUA
						ATCCAAGTTCTA			AACUAUCCAAG
						CCTCTGAATAGA			UUCUACCUCUG
						TAGCTACTGCTA			AAUAGAUAGCU
						GGCAATCCTTCC			ACUGCUAGGCA
						CTCGATAAATGT			AUCCUUCCCUC
						CTTGGCATCGTT			GAUAAAUGUCU
						TGCTTTG			UGGCAUCGUUU
						[SEQ ID NO: 1777]			GCUUUG
									[SEQ ID NO: 1974]
1755	miR-	-2.34216	0.272299	TCGGGTTGA	CACTTCAGA	GAGCTCAGTCA	UCGGGUUG	CACUUCAG	GAGCUCAGUCA
	190a_M			AATCTGAA	TTTCAACCC	AACCTGGATGCC	AAAUCUGA	AUUUCAAC	AACCUGGAUGC
				GTGTG	AC	TTTTCTGCAGGC	AGUGUG	CCAC	CUUUUCUGCAG
				[SEQ ID	[SEQ ID	GTCTGTGTCGGG	[SEQ ID	[SEQ ID	GCGUCUGUGUC
				NO: 657]	NO: 1680]	TTGAAATCTGAA	NO: 1185]	NO: 1875]	GGGUUGAAAUC
						GTGTGTGTTATT			UGAAGUGUGUG
						TAATCCACACTT			UUAUUUAAUCC
						CAGATTTCAACC			ACACUUCAGAU
						CACCTACAGTCT			UUCAACCCACC
						CTTGCCCTGTCT			UACAGUCUCUU
						CCGGGGGTTCCT			GCCCUGUCUCC
						AATAAAG			GGGGGUUCCUA
						[SEQ ID NO: 1778]			AUAAAG
									[SEQ ID NO: 1975]
1784	miR-132	-2.34143	-0.26117	ATTAACTAC	TCCAGACC	GCCGTCCGCGCG	AUUAACUA	UCCAGACC	GCCGUCCGCGC
				TCTTTGGTC	AAATATTA	CCCCGCCCCCGC	CUCUUUGG	AAAUAUUA	GCCCCGCCCCC
				TGAA	GTTAAT	GTCTCCAGGGTC	UCUGAA	GUUAAU	GCGUCUCCAGG
				[SEQ ID	[SEQ ID	CAGACCAAATA	[SEQ ID	[SEQ ID	GUCCAGACCAA
				NO: 608]	NO: 1686]	TTAGTTAATCTG	NO: 112]	NO: 1881]	AUAUUAGUUAA
						TGGGAACTGGA	(Same guide as		UCUGUGGGAAC
						GGATTAACTACT	XD-14792)		UGGAGGAUUAA
						CTTTGGTCTGAA			CUACUCUUUGG
						CCCCGCAGCAC			UCUGAACCCCG
						GCCCACGCGCC			CAGCACGCCCA
						GCGCCACGCCG			CGCGCCGCGCC
						CGCCCCGAGCC			ACGCCGCGCCC
						[SEQ ID NO: 1779]			CGAGCC
									[SEQ ID NO: 1976]
3272	miR-155E	-2.34055	-0.19485	AGGACTGT	GCAATATGT	CTGGAGGCTTGC	AGGACUGU	GCAAUAUG	CUGGAGGCUUG
				AGGCAACA	GCTACAGTC	TTTGGGCTGTAT	AGGCAACA	UGCUACAG	CUUUGGGCUGU
				TATTGC	CT	GCTGAGGACTGT	UAUUGC	UCCU	AUGCUGAGGAC
				[SEQ ID	[SEQ ID	AGGCAACATATT	[SEQ ID	[SEQ ID	UGUAGGCAACA
				NO: 1618]	NO: 1687]	GCTTTTGGCCTC	NO: 1814]	NO: 1882]	UAUUGCUUUUG
						TGACTGAGCAAT			GCCUCUGACUG
						ATGTGCTACAGT			AGCAAUAUGUG
						CCTCAGGACAA			CUACAGUCCUC
						GGCCCTTTATCA			AGGACAAGGCC
						GCACTCACATGG			CUUUAUCAGCA
						AACAAATGGCC			CUCACAUGGAA
						ACCGTGGG			CAAAUGGCCAC
						[SEQ ID NO: 1780]			CGUGGG
									[SEQ ID NO: 1977]
1159	miR-1-1_M	-2.33035	0.359288	TGTACCACA	GATCAGAC	CATGCAGACTGC	UGUACCAC	GAUCAGAC	CAUGCAGACUG
				ACAAAGTC	TTTGTTGTG	CTGCTTGGGGAT	AACAAAGU	UUUGUUGU	CCUGCUUGGGG
				TGAAC	GCGACA	CAGACTTTGTTG	CUGAAC	GGCGACA	AUCAGACUUUG
				[SEQ ID	[SEQ ID	TGGCGACATATG	[SEQ ID	[SEQ ID	UUGUGGCGACA
				NO: 603]	NO: 1688]	GACCTGCTAAGC	NO: 40]	NO: 1883]	UAUGGACCUGC
						TATGTACCACAA	(Same guide as		UAAGCUAUGUA
						CAAAGTCTGAA	XD-14756)		CCACAACAAAG
						CCTCAGGCCGG			UCUGAACCUCA
						GACCTCTTCCGC			GGCCGGGACCU
						CGCACTGAGGG			CUUCCGCCGCA
						GCACTCCACACC			CUGAGGGGCAC
						ACGGGGGCC			UCCACACCACG
						[SEQ ID NO: 1781]			GGGGCC
									[SEQ ID NO: 1978]
3269	miR-	-2.3297	0.037293	ACTGTAGG	CACAATAT	GAGCTCAGTCA	ACUGUAGG	CACAAUAU	GAGCUCAGUCA
	190a_M			CAACATATT	GTTGCCTAC	AACCTGGATGCC	CAACAUAU	GUUGCCUA	AACCUGGAUGC
				GCGTG	AAG	TTTTCTGCAGGC	UGCGUG	CAAG	CUUUUCUGCAG
				SEQ ID	[SEQ ID	GTCTGTGACTGT	[SEQ ID	[SEQ ID	GCGUCUGUGAC
				NO: 1629]	NO: 1689]	AGGCAACATATT	NO: 1822]	NO: 1884]	UGUAGGCAACA
						GCGTGTGTTATT			UAUUGCGUGUG
						TAATCCACACAA			UUAUUUAAUCC
						TATGTTGCCTAC			ACACAAUAUGU
						AAGCTACAGTCT			UGCCUACAAGC
						CTTGCCCTGTCT			UACAGUCUCUU
						CCGGGGGTTCCT			GCCCUGUCUCC
						AATAAAG			GGGGGUUCCUA
						[SEQ ID NO: 1782]			AUAAAG
									[SEQ ID NO: 1979]
3043	miR-1-1	-2.32683	0.097086	TTTGGTGCA	ACCCTGTTT	CATGCAGACTGC	UUUGGUGC	ACCCUGUU	CAUGCAGACUG
				AAACAAAC	GTTTTGCAC	CTGCTTGGGACC	AAAACAAA	UGUUUUGC	CCUGCUUGGGA
				AGGCT	GAAAA	CTGTTTGTTTTG	CAGGCU	ACGAAAA	CCCUGUUUGUU
				[SEQ ID	[SEQ ID	CACGAAAATAT	[SEQ ID	[SEQ ID	UUGCACGAAAA
				NO: 1615]	NO: 1690]	GGACCTGCTAA	NO: 1812]	NO: 1885]	UAUGGACCUGC
						GCTATTTGGTGC			UAAGCUAUUUG
						AAAACAAACAG			GUGCAAAACAA
						GCTCTCAGGCCG			ACAGGCUCUCA
						GGACCTCTCTCG			GGCCGGGACCU
						CCGCACTGAGG			CUCUCGCCGCA
						GGCACTCCACAC			CUGAGGGGCAC
						CACGGGGGCC			UCCACACCACG
						[SEQ ID NO: 1783]			GGGGCC
									[SEQ ID NO: 1980]
3273	miR-1-1	-2.32464	-0.13929	TAGGACTGT	CTATATGTT	CATGCAGACTGC	UAGGACUG	CUAUAUGU	CAUGCAGACUG
				AGGCAACA	GCCTACAGT	CTGCTTGGGCTA	UAGGCAAC	UGCCUACA	CCUGCUUGGGC
				TATTG	GACTA	TATGTTGCCTAC	AUAUUG	GUGACUA	UAUAUGUUGCC
				[SEQ ID	[SEQ ID	AGTGACTATATG	[SEQ ID	[SEQ ID	UACAGUGACUA
				NO: 1628]	NO: 1691]	GACCTGCTAAGC	NO: 1821]	NO: 1886]	UAUGGACCUGC
						TATAGGACTGTA			UAAGCUAUAGG
						GGCAACATATTG			ACUGUAGGCAA
						CTCAGGCCGGG			CAUAUUGCUCA
						ACCTCTCTCGCC			GGCCGGGACCU
						GCACTGAGGGG			CUCUCGCCGCA
						CACTCCACACCA			CUGAGGGGCAC
						CGGGGGCC			UCCACACCACG
						[SEQ ID NO: 1784]			GGGGCC
									[SEQ ID NO: 1981]
2586	miR-100	-2.32164	-0.18294	TAGATTCAG	CCAAGTCCT	CCCAAAAGAGA	UAGAUUCA	CCAAGUCC	CCCAAAAGAGA
				AAGTAGAA	AATTCTGAC	GAAGATATTGA	GAAGUAGA	UAAUUCUG	GAAGAUAUUGA
				CTTGG	TCTT	GGCCTGTTGCCA	ACUUGG	ACUCUU	GGCCUGUUGCC
				[SEQ ID	[SEQ ID	CATAGATTCAGA	[SEQ ID	[SEQ ID	ACAUAGAUUCA
				NO: 1621]	NO: 1692]	AGTAGAACTTG	NO: 1816]	NO: 1887]	GAAGUAGAACU
						GGTATTAGTCCG			UGGGUAUUAGU
						CCAAGTCCTAAT			CCGCCAAGUCC
						TCTGACTCTTTG			UAAUUCUGACU
						TGTCTGTTAGGC			CUUUGUGUCUG
						AATCTCACGGAC			UUAGGCAAUCU
						CTGGGGCTTTGC			CACGGACCUGG
						TTATATGCC			GGCUUUGCUUA
						[SEQ ID NO: 1785]			UAUGCC
									[SEQ ID NO: 1982]
2585	miR-1-1_M	-2.31178	0.12336	AGATTCAG	GGCAAGTT	CATGCAGACTGC	AGAUUCAG	GGCAAGUU	CAUGCAGACUG
				AAGTAGAA	CTACTTCTG	CTGCTTGGGGGC	AAGUAGAA	CUACUUCU	CCUGCUUGGGG
				CTTGGC	ACCTCT	AAGTTCTACTTC	CUUGGC	GACCUCU	GCAAGUUCUAC
				[SEQ ID	[SEQ ID	TGACCTCTTATG	[SEQ ID	[SEQ ID	UUCUGACCUCU
				NO: 1630]	NO: 1693]	GACCTGCTAAGC	NO: 1823]	NO: 1888]	UAUGGACCUGC
						TAAGATTCAGA			UAAGCUAAGAU
						AGTAGAACTTG			UCAGAAGUAGA
						GCCTCAGGCCG			ACUUGGCCUCA
						GGACCTCTTCCG			GGCCGGGACCU
						CCGCACTGAGG			CUUCCGCCGCA
						GGCACTCCACAC			CUGAGGGGCAC
						CACGGGGGCC			UCCACACCACG
						[SEQ ID NO: 1786]			GGGGCC
									[SEQ ID NO: 1983]
3273	miR-122	-2.30907	-0.11515	TAGGACTGT	CAATATGTT	GGCTACAGAGTT	UAGGACUG	CAAUAUGU	GGCUACAGAGU
				AGGCAACA	GCATACAG	TCCTTAGCAGAG	UAGGCAAC	UGCAUACA	UUCCUUAGCAG
				TATTG	TCAGA	CTGTAGGACTGT	AUAUUG	GUCAGA	AGCUGUAGGAC
				[SEQ ID	[SEQ ID	AGGCAACATATT	[SEQ ID	[SEQ ID	UGUAGGCAACA
				NO: 1628]	NO: 1694]	GTGTCTAAACTA	NO: 1821]	NO: 1889]	UAUUGUGUCUA
						TCAATATGTTGC			AACUAUCAAUA
						ATACAGTCAGAT			UGUUGCAUACA
						AGCTACTGCTAG			GUCAGAUAGCU
						GCAATCCTTCCC			ACUGCUAGGCA
						TCGATAAATGTC			AUCCUUCCCUC
						TTGGCATCGTTT			GAUAAAUGUCU
						GCTTTG			UGGCAUCGUUU
						[SEQ ID NO: 1787]			GCUUUG
									[SEQ ID NO: 1984]
3301	miR-100_M	-2.30882	-0.2279	TGAACAAG	TCCCAACTC	CCCAAAAGAGA	UGAACAAG	UCCCAACU	CCCAAAAGAGA
				GGGCTGATT	ATCCCCTTA	GAAGATATTGAT	GGGCUGAU	CAUCCCCU	GAAGAUAUUGA
				TGGGA	TTCT	GCCTGTTGCCAC	UUGGGA	UAUUCU	UGCCUGUUGCC
				[SEQ ID	[SEQ ID	ATGAACAAGGG	[SEQ ID	[SEQ ID	ACAUGAACAAG
				NO: 687]	NO: 1695]	GCTGATTTGGGA	NO: 1215]	NO: 1890]	GGGCUGAUUUG
						GTATTAGTCCGT			GGAGUAUUAGU
						CCCAACTCATCC			CCGUCCCAACU
						CCTTATTCTTGT			CAUCCCCUUAU
						GTCTGTTAGGCT			UCUUGUGUCUG
						ATTCCACGGACC			UUAGGCUAUUC
						TGGGGCTTTGCT			CACGGACCUGG
						TATATGCC			GGCUUUGCUUA
						[SEQ ID NO: 1788]			UAUGCC
									[SEQ ID NO: 1985]
2602	miR-130a	-2.30701	-0.14983	TTTAGTAGT	ATTCTATGG	GCAGGGCCGGC	UUUAGUAG	AUUCUAUG	GCAGGGCCGGC
				TGATCCATA	AGCAACTA	ATGCCTCTGCTG	UUGAUCCA	GAGCAACU	AUGCCUCUGCU
				GATT	TTAAC	CTGGCCAATTCT	UAGAUU	AUUAAC	GCUGGCCAAUU
				[SEQ ID	[SEQ ID	ATGGAGCAACT	[SEQ ID	[SEQ ID	CUAUGGAGCAA
				NO: 1616]	NO: 1696]	ATTAACCTGTCT	NO: 202]	NO: 1891]	CUAUUAACCUG
						GCACCTGTCACT	(Same guide as		UCUGCACCUGU
						AGTTTAGTAGTT	XD-14837)		CACUAGUUUAG
						GATCCATAGATT			UAGUUGAUCCA
						TGGCCGTGTAGT			UAGAUUUGGCC
						GCTACCCAGCGC			GUGUAGUGCUA
						TGGCTGCCTCCT			CCCAGCGCUGG
						CAGCATTG			CUGCCUCCUCA
						[SEQ ID NO: 1789]			GCAUUG
									SEQ ID NO: 1986]
3255	miR-	-2.30348	0.418205	ATTGCGTGG	CCAAGCTTA	GAGCTCAGTCA	AUUGCGUG	CCAAGCUU	GAGCUCAGUCA
	190a_M			AGTAAGCT	CTCCACGCA	AACCTGGATGCC	GAGUAAGC	ACUCCACG	AACCUGGAUGC
				GGTGG	CG	TTTTCTGCAGGC	UGGUGG	CACG	CUUUUCUGCAG
				[SEQ ID	[SEQ ID	GTCTGTGATTGC	[SEQ ID	[SEQ ID	GCGUCUGUGAU
				NO: 617]	NO: 1697]	GTGGAGTAAGC	NO: 306]	NO: 1892]	UGCGUGGAGUA
						TGGTGGTGTTAT	(Same guide as		AGCUGGUGGUG
						TTAATCCACCAA	XD-14889)		UUAUUUAAUCC
						GCTTACTCCACG			ACCAAGCUUAC
						CACGCTACAGTC			UCCACGCACGC
						TCTTGCCCTGTC			UACAGUCUCUU
						TCCGGGGGTTCC			GCCCUGUCUCC
						TAATAAAG			GGGGGUUCCUA
						[SEQ ID NO: 1790]			AUAAAG
									[SEQ ID NO: 1987]
3338	miR-132	-2.2984	-0.03531	TACATGAG	TAAGTCTCA	GCCGTCCGCGCG	UACAUGAG	UAAGUCUC	GCCGUCCGCGC
				GATGCTGA	GCCTACTCA	CCCCGCCCCCGC	GAUGCUGA	AGCCUACU	GCCCCGCCCCC
				GACTGA	TGTA	GTCTCCAGGGTA	GACUGA	CAUGUA	GCGUCUCCAGG
				[SEQ ID	[SEQ ID	AGTCTCAGCCTA	[SEQ ID	[SEQ ID	GUAAGUCUCAG
				NO: 1620]	NO: 1698]	CTCATGTACTGT	NO: 314]	NO: 1893]	CCUACUCAUGU
						GGGAACTGGAG	(Same guide as		ACUGUGGGAAC
						GTACATGAGGA	XD-14893)		UGGAGGUACAU
						TGCTGAGACTGA			GAGGAUGCUGA
						CCCCGCAGCAC			GACUGACCCCG
						GCCCACGCGCC			CAGCACGCCCA
						GCGCCACGCCG			CGCGCCGCGCC
						CGCCCCGAGCC			ACGCCGCGCCC
						[SEQ ID NO: 1791]			CGAGCC
									[SEQ ID NO: 1988]
3842	miR-1-1	-2.27963	-0.68707	AACGTGAG	TTCGATCCA	CATGCAGACTGC	AACGUGAG	UUCGAUCC	CAUGCAGACUG
				AAGGATGG	TCCTTCTCA	CTGCTTGGGTTC	AAGGAUGG	AUCCUUCU	CCUGCUUGGGU
				ATCGTA	GAGTT	GATCCATCCTTC	AUCGUA	CAGAGUU	UCGAUCCAUCC
				[SEQ ID	[SEQ ID	TCAGAGTTTATG	[SEQ ID	[SEQ ID	UUCUCAGAGUU
				NO: 1625]	NO: 1699]	GACCTGCTAAGC	NO: 1824]	NO: 1894]	UAUGGACCUGC
						TAAACGTGAGA			UAAGCUAAACG
						AGGATGGATCG			UGAGAAGGAUG
						TACTCAGGCCGG			GAUCGUACUCA
						GACCTCTCTCGC			GGCCGGGACCU
						CGCACTGAGGG			CUCUCGCCGCA
						GCACTCCACACC			CUGAGGGGCAC
						ACGGGGGCC			UCCACACCACG
						[SEQ ID NO: 1792]			GGGGCC
									[SEQ ID NO: 1989]
3330	miR-155E	-2.26554	0.033188	TATGCTGAG	CCACATTAC	CTGGAGGCTTGC	UAUGCUGA	CCACAUUA	CUGGAGGCUUG
				ACTGATAAT	AGCTCAGC	TTTGGGCTGTAT	GACUGAUA	CAGCUCAG	CUUUGGGCUGU
				GTGG	ATA	GCTGTATGCTGA	AUGUGG	CAUA	AUGCUGUAUGC
				SEQ ID	[SEQ ID	GACTGATAATGT	[SEQ ID	SEQ ID	UGAGACUGAUA
				NO: 1614]	NO: 1700]	GGTTTTGGCCTC	NO: 1811]	NO: 1895]	AUGUGGUUUUG
						TGACTGACCACA			GCCUCUGACUG
						TTACAGCTCAGC			ACCACAUUACA
						ATACAGGACAA			GCUCAGCAUAC
						GGCCCTTTATCA			AGGACAAGGCC
						GCACTCACATGG			CUUUAUCAGCA
						AACAAATGGCC			CUCACAUGGAA
						ACCGTGGG			CAAAUGGCCAC
						[SEQ ID NO: 1793]			CGUGGG
									[SEQ ID NO: 1990]
1162	miR-122	-2.2647	-0.33024	AACTGTACC	CAGACTTTG	GGCTACAGAGTT	AACUGUAC	CAGACUUU	GGCUACAGAGU
				ACAACAAA	TTATGGTAC	TCCTTAGCAGAG	CACAACAA	GUUAUGGU	UUCCUUAGCAG
				GTCTG	ATGT	CTGAACTGTACC	AGUCUG	ACAUGU	AGCUGAACUGU
				[SEQ ID	[SEQ ID	ACAACAAAGTC	[SEQ ID	[SEQ ID	ACCACAACAAA
				NO: 652]	NO: 1701]	TGTGTCTAAACT	NO: 1180]	NO: 1896]	GUCUGUGUCUA
						ATCAGACTTTGT			AACUAUCAGAC
						TATGGTACATGT			UUUGUUAUGGU
						TAGCTACTGCTA			ACAUGUUAGCU
						GGCAATCCTTCC			ACUGCUAGGCA
						CTCGATAAATGT			AUCCUUCCCUC
						CTTGGCATCGTT			GAUAAAUGUCU
						TGCTTTG			UGGCAUCGUUU
						[SEQ ID NO: 1794]			GCUUUG
									[SEQ ID NO: 1991]
3301	miR-122_M	-2.26423	0.053866	TGAACAAG	TCCCAAATC	GGCTACAGAGTT	UGAACAAG	UCCCAAAU	GGCUACAGAGU
				GGGCTGATT	AGACCCTTG	TGCTTAGCAGAG	GGGCUGAU	CAGACCCU	UUGCUUAGCAG
				TGGGA	TGAA	CTGTGAACAAG	UUGGGA	UGUGAA	AGCUGUGAACA
				[SEQ ID	[SEQ ID	GGGCTGATTTGG	[SEQ ID	SEQ ID	AGGGGCUGAUU
				NO: 687]	NO: 1702]	GATGTCTAAACT	NO: 1215]	NO: 1897]	UGGGAUGUCUA
						ATTCCCAAATCA			AACUAUUCCCA
						GACCCTTGTGAA			AAUCAGACCCU
						TAGCTACTGCTA			UGUGAAUAGCU
						GGCCATCCTTCC			ACUGCUAGGCC
						CTCGATAAATGT			AUCCUUCCCUC
						CTTGGCATCGTT			GAUAAAUGUCU
						TGCTTTG			UGGCAUCGUUU
						[SEQ ID NO: 1795]			GCUUUG
									[SEQ ID NO: 1992]
3341	miR-	-2.26383	0.394316	ATAGACAT	GTCAGCATC	GAGCTCAGTCA	AUAGACAU	GUCAGCAU	GAGCUCAGUCA
	190a_M			GAGGATGC	CTCATGTCT	AACCTGGATGCC	GAGGAUGC	CCUCAUGU	AACCUGGAUGC
				TGAGAC	CG	TTTTCTGCAGGC	UGAGAC	CUCG	CUUUUCUGCAG
				[SEQ ID	[SEQ ID	GTCTGTGATAGA	[SEQ ID	[SEQ ID	GCGUCUGUGAU
				NO: 1617]	NO: 1703]	CATGAGGATGCT	NO: 1813]	NO: 1898]	AGACAUGAGGA
						GAGACTGTTATT			UGCUGAGACUG
						TAATCCAGTCAG			UUAUUUAAUCC
						CATCCTCATGTC			AGUCAGCAUCC
						TCGCTACAGTCT			UCAUGUCUCGC
						CTTGCCCTGTCT			UACAGUCUCUU
						CCGGGGGTTCCT			GCCCUGUCUCC
						AATAAAG			GGGGGUUCCUA
						[SEQ ID NO: 1796]			AUAAAG
									[SEQ ID NO: 1993]
2945	miR-100_M	-2.26187	0.044364	TGTAGTAG	TCAGCCCA	CCCAAAAGAGA	UGUAGUAG	UCAGCCCA	CCCAAAAGAGA
				AAGGCTTTG	AGACTTCTA	GAAGATATTGAT	AAGGCUUU	AGACUUCU	GAAGAUAUUGA
				GCTGA	ATACT	GCCTGTTGCCAC	GGCUGA	AAUACU	UGCCUGUUGCC
				[SEQ ID	[SEQ ID	ATGTAGTAGAA	[SEQ ID	[SEQ ID	ACAUGUAGUAG
				NO: 685]	NO: 1678]	GGCTTTGGCTGA	NO: 1213]	NO: 1873]	AAGGCUUUGGC
						GTATTAGTCCGT	(Same guide as		UGAGUAUUAGU
						CAGCCCAAGAC	XD-14860)		CCGUCAGCCCA
						TTCTAATACTTG			AGACUUCUAAU
						TGTCTGTTAGGC			ACUUGUGUCUG
						TATTCCACGGAC			UUAGGCUAUUC
						CTGGGGCTTTGC			CACGGACCUGG
						TTATATGCC			GGCUUUGCUUA
						[SEQ ID NO: 1797]			UAUGCC
									[SEQ ID NO: 1994]
1756	miR-1-1_M	-2.26119	0.153277	TTCGGGTTG	AGACTTCA	CATGCAGACTGC	UUCGGGUU	AGACUUCA	CAUGCAGACUG
				AAATCTGA	GATTTCAAC	CTGCTTGGGAGA	GAAAUCUG	GAUUUCAA	CCUGCUUGGGA
				AGTGT	CGAGAA	CTTCAGATTTCA	AAGUGU	CCGAGAA	GACUUCAGAUU
				[SEQ ID	[SEQ ID	ACCGAGAATAT	[SEQ ID	[SEQ ID	UCAACCGAGAA
				NO: 607]	NO: 1704]	GGACCTGCTAA	NO: 108]	NO: 1907]	UAUGGACCUGC
						GCTATTCGGGTT	(Same guide as		UAAGCUAUUCG
						GAAATCTGAAG	XD-14790)		GGUUGAAAUCU
						TGTCTCAGGCCG			GAAGUGUCUCA
						GGACCTCTTCCG			GGCCGGGACCU
						CCGCACTGAGG			CUUCCGCCGCA
						GGCACTCCACAC			CUGAGGGGCAC
						CACGGGGGCC			UCCACACCACG
						[SEQ ID NO: 1798]			GGGGCC
									[SEQ ID NO: 1995]
3845	miR-1-1_M	-2.26073	-0.20695	TTGAACGTG	GTTCCATCC	CATGCAGACTGC	UUGAACGU	GUUCCAUC	CAUGCAGACUG
				AGAAGGAT	TTCTCACGT	CTGCTTGGGGTT	GAGAAGGA	CUUCUCAC	CCUGCUUGGGG
				GGATC	CGCAA	CCATCCTTCTCA	UGGAUC	GUCGCAA	UUCCAUCCUUC
				[SEQ ID	[SEQ ID	CGTCGCAATATG	[SEQ ID	[SEQ ID	UCACGUCGCAA
				NO: 696]	NO: 1705]	GACCTGCTAAGC	NO: 1224]	NO: 1906]	UAUGGACCUGC
						TATTGAACGTGA			UAAGCUAUUGA
						GAAGGATGGAT			ACGUGAGAAGG
						CCTCAGGCCGG			AUGGAUCCUCA
						GACCTCTTCCGC			GGCCGGGACCU
						CGCACTGAGGG			CUUCCGCCGCA
						GCACTCCACACC			CUGAGGGGCAC
						ACGGGGGCC			UCCACACCACG
						[SEQ ID NO: 1799]			GGGGCC
									[SEQ ID NO: 1996]
3043	miR-190a	-2.26041	0.277711	TTTGGTGCA	AGCGTTTGT	GAGCTCAGTCA	UUUGGUGC	AGCGUUUG	GAGCUCAGUCA
				AAACAAAC	TTTGCACCA	AACCTGGATGCC	AAAACAAA	UUUUGCAC	AACCUGGAUGC
				AGGCT	CC	TTTTCTGCAGGC	CAGGCU	CACC	CUUUUCUGCAG
				[SEQ ID	[SEQ ID	CTCTGTGTTTGG	[SEQ ID	[SEQ ID	GCCUCUGUGUU
				NO: 1615]	NO: 1664]	TGCAAAACAAA	NO: 1812]	NO: 1859]	UGGUGCAAAAC
						CAGGCTTGTTAT			AAACAGGCUUG
						TTAATCCAAGCG			UUAUUUAAUCC
						TTTGTTTTGCAC			AAGCGUUUGUU
						CACCCTACAGTG			UUGCACCACCC
						TCTTGCCCTGTC			UACAGUGUCUU
						TCCGGGGGTTCC			GCCCUGUCUCC
						TAATAAAG			GGGGGUUCCUA
						[SEQ ID NO: 1800]			AUAAAG
									[SEQ ID NO: 1997]
1162	miR-155E	-2.26036	-0.20831	AACTGTACC	CAGACTTTT	CTGGAGGCTTGC	AACUGUAC	CAGACUUU	CUGGAGGCUUG
				ACAACAAA	TGGGTACA	TTTGGGCTGTAT	CACAACAA	UUGGGUAC	CUUUGGGCUGU
				GTCTG	GTT	GCTGAACTGTAC	AGUCUG	AGUU	AUGCUGAACUG
				[SEQ ID	[SEQ ID	CACAACAAAGT	[SEQ ID	[SEQ ID	UACCACAACAA
				NO: 652]	NO: 1706]	CTGTTTTGGCCT	NO: 1180]	NO: 1905]	AGUCUGUUUUG
						CTGACTGACAG			GCCUCUGACUG
						ACTTTTTGGGTA			ACAGACUUUUU
						CAGTTCAGGAC			GGGUACAGUUC
						AAGGCCCTTTAT			AGGACAAGGCC
						CAGCACTCACAT			CUUUAUCAGCA
						GGAACAAATGG			CUCACAUGGAA
						CCACCGTGGG			CAAAUGGCCAC
						[SEQ ID NO: 1801]			CGUGGG
									[SEQ ID NO: 1998]
3273	miR-1-1_M	-2.24883	0.09481	TAGGACTGT	CTATATGTT	CATGCAGACTGC	UAGGACUG	CUAUAUGU	CAUGCAGACUG
				AGGCAACA	GCCTACAGT	CTGCTTGGGCTA	UAGGCAAC	UGCCUACA	CCUGCUUGGGC
				TATTG	GACTA	TATGTTGCCTAC	AUAUUG	GUGACUA	UAUAUGUUGCC
				[SEQ ID	[SEQ ID	AGTGACTATATG	[SEQ ID	[SEQ ID	UACAGUGACUA
				NO: 1628]	NO: 1691]	GACCTGCTAAGC	NO: 1821]	NO: 1886]	UAUGGACCUGC
						TATAGGACTGTA			UAAGCUAUAGG
						GGCAACATATTG			ACUGUAGGCAA
						CTCAGGCCGGG			CAUAUUGCUCA
						ACCTCTTCCGCC			GGCCGGGACCU
						GCACTGAGGGG			CUUCCGCCGCA
						CACTCCACACCA			CUGAGGGGCAC
						CGGGGGCC			UCCACACCACG
						[SEQ ID NO: 1802]			GGGGCC
									[SEQ ID NO: 1999]
2586	miR-190a	-2.24846	0.172174	TAGATTCAG	CCATTCTAC	GAGCTCAGTCA	UAGAUUCA	CCAUUCUA	GAGCUCAGUCA
				AAGTAGAA	TTCTGAATC	AACCTGGATGCC	GAAGUAGA	CUUCUGAA	AACCUGGAUGC
				CTTGG	CC	TTTTCTGCAGGC	ACUUGG	UCCC	CUUUUCUGCAG
				[SEQ ID	[SEQ ID	CTCTGTGTAGAT	SEQ ID	[SEQ ID	GCCUCUGUGUA
				NO: 1621]	NO: 1661]	TCAGAAGTAGA	NO: 1816]	NO: 1856]	GAUUCAGAAGU
						ACTTGGTGTTAT			AGAACUUGGUG
						TTAATCCACCAT			UUAUUUAAUCC
						TCTACTTCTGAA			ACCAUUCUACU
						TCCCCTACAGTG			UCUGAAUCCCC
						TCTTGCCCTGTC			UACAGUGUCUU
						TCCGGGGGTTCC			GCCCUGUCUCC
						TAATAAAG
						[SEQ ID NO: 1803]			GGGGGUUCCUA
									AUAAAG
									[SEQ ID NO: 2000]
3132	miR-122 M	-2.24518	0.049515	ATGTCTTGG	CCAGTGAA	GGCTACAGAGTT	AUGUCUUG	CCAGUGAA	GGCUACAGAGU
				CTTGATTCA	TCACGCCA	TGCTTAGCAGAG	GCUUGAUU	UCACGCCA	UUGCUUAGCAG
				CTGG	AGAACT	CTGATGTCTTGG	CACUGG	AGAACU	AGCUGAUGUCU
				[SEQ ID	[SEQ ID	CTTGATTCACTG	[SEQ ID	[SEQ ID	UGGCUUGAUUC
				NO: 1631]	NO: 1707]	GTGTCTAAACTA	NO: 1826]	NO: 1904]	ACUGGUGUCUA
						TCCAGTGAATCA			AACUAUCCAGU
						CGCCAAGAACTT			GAAUCACGCCA
						AGCTACTGCTAG			AGAACUUAGCU
						GCCATCCTTCCC			ACUGCUAGGCC
						TCGATAAATGTC			AUCCUUCCCUC
						TTGGCATCGTTT			GAUAAAUGUCU
						GCTTTG			UGGCAUCGUUU
						[SEQ ID NO: 1804]			GCUUUG
									[SEQ ID NO: 2001]
3842	miR-1-1_M	-2.24315	-0.38269	AACGTGAG	TTCGATCCA	CATGCAGACTGC	AACGUGAG	UUCGAUCC	CAUGCAGACUG
				AAGGATGG	TCCTTCTCA	CTGCTTGGGTTC	AAGGAUGG	AUCCUUCU	CCUGCUUGGGU
				ATCGTA	GAGTT	GATCCATCCTTC	AUCGUA	CAGAGUU	UCGAUCCAUCC
				[SEQ ID	[SEQ ID	TCAGAGTTTATG	[SEQ ID	[SEQ ID	UUCUCAGAGUU
				NO: 1625]	NO: 1699]	GACCTGCTAAGC	NO: 1824]	NO: 1894]	UAUGGACCUGC
						TAAACGTGAGA			UAAGCUAAACG
						AGGATGGATCG			UGAGAAGGAUG
						TACTCAGGCCGG			GAUCGUACUCA
						GACCTCTTCCGC			GGCCGGGACCU
						CGCACTGAGGG			CUUCCGCCGCA
						GCACTCCACACC			CUGAGGGGCAC
						ACGGGGGCC			UCCACACCACG
						[SEQ ID NO: 1805]			GGGGCC
									[SEQ ID NO: 2002]
2928	miR-1-1	-2.24149	0.19907	TCTGAGAG	ATCCCACGT	CATGCAGACTGC	UCUGAGAG	AUCCCACG	CAUGCAGACUG
				AAGGAACG	TCCTTCTCT	CTGCTTGGGATC	AAGGAACG	UUCCUUCU	CCUGCUUGGGA
				TGGGTT	GAAGA	CCACGTTCCTTC	UGGGUU	CUGAAGA	UCCCACGUUCC
				[SEQ ID	[SEQ ID	TCTGAAGATATG	[SEQ ID	[SEQ ID	UUCUCUGAAGA
				NO: 668]	NO: 1708]	GACCTGCTAAGC	NO: 1196]	NO: 1902]	UAUGGACCUGC
						TATCTGAGAGA			UAAGCUAUCUG
						AGGAACGTGGG			AGAGAAGGAAC
						TTCTCAGGCCGG			GUGGGUUCUCA
						GACCTCTCTCGC			GGCCGGGACCU
						CGCACTGAGGG			CUCUCGCCGCA
						GCACTCCACACC			CUGAGGGGCAC
						ACGGGGGCC			UCCACACCACG
						[SEQ ID NO: 1806]			GGGGCC
									[SEQ ID NO: 2003]
1231	miR-1-1_M	-2.24102	0.10817	TTCACTTTA	CAGCTATCA	CATGCAGACTGC	UUCACUUU	CAGCUAUC	CAUGCAGACUG
				GCACTGAT	GTGCTAAA	CTGCTTGGGCAG	AGCACUGA	AGUGCUAA	CCUGCUUGGGC
				AGCAG	GCGGAA	CTATCAGTGCTA	UAGCAG	AGCGGAA	AGCUAUCAGUG
				[SEQ ID	[SEQ ID	AAGCGGAATAT	[SEQ ID	[SEQ ID	CUAAAGCGGAA
				NO: 1627]	NO: 1709]	GGACCTGCTAA	NO: 1825]	NO: 1903]	UAUGGACCUGC
						GCTATTCACTTT			UAAGCUAUUCA
						AGCACTGATAG			CUUUAGCACUG
						CAGCTCAGGCC			AUAGCAGCUCA
						GGGACCTCTTCC			GGCCGGGACCU
						GCCGCACTGAG			CUUCCGCCGCA
						GGGCACTCCAC			CUGAGGGGCAC
						ACCACGGGGGC			UCCACACCACG
						C			GGGGCC
						[SEQ ID NO: 1807]			[SEQ ID NO: 2004]
1578	miR-100 M	-2.24051	-0.33444	TGGAATTTC	ACAGCAAT	CCCAAAAGAGA	UGGAAUUU	ACAGCAAU	CCCAAAAGAGA
				TCTGAACTG	TCCGAGAA	GAAGATATTGAT	CUCUGAAC	UCCGAGAA	GAAGAUAUUGA
				CTGT	ACTCCT	GCCTGTTGCCAC	UGCUGU	ACUCCU	UGCCUGUUGCC
				[SEQ ID	[SEQ ID	ATGGAATTTCTC	[SEQ ID	[SEQ ID	ACAUGGAAUUU
				NO: 1626]	NO: 1665]	TGAACTGCTGTG	NO: 1820]	NO: 1860]	CUCUGAACUGC
						TATTAGTCCGAC			UGUGUAUUAGU
						AGCAATTCCGA			CCGACAGCAAU
						GAAACTCCTTGT			UCCGAGAAACU
						GTCTGTTAGGCT			CCUUGUGUCUG
						ATTCCACGGACC			UUAGGCUAUUC
						TGGGGCTTTGCT			CACGGACCUGG
						TATATGCC			GGCUUUGCUUA
						[SEQ ID NO: 1808]			UAUGCC
									[SEQ ID NO: 2005]
967	miR-190a	-2.23671	-0.08895	ACTGATGTA	TGGCATATA	GAGCTCAGTCA	ACUGAUGU	UGGCAUAU	GAGCUCAGUCA
				AGTATATG	CTTACATCA	AACCTGGATGCC	AAGUAUAU	ACUUACAU	AACCUGGAUGC
				AACCA	AG	TTTTCTGCAGGC	GAACCA	CAAG	CUUUUCUGCAG
				[SEQ ID	[SEQ ID	CTCTGTGACTGA	[SEQ ID	[SEQ ID	GCCUCUGUGAC
				NO: 1619]	NO: 1642]	TGTAAGTATATG	NO: 1815]	NO: 1837]	UGAUGUAAGUA
						AACCATGTTATT			UAUGAACCAUG
						TAATCCATGGCA			UUAUUUAAUCC
						TATACTTACATC			AUGGCAUAUAC
						AAGCTACAGTGT			UUACAUCAAGC
						CTTGCCCTGTCT			UACAGUGUCUU
						CCGGGGGTTCCT			GCCCUGUCUCC
						AATAAAG			GGGGGUUCCUA
						[SEQ ID NO: 1809]			AUAAAG
									[SEQ ID NO: 2006]
1436	miR155-M	-2.22884	0.141256	TGAGTTATC	GCCCTTAGA	CCTGGAGGCTTG	UGAGUUAU	GCCCUUAG	CCUGGAGGCUU
				TCTTTCTAA	AGGATAAC	CTGAAGGCTGTA	CUCUUUCU	AAGGAUAA	GCUGAAGGCUG
				GGGC	TCA	TGCTGTGAGTTA	AAGGGC	CUCA	UAUGCUGUGAG
				[SEQ ID	[SEQ ID	TCTCTTTCTAAG	[SEQ ID	[SEQ ID	UUAUCUCUUUC
				NO: 1632]	NO: 1710]	GGCTTTTGGCCA	NO: 1827]	NO: 1900]	UAAGGGCUUUU
						CTGACTGAGCCC			GGCCACUGACU
						TTAGAAGGATA			GAGCCCUUAGA
						ACTCACAGGAC			AGGAUAACUCA
						ACAAGGCCTGTT			CAGGACACAAG
						ACTAGCACTCAC			GCCUGUUACUA
						ATGGAACAAAT			GCACUCACAUG
						GGCCACCGG			GAACAAAUGGC
						[SEQ ID NO: 1810]			CACCGG
									[SEQ ID NO: 2007]

TABLE 24
Top 10 miRNAs for each miR backbone
	Atxn2
	Tar-	Atxn2
	get-	low/		Guide
miR	ing	unsort	T1/T0	Se-	Passenger	MiR	Guide	Passenger	MiR
Back-	Posi-	log2	log2	quence	Sequence	Cassette	Sequence	Sequence	Cassette
bone	tion	FC	FC	(DNA)	(DNA)	(DNA)	(RNA)	(RNA)	(RNA)
miR-	1755	−2.5734	−0.10352	TCGGGTTGAA	CTCACTTCAG	CATGCAGACT	UCGGGUUGAA	CUCACUUCAG	CAUGCAGACU
1-1				ATCTGAAGTG	ATTTCAACGA	GCCTGCTTGG	AUCUGAAGUG	AUUUCAACGA	GCCUGCUUGG
				TG	CGA	GCTCACTTCA	UG	CGA	GCUCACUUCA
				[SEQ ID	[SEQ ID	GATTTCAACG	[SEQ ID	[SEQ ID	GAUUUCAACG
				NO:	NO:	ACGATATGGA	NO:	NO:	ACGAUAUGGA
				657]	1645]	CCTGCTAAGC	1185]	1840]	CCUGCUAAGC
						TATCGGGTTG			UAUCGGGUUG
						AAATCTGAAG			AAAUCUGAAG
						TGTGCTCAGG			UGUGCUCAGG
						CCGGGACCTC			CCGGGACCUC
						TCTCGCCGCA			UCUCGCCGCA
						CTGAGGGGCA			CUGAGGGGCA
						CTCCACACCA			CUCCACACCA
						CGGGGGCC			CGGGGGCC
						[SEQ ID			[SEQ ID
						NO:			NO:
						1726]			1923]
miR-	2943	−2.54936	0.148183	TAGTAGAAGG	TGTCAGCCAA	CATGCAGACT	UAGUAGAAGG	UGUCAGCCAA	CAUGCAGACU
1-1				CTTTGGCTGA	AGCCTTCTCC	GCCTGCTTGG	CUUUGGCUGA	AGCCUUCUCC	GCCUGCUUGG
				GA	CTA	GTGTCAGCCA	GA	CUA	GUGUCAGCCA
				[SEQ ID	[SEQ ID	AAGCCTTCTC	[SEQ ID	[SEQ ID	AAGCCUUCUC
				NO:	NO:	CCTATATGGA	NO:	NO:	CCUAUAUGGA
				683]	1650]	CCTGCTAAGC	1211]	1845]	CCUGCUAAGC
						TATAGTAGAA			UAUAGUAGAA
						GGCTTTGGCT			GGCUUUGGCU
						GAGACTCAGG			GAGACUCAGG
						CCGGGACCTC			CCGGGACCUC
						TCTCGCCGCA			UCUCGCCGCA
						CTGAGGGGCA			CUGAGGGGCA
						CTCCACACCA			CUCCACACCA
						CGGGGGCC			CGGGGGCC
						[SEQ ID			[SEQ ID
						NO:			NO:
						1731]			1928]
miR-	3338	−2.52716	−0.2585	TACATGAGGA	TGAGTCTCAG	CATGCAGACT	UACAUGAGGA	UGAGUCUCAG	CAUGCAGACU
1-1				TGCTGAGACT	CATCCTCACG	GCCTGCTTGG	UGCUGAGACU	CAUCCUCACG	GCCUGCUUGG
				GA	GTA	GTGAGTCTCA	GA	GUA	GUGAGUCUCA
				[SEQ ID	[SEQ ID	GCATCCTCAC	[SEQ ID	[SEQ ID	GCAUCCUCAC
				NO:	NO:	GGTATATGGA	NO:	NO:	GGUAUAUGGA
				1620]	1651]	CCTGCTAAGC	314]	1846]	CCUGCUAAGC
						TATACATGAG	(Same		UAUACAUGAG
						GATGCTGAGA	guide		GAUGCUGAGA
						CTGACTCAGG	as		CUGACUCAGG
						CCGGGAC	XD-		CCGGGACCUC
						CTCT	14893		U
						CTCGCC			CUCGCCGCAC
						GCACTGAGGG			UGAGGGGCAC
						GCACTCCACA			UCCACACCAC
						CCACGGGGGC			GGGGGCC
						C			[SEQ ID
						[SEQ ID			NO:
						NO:			1929]
						1732]
miR-	3302	−2.51939	−0.06675	TTGAACAAGG	CGCAAATCAG	CATGCAGACT	UUGAACAAGG	CGCAAAUCAG	CAUGCAGACU
1-1				GGCTGATTTG	CCCCTTGTCG	GCCTGCTTGG	GGCUGAUUUG	CCCCUUGUCG	GCCUGCUUGG
				GG	CAA	GCGCAAATCA	GG	CAA	GCGCAAAUCA
				[SEQ ID	[SEQ ID	GCCCCTTGTC	[SEQ ID	[SEQ ID	GCCCCUUGUC
				NO:	NO:	GCAATATGGA	NO:	NO:	GCAAUAUGGA
				688]	1647]	CCTGCTAAGC	1216]	1842]	CCUGCUAAGC
						TATTGAACAA			UAUUGAACAA
						GGGGCTGATT			GGGGCUGAUU
						TGGGCTCAGG			UGGGCUCAGG
						CCGGGACCTC			CCGGGACCUC
						TCTCGCCGCA			UCUCGCCGCA
						CTGAGGGGCA			CUGAGGGGCA
						CTCCACACCA			CUCCACACCA
						CGGGGGCC			CGGGGGCC
						[SEQ ID			[SEQ ID
						NO:			NO:
						1735]			1932]
miR-	3341	−2.50291	−0.1115	ATAGACATGA	GACTCAGCAT	CATGCAGACT	AUAGACAUGA	GACUCAGCAU	CAUGCAGACU
1-1				GGATGCTGAG	CCTCATGTGA	GCCTGCTTGG	GGAUGCUGAG	CCUCAUGUGA	GCCUGCUUGG
				AC	TAT	GGACTCAGCA	AC	UAU	GGACUCAGCA
				[SEQ ID	[SEQ ID	TCCTCATGTG	[SEQ ID	[SEQ ID	UCCUCAUGUG
				NO:	NO:	ATATTATGGA	NO:	NO:	AUAUUAUGGA
				1617]	1637]	CCTGCTAAGC	1813]	1832]	CCUGCUAAGC
						TAATAGACAT			UAAUAGACAU
						GAGGATGCTG			GAGGAUGCUG
						AGACCTCAGG			AGACCUCAGG
						CCGGGACCTC			CCGGGACCUC
						TCTCGCCGCA			UCUCGCCGCA
						CTGAGGGGCA			CUGAGGGGCA
						CTCCACACCA			CUCCACACCA
						CGGGGGCC			CGGGGGCC
						[SEQ ID			[SEQ ID
						NO:			NO:
						1737]			1934]
miR-	2945	−2.42094	−0.06694	TGTAGTAGAA	TGAGCCAAAG	CATGCAGACT	UGUAGUAGAA	UGAGCCAAAG	CAUGCAGACU
1-1				GGCTTTGGCT	CCTTCTACCG	GCCTGCTTGG	GGCUUUGGCU	CCUUCUACCG	GCCUGCUUGG
				GA	ACA	GTGAGCCAAA	GA	ACA	GUGAGCCAAA
				[SEQ ID	[SEQ ID	GCCTTCTACC	[SEQ ID	[SEQ ID	GCCUUCUACC
				NO:	NO:	GACATATGGA	NO:	NO:	GACAUAUGGA
				685]	1633]	CCTGCTAAGC	1213]	1828]	CCUGCUAAGC
						TATGTAGTAG	(Same		UAUGUAGUAG
						AAGGCTTTGG	guide		AAGGCUUUGG
						CTGACTCAGG	as		CUGACUCAGG
						CCGGGACCTC	XD-		CCGGGACCUC
						TCTCGCCGCA	14860)		UCUCGCCGCA
						C			CUGAG
						TGAGGGGCAC			GGGCACUCCA
						TCCACACCAC			CACCACGGGG
						GGGGGCC			GCC
						[SEQ ID			[SEQ ID
						NO:			NO:
						1761]			1958]
miR-	3301	−2.41082	0.051034	TGAACAAGGG	TGCCAAATCA	CATGCAGACT	UGAACAAGGG	UGCCAAAUCA	CAUGCAGACU
1-1				GCTGATTTGG	GCCCCTTGCG	GCCTGCTTGG	GCUGAUUUGG	GCCCCUUGCG	GCCUGCUUGG
				GA	TCA	GTGCCAAATC	GA	UCA	GUGCCAAAUC
				[SEQ ID	[SEQ ID	AGCCCCTTGC	[SEQ ID	[SEQ ID	AGCCCCUUGC
				NO:	NO:	GTCATATGGA	NO:	NO:	GUCAUAUGGA
				687]	1673]	CCTGCTAAGC	1215]	1868]	CCUGCUAAGC
						TATGAACAAG			UAUGAACAAG
						GGGCTGATTT			GGGCUGAUUU
						GGGACTCAGG			GGGACUCAGG
						CCGGGACCTC			CCGGGACCUC
						TCTCGCCGCA			UCUCGCCGCA
						CTGAGGGGCA			CUGAGGGGCA
						CTCCACACCA			CUCCACACCA
						CGGGGGCC			CGGGGGCC
						[SEQ ID			[SEQ ID
						NO:			NO:
						1763]			1960]
miR-	3043	−2.32683	0.097086	TTTGGTGCAA	ACCCTGTTTG	CATGCAGACT	UUUGGUGCAA	ACCCUGUUUG	CAUGCAGACU
1-1				AACAAACAGG	TTTTGCACGA	GCCTGCTTGG	AACAAACAGG	UUUUGCACGA	GCCUGCUUGG
				CT	AAA	GACCCTGTTT	CU	AAA	GACCCUGUUU
				[SEQ ID	[SEQ ID	GTTTTGCACG	[SEQ ID	[SEQ ID	GUUUUGCACG
				NO:	NO:	AAAATATGGA	NO:	NO:	AAAAUAUGGA
				1615]	1690]	CCTGCTAAGC	1812]	1885]	CCUGCUAAGC
						TATTTGGTGC			UAUUUGGUGC
						AAAACAAACA			AAAACAAACA
						GGCTCTCAGG			GGCUCUCAGG
						CCGGGACCTC			CCGGGACCUC
						TCTCGCCGCA			UCUCGCCGCA
						CTGAGGGGCA			CUGAGGGGCA
						CTCCACACCA			CUCCACACCA
						CGGGGGcc			CGGGGGCC
						[SEQ ID			[SEQ ID
						NO:			NO:
						1783]			1980]
miR-	3273	−2.32464	−0.13929	TAGGACTGTA	CTATATGTTG	CATGCAGACT	UAGGACUGUA	CUAUAUGUUG	CAUGCAGACU
1-1				GGCAACATAT	CCTACAGTGA	GCCTGCTTGG	GGCAACAUAU	CCUACAGUGA	GCCUGCUUGG
				TG	CTA	GCTATATGTT	UG	CUA	GCUAUAUGUU
				[SEQ ID	[SEQ ID	GCCTACAGTG	[SEQ ID	[SEQ ID	GCCUACAGUG
				NO:	NO:	ACTATATGGA	NO:	NO:	ACUAUAUGGA
				1628]	1691]	CCTGCTAAGC	1821]	1886]	CCUGCUAAGC
						TATAGGACTG			UAUAGGACUG
						TAGGCAACAT			UAGGCAACAU
						ATTGCTCAGG			AUUGCUCAGG
						CCGGGACCTC			CCGGGACCUC
						TCTCGCCGCA			UCUCGCCGCA
						CTGAGGGGCA			CUGA
						CTCCA
						CACCACGGGG			GGGGCACUCC
						GCC			ACACCACGGG
						[SEQ ID			GGCC
						NO:			[SEQ ID
						1784]			NO:
									1981]
miR-	3842	−2.27963	−0.68707	AACGTGAGAA	TTCGATCCAT	CATGCAGACT	AACGUGAGAA	UUCGAUCCAU	CAUGCAGACU
1-1				GGATGGATCG	CCTTCTCAGA	GCCTGCTTGG	GGAUGGAUCG	CCUUCUCAGA	GCCUGCUUGG
				TA	GTT	GTTCGATCCA	UA	GUU	GUUCGAUCCA
				[SEQ ID	[SEQ ID	TCCTTCTCAG	[SEQ ID	[SEQ ID	UCCUUCUCAG
				NO:	NO:	AGTTTATGGA	NO:	NO:	AGUUUAUGGA
				1625]	1699]	CCTGCTAAGC	1824]	1894]	CCUGCUAAGC
						TAAACGTGAG			UAAACGUGAG
						AAGGATGGAT			AAGGAUGGAU
						CGTACTCAGG			CGUACUCAGG
						CCGGGACCTC			CCGGGACCUC
						TCTCGCCGCA			UCUCGCCGCA
						CTGAGGGGCA			CUGAGGGGCA
						CTCCACACCA			CUCCACACCA
						CGGGGGCC			CGGGGGCC
						[SEQ ID			[SEQ ID
						NO:			NO:
						1792]			1989]
miR-	2945	−2.89804	0.152222	TGTAGTAGAA	TGAGCCAAAG	CATGCAGACT	UGUAGUAGAA	UGAGCCAAAG	CAUGCAGACU
1-1_M				GGCTTTGGCT	CCTTCTACCG	GCCTGCTTGG	GGCUUUGGCU	CCUUCUACCG	GCCUGCUUGG
				GA	ACA	GTGAGCCAAA	GA	ACA	GUGAGCCAAA
				[SEQ ID	[SEQ ID	GCCTTCTACC	[SEQ ID	[SEQ ID	GCCUUCUACC
				NO:	NO:	GACATATGGA	NO:	NO:	GACAUAUGGA
				685]	1633]	CCTGCTAAGC	1213]	1828]	CCUGCUAAGC
						TATGTAGTAG	(Same		UAUGUAGUAG
						AAGGCTTTGG	guide		AAGGCUUUGG
						CTGACTCAGG	as		CUGACUCAGG
						CCGGGACCTC	XD-		CCGGGACCUC
						TTCCGCCGCA	14860)		UUCCGCCGCA
						CTGAGGGGCA			CUGAGGGGCA
						CTCCACACCA			CUCCACACCA
						CGGGGGCC			CGGGGGCC
						[SEQ ID			[SEQ ID
						NO:			NO:
						1711]			1908]
miR-	3341	−2.70967	0.193529	ATAGACATGA	GACTCAGCAT	CATGCAGACT	AUAGACAUGA	GACUCAGCAU	CAUGCAGACU
1-1_M				GGATGCTGAG	CCTCATGTGA	GCCTGCTTGG	GGAUGCUGAG	CCUCAUGUGA	GCCUGCUUGG
				AC	TAT	GGACTCAGCA	AC	UAU	GGACUCAGCA
				[SEQ ID	[SEQ ID	TCCTCATGTG	[SEQ ID	[SEQ ID	UCCUCAUGUG
				NO:	NO:	ATATTATGGA	NO:	NO:	AUAUUAUGGA
				1617]	1637]	CCTGCTAAGC	1813]	1832]	CCUGCUAAGC
						TAATAGACAT			UAAUAGACAU
						GAGGATGCTG			GAGGAUGCUG
						AGACCTCAGG			AGACCUCAGG
						CCGGGACCTC			CCGGGACCUC
						TTCCGCCGCA			UUCCGCCGCA
						CTGAGGGGCA			CUGAGGGGCA
						CTCCA			CUCCACACCA
						CACCACGGGG			CGGGGGCC
						GCC			[SEQ ID
						[SEQ ID			NO:
						NO:			1912]
						1715]
miR-	1755	−2.62482	0.169485	TCGGGTTGAA	CTCACTTCAG	CATGCAGACT	UCGGGUUGAA	CUCACUUCAG	CAUGCAGACU
1-1_M				ATCTGAAGTG	ATTTCAACGA	GCCTGCTTGG	AUCUGAAGUG	AUUUCAACGA	GCCUGCUUGG
				TG	CGA	GCTCACTTCA	UG	CGA	GCUCACUUCA
				[SEQ ID	[SEQ ID	GATTTCAACG	[SEQ ID	[SEQ ID	GAUUUCAACG
				NO:	NO:	ACGATATGGA	NO:	NO:	ACGAUAUGGA
				657]	1645]	CCTGCTAAGC	1185]	1840]	CCUGCUAAGC
						TATCGGGTTG			UAUCGGGUUG
						AAATCTGAAG			AAAUCUGAAG
						TGTGCTCAGG			UGUGCUCAGG
						CCGGGACCTC			CCGGGACCUC
						TTCCGCCGCA			UUCCGCCGCA
						CTGAGGGGCA			CUGAGGGGCA
						CTCCACACCA			CUCCACACCA
						CGGGGGCC			CGGGGGCC
						[SEQ ID			[SEQ ID
						NO:			NO:
						1723]			1920]
miR-	3302	−2.57066	0.05742	TTGAACAAGG	CGCAAATCAG	CATGCAGACT	UUGAACAAGG	CGCAAAUCAG	CAUGCAGACU
1-1_M				GGCTGATTTG	CCCCTTGTCG	GCCTGCTTGG	GGCUGAUUUG	CCCCUUGUCG	GCCUGCUUGG
				GG	CAA	GCGCAAATCA	GG	CAA	GCGCAAAUCA
				[SEQ ID	[SEQ ID	GCCCCTTGTC	[SEQ ID	[SEQ ID	GCCCCUUGUC
				NO:	NO:	GCAATATGGA	NO:	NO:	GCAAUAUGGA
				688]	1647]	CCTGCTAAGC	1216]	1842]	CCUGCUAAGC
						TATTGAACAA			UAUUGAACAA
						GGGGCTGATT			GGGGCUGAUU
						TGGGCTCAGG			UGGGCUCAGG
						CCGGGACCTC			CCGGGACCUC
						TTCCGCCGCA			UUCCGCCGCA
						CTGAGGGGCA			CUGAGGGGCA
						CTCCACACCA			CUCCACACCA
						CGGGGGCC			CGGGGGCC
						[SEQ ID			[SEQ ID
						NO:			NO:
						1727]			1924]
miR-	3330	−2.47751	0.392579	TATGCTGAGA	CGACATTATC	CATGCAGACT	UAUGCUGAGA	CGACAUUAUC	CAUGCAGACU
1-1_M				CTGATAATGT	AGTCTCAGGA	GCCTGCTTGG	CUGAUAAUGU	AGUCUCAGGA	GCCUGCUUGG
				GG	ATA	GCGACATTAT	GG	AUA	GCGACAUUAU
				[SEQ ID	[SEQ ID	CAGTCTCAGG	[SEQ ID	[SEQ ID	CAGUCUCAGG
				NO:	NO:	AATATATGGA	NO:	NO:	AAUAUAUGGA
				1614]	1659]	CCTGCTAAGC	1811]	1854]	CCUGCUAAGC
						TATATGCTGA			UAUAUGCUGA
						GACTGATAAT			GACUGAUAAU
						GTGGCTCAGG			GUGGCUCAGG
						CCGGGACCTC			CCGGGACCUC
						TTCCGCCGCA			UUCCGCCGCA
						CTGAGGGGCA			CUGAGGGGCA
						CTC			CUCCACACCA
						CACACCACGG			CGGGGGCC
						GGGCC			[SEQ ID
						[SEQ ID			NO:
						NO:			1940]
						1743]
miR-	2586	−2.46317	0.179187	TAGATTCAGA	CGAAGTTCTA	CATGCAGACT	UAGAUUCAGA	CGAAGUUCUA	CAUGCAGACU
1-1M				AGTAGAACTT	CTTCTGAACG	GCCTGCTTGG	AGUAGAACUU	CUUCUGAACG	GCCUGCUUGG
				GG	CTA	GCGAAGTTCT	GG	CUA	GCGAAGUUCU
				[SEQ ID	[SEQ ID	ACTTCTGAAC	[SEQ ID	[SEQ ID	ACUUCUGAAC
				NO:	NO:	GCTATATGGA	NO:	NO:	GCUAUAUGGA
				1621]	1662]	CCTGCTAAGC	1816]	1857]	CCUGCUAAGC
						TATAGATTCA			UAUAGAUUCA
						GAAGTAGAAC			GAAGUAGAAC
						TTGGCTCAGG			UUGGCUCAGG
						CCGGGACCTC			CCGGGACCUC
						TTCCGCCGCA			UUCCGCCGCA
						CTGAGGGGCA			CUGAGGGGCA
						CTCCACACCA			CUCCACACCA
						CGGGGGCC			CGGGGGCC
						[SEQ ID			[SEQ ID
						NO:			NO:
						1747]			1944]
miR-	3272	−2.45517	0.050153	AGGACTGTAG	GGAATATGTT	CATGCAGACT	AGGACUGUAG	GGAAUAUGUU	CAUGCAGACU
1-1_M				GCAACATATT	GCCTACAGCG	GCCTGCTTGG	GCAACAUAUU	GCCUACAGCG	GCCUGCUUGG
				GC	CCT	GGGAATATGT	GC	CCU	GGGAAUAUGU
				[SEQ ID	[SEQ ID	TGCCTACAGC	[SEQ ID	[SEQ ID	UGCCUACAGC
				NO:	NO:	GCCTTATGGA	NO:	NO:	GCCUUAUGGA
				1618]	1663]	CCTGCTAAGC	1814]	1858]	CCUGCUAAGC
						TAAGGACTGT			UAAGGACUGU
						AGGCAACATA			AGGCAACAUA
						TTGCCTCAGG			UUGCCUCAGG
						CCGGGACCTC			CCGGGACCUC
						TTCCGCCGCA			UUCCGCCGCA
						CTGAGGGGCA			CUGAGGGGCA
						CTCCACACCA			CUCCACACCA
						CGGGGGCC			CGGGGGCC
						[SEQ ID			[SEQ ID
						NO:			NO:
						1748]			1945]
miR-	2943	−2.45216	0.338577	TAGTAGAAGG	TGTCAGCCAA	CATGCAGACT	UAGUAGAAGG	UGUCAGCCAA	CAUGCAGACU
1-1_M				CTTTGGCTGA	AGCCTTCTCC	GCCTGCTTGG	CUUUGGCUGA	AGCCUUCUCC	GCCUGCUUGG
				GA	CTA	GTGTCAGCCA	GA	CUA	GUGUCAGCCA
				[SEQ ID	[SEQ ID	AAGCCTTCTC	[SEQ ID	[SEQ ID	AAGCCUUCUC
				NO:	NO:	CCTATATGGA	NO:	NO:	CCUAUAUGGA
				683]	1650]	CCTGCTAAGC	1211]	1845]	CCUGCUAAGC
						TATAGTAGAA			UAUAGUAGAA
						GGCTTTGGCT			GGCUUUGGCU
						GAGACTCAGG			GAGACUCAGG
						CCGGGACCTC			CCGGGACCUC
						TTCCGCCGCA			UUCCGCCGCA
						CTGAGGGGCA			CUGAGGGGCA
						CTCC			CUCCACACCA
						ACACCACGGG			CGGGGGCC
						GGCC			[SEQ ID
						[SEQ ID			NO:
						NO:			1946]
						1749]
miR-	3301	−2.38494	0.327128	TGAACAAGGG	TGCCAAATCA	CATGCAGACT	UGAACAAGGG	UGCCAAAUCA	CAUGCAGACU
1-1_M				GCTGATTTGG	GCCCCTTGCG	GCCTGCTTGG	GCUGAUUUGG	GCCCCUUGCG	GCCUGCUUGG
				GA	TCA	GTGCCAAATC	GA	UCA	GUGCCAAAUC
				[SEQ ID	[SEQ ID	AGCCCCTTGC	[SEQ ID	[SEQ ID	AGCCCCUUGC
				NO:	NO:	GTCATATGGA	NO:	NO:	GUCAUAUGGA
				687]	1673]	CCTGCTAAGC	1215]	1868]	CCUGCUAAGC
						TATGAACAAG			UAUGAACAAG
						GGGCTGATTT			GGGCUGAUUU
						GGGACTCAGG			GGGACUCAGG
						CCGGGACCTC			CCGGGACCUC
						TTCCGCCGCA			UUCCGCCGCA
						CTGAGGGGCA			CUGAGGGGCA
						CTCCACACCA			CUCCACACCA
						CGGGGGcc			CGGGGGCC
						[SEQ ID			[SEQ ID
						NO:			NO:
						1770]			1967]
miR-	2602	−2.37919	0.040602	TTTAGTAGTT	ATTCTATGGA	CATGCAGACT	UUUAGUAGUU	AUUCUAUGGA	CAUGCAGACU
1-1_M				GATCCATAGA	TCAACTACCG	GCCTGCTTGG	GAUCCAUAGA	UCAACUACCG	GCCUGCUUGG
				TT	AAA	GATTCTATGG	UU	AAA	GAUUCUAUGG
				[SEQ ID	[SEQ ID	ATCAACTACC	[SEQ ID	[SEQ ID	AUCAACUACC
				NO:	NO:	GAAATATGGA	NO:	NO:	GAAAUAUGGA
				1616]	1681]	CCTGCTAAGC	202]	1876]	CCUGCUAAGC
						TATTTAGTAG	(Same		UAUUUAGUAG
						TTGATCCATA	guide		UUGAUCCAUA
						GATTCTCAGG	as		GAUUCUCAGG
						CCGGGACCTC	XD-		CCGGGACCUC
						TTCCGCCGCA	14837)		UUCCGCCGCA
						CTGAGGGGCA			CUGAGGGGCA
						CTCCACACCA			CUCCACACCA
						CGGGGGCC			CGGGGGCC
						[SEQ ID			[SEQ ID
						NO:			NO:
						1772]			1969]
miR-	3302	−2.68521	−0.3818	TTGAACAAGG	CCCAAACCAG	CCCAAAAGAG	UUGAACAAGG	CCCAAACCAG	CCCAAAAGAG
100				GGCTGATTTG	ACCCTTGCTC	AGAAGATATT	GGCUGAUUUG	ACCCUUGCUC	AGAAGAUAUU
				GG	AT	GAGGCCTGTT	GG	AU	GAGGCCUGUU
				[SEQ ID	[SEQ ID	GCCACATTGA	[SEQ ID	[SEQ ID	GCCACAUUGA
				NO:	NO:	ACAAGGGGCT	NO:	NO:	ACAAGGGGCU
				688]	1638]	GATTTGGGGT	1216]	1833]	GAUUUGGGGU
						ATTAGTCCGC			AUUAGUCCGC
						CCAAACCAGA			CCAAACCAGA
						CCCTTGCTCA			CCCUUGCUCA
						TTGTGTCTGT			UUGUGUCUGU
						TAGGCAATCT			UAGGCAAUCU
						CACGGACCTG			CACGGACCUG
						GGGC			GGGCUUUGCU
						TTTGCTTATA			UAUAUGCC
						TGCC			[SEQ ID
						[SEQ ID			NO:
						NO:			1913]
						1716]
miR-	3043	−2.67985	−0.0584	TTTGGTGCAA	AGCCTGCTTG	CCCAAAAGAG	UUUGGUGCAA	AGCCUGCUUG	CCCAAAAGAG
100				AACAAACAGG	GTTTGCAACA	AGAAGATATT	AACAAACAGG	GUUUGCAACA	AGAAGAUAUU
				CT	AT	GAGGCCTGTT	CU	AU	GAGGCCUGUU
				[SEQ ID	[SEQ ID	GCCACATTTG	[SEQ ID	[SEQ ID	GCCACAUUUG
				NO:	NO:	GTGCAAAACA	NO:	NO:	GUGCAAAACA
				1615]	1639]	AACAGGCTGT	1812]	1834]	AACAGGCUGU
						ATTAGTCCGA			AUUAGUCCGA
						GCCTGCTTGG			GCCUGCUUGG
						TTTGCAACAA			UUUGCAACAA
						TTGTGTCTGT			UUGUGUCUGU
						TAGGCAATCT			UAGGCAAUCU
						CACGGACCTG			CACGGACCUG
						GGGCTTTGCT			GGGCUUUGCU
						TATATGCC			UAUAUGCC
						[SEQ ID			[SEQ ID
						NO:			NO:
						1717]			1914]
miR-	1755	−2.66915	−0.04161	TCGGGTTGAA	CACACTCCAG	CCCAAAAGAG	UCGGGUUGAA	CACACUCCAG	CCCAAAAGAG
100				ATCTGAAGTG	CTTTCAAACC	AGAAGATATT	AUCUGAAGUG	CUUUCAAACC	AGAAGAUAUU
				TG	GT	GAGGCCTGTT	UG	GU	GAGGCCUGUU
				[SEQ ID	[SEQ ID	GCCACATCGG	[SEQ ID	[SEQ ID	GCCACAUCGG
				NO:	NO:	GTTGAAATCT	NO:	NO:	GUUGAAAUCU
				657]	1640]	GAAGTGTGGT	1185]	1835]	GAAGUGUGGU
						ATTAGTCCGC			AUUAGUCCGC
						ACACTCCAGC			ACACUCCAGC
						TTTCAAACCG			UUUCAAACCG
						TTGTGTCTGT			UUGUGUCUGU
						TAGGCAATCT			UAGGCAAUCU
						CACGGACCTG			CACGGACCUG
						GGGCTTTGCT			GGGCUUUGCU
						TATATGCC			UAUAUGCC
						[SEQ ID			[SEQ ID
						NO:			NO:
						1718]			1915]
miR-	3272	−2.65898	−0.1606	AGGACTGTAG	GCAATACGTT	CCCAAAAGAG	AGGACUGUAG	GCAAUACGUU	CCCAAAAGAG
100				GCAACATATT	TCCTACAATC	AGAAGATATT	GCAACAUAUU	UCCUACAAUC	AGAAGAUAUU
				GC	CA	GAGGCCTGTT	GC	CA	GAGGCCUGUU
				[SEQ ID	[SEQ ID	GCCACAAGGA	[SEQ ID	[SEQ ID	GCCACAAGGA
				NO:	NO:	CTGTAGGCAA	NO:	NO:	CUGUAGGCAA
				1618]	1641]	CATATTGCGT	1814]	1836]	CAUAUUGCGU
						ATTAGTCCGG			AUUAGUCCGG
						CAATACGTTT			CAAUACGUUU
						CCTACAATCC			CCUACAAUCC
						ATGTGTCTGT			AUGUGUCUGU
						TAGGCAATCT			UAGGCAAUCU
						CACGGACCTG			CACGGACCUG
						GGGC			GGGCUUUGCU
						TTTGCTTATA			UAUAUGCC
						TGCC			[SEQ ID
						[SEQ ID			NO:
						NO:			1916]
						1719]
miR-	1578	−2.43871	−0.43493	TGGAATTTCT	ACAGCAATTC	CCCAAAAGAG	UGGAAUUUCU	ACAGCAAUUC	CCCAAAAGAG
100				CTGAACTGCT	CGAGAAACTC	AGAAGATATT	CUGAACUGCU	CGAGAAACUC	AGAAGAUAUU
				GT	CT	GAGGCCTGTT	GU	CU	GAGGCCUGUU
				[SEQ ID	[SEQ ID	GCCACATGGA	[SEQ ID	[SEQ ID	GCCACAUGGA
				NO:	NO:	ATTTCTCTGA	NO:	NO:	AUUUCUCUGA
				1626]	1665]	ACTGCTGTGT	1820]	1860]	ACUGCUGUGU
						ATTAGTCCGA			AUUAGUCCGA
						CAGCAATTCC			CAGCAAUUCC
						GAGAAACTCC			GAGAAACUCC
						TTGTGTCTGT			UUGUGUCUGU
						TAGGCAATCT			UAGGCAAUCU
						CACGGACCTG			CACGGACCUG
						GGGCTTTGCT			GGGCUUUGCU
						TATATGCC			UAUAUGCC
						[SEQ ID			[SEQ ID
						NO:			NO:
						1751]			1948]
miR-	2945	−2.38747	−0.10088	TGTAGTAGAA	TCAGCCCAAG	CCCAAAAGAG	UGUAGUAGAA	UCAGCCCAAG	CCCAAAAGAG
100				GGCTTTGGCT	ACTTCTAATA	AGAAGATATT	GGCUUUGGCU	ACUUCUAAUA	AGAAGAUAUU
				GA	CT	GAGGCCTGTT	GA	CU	GAGGCCUGUU
				[SEQ ID	[SEQ ID	GCCACATGTA	[SEQ ID	[SEQ ID	GCCACAUGUA
				NO:	NO:	GTAGAAGGCT	NO:	NO:	GUAGAAGGCU
				685]	1678]	TTGGCTGAGT	1213]	1873]	UUGGCUGAGU
						ATTAGTCCGT	(Same		AUUAGUCCGU
						CAGCCCAAGA	guide		CAGCCCAAGA
						CTTCTAATAC	as		CUUCUAAUAC
						TTGTGTCTGT	XD-		UUGUGUCUGU
						TAGGCAATCT	14860)		UAGGCAAUCU
						CACGGACCTG			CACGGACCUG
						GGGCTTTGCT			GGGCUUUGCU
						TATATGCC			UAUAUGCC
						[SEQ ID			[SEQ ID
						NO:			NO:
						1768]			1965]
miR-	2586	−2.32164	−0.18294	TAGATTCAGA	CCAAGTCCTA	CCCAAAAGAG	UAGAUUCAGA	CCAAGUCCUA	CCCAAAAGAG
100				AGTAGAACTT	ATTCTGACTC	AGAAGATATT	AGUAGAACUU	AUUCUGACUC	AGAAGAUAUU
				GG	TT	GAGGCCTGTT	GG	UU	GAGGCCUGUU
				[SEQ ID	[SEQ ID	GCCACATAGA	[SEQ ID	[SEQ ID	GCCACAUAGA
				NO:	NO:	TTCAGAAGTA	NO:	NO:	UUCAGAAGUA
				1621]	1692]	GAACTTGGGT	1816]	1887]	GAACUUGGGU
						ATTAGTCCGC			AUUAGUCCGC
						CAAGTCCTAA			CAAGUCCUAA
						TTCTGACTCT			UUCUGACUCU
						TTGTGTCTGT			UUGUGUCUGU
						TAGGCAATCT			UAGGCAAUCU
						CACGGACCTG			CACGGACCUG
						GGGCTTTGCT			GGGCUUUGCU
						TATATGCC			UAUAUGCC
						[SEQ ID			[SEQ ID
						NO:			NO:
						1785]			1982]
miR-	3341	−2.20897	0.022563	ATAGACATGA	GTCTCAACAT	CCCAAAAGAG	AUAGACAUGA	GUCUCAACAU	CCCAAAAGAG
100				GGATGCTGAG	ACTCATGCCT	AGAAGATATT	GGAUGCUGAG	ACUCAUGCCU	AGAAGAUAUU
				AC	AA	GAGGCCTGTT	AC	AA	GAGGCCUGUU
				[SEQ ID	[SEQ ID	GCCACAATAG	[SEQ ID	[SEQ ID	GCCACAAUAG
				NO:	NO:	ACATGAGGAT	NO:	NO:	ACAUGAGGAU
				1617]	2008]	GCTGAGACGT	1813]	2010]	GCUGAGACGU
						ATTAGTCCGG			AUUAGUCCGG
						TCTCAACATA			UCUCAACAUA
						CTCATGCCTA			CUCAUGCCUA
						ATGTGTCTGT			AUGUGUCUGU
						TAGGCAATCT			UAGGCAAUCU
						CACGGACCTG			CACGGACCUG
						GGGCTTTGCT			GGGCUUUGCU
						TATATGCC			UAUAUGCC
						[SEQ ID			[SEQ ID
						NO:			NO:
						2009]			2011]
miR-	893	−2.15837	−0.34705	TTTGTTACTG	CAGAGGCCGA	CCCAAAAGAG	UUUGUUACUG	CAGAGGCCGA	CCCAAAAGAG
100				TTTCGACCTC	CACAGTACCA	AGAAGATATT	UUUCGACCUC	CACAGUACCA	AGAAGAUAUU
				TG	AT	GAGGCCTGTT	UG	AU	GAGGCCUGUU
				[SEQ ID	[SEQ ID	GCCACATTTG	[SEQ ID	[SEQ ID	GCCACAUUUG
				NO:	NO:	TTACTGTTTC	NO:	NO:	UUACUGUUUC
				2012]	2013]	GACCTCTGGT	2015]	2016]	GACCUCUGGU
						ATTAGTCCGC			AUUAGUCCGC
						AGAGGCCGAC			AGAGGCCGAC
						ACAGTACCAA			ACAGUACCAA
						TTGTGTCTGT			UUGUGUCUGU
						TAGGCAATCT			UAGGCAAUCU
						CACGGACCTG			CACGGACCUG
						GGGCTTTGCT			GGGCUUUGCU
						TATATGCC			UAUAUGCC
						[SEQ ID			[SEQ ID
						NO:			NO:
						2014]			2017]
miR-	3330	−2.15241	0.12385	TATGCTGAGA	CCACATCATC	CCCAAAAGAG	UAUGCUGAGA	CCACAUCAUC	CCCAAAAGAG
100				CTGATAATGT	CGTCTCAACA	AGAAGATATT	CUGAUAAUGU	CGUCUCAACA	AGAAGAUAUU
				GG	TT	GAGGCCTGTT	GG	UU	GAGGCCUGUU
				[SEQ ID	[SEQ ID	GCCACATATG	[SEQ ID	[SEQ ID	GCCACAUAUG
				NO:	NO:	CTGAGACTGA	NO:	NO:	CUGAGACUGA
				1614]	2018]	TAATGTGGGT	1811]	2020]	UAAUGUGGGU
						ATTAGTCCGC			AUUAGUCCGC
						CACATCATCC			CACAUCAUCC
						GTCTCAACAT			GUCUCAACAU
						TTGTGTCTGT			UUGUGUCUGU
						TAGGCAATCT			UAGGCAAUCU
						CACGGACCTG			CACGGACCUG
						GGGCTTTGCT			GGGCUUUGCU
						TATATGCC			UAUAUGCC
						[SEQ ID			[SEQ ID
						NO:			NO:
						2019]			2021]
miR-	3302	−2.62238	−0.19177	TTGAACAAGG	CCCAAACCAG	CCCAAAAGAG	UUGAACAAGG	CCCAAACCAG	CCCAAAAGAG
100M				GGCTGATTTG	ACCCTTGCTC	AGAAGATATT	GGCUGAUUUG	ACCCUUGCUC	AGAAGAUAUU
				GG	AT	GATGCCTGTT	GG	AU	GAUGCCUGUU
				[SEQ ID	[SEQ ID	GCCACATTGA	[SEQ ID	[SEQ ID	GCCACAUUGA
				NO:	NO:	ACAAGGGGCT	NO:	NO:	ACAAGGGGCU
				688]	1638]	GATTTGGGGT	1216]	1833]	GAUUUGGGGU
						ATTAGTCCGC			AUUAGUCCGC
						CCAAACCAGA			CCAAACCAGA
						CCCTTGCTCA			CCCUUGCUCA
						TTGTGTCTGT			UUGUGUCUGU
						TAGGCTATTC			UAGGCUAUUC
						CACGGACCTG			CACGGACCUG
						GGGCTTTGCT			GGGCUUUGCU
						TATATGCC			UAUAUGCC
						[SEQ ID			[SEQ ID
						NO:			NO:
						1724]			1921]
miR-	3272	−2.56927	−0.01914	AGGACTGTAG	GCAATACGTT	CCCAAAAGAG	AGGACUGUAG	GCAAUACGUU	CCCAAAAGAG
100_M				GCAACATATT	TCCTACAATC	AGAAGATATT	GCAACAUAUU	UCCUACAAUC	AGAAGAUAUU
				GC	CA	GATGCCTGTT	GC	CA	GAUGCCUGUU
				[SEQ ID	[SEQ ID	GCCACAAGGA	[SEQ ID	[SEQ ID	GCCACAAGGA
				NO:	NO:	CTGTAGGCAA	NO:	NO:	CUGUAGGCAA
				1618]	1641]	CATATTGCGT	1814]	1836]	CAUAUUGCGU
						ATTAGTCCGG			AUUAGUCCGG
						CAATACGTTT			CAAUACGUUU
						CCTACAATCC			CCUACAAUCC
						ATGTGTCTGT			AUGUGUCUGU
						TAGGCTATTC			UAGGCUAUUC
						CACGGACCTG			CACGGACCUG
						GGGCTTTGCT			GGGCUUUGCU
						TATATGCC			UAUAUGCC
						[SEQ ID			[SEQ ID
						NO:			NO:
						17291			1926]
miR-	3133	−2.49525	0.238806	TATGTCTTGG	CAGTGACTCA	CCCAAAAGAG	UAUGUCUUGG	CAGUGACUCA	CCCAAAAGAG
100_M				CTTGATTCAC	CGCCAAGCCA	AGAAGATATT	CUUGAUUCAC	CGCCAAGCCA	AGAAGAUAUU
				TG	TT	GATGCCTGTT	UG	UU	GAUGCCUGUU
				[SEQ ID	[SEQ ID	GCCACATATG	[SEQ ID	[SEQ ID	GCCACAUAUG
				NO:	NO:	TCTTGGCTTG	NO:	NO:	UCUUGGCUUG
				1624]	1656]	ATTCACTGGT	1819]	1851]	AUUCACUGGU
						ATTAGTCCGC			AUUAGUCCGC
						AGTGACTCAC			AGUGACUCAC
						GCCAAGCCAT			GCCAAGCCAU
						TTGTGTCTGT			UUGUGUCUGU
						TAGGCTATTC			UAGGCUAUUC
						CACGGACCTG			CACGGACCUG
						GGGCTTTGCT			GGGCUUUGCU
						TATATGCC			UAUAUGCC
						[SEQ ID			[SEQ ID
						NO:			NO:
						1740]			1937]
miR-	1755	−2.4723	−0.07055	TCGGGTTGAA	CACACTCCAG	CCCAAAAGAG	UCGGGUUGAA	CACACUCCAG	CCCAAAAGAG
100_M				ATCTGAAGTG	CTTTCAAACC	AGAAGATATT	AUCUGAAGUG	CUUUCAAACC	AGAAGAUAUU
				TG	GT	GATGCCTGTT	UG	GU	GAUGCCUGUU
				[SEQ ID	[SEQ ID	GCCACAT	[SEQ ID	[SEQ ID	GCCACAUC
				NO:	NO:	CGGGTTGAAA	NO:	NO:	GGGUUGAAAU
				657]	1640]	TCTGAAGTGT	1185]	1835]	CUGAAGUGUG
						GGTATTAGTC			GUAUUAGUCC
						CGCACACTCC			GCACACUCCA
						AGCTTTCAAA			GCUUUCAAAC
						CCGTTGTGTC			CGUUGUGUCU
						TGTTAGGCTA			GUUAGGCUAU
						TTCCACGGAC			UCCACGGACC
						CTGGGGCTTT			UGGGGCUUUG
						GCTTATATGC			CUUAUAUGCC
						C			[SEQ ID
						[SEQ ID			NO:
						NO:			1942]
						1745]
miR-	1231	−2.37228	−0.26919	TTCACTTTAG	CTGCTACCAG	CCCAAAAGAG	UUCACUUUAG	CUGCUACCAG	CCCAAAAGAG
100_M				CACTGATAGC	GGCTAAAATG	AGAAGATATT	CACUGAUAGC	GGCUAAAAUG	AGAAGAUAUU
				AG	AT	GATGCCTGTT	AG	AU	GAUGCCUGUU
				[SEQ ID	[SEQ ID	GCCACATTCA	[SEQ ID	[SEQ ID	GCCACAUUCA
				NO:	NO:	CTTTAGCACT	NO:	NO:	CUUUAGCACU
				1627]	1682]	GATAGCAGGT	1825]	1877]	GAUAGCAGGU
						ATTAGTCCGC			AUUAGUCCGC
						TGCTACCAGG			UGCUACCAGG
						GCTAAAATGA			GCUAAAAUGA
						TTGTGTCTGT			UUGUGUCUGU
						TAGGCTATTC			UAGGCUAUUC
						CACGGACCTG			CACGGACCUG
						GGGCTTTGCT			GGGCUUUGCU
						TATATGCC			UAUAUGCC
						[SEQ ID			[SEQ ID
						NO:			NO:
						1773]			1970]
miR-	3043	−2.35911	0.045827	TTTGGTGCAA	AGCCTGCTTG	CCCAAAAGAG	UUUGGUGCAA	AGCCUGCUUG	CCCAAAAGAG
100_M				AACAAACAGG	GTTTGCAACA	AGAAGATATT	AACAAACAGG	GUUUGCAACA	AGAAGAUAUU
				CT	AT	GATGCCTGTT	CU	AU	GAUGCCUGUU
				[SEQ ID	[SEQ ID	GCCACATTTG	[SEQ ID	[SEQ ID	GCCACAUUUG
				NO:	NO:	GTGCAAAACA	NO:	NO:	GUGCAAAACA
				1615]	1639]	AACAGGCTGT	1812]	1834]	AACAGGCUGU
						ATTAGTCCGA			AUUAGUCCGA
						GCCTGCTTGG			GCCUGCUUGG
						TTTGCAACAA			UUUGCAACAA
						TTGTGTCTGT			UUGUGUCUGU
						TAGGCTATTC			UAGGCUAUUC
						CACGGACCTG			CACGGACCUG
						GGGCTTTGCT			GGGCUUUGCU
						TATATGCC			UAUAUGCC
						[SEQ ID			[SEQ ID
						NO:			NO:
						1776]			1973]
miR-	3301	−2.30882	−0.2279	TGAACAAGGG	TCCCAACTCA	CCCAAAAGAG	UGAACAAGGG	UCCCAACUCA	CCCAAAAGAG
100_M				GCTGATTTGG	TCCCCTTATT	AGAAGATATT	GCUGAUUUGG	UCCCCUUAUU	AGAAGAUAUU
				GA	CT	GATGCCTGTT	GA	CU	GAUGCCUGUU
				[SEQ ID	[SEQ ID	GCCACATGAA	[SEQ ID	[SEQ ID	GCCACAUGAA
				NO:	NO:	CAAGGGGCTG	NO:	NO:	CAAGGGGCUG
				687]	1695]	ATTTGGGAGT	1215]	1890]	AUUUGGGAGU
						ATTAGTCCGT			AUUAGUCCGU
						CCCAACTCAT			CCCAACUCAU
						CCCCTTA			CCCCUUAU
						TTCTTGTGTC			UCUUGUGUCU
						TGTTAGGCTA			GUUAGGCUAU
						TTCCACGGAC			UCCACGGACC
						CTGGGGCTTT			UGGGGCUUUG
						GCTTATATGC			CUUAUAUGCC
						C			[SEQ ID
						[SEQ ID			NO:
						NO:			1985]
						1788]
miR-	2945	−2.26187	0.044364	TGTAGTAGAA	TCAGCCCAAG	CCCAAAAGAG	UGUAGUAGAA	UCAGCCCAAG	CCCAAAAGAG
100_M				GGCTTTGGCT	ACTTCTAATA	AGAAGATATT	GGCUUUGGCU	ACUUCUAAUA	AGAAGAUAUU
				GA	CT	GATGCCTGTT	GA	CU	GAUGCCUGUU
				[SEQ ID	[SEQ ID	GCCACATGTA	[SEQ ID	[SEQ ID	GCCACAUGUA
				NO:	NO:	GTAGAAGGCT	NO:	NO:	GUAGAAGGCU
				685]	1678]	TTGGCTGAGT	1213]	1873]	UUGGCUGAGU
						ATTAGTCCGT	(Same		AUUAGUCCGU
						CAGCCCAAGA	guide		CAGCCCAAGA
						CTTCTAATAC	as		CUUCUAAUAC
						TTGTGTCTGT	XD-		UUGUGUCUGU
						TAGGCTATTC	14860)		UAGGCUAUUC
						CACGGACCTG			CACGGACCUG
						GGGCTTTGCT			GGGCUUUGCU
						TATATGCC			UAUAUGCC
						[SEQ ID			[SEQ ID
						NO:			NO:
						1797]			1994]
miR-	1578	−2.24051	−0.33444	TGGAATTTCT	ACAGCAATTC	CCCAAAAGAG	UGGAAUUUCU	ACAGCAAUUC	CCCAAAAGAG
100_M				CTGAACTGCT	CGAGAAACTC	AGAAGATATT	CUGAACUGCU	CGAGAAACUC	AGAAGAUAUU
				GT	CT	GATGCCTGTT	GU	CU	GAUGCCUGUU
				[SEQ ID	[SEQ ID	GCCACATGGA	[SEQ ID	[SEQ ID	GCCACAUGGA
				NO:	NO:	ATTTCTCTGA	NO:	NO:	AUUUCUCUGA
				1626]	1665]	ACTGCTGTGT	1820]	1860]	ACUGCUGUGU
						ATTAGTCCGA			AUUAGUCCGA
						CAGCAATTCC			CAGCAAUUCC
						GAGAAACTCC			GAGAAACUCC
						TTGTGTCTGT			UUGUGUCUGU
						TAGGCTATTC			UAGGCUAUUC
						CACGGACCTG			CACGGACCUG
						GGGCTTTGCT			GGGCUUUGCU
						TATATGCC			UAUAUGCC
						[SEQ ID			[SEQ ID
						NO:			NO:
						1808]			2005]
miR-	1580	−2.12895	−0.08237	ACTGGAATTT	AGCAGTCCAG	CCCAAAAGAG	ACUGGAAUUU	AGCAGUCCAG	CCCAAAAGAG
100_M				CTCTGAACTG	CGAAATTACA	AGAAGATATT	CUCUGAACUG	CGAAAUUACA	AGAAGAUAUU
				CT	GA	GATGCCTGTT	CU	GA	GAUGCCUGUU
				[SEQ ID	[SEQ ID	GCCACAACTG	[SEQ ID	[SEQ ID	GCCACAACUG
				NO:	NO:	GAATTTCTCT	NO:	NO:	GAAUUUCUCU
				1622]	2022]	GAACTGCTGT	1817]	2024]	GAACUGCUGU
						ATTAGTCCGA			AUUAGUCCGA
						GCAGTCCAGC			GCAGUCCAGC
						GAAATTACAG			GAAAUUACAG
						ATGTGTCTGT			AUGUGUCUGU
						TAGGCTATTC			UAGGCUAUUC
						CACGGACCTG			CACGGACCUG
						GGGCTTTGCT			GGGCUUUGCU
						TATATGCC			UAUAUGCC
						[SEQ ID			[SEQ ID
						NO:			NO:
						2023]			2025]
miR-	3332	−2.50104	−0.209	AGGATGCTGA	ACATTATCAG	GGCTACAGAG	AGGAUGCUGA	ACAUUAUCAG	GGCUACAGAG
122				GACTGATAAT	TATCAGCATA	TTTCCTTAGC	GACUGAUAAU	UAUCAGCAUA	UUUCCUUAGC
				GT	AT	AGAGCTGAGG	GU	AU	AGAGCUGAGG
				[SEQ ID	[SEQ ID	ATGCTGAGAC	[SEQ ID	[SEQ ID	AUGCUGAGAC
				NO:	NO:	TGATAATGTT	NO:	NO:	UGAUAAUGUU
				1623]	1655]	GTCTAAACTA	1818]	1850]	GUCUAAACUA
						TACATTATCA			UACAUUAUCA
						GTATCAGCAT			GUAUCAGCAU
						AATTAGCTAC			AAUUAGCUAC
						TGCTAGGCAA			UGCUAGGCAA
						TCCTTCCCTC			UCCUUCCCUC
						GATAAATGTC			GAUAAAUGUC
						TTGGCATCGT			UUGGCAUCGU
						TTGCTTTG			UUGCUUUG
						[SEQ ID			[SEQ ID
						NO:			NO:
						1739]			1936]
miR-	3341	−2.4857	0.187025	ATAGACATGA	GTCTCAGCAT	GGCTACAGAG	AUAGACAUGA	GUCUCAGCAU	GGCUACAGAG
122				GGATGCTGAG	CATCATGTCG	TTTCCTTAGC	GGAUGCUGAG	CAUCAUGUCG	UUUCCUUAGC
				AC	CT	AGAGCTGATA	AC	CU	AGAGCUGAUA
				[SEQ ID	[SEQ ID	GACATGAGGA	[SEQ ID	[SEQ ID	GACAUGAGGA
				NO:	NO:	TGCTGAGACT	NO:	NO:	UGCUGAGACU
				1617]	1657]	GTCTAAACTA	1813]	1852]	GUCUAAACUA
						TGTCTCAGCA			UGUCUCAGCA
						TCATCATGTC			UCAUCAUGUC
						GCTTAGCTAC			GCUUAGCUAC
						TGCTAGGCAA			UGCUAGGCAA
						TCCTTCCCTC			UCCUUCCCUC
						GATAAATGTC			GAUAAAUGUC
						TTGGCATCGT			UUGGCAUCGU
						TTGCTTTG			UUGCUUUG
						[SEQ ID			[SEQ ID
						NO:			NO:
						1741]			1938]
miR-	1755	−2.42198	0.034447	TCGGGTTGAA	CACACTTCAG	GGCTACAGAG	UCGGGUUGAA	CACACUUCAG	GGCUACAGAG
122				ATCTGAAGTG	ACTTCAACCA	TTTCCTTAGC	AUCUGAAGUG	ACUUCAACCA	UUUCCUUAGC
				TG	TA	AGAGCTGTCG	UG	UA	AGAGCUGUCG
				[SEQ ID	[SEQ ID	GGTTGAAATC	[SEQ ID	[SEQ ID	GGUUGAAAUC
				NO:	NO:	TGAAGTGTGT	NO:	NO:	UGAAGUGUGU
				657]	1646]	GTCTAAACTA	1185]	1841]	GUCUAAACUA
						TCACACTTCA			UCACACUUCA
						GACTTCAACC			GACUUCAACC
						ATATAGCTAC			AUAUAGCUAC
						TGCTAGGCAA			UGCUAGGCAA
						TCCTTCCCTC			UCCUUCCCUC
						GATAAATGTC			GAUAAAUGUC
						TTGGCATCGT			UUGGCAUCGU
						TTGCTTTG			UUGCUUUG
						[SEQ ID			[SEQ ID
						NO:			NO:
						1760]			1957]
miR-	2586	−2.35707	0.153236	TAGATTCAGA	CCAAGTTCTA	GGCTACAGAG	UAGAUUCAGA	CCAAGUUCUA	GGCUACAGAG
122				AGTAGAACTT	CCTCTGAATA	TTTCCTTAGC	AGUAGAACUU	CCUCUGAAUA	UUUCCUUAGC
				GG	GA	AGAGCTGTAG	GG	GA	AGAGCUGUAG
				[SEQ ID	[SEQ ID	ATTCAGAAGT	[SEQ ID	[SEQ ID	AUUCAGAAGU
				NO:	NO:	AGAACTTGGT	NO:	NO:	AGAACUUGGU
				1621]	1685]	GTCTAAACTA	1816]	1880]	GUCUAAACUA
						TCCAAGTTCT			UCCAAGUUCU
						ACCTCTGAAT			ACCUCUGAAU
						AGATAGCTAC			AGAUAGCUAC
						TGCTAGGCAA			UGCUAGGCAA
						TCCTTCCCTC			UCCUUCCCUC
						GATAAATGTC			GAUAAAUGUC
						TTGGCATCGT			UUGGCAUCGU
						TTGCTTTG			UUGCUUUG
						[SEQ ID			[SEQ ID
						NO:			NO:
						1777]			1974]
miR-	3273	−2.30907	−0.11515	TAGGACTGTA	CAATATGTTG	GGCTACAGAG	UAGGACUGUA	CAAUAUGUUG	GGCUACAGAG
122				GGCAACATAT	CATACAGTCA	TTTCCTTAGC	GGCAACAUAU	CAUACAGUCA	UUUCCUUAGC
				TG	GA	AGAGCTGTAG	UG	GA	AGAGCUGUAG
				[SEQ ID	[SEQ ID	GACTGTAGGC	[SEQ ID	[SEQ ID	GACUGUAGGC
				NO:	NO:	AACATATTGT	NO:	NO:	AACAUAUUGU
				1628]	1694]	GTCTAAACTA	1821]	1889]	GUCUAAACUA
						TCAATATGTT			UCAAUAUGUU
						GCATACAGTC			GCAUACAGUC
						AGATAGCTAC			AGAUAGCUAC
						TGCTAGGCAA			UGCUAGGCAA
						TCCTTCCCTC			UCCUUCCCUC
						GATAAATGTC			GAUAAAUGUC
						TTGGCATCGT			UUGGCAUCGU
						TTGCTTTG			UUGCUUUG
						[SEQ ID			[SEQ ID
						NO:			NO:
						1787]			1984]
miR-	1162	−2.2647	−0.33024	AACTGTACCA	CAGACTTTGT	GGCTACAGAG	AACUGUACCA	CAGACUUUGU	GGCUACAGAG
122				CAACAAAGTC	TATGGTACAT	TTTCCTTAGC	CAACAAAGUC	UAUGGUACAU	UUUCCUUAGC
				TG	GT	AGAGCTGAAC	UG	GU	AGAGCUGAAC
				[SEQ ID	[SEQ ID	TGTACCACAA	[SEQ ID	[SEQ ID	UGUACCACAA
				NO:	NO:	CAAAGTCTGT	NO:	NO:	CAAAGUCUGU
				652]	1701]	GTCTAAACTA	1180]	1896]	GUCUAAACUA
						TCAGACTTTG			UCAGACUUUG
						TTATGGTACA			UUAUGGUACA
						TGTTAGCTAC			UGUUAGCUAC
						TGCTAGGCAA			UGCUAGGCAA
						TCCTTCCCTC			UCCUUCCCUC
						GATAAATGTC			GAUAAAUGUC
						TTGGCATCGT			UUGGCAUCGU
						TTGCTTTG			UUGCUUUG
						[SEQ ID			[SEQ ID
						NO:			NO:
						1794]			1991]
miR-	3301	−2.1436	0.009402	TGAACAAGGG	TCCCAAATCA	GGCTACAGAG	UGAACAAGGG	UCCCAAAUCA	GGCUACAGAG
122				GCTGATTTGG	GACCCTTGTG	TTTCCTTAGC	GCUGAUUUGG	GACCCUUGUG	UUUCCUUAGC
				GA	AA	AGAGCTGTGA	GA	AA	AGAGCUGUGA
				[SEQ ID	[SEQ ID	ACAAGGGGCT	[SEQ ID	[SEQ ID	ACAAGGGGCU
				NO:	NO:	GATTTGGGAT	NO:	NO:	GAUUUGGGAU
				687]	1702]	GTCTAAACTA	1215]	1897]	GUCUAAACUA
						TTCCCAAATC			UUCCCAAAUC
						AGACCCTTGT			AGACCCUUGU
						GAATAGCTAC			GAAUAGCUAC
						TGCTAGGCAA			UGCUAGGCAA
						TCCTTCCCTC			UCCUUCCCUC
						GATAAATGTC			GAUAAAUGUC
						TTGGCATCGT			UUGGCAUCGU
						TTGCTTTG			UUGCUUUG
						[SEQ ID			[SEQ ID
						NO:			NO:
						2026]			2027]
miR-	3330	−2.06194	0.224544	TATGCTGAGA	CCACATTATC	GGCTACAGAG	UAUGCUGAGA	CCACAUUAUC	GGCUACAGAG
122				CTGATAATGT	AATCTCAGCC	TTTCCTTAGC	CUGAUAAUGU	AAUCUCAGCC	UUUCCUUAGC
				GG	GA	AGAGCTGTAT	GG	GA	AGAGCUGUAU
				[SEQ ID	[SEQ ID	GCTGAGACTG	[SEQ ID	[SEQ ID	GCUGAGACUG
				NO:	NO:	ATAATGTGGT	NO:	NO:	AUAAUGUGGU
				1614]	2028]	GTCTAAACTA	1811]	2030]	GUCUAAACUA
						TCCACATTAT			UCCACAUUAU
						CAATCTCAGC			CAAUCUCAGC
						CGATAGCTAC			CGAUAGCUAC
						TGCTAGGCAA			UGCUAGGCAA
						TCCTTCCCTC			UCCUUCCCUC
						GATAAATGTC			GAUAAAUGUC
						TTGGCATCGT			UUGGCAUCGU
						TTGCTTTG			UUGCUUUG
						[SEQ ID			[SEQ ID
						NO:			NO:
						2029]			2031]
miR-	3272	−2.04615	−0.54779	AGGACTGTAG	GCAATATGTT	GGCTACAGAG	AGGACUGUAG	GCAAUAUGUU	GGCUACAGAG
122				GCAACATATT	GACTACAGTA	TTTCCTTAGC	GCAACAUAUU	GACUACAGUA	UUUCCUUAGC
				GC	AT	AGAGCTGAGG	GC	AU	AGAGCUGAGG
				[SEQ ID	[SEQ ID	ACTGTAGGCA	[SEQ ID	[SEQ ID	ACUGUAGGCA
				NO:	NO:	ACATATTGCT	NO:	NO:	ACAUAUUGCU
				1618]	2032]	GTCTAAACTA	1814]	2034]	GUCUAAACUA
						TGCAATATGT			UGCAAUAUGU
						TGACTACAGT			UGACUACAGU
						AATTAGCTAC			AAUUAGCUAC
						TGCTAGGCAA			UGCUAGGCAA
						TCCTTCCCTC			UCCUUCCCUC
						GATAAATGTC			GAUAAAUGUC
						TTGGCATCGT			UUGGCAUCGU
						TTGCTTTG			UUGCUUUG
						[SEQ ID			[SEQ ID
						NO:			NO:
						2033]			2261
miR-	3133	−2.03169	0.05703	TATGTCTTGG	CAGTGAATCA	GGCTACAGAG	UAUGUCUUGG	CAGUGAAUCA	GGCUACAGAG
122				CTTGATTCAC	AACCAAGACC	TTTCCTTAGC	CUUGAUUCAC	AACCAAGACC	UUUCCUUAGC
				TG	GA	AGAGCTGTAT	UG	GA	AGAGCUGUAU
				[SEQ ID	[SEQ ID	GTCTTGGCTT	[SEQ ID	[SEQ ID	GUCUUGGCUU
				NO:	NO:	GATTCACTGT	NO:	NO:	GAUUCACUGU
				1624]	1666]	GTCTAAACTA	1819]	1861]	GUCUAAACUA
						TCAGTGAATC			UCAGUGAAUC
						AAACCAAGAC			AAACCAAGAC
						CGATAGCTAC			CGAUAGCUAC
						TGCTAGGCAA			UGCUAGGCAA
						TCCTTCCCTC			UCCUUCCCUC
						GATAAATGTC			GAUAAAUGUC
						TTGGCATCGT			UUGGCAUCGU
						TTGCTTTG			UUGCUUUG
						[SEQ ID			[SEQ ID
						NO:			NO:
						2262			2263
miR-	1755	−2.60043	−0.03541	TCGGGTTGAA	CACACTTCAG	GGCTACAGAG	UCGGGUUGAA	CACACUUCAG	GGCUACAGAG
122_M				ATCTGAAGTG	ACTTCAACCA	TTTGCTTAGC	AUCUGAAGUG	ACUUCAACCA	UUUGCUUAGC
				TG	TA	AGAGCTGTCG	UG	UA	AGAGCUGUCG
				[SEQ ID	[SEQ ID	GGTTGAAATC	[SEQ ID	[SEQ ID	GGUUGAAAUC
				NO:	NO:	TGAAGTGTGT	NO:	NO:	UGAAGUGUGU
				657]	1646]	GTCTAAACTA	1185]	1841]	GUCUAAACUA
						TCACACTTCA			UCACACUUCA
						GACTTCAACC			GACUUCAACC
						ATATAGCTAC			AUAUAGCUAC
						TGCTAGGCCA			UGCUAGGCCA
						TCCTTCCCTC			UCCUUCCCUC
						GATAAATGTC			GAUAAAUGUC
						TTGGCATCGT			UUGGCAUCGU
						TTGCTTTG			UUGCUUUG
						[SEQ ID			[SEQ ID
						NO:			NO:
						1725]			1922]
miR-	3133	−2.43848	0.197681	TATGTCTTGG	CAGTGAATCA	GGCTACAGAG	UAUGUCUUGG	CAGUGAAUCA	GGCUACAGAG
122_M				CTTGATTCAC	AACCAAGACC	TTTGCTTAGC	CUUGAUUCAC	AACCAAGACC	UUUGCUUAGC
				TG	GA	AGAGCTGTAT	UG	GA	AGAGCUGUAU
				[SEQ ID	[SEQ ID	GTCTTGGCTT	[SEQ ID	[SEQ ID	GUCUUGGCUU
				NO:	NO:	GATTCACTGT	NO:	NO:	GAUUCACUGU
				1624]	1666]	GTCTAAACTA	1819]	1861]	GUCUAAACUA
						TCAGTGAATC			UCAGUGAAUC
						AAACCAAGAC			AAACCAAGAC
						CGATAGCTAC			CGAUAGCUAC
						TGCTAGGCCA			UGCUAGGCCA
						TCCTTCCCTC			UCCUUCCCUC
						GATAAATGTC			GAUAAAUGUC
						TTGGCATCGT			UUGGCAUCGU
						TTGCTTTG			UUGCUUUG
						[SEQ ID			[SEQ ID
						NO:			NO:
						1752]			1949]
miR-	3302	−2.40308	−0.14782	TTGAACAAGG	CCCAAATCAG	GGCTACAGAG	UUGAACAAGG	CCCAAAUCAG	GGCUACAGAG
122_M				GGCTGATTTG	CACCTTGTTA	TTTGCTTAGC	GGCUGAUUUG	CACCUUGUUA	UUUGCUUAGC
				GG	CA	AGAGCTGTTG	GG	CA	AGAGCUGUUG
				[SEQ ID	[SEQ ID	AACAAGGGGC	[SEQ ID	[SEQ ID	AACAAGGGGC
				NO:	NO:	TGATTTGG	NO:	NO:	UGAUUUGGG
				688]	1675]	GTGTCTAAAC	1216]	1870]	UGUCUAAACU
						TATCCCAAAT			AUCCCAAAUC
						CAGCACCTTG			AGCACCUUGU
						TTACATAGCT			UACAUAGCUA
						ACTGCTAGGC			CUGCUAGGCC
						CATCCTTCCC			AUCCUUCCCU
						TCGATAAATG			CGAUAAAUGU
						TCTTGGCATC			CUUGGCAUCG
						GTTTGCTT			UUUGCUUUG
						TG			[SEQ ID
						[SEQ ID			NO:
						NO:			1962]
						1765]
miR-	3301	−2.26423	0.053866	TGAACAAGGG	TCCCAAATCA	GGCTACAGAG	UGAACAAGGG	UCCCAAAUCA	GGCUACAGAG
122_M				GCTGATTTGG	GACCCTTGTG	TTTGCTTAGC	GCUGAUUUGG	GACCCUUGUG	UUUGCUUAGC
				GA	AA	AGAGCTGTGA	GA	AA	AGAGCUGUGA
				[SEQ ID	[SEQ ID	ACAAGGGGCT	[SEQ ID	[SEQ ID	ACAAGGGGCU
				NO:	NO:	GATTTGGGAT	NO:	NO:	GAUUUGGGAU
				687]	1702]	GTCTAAACTA	1215]	1897]	GUCUAAACUA
						TTCCCAAATC			UUCCCAAAUC
						AGACCCTTGT			AGACCCUUGU
						GAATAGCTAC			GAAUAGCUAC
						TGCTAGGCCA			UGCUAGGCCA
						TCCTTCCCTC			UCCUUCCCUC
						GATAAATGTC			GAUAAAUGUC
						TTGGCATCGT			UUGGCAUCGU
						TTGCTTTG			UUGCUUUG
						[SEQ ID			[SEQ ID
						NO:			NO:
						1795]			1992]
miR-	3132	−2.24518	0.049515	ATGTCTTGGC	CCAGTGAATC	GGCTACAGAG	AUGUCUUGGC	CCAGUGAAUC	GGCUACAGAG
122M				TTGATTCACT	ACGCCAAGAA	TTTGCTTAGC	UUGAUUCACU	ACGCCAAGAA	UUUGCUUAGC
				GG	CT	AGAGCTGATG	GG	CU	AGAGCUGAUG
				[SEQ ID	[SEQ ID	TCTTGGCTTG	[SEQ ID	[SEQ ID	UCUUGGCUUG
				NO:	NO:	ATTCACTGGT	NO:	NO:	AUUCACUGGU
				1631]	1707]	GTCTAAACTA	1826]	1904]	GUCUAAACUA
						TCCAGTGAAT			UCCAGUGAAU
						CACGCCAAGA			CACGCCAAGA
						ACTTAGCTAC			ACUUAGCUAC
						TGCTAGGCCA			UGCUAGGCCA
						TCCTTCCCTC			UCCUUCCCUC
						GATAAATGTC			GAUAAAUGUC
						TTGGCATCGT			UUGGCAUCGU
						TTGCTTTG			UUGCUUUG
						[SEQ ID			[SEQ ID
						NO:			NO:
						1804]			2001]
miR-	3341	−2.10774	0.117999	ATAGACATGA	GTCTCAGCAT	GGCTACAGAG	AUAGACAUGA	GUCUCAGCAU	GGCUACAGAG
122_M				GGATGCTGAG	CATCATGTCG	TTTGCTTAGC	GGAUGCUGAG	CAUCAUGUCG	UUUGCUUAGC
				AC	CT	AGAGCTGATA	AC	CU	AGAGCUGAUA
				[SEQ ID	[SEQ ID	GACATGAGGA	[SEQ ID	[SEQ ID	GACAUGAGGA
				NO:	NO:	TGCTGAGACT	NO:	NO:	UGCUGAGACU
				1617]	1657]	GTCTAAACTA	1813]	1852]	GUCUAAACUA
						TGTCTCAGCA			UGUCUCAGCA
						TCAT			UCAU
						CATGTCGCTT			CAUGUCGCUU
						AGCTACTGCT			AGCUACUGCU
						AGGCCATCCT			AGGCCAUCCU
						TCCCTCGATA			UCCCUCGAUA
						AATGTCTTGG			AAUGUCUUGG
						CATCGTTTGC			CAUCGUUUGC
						TTTG			UUUG
						[SEQ ID			[SEQ ID
						NO:			NO:
						2264]			2265]
miR-	3332	−2.06545	−0.17324	AGGATGCTGA	ACATTATCAG	GGCTACAGAG	AGGAUGCUGA	ACAUUAUCAG	GGCUACAGAG
122M				GACTGATAAT	TATCAGCATA	TTTGCTTAGC	GACUGAUAAU	UAUCAGCAUA	UUUGCUUAGC
				GT	AT	AGAGCTGAGG	GU	AU	AGAGCUGAGG
				[SEQ ID	[SEQ ID	ATGCTGAGAC	[SEQ ID	[SEQ ID	AUGCUGAGAC
				NO:	NO:	TGATAATGTT	NO:	NO:	UGAUAAUGUU
				1623]	1655]	GTCTAAACTA	1818]	1850]	GUCUAAACUA
						TACATTATCA			UACAUUAUCA
						GTATCAGCAT			GUAUCAGCAU
						AATTAGCTAC			AAUUAGCUAC
						TGCTAGGCCA			UGCUAGGCCA
						TCCTTCCCTC			UCCUUCCCUC
						GATAAATGTC			GAUAAAUGUC
						TTGGCATCGT			UUGGCAUCGU
						TTGCTTTG			UUGCUUUG
						[SEQ ID			[SEQ ID
						NO:			NO:
						2266]			2267]
miR-	3272	−2.0434	−0.02715	AGGACTGTAG	GCAATATGTT	GGCTACAGAG	AGGACUGUAG	GCAAUAUGUU	GGCUACAGAG
122M				GCAACATATT	GACTACAGTA	TTTGCTTAGC	GCAACAUAUU	GACUACAGUA	UUUGCUUAGC
				GC	AT	AGAGCTGAGG	GC	AU	AGAGCUGAGG
				[SEQ ID	[SEQ ID	ACTGTAGGCA	[SEQ ID	[SEQ ID	ACUGUAGGCA
				NO:	NO:	ACATATTGCT	NO:	NO:	ACAUAUUGCU
				1618]	2032]	GTCTAAACTA	1814]	2034]	GUCUAAACUA
						TGCAATATGT			UGCAAUAUGU
						TGACTACAGT			UGACUACAGU
						AATTAGCTAC			AAUUAGCUAC
						TGCTAGGCCA			UGCUAGGCCA
						TCCTTCCCTC			UCCUUCCCUC
						GATAAATGTC			GAUAAAUGUC
						TTGGCATCGT			UUGGCAUCGU
						TTGCTTTG			UUGCUUUG
						[SEQ ID			[SEQ ID
						NO:			NO:
						2268]			2035]
miR-	3338	−1.99732	−0.07815	TACATGAGGA	TCAGTCTCAG	GGCTACAGAG	UACAUGAGGA	UCAGUCUCAG	GGCUACAGAG
122_M				TGCTGAGACT	CCTCCTCATT	TTTGCTTAGC	UGCUGAGACU	CCUCCUCAUU	UUUGCUUAGC
				GA	GA	AGAGCTGTAC	GA	GA	AGAGCUGUAC
				[SEQ ID	[SEQ ID	ATGAGGATGC	[SEQ ID	[SEQ ID	AUGAGGAUGC
				NO:	NO:	TGAGACTGAT	NO:	NO:	UGAGACUGAU
				1620]	2036]	GTCTAAACTA	314]	2038]	GUCUAAACUA
						TTCAGTCTCA	(Same		UUCAGUCUCA
						GCCTCCTCAT	guide		GCCUCCUCAU
						TGATAGC	as		UGAUAGC
						TACTGCTAGG	XD-		UACUGCUAGG
						CCATCCTTCC	14893)		CCAUCCUUCC
						CTCGATAAAT			CUCGAUAAAU
						GTCTTGGCAT			GUCUUGGCAU
						CGTTTGCTTT			CGUUUGCUUU
						G			G
						[SEQ ID			[SEQ ID
						NO:			NO:
						2037]			2039]
miR-	1162	−1.99443	−0.12785	AACTGTACCA	CAGACTTTGT	GGCTACAGAG	AACUGUACCA	CAGACUUUGU	GGCUACAGAG
122M				CAACAAAGTC	TATGGTACAT	TTTGCTTAGC	CAACAAAGUC	UAUGGUACAU	UUUGCUUAGC
				TG	GT	AGAGCTGAAC	UG	GU	AGAGCUGAAC
				[SEQ ID	[SEQ ID	TGTACCACAA	[SEQ ID	[SEQ ID	UGUACCACAA
				NO:	NO:	CAAAGTCTGT	NO:	NO:	CAAAGUCUGU
				652]	1701]	GTCTAAACTA	1180]	1896]	GUCUAAACUA
						TCAGACTTTG			UCAGACUUUG
						TTATGGTACA			UUAUGGUACA
						TGTTAGCTAC			UGUUAGCUAC
						TGCTAGGCCA			UGCUAGGCCA
						TCCTTCCCTC			UCCUUCCCUC
						GATAAATGTC			GAUAAAUGUC
						TTGGCATCGT			UUGGCAUCGU
						TTGCTTTG			UUGCUUUG
						[SEQ ID			[SEQ ID
						NO:			NO:
						2040]			2041]
miR-	2928	−1.82454	0.049772	TCTGAGAGAA	ACGCCACGTT	TTCCTTCCTC	UCUGAGAGAA	ACGCCACGUU	UUCCUUCCUC
124				GGAACGTGGG	CATTCGATCA	AGGAGAAAGG	GGAACGUGGG	CAUUCGAUCA	AGGAGAAAGG
				TT	GA	CCTCTCTCAC	UU	GA	CCUCUCUCAC
				[SEQ ID	[SEQ ID	GCCACGTTCA	[SEQ ID	[SEQ ID	GCCACGUUCA
				NO:	NO:	TTCGATCAGA	NO:	NO:	UUCGAUCAGA
				668]	2042]	ATTTAAATGT	1196]	2044]	AUUUAAAUGU
						CCATACAATT			CCAUACAAUU
						CTGAGAGAAG			CUGAGAGAAG
						GAACGTGGGT			GAACGUGGGU
						TGAATGGGGC			UGAAUGGGGC
						TGGCTGAGCA			UGGCUGAGCA
						CCGTGGGTCG			CCGUGGGUCG
						GCGAGGGCCC			GCGAGGGCCC
						GCCAAGGA			GCCAAGGA
						[SEQ ID			[SEQ ID
						NO:			NO:
						2043]			2045]
miR-	3043	−1.7881	0.031105	TTTGGTGCAA	AAGCTGTTTG	TTCCTTCCTC	UUUGGUGCAA	AAGCUGUUUG	UUCCUUCCUC
124				AACAAACAGG	TGTTGACCCA	AGGAGAAAGG	AACAAACAGG	UGUUGACCCA	AGGAGAAAGG
				CT	AA	CCTCTCTCAA	CU	AA	CCUCUCUCAA
				[SEQ ID	[SEQ ID	GCTGTTTGTG	[SEQ ID	[SEQ ID	GCUGUUUGUG
				NO:	NO:	TTGACCCAAA	NO:	NO:	UUGACCCAAA
				1615]	2046]	ATTTAAATGT	1812]	2048]	AUUUAAAUGU
						CCATACAATT			CCAUACAAUU
						TTGGTGCAAA			UUGGUGCAAA
						ACAAACAGGC			ACAAACAGGC
						TGAATGGGGC			UGAAUGGGGC
						TGGCTGAGCA			UGGCUGAGCA
						CCGTGGGTCG			CCGUGGGUCG
						GCGAGGGCCC			GCGAGGGCCC
						GCCAAGGA			GCCAAGGA
						[SEQ ID			[SEQ ID
						NO:			NO:
						2047]			2049]
miR-	3256	−1.65786	0.035613	TATTGCGTGG	CCGCAGCTTA	TTCCTTCCTC	UAUUGCGUGG	CCGCAGCUUA	UUCCUUCCUC
124				AGTAAGCTGG	CGCCAAACAA	AGGAGAAAGG	AGUAAGCUGG	CGCCAAACAA	AGGAGAAAGG
				TG	TA	CCTCTCTCCC	UG	UA	CCUCUCUCCC
				[SEQ ID	[SEQ ID	GCAGCTTACG	[SEQ ID	[SEQ ID	GCAGCUUACG
				NO:	NO:	CCAAACAATA	NO:	NO:	CCAAACAAUA
				618]	2050]	ATTTAAATGT	308]	2052]	AUUUAAAUGU
						CCATACAATT	(Same		CCAUACAAUU
						ATTGCGTGGA	as		AUUGCGUGGA
						GTAAGCTGGT	XD-		GUAAGCUGGU
						GGAATGGGGC	14890)		GGAAUGGGGC
						TGGCTGAGCA			UGGCUGAGCA
						CCGTGGGTCG			CCGUGGGUCG
						GCGAGGGCCC			GCGAGGGCCC
						GCCAAGGA			GCCAAGGA
						[SEQ ID			[SEQ ID
						NO:			NO:
						2051]			2053]
miR-	2353	−1.64493	0.114403	TTCGATGCAG	CAGCTGCTAG	TTCCTTCCTC	UUCGAUGCAG	CAGCUGCUAG	UUCCUUCCUC
124				GACTAGCAGG	TACTGACTCG	AGGAGAAAGG	GACUAGCAGG	UACUGACUCG	AGGAGAAAGG
				CG	AA	CCTCTCTCCA	CG	AA	CCUCUCUCCA
				[SEQ ID	[SEQ ID	GCTGCTAGTA	[SEQ ID	[SEQ ID	GCUGCUAGUA
				NO:	NO:	CTGACTCGAA	NO:	NO:	CUGACUCGAA
				612]	2054]	ATTTAAATGT	166]	2056]	AUUUAAAUGU
						CCATACAATT	(Same		CCAUACAAUU
						TCGATGCAGG	as		UCGAUGCAGG
						ACTAGCAGGC	XD-		ACUAGCAGGC
						GGAATGGGGC	14819)		GGAAUGGGGC
						TGGCTGAGCA			UGGCUGAGCA
						CCGTGGGTCG			CCGUGGGUCG
						GCGAGGGCCC			GCGAGGGCCC
						GCCAAGGA			GCCAAGGA
						[SEQ ID			[SEQ ID
						NO:			NO:
						2055]			2057]
miR-	1231	−1.5099	−0.27848	TTCACTTTAG	CCCCTATCAG	TTCCTTCCTC	UUCACUUUAG	CCCCUAUCAG	UUCCUUCCUC
124				CACTGATAGC	TTCTACCGTG	AGGAGAAAGG	CACUGAUAGC	UUCUACCGUG	AGGAGAAAGG
				AG	AA	CCTCTCTCCC	AG	AA	CCUCUCUCCC
				[SEQ ID	[SEQ ID	CCTATCAGTT	[SEQ ID	[SEQ ID	CCUAUCAGUU
				NO:	NO:	CTACCGTGAA	NO:	NO:	CUACCGUGAA
				1627]	2058]	ATTTAAATGT	1825]	2060]	AUUUAAAUGU
						CCATACAATT			CCAUACAAUU
						TCACTTTAGC			UCACUUUAGC
						ACTGATAGCA			ACUGAUAGCA
						GGAATGGGGC			GGAAUGGGGC
						TGGCTGAGCA			UGGCUGAGCA
						CCG			CCGUG
						TGGGTCGGCG			GGUCGGCGAG
						AGGGCCCGCC			GGCCCGCCAA
						AAGGA			GGA
						[SEQ ID			[SEQ ID
						NO:			NO:
						2059]			2061]
miR-	3546	−1.46171	−0.28183	TCGCATACTG	TAGCTTGCTC	TTCCTTCCTC	UCGCAUACUG	UAGCUUGCUC	UUCCUUCCUC
124				CTGAGCAAGG	ATCAGGCTGC	AGGAGAAAGG	CUGAGCAAGG	AUCAGGCUGC	AGGAGAAAGG
				GA	GA	CCTCTCTCTA	GA	GA	CCUCUCUCUA
				[SEQ ID	[SEQ ID	GCTTGCTCAT	[SEQ ID	[SEQ ID	GCUUGCUCAU
				NO:	NO:	CAGGCTGCGA	NO:	NO:	CAGGCUGCGA
				2062]	2063]	ATTTAAATGT	2065]	2066]	AUUUAAAUGU
						CCATACAATT			CCAUACAAUU
						CGCATACTGC			CGCAUACUGC
						TGAGCAAGGG			UGAGCAAGGG
						AGAATGGGGC			AGAAUGGGGC
						TGGCTGAGCA			UGGCUGAGCA
						CCGTGGGTCG			CCGUGGGUCG
						GCGAGGGCCC			GCGAGGGCCC
						GCCAAGGA			GCCAAGGA
						[SEQ ID			[SEQ ID
						NO:			NO:
						2064]			2067]
miR-	893	−1.42389	−0.31114	TTTGTTACTG	CCCAGGTCGA	TTCCTTCCTC	UUUGUUACUG	CCCAGGUCGA	UUCCUUCCUC
124				TTTCGACCTC	ACCAGGCACA	AGGAGAAAGG	UUUCGACCUC	ACCAGGCACA	AGGAGAAAGG
				TG	AA	CCTCTCTCCC	UG	AA	CCUCUCUCCC
				[SEQ ID	[SEQ ID	CAGGTCGAAC	[SEQ ID	[SEQ ID	CAGGUCGAAC
				NO:	NO:	CAGGCACAAA	NO:	NO:	CAGGCACAAA
				2012]	2068]	ATTTAAATGT	2015]	2070]	AUUUAAAUGU
						CCATACAATT			CCAUACAAUU
						TTGTTACTGT			UUGUUACUGU
						TTCGACCTCT			UUCGACCUCU
						GGAATGGGGC			GGAAUGGGGC
						TGGCTGAGCA			UGGCUGAGCA
						CCGTGGGTCG			CCGUGGGUCG
						GCGAGGGCCC			GCGAGGGCCC
						GCCAAGGA			GCCAAGGA
						[SEQ ID			[SEQ ID
						NO:			NO:
						2069]			2071]
miR-	2602	−1.34373	0.049733	TTTAGTAGTT	ACACTATGGA	TTCCTTCCTC	UUUAGUAGUU	ACACUAUGGA	UUCCUUCCUC
124				GATCCATAGA	TAAACGCCTA	AGGAGAAAGG	GAUCCAUAGA	UAAACGCCUA	AGGAGAAAGG
				TT	AA	CCTCTCTCAC	UU	AA	CCUCUCUCAC
				[SEQ ID	[SEQ ID	ACTATGGATA	[SEQ ID	[SEQ ID	ACUAUGGAUA
				NO:	NO:	AACGCCTAAA	NO:	NO:	AACGCCUAAA
				1616]	2072]	ATTTAAATGT	202]	2074]	AUUUAAAUGU
						CCATACAATT	(Same		CCAUACAAUU
						TTAGTAGTTG	guide		UUAGUAGUUG
						ATCCATAGAT	as		AUCCAUAGAU
						TGAATGGGGC	XD-		UGAAUGGGGC
						TGGCTGAGCA	14837)		UGGCUGAGCA
						CCGTGGGTCG			CCG
						GCG			UGGGU
						AGGGCCCGCC			CGGCG
						AAGGA			AGGGCCCGCC
						[SEQ ID			AAGGA
						NO:			[SEQ ID
						2073]			NO:
									2075]
miR-	2944	−1.33679	0.075742	TTAGTAGAAG	CCGAGCCAAA	TTCCTTCCTC	UUAGUAGAAG	CCGAGCCAAA	UUCCUUCCUC
124				GCTTTGGCTG	GACTTACACT	AGGAGAAAGG	GCUUUGGCUG	GACUUACACU	AGGAGAAAGG
				AG	AA	CCTCTCTCCC	AG	AA	CCUCUCUCCC
				[SEQ ID	[SEQ ID	GAGCCAAAGA	[SEQ ID	[SEQ ID	GAGCCAAAGA
				NO:	NO:	CTTACACTAA	NO:	NO:	CUUACACUAA
				684]	2076]	ATTTAAATGT	246]	2078]	AUUUAAAUGU
						CCATACAATT	(Same		CCAUACAAUU
						TAGTAGAAGG	guide		UAGUAGAAGG
						CTTTGGCTGA	as		CUUUGGCUGA
						GGAATGGGGC	XD-		GGAAUGGGGC
						TGGCTGAGCA	14859)		UGGCUGAGCA
						CCGTGGGTCG			CCGUGGGUCG
						GCGAGGGCCC			GCGAGGGCCC
						GCCAAGGA			GCCAAGGA
						[SEQ ID			[SEQ ID
						NO:			NO:
						2077]			2079]
miR-	3270	−1.2645	0.050943	TACTGTAGGC	AACCAATATG	TTCCTTCCTC	UACUGUAGGC	AACCAAUAUG	UUCCUUCCUC
124				AACATATTGC	TGGCCGCCAG	AGGAGAAAGG	AACAUAUUGC	UGGCCGCCAG	AGGAGAAAGG
				GT	TA	CCTCTCTCAA	GU	UA	CCUCUCUCAA
				[SEQ ID	[SEQ ID	CCAATATGTG	[SEQ ID	[SEQ ID	CCAAUAUGUG
				NO:	NO:	GCCGCCAGTA	NO:	NO:	GCCGCCAGUA
				2080]	2081]	ATTTAAATGT	2083]	2084]	AUUUAAAUGU
						CCATACAATT			CCAUACAAUU
						ACTGTAGGCA			ACUGUAGGCA
						ACATATTGCG			ACAUAUUGCG
						TGAATGGGGC			UGAAUGGGGC
						TGGCTGAGCA			UGGCUGAGCA
						CCGTGGGTCG			CCGUGGGUCG
						GCGAGGGCCC			GCGAGGGCCC
						GCCAAGGA			GCCAAGGA
						[SEQ ID			[SEQ ID
						NO:			NO:
						2082]			2085]
miR-	3302	−2.62809	0.136636	TTGAACAAGG	CGCAAATCAG	GCAGGGCCGG	UUGAACAAGG	CGCAAAUCAG	GCAGGGCCGG
130a				GGCTGATTTG	ACCCTTGTTC	CATGCCTCTG	GGCUGAUUUG	ACCCUUGUUC	CAUGCCUCUG
				GG	AC	CTGCTGGCCA	GG	AC	CUGCUGGCCA
				[SEQ ID	[SEQ ID	CGCAAATCAG	[SEQ ID	[SEQ ID	CGCAAAUCAG
				NO:	NO:	ACCCTTGTTC	NO:	NO:	ACCCUUGUUC
				688]	1644]	ACCTGTCTGC	1216]	1839]	ACCUGUCUGC
						ACCTGTCACT			ACCUGUCACU
						AGTTGAACAA			AGUUGAACAA
						GGGGCTGATT			GGGGCUGAUU
						TGGGTGGCCG			UGGGUGGCCG
						TGTAGTGCTA			UGUAGUGCUA
						CCCAGCGCTG			CCCAGCGCUG
						GCTGCCTCCT			GCUGCCUCCU
						CAGCATTG			CAGCAUUG
						[SEQ ID			[SEQ ID
						NO:			NO:
						1722]			1919]
miR-	1755	−2.56989	0.011195	TCGGGTTGAA	CTCACTTCAG	GCAGGGCCGG	UCGGGUUGAA	CUCACUUCAG	GCAGGGCCGG
130a				ATCTGAAGTG	CTTTCAATTC	CATGCCTCTG	AUCUGAAGUG	CUUUCAAUUC	CAUGCCUCUG
				TG	GC	CTGCTGGCCA	UG	GC	CUGCUGGCCA
				[SEQ ID	[SEQ ID	CTCACTTCAG	[SEQ ID	[SEQ ID	CUCACUUCAG
				NO:	NO:	CTTTCAATTC	NO:	NO:	CUUUCAAUUC
				657]	1648]	GCCTGTCTGC	1185]	1843]	GCCUGUCUGC
						ACCTGTCACT			ACCUGUCACU
						AGTCGGGTTG			AGUCGGGUUG
						AAATCTGAAG			AAAUCUGAAG
						TGTGTGGCCG			UGUGUGGCCG
						TGTAGTGCTA			UGUAGUGCUA
						CCCAGCGCTG			CCCAGCGCUG
						GCTGCCTCCT			GCUGCCUCCU
						CAGCATTG			CAGCAUUG
						[SEQ ID			[SEQ ID
						NO:			NO:
						1728]			1925]
miR-	3301	−2.55451	−0.07529	TGAACAAGGG	TGCCAAATCA	GCAGGGCCGG	UGAACAAGGG	UGCCAAAUCA	GCAGGGCCGG
130a				GCTGATTTGG	TCCCCTTGTT	CATGCCTCTG	GCUGAUUUGG	UCCCCUUGUU	CAUGCCUCUG
				GA	CC	CTGCTGGCCA	GA	CC	CUGCUGGCCA
				[SEQ ID	[SEQ ID	TGCCAAATCA	[SEQ ID	[SEQ ID	UGCCAAAUCA
				NO:	NO:	TCCCCTTGTT	NO:	NO:	UCCCCUUGUU
				687]	1649]	CCCTGTCTGC	1215]	1844]	CCCUGUCUGC
						ACCTGTCACT			ACCUGUCACU
						AGTGAACAAG			AGUGAACAAG
						GGGCTGATTT			GGGCUGAUUU
						GGGATGGCCG			GGGAUGGCCG
						TGTAGTGCTA			UGUAGUGCUA
						CCCAGCGCTG			CCCAGCGCUG
						GCTGCCTCCT			GCUGCCUCCU
						CAGCATTG			CAGCAUUG
						[SEQ ID			[SEQ ID
						NO:			NO:
						1730]			1927]
miR-	3341	−2.43802	0.12379	ATAGACATGA	GACTCAGCAT	GCAGGGCCGG	AUAGACAUGA	GACUCAGCAU	GCAGGGCCGG
130a				GGATGCTGAG	ACTCATGTTT	CATGCCTCTG	GGAUGCUGAG	ACUCAUGUUU	CAUGCCUCUG
				AC	AC	CTGCTGGCCA	AC	AC	CUGCUGGCCA
				[SEQ ID	[SEQ ID	GACTCAGCAT	[SEQ ID	[SEQ ID	GACUCAGCAU
				NO:	NO:	ACTCATGTTT	NO:	NO:	ACUCAUGUUU
				1617]	1667]	ACCTGTCTGC	1813]	1862]	ACCUGUCUGC
						ACCTGTCACT			ACCUGUCACU
						AGATAGACAT			AGAUAGACAU
						GAGGATGCTG			GAGGAUGCUG
						AGACTGGCCG			AGACUGGCCG
						TGTAGTGCTA			UGUAGUGCUA
						CCCAGCGCTG			CCCAGCGCUG
						GCTGCCTCCT			GCUGCCUCCU
						CAGCATTG			CAGCAUUG
						[SEQ ID			[SEQ ID
						NO:			NO:
						1753]			1950]
miR-	3330	−2.43606	0.221874	TATGCTGAGA	CGACATTATC	GCAGGGCCGG	UAUGCUGAGA	CGACAUUAUC	GCAGGGCCGG
130a				CTGATAATGT	CGTCTCAGTA	CATGCCTCTG	CUGAUAAUGU	CGUCUCAGUA	CAUGCCUCUG
				GG	TC	CTGCTGGCCA	GG	UC	CUGCUGGCCA
				[SEQ ID	[SEQ ID	CGACATTATC	[SEQ ID	[SEQ ID	CGACAUUAUC
				NO:	NO:	CGTCTCAGTA	NO:	NO:	CGUCUCAGUA
				1614]	1668]	TCCTGTCTGC	1811]	1863]	UCCUGUCUGC
						ACCTGTCACT			ACCUGUCACU
						AGTATGCTGA			AGUAUGCUGA
						GACTGATAAT			GACUGAUAAU
						GTGGTGGCCG			GUGGUGGCCG
						TGTAGTGCTA			UGUAGUGCUA
						CCCAGCGCTG			CCCAGCGCUG
						GCTGCCTCCT			GCUGCCUCCU
						CAGCATTG			CAGCAUUG
						[SEQ ID			[SEQ ID
						NO:			NO:
						1754]			1951]
miR-	3255	−2.43262	0.01754	ATTGCGTGGA	CGACCAGCTT	GCAGGGCCGG	AUUGCGUGGA	CGACCAGCUU	GCAGGGCCGG
130a				GTAAGCTGGT	CCTCCACGTA	CATGCCTCTG	GUAAGCUGGU	CCUCCACGUA	CAUGCCUCUG
				GG	AC	CTGCTGGCCA	GG	AC	CUGCUGGCCA
				[SEQ ID	[SEQ ID	CGACCAGCTT	[SEQ ID	[SEQ ID	CGACCAGCUU
				NO:	NO:	CCTCCACGTA	NO:	NO:	CCUCCACGUA
				617]	1669]	ACCTGTCTGC	306]	1864]	ACCUGUCUGC
						ACCTGTCACT	(Same		ACCUGUCACU
						AGATTGCGTG	guide		AGAUUGCGUG
						GAGTAAGCTG	as		GAGUAAGCUG
						GTGGTGGCCG	XD-		GUGGUGGCCG
						TGTAGTGCTA	14889)		UGUAGUGCUA
						CCCAGCGCTG			CCCAGCGCUG
						GCTGCCTCCT			GCUGCCUCCU
						CAGCATTG			CAGCAUUG
						[SEQ ID			[SEQ ID
						NO:			NO:
						1755]			1952]
miR-	3133	−2.42664	0.412413	TATGTCTTGG	CTGTGAATCA	GCAGGGCCGG	UAUGUCUUGG	CUGUGAAUCA	GCAGGGCCGG
130a				CTTGATTCAC	CGCCAAGATA	CATGCCTCTG	CUUGAUUCAC	CGCCAAGAUA	CAUGCCUCUG
				TG	TC	CTGCTGGCCA	UG	UC	CUGCUGGCCA
				[SEQ ID	[SEQ ID	CTGTGAATCA	[SEQ ID	[SEQ ID	CUGUGAAUCA
				NO:	NO:	CGCCAAGATA	NO:	NO:	CGCCAAGAUA
				1624]	1671]	TCCTGTCTGC	1819]	1866]	UCCUGUCUGC
						ACCTGTCACT			ACCUGUCACU
						AGTATGTCTT			AGUAUGUCUU
						GGCTTGATTC			GGCUUGAUUC
						ACTGTGGCCG			ACUGUGGCCG
						TGTAGTGCTA			UGUAGUGCUA
						CCCAGCGCTG			CCCAGCGCUG
						GCTGCCTCCT			GCUGCCUCCU
						CAGCATTG			CAGCAUUG
						[SEQ ID			[SEQ ID
						NO:			NO:
						1757]			1954]
miR-	3842	−2.39495	−0.55009	AACGTGAGAA	TTCGATCCAT	GCAGGGCCGG	AACGUGAGAA	UUCGAUCCAU	GCAGGGCCGG
130a				GGATGGATCG	ACTTCTCATG	CATGCCTCTG	GGAUGGAUCG	ACUUCUCAUG	CAUGCCUCUG
				TA	TC	CTGCTGGCCA	UA	UC	CUGCUGGCCA
				[SEQ ID	[SEQ ID	TTCGATCCAT	[SEQ ID	[SEQ ID	UUCGAUCCAU
				NO:	NO:	ACTTCTCATG	NO:	NO:	ACUUCUCAUG
				1625]	1677]	T	1824]	1872]	UC
						CCTGTCTGCA			CUGUCUGCAC
						CCTGTCACTA			CUGUCACUAG
						GAACGTGAGA			AACGUGAGAA
						AGGATGGATC			GGAUGGAUCG
						GTATGGCCGT			UAUGGCCGUG
						GTAGTGCTAC			UAGUGCUACC
						CCAGCGCTGG			CAGCGCUGGC
						CTGCCTCCTC			UGCCUCCUCA
						AGCATTG			GCAUUG
						[SEQ ID			[SEQ ID
						NO:			NO:
						1767]			1964]
miR-	2586	−2.38521	0.042149	TAGATTCAGA	CGAAGTTCTA	GCAGGGCCGG	UAGAUUCAGA	CGAAGUUCUA	GCAGGGCCGG
130a				AGTAGAACTT	ATTCTGAATC	CATGCCTCTG	AGUAGAACUU	AUUCUGAAUC	CAUGCCUCUG
				GG	TC	CTGCTGGCCA	GG	UC	CUGCUGGCCA
				[SEQ ID	[SEQ ID	CGAAGTTCTA	[SEQ ID	[SEQ ID	CGAAGUUCUA
				NO:	NO:	ATTCTGAATC	NO:	NO:	AUUCUGAAUC
				1621]	1679]	TCCTGTCTGC	1816]	1874]	UCCUGUCUGC
						ACCTGTCACT			ACCUGUCACU
						AGTAGATTCA			AGUAGAUUCA
						GAAGTAGAAC			GAAGUAGAAC
						TTGGTGGCCG			UUGGUGGCCG
						TGTAGTGCTA			UGUAGUGCUA
						CCCAGCGCTG			CCCAGCGCUG
						GCTGCCTCCT			GCUGCCUCCU
						CAGCATTG			CAGCAUUG
						[SEQ ID			[SEQ ID
						NO:			NO:
						1769]			1966]
miR-	2602	−2.30701	−0.14983	TTTAGTAGTT	ATTCTATGGA	GCAGGGCCGG	UUUAGUAGUU	AUUCUAUGGA	GCAGGGCCGG
130a				GATCCATAGA	GCAACTATTA	CATGCCTCTG	GAUCCAUAGA	GCAACUAUUA	CAUGCCUCUG
				TT	AC	CTGCTGGCCA	UU	AC	CUGCUGGCCA
				[SEQ ID	[SEQ ID	ATTCTATGGA	[SEQ ID	[SEQ ID	AUUCUAUGGA
				NO:	NO:	GCAACTATTA	NO:	NO:	GCAACUAUUA
				1616]	1696]	ACCTGTCTGC	202]	1891]	ACCUGUCUGC
						ACCTGTCACT	(Same		ACCUGUCACU
						AGTTTAGTAG	guide		AGUUUAGUAG
						TTGATCCATA	as		UUGAUCCAUA
						GATTTGGCCG	XD-		GAUUUGGCCG
						TGTAGTGCTA	14837)		UGUAGUGCUA
						CCCAGCGCTG			CCCAGCGCUG
						GCTGCCTCCT			GCUGCCUCCU
						CAGCATTG			CAGCAUUG
						[SEQ ID			[SEQ ID
						NO:			NO:
						1789]			1986]
miR-	3341	−2.48196	0.14613	ATAGACATGA	GCCTCAGCAT	GCCGTCCGCG	AUAGACAUGA	GCCUCAGCAU	GCCGUCCGCG
132				GGATGCTGAG	CATAATGTCT	CGCCCCGCCC	GGAUGCUGAG	CAUAAUGUCU	CGCCCCGCCC
				AC	AT	CCGCGTCTCC	AC	AU	CCGCGUCUCC
				[SEQ ID	[SEQ ID	AGGGGCCTCA	[SEQ ID	[SEQ ID	AGGGGCCUCA
				NO:	NO:	GCATCATAAT	NO:	NO:	GCAUCAUAAU
				1617]	1658]	GTCTATCTGT	1813]	1853]	GUCUAUCUGU
						GGGAACTGGA			GGGAACUGGA
						GGATAGACAT			GGAUAGACAU
						GAGGA			GAGGAUG
						TGCTGAGACC			CUGAGACCCC
						CCCGCAGCAC			CGCAGCACGC
						GCCCACGCGC			CCACGCGCCG
						CGCGCCACGC			CGCCACGCCG
						CGCGCCCCGA			CGCCCCGAGC
						GCC			C
						[SEQ ID			[SEQ ID
						NO:			NO:
						1742]			1939]
miR-	3302	−2.43028	0.018802	TTGAACAAGG	CACAAATCAG	GCCGTCCGCG	UUGAACAAGG	CACAAAUCAG	GCCGUCCGCG
132				GGCTGATTTG	CACATTGTTC	CGCCCCGCCC	GGCUGAUUUG	CACAUUGUUC	CGCCCCGCCC
				GG	AA	CCGCGTCTCC	GG	AA	CCGCGUCUCC
				[SEQ ID	[SEQ ID	AGGGCACAAA	[SEQ ID	[SEQ ID	AGGGCACAAA
				NO:	NO:	TCAGCACATT	NO:	NO:	UCAGCACAUU
				688]	1670]	GTTCAACTGT	1216]	1865]	GUUCAACUGU
						GGGAACTGGA			GGGAACUGGA
						GGTTGAACAA			GGUUGAACAA
						GGGGCTGATT			GGGGCUGAUU
						TGGGCCCCGC			UGGGCCCCGC
						AGCACGCCCA			AGCACGCCCA
						CGCGCCGCGC			CGCGCCGCGC
						CACGCCGCGC			CACGCCGCGC
						CCCGAGCC			CCCGAGCC
						[SEQ ID			[SEQ ID
						NO:			NO:
						1756]			1953]
miR-	1755	−2.41844	0.196175	TCGGGTTGAA	CCCACTTCAG	GCCGTCCGCG	UCGGGUUGAA	CCCACUUCAG	GCCGUCCGCG
132				ATCTGAAGTG	AGTGCAACCC	CGCCCCGCCC	AUCUGAAGUG	AGUGCAACCC	CGCCCCGCCC
				TG	GA	CCGCGTCTCC	UG	GA	CCGCGUCUCC
				[SEQ ID	[SEQ ID	AGGGCCCACT	[SEQ ID	[SEQ ID	AGGGCCCACU
				NO:	NO:	TCAGAGTGCA	NO:	NO:	UCAGAGUGCA
				657]	1672]	ACCCGACTGT	1185]	1867]	ACCCGACUGU
						GGGAACTGGA			GGGAACUGGA
						GGTCGGGTTG			GGUCGGGUUG
						AAATCTGAAG			AAAUCUGAAG
						TGTGCCCCGC			UGUGCCCCGC
						AGCACGCCCA			AGCACGCCCA
						CGCGCCGCGC			CGCGCCGCGC
						CACGCCGCGC			CACGCCGCGC
						CCCGAGCC			CCCGAGCC
						[SEQ ID			[SEQ ID
						NO:			NO:
						1762]			1959]
miR-	2602	−2.40987	−0.18915	TTTAGTAGTT	ACTCTATGGA	GCCGTCCGCG	UUUAGUAGUU	ACUCUAUGGA	GCCGUCCGCG
132				GATCCATAGA	TAACCTACTA	CGCCCCGCCC	GAUCCAUAGA	UAACCUACUA	CGCCCCGCCC
				TT	AA	CCGCGTCTCC	UU	AA	CCGCGUCUCC
				[SEQ ID	[SEQ ID	AGGGACTCTA	[SEQ ID	[SEQ ID	AGGGACUCUA
				NO:	NO:	TGGATAACCT	NO:	NO:	UGGAUAACCU
				1616]	1674]	ACTAAACTGT	202]	1869]	ACUAAACUGU
						GGGAACTGGA	(Same		GGGAACUGGA
						GGTTTAGTAG	guide		GGUUUAGUAG
						TTGATCCATA	as		UUGAUCCAUA
						GATTCCCCG	XD-		GAUUCCCCGC
						CAGCACGCCC	14837)		A
						ACGCGCCGCG			GCACGCCCAC
						CCACGCCGCG			GCGCCGCGCC
						CCCCGAGCC			ACGCCGCGCC
						[SEQ ID			CCGAGCC
						NO:			[SEQ ID
						1764]			NO:
									1961]
miR-	1784	−2.34143	−0.26117	ATTAACTACT	TCCAGACCAA	GCCGTCCGCG	AUUAACUACU	UCCAGACCAA	GCCGUCCGCG
132				CTTTGGTCTG	ATATTAGTTA	CGCCCCGCCC	CUUUGGUCUG	AUAUUAGUUA	CGCCCCGCCC
				AA	AT	CCGCGTCTCC	AA	AU	CCGCGUCUCC
				[SEQ ID	[SEQ ID	AGGGTCCAGA	[SEQ ID	[SEQ ID	AGGGUCCAGA
				NO:	NO:	CCAAATATTA	NO:	NO:	CCAAAUAUUA
				608]	1686]	GTTAATCTGT	112]	1881]	GUUAAUCUGU
						GGGAACTGGA	(Same		GGGAACUGGA
						GGATTAACTA	guide		GGAUUAACUA
						CTCTTTGGTC	as		CUCUUUGGUC
						TGAACCCCGC	XD-		UGAACCCCGC
						AGCACGCCCA	14792)		AGCACGCCCA
						CGCGCCGCGC			CGCGCCGCGC
						CACGCCGCGC			CACGCCGCGC
						CCCGAGCC			CCCGAGCC
						[SEQ ID			[SEQ ID
						NO:			NO:
						1779]			1976]
miR-	3338	−2.2984	−0.03531	TACATGAGGA	TAAGTCTCAG	GCCGTCCGCG	UACAUGAGGA	UAAGUCUCAG	GCCGUCCGCG
132				TGCTGAGACT	CCTACTCATG	CGCCCCGCCC	UGCUGAGACU	CCUACUCAUG	CGCCCCGCCC
				GA	TA	CCGCGTCTCC	GA	UA	CCGCGUCUCC
				[SEQ ID	[SEQ ID	AGGGTAAGTC	[SEQ ID	[SEQ ID	AGGGUAAGUC
				NO:	NO:	TCAGCCTACT	NO:	NO:	UCAGCCUACU
				1620]	1698]	CATGTACTGT	314]	1893]	CAUGUACUGU
						GGGAACTGGA	(Same		GGGAACUGGA
						GGTACATGAG	guide		GGUACAUGAG
						GATGCTGAGA	as		GAUGCUGAGA
						CTGACCCCGC	XD-		CUGACCCCGC
						AGCACGCCCA	14893)		AGCACGCCCA
						CGCGCCGCGC			CGCGCCGCGC
						CACGCCGCGC			CACGCCGCGC
						CCCGAGCC			CCCGAGCC
						[SEQ ID			[SEQ ID
						NO:			NO:
						1791]			1988]
miR-	2945	−2.18195	0.054975	TGTAGTAGAA	TAAGCCAAAG	GCCGTCCGCG	UGUAGUAGAA	UAAGCCAAAG	GCCGUCCGCG
132				GGCTTTGGCT	CATGCTACTA	CGCCCCGCCC	GGCUUUGGCU	CAUGCUACUA	CGCCCCGCCC
				GA	CA	CCGCGTCTCC	GA	CA	CCGCGUCUCC
				[SEQ ID	[SEQ ID	AGGGTAAGCC	[SEQ ID	[SEQ ID	AGGGUAAGCC
				NO:	NO:	AAAGCATGCT	NO:	NO:	AAAGCAUGCU
				685]	2086]	ACTACACTGT	1213]	2088]	ACUACACUGU
						GGGAACTGGA	(Same		GGGAACUGGA
						GGTGTAGTAG	guide		GGUGUAGUAG
						AAGGCTTTGG	as		AAGGCUUUGG
						CTGACCCCGC	XD-		CUGACCCCGC
						AGCACGCCCA	14860)		AGCACGCCCA
									CGCG
						CGCGCCGCGC			CCGCGCCACG
						CACGCCGCGC			CCGCGCCCCG
						CCCGAGCC			AGCC
						[SEQ ID			[SEQ ID
						NO:			NO:
						2087]			2089]
miR-	3256	−2.15429	0.256242	TATTGCGTGG	CCCCAGCTTA	GCCGTCCGCG	UAUUGCGUGG	CCCCAGCUUA	GCCGUCCGCG
132				AGTAAGCTGG	CGCAACGCAA	CGCCCCGCCC	AGUAAGCUGG	CGCAACGCAA	CGCCCCGCCC
				TG	TA	CCGCGTCTCC	UG	UA	CCGCGUCUCC
				[SEQ ID	[SEQ ID	AGGGCCCCAG	[SEQ ID	[SEQ ID	AGGGCCCCAG
				NO:	NO:	CTTACGCAAC	NO:	NO:	CUUACGCAAC
				618]	2090]	GCAATACTGT	308]	2092]	GCAAUACUGU
						GGGAACTGGA	(Same		GGGAACUGGA
						GGTATTGCGT	as		GGUAUUGCGU
						GGAGTAAGCT	XD-		GGAGUAAGCU
						GGTGCCCCGC	14890)		GGUGCCCCGC
						AGCACGCCCA			AGCACGCCCA
						CGCGCCGCGC			CGCGCCGCGC
						CACGCCGCGC			CACGCCGCGC
						CCCGAGCC			CCCGAGCC
						[SEQ ID			[SEQ ID
						NO:			NO:
						2091]			2093]
miR-	3255	−2.09429	0.188946	ATTGCGTGGA	CAACCAGCTT	GCCGTCCGCG	AUUGCGUGGA	CAACCAGCUU	GCCGUCCGCG
132				GTAAGCTGGT	AATACACGCA	CGCCCCGCCC	GUAAGCUGGU	AAUACACGCA	CGCCCCGCCC
				GG	AT	CCGCGTCTCC	GG	AU	CCGCGUCUCC
				[SEQ ID	[SEQ ID	AGGGCAACCA	[SEQ ID	[SEQ ID	AGGGCAACCA
				NO:	NO:	GCTTAATACA	NO:	NO:	GCUUAAUACA
				617]	2094]	CGCAATCTGT	306]	2096]	CGCAAUCUGU
						GGGAACTGGA	(Same		GGGAACUGGA
						GGATTGCGTG	guide		GGAUUGCGUG
						GAGTAAGCTG	as		GAGUAAGCUG
						GTGGCCCCGC	XD-		GUGGCCCCGC
						AGCACGCCCA	14889)		AGCACGCCCA
						CGCGCCGCGC			CGCGCCGCGC
						CACGCCGCGC			CACGCCGCGC
						CCCGAGCC			CCCGAGCC
						[SEQ ID			[SEQ ID
						NO:			NO:
						2095]			2097]
miR-	2928	−2.08992	0.223976	TCTGAGAGAA	ACCCCACGTT	GCCGTCCGCG	UCUGAGAGAA	ACCCCACGUU	GCCGUCCGCG
132				GGAACGTGGG	CATGCTCTCA	CGCCCCGCCC	GGAACGUGGG	CAUGCUCUCA	CGCCCCGCCC
				TT	GA	CCGCGTCTCC	UU	GA	CCGCGUCUCC
				[SEQ ID	[SEQ ID	AGGGACCCCA	[SEQ ID	[SEQ ID	AGGGACCCCA
				NO:	NO:	CGTTCATGCT	NO:	NO:	CGUUCAUGCU
				668]	2098]	CTCAGACTGT	1196]	2100]	CUCAGACUGU
						GGGAACTGGA			GGGAACUGGA
						GGTCTGAGAG			GGUCUGAGAG
						AAGGAACGTG			AAGGAACGUG
						GGTTCCCCGC			GGUUCCCCGC
						AGCACGCCCA			AGCACGCCCA
						CGCGCCGCGC			CGCG
						CAC			CCGCGCCACG
						GCCGCGCCCC			CCGCGCCCCG
						GAGCC			AGCC
						[SEQ ID			[SEQ ID
						NO:			NO:
						2099]			2101]
miR-	3338	−2.01915	−0.127	TACATGAGGA	TCAGAACAGC	GCCGGCGGAG	UACAUGAGGA	UCAGAACAGC	GCCGGCGGAG
138-2				TGCTGAGACT	ATCCTCATGA	TTCTGGTATC	UGCUGAGACU	AUCCUCAUGA	UUCUGGUAUC
				GA	[SEQ ID	GTTGCTGCTA	GA	[SEQ ID	GUUGCUGCUA
				[SEQ ID	NO:	CATGAGGATG	[SEQ ID	NO:	CAUGAGGAUG
				NO:	2102]	CTGAGACTGA	NO:	2104]	CUGAGACUGA
				1620]		GACGAGCAGC	314]		GACGAGCAGC
						GCATCCTCTT	(Same		GCAUCCUCUU
						ACCCTCAGAA	guide		ACCCUCAGAA
						CAGCATCCTC	as		CAGCAUCCUC
						ATGAGTTGCA	XD-		AUGAGUUGCA
						TCATACCCAT	14893)		UCAUACCCAU
						CCTCTCCAGG			CCUCUCCAGG
						CGAGCCTCGT			CGAGCCUCGU
						GGGACCGG			GGGACCGG
						[SEQ ID			[SEQ ID
						NO:			NO:
						2103]			2105]
miR-	3330	−1.83218	0.060443	TATGCTGAGA	CCACTGATCA	GCCGGCGGAG	UAUGCUGAGA	CCACUGAUCA	GCCGGCGGAG
138-2				CTGATAATGT	GTCTCAGCAA	TTCTGGTATC	CUGAUAAUGU	GUCUCAGCAA	UUCUGGUAUC
				GG	[SEQ ID	GTTGCTGCTA	GG	[SEQ ID	GUUGCUGCUA
				[SEQ ID	NO:	TGCTGAGACT	[SEQ ID	NO:	UGCUGAGACU
				NO:	2106]	GATAATGTGG	NO:	2108]	GAUAAUGUGG
				1614]		GACGAGCAGC	1811]		GACGAGCAGC
						GCATCCTCTT			GCAUCCUCUU
						ACCCCCACTG			ACCCCCACUG
						ATCAGTCTCA			AUCAGUCUCA
						GCAAGTTGCA			GCAAGUUGCA
						TCATACCCAT			UCAUACCCAU
						CCTCTCCAGG			CCUCUCCAGG
						CGAGCCTCGT			CGAGCCUCGU
						GGGACCGG			GGGACCGG
						[SEQ ID			[SEQ ID
						NO:			NO:
						2107]			2109]
miR-	3043	−1.6949	0.083253	TTTGGTGCAA	AGCCATTTGT	GCCGGCGGAG	UUUGGUGCAA	AGCCAUUUGU	GCCGGCGGAG
138-2				AACAAACAGG	TTTGCACCAT	TTCTGGTATC	AACAAACAGG	UUUGCACCAU	UUCUGGUAUC
				CT	[SEQ ID	GTTGCTGCTT	CU	[SEQ ID	GUUGCUGCUU
				[SEQ ID	NO:	TGGTGCAAAA	[SEQ ID	NO:	UGGUGCAAAA
				NO:	2110]	CAAACAGGCT	NO:	2112]	CAAACAGGCU
				1615]		GACGAGCAGC	1812]		GACGAGCAGC
						GCATCCTCTT			GCAUCCUCUU
						ACCCAGCCAT			ACCCAGCCAU
						TTGTTTTGCA			UUGUUUUGCA
						CCATGTTGCA			CCAUGUUGCA
						TCATACCCAT			UCAUACCCAU
						CCTCTCCAGG			CCUCUCCAGG
						CGAGCCTCGT			CGAGCCUCGU
						GGGACCGG			GGGACCGG
						[SEQ ID			[SEQ ID
						NO:			NO:
						2111]			2113]
miR-	1689	−1.57706	−0.53719	TACGCGGTGA	GGGACCAGAA	GCCGGCGGAG	UACGCGGUGA	GGGACCAGAA	GCCGGCGGAG
138-2				ATTCTGTCTC	TTCACCGCGA	TTCTGGTATC	AUUCUGUCUC	UUCACCGCGA	UUCUGGUAUC
				CC	[SEQ ID	GTTGCTGCTA	CC	[SEQ ID	GUUGCUGCUA
				[SEQ ID	NO:	CGCGGTGAAT	[SEQ ID	NO:	CGCGGUGAAU
				NO:	2114]	TCTGTCTCCC	NO:	2116]	UCUGUCUCCC
				605]		GACGAGCAGC	100]		GACGAGCAGC
						GCATCCTCTT	(Same		GCAUCCUCUU
						ACCCGGGACC	guide		ACCCGGGACC
						AGAATTCACC	as		AGAAUUCACC
						GCGAGTTGCA	XD-		GCGAGUUGCA
						TCATACCCAT	14786)		UCAUACCCAU
						CCTCTCCAGG			CCUCUCCAGG
						CGAGCCTCGT			CGAGCCUCGU
						GGGACCGG			GGGACCGG
						[SEQ ID			[SEQ ID
						NO:			NO:
						2115]			2117]
miR-	3270	−1.41498	0.087791	TACTGTAGGC	ACGCTCATGT	GCCGGCGGAG	UACUGUAGGC	ACGCUCAUGU	GCCGGCGGAG
138-2				AACATATTGC	TGCCTACAGA	TTCTGGTATC	AACAUAUUGC	UGCCUACAGA	UUCUGGUAUC
				GT	[SEQ ID	GTTGCTGCTA	GU	[SEQ ID	GUUGCUGCUA
				[SEQ ID	NO:	CTGTAGGCAA	[SEQ ID	NO:	CUGUAGGCAA
				NO:	1901]	CATATTGCGT	NO:	2119]	CAUAUUGCGU
				2080]		GACGAGCAGC	2083]		GACGAGCAGC
						GCATCCTCTT			GCAUCCUCUU
						ACCCACGCTC			ACCCACGCUC
						ATGTTGCCTA			AUGUUGCCUA
						CAGAGTTGCA			CAGAGUUGCA
						TCATACCCAT			UCAUACCCAU
						CCTCTCCAGG			CCUCUCCAGG
						CGAGCCTCGT			CGAGCCUCGU
						GGGACCGG			GGGACCGG
						[SEQ ID			[SEQ ID
						NO:			NO:
						2118]			2120]
miR-	2928	−1.36141	0.122452	TCTGAGAGAA	AACCGCGTTC	GCCGGCGGAG	UCUGAGAGAA	AACCGCGUUC	GCCGGCGGAG
138-2				GGAACGTGGG	CTTCTCTCAC	TTCTGGTATC	GGAACGUGGG	CUUCUCUCAC	UUCUGGUAUC
				TT	[SEQ ID	GTTGCTGCTC	UU	[SEQ ID	GUUGCUGCUC
				[SEQ ID	NO:	TGAGAGAAGG	[SEQ ID	NO:	UGAGAGAAGG
				NO:	2121]	AACGTGGGTT	NO:	2123]	AACGUGGGUU
				668]		GACGAGCAGC	1196]		GACGAGCAGC
						GCATCCTCTT			GCAUCCUCUU
						ACCCAACCGC			ACCCAACCGC
						GTTCCTTCTC			GUUCCUUCUC
						TCACGTTGCA			UCACGUUGCA
						TCATACCCAT			UCAUACCCAU
						CCTCTCCAGG			CCUCUCCAGG
						CGAGCCTCGT			CGAGCCUCGU
						GGGACCGG			GGGACCGG
						[SEQ ID			[SEQ ID
						NO:			NO:
						2122]			2124]
miR-	3273	−1.32764	0.033491	TAGGACTGTA	CAATTGTTGC	GCCGGCGGAG	UAGGACUGUA	CAAUUGUUGC	GCCGGCGGAG
138-2				GGCAACATAT	CTACAGTCCA	TTCTGGTATC	GGCAACAUAU	CUACAGUCCA	UUCUGGUAUC
				TG	[SEQ ID	GTTGCTGCTA	UG	[SEQ ID	GUUGCUGCUA
				[SEQ ID	NO:	GGACTGTAGG	[SEQ ID	NO:	GGACUGUAGG
				NO:	2125]	CAACATATTG	NO:	2127]	CAACAUAUUG
				1628]		GACGAGCAGC	1821]		GACGAGCAGC
						GCATCCTCTT			GCAUCCUCUU
						ACCCCAATTG			ACCCCAAUUG
						TTGCCTACAG			UUGCCUACAG
						TCCAGTTGCA			UCCAGUUGCA
						TCATACCCAT			UCAUACCCAU
						CCTCTCCAGG			CCUCUCCAGG
						CGAGCCTCGT			CGAGCCUCGU
						GGGACCGG			GGGACCGG
						[SEQ ID			[SEQ ID
						NO:			NO:
						2126]			2128]
miR-	3255	−1.19435	0.020971	ATTGCGTGGA	CCACGCCTTA	GCCGGCGGAG	AUUGCGUGGA	CCACGCCUUA	GCCGGCGGAG
138-2				GTAAGCTGGT	CTCCACGCAT	TTCTGGTATC	GUAAGCUGGU	CUCCACGCAU	UUCUGGUAUC
				GG	[SEQ ID	GTTGCTGCAT	GG	[SEQ ID	GUUGCUGCAU
				[SEQ ID	NO:	TGCGTGGAGT	[SEQ ID	NO:	UGCGUGGAGU
				NO:	2129]	AAGCTGGTGG	NO:	2131]	AAGCUGGUGG
				617]		GACGAGCAGC	306]		GACGAGCAGC
						GCATCCTCTT	(Same		GCAUCCUCUU
						ACCCCCACGC	guide		ACCCCCACGC
						CTTACTCCAC	as		CUUACUCCAC
						GCATGTTGCA	XD-		GCAUGUUGCA
						TCATACCCAT	14889)		UCAUACCCAU
						CCTCTCCAGG			CCUCUCCAGG
						CGAGCCTCGT			CGAGCCUCGU
						GGGACCGG			GGGACCGG
						[SEQ ID			[SEQ ID
						NO:			NO:
						2130]			2132]
miR-	3302	−1.02575	−0.2011	TTGAACAAGG	CCCATCCAGC	GCCGGCGGAG	UUGAACAAGG	CCCAUCCAGC	GCCGGCGGAG
138-2				GGCTGATTTG	CTCTTGTTCT	TTCTGGTATC	GGCUGAUUUG	CUCUUGUUCU	UUCUGGUAUC
				GG	[SEQ ID	GTTGCTGCTT	GG	[SEQ ID	GUUGCUGCUU
				[SEQ ID	NO:	GAACAAGGGG	[SEQ ID	NO:	GAACAAGGGG
				NO:	2133]	CTGATTTGGG	NO:	2135]	CUGAUUUGGG
				688]		GACGAGCAGC	1216]		GACGAGCAGC
						GCATCCTCTT			GCAUCCUCUU
						ACCCCCCATC			ACCCCCCAUC
						CAGCCTCTTG			CAGCCUCUUG
						TTCTGTTGCA			UUCUGUUGCA
						TCATACCCAT			UCAUACCCAU
						CCTCTCCAGG			CCUCUCCAGG
						CGAGCCTCGT			CGAGCCUCGU
						GGGACCGG			GGGACCGG
						[SEQ ID			[SEQ ID
						NO:			NO:
						2134]			2136]
miR-	1436	−0.97653	0.111912	TGAGTTATCT	GCCCAGGAAA	GCCGGCGGAG	UGAGUUAUCU	GCCCAGGAAA	GCCGGCGGAG
138-2				CTTTCTAAGG	GAGATAACTG	TTCTGGTATC	CUUUCUAAGG	GAGAUAACUG	UUCUGGUAUC
				GC	[SEQ ID	GTTGCTGCTG	GC	[SEQ ID	GUUGCUGCUG
				[SEQ ID	NO:	AGTTATCTCT	[SEQ ID	NO:	AGUUAUCUCU
				NO:	2137]	TTCTAAGGGC	NO:	2139]	UUCUAAGGGC
				1632]		GACGAGCAGC	1827]		GACGAGCAGC
						GCATCCTCTT			GCAUCCUCUU
						ACCCGCCCAG			ACCCGCCCAG
						GAAAGAGATA			GAAAGAGAUA
						ACTGGTTGCA			ACUGGUUGCA
						TCATACCCAT			UCAUACCCAU
						CCTCTCCAGG			CCUCUCCAGG
						CGAGCCTCGT			CGAGCCUCGU
						GGGACCGG			GGGACCGG
						[SEQ ID			[SEQ ID
						NO:			NO:
						2138]			2140]
miR-	3043	−2.80802	0.325474	TTTGGTGCAA	CCGGTTTGTT	TCAAGCCATG	UUUGGUGCAA	CCGGUUUGUU	UCAAGCCAUG
144				AACAAACAGG	TATGCACCAA	CTTCCTGTGC	AACAAACAGG	UAUGCACCAA	CUUCCUGUGC
				CT	A	CCCCAGTGGG	CU	A	CCCCAGUGGG
				[SEQ ID	[SEQ ID	GCCCTGGCTC	[SEQ ID	[SEQ ID	GCCCUGGCUC
				NO:	NO:	CGGTTTGTTT	NO:	NO:	CGGUUUGUUU
				1615]	1635]	ATGCACCAAA	1812]	1830]	AUGCACCAAA
						AGTTTGCGAT			AGUUUGCGAU
						GAGACACTTT			GAGACACUUU
						GGTGCAAAAC			GGUGCAAAAC
						AAACAGGAGT			AAACAGGAGU
						CCGGGCACCC			CCGGGCACCC
						CCAGCTCTGG			CCAGCUCUGG
						AGCCTGACAA			AGCCUGACAA
						GGAGGACA			GGAGGACA
						[SEQ ID			[SEQ ID
						NO:			NO:
						1713]			1910]
miR-	2602	−2.76068	−0.25422	TTTAGTAGTT	TCGATGGATC	TCAAGCCATG	UUUAGUAGUU	UCGAUGGAUC	UCAAGCCAUG
144				GATCCATAGA	ATACTACTAA	CTTCCTGTGC	GAUCCAUAGA	AUACUACUAA	CUUCCUGUGC
				TT	A	CCCCAGTGGG	UU	A	CCCCAGUGGG
				[SEQ ID	[SEQ ID	GCCCTGGCTT	[SEQ ID	[SEQ ID	GCCCUGGCUU
				NO:	NO:	CGATGGATCA	NO:	NO:	CGAUGGAUCA
				1616]	1636]	TACTACTAAA	202]	1831]	UACUACUAAA
						AGTTTGCGAT	(Same		AGUUUGCGAU
						GAGACACTTT	guide		GAGACACUUU
						AGTAGTTGAT	as		AGUAGUUGAU
						CCATAGAAGT	XD-		CCAUAGAAGU
						CCGGGCACCC	14837)		CCGGGCACCC
						CCAGCTCTGG			CCAGCUCUGG
						AGCCTGACAA			AGCCUGACAA
						GGAGGACA			GGAGGACA
						[SEQ ID			[SEQ ID
						NO:			NO:
						1714]			1911]
miR-	3255	−2.47679	−0.08595	ATTGCGTGGA	ACAAGCTTAC	TCAAGCCATG	AUUGCGUGGA	ACAAGCUUAC	UCAAGCCAUG
144				GTAAGCTGGT	TGCCACGCAA	CTTCCTGTGC	GUAAGCUGGU	UGCCACGCAA	CUUCCUGUGC
				GG	T	CCCCAGTGGG	GG	U	CCCCAGUGGG
				[SEQ ID	[SEQ ID	GCCCTGGCTA	[SEQ ID	[SEQ ID	GCCCUGGCUA
				NO:	NO:	CAAGCTTACT	NO:	NO:	CAAGCUUACU
				617]	1660]	GCCACGCAAT	306]	1855]	GCCACGCAAU
						AGTTTGCGAT	(Same		AGUUUGCGAU
						GAGACACATT	guide		GAGACACAUU
						GCGTGGAGTA	as		GCGUGGAGUA
						AGCTGGTAGT	XD-		AGCUGGUAGU
						CCGGGCACCC	14889)		CCGGGCACCC
						CCAGCTCTGG			CCAGCUCUGG
						AGCCTGACAA			AGCCUGACAA
						GGAGGACA			GGAGGACA
						[SEQ ID			[SEQ ID
						NO:			NO:
						1744]			1941]
miR-	1231	−2.16776	−0.36035	TTCACTTTAG	GCGATCAGTG	TCAAGCCATG	UUCACUUUAG	GCGAUCAGUG	UCAAGCCAUG
144				CACTGATAGC	CATAAAGTGA	CTTCCTGTGC	CACUGAUAGC	CAUAAAGUGA	CUUCCUGUGC
				AG	A	CCCCAGTGGG	AG	A	CCCCAGUGGG
				[SEQ ID	[SEQ ID	GCCCTGGCTG	[SEQ ID	[SEQ ID	GCCCUGGCUG
				NO:	NO:	CGATCAGTGC	NO:	NO:	CGAUCAGUGC
				1627]	2141]	ATAAAGTGAA	1825]	2143]	AUAAAGUGAA
						AGTTTGCGAT			AGUUUGCGAU
						GAGACACTTC			GAGACACUUC
						ACTTTAGCAC			ACUUUAGCAC
						TGATAGCAGT			UGAUAGCAGU
						CCGGGCACCC			CCGGGCACCC
						CCAGCTCTGG			CCAGCUCUGG
						AGCCTGACAA			AGCCUGACAA
						GGAGGACA			GGAGGACA
						[SEQ ID			[SEQ ID
						NO:			NO:
						2142]			2144]
miR-	3845	−2.11363	−0.36405	TTGAACGTGA	TCAATCCTTC	TCAAGCCATG	UUGAACGUGA	UCAAUCCUUC	UCAAGCCAUG
144				GAAGGATGGA	TGCACGTTCA	CTTCCTGTGC	GAAGGAUGGA	UGCACGUUCA	CUUCCUGUGC
				TC	A	CCCCAGTGGG	UC	A	CCCCAGUGGG
				[SEQ ID	[SEQ ID	GCCCTGGCTT	[SEQ ID	[SEQ ID	GCCCUGGCUU
				NO:	NO:	CAATCCTTCT	NO:	NO:	CAAUCCUUCU
				696]	2145]	GCACGTTCAA	1224]	2147]	GCACGUUCAA
						AGTTTGCGAT			AGUUUGCGAU
						GAGACACTTG			GAGACACUUG
						AACGTGAGAA			AACGUGAGAA
						GGATGGAAGT			GGAUGGAAGU
						CCGGGCACCC			CCGGGCACCC
						CCAGCTCTGG			CCAGCUCUGG
						AGCCTGACAA			AGCCUGACAA
						GGAGGACA			GGAGGACA
						[SEQ ID			[SEQ ID
						NO:			NO:
						2146]			2148]
miR-	915	−2.04132	−0.09574	AAATCGTAGA	ACGGCCTCAG	TCAAGCCATG	AAAUCGUAGA	ACGGCCUCAG	UCAAGCCAUG
144				CTGAGGCAGT	TGCTACGATT	CTTCCTGTGC	CUGAGGCAGU	UGCUACGAUU	CUUCCUGUGC
				CC	T	CCCCAGTGGG	CC	U	CCCCAGUGGG
				[SEQ ID	[SEQ ID	GCCCTGGCTA	[SEQ ID	[SEQ ID	GCCCUGGCUA
				NO:	NO:	CGGCCTCAGT	NO:	NO:	CGGCCUCAGU
				2149]	2150]	GCTACGATTT	2152]	2153]	GCUACGAUUU
						AGTTTGCGAT			AGUUUGCGAU
						GAGACACAAA			GAGACACAAA
						TCGTAGACTG			UCGUAGACUG
						AGGCAGTAGT			AGGCAGUAGU
						CCGGGCACCC			CCGGGCACCC
						CCAGCTCTGG			CCAGCUCUGG
						AGCCTGACAA			AGCCUGACAA
						GGAGGACA			GGAGGACA
						[SEQ ID			[SEQ ID
						NO:			NO:
						2151]			2154]
miR-	3301	−1.88293	0.071952	TGAACAAGGG	CCCAATCAGC	TCAAGCCATG	UGAACAAGGG	CCCAAUCAGC	UCAAGCCAUG
144				GCTGATTTGG	CGCCTTGTTC	CTTCCTGTGC	GCUGAUUUGG	CGCCUUGUUC	CUUCCUGUGC
				GA	A	CCCCAGTGGG	GA	A	CCCCAGUGGG
				[SEQ ID	[SEQ ID	GCCCTGGCTC	[SEQ ID	[SEQ ID	GCCCUGGCUC
				NO:	NO:	CCAATCAGCC	NO:	NO:	CCAAUCAGCC
				687]	2155]	GCCTTGTTCA	1215]	2157]	GCCUUGUUCA
						AGTTTGCGAT			AGUUUGCGAU
						GAGACACTGA			GAGACACUGA
						ACAAGGGGCT			ACAAGGGGCU
						GATTTGGAGT			GAUUUGGAGU
						CCGGGCACCC			CCGGGCACCC
						CCAGCTCTGG			CCAGCUCUGG
						AGCCTGACAA			AGCCUGACAA
						GGAGGACA			GGAGGACA
						[SEQ ID			[SEQ ID
						NO:			NO:
						2156]			2158]
miR-	2932	−1.63	0.064183	TTTGGCTGAG	CGGTCCTTCT	TCAAGCCATG	UUUGGCUGAG	CGGUCCUUCU	UCAAGCCAUG
144				AGAAGGAACG	CATCAGCCAA	CTTCCTGTGC	AGAAGGAACG	CAUCAGCCAA	CUUCCUGUGC
				TG	A	CCCCAGTGGG	UG	A	CCCCAGUGGG
				[SEQ ID	[SEQ ID	GCCCTGGCTC	[SEQ ID	[SEQ ID	GCCCUGGCUC
				NO:	NO:	GGTCCTTCTC	NO:	NO:	GGUCCUUCUC
				672]	2159]	ATCAGCCAAA	1200]	2161]	AUCAGCCAAA
						AGTTTGCGAT			AGUUUGCGAU
						GAGACACTTT			GAGACACUUU
						GGCTGAGAGA			GGCUGAGAGA
						AGGAACGAGT			AGGAACGAGU
						CCGGGCACCC			CCGGGCACCC
						CCAGCTCTGG			CCAGCUCUGG
						AGCCTGACAA			AGCCUGACAA
						GGAGGACA			GGAGGACA
						[SEQ ID			[SEQ ID
						NO:			NO:
						2160]			2162]
miR-	3842	−1.57752	−0.42898	AACGTGAGAA	CGCTCCATCC	TCAAGCCATG	AACGUGAGAA	CGCUCCAUCC	UCAAGCCAUG
144				GGATGGATCG	TATCTCACGT	CTTCCTGTGC	GGAUGGAUCG	UAUCUCACGU	CUUCCUGUGC
				TA	T	CCCCAGTGGG	UA	U	CCCCAGUGGG
				[SEQ ID	[SEQ ID	GCCCTGGCTC	[SEQ ID	[SEQ ID	GCCCUGGCUC
				NO:	NO:	GCTCCATCCT	NO:	NO:	GCUCCAUCCU
				1625]	2163]	ATCTCACGTT	1824]	2165]	AUCUCACGUU
						AGTTTGCGAT			AGUUUGCGAU
						GAGACACAAC			GAGACACAAC
						GTGAGAAGGA			GUGAGAAGGA
						TGGATCGAGT			UGGAUCGAGU
						CCGGGCACCC			CCGGGCACCC
						CCAGCTCTGG			CCAGCUCUGG
						AGCCTGACAA			AGCCUGACAA
						GGAGGACA			GGAGGACA
						[SEQ ID			[SEQ ID
						NO:			NO:
						2164]			2166]
miR-	2353	−1.4808	0.451244	TTCGATGCAG	CCGGCTAGTC	TCAAGCCATG	UUCGAUGCAG	CCGGCUAGUC	UCAAGCCAUG
144				GACTAGCAGG	CATGCATCGA	CTTCCTGTGC	GACUAGCAGG	CAUGCAUCGA	CUUCCUGUGC
				CG	A	CCCCAGTGGG	CG	A	CCCCAGUGGG
				[SEQ ID	[SEQ ID	GCCCTGGCTC	[SEQ ID	[SEQ ID	GCCCUGGCUC
				NO:	NO:	CGGCTAGTCC	NO:	NO:	CGGCUAGUCC
				612]	2167]	ATGCATCGAA	166]	2169]	AUGCAUCGAA
						AGTTTGCGAT	(Same		AGUUUGCGAU
						GAGACACTTC	as		GAGACACUUC
						GATGCAGGAC	XD-		GAUGCAGGAC
						TAGCAGGAGT	14819)		UAGCAGGAGU
						CCGGGCACCC			CCGGGCACCC
						CCAGCTCTGG			CCAGCUCUGG
						AGCCTGACAA			AGCCUGACAA
						GGAGGACA			GGAGGACA
						[SEQ ID			[SEQ ID
						NO:			NO:
						2168]			2170]
miR-	3302	−2.52291	0.018122	TTGAACAAGG	CCCAAATCGC	CTGGAGGCTT	UUGAACAAGG	CCCAAAUCGC	CUGGAGGCUU
155E				GGCTGATTTG	CCTTGTTCAA	GCTTTGGGCT	GGCUGAUUUG	CCUUGUUCAA	GCUUUGGGCU
				GG	[SEQ ID	GTATGCTGTT	GG	[SEQ ID	GUAUGCUGUU
				[SEQ ID	NO:	GAACAAGGGG	[SEQ ID	NO:	GAACAAGGGG
				NO:	1652]	CTGATTTGGG	NO:	1847]	CUGAUUUGGG
				688]		TTTTGGCCTC	1216]		UUUUGGCCUC
						TGACTGACCC			UGACUGACCC
						AAATCGCCCT			AAAUCGCCCU
						TGTTCAACAG			UGUUCAACAG
						GACAAGGCCC			GACAAGGCCC
						TTTATCAGCA			UUUAUCAGCA
						CTCACATGGA			CUCACAUGGA
						ACAAATGGCC			ACAAAUGGCC
						ACCGTGGG			ACCGUGGG
						[SEQ ID			[SEQ ID
						NO:			NO:
						1734]			1931]
miR-	2586	−2.5179	−0.10172	TAGATTCAGA	CCAAGTTCAC	CTGGAGGCTT	UAGAUUCAGA	CCAAGUUCAC	CUGGAGGCUU
155E				AGTAGAACTT	TCTGAATCTA	GCTTTGGGCT	AGUAGAACUU	UCUGAAUCUA	GCUUUGGGCU
				GG	[SEQ ID	GTATGCTGTA	GG	[SEQ ID	GUAUGCUGUA
				[SEQ ID	NO:	GATTCAGAAG	[SEQ ID	NO:	GAUUCAGAAG
				NO:	1653]	TAGAACTTGG	NO:	1848]	UAGAACUUGG
				1621]		TTTTGGCCTC	1816]		UUUUGGCCUC
						TGACTGACCA			UGACUGACCA
						AGTTCACTCT			AGUUCACUCU
						GAATCTACAG			GAAUCUACAG
						GACAAGGCCC			GACAAGGCCC
						TTTATCAGCA			UUUAUCAGCA
						CTCACATGGA			CUCACAUGGA
						ACAAATGGCC			ACAAAUGGCC
						ACCGTGGG			ACCGUGGG
						[SEQ ID			[SEQ ID
						NO:			NO:
						1736]			1933]
miR-	1755	−2.39604	−0.03915	TCGGGTTGAA	CACACTTCGA	CTGGAGGCTT	UCGGGUUGAA	CACACUUCGA	CUGGAGGCUU
155E				ATCTGAAGTG	TTCAACCCGA	GCTTTGGGCT	AUCUGAAGUG	UUCAACCCGA	GCUUUGGGCU
				TG	[SEQ ID	GTATGCTGTC	UG	[SEQ ID	GUAUGCUGUC
				[SEQ ID	NO:	GGGTTGAAAT	[SEQ ID	NO:	GGGUUGAAAU
				NO:	1676]	CTGAAGTGTG	NO:	1871]	CUGAAGUGUG
				657]		TTTTGGCCTC	1185]		UUUUGGCCUC
						TGACTGACAC			UGACUGACAC
						ACTTCGATTC			ACUUCGAUUC
						AACCCGACAG			AACCCGACAG
						GACAAGGCCC			GACAAGGCCC
						TTTATCAGCA			UUUAUCAGCA
						CTCACATGGA			CUCACAUGGA
						ACAAATGGCC			ACAAAUGGCC
						ACCGTGGG			ACCGUGGG
						[SEQ ID			[SEQ ID
						NO:			NO:
						1766]			1963]
miR-	3273	−2.36984	−0.0801	TAGGACTGTA	CAATATGTGC	CTGGAGGCTT	UAGGACUGUA	CAAUAUGUGC	CUGGAGGCUU
155E				GGCAACATAT	CACAGTCCTA	GCTTTGGGCT	GGCAACAUAU	CACAGUCCUA	GCUUUGGGCU
				TG	[SEQ ID	GTATGCTGTA	UG	[SEQ ID	GUAUGCUGUA
				[SEQ ID	NO:	GGACTGTAGG	[SEQ ID	NO:	GGACUGUAGG
				NO:	1683]	CAACATATTG	NO:	1878]	CAACAUAUUG
				1628]		TTTTGGCCTC	1821]		UUUUGGCCUC
						TGACTGACAA			UGACUGACAA
						TATGTGCCAC			UAUGUGCCAC
						AGTCCTACAG			AGUCCUACAG
						GACAAGGCCC			GACAAGGCCC
						TTTATCAGCA			UUUAUCAGCA
						CTCACATGGA			CUCACAUGGA
						ACAAATGGCC			ACAAAUGGCC
						ACCGTGGG			ACCGUGGG
						[SEQ ID			[SEQ ID
						NO:			NO:
						1774]			1971]
miR-	3272	−2.34055	−0.19485	AGGACTGTAG	GCAATATGTG	CTGGAGGCTT	AGGACUGUAG	GCAAUAUGUG	CUGGAGGCUU
155E				GCAACATATT	CTACAGTCCT	GCTTTGGGCT	GCAACAUAUU	CUACAGUCCU	GCUUUGGGCU
				GC	[SEQ ID	GTATGCTGAG	GC	[SEQ ID	GUAUGCUGAG
				[SEQ ID	NO:	GACTGTAGGC	[SEQ ID	NO:	GACUGUAGGC
				NO:	1687]	AACATATTGC	NO:	1882]	AACAUAUUGC
				1618]		TTTTGGCCTC	1814]		UUUUGGCCUC
						TGACTGAGCA			UGACUGAGCA
						ATATGTGCTA			AUAUGUGCUA
						CAGTCCTCAG			CAGUCCUCAG
						GACAAGGCCC			GACAAGGCCC
						TTTATCAGCA			UUUAUCAGCA
						CTCACATGGA			CUCACAUGGA
						ACAAATGGCC			ACAAAUGGCC
						ACCGTGGG			ACCGUGGG
						[SEQ ID			[SEQ ID
						NO:			NO:
						1780]			1977]
miR-	3330	−2.26554	0.033188	TATGCTGAGA	CCACATTACA	CTGGAGGCTT	UAUGCUGAGA	CCACAUUACA	CUGGAGGCUU
155E				CTGATAATGT	GCTCAGCATA	GCTTTGGGCT	CUGAUAAUGU	GCUCAGCAUA	GCUUUGGGCU
				GG	[SEQ ID	GTATGCTGTA	GG	[SEQ ID	GUAUGCUGUA
				[SEQ ID	NO:	TGCTGAGACT	[SEQ ID	NO:	UGCUGAGACU
				NO:	1700]	GATAATGTGG	NO:	1895]	GAUAAUGUGG
				1614]		TTTTGGCCTC	1811]		UUUUGGCCUC
						TGACTGACCA			UGACUGACCA
						CATTACAGCT			CAUUACAGCU
						CAGCATACAG			CAGCAUACAG
						GACAAGGCCC			GACAAGGCCC
						TTTATCAGCA			UUUAUCAGCA
						CTCACATGGA			CUCACAUGGA
						ACAAATGGCC			ACAAAUGGCC
						ACCGTGGG			ACCGUGGG
						[SEQ ID			[SEQ ID
						NO:			NO:
						1793]			1990]
miR-	1162	−2.26036	−0.20831	AACTGTACCA	CAGACTTTTT	CTGGAGGCTT	AACUGUACCA	CAGACUUUUU	CUGGAGGCUU
155E				CAACAAAGTC	GGGTACAGTT	GCTTTGGGCT	CAACAAAGUC	GGGUACAGUU	GCUUUGGGCU
				TG	[SEQ ID	GTATGCTGAA	UG	[SEQ ID	GUAUGCUGAA
				[SEQ ID	NO:	CTGTACCACA	[SEQ ID	NO:	CUGUACCACA
				NO:	1706]	ACAAAGTCTG	NO:	1905]	ACAAAGUCUG
				652]		TTTTGGCCTC	1180]		UUUUGGCCUC
						TGACTGACAG			UGACUGACAG
						ACTTTTTGGG			ACUUUUUGGG
						TACAGTTCAG			UACAGUUCAG
						GACAAGGCCC			GACAAGGCCC
						TTTATCAGCA			UUUAUCAGCA
						CTCACATGGA			CUCACAUGGA
						ACAAATGGCC			ACAAAUGGCC
						ACCGTGGG			ACCGUGGG
						[SEQ ID			[SEQ ID
						NO:			NO:
						1801]			1998]
miR-	1580	−2.18059	−0.26086	ACTGGAATTT	AGCAGTTCGA	CTGGAGGCTT	ACUGGAAUUU	AGCAGUUCGA	CUGGAGGCUU
155E				CTCTGAACTG	GAATTCCAGT	GCTTTGGGCT	CUCUGAACUG	GAAUUCCAGU	GCUUUGGGCU
				CT	[SEQ ID	GTATGCTGAC	CU	[SEQ ID	GUAUGCUGAC
				[SEQ ID	NO:	TGGAATTTCT	[SEQ ID	NO:	UGGAAUUUCU
				NO:	2171]	CTGAACTGCT	NO:	2173]	CUGAACUGCU
				1622]		TTTTGGCCTC	1817]		UUUUGGCCUC
						TGACTGAAGC			UGACUGAAGC
						AGTTCGAGAA			AGUUCGAGAA
						TTCCAGTCAG			UUCCAGUCAG
						GACAAGGCCC			GACAAGGCCC
						TTTATCAGCA			UUUAUCAGCA
						CTCACATGGA			CUCACAUGGA
						ACAAATGGCC			ACAAAUGGCC
						ACCGTGGG			ACCGUGGG
						[SEQ ID			[SEQ ID
						NO:			NO:
						2172]			2174]
miR-	1436	−2.08545	0.133624	TGAGTTATCT	GCCCTTAGAA	CTGGAGGCTT	UGAGUUAUCU	GCCCUUAGAA	CUGGAGGCUU
155E				CTTTCTAAGG	GGATAACTCA	GCTTTGGGCT	CUUUCUAAGG	GGAUAACUCA	GCUUUGGGCU
				GC	[SEQ ID	GTATGCTGTG	GC	[SEQ ID	GUAUGCUGUG
				[SEQ ID	NO:	AGTTATCTCT	[SEQ ID	NO:	AGUUAUCUCU
				NO:	1710]	TTCTAAGGGC	NO:	1900]	UUCUAAGGGC
				1632]		TTTTGGCCTC	1827]		UUUUGGCCUC
						TGACTGAGCC			UGACUGAGCC
						CTTAGAAGGA			CUUAGAAGGA
						TAACTCACAG			UAACUCACAG
						GACAAGGCCC			GACAAGGCCC
						TTTATCAGCA			UUUAUCAGCA
						CTCACATGGA			CUCACAUGGA
						ACAAATGGCC			ACAAAUGGCC
						ACCGTGGG			ACCGUGGG
						[SEQ ID			[SEQ ID
						NO:			NO:
						2175]			2176]
miR-	3270	−2.03222	0.002948	TACTGTAGGC	ACGCAATAGT	CTGGAGGCTT	UACUGUAGGC	ACGCAAUAGU	CUGGAGGCUU
155E				AACATATTGC	TCCTACAGTA	GCTTTGGGCT	AACAUAUUGC	UCCUACAGUA	GCUUUGGGCU
				GT		GTATGCTGTA	GU	[SEQ ID	GUAUGCUGUA
				[SEQ ID	[SEQ ID	CTGTAGGCAA	[SEQ ID	NO:	CUGUAGGCAA
				NO:	NO:	CATATTGCGT	NO:	2179]	CAUAUUGCGU
				2080]	2177]	TTTTGGCCTC	2083]		UUUUGGCCUC
						TGACTGAACG			UGACUGAACG
						CAATAGTTCC			CAAUAGUUCC
						TACAGTACAG			UACAGUACAG
						GACAAGGCCC			GACAAGGCCC
						TTTATCAGCA			UUUAUCAGCA
						CTCACATGGA			CUCACAUGGA
						ACAAATGGCC			ACAAAUGGCC
						ACCGTGGG			ACCGUGGG
						[SEQ ID			[SEQ ID
						NO:			NO:
						2178]			2180]
miR-	3330	−2.86183	0.46905	TATGCTGAGA	CCATTATCAG	GAGCTCAGTC	UAUGCUGAGA	CCAUUAUCAG	GAGCUCAGUC
190a				CTGATAATGT	TCTCAGCACC	AAACCTGGAT	CUGAUAAUGU	UCUCAGCACC	AAACCUGGAU
				GG	[SEQ ID	GCCTTTTCTG	GG	[SEQ ID	GCCUUUUCUG
				[SEQ ID	NO:	CAGGCCTCTG	[SEQ ID	NO:	CAGGCCUCUG
				NO:	1634]	TGTATGCTGA	NO:	1829]	UGUAUGCUGA
				1614]		G	1811]		G
						ACTGATAATG			ACUGAUAAUG
						TGGTGTTATT			UGGUGUUAUU
						TAATCCACCA			UAAUCCACCA
						TTATCAGTCT			UUAUCAGUCU
						CAGCACCCTA			CAGCACCCUA
						CAGTGTCTTG			CAGUGUCUUG
						CCCTGTCTCC			CCCUGUCUCC
						GGGGGTTCCT			GGGGGUUCCU
						AATAAAG			AAUAAAG
						[SEQ ID			[SEQ ID
						NO:			NO:
						1712]			1909]
miR-	3302	−2.64186	0.124524	TTGAACAAGG	CCCATCAGCC	GAGCTCAGTC	UUGAACAAGG	CCCAUCAGCC	GAGCUCAGUC
190a				GGCTGATTTG	CCTTGTTCCC	AAACCTGGAT	GGCUGAUUUG	CCUUGUUCCC	AAACCUGGAU
				GG	[SEQ ID	GCCTTTTCTG	GG	[SEQ ID	GCCUUUUCUG
				[SEQ ID	NO:	CAGGCCTCTG	[SEQ ID	NO:	CAGGCCUCUG
				NO:	1643]	TGTTGAACAA	NO:	1838]	UGUUGAACAA
				688]		GGGGCTGATT	1216]		GGGGCUGAUU
						TGGGTGTTAT			UGGGUGUUAU
						TTAATCCACC			UUAAUCCACC
						CATCAGCCCC			CAUCAGCCCC
						TTGTTCCCCT			UUGUUCCCCU
						ACAGTGTCTT			ACAGUGUCUU
						GCCCTGTCTC			GCCCUGUCUC
						CGGGGGTTCC			CGGGGGUUCC
						TAATAAAG			UAAUAAAG
						[SEQ ID			[SEQ ID
						NO:			NO:
						1721]			1918]
miR-	1580	−2.50168	−0.06321	ACTGGAATTT	AGCTTCAGAG	GAGCTCAGTC	ACUGGAAUUU	AGCUUCAGAG	GAGCUCAGUC
190a				CTCTGAACTG	AAATTCCAAG	AAACCTGGAT	CUCUGAACUG	AAAUUCCAAG	AAACCUGGAU
				CT	[SEQ ID	GCCTTTTCTG	CU	[SEQ ID	GCCUUUUCUG
				[SEQ ID	NO:	CAGGCCTCTG	[SEQ ID	NO:	CAGGCCUCUG
				NO:	1654]	TGACTGGAAT	NO:	1849]	UGACUGGAAU
				1622]		TTCTCTGAAC	1817]		UUCUCUGAAC
						TGCTTGTTAT			UGCUUGUUAU
						TTAATCCAAG			UUAAUCCAAG
						CTTCAGAGAA			CUUCAGAGAA
						ATTCCAAGCT			AUUCCAAGCU
						ACAGTGTCTT			ACAGUGUCUU
						GCCCTGTCTC			GCCCUGUCUC
						CGGGGGTTCC			CGGGGGUUCC
						TAATAAAG			UAAUAAAG
						[SEQ ID			[SEQ ID
						NO:			NO:
						1738]			1911]
miR-	1755	−2.38113	0.329455	TCGGGTTGAA	CACTTCAGAT	GAGCTCAGTC	UCGGGUUGAA	CACUUCAGAU	GAGCUCAGUC
190a				ATCTGAAGTG	TTCAACCCAC	AAACCTGGAT	AUCUGAAGUG	UUCAACCCAC	AAACCUGGAU
				TG	[SEQ ID	GCCTTTTCTG	UG	[SEQ ID	GCCUUUUCUG
				[SEQ ID	NO:	CAGGCCTCTG	[SEQ ID	NO:	CAGGCCUCUG
				NO:	1680]	TGTCGGGTTG	NO:	1875]	UGUCGGGUUG
				657]		AAATCTGAAG	1185]		AAAUCUGAAG
						TGTGTGTTAT			UGUGUGUUAU
						TTAATCCACA			UUAAUCCACA
						CTTCAGATT			CUUCAGAU
						TCAACCCACC			UUCAACCCAC
						TACAGTGTCT			CUACAGUGUC
						TGCCCTGTCT			UUGCCCUGUC
						CCGGGGGTTC			UCCGGGGGUU
						CTAATAAAG			CCUAAUAAAG
						[SEQ ID			[SEQ ID
						NO:			NO:
						1771]			1968]
miR-	3301	−2.36785	0.195332	TGAACAAGGG	TCCAATCAGC	GAGCTCAGTC	UGAACAAGGG	UCCAAUCAGC	GAGCUCAGUC
190a				GCTGATTTGG	CCCTTGTTAC	AAACCTGGAT	GCUGAUUUGG	CCCUUGUUAC	AAACCUGGAU
				GA	[SEQ ID	GCCTTTTCTG	GA	[SEQ ID	GCCUUUUCUG
				[SEQ ID	NO:	CAGGCCTCTG	[SEQ ID	NO:	CAGGCCUCUG
				NO:	1684]	TGTGAACAAG	NO:	1879]	UGUGAACAAG
				687]		GGGCTGATTT	1215]		GGGCUGAUUU
						GGGATGTTAT			GGGAUGUUAU
						TTAATCCATC			UUAAUCCAUC
						CAATCAGCCC			CAAUCAGCCC
						CTTGTTACCT			CUUGUUACCU
						ACAGTGTCTT			ACAGUGUCUU
						GCCCTGTCTC			GCCCUGUCUC
						CGGGGGTTCC			CGGGGGUUCC
						TAATAAAG			UAAUAAAG
						[SEQ ID			[SEQ ID
						NO:			NO:
						1775]			1972]
miR-	3043	−2.26041	0.277711	TTTGGTGCAA	AGCGTTTGTT	GAGCTCAGTC	UUUGGUGCAA	AGCGUUUGUU	GAGCUCAGUC
190a				AACAAACAGG	TTGCACCACC	AAACCTGGAT	AACAAACAGG	UUGCACCACC	AAACCUGGAU
				CT	[SEQ ID	GCCTTTTCTG	CU	[SEQ ID	GCCUUUUCUG
				[SEQ ID	NO:	CAGGCCTCTG	[SEQ ID	NO:	CAGGCCUCUG
				NO:	1664]	TGTTTGGTGC	NO:	1859]	UGUUUGGUGC
				1615]		AAAACAAACA	1812]		AAAACAAACA
						GGCTTGTTAT			GGCUUGUUAU
						TTAATCCAAG			UUAAUCCAAG
						CGTTTGTTTT			CGUUUGUUUU
						GCACCACCCT			GCACCACCCU
						ACAGTGTCTT			ACAGUGUCUU
						GCCCTGTCTC			GCCCUGUCUC
						CGGGGGTTCC			CGGGGGUUCC
						TAATAAAG			UAAUAAAG
						[SEQ ID			[SEQ ID
						NO:			NO:
						1800]			1997]
miR-	2586	−2.24846	0.172174	TAGATTCAGA	CCATTCTACT	GAGCTCAGTC	UAGAUUCAGA	CCAUUCUACU	GAGCUCAGUC
190a				AGTAGAACTT	TCTGAATCCC	AAACCTGGAT	AGUAGAACUU	UCUGAAUCCC	AAACCUGGAU
				GG	[SEQ ID	GCCTTTTCTG	GG	[SEQ ID	GCCUUUUCUG
				[SEQ ID	NO:	CAGGCCTCTG	[SEQ ID	NO:	CAGGCCUCUG
				NO:	1661]	TGTAGATTCA	NO:	1856]	UGUAGAUUCA
				1621]		GAAGTAGAAC	1816]		GAAGUAGAAC
						TTGGTGTTAT			UUGGUGUUAU
						TTAATCCACC			UUAAUCCACC
						ATTCTACTTC			AUUCUACUUC
						TGAATCCCCT			UGAAUCCCCU
						ACAGTGTCTT			ACAGUGUCUU
						GCCCT			GCCC
						GTCTCCGGGG			UGUCUCCGGG
						GTTCCTAATA			GGUUCCUAAU
						AAG			AAAG
						[SEQ ID			[SEQ ID
						NO:			NO:
						1803]			2000]
miR-	967	−2.23671	−0.08895	ACTGATGTAA	TGGCATATAC	GAGCTCAGTC	ACUGAUGUAA	UGGCAUAUAC	GAGCUCAGUC
190a				GTATATGAAC	TTACATCAAG	AAACCTGGAT	GUAUAUGAAC	UUACAUCAAG	AAACCUGGAU
				CA	[SEQ ID	GCCTTTTCTG	CA	[SEQ ID	GCCUUUUCUG
				[SEQ ID	NO:	CAGGCCTCTG	[SEQ ID	NO:	CAGGCCUCUG
				NO:	1642]	TGACTGATGT	NO:	1837]	UGACUGAUGU
				1619]		AAGTATATGA	1815]		AAGUAUAUGA
						ACCATGTTAT			ACCAUGUUAU
						TTAATCCATG			UUAAUCCAUG
						GCATATACTT			GCAUAUACUU
						ACATCAAGCT			ACAUCAAGCU
						ACAGTGTCTT			ACAGUGUCUU
						GCCCTGTCTC			GCCCUGUCUC
						CGGGGGTTCC			CGGGGGUUCC
						TAATAAAG			UAAUAAAG
						[SEQ ID			[SEQ ID
						NO:			NO:
						1809]			2006]
miR-	3255	−2.21465	0.46382	ATTGCGTGGA	CCAAGCTTAC	GAGCTCAGTC	AUUGCGUGGA	CCAAGCUUAC	GAGCUCAGUC
190a				GTAAGCTGGT	TCCACGCACG	AAACCTGGAT	GUAAGCUGGU	UCCACGCACG	AAACCUGGAU
				GG	[SEQ ID	GCCTTTTCTG	GG	[SEQ ID	GCCUUUUCUG
				[SEQ ID	NO:	CAGGCCTCTG	[SEQ ID	NO:	CAGGCCUCUG
				NO:	1697]	TGATTGCGTG	NO:	1892]	UGAUUGCGUG
				617]		GAGTAAGCTG	306]		GAGUAAGCUG
						GTGGTGTTAT	(Same		GUGGUGUUAU
						TTAATCCACC	guide		UUAAUCCACC
						AAGCTTACTC	as		AAGCUUACUC
						CACGCACGCT	XD-		CACGCACGCU
						ACAGTGTCTT	14889)		ACAGUGUCUU
						GCCCTGTCTC			GCCCUGUCUC
						CGGGGGTTCC			CGGGGGUUCC
						TAATAAAG			UAAUAAAG
						[SEQ ID			[SEQ ID
						NO:			NO:
						2181]			2182]
miR-	3341	−2.17489	0.322497	ATAGACATGA	GTCAGCATCC	GAGCTCAGTC	AUAGACAUGA	GUCAGCAUCC	GAGCUCAGUC
190a				GGATGCTGAG	TCATGTCTCG	AAACCTGGAT	GGAUGCUGAG	UCAUGUCUCG	AAACCUGGAU
				AC	[SEQ ID	GCCTTTTCTG	AC	[SEQ ID	GCCUUUUCUG
				[SEQ ID	NO:	CAGGCCTCTG	[SEQ ID	NO:	CAGGCCUCUG
				NO:	1703]	TGATAGACAT	NO:	1898]	UGAUAGACAU
				1617]		GAGGATGCTG	1813]		GAGGAUGCUG
						AGACTGTTAT			AGACUGUUAU
						TTAATCCAGT			UUAAUCCAGU
						CAGCATCCTC			CAGCAUCCUC
						ATGTCTCGCT			AUGUCUCGCU
						ACAGTGTCTT			ACAGUGUCUU
						GCCCTGTCTC			GCCCUGUCUC
						CGGGGGTTCC			CGGGGGUUCC
						TAATAAAG			UAAUAAAG
						[SEQ ID			[SEQ ID
						NO:			NO:
						2183]			2184]
miR-	967	−2.64307	0.113083	ACTGATGTAA	TGGCATATAC	GAGCTCAGTC	ACUGAUGUAA	UGGCAUAUAC	GAGCUCAGUC
190a_				GTATATGAAC	TTACATCAAG	AAACCTGGAT	GUAUAUGAAC	UUACAUCAAG	AAACCUGGAU
M				CA	[SEQ ID	GCCTTTTCTG	CA	[SEQ ID	GCCUUUUCUG
				[SEQ ID	NO:	CAGGCGTCTG	[SEQ ID	NO:	CAGGCGUCUG
				NO:	1642]	TGACTGATGT	NO:	1837]	UGACUGAUGU
				1619]		AAGTATATGA	1815]		AAGUAUAUGA
						ACCATGTTAT			ACCAUGUUAU
						TTAATCCATG			UUAAUCCAUG
						GCATATACTT			GCAUAUACUU
						ACATCAAGCT			ACAUCAAGCU
						ACAGTCTCTT			ACAGUCUCUU
						GCCCTGTCTC			GCCCUGUCUC
						CGGGGGTTCC			CGGGGGUUCC
						TAATAAAG			UAAUAAAG
						[SEQ ID			[SEQ ID
						NO:			NO:
						1720]			1917]
miR-	3302	−2.52395	0.249656	TTGAACAAGG	CCCATCAGCC	GAGCTCAGTC	UUGAACAAGG	CCCAUCAGCC	GAGCUCAGUC
190a_				GGCTGATTTG	CCTTGTTCCC	AAACCTGGAT	GGCUGAUUUG	CCUUGUUCCC	AAACCUGGAU
M				GG	[SEQ ID	GCCTTTTCTG	GG	[SEQ ID	GCCUUUUCUG
				[SEQ ID	NO:	CAGGCGTCTG	[SEQ ID	NO:	CAGGCGUCUG
				NO:	1643]	TGTTGAACAA	NO:	1838]	UGUUGAACAA
				688]		GGGGCTGATT	1216]		GGGGCUGAUU
						TGGGTGTTAT			UGGGUGUUAU
						TTAATCCACC			UUAAUCCACC
						CATCAGCCCC			CAUCAGCCCC
						TTGTTCCCCT			UUGUUCCCCU
						ACAGTCTCTT			ACAGUCUCUU
						GCCCTGTCTC			GCCCUGUCUC
						CGGGGGTTCC			CGGGGGUUCC
						TAATAAAG			UAAUAAAG
						[SEQ ID			[SEQ ID
						NO:			NO:
						1733]			1930]
miR-	2586	−2.46486	0.014777	TAGATTCAGA	CCATTCTACT	GAGCTCAGTC	UAGAUUCAGA	CCAUUCUACU	GAGCUCAGUC
190a_				AGTAGAACTT	TCTGAATCCC	AAACCTGGAT	AGUAGAACUU	UCUGAAUCCC	AAACCUGGAU
M				GG	[SEQ ID	GCCTTTTCTG	GG	[SEQ ID	GCCUUUUCUG
				[SEQ ID	NO:	CAGGCGTCTG	[SEQ ID	NO:	CAGGCGUCUG
				NO:	1661]	TGTAGATTCA	NO:	1856]	UGUAGAUUCA
				1621]		GAAGTAGAAC	1816]		GAAGUAGAAC
						TTGGTGTTAT			UUGGUGUUAU
						TTAATCCACC			UUAAUCCACC
						ATTCTACTTC			AUUCUACUUC
						TGAATCCCCT			UGAAUCCCCU
						ACAGTCTCTT			ACAGUCUCUU
						GCCCTGTCTC			GCCCUGUCUC
						CGGGGGTTCC			CGGGGGUUCC
						TAATAAAG			UAAUAAAG
						[SEQ ID			[SEQ ID
						NO:			NO:
						1746]			1943]
miR-	3043	−2.44458	0.289334	TTTGGTGCAA	AGCGTTTGTT	GAGCTCAGTC	UUUGGUGCAA	AGCGUUUGUU	GAGCUCAGUC
190a_				AACAAACAGG	TTGCACCACC	AAACCTGGAT	AACAAACAGG	UUGCACCACC	AAACCUGGAU
M				CT	[SEQ ID	GCCTTTTCTG	CU	[SEQ ID	GCCUUUUCUG
				[SEQ ID	NO:	CAGGCGTC	[SEQ ID	NO:	CAGGCGUCU
				NO:	1664]	TGTGTTTGGT	NO:	1859]	GUGUUUGGUG
				1615]		GCAAAACAAA	1812]		CAAAACAAAC
						CAGGCTTGTT			AGGCUUGUUA
						ATTTAATCCA			UUUAAUCCAA
						AGCGTTTGTT			GCGUUUGUUU
						TTGCACCACC			UGCACCACCC
						CTACAGTCTC			UACAGUCUCU
						TTGCCCTGTC			UGCCCUGUCU
						TCCGGGGGTT			CCGGGGGUUC
						CCTAATAAAG			CUAAUAAAG
						[SEQ ID			[SEQ ID
						NO:			NO:
						1750]			1947]
miR-	1755	−2.34216	0.272299	TCGGGTTGAA	CACTTCAGAT	GAGCTCAGTC	UCGGGUUGAA	CACUUCAGAU	GAGCUCAGUC
190a_				ATCTGAAGTG	TTCAACCCAC	AAACCTGGAT	AUCUGAAGUG	UUCAACCCAC	AAACCUGGAU
M				TG	[SEQ ID	GCCTTTTCTG	UG	[SEQ ID	GCCUUUUCUG
				[SEQ ID	NO:	CAGGCGTCTG	[SEQ ID	NO:	CAGGCGUCUG
				NO:	1680]	TGTCGGGTTG	NO:	1875]	UGUCGGGUUG
				657]		AAATCTGAAG	1185]		AAAUCUGAAG
						TGTGTGTTAT			UGUGUGUUAU
						TTAATCCACA			UUAAUCCACA
						CTTCAGATTT			CUUCAGAUUU
						CAACCCACCT			CAACCCACCU
						ACAGTCTCTT			ACAGUCUCUU
						GCCCTGTCTC			GCCCUGUCUC
						CGGGGGTTCC			CGGGGGUUCC
						TAATAAAG			UAAUAAAG
						[SEQ ID			[SEQ ID
						NO:			NO:
						1778]			1975]
miR-	3269	−2.3297	0.037293	ACTGTAGGCA	CACAATATGT	GAGCTCAGTC	ACUGUAGGCA	CACAAUAUGU	GAGCUCAGUC
190a_				ACATATTGCG	TGCCTACAAG	AAACCTGGAT	ACAUAUUGCG	UGCCUACAAG	AAACCUGGAU
M				TG	[SEQ ID	GCCTTTTCTG	UG	[SEQ ID	GCCUUUUCUG
				[SEQ ID	NO:	CAGGCGTCTG	[SEQ ID	NO:	CAGGCGUCUG
				NO:	1689]	TGACTGTAGG	NO:	1884]	UGACUGUAGG
				1629]		CAACATATTG	1822]		CAACAUAUUG
						CGTGTGTTAT			CGUGUGUUAU
						TTAATCCACA			UUAAUCCACA
						CAATATGTTG			CAAUAUGUUG
						CCTACAAGCT			CCUACAAGCU
						ACAGTCTCTT			ACAGUCUCUU
						GCCCTGTCTC			GCCCUGUCUC
						CGGGGGTTCC			CGGGGGUUCC
						TAATAAAG			UAAUAAAG
						[SEQ ID			[SEQ ID
						NO:			NO:
						1782]			1979]
miR-	3255	−2.30348	0.418205	ATTGCGTGGA	CCAAGCTTAC	GAGCTCAGTC	AUUGCGUGGA	CCAAGCUUAC	GAGCUCAGUC
190a_				GTAAGCTGGT	TCCACGCACG	AAACCTGGAT	GUAAGCUGGU	UCCACGCACG	AAACCUGGAU
M				GG	[SEQ ID	GCCTTTTCTG	GG	[SEQ ID	GCCUUUUCUG
				[SEQ ID	NO:	CAGGCGTCTG	[SEQ ID	NO:	CAGGCGUCUG
				NO:	1697]	TGATTGCGTG	NO:	1892]	UGAUUGCGUG
				617]		GAGTAAGCTG	306]		GAGUAAGCUG
						GTGGTGTTAT	(Same		GUGGUGUUAU
						TTAATCCACC	guide		UUAAUCCACC
						AAGCTTACT	as		AAGCUUAC
						CCACGCACGC	XD-		UCCACGCACG
						TACAGTCTCT	14889)		CUACAGUCUC
						TGCCCTGTCT			UUGCCCUGUC
						CCGGGGGTTC			UCCGGGGGUU
						CTAATAAAG			CCUAAUAAAG
						[SEQ ID			[SEQ ID
						NO:			NO:
						1790]			1987]
miR-	3341	−2.26383	0.394316	ATAGACATGA	GTCAGCATCC	GAGCTCAGTC	AUAGACAUGA	GUCAGCAUCC	GAGCUCAGUC
190a_				GGATGCTGAG	TCATGTCTCG	AAACCTGGAT	GGAUGCUGAG	UCAUGUCUCG	AAACCUGGAU
M				AC	[SEQ ID	GCCTTTTCTG	AC	[SEQ ID	GCCUUUUCUG
				[SEQ ID	NO:	CAGGCGTCTG	[SEQ ID	NO:	CAGGCGUCUG
				NO:	1703]	TGATAGACAT	NO:	1898]	UGAUAGACAU
				1617]		GAGGATGCTG	1813]		GAGGAUGCUG
						AGACTGTTAT			AGACUGUUAU
						TTAATCCAGT			UUAAUCCAGU
						CAGCATCCTC			CAGCAUCCUC
						ATGTCTCGCT			AUGUCUCGCU
						ACAGTCTCTT			ACAGUCUCUU
						GCCCTGTCTC			GCCCUGUCUC
						CGGGGGTTCC			CGGGGGUUCC
						TAATAAAG			UAAUAAAG
						[SEQ ID			[SEQ ID
						NO:			NO:
						1796]			1993]
miR-	1436	−2.18684	0.218825	TGAGTTATCT	GCCTAGAAAG	GAGCTCAGTC	UGAGUUAUCU	GCCUAGAAAG	GAGCUCAGUC
190a_				CTTTCTAAGG	AGATAACTAC	AAACCTGGAT	CUUUCUAAGG	AGAUAACUAC	AAACCUGGAU
M				GC	[SEQ ID	GCCTTTTCTG	GC	[SEQ ID	GCCUUUUCUG
				[SEQ ID	NO:	CAGGCGTCTG	[SEQ ID	NO:	CAGGCGUCUG
				NO:	2185]	TGTGAGTTAT	NO:	1899]	UGUGAGUUAU
				1632]		CTCTTTCTAA	1827]		CUCUUUCUAA
						GGGCTGTTAT			GGGCUGUUAU
						TTAATCCAGC			UUAAUCCAGC
						CTAGAAAGAG			CUAGAAAGAG
						ATAACTACCT			AUAACUACCU
						ACAGTCTCTT			ACAGUCUCUU
						GCCCTGTCTC			GCCCUGUCUC
						CGGGGGTTCC			CGGGGGUUCC
						TAATAAAG			UAAUAAAG
						[SEQ ID			[SEQ ID
						NO:			NO:
						2186]			2187]
miR-	3330	−2.17382	0.344749	TATGCTGAGA	CCATTATCAG	GAGCTCAGTC	UAUGCUGAGA	CCAUUAUCAG	GAGCUCAGUC
190a_				CTGATAATGT	TCTCAGCACC	AAACCTGGAT	CUGAUAAUGU	UCUCAGCACC	AAACCUGGAU
M				GG	[SEQ ID	GCCTTTTCTG	GG	[SEQ ID	GCCUUUUCUG
				[SEQ ID	NO:	CAGGCGTCTG	[SEQ ID	NO:	CAGGCGUCUG
				NO:	1634]	TGTATGCTGA	NO:	1829]	UGUAUGCUGA
				1614]		GACTGATAAT	1811]		GACUGAUAAU
						GTGGTGTTAT			GUGGUGUUAU
						TTAATCCACC			UUAAUCCACC
						ATTATCAGTC			AUUAUCAGUC
						TCAGCACCCT			UCAGCACCCU
						ACAGTCTCTT			ACAGUCUCUU
						GCCCT			GCCC
						GTCTCCGGGG			UGUCUCCGGG
						GTTCCTAATA			GGUUCCUAAU
						AAG			AAAG
						[SEQ ID			[SEQ ID
						NO:			NO:
						2188]			2189]
miR	3302	−2.42428	0.285147	TTGAACAAGG	CCCAAATCGC	CCTGGAGGCT	UUGAACAAGG	CCCAAAUCGC	CCUGGAGGCU
155-M				GGCTGATTTG	CCTTGTTCAA	TGCTGAAGGC	GGCUGAUUUG	CCUUGUUCAA	UGCUGAAGGC
				GG	[SEQ ID	TGTATGCTGT	GG	[SEQ ID	UGUAUGCUGU
				[SEQ ID	NO:	TGAACAAGGG	[SEQ ID	NO:	UGAACAAGGG
				NO:	1652]	GCTGATTTGG	NO:	1847]	GCUGAUUUGG
				688]		GTTTTGGCCA	1216]		GUUUUGGCCA
						CTGACTGACC			CUGACUGACC
						CAAATCGCCC			CAAAUCGCCC
						TTGTTCAACA			UUGUUCAACA
						GGACACAAGG			GGACACAAGG
						CCTGTTACTA			CCUGUUACUA
						GCACTCACAT			GCACUCACAU
						GGAACAAATG			GGAACAAAUG
						GCCACCGG			GCCACCGG
						[SEQ ID			[SEQ ID
						NO:			NO:
						1758]			1955]
miR	2586	−2.4228	−0.13209	TAGATTCAGA	CCAAGTTCAC	CCTGGAGGCT	UAGAUUCAGA	CCAAGUUCAC	CCUGGAGGCU
155-M				AGTAGAACTT	TCTGAATCTA	TGCTGAAGGC	AGUAGAACUU	UCUGAAUCUA	UGCUGAAGGC
				GG	[SEQ ID	TGTATGCTGT	GG	[SEQ ID	UGUAUGCUGU
				[SEQ ID	NO:	AGATTCAGAA	[SEQ ID	NO:	AGAUUCAGAA
				NO:	1653]	GTAGAACTTG	NO:	1848]	GUAGAACUUG
				1621]		GTTTTGGCCA	1816]		GUUUUGGCCA
						CTGACTGACC			CUGACUGACC
						AAGTTCACTC			AAGUUCACUC
						TGAATCTACA			UGAAUCUACA
						GGACACAAGG			GGACACAAGG
						CCTGTTACTA			CCUGUUACUA
						GCACTCACAT			GCACUCACAU
						GGAACAAATG			GGAACAAAUG
						GCCACCGG			GCCACCGG
						[SEQ ID			[SEQ ID
						NO:			NO:
						1759]			1956]
miR	1436	−2.22884	0.141256	TGAGTTATCT	GCCCTTAGAA	CCTGGAGGCT	UGAGUUAUCU	GCCCUUAGAA	CCUGGAGGCU
155-M				CTTTCTAAGG	GGATAACTCA	TGCTGAAGGC	CUUUCUAAGG	GGAUAACUCA	UGCUGAAGGC
				GC	[SEQ ID	TGTATGCTGT	GC	[SEQ ID	UGUAUGCUGU
				[SEQ ID	NO:	GAGTTATCTC	[SEQ ID	NO:	GAGUUAUCUC
				NO:	1710]	TTTCTAAGGG	NO:	1900]	UUUCUAAGGG
				1632]		CTTTTGGCCA	1827]		CUUUUGGCCA
						CTGACTGAGC			CUGACUGAGC
						CCTTAGAAGG			CCUUAGAAGG
						ATAACTCACA			AUAACUCACA
						GGACACAAGG			GGACACAAGG
						CCTGTTACTA			CCUGUUACUA
						GCACTCACAT			GCACUCACAU
						GGAA			GGAACAAAUG
						CAAATGGCCA			GCCACCGG
						CCGG			[SEQ ID
						[SEQ ID			NO:
						NO:			2007]
						1810]
miR	3272	−2.02279	−0.09047	AGGACTGTAG	GCAATATGTG	CCTGGAGGCT	AGGACUGUAG	GCAAUAUGUG	CCUGGAGGCU
155-M				GCAACATATT	CTACAGTCCT	TGCTGAAGGC	GCAACAUAUU	CUACAGUCCU	UGCUGAAGGC
				GC	[SEQ ID	TGTATGCTGA	GC	[SEQ ID	UGUAUGCUGA
				[SEQ ID	NO:	GGACTGTAGG	[SEQ ID	NO:	GGACUGUAGG
				NO:	1687]	CAACATATTG	NO:	1882]	CAACAUAUUG
				1618]		CTTTTGGCCA	1814]		CUUUUGGCCA
						CTGACTGAGC			CUGACUGAGC
						AATATGTGCT			AAUAUGUGCU
						ACAGTCCTCA			ACAGUCCUCA
						GGACACAAGG			GGACACAAGG
						CCTGTTACTA			CCUGUUACUA
						GCACTCACAT			GCACUCACAU
						GGAACAAATG			GGAACAAAUG
						GCCACCGG			GCCACCGG
						[SEQ ID			[SEQ ID
						NO:			NO:
						2190]			2191]
miR	1755	−1.98201	−0.05599	TCGGGTTGAA	CACACTTCGA	CCTGGAGGCT	UCGGGUUGAA	CACACUUCGA	CCUGGAGGCU
155-M				ATCTGAAGTG	TTCAACCCGA	TGCTGAAGGC	AUCUGAAGUG	UUCAACCCGA	UGCUGAAGGC
				TG	[SEQ ID	TGTATGCTGT	UG	[SEQ ID	UGUAUGCUGU
				[SEQ ID	NO:	CGGGTTGAAA	[SEQ ID	NO:	CGGGUUGAAA
				NO:	1676]	TCTGAAGTGT	NO:	1871]	UCUGAAGUGU
				657]		GTTTTGGCCA	1185]		GUUUUGGCCA
						CTGACTGACA			CUGACUGACA
						CACTTCGATT			CACUUCGAUU
						CAACCCGACA			CAACCCGACA
						GGACACAAGG			GGACACAAGG
						CCTGTTACTA			CCUGUUACUA
						GCACTCACAT			GCACUCACAU
						GGAACAAATG			GGAACAAAUG
						GCCACCGG			GCCACCGG
						[SEQ ID			[SEQ ID
						NO:			NO:
						2192]			2193]
miR	1162	−1.86763	−0.12823	AACTGTACCA	CAGACTTTTT	CCTGGAGGCT	AACUGUACCA	CAGACUUUUU	CCUGGAGGCU
155-M				CAACAAAGTC	GGGTACAGTT	TGCTGAAGGC	CAACAAAGUC	GGGUACAGUU	UGCUGAAGGC
				TG	[SEQ ID	TGTATGCTGA	UG	[SEQ ID	UGUAUGCUGA
				[SEQ ID	NO:	ACTGTACCAC	[SEQ ID	NO:	ACUGUACCAC
				NO:	1706]	AACAAAGTCT	NO:	1905]	AACAAAGUCU
				652]		GTTTTGGCCA	1180]		GUUUUGGCCA
						CTGACTGACA			CUGACUGACA
						GACTTTTTGG			GACUUUUUGG
						GTACAGTTCA			GUACAGUUCA
						GGACACAAGG			GGACACAAGG
						CCTGTTACTA			CCUGUUACUA
						GCACTCACAT			GCACUCACAU
						GGAA			GGAACAAAUG
						CAAATGGCCA			GCCACCGG
						CCGG			[SEQ ID
						[SEQ ID			NO:
						NO:			2195]
						2194]
miR	1580	−1.73791	−0.21175	ACTGGAATTT	AGCAGTTCGA	CCTGGAGGCT	ACUGGAAUUU	AGCAGUUCGA	CCUGGAGGCU
155-M				CTCTGAACTG	GAATTCCAGT	TGCTGAAGGC	CUCUGAACUG	GAAUUCCAGU	UGCUGAAGGC
				CT	[SEQ ID	TGTATGCTGA	CU	[SEQ ID	UGUAUGCUGA
				[SEQ ID	NO:	CTGGAATTTC	[SEQ ID	NO:	CUGGAAUUUC
				NO:	2171]	TCTGAACTGC	NO:	2173]	UCUGAACUGC
				1622]		TTTTTGGCCA	1817]		UUUUUGGCCA
						CTGACTGAAG			CUGACUGAAG
						CAGTTCGAGA			CAGUUCGAGA
						ATTCCAGTCA			AUUCCAGUCA
						GGACACAAGG			GGACACAAGG
						CCTGTTACTA			CCUGUUACUA
						GCACTCACAT			GCACUCACAU
						GGAACAAATG			GGAACAAAUG
						GCCACCGG			GCCACCGG
						[SEQ ID			[SEQ ID
						NO:			NO:
						2196]			2197]
miR	3273	−1.30321	−0.03402	TAGGACTGTA	CAATATGTGC	CCTGGAGGCT	UAGGACUGUA	CAAUAUGUGC	CCUGGAGGCU
155-M				GGCAACATAT	CACAGTCCTA	TGCTGAAGGC	GGCAACAUAU	CACAGUCCUA	UGCUGAAGGC
				TG	[SEQ ID	TGTATGCTGT	UG	[SEQ ID	UGUAUGCUGU
				[SEQ ID	NO:	AGGACTGTAG	[SEQ ID	NO:	AGGACUGUAG
				NO:	1683]	GCAACATATT	NO:	1878]	GCAACAUAUU
				1628]		GTTTTGGCCA	1821]		GUUUUGGCCA
						CTGACTGACA			CUGACUGACA
						ATATGTGCCA			AUAUGUGCCA
						CAGTCCTACA			CAGUCCUACA
						GGACACAAGG			GGACACAAGG
						CCTGTTACTA			CCUGUUACUA
						GCACTCACAT			GCACUCACAU
						GGAACAAATG			GGAACAAAUG
						GCCACCGG			GCCACCGG
						[SEQ ID			[SEQ ID
						NO:			NO:
						2198]			2199]
miR	3291	−1.28366	0.074799	TCTGATTTGG	CCTCAGCATT	CCTGGAGGCT	UCUGAUUUGG	CCUCAGCAUU	CCUGGAGGCU
155-M				GAACTGCTGA	CCAAATCAGA	TGCTGAAGGC	GAACUGCUGA	CCAAAUCAGA	UGCUGAAGGC
				GG	[SEQ ID	TGTATGCTGT	GG	[SEQ ID	UGUAUGCUGU
				[SEQ ID	NO:	CTGATTTGGG	[SEQ ID	NO:	CUGAUUUGGG
				NO:	2201]	AACTGCTGAG	NO:	2204]	AACUGCUGAG
				2200]		GTTTTGGCCA	2203]		GUUUUGGCCA
						CTGACTGACC			CUGACUGACC
						TCAGCATTCC			UCAGCAUUCC
						AAATCAGACA			AAAUCAGACA
						GGACACAAGG			GGACACAAGG
						CCTGTTACTA			CCUGUUACUA
						GCACTCACAT			GCACUCACAU
						GGA			GGAACAAAUG
						ACAAATGGCC			GCCACCGG
						ACCGG			[SEQ ID
						[SEQ ID			NO:
						NO:			2205]
						2202]
miR	2914	−1.18865	0.250219	TGTGGGTTGA	ATGCAAAGAG	CCTGGAGGCT	UGUGGGUUGA	AUGCAAAGAG	CCUGGAGGCU
155-M				ACTCCTTTGC	TCAACCCACA	TGCTGAAGGC	ACUCCUUUGC	UCAACCCACA	UGCUGAAGGC
				AT	[SEQ ID	TGTATGCTGT	AU	[SEQ ID	UGUAUGCUGU
				[SEQ ID	NO:	GTGGGTTGAA	[SEQ ID	NO:	GUGGGUUGAA
				NO:	2207]	CTCCTTTGCA	NO:	2210]	CUCCUUUGCA
				2206]		TTTTTGGCCA	2209]		UUUUUGGCCA
						CTGACTGAAT			CUGACUGAAU
						GCAAAGAGTC			GCAAAGAGUC
						AACCCACACA			AACCCACACA
						GGACACAAGG			GGACACAAGG
						CCTGTTACTA			CCUGUUACUA
						GCACTCACAT			GCACUCACAU
						GGAACAAATG			GGAACAAAUG
						GCCACCGG			GCCACCGG
						[SEQ ID			[SEQ ID
						NO:			NO:
						2208]			2211]

Methods

Oligo Pool Design and Synthesis:

A total of 7500 elements of 210 bp length were designed for synthesis, split approximately evenly across 20 miRNA backbones. There were more elements in the miR-1-1, miR-155, and miR-16-2 backbones as elements that had been tested in arrayed experiments were also included in this screen. ATXN2 targeting sequences accounted for about 60% of the library.

Each element included the 138 nt pri-miRNA, flanked by dual 18 nt adapter pairs. The outer adapter pair was miR-specific and the inside adapter pair was universal.

Full DS2 Library Cloning Strategy

Oligonucleotide pools were synthesized (Twist Bioscience) and were reconstituted in nuclease free water. For cloning the EF1A oligo pool into pLVX_EF1A-MCS-WPRE-CMV-Puro, the vector was first linearized by XbaI and EcoRI restriction digest and gel purified. The primers DS2_EF1A_fw and DS2_EF1A_rv were used to amplify the oligo pool through 10 cycles of PCR and purified. The purified pooled insert and purified linearized vector were assembled with NEB HiFi assembly, precipitated, concentrated, and electroporated into Lucigen Endura electrocompetent cells, recovered and maxiprepped. Oligo pools were PCR amplified with the following conditions.

The PCR mix consisted of:

Component                  Volume (ul)
NEBNext 2 × mix (M0541L)   50
DMSO (D9170-5VL)           2
Betaine (Sigma, B0300-1VL) 10
100uM FW primer            0.5
(DS2_EF1A_fw)
100uM RV primer            0.5
(DS2_EF1A_rv)
1ng EF1A oligo pool in     37
nuclease free water
Total                      100

The PCR cycling parameters were:

	STEP	TEMP	TIME
	Initial Denaturation	98° C.	30	s
	10 cycles	98° C.	10	s
		64° C.	30	s
		72° C.	15	s
	Final Extension	72° C.	2	min
	Hold	4-10° C.

PCR products of 210 bp length were purified by agarose gel extraction (Zymoclean gel DNA recovery kit, D4002). Agilent Tapestation High Sensitivity D1000 was used to quantify the molarity of the 210 bp peak and to confirm removal of contaminating bands.

HiFi assembly of the pooled library was performed by assembling at 5 to 1 insert to backbone molar ratio. 15 ul of 2×HiFi assembly master mix (NEB, E2621L) and 15 ul of insert and backbone (about 0.375 pmol purified miR library insert to 0.075 pmol purified backbone) and incubating for 1 hr at 50° C.

Assembled DNA was precipitated by adding 1 ul of 20 mg/mL glycogen, one-tenth volume of 3M sodium acetate pH 5.5, and 2.2× volume of ethanol, mixed and stored overnight at −80° C.

Samples were spun at 16,000×g for 15 min at 4° C. Supernatant was removed and discarded. Pellets were washed twice with 1 ml of 70% ice cold ethanol and let to dry, then dissolved in 4 ul nuclease free water.

Purified DNA and 0.1 cm Gene Pulser Cuvettes (Bio-rad, 165-2089) were placed on ice for 10 min. 50 ul of Lucigen Endura electrocompetent cells were thawed briefly on ice. 4 ul of precipitated HiFi reaction was added to 25 ul of electrocompetent cells, mixed, and transferred to the cuvette. DNA and cell mixes were electroporated with the following parameters: 1800 Volts, 10 uF, 600 Ohms, 0.1 cm cuvette. Cuvettes were immediately flushed twice with 1 mL Lucigen recovery media. Cells were recovered at 37° C. for 1 hour at 230 rpm.

To titer the transformed bacteria, 2 uL of each culture was diluted into 200 uL of LB and 100 ul or 10 ul of this plated at a 1:100 dilution onto LB agar plates plus appropriate antibiotic. The number of colonies were counted the next day to determine total number of transformants.

Liquid cultures were inoculated into the appropriate amount of LB with antibiotic for maxi prep. Pooled plasmid libraries were prepared with a Qiagen Plasmid Maxi Kit following the manufacturer's instructions.

Preparation and Titering of Pooled EF1A Library

Lentivirus was produced with the Takara packaging plasmid system in Lenti-X 293T cells. Functional titers were determined by Cell Titer Glo following infection and puromycin selection for 3 days to identify conditions to achieve MOI=0.1.

Execution of Full Library Screens for Atxn2 Levels and Dropout

Concurrent ATXN2 levels and dropout screens were conducted similarly to DS1. U2OS cells were infected at day 0 with the lentiviral pooled EF1A library at 2000× coverage and MOI=0.1.

For the dropout screen, a T0 baseline sample was collected at day 1. Puromycin was added on day 2 and MOI was confirmed by plating cells for Cell Titer Glo titer assessment at day 5. After day 7, puromycin was removed and cells were passaged at a minimum of 20 million cells to day 18, upon which the T1 final cell population was collected.

For the ATXN2 protein levels screen, on day 7 cells were harvested and fixed in 6% sucrose/8% PFA for 10-15 min at room temperature, centrifuged 600×g for 3 minutes, washed thrice using the permeabilization buffer (eBioscience, 00-5523-00), mixed with wash buffer and incubated for 15-20 min at room temperature. Anti-ATXN2 primary antibody (1:200, BD, 611378) was incubated for 30-60 min at RT. Cells were washed thrice and AF647 secondary (1:200, Biolegend, 405322) was added and incubated for 45 min. After three washes, cells were resuspended in FACS buffer and sorted on a BD FACSAria Fusion. After gating for singlets, 25% high and low Atxn2 gates were drawn, adjusting for cell size by sorting on an APC/SSC ratiometric gate. Once 3-3.5 million cells were collected for the 25% high and low sort gates, remaining cells were sorted on a 10% low gate (1 million cells collected) to further enrich for high performing guides. The reference population was collected by sorting for singlets.

Fixed populations of sorted cells were decrosslinked with 1% SDS/1% sodium bicarbonate and incubated overnight at 65 C. Genomic DNA was extracted with Machery Nagel NucleoSpin L kit and proceeded to nested PCR to prepare sequencing libraries.

Sequencing Library Preparation

Nested PCR was performed to produce Illumina adapted sequencing amplicons. The first PCR reaction was performed on all genomic DNA extracted from each cell pellet. A maximum of 5 ug genomic DNA was used per 100 ul PCR reaction using the conditions listed below.

Component                    Volume (ul)
NEBNext 2 × mix (M0541L)     50
DMSO (D9170-5VL)             2
Betaine (Sigma, B0300-1VL)   10
100uM FW primer              0.5
(EF1A_F_intron)
100uM RV primer (WPRE_R_ CG) 0.5
5ug genomic DNA and nuclease 37
free water
Total                        100

	STEP	TEMP	TIME
	Initial Denaturation	98°	C.	30	s
	20 cycles	98°	C.	10	s
		64	C.	30	s
		72°	C.	20	s
	Final Extension	72°	C.	2	minutes
	Hold	4-10°	C.

Following PCR, all reactions from a given sample were consolidated into a single tube.

Bead purification of the first PCR product of 564 bp expected size was performed with 0.5× and 0.9× double sided SPRI bead ratios. Specifically, 25 ul of SPRIselect (Beckman, B23318) was added to 50 ul first PCR product, mixed well by pipetting, and incubated at room temperature for 10 min. Samples were placed on a magnetic stand for 5 min. The supernatant was transferred to a new tube. 45 ul SPRIselect was added to the transferred supernatant, mixed well by pipetting, and incubated at room temperature for 10 min. Samples were placed on a magnetic stand for 5 min. Supernatant was then removed. Beads were washed twice with 1 ml fresh 80% ethanol over 2 min incubations. Beads with bound DNA were air dried for 5-10 min and eluted with 20 ul elution buffer from the Machery Nagel kit.

A second PCR was performed to add sample barcodes and Illumina adapters with the following conditions:

Component                        Volume (ul)
NEBNext 2 × mix (M0541L)         50
DMSO (D9170-5VL)                 2
Betaine (Sigma, B0300-1VL)       10
100uM FW primer (P5-DS2-FW)      0.5
100uM RV primer                  0.5
(RV primer P7-DS2-RV-1 to 12 for
multiplexing onto MiSeq run)
1st PCR bead purification product 5
Nuclease free water              32
Total                            100

	STEP	TEMP	TIME
	Initial Denaturation	98°	C.	30	s
	10 cycles	98°	C.	10	s
		64	C	30	s
		72°	C.	20	s
	Final Extension	72°	C.	2	minutes
	Hold	4-10°	C.

Bead purification of the second PCR product with 300 bp expected size was performed with 0.7× and 1.2× double sided SPRI bead ratios. Specifically, 35 ul of SPRIselect (Beckman, B23318) was added to 50 ul first PCR product, mixed well by pipetting, and incubated at room temperature for 10 min. Samples were placed on a magnetic stand for 5 min. The supernatant was transferred to a new tube. 60 ul SPRIselect was added to the transferred supernatant, mixed well by pipetting, and incubated at room temperature for 10 min. Samples were placed on a magnetic stand for 5 min. Supernatant was then removed. Beads were washed twice with 1 ml fresh 80% ethanol over 2 min incubations. Beads with bound DNA were air dried for 5-10 min and eluted with 20 ul elution buffer from the Machery Nagel kit.

Final bead purified 2^nd PCR product was quantified by Tapestation High Sensitivity D1000 (Agilent) and multiplexed at equimolar ratio for sequencing on a MiSeq (Illumina). Using manufacturer's protocols, 15 pM libraries were denatured and mixed with 2% PhiX control. DS2-EF1A-READ1 primer was spiked into position 12 of the MiSeq v3 cartridge (Illumina). Read 1 was set to 139 cycles and index reads was set to 6 cycles.

Data were demultiplexed using the fastq generation module and analyzed.

Primers

Name      Sequence
WPRE_R_CG Catagcgtaaaaggagcaaca
          (SEQ ID NO: 628)
EF1A_F_   Ccaggcacctcgattagttc
intron    (SEQ ID NO: 2212)
DS2-EF1A- AAGTAAGcctgcaggAATTgCCTAGGgt
READ1     (SEQ ID NO: 2213)
P5-DS2-FW aatgatacggcgaccaccgagatctaca
          cAAGTAAGcctgcaggAATTgCCTAGGg
          t
          (SEQ ID NO: 2214)
P7-DS2-   CAAGCAGAAGACGGCATACGAGATCTTG
RV_1      TAGTGACTGGAGTTCAGACGTGTGCTCT
          TCCGATCTACATGtctcgacctggctta
          ctagtG
          (SEQ ID NO: 2215)
P7-DS2-   CAAGCAGAAGACGGCATACGAGATGCCA
RV_2      ATGTGACTGGAGTTCAGACGTGTGCTCT
          TCCGATCTACATGtctcgacctggctta
          ctagtG
          (SEQ ID NO: 2216)
P7-DS2-   CAAGCAGAAGACGGCATACGAGATAGTT
RV_3      CCGTGACTGGAGTTCAGACGTGTGCTCT
          TCCGATCTACATGtctcgacctggctta
          ctagtG
          (SEQ ID NO: 2217)
P7-DS2-   CAAGCAGAAGACGGCATACGAGATTAGC
RV_4      TTGTGACTGGAGTTCAGACGTGTGCTCT
          TCCGATCTACATGtctcgacctggctta
          ctagtG
          (SEQ ID NO: 2218)
P7-DS2-   CAAGCAGAAGACGGCATACGAGATTTAG
RV_5      GCGTGACTGGAGTTCAGACGTGTGCTCT
          TCCGATCTACATGtctcgacctggctta
          ctagtG
          (SEQ ID NO: 2219)
P7-DS2-   CAAGCAGAAGACGGCATACGAGATATCA
RV_6      CGGTGACTGGAGTTCAGACGTGTGCTCT
          TCCGATCTACATGtctcgacctggctta
          ctagtG
          (SEQ ID NO: 2220)
P7-DS2-   CAAGCAGAAGACGGCATACGAGATGAGT
RV_7      GGGTGACTGGAGTTCAGACGTGTGCTCT
          TCCGATCTACATGtctcgacctggctta
          ctagtG
          (SEQ ID NO: 2221)
P7-DS2-   CAAGCAGAAGACGGCATACGAGATAGTC
RV_8      AAGTGACTGGAGTTCAGACGTGTGCTCT
          TCCGATCTACATGtctcgacctggctta
          ctagtG
          (SEQ ID NO: 2222)
P7-DS2-   CAAGCAGAAGACGGCATACGAGATACAG
RV_9      TGGTGACTGGAGTTCAGACGTGTGCTCT
          TCCGATCTACATGtctcgacctggctta
          ctagtG
          (SEQ ID NO: 2223)
P7-DS2-   CAAGCAGAAGACGGCATACGAGATTGAC
RV_10     CAGTGACTGGAGTTCAGACGTGTGCTCT
          TCCGATCTACATGtctcgacctggctta
          ctagtG
          (SEQ ID NO: 2224)
P7-DS2-   CAAGCAGAAGACGGCATACGAGATCAGA
RV_11     TCGTGACTGGAGTTCAGACGTGTGCTCT
          TCCGATCTACATGtctcgacctggctta
          ctagtG
          (SEQ ID NO: 2225)
P7-DS2-   CAAGCAGAAGACGGCATACGAGATGGCT
RV_12     ACGTGACTGGAGTTCAGACGTGTGCTCT
          TCCGATCTACATGtctcgacctggctta
          ctagtG
          (SEQ ID NO: 2226)

Example 6: Evaluation of miR Backbones in AAV Plasmids

A subset of these miR backbones were subsequently evaluated in cis plasmids for AAV production. As described in Example 4 for AAV packaging of miR-16-2 backbone containing amiRNA vectors, cis plasmids containing an H1 promoter (nucleotides 113-203 of SEQ ID NO:1522) and a stuffer sequence (“AMELY_ITR_Stuffer_V1”—nucleotides 348-2228 of SEQ ID NO:1522) and various miR backbones were used to package AAV, and then the uniformity of vector genomes produced was assessed by agarose gel electrophoresis. SEQ ID NO:1522 provides an example of such a sequence from 5′ ITR to 3′ ITR, where for each library element the plasmid would be as shown but with the bases denoted with ‘n’ in the miR backbone insert (nucleotides 204-341 of SEQ ID NO:1522) replaced by the appropriate 138-bp artificial miRNA sequence (backbone, guide, and passenger insert. FIG. 35 shows the indicated set of AAVs, with indicated ATXN2 guide sequence (targeting position 4402 in ATXN2 transcript, —SEQ ID NO:1279 (RNA)), and overall miR cassette sequences constructed from the rules in Table 8. Among the miR backbones assessed, miR-100 and miR-128 backbone-embedded miRs had more uniform gel patterns. To more generally assess the vector integrity of AAV containing different miR backbones, libraries of cis plasmids, each containing the complete set of ATXN2 targeting amiRNA guide sequences as in Deep Screen 2, were used to package AAV as before. The oligonucleotide amplification strategy used in this experiment does not distinguish between parent and “_M” forms of the miR backbones where both were originally present in the Deep Screen 2 library, so the libraries include mixtures of, for example, miR-100 and miR-100_M backbone containing elements; miR-1-1 and mir-1-1_M backbones. FIG. 36 shows that, as with AAVs containing a miR-100 backbone and the specific guide sequence 4402 (SEQ ID NO:751 (DNA); SEQ ID NO:1279 (RNA)), AAVs derived from a library of miRs embedded in the miR-100 and miR-100_M backbones exhibit a more uniform gel electrophoresis pattern than AAVs with other miR backbones. Although the specific composition of the cis plasmid libraries was not assessed after packaging and confirmed to be consistent across libraries with different miR backbones, the simplest interpretation of this data is that on average, across a range of specific miRs, AAV vector genomes with a miR-100 backbone exhibit more uniform, full-length size, than other backbones.

Based on the combined properties of good knockdown performance and good AAV vector genome uniformity, miR-100 and the slightly modified miR-100_M were prioritized as backbones for advancement. ‘Micropool’ plasmid libraries comprising amiRNAs inserted into unpackaged AAV cis plasmid scAAV_AMELY_V1_H1 (SEQ ID NO:1522; amiRNA insert located at nucleotides 204-341) were tested by transfecting plasmid library into HEK293T cells and harvesting small RNA. As above the oligonucleotide amplification strategy to construct the plasmid library did not distinguish between the miR100 and miR100_M backbones, and so the library represents a mix of both; however, given the similar performance overall of miRs from parent and _M form backbones, the mix of backbones in the library is unlikely to degrade the overall ability to detect precisely processed miRNAs. This small RNAseq data was integrated to evaluate processing precision of individual amiRNAs within the library, as in the below examples.

Methods

AAV Micropool Cloning

To clone micropools into the scAAV_AMELY_V1_H1 backbone (to yield plasmids as set forth in SEQ ID NO:1522), the backbone was first linearized by AarI digestion of a cloning site region and agarose gel purified.

Micropools were amplified using the following conditions, using miR-1-1 as an example. All miRNA backbone specific primer pairs are listed in the table below.

Component	Volume (ul)
NEBNext 2 × mix (M0541L)	50
DMSO (D9170-5VL)	2
Betaine (Sigma, B0300-1VL)	10
100uM FW primer (miR-1-1-	0.5
AAV-H1-AMELY-V1-AarI-
FW)
100uM RV primer (miR-1-1-	0.5
AAV-H1-AMELY-V1-AarI-
RV)
1ng EF1A oligo pool from	37
Twist and nuclease free water
Total	100
STEP	TEMP	TIME
Initial Denaturation	98°	C.	30	s
20 cycles	98°	C.	10	s
	64	C	30	s
	72°	C.	15	s
Final Extension	72°	C.	2	minutes
Hold	4-10°	C.

Double sided bead purification with 0.7×SPRI beads and 1.2×SPRI beads ratios was used on the PCR product, which was in turn used as the insert in the HiFi assembly.

HiFi assembly of the pooled library was performed by assembling at 5 to 1 insert to backbone molar ratio. 15 ul of 2×HiFi assembly master mix (NEB, E2621L) and 15 ul of insert and backbone (about 0.375 pmol purified miR library insert to 0.075 pmol purified backbone) and incubating for 1 hr at 50° C.

Assembled DNA was precipitated by adding 1 ul of 20 mg/mL glycogen, one-tenth volume of 3M sodium acetate pH 5.5, and 2.2× volume of ethanol, mixed and stored overnight at −80° C.

Samples were spun at 16,000×g for 15 min at 4° C. Supernatant was removed and discarded. Pellets were washed twice with 1 ml of 70% ice cold ethanol and let to dry, then dissolved in 4 ul nuclease free water.

Purified DNA and 0.1 cm Gene Pulser Cuvettes (Bio-rad, 165-2089) were placed on ice for 10 min. 50 ul of Lucigen Endura electrocompetent cells were thawed briefly on ice. 4 ul of precipitated HiFi reaction was added to 25 ul of electrocompetent cells, mixed, and transferred to the cuvette. DNA and cell mixes were electroporated with the following parameters: 1800 Volts, 10 uF, 600 Ohms, 0.1 cm cuvette. Cuvettes were immediately flushed 2× with 1 mL Lucigen recovery media. Cells were recovered at 37° C. for 1 hour at 230 rpm.

To titer the transformed bacteria, 2 uL of each culture was diluted into 200 uL of LB and plated 100 ul and 10 ul of this 1:100 dilution onto LB agar plates plus appropriate antibiotic. The number of colonies were counted the next day to determine total number of transformants.

Liquid cultures were inoculated into the appropriate amount of LB with antibiotic for maxi prep. Pooled plasmid libraries were prepared with a Qiagen Plasmid Maxi Kit following the manufacturer's instructions.

Primers

Name      Sequence
miR-1-1-  Taagttctgtatgagaccaccatgcagactg
AAV-H1-   cctgctTGG
AMELY-V1- (SEQ ID NO: 2227)
AarI-FW
miR-100-  taagttctgtatgagaccacCCCAAAAGAGA
AAV-H1-   GAAGATATT
AMELY-V1- (SEQ ID NO: 2228)
AarI-FW
miR-124-  taagttctgtatgagaccacTTCCTTCCTCA
AAV-H1-   GGAGAAAGG
AMELY-V1- (SEQ ID NO: 2229)
AarI-FW
miR-128-  taagttctgtatgagaccacATTTtgcaata
AAV-H1-   attggcctt
AMELY-V1- (SEQ ID NO: 2230)
AarI-FW
miR-122-  taagttctgtatgagaccacggctacagagt
AAV-H1-   ttCCTTAGC
AMELY-V1- (SEQ ID NO: 2231)
AarI-FW
miR-130a- taagttctgtatgagaccacgcagggccggc
AAV-H1-   atgcctcTG
AMELY-V1- (SEQ ID NO: 2232)
AarI-FW
miR-132-  taagttctgtatgagaccacGCCGTCCGCGC
AAV-H1-   GCCCCGCCC
AMELY-V1- (SEQ ID NO: 2233)
AarI-FW
miR-138-  taagttctgtatgagaccacgccggcggagt
2-AAV-H1- tctggtatC
AMELY-V1- (SEQ ID NO: 2234)
AarI-FW
miR-144-  taagttctgtatgagaccacTCAAGCCATGC
AAV-H1-   TTCCTGTGC
AMELY-V1- (SEQ ID NO: 2235)
AarI-FW
miR-155-  taagttctgtatgagaccacCCTGGAGGCTT
M-AAV-H1- GCTGAAGGC
AMELY-V1- (SEQ ID NO: 2236)
AarI-FW
miR-155E- taagttctgtatgagaccacCTGGAGGCTTG
AAV-H1-   CTTTGGGCT
AMELY-V1- (SEQ ID NO: 2237)
AarI-FW
miR-16-2- taagttctgtatgagaccacTTATGTTTGGA
AAV-H1-   TGAACTGAC
AMELY-V1- (SEQ ID NO: 2238)
AarI-FW
miR-190a- taagttctgtatgagaccacGAGCTCAGTCA
AAV-H1-   AACCTGGAT
AMELY-V1- (SEQ ID NO: 2239)
AarI-FW
miR-223-  taagttctgtatgagaccacTCCCCACAGAA
AAV-H1-   GCTCTTGGC
AMELY-V1- (SEQ ID NO: 2240)
AarI-FW
miR-451a- taagttctgtatgagaccacGCTCTCTGCTC
AAV-H1-   AGCCTGTCA
AMELY-V1- (SEQ ID NO: 2241)
AarI-FW
miR-1-1-  TATGTGATATGCATAATAaaaaaaggccccc
AAV-H1-   gtggtgtggagtg
AMELY-V1- (SEQ ID NO: 2242)
AarI-RV
miR-100-  TATGTGATATGCATAATAaaaaaaGGCATAT
AAV-H1-   AAGCAAAGCCCCA
AMELY-V1- (SEQ ID NO: 2243)
AarI-RV
miR-124-  TATGTGATATGCATAATAaaaaaatcctTGG
AAV-H1-   CGGGCCCTCGCCG
AMELY-V1- (SEQ ID NO: 2244)
AarI-RV
miR-128-  TATGTGATATGCATAATAaaaaaaagcagtg
AAV-H1-   gaaacctgagtaa
AMELY-V1- (SEQ ID NO: 2245)
AarI-RV
miR-122-  TATGTGATATGCATAATAaaaaaacaaagca
AAV-H1-   aacgatgccaaga
AMELY-V1- (SEQ ID NO: 2246)
AarI-RV
miR-130a- TATGTGATATGCATAATAaaaaaacaatgct
AAV-H1-   gaggaggcagcca
AMELY-V1- (SEQ ID NO: 2247)
AarI-RV
miR-132-  TATGTGATATGCATAATAaaaaaaGGCTCGG
AAV-H1-   GGCGCGGCGTGGC
AMELY-V1- (SEQ ID NO: 2248)
AarI-RV
miR-138-  TATGTGATATGCATAATAaaaaaaCCggtcc
2-AAV-H1- cacgaggctcgcc
AMELY-V1- (SEQ ID NO: 2249)
AarI-RV
miR-144-  TATGTGATATGCATAATAaaaaaatgtccTC
AAV-H1-   CTTGTCAGGCTCC
AMELY-V1- (SEQ ID NO: 2250)
AarI-RV
miR-155-  TATGTGATATGCATAATAaaaaaaCCGGTGG
M-AAV-H1- CCATTTGTTCCAT
AMELY-V1- (SEQ ID NO: 2251)
AarI-RV
miR-155E- TATGTGATATGCATAATAaaaaaaCCCACGG
AAV-H1-   TGGCCATTTGTTC
AMELY-V1- (SEQ ID NO: 2252)
AarI-RV
miR-16-2- TATGTGATATGCATAATAaaaaaaAAACAAT
AAV-H1-   TGATAAAATAGTT
AMELY-V1- (SEQ ID NO: 2253)
AarI-RV
miR-190a- TATGTGATATGCATAATAaaaaaaCTTTATT
AAV-H1-   AGGAACCCCCGGA
AMELY-V1- (SEQ ID NO: 2254)
AarI-RV
miR-223-  TATGTGATATGCATAATAaaaaaaGGCCTAG
AAV-H1-   AGCTGGTAAGCAT
AMELY-V1- (SEQ ID NO: 2255)
AarI-RV
miR-451a- TATGTGATATGCATAATAaaaaaaCTGAGTT
AAV-H1-   CTCTTCCTGGCAC
AMELY-V1- (SEQ ID NO: 2256)
AarI-RV

Pooled AAV Production

AAV micropools served as cis-plasmids to package with Ad helper and AAV9 RepCap using standard three plasmid AAV packaging methods at Vector BioLabs.

Crude Lysate Processing and Gel Visualization

To extract vector genomes, crude lysates underwent 4 freeze thaw cycles (37° C. and dry ice-ethanol bath) and were passed through a 0.45 um filter (Chemglass, CLS-2005-017). Each 100 ul of passthrough was treated with 2 ul DNAse 1 (NEB, M0303L) and 0.2 ul RNAse A (ThermoScientific, EN0531) for 30 min at 37° C. Vector genomes were extracted with the Quick Viral DNA kit (Zymo, D3015). 1.5% agarose gels with either SYBRsafe or SYBRgold to stain DNA were used for visualization.

Pooled Expression of Micropools for Small RNAseq

Micropools of miR100 and miR100_M backbone miRs, embedded in the plasmid scAAV_AMELY_V1_H1, were transfected into HEK293 cells using a lipid based method (Lipofectamine LTX, ThermoFisher) in cells grown in 6 well plates. 600,000 cells were seeded per well and were transfected the following day in duplicate, with 2.5 micrograms of micropool library transfected per well. Media was changed at day 2 and collected in Trizol at day 3. Total RNA was extracted by chloroform phase separation and purification by Zymo Direct-zol column elution using manufacturer's protocols.

Small RNAseq

Small RNAseq libraries were prepared using the Nextflex v3 small RNA seq kit (Bioo Scientific Corp, NOVA-5132-05). Briefly, library preparation was initiated with 0.5-2 ug of RNA input. 14-18 cycles of PCR were performed for each sample. Two rounds of double-sided bead cleanup were performed prior to pooling samples based on Tapestation High Sensitivity D1000 quantitation of the 150 bp band. Illumina adapted libraries were multiplexed and loaded onto a MiSeq (Illumina), loading the library at 9 pM with 10% phiX on a MiSeq v3 kit and with read 1 set to 75 cycles and index set to 6 cycles.

Example 7: Ranking of Top Artificial miRNAs Embedded in miR-100 and miR-100 M Backbones

Top amiRNAs embedded in miR-100 and miR-100_M backbones were ranked by knockdown performance in Deep Screen 2; by guide to passenger ratio; and by minimal depletion at late (T₁, 18 day) versus early (T₀) timepoints (dropout). (Noting, as above, that the guide:passenger ratios are from a small RNAseq library including a mix of miR100 and miR100_M backbones). Additionally, the set of potential off-target transcripts with 1 or 2 bp mismatches was assessed for each ranked candidate. After eliminating candidates with low guide:passenger ratios, low T₁/T₀ ratios, and candidates with CNS expressed transcripts with near-complementarity of only 1 bp mismatch, a set of 9 active miRNAs, and 2 911 control miRNAs, were cloned into cis plasmids downstream of an H1 promoter, and packaged with a Rep/Cap helper plasmid encoding for AAV-DJ capsid components. Data from Deep Screen 2 (FIG. 37) and small RNAseq profiling for these candidates are listed in Table 25. For these selected hits, the mean of replicate 1 and replicate 2 T₁/T₀ log₂ ratios were all within 1 standard deviation (0.22) of the median (−0.07)log₂ ratio of miR100 and miR100_M amiRNAs targeting ATXN2.

TABLE 25
Data from Deep Screen 2 and small RNAseq
profiling
	Rep. 1	Rep. 2	Rep.	Rep.	miR_	Guide	Guide:
Po-	lo10/	lo10/	1	2	with_	Se-	Pas-
si-	unsort	unsort	T1/T0	T1/T0	suf-	quences	senger
tion	log2FC	log2FC	log2FC	log2FC	fix	(DNA)	ratio
1755	−2.70	−2.64	0.04	−0.12	miR-	TCGGGTT	161
					100	GAAATCT
						GAAGTGT
						G
						[SEQ ID
						NO:
						657]
2586	−2.40	−2.24	−0.28	−0.08	miR-	TAGATTC	390
					100	AGAAGTA
						GAACTTG
						G
						[SEQ ID
						NO:
						1621]
2945	−2.61	−2.17	0.10	−0.31	miR-	TGTAGTA	>778:0
					100	GAAGGCT
						TTGGCTG
						A
						[SEQ ID
						NO:
						685]
3133	−2.66	−2.33	0.21	0.27	miR-	TATGTCT	78
					100_M	TGGCTTG
						ATTCACT
						G
						[SEQ ID
						NO:
						1624]
3270	−2.09	−1.46	0.05	0.03	miR-	TACTGTA	>334:0
					100	GGCAACA
						TATTGCG
						T
						[SEQ ID
						NO:
						2080]
3301	−2.52	−2.09	−0.19	−0.27	miR-	TGAACAA	92
					100_M	GGGGCTG
						ATTTGGG
						A
						[SEQ ID
						NO:
						687]
3302	−2.89	−2.36	−0.09	−0.30	miR-	TTGAACA	41
					100_M	AGGGGCT
						GATTTGG
						G
						[SEQ ID
						NO:
						688]
3330	−2.18	−2.12	0.30	−0.06	miR-	TATGCTG	39
					100	AGACTGA
						TAATGTG
						G
						[SEQ ID
						NO:
						1614]
3338	−1.91	−2.25	0.03	−0.15	miR-	TACATGA	212
					100	GGATGCT
						GAGACTG
						A
						[SEQ ID
						NO:
						1620]

The above miRNAs as well as 911 controls for 1755 (guide sequence SEQ ID NO:1185) and 2945 (guide sequence SEQ ID NO:1213) were tested for knockdown of ATXN2 in stem-cell derived motor neurons. amiRNAs were packaged in cis plasmids to generate self-complementary AAV-DJ vectors containing a long H1 promoter (nucleotides 113-343 of SEQ ID NO:2257), and a stuffer sequence “PSG11_V5” (nucleotides 489-2185 of SEQ ID NO:2257). Sequences for vectors encoding amiRNAs miR100_1755 (SEQ ID NO:1915), miR100_2586 (SEQ ID NO:1982), miR100_2945 (SEQ ID NO:1965), and miR100_3330 (SEQ ID NO:2021) from 5′ ITR to 3′ ITR are provided in SEQ ID NO:2257, SEQ ID NO:2258, SEQ ID NO:2259, and SEQ ID NO:2260, respectively. After titering each vector, and based on hemacytometer based quantification of number of cells plated, vectors were added at intended doses of 3.16E3 and 3.16E4 vector genomes per cell. 7 days after addition of vectors, neurons were harvested and RNA isolated with miRNeasy Tissue/Cells Advanced Mini Kit (Qiagen, P/N 217604) ATXN2 knockdown was assessed by digital droplet RT-PCR, measuring the ratio of ATXN2 expression to housekeeping controls GUSB and B2M.

FIG. 38 shows individual data points, and Table 26 shows mean and standard deviation of knockdown across these constructs, at the two doses of 3.16E3 vg/cell and 3.16E4 vg/cell, normalized to ATXN2 expression values from untransduced cells, which were treated with an equivalent volume of AAV diluent.

TABLE 26
ATXN2 Knockdown by amiRNAs in stem-cell derived
motor neurons at two different doses
miR	mean_ATXN2_3160	SD	N	mean_ATXN2_31600	SD	N
miR100_1755	26.8	1.7	6	14.0	2.1	6
miR100_2586	41.8	2.9	6	29.3	1.0	6
miR100_2945	36.7	1.7	6	26.8	2.6	6
miR100_3270	72.1	5.1	6	47.9	2.2	6
miR100_3330	44.1	6.3	6	32.2	2.7	6
miR100_3338	36.5	3.6	6	23.6	5.5	6
miR100_M_3133	30.6	1.0	5	23.8	1.5	6
miR100_M_3301	83.7	9.1	6	49.1	3.7	6
miR100_M_3302	38.0	3.0	6	32.3	3.3	6
Untransduced	100	9.5	12

Dose Response Studies

The candidates AAVs were also tested at a more extensive range of doses in motor neurons. As before, RNA was isolated from the cultures after 7 days of culture, and ATXN2 knockdown assessed. FIG. 39 shows plots of knockdown across different concentrations of each vector added. Concentration of ATXN2 mRNA, normalized for each data point by B2M expression, and collectively to the ATXN2 expression level in neurons treated with vehicle (PBS+0.001% PF-68) was measured by digital droplet RT-ddPCR. By examination, differences in potencies of amiRNAs can be observed; for example miR100_1755 exhibits knockdown at lower vector genome exposures than other amiRs; mir100_3301 and miR100_3270 appear to exhibit reduced potency relative to other vectors.

Neurons dosed at 3.16E3 vector genomes per cell were additionally subject to small RNA sequencing. Table 27 shows the abundance of the amiRNA, as a fraction of total miRNA. There was a surprising range of expression levels, and several amiRs (1755, 2586, 2945, and 3270) had considerably less amiRNA detected than other amiRNAs.

For these small RNA experiments, reads were not ‘deduplicated’ (by eliminating reads with identical flanking 5′ and 3′ 4-mer random adapters) as in small RNA analysis for deep screen 1 libraries, because the number of reads of the artificial miRNAs in some cases approached the number of unique combinations of nucleotides in the adapters.

TABLE 27
Abundance of amiRNA, as a fraction of total miRNA
		Rep1 amiRNA/	Rep2 amiRNA/
	Guide	total miRNA (%)	total miRNA (%)
	mir100_1755	1.80	2.77
	mir100_2586	2.53	2.73
	mir100_2945	3.20	3.30
	mir100_3270	1.21	1.43
	mir100_3330	16.83	17.75
	mir100_3338	23.84	25.00
	mir100M_3133	22.82	22.57
	mir100M_3301	22.07	17.53
	mir100M_3302	38.04	36.78

To assess whether AAV amiRNA treatment had any obvious impact on neuronal morphology or cell counts, neurons grown in 96-well format were treated with AAV or vehicle at a dose of 1E4 vector genomes/cell, and 7 days later fixed and stained with Hoechst, anti-Isl1, and anti-Beta3 tubulin antibodies to visualize nuclei, a motor neuron marker, and neuronal processes respectively. FIG. 40 shows representative images from cultures treated with indicated amiRNA AAVs and controls, demonstrating that no AAV miRNA exhibited obvious impacts on neuronal morphology. FIG. 41A shows zoomed in images comparing miR100_1755 and miR100_1755_911 (a 911 control, rendered inactive for slicing Atxn2 by complementing bases 9, 10 and 11 of the 1755 amiRNA). No obvious differences can be seen, suggesting that Atxn2 knockdown does not cause dramatic changes in neuronal process or nuclear morphology. Panels on right quantify the total number of Hoechst+ nuclei (FIG. 41B) and the % of total nuclei that are Isl1+ (FIG. 41C). Compared to vehicle-treated (PBS+0.001% PF-68) wells, significant differences (p<0.05) were observed for a few of the AAV-amiRNA treatments, with a reduction in total number of nuclei per field. However, one of these treatments (miR100_1755) were also showed with a significant increase in the fraction of cells that were Isl1+, and an apparent trend toward increasing Isl+ neurons was apparent for other AAV-DJ amiRNAs, arguing against any alteration in total motor neuron numbers. There were no significant differences between neurons transduced with any of the active AAV amiRNAs and the inactive 911 control AAV amiRNAs.

RNAseq Studies

RNA was collected from motor neurons 7 days after dosing with 1E4 vector genomes/cell of the above AAVs. There were 6 replicates per condition, except miR100_1755_911, which had 5. To determine if ATXN2 knockdown from AAV expression impacts the transcriptome in neurons, RNA expression was compared between neurons transduced with active amiRNA-expressing vectors and vectors expressing a cognate 911 control. FIG. 42 shows ‘volcano plots’ of differential expression for miR100_1755 vs. miR100_1755_911 and miR100_2945 and miR100_2945_911. A large separation can be seen in nominal p-values for the differential expression calculated for ATXN2 versus all other genes. Remarkably, after adjustment of nominal p values for multiple comparisons using the Benjamini-Hochberg procedure, only ATXN2 or one other gene exceeded a 10% false discovery rate threshold for 1755 and 2945, respectively.

To further investigate whether there was any impact on any of the predicted off-target genes (the set of transcripts with 2 or fewer mismatches to bases 2-18 of each amiRNA), each amiRNA was compared to data from all other active amiRNAs (FIG. 43). For this set of selected amiRNAs, few of the predicted off-targets exceed the 10% false discovery rate threshold. This suggests that these amiRNAs yield specific knockdown of ATXN2.

Methods

ddPCR AAV Titering

To titer AAVs, each vector was serially diluted in Salmon Sperm DNA solution (20 ng/ul Salmon Sperm DNA, 0.001% PF-68, 10 mM Tris-HCl pH 7.5, 50 mM KCl, 1.5 mM MgCl₂) and subsequently heated at 95° C. for 10 minutes to release the vector genome from the AAV9 capsid. After an incubation with SmaI to reduce secondary structure, known to inhibit the rAAV PCR reactions, (NEB, R0141L), droplets were generated using DG32 Automated Droplet Generator (Bio-Rad), followed by a PCR amplification with vector-specific primer/probe sets. Once complete, droplets were analyzed using QX200 Droplet Digital PCR System (Bio-Rad), and positive and negative populations were definded, and the dilution factor applied to determine the concentration of the undiluted vector stock.

Motor Neuron Immunocytochemistry

Motor neuron cultures were fixed in 4% Paraformaldehyde for 10 minutes at room temperature. Fixed cultures were permeabilized and blocked in PBS containing 0.2% Triton-X-100 and 10% donkey serum solution for 45 minutes at room temperature. Cells were then incubated in blocking solution (10% donkey serum in 0.1% Tween-PBS) containing primary antibody overnight at 4 C. Cells were washed 3 times with PBS-0.1% Tween and then incubated in blocking solution containing secondary antibodies for 1-2 hours at room temperature followed by 3 washes with PBS-0.1% Tween and a rinse with a PBS solution containing Dapi. Stained cultures were imaged on the Perkin Elmer Operetta high content imager with 20× water objective. 40-60 fields were imaged for every well. Cell quantifications were done using the Perkin Elmer Harmony software. Statistical analysis was done using GraphPad Prism software. Primary antibodies used: TUJ1 (1:500 dilution) ISL1 (1:200 dilution) secondary antibodies: AlexaFluor 488 and AlexFluor 647 (1:500 dilutions).

Reagent	Vendor	Cat. No.
32% Paraformaldehyde	Fisher Scientific	50-980-495
Triton X-100	Sigma	T8787-100ML
Tween 20	Sigma	P1379-100ML
Donkey Serum	Jackson Immuno	017-000-121
	Research
Donkey anti-chicken secondary antibody-	Jackson Immuno	703-606-155
Alexa fluor 647	Research
ISL1 antibody	Abcam	ab 109517
TUJ1 antibody	Abcam	ab41489
Donkey anti-rabbit secondary antibody-	ThermoFisher	A32790
Alexa fluor 488
DAPI solution	ThermoFisher	62248
Phosphate-Buffered Saline (PBS) pH 7.4	ThermoFisher	10010023

Off-Target Prediction

To generate a set of predicted off-targets, bases 2-18 of amiRNAs were aligned to the human transcriptome using bowtie commands:

bowtie -n 2 -l 17 -e 81 -seed [pseudorandom number to enforce reproducibility]-nomaqround -tryhard -chunkmbs 256 --all --time (and additional commands for input/output handling). To ensure that only 2 or fewer mismatches occurred, fastq file inputs to the bowtie alignment containing amiRNAs to be tested were constructed in which each amiRNA was given a phred score ‘mask’ of IIIIIIIIIIIIIIIII, such that alignments of the amiRNA with transcripts where more than 2 mismatches occurred would exceed the weight threshold. The amiRNas were aligned to the build Homo_sapiens.GRCh38.cdna.all, Macaca_fascicularis.Macaca_fascicularis_5.0.cdna.all, or Mus_musculus.GRCm38.cdna.all.

RNAseq

Stem-cell derived motor neuron cultures were plated at a density of 200,000 cells per well of 6-well plates. 6 days after plating, cells were transduced with AAV vectors at a dose of 10,000 vector genomes (calculated by titering method described above) per cell. 7 days later, cells were harvested for RNA._Lysis of transduced samples was conducted by addition of 300 ul of Buffer RLT Plus, followed by overnight freeze at −80. Samples were thawed on ice and processed according to the remainder of the RNeasy Plus standard protocol. (Qiagen RNeasy Plus Micro Kit (Catalog 74034)), according to manufacturer's instructions. All purified RNA samples were quantified by Qubit (using RNA HS standard). A selection of samples with low, mid, and high RNA concentrations (16 in total) were further characterized by Tapestation (High Sensitivity RNA) to check purity (RINe score) and verify Qubit quantification. All RINe scores were in the 9.9-10 range, near maximal.

Purified RNAs were then used as input into QuantSeq [Lexogen catalog #015 (QuantSeq 3′ mRNA-Seq Library Prep Kit for Illumina (FWD)]. Target RNA input was 100 ng per reaction (for lower concentration samples, the maximum input volume of 5 ul was used). The standard Quantseq protocol was followed with the following modifications: (1) UMI addition at step 7 using the “UMI Second Strand Synthesis Module” (Lexogen Cat. No. 081). (2) 20 cycles for library amplification. Resulting libraries were quantified by Qubit (DNA HS) and QC spot-checked on Tapestation (HS D5000). Libraries were pooled based on Qubit quantifications and sequenced on an Illumin NovaSeq (Seqmatic). Sequencing parameters were as follows: NovaSeq S1 run, single-read 100 bp, single index 6 bp.

RNAseq Analysis

To analyze RNAseq data, SeqTK was used to split each of the single-end reads obtained from each sample into fastq files containing the UMI and read sequence, respectively:

seqtk trimfq -b 10 raw.fastq > sequence.fastq
seqtk trimfq -e [readsize - 6] raw.fastq > umi.fastq

The resulting sequences were then pseudoaligned with kallsito version 0.46.0 (Bray et al., Nature Biotechnology 2016 34: 525-527). in batch mode to a transcriptome assembly derived from the the trailing 600 bp of all cdnas present in Ensembl release 96 (kmer length=19) using the following command:

kallisto pseudo --umi --quant --single -t 8 -i [kallisto_index] -o
[output_file] -  b [batch_file.txt]

Aligning reads were summed across all transcripts annotated to each gene to generate gene-level count matrices. Genes with five or more counts observed in all replicates of at least one experimental condition were considered in downstream analyses. Sample read counts were converted to base-2-log(CPM) and normalized via TMM (edgeR::calcNormFactors) prior to probability weight estimation via limma::voom. (Law et al., Genome Biology (2014) 15:R29). Evidence for differential expression was quantified by fitting a genewise linear model on the normalized expression values, with fold changes extracted from the model coefficients and associated P-values estimated using a Wald test. Genewise P-values were corrected for multiple testing using the FDR approach.

Example 8: In Vivo Testing of Candidate amiRNAs in Wild-Type Mouse

Two additional studies of in vivo performance of amiRNAs embedded in self-complementary AAV9 vectors were conducted. In a first study, amiRNA 1784 and 3330, in the miR1-1 or miR100 backbone, respectively, were tested in a variety of vector genomes containing different promoters and stuffers. The specific miR cassettes used for in vivo testing are provided in Table 28.

TABLE 28
Specific miR cassettes used in vivo
Cassette Sequence
miR1-    CATGCAGACTGCCTGCTTGGGTACAGACCAAAGAGTA
1.1784   GTCGAATTATGGACCTGCTAAGCTAATTAACTACTCTT
         TGGTCTGAACTCAGGCCGGGACCTCTCTCGCCGCACTG
         AGGGGCACTCCACACCACGGGGGCC
         (SEQ ID NO: 538)
miR      CCCAAAAGAGAGAAGATATTGAGGCCTGTTGCCACAT
100.3330 ATGCTGAGACTGATAATGTGGGTATTAGTCCGCCACAT
         CATCCGTCTCAACATTTGTGTCTGTTAGGCAATCTCAC
         GGACCTGGGGCTTTGCTTATATGCC
         (SEQ ID NO: 2019)

The vector designs, including specific promoter and stuffer, are described separately. Here the performance of the amiRNAs is compared in several overall vector formats and promoters. AAV was dosed to wild-type mice either intravenously (dose: 3.21E9 vg/gram mouse) or by intrastriatal injection (dose: 7.5E9 vg total).

Table 29 shows mean ATXN2 knockdown as assessed in liver 3 weeks after intravenous dosing, relative to animals dosed with vehicle (PBS with 0.001% PF-68). Atxn2 expression was assessed by digital droplet RT-PCR, and knockdown was taken as the mean of Atxn2/Hprt and Atxn2/Gusb ratios, as measured by ddPCR.

TABLE 29
ATXN2 Knock-down in Liver After I.V. amiRNA Dosing
		mean Atxn2
	miR	(% Vehicle injected)	SD	N
	miR1-1.1784	10.8	6.5	12
	miR100.3330	14.7	4.7	12

For striatal samples, vector biodistribution after collection of punch biopsies was more variable from sample to sample. Vector distribution was assessed by digital droplet PCR, measuring the relevant number of droplets amplifying for primer/probesets recognizing the AAV vector genome versus primer/probesets recognizing the Tert gene in the mouse genome. Because there are a fixed number of copies of the Tert gene per cell (2), the number of vector genomes per cell (diploid genome) can be measured in this way. By assessing AAV vector distribution in the same biopsies as ATXN2 mRNA was quantified, a clear dose response trend can be seen (FIGS. 44, 45A-45B). It should be noted that the amount of nuclear vector genomes versus cytoplasmic or extracellular vector was not assessed, such as by histological methods; it is possible that vector introduced by intraparenchymal injections may accumulate extracellularly. Nonetheless, the clear dose response shows that even if not all of the vector genomes measured are in the nucleus, available to express the amiRNA, there is a clear correlation between any such total vector genome exposure and functionally active vector genomes.

To determine the relationship between amiRNA expressed and knockdown, amiRNA was quantified in two ways. First, libraries using TaqMan Advanced miRNA cDNA Synthesis Kit (Thermo, P/N A28007) were generated for all striatal punch biopsy samples, using RNA isolated with a kit which enriches for small RNAs (Qiagen, P/N 217604). To generate a cDNA library for TaqMan qPCR, 3′ poly-A tailing is first complete, then 5′ ligation to add on an adaptor. After reverse transcription, the cDNA is PCR amplified for 14 cycles, then a dilution of the final amplification product is subject to qPCR with primer probe sets specific to exogenous and endogenous miRNAs. Primer/probesets designed to target exogenous amiRNAs were used (Thermo), as well as primer/probesets targeting endogenous miRNAs miR-21a-5p (Thermo, P/N mmu482709_mir) and miR-124-3p (Thermo, P/N mmu480901_mir) as controls. The abundance of miRNA is assessed by the qPCR cycle number at which target amplification occurs. Comparing the qPCR cycle where amplification occurs (CT) between primer/probesets targeting different miRNAs allows assessing the relative abundance of miRNAs.

FIGS. 45A-45B plots the difference in CT value between amiRNA and endogenous control, as well as the difference between two endogenous miRNAs (miR-21 and miR-124), against the vector biodistribution in the same sample. As can be seen, there is no obvious change in the difference in CT thresholds between endogenous miRNAs with increasing detection of AAV vector genome. By contrast, there is what appears to be a log-linear relationship between the expected increase in the CT separation between the amiRNA and endogenous miRNA and vector exposure, consistent with greater amiRNA expression with increased exposure to AAV.

For a subset of samples, small RNAseq was additionally conducted. As above, amiRNA expression normalized by total miRNA expression was quantified for each sample. Since for these samples amiRNA expression was quantified both by small RNAseq and qPCR, a model could be fit to establish how qPCR predicts amiRNA expression as a function of total miRNA. Therefore a linear model was fit (FIG. 46), with good explanation of the variance (R²>0.89) for both amiRNAs.

Using this model, the qPCR-assessed amiRNA expression values for miR100_3330 and miR1.1.1784 in all samples could be converted to an absolute scale, of amiRNA/total miRNA. Plotting ATXN2 mRNA in striatal biopsies versus this metric of predicted amiRNA expression, there was considerably greater knockdown per miRNA expressed in samples expressing the miR100-3330 amiRNA versus samples expressing the miR1.1.1784 amiRNA (FIG. 47). Therefore, although as a function of vector dosed, more knockdown was induced by vectors expressing miR1.1.1784, as a function of amiRNA expressed, more knockdown was induced by miR100.3330. This suggests that in vivo the potency of the approximately 22 nucleotide final product of pri-miRNA processing is higher for miR100.3330 than for miR1.1.1784.

In a second study, self-complementary vectors expressing amiRNAs miR100_1755 (SEQ ID NO:1915), miR100_2945 (SEQ ID NO:1965), miR100_3330 (SEQ ID NO:2021), and miR100_2586 (SEQ ID NO:1982) were packaged in AAV9 with a cis plasmid as described above containing a stuffer sequence “PSG11_V5” (nucleotides 489-2185 of SEQ ID NO:2257), a long H1 promoter (nucleotides 113-343 of SEQ ID NO:2257) and dosed intravenously or intrastriatally in adult wild-type mice. 5′ ITR to 3′ ITR sequences for these vectors, as described in Example 7, are provided in SEQ ID NO:2257 (scAAV_H1_long_miR100_1755_PSG11_V5_ITR_to_ITR.gb), SEQ ID NO:2258 (scAAV_H1_long_miR100_2586_PSG11_V5_ITR_to_ITR.gb), SEQ ID NO:2259 (scAAV_H1_long_miR100_2945_PSG11_V5_ITR_to_ITR.gb), and SEQ ID NO:2260 (scAAV_H1_long_miR100_3330_PSG11_V5_ITR_to_ITR.gb). Because the mouse Atxn2 transcript has several mismatches to 2586, no knockdown of mouse Atxn2 transcript is expected.

During the intravenous study, 4 animals were dosed per group for a 3-week study. There were no clinical observations noted during weekly observation. For ALT and AST analysis, blood was collected via submandibular vein into serum tubes and allowed to clot for 30 minutes. Samples were centrifuged at 12,000 rpm for 5 minutes at 4° C. Serum was collected into clean Eppendorf tubes and stored at −80° C. until further analysis at IDEXX. Results were reported as AST (U/L) and ALT (U/L). FIG. 48 shows liver enzyme data at 2 and 3 weeks post-dosing. All ALT and AST values were within normal ranges at these timepoints.

During the intrastriatal study, 6 animals were dosed 4 microliters per striatum per group for a 3-week study. There were no group wide clinical observations noted for 7 days following injection and during weekly observation and there were no unscheduled deaths. For one cage dosed with miR100-2586, fighting was observed but the bully was separated, and all animals completed the study.

Knockdown performance of vectors was tested in liver. Table 30 quantifies remaining Atxn2, normalizing Atxn2 to two different control genes (Hprt and Gusb) and further normalized to Atxn2 expression levels in naïve animals. From the same liver samples, as above biodistribution was measured. Samples treated with different vectors had highly similar exposures in liver.

TABLE 30
Atxn2 knockdown of amiRNA containing vectors in liver
	Mean %
	Control		Mean
	Atxn2	mRNA	Biodistribution	SD
Treatment	mRNA	SD	(VG/DG)	(VG/DG)	N
miR100_1755	22.1	3.3	5.93	1.70	4
miR100_2945	29.3	5.0	5.77	0.50	4
miR100_3330	27.9	0.8	5.71	0.36	4
None	100.0	10.5	0.01	0.01	4

Knockdown performance of these vectors was further assessed in brain after intrastriatal injections. As in the study described above DNA, mRNA and small RNA were isolated from punch biopsies in order to simultaneously monitor vector biodistribution, Atxn2 knockdown, and amiRNA expression. Although in this in vivo study exposure levels were lower than in the above study with miR1.1.1784 and miR100_3330, for unknown reasons, a clear dose response is visible (FIG. 49).

amiRNA expression versus total miRNA expression was assessed in a subset of samples in both liver and striatal punch biopsies. FIG. 50 shows, for each tissue, vector biodistribution-normalized miRNA expression. In both tissues, miR100_1755 has the lowest miRNA expression, followed by miR100_2945, miR100_3330, and lastly miR100_2586.

Guide processing precision was also assessed in vivo, by counting reads that initiated at each position of the guide and predicted passenger sequences. FIGS. 51, and 52A-51D shows the count of reads aligning to the miR at the start position, where +0 is the expected typical cut position. Table 31 quantifies the proportion out of all reads (including all guide and passenger strand reads) initiating at the +0 and +1 positions for each amiR. FIGS. 52A-52D show the read counts for the top 20 most common sequences for miR100_1755, miR100_2586, miR100_2945 and miR100_3330. Interestingly, by comparing the observed reads versus the pri-miRNA sequence, post-translational modifications such as 3′ monoadenylation or monouridylation can be noted. Since 3′ mismatches to the target transcript have in some cases been reported to increase the knockdown performance of miRNAs (Becker et al., Molecular Cell (2019) 75:741-755), it may be the case that this 3′ modification of these amiRs may contribute to the high knockdown performance of Atxn2.

TABLE 31
Proportion of amiRNAs Initiating at Position + 0 or + 1
amiRNA	Proportion cut at + 0	Proportion cut at + 1
mir100_1755	97.67	1.07
mir100_2586	98.51	0.99
mir100_2945	98.34	0.94
mir100_3330	97.08	2.07

Table 32 quantifies the ratio of guide to passenger strand reads. The ratio of reads detected from guide versus passenger strand for all of these miR100 backbone amiRs exceeded 300:1 in vivo. This high processing ratio may reduce the likelihood of off-target effects.

amiRNA      Ratio guide:passenger
mir100_1755 399.8
mir100_2586 1061.7
mir100_2945 651.3
mir100_3330 508.3

Methods

For intravenous injection, vector was diluted in PBS with 0.001% PF-68 at 3.21E9vg/10 microliters, and mice were injected via tail vein based on weight (average total dose of 8.5E10 VG). Mice are placed in a restrainer and the tail is swabbed with a sterile alcohol wipe to increase vein visibility. Once a lateral tail vein is located, a 32-gauge insulin syringe is used to administer the solution. 3-weeks post-injection, mice were fasted for 4 hours, blood collected via vena cava and serum processed for AST and ALT analysis. Following PBS perfusion, liver was cut into sections and placed in a homogenizing tube (Precellys, P/N P000933-LYSK0-A) and snap frozen in liquid nitrogen. For tissue homogenization, Buffer RLT supplemented with beta-Mercaptoethanol was added to the sample and a Precellys Cryolys Evolution (Bertin Instruments) with program setting 3×45 s at 5000 rpm with 15 s pauses was performed. Samples tubes were centrifuged at 18,000×g for 3 minutes and a fraction of the homogenate was used for DNA, RNA, and protein purification using the AllPrep DNA/RNA/Protein Mini Kit (Qiagen, P/N 80004) and the other fraction of the homogenate was used for small RNA purification using the miRNeasy Tissue/Cells Advanced Mini Kit (Qiagen, P/N 217604).

For intrastriatal injections, vector was diluted in PBS with 0.001% PF-68 at 7.5E9vg/4 microliters, and mice were injected at coordinates (relative to Bregma) 1.5 mm anterior, +/−1.6 mm lateral, and −4.0 mm ventral with 4 uL per hemisphere (Hamilton P/N 7635-01) over 5 minutes. After 3-weeks post-injection, mice were perfused transcardially with cold PBS and the brain placed in a matrix (CellPoint Scientific, Alto Acrylic 1 mm Mouse Brain Coronal 40-75 gm), and a 2 mm cornal section containing the injection site was excised. Within the coronal section, a 2 mm punch biopsy of both the left and right striatum was collected and placed into separate homogenizing tubes (Precellys, P/N P000933-LYSK0-A) then snap frozen in liquid nitrogen. For tissue homogenization, Buffer RLT supplemented with beta-Mercaptoethanol was added to the sample and homogenization with a Precellys Cryolys Evolution (Bertin Instruments) with program setting 3×45 second at 5000 rpm with 15 second pauses was performed. Samples tubes were centrifuged at 18,000×g for 3 minutes and a fraction of the homogenate was used for DNA, RNA, and protein purification using the AllPrep DNA/RNA/Protein Mini Kit (Qiagen, P/N 80004) and the other fraction of the homogenate was used for small RNA purification using the miRNeasy Tissue/Cells Advanced Mini Kit (Qiagen, P/N 217604).

Example 9: Pharmacology Study of AAV Vector Expressed amiRNA Targeting ATXN2 in Non-Human Primates

Testing in non-human primates is conducted to establish knockdown of ATXN2 by ATXN2-targeting amiRNA rAAV vectors in tissues relevant to neurodegenerative disease, via clinically relevant routes of administration. Tissues to be assessed include spinal cord ventral horn, motor cortex, and cerebellum, which are relevant to neurodegenerative diseases such as ALS or Spinocerebellar ataxia-2.

In non-human primates, test articles (1×10¹²-1×10¹⁴ vg of amiRNA expressed with a H1 promoter and packaged in AAV9) or vehicle are administered into the cisterna magna by intrathecal cervical (IT-C) catheter. Male and female cynomolgus monkeys (Macaca fascicularis) of approximately 2.5-4 kg body weight, are implanted with an intrathecal cervical catheter for dose administration and sample collection. Test articles are administered (4 animals per test article) comprising a single 2.5 mL dose of vehicle or test article via the implanted intrathecal catheter at a rate of 0.3 mL/minute, followed by 0.1 mL of vehicle to flush the dose from the catheter. At 5 to 26 weeks following the administration, animals are sacrificed, and selected tissues harvested for bioanalytical and histological evaluation. ATXN2 protein and mRNA levels are assessed for suppression after treatment with ATXN2 amiRNA packaged in AAV9 with a H1 promoter, relative to the vehicle group.

Vector Assessment

Test articles for dosing in non-human primates are assessed by multiple assays. One assessment is analytical ultracentrifugation (AUC) for empty and full capsids and quantification of aggregates. Absorbance scans are collected as the material sediments under the force of a gravitational field. Sample sedimentation profile is monitored in real time during centrifugation, which gives an absolute measurement of molecule size and shape. The distribution movement over time is used to calculate the sedimentation coefficient. Fitting the raw data to the Lamm equation results in a continuous distribution, and area under each peak is proportional to the amount present in solution. Empty capsids are expected to sediment at 65S, partial capsids between 65 and 95S, full capsids at 95S, and aggregates at >110S. Measurements indicating majority full capsids are desirable.

AAV9 capsid ELISA is used to assess intact AAV9 capsids. The capture-antibody detects a conformational epitope that is not present on unassembled capsid proteins. The ADK9 antibody is used as capture and detection antibody in the AAV9 titration ELISA. Assay results are expected to corroborate AUC assessment, by comparing AAV9 capsid ELISA with vector genome titers.

Endotoxin is assessed by Limulus amebocyte lysate (LAL). Detection and quantification of bacterial endotoxins less than 10 EU/mL is desired.

Bioburden is assessed by direct inoculation, and less than 10 CFU/100 mL is desired.

For lot release and stability, an in vitro potency assay for gene therapy product potency is performed. In vitro potency is assessed by amiRNA expression by RT-qPCR, ATXN2 mRNA levels by RT-ddPCR, and ATXN2 protein levels by ATXN2 protein FACS in 2v6.11 or Lec2 cells. Cells may be pre-treated with 1 ug/mL ponasterone A (Invitrogen, H10101), 50 mU/mL neuraminidase (Sigma, N7885), and 2 mM hydroxyurea (Sigma, H8627) prior to transduction. Serial dilutions of vector are used to treat cells in a 96 well format, incubating at 4° C. for 30 min, and then 90 min following application of vector. Plates are then transferred to 37° C. After 2-3 days amiRNA, ATXN2 mRNA, and ATXN2 protein are assessed at each dose.

In vivo potency in some experiments is tested prior to dosing non-human primates and is assessed by single dose (such as 8.5E10 vg/gram) administration of test article intravenously into wild-type C57Bl/6 mice. Liver biopsy is collected, homogenized, and DNA and RNA are extracted by Allprep DNA/RNA/Protein mini kit (Qiagen, 80004) for assessment of vector distribution and ATXN2 mRNA knockdown in liver.

Median Tissue Culture Infectious Dose (TCID50) to assess vector infectivity is performed in HelaRC32 cells. HelaRC32 stably express AAV2 rep and cap genes, and the assay involves serial dilutions of vector in a 96 well plate and co-infection with Adenovirus 5 helper virus, lysing cells, extracting DNA, performing qPCR or ddPCR on the vector genome to assess number of infected cells per well across the dilution range.

Biodistribution and Pharmacodynamic Activity

Non-human primate brain and spinal cord tissue from rAAV vector and control treated animals are collected by punch biopsy or as slabs at necropsy and snap frozen. Samples are homogenized by addition of Buffer RLT (Qiagen) supplemented with beta-mercaptoethanol. Ceramic bead-based homogenization (Precellys, CK14 2 mL) is performed using 3 cycles of 15 s at 6500 rpm and 10 s break. DNA, RNA and protein are extracted with Allprep DNA/RNA/Protein Mini kit (Qiagen, 80004) and small RNA are extracted with miRNEasy Tissue/Cells Advanced Mini kit (Qiagen, 217604).

For isolation of motor neurons from rAAV dosed non-human primates, spinal cord tissues are frozen in liquid nitrogen at necropsy. Cryosections are generated and stained with ARCTURUS HistoGene Quick H&E Stain for LCM, and motor neurons are dissected from each section with the ARCTURURS XT LCM System. DNA and RNA from LCM samples are extracted with PicoPure kits (Thermo Fisher).

For histological evaluations, non-human primate brain and spinal cord tissue are collected at necropsy and fixed with 10% neutral buffered formalin for 24 hr, transferred to 70% ethanol for 3-10 days and embedded into paraffin blocks. Five-micron sections are cut, mounted onto glass slides, and stained for hematoxylin and eosin for histology, or stained in separate protocols for immunohistochemistry or in-situ hybridization.

Vector biodistribution in tissues from animals dosed with rAAV is assessed by ddPCR. Specifically, primer probes that amplify promoter and/or stuffer regions of the vector are used and compared to primer probes specific to host genome and results are expressed as vector genomes per diploid genome.

To isolate biodistribution in tissue material enriched for motor neurons, vector biodistribution is assessed by ddPCR on DNA isolated from spinal cord neurons captured by laser capture microdissection (LCM). Specifically, primer probes that amplify promoter and/or stuffer regions of the vector are used and compared to host diploid genome and results are expressed as vector genomes per diploid genome. Biodistribution in tissue material enriched for other disease-relevant cell types such as motor cortex, containing motor neurons, and cerebellum, containing Purkinje cells, can be assessed by the same ddPCR method in tissue punches from those brain regions.

To measure ATXN2 knockdown in spinal cord motor neurons, ATXN2 mRNA is assessed by RT-ddPCR in spinal cord neurons captured by laser capture microdissection. Knockdown of ATXN2 mRNA is assessed by comparison of spinal cord neurons in amiRNA treated subjects relative to the vehicle treated group, using the ratio of ATXN2 positive droplets to housekeeping genes (GUSB, B2M, TBP, or others). Significant knockdown of ATXN2 in spinal cord neurons in animals dosed with ATXN2 targeting amiRNAs relative to vehicle dosed animals is desirable.

ATXN2 mRNA in spinal cord neurons, cortical motor neurons, cerebellar purkinje cells and other relevant tissues is also assessed by in situ hybridization (ISH) in tissue sections, and by RT-ddPCR in tissue punches. By in situ hybridization, knockdown of ATXN2 mRNA is semi-quantitatively assessed by comparison of amiRNA treated subjects relative to the vehicle group. Significant knockdown in these tissues is desirable, with reductions in ATXN2 mRNA in spinal and cortical motor neurons particularly relevant for ALS and knockdown in Purkinje cells particularly relevant for SCA2. By RT-ddPCR, knockdown is assessed as described above.

ATXN2 protein in spinal cord neurons, cortical motor neurons, cerebellum, and other brain tissues is assessed by immunohistochemistry. Fixed slides are stained with monoclonal ATXN2 antibody (BD, 611378) or polyclonal ATXN2 antibody (Sigma, HPA018295-100UL) using standard protocols. Immunohistochemistry is used to semi-quantitatively assess knockdown of ATXN2 protein, and significant reduction in ATXN2 levels relative to vehicle treated animals is desirable.

Other assays for the pharmacology of ATXN2 amiRNA vectors dosed via administration into the cerebrospinal fluid in non-human primates that may be conducted include ATXN2 assays using alphaLISA® or Simoa® bead technology; or amiRNA detection assays from tissue or body fluids using miRNA-ISH or miRNA RT-qPCR.

ATXN2 protein in bulk tissue is assessed by alphaLISA. The capture antibody is monoclonal ATXN2 antibody (BD, 611378) and detection antibody is polyclonal ATXN2 antibody (ProteinTech, 21776-1-AP). ATXN2 protein in CSF is assessed by custom ATXN2 Simoa assay (Quanterix).

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The various embodiments described above can be combined to provide further embodiments. All of the U.S. patents, U.S. patent application publications, U.S. patent applications, foreign patents, foreign patent applications and non-patent publications referred to in this specification and/or listed in the Application Data Sheet, including but not limited to U.S. Provisional Application No. 62/971,873 filed on Feb. 7, 2020, are incorporated herein by reference, in their entirety. Aspects of the embodiments can be modified, if necessary to employ concepts of the various patents, applications and publications to provide yet further embodiments.

These and other changes can be made to the embodiments in light of the above-detailed description. In general, in the following claims, the terms used should not be construed to limit the claims to the specific embodiments disclosed in the specification and the claims, but should be construed to include all possible embodiments along with the full scope of equivalents to which such claims are entitled. Accordingly, the claims are not limited by the disclosure.

Claims

1. An isolated nucleic acid comprising an expression construct encoding an inhibitory nucleic acid that inhibits expression or activity of ATXN2, wherein the inhibitory nucleic acid comprises a guide strand sequence comprising the nucleic acid sequence set forth in any one of SEQ ID NOS: 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 124, 126, 128, 130, 132, 134, 136, 138, 140, 142, 144, 146, 148, 150, 152, 154, 156, 158, 160, 162, 164, 166, 168, 170, 172, 174, 176, 178, 180, 182, 184, 186, 188, 190, 192, 194, 196, 198, 200, 202, 204, 206, 208, 210, 212, 214, 216, 218, 220, 222, 224, 226, 228, 230, 232, 234, 236, 238, 240, 242, 244, 246, 248, 250, 252, 254, 256, 258, 260, 262, 264, 266, 268, 270, 272, 274, 276, 278, 280, 282, 284, 286, 288, 290, 292, 294, 296, 298, 300, 302, 304, 306, 308, 310, 312, 314, 316, 318, 320, 324, 326, 328, 330, 332, 334, 336, 338, 340, 342, 344, 346, 348, 350, 352, 354, 356, 358, 360, 362, 364, 366, 368, 370, 372, 374, 376, 378, 380, 382, 384, 386, 388, 390, 392, 394, 396, 398, 400, 402, 404, 406, 408, 410, 412, 414, 416, 418, 420, 422, 424, 426, 428, 430, 432, 434, 436, 1176-1288, 1811-1827, 2015, 2065, 2083, 2152, 2203, and 2209.

2. (canceled)

3. The isolated nucleic acid molecule of claim 1, wherein the inhibitory nucleic acid is a siRNA duplex, shRNA, miRNA, or dsRNA.

4. The isolated nucleic acid molecule of claim 1, wherein the inhibitory nucleic acid further comprises a passenger strand sequence, optionally wherein the passenger strand sequence is selected from Tables 1, 19, 23, and 24, or a passenger strand sequence selected from Tables 1, 19, 23, and 24 and having 1-10 insertions, deletions, substitutions, mismatches, wobbles, or any combination thereof.

5. The isolated nucleic acid molecule of claim 4, wherein the inhibitory nucleic acid is an artificial miRNA, wherein the guide strand sequence and passenger strand sequence are contained within a miRNA backbone sequence.

6. (canceled)

7. The isolated nucleic acid molecule of claim 5, wherein the miRNA backbone sequence is a miR-155 backbone sequence, a miR-155E backbone sequence, a miR-155M backbone sequence, a miR1-1 backbone sequence, a miR-1-1_M backbone sequence, a miR-100 backbone sequence, a miR-100_M backbone sequence, a miR-190a backbone sequence, a miR-190a_M backbone sequence, a miR-124 backbone sequence, a miR-124_M backbone sequence, a miR-132 backbone sequence, a miR-9 backbone sequence, a miR-138-2 backbone sequence, a miR-122 backbone sequence, a miR-122_M backbone sequence, a miR-130a backbone sequence, a miR-16-2 backbone sequence, a miR-128 backbone sequence, a miR-144 backbone sequence, a miR-451a backbone sequence, or a miR-223 backbone sequence.

8. (canceled)

9. (canceled)

10. (canceled)

11. The isolated nucleic acid molecule of claim 1, wherein the inhibitory nucleic acid is a miRNA comprising the nucleic acid sequence set forth in any one of SEQ ID NOS: 443-490, 1109-1111, 1114, 1121-1168, 1405-1520, 1908-2007, 2011, 2017, 2021, 2025, 2027, 2031, 2035, 2039, 2041, 2045, 2049, 2053, 2057, 2061, 2067, 2071, 2075, 2079, 2085, 2089, 2093, 2097, 2101, 2105, 2109, 2113, 2117, 2120, 2124, 2128, 2132, 2136, 2140, 2144, 2148, 2154, 2158, 2162, 2166, 2170, 2174, 2176, 2180, 2182, 2184, 2187, 2189, 2191, 2193, 2195, 2197, 2199, 2205, 2211, 2261, 2263, 2265, and 2267.

12. (canceled)

13. The isolated nucleic acid molecule of claim 1, wherein the nucleic acid sequence encoding the inhibitory nucleic acid is located in an untranslated region of the expression construct.

14. (canceled)

15. The isolated nucleic acid molecule of claim 1, further comprising a promoter operably linked to the nucleic acid sequence encoding the inhibitory nucleic acid.

16. The isolated nucleic acid molecule of claim 15, wherein the promoter is a RNA pol III promoter, U6 promoter, H1 promoter, a chicken-beta actin (CBA) promoter, a CAG promoter, a H1 promoter, a CD68 promoter, a human synapsin promoter, or a JeT promoter.

17. The isolated nucleic acid molecule of claim 15, wherein the promoter is an H1 promoter comprising nucleotides 113-203 of SEQ ID NO:1522, nucleotides 1798-1888 of SEQ ID NO:1521, nucleotides 244-343 of SEQ ID NO:2257, or nucleotides 113-343 of SEQ ID NO:2257.

18. The isolated nucleic acid molecule of claim 1, wherein the expression construct is flanked by a 5′ adeno-associated virus (AAV) inverted terminal repeat (ITR) sequence and a 3′ AAV ITR sequence, or variants thereof.

19. The isolated nucleic acid molecule of claim 18, wherein one of the ITR sequences lacks a functional terminal resolution site.

20. The isolated nucleic acid molecule of claim 18, wherein the 5′ and 3′ ITRs are derived from an AAV serotype selected from the group consisting of: AAV1, AAV2, AAV5, AAV6, AAV6.2, AAV7, AAV8, AAV9, AAVRh10, AAV11, and variants thereof.

21. The isolated nucleic acid molecule of claim 18, wherein the 5′ ITR comprises nucleotides 1-106 of SEQ ID NO:2257 and the 3′ ITR comprises nucleotides 2192-2358 of SEQ ID NO:2257.

22. A vector comprising the isolated nucleic acid molecule of claim 1.

23. The vector of claim 16, wherein the vector is a plasmid or viral vector.

24. The vector of claim 23, wherein the viral vector is a recombinant adeno-associated virus (rAAV) vector or a Baculovirus vector.

25. The vector of claim 24, wherein the vector is a self-complementary rAAV vector.

26. The vector of claim 24, wherein the rAAV vector further comprises a stuffer sequence.

27. (canceled)

28. (canceled)

29. A recombinant adeno-associated (rAAV) particle comprising the isolated nucleic acid molecule of claim 1.

30. The rAAV particle of claim 29, wherein the rAAV particle comprises a capsid protein.

31. The rAAV particle of claim 30, wherein the capsid protein is capable of crossing the blood-brain barrier.

32. The rAAV particle of claim 30, wherein the capsid protein is an AAV9 capsid protein or AAVrh.10 capsid protein.

33. (canceled)

34. A pharmaceutical composition comprising the isolated nucleic acid molecule of claim 1, and optionally a pharmaceutically acceptable carrier.

35. A host cell comprising the isolated nucleic acid molecule of claim 1.

36. A method for treating a subject having or suspected of having a neurodegenerative disease, the method comprising administering to the subject the isolated nucleic acid molecule of claim 1.

37. The method of claim 36, wherein the administration comprises direct injection to the CNS of the subject.

38. The method of claim 37, wherein the direct injection is intracerebral injection, intraparenchymal injection, intrathecal injection, intrastriatal injection, subpial injection, direct injection to the cerebrospinal fluid (CSF) of the subject, intracistemal injection, intraventricular injection, intralumbar injection, or any combination thereof.

39. (canceled)

40. The method of claim 36, wherein the subject is characterized as having an ATXN2 allele having at least 22 CAG trinucleotide repeats, optionally wherein the ATXN2 allele has at least 24 CAG trinucleotide repeats, at least 27 CAG trinucleotide repeats, at least 30 CAG trinucleotide repeats, or at least 33 or more CAG trinucleotide repeats.

41. The method of claim 36, wherein the neurodegenerative disease is spinocerebellar ataxia-2, amyotrophic lateral sclerosis, frontotemporal dementia, primary lateral sclerosis, progressive muscular atrophy, limbic-predominant age-related TDP-43 encephalopathy, chronic traumatic encephalopathy, dementia with Lewy bodies, corticobasal degeneration, progressive supranuclear palsy (PSP), dementia Parkinsonism ALS complex of guam (G-PDC), Pick's disease, hippocampal sclerosis, Huntington's disease, Parkinson's disease, or Alzheimer's disease.

42. A method of inhibiting ATXN2 expression in a cell, the method comprising delivering to the cell the isolated nucleic acid of claim 1.

43. (canceled)

44. (canceled)

45. (canceled)

46. (canceled)

47. (canceled)

48. (canceled)

49. A method of inhibiting ATXN2 expression in the central nervous system of a subject, the method comprising administering to the subject the isolated nucleic acid of claim 1.

50. (canceled)

51. (canceled)

52. (canceled)

53. (canceled)

54. An artificial miRNA comprising a guide strand sequence and a passenger strand sequence, wherein the guide strand sequence comprises any one of SEQ ID NOS: 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 124, 126, 128, 130, 132, 134, 136, 138, 140, 142, 144, 146, 148, 150, 152, 154, 156, 158, 160, 162, 164, 166, 168, 170, 172, 174, 176, 178, 180, 182, 184, 186, 188, 190, 192, 194, 196, 198, 200, 202, 204, 206, 208, 210, 212, 214, 216, 218, 220, 222, 224, 226, 228, 230, 232, 234, 236, 238, 240, 242, 244, 246, 248, 250, 252, 254, 256, 258, 260, 262, 264, 266, 268, 270, 272, 274, 276, 278, 280, 282, 284, 286, 288, 290, 292, 294, 296, 298, 300, 302, 304, 306, 308, 310, 312, 314, 316, 318, 320, 324, 326, 328, 330, 332, 334, 336, 338, 340, 342, 344, 346, 348, 350, 352, 354, 356, 358, 360, 362, 364, 366, 368, 370, 372, 374, 376, 378, 380, 382, 384, 386, 388, 390, 392, 394, 396, 398, 400, 402, 404, 406, 408, 410, 412, 414, 416, 418, 420, 422, 424, 426, 428, 430, 432, 434, 436, 1176-1288, 1811-1827, 2015, 2065, 2083, 2152, 2203, and 2209.

55. (canceled)

56. The artificial miRNA of claim 54, wherein the guide strand sequence and passenger strand sequence are contained within a miR backbone sequence.

57. The artificial miRNA of claim 56, wherein the miR backbone sequence is a miR-155 backbone sequence, a miR-155E backbone sequence, a miR-155M backbone sequence, a miR1-1 backbone sequence, a miR-1-1_M backbone sequence, a miR-16-2 backbone sequence, a miR-100 backbone sequence, a miR-100_M backbone sequence, a miR-190a backbone sequence, a miR-190a_M backbone sequence, a miR-124 backbone sequence, a miR-124_M backbone sequence, a miR-132 backbone sequence, a miR-9 backbone sequence, a miR-138-2 backbone sequence, a miR-122 backbone sequence, a miR-122_M backbone sequence, a miR-130a backbone sequence, or a miR-128 backbone sequence, a miR-144 backbone sequence, a miR-451a backbone sequence, or a miR-223 backbone sequence.

58. (canceled)

59. (canceled)

60. (canceled)

61. The artificial miRNA of claim 54, wherein the artificial miRNA comprises the sequence as set forth in any one of SEQ ID NOS: 443-490, 1109-1111, 1114, 1121-1168, 1405-1520, 1908-2007, 2011, 2017, 2021, 2025, 2027, 2031, 2035, 2039, 2041, 2045, 2049, 2053, 2057, 2061, 2067, 2071, 2075, 2079, 2085, 2089, 2093, 2097, 2101, 2105, 2109, 2113, 2117, 2120, 2124, 2128, 2132, 2136, 2140, 2144, 2148, 2154, 2158, 2162, 2166, 2170, 2174, 2176, 2180, 2182, 2184, 2187, 2189, 2191, 2193, 2195, 2197, 2199, 2205, 2211, 2261, 2263, 2265, and 2267.

62. An isolated RNA duplex comprising a guide strand sequence and a passenger strand sequence, wherein the guide strand sequence comprises the nucleic acid sequence set forth in any one of SEQ ID NOS: 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 124, 126, 128, 130, 132, 134, 136, 138, 140, 142, 144, 146, 148, 150, 152, 154, 156, 158, 160, 162, 164, 166, 168, 170, 172, 174, 176, 178, 180, 182, 184, 186, 188, 190, 192, 194, 196, 198, 200, 202, 204, 206, 208, 210, 212, 214, 216, 218, 220, 222, 224, 226, 228, 230, 232, 234, 236, 238, 240, 242, 244, 246, 248, 250, 252, 254, 256, 258, 260, 262, 264, 266, 268, 270, 272, 274, 276, 278, 280, 282, 284, 286, 288, 290, 292, 294, 296, 298, 300, 302, 304, 306, 308, 310, 312, 314, 316, 318, 320, 324, 326, 328, 330, 332, 334, 336, 338, 340, 342, 344, 346, 348, 350, 352, 354, 356, 358, 360, 362, 364, 366, 368, 370, 372, 374, 376, 378, 380, 382, 384, 386, 388, 390, 392, 394, 396, 398, 400, 402, 404, 406, 408, 410, 412, 414, 416, 418, 420, 422, 424, 426, 428, 430, 432, 434, 436, and 1176-1288, 1811-1827, 2015, 2065, 2083, 2152, 2203, and 2209, optionally wherein the guide strand sequence and passenger strand sequence are linked by a loop region to form a hairpin structure comprising a duplex structure and a loop region.

63. (canceled)