COMPOUNDS AND METHODS FOR MODULATING PROGRANULIN EXPRESSION

Abstract
Provided are oligomeric compounds, methods, and pharmaceutical compositions for modulating expression of progranulin RNA, or modulating expression of progranulin protein in a cell or subject. Such compounds, methods, and pharmaceutical compositions are useful to ameliorate at least one symptom or hallmark of a neurological disease or disorder. Such neurological diseases or disorders include those associated with insufficient expression of progranulin, including frontotemporal dementia, frontotemporal lobar degeneration, Alzheimer's disease, amyotrophic lateral sclerosis and neuronal ceroid lipofuscinosis. Such symptoms or hallmarks include deterioration in behavior and personality, language impairment, disturbances or alterations in muscle or motor functions, memory loss, cognitive dysfunction, tremor, seizures, or dizziness.
Description
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 BIOL0452WOSEQ.xml, created on Nov. 17, 2022, which is 839 KB in size. The information in the electronic format of the sequence listing is incorporated herein by reference in its entirety.


FIELD

Provided are oligomeric compounds, methods, and pharmaceutical compositions for modulating expression of progranulin RNA, or modulating expression of progranulin protein in a cell or subject. Such compounds, methods, and pharmaceutical compositions are useful to ameliorate at least one symptom or hallmark of a neurological disease or disorder. Such neurological diseases or disorders include those associated with insufficient expression of progranulin, including frontotemporal dementia, frontotemporal lobar degeneration, Alzheimer's disease, amyotrophic lateral sclerosis, and neuronal ceroid lipofuscinosis. Such symptoms or hallmarks include deterioration in behavior and personality, language impairment, disturbances or alterations in muscle or motor functions, memory loss, cognitive dysfunction, tremor, seizures, or dizziness.


BACKGROUND

The human gene GRN encodes human progranulin protein. Mutations in GRN lead to neurological diseases and disorders, including frontotemporal dementia (FTD), frontotemporal lobar degeneration (FTLD) neuronal ceroid lipofuscinosis (NCL), lysosomal storage diseases, Alzheimer's disease (AD), and amyotrophic lateral sclerosis (ALS). These diseases are associated with GRN and/or progranulin haploinsufficiency. FTD refers to a group of disorders caused by progressive nerve cell loss in the brain's frontal lobes or temporal lobes. Nerve cell damage caused by FTD leads to loss of function in the frontal lobes or temporal lobes, and is associated with deterioration in behavior and personality, language impairment, disturbances or alterations in muscle or motor functions, memory loss, cognitive dysfunction, tremor, seizures, or dizziness. FTD is associated with TDP-43 proteinopathies.


Currently there remains a need for therapies to treat neurological diseases and disorders associated with an insufficient amount of progranulin. It is therefore an object herein to provide oligomeric compounds, methods, and pharmaceutical compositions for the treatment of such diseases or disorders.





BRIEF DESCRIPTION OF THE DRAWINGS


FIG. 1A, FIG. 1B, FIG. 1C, FIG. 1D, and FIG. 1E show human progranulin levels in H4 cell lysates or conditioned media following treatment with modified oligonucleotides. FIG. 1A shows the results of an ELISA assay in cell lysates, following treatment with (from left to right): control scrambled ASO, water, Compounds 1212637, 1212638, 1212639, 1212641, 1212642, 1212646, 1212647, 1212648, 1212660, 1212661, 1212664, 1212665, 1212667, 1212671, 1212672, 1212673, 1212674, 1212675, 1212676, 1212684. FIG. 1B is a Western blot of H4 cell lysates, following treatment of the cells with (from left to right) water, control scrambled ASO, Compounds 1212637, 1212638, 1212639, 1212640, 1212641, 1212642, 1212646, 1212647, 1212672, 1212674 1212675, 1212684. FIG. 1C is a Western blot of H4 cell lysates, following treatment of the cells with (from left to right) water, control scrambled ASO, Compounds 1212646, 1212647, 1212660, 1212661, 1212664, 1212665, 1212667, 1212672, 1212674. FIG. 1D is a Western blot of H4 cell lysates following treatment of the cells with (from left to right) water, control scrambled ASO, Compound 1212676. FIG. 1E is a Western blot of H4 cell lysates (top panel) or conditioned media (bottom panel), following treatment of the cells with (from left to right) water, control scrambled ASO, Compounds 1212637, 1212638, 1212639, 1212640, 1212641, 1212642.



FIG. 2A, FIG. 2B, FIG. 2C, FIG. 2D, FIG. 2E, and FIG. 2F are dose response curves of progranulin levels, presented as percent of GRN level in untreated cells as measured by ELISA, following treatment of H4 cells with either control scrambled ASO or Compounds 1212637, 1212638, 1212641, 1212646, 1212647, 1212672, respectively.



FIG. 3 is a Western blot of iPSC-derived neuron cell lysates, following treatment with control scrambled ASO or Compounds 1212638, 1212641, 1212646, 1212647, and 1212672, at the indicated concentrations (μM).



FIG. 4A and FIG. 4B show the results of ELISA assays of progranulin levels (ng/mg protein) in the cortex of male (FIG. 4A) and female (FIG. 4B) following administration of modified oligonucleotides. GRN levels in treated transgenic mice homozygous for human GRN are compared to GRN levels in saline treated transgenic GRN mice and to GRN levels in untreated non-transgenic mice. FIG. 4A (left to right): untreated non-transgenic mice, transgenic GRN mice: saline, control scrambled ASO, Compounds 1557990, 1557993, 1212647, 1557987. FIG. 4B (left to right) transgenic GRN mice: saline, control scrambled ASO, Compounds 1212640, 1557993, 1557994, 1212647, 1557987. FIG. 4C is a Western blot of progranulin in the cortex, thalamus, or hippocampus of untreated non-transgenic mice, or in transgenic GRN mice treated with saline control or Compound 1557993.





SUMMARY

Provided herein are compounds, methods, and pharmaceutical compositions for modulating the amount of progranulin RNA and/or modulating the amount of progranulin protein in a cell or a subject. In certain embodiments, compounds useful for modulating the amount of progranulin RNA and/or progranulin protein are oligomeric compounds. In certain embodiments, oligomeric compounds increase the amount of progranulin RNA in a cell. In certain embodiments, oligomeric compounds increase the amount of progranulin protein in a cell. In certain embodiments, the oligomeric compound comprises a modified oligonucleotide. In certain embodiments, the subject has a neurological disease or disorder. In certain embodiments, the subject has a neurological disease or disorder associated with an insufficient amount of progranulin protein. In certain embodiments, the subject has FTD. In certain embodiments the subject has FTLD. In certain embodiments, the subject has NCL. In certain embodiments, the subject has a TDP-43 proteinopathy. In certain embodiments, the subject has a lysosomal storage disorder. In some embodiments, the subject has Alzheimer's disease (AD). In certain embodiments, the subject has amyotrophic lateral sclerosis (ALS).


Also provided are methods useful for ameliorating at least one symptom or hallmark of a neurological disease or disorder. In certain embodiments, the neurological disease or disorder is associated with an insufficient amount of progranulin protein. In certain embodiments, the neurological disease or disorder is FTD. In certain embodiments, the neurological disease or disorder is FTLD. In certain embodiments, the neurological disease or disorder is NCL. In certain embodiments, the neurological disease or disorder is a TDP-43 proteinopathy. In certain embodiments, the disease or disorder is a lysosomal storage disorder. In certain embodiments, the disease or disorder is ALS. In certain embodiments, the disease or disorder is AD. In certain embodiments, symptoms or hallmarks include deterioration in behavior and personality, language impairment, disturbances or alterations in muscle or motor functions, memory loss, cognitive dysfunction, tremor, seizures, or dizziness.


DETAILED DESCRIPTION

It is to be understood that both the foregoing general description and the following detailed description are exemplary and explanatory only and are not restrictive. Herein, the use of the singular includes the plural unless specifically stated otherwise. As used herein, the use of “or” means “and/or” unless stated otherwise. Furthermore, the use of the term “including” as well as other forms, such as “includes” and “included”, is not limiting. Also, terms such as “element” or “component” encompass both elements and components comprising one unit and elements and components that comprise more than one subunit, unless specifically stated otherwise.


The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described. All documents, or portions of documents, cited in this application, including, but not limited to, patents, patent applications, articles, books, and treatises, and GenBank, ENSEMBL, and NCBI reference sequence records are hereby expressly incorporated-by-reference for the portions of the document discussed herein, as well as in their entirety.


Definitions

Unless specific definitions are provided, the nomenclature used in connection with, and the procedures and techniques of, analytical chemistry, synthetic organic chemistry, and medicinal and pharmaceutical chemistry described herein are those well-known and commonly used in the art. Where permitted, all patents, applications, published applications and other publications and other data referred to throughout in the disclosure are incorporated by reference herein in their entirety.


Unless otherwise indicated, the following terms have the following meanings:


As used herein, “2′-deoxyribonucleoside” means a nucleoside comprising a 2′-H(H) deoxyribosyl sugar moiety. In certain embodiments, a 2′-deoxyribonucleoside is a 2′-β-D deoxyribonucleoside and comprises a 2′-β-D-deoxyribosyl sugar moiety, which has the β-D configuration as found in naturally occurring deoxyribonucleic acids (DNA). In certain embodiments, a 2′-deoxyribonucleoside may comprise a modified nucleobase or may comprise an RNA nucleobase (uracil).


As used herein, “2′-MOE” means a 2′-O(CH2)2OCH3 group in place of the 2′—OH group of a ribosyl sugar moiety. A “2′-MOE sugar moiety” or a “2′-O-methoxyethyl sugar moiety” or a “2′-MOE modified sugar moiety” means a sugar moiety with a 2′-O(CH2)2OCH3 group in place of the 2′—OH group of a ribosyl sugar moiety. Unless otherwise indicated, a 2′-MOE sugar moiety is in the β-D configuration. “MOE” means O-methoxyethyl.


As used herein, “2′-MOE nucleoside” means a nucleoside comprising a 2′-MOE sugar moiety.


As used herein, “2′-NMA” means a —O—CH2—C(═O)—NH—CH3 group in place of the 2′—OH group of a ribosyl sugar moiety. A “2′-NMA sugar moiety” or a “2′-NMA modified sugar moiety” is a sugar moiety with a 2′-O—CH2—C(═O)—NH—CH3 group in place of the 2′—OH group of a ribosyl sugar moiety. Unless otherwise indicated, a 2′-NMA sugar moiety is in the β-D configuration. “NMA” means O—N-methyl acetamide.


As used herein, “2′-NMA nucleoside” means a nucleoside comprising a 2′-NMA sugar moiety.


As used herein, “2′-OMe” means a 2′-OCH3 group in place of the 2′—OH group of a ribosyl sugar moiety. A “2′-OMe sugar moiety” or a “2′-OMe modified sugar moiety” is a sugar moiety with a 2′-OCH3 group in place of the 2′—OH group of a ribosyl sugar moiety. Unless otherwise indicated, a 2′-OMe sugar moiety is in the β-D configuration. “OMe” means O-methyl.


As used herein, “2′-OMe nucleoside” means a nucleoside comprising a 2′-OMe sugar moiety.


As used herein, “2′-substituted nucleoside” means a nucleoside comprising a 2′-substituted sugar moiety. As used herein, “2′-substituted” in reference to a sugar moiety means a sugar moiety comprising at least one 2′-substituent group other than H or OH.


As used herein, “5-methyl cytosine” means a cytosine modified with a methyl group attached to the 5 position.


A 5-methyl cytosine is a modified nucleobase.


As used herein, “administering” means providing a pharmaceutical agent to a subject.


As used herein, “ameliorate” in reference to a treatment means improvement in at least one symptom or hallmark relative to the same symptom or hallmark in the absence of the treatment. In certain embodiments, amelioration is the reduction in the severity or frequency of a symptom or hallmark, or the delayed onset or slowing of progression in the severity or frequency of a symptom or hallmark. In certain embodiments, the symptom or hallmark is deterioration in behavior and personality, language impairment, disturbances or alterations in muscle or motor functions, memory loss, cognitive dysfunction, tremor, seizures, or dizziness.


As used herein, “antisense activity” means any detectable and/or measurable change attributable to the hybridization of an antisense compound to its target nucleic acid. In certain embodiments, antisense activity is an increase in the amount or expression of a target nucleic acid or protein encoded by such target nucleic acid compared to amount or expression of the target nucleic acid or target protein in the absence of the antisense compound.


As used herein, “antisense agent” means an antisense compound and optionally one or more additional features, such as a sense compound.


As used herein, “antisense compound” means an antisense oligonucleotide and optionally one or more additional features, such as a conjugate group. An “antisense compound” may also be an oligomeric duplex capable of achieving at least one antisense activity.


As used herein, “antisense oligonucleotide” means an oligonucleotide, including the oligonucleotide portion of an antisense compound, that is capable of hybridizing to a target nucleic acid and is capable of at least one antisense activity.


As used herein, “bicyclic nucleoside” or “BNA” means a nucleoside comprising a bicyclic sugar moiety.


As used herein, “bicyclic sugar” or “bicyclic sugar moiety” means a modified sugar moiety comprising two rings, wherein the second ring is formed via a bridge connecting two of the atoms in the first ring thereby forming a bicyclic structure. In certain embodiments, the first ring of the bicyclic sugar moiety is a furanosyl moiety. In certain embodiments, the furanosyl moiety is a ribosyl moiety. In certain embodiments, the bicyclic sugar moiety does not comprise a furanosyl moiety.


As used herein, “cerebrospinal fluid” or “CSF” means the fluid filling the space around the brain and spinal cord. “Artificial cerebrospinal fluid” or “aCSF” means a prepared or manufactured fluid that has certain properties (e.g., osmolarity, pH, and/or electrolytes) of cerebrospinal fluid and is biocompatible with CSF.


As used herein, “cEt” means a 4′ to 2′ bridge in place of the 2′OH-group of a ribosyl sugar moiety, wherein the bridge has the formula of 4′-CH(CH3)—O-2′, and wherein the methyl group of the bridge is in the S configuration. A “cEt sugar moiety” is a bicyclic sugar moiety with a 4′ to 2′ bridge in place of the 2′OH-group of a ribosyl sugar moiety, wherein the bridge has the formula of 4′-CH(CH3)—O-2′, and wherein the methyl group of the bridge is in the S configuration. “cEt” means constrained ethyl.


As used herein, “cEt nucleoside” means a nucleoside comprising a cEt sugar moiety.


As used herein, “chirally enriched” in reference to a population means a plurality of molecules of identical molecular formula, wherein the number or percentage of molecules within the population that contain a particular stereochemical configuration at a particular chiral center is greater than the number or percentage of molecules expected to contain the same particular stereochemical configuration at the same particular chiral center within the population if the particular chiral center were stereorandom as defined herein. Chirally enriched populations of molecules having multiple chiral centers within each molecule may contain one or more stereorandom chiral centers. In certain embodiments, the molecules are modified oligonucleotides. In certain embodiments, the molecules are modified oligonucleotides. In certain embodiments, the molecules are oligomeric compounds comprising modified oligonucleotides. In certain embodiments, the chiral center is at the phosphorous atom of a phosphorothioate internucleoside linkage. As used herein, “chirally controlled” in reference to an internucleoside linkage means chirality at that linkage is enriched for a particular stereochemical configuration.


As used herein, “complementary” in reference to an oligonucleotide means that at least 70% of the nucleobases of the oligonucleotide or one or more portions thereof and the nucleobases of another nucleic acid or one or more portions thereof are capable of hydrogen bonding with one another when the nucleobase sequence of the oligonucleotide and the other nucleic acid are aligned in opposing directions. Complementary nucleobases means nucleobases that are capable of forming hydrogen bonds with one another. Complementary nucleobase pairs include adenine (A) and thymine (T), adenine (A) and uracil (U), cytosine (C) and guanine (G), 5-methyl cytosine (mC) and guanine (G), and hypoxanthine (of inosine (I)) and cytosine (C), 5-methyl cytosine (mC), uracil (U), or adenine (A). Complementary oligonucleotides and/or target nucleic acids need not have nucleobase complementarity at each nucleoside. Rather, some mismatches are tolerated. As used herein, “fully complementary” or “100% complementary” in reference to an oligonucleotide, or a portion thereof, means that the oligonucleotide, or portion thereof, is complementary to another oligonucleotide or target nucleic acid at each nucleobase of the shorter of the two oligonucleotides, or at each nucleoside if the oligonucleotides are the same length.


As used herein, “conjugate group” means a group of atoms that is directly or indirectly attached to an oligonucleotide. Conjugate groups include a conjugate moiety and a conjugate linker that attaches the conjugate moiety to the oligonucleotide.


As used herein, “conjugate linker” means a single bond or a group of atoms comprising at least one bond that connects a conjugate moiety to an oligonucleotide.


As used herein, “conjugate moiety” means a covalently bound group of atoms that modifies one or more pharmacological properties of a molecule compared to the identical molecule lacking the conjugate moiety, including but not limited to pharmacodynamics, pharmacokinetics, stability, binding, absorption, tissue distribution, cellular distribution, cellular uptake, charge and clearance.


As used herein, “contiguous” in the context of an oligonucleotide refers to nucleosides, nucleobases, sugar moieties, or internucleoside linkages that are immediately adjacent to each other. For example, “contiguous nucleobases” means nucleobases that are immediately adjacent to each other in a sequence.


As used herein, “hotspot region” is a range of nucleobases on a target nucleic acid that is amenable to an oligomeric compound-mediated increase in the amount or activity of the target nucleic acid.


As used herein, “hybridization” means the pairing or annealing of complementary oligonucleotides and/or nucleic acids. While not limited to a particular mechanism, the most common mechanism of hybridization involves hydrogen bonding, which may be Watson-Crick, Hoogsteen or reversed Hoogsteen hydrogen bonding, between complementary nucleobases. In certain embodiments, complementary nucleic acid molecules include, but are not limited to, an antisense compound and a nucleic acid target. In certain embodiments, complementary nucleic acid molecules include, but are not limited to, an oligonucleotide and a nucleic acid target.


As used herein, “inosine” (I) or 9-β-D-Ribosylhypoxanthine means a nucleoside that contains a hypoxanthine nucleobase.


As used herein, “internucleoside linkage” is the covalent linkage between adjacent nucleosides in an oligonucleotide. As used herein, “modified internucleoside linkage” means any internucleoside linkage other than a phosphodiester internucleoside linkage. “Phosphorothioate internucleoside linkage” or “PS internucleoside linkage” is a modified internucleoside linkage in which one of the non-bridging oxygen atoms of a phosphodiester internucleoside linkage is replaced with a sulfur atom.


As used herein, “linked nucleosides” are nucleosides that are connected in a contiguous sequence (i.e., no additional nucleosides are presented between those that are linked).


As used herein, “linker-nucleoside” means a nucleoside that links, either directly or indirectly, an oligonucleotide to a conjugate moiety. Linker-nucleosides are located within the conjugate linker of an oligomeric compound. Linker-nucleosides are not considered part of the oligonucleotide portion of an oligomeric compound even if they are contiguous with the oligonucleotide.


As used herein, “mismatch” or “non-complementary” means a nucleobase of a first oligonucleotide that is not complementary with the corresponding nucleobase of a second oligonucleotide or target nucleic acid when the first and second oligonucleotide are aligned.


As used herein, “modified nucleoside” means a nucleoside comprising a modified nucleobase and/or a modified sugar moiety.


As used herein, “motif” means the pattern of unmodified and/or modified sugar moieties, nucleobases, and/or internucleoside linkages, in an oligonucleotide.


As used herein, “non-bicyclic modified sugar moiety” means a modified sugar moiety that comprises a modification, such as a substituent, that does not form a bridge between two atoms of the sugar to form a second ring.


As used herein, “nucleobase” means an unmodified nucleobase or a modified nucleobase. A nucleobase is a heterocyclic moiety. As used herein an “unmodified nucleobase” is adenine (A), thymine (T), cytosine (C), uracil (U), or guanine (G). As used herein, a “modified nucleobase” is a group of atoms other than unmodified A, T, C, U, or G capable of pairing with at least one other nucleobase. A “5-methyl cytosine” is a modified nucleobase. A universal base is a modified nucleobase that can pair with any one of the five unmodified nucleobases. As used herein, “nucleobase sequence” means the order of contiguous nucleobases in a target nucleic acid or oligonucleotide independent of any sugar or internucleoside linkage modification.


As used herein, “nucleoside” means a compound or fragment of a compound comprising a nucleobase and a sugar moiety. The nucleobase and sugar moiety are each, independently, unmodified or modified.


As used herein, “oligomeric compound” means an oligonucleotide and optionally one or more additional features, such as a conjugate group or terminal group. An oligomeric compound may be paired with a second oligomeric compound that is complementary to the first oligomeric compound or may be unpaired. A “singled-stranded oligomeric compound” is an unpaired oligomeric compound. The term “oligomeric duplex” means a duplex formed by two oligomeric compounds having complementary nucleobase sequences. Each oligomeric compound of an oligomeric duplex may be referred to as a “duplexed oligomeric compound.” As used herein, “oligonucleotide” means a strand of linked nucleosides connected via internucleoside linkages, wherein each nucleoside and internucleoside linkage may be modified or unmodified. Unless otherwise indicated, oligonucleotides consist of 8-50 linked nucleosides. As used herein, “modified oligonucleotide” means an oligonucleotide, wherein at least one nucleoside or internucleoside linkage is modified. As used herein, “unmodified oligonucleotide” means an oligonucleotide that does not comprise any nucleoside modifications or internucleoside modifications.


As used herein, “pharmaceutical composition” means a mixture of substances suitable for administering to a subject. For example, a pharmaceutical composition may comprise an oligomeric compound and a sterile aqueous solution.


As used herein, “pharmaceutically acceptable carrier or diluent” means any substance suitable for use in administering to an animal. Certain such carriers enable pharmaceutical compositions to be formulated as, for example, tablets, pills, dragees, capsules, liquids, gels, syrups, slurries, suspension, and lozenges for the oral ingestion by a subject. In certain embodiments, a pharmaceutically acceptable diluent is sterile water, sterile saline, sterile buffer solution, or sterile artificial cerebrospinal fluid.


As used herein, “pharmaceutically acceptable salts” means physiologically and pharmaceutically acceptable salts of compounds. Pharmaceutically acceptable salts retain the desired biological activity of the parent compound and do not impart undesired toxicological effects thereto.


As used herein “pharmaceutical composition” means a mixture of substances suitable for administering to a subject. For example, a pharmaceutical composition may comprise an oligomeric compound and a sterile aqueous solution. In certain embodiments, a pharmaceutical composition shows activity in free uptake assay in certain cell lines.


As used herein, “RNA” means an RNA transcript and includes pre-mRNA and mature mRNA unless otherwise specified.


As used herein “progranulin RNA” means an RNA transcript encoded by the progranulin gene, GRN, and includes pre-mRNA and mRNA unless otherwise specified. Progranulin nucleic acid and GRN nucleic acid are used interchangeably and refer to nucleic acid that encodes the progranulin protein. As used herein, “progranulin nucleic acid” and “GRN nucleic acid” are interchangeable and include progranulin DNA and progranulin RNA unless otherwise specified.


As used herein, “steric-blocking agent” means an antisense agent that acts, at least in part, due to directly binding to a target nucleic acid, thus blocking the interaction of the target nucleic acid with other nucleic acids or proteins.


As used herein, “stereorandom” or “stereorandom chiral center” in the context of a population of molecules of identical molecular formula means a chiral center that is not controlled during synthesis, or enriched following synthesis, for a particular absolute stereochemical configuration. The stereochemical configuration of a chiral center is random when it is the result of a synthetic method that is not designed to control the stereochemical configuration. For example, in a population of molecules comprising a stereorandom chiral center, the number of molecules having the (S) configuration of the stereorandom chiral center may be but is not necessarily the same as the number of molecules having the (R) configuration of the stereorandom chiral center (“racemic”). In certain embodiments, the stereorandom chiral center is not racemic because one absolute configuration predominates following synthesis, e.g., due to the action of non-chiral reagents near the enriched stereochemistry of an adjacent sugar moiety. In certain embodiments, a stereorandom chiral center is a stereorandom phosphorothioate internucleoside linkage.


As used herein, “subject” means a human or non-human animal.


As used herein, “sugar moiety” means an unmodified sugar moiety or a modified sugar moiety. As used herein, “unmodified sugar moiety” means a 2′-OH(H) β-D ribosyl sugar moiety, as found in RNA (an “unmodified RNA sugar moiety”), or a 2′-H(H) β-D deoxyribosyl sugar moiety, as found in DNA (an “unmodified DNA sugar moiety”).


Unmodified sugar moieties have one hydrogen at each of the 1′, 3′, and 4′ positions, an oxygen at the 3′ position, and two hydrogens at the 5′ position. As used herein, “modified sugar moiety” or “modified sugar” means a modified furanosyl sugar moiety or a sugar surrogate.


As used herein, ““sugar surrogate”” means a modified sugar moiety having other than a furanosyl moiety that can link a nucleobase to another group, such as an internucleoside linkage, conjugate group, or terminal group in an oligonucleotide, but which is not a furanosyl sugar moiety or a bicyclic sugar moiety. Modified nucleosides comprising sugar surrogates can be incorporated into one or more positions within an oligonucleotide and such oligonucleotides are capable of hybridizing to complementary oligomeric compounds or target nucleic acids. Examples of sugar surrogates include GNA (glycol nucleic acid), FHNA (fluoro hexitol nucleic acid), morpholino, and other structures described herein and known in the art. As used herein, “standard in vitro assay” means the assay described in Example 1 and reasonable variations thereof.


As used herein, “symptom or hallmark” means any physical feature or test result that indicates the existence or extent of a disease or disorder. In certain embodiments, a symptom is apparent to a subject or to a medical professional examining or testing the subject. In certain embodiments, a hallmark is apparent upon invasive diagnostic testing, including, but not limited to, post-mortem tests. In certain embodiments, a hallmark is apparent on a brain MRI scan.


As used herein, “target nucleic acid” and “target RNA” mean a nucleic acid that an antisense oligomeric or an oligomeric compound is designed to affect. Target RNA means an RNA transcript and includes pre-mRNA and mRNA unless otherwise specified.


As used herein, “target region” means a portion of a target nucleic acid to which an oligomeric compound is designed to hybridize.


As used herein, “terminal group” means a chemical group or group of atoms that is covalently linked to a terminus of an oligonucleotide.


As used herein, “therapeutically effective amount” means an amount of a pharmaceutical agent that provides a therapeutic benefit to a subject. For example, a therapeutically effective amount improves a symptom or hallmark of a disease or disorder.


As used herein, “treating” means improving a subject's disease or disorder by administering an oligomeric compound described herein. In certain embodiments, treating a subject improves a symptom or hallmark relative to the same symptom in the absence of the treatment. In certain embodiments, treatment reduces in the severity or frequency of a symptom or hallmark, or delays the onset of a symptom or hallmark, slows the progression of a symptom or hallmark, or slows the severity or frequency of a symptom or hallmark.


CERTAIN EMBODIMENTS

The present disclosure provides the following non-limiting numbered embodiments:


Embodiment 1. An oligomeric compound comprising a modified oligonucleotide consisting of 12 to 50 linked nucleosides, wherein the nucleobase sequence of the modified oligonucleotide is at least 80% complementary to an equal length portion of a progranulin nucleic acid, and wherein the modified oligonucleotide has at least one modification selected from a modified sugar moiety and a modified internucleoside linkage.


Embodiment 2. The oligomeric compound of embodiment 1, wherein the progranulin nucleic acid has the nucleobase sequence of SEQ ID NO: 1 or SEQ ID NO: 2.


Embodiment 3. An oligomeric compound comprising a modified oligonucleotide consisting of 18, 19, or 20 linked nucleosides, wherein the nucleobase sequence of the modified oligonucleotide comprises at least 16, at least 17, or 18 contiguous nucleobases of any of the nucleobase sequences of SEQ ID NOs:12-854, and wherein the modified oligonucleotide comprises at least one modification selected from a modified sugar moiety and a modified internucleoside linkage.


Embodiment 4. The oligomeric compound of any of embodiments 1-3, wherein the nucleobase sequence of the modified oligonucleotide comprises at least 16, at least 17, or 18 contiguous nucleobases of any of the nucleobase sequences of SEQ ID NOs: 34, 268, 345, 424, 501, 579, 657, 735, 800, 801, 806, 812, 813, 816, 821, 828, 844, and 847.


Embodiment 5. An oligomeric compound comprising a modified oligonucleotide consisting of 18 linked nucleosides, wherein the nucleobase sequence of the modified oligonucleotide consists of the nucleobase sequence of any of SEQ ID NOs: 12-854, and wherein the modified oligonucleotide comprises at least one modification selected from a modified sugar moiety and a modified internucleoside linkage.


Embodiment 6. The oligomeric compound of embodiment 5, wherein the nucleobase sequence of the modified oligonucleotide consists of 18 contiguous nucleobases of any of the nucleobase sequences of SEQ ID NOs: 34, 268, 345, 424, 501, 579, 657, 735, 800, 801, 806, 812, 813, 816, 821, 828, 844, and 847.


Embodiment 7. An oligomeric compound comprising a modified oligonucleotide consisting of 12 to 50 linked nucleosides, wherein the nucleobase sequence of the modified oligonucleotide comprises at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, or 18 contiguous nucleobases complementary to an equal length portion within nucleobases 8497-8552 of SEQ ID NO: 1.


Embodiment 8. The oligomeric compound of embodiment 7, wherein the nucleobase sequence of the modified oligonucleotide consists of the nucleobase sequence of any of SEQ ID NOs: 34, 268, 345, 424, 501, 579, 657, 735, 800, 801, 806, 812, 813, 816, 821, 828, 844, and 847.


Embodiment 9. The oligomeric compound of any of embodiments 1-8, wherein the nucleobase sequence of the modified oligonucleotide is at least 85%, at least 90%, at least 95%, or is 100% complementary to an equal length portion within the nucleobase sequence of any of SEQ ID NO: 1 or SEQ ID NO: 2 when measured across the entire nucleobase sequence of the modified oligonucleotide.


Embodiment 10. The oligomeric compound of any of embodiments 1-9, wherein at least one nucleoside of the modified oligonucleotide comprises a modified sugar moiety.


Embodiment 11. The oligomeric compound of embodiment 10, wherein the modified sugar moiety comprises a bicyclic sugar moiety.


Embodiment 12. The oligomeric compound of embodiment 11, wherein the bicyclic sugar moiety comprises a 4′-2′ bridge selected from —CH2—O— and —CH(CH3)—O—.


Embodiment 13. The oligomeric compound of any of embodiments 10-12, wherein the modified sugar moiety comprises a non-bicyclic modified sugar moiety.


Embodiment 14. The oligomeric compound of embodiment 13, wherein the non-bicyclic modified sugar moiety is a 2′-MOE modified sugar moiety, a 2′-NMA modified sugar moiety, a 2′-OMe modified sugar moiety, or a 2′-F modified sugar moiety.


Embodiment 15. The oligomeric compound of embodiment 10, wherein the modified sugar moiety is a sugar surrogate.


Embodiment 16. The oligomeric compound of embodiment 15, wherein the sugar surrogate is a morpholino, modified morpholino, PNA, THP, or F-HNA.


Embodiment 17. The oligomeric compound of embodiment 10, wherein each nucleoside of the modified oligonucleotide comprises a modified sugar moiety.


Embodiment 18. The oligomeric compound of embodiment 17, wherein each modified sugar moiety is a 2′-MOE modified sugar moiety, a 2′-NMA modified sugar moiety, a 2′-OMe modified sugar moiety, or a 2′-F modified sugar moiety.


Embodiment 19. The oligomeric compound of embodiment 17, wherein each modified sugar moiety is a 2′-MOE modified sugar moiety.


Embodiment 20. The oligomeric compound of embodiment 17, wherein each modified sugar moiety is a sugar surrogate.


Embodiment 21. The oligomeric compound of embodiment 20, wherein each modified sugar moiety is a morpholino, modified morpholino, PNA, THP, or F-HNA.


Embodiment 22. The oligomeric compound of any of embodiments 1-21, wherein the modified oligonucleotide comprises at least one modified internucleoside linkage.


Embodiment 23. The oligomeric compound of embodiment 22, wherein the at least one modified internucleoside linkage is a phosphorothioate internucleoside linkage.


Embodiment 24. The oligomeric compound of embodiment 22, wherein each internucleoside linkage of the modified oligonucleotide is a modified internucleoside linkage.


Embodiment 25. The oligomeric compound of embodiment 22, wherein each internucleoside linkage is a phosphorothioate internucleoside linkage.


Embodiment 26. The oligomeric compound of any of embodiments 1-23, wherein at least one internucleoside linkage of the modified oligonucleotide is a phosphodiester internucleoside linkage.


Embodiment 27. The oligomeric compound of any of embodiments 22-23 or 26, wherein each internucleoside linkage of the modified oligonucleotide is independently selected from a phosphodiester internucleoside linkage and a phosphorothioate internucleoside linkage.


Embodiment 28. The oligomeric compound of any of embodiments 22-24 or 26-27, wherein at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, or at least 17 internucleoside linkages of the modified oligonucleotide are phosphorothioate internucleoside linkages.


Embodiment 29. The oligomeric compound of any of embodiments 1-23, wherein the modified oligonucleotide consists of 18 linked nucleosides and has an internucleoside linkage motif selected from sssssssssssssssss and sosssssssosssssss.


Embodiment 30. The oligomeric compound of embodiment 19, wherein the modified oligonucleotide consists of 18 linked nucleosides, wherein the internucleotide linkage motif of the modified oligonucleotide is sssssssssssssssss.


Embodiment 31. The oligomeric compound of embodiment 19, wherein the modified oligonucleotide consists of 18 linked nucleosides, wherein the internucleotide linkage motif of the modified oligonucleotide is sosssssssosssssss.


Embodiment 32. The oligomeric compound of any of embodiments 1-31, wherein the modified oligonucleotide comprises at least one modified nucleobase.


Embodiment 33. The oligomeric compound of embodiment 32, wherein the modified nucleobase is a 5-methyl cytosine or hypoxanthine.


Embodiment 34. The oligomeric compound of embodiment 33, wherein each cytosine is a 5-methyl cytosine.


Embodiment 35. An oligomeric compound comprising a modified oligonucleotide according to the following chemical notation 5′ to 3′):











(SEQ ID NO: 855)



GesTeomCesmCesAesGesGesGesAesGeoAesAesTesTesTesGesGesTe;







(SEQ ID NO: 856)



GesTeoGesGesAesTesAesGesGesGeoAesAesAesAesGesmCesAesmCe;







(SEQ ID NO: 857)



GesGeoGesTesmCesmCesAesmCesTesGeoAesAesAesmCesGesGesGesGe;







(SEQ ID NO: 858)



GesGeoAesTesAesGesGesGesAesAeoAesAesGesmCesAesmCesmCesTe;







(SEQ ID NO: 859)



GesGeoTesmCesmCesAesGesGesGesAeoGesAesAesTesTesTesGesGe;







(SEQ ID NO: 860)



AesAeoAesmCesGesGesGesGesAesGeoGesGesGesAesTesGesGesmCe;







(SEQ ID NO: 861)



GesAeoAesAesmCesGesGesGesGesAeoGesGesGesGesAesTesGesGe;







(SEQ ID NO: 862)



TesGeoAesAesAesmCesGesGesGesGeoAesGesGesGesGesAesTesGe;







(SEQ ID NO: 863)




mCesTeoGesAesAesAesmCesGesGesGeoGesAesGesGesGesGesAesTe;








(SEQ ID NO: 864)



AesmCeoTesGesAesAesAesmCesGesGeoGesGesAesGesGesGesGesAe;







(SEQ ID NO: 865)




mCesAeomCesTesGesAesAesAesmCesGeoGesGesGesAesGesGesGesGe;




or







(SEQ ID NO: 866)



GesGeoTesmCesmCesAesmCesTesGesAeoAesAesmCesGesGesGesGesAe);







wherein:
    • A=an adenine nucleobase,
    • mC=a 5-methyl cytosine nucleobase,
    • G=a guanine nucleobase,
    • T=a thymine nucleobase,
    • e=a 2′-MOE sugar moiety,
    • s=a phosphorothioate internucleoside linkage, and
    • o=a phosphodiester internucleoside linkage.


      Embodiment 36. The oligomeric compound of any of embodiments 1-35, wherein the modified oligonucleotide is a pharmaceutically acceptable salt.


      Embodiment 37. The oligomeric compound of embodiment 36, wherein the modified oligonucleotide is a pharmaceutically acceptable salt comprising one or more cations selected from sodium, potassium, calcium, and magnesium.


      Embodiment 38. The oligomeric compound of any of embodiments 1-37, consisting of the modified oligonucleotide.


      Embodiment 39. The oligomeric compound of any of embodiments 1-37, wherein the oligomeric compound comprises a conjugate group.


      Embodiment 40. The oligomeric compound of embodiment 39, wherein the conjugate group comprises a conjugate linker and a conjugate moiety.


      Embodiment 41. The oligomeric compound of embodiment 40, wherein the conjugate linker consists of a single bond.


      Embodiment 42. The oligomeric compound of embodiment 40 or embodiment 41, wherein the conjugate linker is cleavable.


      Embodiment 43. The oligomeric compound of embodiment 40 or embodiment 41, wherein the conjugate linker comprises 1-3 linker nucleosides.


      Embodiment 44. The oligomeric compound of any of embodiments 40-42, wherein the conjugate linker does not comprise any linker nucleosides.


      Embodiment 45. The oligomeric compound of any of embodiments 39-44, wherein the conjugate group is attached to the 5′-end of the modified oligonucleotide.


      Embodiment 46. The oligomeric compound of any of embodiments 39-44, wherein the conjugate group is attached to the 3′-end of the modified oligonucleotide.


      Embodiment 47. The oligomeric compound of any of embodiments 39-44, wherein the oligomeric compound comprises a terminal group.


      Embodiment 48. The oligomeric compound of embodiment 47, wherein the terminal group is an abasic sugar moiety.


      Embodiment 49. The oligomeric compound of any of embodiments 1-48, wherein the oligomeric compound is a single-stranded oligomeric compound.


      Embodiment 50. An oligomeric compound comprising a modified oligonucleotide according to the following chemical notation: AesmCeoTesGesAesAesAesmCesGesGeoGesGesAesGesGesGesGesAe (SEQ ID NO 864), wherein:
    • A=an adenine nucleobase,
    • mC=a 5-methyl cytosine nucleobase,
    • G=a guanine nucleobase,
    • T=a thymine nucleobase,
    • e=a 2′-MOE sugar moiety,
    • s=a phosphorothioate internucleoside linkage, and
    • o=a phosphodiester internucleoside linkage.


      Embodiment 51. An oligomeric compound comprising a modified oligonucleotide according to the following chemical notation: mCesAeomCesTesGesAesAesAesmCesGeoGesGesGesAesGesGesGesGe (SEQ ID NO 865), wherein:
    • A=an adenine nucleobase,
    • mC=a 5-methyl cytosine nucleobase,
    • G=a guanine nucleobase,
    • T=a thymine nucleobase,
    • e=a 2′-MOE sugar moiety,
    • s=a phosphorothioate internucleoside linkage, and
    • o=a phosphodiester internucleoside linkage.


      Embodiment 52. A modified oligonucleotide according to the following chemical structure:




embedded image


Embodiment 53. The modified oligonucleotide of embodiment 52, which is the sodium salt or the potassium salt.


Embodiment 54. A modified oligonucleotide according to the following chemical structure:




embedded image


Embodiment 55. A modified oligonucleotide according to the following chemical structure:




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Embodiment 56. The modified oligonucleotide of embodiment 55, which is the sodium salt or the potassium salt.


Embodiment 57. A modified oligonucleotide according to the following chemical structure:




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Embodiment 58. A chirally enriched population of oligomeric compounds of any of embodiments 1-51, or a chirally enriched population of modified oligonucleotides ofany of embodiments 52-57, wherein the population is enriched for oligomeric compounds comprising at least one particular phosphorothioate internucleoside linkage having a particular stereochemical configuration.


Embodiment 59. The chirally enriched population of embodiment 58, wherein the population is enriched for modified oligonucleotides comprising at least one particular phosphorothioate internucleoside linkage having the (Sp) configuration.


Embodiment 60. The chirally enriched population of embodiment 58, wherein the population is enriched for modified oligonucleotides comprising at least one particular phosphorothioate internucleoside linkage having the (Rp) configuration.


Embodiment 61. The chirally enriched population of embodiment 58, wherein the population is enriched for modified oligonucleotides having a particular, independently selected stereochemical configuration at each phosphorothioate internucleoside linkage.


Embodiment 62. The chirally enriched population of embodiment 61, wherein the population is enriched for modified oligonucleotides having the (Sp) configuration at each phosphorothioate internucleoside linkage or for modified oligonucleotides having the (Rp) configuration at each phosphorothioate internucleoside linkage.


Embodiment 63. The chirally enriched population of embodiment 61, wherein the population is enriched for modified oligonucleotides having the (Rp) configuration at one particular phosphorothioate internucleoside linkage and the (Sp) configuration at each of the remaining phosphorothioate internucleoside linkages.


Embodiment 64. The chirally enriched population of embodiment 61, wherein the population is enriched for modified oligonucleotides having at least 3 contiguous phosphorothioate internucleoside linkages in the Sp, Sp, and Rp configurations, in the 5′ to 3′ direction.


Embodiment 65. A population of oligomeric compounds of any of embodiments 1-51, or a population of modified oligonucleotides of any of embodiments 52-57, wherein all of the phosphorothioate internucleoside linkages of the modified oligonucleotide are stereorandom.


Embodiment 66. An antisense agent comprising an antisense compound, wherein the antisense compound is the oligomeric compound of any of embodiments 1-51.


Embodiment 67. The antisense agent of embodiment 66, wherein the antisense agent comprises a conjugate group, wherein the conjugate group comprises a cell-targeting moiety.


Embodiment 68. A pharmaceutical composition comprising an oligomeric compound of any of embodiments 1-51, a modified oligonucleotide of any of embodiments 52-57, a population of any of embodiments 58-65, or an antisense agent of embodiment 66 or embodiment 67, and a pharmaceutically acceptable diluent.


Embodiment 69. The pharmaceutical composition of embodiment 68, wherein the pharmaceutically acceptable diluent is artificial CSF (aCSF) or phosphate-buffered saline (PBS).


Embodiment 70. The pharmaceutical composition of embodiment 69, wherein the pharmaceutical composition consists essentially of the oligomeric compound of any of embodiments 1-51, the modified oligonucleotide of any of embodiments 52-57, the population of any of embodiments 58-65, the or the antisense agent of embodiment 66 or embodiment 67, and aCSF.


Embodiment 71. The pharmaceutical composition of embodiment 69, wherein the pharmaceutical composition consists essentially of the oligomeric compound of any of embodiments 1-51, the modified oligonucleotide of any of embodiments 52-57, the population of any of embodiments 58-65, or the antisense agent of embodiment 66 or embodiment 67, and PBS.


Embodiment 72. A method comprising administering to a subject an oligomeric compound of any of embodiments 1-51, a modified oligonucleotide of any of embodiments 52-57, a population of any of embodiments 58-65, an antisense agent of embodiment 66 or embodiment 67, or a pharmaceutical composition of any of embodiments 68-71.


Embodiment 73. A method of treating a disease or disorder associated with an insufficient expression of progranulin, comprising administering to a subject having or at risk for developing a disease or disorder associated with insufficient expression of progranulin a therapeutically effective amount of an oligomeric compound of any of embodiments 1-51, a modified oligonucleotide of any of embodiments 52-57, a population of any of embodiments 58-65, an antisense agent of embodiment 66 or embodiment 67, or a pharmaceutical composition of any of embodiments 68-71, thereby treating the disease or disorder associated with an insufficient expression of progranulin.


Embodiment 74. The method of embodiment 73, wherein the disease or disorder associated with an insufficient expression of progranulin is a neurological disease or disorder.


Embodiment 75. The method of embodiment 73 or embodiment 74, wherein the disease or disorder associated with an insufficient expression of progranulin is a lysosomal storage disorder or a TDP-43 proteinopathy.


Embodiment 76. The method of any of embodiments 73-75, wherein the disease or disorder associated with insufficient expression of progranulin is frontotemporal dementia (FTD), frontotemporal lobar degeneration (FTLD), neuronal ceroid lipofuscinosis (NCL), Alzheimer's disease (AD), or amyotrophic lateral sclerosis (ALS).


Embodiment 77. The method of any of embodiments 73-77, wherein at least one symptom or hallmark of the disease or disorder associated with insufficient expression of progranulin is ameliorated.


Embodiment 78. The method of embodiment 77, wherein the at least one symptom or hallmark is deterioration in behavior and personality, language impairment, alterations in muscle or motor functions, memory loss, cognitive dysfunction, tremor, seizures, or dizziness.


Embodiment 79. The method of embodiment 77 or embodiment 78, wherein administration of the oligomeric compound of any of embodiments 1-51, the modified oligonucleotide of any of embodiments 52-57, the population of any of embodiments 58-65, the antisense agent of embodiment 66 or embodiment 67, or the pharmaceutical composition of any of embodiments 68-71 improves behavior or personality, slows deterioration in behavior or personality, improves language ability, slows deterioration of language ability, improves muscle or motor function, slows deterioration in muscle or motor function, improves memory, slows deterioration in memory, improves cognitive function, slows deterioration of cognitive function, reduces tremors, reduces seizures, or reduces dizziness.


Embodiment 80. The method of any of embodiments 73-79, wherein the oligomeric compound of any of embodiments 1-51, the modified oligonucleotide of any of embodiments 52-57, the population of any of embodiments 58-65, the antisense agent of embodiment 66 or embodiment 67, or the pharmaceutical composition of any of embodiments 68-71 is administered to the central nervous system or systemically.


Embodiment 81. The method of any of embodiments 73-79, wherein the oligomeric compound of any of embodiments 1-51, the modified oligonucleotide of any of embodiments 52-57, the population of any of embodiments 58-65, the antisense agent of embodiment 66 or embodiment 67, or the pharmaceutical composition of any of embodiments 68-71 is administered any of intrathecally, systemically, subcutaneously, or intramuscularly.


Embodiment 82. The method of any of embodiments 73-81, wherein the subject is a human.


Embodiment 83. A method of increasing progranulin RNA or one or more splice variants of said progranulin RNA in a cell, comprising contacting the cell with an oligomeric compound of any of embodiments 1-51, a modified oligonucleotide of any of embodiments 52-57, a population of any of embodiments 58-65, or an antisense agent of embodiment 66 or embodiment 67.


Embodiment 84. A method of increasing progranulin protein in a cell, comprising contacting the cell with an oligomeric compound of any of embodiments 1-51, a modified oligonucleotide of any of embodiments 52-57, a population of any of embodiments 58-65, or an antisense agent of embodiment 66 or embodiment 67.


Embodiment 85. The method of embodiment 83 or embodiment 84, wherein the cell is a neuron.


Embodiment 86. The method of any of embodiments 83-85, wherein the cell is a human cell.


Embodiment 87. Use of an oligomeric compound of any of embodiments 1-51, a modified oligonucleotide of any of embodiments 52-57, a population of any of embodiments 58-65, an antisense agent of embodiment 66 or embodiment 67, or a pharmaceutical composition of any of embodiments 68-71 for treating a disease or disorder associated with an insufficient expression of progranulin.


Embodiment 88. Use of an oligomeric compound of any of embodiments 1-51, a modified oligonucleotide of any of embodiments 52-57, a population of any of embodiments 58-65, an antisense agent of embodiment 66 or embodiment 67, or a pharmaceutical composition of any of embodiments 68-71 in the manufacture of a medicament for treating a disease or disorder associated with an insufficient expression of progranulin.


Embodiment 89. The use of embodiment 87 or embodiment 88, wherein the disease or disorder associated with an insufficient expression of progranulin is a neurological disease or disorder.


Embodiment 90. The use of embodiment 87 or embodiment 88, wherein the disease or disorder associated with an insufficient expression of progranulin is a lysosomal storage disorder or a TDP-43 proteinopathy.


Embodiment 91. The use of any of embodiments 87-90, wherein the disease or disorder associated with insufficient expression of progranulin is frontotemporal dementia (FTD), frontotemporal lobar degeneration (FTLD), neuronal ceroid lipofuscinosis (NCL), Alzheimer's disease (AD), or amyotrophic lateral sclerosis (ALS).


Certain Compositions
1. Compound No. 1557993

In certain embodiments, Compound No. 1557993 is characterized as a modified oligonucleotide having a nucleobase sequence (from 5′ to 3′) of ACTGAAACGGGGAGGGGA (SEQ ID NO 781), wherein each nucleoside comprises a 2′-MOE sugar moiety, wherein the internucleoside linkages between nucleosides 2 to 3, and 10 to 11 are phosphodiester internucleoside linkages, the internucleoside linkages between nucleosides 1 to 2, 3 to 4, 4 to 5, 5 to 6, 6 to 7, 7 to 8, 8 to 9, 9 to 10, 11 to 12, 12 to 13, 13 to 14, 14 to 15, 15 to 16, 16 to 17, and 17 to 18 are phosphorothioate internucleoside linkages, and wherein each cytosine is a 5-methyl cytosine.


In certain embodiments, Compound No. 1557993 is represented by the following chemical notation:











(SEQ ID NO 864)



AesmCeoTesGesAesAesAesmCesGesGeoGesGesAesGesGesGesGesAe,







wherein
    • A=an adenine nucleobase,
    • mC=a 5-methyl cytosine nucleobase,
    • G=a guanine nucleobase,
    • T=a thymine nucleobase,
    • e=a 2′-MOE sugar moiety,
    • s=a phosphorothioate internucleoside linkage, and
    • o=a phosphodiester internucleoside linkage.


In certain embodiments Compound No. 1557993 is represented by the following chemical structure:




embedded image


Structure 1. Compound No. 1557993

In certain embodiments, an oligomeric compound comprises the sodium salt or the potassium salt of the modified oligonucleotide represented by Structure 1.


In certain embodiments the sodium salt of Compound No. 1557993 is represented by the following chemical structure:




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Structure 2. The Sodium Salt of Compound No. 1557993
2. Compound No. 1557994

In certain embodiments, Compound No. 1557994 is characterized as a modified oligonucleotide having a sequence (from 5′ to 3′) of CACTGAAACGGGGAGGGG (SEQ ID NO 80), wherein each nucleoside comprises a 2′-MOE sugar moiety, wherein the internucleoside linkages between nucleosides 2 to 3, and 10 to 11 are phosphodiester internucleoside linkages, the internucleoside linkages between nucleosides 1 to 2, 3 to 4, 4 to 5, 5 to 6, 6 to 7, 7 to 8, 8 to 9, 9 to 10, 11 to 12, 12 to 13, 13 to 14, 14 to 15, 15 to 16, 16 to 17, and 17 to 18 are phosphorothioate internucleoside linkages, and wherein each cytosine is a 5-methyl cytosine.


In certain embodiments, Compound No. 1557994 is represented by the following chemical notation:











(SEQ ID NO 865)




mCesAeomCesTesGesAesAesAesmCesGeoGesGesGesAesGesGesGesGe,









    • A=an adenine nucleobase,


    • mC=a 5-methyl cytosine nucleobase,

    • G=a guanine nucleobase,

    • T=a thymine nucleobase,

    • e=a 2′-MOE sugar moiety,

    • s=a phosphorothioate internucleoside linkage, and

    • o=a phosphodiester internucleoside linkage.





In certain embodiments Compound No. 1557994 is represented by the following chemical structure:




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Structure 3. Compound No. 1557994

In certain embodiments, an oligomeric compound comprises the sodium salt or the potassium salt of the modified oligonucleotide represented by Structure 3.


In certain embodiments the sodium salt of Compound No. 1557994 is represented by the following chemical structure:




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Structure 4. The sodium salt of Compound No. 1557994


Certain Oligonucleotides

In certain embodiments, provided herein are oligomeric compounds comprising oligonucleotides, which consist of linked nucleosides. Oligonucleotides may be unmodified oligonucleotides (RNA or DNA) or may be modified oligonucleotides. Modified oligonucleotides comprise at least one modification relative to unmodified RNA or DNA. That is, modified oligonucleotides comprise at least one modified nucleoside (comprising a modified sugar moiety and/or a modified nucleobase) and/or at least one modified internucleoside linkage.


A. Certain Modified Nucleosides

Modified nucleosides comprise a modified sugar moiety or a modified nucleobase or both a modified sugar moiety and a modified nucleobase. In certain embodiments, modified nucleosides comprising the following modified sugar moieties and/or the following modified nucleobases may be incorporated into modified oligonucleotides.


1. Certain Sugar Moieties

In certain embodiments, modified sugar moieties are non-bicyclic modified sugar moieties. In certain embodiments, modified sugar moieties are bicyclic or tricyclic sugar moieties. In certain embodiments, modified sugar moieties are sugar surrogates. Such sugar surrogates may comprise one or more substitutions corresponding to those of other types of modified sugar moieties.


In certain embodiments, modified sugar moieties are non-bicyclic modified sugar moieties comprising a furanosyl ring with one or more substituent groups none of which bridges two atoms of the furanosyl ring to form a bicyclic structure. Such non bridging substituents may be at any position of the furanosyl, including but not limited to substituents at the 2′, 3′, 4′, and/or 5′ positions. Examples of 2′-substituent groups suitable for non-bicyclic modified sugar moieties include but are not limited to: 2′-F, 2′-OCH3 (“OMe” or “O-methyl”), and 2′-O(CH2)2OCH3 (“MOE” or “O-methoxyethyl”), and 2′-O—N-alkyl acetamide, e.g., 2′-O—N-methyl acetamide (“NMA”), 2′-O—N-dimethyl acetamide, 2′-O—N-ethyl acetamide, or 2′-O—N-propyl acetamide. For example, see U.S. Pat. No. 6,147,200, Prakash et al., 2003, Org. Lett., 5, 403-6.


A “2′-O—N-methyl acetamide nucleoside” or “2′-NMA nucleoside” is shown below:




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In certain embodiments, modified furanosyl sugar moieties and nucleosides incorporating such modified furanosyl sugar moieties are further defined by isomeric configuration. For example, a 2′-deoxyfuranosyl sugar moiety may be in seven isomeric configurations other than the naturally occurring β-D-deoxyribosyl configuration. Such modified sugar moieties are described in, e.g., WO 2019/157531, incorporated by reference herein. A 2′-modified sugar moiety has an additional stereocenter at the 2′-position relative to a 2′-deoxyfuranosyl sugar moiety; therefore, such sugar moieties have a total of sixteen possible isomeric configurations. 2′-modified sugar moieties described herein are in the β-D-ribosyl isomeric configuration unless otherwise specified.


In certain embodiments, 2′-substituent groups are selected from among: halo, allyl, amino, azido, SH, CN, OCN, CF3, OCF3, O—C1-C10 alkoxy, O—C1-C10 substituted alkoxy, O—C1-C10 alkyl, O—C1-C10 substituted alkyl, S-alkyl, N(Rm)-alkyl, O-alkenyl, S-alkenyl, N(Rm)-alkenyl, O-alkynyl, S-alkynyl, N(Rm)-alkynyl, O-alkylenyl-O-alkyl, alkynyl, alkaryl, aralkyl, O-alkaryl, O-aralkyl, O(CH2)2SCH3, O(CH2)2ON(Rm)(Rn) or OCH2C(═O)—N(Rm)(Rn), where each Rm and Rn is, independently, H, an amino protecting group, or substituted or unsubstituted C1-C10 alkyl, and the 2′-substituent groups described in Cook et al., U.S. Pat. No. 6,531,584; Cook et al., U.S. Pat. No. 5,859,221; and Cook et al., U.S. Pat. No. 6,005,087. Certain embodiments of these 2′-substituent groups can be further substituted with one or more substituent groups independently selected from among: hydroxyl, amino, alkoxy, carboxy, benzyl, phenyl, nitro (NO2), thiol, thioalkoxy, thioalkyl, halogen, alkyl, aryl, alkenyl and alkynyl. Examples of 4′-substituent groups suitable for non-bicyclic modified sugar moieties include but are not limited to alkoxy (e.g., methoxy), alkyl, and those described in Manoharan et al., WO 2015/106128. Examples of 5′-substituent groups suitable for non-bicyclic modified sugar moieties include but are not limited to: 5′-methyl (R or S), 5′-vinyl, and 5′-methoxy. In certain embodiments, non-bicyclic modified sugar moieties comprise more than one non-bridging sugar substituent, for example, 2′-F-5′-methyl sugar moieties and the modified sugar moieties and modified nucleosides described in Migawa et al., WO 2008/101157 and Rajeev et al., US2013/0203836.


In certain embodiments, a 2′-substituted non-bicyclic modified nucleoside comprises a sugar moiety comprising a non-bridging 2′-substituent group selected from: F, NH2, N3, OCF3, OCH3, O(CH2)3NH2, CH2CH═CH2, OCH2CH═CH2, OCH2CH2OCH3, O(CH2)2SCH3, O(CH2)2ON(Rm)(Rn), O(CH2)ON(CH3)2 (DMAOE), O(CH2)2O(CH2)2N(CH3)2 (DMAEOE), and N-substituted acetamide (OCH2C(═O)—N(Rm)(Rn)), where each Rm and Rn is, independently, H, an amino protecting group, or substituted or unsubstituted C1-C10 alkyl, e.g., for example, OCH2C(═O)—N(H)CH3 (“NMA”).


In certain embodiments, a 2′-substituted non-bicyclic modified nucleoside comprises a sugar moiety comprising a non-bridging 2′-substituent group selected from: F, OCF3, OCH3, OCH2CH2OCH3, O(CH2)2SCH3, O(CH2)2ON(CH3)2, O(CH2)2O(CH2)2N(CH3)2, and OCH2C(═O)—N(H)CH3 (“NMA”).


In certain embodiments, a 2′-substituted non-bicyclic modified nucleoside comprises a sugar moiety comprising a non-bridging 2′-substituent group selected from: F, OCH3, OCH2CH2OCH3, and OCH2C(═O)—N(H)CH3.


Certain modified sugar moieties comprise a substituent that bridges two atoms of the furanosyl ring to form a second ring, resulting in a bicyclic sugar moiety. Nucleosides comprising such bicyclic sugar moieties have been referred to as bicyclic nucleosides (BNAs), locked nucleosides, or conformationally restricted nucleotides (CRN). Certain such compounds are described in US Patent Publication No. 2013/0190383; and PCT publication WO 2013/036868. In certain such embodiments, the bicyclic sugar moiety comprises a bridge between the 4′ and the 2′ furanose ring atoms. In certain such embodiments, the furanose ring is a ribose ring. Examples of such 4′ to 2′ bridging sugar substituents include but are not limited to: 4′-CH2-2′, 4′—(CH2)2-2′, 4′—(CH2)3-2′, 4′—CH2—O-2′ (“LNA”), 4′-CH2—S-2′, 4′—(CH2)2—O-2′ (“ENA”), 4′-CH(CH3)—O-2′ (referred to as “constrained ethyl” or “cEt” when in the S configuration), 4′-CH2—O—CH2-2′, 4′—CH2—N(R)-2′, 4′—CH(CH2OCH3)—O-2′ (“constrained MOE” or “cMOE”) and analogs thereof (see, e.g., Seth et al., U.S. Pat. No. 7,399,845, Bhat et al., U.S. Pat. No. 7,569,686, Swayze et al., U.S. Pat. No. 7,741,457, and Swayze et al., U.S. Pat. No. 8,022,193), 4′-C(CH3)(CH3)—O-2′ and analogs thereof (see, e.g., Seth et al., U.S. Pat. No. 8,278,283), 4′-CH2—N(OCH3)-2′ and analogs thereof (see, e.g., Prakash et al., U.S. Pat. No. 8,278,425), 4′-CH2—O—N(CH3)-2′ (see, e.g., Allerson et al., U.S. Pat. No. 7,696,345 and Allerson et al., U.S. Pat. No. 8,124,745), 4′-CH2—C(H)(CH3)-2′ (see, e.g., Zhou, et al., J. Org. Chem., 2009, 74, 118-134), 4′-CH2—C(═CH2)-2′ and analogs thereof (see e.g., Seth et al., U.S. Pat. No. 8,278,426), 4′-C(RaRb)—N(R)—O-2′, 4′—C(RaRb)—O—N(R)-2′, 4′—CH2—O—N(R)-2′, and 4′-CH2—N(R)—O-2′, wherein each R, Ra, and Rb is, independently, H, a protecting group, or C1-C12 alkyl (see, e.g. Imanishi et al., U.S. Pat. No. 7,427,672).


In certain embodiments, such 4′ to 2′ bridges independently comprise from 1 to 4 linked groups independently selected from: —[C(Ra)(Rb)]n—, —[C(Ra)(Rb)]nO—, —C(Ra)═C(Rb)—, —C(Ra)═N—, —C(═NRa)—, —C(═O)—, —C(═S)—, —O—, —Si(Ra)2—, —S(═O)x—, and —N(Ra)—;

    • wherein:
    • x is 0, 1, or 2;
    • n is 1, 2, 3, or 4;


each Ra and Rb is, independently, H, a protecting group, hydroxyl, C1-C12 alkyl, substituted C1-C12 alkyl, C2-C12 alkenyl, substituted C2-C12 alkenyl, C2-C12 alkynyl, substituted C2-C12 alkynyl, C5-C20 aryl, substituted C5-C20 aryl, heterocycle radical, substituted heterocycle radical, heteroaryl, substituted heteroaryl, C5-C7 alicyclic radical, substituted C5-C7 alicyclic radical, halogen, OJ1, NJ1J2, SJ1, N3, COOJ1, acyl (C(═O)—H), substituted acyl, CN, sulfonyl (S(═O)2-J1), or sulfoxyl (S(═O)-J1); and


each J1 and J2 is, independently, H, C1-C12 alkyl, substituted C1-C12 alkyl, C2-C12 alkenyl, substituted C2-C12 alkenyl, C2-C12 alkynyl, substituted C2-C12 alkynyl, C5-C20 aryl, substituted C5-C20 aryl, acyl (C(═O)—H), substituted acyl, a heterocycle radical, a substituted heterocycle radical, C1-C12 aminoalkyl, substituted C1-C12 aminoalkyl, or a protecting group.


Additional bicyclic sugar moieties are known in the art, see, for example: Freier et al., Nucleic Acids Research, 1997, 25(22), 4429-4443, Albaek et al., J. Org. Chem., 2006, 71, 7731-7740, Singh et al., Chem. Commun., 1998, 4, 455-456; Koshkin et al., Tetrahedron, 1998, 54, 3607-3630; Wahlestedt et al., Proc. Natl. Acad. Sci. U.S.A, 2000, 97, 5633-5638; Kumar et al., Bioorg. Med. Chem. Lett., 1998, 8, 2219-2222; Singh et al., J. Org. Chem., 1998, 63, 10035-10039; Srivastava et al., J. Am. Chem. Soc., 2007, 129, 8362-8379; Elayadi et al., Curr. Opinion Invens. Drugs, 2001, 2, 558-561; Braasch et al., Chem. Biol., 2001, 8, 1-7; Orum et al., Curr. Opinion Mol. Ther., 2001, 3, 239-243; Wengel et al., U.S. Pat. No. 7,053,207, Imanishi et al., U.S. Pat. No. 6,268,490, Imanishi et al. U.S. Pat. No. 6,770,748, Imanishi et al., U.S. RE44,779; Wengel et al., U.S. Pat. No. 6,794,499, Wengel et al., U.S. Pat. No. 6,670,461; Wengel et al., U.S. Pat. No. 7,034,133, Wengel et al., U.S. Pat. No. 8,080,644; Wengel et al., U.S. Pat. No. 8,034,909; Wengel et al., U.S. Pat. No. 8,153,365; Wengel et al., U.S. Pat. No. 7,572,582; and Ramasamy et al., U.S. Pat. No. 6,525,191, Torsten et al., WO 2004/106356, Wengel et al., WO 1999/014226; Seth et al., WO 2007/134181; Seth et al., U.S. Pat. No. 7,547,684; Seth et al., U.S. Pat. No. 7,666,854; Seth et al., U.S. Pat. No. 8,088,746; Seth et al., U.S. Pat. No. 7,750,131; Seth et al., U.S. Pat. No. 8,030,467; Seth et al., U.S. Pat. No. 8,268,980; Seth et al., U.S. Pat. No. 8,546,556; Seth et al., U.S. Pat. No. 8,530,640; Migawa et al., U.S. Pat. No. 9,012,421; Seth et al., U.S. Pat. No. 8,501,805; Allerson et al., US2008/0039618; and Migawa et al., US2015/0191727. In certain embodiments, bicyclic sugar moieties and nucleosides incorporating such bicyclic sugar moieties are further defined by isomeric configuration. For example, an LNA nucleoside (described herein) may be in the α-L configuration or in the β-D configuration.




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α-L-methyleneoxy (4′-CH2—O-2′) or α-L-LNA bicyclic nucleosides have been incorporated into oligonucleotides that showed antisense activity (Frieden et al., Nucleic Acids Research, 2003, 21, 6365-6372). Herein, general descriptions of bicyclic nucleosides include both isomeric configurations. When the positions of specific bicyclic nucleosides (e.g., LNA or cEt) are identified in exemplified embodiments herein, they are in the β-D configuration, unless otherwise specified.


In certain embodiments, modified sugar moieties comprise one or more non-bridging sugar substituent and one or more bridging sugar substituent (e.g., 5′-substituted and 4′-2′ bridged sugars).


In certain embodiments, modified sugar moieties are sugar surrogates. In certain such embodiments, the oxygen atom of the sugar moiety is replaced, e.g., with a sulfur, carbon or nitrogen atom. In certain such embodiments, such modified sugar moieties also comprise bridging and/or non-bridging substituents as described herein. For example, certain sugar surrogates comprise a 4′-sulfur atom and a substitution at the 2-position (see, e.g., Bhat et al., U.S. Pat. No. 7,875,733 and Bhat et al., U.S. Pat. No. 7,939,677) and/or the 5′ position.


In certain embodiments, sugar surrogates comprise rings having other than 5 atoms. For example, in certain embodiments, a sugar surrogate comprises a six-membered tetrahydropyran (“THP”). Such tetrahydropyrans may be further modified or substituted. Nucleosides comprising such modified tetrahydropyrans include but are not limited to hexitol nucleic acid (“HNA”), anitol nucleic acid (“ANA”), manitol nucleic acid (“NINA”) (see, e.g., Leumann, CJ. Bioorg. & Med. Chem. 2002, 10, 841-854), fluoro HNA:




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(“F-HNA”, see e.g. Swayze et al., U.S. Pat. No. 8,088,904; Swayze et al., U.S. Pat. No. 8,440,803; Swayze et al., U.S. Pat. No. 8,796,437; and Swayze et al., U.S. Pat. No. 9,005,906; F-HNA can also be referred to as a F-THP or 3′-fluoro tetrahydropyran), and nucleosides comprising additional modified THP compounds having the formula:




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wherein, independently, for each of the modified THP nucleosides:


Bx is a nucleobase moiety;


T3 and T4 are each, independently, an internucleoside linking group linking the modified THP nucleoside to the remainder of an oligonucleotide or one of T3 and T4 is an internucleoside linking group linking the modified THP nucleoside to the remainder of an oligonucleotide and the other of T3 and T4 is H, a hydroxyl protecting group, a linked conjugate group, or a 5′ or 3′-terminal group;


q1, q2, q3, q4, q5, q6 and q7 are each, independently, H, C1-C6 alkyl, substituted C1-C6 alkyl, C2-C6 alkenyl, substituted C2-C6 alkenyl, C2-C6 alkynyl, or substituted C2-C6 alkynyl; and


each of R1 and R2 is independently selected from among: hydrogen, halogen, substituted or unsubstituted alkoxy, NJ1J2, SJ1, N3, OC(═X)J1, OC(═X)NJ1J2, NJ3C(═X)NJ1J2, and CN, wherein X is O, S or NJ1, and each J1, J2, and J3 is, independently, H or C1-C6 alkyl.


In certain embodiments, modified THP nucleosides are provided wherein q1, q2, q3, q4, q5, q6 and q are each H. In certain embodiments, at least one of q1, q2, q3, q4, q5, q6 and q is other than H. In certain embodiments, at least one of q1, q2, q3, q4, q5, q6 and q7 is methyl. In certain embodiments, modified THP nucleosides are provided wherein one of R1 and R2 is F. In certain embodiments, R1 is F and R2 is H, in certain embodiments, R1 is methoxy and R2 is H, and in certain embodiments, R1 is methoxyethoxy and R2 is H.


In certain embodiments, sugar surrogates comprise rings having more than 5 atoms and more than one heteroatom. For example, nucleosides comprising morpholino sugar moieties and their use in oligonucleotides have been reported (see, e.g., Braasch et al., Biochemistry, 2002, 41, 4503-4510 and Summerton et al., U.S. Pat. No. 5,698,685; Summerton et al., U.S. Pat. No. 5,166,315; Summerton et al., U.S. Pat. No. 5,185,444; and Summerton et al., U.S. Pat. No. 5,034,506). As used here, the term “morpholino” means a sugar surrogate having the following structure:




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In certain embodiments, morpholinos may be modified, for example by adding or altering various substituent groups from the above morpholino structure. Such sugar surrogates are referred to herein as “modified morpholinos.”


In certain embodiments, sugar surrogates comprise acyclic moieties. Examples of nucleosides and oligonucleotides comprising such acyclic sugar surrogates include but are not limited to: peptide nucleic acid (“PNA”), acyclic butyl nucleic acid (see, e.g., Kumar et al., Org. Biomol. Chem., 2013, 11, 5853-5865), and nucleosides and oligonucleotides described in Manoharan et al., WO2011/133876.


Many other bicyclic and tricyclic sugar and sugar surrogate ring systems are known in the art that can be used in modified nucleosides.


2. Certain Modified Nucleobases

In certain embodiments, modified oligonucleotides comprise one or more nucleosides comprising an unmodified nucleobase. In certain embodiments, modified oligonucleotides comprise one or more nucleosides comprising a modified nucleobase. In certain embodiments, modified oligonucleotides comprise one or more nucleosides that does not comprise a nucleobase, referred to as an abasic nucleoside. Examples of modified nucleobases include 5-methyl cytosine.


In certain embodiments, modified nucleobases are selected from: 5-substituted pyrimidines, 6-azapyrimidines, alkyl or alkynyl substituted pyrimidines, alkyl substituted purines, and N-2, N-6 and O-6 substituted purines. In certain embodiments, modified nucleobases are selected from: 2-aminopropyladenine, 5-hydroxymethyl cytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-N-methylguanine, 6-N-methyladenine, 2-propyl adenine, 2-thiouracil, 2-thiothymine and 2-thiocytosine, 5-propynyl (—C≡C—CH3) uracil, 5-propynylcytosine, 6-azouracil, 6-azocytosine, 6-azothymine, 5-ribosyluracil (pseudouracil), 4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl, 8-aza and other 8-substituted purines, 5-halo, particularly 5-bromo, 5-trifluoromethyl, 5-halouracil, and 5-halocytosine, 7-methylguanine, 7-methyladenine, 2-F-adenine, 2-aminoadenine, 7-deazaguanine, 7-deazaadenine, 3-deazaguanine, 3-deazaadenine, 6-N-benzoyladenine, 2-N-isobutyrylguanine, 4-N-benzoylcytosine, 4-N-benzoyluracil, 5-methyl 4-N-benzoylcytosine, 5-methyl 4-N-benzoyluracil, universal bases, hydrophobic bases, promiscuous bases, size-expanded bases, and fluorinated bases. Further modified nucleobases include tricyclic pyrimidines, such as 1,3-diazaphenoxazine-2-one, 1,3-diazaphenothiazine-2-one and 9-(2-aminoethoxy)-1,3-diazaphenoxazine-2-one (G-clamp). Modified nucleobases may also include those in which the purine or pyrimidine base is replaced with other heterocycles, for example 7-deazaadenine, 7-deazaguanosine, 2-aminopyridine and 2-pyridone. Further nucleobases include those disclosed in Merigan et al., U.S. Pat. No. 3,687,808, those disclosed in The Concise Encyclopedia Of Polymer Science And Engineering, Kroschwitz, J. I., Ed., John Wiley & Sons, 1990, 858-859; Englisch et al., Angewandte Chemie, International Edition, 1991, 30, 613; Sanghvi, Y. S., Chapter 15, Antisense Research and Applications, Crooke, S. T. and Lebleu, B., Eds., CRC Press, 1993, 273-288; and those disclosed in Chapters 6 and 15, Antisense Drug Technology, Crooke S. T., Ed., CRC Press, 2008, 163-166 and 442-443.


Publications that teach the preparation of certain of the above noted modified nucleobases as well as other modified nucleobases include without limitation, Manoharan et al., US2003/0158403; Manoharan et al., US2003/0175906; Dinh et al., U.S. Pat. No. 4,845,205; Spielvogel et al., U.S. Pat. No. 5,130,302; Rogers et al., U.S. Pat. No. 5,134,066; Bischofberger et al., U.S. Pat. No. 5,175,273; Urdea et al., U.S. Pat. No. 5,367,066; Benner et al., U.S. Pat. No. 5,432,272; Matteucci et al., U.S. Pat. No. 5,434,257; Gmeiner et al., U.S. Pat. No. 5,457,187; Cook et al., U.S. Pat. No. 5,459,255; Froehler et al., U.S. Pat. No. 5,484,908; Matteucci et al., U.S. Pat. No. 5,502,177; Hawkins et al., U.S. Pat. No. 5,525,711; Haralambidis et al., U.S. Pat. No. 5,552,540; Cook et al., U.S. Pat. No. 5,587,469; Froehler et al., U.S. Pat. No. 5,594,121; Switzer et al., U.S. Pat. No. 5,596,091; Cook et al., U.S. Pat. No. 5,614,617; Froehler et al., U.S. Pat. No. 5,645,985; Cook et al., U.S. Pat. No. 5,681,941; Cook et al., U.S. Pat. No. 5,811,534; Cook et al., U.S. Pat. No. 5,750,692; Cook et al., U.S. Pat. No. 5,948,903; Cook et al., U.S. Pat. No. 5,587,470; Cook et al., U.S. Pat. No. 5,457,191; Matteucci et al., U.S. Pat. No. 5,763,588; Froehler et al., U.S. Pat. No. 5,830,653; Cook et al., U.S. Pat. No. 5,808,027; Cook et al., 6,166,199; and Matteucci et al., U.S. Pat. No. 6,005,096.


3. Certain Modified Internucleoside Linkages

The naturally occurring internucleoside linkage of RNA and DNA is a 3′ to 5′ phosphodiester linkage. In certain embodiments, nucleosides of modified oligonucleotides may be linked together using one or more modified internucleoside linkages. The two main classes of internucleoside linking groups are defined by the presence or absence of a phosphorus atom. Representative phosphorus-containing internucleoside linkages include but are not limited to phosphodiesters, which contain a phosphodiester bond, P(O2)═O, (also referred to as unmodified or naturally occurring linkages); phosphotriesters; methylphosphonates; methoxypropylphosphonates (“MOP”); phosphoramidates; phosphorothioates (P(O2)═S); and phosphorodithioates (HS—P═S). Representative non-phosphorus containing internucleoside linking groups include but are not limited to methylenemethylimino (—CH2—N(CH3)—O—CH2—); thiodiester, thionocarbamate (—O—C(═O)(NH)—S—); siloxane (—O—SiH2—O—); and N,N′-dimethylhydrazine (—CH2—N(CH3)—N(CH3)—). Modified internucleoside linkages, compared to naturally occurring phosphodiester internucleoside linkages, can be used to alter, typically increase, nuclease resistance of the oligonucleotide. In certain embodiments, internucleoside linkages having a chiral atom can be prepared as a racemic mixture, or as separate enantiomers. Methods of preparation of phosphorous-containing and non-phosphorous-containing internucleoside linkages are well known to those skilled in the art.


Representative internucleoside linkages having a chiral center include but are not limited to alkylphosphonates and phosphorothioates. Modified oligonucleotides comprising internucleoside linkages having a chiral center can be prepared as populations of modified oligonucleotides comprising stereorandom internucleoside linkages, or as populations of modified oligonucleotides comprising phosphorothioate or other internucleoside linkages in particular stereochemical configurations. In certain embodiments, populations of modified oligonucleotides comprise phosphorothioate internucleoside linkages wherein all of the phosphorothioate internucleoside linkages are stereorandom. Such modified oligonucleotides can be generated using synthetic methods that result in random selection of the stereochemical configuration of each phosphorothioate internucleoside linkage. Nonetheless, each individual phosphorothioate of each individual oligonucleotide molecule has a defined stereoconfiguration. In certain embodiments, populations of modified oligonucleotides are enriched for modified oligonucleotides comprising one or more particular phosphorothioate internucleoside linkages in a particular, independently selected stereochemical configuration. In certain embodiments, the particular configuration of the particular phosphorothioate internucleoside linkage is present in at least 65% of the molecules in the population. In certain embodiments, the particular configuration of the particular phosphorothioate internucleoside linkage is present in at least 70% of the molecules in the population. In certain embodiments, the particular configuration of the particular phosphorothioate internucleoside linkage is present in at least 80% of the molecules in the population. In certain embodiments, the particular configuration of the particular phosphorothioate internucleoside linkage is present in at least 90% of the molecules in the population. In certain embodiments, the particular configuration of the particular phosphorothioate internucleoside linkage is present in at least 99% of the molecules in the population. Such chirally enriched populations of modified oligonucleotides can be generated using synthetic methods known in the art, e.g., methods described in Oka et al., JACS, 2003, 125, 8307, Wan et al. Nuc. Acid. Res., 2014, 42, 13456, and WO 2017/015555. In certain embodiments, a population of modified oligonucleotides is enriched for modified oligonucleotides having at least one indicated phosphorothioate in the (Sp) configuration. In certain embodiments, a population of modified oligonucleotides is enriched for modified oligonucleotides having at least one phosphorothioate in the (Rp) configuration. In certain embodiments, modified oligonucleotides comprising (Rp) and/or (Sp) phosphorothioates comprise one or more of the following formulas, respectively, wherein “B” indicates a nucleobase:




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Unless otherwise indicated, chiral internucleoside linkages of modified oligonucleotides described herein can be stereorandom or in a particular stereochemical configuration.


Neutral internucleoside linkages include, without limitation, phosphotriesters, methylphosphonates, MMI (3′-CH2—N(CH3)—O-5′), amide-3 (3′-CH2—C(═O)—N(H)-5′), amide-4 (3′-CH2—N(H)—C(═O)-5′), formacetal (3′-O—CH2—O-5′), methoxypropyl (MOP), and thioformacetal (3′-S—CH2—O-5′). Further neutral internucleoside linkages include nonionic linkages comprising siloxane (dialkylsiloxane), carboxylate ester, carboxamide, sulfide, sulfonate ester and amides (see for example, Carbohydrate Modifications in Antisense Research; Y. S. Sanghvi and P. D. Cook, Eds., ACS Symposium Series 580; Chapters 3 and 4, 40-65). Further neutral internucleoside linkages include nonionic linkages comprising mixed N, O, S and CH2 component parts.


B. Certain Motifs

In certain embodiments, modified oligonucleotides comprise one or more modified nucleosides comprising a modified sugar moiety. In certain embodiments, modified oligonucleotides comprise one or more modified nucleosides comprising a modified nucleobase. In certain embodiments, modified oligonucleotides comprise one or more modified internucleoside linkage. In such embodiments, the modified, unmodified, and differently modified sugar moieties, nucleobases, and/or internucleoside linkages of a modified oligonucleotide define a pattern or motif. In certain embodiments, the patterns of sugar moieties, nucleobases, and internucleoside linkages are each independent of one another. Thus, a modified oligonucleotide may be described by its sugar motif, nucleobase motif and/or internucleoside linkage motif (as used herein, nucleobase motif describes the modifications to the nucleobases independent of the sequence of nucleobases).


1. Certain Sugar Motifs

In certain embodiments, oligonucleotides comprise one or more type of modified sugar and/or unmodified sugar moiety arranged along the oligonucleotide, or portion thereof, in a defined pattern or sugar motif. In certain instances, such sugar motifs include but are not limited to any of the sugar modifications discussed herein.


In certain embodiments, each nucleoside of a modified oligonucleotide, or portion thereof, comprises a 2′-substituted sugar moiety, a bicyclic sugar moiety, a sugar surrogate, or a 2′-deoxyribosyl sugar moiety. In certain embodiments, the 2′-substituted sugar moiety is selected from a 2′-MOE modified sugar moiety, a 2′-NMA modified sugar moiety, a 2′-OMe modified sugar moiety, and a 2′-F modified sugar moiety. In certain embodiments, the bicyclic sugar moiety is selected from a cEt sugar moiety and an LNA sugar moiety. In certain embodiments, the sugar surrogate is selected from morpholino, modified morpholino, PNA, THP, and F-HNA.


In certain embodiments, modified oligonucleotides comprise at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, or at least 20 nucleosides comprising a modified sugar moiety. In certain embodiments, the modified sugar moiety is selected independently from a 2′-substituted sugar moiety, a bicyclic sugar moiety, or a sugar surrogate. In certain embodiments, the 2′-substituted sugar moiety is selected from a 2′-MOE modified sugar moiety, a 2′-NMA modified sugar moiety, a 2′-OMe modified sugar moiety, and a 2′-F modified sugar moiety. In certain embodiments, the bicyclic sugar moiety is selected from a cEt sugar moiety and an LNA sugar moiety. In certain embodiments, the sugar surrogate is selected from morpholino, modified morpholino, THP, and F-HNA.


In certain embodiments, each nucleoside of a modified oligonucleotide comprises a modified sugar moiety (“fully modified oligonucleotide”). In certain embodiments, each nucleoside of a fully modified oligonucleotide comprises a 2′-substituted sugar moiety, a bicyclic sugar moiety, or a sugar surrogate. In certain embodiments, the 2′-substituted sugar moiety is selected from a 2′-MOE modified sugar moiety, a 2′-NMA modified sugar moiety, a 2′-OMe modified sugar moiety, and a 2′-F modified sugar moiety. In certain embodiments, the bicyclic sugar moiety is selected from a cEt sugar moiety and an LNA sugar moiety. In certain embodiments, the sugar surrogate is selected from morpholino, modified morpholino, THP, and F-HNA. In certain embodiments, each nucleoside of a fully modified oligonucleotide comprises the same modified sugar moiety (“uniformly modified sugar motif”). In certain embodiments, the uniformly modified sugar motif is 7 to 20 nucleosides in length. In certain embodiments, each nucleoside of the uniformly modified sugar motif comprises a 2′-substituted sugar moiety, a bicyclic sugar moiety, or a sugar surrogate. In certain embodiments, the 2′-substituted sugar moiety is selected from a 2′-MOE modified sugar moiety, a 2′-NMA modified sugar moiety, a 2′-OMe modified sugar moiety, and a 2′-F modified sugar moiety. In certain embodiments, the bicyclic sugar moiety is selected from a cEt sugar moiety and an LNA sugar moiety. In certain embodiments, the sugar surrogate is selected from morpholino, modified morpholino, THP, and F-HNA. In certain embodiments, modified oligonucleotides having at least one fully modified sugar motif may also comprise at least 1, at least 2, at least 3, or at least 4 2′-deoxyribonucleosides.


2. Certain Nucleobase Motifs

In certain embodiments, oligonucleotides comprise modified and/or unmodified nucleobases arranged along the oligonucleotide, or portion thereof, in a defined pattern or motif. In certain embodiments, each nucleobase is modified. In certain embodiments, none of the nucleobases are modified. In certain embodiments, each purine or each pyrimidine is modified. In certain embodiments, each adenine is modified. In certain embodiments, each guanine is modified. In certain embodiments, each thymine is modified. In certain embodiments, each uracil is modified. In certain embodiments, each cytosine is modified. In certain embodiments, some or all of the cytosine nucleobases in a modified oligonucleotide are 5-methyl cytosines. In certain embodiments, all of the cytosine nucleobases are 5-methyl cytosines and all of the other nucleobases of the modified oligonucleotide are unmodified nucleobases.


In certain embodiments, modified oligonucleotides comprise a block of modified nucleobases. In certain such embodiments, the block is at the 3′-end of the oligonucleotide. In certain embodiments the block is within 3 nucleosides of the 3′-end of the oligonucleotide. In certain embodiments, the block is at the 5′-end of the oligonucleotide. In certain embodiments the block is within 3 nucleosides of the 5′-end of the oligonucleotide.


3. Certain Internucleoside Linkage Motifs

In certain embodiments, oligonucleotides comprise modified and/or unmodified internucleoside linkages arranged along the oligonucleotide, or portion thereof, in a defined pattern or motif. In certain embodiments, each internucleoside linking group is a phosphodiester internucleoside linkage. In certain embodiments, each internucleoside linking group of a modified oligonucleotide is a phosphorothioate internucleoside linkage. In certain embodiments, each internucleoside linkage of a modified oligonucleotide is independently selected from a phosphorothioate internucleoside linkage and phosphodiester internucleoside linkage. In certain embodiments, each phosphorothioate internucleoside linkage is independently selected from a stereorandom phosphorothioate, a (Sp) phosphorothioate, and a (Rp) phosphorothioate.


In certain embodiments, modified oligonucleotides comprise at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, or at least 19 phosphodiester internucleoside linkages. In certain embodiments, modified oligonucleotides comprise at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, or at least 19 phosphorothioate internucleoside linkages. In certain embodiments, modified oligonucleotides comprise at least 1, at least 2, at least 3, at least 4, or at least 5 phosphodiester internucleoside linkages and the remainder of the internucleoside linkages are phosphorothioate internucleoside linkages. In certain embodiments, modified oligonucleotides have an internucleoside linkage motif of (5′ to 3′): sssssssssssssssss, wherein each “s” represents a phosphorothioate internucleoside linkage. In certain embodiments, modified oligonucleotides have an internucleoside linkage motif of (5′ to 3′): sosssssssosssssss wherein each “s” represents a phosphorothioate internucleoside linkage and each “o” represents a phosphodiester internucleoside linkage.


C. Certain Lengths

It is possible to increase or decrease the length of an oligonucleotide without eliminating activity. For example, in Woolf et al., Proc. Natl. Acad. Sci. USA, 1992, 89, 7305-7309, 1992), a series of oligonucleotides 13-25 nucleobases in length were tested for their ability to induce cleavage of a target nucleic acid in an oocyte injection model. Oligonucleotides 25 nucleobases in length with 8 or 11 mismatch bases near the ends of the oligonucleotides were able to direct specific cleavage of the target nucleic acid, albeit to a lesser extent than the oligonucleotides that contained no mismatches. Similarly, target specific cleavage was achieved using 13 nucleobase oligonucleotides, including those with 1 or 3 mismatches.


In certain embodiments, oligonucleotides (including modified oligonucleotides) can have any of a variety of ranges of lengths. In certain embodiments, oligonucleotides consist of X to Y linked nucleosides, where X represents the fewest number of nucleosides in the range and Y represents the largest number nucleosides in the range. In certain such embodiments, X and Y are each independently selected from 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, and 50; provided that X≤Y. For example, in certain embodiments, oligonucleotides consist of 12 to 13, 12 to 14, 12 to 15, 12 to 16, 12 to 17, 12 to 18, 12 to 19, 12 to 20, 12 to 21, 12 to 22, 12 to 23, 12 to 24, 12 to 25, 12 to 26, 12 to 27, 12 to 28, 12 to 29, 12 to 30, 13 to 14, 13 to 15, 13 to 16, 13 to 17, 13 to 18, 13 to 19, 13 to 20, 13 to 21, 13 to 22, 13 to 23, 13 to 24, 13 to 25, 13 to 26, 13 to 27, 13 to 28, 13 to 29, 13 to 30, 14 to 15, 14 to 16, 14 to 17, 14 to 18, 14 to 19, 14 to 20, 14 to 21, 14 to 22, 14 to 23, 14 to 24, 14 to 25, 14 to 26, 14 to 27, 14 to 28, 14 to 29, 14 to 30, 15 to 16, 15 to 17, 15 to 18, 15 to 19, 15 to 20, 15 to 21, 15 to 22, 15 to 23, 15 to 24, 15 to 25, 15 to 26, 15 to 27, 15 to 28, 15 to 29, 15 to 30, 16 to 17, 16 to 18, 16 to 19, 16 to 20, 16 to 21, 16 to 22, 16 to 23, 16 to 24, 16 to 25, 16 to 26, 16 to 27, 16 to 28, 16 to 29, 16 to 30, 17 to 18, 17 to 19, 17 to 20, 17 to 21, 17 to 22, 17 to 23, 17 to 24, 17 to 25, 17 to 26, 17 to 27, 17 to 28, 17 to 29, 17 to 30, 18 to 19, 18 to 20, 18 to 21, 18 to 22, 18 to 23, 18 to 24, 18 to 25, 18 to 26, 18 to 27, 18 to 28, 18 to 29, 18 to 30, 19 to 20, 19 to 21, 19 to 22, 19 to 23, 19 to 24, 19 to 25, 19 to 26, 19 to 27, 19 to 28, 19 to 29, 19 to 30, 20 to 21, 20 to 22, 20 to 23, 20 to 24, 20 to 25, 20 to 26, 20 to 27, 20 to 28, 20 to 29, 20 to 30, 21 to 22, 21 to 23, 21 to 24, 21 to 25, 21 to 26, 21 to 27, 21 to 28, 21 to 29, 21 to 30, 22 to 23, 22 to 24, 22 to 25, 22 to 26, 22 to 27, 22 to 28, 22 to 29, 22 to 30, 23 to 24, 23 to 25, 23 to 26, 23 to 27, 23 to 28, 23 to 29, 23 to 30, 24 to 25, 24 to 26, 24 to 27, 24 to 28, 24 to 29, 24 to 30, 25 to 26, 25 to 27, 25 to 28, 25 to 29, 25 to 30, 26 to 27, 26 to 28, 26 to 29, 26 to 30, 27 to 28, 27 to 29, 27 to 30, 28 to 29, 28 to 30, or 29 to 30 linked nucleosides.


In certain embodiments, oligonucleotides consist of 16 linked nucleosides. In certain embodiments, oligonucleotides consist of 17 linked nucleosides. In certain embodiments, oligonucleotides consist of 18 linked nucleosides. In certain embodiments, oligonucleotides consist of 19 linked nucleosides. In certain embodiments, oligonucleotides consist of 20 linked nucleosides.


D. Certain Modified Oligonucleotides

In certain embodiments, the above modifications (sugar, nucleobase, internucleoside linkage) are incorporated into a modified oligonucleotide. In certain embodiments, modified oligonucleotides are characterized by their modification motifs and overall lengths. In certain embodiments, such parameters are each independent of one another. Thus, unless otherwise indicated, each internucleoside linkage of an oligonucleotide having a particular sugar motif may be modified or unmodified and may or may not follow the modification pattern of the sugar modifications. Unless otherwise indicated, all modifications are independent of nucleobase sequence.


E. Certain Populations of Modified Oligonucleotides

Populations of modified oligonucleotides in which all of the modified oligonucleotides of the population have the same molecular formula can be stereorandom populations or chirally enriched populations. All of the chiral centers of all of the modified oligonucleotides are stereorandom in a stereorandom population. In a chirally enriched population, at least one particular chiral center is not stereorandom in the modified oligonucleotides of the population. In certain embodiments, the modified oligonucleotides of a chirally enriched population are enriched for β-D ribosyl sugar moieties, and all of the phosphorothioate internucleoside linkages are stereorandom. In certain embodiments, the modified oligonucleotides of a chirally enriched population are enriched for both β-D ribosyl sugar moieties and at least one, particular phosphorothioate internucleoside linkage in a particular stereochemical configuration.


F. Nucleobase Sequence

In certain embodiments, oligonucleotides (unmodified or modified oligonucleotides) are further described by their nucleobase sequence. In certain embodiments oligonucleotides have a nucleobase sequence that is complementary to a second oligonucleotide or an identified reference nucleic acid, such as a target nucleic acid. In certain such embodiments, a portion of an oligonucleotide has a nucleobase sequence that is complementary to a second oligonucleotide or an identified reference nucleic acid, such as a target nucleic acid. In certain such embodiments, a portion of an oligonucleotide has a nucleobase sequence that is complementary to a second oligonucleotide or an identified reference nucleic acid, such as a target nucleic acid. In certain embodiments, the nucleobase sequence of a portion or entire length of an oligonucleotide is at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% complementary to the second oligonucleotide or nucleic acid, such as a target nucleic acid.


II. Certain Oligomeric Compounds

In certain embodiments, provided herein are oligomeric compounds, which consist of an oligonucleotide (modified or unmodified) and optionally one or more conjugate groups and/or terminal groups. Conjugate groups consist of one or more conjugate moiety and a conjugate linker which links the conjugate moiety to the oligonucleotide. Conjugate groups may be attached to either or both ends of an oligonucleotide and/or at any internal position. In certain embodiments, conjugate groups are attached to the 2′-position of a nucleoside of a modified oligonucleotide. In certain embodiments, conjugate groups that are attached to either or both ends of an oligonucleotide are terminal groups. In certain such embodiments, conjugate groups or terminal groups are attached at the 3′ and/or 5′-end of oligonucleotides. In certain such embodiments, conjugate groups (or terminal groups) are attached at the 3′-end of oligonucleotides. In certain embodiments, conjugate groups are attached near the 3′-end of oligonucleotides. In certain embodiments, conjugate groups (or terminal groups) are attached at the 5′-end of oligonucleotides. In certain embodiments, conjugate groups are attached near the 5′-end of oligonucleotides.


Examples of terminal groups include but are not limited to conjugate groups, capping groups, phosphate moieties, protecting groups, abasic nucleosides, modified or unmodified nucleosides, and two or more nucleosides that are independently modified or unmodified.


A. Certain Conjugate Groups

In certain embodiments, oligonucleotides are covalently attached to one or more conjugate groups. In certain embodiments, conjugate groups modify one or more properties of the attached oligonucleotide, including but not limited to pharmacodynamics, pharmacokinetics, stability, binding, absorption, tissue distribution, cellular distribution, cellular uptake, charge, and clearance.


In certain embodiments, conjugation of one or more carbohydrate moieties to a modified oligonucleotide can optimize one or more properties of the modified oligonucleotide. In certain embodiments, the carbohydrate moiety is attached to a modified subunit of the modified oligonucleotide. For example, the ribose sugar of one or more ribonucleotide subunits of a modified oligonucleotide can be replaced with another moiety, e.g. a non-carbohydrate (preferably cyclic) carrier to which is attached a carbohydrate ligand. A ribonucleotide subunit in which the ribose sugar of the subunit has been so replaced is referred to herein as a ribose replacement modification subunit (RRMS), which is a modified sugar moiety. A cyclic carrier may be a carbocyclic ring system, i.e., one or more ring atoms may be a heteroatom, e.g., nitrogen, oxygen, sulphur. The cyclic carrier may be a monocyclic ring system, or may contain two or more rings, e.g. fused rings. The cyclic carrier may be a fully saturated ring system, or it may contain one or more double bonds.


In certain embodiments, conjugate groups impart a new property on the attached oligonucleotide, e.g., fluorophores or reporter groups that enable detection of the oligonucleotide. Certain conjugate groups and conjugate moieties have been described previously, for example: cholesterol moiety (Letsinger et al., Proc. Natl. Acad. Sci. USA, 1989, 86, 6553-6556), cholic acid (Manoharan et al., Bioorg. Med. Chem. Lett., 1994, 4, 1053-1060), a thioether, e.g., hexyl-S-tritylthiol (Manoharan et al., Ann. N. Y. Acad. Sci., 1992, 660, 306-309; Manoharan et al., Bioorg. Med. Chem. Lett., 1993, 3, 2765-2770), a thiocholesterol (Oberhauser et al., Nucl. Acids Res., 1992, 20, 533-538), an aliphatic chain, e.g., do-decan-diol or undecyl residues (Saison-Behmoaras et al., EMBO J., 1991, 10, 1111-1118; Kabanov et al., FEBS Lett., 1990, 259, 327-330; Svinarchuk et al., Biochimie, 1993, 75, 49-54), a phospholipid, e.g., di-hexadecyl-rac-glycerol or triethyl-ammonium 1,2-di-O-hexadecyl-rac-glycero-3-H-phosphonate (Manoharan et al., Tetrahedron Lett., 1995, 36, 3651-3654; Shea et al., Nucl. Acids Res., 1990, 18, 3777-3783), a polyamine or a polyethylene glycol chain (Manoharan et al., Nucleosides & Nucleotides, 1995, 14, 969-973), or adamantane acetic acid a palmityl moiety (Mishra et al., Biochim. Biophys. Acta, 1995, 1264, 229-237), an octadecylamine or hexylamino-carbonyl-oxycholesterol moiety (Crooke et al., J. Pharmacol. Exp. Ther., 1996, 277, 923-937), a tocopherol group (Nishina et al., Molecular Therapy Nucleic Acids, 2015, 4, e220; and Nishina et al., Molecular Therapy, 2008, 16, 734-740), or a GalNAc cluster (e.g., WO2014/179620).


In certain embodiments, the conjugate group may comprise a conjugate moiety selected from any of a C22 alkyl, C20 alkyl, C16 alkyl, C10 alkyl, C21 alkyl, C19 alkyl, C18 alkyl, C17 alkyl, C15 alkyl, C14 alkyl, C13 alkyl, C12 alkyl, C11 alkyl, C9 alkyl, C8 alkyl, C7 alkyl, C6 alkyl, C5 alkyl, C22 alkenyl, C20 alkenyl, C16 alkenyl, C10 alkenyl, C21 alkenyl, C19 alkenyl, C18 alkenyl, C17 alkenyl, C15 alkenyl, C14 alkenyl, C13 alkenyl, C12 alkenyl, C11 alkenyl, C9 alkenyl, C8 alkenyl, C7 alkenyl, C6 alkenyl, or C5 alkenyl.


In certain embodiments, the conjugate group may comprise a conjugate moiety selected from any of a C22 alkyl, C20 alkyl, C17 alkyl, C16 alkyl, C10 alkyl, C21 alkyl, C19 alkyl, C18 alkyl, C15 alkyl, C14 alkyl, C13 alkyl, C12 alkyl, C11 alkyl, C9 alkyl, C8 alkyl, C7 alkyl, C6 alkyl, or C5 alkyl, where the alkyl chain has one or more unsaturated bonds.


In certain embodiments, a conjugate group is a lipid having the following structure:




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1. Conjugate Moieties

Conjugate moieties include, without limitation, intercalators, reporter molecules, polyamines, polyamides, peptides, carbohydrates, vitamin moieties, polyethylene glycols, thioethers, polyethers, cholesterols, thiocholesterols, cholic acid moieties, folate, lipids, lipophilic groups, phospholipids, biotin, phenazine, phenanthridine, anthraquinone, adamantane, acridine, fluoresceins, rhodamines, coumarins, fluorophores, and dyes.


In certain embodiments, a conjugate moiety comprises an active drug substance, for example, aspirin, warfarin, phenylbutazone, ibuprofen, suprofen, fen-bufen, ketoprofen, (S)-(+)-pranoprofen, carprofen, dansylsarcosine, 2,3,5-triiodobenzoic acid, fingolimod, flufenamic acid, folinic acid, a benzothiadiazide, chlorothiazide, a diazepine, indo-methicin, a barbiturate, a cephalosporin, a sulfa drug, an antidiabetic, an antibacterial, or an antibiotic.


2. Conjugate Linkers

Conjugate moieties are attached to oligonucleotides through conjugate linkers. In certain oligomeric compounds, the conjugate linker is a single chemical bond (i.e., the conjugate moiety is attached directly to an oligonucleotide through a single bond). In certain oligomeric compounds, a conjugate moiety is attached to an oligonucleotide via a more complex conjugate linker comprising one or more conjugate linker moieties, which are subunits making up a conjugate linker. In certain embodiments, the conjugate linker comprises a chain structure, such as a hydrocarbyl chain, or an oligomer of repeating units such as ethylene glycol, nucleosides, or amino acid units. In certain embodiments, a conjugate linker comprises pyrrolidine.


In certain embodiments, a conjugate linker comprises one or more groups selected from alkyl, amino, oxo, amide, disulfide, polyethylene glycol, ether, thioether, and hydroxylamino. In certain such embodiments, the conjugate linker comprises groups selected from alkyl, amino, oxo, amide and ether groups. In certain embodiments, the conjugate linker comprises groups selected from alkyl and amide groups. In certain embodiments, the conjugate linker comprises groups selected from alkyl and ether groups. In certain embodiments, the conjugate linker comprises at least one phosphorus moiety. In certain embodiments, the conjugate linker comprises at least one phosphate group. In certain embodiments, the conjugate linker includes at least one neutral linking group.


In certain embodiments, conjugate linkers, including the conjugate linkers described above, are bifunctional linking moieties, e.g., those known in the art to be useful for attaching conjugate moieties to compounds, such as the oligonucleotides provided herein. In general, a bifunctional linking moiety comprises at least two functional groups. One of the functional groups is selected to react with a particular site on a compound and the other is selected to react with a conjugate moiety. Examples of functional groups used in a bifunctional linking moiety include but are not limited to electrophiles for reacting with nucleophilic groups and nucleophiles for reacting with electrophilic groups. In certain embodiments, bifunctional linking moieties comprise one or more groups selected from amino, hydroxyl, carboxylic acid, thiol, alkyl, alkenyl, and alkynyl.


Examples of conjugate linkers include but are not limited to pyrrolidine, 8-amino-3,6-dioxaoctanoic acid (ADO), succinimidyl 4-(N-maleimidomethyl) cyclohexane-1-carboxylate (SMCC) and 6-aminohexanoic acid (AHEX or AHA). Other conjugate linkers include but are not limited to substituted or unsubstituted C1-C10 alkyl, substituted or unsubstituted C2-C10 alkenyl or substituted or unsubstituted C2-C10 alkynyl, wherein a nonlimiting list of preferred substituent groups includes hydroxyl, amino, alkoxy, carboxy, benzyl, phenyl, nitro, thiol, thioalkoxy, halogen, alkyl, aryl, alkenyl and alkynyl.


In certain embodiments, conjugate linkers comprise 1-10 linker-nucleosides. In certain embodiments, conjugate linkers comprise 2-5 linker-nucleosides. In certain embodiments, conjugate linkers comprise 1-3 linker nucleosides. In certain embodiments, conjugate linkers comprise exactly 3 linker-nucleosides. In certain embodiments, conjugate linkers comprise the TCA motif. In certain embodiments, such linker-nucleosides are modified nucleosides. In certain embodiments such linker-nucleosides comprise a modified sugar moiety. In certain embodiments, linker-nucleosides are unmodified. In certain embodiments, linker-nucleosides comprise an optionally protected heterocyclic base selected from a purine, substituted purine, pyrimidine or substituted pyrimidine. In certain embodiments, a cleavable moiety is a nucleoside selected from uracil, thymine, cytosine, 4-N-benzoylcytosine, 5-methyl cytosine, 4-N-benzoyl-5-methyl cytosine, adenine, 6-N-benzoyladenine, guanine and 2-N-isobutyrylguanine. It is typically desirable for linker-nucleosides to be cleaved from the oligomeric compound after it reaches a target tissue. Accordingly, linker-nucleosides are typically linked to one another and to the remainder of the oligomeric compound through cleavable bonds. In certain embodiments, such cleavable bonds are phosphodiester bonds.


Herein, linker-nucleosides are not considered to be part of the oligonucleotide. Accordingly, in embodiments in which an oligomeric compound comprises an oligonucleotide consisting of a specified number or range of linked nucleosides and/or a specified percent complementarity to a reference nucleic acid and the oligomeric compound also comprises a conjugate group comprising a conjugate linker comprising linker-nucleosides, those linker-nucleosides are not counted toward the length of the oligonucleotide and are not used in determining the percent complementarity of the oligonucleotide for the reference nucleic acid. For example, an oligomeric compound may comprise (1) a modified oligonucleotide consisting of 8-30 nucleosides and (2) a conjugate group comprising 1-10 linker-nucleosides that are contiguous with the nucleosides of the modified oligonucleotide. The total number of contiguous linked nucleosides in such an oligomeric compound is more than 30. Alternatively, an oligomeric compound may comprise a modified oligonucleotide consisting of 8-30 nucleosides and no conjugate group. The total number of contiguous linked nucleosides in such an oligomeric compound is no more than 30. Unless otherwise indicated conjugate linkers comprise no more than 10 linker-nucleosides. In certain embodiments, conjugate linkers comprise no more than 5 linker-nucleosides. In certain embodiments, conjugate linkers comprise no more than 3 linker-nucleosides. In certain embodiments, conjugate linkers comprise no more than 2 linker-nucleosides. In certain embodiments, conjugate linkers comprise no more than 1 linker-nucleoside.


In certain embodiments, it is desirable for a conjugate group to be cleaved from the oligonucleotide. For example, in certain circumstances oligomeric compounds comprising a particular conjugate moiety are better taken up by a particular cell type, but once the oligomeric compound has been taken up, it is desirable that the conjugate group be cleaved to release the unconjugated or parent oligonucleotide. Thus, certain conjugate linkers may comprise one or more cleavable moieties. In certain embodiments, a cleavable moiety is a cleavable bond. In certain embodiments, a cleavable moiety is a group of atoms comprising at least one cleavable bond. In certain embodiments, a cleavable moiety comprises a group of atoms having one, two, three, four, or more than four cleavable bonds. In certain embodiments, a cleavable moiety is selectively cleaved inside a cell or subcellular compartment, such as a lysosome. In certain embodiments, a cleavable moiety is selectively cleaved by endogenous enzymes, such as nucleases.


In certain embodiments, a cleavable bond is selected from among: an amide, an ester, an ether, one or both esters of a phosphodiester, a phosphate ester, a carbamate, or a disulfide. In certain embodiments, a cleavable bond is one or both of the esters of a phosphodiester. In certain embodiments, a cleavable moiety comprises a phosphate or phosphodiester. In certain embodiments, the cleavable moiety is a phosphate linkage between an oligonucleotide and a conjugate moiety or conjugate group.


In certain embodiments, a cleavable moiety comprises or consists of one or more linker-nucleosides. In certain such embodiments, the one or more linker-nucleosides are linked to one another and/or to the remainder of the oligomeric compound through cleavable bonds. In certain embodiments, such cleavable bonds are unmodified phosphodiester bonds. In certain embodiments, a cleavable moiety is 2′-deoxyribonucleoside that is attached to either the 3′ or 5-terminal nucleoside of an oligonucleotide by a phosphate internucleoside linkage and covalently attached to the remainder of the conjugate linker or conjugate moiety by a phosphodiester or phosphorothioate internucleoside linkage. In certain such embodiments, the cleavable moiety is 2′-deoxyadenosine.


1. Cell-Targeting Moieties

In certain embodiments, a conjugate group comprises a cell-targeting moiety. In certain embodiments, a conjugate group has the general formula:




embedded image


wherein n is from 1 to about 3, m is 0 when n is 1, m is 1 when n is 2 or greater, j is 1 or 0, and k is 1 or 0.


In certain embodiments, n is 1, j is 1 and k is 0. In certain embodiments, n is 1, j is 0 and k is 1. In certain embodiments, n is 1, j is 1 and k is 1. In certain embodiments, n is 2, j is 1 and k is 0. In certain embodiments, n is 2, j is 0 and k is 1. In certain embodiments, n is 2, j is 1 and k is 1. In certain embodiments, n is 3, j is 1 and k is 0. In certain embodiments, n is 3, j is 0 and k is 1. In certain embodiments, n is 3, j is 1 and k is 1.


In certain embodiments, conjugate groups comprise cell-targeting moieties that have at least one tethered ligand. In certain embodiments, cell-targeting moieties comprise two tethered ligands covalently attached to a branching group. In certain embodiments, cell-targeting moieties comprise three tethered ligands covalently attached to a branching group.


In certain embodiments, each ligand of a cell-targeting moiety has an affinity for at least one type of receptor on a target cell. In certain embodiments, each ligand has an affinity for at least one type of receptor on the surface of a mammalian liver cell. In certain embodiments, each ligand has an affinity for the hepatic asialoglycoprotein receptor (ASGP-R). In certain embodiments, each ligand is a carbohydrate.


In certain embodiments, a conjugate group comprises a cell-targeting conjugate moiety. In certain embodiments, a conjugate group has the general formula:




embedded image


wherein n is from 1 to about 3, m is 0 when n is 1, m is 1 when n is 2 or greater, j is 1 or 0, and k is 1 or 0.


In certain embodiments, n is 1, j is 1 and k is 0. In certain embodiments, n is 1, j is 0 and k is 1. In certain embodiments, n is 1, j is 1 and k is 1. In certain embodiments, n is 2, j is 1 and k is 0. In certain embodiments, n is 2, j is 0 and k is 1. In certain embodiments, n is 2, j is 1 and k is 1. In certain embodiments, n is 3, j is 1 and k is 0. In certain embodiments, n is 3, j is 0 and k is 1. In certain embodiments, n is 3, j is 1 and k is 1.


In certain embodiments, conjugate groups comprise cell-targeting moieties that have at least one tethered ligand. In certain embodiments, cell-targeting moieties comprise two tethered ligands covalently attached to a branching group. In certain embodiments, cell-targeting moieties comprise three tethered ligands covalently attached to a branching group.


B. Certain Terminal Groups

In certain embodiments, oligomeric compounds comprise one or more terminal groups. In certain such embodiments, oligomeric compounds comprise a stabilized 5′-phosphate. Stabilized 5′-phosphates include, but are not limited to 5′-phosphanates, including, but not limited to 5′-vinylphosphonates. In certain embodiments, terminal groups comprise one or more abasic sugar moieties and/or inverted nucleosides. In certain embodiments, terminal groups comprise one or more 2′-linked nucleosides or sugar moieties. In certain such embodiments, the 2′-linked group is an abasic sugar moiety.


C. Oligomeric Duplexes

In certain embodiments, oligomeric compounds described herein comprise an oligonucleotide, having a nucleobase sequence complementary to that of a target nucleic acid. In certain embodiments, an oligomeric compound is paired with a second oligomeric compound to form an oligomeric duplex. Such oligomeric duplexes comprise a first oligomeric compound having a portion complementary to a target nucleic acid and a second oligomeric compound having a portion complementary to the first oligomeric compound. In certain embodiments, the first oligomeric compound of an oligomeric duplex comprises or consists of (1) a modified or unmodified oligonucleotide and optionally a conjugate group and (2) a second modified or unmodified oligonucleotide and optionally a conjugate group. Either or both oligomeric compounds of an oligomeric duplex may comprise a conjugate group. The oligonucleotides of each oligomeric compound of an oligomeric duplex may include non-complementary overhanging nucleosides.


D. Antisense Activity

In certain embodiments, oligomeric compounds and oligomeric duplexes are capable of hybridizing to a target nucleic acid, resulting in at least one antisense activity; such oligomeric compounds and oligomeric duplexes are antisense compounds. In certain embodiments, antisense compounds have antisense activity when they reduce, modulate, or increase the amount or activity of a target nucleic acid by 25% or more in the standard in vitro assay. In certain embodiments, antisense compounds selectively affect one or more target nucleic acid. Such antisense compounds comprise a nucleobase sequence that hybridizes to one or more target nucleic acid, resulting in one or more desired antisense activity and does not hybridize to one or more non-target nucleic acid or does not hybridize to one or more non-target nucleic acid in such a way that results in significant undesired antisense activity.


In certain antisense activities, hybridization of an antisense compound to a target nucleic acid results in recruitment of a protein that cleaves the target nucleic acid. For example, certain antisense compounds result in RNase H mediated cleavage of the target nucleic acid. RNase H is a cellular endonuclease that cleaves the RNA strand of an RNA:DNA duplex. The DNA in such an RNA:DNA duplex need not be unmodified DNA. In certain embodiments, provided herein are antisense compounds that are sufficiently “DNA-like” to elicit RNase H activity. In certain embodiments, one or more non-DNA-like nucleoside in the gap of a gapmer is tolerated.


In certain antisense activities, an antisense compound or a portion of an antisense compound is loaded into an RNA-induced silencing complex (RISC), ultimately resulting in cleavage of the target nucleic acid. For example, certain antisense compounds result in cleavage of the target nucleic acid by Argonaute. Antisense compounds that are loaded into RISC are RNAi compounds. RNAi compounds may be double-stranded (siRNA or dsRNAi) or single-stranded (ssRNA).


In certain embodiments, hybridization of an antisense compound to a target nucleic acid does not result in recruitment of a protein that cleaves that target nucleic acid. In certain embodiments, hybridization of the antisense compound to the target nucleic acid results in alteration of splicing of the target nucleic acid. In certain embodiments, hybridization of an antisense compound to a target nucleic acid results in inhibition of a binding interaction between the target nucleic acid and a protein or other nucleic acid. In certain embodiments, hybridization of an antisense compound to a target nucleic acid results in alteration of translation of the target nucleic acid. In certain embodiments, hybridization of an antisense compound to a target nucleic acid results in exon inclusion. In certain embodiments, hybridization of an antisense compound to a target nucleic acid results in an increase in the amount or activity of a target nucleic acid. In certain embodiments, hybridization of an antisense compound complementary to a target nucleic acid results in alteration of splicing, leading to the inclusion or the exclusion of an exon in the mRNA.


Antisense activities may be observed directly or indirectly. In certain embodiments, observation or detection of an antisense activity involves observation or detection of a change in an amount of a target nucleic acid or protein encoded by such target nucleic acid, a change in the ratio of splice variants of a nucleic acid or protein and/or a phenotypic change in a cell or subject.


III. Certain Target Nucleic Acids

In certain embodiments, oligomeric compounds comprise or consist of an oligonucleotide comprising a portion that is complementary to a target nucleic acid. In certain embodiments, the target nucleic acid is an endogenous RNA molecule. In certain embodiments, the target nucleic acid encodes a protein. In certain such embodiments, the target nucleic acid is selected from: a mature mRNA and a pre-mRNA, including intronic, exonic and untranslated regions. In certain embodiments, the target nucleic acid is a mature mRNA. In certain embodiments, the target nucleic acid is a pre-mRNA. In certain embodiments, the target region is entirely within an intron. In certain embodiments, the target region spans an intron/exon junction. In certain embodiments, the target region is at least 50% within an intron.


A. Complementarity/Mismatches to the Target Nucleic Acid It is possible to introduce mismatch bases without eliminating activity. For example, Gautschi et al (J. Natl. Cancer Inst. 93:463-471, March 2001) demonstrated the ability of an oligonucleotide having 100% complementarity to the bcl-2 mRNA and having 3 mismatches to the bcl-xL mRNA to reduce the expression of both bcl-2 and bcl-xL in vitro and in vivo. Furthermore, this oligonucleotide demonstrated potent anti-tumor activity in vivo. Maher and Dolnick (Nuc. Acid. Res. 16:3341-3358, 1988) tested a series of tandem 14 nucleobase oligonucleotides, and a 28 and 42 nucleobase oligonucleotides comprised of the sequence of two or three of the tandem oligonucleotides, respectively, for their ability to arrest translation of human DHFR in a rabbit reticulocyte assay. Each of the three 14 nucleobase oligonucleotides alone was able to inhibit translation, albeit at a more modest level than the 28 or 42 nucleobase oligonucleotides.


In certain embodiments, oligonucleotides are complementary to the target nucleic acid over the entire length of the oligonucleotide. In certain embodiments, oligonucleotides are 99%, 95%, 90%, 85%, or 80% complementary to the target nucleic acid. In certain embodiments, oligonucleotides are at least 80% complementary to the target nucleic acid over the entire length of the oligonucleotide and comprise a portion that is 100% or fully complementary to a target nucleic acid. In certain embodiments, the portion of full complementarity is 6 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleobases in length.


In certain embodiments, oligonucleotides comprise one or more mismatched nucleobases relative to the target nucleic acid. In certain embodiments, the mismatch is at position 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 from the 5′-end of the oligonucleotide.


B. Progranulin

In certain embodiments, oligomeric compounds comprise or consist of a modified oligonucleotide that is complementary to a target nucleic acid encoding progranulin, or a portion thereof. In certain embodiments, the target nucleic acid has the sequence set forth in SEQ ID NO: 1 (GENBANK Accession No. NC_000017.11 truncated from nucleotides 44342001 to 44356000), to SEQ ID NO: 2 (GENBANK Accession No. NM_002087.3), or to both. In certain embodiments, oligomeric compounds comprise or consist of a modified oligonucleotide consisting of 18, 19, or 20 linked nucleosides, wherein the nucleobase sequence of the modified oligonucleotide comprises at least 16, at least 17, or 18 contiguous nucleobases of any of the nucleobase sequences of SEQ ID NOs:12-854.


In certain embodiments, contacting a cell with the oligomeric compound modulates the amount of progranulin RNA in a cell. In certain embodiments, contacting a cell with the oligomeric compound increases the amount of progranulin RNA in a cell. In certain embodiments, contacting a cell with the oligomeric compound modulates the amount of progranulin protein in a cell. In certain embodiments, contacting a cell with the oligomeric compound increases the amount of progranulin protein in a cell. In certain embodiments, the oligomeric compound consists of a modified oligonucleotide.


In certain embodiments, contacting a cell in a subject with the oligomeric compound ameliorates one or more symptom or hallmark of a neurological disease or disorder. In certain embodiments, the neurological disease or disorder is FTD. In certain embodiments the neurological disease or disorder is FTLD. In certain embodiments, the neurological disease or disorder is NCL. In certain embodiments, the neurological disease or disorder is a TDP-43 proteinopathy. In certain embodiments, the disease or disorder is a lysosomal storage disorder. In some embodiments, the disease or disorder is ALS. In certain embodiments, the symptom or hallmark is any of deterioration in behavior and personality, language impairment, disturbances or alterations in muscle or motor functions, memory loss, cognitive dysfunction, tremor, seizures, or dizziness.


In certain embodiments, the oligomeric compound is capable of increasing progranulin RNA in vitro by at least 0.5 fold, at least 1 fold, at least 2 fold, or at least 3 fold when tested according to the standard in vitro assay.


C. Certain Target Nucleic Acids in Certain Tissues

In certain embodiments, oligomeric compounds comprise or consist of a modified oligonucleotide comprising a portion that is complementary to a target nucleic acid, wherein the target nucleic acid is expressed in a pharmacologically relevant tissue. In certain embodiments, the pharmacologically relevant tissues are the cells and tissues that comprise the central nervous system (CNS). Such tissues include brain tissues, such as, cortex, hypothalamus, hippocampus, cerebellum, and coronal brain tissue.


IV. Certain Pharmaceutical Compositions

In certain embodiments, described herein are pharmaceutical compositions comprising one or more oligomeric compounds. In certain embodiments, the one or more oligomeric compounds each consists of a modified oligonucleotide. In certain embodiments, the pharmaceutical composition comprises a pharmaceutically acceptable diluent or carrier. In certain embodiments, a pharmaceutical composition comprises or consists of a sterile saline solution and one or more oligomeric compound. In certain embodiments, the sterile saline is pharmaceutical grade saline. In certain embodiments, a pharmaceutical composition comprises or consists of one or more oligomeric compound and sterile water. In certain embodiments, the sterile water is pharmaceutical grade water. In certain embodiments, a pharmaceutical composition comprises or consists of one or more oligomeric compound and phosphate-buffered saline (PBS). In certain embodiments, the sterile PBS is pharmaceutical grade PBS. In certain embodiments, a pharmaceutical composition comprises or consists of one or more oligomeric compound and artificial cerebrospinal fluid (“artificial CSF” or “aCSF”). In certain embodiments, the artificial cerebrospinal fluid is pharmaceutical grade.


In certain embodiments, a pharmaceutical composition comprises a modified oligonucleotide and artificial cerebrospinal fluid. In certain embodiments, a pharmaceutical composition consists of a modified oligonucleotide and artificial cerebrospinal fluid. In certain embodiments, a pharmaceutical composition consists essentially of a modified oligonucleotide and artificial cerebrospinal fluid. In certain embodiments, the artificial cerebrospinal fluid is pharmaceutical grade artificial cerebrospinal fluid.


In certain embodiments, a pharmaceutical composition comprises a modified oligonucleotide and PBS. In certain embodiments, a pharmaceutical composition consists of a modified oligonucleotide and PBS. In certain embodiments, a pharmaceutical composition consists essentially of a modified oligonucleotide and PBS. In certain embodiments, the PBS is pharmaceutical grade PBS.


In certain embodiments, aCSF comprises sodium chloride, potassium chloride, sodium dihydrogen phosphate dihydrate, sodium phosphate dibasic anhydrous, calcium chloride dihydrate, and magnesium chloride hexahydrate. In certain embodiments, the pH of an aCSF solution is modulated with a suitable pH-adjusting agent, for example, with acids such as hydrochloric acid and alkalis such as sodium hydroxide, to a range of from about 7.1-7.3, or to about 7.2.


In certain embodiments, pharmaceutical compositions comprise one or more oligomeric compound and one or more excipients. In certain embodiments, excipients are selected from water, salt solutions, alcohol, polyethylene glycols, gelatin, lactose, amylase, magnesium stearate, talc, silicic acid, viscous paraffin, hydroxymethylcellulose and polyvinylpyrrolidone.


In certain embodiments, oligomeric compounds may be admixed with pharmaceutically acceptable active and/or inert substances for the preparation of pharmaceutical compositions or formulations. Compositions and methods for the formulation of pharmaceutical compositions depend on a number of criteria, including, but not limited to, route of administration, extent of disease, or dose to be administered.


In certain embodiments, pharmaceutical compositions comprising an oligomeric compound encompass any pharmaceutically acceptable salts of the oligomeric compound, esters of the oligomeric compound, or salts of such esters. In certain embodiments, pharmaceutical compositions comprising oligomeric compounds comprising one or more oligonucleotide, upon administration to a subject, including a human, are capable of providing (directly or indirectly) the biologically active metabolite or residue thereof. Accordingly, for example, the disclosure is also drawn to pharmaceutically acceptable salts of oligomeric compounds, prodrugs, pharmaceutically acceptable salts of such prodrugs, and other bioequivalents. In certain embodiments, pharmaceutically acceptable salts comprise inorganic salts, such as monovalent or divalent inorganic salts. Suitable pharmaceutically acceptable salts include, but are not limited to, sodium, potassium, calcium, and magnesium salts. In certain embodiments, prodrugs comprise one or more conjugate group attached to a modified oligonucleotide, wherein the conjugate group is cleaved by endogenous nucleases within the body. In certain embodiments, prodrugs comprise one or more conjugate group attached to a modified oligonucleotide, wherein the conjugate group is cleaved by endogenous nucleases within the body.


In certain embodiments, oligomeric compounds are lyophilized and isolated as sodium salts. In certain embodiments, the sodium salt of an oligomeric compound is mixed with a pharmaceutically acceptable diluent. In certain embodiments, the pharmaceutically acceptable diluent comprises sterile saline, sterile water, PBS, or aCSF. In certain embodiments, the sodium salt of an oligomeric compound is mixed with PBS. In certain embodiments, the sodium salt of an oligomeric compound is mixed with aCSF.


Lipid moieties have been used in nucleic acid therapies in a variety of methods. In certain such methods, the nucleic acid, such as an oligomeric compound, is introduced into preformed liposomes or lipoplexes made of mixtures of cationic lipids and neutral lipids. In certain methods, DNA complexes with mono- or poly-cationic lipids are formed without the presence of a neutral lipid. In certain embodiments, a lipid moiety is selected to increase distribution of a pharmaceutical agent to a particular cell or tissue. In certain embodiments, a lipid moiety is selected to increase distribution of a pharmaceutical agent to fat tissue. In certain embodiments, a lipid moiety is selected to increase distribution of a pharmaceutical agent to muscle tissue.


In certain embodiments, pharmaceutical compositions comprise a delivery system. Examples of delivery systems include, but are not limited to, liposomes and emulsions. Certain delivery systems are useful for preparing certain pharmaceutical compositions including those comprising hydrophobic compounds. In certain embodiments, certain organic solvents such as dimethylsulfoxide are used.


In certain embodiments, pharmaceutical compositions comprise one or more tissue-specific delivery molecules designed to deliver the one or more pharmaceutical agents comprising an oligomeric compound provided herein to specific tissues or cell types. For example, in certain embodiments, pharmaceutical compositions include liposomes coated with a tissue-specific antibody.


In certain embodiments, pharmaceutical compositions comprise a co-solvent system. Certain of such co-solvent systems comprise, for example, benzyl alcohol, a nonpolar surfactant, a water-miscible organic polymer, and an aqueous phase. In certain embodiments, such co-solvent systems are used for hydrophobic compounds. A non-limiting example of such a co-solvent system is the VPD co-solvent system, which is a solution of absolute ethanol comprising 3% w/v benzyl alcohol, 8% w/v of the nonpolar surfactant Polysorbate 80™ and 65% w/v polyethylene glycol 300. The proportions of such co-solvent systems may be varied considerably without significantly altering their solubility and toxicity characteristics. Furthermore, the identity of co-solvent components may be varied: for example, other surfactants may be used instead of Polysorbate 80™; the fraction size of polyethylene glycol may be varied; other biocompatible polymers may replace polyethylene glycol, e.g., polyvinyl pyrrolidone; and other sugars or polysaccharides may substitute for dextrose.


In certain embodiments, pharmaceutical compositions are prepared for oral administration. In certain embodiments, pharmaceutical compositions are prepared for buccal administration. In certain embodiments, a pharmaceutical composition is prepared for administration by injection (e.g., intravenous, subcutaneous, intramuscular, intrathecal (IT), intracerebroventricular (ICV), intraneural, perineural, etc.). In certain of such embodiments, a pharmaceutical composition comprises a carrier and is formulated in aqueous solution, such as water or physiologically compatible buffers such as Hanks's solution, Ringer's solution, or physiological saline buffer. In certain embodiments, other ingredients are included (e.g., ingredients that aid in solubility or serve as preservatives). In certain embodiments, injectable suspensions are prepared using appropriate liquid carriers, suspending agents and the like. Certain pharmaceutical compositions for injection are presented in unit dosage form, e.g., in ampoules or in multi-dose containers. Certain pharmaceutical compositions for injection are suspensions, solutions or emulsions in oily or aqueous vehicles, and may contain formulatory agents such as suspending, stabilizing and/or dispersing agents. Certain solvents suitable for use in pharmaceutical compositions for injection include, but are not limited to, lipophilic solvents and fatty oils, such as sesame oil, synthetic fatty acid esters, such as ethyl oleate or triglycerides, and liposomes.


Under certain conditions, certain compounds disclosed herein act as acids. Although such compounds may be drawn or described in protonated (free acid) form, or ionized and in association with a cation (salt) form, aqueous solutions of such compounds exist in equilibrium among such forms. For example, a phosphate linkage of an oligonucleotide in aqueous solution exists in equilibrium among free acid, anion and salt forms. Unless otherwise indicated, compounds described herein are intended to include all such forms. Moreover, certain oligonucleotides have several such linkages, each of which is in equilibrium. Thus, oligonucleotides in solution exist in an ensemble of forms at multiple positions all at equilibrium. The term “oligonucleotide” is intended to include all such forms. Drawn structures necessarily depict a single form. Nevertheless, unless otherwise indicated, such drawings are likewise intended to include corresponding forms. Herein, a structure depicting the free acid of a compound followed by the term “or salt thereof” or “or a pharmaceutically acceptable salt thereof” expressly includes all such forms that may be fully or partially protonated/de-protonated/in association with a cation or a combination of cations. In certain embodiments, one or more specific cation is identified. The cations include, but are not limited to, sodium, potassium, calcium, and magnesium. In certain embodiments, a structure depicting the free acid of a compound followed by the term “or a pharmaceutically acceptable salt thereof” expressly includes all such forms that may be fully or partially protonated/de-protonated/in association with one or more cations selected from sodium, potassium, calcium, and magnesium.


In certain embodiments, modified oligonucleotides or oligomeric compounds are in aqueous solution with sodium. In certain embodiments, modified oligonucleotides or oligomeric compounds are in aqueous solution with potassium. In certain embodiments, modified oligonucleotides or oligomeric compounds are in PBS. In certain embodiments, modified oligonucleotides or oligomeric compounds are in water. In certain such embodiments, the pH of the solution is adjusted with NaOH and/or HCl to achieve a desired pH.


Herein, certain specific doses are described. A dose may be in the form of a dosage unit. For clarity, a dose (or dosage unit) of a modified oligonucleotide or an oligomeric compound in milligrams indicates the mass of the free acid form of the modified oligonucleotide or oligomeric compound. As described above, in aqueous solution, the free acid is in equilibrium with anionic and salt forms. However, for the purpose of calculating dose, it is assumed that the modified oligonucleotide or oligomeric compound exists as a solvent-free, sodium-acetate free, anhydrous, free acid. For example, where a modified oligonucleotide or an oligomeric compound is in solution comprising sodium (e.g., saline), the modified oligonucleotide or oligomeric compound may be partially or fully de-protonated and in association with Na+ ions. However, the mass of the protons are nevertheless counted toward the weight of the dose, and the mass of the Na+ ions are not counted toward the weight of the dose. Thus, for example, a dose, or dosage unit, of 10 mg of Compound No. 1557993, equals the number of fully protonated molecules that weighs 10 mg. This would be equivalent to 10.51 mg of solvent-free, sodium acetate-free, anhydrous sodiated Compound No. 1557993. When an oligomeric compound comprises a conjugate group, the mass of the conjugate group is included in calculating the dose of such oligomeric compound. If the conjugate group also has an acid, the conjugate group is likewise assumed to be fully protonated for the purpose of calculating dose.


In certain embodiments, where a modified oligonucleotide or oligomeric compound is in a solution, such as aCSF, comprising sodium, potassium, calcium, and magnesium, the modified oligonucleotide or oligomeric compound may be partially or fully de-protonated and in association with sodium, potassium, calcium, and/or magnesium.


However, the mass of the protons is nevertheless counted toward the weight of the dose, and the mass of the sodium, potassium, calcium, and magnesium ions is not counted toward the weight of the dose.


In certain embodiments, when an oligomeric compound comprises a conjugate group, the mass of the conjugate group is included in calculating the dose of such oligomeric compound. If the conjugate group also has an acid, the conjugate group is likewise assumed to be fully protonated for the purpose of calculating dose.


V. Certain Hotspot Regions

In certain embodiments, nucleobases 8,497-8,552 of SEQ ID NO: 1 comprise a hotspot region. In certain embodiments, modified oligonucleotides are complementary to a portion within nucleobases 8,497-8,552 of SEQ ID NO: 1. In certain embodiments, modified oligonucleotides are 18 nucleobases in length. In certain embodiments, each nucleoside of the modified oligonucleotide comprises a 2′-MOE sugar moiety. In certain embodiments, all of the internucleoside linkages of the modified oligonucleotides are phosphorothioate internucleoside linkages. In certain embodiments, the modified oligonucleotide has an internucleoside linkage motif of (from 5′ to 3′): sosssssssosssssss.


The nucleobase sequences of SEQ ID NOs: 34, 268, 345, 424, 501, 579, 657, 735, 800, 801, 806, 812, 813, 816, 821, 828, 844, and 847 are complementary to a portion of nucleobases 8,497-8,552 of SEQ ID NO: 1.


The nucleobase sequence of Compound Nos.: 1212177, 1212178, 1212179, 1212180, 1212181, 1212182, 1212183, 1212184, 1366757, 1366758, 1366763, 1366769, 1366770, 1366773, 1366778, 1366785, 1366801, and 1366804 are complementary to a portion within nucleobases 8,497-8,552 of SEQ ID NO: 1.


In certain embodiments, modified oligonucleotides complementary to a portion within nucleobases 8,497-8,552 of SEQ ID NO: 1 achieve at least 100% expression of progranulin RNA in the standard in vitro assay. In certain embodiments, modified oligonucleotides complementary to a portion within nucleobases 8,497-8,552 of SEQ ID NO: 1 achieve an average of 147% expression of progranulin RNA in the standard in vitro assay.


NONLIMITING DISCLOSURE AND INCORPORATION BY REFERENCE

Each of the literature and patent publications listed herein is incorporated by reference in its entirety. While certain compounds, compositions and methods described herein have been described with specificity in accordance with certain embodiments, the following examples serve only to illustrate the compounds described herein and are not intended to limit the same. Each of the references, GenBank accession numbers, ENSEMBL identifiers, and the like recited in the present application is incorporated herein by reference in its entirety.


Although the sequence listing accompanying this filing identifies each sequence as either “RNA” or “DNA” as required, in reality, those sequences may be modified with any combination of chemical modifications. One of skill in the art will readily appreciate that such designation as “RNA” or “DNA” to describe modified oligonucleotides is, in certain instances, arbitrary. For example, an oligonucleotide comprising a nucleoside comprising a 2′-OH sugar moiety and a thymine base could be described as a DNA having a modified sugar moiety (2′-OH in place of one 2′-H of DNA) or as an RNA having a modified base (thymine (methylated uracil) in place of a uracil of RNA). Accordingly, nucleic acid sequences provided herein, including, but not limited to those in the sequence listing, are intended to encompass nucleic acids containing any combination of natural or modified RNA and/or DNA, unless otherwise stated, including, but not limited to such nucleic acids having modified nucleobases. By way of further example and without limitation, an oligomeric compound having the nucleobase sequence “ATCGATCG” encompasses any oligomeric compounds having such nucleobase sequence, whether modified or unmodified, including, but not limited to, such compounds comprising RNA bases, such as those having sequence “AUCGAUCG” and those having some DNA bases and some RNA bases such as “AUCGATCG” and oligomeric compounds having other modified nucleobases, such as “ATmCGAUCG,” wherein mC indicates a cytosine base comprising a methyl group at the 5-position. Finally, for clarity, unless otherwise indicated, the phrase “nucleobase sequence of SEQ ID NO: X”, refers only to the sequence of nucleobases in that SEQ ID NO: X, independent of any sugar modifications or internucleoside linkage modifications also described in such SEQ ID NO: X.


Certain compounds described herein (e.g., modified oligonucleotides) have one or more asymmetric center and thus give rise to enantiomers, diastereomers, and other stereoisomeric configurations that may be defined, in terms of absolute stereochemistry, as (R) or (S), as a or $ such as for sugar anomers, or as (D) or (L), such as for amino acids, etc. Compounds provided herein that are drawn or described as having certain stereoisomeric configurations include only the indicated compounds. Compounds provided herein that are drawn or described with undefined stereochemistry include all such possible isomers, including their stereorandom and optically pure forms, unless specified otherwise. Likewise, all cis- and trans-isomers and tautomeric forms of the compounds herein are also included unless otherwise indicated. Oligomeric compounds described herein include chirally pure or enriched mixtures as well as racemic mixtures. For example, oligomeric compounds having a plurality of phosphorothioate internucleoside linkages include such compounds in which chirality of the phosphorothioate internucleoside linkages is controlled or is random. Unless otherwise indicated, compounds described herein are intended to include corresponding salt forms.


The compounds described herein include variations in which one or more atoms are replaced with a non-radioactive isotope or radioactive isotope of the indicated element. For example, compounds herein that comprise hydrogen atoms encompass all possible deuterium substitutions for each of the 1H hydrogen atoms. Isotopic substitutions encompassed by the compounds herein include but are not limited to: 2H or 3H in place of 1H, 13C or 14C in place of 12C, 15N in place of 14N, 17O or 18O in place of 16O and 33S, 34S, 35S, or 36S in place of 32S. In certain embodiments, non-radioactive isotopic substitutions may impart new properties on the oligomeric compound that are beneficial for use as a therapeutic or research tool. In certain embodiments, radioactive isotopic substitutions may make the compound suitable for research or diagnostic purposes such as imaging.


Examples

The following examples illustrate certain embodiments of the present disclosure and are not limiting. Moreover, where specific embodiments are provided, the inventors have contemplated generic application of those specific embodiments. For example, disclosure of an oligonucleotide having a particular motif provides reasonable support for additional oligonucleotides having the same or similar motif. And, for example, where a particular high-affinity modification appears at a particular position, other high-affinity modifications at the same position are considered suitable, unless otherwise indicated.


Example 1: Effect of Uniform MOE Modified Oligonucleotides with Uniform Phosphorothioate Internucleoside Linkages on Human Progranulin In Vitro, Single Dose

Modified oligonucleotides complementary to a human progranulin nucleic acid were designed and tested for their effect on progranulin RNA in vitro. The modified oligonucleotides were tested in a series of experiments that had the same culture conditions.


“Start site” indicates the 5′-most nucleoside to which the modified oligonucleotide is complementary in the target nucleic acid sequence. “Stop site” indicates the 3′-most nucleoside to which the modified oligonucleotide is complementary in the target nucleic acid sequence. Each modified oligonucleotide listed in the tables below is 100% complementary to SEQ ID NO: 1 (GENBANK Accession No. NC_000017.11 truncated from nucleotides 44342001 to 44356000), to SEQ ID NO: 2 (GENBANK Accession No. NM_002087.3), or to both. ‘N/A’ indicates that the modified oligonucleotide is not 100% complementary to that particular target nucleic acid sequence.


The modified oligonucleotides in the table below are 18 nucleosides in length, the sugar motif for the modified oligonucleotides is (from 5′ to 3′): eeeeeeeeeeeeeeeeee; wherein “e” represents a 2′-MOE sugar moiety. The internucleoside linkage motif for the modified oligonucleotides is (from 5′ to 3′): sssssssssssssssss; wherein each “s” represents a phosphorothioate internucleoside linkage. Each cytosine residue is a 5-methyl cytosine.


Cultured A-431 cells were treated with modified oligonucleotide at a concentration of 6,000 nM by free uptake at a density of 10,000 cells per well. After a treatment period of approximately 48 hours, RNA was isolated from the cells and progranulin RNA levels were measured by quantitative real-time PCR. Human primer probe set RTS42426 (forward sequence AGGACTAACAGGGCAGTGG, designated herein as SEQ ID NO: 3; reverse sequence CAGCAGCCATACTTCCCA, designated herein as SEQ ID NO: 4; probe sequence TTGTCCAGCTCGGTCATGTGTCC designated herein as SEQ ID NO: 5) was used to measure upregulation of progranulin RNA. Progranulin RNA levels were normalized to total RNA content, as measured by RIBOGREEN®. Results are presented as percent progranulin RNA, relative to the amount in untreated control cells (% UTC). Each separate experiment described in this example is identified by an Assay Identification letter in the table column labeled “AID”.


The values marked with a “T” indicate that the RNAi compound is complementary to the amplicon region of the primer probe set. Values marked as “N.D.” are not defined. Each separate experiment described in this example is identified by an Assay Identification letter in the table column labeled “AID”.









TABLE 1







Uniform MOE modified oligonucleotides with uniform


PS linkages complementary to human progranulin
















SEQ
SEQ
SEQ
SEQ







ID
ID
ID
ID







NO:
NO:
NO:
NO:







1
1
2
2



SEQ


Compound
Start
Stop
Start
Stop

progranulin

ID


No.
Site
Site
Site
Site
Sequence (5′ to 3′)
(% UTC)
AID
NO


















1211964
3086
3103
1
18
ATCATGTGATTGGAGAAT
98
A
12





1211974
3126
3143
41
58
TCCTCCCTGCTTCCTCTC
78
A
13





1211984
3166
3183
81
98
CAGCCTGGAATGCTGTGT
76
A
14





1211994
3206
3223
121
138
ACCCGCTCCCATTGGCTA
77
A
15





1212004
3246
3263
161
178
ACCCGCCTACCTCAGTTT
76
A
16





1212014
3283
3300
198
215
ACCGGGTAGCGCTCAGAC
101
A
17





1212024
3293
3310
208
225
GGCAGCAGCAACCGGGTA
54
A
18





1212034
3303
3320
218
235
GCGGTCCTTGGGCAGCAG
117
A
19





1212044
3313
3330
228
245
GTCCGACTCCGCGGTCCT
105
A
20





1212054
N/A
N/A
238
255
GTCTGCCTGCGTCCGACT
94
A
21





1212064
7156
7173
248
265
GGTCCACATGGTCTGCCT
88
A
22





1212074
7170
7187
262
279
ACCCAGCTCACCAGGGTC
99
A
23





1212084
7220
7237
312
329
ACTGACCATCTGGGCACC
54
A
24





1212094
7270
7287
362
379
GCAGCTGTAGCTGGCTCC
80
A
25





1212104
7443
7460
412
429
CCCAGATGCCTGCTCAGT
105
A
26





1212114
7493
7510
462
479
AGATGCAGGAGTGGCCGG
95
A
27





1212124
N/A
N/A
512
529
TGCCACGGCCTCTGGGAA
103
A
28





1212134
7708
7725
562
579
TCTGCACTGCAGTGGAAG
90
A
29





1212144
8234
8251
612
629
GGATGGCACCCACGGAGT
94
A
30





1212154
8284
8301
662
679
AACACAGCACGTGGAGAA
91
A
31





1212164
N/A
N/A
712
729
CAGCAGGAAGCCTGGGGC
92
A
32





1212174
8485
8502
762
779
GAACCAGGTCGCAGAAGG
97
A
33





1212184
8535
8552
812
829
GAGCTTCTTTGCCAGGGG
126
A
34





1212194
8698
8715
862
879
CACATGACCGAGCTGGAC
13†
A
35





1212204
8748
8765
912
929
GCAGCTCACAGCAGGTAG
11†
A
36





1212214
N/A
N/A
962
979
GGAGCAGCAGGTGGCGTT
94
A
37





1212224
9084
9101
1012
1029
CTCTGGATCAGGTCACAC
112
A
38





1212234
9134
9151
1062
1079
GCAGCTTAGTGAGGAGGT
89
A
39





1212244
9383
9400
1112
1129
TGGGCAGCTCACCTCCAT
40
A
40





1212254
9433
9450
1162
1179
GGGCAGCAGCCCCAGGCC
69
A
41





1212264
9572
9589
1212
1229
CCGCGGGACAGCAGTGTA
N.D.
A
42





1212274
9622
9639
1262
1279
CTGGTGGGGCCCCTGTTC
94
A
43





1212284
9672
9689
1312
1329
TGTGGGTCTGGCAGGCTG
79
A
44





1212294
9722
9739
1362
1379
AGGAGGGACAGCTGCTGA
87
A
45





1212304
9772
9789
1412
1429
GATTGGACAGCAGCCCCA
56
A
46





1212314
10041
10058
1462
1479
TAGCCCTGGGGGCAGCAG
111
A
47





1212324
10091
10108
1512
1529
CAGCCACGATCTCGCTTC
74
A
48





1212334
10141
10158
1562
1579
GTCTCTGGGGTGGGATAA
56
A
49





1212344
10191
10208
1612
1629
GGGCAGCAGGTCTGCCCC
97
A
50





1212354
N/A
N/A
1662
1679
AGCACACAGCATGGGGCA
80
A
51





1212364
10383
10400
1712
1729
CTTCACGTTGCAGGTGTA
106
A
52





1212374
10433
10450
1762
1779
AGGAAGGTGGCAGGCTGG
131
A
53





1212384
10483
10500
1812
1829
GTCCTTCCCCACACTCCA
88
A
54





1212393
10533
10550
1862
1879
CCAGCCCTGTCGGTTGTC
86
A
55





1212402
10672
10689
1912
1929
CAGTGGCGCCGATCAGCA
90
A
56





1212412
10722
10739
1962
1979
TGCGCAAACACTTGGTAC
110
A
57





1212422
10772
10789
2012
2029
CTGTCTCAAGGCTGGGTC
69
A
58





1212432
10790
10807
2030
2047
TACTGTCCCTCACAGCAG
N.D.
A
59





1212442
10800
10817
2040
2057
GAGTCTTCAGTACTGTCC
92
A
60





1212452
10810
10827
2050
2067
GAGGGCTGCAGAGTCTTC
101
A
61





1212462
10820
10837
2060
2077
GTGGGGTCCCGAGGGCTG
80
A
62





1212472
10830
10847
2070
2087
CACCCTCCGAGTGGGGTC
124
A
63





1212482
10840
10857
2080
2097
GAGCAGAGGGCACCCTCC
94
A
64





1212492
10850
10867
2090
2107
AGGGAGGCCTGAGCAGAG
91
A
65





1212502
10860
10877
2100
2117
GGGAGGTGCTAGGGAGGC
62
A
66





1212512
10870
10887
2110
2127
TTTGGTTAGGGGGAGGTG
107
A
67





1212522
10880
10897
2120
2137
CCAGGGAGAATTTGGTTA
96
A
68





1212532
10890
10907
2130
2147
AGAATGGGGTCCAGGGAG
63
A
69





1212542
10900
10917
2140
2157
TGGGGAGCTCAGAATGGG
120
A
70





1212552
10910
10927
2150
2167
CCCATGGTGATGGGGAGC
68
A
71





1212562
10920
10937
2160
2177
GGCCCCACCTCCCATGGT
88
A
72





1212572
10930
10947
2170
2187
CTTAGATTGAGGCCCCAC
87
A
73





1212582
10940
10957
2180
2197
CAGGGAAGGCCTTAGATT
114
A
74





1212592
10950
10967
2190
2207
CCCCTTCTGACAGGGAAG
59
A
75





1212602
10960
10977
2200
2217
TTGCCACAACCCCCTTCT
68
A
76





1212612
10970
10987
2210
2227
AATGTGGCTTTTGCCACA
85
A
77





1212622
10980
10997
2220
2237
GGCAGCTTGTAATGTGGC
86
A
78





1212632
10990
11007
2230
2247
GGGAGGGGATGGCAGCTT
138
A
79





1212642
11000
11017
2240
2257
CACTGAAACGGGGAGGGG
84
A
80





1212652
11010
11027
2250
2267
CCACAGGGTCCACTGAAA
98
A
81





1212662
11020
11037
2260
2277
AAGCACCTGGCCACAGGG
128
A
82





1212672
11030
11047
2270
2287
GGATAGGGAAAAGCACCT
82
A
83





1212682
11040
11057
2280
2297
ACACCCCTGTGGATAGGG
86
A
84





1212692
11050
11067
2290
2307
ACACACACAAACACCCCT
83
A
85





1212702
11060
11077
2300
2317
GCACACGCGCACACACAC
92
A
86





1212712
11070
11087
2310
2327
TTATTGAAACGCACACGC
94
A
87





1212722
11080
11097
2320
2337
TGTACAAACTTTATTGAA
76
A
88





1212732
N/A
N/A
2330
2347
TTTAAGAAAGTGTACAAA
62
A
89





1211965
3090
3107
5
22
AGGGATCATGTGATTGGA
85
B
90





1211975
3130
3147
45
62
ACTCTCCTCCCTGCTTCC
79
B
91





1211985
3170
3187
85
102
GGGCCAGCCTGGAATGCT
91
B
92





1211995
3210
3227
125
142
GGCTACCCGCTCCCATTG
77
B
93





1212005
3250
3267
165
182
GATGACCCGCCTACCTCA
84
B
94





1212015
3284
3301
199
216
AACCGGGTAGCGCTCAGA
75
B
95





1212025
3294
3311
209
226
GGGCAGCAGCAACCGGGT
57
B
96





1212035
3304
3321
219
236
CGCGGTCCTTGGGCAGCA
193
B
97





1212045
3314
3331
229
246
CGTCCGACTCCGCGGTCC
87
B
98





1212055
N/A
N/A
239
256
GGTCTGCCTGCGTCCGAC
75
B
99





1212065
7157
7174
249
266
GGGTCCACATGGTCTGCC
100
B
100





1212075
7175
7192
267
284
AGGCCACCCAGCTCACCA
88
B
101





1212085
7225
7242
317
334
GCAGAACTGACCATCTGG
91
B
102





1212095
7275
7292
367
384
CGGCAGCAGCTGTAGCTG
66
B
103





1212105
7448
7465
417
434
GGCCACCCAGATGCCTGC
101
B
104





1212115
7498
7515
467
484
GGTAAAGATGCAGGAGTG
82
B
105





1212125
N/A
N/A
517
534
CCGCATGCCACGGCCTCT
89
B
106





1212135
7713
7730
567
584
GCCCGTCTGCACTGCAGT
140
B
107





1212145
8239
8256
617
634
GCACTGGATGGCACCCAC
108
B
108





1212155
8289
8306
667
684
ACCATAACACAGCACGTG
88
B
109





1212165
8440
8457
717
734
CTTCACAGCAGGAAGCCT
126
B
110





1212175
8490
8507
767
784
GGTGTGAACCAGGTCGCA
116
B
111





1212185
8540
8557
817
834
GCAGGGAGCTTCTTTGCC
85
B
112





1212195
8703
8720
867
884
CCGGACACATGACCGAGC
30†
B
113





1212205
8753
8770
917
934
ACTGGGCAGCTCACAGCA
7†
B
114





1212215
9039
9056
967
984
TGATCGGAGCAGCAGGTG
114
B
115





1212225
9089
9106
1017
1034
ACTTACTCTGGATCAGGT
98
B
116





1212235
9139
9156
1067
1084
CGCAGGCAGCTTAGTGAG
96
B
117





1212245
9388
9405
1117
1134
CCATCTGGGCAGCTCACC
120
B
118





1212255
9438
9455
1167
1184
TAAAAGGGCAGCAGCCCC
85
B
119





1212265
9577
9594
1217
1234
AAACCCCGCGGGACAGCA
94
B
120





1212275
9627
9644
1267
1284
GGCACCTGGTGGGGCCCC
63
B
121





1212285
9677
9694
1317
1334
AGGCTTGTGGGTCTGGCA
111
B
122





1212295
9727
9744
1367
1384
ATCGGAGGAGGGACAGCT
81
B
123





1212305
9777
9794
1417
1434
TCTGGGATTGGACAGCAG
114
B
124





1212315
10046
10063
1467
1484
ACGTGTAGCCCTGGGGGC
104
B
125





1212325
10096
10113
1517
1534
CAGTCCAGCCACGATCTC
106
B
126





1212335
10146
10163
1567
1584
CCGATGTCTCTGGGGTGG
87
B
127





1212345
10196
10213
1617
1634
GGCTCGGGCAGCAGGTCT
163
B
128





1212355
N/A
N/A
1667
1684
CTCGCAGCACACAGCATG
82
B
129





1212365
10388
10405
1717
1734
CGAGCCTTCACGTTGCAG
54
B
130





1212375
10438
10455
1767
1784
GGGCCAGGAAGGTGGCAG
73
B
131





1212385
10488
10505
1817
1834
GAAGTGTCCTTCCCCACA
81
B
132





1212394
10538
10555
1867
1884
CAGGCCCAGCCCTGTCGG
96
B
133





1212403
10677
10694
1917
1934
GACAGCAGTGGCGCCGAT
80
B
134





1212413
10727
10744
1967
1984
CTCCCTGCGCAAACACTT
66
B
135





1212423
10777
10794
2017
2034
AGCAGCTGTCTCAAGGCT
69
B
136





1212433
10791
10808
2031
2048
GTACTGTCCCTCACAGCA
89
B
137





1212443
10801
10818
2041
2058
AGAGTCTTCAGTACTGTC
123
B
138





1212453
10811
10828
2051
2068
CGAGGGCTGCAGAGTCTT
73
B
139





1212463
10821
10838
2061
2078
AGTGGGGTCCCGAGGGCT
100
B
140





1212473
10831
10848
2071
2088
GCACCCTCCGAGTGGGGT
69
B
141





1212483
10841
10858
2081
2098
TGAGCAGAGGGCACCCTC
112
B
142





1212493
10851
10868
2091
2108
TAGGGAGGCCTGAGCAGA
100
B
143





1212503
10861
10878
2101
2118
GGGGAGGTGCTAGGGAGG
93
B
144





1212513
10871
10888
2111
2128
ATTTGGTTAGGGGGAGGT
58
B
145





1212523
10881
10898
2121
2138
TCCAGGGAGAATTTGGTT
108
B
146





1212533
10891
10908
2131
2148
CAGAATGGGGTCCAGGGA
100
B
147





1212543
10901
10918
2141
2158
ATGGGGAGCTCAGAATGG
73
B
148





1212553
10911
10928
2151
2168
TCCCATGGTGATGGGGAG
93
B
149





1212563
10921
10938
2161
2178
AGGCCCCACCTCCCATGG
104
B
150





1212573
10931
10948
2171
2188
CCTTAGATTGAGGCCCCA
65
B
151





1212583
10941
10958
2181
2198
ACAGGGAAGGCCTTAGAT
78
B
152





1212593
10951
10968
2191
2208
CCCCCTTCTGACAGGGAA
67
B
153





1212603
10961
10978
2201
2218
TTTGCCACAACCCCCTTC
98
B
154





1212613
10971
10988
2211
2228
TAATGTGGCTTTTGCCAC
94
B
155





1212623
10981
10998
2221
2238
TGGCAGCTTGTAATGTGG
79
B
156





1212633
10991
11008
2231
2248
GGGGAGGGGATGGCAGCT
149
B
157





1212643
11001
11018
2241
2258
CCACTGAAACGGGGAGGG
65
B
158





1212653
11011
11028
2251
2268
GCCACAGGGTCCACTGAA
110
B
159





1212663
11021
11038
2261
2278
AAAGCACCTGGCCACAGG
101
B
160





1212673
11031
11048
2271
2288
TGGATAGGGAAAAGCACC
79
B
161





1212683
11041
11058
2281
2298
AACACCCCTGTGGATAGG
84
B
162





1212693
11051
11068
2291
2308
CACACACACAAACACCCC
73
B
163





1212703
11061
11078
2301
2318
CGCACACGCGCACACACA
83
B
164





1212713
11071
11088
2311
2328
TTTATTGAAACGCACACG
97
B
165





1212723
11081
11098
2321
2338
GTGTACAAACTTTATTGA
89
B
166





1212733
N/A
N/A
2331
2348
TTTTAAGAAAGTGTACAA
68
B
167





1211966
3094
3111
9
26
TTCTAGGGATCATGTGAT
83
C
168





1211976
3134
3151
49
66
AATCACTCTCCTCCCTGC
92
C
169





1211986
3174
3191
89
106
GGTGGGGCCAGCCTGGAA
81
C
170





1211996
3214
3231
129
146
TCAGGGCTACCCGCTCCC
73
C
171





1212006
3254
3271
169
186
GCGCGATGACCCGCCTAC
87
C
172





1212016
3285
3302
200
217
CAACCGGGTAGCGCTCAG
75
C
173





1212026
3295
3312
210
227
TGGGCAGCAGCAACCGGG
38
C
174





1212036
3305
3322
220
237
CCGCGGTCCTTGGGCAGC
151
C
175





1212046
3315
3332
230
247
GCGTCCGACTCCGCGGTC
84
C
176





1212056
N/A
N/A
240
257
TGGTCTGCCTGCGTCCGA
63
C
177





1212066
7158
7175
250
267
AGGGTCCACATGGTCTGC
55
C
178





1212076
7180
7197
272
289
TGTTAAGGCCACCCAGCT
98
C
179





1212086
7230
7247
322
339
ACAGGGCAGAACTGACCA
114
C
180





1212096
7280
7297
372
389
GGGGACGGCAGCAGCTGT
79
C
181





1212106
7453
7470
422
439
GCAGGGGCCACCCAGATG
103
C
182





1212116
7503
7520
472
489
GAGACGGTAAAGATGCAG
85
C
183





1212126
7668
7685
522
539
CATCCCCGCATGCCACGG
83
C
184





1212136
7718
7735
572
589
GGATCGCCCGTCTGCACT
91
C
185





1212146
8244
8261
622
639
TCAGGGCACTGGATGGCA
102
C
186





1212156
8294
8311
672
689
CATCGACCATAACACAGC
78
C
187





1212166
8445
8462
722
739
CCTGTCTTCACAGCAGGA
73
C
188





1212176
8495
8512
772
789
CAGCGGGTGTGAACCAGG
61
C
189





1212186
8545
8562
822
839
TCTGGGCAGGGAGCTTCT
135†
C
190





1212196
8708
8725
872
889
TGCGTCCGGACACATGAC
87†
C
191





1212206
8758
8775
922
939
TTCCCACTGGGCAGCTCA
32†
C
192





1212216
9044
9061
972
989
GCAGGTGATCGGAGCAGC
92
C
193





1212226
9094
9111
1022
1039
GAGGCACTTACTCTGGAT
89
C
194





1212236
9144
9161
1072
1089
GTGTGCGCAGGCAGCTTA
92
C
195





1212246
9393
9410
1122
1139
TATAGCCATCTGGGCAGC
143
C
196





1212256
9443
9460
1172
1189
CTGGGTAAAAGGGCAGCA
171
C
197





1212266
9582
9599
1222
1239
CACGTAAACCCCGCGGGA
101
C
198





1212276
9632
9649
1272
1289
TCCAGGGCACCTGGTGGG
116
C
199





1212286
9682
9699
1322
1339
CTTCAAGGCTTGTGGGTC
110
C
200





1212296
9732
9749
1372
1389
CAGGTATCGGAGGAGGGA
99
C
201





1212306
N/A
N/A
1422
1439
CAGCCTCTGGGATTGGAC
105
C
202





1212316
10051
10068
1472
1489
TACACACGTGTAGCCCTG
108
C
203





1212326
10101
10118
1522
1539
TTCTCCAGTCCAGCCACG
67
C
204





1212336
10151
10168
1572
1589
CACAGCCGATGTCTCTGG
85
C
205





1212346
10201
10218
1622
1639
ACCCAGGCTCGGGCAGCA
108
C
206





1212356
10343
10360
1672
1689
CGATCCTCGCAGCACACA
103
C
207





1212366
10393
10410
1722
1739
AGGATCGAGCCTTCACGT
123
C
208





1212376
10443
10460
1772
1789
GCTACGGGCCAGGAAGGT
106
C
209





1212386
10493
10510
1822
1839
TGGCAGAAGTGTCCTTCC
96
C
210





1212395
10543
10560
1872
1889
GACAGCAGGCCCAGCCCT
91
C
211





1212404
10682
10699
1922
1939
AGCAGGACAGCAGTGGCG
82
C
212





1212414
10732
10749
1972
1989
GGGGCCTCCCTGCGCAAA
98
C
213





1212424
10782
10799
2022
2039
CTCACAGCAGCTGTCTCA
86
C
214





1212434
10792
10809
2032
2049
AGTACTGTCCCTCACAGC
102
C
215





1212444
10802
10819
2042
2059
CAGAGTCTTCAGTACTGT
74
C
216





1212454
10812
10829
2052
2069
CCGAGGGCTGCAGAGTCT
106
C
217





1212464
10822
10839
2062
2079
GAGTGGGGTCCCGAGGGC
92
C
218





1212474
10832
10849
2072
2089
GGCACCCTCCGAGTGGGG
108
C
219





1212484
10842
10859
2082
2099
CTGAGCAGAGGGCACCCT
72
C
220





1212494
10852
10869
2092
2109
CTAGGGAGGCCTGAGCAG
80
C
221





1212504
10862
10879
2102
2119
GGGGGAGGTGCTAGGGAG
112
C
222





1212514
10872
10889
2112
2129
AATTTGGTTAGGGGGAGG
73
C
223





1212524
10882
10899
2122
2139
GTCCAGGGAGAATTTGGT
69
C
224





1212534
10892
10909
2132
2149
TCAGAATGGGGTCCAGGG
63
C
225





1212544
10902
10919
2142
2159
GATGGGGAGCTCAGAATG
70
C
226





1212554
10912
10929
2152
2169
CTCCCATGGTGATGGGGA
92
C
227





1212564
10922
10939
2162
2179
GAGGCCCCACCTCCCATG
69
C
228





1212574
10932
10949
2172
2189
GCCTTAGATTGAGGCCCC
114
C
229





1212584
10942
10959
2182
2199
GACAGGGAAGGCCTTAGA
110
C
230





1212594
10952
10969
2192
2209
ACCCCCTTCTGACAGGGA
91
C
231





1212604
10962
10979
2202
2219
TTTTGCCACAACCCCCTT
73
C
232





1212614
10972
10989
2212
2229
GTAATGTGGCTTTTGCCA
126
C
233





1212624
10982
10999
2222
2239
ATGGCAGCTTGTAATGTG
88
C
234





1212634
10992
11009
2232
2249
CGGGGAGGGGATGGCAGC
95
C
235





1212644
11002
11019
2242
2259
TCCACTGAAACGGGGAGG
88
C
236





1212654
11012
11029
2252
2269
GGCCACAGGGTCCACTGA
81
C
237





1212664
11022
11039
2262
2279
AAAAGCACCTGGCCACAG
93
C
238





1212674
11032
11049
2272
2289
GTGGATAGGGAAAAGCAC
113
C
239





1212684
11042
11059
2282
2299
AAACACCCCTGTGGATAG
84
C
240





1212694
11052
11069
2292
2309
GCACACACACAAACACCC
97
C
241





1212704
11062
11079
2302
2319
ACGCACACGCGCACACAC
75
C
242





1212714
11072
11089
2312
2329
CTTTATTGAAACGCACAC
74
C
243





1212724
11082
11099
2322
2339
AGTGTACAAACTTTATTG
87
C
244





1212734
N/A
N/A
2332
2349
TTTTTAAGAAAGTGTACA
79
C
245





887390
10548
10565
1877
1894
GTAGGGACAGCAGGCCCA
85
D
246





1211967
3098
3115
13
30
CCATTTCTAGGGATCATG
80
D
247





1211977
3138
3155
53
70
CTCAAATCACTCTCCTCC
76
D
248





1211987
3178
3195
93
110
TAGAGGTGGGGCCAGCCT
97
D
249





1211997
3218
3235
133
150
GGGATCAGGGCTACCCGC
99
D
250





1212007
3258
3275
173
190
CCCAGCGCGATGACCCGC
108
D
251





1212017
3286
3303
201
218
GCAACCGGGTAGCGCTCA
72
D
252





1212027
3296
3313
211
228
TTGGGCAGCAGCAACCGG
42
D
253





1212037
3306
3323
221
238
TCCGCGGTCCTTGGGCAG
114
D
254





1212047
3316
3333
231
248
TGCGTCCGACTCCGCGGT
87
D
255





1212057
N/A
N/A
241
258
ATGGTCTGCCTGCGTCCG
76
D
256





1212067
7159
7176
251
268
CAGGGTCCACATGGTCTG
73
D
257





1212077
7185
7202
277
294
CCTGCTGTTAAGGCCACC
55
D
258





1212087
7235
7252
327
344
AGGCCACAGGGCAGAACT
86
D
259





1212097
7285
7302
377
394
CAGAAGGGGACGGCAGCA
80
D
260





1212107
7458
7475
427
444
ACCTGGCAGGGGCCACCC
102
D
261





1212117
7508
7525
477
494
TCCCTGAGACGGTAAAGA
102
D
262





1212127
7673
7690
527
544
ATGGCCATCCCCGCATGC
92
D
263





1212137
7723
7740
577
594
AAGCAGGATCGCCCGTCT
120
D
264





1212147
8249
8266
627
644
GACTATCAGGGCACTGGA
62
D
265





1212157
8299
8316
677
694
GGAGCCATCGACCATAAC
114
D
266





1212167
8450
8467
727
744
TGCACCCTGTCTTCACAG
101
D
267





1212177
8500
8517
777
794
TGATGCAGCGGGTGTGAA
88
D
268





1212187
8550
8567
827
844
AGTCCTCTGGGCAGGGAG
114†
D
269





1212197
8713
8730
877
894
GACCGTGCGTCCGGACAC
4†
D
270





1212207
8763
8780
927
944
CATACTTCCCACTGGGCA
69†
D
271





1212217
9049
9066
977
994
GCAGTGCAGGTGATCGGA
84
D
272





1212227
9099
9116
1027
1044
TTGGAGAGGCACTTACTC
74
D
273





1212237
N/A
N/A
1077
1094
CCACTGTGTGCGCAGGCA
97
D
274





1212247
9398
9415
1127
1144
GCAGGTATAGCCATCTGG
100
D
275





1212257
N/A
N/A
1177
1194
ACAGCCTGGGTAAAAGGG
163
D
276





1212267
9587
9604
1227
1244
TGTCACACGTAAACCCCG
73
D
277





1212277
9637
9654
1277
1294
CTCCATCCAGGGCACCTG
80
D
278





1212287
9687
9704
1327
1344
TCTCTCTTCAAGGCTTGT
84
D
279





1212297
9737
9754
1377
1394
GGCAGCAGGTATCGGAGG
110
D
280





1212307
N/A
N/A
1427
1444
GCAGACAGCCTCTGGGAT
81
D
281





1212317
10056
10073
1477
1494
TCAGCTACACACGTGTAG
104
D
282





1212327
10106
10123
1527
1544
GCATCTTCTCCAGTCCAG
113
D
283





1212337
10156
10173
1577
1594
CTGGTCACAGCCGATGTC
70
D
284





1212347
10206
10223
1627
1644
CTCCCACCCAGGCTCGGG
93
D
285





1212357
10348
10365
1677
1694
GCTGGCGATCCTCGCAGC
85
D
286





1212367
10398
10415
1727
1744
CTCGCAGGATCGAGCCTT
71
D
287





1212377
10448
10465
1777
1794
TGAGGGCTACGGGCCAGG
86
D
288





1212387
10498
10515
1827
1844
TATCATGGCAGAAGTGTC
71
D
289





1212405
10687
10704
1927
1944
AAGCCAGCAGGACAGCAG
119
D
290





1212415
10737
10754
1977
1994
AGCGCGGGGCCTCCCTGC
96
D
291





1212425
10783
10800
2023
2040
CCTCACAGCAGCTGTCTC
74
D
292





1212435
10793
10810
2033
2050
CAGTACTGTCCCTCACAG
94
D
293





1212445
10803
10820
2043
2060
GCAGAGTCTTCAGTACTG
97
D
294





1212455
10813
10830
2053
2070
CCCGAGGGCTGCAGAGTC
74
D
295





1212465
10823
10840
2063
2080
CGAGTGGGGTCCCGAGGG
79
D
296





1212475
10833
10850
2073
2090
GGGCACCCTCCGAGTGGG
78
D
297





1212485
10843
10860
2083
2100
CCTGAGCAGAGGGCACCC
88
D
298





1212495
10853
10870
2093
2110
GCTAGGGAGGCCTGAGCA
76
D
299





1212505
10863
10880
2103
2120
AGGGGGAGGTGCTAGGGA
153
D
300





1212515
10873
10890
2113
2130
GAATTTGGTTAGGGGGAG
97
D
301





1212525
10883
10900
2123
2140
GGTCCAGGGAGAATTTGG
112
D
302





1212535
10893
10910
2133
2150
CTCAGAATGGGGTCCAGG
108
D
303





1212545
10903
10920
2143
2160
TGATGGGGAGCTCAGAAT
80
D
304





1212555
10913
10930
2153
2170
CCTCCCATGGTGATGGGG
105
D
305





1212565
10923
10940
2163
2180
TGAGGCCCCACCTCCCAT
78
D
306





1212575
10933
10950
2173
2190
GGCCTTAGATTGAGGCCC
62
D
307





1212585
10943
10960
2183
2200
TGACAGGGAAGGCCTTAG
89
D
308





1212595
10953
10970
2193
2210
AACCCCCTTCTGACAGGG
89
D
309





1212605
10963
10980
2203
2220
CTTTTGCCACAACCCCCT
101
D
310





1212615
10973
10990
2213
2230
TGTAATGTGGCTTTTGCC
110
D
311





1212625
10983
11000
2223
2240
GATGGCAGCTTGTAATGT
135
D
312





1212635
10993
11010
2233
2250
ACGGGGAGGGGATGGCAG
135
D
313





1212645
11003
11020
2243
2260
GTCCACTGAAACGGGGAG
103
D
314





1212655
11013
11030
2253
2270
TGGCCACAGGGTCCACTG
83
D
315





1212665
11023
11040
2263
2280
GAAAAGCACCTGGCCACA
123
D
316





1212675
11033
11050
2273
2290
TGTGGATAGGGAAAAGCA
115
D
317





1212685
11043
11060
2283
2300
CAAACACCCCTGTGGATA
88
D
318





1212695
11053
11070
2293
2310
CGCACACACACAAACACC
74
D
319





1212705
11063
11080
2303
2320
AACGCACACGCGCACACA
82
D
320





1212715
11073
11090
2313
2330
ACTTTATTGAAACGCACA
49
D
321





1212725
11083
11100
2323
2340
AAGTGTACAAACTTTATT
80
D
322





1212735
N/A
N/A
2333
2350
TTTTTTAAGAAAGTGTAC
84
D
323





1211968
3102
3119
17
34
CACCCCATTTCTAGGGAT
94
E
324





1211978
3142
3159
57
74
TCTACTCAAATCACTCTC
119
E
325





1211988
3182
3199
97
114
AATATAGAGGTGGGGCCA
103
E
326





1211998
3222
3239
137
154
GCCAGGGATCAGGGCTAC
100
E
327





1212008
3262
3279
177
194
AGACCCCAGCGCGATGAC
116
E
328





1212018
3287
3304
202
219
AGCAACCGGGTAGCGCTC
99
E
329





1212028
3297
3314
212
229
CTTGGGCAGCAGCAACCG
71
E
330





1212038
3307
3324
222
239
CTCCGCGGTCCTTGGGCA
96
E
331





1212048
3317
3334
232
249
CTGCGTCCGACTCCGCGG
96
E
332





1212058
N/A
N/A
242
259
CATGGTCTGCCTGCGTCC
107
E
333





1212068
7160
7177
252
269
CCAGGGTCCACATGGTCT
81
E
334





1212078
7190
7207
282
299
CCAGCCCTGCTGTTAAGG
100
E
335





1212088
7240
7257
332
349
GCAGCAGGCCACAGGGCA
133
E
336





1212098
N/A
N/A
382
399
TTGTCCAGAAGGGGACGG
120
E
337





1212108
7463
7480
432
449
CATCAACCTGGCAGGGGC
99
E
338





1212118
7513
7530
482
499
GGAAGTCCCTGAGACGGT
87
E
339





1212128
7678
7695
532
549
CAGTGATGGCCATCCCCG
130
E
340





1212138
7728
7745
582
599
TTTGGAAGCAGGATCGCC
112
E
341





1212148
8254
8271
632
649
GAACTGACTATCAGGGCA
90
E
342





1212158
8304
8321
682
699
CCCCAGGAGCCATCGACC
100
E
343





1212168
8455
8472
732
749
AGCAGTGCACCCTGTCTT
97
E
344





1212178
8505
8522
782
799
GGGTGTGATGCAGCGGGT
104
E
345





1212188
8555
8572
832
849
CTGTTAGTCCTCTGGGCA
42†
E
346





1212198
8718
8735
882
899
ACCGGGACCGTGCGTCCG
27†
E
347





1212208
8768
8785
932
949
GCAGCCATACTTCCCACT
15†
E
348





1212218
9054
9071
982
999
GGGCAGCAGTGCAGGTGA
73
E
349





1212228
9104
9121
1032
1049
TCTCCTTGGAGAGGCACT
140
E
350





1212238
N/A
N/A
1082
1099
ATCCCCCACTGTGTGCGC
122
E
351





1212248
9403
9420
1132
1149
CGGCAGCAGGTATAGCCA
144
E
352





1212258
N/A
N/A
1182
1199
AGCACACAGCCTGGGTAA
94
E
353





1212268
9592
9609
1232
1249
CTGCGTGTCACACGTAAA
88
E
354





1212278
9642
9659
1282
1299
GCCTTCTCCATCCAGGGC
97
E
355





1212288
9692
9709
1332
1349
GGACATCTCTCTTCAAGG
90
E
356





1212298
9742
9759
1382
1399
GAGTTGGCAGCAGGTATC
158
E
357





1212308
N/A
N/A
1432
1449
GAGCAGCAGACAGCCTCT
104
E
358





1212318
10061
10078
1482
1499
GCCCCTCAGCTACACACG
88
E
359





1212328
10111
10128
1532
1549
GGCAGGCATCTTCTCCAG
87
E
360





1212338
10161
10178
1582
1599
GTGTGCTGGTCACAGCCG
110
E
361





1212348
10211
10228
1632
1649
CCCAGCTCCCACCCAGGC
107
E
362





1212358
10353
10370
1682
1699
GCAGTGCTGGCGATCCTC
82
E
363





1212368
10403
10420
1732
1749
TCCTTCTCGCAGGATCGA
93
E
364





1212378
10453
10470
1782
1799
CCACGTGAGGGCTACGGG
115
E
365





1212388
10503
10520
1832
1849
CTGGTTATCATGGCAGAA
85
E
366





1212396
10553
10570
1882
1899
TGGCGGTAGGGACAGCAG
130
E
367





1212406
10692
10709
1932
1949
AGCGGAAGCCAGCAGGAC
93
E
368





1212416
10742
10759
1982
1999
GTCCCAGCGCGGGGCCTC
113
E
369





1212426
10784
10801
2024
2041
CCCTCACAGCAGCTGTCT
113
E
370





1212436
10794
10811
2034
2051
TCAGTACTGTCCCTCACA
96
E
371





1212446
10804
10821
2044
2061
TGCAGAGTCTTCAGTACT
112
E
372





1212456
10814
10831
2054
2071
TCCCGAGGGCTGCAGAGT
104
E
373





1212466
10824
10841
2064
2081
CCGAGTGGGGTCCCGAGG
75
E
374





1212476
10834
10851
2074
2091
AGGGCACCCTCCGAGTGG
104
E
375





1212486
10844
10861
2084
2101
GCCTGAGCAGAGGGCACC
122
E
376





1212496
10854
10871
2094
2111
TGCTAGGGAGGCCTGAGC
86
E
377





1212506
10864
10881
2104
2121
TAGGGGGAGGTGCTAGGG
139
E
378





1212516
10874
10891
2114
2131
AGAATTTGGTTAGGGGGA
117
E
379





1212526
10884
10901
2124
2141
GGGTCCAGGGAGAATTTG
90
E
380





1212536
10894
10911
2134
2151
GCTCAGAATGGGGTCCAG
110
E
381





1212546
10904
10921
2144
2161
GTGATGGGGAGCTCAGAA
88
E
382





1212556
10914
10931
2154
2171
ACCTCCCATGGTGATGGG
83
E
383





1212566
10924
10941
2164
2181
TTGAGGCCCCACCTCCCA
101
E
384





1212576
10934
10951
2174
2191
AGGCCTTAGATTGAGGCC
75
E
385





1212586
10944
10961
2184
2201
CTGACAGGGAAGGCCTTA
98
E
386





1212596
10954
10971
2194
2211
CAACCCCCTTCTGACAGG
93
E
387





1212606
10964
10981
2204
2221
GCTTTTGCCACAACCCCC
104
E
388





1212616
10974
10991
2214
2231
TTGTAATGTGGCTTTTGC
98
E
389





1212626
10984
11001
2224
2241
GGATGGCAGCTTGTAATG
135
E
390





1212636
10994
11011
2234
2251
AACGGGGAGGGGATGGCA
85
E
391





1212646
11004
11021
2244
2261
GGTCCACTGAAACGGGGA
124
E
392





1212656
11014
11031
2254
2271
CTGGCCACAGGGTCCACT
84
E
393





1212666
11024
11041
2264
2281
GGAAAAGCACCTGGCCAC
110
E
394





1212676
11034
11051
2274
2291
CTGTGGATAGGGAAAAGC
96
E
395





1212686
11044
11061
2284
2301
ACAAACACCCCTGTGGAT
81
E
396





1212696
11054
11071
2294
2311
GCGCACACACACAAACAC
108
E
397





1212706
11064
11081
2304
2321
AAACGCACACGCGCACAC
109
E
398





1212716
11074
11091
2314
2331
AACTTTATTGAAACGCAC
107
E
399





1212726
11084
11101
2324
2341
AAAGTGTACAAACTTTAT
112
E
400





1212736
N/A
N/A
2334
2351
TTTTTTTAAGAAAGTGTA
94
E
401





887369
10508
10525
1837
1854
CAGGTCTGGTTATCATGG
98
F
402





1211969
3106
3123
21
38
CCCACACCCCATTTCTAG
115
F
403





1211979
3146
3163
61
78
CTTTTCTACTCAAATCAC
94
F
404





1211989
3186
3203
101
118
TATCAATATAGAGGTGGG
110
F
405





1211999
3226
3243
141
158
ATTGGCCAGGGATCAGGG
105
F
406





1212009
3266
3283
181
198
CTACAGACCCCAGCGCGA
98
F
407





1212019
3288
3305
203
220
CAGCAACCGGGTAGCGCT
106
F
408





1212029
3298
3315
213
230
CCTTGGGCAGCAGCAACC
75
F
409





1212039
3308
3325
223
240
ACTCCGCGGTCCTTGGGC
108
F
410





1212049
3318
3335
233
250
CCTGCGTCCGACTCCGCG
102
F
411





1212059
N/A
N/A
243
260
ACATGGTCTGCCTGCGTC
97
F
412





1212069
7161
7178
253
270
ACCAGGGTCCACATGGTC
95
F
413





1212079
7195
7212
287
304
AGCCACCAGCCCTGCTGT
98
F
414





1212089
7245
7262
337
354
TCCAGGCAGCAGGCCACA
112
F
415





1212099
N/A
N/A
387
404
GCCATTTGTCCAGAAGGG
109
F
416





1212109
7468
7485
437
454
GTGGGCATCAACCTGGCA
99
F
417





1212119
7518
7535
487
504
CAACTGGAAGTCCCTGAG
90
F
418





1212129
7683
7700
537
554
GGCAGCAGTGATGGCCAT
132
F
419





1212139
7733
7750
587
604
TGATCTTTGGAAGCAGGA
93
F
420





1212149
8259
8276
637
654
CATTCGAACTGACTATCA
99
F
421





1212159
8309
8326
687
704
AGCACCCCCAGGAGCCAT
84
F
422





1212169
8460
8477
737
754
CGGACAGCAGTGCACCCT
129
F
423





1212179
8510
8527
787
804
CCCGTGGGTGTGATGCAG
238
F
424





1212189
8560
8577
837
854
CTGCCCTGTTAGTCCTCT
8†
F
425





1212199
8723
8740
887
904
AGGGCACCGGGACCGTGC
8†
F
426





1212209
8773
8790
937
954
GGGCAGCAGCCATACTTC
22†
F
427





1212219
9059
9076
987
1004
CTTGGGGGCAGCAGTGCA
151
F
428





1212229
9109
9126
1037
1054
AGCGTTCTCCTTGGAGAG
115
F
429





1212239
9358
9375
1087
1104
TTCACATCCCCCACTGTG
91
F
430





1212249
9408
9425
1137
1154
GTAGACGGCAGCAGGTAT
135
F
431





1212259
N/A
N/A
1187
1204
CTCACAGCACACAGCCTG
85
F
432





1212269
9597
9614
1237
1254
CCCTTCTGCGTGTCACAC
113
F
433





1212279
9647
9664
1287
1304
CTGGGGCCTTCTCCATCC
110
F
434





1212289
9697
9714
1337
1354
ACAGGGGACATCTCTCTT
92
F
435





1212299
9747
9764
1387
1404
GACGTGAGTTGGCAGCAG
116
F
436





1212309
10016
10033
1437
1454
GGTCCGAGCAGCAGACAG
105
F
437





1212319
10066
10083
1487
1504
ACACTGCCCCTCAGCTAC
96
F
438





1212329
10116
10133
1537
1554
CGGCGGGCAGGCATCTTC
91
F
439





1212339
10166
10183
1587
1604
AGCTGGTGTGCTGGTCAC
103
F
440





1212349
10216
10233
1637
1654
GCAGGCCCAGCTCCCACC
97
F
441





1212359
10358
10375
1687
1704
GGGCAGCAGTGCTGGCGA
133
F
442





1212369
10408
10425
1737
1754
CCACTTCCTTCTCGCAGG
97
F
443





1212379
10458
10475
1787
1804
CACACCCACGTGAGGGCT
100
F
444





1212397
N/A
N/A
1887
1904
CGCCCTGGCGGTAGGGAC
74
F
445





1212407
10697
10714
1937
1954
TGCGCAGCGGAAGCCAGC
114
F
446





1212417
10747
10764
1987
2004
GGGGCGTCCCAGCGCGGG
99
F
447





1212427
10785
10802
2025
2042
TCCCTCACAGCAGCTGTC
105
F
448





1212437
10795
10812
2035
2052
TTCAGTACTGTCCCTCAC
111
F
449





1212447
10805
10822
2045
2062
CTGCAGAGTCTTCAGTAC
113
F
450





1212457
10815
10832
2055
2072
GTCCCGAGGGCTGCAGAG
126
F
451





1212467
10825
10842
2065
2082
TCCGAGTGGGGTCCCGAG
113
F
452





1212477
10835
10852
2075
2092
GAGGGCACCCTCCGAGTG
112
F
453





1212487
10845
10862
2085
2102
GGCCTGAGCAGAGGGCAC
97
F
454





1212497
10855
10872
2095
2112
GTGCTAGGGAGGCCTGAG
103
F
455





1212507
10865
10882
2105
2122
TTAGGGGGAGGTGCTAGG
139
F
456





1212517
10875
10892
2115
2132
GAGAATTTGGTTAGGGGG
99
F
457





1212527
10885
10902
2125
2142
GGGGTCCAGGGAGAATTT
97
F
458





1212537
10895
10912
2135
2152
AGCTCAGAATGGGGTCCA
84
F
459





1212547
10905
10922
2145
2162
GGTGATGGGGAGCTCAGA
86
F
460





1212557
10915
10932
2155
2172
CACCTCCCATGGTGATGG
114
F
461





1212567
10925
10942
2165
2182
ATTGAGGCCCCACCTCCC
96
F
462





1212577
10935
10952
2175
2192
AAGGCCTTAGATTGAGGC
97
F
463





1212587
10945
10962
2185
2202
TCTGACAGGGAAGGCCTT
84
F
464





1212597
10955
10972
2195
2212
ACAACCCCCTTCTGACAG
90

465





1212607
10965
10982
2205
2222
GGCTTTTGCCACAACCCC
102
F
466





1212617
10975
10992
2215
2232
CTTGTAATGTGGCTTTTG
113
F
467





1212627
10985
11002
2225
2242
GGGATGGCAGCTTGTAAT
128
F
468





1212637
10995
11012
2235
2252
AAACGGGGAGGGGATGGC
106
F
469





1212647
11005
11022
2245
2262
GGGTCCACTGAAACGGGG
131
F
470





1212657
11015
11032
2255
2272
CCTGGCCACAGGGTCCAC
114
F
471





1212667
11025
11042
2265
2282
GGGAAAAGCACCTGGCCA
87
F
472





1212677
11035
11052
2275
2292
CCTGTGGATAGGGAAAAG
108
F
473





1212687
11045
11062
2285
2302
CACAAACACCCCTGTGGA
95
F
474





1212697
11055
11072
2295
2312
CGCGCACACACACAAACA
117
F
475





1212707
11065
11082
2305
2322
GAAACGCACACGCGCACA
87
F
476





1212717
11075
11092
2315
2332
AAACTTTATTGAAACGCA
77
F
477





1212727
11085
11102
2325
2342
GAAAGTGTACAAACTTTA
90
F
478





1212737
N/A
N/A
2335
2352
TTTTTTTTAAGAAAGTGT
111
F
479





1211970
3110
3127
25
42
TCGCCCCACACCCCATTT
78
G
480





1211980
3150
3167
65
82
GTTTCTTTTCTACTCAAA
106
G
481





1211990
3190
3207
105
122
TACTTATCAATATAGAGG
105
G
482





1212000
3230
3247
145
162
TTCCATTGGCCAGGGATC
196
G
483





1212010
3270
3287
185
202
CAGACTACAGACCCCAGC
104
G
484





1212020
3289
3306
204
221
GCAGCAACCGGGTAGCGC
96
G
485





1212030
3299
3316
214
231
TCCTTGGGCAGCAGCAAC
78
G
486





1212040
3309
3326
224
241
GACTCCGCGGTCCTTGGG
112
G
487





1212050
N/A
N/A
234
251
GCCTGCGTCCGACTCCGC
129
G
488





1212060
N/A
N/A
244
261
CACATGGTCTGCCTGCGT
102
G
489





1212070
7162
7179
254
271
CACCAGGGTCCACATGGT
87
G
490





1212080
7200
7217
292
309
GTTCCAGCCACCAGCCCT
261
G
491





1212090
7250
7267
342
359
CGGGGTCCAGGCAGCAGG
123
G
492





1212100
N/A
N/A
392
409
TGTGGGCCATTTGTCCAG
85
G
493





1212110
7473
7490
442
459
GAGCAGTGGGCATCAACC
124
G
494





1212120
7523
7540
492
509
GGCAGCAACTGGAAGTCC
94
G
495





1212130
7688
7705
542
559
CCGTGGGCAGCAGTGATG
133
G
496





1212140
N/A
N/A
592
609
TTACCTGATCTTTGGAAG
122
G
497





1212150
8264
8281
642
659
CCGGGCATTCGAACTGAC
81
G
498





1212160
8314
8331
692
709
GGGGCAGCACCCCCAGGA
114
G
499





1212170
8465
8482
742
759
CCGTGCGGACAGCAGTGC
100
G
500





1212180
8515
8532
792
809
GGGTGCCCGTGGGTGTGA
294
G
501





1212190
N/A
N/A
842
859
GGCCACTGCCCTGTTAGT
83+
G
502





1212200
8728
8745
892
909
CCATCAGGGCACCGGGAC
7+
G
503





1212210
8778
8795
942
959
GCATTGGGCAGCAGCCAT
35+
G
504





1212220
9064
9081
992
1009
AGTGTCTTGGGGGCAGCA
123
G
505





1212230
9114
9131
1042
1059
GTGGTAGCGTTCTCCTTG
90
G
506





1212240
9363
9380
1092
1109
CACATTTCACATCCCCCA
117
G
507





1212250
9413
9430
1142
1159
CGACTGTAGACGGCAGCA
107
G
508





1212260
9552
9569
1192
1209
TGGTCCTCACAGCACACA
99
G
509





1212270
9602
9619
1242
1259
AGGTACCCTTCTGCGTGT
91
G
510





1212280
9652
9669
1292
1309
GTGAGCTGGGGCCTTCTC
86
G
511





1212290
9702
9719
1342
1359
TTATCACAGGGGACATCT
82
G
512





1212300
9752
9769
1392
1409
CCCCAGACGTGAGTTGGC
104
G
513





1212310
10021
10038
1442
1459
CTGGTGGTCCGAGCAGCA
102
G
514





1212320
10071
10088
1492
1509
CGCTGACACTGCCCCTCA
97
G
515





1212330
10121
10138
1542
1559
AAGCCCGGCGGGCAGGCA
78
G
516





1212340
10171
10188
1592
1609
CGGGCAGCTGGTGTGCTG
97
G
517





1212350
10221
10238
1642
1659
TGGCAGCAGGCCCAGCTC
113
G
518





1212360
10363
10380
1692
1709
CAGCCGGGCAGCAGTGCT
106
G
519





1212370
10413
10430
1742
1759
AGAGACCACTTCCTTCTC
91
G
520





1212380
10463
10480
1792
1809
TCCTTCACACCCACGTGA
92
G
521





1212389
10513
10530
1842
1859
GGCAGCAGGTCTGGTTAT
78
G
522





1212398
N/A
N/A
1892
1909
ACAGACGCCCTGGCGGTA
105
G
523





1212408
10702
10719
1942
1959
CTGGCTGCGCAGCGGAAG
102
G
524





1212418
10752
10769
1992
2009
TCAAAGGGGCGTCCCAGC
87
G
525





1212428
10786
10803
2026
2043
GTCCCTCACAGCAGCTGT
76
G
526





1212438
10796
10813
2036
2053
CTTCAGTACTGTCCCTCA
100
G
527





1212448
10806
10823
2046
2063
GCTGCAGAGTCTTCAGTA
87
G
528





1212458
10816
10833
2056
2073
GGTCCCGAGGGCTGCAGA
97
G
529





1212468
10826
10843
2066
2083
CTCCGAGTGGGGTCCCGA
78
G
530





1212478
10836
10853
2076
2093
AGAGGGCACCCTCCGAGT
132
G
531





1212488
10846
10863
2086
2103
AGGCCTGAGCAGAGGGCA
81
G
532





1212498
10856
10873
2096
2113
GGTGCTAGGGAGGCCTGA
102
G
533





1212508
10866
10883
2106
2123
GTTAGGGGGAGGTGCTAG
140
G
534





1212518
10876
10893
2116
2133
GGAGAATTTGGTTAGGGG
106
G
535





1212528
10886
10903
2126
2143
TGGGGTCCAGGGAGAATT
85
G
536





1212538
10896
10913
2136
2153
GAGCTCAGAATGGGGTCC
96
G
537





1212548
10906
10923
2146
2163
TGGTGATGGGGAGCTCAG
109
G
538





1212558
10916
10933
2156
2173
CCACCTCCCATGGTGATG
123
G
539





1212568
10926
10943
2166
2183
GATTGAGGCCCCACCTCC
100
G
540





1212578
10936
10953
2176
2193
GAAGGCCTTAGATTGAGG
76
G
541





1212588
10946
10963
2186
2203
TTCTGACAGGGAAGGCCT
104
G
542





1212598
10956
10973
2196
2213
CACAACCCCCTTCTGACA
98
G
543





1212608
10966
10983
2206
2223
TGGCTTTTGCCACAACCC
100
G
544





1212618
10976
10993
2216
2233
GCTTGTAATGTGGCTTTT
68
G
545





1212628
10986
11003
2226
2243
GGGGATGGCAGCTTGTAA
145
G
546





1212638
10996
11013
2236
2253
GAAACGGGGAGGGGATGG
92
G
547





1212648
11006
11023
2246
2263
AGGGTCCACTGAAACGGG
110
G
548





1212658
11016
11033
2256
2273
ACCTGGCCACAGGGTCCA
163
G
549





1212668
11026
11043
2266
2283
AGGGAAAAGCACCTGGCC
102
G
550





1212678
11036
11053
2276
2293
CCCTGTGGATAGGGAAAA
127
G
551





1212688
11046
11063
2286
2303
ACACAAACACCCCTGTGG
106
G
552





1212698
11056
11073
2296
2313
ACGCGCACACACACAAAC
115
G
553





1212708
11066
11083
2306
2323
TGAAACGCACACGCGCAC
81
G
554





1212718
11076
11093
2316
2333
CAAACTTTATTGAAACGC
100
G
555





1212728
11086
11103
2326
2343
AGAAAGTGTACAAACTTT
111
G
556





1212738
N/A
N/A
2336
2353
TTTTTTTTTAAGAAAGTG
100
G
557





1211971
3114
3131
29
46
CCTCTCGCCCCACACCCC
65
H
558





1211981
3154
3171
69
86
CTGTGTTTCTTTTCTACT
90
H
559





1211991
3194
3211
109
126
TGGCTACTTATCAATATA
95
H
560





1212001
3234
3251
149
166
CAGTTTCCATTGGCCAGG
104
H
561





1212011
3274
3291
189
206
CGCTCAGACTACAGACCC
100
H
562





1212021
3290
3307
205
222
AGCAGCAACCGGGTAGCG
82
H
563





1212031
3300
3317
215
232
GTCCTTGGGCAGCAGCAA
73
H
564





1212041
3310
3327
225
242
CGACTCCGCGGTCCTTGG
57
H
565





1212051
N/A
N/A
235
252
TGCCTGCGTCCGACTCCG
86
H
566





1212061
N/A
N/A
245
262
CCACATGGTCTGCCTGCG
101
H
567





1212071
7163
7180
255
272
TCACCAGGGTCCACATGG
151
H
568





1212081
7205
7222
297
314
ACCGCGTTCCAGCCACCA
103
H
569





1212091
7255
7272
347
364
TCCTCCGGGGTCCAGGCA
81
H
570





1212101
7428
7445
397
414
AGTGTTGTGGGCCATTTG
77
H
571





1212111
7478
7495
447
464
CGGCAGAGCAGTGGGCAT
115
H
572





1212121
7528
7545
497
514
GAAGGGGCAGCAACTGGA
115
H
573





1212131
7693
7710
547
564
AAGCCCCGTGGGCAGCAG
298
H
574





1212141
N/A
N/A
597
614
AGTTGTTACCTGATCTTT
103
H
575





1212151
8269
8286
647
664
GAAGTCCGGGCATTCGAA
68
H
576





1212161
8319
8336
697
714
GGCATGGGGCAGCACCCC
143
H
577





1212171
8470
8487
747
764
AGGCACCGTGCGGACAGC
78
H
578





1212181
8520
8537
797
814
GGGGTGGGTGCCCGTGGG
228
H
579





1212191
N/A
N/A
847
864
GACAAGGCCACTGCCCTG
77†
H
580





1212201
8733
8750
897
914
TAGAACCATCAGGGCACC
13†
H
581





1212211
8783
8800
947
964
GTTGGGCATTGGGCAGCA
361+
H
582





1212221
9069
9086
997
1014
CACACAGTGTCTTGGGGG
73
H
583





1212231
9119
9136
1047
1064
GGTCCGTGGTAGCGTTCT
102
H
584





1212241
9368
9385
1097
1114
CATGTCACATTTCACATC
114
H
585





1212251
9418
9435
1147
1164
GCCCCCGACTGTAGACGG
104
H
586





1212261
9557
9574
1197
1214
GTATGTGGTCCTCACAGC
114
H
587





1212271
9607
9624
1247
1264
TTCACAGGTACCCTTCTG
62
H
588





1212281
9657
9674
1297
1314
CTGAGGTGAGCTGGGGCC
76
H
589





1212291
9707
9724
1347
1364
TGACATTATCACAGGGGA
73
H
590





1212301
9757
9774
1397
1414
CCACTCCCCAGACGTGAG
91
H
591





1212311
10026
10043
1447
1464
CAGTGCTGGTGGTCCGAG
77
H
592





1212321
10076
10093
1497
1514
TTCCTCGCTGACACTGCC
94
H
593





1212331
10126
10143
1547
1564
TAAGGAAGCCCGGCGGGC
85
H
594





1212341
10176
10193
1597
1614
CCCACCGGGCAGCTGGTG
78
H
595





1212351
10226
10243
1647
1664
GCAACTGGCAGCAGGCCC
121
H
596





1212361
10368
10385
1697
1714
GTAGCCAGCCGGGCAGCA
94
H
597





1212371
10418
10435
1747
1764
TGGGCAGAGACCACTTCC
98
H
598





1212381
10468
10485
1797
1814
CCACGTCCTTCACACCCA
84
H
599





1212390
10518
10535
1847
1864
GTCTCGGCAGCAGGTCTG
141
H
600





1212399
10657
10674
1897
1914
GCACAACAGACGCCCTGG
73
H
601





1212409
10707
10724
1947
1964
TACCCCTGGCTGCGCAGC
77
H
602





1212419
10757
10774
1997
2014
GTCCCTCAAAGGGGCGTC
83
H
603





1212429
10787
10804
2027
2044
TGTCCCTCACAGCAGCTG
76
H
604





1212439
10797
10814
2037
2054
TCTTCAGTACTGTCCCTC
76
H
605





1212449
10807
10824
2047
2064
GGCTGCAGAGTCTTCAGT
95
H
606





1212459
10817
10834
2057
2074
GGGTCCCGAGGGCTGCAG
24
H
607





1212469
10827
10844
2067
2084
CCTCCGAGTGGGGTCCCG
92
H
608





1212479
10837
10854
2077
2094
CAGAGGGCACCCTCCGAG
61
H
609





1212489
10847
10864
2087
2104
GAGGCCTGAGCAGAGGGC
72
H
610





1212499
10857
10874
2097
2114
AGGTGCTAGGGAGGCCTG
69
H
611





1212509
10867
10884
2107
2124
GGTTAGGGGGAGGTGCTA
206
H
612





1212519
10877
10894
2117
2134
GGGAGAATTTGGTTAGGG
57
H
613





1212529
10887
10904
2127
2144
ATGGGGTCCAGGGAGAAT
73
H
614





1212539
10897
10914
2137
2154
GGAGCTCAGAATGGGGTC
90
H
615





1212549
10907
10924
2147
2164
ATGGTGATGGGGAGCTCA
85
H
616





1212559
10917
10934
2157
2174
CCCACCTCCCATGGTGAT
105
H
617





1212569
10927
10944
2167
2184
AGATTGAGGCCCCACCTC
56
H
618





1212579
10937
10954
2177
2194
GGAAGGCCTTAGATTGAG
104
H
619





1212589
10947
10964
2187
2204
CTTCTGACAGGGAAGGCC
91
H
620





1212599
10957
10974
2197
2214
CCACAACCCCCTTCTGAC
70
H
621





1212609
10967
10984
2207
2224
GTGGCTTTTGCCACAACC
88
H
622





1212619
10977
10994
2217
2234
AGCTTGTAATGTGGCTTT
82
H
623





1212629
10987
11004
2227
2244
AGGGGATGGCAGCTTGTA
222
H
624





1212639
10997
11014
2237
2254
TGAAACGGGGAGGGGATG
79
H
625





1212649
11007
11024
2247
2264
CAGGGTCCACTGAAACGG
74
H
626





1212659
11017
11034
2257
2274
CACCTGGCCACAGGGTCC
55
H
627





1212669
11027
11044
2267
2284
TAGGGAAAAGCACCTGGC
74
H
628





1212679
11037
11054
2277
2294
CCCCTGTGGATAGGGAAA
167
H
629





1212689
11047
11064
2287
2304
CACACAAACACCCCTGTG
89
H
630





1212699
11057
11074
2297
2314
CACGCGCACACACACAAA
99
H
631





1212709
11067
11084
2307
2324
TTGAAACGCACACGCGCA
105
H
632





1212719
11077
11094
2317
2334
ACAAACTTTATTGAAACG
87
H
633





1212729
11087
11104
2327
2344
AAGAAAGTGTACAAACTT
84
H
634





1212739
N/A
N/A
2337
2354
TTTTTTTTTTAAGAAAGT
72
H
635





1211972
3118
3135
33
50
GCTTCCTCTCGCCCCACA
85
I
636





1211982
3158
3175
73
90
AATGCTGTGTTTCTTTTC
86
I
637





1211992
3198
3215
113
130
CCATTGGCTACTTATCAA
81
I
638





1212002
3238
3255
153
170
ACCTCAGTTTCCATTGGC
89
I
639





1212012
3278
3295
193
210
GTAGCGCTCAGACTACAG
93
I
640





1212022
3291
3308
206
223
CAGCAGCAACCGGGTAGC
82
I
641





1212032
3301
3318
216
233
GGTCCTTGGGCAGCAGCA
69
I
642





1212042
3311
3328
226
243
CCGACTCCGCGGTCCTTG
95
I
643





1212052
N/A
N/A
236
253
CTGCCTGCGTCCGACTCC
87
I
644





1212062
7154
7171
246
263
TCCACATGGTCTGCCTGC
90
I
645





1212072
7164
7181
256
273
CTCACCAGGGTCCACATG
117
I
646





1212082
7210
7227
302
319
TGGGCACCGCGTTCCAGC
86
I
647





1212092
7260
7277
352
369
CTGGCTCCTCCGGGGTCC
80
I
648





1212102
7433
7450
402
419
TGCTCAGTGTTGTGGGCC
107
I
649





1212112
7483
7500
452
469
GTGGCCGGCAGAGCAGTG
106
I
650





1212122
7533
7550
502
519
TCTGGGAAGGGGCAGCAA
187
I
651





1212132
7698
7715
552
569
AGTGGAAGCCCCGTGGGC
108
I
652





1212142
N/A
N/A
602
619
CACGGAGTTGTTACCTGA
86
I
653





1212152
8274
8291
652
669
GTGGAGAAGTCCGGGCAT
90
I
654





1212162
8324
8341
702
719
CCTGGGGCATGGGGCAGC
220
I
655





1212172
8475
8492
752
769
GCAGAAGGCACCGTGCGG
121
I
656





1212182
8525
8542
802
819
GCCAGGGGGGGGTGCCC
172
I
657





1212192
8688
8705
852
869
AGCTGGACAAGGCCACTG
27†
I
658





1212202
8738
8755
902
919
GCAGGTAGAACCATCAGG
10†
I
659





1212212
N/A
N/A
952
969
GTGGCGTTGGGCATTGGG
391
I
660





1212222
9074
9091
1002
1019
GGTCACACACAGTGTCTT
90
I
661





1212232
9124
9141
1052
1069
GAGGAGGTCCGTGGTAGC
118
I
662





1212242
9373
9390
1102
1119
ACCTCCATGTCACATTTC
87
I
663





1212252
9423
9440
1152
1169
CCCAGGCCCCCGACTGTA
97
I
664





1212262
9562
9579
1202
1219
GCAGTGTATGTGGTCCTC
111
I
665





1212272
9612
9629
1252
1269
CCCTGTTCACAGGTACCC
119
I
666





1212282
9662
9679
1302
1319
GCAGGCTGAGGTGAGCTG
239
I
667





1212292
9712
9729
1352
1369
GCTGCTGACATTATCACA
117
I
668





1212302
9762
9779
1402
1419
CAGCCCCACTCCCCAGAC
90
I
669





1212312
10031
10048
1452
1469
GGCAGCAGTGCTGGTGGT
122
I
670





1212322
10081
10098
1502
1519
CTCGCTTCCTCGCTGACA
77
I
671





1212332
10131
10148
1552
1569
TGGGATAAGGAAGCCCGG
85
I
672





1212342
10181
10198
1602
1619
TCTGCCCCACCGGGCAGC
103
I
673





1212352
10231
10248
1652
1669
ATGGGGCAACTGGCAGCA
153
I
674





1212362
10373
10390
1702
1719
CAGGTGTAGCCAGCCGGG
80
I
675





1212372
10423
10440
1752
1769
CAGGCTGGGCAGAGACCA
83
I
676





1212382
10473
10490
1802
1819
ACACTCCACGTCCTTCAC
86
I
677





1212391
10523
10540
1852
1869
CGGTTGTCTCGGCAGCAG
92
I
678





1212400
10662
10679
1902
1919
GATCAGCACAACAGACGC
85
I
679





1212410
10712
10729
1952
1969
CTTGGTACCCCTGGCTGC
123
I
680





1212420
10762
10779
2002
2019
GCTGGGTCCCTCAAAGGG
88
I
681





1212430
10788
10805
2028
2045
CTGTCCCTCACAGCAGCT
79
I
682





1212440
10798
10815
2038
2055
GTCTTCAGTACTGTCCCT
109
I
683





1212450
10808
10825
2048
2065
GGGCTGCAGAGTCTTCAG
81
I
684





1212460
10818
10835
2058
2075
GGGGTCCCGAGGGCTGCA
89
I
685





1212470
10828
10845
2068
2085
CCCTCCGAGTGGGGTCCC
72
I
686





1212480
10838
10855
2078
2095
GCAGAGGGCACCCTCCGA
87
I
687





1212490
10848
10865
2088
2105
GGAGGCCTGAGCAGAGGG
88
I
688





1212500
10858
10875
2098
2115
GAGGTGCTAGGGAGGCCT
94
I
689





1212510
10868
10885
2108
2125
TGGTTAGGGGGAGGTGCT
75
I
690





1212520
10878
10895
2118
2135
AGGGAGAATTTGGTTAGG
73
I
691





1212530
10888
10905
2128
2145
AATGGGGTCCAGGGAGAA
158
I
692





1212540
10898
10915
2138
2155
GGGAGCTCAGAATGGGGT
92
I
693





1212550
10908
10925
2148
2165
CATGGTGATGGGGAGCTC
87
I
694





1212560
10918
10935
2158
2175
CCCCACCTCCCATGGTGA
115
I
695





1212570
10928
10945
2168
2185
TAGATTGAGGCCCCACCT
85
I
696





1212580
10938
10955
2178
2195
GGGAAGGCCTTAGATTGA
68
I
697





1212590
10948
10965
2188
2205
CCTTCTGACAGGGAAGGC
105
I
698





1212600
10958
10975
2198
2215
GCCACAACCCCCTTCTGA
88
I
699





1212610
10968
10985
2208
2225
TGTGGCTTTTGCCACAAC
102
I
700





1212620
10978
10995
2218
2235
CAGCTTGTAATGTGGCTT
84
I
701





1212630
10988
11005
2228
2245
GAGGGGATGGCAGCTTGT
153
I
702





1212640
10998
11015
2238
2255
CTGAAACGGGGAGGGGAT
87
I
703





1212650
11008
11025
2248
2265
ACAGGGTCCACTGAAACG
96
I
704





1212660
11018
11035
2258
2275
GCACCTGGCCACAGGGTC
89
I
705





1212670
11028
11045
2268
2285
ATAGGGAAAAGCACCTGG
93
I
706





1212680
11038
11055
2278
2295
ACCCCTGTGGATAGGGAA
90
I
707





1212690
11048
11065
2288
2305
ACACACAAACACCCCTGT
94
I
708





1212700
11058
11075
2298
2315
ACACGCGCACACACACAA
143
I
709





1212710
11068
11085
2308
2325
ATTGAAACGCACACGCGC
100
I
710





1212720
11078
11095
2318
2335
TACAAACTTTATTGAAAC
93
I
711





1212730
11088
11105
2328
2345
TAAGAAAGTGTACAAACT
80
I
712





1212740
N/A
N/A
2338
2355
TTTTTTTTTTTAAGAAAG
81
I
713





1211973
3122
3139
37
54
CCCTGCTTCCTCTCGCCC
53
J
714





1211983
3162
3179
77
94
CTGGAATGCTGTGTTTCT
132
J
715





1211993
3202
3219
117
134
GCTCCCATTGGCTACTTA
170
J
716





1212003
3242
3259
157
174
GCCTACCTCAGTTTCCAT
81
J
717





1212013
3282
3299
197
214
CCGGGTAGCGCTCAGACT
100
J
718





1212023
3292
3309
207
224
GCAGCAGCAACCGGGTAG
111
J
719





1212033
3302
3319
217
234
CGGTCCTTGGGCAGCAGC
101
J
720





1212043
3312
3329
227
244
TCCGACTCCGCGGTCCTT
109
J
721





1212053
N/A
N/A
237
254
TCTGCCTGCGTCCGACTC
85
J
722





1212063
7155
7172
247
264
GTCCACATGGTCTGCCTG
86
J
723





1212073
7165
7182
257
274
GCTCACCAGGGTCCACAT
84
J
724





1212083
7215
7232
307
324
CCATCTGGGCACCGCGTT
46
J
725





1212093
7265
7282
357
374
TGTAGCTGGCTCCTCCGG
90
J
726





1212103
7438
7455
407
424
ATGCCTGCTCAGTGTTGT
83
J
727





1212113
7488
7505
457
474
CAGGAGTGGCCGGCAGAG
82
J
728





1212123
N/A
N/A
507
524
CGGCCTCTGGGAAGGGGC
93
J
729





1212133
7703
7720
557
574
ACTGCAGTGGAAGCCCCG
122
J
730





1212143
8229
8246
607
624
GCACCCACGGAGTTGTTA
75
J
731





1212153
8279
8296
657
674
AGCACGTGGAGAAGTCCG
93
J
732





1212163
N/A
N/A
707
724
GGAAGCCTGGGGCATGGG
100
J
733





1212173
8480
8497
757
774
AGGTCGCAGAAGGCACCG
85
J
734





1212183
8530
8547
807
824
TCTTTGCCAGGGGGTGGG
153
J
735





1212193
8693
8710
857
874
GACCGAGCTGGACAAGGC
10†
J
736





1212203
8743
8760
907
924
TCACAGCAGGTAGAACCA
18†
J
737





1212213
N/A
N/A
957
974
AGCAGGTGGCGTTGGGCA
108
J
738





1212223
9079
9096
1007
1024
GATCAGGTCACACACAGT
79
J
739





1212233
9129
9146
1057
1074
TTAGTGAGGAGGTCCGTG
83
J
740





1212243
9378
9395
1107
1124
AGCTCACCTCCATGTCAC
108
J
741





1212253
9428
9445
1157
1174
GCAGCCCCAGGCCCCCGA
83
J
742





1212263
9567
9584
1207
1224
GGACAGCAGTGTATGTGG
93
J
743





1212273
9617
9634
1257
1274
GGGGCCCCTGTTCACAGG
71
J
744





1212283
9667
9684
1307
1324
GTCTGGCAGGCTGAGGTG
98
J
745





1212293
9717
9734
1357
1374
GGACAGCTGCTGACATTA
102
J
746





1212303
9767
9784
1407
1424
GACAGCAGCCCCACTCCC
62
J
747





1212313
10036
10053
1457
1474
CTGGGGGCAGCAGTGCTG
158
J
748





1212323
10086
10103
1507
1524
ACGATCTCGCTTCCTCGC
110
J
749





1212333
10136
10153
1557
1574
TGGGGTGGGATAAGGAAG
107
J
750





1212343
10186
10203
1607
1624
GCAGGTCTGCCCCACCGG
83
J
751





1212353
N/A
N/A
1657
1674
ACAGCATGGGGCAACTGG
82
J
752





1212363
10378
10395
1707
1724
CGTTGCAGGTGTAGCCAG
93
J
753





1212373
10428
10445
1757
1774
GGTGGCAGGCTGGGCAGA
215
J
754





1212383
10478
10495
1807
1824
TCCCCACACTCCACGTCC
83
J
755





1212392
10528
10545
1857
1874
CCTGTCGGTTGTCTCGGC
101
J
756





1212401
10667
10684
1907
1924
GCGCCGATCAGCACAACA
86
J
757





1212411
10717
10734
1957
1974
AAACACTTGGTACCCCTG
94
J
758





1212421
10767
10784
2007
2024
TCAAGGCTGGGTCCCTCA
86
J
759





1212431
10789
10806
2029
2046
ACTGTCCCTCACAGCAGC
82
J
760





1212441
10799
10816
2039
2056
AGTCTTCAGTACTGTCCC
97
J
761





1212451
10809
10826
2049
2066
AGGGCTGCAGAGTCTTCA
88
J
762





1212461
10819
10836
2059
2076
TGGGGTCCCGAGGGCTGC
90
J
763





1212471
10829
10846
2069
2086
ACCCTCCGAGTGGGGTCC
78
J
764





1212481
10839
10856
2079
2096
AGCAGAGGGCACCCTCCG
97
J
765





1212491
10849
10866
2089
2106
GGGAGGCCTGAGCAGAGG
75
J
766





1212501
10859
10876
2099
2116
GGAGGTGCTAGGGAGGCC
64
J
767





1212511
10869
10886
2109
2126
TTGGTTAGGGGGAGGTGC
102
J
768





1212521
10879
10896
2119
2136
CAGGGAGAATTTGGTTAG
85
J
769





1212531
10889
10906
2129
2146
GAATGGGGTCCAGGGAGA
71
J
770





1212541
10899
10916
2139
2156
GGGGAGCTCAGAATGGGG
102
J
771





1212551
10909
10926
2149
2166
CCATGGTGATGGGGAGCT
72
J
772





1212561
10919
10936
2159
2176
GCCCCACCTCCCATGGTG
92
J
773





1212571
10929
10946
2169
2186
TTAGATTGAGGCCCCACC
89
J
774





1212581
10939
10956
2179
2196
AGGGAAGGCCTTAGATTG
95
J
775





1212591
10949
10966
2189
2206
CCCTTCTGACAGGGAAGG
84
J
776





1212601
10959
10976
2199
2216
TGCCACAACCCCCTTCTG
80
J
777





1212611
10969
10986
2209
2226
ATGTGGCTTTTGCCACAA
144
J
778





1212621
10979
10996
2219
2236
GCAGCTTGTAATGTGGCT
82
J
779





1212631
10989
11006
2229
2246
GGAGGGGATGGCAGCTTG
89
J
780





1212641
10999
11016
2239
2256
ACTGAAACGGGGAGGGGA
97
J
781





1212651
11009
11026
2249
2266
CACAGGGTCCACTGAAAC
76
J
782





1212661
11019
11036
2259
2276
AGCACCTGGCCACAGGGT
88
J
783





1212671
11029
11046
2269
2286
GATAGGGAAAAGCACCTG
86
J
784





1212681
11039
11056
2279
2296
CACCCCTGTGGATAGGGA
95
J
785





1212691
11049
11066
2289
2306
CACACACAAACACCCCTG
78
J
786





1212701
11059
11076
2299
2316
CACACGCGCACACACACA
145
J
787





1212711
11069
11086
2309
2326
TATTGAAACGCACACGCG
86
J
788





1212721
11079
11096
2319
2336
GTACAAACTTTATTGAAA
142
J
789





1212731
11089
11106
2329
2346
TTAAGAAAGTGTACAAAC
99
J
790





1212741
N/A
N/A
2339
2356
TTTTTTTTTTTTAAGAAA
95
J
791









Example 2: Effect of Uniform MOE Modified Oligonucleotides with Uniform Phosphorothioate Internucleoside Linkages on Human Progranulin In Vitro, Single Dose

Modified oligonucleotides complementary to a human progranulin nucleic acid were designed and tested for their effect on progranulin RNA in vitro. The modified oligonucleotides were tested in a series of experiments that had the same culture conditions.


“Start site” indicates the 5′-most nucleoside to which the modified oligonucleotide is complementary in the target nucleic acid sequence. “Stop site” indicates the 3′-most nucleoside to which the modified oligonucleotide is complementary in the target nucleic acid sequence. Each modified oligonucleotide listed in the tables below is 100% complementary to SEQ ID NO: 1 (GENBANK Accession No. NC_000017.11 truncated from nucleotides 44342001 to 44356000), to SEQ ID NO: 2 (GENBANK Accession No. NM_002087.3), or to both. ‘N/A’ indicates that the modified oligonucleotide is not 100% complementary to that particular target nucleic acid sequence.


The modified oligonucleotides in the table below are 18 nucleosides in length, wherein the sugar motif for the modified oligonucleotides is (from 5′ to 3′): eeeeeeeeeeeeeeeeee; wherein “e” represents a 2′-MOE sugar moiety. The internucleoside linkage motif for the modified oligonucleotides is (from 5′ to 3′): sssssssssssssssss; wherein each “s” represents a phosphorothioate internucleoside linkage. Each cytosine residue is a 5-methyl cytosine.


Cultured A-431 cells were treated with modified oligonucleotide at a concentration of 4,000 nM by free uptake at a density of 10,000 cells per well. After a treatment period of approximately 48 hours, RNA was isolated from the cells and progranulin RNA levels were measured by quantitative real-time PCR. Human primer probe set RTS42426 (described herein above) was used to measure upregulation of progranulin RNA. Progranulin RNA levels were normalized to total RNA content, as measured by RIBOGREEN®. Results are presented as percent progranulin RNA, relative to the amount in untreated control cells (% UTC).









TABLE 2







Uniform MOE modified oligonucleotides with uniform PS linkages


 complementary to human progranulin
















SEQ
SEQ
SEQ
SEQ







ID
ID
ID
ID







NO: 1
NO: 1
NO: 2
NO: 2



SEQ


Compound
Start
Stop
Start
Stop

progranulin

ID


No.
Site
Site
Site
Site
Sequence (5′ to 3′)
(% UTC)
AID
NO





1212165
8440
8457
717
734
CTTCACAGCAGGAAGCCT
109
K
110





1212166
8445
8462
722
739
CCTGTCTTCACAGCAGGA
101
K
188





1212167
8450
8467
727
744
TGCACCCTGTCTTCACAG
135
K
267





1212168
8455
8472
732
749
AGCAGTGCACCCTGTCTT
 91
K
344





1212169
8460
8477
737
754
CGGACAGCAGTGCACCCT
119
K
423





1212170
8465
8482
742
759
CCGTGCGGACAGCAGTGC
 88
K
500





1212171
8470
8487
747
764
AGGCACCGTGCGGACAGC
 66
K
578





1212172
8475
8492
752
769
GCAGAAGGCACCGTGCGG
 96
K
656





1212173
8480
8497
757
774
AGGTCGCAGAAGGCACCG
 79
K
734





1212174
8485
8502
762
779
GAACCAGGTCGCAGAAGG
100
K
 33





1212175
8490
8507
767
784
GGTGTGAACCAGGTCGCA
106
K
111





1212176
8495
8512
772
789
CAGCGGGTGTGAACCAGG
 74
K
189





1212177
8500
8517
777
794
TGATGCAGCGGGTGTGAA
131
K
268





1212178
8505
8522
782
799
GGGTGTGATGCAGCGGGT
115
K
345





1366749
8447
8464
724
741
ACCCTGTCTTCACAGCAG
112
K
792





1366750
8452
8469
729
746
AGTGCACCCTGTCTTCAC
 90
K
793





1366751
8432
8449
N/A
N/A
CAGGAAGCCTGAGAAAAA
 75
K
794





1366752
8489
8506
766
783
GTGTGAACCAGGTCGCAG
 91
K
795





1366753
8474
8491
751
768
CAGAAGGCACCGTGCGGA
 81
K
796





1366754
8492
8509
769
786
CGGGTGTGAACCAGGTCG
 84
K
797





1366755
8436
8453
N/A
N/A
ACAGCAGGAAGCCTGAGA
107
K
798





1366756
8441
8458
718
735
TCTTCACAGCAGGAAGCC
100
K
799





1366757
8502
8519
779
796
TGTGATGCAGCGGGTGTG
109
K
800





1366758
8497
8514
774
791
TGCAGCGGGTGTGAACCA
122
K
801





1366759
8457
8474
734
751
ACAGCAGTGCACCCTGTC
 81
K
802





1366760
8467
8484
744
761
CACCGTGCGGACAGCAGT
106
K
803





1366761
8437
8454
N/A
N/A
CACAGCAGGAAGCCTGAG
 66
K
804





1366762
8444
8461
721
738
CTGTCTTCACAGCAGGAA
 96
K
805





1366763
8501
8518
778
795
GTGATGCAGCGGGTGTGA
151
K
806





1366764
8466
8483
743
760
ACCGTGCGGACAGCAGTG
 74
K
807





1366765
8438
8455
N/A
N/A
TCACAGCAGGAAGCCTGA
 81
K
808





1366766
8472
8489
749
766
GAAGGCACCGTGCGGACA
 78
K
809





1366767
8446
8463
723
740
CCCTGTCTTCACAGCAGG
 71
K
810





1366768
8443
8460
720
737
TGTCTTCACAGCAGGAAG
 79
K
811





1366769
8498
8515
775
792
ATGCAGCGGGTGTGAACC
118
K
812





1366770
8504
8521
781
798
GGTGTGATGCAGCGGGTG
114
K
813





1366771
8482
8499
759
776
CCAGGTCGCAGAAGGCAC
 87
K
814





1366772
8483
8500
760
777
ACCAGGTCGCAGAAGGCA
118
K
815





1366773
8507
8524
784
801
GTGGGTGTGATGCAGCGG
103
K
816





1366774
8442
8459
719
736
GTCTTCACAGCAGGAAGC
 87
K
817





1366775
8464
8481
741
758
CGTGCGGACAGCAGTGCA
 97
K
818





1366776
8448
8465
725
742
CACCCTGTCTTCACAGCA
 82
K
819





1366777
8476
8493
753
770
CGCAGAAGGCACCGTGCG
102
K
820





1366778
8508
8525
785
802
CGTGGGTGTGATGCAGCG
100
K
821





1366779
8494
8511
771
788
AGCGGGTGTGAACCAGGT
 85
K
822





1366780
8453
8470
730
747
CAGTGCACCCTGTCTTCA
170
K
823





1366781
8477
8494
754
771
TCGCAGAAGGCACCGTGC
 87
K
824





1366782
8469
8486
746
763
GGCACCGTGCGGACAGCA
 56
K
825





1366783
8435
8452
N/A
N/A
CAGCAGGAAGCCTGAGAA
132
K
826





1366784
8484
8501
761
778
AACCAGGTCGCAGAAGGC
 81
K
827





1366785
8499
8516
776
793
GATGCAGCGGGTGTGAAC
132
K
828





1366786
8496
8513
773
790
GCAGCGGGTGTGAACCAG
 76
K
829





1366787
8493
8510
770
787
GCGGGTGTGAACCAGGTC
 95
K
830





1366788
8439
8456
716
733
TTCACAGCAGGAAGCCTG
 93
K
831





1366789
8468
8485
745
762
GCACCGTGCGGACAGCAG
 95
K
832





1366790
8459
8476
736
753
GGACAGCAGTGCACCCTG
153
K
833





1366791
8454
8471
731
748
GCAGTGCACCCTGTCTTC
 94
K
834





1366792
8471
8488
748
765
AAGGCACCGTGCGGACAG
 65
K
835





1366793
8433
8450
N/A
N/A
GCAGGAAGCCTGAGAAAA
 87
K
836





1366794
8462
8479
739
756
TGCGGACAGCAGTGCACC
119
K
837





1366795
8451
8468
728
745
GTGCACCCTGTCTTCACA
 88
K
838





1366796
8458
8475
735
752
GACAGCAGTGCACCCTGT
 90
K
839





1366797
8479
8496
756
773
GGTCGCAGAAGGCACCGT
 97
K
840





1366798
8486
8503
763
780
TGAACCAGGTCGCAGAAG
108
K
841





1366799
8491
8508
768
785
GGGTGTGAACCAGGTCGC
105
K
842





1366800
8473
8490
750
767
AGAAGGCACCGTGCGGAC
 69
K
843





1366801
8506
8523
783
800
TGGGTGTGATGCAGCGGG
129
K
844





1366802
8488
8505
765
782
TGTGAACCAGGTCGCAGA
182
K
845





1366803
8456
8473
733
750
CAGCAGTGCACCCTGTCT
118
K
846





1366804
8503
8520
780
797
GTGTGATGCAGCGGGTGT
116
K
847





1366805
8481
8498
758
775
CAGGTCGCAGAAGGCACC
112
K
848





1366806
8461
8478
738
755
GCGGACAGCAGTGCACCC
 92
K
849





1366807
8463
8480
740
757
GTGCGGACAGCAGTGCAC
 81
K
850





1366808
8487
8504
764
781
GTGAACCAGGTCGCAGAA
124
K
851





1366809
8478
8495
755
772
GTCGCAGAAGGCACCGTG
114
K
852





1366810
8434
8451
N/A
N/A
AGCAGGAAGCCTGAGAAA
 97
K
853





1366811
8449
8466
726
743
GCACCCTGTCTTCACAGC
 82
K
854









Example 3: Effect of Modified Oligonucleotides on Human Progranulin In Vitro, Multiple Doses

Modified oligonucleotides selected from the example above were tested in A-431 cells. The modified oligonucleotides were tested in a series of experiments using the same culture conditions. The results for each experiment are presented in separate tables shown below.


Cultured A-431 cells were plated at a density of 10,000 cells per well and were treated by free uptake at various doses, as specified in the tables below. After a treatment period of approximately 48 hours, total RNA was isolated from the cells and progranulin RNA levels were measured by quantitative real-time PCR. Human progranulin primer probe set RTS42426 (described herein above) was used to measure progranulin RNA. Progranulin RNA levels were normalized to total RNA content, as measured by RIBOGREEN®. Results are presented in the tables below as percent progranulin RNA, relative to the amount in untreated control cells (% UTC). Fold increase in the amount of progranulin RNA at 20 μM relative to untreated control cells was calculated and is presented in the tables below.









TABLE 3







Dose-dependent increase in human progranulin


RNA by modified oligonucleotides









Compound
progranulin RNA (% UTC)
Fold Increase












No.
313 nM
1250 nM
5000 nM
20000 nM
@ 20 μM















1212035
175
221
271
334
3.34


1212135
122
123
131
146
1.46


1212165
144
147
154
156
1.56


1212175
133
127
155
166
1.66


1212184
135
141
144
189
1.89


1212245
131
147
141
137
1.37


1212246
184
185
216
254
2.54


1212264
111
115
133
127
1.27


1212345
137
173
200
250
2.50


1212366
104
104
112
122
1.22


1212373
289
312
328
284
2.84


1212374
120
126
131
194
1.94


1212432
104
98
107
131
1.31


1212472
117
138
181
210
2.10


1212542
130
143
190
248
2.48


1212614
174
167
171
261
2.61


1212632
122
122
143
166
1.66


1212633
121
135
161
182
1.82


1212662
110
104
97
111
1.11
















TABLE 4







Dose-dependent increase in human progranulin RNA


by modified oligonucleotides

















Fold









Compound
progranulin RNA (% UTC)
Increase @












No.
313 nM
1250 nM
5000 nM
20000 nM
20 μM















1212036
107
231
267
337
3.37


1212088
104
110
129
128
1.28


1212128
153
143
155
152
1.52


1212169
189
162
166
210
2.10


1212228
167
194
196
208
2.08


1212248
123
136
150
157
1.57


1212256
171
211
249
278
2.78


1212257
115
120
150
145
1.45


1212298
138
136
146
163
1.63


1212373
293
315
339
307
3.07


1212396
119
118
120
196
1.96


1212505
122
144
157
178
1.78


1212506
152
177
205
239
2.39


1212625
101
106
127
126
1.26


1212626
110
121
136
152
1.52


1212627
143
152
148
131
1.31


1212635
115
103
103
186
1.86


1212647
184
162
153
197
1.97


1212665
104
103
107
118
1.18
















TABLE 5







Dose-dependent increase in human progranulin


RNA by modified oligonucleotides












progranulin RNA (% UTC)
Fold














Compound
313
1250
5000

Increase



No.
nM
nM
nM
20000 nM
@ 20 μM


















1212000
94
108
109
127
1.27



1212050
112
108
102
98
0.98



1212071
171
174
147
145
1.45



1212080
100
125
141
237
2.37



1212121
113
118
150
184
1.84



1212129
140
154
162
186
1.86



1212161
151
165
189
223
2.23



1212179
229
222
215
238
2.38



1212180
359
365
405
427
4.27



1212219
194
182
179
196
1.96



1212249
138
133
158
241
2.41



1212359
147
159
164
217
2.17



1212373
266
286
293
263
2.63



1212390
130
160
255
281
2.81



1212507
113
130
170
268
2.68



1212508
129
120
147
217
2.17



1212628
143
152
213
219
2.19



1212658
102
104
136
200
2.00



1212679
185
175
137
167
1.67

















TABLE 6







Dose-dependent increase in human progranulin


RNA by modified oligonucleotides

















Fold









Compound
progranulin RNA (% UTC)
Increase @












No.
313 nM
1250 nM
5000 nM
20000 nM
20 μM















1211983
162
205
177
194
1.94


1211993
200
179
162
183
1.83


1212122
137
171
206
209
2.09


1212131
146
173
280
352
3.52


1212181
231
243
174
179
1.79


1212182
264
285
265
270
2.70


1212183
224
240
246
293
2.93


1212282
92
117
116
154
1.54


1212313
120
166
158
157
1.57


1212352
145
157
188
217
2.17


1212373
274
275
252
244
2.44


1212509
144
173
239
336
3.36


1212530
70
105
118
132
1.32


1212611
100
108
135
207
2.07


1212629
125
146
177
233
2.33


1212630
106
116
106
140
1.40


1212700
132
185
207
282
2.82


1212701
157
212
235
297
2.97


1212721
135
201
255
350
3.50
















TABLE 7







Dose-dependent increase in human progranulin


RNA by modified oligonucleotides












progranulin RNA (% UTC)
Fold














Compound
313
1250
5000

Increase



No.
nM
nM
nM
20000 nM
@ 20 μM


















1212167
90
98
111
108
1.08



1212169
93
88
97
87
0.87



1212177
126
131
144
172
1.72



1212178
107
98
91
100
1.00



1366758
125
103
106
139
1.39



1366763
113
160
169
172
1.72



1366769
144
123
104
102
1.02



1366772
87
111
100
115
1.15



1366780
110
108
124
142
1.42



1366783
101
123
105
153
1.53



1366785
134
134
143
169
1.69



1366790
140
130
142
136
1.36



1366794
93
103
102
118
1.18



1366801
100
107
122
142
1.42



1366802
118
136
129
148
1.48



1366803
109
105
113
107
1.07



1366804
117
108
105
105
1.05



1366808
94
110
97
111
1.11










Example 4: Design of Uniform MOE Modified Oligonucleotides with Mixed PS/PO Internucleoside Linkages Complementary to Human Progranulin RNA

Modified oligonucleotides complementary to a human progranulin RNA were designed as described in Table 8 below.


The modified oligonucleotides in the table below are 18 nucleosides in length, and the sugar motif for the modified oligonucleotides is (from 5′ to 3′): eeeeeeeeeeeeeeeeee; wherein “e” represents a 2′-MOE sugar moiety. The internucleoside linkage motif for the modified oligonucleotides is (from 5′ to 3′): sosssssssosssssss; wherein each “s” represents a phosphorothioate internucleoside linkage, and each “o” represents a phosphodiester internucleoside linkage.


Each cytosine residue is a 5-methyl cytosine. “Start site” indicates the 5′-most nucleoside to which the modified oligonucleotide is complementary in the target nucleic acid sequence. “Stop site” indicates the 3′-most nucleoside to which the modified oligonucleotide is complementary in the target nucleic acid sequence. Each modified oligonucleotide listed in the tables below is 100% complementary to SEQ ID NO: 1 (described herein above) to SEQ ID NO: 2 (described herein above), or to both. ‘N/A’ indicates that the modified oligonucleotide is not 100% complementary to that particular target nucleic acid sequence.









TABLE 8







Uniform 2′-MOE modified oligonucleotides with mixed PS/PO 


linkages complementary to human GRN














SEQ ID
SEQ ID
SEQ ID
SEQ ID





NO: 1
NO: 1
NO: 2
NO: 2

SEQ


Compound
Start
Stop
Start
Stop

ID


No.
Site
Site
Site
Site
Sequence (5′ to 3′)
NO





1557984
10882
10899
2122
2139
GTCCAGGGAGAATTTGGT
224





1557988
10883
10900
2123
2140
GGTCCAGGGAGAATTTGG
302





1557989
10995
11012
2235
2252
AAACGGGGAGGGGATGGC
469





1557990
10996
11013
2236
2253
GAAACGGGGAGGGGATGG
547





1557991
10997
11014
2237
2254
TGAAACGGGGAGGGGATG
625





1557992
10998
11015
2238
2255
CTGAAACGGGGAGGGGAT
703





1557993
10999
11016
2239
2256
ACTGAAACGGGGAGGGGA
781





1557994
11000
11017
2240
2257
CACTGAAACGGGGAGGGG
 80





1557995
11004
11021
2244
2261
GGTCCACTGAAACGGGGA
392





1557986
11005
11022
2245
2262
GGGTCCACTGAAACGGGG
470





1557987
11030
11047
2270
2287
GGATAGGGAAAAGCACCT
 83





1557985
11032
11049
2272
2289
GTGGATAGGGAAAAGCAC
239









Example 5: Effect of Modified Oligonucleotides Complementary to Human GRN in H4 Cells; Single Dose

Modified oligonucleotides selected from the examples above were tested for their effect on progranulin protein levels in vitro. Also tested was a control scrambled modified oligonucleotide (“control scrambled ASO”) (uniformly 2′MOE-modified, phosphorothioate backbone having a nucleobase sequence (from 5′ to 3′):











(SEQ ID NO: 10))



TTAGTTTAATCACGCTCG.






H4 human neuroglioma cells (ATCC HTB-148) were treated with modified oligonucleotide at a concentration of 5000 nM for 24 hours. Progranulin levels were measured in cell lysates by ELISA. Progranulin concentrations were determined in duplicate using 10-15 μl of lysates per well (typically 8-20 μg of total protein per well) using a sandwich ELISA assay (R&D Systems, DPGRN0). Data are presented in FIG. 1A as means±/−SD, * indicates p<0.05; ** indicates p<0.01, *** indicates p<0.001, **** indicates p<0.0002, as determined by one-way ANOVA with Dunnett post hoc test.


H4 human neuroglioma cells were treated with modified oligonucleotide at a concentration of 10 μM for 24 hours. Progranulin levels were measured in cell lysates by Western blot (FIG. 1B, FIG. 1C, and FIG. 1D). In a separate experiment, H4 cells were treated with 10 μM modified oligonucleotide for 24 hours; progranulin levels in the cell lysates, as well as secreted progranulin levels in the conditioned media were assessed by Western blot. Modified oligonucleotides that increased cellular progranulin levels (FIG. 1E top panel) also increased secreted progranulin levels in the media (FIG. 1E bottom panel).


Primary antibodies used for immunoblot analysis include: an anti-human progranulin linker 5 polyclonal antibody #614 that recognizes an epitope between amino acids 497-515 (Nguyen, A. D., et al., J. Biol. Chem. 288, 8627-8635 (2013)), an anti-human vinculin monoclonal antibody (loading control for protein level) (Cell Signaling Technology, 13901).


Example 6: Effect of Modified Oligonucleotides Complementary to Human GRN In Vitro; Multiple Dose

Modified oligonucleotides selected from the examples above, and the control scrambled ASO described above, were tested for their effect on progranulin protein levels in H4 cells. Cells were treated with modified oligonucleotide at the doses indicated in FIGS. 2A-2F for 24 hours. Progranulin levels were measured in cell lysates by ELISA, as in Example 5. Data are presented as means±SD, with EC50 values as indicated.


Example 7: Effect of Modified Oligonucleotides Complementary to Human GRN in iPSC-Derived Neurons

Modified oligonucleotides selected from the examples above, and the control scrambled ASO described above, were tested for their effect on progranulin protein levels in iPSC-derived neurons. Human iPSCs harboring doxycycline-responsive Neurogenin-2 (NGN2) expression for differentiation into i3 cortical neurons were prepared as described (Fernandopulle, M. S., et al., Curr. Protoc. Cell Biol. 79, e51 (2018)). After 2 weeks of differentiation, the i3 neurons were treated with modified oligonucleotide at the indicated concentrations (μM) for 3-4 days. Western blot analysis was conducted as in Example 5, with the data shown in FIG. 3.


Example 8: Effect of Modified Oligonucleotides Complementary to Human GRN In Vivo

Homozygous knock in mice for human GRN were prepared from a heterozygous C57BL/6J breeder pair (Petkau et al., Neurobiol. Dis. 153, 105314 (2021)). These transgenic GRN mice were treated with 500 μg modified oligonucleotide by bolus intracerebroventricular injection into the right lateral ventricle (Jafar-Nejad, P., et al., Nucleic Acids Res. 49, 657-673 (2021); Farr, S. A., et al., J. Alzheimers Dis. 40, 1005-1016 (2014)). Three weeks post administration, mice were sacrificed and brain tissues were collected for protein analysis. Human progranulin levels in the cortex of mice treated with the indicated modified oligonucleotide, the control scrambled ASO described above, and saline-treated mice as determined by ELISA (progranulin levels determined in duplicate), are shown in FIG. 4B and FIG. 4C. Human progranulin levels were increased by 53% and 55% in male and female mice, respectively, that were treated with Compound 1557993, compared to mice of the same sex that received the scrambled control ASO. Data are presented as means±SEM; *indicates p<0.05, as determined by one-way ANOVA with Dunnett post hoc test. Non-Tg=untreated non-transgenic mice, which are littermates of the transgenic mice and do not carry the human GRN alleles. A Western blot of human progranulin in cortex, thalamus, and hippocampus of male mice treated with control, or with Compound 1557993 (FIG. 4C) was conducted as in Example 5, and shows increased human progranulin in the cortex, thalamus, and hippocampus of male mice treated with the compound.

Claims
  • 1. An oligomeric compound comprising a modified oligonucleotide consisting of 12 to 50 linked nucleosides, wherein the nucleobase sequence of the modified oligonucleotide is at least 80% complementary to an equal length portion of a progranulin nucleic acid, and wherein the modified oligonucleotide has at least one modification selected from a modified sugar moiety and a modified internucleoside linkage.
  • 2. The oligomeric compound of claim 1, wherein the progranulin nucleic acid has the nucleobase sequence of SEQ ID NO: 1 or SEQ ID NO: 2.
  • 3. An oligomeric compound comprising a modified oligonucleotide consisting of 18, 19, or 20 linked nucleosides, wherein the nucleobase sequence of the modified oligonucleotide comprises at least 16, at least 17, or 18 contiguous nucleobases of any of the nucleobase sequences of SEQ ID NOs:12-854, and wherein the modified oligonucleotide comprises at least one modification selected from a modified sugar moiety and a modified internucleoside linkage.
  • 4. The oligomeric compound of any of claims 1-3, wherein the nucleobase sequence of the modified oligonucleotide comprises at least 16, at least 17, or 18 contiguous nucleobases of any of the nucleobase sequences of SEQ ID NOs: 34, 268, 345, 424, 501, 579, 657, 735, 800, 801, 806, 812, 813, 816, 821, 828, 844, and 847.
  • 5. An oligomeric compound comprising a modified oligonucleotide consisting of 18 linked nucleosides, wherein the nucleobase sequence of the modified oligonucleotide consists of the nucleobase sequence of any of SEQ ID NOs: 12-854, and wherein the modified oligonucleotide comprises at least one modification selected from a modified sugar moiety and a modified internucleoside linkage.
  • 6. The oligomeric compound of claim 5, wherein the nucleobase sequence of the modified oligonucleotide consists of 18 contiguous nucleobases of any of the nucleobase sequences of SEQ ID NOs: 34, 268, 345, 424, 501, 579, 657, 735, 800, 801, 806, 812, 813, 816, 821, 828, 844, and 847.
  • 7. An oligomeric compound comprising a modified oligonucleotide consisting of 12 to 50 linked nucleosides, wherein the nucleobase sequence of the modified oligonucleotide comprises at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, or 18 contiguous nucleobases complementary to an equal length portion within nucleobases 8497-8552 of SEQ ID NO: 1.
  • 8. The oligomeric compound of claim 7, wherein the nucleobase sequence of the modified oligonucleotide consists of the nucleobase sequence of any of SEQ ID NOs: 34, 268, 345, 424, 501, 579, 657, 735, 800, 801, 806, 812, 813, 816, 821, 828, 844, and 847.
  • 9. The oligomeric compound of any of claims 1-8, wherein the nucleobase sequence of the modified oligonucleotide is at least 85%, at least 90%, at least 95%, or is 100% complementary to an equal length portion within the nucleobase sequence of any of SEQ ID NO: 1 or SEQ ID NO: 2 when measured across the entire nucleobase sequence of the modified oligonucleotide.
  • 10. The oligomeric compound of any of claims 1-9, wherein at least one nucleoside of the modified oligonucleotide comprises a modified sugar moiety.
  • 11. The oligomeric compound of claim 10, wherein the modified sugar moiety comprises a bicyclic sugar moiety.
  • 12. The oligomeric compound of claim 11, wherein the bicyclic sugar moiety comprises a 4′-2′ bridge selected from —CH2—O— and —CH(CH3)—O—.
  • 13. The oligomeric compound of any of claims 10-12, wherein the modified sugar moiety comprises a non-bicyclic modified sugar moiety.
  • 14. The oligomeric compound of claim 13, wherein the non-bicyclic modified sugar moiety is a 2′-MOE modified sugar moiety, a 2′-NMA modified sugar moiety, a 2′-OMe modified sugar moiety, or a 2′-F modified sugar moiety.
  • 15. The oligomeric compound of claim 10, wherein the modified sugar moiety is a sugar surrogate.
  • 16. The oligomeric compound of claim 15, wherein the sugar surrogate is a morpholino, modified morpholino, PNA, THP, or F-HNA.
  • 17. The oligomeric compound of claim 10, wherein each nucleoside of the modified oligonucleotide comprises a modified sugar moiety.
  • 18. The oligomeric compound of claim 17, wherein each modified sugar moiety is a 2′-MOE modified sugar moiety, a 2′-NMA modified sugar moiety, a 2′-OMe modified sugar moiety, or a 2′-F modified sugar moiety.
  • 19. The oligomeric compound of claim 17, wherein each modified sugar moiety is a 2′-MOE modified sugar moiety.
  • 20. The oligomeric compound of claim 17, wherein each modified sugar moiety is a sugar surrogate.
  • 21. The oligomeric compound of claim 20, wherein each modified sugar moiety is a morpholino, modified morpholino, PNA, THP, or F-HNA.
  • 22. The oligomeric compound of any of claims 1-21, wherein the modified oligonucleotide comprises at least one modified internucleoside linkage.
  • 23. The oligomeric compound of claim 22, wherein the at least one modified internucleoside linkage is a phosphorothioate internucleoside linkage.
  • 24. The oligomeric compound of claim 22, wherein each internucleoside linkage of the modified oligonucleotide is a modified internucleoside linkage.
  • 25. The oligomeric compound of claim 22, wherein each internucleoside linkage is a phosphorothioate internucleoside linkage.
  • 26. The oligomeric compound of any of claims 1-23, wherein at least one internucleoside linkage of the modified oligonucleotide is a phosphodiester internucleoside linkage.
  • 27. The oligomeric compound of any of claims 22-23 or 26, wherein each internucleoside linkage of the modified oligonucleotide is independently selected from a phosphodiester internucleoside linkage and a phosphorothioate internucleoside linkage.
  • 28. The oligomeric compound of any of claims 22-24 or 26-27, wherein at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, or at least 17 internucleoside linkages of the modified oligonucleotide are phosphorothioate internucleoside linkages.
  • 29. The oligomeric compound of any of claims 1-23, wherein the modified oligonucleotide consists of 18 linked nucleosides and has an internucleoside linkage motif selected from sssssssssssssssss and sosssssssosssssss.
  • 30. The oligomeric compound of claim 19, wherein the modified oligonucleotide consists of 18 linked nucleosides, wherein the internucleotide linkage motif of the modified oligonucleotide is sssssssssssssssss.
  • 31. The oligomeric compound of claim 19, wherein the modified oligonucleotide consists of 18 linked nucleosides, wherein the internucleotide linkage motif of the modified oligonucleotide is sosssssssosssssss.
  • 32. The oligomeric compound of any of claims 1-31, wherein the modified oligonucleotide comprises at least one modified nucleobase.
  • 33. The oligomeric compound of claim 32, wherein the modified nucleobase is a 5-methyl cytosine or hypoxanthine.
  • 34. The oligomeric compound of claim 33, wherein each cytosine is a 5-methyl cytosine.
  • 35. An oligomeric compound comprising a modified oligonucleotide according to any one of the following chemical notations (5′ to 3′):
  • 36. The oligomeric compound of any of claims 1-35, wherein the modified oligonucleotide is a pharmaceutically acceptable salt.
  • 37. The oligomeric compound of claim 36, wherein the modified oligonucleotide is a pharmaceutically acceptable salt comprising one or more cations selected from sodium, potassium, calcium, and magnesium.
  • 38. The oligomeric compound of any of claims 1-37, consisting of the modified oligonucleotide.
  • 39. The oligomeric compound of any of claims 1-37, wherein the oligomeric compound comprises a conjugate group.
  • 40. The oligomeric compound of claim 39, wherein the conjugate group comprises a conjugate linker and a conjugate moiety.
  • 41. The oligomeric compound of claim 40, wherein the conjugate linker consists of a single bond.
  • 42. The oligomeric compound of claim 40 or claim 41, wherein the conjugate linker is cleavable.
  • 43. The oligomeric compound of claim 40 or claim 41, wherein the conjugate linker comprises 1-3 linker nucleosides.
  • 44. The oligomeric compound of any of claims 40-42, wherein the conjugate linker does not comprise any linker nucleosides.
  • 45. The oligomeric compound of any of claims 39-44, wherein the conjugate group is attached to the 5′-end of the modified oligonucleotide.
  • 46. The oligomeric compound of any of claims 39-44, wherein the conjugate group is attached to the 3′-end of the modified oligonucleotide.
  • 47. The oligomeric compound of any of claims 39-44, wherein the oligomeric compound comprises a terminal group.
  • 48. The oligomeric compound of claim 47, wherein the terminal group is an abasic sugar moiety.
  • 49. The oligomeric compound of any of claims 1-48, wherein the oligomeric compound is a single-stranded oligomeric compound.
  • 50. An oligomeric compound comprising a modified oligonucleotide according to the following chemical notation:
  • 51. An oligomeric compound comprising a modified oligonucleotide according to the following chemical notation:
  • 52. A modified oligonucleotide according to the following chemical structure:
  • 53. The modified oligonucleotide of claim 52, which is the sodium salt or the potassium salt.
  • 54. A modified oligonucleotide according to the following chemical structure:
  • 55. A modified oligonucleotide according to the following chemical structure:
  • 56. The modified oligonucleotide of claim 55, which is the sodium salt or the potassium salt.
  • 57. A modified oligonucleotide according to the following chemical structure:
  • 58. A chirally enriched population of oligomeric compounds of any of claims 1-51, or a chirally enriched population of modified oligonucleotides ofany of claims 52-57, wherein the population is enriched for oligomeric compounds comprising at least one particular phosphorothioate internucleoside linkage having a particular stereochemical configuration.
  • 59. The chirally enriched population of claim 58, wherein the population is enriched for modified oligonucleotides comprising at least one particular phosphorothioate internucleoside linkage having the (Sp) configuration.
  • 60. The chirally enriched population of claim 58, wherein the population is enriched for modified oligonucleotides comprising at least one particular phosphorothioate internucleoside linkage having the (Rp) configuration.
  • 61. The chirally enriched population of claim 58, wherein the population is enriched for modified oligonucleotides having a particular, independently selected stereochemical configuration at each phosphorothioate internucleoside linkage.
  • 62. The chirally enriched population of claim 61, wherein the population is enriched for modified oligonucleotides having the (Sp) configuration at each phosphorothioate internucleoside linkage or for modified oligonucleotides having the (Rp) configuration at each phosphorothioate internucleoside linkage.
  • 63. The chirally enriched population of claim 61, wherein the population is enriched for modified oligonucleotides having the (Rp) configuration at one particular phosphorothioate internucleoside linkage and the (Sp) configuration at each of the remaining phosphorothioate internucleoside linkages.
  • 64. The chirally enriched population of claim 61, wherein the population is enriched for modified oligonucleotides having at least 3 contiguous phosphorothioate internucleoside linkages in the Sp, Sp, and Rp configurations, in the 5′ to 3′ direction.
  • 65. A population of oligomeric compounds of any of claims 1-51, or a population of modified oligonucleotides of any of claims 52-57, wherein all of the phosphorothioate internucleoside linkages of the modified oligonucleotide are stereorandom.
  • 66. An antisense agent comprising an antisense compound, wherein the antisense compound is the oligomeric compound of any of claims 1-51.
  • 67. The antisense agent of claim 66, wherein the antisense agent comprises a conjugate group, wherein the conjugate group comprises a cell-targeting moiety.
  • 68. A pharmaceutical composition comprising an oligomeric compound of any of claims 1-51, a modified oligonucleotide of any of claims 52-57, a population of any of claims 58-65, or an antisense agent of claim 66 or claim 67, and a pharmaceutically acceptable diluent.
  • 69. The pharmaceutical composition of claim 68, wherein the pharmaceutically acceptable diluent is artificial CSF (aCSF) or phosphate-buffered saline (PBS).
  • 70. The pharmaceutical composition of claim 69, wherein the pharmaceutical composition consists essentially of the oligomeric compound of any of claims 1-51, the modified oligonucleotide of any of claims 52-57, the population of any of claims 58-65, the or the antisense agent of claim 66 or claim 67, and aCSF.
  • 71. The pharmaceutical composition of claim 69, wherein the pharmaceutical composition consists essentially of the oligomeric compound of any of claims 1-51, the modified oligonucleotide of any of claims 52-57, the population of any of claims 58-65, or the antisense agent of claim 66 or claim 67, and PBS.
  • 72. A method comprising administering to a subject an oligomeric compound of any of claims 1-51, a modified oligonucleotide of any of claims 52-57, a population of any of claims 58-65, an antisense agent of claim 66 or claim 67, or a pharmaceutical composition of any of claims 68-71.
  • 73. A method of treating a disease or disorder associated with an insufficient expression of progranulin, comprising administering to a subject having or at risk for developing a disease or disorder associated with insufficient expression of progranulin a therapeutically effective amount of an oligomeric compound of any of claims 1-51, a modified oligonucleotide of any of claims 52-57, a population of any of claims 58-65, an antisense agent of claim 66 or claim 67, or a pharmaceutical composition of any of claims 68-71, thereby treating the disease or disorder associated with an insufficient expression of progranulin.
  • 74. The method of claim 73, wherein the disease or disorder associated with an insufficient expression of progranulin is a neurological disease or disorder.
  • 75. The method of claim 73 or claim 74, wherein the disease or disorder associated with an insufficient expression of progranulin is a lysosomal storage disorder or a TDP-43 proteinopathy.
  • 76. The method of any of claims 73-75, wherein the disease or disorder associated with insufficient expression of progranulin is frontotemporal dementia (FTD), frontotemporal lobar degeneration (FTLD), neuronal ceroid lipofuscinosis (NCL), Alzheimer's disease (AD), or amyotrophic lateral sclerosis (ALS).
  • 77. The method of any of claims 73-77, wherein at least one symptom or hallmark of the disease or disorder associated with insufficient expression of progranulin is ameliorated.
  • 78. The method of claim 77, wherein the at least one symptom or hallmark is deterioration in behavior and personality, language impairment, alterations in muscle or motor functions, memory loss, cognitive dysfunction, tremor, seizures, or dizziness.
  • 79. The method of claim 77 or claim 78, wherein administration of the oligomeric compound of any of claims 1-51, the modified oligonucleotide of any of claims 52-57, the population of any of claims 58-65, the antisense agent of claim 66 or claim 67, or the pharmaceutical composition of any of claims 68-71 improves behavior or personality, slows deterioration in behavior or personality, improves language ability, slows deterioration of language ability, improves muscle or motor function, slows deterioration in muscle or motor function, improves memory, slows deterioration in memory, improves cognitive function, slows deterioration of cognitive function, reduces tremors, reduces seizures, or reduces dizziness.
  • 80. The method of any of claims 73-79, wherein the oligomeric compound of any of claims 1-51, the modified oligonucleotide of any of claims 52-57, the population of any of claims 58-65, the antisense agent of claim 66 or claim 67, or the pharmaceutical composition of any of claims 68-71 is administered to the central nervous system or systemically.
  • 81. The method of any of claims 73-79, wherein the oligomeric compound of any of claims 1-51, the modified oligonucleotide of any of claims 52-57, the population of any of claims 58-65, the antisense agent of claim 66 or claim 67, or the pharmaceutical composition of any of claims 68-71 is administered any of intrathecally, systemically, subcutaneously, or intramuscularly.
  • 82. The method of any of claims 73-81, wherein the subject is a human.
  • 83. A method of increasing progranulin RNA or one or more splice variants of said progranulin RNA in a cell, comprising contacting the cell with an oligomeric compound of any of claims 1-51, a modified oligonucleotide of any of claims 52-57, a population of any of claims 58-65, or an antisense agent of claim 66 or claim 67.
  • 84. A method of increasing progranulin protein in a cell, comprising contacting the cell with an oligomeric compound of any of claims 1-51, a modified oligonucleotide of any of claims 52-57, a population of any of claims 58-65, or an antisense agent of claim 66 or claim 67.
  • 85. The method of claim 83 or claim 84, wherein the cell is a neuron.
  • 86. The method of any of claims 83-85, wherein the cell is a human cell.
  • 87. Use of an oligomeric compound of any of claims 1-51, a modified oligonucleotide of any of claims 52-57, a population of any of claims 58-65, an antisense agent of claim 66 or claim 67, or a pharmaceutical composition of any of claims 68-71 for treating a disease or disorder associated with an insufficient expression of progranulin.
  • 88. Use of an oligomeric compound of any of claims 1-51, a modified oligonucleotide of any of claims 52-57, a population of any of claims 58-65, an antisense agent of claim 66 or claim 67, or a pharmaceutical composition of any of claims 68-71 in the manufacture of a medicament for treating a disease or disorder associated with an insufficient expression of progranulin.
  • 89. The use of claim 87 or claim 88, wherein the disease or disorder associated with an insufficient expression of progranulin is a neurological disease or disorder.
  • 90. The use of claim 87 or claim 88, wherein the disease or disorder associated with an insufficient expression of progranulin is a lysosomal storage disorder or a TDP-43 proteinopathy.
  • 91. The use of any of claims 87-90, wherein the disease or disorder associated with insufficient expression of progranulin is frontotemporal dementia (FTD), frontotemporal lobar degeneration (FTLD), neuronal ceroid lipofuscinosis (NCL), Alzheimer's disease (AD), or amyotrophic lateral sclerosis (ALS).
STATEMENT OF GOVERNMENT SUPPORT

This invention was made with government support under AG064069 awarded by National Institute of Health/National Institute on Aging; and UL 1TR002345 awarded by National Institute of Health/National Center for Advancing Translational Sciences. The government has certain rights in the invention.

PCT Information
Filing Document Filing Date Country Kind
PCT/US2022/080113 11/18/2022 WO
Provisional Applications (1)
Number Date Country
63281057 Nov 2021 US