REDIRECTION OF TROPISM OF AAV CAPSIDS

Abstract

The disclosure relates to compositions, methods, and processes for the preparation, use, and/or formulation of adeno-associated virus capsid proteins, wherein the capsid proteins comprise targeting peptide inserts for enhanced tropism to a target tissue.

Description

REFERENCE TO THE SEQUENCE LISTING

The present application is being filed along with a Sequence Listing in electronic format. The Sequence Listing is provided as a file entitled 20571060PCTSL.txt, created on Oct. 2, 2019, which is 428,491 bytes in size. The information in the electronic format of the sequence listing is incorporated herein by reference in its entirety.

FIELD OF THE DISCLOSURE

The disclosure relates to compositions, methods, and processes for the preparation, use, and/or formulation of adeno-associated virus capsid proteins, wherein the capsid proteins comprise targeting peptide inserts for enhanced tropism to a target tissue.

BACKGROUND

Gene delivery to the adult central nervous system (CNS) remains a major challenge in gene therapy, and engineered AAV capsids with improved brain tropism represent an attractive solution.

Adeno-associated virus (AAV)-derived vectors are promising tools for clinical gene transfer because of their non-pathogenic nature, their low immunogenic profile, low rate of integration into the host genome and long-term transgene expression in non-dividing cells. However, the transduction efficiency of AAV natural variants in certain organs is too low for clinical applications, and capsid neutralization by pre-existing neutralizing antibodies may prevent treatment of a large proportion of patients. For these reasons, major efforts have been devoted to obtaining novel capsid variants with enhanced properties. Of many approaches tested so far, the most significant advances have resulted from directed evolution of AAV capsids using in vitro or in vivo selection of capsid variants created by capsid sequence randomization using either error-prone PCR, shuffling of various parent serotypes or insertion of fully randomized short peptides at defined positions.

In order to perform directed evolution of AAV capsids, the sequence encoding the viral capsid is itself flanked by inverted terminal repeats (ITR) so it can be packaged into its own capsid shell. Following infection of cultured cells or animals by the mixed population of capsids, the DNA encoding capsids variants that have successfully homed into the tissue of interest is recovered by PCR for further rounds of selection. In this approach, all viral DNA species present in a given tissue are recovered, with no discrimination for specific cell types or for vectors able to perform complete transduction (cell surface binding, endocytosis, trafficking, nuclear import, uncoating, second-strand synthesis, transcription). For example, in the case of highly complex tissues containing multiple cell types, such as the central nervous system (CNS), it would be highly preferable to apply a more stringent selective pressure aimed at recovering capsid variants capable of transducing neuron and/or astrocyte rather than microglia or blood vessel endothelial cells.

Attempts at improving the CNS tropism of AAV capsids upon systemic administration have been met with limited success.

Two previous approaches have been used to address this issue. The first strategy used co-infection of cultured cells (Grimm et al., 2008) or in situ animal tissue (Lisowski et al., 2014) with adenovirus, in order to trigger exponential replication of infectious AAV DNA. Another successful approach involved the use of cell-specific CRE transgenic mice (Deverman et al., 2016) allowing viral DNA recombination specifically in astrocytes, followed by recovery of CRE-recombined capsid variants. Both approaches proved successful, allowing the isolation of several capsid variants with enhanced transduction of target cell populations.

This finding suggested that cell type-specific library selection could improve the outcome of directed evolution. However, the transgenic CRE system used by Deverman et al. is not tractable in other animal species and AAV variants selected by directed evolution in mouse tissue do not show similar properties in large animals. Therefore, it would be necessary to perform the entire directed evolution process directly in non-human primates to increase the probability of translatability in human subjects. None of the previously described transduction-specific approaches are amenable to large animal studies because: 1) many tissues of interest (e.g. CNS) are not readily accessible to adenovirus co-infection, 2) the specific Ad tropism itself would bias the library distribution, and 3) large animals are typically not amenable to transgenesis and cannot be genetically engineered to express CRE recombinase in defined cell types.

To address this problem, we have developed a broadly-applicable functional AAV capsid library screening platform for cell type-specific biopanning in non-transgenic animals. In the TRACER (Tropism Redirection of AAV by Cell type-specific Expression of RNA) platform system, the capsid gene is placed under the control of a cell type-specific promoter to drive capsid mRNA expression in the absence of helper virus co-infection. This RNA-driven screen increases the selective pressure in favor of capsid variants which transduce a specific cell type.

The TRACER platform allows generation of AAV capsid libraries whereby specific recovery and subcloning of capsid mRNA expressed in transduced cells is achieved with no need for transgenic animals or helper virus co-infection. Since mRNA transcription is a hallmark of full transduction, these methods will allow identification of fully infectious AAV capsid mutants. In addition to its higher stringency, this method allows identification of capsids with high tropism for particular cell types using libraries designed to express CAP mRNA under the control of any cell-specific promoter such as, but not limited to, synapsin-1 promoter (neurons), GFAP promoter (astrocytes), TBG promoter (liver), CAMK promoter (skeletal muscle), MYH6 promoter (cardiomyocytes).

SUMMARY OF THE DISCLOSURE

The present disclosure provides compositions and methods for the engineering and/or redirecting the tropism of AAV capsids. Also provided herein are peptides which may be inserted into AAV capsid sequences to increase the tropism of the capsid for a particular tissue. In one aspect, the peptides may be used to target the capsids to brain or regions of the brain or the spinal cord.

The present disclosure presents methods for generating one or more variant AAV capsid polypeptides. In certain embodiments, the variant AAV capsid polypeptides exhibit at least one of improved transduction or increased cell or tissue specificity, relative to a parental AAV capsid polypeptide. In certain embodiments, the method includes: (a) generating a library of variant AAV capsid polypeptides, wherein said library includes (i) a plurality of capsid polypeptides having a region of randomized sequence of 2, 3, 4, 5, 6, 7, 8, or 9 consecutive amino acids, or (ii) a plurality of capsid polypeptides from more than one parental AAV capsid polypeptide; (b) generating an AAV vector library by cloning the capsid polypeptides of libraries (a)(i) or (a)(ii) into AAV vectors, wherein the AAV vectors include a first promoter and a second promoter, wherein said second promoter drives capsid mRNA expression in the absence of helper virus co-infection.

In certain embodiments, the first promoter is AAV2 P40. In certain embodiments, the second promoter is a ubiquitous promoter. In certain embodiments, the first promoter is AAV2 P40 and the second promoter is a ubiquitous promoter.

In certain embodiments, the first promoter is AAV2 P40. In certain embodiments, the second promoter is a cell-type-specific promoter. In certain embodiments, the first promoter is AAV2 P40 and the second promoter is a cell-type-specific promoter.

In certain embodiments, the promoter is selected from any promoter listed in Table 3. In certain embodiments, the ubiquitous or cell-specific promoter allows the expression of RNA encoding the capsid polypeptides.

In certain embodiments, the method includes recovery of the RNA encoding the capsid polypeptides. In certain embodiments, the method includes determining the sequence of the capsid polypeptides. In certain embodiments, the capsid polypeptides recovered exhibit increased target cell transduction or target cell specificity (tropism) as compared to a parental capsid polypeptide.

In certain embodiments, the target cell is a neuronal cell, a neural stem cell, an astrocyte, an oligodendrocyte, a microglia cell, a retinal cell, a tumor cell, a hematopoietic stem cell, an insulin producing beta cell, a lung epithelium cell, an endothelial cell, a liver cell, a skeletal muscle cell, a muscle stem cell, a muscle satellite cell, or a cardiac muscle cell.

In certain embodiments, the AAV vectors comprise a first promoter and a second promoter, wherein the second promoter is located the downstream of the capsid gene and drives its anti-sense RNA expression in the absence of helper virus co-infection.

In certain embodiments, the first promoter is AAV2 P40 and the second promoter is a ubiquitous promoter. In certain embodiments, the first promoter is AAV2 P40 and the second promoter is a cell-specific promoter. In certain embodiments, the ubiquitous or cell-specific promoter allows the expression of gene encoding the capsid polypeptide of variant AAV in an anti-sense direction, resulting in the anti-sense RNA. In certain embodiments, the method included the recovery of the anti-sense RNA that can be converted to RNA encoding the variant AAV capsid polypeptide that is used to determine the sequence of the variant AAV capsid polypeptides.

In certain embodiments, the variant AAV capsid polypeptide exhibits increased target cell transduction or target cell specificity (tropism) as compared to a parental capsid polypeptide.

BRIEF DESCRIPTION OF THE DRAWINGS

The foregoing and other objects, features, and advantages will be apparent from the following description of particular embodiments of the disclosure, as illustrated in the accompanying drawings. The drawings are not necessarily to scale, emphasis instead being placed upon illustrating the principles of various embodiments of the disclosure.

FIG. 1A and FIG. 1B are maps of wild-type AAV capsid gene transcription and CMV-CAP vectors. FIG. 1A shows transcription of VP1, VP2 and VP3 AAV transcripts from wildtype AAV genome. Transcription start sites of each viral promoter are indicated. SD, splice donor, SA, splice acceptor. Sequence of start codons for each reading frame is indicated. Translation of AAP and VP3 is performed by leaky scanning of the major mRNA. FIG. 1B shows the structure of the CMV-p40 dual promoter vectors used to determine the minimal regulatory sequences necessary for efficient virus production. The pREP2ΔCAP vector shown at the bottom is obtained by deletion of most CAP reading frame and is used to provide the REP protein in trans.

FIG. 2A and FIG. 2B are histogram representations of the data and show the effect of CMV promoter position on virus yield and CAP mRNA splicing. FIG. 2A shows average yield of AAV9 produced in HEK-293T cells using the constructs described in FIG. 1, co-transfected with an Ad Helper vector. Wild-type AAV9 plasmid (pAV9) is used as a positive control. Y-axis values indicate AAV DNA copies per ul from each 15-cm plate (˜1000 ul total, left panel) or the percentage of wtAAV9 (right panel). FIG. 2B shows evidence for expression of CAP transcripts in transfected cells. mRNA from transfected 293T cells was subjected to RT-PCR using primers specific for the major spliced CAP transcript. Note the lack of p40-driven transcription in the absence of Ad Helper vector (lane 2).

FIG. 3A, FIG. 3B and FIG. 3C show the effect of REP helper plasmid optimization on virus yield. FIG. 3A shows the design of improved pREP helper vectors. The MscI fragment deletion removes the C-terminal part of VP proteins, which is necessary for capsid formation. Asterisks represent early stop codons introduced to disrupt the coding potential of VP1, VP2 and VP3 reading frames. FIG. 3B shows the yield of Synapsin-p40-CAP9 AAV produced with various REP plasmid architectures. Values on the Y-axis represent the percentage of VG relative to wild-type AAV9. FIG. 3C shows the quantification of recombination and/or illegitimate packaging of full-length REP from the pREP plasmids. Virus stocks produced were subjected to qPCR using Taqman probes located in the N-terminal part of REP absent from the ITR-containing vectors.

FIG. 4A, FIG. 4B, FIG. 4C and FIG. 4D describe the in vivo analysis of the second-generation vectors. FIG. 4A shows the design of Pro9 vectors. Architecture of all three vectors is based on the BstEII construct. AAV9 capsid RNA is placed under control of P40 and CMV, hSyn1 or GFAP promoters, respectively. FIG. 4B shows the silver stain of SDS-PAGE gel obtained by running 1e10 VG of each vector, after double iodixanol purification. FIG. 4C shows the biodistribution of viral DNA in mouse brain (cortex), liver and heart following tail-vein injection of 1e12 VG per mouse. AAV9 VP3 DNA is quantified by Taqman PCR and normalized to mouse transferrin receptor gene. FIG. 4D shows the capsid RNA recovery from mouse tissues. Total RNA was reverse transcribed and Taqman PCR was performed with capsid-specific Taqman primers and probe. Values represent VP3 cDNA copies normalized to TBP housekeeping gene.

FIG. 5A, FIG. 5B, FIG. 5C, FIG. 5D and FIG. 5E describes in vitro analysis of intronic second generation vectors. FIG. 5A shows the design of intronic Pro9 vectors harboring a hybrid CMV/Globin intron. AAV9 capsid RNA is placed under control of P40 and CBA, hSyn1 or GFAP promoters in a tandem configuration (top) or in an inverted configuration (bottom). In the inverted promoter vectors, an extra SV40 polyadenylation site (orange) is added at the 3′ extremity to allow polyadenylation of antisense CAPS transcripts. FIG. 5B shows the AAV9 CAP cDNA amplification. All vectors depicted were produced using triple transfection with pHelper and pREP-3stops and resulting viruses were used to infect HEK-293T cells at a MOI of 1e4 VG per cell. RNA was extracted 48 hours post-infection and subjected to RT-PCR with primers amplifying full capsid (top) or a C-terminal fragment (bottom). FIG. 5C shows the AAV9 VP3 cDNA from cells infected with intronless or intronic viruses with tandem promoters in forward orientation was quantified by Taqman PCR and normalized to GAPDH housekeeping gene. Values indicate the ratio of VP3 to GAPDH cDNA. FIG. 5D shows the mapping of capsid RNA recovery from cells infected with tandem or inverted constructs. Total RNA was reverse transcribed and PCR was performed with primers flanking the entire capsid gene. White arrowheads represent VP3 size variants resulting from aberrant splicing of antisense CAP mRNA. FIG. 5E shows the analysis of Globin intron splicing. CAG9 plasmid (left) or cDNA from HEK-293T cells transduced by CAG9 virus was submitted to PCR with forward primers located before (Glo ex1) or within (GloSpliceF4 (SEQ ID NO: 26) and GloSpliceF6 (SEQ ID NO: 13)) the Globin exon-exon junction. Primers spanning junction between exon 1 (no underline) and exon 2 (underline) are described at the bottom.

FIG. 6 provides in vitro evidence that the presence of the P40 promoter downstream of Synapsin or Gfabc1D promoters does not relieve the repression of either promoter in HEK-293T cells.

FIG. 7 illustrates the basic tenets of the TRACER platform.

FIG. 8 illustrates features of the TRACER platform including the use of a tissue specific promoter and RNA recovery.

FIG. 9 provides one embodiment of the TRACER production architecture.

FIG. 10 provides a comparison between traditional vDNA recovery and 2^nd generation vRNA recovery.

FIG. 11 provides an overview of the use of cell-specific RNA expression for targeted evolution.

FIG. 12A and FIG. 12B provide diagrams representing capsid gene transcription of natural AAV (FIG. 12A) and TRACER libraries (FIG. 12B).

FIG. 13 is a diagram of the AAV6, AAV5 and AAV-DJ capsid peptide display libraries used for in vivo evolution (SEQ ID NOS 27-32, respectively, in order of appearance).

FIG. 14 is a diagram of the AAV9 capsid peptide display libraries used for in vivo evolution (SEQ ID NOS 33-42, respectively, in order of appearance).

FIG. 15A and FIG. 15B present the method used for library construction. FIG. 15A shows the sequence of the insertion site used to introduce random libraries (SEQ ID NOS 43-46, respectively, in order of appearance). FIG. 15B provides a description of the assembly procedure.

FIG. 16 provides an exemplary diagram of cloning-free rolling circle procedure used for library amplification (SEQ ID NO 47; NNK₇).

FIG. 17 provides the sequence of the codon-mutant AAV9 library shuttle designed to minimize wild-type contamination (SEQ ID NOS 33-34 and 48-52, respectively, in order of appearance).

FIG. 18 provides a description of AAV9 peptide libraries biopanning.

FIG. 19 illustrates the recovery process from an initial pool with recovery at 50%.

FIG. 20 provides an example of the cDNA recovery and amplification from GFAP-driven libraries (B group and F group).

FIG. 21A, FIG. 21B and FIG. 21C show the progression of AAV9 peptide library diversity throughout the biopanning process. FIG. 21A describes RNA library evolution. FIG. 21B and FIG. 21C show the amino acid distribution of NNK machine mix preparations for P0 and P1 virus.

FIG. 22 provides neuron (SYN)-AAV9 Peptide Libraries Composition at P2.

FIG. 23 provides astrocyte (GFAP)-AAV9 Peptide Libraries Composition at P2.

FIG. 24 provides an estimation of brain/liver specificity in GFAP-AAV9 peptide library candidates.

FIG. 25 provides an estimation of brain/liver specificity in GFAP-AAV9 peptide library candidates.

FIG. 26 provide an example subpopulation selection of variants.

FIG. 27 provides an exemplary design of a library generation and cloning procedure.

FIG. 28 provides the NNK/NNM codon distribution (covariance of codon mutants) of AAV produced with a synthetic library of 666 sequence variants (GFAP promoter).

FIG. 29 provides the NNK/NNM codon distribution (covariance of codon mutants) of AAV produced with a synthetic library of 666 sequence variants (SYN9 promoter).

FIG. 30 provides the data from the tissue recovery, one-month post injection, from brain and a liver punch.

FIG. 31A, FIG. 31B, FIG. 31C and FIG. 31D provide results of control capsids from the Syn-driven synthetic library NGS analysis. FIG. 31A shows the enrichment analysis of internal AAV9, PHP.B and PHP.eB controls (SEQ ID NOS 53-58 and 53-58, respectively, in order of appearance). FIG. 31B, FIG. 31C and FIG. 31D show the NNK/NNM codon distribution in mRNA from mouse brain tissue.

FIG. 32A and FIG. 32B provide the results of the neuron synthetic library NGS analysis (SEQ ID NOS 59-60, 59-61, 61-63, 62, 64, 64, 63, 65-67, 67, 65, 68, 66, 69, 70-71, and 70-74, respectively, in order of appearance).

FIG. 33 provides the results of the astrocyte synthetic library NGS analysis (SEQ ID NOS 53-58, 53-58, and 53-58, respectively, in order of appearance).

FIG. 34A and FIG. 34B provide astrocyte synthetic library codon mutants covariance.

FIG. 35 provides the results of the astrocyte synthetic library NGS analysis (SEQ ID NOS 75, 75-78, 76-77, 79-83, 65, 78, 84, 80, 85, 70, 86, 82, 81, 79, 87, 65, 85, 84, 70, 86, 88-90, 87, 91, 83, 88, 63, 89-90, 92-93, 91, 94-97, 93, 95, 98, 98, 97, 63, 92, 94, 99-101, 75, 75-78, 76-77, 79-83, 65, 78, 84, 80, 85, 70, 86, 82, 81, 79, 87, 65, 85, 84, 70, 86, 88-90, 87, 91, 83, 88, 63, 89-90, 92-93, 91, 94-97, 93, 95, 98, 98, 97, 63, 92, 94, 99-102, 99, 103, 103-104, 96, 105-106, 101, 100, 102, 107, 104-105, 108-113, 106, 60, 66, 114-117, 109, 113, 72, 108, 110, 67, 118-119, 116, 120, 120, 107, 112, 121-123, 66, 124-125, 115, 118, 126, 121, 127-128, 60, 129, 119, 130-132, 72, 133, 123, 125, 69, 134-139, 62, 124, 67, 111, 114, 126, 140-141, 122, 142, 128-129, 143, 138, 144, 134, 62, 136, 145, 141, 146-153, 127, 154, 69, 144, 155, 71, 156, 133, 132, 137, 147, 157-158, 135, 159, 140, 117, 160, 139, 161-162, 130, 163, 143, 164, 152, 151, 165-167, 155, 168, 71, 169, and 146, respectively, in order of appearance).

FIG. 36 provides the GFAP synthetic library NGS analysis.

FIG. 37A and FIG. 37B provides the top 38 variants from the synthetic library screen. FIG. 37A shows the phylogenetic analysis of 9-mer peptide sequences, and also shows the sequence of the peptide variants (SEQ ID NOS 67, 59, 64, 61, 77, 84, 96, 60, 80, 82, 66, 62, 83, 85, 106, 131, 94, 90, 76, 68-69, 79, 75, 81, 88, 139, 78, 155, 102, 63, 140, 87, 70, 105, 120, 89, 65, and 109, respectively, in order of appearance). Highlighted sequences represent the peptides that were selected for individual transduction assay. FIG. 37B shows the graphic representation of the neuron and astrocyte tropism of each peptide, both axis indicate the inverted rank in Synapsin and GFAP screen.

FIG. 38 provides the top consensus sequences as compared to PHP.N and PHP.B (SEQ ID NOS 168 and 71, respectively, in order of appearance).

FIG. 39 is a diagram of the Gibson assembly library cloning procedure.

FIG. 40 provides an example of TRIM/NNK peptide prevalence (SEQ ID NOS 170-171, respectively, in order of appearance).

FIG. 41 provides peptide diversity statistics from a study using the Illumina adapter having 42 million bacterial transformants, 81 million sequence reads and 12 million sequence variants (SEQ ID NOS 172-173, 48-49, and 174-175, respectively, in order of appearance).

FIG. 42 provides an exemplary diagram of cloning-free DNA amplification by rolling circle amplification.

FIG. 43 provides a diagram of protelomerase monomer processing (SEQ ID NOS 176-178, respectively, in order of appearance).

FIG. 44 provides a diagram comparing the traditional and cloning-free methods.

FIG. 45A and FIG. 45C provide the full ranking of Syn-driven (FIG. 45A) and GFAP-driven (FIG. 45B) 333 variants in the brain, spinal cord, liver and heart tissues. Capsid variants are ranked by their average brain RNA enrichment score (average of NNK and NNM codons). The rank of internal control capsids PHP.B, PHP.eB and AAV9 is indicated (FIG. 45A and FIG. 45B). A comparison of combined Syn-driven results and GFAP-driven results is provided (FIG. 45C). Only 4 animals were represented for the GFAP-driven libraries because 2/6 mice showed a very different ranking profile and were considered as outliers.

FIG. 46A and FIG. 46B provide the comparison of results of the neuron and astrocyte synthetic library NGS analysis. FIG. 46A shows the ranking of capsids using SYN or GFAP promoters; FIG. 46B shows the scatter plot showing the correlation of Syn-versus GFAP-driven libraries.

FIG. 47 illustrates one embodiment of a multi-species (e.g., rodent) study followed by next generation sequencing (NGS).

FIG. 48A, FIG. 48B and FIG. 48C provide results from a multi-strain/species comparison of 333 capsid variants. FIG. 48A shows the ranking of 333 capsids by brain RNA enrichment score in C57BL/6 mice, BALB/C mice and rats. Capsids are ranked according to Syn-driven brain enrichment score in C57BL/6 mice. FIG. 48B shows the scatter plots showing the correlation between C57BL/6 and BALB/C enrichment scores from Syn- and GFAP-driven pools. FIG. 48C shows the Venn diagram showing the intersection and consensus sequence of capsids with a brain enrichment score >10-fold higher than AAV9 (either Syn- or GFAP-driven) in C57BL/6 and BALB/C strains. In rats, no capsid showed an enrichment score >10-fold versus AAV9.

FIG. 49A, FIG. 49B, FIG. 49C and FIG. 49D provide transduction (RNA) and biodistribution (DNA) analysis of 10 capsid variants indicated in FIG. 49A (SEQ ID NOS 179-188, respectively, in order of appearance). Individual capsids were used to package self-complementary CBA-EGFP genomes (FIG. 49B) and injected intravenously to C57BL/6 mice. FIG. 49C shows the RNA expression in brain and spinal cord samples. FIG. 49D shows the DNA distribution in brain and spinal cord samples.

FIG. 50A, FIG. 50B and FIG. 50C provide the results of testing of individual capsids and their mRNA expression in brain, spinal cord and liver. EGFP mRNA expression results are shown for the brain (FIG. 50A), the spinal cord (FIG. 50B) and the liver (FIG. 50C).

FIG. 51 provides results for NGS screening using neuronal NeuN marker (FIG. 51) for both GFAP screening and SYN screening.

FIG. 52 provides the results of testing of individual capsids in whole brain.

FIG. 53 provides the results of testing of additional individual capsids in whole brain.

FIG. 54 provides the results of testing of individual capsids in cerebellum.

FIG. 55 provides the results of testing of individual capsids in cortex.

FIG. 56 provides the results of testing of individual capsids in hippocampus.

FIG. 57A and FIG. 57B provide transduction data of 10 capsid variants in mouse liver (FIG. 57B), analyzed by EGFP RNA expression and whole tissue fluorescence (FIG. 57A).

FIG. 58A and FIG. 58B provide results for comparison studies on the efficacy of the 333 capsid variants to transduce CNS for C57BL/6 mice BMVEC (FIG. 58A) and Human BMVEC (FIG. 58B).

FIG. 59A, FIG. 59B and FIG. 57C provide diagrams of external barcoding for NGS analysis and recovery of full-length capsid variants. A general barcode pair is shown (FIG. 59C). Full ITR-to-ITR constructs are shown with the barcode pair 5′ of the CAP sequence (FIG. 59A) and 3′ of the CAP sequence (FIG. 59B).

FIG. 60A, FIG. 60B and FIG. 60C provide detailed analysis of virus production and RNA splicing with several configurations of intronic barcoded platforms. A general ITR-to-ITR construct is shown in FIG. 60A (SEQ ID NOS 189-193, respectively, in order of appearance), with intronic barcode yields (FIG. 60B) and gel columns showing AAV intron splicing and Globin intron splicing results (FIG. 60C).

DETAILED DESCRIPTION OF THE DISCLOSURE

The details of one or more embodiments of the disclosure are set forth in the accompanying description below. Although any materials and methods similar or equivalent to those described herein can be used in the practice or testing of the present disclosure, the preferred materials and methods are now described. Other features, objects and advantages of the disclosure will be apparent from the description. In the description, the singular forms also include the plural unless the context clearly dictates otherwise. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. In the case of conflict, the present description will control.

According to the present disclosure, AAV particles with enhanced tropism for a target tissue (e.g., CNS) are provided, as well as associated processes for their targeting, preparation, formulation and use. Targeting peptides and nucleic acid sequences encoding the targeting peptides are provided. These targeting peptides may be inserted into an AAV capsid protein sequence to alter tropism to a particular cell-type, tissue, organ or organism, in vivo, ex vivo or in vitro.

As used herein, an “AAV particle” or “AAV vector” comprises a capsid protein and a viral genome, wherein the viral genome comprises at least one payload region and at least one inverted terminal repeat (ITR). The AAV particle and/or its component capsid and viral genome may be engineered to alter tropism to a particular cell-type, tissue, organ or organism.

As used herein, “viral genome” or “vector genome” refers to the nucleic acid sequence(s) encapsulated in an AAV particle. A viral genome comprises a nucleic acid sequence with at least one payload region encoding a payload and at least one ITR.

As used herein, a “payload region” is any nucleic acid molecule which encodes one or more “payloads” of the disclosure. As non-limiting examples, a payload region may be a nucleic acid sequence encoding a payload comprising an RNAi agent or a polypeptide.

As used herein, a “targeting peptide” refers to a peptide of 3-20 amino acids in length. These targeting peptides may be inserted into, or attached to, a parent amino acid sequence to alter the characteristics (e.g., tropism) of the parent protein. As a non-limiting example, the targeting peptide can be inserted into an AAV capsid sequence for enhanced targeting to a desired cell-type, tissue, organ or organism.

The AAV particles and payloads of the disclosure may be delivered to one or more target cells, tissues, organs, or organisms. In a preferred embodiment, the AAV particles of the disclosure demonstrate enhanced tropism for a target cell type, tissue or organ. As a non-limiting example, the AAV particle may have enhanced tropism for cells and tissues of the central or peripheral nervous systems (CNS and PNS, respectively). The AAV particles of the disclosure may, in addition, or alternatively, have decreased tropism for an undesired target cell-type, tissue or organ.

Adeno-associated viruses (AAV) are small non-enveloped icosahedral capsid viruses of the Parvoviridae family characterized by a single stranded DNA viral genome. Parvoviridae family viruses consist of two subfamilies: Parvovirinae, which infect vertebrates, and Densovirinae, which infect invertebrates. The Parvoviridae family comprises the Dependovirus genus which includes AAV, capable of replication in vertebrate hosts including, but not limited to, human, primate, bovine, canine, equine, and ovine species.

The parvoviruses and other members of the Parvoviridae family are generally described in Kenneth I. Berns, “Parvoviridae: The Viruses and Their Replication,” Chapter 69 in FIELDS VIROLOGY (3d Ed. 1996), the contents of which are incorporated by reference in their entirety.

AAV have proven to be useful as a biological tool due to their relatively simple structure, their ability to infect a wide range of cells (including quiescent and dividing cells) without integration into the host genome and without replicating, and their relatively benign immunogenic profile. The genome of the virus may be manipulated to contain a minimum of components for the assembly of a functional recombinant virus, or viral particle, which is loaded with or engineered to target a particular tissue and express or deliver a desired payload.

The wild-type AAV vector genome is a linear, single-stranded DNA (ssDNA) molecule approximately 5,000 nucleotides (nt) in length. Inverted terminal repeats (ITRs) traditionally cap the viral genome at both the 5′ and the 3′ end, providing origins of replication for the viral genome. While not wishing to be bound by theory, an AAV viral genome typically comprises two ITR sequences. These ITRs have a characteristic T-shaped hairpin structure defined by a self-complementary region (145nt in wild-type AAV) at the 5′ and 3′ ends of the ssDNA which form an energetically stable double stranded region. The double stranded hairpin structures comprise multiple functions including, but not limited to, acting as an origin for DNA replication by functioning as primers for the endogenous DNA polymerase complex of the host viral replication cell.

The wild-type AAV viral genome further comprises nucleotide sequences for two open reading frames, one for the four non-structural Rep proteins (Rep78, Rep68, Rep52, Rep40, encoded by Rep genes) and one for the three capsid, or structural, proteins (VP1, VP2, VP3, encoded by capsid genes or Cap genes). The Rep proteins are important for replication and packaging, while the capsid proteins are assembled to create the protein shell of the AAV, or AAV capsid. Alternative splicing and alternate initiation codons and promoters result in the generation of four different Rep proteins from a single open reading frame and the generation of three capsid proteins from a single open reading frame. Though it varies by AAV serotype, as a non-limiting example, for AAV9/hu.14 (SEQ ID NO: 123 of U.S. Pat. No. 7,906,111, the contents of which are herein incorporated by reference in their entirety) VP1 refers to amino acids 1-736, VP2 refers to amino acids 138-736, and VP3 refers to amino acids 203-736. In other words, VP1 is the full-length capsid sequence, while VP2 and VP3 are shorter components of the whole. As a result, changes in the sequence in the VP3 region, are also changes to VP1 and VP2, however, the percent difference as compared to the parent sequence will be greatest for VP3 since it is the shortest sequence of the three. Though described here in relation to the amino acid sequence, the nucleic acid sequence encoding these proteins can be similarly described. Together, the three capsid proteins assemble to create the AAV capsid protein. While not wishing to be bound by theory, the AAV capsid protein typically comprises a molar ratio of 1:1:10 of VP1:VP2:VP3. As used herein, an “AAV serotype” is defined primarily by the AAV capsid. In some instances, the ITRs are also specifically described by the AAV serotype (e.g., AAV2/9).

AAV vectors of the present disclosure may be produced recombinantly and may be based on adeno-associated virus (AAV) parent or reference sequences. As used herein, a “vector” is any molecule or moiety which transports, transduces, or otherwise acts as a carrier of a heterologous molecule such as the nucleic acids described herein.

In addition to single stranded AAV viral genomes (e.g., ssAAVs), the present disclosure also provides for self-complementary AAV (scAAVs) viral genomes. scAAV vector genomes contain DNA strands which anneal together to form double stranded DNA. By skipping second strand synthesis, scAAVs allow for rapid expression in the transduced cell.

In one embodiment, the AAV particle of the present disclosure is an scAAV.

In one embodiment, the AAV particle of the present disclosure is an ssAAV.

Methods for producing and/or modifying AAV particles are disclosed in the art such as pseudotyped AAV vectors (PCT Patent Publication Nos. WO200028004; WO200123001; WO2004112727; WO2005005610; and WO2005072364, the content of each of which is incorporated herein by reference in its entirety).

In one embodiment, the AAV particles of the disclosure comprising a capsid with an inserted targeting peptide and a viral genome, may have enhanced tropism for a cell-type or tissue of the human CNS.

AAV Capsids

AAV particles of the present disclosure may comprise or be derived from any natural or recombinant AAV serotype. AAV serotypes may differ in characteristics such as, but not limited to, packaging, tropism, transduction and immunogenic profiles. While not wishing to be bound by theory, the AAV capsid protein is often considered to be the driver of AAV particle tropism to a particular tissue.

In one embodiment, an AAV particle may have a capsid protein and ITR sequences derived from the same parent serotype (e.g., AAV2 capsid and AAV2 ITRs). In another embodiment, the AAV particle may be a pseudo-typed AAV particle, wherein the capsid protein and ITR sequences are derived from different parent serotypes (e.g., AAV9 capsid and AAV2 ITRs; AAV2/9).

The AAV particles of the present disclosure may comprise an AAV capsid protein with a targeting peptide inserted into the parent sequence. The parent capsid or serotype may comprise or be derived from any natural or recombinant AAV serotype. As used herein, a “parent” sequence is a nucleotide or amino acid sequence into which a targeting sequence is inserted (i.e., nucleotide insertion into nucleic acid sequence or amino acid sequence insertion into amino acid sequence).

In a preferred embodiment, the parent AAV capsid nucleotide sequence is as set forth in SEQ ID NO: 1.

In another embodiment, the parent AAV capsid nucleotide sequence is a K449R variant of SEQ ID NO: 1, wherein the codon encoding a lysine (e.g., AAA or AAG) at position 449 in the amino acid sequence (nucleotides 1345-1347) is exchanged for one encoding an arginine (CGT, CGC, CGA, CGG, AGA, AGG). The K449R variant has the same function as wild-type AAV9.

In one embodiment, the parent AAV capsid amino acid sequence is as set forth in SEQ ID NO: 2.

In another embodiment, the parent AAV capsid amino acid sequence is as set forth in SEQ ID NO: 3.

In one embodiment the parent AAV capsid sequence is any of those shown in Table 1.

TABLE 1
AAV Capsid Sequences
	SEQ
Serotype	ID NO	Reference Information
AAV9/hu.14 (nt)	1	U.S. Pat. No. 7,906,111 SEQ ID NO:
		3; WO2015038958 SEQ ID NO: 11
AAV9/hu.14 (aa)	2	U.S. Pat. No. 7,906,111 SEQ ID NO:
		123; WO2015038958 SEQ ID NO: 2
AAV9/hu.14 K449R (aa)	3	WO2017100671 SEQ ID NO: 45

Each of the patents, applications and or publications listed in Table 1 are hereby incorporated by reference in their entirety.

The parent AAV serotype and associated capsid sequence may be any of those known in the art. Non-limiting examples of such AAV serotypes include, AAV9, AAV9 K449R (or K449R AAV9), AAV1, AAVrh10, AAV-DJ, AAV-DJ8, AAV5, AAVPHP.B (PHP.B), AAVPHP.A (PHP.A), AAVG2B-26, AAVG2B-13, AAVTH1.1-32, AAVTH1.1-35, AAVPHP.B2 (PHP.B2), AAVPHP.B3 (PHP.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, AAVG2A15/G2A3 (G2A3), AAVG2B4 (G2B4), AAVG2B5 (G2B5), PHP.S, AAV2, AAV2G9, AAV3, AAV3a, AAV3b, AAV3-3, AAV4, AAV4-4, AAV6, AAV6.1, AAV6.2, AAV6.1.2, AAV7, AAV7.2, AAV8, AAV9.11, AAV9.13, AAV9.16, AAV9.24, AAV9.45, AAV9.47, AAV9.61, AAV9.68, AAV9.84, AAV9.9, AAV10, AAV11, AAV12, AAV16.3, AAV24.1, AAV27.3, AAV42.12, AAV42-1b, AAV42-2, AAV42-3a, AAV42-3b, AAV42-4, AAV42-5a, AAV42-5b, AAV42-6b, AAV42-8, AAV42-10, AAV42-11, AAV42-12, AAV42-13, AAV42-15, AAV42-aa, AAV43-1, AAV43-12, AAV43-20, AAV43-21, AAV43-23, AAV43-25, AAV43-5, AAV44.1, AAV44.2, AAV44.5, AAV223.1, AAV223.2, AAV223.4, AAV223.5, AAV223.6, AAV223.7, AAV1-7/rh.48, AAV1-8/rh.49, AAV2-15/rh.62, AAV2-3/rh.61, AAV2-4/rh.50, AAV2-5/rh.51, AAV3.1/hu.6, AAV3.1/hu.9, AAV3-9/rh.52, AAV3-11/rh.53, AAV4-8/r11.64, AAV4-9/rh.54, AAV4-19/rh.55, AAVS-3/rh.57, AAVS-22/rh.58, AAV7.3/hu.7, AAV16.8/hu.10, AAV16.12/hu.11, AAV29.3/bb .1, AAV29.5/bb .2, AAV106.1/hu.37, AAV114.3/hu.40, AAV127.2/hu.41, AAV127.5/hu.42, AAV128.3/hu.44, AAV130.4/hu.48, AAV145.1/hu.53, AAV145.5/hu.54, AAV145.6/hu.55, AAV161.10/hu.60, AAV161.6/hu.61, AAV33.12/hu.17, AAV33 .4/hu. 15, AAV33.8/hu.16, AAV52/hu.19, AAV52.1/hu.20, AAV58.2/hu.25, AAVA3.3, AAVA3.4, AAVA3.5, AAVA3.7, AAVC1, AAVC2, AAVCS, AAVF3, AAVFS, AAVH2, AAVrh.72, AAVhu.8, AAVrh.68, AAVrh.70, AAVpi.1, AAVpi.3, AAVpi.2, AAVrh.60, AAVrh.44, AAVrh.65, AAVrh.55, AAVrh.47, AAVrh.69, AAVrh.45, AAVrh.59, AAVhu.12, AAVH6, AAVH-1/hu.1, AAVH-5/hu.3, AAVLG-10/rh.40, AAVLG-4/rh.38, AAVLG-9/hu.39, AAVN721-8/rh.43, AAVCh.5, AAVCh.5R1, AAVcy.2, AAVcy.3, AAVcy.4, AAVcy.5, AAVCy.5R1, AAVCy.5R2, AAVCy.5R3, AAVCy.5R4, AAVcy.6, AAVhu.1, AAVhu.2, AAVhu.3, AAVhu.4, AAVhu.5, AAVhu.6, AAVhu.7, AAVhu.9, AAVhu.10, AAVhu.11, AAVhu.13, AAVhu.15, AAVhu.16, AAVhu.17, AAVhu.18, AAVhu.20, AAVhu.21, AAVhu.22, AAVhu.23.2, AAVhu.24, AAVhu.25, AAVhu.27, AAVhu.28, AAVhu.29, AAVhu.29R, AAVhu.31, AAVhu.32, AAVhu.34, AAVhu.35, AAVhu.37, AAVhu.39, AAVhu.40, AAVhu.41, AAVhu.42, AAVhu.43, AAVhu.44, AAVhu.44R1, AAVhu.44R2, AAVhu.44R3, AAVhu.45, AAVhu.46, AAVhu.47, AAVhu.48, AAVhu.48R1, AAVhu.48R2, AAVhu.48R3, AAVhu.49, AAVhu.51, AAVhu.52, AAVhu.54, AAVhu.55, AAVhu.56, AAVhu.57, AAVhu.58, AAVhu.60, AAVhu.61, AAVhu.63, AAVhu.64, AAVhu.66, AAVhu.67, AAVhu.14/9, AAVhu.t 19, AAVrh.2, AAVrh.2R, AAVrh.8, AAVrh.8R, AAVrh.10, AAVrh.12, AAVrh.13, AAVrh.13R, AAVrh.14, AAVrh.17, AAVrh.18, AAVrh.19, AAVrh.20, AAVrh.21, AAVrh.22, AAVrh.23, AAVrh.24, AAVrh.25, AAVrh.31, AAVrh.32, AAVrh.33, AAVrh.34, AAVrh.35, AAVrh.36, AAVrh.37, AAVrh.37R2, AAVrh.38, AAVrh.39, AAVrh.40, AAVrh.46, AAVrh.48, AAVrh.48.1, AAVrh.48.1.2, AAVrh.48.2, AAVrh.49, AAVrh.51, AAVrh.52, AAVrh.53, AAVrh.54, AAVrh.56, AAVrh.57, AAVrh.58, AAVrh.61, AAVrh.64, AAVrh.64R1, AAVrh.64R2, AAVrh.67, AAVrh.73, AAVrh.74, AAVrh8R, AAVrh8R A586R mutant, AAVrh8R R533A mutant, AAAV, BAAV, caprine AAV, bovine AAV, AAVhE1.1, AAVhEr1.5, AAVhER1.14, AAVhEr1.8, AAVhEr1.16, AAVhEr1.18, AAVhEr1.35, AAVhEr1.7, AAVhEr1.36, AAVhEr2.29, AAVhEr2.4, AAVhEr2.16, AAVhEr2.30, AAVhEr2.31, AAVhEr2.36, AAVhER1.23, AAVhEr3.1, AAV2.5T , AAV-PAEC, AAV-LK01, AAV-LK02, AAV-LK03, AAV-LK04, AAV-LK05, AAV-LK06, AAV-LK07, AAV-LK08, AAV-LK09, AAV-LK10, AAV-LK11, AAV-LK12, AAV-LK13, AAV-LK14, AAV-LK15, AAV-LK16, AAV-LK17, AAV-LK18, AAV-LK19, AAV-PAEC2, AAV-PAEC4, AAV-PAEC6, AAV-PAEC7, AAV-PAEC8, AAV-PAEC11, AAV-PAEC12, AAV-2-pre-miRNA-101 , AAV-8h, AAV-8b, AAV-h, AAV-b, AAV SM 10-2 , AAV Shuffle 100-1 , AAV Shuffle 100-3, AAV Shuffle 100-7, AAV Shuffle 10-2, AAV Shuffle 10-6, AAV Shuffle 10-8, AAV Shuffle 100-2, AAV SM 10-1, AAV SM 10-8 , AAV SM 100-3, AAV SM 100-10, BNP61 AAV, BNP62 AAV, BNP63 AAV, AAVrh.50, AAVrh.43, AAVrh.62, AAVrh.48, AAVhu.19, AAVhu.11, AAVhu.53, AAV4-8/rh.64, AAVLG-9/hu.39, AAV54.5/hu.23, AAV54.2/hu.22, AAV54.7/hu.24, AAV54.1/hu.21, AAV54.4R/hu.27, AAV46.2/hu.28, AAV46.6/hu.29, AAV128.1/hu.43, true type AAV (ttAAV), UPENN AAV 10, Japanese AAV 10 serotypes, AAV CBr-7.1, AAV CBr-7.10, AAV CBr-7.2, AAV CBr-7.3, AAV CBr-7.4, AAV CBr-7.5, AAV CBr-7.7, AAV CBr-7.8, AAV CBr-B7.3, AAV CBr-B7.4, AAV CBr-E1, AAV CBr-E2, AAV CBr-E3, AAV CBr-E4, AAV CBr-E5, AAV CBr-e5, AAV CBr-E6, AAV CBr-E7, AAV CBr-E8, AAV CHt-1, AAV CHt-2, AAV CHt-3, AAV CHt-6.1, AAV CHt-6.10, AAV CHt-6.5, AAV CHt-6.6, AAV CHt-6.7, AAV CHt-6.8, AAV CHt-P1, AAV CHt-P2, AAV CHt-P5, AAV CHt-P6, AAV CHt-P8, AAV CHt-P9, AAV CKd-1, AAV CKd-10, AAV CKd-2, AAV CKd-3, AAV CKd-4, AAV CKd-6, AAV CKd-7, AAV CKd-8, AAV CKd-B1, AAV CKd-B2, AAV CKd-B3, AAV CKd-B4, AAV CKd-B5, AAV CKd-B6, AAV CKd-B7, AAV CKd-B8, AAV CKd-H1, AAV CKd-H2, AAV CKd-H3, AAV CKd-H4, AAV CKd-H5, AAV CKd-H6, AAV CKd-N3, AAV CKd-N4, AAV CKd-N9, AAV CLg-F1, AAV CLg-F2, AAV CLg-F3, AAV CLg-F4, AAV CLg-F5, AAV CLg-F6, AAV CLg-F7, AAV CLg-F8, AAV CLv-1, AAV CLv1-1, AAV Clv1-10, AAV CLv1-2, AAV CLv-12, AAV CLv1-3, AAV CLv-13, AAV CLv1-4, AAV Clv1-7, AAV Clv1-8, AAV Clv1-9, AAV CLv-2, AAV CLv-3, AAV CLv-4, AAV CLv-6, AAV CLv-8, AAV CLv-D1, AAV CLv-D2, AAV CLv-D3, AAV CLv-D4, AAV CLv-D5, AAV CLv-D6, AAV CLv-D7, AAV CLv-D8, AAV CLv-E1, AAV CLv-K1, AAV CLv-K3, AAV CLv-K6, AAV CLv-L4, AAV CLv-L5, AAV CLv-L6, AAV CLv-M1, AAV CLv-M11, AAV CLv-M2, AAV CLv-M5, AAV CLv-M6, AAV CLv-M7, AAV CLv-M8, AAV CLv-M9, AAV CLv-R1, AAV CLv-R2, AAV CLv-R3, AAV CLv-R4, AAV CLv-R5, AAV CLv-R6, AAV CLv-R7, AAV CLv-R8, AAV CLv-R9, AAV CSp-1, AAV CSp-10, AAV CSp-11, AAV CSp-2, AAV CSp-3, AAV CSp-4, AAV CSp-6, AAV CSp-7, AAV CSp-8, AAV CSp-8.10, AAV CSp-8.2, AAV CSp-8.4, AAV CSp-8.5, AAV CSp-8.6, AAV CSp-8.7, AAV CSp-8.8, AAV CSp-8.9, AAV CSp-9, AAV.hu.48R3, AAV.VR-355, AAV3B, AAV4, AAV5, AAVF1/HSC1, AAVF11/HSC11, AAVF12/HSC12, AAVF13/HSC13, AAVF14/HSC14, AAVF15/HSC15, AAVF16/HSC16, AAVF17/HSC17, AAVF2/HSC2, AAVF3/HSC3, AAVF4/HSC4, AAVF5/HSC5, AAVF6/HSC6, AAVF7/HSC7, AAVF8/HSC8, and/or AAVF9/HSC9 and variants thereof.

In some embodiments, the serotype may be AAVDJ or a variant thereof, such as AAVDJ8 (or AAV-DJ8), as described by Grimm et al. (Journal of Virology 82(12): 5887-5911 (2008), US Publication US20140359799 and U.S. Pat. No. 7,588,772, each of which is herein incorporated by reference in its entirety). The amino acid sequence of AAVDJ8 may comprise two or more mutations in order to remove the heparin binding domain (HBD). As a non-limiting example, the AAV-DJ sequence is as described by SEQ ID NO: 1 in U.S. Pat. No. 7,588,772, the contents of which are herein incorporated by reference in their entirety, and the AAVDJ8 sequence may comprise two mutations: (1) R587Q where arginine (R; Arg) at amino acid 587 is changed to glutamine (Q; Gln) and (2) R590T where arginine (R; Arg) at amino acid 590 is changed to threonine (T; Thr). As another non-limiting example, the AAVDJ8 sequence may comprise three mutations: (1) K406R where lysine (K; Lys) at amino acid 406 is changed to arginine (R; Arg), (2) R587Q where arginine (R; Arg) at amino acid 587 is changed to glutamine (Q; Gln) and (3) R590T where arginine (R; Arg) at amino acid 590 is changed to threonine (T; Thr).

In one embodiment, the parent AAV capsid sequence comprises an AAV9 sequence.

In one embodiment, the parent AAV capsid sequence comprises an K449R AAV9 sequence.

In one embodiment, the parent AAV capsid sequence comprises an AAVDJ sequence.

In one embodiment, the parent AAV capsid sequence comprises an AAVDJ8 sequence.

In one embodiment, the parent AAV capsid sequence comprises an AAVrh10 sequence.

In one embodiment, the parent AAV capsid sequence comprises an AAV1 sequence.

In one embodiment, the parent AAV capsid sequence comprises an AAV5 sequence.

While not wishing to be bound by theory, it is understood that a parent AAV capsid sequence comprises a VP1 region. In one embodiment, a parent AAV capsid sequence comprises a VP1, VP2 and/or VP3 region, or any combination thereof. A parent VP1 sequence may be considered synonymous with a parent AAV capsid sequence.

The present disclosure refers to structural capsid proteins (including VP1, VP2 and VP3) which are encoded by capsid (Cap) genes. These capsid proteins form an outer protein structural shell (i.e. capsid) of a viral vector such as AAV. VP capsid proteins synthesized from Cap polynucleotides generally include a methionine as the first amino acid in the peptide sequence (Met1), which is associated with the start codon (AUG or ATG) in the corresponding Cap nucleotide sequence. However, it is common for a first-methionine (Met1) residue or generally any first amino acid (AA1) to be cleaved off after or during polypeptide synthesis by protein processing enzymes such as Met-aminopeptidases. This “Met/AA-clipping” process often correlates with a corresponding acetylation of the second amino acid in the polypeptide sequence (e.g., alanine, valine, serine, threonine, etc.). Met-clipping commonly occurs with VP1 and VP3 capsid proteins but can also occur with VP2 capsid proteins.

Where the Met/AA-clipping is incomplete, a mixture of one or more (one, two or three) VP capsid proteins comprising the viral capsid may be produced, some of which may include a Met1/AA1 amino acid (Met+/AA+) and some of which may lack a Met1/AA1 amino acid as a result of Met/AA-clipping (Met−/AA−). For further discussion regarding Met/AA-clipping in capsid proteins, see Jin, et al. Direct Liquid Chromatography/Mass Spectrometry Analysis for Complete Characterization of Recombinant Adeno-Associated Virus Capsid Proteins. Hum Gene Ther Methods. 2017 October 28(5):255-267; Hwang, et al. N-Terminal Acetylation of Cellular Proteins Creates Specific Degradation Signals. Science. 2010 Feb. 19. 327(5968): 973-977; the contents of which are each incorporated herein by reference in its entirety.

According to the present disclosure, references to capsid proteins is not limited to either clipped (Met−/AA−) or unclipped (Met+/AA+) and may, in context, refer to independent capsid proteins, viral capsids comprised of a mixture of capsid proteins, and/or polynucleotide sequences (or fragments thereof) which encode, describe, produce or result in capsid proteins of the present disclosure. A direct reference to a “capsid protein” or “capsid polypeptide” (such as VP1, VP2 or VP2) may also comprise VP capsid proteins which include a Met1/AA1 amino acid (Met+/AA+) as well as corresponding VP capsid proteins which lack the Met1/AA1 amino acid as a result of Met/AA-clipping (Met−/AA−).

Further according to the present disclosure, a reference to a specific SEQ ID NO: (whether a protein or nucleic acid) which comprises or encodes, respectively, one or more capsid proteins which include a Met1/AA1 amino acid (Met+/AA+) should be understood to teach the VP capsid proteins which lack the Met1/AA1 amino acid as upon review of the sequence, it is readily apparent any sequence which merely lacks the first listed amino acid (whether or not Met1/AA1).

As a non-limiting example, reference to a VP1 polypeptide sequence which is 736 amino acids in length and which includes a “Met1” amino acid (Met+) encoded by the AUG/ATG start codon may also be understood to teach a VP1 polypeptide sequence which is 735 amino acids in length and which does not include the “Met1” amino acid (Met−) of the 736 amino acid Met+ sequence. As a second non-limiting example, reference to a VP1 polypeptide sequence which is 736 amino acids in length and which includes an “AA1” amino acid (AA1+) encoded by any NNN initiator codon may also be understood to teach a VP1 polypeptide sequence which is 735 amino acids in length and which does not include the “AA1” amino acid (AA1−) of the 736 amino acid AA1+sequence.

References to viral capsids formed from VP capsid proteins (such as reference to specific AAV capsid serotypes), can incorporate VP capsid proteins which include a Met1/AA1 amino acid (Met+/AA1+), corresponding VP capsid proteins which lack the Met1/AA1 amino acid as a result of Met/AA1-clipping (Met−/AA1−), and combinations thereof (Met+/AA1+ and Met−/AA1−).

As a non-limiting example, an AAV capsid serotype can include VP1 (Met+/AA1+), VP1 (Met−/AA1−), or a combination of VP1 (Met+/AA1+) and VP1 (Met−/AA1−). An AAV capsid serotype can also include VP3 (Met+/AA1+), VP3 (Met−/AA1−), or a combination of VP3 (Met+/AA1+) and VP3 (Met−/AA1−); and can also include similar optional combinations of VP2 (Met+/AA1) and VP2 (Met−/AA1−).

In one embodiment, the parent AAV capsid sequence may comprise an amino acid sequence with 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to any of the those described above.

In one embodiment, the parent AAV capsid sequence may be encoded by a nucleotide sequence with 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to any of those described above.

In one embodiment, the parent sequence is not an AAV capsid sequence and is instead a different vector (e.g., lentivirus, plasmid, etc.). In another embodiment, the parent sequence is a delivery vehicle (e.g., a nanoparticle) and the targeting peptide is attached thereto.

Targeting Peptides

Disclosed herein are targeting peptides and associated AAV particles comprising a capsid protein with one or more targeting peptide inserts, for enhanced or improved transduction of a target tissue (e.g., cells of the CNS or PNS).

In one embodiment, the targeting peptide may direct an AAV particle to a cell or tissue of the CNS. The cell of the CNS may be, but is not limited to, neurons (e.g., excitatory, inhibitory, motor, sensory, autonomic, sympathetic, parasympathetic, Purkinje, Betz, etc.), glial cells (e.g., microglia, astrocytes, oligodendrocytes) and/or supporting cells of the brain such as immune cells (e.g., T cells). The tissue of the CNS may be, but is not limited to, the cortex (e.g., frontal, parietal, occipital, temporal), thalamus, hypothalamus, striatum, putamen, caudate nucleus, hippocampus, entorhinal cortex, basal ganglia, or deep cerebellar nuclei.

In one embodiment, the targeting peptide may direct an AAV particle to a cell or tissue of the PNS. The cell or tissue of the PNS may be, but is not limited to, a dorsal root ganglion (DRG).

The targeting peptide may direct an AAV particle to the CNS (e.g., the cortex) after intravenous administration.

The targeting peptide may direct and AAV particle to the PNS (e.g., DRG) after intravenous administration.

A targeting peptide may vary in length. In one embodiment, the targeting peptide is 3-20 amino acids in length. As non-limiting examples, the targeting peptide may be 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or 3-5, 3-8, 3-10, 3-12, 3-15, 3-18, 3-20, 5-10, 5-15, 5-20, 10-12, 10-15, 10-20, 12-20, or 15-20 amino acids in length.

Targeting peptides of the present disclosure may be identified and/or designed by any method known in the art. As a non-limiting example, the CREATE system as described in Deverman et al., (Nature Biotechnology 34(2):204-209 (2016)), Chan et al., (Nature Neuroscience 20(8):1172-1179 (2017)), and in International Patent Application Publication Nos. WO2015038958 and WO2017100671, the contents of each of which are herein incorporated by reference in their entirety, may be used as a means of identifying targeting peptides, in either mice or other research animals, such as, but not limited to, non-human primates.

Targeting peptides and associated AAV particles may be identified from libraries of AAV capsids comprised of targeting peptide variants. In one embodiment, the targeting peptides may be 7 amino acid sequences (7-mers). In another embodiment, the targeting peptides may be 9 amino acid sequences (9-mers). The targeting peptides may also differ in their method of creation or design, with non-limiting examples including, random peptide selection, site saturation mutagenesis, and/or optimization of a particular region of the peptide (e.g., flanking regions or central core).

In one embodiment, a targeting peptide library comprises targeting peptides of 7 amino acids (7-mer) in length randomly generated by PCR.

In one embodiment, a targeting peptide library comprises targeting peptides with 3 mutated amino acids. In one embodiment, these 3 mutated amino acids are consecutive amino acids. In another embodiment, these 3 mutated amino acids are not consecutive amino acids. In one embodiment, the parent targeting peptide is a 7-mer. In another embodiment, the parent peptide is a 9-mer.

In one embodiment, a targeting peptide library comprises 7-mer targeting peptides, wherein the amino acids of the targeting peptide and/or the flanking sequences are evolved through site saturation mutagenesis of 3 consecutive amino acids. In one embodiment, NNK (N=any base; K=G or T) codons are used to generate the site saturated mutation sequences.

AAV particles comprising capsid proteins with targeting peptide inserts are generated and viral genomes encoding a reporter (e.g., GFP) encapsulated within. These AAV particles (or AAV capsid library) are then administered to a transgenic mouse by intravenous delivery to the tail vein. Administration of these capsid libraries to cre-expressing mice results in expression of the reporter payload in the target tissue, due to the expression of Cre.

AAV particles and/or viral genomes may be recovered from the target tissue for identification of targeting peptides and associated AAV particles that are enriched, indicating enhanced transduction of target tissue. Standard methods in the art, such as, but not limited to next generation sequencing (NGS), viral genome quantification, biochemical assays, immunohistochemistry and/or imaging of target tissue samples may be used to determine enrichment.

A target tissue may be any cell, tissue or organ of a subject. As non-limiting examples, samples may be collected from brain, spinal cord, dorsal root ganglia and associated roots, liver, heart, gastrocnemius muscle, soleus muscle, pancreas, kidney, spleen, lung, adrenal glands, stomach, sciatic nerve, saphenous nerve, thyroid gland, eyes (with or without optic nerve), pituitary gland, skeletal muscle (rectus femoris), colon, duodenum, ileum, jejunum, skin of the leg, superior cervical ganglia, urinary bladder, ovaries, uterus, prostate gland, testes, and/or any sites identified as having a lesion, or being of interest.

Targeting Peptide Sequences

In one embodiment the targeting peptide may comprise a sequence as set forth in Table 2. In Table 2, “_1” refers to NNM codons where A or C is in the third position and “_2” refers to NNK codons where G or T is in the third position. Additionally, the NNM codons cannot cover the entire repertoire of amino acids since Met or Trp can only be encoded by codons ATG and TGG, respectively. Therefore, some “NNM” sequences also contain some codons ending in G.

TABLE 2
Peptides
Peptide	SEQ	Peptide	SEQ
Sequence_ID	ID NO:	Sequence_ID	ID NO:
AQAGAGSER_1	194	DGTGQVTGW_1	68
AQAGAGSER_2	194	DGTGQVTGW_2	68
AQDQNPGRW_1	195	DGTGRLTGW_1	159
AQDQNPGRW_2	195	DGTGRLTGW_2	159
AQELTRPFL_1	144	DGTGRTVGW_1	117
AQELTRPFL_2	144	DGTGRTVGW_2	117
AQEVPGYRW_1	196	DGTGSGMMT_1	306
AQEVPGYRW_2	196	DGTGSGMMT_2	306
AQFPTNYDS_1	66	DGTGSISGW_1	307
AQFPTNYDS_2	66	DGTGSISGW_2	307
AQFVVGQQY_1	95	DGTGSLAGW_1	308
AQFVVGQQY_2	95	DGTGSLAGW_2	308
AQGASPGRW_1	149	DGTGSLNGW_1	309
AQGASPGRW_2	149	DGTGSLNGW_2	309
AQGENPGRW_1	96	DGTGSLQGW_1	310
AQGENPGRW_2	96	DGTGSLQGW_2	310
AQGGNPGRW_1	91	DGTGSLSGW_1	311
AQGGNPGRW_2	91	DGTGSLSGW_2	311
AQGGSTGSN_1	197	DGTGSLVGW_1	312
AQGGSTGSN_2	197	DGTGSLVGW_2	312
AQGPTRPFL_1	125	DGTGSTHGW_1	119
AQGPTRPFL_2	125	DGTGSTHGW_2	119
AQGRDGWAA_1	198	DGTGSTKGW_1	313
AQGRDGWAA_2	198	DGTGSTKGW_2	313
AQGRMTDSQ_1	199	DGTGSTMGW_1	314
AQGRMTDSQ_2	199	DGTGSTMGW_2	314
AQGSDVGRW_1	128	DGTGSTQGW_1	315
AQGSDVGRW_2	128	DGTGSTQGW_2	315
AQGSNPGRW_1	103	DGTGSTSGW_1	316
AQGSNPGRW_2	103	DGTGSTSGW_2	316
AQGSNSPQV_1	200	DGTGSTTGW_1	134
AQGSNSPQV_2	200	DGTGSTTGW_2	134
AQGSWNPPA_1	80	DGTGSVMGW_1	317
AQGSWNPPA_2	80	DGTGSVMGW_2	317
AQGTWNPPA_1	82	DGTGSVTGW_1	318
AQGTWNPPA_2	82	DGTGSVTGW_2	318
AQGVFIPPK_1	201	DGTGTLAGW_1	319
AQGVFIPPK_2	201	DGTGTLAGW_2	319
AQHVNASQS_1	202	DGTGTLHGW_1	320
AQHVNASQS_2	202	DGTGTLHGW_2	320
AQIKAGWAQ_1	203	DGTGTLKGW_1	321
AQIKAGWAQ_2	203	DGTGTLKGW_2	321
AQIMSGYAQ_1	204	DGTGTLSGW_1	322
AQIMSGYAQ_2	204	DGTGTLSGW_2	322
AQKSVGSVY_1	205	DGTGTTLGW_1	323
AQKSVGSVY_2	205	DGTGTTLGW_2	323
AQLEHGFAQ_1	206	DGTGTTMGW_1	324
AQLEHGFAQ_2	206	DGTGTTMGW_2	324
AQLGGVLSA_1	207	DGTGTTTGW_1	130
AQLGGVLSA_2	207	DGTGTTTGW_2	130
AQLGLSQGR_1	208	DGTGTTVGW_1	74
AQLGLSQGR_2	208	DGTGTTVGW_2	74
AQLGYGFAQ_1	209	DGTGTTYGW_1	325
AQLGYGFAQ_2	209	DGTGTTYGW_2	325
AQLKYGLAQ_1	115	DGTGTVHGW_1	326
AQLKYGLAQ_2	115	DGTGTVHGW_2	326
AQLRIGFAQ_1	210	DGTGTVQGW_1	327
AQLRIGFAQ_2	210	DGTGTVQGW_2	327
AQLRMGYSQ_1	211	DGTGTVSGW_1	328
AQLRMGYSQ_2	211	DGTGTVSGW_2	328
AQLRQGYAQ_1	212	DGTGTVTGW_1	329
AQLRQGYAQ_2	212	DGTGTVTGW_2	329
AQLRVGFAQ_1	123	DGTHARLSS_1	330
AQLRVGFAQ_2	123	DGTHARLSS_2	330
AQLSCRSQM_1	213	DGTHAYMAS_1	153
AQLSCRSQM_2	213	DGTHAYMAS_2	153
AQLTYSQSL_1	214	DGTHFAPPR_1	112
AQLTYSQSL_2	214	DGTHFAPPR_2	112
AQLYKGYSQ_1	215	DGTHIHLSS_1	162
AQLYKGYSQ_2	215	DGTHIHLSS_2	162
AQMPQRPFL_1	216	DGTHIRALS_1	331
AQMPQRPFL_2	216	DGTHIRALS_2	331
AQNGNPGRW_1	84	DGTHIRLAS_1	332
AQNGNPGRW_2	84	DGTHIRLAS_2	332
AQPEGSARW_1	60	DGTHLQPFR_1	333
AQPEGSARW_2	60	DGTHLQPFR_2	333
AQPLAVYGA_1	217	DGTHSFYDA_1	334
AQPLAVYGA_2	217	DGTHSFYDA_2	334
AQPQSSSMS_1	218	DGTHSTTGW_1	145
AQPQSSSMS_2	218	DGTHSTTGW_2	145
AQPSVGGYW_1	219	DGTHTRTGW_1	90
AQPSVGGYW_2	219	DGTHTRTGW_2	90
AQQAVGQSW_1	220	DGTHVRALS_1	335
AQQAVGQSW_2	220	DGTHVRALS_2	335
AQQRSLASG_1	221	DGTHVYMAS_1	336
AQQRSLASG_2	221	DGTHVYMAS_2	336
AQQVMNSQG_1	222	DGTHVYMSS_1	337
AQQVMNSQG_2	222	DGTHVYMSS_2	337
AQRGVGLSQ_1	223	DGTIALPFK_1	338
AQRGVGLSQ_2	223	DGTIALPFK_2	338
AQRHDAEGS_1	224	DGTIALPFR_1	339
AQRHDAEGS_2	224	DGTIALPFR_2	339
AQRKGEPHY_1	225	DGTIATRYV_1	340
AQRKGEPHY_2	225	DGTIATRYV_2	340
AQRYTGDSS_1	138	DGTIERPFR_1	87
AQRYTGDSS_2	138	DGTIERPFR_2	87
AQSAMAAKG_1	226	DGTIGYAYV_1	341
AQSAMAAKG_2	226	DGTIGYAYV_2	341
AQSGGLTGS_1	227	DGTIQAPFK_1	342
AQSGGLTGS_2	227	DGTIQAPFK_2	342
AQSGGVGQV_1	228	DGTIRLPFK_1	343
AQSGGVGQV_2	228	DGTIRLPFK_2	343
AQSLATPFR_1	169	DGTISKEVG_1	344
AQSLATPFR_2	169	DGTISKEVG_2	344
AQSMSRPFL_1	229	DGTISQPFK_1	105
AQSMSRPFL_2	229	DGTISQPFK_2	105
AQSQLRPFL_1	230	DGTKIQLSS_1	146
AQSQLRPFL_2	230	DGTKIQLSS_2	146
AQSVAKPFL_1	231	DGTKIRLSS_1	111
AQSVAKPFL_2	231	DGTKIRLSS_2	111
AQSVSQPFR_1	232	DGTKLMLSS_1	157
AQSVSQPFR_2	232	DGTKLMLSS_2	157
AQSVVRPFL_1	233	DGTKLRLSS_1	118
AQSVVRPFL_2	233	DGTKLRLSS_2	118
AQTALSSST_1	234	DGTKMVLQL_1	142
AQTALSSST_2	234	DGTKMVLQL_2	142
AQTEMGGRC_1	235	DGTKSLVQL_1	345
AQTEMGGRC_2	235	DGTKSLVQL_2	345
AQTGFAPPR_1	161	DGTKVLVQL_1	122
AQTGFAPPR_2	161	DGTKVLVQL_2	122
AQTIRGYSS_1	236	DGTLAAPFK_1	120
AQTIRGYSS_2	236	DGTLAAPFK_2	120
AQTISNYHT_1	237	DGTLAVNFK_1	346
AQTISNYHT_2	237	DGTLAVNFK_2	346
AQTLARPFV_1	98	DGTLAVPFK_1	71
AQTLARPFV_2	98	DGTLAVPFK_2	71
AQTLAVPFK_1	168	DGTLAYPFK_1	347
AQTLAVPFK_2	168	DGTLAYPFK_2	347
AQTPDRPWL_1	238	DGTLERPFR_1	156
AQTPDRPWL_2	238	DGTLERPFR_2	156
AQTRAGYAQ_1	126	DGTLEVHFK_1	348
AQTRAGYAQ_2	126	DGTLEVHFK_2	348
AQTRAGYSQ_1	141	DGTLLRLSS_1	121
AQTRAGYSQ_2	141	DGTLLRLSS_2	121
AQTREYLLG_1	93	DGTLNNPFR_1	109
AQTREYLLG_2	93	DGTLNNPFR_2	109
AQTSAKPFL_1	163	DGTLQQPFR_1	89
AQTSAKPFL_2	163	DGTLQQPFR_2	89
AQTSARPFL_1	100	DGTLSQPFR_1	65
AQTSARPFL_2	100	DGTLSQPFR_2	65
AQTTDRPFL_1	85	DGTLSRTLW_1	349
AQTTDRPFL_2	85	DGTLSRTLW_2	349
AQTTEKPWL_1	83	DGTLSSPFR_1	350
AQTTEKPWL_2	83	DGTLSSPFR_2	350
AQTVARPFY_1	239	DGTLTVPFR_1	351
AQTVARPFY_2	239	DGTLTVPFR_2	351
AQTVATPFR_1	240	DGTLVAPFR_1	352
AQTVATPFR_2	240	DGTLVAPFR_2	352
AQTVTQLFK_1	241	DGTMDKPFR_1	70
AQTVTQLFK_2	241	DGTMDKPFR_2	70
AQVHVGSVY_1	165	DGTMDRPFK_1	102
AQVHVGSVY_2	165	DGTMDRPFK_2	102
AQVLAGYNM_1	242	DGTMLRLSS_1	148
AQVLAGYNM_2	242	DGTMLRLSS_2	148
AQVSEARVR_1	243	DGTMQLTGW_1	353
AQVSEARVR_2	243	DGTMQLTGW_2	353
AQVVVGYSQ_1	244	DGTNGLKGW_1	76
AQVVVGYSQ_2	244	DGTNGLKGW_2	76
AQWAAGYNV_1	245	DGTNSISGW_1	354
AQWAAGYNV_2	245	DGTNSISGW_2	354
AQWELSNGY_1	246	DGTNSLSGW_1	355
AQWELSNGY_2	246	DGTNSLSGW_2	355
AQWEVKGGY_1	247	DGTNSTTGW_1	143
AQWEVKGGY_2	247	DGTNSTTGW_2	143
AQWEVKRGY_1	248	DGTNSVTGW_1	356
AQWEVKRGY_2	248	DGTNSVTGW_2	356
AQWEVQSGF_1	249	DGTNTINGW_1	124
AQWEVQSGF_2	249	DGTNTINGW_2	124
AQWEVRGGY_1	250	DGTNTLGGW_1	357
AQWEVRGGY_2	250	DGTNTLGGW_2	357
AQWEVTSGW_1	251	DGTNTTHGW_1	113
AQWEVTSGW_2	251	DGTNTTHGW_2	113
AQWGAPSHG_1	252	DGTNYRLSS_1	358
AQWGAPSHG_2	252	DGTNYRLSS_2	358
AQWMELGSS_1	253	DGTQALSGW_1	359
AQWMELGSS_2	253	DGTQALSGW_2	359
AQWMFGGSG_1	254	DGTQFRLSS_1	129
AQWMFGGSG_2	254	DGTQFRLSS_2	129
AQWMLGGAQ_1	255	DGTQFSPPR_1	108
AQWMLGGAQ_2	255	DGTQFSPPR_2	108
AQWPTAYDA_1	256	DGTQGLKGW_1	158
AQWPTAYDA_2	256	DGTQGLKGW_2	158
AQWPTSYDA_1	62	DGTQTTSGW_1	360
AQWPTSYDA_2	62	DGTQTTSGW_2	360
AQWQVQTGF_1	257	DGTRALTGW_1	361
AQWQVQTGF_2	257	DGTRALTGW_2	361
AQWSTEGGY_1	258	DGTRFSLSS_1	362
AQWSTEGGY_2	258	DGTRFSLSS_2	362
AQWTAAGGY_1	259	DGTRGLSGW_1	363
AQWTAAGGY_2	259	DGTRGLSGW_2	363
AQWTTESGY_1	260	DGTRIGLSS_1	364
AQWTTESGY_2	260	DGTRIGLSS_2	364
AQWVYGSSH_1	261	DGTRLHLAS_1	365
AQWVYGSSH_2	261	DGTRLHLAS_2	365
AQYLAGYTV_1	262	DGTRLHLSS_1	366
AQYLAGYTV_2	262	DGTRLHLSS_2	366
AQYLKGYSV_1	152	DGTRLLLSS_1	367
AQYLKGYSV_2	152	DGTRLLLSS_2	367
AQYLSGYNT_1	263	DGTRLMLSS_1	368
AQYLSGYNT_2	263	DGTRLMLSS_2	368
DGAAATTGW_1	264	DGTRLNLSS_1	369
DGAAATTGW_2	264	DGTRLNLSS_2	369
DGAGGTSGW_1	151	DGTRMVVQL_1	370
DGAGGTSGW_2	151	DGTRMVVQL_2	370
DGAGTTSGW_1	265	DGTRNMYEG_1	135
DGAGTTSGW_2	265	DGTRNMYEG_2	135
DGAHGLSGW_1	266	DGTRSITGW_1	371
DGAHGLSGW_2	266	DGTRSITGW_2	371
DGAHVGLSS_1	267	DGTRSLHGW_1	372
DGAHVGLSS_2	267	DGTRSLHGW_2	372
DGARTVLQL_1	268	DGTRSTTGW_1	373
DGARTVLQL_2	268	DGTRSTTGW_2	373
DGEYQKPFR_1	269	DGTRTTTGW_1	106
DGEYQKPFR_2	269	DGTRTTTGW_2	106
DGGGTTTGW_1	270	DGTRTVTGW_1	374
DGGGTTTGW_2	270	DGTRTVTGW_2	374
DGHATSMGW_1	271	DGTRTVVQL_1	375
DGHATSMGW_2	271	DGTRTVVQL_2	375
DGKGSTQGW_1	272	DGTRVHLSS_1	376
DGKGSTQGW_2	272	DGTRVHLSS_2	376
DGKQYQLSS_1	92	DGTSFPYAR_1	86
DGKQYQLSS_2	92	DGTSFPYAR_2	86
DGNGGLKGW_1	167	DGTSFTPPK_1	81
DGNGGLKGW_2	167	DGTSFTPPK_2	81
DGQGGLSGW_1	273	DGTSFTPPR_1	88
DGQGGLSGW_2	273	DGTSFTPPR_2	88
DGQHFAPPR_1	110	DGTSGLHGW_1	377
DGQHFAPPR_2	110	DGTSGLHGW_2	377
DGRATKTLY_1	274	DGTSGLKGW_1	101
DGRATKTLY_2	274	DGTSGLKGW_2	101
DGRNALTGW_1	275	DGTSIHLSS_1	378
DGRNALTGW_2	275	DGTSIHLSS_2	378
DGRRQVIQL_1	276	DGTSIMLSS_1	379
DGRRQVIQL_2	276	DGTSIMLSS_2	379
DGRVYGLSS_1	277	DGTSLRLSS_1	166
DGRVYGLSS_2	277	DGTSLRLSS_2	166
DGSGRTTGW_1	147	DGTSNYGAR_1	380
DGSGRTTGW_2	147	DGTSNYGAR_2	380
DGSGTTRGW_1	114	DGTSSYYDA_1	381
DGSGTTRGW_2	114	DGTSSYYDA_2	381
DGSGTVSGW_1	278	DGTSSYYDS_1	59
DGSGTVSGW_2	278	DGTSSYYDS_2	59
DGSPEKPFR_1	160	DGTSTISGW_1	382
DGSPEKPFR_2	160	DGTSTISGW_2	382
DGSQSTTGW_1	136	DGTSTITGW_1	383
DGSQSTTGW_2	136	DGTSTITGW_2	383
DGSSFYPPK_1	127	DGTSTLHGW_1	384
DGSSFYPPK_2	127	DGTSTLHGW_2	384
DGSSSYYDA_1	64	DGTSTLRGW_1	385
DGSSSYYDA_2	64	DGTSTLRGW_2	385
DGSIERPFR_1	99	DGTSTLSGW_1	386
DGSIERPFR_2	99	DGTSTLSGW_2	386
DGTAARLSS_1	132	DGTSYVPPK_1	97
DGTAARLSS_2	132	DGTSYVPPK_2	97
DGTADKPFR_1	63	DGTSYVPPR_1	78
DGTADKPFR_2	63	DGTSYVPPR_2	78
DGTADRPFR_1	155	DGTTATYYK_1	387
DGTADRPFR_2	155	DGTTATYYK_2	387
DGTAERPFR_1	140	DGTTFTPPR_1	79
DGTAERPFR_2	140	DGTTFTPPR_2	79
DGTAIHLSS_1	67	DGTTLAPFR_1	388
DGTAIHLSS_2	67	DGTTLAPFR_2	388
DGTAIYLSS_1	279	DGTTLVPPR_1	116
DGTAIYLSS_2	279	DGTTLVPPR_2	116
DGTALMLSS_1	280	DGTTSKTLW_1	389
DGTALMLSS_2	280	DGTTSKTLW_2	389
DGTASISGW_1	281	DGTTSRTLW_1	390
DGTASISGW_2	281	DGTTSRTLW_2	390
DGTASTSGW_1	282	DGTTTRSLY_1	391
DGTASTSGW_2	282	DGTTTRSLY_2	391
DGTASVTGW_1	283	DGTTTTTGW_1	392
DGTASVTGW_2	283	DGTTTTTGW_2	392
DGTASYYDS_1	61	DGTTTYGAR_1	77
DGTASYYDS_2	61	DGTTTYGAR_2	77
DGTATTMGW_1	284	DGTTWTPPR_1	139
DGTATTMGW_2	284	DGTTWTPPR_2	139
DGTATTTGW_1	285	DGTTYMLSS_1	393
DGTATTTGW_2	285	DGTTYMLSS_2	393
DGTAYRLSS_1	286	DGTTYVPPR_1	75
DGTAYRLSS_2	286	DGTTYVPPR_2	75
DGTDKMWSL_1	287	DGTVANPFR_1	394
DGTDKMWSL_2	287	DGTVANPFR_2	394
DGTGGIKGW_1	131	DGTVDRPFK_1	395
DGTGGIKGW_2	131	DGTVDRPFK_2	395
DGTGGIMGW_1	288	DGTVIHLSS_1	73
DGTGGIMGW_2	288	DGTVIHLSS_2	73
DGTGGISGW_1	289	DGTVILLSS_1	396
DGTGGISGW_2	289	DGTVILLSS_2	396
DGTGGLAGW_1	290	DGTVIMLSS_1	397
DGTGGLAGW_2	290	DGTVIMLSS_2	397
DGTGGLHGW_1	291	DGTVLHLSS_1	398
DGTGGLHGW_2	291	DGTVLHLSS_2	398
DGTGGLQGW_1	292	DGTVLMLSS_1	399
DGTGGLQGW_2	292	DGTVLMLSS_2	399
DGTGGLRGW_1	154	DGTVLVPFR_1	150
DGTGGLRGW_2	154	DGTVLVPFR_2	150
DGTGGLSGW_1	293	DGTVPYLAS_1	400
DGTGGLSGW_2	293	DGTVPYLAS_2	400
DGTGGLTGW_1	294	DGTVPYLSS_1	401
DGTGGLTGW_2	294	DGTVPYLSS_2	401
DGTGGTKGW_1	107	DGTVRVPFR_1	164
DGTGGTKGW_2	107	DGTVRVPFR_2	164
DGTGGTSGW_1	295	DGTVSMPFK_1	402
DGTGGTSGW_2	295	DGTVSMPFK_2	402
DGTGGVHGW_1	296	DGTVSNPFR_1	403
DGTGGVHGW_2	296	DGTVSNPFR_2	403
DGTGGVMGW_1	297	DGTVSTRWV_1	404
DGTGGVMGW_2	297	DGTVSTRWV_2	404
DGTGGVSGW_1	298	DGTVTTTGW_1	405
DGTGGVSGW_2	298	DGTVTTTGW_2	405
DGTGGVTGW_1	299	DGTVTVTGW_1	406
DGTGGVTGW_2	299	DGTVTVTGW_2	406
DGTGGVYGW_1	300	DGTVWVPPR_1	407
DGTGGVYGW_2	300	DGTVWVPPR_2	407
DGTGNLQGW_1	301	DGTVYRLSS_1	408
DGTGNLQGW_2	301	DGTVYRLSS_2	408
DGTGNLRGW_1	133	DGTYARLSS_1	409
DGTGNLRGW_2	133	DGTYARLSS_2	409
DGTGNLSGW_1	302	DGTYGNKLW_1	410
DGTGNLSGW_2	302	DGTYGNKLW_2	410
DGTGNTHGW_1	72	DGTYIHLSS_1	411
DGTGNTHGW_2	72	DGTYIHLSS_2	411
DGTGNTRGW_1	94	DGTYSTSGW_1	412
DGTGNTRGW_2	94	DGTYSTSGW_2	412
DGTGNTSGW_1	137	DGVHPGLSS_1	104
DGTGNTSGW_2	137	DGVHPGLSS_2	104
DGTGNVSGW_1	303	DGVVALLAS_1	413
DGTGNVSGW_2	303	DGVVALLAS_2	413
DGTGNVTGW_1	69	DGYVGVGSL_1	414
DGTGNVTGW_2	69	DGYVGVGSL_2	414
DGTGQLVGW_1	304	control
		(wtAAV9-
		NNM)
DGTGQLVGW_2	304	control
		(wtAAV9-
		NNK)
DGTGQTIGW_1	305
DGTGQTIGW_2	305

In one embodiment, the targeting peptide may comprise an amino acid sequence with 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to any of the sequences shown in Table 2.

In one embodiment, a targeting peptide may comprise 4 or more contiguous amino acids of any of the targeting peptides disclosed herein. In one embodiment the targeting peptide may comprise 4 contiguous amino acids of any of the sequences as set forth in Table 2. In one embodiment the targeting peptide may comprise 5 contiguous amino acids of any of the sequences as set forth in Table 2. In one embodiment the targeting peptide may comprise 6 contiguous amino acids of any of the sequences as set forth in Table 2.

In one embodiment, the AAV particle of the disclosure comprises an AAV capsid with a targeting peptide insert, wherein the targeting peptide has an amino acid sequence as set forth in any of Table 2.

In one embodiment, the AAV particle of the disclosure comprises an AAV capsid with a targeting peptide insert, wherein the targeting peptide has an amino acid sequence comprising at least 4 contiguous amino acids of any of the sequences as set forth in any of Table 2.

In one embodiment, the AAV particle of the disclosure comprises an AAV capsid with a targeting peptide insert, wherein the targeting peptide has an amino acid sequence substantially comprising any of the sequences as set forth in any of Table 2.

In one embodiment, the AAV particle of the disclosure comprises an AAV capsid polynucleotide with a targeting nucleic acid insert, wherein the targeting nucleic acid insert has a nucleotide sequence substantially comprising any of those set forth as Table 2.

The AAV particle of the disclosure comprising a targeting nucleic acid insert, may have a polynucleotide sequence with 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more, identity to the parent capsid sequence.

The AAV particle of the disclosure comprising a targeting peptide insert, may have an amino acid sequence with 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more, identity to the parent capsid sequence.

In any of the DNA and RNA sequences referenced and/or described herein, the single letter symbol has the following description: A for adenine; C for cytosine; G for guanine; T for thymine; U for Uracil; W for weak bases such as adenine or thymine; S for strong nucleotides such as cytosine and guanine; M for amino nucleotides such as adenine and cytosine; K for keto nucleotides such as guanine and thymine; R for purines adenine and guanine; Y for pyrimidine cytosine and thymine; B for any base that is not A (e.g., cytosine, guanine, and thymine); D for any base that is not C (e.g., adenine, guanine, and thymine); H for any base that is not G (e.g., adenine, cytosine, and thymine); V for any base that is not T (e.g., adenine, cytosine, and guanine); N for any nucleotide (which is not a gap); and Z is for zero.

In any of the amino acid sequences referenced and/or described herein, the single letter symbol has the following description: G (Gly) for Glycine; A (Ala) for Alanine; L (Leu) for Leucine; M (Met) for Methionine; F (Phe) for Phenylalanine; W (Trp) for Tryptophan; K (Lys) for Lysine; Q (Gln) for Glutamine; E (Glu) for Glutamic Acid; S (Ser) for Serine; P (Pro) for Proline; V (Val) for Valine; I (Ile) for Isoleucine; C (Cys) for Cysteine; Y (Tyr) for Tyrosine; H (His) for Histidine; R (Arg) for Arginine; N (Asn) for Asparagine; D (Asp) for Aspartic Acid; T (Thr) for Threonine; B (Asx) for Aspartic acid or Asparagine; J (Xle) for Leucine or Isoleucine; O (Pyl) for Pyrrolysine; U (Sec) for Selenocysteine; X (Xaa) for any amino acid; and Z (Glx) for Glutamine or Glutamic acid.

Use of Targeting Peptides in AAV Particles

Targeting peptides may be stand-alone peptides or may be inserted into or conjugated to a parent sequence. In one embodiment, the targeting peptides are inserted into the capsid protein of an AAV particle.

One or more targeting peptides may be inserted into a parent AAV capsid sequence to generate the AAV particles of the disclosure.

Targeting peptides may be inserted into a parent AAV capsid sequence in any location that results in fully functional AAV particles. The targeting peptide may be inserted in VP1, VP2 and/or VP3. Numbering of the amino acid residues differs across AAV serotypes, and so the exact amino acid position of the targeting peptide insertion may not be critical. As used herein, amino acid positions of the parent AAV capsid sequence are described using AAV9 (SEQ ID NO: 2) as reference.

In one embodiment, the targeting peptides are inserted in a hypervariable region of the AAV capsid sequence. Non-limiting examples of such hypervariable regions include Loop IV and Loop VIII of the parent AAV capsid. While not wishing to be bound by theory, these surface exposed loops are unstructured and poorly conserved, making them ideal regions for insertion of targeting peptides.

In one embodiment, the targeting peptide is inserted into Loop IV. In another embodiment, the targeting peptide is used to replace a portion, or all of Loop IV. As a non-limiting example, addition of the targeting peptide to the parent AAV capsid sequence may result in the replacement or mutation of at least one amino acid of the parent AAV capsid.

In one embodiment, the targeting peptide is inserted into Loop VIII. In another embodiment, the targeting peptide is used to replace a portion, or all of Loop VIII. As a non-limiting example, addition of the targeting peptide to the parent AAV capsid sequence may result in the replacement or mutation of at least one amino acid of the parent AAV capsid.

In one embodiment, more than one targeting peptide is inserted into a parent AAV capsid sequence. As a non-limiting example, targeting peptides may be inserted at both Loop IV and Loop VIII in the same parent AAV capsid sequence.

Targeting peptides may be inserted at any amino acid position of the parent AAV capsid sequence, such as, but not limited to, between amino acids at positions 586-592, 588-589, 586-589, 452-458, 262-269, 464-473, 491-495, 546-557 and/or 659-668.

In a preferred embodiment, the targeting peptides are inserted into a parent AAV capsid sequence between amino acids at positions 588 and 589 (Loop VIII). In one embodiment, the parent AAV capsid is AAV9 (SEQ ID NO: 2). In a second embodiment, the parent AAV capsid is K449R AAV9 (SEQ ID NO: 3).

The targeting peptides described herein may increase the transduction of the AAV particles of the disclosure to a target tissue as compared to the parent AAV particle lacking a targeting peptide insert. In one embodiment, the targeting peptide increases the transduction of an AAV particle to a target tissue by at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 125%, 150%, 200%, 300%, 400%, 500%, or more as compared to a parent AAV particle lacking a targeting peptide insert.

In one embodiment, the targeting peptide increases the transduction of an AAV particle to a cell or tissue of the CNS by at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 125%, 150%, 200%, 300%, 400%, 500%, or more as compared to a parent AAV particle lacking a targeting peptide insert.

In one embodiment, the targeting peptide increases the transduction of an AAV particle to a cell or tissue of the PNS by at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 125%, 150%, 200%, 300%, 400%, 500%, or more as compared to a parent AAV particle lacking a targeting peptide insert.

In one embodiment, the targeting peptide increases the transduction of an AAV particle to a cell or tissue of the DRG by at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 125%, 150%, 200%, 300%, 400%, 500%, or more as compared to a parent AAV particle lacking a targeting peptide insert.

AAV Production

Viral production disclosed herein describes processes and methods for producing AAV particles (with enhanced, improved and/or increased tropism for a target tissue) that may be used to contact a target cell to deliver a payload.

The present disclosure provides methods for the generation of AAV particles comprising targeting peptides. In one embodiment, the AAV particles are prepared by viral genome replication in a viral replication cell. Any method known in the art may be used for the preparation of AAV particles. In one embodiment, AAV particles are produced in mammalian cells (e.g., HEK293). In another embodiment, AAV particles are produced in insect cells (e.g., Sf9)

Methods of making AAV particles are well known in the art and are described in e.g., U.S. Pat. Nos. 6,204,059, 5,756,283, 6,258,595, 6,261,551, 6,270,996, 6,281,010, 6,365,394, 6,475,769, 6,482,634, 6,485,966, 6,943,019, 6,953,690, 7,022,519, 7,238,526, 7,291,498 and 7,491,508, 5,064,764, 6,194,191, 6,566,118, 8,137,948; or International Publication Nos. WO1996039530, WO1998010088, WO1999014354, WO1999015685, WO1999047691, WO2000055342, WO2000075353 and WO2001023597; Methods In Molecular Biology, ed. Richard, Humana Press, NJ (1995); O'Reilly et al., Baculovirus Expression Vectors, A Laboratory Manual, Oxford Univ. Press (1994); Samulski et al., J. Vir. 63:3822-8 (1989); Kajigaya et al., Proc. Nat'l. Acad. Sci. USA 88: 4646-50 (1991); Ruffing et al., J. Vir. 66:6922-30 (1992); Kimbauer et al., Vir., 219:37-44 (1996); Zhao et al., Vir. 272:382-93 (2000); the contents of each of which are herein incorporated by reference in their entirety. In one embodiment, the AAV particles are made using the methods described in International Patent Publication WO2015191508, the contents of which are herein incorporated by reference in their entirety.

Therapeutic Applications

The present disclosure provides a method for treating a disease, disorder and/or condition in a mammalian subject, including a human subject, comprising administering to the subject an AAV particle described herein where the AAV particle comprises the novel capsids (“TRACER AAV particles”) defined by the present disclosure or administering to the subject any of the described compositions, including pharmaceutical compositions, described herein.

In one embodiment, the TRACER AAV particles of the present disclosure are administered to a subject prophylactically, to prevent on-set of disease. In another embodiment, the TRACER AAV particles of the present disclosure are administered to treat (lessen the effects of) a disease or symptoms thereof. In yet another embodiment, the TRACER AAV particles of the present disclosure are administered to cure (eliminate) a disease. In another embodiment, the TRACER AAV particles of the present disclosure are administered to prevent or slow progression of disease. In yet another embodiment, the TRACER AAV particles of the present disclosure are used to reverse the deleterious effects of a disease. Disease status and/or progression may be determined or monitored by standard methods known in the art.

In some embodiments, the TRACER AAV particles of the disclosure are useful in the field of medicine for the treatment, prophylaxis, palliation or amelioration of neurological diseases and/or disorders.

In some embodiments, the TRACER AAV particles of the disclosure are useful in the field of medicine for the treatment, prophylaxis, palliation or amelioration of tauopathy.

In some embodiments, the TRACER AAV particles of the disclosure are useful in the field of medicine for the treatment, prophylaxis, palliation or amelioration of Alzheimer's Disease.

In some embodiments, the TRACER AAV particles of the disclosure are useful in the field of medicine for the treatment, prophylaxis, palliation or amelioration of Friedreich's ataxia, or any disease stemming from a loss or partial loss of frataxin protein.

In some embodiments, the TRACER AAV particles of the disclosure are useful in the field of medicine for the treatment, prophylaxis, palliation or amelioration of Parkinson's Disease.

In some embodiments, the TRACER AAV particles of the disclosure are useful in the field of medicine for the treatment, prophylaxis, palliation or amelioration of Amyotrophic lateral sclerosis.

In some embodiments, the TRACER AAV particles of the disclosure are useful in the field of medicine for the treatment, prophylaxis, palliation or amelioration of Huntington's Disease.

In some embodiments, the TRACER AAV particles of the disclosure are useful in the field of medicine for the treatment, prophylaxis, palliation or amelioration of chronic or neuropathic pain.

In some embodiments, the TRACER AAV particles of the disclosure are useful in the field of medicine for treatment, prophylaxis, palliation or amelioration of a disease associated with the central nervous system.

In some embodiments, the TRACER AAV particles of the disclosure are useful in the field of medicine for treatment, prophylaxis, palliation or amelioration of a disease associated with the peripheral nervous system.

In one embodiment, the TRACER AAV particles of the present disclosure are administered to a subject having at least one of the diseases or symptoms described herein.

As used herein, any disease associated with the central or peripheral nervous system and components thereof (e.g., neurons) may be considered a “neurological disease”.

Any neurological disease may be treated with the TRACER AAV particles of the disclosure, or pharmaceutical compositions thereof, including but not limited to, Absence of the Septum Pellucidum, Acid Lipase Disease, Acid Maltase Deficiency, Acquired Epileptiform Aphasia, Acute Disseminated Encephalomyelitis, Attention Deficit-Hyperactivity Disorder (ADHD), Adie's Pupil, Adie's Syndrome, Adrenoleukodystrophy, Agenesis of the Corpus Callosum, Agnosia, Aicardi Syndrome, Aicardi-Goutieres Syndrome Disorder, AIDS—Neurological Complications, Alexander Disease, Alpers' Disease, Alternating Hemiplegia, Alzheimer's Disease, Amyotrophic Lateral Sclerosis (ALS), Anencephaly, Aneurysm, Angelman Syndrome, Angiomatosis, Anoxia, Antiphospholipid Syndrome, Aphasia, Apraxia, Arachnoid Cysts, Arachnoiditis, Arnold-Chiari Malformation, Arteriovenous Malformation, Asperger Syndrome, Ataxia, Ataxia Telangiectasia, Ataxias and Cerebellar or Spinocerebellar Degeneration, Atrial Fibrillation and Stroke, Attention Deficit-Hyperactivity Disorder, Autism Spectrum Disorder, Autonomic Dysfunction, Back Pain, Barth Syndrome, Batten Disease, Becker's Myotonia, Bechet's Disease, Bell's Palsy, Benign Essential Blepharospasm, Benign Focal Amyotrophy, Benign Intracranial Hypertension, Bernhardt-Roth Syndrome, Binswanger's Disease, Blepharospasm, Bloch-Sulzberger Syndrome, Brachial Plexus Birth Injuries, Brachial Plexus Injuries, Bradbury-Eggleston Syndrome, Brain and Spinal Tumors, Brain Aneurysm, Brain Injury, Brown-Sequard Syndrome, Bulbar palsy, Bulbospinal Muscular Atrophy, Cerebral Autosomal Dominant Arteriopathy with Sub-cortical Infarcts and Leukoencephalopathy (CADASIL), Canavan Disease, Carpal Tunnel Syndrome, Causalgia, Cavernomas, Cavernous Angioma, Cavernous Malformation, Central Cervical Cord Syndrome, Central Cord Syndrome, Central Pain Syndrome, Central Pontine Myelinolysis, Cephalic Disorders, Ceramidase Deficiency, Cerebellar Degeneration, Cerebellar Hypoplasia, Cerebral Aneurysms, Cerebral Arteriosclerosis, Cerebral Atrophy, Cerebral Beriberi, Cerebral Cavernous Malformation, Cerebral Gigantism, Cerebral Hypoxia, Cerebral Palsy, Cerebro-Oculo-Facio-Skeletal Syndrome (COFS), Charcot-Marie-Tooth Disease, Chiari Malformation, Cholesterol Ester Storage Disease, Chorea, Choreoacanthocytosis, Chronic Inflammatory Demyelinating Polyneuropathy (CIDP), Chronic Orthostatic Intolerance, Chronic Pain, Cockayne Syndrome Type II, Coffin Lowry Syndrome, Colpocephaly, Coma, Complex Regional Pain Syndrome, Concentric sclerosis (Balo's sclerosis), Congenital Facial Diplegia, Congenital Myasthenia, Congenital Myopathy, Congenital Vascular Cavernous Malformations, Corticobasal Degeneration, Cranial Arteritis, Craniosynostosis, Cree encephalitis, Creutzfeldt-Jakob Disease, Chronic progressive external ophtalmoplegia, Cumulative Trauma Disorders, Cushing's Syndrome, Cytomegalic Inclusion Body Disease, Cytomegalovirus Infection, Dancing Eyes-Dancing Feet Syndrome, Dandy-Walker Syndrome, Dawson Disease, De Morsier's Syndrome, Dejerine-Klumpke Palsy, Dementia, Dementia—Multi-Infarct, Dementia—Semantic, Dementia—Subcortical, Dementia With Lewy Bodies, Demyelination diseases, Dentate Cerebellar Ataxia, Dentatorubral Atrophy, Dermatomyositis, Developmental Dyspraxia, Devic's Syndrome, Diabetic Neuropathy, Diffuse Sclerosis, Distal hereditary motor neuronopathies, Dravet Syndrome, Dysautonomia, Dysgraphia, Dyslexia, Dysphagia, Dyspraxia, Dyssynergia Cerebellaris Myoclonica, Dyssynergia Cerebellaris Progressiva, Dystonias, Early Infantile Epileptic Encephalopathy, Empty Sella Syndrome, Encephalitis, Encephalitis Lethargica, Encephaloceles, Encephalomyelitis, Encephalopathy, Encephalopathy (familial infantile), Encephalotrigeminal Angiomatosis, Epilepsy, Epileptic Hemiplegia, Episodic ataxia, Erb's Palsy, Erb-Duchenne and Dejerine-Klumpke Palsies, Essential Tremor, Extrapontine Myelinolysis, Faber's disease, Fabry Disease, Fahr's Syndrome, Fainting, Familial Dysautonomia, Familial Hemangioma, Familial Idiopathic Basal Ganglia Calcification, Familial Periodic Paralyses, Familial Spastic Paralysis, Farber's Disease, Febrile Seizures, Fibromuscular Dysplasia, Fisher Syndrome, Floppy Infant Syndrome, Foot Drop, Friedreich's Ataxia, Frontotemporal Dementia, Gaucher Disease, Generalized Gangliosidoses (GM1, GM2), Gerstmann's Syndrome, Gerstmann-Straussler-Scheinker Disease, Giant Axonal Neuropathy, Giant Cell Arteritis, Giant Cell Inclusion Disease, Globoid Cell Leukodystrophy, Glossopharyngeal Neuralgia, Glycogen Storage Disease, Guillain-Barré Syndrome, Hallervorden-Spatz Disease, Head Injury, Headache, Hemicrania Continua, Hemifacial Spasm, Hemiplegia Alterans, Hereditary Neuropathies, Hereditary Spastic Paraplegia, Heredopathia Atactica Polyneuritiformis, Herpes Zoster, Herpes Zoster Oticus, Hirayama Syndrome, Holmes-Adie syndrome, Holoprosencephaly, HTLV-1 Associated Myelopathy, Hughes Syndrome, Huntington's Disease, Hurler syndrome, Hydranencephaly, Hydrocephalus, Hydrocephalus—Normal Pressure, Hydromyelia, Hypercortisolism, Hypersomnia, Hypertonia, Hypotonia, Hypoxia, Immune-Mediated Encephalomyelitis, Inclusion Body Myositis, Incontinentia Pigmenti, Infantile Hypotonia, Infantile Neuroaxonal Dystrophy, Infantile Phytanic Acid Storage Disease, Infantile Refsum Disease, Infantile Spasms, Inflammatory Myopathies, Iniencephaly, Intestinal Lipodystrophy, Intracranial Cysts, Intracranial Hypertension, Isaacs' Syndrome, Joubert Syndrome, Kearns-Sayre Syndrome, Kennedy's Disease, Kinsbourne syndrome, Kleine-Levin Syndrome, Klippel-Feil Syndrome, Klippel-Trenaunay Syndrome (KTS), Kluver-Bucy Syndrome, Korsakoff s Amnesic Syndrome, Krabbe Disease, Kugelberg-Welander Disease, Kuru, Lambert-Eaton Myasthenic Syndrome, Landau-Kleffner Syndrome, Lateral Femoral Cutaneous Nerve Entrapment, Lateral Medullary Syndrome, Learning Disabilities, Leigh's Disease, Lennox-Gastaut Syndrome, Lesch-Nyhan Syndrome, Leukodystrophy, Levine-Critchley Syndrome, Lewy Body Dementia, Lichtheim's disease, Lipid Storage Diseases, Lipoid Proteinosis, Lissencephaly, Locked-In Syndrome, Lou Gehrig's Disease, Lupus—Neurological Sequelae, Lyme Disease—Neurological Complications, Lysosomal storage disorders, Machado-Joseph Disease, Macrencephaly, Megalencephaly, Melkersson-Rosenthal Syndrome, Meningitis, Meningitis and Encephalitis, Menkes Disease, Meralgia Paresthetica, Metachromatic Leukodystrophy, Microcephaly, Migraine, Miller Fisher Syndrome, Mini Stroke, Mitochondrial Myopathy, Mitochondrial DNA depletion syndromes, Moebius Syndrome, Monomelic Amyotrophy, Morvan Syndrome, Motor Neuron Diseases, Moyamoya Disease, Mucolipidoses, Mucopolysaccharidoses, Multi-Infarct Dementia, Multifocal Motor Neuropathy, Multiple Sclerosis, Multiple System Atrophy, Multiple System Atrophy with Orthostatic Hypotension, Muscular Dystrophy, Myasthenia—Congenital, Myasthenia Gravis, Myelinoclastic Diffuse Sclerosis, Myelitis, Myoclonic Encephalopathy of Infants, Myoclonus, Myoclonus epilepsy, Myopathy, Myopathy—Congenital, Myopathy—Thyrotoxic, Myotonia, Myotonia Congenita, Narcolepsy, NARP (neuropathy, ataxia and retinitis pigmentosa), Neuroacanthocytosis, Neurodegeneration with Brain Iron Accumulation, Neurodegenerative disease, Neurofibromatosis, Neuroleptic Malignant Syndrome, Neurological Complications of AIDS, Neurological Complications of Lyme Disease, Neurological Consequences of Cytomegalovirus Infection, Neurological Manifestations of Pompe Disease, Neurological Sequelae Of Lupus, Neuromyelitis Optica, Neuromyotonia, Neuronal Ceroid Lipofuscinosis, Neuronal Migration Disorders, Neuropathic pain, Neuropathy—Hereditary, Neuropathy, Neurosarcoidosis, Neurosyphilis, Neurotoxicity, Nevus Cavernosus, Niemann-Pick Disease, O'Sullivan-McLeod Syndrome, Occipital Neuralgia, Ohtahara Syndrome, Olivopontocerebellar Atrophy, Opsoclonus Myoclonus, Orthostatic Hypotension, Overuse Syndrome, Pain—Chronic, Pantothenate Kinase-Associated Neurodegeneration, Paraneoplastic Syndromes, Paresthesia, Parkinson's Disease, Paroxysmal Choreoathetosis, Paroxysmal Hemicrania, Parry-Romberg, Pelizaeus-Merzbacher Disease, Pena Shokeir II Syndrome, Perineural Cysts, Peroneal muscular atrophy, Periodic Paralyses, Peripheral Neuropathy, Periventricular Leukomalacia, Persistent Vegetative State, Pervasive Developmental Disorders, Phytanic Acid Storage Disease, Pick's Disease, Pinched Nerve, Piriformis Syndrome, Pituitary Tumors, Polymyositis, Pompe Disease, Porencephaly, Post-Polio Syndrome, Postherpetic Neuralgia, Postinfectious Encephalomyelitis, Postural Hypotension, Postural Orthostatic Tachycardia Syndrome, Postural Tachycardia Syndrome, Primary Dentatum Atrophy, Primary Lateral Sclerosis, Primary Progressive Aphasia, Prion Diseases, Progressive bulbar palsy, Progressive Hemifacial Atrophy, Progressive Locomotor Ataxia, Progressive Multifocal Leukoencephalopathy, Progressive Muscular Atrophy, Progressive Sclerosing Poliodystrophy, Progressive Supranuclear Palsy, Prosopagnosia, Pseudobulbar palsy, Pseudo-Torch syndrome, Pseudotoxoplasmosis syndrome, Pseudotumor Cerebri, Psychogenic Movement, Ramsay Hunt Syndrome I, Ramsay Hunt Syndrome II, Rasmussen's Encephalitis, Reflex Sympathetic Dystrophy Syndrome, Refsum Disease, Refsum Disease—Infantile, Repetitive Motion Disorders, Repetitive Stress Injuries, Restless Legs Syndrome, Retrovirus-Associated Myelopathy, Rett Syndrome, Reye's Syndrome, Rheumatic Encephalitis, Riley-Day Syndrome, Sacral Nerve Root Cysts, Saint Vitus Dance, Salivary Gland Disease, Sandhoff Disease, Schilder's Disease, Schizencephaly, Seitelberger Disease, Seizure Disorder, Semantic Dementia, Septo-Optic Dysplasia, Severe Myoclonic Epilepsy of Infancy (SMEI), Shaken Baby Syndrome, Shingles, Shy-Drager Syndrome, Sjögren's Syndrome, Sleep Apnea, Sleeping Sickness, Sotos Syndrome, Spasticity, Spina Bifida, Spinal Cord Infarction, Spinal Cord Injury, Spinal Cord Tumors, Spinal Muscular Atrophy, Spinocerebellar Ataxia, Spinocerebellar Atrophy, Spinocerebellar Degeneration, Sporadic ataxia, Steele-Richardson-Olszewski Syndrome, Stiff-Person Syndrome, Striatonigral Degeneration, Stroke, Sturge-Weber Syndrome, Subacute Sclerosing Panencephalitis, Subcortical Arteriosclerotic Encephalopathy, Short-lasting, Unilateral, Neuralgiform (SUNCT) Headache, Swallowing Disorders, Sydenham Chorea, Syncope, Syphilitic Spinal Sclerosis, Syringohydromyelia, Syringomyelia, Systemic Lupus Erythematosus, Tabes Dorsalis, Tardive Dyskinesia, Tarlov Cysts, Tay-Sachs Disease, Temporal Arteritis, Tethered Spinal Cord Syndrome, Thomsen's Myotonia, Thoracic Outlet Syndrome, Thyrotoxic Myopathy, Tic Douloureux, Todd's Paralysis, Tourette Syndrome, Transient Ischemic Attack, Transmissible Spongiform Encephalopathies, Transverse Myelitis, Traumatic Brain Injury, Tremor, Trigeminal Neuralgia, Tropical Spastic Paraparesis, Troyer Syndrome, Tuberous Sclerosis, Vascular Erectile Tumor, Vasculitis Syndromes of the Central and Peripheral Nervous Systems, Vitamin B12 deficiency, Von Economo's Disease, Von Hippel-Lindau Disease (VHL), Von Recklinghausen's Disease, Wallenberg's Syndrome, Werdnig-Hoffman Disease, Wernicke-Korsakoff Syndrome, West Syndrome, Whiplash, Whipple's Disease, Williams Syndrome, Wilson Disease, Wolman's Disease, X-Linked Spinal and Bulbar Muscular Atrophy.

Methods of Treatment of Neurological Disease

TRACER AAV Particles Encoding Protein Payloads

Provided in the present disclosure are methods for introducing the TRACER AAV particles of the present disclosure into cells, the method comprising introducing into said cells any of the vectors in an amount sufficient for an increase in the production of target mRNA and protein to occur. In some aspects, the cells may be neurons such as but not limited to, motor, hippocampal, entorhinal, thalamic, cortical, sensory, sympathetic, or parasympathetic neurons, and glial cells such as astrocytes, microglia, and/or oligodendrocytes.

Disclosed in the present disclosure are methods for treating neurological disease associated with insufficient function/presence of a target protein (e.g., ApoE, FXN) in a subject in need of treatment. The method optionally comprises administering to the subject a therapeutically effective amount of a composition comprising TRACER AAV particles of the present disclosure. As a non-limiting example, the TRACER AAV particles can increase target gene expression, increase target protein production, and thus reduce one or more symptoms of neurological disease in the subject such that the subject is therapeutically treated.

In one embodiment, the composition comprising the TRACER AAV particles of the present disclosure is administered to the central nervous system of the subject via systemic administration. In one embodiment, the systemic administration is intravenous injection.

In some embodiments, the composition comprising the TRACER AAV particles of the present disclosure is administered to the central nervous system of the subject. In other embodiments, the composition comprising the TRACER AAV particles of the present disclosure is administered to a CNS tissue of a subject (e.g., putamen, thalamus or cortex of the subject).

In one embodiment, the composition comprising the TRACER AAV particles of the present disclosure is administered to the central nervous system of the subject via intraparenchymal injection. Non-limiting examples of intraparenchymal injections include intraputamenal, intracortical, intrathalamic, intrastriatal, intrahippocampal or into the entorhinal cortex.

In one embodiment, the composition comprising the TRACER AAV particles of the present disclosure is administered to the central nervous system of the subject via intraparenchymal injection and intravenous injection.

In one embodiment, the TRACER AAV particles of the present disclosure may be delivered into specific types of targeted cells, including, but not limited to, thalamic, hippocampal, entorhinal, cortical, motor, sensory, excitatory, inhibitory, sympathetic, or parasympathetic neurons; glial cells including oligodendrocytes, astrocytes and microglia; and/or other cells surrounding neurons such as T cells.

In one embodiment, the TRACER AAV particles of the present disclosure may be delivered to neurons in the putamen, thalamus and/or cortex.

In some embodiments, the TRACER AAV particles of the present disclosure may be used as a therapy for neurological disease.

In some embodiments, the TRACER AAV particles of the present disclosure may be used as a therapy for tauopathies.

In some embodiments, the TRACER AAV particles of the present disclosure may be used as a therapy for Alzheimer's Disease.

In some embodiments, the TRACER AAV particles of the present disclosure may be used as a therapy for Amyotrophic Lateral Sclerosis.

In some embodiments, the TRACER AAV particles of the present disclosure may be used as a therapy for Huntington's Disease.

In some embodiments, the TRACER AAV particles of the present disclosure may be used as a therapy for Parkinson's Disease.

In some embodiments, the TRACER AAV particles of the present disclosure may be used as a therapy for Friedreich's Ataxia.

In some embodiments, the TRACER AAV particles of the present disclosure may be used as a therapy for chronic or neuropathic pain.

In one embodiment, administration of the TRACER AAV particles described herein to a subject may increase target protein levels in a subject. The target protein levels may be increased by about 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95% and 100%, or at least 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% or 95-100% in a subject such as, but not limited to, the CNS, a region of the CNS, or a specific cell of the CNS of a subject. As a non-limiting example, the TRACER AAV particles may increase the protein levels of a target protein by at least 50%. As a non-limiting example, the TRACER AAV particles may increase the proteins levels of a target protein by at least 40%. As a non-limiting example, a subject may have an increase of 10% of target protein. As a non-limiting example, the TRACER AAV particles may increase the protein levels of a target protein by fold increases over baseline. In one embodiment, TRACER AAV particles lead to 5-6 times higher levels of a target protein.

In one embodiment, administration of the TRACER AAV particles described herein to a subject may increase the expression of a target protein in a subject. The expression of the target protein may be increased by about 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95% and 100%, or at least 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% or 95-100% in a subject such as, but not limited to, the CNS, a region of the CNS, or a specific cell of the CNS of a subject. As a non-limiting example, the TRACER AAV particles may increase the expression of a target protein by at least 50%. As a non-limiting example, the TRACER AAV particles may increase the expression of a target protein by at least 40%.

In one embodiment, intravenous administration of the TRACER AAV particles described herein to a subject may increase the CNS expression of a target protein in a subject. The expression of the target protein may be increased by about 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95% and 100%, or at least 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% or 95-100% in a subject such as, but not limited to, the CNS, a region of the CNS, or a specific cell of the CNS of a subject. As a non-limiting example, the TRACER AAV particles may increase the expression of a target protein in the CNS by at least 50%. As a non-limiting example, the TRACER AAV particles may increase the expression of a target protein in the CNS by at least 40%.

In some embodiments, the TRACER AAV particles of the present disclosure may be used to increase target protein expression in astrocytes in order to treat a neurological disease. Target protein in astrocytes may be increased by 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or more than 95%, 5-15%, 5-20%, 5-25%, 5-30%, 5-35%, 5-40%, 5-45%, 5-50%, 5-55%, 5-60%, 5-65%, 5-70%, 5-75%, 5-80%, 5-85%, 5-90%, 5-95%, 10-20%, 10-25%, 10-30%, 10-35%, 10-40%, 10-45%, 10-50%, 10-55%, 10-60%, 10-65%, 10-70%, 10-75%, 10-80%, 10-85%, 10-90%, 10-95%, 15-25%, 15-30%, 15-35%, 15-40%, 15-45%, 15-50%, 15-55%, 15-60%, 15-65%, 15-70%, 15-75%, 15-80%, 15-85%, 15-90%, 15-95%, 20-30%, 20-35%, 20-40%, 20-45%, 20-50%, 20-55%, 20-60%, 20-65%, 20-70%, 20-75%, 20-80%, 20-85%, 20-90%, 20-95%, 25-35%, 25-40%, 25-45%, 25-50%, 25-55%, 25-60%, 25-65%, 25-70%, 25-75%, 25-80%, 25-85%, 25-90%, 25-95%, 30-40%, 30-45%, 30-50%, 30-55%, 30-60%, 30-65%, 30-70%, 30-75%, 30-80%, 30-85%, 30-90%, 30-95%, 35-45%, 35-50%, 35-55%, 35-60%, 35-65%, 35-70%, 35-75%, 35-80%, 35-85%, 35-90%, 35-95%, 40-50%, 40-55%, 40-60%, 40-65%, 40-70%, 40-75%, 40-80%, 40-85%, 40-90%, 40-95%, 45-55%, 45-60%, 45-65%, 45-70%, 45-75%, 45-80%, 45-85%, 45-90%, 45-95%, 50-60%, 50-65%, 50-70%, 50-75%, 50-80%, 50-85%, 50-90%, 50-95%, 55-65%, 55-70%, 55-75%, 55-80%, 55-85%, 55-90%, 55-95%, 60-70%, 60-75%, 60-80%, 60-85%, 60-90%, 60-95%, 65-75%, 65-80%, 65-85%, 65-90%, 65-95%, 70-80%, 70-85%, 70-90%, 70-95%, 75-85%, 75-90%, 75-95%, 80-90%, 80-95%, or 90-95%.

In some embodiments, the TRACER AAV particles may be used to increase target protein in microglia. The increase of target protein in microglia may be, independently, increased by 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or more than 95%, 5-15%, 5-20%, 5-25%, 5-30%, 5-35%, 5-40%, 5-45%, 5-50%, 5-55%, 5-60%, 5-65%, 5-70%, 5-75%, 5-80%, 5-85%, 5-90%, 5-95%, 10-20%, 10-25%, 10-30%, 10-35%, 10-40%, 10-45%, 10-50%, 10-55%, 10-60%, 10-65%, 10-70%, 10-75%, 10-80%, 10-85%, 10-90%, 10-95%, 15-25%, 15-30%, 15-35%, 15-40%, 15-45%, 15-50%, 15-55%, 15-60%, 15-65%, 15-70%, 15-75%, 15-80%, 15-85%, 15-90%, 15-95%, 20-30%, 20-35%, 20-40%, 20-45%, 20-50%, 20-55%, 20-60%, 20-65%, 20-70%, 20-75%, 20-80%, 20-85%, 20-90%, 20-95%, 25-35%, 25-40%, 25-45%, 25-50%, 25-55%, 25-60%, 25-65%, 25-70%, 25-75%, 25-80%, 25-85%, 25-90%, 25-95%, 30-40%, 30-45%, 30-50%, 30-55%, 30-60%, 30-65%, 30-70%, 30-75%, 30-80%, 30-85%, 30-90%, 30-95%, 35-45%, 35-50%, 35-55%, 35-60%, 35-65%, 35-70%, 35-75%, 35-80%, 35-85%, 35-90%, 35-95%, 40-50%, 40-55%, 40-60%, 40-65%, 40-70%, 40-75%, 40-80%, 40-85%, 40-90%, 40-95%, 45-55%, 45-60%, 45-65%, 45-70%, 45-75%, 45-80%, 45-85%, 45-90%, 45-95%, 50-60%, 50-65%, 50-70%, 50-75%, 50-80%, 50-85%, 50-90%, 50-95%, 55-65%, 55-70%, 55-75%, 55-80%, 55-85%, 55-90%, 55-95%, 60-70%, 60-75%, 60-80%, 60-85%, 60-90%, 60-95%, 65-75%, 65-80%, 65-85%, 65-90%, 65-95%, 70-80%, 70-85%, 70-90%, 70-95%, 75-85%, 75-90%, 75-95%, 80-90%, 80-95%, or 90-95%.

In some embodiments, the TRACER AAV particles may be used to increase target protein in cortical neurons. The increase of target protein in the cortical neurons may be, independently, increased by 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or more than 95%, 5-15%, 5-20%, 5-25%, 5-30%, 5-35%, 5-40%, 5-45%, 5-50%, 5-55%, 5-60%, 5-65%, 5-70%, 5-75%, 5-80%, 5-85%, 5-90%, 5-95%, 10-20%, 10-25%, 10-30%, 10-35%, 10-40%, 10-45%, 10-50%, 10-55%, 10-60%, 10-65%, 10-70%, 10-75%, 10-80%, 10-85%, 10-90%, 10-95%, 15-25%, 15-30%, 15-35%, 15-40%, 15-45%, 15-50%, 15-55%, 15-60%, 15-65%, 15-70%, 15-75%, 15-80%, 15-85%, 15-90%, 15-95%, 20-30%, 20-35%, 20-40%, 20-45%, 20-50%, 20-55%, 20-60%, 20-65%, 20-70%, 20-75%, 20-80%, 20-85%, 20-90%, 20-95%, 25-35%, 25-40%, 25-45%, 25-50%, 25-55%, 25-60%, 25-65%, 25-70%, 25-75%, 25-80%, 25-85%, 25-90%, 25-95%, 30-40%, 30-45%, 30-50%, 30-55%, 30-60%, 30-65%, 30-70%, 30-75%, 30-80%, 30-85%, 30-90%, 30-95%, 35-45%, 35-50%, 35-55%, 35-60%, 35-65%, 35-70%, 35-75%, 35-80%, 35-85%, 35-90%, 35-95%, 40-50%, 40-55%, 40-60%, 40-65%, 40-70%, 40-75%, 40-80%, 40-85%, 40-90%, 40-95%, 45-55%, 45-60%, 45-65%, 45-70%, 45-75%, 45-80%, 45-85%, 45-90%, 45-95%, 50-60%, 50-65%, 50-70%, 50-75%, 50-80%, 50-85%, 50-90%, 50-95%, 55-65%, 55-70%, 55-75%, 55-80%, 55-85%, 55-90%, 55-95%, 60-70%, 60-75%, 60-80%, 60-85%, 60-90%, 60-95%, 65-75%, 65-80%, 65-85%, 65-90%, 65-95%, 70-80%, 70-85%, 70-90%, 70-95%, 75-85%, 75-90%, 75-95%, 80-90%, 80-95%, or 90-95%.

In some embodiments, the TRACER AAV particles may be used to increase target protein in hippocampal neurons. The increase of target protein in the hippocampal neurons may be, independently, increased by 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or more than 95%, 5-15%, 5-20%, 5-25%, 5-30%, 5-35%, 5-40%, 5-45%, 5-50%, 5-55%, 5-60%, 5-65%, 5-70%, 5-75%, 5-80%, 5-85%, 5-90%, 5-95%, 10-20%, 10-25%, 10-30%, 10-35%, 10-40%, 10-45%, 10-50%, 10-55%, 10-60%, 10-65%, 10-70%, 10-75%, 10-80%, 10-85%, 10-90%, 10-95%, 15-25%, 15-30%, 15-35%, 15-40%, 15-45%, 15-50%, 15-55%, 15-60%, 15-65%, 15-70%, 15-75%, 15-80%, 15-85%, 15-90%, 15-95%, 20-30%, 20-35%, 20-40%, 20-45%, 20-50%, 20-55%, 20-60%, 20-65%, 20-70%, 20-75%, 20-80%, 20-85%, 20-90%, 20-95%, 25-35%, 25-40%, 25-45%, 25-50%, 25-55%, 25-60%, 25-65%, 25-70%, 25-75%, 25-80%, 25-85%, 25-90%, 25-95%, 30-40%, 30-45%, 30-50%, 30-55%, 30-60%, 30-65%, 30-70%, 30-75%, 30-80%, 30-85%, 30-90%, 30-95%, 35-45%, 35-50%, 35-55%, 35-60%, 35-65%, 35-70%, 35-75%, 35-80%, 35-85%, 35-90%, 35-95%, 40-50%, 40-55%, 40-60%, 40-65%, 40-70%, 40-75%, 40-80%, 40-85%, 40-90%, 40-95%, 45-55%, 45-60%, 45-65%, 45-70%, 45-75%, 45-80%, 45-85%, 45-90%, 45-95%, 50-60%, 50-65%, 50-70%, 50-75%, 50-80%, 50-85%, 50-90%, 50-95%, 55-65%, 55-70%, 55-75%, 55-80%, 55-85%, 55-90%, 55-95%, 60-70%, 60-75%, 60-80%, 60-85%, 60-90%, 60-95%, 65-75%, 65-80%, 65-85%, 65-90%, 65-95%, 70-80%, 70-85%, 70-90%, 70-95%, 75-85%, 75-90%, 75-95%, 80-90%, 80-95%, or 90-95%.

In some embodiments, the TRACER AAV particles may be used to increase target protein in DRG and/or sympathetic neurons. The increase of target protein in the DRG and/or sympathetic neurons may be, independently, increased by 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or more than 95%, 5-15%, 5-20%, 5-25%, 5-30%, 5-35%, 5-40%, 5-45%, 5-50%, 5-55%, 5-60%, 5-65%, 5-70%, 5-75%, 5-80%, 5-85%, 5-90%, 5-95%, 10-20%, 10-25%, 10-30%, 10-35%, 10-40%, 10-45%, 10-50%, 10-55%, 10-60%, 10-65%, 10-70%, 10-75%, 10-80%, 10-85%, 10-90%, 10-95%, 15-25%, 15-30%, 15-35%, 15-40%, 15-45%, 15-50%, 15-55%, 15-60%, 15-65%, 15-70%, 15-75%, 15-80%, 15-85%, 15-90%, 15-95%, 20-30%, 20-35%, 20-40%, 20-45%, 20-50%, 20-55%, 20-60%, 20-65%, 20-70%, 20-75%, 20-80%, 20-85%, 20-90%, 20-95%, 25-35%, 25-40%, 25-45%, 25-50%, 25-55%, 25-60%, 25-65%, 25-70%, 25-75%, 25-80%, 25-85%, 25-90%, 25-95%, 30-40%, 30-45%, 30-50%, 30-55%, 30-60%, 30-65%, 30-70%, 30-75%, 30-80%, 30-85%, 30-90%, 30-95%, 35-45%, 35-50%, 35-55%, 35-60%, 35-65%, 35-70%, 35-75%, 35-80%, 35-85%, 35-90%, 35-95%, 40-50%, 40-55%, 40-60%, 40-65%, 40-70%, 40-75%, 40-80%, 40-85%, 40-90%, 40-95%, 45-55%, 45-60%, 45-65%, 45-70%, 45-75%, 45-80%, 45-85%, 45-90%, 45-95%, 50-60%, 50-65%, 50-70%, 50-75%, 50-80%, 50-85%, 50-90%, 50-95%, 55-65%, 55-70%, 55-75%, 55-80%, 55-85%, 55-90%, 55-95%, 60-70%, 60-75%, 60-80%, 60-85%, 60-90%, 60-95%, 65-75%, 65-80%, 65-85%, 65-90%, 65-95%, 70-80%, 70-85%, 70-90%, 70-95%, 75-85%, 75-90%, 75-95%, 80-90%, 80-95%, or 90-95%.

In some embodiments, the TRACER AAV particles of the present disclosure may be used to increase target protein in sensory neurons in order to treat neurological disease. Target protein in sensory neurons may be increased by 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or more than 95%, 5-15%, 5-20%, 5-25%, 5-30%, 5-35%, 5-40%, 5-45%, 5-50%, 5-55%, 5-60%, 5-65%, 5-70%, 5-75%, 5-80%, 5-85%, 5-90%, 5-95%, 10-20%, 10-25%, 10-30%, 10-35%, 10-40%, 10-45%, 10-50%, 10-55%, 10-60%, 10-65%, 10-70%, 10-75%, 10-80%, 10-85%, 10-90%, 10-95%, 15-25%, 15-30%, 15-35%, 15-40%, 15-45%, 15-50%, 15-55%, 15-60%, 15-65%, 15-70%, 15-75%, 15-80%, 15-85%, 15-90%, 15-95%, 20-30%, 20-35%, 20-40%, 20-45%, 20-50%, 20-55%, 20-60%, 20-65%, 20-70%, 20-75%, 20-80%, 20-85%, 20-90%, 20-95%, 25-35%, 25-40%, 25-45%, 25-50%, 25-55%, 25-60%, 25-65%, 25-70%, 25-75%, 25-80%, 25-85%, 25-90%, 25-95%, 30-40%, 30-45%, 30-50%, 30-55%, 30-60%, 30-65%, 30-70%, 30-75%, 30-80%, 30-85%, 30-90%, 30-95%, 35-45%, 35-50%, 35-55%, 35-60%, 35-65%, 35-70%, 35-75%, 35-80%, 35-85%, 35-90%, 35-95%, 40-50%, 40-55%, 40-60%, 40-65%, 40-70%, 40-75%, 40-80%, 40-85%, 40-90%, 40-95%, 45-55%, 45-60%, 45-65%, 45-70%, 45-75%, 45-80%, 45-85%, 45-90%, 45-95%, 50-60%, 50-65%, 50-70%, 50-75%, 50-80%, 50-85%, 50-90%, 50-95%, 55-65%, 55-70%, 55-75%, 55-80%, 55-85%, 55-90%, 55-95%, 60-70%, 60-75%, 60-80%, 60-85%, 60-90%, 60-95%, 65-75%, 65-80%, 65-85%, 65-90%, 65-95%, 70-80%, 70-85%, 70-90%, 70-95%, 75-85%, 75-90%, 75-95%, 80-90%, 80-95%, or 90-95%.

In some embodiments, the TRACER AAV particles of the present disclosure may be used to increase target protein and reduce symptoms of neurological disease in a subject. The increase of target protein and/or the reduction of symptoms of neurological disease may be, independently, altered (increased for the production of target protein and reduced for the symptoms of neurological disease) by 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or more than 95%, 5-15%, 5-20%, 5-25%, 5-30%, 5-35%, 5-40%, 5-45%, 5-50%, 5-55%, 5-60%, 5-65%, 5-70%, 5-75%, 5-80%, 5-85%, 5-90%, 5-95%, 10-20%, 10-25%, 10-30%, 10-35%, 10-40%, 10-45%, 10-50%, 10-55%, 10-60%, 10-65%, 10-70%, 10-75%, 10-80%, 10-85%, 10-90%, 10-95%, 15-25%, 15-30%, 15-35%, 15-40%, 15-45%, 15-50%, 15-55%, 15-60%, 15-65%, 15-70%, 15-75%, 15-80%, 15-85%, 15-90%, 15-95%, 20-30%, 20-35%, 20-40%, 20-45%, 20-50%, 20-55%, 20-60%, 20-65%, 20-70%, 20-75%, 20-80%, 20-85%, 20-90%, 20-95%, 25-35%, 25-40%, 25-45%, 25-50%, 25-55%, 25-60%, 25-65%, 25-70%, 25-75%, 25-80%, 25-85%, 25-90%, 25-95%, 30-40%, 30-45%, 30-50%, 30-55%, 30-60%, 30-65%, 30-70%, 30-75%, 30-80%, 30-85%, 30-90%, 30-95%, 35-45%, 35-50%, 35-55%, 35-60%, 35-65%, 35-70%, 35-75%, 35-80%, 35-85%, 35-90%, 35-95%, 40-50%, 40-55%, 40-60%, 40-65%, 40-70%, 40-75%, 40-80%, 40-85%, 40-90%, 40-95%, 45-55%, 45-60%, 45-65%, 45-70%, 45-75%, 45-80%, 45-85%, 45-90%, 45-95%, 50-60%, 50-65%, 50-70%, 50-75%, 50-80%, 50-85%, 50-90%, 50-95%, 55-65%, 55-70%, 55-75%, 55-80%, 55-85%, 55-90%, 55-95%, 60-70%, 60-75%, 60-80%, 60-85%, 60-90%, 60-95%, 65-75%, 65-80%, 65-85%, 65-90%, 65-95%, 70-80%, 70-85%, 70-90%, 70-95%, 75-85%, 75-90%, 75-95%, 80-90%, 80-95%, or 90-95%.

In one embodiment, the TRACER AAV particles of the present disclosure may be used to reduce the decline of functional capacity and activities of daily living as measured by a standard evaluation system such as, but not limited to, the total functional capacity (TFC) scale.

In one embodiment, the TRACER AAV particles of the present disclosure may be used to improve performance on any assessment used to measure symptoms of neurological disease. Such assessments include, but are not limited to ADAS-cog (Alzheimer Disease Assessment Scale—cognitive), MNISE (Mini-Mental State Examination), GDS (Geriatric Depression Scale), FAQ (Functional Activities Questionnaire), ADL (Activities of Daily Living), GPCOG (General Practitioner Assessment of Cognition), Mini-Cog, AMTS (Abbreviated Mental Test Score), Clock-drawing test, 6-CIT (6-item Cognitive Impairment Test), TYM (Test Your Memory), MoCa (Montreal Cognitive Assessment), ACE-R (Addenbrookes Cognitive Assessment), MIS (Memory Impairment Screen), BADLS (Bristol Activities of Daily Living Scale), Barthel Index, Functional Independence Measure, Instrumental Activities of Daily Living, IQCODE (Informant Questionnaire on Cognitive Decline in the Elderly), Neuropsychiatric Inventory, The Cohen-Mansfield Agitation Inventory, BEHAVE-AD, EuroQol, Short Form-36 and/or MBR Caregiver Strain Instrument, or any of the other tests as described in Sheehan B (Ther Adv Neurol Disord. 5(6):349-358 (2012)), the contents of which are herein incorporated by reference in their entirety.

In some embodiments, the present composition is administered as a solo therapeutic or as combination therapeutic for the treatment of neurological disease.

The TRACER AAV particles encoding the target protein may be used in combination with one or more other therapeutic agents. By “in combination with,” it is not intended to imply that the agents must be administered at the same time and/or formulated for delivery together, although these methods of delivery are within the scope of the present disclosure. Compositions can be administered concurrently with, prior to, or subsequent to, one or more other desired therapeutics or medical procedures. In general, each agent will be administered at a dose and/or on a time schedule determined for that agent.

Therapeutic agents that may be used in combination with the TRACER AAV particles of the present disclosure can be small molecule compounds which are antioxidants, anti-inflammatory agents, anti-apoptosis agents, calcium regulators, antiglutamatergic agents, structural protein inhibitors, compounds involved in muscle function, and compounds involved in metal ion regulation. As a non-limiting example, the combination therapy may be in combination with one or more neuroprotective agents such as small molecule compounds, growth factors and hormones which have been tested for their neuroprotective effect on motor neuron degeneration.

Compounds tested for treating neurological disease which may be used in combination with the TRACER AAV particles described herein include, but are not limited to, cholinesterase inhibitors (donepezil, rivastigmine, galantamine), NMDA receptor antagonists such as memantine, anti-psychotics, anti-depressants, anti-convulsants (e.g., sodium valproate and levetiracetam for myoclonus), secretase inhibitors, amyloid aggregation inhibitors, copper or zinc modulators, BACE inhibitors, inhibitors of tau aggregation, such as Methylene blue, phenothiazines, anthraquinones, n-phenylamines or rhodamines, microtubule stabilizers such as NAP, taxol or paclitaxel, kinase or phosphatase inhibitors such as those targeting GSK3 (3 (lithium) or PP2A, immunization with Aβ peptides or tau phospho-epitopes, anti-tau or anti-amyloid antibodies, dopamine-depleting agents (e.g., tetrabenazine for chorea), benzodiazepines (e.g., clonazepam for myoclonus, chorea, dystonia, rigidity, and/or spasticity), amino acid precursors of dopamine (e.g., levodopa for rigidity), skeletal muscle relaxants (e.g., baclofen, tizanidine for rigidity and/or spasticity), inhibitors for acetylcholine release at the neuromuscular junction to cause muscle paralysis (e.g., botulinum toxin for bruxism and/or dystonia), atypical neuroleptics (e.g., olanzapine and quetiapine for psychosis and/or irritability, risperidone, sulpiride and haloperidol for psychosis, chorea and/or irritability, clozapine for treatment-resistant psychosis, aripiprazole for psychosis with prominent negative symptoms), selective serotonin reuptake inhibitors (SSRIs) (e.g., citalopram, fluoxetine, paroxetine, sertraline, mirtazapine, venlafaxine for depression, anxiety, obsessive compulsive behavior and/or irritability), hypnotics (e.g., xopiclone and/or zolpidem for altered sleep-wake cycle), anticonvulsants (e.g., sodium valproate and carbamazepine for mania or hypomania) and mood stabilizers (e.g., lithium for mania or hypomania).

Neurotrophic factors may be used in combination therapy with the TRACER AAV particles of the present disclosure for treating neurological disease. Generally, a neurotrophic factor is defined as a substance that promotes survival, growth, differentiation, proliferation and/or maturation of a neuron, or stimulates increased activity of a neuron. In some embodiments, the present methods further comprise delivery of one or more trophic factors into the subject in need of treatment. Trophic factors may include, but are not limited to, IGF-I, GDNF, BDNF, CTNF, VEGF, Colivelin, Xaliproden, Thyrotrophin-releasing hormone and ADNF, and variants thereof.

In one aspect, the TRACER AAV particle described herein may be co-administered with TRACER AAV particles expressing neurotrophic factors such as AAV-IGF-I (See e.g., Vincent et al., Neuromolecular medicine, 2004, 6, 79-85; the contents of which are incorporated herein by reference in their entirety) and AAV-GDNF (See e.g., Wang et al., J Neurosci., 2002, 22, 6920-6928; the contents of which are incorporated herein by reference in their entirety).

In one embodiment, administration of the TRACER AAV particles to a subject will increase the expression of a target protein in a subject and the increase of the expression of the target protein will reduce the effects and/or symptoms of neurological disease in a subject.

As a non-limiting example, the target protein may be an antibody, or fragment thereof.

TRACER AAV Particles Comprising RNAi Agents or Modulatory Polynucleotides

Provided in the present disclosure are methods for introducing the TRACER AAV particles of the disclosure, comprising a viral genome with a nucleic acid sequence encoding one or more siRNA molecules into cells, the method comprising introducing into said cells any of the vectors in an amount sufficient for degradation of a target mRNA to occur, thereby activating target-specific RNAi in the cells. In some aspects, the cells may be neurons such as but not limited to, motor, hippocampal, entorhinal, thalamic, cortical, sensory, sympathetic, or parasympathetic neurons, and glial cells such as astrocytes, microglia, and/or oligodendrocytes.

Disclosed in the present disclosure are methods for treating neurological diseases associated with dysfunction of a target protein in a subject in need of treatment. The method optionally comprises administering to the subject a therapeutically effective amount of a composition comprising TRACER AAV particles comprising a viral genome with a nucleic acid sequence encoding one or more siRNA molecules. As a non-limiting example, the siRNA molecules can silence target gene expression, inhibit target protein production, and reduce one or more symptoms of neurological disease in the subject such that the subject is therapeutically treated.

In some embodiments, the composition comprising the TRACER AAV particles of the present disclosure comprising a viral genome encoding one or more siRNA molecules comprise an AAV capsid that allows for enhanced transduction of CNS and/or PNS cells after intravenous administration.

In some embodiments, the composition comprising the TRACER AAV particles of the present disclosure with a viral genome encoding at least one siRNA molecule is administered to the central nervous system of the subject. In other embodiments, the composition comprising the TRACER AAV particles of the present disclosure is administered to a tissue of a subject (e.g., putamen, thalamus or cortex of the subject).

In one embodiment, the composition comprising the TRACER AAV particles of the disclosure, comprising a viral genome with a nucleic acid sequence encoding one or more siRNA molecules is administered to the central nervous system of the subject via systemic administration. In one embodiment, the systemic administration is intravenous injection.

In one embodiment, the composition comprising the TRACER AAV particles of the disclosure comprising a viral genome with a nucleic acid sequence encoding one or more siRNA molecules is administered to the central nervous system of the subject via intraparenchymal injection. Non-limiting examples of intraparenchymal injections include intraputamenal, intracortical, intrathalamic, intrastriatal, intrahippocampal or into the entorhinal cortex.

In one embodiment, the composition comprising the TRACER AAV particles comprising a viral genome with a nucleic acid sequence encoding one or more siRNA molecules is administered to the central nervous system of the subject via intraparenchymal injection and intravenous injection.

In one embodiment, the TRACER AAV particles comprising a viral genome with a nucleic acid sequence encoding one or more siRNA molecules may be delivered into specific types or targeted cells, including, but not limited to, thalamic, hippocampal, entorhinal, cortical, motor, sensory, excitatory, inhibitory, sympathetic, or parasympathetic neurons; glial cells including oligodendrocytes, astrocytes and microglia; and/or other cells surrounding neurons such as T cells.

In one embodiment, the TRACER AAV particles comprising a viral genome with a nucleic acid sequence encoding one or more siRNA molecules may be delivered to neurons in the putamen, thalamus, and/or cortex.

In one embodiment, the TRACER AAV particles comprising a viral genome with a nucleic acid sequence encoding one or more siRNA molecules may be used as a therapy for neurological disease.

In one embodiment, the TRACER AAV particles comprising a viral genome with a nucleic acid sequence encoding one or more siRNA molecules may be used as a therapy for tauopathies.

In one embodiment, the TRACER AAV particles comprising a viral genome with a nucleic acid sequence encoding one or more siRNA molecules may be used as a therapy for Alzheimer' s Disease.

In one embodiment, the TRACER AAV particles comprising a viral genome with a nucleic acid sequence encoding one or more siRNA molecules may be used as a therapy for Amyotrophic Lateral Sclerosis.

In one embodiment, the TRACER AAV particles comprising a viral genome with a nucleic acid sequence encoding one or more siRNA molecules may be used as a therapy for Huntington's Disease.

In one embodiment, the TRACER AAV particles comprising a viral genome with a nucleic acid sequence encoding one or more siRNA molecules may be used as a therapy for Parkinson's Disease.

In one embodiment, the TRACER AAV particles comprising a viral genome with a nucleic acid sequence encoding one or more siRNA molecules may be used as a therapy for Friedreich's Ataxia.

In one embodiment, the administration of TRACER AAV particles comprising a viral genome with a nucleic acid sequence encoding one or more siRNA molecules to a subject may lower target protein levels in a subject. The target protein levels may be lowered by about 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95% and 100%, or at least 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% or 95-100% in a subject such as, but not limited to, the CNS, a region of the CNS, or a specific cell of the CNS of a subject. As a non-limiting example, the TRACER AAV particles may lower the protein levels of a target protein by at least 50%. As a non-limiting example, the TRACER AAV particles may lower the proteins levels of a target protein by at least 40%.

In one embodiment, the administration of TRACER AAV particles comprising a viral genome with a nucleic acid sequence encoding one or more siRNA molecules to a subject may lower the expression of a target protein in a subject. The expression of a target protein may be lowered by about 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95% and 100%, or at least 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% or 95-100% in a subject such as, but not limited to, the CNS, a region of the CNS, or a specific cell of the CNS of a subject. As a non-limiting example, the TRACER AAV particles may lower the expression of a target protein by at least 50%. As a non-limiting example, the TRACER AAV particles may lower the expression of a target protein by at least 40%.

In one embodiment, the administration of TRACER AAV particles comprising a viral genome with a nucleic acid sequence encoding one or more siRNA molecules to a subject may lower the expression of a target protein in the CNS of a subject. The expression of a target protein may be lowered by about 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95% and 100%, or at least 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% or 95-100% in a subject such as, but not limited to, the CNS, a region of the CNS, or a specific cell of the CNS of a subject. As a non-limiting example, the TRACER AAV particles may lower the expression of a target protein by at least 50%. As a non-limiting example, the TRACER AAV particles may lower the expression of a target protein by at least 40%.

In one embodiment, the TRACER AAV particles comprising a viral genome with a nucleic acid sequence encoding one or more siRNA molecules may be used to suppress a target protein in astrocytes in order to treat neurological disease. Target protein in astrocytes may be suppressed by 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or more than 95%, 5-15%, 5-20%, 5-25%, 5-30%, 5-35%, 5-40%, 5-45%, 5-50%, 5-55%, 5-60%, 5-65%, 5-70%, 5-75%, 5-80%, 5-85%, 5-90%, 5-95%, 10-20%, 10-25%, 10-30%, 10-35%, 10-40%, 10-45%, 10-50%, 10-55%, 10-60%, 10-65%, 10-70%, 10-75%, 10-80%, 10-85%, 10-90%, 10-95%, 15-25%, 15-30%, 15-35%, 15-40%, 15-45%, 15-50%, 15-55%, 15-60%, 15-65%, 15-70%, 15-75%, 15-80%, 15-85%, 15-90%, 15-95%, 20-30%, 20-35%, 20-40%, 20-45%, 20-50%, 20-55%, 20-60%, 20-65%, 20-70%, 20-75%, 20-80%, 20-85%, 20-90%, 20-95%, 25-35%, 25-40%, 25-45%, 25-50%, 25-55%, 25-60%, 25-65%, 25-70%, 25-75%, 25-80%, 25-85%, 25-90%, 25-95%, 30-40%, 30-45%, 30-50%, 30-55%, 30-60%, 30-65%, 30-70%, 30-75%, 30-80%, 30-85%, 30-90%, 30-95%, 35-45%, 35-50%, 35-55%, 35-60%, 35-65%, 35-70%, 35-75%, 35-80%, 35-85%, 35-90%, 35-95%, 40-50%, 40-55%, 40-60%, 40-65%, 40-70%, 40-75%, 40-80%, 40-85%, 40-90%, 40-95%, 45-55%, 45-60%, 45-65%, 45-70%, 45-75%, 45-80%, 45-85%, 45-90%, 45-95%, 50-60%, 50-65%, 50-70%, 50-75%, 50-80%, 50-85%, 50-90%, 50-95%, 55-65%, 55-70%, 55-75%, 55-80%, 55-85%, 55-90%, 55-95%, 60-70%, 60-75%, 60-80%, 60-85%, 60-90%, 60-95%, 65-75%, 65-80%, 65-85%, 65-90%, 65-95%, 70-80%, 70-85%, 70-90%, 70-95%, 75-85%, 75-90%, 75-95%, 80-90%, 80-95%, or 90-95%. Target protein in astrocytes may be reduced may be 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or more than 95%, 5-15%, 5-20%, 5-25%, 5-30%, 5-35%, 5-40%, 5-45%, 5-50%, 5-55%, 5-60%, 5-65%, 5-70%, 5-75%, 5-80%, 5-85%, 5-90%, 5-95%, 10-20%, 10-25%, 10-30%, 10-35%, 10-40%, 10-45%, 10-50%, 10-55%, 10-60%, 10-65%, 10-70%, 10-75%, 10-80%, 10-85%, 10-90%, 10-95%, 15-25%, 15-30%, 15-35%, 15-40%, 15-45%, 15-50%, 15-55%, 15-60%, 15-65%, 15-70%, 15-75%, 15-80%, 15-85%, 15-90%, 15-95%, 20-30%, 20-35%, 20-40%, 20-45%, 20-50%, 20-55%, 20-60%, 20-65%, 20-70%, 20-75%, 20-80%, 20-85%, 20-90%, 20-95%, 25-35%, 25-40%, 25-45%, 25-50%, 25-55%, 25-60%, 25-65%, 25-70%, 25-75%, 25-80%, 25-85%, 25-90%, 25-95%, 30-40%, 30-45%, 30-50%, 30-55%, 30-60%, 30-65%, 30-70%, 30-75%, 30-80%, 30-85%, 30-90%, 30-95%, 35-45%, 35-50%, 35-55%, 35-60%, 35-65%, 35-70%, 35-75%, 35-80%, 35-85%, 35-90%, 35-95%, 40-50%, 40-55%, 40-60%, 40-65%, 40-70%, 40-75%, 40-80%, 40-85%, 40-90%, 40-95%, 45-55%, 45-60%, 45-65%, 45-70%, 45-75%, 45-80%, 45-85%, 45-90%, 45-95%, 50-60%, 50-65%, 50-70%, 50-75%, 50-80%, 50-85%, 50-90%, 50-95%, 55-65%, 55-70%, 55-75%, 55-80%, 55-85%, 55-90%, 55-95%, 60-70%, 60-75%, 60-80%, 60-85%, 60-90%, 60-95%, 65-75%, 65-80%, 65-85%, 65-90%, 65-95%, 70-80%, 70-85%, 70-90%, 70-95%, 75-85%, 75-90%, 75-95%, 80-90%, 80-95%, or 90-95%.

In one embodiment, the TRACER AAV particles comprising a viral genome with a nucleic acid sequence encoding one or more siRNA molecules may be used to suppress a target protein in microglia. The suppression of the target protein in microglia may be, independently, suppressed by 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or more than 95%, 5-15%, 5-20%, 5-25%, 5-30%, 5-35%, 5-40%, 5-45%, 5-50%, 5-55%, 5-60%, 5-65%, 5-70%, 5-75%, 5-80%, 5-85%, 5-90%, 5-95%, 10-20%, 10-25%, 10-30%, 10-35%, 10-40%, 10-45%, 10-50%, 10-55%, 10-60%, 10-65%, 10-70%, 10-75%, 10-80%, 10-85%, 10-90%, 10-95%, 15-25%, 15-30%, 15-35%, 15-40%, 15-45%, 15-50%, 15-55%, 15-60%, 15-65%, 15-70%, 15-75%, 15-80%, 15-85%, 15-90%, 15-95%, 20-30%, 20-35%, 20-40%, 20-45%, 20-50%, 20-55%, 20-60%, 20-65%, 20-70%, 20-75%, 20-80%, 20-85%, 20-90%, 20-95%, 25-35%, 25-40%, 25-45%, 25-50%, 25-55%, 25-60%, 25-65%, 25-70%, 25-75%, 25-80%, 25-85%, 25-90%, 25-95%, 30-40%, 30-45%, 30-50%, 30-55%, 30-60%, 30-65%, 30-70%, 30-75%, 30-80%, 30-85%, 30-90%, 30-95%, 35-45%, 35-50%, 35-55%, 35-60%, 35-65%, 35-70%, 35-75%, 35-80%, 35-85%, 35-90%, 35-95%, 40-50%, 40-55%, 40-60%, 40-65%, 40-70%, 40-75%, 40-80%, 40-85%, 40-90%, 40-95%, 45-55%, 45-60%, 45-65%, 45-70%, 45-75%, 45-80%, 45-85%, 45-90%, 45-95%, 50-60%, 50-65%, 50-70%, 50-75%, 50-80%, 50-85%, 50-90%, 50-95%, 55-65%, 55-70%, 55-75%, 55-80%, 55-85%, 55-90%, 55-95%, 60-70%, 60-75%, 60-80%, 60-85%, 60-90%, 60-95%, 65-75%, 65-80%, 65-85%, 65-90%, 65-95%, 70-80%, 70-85%, 70-90%, 70-95%, 75-85%, 75-90%, 75-95%, 80-90%, 80-95%, or 90-95%. The reduction may be 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or more than 95%, 5-15%, 5-20%, 5-25%, 5-30%, 5-35%, 5-40%, 5-45%, 5-50%, 5-55%, 5-60%, 5-65%, 5-70%, 5-75%, 5-80%, 5-85%, 5-90%, 5-95%, 10-20%, 10-25%, 10-30%, 10-35%, 10-40%, 10-45%, 10-50%, 10-55%, 10-60%, 10-65%, 10-70%, 10-75%, 10-80%, 10-85%, 10-90%, 10-95%, 15-25%, 15-30%, 15-35%, 15-40%, 15-45%, 15-50%, 15-55%, 15-60%, 15-65%, 15-70%, 15-75%, 15-80%, 15-85%, 15-90%, 15-95%, 20-30%, 20-35%, 20-40%, 20-45%, 20-50%, 20-55%, 20-60%, 20-65%, 20-70%, 20-75%, 20-80%, 20-85%, 20-90%, 20-95%, 25-35%, 25-40%, 25-45%, 25-50%, 25-55%, 25-60%, 25-65%, 25-70%, 25-75%, 25-80%, 25-85%, 25-90%, 25-95%, 30-40%, 30-45%, 30-50%, 30-55%, 30-60%, 30-65%, 30-70%, 30-75%, 30-80%, 30-85%, 30-90%, 30-95%, 35-45%, 35-50%, 35-55%, 35-60%, 35-65%, 35-70%, 35-75%, 35-80%, 35-85%, 35-90%, 35-95%, 40-50%, 40-55%, 40-60%, 40-65%, 40-70%, 40-75%, 40-80%, 40-85%, 40-90%, 40-95%, 45-55%, 45-60%, 45-65%, 45-70%, 45-75%, 45-80%, 45-85%, 45-90%, 45-95%, 50-60%, 50-65%, 50-70%, 50-75%, 50-80%, 50-85%, 50-90%, 50-95%, 55-65%, 55-70%, 55-75%, 55-80%, 55-85%, 55-90%, 55-95%, 60-70%, 60-75%, 60-80%, 60-85%, 60-90%, 60-95%, 65-75%, 65-80%, 65-85%, 65-90%, 65-95%, 70-80%, 70-85%, 70-90%, 70-95%, 75-85%, 75-90%, 75-95%, 80-90%, 80-95%, or 90-95%.

In one embodiment, the TRACER AAV particles comprising a viral genome with a nucleic acid sequence encoding one or more siRNA molecules may be used to suppress target protein in cortical neurons. The suppression of a target protein in cortical neurons may be, independently, suppressed by 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or more than 95%, 5-15%, 5-20%, 5-25%, 5-30%, 5-35%, 5-40%, 5-45%, 5-50%, 5-55%, 5-60%, 5-65%, 5-70%, 5-75%, 5-80%, 5-85%, 5-90%, 5-95%, 10-20%, 10-25%, 10-30%, 10-35%, 10-40%, 10-45%, 10-50%, 10-55%, 10-60%, 10-65%, 10-70%, 10-75%, 10-80%, 10-85%, 10-90%, 10-95%, 15-25%, 15-30%, 15-35%, 15-40%, 15-45%, 15-50%, 15-55%, 15-60%, 15-65%, 15-70%, 15-75%, 15-80%, 15-85%, 15-90%, 15-95%, 20-30%, 20-35%, 20-40%, 20-45%, 20-50%, 20-55%, 20-60%, 20-65%, 20-70%, 20-75%, 20-80%, 20-85%, 20-90%, 20-95%, 25-35%, 25-40%, 25-45%, 25-50%, 25-55%, 25-60%, 25-65%, 25-70%, 25-75%, 25-80%, 25-85%, 25-90%, 25-95%, 30-40%, 30-45%, 30-50%, 30-55%, 30-60%, 30-65%, 30-70%, 30-75%, 30-80%, 30-85%, 30-90%, 30-95%, 35-45%, 35-50%, 35-55%, 35-60%, 35-65%, 35-70%, 35-75%, 35-80%, 35-85%, 35-90%, 35-95%, 40-50%, 40-55%, 40-60%, 40-65%, 40-70%, 40-75%, 40-80%, 40-85%, 40-90%, 40-95%, 45-55%, 45-60%, 45-65%, 45-70%, 45-75%, 45-80%, 45-85%, 45-90%, 45-95%, 50-60%, 50-65%, 50-70%, 50-75%, 50-80%, 50-85%, 50-90%, 50-95%, 55-65%, 55-70%, 55-75%, 55-80%, 55-85%, 55-90%, 55-95%, 60-70%, 60-75%, 60-80%, 60-85%, 60-90%, 60-95%, 65-75%, 65-80%, 65-85%, 65-90%, 65-95%, 70-80%, 70-85%, 70-90%, 70-95%, 75-85%, 75-90%, 75-95%, 80-90%, 80-95%, or 90-95%. The reduction may be 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or more than 95%, 5-15%, 5-20%, 5-25%, 5-30%, 5-35%, 5-40%, 5-45%, 5-50%, 5-55%, 5-60%, 5-65%, 5-70%, 5-75%, 5-80%, 5-85%, 5-90%, 5-95%, 10-20%, 10-25%, 10-30%, 10-35%, 10-40%, 10-45%, 10-50%, 10-55%, 10-60%, 10-65%, 10-70%, 10-75%, 10-80%, 10-85%, 10-90%, 10-95%, 15-25%, 15-30%, 15-35%, 15-40%, 15-45%, 15-50%, 15-55%, 15-60%, 15-65%, 15-70%, 15-75%, 15-80%, 15-85%, 15-90%, 15-95%, 20-30%, 20-35%, 20-40%, 20-45%, 20-50%, 20-55%, 20-60%, 20-65%, 20-70%, 20-75%, 20-80%, 20-85%, 20-90%, 20-95%, 25-35%, 25-40%, 25-45%, 25-50%, 25-55%, 25-60%, 25-65%, 25-70%, 25-75%, 25-80%, 25-85%, 25-90%, 25-95%, 30-40%, 30-45%, 30-50%, 30-55%, 30-60%, 30-65%, 30-70%, 30-75%, 30-80%, 30-85%, 30-90%, 30-95%, 35-45%, 35-50%, 35-55%, 35-60%, 35-65%, 35-70%, 35-75%, 35-80%, 35-85%, 35-90%, 35-95%, 40-50%, 40-55%, 40-60%, 40-65%, 40-70%, 40-75%, 40-80%, 40-85%, 40-90%, 40-95%, 45-55%, 45-60%, 45-65%, 45-70%, 45-75%, 45-80%, 45-85%, 45-90%, 45-95%, 50-60%, 50-65%, 50-70%, 50-75%, 50-80%, 50-85%, 50-90%, 50-95%, 55-65%, 55-70%, 55-75%, 55-80%, 55-85%, 55-90%, 55-95%, 60-70%, 60-75%, 60-80%, 60-85%, 60-90%, 60-95%, 65-75%, 65-80%, 65-85%, 65-90%, 65-95%, 70-80%, 70-85%, 70-90%, 70-95%, 75-85%, 75-90%, 75-95%, 80-90%, 80-95%, or 90-95%.

In one embodiment, the TRACER AAV particles comprising a viral genome with a nucleic acid sequence encoding one or more siRNA molecules may be used to suppress a target protein in hippocampal neurons. The suppression of a target protein in the hippocampal neurons may be, independently, suppressed by 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or more than 95%, 5-15%, 5-20%, 5-25%, 5-30%, 5-35%, 5-40%, 5-45%, 5-50%, 5-55%, 5-60%, 5-65%, 5-70%, 5-75%, 5-80%, 5-85%, 5-90%, 5-95%, 10-20%, 10-25%, 10-30%, 10-35%, 10-40%, 10-45%, 10-50%, 10-55%, 10-60%, 10-65%, 10-70%, 10-75%, 10-80%, 10-85%, 10-90%, 10-95%, 15-25%, 15-30%, 15-35%, 15-40%, 15-45%, 15-50%, 15-55%, 15-60%, 15-65%, 15-70%, 15-75%, 15-80%, 15-85%, 15-90%, 15-95%, 20-30%, 20-35%, 20-40%, 20-45%, 20-50%, 20-55%, 20-60%, 20-65%, 20-70%, 20-75%, 20-80%, 20-85%, 20-90%, 20-95%, 25-35%, 25-40%, 25-45%, 25-50%, 25-55%, 25-60%, 25-65%, 25-70%, 25-75%, 25-80%, 25-85%, 25-90%, 25-95%, 30-40%, 30-45%, 30-50%, 30-55%, 30-60%, 30-65%, 30-70%, 30-75%, 30-80%, 30-85%, 30-90%, 30-95%, 35-45%, 35-50%, 35-55%, 35-60%, 35-65%, 35-70%, 35-75%, 35-80%, 35-85%, 35-90%, 35-95%, 40-50%, 40-55%, 40-60%, 40-65%, 40-70%, 40-75%, 40-80%, 40-85%, 40-90%, 40-95%, 45-55%, 45-60%, 45-65%, 45-70%, 45-75%, 45-80%, 45-85%, 45-90%, 45-95%, 50-60%, 50-65%, 50-70%, 50-75%, 50-80%, 50-85%, 50-90%, 50-95%, 55-65%, 55-70%, 55-75%, 55-80%, 55-85%, 55-90%, 55-95%, 60-70%, 60-75%, 60-80%, 60-85%, 60-90%, 60-95%, 65-75%, 65-80%, 65-85%, 65-90%, 65-95%, 70-80%, 70-85%, 70-90%, 70-95%, 75-85%, 75-90%, 75-95%, 80-90%, 80-95%, or 90-95%. The reduction may be 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or more than 95%, 5-15%, 5-20%, 5-25%, 5-30%, 5-35%, 5-40%, 5-45%, 5-50%, 5-55%, 5-60%, 5-65%, 5-70%, 5-75%, 5-80%, 5-85%, 5-90%, 5-95%, 10-20%, 10-25%, 10-30%, 10-35%, 10-40%, 10-45%, 10-50%, 10-55%, 10-60%, 10-65%, 10-70%, 10-75%, 10-80%, 10-85%, 10-90%, 10-95%, 15-25%, 15-30%, 15-35%, 15-40%, 15-45%, 15-50%, 15-55%, 15-60%, 15-65%, 15-70%, 15-75%, 15-80%, 15-85%, 15-90%, 15-95%, 20-30%, 20-35%, 20-40%, 20-45%, 20-50%, 20-55%, 20-60%, 20-65%, 20-70%, 20-75%, 20-80%, 20-85%, 20-90%, 20-95%, 25-35%, 25-40%, 25-45%, 25-50%, 25-55%, 25-60%, 25-65%, 25-70%, 25-75%, 25-80%, 25-85%, 25-90%, 25-95%, 30-40%, 30-45%, 30-50%, 30-55%, 30-60%, 30-65%, 30-70%, 30-75%, 30-80%, 30-85%, 30-90%, 30-95%, 35-45%, 35-50%, 35-55%, 35-60%, 35-65%, 35-70%, 35-75%, 35-80%, 35-85%, 35-90%, 35-95%, 40-50%, 40-55%, 40-60%, 40-65%, 40-70%, 40-75%, 40-80%, 40-85%, 40-90%, 40-95%, 45-55%, 45-60%, 45-65%, 45-70%, 45-75%, 45-80%, 45-85%, 45-90%, 45-95%, 50-60%, 50-65%, 50-70%, 50-75%, 50-80%, 50-85%, 50-90%, 50-95%, 55-65%, 55-70%, 55-75%, 55-80%, 55-85%, 55-90%, 55-95%, 60-70%, 60-75%, 60-80%, 60-85%, 60-90%, 60-95%, 65-75%, 65-80%, 65-85%, 65-90%, 65-95%, 70-80%, 70-85%, 70-90%, 70-95%, 75-85%, 75-90%, 75-95%, 80-90%, 80-95%, or 90-95%.

In one embodiment, the TRACER AAV particles comprising a viral genome with a nucleic acid sequence encoding one or more siRNA molecules may be used to suppress a target protein in DRG and/or sympathetic neurons. The suppression of a target protein in the DRG and/or sympathetic neurons may be, independently, suppressed by 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or more than 95%, 5-15%, 5-20%, 5-25%, 5-30%, 5-35%, 5-40%, 5-45%, 5-50%, 5-55%, 5-60%, 5-65%, 5-70%, 5-75%, 5-80%, 5-85%, 5-90%, 5-95%, 10-20%, 10-25%, 10-30%, 10-35%, 10-40%, 10-45%, 10-50%, 10-55%, 10-60%, 10-65%, 10-70%, 10-75%, 10-80%, 10-85%, 10-90%, 10-95%, 15-25%, 15-30%, 15-35%, 15-40%, 15-45%, 15-50%, 15-55%, 15-60%, 15-65%, 15-70%, 15-75%, 15-80%, 15-85%, 15-90%, 15-95%, 20-30%, 20-35%, 20-40%, 20-45%, 20-50%, 20-55%, 20-60%, 20-65%, 20-70%, 20-75%, 20-80%, 20-85%, 20-90%, 20-95%, 25-35%, 25-40%, 25-45%, 25-50%, 25-55%, 25-60%, 25-65%, 25-70%, 25-75%, 25-80%, 25-85%, 25-90%, 25-95%, 30-40%, 30-45%, 30-50%, 30-55%, 30-60%, 30-65%, 30-70%, 30-75%, 30-80%, 30-85%, 30-90%, 30-95%, 35-45%, 35-50%, 35-55%, 35-60%, 35-65%, 35-70%, 35-75%, 35-80%, 35-85%, 35-90%, 35-95%, 40-50%, 40-55%, 40-60%, 40-65%, 40-70%, 40-75%, 40-80%, 40-85%, 40-90%, 40-95%, 45-55%, 45-60%, 45-65%, 45-70%, 45-75%, 45-80%, 45-85%, 45-90%, 45-95%, 50-60%, 50-65%, 50-70%, 50-75%, 50-80%, 50-85%, 50-90%, 50-95%, 55-65%, 55-70%, 55-75%, 55-80%, 55-85%, 55-90%, 55-95%, 60-70%, 60-75%, 60-80%, 60-85%, 60-90%, 60-95%, 65-75%, 65-80%, 65-85%, 65-90%, 65-95%, 70-80%, 70-85%, 70-90%, 70-95%, 75-85%, 75-90%, 75-95%, 80-90%, 80-95%, or 90-95%. The reduction may be 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or more than 95%, 5-15%, 5-20%, 5-25%, 5-30%, 5-35%, 5-40%, 5-45%, 5-50%, 5-55%, 5-60%, 5-65%, 5-70%, 5-75%, 5-80%, 5-85%, 5-90%, 5-95%, 10-20%, 10-25%, 10-30%, 10-35%, 10-40%, 10-45%, 10-50%, 10-55%, 10-60%, 10-65%, 10-70%, 10-75%, 10-80%, 10-85%, 10-90%, 10-95%, 15-25%, 15-30%, 15-35%, 15-40%, 15-45%, 15-50%, 15-55%, 15-60%, 15-65%, 15-70%, 15-75%, 15-80%, 15-85%, 15-90%, 15-95%, 20-30%, 20-35%, 20-40%, 20-45%, 20-50%, 20-55%, 20-60%, 20-65%, 20-70%, 20-75%, 20-80%, 20-85%, 20-90%, 20-95%, 25-35%, 25-40%, 25-45%, 25-50%, 25-55%, 25-60%, 25-65%, 25-70%, 25-75%, 25-80%, 25-85%, 25-90%, 25-95%, 30-40%, 30-45%, 30-50%, 30-55%, 30-60%, 30-65%, 30-70%, 30-75%, 30-80%, 30-85%, 30-90%, 30-95%, 35-45%, 35-50%, 35-55%, 35-60%, 35-65%, 35-70%, 35-75%, 35-80%, 35-85%, 35-90%, 35-95%, 40-50%, 40-55%, 40-60%, 40-65%, 40-70%, 40-75%, 40-80%, 40-85%, 40-90%, 40-95%, 45-55%, 45-60%, 45-65%, 45-70%, 45-75%, 45-80%, 45-85%, 45-90%, 45-95%, 50-60%, 50-65%, 50-70%, 50-75%, 50-80%, 50-85%, 50-90%, 50-95%, 55-65%, 55-70%, 55-75%, 55-80%, 55-85%, 55-90%, 55-95%, 60-70%, 60-75%, 60-80%, 60-85%, 60-90%, 60-95%, 65-75%, 65-80%, 65-85%, 65-90%, 65-95%, 70-80%, 70-85%, 70-90%, 70-95%, 75-85%, 75-90%, 75-95%, 80-90%, 80-95%, or 90-95%.

In one embodiment, the TRACER AAV particles comprising a viral genome with a nucleic acid sequence encoding one or more siRNA molecules may be used to suppress a target protein in sensory neurons in order to treat neurological disease. Target protein in sensory neurons may be suppressed by 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or more than 95%, 5-15%, 5-20%, 5-25%, 5-30%, 5-35%, 5-40%, 5-45%, 5-50%, 5-55%, 5-60%, 5-65%, 5-70%, 5-75%, 5-80%, 5-85%, 5-90%, 5-95%, 10-20%, 10-25%, 10-30%, 10-35%, 10-40%, 10-45%, 10-50%, 10-55%, 10-60%, 10-65%, 10-70%, 10-75%, 10-80%, 10-85%, 10-90%, 10-95%, 15-25%, 15-30%, 15-35%, 15-40%, 15-45%, 15-50%, 15-55%, 15-60%, 15-65%, 15-70%, 15-75%, 15-80%, 15-85%, 15-90%, 15-95%, 20-30%, 20-35%, 20-40%, 20-45%, 20-50%, 20-55%, 20-60%, 20-65%, 20-70%, 20-75%, 20-80%, 20-85%, 20-90%, 20-95%, 25-35%, 25-40%, 25-45%, 25-50%, 25-55%, 25-60%, 25-65%, 25-70%, 25-75%, 25-80%, 25-85%, 25-90%, 25-95%, 30-40%, 30-45%, 30-50%, 30-55%, 30-60%, 30-65%, 30-70%, 30-75%, 30-80%, 30-85%, 30-90%, 30-95%, 35-45%, 35-50%, 35-55%, 35-60%, 35-65%, 35-70%, 35-75%, 35-80%, 35-85%, 35-90%, 35-95%, 40-50%, 40-55%, 40-60%, 40-65%, 40-70%, 40-75%, 40-80%, 40-85%, 40-90%, 40-95%, 45-55%, 45-60%, 45-65%, 45-70%, 45-75%, 45-80%, 45-85%, 45-90%, 45-95%, 50-60%, 50-65%, 50-70%, 50-75%, 50-80%, 50-85%, 50-90%, 50-95%, 55-65%, 55-70%, 55-75%, 55-80%, 55-85%, 55-90%, 55-95%, 60-70%, 60-75%, 60-80%, 60-85%, 60-90%, 60-95%, 65-75%, 65-80%, 65-85%, 65-90%, 65-95%, 70-80%, 70-85%, 70-90%, 70-95%, 75-85%, 75-90%, 75-95%, 80-90%, 80-95%, or 90-95%. Target protein in the sensory neurons may be reduced may be 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or more than 95%, 5-15%, 5-20%, 5-25%, 5-30%, 5-35%, 5-40%, 5-45%, 5-50%, 5-55%, 5-60%, 5-65%, 5-70%, 5-75%, 5-80%, 5-85%, 5-90%, 5-95%, 10-20%, 10-25%, 10-30%, 10-35%, 10-40%, 10-45%, 10-50%, 10-55%, 10-60%, 10-65%, 10-70%, 10-75%, 10-80%, 10-85%, 10-90%, 10-95%, 15-25%, 15-30%, 15-35%, 15-40%, 15-45%, 15-50%, 15-55%, 15-60%, 15-65%, 15-70%, 15-75%, 15-80%, 15-85%, 15-90%, 15-95%, 20-30%, 20-35%, 20-40%, 20-45%, 20-50%, 20-55%, 20-60%, 20-65%, 20-70%, 20-75%, 20-80%, 20-85%, 20-90%, 20-95%, 25-35%, 25-40%, 25-45%, 25-50%, 25-55%, 25-60%, 25-65%, 25-70%, 25-75%, 25-80%, 25-85%, 25-90%, 25-95%, 30-40%, 30-45%, 30-50%, 30-55%, 30-60%, 30-65%, 30-70%, 30-75%, 30-80%, 30-85%, 30-90%, 30-95%, 35-45%, 35-50%, 35-55%, 35-60%, 35-65%, 35-70%, 35-75%, 35-80%, 35-85%, 35-90%, 35-95%, 40-50%, 40-55%, 40-60%, 40-65%, 40-70%, 40-75%, 40-80%, 40-85%, 40-90%, 40-95%, 45-55%, 45-60%, 45-65%, 45-70%, 45-75%, 45-80%, 45-85%, 45-90%, 45-95%, 50-60%, 50-65%, 50-70%, 50-75%, 50-80%, 50-85%, 50-90%, 50-95%, 55-65%, 55-70%, 55-75%, 55-80%, 55-85%, 55-90%, 55-95%, 60-70%, 60-75%, 60-80%, 60-85%, 60-90%, 60-95%, 65-75%, 65-80%, 65-85%, 65-90%, 65-95%, 70-80%, 70-85%, 70-90%, 70-95%, 75-85%, 75-90%, 75-95%, 80-90%, 80-95%, or 90-95%.

In one embodiment, the TRACER AAV particles comprising a viral genome with a nucleic acid sequence encoding one or more siRNA molecules may be used to suppress a target protein and reduce symptoms of neurological disease in a subject. The suppression of target protein and/or the reduction of symptoms of neurological disease may be, independently, reduced or suppressed by 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or more than 95%, 5-15%, 5-20%, 5-25%, 5-30%, 5-35%, 5-40%, 5-45%, 5-50%, 5-55%, 5-60%, 5-65%, 5-70%, 5-75%, 5-80%, 5-85%, 5-90%, 5-95%, 10-20%, 10-25%, 10-30%, 10-35%, 10-40%, 10-45%, 10-50%, 10-55%, 10-60%, 10-65%, 10-70%, 10-75%, 10-80%, 10-85%, 10-90%, 10-95%, 15-25%, 15-30%, 15-35%, 15-40%, 15-45%, 15-50%, 15-55%, 15-60%, 15-65%, 15-70%, 15-75%, 15-80%, 15-85%, 15-90%, 15-95%, 20-30%, 20-35%, 20-40%, 20-45%, 20-50%, 20-55%, 20-60%, 20-65%, 20-70%, 20-75%, 20-80%, 20-85%, 20-90%, 20-95%, 25-35%, 25-40%, 25-45%, 25-50%, 25-55%, 25-60%, 25-65%, 25-70%, 25-75%, 25-80%, 25-85%, 25-90%, 25-95%, 30-40%, 30-45%, 30-50%, 30-55%, 30-60%, 30-65%, 30-70%, 30-75%, 30-80%, 30-85%, 30-90%, 30-95%, 35-45%, 35-50%, 35-55%, 35-60%, 35-65%, 35-70%, 35-75%, 35-80%, 35-85%, 35-90%, 35-95%, 40-50%, 40-55%, 40-60%, 40-65%, 40-70%, 40-75%, 40-80%, 40-85%, 40-90%, 40-95%, 45-55%, 45-60%, 45-65%, 45-70%, 45-75%, 45-80%, 45-85%, 45-90%, 45-95%, 50-60%, 50-65%, 50-70%, 50-75%, 50-80%, 50-85%, 50-90%, 50-95%, 55-65%, 55-70%, 55-75%, 55-80%, 55-85%, 55-90%, 55-95%, 60-70%, 60-75%, 60-80%, 60-85%, 60-90%, 60-95%, 65-75%, 65-80%, 65-85%, 65-90%, 65-95%, 70-80%, 70-85%, 70-90%, 70-95%, 75-85%, 75-90%, 75-95%, 80-90%, 80-95%, or 90-95%.

In one embodiment, the TRACER AAV particles comprising a viral genome with a nucleic acid sequence encoding one or more siRNA molecules may be used to reduce the decline of functional capacity and activities of daily living as measured by a standard evaluation system such as, but not limited to, the total functional capacity (TFC) scale.

In some embodiments, the present composition is administered as a solo therapeutic or as combination therapeutic for the treatment of neurological disease.

The TRACER AAV particles comprising a viral genome with a nucleic acid sequence encoding one or more siRNA molecules may be used in combination with one or more other therapeutic agents. By “in combination with,” it is not intended to imply that the agents must be administered at the same time and/or formulated for delivery together, although these methods of delivery are within the scope of the present disclosure. Compositions can be administered concurrently with, prior to, or subsequent to, one or more other desired therapeutics or medical procedures. In general, each agent will be administered at a dose and/or on a time schedule determined for that agent.

Therapeutic agents that may be used in combination with the TRACER AAV particles comprising a viral genome with a nucleic acid sequence encoding one or more siRNA molecules can be small molecule compounds which are antioxidants, anti-inflammatory agents, anti-apoptosis agents, calcium regulators, antiglutamatergic agents, structural protein inhibitors, compounds involved in muscle function, and compounds involved in metal ion regulation.

Compounds tested for treating neurological disease which may be used in combination with the TRACER AAV particles comprising a viral genome with a nucleic acid sequence encoding one or more siRNA molecules include, but are not limited to, cholinesterase inhibitors (donepezil, rivastigmine, galantamine), NMDA receptor antagonists such as memantine, anti-psychotics, anti-depressants, anti-convulsants (e.g., sodium valproate and levetiracetam for myoclonus), secretase inhibitors, amyloid aggregation inhibitors, copper or zinc modulators, BACE inhibitors, inhibitors of tau aggregation, such as Methylene blue, phenothiazines, anthraquinones, n-phenylamines or rhodamines, microtubule stabilizers such as NAP, taxol or paclitaxel, kinase or phosphatase inhibitors such as those targeting GSK3β (lithium) or PP2A, immunization with Aβ peptides or tau phospho-epitopes, anti-tau or anti-amyloid antibodies, dopamine-depleting agents (e.g., tetrabenazine for chorea), benzodiazepines (e.g., clonazepam for myoclonus, chorea, dystonia, rigidity, and/or spasticity), amino acid precursors of dopamine (e.g., levodopa for rigidity), skeletal muscle relaxants (e.g., baclofen, tizanidine for rigidity and/or spasticity), inhibitors for acetylcholine release at the neuromuscular junction to cause muscle paralysis (e.g., botulinum toxin for bruxism and/or dystonia), atypical neuroleptics (e.g., olanzapine and quetiapine for psychosis and/or irritability, risperidone, sulpiride and haloperidol for psychosis, chorea and/or irritability, clozapine for treatment-resistant psychosis, aripiprazole for psychosis with prominent negative symptoms), selective serotonin reuptake inhibitors (SSRIs) (e.g., citalopram, fluoxetine, paroxetine, sertraline, mirtazapine, venlafaxine for depression, anxiety, obsessive compulsive behavior and/or irritability), hypnotics (e.g., xopiclone and/or zolpidem for altered sleep-wake cycle), anticonvulsants (e.g., sodium valproate and carbamazepine for mania or hypomania) and mood stabilizers (e.g., lithium for mania or hypomania).

Neurotrophic factors may be used in combination therapy with the TRACER AAV particles comprising a viral genome with a nucleic acid sequence encoding one or more siRNA molecules for treating neurological disease. Generally, a neurotrophic factor is defined as a substance that promotes survival, growth, differentiation, proliferation and/or maturation of a neuron, or stimulates increased activity of a neuron. In some embodiments, the present methods further comprise delivery of one or more trophic factors into the subject in need of treatment. Trophic factors may include, but are not limited to, IGF-I, GDNF, BDNF, CTNF, VEGF, Colivelin, Xaliproden, Thyrotrophin-releasing hormone and ADNF, and variants thereof.

In one aspect, the TRACER AAV particle encoding the nucleic acid sequence for the at least one siRNA duplex targeting the gene of interest may be co-administered with TRACER AAV particles expressing neurotrophic factors such as AAV-IGF-I (See e.g., Vincent et al., Neuromolecular medicine, 2004, 6, 79-85; the content of which is incorporated herein by reference in its entirety) and AAV-GDNF (See e.g., Wang et al., J Neurosci., 2002, 22, 6920-6928; the contents of which are incorporated herein by reference in their entirety).

In one embodiment, administration of the TRACER AAV particles to a subject will reduce the expression of a target protein in a subject and the reduction of expression of the target protein will reduce the effects and/or symptoms of neurological disease in a subject.

DEFINITIONS

Adeno-associated virus: As used herein, the term “adeno-associated virus” or “AAV” refers to members of the Dependovirus genus comprising any particle, sequence, gene, protein, or component derived therefrom.

AAV Particle: As used herein, an “AAV particle” is a virus which comprises a capsid and a viral genome with at least one payload region and at least one ITR. As used herein “AAV particles of the disclosure” are AAV particles comprising a parent capsid sequence with at least one targeting peptide insert. AAV particles of the present disclosure may be produced recombinantly and may be based on adeno-associated virus (AAV) parent or reference sequences. AAV particle may be derived from any serotype, described herein or known in the art, including combinations of serotypes (i.e., “pseudotyped” AAV) or from various genomes (e.g., single stranded or self-complementary). In addition, the AAV particle may be replication defective and/or targeted. In one embodiment, the AAV particle may have a targeting peptide inserted into the capsid to enhance tropism for a desired target tissue. It is to be understood that reference to the AAV particles of the disclosure also includes pharmaceutical compositions thereof, even if not explicitly recited.

Administering: As used herein, the term “administering” refers to providing a pharmaceutical agent or composition to a subject.

Amelioration: As used herein, the term “amelioration” or “ameliorating” refers to a lessening of severity of at least one indicator of a condition or disease. For example, in the context of neurodegeneration disorder, amelioration includes the reduction of neuron loss.

Animal: As used herein, the term “animal” refers to any member of the animal kingdom. In some embodiments, “animal” refers to humans at any stage of development. In some embodiments, “animal” refers to non-human animals at any stage of development. In certain embodiments, the non-human animal is a mammal (e.g., a rodent, a mouse, a rat, a rabbit, a monkey, a dog, a cat, a sheep, cattle, a primate, or a pig). In some embodiments, animals include, but are not limited to, mammals, birds, reptiles, amphibians, fish, and worms. In some embodiments, the animal is a transgenic animal, genetically engineered animal, or a clone.

Antisense strand: As used herein, the term “the antisense strand” or “the first strand” or “the guide strand” of a siRNA molecule refers to a strand that is substantially complementary to a section of about 10-50 nucleotides, e.g., about 15-30, 16-25, 18-23 or 19-22 nucleotides of the mRNA of a gene targeted for silencing. The antisense strand or first strand has sequence sufficiently complementary to the desired target mRNA sequence to direct target-specific silencing, e.g., complementarity sufficient to trigger the destruction of the desired target mRNA by the RNAi machinery or process.

Approximately: As used herein, the term “approximately” or “about,” as applied to one or more values of interest, refers to a value that is similar to a stated reference value. In certain embodiments, the term “approximately” or “about” refers to a range of values that fall within 25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or less in either direction (greater than or less than) of the stated reference value unless otherwise stated or otherwise evident from the context (except where such number would exceed 100% of a possible value).

Capsid: As used herein, the term “capsid” refers to the protein shell of a virus particle.

Complementary and substantially complementary: As used herein, the term “complementary” refers to the ability of polynucleotides to form base pairs with one another. Base pairs are typically formed by hydrogen bonds between nucleotide units in antiparallel polynucleotide strands. 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 persons skilled in the art are aware, when using RNA as opposed to DNA, uracil rather than thymine is the base that is considered to be complementary to adenine. However, when a U is denoted in the context of the present disclosure, the ability to substitute a T is implied, unless otherwise stated. Perfect complementarity or 100% complementarity refers to the situation in which each nucleotide unit of one polynucleotide strand can form a hydrogen bond with a nucleotide unit of a second polynucleotide strand. Less than perfect complementarity refers to the situation in which some, but not all, nucleotide units of two strands can form hydrogen bond with each other. For example, for two 20-mers, if only two base pairs on each strand can form a hydrogen bond with each other, the polynucleotide strands exhibit 10% complementarity. In the same example, if 18 base pairs on each strand can form hydrogen bonds with each other, the polynucleotide strands exhibit 90% complementarity. As used herein, the term “substantially complementary” means that the siRNA has a sequence (e.g., in the antisense strand) which is sufficient to bind the desired target mRNA, and to trigger the RNA silencing of the target mRNA.

Control Elements: As used herein, “control elements”, “regulatory control elements” or “regulatory sequences” refers to promoter regions, polyadenylation signals, transcription termination sequences, upstream regulatory domains, origins of replication, internal ribosome entry sites (“IRES”), enhancers, and the like, which provide for the replication, transcription and translation of a coding sequence in a recipient cell. Not all of these control elements need always be present as long as the selected coding sequence is capable of being replicated, transcribed and/or translated in an appropriate host cell.

Delivery: As used herein, “delivery” refers to the act or manner of delivering an AAV particle, a compound, substance, entity, moiety, cargo or payload.

Element: As used herein, the term “element” refers to a distinct portion of an entity. In some embodiments, an element may be a polynucleotide sequence with a specific purpose, incorporated into a longer polynucleotide sequence.

Encapsulate: As used herein, the term “encapsulate” means to enclose, surround or encase. As an example, a capsid protein often encapsulates a viral genome.

Engineered: As used herein, embodiments of the disclosure are “engineered” when they are designed to have a feature or property, whether structural or chemical, that varies from a starting point, wild type or native molecule.

Effective Amount: As used herein, the term “effective amount” of an agent is that amount sufficient to effect beneficial or desired results, for example, clinical results, and, as such, an “effective amount” depends upon the context in which it is being applied. For example, in the context of administering an agent that treats cancer, an effective amount of an agent is, for example, an amount sufficient to achieve treatment, as defined herein, of cancer, as compared to the response obtained without administration of the agent.

Expression: As used herein, “expression” of a nucleic acid sequence refers to one or more of the following events: (1) production of an RNA template from a DNA sequence (e.g., by transcription); (2) processing of an RNA transcript (e.g., by splicing, editing, 5′ cap formation, and/or 3′ end processing); (3) translation of an RNA into a polypeptide or protein; and (4) post-translational modification of a polypeptide or protein.

Feature: As used herein, a “feature” refers to a characteristic, a property, or a distinctive element.

Formulation: As used herein, a “formulation” includes at least one AAV particle (active ingredient) and an excipient, and/or an inactive ingredient.

Fragment: A “fragment,” as used herein, refers to a portion. For example, an antibody fragment may comprise a CDR, or a heavy chain variable region, or a scFv, etc.

Functional: As used herein, a “functional” biological molecule is a biological molecule in a form in which it exhibits a property and/or activity by which it is characterized.

Gene expression: The term “gene expression” refers to the process by which a nucleic acid sequence undergoes successful transcription and in most instances translation to produce a protein or peptide. For clarity, when reference is made to measurement of “gene expression”, this should be understood to mean that measurements may be of the nucleic acid product of transcription, e.g., RNA or mRNA or of the amino acid product of translation, e.g., polypeptides or peptides. Methods of measuring the amount or levels of RNA, mRNA, polypeptides and peptides are well known in the art.

Homology: As used herein, the term “homology” refers to the overall relatedness between polymeric molecules, e.g. between polynucleotide molecules (e.g. DNA molecules and/or RNA molecules) and/or between polypeptide molecules. In some embodiments, polymeric molecules are considered to be “homologous” to one another if their sequences are at least 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% identical or similar. The term “homologous” necessarily refers to a comparison between at least two sequences (polynucleotide or polypeptide sequences). In accordance with the disclosure, two polynucleotide sequences are considered to be homologous if the polypeptides they encode are at least about 50%, 60%, 70%, 80%, 90%, 95%, or even 99% for at least one stretch of at least about 20 amino acids. In some embodiments, homologous polynucleotide sequences are characterized by the ability to encode a stretch of at least 4-5 uniquely specified amino acids. For polynucleotide sequences less than 60 nucleotides in length, homology is determined by the ability to encode a stretch of at least 4-5 uniquely specified amino acids. In accordance with the disclosure, two protein sequences are considered to be homologous if the proteins are at least about 50%, 60%, 70%, 80%, or 90% identical for at least one stretch of at least about 20 amino acids.

Identity: As used herein, the term “identity” refers to the overall relatedness between polymeric molecules, e.g., between polynucleotide molecules (e.g. DNA molecules and/or RNA molecules) and/or between polypeptide molecules. Calculation of the percent identity of two polynucleotide sequences, for example, can be performed by aligning the two sequences for optimal comparison purposes (e.g., gaps can be introduced in one or both of a first and a second nucleic acid sequences for optimal alignment and non-identical sequences can be disregarded for comparison purposes). In certain embodiments, the length of a sequence aligned for comparison purposes is at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or 100% of the length of the reference sequence. The nucleotides at corresponding nucleotide positions are then compared. When a position in the first sequence is occupied by the same nucleotide as the corresponding position in the second sequence, then the molecules are identical at that position. The percent identity between the two sequences is a function of the number of identical positions shared by the sequences, taking into account the number of gaps, and the length of each gap, which needs to be introduced for optimal alignment of the two sequences. The comparison of sequences and determination of percent identity between two sequences can be accomplished using a mathematical algorithm. For example, the percent identity between two nucleotide sequences can be determined using methods such as those described in Computational Molecular Biology, Lesk, A. M., ed., Oxford University Press, New York, 1988; Biocomputing: Informatics and Genome Projects, Smith, D. W., ed., Academic Press, New York, 1993; Sequence Analysis in Molecular Biology, von Heinje, G., Academic Press, 1987; Computer Analysis of Sequence Data, Part I, Griffin, A. M., and Griffin, H. G., eds., Humana Press, New Jersey, 1994; and Sequence Analysis Primer, Gribskov, M. and Devereux, J., eds., M Stockton Press, New York, 1991; the contents of each of which are incorporated herein by reference in their entirety. For example, the percent identity between two nucleotide sequences can be determined using the algorithm of Meyers and Miller (CABIOS, 1989, 4:11-17), which has been incorporated into the ALIGN program (version 2.0) using a PAM120 weight residue table, a gap length penalty of 12 and a gap penalty of 4. The percent identity between two nucleotide sequences can, alternatively, be determined using the GAP program in the GCG software package using an NWSgapdna.CMP matrix. Methods commonly employed to determine percent identity between sequences include, but are not limited to those disclosed in Carillo, H., and Lipman, D., SIAM J Applied Math., 48:1073 (1988); incorporated herein by reference. Techniques for determining identity are codified in publicly available computer programs. Exemplary computer software to determine homology between two sequences include, but are not limited to, GCG program package, Devereux, J., et al., Nucleic Acids Research, 12(1), 387 (1984)), BLASTP, BLASTN, and FASTA Altschul, S. F. et al., J. Molec. Biol., 215, 403 (1990)).

Inhibit expression of a gene: As used herein, the phrase “inhibit expression of a gene” means to cause a reduction in the amount of an expression product of the gene. The expression product can be an RNA 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.

Insert: As used herein the term “insert” may refer to the addition of a targeting peptide sequence to a parent AAV capsid sequence. An “insertion” may result in the replacement of one or more amino acids of the parent AAV capsid sequence. Alternatively, an insertion may result in no changes to the parent AAV capsid sequence beyond the addition of the targeting peptide sequence.

Inverted terminal repeat: As used herein, the term “inverted terminal repeat” or “ITR” refers to a cis-regulatory element for the packaging of polynucleotide sequences into viral capsids.

Library: As used herein, the term “library” refers to a diverse collection of linear polypeptides, polynucleotides, viral particles, or viral vectors. As examples, a library may be a DNA library or an AAV capsid library.

Neurological disease: As used herein, a “neurological disease” is any disease associated with the central or peripheral nervous system and components thereof (e.g., neurons).

Naturally Occurring: As used herein, “naturally occurring” or “wild-type” means existing in nature without artificial aid, or involvement of the hand of man.

Open reading frame: As used herein, “open reading frame” or “ORF” refers to a sequence which does not contain a stop codon in a given reading frame.

Parent sequence: As used herein, a “parent sequence” is a nucleic acid or amino acid sequence from which a variant is derived. In one embodiment, a parent sequence is a sequence into which a heterologous sequence is inserted. In other words, a parent sequence may be considered an acceptor or recipient sequence. In one embodiment, a parent sequence is an AAV capsid sequence into which a targeting sequence is inserted.

Particle: As used herein, a “particle” is a virus comprised of at least two components, a protein capsid and a polynucleotide sequence enclosed within the capsid.

Patient: As used herein, “patient” refers to a subject who may seek or be in need of treatment, requires treatment, is receiving treatment, will receive treatment, or a subject who is under care by a trained professional for a particular disease or condition.

Payload region: As used herein, a “payload region” is any nucleic acid sequence (e.g., within the viral genome) which encodes one or more “payloads” of the disclosure. As non-limiting examples, a payload region may be a nucleic acid sequence within the viral genome of an AAV particle, which encodes a payload, wherein the payload is an RNAi agent or a polypeptide. Payloads of the present disclosure may be, but are not limited to, peptides, polypeptides, proteins, antibodies, RNAi agents, etc.

Peptide: As used herein, “peptide” is less than or equal to 50 amino acids long, e.g., about 5, 10, 15, 20, 25, 30, 35, 40, 45, or 50 amino acids long.

Pharmaceutically acceptable: The phrase “pharmaceutically acceptable” is employed herein to refer to those compounds, materials, compositions, and/or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit/risk ratio.

Preventing: As used herein, the term “preventing” or “prevention” refers to partially or completely delaying onset of an infection, disease, disorder and/or condition; partially or completely delaying onset of one or more symptoms, features, or clinical manifestations of a particular infection, disease, disorder, and/or condition; partially or completely delaying onset of one or more symptoms, features, or manifestations of a particular infection, disease, disorder, and/or condition; partially or completely delaying progression from an infection, a particular disease, disorder and/or condition; and/or decreasing the risk of developing pathology associated with the infection, the disease, disorder, and/or condition.

Prophylactic: As used herein, “prophylactic” refers to a therapeutic or course of action used to prevent the spread of disease.

Prophylaxis: As used herein, a “prophylaxis” refers to a measure taken to maintain health and prevent the spread of disease.

Region: As used herein, the term “region” refers to a zone or general area. In some embodiments, when referring to a protein or protein module, a region may comprise a linear sequence of amino acids along the protein or protein module or may comprise a three-dimensional area, an epitope and/or a cluster of epitopes. In some embodiments, regions comprise terminal regions. As used herein, the term “terminal region” refers to regions located at the ends or termini of a given agent. When referring to proteins, terminal regions may comprise N- and/or C-termini.

In some embodiments, when referring to a polynucleotide, a region may comprise a linear sequence of nucleic acids along the polynucleotide or may comprise a three-dimensional area, secondary structure, or tertiary structure. In some embodiments, regions comprise terminal regions. As used herein, the term “terminal region” refers to regions located at the ends or termini of a given agent. When referring to polynucleotides, terminal regions may comprise 5′ and/or 3′ termini.

RNA or RNA molecule: As used herein, the term “RNA” or “RNA molecule” or “ribonucleic acid molecule” refers to a polymer of ribonucleotides; the term “DNA” or “DNA molecule” or “deoxyribonucleic acid molecule” refers to a polymer of deoxyribonucleotides. DNA and RNA can be synthesized naturally, e.g., by DNA replication and transcription of DNA, respectively; or be chemically synthesized. DNA and RNA can be single-stranded (i.e., ssRNA or ssDNA, respectively) or multi-stranded (e.g., double stranded, i.e., dsRNA and dsDNA, respectively). The term “mRNA” or “messenger RNA”, as used herein, refers to a single stranded RNA that encodes the amino acid sequence of one or more polypeptide chains.

RNA interfering or RNAi: As used herein, the term “RNA interfering” or “RNAi” refers to a sequence specific regulatory mechanism mediated by RNA molecules which results in the inhibition or interfering or “silencing” of the expression of a corresponding protein-coding gene. RNAi has been observed in many types of organisms, including plants, animals and fungi. RNAi occurs in cells naturally to remove foreign RNAs (e.g., viral RNAs). Natural RNAi proceeds via fragments cleaved from free dsRNA which direct the degradative mechanism to other similar RNA sequences. RNAi is controlled by the RNA-induced silencing complex (RISC) and is initiated by short/small dsRNA molecules in cell cytoplasm, where they interact with the catalytic RISC component argonaute. The dsRNA molecules can be introduced into cells exogenously. Exogenous dsRNA initiates RNAi by activating the ribonuclease protein Dicer, which binds and cleaves dsRNAs to produce double-stranded fragments of 21-25 base pairs with a few unpaired overhang bases on each end. These short double stranded fragments are called small interfering RNAs (siRNAs).

RNAi agent: As used herein, the term “RNAi agent” refers to an RNA molecule, or its derivative, that can induce inhibition, interfering, or “silencing” of the expression of a target gene and/or its protein product. An RNAi agent may knock-out (virtually eliminate or eliminate) expression, or knock-down (lessen or decrease) expression. The RNAi agent may be, but is not limited to, dsRNA, siRNA, shRNA, pre-miRNA, pri-miRNA, miRNA, stRNA, lncRNA, piRNA, or snoRNA.

Sample: As used herein, the term “sample” or “biological sample” refers to a subset of its tissues, cells or component parts (e.g. body fluids, including but not limited to blood, serum, mucus, lymphatic fluid, synovial fluid, cerebrospinal fluid, saliva, amniotic fluid, amniotic cord blood, urine, vaginal fluid and semen). A sample further may include a homogenate, lysate or extract prepared from a whole organism or a subset of its tissues, cells or component parts, or a fraction or portion thereof, including but not limited to, for example, plasma, serum, spinal fluid, lymph fluid, the external sections of the skin, respiratory, intestinal, and genitourinary tracts, tears, saliva, milk, blood cells, tumors, organs. A sample further refers to a medium, such as a nutrient broth or gel, which may contain cellular components, such as proteins or nucleic acid molecule.

Self-complementary viral particle: As used herein, a “self-complementary viral particle” is a particle comprised of at least two components, a protein capsid and a self-complementary viral genome enclosed within the capsid.

Sense Strand: As used herein, the term “the sense strand” or “the second strand” or “the passenger strand” of a siRNA molecule refers to a strand that is complementary to the antisense strand or first strand. The antisense and sense strands of a siRNA molecule are hybridized to form a duplex structure. As used herein, a “siRNA duplex” includes a siRNA strand having sufficient complementarity to a section of about 10-50 nucleotides of the mRNA of the gene targeted for silencing and a siRNA strand having sufficient complementarity to form a duplex with the other siRNA strand.

Similarity: As used herein, the term “similarity” refers to the overall relatedness between polymeric molecules, e.g. between polynucleotide molecules (e.g. DNA molecules and/or RNA molecules) and/or between polypeptide molecules. Calculation of percent similarity of polymeric molecules to one another can be performed in the same manner as a calculation of percent identity, except that calculation of percent similarity takes into account conservative substitutions as is understood in the art.

Short interfering RNA or siRNA: As used herein, the terms “short interfering RNA,” “small interfering RNA” or “siRNA” refer to an RNA molecule (or RNA analog) comprising between about 5-60 nucleotides (or nucleotide analogs) which is capable of directing or mediating RNAi. Preferably, a siRNA molecule comprises between about 15-30 nucleotides or nucleotide analogs, such as between about 16-25 nucleotides (or nucleotide analogs), between about 18-23 nucleotides (or nucleotide analogs), between about 19-22 nucleotides (or nucleotide analogs) (e.g., 19, 20, 21 or 22 nucleotides or nucleotide analogs), between about 19-25 nucleotides (or nucleotide analogs), and between about 19-24 nucleotides (or nucleotide analogs). The term “short” siRNA refers to a siRNA comprising 5-23 nucleotides, preferably 21 nucleotides (or nucleotide analogs), for example, 19, 20, 21 or 22 nucleotides. The term “long” siRNA refers to a siRNA comprising 24-60 nucleotides, preferably about 24-25 nucleotides, for example, 23, 24, 25 or 26 nucleotides. Short siRNAs may, in some instances, include fewer than 19 nucleotides, e.g., 16, 17 or 18 nucleotides, or as few as 5 nucleotides, provided that the shorter siRNA retains the ability to mediate RNAi. Likewise, long siRNAs may, in some instances, include more than 26 nucleotides, e.g., 27, 28, 29, 30, 35, 40, 45, 50, 55, or even 60 nucleotides, provided that the longer siRNA retains the ability to mediate RNAi or translational repression absent further processing, e.g., enzymatic processing, to a short siRNA. siRNAs can be single stranded RNA molecules (ss-siRNAs) or double stranded RNA molecules (ds-siRNAs) comprising a sense strand and an antisense strand which hybridized to form a duplex structure called an siRNA duplex.

Subject: As used herein, the term “subject” or “patient” refers to any organism to which a composition in accordance with the disclosure may be administered, e.g., for experimental, diagnostic, prophylactic, and/or therapeutic purposes. Typical subjects include animals (e.g., mammals such as mice, rats, rabbits, non-human primates, and humans) and/or plants.

Substantially: As used herein, the term “substantially” refers to the qualitative condition of exhibiting total or near-total extent or degree of a characteristic or property of interest. One of ordinary skill in the biological arts will understand that biological and chemical phenomena rarely, if ever, go to completion and/or proceed to completeness or achieve or avoid an absolute result. The term “substantially” is therefore used herein to capture the potential lack of completeness inherent in many biological and chemical phenomena.

Targeting peptide: As used herein, a “targeting peptide” refers to a peptide of 3-20 amino acids in length. These targeting peptides may be inserted into, or attached to, a parent amino acid sequence to alter the characteristics (e.g., tropism) of the parent protein. As a non-limiting example, the targeting peptide can be inserted into an AAV capsid sequence for enhanced targeting to a desired cell-type, tissue, organ or organism. It is to be understood that a targeting peptide is encoded by a targeting polynucleotide which may similarly be inserted into a parent polynucleotide sequence. Therefore, a “targeting sequence” refers to a peptide or polynucleotide sequence for insertion into an appropriate parent sequence (amino acid or polynucleotide, respectively).

Target Cells: As used herein, “target cells” or “target tissue” refers to any one or more cells of interest. The cells may be found in vitro, in vivo, in situ or in the tissue or organ of an organism. The organism may be an animal, preferably a mammal, more preferably a human and most preferably a patient.

Therapeutic Agent: The term “therapeutic agent” refers to any agent that, when administered to a subject, has a therapeutic, diagnostic, and/or prophylactic effect and/or elicits a desired biological and/or pharmacological effect.

Therapeutically effective amount: As used herein, the term “therapeutically effective amount” means an amount of an agent to be delivered (e.g., nucleic acid, drug, therapeutic agent, diagnostic agent, prophylactic agent, etc.) that is sufficient, when administered to a subject suffering from or susceptible to an infection, disease, disorder, and/or condition, to treat, improve symptoms of, diagnose, prevent, and/or delay the onset of the infection, disease, disorder, and/or condition. In some embodiments, a therapeutically effective amount is provided in a single dose.

Therapeutically effective outcome: As used herein, the term “therapeutically effective outcome” means an outcome that is sufficient in a subject suffering from or susceptible to an infection, disease, disorder, and/or condition, to treat, improve symptoms of, diagnose, prevent, and/or delay the onset of the infection, disease, disorder, and/or condition.

Treating: As used herein, the term “treating” refers to partially or completely alleviating, ameliorating, improving, relieving, delaying onset of, inhibiting progression of, reducing severity of, and/or reducing incidence of one or more symptoms or features of a particular infection, disease, disorder, and/or condition. For example, “treating” cancer may refer to inhibiting survival, growth, and/or spread of a tumor. Treatment may be administered to a subject who does not exhibit signs of a disease, disorder, and/or condition and/or to a subject who exhibits only early signs of a disease, disorder, and/or condition for the purpose of decreasing the risk of developing pathology associated with the disease, disorder, and/or condition.

Vector: As used herein, the term “vector” refers to any molecule or moiety which transports, transduces or otherwise acts as a carrier of a heterologous molecule. In some embodiments, vectors may be plasmids. In some embodiments, vectors may be viruses. An AAV particle is an example of a vector. Vectors of the present disclosure may be produced recombinantly and may be based on and/or may comprise adeno-associated virus (AAV) parent or reference sequences. The heterologous molecule may be a polynucleotide and/or a polypeptide.

Viral Genome: As used herein, the terms “viral genome” or “vector genome” refer to the nucleic acid sequence(s) encapsulated in an AAV particle. A viral genome comprises a nucleic acid sequence with at least one payload region encoding a payload and at least one ITR.

Equivalents and Scope

Those skilled in the art will recognize or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments in accordance with the disclosure described herein. The scope of the present disclosure is not intended to be limited to the above Description, but rather is as set forth in the appended claims.

In the claims, articles such as “a,” “an,” and “the” may mean one or more than one unless indicated to the contrary or otherwise evident from the context. Claims or descriptions that include “or” between one or more members of a group are considered satisfied if one, more than one, or all of the group members are present in, employed in, or otherwise relevant to a given product or process unless indicated to the contrary or otherwise evident from the context. The disclosure includes embodiments in which exactly one member of the group is present in, employed in, or otherwise relevant to a given product or process. The disclosure includes embodiments in which more than one, or the entire group members are present in, employed in, or otherwise relevant to a given product or process.

It is also noted that the term “comprising” is intended to be open and permits but does not require the inclusion of additional elements or steps. When the term “comprising” is used herein, the term “consisting of” is thus also encompassed and disclosed.

Where ranges are given, endpoints are included. Furthermore, it is to be understood that unless otherwise indicated or otherwise evident from the context and understanding of one of ordinary skill in the art, values that are expressed as ranges can assume any specific value or subrange within the stated ranges in different embodiments of the disclosure, to the tenth of the unit of the lower limit of the range, unless the context clearly dictates otherwise.

In addition, it is to be understood that any particular embodiment of the present disclosure that falls within the prior art may be explicitly excluded from any one or more of the claims. Since such embodiments are deemed to be known to one of ordinary skill in the art, they may be excluded even if the exclusion is not set forth explicitly herein. Any particular embodiment of the compositions of the disclosure (e.g., any antibiotic, therapeutic or active ingredient; any method of production; any method of use; etc.) can be excluded from any one or more claims, for any reason, whether or not related to the existence of prior art.

It is to be understood that the words which have been used are words of description rather than limitation, and that changes may be made within the purview of the appended claims without departing from the true scope and spirit of the disclosure in its broader aspects.

While the present disclosure has been described at some length and with some particularity with respect to the several described embodiments, it is not intended that it should be limited to any such particulars or embodiments or any particular embodiment, but it is to be construed with references to the appended claims so as to provide the broadest possible interpretation of such claims in view of the prior art and, therefore, to effectively encompass the intended scope of the disclosure.

The present disclosure is further illustrated by the following non-limiting examples.

EXAMPLES

Example 1. TRACER Proof of Concept: Promoter Selection

Proof-of-concept experiments were conducted by placing the genes encoding an AAV9 peptide display capsid library under the control of either the neuron-specific synapsin promoter (SYN) or the astrocyte-specific GFAP promoter. Following intravenous administration to C57BL/6 mice, RNA was recovered from brain tissue and used for further library evolution. Next-generation sequencing (NGS) showed sequence convergence between animals after only two rounds of selection. Interestingly, several variants highly similar to the PHP.eB capsid were recovered, suggesting that our method allowed a rapid selection of high-performance capsids. A subset of capsids having peptide sequences with high CNS enrichment was selected for further study. It is understood that any promoter may be selected depending on the desired tropism. Examples of such promoters are found in Table 3.

TABLE 3
Promoters, tissue and cell type
Promoter name	Tissue	Cell type
B29 promoter	Blood	B cells
Immunoglobulin heavy chain	Blood	B cells
promoter
CD45 promoter	Blood	Hematopoietic
Mouse INF-β promoter	Blood	Hematopoietic
CD45 SV40/CD45 promoter	Blood	Hematopoietic
WASP promoter	Blood	Hematopoietic
CD43 promoter	Blood	Leuko & Platelets
CD43 SV40/CD43 promoter	Blood	Leuko & Platelets
CD68 promoter	Blood	Macrophages
GPIIb promoter	Blood	Megakaryocyte
CD14 promoter	Blood	Monocytes
CD2 promoter	Blood	T cells
Osteocalcin	Bone	Osteoblasts
Bone sialoprotein	Bone	Osteoblasts
OG-2 promoter	Bone	Osteoblasts, odontoblasts
GFAP promoter	Brain	Astrocytes
Vga	Brain	GABAergic neurons
Vglut2	Brain	glutamatergic neurons
NSE/RU5′ promoter	Brain	Neurons
SYN1 promoter	Brain	Neurons
Neurofilament light chain	Brain	Neurons
VGF	Brain	Neurons
Nestin	Brain	NSC
Chx10	Eye	All retinal neurons
PrP	Eye	All retinal neurons
Dkk3	Eye	All retinal neurons
Math5	Eye	Amacrine and horizontal
		cells
Ptf1a	Eye	Amacrine and horizontal
		cells
Pcp2	Eye	Bipolar cells
Nefh	Eye	Ganglion cells
gamma-synuclein gene	Eye	ganglion cells
(SNCG)
Grik4	Eye	GC
Pdgfra	Eye	GC and ONL Müller cells
Chat	Eye	GC/Amacrine cells
Thy 1.2	Eye	GC/neural retina
hVmd2	Eye	INL Müller cells
Thy 1	Eye	INL Müller cells
Modified αA-crystallin	Eye	Lens/neural retina
hRgp	Eye	M- and S-cone
mMo	Eye	M-cone
Opn4	Eye	Melanopsin-expressing GC
RLBP1	Eye	Muller cells
Glast	Eye	Müller cells
Foxg1	Eye	Müller cells
hVmd2	Eye	Müller cells/optic nerve/
		INL
Trp1	Eye	Neural retina
Six3	Eye	Neural retina
cx36	Eye	Neurons
Grm6 - SV40 eukaryotic	Eye	ON bipolar
promoter
hVmd2	Eye	Optic nerve
Dct	Eye	Pigmented cells
Rpc65	Eye	Retinal pigment epithelium
mRho	Eye	Rod
Irbp	Eye	Rod
hRho	Eye	Rod
Pcp2	Eye	Rod bipolar cells
Rhodopsin	Eye	Rod Photoreceptors
mSo	Eye	S-cone
MLC2v promoter	Heart	Cardiomyocyte
αMHC promoter	Heart	Cardiomyocyte
rat troponin T (Tnnt2)	Heart	Cardiomyocyte
Tie2	Heart	Endothelial
Tcf21	Heart	Fibroblasts
ECAD	Kidney	Collecting duct
NKCC2	Kidney	Loop of Henle
KSPC	Kidney	Nephron
NPHS1	Kidney	Podocyte
SGLT2	Kidney	Proximal tubular cells
SV40/bAlb promoter	Liver	hepatocytes
SV40/hAlb promoter	Liver	hepatocytes
Hepatitis B virus core	Liver	hepatocytes
promoter
Alpha fetoprotein	Liver	hepatocytes
Surfactant protein B promoter	Lung	AT II cells and Clara cells
Surfactant protein C promoter	Lung	AT II cells and Clara cells
Desmin	Muscle	Muscle stem cells +
		Myocytes
Mb promoter	Muscle	Myocyte
Myosin	Muscle	Myocyte
Dystrophin	Muscle	Myocyte
dMCK and tMCK	Muscle	Myocytes
Elastase-1 promoter	Pancreas	Acinar cells
PDX1 promoter	Pancreas	Beta cells
Insulin promoter	Pancreas	langherans
Slco1c1	Vasculature	BBB Endothelial
tie	Vasculature	Endothelial
cadherin	Vasculature	Endothelial
ICAM-2	Vasculature	Endothelial
claudin 1	Vasculature	Endothelial
Cldn5	Vasculature	Endothelial
Flt-1 promoter	Vasculature	Endothelial
Endoglin promoter	Vasculature	Endothelial

Capsid pools were injected to three rodent species, followed by RNA enrichment analysis for characterization of transduction efficiency in neurons or astrocytes and cross-species performance. Top-ranking capsids were then individually tested and several variants showed CNS transduction similar to or higher than the PHP.eB benchmark. These results suggest that the TRACER platform allows rapid in vivo evolution of AAV capsids in non-transgenic animals with a high degree of tropism improvement. The following examples illustrate the findings in more detail.

Example 2. Generation of an AAV Vectors Capable of Capsid mRNA Expression in the Absence of Helper Virus

In order to perform cell type- and transduction-restricted in vivo evolution of AAV capsid libraries, a capsid library system was engineered in which the capsid mutant gene can be transcribed in the absence of a helper virus, in a specific cell type. In the wild-type AAV virus, the mRNA encoding the capsid proteins VP1, VP2 and VP3, as well as the AAP accessory protein, are expressed by the P40 promoter located in the 3′ region of the REP gene (FIG. 1A), that is only active in the presence of the REP protein as well as the helper virus functions (Berns et al., 1996). In order to allow expression of the capsid mRNA in animal tissue or in cultured cells, another promoter must be inserted upstream or downstream of the CAP gene. Because of the limited packaging capacity of the AAV capsid, a portion of the REP gene must be deleted to accommodate the extra promoter insertion, and the REP gene has to be provided in trans by another plasmid to allow virus production. The minimal viral sequence required for high titer AAV production was determined by introducing a CMV promoter at various locations upstream of the CAP gene of AAV9 (FIG. 1B). The REP protein was provided in trans by the pREP2 plasmid obtained by deleting the CAP gene from a REP2-CAP2 packaging vector using EcoNI and ClaI (SEQ. ID NO:4). For small-scale virus production test, HEK-293T cells grown in DMEM supplemented with 5% FBS and 1× pen/strep were plated in 15-cm dishes and co-transfected with 15 ug of pHelper (pFdelta6) plasmid, 10 ug pREP2 plasmid and lug ITR-CMV-CAP plasmid using calcium phosphate transfection. After 72 hours, cells were harvested by scraping, pelleted by a brief centrifugation and suspended in 1 ml of a buffer containing 10 mM Tris and 2 mM MgCl2. Cells were lysed by addition of triton X-100 to 0.1% final concentration and treated with 50U of benzonase for 1 hour. Virus from the supernatants was precipitated with 8% polyethylene glycol and 0.5M NaCl, suspended in 1 ml of 10 mM TRIS-2mM MgCl2 and combined with the cell lysate. The pooled virus was adjusted to 0.5M NaCl, cleared by centrifugation for 15 minutes at 4,000×g and fractionated on a step iodixanol gradient of 15%, 25%, 40% and 60% for 3 hours at 40,000prm (Zolotukhin et al., 1999). The 40% fraction containing the purified AAV particles was harvested and viral titers were measured by real-time PCR using a Taqman primer/probe mix specific for the 3′-end of REP, shared by all the constructs. Virus yields were significantly lower than the fully wild-type ITR-REP2-CAP9-ITR used as a reference (1.7% to 8.8%), but the CMV-BstEII construct allowed the highest yields of all three CMV constructs. See FIG. 2. The CMV-HindIII construct, in which most of the P40 promoter sequence is deleted, generated the lowest yield (1.7% of wtAAV9), indicating that even the potent CMV promoter cannot replace the P40 promoter without a severe drop in virus yields. Following these observations, the BstEII architecture (SEQ. ID NO:5), which preserves the minimal P40 sequence and the CAP mRNA splice donor, was used in all further experiments.

The REP-expressing plasmid was then improved by preserving the AAP reading frame together with a large portion of the capsid gene from the REP2-CAP9 helper vector, which may contain sequences necessary for the regulation of CAP transcription and/or splicing. In order to eliminate the capsid coding potential of the vector, a C-terminus fragment of the capsid gene was deleted by a triple cut with the MscI restriction enzyme followed by self-ligation, in order to obtain the pREP-AAP plasmid (FIG. 3A, SEQ. ID NO:6).

An iteration of this construct was engineered by introducing premature stop codons immediately after the start codons of VP1, VP2 and VP3, without perturbing the amino acid sequence of the colinear AAP reading frame (FIG. 3A). This construct was named pREP-3stop (SEQ. ID NO:7). A neuron-specific syn-CAPS vector (SEQ. ID NO:8) was derived from the CMV9-BstEII plasmid by swapping the CMV promoter with the neuron-specific human synapsin 1 promoter.

Production efficiency of this Syn-CAPS was tested as described previously using pREP, pREP-AAP or pREP-3stop plasmid to supply REP in trans. As shown in FIG. 3B, the REP plasmids harboring a longer capsid sequence as well as AAP increased virus yields by approximately 3-fold compared to the pREP plasmid. Virus titers obtained with the pREP-AAP or pREP-3stop vectors reached ˜30% of wild-type AAV9. An important concern with plasmids harboring long homologous regions is the potential for unwanted recombination with the ITR-CAP vector, that would reconstitute a wild-type ITR-REP-CAP vector and contaminate combinatorial libraries.

To evaluate the risk of wild-type virus reconstitution, the viral preparations obtained in FIG. 3B were subjected to real-time PCR with a Taqman probe located in the N terminus of REP. The percentage of capsids containing a detectable full-length REP was less than 0.03% of wild-type virus (FIG. 3C), which was even lower than the routinely detected 0.1% illegitimate REP-CAP packaging occurring in most recombinant AAV preparations obtained from 293T cell transfection (FIG. 3C, our unpublished observations). Because the premature stop codons of the pREP-3 stop vector offer an extra layer of safety against potential reconstitution of wild-type capsids and prevents the translation of truncated capsid proteins, the 3stop plasmid was used for all subsequent studies.

Following this, the feasibility of RNA-driven biopanning in C57BL/6 mice using AAV9-packaged vectors where the AAV9 capsid gene is driven by the CMV promoter, the Synapsin promoter or the astrocyte-specific GFabc1D promoter (SEQ. ID NO:9), thereafter referred to as GFAP promoter (Brenner et al., 2008) was tested (FIG. 4A). The three vectors were produced in HEK-293T cells as previously described and analyzed by PAGE-silver stain. As shown in FIG. 4B, all vectors showed a normal ratio of VP1, VP2 and VP3 capsid proteins, indicating that the particular promoter architecture does not disrupt the balance of capsid protein expression. Six-week old male C57BL/6 mice were injected intravenously with 1e12 VG per mouse and sacrificed after 28 days. DNA biodistribution and capsid mRNA expression were tested in the brain, liver and heart tissues.

Total DNA was extracted from brain, liver and heart tissues using Qiagen DNeasy Blood and Tissue columns, and viral DNA was quantified by real-time PCR using a Taqman probe located in the VP3 N-terminal region. DNA abundance was normalized using a pre-designed probe detecting the single-copy transferrin receptor gene (Life Technologies ref. 4458366). Viral DNA was highly abundant in the liver and to a lower extent in the heart. The DNA distribution did not show any noticeable difference between the three vectors (FIG. 4C). RNA was extracted with Qiagen RNeasy plus universal kit following manufacturer's instructions, then treated with ezDNAse (Qiagen) to remove residual DNA, and reverse transcribed with Superscript IV (Life technologies).

RNA expression was evaluated using the same VP3 probe used to quantify viral DNA and normalized using TBP as a reference RNA (Life technologies Mm01277042 m1). In the brain, the GFAP promoter allowed the strongest expression level, and the Synapsin promoter allowed a comparable expression as the potent CMV promoter. In the liver, all promoters resulted in a similar expression level, which could be the result of a leaky expression at very high copy number (FIG. 4D). In the heart, the cell type specificity of the Syn and GFAP promoters was evident, since they allowed only ˜3 and 10% of CMV expression, respectively despite of a similar DNA biodistribution.

Overall the experiment showed that mRNA from transduction-competent capsids could be recovered from various animal organs, including weakly transduced tissues such as the brain.

Example 3. AAV Vector Configuration

Various vector configurations were explored toward increasing RNA expression to maximize library recovery. The CMV promoter was replaced by a hybrid CMV enhancer/Chicken beta-actin promoter sequence (Niwa et al., 1991) and a potent cytomegalovirus-beta-globin hybrid intron derived from the AAV-MCS cloning vector (Stratagene) was inserted between the promoter sequence and the capsid gene, as introns have been shown to increase mRNA processing and stability (Powell et al., 2015). This resulted in the constructs CAG9 (SEQ. ID NO:10), SYNG9 (SEQ. ID NO:11) and GFAPG (SEQ. ID NO:12).

An inverted vector configuration was also tested where the helper-independent promoter was placed downstream of the capsid gene in reverse orientation, in order to avoid potential interference with the P40 promoter (FIG. 5A). This configuration allows the expression of an antisense capsid transcript in animal tissue. Because most polyadenylation signals (AATAAA) are orientation-dependent, it was hypothesized that the natural AAV capsid polyA would not prematurely terminate transcription when placed in reverse orientation. All constructs were co-transfected with pHelper and pREP-3 stop plasmids to generate AAV9-packaged virions that were used to transduce HEK-293T cells at a MOI of 1e4 VG per cell. RNA was extracted 48 hours post-transfection and reverse transcribed using the Quantitect kit (Qiagen).

PCR was performed with primers allowing amplification of the full-length capsid or a partial sequence localized close to the C-terminus (FIG. 5B). Overall, the presence of an intron had little influence on the expression from low-activity promoters Syn and GFAP, which indicates that mRNA splicing did not alleviate promoter repression in nonpermissive cells. The combination of the CMV enhancer with a Chicken beta-actin promoter and the hybrid intron allowed a significantly higher (>10-fold) mRNA expression compared to CMV promoter alone (FIGS. 5B, C).

When comparing endpoint PCR amplification between forward and inverted intronic vectors, a discrepancy was obvious between full-length and partial capsid amplicons (FIG. 5B, right-hand lanes), which led us to question the integrity of capsid RNA. When cDNA from inverted iCAG9 genome was amplified using primers flanking the full-length capsid, multiple low-molecular weight bands were detected, whereas the forward orientation vector allowed amplification of a single product with the expected length (FIG. 5D). Sanger sequencing of low-molecular weight amplicons showed that each band corresponded to an illegitimate splicing product from the antisense capsid RNA.

In light of these results, the forward tandem promoter architecture for subsequent experiments.

Splice-specific PCR amplification was tested to avoid amplification of residual DNA present in RNA preparations. Two candidate PCR primers overlapping the CMV/Globin exon-exon junction were designed and tested them for amplification of cDNA (spliced) or plasmid DNA (still containing the intron sequence). As shown in FIG. 5E, the GloSpliceF6 primer (SEQ. ID NO:13) allowed a fully specific amplification from cDNA without producing a detectable amplicon from the plasmid DNA sequence. This primer was used in subsequent assays to ascertain the absence of amplification from contaminating DNA.

Tandem constructs were then tested for potential interference of the P40 promoter with the cell-specific promoter placed upstream. For this, two series of AAV genomes were tested for transgene mRNA expression in HEK-293T cells. A series of transgenes where the GFP gene was placed immediately downstream of the CAG, SYNG or GFAPG promoter without P40 sequence were tested, and compared to the library constructs where AAV9 capsid was placed downstream of the P40 promoter (FIG. 6A). All genomes were packaged into the AAV9 capsid and used to infect HEK-293T at a MOI of 1e4 VG per cell. RNA was extracted 48 hours post-infection and transgene RNA was quantified by using a Taqman primer/probe mix specific for the spliced globin exon-exon junction. As shown in FIG. 6B, the expression from the CAG promoter was similar between the GFP and the P40-CAP9 constructs (2-fold lower in p40-CAP9, within the error margin of AAV titration). Expression from the synapsin promoter was drastically lower with both constructs and even lower for GFAP-driven mRNA (FIG. 6B). This was expected since HEK-293T cells are not permissive to Synapsin or GFAP promoter expression. Overall, this experiment confirmed that the presence of the P40 sequence did not alter the cell type specificity of synapsin or GFAP promoters.

This novel platform was termed TRACER (Tropism Redirection of AAV by Cell type-specific Expression of RNA). The TRACER platform solves the problems of standard methods including transduction and cell-type restrictions. (FIG. 7). Use of the TRACER system is well suited to capsid discovery where targeting peptide libraries are utilized. Screening of such a library may be conducted as outlined in FIG. 8.

While several variations of the AAV vectors which encode the capsids as payloads are taught herein, one canonical design is shown in FIG. 9B and in FIG. 12A and FIG. 12B.

Further advantages of the TRACER platform relate to the nature of the virus pool and the recovery of RNA only from fully transduced cells (FIG. 10). Consequently, capsid discovery can be accelerated in a manner that results in cell and/or tissue specific tropism (FIG. 11).

Example 4. Generation of Peptide Display Libraries and Cloning-Free Amplification

Several peptide display capsid libraries were generated by insertion of seven contiguous randomized amino acids into the surface-exposed hypervariable loop VIII region of AAV5, AAV6, or AAV-DJ8 capsids (FIG. 13 and FIG. 39) as well as AAV9 (FIG. 14). For AAV9 libraries, two extra libraries by modifying residues at positions −2, −1 and +1 of the insertion to match the flanking sequence of the highly neurotrophic PHP.eB vector (Chan et al., 2018). In order to facilitate the insertion of various loops and to prevent contamination by wild-type capsids, defective shuttle vectors were generated in which the C-terminal region of the capsid gene comprised between the loop VIII and the stop codon was deleted and replaced by a unique BsrGI restriction site (FIGS. 15A, B). Degenerate primers containing randomized NNK (K=T or G) sequences able to encode all amino acids were synthesized by IDT and used to amplify the missing capsid fragment using gBlock (IDT) double-stranded linear DNA as templates (SEQ. ID NO 14, 15, 16, 17). Linear PCR templates were preferred to plasmids in order to completely prevent the possibility of plasmid carryover in the PCR reaction. Amplicons containing the random library sequence (500 ng) were inserted in the shuttle plasmid linearized by BsrGI (2 ug) using 100 ul of NEBuilder HiFi DNA assembly master mix (NEB) during 30 minutes at 50° C. Unassembled linear templates were eliminated by addition of 5 ul of T5 exonuclease to the reaction and digestion for 30 minutes at 37° C. The entire reaction was purified with DNA Clean and Concentrator-5 and quantified with a nanodrop to estimate the efficiency of assembly. This method routinely allows the recovery of 0.5-1 ug assembled material.

gBlock templates were engineered by introducing silent mutations to remove unique restriction sites, to allow selective elimination of wild-type virus contaminants from the libraries by restriction enzyme treatment. As an example, AAV9 gBlock was engineered to remove BamHI and AfeI sites present in the parental sequence (SEQ. ID NO 17).

Example 5. Cloning Free Amplification

Transformation of assembled library DNA into competent bacteria represents a major bottleneck in library diversity, since even highly competent strains rarely exceed 1e7-1e8 colonies per transformation. By comparison, 100 nanograms of a 6-kilobase plasmid contain 1.5e10 DNA molecules. Therefore, bacterial transformation arbitrarily eliminates more than 99% of DNA species in a given pool. A cloning-free method was therefore created that allows >100-fold amplification of Gibson-assembled DNA while bypassing the bacterial transformation bottleneck (FIG. 16). A protocol based on rolling-circle amplification was optimized, which allows unbiased exponential amplification of circular DNA templates with an extremely low error rate (Hutchinson et al., 2005). One issue with rolling circle amplification is that it produces very large (˜70 kilobases on average) heavily branched concatemers that have to be cleaved into monomers for efficient cell transfection. This process can be accomplished by several methods, for example by using restriction enzymes to generate open-ended linear templates (Hutchinson et al., 2005, Huovinen, 2012), or CRE-Lox recombination to generates self-ligated circular templates (Huovinen et al., 2011). However, open-ended DNA is sensitive to degradation by cytoplasmic exonucleases, and the CRE recombination method showed relatively low efficiency (our unpublished observations). Therefore, an alternative monomer resolution method was chosen based on the use of TelN protelomerase (Rybchin et al., 1999), an enzyme that catalyzes the formation of closed-ended linear “dogbone” DNA monomers that are highly suitable for mammalian cell transfection (Heinrich et al., 2002).

To that end, the protelomerase recognition sequence TATCAGCACACAATTGCCCATTATACGC*GCGTATAATGGACTATTGTGTGCTGATA (SEQ ID NO: 176) was introduced outside both ITRs in all the BsrGI shuttle vectors used for capsid library insertion (the asterisk depicts the position were the two complementary strands get covalently linked to each other), in order to obtain the following plasmids: TelN-Syn9-BsrGI (SEQ ID NO 18), TelN-GFAP9-BsrGI (SEQ ID NO 19), TelN-Syn5-BsrGI (SEQ ID NO 20), TelN-GFAP5-BsrGI (SEQ ID NO 21), TelN-Syn6-BsrGI (SEQ ID NO 22), TelN-GFAP6-BsrGI (SEQ ID NO 23), TelN-SynDJ8-BsrGI (SEQ ID NO 24), TelN-GFAPDJ8-BsrGI (SEQ ID NO 25). Several methods for rolling circle amplification were tested, and the best results (high yield and low non-specific amplification) were obtained with the TruePrime technology (Expedeon), which relies on primerless amplification (Picher et al., 2016).

Briefly, the entire column-purified assembly reaction was used in a 900-ul TruePrime reaction following the manufacturer's instructions and incubated overnight at 30° C. The following day, the rolling circle reaction product was incubated 10 minutes at 65° C. to inactivate the enzymes and was diluted 5-fold in 1× thermoPol buffer with 50 ul protelomerase (NEB) in a 4.5-ml reaction. After 1 hour at 30° C., the reaction was heat-treated for 10 minutes at 70° C. to inactivate the protelomerase, and a 4.5-ul aliquot was run on an agarose gel. The entire reaction was then purified on multiple (10-12) Qiagen QiaPrep 2.0 columns following manufacturer's instructions. The typical yield obtained with this method was 160-180 ug DNA, which indicates >100-fold amplification of the starting material (typically 0.5-1 ug) and provides enough DNA for transfection of 200 cell culture dishes (FIG. 16).

The composition of all libraries was tested by next-gen sequencing with an Illumina NextSeq sequencing platform to estimate the number of variants and the eventual contamination by wild-type viruses. Amplicons were generated by PCR with Q5 polymerase (NEB) using primers containing Illumina TruSeq adapters and index barcodes. Amplicons were obtained by low-cycle PCR amplification (15 cycles), ran on 3% agarose gels and purified using Zymo gel extraction reagents. Libraries were quantified using a nanodrop, pooled into equimolar mixes and re-quantified with a KAPA library quantification kit following manufacturer's instruction. Libraries were mixed with 20-40% of PhiX control library to increase sequence diversity.

All DNA libraries generated by rolling circle showed a high sequence diversity (typically >1e8 unique variants, beyond the limits of NextSeq sequencing). By comparison, plasmid libraries generated by bacterial transformation rarely exceeded 1-2e7 variants.

Example 6. Prevention and/or Reduction of Contamination

In another embodiment, a primer/vector system aimed at completely preventing contamination of AAV9 libraries by wild-type virus possibly recovered from environmental contamination or from naturally infected primate animal tissues was created. This was achieved by introducing a maximum number of silent mutations in the sequences surrounding the library insertion site, as well as the sequence immediately before the CAP stop codon, used for PCR amplification (FIG. 17). These libraries showed an extremely low number of wild-type AAV9 detection by NGS (<2 AAV9 reads per 5e7 total reads), suggesting that the alteration of codons surrounding the library amplification and cloning sites is a very efficient way to preserve libraries from environmental or experimental contaminations.

Libraries were produced as described previously by calcium phosphate transfection of HEK-293T cells, dual iodixanol gradient fractionation and membrane ultrafiltration using 100,000 Da MWCO Amicon-15 membranes (Millipore), quantified by real-time PCR and an aliquot was used for NGS amplicon generation and NextSeq sequencing. The diversity of viral libraries was significantly lower than that of DNA libraries (typically ˜1-2e7 unique variants) and showed a very strong counter-selection of variants containing stop codons (from 20% in DNA libraries to ˜1% in virus libraries), evincing a very high rate of cis-packaging, as observed in previous studies (Nonnenmacher et al., 2014).

Example 7. In Vivo Selection of AAV9 Libraries for Mouse Brain Transduction

An RNA-driven library selection for increased brain transduction in a murine model was then developed. AAV9 libraries generated as described above were intravenously injected to male C57BL/6 mice at a dose 2e12 VG per mouse. Two groups of mice were injected with a single SYN-driven or GFAP-driven libraries derived from wild-type AAV9 flanking sequences, and two other groups received pooled libraries containing wild-type and PHP.eB-derived flanking sequences (FIG. 18). After one month, RNA was extracted from 200 mg of brain tissue corresponding to a whole hemisphere using RNeasy Universal Plus procedure (Qiagen). In order to minimize the possibility of RNA under sampling, the entire RNA preparation (˜200 ug) was subjected to mRNA enrichment using Oligotex beads (Qiagen) as recommended by the manufacturer. The entire preparation of enriched mRNA (˜5 ug, equivalent to 2% of total RNA) was then reverse transcribed in a 40-ul Superscript IV reaction (Life Technologies) using a library-specific primer with the following sequence: 5′-GAAACGAATTAAACGGTTTATTGATTAACAATCGATTA-3′ (SEQ ID NO: 415) (CAP stop codon is underlined) (FIG. 19). The entire pool of cDNA was then amplified 30 cycles with 55° C. annealing temperature and 2 minutes elongation in a 500-ul PCR reaction assembled with Q5 master mix, GloSpliceF6 forward primer and a CAP9-specific reverse primer: 5′-CGGTTTATTGATTAACAATCGATTACAGATTACGAGTCAGGTATC-3′ (SEQ ID NO: 416) (CAP stop codon is underlined). This method allowed recovery of abundant amplicons from all brain samples (FIG. 20).

Full-length capsid amplicons were then used as templates for NGS library generation, as well as cloning into a P1 DNA library for the next round of biopanning, using the exact same assembly and cloning-free procedure. NGS analysis performed on PCR amplicons indicated that the library diversity dropped ˜25-fold (from 1e7 to 4e5) after the first round of biopanning for both Syn-driven and GFAP-driven libraries (FIG. 21). The number of 1s^t pass variants (P1) recovered was too high to show any significant sequence convergence at this point, and there was very little overlap between the composition of pools recovered from individual animals. Therefore, a second round of selection was performed. After the second biopanning (P2), the total number of unique variants further dropped by 4-5-fold, down to <1e5 peptides. Importantly, some libraries recovered after the first round of biopanning showed significant counts of wild-type AAV9 and AAV-PHP.eB sequences, presumably from environmental contamination. These later became useful benchmarks in the second round of enrichment.

Following RNA recovery and PCR amplification, a systematic enrichment analysis by NGS was performed by calculating the ratio of P2/P1 reads and comparing it to AAV9 or PHP.eB P2/P1 ratio. As shown in FIG. 22, Table 4, FIG. 23 and Table 5, several capsids showed a higher enrichment ratio than the benchmark PHP.eB in both Syn-driven and GFAP-driven libraries, and sequence convergence was obvious, as represented by consensus sequence generation.

TABLE 4
Capsid analysis results
Rank			SEQ			Brain/
(enrichment	Ranking		ID	Average	P1	virus
factor)	(count)	Peptide	NO	of brain	AEvirus_S11	stock
1	136	DGTLAVHFK	417	2546.3	6	254.6
2	153	DGTFAVPFK	418	2321.7	6	232.2
3	155	EGTLAVPFK	419	2351.0	7	201.5
4	147	DGTMAVPFK	420	2547.0	8	191.0
5	32	DGTGGTKGW	107	11116.0	35	190.6
6	3	AQWPTSYDA	62	119359.7	512	139.9
7	99	DGTLAVTFK	421	3779.7	19	119.4
8	176	DGTLAVPIK	422	1882.0	13	86.9
9	36	AQTTEKPWL	83	10192.0	76	80.5
10	165	DGTAIHLSS	67	2885.0	23	75.3
11	13	DGTLSQPFR	65	42145.7	344	73.5
12	2	DGTLAAPFK	120	157129.3	1,300	72.5
13	8	AQPEGSARW	60	70884.0	594	71.6
14	48	AQWPTAYDA	256	5934.0	53	67.2
15	198	DGTLQQPFR	89	2793.3	25	67.0
16	104	DGTLAVNFK	346	3511.0	32	65.8
17	31	DGTGNLSGW	302	14521.3	133	65.5
18	158	DGTLEVTFK	423	2337.7	22	63.8
19	51	DGTMDKPFR	70	23962.3	234	61.4
20	80	DGTGQVTGW	68	6242.7	62	60.4
21	42	AQFPTNYDS	66	8640.0	86	60.3
22	127	ERTLAVPFK	424	2873.3	31	55.6
23	1	DGTLAVPFK	71	9885065.7	110,785	53.5
24	61	DGTGTTMGW	324	6753.0	76	53.3
25	69	DGSQSTTGW	136	7227.7	82	52.9
26	186	DGTVSNPFR	403	2074.3	24	51.9
27	160	DGTLEVHFK	348	2245.0	26	51.8
28	29	DGTISQPFK	105	20505.7	243	50.6
29	102	AQGSWNPPA	80	3746.0	45	49.9
30	59	DGTHSTTGW	145	7499.0	91	49.4
31	23	DGTGSTTGW	134	21582.0	272	47.6
32	142	DGTGTTTGW	130	3077.3	39	47.3
33	74	DGTVTTTGW	405	5088.7	66	46.3
34	35	DGTTYVPPR	75	9614.7	126	45.8
35	40	DGTMDRPFK	102	7868.3	104	45.4
36	4	DGTGTTLGW	323	88397.3	1,169	45.4
37	156	DGTALMLSS	280	2444.0	34	43.1
38	116	DGTNTTHGW	113	3065.0	43	42.8
39	98	SGSLAVPFK	425	4107.3	58	42.5
40	38	DGTATTTGW	285	10529.7	150	42.1
41	11	DGTSYVPPR	78	36293.3	526	41.4
42	89	DGTGNTHGW	72	3399.3	50	40.8
43	129	DGTASVTGW	283	4824.3	71	40.8
44	12	AQWELSNGY	246	40837.0	611	40.1
45	115	DGTGNTSGW	137	3405.0	51	40.1
46	67	DGKGSTQGW	272	5818.0	88	39.7
47	137	DGTVIMLSS	397	3781.0	58	39.1
48	119	DGTGGVMGW	297	2302.3	36	38.4
49	58	DGGGTTTGW	270	11174.3	175	38.3
50	71	DGTSIHLSS	378	5703.7	90	38.0

TABLE 5
Capsid analysis results
Rank			SEQ			Brain/
(enrichment	Ranking		ID	Average	p1	virus
factor)	(count)	Peptide	NO	of brain	AEvirus_S11	stock
1	106	DGTGGTKGW	107	3620.7	0	NA
2	264	GGTRNTAPM	426	831.0	0	NA
3	295	AQGRMTDSQ	199	716.0	0	NA
4	677	DGNSYVPPR	427	474.3	0	NA
5	700	AQAGVSGQR	428	456.0	0	NA
6	731	AQAGNSNAV	429	844.0	0	NA
7	181	DGTGGLTGW	294	4044.3	4	606.7
8	558	AQWVYGQTV	430	977.7	1	586.6
9	123	DGTSFSPPK	431	4227.3	10	253.6
10	35	DGTIERPFR	87	29872.0	92	194.8
11	105	DGTTLVPPR	116	5597.3	19	176.8
12	18	DGTADKPFR	63	103305.3	363	170.8
13	22	DGTASYYDS	61	61841.3	233	159.2
14	26	AQTTDRPFL	85	38893.7	147	158.7
15	8	DGTQFSPPR	108	206660.7	801	154.8
16	169	DGTTTYGAR	77	4237.3	17	149.6
17	11	AQFVVGQQY	95	152965.0	625	146.8
18	61	DGTSYVPPR	78	13968.0	58	144.5
19	16	DGTAERPFR	140	134132.7	565	142.4
20	21	AQGENPGRW	96	68919.7	292	141.6
21	157	DGTSFTPPR	88	3210.0	14	137.6
22	73	AQTLARPFV	98	5947.7	26	137.3
23	9	DGTTWTPPR	139	184936.7	825	134.5
24	721	DGTATTMGW	284	5562.3	25	133.5
25	129	AQGTWNPPA	82	12379.3	57	130.3
26	215	DGTRLMLSS	368	2505.0	12	125.3
27	60	AQPLAVYGA	217	13419.3	66	122.0
28	909	AQGLDLGRW	432	405.0	2	121.5
29	53	DGTSFTPPK	81	13673.3	68	120.6
30	412	AQVMSGVGQ	433	583.0	3	116.6
31	390	AQKSVGSVY	205	4415.7	23	115.2
32	70	AQTREYLLG	93	5752.7	30	115.1
33	43	DGTNGLKGW	76	15068.7	79	114.4
34	93	AQYLAGYTV	262	6223.3	33	113.2
35	54	AQTGFAPPR	161	14611.3	78	112.4
36	115	DGTLNNPFR	109	4719.7	26	108.9
37	968	DGNGGLKGW	167	3199.0	18	106.6
38	120	AQSVAKPFL	231	6929.7	39	106.6
39	544	DGTHGLRGW	434	528.0	3	105.6
40	159	AQSVVRPFL	233	2457.3	14	105.3
41	65	DGTRNMYEG	135	21086.3	124	102.0
42	556	AQRWAADSS	435	500.7	3	100.1
43	30	AQGPTRPFL	125	46225.3	279	99.4
44	64	DGTVPYLSS	401	22384.3	137	98.0
45	870	AQTGASGAT	436	473.7	3	94.7
46	341	AQLVAGYSQ	437	1240.0	8	93.0
47	375	AQSGGVGQV	228	768.3	5	92.2
48	145	AQSLARLFP	438	4435.3	29	91.8
49	1	DGTLAVPFK	71	1445517.0	9453	91.7
50	124	DGTGNVTGW	69	5424.3	36	90.4

Importantly, there was also a strong sequence convergence between different animals, suggesting an efficient selection after only two passages. FIG. 24 and FIG. 25 provide an estimation of brain/liver specificity in GFAP-AAV9 peptide library candidates.

Example 8. Multiplexing Selections

For the final multiplex in vivo screen by individual variant pooling in equimolar library, a subpopulation of variants with promising properties (such as, but not limited to, enrichment factor, liver detargeting, high counts in more than one mouse, etc.) may be selected as shown in FIG. 26 and then an equimolar pool of primers encoding all the 7-mers (microchip solid-phase synthesis, up to 3,800 primers per chip) can be synthesized. The limited diversity library may be produced including internal controls such as, but not limited to, PHP.N, PHP.B, wild-type AAV9 (wtAAV9) and/or any other serotype including those taught herein. The mice are injected and then the RNA enrichment is compared to internal controls in a similar manner to a barcoding study, which is known in the art and described herein.

Example 9. Codon Optimization

Codon variants may be used to improve data strength when using synthesized libraries. A listing of NNK codons, NNM codons and the most favorable NNM codons in mammals for various amino acids is provided in Table 6. In Table 6, * means that no NNM codon was available and ** means “avoid homopolymeric stretches if possible.”

TABLE 6
Codon Variants
				Most
				favorable
				NNM
	Amino	NNK	NNM	codon in
	acid	codon	codons	mammals
	F	TTT	TTC	TTC
	L	TTG, CTT, CTG	TTA, CTC, CTA	CTC
	S	TCT, TCG, AGT	TCC, TCA, AGC	AGC
	Y	TAT	TAC	TAC
	C	TGT	TGC	TGC
	W	TGG		TGG*
	P	CCT, CCG	CCC, CCA	CCA**
	H	CAT	CAC	CAC
	Q	CAG	CAA	CAA
	R	CGT, CGG, AGG	CGC, CGA, AGA	AGA
	I	ATT	ATC, ATA	ATC
	M	ATG		ATT*
	T	ACT, ACG	ACC, ACA	ACC
	N	AAT	AAC	AAC
	K	AAG	AAA	AAA
	V	GTT, GTG	GTC, GTA	GTC
	A	GCT, GCG	GCC, GCA	GCC
	D	GAT	GAC	GAC
	E	GAG	GAA	GAA
	G	GGT, GGG	GGC, GGA	GGC
	stop	TAG	TAC, TAA	n/a
*no NNM codon available
**avoid homopolymeric stretches if possible

In order to have a balanced library it is recommended to establish a list of potential candidates. Then, using Table 6, a pooled primer library containing every peptide variant with encoded by NNK codons (original from library) and non-NNK codons (maximum variation). If similar behavior is seen between the two variants of the same peptide, this would strengthen the analysis of that peptide. Additionally, it is recommended to choose the most favorable NNM codons (M=A or C).

Example 10. Library Generation

The top-ranking 330 peptide variants from SYN-driven and GFAP-driven libraries that showed enhanced performance relative to the parental AAV9 were selected. A de novo library by pooled primer synthesis of all 330 peptide sequences plus AAV9, AAV-PHP.B and AAV-PHP.eB controls was generated (Table 7). In order to exclude potential artifacts due to the DNA sequence and to increase the robustness of the assay, each peptide variant was encoded by two different DNA sequences, one where all amino acids were encoded by NNK codons (identical to the original library) and another one where NNM codons were used whenever possible (M=C or A, Table 6).

TABLE 7
Peptide variants selected after 2 rounds
of RNA-driven mouse brain biopanning
	SEQ	Nucleotide	SEQ	Nucleotide	SEQ
Peptide	ID	sequence	ID	sequence	ID
Sequence	NO:	(NNK codons)	NO:	(NNM codons)	NO:
AQ (AAV9)		CAGAGTGCTCAG	439	CAGAGTGCCCAA	772
		GCACAG		GCACAG
AQAGAGSER	194	CAGAGTGCCCAA	440	CAGAGTGCACAA	773
		GCGGGTGCGGGG		GCAGGAGCAGGA
		TCGGAGCGGGCA		AGCGAAAGAGCA
		CAG		CAG
AQDQNPGRW	195	CAGAGTGCCCAA	441	CAGAGTGCACAA	774
		GATCAGAATCCG		GACCAAAACCCA
		GGGCGTTGGGCA		GGAAGATGGGCA
		CAG		CAG
AQELTRPFL	144	CAGAGTGCCCAA	442	CAGAGTGCACAA	775
		GAGTTGACGCGT		GAACTCACAAGA
		CCGTTTTTGGCAC		CCATTCCTCGCAC
		AG		AG
AQEVPGYRW	196	CAGAGTGCCCAA	443	CAGAGTGCACAA	776
		GAGGTGCCTGGG		GAAGTCCCAGGA
		TATAGGTGGGCA		TACAGATGGGCA
		CAG		CAG
AQFPTNYDS	66	CAGAGTGCCCAA	444	CAGAGTGCACAA	777
		TTTCCTACGAATT		TTCCCAACAAACT
		ATGATTCTGCACA		ACGACAGCGCAC
		G		AG
AQFVVGQQY	95	CAGAGTGCCCAA	445	CAGAGTGCACAA	778
		TTTGTGGTTGGTC		TTCGTCGTCGGAC
		AGCAGTATGCAC		AACAATACGCAC
		AG		AG
AQGASPGRW	149	CAGAGTGCCCAA	446	CAGAGTGCACAA	779
		GGGGCTAGTCCG		GGAGCAAGCCCA
		GGGCGGTGGGCA		GGAAGATGGGCA
		CAG		CAG
AQGENPGRW	96	CAGAGTGCCCAA	447	CAGAGTGCACAA	780
		GGGGAGAATCCG		GGAGAAAACCCA
		GGTAGGTGGGCA		GGAAGATGGGCA
		CAG		CAG
AQGGNPGRW	91	CAGAGTGCCCAA	448	CAGAGTGCACAA	781
		GGGGGGAATCCG		GGAGGAAACCCA
		GGTCGGTGGGCA		GGAAGATGGGCA
		CAG		CAG
AQGGSTGSN	197	CAGAGTGCCCAA	449	CAGAGTGCACAA	782
		GGTGGTTCTACG		GGAGGAAGCACA
		GGGTCGAATGCA		GGAAGCAACGCA
		CAG		CAG
AQGPTRPFL	125	CAGAGTGCCCAA	450	CAGAGTGCACAA	783
		GGGCCGACTAGG		GGACCAACAAGA
		CCGTTTTTGGCAC		CCATTCCTCGCAC
		AG		AG
AQGRDGWAA	198	CAGAGTGCCCAA	451	CAGAGTGCACAA	784
		GGTCGGGATGGT		GGAAGAGACGGA
		TGGGCGGCGGCA		TGGGCAGCAGCA
		CAG		CAG
AQGRMTDSQ	199	CAGAGTGCCCAA	452	CAGAGTGCACAA	785
		GGTCGTATGACT		GGAAGAATGACA
		GATTCGCAGGCA		GACAGCCAAGCA
		CAG		CAG
AQGSDVGRW	128	CAGAGTGCCCAA	453	CAGAGTGCACAA	786
		GGTAGTGATGTG		GGAAGCGACGTC
		GGGCGGTGGGCA		GGAAGATGGGCA
		CAG		CAG
AQGSNPGRW	103	CAGAGTGCCCAA	454	CAGAGTGCACAA	787
		GGTAGTAATCCG		GGAAGCAACCCA
		GGGAGGTGGGCA		GGAAGATGGGCA
		CAG		CAG
AQGSNSPQV	200	CAGAGTGCCCAA	455	CAGAGTGCACAA	788
		GGGTCTAATTCGC		GGAAGCAACAGC
		CTCAGGTGGCAC		CCACAAGTCGCA
		AG		CAG
AQGSWNPPA	80	CAGAGTGCCCAA	456	CAGAGTGCACAA	789
		GGTTCGTGGAAT		GGAAGCTGGAAC
		CCGCCGGCGGCA		CCACCAGCAGCA
		CAG		CAG
AQGTWNPPA	82	CAGAGTGCCCAA	457	CAGAGTGCACAA	790
		GGTACTTGGAAT		GGAACATGGAAC
		CCGCCGGCTGCA		CCACCAGCAGCA
		CAG		CAG
AQGVFIPPK	201	CAGAGTGCCCAA	458	CAGAGTGCACAA	791
		GGTGTTTTTATTC		GGAGTCTTCATCC
		CGCCGAAGGCAC		CACCAAAAGCAC
		AG		AG
AQHVNASQS	202	CAGAGTGCCCAA	459	CAGAGTGCACAA	792
		CATGTGAATGCTT		CACGTCAACGCA
		CTCAGTCTGCACA		AGCCAAAGCGCA
		G		CAG
AQIKAGWAQ	203	CAGAGTGCCCAA	460	CAGAGTGCACAA	793
		ATTAAGGCGGGG		ATCAAAGCAGGA
		TGGGCGCAGGCA		TGGGCACAAGCA
		CAG		CAG
AQIMSGYAQ	204	CAGAGTGCCCAA	461	CAGAGTGCACAA	794
		ATTATGAGTGGG		ATCATGAGCGGA
		TATGCTCAGGCA		TACGCACAAGCA
		CAG		CAG
AQKSVGSVY	205	CAGAGTGCCCAA	462	CAGAGTGCACAA	795
		AAGAGTGTGGGT		AAAAGCGTCGGA
		AGTGTTTATGCAC		AGCGTCTACGCA
		AG		CAG
AQLEHGFAQ	206	CAGAGTGCCCAA	463	CAGAGTGCACAA	796
		CTTGAGCATGGG		CTCGAACACGGA
		TTTGCTCAGGCAC		TTCGCACAAGCA
		AG		CAG
AQLGGVLSA	207	CAGAGTGCCCAA	464	CAGAGTGCACAA	797
		CTGGGTGGGGTG		CTCGGAGGAGTC
		TTGAGTGCTGCAC		CTCAGCGCAGCA
		AG		CAG
AQLGLSQGR	208	CAGAGTGCCCAA	465	CAGAGTGCACAA	798
		CTGGGGCTTTCGC		CTCGGACTCAGC
		AGGGGCGGGCAC		CAAGGAAGAGCA
		AG		CAG
AQLGYGFAQ	209	CAGAGTGCCCAA	466	CAGAGTGCACAA	799
		TTGGGGTATGGG		CTCGGATACGGA
		TTTGCTCAGGCAC		TTCGCACAAGCA
		AG		CAG
AQLKYGLAQ	115	CAGAGTGCCCAA	467	CAGAGTGCACAA	800
		TTGAAGTATGGTC		CTCAAATACGGA
		TTGCGCAGGCAC		CTCGCACAAGCA
		AG		CAG
AQLRIGFAQ	210	CAGAGTGCCCAA	468	CAGAGTGCACAA	801
		CTTCGGATTGGTT		CTCAGAATCGGA
		TTGCTCAGGCAC		TTCGCACAAGCA
		AG		CAG
AQLRMGYSQ	211	CAGAGTGCCCAA	469	CAGAGTGCACAA	802
		TTGCGTATGGGTT		CTCAGAATGGGA
		ATAGTCAGGCAC		TACAGCCAAGCA
		AG		CAG
AQLRQGYAQ	212	CAGAGTGCCCAA	470	CAGAGTGCACAA	803
		CTGAGGCAGGGG		CTCAGACAAGGA
		TATGCTCAGGCA		TACGCACAAGCA
		CAG		CAG
AQLRVGFAQ	123	CAGAGTGCCCAA	471	CAGAGTGCACAA	804
		TTGCGTGTTGGTT		CTCAGAGTCGGA
		TTGCGCAGGCAC		TTCGCACAAGCA
		AG		CAG
AQLSCRSQM	213	CAGAGTGCCCAA	472	CAGAGTGCACAA	805
		CTGTCGTGTCGGA		CTCAGCTGCAGA
		GTCAGATGGCAC		AGCCAAATGGCA
		AG		CAG
AQLTYSQSL	214	CAGAGTGCCCAA	473	CAGAGTGCACAA	806
		TTGACGTATAGTC		CTCACATACAGC
		AGTCGCTGGCAC		CAAAGCCTCGCA
		AG		CAG
AQLYKGYSQ	215	CAGAGTGCCCAA	474	CAGAGTGCACAA	807
		CTGTATAAGGGTT		CTCTACAAAGGA
		ATAGTCAGGCAC		TACAGCCAAGCA
		AG		CAG
AQMPQRPFL	216	CAGAGTGCCCAA	475	CAGAGTGCACAA	808
		ATGCCTCAGCGG		ATGCCACAAAGA
		CCGTTTTTGGCAC		CCATTCCTCGCAC
		AG		AG
AQNGNPGRW	84	CAGAGTGCCCAA	476	CAGAGTGCACAA	809
		AATGGTAATCCG		AACGGAAACCCA
		GGGCGGTGGGCA		GGAAGATGGGCA
		CAG		CAG
AQPEGSARW	60	CAGAGTGCCCAA	477	CAGAGTGCACAA	810
		CCTGAGGGTAGT		CCAGAAGGAAGC
		GCGAGGTGGGCA		GCAAGATGGGCA
		CAG		CAG
AQPLAVYGA	217	CAGAGTGCCCAA	478	CAGAGTGCACAA	811
		CCGTTGGCTGTTT		CCACTCGCAGTCT
		ATGGGGCGGCAC		ACGGAGCAGCAC
		AG		AG
AQPQSSSMS	218	CAGAGTGCCCAA	479	CAGAGTGCACAA	812
		CCGCAGTCGTCGT		CCACAAAGCAGC
		CGATGAGTGCAC		AGCATGAGCGCA
		AG		CAG
AQPSVGGYW	219	CAGAGTGCCCAA	480	CAGAGTGCACAA	813
		CCGAGTGTGGGT		CCAAGCGTCGGA
		GGGTATTGGGCA		GGATACTGGGCA
		CAG		CAG
AQQAVGQSW	220	CAGAGTGCCCAA	481	CAGAGTGCACAA	814
		CAGGCTGTGGGT		CAAGCAGTCGGA
		CAGTCTTGGGCA		CAAAGCTGGGCA
		CAG		CAG
AQQRSLASG	221	CAGAGTGCCCAA	482	CAGAGTGCACAA	815
		CAGCGTTCGCTG		CAAAGAAGCCTC
		GCTTCGGGTGCA		GCAAGCGGAGCA
		CAG		CAG
AQQVMNSQG	222	CAGAGTGCCCAA	483	CAGAGTGCACAA	816
		CAGGTGATGAAT		CAAGTCATGAAC
		AGTCAGGGGGCA		AGCCAAGGAGCA
		CAG		CAG
AQRGVGLSQ	223	CAGAGTGCCCAA	484	CAGAGTGCACAA	817
		CGTGGGGTTGGG		AGAGGAGTCGGA
		TTGAGTCAGGCA		CTCAGCCAAGCA
		CAG		CAG
AQRHDAEGS	224	CAGAGTGCCCAA	485	CAGAGTGCACAA	818
		AGGCATGATGCG		AGACACGACGCA
		GAGGGTAGTGCA		GAAGGAAGCGCA
		CAG		CAG
AQRKGEPHY	225	CAGAGTGCCCAA	486	CAGAGTGCACAA	819
		CGTAAGGGGGAG		AGAAAAGGAGAA
		CCTCATTATGCAC		CCACACTACGCA
		AG		CAG
AQRYTGDSS	138	CAGAGTGCCCAA	487	CAGAGTGCACAA	820
		AGGTATACGGGG		AGATACACAGGA
		GATTCTAGTGCAC		GACAGCAGCGCA
		AG		CAG
AQSAMAAKG	226	CAGAGTGCCCAA	488	CAGAGTGCACAA	821
		TCGGCGATGGCT		AGCGCAATGGCA
		GCGAAGGGTGCA		GCAAAAGGAGCA
		CAG		CAG
AQSGGLTGS	227	CAGAGTGCCCAA	489	CAGAGTGCACAA	822
		TCTGGGGGTCTTA		AGCGGAGGACTC
		CGGGGAGTGCAC		ACAGGAAGCGCA
		AG		CAG
AQSGGVGQV	228	CAGAGTGCCCAA	490	CAGAGTGCACAA	823
		TCGGGTGGGGTG		AGCGGAGGAGTC
		GGGCAGGTGGCA		GGACAAGTCGCA
		CAG		CAG
AQSLATPFR	169	CAGAGTGCCCAA	491	CAGAGTGCACAA	824
		TCTCTGGCGACGC		AGCCTCGCAACA
		CTTTTCGTGCACA		CCATTCAGAGCA
		G		CAG
AQSMSRPFL	229	CAGAGTGCCCAA	492	CAGAGTGCACAA	825
		AGTATGTCGCGTC		AGCATGAGCAGA
		CGTTTCTGGCACA		CCATTCCTCGCAC
		G		AG
AQSQLRPFL	230	CAGAGTGCCCAA	493	CAGAGTGCACAA	826
		AGTCAGCTTAGG		AGCCAACTCAGA
		CCGTTTCTTGCAC		CCATTCCTCGCAC
		AG		AG
AQSVAKPFL	231	CAGAGTGCCCAA	494	CAGAGTGCACAA	827
		TCTGTGGCTAAGC		AGCGTCGCAAAA
		CTTTTTTGGCACA		CCATTCCTCGCAC
		G		AG
AQSVSQPFR	232	CAGAGTGCCCAA	495	CAGAGTGCACAA	828
		TCGGTTTCGCAGC		AGCGTCAGCCAA
		CGTTTAGGGCAC		CCATTCAGAGCA
		AG		CAG
AQSVVRPFL	233	CAGAGTGCCCAA	496	CAGAGTGCACAA	829
		TCTGTGGTGCGTC		AGCGTCGTCAGA
		CTTTTCTGGCACA		CCATTCCTCGCAC
		G		AG
AQTALSSST	234	CAGAGTGCCCAA	497	CAGAGTGCACAA	830
		ACTGCGCTTTCGT		ACAGCACTCAGC
		CGTCGACGGCAC		AGCAGCACAGCA
		AG		CAG
AQTEMGGRC	235	CAGAGTGCCCAA	498	CAGAGTGCACAA	831
		ACGGAGATGGGT		ACAGAAATGGGA
		GGGAGGTGTGCA		GGAAGATGCGCA
		CAG		CAG
AQTGFAPPR	161	CAGAGTGCCCAA	499	CAGAGTGCACAA	832
		ACGGGGTTTGCTC		ACAGGATTCGCA
		CGCCGCGTGCAC		CCACCAAGAGCA
		AG		CAG
AQTIRGYSS	236	CAGAGTGCCCAA	500	CAGAGTGCACAA	833
		ACGATTCGGGGG		ACAATCAGAGGA
		TATTCGTCTGCAC		TACAGCAGCGCA
		AG		CAG
AQTISNYHT	237	CAGAGTGCCCAA	501	CAGAGTGCACAA	834
		ACTATTTCTAATT		ACAATCAGCAAC
		ATCATACGGCAC		TACCACACAGCA
		AG		CAG
AQTLARPFV	98	CAGAGTGCCCAA	502	CAGAGTGCACAA	835
		ACTTTGGCGCGTC		ACACTCGCAAGA
		CGTTTGTGGCACA		CCATTCGTCGCAC
		G		AG
AQTLAVPFK	168	CAGAGTGCCCAA	503	CAGAGTGCACAA	836
(PHP.B)		ACTTTGGCGGTGC		ACACTCGCAGTC
		CTTTTAAGGCACA		CCATTCAAAGCA
		G		CAG
AQTPDRPWL	238	CAGAGTGCCCAA	504	CAGAGTGCACAA	837
		ACTCCTGATCGTC		ACACCAGACAGA
		CTTGGTTGGCACA		CCATGGCTCGCA
		G		CAG
AQTRAGYAQ	126	CAGAGTGCCCAA	505	CAGAGTGCACAA	838
		ACTCGGGCTGGG		ACAAGAGCAGGA
		TATGCTCAGGCA		TACGCACAAGCA
		CAG		CAG
AQTRAGYSQ	141	CAGAGTGCCCAA	506	CAGAGTGCACAA	839
		ACTAGGGCGGGG		ACAAGAGCAGGA
		TATTCTCAGGCAC		TACAGCCAAGCA
		AG		CAG
AQTREYLLG	93	CAGAGTGCCCAA	507	CAGAGTGCACAA	840
		ACGCGTGAGTAT		ACAAGAGAATAC
		CTGCTGGGGGCA		CTCCTCGGAGCA
		CAG		CAG
AQTSAKPFL	163	CAGAGTGCCCAA	508	CAGAGTGCACAA	841
		ACTTCTGCGAAG		ACAAGCGCAAAA
		CCGTTTCTTGCAC		CCATTCCTCGCAC
		AG		AG
AQTSARPFL	100	CAGAGTGCCCAA	509	CAGAGTGCACAA	842
		ACTTCTGCTAGGC		ACAAGCGCAAGA
		CTTTTCTGGCACA		CCATTCCTCGCAC
		G		AG
AQTTDRPFL	85	CAGAGTGCCCAA	510	CAGAGTGCACAA	843
		ACTACTGATAGG		ACAACAGACAGA
		CCTTTTTTGGCAC		CCATTCCTCGCAC
		AG		AG
AQTTEKPWL	83	CAGAGTGCCCAA	511	CAGAGTGCACAA	844
		ACGACTGAGAAG		ACAACAGAAAAA
		CCGTGGCTGGCA		CCATGGCTCGCA
		CAG		CAG
AQTVARPFY	239	CAGAGTGCCCAA	512	CAGAGTGCACAA	845
		ACGGTTGCGCGG		ACAGTCGCAAGA
		CCTTTTTATGCAC		CCATTCTACGCAC
		AG		AG
AQTVATPFR	240	CAGAGTGCCCAA	513	CAGAGTGCACAA	846
		ACTGTTGCTACGC		ACAGTCGCAACA
		CGTTTAGGGCAC		CCATTCAGAGCA
		AG		CAG
AQTVTQLFK	241	CAGAGTGCCCAA	514	CAGAGTGCACAA	847
		ACGGTGACGCAG		ACAGTCACACAA
		TTGTTTAAGGCAC		CTCTTCAAAGCAC
		AG		AG
AQVHVGSVY	165	CAGAGTGCCCAA	515	CAGAGTGCACAA	848
		GTTCATGTTGGGA		GTCCACGTCGGA
		GTGTTTATGCACA		AGCGTCTACGCA
		G		CAG
AQVLAGYNM	242	CAGAGTGCCCAA	516	CAGAGTGCACAA	849
		GTTCTTGCTGGGT		GTCCTCGCAGGA
		ATAATATGGCAC		TACAACATGGCA
		AG		CAG
AQVSEARVR	243	CAGAGTGCCCAA	517	CAGAGTGCACAA	850
		GTTTCTGAGGCG		GTCAGCGAAGCA
		AGGGTTAGGGCA		AGAGTCAGAGCA
		CAG		CAG
AQVVVGYSQ	244	CAGAGTGCCCAA	518	CAGAGTGCACAA	851
		GTTGTGGTGGGTT		GTCGTCGTCGGAT
		ATAGTCAGGCAC		ACAGCCAAGCAC
		AG		AG
AQWAAGYNV	245	CAGAGTGCCCAA	519	CAGAGTGCACAA	852
		TGGGCTGCTGGG		TGGGCAGCAGGA
		TATAATGTGGCA		TACAACGTCGCA
		CAG		CAG
AQWELSNGY	246	CAGAGTGCCCAA	520	CAGAGTGCACAA	853
		TGGGAGCTGAGT		TGGGAACTCAGC
		AATGGGTATGCA		AACGGATACGCA
		CAG		CAG
AQWEVKGGY	247	CAGAGTGCCCAA	521	CAGAGTGCACAA	854
		TGGGAGGTGAAG		TGGGAAGTCAAA
		GGGGGTTATGCA		GGAGGATACGCA
		CAG		CAG
AQWEVKRGY	248	CAGAGTGCCCAA	522	CAGAGTGCACAA	855
		TGGGAGGTGAAG		TGGGAAGTCAAA
		CGGGGGTATGCA		AGAGGATACGCA
		CAG		CAG
AQWEVQSGF	249	CAGAGTGCCCAA	523	CAGAGTGCACAA	856
		TGGGAGGTTCAG		TGGGAAGTCCAA
		TCTGGGTTTGCAC		AGCGGATTCGCA
		AG		CAG
AQWEVRGGY	250	CAGAGTGCCCAA	524	CAGAGTGCACAA	857
		TGGGAGGTTCGT		TGGGAAGTCAGA
		GGTGGTTATGCA		GGAGGATACGCA
		CAG		CAG
AQWEVTSGW	251	CAGAGTGCCCAA	525	CAGAGTGCACAA	858
		TGGGAGGTGACG		TGGGAAGTCACA
		AGTGGTTGGGCA		AGCGGATGGGCA
		CAG		CAG
AQWGAPSHG	252	CAGAGTGCCCAA	526	CAGAGTGCACAA	859
		TGGGGGGCGCCG		TGGGGAGCACCA
		AGTCATGGGGCA		AGCCACGGAGCA
		CAG		CAG
AQWMELGSS	253	CAGAGTGCCCAA	527	CAGAGTGCACAA	860
		TGGATGGAGCTT		TGGATGGAACTC
		GGTAGTTCGGCA		GGAAGCAGCGCA
		CAG		CAG
AQWMFGGSG	254	CAGAGTGCCCAA	528	CAGAGTGCACAA	861
		TGGATGTTTGGG		TGGATGTTCGGA
		GGTAGTGGGGCA		GGAAGCGGAGCA
		CAG		CAG
AQWMLGGAQ	255	CAGAGTGCCCAA	529	CAGAGTGCACAA	862
		TGGATGCTGGGG		TGGATGCTCGGA
		GGGGCGCAGGCA		GGAGCACAAGCA
		CAG		CAG
AQWPTAYDA	256	CAGAGTGCCCAA	530	CAGAGTGCACAA	863
		TGGCCGACTGCTT		TGGCCAACAGCA
		ATGATGCGGCAC		TACGACGCAGCA
		AG		CAG
AQWPTSYDA	62	CAGAGTGCCCAA	531	CAGAGTGCACAA	864
		TGGCCTACGAGTT		TGGCCAACAAGC
		ATGATGCTGCAC		TACGACGCAGCA
		AG		CAG
AQWQVQTGF	257	CAGAGTGCCCAA	532	CAGAGTGCACAA	865
		TGGCAGGTTCAG		TGGCAAGTCCAA
		ACGGGGTTTGCA		ACAGGATTCGCA
		CAG		CAG
AQWSTEGGY	258	CAGAGTGCCCAA	533	CAGAGTGCACAA	866
		TGGTCGACTGAG		TGGAGCACAGAA
		GGTGGGTATGCA		GGAGGATACGCA
		CAG		CAG
AQWTAAGGY	259	CAGAGTGCCCAA	534	CAGAGTGCACAA	867
		TGGACTGCTGCG		TGGACAGCAGCA
		GGTGGTTATGCA		GGAGGATACGCA
		CAG		CAG
AQWTTESGY	260	CAGAGTGCCCAA	535	CAGAGTGCACAA	868
		TGGACGACGGAG		TGGACAACAGAA
		TCGGGTTATGCAC		AGCGGATACGCA
		AG		CAG
AQWVYGSSH	261	CAGAGTGCCCAA	536	CAGAGTGCACAA	869
		TGGGTTTATGGG		TGGGTCTACGGA
		AGTTCGCATGCA		AGCAGCCACGCA
		CAG		CAG
AQYLAGYTV	262	CAGAGTGCCCAA	537	CAGAGTGCACAA	870
		TATTTGGCGGGGT		TACCTCGCAGGA
		ATACGGTGGCAC		TACACAGTCGCA
		AG		CAG
AQYLKGYSV	152	CAGAGTGCCCAA	538	CAGAGTGCACAA	871
		TATCTGAAGGGG		TACCTCAAAGGA
		TATTCTGTGGCAC		TACAGCGTCGCA
		AG		CAG
AQYLSGYNT	263	CAGAGTGCCCAA	539	CAGAGTGCACAA	872
		TATTTGTCGGGTT		TACCTCAGCGGA
		ATAATACGGCAC		TACAACACAGCA
		AG		CAG
DGAAATTGW	264	CAGAGTGATGGC	540	CAGAGTGACGGA	873
		GCTGCGGCGACT		GCAGCAGCAACA
		ACTGGGTGGGCA		ACAGGATGGGCA
		CAG		CAG
DGAGGTSGW	151	CAGAGTGATGGC	541	CAGAGTGACGGA	874
		GCGGGTGGGACG		GCAGGAGGAACA
		AGTGGTTGGGCA		AGCGGATGGGCA
		CAG		CAG
DGAGTTSGW	265	CAGAGTGATGGC	542	CAGAGTGACGGA	875
		GCGGGTACTACTT		GCAGGAACAACA
		CGGGTTGGGCAC		AGCGGATGGGCA
		AG		CAG
DGAHGLSGW	266	CAGAGTGATGGC	543	CAGAGTGACGGA	876
		GCTCATGGGCTGT		GCACACGGACTC
		CGGGGTGGGCAC		AGCGGATGGGCA
		AG		CAG
DGAHVGLSS	267	CAGAGTGATGGC	544	CAGAGTGACGGA	877
		GCTCATGTTGGGC		GCACACGTCGGA
		TGTCGTCGGCAC		CTCAGCAGCGCA
		AG		CAG
DGARTVLQL	268	CAGAGTGATGGC	545	CAGAGTGACGGA	878
		GCTCGGACGGTG		GCAAGAACAGTC
		CTTCAGTTGGCAC		CTCCAACTCGCAC
		AG		AG
DGEYQKPFR	269	CAGAGTGATGGC	546	CAGAGTGACGGA	879
		GAGTATCAGAAG		GAATACCAAAAA
		CCGTTTAGGGCA		CCATTCAGAGCA
		CAG		CAG
DGGGTTTGW	270	CAGAGTGATGGC	547	CAGAGTGACGGA	880
		GGTGGGACTACG		GGAGGAACAACA
		ACGGGGTGGGCA		ACAGGATGGGCA
		CAG		CAG
DGHATSMGW	271	CAGAGTGATGGC	548	CAGAGTGACGGA	881
		CATGCGACGAGT		CACGCAACAAGC
		ATGGGTTGGGCA		ATGGGATGGGCA
		CAG		CAG
DGKGSTQGW	272	CAGAGTGATGGC	549	CAGAGTGACGGA	882
		AAGGGTTCGACG		AAAGGAAGCACA
		CAGGGGTGGGCA		CAAGGATGGGCA
		CAG		CAG
DGKQYQLSS	92	CAGAGTGATGGC	550	CAGAGTGACGGA	883
		AAGCAGTATCAG		AAACAATACCAA
		CTGTCTTCGGCAC		CTCAGCAGCGCA
		AG		CAG
DGNGGLKGW	167	CAGAGTGATGGC	551	CAGAGTGACGGA	884
		AATGGTGGGTTG		AACGGAGGACTC
		AAGGGGTGGGCA		AAAGGATGGGCA
		CAG		CAG
DGQGGLSGW	273	CAGAGTGATGGC	552	CAGAGTGACGGA	885
		CAGGGGGGTTTG		CAAGGAGGACTC
		TCTGGGTGGGCA		AGCGGATGGGCA
		CAG		CAG
DGQHFAPPR	110	CAGAGTGATGGC	553	CAGAGTGACGGA	886
		CAGCATTTTGCTC		CAACACTTCGCA
		CGCCGCGGGCAC		CCACCAAGAGCA
		AG		CAG
DGRATKTLY	274	CAGAGTGATGGC	554	CAGAGTGACGGA	887
		CGTGCGACTAAG		AGAGCAACAAAA
		ACGCTTTATGCAC		ACACTCTACGCA
		AG		CAG
DGRNALTGW	275	CAGAGTGATGGC	555	CAGAGTGACGGA	888
		CGTAATGCGTTG		AGAAACGCACTC
		ACGGGGTGGGCA		ACAGGATGGGCA
		CAG		CAG
DGRRQVIQL	276	CAGAGTGATGGC	556	CAGAGTGACGGA	889
		AGGAGGCAGGTG		AGAAGACAAGTC
		ATTCAGCTGGCA		ATCCAACTCGCA
		CAG		CAG
DGRVYGLSS	277	CAGAGTGATGGC	557	CAGAGTGACGGA	890
		AGGGTTTATGGTC		AGAGTCTACGGA
		TTTCGTCGGCACA		CTCAGCAGCGCA
		G		CAG
DGSGRTTGW	147	CAGAGTGATGGC	558	CAGAGTGACGGA	891
		AGTGGGCGTACG		AGCGGAAGAACA
		ACGGGTTGGGCA		ACAGGATGGGCA
		CAG		CAG
DGSGTTRGW	114	CAGAGTGATGGC	559	CAGAGTGACGGA	892
		TCTGGTACGACG		AGCGGAACAACA
		CGGGGTTGGGCA		AGAGGATGGGCA
		CAG		CAG
DGSGTVSGW	278	CAGAGTGATGGC	560	CAGAGTGACGGA	893
		TCGGGTACGGTT		AGCGGAACAGTC
		AGTGGGTGGGCA		AGCGGATGGGCA
		CAG		CAG
DGSPEKPFR	160	CAGAGTGATGGC	561	CAGAGTGACGGA	894
		AGTCCGGAGAAG		AGCCCAGAAAAA
		CCGTTTCGGGCAC		CCATTCAGAGCA
		AG		CAG
DGSQSTTGW	136	CAGAGTGATGGC	562	CAGAGTGACGGA	895
		AGTCAGTCTACTA		AGCCAAAGCACA
		CGGGGTGGGCAC		ACAGGATGGGCA
		AG		CAG
DGSSFYPPK	127	CAGAGTGATGGC	563	CAGAGTGACGGA	896
		AGTAGTTTTTATC		AGCAGCTTCTACC
		CTCCTAAGGCAC		CACCAAAAGCAC
		AG		AG
DGSSSYYDA	64	CAGAGTGATGGC	564	CAGAGTGACGGA	897
		AGTAGTTCTTATT		AGCAGCAGCTAC
		ATGATGCGGCAC		TACGACGCAGCA
		AG		CAG
DGSTERPFR	99	CAGAGTGATGGC	565	CAGAGTGACGGA	898
		TCTACGGAGAGG		AGCACAGAAAGA
		CCGTTTAGGGCA		CCATTCAGAGCA
		CAG		CAG
DGTAARLSS	132	CAGAGTGATGGC	566	CAGAGTGACGGA	899
		ACCGCGGCTCGG		ACAGCAGCAAGA
		CTGTCGTCGGCAC		CTCAGCAGCGCA
		AG		CAG
DGTADKPFR	63	CAGAGTGATGGC	567	CAGAGTGACGGA	900
		ACCGCTGATAAG		ACAGCAGACAAA
		CCGTTTCGGGCAC		CCATTCAGAGCA
		AG		CAG
DGTADRPFR	155	CAGAGTGATGGC	568	CAGAGTGACGGA	901
		ACGGCGGATCGT		ACAGCAGACAGA
		CCTTTTCGGGCAC		CCATTCAGAGCA
		AG		CAG
DGTAERPFR	140	CAGAGTGATGGC	569	CAGAGTGACGGA	902
		ACCGCGGAGAGG		ACAGCAGAAAGA
		CCTTTTAGGGCAC		CCATTCAGAGCA
		AG		CAG
DGTAIHLSS	67	CAGAGTGATGGC	570	CAGAGTGACGGA	903
		ACCGCGATTCATC		ACAGCAATCCAC
		TTTCGTCTGCACA		CTCAGCAGCGCA
		G		CAG
DGTAIYLSS	279	CAGAGTGATGGC	571	CAGAGTGACGGA	904
		ACCGCGATTTATC		ACAGCAATCTAC
		TGTCTTCTGCACA		CTCAGCAGCGCA
		G		CAG
DGTALMLSS	280	CAGAGTGATGGC	572	CAGAGTGACGGA	905
		ACCGCTCTTATGT		ACAGCACTCATG
		TGTCGTCTGCACA		CTCAGCAGCGCA
		G		CAG
DGTASISGW	281	CAGAGTGATGGC	573	CAGAGTGACGGA	906
		ACCGCGAGTATT		ACAGCAAGCATC
		AGTGGTTGGGCA		AGCGGATGGGCA
		CAG		CAG
DGTASTSGW	282	CAGAGTGATGGC	574	CAGAGTGACGGA	907
		ACCGCGTCGACG		ACAGCAAGCACA
		AGTGGGTGGGCA		AGCGGATGGGCA
		CAG		CAG
DGTASVTGW	283	CAGAGTGATGGC	575	CAGAGTGACGGA	908
		ACCGCGTCGGTG		ACAGCAAGCGTC
		ACGGGGTGGGCA		ACAGGATGGGCA
		CAG		CAG
DGTASYYDS	61	CAGAGTGATGGC	576	CAGAGTGACGGA	909
		ACCGCGAGTTATT		ACAGCAAGCTAC
		ATGATTCTGCACA		TACGACAGCGCA
		G		CAG
DGTATTMGW	284	CAGAGTGATGGC	577	CAGAGTGACGGA	910
		ACCGCGACGACG		ACAGCAACAACA
		ATGGGGTGGGCA		ATGGGATGGGCA
		CAG		CAG
DGTATTTGW	285	CAGAGTGATGGC	578	CAGAGTGACGGA	911
		ACCGCGACGACG		ACAGCAACAACA
		ACGGGTTGGGCA		ACAGGATGGGCA
		CAG		CAG
DGTAYRLSS	286	CAGAGTGATGGC	579	CAGAGTGACGGA	912
		ACCGCGTATCGTT		ACAGCATACAGA
		TGTCGTCTGCACA		CTCAGCAGCGCA
		G		CAG
DGTDKMWSI	287	CAGAGTGATGGC	580	CAGAGTGACGGA	913
		ACCGATAAGATG		ACAGACAAAATG
		TGGAGTATTGCA		TGGAGCATCGCA
		CAG		CAG
DGTGGIKGW	131	CAGAGTGATGGC	581	CAGAGTGACGGA	914
		ACCGGTGGTATT		ACAGGAGGAATC
		AAGGGGTGGGCA		AAAGGATGGGCA
		CAG		CAG
DGTGGIMGW	288	CAGAGTGATGGC	582	CAGAGTGACGGA	915
		ACCGGGGGGATT		ACAGGAGGAATC
		ATGGGTTGGGCA		ATGGGATGGGCA
		CAG		CAG
DGTGGISGW	289	CAGAGTGATGGC	583	CAGAGTGACGGA	916
		ACCGGTGGGATT		ACAGGAGGAATC
		TCGGGGTGGGCA		AGCGGATGGGCA
		CAG		CAG
DGTGGLAGW	290	CAGAGTGATGGC	584	CAGAGTGACGGA	917
		ACCGGGGGTCTT		ACAGGAGGACTC
		GCTGGTTGGGCA		GCAGGATGGGCA
		CAG		CAG
DGTGGLHGW	291	CAGAGTGATGGC	585	CAGAGTGACGGA	918
		ACCGGGGGGTTG		ACAGGAGGACTC
		CATGGTTGGGCA		CACGGATGGGCA
		CAG		CAG
DGTGGLQGW	292	CAGAGTGATGGC	586	CAGAGTGACGGA	919
		ACCGGGGGTTTG		ACAGGAGGACTC
		CAGGGTTGGGCA		CAAGGATGGGCA
		CAG		CAG
DGTGGLRGW	154	CAGAGTGATGGC	587	CAGAGTGACGGA	920
		ACCGGGGGTTTG		ACAGGAGGACTC
		CGTGGTTGGGCA		AGAGGATGGGCA
		CAG		CAG
DGTGGLSGW	293	CAGAGTGATGGC	588	CAGAGTGACGGA	921
		ACCGGTGGGTTG		ACAGGAGGACTC
		TCGGGTTGGGCA		AGCGGATGGGCA
		CAG		CAG
DGTGGLTGW	294	CAGAGTGATGGC	589	CAGAGTGACGGA	922
		ACCGGGGGGTTG		ACAGGAGGACTC
		ACGGGTTGGGCA		ACAGGATGGGCA
		CAG		CAG
DGTGGTKGW	107	CAGAGTGATGGC	590	CAGAGTGACGGA	923
		ACCGGTGGGACT		ACAGGAGGAACA
		AAGGGTTGGGCA		AAAGGATGGGCA
		CAG		CAG
DGTGGTSGW	295	CAGAGTGATGGC	591	CAGAGTGACGGA	924
		ACCGGGGGGACG		ACAGGAGGAACA
		AGTGGTTGGGCA		AGCGGATGGGCA
		CAG		CAG
DGTGGVHGW	296	CAGAGTGATGGC	592	CAGAGTGACGGA	925
		ACCGGTGGGGTG		ACAGGAGGAGTC
		CATGGTTGGGCA		CACGGATGGGCA
		CAG		CAG
DGTGGVMGW	297	CAGAGTGATGGC	593	CAGAGTGACGGA	926
		ACCGGTGGTGTT		ACAGGAGGAGTC
		ATGGGGTGGGCA		ATGGGATGGGCA
		CAG		CAG
DGTGGVSGW	298	CAGAGTGATGGC	594	CAGAGTGACGGA	927
		ACCGGGGGGGTG		ACAGGAGGAGTC
		TCTGGTTGGGCAC		AGCGGATGGGCA
		AG		CAG
DGTGGVTGW	299	CAGAGTGATGGC	595	CAGAGTGACGGA	928
		ACCGGTGGTGTG		ACAGGAGGAGTC
		ACGGGGTGGGCA		ACAGGATGGGCA
		CAG		CAG
DGTGGVYGW	300	CAGAGTGATGGC	596	CAGAGTGACGGA	929
		ACCGGTGGTGTG		ACAGGAGGAGTC
		TATGGGTGGGCA		TACGGATGGGCA
		CAG		CAG
DGTGNLQGW	301	CAGAGTGATGGC	597	CAGAGTGACGGA	930
		ACCGGTAATTTGC		ACAGGAAACCTC
		AGGGTTGGGCAC		CAAGGATGGGCA
		AG		CAG
DGTGNLRGW	133	CAGAGTGATGGC	598	CAGAGTGACGGA	931
		ACCGGGAATCTT		ACAGGAAACCTC
		AGGGGGTGGGCA		AGAGGATGGGCA
		CAG		CAG
DGTGNLSGW	302	CAGAGTGATGGC	599	CAGAGTGACGGA	932
		ACCGGGAATTTG		ACAGGAAACCTC
		AGTGGGTGGGCA		AGCGGATGGGCA
		CAG		CAG
DGTGNTHGW	72	CAGAGTGATGGC	600	CAGAGTGACGGA	933
		ACCGGGAATACT		ACAGGAAACACA
		CATGGGTGGGCA		CACGGATGGGCA
		CAG		CAG
DGTGNTRGW	94	CAGAGTGATGGC	601	CAGAGTGACGGA	934
		ACCGGGAATACT		ACAGGAAACACA
		CGGGGGTGGGCA		AGAGGATGGGCA
		CAG		CAG
DGTGNTSGW	137	CAGAGTGATGGC	602	CAGAGTGACGGA	935
		ACCGGTAATACT		ACAGGAAACACA
		AGTGGTTGGGCA		AGCGGATGGGCA
		CAG		CAG
DGTGNVSGW	303	CAGAGTGATGGC	603	CAGAGTGACGGA	936
		ACCGGGAATGTG		ACAGGAAACGTC
		TCGGGGTGGGCA		AGCGGATGGGCA
		CAG		CAG
DGTGNVTGW	69	CAGAGTGATGGC	604	CAGAGTGACGGA	937
		ACCGGTAATGTG		ACAGGAAACGTC
		ACGGGGTGGGCA		ACAGGATGGGCA
		CAG		CAG
DGTGQLVGW	304	CAGAGTGATGGC	605	CAGAGTGACGGA	938
		ACCGGGCAGCTT		ACAGGACAACTC
		GTGGGTTGGGCA		GTCGGATGGGCA
		CAG		CAG
DGTGQTIGW	305	CAGAGTGATGGC	606	CAGAGTGACGGA	939
		ACCGGTCAGACG		ACAGGACAAACA
		ATTGGTTGGGCA		ATCGGATGGGCA
		CAG		CAG
DGTGQVTGW	68	CAGAGTGATGGC	607	CAGAGTGACGGA	940
		ACCGGGCAGGTG		ACAGGACAAGTC
		ACTGGGTGGGCA		ACAGGATGGGCA
		CAG		CAG
DGTGRLTGW	159	CAGAGTGATGGC	608	CAGAGTGACGGA	941
		ACCGGTCGGTTG		ACAGGAAGACTC
		ACGGGTTGGGCA		ACAGGATGGGCA
		CAG		CAG
DGTGRTVGW	117	CAGAGTGATGGC	609	CAGAGTGACGGA	942
		ACCGGTCGGACT		ACAGGAAGAACA
		GTTGGGTGGGCA		GTCGGATGGGCA
		CAG		CAG
DGTGSGMMT	306	CAGAGTGATGGC	610	CAGAGTGACGGA	943
		ACCGGTTCGGGT		ACAGGAAGCGGA
		ATGATGACGGCA		ATGATGACAGCA
		CAG		CAG
DGTGSISGW	307	CAGAGTGATGGC	611	CAGAGTGACGGA	944
		ACCGGGTCGATT		ACAGGAAGCATC
		AGTGGGTGGGCA		AGCGGATGGGCA
		CAG		CAG
DGTGSLAGW	308	CAGAGTGATGGC	612	CAGAGTGACGGA	945
		ACCGGTTCTTTGG		ACAGGAAGCCTC
		CGGGGTGGGCAC		GCAGGATGGGCA
		AG		CAG
DGTGSLNGW	309	CAGAGTGATGGC	613	CAGAGTGACGGA	946
		ACCGGGTCTTTGA		ACAGGAAGCCTC
		ATGGGTGGGCAC		AACGGATGGGCA
		AG		CAG
DGTGSLQGW	310	CAGAGTGATGGC	614	CAGAGTGACGGA	947
		ACCGGGTCGCTG		ACAGGAAGCCTC
		CAGGGTTGGGCA		CAAGGATGGGCA
		CAG		CAG
DGTGSLSGW	311	CAGAGTGATGGC	615	CAGAGTGACGGA	948
		ACCGGGAGTCTG		ACAGGAAGCCTC
		TCGGGGTGGGCA		AGCGGATGGGCA
		CAG		CAG
DGTGSLVGW	312	CAGAGTGATGGC	616	CAGAGTGACGGA	949
		ACCGGGTCGTTG		ACAGGAAGCCTC
		GTGGGTTGGGCA		GTCGGATGGGCA
		CAG		CAG
DGTGSTHGW	119	CAGAGTGATGGC	617	CAGAGTGACGGA	950
		ACCGGGAGTACG		ACAGGAAGCACA
		CATGGGTGGGCA		CACGGATGGGCA
		CAG		CAG
DGTGSTKGW	313	CAGAGTGATGGC	618	CAGAGTGACGGA	951
		ACCGGGAGTACT		ACAGGAAGCACA
		AAGGGGTGGGCA		AAAGGATGGGCA
		CAG		CAG
DGTGSTMGW	314	CAGAGTGATGGC	619	CAGAGTGACGGA	952
		ACCGGTTCTACTA		ACAGGAAGCACA
		TGGGTTGGGCAC		ATGGGATGGGCA
		AG		CAG
DGTGSTQGW	315	CAGAGTGATGGC	620	CAGAGTGACGGA	953
		ACCGGTAGTACG		ACAGGAAGCACA
		CAGGGTTGGGCA		CAAGGATGGGCA
		CAG		CAG
DGTGSTSGW	316	CAGAGTGATGGC	621	CAGAGTGACGGA	954
		ACCGGGAGTACT		ACAGGAAGCACA
		TCGGGGTGGGCA		AGCGGATGGGCA
		CAG		CAG
DGTGSTTGW	134	CAGAGTGATGGC	622	CAGAGTGACGGA	955
		ACCGGGAGTACG		ACAGGAAGCACA
		ACGGGGTGGGCA		ACAGGATGGGCA
		CAG		CAG
DGTGSVMGW	317	CAGAGTGATGGC	623	CAGAGTGACGGA	956
		ACCGGTTCGGTTA		ACAGGAAGCGTC
		TGGGGTGGGCAC		ATGGGATGGGCA
		AG		CAG
DGTGSVTGW	318	CAGAGTGATGGC	624	CAGAGTGACGGA	957
		ACCGGGTCTGTG		ACAGGAAGCGTC
		ACTGGGTGGGCA		ACAGGATGGGCA
		CAG		CAG
DGTGTLAGW	319	CAGAGTGATGGC	625	CAGAGTGACGGA	958
		ACCGGGACGCTT		ACAGGAACACTC
		GCGGGGTGGGCA		GCAGGATGGGCA
		CAG		CAG
DGTGTLHGW	320	CAGAGTGATGGC	626	CAGAGTGACGGA	959
		ACCGGTACTTTGC		ACAGGAACACTC
		ATGGTTGGGCAC		CACGGATGGGCA
		AG		CAG
DGTGTLKGW	321	CAGAGTGATGGC	627	CAGAGTGACGGA	960
		ACCGGTACTCTTA		ACAGGAACACTC
		AGGGTTGGGCAC		AAAGGATGGGCA
		AG		CAG
DGTGTLSGW	322	CAGAGTGATGGC	628	CAGAGTGACGGA	961
		ACCGGGACTCTG		ACAGGAACACTC
		TCGGGTTGGGCA		AGCGGATGGGCA
		CAG		CAG
DGTGTTLGW	323	CAGAGTGATGGC	629	CAGAGTGACGGA	962
		ACCGGGACTACG		ACAGGAACAACA
		CTGGGGTGGGCA		CTCGGATGGGCA
		CAG		CAG
DGTGTTMGW	324	CAGAGTGATGGC	630	CAGAGTGACGGA	963
		ACCGGGACTACT		ACAGGAACAACA
		ATGGGTTGGGCA		ATGGGATGGGCA
		CAG		CAG
DGTGTTTGW	130	CAGAGTGATGGC	631	CAGAGTGACGGA	964
		ACCGGGACTACT		ACAGGAACAACA
		ACGGGGTGGGCA		ACAGGATGGGCA
		CAG		CAG
DGTGTTVGW	74	CAGAGTGATGGC	632	CAGAGTGACGGA	965
		ACCGGTACTACG		ACAGGAACAACA
		GTGGGGTGGGCA		GTCGGATGGGCA
		CAG		CAG
DGTGTTYGW	325	CAGAGTGATGGC	633	CAGAGTGACGGA	966
		ACCGGGACGACG		ACAGGAACAACA
		TATGGTTGGGCA		TACGGATGGGCA
		CAG		CAG
DGTGTVHGW	326	CAGAGTGATGGC	634	CAGAGTGACGGA	967
		ACCGGTACGGTT		ACAGGAACAGTC
		CATGGTTGGGCA		CACGGATGGGCA
		CAG		CAG
DGTGTVQGW	327	CAGAGTGATGGC	635	CAGAGTGACGGA	968
		ACCGGGACTGTG		ACAGGAACAGTC
		CAGGGGTGGGCA		CAAGGATGGGCA
		CAG		CAG
DGTGTVSGW	328	CAGAGTGATGGC	636	CAGAGTGACGGA	969
		ACCGGTACTGTTT		ACAGGAACAGTC
		CTGGTTGGGCAC		AGCGGATGGGCA
		AG		CAG
DGTGTVTGW	329	CAGAGTGATGGC	637	CAGAGTGACGGA	970
		ACCGGTACTGTTA		ACAGGAACAGTC
		CTGGGTGGGCAC		ACAGGATGGGCA
		AG		CAG
DGTHARLSS	330	CAGAGTGATGGC	638	CAGAGTGACGGA	971
		ACCCATGCGAGG		ACACACGCAAGA
		TTGTCTTCGGCAC		CTCAGCAGCGCA
		AG		CAG
DGTHAYMAS	153	CAGAGTGATGGC	639	CAGAGTGACGGA	972
		ACCCATGCTTATA		ACACACGCATAC
		TGGCGTCTGCAC		ATGGCAAGCGCA
		AG		CAG
DGTHFAPPR	112	CAGAGTGATGGC	640	CAGAGTGACGGA	973
		ACCCATTTTGCGC		ACACACTTCGCA
		CGCCGCGTGCAC		CCACCAAGAGCA
		AG		CAG
DGTHIHLSS	162	CAGAGTGATGGC	641	CAGAGTGACGGA	974
		ACCCATATTCATC		ACACACATCCAC
		TGAGTAGTGCAC		CTCAGCAGCGCA
		AG		CAG
DGTHIRALS	331	CAGAGTGATGGC	642	CAGAGTGACGGA	975
		ACCCATATTAGG		ACACACATCAGA
		GCTCTGAGTGCA		GCACTCAGCGCA
		CAG		CAG
DGTHIRLAS	332	CAGAGTGATGGC	643	CAGAGTGACGGA	976
		ACCCATATTCGTT		ACACACATCAGA
		TGGCGAGTGCAC		CTCGCAAGCGCA
		AG		CAG
DGTHLQPFR	333	CAGAGTGATGGC	644	CAGAGTGACGGA	977
		ACCCATCTGCAG		ACACACCTCCAA
		CCGTTTAGGGCA		CCATTCAGAGCA
		CAG		CAG
DGTHSFYDA	334	CAGAGTGATGGC	645	CAGAGTGACGGA	978
		ACCCATAGTTTTT		ACACACAGCTTCT
		ATGATGCGGCAC		ACGACGCAGCAC
		AG		AG
DGTHSTTGW	145	CAGAGTGATGGC	646	CAGAGTGACGGA	979
		ACCCATTCTACTA		ACACACAGCACA
		CGGGTTGGGCAC		ACAGGATGGGCA
		AG		CAG
DGTHTRTGW	90	CAGAGTGATGGC	647	CAGAGTGACGGA	980
		ACCCATACGCGG		ACACACACAAGA
		ACGGGTTGGGCA		ACAGGATGGGCA
		CAG		CAG
DGTHVRALS	335	CAGAGTGATGGC	648	CAGAGTGACGGA	981
		ACCCATGTTAGG		ACACACGTCAGA
		GCGTTGTCGGCA		GCACTCAGCGCA
		CAG		CAG
DGTHVYMAS	336	CAGAGTGATGGC	649	CAGAGTGACGGA	982
		ACCCATGTTTATA		ACACACGTCTAC
		TGGCTAGTGCAC		ATGGCAAGCGCA
		AG		CAG
DGTHVYMSS	337	CAGAGTGATGGC	650	CAGAGTGACGGA	983
		ACCCATGTGTATA		ACACACGTCTAC
		TGTCTAGTGCACA		ATGAGCAGCGCA
		G		CAG
DGTIALPFK	338	CAGAGTGATGGC	651	CAGAGTGACGGA	984
		ACCATTGCGCTTC		ACAATCGCACTC
		CGTTTAAGGCAC		CCATTCAAAGCA
		AG		CAG
DGTIALPFR	339	CAGAGTGATGGC	652	CAGAGTGACGGA	985
		ACCATTGCTTTGC		ACAATCGCACTC
		CGTTTAGGGCAC		CCATTCAGAGCA
		AG		CAG
DGTIATRYV	340	CAGAGTGATGGC	653	CAGAGTGACGGA	986
		ACCATTGCGACG		ACAATCGCAACA
		CGGTATGTGGCA		AGATACGTCGCA
		CAG		CAG
DGTIERPFR	87	CAGAGTGATGGC	654	CAGAGTGACGGA	987
		ACCATTGAGCGG		ACAATCGAAAGA
		CCTTTTCGTGCAC		CCATTCAGAGCA
		AG		CAG
DGTIGYAYV	341	CAGAGTGATGGC	655	CAGAGTGACGGA	988
		ACCATTGGTTATG		ACAATCGGATAC
		CGTATGTTGCACA		GCATACGTCGCA
		G		CAG
DGTIQAPFK	342	CAGAGTGATGGC	656	CAGAGTGACGGA	989
		ACCATTCAGGCTC		ACAATCCAAGCA
		CGTTTAAGGCAC		CCATTCAAAGCA
		AG		CAG
DGTIRLPFK	343	CAGAGTGATGGC	657	CAGAGTGACGGA	990
		ACCATTCGTCTTC		ACAATCAGACTC
		CTTTTAAGGCACA		CCATTCAAAGCA
		G		CAG
DGTISKEVG	344	CAGAGTGATGGC	658	CAGAGTGACGGA	991
		ACCATTTCTAAGG		ACAATCAGCAAA
		AGGTGGGGGCAC		GAAGTCGGAGCA
		AG		CAG
DGTISQPFK	105	CAGAGTGATGGC	659	CAGAGTGACGGA	992
		ACCATTTCGCAGC		ACAATCAGCCAA
		CTTTTAAGGCACA		CCATTCAAAGCA
		G		CAG
DGTKIQLSS	146	CAGAGTGATGGC	660	CAGAGTGACGGA	993
		ACCAAGATTCAG		ACAAAAATCCAA
		CTGTCTAGTGCAC		CTCAGCAGCGCA
		AG		CAG
DGTKIRLSS	111	CAGAGTGATGGC	661	CAGAGTGACGGA	994
		ACCAAGATTCGG		ACAAAAATCAGA
		TTGTCGTCTGCAC		CTCAGCAGCGCA
		AG		CAG
DGTKLMLSS	157	CAGAGTGATGGC	662	CAGAGTGACGGA	995
		ACCAAGCTGATG		ACAAAACTCATG
		TTGAGTAGTGCA		CTCAGCAGCGCA
		CAG		CAG
DGTKLRLSS	118	CAGAGTGATGGC	663	CAGAGTGACGGA	996
		ACCAAGTTGAGG		ACAAAACTCAGA
		CTTAGTTCTGCAC		CTCAGCAGCGCA
		AG		CAG
DGTKMVLQL	142	CAGAGTGATGGC	664	CAGAGTGACGGA	997
		ACCAAGATGGTG		ACAAAAATGGTC
		TTGCAGCTGGCA		CTCCAACTCGCAC
		CAG		AG
DGTKSLVQL	345	CAGAGTGATGGC	665	CAGAGTGACGGA	998
		ACCAAGAGTCTT		ACAAAAAGCCTC
		GTGCAGCTTGCA		GTCCAACTCGCA
		CAG		CAG
DGTKVLVQL	122	CAGAGTGATGGC	666	CAGAGTGACGGA	999
		ACCAAGGTGCTG		ACAAAAGTCCTC
		GTGCAGTTGGCA		GTCCAACTCGCA
		CAG		CAG
DGTLAAPFK	120	CAGAGTGATGGC	667	CAGAGTGACGGA	1000
		ACCTTGGCTGCTC		ACACTCGCAGCA
		CTTTTAAGGCACA		CCATTCAAAGCA
		G		CAG
DGTLAVNFK	346	CAGAGTGATGGG	668	CAGAGTGACGGA	1001
		ACTTTGGCGGTG		ACACTCGCAGTC
		AATTTTAAGGCA		AACTTCAAAGCA
		CAG		CAG
DGTLAVPFK	71	CAGAGTGATGGG	669	CAGAGTGACGGA	1002
(PHP.eB)		ACTTTGGCGGTGC		ACACTCGCAGTC
		CTTTTAAGGCACA		CCATTCAAAGCA
		G		CAG
DGTLAYPFK	347	CAGAGTGATGGC	670	CAGAGTGACGGA	1003
		ACCCTTGCGTATC		ACACTCGCATAC
		CTTTTAAGGCACA		CCATTCAAAGCA
		G		CAG
DGTLERPFR	156	CAGAGTGATGGC	671	CAGAGTGACGGA	1004
		ACCCTGGAGAGG		ACACTCGAAAGA
		CCGTTTCGGGCAC		CCATTCAGAGCA
		AG		CAG
DGTLEVHFK	348	CAGAGTGATGGG	672	CAGAGTGACGGA	1005
		ACTTTGGAGGTG		ACACTCGAAGTC
		CATTTTAAGGCAC		CACTTCAAAGCA
		AG		CAG
DGTLLRLSS	121	CAGAGTGATGGC	673	CAGAGTGACGGA	1006
		ACCTTGCTGAGG		ACACTCCTCAGA
		CTGAGTAGTGCA		CTCAGCAGCGCA
		CAG		CAG
DGTLNNPFR	109	CAGAGTGATGGC	674	CAGAGTGACGGA	1007
		ACCTTGAATAATC		ACACTCAACAAC
		CGTTTAGGGCAC		CCATTCAGAGCA
		AG		CAG
DGTLQQPFR	89	CAGAGTGATGGC	675	CAGAGTGACGGA	1008
		ACCTTGCAGCAG		ACACTCCAACAA
		CCGTTTCGGGCAC		CCATTCAGAGCA
		AG		CAG
DGTLSQPFR	65	CAGAGTGATGGC	676	CAGAGTGACGGA	1009
		ACCCTGTCTCAGC		ACACTCAGCCAA
		CTTTTAGGGCACA		CCATTCAGAGCA
		G		CAG
DGTLSRTLW	349	CAGAGTGATGGC	677	CAGAGTGACGGA	1010
		ACCTTGTCGCGTA		ACACTCAGCAGA
		CGCTTTGGGCAC		ACACTCTGGGCA
		AG		CAG
DGTLSSPFR	350	CAGAGTGATGGC	678	CAGAGTGACGGA	1011
		ACCCTGTCTAGTC		ACACTCAGCAGC
		CGTTTAGGGCAC		CCATTCAGAGCA
		AG		CAG
DGTLTVPFR	351	CAGAGTGATGGC	679	CAGAGTGACGGA	1012
		ACCTTGACGGTTC		ACACTCACAGTC
		CTTTTCGGGCACA		CCATTCAGAGCA
		G		CAG
DGTLVAPFR	352	CAGAGTGATGGC	680	CAGAGTGACGGA	1013
		ACCCTTGTTGCGC		ACACTCGTCGCA
		CGTTTAGGGCAC		CCATTCAGAGCA
		AG		CAG
DGTMDKPFR	70	CAGAGTGATGGC	681	CAGAGTGACGGA	1014
		ACGATGGATAAG		ACAATGGACAAA
		CCTTTTAGGGCAC		CCATTCAGAGCA
		AG		CAG
DGTMDRPFK	102	CAGAGTGATGGC	682	CAGAGTGACGGA	1015
		ACCATGGATAGG		ACAATGGACAGA
		CCGTTTAAGGCA		CCATTCAAAGCA
		CAG		CAG
DGTMLRLSS	148	CAGAGTGATGGC	683	CAGAGTGACGGA	1016
		ACCATGTTGCGTC		ACAATGCTCAGA
		TTAGTTCGGCACA		CTCAGCAGCGCA
		G		CAG
DGTMQLTGW	353	CAGAGTGATGGC	684	CAGAGTGACGGA	1017
		ACCATGCAGCTT		ACAATGCAACTC
		ACGGGGTGGGCA		ACAGGATGGGCA
		CAG		CAG
DGTNGLKGW	76	CAGAGTGATGGC	685	CAGAGTGACGGA	1018
		ACCAATGGTCTG		ACAAACGGACTC
		AAGGGGTGGGCA		AAAGGATGGGCA
		CAG		CAG
DGTNSISGW	354	CAGAGTGATGGC	686	CAGAGTGACGGA	1019
		ACCAATAGTATT		ACAAACAGCATC
		AGTGGGTGGGCA		AGCGGATGGGCA
		CAG		CAG
DGTNSLSGW	355	CAGAGTGATGGC	687	CAGAGTGACGGA	1020
		ACCAATTCTCTGT		ACAAACAGCCTC
		CGGGTTGGGCAC		AGCGGATGGGCA
		AG		CAG
DGTNSTTGW	143	CAGAGTGATGGC	688	CAGAGTGACGGA	1021
		ACCAATTCTACG		ACAAACAGCACA
		ACGGGTTGGGCA		ACAGGATGGGCA
		CAG		CAG
DGTNSVTGW	356	CAGAGTGATGGC	689	CAGAGTGACGGA	1022
		ACCAATAGTGTT		ACAAACAGCGTC
		ACGGGTTGGGCA		ACAGGATGGGCA
		CAG		CAG
DGTNTINGW	124	CAGAGTGATGGC	690	CAGAGTGACGGA	1023
		ACCAATACTATTA		ACAAACACAATC
		ATGGGTGGGCAC		AACGGATGGGCA
		AG		CAG
DGTNTLGGW	357	CAGAGTGATGGC	691	CAGAGTGACGGA	1024
		ACCAATACGTTG		ACAAACACACTC
		GGGGGGTGGGCA		GGAGGATGGGCA
		CAG		CAG
DGTNTTHGW	113	CAGAGTGATGGC	692	CAGAGTGACGGA	1025
		ACCAATACTACTC		ACAAACACAACA
		ATGGGTGGGCAC		CACGGATGGGCA
		AG		CAG
DGTNYRLSS	358	CAGAGTGATGGC	693	CAGAGTGACGGA	1026
		ACCAATTATAGG		ACAAACTACAGA
		CTGTCGAGTGCA		CTCAGCAGCGCA
		CAG		CAG
DGTQALSGW	359	CAGAGTGATGGC	694	CAGAGTGACGGA	1027
		ACCCAGGCGCTG		ACACAAGCACTC
		TCGGGGTGGGCA		AGCGGATGGGCA
		CAG		CAG
DGTQFRLSS	129	CAGAGTGATGGC	695	CAGAGTGACGGA	1028
		ACCCAGTTTAGGT		ACACAATTCAGA
		TGTCTTCGGCACA		CTCAGCAGCGCA
		G		CAG
DGTQFSPPR	108	CAGAGTGATGGC	696	CAGAGTGACGGA	1029
		ACCCAGTTTAGTC		ACACAATTCAGC
		CTCCGCGTGCAC		CCACCAAGAGCA
		AG		CAG
DGTQGLKGW	158	CAGAGTGATGGC	697	CAGAGTGACGGA	1030
		ACCCAGGGGCTG		ACACAAGGACTC
		AAGGGGTGGGCA		AAAGGATGGGCA
		CAG		CAG
DGTQTTSGW	360	CAGAGTGATGGC	698	CAGAGTGACGGA	1031
		ACCCAGACTACG		ACACAAACAACA
		AGTGGGTGGGCA		AGCGGATGGGCA
		CAG		CAG
DGTRALTGW	361	CAGAGTGATGGC	699	CAGAGTGACGGA	1032
		ACCAGGGCTCTT		ACAAGAGCACTC
		ACGGGTTGGGCA		ACAGGATGGGCA
		CAG		CAG
DGTRFSLSS	362	CAGAGTGATGGC	700	CAGAGTGACGGA	1033
		ACCCGGTTTTCGC		ACAAGATTCAGC
		TTTCGAGTGCACA		CTCAGCAGCGCA
		G		CAG
DGTRGLSGW	363	CAGAGTGATGGC	701	CAGAGTGACGGA	1034
		ACCAGGGGGTTG		ACAAGAGGACTC
		TCGGGGTGGGCA		AGCGGATGGGCA
		CAG		CAG
DGTRIGLSS	364	CAGAGTGATGGC	702	CAGAGTGACGGA	1035
		ACCAGGATTGGG		ACAAGAATCGGA
		CTGAGTAGTGCA		CTCAGCAGCGCA
		CAG		CAG
DGTRLHLAS	365	CAGAGTGATGGC	703	CAGAGTGACGGA	1036
		ACCAGGCTTCATC		ACAAGACTCCAC
		TGGCGAGTGCAC		CTCGCAAGCGCA
		AG		CAG
DGTRLHLSS	366	CAGAGTGATGGC	704	CAGAGTGACGGA	1037
		ACCAGGCTTCATC		ACAAGACTCCAC
		TGTCGTCGGCAC		CTCAGCAGCGCA
		AG		CAG
DGTRLLLSS	367	CAGAGTGATGGC	705	CAGAGTGACGGA	1038
		ACCCGTTTGCTGC		ACAAGACTCCTC
		TGTCGAGTGCAC		CTCAGCAGCGCA
		AG		CAG
DGTRLMLSS	368	CAGAGTGATGGC	706	CAGAGTGACGGA	1039
		ACCCGTTTGATGC		ACAAGACTCATG
		TTTCTAGTGCACA		CTCAGCAGCGCA
		G		CAG
DGTRLNLSS	369	CAGAGTGATGGC	707	CAGAGTGACGGA	1040
		ACCCGTTTGAATC		ACAAGACTCAAC
		TTAGTTCGGCACA		CTCAGCAGCGCA
		G		CAG
DGTRMVVQL	370	CAGAGTGATGGC	708	CAGAGTGACGGA	1041
		ACCCGGATGGTT		ACAAGAATGGTC
		GTTCAGCTTGCAC		GTCCAACTCGCA
		AG		CAG
DGTRNMYEG	135	CAGAGTGATGGC	709	CAGAGTGACGGA	1042
		ACCCGTAATATGT		ACAAGAAACATG
		ATGAGGGGGCAC		TACGAAGGAGCA
		AG		CAG
DGTRSITGW	371	CAGAGTGATGGC	710	CAGAGTGACGGA	1043
		ACCAGGAGTATT		ACAAGAAGCATC
		ACGGGGTGGGCA		ACAGGATGGGCA
		CAG		CAG
DGTRSLHGW	372	CAGAGTGATGGC	711	CAGAGTGACGGA	1044
		ACCAGGAGTTTG		ACAAGAAGCCTC
		CATGGGTGGGCA		CACGGATGGGCA
		CAG		CAG
DGTRSTTGW	373	CAGAGTGATGGC	712	CAGAGTGACGGA	1045
		ACCCGGAGTACT		ACAAGAAGCACA
		ACGGGTTGGGCA		ACAGGATGGGCA
		CAG		CAG
DGTRTTTGW	106	CAGAGTGATGGC	713	CAGAGTGACGGA	1046
		ACCCGTACTACG		ACAAGAACAACA
		ACGGGTTGGGCA		ACAGGATGGGCA
		CAG		CAG
DGTRTVTGW	374	CAGAGTGATGGC	714	CAGAGTGACGGA	1047
		ACCCGGACGGTG		ACAAGAACAGTC
		ACTGGTTGGGCA		ACAGGATGGGCA
		CAG		CAG
DGTRTVVQL	375	CAGAGTGATGGC	715	CAGAGTGACGGA	1048
		ACCCGTACTGTG		ACAAGAACAGTC
		GTGCAGTTGGCA		GTCCAACTCGCA
		CAG		CAG
DGTRVHLSS	376	CAGAGTGATGGC	716	CAGAGTGACGGA	1049
		ACCCGGGTGCAT		ACAAGAGTCCAC
		CTTTCTAGTGCAC		CTCAGCAGCGCA
		AG		CAG
DGTSFPYAR	86	CAGAGTGATGGC	717	CAGAGTGACGGA	1050
		ACCTCGTTTCCGT		ACAAGCTTCCCAT
		ATGCTCGGGCAC		ACGCAAGAGCAC
		AG		AG
DGTSFTPPK	81	CAGAGTGATGGC	718	CAGAGTGACGGA	1051
		ACCTCGTTTACGC		ACAAGCTTCACA
		CGCCTAAGGCAC		CCACCAAAAGCA
		AG		CAG
DGTSFTPPR	88	CAGAGTGATGGC	719	CAGAGTGACGGA	1052
		ACCTCGTTTACTC		ACAAGCTTCACA
		CGCCGCGGGCAC		CCACCAAGAGCA
		AG		CAG
DGTSGLHGW	377	CAGAGTGATGGC	720	CAGAGTGACGGA	1053
		ACCTCTGGGTTGC		ACAAGCGGACTC
		ATGGGTGGGCAC		CACGGATGGGCA
		AG		CAG
DGTSGLKGW	101	CAGAGTGATGGC	721	CAGAGTGACGGA	1054
		ACCAGTGGGCTT		ACAAGCGGACTC
		AAGGGGTGGGCA		AAAGGATGGGCA
		CAG		CAG
DGTSIHLSS	378	CAGAGTGATGGC	722	CAGAGTGACGGA	1055
		ACCTCGATTCATT		ACAAGCATCCAC
		TGAGTAGTGCAC		CTCAGCAGCGCA
		AG		CAG
DGTSIMLSS	379	CAGAGTGATGGC	723	CAGAGTGACGGA	1056
		ACCTCGATTATGT		ACAAGCATCATG
		TGAGTTCTGCACA		CTCAGCAGCGCA
		G		CAG
DGTSLRLSS	166	CAGAGTGATGGC	724	CAGAGTGACGGA	1057
		ACCTCTTTGCGGC		ACAAGCCTCAGA
		TTTCTTCTGCACA		CTCAGCAGCGCA
		G		CAG
DGTSNYGAR	380	CAGAGTGATGGC	725	CAGAGTGACGGA	1058
		ACCTCTAATTATG		ACAAGCAACTAC
		GGGCGCGGGCAC		GGAGCAAGAGCA
		AG		CAG
DGTSSYYDA	381	CAGAGTGATGGC	726	CAGAGTGACGGA	1059
		ACCAGTTCGTATT		ACAAGCAGCTAC
		ATGATGCGGCAC		TACGACGCAGCA
		AG		CAG
DGTSSYYDS	59	CAGAGTGATGGC	727	CAGAGTGACGGA	1060
		ACCTCGAGTTATT		ACAAGCAGCTAC
		ATGATTCTGCACA		TACGACAGCGCA
		G		CAG
DGTSTISGW	382	CAGAGTGATGGC	728	CAGAGTGACGGA	1061
		ACCTCTACGATTT		ACAAGCACAATC
		CTGGTTGGGCAC		AGCGGATGGGCA
		AG		CAG
DGTSTITGW	383	CAGAGTGATGGC	729	CAGAGTGACGGA	1062
		ACCAGTACTATTA		ACAAGCACAATC
		CGGGTTGGGCAC		ACAGGATGGGCA
		AG		CAG
DGTSTLHGW	384	CAGAGTGATGGC	730	CAGAGTGACGGA	1063
		ACCTCGACGTTGC		ACAAGCACACTC
		ATGGGTGGGCAC		CACGGATGGGCA
		AG		CAG
DGTSTLRGW	385	CAGAGTGATGGC	731	CAGAGTGACGGA	1064
		ACCTCTACTCTGC		ACAAGCACACTC
		GTGGGTGGGCAC		AGAGGATGGGCA
		AG		CAG
DGTSTLSGW	386	CAGAGTGATGGC	732	CAGAGTGACGGA	1065
		ACCTCGACGCTGT		ACAAGCACACTC
		CGGGGTGGGCAC		AGCGGATGGGCA
		AG		CAG
DGTSYVPPK	97	CAGAGTGATGGC	733	CAGAGTGACGGA	1066
		ACCTCTTATGTGC		ACAAGCTACGTC
		CGCCGAAGGCAC		CCACCAAAAGCA
		AG		CAG
DGTSYVPPR	78	CAGAGTGATGGC	734	CAGAGTGACGGA	1067
		ACCAGTTATGTGC		ACAAGCTACGTC
		CGCCTCGGGCAC		CCACCAAGAGCA
		AG		CAG
DGTTATYYK	387	CAGAGTGATGGC	735	CAGAGTGACGGA	1068
		ACCACGGCGACT		ACAACAGCAACA
		TATTATAAGGCA		TACTACAAAGCA
		CAG		CAG
DGTTFTPPR	79	CAGAGTGATGGC	736	CAGAGTGACGGA	1069
		ACCACTTTTACTC		ACAACATTCACA
		CTCCTCGGGCAC		CCACCAAGAGCA
		AG		CAG
DGTTLAPFR	388	CAGAGTGATGGC	737	CAGAGTGACGGA	1070
		ACCACTCTGGCTC		ACAACACTCGCA
		CTTTTAGGGCACA		CCATTCAGAGCA
		G		CAG
DGTTLVPPR	116	CAGAGTGATGGC	738	CAGAGTGACGGA	1071
		ACCACTTTGGTTC		ACAACACTCGTC
		CGCCGCGTGCAC		CCACCAAGAGCA
		AG		CAG
DGTTSKTLW	389	CAGAGTGATGGC	739	CAGAGTGACGGA	1072
		ACCACGAGTAAG		ACAACAAGCAAA
		ACGCTTTGGGCA		ACACTCTGGGCA
		CAG		CAG
DGTTSRTLW	390	CAGAGTGATGGC	740	CAGAGTGACGGA	1073
		ACCACTTCTAGG		ACAACAAGCAGA
		ACTTTGTGGGCAC		ACACTCTGGGCA
		AG		CAG
DGTTTRSLY	391	CAGAGTGATGGC	741	CAGAGTGACGGA	1074
		ACCACGACTCGT		ACAACAACAAGA
		AGTTTGTATGCAC		AGCCTCTACGCA
		AG		CAG
DGTTTTTGW	392	CAGAGTGATGGC	742	CAGAGTGACGGA	1075
		ACCACTACGACT		ACAACAACAACA
		ACGGGTTGGGCA		ACAGGATGGGCA
		CAG		CAG
DGTTTYGAR	77	CAGAGTGATGGC	743	CAGAGTGACGGA	1076
		ACCACTACGTAT		ACAACAACATAC
		GGGGCTCGTGCA		GGAGCAAGAGCA
		CAG		CAG
DGTTWTPPR	139	CAGAGTGATGGC	744	CAGAGTGACGGA	1077
		ACCACTTGGACG		ACAACATGGACA
		CCGCCGCGTGCA		CCACCAAGAGCA
		CAG		CAG
DGTTYMLSS	393	CAGAGTGATGGC	745	CAGAGTGACGGA	1078
		ACCACGTATATG		ACAACATACATG
		CTTAGTAGTGCAC		CTCAGCAGCGCA
		AG		CAG
DGTTYVPPR	75	CAGAGTGATGGC	746	CAGAGTGACGGA	1079
		ACCACGTATGTTC		ACAACATACGTC
		CTCCGCGGGCAC		CCACCAAGAGCA
		AG		CAG
DGTVANPFR	394	CAGAGTGATGGC	747	CAGAGTGACGGA	1080
		ACCGTGGCGAAT		ACAGTCGCAAAC
		CCTTTTCGGGCAC		CCATTCAGAGCA
		AG		CAG
DGTVDRPFK	395	CAGAGTGATGGC	748	CAGAGTGACGGA	1081
		ACCGTGGATCGG		ACAGTCGACAGA
		CCTTTTAAGGCAC		CCATTCAAAGCA
		AG		CAG
DGTVIHLSS	73	CAGAGTGATGGC	749	CAGAGTGACGGA	1082
		ACCGTTATTCATC		ACAGTCATCCAC
		TGAGTAGTGCAC		CTCAGCAGCGCA
		AG		CAG
DGTVILLSS	396	CAGAGTGATGGC	750	CAGAGTGACGGA	1083
		ACCGTTATTCTGT		ACAGTCATCCTCC
		TGTCGAGTGCAC		TCAGCAGCGCAC
		AG		AG
DGTVIMLSS	397	CAGAGTGATGGC	751	CAGAGTGACGGA	1084
		ACCGTGATTATGC		ACAGTCATCATG
		TGTCGAGTGCAC		CTCAGCAGCGCA
		AG		CAG
DGTVLHLSS	398	CAGAGTGATGGC	752	CAGAGTGACGGA	1085
		ACCGTGCTTCATT		ACAGTCCTCCACC
		TGTCGTCTGCACA		TCAGCAGCGCAC
		G		AG
DGTVLMLSS	399	CAGAGTGATGGC	753	CAGAGTGACGGA	1086
		ACCGTTTTGATGC		ACAGTCCTCATGC
		TGAGTAGTGCAC		TCAGCAGCGCAC
		AG		AG
DGTVLVPFR	150	CAGAGTGATGGC	754	CAGAGTGACGGA	1087
		ACCGTGTTGGTGC		ACAGTCCTCGTCC
		CGTTTAGGGCAC		CATTCAGAGCAC
		AG		AG
DGTVPYLAS	400	CAGAGTGATGGC	755	CAGAGTGACGGA	1088
		ACCGTTCCGTATC		ACAGTCCCATAC
		TTGCTTCTGCACA		CTCGCAAGCGCA
		G		CAG
DGTVPYLSS	401	CAGAGTGATGGC	756	CAGAGTGACGGA	1089
		ACCGTGCCGTATT		ACAGTCCCATAC
		TGTCTTCGGCACA		CTCAGCAGCGCA
		G		CAG
DGTVRVPFR	164	CAGAGTGATGGC	757	CAGAGTGACGGA	1090
		ACCGTTCGTGTGC		ACAGTCAGAGTC
		CGTTTAGGGCAC		CCATTCAGAGCA
		AG		CAG
DGTVSMPFK	402	CAGAGTGATGGC	758	CAGAGTGACGGA	1091
		ACCGTGTCGATG		ACAGTCAGCATG
		CCGTTTAAGGCA		CCATTCAAAGCA
		CAG		CAG
DGTVSNPFR	403	CAGAGTGATGGC	759	CAGAGTGACGGA	1092
		ACCGTGTCTAATC		ACAGTCAGCAAC
		CGTTTAGGGCAC		CCATTCAGAGCA
		AG		CAG
DGTVSTRWV	404	CAGAGTGATGGC	760	CAGAGTGACGGA	1093
		ACCGTTTCTACGC		ACAGTCAGCACA
		GTTGGGTGGCAC		AGATGGGTCGCA
		AG		CAG
DGTVTTTGW	405	CAGAGTGATGGC	761	CAGAGTGACGGA	1094
		ACCGTGACGACG		ACAGTCACAACA
		ACTGGGTGGGCA		ACAGGATGGGCA
		CAG		CAG
DGTVTVTGW	406	CAGAGTGATGGC	762	CAGAGTGACGGA	1095
		ACCGTGACGGTT		ACAGTCACAGTC
		ACGGGGTGGGCA		ACAGGATGGGCA
		CAG		CAG
DGTVWVPPR	407	CAGAGTGATGGC	763	CAGAGTGACGGA	1096
		ACCGTTTGGGTGC		ACAGTCTGGGTC
		CTCCTAGGGCAC		CCACCAAGAGCA
		AG		CAG
DGTVYRLSS	408	CAGAGTGATGGC	764	CAGAGTGACGGA	1097
		ACCGTTTATAGGT		ACAGTCTACAGA
		TGTCGAGTGCAC		CTCAGCAGCGCA
		AG		CAG
DGTYARLSS	409	CAGAGTGATGGC	765	CAGAGTGACGGA	1098
		ACCTATGCGCGTT		ACATACGCAAGA
		TGTCTTCTGCACA		CTCAGCAGCGCA
		G		CAG
DGTYGNKLW	410	CAGAGTGATGGC	766	CAGAGTGACGGA	1099
		ACCTATGGTAAT		ACATACGGAAAC
		AAGTTGTGGGCA		AAACTCTGGGCA
		CAG		CAG
DGTYIHLSS	411	CAGAGTGATGGC	767	CAGAGTGACGGA	1100
		ACCTATATTCATC		ACATACATCCAC
		TGTCTTCGGCACA		CTCAGCAGCGCA
		G		CAG
DGTYSTSGW	412	CAGAGTGATGGC	768	CAGAGTGACGGA	1101
		ACCTATTCGACG		ACATACAGCACA
		AGTGGGTGGGCA		AGCGGATGGGCA
		CAG		CAG
DGVHPGLSS	104	CAGAGTGATGGC	769	CAGAGTGACGGA	1102
		GTGCATCCTGGG		GTCCACCCAGGA
		CTTTCGAGTGCAC		CTCAGCAGCGCA
		AG		CAG
DGVVALLAS	413	CAGAGTGATGGC	770	CAGAGTGACGGA	1103
		GTGGTTGCGTTGC		GTCGTCGCACTCC
		TTGCTAGTGCACA		TCGCAAGCGCAC
		G		AG
DGYVGVGSL	414	CAGAGTGATGGC	771	CAGAGTGACGGA	1104
		TATGTGGGTGTTG		TACGTCGGAGTC
		GTAGTTTGGCAC		GGAAGCCTCGCA
		AG		CAG

Primer pools were produced by Twist biosciences using solid-phase synthesis and were used to generate a balanced library of 666 nucleotide variants by PCR amplification of CAP C-terminus and Gibson assembly as described in FIG. 27. 666 primers were provided a 1 fmole each, resulting in 0.6 pmole (regular PCR requires ˜25 pmole of primer). Primerless amplification on capsid gBlock template was performed over 10 cycles. Forward and reverse primers were added, followed by an additional 10, 15 or 20 PCR cycles. Constructs were then cloned into AAV9 backbone plasmids by Gibson/RCA (like regular libraries).

NGS analysis of SYN- and GFAP-driven AAV libraries produced with the pooled DNA showed a good correlation between the codon variants of each peptide, suggesting that the DNA sequence itself had little influence on virus production (FIG. 28 and FIG. 29). The pooled synthetic library was injected intravenously to C57BL/6 mice (5e11 VG per mouse, N=9), BALB/C mice (5e11 VG per mouse, N=6) and to rats (5e12 VG per rat, N=6), and after one month in-life RNA was extracted from the brain and spinal cord, and DNA was extracted from liver and heart tissue samples for biodistribution analysis (FIG. 30). Because the Synapsin and GFAP promoters are not fully active in non-CNS tissue, DNA was analyzed instead of RNA in peripheral organs. The initial focus was on the C57BL/6 mouse analysis because this is the mouse strain in which library evolution was performed.

The enrichment score of each capsid was determined by NGS analysis and defined as the ratio of reads per million (RPM) in the target tissue versus RPM in the inoculum. An example of analysis performed on the control capsids is shown in FIG. 31A. As expected from the published data, the PHP.B and PHP.eB (aka, PHP.N) capsids allowed significantly higher RNA expression in neurons compared to the AAV9 parental capsid (8-fold and 25-fold, respectively). There was a very high correlation between the codon variants of each peptide species in each animal (r=0.92, 0.93 and 0.95), confirming the robustness of the NGS assay (FIG. 31B-FIG. 31D).

An example of enrichment analysis is presented in FIG. 32A-FIG. 36. The 333 capsid variants are ranked by average brain enrichment score from all animals, and the individual enrichment values are indicated by a color scale. As indicated by the position of the reference capsids, a group of novel variants showed a higher enrichment score than the PHP.eB benchmark capsid in both neurons (Syn-driven) and astrocytes (GFAP-driven). Interestingly, many variants showed a different enrichment score in neurons vs. astrocytes, as indicated by the medium level of correlation between Syn- and GFAP-driven RNA. This suggests that certain capsids display an enhanced tropism for neurons, and others for astrocytes (FIG. 33).

A group of 38 capsids showed potentially interesting properties based on their tropism for neurons, astrocytes or both (Table 8A and Table 8B) (FIG. 38) and showed a strong consensus peptide sequence similarity, different between neuron- and astrocyte-targeting variants (FIG. 45A-FIG. 45C and FIG. 46A-FIG. 46B).

TABLE 8A
TOP 38 candidates from C57BL/6 screen #1 (N =3)
			SEQ ID	SYN	GFAP
Groups	variant	peptide	NO:	ranking	ranking
A	9p32	DGTAIHLSS	67	15, 16	113, 133
	9p35	DGTSSYYDS	59	1, 3	565, 581
B	9p36	DGSSSYYDA	64	10, 11	591, 594
	9p37	DGTASYYDS	61	5, 6	553, 560
C	9p26	DGTTTYGAR	77	225, 262	49, 56
D	9p2	AQNGNPGRW	84	156, 160	38, 44
	9p13	AQGENPGRW	96	77, 87	7, 13
	9p30	AQPEGSARW	60	2, 4	154, 160
E	9p1	AQGSWNPPA	80	348, 361	8, 15
	9p14	AQGTWNPPA	82	448, 467	14, 17
F	9p29	AQFPTNYDS	66	14, 19	490, 537
	9p31	AQWPTSYDA	62	7, 9	290, 304
G	9p3	AQTTEKPWL	83	53, 72	35, 70
	9p15	AQTTDRPFL	85	206, 219	26, 43
H	9p10	DGTRTTTGW	106	161, 220	10, 22
	9p18	DGTGGIKGW	131	346, 388	41, 68
	9p19	DGTGNTRGW	94	322, 340	45, 54
	9p20	DGTHTRTGW	90	380, 427	31, 39
	9p23	DGTNGLKGW	76	132, 153	5, 16
	9p33	DGTGQVTGW	68	18, 33	172, 213
	9p38	DGTGNVTGW	69	20, 31	117, 137
I	9p11	DGTTFTPPR	79	183, 199	11, 19
	9p12	DGTTYVPPR	75	146, 154	4, 9
	9p24	DGTSFTPPK	81	210, 243	29, 40
	9p25	DGTSFTPPR	88	250, 273	28, 37
	9p27	DGTTWTPPR	139	567, 570	46, 59
	9p28	DGTSYVPPR	78	162, 179	20, 25
J	9p4	DGTADRPFR	155	109, 118	48, 57
	9p9	DGTMDRPFK	102	102, 113	23 ,34
	9p16	DGTADKPFR	63	8, 12	1, 6
	9p17	DGTAERPFR	140	106, 138	42, 50
	9p21	DGTIERPFR	87	186, 235	21, 33
	9p34	DGTMDKPFR	70	21, 23	107, 112
K	9p5	DGTISQPFK	105	184, 193	12, 18
	9p6	DGTLAAPFK	120	110, 112	27, 30
	9p7	DGTLQQPFR	89	46, 57	32, 47
	9p8	DGTLSQPFR	65	13, 17	2, 3
	9p22	DGTLNNPFR	109	30, 41	24, 36
Ref.	PHPN	DGTLAVPFK	71	22, 24	51, 60
	PHPB	AQTLAVPFK	168	253, 261	61, 62
	wtAAV9	AQ		630, 631	611, 620

TABLE 8B
Variant 9mer and encoding sequences
		SEQ	NNK	SEQ	NNM	SEQ
	9mer	ID	nucleotide	ID	nucleotide	ID
variant	peptide	NO:	sequences	NO:	sequences	NO:
9p1	AQGSWNPPA	80	GCCCAAGGTT	1105	GCACAAGGAAG	1143
			CGTGGAATCC		CTGGAACCCACC
			GCCGGCG		AGCA
9p2	AQNGNPGRW	84	GCCCAAAATG	1106	GCACAAAACGG	1144
			GTAATCCGGG		AAACCCAGGAA
			GCGGTGG		GATGG
9p3	AQTIEKPWL	83	GCCCAAACGA	1107	GCACAAACAAC	1145
			CTGAGAAGCC		AGAAAAACCAT
			GTGGCTG		GGCTC
9p4	DGTADRPFR	155	GATGGCACGG	1108	GACGGAACAGC	1146
			CGGATCGTCCT		AGACAGACCATT
			TTTCGG		CAGA
9p5	DGTISQPFK	105	GATGGCACCA	1109	GACGGAACAAT	1147
			TTTCGCAGCCT		CAGCCAACCATT
			TTTAAG		CAAA
9p6	DGTLAAPFK	120	GATGGCACCTT	1110	GACGGAACACTC	1148
			GGCTGCTCCTT		GCAGCACCATTC
			TTAAG		AAA
9p7	DGTLQQPFR	89	GATGGCACCTT	1111	GACGGAACACTC	1149
			GCAGCAGCCG		CAACAACCATTC
			TTTCGG		AGA
9p8	DGTLSQPFR	65	GATGGCACCC	1112	GACGGAACACTC	1150
			TGTCTCAGCCT		AGCCAACCATTC
			TTTAGG		AGA
9p9	DGTMDRPFK	102	GATGGCACCA	1113	GACGGAACAAT	1151
			TGGATAGGCC		GGACAGACCATT
			GTTTAAG		CAAA
9p10	DGTRTTTGW	106	GATGGCACCC	1114	GACGGAACAAG	1152
			GTACTACGAC		AACAACAACAG
			GGGTTGG		GATGG
9p11	DGTTFTPPR	79	GATGGCACCA	1115	GACGGAACAAC	1153
			CTTTTACTCCT		ATTCACACCACC
			CCTCGG		AAGA
9p12	DGTTYVPPR	75	GATGGCACCA	1116	GACGGAACAAC	1154
			CGTATGTTCCT		ATACGTCCCACC
			CCGCGG		AAGA
9p13	AQGENPGRW	96	GCCCAAGGGG	1117	GCACAAGGAGA	1155
			AGAATCCGGG		AAACCCAGGAA
			TAGGTGG		GATGG
9p14	AQGTWNPPA	82	GCCCAAGGTA	1118	GCACAAGGAAC	1156
			CTTGGAATCCG		ATGGAACCCACC
			CCGGCT		AGCA
9p15	AQTTDRPFL	85	GCCCAAACTA	1119	GCACAAACAAC	1157
			CTGATAGGCCT		AGACAGACCATT
			TTTTTG		CCTC
9p16	DGTADKPFR	63	GATGGCACCG	1120	GACGGAACAGC	1158
			CTGATAAGCC		AGACAAACCATT
			GTTTCGG		CAGA
9p17	DGTAERPFR	140	GATGGCACCG	1121	GACGGAACAGC	1159
			CGGAGAGGCC		AGAAAGACCATT
			TTTTAGG		CAGA
9p18	DGTGGIKGW	131	GATGGCACCG	1122	GACGGAACAGG	1160
			GTGGTATTAA		AGGAATCAAAG
			GGGGTGG		GATGG
9p19	DGTGNTRGW	94	GATGGCACCG	1123	GACGGAACAGG	1161
			GGAATACTCG		AAACACAAGAG
			GGGGTGG		GATGG
9p20	DGTHTRTGW	90	GATGGCACCC	1124	GACGGAACACA	1162
			ATACGCGGAC		CACAAGAACAG
			GGGTTGG		GATGG
9p21	DGTIERPFR	87	GATGGCACCA	1125	GACGGAACAAT	1163
			TTGAGCGGCCT		CGAAAGACCATT
			TTTCGT		CAGA
9p22	DGTLNNPFR	109	GATGGCACCTT	1126	GACGGAACACTC	1164
			GAATAATCCG		AACAACCCATTC
			TTTAGG		AGA
9p23	DGTNGLKGW	76	GATGGCACCA	1127	GACGGAACAAA	1165
			ATGGTCTGAA		CGGACTCAAAG
			GGGGTGG		GATGG
9p24	DGTSFTPPK	81	GATGGCACCT	1128	GACGGAACAAG	1166
			CGTTTACGCCG		CTTCACACCACC
			CCTAAG		AAAA
9p25	DGTSFTPPR	88	GATGGCACCT	1129	GACGGAACAAG	1167
			CGTTTACTCCG		CTTCACACCACC
			CCGCGG		AAGA
9p26	DGTTTYGAR	77	GATGGCACCA	1130	GACGGAACAAC	1168
			CTACGTATGG		AACATACGGAG
			GGCTCGT		CAAGA
9p27	DGTTWTPPR	139	GATGGCACCA	1131	GACGGAACAAC	1169
			CTTGGACGCC		ATGGACACCACC
			GCCGCGT		AAGA
9p28	DGTSYVPPR	78	GATGGCACCA	1132	GACGGAACAAG	1170
			GTTATGTTCCT		CTACGTCCCACC
			CCGAGG		AAGA
9p29	AQFPTNYDS	66	GCCCAATTTCC	1133	GCACAATTCCCA	1171
			TACGAATTATG		ACAAACTACGAC
			ATTCT		AGC
9p30	AQPEGSARW	60	GCCCAACCTG	1134	GCACAACCAGA	1172
			AGGGTAGTGC		AGGAAGCGCAA
			GAGGTGG		GATGG
9p31	AQWPTSYDA	62	GCCCAATGGC	1135	GCACAATGGCCA	1173
			CTACGAGTTAT		ACAAGCTACGAC
			GATGCT		GCA
9p32	DGTAIHLSS	67	GATGGCACCG	1136	GACGGAACAGC	1174
			CGATTCATCTT		AATCCACCTCAG
			TCGTCT		CAGC
9p33	DGTGQVTGW	68	GATGGCACCG	1137	GACGGAACAGG	1175
			GGCAGGTGAC		ACAAGTCACAG
			TGGGTGG		GATGG
9p34	DGTMDKPFR	70	GATGGCACGA	1138	GACGGAACAAT	1176
			TGGATAAGCC		GGACAAACCATT
			TTTTAGG		CAGA
9p35	DGTSSYYDS	59	GATGGCACCT	1139	GACGGAACAAG	1177
			CGAGTTATTAT		CAGCTACTACGA
			GATTCT		CAGC
9p36	DGSSSYYDA	64	GATGGCAGTA	1140	GACGGAAGCAG	1178
			GTTCTTATTAT		CAGCTACTACGA
			GATGCG		CGCA
9p37	DGTASYYDS	61	GATGGCACCG	1141	GACGGAACAGC	1179
			CGAGTTATTAT		AAGCTACTACGA
			GATTCT		CAGC
9p38	DGTGNVTGW	69	GATGGCACCG	1142	GACGGAACAGG	1180
			GTAATGTGAC		AAACGTCACAG
			GGGGTGG		GATGG
AAV9	AQ		AGTGCTCAGG	54	AGTGCCCAAGCA	53
			CACAGGCGCA		CAGGCGCAGAC
			GACC		C
PHPN	DGTLAVPFK	71	GATGGGACTTT	56	GACGGAACACTC	55
			GGCGGTGCCTT		GCAGTCCCATTC
			TTAAG		AAA
PHPB	AQTLAVPFK	168	GCCCAAACTTT	58	GCACAAACACTC	57
			GGCGGTGCCTT		GCAGTCCCATTC
			TTAAG		AAA

Example 11. Phylogenetic Grouping

Phylogenetic grouping of peptide sequences showed an evident correlation between sequence homology clusters and capsid phenotypes (FIG. 37). For example, 9-mer variants with the sequence DGTxxxPFK/R (SEQ ID NO: 1181) presented a similar behavior as PHP.eB capsid (high transduction of both neurons and astrocytes), whereas variants harboring the sequence DGTxxxYDS/A (SEQ ID NO: 1182) showed a preference for neuron transduction. By contrast, peptides with the consensus DGTxxxxGW (SEQ ID NO: 1183) or CGTxxxPPR/K (SEQ ID NO: 1184) presented a higher tropism for astrocytes.

Example 12. Capsid Testing

Capsid variants representative of distinct sequence clusters (highlighted in FIG. 37B) were chosen for individual transduction analysis in C57BL/6 mice. Each capsid was produced as a recombinant AAV packaging a self-complementary EGFP transgene driven by the ubiquitous promoter (FIGS. 49A, B). Mouse groups (N=3) were injected intravenously with 6e10 VG and transduction efficiency was assessed after 1 month by quantifying EGFP mRNA in the brain, spinal cord, and liver tissue. EGFP mRNA expression was normalized using mouse TBP as a housekeeping gene, and DNA biodistribution was normalized to the single-copy mouse TfR gene (FIG. 50A-FIG. 50C). Reverse transcription was performed with the Quantitect kit and included a DNA removal treatment. All capsid variants showed a significant improvement in brain and spinal cord mRNA expression by comparison to the parent AAV9 capsid, and 3 out of 7 variants (9P16, 9P31 and 9P35) showed similar or higher transduction than the PHP.eB benchmark capsid (FIG. 49C, Table 10). The viral DNA biodistribution showed a very strong tropism of 9P31 and 9P35 for the brain and spinal cord, but all the variants showed a 40- to 260-fold increase of biodistribution compared to AAV9 (FIG. 49D, Table 10).

Expected cellular tropism was tested using an NGS screen by labeling the neuronal NeuN marker (FIG. 51). Within the cortex, the top capsids in the GFAP screen showed mostly GFP expression in NeuN-negative cells with glial morphology. Conversely, top capsids in the SYN screen showed a very high transduction of NeuN-positive cells, and the dual-specificity capsids 9P08 and 9P16—ranking high in both assays—showed mixed cell preference with multiple NeuN+ cells and glial cells.

Cellular tropism was also tested using mouse brain microvascular EC (mBMVEC) binding relative to AAV9. Results are shown in Table 9.

TABLE 9
mBMVEC binding results
			BINDING TO
		SEQUENCE	mBMVEC (fold
PEPTIDE	SEQUENCE	ID	over AAV9)
AAV9	AQ		1
PHP.eB	DGTLAVPFK	71	153
9P03	AQTTEKPWL	83	170
9P08	DGTLSQPFR	65	349
9P09	DGTMDRPFK	102	222
9P13	AQGENPGRW	96	2.5
9P16	DGTADKPFR	63	176
9P31	AQWPTSYDA	62	2
9P32	DGTAIHLSS	67	16
9P33	DGTGQVTGW	68	5
9P36	DGSSSYYDA	64	0
9P39	DGTGSTTGW	134	2

Fluorescent EGFP expression in tissues of whole brain, cerebellum, cortex, and hippocampus revealed transduction patterns across a spectrum and demonstrate the identification of tissue-specific capsids (FIG. 52-FIG. 56).

The liver transduction, measured by mRNA expression and by whole tissue GFP expression, showed that several variants outperformed AAV9, which was unexpected in light of the NGS results. Some variants, such as 9P08 or 9P23, showed a relative liver detargeting by comparison with AAV9 (FIG. 57A-FIG. 57B).

TABLE 10
Brain and Spinal cord tropism
	BRAIN EGFP mRNA*
	EGFP/TBP	EGFP/TBP	EGFP/TBP	group	group	Mean Fold	Fold
CAPSID	m1	m2	m3	mean	SD	over AAV9	SDEV
AAV9	0.11	0.1	0.15	0.12	0.03	1	0.21
PHPN	2.94	4.44	3.42	3.6	0.77	30	6.38
9P08	2.46	3.47	2.73	2.89	0.53	24	4.38
9P12	3.07	2.27	2.98	2.77	0.44	23	3.65
9P16	4.31	4.75	5.28	4.78	0.49	39	4.06
9P23	3.28	2.37	2.79	2.81	0.46	23	3.79
9P30	1.06	1.7	1.32	1.36	0.32	11	2.66
9P31	4.87	5.53	4.2	4.87	0.66	40	5.54
9P35	3.9	3.24	3.45	3.53	0.33	29	2.78
PHPB***	2.68	2.68	2.68	2.68	0	22	0
ctrl	0	0	0	0	0	0	0
	SPINAL CORD EGFP mRNA*
	EGFP/TBP	EGFP/TBP	EGFP/TBP	group	group	Mean Fold	Fold
CAPSID	m1	m2	m3	mean	SD	over AAV9	SD
AAV9	0.84	0.29	0.3	0.48	0.31	1	0.66
PHPN	3.36	5.8	5.4	4.86	1.31	10.22	2.75
9P08	4.3	5.62	4.65	4.86	0.68	10.22	1.43
9P12	6.09	5.94	5.78	5.94	0.16	12.49	0.33
9P16	4.42	5.31	5.37	5.04	0.53	10.6	1.12
9P23	5.41	5.95	5.04	5.47	0.46	11.5	0.96
9P30	1.53	1.83	2.11	1.82	0.29	3.84	0.61
9P31	6.92	7.06	6.94	6.98	0.08	14.68	0.16
9P35	4.68	4.81	4.79	4.76	0.07	10.02	0.15
PHPB	3.84	3.84	3.84	3.84	0	8.09	0
ctrl	0	0	0	0	0	0	0
	BRAIN EGFP DNA** (VG/Cell)
	EGFP/TERT	EGFP/TERT	EGFP/TERT	Group	Group	Mean Fold	Fold
CAPSID	m1	m2	m3	mean	SD	over AAV9	SDEV
AAV9	0.03	0.04	0.01	0.03	0.01	1	0
PHPN	2.07	2.79	1.94	2.27	0.46	87	18
P08	1.25	1.62	5.47	2.78	2.34	107	90
P12	1.43	0.94	1.41	1.26	0.27	48	10
P16	4.13	1.15	3.56	2.95	1.58	113	60
P23	1.34	2.68	1.87	1.96	0.68	75	26
P30	0.59	1.42	1.21	1.08	0.43	41	17
P31	6.47	5.6	8.81	6.96	1.66	267	64
P35	4.62	5.55	2.52	4.23	1.55	162	59
PHPB	1.5	1.5	1.5	1.5	0	58	0
ctrl	0	0	0	0	0	0	0
	SPINAL CORD EGFP DNA** (VG/Cell)
	EGFP/TERT	EGFP/TERT	EGFP/TERT	Group	Group	Mean Fold	Fold
CAPSID	m 1	m 2	m 3	AVG	SD	over AAV9	SDEV
AAV9	0.03	0.04	0.04	0.03	0.007	1	0.2
PHPN	1.75	2.96	3.14	2.62	0.752	75	21.7
P08	3.81	3.47	3.66	3.65	0.174	105	5
P12	1.62	3.31	2.87	2.6	0.873	75	25.2
P16	3.3	3.34	2.96	3.2	0.211	92	6.1
P23	2.63	2.47	3.1	2.73	0.322	79	9.3
P30	0.8	1.8	1.43	1.34	0.507	39	14.6
P31	9.88	6.19	5.47	7.18	2.366	207	68.2
P35	2.95	3.92	2.41	3.1	0.765	89	22
PHPB	1.34	1.34	1.34	1.34	0	39	0
ctrl	0	0	0	0	0	0	0
*EGFP mRNA expression was normalized to TBP as a housekeeping marker
**GFP DNA was normalized to single-copy TfR DNA
***N = 1

Example 13. Multi-Rodent Testing (Cross Species)

The efficacy of the 333 capsid variants to transduce CNS was tested in other rodent strains or species (FIG. 47). Side-by-side comparison of neuron and astrocyte transduction in C57BL/6 mice, BALB/C mice and rats showed major differences in the enrichment scores of multiple variants between the two mouse strains, and even more pronounced differences between mice and rats (FIG. 48A-FIG. 48C). Strikingly, the most efficient capsid for rat brain transduction was the parental AAV9, which suggests that directed evolution “bottlenecks” capsid variants that are highly performant in one given species, as opposed to the versatility of wild-type AAV capsids.

Correlation analysis showed that some capsids shared high CNS transduction between C57BL/6 and BALB/C mice, whereas others were restricted to only one strain (FIG. 48B).

Interestingly, the PHP.B and PHP.eB capsid showed poor brain transduction in BALB/C mice, in line with a recent publication (Hordeaux et al., 2018). When focusing on the capsids that showed >10-fold increase in brain transduction, 62 variants were improved only in C57BL/6 mice, 28 variants were improved only in BALB/C mice and 30 variants showed improved brain transduction in both strains (Table 11). Consensus sequence analysis showed a “C57BL/6 signature” closely resembling the PHP.eB peptide (DGTxxxPFR (SEQ ID NO: 1185)) whereas the BALB/C signature showed a different consensus (DGTxxxxGW (SEQ ID NO: 1183)), suggesting the use of a different cellular receptor (FIG. 48C).

TABLE 11
TOP 30 candidates from C57BL/6 and BALB/C mouse screen
SYNAPSIN PROMOTER
C57BL/6	BALB/C
REPLICATE 1 (N = 3)	REPLICATE 2 (N = 6)	REPLICATE 1 (N = 6)
	Brain		Brain		Brain
	Enrichment		Enrichment		Enrichment
9-mer peptide	Factor (fold	9-mer peptide	Factor (fold	9-mer peptide	Factor (fold
insert	over AAV9)	insert	over AAV9)	insert	over AAV9)
DGTSSYYDS	36.40	AQWPTSYDA	39.97	DGTGSTTGW	57.05
(SEQ ID NO:		(SEQ ID NO:		(SEQ ID NO:
59)		62)		134)
AQPEGSARW	35.95	AQPEGSARW	31.83	DGTGQVTGW	49.87
(SEQ ID NO:		(SEQ ID NO:		(SEQ ID NO:
60)		60)		68)
DGTASYYDS	32.34	DGTGQVTGW	20.35	DGTGSTHGW	43.08
(SEQ ID NO:		(SEQ ID NO:		(SEQ ID NO:
61)		68)		119)
AQWPTSYDA	30.81	DGTAIHLSS	19.55	DGTGSTQGW	38.31
(SEQ ID NO:		(SEQ ID NO:		(SEQ ID NO:
62)		67)		315)
DGTADKPFR	29.30	DGTMDRPFK	19.48	DGTGTTTGW	37.29
(SEQ ID NO:		(SEQ ID NO:		(SEQ ID NO:
63)		102)		130)
DGSSSYYDA	28.05	DGTGSTTGW	19.20	AQWAAGYNV	34.57
(SEQ ID NO:		(SEQ ID NO:		(SEQ ID NO:
64)		134)		245)
DGTLSQPFR	26.73	DGSSSYYDA	18.08	DGTGGTKGW	33.59
(SEQ ID NO:		(SEQ ID NO:		(SEQ ID NO:
65)		64)		107)
DGTAIHLSS	26.23	DGTSSYYDA	17.93	DGTGSTKGW	29.64
(SEQ ID NO:		(SEQ ID NO:		(SEQ ID NO:
67)		381)		313)
AQFPTNYDS	26.07	DGSQSTTGW	17.59	DGSQSTTGW	25.19
(SEQ ID NO:		(SEQ ID NO:		(SEQ ID NO:
66)		136)		136)
DGTMDKPFR	25.05	DGTGSTQGW	17.24	AQWEVKGGY	23.44
(SEQ ID NO:		(SEQ ID NO:		(SEQ ID NO:
70)		315)		247)
DGTLAVPFK	24.62	DGTGTTTGW	17.00	DGTAIHLSS	22.81
(SEQ ID NO:		(SEQ ID NO:		(SEQ ID NO:
71)		130)		67)
DGTGNVTGW	24.05	DGTLAVPFK	16.84	DGGGTTTGW	22.62
(SEQ ID NO:		(SEQ ID NO:		(SEQ ID NO:
69)		71)		270)
DGTGQVTGW	23.83	DGTASYYDS	16.68	DGTGGLTGW	22.42
(SEQ ID NO:		(SEQ ID NO:		(SEQ ID NO:
68)		61)		294)
DGTHIHLSS	22.93	DGTMDKPFR	16.68	DGTNTINGW	20.76
(SEQ ID NO:		(SEQ ID NO:		(SEQ ID NO:
162)		70)		124)
DGTGNTHGW	22.63	DGTVANPFR	16.32	DGAGGTSGW	19.55
(SEQ ID NO:		(SEQ ID NO:		(SEQ ID NO:
72)		394)		151)
DGTVIHLSS	22.62	DGTLNNPFR	16.24	DGTNTTHGW	18.99
(SEQ ID NO:		(SEQ ID NO:		(SEQ ID NO:
73)		109)		113)
DGTLNNPFR	22.33	DGTLAAPFK	15.96	DGTGTVQGW	18.84
(SEQ ID NO:		(SEQ ID NO:		(SEQ ID NO:
109)		120)		327)
DGTGNTSGW	22.10	DGTLSQPFR	15.43	DGTGQTIGW	18.55
(SEQ ID NO:		(SEQ ID NO:		(SEQ ID NO:
137)		65)		305)
DGTGTTVGW	21.72	DGTHIHLSS	15.11	AQWELSNGY	18.13
(SEQ ID NO:		(SEQ ID NO:		(SEQ ID NO:
74)		162)		246)
DGTSSYYDA	20.94	AQTTEKPWL	15.00	DGTGSLNGW	17.93
(SEQ ID NO:		(SEQ ID NO:		(SEQ ID NO:
381)		83)		309)
DGAGTTSGW	20.42	DGTGNVTGW	14.90	DGTGTTLGW	17.48
(SEQ ID NO:		(SEQ ID NO:		(SEQ ID NO:
265)		69)		323)
DGGGTTTGW	20.27	DGTGGVTGW	14.89	AQPEGSARW	17.11
(SEQ ID NO:		(SEQ ID NO:		(SEQ ID NO:
270)		299)		60)
DGTLQQPFR	19.88	DGTSSYYDS	14.80	DGTGSTMGW	16.91
(SEQ ID NO:		(SEQ ID NO:		(SEQ ID NO:
89)		59)		314)
DGTGQTIGW	19.52	DGTGNTSGW	14.48	DGTGNTHGW	16.47
(SEQ ID NO:		(SEQ ID NO:		(SEQ ID NO:
305)		137)		72)
DGTVTTTGW	19.49	AQWPTAYDA	14.48	DGSGTTRGW	15.83
(SEQ ID NO:		(SEQ ID NO:		(SEQ ID NO:
405)		256)		114)
DGTSIHLSS	19.45	AQGENPGRW	14.41	DGTNSTTGW	15.48
(SEQ ID NO:		(SEQ ID NO:		(SEQ ID NO:
378)		96)		143)
DGTGSTTGW	19.45	DGTADKPFR	14.32	DGRNALTGW	15.13
(SEQ ID NO:		(SEQ ID NO:		(SEQ ID NO:
134)		63)		275)
DGTGGVTGW	19.44	DGTGQTIGW	14.27	DGAAATTGW	15.02
(SEQ ID NO:		(SEQ ID NO:		(SEQ ID NO:
299)		305)		264)
DGTVANPFR	19.42	DGTISQPFK	13.84	DGTATTMGW	14.54
(SEQ ID NO:		(SEQ ID NO:		(SEQ ID NO:
394)		105)		284)
DGTGTTTGW	19.16	DGTKLMLSS	13.71	AQRYTGDSS	14.35
(SEQ ID NO:		(SEQ ID NO:		(SEQ ID NO:
130)		157)		138)
DGAGGTSGW	18.99	AQTLAVPFK	13.69	DGAGTTSGW	14.29
(SEQ ID NO:		(SEQ ID NO:		(SEQ ID NO:
151)		168)		265)
GFAP PROMOTER
C57BL/6	BALB/C
REPLICATE 1 (N = 2)	REPLICATE 2 (N = 6)	REPLICATE 1 (N = 6)
	Brain		Brain		Brain
	Enrichment		Enrichment		Enrichment
9-mer peptide	Factor (fold	9-mer peptide	Factor (fold	9-mer peptide	Factor (fold
insert	over AAV9)	insert	over AAV9)	insert	over AAV9)
DGTADKPFR	37.60	DGTMDRPFK	24.89	DGTGSTTGW	21.03
(SEQ ID NO:		(SEQ ID NO:		(SEQ ID NO:
63)		102)		134)
DGTLSQPFR	35.97	DGTAERPFR	24.66	DGTGQVTGW	19.24
(SEQ ID NO:		(SEQ ID NO:		(SEQ ID NO:
65)		140)		68)
DGTTYVPPR	33.09	DGTADKPFR	23.03	DGTGTTTGW	15.56
(SEQ ID NO:		(SEQ ID NO:		(SEQ ID NO:
75)		63)		130)
DGTNGLKGW	32.14	DGTLNNPFR	22.91	DGTGSTHGW	14.45
(SEQ ID NO:		(SEQ ID NO:		(SEQ ID NO:
76)		109)		119)
AQGENPGRW	31.99	DGTLSQPFR	21.60	DGTAIHLSS	11.74
(SEQ ID NO:		(SEQ ID NO:		(SEQ ID NO:
96)		65)		67)
AQGSWNPPA	30.78	DGTMDKPFR	20.52	DGTGSTQGW	11.40
(SEQ ID NO:		(SEQ ID NO:		(SEQ ID NO:
80)		70)		315)
AQGTWNPPA	29.19	DGTISQPFK	20.47	DGTGGLTGW	8.87
(SEQ ID NO:		(SEQ ID NO:		(SEQ ID NO:
82)		105)		294)
DGTISQPFK	29.01	AQGENPGRW	20.09	AQNGNPGRW	8.82
(SEQ ID NO:		(SEQ ID NO:		(SEQ ID NO:
105)		96)		84)
DGTTFTPPR	28.94	AQTTEKPWL	18.04	DGTGGIKGW	8.62
(SEQ ID NO:		(SEQ ID NO:		(SEQ ID NO:
79)		83)		131)
DGTRTTTGW	28.59	DGTVANPFR	16.87	DGRNALTGW	8.39
(SEQ ID NO:		(SEQ ID NO:		(SEQ ID NO:
106)		394)		275)
DGTSYVPPR	26.17	DGTTYVPPR	16.31	DGTGSTKGW	8.38
(SEQ ID NO:		(SEQ ID NO:		(SEQ ID NO:
78)		75)		313)
DGTIERPFR	25.37	AQTTDRPFL	16.27	AQRYTGDSS	8.13
(SEQ ID NO:		(SEQ ID NO:		(SEQ ID NO:
87)		85)		138)
DGTMDRPFK	24.85	DGTTTYGAR	15.62	DGTGGTKGW	8.06
(SEQ ID NO:		(SEQ ID NO:		(SEQ ID NO:
102)		77)		107)
DGTLAAPFK	24.67	DGTADRPFR	15.60	DGTATTTGW	8.04
(SEQ ID NO:		(SEQ ID NO:		(SEQ ID NO:
120)		155)		285)
DGTLNNPFR	24.62	DGTIERPFR	15.11	DGTKMVLQL	7.87
(SEQ ID NO:		(SEQ ID NO:		(SEQ ID NO:
109)		87)		142)
DGTSFTPPR	24.14	AQGSWNPPA	15.11	DGTGSLNGW	7.71
(SEQ ID NO:		(SEQ ID NO:		(SEQ ID NO:
88)		80)		309)
AQTTDRPFL	23.85	AQGTWNPPA	15.03	DGTNTINGW	7.59
(SEQ ID NO:		(SEQ ID NO:		(SEQ ID NO:
85)		82)		124)
DGTSFTPPK	23.75	DGSTERPFR	15.01	AQWELSNGY	7.57
(SEQ ID NO:		(SEQ ID NO:		(SEQ ID NO:
81)		99)		246)
DGTHTRTGW	23.54	AQSVAKPFL	14.90	DGTNGLKGW	7.50
(SEQ ID NO:		(SEQ ID NO:		(SEQ ID NO:
90)		231)		76)
DGTLQQPFR	22.94	DGTVDRPFK	14.74	DGTNTTHGW	7.25
(SEQ ID NO:		(SEQ ID NO:		(SEQ ID NO:
89)		395)		113)
AQNGNPGRW	22.80	DGTTFTPPR	14.56	DGTRMVVQL	7.25
(SEQ ID NO:		(SEQ ID NO:		(SEQ ID NO:
84)		79)		370)
DGTAERPFR	21.65	AQTLARPFV	14.51	DGTNSTTGW	6.41
(SEQ ID NO:		(SEQ ID NO:		(SEQ ID NO:
140)		98)		143)
DGTGNTRGW	21.12	DGTGGTKGW	14.13	DGSQSTTGW	6.29
(SEQ ID NO:		(SEQ ID NO:		(SEQ ID NO:
94)		107)		136)
AQTTEKPWL	20.58	AQGPTRPFL	13.47	AQPEGSARW	6.23
(SEQ ID NO:		(SEQ ID NO:		(SEQ ID NO:
83)		125)		60)
DGTADRPFR	20.49	DGTRTTTGW	13.39	DGTGQTIGW	6.16
(SEQ ID NO:		(SEQ ID NO:		(SEQ ID NO:
155)		106)		305)
DGTTWTPPR	20.44	AQNGNPGRW	13.09	DGTGGVTGW	6.07
(SEQ ID NO:		(SEQ ID NO:		(SEQ ID NO:
139)		84)		299)
DGTTTYGAR	20.43	DGTVSNPFR	12.77	DGTVTTTGW	6.04
(SEQ ID NO:		(SEQ ID NO:		(SEQ ID NO:
77)		403)		405)
DGTGGIKGW	20.20	AQGGNPGRW	12.21	DGKGSTQGW	5.97
(SEQ ID NO:		(SEQ ID NO:		(SEQ ID NO:
131)		91)		272)
DGTLAVPFK	19.43	AQWPTSYDA	11.93	AQGENPGRW	5.88
(SEQ ID NO:		(SEQ ID NO:		(SEQ ID NO:
71)		62)		96)
DGKQYQLSS	18.74	DGTLQQPFR	11.92	DGNGGLKGW	5.82
(SEQ ID NO:		(SEQ ID NO:		(SEQ ID NO:
92)		89)		167)
DGSPEKPFR	18.73	DGTNGLKGW	11.53	DGTGTVHGW	5.82
(SEQ ID NO:		(SEQ ID NO:		(SEQ ID NO:
160)		76)		326)

The efficacy of the 333 capsid variants to transduce CNS was also compared for C57BL/6 mice BMVEC and Human BMVEC (FIG. 58A and FIG. 58B).

Example 14. Engineering of a NGS-Driven Selection System for Full-Length Capsid Variants

A barcode system was engineered to allow enrichment studies with full capsid length modifications. While the TRACER platform described here was initially developed for the use of peptide display libraries, where the randomized peptide sequence itself can be used for Illumina NGS analysis due to its short size, the Illumina sequencing technology does not typically allow sequencing of more than 300 contiguous bases, and therefore our platform cannot be used for NGS analysis of full-length capsid variants, such as those generated by DNA shuffling technology or error-prone PCR.

An alternative RNA-driven platform for full-length capsid libraries in which a unique molecular identified (UMI) is placed outside the capsid gene and can be used for NGS enrichment analysis was designed (FIG. 59A-FIG. 59C). Once the variants with desired properties are identified by UMI enrichment analysis from animal tissue, the UMI sequence must allow highly specific recovery of the full-length capsid from the starting material with a minimal error rate. The system should have one or more of the following properties to be effective: 1) the UMI should be transcribed under control of a cell type-specific promoter, 2) the UMI should not interfere with capsid expression or splicing during virus production, 3) the UMI should be short enough for Illumina NGS sequencing (typically less than 60nt for standard single-end 75 nt sequencing), and 4) the UMI should allow sequence-specific recovery of full-length capsids of interest from the starting DNA/virus library with a minimal error rate.

To address these properties: 1) the UMI was placed in the transcribed region of capsid library (i.e., anywhere between the transcription start site and the polyadenylation signal), 2) the UMI was placed either in various locations of the AAV intron (which mostly unspliced in the absence of helper functions) or between the capsid stop codon and the polyadenylation signal, 3) the UMI cassette was composed of two randomized 21-nt sequences separated by a 15-nt spacer, for a total length of 57 nt, which allows 18 extra nucleotides for primer annealing, and 4) the UMI randomized sequences were formed of NSW triplets (N=A, T, G, C; S=G, C; W=A, T) to prevent large variations in annealing temperature with amplification primers, avoid homopolymeric stretches and prevent the formation of a premature polyA signal (AATAAA).

Importantly, the UMI cassette contained two random sequences in tandem. The first sequence (outermost) is used to design a matching capsid recovery primer, and the second sequence (innermost) to confirm the identity of the capsid amplicon after cloning. This method should allow to eliminate all clones containing non-specific amplification products. In an alternative embodiment, the innermost sequence can also be used to design a nested PCR primer in order to increase the specificity of amplification (FIG. 59A-FIG. 59C).

Several insertion sites of the tandem barcode to test the impact on virus viability and titers were explored. A series of constructs were engineered with the barcode inserted in the AAV intron of the CAG9 plasmid (FIG. 60A). Since AAV intron is spliced during virus production, the presence of the barcode should have only a minimal impact on the yields. Conversely, the AAV splicing is very ineffective in the absence of helper functions (Mouw et al., 2000), therefore the barcode sequence will be preserved in the RNA recovered from animal tissue. All intronic barcode constructs were tested for their ability to produce high titer AAV progeny by cotransfecting them with pHelper and pREP3 stop plasmids. All constructs allowed high titer AAV production going from 50% to 80% of non-barcoded CAG9 virus (FIG. 60B).

RNA splicing analysis from transfected cells showed that the rate of AAV intron splicing was slightly different between constructs and was more efficient when the intronic barcode was inserted after a conserved intervening sequence downstream of the splice donor (FIG. 58C, upper panel).

Globin intron splicing was 100% effective in all tested conditions (FIG. 60C, lower panel). As expected, AAV intron splicing was almost undetectable in the absence of helper functions.

An alternative platform was tested where the tandem barcode was placed between the capsid stop codon and the polyadenylation signal (FIG. 59B). Titers produced by the 3′-barcoded constructs were identical to the non-barcoded CAG9 construct.

Overall, external barcoding of full-length capsid allows highly efficient AAV production, and the novel tandem barcode platform allows NGS-driven sequence-specific recovery from library preparations with high confidence.

TABLE 12
Sequences
DESCRIPTION
SEQ ID NO:    NUCLEIC ACID SEQUENCE
PREP2 SEQ ID  CGCAGGGTCTCCATTTTGAAGCGGGAGGTTTGAACGCGCAGCCGCCATGCCGGGGTTTTA
NO: 4         CGAGATTGTGATTAAGGTCCCCAGCGACCTTGACGAGCATCTGCCCGGCATTTCTGACAG
              CTTTGTGAACTGGGTGGCCGAGAAGGAATGGGAGTTGCCGCCAGATTCTGACATGGATCT
              GAATCTGATTGAGCAGGCACCCCTGACCGTGGCCGAGAAGCTGCAGCGCGACTTTCTGAC
              GGAATGGCGCCGTGTGAGTAAGGCCCCGGAGGCTCTTTTCTTTGTGCAATTTGAGAAGGG
              AGAGAGCTACTTCCACATGCACGTGCTCGTGGAAACCACCGGGGTGAAATCCATGGTTTT
              GGGACGTTTCCTGAGTCAGATTCGCGAAAAACTGATTCAGAGAATTTACCGCGGGATCGA
              GCCGACTTTGCCAAACTGGTTCGCGGTCACAAAGACCAGAAATGGCGCCGGAGGCGGGA
              ACAAGGTGGTGGATGAGTGCTACATCCCCAATTACTTGCTCCCCAAAACCCAGCCTGAGC
              TCCAGTGGGCGTGGACTAATATGGAACAGTATTTAAGCGCCTGTTTGAATCTCACGGAGC
              GTAAACGGTTGGTGGCGCAGCATCTGACGCACGTGTCGCAGACGCAGGAGCAGAACAAA
              GAGAATCAGAATCCCAATTCTGATGCGCCGGTGATCAGATCAAAAACTTCAGCCAGGTAC
              ATGGAGCTGGTCGGGTGGCTCGTGGACAAGGGGATTACCTCGGAGAAGCAGTGGATCCA
              GGAGGACCAGGCCTCATACATCTCCTTCAATGCGGCCTCCAACTCGCGGTCCCAAATCAA
              GGCTGCCTTGGACAATGCGGGAAAGATTATGAGCCTGACTAAAACCGCCCCCGACTACCT
              GGTGGGCCAGCAGCCCGTGGAGGACATTTCCAGCAATCGGATTTATAAAATTTTGGAACT
              AAACGGGTACGATCCCCAATATGCGGCTTCCGTCTTTCTGGGATGGGCCACGAAAAAGTT
              CGGCAAGAGGAACACCATCTGGCTGTTTGGGCCTGCAACTACCGGGAAGACCAACATCG
              CGGAGGCCATAGCCCACACTGTGCCCTTCTACGGGTGCGTAAACTGGACCAATGAGAACT
              TTCCCTTCAACGACTGTGTCGACAAGATGGTGATCTGGTGGGAGGAGGGGAAGATGACC
              GCCAAGGTCGTGGAGTCGGCCAAAGCCATTCTCGGAGGAAGCAAGGTGCGCGTGGACCA
              GAAATGCAAGTCCTCGGCCCAGATAGACCCGACTCCCGTGATCGTCACCTCCAACACCAA
              CATGTGCGCCGTGATTGACGGGAACTCAACGACCTTCGAACACCAGCAGCCGTTGCAAGA
              CCGGATGTTCAAATTTGAACTCACCCGCCGTCTGGATCATGACTTTGGGAAGGTCACCAA
              GCAGGAAGTCAAAGACTTTTTCCGGTGGGCAAAGGATCACGTGGTTGAGGTGGAGCATG
              AATTCTACGTCAAAAAGGGTGGAGCCAAGAAAAGACCCGCCCCCAGTGACGCAGATATA
              AGTGAGCCCAAACGGGTGCGCGAGTCAGTTGCGCAGCCATCGACGTCAGACGCGGAAGC
              TTCGATCAACTACGCAGACAGGTACCAAAACAAATGTTCTCGTCACGTGGGCATGAATCT
              GATGCTGTTTCCCTGCAGACAATGCGAGAGAATGAATCAGAATTCAAATATCTGCTTCAC
              TCACGGACAGAAAGACTGTTTAGAGTGCTTTCCCGTGTCAGAATCTCAACCCGTTTCTGTC
              GTCAAAAAGGCGTATCAGAAACTGTGCTACATTCATCATATCATGGGAAAGGTGCCAGAC
              GCTTGCACTGCCTGCGATCTGGTCAATGTGGATTTGGATGACTGCATCTTTGAACAATAA
              ATGATTTAAATCAGGTATGGCTGCCGATGGTTATCTTCCAGATTGGCTCGAGGACACTCTC
              TCTGAAGGAATAAGACAGTGGTGGAAGCTCAAACCTGGCCCACCACCACCAAAGCCCGC
              AGAGCGGCATAAGGACGACAGCAGGGGTCTTGTGCTTCCTGGGTACAAGTACCTCGGAC
              CCTTCAACGGACTCGACAAGGGAGAGCCGGTCAACGAGGCAGACGCCGCGGCCCTCGAG
              CACGACAAAGCCTACGACCGGCAGCTCGACAGCGGAGACAACCCGTACCTCAAGTACAA
              CCACGCCGACGCGGAGTTTCAGGAGCGCCTTAAAGAAGATACGTCTTTTGGGGGCAACCT
              CGGACGAGCAGTCTTCCAGGCGAAAAAGAGGGTTCTTGAACCTCTGGGCCTGGTCCACCA
              TACCTTCGATTATCCGATTTGCTTGTTAATCAATAAACCGTTTAATTCGTTTCAGTTGAACT
              TTGGTCTCTGCGTATTTCTTTCTTATCTAGTTTCCATGCTCTAGAGCGGCCGCCACCGCGGT
              GGAGCTCCAGCTTTTGT
CMV9-BSTEII   TTGGCCACTCCCTCTCTGCGCGCTCGCTCGCTCACTGAGGCCGGGCGACCAAAGGTCGCC
SEQ ID NO: 5  CGACGCCCGGGCTTTGCCCGGGCGGCCTCAGTGAGCGAGCGAGCGCGCAGAGAGGGAGT
              GGCCAACTCCATCACTAGGGGTTCCTGGAGGGGTGGAGTCGTGACGATATCGTTTAAACC
              GCGTCGTTACATAACTTACGGTAAATGGCCCGCCTGGCTGACCGCCCAACGACCCCCGCC
              CATTGACGTCAATAATGACGTATGTTCCCATAGTAACGCCAATAGGGACTTTCCATTGAC
              GTCAATGGGTGGAGTATTTACGGTAAACTGCCCACTTGGCAGTACATCAAGTGTATCATA
              TGCCAAGTACGCCCCCTATTGACGTCAATGACGGTAAATGGCCCGCCTGGCATTATGCCC
              AGTACATGACCTTATGGGACTTTCCTACTTGGCAGTACATCTACGTATTAGTCATCGCTAT
              TACCATGGTGATGCGGTTTTGGCAGTACATCAATGGGCGTGGATAGCGGTTTGACTCACG
              GGGATTTCCAAGTCTCCACCCCATTGACGTCAATGGGAGTTTGTTTTGGCACCAAAATCA
              ACGGGACTTTCCAAAATGTCGTAACAACTCCGCCCCATTGACGCAAATGGGCGGTAGGCG
              TGTACGGTGGGAGGTCTATATAAGCAGAGCTCGGGAGCGGTCACCAAGCAGGAAGTCAA
              AGACTTTTTCCGGTGGGCAAAGGATCACGTGGTTGAGGTGGAGCATGAATTCTACGTCAA
              AAAGGGTGGAGCCAAGAAAAGACCCGCCCCCAGTGACGCAGATATAAGTGAGCCCAAAC
              GGGTGCGCGAGTCAGTTGCGCAGCCATCGACGTCAGACGCGGAAGCTTCGATCAACTAC
              GCGGACAGGTACCAAAACAAATGTTCTCGTCACGTGGGCATGAATCTGATGCTGTTTCCC
              TGCAGACAATGCGAGAGACTGAATCAGAATTCAAATATCTGCTTCACTCACGGTGTCAAA
              GACTGTTTAGAGTGCTTTCCCGTGTCAGAATCTCAACCCGTTTCTGTCGTCAAAAAGGCGT
              ATCAGAAACTGTGCTACATTCATCACATCATGGGAAAGGTGCCAGACGCTTGCACTGCTT
              GCGACCTGGTCAATGTGGACTTGGATGACTGTGTTTCTGAACAATAAATGACTTAAACCA
              GGTATGGCTGCCGATGGTTATCTTCCAGATTGGCTCGAGGACAACCTTAGTGAAGGAATT
              CGCGAGTGGTGGGCTTTGAAACCTGGAGCCCCTCAACCCAAGGCAAATCAACAACATCA
              AGACAACGCTCGAGGTCTTGTGCTTCCGGGTTACAAATACCTTGGACCCGGCAACGGACT
              CGACAAGGGGGAGCCGGTCAACGCAGCAGACGCGGCGGCCCTCGAGCACGACAAGGCCT
              ACGACCAGCAGCTCAAGGCCGGAGACAACCCGTACCTCAAGTACAACCACGCCGACGCC
              GAGTTCCAGGAGCGGCTCAAAGAAGATACGTCTTTTGGGGGCAACCTCGGGCGAGCAGT
              CTTCCAGGCCAAAAAGAGGCTTCTTGAACCTCTTGGTCTGGTTGAGGAAGCGGCTAAGAC
              GGCTCCTGGAAAGAAGAGGCCTGTAGAGCAGTCTCCTCAGGAACCGGACTCCTCCGCGG
              GTATTGGCAAATCGGGTGCACAGCCCGCTAAAAAGAGACTCAATTTCGGTCAGACTGGCG
              ACACAGAGTCAGTCCCAGACCCTCAACCAATCGGAGAACCTCCCGCAGCCCCCTCAGGTG
              TGGGATCTCTTACAATGGCTTCAGGTGGTGGCGCACCAGTGGCAGACAATAACGAAGGTG
              CCGATGGAGTGGGTAGTTCCTCGGGAAATTGGCATTGCGATTCCCAATGGCTGGGGGACA
              GAGTCATCACCACCAGCACCCGAACCTGGGCCCTGCCCACCTACAACAATCACCTCTACA
              AGCAAATCTCCAACAGCACATCTGGAGGATCTTCAAATGACAACGCCTACTTCGGCTACA
              GCACCCCCTGGGGGTATTTTGACTTCAACAGATTCCACTGCCACTTCTCACCACGTGACTG
              GCAGCGACTCATCAACAACAACTGGGGATTCCGGCCTAAGCGACTCAACTTCAAGCTCTT
              CAACATTCAGGTCAAAGAGGTTACGGACAACAATGGAGTCAAGACCATCGCCAATAACC
              TTACCAGCACGGTCCAGGTCTTCACGGACTCAGACTATCAGCTCCCGTACGTGCTCGGGT
              CGGCTCACGAGGGCTGCCTCCCGCCGTTCCCAGCGGACGTTTTCATGATTCCTCAGTACG
              GGTATCTGACGCTTAATGATGGAAGCCAGGCCGTGGGTCGTTCGTCCTTTTACTGCCTGG
              AATATTTCCCGTCGCAAATGCTAAGAACGGGTAACAACTTCCAGTTCAGCTACGAGTTTG
              AGAACGTACCTTTCCATAGCAGCTACGCTCACAGCCAAAGCCTGGACCGACTAATGAATC
              CACTCATCGACCAATACTTGTACTATCTCTCAAAGACTATTAACGGTTCTGGACAGAATC
              AACAAACGCTAAAATTCAGTGTGGCCGGACCCAGCAACATGGCTGTCCAGGGAAGAAAC
              TACATACCTGGACCCAGCTACCGACAACAACGTGTCTCAACCACTGTGACTCAAAACAAC
              AACAGCGAATTTGCTTGGCCTGGAGCTTCTTCTTGGGCTCTCAATGGACGTAATAGCTTGA
              TGAATCCTGGACCTGCTATGGCCAGCCACAAAGAAGGAGAGGACCGTTTCTTTCCTTTGT
              CTGGATCTTTAATTTTTGGCAAACAAGGAACTGGAAGAGACAACGTGGATGCGGACAAA
              GTCATGATAACCAACGAAGAAGAAATTAAAACTACTAACCCGGTAGCAACGGAGTCCTA
              TGGACAAGTGGCCACAAACCACCAGAGTGCCCAAGCACAGGCGCAGACCGGCTGGGTTC
              AAAACCAAGGAATACTTCCGGGTATGGTTTGGCAGGACAGAGATGTGTACCTGCAAGGA
              CCCATTTGGGCCAAAATTCCTCACACGGACGGCAACTTTCACCCTTCTCCGCTGATGGGA
              GGGTTTGGAATGAAGCACCCGCCTCCTCAGATCCTCATCAAAAACACACCTGTACCTGCG
              GATCCTCCAACGGCCTTCAACAAGGACAAGCTGAACTCTTTCATCACCCAGTATTCTACT
              GGCCAAGTCAGCGTGGAGATCGAGTGGGAGCTGCAGAAGGAAAACAGCAAGCGCTGGA
              ACCCGGAGATCCAGTACACTTCCAACTATTACAAGTCTAATAATGTTGAATTTGCTGTTAA
              TACTGAAGGTGTATATAGTGAACCCCGCCCCATTGGCACCAGATACCTGACTCGTAATCT
              GTAATCGATTGTTAATCAATAAACCGTTTAATTCGTTTCAGTTGAACTTTGGTCTCTGCGT
              ATTTCTTTCTTATCTAGTTTCCATGGCTACGTAGATAAGTAGCATGGCGGGTTAATCATTA
              ACTACAAGGAACCCCTAGTGATGGAGTTGGCCACTCCCTCTCTGCGCGCTCGCTCGCTCA
              CTGAGGCCGGGCGACCAAAGGTCGCCCGACGCCCGGGCTTTGCCCGGGCGGCCTCAGTG
              AGCGAGCGAGCGCGCAGAGAGGGAGTGGCCAA
PREP-AAP      GTCGACGGTATCGGGGGAGCTCGCAGGGTCTCCATTTTGAAGCGGGAGGTTTGAACGCGCAGC
SEQ ID NO: 6  CGCCATGCCGGGGTTTTACGAGATTGTGATTAAGGTCCCCAGCGACCTTGACGAGCATCTGCCC
              GGCATTTCTGACAGCTTTGTGAACTGGGTGGCCGAGAAGGAATGGGAGTTGCCGCCAGATTCT
              GACATGGATCTGAATCTGATTGAGCAGGCACCCCTGACCGTGGCCGAGAAGCTGCAGCGCGAC
              TTTCTGACGGAATGGCGCCGTGTGAGTAAGGCCCCGGAGGCTCTTTTCTTTGTGCAATTTGAGA
              AGGGAGAGAGCTACTTCCACATGCACGTGCTCGTGGAAACCACCGGGGTGAAATCCATGGTTT
              TGGGACGTTTCCTGAGTCAGATTCGCGAAAAACTGATTCAGAGAATTTACCGCGGGATCGAGC
              CGACTTTGCCAAACTGGTTCGCGGTCACAAAGACCAGAAATGGCGCCGGAGGCGGGAACAAG
              GTGGTGGATGAGTGCTACATCCCCAATTACTTGCTCCCCAAAACCCAGCCTGAGCTCCAGTGGG
              CGTGGACTAATATGGAACAGTATTTAAGCGCCTGTTTGAATCTCACGGAGCGTAAACGGTTGGT
              GGCGCAGCATCTGACGCACGTGTCGCAGACGCAGGAGCAGAACAAAGAGAATCAGAATCCCA
              ATTCTGATGCGCCGGTGATCAGATCAAAAACTTCAGCCAGGTACATGGAGCTGGTCGGGTGGC
              TCGTGGACAAGGGGATTACCTCGGAGAAGCAGTGGATCCAGGAGGACCAGGCCTCATACATCT
              CCTTCAATGCGGCCTCCAACTCGCGGTCCCAAATCAAGGCTGCCTTGGACAATGCGGGAAAGA
              TTATGAGCCTGACTAAAACCGCCCCCGACTACCTGGTGGGCCAGCAGCCCGTGGAGGACATTT
              CCAGCAATCGGATTTATAAAATTTTGGAACTAAACGGGTACGATCCCCAATATGCGGCTTCCGT
              CTTTCTGGGATGGGCCACGAAAAAGTTCGGCAAGAGGAACACCATCTGGCTGTTTGGGCCTGC
              AACTACCGGGAAGACCAACATCGCGGAGGCCATAGCCCACACTGTGCCCTTCTACGGGTGCGT
              AAACTGGACCAATGAGAACTTTCCCTTCAACGACTGTGTCGACAAGATGGTGATCTGGTGGGA
              GGAGGGGAAGATGACCGCCAAGGTCGTGGAGTCGGCCAAAGCCATTCTCGGAGGAAGCAAGG
              TGCGCGTGGACCAGAAATGCAAGTCCTCGGCCCAGATAGACCCGACTCCCGTGATCGTCACCT
              CCAACACCAACATGTGCGCCGTGATTGACGGGAACTCAACGACCTTCGAACACCAGCAGCCGT
              TGCAAGACCGGATGTTCAAATTTGAACTCACCCGCCGTCTGGATCATGACTTTGGGAAGGTCAC
              CAAGCAGGAAGTCAAAGACTTTTTCCGGTGGGCAAAGGATCACGTGGTTGAGGTGGAGCATGA
              ATTCTACGTCAAAAAGGGTGGAGCCAAGAAAAGACCCGCCCCCAGTGACGCAGATATAAGTGA
              GCCCAAACGGGTGCGCGAGTCAGTTGCGCAGCCATCGACGTCAGACGCGGAAGCTTCGATCAA
              CTACGCGGACAGGTACCAAAACAAATGTTCTCGTCACGTGGGCATGAATCTGATGCTGTTTCCC
              TGCAGACAATGCGAGAGACTGAATCAGAATTCAAATATCTGCTTCACTCACGGTGTCAAAGAC
              TGTTTAGAGTGCTTTCCCGTGTCAGAATCTCAACCCGTTTCTGTCGTCAAAAAGGCGTATCAGA
              AACTGTGCTACATTCATCACATCATGGGAAAGGTGCCAGACGCTTGCACTGCTTGCGACCTGGT
              CAATGTGGACTTGGATGACTGTGTTTCTGAACAATAAATGACTTAAACCAGGTATGGCTGCCGA
              TGGTTATCTTCCAGATTGGCTCGAGGACAACCTTAGTGAAGGAATTCGCGAGTGGTGGGCTTTG
              AAACCTGGAGCCCCTCAACCCAAGGCAAATCAACAACATCAAGACAACGCTCGAGGTCTTGTG
              CTTCCGGGTTACAAATACCTTGGACCCGGCAACGGACTCGACAAGGGGGAGCCGGTCAACGCA
              GCAGACGCGGCGGCCCTCGAGCACGACAAGGCCTACGACCAGCAGCTCAAGGCCGGAGACAA
              CCCGTACCTCAAGTACAACCACGCCGACGCCGAGTTCCAGGAGCGGCTCAAAGAAGATACGTC
              TTTTGGGGGCAACCTCGGGCGAGCAGTCTTCCAGGCCAAAAAGAGGCTTCTTGAACCTCTTGGT
              CTGGTTGAGGAAGCGGCTAAGACGGCTCCTGGAAAGAAGAGGCCTGTAGAGCAGTCTCCTCAG
              GAACCGGACTCCTCCGCGGGTATTGGCAAATCGGGTGCACAGCCCGCTAAAAAGAGACTCAAT
              TTCGGTCAGACTGGCGACACAGAGTCAGTCCCAGACCCTCAACCAATCGGAGAACCTCCCGCA
              GCCCCCTCAGGTGTGGGATCTCTTACAATGGCTTCAGGTGGTGGCGCACCAGTGGCAGACAAT
              AACGAAGGTGCCGATGGAGTGGGTAGTTCCTCGGGAAATTGGCATTGCGATTCCCAATGGCTG
              GGGGACAGAGTCATCACCACCAGCACCCGAACCTGGGCCCTGCCCACCTACAACAATCACCTC
              TACAAGCAAATCTCCAACAGCACATCTGGAGGATCTTCAAATGACAACGCCTACTTCGGCTAC
              AGCACCCCCTGGGGGTATTTTGACTTCAACAGATTCCACTGCCACTTCTCACCACGTGACTGGC
              AGCGACTCATCAACAACAACTGGGGATTCCGGCCTAAGCGACTCAACTTCAAGCTCTTCAACAT
              TCAGGTCAAAGAGGTTACGGACAACAATGGAGTCAAGACCATCGCCAATAACCTTACCAGCAC
              GGTCCAGGTCTTCACGGACTCAGACTATCAGCTCCCGTACGTGCTCGGGTCGGCTCACGAGGGC
              TGCCTCCCGCCGTTCCCAGCGGACGTTTTCATGATTCCTCAGTACGGGTATCTGACGCTTAATG
              ATGGAAGCCAGGCCGTGGGTCGTTCGTCCTTTTACTGCCTGGAATATTTCCCGTCGCAAATGCT
              AAGAACGGGTAACAACTTCCAGTTCAGCTACGAGTTTGAGAACGTACCTTTCCATAGCAGCTAC
              GCTCACAGCCAAAGCCTGGACCGACTAATGAATCCACTCATCGACCAATACTTGTACTATCTCT
              CAAAGACTATTAACGGTTCTGGACAGAATCAACAAACGCTAAAATTCAGTGTGGCCGGACCCA
              GCAACATGGCTGTCCAGGGAAGAAACTACATACCTGGACCCAGCTACCGACAACAACGTGTCT
              CAACCACTGTGACTCAAAACAACAACAGCGAATTTGCTTGGCCTGGAGCTTCTTCTTGGGCTCT
              CAATGGACGTAATAGCTTGATGAATCCTGGACCTGCTATGGCCAAGTCAGCGTGGAGATCGAG
              TGGGAGCTGCAGAAGGAAAACAGCAAGCGCTGGAACCCGGAGATCCAGTACACTTCCAACTAT
              TACAAGTCTAATAATGTTGAATTTGCTGTTAATACTGAAGGTGTATATAGTGAACCCCGCCCCA
              TTGGCACCAGATACCTGACTCGTAATCTGTAATTGCTTGTTAATCAATAAACCGTTTAATTCGTT
              TCAGTTGAACTTTGGTCTC
PREP3 STOP    GTCGACGGTATCGGGGGAGCTCGCAGGGTCTCCATTTTGAAGCGGGAGGTTTGAACGCGCAGC
SEQ ID NO: 7  CGCCATGCCGGGGTTTTACGAGATTGTGATTAAGGTCCCCAGCGACCTTGACGAGCATCTGCCC
              GGCATTTCTGACAGCTTTGTGAACTGGGTGGCCGAGAAGGAATGGGAGTTGCCGCCAGATTCT
              GACATGGATCTGAATCTGATTGAGCAGGCACCCCTGACCGTGGCCGAGAAGCTGCAGCGCGAC
              TTTCTGACGGAATGGCGCCGTGTGAGTAAGGCCCCGGAGGCTCTTTTCTTTGTGCAATTTGAGA
              AGGGAGAGAGCTACTTCCACATGCACGTGCTCGTGGAAACCACCGGGGTGAAATCCATGGTTT
              TGGGACGTTTCCTGAGTCAGATTCGCGAAAAACTGATTCAGAGAATTTACCGCGGGATCGAGC
              CGACTTTGCCAAACTGGTTCGCGGTCACAAAGACCAGAAATGGCGCCGGAGGCGGGAACAAG
              GTGGTGGATGAGTGCTACATCCCCAATTACTTGCTCCCCAAAACCCAGCCTGAGCTCCAGTGGG
              CGTGGACTAATATGGAACAGTATTTAAGCGCCTGTTTGAATCTCACGGAGCGTAAACGGTTGGT
              GGCGCAGCATCTGACGCACGTGTCGCAGACGCAGGAGCAGAACAAAGAGAATCAGAATCCCA
              ATTCTGATGCGCCGGTGATCAGATCAAAAACTTCAGCCAGGTACATGGAGCTGGTCGGGTGGC
              TCGTGGACAAGGGGATTACCTCGGAGAAGCAGTGGATCCAGGAGGACCAGGCCTCATACATCT
              CCTTCAATGCGGCCTCCAACTCGCGGTCCCAAATCAAGGCTGCCTTGGACAATGCGGGAAAGA
              TTATGAGCCTGACTAAAACCGCCCCCGACTACCTGGTGGGCCAGCAGCCCGTGGAGGACATTT
              CCAGCAATCGGATTTATAAAATTTTGGAACTAAACGGGTACGATCCCCAATATGCGGCTTCCGT
              CTTTCTGGGATGGGCCACGAAAAAGTTCGGCAAGAGGAACACCATCTGGCTGTTTGGGCCTGC
              AACTACCGGGAAGACCAACATCGCGGAGGCCATAGCCCACACTGTGCCCTTCTACGGGTGCGT
              AAACTGGACCAATGAGAACTTTCCCTTCAACGACTGTGTCGACAAGATGGTGATCTGGTGGGA
              GGAGGGGAAGATGACCGCCAAGGTCGTGGAGTCGGCCAAAGCCATTCTCGGAGGAAGCAAGG
              TGCGCGTGGACCAGAAATGCAAGTCCTCGGCCCAGATAGACCCGACTCCCGTGATCGTCACCT
              CCAACACCAACATGTGCGCCGTGATTGACGGGAACTCAACGACCTTCGAACACCAGCAGCCGT
              TGCAAGACCGGATGTTCAAATTTGAACTCACCCGCCGTCTGGATCATGACTTTGGGAAGGTCAC
              CAAGCAGGAAGTCAAAGACTTTTTCCGGTGGGCAAAGGATCACGTGGTTGAGGTGGAGCATGA
              ATTCTACGTCAAAAAGGGTGGAGCCAAGAAAAGACCCGCCCCCAGTGACGCAGATATAAGTGA
              GCCCAAACGGGTGCGCGAGTCAGTTGCGCAGCCATCGACGTCAGACGCGGAAGCTTCGATCAA
              CTACGCGGACAGGTACCAAAACAAATGTTCTCGTCACGTGGGCATGAATCTGATGCTGTTTCCC
              TGCAGACAATGCGAGAGACTGAATCAGAATTCAAATATCTGCTTCACTCACGGTGTCAAAGAC
              TGTTTAGAGTGCTTTCCCGTGTCAGAATCTCAACCCGTTTCTGTCGTCAAAAAGGCGTATCAGA
              AACTGTGCTACATTCATCACATCATGGGAAAGGTGCCAGACGCTTGCACTGCTTGCGACCTGGT
              CAATGTGGACTTGGATGACTGTGTTTCTGAACAATAAATGACTTAAACCAGGTATGGCTGCCGA
              TGGTTAGCTTCCAGATTGGCTCGAGGACAACCTTAGTGAAGGAATTCGCGAGTGGTGGGCTTTG
              AAACCTGGAGCCCCTCAACCCAAGGCAAATCAACAACATCAAGACAACGCTCGAGGTCTTGTG
              CTTCCGGGTTACAAATACCTTGGACCCGGCAACGGACTCGACAAGGGGGAGCCGGTCAACGCA
              GCAGACGCGGCGGCCCTCGAGCACGACAAGGCCTACGACCAGCAGCTCAAGGCCGGAGACAA
              CCCGTACCTCAAGTACAACCACGCCGACGCCGAGTTCCAGGAGCGGCTCAAAGAAGATACGTC
              TTTTGGGGGCAACCTCGGGCGAGCAGTCTTCCAGGCCAAAAAGAGGCTTCTTGAACCTCTTGGT
              CTGGTTGAGGAAGCGGCTAAGACGGCTCCTGGAAAGTAGAGGCCTGTAGAGCAGTCTCCTCAG
              GAACCGGACTCCTCCGCGGGTATTGGCAAATCGGGTGCACAGCCCGCTAAAAAGAGACTCAAT
              TTCGGTCAGACTGGCGACACAGAGTCAGTCCCAGACCCTCAACCAATCGGAGAACCTCCCGCA
              GCCCCCTCAGGTGTGGGATCTCTTACAATGGCTTCAGGTGGTGGCGCACCAGTGGCAGACAAT
              AACTAAGGTGCCGATGGAGTGGGTAGTTCCTCGGGAAATTGGCATTGCGATTCCCAATGGCTG
              GGGGACAGAGTCATCACCACCAGCACCCGAACCTGGGCCCTGCCCACCTACAACAATCACCTC
              TACAAGCAAATCTCCAACAGCACATCTGGAGGATCTTCAAATGACAACGCCTACTTCGGCTAC
              AGCACCCCCTGGGGGTATTTTGACTTCAACAGATTCCACTGCCACTTCTCACCACGTGACTGGC
              AGCGACTCATCAACAACAACTGGGGATTCCGGCCTAAGCGACTCAACTTCAAGCTCTTCAACAT
              TCAGGTCAAAGAGGTTACGGACAACAATGGAGTCAAGACCATCGCCAATAACCTTACCAGCAC
              GGTCCAGGTCTTCACGGACTCAGACTATCAGCTCCCGTACGTGCTCGGGTCGGCTCACGAGGGC
              TGCCTCCCGCCGTTCCCAGCGGACGTTTTCATGATTCCTCAGTACGGGTATCTGACGCTTAATG
              ATGGAAGCCAGGCCGTGGGTCGTTCGTCCTTTTACTGCCTGGAATATTTCCCGTCGCAAATGCT
              AAGAACGGGTAACAACTTCCAGTTCAGCTACGAGTTTGAGAACGTACCTTTCCATAGCAGCTAC
              GCTCACAGCCAAAGCCTGGACCGACTAATGAATCCACTCATCGACCAATACTTGTACTATCTCT
              CAAAGACTATTAACGGTTCTGGACAGAATCAACAAACGCTAAAATTCAGTGTGGCCGGACCCA
              GCAACATGGCTGTCCAGGGAAGAAACTACATACCTGGACCCAGCTACCGACAACAACGTGTCT
              CAACCACTGTGACTCAAAACAACAACAGCGAATTTGCTTGGCCTGGAGCTTCTTCTTGGGCTCT
              CAATGGACGTAATAGCTTGATGAATCCTGGACCTGCTATGGCCAAGTCAGCGTGGAGATCGAG
              TGGGAGCTGCAGAAGGAAAACAGCAAGCGCTGGAACCCGGAGATCCAGTACACTTCCAACTAT
              TACAAGTCTAATAATGTTGAATTTGCTGTTAATACTGAAGGTGTATATAGTGAACCCCGCCCCA
              TTGGCACCAGATACCTGACTCGTAATCTGTAATTGCTTGTTAATCAATAAACCGTTTAATTCGTT
              TCAGTTGAACTTTGGTCTC
SYN-CAP9      TTGGCCACTCCCTCTCTGCGCGCTCGCTCGCTCACTGAGGCCGGGCGACCAAAGGTCGCCCGAC
SEQ ID NO: 8  GCCCGGGCTTTGCCCGGGCGGCCTCAGTGAGCGAGCGAGCGCGCAGAGAGGGAGTGGCCAACT
              CCATCACTAGGGGTTCCTGGAGGGGTGGAGTCGTGACGATATCTAGTATCTGCAGAGGGCCCT
              GCGTATGAGTGCAAGTGGGTTTTAGGACCAGGATGAGGCGGGGTGGGGGTGCCTACCTGACGA
              CCGACCCCGACCCACTGGACAAGCACCCAACCCCCATTCCCCAAATTGCGCATCCCCTATCAGA
              GAGGGGGAGGGGAAACAGGATGCGGCGAGGCGCGTGCGCACTGCCAGCTTCAGCACCGCGGA
              CAGTGCCTTCGCCCCCGCCTGGCGGCGCGCGCCACCGCCGCCTCAGCACTGAAGGCGCGCTGA
              CGTCACTCGCCGGTCCCCCGCAAACTCCCCTTCCCGGCCACCTTGGTCGCGTCCGCGCCGCCGC
              CGGCCCAGCCGGACCGCACCACGCGAGGCGCGAGATAGGGGGGCACGGGCGCGACCATCTGC
              GCTGCGGCGCCGGCGACTCAGCGCTGCCTCAGTCTGCGGTGGGCAGCGGAGGAGTCGTGTCGT
              GCCTGAGAGCGCAGCTGTGCTCCTGGGCACCGCGCAGTCCGCCCCCGCGGCTCCTGGCCAGAC
              CACCCCTAGGACCCCCTGCCCCAAGTCGCAGCCGGTCACCAAGCAGGAAGTCAAAGACTTTTT
              CCGGTGGGCAAAGGATCACGTGGTTGAGGTGGAGCATGAATTCTACGTCAAAAAGGGTGGAGC
              CAAGAAAAGACCCGCCCCCAGTGACGCAGATATAAGTGAGCCCAAACGGGTGCGCGAGTCAG
              TTGCGCAGCCATCGACGTCAGACGCGGAAGCTTCGATCAACTACGCGGACAGGTACCAAAACA
              AATGTTCTCGTCACGTGGGCATGAATCTGATGCTGTTTCCCTGCAGACAATGCGAGAGACTGAA
              TCAGAATTCAAATATCTGCTTCACTCACGGTGTCAAAGACTGTTTAGAGTGCTTTCCCGTGTCA
              GAATCTCAACCCGTTTCTGTCGTCAAAAAGGCGTATCAGAAACTGTGCTACATTCATCACATCA
              TGGGAAAGGTGCCAGACGCTTGCACTGCTTGCGACCTGGTCAATGTGGACTTGGATGACTGTGT
              TTCTGAACAATAAATGACTTAAACCAGGTATGGCTGCCGATGGTTATCTTCCAGATTGGCTCGA
              GGACAACCTTAGTGAAGGAATTCGCGAGTGGTGGGCTTTGAAACCTGGAGCCCCTCAACCCAA
              GGCAAATCAACAACATCAAGACAACGCTCGAGGTCTTGTGCTTCCGGGTTACAAATACCTTGG
              ACCCGGCAACGGACTCGACAAGGGGGAGCCGGTCAACGCAGCAGACGCGGCGGCCCTCGAGC
              ACGACAAGGCCTACGACCAGCAGCTCAAGGCCGGAGACAACCCGTACCTCAAGTACAACCACG
              CCGACGCCGAGTTCCAGGAGCGGCTCAAAGAAGATACGTCTTTTGGGGGCAACCTCGGGCGAG
              CAGTCTTCCAGGCCAAAAAGAGGCTTCTTGAACCTCTTGGTCTGGTTGAGGAAGCGGCTAAGA
              CGGCTCCTGGAAAGAAGAGGCCTGTAGAGCAGTCTCCTCAGGAACCGGACTCCTCCGCGGGTA
              TTGGCAAATCGGGTGCACAGCCCGCTAAAAAGAGACTCAATTTCGGTCAGACTGGCGACACAG
              AGTCAGTCCCAGACCCTCAACCAATCGGAGAACCTCCCGCAGCCCCCTCAGGTGTGGGATCTCT
              TACAATGGCTTCAGGTGGTGGCGCACCAGTGGCAGACAATAACGAAGGTGCCGATGGAGTGGG
              TAGTTCCTCGGGAAATTGGCATTGCGATTCCCAATGGCTGGGGGACAGAGTCATCACCACCAG
              CACCCGAACCTGGGCCCTGCCCACCTACAACAATCACCTCTACAAGCAAATCTCCAACAGCAC
              ATCTGGAGGATCTTCAAATGACAACGCCTACTTCGGCTACAGCACCCCCTGGGGGTATTTTGAC
              TTCAACAGATTCCACTGCCACTTCTCACCACGTGACTGGCAGCGACTCATCAACAACAACTGGG
              GATTCCGGCCTAAGCGACTCAACTTCAAGCTCTTCAACATTCAGGTCAAAGAGGTTACGGACA
              ACAATGGAGTCAAGACCATCGCCAATAACCTTACCAGCACGGTCCAGGTCTTCACGGACTCAG
              ACTATCAGCTCCCGTACGTGCTCGGGTCGGCTCACGAGGGCTGCCTCCCGCCGTTCCCAGCGGA
              CGTTTTCATGATTCCTCAGTACGGGTATCTGACGCTTAATGATGGAAGCCAGGCCGTGGGTCGT
              TCGTCCTTTTACTGCCTGGAATATTTCCCGTCGCAAATGCTAAGAACGGGTAACAACTTCCAGT
              TCAGCTACGAGTTTGAGAACGTACCTTTCCATAGCAGCTACGCTCACAGCCAAAGCCTGGACCG
              ACTAATGAATCCACTCATCGACCAATACTTGTACTATCTCTCAAAGACTATTAACGGTTCTGGA
              CAGAATCAACAAACGCTAAAATTCAGTGTGGCCGGACCCAGCAACATGGCTGTCCAGGGAAGA
              AACTACATACCTGGACCCAGCTACCGACAACAACGTGTCTCAACCACTGTGACTCAAAACAAC
              AACAGCGAATTTGCTTGGCCTGGAGCTTCTTCTTGGGCTCTCAATGGACGTAATAGCTTGATGA
              ATCCTGGACCTGCTATGGCCAGCCACAAAGAAGGAGAGGACCGTTTCTTTCCTTTGTCTGGATC
              TTTAATTTTTGGCAAACAAGGAACTGGAAGAGACAACGTGGATGCGGACAAAGTCATGATAAC
              CAACGAAGAAGAAATTAAAACTACTAACCCGGTAGCAACGGAGTCCTATGGACAAGTGGCCAC
              AAACCACCAGAGTGCCCAAGCACAGGCGCAGACCGGCTGGGTTCAAAACCAAGGAATACTTCC
              GGGTATGGTTTGGCAGGACAGAGATGTGTACCTGCAAGGACCCATTTGGGCCAAAATTCCTCA
              CACGGACGGCAACTTTCACCCTTCTCCGCTGATGGGAGGGTTTGGAATGAAGCACCCGCCTCCT
              CAGATCCTCATCAAAAACACACCTGTACCTGCGGATCCTCCAACGGCCTTCAACAAGGACAAG
              CTGAACTCTTTCATCACCCAGTATTCTACTGGCCAAGTCAGCGTGGAGATCGAGTGGGAGCTGC
              AGAAGGAAAACAGCAAGCGCTGGAACCCGGAGATCCAGTACACTTCCAACTATTACAAGTCTA
              ATAATGTTGAATTTGCTGTTAATACTGAAGGTGTATATAGTGAACCCCGCCCCATTGGCACCAG
              ATACCTGACTCGTAATCTGTAATCGATTGTTAATCAATAAACCGTTTAATTCGTTTCAGTTGAAC
              TTTGGTCTCTGCGTATTTCTTTCTTATCTAGTTTCCATGGCTACGTAGATAAGTAGCATGGCGGG
              TTAATCATTAACTACAAGGAACCCCTAGTGATGGAGTTGGCCACTCCCTCTCTGCGCGCTCGCT
              CGCTCACTGAGGCCGGGCGACCAAAGGTCGCCCGACGCCCGGGCTTTGCCCGGGCGGCCTCAG
              TGAGCGAGCGAGCGCGCAGAGAGGGAGTGGCCAA
GFAP-CAP9     TTGGCCACTCCCTCTCTGCGCGCTCGCTCGCTCACTGAGGCCGGGCGACCAAAGGTCGCCCGAC
SEQ ID NO: 9  GCCCGGGCTTTGCCCGGGCGGCCTCAGTGAGCGAGCGAGCGCGCAGAGAGGGAGTGGCCAACT
              CCATCACTAGGGGTTCCTGGAGGGGTGGAGTCGTGACGATATCGATCTAACATATCCTGGTGTG
              GAGTAGCGGACGCTGCTATGACAGAGGCTCGGGGGCCTGAGCTGGCTCTGTGAGCTGGGGAGG
              AGGCAGACAGCCAGGCCTTGTCTGCAAGCAGACCTGGCAGCATTGGGCTGGCCGCCCCCCAGG
              GCCTCCTCTTCATGCCCAGTGAATGACTCACCTTGGCACAGACACAATGTTCGGGGTGGGCACA
              GTGCCTGCTTCCCGCCGCACCCCAGCCCCCCTCAAATGCCTTCCGAGAAGCCCATTGAGCAGGG
              GGCTTGCATTGCACCCCAGCCTGACAGCCTGGCATCTTGGGATAAAAGCAGCACAGCCCCCTA
              GGGGCTGCCCTTGCTGTGTGGCGCCACCGGCGGTGGAGAACAAGGCTCTATTCAGCCTGTGCCC
              AGGAAAGGGGATCAGGGGATGCCCAGGCATGGACAGTGGGTGGCAGGGGGGGAGAGGAGGG
              CTGTCTGCTTCCCAGAAGTCCAAGGACACAAATGGGTGAGGGGAGAGCTCTCCCCATAGCTGG
              GCTGCGGCCCAACCCCACCCCCTCAGGCTATGCCAGGGGGTGTTGCCAGGGGCACCCGGGCAT
              CGCCAGTCTAGCCCACTCCTTCATAAAGCCCTCGCATCCCAGGAGCGAGCAGAGCCAGAGCAG
              GTTGGAGAGGAGACGCATCACCTCCGCTGCTCGCGGGGATCCTCTAGGGTCACCAAGCAGGAA
              GTCAAAGACTTTTTCCGGTGGGCAAAGGATCACGTGGTTGAGGTGGAGCATGAATTCTACGTC
              AAAAAGGGTGGAGCCAAGAAAAGACCCGCCCCCAGTGACGCAGATATAAGTGAGCCCAAACG
              GGTGCGCGAGTCAGTTGCGCAGCCATCGACGTCAGACGCGGAAGCTTCGATCAACTACGCGGA
              CAGGTACCAAAACAAATGTTCTCGTCACGTGGGCATGAATCTGATGCTGTTTCCCTGCAGACAA
              TGCGAGAGACTGAATCAGAATTCAAATATCTGCTTCACTCACGGTGTCAAAGACTGTTTAGAGT
              GCTTTCCCGTGTCAGAATCTCAACCCGTTTCTGTCGTCAAAAAGGCGTATCAGAAACTGTGCTA
              CATTCATCACATCATGGGAAAGGTGCCAGACGCTTGCACTGCTTGCGACCTGGTCAATGTGGAC
              TTGGATGACTGTGTTTCTGAACAATAAATGACTTAAACCAGGTATGGCTGCCGATGGTTATCTT
              CCAGATTGGCTCGAGGACAACCTTAGTGAAGGAATTCGCGAGTGGTGGGCTTTGAAACCTGGA
              GCCCCTCAACCCAAGGCAAATCAACAACATCAAGACAACGCTCGAGGTCTTGTGCTTCCGGGT
              TACAAATACCTTGGACCCGGCAACGGACTCGACAAGGGGGAGCCGGTCAACGCAGCAGACGC
              GGCGGCCCTCGAGCACGACAAGGCCTACGACCAGCAGCTCAAGGCCGGAGACAACCCGTACCT
              CAAGTACAACCACGCCGACGCCGAGTTCCAGGAGCGGCTCAAAGAAGATACGTCTTTTGGGGG
              CAACCTCGGGCGAGCAGTCTTCCAGGCCAAAAAGAGGCTTCTTGAACCTCTTGGTCTGGTTGAG
              GAAGCGGCTAAGACGGCTCCTGGAAAGAAGAGGCCTGTAGAGCAGTCTCCTCAGGAACCGGA
              CTCCTCCGCGGGTATTGGCAAATCGGGTGCACAGCCCGCTAAAAAGAGACTCAATTTCGGTCA
              GACTGGCGACACAGAGTCAGTCCCAGACCCTCAACCAATCGGAGAACCTCCCGCAGCCCCCTC
              AGGTGTGGGATCTCTTACAATGGCTTCAGGTGGTGGCGCACCAGTGGCAGACAATAACGAAGG
              TGCCGATGGAGTGGGTAGTTCCTCGGGAAATTGGCATTGCGATTCCCAATGGCTGGGGGACAG
              AGTCATCACCACCAGCACCCGAACCTGGGCCCTGCCCACCTACAACAATCACCTCTACAAGCA
              AATCTCCAACAGCACATCTGGAGGATCTTCAAATGACAACGCCTACTTCGGCTACAGCACCCCC
              TGGGGGTATTTTGACTTCAACAGATTCCACTGCCACTTCTCACCACGTGACTGGCAGCGACTCA
              TCAACAACAACTGGGGATTCCGGCCTAAGCGACTCAACTTCAAGCTCTTCAACATTCAGGTCAA
              AGAGGTTACGGACAACAATGGAGTCAAGACCATCGCCAATAACCTTACCAGCACGGTCCAGGT
              CTTCACGGACTCAGACTATCAGCTCCCGTACGTGCTCGGGTCGGCTCACGAGGGCTGCCTCCCG
              CCGTTCCCAGCGGACGTTTTCATGATTCCTCAGTACGGGTATCTGACGCTTAATGATGGAAGCC
              AGGCCGTGGGTCGTTCGTCCTTTTACTGCCTGGAATATTTCCCGTCGCAAATGCTAAGAACGGG
              TAACAACTTCCAGTTCAGCTACGAGTTTGAGAACGTACCTTTCCATAGCAGCTACGCTCACAGC
              CAAAGCCTGGACCGACTAATGAATCCACTCATCGACCAATACTTGTACTATCTCTCAAAGACTA
              TTAACGGTTCTGGACAGAATCAACAAACGCTAAAATTCAGTGTGGCCGGACCCAGCAACATGG
              CTGTCCAGGGAAGAAACTACATACCTGGACCCAGCTACCGACAACAACGTGTCTCAACCACTG
              TGACTCAAAACAACAACAGCGAATTTGCTTGGCCTGGAGCTTCTTCTTGGGCTCTCAATGGACG
              TAATAGCTTGATGAATCCTGGACCTGCTATGGCCAGCCACAAAGAAGGAGAGGACCGTTTCTTT
              CCTTTGTCTGGATCTTTAATTTTTGGCAAACAAGGAACTGGAAGAGACAACGTGGATGCGGAC
              AAAGTCATGATAACCAACGAAGAAGAAATTAAAACTACTAACCCGGTAGCAACGGAGTCCTAT
              GGACAAGTGGCCACAAACCACCAGAGTGCCCAAGCACAGGCGCAGACCGGCTGGGTTCAAAA
              CCAAGGAATACTTCCGGGTATGGTTTGGCAGGACAGAGATGTGTACCTGCAAGGACCCATTTG
              GGCCAAAATTCCTCACACGGACGGCAACTTTCACCCTTCTCCGCTGATGGGAGGGTTTGGAATG
              AAGCACCCGCCTCCTCAGATCCTCATCAAAAACACACCTGTACCTGCGGATCCTCCAACGGCCT
              TCAACAAGGACAAGCTGAACTCTTTCATCACCCAGTATTCTACTGGCCAAGTCAGCGTGGAGAT
              CGAGTGGGAGCTGCAGAAGGAAAACAGCAAGCGCTGGAACCCGGAGATCCAGTACACTTCCA
              ACTATTACAAGTCTAATAATGTTGAATTTGCTGTTAATACTGAAGGTGTATATAGTGAACCCCG
              CCCCATTGGCACCAGATACCTGACTCGTAATCTGTAATCGATTGTTAATCAATAAACCGTTTAA
              TTCGTTTCAGTTGAACTTTGGTCTCTGCGTATTTCTTTCTTATCTAGTTTCCATGGCTACGTAGAT
              AAGTAGCATGGCGGGTTAATCATTAACTACAAGGAACCCCTAGTGATGGAGTTGGCCACTCCC
              TCTCTGCGCGCTCGCTCGCTCACTGAGGCCGGGCGACCAAAGGTCGCCCGACGCCCGGGCTTTG
              CCCGGGCGGCCTCAGTGAGCGAGCGAGCGCGCAGAGAGGGAGTGGCCAA
CAG-CAP9      TTGGCCACTCCCTCTCTGCGCGCTCGCTCGCTCACTGAGGCCGGGCGACCAAAGGTCGCC
SEQ ID NO: 10 CGACGCCCGGGCTTTGCCCGGGCGGCCTCAGTGAGCGAGCGAGCGCGCAGAGAGGGAGT
              GGCCAACTCCATCACTAGGGGTTCCTGGAGGGGTGGAGTCGTGACGATATCCATGCGTCG
              ACATAACGCGTCGACATTGATTATTGACTAGTTATTAATAGTAATCAATTACGGGGTCATT
              AGTTCATAGCCCATATATGGAGTTCCGCGTTACATAACTTACGGTAAATGGCCCGCCTGG
              CTGACCGCCCAACGACCCCCGCCCATTGACGTCAATAATGACGTATGTTCCCATAGTAAC
              GCCAATAGGGACTTTCCATTGACGTCAATGGGTGGAGTATTTACGGTAAACTGCCCACTT
              GGCAGTACATCAAGTGTATCATATGCCAAGTACGCCCCCTATTGACGTCAATGACGGTAA
              ATGGCCCGCCTGGCATTATGCCCAGTACATGACCTTATGGGACTTTCCTACTTGGCAGTAC
              ATCTACGTATTAGTCATCGCTATTACCATGTCGAGGCCACGTTCTGCTTCACTCTCCCCAT
              CTCCCCCCCCTCCCCACCCCCAATTTTGTATTTATTTATTTTTTAATTATTTTGTGCAGCGA
              TGGGGGCGGGGGGGGGGGGCGCGCGCCAGGCGGGGCGGGGCGGGGCGAGGGGCGGGGC
              GGGGCGAGGCGGAGAGGTGCGGCGGCAGCCAATCAGAGCGGCGCGCTCCGAAAGTTTCC
              TTTTATGGCGAGGCGGCGGCGGCGGCGGCCCTATAAAAAGCGAAGCGCGCGGCGGGCGG
              GAGCAAGCTTCGTTTAGTGAACCGTCAGATCGCCTGGAGACGCCATCCACGCTGTTTTGA
              CCTCCATAGAAGACACCGGGACCGATCCAGCCTCCGCGGATTCGAATCCCGGCCGGGAA
              CGGTGCATTGGAACGCGGATTCCCCGTGCCAAGAGTGACGTAAGTACCGCCTATAGAGTC
              TATAGGCCCACAAAAAATGCTTTCTTCTTTTAATATACTTTTTTGTTTATCTTATTTCTAAT
              ACTTTCCCTAATCTCTTTCTTTCAGGGCAATAATGATACAATGTATCATGCCTCTTTGCAC
              CATTCTAAAGAATAACAGTGATAATTTCTGGGTTAAGGCAATAGCAATATTTCTGCATAT
              AAATATTTCTGCATATAAATTGTAACTGATGTAAGAGGTTTCATATTGCTAATAGCAGCTA
              CAATCCAGCTACCATTCTGCTTTTATTTTATGGTTGGGATAAGGCTGGATTATTCTGAGTC
              CAAGCTAGGCCCTTTTGCTAATCATGTTCATACCTCTTATCTTCCTCCCACAGCTCCTGGG
              CAACGTGCTGGTCTGTGTGCTGGCCCATCACTTTGGCAAAGAATTGGGATTCGAACCGGT
              CACCAAGCAGGAAGTCAAAGACTTTTTCCGGTGGGCAAAGGATCACGTGGTTGAGGTGG
              AGCATGAATTCTACGTCAAAAAGGGTGGAGCCAAGAAAAGACCCGCCCCCAGTGACGCA
              GATATAAGTGAGCCCAAACGGGTGCGCGAGTCAGTTGCGCAGCCATCGACGTCAGACGC
              GGAAGCTTCGATCAACTACGCGGACAGGTACCAAAACAAATGTTCTCGTCACGTGGGCAT
              GAATCTGATGCTGTTTCCCTGCAGACAATGCGAGAGACTGAATCAGAATTCAAATATCTG
              CTTCACTCACGGTGTCAAAGACTGTTTAGAGTGCTTTCCCGTGTCAGAATCTCAACCCGTT
              TCTGTCGTCAAAAAGGCGTATCAGAAACTGTGCTACATTCATCACATCATGGGAAAGGTG
              CCAGACGCTTGCACTGCTTGCGACCTGGTCAATGTGGACTTGGATGACTGTGTTTCTGAAC
              AATAAATGACTTAAACCAGGTATGGCTGCCGATGGTTATCTTCCAGATTGGCTCGAGGAC
              AACCTTAGTGAAGGAATTCGCGAGTGGTGGGCTTTGAAACCTGGAGCCCCTCAACCCAAG
              GCAAATCAACAACATCAAGACAACGCTCGAGGTCTTGTGCTTCCGGGTTACAAATACCTT
              GGACCCGGCAACGGACTCGACAAGGGGGAGCCGGTCAACGCAGCAGACGCGGCGGCCCT
              CGAGCACGACAAGGCCTACGACCAGCAGCTCAAGGCCGGAGACAACCCGTACCTCAAGT
              ACAACCACGCCGACGCCGAGTTCCAGGAGCGGCTCAAAGAAGATACGTCTTTTGGGGGC
              AACCTCGGGCGAGCAGTCTTCCAGGCCAAAAAGAGGCTTCTTGAACCTCTTGGTCTGGTT
              GAGGAAGCGGCTAAGACGGCTCCTGGAAAGAAGAGGCCTGTAGAGCAGTCTCCTCAGGA
              ACCGGACTCCTCCGCGGGTATTGGCAAATCGGGTGCACAGCCCGCTAAAAAGAGACTCA
              ATTTCGGTCAGACTGGCGACACAGAGTCAGTCCCAGACCCTCAACCAATCGGAGAACCTC
              CCGCAGCCCCCTCAGGTGTGGGATCTCTTACAATGGCTTCAGGTGGTGGCGCACCAGTGG
              CAGACAATAACGAAGGTGCCGATGGAGTGGGTAGTTCCTCGGGAAATTGGCATTGCGATT
              CCCAATGGCTGGGGGACAGAGTCATCACCACCAGCACCCGAACCTGGGCCCTGCCCACCT
              ACAACAATCACCTCTACAAGCAAATCTCCAACAGCACATCTGGAGGATCTTCAAATGACA
              ACGCCTACTTCGGCTACAGCACCCCCTGGGGGTATTTTGACTTCAACAGATTCCACTGCCA
              CTTCTCACCACGTGACTGGCAGCGACTCATCAACAACAACTGGGGATTCCGGCCTAAGCG
              ACTCAACTTCAAGCTCTTCAACATTCAGGTCAAAGAGGTTACGGACAACAATGGAGTCAA
              GACCATCGCCAATAACCTTACCAGCACGGTCCAGGTCTTCACGGACTCAGACTATCAGCT
              CCCGTACGTGCTCGGGTCGGCTCACGAGGGCTGCCTCCCGCCGTTCCCAGCGGACGTTTT
              CATGATTCCTCAGTACGGGTATCTGACGCTTAATGATGGAAGCCAGGCCGTGGGTCGTTC
              GTCCTTTTACTGCCTGGAATATTTCCCGTCGCAAATGCTAAGAACGGGTAACAACTTCCA
              GTTCAGCTACGAGTTTGAGAACGTACCTTTCCATAGCAGCTACGCTCACAGCCAAAGCCT
              GGACCGACTAATGAATCCACTCATCGACCAATACTTGTACTATCTCTCAAAGACTATTAA
              CGGTTCTGGACAGAATCAACAAACGCTAAAATTCAGTGTGGCCGGACCCAGCAACATGG
              CTGTCCAGGGAAGAAACTACATACCTGGACCCAGCTACCGACAACAACGTGTCTCAACCA
              CTGTGACTCAAAACAACAACAGCGAATTTGCTTGGCCTGGAGCTTCTTCTTGGGCTCTCA
              ATGGACGTAATAGCTTGATGAATCCTGGACCTGCTATGGCCAGCCACAAAGAAGGAGAG
              GACCGTTTCTTTCCTTTGTCTGGATCTTTAATTTTTGGCAAACAAGGAACTGGAAGAGACA
              ACGTGGATGCGGACAAAGTCATGATAACCAACGAAGAAGAAATTAAAACTACTAACCCG
              GTAGCAACGGAGTCCTATGGACAAGTGGCCACAAACCACCAGAGTGCCCAAGCACAGGC
              GCAGACCGGCTGGGTTCAAAACCAAGGAATACTTCCGGGTATGGTTTGGCAGGACAGAG
              ATGTGTACCTGCAAGGACCCATTTGGGCCAAAATTCCTCACACGGACGGCAACTTTCACC
              CTTCTCCGCTGATGGGAGGGTTTGGAATGAAGCACCCGCCTCCTCAGATCCTCATCAAAA
              ACACACCTGTACCTGCGGATCCTCCAACGGCCTTCAACAAGGACAAGCTGAACTCTTTCA
              TCACCCAGTATTCTACTGGCCAAGTCAGCGTGGAGATCGAGTGGGAGCTGCAGAAGGAA
              AACAGCAAGCGCTGGAACCCGGAGATCCAGTACACTTCCAACTATTACAAGTCTAATAAT
              GTTGAATTTGCTGTTAATACTGAAGGTGTATATAGTGAACCCCGCCCCATTGGCACCAGA
              TACCTGACTCGTAATCTGTAATCGATTGTTAATCAATAAACCGTTTAATTCGTTTCAGTTG
              AACTTTGGTCTCTGCGTATTTCTTTCTTATCTAGTTTCCATGGCTACGTAGATAAGTAGCAT
              GGCGGGTTAATCATTAACTACAAGGAACCCCTAGTGATGGAGTTGGCCACTCCCTCTCTG
              CGCGCTCGCTCGCTCACTGAGGCCGGGCGACCAAAGGTCGCCCGACGCCCGGGCTTTGCC
              CGGGCGGCCTCAGTGAGCGAGCGAGCGCGCAGAGAGGGAGTGGCCAA
SYNG-CAP9     TTGGCCACTCCCTCTCTGCGCGCTCGCTCGCTCACTGAGGCCGGGCGACCAAAGGTCGCC
SEQ ID NO: 11 CGACGCCCGGGCTTTGCCCGGGCGGCCTCAGTGAGCGAGCGAGCGCGCAGAGAGGGAGT
              GGCCAACTCCATCACTAGGGGTTCCTGGAGGGGTGGAGTCGTGACGATATCCATGCGTCG
              ACATAACGCGTGATCTAACATATCCTGGTGTGGAGTAGCGGACGCTGCTATGACAGAGGC
              TCGGGGGCCTGAGCTGGCTCTGTGAGCTGGGGAGGAGGCAGACAGCCAGGCCTTGTCTG
              CAAGCAGACCTGGCAGCATTGGGCTGGCCGCCCCCCAGGGCCTCCTCTTCATGCCCAGTG
              AATGACTCACCTTGGCACAGACACAATGTTCGGGGTGGGCACAGTGCCTGCTTCCCGCCG
              CACCCCAGCCCCCCTCAAATGCCTTCCGAGAAGCCCATTGAGCAGGGGGCTTGCATTGCA
              CCCCAGCCTGACAGCCTGGCATCTTGGGATAAAAGCAGCACAGCCCCCTAGGGGCTGCCC
              TTGCTGTGTGGCGCCACCGGCGGTGGAGAACAAGGCTCTATTCAGCCTGTGCCCAGGAAA
              GGGGATCAGGGGATGCCCAGGCATGGACAGTGGGTGGCAGGGGGGGAGAGGAGGGCTG
              TCTGCTTCCCAGAAGTCCAAGGACACAAATGGGTGAGGGGAGAGCTCTCCCCATAGCTGG
              GCTGCGGCCCAACCCCACCCCCTCAGGCTATGCCAGGGGGTGTTGCCAGGGGCACCCGGG
              CATCGCCAGTCTAGCCCACTCCTTCATAAAGCCCTCGCATCCCAGGAGCGAGCAGAGCCA
              GAGCAGGTTGGAGAGGAGACGCATCACCTCCGCTGCTCGCGGGGATCCTCTAGAAGCTTC
              GTTTAGTGAACCGTCAGATCGCCTGGAGACGCCATCCACGCTGTTTTGACCTCCATAGAA
              GACACCGGGACCGATCCAGCCTCCGCGGATTCGAATCCCGGCCGGGAACGGTGCATTGG
              AACGCGGATTCCCCGTGCCAAGAGTGACGTAAGTACCGCCTATAGAGTCTATAGGCCCAC
              AAAAAATGCTTTCTTCTTTTAATATACTTTTTTGTTTATCTTATTTCTAATACTTTCCCTAAT
              CTCTTTCTTTCAGGGCAATAATGATACAATGTATCATGCCTCTTTGCACCATTCTAAAGAA
              TAACAGTGATAATTTCTGGGTTAAGGCAATAGCAATATTTCTGCATATAAATATTTCTGCA
              TATAAATTGTAACTGATGTAAGAGGTTTCATATTGCTAATAGCAGCTACAATCCAGCTAC
              CATTCTGCTTTTATTTTATGGTTGGGATAAGGCTGGATTATTCTGAGTCCAAGCTAGGCCC
              TTTTGCTAATCATGTTCATACCTCTTATCTTCCTCCCACAGCTCCTGGGCAACGTGCTGGTC
              TGTGTGCTGGCCCATCACTTTGGCAAAGAATTGGGATTCGAACCGGTCGCCACCGGTCAC
              CAAGCAGGAAGTCAAAGACTTTTTCCGGTGGGCAAAGGATCACGTGGTTGAGGTGGAGC
              ATGAATTCTACGTCAAAAAGGGTGGAGCCAAGAAAAGACCCGCCCCCAGTGACGCAGAT
              ATAAGTGAGCCCAAACGGGTGCGCGAGTCAGTTGCGCAGCCATCGACGTCAGACGCGGA
              AGCTTCGATCAACTACGCGGACAGGTACCAAAACAAATGTTCTCGTCACGTGGGCATGAA
              TCTGATGCTGTTTCCCTGCAGACAATGCGAGAGACTGAATCAGAATTCAAATATCTGCTT
              CACTCACGGTGTCAAAGACTGTTTAGAGTGCTTTCCCGTGTCAGAATCTCAACCCGTTTCT
              GTCGTCAAAAAGGCGTATCAGAAACTGTGCTACATTCATCACATCATGGGAAAGGTGCCA
              GACGCTTGCACTGCTTGCGACCTGGTCAATGTGGACTTGGATGACTGTGTTTCTGAACAAT
              AAATGACTTAAACCAGGTATGGCTGCCGATGGTTATCTTCCAGATTGGCTCGAGGACAAC
              CTTAGTGAAGGAATTCGCGAGTGGTGGGCTTTGAAACCTGGAGCCCCTCAACCCAAGGCA
              AATCAACAACATCAAGACAACGCTCGAGGTCTTGTGCTTCCGGGTTACAAATACCTTGGA
              CCCGGCAACGGACTCGACAAGGGGGAGCCGGTCAACGCAGCAGACGCGGCGGCCCTCGA
              GCACGACAAGGCCTACGACCAGCAGCTCAAGGCCGGAGACAACCCGTACCTCAAGTACA
              ACCACGCCGACGCCGAGTTCCAGGAGCGGCTCAAAGAAGATACGTCTTTTGGGGGCAAC
              CTCGGGCGAGCAGTCTTCCAGGCCAAAAAGAGGCTTCTTGAACCTCTTGGTCTGGTTGAG
              GAAGCGGCTAAGACGGCTCCTGGAAAGAAGAGGCCTGTAGAGCAGTCTCCTCAGGAACC
              GGACTCCTCCGCGGGTATTGGCAAATCGGGTGCACAGCCCGCTAAAAAGAGACTCAATTT
              CGGTCAGACTGGCGACACAGAGTCAGTCCCAGACCCTCAACCAATCGGAGAACCTCCCG
              CAGCCCCCTCAGGTGTGGGATCTCTTACAATGGCTTCAGGTGGTGGCGCACCAGTGGCAG
              ACAATAACGAAGGTGCCGATGGAGTGGGTAGTTCCTCGGGAAATTGGCATTGCGATTCCC
              AATGGCTGGGGGACAGAGTCATCACCACCAGCACCCGAACCTGGGCCCTGCCCACCTAC
              AACAATCACCTCTACAAGCAAATCTCCAACAGCACATCTGGAGGATCTTCAAATGACAAC
              GCCTACTTCGGCTACAGCACCCCCTGGGGGTATTTTGACTTCAACAGATTCCACTGCCACT
              TCTCACCACGTGACTGGCAGCGACTCATCAACAACAACTGGGGATTCCGGCCTAAGCGAC
              TCAACTTCAAGCTCTTCAACATTCAGGTCAAAGAGGTTACGGACAACAATGGAGTCAAGA
              CCATCGCCAATAACCTTACCAGCACGGTCCAGGTCTTCACGGACTCAGACTATCAGCTCC
              CGTACGTGCTCGGGTCGGCTCACGAGGGCTGCCTCCCGCCGTTCCCAGCGGACGTTTTCA
              TGATTCCTCAGTACGGGTATCTGACGCTTAATGATGGAAGCCAGGCCGTGGGTCGTTCGT
              CCTTTTACTGCCTGGAATATTTCCCGTCGCAAATGCTAAGAACGGGTAACAACTTCCAGTT
              CAGCTACGAGTTTGAGAACGTACCTTTCCATAGCAGCTACGCTCACAGCCAAAGCCTGGA
              CCGACTAATGAATCCACTCATCGACCAATACTTGTACTATCTCTCAAAGACTATTAACGGT
              TCTGGACAGAATCAACAAACGCTAAAATTCAGTGTGGCCGGACCCAGCAACATGGCTGTC
              CAGGGAAGAAACTACATACCTGGACCCAGCTACCGACAACAACGTGTCTCAACCACTGT
              GACTCAAAACAACAACAGCGAATTTGCTTGGCCTGGAGCTTCTTCTTGGGCTCTCAATGG
              ACGTAATAGCTTGATGAATCCTGGACCTGCTATGGCCAGCCACAAAGAAGGAGAGGACC
              GTTTCTTTCCTTTGTCTGGATCTTTAATTTTTGGCAAACAAGGAACTGGAAGAGACAACGT
              GGATGCGGACAAAGTCATGATAACCAACGAAGAAGAAATTAAAACTACTAACCCGGTAG
              CAACGGAGTCCTATGGACAAGTGGCCACAAACCACCAGAGTGCCCAAGCACAGGCGCAG
              ACCGGCTGGGTTCAAAACCAAGGAATACTTCCGGGTATGGTTTGGCAGGACAGAGATGT
              GTACCTGCAAGGACCCATTTGGGCCAAAATTCCTCACACGGACGGCAACTTTCACCCTTC
              TCCGCTGATGGGAGGGTTTGGAATGAAGCACCCGCCTCCTCAGATCCTCATCAAAAACAC
              ACCTGTACCTGCGGATCCTCCAACGGCCTTCAACAAGGACAAGCTGAACTCTTTCATCAC
              CCAGTATTCTACTGGCCAAGTCAGCGTGGAGATCGAGTGGGAGCTGCAGAAGGAAAACA
              GCAAGCGCTGGAACCCGGAGATCCAGTACACTTCCAACTATTACAAGTCTAATAATGTTG
              AATTTGCTGTTAATACTGAAGGTGTATATAGTGAACCCCGCCCCATTGGCACCAGATACC
              TGACTCGTAATCTGTAATCGATTGTTAATCAATAAACCGTTTAATTCGTTTCAGTTGAACT
              TTGGTCTCTGCGTATTTCTTTCTTATCTAGTTTCCATGGCTACGTAGATAAGTAGCATGGC
              GGGTTAATCATTAACTACAAGGAACCCCTAGTGATGGAGTTGGCCACTCCCTCTCTGCGC
              GCTCGCTCGCTCACTGAGGCCGGGCGACCAAAGGTCGCCCGACGCCCGGGCTTTGCCCGG
              GCGGCCTCAGTGAGCGAGCGAGCGCGCAGAGAGGGAGTGGCCAA
GFAPG-CAP9    TTGGCCACTCCCTCTCTGCGCGCTCGCTCGCTCACTGAGGCCGGGCGACCAAAGGTCGCC
SEQ ID NO: 12 CGACGCCCGGGCTTTGCCCGGGCGGCCTCAGTGAGCGAGCGAGCGCGCAGAGAGGGAGT
              GGCCAACTCCATCACTAGGGGTTCCTGGAGGGGTGGAGTCGTGACGATATCCATGCGTCG
              ACATAACGCGTTAGTATCTGCAGAGGGCCCTGCGTATGAGTGCAAGTGGGTTTTAGGACC
              AGGATGAGGCGGGGTGGGGGTGCCTACCTGACGACCGACCCCGACCCACTGGACAAGCA
              CCCAACCCCCATTCCCCAAATTGCGCATCCCCTATCAGAGAGGGGGAGGGGAAACAGGA
              TGCGGCGAGGCGCGTGCGCACTGCCAGCTTCAGCACCGCGGACAGTGCCTTCGCCCCCGC
              CTGGCGGCGCGCGCCACCGCCGCCTCAGCACTGAAGGCGCGCTGACGTCACTCGCCGGTC
              CCCCGCAAACTCCCCTTCCCGGCCACCTTGGTCGCGTCCGCGCCGCCGCCGGCCCAGCCG
              GACCGCACCACGCGAGGCGCGAGATAGGGGGGCACGGGCGCGACCATCTGCGCTGCGGC
              GCCGGCGACTCAGCGCTGCCTCAGTCTGCGGTGGGCAGCGGAGGAGTCGTGTCGTGCCTG
              AGAGCGCAGCTGTGCTCCTGGGCACCGCGCAGTCCGCCCCCGCGGCTCCTGGCCAGACCA
              CCCCTAGGACCCCCTGCCCCAAGTCGCAGCCAAGCTTCGTTTAGTGAACCGTCAGATCGC
              CTGGAGACGCCATCCACGCTGTTTTGACCTCCATAGAAGACACCGGGACCGATCCAGCCT
              CCGCGGATTCGAATCCCGGCCGGGAACGGTGCATTGGAACGCGGATTCCCCGTGCCAAG
              AGTGACGTAAGTACCGCCTATAGAGTCTATAGGCCCACAAAAAATGCTTTCTTCTTTTAAT
              ATACTTTTTTGTTTATCTTATTTCTAATACTTTCCCTAATCTCTTTCTTTCAGGGCAATAATG
              ATACAATGTATCATGCCTCTTTGCACCATTCTAAAGAATAACAGTGATAATTTCTGGGTTA
              AGGCAATAGCAATATTTCTGCATATAAATATTTCTGCATATAAATTGTAACTGATGTAAG
              AGGTTTCATATTGCTAATAGCAGCTACAATCCAGCTACCATTCTGCTTTTATTTTATGGTT
              GGGATAAGGCTGGATTATTCTGAGTCCAAGCTAGGCCCTTTTGCTAATCATGTTCATACCT
              CTTATCTTCCTCCCACAGCTCCTGGGCAACGTGCTGGTCTGTGTGCTGGCCCATCACTTTG
              GCAAAGAATTGGGATTCGAACCGGTCGCCACCGGTCACCAAGCAGGAAGTCAAAGACTT
              TTTCCGGTGGGCAAAGGATCACGTGGTTGAGGTGGAGCATGAATTCTACGTCAAAAAGG
              GTGGAGCCAAGAAAAGACCCGCCCCCAGTGACGCAGATATAAGTGAGCCCAAACGGGTG
              CGCGAGTCAGTTGCGCAGCCATCGACGTCAGACGCGGAAGCTTCGATCAACTACGCGGA
              CAGGTACCAAAACAAATGTTCTCGTCACGTGGGCATGAATCTGATGCTGTTTCCCTGCAG
              ACAATGCGAGAGACTGAATCAGAATTCAAATATCTGCTTCACTCACGGTGTCAAAGACTG
              TTTAGAGTGCTTTCCCGTGTCAGAATCTCAACCCGTTTCTGTCGTCAAAAAGGCGTATCAG
              AAACTGTGCTACATTCATCACATCATGGGAAAGGTGCCAGACGCTTGCACTGCTTGCGAC
              CTGGTCAATGTGGACTTGGATGACTGTGTTTCTGAACAATAAATGACTTAAACCAGGTAT
              GGCTGCCGATGGTTATCTTCCAGATTGGCTCGAGGACAACCTTAGTGAAGGAATTCGCGA
              GTGGTGGGCTTTGAAACCTGGAGCCCCTCAACCCAAGGCAAATCAACAACATCAAGACA
              ACGCTCGAGGTCTTGTGCTTCCGGGTTACAAATACCTTGGACCCGGCAACGGACTCGACA
              AGGGGGAGCCGGTCAACGCAGCAGACGCGGCGGCCCTCGAGCACGACAAGGCCTACGAC
              CAGCAGCTCAAGGCCGGAGACAACCCGTACCTCAAGTACAACCACGCCGACGCCGAGTT
              CCAGGAGCGGCTCAAAGAAGATACGTCTTTTGGGGGCAACCTCGGGCGAGCAGTCTTCCA
              GGCCAAAAAGAGGCTTCTTGAACCTCTTGGTCTGGTTGAGGAAGCGGCTAAGACGGCTCC
              TGGAAAGAAGAGGCCTGTAGAGCAGTCTCCTCAGGAACCGGACTCCTCCGCGGGTATTG
              GCAAATCGGGTGCACAGCCCGCTAAAAAGAGACTCAATTTCGGTCAGACTGGCGACACA
              GAGTCAGTCCCAGACCCTCAACCAATCGGAGAACCTCCCGCAGCCCCCTCAGGTGTGGGA
              TCTCTTACAATGGCTTCAGGTGGTGGCGCACCAGTGGCAGACAATAACGAAGGTGCCGAT
              GGAGTGGGTAGTTCCTCGGGAAATTGGCATTGCGATTCCCAATGGCTGGGGGACAGAGTC
              ATCACCACCAGCACCCGAACCTGGGCCCTGCCCACCTACAACAATCACCTCTACAAGCAA
              ATCTCCAACAGCACATCTGGAGGATCTTCAAATGACAACGCCTACTTCGGCTACAGCACC
              CCCTGGGGGTATTTTGACTTCAACAGATTCCACTGCCACTTCTCACCACGTGACTGGCAGC
              GACTCATCAACAACAACTGGGGATTCCGGCCTAAGCGACTCAACTTCAAGCTCTTCAACA
              TTCAGGTCAAAGAGGTTACGGACAACAATGGAGTCAAGACCATCGCCAATAACCTTACC
              AGCACGGTCCAGGTCTTCACGGACTCAGACTATCAGCTCCCGTACGTGCTCGGGTCGGCT
              CACGAGGGCTGCCTCCCGCCGTTCCCAGCGGACGTTTTCATGATTCCTCAGTACGGGTATC
              TGACGCTTAATGATGGAAGCCAGGCCGTGGGTCGTTCGTCCTTTTACTGCCTGGAATATTT
              CCCGTCGCAAATGCTAAGAACGGGTAACAACTTCCAGTTCAGCTACGAGTTTGAGAACGT
              ACCTTTCCATAGCAGCTACGCTCACAGCCAAAGCCTGGACCGACTAATGAATCCACTCAT
              CGACCAATACTTGTACTATCTCTCAAAGACTATTAACGGTTCTGGACAGAATCAACAAAC
              GCTAAAATTCAGTGTGGCCGGACCCAGCAACATGGCTGTCCAGGGAAGAAACTACATAC
              CTGGACCCAGCTACCGACAACAACGTGTCTCAACCACTGTGACTCAAAACAACAACAGC
              GAATTTGCTTGGCCTGGAGCTTCTTCTTGGGCTCTCAATGGACGTAATAGCTTGATGAATC
              CTGGACCTGCTATGGCCAGCCACAAAGAAGGAGAGGACCGTTTCTTTCCTTTGTCTGGAT
              CTTTAATTTTTGGCAAACAAGGAACTGGAAGAGACAACGTGGATGCGGACAAAGTCATG
              ATAACCAACGAAGAAGAAATTAAAACTACTAACCCGGTAGCAACGGAGTCCTATGGACA
              AGTGGCCACAAACCACCAGAGTGCCCAAGCACAGGCGCAGACCGGCTGGGTTCAAAACC
              AAGGAATACTTCCGGGTATGGTTTGGCAGGACAGAGATGTGTACCTGCAAGGACCCATTT
              GGGCCAAAATTCCTCACACGGACGGCAACTTTCACCCTTCTCCGCTGATGGGAGGGTTTG
              GAATGAAGCACCCGCCTCCTCAGATCCTCATCAAAAACACACCTGTACCTGCGGATCCTC
              CAACGGCCTTCAACAAGGACAAGCTGAACTCTTTCATCACCCAGTATTCTACTGGCCAAG
              TCAGCGTGGAGATCGAGTGGGAGCTGCAGAAGGAAAACAGCAAGCGCTGGAACCCGGA
              GATCCAGTACACTTCCAACTATTACAAGTCTAATAATGTTGAATTTGCTGTTAATACTGAA
              GGTGTATATAGTGAACCCCGCCCCATTGGCACCAGATACCTGACTCGTAATCTGTAATCG
              ATTGTTAATCAATAAACCGTTTAATTCGTTTCAGTTGAACTTTGGTCTCTGCGTATTTCTTT
              CTTATCTAGTTTCCATGGCTACGTAGATAAGTAGCATGGCGGGTTAATCATTAACTACAA
              GGAACCCCTAGTGATGGAGTTGGCCACTCCCTCTCTGCGCGCTCGCTCGCTCACTGAGGC
              CGGGCGACCAAAGGTCGCCCGACGCCCGGGCTTTGCCCGGGCGGCCTCAGTGAGCGAGC
              GAGCGCGCAGAGAGGGAGTGGCCAA
GLOSPLICEF6   GTGCCAAGAGTGACCTCCTG
SEQ ID NO: 13
CAP5L8        ACTGCCCCCGCGACCGGCACGTACAACCTCCAGGAAATCGTGCCCGGCAGCGTGTGGATG
GBLOCKSEQ     GAGAGGGACGTGTACCTCCAAGGACCCATCTGGGCCAAGATCCCAGAGACGGGGGCGCA
ID NO: 14     CTTTCACCCCTCTCCGGCTATGGGCGGATTCGGACTCAAACACCCACCGCCCATGATGCTC
              ATCAAGAACACGCCTGTGCCCGGAAATATCACCAGCTTCTCGGACGTGCCCGTCAGCAGC
              TTCATCACCCAGTACAGCACCGGGCAGGTCACCGTGGAGATGGAGTGGGAGCTCAAGAA
              GGAAAACTCCAAGAGGTGGAACCCAGAGATCCAGTACACAAACAACTACAACGACCCCC
              AGTTTGTGGACTTTGCCCCGGACAGCACCGGGGAATACAGAACCACCAGACCTATCGGA
              ACCCGATACCTTACCCGACCCCTTTAA
CAP6L8        ACCGGAGATGTGCATGTTATGGGAGCCTTACCTGGAATGGTGTGGCAAGACAGGGACGT
GBLOCKSEQ     CTACCTGCAGGGTCCTATTTGGGCCAAAATTCCTCACACGGATGGACACTTTCACCCATCT
ID NO: 15     CCTCTCATGGGCGGCTTTGGACTTAAGCACCCGCCTCCTCAGATCCTCATCAAAAACACG
              CCTGTTCCTGCGAATCCTCCGGCAGAGTTTTCGGCTACAAAGTTTGCTTCATTCATCACCC
              AGTATTCCACAGGACAAGTGAGCGTGGAGATTGAATGGGAGCTGCAGAAAGAAAACAGC
              AAACGCTGGAATCCCGAAGTGCAATATACATCTAACTATGCAAAATCTGCCAACGTTGAT
              TTCACTGTGGACAACAATGGACTTTATACTGAGCCTCGCCCCATTGGCACCCGTTACCTCA
              CCCGTCCCCTGTAATCGAT
CAPDJ8L8      ACACAAGCAGCTACCGCAGATGTCAACACACAAGGCGTTCTTCCAGGCATGGTCTGGCAG
GBLOCKSEQ     GACAGAGATGTGTACCTTCAGGGGCCCATCTGGGCAAAGATTCCACACACGGACGGACA
ID NO: 16     TTTTCACCCCTCTCCCCTCATGGGTGGATTCGGACTTAAACACCCTCCGCCTCAGATCCTG
              ATCAAGAACACGCCTGTACCTGCGGACCCTCCGACCACCTTCAACCAGTCAAAGCTGAAC
              TCTTTCATCACCCAGTATTCTACTGGCCAAGTCAGCGTGGAGATCGAGTGGGAGCTGCAG
              AAGGAAAACAGCAAGCGCTGGAACCCCGAGATCCAGTACACCTCCAACTACTACAAATC
              TACAAGTGTGGACTTTGCTGTTAATACAGAAGGCGTGTACTCTGAACCCCGCCCCATTGG
              CACCCGTTACCTCACCCGTAATCTGTAA
CAP9L8M       GCACAGGCGCAGACCGGCTGGGTTCAAAACCAAGGAATACTTCCGGGTATGGTTTGGCA
GBLOCKSEQ     GGACAGAGATGTGTACCTGCAAGGACCCATTTGGGCCAAAATTCCTCACACGGACGGCA
ID NO: 17     ACTTTCACCCTTCTCCGCTGATGGGAGGGTTTGGAATGAAGCACCCGCCTCCTCAGATCCT
              CATCAAAAACACACCTGTACCTGCCGATCCTCCAACGGCCTTCAACAAGGACAAGCTGAA
              CTCTTTCATCACCCAGTATTCTACTGGCCAAGTCAGCGTGGAGATCGAGTGGGAGCTGCA
              GAAGGAAAACAGCAAGCGGTGGAACCCGGAGATCCAGTACACTTCCAACTATTACAAGT
              CTAATAATGTTGAATTTGCTGTTAATACTGAAGGTGTATATAGTGAACCCCGCCCCATTGG
              CACCAGATACCTGACTCGTAATCTGTAA
TELN-SYNG9-   TATCAGCACACAATAGTCCATTATACGCGCGTATAATGGGCAATTGTGTGCTGATACAGC
BSRGI SEQ ID  TGGCACGACAGGTTTCCCGACTGGAAAGCGGGCAGTGAGCGCAACGCAATTAATGTGAG
NO: 18        TTAGCTCACTCATTAGGCACCCCAGGCTTTACACTTTATGCTTCCGGCTCGTATGTTGTGT
              GGAATTGTGAGCGGATAACAATTTCACACAGGAAACAGCTATGACCATGATTACGCCAG
              ATTTAATTAAGGCCTTAATTAGGCTAGCTTGGCCACTCCCTCTCTGCGCGCTCGCTCGCTC
              ACTGAGGCCGGGCGACCAAAGGTCGCCCGACGCCCGGGCTTTGCCCGGGCGGCCTCAGT
              GAGCGAGCGAGCGCGCAGAGAGGGAGTGGCCAACTCCATCACTAGGGGTTCCTGGAGGG
              GTGGAGTCGTGACGATATCCATGCGTCGACATAACGCGTTAGTATCTGCAGAGGGCCCTG
              CGTATGAGTGCAAGTGGGTTTTAGGACCAGGATGAGGCGGGGTGGGGGTGCCTACCTGA
              CGACCGACCCCGACCCACTGGACAAGCACCCAACCCCCATTCCCCAAATTGCGCATCCCC
              TATCAGAGAGGGGGAGGGGAAACAGGATGCGGCGAGGCGCGTGCGCACTGCCAGCTTCA
              GCACCGCGGACAGTGCCTTCGCCCCCGCCTGGCGGCGCGCGCCACCGCCGCCTCAGCACT
              GAAGGCGCGCTGACGTCACTCGCCGGTCCCCCGCAAACTCCCCTTCCCGGCCACCTTGGT
              CGCGTCCGCGCCGCCGCCGGCCCAGCCGGACCGCACCACGCGAGGCGCGAGATAGGGGG
              GCACGGGCGCGACCATCTGCGCTGCGGCGCCGGCGACTCAGCGCTGCCTCAGTCTGCGGT
              GGGCAGCGGAGGAGTCGTGTCGTGCCTGAGAGCGCAGCTGTGCTCCTGGGCACCGCGCA
              GTCCGCCCCCGCGGCTCCTGGCCAGACCACCCCTAGGACCCCCTGCCCCAAGTCGCAGCC
              AAGCTTCGTTTAGTGAACCGTCAGATCGCCTGGAGACGCCATCCACGCTGTTTTGACCTCC
              ATAGAAGACACCGGGACCGATCCAGCCTCCGCGGATTCGAATCCCGGCCGGGAACGGTG
              CATTGGAACGCGGATTCCCCGTGCCAAGAGTGACGTAAGTACCGCCTATAGAGTCTATAG
              GCCCACAAAAAATGCTTTCTTCTTTTAATATACTTTTTTGTTTATCTTATTTCTAATACTTTC
              CCTAATCTCTTTCTTTCAGGGCAATAATGATACAATGTATCATGCCTCTTTGCACCATTCT
              AAAGAATAACAGTGATAATTTCTGGGTTAAGGCAATAGCAATATTTCTGCATATAAATAT
              TTCTGCATATAAATTGTAACTGATGTAAGAGGTTTCATATTGCTAATAGCAGCTACAATCC
              AGCTACCATTCTGCTTTTATTTTATGGTTGGGATAAGGCTGGATTATTCTGAGTCCAAGCT
              AGGCCCTTTTGCTAATCATGTTCATACCTCTTATCTTCCTCCCACAGCTCCTGGGCAACGT
              GCTGGTCTGTGTGCTGGCCCATCACTTTGGCAAAGAATTGGGATTCGAACCGGTCGCCAC
              CGGTCACCAAGCAGGAAGTCAAAGACTTTTTCCGGTGGGCAAAGGATCACGTGGTTGAG
              GTGGAGCATGAATTCTACGTCAAAAAGGGTGGAGCCAAGAAAAGACCCGCCCCCAGTGA
              CGCAGATATAAGTGAGCCCAAACGGGTGCGCGAGTCAGTTGCGCAGCCATCGACGTCAG
              ACGCGGAAGCTTCGATCAACTACGCGGACAGGTACCAAAACAAATGTTCTCGTCACGTGG
              GCATGAATCTGATGCTGTTTCCCTGCAGACAATGCGAGAGACTGAATCAGAATTCAAATA
              TCTGCTTCACTCACGGTGTCAAAGACTGTTTAGAGTGCTTTCCCGTGTCAGAATCTCAACC
              CGTTTCTGTCGTCAAAAAGGCGTATCAGAAACTGTGCTACATTCATCACATCATGGGAAA
              GGTGCCAGACGCTTGCACTGCTTGCGACCTGGTCAATGTGGACTTGGATGACTGTGTTTCT
              GAACAATAAATGACTTAAACCAGGTATGGCTGCCGATGGTTATCTTCCAGATTGGCTCGA
              GGACAACCTTAGTGAAGGAATTCGCGAGTGGTGGGCTTTGAAACCTGGAGCCCCTCAACC
              CAAGGCAAATCAACAACATCAAGACAACGCTCGAGGTCTTGTGCTTCCGGGTTACAAATA
              CCTTGGACCCGGCAACGGACTCGACAAGGGGGAGCCGGTCAACGCAGCAGACGCGGCGG
              CCCTCGAGCACGACAAGGCCTACGACCAGCAGCTCAAGGCCGGAGACAACCCGTACCTC
              AAGTACAACCACGCCGACGCCGAGTTCCAGGAGCGGCTCAAAGAAGATACGTCTTTTGG
              GGGCAACCTCGGGCGAGCAGTCTTCCAGGCCAAAAAGAGGCTTCTTGAACCTCTTGGTCT
              GGTTGAGGAAGCGGCTAAGACGGCTCCTGGAAAGAAGAGGCCTGTAGAGCAGTCTCCTC
              AGGAACCGGACTCCTCCGCGGGTATTGGCAAATCGGGTGCACAGCCCGCTAAAAAGAGA
              CTCAATTTCGGTCAGACTGGCGACACAGAGTCAGTCCCAGACCCTCAACCAATCGGAGAA
              CCTCCCGCAGCCCCCTCAGGTGTGGGATCTCTTACAATGGCTTCAGGTGGTGGCGCACCA
              GTGGCAGACAATAACGAAGGTGCCGATGGAGTGGGTAGTTCCTCGGGAAATTGGCATTG
              CGATTCCCAATGGCTGGGGGACAGAGTCATCACCACCAGCACCCGAACCTGGGCCCTGCC
              CACCTACAACAATCACCTCTACAAGCAAATCTCCAACAGCACATCTGGAGGATCTTCAAA
              TGACAACGCCTACTTCGGCTACAGCACCCCCTGGGGGTATTTTGACTTCAACAGATTCCA
              CTGCCACTTCTCACCACGTGACTGGCAGCGACTCATCAACAACAACTGGGGATTCCGGCC
              TAAGCGACTCAACTTCAAGCTCTTCAACATTCAGGTCAAAGAGGTTACGGACAACAATGG
              AGTCAAGACCATCGCCAATAACCTTACCAGCACGGTCCAGGTCTTCACGGACTCAGACTA
              TCAGCTCCCGTACGTGCTCGGGTCGGCTCACGAGGGCTGCCTCCCGCCGTTCCCAGCGGA
              CGTTTTCATGATTCCTCAGTACGGGTATCTGACGCTTAATGATGGAAGCCAGGCCGTGGG
              TCGTTCGTCCTTTTACTGCCTGGAATATTTCCCGTCGCAAATGCTAAGAACGGGTAACAAC
              TTCCAGTTCAGCTACGAGTTTGAGAACGTACCTTTCCATAGCAGCTACGCTCACAGCCAA
              AGCCTGGACCGACTAATGAATCCACTCATCGACCAATACTTGTACTATCTCTCAAAGACT
              ATTAACGGTTCTGGACAGAATCAACAAACGCTAAAATTCAGTGTGGCCGGACCCAGCAA
              CATGGCTGTCCAGGGAAGAAACTACATACCTGGACCCAGCTACCGACAACAACGTGTCTC
              AACCACTGTGACTCAAAACAACAACAGCGAATTTGCTTGGCCTGGAGCTTCTTCTTGGGC
              TCTCAATGGACGTAATAGCTTGATGAATCCTGGACCTGCTATGGCCAGCCACAAAGAAGG
              AGAGGACCGTTTCTTTCCTTTGTCTGGATCTTTAATTTTTGGCAAACAAGGAACTGGAAGA
              GACAACGTGGATGCGGACAAAGTCATGATAACCAACGAAGAAGAAATTAAAACTACTAA
              CCCGGTAGCAACGGAGTCCTATGGACAAGTGGCCACAAACCACCAGAGTGTACATCGAT
              TGTTAATCAATAAACCGTTTAATTCGTTTCAGTTGAACTTTGGTCTCTGCGTATTTCTTTCT
              TATCTAGTTTCCATGGCTACGTAGATAAGTAGCATGGCGGGTTAATCATTAACTACAAGG
              AACCCCTAGTGATGGAGTTGGCCACTCCCTCTCTGCGCGCTCGCTCGCTCACTGAGGCCG
              GGCGACCAAAGGTCGCCCGACGCCCGGGCTTTGCCCGGGCGGCCTCAGTGAGCGAGCGA
              GCGCGCAGAGAGGGAGTGGCCAAGCATGCAATTAACTGGCCGTCGTTTTACAACGTCGTG
              ACTGGGAAAACCCTGGCGTTACCCAACTTAATCGCCTTGCAGCACATCCCCCTTTCGCCA
              GCTGTATCAGCACACAATTGCCCATTATACGCGCGTATAATGGACTATTGTGTGCTGATA
TELN-         TATCAGCACACAATAGTCCATTATACGCGCGTATAATGGGCAATTGTGTGCTGATACAGC
GFAPG9-       TGGCACGACAGGTTTCCCGACTGGAAAGCGGGCAGTGAGCGCAACGCAATTAATGTGAG
BSRGI SEQ ID  TTAGCTCACTCATTAGGCACCCCAGGCTTTACACTTTATGCTTCCGGCTCGTATGTTGTGT
NO: 19        GGAATTGTGAGCGGATAACAATTTCACACAGGAAACAGCTATGACCATGATTACGCCAG
              ATTTAATTAAGGCCTTAATTAGGCTAGCTTGGCCACTCCCTCTCTGCGCGCTCGCTCGCTC
              ACTGAGGCCGGGCGACCAAAGGTCGCCCGACGCCCGGGCTTTGCCCGGGCGGCCTCAGT
              GAGCGAGCGAGCGCGCAGAGAGGGAGTGGCCAACTCCATCACTAGGGGTTCCTGGAGGG
              GTGGAGTCGTGACGATATCCATGCGTCGACATAACGCGTGATCTAACATATCCTGGTGTG
              GAGTAGCGGACGCTGCTATGACAGAGGCTCGGGGGCCTGAGCTGGCTCTGTGAGCTGGG
              GAGGAGGCAGACAGCCAGGCCTTGTCTGCAAGCAGACCTGGCAGCATTGGGCTGGCCGC
              CCCCCAGGGCCTCCTCTTCATGCCCAGTGAATGACTCACCTTGGCACAGACACAATGTTC
              GGGGTGGGCACAGTGCCTGCTTCCCGCCGCACCCCAGCCCCCCTCAAATGCCTTCCGAGA
              AGCCCATTGAGCAGGGGGCTTGCATTGCACCCCAGCCTGACAGCCTGGCATCTTGGGATA
              AAAGCAGCACAGCCCCCTAGGGGCTGCCCTTGCTGTGTGGCGCCACCGGCGGTGGAGAA
              CAAGGCTCTATTCAGCCTGTGCCCAGGAAAGGGGATCAGGGGATGCCCAGGCATGGACA
              GTGGGTGGCAGGGGGGGAGAGGAGGGCTGTCTGCTTCCCAGAAGTCCAAGGACACAAAT
              GGGTGAGGGGAGAGCTCTCCCCATAGCTGGGCTGCGGCCCAACCCCACCCCCTCAGGCTA
              TGCCAGGGGGTGTTGCCAGGGGCACCCGGGCATCGCCAGTCTAGCCCACTCCTTCATAAA
              GCCCTCGCATCCCAGGAGCGAGCAGAGCCAGAGCAGGTTGGAGAGGAGACGCATCACCT
              CCGCTGCTCGCGGGGATCCTCTAGAAGCTTCGTTTAGTGAACCGTCAGATCGCCTGGAGA
              CGCCATCCACGCTGTTTTGACCTCCATAGAAGACACCGGGACCGATCCAGCCTCCGCGGA
              TTCGAATCCCGGCCGGGAACGGTGCATTGGAACGCGGATTCCCCGTGCCAAGAGTGACGT
              AAGTACCGCCTATAGAGTCTATAGGCCCACAAAAAATGCTTTCTTCTTTTAATATACTTTT
              TTGTTTATCTTATTTCTAATACTTTCCCTAATCTCTTTCTTTCAGGGCAATAATGATACAAT
              GTATCATGCCTCTTTGCACCATTCTAAAGAATAACAGTGATAATTTCTGGGTTAAGGCAAT
              AGCAATATTTCTGCATATAAATATTTCTGCATATAAATTGTAACTGATGTAAGAGGTTTCA
              TATTGCTAATAGCAGCTACAATCCAGCTACCATTCTGCTTTTATTTTATGGTTGGGATAAG
              GCTGGATTATTCTGAGTCCAAGCTAGGCCCTTTTGCTAATCATGTTCATACCTCTTATCTTC
              CTCCCACAGCTCCTGGGCAACGTGCTGGTCTGTGTGCTGGCCCATCACTTTGGCAAAGAA
              TTGGGATTCGAACCGGTCGCCACCGGTCACCAAGCAGGAAGTCAAAGACTTTTTCCGGTG
              GGCAAAGGATCACGTGGTTGAGGTGGAGCATGAATTCTACGTCAAAAAGGGTGGAGCCA
              AGAAAAGACCCGCCCCCAGTGACGCAGATATAAGTGAGCCCAAACGGGTGCGCGAGTCA
              GTTGCGCAGCCATCGACGTCAGACGCGGAAGCTTCGATCAACTACGCGGACAGGTACCA
              AAACAAATGTTCTCGTCACGTGGGCATGAATCTGATGCTGTTTCCCTGCAGACAATGCGA
              GAGACTGAATCAGAATTCAAATATCTGCTTCACTCACGGTGTCAAAGACTGTTTAGAGTG
              CTTTCCCGTGTCAGAATCTCAACCCGTTTCTGTCGTCAAAAAGGCGTATCAGAAACTGTGC
              TACATTCATCACATCATGGGAAAGGTGCCAGACGCTTGCACTGCTTGCGACCTGGTCAAT
              GTGGACTTGGATGACTGTGTTTCTGAACAATAAATGACTTAAACCAGGTATGGCTGCCGA
              TGGTTATCTTCCAGATTGGCTCGAGGACAACCTTAGTGAAGGAATTCGCGAGTGGTGGGC
              TTTGAAACCTGGAGCCCCTCAACCCAAGGCAAATCAACAACATCAAGACAACGCTCGAG
              GTCTTGTGCTTCCGGGTTACAAATACCTTGGACCCGGCAACGGACTCGACAAGGGGGAGC
              CGGTCAACGCAGCAGACGCGGCGGCCCTCGAGCACGACAAGGCCTACGACCAGCAGCTC
              AAGGCCGGAGACAACCCGTACCTCAAGTACAACCACGCCGACGCCGAGTTCCAGGAGCG
              GCTCAAAGAAGATACGTCTTTTGGGGGCAACCTCGGGCGAGCAGTCTTCCAGGCCAAAA
              AGAGGCTTCTTGAACCTCTTGGTCTGGTTGAGGAAGCGGCTAAGACGGCTCCTGGAAAGA
              AGAGGCCTGTAGAGCAGTCTCCTCAGGAACCGGACTCCTCCGCGGGTATTGGCAAATCGG
              GTGCACAGCCCGCTAAAAAGAGACTCAATTTCGGTCAGACTGGCGACACAGAGTCAGTC
              CCAGACCCTCAACCAATCGGAGAACCTCCCGCAGCCCCCTCAGGTGTGGGATCTCTTACA
              ATGGCTTCAGGTGGTGGCGCACCAGTGGCAGACAATAACGAAGGTGCCGATGGAGTGGG
              TAGTTCCTCGGGAAATTGGCATTGCGATTCCCAATGGCTGGGGGACAGAGTCATCACCAC
              CAGCACCCGAACCTGGGCCCTGCCCACCTACAACAATCACCTCTACAAGCAAATCTCCAA
              CAGCACATCTGGAGGATCTTCAAATGACAACGCCTACTTCGGCTACAGCACCCCCTGGGG
              GTATTTTGACTTCAACAGATTCCACTGCCACTTCTCACCACGTGACTGGCAGCGACTCATC
              AACAACAACTGGGGATTCCGGCCTAAGCGACTCAACTTCAAGCTCTTCAACATTCAGGTC
              AAAGAGGTTACGGACAACAATGGAGTCAAGACCATCGCCAATAACCTTACCAGCACGGT
              CCAGGTCTTCACGGACTCAGACTATCAGCTCCCGTACGTGCTCGGGTCGGCTCACGAGGG
              CTGCCTCCCGCCGTTCCCAGCGGACGTTTTCATGATTCCTCAGTACGGGTATCTGACGCTT
              AATGATGGAAGCCAGGCCGTGGGTCGTTCGTCCTTTTACTGCCTGGAATATTTCCCGTCGC
              AAATGCTAAGAACGGGTAACAACTTCCAGTTCAGCTACGAGTTTGAGAACGTACCTTTCC
              ATAGCAGCTACGCTCACAGCCAAAGCCTGGACCGACTAATGAATCCACTCATCGACCAAT
              ACTTGTACTATCTCTCAAAGACTATTAACGGTTCTGGACAGAATCAACAAACGCTAAAAT
              TCAGTGTGGCCGGACCCAGCAACATGGCTGTCCAGGGAAGAAACTACATACCTGGACCC
              AGCTACCGACAACAACGTGTCTCAACCACTGTGACTCAAAACAACAACAGCGAATTTGCT
              TGGCCTGGAGCTTCTTCTTGGGCTCTCAATGGACGTAATAGCTTGATGAATCCTGGACCTG
              CTATGGCCAGCCACAAAGAAGGAGAGGACCGTTTCTTTCCTTTGTCTGGATCTTTAATTTT
              TGGCAAACAAGGAACTGGAAGAGACAACGTGGATGCGGACAAAGTCATGATAACCAACG
              AAGAAGAAATTAAAACTACTAACCCGGTAGCAACGGAGTCCTATGGACAAGTGGCCACA
              AACCACCAGAGTGTACATCGATTGTTAATCAATAAACCGTTTAATTCGTTTCAGTTGAACT
              TTGGTCTCTGCGTATTTCTTTCTTATCTAGTTTCCATGGCTACGTAGATAAGTAGCATGGC
              GGGTTAATCATTAACTACAAGGAACCCCTAGTGATGGAGTTGGCCACTCCCTCTCTGCGC
              GCTCGCTCGCTCACTGAGGCCGGGCGACCAAAGGTCGCCCGACGCCCGGGCTTTGCCCGG
              GCGGCCTCAGTGAGCGAGCGAGCGCGCAGAGAGGGAGTGGCCAAGCATGCAATTAACTG
              GCCGTCGTTTTACAACGTCGTGACTGGGAAAACCCTGGCGTTACCCAACTTAATCGCCTT
              GCAGCACATCCCCCTTTCGCCAGCTGTATCAGCACACAATTGCCCATTATACGCGCGTAT
              AATGGACTATTGTGTGCTGATA
TELN-SYNG5-   TATCAGCACACAATAGTCCATTATACGCGCGTATAATGGGCAATTGTGTGCTGATACAGC
BSRGI SEQ ID  TGGCACGACAGGTTTCCCGACTGGAAAGCGGGCAGTGAGCGCAACGCAATTAATGTGAG
NO: 20        TTAGCTCACTCATTAGGCACCCCAGGCTTTACACTTTATGCTTCCGGCTCGTATGTTGTGT
              GGAATTGTGAGCGGATAACAATTTCACACAGGAAACAGCTATGACCATGATTACGCCAG
              ATTTAATTAAGGCCTTAATTAGGCTAGCTTGGCCACTCCCTCTCTGCGCGCTCGCTCGCTC
              ACTGAGGCCGGGCGACCAAAGGTCGCCCGACGCCCGGGCTTTGCCCGGGCGGCCTCAGT
              GAGCGAGCGAGCGCGCAGAGAGGGAGTGGCCAACTCCATCACTAGGGGTTCCTGGAGGG
              GTGGAGTCGTGACGATATCCATGCGTCGACATAACGCGTTAGTATCTGCAGAGGGCCCTG
              CGTATGAGTGCAAGTGGGTTTTAGGACCAGGATGAGGCGGGGTGGGGGTGCCTACCTGA
              CGACCGACCCCGACCCACTGGACAAGCACCCAACCCCCATTCCCCAAATTGCGCATCCCC
              TATCAGAGAGGGGGAGGGGAAACAGGATGCGGCGAGGCGCGTGCGCACTGCCAGCTTCA
              GCACCGCGGACAGTGCCTTCGCCCCCGCCTGGCGGCGCGCGCCACCGCCGCCTCAGCACT
              GAAGGCGCGCTGACGTCACTCGCCGGTCCCCCGCAAACTCCCCTTCCCGGCCACCTTGGT
              CGCGTCCGCGCCGCCGCCGGCCCAGCCGGACCGCACCACGCGAGGCGCGAGATAGGGGG
              GCACGGGCGCGACCATCTGCGCTGCGGCGCCGGCGACTCAGCGCTGCCTCAGTCTGCGGT
              GGGCAGCGGAGGAGTCGTGTCGTGCCTGAGAGCGCAGCTGTGCTCCTGGGCACCGCGCA
              GTCCGCCCCCGCGGCTCCTGGCCAGACCACCCCTAGGACCCCCTGCCCCAAGTCGCAGCC
              AAGCTTCGTTTAGTGAACCGTCAGATCGCCTGGAGACGCCATCCACGCTGTTTTGACCTCC
              ATAGAAGACACCGGGACCGATCCAGCCTCCGCGGATTCGAATCCCGGCCGGGAACGGTG
              CATTGGAACGCGGATTCCCCGTGCCAAGAGTGACGTAAGTACCGCCTATAGAGTCTATAG
              GCCCACAAAAAATGCTTTCTTCTTTTAATATACTTTTTTGTTTATCTTATTTCTAATACTTTC
              CCTAATCTCTTTCTTTCAGGGCAATAATGATACAATGTATCATGCCTCTTTGCACCATTCT
              AAAGAATAACAGTGATAATTTCTGGGTTAAGGCAATAGCAATATTTCTGCATATAAATAT
              TTCTGCATATAAATTGTAACTGATGTAAGAGGTTTCATATTGCTAATAGCAGCTACAATCC
              AGCTACCATTCTGCTTTTATTTTATGGTTGGGATAAGGCTGGATTATTCTGAGTCCAAGCT
              AGGCCCTTTTGCTAATCATGTTCATACCTCTTATCTTCCTCCCACAGCTCCTGGGCAACGT
              GCTGGTCTGTGTGCTGGCCCATCACTTTGGCAAAGAATTGGGATTCGAACCGGTCGCCAC
              CGGTCACAAGCAGGAAGTCAAAGACTTTTTCCGGTGGGCAAAGGATCACGTGGTTGAGG
              TGGAGCATGAATTCTACGTCAAAAAGGGTGGAGCCAAGAAAAGACCCGCCCCCAGTGAC
              GCAGATATAAGTGAGCCCAAACGGGTGCGCGAGTCAGTTGCGCAGCCATCGACGTCAGA
              CGCGGAAGCTTCGATCAACTACGCGGACAGGTACCAAAACAAATGTTCTCGTCACGTGGG
              CATGAATCTGATGCTGTTTCCCTGCAGACAATGCGAGAGAATGAATCAGAATTCAAATAT
              CTGCTTCACTCACGGACAGAAAGACTGTTTAGAGTGCTTTCCCGTGTCAGAATCTCAACC
              CGTTTCTGTCGTCAAAAAGGCGTATCAGAAACTGTGCTACATTCATCATATCATGGGAAA
              GGTGCCAGACGCTTGCACTGCCTGCGATCTGGTCAATGTGGATTTGGATGACTGCATCTTT
              GAACAATAAATGATTTAAATCAGGTATGTCTTTTGTTGATCACCCTCCAGATTGGTTGGAA
              GAAGTTGGTGAAGGTCTTCGCGAGTTTTTGGGCCTTGAAGCGGGCCCACCGAAACCAAAA
              CCCAATCAGCAGCATCAAGATCAAGCCCGTGGTCTTGTGCTGCCTGGTTATAACTATCTC
              GGACCCGGAAACGGTCTCGATCGAGGAGAGCCTGTCAACAGGGCAGACGAGGTCGCGCG
              AGAGCACGACATCTCGTACAACGAGCAGCTTGAGGCGGGAGACAACCCCTACCTCAAGT
              ACAACCACGCGGACGCCGAGTTTCAGGAGAAGCTCGCCGACGACACATCCTTCGGGGGA
              AACCTCGGAAAGGCAGTCTTTCAGGCCAAGAAAAGGGTTCTCGAACCTTTTGGCCTGGTT
              GAAGAGGGTGCTAAGACGGCCCCTACCGGAAAGCGGATAGACGACCACTTTCCAAAAAG
              AAAGAAGGCCCGGACCGAAGAGGACTCCAAGCCTTCCACCTCGTCAGACGCCGAAGCTG
              GACCCAGCGGATCCCAGCAGCTGCAAATCCCAGCCCAACCAGCCTCAAGTTTGGGAGCTG
              ATACAATGTCTGCGGGAGGTGGCGGCCCATTGGGCGACAATAACCAAGGTGCCGATGGA
              GTGGGCAATGCCTCGGGAGATTGGCATTGCGATTCCACGTGGATGGGGGACAGAGTCGTC
              ACCAAGTCCACCCGAACCTGGGTGCTGCCCAGCTACAACAACCACCAGTACCGAGAGAT
              CAAAAGCGGCTCCGTCGACGGAAGCAACGCCAACGCCTACTTTGGATACAGCACCCCCTG
              GGGGTACTTTGACTTTAACCGCTTCCACAGCCACTGGAGCCCCCGAGACTGGCAAAGACT
              CATCAACAACTACTGGGGCTTCAGACCCCGGTCCCTCAGAGTCAAAATCTTCAACATTCA
              AGTCAAAGAGGTCACGGTGCAGGACTCCACCACCACCATCGCCAACAACCTCACCTCCAC
              CGTCCAAGTGTTTACGGACGACGACTACCAGCTGCCCTACGTCGTCGGCAACGGGACCGA
              GGGATGCCTGCCGGCCTTCCCTCCGCAGGTCTTTACGCTGCCGCAGTACGGTTACGCGAC
              GCTGAACCGCGACAACACAGAAAATCCCACCGAGAGGAGCAGCTTCTTCTGCCTAGAGT
              ACTTTCCCAGCAAGATGCTGAGAACGGGCAACAACTTTGAGTTTACCTACAACTTTGAGG
              AGGTGCCCTTCCACTCCAGCTTCGCTCCCAGTCAGAACCTCTTCAAGCTGGCCAACCCGCT
              GGTGGACCAGTACTTGTACCGCTTCGTGAGCACAAATAACACTGGCGGAGTCCAGTTCAA
              CAAGAACCTGGCCGGGAGATACGCCAACACCTACAAAAACTGGTTCCCGGGGCCCATGG
              GCCGAACCCAGGGCTGGAACCTGGGCTCCGGGGTCAACCGCGCCAGTGTCAGCGCCTTCG
              CCACGACCAATAGGATGGAGCTCGAGGGCGCGAGTTACCAGGTGCCCCCGCAGCCGAAC
              GGCATGACCAACAACCTCCAGGGCAGCAACACCTATGCCCTGGAGAACACTATGATCTTC
              AACAGCCAGCCGGCGAACCCGGGCACCACCGCCACGTACCTCGAGGGCAACATGCTCAT
              CACCAGCGAGAGCGAGACGCAGCCGGTGAACCGCGTGGCGTACAACGTCGGCGGGCAGA
              TGGCCACCAACAACCAGAGCTCTGTACATCGATTGTTAATCAATAAACCGTTTAATTCGTT
              TCAGTTGAACTTTGGTCTCTGCGTATTTCTTTCTTATCTAGTTTCCATGGCTACGTAGATAA
              GTAGCATGGCGGGTTAATCATTAACTACAAGGAACCCCTAGTGATGGAGTTGGCCACTCC
              CTCTCTGCGCGCTCGCTCGCTCACTGAGGCCGGGCGACCAAAGGTCGCCCGACGCCCGGG
              CTTTGCCCGGGCGGCCTCAGTGAGCGAGCGAGCGCGCAGAGAGGGAGTGGCCAAGCATG
              CAATTAACTGGCCGTCGTTTTACAACGTCGTGACTGGGAAAACCCTGGCGTTACCCAACT
              TAATCGCCTTGCAGCACATCCCCCTTTCGCCAGCTGTATCAGCACACAATTGCCCATTATA
              CGCGCGTATAATGGACTATTGTGTGCTGATA
TELN-         TATCAGCACACAATAGTCCATTATACGCGCGTATAATGGGCAATTGTGTGCTGATACAGC
GFAPG5-       TGGCACGACAGGTTTCCCGACTGGAAAGCGGGCAGTGAGCGCAACGCAATTAATGTGAG
BSRGI SEQ ID  TTAGCTCACTCATTAGGCACCCCAGGCTTTACACTTTATGCTTCCGGCTCGTATGTTGTGT
NO: 21        GGAATTGTGAGCGGATAACAATTTCACACAGGAAACAGCTATGACCATGATTACGCCAG
              ATTTAATTAAGGCCTTAATTAGGCTAGCTTGGCCACTCCCTCTCTGCGCGCTCGCTCGCTC
              ACTGAGGCCGGGCGACCAAAGGTCGCCCGACGCCCGGGCTTTGCCCGGGCGGCCTCAGT
              GAGCGAGCGAGCGCGCAGAGAGGGAGTGGCCAACTCCATCACTAGGGGTTCCTGGAGGG
              GTGGAGTCGTGACGATATCCATGCGTCGACATAACGCGTGATCTAACATATCCTGGTGTG
              GAGTAGCGGACGCTGCTATGACAGAGGCTCGGGGGCCTGAGCTGGCTCTGTGAGCTGGG
              GAGGAGGCAGACAGCCAGGCCTTGTCTGCAAGCAGACCTGGCAGCATTGGGCTGGCCGC
              CCCCCAGGGCCTCCTCTTCATGCCCAGTGAATGACTCACCTTGGCACAGACACAATGTTC
              GGGGTGGGCACAGTGCCTGCTTCCCGCCGCACCCCAGCCCCCCTCAAATGCCTTCCGAGA
              AGCCCATTGAGCAGGGGGCTTGCATTGCACCCCAGCCTGACAGCCTGGCATCTTGGGATA
              AAAGCAGCACAGCCCCCTAGGGGCTGCCCTTGCTGTGTGGCGCCACCGGCGGTGGAGAA
              CAAGGCTCTATTCAGCCTGTGCCCAGGAAAGGGGATCAGGGGATGCCCAGGCATGGACA
              GTGGGTGGCAGGGGGGGAGAGGAGGGCTGTCTGCTTCCCAGAAGTCCAAGGACACAAAT
              GGGTGAGGGGAGAGCTCTCCCCATAGCTGGGCTGCGGCCCAACCCCACCCCCTCAGGCTA
              TGCCAGGGGGTGTTGCCAGGGGCACCCGGGCATCGCCAGTCTAGCCCACTCCTTCATAAA
              GCCCTCGCATCCCAGGAGCGAGCAGAGCCAGAGCAGGTTGGAGAGGAGACGCATCACCT
              CCGCTGCTCGCGGGGATCCTCTAGAAGCTTCGTTTAGTGAACCGTCAGATCGCCTGGAGA
              CGCCATCCACGCTGTTTTGACCTCCATAGAAGACACCGGGACCGATCCAGCCTCCGCGGA
              TTCGAATCCCGGCCGGGAACGGTGCATTGGAACGCGGATTCCCCGTGCCAAGAGTGACGT
              AAGTACCGCCTATAGAGTCTATAGGCCCACAAAAAATGCTTTCTTCTTTTAATATACTTTT
              TTGTTTATCTTATTTCTAATACTTTCCCTAATCTCTTTCTTTCAGGGCAATAATGATACAAT
              GTATCATGCCTCTTTGCACCATTCTAAAGAATAACAGTGATAATTTCTGGGTTAAGGCAAT
              AGCAATATTTCTGCATATAAATATTTCTGCATATAAATTGTAACTGATGTAAGAGGTTTCA
              TATTGCTAATAGCAGCTACAATCCAGCTACCATTCTGCTTTTATTTTATGGTTGGGATAAG
              GCTGGATTATTCTGAGTCCAAGCTAGGCCCTTTTGCTAATCATGTTCATACCTCTTATCTTC
              CTCCCACAGCTCCTGGGCAACGTGCTGGTCTGTGTGCTGGCCCATCACTTTGGCAAAGAA
              TTGGGATTCGAACCGGTCGCCACCGGTCACCAAGCAGGAAGTCAAAGACTTTTTCCGGTG
              GGCAAAGGATCACGTGGTTGAGGTGGAGCATGAATTCTACGTCAAAAAGGGTGGAGCCA
              AGAAAAGACCCGCCCCCAGTGACGCAGATATAAGTGAGCCCAAACGGGTGCGCGAGTCA
              GTTGCGCAGCCATCGACGTCAGACGCGGAAGCTTCGATCAACTACGCGGACAGGTACCA
              AAACAAATGTTCTCGTCACGTGGGCATGAATCTGATGCTGTTTCCCTGCAGACAATGCGA
              GAGAATGAATCAGAATTCAAATATCTGCTTCACTCACGGACAGAAAGACTGTTTAGAGTG
              CTTTCCCGTGTCAGAATCTCAACCCGTTTCTGTCGTCAAAAAGGCGTATCAGAAACTGTGC
              TACATTCATCATATCATGGGAAAGGTGCCAGACGCTTGCACTGCCTGCGATCTGGTCAAT
              GTGGATTTGGATGACTGCATCTTTGAACAATAAATGATTTAAATCAGGTATGTCTTTTGTT
              GATCACCCTCCAGATTGGTTGGAAGAAGTTGGTGAAGGTCTTCGCGAGTTTTTGGGCCTT
              GAAGCGGGCCCACCGAAACCAAAACCCAATCAGCAGCATCAAGATCAAGCCCGTGGTCT
              TGTGCTGCCTGGTTATAACTATCTCGGACCCGGAAACGGTCTCGATCGAGGAGAGCCTGT
              CAACAGGGCAGACGAGGTCGCGCGAGAGCACGACATCTCGTACAACGAGCAGCTTGAGG
              CGGGAGACAACCCCTACCTCAAGTACAACCACGCGGACGCCGAGTTTCAGGAGAAGCTC
              GCCGACGACACATCCTTCGGGGGAAACCTCGGAAAGGCAGTCTTTCAGGCCAAGAAAAG
              GGTTCTCGAACCTTTTGGCCTGGTTGAAGAGGGTGCTAAGACGGCCCCTACCGGAAAGCG
              GATAGACGACCACTTTCCAAAAAGAAAGAAGGCCCGGACCGAAGAGGACTCCAAGCCTT
              CCACCTCGTCAGACGCCGAAGCTGGACCCAGCGGATCCCAGCAGCTGCAAATCCCAGCCC
              AACCAGCCTCAAGTTTGGGAGCTGATACAATGTCTGCGGGAGGTGGCGGCCCATTGGGCG
              ACAATAACCAAGGTGCCGATGGAGTGGGCAATGCCTCGGGAGATTGGCATTGCGATTCC
              ACGTGGATGGGGGACAGAGTCGTCACCAAGTCCACCCGAACCTGGGTGCTGCCCAGCTA
              CAACAACCACCAGTACCGAGAGATCAAAAGCGGCTCCGTCGACGGAAGCAACGCCAACG
              CCTACTTTGGATACAGCACCCCCTGGGGGTACTTTGACTTTAACCGCTTCCACAGCCACTG
              GAGCCCCCGAGACTGGCAAAGACTCATCAACAACTACTGGGGCTTCAGACCCCGGTCCCT
              CAGAGTCAAAATCTTCAACATTCAAGTCAAAGAGGTCACGGTGCAGGACTCCACCACCAC
              CATCGCCAACAACCTCACCTCCACCGTCCAAGTGTTTACGGACGACGACTACCAGCTGCC
              CTACGTCGTCGGCAACGGGACCGAGGGATGCCTGCCGGCCTTCCCTCCGCAGGTCTTTAC
              GCTGCCGCAGTACGGTTACGCGACGCTGAACCGCGACAACACAGAAAATCCCACCGAGA
              GGAGCAGCTTCTTCTGCCTAGAGTACTTTCCCAGCAAGATGCTGAGAACGGGCAACAACT
              TTGAGTTTACCTACAACTTTGAGGAGGTGCCCTTCCACTCCAGCTTCGCTCCCAGTCAGAA
              CCTCTTCAAGCTGGCCAACCCGCTGGTGGACCAGTACTTGTACCGCTTCGTGAGCACAAA
              TAACACTGGCGGAGTCCAGTTCAACAAGAACCTGGCCGGGAGATACGCCAACACCTACA
              AAAACTGGTTCCCGGGGCCCATGGGCCGAACCCAGGGCTGGAACCTGGGCTCCGGGGTC
              AACCGCGCCAGTGTCAGCGCCTTCGCCACGACCAATAGGATGGAGCTCGAGGGCGCGAG
              TTACCAGGTGCCCCCGCAGCCGAACGGCATGACCAACAACCTCCAGGGCAGCAACACCT
              ATGCCCTGGAGAACACTATGATCTTCAACAGCCAGCCGGCGAACCCGGGCACCACCGCC
              ACGTACCTCGAGGGCAACATGCTCATCACCAGCGAGAGCGAGACGCAGCCGGTGAACCG
              CGTGGCGTACAACGTCGGCGGGCAGATGGCCACCAACAACCAGAGCTCTGTACATCGATT
              GTTAATCAATAAACCGTTTAATTCGTTTCAGTTGAACTTTGGTCTCTGCGTATTTCTTTCTT
              ATCTAGTTTCCATGGCTACGTAGATAAGTAGCATGGCGGGTTAATCATTAACTACAAGGA
              ACCCCTAGTGATGGAGTTGGCCACTCCCTCTCTGCGCGCTCGCTCGCTCACTGAGGCCGG
              GCGACCAAAGGTCGCCCGACGCCCGGGCTTTGCCCGGGCGGCCTCAGTGAGCGAGCGAG
              CGCGCAGAGAGGGAGTGGCCAAGCATGCAATTAACTGGCCGTCGTTTTACAACGTCGTGA
              CTGGGAAAACCCTGGCGTTACCCAACTTAATCGCCTTGCAGCACATCCCCCTTTCGCCAG
              CTGTATCAGCACACAATTGCCCATTATACGCGCGTATAATGGACTATTGTGTGCTGATA
TELN-SYNG6-   TATCAGCACACAATAGTCCATTATACGCGCGTATAATGGGCAATTGTGTGCTGATACAGC
BSRGI SEQ ID  TGGCACGACAGGTTTCCCGACTGGAAAGCGGGCAGTGAGCGCAACGCAATTAATGTGAG
NO: 22        TTAGCTCACTCATTAGGCACCCCAGGCTTTACACTTTATGCTTCCGGCTCGTATGTTGTGT
              GGAATTGTGAGCGGATAACAATTTCACACAGGAAACAGCTATGACCATGATTACGCCAG
              ATTTAATTAAGGCCTTAATTAGGCTAGCTTGGCCACTCCCTCTCTGCGCGCTCGCTCGCTC
              ACTGAGGCCGGGCGACCAAAGGTCGCCCGACGCCCGGGCTTTGCCCGGGCGGCCTCAGT
              GAGCGAGCGAGCGCGCAGAGAGGGAGTGGCCAACTCCATCACTAGGGGTTCCTGGAGGG
              GTGGAGTCGTGACGATATCCATGCGTCGACATAACGCGTTAGTATCTGCAGAGGGCCCTG
              CGTATGAGTGCAAGTGGGTTTTAGGACCAGGATGAGGCGGGGTGGGGGTGCCTACCTGA
              CGACCGACCCCGACCCACTGGACAAGCACCCAACCCCCATTCCCCAAATTGCGCATCCCC
              TATCAGAGAGGGGGAGGGGAAACAGGATGCGGCGAGGCGCGTGCGCACTGCCAGCTTCA
              GCACCGCGGACAGTGCCTTCGCCCCCGCCTGGCGGCGCGCGCCACCGCCGCCTCAGCACT
              GAAGGCGCGCTGACGTCACTCGCCGGTCCCCCGCAAACTCCCCTTCCCGGCCACCTTGGT
              CGCGTCCGCGCCGCCGCCGGCCCAGCCGGACCGCACCACGCGAGGCGCGAGATAGGGGG
              GCACGGGCGCGACCATCTGCGCTGCGGCGCCGGCGACTCAGCGCTGCCTCAGTCTGCGGT
              GGGCAGCGGAGGAGTCGTGTCGTGCCTGAGAGCGCAGCTGTGCTCCTGGGCACCGCGCA
              GTCCGCCCCCGCGGCTCCTGGCCAGACCACCCCTAGGACCCCCTGCCCCAAGTCGCAGCC
              AAGCTTCGTTTAGTGAACCGTCAGATCGCCTGGAGACGCCATCCACGCTGTTTTGACCTCC
              ATAGAAGACACCGGGACCGATCCAGCCTCCGCGGATTCGAATCCCGGCCGGGAACGGTG
              CATTGGAACGCGGATTCCCCGTGCCAAGAGTGACGTAAGTACCGCCTATAGAGTCTATAG
              GCCCACAAAAAATGCTTTCTTCTTTTAATATACTTTTTTGTTTATCTTATTTCTAATACTTTC
              CCTAATCTCTTTCTTTCAGGGCAATAATGATACAATGTATCATGCCTCTTTGCACCATTCT
              AAAGAATAACAGTGATAATTTCTGGGTTAAGGCAATAGCAATATTTCTGCATATAAATAT
              TTCTGCATATAAATTGTAACTGATGTAAGAGGTTTCATATTGCTAATAGCAGCTACAATCC
              AGCTACCATTCTGCTTTTATTTTATGGTTGGGATAAGGCTGGATTATTCTGAGTCCAAGCT
              AGGCCCTTTTGCTAATCATGTTCATACCTCTTATCTTCCTCCCACAGCTCCTGGGCAACGT
              GCTGGTCTGTGTGCTGGCCCATCACTTTGGCAAAGAATTGGGATTCGAACCGGTCGCCAC
              CGGTCACAAGCAGGAAGTCAAAGACTTTTTCCGGTGGGCAAAGGATCACGTGGTTGAGG
              TGGAGCATGAATTCTACGTCAAAAAGGGTGGAGCCAAGAAAAGACCCGCCCCCAGTGAC
              GCAGATATAAGTGAGCCCAAACGGGTGCGCGAGTCAGTTGCGCAGCCATCGACGTCAGA
              CGCGGAAGCTTCGATCAACTACGCGGACAGGTACCAAAACAAATGTTCTCGTCACGTGGG
              CATGAATCTGATGCTGTTTCCCTGCAGACAATGCGAGAGAATGAATCAGAATTCAAATAT
              CTGCTTCACTCACGGACAGAAAGACTGTTTAGAGTGCTTTCCCGTGTCAGAATCTCAACC
              CGTTTCTGTCGTCAAAAAGGCGTATCAGAAACTGTGCTACATTCATCATATCATGGGAAA
              GGTGCCAGACGCTTGCACTGCCTGCGATCTGGTCAATGTGGATTTGGATGACTGCATCTTT
              GAACAATAAATGATTTAAATCAGGTATGGCTGCCGATGGTTATCTTCCAGATTGGCTCGA
              GGACAACCTCTCTGAGGGCATTCGCGAGTGGTGGGACTTGAAACCTGGAGCCCCGAAAC
              CCAAAGCCAACCAGCAAAAGCAGGACGACGGCCGGGGTCTGGTGCTTCCTGGCTACAAG
              TACCTCGGACCCTTCAACGGACTCGACAAGGGGGAGCCCGTCAACGCGGCGGATGCAGC
              GGCCCTCGAGCACGACAAGGCCTACGACCAGCAGCTCAAAGCGGGTGACAATCCGTACC
              TGCGGTATAACCACGCCGACGCCGAGTTTCAGGAGCGTCTGCAAGAAGATACGTCTTTTG
              GGGGCAACCTCGGGCGAGCAGTCTTCCAGGCCAAGAAGAGGGTTCTCGAACCTTTTGGTC
              TGGTTGAGGAAGGTGCTAAGACGGCTCCTGGAAAGAAACGTCCGGTAGAGCAGTCGCCA
              CAAGAGCCAGACTCCTCCTCGGGCATTGGCAAGACAGGCCAGCAGCCCGCTAAAAAGAG
              ACTCAATTTTGGTCAGACTGGCGACTCAGAGTCAGTCCCCGACCCACAACCTCTCGGAGA
              ACCTCCAGCAACCCCCGCTGCTGTGGGACCTACTACAATGGCTTCAGGCGGTGGCGCACC
              AATGGCAGACAATAACGAAGGCGCCGACGGAGTGGGTAATGCCTCAGGAAATTGGCATT
              GCGATTCCACATGGCTGGGCGACAGAGTCATCACCACCAGCACCCGAACATGGGCCTTGC
              CCACCTATAACAACCACCTCTACAAGCAAATCTCCAGTGCTTCAACGGGGGCCAGCAACG
              ACAACCACTACTTCGGCTACAGCACCCCCTGGGGGTATTTTGATTTCAACAGATTCCACTG
              CCATTTCTCACCACGTGACTGGCAGCGACTCATCAACAACAATTGGGGATTCCGGCCCAA
              GAGACTCAACTTCAAGCTCTTCAACATCCAAGTCAAGGAGGTCACGACGAATGATGGCGT
              CACGACCATCGCTAATAACCTTACCAGCACGGTTCAAGTCTTCTCGGACTCGGAGTACCA
              GTTGCCGTACGTCCTCGGCTCTGCGCACCAGGGCTGCCTCCCTCCGTTCCCGGCGGACGTG
              TTCATGATTCCGCAGTACGGCTACCTAACGCTCAACAATGGCAGCCAGGCAGTGGGACGG
              TCATCCTTTTACTGCCTGGAATATTTCCCATCGCAGATGCTGAGAACGGGCAATAACTTTA
              CCTTCAGCTACACCTTCGAGGACGTGCCTTTCCACAGCAGCTACGCGCACAGCCAGAGCC
              TGGACCGGCTGATGAATCCTCTCATCGACCAGTACCTGTATTACCTGAACAGAACTCAGA
              ATCAGTCCGGAAGTGCCCAAAACAAGGACTTGCTGTTTAGCCGGGGGTCTCCAGCTGGCA
              TGTCTGTTCAGCCCAAAAACTGGCTACCTGGACCCTGTTACCGGCAGCAGCGCGTTTCTA
              AAACAAAAACAGACAACAACAACAGCAACTTTACCTGGACTGGTGCTTCAAAATATAAC
              CTTAATGGGCGTGAATCTATAATCAACCCTGGCACTGCTATGGCCTCACACAAAGACGAC
              AAAGACAAGTTCTTTCCCATGAGCGGTGTCATGATTTTTGGAAAGGAGAGCGCCGGAGCT
              TCAAACACTGCATTGGACAATGTCATGATCACAGACGAAGAGGAAATCAAAGCCACTAA
              CCCCGTGGCCACCGAAAGATTTGGGACTGTGGCAGTCAATCTCCAGAGTGTACATCGATT
              GTTAATCAATAAACCGTTTAATTCGTTTCAGTTGAACTTTGGTCTCTGCGTATTTCTTTCTT
              ATCTAGTTTCCATGGCTACGTAGATAAGTAGCATGGCGGGTTAATCATTAACTACAAGGA
              ACCCCTAGTGATGGAGTTGGCCACTCCCTCTCTGCGCGCTCGCTCGCTCACTGAGGCCGG
              GCGACCAAAGGTCGCCCGACGCCCGGGCTTTGCCCGGGCGGCCTCAGTGAGCGAGCGAG
              CGCGCAGAGAGGGAGTGGCCAAGCATGCAATTAACTGGCCGTCGTTTTACAACGTCGTGA
              CTGGGAAAACCCTGGCGTTACCCAACTTAATCGCCTTGCAGCACATCCCCCTTTCGCCAG
              CTGTATCAGCACACAATTGCCCATTATACGCGCGTATAATGGACTATTGTGTGCTGATA
TELN-         TATCAGCACACAATAGTCCATTATACGCGCGTATAATGGGCAATTGTGTGCTGATACAGC
GFAPG6-       TGGCACGACAGGTTTCCCGACTGGAAAGCGGGCAGTGAGCGCAACGCAATTAATGTGAG
BSRGI SEQ ID  TTAGCTCACTCATTAGGCACCCCAGGCTTTACACTTTATGCTTCCGGCTCGTATGTTGTGT
NO: 23        GGAATTGTGAGCGGATAACAATTTCACACAGGAAACAGCTATGACCATGATTACGCCAG
              ATTTAATTAAGGCCTTAATTAGGCTAGCTTGGCCACTCCCTCTCTGCGCGCTCGCTCGCTC
              ACTGAGGCCGGGCGACCAAAGGTCGCCCGACGCCCGGGCTTTGCCCGGGCGGCCTCAGT
              GAGCGAGCGAGCGCGCAGAGAGGGAGTGGCCAACTCCATCACTAGGGGTTCCTGGAGGG
              GTGGAGTCGTGACGATATCCATGCGTCGACATAACGCGTGATCTAACATATCCTGGTGTG
              GAGTAGCGGACGCTGCTATGACAGAGGCTCGGGGGCCTGAGCTGGCTCTGTGAGCTGGG
              GAGGAGGCAGACAGCCAGGCCTTGTCTGCAAGCAGACCTGGCAGCATTGGGCTGGCCGC
              CCCCCAGGGCCTCCTCTTCATGCCCAGTGAATGACTCACCTTGGCACAGACACAATGTTC
              GGGGTGGGCACAGTGCCTGCTTCCCGCCGCACCCCAGCCCCCCTCAAATGCCTTCCGAGA
              AGCCCATTGAGCAGGGGGCTTGCATTGCACCCCAGCCTGACAGCCTGGCATCTTGGGATA
              AAAGCAGCACAGCCCCCTAGGGGCTGCCCTTGCTGTGTGGCGCCACCGGCGGTGGAGAA
              CAAGGCTCTATTCAGCCTGTGCCCAGGAAAGGGGATCAGGGGATGCCCAGGCATGGACA
              GTGGGTGGCAGGGGGGGAGAGGAGGGCTGTCTGCTTCCCAGAAGTCCAAGGACACAAAT
              GGGTGAGGGGAGAGCTCTCCCCATAGCTGGGCTGCGGCCCAACCCCACCCCCTCAGGCTA
              TGCCAGGGGGTGTTGCCAGGGGCACCCGGGCATCGCCAGTCTAGCCCACTCCTTCATAAA
              GCCCTCGCATCCCAGGAGCGAGCAGAGCCAGAGCAGGTTGGAGAGGAGACGCATCACCT
              CCGCTGCTCGCGGGGATCCTCTAGAAGCTTCGTTTAGTGAACCGTCAGATCGCCTGGAGA
              CGCCATCCACGCTGTTTTGACCTCCATAGAAGACACCGGGACCGATCCAGCCTCCGCGGA
              TTCGAATCCCGGCCGGGAACGGTGCATTGGAACGCGGATTCCCCGTGCCAAGAGTGACGT
              AAGTACCGCCTATAGAGTCTATAGGCCCACAAAAAATGCTTTCTTCTTTTAATATACTTTT
              TTGTTTATCTTATTTCTAATACTTTCCCTAATCTCTTTCTTTCAGGGCAATAATGATACAAT
              GTATCATGCCTCTTTGCACCATTCTAAAGAATAACAGTGATAATTTCTGGGTTAAGGCAAT
              AGCAATATTTCTGCATATAAATATTTCTGCATATAAATTGTAACTGATGTAAGAGGTTTCA
              TATTGCTAATAGCAGCTACAATCCAGCTACCATTCTGCTTTTATTTTATGGTTGGGATAAG
              GCTGGATTATTCTGAGTCCAAGCTAGGCCCTTTTGCTAATCATGTTCATACCTCTTATCTTC
              CTCCCACAGCTCCTGGGCAACGTGCTGGTCTGTGTGCTGGCCCATCACTTTGGCAAAGAA
              TTGGGATTCGAACCGGTCGCCACCGGTCACCAAGCAGGAAGTCAAAGACTTTTTCCGGTG
              GGCAAAGGATCACGTGGTTGAGGTGGAGCATGAATTCTACGTCAAAAAGGGTGGAGCCA
              AGAAAAGACCCGCCCCCAGTGACGCAGATATAAGTGAGCCCAAACGGGTGCGCGAGTCA
              GTTGCGCAGCCATCGACGTCAGACGCGGAAGCTTCGATCAACTACGCGGACAGGTACCA
              AAACAAATGTTCTCGTCACGTGGGCATGAATCTGATGCTGTTTCCCTGCAGACAATGCGA
              GAGAATGAATCAGAATTCAAATATCTGCTTCACTCACGGACAGAAAGACTGTTTAGAGTG
              CTTTCCCGTGTCAGAATCTCAACCCGTTTCTGTCGTCAAAAAGGCGTATCAGAAACTGTGC
              TACATTCATCATATCATGGGAAAGGTGCCAGACGCTTGCACTGCCTGCGATCTGGTCAAT
              GTGGATTTGGATGACTGCATCTTTGAACAATAAATGATTTAAATCAGGTATGGCTGCCGA
              TGGTTATCTTCCAGATTGGCTCGAGGACAACCTCTCTGAGGGCATTCGCGAGTGGTGGGA
              CTTGAAACCTGGAGCCCCGAAACCCAAAGCCAACCAGCAAAAGCAGGACGACGGCCGGG
              GTCTGGTGCTTCCTGGCTACAAGTACCTCGGACCCTTCAACGGACTCGACAAGGGGGAGC
              CCGTCAACGCGGCGGATGCAGCGGCCCTCGAGCACGACAAGGCCTACGACCAGCAGCTC
              AAAGCGGGTGACAATCCGTACCTGCGGTATAACCACGCCGACGCCGAGTTTCAGGAGCG
              TCTGCAAGAAGATACGTCTTTTGGGGGCAACCTCGGGCGAGCAGTCTTCCAGGCCAAGAA
              GAGGGTTCTCGAACCTTTTGGTCTGGTTGAGGAAGGTGCTAAGACGGCTCCTGGAAAGAA
              ACGTCCGGTAGAGCAGTCGCCACAAGAGCCAGACTCCTCCTCGGGCATTGGCAAGACAG
              GCCAGCAGCCCGCTAAAAAGAGACTCAATTTTGGTCAGACTGGCGACTCAGAGTCAGTCC
              CCGACCCACAACCTCTCGGAGAACCTCCAGCAACCCCCGCTGCTGTGGGACCTACTACAA
              TGGCTTCAGGCGGTGGCGCACCAATGGCAGACAATAACGAAGGCGCCGACGGAGTGGGT
              AATGCCTCAGGAAATTGGCATTGCGATTCCACATGGCTGGGCGACAGAGTCATCACCACC
              AGCACCCGAACATGGGCCTTGCCCACCTATAACAACCACCTCTACAAGCAAATCTCCAGT
              GCTTCAACGGGGGCCAGCAACGACAACCACTACTTCGGCTACAGCACCCCCTGGGGGTAT
              TTTGATTTCAACAGATTCCACTGCCATTTCTCACCACGTGACTGGCAGCGACTCATCAACA
              ACAATTGGGGATTCCGGCCCAAGAGACTCAACTTCAAGCTCTTCAACATCCAAGTCAAGG
              AGGTCACGACGAATGATGGCGTCACGACCATCGCTAATAACCTTACCAGCACGGTTCAAG
              TCTTCTCGGACTCGGAGTACCAGTTGCCGTACGTCCTCGGCTCTGCGCACCAGGGCTGCCT
              CCCTCCGTTCCCGGCGGACGTGTTCATGATTCCGCAGTACGGCTACCTAACGCTCAACAA
              TGGCAGCCAGGCAGTGGGACGGTCATCCTTTTACTGCCTGGAATATTTCCCATCGCAGAT
              GCTGAGAACGGGCAATAACTTTACCTTCAGCTACACCTTCGAGGACGTGCCTTTCCACAG
              CAGCTACGCGCACAGCCAGAGCCTGGACCGGCTGATGAATCCTCTCATCGACCAGTACCT
              GTATTACCTGAACAGAACTCAGAATCAGTCCGGAAGTGCCCAAAACAAGGACTTGCTGTT
              TAGCCGGGGGTCTCCAGCTGGCATGTCTGTTCAGCCCAAAAACTGGCTACCTGGACCCTG
              TTACCGGCAGCAGCGCGTTTCTAAAACAAAAACAGACAACAACAACAGCAACTTTACCT
              GGACTGGTGCTTCAAAATATAACCTTAATGGGCGTGAATCTATAATCAACCCTGGCACTG
              CTATGGCCTCACACAAAGACGACAAAGACAAGTTCTTTCCCATGAGCGGTGTCATGATTT
              TTGGAAAGGAGAGCGCCGGAGCTTCAAACACTGCATTGGACAATGTCATGATCACAGAC
              GAAGAGGAAATCAAAGCCACTAACCCCGTGGCCACCGAAAGATTTGGGACTGTGGCAGT
              CAATCTCCAGAGTGTACATCGATTGTTAATCAATAAACCGTTTAATTCGTTTCAGTTGAAC
              TTTGGTCTCTGCGTATTTCTTTCTTATCTAGTTTCCATGGCTACGTAGATAAGTAGCATGGC
              GGGTTAATCATTAACTACAAGGAACCCCTAGTGATGGAGTTGGCCACTCCCTCTCTGCGC
              GCTCGCTCGCTCACTGAGGCCGGGCGACCAAAGGTCGCCCGACGCCCGGGCTTTGCCCGG
              GCGGCCTCAGTGAGCGAGCGAGCGCGCAGAGAGGGAGTGGCCAAGCATGCAATTAACTG
              GCCGTCGTTTTACAACGTCGTGACTGGGAAAACCCTGGCGTTACCCAACTTAATCGCCTT
              GCAGCACATCCCCCTTTCGCCAGCTGTATCAGCACACAATTGCCCATTATACGCGCGTAT
              AATGGACTATTGTGTGCTGATA
TELN-         TATCAGCACACAATAGTCCATTATACGCGCGTATAATGGGCAATTGTGTGCTGATACAGC
SYNGDJ8-      TGGCACGACAGGTTTCCCGACTGGAAAGCGGGCAGTGAGCGCAACGCAATTAATGTGAG
BSRGI SEQ ID  TTAGCTCACTCATTAGGCACCCCAGGCTTTACACTTTATGCTTCCGGCTCGTATGTTGTGT
NO: 24        GGAATTGTGAGCGGATAACAATTTCACACAGGAAACAGCTATGACCATGATTACGCCAG
              ATTTAATTAAGGCCTTAATTAGGCTAGCTTGGCCACTCCCTCTCTGCGCGCTCGCTCGCTC
              ACTGAGGCCGGGCGACCAAAGGTCGCCCGACGCCCGGGCTTTGCCCGGGCGGCCTCAGT
              GAGCGAGCGAGCGCGCAGAGAGGGAGTGGCCAACTCCATCACTAGGGGTTCCTGGAGGG
              GTGGAGTCGTGACGATATCCATGCGTCGACATAACGCGTTAGTATCTGCAGAGGGCCCTG
              CGTATGAGTGCAAGTGGGTTTTAGGACCAGGATGAGGCGGGGTGGGGGTGCCTACCTGA
              CGACCGACCCCGACCCACTGGACAAGCACCCAACCCCCATTCCCCAAATTGCGCATCCCC
              TATCAGAGAGGGGGAGGGGAAACAGGATGCGGCGAGGCGCGTGCGCACTGCCAGCTTCA
              GCACCGCGGACAGTGCCTTCGCCCCCGCCTGGCGGCGCGCGCCACCGCCGCCTCAGCACT
              GAAGGCGCGCTGACGTCACTCGCCGGTCCCCCGCAAACTCCCCTTCCCGGCCACCTTGGT
              CGCGTCCGCGCCGCCGCCGGCCCAGCCGGACCGCACCACGCGAGGCGCGAGATAGGGGG
              GCACGGGCGCGACCATCTGCGCTGCGGCGCCGGCGACTCAGCGCTGCCTCAGTCTGCGGT
              GGGCAGCGGAGGAGTCGTGTCGTGCCTGAGAGCGCAGCTGTGCTCCTGGGCACCGCGCA
              GTCCGCCCCCGCGGCTCCTGGCCAGACCACCCCTAGGACCCCCTGCCCCAAGTCGCAGCC
              AAGCTTCGTTTAGTGAACCGTCAGATCGCCTGGAGACGCCATCCACGCTGTTTTGACCTCC
              ATAGAAGACACCGGGACCGATCCAGCCTCCGCGGATTCGAATCCCGGCCGGGAACGGTG
              CATTGGAACGCGGATTCCCCGTGCCAAGAGTGACGTAAGTACCGCCTATAGAGTCTATAG
              GCCCACAAAAAATGCTTTCTTCTTTTAATATACTTTTTTGTTTATCTTATTTCTAATACTTTC
              CCTAATCTCTTTCTTTCAGGGCAATAATGATACAATGTATCATGCCTCTTTGCACCATTCT
              AAAGAATAACAGTGATAATTTCTGGGTTAAGGCAATAGCAATATTTCTGCATATAAATAT
              TTCTGCATATAAATTGTAACTGATGTAAGAGGTTTCATATTGCTAATAGCAGCTACAATCC
              AGCTACCATTCTGCTTTTATTTTATGGTTGGGATAAGGCTGGATTATTCTGAGTCCAAGCT
              AGGCCCTTTTGCTAATCATGTTCATACCTCTTATCTTCCTCCCACAGCTCCTGGGCAACGT
              GCTGGTCTGTGTGCTGGCCCATCACTTTGGCAAAGAATTGGGATTCGAACCGGTCGCCAC
              CGGTCACAAGCAGGAAGTCAAAGACTTTTTCCGGTGGGCAAAGGATCACGTGGTTGAGG
              TGGAGCATGAATTCTACGTCAAAAAGGGTGGAGCCAAGAAAAGACCCGCCCCCAGTGAC
              GCAGATATAAGTGAGCCCAAACGGGTGCGCGAGTCAGTTGCGCAGCCATCGACGTCAGA
              CGCGGAAGCTTCGATCAACTACGCGGACAGGTACCAAAACAAATGTTCTCGTCACGTGGG
              CATGAATCTGATGCTGTTTCCCTGCAGACAATGCGAGAGAATGAATCAGAATTCAAATAT
              CTGCTTCACTCACGGACAGAAAGACTGTTTAGAGTGCTTTCCCGTGTCAGAATCTCAACC
              CGTTTCTGTCGTCAAAAAGGCGTATCAGAAACTGTGCTACATTCATCATATCATGGGAAA
              GGTGCCAGACGCTTGCACTGCCTGCGATCTGGTCAATGTGGATTTGGATGACTGCATCTTT
              GAACAATAAATGATTTAAATCAGGTATGGCTGCCGATGGTTATCTTCCAGATTGGCTCGA
              GGACACTCTCTCTGAAGGAATAAGACAGTGGTGGAAGCTCAAACCTGGCCCACCACCAC
              CAAAGCCCGCAGAGCGGCATAAGGACGACAGCAGGGGTCTTGTGCTTCCTGGGTACAAG
              TACCTCGGACCCTTCAACGGACTCGACAAGGGAGAGCCGGTCAACGAGGCAGACGCCGC
              GGCCCTCGAGCACGACAAAGCCTACGACCGGCAGCTCGACAGCGGAGACAACCCGTACC
              TCAAGTACAACCACGCCGACGCCGAGTTCCAGGAGCGGCTCAAAGAAGATACGTCTTTTG
              GGGGCAACCTCGGGCGAGCAGTCTTCCAGGCCAAAAAGAGGCTTCTTGAACCTCTTGGTC
              TGGTTGAGGAAGCGGCTAAGACGGCTCCTGGAAAGAAGAGGCCTGTAGAGCACTCTCCT
              GTGGAGCCAGACTCCTCCTCGGGAACCGGAAAGGCGGGCCAGCAGCCTGCAAGAAAAAG
              ATTGAATTTTGGTCAGACTGGAGACGCAGACTCAGTCCCAGACCCTCAACCAATCGGAGA
              ACCTCCCGCAGCCCCCTCAGGTGTGGGATCTCTTACAATGGCTGCAGGCGGTGGCGCACC
              AATGGCAGACAATAACGAGGGCGCCGACGGAGTGGGTAATTCCTCGGGAAATTGGCATT
              GCGATTCCACATGGATGGGCGACAGAGTCATCACCACCAGCACCCGAACCTGGGCCCTGC
              CCACCTACAACAACCACCTCTACAAGCAAATCTCCAACAGCACATCTGGAGGATCTTCAA
              ATGACAACGCCTACTTCGGCTACAGCACCCCCTGGGGGTATTTTGACTTTAACAGATTCC
              ACTGCCACTTTTCACCACGTGACTGGCAGCGACTCATCAACAACAACTGGGGATTCCGGC
              CCAAGAGACTCAGCTTCAAGCTCTTCAACATCCAGGTCAAGGAGGTCACGCAGAATGAA
              GGCACCAAGACCATCGCCAATAACCTCACCAGCACCATCCAGGTGTTTACGGACTCGGAG
              TACCAGCTGCCGTACGTTCTCGGCTCTGCCCACCAGGGCTGCCTGCCTCCGTTCCCGGCGG
              ACGTGTTCATGATTCCCCAGTACGGCTACCTAACACTCAACAACGGTAGTCAGGCCGTGG
              GACGCTCCTCCTTCTACTGCCTGGAATACTTTCCTTCGCAGATGCTGAGAACCGGCAACA
              ACTTCCAGTTTACTTACACCTTCGAGGACGTGCCTTTCCACAGCAGCTACGCCCACAGCCA
              GAGCTTGGACCGGCTGATGAATCCTCTGATTGACCAGTACCTGTACTACTTGTCTCGGACT
              CAAACAACAGGAGGCACGACAAATACGCAGACTCTGGGCTTCAGCCAAGGTGGGCCTAA
              TACAATGGCCAATCAGGCAAAGAACTGGCTGCCAGGACCCTGTTACCGCCAGCAGCGAG
              TATCAAAGACATCTGCGGATAACAACAACAGTGAATACTCGTGGACTGGAGCTACCAAG
              TACCACCTCAATGGCAGAGACTCTCTGGTGAATCCGGGCCCGGCCATGGCAAGCCACAAG
              GACGATGAAGAAAAGTTTTTTCCTCAGAGCGGGGTTCTCATCTTTGGGAAGCAAGGCTCA
              GAGAAAACAAATGTGGACATTGAAAAGGTCATGATTACAGACGAAGAGGAAATCAGGAC
              AACCAATCCCGTGGCTACGGAGCAGTATGGTTCTGTATCTACCAACCTCCAGCAAGGTGT
              ACATCGATTGTTAATCAATAAACCGTTTAATTCGTTTCAGTTGAACTTTGGTCTCTGCGTA
              TTTCTTTCTTATCTAGTTTCCATGGCTACGTAGATAAGTAGCATGGCGGGTTAATCATTAA
              CTACAAGGAACCCCTAGTGATGGAGTTGGCCACTCCCTCTCTGCGCGCTCGCTCGCTCACT
              GAGGCCGGGCGACCAAAGGTCGCCCGACGCCCGGGCTTTGCCCGGGCGGCCTCAGTGAG
              CGAGCGAGCGCGCAGAGAGGGAGTGGCCAAGCATGCAATTAACTGGCCGTCGTTTTACA
              ACGTCGTGACTGGGAAAACCCTGGCGTTACCCAACTTAATCGCCTTGCAGCACATCCCCC
              TTTCGCCAGCTGTATCAGCACACAATTGCCCATTATACGCGCGTATAATGGACTATTGTGT
              GCTGATA
TELN-GFAPG-   TATCAGCACACAATAGTCCATTATACGCGCGTATAATGGGCAATTGTGTGCTGATACAGC
DJ8-BSRGI     TGGCACGACAGGTTTCCCGACTGGAAAGCGGGCAGTGAGCGCAACGCAATTAATGTGAG
SEQ ID NO: 25 TTAGCTCACTCATTAGGCACCCCAGGCTTTACACTTTATGCTTCCGGCTCGTATGTTGTGT
              GGAATTGTGAGCGGATAACAATTTCACACAGGAAACAGCTATGACCATGATTACGCCAG
              ATTTAATTAAGGCCTTAATTAGGCTAGCTTGGCCACTCCCTCTCTGCGCGCTCGCTCGCTC
              ACTGAGGCCGGGCGACCAAAGGTCGCCCGACGCCCGGGCTTTGCCCGGGCGGCCTCAGT
              GAGCGAGCGAGCGCGCAGAGAGGGAGTGGCCAACTCCATCACTAGGGGTTCCTGGAGGG
              GTGGAGTCGTGACGATATCCATGCGTCGACATAACGCGTGATCTAACATATCCTGGTGTG
              GAGTAGCGGACGCTGCTATGACAGAGGCTCGGGGGCCTGAGCTGGCTCTGTGAGCTGGG
              GAGGAGGCAGACAGCCAGGCCTTGTCTGCAAGCAGACCTGGCAGCATTGGGCTGGCCGC
              CCCCCAGGGCCTCCTCTTCATGCCCAGTGAATGACTCACCTTGGCACAGACACAATGTTC
              GGGGTGGGCACAGTGCCTGCTTCCCGCCGCACCCCAGCCCCCCTCAAATGCCTTCCGAGA
              AGCCCATTGAGCAGGGGGCTTGCATTGCACCCCAGCCTGACAGCCTGGCATCTTGGGATA
              AAAGCAGCACAGCCCCCTAGGGGCTGCCCTTGCTGTGTGGCGCCACCGGCGGTGGAGAA
              CAAGGCTCTATTCAGCCTGTGCCCAGGAAAGGGGATCAGGGGATGCCCAGGCATGGACA
              GTGGGTGGCAGGGGGGGAGAGGAGGGCTGTCTGCTTCCCAGAAGTCCAAGGACACAAAT
              GGGTGAGGGGAGAGCTCTCCCCATAGCTGGGCTGCGGCCCAACCCCACCCCCTCAGGCTA
              TGCCAGGGGGTGTTGCCAGGGGCACCCGGGCATCGCCAGTCTAGCCCACTCCTTCATAAA
              GCCCTCGCATCCCAGGAGCGAGCAGAGCCAGAGCAGGTTGGAGAGGAGACGCATCACCT
              CCGCTGCTCGCGGGGATCCTCTAGAAGCTTCGTTTAGTGAACCGTCAGATCGCCTGGAGA
              CGCCATCCACGCTGTTTTGACCTCCATAGAAGACACCGGGACCGATCCAGCCTCCGCGGA
              TTCGAATCCCGGCCGGGAACGGTGCATTGGAACGCGGATTCCCCGTGCCAAGAGTGACGT
              AAGTACCGCCTATAGAGTCTATAGGCCCACAAAAAATGCTTTCTTCTTTTAATATACTTTT
              TTGTTTATCTTATTTCTAATACTTTCCCTAATCTCTTTCTTTCAGGGCAATAATGATACAAT
              GTATCATGCCTCTTTGCACCATTCTAAAGAATAACAGTGATAATTTCTGGGTTAAGGCAAT
              AGCAATATTTCTGCATATAAATATTTCTGCATATAAATTGTAACTGATGTAAGAGGTTTCA
              TATTGCTAATAGCAGCTACAATCCAGCTACCATTCTGCTTTTATTTTATGGTTGGGATAAG
              GCTGGATTATTCTGAGTCCAAGCTAGGCCCTTTTGCTAATCATGTTCATACCTCTTATCTTC
              CTCCCACAGCTCCTGGGCAACGTGCTGGTCTGTGTGCTGGCCCATCACTTTGGCAAAGAA
              TTGGGATTCGAACCGGTCGCCACCGGTCACCAAGCAGGAAGTCAAAGACTTTTTCCGGTG
              GGCAAAGGATCACGTGGTTGAGGTGGAGCATGAATTCTACGTCAAAAAGGGTGGAGCCA
              AGAAAAGACCCGCCCCCAGTGACGCAGATATAAGTGAGCCCAAACGGGTGCGCGAGTCA
              GTTGCGCAGCCATCGACGTCAGACGCGGAAGCTTCGATCAACTACGCGGACAGGTACCA
              AAACAAATGTTCTCGTCACGTGGGCATGAATCTGATGCTGTTTCCCTGCAGACAATGCGA
              GAGAATGAATCAGAATTCAAATATCTGCTTCACTCACGGACAGAAAGACTGTTTAGAGTG
              CTTTCCCGTGTCAGAATCTCAACCCGTTTCTGTCGTCAAAAAGGCGTATCAGAAACTGTGC
              TACATTCATCATATCATGGGAAAGGTGCCAGACGCTTGCACTGCCTGCGATCTGGTCAAT
              GTGGATTTGGATGACTGCATCTTTGAACAATAAATGATTTAAATCAGGTATGGCTGCCGA
              TGGTTATCTTCCAGATTGGCTCGAGGACACTCTCTCTGAAGGAATAAGACAGTGGTGGAA
              GCTCAAACCTGGCCCACCACCACCAAAGCCCGCAGAGCGGCATAAGGACGACAGCAGGG
              GTCTTGTGCTTCCTGGGTACAAGTACCTCGGACCCTTCAACGGACTCGACAAGGGAGAGC
              CGGTCAACGAGGCAGACGCCGCGGCCCTCGAGCACGACAAAGCCTACGACCGGCAGCTC
              GACAGCGGAGACAACCCGTACCTCAAGTACAACCACGCCGACGCCGAGTTCCAGGAGCG
              GCTCAAAGAAGATACGTCTTTTGGGGGCAACCTCGGGCGAGCAGTCTTCCAGGCCAAAA
              AGAGGCTTCTTGAACCTCTTGGTCTGGTTGAGGAAGCGGCTAAGACGGCTCCTGGAAAGA
              AGAGGCCTGTAGAGCACTCTCCTGTGGAGCCAGACTCCTCCTCGGGAACCGGAAAGGCG
              GGCCAGCAGCCTGCAAGAAAAAGATTGAATTTTGGTCAGACTGGAGACGCAGACTCAGT
              CCCAGACCCTCAACCAATCGGAGAACCTCCCGCAGCCCCCTCAGGTGTGGGATCTCTTAC
              AATGGCTGCAGGCGGTGGCGCACCAATGGCAGACAATAACGAGGGCGCCGACGGAGTGG
              GTAATTCCTCGGGAAATTGGCATTGCGATTCCACATGGATGGGCGACAGAGTCATCACCA
              CCAGCACCCGAACCTGGGCCCTGCCCACCTACAACAACCACCTCTACAAGCAAATCTCCA
              ACAGCACATCTGGAGGATCTTCAAATGACAACGCCTACTTCGGCTACAGCACCCCCTGGG
              GGTATTTTGACTTTAACAGATTCCACTGCCACTTTTCACCACGTGACTGGCAGCGACTCAT
              CAACAACAACTGGGGATTCCGGCCCAAGAGACTCAGCTTCAAGCTCTTCAACATCCAGGT
              CAAGGAGGTCACGCAGAATGAAGGCACCAAGACCATCGCCAATAACCTCACCAGCACCA
              TCCAGGTGTTTACGGACTCGGAGTACCAGCTGCCGTACGTTCTCGGCTCTGCCCACCAGG
              GCTGCCTGCCTCCGTTCCCGGCGGACGTGTTCATGATTCCCCAGTACGGCTACCTAACACT
              CAACAACGGTAGTCAGGCCGTGGGACGCTCCTCCTTCTACTGCCTGGAATACTTTCCTTCG
              CAGATGCTGAGAACCGGCAACAACTTCCAGTTTACTTACACCTTCGAGGACGTGCCTTTC
              CACAGCAGCTACGCCCACAGCCAGAGCTTGGACCGGCTGATGAATCCTCTGATTGACCAG
              TACCTGTACTACTTGTCTCGGACTCAAACAACAGGAGGCACGACAAATACGCAGACTCTG
              GGCTTCAGCCAAGGTGGGCCTAATACAATGGCCAATCAGGCAAAGAACTGGCTGCCAGG
              ACCCTGTTACCGCCAGCAGCGAGTATCAAAGACATCTGCGGATAACAACAACAGTGAAT
              ACTCGTGGACTGGAGCTACCAAGTACCACCTCAATGGCAGAGACTCTCTGGTGAATCCGG
              GCCCGGCCATGGCAAGCCACAAGGACGATGAAGAAAAGTTTTTTCCTCAGAGCGGGGTT
              CTCATCTTTGGGAAGCAAGGCTCAGAGAAAACAAATGTGGACATTGAAAAGGTCATGAT
              TACAGACGAAGAGGAAATCAGGACAACCAATCCCGTGGCTACGGAGCAGTATGGTTCTG
              TATCTACCAACCTCCAGCAAGGTGTACATCGATTGTTAATCAATAAACCGTTTAATTCGTT
              TCAGTTGAACTTTGGTCTCTGCGTATTTCTTTCTTATCTAGTTTCCATGGCTACGTAGATAA
              GTAGCATGGCGGGTTAATCATTAACTACAAGGAACCCCTAGTGATGGAGTTGGCCACTCC
              CTCTCTGCGCGCTCGCTCGCTCACTGAGGCCGGGCGACCAAAGGTCGCCCGACGCCCGGG
              CTTTGCCCGGGCGGCCTCAGTGAGCGAGCGAGCGCGCAGAGAGGGAGTGGCCAAGCATG
              CAATTAACTGGCCGTCGTTTTACAACGTCGTGACTGGGAAAACCCTGGCGTTACCCAACT
              TAATCGCCTTGCAGCACATCCCCCTTTCGCCAGCTGTATCAGCACACAATTGCCCATTATA
              CGCGCGTATAATGGACTATTGTGTGCTGATA

LITERATURE CITED

Berns K I, Giraud C. Biology of adeno-associated virus. Curr Top Microbiol Immunol. 1996; 218:1-23.

Chan K Y, Jang M J, Yoo B B, Greenbaum A, Ravi N, Wu W L, Sánchez-Guardado L, Lois C, Mazmanian S K, Deverman B E, Gradinaru V. Engineered AAVs for efficient noninvasive gene delivery to the central and peripheral nervous systems. Nat Neurosci. 2017 August; 20(8):1172-1179.

Heinrich J, Schultz J, Bosse M, Ziegelin G, Lanka E, Moelling K. Linear closed mini DNA generated by the prokaryotic cleaving-joining enzyme TelN is functional in mammalian cells. J Mol Med (Berl). 2002 October; 80(10):648-54.

Hordeaux J, Wang Q, Katz N, Buza E L, Bell P, Wilson J M. The Neurotropic Properties of AAV-PHP.B Are Limited to C57BL/6J Mice. Mol Ther. 2018 Mar. 7; 26(3):664-668.

Huovinen T, Brockmann E C, Akter S, Perez-Gamarra S, Ylä-Pelto J, Liu Y, Lamminmäki U. Primer extension mutagenesis powered by selective rolling circle amplification. PLoS One. 2012; 7(2):e31817.

Huovinen T, Julin M, Sanmark H, Lamminmäki U. Enhanced error-prone RCA mutagenesis by concatemer resolution. Plasmid. 2011 October; 66(1):47-51.

Hutchison C A 3rd, Smith H O, Pfannkoch C, Venter J C. Cell-free cloning using phi29 DNA polymerase. Proc Natl Acad Sci USA. 2005 Nov. 29; 102(48):17332-6.

Miyazaki J, Takaki S, Araki K, Tashiro F, Tominaga A, Takatsu K, Yamamura K. Expression vector system based on the chicken beta-actin promoter directs efficient production of interleukin-5. Gene. 1989 Jul. 15; 79(2):269-77.

Mouw M B, Pintel D J. Adeno-associated virus RNAs appear in a temporal order and their splicing is stimulated during coinfection with adenovirus. J Virol. 2000 November; 74(21):9878-88.

Niwa H, Yamamura K, Miyazaki J. Efficient selection for high-expression transfectants with a novel eukaryotic vector. Gene. 1991 Dec. 15; 108(2):193-9.

Nonnenmacher M, van Bakel H, Hajjar R J, Weber T. High capsid-genome correlation facilitates creation of AAV libraries for directed evolution. Mol Ther. 2015 April; 23(4):675-82.

Picher ÁJ, Budeus B, Wafzig O, Krüger C, García-Gómez S, Martínez-Jiménez M I, Díaz-Talavera A, Weber D, Blanco L, Schneider A. TruePrime is a novel method for whole-genome amplification from single cells based on TthPrimPol. Nat Commun. 2016 Nov. 29; 7:13296.

Powell S K, Rivera-Soto R, Gray S J. Viral expression cassette elements to enhance transgene target specificity and expression in gene therapy. Discov Med. 2015 January; 19(102):49-57.

Rybchin V N, Svarchevsky A N. The plasmid prophage N15: a linear DNA with covalently closed ends. Mol Microbiol. 1999 September; 33(5):895-903.

Zolotukhin S, Byrne B J, Mason E, Zolotukhin I, Potter M, Chesnut K, Summerford C, Samulski R J, Muzyczka N. Recombinant adeno-associated virus purification using novel methods improves infectious titer and yield. Gene Ther. 1999 June; 6(6):973-85.

Claims

1. A method for generating a variant AAV capsid polypeptides, wherein relative to a parental AAV capsid polypeptide said variant AAV capsid polypeptides exhibit at least one of improved transduction or increased cell or tissue specificity, said method comprising: a) generating a library of variant AAV capsid polypeptides, wherein said library comprises i) a plurality of capsid polypeptides having a region of randomized sequence of 2, 3, 4, 5, 6, 7, 8, or 9 consecutive amino acids, orii) a plurality of capsid polypeptides from more than one parental AAV capsid polypeptide;b) generating an AAV vector library by cloning the capsid polypeptides of libraries (i) or (ii) into AAV vectors, wherein said AAV vectors comprise a first promoter and a second promoter, wherein said second promoter drives capsid mRNA expression in the absence of helper virus co-infection.

2. The method of claim 1, wherein the first promoter is AAV2 P40 and the second promoter is a ubiquitous promoter.

3. The method of claim 1, wherein the first promoter is AAV2 P40 and the second promoter is a cell-type-specific promoter.

4. The method of claim 2 or claim 3, wherein the promoter is selected from any of those listed in Table 3.

5. The method of claim 4, wherein the ubiquitous or cell-specific promoter allows the expression of RNA encoding the capsid polypeptides.

6. The method of claim 5, further comprising the recovery of said RNA encoding the capsid polypeptides and determining the sequence of said capsid polypeptides.

7. The method of claim 6, wherein the capsid polypeptides recovered exhibit increased target cell transduction or target cell specificity (tropism) as compared to a parental capsid polypeptide.

8. The method of claim 7, wherein the target cell is a neuronal cell, a neural stem cell, an astrocyte, an oligodendrocyte, a microglia cell, a retinal cell, a tumor cell, a hematopoietic stem cell, an insulin producing beta cell, a lung epithelium cell, an endothelial cell, a liver cell, a skeletal muscle cell, a muscle stem cell, a muscle satellite cell, or a cardiac muscle cell.

9. The method of claim 1, wherein said AAV vectors comprise a first promoter and a second promoter, wherein said second promoter is located at the downstream of the capsid gene and drives its anti-sense RNA expression in the absence of helper virus co-infection.

10. The method of claim 9, wherein the first promoter is AAV2 P40 and the second promoter is a ubiquitous promoter.

11. The method of claim 9, wherein the first promoter is AAV2 P40 and the second promoter is a cell-specific promoter.

12. The method of claim 10 or claim 11, wherein the ubiquitous or cell-specific promoter allows the expression of gene encoding the capsid polypeptide of variant AAV in an anti-sense direction, resulting in the anti-sense RNA.

13. The method of claim 12, wherein said method further comprises the recovery of said anti-sense RNA that can be converted to RNA encoding said variant AAV capsid polypeptide that is used to determining the sequence of said variant AAV capsid polypeptides.

14. The method of claim 13, wherein said variant AAV capsid polypeptide exhibits increased target cell transduction or target cell specificity (tropism) as compared to a parental capsid polypeptide.

15. The method of claim 14, wherein the target cell is a neuronal cell, a neural stem cell, an astrocyte, a oligodendrocyte, a microglia cell, a retinal cell, a tumor cell, a hematopoietic stem cell, an insulin producing beta cell, a lung epithelium cell, an endothelial cell, a liver cell, a skeletal muscle cell, a muscle stem cell, a muscle satellite cell, or a cardiac muscle cell.