Modulation of SYNGAP1 gene transcription using antisense oligonucleotides targeting regulatory RNAs

Abstract

Described herein are methods for modulating SYNGAP1 gene transcription using antisense oligonucleotides (ASOs) that target regulatory RNAs, such as promoter-associated RNAs, enhancer RNAs, and natural antisense transcripts (NATs). These methods increase SYNGAP1 mRNA and protein expression, and are therefore useful for treating diseases associated with SYNGAP1 mutations.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Provisional Application No. 63 / 385,695, filed December 1, 2022, which is incorporated herein by reference in its entirety.

[0002] Sequence Listing This application contains a Sequence Listing, which is incorporated herein by reference in its entirety. The XML file, created on XX / 20XX, is named CTC-032WO_SL.xml and is X,XXX,XXX bytes in size. [Background technology]

[0003] background Transcription factors bind to specific sequences in promoter and enhancer DNA elements to regulate gene transcription. It has recently been reported that active promoter and enhancer elements are themselves transcribed to generate non-coding regulatory RNAs (regRNAs), such as promoter-associated RNAs (paRNAs) and enhancer RNAs (eRNAs) (see Sartorelli and Lauberth, Nat. Struct. Mol. Biol. (2020) 27:521-28). Unlike coding RNAs, regRNAs are transcribed bidirectionally. Various models for the function of regRNAs have been proposed, including nucleosome remodeling (see Mousavi et al., Mol. Cell (2013) 51(5):606-17 (Non-Patent Document 2)), regulation of enhancer-promoter loops (see Lai et al., Nature (2013) 494(7438):497-501 (Non-Patent Document 3)), and direct interaction with transcriptional regulators (see Sigova et al., Science (2015) 350,978-81 (Non-Patent Document 4)).

[0004] Approximately 1-2% of all cases of intellectual disability are due to mutations in the SYNGAP1 gene. SynGAP1-associated intellectual disability (SynGAP1-ID) is a neurological disorder characterized by moderate to severe impairment in intellectual development accompanied by delayed psychomotor development. Mental retardation autosomal dominant 5 (MRD5), also known as intellectual disability autosomal dominant 5, is SYNGAP1-ID caused by autosomal recessive mutations in the SYNGAP1 gene. SynGAP1-associated nonsyndromic intellectual disability (NSID) is the result of heterozygous pathogenic variants in SYNGAP1 (approximately 89% of cases) or deletions of 6p21.3 (approximately 11% of cases). SYNGAP1-associated NSID presents with moderate to severe cognitive impairment, mild hypotonia, global developmental delay, delayed language development, sleep disturbances, oral apraxia, inattention, impulsivity, physical aggression, mood swings, irritability, and rigidity. Furthermore, 94-98% of cases of MRD5- and SYNGAP1-related NSIDs also present with epilepsy. There is no therapy or treatment for MRD5- or SYNGAP1-related NSIDs. Patient treatment is limited to epilepsy treatment and behavioral management. Therefore, additional therapeutic agents are needed.

[0005] Gene expression is generally known as an undruggable biological process. Despite efforts to understand the biology of gene transcription and regRNA, clinically relevant methods for regulating gene expression are limited. There remains a need for novel and useful methods for treating diseases associated with abnormal gene expression. [Prior art documents] [Non-patent literature]

[0006] [Non-Patent Document 1] Sartorelli and Lauberth, Nat. Structure. Mol. Biol. (2020) 27:521-28 [Non-patent document 2] Mousavi et al.,Mol.Cell(2013)51(5):606-17 [Non-patent document 3] Lai et al.,Nature(2013)494(7438):497-501 [Non-patent document 4] Sigova et al.,Science(2015)350,978-81 Summary of the Invention

[0007] overview In one aspect, provided herein is an antisense oligonucleotide (ASO) complementary to at least 8 consecutive nucleotides of a regulatory RNA of human SYNGAP1, wherein the regulatory RNA has a nucleotide sequence selected from the group consisting of SEQ ID NOs: 1-7.

[0008] In another aspect, provided herein is an antisense oligonucleotide (ASO) complementary to at least 8 consecutive nucleotides of a regulatory RNA of human SYNGAP1, wherein the regulatory RNA has a nucleotide sequence selected from the group consisting of SEQ ID NO: 4, 5, or 6.

[0009] In some embodiments, the ASO is complementary to a sequence in the regRNA that is no more than 200 nucleotides from the 3' end of the regRNA.

[0010] In some embodiments, the ASO is complementary to a sequence in the regRNA that is no more than 200 nucleotides from the 5' end of the regRNA.

[0011] In some embodiments, the regRNA is not polyadenylated RNA.

[0012] In some embodiments, the regulatory RNA has the nucleotide sequence of SEQ ID NO: 1 and the ASO comprises a nucleotide sequence selected from the group consisting of SEQ ID NOs: 10-59, 220-250, 261-267, 272-278, 526-528, 542-591, 702-728, 729-735-741, 988-990, and 1004-2961.

[0013] In some embodiments, the regulatory RNA has the nucleotide sequence of SEQ ID NO: 2 and the ASO comprises a nucleotide sequence selected from the group consisting of SEQ ID NOs: 60-73.

[0014] In some embodiments, the regulatory RNA has the nucleotide sequence of SEQ ID NO: 3 and the ASO comprises a nucleotide sequence selected from the group consisting of SEQ ID NOs: 74-109, 251-260, 268-271, and 279.

[0015] In some embodiments, the regulatory RNA has the nucleotide sequence of SEQ ID NO: 4 or 6, and the ASO comprises a nucleotide sequence selected from the group consisting of SEQ ID NOs: 110-219, 280-525, 529-541, 592-701, 742-891, 906-987, 991-1003, and 2962-4852.

[0016] In some embodiments, the regulatory RNA has the nucleotide sequence of SEQ ID NO: 5 and the ASO comprises a nucleotide sequence selected from the group consisting of SEQ ID NOs: 14-59, 220-250, 261-267, 272-278, 526-528, 542-591, 702-728, 729-735-741, 988-990, and 1007-2961.

[0017] In some embodiments, the ASO comprises a nucleotide sequence of at least 8 consecutive nucleotides from chr6:33419695 to 33419939.

[0018] In some embodiments, the ASO comprises a nucleotide sequence of at least 8 contiguous nucleotides from chr6:33453987 to 33454269.

[0019] In some embodiments, the ASO comprises a nucleotide sequence of at least 8 consecutive nucleotides from chr6:33419674 to 33419940.

[0020] In some embodiments, the ASO is no more than 50, 40, 30, 25, 20, 18, or 16 nucleotides in length.

[0021] In some embodiments, the ASO comprises an RNA polynucleotide that includes one or more chemical modifications.

[0022] In some embodiments, at least 3, 4, or 5 nucleotides at the 5' end and at least 3, 4, or 5 nucleotides at the 3' end of the ASO comprise ribonucleotides having one or more chemical modifications.

[0023] In some embodiments, the one or more chemical modifications are 2'-OC 1-4 Alkyl, e.g., 2'-O-methyl (2'-OMe), 2'-deoxy (2'-H), 2'-OC 1-3 Alkyl-OC 1-3 Included are nucleotide sugar modifications including one or more of alkyl, e.g., 2'-methoxyethyl ("2'-MOE"), 2'-fluoro ("2'-F"), 2'-amino ("2'-NH"), 2'-arabinosyl ("2'-arabino") nucleotides, 2'-F-arabinosyl ("2'-F-arabino") nucleotides, 2'-locked nucleic acid ("LNA") nucleotides, 2'-amide bridged nucleic acid (AmNA), 2'-unlocked nucleic acid ("ULNA") nucleotides, L-form sugars ("L-sugars"), 4'-thioribosyl nucleotides, constrained ethyl (cET), 2'-fluoro-arabino (FANA), or thiomorpholino.

[0024] In some embodiments, the one or more chemical modifications are phosphorothioates ("PS" or (P(S))), phosphoramidates (P(NR1R2), e.g., dimethylaminophosphoramidate (P(N(CH3)2)), phosphonocarboxylates (P(CH2) n COOR), e.g., phosphonoacetate "PACE" (P(CHCOO - )), thiophosphonocarboxylate ((S)P(CH2) nCOOR), e.g., thiophosphonoacetate "thioPACE" ((S)P(CHCOO - )), alkyl phosphonates (P(C 1-3 The internucleotide linkage modifications include one or more of the following: alkyl (P(CH), methylphosphonate (P(CH), boranophosphonate (P(BH), or phosphorodithioate (P(S)).

[0025] In some embodiments, the one or more chemical modifications are 2-thiouracil ("2-thioU"), 2-thiocytosine ("2-thioC"), 4-thiouracil ("4-thioU"), 6-thioguanine ("6-thioG"), 2-aminoadenine ("2-aminoA"), 2-aminopurine, pseudouracil, hypoxanthine, 7-deazaguanine, 7-deaza-8-azaguanine, 7-deazaadenine, 7-deaza-8-azaadenine, 5-methylcytosine ("5-methylC"), 5-methyluracil ("5-methylU"), 5-hydroxymethylcytosine, 5-hydroxymethyluracil, 5,6-dehydrouracil , 5-propynylcytosine, 5-propynyluracil, 5-ethynylcytosine, 5-ethynyluracil, 5-allyluracil ("5-allylU"), 5-allylcytosine ("5-allylC"), 5-aminoallyluracil ("5-aminoallylU"), 5-aminoallyl-cytosine ("5-aminoallylC"), abasic nucleotides, Z bases, P bases, unstructured nucleic acids ("UNA"), isoguanine ("isoG"), isocytosine ("isoC"), glycerol nucleic acid (GNA), or thiophosphoramidate morpholino (TMO).

[0026] In some embodiments, the one or more chemical modifications include 2'-O-methoxyethyl, 5-methyl on cytidine, locked nucleic acid (LNA), phosphodiester (PO) internucleotide linkage, or phosphorothioate (PS) internucleotide linkage.

[0027] In some embodiments, the ASO further comprises a GalNAc moiety, optionally a GalNAc3 moiety.

[0028] In some embodiments, the ASO does not contain 10 or more contiguous nucleotides of unmodified DNA.

[0029] In some embodiments, the ASO does not contain deoxyribonucleotides.

[0030] In some embodiments, the ASO does not include unmodified ribonucleotides.

[0031] In some embodiments, the length of the ASO is 5×n+5 nucleotides (n is an integer greater than or equal to 3), the nucleotide at position 5×m is an LNA-modified ribonucleotide (m is an integer from 1 to n), and the nucleotides at the remaining positions are 2′-O-methoxyethyl-modified ribonucleotides.

[0032] In some embodiments, the length of the ASO is 3×n+2 nucleotides (n is an integer greater than or equal to 6), the nucleotide at position 3×m is an LNA-modified ribonucleotide (m is an integer from 1 to n), and the nucleotides at the remaining positions are 2′-O-methoxyethyl-modified ribonucleotides.

[0033] In some embodiments, each ribonucleotide of the ASO is modified with 2'-O-methoxyethyl.

[0034] In some embodiments, each nucleotide of the ASO is a ribonucleotide that is modified with 2'-O-methoxyethyl.

[0035] In some embodiments, the ASO comprises 10 or more contiguous nucleotides of unmodified DNA flanked at each of the 5' and 3' ends by at least 3 nucleotides of modified ribonucleotides.

[0036] In some embodiments, each cytidine in the ASO is modified with a 5-methyl.

[0037] In some embodiments, the regRNA is a natural antisense transcript (NAT).

[0038] In some embodiments, the regRNA is a paRNA.

[0039] In another aspect, provided herein is a pharmaceutical composition comprising an ASO disclosed herein and a pharmaceutically acceptable carrier or excipient carrier.

[0040] In another aspect, provided herein is a method for increasing transcription of SYNGAP1 in a human cell, the method comprising contacting the cell with an ASO disclosed herein or a pharmaceutical composition disclosed herein.

[0041] In some embodiments, the cell is a neuron.

[0042] In some embodiments, the ASO increases the amount of a regulatory RNA in a cell.

[0043] In some embodiments, the ASO increases the stability of a regulatory RNA in a cell.

[0044] In some embodiments, the method results in an increase in SYNGAP1 mRNA in the cell.

[0045] In some embodiments, the method results in an increase in SYNGAP1 protein in the cell.

[0046] In one aspect, provided herein is a method of treating a disease or disorder, comprising administering to a subject in need thereof an effective amount of an ASO disclosed herein or a pharmaceutical composition disclosed herein.

[0047] In some embodiments, the disease or disorder is a SYNGAP1-associated disease or disorder.

[0048] In some embodiments, the SYNGAP1-associated disorder is SynGAP1-associated intellectual disability (ID), mental retardation, autosomal dominant 5 (MRD5), or SynGAP1-associated non-syndromic intellectual disability (NSID).

[0049] In some embodiments, the disease or disorder is a disorder of the central nervous system (CNS) or a disorder of the peripheral nervous system (PNS).

[0050] In some embodiments, the disease or disorder is an affective disorder (e.g., depression), schizophrenia, Alzheimer's disease, Parkinson's disease, Huntington's disease, an autism spectrum disorder (ASD) (e.g., Asperger's syndrome, autism, pervasive developmental disorder not otherwise specified (PDD-NOS)), or a CNS or PNS trauma (e.g., a neurological abnormality associated with brain or spinal cord ischemia or trauma, stroke, or surgery or anesthesia).

[0051] In some embodiments, administration of the ASO modulates SYNGAP1 gene expression in a subject (e.g., in cells or tissues of the subject) compared to baseline levels before administration.

[0052] In some embodiments, the ASO increases the amount of a regulatory RNA in the subject's cells.

[0053] In some embodiments, the ASO increases the stability of a regulatory RNA in a cell of a subject.

[0054] In some embodiments, administration of the ASO increases SYNGAP1 gene expression in the subject's cells compared to baseline levels before administration.

[0055] In some embodiments, the cell is a neuron. [Brief explanation of the drawings]

[0056] [Figure 1A]

[0013] Figure 1 shows an exemplary schematic diagram of the eRNA, paRNA, mRNA, and natural antisense transcript (NAT) of a gene on a chromosome. eRNA, paRNA, and NAT are all non-coding RNAs. eRNA is transcribed bidirectionally from the enhancer of a gene. paRNA is transcribed from the promoter of a gene in the same direction as the mRNA but in the antisense direction. NAT is transcribed in the antisense direction from its own downstream promoter, resulting in a transcript that at least partially overlaps with the mRNA. [Figure 1B] FIG. 1 shows an exemplary schematic of the interaction of regRNA with enhancer and promoter regions to recruit transcription and regulatory factors that regulate gene expression. [Figure 2-1] Exemplary ASO sequences and chemistries targeting human SYNGAP1 regRNA are provided. Light gray shading indicates 2'-MOE; * indicates 5Me-C; dark gray shading indicates LNA; dark gray lines indicate phosphodiester (PO) linkages; and white indicates DNA. [Figure 2-2] See description of Figure 2-1. [Figure 2-3] See description of Figure 2-1. [Figure 2-4] See description of Figure 2-1. [Figure 2-5] See description of Figure 2-1. [Figure 2-6] See description of Figure 2-1. [Figure 2-7] See description of Figure 2-1. [Figure 3] 1 shows that SYNGAP1 regRNAs RR86 and RR93 were detected in HEK293 and SK-N-AS cells, as well as human brain samples, by RNA capture sequencing and qPCR. [Figure 4A] Shown are SYNGAP1 mRNA levels in HEK293 cells after treatment with the indicated SYNGAP1 regRNA-targeting ASOs, or a gapmer non-targeting control (NTC) ASO (CO-1588), a stereogenic NTC ASO (CO-1589), or an untreated control ("UTC" or "no ASO"). [Figure 4B] Shown are SYNGAP1 mRNA levels in SK-N-AS cells after treatment with the indicated SYNGAP1 regRNA-targeting ASOs, or a gapmer non-targeting control (NTC) ASO (CO-1588), a stereogenic NTC ASO (CO-1589), or an untreated control ("UTC" or "no ASO"). [Figure 4C] Shows a dose-dependent increase in SYNGAP1 mRNA levels in HEK293 cells after treatment with the indicated SYNGAP1 regRNA-targeting ASOs compared to cells treated with a gapmer NTC ASO (control; CO-1588). [Figure 4D] Shows a dose-dependent increase in SYNGAP1 mRNA levels in SK-N-AS cells after treatment with the indicated SYNGAP1 regRNA-targeting ASOs compared to cells treated with a gapmer NTC ASO (control; CO-1588). [Figure 4E] 1 shows a dose-dependent increase in SYNGAP1 mRNA levels in HEK293 cells after treatment with the indicated ASOs compared to cells treated with a gapmer NTC ASO (control; CO-1588). [Figure 4F] 1 shows a dose-dependent increase in SYNGAP1 mRNA levels in SK-N-AS cells after treatment with the indicated ASOs compared to cells treated with a gapmer NTC ASO (control; CO-1588). [Figure 5] Shown are SYNGAP1 mRNA levels in SK-N-AS and HEK293 cells after treatment with the indicated SYNGAP1 regRNA-targeting ASOs, or a gapmer non-targeting control (NTC) ASO (CO-1588), a stereogenic NTC ASO (CO-1589), or an untreated control ("UTC"). [Figure 6] Shows dose-dependent upregulation of SYNGAP1 mRNA levels in both SK-N-AS and HEK293 cells after treatment with the indicated SYNGAP1 regRNA-targeting ASOs compared to cells treated with an untreated control ("UTC") or a gapmer NTC ASO (control; CO-1588). [Figure 7] SYNGAP1 mRNA levels in neurons differentiated from human induced pluripotent stem cells after treatment with the indicated concentrations of SYNGAP1 regRNA-targeting ASO or gapmer NTC ASO (CO-1588; control). DETAILED DESCRIPTION OF THE INVENTION

[0057] Detailed Description The present disclosure provides antisense oligonucleotides (ASOs) that target regulatory RNAs, such as promoter-associated (paRNA) RNAs and enhancer RNAs (eRNAs), and methods of using these ASOs to regulate gene expression. These methods are useful for regulating the level of a gene product, for example, regulating the expression level of SYNGAP1, thereby treating SYNGAP1-associated disorders (e.g., diseases associated with SYNGAP1 mutations), such as mental retardation, autosomal dominant 5 (MRD5), and SynGAP1-associated nonsyndromic intellectual disability (NSID), or other diseases or disorders.

[0058] Various aspects of the compositions and methods described in this application are described in the following sections.

[0059] I. Definition To facilitate understanding of this application, a number of terms and phrases are defined below.

[0060] As used herein, the terms "a" and "an" mean "one or more" and include pluralities unless the context is inappropriate.

[0061] As used herein, the term "SYNGAP1" or "synaptic Ras GTPase-activating protein 1" refers, when used in reference to the human gene, to the gene with NCBI Gene ID: 8831 or Hugo Gene Nomenclature Committee (HGNC) ID: 11497, or, when used in reference to the human version of the protein, to the protein with UniProt accession number Q96PV0 (human), and, when used in reference to the mouse gene, to the gene with NCBI Gene ID: 240057, or, when used in reference to the mouse version of the protein, to the protein with UniProt accession number J3QQ18 (mouse), as well as related isoforms and orthologs of the foregoing. SYNGAP1 is a protein in the postsynaptic density (PSD) of glutamatergic neurons that interacts with PSD95 and SAP102 and can positively or negatively regulate the density of N-methyl-D-aspartate (NMDA) and α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors at glutamatergic synapses, and negatively regulates small G protein signaling downstream of glutamate receptor activation (see, e.g., Jeyabalan et al. (2016) Front. Cell Neurosci. 10:32, which is incorporated herein by reference). In some embodiments, the SYNGAP1 protein comprises a SYNGAP1 isoform (e.g., N-terminal isoforms A, B, and C of human SYNGAP1 and / or C-terminal isoforms alpha 1 (α1), alpha 2 (α2), beta (β), or gamma (γ)). In some embodiments, the SYNGAP1 protein comprises an isoform selected from SYNGAP1 Aα1, SYNGAP1 Aα2, SYNGAP1 Aβ, SYNGAP1 Aγ, SYNGAP1 Bα1, SYNGAP1 Bα2, SYNGAP1 Bβ, SYNGAP1 Bγ, SYNGAP1 Cα1, SYNGAP1 Cα2, SYNGAP1 Cβ, SYNGAP1 Cγ, or any combination of the foregoing isoforms.

[0062] As used herein, the terms "regulatory RNA" and "regRNA" are used interchangeably to refer to non-coding RNA transcribed from a regulatory element of a gene (e.g., a protein-coding gene), where the gene is not itself a non-coding RNA. Exemplary regulatory elements include, but are not limited to, promoters, enhancers, super-enhancers, and natural antisense transcripts. Non-coding RNA transcribed in the antisense direction from a promoter is also referred to as "promoter RNA" or "paRNA." Non-coding RNA transcribed in either the sense or antisense direction from an enhancer or super-enhancer is also referred to as "enhancer RNA" or "eRNA."

[0063] As used herein, the term "nascent RNA" refers to RNA that is still being transcribed or has just been transcribed by RNA polymerase and remains tethered to the DNA that it was transcribed from. RNA that has dissociated from the DNA that it is transcribed from is also called "untethered RNA."

[0064] As used herein, the term "antisense oligonucleotide" or "ASO" refers to a single-stranded oligonucleotide having a nucleotide sequence that hybridizes to a target nucleic acid under appropriate conditions, or a conjugate comprising such a single-stranded oligonucleotide. In some embodiments, the present disclosure encompasses pharmaceutically acceptable salts of any of the ASOs described herein. Suitable pharmaceutically acceptable salts include, but are not limited to, sodium, potassium, calcium, and magnesium salts. In some embodiments, the ASOs provided herein are lyophilized and isolated as a salt (e.g., sodium salt).

[0065] As used herein, in some embodiments, regRNA stability is inversely correlated with the rate of regRNA degradation. In some embodiments, if an ASO increases regRNA stability, it decreases the rate of regRNA degradation. In some embodiments, if an ASO reduces regRNA stability, it increases the rate of regRNA degradation. In some embodiments, the rate of regRNA degradation can be measured by blocking the synthesis of new regRNA and assessing the half-life of existing regRNA.

[0066] As used herein, the terms "subject" and "patient" refer to an organism treated by the methods and compositions described herein. Such organisms preferably include, but are not limited to, mammals (e.g., rodents (e.g., mice), primates, monkeys, horses, cows, pigs, dogs, cats, etc.), and more preferably, humans.

[0067] As used herein, the term "effective amount" refers to an amount of a compound (e.g., a compound of the present application) sufficient to produce a beneficial or desired result. An effective amount can be administered in one or more administrations, applications, or dosages, and is not intended to be limited to a particular formulation or route of administration. As used herein, the term "treating" includes any effect that results in the improvement of a condition, disease, disorder, etc., or the amelioration of symptoms thereof, such as, for example, lowering, reducing, modulating, ameliorating, or eliminating.

[0068] As used herein, the term "pharmaceutical composition" refers to a combination of an active agent with an inert or active carrier that inherently renders the composition suitable for diagnostic or therapeutic use in vivo or ex vivo.

[0069] As used herein, the term "pharmaceutically acceptable carrier" refers to standard pharmaceutical carriers, such as phosphate-buffered saline, water, emulsions (e.g., oil-in-water or water-in-oil emulsions), and any of various types of wetting agents. The compositions may also contain stabilizers and preservatives. For examples of carriers, stabilizers, and adjuvants, see, for example, Martin, Remington's Pharmaceutical Sciences, 15th Ed., Mack Publ. Co., Easton, PA (1975).

[0070] Throughout this description, where compositions are described as having, containing, or comprising particular components, or processes and methods are described as having, containing, or comprising particular steps, it is contemplated that there are also compositions described herein that consist essentially of, or consist of, the recited components, and processes and methods according to this application that consist essentially of, or consist of, the recited process steps.

[0071] As a general matter, compositions specifying percentages are by weight unless otherwise specified. Further, if a variable is not accompanied by a definition, the previous definition of that variable takes precedence.

[0072] II. Antisense Oligonucleotides In some embodiments, the antisense oligonucleotides (ASOs) disclosed herein hybridize to or target regRNA (e.g., eRNA, paRNA, or NAT) transcribed from a regulatory element of the SYNGAP1 gene (also referred to herein as "SYNGAP1 regRNA"). NATe, RNA, and paRNA are understood to be regRNAs that regulate (e.g., promote or upregulate) gene expression (FIG. 1). In some embodiments, the SYNGAP1 regRNA is human SYNGAP1 regRNA. In some embodiments, the SYNGAP1 regRNA is mouse SYNGAP1 regRNA. In certain embodiments, the SYNGAP1 regRNA is eRNA. In certain embodiments, the SYNGAP1 regRNA is paRNA. In certain embodiments, the SYNGAP1 regRNA is NAT. In certain embodiments, the SYNGAP1 regRNA is not polyadenylated RNA.

[0073] eRNAs can be identified using methods known in the art, such as assays for transposase-accessible chromatin using sequencing (ATAC-seq), global run-on sequencing, precision run-on sequencing, cap analysis gene expression, and histone modification analysis (see, e.g., Sartorelli & Lauberth, Nat. Struct Mol. Biol. (2020) 27:521-28; PCT Application Publication No. WO2013 / 177248). paRNAs are RNAs transcribed from the promoter of a target gene in the antisense orientation (the transcript in the sense orientation is the mRNA of the target gene). They can be identified by similar methods, taking into account their specific location and orientation. The nucleotide sequences of exemplary human and mouse SYNGAP1 regRNAs are shown in Table 1 below. Both of these human and mouse SYNGAP1 regRNAs are contemplated as target regRNAs for the ASOs disclosed herein.

[0074] [Table 1] TIFF2025540101000002.tif133165TIFF2025540101000003.tif231165TIFF2025540101000004.tif190165TIFF2025540101000005.tif213165

[0075] The present disclosure describes ASOs that can be used to increase the expression of a target gene, SYNGAP1 (e.g., human SYNGAP1 or mouse SYNGAP1). Without wishing to be bound by theory, this increase in gene expression may result from an increase in the amount or stability of the targeted SYNGAP1 regRNA, or from interference with a regRNA-associated repressor that inhibits gene expression, thereby increasing SYNGAP1 gene expression. These ASOs differ from previously described ASOs designed to inhibit eRNA (see, e.g., PCT Application Publication Nos. WO2013 / 177248 and WO2017 / 075406). Without wishing to be bound by theory, it is hypothesized that the ability of the ASOs to upregulate SYNGAP1 gene expression results from the selection of the target sequence in the regRNA and / or chemical modification of the ASO.

[0076] The increase in SYNGAP1 gene expression is at least about 0.1%, 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 105%, 110%, 115%, 120%, 125%, 130%, 135%, 140%, 145%, 150%, 160%, 165%, 170%, 175%, 180%, 185%, 190%, 200%, 210%, 215%, 220%, 225%, 230%, 235%, 240%, 245%, 250%, 255%, 260%, 265%, 270%, 275%, 280%, 285%, 290%, 300%, 310%, 315%, 320%, 325%, 330%, 335%, 340%, 345%, 350%, 360%, 365%, 370%, 375%, 380%, 385%, 390%, 400%, 410%, 415%, 420%, 425%, 430%, 435%, 440%, 445%, 450%, 455%, 460%, 465%, 470%, 475%, 480%, 485%, 490%, 500%, 510%, 515%, 520%, 525%, 530%, 53 The increase in expression may be 155%, 160%, 165%, 170%, 175%, 180%, 185%, 190%, 195%, 200%, 205%, 210%, 215%, 220%, 225%, 230%, 235%, 240%, 245%, 250%, 255%, 260%, 265%, 270%, 275%, 280%, 285%, 290%, 295%, 300%, 350%, 400%, 450%, 500%, 550%, 600%, 650%, 700%, 750%, 800%, 850%, 900%, 950%, or 1000% or more. The increase in SYNGAP1 gene expression can be at least about 0.1-fold, 0.5-fold, 1-fold, 1.5-fold, 2-fold, 2.5-fold, 3-fold, 3.5-fold, 4-fold, 4.5-fold, 5-fold, 6-fold, 5.5-fold, 6-fold, 6.5-fold, 7-fold, 7.5-fold, 8-fold, 8.5-fold, 9-fold, 9.5-fold, 10-fold or more increase in expression compared to baseline gene expression, gene expression before treatment, or gene expression after treatment with a control ASO.

[0077] ASOs that hybridize to (e.g., are complementary to) a portion of any of the regulatory RNAs provided herein (e.g., as described in Table 1 above) are contemplated by the present disclosure. In some embodiments, the regulatory RNA has the nucleotide sequence of any one of SEQ ID NOs: 1, 2, 3, 4, 5, 6, and 7.

[0078] ASO sequence In certain embodiments, the ASO disclosed herein is complementary to a sequence in SYNGAP1 regRNA (e.g., a SYNGAP1 regRNA provided in Table 1) that is no more than 300, 250, 200, 150, 100, 50, 40, 30, 20, 10, 8, 5, or 1 nucleotide from the 5' or 3' end of the SYNGAP1 regRNA. In certain embodiments, the ASO disclosed herein is complementary to a sequence in SYNGAP1 regRNA that is no more than 300, 250, 200, 150, 100, 50, 40, 30, 20, or 10 nucleotides from the 5' end of the SYNGAP1 regRNA (i.e., the 5'-most nucleotide of the regRNA sequence that forms a duplex with the ASO is no more than 300, 250, 200, 150, 100, 50, 40, 30, 20, 10, 8, 5, or 1 nucleotide from the 5' end of the SYNGAP1 regRNA). In certain embodiments, the ASO disclosed herein is complementary to a sequence in SYNGAP1 regRNA that is no more than 300, 250, 200, 150, 100, 50, 40, 30, 20, 10, 8, 5, or 1 nucleotide from the 3' end of the SYNGAP1 regRNA (i.e., the 3'-most nucleotide of the regRNA sequence that forms a duplex with the ASO is no more than 300, 250, 200, 150, 100, 50, 40, 30, 20, or 10 nucleotides from the 3' end of the SYNGAP1 regRNA). In some embodiments, provided herein are ASOs comprising a nucleotide sequence that is complementary to at least 8 nucleotides (e.g., 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, or 40 nucleotides) of a portion of a SYNGAP1 regRNA provided herein (e.g., a regRNA that includes or consists of a portion of the full-length nucleotide sequence provided in any one of SEQ ID NOs: 1, 2, 3, 4, 5, 6, and 7).In some embodiments, provided herein is an ASO comprising or consisting of a nucleotide sequence that is complementary to at least 8 nucleotides (e.g., 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, or 40 nucleotides) of the SYNGAP1 regRNA identified herein as RR86_v1 (SEQ ID NO: 1). In some embodiments, the 3' portion of SEQ ID NO: 1 (e.g., nucleotides 185 to 467, 186 to 467, 187 to 467, 188 to 467, 189 to 467, 190 to 467, 191 to 467, 192 to 467, 193 to 467, 194 to 467, 195 to 467, 196 to 467, 197 to 467, 198 to 467, 199 to 467, 200 to 467, 201 to 467, 202 to 467, 203 to 467, 204 to 467, 205 to 467, 206 to 467, 207 to 467, 208 to 467, 209 to 467, 300 to 467, 301 to 467, 302 to 467, 303 to 467, 304 to 467, 305 to 467, 306 to 467, 307 to 467, 308 to 467, 309 to 467, 310 to 467, 311 to 467, 312 to 467, 313 to 467, 314 to 467, 315 to 467, 316 to 467, 317 to 467, 318 to 467, 319 to 467, 320 to 467, 321 to 467, 322 to 467, 323 to Provided herein are ASOs comprising or consisting of a nucleotide sequence that is complementary to at least 8 nucleotides (e.g., 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, or 40 nucleotides) of (a) nucleotide sequence(s) 05-467, 210-467, 215-467, 220-467, or 220-467). In some embodiments, the 5' portion of SEQ ID NO: 1 (e.g., nucleotides 1-184, 1-183, 1-182, 1-181, 1-180, 1-179, 1-178, 1-177, 1-176, 1-175, 1-174, 1-173, 1-172, 1-171, 1-170, 1-169, 1-168, 1-167, 1-166, 1-165, 1-164, 1-163, 1-162 of SEQ ID NO: 1) Provided herein are ASOs comprising or consisting of a nucleotide sequence that is complementary to at least 8 nucleotides (e.g., 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, or 40 nucleotides) of a sequence of nucleotides 1-161 of the sequence.

[0079] In some embodiments, provided herein is an ASO comprising or consisting of a nucleotide sequence that is complementary to at least 8 nucleotides (e.g., 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, or 40 nucleotides) of the SYNGAP1 regRNA identified herein as RR87 (SEQ ID NO: 2).

[0080] In some embodiments, provided herein is an ASO comprising or consisting of a nucleotide sequence that is complementary to at least 8 nucleotides (e.g., 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, or 40 nucleotides) of the SYNGAP1 regRNA identified herein as RR88 (SEQ ID NO: 3).

[0081] In some embodiments, provided herein is an ASO comprising or consisting of a nucleotide sequence that is complementary to at least 8 nucleotides (e.g., 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, or 40 nucleotides) of the SYNGAP1 regRNA identified herein as RR93_v1 (SEQ ID NO: 4).

[0082] In some embodiments, provided herein is an ASO comprising or consisting of a nucleotide sequence that is complementary to at least 8 nucleotides (e.g., 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, or 40 nucleotides) of the SYNGAP1 regRNA identified herein as RR86_v2 (SEQ ID NO: 5).

[0083] In some embodiments, provided herein is an ASO comprising or consisting of a nucleotide sequence that is complementary to at least 8 nucleotides (e.g., 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, or 40 nucleotides) of the SYNGAP1 regRNA identified herein as RR93_v2 (SEQ ID NO: 6).

[0084] In some embodiments, provided herein is an ASO comprising or consisting of a nucleotide sequence that is complementary to at least 8 nucleotides (e.g., 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, or 40 nucleotides) of the SYNGAP1 regRNA identified herein as RR121 ​​(SEQ ID NO: 7).

[0085] In certain embodiments, the ASO is at least 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, 50, 60, 70, 80, 90, or 100 nucleotides in length. In certain embodiments, the ASO is at least 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleotides in length. In certain embodiments, the ASO is at least 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, or 40 nucleotides in length.

[0086] In certain embodiments, ASOs are designed to lack stable secondary structures within themselves or between each other, thereby increasing the amount of ASOs in single-stranded form ready to hybridize with SYNGAP1 regRNA. Methods for predicting secondary structure are known in the art (see, e.g., Seetin and Mathews, Methods Mol. Biol. (2012) 905:99-122; Zhao et al., PLoS Comput. Biol. (2021) 17(8):e1009291), and web-based programs (e.g., RNAfold) are available to the public.

[0087] For example, ASOs have been designed to target human SYNGAP1 paRNA (e.g., eRNA, NAT, or paRNA). The nucleotide sequences of some of these ASOs are shown in Table 2 below. In some embodiments, an ASO of the disclosure comprises or consists of a nucleotide sequence provided in any one of Tables 2-4. In some embodiments, an ASO of the disclosure comprises or consists of a nucleotide sequence and / or chemical modifications provided in Table 2. Any chemical modification or combination of chemical modifications described herein can be applied to any ASO sequence provided herein (e.g., those in Tables 2, 3, or 4). In some embodiments, an ASO comprises or consists of the nucleotide sequence and / or chemical modifications of any one of SEQ ID NOs: 10-4852. In some embodiments, an ASO comprises or consists of the nucleotide sequence and / or chemical modifications of any one of SEQ ID NOs: 10-1003, 1004-2961, or 2962-4852.

[0088] Additional ASO sequences targeting SYNGAP1 regRNA RR86_v1, RR86_v2, RR93_v1, and RR93_v2 are provided in Tables 3 and 4. In some embodiments, an ASO of the disclosure comprises or consists of a nucleotide sequence selected from any one of the ASOs presented in Table 2. In some embodiments, the ASO comprises or consists of a nucleotide sequence set forth in any one of SEQ ID NOs: 1004-2961 or 2962-4852.

[0089] [Table 2] TIFF2025540101000007.tif234165TIFF2025540101000008.tif234165TIFF2025540101000009.tif234165TIFF2025540101000010.tif234165TIFF2025540101000011.tif234165TIFF2025540101000012.tif234165TIFF2025540101000013.tif234165TIFF2025540101000014.tif234165TIFF2025540101000015.tif234165TIFF2025540101000016.tif234165TIFF2025540101000017.tif234165TIFF2025540101000018.tif234165TIFF2025540101000019.tif234165TIFF2025540101000020.tif234165TIFF2025540101000021.tif234165TIFF2025540101000022.tif234165TIFF2025540101000023.tif234165TIFF2025540101000024.tif234165TIFF2025540101000025.tif234165TIFF2025540101000026.tif234165TIFF2025540101000027.tif234165TIFF2025540101000028.tif234165TIFF2025540101000029.tif234165TIFF2025540101000030.tif234165

[0090]

Table 3

[0091]

Table 4

[0092] In some embodiments, the ASOs provided herein comprise 16, 17, 18, 19, 20, 21, 22, 23, 24, 26, or 26 of the nucleotide sequences presented in Tables 2-4 below. For example, the ASO can comprise the first (5' to 3') 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, or 26 nucleotides of any one of SEQ ID NOs: 10-4852, e.g., nucleotides from position 1 to any one of positions 16, 17, 18, 19, 21, 22, 23, 24, 25, or 26 of any one of SEQ ID NOs: 10-4852. Alternatively, the ASO may include the last 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 nucleotides of any one of SEQ ID NOs: 10-4852, for example, nucleotides from position 2, 3, 4, 5, 6, 7, 8, 9, or 10 to position 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, or 26 of any one of SEQ ID NOs: 10-4852.For example, the ASOs provided herein can be any of positions 1 to 16, 1 to 17, 1 to 18, 1 to 19, 1 to 20, 1 to 21, 1 to 22, 1 to 23, 1 to 24, 1 to 25, 1 to 26, 2 to 17, 2 to 18, 2 to 19, 2 to 20, 2 to 21, 2 to 22, 2 to 23, 2 to 24, 2 to 25, 2 to 26, 3 to 18, 3 to 19, 3 to 20, 3 to 21, 3 to 22, 3 to 23, 3 to 24, 3 to 25, 3 to 26, 4 to 19, 4 to 20, 4 to 21, 4 to 22, 4 to 23, 4 to 24, 4 to 25, 4 to 26, 5 to 19, 5 to 20, 5 to 26, 6 to 10, 6 to 28, 7 to 29, 8 to 30, 8 to 31, 8 to 32, 8 to 33, 8 to 34, 8 to 35, 8 to 36, 9 to 37, 9 to 38, 10 to 14, 10 to 16, 10 to 18, 10 to 19, 10 to 20, 10 to 21, 10 to 22, 10 to 23, 10 to 24, 10 to 25, 10 to 26, 11 to 12, 11 to 14, 11 to 15, 11 to 16, 11 to 1 positions, 4 to 21, 4 to 22, 4 to 23, 4 to 24, 4 to 25, 4 to 26, 5 to 20, 5 to 21, 5 to 22, 5 to 23, 5 to 24, 5 to 25, 5 to 26, 6 to 21, 6 to 22, 6 to 23, 6 to 24, 6 to 25, 6 to 26, 7 to 22, 7 to 23, 7 to 24, 7 to 25, 7 to 26, 8 to 23, 8 to 24, 8 to 25, 8 to 26, 9 to 24, 9 to 25, 9 to 26, 10 to 25, or 10 to 26. In some embodiments, the ASO provided herein comprises the nucleotide sequence of nucleotides 1 to 16, 2 to 17, 3 to 18, 4 to 19, 5 to 20, 6 to 21, 7 to 23, 8 to 24, 9 to 25, or 10 to 26 of any one of SEQ ID NOs: 10 to 4852. In such embodiments, the ASO is at least 16, 17, 18, 19, or 20 nucleotides in length.

[0093] In some embodiments, the ASO comprises a nucleotide sequence of at least 8 contiguous nucleotides from chr6:33419695 to 33419939. In some embodiments, the ASO comprises a nucleotide sequence of at least 8 contiguous nucleotides from chr6:33453987 to 33454269. In some embodiments, the ASO comprises a nucleotide sequence of at least 8 contiguous nucleotides from chr6:33419674 to 33419940. In such embodiments, the at least 8 contiguous nucleotides of chromosome 6 (chr6) are positive-strand nucleotides of chromosome 6 compared to a reference genome.

[0094] In some embodiments, the ASOs provided herein comprise, in addition to the nucleotide sequence of any one of SEQ ID NOs: 10-4852, up to four additional nucleotides at the 5' end of the nucleotide sequence that are complementary to the target SYNGAP1 regRNA. For example, the ASO can comprise up to one, two, three, or four additional nucleotides at the 5' end of any one of SEQ ID NOs: 10-4852 that are complementary to a human SYNGAP1 regRNA (e.g., any one of the regRNAs listed in Table 1, e.g., RR86_v1 (SEQ ID NO: 1), RR87 (SEQ ID NO: 2), RR88 (SEQ ID NO: 3), RR93_v1 (SEQ ID NO: 4), RR86_v2 (SEQ ID NO: 5), RR93_v1 (SEQ ID NO: 6), or mouse SYNGAP1 regRNA (the regRNA listed in Table 1 as RR121 ​​(SEQ ID NO: 7))). In some embodiments, if an ASO includes up to four (e.g., 1, 2, 3, or 4) additional nucleotides at the 5' end of the nucleotide sequence of any one of SEQ ID NOs: 10-4852 that are complementary to the target SYNGAP1 regRNA, the ASO may also exclude up to four (e.g., 1, 2, 3, or 4) nucleotides from the 3' end of the nucleotide sequence of any one of SEQ ID NOs: 10-4852. For example, if an ASO includes 1, 2, 3, or 4 5' nucleotides that are complementary to the target SYNGAP1 regRNA, the ASO may exclude 1, 2, 3, or 4 3' terminal nucleotides from the nucleotide sequence of any one of SEQ ID NOs: 10-4852.

[0095] In some embodiments, the ASOs provided herein comprise, in addition to the nucleotide sequence of any one of SEQ ID NOs: 10 to 4852, up to four additional nucleotides at the 3' end of the nucleotide sequence that are complementary to the target SYNGAP1 regRNA. For example, an ASO can include up to 1, 2, 3, or 4 additional nucleotides at the 3' end of any one of SEQ ID NOs: 10 to 4852 that are complementary to the target SYNGAP1 regRNA (e.g., any one of the regRNAs listed in Table 1, e.g., RR86_v1 (SEQ ID NO: 1), RR87 (SEQ ID NO: 2), RR88 (SEQ ID NO: 3), RR93_v1 (SEQ ID NO: 4), RR86_v2 (SEQ ID NO: 5), or RR93_v1 (SEQ ID NO: 6). In some embodiments, when an ASO includes up to four (e.g., 1, 2, 3, or 4) additional nucleotides at the 3' end of the nucleotide sequence of any one of SEQ ID NOs: 10 to 4852 that are complementary to the target SYNGAP1 regRNA, the ASO can also exclude up to four (e.g., 1, 2, 3, or 4) nucleotides from the 5' end of the nucleotide sequence of any one of SEQ ID NOs: 10 to 4852. For example, an ASO can exclude up to four (e.g., 1, 2, 3, or 4) nucleotides from the 5' end of the nucleotide sequence of any one of SEQ ID NOs: 10 to 4852 that are complementary to the target SYNGAP1 regRNA. When including 1, 2, 3, or 4 3' nucleotides complementary to the regRNA, the ASO may exclude 1, 2, 3, or 4 5' terminal nucleotides from the nucleotide sequence of any one of SEQ ID NOs: 10-4852.

[0096] In some embodiments, the regulatory RNA has the nucleotide sequence of SEQ ID NO: 1 and the ASO comprises a nucleotide sequence selected from the group consisting of SEQ ID NOs: 10-59, 220-250, 261-267, 272-278, 526-528, 542-591, 702-728, 729-735-741, 988-990, and 1004-2961.

[0097] In some embodiments, the regulatory RNA has the nucleotide sequence of SEQ ID NO: 1, and the ASO comprises a nucleotide sequence selected from the group consisting of SEQ ID NOs: 14-59, 220-250, 261-267, 272-278, 526-528, 542-591, 702-728, 729-735-741, 988-990, and 1007-2961. In some embodiments, the regulatory RNA has a nucleotide sequence comprising nucleotides 185-467 of SEQ ID NO: 1, and the ASO comprises a nucleotide sequence selected from the group consisting of SEQ ID NOs: 14-59, 220-250, 261-267, 272-278, 526-528, 542-591, 702-728, 729-735-741, 988-990, and 1007-2961. In some embodiments, the regulatory RNA does not comprise or consist of a nucleotide sequence comprising nucleotides 1-184 of SEQ ID NO:1, and the ASO comprises a nucleotide sequence selected from the group consisting of SEQ ID NOs:14-59, 220-250, 261-267, 272-278, 526-528, 542-591, 702-728, 729-735-741, 988-990, and 1007-2961.

[0098] In some embodiments, the regulatory RNA has the nucleotide sequence of SEQ ID NO: 5 and the ASO comprises a nucleotide sequence selected from the group consisting of SEQ ID NOs: 14-59, 220-250, 261-267, 272-278, 526-528, 542-591, 702-728, 729-735-741, 988-990, and 1007-2961.

[0099] In some embodiments, the regulatory RNA has the nucleotide sequence of SEQ ID NO: 2 and the ASO comprises a nucleotide sequence selected from the group consisting of SEQ ID NOs: 60-73.

[0100] In some embodiments, the regulatory RNA has the nucleotide sequence of SEQ ID NO: 3 and the ASO comprises a nucleotide sequence selected from the group consisting of SEQ ID NOs: 74-109, 251-260, 268-271, and 279.

[0101] In some embodiments, the regulatory RNA has the nucleotide sequence of SEQ ID NO: 4 or 6, and the ASO comprises a nucleotide sequence selected from the group consisting of SEQ ID NOs: 110-219, 280-525, 529-541, 592-701, 742-987, and 991-1003.

[0102] In some embodiments, the regulatory RNA has the nucleotide sequence of SEQ ID NO: 7 and the ASO comprises a nucleotide sequence selected from the group consisting of SEQ ID NOs: 430-444 and 892-905.

[0103] Hybridization and ΔG As used herein, the term "hybridizing" or "hybridizing" should be understood to mean that two nucleic acid strands (e.g., an oligonucleotide and a target nucleic acid) form hydrogen bonds between base pairs on opposing strands, thereby forming a duplex. The affinity of the binding between two nucleic acid strands is the strength of hybridization. This can be measured by the melting temperature (T), which is defined as the temperature at which half of the oligonucleotide forms a duplex with the target nucleic acid. m ) is often described in terms of physiological conditions T m is not strictly proportional to affinity (Mergny and Lacroix, 2003, Oligonucleotides 13:515-537). The standard state Gibbs free energy, ΔG°, is a more accurate representation of binding affinity, ΔG° = -RTIn(K d ) dissociation constant (K d) (where R is the gas constant and T is the absolute temperature). Therefore, a very low ΔG° of the reaction between an oligonucleotide and a target nucleic acid reflects strong hybridization between the oligonucleotide and the target nucleic acid. ΔG° is the free energy associated with the reaction when the aqueous solution concentration is 1 M, pH is 7, and temperature is 37°C. The hybridization of an oligonucleotide to a target nucleic acid is a spontaneous reaction, and in the case of a spontaneous reaction, ΔG° is less than zero. ΔG° can be experimentally measured using, for example, the isothermal titration calorimetry (ITC) method described in Hansen et al., 1965, Chem. Comm. 36-38 and Holdgate et al., 2005, Drug Discovery Today. Those skilled in the art will be aware that commercially available devices are available for measuring ΔG°. ΔG° can be numerically estimated using the nearest neighbor model described in Sugimoto et al., 1995, Biochemistry 34:11211-11216 and McTigue et al., 2004, Biochemistry 43:5388-5405, Santa Lucia, 1998, Proc Natl Aced Sci USA 95:1460-1465, using appropriately derived thermodynamic parameters. To have the potential to modulate their intended nucleic acid targets through hybridization, oligonucleotides of the present disclosure hybridize to target nucleic acids with estimated ΔG° values ​​of less than -10 kcal / mol for oligonucleotides that are 10-30 nucleotides in length. In some embodiments, the degree or strength of hybridization is measured by the standard-state Gibbs free energy ΔG°. The oligonucleotide may hybridize to the target nucleic acid with an estimated ΔG° value below the range of −10 kcal / mol, such as less than −15 kcal / mol, e.g., less than −20 kcal / mol, e.g., less than −25 kcal / mol, for oligonucleotides 8 to 30 nucleotides in length.In some embodiments, the oligonucleotide hybridizes to the target nucleic acid with an estimated ΔG° value of −10 to −60 kcal / mol, e.g., −12 to −40 kcal / mol, −15 to −30 kcal / mol, −16 to −27 kcal / mol, or −18 to −25 kcal / mol.

[0104] double stranded region The phrase "duplex region" refers to a region in two complementary or substantially complementary polynucleotides that base-pair with each other by Watson-Crick base pairing or any other manner that allows for a stabilized duplex between complementary or substantially complementary polynucleotide strands. For example, a polynucleotide strand having 21 nucleotide units can base-pair with another 21 nucleotide unit polynucleotide, yet only 19 bases on each strand are complementary or fully complementary, so that the "duplex region" is 19 base pairs. The remaining bases may be present, for example, as 5' and / or 3' overhangs. Furthermore, 100% complementarity within a duplex is not required; substantial complementarity is acceptable within a duplex. Substantial complementarity refers to 70% or greater complementarity. For example, a mismatch in a 19-base pair duplex results in 94.7% complementarity, making the duplex region fully complementary. The double-stranded region can be formed by two separate oligonucleotide strands as well as by a single oligonucleotide strand that can form a hairpin structure that includes the double-stranded region.

[0105] dsRNA comprises two RNA strands that are complementary and hybridize to form a duplex structure under the conditions in which the dsRNA is used. One strand of the dsRNA (antisense strand) comprises a region of complementarity that is substantially complementary to the target sequence and is generally completely complementary. The target sequence can be derived from the sequence of a SYNGAP1 regRNA, such as eRNA or paRNA. The other strand (sense strand) comprises a region that is complementary to the antisense strand, so that when the two strands are combined under appropriate conditions, they hybridize and form a duplex structure. As described elsewhere herein and known in the art, the complementary sequences of dsRNA may be contained as self-complementary regions of a single nucleic acid molecule, as opposed to being on separate oligonucleotides. Generally, the duplex structure has a length of 5 to 50 base pairs, e.g., 5 to 50, 5 to 49, 5 to 48, 5 to 47, 5 to 46, 5 to 45, 5 to 44, 5 to 43, 5 to 42, 5 to 41, 5 to 40, 5 to 39, 5 to 38, 5 to 37, 5 to 36, 5 to 35, 5 to 34, 5 to 33, 5 to 32, 5 to 31, 5 to 30, 5 to 29, 5 to 28, 5 to 27, 5 to 26, 5 to 25, 5 to 24, 5 to 23, 5-22, 5-21, 5-20, 5-19, 5-18, 5-17, 5-16, 5-15, 5-14, 5-13, 5-12, 5-11, 5-10, 5-9, 5-8, 5-7, 5-6, 6-50, 6-49, 6-48, 6-47, 6-46, 6-45, 6-44, 6-43, 6-42, 6-41, 6-40, 6-39, 6-38, 6-37, 6-36, 6-35, 6-34, 6-33, 6-32, 6-31, 6-30, 6-29, 6-28, 6-27, 6-26, 6-25, 6-24, 6-23, 6-22, 6-21, 6-20, 6-19, 6-18, 6-17, 6-16, 6-15, 6-14, 6-13, 6-12, 6-11, 6-10, 6-9, 6-8, 6-7, 8-50, 8-49, 8-48, 8-47, 8-46, 8 ~45, 8~44, 8~43, 8~42, 8~41, 8~40, 8~39, 8~38, 8~37, 8~36, 8~35, 8~34, 8~33, 8~32, 8~31, 8~30, 8~29, 8~28, 8~27, 8~26, 8~25, 8~24, 8~23, 8~22, 8~21, 8~20, 8~19, 8~18, 8~17, 8~16, 8~15, 8~14, 8~13,8~12、8~11、8~10、8~9、10~50、10~49、10~48、10~47、10~46、10~45、10~44、10~43、10~42、10~41、10~40、10~39、10~38、10~37、10~36、10~35、10~34、10~33、10~32、10~31、10~30、10~29、10~28、10~27、10~26、10~25、10~24、10~23、10~22、10~21、10~20、10~19、10~18、10~17、10~16、10~15、10~14、10~13、10~12、10~11、10~10、10~9、12~50、12~49、12~48、12~47、12~46、12~45、12~44、12~43、12~42、12~41、12~40、12~39、12~38、12~37、12~36、12~35、12~34、12~33、12~32、12~31、12~30、12~29、12~28、12~27、12~26、12~25、12~24、12~23、12~22、12~21、12~20、12~19、12~18、12~17、12~16、12~15、12~14、12~13、15~50、15~49、15~48、15~47、15~46、15~45、15~44、15~43、15~42、15~41、15~40、15~39、15~38、15~37、15~36、15~35、15~34、15~33、15~32、15~31、15~30、15~29、15~28、15~27、15~26、15~25、15~24、15~23、15~22、15~21、15~20、15~19、15~18、15~17、18~50、18~49、18~48、18~47、18~46、18~45、18~44、18~43、18~42、18~41、18~40、18~39、18~38、18~37、18~36、18~35、18~34、18~33、18~32、18~31、18~30、18~30、18~29、18~28、18~27、18~26、18~25、18~24、18~23、18~22、18~21、18~20、19~50、19~49、19~48、19~47、19~46、19~45、19~44、19~43、19~42、19~41、19~40、19~39、19~38、19~37、19~36、19~35、19~34、19-33, 19-32, 19-31, 19-30, 19-30, 19-29, 19-28, 19-27, 19-26, 19-25, 19-24, 19-23, 19-22, 19-21, 19-20, 20-50, 20-49, 20-48, 20-47, 20-46, 20-45, 20-44, 20-43, 20-42, 20-41, 20-40, 20-39, 20-38, 20-37, 20-36, 20-35, 20-34, 20-33, 20-32, 20-31, 20-30, 20-30, 20-29, 20-28, 20-27, 20-26, 20-25, 20-24, 20-23, 20-22, 20-21, 21-50, 21-49, 21-48, 21-47, 21-46, 21-45, 21-44, 21-43, 21-42, 21-41, 21-40, 21-39, 21-38, 21-37, 21-36, 21-35, 21-34, 21-33, 21-32, 21-31, 21-32 1-30, 21-29, 21-28, 21-27, 21-26, 21-25, 21-24, 21-23, 21-22, 22-50, 22-49, 22-48, 22-47, 22-46, 22-45, 22-44, 22-43, 22-42, 22-41, 22-40, 22-39, 22-38, 22-37, 22-36, 22-35, 22-34, 22-33, 22-32, 22-31, 22-30, 22-29, 22-28, 22-27, 22 The length is between 26, 22-25, 22-24, 22-23, 23-50, 23-49, 23-48, 23-47, 23-46, 23-45, 23-44, 23-43, 23-42, 23-41, 23-40, 23-39, 23-38, 23-37, 23-36, 23-35, 23-34, 23-33, 23-32, 23-31, 23-30, 23-29, 23-28, 23-27, 23-26, 23-25, or 23-24 base pairs. Ranges and lengths intermediate to the above ranges and lengths are also contemplated as part of the present disclosure.

[0106] Similarly, the region of complementarity to the target sequence may be 5-50 nucleotides in length, e.g., 5-50, 5-49, 5-48, 5-47, 5-46, 5-45, 5-44, 5-43, 5-42, 5-41, 5-40, 5-39, 5-38, 5-37, 5-36, 5-35, 5-34, 5-33, 5-32, 5-31, 5-30, 5-29, 5-28, 5-27, 5-26, 5-25, 5-24, 5-23, 5-22, 5-21, 5-20, 5-19, 5-18, 5-17, 5-16, 5-15, 5-14, 5-13, 5-12, 5-11, 5-10, 5-9, 5-11, 5-12, 5-13, 5-14, 5-15, 5-16, 5-17, 5-18, 5-19, 5-20, 5-21, 5-22, 5-23, 5-24, 5-25, 5-26, 5-27, 5-28, 5-29, 5-30, 5-31, 5-32, 5-33, 5-34, 5-35, 5-36, 5-37, 5-38, 5-39, 5-40, 5-41, 5-42, 5-43, 5-44, 5-45, 5-46, 5-47, 5-48, 5- 8, 5-7, 5-6, 6-50, 6-49, 6-48, 6-47, 6-46, 6-45, 6-44, 6-43, 6-42, 6-41, 6-40, 6-39, 6-38, 6-37, 6-36, 6-35, 6-34, 6-33, 6-32, 6-31, 6-30, 6-29, 6-28 , 6-27, 6-26, 6-25, 6-24, 6-23, 6-22, 6-21, 6-20, 6-19, 6-18, 6-17, 6-16, 6-15, 6-14, 6-13, 6-12, 6-11, 6-10, 6-9, 6-8, 6-7, 8-50, 8-49, 8-48, 8-47, 8- 46, 8-45, 8-44, 8-43, 8-42, 8-41, 8-40, 8-39, 8-38, 8-37, 8-36, 8-35, 8-34, 8-33, 8-32, 8-31, 8-30, 8-29, 8-28, 8-27, 8-26, 8-25, 8-24, 8-23, 8-22, 8 ~21, 8~20, 8~19, 8~18, 8~17, 8~16, 8~15, 8~14, 8~13, 8~12, 8~11, 8~10, 8~9, 10~50, 10~49, 10~48, 10~47, 10~46, 10~45, 10~44, 10~43, 10~42, 10~41, 10 ~40, 10~39, 10~38, 10~37, 10~36, 10~35, 10~34, 10~33, 10~32, 10~31, 10~30, 10~29, 10~28, 10~27, 10~26, 10~25, 10~24, 10~23, 10~22, 10~21, 10~20, 10-19, 10-18, 10-17, 10-16, 10-15, 10-14, 10-13, 10-12, 10-11, 10-10, 10-9, 12-50, 12-49, 12-48, 12-47, 12-46, 12-45, 12-44, 12-43, 12-42, 12-41,12~40、12~39、12~38、12~37、12~36、12~35、12~34、12~33、12~32、12~31、12~30、12~29、12~28、12~27、12~26、12~25、12~24、12~23、12~22、12~21、12~20、12~19、12~18、12~17、12~16、12~15、12~14、12~13、15~50、15~49、15~48、15~47、15~46、15~45、15~44、15~43、15~42、15~41、15~40、15~39、15~38、15~37、15~36、15~35、15~34、15~33、15~32、15~31、15~30、15~29、15~28、15~27、15~26、15~25、15~24、15~23、15~22、15~21、15~20、15~19、15~18、15~17、18~50、18~49、18~48、18~47、18~46、18~45、18~44、18~43、18~42、18~41、18~40、18~39、18~38、18~37、18~36、18~35、18~34、18~33、18~32、18~31、18~30、18~30、18~29、18~28、18~27、18~26、18~25、18~24、18~23、18~22、18~21、18~20、19~50、19~49、19~48、19~47、19~46、19~45、19~44、19~43、19~42、19~41、19~40、19~39、19~38、19~37、19~36、19~35、19~34、19~33、19~32、19~31、19~30、19~30、19~29、19~28、19~27、19~26、19~25、19~24、19~23、19~22、19~21、19~20、20~50、20~49、20~48、20~47、20~46、20~45、20~44、20~43、20~42、20~41、20~40、20~39、20~38、20~37、20~36、20~35、20~34、20~33、20~32、20~31、20~30、20~30、20~29、20~28、20~27、20~26、20~25、20~24、20~23、20~22、20~21、21~50、21~49、21~48、21~47、21~46、21~45、21~44、21~43、21~42、21-41, 21-40, 21-39, 21-38, 21-37, 21-36, 21-35, 21-34, 21-33, 21-32, 21-31, 21-30, 21-29, 21-28, 21-27, 21-26, 21-25, 21-24, 21-23, 21-22, 22-50, 22-49, 22-48, 22-47, 22-46, 22-45, 22-44, 22-43, 22-42, 22-41, 22-40, 22-39, 22-38, 22-37, 22-36, 22-35, 22-34, 22-33, 22-32, 22 The length may be between 23-31, 22-30, 22-29, 22-28, 22-27, 22-26, 22-25, 22-24, 22-23, 23-50, 23-49, 23-48, 23-47, 23-46, 23-45, 23-44, 23-43, 23-42, 23-41, 23-40, 23-39, 23-38, 23-37, 23-36, 23-35, 23-34, 23-33, 23-32, 23-31, 23-30, 23-29, 23-28, 23-27, 23-26, 23-25, or 23-24 nucleotides. Ranges and lengths intermediate to the above ranges and lengths are also contemplated as part of this disclosure.

[0107] Chemical modification of ASOs In certain embodiments, the ASO does not consist solely of DNA. In certain embodiments, the ASO comprises at least one chemical modification to a natural nucleotide (e.g., a ribonucleotide (e.g., 2'-deoxy-2'-ribonucleotide)). Various chemical modifications can be included in the ASOs of the present disclosure. Modifications can include one or more modifications in the sugar group (e.g., ribose), one or more modifications in the phosphate group, one or more modifications in the nucleobase, one or more terminal modifications, or a combination thereof. In some embodiments, exemplary ASO sequences comprising or consisting of nucleotides targeting regRNAs set forth in any one of Tables 2-4 are chemically modified. Such modifications can be, but are not limited to, 2'-O-(2-methoxyethyl) (2'-MOE), locked nucleic acid (LNA), 5-methyl on cytidine, constrained ethyl (cET), phosphorothioate (PS) linkages, and / or phosphodiester (PO) linkages, or any combination thereof. Chemical modifications of RNA are known in the art and are described, for example, in PCT Application Publication No. WO2013 / 177248, which is incorporated herein by reference. In certain embodiments, each cytidine in the ASOs provided herein is modified with a 5-methyl.

[0108] Various chemical modifications for use with the ASOs of the present disclosure include, but are not limited to, 3'-terminal deoxythymine (dT) nucleotides, 2'-O-methyl modified nucleotides, 2'-fluoro modified nucleotides, 2'-deoxy modified nucleotides, locked nucleotides, unlocked nucleotides, conformationally restricted nucleotides, constrained ethyl nucleotides, abasic nucleotides, 2'-amino modified nucleotides, 2'-O-allyl modified nucleotides, 2'-C-alkyl modified nucleotides, 2'-hydroxyl modified nucleotides, 2'-methoxyethyl modified nucleotides, 2'-O-alkyl modified nucleotides, morpholino nucleotides, phosphoramidates, non-natural base containing nucleotides, tetrahydropyran modified nucleotides, 1,5-anhydrohexitol modified nucleotides, cyclohexenyl modified nucleotides, nucleotides containing phosphorothioate groups, nucleotides containing methylphosphonate groups, nucleotides containing 5'-phosphates, and nucleotides containing 5'-phosphate mimetics.

[0109] In certain embodiments, the ASO comprises an RNA polynucleotide that has been chemically modified to be resistant to one or more nucleases (e.g., nuclear RNases (e.g., exosome complexes or RNase H)). In some embodiments, all nucleotide bases are modified in the ASO. In certain embodiments, the chemical modifications include β-D-ribonucleotides, 2'-modified nucleotides (e.g., 2'-O-(2-methoxyethyl) (2'-MOE), 2'-O-CH3, or 2'-fluoro-arabino (FANA)), bicyclic sugar-modified nucleotides (e.g., having constrained ethyl or locked nucleic acids (LNA)), and / or one or more modified internucleotide linkages (e.g., phosphorothioate internucleotide linkages). In certain embodiments, the chemical modifications include 2'-MOE and phosphorothioate internucleotide linkages. In certain embodiments, at least 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more consecutive nucleotides of the ASO are modified with 2'-MOE. In certain embodiments, each nucleotide of the ASO is modified with 2'-MOE. In certain embodiments, at least 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more consecutive internucleotide linkages of the ASO are phosphorothioate internucleotide linkages. In certain embodiments, each internucleotide linkage of the ASO is a phosphorothioate internucleotide linkage.

[0110] Internucleotide linkage modifications that can be used with the ASOs of the present disclosure include, but are not limited to, phosphorothioate "PS" (P(S)), phosphoramidate (P(NR1R2), e.g., dimethylaminophosphoramidate (P(N(CH3)2)), phosphonocarboxylate (P(CH2) n COOR), e.g., phosphonoacetate "PACE" (P(CHCOO - )), thiophosphonocarboxylates ((S)P(CH2)nCOOR), e.g., thiophosphonoacetates, "thioPACE" ((S)P(CH2COO - )), alkyl phosphonates (P(C 1-3alkyl), such as methylphosphonate -P(CH), borane phosphonate (P(BH)), and phosphorodithioate (P(S)).

[0111] In some embodiments, the ASOs provided herein contain at least 1, 2, 3, 4, 5, 6, 7, 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, or more PO linkages. In some embodiments, all internucleotide linkages in the ASOs provided herein are PO internucleotide linkages. In some embodiments, the ASOs provided herein do not contain any PO internucleotide linkages. In some embodiments, the ASOs provided herein contain at least 1, 2, 3, 4, 5, 6, 7, 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, or more PS internucleotide linkages. In some embodiments, all internucleotide linkages in the ASOs provided herein are PS linkages. In some embodiments, the ASOs provided herein do not contain PS internucleotide linkages.

[0112] In some embodiments, the ASOs provided herein comprise at least 1, 2, 3, 4, 5, 6, 7, 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, or more PS linkages. In some embodiments, all internucleotide linkages in the ASOs provided herein are PS internucleotide linkages. In some embodiments, the ASOs provided herein do not comprise PS internucleotide linkages. In some embodiments, the ASOs provided herein contain at least 1, 2, 3, 4, 5, 6, 7, 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, or more PS internucleotide linkages. In some embodiments, all internucleotide linkages in the ASOs provided herein are PO linkages. In some embodiments, the ASOs provided herein do not contain PO internucleotide linkages.

[0113] In certain embodiments, the ASO contains one or more chemical modifications at the 5' end, the 3' end, or both. Without wishing to be bound by theory, chemical modifications at one or both ends of a polynucleotide (e.g., polyribonucleotides) can stabilize the polynucleotide. In certain embodiments, the ASO contains one or more chemical modifications at at least 1, 2, 3, 4, or 5 nucleotides at the 5' end of the ASO. In certain embodiments, the ASO contains one or more chemical modifications at at least 1, 2, 3, 4, or 5 nucleotides at the 3' end of the ASO. In certain embodiments, the ASO contains one or more chemical modifications at at least 1, 2, 3, 4, or 5 nucleotides at the 5' end of the ASO and one or more chemical modifications at at least 1, 2, 3, 4, or 5 nucleotides at the 3' end of the ASO.

[0114] Chemical structures may also be written in letters, where "M" indicates MOE, "d" indicates DNA, "L" indicates LNA, "m" indicates 2'O-methyl, "=" indicates a phosphorothioate (PS) bond, "-" indicates a phosphodiester (PO) bond; "*" or "5C" indicates 5-methylcytosine, "ag" indicates GalNAc, "tg" or "teg" indicates Teg-GalNAc, "^" indicates FANA, "BioTeg" indicates biotin, "Palm" indicates palmitic acid; and "C18" indicates a spacer 18 moiety.

[0115] For the avoidance of doubt, this LNA has the following formula: TIFF2025540101000079.tif60165where B is a specific designated base.

[0116] An exemplary visual representation of an ASO with chemical modifications is provided in Figure 2. Additional exemplary ASOs with chemical modifications are provided in Table 2. In some embodiments, the ASOs provided herein comprise the nucleotide sequence and / or chemical modifications of any one of the ASOs provided in Tables 2-4.

[0117] In some embodiments, the ASO comprises a sequence selected from the group consisting of SEQ ID NOs: 10-4852. In some embodiments, the ASO comprises a sequence selected from the group consisting of SEQ ID NOs: 542-1003 and a chemical modification.

[0118] In some embodiments, the ASO provided herein comprises the nucleotide sequence of any one of the ASOs presented in Tables 2-4 below. In some embodiments, the ASO comprises a sequence and / or a chemical modification selected from the group consisting of SEQ ID NOs: 10-4852. In some embodiments, the ASO comprising a sequence selected from the group consisting of SEQ ID NOs: 10-541 or 1004-4852 further comprises any chemical modification disclosed herein.

[0119] High-affinity modified nucleotides A high-affinity modified nucleotide is a modified nucleotide that, when incorporated into an oligonucleotide, enhances the affinity of the oligonucleotide for its complementary target, e.g., as measured by melting temperature (Tm). The high-affinity modified nucleotides of the present disclosure preferably result in an increase in melting temperature of +0.5 to +12°C, e.g., +1.5 to +10°C, or +3 to +8°C, per modified nucleotide. Many high-affinity modified nucleotides are known in the art, including, for example, many 2'-substituted nucleotides and locked nucleic acids (LNAs) (see, e.g., Freier & Altmann (1997) Nucl. Acid Res. 25:4429-4443 and Uhlmann (2000) Curr. Opinion in Drug Development 3(2):203-213, each of which is incorporated herein by reference).

[0120] sugar modification The ASOs described herein may contain one or more nucleotides with modified sugar moieties, i.e., modifications to the sugar moiety compared to the ribose sugar moiety found in DNA and RNA. Numerous nucleotides with modified ribose sugar moieties have been created, primarily for the purpose of improving certain properties of the oligonucleotide, such as affinity and / or nuclease resistance. Such modifications include those in which the ribose ring structure is modified by substitution with, for example, a hexose ring (HNA) or a bicyclic ring (which typically has a biradical bridge between the C2 and C4 carbons on the ribose ring (LNA)), or an unlinked ribose ring (which typically lacks a bond between the C2 and C3 carbons) (e.g., UNA). Other sugar-modified nucleotides include, for example, bicyclohexose nucleic acids (WO 2011 / 017521) or tricyclic nucleic acids (WO 2013 / 154798), both of which are incorporated herein by reference. Modified nucleotides also include nucleotides in which a sugar moiety is replaced with a non-sugar moiety, for example, in the case of peptide nucleic acids (PNAs) or morpholino nucleic acids.

[0121] Sugar modifications also include modifications made by changing the substituent on the ribose ring to a group other than hydrogen or to the 2'-OH group naturally found in RNA nucleotides. Substituents may be introduced, for example, at the 2', 3', 4', or 5' position.

[0122] In some embodiments, the oligonucleotide comprises a 2'-O-methyl (2'OMe) moiety, a 2'-O-methoxyethyl moiety, a bicyclic sugar moiety, a PNA (e.g., an oligonucleotide comprising one or more N-(2-aminoethyl)-glycine units linked by amide or carbonyl methylene bonds as repeating units instead of a sugar phosphate backbone), a locked nucleotide (LNA) (e.g., an oligonucleotide comprising one or more locked riboses, which may be a mixture of 2'-deoxynucleotides or 2'OMe nucleotides), a cET (e.g., an oligonucleotide comprising one or more cET sugars), a cMOE (e.g., an oligonucleotide comprising one or more cMOE sugars), a morpholino oligomer (e.g., one or more oligonucleotides comprising a backbone comprising a phosphorodiamidate morpholino oligomer of 2'-diaminodiisopropyl ...

[0123] In some embodiments, the oligonucleotide comprises 2-thiouracil ("2-thioU"), 2-thiocytosine ("2-thioC"), 4-thiouracil ("4-thioU"), 6-thioguanine ("6-thioG"), 2-aminoadenine ("2-aminoA"), 2-aminopurine, pseudouracil, hypoxanthine, 7-deazaguanine, 7-deaza-8-azaguanine, 7-deazaadenine, 7-deaza-8-azaadenine, 5-methylcytosine ("5-methylC"), 5-methyluracil ("5-methylU"), 5-hydroxymethylcytosine, 5-hydroxymethyluracil, 5,6-dehydrouracil, 5 nucleobase modifications selected from the group consisting of 5-propynylcytosine, 5-propynyluracil, 5-ethynylcytosine, 5-ethynyluracil, 5-allyluracil ("5-allylU"), 5-allylcytosine ("5-allylC"), 5-aminoallyluracil ("5-aminoallylU"), 5-aminoallyl-cytosine ("5-aminoallylC"), abasic nucleotides, Z bases, P bases, unstructured nucleic acids ("UNA"), isoguanine ("isoG"), and isocytosine ("isoC"), glycerol nucleic acid (GNA), thiomorpholino (CHNS) or thiophosphoramidate morpholino (TMO). The synthesis of glycerol nucleic acids (GNAs) (also known as glycol nucleic acids) is described in Zhang et al. (2010) Current Protocols in Nucleic Acid Chemistry 4.40.1-4.40.18, which is incorporated herein by reference. The synthesis of thiophosphoramidate morpholino oligonucleotides is described in Langer et al. J. Am. Chem. Soc. 2020, 142(38):16240-53.

[0124] 2' sugar-modified nucleotides 2' sugar-modified nucleotides are nucleotides having a substituent other than H or -OH at the 2' position (2' substituted nucleotides) or include 2' linked bicyclic nucleotides that can form a bridge between the 2' carbon and the second carbon in the ribose ring, such as LNA (2'-4' bridged bicyclic) nucleotides.

[0125] Without wishing to be bound by theory, 2'-modified sugars may provide enhanced binding affinity and / or increased nuclease resistance to oligonucleotides. Examples of 2'-substituted modified nucleotides are 2'-O-alkyl-RNA, 2'-O-methyl-RNA, 2'-alkoxy-RNA, 2'-O-methoxyethyl-RNA (MOE), 2'-amino-DNA, 2'-fluoro-RNA, and 2'-F-ANA nucleotides. For further examples, see, for example, Freier & Altmann (1997) Nucl. Acid Res., 1997, 25, 4429-4443 and Uhlmann (2000) Curr. Opinion in Drug Development, 2000, 3(2), 293-213, and Deleavy and Damha, Chemistry and Biology 2012, 19, 937 (each of which is incorporated herein by reference).

[0126] Locked Nucleic Acid Nucleotides (LNA Nucleotides) An "LNA nucleotide" is a 2'-sugar-modified nucleotide containing a biradical (also called a "2'-4' bridge") linking the C2' and C4' ends of the ribose sugar ring of the nucleotide, which restricts or locks the conformation of the ribose ring. In other words, a locked nucleotide is a nucleotide containing a bicyclic sugar moiety containing a 4'-CH2-O-2' bridge. This structure effectively "locks" the ribose in a 3'-endo conformation. The addition of locked nucleotides to oligonucleotides has been shown to increase the stability of oligonucleotides in serum and reduce off-target effects (Grunweller, A. et al., (2003) Nucleic Acids Research 31(12):3185-3193). These nucleotides are also sometimes called bridged nucleic acids or bicyclic nucleic acids (BNAs). Locking the conformation of the ribose is associated with increased hybridization affinity (duplex stabilization) when LNAs are incorporated into oligonucleotides complementary to RNA or DNA molecules. This can be routinely determined by measuring the melting temperature of the oligonucleotide / complement duplex. Exemplary LNA nucleotides include beta-D-oxy-LNA, 6'-methyl-beta-D-oxy-LNA, such as (S)-6'-methyl-beta-D-oxy-LNA (ScET), and ENA.

[0127] Examples of bicyclic nucleotides for use in polynucleotides of the present disclosure include, but are not limited to, nucleotides comprising a bridge between the 4' and 2' ribosyl ring atoms. In certain embodiments, polynucleotide agents of the present disclosure comprise one or more bicyclic nucleotides comprising a 4' to 2' bridge. Examples of such 4' to 2' bridged bicyclic nucleotides include, but are not limited to, 4'-(CH2)-O-2' (LNA); 4'-(CH2)-S-2'; 4'-(CH2)2-O-2' (ENA); 4'-CH(CH3)-O-2' (also referred to as "constrained ethyl" or "cEt") and 4'-CH(CHOCH3)-O-2' (and analogs thereof; see, e.g., U.S. Pat. No. 7,399,845). 4'-C(CH3)(CH3)-O-2' (and analogs thereof; see, e.g., U.S. Pat. No. 8,278,283); 4'-CH2-N(OCH3)-2' (and analogs thereof; see, e.g., U.S. Pat. No. 8,278,425); 4'-CH2-ON(CH3)2-2' (see, e.g., U.S. Patent Publication No. 2004 / 0171570); 4'-CH2-N(R)-O-2' (wherein R is H, C1-C 12 alkyl, or a protecting group] (see, e.g., U.S. Pat. No. 7,427,672); 4'-CH2-C(H)(CH3)-2' (see, e.g., Chattopadhyaya et al., J. Org. Chem., 2009, 74, 118-134); and 4'-CH2-C(=CH2)-2' (and analogs thereof; see, e.g., U.S. Pat. No. 8,278,426). The entire contents of each of the foregoing are incorporated herein by reference.

[0128] Additional representative United States patents and published United States patent applications that teach the preparation of locked nucleic acid nucleotides include, but are not limited to, the following, the entire contents of each of which are incorporated herein by reference: U.S. Patent Nos. 6,268,490, 6,525,191, 6,670,461, 6,770,748, 6,794,499, 6,998,484, 7,053,207, 7,034 ,133, 7,084,125, 7,399,845, 7,427,672, 7,569,686, 7,741,457, 8,022,193, 8,030,467, 8,278,425, 8,278,426, 8,278,283, U.S. Patent Application Publication No. 2008 / 0039618, and U.S. Patent Application Publication No. 2009 / 0012281.

[0129] Any of the aforementioned bicyclic nucleotides can be prepared with one or more stereochemical sugar configurations, such as, for example, α-L-ribofuranose and β-D-ribofuranose (see International Publication No. WO 99 / 14226, the contents of which are incorporated herein by reference).

[0130] The oligonucleotides of the present disclosure can also be modified to contain one or more constrained ethyl nucleotides. As used herein, a "constrained ethyl nucleotide" or "cEt" is a locked nucleotide containing a bicyclic sugar moiety containing a 4'-CH(CH3)-O-2' bridge. In one embodiment, the constrained ethyl nucleotide is in the S conformation, referred to herein as an "S-cEt."

[0131] The oligonucleotides of the present disclosure may also contain one or more "conformationally restricted nucleotides" ("CRNs"). A CRN is a nucleotide analogue with a linker connecting the C2' and C4' carbons of ribose or the C3 and C5' carbons of ribose. The CRN locks the ribose ring into a stable conformation, increasing hybridization affinity to RNA (e.g., regRNA or mRNA). The linker is long enough to optimally position the oxygen for stability and affinity, reducing ribose ring puckering.

[0132] Representative publications that teach the preparation of the specific CRNs described above include, but are not limited to, U.S. Patent Application Publication No. 2013 / 0190383 and PCT Publication WO2013 / 036868, the entire contents of each of which are incorporated herein by reference.

[0133] In some embodiments, the oligonucleotides of the present disclosure include one or more monomers that are UNA (unlocked nucleotide) nucleotides. UNAs are unlocked acyclic nucleotides in which the sugar bond has been removed to form an unlocked "sugar" residue. In one example, UNAs also encompass monomers in which the C1'-C4' bond (i.e., the covalent carbon-oxygen-carbon bond between the C1' and C4' carbons) has been removed. In another example, the C2'-C3' bond (i.e., the covalent carbon-carbon bond between the C2' and C3' carbons) of the sugar has been removed (see Nuc. Acids Symp. Series, 52, 133-134 (2008) and Fluiter et al., Mol. Biosyst., 2009, 10, 1039, which are incorporated herein by reference).

[0134] Representative U.S. patent publications that teach the preparation of UNAs include, but are not limited to, U.S. Patent No. 8,314,227, and U.S. Patent Application Publication Nos. 2013 / 0096289, 2013 / 0011922, and 2011 / 0313020, the entire contents of each of which are incorporated herein by reference.

[0135] The ribose molecule can also be modified with a cyclopropane ring to produce tricyclodeoxynucleic acid (tricycloDNA). The ribose moiety can be replaced with another sugar, such as 1,5-anhydrohexitol, threose to produce threose nucleotides (TNA), or arabinose to produce arabinonucleotides. The ribose molecule can also be replaced with a non-sugar, such as cyclohexene to produce cyclohexene nucleotides, or glycol to produce glycol nucleotides.

[0136] Potentially stabilizing modifications to the termini of nucleotide molecules can include N-(acetylaminocaproyl)-4-hydroxyprolinol (Hyp-C6-NHAc), N-(caproyl-4-hydroxyprolinol (Hyp-C6), N-(acetyl-4-hydroxyprolinol (Hyp-NHAc), thymidine-2'-O-deoxythymidine (ether), N-(aminocaproyl)-4-hydroxyprolinol (Hyp-C6-amino), 2-docosanoyl-uridine-3'-phosphate, inverted base dT (idT), and the like. Disclosure of this modification can be found in PCT Publication No. WO2011 / 005861.

[0137] Other alternative chemistry of the oligonucleotide of the present disclosure includes 5' phosphate or 5' phosphate mimic of oligonucleotide, for example, 5'-terminal phosphate or phosphate mimic.Suitable phosphate mimic is disclosed in, for example, US Patent Publication No. 2012 / 0157511, the entire content of which is incorporated herein by reference.

[0138] Additional non-limiting exemplary LNA nucleotides are described in WO99 / 014226, WO00 / 66604, WO98 / 039352, WO2004 / 046160, WO00 / 047599, WO2007 / 134181, WO2010 / 077578, WO2010 / 036698, WO2007 / 090071, WO2009 / 006478, WO2011 / 156202, WO2008 / 154401, WO2009 / 067647, WO2008 / 150729, Morita et al., Bioorganic & Med. Chem. Lett. 12, 73-76, Seth et al. al. J. Org. Chem. 2010, Vol 75(5) pp. 1569-81, Mitsuoka et al., Nucleic Acids Research 2009, 37(4), 1225-1238, and Wan and Seth, J. Medical Chemistry 2016, 59, 9645-9667 (each of which is incorporated herein by reference).

[0139] In some embodiments, the length of the ASO is 5×n+5 nucleotides (n is an integer greater than or equal to 3), the nucleotide at position 5×m is an LNA-modified ribonucleotide (m is an integer from 1 to n), and the nucleotides at the remaining positions are 2′-O-methoxyethyl-modified ribonucleotides.

[0140] In some embodiments, the nucleotide sugar modification is 2'-O-Ci-4 alkyl, e.g., 2'-O-methyl (2'-OMe), 2'-deoxy (2'-H), 2'-O-Ci-3 alkyl-O-Ci-3 alkyl, e.g., 2'-methoxyethyl ("2'-MOE"), 2'-fluoro ("2'-F"), 2'-amino ("2'-NH"), 2'-arabinosyl ("2'-arabino") nucleotide, 2'-F-arabinosyl ("2'-F-arabino") nucleotide, 2'-locked nucleic acid ("LNA") nucleotide, 2'-amide bridged nucleic acid (AmNA), 2'-unlocked nucleic acid ("ULNA") nucleotide, L-form sugar ("L-sugar"), or 4'-thioribosyl nucleotide.

[0141] Mixmers and Gapmers The ASO may have a mixmer and / or gapmer structure, for example, in the pattern disclosed by the ASO of FIG.

[0142] In certain embodiments, the ASO is a mixmer. As used herein, the term "mixmer" refers to an oligonucleotide containing an alternating composition of DNA monomers and nucleotide analog monomers across at least a portion of the oligonucleotide sequence. In certain embodiments, the ASO is a mixmer based on a gapmer structure, containing a mixture of DNA nucleotides and 2'-MOE nucleotides in the gap and flanked by RNA sequences (e.g., 2'-modified RNA sequences) in the wings. The mixmer can be designed to contain a mixture of affinity-enhancing nucleotide analogs, for example, in non-limiting examples, 2'-O-alkyl-RNA monomers, 2'-amino-DNA monomers, 2'-fluoro-DNA monomers, LNA monomers, arabinonucleic acid (ANA) monomers, 2'-fluoro-ANA monomers, HNA monomers, INA monomers, 2'-MOE-RNA (2'-O-methoxyethyl-RNA), 2'fluoro-DNA, and LNA. In some embodiments, the mixmer is unable to recruit RNase H. In some embodiments, the mixmer contains one type of affinity-enhancing nucleotide analog along with DNA and / or RNA.

[0143] Multiple different modifications can be spaced apart within the mixmer.For example, ASO can comprise LNA modification in multiple nucleotides, and can comprise different modifications in some or all of the remaining nucleotides.In some embodiments, any two adjacent LNA modified nucleotides are separated by at least 1, 2, 3, 4, or 5 nucleotides.Throughout ASO, the distance between adjacent LNA modified nucleotides can be constant (for example, any two adjacent LNA modified nucleotides are separated by 1, 2, 3, 4, or 5 nucleotides), or can be variable. In some embodiments, the length of the ASO is 3xn, 3xn-1, or 3xn-2 nucleotides (n is an integer greater than or equal to 6), and (a) (i) the nucleotide at position 3xm-2 (m is an integer from 1 to n) is a nucleotide (e.g., a ribonucleotide or deoxyribonucleotide) that includes a first modification (e.g., LNA), (ii) the nucleotide at position 3xm-1 (m is an integer from 1 to n) is a nucleotide (e.g., a ribonucleotide or deoxyribonucleotide) that includes a first modification (e.g., LNA), or (iii) the nucleotide at position 3xm (m is an integer from 1 to n) is a nucleotide (e.g., a ribonucleotide or deoxyribonucleotide) that includes a first modification (e.g., LNA); and (b) the nucleotides at the remaining positions include a second, different modification (e.g., 2'-O-methoxyethyl). In some embodiments, the length of the ASO is 2xn or 2xn-1 nucleotides (n is an integer greater than or equal to 9), where (a) (i) the nucleotide at position 2xm-1 (m is an integer from 1 to n) is a nucleotide (e.g., a ribonucleotide or deoxyribonucleotide) containing a first modification (e.g., LNA), or (ii) the nucleotide at position 2xm (m is an integer from 1 to n) is a nucleotide (e.g., a ribonucleotide or deoxyribonucleotide) containing a first modification (e.g., LNA); and (b) the nucleotides at the remaining positions contain a second, different modification (e.g., 2'-O-methoxyethyl). Similar modification patterns are also contemplated, e.g., patterns in which the first modification is repeated in exactly 4, 5, or more nucleotides.In some embodiments, the length of the ASO is 4xn, 4xn-1, or 4xn-2 nucleotides (n is an integer greater than or equal to 6), and (a) (i) the nucleotide at position 4xm-2 (m is an integer from 1 to n) is a nucleotide (e.g., a ribonucleotide or deoxyribonucleotide) that includes a first modification (e.g., LNA), (ii) the nucleotide at position 4xm-1 (m is an integer from 1 to n) is a nucleotide (e.g., a ribonucleotide or deoxyribonucleotide) that includes a first modification (e.g., LNA), or (iii) the nucleotide at position 3xm (m is an integer from 1 to n) is a nucleotide (e.g., a ribonucleotide or deoxyribonucleotide) that includes a first modification (e.g., LNA); and (b) the nucleotides at the remaining positions include a second, different modification (e.g., 2'-O-methoxyethyl). In some embodiments, the length of the ASO is 5xn, 5xn-1, or 5xn-2 nucleotides (n is an integer greater than or equal to 6), and (a) (i) the nucleotide at position 5xm-2 (m is an integer from 1 to n) is a nucleotide (e.g., a ribonucleotide or deoxyribonucleotide) that includes a first modification (e.g., LNA), (ii) the nucleotide at position 5xm-1 (m is an integer from 1 to n) is a nucleotide (e.g., a ribonucleotide or deoxyribonucleotide) that includes a first modification (e.g., LNA), or (iii) the nucleotide at position 5xm (m is an integer from 1 to n) is a nucleotide (e.g., a ribonucleotide or deoxyribonucleotide) that includes a first modification (e.g., LNA); and (b) the nucleotides at the remaining positions include a second, different modification (e.g., 2'-O-methoxyethyl).

[0144] In some embodiments, the ASO further comprises a GalNAc or Teg-GalNAc moiety at the 5' or 3' end of the ASO.

[0145] In certain embodiments, the ASO comprises a DNA sequence (e.g., having at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 contiguous nucleotides of unmodified DNA) flanking an RNA sequence on both sides. Such structures are known as "gapmers," with the DNA region referred to as the "gap" and the RNA region referred to as the "wings" (see, e.g., PCT Application Publication No. WO2013 / 177248). Gapmers are known to promote degradation of target RNA by recruiting nucleases (e.g., nuclear RNAses (e.g., RNase H)). Surprisingly, in some embodiments of the present disclosure, it has been discovered that gapmers (having the same sequence as the parent ASO but with different chemical modifications) that bind to regRNA can also increase target gene expression. In certain embodiments, the ASO comprises a DNA sequence flanking an RNA sequence and does not induce RNAse or RNAse H-mediated degradation.

[0146] In some embodiments, an ASO gapmer comprises an internal DNA region flanked by two external RNA "wings." For example, the internal DNA gap may comprise at least 49, 48, 47, 46, 45, 44, 43, 42, 41, 40, 39, 38, 37, 36, 35, 34, 33, 32, 31, 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 nucleotide(s), while the external RNA "wings" may comprise at least 49, 48, 47, 46, 45, 44, 43, 42, 41, 40, 39, 38, 37, 36, 35, 34, 33, 32, 31, 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 nucleotide(s). Each of the RNA wing(s) may independently comprise 1, 2, 3, 4, 5, 6, 7, 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, or more nucleotides. Exemplary gapmer structures include, but are not limited to, 1-10-9, 2-10-8, 3-10-7, 4-10-6, 6-10-4, 7-10-3, 8-10-2, 9-10-1, 1-18-1, 2-16-2, 3-14-3, 4-12-4, 5-10-5, 6-8-6, 7-6-7, 8-5-7, 7-5-8, 8-4-8, or 9-2-9 structures, where the first and third numbers indicate the number of external RNA nucleotides and the second number indicates the number of internal DNA nucleotides.

[0147] The ASO may also be a mixmer, which comprises a DNA region linked to an RNA region. In some embodiments, the mixmer comprises at least 10 DNA nucleotides linked to at least 10 RNA nucleotides, the DNA nucleotides being at the 5' end of the mixmer or the 3' end of the mixmer. In some embodiments, a mixmer comprises at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, or 19 DNA nucleotide(s) linked to at least 49, 48, 47, 46, 45, 44, 43, 42, 41, 40, 39, 38, 37, 36, 35, 34, 33, 32, 31, 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 RNA nucleotide(s), where the DNA nucleotide is at the 5' end of the mixmer or the 3' end of the mixmer. In some embodiments, the RNA region of the gapmer or mixmer may include any additional chemical modifications disclosed herein.

[0148] In certain embodiments, the ASO (e.g., a gapmer or mixmer) is about 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, or more nucleotides in length. In certain embodiments, the gap is about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or more nucleotides in length. In certain embodiments, one or both wings are about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, or 19 or more nucleotides in length. In certain embodiments, one or both wings comprise RNA modifications, such as β-D-ribonucleotides, 2'-modified nucleotides (e.g., 2'-O-(2-methoxyethyl) (2'-MOE), 2'-O-CH3, or 2'-fluoro-arabino (FANA)), and bicyclic sugar-modified nucleotides (e.g., having a constrained ethyl or locked nucleic acid (LNA)). In certain embodiments, each ribonucleotide in a mixmer or gapmer is modified with 2'-MOE. In certain embodiments, a mixmer or gapmer comprises one or more modified internucleotide linkages, such as a phosphorothioate (PS) internucleotide linkage. In certain embodiments, every two adjacent nucleotides in a mixmer or gapmer are linked by a phosphorothioate internucleotide bond.

[0149] In certain embodiments, the ASO does not contain 1 or more, 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, 10 or more, 11 or more, 12 or more, 13 or more, 14 or more, 15 or more, 20 or more, 25 or more, 30 or more, 35 or more, 40 or more, or 45 or more consecutive nucleotides of unmodified DNA. In some embodiments, such DNA sequences are disrupted every 2, 3, 4, 5, or more nucleotides by modified (e.g., 2'-MOE-modified) ribonucleotides. In some embodiments, the ASO contains only ribonucleotides and no deoxyribonucleotides.

[0150] The structural features of mixmers and gapmers may be combined. In certain embodiments, the ASO has a structure similar to that of a mixmer disclosed herein (e.g., a structure with intervening modifications), except that the second modification in the gap is changed to a third modification (e.g., a deoxyribonucleotide). In certain embodiments, the ASO has a structure similar to that of a gapmer disclosed herein, except that the nucleotide in the gap is modified in a mixmer pattern.

[0151] In certain embodiments, the ASO further comprises a ligand moiety, e.g., a ligand moiety that specifically targets a tissue or organ of interest. For example, N-acetylgalactosamine (GalNAc) specifically targets the liver. In certain embodiments, the ligand moiety comprises GalNAc. In certain embodiments, the ligand moiety comprises a 3-cluster GalNAc moiety (commonly designated GALNAc3). Other types of GalNAc moieties are 1-cluster, 2-cluster, or 4-cluster GalNAc, designated GalNAc1, GalNAc2, or GalNAc4. In certain embodiments, the ligand moiety comprises GalNAc1, GalNAc2, GalNAc3, or GalNAc4.

[0152] In certain embodiments, the ligand moiety comprises biotin. In certain embodiments, the ligand moiety comprises palmitic acid. In certain embodiments, the ligand moiety comprises a spacer 18 moiety (C18).

[0153] III. Pharmaceutical Compositions In certain embodiments, the ASOs disclosed herein can be present in pharmaceutical compositions. Pharmaceutical compositions can be formulated for use in various drug delivery systems. One or more pharmaceutically acceptable excipients or carriers may also be included in the composition for appropriate formulation. In some embodiments, pharmaceutically acceptable carriers include sterile saline, sterile water, or phosphate-buffered saline (PBS). Suitable formulations for use in the present disclosure can be found in Remington's Pharmaceutical Sciences, Mack Publishing Company, Philadelphia, Pa., 17th ed., 1985. For a brief review of methods for drug delivery, see, for example, Langer (Science 249:1527-1533, 1990).

[0154] Exemplary carriers and pharmaceutical formulations suitable for delivering nucleic acids are described in Darymanov and Reineke (2018) Front. Pharmacol. 9:971; Barba et al. (2019) Pharmaceutics 11(8):360; Ni et al. (2019) Life (Basel) 9(3):59 (each incorporated herein by reference). It will be appreciated that the presence of a ligand moiety conjugated to the ASO may obviate the need for a carrier for delivery to the tissue or organ targeted by the ligand moiety.

[0155] Delivery of the oligonucleotides of the present disclosure to cells, for example, cells in a subject, for example, a human subject, for example, a subject in need thereof, for example, a subject having or at risk of developing a SYNGAP1-associated disorder, can be achieved in many different ways. For example, delivery can be carried out by contacting the oligonucleotides of the present disclosure with cells either in vitro or in vivo. In vivo delivery can also be carried out directly by administering a composition comprising the oligonucleotide to a subject. These alternatives are further discussed below.

[0156] Generally, any method of delivering nucleic acid molecules (in vitro or in vivo) may be adapted for use with the oligonucleotides of the present disclosure (see, for example, Akhtar S. and Julian R L., (1992) Trends Cell. Biol. 2(5):139-144 and WO94 / 02595, which are incorporated herein by reference in their entireties). For in vivo delivery, factors to consider when delivering oligonucleotide molecules include, for example, the biological stability of the delivered molecule, the prevention of non-specific effects, and the accumulation of the delivered molecule in the target tissue. Non-specific effects of oligonucleotides may be minimized by local administration, for example, by direct injection or implantation into the tissue, or by local administration of a preparation. Local administration to the treatment site maximizes the local concentration of the drug, limits exposure of the drug to systemic tissues that may otherwise be harmed by or degrade the drug, and allows for the administration of a lower total dose of the oligonucleotide molecule.

[0157] To administer oligonucleotides systemically for the treatment of disease, the oligonucleotides may contain alternative nucleobases, alternative sugar moieties, and / or alternative internucleotide linkages, or alternatively, be delivered using a drug delivery system; both methods act to prevent rapid degradation of the oligonucleotide by endo- and exonucleases in vivo. Modification of the oligonucleotide or pharmaceutical carrier may also enable targeting of the oligonucleotide composition to target tissues and avoid undesirable off-target effects. Oligonucleotide molecules may be modified by chemical conjugation to lipophilic groups, such as cholesterol, to promote cellular uptake and prevent degradation. In alternative embodiments, oligonucleotides may be delivered using drug delivery systems such as nanoparticles, lipid nanoparticles, polyplex nanoparticles, lipoplex nanoparticles, dendrimers, polymers, liposomes, or cationic delivery systems. Positively charged cationic delivery systems promote binding of oligonucleotide molecules (which are negatively charged) and also enhance interactions with negatively charged cell membranes, allowing for efficient uptake of the oligonucleotides by cells. Cationic lipids, dendrimers, or polymers can be derivatized to bind to oligonucleotides or form vesicles or micelles that encase the oligonucleotides. The formation of vesicles or micelles further prevents the degradation of the oligonucleotides when administered systemically. Generally, any method of nucleic acid delivery known in the art can be adapted to deliver the oligonucleotides of the present disclosure. Methods for creating and administering cationic oligonucleotide complexes are within the capabilities of those skilled in the art (see, for example, Sorensen, D R., et al. (2003) J. Mol. Biol 327:761-766; Verma, U N. et al., (2003) Clin. Cancer Res. 9:1291-1300; Arnold, A S et al., (2007) J. Hypertens. 25:197-205, the entire contents of which are incorporated herein by reference).Some non-limiting examples of drug delivery systems useful for systemic delivery of oligonucleotides include DOTAP (Sorensen, D R., et al. (2003), supra; Verma, U N. et al., (2003), supra), oligofectamine, "solid nucleic acid lipid particles" (Zimmermann, T S. et al., (2006) Nature 441:111-114), cardiolipin (Chien, P Y. et al., (2005) Cancer Gene Ther. 12:321-328; Pal, A. et al., (2005) Int J. Oncol. 26:1087-1091), polyethyleneimine (Bonnet M E. et al., (2008) Pharm. Res. Aug 16 Epub ahead of print; Aigner, A. (2006) J. Biomed. Biotechnol. 71659), Arg-Gly-Asp (RGD) peptides (Liu, S. (2006) Mol. Pharm. 3:472-487), and polyamidoamines (Tomalia, D A. et al., (2007) Biochem. Soc. Trans. 35:61-67; Yoo, H. et al., (1999) Pharm. Res. 16:1799-1804). In some embodiments, oligonucleotides are complexed with cyclodextrins for systemic administration. Methods and pharmaceutical compositions for administering oligonucleotides and cyclodextrins can be found in U.S. Pat. No. 7,427,605, which is incorporated herein by reference in its entirety. In some embodiments, the oligonucleotides of the present disclosure are delivered via polyplex or lipoplex nanoparticles.Methods of administration and pharmaceutical compositions for oligonucleotides and polyplex nanoparticles and lipoplex nanoparticles can be found in U.S. Patent Application Nos. 2017 / 0121454; 2016 / 0369269; 2016 / 0279256; 2016 / 0251478; 2016 / 0230189; 2015 / 0335764; 2015 / 0307554; 2015 / 0174549; 2014 / 0342003; 2014 / 0135376; and 2013 / 0317086, which are incorporated by reference herein in their entireties.

[0158] In some embodiments, the compounds described herein can be administered in combination with additional therapeutic agents (e.g., using simultaneous or alternating regimens). Examples of additional therapeutic agents include antiepileptic drugs such as quinidine and / or sodium channel blockers, anticonvulsants, cholinesterase inhibitors, dopamine agonists, levodopa, dopamine reuptake inhibitors (SSRIs), selective serotonin reuptake inhibitors (NRIs), norepinephrine-dopamine reuptake inhibitors (NDRIs), and serotonin-norepinephrine-dopamine reuptake inhibitors (SNDRIs). Additionally, the compounds described herein can be administered in combination with recommended lifestyle changes.

[0159] Methods for delivering membrane molecular assemblies The oligonucleotides of the present disclosure may also be delivered using various membrane molecular assembly delivery methods, including polymer, biodegradable microparticle, or microcapsule delivery devices known in the art. For example, colloidal dispersion systems may be used for targeted delivery of the oligonucleotide agents described herein. Colloidal dispersion systems include macromolecular complexes, nanocapsules, microspheres, beads, and lipid-based systems, including oil-in-water emulsions, micelles, mixed micelles, and liposomes. Liposomes are artificial membrane vesicles useful as delivery vehicles in vitro and in vivo. Large unilamellar vesicles (LUVs), ranging in size from 0.2 to 4.0 μm, have been shown to be capable of encapsulating a significant fraction of aqueous buffer solutions containing large macromolecules. Liposomes are useful for the introduction and delivery of active ingredients to their site of action. Because liposome membranes are structurally similar to biological membranes, upon application of liposomes to tissues, the liposome bilayer fuses with the cell membrane bilayer. As liposome fusion with the cell progresses, the aqueous contents, including the oligonucleotide, are delivered to the cell, where the oligonucleotide may specifically bind to the target RNA. In some cases, liposomes are also specifically targeted, for example, to deliver oligonucleotides to specific cell types. Liposome compositions are usually a combination of phospholipids, usually in combination with steroids, especially cholesterol. Other phospholipids or other lipids can also be used. The physical properties of liposomes depend on pH, ionic strength, and the presence of divalent cations.

[0160] Liposomes containing oligonucleotides can be prepared in various ways. In one example, the lipid components of the liposome are dissolved in a detergent to form micelles with the lipid components. For example, the lipid components may be amphipathic cationic lipids or lipid conjugates. The detergent may have a high critical micelle concentration and be non-ionic. Exemplary detergents include cholate, CHAPS, octylglucoside, deoxycholate, and lauroyl sarcosine. The oligonucleotide formulation is then added to the micelles containing the lipid components. The cationic groups on the lipids interact with the oligonucleotides and condense around the oligonucleotides to form liposomes. After condensation, the detergent is removed, for example, by dialysis, to obtain a liposome formulation of the oligonucleotide.

[0161] If necessary, a carrier compound that aids in condensation may be added during the condensation reaction, for example, by controlled addition. For example, the carrier compound may be a polymer other than nucleic acid (e.g., spermine or spermidine). The pH may be adjusted to promote condensation.

[0162] Methods for producing stable polynucleotide delivery vehicles incorporating polynucleotide / cationic lipid complexes as structural components of the delivery vehicle are further described, for example, in WO 96 / 37194, the entire contents of which are incorporated herein by reference. Liposome formation has also been described by Feigner, PLet al., (1987) Proc. Natl. Acad. Sci. USA 8:7413-7417; U.S. Patent No. 4,897,355; al.,(1979)Biochim. Biophys. Acta 557:9;Szoka et al.,(1978)Proc.Natl.Acad.Sci.75:4194;Mayhew et al.,(1984)Biochim. Biophys. Acta 775:169;Kim et al.,(1983)Biochim. Biophys. Acta 728:339; and Fukunaga et The method may include one or more embodiments of the exemplary method described in

[0010] et al., (1984) Endocrinol. 115:757. Commonly used techniques for preparing lipid aggregates of appropriate size for use as delivery vehicles include sonication and freeze-thaw plus extrusion (see, e.g., Mayer et al., (1986) Biochim. Biophys. Acta 858:161). Microfluidization can be used when consistently small (50-200 nm), relatively uniform aggregates are required (Mayhew et al., (1984) Biochim. Biophys. Acta 775:169). These methods are easily adapted to packaging oligonucleotide formulations into liposomes.

[0163] Liposomes are broadly divided into two classes. Cationic liposomes are positively charged liposomes that interact with negatively charged nucleic acid molecules to form stable complexes. The positively charged nucleic acid / liposome complexes bind to the negatively charged cell surface and are taken up into endosomes. Due to the acidic pH within the endosome, the liposomes rupture, releasing their contents into the cytoplasm (Wang et al. (1987) Biochem. Biophys. Res. Commun., 147:980-985).

[0164] pH-sensitive or negatively charged liposomes entrap nucleic acids rather than complexing them. Because both the nucleic acid and the lipid are similarly charged, repulsion occurs rather than complexation. Nevertheless, some nucleic acid is trapped within the aqueous interior of these liposomes. pH-sensitive liposomes have been used to deliver nucleic acids encoding the thymidine kinase gene to cell monolayers in culture. Expression of the exogenous gene was detected in the target cells (Zhou et al. (1992) Journal of Controlled Release, 19:269-274).

[0165] One major type of liposome composition contains phospholipids other than naturally occurring phosphatidylcholine. Neutral liposome compositions can be formed, for example, from dimyristoylphosphatidylcholine (DMPC) or dipalmitoylphosphatidylcholine (DPPC). Anionic liposome compositions are generally formed from dimyristoylphosphatidylglycerol, while anionic fusogenic liposomes are primarily formed from dioleoylphosphatidylethanolamine (DOPE). Another type of liposome composition is formed from phosphatidylcholine (PC), such as soybean PC and egg PC. Another type is formed from a mixture of phospholipids and / or phosphatidylcholine and / or cholesterol.

[0166] Other examples of methods for introducing liposomes into cells in vitro and in vivo include U.S. Pat. No. 5,283,185; U.S. Pat. No. 5,171,678; WO 94 / 00569; WO 93 / 24640; WO 91 / 16024; Feigner, (1994) J. Biol. Chem. 269:2550; Nabel, (1993) Proc. Natl. Acad. Sci. 90:11307; Nabel, (1992) Human Gene Ther. 3:649; Gershon, (1993) Biochem. 32:7143; and Strauss, (1992) EMBO J. 11:417.

[0167] Nonionic liposomal systems, particularly those containing nonionic surfactants and cholesterol, have also been investigated to determine their usefulness in delivering drugs to the skin. Nonionic liposomal formulations containing NOVASOME™ I (glyceryl dilaurate / cholesterol / polyoxyethylene-10-stearyl ether) and NOVASOME™ II (glyceryl distearate / cholesterol / polyoxyethylene-10-stearyl ether) were used to deliver cyclosporine A to the dermis of mouse skin. The results showed that such nonionic liposomal systems were effective in promoting the deposition of cyclosporine A into different layers of the skin (Hu et al., (1994) STP Pharma. Sci., 4(6):466).

[0168] Liposomes may also be sterically stabilized liposomes, which contain one or more specialized lipids that provide extended circulation life compared to liposomes lacking such specialized lipids. An example of a sterically stabilized liposome is one in which a portion of the vesicle-forming lipid portion of the liposome is monosialoganglioside G M1or (B) liposomes that are derivatized with one or more hydrophilic polymers, such as polyethylene glycol (PEG) moieties. Without wishing to be bound by any particular theory, it is believed in the art that the enhanced circulation half-life of sterically stabilized liposomes, at least those containing gangliosides, sphingomyelin, or PEG-derivatized lipids, is due to reduced uptake into cells of the reticuloendothelial system (RES) (Allen et al., (1987) FEBS Letters, 223:42; Wu et al., (1993) Cancer Research, 53:3765).

[0169] Various liposomes containing one or more glycolipids are known in the art. Papahadjopoulos et al. (Ann. NY Acad. Sci., (1987), 507:64) reported that monosialoganglioside G improves the blood half-life of liposomes. M1 reported the ability of (1) sphingomyelin and (2) ganglioside G to bind to sphingomyelin. These findings were expanded upon by Gabizon et al. (Proc. Natl. Acad. Sci. USA, (1988), 85:6949). U.S. Patent No. 4,837,028 and WO 88 / 04924, both to Allen et al., reported the ability of (1) sphingomyelin and (2) ganglioside G to bind to sphingomyelin. M1 or galactocerebroside sulfate esters. U.S. Patent No. 5,543,152 (Webb et al.) discloses liposomes containing sphingomyelin. WO 97 / 13499 (Lim et al.) discloses liposomes containing 1,2-sn-dimyristoylphosphatidylcholine.

[0170] In one embodiment, cationic liposomes are used. Cationic liposomes have the advantage of being able to fuse with the cell membrane. Non-cationic liposomes cannot fuse efficiently with the plasma membrane, but they can be taken up by macrophages in vivo and used to deliver oligonucleotides to macrophages.

[0171] Additional advantages of liposomes include that liposomes derived from natural phospholipids are biocompatible and biodegradable; liposomes can incorporate a wide range of water-soluble and lipid-soluble drugs; and liposomes can protect oligonucleotides encapsulated in their internal compartments from metabolism and degradation (Rosoff, in "Pharmaceutical Dosage Forms," ​​Lieberman, Rieger and Banker (Eds.), 1988, volume 1, p. 245). Important considerations in the preparation of liposome formulations are the surface charge of the lipid, the size of the vesicle, and the water content of the liposomes.

[0172] A positively charged synthetic cationic lipid, N-[1-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTMA), can be used to form small liposomes that spontaneously interact with nucleic acids, forming lipid-nucleic acid complexes that can fuse with negatively charged lipids in the plasma membrane of tissue culture cells to deliver oligonucleotides (see, e.g., Feigner, PL et al., (1987) Proc. Natl. Acad. Sci. USA 8:7413-7417, and U.S. Pat. No. 4,897,355 for a description of DOTMA and its use with DNA).

[0173] The DOTMA analog 1,2-bis(oleoyloxy)-3-(trimethylammonia)propane (DOTAP) can be used in combination with phospholipids to form DNA-complexed vesicles. LIPOFECTIN™ (Bethesda Research Laboratories, Gaithersburg, Md.) is an effective agent for delivering highly anionic nucleic acids to living tissue culture cells, containing positively charged DOTMA liposomes that spontaneously interact with negatively charged polynucleotides to form complexes. If sufficient positively charged liposomes are used, the net charge of the resulting complexes will also be positive. The positively charged complexes thus prepared spontaneously adhere to the negatively charged cell surface, fuse with the plasma membrane, and efficiently deliver functional nucleic acids to, for example, tissue culture cells. Another commercially available cationic lipid, 1,2-bis(oleoyloxy)-3,3-(trimethylammonia)propane ("DOTAP") (Boehringer Mannheim, Indianapolis, Ind.), differs from DOTMA in that the oleoyl moieties are linked by ester rather than ether bonds.

[0174] Other reported cationic lipid compounds include compounds conjugated to various moieties, including, for example, carboxyspermine conjugated to either of two types of lipids, including compounds such as 5-carboxyspermylglycine dioctaoleoylamide ("DOGS") (TRANSFECTAM™, Promega, Madison, Wis.), and dipalmitoylphosphatidylethanolamine 5-carboxyspermylamide ("DPPES") (see, e.g., U.S. Pat. No. 5,171,678).

[0175] Another cationic lipid conjugate involves derivatizing lipids with cholesterol ("DC-Chol") formulated into liposomes in combination with DOPE (see Gao, X. and Huang, L., (1991) Biochim. Biophys. Res. Commun. 179:280). Lipopolylysine, prepared by conjugating polylysine to DOPE, has been reported to be effective for transfection in the presence of serum (Zhou, X. et al., (1991) Biochim. Biophys. Acta 1065:8). In certain cell lines, these liposomes containing conjugated cationic lipids are said to be less toxic and provide more efficient transfection than compositions containing DOTMA. Other commercially available cationic lipid products include DMRIE and DMRIE-HP (Vical, La Jolla, Calif.) and Lipofectamine (DOSPA) (Life Technology, Inc., Gaithersburg, Md.). Other cationic lipids suitable for delivery of oligonucleotides are described in WO98 / 39359 and WO96 / 37194.

[0176] Liposomal formulations are particularly suitable for topical administration, and liposomes offer several advantages over other formulations. Such advantages include reduced side effects associated with high systemic absorption of the administered drug, increased accumulation of the administered drug at the desired target, and the ability to administer oligonucleotides to the skin. In some implementations, liposomes are used to deliver oligonucleotides to epidermal cells and also to enhance the penetration of oligonucleotides into skin tissues, such as the skin. For example, liposomes may be applied topically. Topical delivery of drugs formulated as liposomes to the skin has been documented (e.g., Weiner et al., (1992) Journal of Drug Targeting, vol. 2, 405-410 and du Plessis et al., (1992) Antiviral Research, 18:259-265; Mannino, RJ and Fould-Fogerite, S., (1998) Biotechniques 6:682-690; Itani, T. et al., (1987) Gene 56:267-276; Nicolau, C. et al. (1987) Meth. Enzymol. 149:157-176; Straubinger, R M and Papahadjopoulos, D. (1983) Meth. Enzymol. 101:512-527; Wang, C Y and See Huang, L., (1987) Proc. Natl. Acad. Sci. USA 84:7851-7855).

[0177] Nonionic liposome systems, particularly those containing nonionic surfactants and cholesterol, have also been investigated to determine their usefulness in delivering drugs to the skin. Nonionic liposome formulations containing NOVASOME I (glyceryl dilaurate / cholesterol / polyoxyethylene-10-stearyl ether) and NOVASOME II (glyceryl distearate / cholesterol / polyoxyethylene-10-stearyl ether) were used to deliver drugs to the dermis of mouse skin. Such formulations containing oligonucleotides are useful for treating skin disorders.

[0178] Liposome targeting can also be based on, for example, organ specificity, cell specificity, and organelle specificity, and is known in the art.In the case of liposome targeted delivery system, lipid groups can be incorporated into the lipid bilayer of liposome to maintain the targeting ligand in stable association with the liposome bilayer.Various linking groups can be used to connect lipid chains to targeting ligands.Additional methods are known in the art, and are described, for example, in U.S. Patent Application Publication No. 20060058255, and the linking groups are incorporated herein by reference.

[0179] Liposomes containing oligonucleotides can be highly deformable. Such deformability can allow liposomes to penetrate pores smaller than the average radius of the liposome. For example, transfersomes are yet another type of liposome, highly deformable lipid aggregates that are attractive candidates for drug delivery vehicles. Transfersomes can be described as lipid droplets that are so deformable that they can easily penetrate pores smaller than a liquid droplet. Transfersomes can be made by adding a surface edge activator, usually a surfactant, to a standard liposome composition. Oligonucleotide-containing transfersomes can be delivered subcutaneously, for example, via infection, to deliver oligonucleotides to keratinocytes in the skin. To pass through intact mammalian skin, lipid vesicles must pass through a series of minute pores, each with a diameter of less than 50 nm, under the influence of an appropriate transdermal gradient. Furthermore, due to the properties of lipids, these transfersomes can self-optimize (e.g., adapt to the shape of pores in the skin), self-repair, and often self-load to reach their target without fragmentation. Transfersomes have been used to deliver serum albumin to the skin, and transfersome-mediated delivery of serum albumin has been shown to be as effective as subcutaneous injection of a solution containing serum albumin.

[0180] Other formulations suitable for the present disclosure are described in PCT Publication Nos. WO2009 / 088891, WO2009 / 132131, and WO2008 / 042973, which are incorporated by reference in their entireties.

[0181] Surfactants are widely used in formulations such as emulsions (including microemulsions) and liposomes. The most common way to classify and rank the properties of the many different types of surfactants, both natural and synthetic, is by using the hydrophilic / lipophilic balance (HLB). The nature of the hydrophilic group (also known as the "head") provides the most useful means for classifying the various surfactants used in formulations (Rieger, in Pharmaceutical Dosage Forms, Marcel Dekker, Inc., New York, NY, 1988, p. 285).

[0182] If the surfactant molecule is not ionized, it is classified as a nonionic surfactant. Nonionic surfactants are widely used in pharmaceuticals and cosmetics and are usable over a wide range of pH values. Their HLB values ​​generally range from 2 to approximately 18, depending on their structure. Nonionic surfactants include nonionic esters such as ethylene glycol esters, propylene glycol esters, glyceryl esters, polyglyceryl esters, sorbitan esters, sucrose esters, and ethoxylated esters. Nonionic alkanolamides and ethers, such as fatty alcohol ethoxylates, propoxylated alcohols, and ethoxylated / propoxylated block polymers, are also included in this class. Polyoxyethylene surfactants are the most popular members of the nonionic surfactant class.

[0183] If the surfactant molecule carries a negative charge when dissolved or dispersed in water, the surfactant is classified as anionic. Anionic surfactants include carboxylates such as soaps, acyl lactylates, acyl amides of amino acids, esters of sulfuric acid such as alkyl sulfates and ethoxylated alkyl sulfates, sulfonates such as alkyl benzene sulfonates, acyl isethionates, acyltaurates and sulfosuccinates, and phosphates. The most important members of the anionic surfactant class are alkyl sulfates and soaps.

[0184] If the surfactant molecule carries a positive charge when dissolved or dispersed in water, the surfactant is classified as cationic. Cationic surfactants include quaternary ammonium salts and ethoxylated amines. Quaternary ammonium salts are the most commonly used members of this class.

[0185] If the surfactant molecule has the ability to carry either a positive or negative charge, the surfactant is classified as amphoteric. Amphoteric surfactants include acrylic acid derivatives, substituted alkylamides, N-alkylbetaines, and phosphatides.

[0186] The use of surfactants in pharmaceuticals, formulations, and emulsions has been reviewed (Rieger, in Pharmaceutical Dosage Forms, Marcel Dekker, Inc., New York, NY, 1988, p. 285).

[0187] The oligonucleotide for use in the method of the present disclosure can also be provided as a micelle formulation.Micelle is a specific type of molecular assembly in which amphiphilic molecules are arranged in a spherical structure, so that all hydrophobic parts of the molecule are directed inward, and hydrophilic parts remain in contact with the surrounding aqueous phase.When the environment is hydrophobic, the opposite arrangement exists.

[0188] Lipid nanoparticle-based delivery methods The oligonucleotides of the present disclosure can be fully encapsulated in lipid formulations, such as lipid nanoparticles (LNPs) or other nucleic acid-lipid particles. LNPs are useful for systemic administration because they have an extended circulatory life after intravenous (iv) injection and accumulate at distal sites (e.g., sites physically distant from the administration site). LNPs include "pSPLPs," which include the encapsulated condensing agent-nucleic acid complexes described in PCT Publication No. WO 00 / 03683. The particles of the present disclosure typically have an average diameter of about 50 nm to about 150 nm, more typically about 60 nm to about 130 nm, more typically about 70 nm to about 110 nm, and most typically about 70 nm to about 90 nm, and are substantially nontoxic. Furthermore, when present in the nucleic acid-lipid particles of the present disclosure, the nucleic acid is resistant to degradation by nucleases in aqueous solution. Nucleic acid-lipid particles and methods for their preparation are disclosed, for example, in U.S. Patent Nos. 5,976,567; 5,981,501; 6,534,484; 6,586,410; 6,815,432; U.S. Publication No. 2010 / 0324120; and PCT Publication No. WO 96 / 40964.

[0189] Non-limiting examples of cationic lipids include N,N-dioleyl-N,N-dimethylammonium chloride (DODAC), N,N-distearyl-N,N-dimethylammonium bromide (DDAB), N-(I-(2,3-dioleoyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTAP), N-(I-(2,3-dioleyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTMA), N,N-dimethyl-2,3-dioleyloxy)propylamine (DODMA), 1,2-dilinoleyloxy-N,N-dimethylaminopropane (DLinDMA), 1,2-dilinolenyloxy-N,N-dimethylaminopropane (DLenDMA), 1,2-dilinole ... DLin-C-DAP, 1,2-dilinoleoyloxy-3-(dimethylamino)acetoxypropane (DLin-DAC), 1,2-dilinoleoyloxy-3-morpholinopropane (DLin-MA), 1,2-dilinoleoyl-3-dimethylaminopropane (DLin-DAP), 1,2-dilinoleylthio-3-dimethylaminopropane (DLin-S-DMA), 1-linoleoyl-2-linoleyloxy-3-dimethylaminopropane (DLin-2-DMAP), 1,2-dilinoleyloxy-3-trimethylaminopropane chloride salt (DLin-TMA.Cl), 1,2-dilinoleoyl-3-trimethylaminopropane chloride salt (DLin-TAP.Cl), 1,2-dilinoleyloxy-3-(N-methylpiperazino)propane (DLin-MPZ), or 3-(N,N-dilinoleylamino)-1,2-propanediol (DLinAP), 3-(N,N-dioleylamino)-1,2-propanedio (DOAP), 1,2-dilinoleyloxo-3-(2-N,N-dimethylamino)ethoxypropane (DLin-EG-DMA), 1,2-dilinoleyloxy-N,N-dimethylaminopropane (DLinDMA), 2,2-dilinoleyl-4-dimethylaminomethyl-[1,3]-dioxolane (DLin-K-DMA) or Analogs, (3aR,5s,6aS)-N,N-dimethyl-2,2-di((9Z,12Z)-octadeca-9,12-dienietetrahydro-3aH-cyclopenta[d][1,3]dioxol-5-amine (ALN100), (6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-yl 4-(dimethylamino)butanoate (MC3), 1,1'-(2-(4-(2-((2-(bis(2-hydroxydodecyl)amino)ethyl)(2-hydroxydodecyl)amino)ethyl)piperazin-1-ylethylazanediedidodecan-2-ol (Tech G1), or a mixture thereof. The cationic lipid may comprise, for example, about 20 mol % to about 50 mol %, or about 40 mol % of the total lipid present in the particle.

[0190] The ionizable / non-cationic lipids may be anionic or neutral lipids, including distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylglycerol (DOPG), dipalmitoylphosphatidylglycerol (DPPG), dioleoylphosphatidylethanolamine (DOPE), palmitoyloleoylphosphatidylcholine (POPC), palmitoyloleoylphosphatidylethanolamine (POPE), dioleoylphosphatidylcholine (DSPC), dioleoylphosphatidylglycerol (DOPG), dipalmitoylphosphatidylethanolamine (DPPG), dioleoylphosphatidylethanolamine (DOPE), palmitoyloleoylphosphatidylcholine (POPC), palmitoyloleoylphosphatidylethanolamine (POPE), dioleoylphosphatidylethanolamine (DOPE), palmitoyloleoylphosphatidylethanolamine (P ... Non-cationic lipids include, but are not limited to, oleoyl-phosphatidylethanolamine 4-(N-maleimidomethyl)-cyclohexane-1-carboxylate (DOPE-mal), dipalmitoylphosphatidylethanolamine (DPPE), dimyristoylphosphoethanolamine (DMPE), distearoyl-phosphatidylethanolamine (DSPE), 16-O-monomethyl PE, 16-O-dimethyl PE, 18-1-trans PE, 1-stearoyl-2-oleoyl-phosphatidylethanolamine (SOPE), cholesterol, or mixtures thereof. For example, when cholesterol is included, the non-cationic lipid may comprise about 5 mol% to about 90 mol%, about 10 mol%, or about 60 mol% of the total lipid present in the particle.

[0191] The conjugated lipid that inhibits particle aggregation can be, for example, a polyethylene glycol (PEG)-lipid, including, but not limited to, PEG-diacylglycerol (DAG), PEG-dialkyloxypropyl (DAA), PEG-phospholipid, PEG-ceramide (Cer), or a mixture thereof. PEG-DAA conjugates can be, for example, PEG-dilauryloxypropyl (C 12 ), PEG dimyristyloxypropyl (C 14 ), PEG-dipalmityloxypropyl (C 16 ), or PEG-distearyloxypropyl (C 18 The complex lipid that prevents particle aggregation can be, for example, 0 mol % to about 20 mol %, or about 2 mol % of the total lipid present in the particle.

[0192] In some embodiments, the nucleic acid-lipid particles further comprise cholesterol, for example, from about 10 mol % to about 60 mol %, or about 50 mol % of the total lipid present in the particle.

[0193] ASOs may also be delivered in lipidoids. The synthesis of lipidoids has been extensively described, and formulations containing these compounds are particularly suitable for the delivery of modified nucleic acid molecules or ASOs (see Mahon et al., Bioconjug Chem. 2010 21:1448-1454; Schroeder et al., J Intern Med. 2010 267:9-21; Akinc et al., Nat Biotechnol. 2008:26:561-569; Love et al., Proc Natl Acad Sci U A. 2010 107:1864-1869; Siegwart et al., Proc Natl Acad Sci U S A. 2011 108:12996-3001, all of which are incorporated herein in their entireties).

[0194] Lipid compositions for RNA delivery are disclosed in WO2012170930A1, WO2013149141A1, and WO2014152211A1, each of which is incorporated herein by reference.

[0195] IV. Therapeutic uses The present disclosure provides methods for treating or preventing diseases and disorders of the central nervous system (CNS) and peripheral nervous system (PNS), including SYNGAP1-associated disorders (e.g., associated with SYNGAP1 mutations), such as SynGAP1-associated intellectual disability (ID), mental retardation, autosomal dominant 5 (MRD5), or SynGAP1-associated nonsyndromic intellectual disability (NSID), affective disorders (e.g., depression), schizophrenia, Alzheimer's disease, Parkinson's disease, Huntington's disease, and autism spectrum disorders (ASD) (e.g., Asperger's syndrome, autism, and pervasive developmental disorder not otherwise specified (PDD-NOS)). Subjects with CNS or PNS trauma (e.g., neurological abnormalities associated with brain or spinal cord ischemia or trauma, stroke, or surgery or anesthesia) can also be treated according to the methods provided herein. The methods include administering to a subject an ASO or a pharmaceutical composition comprising an ASO as provided herein. Without wishing to be bound by theory, it is believed that the ASOs provided herein exert their desired effects through their ability to modulate (e.g., increase or decrease) the levels of SYNGAP1 protein, SYNGAP1 mRNA, and / or SYNGAP1 activity in the cells of a subject (e.g., a human, mouse, hamster, non-human primate (e.g., a monkey)), for example, by increasing the levels of SYNGAP1 protein in the cells of the subject.

[0196] Another aspect of the present disclosure includes a method of regulating (e.g., increasing or decreasing) the expression of SYNGAP1 in a subject's cell, comprising contacting the cell with an ASO of the present disclosure (or a pharmaceutical composition comprising the ASO), thereby treating a disease or disorder in the subject (e.g., a disease or disorder provided herein).

[0197] Another aspect of the present disclosure includes a method of modulating (e.g., increasing or decreasing) SYNGAP1 mRNA or protein levels in cells of a subject identified as having a disease or disorder (e.g., a SYNGAP1-associated disorder) provided herein.

[0198] Yet another aspect includes a method of modulating (e.g., increasing or decreasing) expression of the SYNGAP1 gene in a cell (e.g., in vivo, ex vivo, or in vitro), comprising contacting the cell with an ASO of the present disclosure (or a pharmaceutical composition comprising the ASO), thereby increasing expression of the SYNGAP1 gene in the cell. In some embodiments, the cell is a mammalian cell (e.g., a human cell, such as a human neuron). The method may include contacting the cell with an ASO of the present disclosure (or a pharmaceutical composition comprising the ASO) in an amount effective to increase expression of the SYNGAP1 gene in the cell, thereby increasing expression of the SYNGAP1 gene in the cell. In some embodiments, contacting the cell with the ASO (or a pharmaceutical composition comprising the ASO) modulates (e.g., increases) the amount of SYNGAP1 mRNA in the cell. In some embodiments, contacting the cell with the ASO (or a pharmaceutical composition comprising the ASO) modulates (e.g., increases or decreases) the amount of SYNGAP1 protein in the cell. In some embodiments, contacting a cell with an ASO (or a pharmaceutical composition comprising the ASO) modulates (e.g., increases or decreases) the amount of SYNGAP1 activity in the cell.

[0199] In yet another aspect, the present disclosure provides an ASO of the present disclosure (or a pharmaceutical composition comprising the ASO) for use as a medicament. Additionally, the present disclosure provides an ASO of the present disclosure (or a pharmaceutical composition comprising the ASO) for use in therapy.

[0200] Contacting a cell with an ASO can be performed in vitro, ex vivo, or in vivo. Contacting a cell with an oligonucleotide in vivo includes contacting a cell or cell population within a subject, e.g., a human subject, with an ASO. A combination of in vitro, ex vivo, and in vivo cell contacting methods is also possible. As described above, contacting a cell can be direct or indirect. Furthermore, contacting a cell can be achieved via a targeting ligand, including any ligand described herein or known in the art. In some embodiments, the targeting ligand is a carbohydrate moiety, e.g., a GalNAc3 ligand, or other ligand that directs the oligonucleotide to a site of interest. In some embodiments, the cell can be a neuron. For example, the neuron can be from the CNS, prefrontal cortex, motor cortex, or hippocampus. In some embodiments, the cell is a neuron. In some embodiments, the neuron is a glutamatergic neuron. In some embodiments, the ASO is administered with one or more agents that can facilitate penetration of the ASO through the blood-brain barrier. For example, in some embodiments, the ASO is conjugated to a composition that facilitates penetration or transport of the ASO across the blood-brain barrier, such as a viral vector or an antibody against the transferrin receptor.

[0201] The ASO or pharmaceutical composition disclosed herein may be administered to a subject intravenously, intraarterially, intraperitoneally, intramuscularly, subcutaneously, transdermally, intrapleurally, intrathecally, intracerebrally, intraventricularly, intracerebroventricularly, intracisternally, intraspinally, perispinally, intracavitary, via perfusion through a catheter, or by direct lesion injection. In certain embodiments, the ASO or pharmaceutical composition is administered using an intracranial or intraspinal needle or catheter. In certain embodiments, the ASO or pharmaceutical composition is administered systemically. In certain embodiments, the ASO or pharmaceutical composition is administered parenterally. For example, in certain embodiments, the ASO or pharmaceutical composition is administered intravenously (e.g., by intravenous infusion), for example, using a prefilled bag, prefilled pen, or prefilled syringe. In other embodiments, the ASO or pharmaceutical composition is administered locally to an organ or tissue in which increased target gene expression is desired (e.g., neuronal cells).

[0202] In some embodiments, the ASO is administered to a subject so that the ASO is delivered to a specific site within the subject. Such targeted delivery can be achieved by either systemic or local administration. Increased expression of SYNGAP1 can be assessed by measuring the level or change in the level of SYNGAP1 mRNA or SYNGAP1 protein in a sample (e.g., blood, tissue (e.g., neural tissue), neuronal cell sample (e.g., hippocampal cells, motor cortex cells, or prefrontal cortex cells), or neural fluid (e.g., cerebrospinal fluid (CSF)) from a specific site within the subject. In certain embodiments, the method includes a clinically relevant increase in SYNGAP1 expression, as indicated, for example, by a clinically relevant outcome after treating the subject with an agent that reduces SYNGAP1 expression.

[0203] In some embodiments, the methods provided herein may ameliorate or prevent the onset of one or more symptoms or conditions associated with a disease or disorder described herein (e.g., a SYNGAP1-associated disorder), including epilepsy, cognitive impairment (e.g., moderate to severe cognitive impairment), hypotonia (e.g., mild hypotonia), global developmental delay, delayed language development, sleep disorders, oral apraxia, inattention, impulsivity, physical aggression, mood swings, irritability, and rigidity. In some embodiments of the methods provided herein, an ASO provided herein (or a pharmaceutical composition comprising the ASO) is administered in an amount and for a time effective to result in a reduction or amelioration (e.g., 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%) of one or more symptoms associated with a disease or disorder described herein (e.g., a SYNGAP1-associated disorder).

[0204] Regulation of SYNGAP1 expression levels In some aspects, the therapeutic methods disclosed herein use ASOs targeting SYNGAP1 regRNA to modulate (e.g., increase or decrease) SYNGAP1 gene expression levels in a subject. Modulated SYNGAP1 gene expression includes any level of SYNGAP1 gene modulation, e.g., at least partial modulation of SYNGAP1 gene expression. Modulation (e.g., increase or decrease) of SYNGAP1 gene expression can be assessed by determining the absolute or relative levels of one or more of these variables compared to a control level. The control level can be any type of control used in the art, such as a baseline level before administration, or a level determined from a similar subject, cell, or untreated or control-treated sample (e.g., a buffer-only (vehicle) control or a non-active agent control). In certain embodiments, the methods provided herein result in clinically relevant modulation (e.g., increase or decrease) of SYNGAP1 expression, as indicated by a clinically relevant outcome after treating a subject with an agent that modulates (e.g., increases) SYNGAP1 expression.

[0205] In certain embodiments, the methods disclosed herein provide for an increase in blood cholesterol levels of at least about 1%, at least about 2%, at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99%, relative to a pre-dosing, pre-administration, or pre-exposure baseline level. In certain embodiments, the methods disclosed herein result in an increase in SYNGAP1 gene expression in at least about 99% of the subject's cells, tissue (e.g., neural tissue), neuronal cell sample (e.g., hippocampal cells, motor cortex cells, or prefrontal cortex cells), or sample (e.g., neural fluid (e.g., cerebrospinal fluid (CSF))). In certain embodiments, the methods disclosed herein increase SYNGAP1 gene expression by at least 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, at least 6-fold, at least 7-fold, at least 8-fold, at least 9-fold, or at least 10-fold relative to pre-dosing baseline levels. In certain embodiments, the subject has a deficiency in SYNGAP1 expression, and the methods disclosed herein increase SYNGAP1 expression levels (e.g., SYNGAP1 protein levels or SYNGAP1 protein levels) in at least about 99% of the subject's cells, tissue (e.g., neural tissue), neuronal cell sample (e.g., hippocampal cells, motor cortex cells, or prefrontal cortex cells), or sample (e.g., neural fluid (e.g., cerebrospinal fluid (CSF)). ... mRNA level) or SYNGAP1 protein activity to at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 100% of the average SYNGAP1 expression level (e.g., SYNGAP1 protein level or SYNGAP1 mRNA level) or SYNGAP1 protein activity in a similar cell, tissue, or subject (e.g., of the same species, similar age, and / or same sex) that does not have a SYNGAP1 expression deficiency.

[0206] In some embodiments, the ASO of the disclosure reduces SYNGAP1 mRNA production (e.g., in a cell or in a cell, tissue, or sample of a subject) by at least about 1%, at least about 2%, at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95 ... The enhancement may be up to about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, at least about 100%, at least about 150%, at least about 200%, at least about 250%, at least about 300%, at least about 350%, at least about 400%, at least about 500%, at least about 600%, at least about 700%, at least about 800%, at least about 900%, or more. In some embodiments, the ASOs of the present disclosure may enhance production of SYNGAP1 mRNA (e.g., in cells or in a subject's cells, tissues, or samples) by at least 1-fold, at least 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, at least 6-fold, at least 7-fold, at least 8-fold, at least 9-fold, or at least 10-fold or more relative to baseline levels before dosing, administration, or exposure.

[0207] In certain embodiments, the methods disclosed herein provide for an improvement in blood cholesterol levels relative to pre-dosing, pre-administration, or pre-exposure baseline levels of at least about 1%, at least about 2%, at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about The method results in a 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% reduction in SYNGAP1 gene expression in a subject's cells, tissue (e.g., neural tissue), neuronal cell sample (e.g., hippocampal cells, motor cortex cells, or prefrontal cortex cells), or sample (e.g., neural fluid (e.g., cerebrospinal fluid (CSF))). In certain embodiments, the methods disclosed herein result in at least a 2-fold, at least a 3-fold, at least a 4-fold, at least a 5-fold, at least a 6-fold, at least a 7-fold, at least a 8-fold, at least a 9-fold, or at least a 10-fold reduction in SYNGAP1 gene expression relative to pre-dosing baseline levels.

[0208] In some embodiments, the ASO of the disclosure reduces SYNGAP1 mRNA production (e.g., in a cell or in a cell, tissue, or sample of a subject) by at least about 1%, at least about 2%, at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95 ... The reduction may be by about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, at least about 100%, at least about 150%, at least about 200%, at least about 250%, at least about 300%, at least about 350%, at least about 400%, at least about 500%, at least about 600%, at least about 700%, at least about 800%, at least about 900%, or more. In some embodiments, the ASOs of the present disclosure may reduce production of SYNGAP1 mRNA (e.g., in cells or in a subject's cells, tissues, or samples) by at least 1-fold, at least 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, at least 6-fold, at least 7-fold, at least 8-fold, at least 9-fold, or at least 10-fold or more relative to pre-dose, pre-administration, or pre-exposure baseline levels.

[0209] In some embodiments, expression of a SYNGAP1 protein (e.g., one or more isoforms of a SYNGAP1 protein, e.g., SYNGAP1 Aα1, SYNGAP1 Aα2, SYNGAP1 Aβ, SYNGAP1 Aγ, SYNGAP1 Bα1, SYNGAP1 Bα2, SYNGAP1 Bβ, SYNGAP1 Bγ, SYNGAP1 Cα1, SYNGAP1 Cα2, SYNGAP1 Cβ, SYNGAP1 Cγ, or any combination thereof) is modulated (e.g., increased or decreased) following treatment with or administration of an ASO of the present disclosure. In some embodiments, expression of SYNGAP1 protein (e.g., in a cell or in a cell, tissue, or sample of a subject (e.g., neural tissue or neural fluid (e.g., cerebrospinal fluid (CSF))))) increases by at least about 1%, at least about 2%, at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85 ... Modulated (e.g., increased or decreased) by 5%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, at least about 100%, at least about 150%, at least about 200%, at least about 250%, at least about 300%, at least about 350%, at least about 400%, at least about 500%, at least about 600%, at least about 700%, at least about 800%, at least about 900%, or more.In some embodiments, expression of SYNGAP1 protein (e.g., in a cell or in a cell, tissue, or sample of a subject (e.g., neural tissue or neural fluid (e.g., cerebrospinal fluid (CSF)))) is increased by at least 1-fold, at least 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, at least 6-fold, at least 7-fold, at least 8-fold, at least 9-fold, or at least 10-fold or more relative to pre-dosing, pre-administration, or pre-exposure baseline levels.

[0210] The expression of the SYNGAP1 gene can be evaluated based on the level of any variable associated with SYNGAP1 gene expression, such as the SYNGAP1 mRNA level or the SYNGAP1 protein level. In certain embodiments, the expression level or activity of SYNGAP1 herein refers to the average expression level or activity of SYNGAP1 in the brain (e.g., in neuronal cells of a mammal or human subject).

[0211] In certain embodiments, surrogate markers may be used to detect modulation (e.g., increase or decrease) of SYNGAP1 expression levels or SYNGAP1 activity. For example, effective treatment of a disease or disorder provided herein (e.g., a SYNGAP1-associated disorder) as indicated by acceptable diagnostic and monitoring criteria with an agent that increases SYNGAP1 expression may be understood to exhibit a clinically relevant increase in SYNGAP1.

[0212] An increase in expression of the SYNGAP1 gene may be evident by an increase in the amount of mRNA expressed by a first cell or group of cells (such cells may be present, for example, in a sample derived from a subject) in which the SYNGAP1 gene is transcribed, processed, or has been treated (e.g., by contacting the cell(s) with an oligonucleotide of the present disclosure or by administering an oligonucleotide of the present disclosure to a subject in which the cells are or were present), such that expression of the SYNGAP1 gene is increased when compared to a second cell or group of cells that is substantially identical to the first cell or group of cells but that has not or has not been so treated (control cell(s) that are not treated with the oligonucleotide or that are not treated with an oligonucleotide targeting the gene of interest). A decrease in expression of the SYNGAP1 gene may be evident by a decrease in the amount of mRNA expressed by a first cell or group of cells (such cells may be present, for example, in a sample derived from a subject) in which the SYNGAP1 gene is transcribed, processed, or has been treated (e.g., by contacting the cell(s) with an oligonucleotide of the present disclosure or by administering an oligonucleotide of the present disclosure to a subject in which the cells are or were present), such that expression of the SYNGAP1 gene is decreased when compared to a second cell or group of cells that is substantially identical to the first cell or group of cells but that has not or has not been so treated (control cell(s) that are not treated with the oligonucleotide or that are not treated with an oligonucleotide targeting the gene of interest).

[0213] In other embodiments, an increase or decrease in SYNGAP1 gene expression can be assessed with respect to a parameter functionally related to SYNGAP1 gene expression, such as an increase in SYNGAP1 protein expression or SYNGAP1 activity. Increases or decreases in SYNGAP1 protein levels, mRNA levels, or activity can be determined in any cell that expresses SYNGAP1, either endogenously or heterologously from an expression construct, using any assay known in the art.

[0214] An increase or decrease in SYNGAP1 expression may be evidenced by an increase or decrease in the level of SYNGAP1 protein expressed by a cell or group of cells (e.g., the level of protein expressed in a sample derived from a subject) compared to a control cell or group of cells. An increase or decrease in SYNGAP1 expression may also be evidenced by an increase in SYNGAP1 mRNA levels in a treated cell or group of cells compared to a control cell or group of cells.

[0215] Control cells or cell groups that can be used to assess the increase or decrease in expression of the SYNGAP1 gene include cells or cell groups that have not yet been contacted with the oligonucleotides of the present disclosure. For example, the control cells or cell groups can be derived from an individual subject (e.g., a human or animal subject) before the subject is treated with the oligonucleotide.

[0216] The level of SYNGAP1 mRNA expressed by a cell or group of cells can be determined using any method known in the art for assessing mRNA expression. In one embodiment, the expression level of SYNGAP1 in a sample is determined by detecting a transcribed polynucleotide, or a portion thereof, such as the mRNA of the SYNGAP1 gene. RNA can be extracted from cells using RNA extraction techniques, including, for example, acid phenol / guanidine isothiocyanate extraction (RNAzol B; Biogenesis), the RNEASY™ RNA preparation kit (Qiagen), or PAXgene (PreAnalytix, Switzerland). Typical assay formats utilizing ribonucleic acid hybridization include nuclear run-on assays, RT-PCR, RNase protection assays, Northern blotting, in situ hybridization, and microarray analysis. Circulating SYNGAP1 mRNA can be detected using the methods described in PCT Publication WO 2012 / 177906, the entire contents of which are incorporated herein by reference. In some embodiments, the level of SYNGAP1 expression is determined using a nucleic acid probe. As used herein, the term "probe" refers to any molecule capable of selectively binding to a specific SYNGAP1 sequence, e.g., to an mRNA or polypeptide. Probes may be synthesized by one skilled in the art or derived from appropriate biological preparations. Probes may also be specifically designed to be labeled. Examples of molecules that can be utilized as probes include, but are not limited to, RNA, DNA, proteins, antibodies, and organic molecules.

[0217] The isolated mRNA can be used in hybridization or amplification assays, including, but not limited to, Southern or Northern analysis, polymerase chain reaction (PCR) analysis, and probe arrays. One method for determining mRNA levels involves contacting the isolated mRNA with a nucleic acid molecule (probe) that can hybridize to SYNGAP1 mRNA. In one embodiment, the mRNA is immobilized on a solid surface and contacted with the probe, for example, by running the isolated mRNA on an agarose gel and transferring the mRNA from the gel to a membrane such as nitrocellulose. In an alternative embodiment, the probe(s) are immobilized on a solid surface and the mRNA is contacted with the probe(s), for example, in an AFFYMETRIX gene chip array. One skilled in the art can readily adapt known mRNA detection methods for use in determining SYNGAP1 mRNA levels.

[0218] Alternative methods for determining the expression level of SYNGAP1 in a sample include, for example, processes of nucleic acid amplification and / or reverse transcription (to prepare cDNA) of mRNA in the sample, such as RT-PCR (Mullis, 1987, experimental embodiment described in U.S. Pat. No. 4,683,202), ligase chain reaction (Barany (1991) Proc. Natl. Acad. Sci. USA 88:189-193), self-sustained sequence replication (Guatelli et al. (1990) Proc. Natl. Acad. Sci. USA 87:1874-1878), transcription amplification systems (Kwoh et al. (1989) Proc. Natl. Acad. Sci. USA 86:1173-1177), Q-beta replicase (Lizardi et al. (1988) Bio / Technology 6:1197), rolling circle replication (Lizardi et al., U.S. Pat. No. 5,854,033), or any other nucleic acid amplification method, followed by detection of the amplified molecules using techniques well known to those of skill in the art. These detection schemes are particularly useful for detecting nucleic acid molecules when such molecules are present in very low numbers. In certain embodiments of the present disclosure, the level of SYNGAP1 expression is determined by quantitative fluorogenic RT-PCR (i.e., TaqMan™ system) or DUAL-GLO® luciferase assay.

[0219] SYNGAP1 mRNA expression levels can be monitored using membrane blots (such as those used in Northern, Southern, dot, and other hybridization analyses), or microwells, sample tubes, gels, beads, or fibers (or any solid support containing bound nucleic acids). See U.S. Patent Nos. 5,770,722, 5,874,219, 5,744,305, 5,677,195, and 5,445,934, which are incorporated herein by reference. Determining SYNGAP1 expression levels can also involve using nucleic acid probes in solution.

[0220] In some embodiments, the level of mRNA expression is assessed using branched DNA (bDNA) assays, quantitative PCR (qPCR), real-time quantitative PCR (RT-qPCR), multiplex qPCR or RT-qPCR, RNA-seq, or microarray analysis. Such methods can also be used to detect SYNGAP1 nucleic acids.

[0221] The level of SYNGAP1 protein expression can be determined using any method known in the art for measuring protein levels.Such methods include, for example, electrophoresis, capillary electrophoresis, high-performance liquid chromatography (HPLC), thin-layer chromatography (TLC), hyperdiffusion chromatography, fluid or gel precipitation reaction, absorption spectroscopy, colorimetry, spectrophotometric analysis, flow cytometry, FACS, immunodiffusion (single or double), immunoelectrophoresis, Western blotting, radioimmunoassay (RIA), enzyme-linked immunosorbent assay (ELISA), immunofluorescence assay, electrochemiluminescence assay, Luminex, MSD, FISH, etc.Such assays can also be used to detect proteins that indicate the presence or replication of SYNGAP1 protein. [Example]

[0222] Below are examples of specific embodiments for carrying out the present disclosure. These examples are presented for illustrative purposes only and are not intended to limit the scope of the present invention in any way. Efforts have been made to ensure accuracy with respect to numbers used (e.g., amounts, temperatures, etc.), but some experimental error and deviation should, of course, be allowed for.

[0223] The practice of the present disclosure will employ, unless otherwise indicated, conventional methods of protein chemistry, biochemistry, recombinant DNA techniques, and pharmacology, within the skill of one in the art. Such techniques are fully explained in the literature. See, e.g., T.E. Creighton, Proteins: Structures and Molecular Properties (W.H. Freeman and Company, 1993); A.L. Lehninger, Biochemistry (Worth Publishers, Inc., current addition); Sambrook, et al., Molecular Cloning: A Laboratory Manual (2nd Edition, 1989); Methods In Enzymology (S. Colowick and N. Kaplan eds., Academic Press, Inc.); Remington's Pharmaceutical Sciences, 18th Edition (Easton, Pennsylvania: Mack Publishing Company, 1990); and Carey and Sundberg Advanced Organic Chemistry, 3rd Ed. (Plenum Press), Vols. A and B (1992).

[0224] Example 1: Modulation of SYNGAP1 expression using SYNGAP1 regRNA-targeting ASO Four human SYNGAP1 regRNA targets were identified in the human genome (RR86, RR87, RR88, and RR93). RNA capture sequencing and real-time quantitative PCR (qPCR) were used to assess the expression of human SYNGAP1 (hSYNGAP1) regRNAs RR86 and RR93 in HEK293 cells, SK-N-AS cells, and human brain tissue. In this analysis, the qPCR reference gene was PPIA. As shown in Figure 3, SYNGAP1 regRNAs RR86 and RR93 were detected in HEK293 and SK-N-AS cells, as well as human brain samples.

[0225] To evaluate the ability of ASOs targeting hSYGNAP1 regRNA to regulate SYNGAP1 expression, 210 ASOs targeting human SYNGAP1 regRNA were synthesized. 105 ASOs were stereogenic oligonucleotides, and 105 ASOs were gapmers. The ASOs were screened at 120 nM in SK-N-AS and HEK293 cells to determine their efficacy in increasing human SYNGAP1 mRNA levels. Briefly, SK-N-AS or HEK293 cells were reverse transfected with 120 nM ASOs on day 0, and cells were harvested on day 2 for mRNA quantification via qPCR. Cells untreated with ASOs or treated with either a gapmer non-targeting control (NTC) ASO (CO-1588) or a stereogenic NTC ASO (CO-1589) served as controls. mRNA was normalized to mRNA from cells treated with the gapmer NTC ASO (CO-1588).

[0226] From this initial screen, 11 ASOs were selected for chemical fine-tuning by varying the chemistry, type, and location of chemical modifications of the selected ASOs. Thirty-nine additional ASOs were synthesized from basewalks and tiling and further tested for dose-dependent efficacy. Forty-four ASOs containing additional chemical modifications were synthesized, including four extended gapmers, 24 LNA gapmers, eight with PO / PS linkages, and eight mixmers. ASOs that demonstrated efficacy in increasing hSYNGAP1 mRNA at 120 nM in SK-N-AS and HEK293 cells (as described above) were further tested for dose-dependent efficacy at 60 nM, 90 nM, or 120 nM in SK-N-AS cells or at 12, 30, 60, 90, 120, or 150 nM in HEK293 cells.

[0227] The ASO sequences and chemical modifications for the above screens are provided in Figure 2 and Table 2. Table 5 provides the SYNGAP1 mRNA fold change for each ASO tested at 120 or 150 nM in HEK293 and SK-N-AS cells. The data in Table 5 are the maximum fold change in either HEK293 or SK-N-AS cells.

[0228] [Table 5] TIFF2025540101000081.tif209165TIFF2025540101000082.tif209165TIFF2025540101000083.tif129165

[0229] Gapmer ASOs CO-7432, CO-7433, CO-7435, CO-7441, CO-7447, CO-7482, and CO-7494 targeting regRNAs RR86, RR87, and RR88 upregulated SYNGAP1 mRNA levels by more than 1.4-fold in HEK293 cells compared with control ASOs CO-1588 and CO-1589.

[0230] As shown in Figures 4A, 4B, 4C, and 4D, a dose-dependent increase in SYNGAP1 mRNA was observed in HEK293 cells after treatment with selected ASOs CO-7435, CO-7447, CO-7494, CO-7512, CO-7432, and CO-7433. Furthermore, the ASOs CO-7432, CO-7433, CO-7435, CO-7436, CO-7447, CO-7482, CO-7494, CO-7498, CO-7512, and CO-7524 increased SYNGAP1 mRNA in SK-N-AS cells. As shown in Figures 4E and 4F, a dose-dependent increase in SYNGAP1 mRNA was observed in HEK293 cells and SK-N-AS after treatment with the selected ASOs CO-9367, CO-9369, CO-9370, CO-9373, and CO-9376.

[0231] A gapmer hotspot at SYNGAP1 chr6:33419695-33419939 was identified between CO-9366 and CO-9376 (Figure 5). SK-N-AS or HEK293 cells were reverse transfected with 120 nM of the selected ASO on day 0. Cells were harvested on day 2 for SYNGAP1 mRNA quantification using qPCR (as described above). The tiled ASO CO-9366-CO-9376, which covers the regRNA RR93 at this hotspot, increased SYNGAP1 mRNA by 1.2- to 2.5-fold in both SK-N-AS and HEK293 cells compared to the control ASOs CO-1588 and CO-1589 (Figure 5).

[0232] Further dose-response characterization of this regRNA hotspot in SK-N-AS and HEK293 cells was also performed by reverse transfecting cells with 7.5, 15, 30, 60, and 120 nM ASO, as previously described. As shown in Figure 6, ASOs targeting the regRNA hotspot induced a dose-dependent upregulation of SYNGAP1 mRNA in both SK-N-AS and HEK293 cells.

[0233] Example 2: ASOs targeting SYNGAP1 regRNAs RR86 and RR96 induce increased expression of SYNGAP1 mRNA in iPSC-differentiated neurons To evaluate the ability of ASOs targeting hSYGNAP1 regRNA to regulate SYNGAP1 expression in a CNS translation model, the following experiment was performed using human iPSC-differentiated neurons. Briefly, human induced pluripotent stem cells (iPSCs) were differentiated into neurons by overexpression of the transcription factor neurogenin 2 via viral transduction as described in Zhang et al. (2013) Neuron 78(5):785-98 (incorporated herein by reference). Differentiated neurons were matured in culture for 7–10 days and transfected using Lipofectamine™ 2000 (INVITROGEN) with 12.5 nM, 25 nM, 50 nM, or 100 nM of one of the ASOs targeting SYNGAP1 regRNA RR86_v2 and RR93: CO-10645, CO-11528, CO-7432, CO-7435, CO-9367, CO-9369, or CO-9370. SYNGAP1 mRNA was detected using qPCR as described above.

[0234] As shown in Figure 7, ASOs targeting RR86v2 and RR93 upregulated SYNGAP1 mRNA by approximately 1.5- to 2.2-fold compared to the control ASO CO-1588, indicating that targeting these regulatory RNAs can increase SYNGAP1 expression in clinically relevant cell models.

[0235] Incorporation by Reference Unless otherwise stated, the entire disclosure of each of the patent documents and scientific articles referred to herein is incorporated by reference for all purposes.

[0236] equivalent The present disclosure may be embodied in other specific forms without departing from its spirit or essential characteristics. Accordingly, the foregoing embodiments are to be considered in all respects as illustrative and not limiting of the present disclosure described herein. The scope of the present disclosure is, therefore, indicated by the appended claims, rather than by the foregoing description, and all changes that come within the meaning and range of equivalency of the claims are intended to be embraced therein.

Claims

1. An antisense oligonucleotide (ASO) complementary to at least 8 consecutive nucleotides of a regulatory RNA of human SYNGAP1, wherein the regulatory RNA has a nucleotide sequence selected from the group consisting of SEQ ID NO: 4, 5, or 6.

2. The ASO of claim 1, wherein the ASO is complementary to a sequence in the regRNA that is no more than 200 nucleotides from the 3' end of the regRNA.

3. The ASO of claim 1, wherein the ASO is complementary to a sequence in the regRNA that is no more than 200 nucleotides from the 5' end of the regRNA.

4. The ASO of any one of claims 1 to 3, wherein the regRNA is not a polyadenylated RNA.

5. 5. The ASO of any one of claims 1 to 4, wherein the regulatory RNA has the nucleotide sequence of SEQ ID NO: 5, and the ASO comprises a nucleotide sequence selected from the group consisting of SEQ ID NOs: 14-59, 220-250, 261-267, 272-278, 526-528, 542-591, 702-728, 729-735-741, 988-990, and 1007-2961.

6. The ASO of any one of claims 1 to 4, wherein the regulatory RNA has the nucleotide sequence of SEQ ID NO: 4 or 6, and the ASO comprises a nucleotide sequence selected from the group consisting of SEQ ID NOs: 110-219, 280-525, 529-541, 592-701, 742-987, 991-1003, and 2962-4852.

7. The ASO of any one of claims 1 to 4 or 6, comprising a nucleotide sequence of at least 8 consecutive nucleotides of chr6:33419695 to 33419939.

8. The ASO of any one of claims 1 to 5, comprising a nucleotide sequence of at least 8 consecutive nucleotides of chr6:33453987 to 33454269.

9. The ASO of any one of claims 1 to 4 or 6, comprising a nucleotide sequence of at least 8 consecutive nucleotides of chr6:33419674 to 33419940.

10. The ASO of any one of claims 1 to 9, which is no more than 50, 40, 30, 25, 20, 18, or 16 nucleotides in length.

11. The ASO of any one of claims 1 to 10, comprising an RNA polynucleotide comprising one or more chemical modifications.

12. The ASO of claim 11, wherein at least 3, 4, or 5 nucleotides at the 5' end and at least 3, 4, or 5 nucleotides at the 3' end of the ASO comprise ribonucleotides having one or more chemical modifications.

13. The one or more chemical modifications may be 2'-O-C1-4 alkyl, e.g., 2'-O-methyl (2'-OMe), 2'-deoxy (2'-H), 2'-O-C1-3 alkyl-O-C1-3 alkyl, e.g., 2'-methoxyethyl ("2'-MOE"), 2'-fluoro ("2'-F"), 2'-amino ("2'-NH2"), 2'-arabinosyl ("2'-arabino") nucleotides, 2'-F-arabinosyl ("2'-F"), 2'-F-arabinosyl ("2'-F"), 2'-F-isopropyl (" ...

13. The ASO of claim 11 or 12, comprising a nucleotide sugar modification comprising one or more of: 2'-amido-arabino ("Arabino") nucleotides, 2'-locked nucleic acid ("LNA") nucleotides, 2'-amide-bridged nucleic acid (AmNA), 2'-unlocked nucleic acid ("ULNA") nucleotides, L-sugars ("L-sugars"), 4'-thioribosyl nucleotides, constrained ethyl (cET), 2'-fluoro-arabino (FANA), or thiomorpholino.

14. 14. The ASO of any one of claims 11-13, wherein the one or more chemical modifications comprise an internucleotide linkage modification comprising one or more of phosphorothioate ("PS" or (P(S))), phosphoramidate (P(NR1R2), e.g., dimethylaminophosphoramidate (P(N(CH3)2)), phosphonocarboxylate (P(CH2)nCOOR), e.g., phosphonoacetate "PACE" (P(CH2COO-)), thiophosphonocarboxylate ((S)P(CH2)nCOOR), e.g., thiophosphonoacetate "thioPACE" ((S)P(CH2COO-)), alkylphosphonate (P(C1-3 alkyl), e.g., methylphosphonate-P(CH3), boranophosphonate (P(BH3)), or phosphorodithioate (P(S)2).

15. The one or more chemical modifications may be 2-thiouracil ("2-thioU"), 2-thiocytosine ("2-thioC"), 4-thiouracil ("4-thioU"), 6-thioguanine ("6-thioG"), 2-aminoadenine ("2-aminoA"), 2-aminopurine, pseudouracil, hypoxanthine, 7-deazaguanine, 7-deaza-8-azaguanine, 7-deazaadenine, 7-deaza-8-azaadenine, 5-methylcytosine ("5-methylC"), 5-methyluracil ("5-methylU"), 5-hydroxymethylcytosine, 5-hydroxymethyluracil, 5,6-dehydrouracil, 5-propynylcytosine ...

15. The ASO of any one of claims 11-14, comprising nucleobase modifications comprising one or more of ropinyluracil, 5-ethynylcytosine, 5-ethynyluracil, 5-allyluracil ("5-allyl U"), 5-allylcytosine ("5-allyl C"), 5-aminoallyluracil ("5-aminoallyl U"), 5-aminoallyl-cytosine ("5-aminoallyl C"), abasic nucleotides, Z bases, P bases, unstructured nucleic acids ("UNA"), isoguanine ("isoG"), isocytosine ("isoC"), glycerol nucleic acid (GNA), glycerol nucleic acid (GNA), or thiophosphoramidate morpholino (TMO).

16. 16. The ASO of any one of claims 11 to 15, wherein the one or more chemical modifications comprise 2'-O-methoxyethyl, 5-methyl on cytidine, locked nucleic acid (LNA), phosphodiester (PO) internucleotide linkage, or phosphorothioate (PS) internucleotide linkage.

17. The ASO of any one of claims 11 to 16, which does not contain 10 or more consecutive nucleotides of unmodified DNA.

18. 18. The ASO of claim 17, which does not contain deoxyribonucleotides.

19. The ASO of any one of claims 11 to 18, which does not contain unmodified ribonucleotides.

20. The ASO of any one of claims 11 to 19, wherein the length of the ASO is 5xn+5 nucleotides (n is an integer of 3 or more), the nucleotide at the 5xm position is a ribonucleotide modified by LNA (m is an integer of 1 to n), and the nucleotides at the remaining positions are ribonucleotides modified by 2'-O-methoxyethyl.

21. The ASO of any one of claims 11 to 19, wherein the length of the ASO is 3xn+2 nucleotides (n is an integer of 6 or more), the nucleotide at the 3xm position is a ribonucleotide modified with LNA (m is an integer of 1 to n), and the nucleotides at the remaining positions are ribonucleotides modified with 2'-O-methoxyethyl.

22. 20. The ASO of any one of claims 11 to 19, wherein each ribonucleotide of the ASO is modified with 2'-O-methoxyethyl.

23. The ASO of any one of claims 11 to 19, wherein each nucleotide of the ASO is a ribonucleotide modified with 2'-O-methoxyethyl.

24. 24. The ASO of any one of claims 11 to 23, comprising 10 or more contiguous nucleotides of unmodified DNA flanked by at least three nucleotides of modified ribonucleotides at each of the 5' and 3' ends.

25. The ASO of any one of claims 11 to 24, wherein each cytidine in the ASO is modified by 5-methyl.

26. The ASO of any one of claims 1 to 25, wherein the regRNA is paRNA.

27. A pharmaceutical composition comprising the ASO of any one of claims 1 to 26 and a pharmaceutically acceptable carrier or excipient carrier.

28. A method for increasing the transcription of SYNGAP1 in a human cell, the method comprising contacting the cell with an ASO of any one of claims 1 to 26 or a pharmaceutical composition of claim 27.

29. 29. The method of claim 28, wherein the cell is a neuron.

30. 30. The method of any one of claims 28 or 29, wherein the ASO increases the amount of the regulatory RNA in the cell.

31. The method of any one of claims 28 to 30, wherein the ASO increases the stability of the regulatory RNA in the cell.

32. The method of any one of claims 28 to 31, which results in an increase in SYNGAP1 mRNA in the cell.

33. The method of any one of claims 28 to 32, which results in an increase in SYNGAP1 protein in the cell.

34. A method for treating a disease or disorder, comprising administering to a subject in need thereof an effective amount of the ASO of any one of claims 1 to 26, or the pharmaceutical composition of claim 27.

35. 35. The method of claim 34, wherein the disease or disorder is a SYNGAP1-related disease or disorder.

36. 38. The method of claim 34 or 37, wherein the SYNGAP1-associated disorder is SYNGAP1-associated intellectual disability (ID), mental retardation, autosomal dominant 5 (MRD5), or SYNGAP1-associated non-syndromic intellectual disability (NSID).

37. 35. The method of claim 34, wherein the disease or disorder is a disorder of the central nervous system (CNS) or a disorder of the peripheral nervous system (PNS).

38. 39. The method of claim 38, wherein the disease or disorder is an affective disorder (e.g., depression), schizophrenia, Alzheimer's disease, Parkinson's disease, Huntington's disease, autism spectrum disorder (ASD) (e.g., Asperger's syndrome, autism, pervasive developmental disorder not otherwise specified (PDD-NOS)), or CNS or PNS trauma (e.g., brain or spinal cord ischemia or trauma, stroke, or neurological abnormalities associated with surgery or anesthesia).

39. The method of any one of claims 34 to 38, wherein administration of the ASO increases SYNGAP1 gene expression in the subject compared to the baseline level before administration.

40. 40. The method of any one of claims 34 to 39, wherein the ASO increases the amount of the regulatory RNA in cells of the subject.

41. The method of any one of claims 34 to 40, wherein the ASO increases the stability of the regulatory RNA in the cells of the subject.

42. The method of any one of claims 34 to 41, wherein administration of the ASO increases SYNGAP1 gene expression in cells of the subject compared to the baseline level before administration.

43. The method of claims 40 to 42, wherein the cell is a neuron.

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