US20230041016A1

US20230041016A1 - Anti-slc6a1 oligonucleotides and related methods

Info

Publication number: US20230041016A1
Application number: US17/781,554
Authority: US
Inventors: Athma A. PAI; Jonathan K. Watts; Kaitlyn VALLA; Eraj Shafiq KHOKHAR
Original assignee: Individual
Current assignee: Individual
Priority date: 2019-12-04
Filing date: 2020-12-04
Publication date: 2023-02-09
Also published as: WO2021113773A3; WO2021113773A2; WO2021113755A2; EP4069844A2; WO2021113755A3; US20230022489A1; EP4069255A2; EP4069255A4; EP4069844A4

Abstract

The present disclosure provides antisense oligonucleotides that bind to a splice modulatory element target region in an SLC6A1 RNA transcript. The present disclosure provides antisense oligonucleotides that increase the expression of a functional protein encoded by the SLC6A1 RNA transcript in a cell (i.e., GABA Transporter 1, GAT-1). The present disclosure also provides methods of treating a disease or disorder associated with non-productive SLC6A1 RNA transcripts.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a 35 U.S.C. § 371 filing of International Patent Application No. PCT/US2020/063469, filed Dec. 4, 2020, which claims the benefit of U.S. Provisional Patent Application Ser. Nos. 62/943,670, and 63/943,672, both filed Dec. 4, 2019, the entire disclosures of which are incorporated herein by reference.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has been submitted electronically in ASCII format and is hereby incorporated by reference in its entirety. Said ASCII copy, created on Jun. 1, 2022, is named 730316_UM9-248US_SL.txt and is 78 kilobytes in size.

BACKGROUND

Myoclonic-atonic epilepsy (MAE) is an idiopathic form of epilepsy characterized by rapid oscillations between muscle contraction/relaxation (myoclonic) and drop seizures (atonic). Patients suffering from MAE may experience absence seizures, which are characterized by a brief loss of consciousness where the individual will appear to be staring off into space. The affected individual has no memory of the absence seizure and, in children, absence seizures are often misdiagnosed as attention-deficit disorder (ADD). Patients also exhibit varying levels of intellectual disability that differ dramatically from individual to individual.
MAE is a primary symptom associated with mutations in the SLC6A1 gene. Human SLC6A1 encodes for the gamma-aminobutyric acid (GABA) transporter protein type 1, GAT-1, which is responsible for the removal of GABA from the synaptic cleft. GABA is the primary inhibitory neurotransmitter and this channel is primarily localized to axons and nerve terminals of GABAergic interneurons. As a member of the neurotransmitter sodium symporters family of proteins, GAT-1 couples the transport of GABA with ion exchange through the GAT-1 channel via the exchange of 1 GABA molecule for 2 sodium ions and 1 chloride ion. The GAT-1 transporter is specifically responsible for the reuptake of GABA into the presynapse following the firing of the interneuron. Without a functional GAT-1 channel, GABA builds up in the synaptic cleft, which can increase the inhibitory activity of the interneuron.
It has been hypothesized that the MAE disease mechanism is caused by a loss-of-function leading to haploinsufficiency of GAT-1. Since mutations in SLC6A1 were only added to genetic testing panels in 2017, SLC6A1-related disorders may be critically underdiagnosed. While mutations in SLC6A1 are not considered diagnostic hallmarks of autism spectrum disorder or epilepsy, recent studies have found that the gene may play a significant role in these disorders. Since SLC6A1 is the sixth most significant gene in genome-wide association studies for epilepsy phenotypes and the tenth most associated gene for autism phenotypes, it is also possible that children with SLC6A1 mutations are often misdiagnosed as having generalized epilepsy or autism spectrum disorder
In the patients with diagnosed mutations in the SLC6A1 gene, the variability in the mutational positions are striking. SLC6A1 mutations include, but are not limited to, missense mutations, splice-site variants, frameshift mutations, nonsense mutations, and in-frame deletions. Given this diversity in the mutational spectrum, there is a need for a treatment which would function in a mutation-agnostic manner.

SUMMARY

In one aspect, the disclosure provides an antisense oligonucleotide that binds to a target region in an SLC6A1 RNA transcript, wherein the target region comprises a splice modulatory element.
In an embodiment, binding of the antisense oligonucleotide to the target region increases the expression of a functional protein encoded by the SLC6A1 RNA transcript in a cell.
In an embodiment, the protein comprises GABA Transporter 1 (GAT-1).
In an embodiment, the cell comprises an SLC6A1 expressing cell. In an embodiment, the cell comprises a neuronal cell and/or an astrocyte. In an embodiment, the neuronal cell comprises a GABAergic neuronal cell.
In an embodiment, the splice modulatory element comprises one or more of a non-productive splice site, a exonic splicing enhancer, an exonic splicing silencer, an intronic splicing enhancer, or an intronic splicing silencer.
In an embodiment, the antisense oligonucleotide comprises a region of complementarity to a target region of an RNA transcript corresponding to a nucleotide sequence of any one of SEQ ID NOs: 1-108.
In an embodiment, the antisense oligonucleotide comprises 8 to 80 nucleotides in length. In an embodiment, the antisense oligonucleotide comprises 15 to 25 nucleotides in length. In an embodiment, the antisense oligonucleotide comprises 18 to 20 nucleotides in length.
In an embodiment, the antisense oligonucleotide comprises one or more modified nucleotides.
In an embodiment, the one or more modified nucleotides comprise a modification of a ribose group, a phosphate group, a nucleobase, or a combination thereof.
In an embodiment, the modification of the ribose group comprises 2′-O-methyl, 2′-fluoro, 2′-deoxy, 2′-O-(2-methoxyethyl) (MOE), 2′-O-alkyl, 2′-O-alkoxy, 2′-O-alkylamino, 2′-NH₂, a constrained nucleotide, or a combination thereof. In an embodiment, the constrained nucleotide comprises a locked nucleic acid (LNA), an ethyl-constrained nucleotide, a 2′-(S)-constrained ethyl (S-cEt) nucleotide, a constrained MOE, a 2′-O,4′-C-aminomethylene bridged nucleic acid (2′,4′-BNA^NC), an alpha-L-locked nucleic acid, a tricyclo-DNA, or a combination thereof. In an embodiment, the modification of the ribose group comprises 2′-O-(2-methoxyethyl) (MOE).
In an embodiment, the modification of the phosphate group comprises a phosphorothioate, a phosphonoacetate (PACE), a thiophosphonoacetate (thioPACE), an amide, a triazole, a phosphonate, a phosphotriester modification, or a combination thereof. In an embodiment, the modification of the phosphate group comprises phosphorothioate.
In an embodiment, the modification of the nucleobase group comprises 2-thiouridine, 4-thiouridine, N⁶-methyladenosine, pseudouridine, 2,6-diaminopurine, inosine, thymidine, 5-methylcytosine, 5-substituted pyrimidine, isoguanine, isocytosine, halogenated aromatic groups, or a combination thereof. In an embodiment, the modification of the nucleobase group comprises 5-methylcytosine.
In an embodiment, the antisense oligonucleotide further comprises a ligand.
In an embodiment, the antisense oligonucleotide comprises a sequence modification pattern of
XsXsXsXsXsXsXsXsXsXsXsXsXsXsXsXsXsXs,

XsXsXsXsXsXsXsXsXsXsXsXsXsXsXsXsXsXsXs,

or

XsXsXsXsXsXsXsXsXsXsXsXsXsXsXsXsXsXsXsXs,

wherein: s represents a phosphorothioate internucleoside linkage; and X represents an adenosine, a guanosine, a cytidine, or a thymine comprising a 2′-O-(2-methoxyethyl) modification.
In an embodiment, the antisense oligonucleotide comprises a nucleotide sequence selected from the group consisting of SEQ ID NOs: 110-127.
In an embodiment, the antisense oligonucleotide increases the level of a functional SLC6A1 RNA transcript in a cell that contains the antisense oligonucleotide by about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 100%, or more, relative to a cell that does not contain the antisense oligonucleotide.
In one aspect, the disclosure provides an antisense oligonucleotide comprising a region of complementarity to a SLC6A1 RNA transcript target region corresponding to a nucleotide sequence of any of SEQ ID NOs: 1-108.
In an embodiment, the target region comprises a non-productive splice site.
In an embodiment, binding of the antisense oligonucleotide to the target region increases the expression of a functional protein encoded by the SLC6A1 RNA transcript in a cell.
In an embodiment, the protein comprises GABA Transporter 1 (GAT-1).
In an embodiment, the cell comprises an SLC6A1 expressing cell. In an embodiment, the cell comprises a neuronal cell and/or an astrocyte. In an embodiment, the neuronal cell comprises a GABAergic neuronal cell.
In an embodiment, the antisense oligonucleotide comprises 8 to 80 nucleotides in length. In an embodiment, the antisense oligonucleotide comprises 15 to 25 nucleotides in length. In an embodiment, the antisense oligonucleotide comprises 18 to 20 nucleotides in length.
In an embodiment, the antisense oligonucleotide comprises one or more modified nucleotides.
In an embodiment, the one or more modified nucleotides comprise a modification of a ribose group, a phosphate group, a nucleobase, or a combination thereof.
In an embodiment, the modification of the ribose group comprises 2′-O-methyl, 2′-fluoro, 2′-deoxy, 2′-O-(2-methoxyethyl) (MOE), 2′-O-alkyl, 2′-O-alkoxy, 2′-O-alkylamino, 2′-NH₂, a constrained nucleotide, or a combination thereof. In an embodiment, the constrained nucleotide comprises a locked nucleic acid (LNA), an ethyl-constrained nucleotide, a 2′-(S)-constrained ethyl (S-cEt) nucleotide, a constrained MOE, a 2′-O,4′-C-aminomethylene bridged nucleic acid (2′,4′-BNA^NC), an alpha-L-locked nucleic acid, a tricyclo-DNA, or a combination thereof. In an embodiment, the modification of the ribose group comprises 2′-O-(2-methoxyethyl) (MOE).
In an embodiment, the modification of the phosphate group comprises a phosphorothioate, a phosphonoacetate (PACE), a thiophosphonoacetate (thioPACE), an amide, a triazole, a phosphonate, a phosphotriester modification, or a combination thereof. In an embodiment, the modification of the phosphate group comprises phosphorothioate.
In an embodiment, the modification of the nucleobase group comprises 2-thiouridine, 4-thiouridine, N⁶-methyladenosine, pseudouridine, 2,6-diaminopurine, inosine, thymidine, 5-methylcytosine, 5-substituted pyrimidine, isoguanine, isocytosine, halogenated aromatic groups, or a combination thereof. In an embodiment, the modification of the nucleobase group comprises 5-methylcytosine.
In an embodiment, the antisense oligonucleotide further comprises a ligand.
In an embodiment, the antisense oligonucleotide comprises a sequence modification pattern of

XsXsXsXsXsXsXsXsXsXsXsXsXsXsXsXsXsXs,

XsXsXsXsXsXsXsXsXsXsXsXsXsXsXsXsXsXsXs, or

XsXsXsXsXsXsXsXsXsXsXsXsXsXsXsXsXsXsXsXs, wherein: s represents a phosphorothioate internucleoside linkage; and X represents an adenosine, a guanosine, a cytidine, or a thymine comprising a 2′-O-(2-methoxyethyl) modification.
In an embodiment, the antisense oligonucleotide comprises a nucleotide sequence selected from the group consisting of SEQ ID NOs: 110-127.
In an embodiment, the antisense oligonucleotide increases the level of a functional SLC6A1 RNA transcript in a cell that contain the antisense oligonucleotide by about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 100%, or more, relative to a cell that does not contain the antisense oligonucleotide.
In one aspect, the disclosure provides a multimeric antisense oligonucleotide compound comprising two or more antisense oligonucleotides as described above, wherein the two or more antisense oligonucleotides are linked together through a linker.
In an embodiment, the linker comprises a cleavable linker. In an embodiment, the cleavable linker degrades when cleaved. In an embodiment, the cleavable linker comprises a nuclease-cleavable linker comprising a phosphodiester linkage. In an embodiment, the nuclease-cleavable linker comprises from about 2 to about 8 nucleotides. In an embodiment, the nuclease-cleavable linker comprises about 6 nucleotides. In an embodiment, the cleavable linker is cleaved under reducing conditions or changing pH conditions. In an embodiment, the cleavable linker is cleaved by an intracellular or endosomal nuclease. In an embodiment, the cleavable linker is cleaved by an intracellular or endosomal protease.
In an embodiment, at least one of the antisense oligonucleotides comprises a nucleotide sequence selected from the group consisting of SEQ ID NOs: 110-127.
In one aspect, the disclosure provides a combination comprising two or more antisense oligonucleotides that bind to two or more target regions in an SLC6A1 RNA transcript, wherein the two or more target regions comprise a splice modulatory element.
In an embodiment, two or more antisense oligonucleotides are linked together through a linker.
In an embodiment, the linker comprises a cleavable linker. In an embodiment, the cleavable linker degrades when cleaved. In an embodiment, the cleavable linker comprises a nuclease-cleavable linker comprising a phosphodiester linkage. In an embodiment, the nuclease-cleavable linker comprises from about 2 to about 8 nucleotides. In an embodiment, the nuclease-cleavable linker comprises about 6 nucleotides. In an embodiment, the cleavable linker is cleaved under reducing conditions or changing pH conditions. In an embodiment, the cleavable linker is cleaved by an intracellular or endosomal nuclease. In an embodiment, the cleavable linker is cleaved by an intracellular or endosomal protease.
In an embodiment, at least one antisense oligonucleotide comprises a region of complementarity to a SLC6A1 RNA transcript target region corresponding to a nucleotide sequence of any of SEQ ID NOs: 1-108.
In an embodiment, at least one of the antisense oligonucleotides comprises a nucleotide sequence selected from the group consisting of SEQ ID NOs: 110-127.
In one aspect, the disclosure provides a method of treating a disease or disorder characterized by haploinsufficiency of a SLC6A1 gene, comprising administering to a subject in need thereof the antisense oligonucleotide, the multimeric antisense oligonucleotide compound, or combination of antisense oligonucleotides as described above, and treating the disease or disorder.
In one aspect, the disclosure provides a method of treating a disease or disorder characterized by reduced expression of a functional GAT-1 protein encoded by a SLC6A1 gene, comprising administering to a subject in need thereof the antisense oligonucleotide, the multimeric antisense oligonucleotide compound, or combination of antisense oligonucleotides as described above, and treating the disease or disorder.
In an embodiment, the disease or disorder comprises myoclonic-atonic epilepsy (MAE), epilepsy, epileptic encephalopathy, seizures, autism spectrum disorders, intellectual disability, or a combination thereof.
In an embodiment, the methods comprise administering the antisense oligonucleotide, the multimeric antisense oligonucleotide compound, or combination of antisense oligonucleotides to a brain of the subject.
In an embodiment, the methods comprise administering the antisense oligonucleotide, the multimeric antisense oligonucleotide compound, or combination of antisense oligonucleotides by intrathecal, intraventricular, intrastriatal injection or infusion, or a combination thereof.
In an embodiment, the injection or infusion comprises administration using an Ommaya reservoir, an intrathecal catheter, or a combination thereof.
In one aspect, the disclosure provides a method of increasing expression of a functional SLC6A1 RNA transcript in a cell, the method comprising contacting the cell with the antisense oligonucleotide, the multimeric antisense oligonucleotide compound, or combination of antisense oligonucleotides as described above, thereby increasing the expression of a functional transcript of the SLC6A1 RNA transcript in a cell.
In an embodiment, expression is increased by about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 100%, or more, relative to a cell that is not contacted with the antisense oligonucleotide, the multimeric antisense oligonucleotide compound, or combination of antisense oligonucleotides.
In one aspect, the disclosure provides a method of increasing expression of a protein encoded by a SLC6A1 RNA transcript in a cell, the method comprising contacting a cell with the antisense oligonucleotide, the multimeric antisense oligonucleotide compound, or combination of antisense oligonucleotides as described above, thereby increasing expression of the protein.
In an embodiment, expression is increased by about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 100%, or more, relative to a cell that is not contacted with the antisense oligonucleotide, the multimeric antisense oligonucleotide compound, or combination of antisense oligonucleotides.
In an embodiment, the protein comprises GABA Transporter 1 (GAT-1).
In an embodiment, the cell comprises an SLC6A1 expressing cell. In an embodiment, the cell comprises a neuronal cell and/or an astrocyte. In an embodiment, the neuronal cell comprises a GABAergic neuronal cell.
In one aspect, the disclosure provides an antisense oligonucleotide that increases expression of a functional protein from the SLC6A1 gene by targeting the 5′- and/or 3′-untranslated regions of the SLC6A1 transcript.
In an embodiment, the antisense oligonucleotide inhibits translation initiation from an upstream open reading frame to increase translation efficiency from the primary open reading frame.
In an embodiment, the antisense oligonucleotide increases mRNA stability.

BRIEF DESCRIPTION OF THE DRAWINGS

The foregoing and other features and advantages of the present invention will be more fully understood from the following detailed description of illustrative embodiments taken in conjunction with the accompanying drawings. The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.

FIG. 1 depicts qPCR-based expression of SLC6A1 in human SH-SY5Y cells.

FIG. 2 depicts the schematic of the SLC6A1 genetic locus on human chromosome 3.

Structures for the 37 most comprehensive annotated isoforms are shown (bottom), with common exons (i.e. represented in at least 50% of isoforms) highlighted in black.
Computationally predicted cryptic splice sites (5′ splice sites and 3′ splice sites) are indicated by dashes at the top.
FIG. 3 depicts a bar graph of SLC6A1 relative mRNA levels in KNS60 neuroblastoma cells transfected with various steric blocking antisense oligonucleotides at 50 nM.

DETAILED DESCRIPTION

The present disclosure provides antisense compounds, methods, and compositions for the treatment, prevention, or amelioration of diseases, disorders, and conditions associated with SLC6A1 in a subject in need thereof. SLC6A1-related diseases, disorders, and conditions include, without limitation, neurological diseases and disorders, such as autism spectrum disorder, epilepsy and attention deficit hyperactivity disorder (ADHD).
It is to be understood that the methods described in this disclosure are not limited to particular methods and experimental conditions disclosed herein as such methods and conditions may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting.
Unless otherwise defined herein, scientific and technical terms used herein have the meanings that are commonly understood by those of ordinary skill in the art. In the event of any latent ambiguity, definitions provided herein take precedent over any dictionary or extrinsic definition. Unless otherwise required by context, singular terms shall include pluralities and plural terms shall include the singular. The use of “or” means “and/or” unless stated otherwise. The use of the term “including,” as well as other forms, such as “includes” and “included,” is not limiting.
Generally, nomenclatures used in connection with cell and tissue culture, molecular biology, immunology, microbiology, genetics, protein, and nucleic acid chemistry, and hybridization described herein are those well-known and commonly used in the art. The nomenclatures used in connection with, and the laboratory 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.
So that the invention may be more readily understood, certain terms are first defined.
The term “nucleoside” refers to a molecule having a purine or pyrimidine base covalently linked to a ribose or deoxyribose sugar. Exemplary nucleosides include adenosine, guanosine, cytidine, uridine and thymidine. Additional exemplary nucleosides include inosine, 1-methyl inosine, pseudouridine, 5,6-dihydrouridine, ribothymidine, 2N-methylguanosine and N²,N²-dimethylguanosine (also referred to as “rare” nucleosides). The term “nucleotide” refers to a nucleoside having one or more phosphate groups joined in ester linkages to the sugar moiety. Exemplary nucleotides include nucleoside monophosphates, diphosphates and triphosphates. The terms “polynucleotide” and “nucleic acid molecule” are used interchangeably herein and refer to a polymer of nucleotides joined together by a phosphodiester or phosphorothioate linkage between 5′ and 3′ carbon atoms.
The term “RNA” or “RNA molecule” or “ribonucleic acid molecule” refers to a polymer of ribonucleotides (e.g., 2, 3, 4, 5, 10, 15, 20, 25, 30, or more ribonucleotides). An RNA nucleotide refers to a single ribonucleotide. The term “DNA” or “DNA molecule” or “deoxyribonucleic acid molecule” refers to a polymer of deoxyribonucleotides. A DNA nucleotide refers to a single deoxyribonucleotide. As used herein, the term “DNA-like” refers to a conformation of, e.g. a modified nucleoside or nucleotide, which is similar to the conformation of a corresponding unmodified DNA unit. For example, a DNA-like nucleotide may refer to a conformation of a modified deoxyribonucleotide similar to a corresponding unmodified deoxyribonucleotide. Examples of DNA-like nucleotides include, without limitation, e.g., 2′-deoxyribonucleotides, 2′-deoxy-2′-substituted arabinonucleotides (e.g., 2′-deoxy-2′-fluoroarabinonucleotides, also known in the art as 2′F-ANA or FANA), and corresponding phosphorothioate analogs. As used herein, the term “RNA-like” refers to a conformation of, e.g. a modified nucleoside or nucleotide which is similar to the conformation of a corresponding unmodified RNA unit. RNA-like conformations may adopt an A-form helix while DNA-like conformations adopt a B-form helix. Examples RNA-like nucleotides include, without limitation, e.g., 2′-substituted-RNA nucleotides (e.g., 2′-fluoro-RNA nucleotides also known in the art as 2′F-RNA), locked nucleic acid (LNA) nucleotides (also known in the art as bridged nucleic acids or bicyclic nucleotides), 2′-fluoro-4′-thioarabinonucleotide (also known in the art as 4'S-FANA nucleotides), 2′-O-alkyl-RNA, and corresponding phosphorothioate analogs.
DNA and RNA can be synthesized naturally (e.g., by DNA replication or transcription of DNA, respectively) or chemically synthesized. RNA can be post-transcriptionally modified. DNA and RNA can be single-stranded (i.e., ssRNA and ssDNA, respectively) or multi-stranded (e.g., double stranded, i.e., dsRNA and dsDNA, respectively). “mRNA” or “messenger RNA” is single-stranded RNA that specifies the amino acid sequence of one or more polypeptide chains. This information is translated during protein synthesis when ribosomes bind to the mRNA.
As used herein, the term “small interfering RNA” (“siRNA”) (also referred to in the art as “short interfering RNAs”) refers to an RNA (or RNA analog) comprising between about 10-50 nucleotides (or nucleotide analogs) which is capable of directing or mediating RNA interference. In one aspect, a siRNA comprises between about 15-30 nucleotides or nucleotide analogs, or between about 16-25 nucleotides (or nucleotide analogs), or between about 18-23 nucleotides (or nucleotide analogs), or between about 19-22 nucleotides (or nucleotide analogs) (e.g., 19, 20, 21 or 22 nucleotides or nucleotide analogs). The term “short” siRNA refers to a siRNA comprising about 21 nucleotides (or nucleotide analogs), for example, 19, 20, 21 or 22 nucleotides. The term “long” siRNA refers to a siRNA comprising about 24-25 nucleotides, for example, 23, 24, 25 or 26 nucleotides. Short siRNAs may, in some instances, include fewer than 19 nucleotides, e.g., 16, 17 or 18 nucleotides, provided that the shorter siRNA retains the ability to mediate RNAi. Likewise, long siRNAs may, in some instances, include more than 26 nucleotides, provided that the longer siRNA retains the ability to mediate RNAi absent further processing, e.g., enzymatic processing, to a short siRNA.
The term “nucleotide analog” or “altered nucleotide” or “modified nucleotide” refers to a non-standard nucleotide, including non-naturally occurring ribonucleotides or deoxyribonucleotides. Exemplary modified nucleotides are modified at any position so as to alter certain chemical properties of the nucleotide yet retain the ability of the modified nucleotide to perform its intended function. Examples of positions of the nucleotide which may be derivatized include the 5 position, e.g., 5-(2-amino)propyl uridine, 5-bromo uridine, 5-propyne uridine, 5-propenyl uridine, etc.; the 6 position, e.g., 6-(2-amino)propyl uridine; the 8-position for adenosine and/or guanosines, e.g., 8-bromo guanosine, 8-chloro guanosine, 8-fluoroguanosine, etc. Modified nucleotides also include deaza nucleotides, e.g., 7-deaza-adenosine; O- and N-modified (e.g., alkylated, e.g., N6-methyl adenosine, or as otherwise known in the art) nucleotides; and other heterocyclically modified nucleotides such as those described in Herdewijn, Antisense Nucleic Acid Drug Dev., 2000 Aug. 10(4):297-310.
Modified nucleotides may also comprise modifications to the sugar portion of the nucleotides. For example, the 2′ OH-group may be replaced by a group selected from H, OR, R, F, Cl, Br, I, SH, SR, NH₂, NHR, NR₂, COOR, or OR, wherein R is substituted or unsubstituted with C₁-C₆alkyl, alkenyl, alkynyl, aryl, etc. As another example, the ribose sugar may be replaced with a bicyclic or tricylic moiety, such as in Locked Nucleic Acid, constrained ethyl, tricycloDNA, or other bridged or bicyclic modifications. Other possible modifications include those described in U.S. Pat. Nos. 5,858,988, and 6,291,438.
The phosphate group of the nucleotide can also be modified, e.g., by substituting one or more of the oxygens of the phosphate group with sulfur (e.g., phosphorothioates), or by making other substitutions which allow the nucleotide to perform its intended function such as described in, Eckstein, Antisense Nucleic Acid Drug Dev. 2000 Apr. 10(2):117-21, Rusckowski et al. Antisense Nucleic Acid Drug Dev. 2000 Oct. 10(5):333-45, Stein, Antisense Nucleic Acid Drug Dev. 2001 Oct. 11(5): 317-25, Vorobjev et al. Antisense Nucleic Acid Drug Dev. 2001 Apr. 11(2):77-85, and U.S. Pat. No. 5,684,143. Certain of the above-referenced modifications (e.g., phosphate group modifications) can decrease the rate of hydrolysis of, for example, polynucleotides comprising said analogs in vivo or in vitro.
As used herein, the terms “unmodified nucleotide” or “non-modified nucleotide” refers to a nucleotide composed of naturally occurring nucleobases, sugar moieties, and internucleoside linkages. In some embodiments, a non-modified nucleotide is an RNA nucleotide (i.e. β-D-ribonucleoside) or a DNA nucleotide (i.e. β-D-deoxyribonucleoside).
The term “oligonucleotide” refers to a short polymer of nucleotides and/or modified nucleotides. As discussed above, the oligonucleotides may be linked with linkages which result in a lower rate of hydrolysis as compared to an oligonucleotide linked with phosphodiester linkages. For example, the nucleotides of the oligonucleotide may comprise triazole, amide, carbamate, methylenediol, ethylene diol, oxymethylthio, oxyethylthio, oxycarbonyloxy, phosphorodiamidate, phosphoroamidate, phosphonate, and/or phosphorothioate linkages. Alterations or modifications of the oligonucleotide can further include addition of non-nucleotide material, such as to the end(s) of the oligonucleotide or internally (at one or more nucleotides of the oligonucleotide).
The term “antisense” refers generally to any approach reliant upon agents, e.g., single-stranded oligonucleotides, that are sufficiently complementary to a target sequence to associate with the target sequence in a sequence-specific manner (e.g., hybridize to the target sequence). Exemplary uses of antisense in the instant application involve use of an oligoribonucleotide agent that hybridizes to a target pre-mRNA molecule and blocks an activity/effect (e.g., splicing pattern and/or blocking of non-productive splice sites) of the targeted pre-mRNA sequence. Antisense approaches commonly are used to target DNA or RNA for transcriptional inhibition, translational inhibition, degradation, etc. Antisense is a technology that can be initiated by the hand of man, for example, to modulate splicing and/or silence the expression of target genes.
As used herein, the term “antisense oligonucleotide” refers to a nucleic acid (e.g., an RNA or analog thereof), having sufficient sequence complementarity to a target RNA (i.e., the RNA for which splice site selection is modulated) to block a region of a target RNA (e.g., pre-mRNA) in an effective manner. In exemplary embodiments of the instant invention, such blocking of non-productive splice sites in SLC6A1 pre-mRNA serves to modulate splicing, either by masking a binding site for a native protein that would otherwise modulate splicing and/or by altering the structure of the targeted RNA. In certain embodiments of the instant invention, the target RNA is a target pre-mRNA (e.g., SLC6A1 pre-mRNA).
An antisense oligonucleotide having a “sequence sufficiently complementary to a target RNA sequence to modulate splicing of the target RNA” means that the antisense agent has a sequence sufficient to trigger the masking of a binding site for a native protein that would otherwise modulate splicing and/or alters the three-dimensional structure of the targeted RNA. Likewise, an oligonucleotide reagent having a “sequence sufficiently complementary to a target RNA sequence to modulate splicing of the target RNA” means that the oligonucleotide reagent has a sequence sufficient to trigger the masking of a binding site for a native protein that would otherwise modulate splicing and/or alters the three-dimensional structure of the targeted RNAs used herein.
The term “target gene” or “target RNA transcript” is a gene or transcript (e.g., a pre-mRNA) whose expression is to be substantially modulated. This modulation can be achieved by steric blocking of a non-productive or cryptic splice site.
The term “non-target gene” is a gene whose expression is not to be substantially modulated. For example, a target gene of the present invention is SLC6A1, and a non-target gene of the present invention is a gene that is not SLC6A1. In one embodiment, the polynucleotide sequences of the target and non-target gene (e.g., mRNA encoded by the target and non-target genes) can differ by one or more nucleotides. In another embodiment, the target and non-target genes can differ by one or more polymorphisms (e.g., Single Nucleotide Polymorphisms or SNPs). In another embodiment, the target and non-target genes can share less than 100% sequence identity. In another embodiment, the non-target gene may be a homologue (e.g., an orthologue or paralogue) of the target gene.
The term “antisense activity” means any detectable or measurable activity attributable to the hybridization of an antisense compound to its target nucleic acid. In some embodiments, antisense activity is an increase in the amount or expression of a target nucleic acid or protein encoded by such target nucleic acid.
The term “target-recognition sequence” refers to the portion of an antisense compound that recognizes a target nucleic acid. The target-recognition sequence has a nucleobase sequence that permits hybridization to a corresponding region or segment of a target nucleic acid.
The term “conserved region” refers to a portion, or portions, of a nucleic acid sequence that is conserved, i.e. a portion, or portions of the nucleic acid sequence having a similar or identical sequence across species. A conserved region may be computationally identified, e.g., using any sequence alignment software available in the art.
As used herein, the term “sufficiently complementary” means that antisense oligonucleotide has a sequence (e.g., an antisense oligonucleotide having a target-recognition sequence), which is sufficient to bind the desired target transcript (e.g., a SLC6A1 transcript), and to trigger the inhibition of non-productive splicing of the target transcript (e.g., steric inhibition of splicing machinery of the target pre-mRNA). For example, a target-recognition sequence with at least 90% complementarity to a target nucleic acid sequence (e.g., a portion of a SLC6A1 transcript) can be sufficiently complementary to trigger modulation of the SLC6A1 transcript. The term “perfectly complementary” refers to, e.g., a target-recognition sequence with 100% complementarity to a target nucleic acid sequence. Complementary nucleic acid molecules hybridize to each other. The term “hybridization” means the annealing of complementary nucleic acid molecules. In certain embodiments, complementary nucleic acid molecules include an antisense compound and a target nucleic acid.
The term “about” or “approximately” means within 20%, within 10%, within 5%, or within 1% or less of a given value or range.
As used herein, “administer” or “administration” refers to the act of injecting or otherwise physically delivering a substance as it exists outside the body (e.g., an antisense compound provided herein) into a patient. The antisense oligonucleotides described herein may be administered to the central nervous system of a patient. The central nervous system includes the brain and spinal cord. Administration methods to the central nervous system include, but are not limited to, intrathecal, intraventricular or intrastriatal infusion or delivery and/or any other method of physical delivery described herein or known in the art. Intraventricular infusion can comprise administration using an Ommaya reservoir.
When a disease, or a symptom thereof, is being managed or treated, administration of the substance typically occurs after the onset of the disease or symptoms thereof. When a disease, or symptom thereof, is being prevented, administration of the substance typically occurs before the onset of the disease or symptoms thereof and can be continued chronically to defer or reduce the appearance or magnitude of disease-associated symptoms, e.g., damage to the involved tissues and airways.
As used herein, the term “composition” is intended to encompass a product containing the specified ingredients (e.g., an antisense compound provided herein) in the specified amounts, as well as any product which results, directly or indirectly, from combination of the specified ingredients in the specified amounts.
“Effective amount” means the amount of active pharmaceutical agent (e.g., an antisense compound of the present disclosure) sufficient to effectuate a desired physiological outcome in an individual in need of the agent. The effective amount can vary among individuals depending on the health and physical condition of the individual to be treated, the taxonomic group of the individuals to be treated, the formulation of the composition, assessment of the individual's medical condition, and other relevant factors.
As used herein, the terms “subject” and “patient” are used interchangeably. For instance, a subject is can be a mammal, such as a non-primate (e.g., cows, pigs, horses, cats, dogs, rats, etc.) or a primate (e.g., monkey and human). In certain embodiments, the term “subject,” as used herein, refers to a vertebrate, such as a mammal. Mammals include, without limitation, humans, non-human primates, wild animals, feral animals, farm animals, sports animals, and pets. In one embodiment, the subject is a mammal, such as a human, having a SLC6A1-related disorder (e.g., myoclonic-atonic epilepsy (MAE)). In another embodiment, the subject is a mammal, such as a human, that is at risk for developing a SLC6A1-related disorder.
As used herein, the term “therapy” refers to any protocol, method, and/or agent that can be used in the prevention, management, treatment and/or amelioration of a disease or a symptom related thereto, such as a SLC6A1-related disorder (e.g., myoclonic-atonic epilepsy (MAE)). In some embodiments, the term “therapy” refers to any protocol, method, and/or agent that can be used in the modulation of an immune response to an infection in a subject or a symptom related thereto. In some embodiments, the terms “therapies” and “therapy” refer to a biological therapy, supportive therapy, and/or other therapies useful in the prevention, management, treatment and/or amelioration of a disease or a symptom related thereto, such as a SLC6A1-related disorder known to one of skill in the art, such as medical personnel. In other embodiments, the terms “therapies” and “therapy” refer to a biological therapy, supportive therapy, and/or other therapies useful in the modulation of an immune response to an infection in a subject or a symptom related thereto known to one of skill in the art, such as medical personnel.
As used herein, the terms “treat,” “treatment” and “treating” refer to the reduction or amelioration of the progression, severity, and/or duration of a disease or a symptom related thereto, such as a SLC6A1-related disorder, by the administration of one or more therapies (including, but not limited to, the administration of one or more prophylactic or therapeutic agents, such as an antisense oligonucleotide provided herein). The term “treating,” as used herein, can also refer to altering the disease course of the subject being treated. Therapeutic effects of treatment include, without limitation, preventing occurrence or recurrence of disease, alleviation of symptom(s), diminishment of direct or indirect pathological consequences of the disease, decreasing the rate of disease progression, amelioration or palliation of the disease state, and remission or improved prognosis.
As used herein, a “splice modulatory element” is a nucleic acid region in a target RNA transcript (e.g., a SLC6A1 transcript), which either enhances or silences the splicing of introns in the pre-mRNA, or in general regulates the constitutive or alternative splicing of the pre-mRNA. Examples of splice modulatory elements include, but are not limited to, non-productive splice sites, exonic splicing enhancers, exonic splicing silencers, intronic splicing enhancers, and intronic splicing silencers.
As used herein, a “non-productive splice site” or “cryptic splice site” is splice site in a pre-mRNA that is used by the cellular splicing machinery that leads to the inappropriate inclusion and/or exclusion of introns and/or exons, thereby producing a non-functional transcript. The non-functional transcript can be rapidly degraded in the cell via one or more mechanisms, such as nonsense-mediated decay (NMD). The non-functional transcript may be translated into a non-functional or deleterious protein.
As used herein, a “functional SLC6A1 RNA transcript” is a SLC6A1 RNA transcript that is translated into a functional protein encoded by SLC6A1 (i.e., GABA Transporter 1, GAT-1).

SLC6A1 Antisense Oligonucleotides

The present disclosure provides antisense oligonucleotides that are capable of sterically blocking splice modulatory elements, such as non-productive splice cites, a exonic splicing enhancer, an exonic splicing silencer, an intronic splicing enhancer, or an intronic splicing silencer.
In certain embodiments, the antisense oligonucleotides are capable of sterically blocking non-productive splice sites in SLC6A1 transcripts (e.g., SLC6A1 pre-mRNA). Cryptic or non-productive splicing occurs when the spliceosome utilizes erroneous splice sites and generates transcripts that then undergo nonsense-mediated mRNA decay (NMD). This may be common in genes with long introns or many introns. While these isoforms are rarely observed in steady-state gene expression measurements, they are likely to represent a large amount of the total transcriptional output of a gene. Without wishing to be bound by theory, blocking non-productive splice sites may lead to an increase in productive mRNA levels as there are fewer molecular resources being wasted on the generation of non-productive transcripts.
Sterically blocking non-productive splice sites in SLC6A1 transcripts may reduce the generation of non-productive splice forms of SCL6A1. Non-productive splice forms of SCL6A1 may be SLC6A1 transcripts that are not translated into a functional protein encoded by SLC6A1 (i.e., GABA Transporter 1, GAT-1) or SLC6A1 transcripts that are translated into non-functional proteins. In certain embodiments, the antisense oligonucleotides of the disclosure reduce the level of SCL6A1 non-productive splice forms by at least about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or about 100%. The % reduction may be in comparison to a non-specific control antisense oligonucleotide or in comparison to the levels of SCL6A1 non-productive splice forms prior to administration of an antisense oligonucleotide. In certain embodiments, sterically blocking non-productive splice sites in SLC6A1 transcripts may increase the generation of productive SCL6A1 mRNA isoforms. Productive SCL6A1 mRNA isoforms are mRNA that are translated into a functional protein encoded by SLC6A1 (i.e., GABA Transporter 1, GAT-1). In certain embodiments, the antisense oligonucleotides of the disclosure increase the level of productive SCL6A1 mRNA isoforms by at least about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or about 100%. The % increase may be in comparison to a non-specific control antisense oligonucleotide or in comparison to the levels of productive SCL6A1 mRNA isoforms forms prior to administration of an antisense oligonucleotide.
In certain embodiments, the antisense oligonucleotides of the disclosure possess complementarity to a target non-productive splice site in an SLC6A1 transcript, thereby sterically blocking the non-productive splice site. In certain embodiments, the antisense oligonucleotides of the disclosure possess complementarity to a target non-productive 5′ splice site (5′ ss). In certain embodiments, the antisense oligonucleotides of the disclosure possess complementarity to a target non-productive 3′ splice site (3′ ss). The antisense oligonucleotides of the disclosure possess a region of complementarity to a target non-productive 5′ ss or 3′ ss sufficient to reduce the level of SCL6A1 non-productive splice forms or increase the generation of productive SCL6A1 mRNA isoforms. In certain embodiments, the antisense oligonucleotides of the disclosure comprise a region of complementarity to a target region of an RNA transcript corresponding to a nucleotide sequence of any one of SEQ ID NOs: 1-108, as recited in Table 1 and Table 2. In other embodiments, the antisense oligonucleotides of the disclosure comprise a nucleotide sequence selected from the group consisting of SEQ ID NOs: 110-127, as recited in Table 4.
In certain embodiments, the antisense oligonucleotides of the disclosure comprise at least one nucleotide that has complementarity to the non-productive splice site. The antisense oligonucleotides of the disclosure need not comprise complementarity to the non-productive splice site to reduce the level of SCL6A1 non-productive splice forms or increase the generation of productive SCL6A1 mRNA isoforms. Rather, the antisense oligonucleotides of the disclosure may comprise complementarity to a region around the non-productive splice site. For example, but in no way limiting, the antisense oligonucleotides may comprise complementarity to a region upstream (5′) of the non-productive splice site or a region downstream (3′) of the non-productive splice site. The antisense oligonucleotides may comprise complementarity to a region 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, or more nucleotides upstream of the non-productive splice site. The antisense oligonucleotides may comprise complementarity to a region about 1 to about 100 nucleotides upstream of the non-productive splice site. The antisense oligonucleotides may comprise complementarity to a region 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, or more nucleotides downstream of the non-productive splice site. The antisense oligonucleotides may comprise complementarity to a region about 1 to about 100 nucleotides downstream of the non-productive splice site.
In certain embodiments, the antisense oligonucleotides of the disclosure may comprise complementarity to a exonic splicing enhancer, an exonic splicing silencer, an intronic splicing enhancer, or an intronic splicing silencer. The antisense oligonucleotides of the disclosure may possess a region of complementarity to a target exonic splicing enhancer, an exonic splicing silencer, an intronic splicing enhancer, or an intronic splicing silencer sufficient to reduce the level of SCL6A1 non-productive splice forms or increase the generation of productive SCL6A1 mRNA isoforms.
In another aspect of the disclosure, a combination comprising two or more antisense oligonucleotides that bind to two or more target regions in an SLC6A1 RNA transcript, is provided. Each antisense oligonucleotide in the combination may comprise complementarity to a region within or around a different splice modulatory element. For example, but in no way limiting, a first antisense oligonucleotide may comprise complementarity to a target region of SEQ ID NO: 1, as recited in Table 1, and a second antisense oligonucleotide may comprise complementarity to a target region of SEQ ID NO: 53, as recited in Table 2. The combination may be administered to a subject in vivo or cells ex vivo or in vitro as separate antisense oligonucleotides (i.e., two or more antisense oligonucleotides in a mixture), or the combination may be administered by linking the two or more antisense oligonucleotides.
In certain embodiments, the antisense oligonucleotides that are capable of sterically blocking non-productive splice sites in SLC6A1 transcripts, have chemically modified subunits arranged in patterns, or motifs, to confer to the antisense compounds properties such as enhanced the inhibitory activity, increased binding affinity for a target nucleic acid, or resistance to degradation by in vivo nucleases.
A nucleoside is a base-sugar combination. The nucleobase (also known as base) portion of the nucleoside is normally a heterocyclic base moiety. Nucleotides are nucleosides that further include a phosphate group covalently linked to the sugar portion of the nucleoside. For those nucleosides that include a pentofuranosyl sugar, the phosphate group can be linked to the 2′, 3′ or 5′ hydroxyl moiety of the sugar. Oligonucleotides are formed through the covalent linkage of adjacent nucleosides to one another, to form a linear polymeric oligonucleotide. Within the oligonucleotide structure, the phosphate groups are commonly referred to as forming the internucleoside linkages of the oligonucleotide.
Modifications to antisense compounds encompass substitutions or changes to internucleoside linkages, sugar moieties, or nucleobases. Modified antisense compounds are often preferred over native forms because of desirable properties such as, for example, enhanced cellular uptake, enhanced affinity for nucleic acid target, increased stability in the presence of nucleases, or increased inhibitory activity.
Chemically modified nucleosides may also be employed to increase the binding affinity of a shortened or truncated antisense oligonucleotide for its target nucleic acid. Consequently, comparable results can often be obtained with shorter antisense compounds that have such chemically modified nucleosides.
The naturally occurring internucleoside linkage of RNA and DNA is a 3′ to 5′ phosphodiester linkage. Antisense compounds having one or more modified, i.e. non-naturally occurring, internucleoside linkages are often selected over antisense compounds having naturally occurring internucleoside linkages because of desirable properties such as, for example, enhanced cellular uptake, enhanced affinity for target nucleic acids, and increased stability in the presence of nucleases.
Oligonucleotides having modified internucleoside linkages include internucleoside linkages that retain a phosphorus atom as well as internucleoside linkages that do not have a phosphorus atom. Representative phosphorus containing internucleoside linkages include, but are not limited to, phosphodiesters, phosphotriesters, methylphosphonates, phosphoramidate, and phosphorothioates. Methods of preparation of phosphorous-containing and non-phosphorous-containing linkages are well known.
In certain embodiments, antisense compounds targeted to a SLC6A1 nucleic acid comprise one or more modified internucleoside linkages. In certain embodiments, the modified internucleoside linkages are phosphorothioate linkages. In certain embodiments, each internucleoside linkage of an antisense compound is a phosphorothioate internucleoside linkage.
Antisense compounds of the invention can optionally contain one or more nucleosides wherein the sugar group has been modified. Such sugar modified nucleosides may impart enhanced nuclease stability, increased binding affinity or some other beneficial biological property to the antisense compounds. In certain embodiments, nucleosides comprise a chemically modified ribofuranose ring moieties. Examples of chemically modified ribofuranose rings include without limitation, addition of substituent groups (including 5′ and 2′ substituent groups, bridging of ring atoms to form bicyclic nucleic acids (BNA), replacement of the ribosyl ring oxygen atom with S, N(R), or C(R¹)(R²) (R═H, C₁-C₁₂alkyl or a protecting group) and combinations thereof. Examples of chemically modified sugars include 2′-F-5′-methyl substituted nucleoside (see PCT International Application WO 2008/101157 Published on Aug. 21, 2008 for other disclosed 5′,2′-bis substituted nucleosides) or replacement of the ribosyl ring oxygen atom with S with further substitution at the 2′-position (see published U.S. Patent Application US2005-0130923, published on Jun. 16, 2005) or alternatively 5′-substitution of a BNA (see PCT International Application WO 2007/134181 Published on Nov. 22, 2007) wherein LNA is substituted with for example a 5′-methyl or a 5′-vinyl group).
Examples of nucleosides having modified sugar moieties include without limitation nucleosides comprising 5′-vinyl, 5′-methyl (R or S), 4′-S, 2′-F (i.e., 2′-fluoro), 2′-OCH₃(i.e., 2′-O-methyl) and 2′-O(CH₂)₂OCH₃(i.e., 2′-O-methoxyethyl) substituent groups. The substituent at the 2′ position can also be selected from allyl, amino, azido, thio, O-allyl, O-C1-C10 alkyl, OCF₃, O(CH₂)₂SCH₃, O(CH₂)₂—O—N(R_m)(R_n), and O—CH₂—C(═O)—N(R_m)(R_n), where each R_mand R_nis, independently, H or substituted or unsubstituted C1-C10 alkyl. 2′-modified nucleotides are useful in the present invention, for example, 2′-O-methyl RNA, 2′-O-methoxyethyl RNA, 2′-fluoro RNA, and others envisioned by one of ordinary skill in the art.
Examples of bicyclic nucleic acids (BNAs) include without limitation nucleosides comprising a bridge between the 4′ and the 2′ ribosyl ring atoms. A BNA comprising a bridge between the 4′ and 2′ ribosyl ring atoms can be referred to as a locked nucleic acid (LNA), and is often referred to as inaccessible RNA. As used herein, the term “locked nucleotide” or “locked nucleic acid (LNA)” comprises nucleotides in which the 2′ deoxy ribose sugar moiety is modified by introduction of a structure containing a heteroatom bridging from the 2′ to the 4′ carbon atoms. The term “non-locked nucleotide” comprises nucleotides that do not contain a bridging structure in the ribose sugar moiety. Thus, the term comprises DNA and RNA nucleotide monomers (phosphorylated adenosine, guanosine, uridine, cytidine, deoxyadenosine, deoxyguanosine, deoxythymidine, deoxycytidine) and derivatives thereof as well as other nucleotides having a 2′-deoxy-erythro-pentofuranosyl sugar moiety or a ribo-pentofuranosyl moiety. In certain embodiments, antisense compounds provided herein include one or more BNA nucleosides wherein the bridge comprises one of the formulas: 4′-(CH₂)—O-2′ (LNA); 4′-(CH₂)—S-2′; 4′-(CH₂)—O-2′ (LNA); 4′-(CH₂)2-O-2′ (ENA); 4′-C(CH₃)2-O-2′ (see PCT/US2008/068922); 4′-CH(CH₃)—O-2′ and 4′-CH(CH₂OCH₃)—O-2′ (see U.S. Pat. No. 7,399,845, issued on Jul. 15, 2008); 4′-CH₂—N(OCH₃)-2′ (see PCT/US2008/064591); 4′-CH₂—O—N(CH₃)-2′ (see published U.S. Patent Application US2004-0171570, published Sep. 2, 2004); 4′-CH₂—N(R)—O-2′ (see U.S. Pat. No. 7,427,672, issued on Sep. 23, 2008); 4′-CH₂—C(CH₃)-2′ and 4′-CH₂—C(═CH₂)-2′ (see PCT/US2008/066154); and wherein R is, independently, H, C1-C12 alkyl, or a protecting group. Each of the foregoing BNAs include various stereochemical sugar configurations including for example α-L-ribofuranose and β-D-ribofuranose (see PCT international application PCT/DK98/00393, published on Mar. 25, 1999 as WO 99/14226).
In some embodiments, antisense compounds provided herein include one or more 2′, 4′-constrained nucleotides. For example, antisense compounds provided by the present disclosure include those having one or more constrained ethyl (cEt) or constrained methoxyethyl (cMOE) nucleotides. In some embodiments, antisense compounds provided herein are antisense oligonucleotides comprising one or more constrained ethyl (cEt) nucleotides. The terms “constrained ethyl” and “ethyl-constrained” are used interchangeably.
In certain embodiments, nucleosides are modified by replacement of the ribosyl ring with a sugar surrogate. Such modification includes without limitation, replacement of the ribosyl ring with a surrogate ring system (sometimes referred to as DNA analogs) such as a morpholino ring, a cyclohexenyl ring, a cyclohexyl ring or a tetrahydropyranyl ring such as one having one of the formula:
Many other bicyclo and tricyclo sugar surrogate ring systems are also known in the art that can be used to modify nucleosides for incorporation into antisense compounds (see for example review article: Leumann, J. C, Bioorganic & Medicinal Chemistry, 2002, 10, 841-854; Ito, K. R.; Obika, S., Recent Advances in Medicinal Chemistry of Antisense Oligonucleotides. In Comprehensive Medicinal Chemistry, 3rd edition, Elsevier: 2017). Such ring systems can undergo various additional substitutions to enhance activity.
Methods for the preparations of modified sugars are well known to those skilled in the art. In nucleotides having modified sugar moieties, the nucleobase moieties (natural, modified or a combination thereof) are maintained for hybridization with an appropriate nucleic acid target.
In certain embodiments, antisense compounds targeted to a SLC6A1 nucleic acid comprise one or more kinds of modified nucleotides. In one embodiment, antisense compounds targeted to a SLC6A1 nucleic acid comprise 2′-modified nucleotides. In one embodiment, antisense compounds targeted to a SLC6A1 nucleic acid comprise a 2′-O-methyl RNA, a 2′-O-methoxyethyl RNA, or a 2′-fluoro RNA. In one embodiment, antisense compounds targeted to a SLC6A1 nucleic acid comprise tricyclo-DNA. Tricyclo-DNA belongs to a class of constrained DNA analogs that display improved hybridizing capacities to complementary RNA, see, e.g., Ittig et al., Nucleic Acids Res. 32:346-353 (2004); Ittig et al., Prague, Academy of Sciences of the Czech Republic. 7:21-26 (Coll. Symp. Series, Hocec, M., 2005); Ivanova et al., Oligonucleotides 17:54-65 (2007); Renneberg et al., Nucleic Acids Res. 30:2751-2757 (2002); Renneberg et al., Chembiochem. 5:1114-1118 (2004); and Renneberg et al., JACS. 124:5993-6002 (2002). In one embodiment, antisense compounds targeted to a SLC6A1 nucleic acid comprise a locked nucleotide, an ethyl-constrained nucleotide, or an alpha-L-locked nucleic acid. Various alpha-L-locked nucleic acids are known by those of ordinary skill in the art, and are described in, e.g., Sorensen et al., J. Am. Chem. Soc. (2002) 124(10):2164-2176.
In certain embodiments, antisense compounds targeted to a mutant SLC6A1 nucleic acid comprise one or more modified nucleotides having modified sugar moieties. In some embodiments, the modified nucleotide is a locked nucleotide. In certain embodiments, the locked nucleotides are arranged in a gapmer motif, e.g. a 3-9-3 gapmer format wherein 9 non-locked nucleotides are flanked by 3 locked nucleotides on each side.
Nucleobase (or base) modifications or substitutions are structurally distinguishable from, yet functionally interchangeable with, naturally occurring or synthetic unmodified nucleobases. Both natural and modified nucleobases are capable of participating in hydrogen bonding. Such nucleobase modifications may impart nuclease stability, binding affinity or some other beneficial biological property to antisense compounds. Modified nucleobases include synthetic and natural nucleobases such as, for example, 5-methylcytosine (5-me-C). Certain nucleobase substitutions, including 5-methylcytosine substitutions, are particularly useful for increasing the binding affinity of an antisense compound for a target nucleic acid. For example, 5-methylcytosine substitutions have been shown to increase nucleic acid duplex stability by 0.6-1.2° C. (Sanghvi, Y. S., Crooke, S. T. and Lebleu, B., eds., Antisense Research and Applications, CRC Press, Boca Raton, 1993, pp. 276-278).
Additional modified nucleobases include 5-hydroxymethyl cytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-methyl and other alkyl derivatives of adenine and guanine, 2-propyl and other alkyl derivatives of adenine and guanine, 2-thiouracil, 2-thiothymine and 2-thiocytosine, 5-halouracil and cytosine, 5-propynyl (—C≡C—CH₃) uracil and cytosine and other alkynyl derivatives of pyrimidine bases, 6-azo uracil, cytosine and thymine, 5-uracil (pseudouracil), 4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl and other 8-substituted adenines and guanines, 5-halo particularly 5-bromo, 5-trifluoromethyl and other 5-substituted uracils and cytosines, 7-methylguanine and 7-methyladenine, 2-F-adenine, 2-amino-adenine, 8-azaguanine and 8-azaadenine, 7-deazaguanine and 7-deazaadenine and 3-deazaguanine and 3-deazaadenine.
Heterocyclic base moieties may also include those in which the purine or pyrimidine base is replaced with other heterocycles, for example 7-deaza-adenine, 7-deazaguanosine, 2-aminopyridine and 2-pyridone. Nucleobases that are particularly useful for increasing the binding affinity of antisense compounds include 5-substituted pyrimidines, 6-azapyrimidines and N-2, N-6 and 0-6 substituted purines, including 2 aminopropyladenine, 5-propynyluracil and 5-propynylcytosine.
In certain embodiments, antisense compounds targeted to a SLC6A1 nucleic acid comprise one or more modified nucleotides having modified sugar moieties. In some embodiments, the modified nucleotide is a locked nucleotide. In certain embodiments, the locked nucleotides are arranged in a gapmer motif, e.g. a 3-9-3 gapmer format wherein 9 non-locked nucleotides are flanked by 3 locked nucleotides on each side. In certain embodiments, antisense compounds targeted to a SLC6A1 nucleic acid comprise one or more modified nucleotides. In some embodiments, the modified nucleotide is 5-methylcytosine. In certain embodiments, each cytosine is a 5-methylcytosine.
In certain embodiments, the antisense oligonucleotides of the disclosure comprise a 2′-0-(2-methoxyethyl) modification at one or more nucleotides. In certain embodiments, the antisense oligonucleotides of the disclosure comprise a 2′-O-(2-methoxyethyl) modification at 20% of the nucleotides, at 30% of the nucleotides, at 40% of the nucleotides, at 50% of the nucleotides, at 60% of the nucleotides, at 70% of the nucleotides, at 80% of the nucleotides, or at 90% of the nucleotides. In certain embodiments, the antisense oligonucleotides of the disclosure comprise a 2′-O-(2-methoxyethyl) modification at every nucleotide (100% 2′-O-(2-methoxyethyl) modification).
In certain embodiments, the antisense oligonucleotides of the disclosure comprise one or more phosphorothioate internucleoside linkages. In certain embodiments, the antisense oligonucleotides of the disclosure comprise one or more phosphorothioate internucleoside linkages and one or more phosphodiester linkages. In certain embodiments, the antisense oligonucleotides of the disclosure comprise phosphorothioate at every internucleoside linkage.
In certain embodiments, the antisense oligonucleotides of the disclosure comprise a sequence modification pattern of
XsXsXsXsXsXsXsXsXsXsXsXsXsXsXsXsXsXs,

XsXsXsXsXsXsXsXsXsXsXsXsXsXsXsXsXsXsXs,

or

XsXsXsXsXsXsXsXsXsXsXsXsXsXsXsXsXsXsXsXs,

wherein: s represents a phosphorothioate internucleoside linkage; and
X is an adenosine, a guanosine, a cytidine, or a thymine comprising a 2′-O-(2-methoxyethyl) modification.
In certain embodiments, an antisense oligonucleotide that targets a SLC6A1 transcript is from about 8 to about 80 nucleotides in length. In other embodiments, the antisense oligonucleotide that targets a SLC6A1 transcript is from about 15 to about 25 nucleotides in length. In other embodiments, the antisense oligonucleotide that targets a SLC6A1 transcript is from about 18 to about 20 nucleotides in length. For example, the antisense oligonucleotides are 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, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, or 80 nucleotides in length, or a range defined by any two of the above values.

Branched Antisense Oligonucleotide Compounds

The present disclosure also provides branched antisense compounds comprising two or more target-recognition sequences that targets a portion of a SLC6A1 RNA transcript. A branched antisense compound of the present disclosure may be, e.g., a branched antisense oligonucleotide compound.
As used herein, the term “branched antisense compound” or “branched antisense oligonucleotide” or “multimeric oligonucleotide compound” refers to two or more antisense compounds or antisense oligonucleotides that are connected together. In certain embodiments, the two or more antisense oligonucleotides are linked together through a linker.
In one embodiment, a branched oligonucleotide compound comprises two or more target-recognition sequences, wherein the target-recognition sequences are connected to one another by one or more moieties selected from a linker, a spacer, and a branching point. Target-recognition sequences are described herein. In some embodiments, the branched oligonucleotide compound comprises 2, 3, 4, 5, 6, 7, 8, or more target-recognition sequences, wherein each target-recognition sequences comprises a 5′ end and a 3′ end, and each target-recognition sequence is independently connected to a linker, a spacer, or a branching point at the 5′ end or the 3′ end. In some embodiments, each target-recognition sequence is connected to a linker, a spacer, or a branching point at the 5′ end. In some embodiments, each target-recognition sequence is connected to a linker, a spacer, or a branching point at the 3′ end. In another embodiment, each target-recognition sequence is connected to a linker, a spacer, or a branching point. In some embodiments, each of the target-recognition sequences are antisense compounds and/or oligonucleotides that target a portion of a SLC6A1 nucleic acid.
In some embodiments, a branched oligonucleotide compound of the present disclosure has the formula
L-(N)_n
wherein N represents a target-recognition sequence of the present disclosure; n represents an integer, e.g., 2, 3, 4, 5, 6, 7, or 8; and L represents a linker selected from an ethylene glycol chain, an alkyl chain, a peptide, RNA, DNA, a phosphate, a phosphonate, a phosphoramidate, an ester, an amide, a triazole, and any combination thereof.
In some embodiments, a branched oligonucleotide compound of the present disclosure has the formula
L-(N)_n
wherein the compound optionally further comprises one or more branching points B, and wherein the compound optionally further comprises one or more spacers S. In such embodiments, each of the one or more branching points B independently represents a polyvalent organic species or derivative thereof, and each of the one or more spacers S is independently selected from an ethylene glycol chain, an alkyl chain, a peptide, RNA, DNA, a phosphate, a phosphonate, a phosphoramidate, an ester, an amide, a triazole, and any combination thereof. For example, a branched oligonucleotide compound of the present disclosure having the formula L-(N)n has a structure, not to be limited in any fashion, e.g.,

Target-Recognition Sequences

The present disclosure provides an antisense oligonucleotide comprising a target-recognition sequence that targets a portion of a SLC6A1 nucleic acid (e.g., a SLC6A1 transcript). In certain embodiments, an antisense oligonucleotide has a nucleobase sequence that, when written in the 5′ to 3′ direction, comprises the reverse complement of a portion of a SLC6A1 nucleic acid. In some embodiments, an antisense oligonucleotide is an antisense oligonucleotide. In certain such embodiments, an antisense oligonucleotide has a nucleobase sequence that, when written in the 5′ to 3′ direction, comprises the reverse complement of a portion of a SLC6A1 nucleic acid.
In certain embodiments, a target region is a structurally defined region of a SLC6A1 nucleic acid. For example, a target region may encompass a 3′ untranslated region (UTR), a 5′ untranslated region (UTR), an exon, an intron, an exon/intron junction, a coding region, a translation initiation region, translation termination region, or other defined nucleic acid region, for example, an open reading frame, or the junction between an open reading frame and an untranslated region and any combinations thereof. The structurally defined regions for SLC6A1 can be obtained by accession number from sequence databases such as NCBI and such information is incorporated herein by reference. In certain embodiments, a target region may encompass the sequence from a 5′ target site of one target segment within the target region to a 3′ target site of another target segment within the same target region.
Targeting includes determination of at least one target segment to which an antisense oligonucleotide hybridizes, such that a desired effect occurs. In certain embodiments, the desired effect is a reduction in non-productive transcript target nucleic acid levels, i.e., a reduction in SLC6A1 non-productive transcript levels through the inhibition ofnon-productive splice sites. In certain embodiments, the desired effect is an increase in the levels of functional protein encoded by the target nucleic acid or a phenotypic change associated with the target nucleic acid, e.g., an increase in the level of GAT-1 protein, encoded by SLC6A1 mRNA.
A target region may contain one or more target segments. Multiple target segments within a target region may be overlapping. Alternatively, they may be non-overlapping. In certain embodiments, target segments within a target region are separated by no more than about 300 nucleotides. In certain embodiments, target segments within a target region are separated by a number of nucleotides that is, is about, is no more than, is no more than about, 250, 200, 150, 100, 90, 80, 70, 60, 50, 40, 30, 20, or 10 nucleotides on the target nucleic acid, or is a range defined by any two of the preceding values. In certain embodiments, target segments within a target region are separated by no more than, or no more than about, 5 nucleotides on the target nucleic acid. In certain embodiments, target segments are contiguous.
Suitable target segments may be found within a 5′ UTR, a coding region, a 3′ UTR, an intron, an exon, and/or an exon/intron junction. Target segments containing a start codon or a stop codon are also suitable target segments. A suitable target segment may specifically exclude a certain structurally defined region such as the start codon or stop codon.
The determination of suitable target segments may include a comparison of the sequence of a target nucleic acid (e.g., SLC6A1) to other sequences throughout the genome. For example, the BLAST algorithm may be used to identify regions of similarity amongst different nucleic acids. This comparison can prevent the selection of antisense oligonucleotide sequences that may hybridize in a non-specific manner to sequences other than a selected target nucleic acid (i.e., non-target or off-target sequences). The determination of suitable target segments may include comparison of the sequences of a target nucleic acid (e.g., a mutant SLC6A1 transcript) across several species. For example, various sequence alignment software are known in the art and can be used to identify regions of similar or identical sequence across species.
There may be variation in activity (e.g., as defined by percent reduction of non-productive target nucleic acid levels or percent increase of productive, functional mRNA) of the antisense oligonucleotides within an active target region. In certain embodiments, reduction in non-productive SLC6A1 transcript levels is indicative of inhibition of non-productive SLC6A1 mRNA expression.
An antisense oligonucleotide and a target nucleic acid (e.g., a SLC6A1 transcript or portion thereof) are complementary to each other when a sufficient number of nucleobases of the antisense oligonucleotide can hydrogen bond with the corresponding nucleobases of the target nucleic acid, such that a desired effect will occur (e.g., antisense inhibition of a non-productive target nucleic acid, such as a SLC6A1 non-productive transcript or portion thereof).
Non-complementary nucleobases between an antisense oligonucleotide and a SLC6A1 nucleic acid may be tolerated provided that the antisense oligonucleotide remains able to specifically hybridize to a target nucleic acid. Moreover, an antisense oligonucleotide may hybridize over one or more segments of a SLC6A1 nucleic acid such that intervening or adjacent segments are not involved in the hybridization event (e.g., a loop structure, mismatch or hairpin structure).
In certain embodiments, the antisense oligonucleotides provided herein, or a specified portion thereof, are, or are at least, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% complementary to a SLC6A1 nucleic acid, a target region, target segment, or specified portion thereof. Percent complementarity of an antisense oligonucleotide with a target nucleic acid can be determined using routine methods.
For example, an antisense oligonucleotide in which 18 of 20 nucleobases of the antisense oligonucleotide are complementary to a target region (e.g., an equal length portion of a SLC6A1 transcript), and would therefore specifically hybridize, would represent 90 percent complementarity. In this example, the remaining noncomplementary nucleobases may be clustered or interspersed with complementary nucleobases and need not be contiguous to each other or to complementary nucleobases. As such, an antisense oligonucleotide which is 18 nucleobases in length having 4 (four) noncomplementary nucleobases which are flanked by two regions of complete complementarity with the target nucleic acid would have 77.8% overall complementarity with the target nucleic acid and would thus fall within the scope of the present invention. Percent complementarity of an antisense oligonucleotide with a region of a target nucleic acid can be determined routinely using BLAST programs (basic local alignment search tools) and PowerBLAST programs known in the art (Altschul et al., J. Mol. Biol., 1990, 215, 403 410; Zhang and Madden, Genome Res., 1997, 7, 649 656). Percent homology, sequence identity or complementarity, can be determined by, for example, the Gap program (Wisconsin Sequence Analysis Package, Version 8 for Unix, Genetics Computer Group, University Research Park, Madison Wis.), using default settings, which uses the algorithm of Smith and Waterman (Adv. Appl. Math., 1981, 2, 482 489).

Conjugated Antisense Oligonucleotides

Antisense oligonucleotides may be covalently linked to one or more moieties, ligands, or conjugates which enhance the activity, cellular distribution or cellular uptake of the resulting antisense oligonucleotides. Antisense oligonucleotides may be covalently linked to one or more moieties, ligands, or conjugates which enhance and/or optimize pharmacokinetic parameters. Various pharmacokinetic parameters are known to a person of ordinary skill in the art, for example, absorbance, concentration of a compound in the body, the degree to which a compound permeates the body, the rate of elimination/clearance of a compound, the volume of plasma cleared of a compound per unit time, and others.
Typical conjugate groups include hydrophobic moieties such as cholesterol and lipid moieties. Such moieties include, but are not limited to, lipid moieties such as a cholesterol moiety (Letsinger et al., Proc. Natl. Acad. Sci. USA, 1989, 86, 6553-6556), cholic acid (Manoharan et al., Bioorg. Med. Chem. Let., 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. Let., 1993, 3, 2765-2770), a thiocholesterol (Oberhauser et al., Nucl. Acids Res., 1992, 20, 533-538), an aliphatic chain, e.g., dodecandiol or undecyl residues (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 triethylammonium 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 (Mancharan et al., Nucleosides & Nucleotides, 1995, 14, 969-973), or adamantane acetic acid (Manoharan et al., Tetrahedron Lett., 1995, 36, 3651-3654), a palmityl moiety (Mishra et al., Biochim. Biophys. Acta, 1995, 1264, 229-237), an unsaturated fatty acid such as docosahexaenoic acid (Nikan et al, Mol Ther Nucleic Acids. 2016, 5, e344), or an octadecylamine or hexylamino-carbonyl-t oxycholesterol moiety (Crooke et al., J. Pharmacol. Exp. Ther., 1996, 277, 923-937). Diverse lipid conjugates can preferentially drive oligonucleotide uptake into different tissues (Biscans et al, Nucleic Acids Res. 2019, 47, 1082-1096). For example, a lipid moiety based on 1-O-hexa-decyloxy-1,3-propanediol can be conjugated to an antisense oligonucleotide of the present disclosure. Such a lipid moiety has previously been shown to increase small molecule uptake and improve the oral bioavailability of nucleoside drugs (see, e.g., Aldem et al., Mol. Pharmacol. 2003, 63:678-681; and Hostetler, Antiviral Res. 2009, 82:A84-A98). Additional conjugate groups include carbohydrates, phospholipids, biotin, phenazine, folate, phenanthridine, anthraquinone, acridine, fluoresceins, rhodamines, coumarins, and dyes. In some embodiments, conjugation of a ligand to an antisense oligonucleotide allows recognition by cell-surface receptors (see, e.g., Wolfrum et al., Nat. Biotechnol. 2007, 25:1149-1157; Hostetler et al., Antiviral Chem. Chemother. 2001, 12:61-70; and Prakash et al., Nucleic Acids Res. 2014, 42:8796-807). Methods of attaching one or more moieties or conjugates are well known in the art.
Antisense oligonucleotides can also be modified to have one or more stabilizing groups that are generally attached to one or both termini of antisense oligonucleotides to enhance properties such as, for example, nuclease stability. Included in stabilizing groups are cap structures. These terminal modifications protect the antisense oligonucleotide having terminal nucleic acid from exonuclease degradation, and can help in delivery and/or localization within a cell. The cap can be present at the 5′-terminus (5′-cap), or at the 3′-terminus (3′-cap), or can be present on both termini. Cap structures are well known in the art and include, for example, inverted deoxy abasic caps. Further 3′ and 5′-stabilizing groups that can be used to cap one or both ends of an antisense oligonucleotide to impart nuclease stability include those disclosed in WO 03/004602 published on Jan. 16, 2003.
In some embodiments, an antisense oligonucleotide of the present disclosure comprises a conjugate. In one embodiment, an antisense oligonucleotide of the present disclosure comprises a antisense oligonucleotide sequence and a conjugate, wherein the conjugate is linked to the antisense oligonucleotide sequence. In some embodiments, the conjugate is selected from any of the conjugates described herein, for example, a hydrophobic conjugate, a tissue-targeting conjugate, or a conjugate designed to optimize pharmacokinetic parameters. A hydrophobic conjugate useful for conjugating to antisense oligonucleotides of the present disclosure, includes a hexadecyloxypropyl conjugate, a cholesterol conjugate, a polyunsaturated fatty acid conjugate, and others known in the art that may improve cellular uptake of a conjugate antisense oligonucleotide. In some embodiments, the conjugate may be a tissue-targeting conjugate, for example, a carbohydrate conjugate, or a peptide conjugate, or any conjugate known in the art that can target an antisense oligonucleotide of the present disclosure to a specific tissue. In some embodiments, an antisense oligonucleotide of the present disclosure is conjugated with a polyethylene glycol conjugate. In one embodiment, a polyethylene glycol conjugate antisense oligonucleotide optimizes pharmacokinetic properties of the antisense oligonucleotide.
In some embodiments, the present disclosure provides biocleavable analogues of antisense oligonucleotides described herein. In such cases, biocleavable analogues comprise a hydrophobic conjugate that leads to stronger association with cell membranes and a linker. In one embodiment, the linker is a cleavable linker that when cleaved, releases the antisense oligonucleotide, e.g., releases the antisense oligonucleotide into endosomes. In some embodiments, an antisense compound comprises a cleavable linker, wherein the cleavable linker degrades when cleaved. In some embodiments, the linker is a nuclease-cleavable linker comprising a phosphodiester linkage. In some embodiments, the nuclease-cleavable linker comprising a phosphodiester linkage is about 2 to about 8 nucleotides. For example, a nuclease-cleavable phosphodiester linker can be 3, 4, 5, 6, 7, 8 nucleotides in length, or longer, e.g., 9, 10, 11, 12, 13, 14, 15, 16, 17, 18 nucleotides in length, or longer. In one embodiment, the nuclease-cleavable linker comprises about 6 nucleotides. In some embodiments, the cleavable linker is cleaved after cellular internalization. In some embodiments, the cleavable linker is cleaved within an endosome. In some embodiments, the cleavable linker is cleaved under reducing conditions. In some embodiments, the cleavable linker is cleaved under changing pH conditions, for example the cleavable linker is cleaved when the pH decreases, or when the pH increases. In some embodiments, the cleavable linker is cleaved by an intracellular nuclease or protease. In some embodiments, the cleavable linker is cleaved by an endosomal nuclease or protease.

Pharmaceutical Compositions and Formulations

Provided herein are pharmaceutical compositions and formulations which include the antisense compounds described herein. For example, the antisense oligonucleotides described herein can be admixed, encapsulated, conjugated or otherwise associated with other molecules, molecule structures or mixtures of compounds. A pharmaceutical composition of the invention is formulated to be compatible with its intended route of administration. Examples of routes of administration include intrathecal administration, intraventricular administration or intrastriatal administration. In some embodiments, the administration may employ an implanted device such as an Ommaya reservoir or implanted intrathecal catheter. Solutions or suspensions used for administration can include the following components: a sterile diluent such as water for injection, saline solution, lactated Ringers solution, Elliotts B solution, fixed oils, polyethylene glycols, glycerine, propylene glycol or other synthetic solvents; antibacterial agents such as benzyl alcohol or methyl parabens; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid; buffers such as acetates, citrates, carbonates or phosphates and agents for the adjustment of tonicity such as sodium chloride or dextrose. pH can be adjusted with acids or bases, such as hydrochloric acid or sodium hydroxide. The pharmaceutical compositions can be enclosed in ampoules, disposable syringes or multiple dose vials made of glass or plastic.
Pharmaceutical compositions suitable for injectable use include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion. In all cases, the composition must be sterile and should be fluid to the extent that easy syringability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyetheylene glycol, and the like), and suitable mixtures thereof. The proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In certain embodiments, isotonic agents may be included, for example, sugars, polyalcohols such as manitol, sorbitol, sodium chloride in the composition. Prolonged absorption of the injectable compositions can be brought about by including in the composition an agent which delays absorption, for example, aluminum monostearate and gelatin.
Sterile injectable solutions can be prepared by incorporating the active compound in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the active compound into a sterile vehicle which contains a basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, certain methods of preparation are vacuum drying and freeze-drying which yields a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.
The pharmaceutical compositions and formulations provided herein can, in some embodiments, be conveniently presented in unit dosage form and can be prepared according to techniques well known in the pharmaceutical industry. Such techniques can include bringing into association the active ingredients with the pharmaceutical carrier(s) or excipient(s). In general, the formulations can be prepared by uniformly and intimately bringing into association the active ingredients with liquid carriers, finely divided solid carriers, or both, and then, if necessary, shaping the product (e.g., into a specific particle size for delivery). In one embodiment, the pharmaceutical formulations are prepared for intrathecal, intraventricular or intrastriatal administration in an appropriate solvent, e.g., water or normal saline.
An agent of the present disclosure, e.g., an antisense compound targeting a SLC6A1 transcript can also be administered by transfection or infection using methods known in the art, including but not limited to the methods described in McCaffrey et al. (2002), Nature, 418(6893), 38-9 (hydrodynamic transfection); Xia et al. (2002), Nature Biotechnol., 20(10), 1006-10 (viral-mediated delivery); or Putnam (1996), Am. J. Health Syst. Pharm. 53(2), 151-160, erratum at Am. J. Health Syst. Pharm. 53(3), 325 (1996).
An agent of the present disclosure, e.g., an antisense compound targeting a SLC6A1 transcript can also be administered by any method suitable for administration of nucleic acid agents, such as a DNA vaccine. These methods include gene guns, bio injectors, and skin patches as well as needle-free methods such as the micro-particle DNA vaccine technology disclosed in U.S. Pat. No. 6,194,389, and the mammalian transdermal needle-free vaccination with powder-form vaccine as disclosed in U.S. Pat. No. 6,168,587. Additionally, intranasal delivery is possible, as described in, inter alia, Hamajima et al. (1998), Clin. Immunol. Immunopathol., 88(2), 205-10. Liposomes (e.g., as described in U.S. Pat. No. 6,472,375) and microencapsulation can also be used. Biodegradable targetable microparticle delivery systems can also be used (e.g., as described in U.S. Pat. No. 6,471,996).
In one embodiment, the active agents are prepared with carriers that will protect the compound against rapid elimination from the body, such as a controlled release formulation, including implants and microencapsulated delivery systems. Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Methods for preparation of such formulations will be apparent to those skilled in the art. The materials can also be obtained commercially from Alza Corporation and Nova Pharmaceuticals, Inc. Liposomal suspensions (including liposomes targeted to infected cells with monoclonal antibodies to viral antigens) can also be used as pharmaceutically acceptable carriers. These can be prepared according to methods known to those skilled in the art, for example, as described in U.S. Pat. No. 4,522,811.
Toxicity and therapeutic efficacy of such compounds can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., for determining the LD50 (the dose lethal to 50% of the population) and the ED50 (the dose therapeutically effective in 50% of the population). The dose ratio between toxic and therapeutic effects is the therapeutic index and it can be expressed as the ratio LD50/ED50. Compounds that exhibit large therapeutic indices are desirable. Although compounds that exhibit toxic side effects may be used, care should be taken to design a delivery system that targets such compounds to the site of affected tissue in order to minimize potential damage to uninfected cells and, thereby, reduce side effects.
The data obtained from the cell culture assays and animal studies can be used in formulating a range of dosage for use in humans. The dosage of such compounds lies preferably within a range of circulating concentrations that include the ED50 with little or no toxicity. The dosage may vary within this range depending upon the dosage form employed and the route of administration utilized. For any compound used in the method of the invention, the therapeutically effective dose can be estimated initially from cell culture assays. A dose may be formulated in animal models to achieve a circulating plasma concentration range that includes the EC50 (i.e., the concentration of the test compound which achieves a half-maximal response) as determined in cell culture. Such information can be used to more accurately determine useful doses in humans. Levels in plasma may be measured, for example, by high performance liquid chromatography.
The pharmaceutical compositions can be included in a container, pack, or dispenser together with instructions for administration.
An antisense compound targeted to a SLC6A1 nucleic acid can be utilized in pharmaceutical compositions by combining the antisense compound with a suitable pharmaceutically acceptable diluent or carrier. A pharmaceutically acceptable diluent includes phosphate-buffered saline (PBS). PBS is a diluent suitable for use in compositions to be delivered parenterally. Accordingly, in one embodiment, employed in the methods described herein is a pharmaceutical composition comprising an antisense compound targeted to a SLC6A1 nucleic acid and a pharmaceutically acceptable diluent. In certain embodiments, the pharmaceutically acceptable diluent is PBS. In certain embodiments, the antisense compound is an antisense oligonucleotide.
In certain embodiments, the pharmaceutically acceptable diluent is designed to mimic the composition of cerebrospinal fluid. As such, it may contain divalent salts such as Mg²⁺ and Ca²⁺. Elliotts B solution is a diluent suitable for use in compositions to be delivered into the cerebrospinal fluid. A person of skill in the art will be able to see that other buffer solutions, with variations in the concentrations of different monovalent and divalent ions, may also be suitable as pharmaceutically acceptable diluents.
Pharmaceutical compositions comprising antisense compounds encompass any pharmaceutically acceptable salts, esters, or salts of such esters, or any other oligonucleotide which, upon administration to an animal, including a human, is 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 antisense compounds, prodrugs, pharmaceutically acceptable salts of such prodrugs, and other bioequivalents. Suitable pharmaceutically acceptable salts include, but are not limited to, sodium and potassium salts. A prodrug can include the incorporation of additional nucleosides at one or both ends of an antisense compound which are cleaved by endogenous nucleases within the body, to form the active antisense compound.

Methods of Treatment

The present disclosure provides a method of treating a subject having a SLC6A1-related disease or disorder. Methods of treatment include administering to the subject in need thereof an effective amount of an antisense oligonucleotide described herein. In some embodiments, the antisense oligonucleotide binds a target region in an SLC6A1 RNA transcript, wherein the target region comprises a splice modulatory element (e.g., a non-productive splice site).
Methods of treating a subject having a SLC6A1-related disease or disorder are useful in treating any SLC6A1-related disease or disorder known to those of ordinary skill in the art. For example, a SLC6A1-related disease or disorder includes, without limitation, e.g., myoclonic-atonic epilepsy (MAE), epilepsy, epileptic encephalopathy, seizures, autism spectrum disorders, intellectual disability, or a combination thereof. In certain embodiments, the SLC6A1-related disease or disorder is a disease or disorder of the central nervous system (CNS).
The contents of the articles, patents, and patent applications, and all other documents and electronically available information mentioned or cited herein, are hereby incorporated by reference in their entirety to the same extent as if each individual publication was specifically and individually indicated to be incorporated by reference. Applicants reserve the right to physically incorporate into this application any and all materials and information from any such articles, patents, patent applications, or other physical and electronic documents.
While the present invention has been described with reference to the specific embodiments thereof, it should be understood by those skilled in the art that various changes may be made and equivalents may be substituted without departing from the true spirit and scope of the invention. It will be readily apparent to those skilled in the art that other suitable modifications and adaptations of the methods described herein may be made using suitable equivalents without departing from the scope of the embodiments disclosed herein. In addition, many modifications may be made to adapt a particular situation, material, composition of matter, process, process step or steps, to the objective, spirit and scope of the present invention. All such modifications are intended to be within the scope of the claims appended hereto. Having now described certain embodiments in detail, the same will be more clearly understood by reference to the following examples, which are included for purposes of illustration only and are not intended to be limiting.

EXAMPLES

The present invention is further illustrated by the following examples which should not be construed as further limiting.

Example 1—Materials and Methods

Antisense Oligonucleotides

All phosphoramidites will be purchased from ChemGenes. 0.1M DDTT (ChemGenes) will be used as the sulfurising reagent and 0.25M BTT (AIC) as the activator. Antisense oligonucleotides will be synthesized on Dr. Oligo 48, ABI394, AKTA Oligopilot10 or AKTA Oligopilot 100 synthesizers, according to the required scale. MOE phosphoramidites will be coupled for 8 minutes. Oligonucleotides will be deprotected in concentrated aqueous ammonia at 55° C. for 18 h and purified using ion-exchange chromatography (eluting with 30% acetonitrile in water containing increasing gradients of NaClO₄). Final purification, desalting, concentration and pH adjustment will be effected by diafiltration in an Amicon centrifugal filter. All oligonucleotides will be characterized by LCMS.

Cell Line Selection

Splice site identification and antisense oligonucleotide testing experiments must be performed in a cell line in which SLC6A1 is transcriptionally active. Furthermore, since SLC6A1 intronic sequences are not well conserved between mouse and human and cryptic splice sites often occur in introns, experiments must be performed in a human cell line. The experimentally tractable human SH-SY5Y cell line (derived from neuroblastoma cells) expresses SLC6A1 (FIG. 1 ). However, it would be more optimal to perform these experiments in human GABAergic interneurons. Given that these are impossible to obtain from a living patient, approaches have been recently developed to differentiate GABAergic inhibitory neurons (iNs) from human embryonic stem cells (hESCs) or induced pluripotent stem cells (iPSCs). Thus, initial experiments and optimizations will be performed in parallel in SH-SY5Y cells and in iNs derived from commercial hESCs and an iPSC line derived from a clinical subject.

4sU Labeling of Nascent RNA Intermediates

Short time point metabolic labelling of SH-SY5Y cells with 4-thiouridine (4sU) will be carried out as described (Dalken et al. 2008; Pai et al., 2017). 4sU incorporates into newly created RNA in the place of standard uridine nucleotides and can be selectively isolated to capture nascent RNA shortly after its biogenesis. SH-SY5Y cells will be cultured in DMEM supplemented with 10% FBS. Newly transcribed RNA from three independent replicates of SH-SY5Y cells will be labeled for various time intervals, for example, 2, 5, 15 or 30 min, using 500 μM 4-thiouridine (Sigma, T4509). Additionally, for analysis of steady-state RNA levels, two independent biological replicates of SH-SY5Y cells will be generated without 4sU labeling. To normalize samples and assess metabolic labeled RNA capture efficiency, several synthetic RNAs will be spiked into the Trizol preparation at specific quantities per 10⁶cells. Quantities will be determined as described previously (Henriques et al., 2013).

RNA Extraction and Quantitative RealTime-PCR

Total RNA will be isolated from SH-SY5Y cells using Trizol (ThermoScientific) and subsequently treated with DNase I (Qiagen). One μg of total RNA will be reverse transcribed into cDNA using random hexamers and MultiScribe reverse transcriptase (ThermoScientific) following the manufacturer's instructions. Quantitative PCR will be performed on a StepOnePlus Real-Time PCR system using SYBR Green Master Mix (Applied Biosystems) and 0.2 μM of forward and reverse primers as described in (Jiang et al., Neuron, 2016, 90, 535-550; Tran et al, 2015, Neuron, 87, 1207-1214). Ct values for each sample and gene will be normalized to GAPDH. The 2(−ΔΔCt) method was used to determine the relative expression of each target gene.
Biotinylation of Nascent 4sU-Labeled RNA Intermediates
To purify metabolic labeled RNA 300 μg total RNA will be used for the biotinylation reaction. Separation of total RNA into newly transcribed and untagged pre-existing RNA will be performed as previously described (Windhager et al., 2012; Cleary et al., 2005). Specifically, 4sU-labeled RNA will be biotinylated using EZ-Link Biotin-HPDP (Thermo Fisher, Waltham Mass.), dissolved in dimethylformamide (DMF) at a concentration of 1 mg/ml. Biotinylation will be done in labeling buffer (10 mM Tris pH 7.4, 1 mM EDTA) and 0.2 mg/ml Biotin-HPDP for 2 hr at 25° C. Unbound Biotin-HPDP will be removed by extraction with chloroform/isoamylalcohol (24:1) using MaXtract (high density) tubes (Qiagen, Germany). RNA will be precipitated at 20,000 g for 20 min with a 1:10 vol of 5 M NaCl and 2.5× volume of ethanol. The pellet will be washed with ice-cold 75% ethanol and precipitated again at 20,000 g for 5 min. The pellet will be resuspended in 1 ml RPB buffer (300 mM NaCl, 10 mM Tris pH 7.5, 1 mM EDTA).

Capturing Biotinylated 4sU RNA

Biotinylated 4sU RNA will be captured using Streptavidin MagneSphere Paramagnetic particles (Promega, Madison Wis.). Before incubation with biotinylated 4sU RNA, streptavidin beads will be washed four times with wash buffer (50 mM NaCl, 10 mM Tris pH 7.5, 1 mM EDTA) and blocked with 1% polyvinylpyrrolidone (Millipore Sigma, Burlington Mass.) for 10 min with rotation. Biotinylated 4sU RNA will then be incubated with 600 μl of beads with rotation for 30 min at 25° C. Beads will be magnetically fixed and washed 5 times with 4TU wash buffer (1 M NaCl, 10 mM Tris pH 7.5, 1 mM EDTA, 0.1% Tween 20). Unlabeled RNA present in the supernatant will be discarded. 4sU RNA will be eluted twice with 75 μl of freshly prepared 100 mM dithiothreitol (DTT). 4sU RNA will be recovered from eluates by ethanol precipitation.

Library Preparation

RNA quality will be assessed using a Bioanalyzer Nano ChIP (Agilent). Ribosomal RNA will be removed prior to library construction by hybridizing to ribo-depletion beads that contain biotinylated capture probes (Ribo-Zero, Epicentre, Madison Wis.). RNA will then be fragmented and libraries will be prepared according to the TruSeq Stranded Total RNA Gold Kit (Illumina, San Diego Calif.) using random hexamer priming. cDNA for the two ‘total’ RNA samples will be prepared using an equal mix of random hexamers and oligo-dT primers (Pai et al., 2017).

Illumina Sequencing

Libraries will be sequenced on an Illumina HiSeq machine with paired-end 150 nucleotide reads (100 nucleotide reads for the ‘total’ RNA samples), for an average of 100 million read pairs per library. Reads for each sample will be filtered, removing pairs where the mean quality score of one or both mates fell below 20. Mean fragment length and standard deviation will be assessed using CollectInsertSizeMetrics, a component of Picard Tools 1.62. All reads will subsequently be aligned to hg38 with STAR. Strand-specific alignments will be performed for the 4sU RNA-seq (--library-type first strand), while unstranded alignments will be performed for the total RNA-seq (--library-type unstranded).

Identification of Non-Productive Splicing

Sites of non-productive splicing will be identified by non-annotated junction reads with canonical or non-canonical splice site sequences within annotated introns using nascent RNA reads from short labeling periods. To do so, the raw 4sU-seq reads will be re-mapped with the STAR v2.5 software (Dobin et al., 2013), with the mapping parameter-outSAMattribute NH HI AS nM jM to mark the intron motif category for each junction read in the final mapped file.
The jM attribute adds a jM:B:c SAM attribute to split reads arising from exon-exon junctions. All junction reads will be first isolated and separated based on the value assigned to the jM:B:c tag. Junction reads spanning splice sites in the following categories will be considered to be annotated or canonical: (1) any annotated splice site [jM:B:c, [20-26]], (2) intron motifs containing “GT-AG” (or the reverse complement) [jM:B:c, 1 or jM:B:c, 2], (3) intron motifs containing “GC-AG” (or the reverse complement) [jM:B:c, 3 or jM:B:c, 4], and (4) intron motifs containing “AT-AC” (or the reverse complement) [jM:B:c, 5 or jM:B:c, 6]. Junction reads with jM:B:c, 0 will be considered to arise from non-canonical non-annotated splice sites.

Statistical Analysis

All data were graphed as mean SEM and analyzed using GraphPad Prism Software (Version7). Tests between two groups used the two-tailed student-t test. Tests between multiple groups used one-way analysis of variance (ANOVA) corrected with Bonferroni multiple comparison post-hoc test. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001, ns not significant

Example 2—Identifying Cryptic Splice Sites by Using Computational Software

Identifying transcripts that are being created but not lasting until maturity would enable targeted optimization of mRNA processing pathways to allow for maturation of these transcripts. The first challenge is to identify sites at which non-productive splicing commonly occurs. Those sites can then be blocked by complementary antisense oligonucleotides to redirect the splicing machinery towards sites promoting productive splicing. SLC6A1 is a 46.5 kb gene with 17 introns and extensive alternative splicing, increasing the probability that many cryptic splice sites exist within this genomic space. Two complementary approaches will be used to identify sites of non-productive splicing in SLC6A1-expressing neurons: (1) computational identification of strong cryptic splice sites and (2) targeted sequencing of SLC6A1 mRNA intermediates (see Example 3). Computational software will be used to identify the sites that may underlie non-productively spliced isoforms. The commonly used maxEnt splice site algorithm will be applied, which uses a maximum entropy model to score sites relative to the entropy of known 5′ or 3′ splice site elements (Yeo and Burge 2004). Publicly available MaxEnt resources are available at: http://hollywood.mit.edu/burgelab/maxent/Xmaxentscan_scoreseq.html; MaxEntScan::score5ss will be used for human 5′ splice sites and MaxEntScan::score3ss will be used for human 3′ splice sites. The SLC6A1 genetic locus on human chromosome 3 is shown in FIG. 2 .
A sliding window algorithm will be used to scan every 9 and 23 nucleotide region segments in the human SLC6A1 gene sequence and the maximum entropy for 5′ and 3′ splice site motifs will be calculated, respectively. After removing annotated splice sites, an entropy threshold to identify high-scoring putative cryptic splice sites will be conditioned on.
Initial analyses of SLC6A1 RNA with maxEnt have identified 34 cryptic 5′ splice sites, depicted as SEQ ID NOs: 1 to 34 in Table 1, and 74 cryptic 3′ splice sites, depicted as SEQ ID NOs: 35-108 in Table 2. SEQ ID No: 109 depicts the entire SLC6A1 RNA sequence. Genomic scanning scripts will be used to identify high-scoring cryptic polyadenylation sites in SLC6A1, the usage of which might lead to truncated isoforms that are similarly targeted for degradation. Once identified, these sites can be targeted with antisense oligonucleotides to block the formation of non-productive, truncated transcripts.
The nucleic acid target sequences of Table 1 and Table 2 correspond to the genomic target sequence. An antisense oligonucleotide is designed to have sufficient complementarity to the corresponding RNA transcript expressed from said genomic target sequence (i.e., the reverse complement of the genomic target sequence, where each T is replaced by a U). For example, but in no way limiting, an antisense oligonucleotide may possess sufficient complementarity to CAGCCUGAUUCUGCCUGUGACUCACUUUGUGACCUCAGGAGAGUCCCUCC (SEQ ID NO: 128) (the RNA transcript sequence corresponding to SEQ ID NO: 1 in Table 1) to block the formation of non-productive, truncated transcripts.

Example 3—Targeted High-Throughput Sequencing of SLC6A1 Nascent RNA Intermediates

Targeted high-throughput sequencing of SLC6A1 nascent RNA intermediates will be performed to experimentally identify short-lived non-productive isoforms. Nascent RNA intermediates will be captured with methods as described in Example 1. To obtain high-resolution information about nonproductive SLC6A1 splicing, biotinylated probes complementary to regions of the gene to selectively isolate SLC6A1 mRNA will be used from the pool of nascent RNA. Probes will be designed to have optimal nucleotide composition and chemistry, match a unique location in the human genome (<70% match to a second location), and be located within exons that are included within 50% of annotated SLC6A1 isoforms (FIG. 1 ). The probes will tile across these exons within SLC6A1, located at the beginning, middle, and end of the gene to enable the most comprehensive capture of entire distribution of possible isoforms. Nascent SLC6A1 RNA across all intermediate lifetimes will be sequenced using a combination of short-read and long-read high-throughput sequencing. Short-read sequencing with the Illumina platform provides the ability to obtain high-resolution information about cryptic splice site usage with higher coverage. A total of 12 libraries (3 replicates for each of the 5, 15, 30-minute nascent RNA timepoints and 3 replicates of the steady state sample) will be sequenced in 1 NextSeq lane, with an estimated 450 million reads across all libraries. These data will be used to comprehensively identify sites of cryptic splicing leading to non-productive isoforms across a range of mRNA intermediate lifetimes. To do so, non-canonical splicing junctions will be identified by specifically analyzing splitreads that do not map to annotated SLC6A1 exon-exon junctions. Cryptic splice sites that recurrently have split-junction reads in multiple samples and after sub-sampling approaches will be considered to be major sites of cryptic splicing in SLC6A1. Open reading frames (ORFs) will be predicted and premature stop codon usage in all isoforms (both annotated and cryptic) expressed in neuronal systems and identified through this analysis. These predictions will be used to quantify the probability that cryptic splice site usage leads to isoform degradation through nonsense mediated decay (NMD) pathways. Splice sites that lead to NMD will be prioritized for downstream antisense oligonucleotide design and targeting approaches (Aim 2). Further Oxford Nanopore Minion cDNA libraries will be generated with the same 12 samples and sequence them across 2 Minion flowcells to assess the long-range connectivity between different cryptic sites. Since long-read datasets are inherently error prone, annotations of cryptic splice sites derived from the short-read Illumina data will be used to refine mapping of the long-read Minion data. These data will provide isoform level insights that may be useful for combinatorial targeting of multiple cryptic splice sites for maximal effect with a minimal number of antisense oligonucleotides.

Example 4—Use of Publicly Available Datasets

For a more comprehensive picture of non-productive splicing isoforms in SLC6A1, publicly available sequencing datasets will be analyzed that are designed to capture total cellular mRNA, as opposed to only polyadenylated mature mRNA (Schwarzl et al. 2015; Rybak-Wolf et al. 2015; Pandey et al. 2014). We will also generate a genome-wide nascent RNA sequencing dataset with SH-SY5Y cells, neurons derived from human embryonic stem cells, and neurons derived from induced-pluripotent stem cells. These datasets will all be enriched for mature polyadenylated RNA but will also contain a small amount of information about intermediate RNA species. In all of these datasets, we will identify novel isoforms of SLC6A1 (using the MAGIQ splicing analysis software), and identify splicing junction reads.

Example 5- Antisense Oligonucleotides Targeting Non-Productive Splice Sites that Increase the Expression of Productive SLC6A1 mRNA Molecules

Computational software was used to identify cryptic splice sites that are likely to underlie non-productively spliced isoforms of SLC6A1. Specifically, the maxEnt splice site algorithm was applied, which uses a maximum entropy model to score sites relative to the entropy of known 5′ or 3′ splice site elements (Yeo and Burge 2004). A sliding window algorithm was used to scan every 9 and 23 nucleotide region segment in the human SLC6A1 gene sequence and calculate the maximum entropy for 5′ and 3′ splice site motifs, respectively. After removing annotated splice sites, an entropy threshold of 8.72 and 7.25 (mean entropy scores for annotated sites) were used to identify high-scoring putative cryptic 5′ and 3′ splice sites, respectively. Initial computational analyses identified 12 cryptic 5′ splice sites and 84 cryptic 3′ splice sites in SLC6A1. The number of predicted sites that would target was narrowed to 15 (5 5′ splice sites and 10 3′ splice sites) based on their position along the SLC6A1 locus (focusing on intron 1) and maximizing the specificity of those sites by selecting antisense oligonucleotides with minimal complementarity to other sites in the transcriptome using NCBI BLAST.
Three antisense oligonucleotides were selected for each of the 15 predicted sites. These antisense oligonucleotides were designed as steric blockers, with each nucleotide comprising a 2′-O-methoxyethyl RNA (MOE) modification and a phosphorothioate backbone. Each antisense oligonucleotide was 20 nucleotides in length. 45 antisense oligonucleotides were synthesized using standard methods on a Dr. Oligo 48 synthesizer, and their identity and purity was confirmed by high performance liquid chromatography coupled to mass spectroscopy (LCMS).
Each antisense oligonucleotide was transfected into KNS60 neuroblastoma cells (JCRB Cell Bank). These cells were cultured in DMEM (Sigma) supplemented with 5% fetal bovine serum at 37° C. with 5% CO2. One day prior to transfection, cells were seeded at a density of 50,000 and incubated overnight. Cells were transfected with 50 nM antisense oligonucleotide using Lipofectamine RNAiMAX (Thermofisher) transfection reagent.
After 24 hours, RNA was collected using TRI Reagent (Sigma) and subjected to reverse transcription using the High-Capacity cDNA Reverse Transcription kit (ThermoFisher) according to the manufacturer's instructions. Resulting cDNA was used for qPCR reaction with IDT PrimeTime Taqman primers for SLC6A1 (Hs.PT.58.40113647) and GAPDH (Hs.PT.39a.22214836). The qPCR was performed in technical duplicate on a Bio-Rad CFX96 Real-Time System Thermal Cycler.
Several of the antisense oligonucleotides produced activation of SLC6A1 as seen by up to 2.5-fold increases in mRNA expression (FIG. 3 ). Sequences, masses, target sites, and percent activation details of active antisense oligonucleotides are presented in Table 4.

Example 6—In Vivo Testing in Mice

For in vivo testing, antisense oligonucleotides will be delivered into wild-type mice at a range of doses, both systemically and by intracerebroventricular (ICV) injection, to rule out any toxic compounds. For intracerebroventricular (ICV) infusion of antisense oligonucleotide or PBS vehicle through a micro-osmotic pump (Alzet pump model 1007D will be attached to Alzet brain infusion kit 3), wild-type C57BL/6 or C9BAC transgenic mice will be anesthetized and maintained on 2.5% isoflurane via a nose cone under a stereotaxic frame. Implantation procedure will be performed as previously described (Devos et al, 2013, J Vis Exp, e50326-e50326), with a 3 mm cannula implantation 0.2 mm posterior and 1.0 mm lateral to the right of Bregma. For ICV bolus injection mice will be anesthetized with isoflurane and placed into a stereotaxic frame. 10 μL of sterile PBS or antisense oligonucleotide was injected into the right lateral ventricle using the following coordinates: 0.2 mm posterior and 1.0 mm lateral to the right from the Bregma and lowered to a depth of 3 mm. Pharmacokinetic properties will be measured (drug levels in tissue at multiple timepoints after dosing) using an assay previously described.

Example 7—Clinical Testing

One or more neurologists will be identified as a clinical partner for IND submission and drug administration. If safety and efficacy data are supportive, permission will be sought to treat a patient by lumbar intrathecal infusion, using a dose-escalation protocol (i.e. starting at a low dose to ensure there is no adverse reaction, then re-treating with an increasing drug dose approximately every 2-4 weeks)

TABLE 1

Target sequences for 34 cryptic 5′ splice sites

SEQ
ID NO.	Target Location	Nucleic acid target sequence

1	>SLC6A1::3:10992186-	GGAGGGACTCTCCTGAGGTCACAAAGTGAG
	11039247; startsite =	TCACAGGCAGAATCAGGCTG
	11029076; maxEnt = 8.4(+)

2	>SLC6A1::3:10992186-	TGACAGGCACCCAGGTAGATACATGGTGAG
	11039247; startsite =	TCATGCTCACTGACCGAGGG
	11018312; maxEnt = 10.13(+)

3	>SLC6A1::3:10992186-	GTCACAGAGATCACATGCTCACAAGGTAAT
	11039247; startsite =	AAAATATCACAAGGCAAATG
	11026999; maxEnt = 8.49(+)

4	>SLC6A1::3:10992186-	ATGGAGGGTTTGGGGGGTTCCACAGGTACC
	11039247; startsite =	CCATTAGAGACAGCAGAGTG
	11008010 ; maxEnt = 8.63(+)

5	>SLC6A1::3:10992186-	GTGGCCAGTCCTTGGAGGACAAACAGTGAG
	11039247; startsite =	TCCCAAGCAGAGAGACGCAG
	10994543; maxEnt = 8.34(+)

6	>SLC6A1::3:10992186-	TGTATTACATATGCATTTTTAGCAGGTTGGT
	11039247; startsite =	TCAGCATAATGACACAGAA
	10999403; maxEnt = 8.08(+)

7	>SLC6A1::3:10992186-	TCCCCCAAATTCCAAAACAAAAGAGGTCAG
	11039247; startsite =	TGAAAGCTACTCGCATTTTG
	10994225; maxEnt = 7.7 (+)

8	>SLC6A1::3:10992186-	TTCCTTCCTCCAGGAGCAAGGGCGGGTGAG
	11039247; startsite =	AGGAAGAGGGCTTATAGAGA
	11016980; maxEnt = 7.62(+)

9	>SLC6A1::3:10992186-	CTGCGCCTGGCAGAGAACAAGCCCTGTAAG
	11039247; startsite =	TGTTTGCTGGTGTCGTTGCA
	11019859; maxEnt = 7.52(+)

10	>SLC6A1::3:10992186-	CTAAAAATACAAAAAAATTAGCCAGGTATG
	11039247; startsite =	GTGGCAGGCACCTGTAGTCC
	11003074; maxEnt = 9.99(+)

11	>SLC6A1::3:10992186-	TATTTAGAGTCACATAAAATTGGAGGTAACC
	11039247; startsite =	TGAATGCATAGCAGTGGGG
	11024724; maxEnt = 8.55(+)

12	>SLC6A1::3:10992186-	AGTTCCATTTCTCGAAAGCAACAAGGTAATA
	11039247; startsite =	TGGATCAAAAGCCTGTGAT
	11024566; maxEnt = 8.49(+)

13	>SLC6A1::3:10992186-	TTTCTTCCGCCTGCTCCACCAGCAGGTAAAG
	11039247; startsite =	GAGGCTGATCACAGGCTGG
	11012797; maxEnt = 9.65 (+)

14	>SLC6A1::3:10992186-	CCTGCTTCTGCAAATTCCCTCTCAGGTACGT
	11039247; startsite =	TGAGGCAGCTGAGGGGTTT
	11009361; maxEnt = 10.65(+)

15	>SLC6A1::3:10992186-	GACCGAGACAGCGGAGAGGTTGCGGGTGAG
	11039247; startsite =	CTGCGCTGAGCCCAGGAGCC
	10992782; maxEnt = 8.19(+)

16	>SLC6A1:3:10992186-	TGTGCCAAGCTCTGGGCACATAACAGTGAG
	11039247; startsite =	TCAGATGGGGTCCCTGCCCT
	11029491; maxEnt = 8.34(+)

17	>SLC6A1::3:10992186-	GTGTGATCATCATTCTTATTACAGGGTAAGA
	11039247; startsite =	TGCGCCCTCTTTTCCTGCA
	11006834; maxEnt = 9.21(+)

18	>SLC6A1::3:10992186-	TACGCAGCACAGTGCGAAGCTCACAGTGAG
	11039247; startsite =	TCCCATGGGATTCCACTGGG
	11022717; maxEnt = 8.34(+)

19	>SLC6A1::3:10992186-	GGGAGAGGGCAGCAGCCACAGGGAGGTGA
	11039247; startsite =	GCAAAGACATTTGGTATCAGT
	10997671; maxEnt = 8.7 (+)

20	>SLC6A1::3:10992186-	ACCTCAGGCTTCTCTTGGCTGAAAGGTAGGC
	11039247; startsite =	TCCTTCCCTCCCTCCTTGG
	11005443; maxEnt = 10.08(+)

21	>SLC6A1::3:10992186-	GGACTGGCATAAGGTCACACAGCTAGTAAG
	11039247; startsite =	TTTAGAGAGAGATTTCAAAC
	11007557; maxEnt = 8.78(+)

22	>SLC6A1::3:10992186-	CATTTAGAGTGGCCCCGGCACATTAGTAAGT
	11039247; startsite =	GTGTCCAGCTCACTTCCTC
	21100816; maxEnt = 7.79(+)

23	>SLC6A1::3:10992186-	GCCATTTCTGGCCAGGTGACCTTGGGTACGT
	11039247; startsite =	TCTTGTTCCTGTTGGAAGG
	10995132; maxEnt = 7.7 (+)

24	>SLC6A1::3:10992186-	CCACGCTGCCCCTGGAAATAAAAAGGTAAG
	11039247; startsite =	AAGGCTGCAGAGTGTCAGTA
	11016045; maxEnt = 10.5 7(+)

25	>SLC6A1::3:10992186-	AATCCCAGCTACTTGGGAGGCTGAGGTAGG
	11039247; startsite =	AGAATTGCTTGAACCCGGGA
	11029877; maxEnt = 8.24(+)

26	>SLC6A1::3:10992186-	ACAGCCCTGGGAACTTCAAGTGAAGGTAAT
	11039247; startsite =	TTTATTGTTATTATTGGTAC
	10998050; maxEnt = 8.83(+)

27	>SLC6A1:3:10992186-	AAATCACATGTATGTCTGCTTTATGGTGAGG
	11039247; startsite =	TCTTCAGAGCAGCCACCGT
	11014026; maxEnt = 7.61 (+)

28	>SLC6A1::3:10992186-	GCACTTCGTGGAGGTGCAGAGTCAGGTGAG
	11039247; startsite =	GAGAGGTGAAGTGACTCATC
	11001471; maxEnt = 10.07(+)

29	>SLC6A1::3:10992186-	GAACGGAGATCAGTGTGGCTGGAAGGTAAA
	11039247; startsite =	GTGGAAAGGGGTGCGAGGAG
	11035089; maxEnt = 9.06(+)

30	>SLC6A1::3:10992186-	CCTTTGTTTTTCTATGACACACAAGGTGGGT
	11039247; startsite =	GTTTGGCAAGAGAGTAGGG
	10994083; maxEnt = 8.23(+)

31	>SLC6A1::3:10992186-	AGGAAAAGTAACAACTTGCAAAAAGGTTGG
	11039247; startsite =	TTCTGCTTCAGAGAAATGTT
	11012956; maxEnt = 8.46(+)

32	>SLC6A1::3:10992186-	GAACTTGTCCAGCTGGGCCCTGACAGTGAGT
	11039247; startsite =	TCAGAGGGCCTGCTGGGCA
	10998840; maxEnt = 8.34(+)

33	>SLC6A1::3:10992186-	GCATTTGGCTGTTTGAAAAGCGCTGGTAAGA
	11039247; startsite =	GCTGGGATCCTGATGATTG
	11002499; maxEnt = 9.45(+)

34	>SLC6A1::3:10992186-	AGCACCAGGGTGGCTTTGCTGCTGTGTAAGT
	11039247; startsite =	CAAAGAGCCTTCTCCGTTC
	11013805; maxEnt = 7.65 (+)

TABLE 2

Target sequences for 74 cryptic 3′ splice sites

SEQ
ID NO.	Target Location	Nucleic acid target sequence

35	>SLC6A1::3:10992186-	TAACCCTACTGTTCTAATTTCCAGGCCAACT
	11039247; startsite =	CTGTCCTATTGTGGGTCTC
	10995350; maxEnt = 8.94(+)

36	>SLC6A1::3:10992186-	TGGCTGCCTTCCTTAATCCTGCAGAATCTCG
	11039247; startsite =	GGGTCAAGTCAGGGAGGTG
	11024297; maxEnt = 9.97(+)

37	>SLC6A1:3:10992186-	GGGGACACCTTTCTCCCCATTCAGCCACAGG
	11039247; startsite =	TGGAGCTTCTTTCTTGCTC
	11016703; maxEnt = 8.34(+)

38	>SLC6A1::3:10992186-	TCAGCCACCTTCTCCATTTTCCAGAAGGGGA
	11039247; startsite =	AACTGATGCCCAGAGGGGA
	11029029; maxEnt = 9.21(+)

39	>SLC6A1::3:10992186-	ATACCATAAAATTCCTTTCCACAGCTAAGTG
	11039247; startsite =	AGTGAGTCAAGAACAGATG
	11003960; maxEnt = 7.75(+)

40	>SLC6A1::3:10992186-	GTCTTGTTCTTACACCTCTTGCAGCACTTATC
	11039247; startsite =	ACAGGGTATTATGATCAG
	11012234; maxEnt = 8.97(+)

41	>SLC6A1::3:10992186-	GGTGCTACTCTCTCCTTCTGCCAGAGAGGAC
	11039247; startsite =	CCTGAGCCCAAGGGTGGTG
	10998924; maxEnt = 8.12(+)

42	>SLC6A1::3:10992186-	CATCACCACTTCCCTTCTCCAAAGCATGCAA
	11039247; startsite =	GTTCAATTTCTAAAATTCA
	11004488; maxEnt = 7.9(+)

43	>SLC6A1::3:10992186-	ATTAATTTCTTTCTTAAACCACAGAGTTTCA
	11039247; startsite =	AAAAGAAATAAAGGCAAAC
	11010695; maxEnt = 8.06(+)

44	>SLC6A1::3:10992186-	AAAGTATCTCTGATCCATTCCTAGGACTAGG
	11039247; startsite =	GACGCCGGAGGAGGGCACA
	51102392; maxEnt = 8.93(+)

45	>SLC6A1::3:10992186-	TGACATCACATATCCATCCACCAGGTGCATA
	11039247; startsite =	TCTGAGCAGAGTGAGGGCT
	11018094; maxEnt = 7.61(+)

46	>SLC6A1::3:10992186-	GCTGGGCCTCCCGTCCTTCCTTAGAGGGCCA
	11039247; startsite =	GGCTTTGGGTGGGTTGGGG
	11029153; maxEnt = 8.31(+)

47	>SLC6A1::3:10992186-	TCACCCCCACCCCCCCCCCACCAGATCCAAA
	11039247; startsite =	TGTAGTTCTGCTCCAGAGT
	11030256; maxEnt = 8.3(+)

48	>SLC6A1:3:10992186-	TCTCCTCTCCTCCTCCCCACACAGCTGCTGG
	11039247; startsite =	GCGGTCTTGCCAAGTCACC
	11006564; maxEnt = 10.01(+)

49	>SLC6A1::3:10992186-	AATAATTTCTTTTCCTTTGGATAGATAGCCA
	11039247; startsite =	GTAGTGGGATTGCTGGATC
	11032621; maxEnt = 7.74(+)

50	>SLC6A1::3:10992186-	CAAGGCTGTTTCCTTATCTGTCAGATGGGTG
	11039247; startsite =	AGGTAGCCCTGGCTTACAT
	11010424; maxEnt = 8.08(+)

51	>SLC6A1::3:10992186-	AGGCAGAAATATTTCTCCTTCTAGGCCATGA
	11039247; startsite =	CCTTGACAAGGGCAAGGGT
	11012288; maxEnt = 8(+)

52	>SLC6A1::3:10992186-	AGGCCGTGCTGATTGTATCTTCAGGGGAGTA
	11039247; startsite =	CCTTGTAGAGATGTGTGTC
	11021265; maxEnt = 8.02(+)

53	>SLC6A1::3:10992186-	CCTGGCCTCTCGGTCTCTGCCTAGGTCCCCA
	11039247; startsite =	CCCCACGCAGCCGCCTGTC
	11010986; maxEnt = 9.85(+)

54	>SLC6A1::3:10992186-	AAAACTGGGTGTCCTTGCCCCTAGGGAAGG
	11039247; startsite =	ACAAATTTTCTTTAAGTCCC
	11003518; maxEnt = 10.44(+)

55	>SLC6A1::3:10992186-	TGACTTTCTTTGCCTCGTGCTCAGTGCCTGA
	11039247; startsite =	CAGGCACCCAGGTAGATAC
	11018284; maxEnt = 7.93(+)

56	>SLC6A1::3:10992186-	TGGAGCGCTCATTCCCTTTCCCAGGAAGCTC
	11039247; startsite =	AGCCTTATCCCCATGAAGA
	10993935; maxEnt = 7.97(+)

57	>SLC6A1::3:10992186-	TAGGTGTTATCACTTCTGTTTTAGACAGAGA
	11039247; startsite =	GAGTAGATGACTAACCTAC
	41100336; maxEnt = 8.89(+)

58	>SLC6A1::3:10992186-	TATTATTATCTTCCCATTTTATAGATGAGGG
	11039247; startsite =	TCAGAGAGGTGAAGTAACT
	11013708; maxEnt = 8.6(+)

59	>SLC6A1:3:10992186-	AAAAATCAGCTTCTCGTTCCACAGGTCTTGA
	11039247; startsite =	GTGGGGCCCAAGATTCTGC
	11010272; maxEnt = 9.79(+)

60	>SLC6A1::3:10992186-	GCTGTATTAATGCGTTTCTTCTAGGCCTCCC
	11039247; startsite =	GTGTCTTGTTCTTACACCT
	11012201; maxEnt = 8.71(+)

61	>SLC6A1::3:10992186-	GGCTCTGTTCCACCTGGCCCACAGGCAGCCA
	11039247; startsite =	GACGTTAAGGTTATCTCCC
	31102798; maxEnt = 8.42(+)

62	>SLC6A1::3:10992186-	CCCCTTGCCTGCCATCTGGTCCAGGGCTGGG
	11039247; startsite =	CTGCTCACAGCCAATCATC
	11011094; maxEnt = 7.54(+)

63	>SLC6A1::3:10992186-	ACTCCACCTTTTCTCCCTTTCAAGCCCTACCC
	11039247; startsite =	CAGGAGCCTGGGGGCAGA
	11023244; maxEnt = 8.27(+)

64	>SLC6A1::3:10992186-	TCTAGTTTTCTCCTTCCTCAGCAGACCAAAT
	11039247; startsite =	CTCACTCTGAGTACAAGAT
	11014323; maxEnt = 8.36(+)

65	>SLC6A1::3:10992186-	CTGTTATCCTGTTTTTTTTCCAAGGTGCCCAG
	11039247; startsite =	ATTTCATATTGTTTAAAC
	11027465; maxEnt = 9.35(+)

66	>SLC6A1::3:10992186-	CCCACTTCCTCCCATCCCACTTAGAATGAAA
	11039247; startsite =	CCTGAATCCTTGCTGTGAC
	11011378; maxEnt = 8.24(+)

67	>SLC6A1::3:10992186-	GCATCTTTCTGACCCTCACTGTAGACCAGGT
	11039247; startsite =	TTGTTGCCAGGGAGAGCTG
	11025669; maxEnt = 9.61(+)

68	>SLC6A1::3:10992186-	GACCTGGGCCCCGTTCTTGCATAGGTGACAG
	11039247; startsite =	TGCAGCTGGGAAGCTAAGA
	11006369; maxEnt = 7.84(+)

69	>SLC6A1::3:10992186-	CTGCACCTTTGATTGTCCCATCAGAGCAAAT
	11039247; startsite =	GTTTTTAAAGAAGCATGAT
	11031715; maxEnt = 8.13(+)

70	>SLC6A1:3:10992186-	GCTGCCCTCATCCCACGCCCACAGCTGTCCC
	11039247; startsite =	GAGGGCAGCGGGCCCCACT
	11015440; maxEnt = 8.64(+)

71	>SLC6A1::3:10992186-	AAGCTAACTGCCCTTCCTCCTCAGGTCAGCT
	11039247; startsite =	CCTCGCAGCAGCTGCAAGG
	11007633; maxEnt = 11.03(+)

72	>SLC6A1::3:10992186-	CTGGGTTTTTTTCTTCATCTATAGAATGCCAT
	11039247; startsite =	GGTAGACCAGGTGCACCA
	11009538; maxEnt = 9.83(+)

73	>SLC6A1::3:10992186-	CTGCTTCTGCAAATTCCCTCTCAGGTACGTT
	11039247; startsite =	GAGGCAGCTGAGGGGTTTA
	11009362; maxEnt = 8.03(+)

74	>SLC6A1::3:10992186-	CACTTTAACCTCTCTGTGCCTCAGTTTACCC
	11039247; startsite =	ATCAATAAAATGGGGGCTA
	11008143; maxEnt = 7.54(+)

75	>SLC6A1::3:10992186-	ATCCTTGCACCATCCCTGTTACAGCAACCTC
	11039247; startsite =	CACCCATGCTCCCTCTCCT
	11006520; maxEnt = 7.77(+)

76	>SLC6A1::3:10992186-	TCTCTGCCTCTAATCTCCTGCCAGCTCCTCCC
	11039247; startsite =	AATGGTCAAACCCAGCTA
	11020973; maxEnt = 7.72(+)

77	>SLC6A1::3:10992186-	GTGCCAGGCTCCTTCCTGCCTCAGGGCCTTT
	11039247; startsite =	GCACTTGCTGCTCCCTCTG
	11011532; maxEnt = 8.06(+)

78	>SLC6A1::3:10992186-	ATCAGCCCTGCCCCATCCCTGCAGCTAGTCC
	11039247; startsite =	CCAGGTTCCTTAGTCCGGT
	91101185; maxEnt = 10.27(+)

79	>SLC6A1::3:10992186-	CAGGGAGCTTTCCCTGACCTCCAGGACAGC
	11039247; startsite =	GCGTGGCAAGCACTGCCCAC
	11009833; maxEnt = 7.67(+)

80	>SLC6A1::3:10992186-	TATTGTCCCACTTTACCGCTGCAGGATCTGG
	11039247; startsite =	GGCTCACCTAGCCAGCATC
	11033989; maxEnt = 8.03(+)

81	>SLC6A1:3:10992186-	GAATGTGTGTTTTCTCTGTGCCAGCTATCTA
	11039247; startsite =	AATGACCCCATGCTGCAAA
	11002235; maxEnt = 8.56(+)

82	>SLC6A1::3:10992186-	GCTGTGTGCTTTATATCGTTGCAGTTAATTTT
	11039247; startsite =	CACAAAACCCTGTGAGAT
	10996585; maxEnt = 7.73(+)

83	>SLC6A1::3:10992186-	TTCCTTTATATTGATTGCCTATAGGTTAAGA
	11039247; startsite =	TAACACTGGGCCTGGCGCA
	11002919; maxEnt = 8.57(+)

84	>SLC6A1::3:10992186-	CGAAGCTCCCTATTCATTCCCCAGGGCATGG
	11039247; startsite =	AGGGGACGCGGAGTGAATG
	10993277; maxEnt = 9.29(+)

85	>SLC6A1::3:10992186-	TGGGCACCCGGACCTGTTCCACAGGGGCTC
	11039247; startsite =	GCCCTCGTGCCCAGCACAGG
	11005217; maxEnt = 7.83(+)

86	>SLC6A1::3:10992186-	GAAGTCCATCTCCTTTGTCCTTAGAAGCCCA
	11039247; startsite =	TCCCTGTTGCCTAGCCCAA
	71099533; maxEnt = 9.08(+)

87	>SLC6A1::3:10992186-	TACACGCCTCCCTCATTCTTGCAGACTATTC
	11039247; startsite =	TAAAGCAGAGTCTCTCTGC
	11004115; maxEnt = 9.68(+)

88	>SLC6A1::3:10992186-	GGTTCGATGTTCCTCATCCTGCAGCAGACGT
	11039247; startsite =	CTCTGCGGGCACCCACCAG
	11005822; maxEnt = 7.69(+)

89	>SLC6A1::3:10992186-	TTTTTTTTTTTTTTTTTTGCTCAGGCCAAATA
	11039247; startsite =	AAACAAGCCCAAGGGCCA
	11011646; maxEnt = 9.18(+)

90	>SLC6A1::3:1O992186-	CTGCACCCCTGCTGCCCTCTGTAGGAGCTGC
	11039247; startsite =	CTGCCTGCCCCATCGCTGC
	11015607; maxEnt = 9.95 (+)

91	>SLC6A1::3:10992186-	GTCCTCGATTCCCTGGCTTTTCAGGGCTCCC
	11039247; startsite =	CACTCACTCCATGGCGGGG
	11010899; maxEnt = 8.83(+)

92	>SLC6A1:3:10992186-	CAAAAGTCACCTTCCTTCCTCCAGGAGCAAG
	11039247; startsite =	GGCGGGTGAGAGGAAGAGG
	11016969; maxEnt = 9.36(+)

93	>SLC6A1::3:10992186-	GATCTCTTTTTATCGCCATTCCAGGGGCCTC
	11039247; startsite =	AGGTCCTACTGGGGAAACT
	11037771; maxEnt = 8.21(+)

94	>SLC6A1::3:10992186-	TGACTCCTCATCTCTGTCCCCTAGTTTCCCA
	11039247; startsite =	GCTGATGAAAATCACCTTT
	10998146; maxEnt = 8.85(+)

95	>SLC6A1::3:10992186-	AACACTGGCCTGCCTTCCACCCAGGACTTTT
	11039247; startsite =	TCCCATCACTAACTAAAAA
	11003262; maxEnt = 8.32(+)

96	>SLC6A1::3:10992186-	TCCGGTTCTAATCTCCCCTTCAAGGGCAGCC
	11039247; startsite =	ACCATCTCGTTTCTCTGCA
	11028119; maxEnt = 7.52(+)

97	>SLC6A1::3:10992186-	TGACTGTTATCTCGGACTTTGCAGGAGTTCC
	11039247; startsite =	TTTCCCTCCGAACGCTGCT
	11037369; maxEnt = 7.54(+)

98	>SLC6A1::3:10992186-	CGAAGTCTCGCTCTTGTTCCCCAGGCTGGAG
	11039247; startsite =	TACAATGGCACGATCTCGG
	11032986; maxEnt = 9.32(+)

99	>SLC6A1::3:10992186-	GGTAGATCATTTTTATCCCGCCAGGGAGTGT
	11039247; startsite =	GATGCAGGAAGACCACATG
	11037267; maxEnt = 7.93(+)

100	>SLC6A1::3:10992186-	AGTCCTGGCCCCCTGGCTTGTCAGATGTACA
	11039247; startsite =	TGACCTTCAGCAAGTCACT
	11018437; maxEnt = 7.6(+)

101	>SLC6A1::3:10992186-	TCTCTACTTCCATCCTTCCTACAGCCTTGTCA
	11039247; startsite =	GAATGGGATACTTCCCTG
	11001422; maxEnt = 8.11(+)

102	>SLC6A1::3:10992186-	TGGCCCATCTTCCTCCAACCTCAGATGAAGA
	11039247; startsite =	AACTGGGGAACCACAGGGG
	11035701; maxEnt = 8.47(+)

103	>SLC6A1::3:10992186-	TCTTCTGGCTCTGCCCTCCTCTAGCTTGCTCT
	11039247; startsite =	TAGGGCCTGTGCATCTGG
	11026090; maxEnt = 8.54(+)

104	>SLC6A1::3:10992186-	CCTAGATTTTAAATTCATTTGCAGCTAGTGC
	11039247; startsite =	TGATGTCATGCACTCAGCC
	10997049; maxEnt = 7.66(+)

105	>SLC6A1::3:10992186-	AGTGGAGTTCTCTCTCTTAACCAGGTTGGCG
	11039247; startsite =	AAAAGCACTCTTGCAGCGA
	11015791; maxEnt = 7.83(+)

106	>SLC6A1::3:10992186-	GGATGCTGTTCCTGCCCTTTGAAGATCCACT
	11039247; startsite =	TGCATTGAAAACCGTAGAT
	11000356; maxEnt = 8.27(+)

107	>SLC6A1::3:10992186-	CTTCCATTTATTCATCTGTCCTAGGACAGTG
	11039247; startsite =	AGGCTAGGCAAACTCATCT
	11008549; maxEnt = 8.67(+)

108	>SLC6A1::3:10992186-	CTCACCCTTGCTCTGCTTTTGAAGCTGGGGA
	11039247; startsite =	ACTTGGAGGAAGGGGTTCC
	11034251; maxEnt = 8.07(+)

TABLE 3

Full length SLC6A1 nucleic acid sequence
SLC6A1 full nucleotide sequence

AGAAACGGAAAGGACAGGCCAACGGAAGCAGTACTGCAAGGCTGGAAGGAG

AAAAGCCAGGAGGGGAGTGCTTGCTGTGAAAGACAGGGAGACAGAGACCAAG

ACGGACAGGCAGACAGGCTGGTGACCCAGGATGAGGCCGGAAAGAGCCATCA

AAGGAAGGAGAAGGAAGGAGAGAGATTGGAGCGGGACGGCGGGGCAGGCGA

GGGAAGGAGGGGGTGGGGAGAGGGAGGGAGGAAGAGAGGGGAGAAAGAGG

GAGGAAGAGAGGGGAGAAGGAGGGAGAAGAGAGCGGGAGAATGCGAGAGGA

AAGAAGGGAGAGGGGAGGCGTAGAAGGGGAGAGGAGGTGAAGGGAAAAGGA

GAGAGCCTGCTGGCGGCGAAGCTGCAAGAGGCAGCTGCGGAGGGAGCGCGCG

GCGGGCCTGGGGGAGCGCTGGGCGGGGGCGGGCGGTGCGGGCAGGGCTATAC

CCGAGCTGGGCGGGCTCCGGGCGCCGCGGGCCCTGCCCTCCCCCTCCATCCCT

CCGGACTCGCTCCCCCCTCCTCTCCCTTCCCCGCGACCCTCCGCCCGCCCTCGG

AAGACCGAGACAGCGGAGAGGTTGCGGGTGAGCTGCGCTGAGCCCAGGAGCC

GAGGAGTCGGGAGCGCAGTAGCGCTGAGCCCGAGCCCGAGCGGCCCCGCGTC

CCGAGCGCATCGGAGCGGCCGAGCCGCCCGGATGCAGCGCCTGTCCCGGGCA

GCGCAGCCCCGGCCGCAGGTAGGAAAGGGCGGCCGGCGTCGGGGCGCGGGGC

GCAGAGCCTGGGATAACGGCGCAGGAACGGCCAGAGCCCCGAGCCGCTGCCG

CGAGCCGGGCCGGGTGGGGGGCGGACACCTCGAGATTCAAGCCCCCGCGCCG

CACCTTCCCTGAGCCACATGAAGGAGCAGAGGCGCCGCTCGGACCCCGACGTG

CGCCCAGGCCATCCGTCCCCTGGGACACTCGGGAGGCAGGAGGGCTGGAACA

GAGGCGAGAGTTGCATGCCCCTTCCCTCCCGCGGTGCCCCACTGGCCCCGCAA

GTTGGCACTGGATTCTGCTGGAGCCGTCGAAGCTCCCTATTCATTCCCCAGGGC

ATGGAGGGGACGCGGAGTGAATGACACGAGCAGCGTTGGGGGGCCGTGCGCC

ACTTTCCCAATCCTGGGGTCAGCTGAGCCGAGTTAGAGCTATGCCCCTCATTTG

TTCTCTGCGGGCTACGGCGGGGTCGGGTGCGACTTGAGGATGGGACGTCTCTG

GGACAGCCCGAGCTCTCAGAAACGTTGGCTTAAAAGCACACCGGGGCTTGGGC

TGAGTGCACCGGAGCTAAGCCTAGTGCACCAGAGCTCGCGCCGCGCTCTCCCA

GCCCCGACTTTCTCCAAGAGATGTGGGGTGCGGGCGGTCCCTTAGAGGGCTGG

AGGACGGTGCCTCCTTGCTGCTTCGGGAACTGCGCTCCCCTTCTTGCTTTTGGT

CTCTGCGTGTACTCCCCGGCGCGACACCGCTCACCACGGTAGCCTCGGGCAGT

GCCCATTGGGTTCTGAGCACACGTCCCCACGGGTGGCACCCACAGATGTCCTG

TTCTAGGCTTGGCTCGGTCTTCAGACAAGAAACTCAGACCGGGCAGTCCCCTA

TTGAGGCTCTGAGCTAATATCCTCCCAAAATAGACATGAACCACAAGGAGAAT

TTTTTAAAAGCCAAATGATAACACCACGTTCTTTCCGGATGTGGGGCTGGAGC

GCTCATTCCCTTTCCCAGGAAGCTCAGCCTTATCCCCATGAAGAGCTGGGGGG

GCGGGGAGGGCGGGAATAAAATGGCTCCTGAGATTTGTGACACCCCAAAAATC

AGAGACGGTATCAGACTGAGTGGCATTGGGGGTCTACCTTTGTTTTTCTATGAC

ACACAAGGTGGGTGTTTGGCAAGAGAGTAGGGATGTTGGGTGCTGGATAGTGC

CTAGATTCTGTGTCTTTGGGAACTTTGGGGGAAGTGTCCTCCTAAGGGAGCCAA

AAAGGCTGCTACCCCAGTCCCCCAAATTCCAAAACAAAAGAGGTCAGTGAAA

GCTACTCGCATTTTGCATGTTATCTCTTTATCTTTGGAGCACATTGGGAAGGAC

TGACCCAGCTCCAGATCAGGGTCTGATTGGGGCAGGACAGACCCTCTCCAAGG

CAGAGAAGGGGAAAAAGAACTTGCCCTTAGAAGTGGGAGTCCCTGGGGGAGT

GGGACAGACCTTTTCCCAGAGCAGCCCTGGCAAAATGGCTGAGCCTCCCTTGA

GCCTCAGAAATAAATCGTCAGGCAGGCAGCAAACAAAGAAAGAGCTCTGTCT

CCAGCCAAGCACTGGCTCAGTGGCCAGTCCTTGGAGGACAAACAGTGAGTCCC

AAGCAGAGAGACGCAGCCCGGCCTGTCTGGCAGAGCATCTGGAAAGTGGGGT

TCCCCTAGGAACACACAGGAGAGAGGCAGCCACTCTGGAGGTAAAATCAGGG

ACCCTCTGTCCTGATGTGGGACACTTTATTTCAGGTCTGGAGGCTTGATGAGAA

GTGGGTCTCCAGGAACTTAGATGGGGCTGGGAAGGATGTCTGGAGACCAAGG

AGTAGGTAGCTTAGAGGGTAAAGATGAGGCAAAATCAAGTCCATCGAAGCCA

AAAGATGCACAGTGGCCACGTCTCTCCTGCCTGCCACGGATTTTACATGCCTGG

TGACCCCGGAGCAGCAGAGGCTATAAATTTTTTTACACGTGGCCCAGCACACA

GCAGGACCTCAATAAACCTCTTAGAATAAAGCCTGCATGGATGGAAGGTGATG

CTATCACTGGCATGGACCTACCTGCAATCTGTACCTGTCTCACTGAGCCAGGAA

GGCTATAAAATGTGGCCCCTTAGTCCATAGCCGGGAATGAACGAGCCCCAGAT

GAGGGTTTGCTTTGAGCTGGGGCATTGCCATTTCTGGCCAGGTGACCTTGGGTA

CGTTCTTGTTCCTGTTGGAAGGAAGGAATGGAGTACCCAAAGCTTCCAATGTG

CCAGGGATTATTCCTACAAGAACCGTGTGGAGCAGGCACTATTCTAATCCCCA

CTTCAGAGGCGATGAAACTGGATGCAGAGAGGGGAAGTGACTTGTCAAAGGT

CACATAGCCAGGAGGAGGAAGGGCAGGGATCTTAACCCTACTGTTCTAATTTC

CAGGCCAACTCTGTCCTATTGTGGGTCTCTGGGCCTCGGTTTTTCCATCTGTAA

AGTGAAGTGATGGGGTGGTAGTTCCCGTGGGCCTGCCATGGGAGCAGTGGCTG

GTCCTCTACCTTGGTGGCATATGACATGTGGAGAGCACCATGAAATAAATAAA

GCACCAATATCAGGCCACTTCTTGCCATCACACAAAGACTCCATTCCTTGGCTT

GGCTGTGAGCTCAGCAGCTGAGATAGAGTTCCATGCTACTTGGAATTTGGAGA

TGTCTTTTCCACCCTCTTGCTTGTCTATGGGTCTCAGGCAGAGTGTTAATGACC

GTTGATTCAGCCTTGCCACCACCTTTAGCCACCTGCATCCTGAAGTCCATCTCC

TTTGTCCTTAGAAGCCCATCCCTGTTGCCTAGCCCAAAATGTGCCCACCTTCCC

CTTGACTACCACCCCCGCCCCAGCAGCCCCTCTCCTCAGCCACAGCCCCAATGC

CAAACCCAGTTAGAGCCACTTCATCCCCTCTCTGCCCAAATGAAAACTAAAAT

GCAAGCGTCAGCAGGCCTGTCTATGCCTGTGTCGGGACTACCCCTTCAATTCAC

CCATGCAACGTCGCCTGAGCTTCTCTGTGCCTGGAGAATGGTGGGCAGGGTAT

AGCCCCGCTGAATGACAGCAACCTCGAATTGACTTCAGTTGCCAAGAGCATTG

GGCACTTCCCCCACAAGGTGAAAAGATCATCCTGTGTCCACTGCTCTGTGGCTT

GGCATGCCTCGATGCTGGCTTCTCAGACAGGCCCTGCACGGGTGGCTCTTCTTG

CCAACTTTACCCCATTCTCTGAAGGGCACTGGGAGATGGAGGGGAAGAGGTTG

GTGCTTGAAAAAGAAAAGATTGGGAGGCCAGATGTGCACCCCAAAATAGGAC

TTCCCAGGAAATGGGTCTCAGTGAGCATATTAACATCCAGCATCTTCATCTACT

CCATGCTCACCTAAGACCTCTGGGGTCAGGGGAAGGGGCTATGGAGTCAGTGG

GAAGTGGGCCAGTAGCAGACAGGAGAGTTGCCTGGCATAGGCAGTAGGTGTG

AATGCAAGCTGTGATATTTCAGCAGAGACCTGGAATTCAGTCATAAACAGAAA

AGAAAGAAAACACTAAATATTGTTCCAATGCAATTGTATCAAAAACAGTAATA

ACTGTCAGTTACTGCATGTTGGCAATATGCCAGGGACTGTGCTGTGTGCTTTAT

ATCGTTGCAGTTAATTTTCACAAAACCCTGTGAGATACGTAGCATAATGATCCC

TGTCAGGGTGAGGAAACTGAGGAGGAGAGGGGCTAAAGCACTTGCTGCAGGA

TCACTGGGCTGGTAAATAGCCTTAAAGCCCAAACTCAGTCACAGCATGCTTAA

ATCTCTGCCACTAAAAGCCCCTGACACTGCTTACTTGGCAATGACCATGAGCG

CCCCCCGCTGCCTCCACCCCAGTTAAGGAGCTGCACAGTTTACAAAGCACTTCT

TCATCCATTCCATCTGTGGAGCTTTGCCACAGCTCTGGGAGGCTGGGTGGCATG

ATAGCTTCTGCGTGATAACTGGGGAAGCTGACTTGCAGAGAGATGAATTTGTG

CCCAAGGTCCCTCAGCTGCTAGGTTGCAGAACTGGGGTTCAAATTGAGATGTC

CAGCCTGTTACAACATATTTGGGACCTAGATTTTAAATTCATTTGCAGCTAGTG

CTGATGTCATGCACTCAGCCACCTTTCCCTAACTCTCGGGTATCCCCCAATTTT

GTCCCCACCATCTTACTGCTCAGGAAGCTATTGAGAGTGCCCTGACAAAGCCA

TCAGCTGCATTTACCGTGATCTGACATGACACAGAAGTGGCATCCGATTCTATG

CAAGCTGCCTGCAGCGAATGGTCTTCTATTTTTACATTTCTCACAAATTGGAAA

TTACAAGGAGGCCAGGTCTTGCAGCGAGATTGGCTGTGGTGCTCTGAAAGCAG

GGGATGTACAACTAGGTGGAACAGGCCACTTTAGGATGCCAGGGGCACTGTTG

GCTGTGAAAGGGGCCCCCTGGCTGCTGGGGCTGGATGGGATCCTGGCACACAG

GCCAGGCTTGGGGATGCAGGAGTTAGTGACTGCCTTGGACGGTGACCTGCAGC

TCTGCCCACCTGTCAGCTGCCTGGTGTCTGCAAGTTCTGTGTCTCACGCCAGCT

CCTGAGATAACTGAGCCCTGGCCAGAGGGACATTCAAGGTCAAAGATGCCCTG

AAACATCGACAGCAGGTTGCTTTCATGGCTGTGTAGTTTGATAAGGAGATTCTG

TGCAGGGAGAGGGCAGCAGCCACAGGGAGGTGAGCAAAGACATTTGGTATCA

GTGTTGCGGTTTGCTGCAACTTAGCGCTGATCACGATACTCCCGGTCATCAGAA

CCGCACTTTATACTTTGCTAAAAATGCTCTTGCAGCTGCTTTTTCTTTTGATCAT

GTATGAAGTATTCCAGGGGAAGAAAATATGGTCCTTGAGATCCTGGAGAAGAA

TCCTCTCTCCAATAAAGAGTGATTTTTTTAAAGCTAGTTTTCTTATAAAGCCCA

AATCAGAATGACCTTGACCCAAATGAGGCTCCTTTGCACACATGTGCGTACAT

GCACACGTTAACACTTATGTAGAATTTCAGTTTTCAAAGTGTTTTCTCAGTCGA

AGCTTGTGACAGCCCTGGGAACTTCAAGTGAAGGTAATTTTATTGTTATTATTG

GTACCATTATTATCCCCATCCAAAAGATGAACCTGAGGCTCAGTGAGCCCTGA

CTCCTCATCTCTGTCCCCTAGTTTCCCAGCTGATGAAAATCACCTTTGCTAGGC

TGAAATCCAAGTTCACCAGGTAGCCCCTTGAGAAGGAGCCCAGATGAGGCCAG

ATCTGGTAGGAGGCAGAGCACAGAGCACTCAAAGATCGAGACTTACAAGAGA

GCGTGGGAAGGCACGGCTGTGAGGCTGGGGAGAAAAGGGCCGTAGCATCTTG

TAACCTTACCTGAGCTCTGGCCAGAGATTAACATCTCCTTGGTTATGCGCAGAG

TTTGGGGTCCAAAGTGTGATCATTTGGCCGGGGGGTCCCAGCAGTAACCAGGC

CTGGAGGGAGAGGAAGGGATGGTTGCTCTCACACACCTATGTGCACACAAGAA

TGCTTTTCTCGGTGAATTGGGGGAGAAATGAACACCAGGGACCCCATGAGATT

TCATAGAAGGATCTAAAGGTCTTGGTAGCCAGGAAGGTTGTGGGGAGTGTTTG

TATGTGAGCCTCAGCTCAGACCCACTGGAGGAGCAGGCTGTGTGCTTCTGAGC

CATGCCCAGGCAAGAGGGCAGGGATCACTCACCTCTGCCTTTGGACTGTTGCC

ATGCTGGAGATTGTGAAATATCATGAGTTCTAGGAGCTTCCCTCTGGGGTGCA

GGGGGAGGCAAGAGACCTGAGGGTTTGACAGTTCCCTGGCTGGATGGTGGGG

AGAGAACTTGTCCAGCTGGGCCCTGACAGTGAGTTCAGAGGGCCTGCTGGGCA

CTGGTCCCTGAGCCAAGGCTGATACTGGATGATAGGTGCTACTCTCTCCTTCTG

CCAGAGAGGACCCTGAGCCCAAGGGTGGTGGAGTCACTTGCCACAGGGTGCA

CAGACCAAGTTTGGTCCCAGGTCTTGGTGATGCCAACCTCATGCTGCTTCTGAA

AAGCACCTGAGACAAGATAACTGTTGGGGAGCCAGGGGCTTTTTCTCTGTATT

CGCCATTTCTGGCTGTGCCATGCAAGGGCTGAGAAAAAGTGAAAGCAATACAA

AATAAAAGTGGCCAAAAGCAAAAGCTACCAGCTTCCTGGGCACCGTCTGAGCT

GCCAGCCTGCAATCCAGGCTTCACTGCCGCCTGAGCTGACCAAGCCCCTTTCCG

CCCAAGTCAGATGTTTCCAGAGCTTAAGATCCAGGACCCACCTATGATTAAAA

AAATAATAATAAAAAGTAGGCCTGGGGAGAGAGAAGAAGTCATCTTACTGAA

GAGTATTTTTGAAAGATGGAATTTAAATGCACGCATGTATTACATATGCATTTT

TAGCAGGTTGGTTCAGCATAATGACACAGAATGATCTGATATGTACATCTCTCA

GTCTTGGGCTCTGCCTGTGACCTAGGCACTGAGCCAGTACTTCACAAGCATTGT

CCCAGGATCCTCAATAAGGAAACTGAGGCTCAGGGAGCTCAAGAAGTTGCCCA

AGGTCATACCACTGACACAGGGCAGAGCTGGGAATCAGATCCAAAGCTGCAG

GGCTCCGAGGCTTGGGCTTGCAGACCTGAGAAAGAAGTGGGAGAGCCGCCTTC

TGCTGGGCCTCAAGCCTGTGGGGGACCGTGGGCAGGTTCTTTCCCCTCTTGGCT

TTGCATGCTCAGTTCATACAGCCAAGAGGTTTGGACCATTGGATAGGATGATC

GGCTGCTCCCCGCACCAGGCGCCAGTTCTCCAGGCAAGGGGAGGGAAGTTGCA

CGCCCCACTTCCTCCTCTCCTCCTCCATTTCCCCCATAATAGCCTCCCCGAGACT

AATGACTGAGGAAGATAAGAGCCCTTTCTCCAAATGGCAGTGAATGCCACTAA

TGACAGAGCAGATGCAGCACTGTTAATTATTCCACGGGTATTAAATGAAAACG

CCAGCCCTGGAGGAGTTAAATAGCACCACTGATCTGGCAACGTCTGTGCTTAA

AAGTGATGCCATCAAACCGGGCCGGGGCAGAGCAGCCCCCTCCCCAAACTTGG

AGCTAGACAGGAGTCACAAAGTAAGGGTCCCCTCATTGGTCACAGACCCCTCT

TCAGCTCAGGGTCCGAAGCAGCTCCTTGGAGACATGGATCGGTGGCTGCTTTG

GGAGATTTTCTTAAAGGCCCCTGGGGGGCCTAGACTAGTTATCCTTAGGGGCA

GATGTGGACAAACAATTGGACCGAATAAAACTCTGAGCACCTACTGTGTGCCA

GGTCCTATTTTGGATTGTAGAAAGAAACTGGATGCTGTTCCTGCCCTTTGAAGA

TCCACTTGCATTGAAAACCGTAGATTCCTTAAGAGCAGGAATGCTGTGTTGTTT

GGAGTCTCTGGTCCCATAGTCAAGAACACAGAGTGGGTACCTCTGTATTACTTT

GATTATCTGCTGGTTCCTTGAATGAGAGCCAGAGGCCACACAGATTGTGGCCA

GAGTGAGATCGGTGGGCAGAGGGACTGGGCCAGGGTCAGGCTGTCTTCTTCAG

GTACAAGCAACATGACCTTCCTGGACAGCTTAGCCCCAGGTTCTGAAACAAGC

CATGACCTGGATGAAGAGCCGGGTTTTCCACTTGCTGAGGCACCACAGGCGAC

TCCTTTGCCCTCTCTGAGCCTCAGTTTGCACATCTGCAAAATGGGGAGGATAAG

GGCCATGATGGCTGCAGGCAAAACAACTGTCTCCTAACCAATCCTGGCCCTGA

ATCTGGAGGATCACAGAGACCTCATGAATGTTTGAAGAGTGACATTCTGGAGG

AAATTCAGCAGAGAGAGAGAGTGTAGTCACCAAAGGCAGCATCTGGTTCTTCC

TGTGGGTCTCACTCAAATCTCACTCCAACCCCCAATTCATCTCCAGCAGCTCAG

GTCACTTGTTCCCAGCTTCCTGGACCTGGAAAGCTGTTTCTCAGGTCTTTGTAG

GCTAGAGAACATCACAAAACCCACCCATTTCAGACACACCTGTGTTATAGATG

GGTAAACTGAGGCCTAGAGAGGAAGAGTCCTAGGTCTTGCAGAGGCTAGGAC

CCAGAGCTTCTAACCCCTAACCCAGTGCTCTTTCCCTTCCACCAAGCCAGCCCA

GCTCCTCCGTTAAGAAATTATTAAATAGAGTTGGTGGAAGGACTCTGGTTGCC

GTGACAACGGAGCCTGTGTCCTCGGGAGATAATGGCGTTGCACAAGGCAGAGC

GATGTGTGTTGAATGCTGATTCAGGACCTGCTAGAGTGAGAGTGAGTACAAGA

GGAGAGTGGAGGGGACCGTGTCTAGAACTCGAGCTTCCCCAAGAAGGAGGTG

CGGGACTGGTAGGGGTGGGGCAAATTGCCTTCTCTACTTCCATCCTTCCTACAG

CCTTGTCAGAATGGGATACTTCCCTGCACTTCGTGGAGGTGCAGAGTCAGGTG

AGGAGAGGTGAAGTGACTCATCCCAGGTCACACAACCAGGAAAGAGCCAGAG

CTGGGATTATACCTCCAACCAACACGGGGGCCTAGGCTATAGGGAGGGCCTGG

AAGGAGGCTCACCTGCAGGGGCAGGGGATGGTTGATGGAGACCTCTCTAAAGT

CTTAAGTGCTTGTGGAGGTCCTGATGGTCCAGGAATGACAATTCCAGCCCAAG

AGGAAGAGAAAACAGAATGTCAGCATAGCTAAAAGCATCAGCTTTGGAGCCA

AAGTCACATCTCTCCCTGCCACTTACTAAACTGTGTAATCCTGGATGAGTCACT

TTCTCTCTGAGCCTCAGTGTCCTCATCTGCAAAATGGGGATAATGCCGGCACCT

ACCTCATAGGATTGTGGTGAGACTGCATATCATGAGGAAGATAAAGCATCGAG

CCCCAATCCTGGCCATAGCTCAATCACCAGCAGCTGTTGTTATGAACCAAAGG

GGAGAGGCTGGGAAAAGGATCCACTCCCAACCATGTATCAGCTTGTAATTTGT

GGTGTTGGAAGAGCGAAACTGGCTAACACAGTACAAGCATGATACATAACAG

GGCTCTTCATTCCAGGCTAGTAAATATCACTAGAACAAATGAACAGCTCCATC

ACCAAAAATATACATAAATATTTTGCATTTGAAGAGTGTGAATGTTGTACCTCA

CTGCAAATGTACTGTCCTTAAAAACCTCCATGCACACCTCACTGAGGGAATGT

GTGTTTTCTCTGTGCCAGCTATCTAAATGACCCCATGCTGCAAACGCTTTTGGA

AAATAAACACTCTCACCCACCCACCTAGTTTCTCTTCTAGAAGCACAGCTGGTT

CAGTGTACTTACCTCCCTAATCAGGGATTGGAGAACATGGGGTTAATACCCTGT

GAAACCCCAAAGTGCATCATTTGCATGCTGGCAAAGATGCAGGCTGGCAGACA

ACCCCATTCCATTTTGGAAGCGGAAAGAGGGTGCTGGACTCAGGCATTTGGCT

GTTTGAAAAGCGCTGGTAAGAGCTGGGATCCTGATGATTGAGATGCAAGCAAC

CATTTTGTTTGTTTCCGCAAACCTGTTTGAGCAAATCCACCCCCAGAGATAGCA

ACCTGTTTCTGGGTCCCCTGAGAGAGAGGGTCATTTGTTTGCTGCTGTTTCTGG

GGCTGGATGCCAAGCTATGAGGCAATGAGCTGACGACCTTGGATCCTGACACC

CCTATTCTATGCAGGCAGGAGAAGGGTCAGGGAGACTGATTCAGTGAACAAAT

AAAGACGGTTGACATTTAACCAGGATTTTAATGGGTGTGCCAAATTGCAAGAT

ACATACACATGTCCACTTTGGGAATCATGTTTAATAAACAATTTGCCCTAAGAA

TTTGATTCAATCAGCATCTCCCAGTGACTCCCACCATTCCTTTATATTGATTGCC

TATAGGTTAAGATAACACTGGGCCTGGCGCAGTGGCTCACGCTTGTAATCCTA

GCATTTTGGGAGGCTGAGGCAGGCGGATCATGAGGTCAGGAGATCGAGACCAT

CCTGGCTAACACGGTGAAACCCCGTCTCTACTAAAAATACAAAAAAATTAGCC

AGGTATGGTGGCAGGCACCTGTAGTCCCAGCTACTCGGGAGGCTGAGGGAGGA

GAATGGCATGAACCCGGGAGGCAGAGCTTGCAGTGAGCTGAGATTGCGCCACT

GCACTCCAGCCTGGGCGACAGAGCAAGACTCCGCCTCAACAACAACAACAAA

AAAAGATAACACTGGCCTGCCTTCCACCCAGGACTTTTTCCCATCACTAACTAA

AAATATAAATGAAAGCCCTGATCAGGTGGTTTATTATAATATCTGCCAGGTATC

AAGCATTTACTATGTCCTAGATGCTGTACTAACTACATATTTCCCCATTTAATC

CTCATAGCAGCCCTAGAAGTAGGTGTTATCACTTCTGTTTTAGACAGAGAGAGT

AGATGACTAACCTACAGTCACACAGCATTGAACCAGAGCTGAAACTCAAAACT

GGGTGTCCTTGCCCCTAGGGAAGGACAAATTTTCTTTAAGTCCCACGAACAAT

GGGCATACTTATGAAGAGCTGCTGCTTGGGATATCATAGAGTGATCAGAAGTT

TGTTTGTCAGGAGCCATGATAGGGGCAGGCAGAAGAGGGCATTGCTCATTGTT

CATGGGACTTAAAGAAAATTTGTTCAATAAAAATCTGATTTTTAGCCAGACAT

GGTGGCACATGCCTGTAGTCCCAGCTACGTAGGGGGCTGAGGTGGGAGGATTG

CTTGAGCCTGAGAGACAGAGGTTACAATGAGCCGAGATCATGCCACTGCACTG

CAGCCTAGGTGACAGAGTGAGACCCTGTCTCAGAAAAAAAAAAAGAATCTGA

TGTTACCAAATGGGTAGACTGTTCTTGCTACATAGGAGTTTCCACTTTACAAAA

ACAAGGGGCTCTATACCATAAAATTCCTTTCCACAGCTAAGTGAGTGAGTCAA

GAACAGATGCACTGGTGAACGTGAGCATTCAAGTCTTTTAAAAATAAAATTAG

CCTTTATATTGTGGGAAAATATTTTAGACCCTGGAATCTGACCCTTAATTGACT

CTGAAAATACACGCCTCCCTCATTCTTGCAGACTATTCTAAAGCAGAGTCTCTC

TGCGGTTTTTTAAATCCAACTGAGCTTTTATTAAGAGACCAATAGGATTTGTTG

TTTCCCAAATATAAAACTATAACACCAAACATGCTTTTTCAAACTTCACCACCC

CCAAGAGTTTGACATATCCCCCAGGAAAGGGGCAATATTAACCCTCATCCATC

TTCCACTGCTTAGAGAAAGAGTTCTACTGAGTGACTAAGATTGTACCGTGTATA

CAAGGATTGCAAATTCAGGGGCCATTGAGGGCCAGACAGGTCACAGAAATGA

GTAAAGAGGTCAGCGATAGGATGACAGTAATGGTGGGACTTGTAGTAAACTAG

AAAACACATCACCACTTCCCTTCTCCAAAGCATGCAAGTTCAATTTCTAAAATT

CAAATCTGATACCACTCAATCAAGCATAGCTGTGGGACCCATTCGGCTCAAAA

CCACCACGTTGAGATCCCTGGTGGTTCTTATTGTGACCTTGTGTGATCTTGATC

CAGACAGTTTCTCTTTGGGGCTCAGTCTGATTGTCTCTAGAAGGGGCTGATAGA

GCCTGCAGAGCTGCTGAGAGAGAAATGCAGTCCCAGGTGTGGACACTTAATAA

GGAGCCATGGCGCTGTCTGCAGCCACTGTCACGGATATGCCTTCTGCGACCCT

AAGCCAGCTCTCTGTTTGGGCTGTGCTCCTGCACGTCACTTATTTAGTTCATCGT

TTAGTTCAGCCAGCCCTTGCAGTGTTATTTTTTCTTTTCCCCAAATCTGAATGCT

TAAAGGGCCTTTATCTTAATGCCCGTCGGAGCCTTCCAAGCACATCTTGAACAG

GAATGAACAGCTATTTTAACCCCACGGGCTTCTTACAAGTGTGTTTTAAAAGGC

AAAGAGCGATGTAAAAGAATGGCAAAAGGTCATCGTGCCGCTCTTATTTCCTG

GTCTAAGTCAGTCCTTTCTGGCCGGTTGAGCACAGGGCTTTTACAGAGGCGTCC

CCTGGAACTGAAGTCCTCCAGAAAGCCCCACACCCCTCAGAGGCAGGAGGAA

GAAAGGAGCCTGGAACAGGTGGTGAGGGTGCAGAGGGTGGGCACCCGGACCT

GTTCCACAGGGGCTCGCCCTCGTGCCCAGCACAGGGTTGGGTGCTCAAGCTGG

CAATGGGAGACAGCTGCCCTGAGTGGAAGGTTGAGACTAATGGGCTGGCCCCC

AGCAAATACAGTCAGAACCAACTGAAGGAAGTCAGTGGAACAGGCCCTGCAG

CCTGGGTTCAAAAATCCCGGCTCCTCAGCCGCTTTTGACCTTGGCCTCCGCCAA

CCTCAGGCTTCTCTTGGCTGAAAGGTAGGCTCCTTCCCTCCCTCCTTGGGTTCA

GTGAGCGACACAGTGGAGCCTTGCCCAGCTCACAAGCAGTGAATGGCGCATCT

GTTTTCTCTTCTGTCCTTCCTTCCTTCTGCCCTTATTTTTCCTCTTTCCCCTTTCTG

ATCCCTTCCTGCGCTCCTCCTTTCTTCCTCCTGTTTTCCTCCTCATCACTCTGCTC

CTTTACCCCAGCCTCCTCCTTGTCTGTTTCTCTCCTCCTCTCCTTCCATACCCTTT

TCTCTCCTTTCCCCTCCTTCCCACCCTCCTCTCTCTCCATCCCTCTCCACTCTTTC

AAATCCACCCAGGGAGATGCCTCTGAGTTCCTGTGTCTGATCCCAGGGTTCGAT

GTTCCTCATCCTGCAGCAGACGTCTCTGCGGGCACCCACCAGCCCCAGCATGC

GCTCCAGGGCCAGCCCAGGGGTTGACATCCAGGGCTTTCCTTACAGGAAGCAA

TCAAGAGAGTTTCCAGCATTAAACCACTCAGGAAGCAAGGCCAGTTTCTGCCC

GAGAGCATGCCTCACTGGCCCCACAAGAAGAAACAGGCCTAAGGCTCAGTGG

TGTGCTGGAGCCTGCTCACAGATGGCACATACCCAATCCTATGACCTCATATTA

GTACCTTGCACAGGCCACAGTGGGAGTGTTTACACCACAGAAATCAGCAGATG

CTGCAAATTGAGTCTTCTGTTTTGTTTCCTGAGAGCCCATTGTTAAAGCTTACC

AGCACACCATTGCTCTGTAGACCTGCCACCTTCTCCCAAGCAAACGTTTCTGTG

CTGAGGCCTTGTAAGAACAATGTCCTCTGCCATAGAGGGCAGAACCCAGAAGA

GCGCATGCATATCTAGCACTGTTCAAGCTGCCTGCTGTCGGGGAAGCAGAGCG

GGTGGAAGACCTGGGCCCCGTTCTTGCATAGGTGACAGTGCAGCTGGGAAGCT

AAGAGAGAAGAATCCGTAATTAGTCACGAGAGAAGCCATGTTGTATAGGGCG

ACCAGGCCTTCCTCCTCGCCCCCACCATGGTCCTGGTTCAGGCGCTGTCCCTTA

TCCTTGCACCATCCCTGTTACAGCAACCTCCACCCATGCTCCCTCTCCTCTCCTC

CTCCCCACACAGCTGCTGGGCGGTCTTGCCAAGTCACCATCACATCAAATCTCC

CCATCTTCCACGGTTCAGGTGGCAGCCCTGGTGACCACCCCATGCCCACAGTG

GAGAGGGATAGTTAAGAACATGGGCTTTGGAATCAAACTGCAGTGTCTGGGAG

CTGGGAAGTTGTTTTACTGTCTGAACTTCGCCTTCCTCATCTGTACAGTGTGCA

GTACAGAAATGCAGTAAATATATTAAGCACTTCCTCAGCCTTCGGTGTGATCAT

CATTCTTATTACAGGGTAAGATGCGCCCTCTTTTCCTGCATCAGAGCTCTTTACT

ACTACTGGCGGCTTCCTGGCCACGTTCCCCTAAATCACCCCAGATTCTAGCCCC

CAACAAGCCTCCTCACTGTCTCCAAATGTGTTCTGTATTTTGGCTTTGTTCTATC

TGCCTGGAATGGACCAGGAGTGGTTAGGGTTGCAGTTAAAAGCAAGGAGTCCG

GTCCTGCTCCTCTAGCTGTGATTATTTGAAAAATATCCCCACTTCTGCTCCCTTT

TCCACCAACCGCAATGCTACCCCTCCCAGTTGCCTGCCTGAACCCAGACCCCG

GGGCAGGGTGCCACCTCTCCTTCTCTGCGTGCTGTGGAAGAACTTACCCCGTGG

ATGTGGATTGTCCATGGCCCATCTCCCAGCTGTCCCGAGAGCTCAGTGGCAGC

AGGACTCAGACTCCAACTTGCATCTTTGTCCCTCAGCACCCAGCATGAGGCCC

GGCTCCCCGGAGGTGCACAGTCGATGCGGATCAGATTGAAAATTGCCTTGACA

AAATGAAGTTCTCAGTTGGGCACTACAGATAATTAGGGGCCAGAACAGACGTT

AGTAAACCACTGATCGTGTGAGCGAGACAAGCAGGACTGGATCAGAGTGAAG

TGGAGCTGGAAATTGGAGTCTGACTGTCCTGCTTGCACACAGAAGAAAGCTGA

GGCACAGAGAGGGGAAGGGACTGGCATAAGGTCACACAGCTAGTAAGTTTAG

AGAGAGATTTCAAACCTGTGTCTACTGGACTTCAGGGCGTCAAGCTAACTGCC

CTTCCTCCTCAGGTCAGCTCCTCGCAGCAGCTGCAAGGCACGAGCCATGTCTGC

AGTTGGAGGAGAGCTGGGGTCTCCAAGAGGGGAGTGCTGCCCCTAGAGGCTG

GCTGCCTCCTGGGGACTCTGATTAGAGCCTGCAGCCCCACAGCTCAGCCAGAA

TCCCACGTGACCTCCCGGGGTCTCCCTGGATACCTGTAGGCTTTGTCCAGCGCA

ACCGTAGAAGCCATTTTGGAGCCAGAACTCAGAGGCTCTCATGCTAGCAGCCT

CTCTTGGGTTTCTTAAAAAGAACAGCAGGCCTTGTCCTTGTGGAGGTTTGTCCT

GGAGGGAGGTGGCTGCTGGGGACGGGCCATACTTGTACACCCCAAATGGAGG

GTTTGGGGGGTTCCACAGGTACCCCATTAGAGACAGCAGAGTGGTTAAGAGTT

TGGACACAGGAGCCCAGCTGCCTGGGTCAAGGCTTGGCATCCTTAGCTCCTAA

CTGGGAGACCTTTGGCACGTCACTTTAACCTCTCTGTGCCTCAGTTTACCCATC

AATAAAATGGGGGCTAAAAGCAATTGAATAGACAGAAAGCATTTAGAGTGGC

CCCGGCACATTAGTAAGTGTGTCCAGCTCACTTCCTCCCTGTACTGAGCTCTTC

TTTTTCTTTCACCCCTTGTTAATGAAAAGTTGGGGAATTTTTCTTTTCTGTAGGC

GGCTGCAGAAGGAGAACCATCCTCCTGGAATCTGTTTCACCATTGGATGGGTC

TGTCATATTCCAGTAGTCGCAAAGTTGAATTTGTTCTCGCCTGGGTTGTCGGGG

TGGGGATGCCCTCTGGCCTTCTTCCACATTGTGGTTTCAGAGGTGTCCTAGAAA

ACCTGACTTCTAGGCTCTCAGAGGCTGAGAGCAGAGGGGAAGGGCTAGAGCTT

CCATTTATTCATCTGTCCTAGGACAGTGAGGCTAGGCAAACTCATCTCCATGCA

GTCACATTTCTCTCCCTCTGGCATCCAGCAGCCATGCTTGGGCAGATCAGATCA

TGAGGCCTGATAGGTCTAATGGTTTCAGGGAATTTCACACAGGCCAGCGCCTG

GGAGGCACTTTCTGGACAAGCAAATAGCACCAGACTATAACTCAGAGGGCCTG

GGTCAAGTCCTGTGTGACCTTGGGTAGGCCATTTGACGTCTCTGGGCCTTAGTG

CCATCATCTGCAAAATGGGACCAATGATCAGTTTAGCGTCAATGTATTTAGCTC

TTGCTGAACTGGGCACGGTGCTAGAAAATTCAAATCCAGTCATCACAGAGTGG

GAAGTGCTTACTATTGACCTCCTTTATTGATGAAGAAACAAAAACTTTTTTTTT

TTTGAGACGAAGTCTCGCTCTGTCATCCAGGCTGGAGTGCAGTGGCGCGATCTC

AGCTCACTGCAAGCTCTGCCTCCCGGGTTCATGCCATTCTCCTGCCTCAGCCTC

CCGAGTGGCTGGGACTACAGACACCCACCACCATGCCCAGCTAATTTTTCGTA

TTTTTTAGTAGAGACGGGTTTTCACCGTGTTAGCCAGGATGGTCTCGATCTCCT

GACCTAGTGATCCGCCCGCCTCGGCCTCCCAAAGTGCTGGGATTACAGGCGTG

AGCCACCGCGCCCGGCCAAAACAAAAACTTTGAGAAAATAAGTAGCTTACTTG

AAAATCACAGTGGGATGTTGTGAACAGTAGGCACGCGATAAATACTACTGACA

AAGACACCTGCTTCTGCAAATTCCCTCTCAGGTACGTTGAGGCAGCTGAGGGG

TTTATACCAAGGCATGAAACTGCTTTCCTCCATCTCTTGAGTAAGAGTATGCGG

CTGGCTGGGAGTTGGGAGTCCATAAGGTTCAGGCCACTCTCAGGCTGTGTGAC

CTTGGGCCAGCCACCAGTCCTTGCTGGGTTTTTTTCTTCATCTATAGAATGCCAT

GGTAGACCAGGTGCACCAGTTTGGCATGCAGCAAGTCTGGACCAGGAAGGAG

ACCAGCTGGGTCGTGGCCCTAAGCACCACTGTGCATGGGGCTGAGGTCCAAGA

AGGGAGGCCTCCCATTTAGACCCCAAGCTGTCCCAACCAGCTCATTTCTCCTGT

AGCCTGCATCCCACCACAGGCCTGGCTGGCCTCTGAGGTCTTGTTAAAATGCTG

GGCCTTGGAGACCCTCTCTGGCGGCTCCCTTTCCATTAGAGCACCTTCTCCAGG

GAGCTTTCCCTGACCTCCAGGACAGCGCGTGGCAAGCACTGCCCACAGCACCG

GCTGTCCCACACAATTCCAACCTCCCTCTAACCTTCTGTACCTGATCATCCACT

ATTAATTGGTCTTCACATTTGTATAACATTTTTCAATTACAAAGCACTTTTGCGG

CCATTCATCTCTTTGAGTGGCAGCCTAAGCTGCTATTTATTGACACTTCCTGTGT

GCCAGGCCCTTGGCTGGCCTCTTTCCATGCATTATCATGCAATTCTCAGAGCAA

TTCTGTGAAATCCACATTTACAGATGAGAAAATTGGGACTCAAGCAAGCCAGA

TATGTAGCCCTATTACACAGAGATTTGAACGAAGTCCCATGAAATAATCTGGG

TCATTCCTAACAACAGCTTCTGAAGCTTTCCTATGTGCCTAAATCACGGGACCT

GTTTAAAAATCAGCTTCTCGTTCCACAGGTCTTGAGTGGGGCCCAAGATTCTGC

ATTTCTGACAGGCTCCCAGGTGATGCTGATGCTGTCTCTTGAAGAACCGCACTT

TGAGAAGCAAGGCCCTGAATCACTGGGCAATTTGAGACCCCAGGCTGGCAAG

GCTGTTTCCTTATCTGTCAGATGGGTGAGGTAGCCCTGGCTTACATAGATGGAA

TGACTCCCGGATGATATAGTGCCTGTGAAATGCCTGGCACACAATAGGCGCTC

AATAAATGCTAGCTCTTCTCCTTCGATAGGAGCTGGCATGAGTCTGGGTACACA

ACGGATGCTCAATAAAGCCTTGTCCAATGGATTGAGCCTAGCATTTTTCCCGAA

GGCAGAGAATAAACTGTATTATGTTTCCCCTTGGAATGAAGCCAAACTCAATA

TTAATTTCTTTCTTAAACCACAGAGTTTCAAAAAGAAATAAAGGCAAACTAGA

GTGATTGCAGTCTGAAGCGAGCCTCTGTAATTTCTGTAATTACAGAACGAGAG

CAAAGCAAAGCAGGCATGAGCACAGTCTCATGGAGGGAGTGGGGTGGGGATA

GGGGGAGGGGAAGAGCAGGCCCTACTCCCAGACCCACTACTCCCCGTCCTCGA

TTCCCTGGCTTTTCAGGGCTCCCCACTCACTCCATGGCGGGGCCATGGCCTGGG

ACAATGGCAGCCCTGAGACTCCAGCCCTGGCCTCTCGGTCTCTGCCTAGGTCCC

CACCCCACGCAGCCGCCTGTCCCTCTGATGCAGTGGGACAGAGGGAGATTGTC

CAAGGCGGTTTCCACACACTTCCCTCCCCCTTGCCTGCCATCTGGTCCAGGGCT

GGGCTGCTCACAGCCAATCATCCAGCAAGGCCCATGGCCCACGGGCCCCACCC

CCAGAATAGATCCTGGGTGTCGGTGACACTTCCCCCAACTTCTGCCCATCCAGC

CACCCCCATGCAAGCCACCATCATCTCTCTGGACTGGCAGCCTCTGATCATGTA

TCCCTGCTTCCCCCTTGCCCCTATGCAGCCTTTTCTGGGCAGCCAGAGGGAGAC

TCTTAAACTAACCGTAAGTCAGATCTCCCACCACTTCAACCCCCACTTCCTCCC

ATCCCACTTAGAATGAAACCTGAATCCTTGCTGTGACCTCTGAGACCTGTGTGA

CCTGGCCCCTGCCCACCACCTCTGCCTGAATCCATTTCCTCTCACACTGGACTC

CAACCACCTTGGGCACCTTGGTGTCTTTCAAAAGTGCCAGGCTCCTTCCTGCCT

CAGGGCCTTTGCACTTGCTGCTCCCTCTGTTTGAAATACTGTATCCCAGAGAGT

CCCATTTCTGGCTCCTAATTCAGACTGAATTTTTTTTTTTTTTTTTTTTTTTTTTTT

GCTCAGGCCAAATAAAACAAGCCCAAGGGCCAGTTTTGACCCATTTTGCCACT

TCTGATGTAGAGCTTCCAAACTGTACATGTTTTCTTTCCCTACAAAGCATCTTGT

CATTCAGAGATTGGCAGAGGAATTCTTTGTGCTTGGTTTGGCCCCATTTTTATC

AATGGCTTTAACACTTGGTCATAGAGCATCCACTGAGGAAGGATGGGATATCC

TGGGAATGTTTTACCATCTTCCTGCCTGGAGTGTCTTTCACTAACTCCGTCCTTC

CCAGAGGCCCCATGAGCGAGTCATCTTCTGGTTTACGAATCTGATCCCATTATC

AGCCCTGCCCCATCCCTGCAGCTAGTCCCCAGGTTCCTTAGTCCGGTGTCCAAG

ACCCTTCATATACTGACCTTCCTAGCTTCACACTGTATACTTTAGTACATGTGTT

TCCCTCCACTTGGAACGCCTTTTCTCTCTTTTTAAACCTGATGAACACCTATTCA

ACCTTCAAAGCCTTGCTCAAAGGACGCCTCGCCTAGGATACTTTCTCTGGCTGT

ATTAATGCGTTTCTTCTAGGCCTCCCGTGTCTTGTTCTTACACCTCTTGCAGCAC

TTATCACAGGGTATTATGATCAGCTATAGGCAGAAATATTTCTCCTTCTAGGCC

ATGACCTTGACAAGGGCAAGGGTCTTGTCTTATGAAATTCTCACACCCTGAGTG

TCCACCCCAGCTCCACCCACATAGGAGGCCTCAGTAAGGATTTACAGAAATGA

ATCTTTCTGCTGGAACCTGTTAGTTCACTGATTCGGATTCTGGTGACCAGGGTG

TAGTTCAGAATACAAAATGCTTGAAGAGACGGCTCCATTGTCGTTTCAGCACA

TAGCATGTGCTCAGTAAATACTTGCAGAATGAATGTGTTAGTGAGTGAGTACA

GAGGACCTGACATGTCATCACTGGTGGTCTCACGTTAATAGTGCCCTTTGAACG

TGGCTTTGGTGGAATTAAGTTAATCGTGGTATAGTATGCACCTGAAGGAATGT

GGAGCCGCATGCTGGGTCTGCAGAATGTTGTCACCTGATGAAGAAGAACTGGA

AGACGCTGGAAGAAGGCTTTGGAAAAATGCCCAGATCATGAGAAGGTGAGAG

GTGTTTCTTCCGCCTGCTCCACCAGCAGGTAAAGGAGGCTGATCACAGGCTGG

CACTCAGGGCGAGGTGGAAGAAATGCGTGGTCTTGCCACTACAGACCCGCAGA

GCTGTCCTCCAACATGATCCAAGAGAGCCACAAAACACAGGGGTGTGTCTTGG

GAAAGGAAAAGTAACAACTTGCAAAAAGGTTGGTTCTGCTTCAGAGAAATGTT

CTAGAAAGACAGCCAAGTTACCCTGCCGTGAACTCACACACACCTGTGCTTTC

TGCATGTTATGTTTCATAGGAGAGGGAAGTGCCGGGAATTAAAATTGCGGATG

CACTGTTCCCCAGGTGTGTGGGTGAGGCACGCTTCCTGGGACCACTCCATTTAA

GCATTGGGTCCCAGGCATGTTCACTACTAGCAGGAGGCTCTTTATCAATTTCCT

CCCGTGGACCAGATGATTGGGATTTTTTTTTCTACCAGACTTTATTACAGAATA

ACTATTTTTGTATTAACTAAGCAAACAAATGTACCGCTCCCAGCTTCTGACTGG

CCCAAGGTGCTGAGCTAGACCTCTTTGCTTTGGTACAAAGAGAGCCCAAAGCC

AAGAGACTTGGCATTTCTGTTAGCTGGTATTCCCCAGGGTGACTAAAAGGATG

ATTTCCATTGGATTTTTTAAAATGCTGAAGACACTGAAAGACCTGAATAGTAA

GCCCGTAATAAACGTTATTCGGTATTATTATTGTTGTTGTTGTAGCCTTCTGGG

AAGTTGCGAATGCCAAATTAAGAACAATGGACCCATACCCAGATGGTGGCGAT

TGCAGTGATAACAATGGCTGAGATTTCCTAAGCCCTAACTATATGCCAGGCAT

AGTTGTACATGCTTTACACACATGATCTCATTTAATCATCACCATAACCCTCGG

AAGGAGTTATTATTATCTTCCCATTTTATAGATGAGGGTCAGAGAGGTGAAGT

AACTCACCCAGCATTAGTAGGTGTTGGAGGGAGCCGAGGTCTGTCTGACTCAG

CACCAGGGTGGCTTTGCTGCTGTGTAAGTCAAAGAGCCTTCTCCGTTCTAAATC

CTCAAAGAAGTATGAGTTATATCAATTCATGGCTTTACTTTGTTTATCTTCATAT

GTGCCACATCTTCTAAAAGGAAAACTCCAAATATAGTCATGTGGCTCCTCCTAG

GGAATTATAGGCATTTGGCTGAATCTGAATACATATCCCCGTATTCCAGAAATT

GGAAATCACATGTATGTCTGCTTTATGGTGAGGTCTTCAGAGCAGCCACCGTCT

CTGATCGTTCAGGGCTTGGCATGAGGAGGTGCTCAATATAGTCATGGAGAAGA

ACTCATGGACTGAACTTCCTGGTGAACCAGAGGGATGGTGCAGAGATTTAAGA

TTGCAACAGGAACCCCCAAGCCAATGTAAGCAAAACATCTATTTCATGACTAT

GGGAATGTTTTGTAAAATCCAAGCACCAGCCTGAAGAAAGCCTGGAACTGCAA

ACTAGGAGCTTTGAGACACTCTATCTTTTGATGTCTAGTTTTCTCCTTCCTCAGC

AGACCAAATCTCACTCTGAGTACAAGATGGGCAGAGGATGGCTGCCCCAGAGT

CCCCAAGTCACTAGCAAAACTGCCCTTCTTCCTGGGTCCCATGCCAAATTCCAG

AGAAGGAATTTGATTGGCTAGCCTGGGTCAGGTGGTCAGTCCTGATTCAAGGT

GATTGGATCATTTTGAACAAATATGGCAGCTGGAGCCCACAAGGGTAGATCAT

GGGACCGTTAAGGGCATCATTGTTACCAGAGTTAATGGCTGAGGCTGCTCTCTC

CTGCGTATCCCAAGAGTCCCTGGTAGACCACAGAATCGGCCGCCTCCTACTAT

CCTGCCATTCTCCAGAAGGGGAGGCAGGCACCCTACTGAGTCCAGAGCTCTCT

CTAACATTGCCATGCCCAGGCCAGGATCTAGATGGGCATCCAAGACCAGCCTG

TGTGCCGTGAAGGAGTTCAAAGTGAGCAGGCCATCCCTGAGAACGAGGAGCA

GGGGTGTTAGGAAAGTTTGGATTATCTGAAGATGGCCTGGTTCTCCGTGAACTC

TGGGAGGAGATGATGGTGTTTGGTAATGGGGACTGCATGCGAGGGGCTGGAG

GGCGAGTGCACAGCAGACTGGGCCCCCGCCGGGCAAAGCTGCGGGGAGGAGG

CCGCGTGGGGAGGGCCAGGGCCACGCCGCACCTTGCTTTGGCGCAGCTCCCAG

CTCTGAGGGAATGCAGACCGCTTCACATGTTCTGGTTTGTTCTGAGGGTTCCAT

AAGTTTATTTTTTGTTATATTATTAATTTAAATCAGTTTTGTAGAGCATTATAGA

AATTAAAAAAGCACAAAGAAGAAAACGAAAGTCACTCAAAAACTAAGCATTG

TAACCATCTTTGGGTATATCCTTTTTTAAAATAGATGTGGGGGGTGGGGGCAAA

ATGGGATAATTCGAAATAAACTGTCTAGAAACCTGCCTTTCTCACCCCAAAAC

AAGGTGCTCTTTCTCGTGTTCTTACGTGGTCTTTTTGTTACATTTCCACAAGTTT

GGATGGAGGACAGAGAAAAGTCTCAGCAGCCCCTCTGTTGGTTTCTCTTTTCTT

CTGATAAAATTATGGAATGTCTTCTCCACGCTGCCCTCATCCCACGCCCACAGC

TGTCCCGAGGGCAGCGGGCCCCACTCCTGCATGTTCCCAGCACCCCAAGGGCC

AGTGCCATGCTGCATGGTCTCGCTGCGGCCACCCTGGCTGGGGACGCTCTGCG

GGACACGCGCACAGCCCAGACGCCCTGGCAGGCTTCCTGCACCCCTGCTGCCC

TCTGTAGGAGCTGCCTGCCTGCCCCATCGCTGCTTCACCATCCCTCCTCTCCTCC

CCTTCTCTGCTTTGCAGGATCTCACCCAGGGTGGCAGAAGGAGGCCTTCTGGA

GCTGACCCACCCCCGACGACCATCAGGTAACGTCAACAGCCCCGGTAACAACA

TCCATGAGTGGAGTTCTCTCTCTTAACCAGGTTGGCGAAAAGCACTCTTGCAGC

GATTACAGCTGAGTTTTGATGCAACTCTACAAGCCAGAGGGGATTGCCCCCAT

TTTATACATGGGGAAACTAAGCACACACAACAGCAGTACTTGGTAGTAGTATT

GAGTAGTAGTACTGAGCAGTAGTACTCAGCTGGTAGAAGGCAGACCGGAGATT

TGAACTCAGGACTAGTCTCAGCCCAATTTCCTCCACTGTACTGTACCCCACGCT

GCCCCTGGAAATAAAAAGGTAAGAAGGCTGCAGAGTGTCAGTAGAATATCTTA

GGGTGATTGAAGGCTGAAAGAGGTCATCTAAACCAGTGGCCCTTAGCTGAGGG

CCAGGGCTGGGCTACCAGTATAAGAATCTCCTTTGGCAGAGCTTGTTAAAAGT

ATAGACTCAGGACTTCCTGCAACCCAGTGAGAGATGTACCAGTGTCATCCTCA

TTTTAAAAGATAGGGGCACACAGAGGTTGAGTGTACTTAAGGTCACACAGCTC

AAAAGTGGTAAAGTCGGGGCAGGGTGCAGTGGCTGGCTCACGCCTGTAATCCC

AGCACTTTGGGAGGCGGAGGCGGAGGCGGGTGGATCACGAGGTCAGGAGATC

GAGACCATGGTGAAACCCCGTCTCTACTAAAAATACAAAAAAAAAATTAGCTG

GGCGTGGTGGTGGGCACCTGTAGTCCCAGCTACTCGGGAGGCTGAGGCAGGAG

AATGGCATGAACCCGGGAGGCAGAGCTTGCAGTGAGCTGAGATGGCACCACT

GCACTCCAGCCTGGGCAACAGAGCAAGACTCCATCTCAAAAAAAAAAAAATG

TGGTAAAGTGGACTGTGATCAGAGGTCGTAACTCCTAGGCTCTGTCGTCTTCAC

CCAGACACCTCCGTAGTGGGGACACCTTTCTCCCCATTCAGCCACAGGTGGAG

CTTCTTTCTTGCTCACCCTGGCGATGGCTAGTATGGCCGCAGAGTGTGACAAGG

CTCCCAATTGCATCCTTGGAGGCAAAGTTAAAGATTTTATGATTAAGTACTTAA

CATTTCCTGTTGGAAGTTTGGTGGCCTCAGATTTCTCCTCTGAGCAGGACATAC

AACAGAGGGTTGGACACAGATGGCCAGAGTTGAAGCTAGGCCAGGAAACATC

GCTGTGTCTTTGGAAACAAAAGTCACCTTCCTTCCTCCAGGAGCAAGGGCGGG

TGAGAGGAAGAGGGCTTATAGAGAGCCTTTTCAAGCCCCTAGCTTGTTTATGG

GTTATTTTGTTTTTGTCATTTTAGGGTGAGGCAACTCCAAGGTCCTACTCTCTTT

CTGTGCCTGTTACCCACCCCGTCCTCCTAGGGTGCCCTTGAGCCGCAAAACTGC

TGTCCACGTGGACCGGGGGTGACATCGCACGTCCATCTGCCAGGACCCCTGCG

TCCAAATTCCGAGACATGGCGACCAACGGCAGCAAGGTGGCCGACGGGCAGA

TCTCCACCGAGGTCAGCGAGGCCCCTGTGGCCAATGACAAGCCCAAAACCTTG

GTGGTCAAGGTGCAGAAGAAGGCGGCAGACCTCCCCGACCGGGACACGTGGA

AGGGCCGCTTCGACTTCCTCATGTCCTGTGTGGGCTATGCCATCGGCCTGGGCA

ACGTCTGGAGGTTCCCCTATCTCTGCGGGAAAAATGGTGGGGGTAGGTGCTGG

CCCGGGGACCTCCTGGCTGGGTCTGGACCCTGCAAAAAGGATCCTGCTTAGGC

ATTTTTCTGGGGCAACTGTAGGAGAAAAGCCCCCCTAAAGACTCCAGAGGGAG

CACCAGGCCCCATTTCTTGTTTCTTGTCCCTTTATCAAAAGATTCCTGAAGGAC

AATTGTCCTCTGTTAATGATACCAAGAAATTCATCTATTCACCTGCAAAAAATG

GGATGTATTTCACATATGGCTTTCCTTGGGCCTGTGGTTGGCTGTGGGTCCTGC

TTTTGAGGCAGGTATAAAGAAATACTGGATGGGGACCCAGACCCACGACACTC

CCTCTACCAGGGCGGGTCTTGAAAGTCTTGGGGCCACGAGCTCTGTCTTCTTTA

CCTGCAGGAGCCTTCCTGATCCCCTATTTCCTGACACTCATCTTTGCGGGGGTC

CCACTCTTCCTGCTGGAGTGCTCCCTGGGCCAGTACACCTCCATCGGGGGGCTA

GGGGTATGGAAGCTGGCTCCTATGTTCAAGGGTAAGTCTGCAGATCAGGGACC

TGGGTGGTGGTGCCCCTGCCCACACACACCTCTTGCCCTCTGGAGAGAGCGAG

CTGTCTACCCAGACCAGGGCTGACATCACATATCCATCCACCAGGTGCATATCT

GAGCAGAGTGAGGGCTGTTGCAAGGGGCCAGGTGGCAGATGACAGTGCTGGT

CAGAAGCTAGGGAGGTGGAGGTGGAGAGGCCCAATCCAGAGTCCCCCGCACA

TCAGCCTGCTCTGTCCTGTTAGCCCATCTATTTCCTCTGCAAGGGCTGGGTGTG

ACTTTCTTTGCCTCGTGCTCAGTGCCTGACAGGCACCCAGGTAGATACATGGTG

AGTCATGCTCACTGACCGAGGGCCTGGGCCTCACCTCGTCCGGCAAGCTGAGA

CCCCAGCCCTGCCTGCCACCAGTCTGCACTGGGCCCCAGACATGAGTCCTGGC

CCCCTGGCTTGTCAGATGTACATGACCTTCAGCAAGTCACTGCCTCTCTCTTAG

CCTCAGTGTCCTCAGTAATGCCTGTCTCCGAGATCAATAAGATTGTAGAGGTCA

TGGAACATGCCACGACAGTTCATGTTAATTCCGTATTAAATGGACGCGTGTCTG

TGTTCCGCTCCCAGGCGTGGGCCTTGCGGCTGCTGTGCTATCATTCTGGCTGAA

CATCTACTACATCGTCATCATCTCCTGGGCCATTTACTACCTGTACAACTCCTTC

ACCACGGTGAGTGGTCCCTTTGACCACCCCATCCAGCAGAAGCCACACCCAGA

GCCCACCCTTGGCCCACCACGGCCACCACTCTGCTGACAAGGCCATTCCTCCA

GGAGTGGGGAACTTGGTTTGTCTTTTGTGGGCCTTGGTCTCCCCACAACAGCCA

TAGCCTCACAAATTTAGCATCTCCTGTGAGACAGGCACTCCTTGCAGGCTCACA

TCCAATCCTCATGACACCCCTCTAAAGTGCGTATTGTTAGCACATTTTATATGG

ACACAGACAGCCTGGGTTCAAATCCTGACTCTACTATATACCAGCTGTGCAAC

TTCAGAAAAGAGACTTAACCTCTCTGAGTCTCAGTGCCCTCATTTATAAAATGG

AAATAAAATTAGAACCTGCCCCAGTTCCTCAAAAAATTAAACATAGAATTACC

ATATGATCCAGCAATTCTGCTTCTGAGTAGATACCTGAAGAATTACAAGCAGC

TCTCAAAGAGATATTTGTACACTCCTGTTCACAGCAGCACTATTCACAGTAGGC

AAAAGGTGGAAGCAAGCCAAGCGTTCACTGATAAATGAATTGGTAAGCAAAA

TGTGGTCTATACATACAATGGAATGTAATTCAGCCTTAAAAGGAAGGAAATTC

TGACGCATGCATCAACATGGATGGACCTTGAGGATATTATGTCAAGTGAAATA

AGCCGGTCACAAAAATACAAATAGTGTTTGATCTTGCTTATATGAGTTCCCTAG

AGTAATCAAATTTATAGAGACAGAAAGTAGAATGATGGTTGCCAGGGGTTGTG

GGTAGGGGGATGAGGAGTTGTTGTTTAGTGGGTGGAGTTGCAGTTTTGCAAAA

TGAAAAAAGTTCTGGAGCTGGATGCTGACAATGTTAGGCAATACATTTCTCAC

AATGTACTCCCATTGTTAAGTGACACATGACTGTATTTGCAGAGGTGTGGCAG

GGTTCAGGGAGCTACATTATGGGTATACTTAATGCTTCTAAATTGTACAGGCAC

CAATAGTTAAGATGGTAAATGTTGTGTGTATTTTATCAAAATTAAAAAATGAA

AGAAAACAAAAAGTGAAACCCACCTCATAGGGTTCTTGTGAGGATTAAGGAGC

AGATATGTGTAAGGAACTTAGAACTGCGCCTGGCAGAGAACAAGCCCTGTAAG

TGTTTGCTGGTGTCGTTGCAGTGAGAGTTAGATGGGCAAGGTCTCTTGCCCAAG

GTCACATGGTGGGGAAGTAGCCCCGGCTGCAGCTTGGAAGGCTGTTCTCACAG

TGAACAGGTTGAGGTTCAAATCTCAGCTCTGGCACTTAGTAGCTGTGCATTCTC

AAGCAAGTCACTGTCCCTCTCTGATCTGTCAAATGGGGATCATAATGCTACTTG

CCTCCCAGAGCTGCTGCGAAGGGGAGGTGTGAGGAGGCTTGGGGACCATGAG

GGTGGGCAGGGTGCACTGGGACCACTCATGCCAGCCCCTGGGAGCCCAGCAG

GATGCGTCCTCATGACTGTGTCCTTGCAGACACTGCCGTGGAAACAGTGCGAC

AACCCCTGGAACACAGACCGCTGCTTCTCCAACTACAGCATGGTCAACACTAC

CAACATGACCAGCGCTGTGGTGGAGTTCTGGGAGTGAGTATGGGGCCATCAGG

GGATGGCAGGGACGGGGCATGTCAGCCAGAGCCCGGCAGGAGACAGAGGCCC

CTCGGAGGGGTAACAGAAGAGGTGGATAAAGGGGCTACAGTCATGTGTCACA

TCATGACATTTTGGTCATCAGTGGACCACATATGCAACGATGGTCCCATAAGAT

TATAATACCATATTTTTACTTTACCTTTTCTACGTCTAGACATGTCTAGTACCGA

AATGCATTGTGTTACAGTTGCGTACAATATTCAGTACAGTCATGTGCTGTACAG

GTTTGTAGTCTAGGAGCAATAGGCCACACCGTATGGCATAGGAGTGTAGCAGG

CTAGACCATCTAGGTTTGTGCAAGTACACGCTATGATATGCACACAACAACAA

AATTGCTTAAGAACGCATTTCTCAGAACATATCCCTGTTGTTAAGTGACACATG

ACTATACTTGCAGAGTTGTGGCAGGGCTAAGGCAGCCACAAGGGATGCTGAAG

ACCTTTTCTCCCCAAAAAGAGTTGGACCACAGATAGAGCCATGAGACACACAA

ACTCTGACCTTTGATAGCAAAACCCAGCTACTCACAAAGCCAAGGTCCAGCAG

GAAGAATACATTCCCCAACATCTCTGCCTCTAATCTCCTGCCAGCTCCTCCCAA

TGGTCAAACCCAGCTAGAAGCCAGAGGGCAGGGGACCAGATAATGTGGCCAT

AGTCGTCAGCATCCTGGGGCCTGGCACAGAGTGGTCAAGGGGAAGAGTGGATC

AGAGAGGCAAACGGAGGGGAATAACCAGCATAGCCAATTAGAATCACCTGGA

GAGGTTGACCACATCCTGATTCATCCTGAAAACCAGCCTGATCTACTTACGAA

CCTAGAGTCACTCGGAATAAACATGCACACACACTCTGGCCATAGTGAAGGCC

GTGCTGATTGTATCTTCAGGGGAGTACCTTGTAGAGATGTGTGTCGGGGGTCAC

TGATGTGACACAATGAGGTGCTGGGGCCCAAATCTCTCTGGCATGAAGGAAGC

ATCCATGCTGGACAGCTCAGAAGGATCCGGGAGCAGTCTAGAAGGTCTCAGAG

CACCTTGGATAAGCTCCCAGGAGGAGCTGTTCTCAGGCACTGTCAGAATCAGA

GTTGCCTGATCTCTTCATGGCCAATGTCCATGGTTACACTGTTTCTTTACAAATA

AAAATCAAATTCTGCTCTGGTGGGTTCTGCTTTCTGGCATCTCCCGCATGCTGC

TCTCAGGCACGTTCGGACTTTTCATTCAACCATTTATTCAGTAAACACTTCTTG

ATCTCCTACTATGTGCCAACTGATGACAGGCACGGACATGCAGACAGGCAATG

GCACCACCAGGCGGCCAATGCGGAGAGGAGGGAAGCCCAGGCAGCTGGGCTG

CGGGGACCTCAGGGAGTCCCCTGGACCACCCCAGGTCGGGAGTGGCATCTAGA

AGAGAAAACTGCAGGGCAGGCAGGTGCTGGAGAGAAACAGTGTGAAGGCTGG

GAGGCAGATGAGGTCAGGCCAGGCACCGGCAAACAGCTGCAGTGAGTGCTGA

GAGGAGAGAGAGATGCAAGGGCCTTGAAAGGCTGGCCAGGGAGTTTGGGCTT

CATTCTGAGAACAAGCGGGTGCTATTCATCGCAGTAAGCAGAGGAGTGACATG

ATCAGACTTGCAATTTAGAAGGCTCTCTGGGGCTGGAGAGGGGATAGAGCAAA

ACTGAAGGCAGGGTCCCACTAGGAGGCCACTGGGCCATCCAGATGAGCAGTA

GTGGTGGCTGACTAAGGCCCTGGCAGGTCCTGTTCTCTTTGATCTCTTGACCAA

ACAGCACTTTCTACTGATTAGTATAATGGGCCTCACAGTCCCCAGCTACCTCTT

GAATCACGGTGATGACGGTGTCAGCACAGTTCTAGGGCGGGGATATCATCTGT

GGCCATGCCCTGGGCACCCCTGGGTCACTCCGGCTCCCACCAGCTCTGTGTAAC

TTTCTCCTCCCTCCACTGTTTGACCAGGCGCAACATGCATCAGATGACGGACGG

GCTGGATAAGCCAGGTCAGATCCGCTGGCCACTGGCCATCACGCTGGCCATCG

CCTGGATCCTTGTGTATTTCTGTATCTGGAAGGGTGTTGGCTGGACTGGAAAGG

TAAGGGATATATGTGCACAGTGGGGACAGGAGGGCACTGGATAGAGGAACAG

GTCTACAAAGGAAGCTCTGCCCACAACTCCCAAGAGTCCCATCCAACCTTGAA

TTAAGCAGGTCCCTTCAACACTCGGTGTGTCCGTTTCTTTTACTGTGAAATGGG

AATAATAACATTACCTTACCTTCTGGGGCAGTTGTGAGGATAAAAGGAAATAG

TGCAAGTCAAGTACGCAGCACAGTGCGAAGCTCACAGTGAGTCCCATGGGATT

CCACTGGGCCTAACAGAGTCATCTCAAAACAGAGGGCAAATGTTGCTCCTGGA

CTGTTGAACAAACTTTTCCAGAGCATCTGCTCTGCACCAAACCTTGTTCTAGGT

GCAGAAACTCCAGAGATGACAGCTTCACATCCTGACCCTTGAGGAACAACAGA

GATGAATAGGTTAATGGTTAGATTCAGGCTCTGTATTAAGCAGTCCATGAGTTA

AGATCCCAACCTCACCATTTACTAGCTGTGAGCTCAGGCAAGTCACTTTGCCTC

CCTGGAACTCAGTTTCCTTCTCTGTAAAACGGGGCTAATAATTATACTTATCTG

TTGGTTTATTATGATAATCAGATGAGATGGTGCTTTTGTCAAGCGCTCAGCACC

CAGCAAGGAGAGTTGTTATTACTCTTGTTTTTATTCTCAGCAAGTGCAGAGCAA

ATGCCAAGCTGGGATGTCTAGCCCAGGGACACCCGGGAGGTGGAAAACTGGC

CTTACTCCACCTTTTCTCCCTTTCAAGCCCTACCCCAGGAGCCTGGGGGCAGAA

GATGGCAGTCTCTCACTGGCCCTGACCTGAAGGGAACCCGGAGGACAAGTGTG

TTGTCTCAGATCTTATGGCAAGTCAAGGGCAGAGAAGGGATTTGTACTGAATA

AGATGGGAGGCAGGAAGGTCAGAACTACCCAAGAAGTGAATGCAAGGTTCTA

GAAGCAGTAGTAGAAATGAACCAAGCAGAACCTTAACAAATTCCACACTTGGG

CAGGGCAGAGCTGATGAAAAGCTACCCTAGTTTCATCTAGTGGGAACTTCTGG

ACCCACTGGGTTCTCAAATCCAAGGCTGGTGTAAAAGTATCTCTGATCCATTCC

TAGGACTAGGGACGCCGGAGGAGGGCACAAGTATCTCCCAGTCTAAATTTCAG

GAGCAGAAAAGTATAGGGCTTATGGGCTTTGGAAGGGTTTGAGTCTCAGTCCC

TGAGCTTCTAGCTGTGTGCACTTGAGCAAGCCATTTCATCTCTCTGAGCCTCAG

TCCCCTCATCTGTTCAATGGTCACAATAACAGTACCTTACAGGGTTGTCCCAAA

GACTGAGGGAGATGAGTAGAATGCTTAGCATATGCCTTACACATAGTAAGCAC

TCAATACATAGTATCAATTATAATCATTTTGGAGTGGTAATTAGAGTTCGTGCT

ACATTAGGAGACTGGCCACTTCTTGGGGACAGAGAGTAGGGCCCCCTCACTGC

ATATATTGAGGATGTCAACCAATCCATCCACAAGCATTTATTGAATACCTACTA

TGTGCCTGGCTTCATGCTGAACACGGAAGGCACAGGGAAGTACGAGACCAAG

GTGACATATAATAAGCCTGCGGCATGTCCTGATGTGACTGACCTTTCTGAGCAG

TAGGTGTGACTGTGCCTGATTCCAGACAAGAGAAAGTGAAGCTAAGAGAAAAT

ACAACATTGACCCTAAGTTACGCAGCTTGTGGGTGACACAGATGAGTTTGAAT

TCCCTTTTCCTAGCTCCAAGTACAATGGGTTTTCTCTTGGATTGTGGCTGCCTTC

CTTAATCCTGCAGAATCTCGGGGTCAAGTCAGGGAGGTGAAGCTAAGCCAGTG

AACTGTCCATGCTGACCGGGAAACAGGCTCACATTAGACTCTGAGATACAAAG

GTCATCAAACTTGAACATTGCTCTTCCATACTAGTGAGCAAAGAAATGCAATTT

TAGACAATGATCATCAGTTTGGCAAAAAATAATTGTTTAAATGATGATACCTA

GTGCTGGCCTTGTGACAGAAAGGCACTCTCTGTAATGCCAGGTGAAGTTCCATT

TCTCGAAAGCAACAAGGTAATATGGATCAAAAGCCTGTGATGAATTTATTCCT

TTTAATTCAGTAGTTCCACCTCTAAAAATTTTTCCCAAGAGTGGTATAATAATT

TTGTAGCTGAAGATTTATATACAGGGGTGTTTAATTTGGTGTTATTTAGAGTCA

CATAAAATTGGAGGTAACCTGAATGCATAGCAGTGGGGAAGCTTAAAAGAGA

ATGGTAGGACCTTCTGAGGGACTGTTTTGTGCAATTAAAACGTCAAATTGTTCA

TGAGGCAATAAAACAACGTTTAGTGAACAAAATGGGAAACAAACCTGTATGTA

CTGCGTTTTTTTTAACTCTAAACATGTTACATGGAAAGGCAAGTACCTAATTTG

AACCTCATTTTGTAAGAAAATGTGATTGCATCTGCAAAGGAGAGTTTCAGAGG

AGCATTAAAGTAGGAACATCCCTGCATTTATGTGTTCGTCAGACAATTCTTGAG

TGTCTACTATATACGGTGGACTGTTCTAGACATGGGGATGTGAACAACACAAG

CCCTGCCCCCCACATAACTTACATTCTAGGTGCTGGTTAAAGGAACTTTCATTT

TATGTGTGTGTGTGCATATGTAATTTAAAAGTTTTCCTGTCACCACATGCAATA

CTTGGATAATTTAGAAGTCAAAAAATAGGAGGGGACAGGGGAAGGGAAAAAC

CAAATGTGAGCTGGTTGGCTCAGACTCCAAGGAGGAGCTTCAGATAAGTGGCC

AAGGAGAGCAAGGATCTGGGAAGGCTTCCTGGAGGCGGAAGAACTGGAATCA

AGCCCAGAGCCACAACAGATACGGATGTAGGGGAAATGGGGGAGGAGGAAGC

AAGGGTGTACAGGAGGAGGGGTCACCAGTCACCCACTTTCCTCCCAGGTGGTC

TACTTTTCAGCCACATACCCCTACATCATGCTGATCATCCTGTTCTTCCGTGGA

GTGACGCTGCCCGGGGCCAAGGAGGGCATCCTCTTCTACATCACACCCAACTT

CCGCAAGCTGTCTGACTCCGAGGTGAGTGCCCCTCCCAGCCCGGATTCTGAGC

CACCACTTAGCAGCTTTGAGATAAGCTTTTGCATCTTTCTGACCCTCACTGTAG

ACCAGGTTTGTTGCCAGGGAGAGCTGGTGGGCACATGGAAAAGTTCCTGGATA

GCTGCGGCTATGGTGATTACTTTTGACAGTCTTTGATAATTCTGCCTATAGGTG

TGGCTGGATGCGGCAACCCAGATCTTCTTCTCATACGGGCTGGGCCTGGGGTC

CCTGATCGCTCTCGGGAGCTACAACTCTTTCCACAACAATGTCTACAGGTTTGA

GAGGACAGCTGCGGGAGCCCCTTCCTTCCTGGCCGAGCCCTTTGAGTCCACAT

CCCACACTCATGTGTTAGGGCCCTCTTTGGCCACTTTGCACTCTCAGCTTAACC

TCTCCCTGAGCCAGTCGTTTGTTTAACTCCTTCTGGAGACACAGACATTCCTGT

TGTCCCCCTTTCCTGACCCTGATCTTCTGGCTCTGCCCTCCTCTAGCTTGCTCTT

AGGGCCTGTGCATCTGGGACCTACCGTGTAGCCCCATTCCCAGCCCCCTCCTCC

GGGCCACCCCTCCATAACCACAGCCAGCAGCTACACCTTCTGCCAGCTGCACC

CTCCTCACCCCACCCCTCCCTCTCTTCAGGGACTCCATCATCGTCTGCTGCATC

AATTCGTGCACCAGCATGTTCGCAGGATTCGTCATCTTCTCCATCGTGGGCTTC

ATGGCCCATGTCACCAAGAGGTCCATTGCTGATGTGGCGGCCTCAGGTCAGTG

CAACACTGTGTGGGGCCGGGCTCCTGGCATGGGGGCACTAACCATGCTGGCCT

TTGCCATCATCACCATTACCACCACCACTGTGGCTCACTGGCCTTGAGCACTCC

CTATGTGCCAGGCAATGCACTGAGCATTTTACATGGACTGTACCCATGATTCTT

CTCAACACTTTGCAATACACGCTATTGTTAGCCCATTTCATAGATGAGAAAACT

GAGACTCACCTCCTAAGGGTATGGAGCTAGAAAGATGGCAGATCTGGAGTTGG

AGCCCAGGTCTGGCTGACTCCCAAGCCTGGGCTCTTAGTCTCTTGGCTATACTA

GCATGTCCAGCACAATCCTGGACAATTTGGGGAGAAGATCCACTCTTGCCTTG

GGAGTATAAACTTTGGTATTGGGGGAAATTCACCCCCGATATTTCACGTAGGTT

CTTTTCTATTTTACCTAAGTGTCAGCTGGTCTGAGAAATAAAGGGAAAGAGTAC

AAAAGAGAGAAATTTTAAAGCTGGATGTCTGGGGGAGACATCACATGATGCCC

CCGAAGCTGTAAAACCAGCAAGTTTTTATTCATGATTTTCAAAAGGGGAGGGA

GTGTATGAATAGGGTGTGGGTCACAGAGATCACATGCTCACAAGGTAATAAAA

TATCACAAGGCAAATGGAGGCAGGGCGAGATCACAGGACCAGGGCAGAATTA

AAATTGCTAATGAAGTTTCGGGCACACATTGTCATTGAAAACATCTTATCAGG

AGACAGGGTTTGAGAGCAGACAACCGGTCTGACTAAAATTTATTAGGCAGGAA

TTTCCTCATCCTAATAAGCCTGGGAGCGCTACGGGAGACCGGGGCTTATTTCAT

CCCTTATCTACAACTGTAAAAGACAGACGTCCCCAAAGCGGCCATTTCAGAGG

CCTCCCCTTAGGGACGCATTCTCTTTCTCAGGGAAGTTCCTTGCTGAGAAAAAG

AATTCAGCGATATTTCTCCTATTTGCTTTTGAAAGAAGAGAAATATGGCTCTGT

TCCACCTGGCCCACAGGCAGCCAGACGTTAAGGTTATCTCCCTTGTTCCCTGGA

CATCACTGTTATCCTGTTTTTTTTCCAAGGTGCCCAGATTTCATATTGTTTAAAC

AATTTGTGCAGTTAATGCAATCATCACAAGGTCCTGTGGCAACATTCATCCTCA

GCTTACAAAGATGATGGGATTAAGATATTAAAGTAAAGACGGGCATAGGAAA

TCACAAGAGTATTGATTGGGGAAGTGATAAGTGTCCATGAAATCTTCACAATT

TATGTTCAGAGATTGCAGTAAAGGCAGGTGTAAGAAATTATAAAAGTATTAAT

TTGGGGAACTAGTGTCAATGAAATCTTCACAATCTATGTTCTTCTGCCATGGCT

TCAGCCGGTCCCTCTGTTTGGGGTCCCTGACCTCCCGCAACACTTTGGCTCAAT

TCAGACATGTTCATCACCATACTACAAAGACCACTAAATCCATGCTTTTGTCTC

AGCTCCAAAAATGTTCTCGCCAAGTGGGCCATGCCCACTCTGCTCACCACACA

GTCATCAATGCCACAGAACCCAAGGAAAACCCCAGACAGCCCTTCGCTGCACA

GCCAGGCAAGCAGCCAACAGGTGGAACCCACTGTATGCAGGGCAGAGGCCCC

TGAGGCTCATGGAGGCCACACGGCTGGTCCCTTGAAGAAGTGGGCTGGTTCCG

TGAAAGGAGCATTCTCTGCTCCGGTTCTAATCTCCCCTTCAAGGGCAGCCACCA

TCTCGTTTCTCTGCAATTCCCCAACCATTCCCCCACCCCCATCCGGAGCCAGAC

CCAGGGAGGTTACAATCTCTTTCCTTCTCATTCCGAGATTTCCTGATCTCTTAGA

CACCCCCACCAACTACTCAAACACAATACATGCCAAACAAGGCTCCAAAGCCT

TCTCCTCCACCATGCTCCCCATCACAGAAGGGTCCCATGGTCCAGTGCCCAGG

GTGGAGACCTCAGCATTGTCCTGGACTCTTCCCTCACCCTCATCCTGCAAACCA

GCCAATACCAGCACCTGCCCCTCCATCCCCTACATATCTCTCTATCTGGTTGCC

TCTCTGCACCTGCACTATCCTCACTACTGGCCAAGCCATGCCATCCCCCATCGA

GATGATGGCCACAGCTCCTCACTGGTCCTCTTGCCTCTGGGCACAATTACTATG

GCCCCACATCCCCCACCCTCTTTCTGCACCTGGAAAAAGGGGCTGAAAACACT

CACTCAAAAGGTTACTCTGAAGATGAAATGAAATAAGGCTTAGAAAATGGGCA

GTGTGCTGGAAAGGCTGTAGGTGATGAGTACCTTTTATTTTTCTTCTTTTTTGCA

CTTCTCTGTTCAGGCCCCGGGCTGGCGTTCCTGGCATACCCAGAGGCGGTGACC

CAGCTGCCTATCTCCCCACTCTGGGCCATCCTCTTCTTCTCCATGCTGTTGATGC

TGGGCATTGACAGCCAGGTGAGGGCGCCCCCCCCGCACCCCAGGAGCTCTCCA

TCCCCAGACTCCTCAGTCCCCGAGTAAACAGGGAGAGGAGGCAACTCTTAGGC

TGCCAAGAAGATGACCAAAAATTCCAGGTCCCTAGAATGTTCCACTGCATGAA

ATTTCAGGAAACAGCTCAGCCACCTTCTCCATTTTCCAGAAGGGGAAACTGAT

GCCCAGAGGGGAGGGACTCTCCTGAGGTCACAAAGTGAGTCACAGGCAGAAT

CAGGCTGGACCCTGGGCTCTCCTGGCTCGGTGCTGCTGGGCCTCCCGTCCTTCC

TTAGAGGGCCAGGCTTTGGGTGGGTTGGGGCTGGGGCTGCTGCAGGTGGCTGA

CCTCTGTTCCCACCTGAAGTTCTGCACTGTGGAGGGCTTCATCACAGCCCTGGT

GGATGAGTACCCCAGGCTCCTCCGCAACCGCAGAGAGCTCTTCATTGCTGCTG

TCTGCATCATCTCCTACCTGATCGGTCTCTCTAACATCACTCAGGTAAGCTCAG

TAAGCACCTGGCACTCCCCCAGCACTGCCCAGGGCCATGCCCAGGGCCTCCTC

TCCCCTCCTCTGTTGTATCCCACCTTTCCTGCCTTCATTGAGCATCATCTCTGTG

CCAAGCTCTGGGCACATAACAGTGAGTCAGATGGGGTCCCTGCCCTCCAGAGG

CTCCCAGGCTGGTGGAGACACAAACATTGAGCTAAGACATATAGGACAGAGCT

AGAGAATGCAGCACCTAACTGGACTCGGAGCACCAGGGACTGCTTCTTGGAGG

AGGGGATGTAGAAATTGAGCTTTGAAAGGCCCACAGAAGGCCTGGCATGGTG

GCTCATGTCTATAATCCCAGCACTTTGGGAGGCAGAGGCCGGCGGATCACCTG

AAGTCAGGAGTTTGAGACCAGCTTGGCCAATGTGGTGAAAACCCGTCTCTACT

TAAAAAAATAAAATAAAGTAAATAAATAATACAAAAATTAGTCAGGCATTGG

GGCAGGTGCCTGTAATCCCAGCTACTTGGGAGGCTGAGGTAGGAGAATTGCTT

GAACCCGGGAGGCAGAGGTTGCAGTGAGCTGAGATCGCGCCATTGCACTCCAG

CCTGGGCAACAAGAGCGAAAAACTCTGTCTCAAAAAAAAAAAAAAAAAAGAA

AGAAAGAAAGAAAGACAGAAAAAGAAAAGAAAGGCCCACAGAAATTAGCCA

GAAAGTCAAATTGGAGTTGAGCCTTTGAAGTTGACACTGCCCAAAGCTTTCTTT

CATTCCCATTTTTCTTTTGTATTGACATGAATGAGAAATGCTCACATAGCTGGT

GAATTGTGCAATAAATATAAACGACTCTAGCAACATGAGTTCAGCACTGCGGG

TCTGATTTTTATAATCCACAAATCACCCCCACCCCCCCCCCACCAGATCCAAAT

GTAGTTCTGCTCCAGAGTTAGTATTCATTCATCCCTTCATTCATAGTTGTCAAAT

ACCTACTATGGGCCTGGCATGACCCCAGAAATTTGGCAATTAATGTGGCAGAT

ACTACCATGTCTGCTGTCTTGGAGCTTACATTTTGGTGGACAAGACTGACAGAC

AAATTAGATGATTTTGGAGAGTGTCAAGTGCCCTGGAGAAAAGAGGACAGCA

GCACAGAGAATGGCCAGTAGCACAGGAGGGGCGCTCCCTCAGACAAGGTAGT

CAAGGGAGGTCTCTCTCTCCAAGAAAGCGACAGTTGAGCTGAGACTTAAATGA

TGAGAAAGAGCCAGCCAGAGAAAGATCTGGGGAAAGAGAGCAGAGTAGGTGC

AAAAGCCCTGAGGCATTTGAGGGACGAGGGAAGCCAGAGTGGTGGAGGCAGA

GGGAGTGAGGCAGTGGGTGGGAGACGAAGAACCAGCCCATGCGGACCTTGCA

GGCCAAGACATTTGGGTGGGATGGGATGGGATGGGATGGGATGGGATGGGAT

TGGATGGCATGGGATTGGATTAGATCTCTCTCTGCTTATTTATGTGTTATATTAT

TGTCTCATTTAAGTCCTGAAAGGTTTTAAGCAGGGGAGAGACCTGACTGATTTA

CACTGTAAAAAGATGGCCCTGGTGACCAACTCCGCTTTCTGCCCTCCAACCTTA

CCAGAAATTGGCATTTTCCTCTACCACCTGGACATTTCCTTCCCAATGCCCGGA

CTGTCAATGTCCACATCCTCTCGTGGTCACAAAGCCCTCAGTGCTTCATCTAAT

CTACCCTGAAGCCTAATCCCAGCCCCATCAGCCTCCAGAGTCCATGTGGTCTGT

GAACTGGATGCAGTGGGCGCCGGGGACAATGCACCTGTTCTCACCTCTTTTCAT

CTTGCAGGGGGGTATTTATGTCTTCAAACTCTTTGACTACTACTCTGCCAGTGG

CATGAGCCTGCTGTTCCTCGTGTTCTTTGAATGTGTCTCTATTTCCTGGTTTTAC

GGTGAGTATCAGCCCCTCATCCCTTATTCATTCACTCATTCATCCATTCATTCCT

TTCTTGAGTCATCATTCAACAAGTGCAATTTGAACCCCAGGTCTTCTCCTTACA

TTTATTTATTTATTTATTCTTCACATTACAATTCAATGAGGTTAGTCCAGGAGTC

CCCCATGTTTAGAAGATATAAGAGCAGTTAAATGAATTCATTTAAAATAAGTG

GAGATTTTATCCAGAAAAGGGAAACCAAAACTACTTTTTAAAGTTTTAAAGTT

ATATATTTAACACAGTCTAGGCTGTAATTAAAAGTGTGCACTATCTTAAAACAA

ATAACAAAAGAAACATTGATTTTGTTTATTTTGTGGCCTAAGTTTCTTGAGCCA

ACTGCATGATAGTAAAAAATGACAACATCCTTTCCTGCACCTTTGATTGTCCCA

TCAGAGCAAATGTTTTTAAAGAAGCATGATATTTTGTGTCCCCCTTTGCAATAT

CTGCCACCTCATCCTCCTTGTCATTGTCCCTTCTTTTTTGTTTGTTTTATATGTAT

AAATTTAAGGGACATGAGTGCAATTTTGTTACCTGGATATCTTGTGTAGTGGTG

AAGTCTGGGCTTTTAGTGTACCCATCACCACATAATGTACATTCTTTTTTTATTT

TTATTTTTATTTATTTTATTTTATTCATTTTTTTGATGGAGTCTTGCTCTGTCACC

AGGCTGGAGTGCAGCGGTGCAAACTCAGCTCACTGCAACCTCTGCCTGCCTGG

TTCAAGCAATTCCCCTACCTCAGCCTCCCAAGTAGCTGGGATTACAGGCATGTG

CCACCACGCCCAGCTAATTTTTTGTATTTTAGTAGAGGCAGGGTTTCACCATGT

TGGCCAAGATGGTCTCGACCTCCTGACCTCATGATCCGCCCACCTCGGCCTCCC

AAAGTGCTGGGATTACAGGCATAAGCCACCAAGCCCAGCAGTACATTCTACCC

ACTAAGTAATTTCTCATCATCCACCCCAGCTCCCATTCCCCCACCCTTCTGAGT

CTCCAGTGTCTATTATTCCACCTGTGCCCAGTGTTTAGCTCCTACCTTTAAGTGA

GAACATGCAGTATTTGACTTCCTGTTTCTGAGTTGTTTTGCTTAAGATGATGGC

CTCCAGTTCCATCCACGTTGCTGCAATAGGCATGACTTCATTCTATTTTATGGCT

GAATAGTATTCCATCGTGTATGTATACCATATTTCCTTTATCCAGCCATCCACG

GATGGACACTTAGGTTGATTCCCTATCTTTGCTATTGTGAATAAACACGAGTGC

AAATATCTTTTTGATATAATAATTTCTTTTCCTTTGGATAGATAGCCAGTAGTG

GGATTGCTGGATCAAATGGTGGTTCTATTTCTGATTCTTTGAGAAATCTCTGTA

CTGGTTTCCATAGAGGTTGCACTAATTTACACTCCCAATAACACCTTCTCTCTCT

AACTGTTCCAACACGGTCAGATCCTCACTGGTCAGTGACTCTGCTTATAATTTG

AGCAGTGCCCGAAGCCGCTCTGGTGAACTTCAAGACACCAGTTTGGGGTTTGG

GGTTTTTCTTTTTCATGAGATTTGCAGTTCTTTGCAATTGATTGTCAATTTCTTTT

TCTTTCATTCATTCATTTATTTATTTATTTATTTATTTATTTATTTATTTTTGAGA

CGAAGTCTCGCTCTTGTTCCCCAGGCTGGAGTACAATGGCACGATCTCGGCTCA

CTGCAACCTCCACCTCCCAGGTTCAAGCAGTTCTCCTGCCTCAGCCTCCCAAGT

AGCTGGGATTACAGGCGCCCGCCACCACACCTGGCTAATGTTTATATTTTTAGT

AGAGACGGAGTTTCACCGTGTTGGCCAGGCTGGTCTCGAACTCCTGACCTCAG

GTGATCCTCCCACCTCGGCCTCCCAAAGTGCTGGGATTACAGGCATGGGCCAC

CGTGTCCGGCAGATTGCCAGTTTCTTATAGCATCTGACCATCAGGGAGAAACA

GCCCCTGCCCAGGAAAGCCTTTGAAGGAATGGCTGGGATAGATGTCGAGTGGG

AGGGACATTGAGAGTGGCAGGGGTGGAGGGGATCATCATGTCAGTCTCCTTTT

GTTGGTAGATATCTTCACATTAAAGTGACTTGCTCACCTACACAACCCACTTGC

TGTGAGAATTGGGCTATTGAAAAATACTTGGTAACGAGGACCCCCAGAGCATA

ACCCCCATGGTACAGACAAGGAAACTGAGACTCAGAGAGGTTAGGTGACTTGC

CCATGGTCACACTGTGAGAAAGTGGTAGAACCAGGCTTTGACCCTGAAGCACC

CACAGCTCATCCATCCCTCCTGCTCCCAATGTCACAGGTGTCAACCGATTCTAT

GACAATATCCAAGAGATGGTTGGATCCAGGCCCTGCATCTGGTGGAAACTCTG

CTGGTCTTTCTTCACACCAATCATTGTGGCGGTAAGAACAAGGCCTGACTAGCC

CTGTTAGGATGAGGCTAGACCAAGCCCTGGGGGGACTCAGGTCCAGGGAGAA

ACTTCTAGAGGCAGAGGCAGGTGGGAGAGGCCCCCAGAAACCCTGTTCCTTAA

TAATACTTCTCTCTACCATTTGTTGGGCATCTATTCTGCACCCTGCATGGTACAA

GACACATTATATCATCCTCCACAAACCTCAGAAAGGAGGCATTATTGTCCCAC

TTTACCGCTGCAGGATCTGGGGCTCACCTAGCCAGCATCACCCAGCTAGTCAG

CACTGGGATTCGAACCAAGCCCAGTCTCTTGGCACTTGGAGACTCTCCTTCTCA

GGGTCCCTCATCCCCACAAGCAGGAGCTGAAGCAGGGGCGGGCCTGGGAGGG

GCCACCCAAGGTTCTTGAGGTGGGGGCAGAGCCACTGCATATGTTGTGTCCAC

ATAAGGGGACCCTGCCAATGGCACATTGGGAGCTGATATCTCACCCTTGCTCT

GCTTTTGAAGCTGGGGAACTTGGAGGAAGGGGTTCCCTTTTCTAATTAGTCTCC

CTAGAGGAGTTGCCTTTTCCTACTGATCTTCCCTGAGAGGGTGTCTTTTTCTATT

ATGCACAAAGGCACCTTATGTGCTAGCAGCGGTGCTGAGTGCAGGACTCCCAA

GACCTTGGGCCTCTGTAAGGGTCAGCCTCGGGGTGAGGGTGGAGGGCAGTCAG

CCTGCTGGCTTCAGGGTGGTTCATCCTGGGTGACTGACTCCTCCCCCTCCCTCC

CCTCCCCTCCACCCTCTCCAGGGCGTGTTCATTTTCAGTGCTGTGCAGATGACG

CCACTCACCATGGGAAACTATGTTTTCCCCAAGTGGGGCCAGGGTGTGGGCTG

GCTGATGGCTCTGTCTTCCATGGTCCTCATCCCCGGGTACATGGCCTACATGTT

CCTCACCTTAAAGGGCTCCCTGAAGCAGGTAAGCCCTTCCCCACCCTTCAAATT

GCCAGTGCCCTTCTCCCACTGTGTCAGGCACAGCCCCACCCCTTGTGAAGCTCA

CAGTCTAGCTGGGGAAACAGACAAGTCAATAGCCAATGACAGTAGAGTATTAG

AGACACTGAGATTAGGGAAAGCCAAGGGGGTGGGAGCAAACAGCCTGCCTTA

GCCCTACTTGGGTTAAGGAGGATTAAAGAAGCTTCCCAGAAGAGGGGATAACA

AAGGAGAGGCCTGGGGATAAACCAAACTTAAAAGTAGAAGCAGAAAGTATGT

TTCTAGAAGAGGGAGGGGAGGAGAGGTTGGAAAGCATGTGCAAAGGTCCAGG

GGTATAAACTGCATTGGGTCAACTGAACGGAGATCAGTGTGGCTGGAAGGTAA

AGTGGAAAGGGGTGCGAGGAGGGGACATGACAAGAGATGAGGTTGGGATGTA

AGAAGGTCCAGCCAGATCATGAAGGGCTTTGAAAACCAGGACAAGAACTTTG

AACTTTATCCTGGCAGCAATGGGGTGTCATGAAGGGCTTGGAACAGACGGAGG

GTGGGATCACATTTGCCCAGGGCTCACTCTAGATCTCATTTGTAGGGTTGATAT

AGAGAAAATGTGGTCTTACCCACAAAGAAAGACAAAGCACAAAAGACCTGCT

GCCTGCAGCAGGGGCCTCACCATCACATAGGGTTCTCTGTCCATGCCCACACTT

TGTTAAATTGCACTAAATTCTTGAGGGAAGCAAAGAAGTGCTGGAGGTGCCAG

GCAGGTGCCTCATTGATGAGGTGTGTGCAGGCTGAAAAGATGCCACGGGGAGA

AGTGGAGAGTCGGAGTGTGAGGGCTGCCCCGGAAGGCCCTGGATGAGTTGCTG

TCCACTATAGTGCCCTGGGAAATGCACCTGCCAGACCCAGGGGAAGCGCTCTC

AGGTCTGAGATACAGGGAAGCCTGGCTGCAGGTTATGTGCTGCCGTGGAGTTA

CTGGCCCATCTTCCTCCAACCTCAGATGAAGAAACTGGGGAACCACAGGGGTT

GAGTAAGCAGGTTCCCAGCTCAGGAAGCTGTGAGCAGAGCTAGGGCTTGCAGC

CCTTTCTTCCCAATGCAAGTCCAAAGCTTTCATTTTCCATGATCCACTGGAGAG

CCTCAAACAGCAGCTGGGTCTTAGCGTGGCAAGAGGTTAGGGGGGTGCCCTTG

CAGAAAACAGCACAGCTTGTGCAAAGGCCCAGCGGTAGGAAGGGCCAAGACA

TACCTGAGGCCAGGGTGTGAGCTGACGGGGCAGGATGGCGAAGGCAGTAGGG

GGTCCATCCATTCAGCACACATGGACGGAACCCTGATGATGTGCCAGACACTA

CTGTCAGTGCTGGGGACACAGCCATGAACAAGATAAGCAGAAATTGCACCCTC

CTGGAGCTCCCATTCAGGGAGAGGAAGATAAGCAACTAACAAAATAAAAGCA

CGTGAGGTGGCGAGGAGCACTATAGAGAAAAATCAAGTAGACAGGGTGTTTA

GGGAGTGCCGGATGAGGCCTCACTGAGAAGGCGAAACATTTGAGCCAAGACC

TGGGGAAGTGGAAAGCGAGCTGTCGCTATCTGAGAGGAAGGCTCTGCCAGAC

AGAGGGCACAGCCTGTGCAAACGCCCTGGAGCGGAAGCGTGCCTGCTGTGTTT

GCGGGGCGTCGAGGAGTCCTATGCAGATGAGGTGGATGTGCGAGGAAGAGGG

GAGGACACTGAGGACTGGGAGGTGGCAGGGGCCAGGTCGTGTAGGGCCTTGA

AGAACTTTGGCTCTGCCTCTTGGTGAAATGGGAGCCTTCATTGGGTCCTGAAGA

GTACAGGGGCATGATCTGAGTCTGGTTTTCATACAACTCTCCTGCTGCTGTGTT

GACGACAGAATGAAGGGGTGAGAGGACAGCATGAAAGTGGGAGTGGAAGCTG

AGGGCTGATTGGGAGTTGTTGCAATAACCCAAGTGAGAGGGGCTGTGGCGTGG

CCCATGTGGGGCAGTGGCAGGGGTGCAGGAGGTGGGGATGTGGCTGGAGTCT

GGGTGTAGTTTGGAGGCAGAGCAGACAGGATGCACTTGAATAGAGAGCAGGA

GAGGGAAGCGGCCAGTTCCTCACCATCCCAGGCTGGGATTTTCCCCAGAGAGC

CTCACACCTTCCTCCCCGGTACCCACAGCGCATCCAAGTCATGGTCCAGCCCAG

CGAAGACATCGTTCGCCCAGAGAATGGTCCTGAGCAGCCCCAGGCGGGCAGCT

CCACCAGCAAGGAGGCCTACATCTAGGGTGGGGGCCACTCACCGACCCGACAC

TCTCACCCCCCGACCTGGCTGAGTGCGACCACCACTTGATGTCTGAGGATACCT

TCCATCTCAACCTACCTCGAGTGGCGAGTCCAGACACCATCACCACGCAGAGA

GGGGAGGTGGGAGGACAGTTAGACCCCTGGGTGGGCCCTGCCGTGGGCAAGG

ATACCCGGTGGCTTCTGGCACCTGGCGGGCTGGTGACCTTTTTAATCCAGGCCC

CATCAGCATCCCACGATCGGCCTTGGTAACCGCCGCGGTAGATCATTTTTATCC

CGCCAGGGAGTGTGATGCAGGAAGACCACATGCGCTCCTGGCTTTTAAACCTG

TTCCTGACTGTTCTCTTACTGCCGAAACCCTTGACTGTTATCTCGGACTTTGCAG

GAGTTCCTTTCCCTCCGAACGCTGCTCCATGCACAGGAAAAGGGCATTTTGTAC

AATGGGGACTTCCCGGGAACGCTTGCTCTTAAGTACCAGAAGCCGGCGGAGCT

CTGGCTTTCGTGTTTTTGGTTTTCTCCTTCCCAAGGCAGCTGGATTGAAAAAAC

AAAACAAAACAAAAAAACCCAGGGGCGTCAGTCGATATTCCCAGGGCCGCTT

CTCCTGCAGTCTGTGGAGCGTCCTTGTCCCCGCCGCCGGAATGAATGAGCATTC

TGCAGCCCGATGTCCCTGTCCCCTCCTCGCCGGGCCATTCTGATTGGACCTGGC

CCAGTGCAATCTGTCCAGACAAGCCCTGCTTGCTGGAAAACTGCCACAAGCAC

AATTGATCTCTTTTTATCGCCATTCCAGGGGCCTCAGGTCCTACTGGGGAAACT

TCCTATACCGGAGCTCCAGTTTCTCTTAAGCTGCCCAATTTCACAGAGTACAAA

ATAGTTGTAGGGGAAATCAAGGTGAAGGATCTGTCCGACAGTCAAGACGGATC

CACAGTAATCTTTCGGTCTCCTTAAACTACCACCCTCGCTGCCACCCACCCCAA

GCTGCTGCCGCCTCACCTTCCTTGAAATTTCTCAGCGGGAGTCTCCTCACTGCC

ACTAAAATCCACCCAGCCCACTAACTGAGGAGCTAGTGTTAATCCAGAGAACC

CCCCGCAATGTGCTTCCGAGATTCAGACTGCTTCATTGGGAAGTATGATTTGTT

CCTTTCTGGAATTGGGCTCCGTGGTGGCGGCGGCACTTCAAGCAAAGACAGTT

TCTTGCAAGCTCCAGTAGCTCCGCGTGTCTCATTTGCCAGGAAGATGGGTTCCC

ACGTAGCAAATCGTACATTGTGCCCTGTAGCTCCTTAGCTAGTTAGCTCACAAG

CCGTGTTTTATGACTAATCCTTAATAACTATGGTAAATAACTGTGACTGTGGGG

TTTTTAATCTCTTGTCATTCTCATCCAAAAGTGACCAGCATACCAGTTCTTGCA

ATAAGATATTACCCTCAGAATATTAAGCACATTATTGTAGAGAAAAAAAAATA

TGTGTACACATATGAACGCACAACATGCACATTCATCCTCACATGTGGCACGT

AAGGTCTCATTTGATATTGTGTAGGAAATCTGAAGCCTTTTCCTGAGGTCATCT

GTAAAATAGTCTCATTGCCAAGGCATCCCCAGTGCCAGCTGGTGAATCCATGA

TCAAAATGCATACGTATTGTTAAATGATAAGGTTTAGAATGACAGGAACCCAT

CACTGTGTCTCATGGTCCCACTTCCCCATCTGTGTGTGAATTCCTTTAGACTAA

GGGCAGGAAGACTTCCAGCTTTCTCTTTGTTCTTCAATGTGAAACTGAGACCAA

GTCTCTCTAAGACAAATGCAGTGTATTTAATGTTTGTAAGCAATTCTAAGTGAG

ATGTTTGGCAAGAAATCCCCTAACTGATTTCCATCCAAACCTACCTTATAGAGC

ACAATATTAAGTGTTGTACAATTACTGTGAGAACTGTGAATATGTGTAACTTTT

TTTTAGTATTTGCCCGGGGGGAAAAAGATATTGTATTATCATATATGCTTTTTT

GCAATAAGGATTTATTCTCAGAACACCAAGTAAATCTATCTCTATATAAAAAA

TATATGTAATATATACATATTCAAAGTATATACAGAGCCTGTTTTAAAAAATAC

AGTATTATTTAGTAAAATTATCTGTTCTATGGACCAAATGTAAAATATTTATAA

ATGAAGATGCATTTTAAATGTCTATAAATGGTGTCATAACTAGAGCACGGGCG

TTATGTAAGTTTCTAAGAATTTAGAGGATAAATAATAAAGGTTCTATGATATAC

AA (SEQ ID NO: 109)

TABLE 4

Sequences and chemical modifications of selected steric blocker antisense
oligonucleotide targeting cryptic splice sites within SLC6A1 introns (showing
compounds that activate the expression of SLC6A1 mRNA in KNS60 neuroblastoma
cells.)

		Molecular		Percent
Oligo Name	Oligo Sequence	Weight (Da)	Target Site	Activation

SLCss0603	TCTTGCAGACTATTCTAAAG	7933.92	Intron 1:	102.81
	(SEQ ID NO: 110)		11004094-11004117

SLCss0701	AGGGTGGGCACCCGGACCTG	8054.98	Intron 1:	120.84
	(SEQ ID NO: 111)		11005196-11005219

SLCss0801	GGAAGACCTGGGCCCCGTTC	8003.97	Intron 1:	209.24
	(SEQ ID NO: 112)		11006348-11006371

SLCss0802	TGGGCCCCGTTCTTGCATAG	7970.93	Intron 1:	136.17
	(SEQ ID NO: 113)		11006348-11006371

SLCss0803	TTGCATAGGTGACAGTGCAG	8025.94	Intron 1:	123.55
	(SEQ ID NO: 114)		11006348-11006371

SLCss0901	TGATCATCATTCTTATTACA	7883.89	Intron 1:	138.59
	(SEQ ID NO: 115)		11006831-11006840

SLCss100l	GCTTCTGCAAATTCCCTCTC	7877.93	Intron 1:	180.25
	(SEQ ID NO: 116)		11009358-11009367

SLCss1002	AATTCCCTCTCAGGTACGTT	7913.92	Intron 1:	228.55
	(SEQ ID NO: 117)		11009358-11009367

SLCss1101	GTTTAAAAATCAGCTTCTCG	7933.92	Intron 1:	114.56
	(SEQ ID NO: 118)		11010251-11010274

SLCss1301	TCTGGCTGTATTAATGCGTT	7957.89	Intron 1:	101.13
	(SEQ ID NO: 119)		11012180-11012203

SLCss1302	TATTAATGCGTTTCTTCTAG	7916.88	Intron 1:	257.13
	(SEQ ID NO: 120)		11012180-11012203

SLCss1303	TCTTCTAGGCCTCCCGTGTC	7909.92	Intron 1:	113.69
	(SEQ ID NO: 121)		11012180-11012203

SLCss1401	CCGTGTCTTGTTCTTACACC	7894.91	Intron 1:	121.89
	(SEQ ID NO: 122)		11012213-11012236

SLCss1402	TGTTCTTACACCTCTTGCAG	7904.91	Intron 1:	160.77
	(SEQ ID NO: 123)		11012213-11012236

SLCss1403	TCTTGCAGCACTTATCACAG	7922.93	Intron 1:	174.72
	(SEQ ID NO: 124)		11012213-11012236

SLCss1501	CTTCCGCCTGCTCCACCAGC	7890.97	Intron 1:	145.5
	(SEQ ID NO: 125)		11012794-11012803

SLCss1502	GCTCCACCAGCAGGTAAAGG	8006.99	Intron 1:	152.72
	(SEQ ID NO: 126)		11012794-11012803

SLCss1503	AGGTAAAGGAGGCTGATCAC	8043.97	Intron 1:	169.43
	(SEQ ID NO: 127)		11012794-11012803

Claims

What is claimed is:

1. An antisense oligonucleotide that binds to a target region in an SLC6A1 RNA transcript, wherein the target region comprises a splice modulatory element.

2. The antisense oligonucleotide of claim 1, wherein binding of the antisense oligonucleotide to the target region increases the expression of a functional protein encoded by the SLC6A1 RNA transcript in a cell.

3. The antisense oligonucleotide of claim 2, wherein the protein comprises GABA Transporter 1 (GAT-1).

4. The antisense oligonucleotide of claim 2, wherein the cell comprises an SLC6A1 expressing cell.

5. The antisense oligonucleotide of claim 4, wherein the cell comprises a neuronal cell and/or an astrocyte.

6. The antisense oligonucleotide of claim 5, wherein the neuronal cell comprises a GABAergic neuronal cell.

7. The antisense oligonucleotide of any of claims 1 or 2, wherein the splice modulatory element comprises one or more of a non-productive splice site, a exonic splicing enhancer, an exonic splicing silencer, an intronic splicing enhancer, or an intronic splicing silencer.

8. The antisense oligonucleotide of any of claims 1-7, comprising a region of complementarity to a target region of an RNA transcript corresponding to a nucleotide sequence of any one of SEQ ID NOs: 1-108.

9. The antisense oligonucleotide of any of claims 1-8, wherein the antisense oligonucleotide comprises 8 to 80 nucleotides in length.

10. The antisense oligonucleotide of any of claims 1-9, wherein the antisense oligonucleotide comprises 15 to 25 nucleotides in length.

11. The antisense oligonucleotide of any of claims 1-10, wherein the antisense oligonucleotide comprises 18 to 20 nucleotides in length.

12. The antisense oligonucleotide of any of claims 1-11, wherein the antisense oligonucleotide comprises one or more modified nucleotides.

13. The antisense oligonucleotide of claim 12, wherein the one or more modified nucleotides comprise a modification of a ribose group, a phosphate group, a nucleobase, or a combination thereof.

14. The antisense oligonucleotide of claim 13, wherein the modification of the ribose group comprises 2′-O-methyl, 2′-fluoro, 2′-deoxy, 2′-O-(2-methoxyethyl) (MOE), 2′-O-alkyl, 2′-O-alkoxy, 2′-O-alkylamino, 2′-NH₂, a constrained nucleotide, or a combination thereof.

15. The antisense oligonucleotide of claim 14, wherein the constrained nucleotide comprises a locked nucleic acid (LNA), an ethyl-constrained nucleotide, a 2′-(S)-constrained ethyl (S-cEt) nucleotide, a constrained MOE, a 2′-O,4′-C-aminomethylene bridged nucleic acid (2′,4′-BNA^NC), an alpha-L-locked nucleic acid, a tricyclo-DNA, or a combination thereof.

16. The antisense oligonucleotide of claim 15, wherein the modification of the ribose group comprises 2′-O-(2-methoxyethyl) (MOE).

17. The antisense oligonucleotide of claim 13, wherein the modification of the phosphate group comprises a phosphorothioate, a phosphonoacetate (PACE), a thiophosphonoacetate (thioPACE), an amide, a triazole, a phosphonate, a phosphotriester modification, or a combination thereof.

18. The antisense oligonucleotide of claim 17, wherein the modification of the phosphate group comprises phosphorothioate.

19. The antisense oligonucleotide of claim 13, wherein the modification of the nucleobase group comprises 2-thiouridine, 4-thiouridine, N⁶-methyladenosine, pseudouridine, 2,6-diaminopurine, inosine, thymidine, 5-methylcytosine, 5-substituted pyrimidine, isoguanine, isocytosine, halogenated aromatic groups, or a combination thereof.

20. The antisense oligonucleotide of claim 19, wherein the modification of the nucleobase group comprises 5-methylcytosine.

21. The antisense oligonucleotide of any of claims 1-20, wherein the antisense oligonucleotide further comprises a ligand.

22. The antisense oligonucleotide of any one of claims 1-21, comprising a sequence modification pattern of

XsXsXsXsXsXsXsXsXsXsXsXsXsXsXsXsXsXs, XsXsXsXsXsXsXsXsXsXsXsXsXsXsXsXsXsXsXs, or XsXsXsXsXsXsXsXsXsXsXsXsXsXsXsXsXsXsXsXs,

wherein:

s represents a phosphorothioate internucleoside linkage; and

X represents an adenosine, a guanosine, a cytidine, or a thymine comprising a 2′-O-(2-methoxyethyl) modification.

23. The antisense oligonucleotide of any one of claims 1-22, comprising a nucleotide sequence selected from the group consisting of SEQ ID NOs: 110-127.

24. The antisense oligonucleotide of any one of claims 1-22, wherein the antisense oligonucleotide increases the level of a functional SLC6A1 RNA transcript in a cell that contains the antisense oligonucleotide by about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 100%, or more, relative to a cell that does not contain the antisense oligonucleotide.

25. An antisense oligonucleotide comprising a region of complementarity to a SLC6A1 RNA transcript target region corresponding to a nucleotide sequence of any of SEQ ID NOs: 1-108.

26. The antisense oligonucleotide of claim 25, wherein the target region comprises a non-productive splice site.

27. The antisense oligonucleotide of claim 25, wherein binding of the antisense oligonucleotide to the target region increases the expression of a functional protein encoded by the SLC6A1 RNA transcript in a cell.

28. The antisense oligonucleotide of claim 27, wherein the protein comprises GABA Transporter 1 (GAT-1).

29. The antisense oligonucleotide of claim 27, wherein the cell comprises an SLC6A1 expressing cell.

30. The antisense oligonucleotide of claim 29, wherein the cell comprises a neuronal cell and/or an astrocyte.

31. The antisense oligonucleotide of claim 30, wherein the neuronal cell comprises a GABAergic neuronal cell.

32. The antisense oligonucleotide of any of claims 25-31, wherein the antisense oligonucleotide comprises 8 to 80 nucleotides in length.

33. The antisense oligonucleotide of any of claims 25-32, wherein the antisense oligonucleotide comprises 15 to 25 nucleotides in length.

34. The antisense oligonucleotide of any of claims 25-33, wherein the antisense oligonucleotide comprises 18 to 20 nucleotides in length.

35. The antisense oligonucleotide of any of claims 25-34, wherein the antisense oligonucleotide comprises one or more modified nucleotides.

36. The antisense oligonucleotide of claim 35, wherein the one or more modified nucleotides comprise a modification of a ribose group, a phosphate group, a nucleobase, or a combination thereof.

37. The antisense oligonucleotide of claim 36, wherein the modification of the ribose group comprises 2′-methyl, 2′-fluoro, 2′-deoxy, 2′-O-(2-methoxyethyl) (MOE), 2′-O-alkyl, 2′-O-alkoxy, 2′-O-alkylamino, 2′-NH₂, a constrained nucleotide, or a combination thereof.

38. The antisense oligonucleotide of claim 37, wherein the constrained nucleotide comprises a locked nucleic acid (LNA), an ethyl-constrained nucleotide, a 2′-(S)-constrained ethyl (S-cEt) nucleotide, a constrained MOE, a 2′-O,4′-C-aminomethylene bridged nucleic acid (2′,4′-BNA^NC), an alpha-L-locked nucleic acid, a tricyclo-DNA, or a combination thereof.

39. The antisense oligonucleotide of claim 38, wherein the modification of the ribose group comprises 2′-O-(2-methoxyethyl) (MOE).

40. The antisense oligonucleotide of claim 36, wherein the modification of the phosphate group comprises a phosphorothioate, a phosphonoacetate (PACE), a thiophosphonoacetate (thioPACE), an amide, a triazole, a phosphonate, a phosphotriester modification, or a combination thereof.

41. The antisense oligonucleotide of claim 40, wherein the modification of the phosphate group comprises phosphorothioate.

42. The antisense oligonucleotide of claim 36, wherein the modification of the nucleobase group comprises 2-thiouridine, 4-thiouridine, N⁶-methyladenosine, pseudouridine, 2,6-diaminopurine, inosine, thymidine, 5-methylcytosine, 5-substituted pyrimidine, isoguanine, isocytosine, halogenated aromatic groups, or a combination thereof.

43. The antisense oligonucleotide of claim 42, wherein the modification of the nucleobase group comprises 5-methylcytosine.

44. The antisense oligonucleotide of any of claims 25-43, wherein the antisense oligonucleotide further comprises a ligand.

45. The antisense oligonucleotide of any of claims 25-44, comprising a sequence modification pattern of

wherein:

s represents a phosphorothioate internucleoside linkage; and

46. The antisense oligonucleotide of any one of claims 25-45, comprising a nucleotide sequence selected from the group consisting of SEQ ID NOs: 110-127.

47. The antisense oligonucleotide of any one of claims 25-46, wherein the antisense oligonucleotide increases the level of a functional SLC6A1 RNA transcript in a cell that contain the antisense oligonucleotide by about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 100%, or more, relative to a cell that does not contain the antisense oligonucleotide.

48. A multimeric antisense oligonucleotide compound comprising two or more antisense oligonucleotides of any of claims 1-47, wherein the two or more antisense oligonucleotides are linked together through a linker.

49. The multimeric antisense oligonucleotide compound of claim 48, wherein the linker comprises a cleavable linker.

50. The multimeric antisense oligonucleotide compound of claim 49, wherein the cleavable linker degrades when cleaved.

51. The multimeric antisense oligonucleotide compound of claims 49 or 50, wherein the cleavable linker comprises a nuclease-cleavable linker comprising a phosphodiester linkage.

52. The multimeric antisense oligonucleotide compound of claim 51, wherein the nuclease-cleavable linker comprises from about 2 to about 8 nucleotides.

53. The multimeric antisense oligonucleotide compound of claims 51 or 52, wherein the nuclease-cleavable linker comprises about 6 nucleotides.

54. The multimeric antisense oligonucleotide compound of any one of claims 48-53, wherein the cleavable linker is cleaved under reducing conditions or changing pH conditions.

55. The multimeric antisense oligonucleotide compound of any one of claims 48-54, wherein the cleavable linker is cleaved by an intracellular or endosomal nuclease.

56. The multimeric antisense oligonucleotide compound of any one of claims of claims 48-55, wherein the cleavable linker is cleaved by an intracellular or endosomal protease.

57. The multimeric antisense oligonucleotide compound of any one of claims of claims 48-55, wherein at least one of the antisense oligonucleotides comprises a nucleotide sequence selected from the group consisting of SEQ ID NOs: 110-127.

58. A combination comprising two or more antisense oligonucleotides that bind to two or more target regions in an SLC6A1 RNA transcript, wherein the two or more target regions comprise a splice modulatory element.

59. The combination of claim 58, wherein two or more antisense oligonucleotides are linked together through a linker.

60. The combination of claim 59, wherein the linker comprises a cleavable linker.

61. The combination of claim 60, wherein the cleavable linker degrades when cleaved.

62. The combination of claims 60 or 61, wherein the cleavable linker comprises a nuclease-cleavable linker comprising a phosphodiester linkage.

63. The combination of claim 62, wherein the nuclease-cleavable linker comprises from about 2 to about 8 nucleotides.

64. The combination of claims 62 or 63, wherein the nuclease-cleavable linker comprises about 6 nucleotides.

65. The combination of any one of claims 58-64, wherein the cleavable linker is cleaved under reducing conditions or changing pH conditions.

66. The combination of any one of claims 58-65, wherein the cleavable linker is cleaved by an intracellular or endosomal nuclease.

67. The combination of any one of claims 58-66, wherein the cleavable linker is cleaved by an intracellular or endosomal protease.

68. The combination of any one of claims 58-67, wherein at least one antisense oligonucleotide comprises a region of complementarity to a SLC6A1 RNA transcript target region corresponding to a nucleotide sequence of any of SEQ ID NOs: 1-108.

69. The combination of any one of claims 58-68, wherein at least one of the antisense oligonucleotides comprises a nucleotide sequence selected from the group consisting of SEQ ID NOs: 110-127.

70. A method of treating a disease or disorder characterized by haploinsufficiency of a SLC6A1 gene, comprising administering to a subject in need thereof the antisense oligonucleotide, the multimeric antisense oligonucleotide compound, or combination of antisense oligonucleotides of any of claims 1-69, and treating the disease or disorder.

71. A method of treating a disease or disorder characterized by reduced expression of a functional GAT-1 protein encoded by a SLC6A1 gene, comprising administering to a subject in need thereof the antisense oligonucleotide, the multimeric antisense oligonucleotide compound, or combination of antisense oligonucleotides of any of claims 1-69, and treating the disease or disorder.

72. The method of claims 70 or 71, wherein the disease or disorder comprises myoclonic-atonic epilepsy (MAE), epilepsy, epileptic encephalopathy, seizures, autism spectrum disorders, intellectual disability, or a combination thereof.

73. The method of claims 70 or 71, comprising administering the antisense oligonucleotide, the multimeric antisense oligonucleotide compound, or combination of antisense oligonucleotides to a brain of the subject.

74. The method of claims 70 or 71, comprising administering the antisense oligonucleotide, the multimeric antisense oligonucleotide compound, or combination of antisense oligonucleotides by intrathecal, intraventricular, intrastriatal injection or infusion, or a combination thereof.

75. The method of claim 74, wherein the injection or infusion comprises administration using an Ommaya reservoir, an intrathecal catheter, or a combination thereof.

76. A method of increasing expression of a functional SLC6A1 RNA transcript in a cell, the method comprising contacting the cell with the antisense oligonucleotide, the multimeric antisense oligonucleotide compound, or combination of antisense oligonucleotides of any of claims 1-69, thereby increasing the expression of a functional transcript of the SLC6A1 RNA transcript in a cell.

77. The method of claim 76, wherein expression is increased by about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 100%, or more, relative to a cell that is not contacted with the antisense oligonucleotide, the multimeric antisense oligonucleotide compound, or combination of antisense oligonucleotides.

78. A method of increasing expression of a protein encoded by a SLC6A1 RNA transcript in a cell, the method comprising contacting a cell with the antisense oligonucleotide, the multimeric antisense oligonucleotide compound, or combination of antisense oligonucleotides of any of claims 1-69, thereby increasing expression of the protein.

79. The method of claim 78, wherein expression is increased by about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 100%, or more, relative to a cell that is not contacted with the antisense oligonucleotide, the multimeric antisense oligonucleotide compound, or combination of antisense oligonucleotides.

80. The method of claim 78, wherein the protein comprises GABA Transporter 1 (GAT-1).

81. The method of any of claims 77-80, wherein the cell comprises an SLC6A1 expressing cell.

82. The method of any of claims 77-80, wherein the cell comprises a neuronal cell and/or an astrocyte.

83. The method of claim 82, wherein the neuronal cell comprises a GABAergic neuronal cell.

84. An antisense oligonucleotide that increases expression of a functional protein from the SLC6A1 gene by targeting the 5′- and/or 3′-untranslated regions of the SLC6A1 transcript.

85. The antisense oligonucleotide of claim 85, wherein the antisense oligonucleotide inhibits translation initiation from an upstream open reading frame to increase translation efficiency from the primary open reading frame.

86. The antisense oligonucleotide of claim 85, wherein the antisense oligonucleotide increases mRNA stability.