Therapeutics for SYNGAP haploinsufficiency

Abstract

The present invention relates to therapeutic compositions for disorders associated with haploinsufficiency. The present invention provides antisense oligonucleotides useful for treating neurodevelopmental disorders associated with mutations in the SYNGAP gene. The present invention provides compositions comprising synthetic antisense oligonucleotides (ASOs) that promote the expression of SynGAP protein by one or any combination of the following steps: (i) preventing one or more miRNAs from interfering with the production of SynGAP protein; (ii) binding to the 5'-UTR of SYNGAP1 transcript and enhancing the translation of Syngap protein; and (iii) promoting the RNAseH cleavage of antisense long non-coding RNA that is anti-correlated with SYNGAP expression. When the compositions are delivered to patients with SYNGAP haploinsufficiency, the ASOs promote the expression of SynGAP protein.

Description

[Technical field]

[0001] Technical Field The present invention relates to therapeutic compositions for disorders associated with haploinsufficiency.

[0002] Sequence Listing This application includes an electronic sequence listing, which has been submitted herewith in ST.26 format and is incorporated by reference. The sequence listing (created on September 22, 2022) is entitled QSTA-045-01WO-Sequence-Listing and is 26 kilobytes in size. [Background technology]

[0003] background Mutations in the SYNGAP1 gene are associated with severe neurodevelopmental disorders, where 50% of patients exhibit treatment-refractory seizures and cognitive impairment, and 50% also meet diagnostic criteria for autism spectrum disorder. SYNGAP has also been associated with schizophrenia and intellectual disability. See Gamache, 2020, Twenty years of SynGAP research: from synapses to cognition, J Neurosci 40(8): 1596-1605 and Jeyabalan, 2016, SYNGAP1 : Mind the Gap. Front Cell Neurosci 10:article 32, both incorporated by reference.

[0004] In particular, SYNGAP1-associated intellectual disability (SYNGAP1-ID) is characterized in 100% of affected individuals by developmental delay (DD) or intellectual disability (ID), generalized epilepsy (approximately 84% affected individuals), autism spectrum disorder (ASD) and other behavioral abnormalities. The disorder typically results from an autosomal dominant disease mechanism caused by de novo mutations. Penetrance is 100% and mutations include deletions, nonsense, frameshift and missense. The majority of mutations are nonsense, truncating or frameshift, including some early truncating mutations. This and the presence of deletions have led to the designation haploinsufficient disease mechanism.

[0005] To date, over 50 individuals with SYNGAP1-ID have been reported. In the majority, DD / ID was moderate to severe; in some, it was mild. Epilepsy is generalized; a subset of individuals with epilepsy has epilepsy with myoclonic atonic seizures (Dawse syndrome) or myoclonic absence epilepsy. Behavioral abnormalities may include stereotypic behaviors (e.g., hand flapping, attachment to specific objects), and poor social development. Feeding difficulties may be prominent in some. The defining features in children are progressive intellectual decline, behavioral changes, myoclonus, and epilepsy. 53% of patients are diagnosed by the age of 4 years. The incidence of ASD may be as high as 50%. This includes stereotypic behaviors (e.g., hand flapping, attachment to specific objects), and poor social development. Additionally, inattention, impulsivity, self- and other-directed aggressive behavior, elevated pain threshold, hyperacusis, and sleep disturbances have been observed. To date, there are no disease-specific or disease-modifying treatments for SYNGAP1 neurodevelopmental disorders. [Prior art documents] [Non-patent literature]

[0006] [Non-Patent Document 1] Gamache, 2020, Twenty years of SynGAP research: from synapses to cognition, J Neurosci 40(8): 1596-1605 [Non-Patent Document 2] Jeyabalan, 2016, SYNGAP1 : Mind the Gap. Front Cell Neurosci 10:article 32 Summary of the Invention [Means for solving the problem]

[0007] Abstract The present invention provides antisense oligonucleotides useful for treating certain neurodevelopmental disorders by promoting the expression of SynGAP protein. Some individuals are born with heterozygous loss-of-function mutations on the SYNGAP gene (also known as SYNGAP1), which encodes SynGAP (a condition known as Syngap haploinsufficiency). In Syngap haploinsufficiency, there is only one fully functional copy of the SYNGAP gene. During neurodevelopment, all SynGAP proteins must be produced by transcribing one copy of the gene into pre-mRNA, splicing the pre-mRNA into mRNA, and translating the mRNA into protein. The present invention provides compositions and methods that use various strategies to upregulate the expression of SynGAP protein from the SYNGAP gene. For example, the compositions and methods of the present invention use the insight that micro-RNAs (miRNAs) may have a role in gene regulation and may interfere with the production of SynGAP protein.

[0008] Specifically, certain miRNAs can bind to SYNGAP RNA and prevent protein production. An embodiment of the present invention provides a composition comprising a synthetic antisense oligonucleotide (ASO) that prevents miRNA from interfering with the production of SynGAP protein.

[0009] Second, the present invention also takes advantage of the insight that features of SYNGAP mRNA can impede translation efficiency. Specifically, the 5' untranslated region (5'UTR) of SYNGAP mRNA can have a negative regulatory effect. Some embodiments of the present invention provide compositions comprising ASOs that alleviate the negative regulatory effect associated with the 5'UTR, thereby improving the translation of SynGAP protein.

[0010] Third, the present invention reflects the insight that antisense long non-coding RNA (IncRNA) to SYNGAP transcripts (e.g., SYNGAP1-AS) can interfere with expression. The present invention provides antisense oligonucleotide (ASO) gapmers that hybridize to SYNGAP1-AS and promote RNaseH-mediated removal of SYNGAP1-AS. When delivered to patients with Syngap haploinsufficiency, the ASO of the present invention prevents miRNAs from downregulating the synthesis of SynGAP protein and / or reduces the negative effects associated with the 5'UTR and / or depletes SYNGAP1-AS IncRNAs that interfere with expression.

[0011] Whereas untreated Syngap haploinsufficiency may result in a lack of protein expressed during neurodevelopment, treatment with the compositions of the present invention increases the production of functional SynGAP protein from non-mutated alleles. Increased SynGAP protein results in a healthy phenotype despite haploinsufficient genotype. Thus, the compositions of the present invention are useful for treating or preventing the occurrence of Syngap haploinsufficiency or its symptoms and associated conditions. Treatment can be delivered, for example, upon detection of any symptoms of haploinsufficiency or detection by genetic screening.

[0012] Without being bound by any mechanism of action, miRNA can bind to sequences within pre-mRNA or mRNA (e.g., 3'UTR of mRNA). This sequence-specific binding can induce translational repression, RNA cleavage, mRNA deadenylation, or mRNA decapping. In the composition of the present invention, ASO comprises a nucleotide sequence that can allow ASO to bind to either SYNGAP mRNA or miRNA, and prevent or inhibit miRNA-mediated translational repression. Preferably, the ASO can bind to SYNGAP mRNA and sterically block the miRNA from binding to its normal target site.

[0013] In certain aspects, the present invention provides compositions that promote expression of a SynGAP protein from a transcript of a SYNGAP gene and include at least one nucleic acid having at least 80% sequence similarity to one of SEQ ID NOs: 1-29. The nucleic acid may have a length between about 10 and about 30 bases. Preferably, the nucleic acid has a region of at least about 10 contiguous bases that has a 100% match to a segment in one of SEQ ID NOs: 1-29. For steric block oligonucleotide (SBO) embodiments, a majority of the bases of the nucleic acid may have 2'-O-methoxyethyl modified ribose. In some embodiments, at least a majority of the interbase linkages in the nucleic acid are phosphorothioates. In certain embodiments, all of the bases in the nucleic acid include 2'-O-methoxyethyl ribose sugars. In certain specific embodiments, the nucleic acid has at least 88% sequence similarity to one of SEQ ID NOs: 1-29 and all of the bases in the nucleic acid include 2'-O-methoxyethyl ribose sugars. For example, the nucleic acid may have at least 94% sequence similarity to one of SEQ ID NOs: 1-29, where all of the bases in the nucleic acid include a 2'-O-methoxyethyl ribose sugar, and optionally, at least about 90% of the interbase linkages in the nucleic acid are phosphorothioate linkages. The nucleic acid may have 100% sequence similarity to one of SEQ ID NOs: 1-29; all of the bases in the nucleic acid may include a 2'-O-methoxyethyl ribose sugar; all instances of U and C may be methylated at the 5-position of the nitrogenous base; and all interbase linkages in the nucleic acid may be phosphorothioate linkages.

[0014] In a miRNA blocking aspect, the nucleic acid has a sequence at least 90% similar to one of the sequences numbered 1-10 and blocks the miRNA from binding to the transcript. Preferably, the nucleic acid binds within the 3' untranslated region (UTR) of the SYNGAP1 transcript and prevents binding of the miRNA, where the miRNA is a member of the hsa-miR-29-3p family. For example, the miRNA can be hsa-miR-29c-3p, hsa-miR-29b-3p, or hsa-miR-29a-3p, and the nucleic acid can hybridize to a position within bases 4520-4526 of the transcript, where the transcript is represented by NCBI reference sequence NM_006772.3. Alternatively, in a related example, the miRNA is hsa-miR-1343-3p or hsa-miR-6783-3p and the nucleic acid hybridizes to a position within bases 4479 to 4485 of the transcript represented by NCBI reference sequence NM_006772.3. Preferably, the nucleic acid is a steric block oligonucleotide (SBO).

[0015] In the secondary structure / cryptic ORF aspect, the nucleic acid has a sequence at least 90% similar to that of SEQ ID NOs: 11-20 and promotes expression by destabilizing a secondary structure or masking a cryptic open reading frame (ORF) in the 5'UTR of the transcript. The composition may include a plurality of the nucleic acids, each having a sequence at least 90% similar to one of SEQ ID NOs: 11-20. For example, the composition may include five or more of the nucleic acids. Preferably, the five nucleic acids hybridize to positions distributed across the 5'UTR of the transcript. The composition may, for example, include all ten nucleic acids described by SEQ ID NOs: 11-20, where the ten nucleic acids tile across substantially all of the 200 nt of the 5'UTR.

[0016] In an antisense IncRNA aspect, the nucleic acid has a sequence at least 90% similar to one of SEQ ID NOs: 21-29 and mediates digestion of long non-coding RNA antisense to a transcript. For example, the composition can include one or more nucleic acids, each having a base sequence at least 94% identical to one of SEQ ID NOs: 21-29, where each of the nucleic acids has a gapmer structure that includes a central DNA segment flanked by modified RNA wings. Preferably, the nucleic acid includes two wings flanking a central region of at least 8 or 9 DNA bases. Either end of the nucleic acid can include modified RNA bases (e.g., 2'-O-methoxyethyl RNA). In certain embodiments, the nucleic acid has a base sequence with at least a 94% match to one of SEQ ID NOs: 21-29, where at least the outermost interbase linkages and all linkages involving DNA bases are phosphorothioate, and where the oligonucleotide further comprises the central 12 DNA bases flanked by a 5' RNA wing and a 3' RNA wing, the 5' wing and the 3' wing each comprising four consecutive 2' modified RNA bases ("4-12-4" gapmer structure; "5-10-5" or similar gapmer structures are equally within the scope of this disclosure). Preferably, the nucleic acid has a sequence of one of SEQ ID NOs: 21-29, where both RNA wings consist of four 2-methoxyethyl-modified RNA bases. The composition may comprise a plurality of the nucleic acids, for example tiled along a long non-coding RNA antisense to a transcript. [Brief description of the drawings]

[0017] [Figure 1] FIG. 1 shows SYNGAP1 transcript expression across ASO-treated samples. [Diagram 2] FIG. 2 shows SYNGAP1 transcript expression across ASO-treated samples. [Diagram 3] Figure 3 shows knockdown of SYNGAP1-AS1 by the ASO of the present disclosure. [Figure 4] Figure 4 shows SYNGAP1 expression in all ASOs in human NGN2 neurons. [Diagram 5] Figure 5 is a Western blot of ASO candidates, ASO-009 to ASO-012. [Figure 6] Figure 6 shows SynGAP protein boost for several of the ASOs normalized to vinculin. [Figure 7] FIG. 7 shows SynGAP protein boost for ASOs normalized to GAPDH. [Figure 8] FIG. 8 graphically depicts SynGAP protein boost quantified by Western blot for ASOs of the disclosure. [Figure 9] FIG. 9 is a schematic graph of protein boost. [Figure 10] FIG. 10 shows the SynGAP protein boost quantified by Western blot. [Figure 11] FIG. 11 shows a western blot showing SynGap expression from neurons in CRISPR edited human iPSC-derived cell lines. [Figure 12] FIG. 12 provides data for SYNGAP1 mutant cell lines (each line represents an individual SYNGAP1 mutant clone) versus the average of all WT clones. [Figure 13] Figure 13 shows rescue of the SYNGAP1 heterozygous phenotype using an ASO of the invention. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0018] Detailed Description The present invention provides antisense oligonucleotides (ASOs) useful for treating SYNGAP1-associated intellectual disability (SYNGAP1-ID). SYNGAP1-ID is characterized by developmental delay (DD) or intellectual disability (ID) (100% of affected individuals), generalized epilepsy (about 84%), and autism spectrum disorder (ASD) and other behavioral abnormalities. The disorders typically result from an autosomal dominant disease mechanism caused by de novo mutations. Penetrance is understood to be 100%, and mutations can include deletions, nonsense, frameshifts, and missenses. The majority of mutations are nonsense, truncating, or frameshift, including some early truncating mutations. This and the presence of deletions have led to the designation haploinsufficient disease mechanism.

[0019] To date, many individuals with SYNGAP1-ID have been reported. In most cases, DD / ID was moderate to severe; in some, it was mild. Epilepsy is generalized; a subset of individuals with epilepsy has epilepsy with myoclonic atonic seizures (Dawse syndrome) or myoclonic absence epilepsy. The compositions of the present invention are useful for treating these conditions by boosting or restoring SYNGAP expression.

[0020] Behavioral abnormalities may include stereotyped behaviors (e.g., hand flapping, attachment to certain objects) as well as poor social development. Feeding difficulties may be prominent in some. The incidence of ASD may be as high as 50%. In addition, inattention, impulsivity, self-directed and other-directed aggressive behavior, elevated pain threshold, hyperacusis, and sleep disorders have been observed.

[0021] The present invention provides disease-specific or disease-modifying treatments for SYNGAP1 neurodevelopmental disorders, thus addressing an unmet need.

[0022] The SYNGAP gene encodes the SynGAP protein, an enzyme abundant at synapses that plays a key role in synaptic physiology and synaptic plasticity, and expression of this protein is highest in the central nervous system (CNS). SynGAP is a Ras and RapGAP; that is, it inhibits the function of ras and rap by promoting the hydrolysis of bound GTP. In healthy neurons, SynGAP is primarily located in the postsynaptic density (PSD) under basal activity conditions. Under increased neuronal activity, SynGap is phosphorylated by CaMKII, decreasing its affinity for the PSD and allowing SynGAP to disperse from the postsynaptic density, disinhibiting ras signaling and enabling AMP AR insertion, which is associated with dendritic spine morphology changes and long-term potentiation (LTP). This process is NMDAR activation dependent.

[0023] Animal models of SYNGAP1 mutations have disease-associated behavioral phenotypes, including memory dysfunction. Loss of ras / rap inhibition of SynGAP is thought to contribute to defects in synapse development, spine formation, and disruptions of synaptic signaling and plasticity.

[0024] SynGAP regulates synaptic strength and AMPAR activity in cultured neurons, a mitogen-activated protein kinase (MAPK) that is implicated in a role in NMDAR / MAPK signaling. It is understood that SynGAP plays a pivotal role in regulating neuronal MAPK signaling, AMPAR membrane trafficking, and excitatory synaptic transmission. SYNGAP activity reduces ERK activation; depresses AMPAR-mediated miniature excitatory postsynaptic currents, reduces synaptic AMPAR surface expression, and reduces AMPAR insertion into the plasma membrane. See Rumbaugh, 2006, SynGAP regulates synaptic strength and mitogen-activated protein kinases in cultured neurons, PNAS 103(12):4344-51 (incorporated by reference).

[0025] Rapid dispersion of SynGAP from synaptic spines induces AMPA receptor insertion and spine expansion during long-term potentiation (LTP) along with its involvement in PSD compartmentalization / regulation. Application of N-methyl-D-aspartate (NMDA) promotes the translocation of SynGAP-α1 and SynGAP-α2 from the PSD core. SynGAP is rapidly dispersed from spines upon LTP induction in hippocampal neurons and depends on phosphorylation of SynGAP by CaMKII (calmodulin-dependent protein kinase II). The extent of acute dispersion estimates the maintenance of spine expansion. Thus, synaptic dispersion of SynGAP by CaMKII phosphorylation during LTP represents a key signaling component that conveys CaMKII activity to small G protein-mediated spine expansion, AMPA receptor synaptic incorporation, and synaptic strengthening. See Araki, 2015, Rapid Dispersion of SynGAP from Synaptic Spines Triggers AMPA Receptor Insertion and Spine Enlargement during LTP, Neuron 85(1): 173-189 (incorporated by reference).

[0026] The present invention provides compositions that promote SynGAP expression. As discussed herein, the effects of the compositions of the present disclosure can be assayed or demonstrated by a variety of techniques, including rt-PCR, qPCR, RNA-Seq, optogenetics, patch clamp, etc.

[0027] For example, the compositions of the present invention and their effects can be evaluated in an Optopatch assay. In general, Optopatch involves the use of an in vitro neuron that includes an optogenetic construct that provides neuronal activation under optical stimuli (e.g., a modified algal channelrhodopsin that causes neurons to fire in response to light) and an optical reporter of neuronal activity (a modified archaerhodopsin that emits light proportional to neuronal membrane voltage, resulting in a signal of neuronal activity). The in vitro neuron can be assayed under a fluorescent microscope and can also be evaluated (e.g., subsequently) by, for example, staining (e.g., immunocytochemistry), RNA-Seq, or other such assays. Any suitable optogenetic construct, optogenetic microscope, or other assay can be used. For example, suitable optogenetic constructs include those described in U.S. Pat. No. 9,594,075 (incorporated by reference). Suitable optogenetic microscopes include those described in U.S. Pat. No. 10,288,863 (incorporated by reference). The compositions of the present invention and their effects can be evaluated using iPSC-derived neuronal cell lines with mutations in the SYNGAP gene (both heterozygous and homozygous loss-of-function mutations).Optopatch phenotyping is performed on such cell lines.

[0028] To provide the compositions and methods of the present invention, the sequence of the ASO can be selected by a process that involves balancing a variety of factors, such as: 1. Determine the target transcript region and generate sequences (all possible N-mers); 2. Elimination based on experimental constraints (isoforms, homology); 3. Elimination based on predicted off-target mRNA, pre-mRNA, miRNA, and IncRNA hits in humans and non-human primates (rhesus and cynomolgus); 4. Elimination / filtering based on sequence characteristics (tetra-G, tetra-C, CpG, palindrome, GC content, and poly-X stretch); 5. Filter based on thermodynamic parameters (Tm, hairpin ΔG, ASO duplex ΔG, ASO:target ΔG); and 7. Select ASOs within the region based on thermodynamic parameters, spacing, and experimental objectives (overall ΔG). To determine the target transcript region, criteria can be applied from the literature, sequencing-based assays, and models that identify miRNA binding sites to block. For miRNA steric blocking oligonucleotides (SBOs), for targets that pass the criteria, predicted miRNA binding sites of 7-8 nt are identified based on a model that includes the seed sequence and various contextual information. Conserved sites are more likely to be functional / relevant.

[0029] Identifying targets to be addressed by the compositions of the present invention can be performed using any suitable technique or combination of techniques. A preferred embodiment uses CLIP-Seq, TargetScan, and miRNA Atlas as tools to identify targets and design sequences of the present invention.

[0030] Cross-linking immunoprecipitation (CLIP) uses UV cross-linking and immunoprecipitation to analyze RNA interactions and modifications, and has been used with sequencing (dubbed CLIP-Seq) to generate genome-wide RNA interaction maps, and to identify microRNA targets by deciphering microRNA-mRNA and protein-RNA interaction maps in tissue and cell cultures and samples. See Thomson, 2011, Experimental strategies for microRNA target identification, Nucleic Acids Res 39(16):6845-53 (incorporated by reference).

[0031] TargetScan is a digital tool that predicts biological targets of miRNAs by searching for the presence of conserved 8-mer, 7-mer, and 6-mer sites that match the seed region of each miRNA. See Lewis, 2005, Conserved Seed Pairing, Often Flanked by Adenosines, Indicates that Thousands of Human Genes are MicroRNA Targets., Cell 120: 15-20, incorporated by reference. TargetScan can be used to identify conserved sites, poorly conserved sites, and sites with mismatches in the seed region that are compensated for by conserved 3' pairing, as well as central sites. See Friedman, 2009, Most mammalian mRNAs are conserved targets of MicroRNAs, Genome Res 19:92-105 and Shin, 2010, Expanding the microRNA targeting code: functional sites with centered pairing, Mol Cell 38(6):789-802, both incorporated by reference. TargetScan ranks predictions based on the predicted efficacy of targeting as calculated using the cumulative weighted context++ score of the sites. Predictions can also be ranked by their probability of conserved targeting. TargetScanHuman considers matches to human 3'UTRs and their orthologs as defined by UCSC whole genome alignment. Conserved targeting has also been detected within open reading frames (ORFs).

[0032] Atlases of miRNAs exist. For example, The Human miRNA Tissue Atlas is a catalog of tissue-specific microRNA (miRNA) expression across 62 tissues. See Ludwig, 2016, Distribution of miRNA expression across human tissues, Nucleic Acids Res 44(8):3865-77 (incorporated by reference). Another example is an integrated expression atlas of miRNAs and their promoters created by deep sequencing 492 short RNA (sRNA) libraries from 396 human and 47 mouse RNA samples with matching Cap Analysis Gene Expression (CAGE) data. See de Rie, 2017, An integrated expression atlas of miRNAs and their promoters in human and mouse, Nat Biotech 35:872-878 (incorporated by reference). For the 2017 atlas, promoters were identified for 1,357 human and 804 mouse miRNAs, showing strong sequence conservation across species. It was found that primary and mature miRNA expression levels are correlated, allowing primary miRNA measurements to be used as a proxy for mature miRNA levels in a total of 1,829 human and 1,029 mouse CAGE libraries. Such tools provide an atlas of miRNA expression and promoters in primary mammalian cells, establishing the basis for detailed analysis of miRNA expression patterns and transcriptional control regions. Such miRNA atlases can be used to identify targets of the present invention.

[0033] The present invention provides compositions that use one of several SYNGAP1 boosting strategies. First, the disclosure provides 10 different sequences (SEQ ID NOs: 11-20) for use in miRNA-blocking steric blocking oligonucleotides (SBOs). Bioinformatics analysis identified promising CLIP-seq peaks in the 3'UTR of SYNGAP1, along with binding sites for members of the hsa-miR-29-3p family expressed in human cortex. Sequences that dilate the CLIP-seq peaks were designed to block interactions between the miRNA and this site, including SBOs that completely occlude the most promising miRNA binding sites, with the goal of eliminating mRNA-mediated negative regulation of SYNGAP1 levels.

[0034] Table 1 shows the sequences of SEQ ID NOs: 1 to 29.

[0035] Second, this disclosure provides nine different sequences (SEQ ID NOs: 21-29) for gapmers targeting SYNGAP1-AS1. There is some evidence in the literature that SYNGAP1 levels in the brain are anti-correlated with short IncRNAs antisense to SYNGAP1. We designed 20-mer gapmers that tile this transcript.

[0036] Finally, third, the disclosure provides 10 different sequences (SEQ ID NOs: 1-10) for SBOs that tile the 5'UTR. It is understood that the SYNGAP transcript may contain novel regulatory features (e.g., secondary structure or upstream open reading frames). Others have tiled non-coding regions of target transcripts to stabilize mRNA levels and boost protein expression of other genes. The disclosure provides 10 sequences that tile most of the 200nt 5'UTR of the SYNGAP transcript to promote expression.

[0037] The compositions of the present disclosure embody results resulting from identifying miRNA binding sites to block. Efforts to identify miRNA binding sites may include multiple avenues of investigation including, for example, 1. AG02 CLIP-seq; 2. TargetScan; and 3. Study of miRNAs in the CNS. Here, AG02 CLIP-seq identified a 50-80 nt region with evidence for an interaction between a silencing complex and a target transcript in the adult or developing cortex. CLIP-Seq identified two peaks in the SYNGAP transcript, one in an intron and one in the 3'UTR. See Nowakowski, 2018, Regulation of cell-type-specific transcriptomes by microRNA networks during human brain development, Nat Neurosci 21(12): 1784-1792 (incorporated by reference). Additionally, TargetScan was used to predict 7-8 nt miRNA binding sites predicted based on a model that included the seed sequence and various contextual information. Conserved sites are more likely to be functional / relevant. TargetScan identified potential miRNA binding sites that are at least well conserved or confidently annotated. For example, TargetScan identified a well conserved site at bases 4520-4526 of the transcript, where the transcript is represented by NCBI reference sequence NM 006772.3, and it is theorized that hsa-miR-29c-3p, hsa-miR-29b-3p, or hsa-miR-29a-3p may bind thereto. TargetScan identified a well annotated position at bases 4479-4485 of the transcript, where the transcript is represented by NCBI reference sequence NM_006772.3, and it is theorized that hsa-miR-1343-3p and hsa-miR-6783-3p may bind thereto.See McCall, 2017, Toward the human cellular microRNAome, Genome Res 27(10): 1769-1781 (incorporated by reference). Finally, studies of miRNAs in the CNS provide evidence that miRNAs are expressed in adult cortex (Boudreau et al. 2014 Neuron), developing cortex (Nowakowski et al. 2018 Nature Neuroscience), and / or CNS tissues from the miRNA atlas (McCall et al. 2017 Genome Research).

[0038] This disclosure uses those insights and provides 10 sequences for SBOs that tile CLIP-seq peaks and miRNA binding sites. The design process generally involves: starting with all 16-21mers that target CLIP-seq peaks; removing sequences with ineligible off-target hits in any human reference (ineligibility criteria may include: edit distance < 2, no consecutive match length filter); requiring GC content: 45-75% GC; requiring certain thermodynamic parameters: duplex DG < -23 (standard), hairpin DG > -5 (usually -2.5), dimer DG > -10 (usually -8); for sequences that completely occlude miRNA binding sites: for the shortest ASOs and other sequences without CpG motifs or GGGG motifs: the shortest sequence with the best thermodynamic properties and the lowest problematic motif occurrence rate that tiles the interval. Their process provided SEQ ID NOs: 11-20. Each of these is useful for generating steric blocking oligonucleotides (SBOs) useful for preventing miRNAs from binding to the SYNGAP1 transcript.

[0039] In certain embodiments, the present disclosure provides sequences useful in generating antisense oligonucleotides that disrupt secondary structures or cryptic open reading frames in SYNGAP1 transcripts. Stable 5' secondary structures can reduce translation efficiency.

[0040] The 5'UTR of the SYNGAP1 transcript, NM_006772.3, is short (200 nt) and therefore amenable to tiling. Using a tiling approach, the inventors empirically discovered regulatory sites in the 5'UTR of the SYNGAP1 transcript, despite the absence of prior indications of hairpins and / or uORFs. Thus, the present disclosure provides 10 sequences useful in ASO (e.g., SBO) tiling across the 5'UTR of the SYNGAP1 transcript. The present invention also provides a method for empirically discovering regulatory sites in the 5' of the SYNGAP1 transcript for tiling and designing ASO sequences.

[0041] In one particular method, the sequences are designed by a process that includes: starting with all 16-21mers targeting the 200nt 5'UTR; eliminating off-target hits (e.g., other exact matches in the human spliced ​​or unspliced ​​transcriptome, no hits with an edit distance of 1 in the human spliced ​​transcriptome); omitting sequence debt (e.g., no polyX stretches >5, no palindromes >8); requiring certain standard thermodynamic parameters (e.g., duplex DG<-23, hairpin DG>-2.5, dimer DG>-8); and minimizing GGGG motifs and CpG dinucleotides. The above design process provided SEQ ID NOs: 1-10.

[0042] In other embodiments, long non-coding RNAs (IncRNAs) understood to be antisense to SYNGAP1 transcripts (SYNGAP1-AS) are degraded using antisense gapmers of the present disclosure (e.g., antisense oligonucleotides having a sequence at least about 75% similar to one of SEQ ID NOs: 21-29). Some antisense RNAs overlapping with suspected autism spectrum disorder (ASD) genes appear to show concordant expression relative to their sense protein-coding genes, while other sense-antisense pairs clearly show discordant relationships. At least one antisense RNA (SYNGAP1-AS) corresponding to the SYNGAP1 locus has been found to be differentially expressed in brain regions of patients with ASD compared to control individuals, and is now targeted for gapmer-mediated degradation. See Velmeshev, 2013, Expression of non-protein-coding antisense RNAs in genomic regions related to autism spectrum disorders, Molecular Autism 4:article 32 (incorporated by reference). The present disclosure provides nine sequences (SEQ ID NOs: 21-29) useful in gapmers targeting SYNGAP1-AS1 (496nt ENST00000630418.1). They are designed by a process that includes: starting with all 20-mers that tile the transcript; removing sequences with unqualified off-target hits (e.g., no edit distance <2, no match length >17 hits in the human transcriptome); removing sequences with known liabilities (e.g., no polyX stretches >5, no palindromes >8, no GGGG, %GC 45-75; no CpG unless at the 5' end of the transcript); applying favorable gapmer thermodynamic parameters (e.g., duplex DG<-23, hairpin DG>-2.5, dimer DG>-8, Tm>67).

[0043] To summarize a particular boosting strategy, the present disclosure uses at least three. First, the compositions of the present disclosure block miRNA binding. The compositions include SBOs based on one of SEQ ID NOs: 11-20 that tile across the identified CLIP-seq peaks. Second, the compositions of the present disclosure mitigate the negative regulatory effect of the 5'UTR of the SYNGAP transcript that may prevent / interfere with translation. The compositions include ASOs based on one of SEQ ID NOs: 1-10. Third and finally, the compositions of the present disclosure provide ASO gapmers that mediate digestion of antisense IncRNA. Specifically, the compositions include gapmers targeting SYNGAP1-AS1 (isoforms characterized in Velmeshev 2013 Molecular Autism) based on SEQ ID NOs: 21-29.

[0044] Table 1 shows the sequences of the ASOs of the present disclosure. [Table 1-1] [Table 1-2]

[0045] The sequences listed in Table 1 may be treated as baseline references, and the nucleic acids in the compositions of the invention (e.g., steric blocking oligonucleotides or SBOs, or gapmers) may be described relative to one of the listed sequences. For example, mismatches are tolerated, i.e., even if the SYNGAP1 transcript contains a reverse complement to one of SEQ ID NOs: 1-29, the nucleic acids of the invention may be found to function well even with less than a 100% match to one of SEQ ID NOs: 1-29. Results suggest that mismatches are most tolerated near the ends of the SBOs, and that it may be most important to block the binding region of the miRNA seed sequence (where the seed may be about 7-8 bases long, or even as short as 5 or 6 bases).

[0046] It is also noted that certain letters used in Table 1 are indicated in the sequence using DNA nomenclature (e.g., using the letters A, T, C, and G) and do not describe ribose sugar composition, nitrogenous base modification, or interbase linkage. It may be found that the nucleic acids of the present invention are most useful with RNA bases (e.g., uracil for the nitrogenous base when T is indicated) and also or alternatively with modified ribose sugars (e.g., 2'-MOE). Indeed, it may be found that the letter T in the recited sequence can exist in the nucleic acids of the present invention as the nucleobases thymine or uracil, and / or even that the bases can be mixed or intermingled along the SBO. In some embodiments, the nucleic acids of the present invention include 2'-MOE "methylated" U (5-methyluridine), which is essentially 2'-MOE-T.

[0047] In particular, 2'-MOE bases may use 5-methylcytosine and 5-methyluridine. Using 5-methylcytosine and avoiding unmethylated CpG may be preferred. Avoiding unmethylated CpG may be found to reduce or avoid inflammatory potential. Furthermore, 5-methylcytosine 2'-MOE bases may be found to be consistent with clinically validated chemistry and may be preferred for this reason, if necessary. 5-methyl-U(T) bases may be used with 2'-MOE chemistry due to ease of manufacture and commercial availability. For sequences using 2'-OMe chemistry, it may be advantageous not to use 5-methyl-C and / or 5-methyl-U(T) due to ease of manufacture and / or commercial availability.

[0048] In certain embodiments, most or all of the bases represented by the letter T may have the nucleobase uracil. In preferred embodiments, most or all of the bases represented by the letter T have 2'-MOE-T. Any, most, or all of the linkages may be phosphorothioate. A preferred embodiment uses all phosphorothioate linkages for SEQ ID NOs: 1-20. A preferred embodiment may include some PO linkages. This appears to be the case for wings in gapmers, and for 2'-MOE SBOs, where some PO linkages are also preferred in each case to reduce protein binding.

[0049] Indeed, one first embodiment provides a composition having a nucleic acid for use as an ASO to promote SYNGAP expression. The nucleic acid has bases with at least 88% match with one of SEQ ID NOs: 1-29 (i.e., no more than two mismatches). All nitrogenous bases are A, T, U, C, or G, and optionally have mC. Optionally, all sugars are 2'-MOE with respect to SEQ ID NOs: 1-29, and all linkages are PS with respect to SEQ ID NOs: 1-29. These first embodiments are attractive for their ease of manufacture. Such ASOs can be synthesized on a standard benchtop RNA synthesis system and / or ordered from a commercial vendor.

[0050] More generally, preferably at least about 6-12 contiguous bases in the nucleic acid have at least 90% sequence identity with the corresponding 12 contiguous bases in one of SEQ ID NOs: 1-29 (to stably bind to a target). Preferably, at least about 6-12 contiguous bases in the nucleic acid have at least 90% sequence identity with the corresponding 12 contiguous bases in one of SEQ ID NOs: 1-10 (e.g., to block the seed region of the miRNA involved).

[0051] Preferably, for steric blocking oligonucleotides, the majority of the bases of the nucleic acid have 2'-O-methoxyethyl modified ribose (in SEQ ID NOs: 1-29), the majority of the interbase linkages in the nucleic acid are phosphorothioate linkages (in SEQ ID NOs: 1-29), and the nitrogenous base is A, T, U, C, G, or mC. As alluded to above, SEQ ID NOs: 11-20 of Table 1 are specifically designed to block specific miRNAs from downregulating SYNGAP transcripts; SEQ ID NOs: 1-10 are designed to mitigate potential negative regulatory effects of the 5'UTR or enhance SynGAP protein expression.

[0052] Figure 1 shows SYNGAP transcript expression across ASO-treated (ASO 006-020) samples. All SYNGAP1-targeting ASOs were screened in vitro by treating two cell lines of interest (HEK-293T and SH-SY5Y) at two concentrations. For HEK-293T cells, the cells were plated with 150 nM and 300 nM ASO at 50K per well of a 96-well plate. ASOs were delivered by transfection using RNAi Max at 0.75 μL per well of a 96-well plate. For SH-SY5Y cells, the cells were plated with 150 nM and 300 nM ASO at 45K per well of a 96-well plate. ASOs were delivered by transfection using RNAi Max at 0.5 μL per well of a 96-well plate. The data shown in Figure 1 shows a summary table of qPCR readouts of SYNGAP1 transcript expression for the first 15 ASOs (out of 29 ASOs) screened in the primary screen. All samples were normalized to vehicle conditions. The cells only condition shows no change in SYNGAP1 expression. Using the publicly disclosed SYNGAP-targeting boost ASO (ASO-005) as a positive control shows approximately 2-4x boost of SYNGAP1 transcript. Using GFP-004 ASO and UBE3A ASO-015 as negative controls shows no boost of SYNGAP1. The top two graphs show data for SYNGAP1 ASOs 006-020 screened at 300 nM and 150 nM in SH-SY5Y cells. The bottom two graphs show data for SYNGAP1 ASOs 006-020 screened at 300 nM and 150 nM in HEK-293T cells. The SH-SY5Y cells were transfected with ASO 24 hours after plating, and the HEK cells were transfected with ASO at the time of plating. For both cell lines, cells were harvested for qPCR 48 hours after ASO transfection. GAPDH was used as a normalization gene for SYNGAP1.Each ASO had two biological replicates shown as two bars, with each bar representing two technical replicates and one biological replicate.

[0053] Figure 2 shows SYNGAP transcript expression across ASO treated samples (ASO 021-034). In Figure 2, all SYNGAP1 targeted ASOs were screened in vitro by treating two cell lines of interest (HEK-293T and SH-SY5Y) at two concentrations. For HEK-293T cells, the cells were plated with 150 nM and 300 nM ASO at 50K per well of a 96-well plate. ASOs were delivered by transfection using RNAi Max at 0.75 μL per well of a 96-well plate. For SH-SY5Y cells, the cells were plated with 150 nM and 300 nM ASO at 45K per well of a 96-well plate. ASOs were delivered by transfection using RNAi Max at 0.5 μL per well of a 96-well plate. The data shown in Figure 2 shows a summary table of qPCR readouts of SYNGAP1 transcript expression for the second set of 14 ASOs (out of 29 ASOs) screened in the primary screen. All samples were normalized to vehicle condition. The cells only condition shows no change in SYNGAP1 expression. The publicly disclosed SYNGAP-targeted boost ASO (ASO-005) was used as a positive control and shows approximately 2-4x boost of SYNGAP1 transcript. The GFP-004 ASO and UBE3A ASO-008 and ASO-015 were used as negative controls and for the most part show no significant boost of SYNGAP1. The top two graphs show data for SYNGAP1 ASOs 021-034 screened in SH-SY5Y at 300nM and 150nM. The bottom two graphs show data for SYNGAP1 ASOs 021-034 screened at 300 nM and 150 nM in HEK-293T cells. The SH-SY5Y cells were transfected with ASOs 24 hours after plating, and the HEK cells were transfected with ASOs at the time of plating. For both cell lines, cells were harvested for qPCR 48 hours after ASO transfection.GAPDH was used as a normalization gene for SYNGAP1. Each ASO condition had two biological replicates shown as two bars, with each bar representing two technical replicates and one biological replicate.

[0054] Figure 3 shows SYNGAP1 antisense transcript expression across relevant ASO-treated samples. For Figure 3, all SYNGAP1-AS targeting ASOs were screened in vitro by treating two cell lines of interest (HEK-293T and SH-SY5Y) at two concentrations. Data shown is from SH-SY5Y cells. For SH-SY5Y cells, the cells were plated with 150 nM and 300 nM ASOs at 45K per well of a 96-well plate. ASOs were delivered by transfection using RNAi Max at 0.5 μL per well of a 96-well plate. Data shown shows a summary plot of qPCR readout of SYNGAP1-AS1 knockdown for SYNGAP1-AS1 specific ASOs screened in the primary screen (along with several neutral ASOs that do not target SYNGAP-AS1). All samples were normalized to vehicle conditions. The cells only condition (white) shows no change in SYNGAP-AS1 expression. All neutral ASOs (SYNGAP ASO 021-024, GFP-004 ASO, UBE3A ASO-008, ASO-015 and the publicly disclosed SYNGAP-targeted boost ASO (ASO-005)) were used as negative controls and show no significant knockdown of SYNGAP1-AS1. The top graph shows data for SYNGAP1-AS1 ASO 025-034 screened at 150 nM in SH-SY5Y cells. The bottom graph shows data for SYNGAP1 ASO 025-034 screened at 300 nM in SH-SY5Y cells. The SH-SY5Y cells were transfected with ASOs 24 hours after plating and the cells were harvested for qPCR 48 hours after ASO transfection. GAPDH was used as a normalization gene for SYNGAP1-AS1. Each ASO condition had two biological replicates shown as two bars, with each bar representing two technical replicates and one biological replicate.

[0055] Figure 4 shows the results from screening all SYNGAP1 ASOs in human NGN2 stem cell-derived neurons. SYNGAP1 is a postsynaptic GTPase activating protein that negatively regulates small GTPases, is crucial for the development of cognition and proper synaptic function, and is highly expressed in human neuronal cells. With reference to Figure 4, to show that the ASOs of the present invention are effective in relevant human cell types, human induced pluripotent stem cell-derived neurons (differentiated via NGN2 overexpression and dual SMAD inhibition) were treated with SYNGAP1 ASOs as described herein. Neurons were plated at 35k per well of a 96-well plate and treated with 200nM SYNGAP1 ASOs. ASOs were transfected into neurons with Endoporter reagent at DIV 20. Cells were harvested for qPCR and Western blots 10 days after treatment at DIV30. The data shown in Figure 4 shows a graph of qPCR readings of SYNGAP1 boost for 29 SYNGAP1 ASO candidates screened in the primary screen. All samples were normalized to vehicle condition. The cell-only condition shows no change in SYNGAP1 expression. SYNGAP1 expression levels were normalized to actin (used as a housekeeping gene). All normalized expressions were then quantified relative to the initial vehicle condition. Each bar represents two technical replicates and one biological replicate.

[0056] Figures 5-7 show exemplary data of SynGAP protein boost using SYNGAP1 ASO. Human induced pluripotent stem cell derived neurons (differentiated via NGN2 overexpression and dual SMAD inhibition) were treated with SYNGAP1 ASO and screened for SynGAP protein boost using Western blot. Neurons were plated at 35k per well of a 96-well plate and treated with 200nM SYNGAP1 ASO. ASO was transfected into neurons with Endoporter reagent (0.6μL / well) at DIV 20. Cells were harvested for Western blot 10 days after treatment at DIV30.

[0057] Figure 5 is a Western blot of four example SYNGAP1 ASO candidates (ASO-009 to ASO-012). SynGAP expression was normalized to vinculin or GAPDH protein levels and then normalized to vehicle average.

[0058] FIG. 6 shows quantification of SynGAP protein boost for seven of our SYNGAP1 candidates normalized to vinculin.

[0059] Figure 7 shows quantification of SynGAP protein boost for seven of our SYNGAP1 candidates normalized to GAPDH. For SynGAP blots, exposure was 300 seconds. For GAPDH, exposure was 20 seconds and for Vinculin, exposure was 20 seconds. 20 μg of protein was loaded per lane and high molecular weight migration was used. SynGAP antibody: anti-SynGAP - (Cell Signaling) - Cat# 5539 (1:1000); GAPDH antibody: Ms β-Actin - (EMD Millipore) - MAB374 (1:1000); Vinculin antibody: Ms Vinculin - (Invitrogen) - VLN01 - MA5-11690 (1:1000).

[0060] Figure 8 is a summary graph of SynGAP protein boost using all SYNGAP1 ASOs (normalized to total protein) quantified by Western blot for all SYNGAP1 ASO candidates (ASO-00 to ASO-034). Human induced pluripotent stem cell derived neurons (differentiated via NGN2 overexpression and dual SMAD inhibition) were treated with our SYNGAP1 ASOs and screened for SynGAP protein boost using a Western blot assay. Neurons were plated at 35k per well of a 96-well plate and treated with 200nM SYNGAP1 ASO. ASOs were transfected into neurons with Endoporter reagent (0.6μL / well) at DIV 20. Cells were harvested for Western blot 10 days after treatment at DIV30. In all experiments, GFP-targeted ASO (GFP-004) and UBE3A-targeted ASO (ASO-015) were used as negative controls. SynGAP expression was normalized to total protein quantification of all lanes and then normalized to vehicle conditions. For SynGAP blots, exposure was 300 seconds. For total protein quantification, blots were activated, imaged in UV exposure, and quantified using Bio-Rad software. 20 μg of protein was loaded per lane and high molecular weight migration was used. SynGAP antibody: Anti-SynGAP - (Cell Signaling) - Cat# 5539 (1:1000).

[0061] Figure 9 includes a summary graph of SynGAP protein boost quantified by Western blot for all SYNGAP1 ASO candidates (ASO-006-034). Human induced pluripotent stem cell derived neurons (differentiated via NGN2 overexpression and dual SMAD inhibition) were treated with the SYNGAP1 ASO of the present invention and screened for SynGAP protein boost using Western blot. Neurons were plated at 35k per well of a 96-well plate and treated with 200nM SYNGAP1 ASO. ASOs were transfected into neurons with Endoporter reagent (0.6μL / well) at DIV 20. Cells were harvested for Western blot 10 days after treatment at DIV30. In all experiments, GFP-targeted ASO (GFP-004) and UBE3A-targeted ASO (ASO-015) were used as negative controls. SynGAP expression was normalized to Vinculin protein and then to vehicle average. For SynGAP blots, exposure was 300 seconds. For Vinculin, exposure was 20 seconds. 20 μg of protein was loaded per lane and high molecular weight migration was used. SynGAP antibodies: Anti-SynGAP - (Cell Signaling) - Cat# 5539 (1:1000); Vinculin antibody Ms Vinculin - (Invitrogen) - VLN01 - MA5-11690 (1:1000).

[0062] Figure 10 shows a summary graph of SynGAP protein boost using all SYNGAP1 ASOs. Figure 10 includes a summary graph of SynGAP protein boost quantified by Western blot for all SYNGAP1 ASO candidates (ASO-006 to ASO-034) when normalized to GAPDH. Human induced pluripotent stem cell derived neurons (differentiated via NGN2 overexpression and dual SMAD inhibition) were treated with our SYNGAP1 ASOs and screened for SYNGAP1 protein boost using a Western blot assay. Neurons were plated at 35k per well of a 96-well plate and treated with 200nM SYNGAP1 ASOs. ASOs were transfected into neurons with Endoporter reagent (0.6μL / well) at DIV 20. Cells were harvested for Western blot 10 days after treatment at DIV30. In all experiments, GFP-targeting ASO (GFP-004) and UBE3A-targeting ASO (ASO-015) were used as negative controls. For SynGAP blots, exposure was 300 sec. For GAPDH, exposure was 20 sec. 20 μg of protein was loaded per lane and high molecular weight migration was used. SynGAP antibodies: anti-SynGAP - (Cell Signaling) - Cat# 5539(1:1000); Ms Vinculin - (Invitrogen) - VLN01 - MA5-11690(1:1000); GAPDH antibodies: Ms β-Actin - (EMD Millipore) - MAB374(1:1000).

[0063] FIG. 11 shows an image of a western blot gel providing data on SYNGAP protein expression from human isogenic CRISPR-edited induced pluripotent stem cell (iPSC)-derived neuronal cell lines that are either wild type (WT) or have heterozygous (Het) or homozygous (knockout; KO) mutations for the SYNGAP1 gene (top left).

[0064] Using these CRISPR-edited human iPSC-derived neuronal lines, we performed all optical electrophysiology and evoked synaptic responses in WT and SYNGAP1 mutant cell lines.

[0065] Figure 12 provides the resulting data, shown for SYNGAP1 mutant cell lines (each line represents an individual SYNGAP1 mutant clone) versus the average of all WT clones. Human iPSC-derived NGN2 neurons were measured at DIV45.

[0066] FIG. 13 shows rescue of SYNGAP1 heterozygous phenotype using two hit ASOs: ASO 5, a published ASO, and ASO 12, a Q-State designed ASO. CRISPR edited SYNGAP1 heterozygous mutants were treated with either ASO 5, ASO 12, or a control ASO that does not modulate SYNGAP expression. Treatment with SYNGAP protein boosting ASOs 5 and 12 rescues the heterozygous functional synaptic phenotype. Cells were treated with 200 nM ASO on day 20 and measured on day 30.

[0067] Oligonucleotides can be made in the laboratory by solid phase chemical synthesis, followed by purification and isolation.When referring to the sequence of an oligonucleotide, reference is made to the sequence or order of the nucleobase moiety of covalently linked nucleotides or nucleosides, or their modification.The oligonucleotides of the present invention can be artificial, i.e., chemically synthesized, and typically purified or isolated.The oligonucleotides of the present invention can include one or more modified nucleosides or nucleotides (e.g., 2' sugar modified nucleosides).

[0068] The modified nucleotides may be independently selected from the group consisting of deoxy-nucleotides, 3'-terminal deoxy-thymine (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, nucleotides containing unnatural bases, 1,5-anhydrohexitol modified nucleotides, cyclohexenyl modified nucleotides, nucleotides containing phosphorothioate groups, nucleotides containing methylphosphonate groups, nucleotides containing 5'-phosphates, nucleotides containing 5'-phosphate mimetics, glycol modified nucleotides, and 2'-O-(N-methylacetamide) modified nucleotides, and combinations thereof.

[0069] The nitrogenous bases of the ASO can be naturally occurring nucleobases (e.g., adenine, guanine, cytosine, thymidine, uracil, xanthine and hypoxanthine), as well as non-naturally occurring variants (e.g., substituted purines or substituted pyrimidines, such as nucleobases selected from isocytosine, pseudoisocytosine, 5-methylcytosine, 5-thiazolo-cytosine, 5-propynyl-cytosine, 5-propynyl-uracil, 5-bromouracil, 5-thiazolo-uracil, 2-thio-uracil, 2'thio-thymine, inosine, diaminopurine, 6-aminopurine, 2-aminopurine, 2,6-diaminopurine and 2-chloro-6-aminopurine).

[0070] The nucleobase moiety can be represented by the letter code of each corresponding nucleobase (e.g., A, T, G, C or U), where each letter can optionally include a modified nucleobase of equivalent function.For example, in the exemplified oligonucleotide, the nucleobase moiety is selected from A, T, G, C and 5-methylcytosine.Optionally, 5-methylcytosine LNA nucleoside can be used.

[0071] The oligonucleotides of the present disclosure can upregulate the expression of SYNGAP.

[0072] The oligonucleotides of the present disclosure may include one or more nucleosides with modified sugar moieties (i.e., modified sugar moieties compared to the ribose sugar moieties found in DNA and RNA). Many nucleosides with modified ribose sugar moieties have been made primarily for the purpose of improving certain properties of oligonucleotides (e.g., affinity and / or nuclease resistance). Such modifications include those in which the ribose ring structure is modified, for example, by replacement with a hexose ring (HNA), or a bicyclic ring (which typically has a bridge between the C2 and C4 carbons on the ribose ring (LNA)), or an unlinked ribose ring (e.g., UNA), which typically lacks a bond between the C2 and C3 carbons. Modified nucleosides also include nucleosides in which the sugar moiety is replaced with a non-sugar moiety, for example, in the case of peptide nucleic acid (PNA) or morpholino nucleic acid.

[0073] Sugar modifications also include modifications made through changing the substituents on the ribose ring to groups other than hydrogen, or the 2'-OH group found naturally in DNA and RNA nucleosides. Substituents can be introduced, for example, at the 2', 3', 4', or 5' positions.

[0074] Oligonucleotides may contain one or more locked nucleic acid (LNA) bases. LNA may include 2'-modified nucleosides that contain a biradical linking the C2' and C4' of the ribose sugar ring of the nucleoside (also referred to as a "2'-4' bridge"), which restricts or locks the conformation of the ribose ring. These nucleosides are also referred to in the literature as bridged nucleic acids or bicyclic nucleic acids (BNA). Locking the conformation of the ribose is associated with enhanced affinity of hybridization (duplex stabilization) when LNA is incorporated into an oligonucleotide for complementary RNA or DNA molecules. This can be routinely determined by measuring the melting temperature of the oligonucleotide / complement duplex. Without limitation, exemplary LNA nucleosides are disclosed in WO 99 / 014226, WO 00 / 66604, WO 98 / 039352, WO 2004 / 046160, WO 00 / 047599, WO 2007 / 134181, WO 2010 / 077578, WO 2010 / 036698, WO 2007 / 090071, WO 2009 / 006478, WO 2011 / 156202, WO 2008 / 154401, WO 2009 / 067647, and WO 2008 / 150729 (all incorporated by reference).

[0075] The pharma- ceutically acceptable salts of the oligonucleotides of the present disclosure include those salts that retain the biological effectiveness and properties of free base or free acid.The above salts are formed with inorganic acids (e.g., hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, especially hydrochloric acid) and organic acids (e.g., acetic acid, propionic acid, glycolic acid, pyruvic acid, oxalic acid, maleic acid, malonic acid, succinic acid, formic acid, tartaric acid, citric acid, benzoic acid, cinnamic acid, sulfonic acid, or salicylic acid).Furthermore, these salts can be prepared from inorganic or organic bases that change to the above free acid.The salts obtained from inorganic bases include, but are not limited to, sodium, potassium, lithium, ammonium, calcium, magnesium salts. Salts derived from organic bases include, but are not limited to, salts of primary, secondary, and tertiary amines, substituted amines, including naturally occurring substituted amines, cyclic amines, and basic ion exchange resins (e.g., isopropylamine, trimethylamine, diethylamine, triethylamine, tripropylamine, ethanolamine, lysine, arginine, N-ethylpiperidine, piperidine, polyamine resins).

[0076] The ASO can include a 2'-substituted nucleoside (e.g., a 2'-substituted nucleoside independently selected from the group consisting of a 2'-O-alkyl-RNA unit, a 2'-O-methyl-RNA, a 2'-amino-DNA unit, a 2'-fluoro-DNA unit, a 2'-alkoxy-RNA, a 2'-MOE unit, an arabinonucleic acid (ANA) unit, a 2'-fluoro-ANA unit, or a combination thereof).

[0077] Conjugation of an oligonucleotide to one or more non-nucleotide moieties can improve the pharmacology of the oligonucleotide, for example, by affecting the activity, cellular distribution, cellular uptake or stability of the oligonucleotide. In some embodiments, the conjugate moiety can modify or enhance the pharmacokinetic properties of the oligonucleotide by improving the cellular distribution, bioavailability, metabolism, excretion, permeability and / or cellular uptake of the oligonucleotide. In particular, the conjugate can target the oligonucleotide to a specific organ, tissue or cell type, thereby enhancing the efficacy of the oligonucleotide in that organ, tissue or cell type. The conjugate can also be useful for reducing the activity of the oligonucleotide in non-target cell types, tissues or organs (e.g., off-target activity or activity in non-target cell types, tissues or organs).

[0078] In one embodiment, the non-nucleotide moiety (conjugate moiety) is selected from the group consisting of carbohydrates, cell surface receptor ligands, drug substances, hormones, lipophilic substances, polymers, proteins, peptides, toxins (e.g., bacterial toxins), vitamins, viral proteins (e.g., capsids), or combinations thereof. In some embodiments, the ASO of the present invention is conjugated to an antibody. For example, an antibody can be conjugated to the ASO to promote or facilitate the delivery of the ASO.

[0079] The composition of the present disclosure can be provided in a pharmaceutical composition comprising any of the above-mentioned nucleic acids or their salts, and pharma- ceutical acceptable diluents, carriers, salts and / or adjuvants. Pharmaceutically acceptable diluents include ACSF artificial cerebrospinal fluid, and pharma-ceutical acceptable salts include, but are not limited to, sodium and potassium salts. In some embodiments, the pharma-ceutical acceptable diluent is sterile phosphate buffered saline or sterile sodium carbonate buffer. In some preferred embodiments, diluents for clinical applications include Elliotts B solution and / or ACSF artificial cerebrospinal fluid.

[0080] In some embodiments, the oligonucleotide of the present invention is in the form of a liquid in a pharma- ceutically acceptable diluent, for example, dissolved in saline, PBS, or sodium carbonate buffer. The oligonucleotide may be preformulated in a liquid, or in some embodiments, may be in the form of a dry powder (e.g., a lyophilized powder) that can be dissolved in a pharma-ceutically acceptable diluent for administration. Suitably, for example, the oligonucleotide may be dissolved at a concentration of 0.1-100 mg / mL (e.g., 1-10 mg / mL).

[0081] The present invention provides a method for treating neurodevelopmental disorders by delivering any of the compositions of the present disclosure to a patient in need. The delivered composition may include one or any combination of the nucleic acids of the present disclosure. By stating "one nucleic acid" it is not meant a single molecule, but rather a composition that includes, for example, millions of chemically identical or essentially identical copies of the molecule. It may be preferable to deliver two or more of the nucleic acids to the same patient, either in combination or at different times. Preferably, the composition is delivered across the blood-brain barrier. The nucleic acid may be delivered using a vector, including, for example, certain viral vectors (e.g., adeno-associated viral vectors) that serve such a purpose, facilitating crossing of the blood-brain barrier. The nucleic acid may be packaged in a particle, such as, for example, a viral vector, a vesicle, or a lipid nanoparticle. The delivery particle may then be modified with a ligand that facilitates delivery, such as, for example, an antibody known to facilitate receptor-mediated transcytosis across the blood-brain barrier. For a review, see Stanimirovic, 2018, Emerging technologies for delivery of biotherapeutics and gene therapy across the blood-brain barrier, BioDrugs 32(6):547-559 (incorporated by reference). The composition can be delivered, for example, systemically or by intrathecal injection. The nucleic acid is delivered to increase expression of SynGAP in the patient. The method can include selecting the patient by identifying that the patient has a heterozygous loss-of-function mutation in the SYNGAP gene.

[0082] Listed below are sequences for order forms from commercial suppliers for certain specific embodiments. The sequences listed below are the same as those shown in Table 1. The sequences are shown below along with the nomenclature (i.e., letters) as they may be used to order such nucleic acids from commercial services (e.g., Integrated DNA Technologies). [ka] [ka] [ka] [ka] [ka]

[0083] The present invention relates to therapeutic compositions for disorders associated with haploinsufficiency. The present invention provides antisense oligonucleotides useful for treating neurodevelopmental disorders associated with mutations in the SYNGAP gene. The present invention provides compositions comprising synthetic antisense oligonucleotides (ASOs) that promote the expression of SynGAP protein by one or any combination of the following steps: (i) preventing one or more miRNAs from interfering with the production of Syngap protein; (ii) binding to the 5'-UTR of SYNGAP1 transcript and enhancing the translation of SynGAP protein; and (iii) promoting RNAseH cleavage of antisense long non-coding RNA that is anti-correlated with SYNGAP expression. When the compositions are delivered to patients with SYNGAP haploinsufficiency, the ASOs promote the expression of SynGAP protein.

Claims

1. At least one nucleic acid that promotes expression of the SynGAP protein from the transcription of the SYNGAP gene and has at least 80% sequence similarity to one of SEQ ID NOs: 1-29; A composition comprising:

2. The composition of claim 1, wherein the nucleic acid has a length between about 10 bases and about 30 bases.

3. 3. The composition of claim 2, wherein the nucleic acid has a region of at least about 10 contiguous bases that has a 100% match to a segment in one of SEQ ID NOs: 1-29.

4. 4. The composition of claim 3, wherein some of the bases of the nucleic acid have a 2'-O-methoxyethyl modified ribose.

5. 5. The composition of claim 4, wherein a majority of the interbase linkages in the nucleic acid are phosphorothioate.

6. 6. The composition of claim 5, wherein at least about half of the bases in the nucleic acid comprise a 2'-O-methoxyethyl ribose sugar.

7. The composition of claim 5, wherein the nucleic acid has at least 88% sequence similarity to one of SEQ ID NOs: 1-29.

8. 6. The composition of claim 5, wherein the nucleic acid has at least 94% sequence similarity to one of SEQ ID NOs: 1-29, and at least about half of the bases in the nucleic acid contain a 2'-O-methoxyethyl ribose sugar.

9. The composition of claim 8 , wherein at least about 90% of the interbase linkages in the nucleic acid are phosphorothioate linkages.

10. 2. The composition of claim 1, wherein the nucleic acid has 100% sequence similarity to one of SEQ ID NOs: 1-29; at least about half of the bases in the nucleic acid contain 2'-O-methoxyethyl ribose sugars; all cases of U and C are methylated at the 5-position of the nitrogenous base; and all interbase linkages in the nucleic acid are phosphorothioate bonds.

11. The composition according to one of claims 1 to 10, wherein the nucleic acid has a sequence at least 90% similar to one of SEQ ID NOs: 11 to 20 and blocks miRNA from binding to the transcript.

12. 12. The composition of claim 11, wherein the nucleic acid binds within the 3' untranslated region (UTR) of a SYNGAP1 transcript and prevents binding of the miRNA, wherein the miRNA is a member of the hsa-miR-29-3p family.

13. 13. The composition of claim 12, wherein the miRNA is selected from the group consisting of hsa-miR-29c-3p, hsa-miR-29b-3p, and hsa-miR-29a-3p, and wherein the nucleic acid hybridizes to a position within bases 4520 to 4526 of the transcript, the transcript being as given by NCBI Reference Sequence NM_006772.

3.

14. 13. The composition of claim 12, wherein the miRNA is selected from the group consisting of hsa-miR-1343-3p and hsa-miR-6783-3p, and wherein the nucleic acid hybridizes to a position within bases 4479-4485 of the transcript, the transcript being given by NCBI Reference Sequence NM_006772.

3.

15. The composition of claim 12, wherein the nucleic acid is a steric blocked oligonucleotide (SBO).

16. The composition according to any one of claims 1 to 10, wherein the nucleic acid has a sequence at least 90% similar to one of sequences numbered 1 to 10 and enhances expression by mitigating the negative effect of the 5'UTR of the transcript.

17. The composition of claim 16, further comprising a plurality of nucleic acids, each having a sequence at least 90% similar to one of SEQ ID NOs: 1-10.

18. 20. The composition of claim 17, further comprising at least about five of said nucleic acids.

19. 19. The composition of claim 18, wherein the five nucleic acids hybridize to positions distributed across the 5'UTR of the transcript.

20. 20. The composition of claim 19, further comprising 10 nucleic acids described by SEQ ID NOs: 1-10, wherein said 10 nucleic acids tile across substantially all 200 nt of the 5'UTR.

21. The composition according to one of claims 1 to 10, wherein the nucleic acid has a sequence at least 90% similar to one of SEQ ID NOs: 21 to 29 and mediates digestion of a long non-coding RNA antisense to the transcript.

22. The composition of claim 21, wherein the composition comprises one or more nucleic acids, each having a base sequence at least 94% identical to one of SEQ ID NOs:21-29, and wherein each of the nucleic acids has a gapmer structure comprising a central DNA segment flanked by modified RNA wings.

23. 22. The composition of claim 21, wherein the nucleic acid comprises two wings flanking a central region of at least 9 DNA bases.

24. 22. The composition of claim 21, wherein at least one end of the nucleic acid comprises a modified RNA base.

25. 25. The composition of claim 24, wherein each modified RNA base is a 2'-O-methoxyethyl RNA.

26. 22. The composition of claim 21, wherein the nucleic acid has a base sequence having at least a 94% match to one of SEQ ID NOs:21-29, where at least the outermost interbase linkages and all linkages involving DNA bases are phosphorothioate, and the oligonucleotide further comprises central 12 DNA bases flanked by 5' and 3' RNA wings, each of the 5' and 3' wings comprising 4 consecutive 2' modified RNA bases.

27. 27. The composition of claim 26, wherein the oligonucleotide has the sequence of one of SEQ ID NOs: 21-29, wherein both RNA wings consist of four 2-methoxyethyl-modified RNA bases.

28. 27. The composition of claim 26, further comprising a plurality of said nucleic acids, wherein said plurality of nucleic acids are tiled along a long non-coding RNA antisense to said transcript.

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