Therapeutic Agents for the Treatment of Neurodegenerative Disorders

Abstract

Provided is antisense oligonucleotide (ASO) that can regulate splicing by preventing the inclusion of UNC13A cryptic exon into UNC13A mature mRNA.Such ASO can be used as medicine to treat, for example, neurodegenerative disorders, particularly those related to TDP-43 pathology.

Description

[Technical Field]

[0001] Sequence List This application is filed together with an electronic sequence listing. The sequence listing file, titled sequence-P141813PC00.txt, was created on December 6, 2021, and is 185 kilobytes in size. The information in the electronic sequence listing is incorporated herein by reference in its entirety.

[0002] This disclosure relates to novel therapeutic agents for the treatment of neurodegenerative disorders, more particularly amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD), or those associated with TDP-43 pathology. [Background technology]

[0003] Amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) are two devastating adult-onset neurodegenerative disorders that can occur simultaneously and are considered part of a single disease spectrum, ALS / FTD. In ALS, neuronal loss primarily affects upper and lower motor neurons (MNs), leading to rapid, progressive deterioration of muscle function and ultimately death due to respiratory failure. In FTD, frontal and temporal cortical neurons are preferentially lost, inducing cognitive impairment accompanied by language and behavioral changes.

[0004] The precise etiology of ALS remains largely unknown. Disease-causing gene mutations are identified in <15% of cases, and a combination of numerous neurological invaders with potential genetic, viral, autoimmune, and neurotoxic origins is thought to underlie the remaining cases. Proposed mechanisms for the pathogenicity of ALS include glutamate excitotoxicity, mitochondrial structural and functional abnormalities, axonal structural defects and transport defects, altered protein handling, and free radical-mediated oxidative stress.

[0005] TDP-43 is an RNA-binding protein that is normally located in the cell nucleus, but is depleted in the nucleus and accumulated in cytoplasmic inclusions in numerous neurodegenerative disorders, including >95% of amyotrophic lateral sclerosis (ALS) cases, approximately 50% of frontotemporal dementia (FTD) cases, approximately 30% of Alzheimer's disease cases, Parkinson's disease, and other rare neurodegenerative disorders. TDP-43 is involved in numerous RNA processing functions, including suppressing the inclusion of unwanted intronic RNA sequences into mature mRNA.

[0006] When TDP-43 is deficient in the nucleus, this repressive effect cannot be carried out, and pseudo-segments of introns, which have been named "latent exons," are included in the mRNA. Latent exons can induce a reduction in protein levels through abnormal RNA degradation or due to the presence of immature stop codons. [Overview of the project] [Problems that the invention aims to solve]

[0007] Further understanding is needed regarding the mechanisms of TDP-43 depletion and TDP-43 pathology, and how this leads to disease development. Furthermore, it is necessary to develop therapeutic strategies to address neurodegenerative disorders and / or diseases associated with TDP-43 depletion. [Means for solving the problem]

[0008] This summary provides various embodiments of the subject matter of this disclosure, thereby further describing herein details, examples and options provided in connection with one or more of the described aspects of the invention, which apply equally to all aspects of the invention. Any combination of all possible variations of the embodiments, examples and options described herein is incorporated into the invention unless otherwise expressly stated herein or otherwise explicitly rejected by the context.

[0009] This invention solves the problem of further understanding the mechanisms of TDP-43 depletion and TDP-43 pathology, and how this leads to the development of disease. Provided herein are polynucleotides, namely UNC13A antisense compounds, which are useful in elucidating TDP-43 pathology and / or UNC13A dysfunction, and their roles in disease. This invention also solves the problem of providing alternative therapeutic strategies to treat neurodegenerative disorders and / or diseases associated with TDP-43 depletion, since it has been surprisingly found that the UNC13A ASOs described herein prevent the splicing mechanism from recognizing latent exons arising from the disease state and incorporating these latent exons into the resulting UNC13A mRNA. Thus, UNC13A antisense compounds with these effects provide a rescue effect on the disease state.

[0010] According to the first embodiment, an antisense oligonucleotide (ASO) is provided, comprising a nucleotide sequence of 13 to 30 nucleotides, wherein the nucleotide sequence has a sequence complementary to SEQ ID NO: 1 (i.e., a portion of the sequence of SEQ ID NO: 1 having the same number of nucleotides). In one embodiment, the nucleotide sequence is 100% complementary to SEQ ID NO: 1. In some embodiments, the ASO is complementary to SEQ ID NO: 1, where the base at position 415 is G or C. In some embodiments, the ASO is complementary to SEQ ID NO: 1, where the base at position 965 is U or G. In some embodiments, SEQ ID NO: 1 has G at position 415 and U at position 965. In some embodiments, SEQ ID NO: 1 has C at position 415 and U at position 965. In some embodiments, SEQ ID NO: 1 has C at position 415 and G at position 965. The ASO is synthetic.

[0011] In one embodiment, ASO can regulate splicing by preventing the inclusion of the UNC13A latent exon into mature UNC13A mRNA.

[0012] According to a second aspect of the present invention, an antisense oligonucleotide (ASO) comprising a nucleotide sequence of 13 to 30 nucleotides is provided, wherein the ASO can modulate splicing by preventing the inclusion of the UNC13A latent exon into mature UNC13A mRNA. The ASO may be synthesized.

[0013] In one embodiment of the first and second aspects, the ASO comprises 20 to 24 nucleotides, preferably 21 or 22 nucleotides, preferably 21 nucleotides.

[0014] In one embodiment of the first and second aspects, the ASO can bind to the UNC13A latent exon or its flanking region. In some embodiments, the ASO is complementary to SEQ ID NO: 1, where the base at position 415 is G or C. In some embodiments, the ASO is complementary to SEQ ID NO: 1, where the base at position 965 is U or G. In one embodiment, the ASO contains a sequence corresponding to one or more SEQ ID NOs from 4 to 546.

[0015] In one embodiment, ASO can bind to the UNC13A latent exon, and the UNC13A latent exon appears in the UNC13A premRNA.

[0016] In one embodiment, the UNC13A latent exon corresponds to SEQ ID NO: 2 or SEQ ID NO: 2. In one embodiment, ASO is complementary to SEQ ID NO: 2 or SEQ ID NO: 3. In SEQ ID NO: 2, the base at position 112 can be either G or C. In SEQ ID NO: 3, the base at position 162 can be either G or C.

[0017] In one embodiment of the first and second aspects, the ASO can bind to the branch point of the UNC13A latent exon. In one embodiment, the ASO is a sequence corresponding to one or more of sequence numbers 4 to 104. These ASOs can target the branch point to reduce the efficiency of splicing.

[0018] In one embodiment of the first and second aspects, the ASO can bind to the splice site of the UNC13A latent exon. In one embodiment, the ASO includes a sequence corresponding to one or more of SEQ ID NOs: 105-189 or 270-352. These ASOs can target the splice site to reduce its availability for splicing.

[0019] In one embodiment of the first and second aspects, the ASO can bind to splice regulatory elements (SREs) associated with UNC13A latent exons in UNC13A premRNA. In one embodiment, the ASO can bind to i) latent exons, ii) SNPs in UNC13A introns, iii) TDP-43 binding sites, or iv) splice enhancers. In one embodiment, the ASO includes i) sequences corresponding to SEQ ID NOs. 190-269 that can bind to latent exons, ii) sequences corresponding to SEQ ID NOs. 353-426 that can bind to SNPs in UNC13A introns, iii) sequences corresponding to SEQ ID NOs. 427-474 that can bind to TDP-43 binding sites in UNC13A premRNA, or iv) sequences corresponding to SEQ ID NOs. 475-546 that can bind to splice enhancers associated with UNC13A latent exons. The ASO of the present invention can target these splicing regulatory elements to control the binding of RNA-binding proteins that enhance / regulate the inclusion of latent exons.

[0020] The ASO is synthetic and preferably chemically modified (i.e., the ASO contains one or more modified nucleotides). In one embodiment, the nucleotide may have a phosphate (i.e., phosphodiester), phosphorothioate, or phosphorodiamidate linkage. In one embodiment of the first and second embodiments, the ASO contains locked, crosslinked, or restricted nucleic acids. In one embodiment of the first and second aspects, the ASO has or contains a skeleton selected from RNA, DNA, LNA (locked nucleic acid), tcDNA (tricycloDNA), HNA (hexitol nucleic acid), TNA (threose nucleic acid), morpholino oligomer (PMO), peptide nucleic acid (PNA), 2'-OMe-RNA, 2'-O,4'-C-ethylene crosslinked nucleic acid (ENA), 2'-O-methoxyethyl (MOE) nucleic acid or 2-O-(2-methylcarbamoyl (MCE)) nucleotide, or any combination thereof. The ASO contains a portion of DNA nucleotides, for example, at least 10%, or at least 20%, or at least 30%, or at least 40%, or less of the DNA nucleotides. It contains at least 50%, or at least 60%, or up to 70%, i.e., in combination with LNA, tcDNA, cET, ENA, HNA, TNA, PMO, PNA, 2'-OMe-RNA, 2'-O-methyloxyethyl (MOE) nucleic acid, or MCE nucleotides. In some examples disclosed herein, the ASO has an LNA or 2-OMe-RNA backbone. In some examples, the ASO contains about 20–60% LNA or preferably about 30–50% LNA (i.e., other bases are chemically unmodified and, for example, formed from DNA). In some embodiments, the ASO contains an LNA base every at least 3 nucleotides or every at least 2 nucleotides. In some examples, the ASO contains 100% 2-OMe-RNA.

[0021] According to a third aspect of the present invention, a guide RNA is provided which includes an ASO and a Cas nuclease scaffold sequence according to the first or second aspect. In one embodiment, the ASO has an RNA backbone. In one embodiment, the scaffold sequence is a Cas13 nuclease scaffold sequence.

[0022] A fourth aspect of the present invention provides a viral vector comprising an ASO of the first or second aspect or a guide RNA of the third aspect. In one embodiment, the viral vector is a retrovirus, lentivirus, adenovirus, or adeno-associated virus.

[0023] According to a fifth aspect of the present invention, a pharmaceutical composition is provided comprising one or more ASOs of the first or second aspect, one or more guide RNAs of the third aspect, or one or more viral vectors of the fourth aspect. In one embodiment, the pharmaceutical composition comprises a pharmaceutical carrier, a diluent, or an excipient. In one embodiment, the pharmaceutical composition comprises a polymer, liposomes, micelles, dendrimers, nanoparticles, or a combination thereof.

[0024] According to a sixth aspect of the present invention, an ASO of the first or second aspect, a guide RNA of the third aspect, a viral vector of the fourth aspect, or a pharmaceutical composition of the fifth aspect is provided for use as a pharmaceutical.

[0025] According to a seventh aspect of the present invention, an ASO of the first or second aspect, a guide RNA of the third aspect, a viral vector of the fourth aspect, or a pharmaceutical composition of the fifth aspect is provided for use in a method for treating a neurodegenerative disorder. In one embodiment, the neurodegenerative disorder is related to TDP-43 pathology. In one embodiment, the neurodegenerative disorder is ALS, frontotemporal dementia (FTD), Alzheimer's disease, Parkinson's disease, FOSMN, Perry syndrome, or any combination thereof. In one embodiment, the neurodegenerative disorder is ALS, where ALS is familial ALS or sporadic ALS.

[0026] According to an eighth aspect of the present invention, a method is provided for delivering an ASO of the first or second aspect, a guide RNA of the third aspect, a viral vector of the fourth aspect, or a pharmaceutical composition of the fifth aspect to a cell, the method comprising contacting the cell with the ASO, wherein the ASO modulates UNC13A splicing to prevent the inclusion of latent exons into mature UNC13A RNA. In one embodiment, this prevents the loss of UNC13A translation protein. In one embodiment, this restores the functionality of the UNC13A protein. This may be an in vitro or in vivo method.

[0027] Also disclosed herein are methods for treating neurodegenerative disorders, comprising administering a therapeutically effective dose of a first or second embodiment of ASO, a third embodiment of guide RNA, a fourth embodiment of a viral vector, or a fifth embodiment of a pharmaceutical composition to a subject in need. Also disclosed herein are methods for treating conditions associated with TDP-43 pathology, comprising administering a therapeutically effective dose of a first or second embodiment of ASO, a third embodiment of guide RNA, a fourth embodiment of a viral vector, or a fifth embodiment of a pharmaceutical composition to a subject in need. In one example, an ASO, guide RNA, viral vector, or pharmaceutical composition can be used to prevent and / or restore the function of the UNC13A protein. In one example, an ASO, guide RNA, viral vector, or pharmaceutical composition can be used to modulate splicing so that the UNC13A latent exon is not included in the UNC13A mature mRNA.

[0028] Also disclosed herein are the use of ASOs of the first or second aspect, guide RNAs of the third aspect, viral vectors of the fourth aspect, or pharmaceutical compositions of the fifth aspect for the manufacture of pharmaceuticals. Pharmaceuticals can be used to treat neurodegenerative disorders and / or disorders related to TDP-pathology as described herein.

[0029] Also disclosed herein are ASOs containing nucleotide sequences of 13 to 30 nucleotides, in which the ASOs are used in a method to prevent the inclusion of the UNC13A latent exon into UNC13A mature RNA.

[0030] Also disclosed herein are ASOs comprising nucleotide sequences of 13 to 30 nucleotides, where the ASO (or at least a portion thereof) comprises sequences corresponding to SEQ ID NOs. 4 to 546. As described elsewhere herein, U can be replaced with T in SEQ ID NOs. These sequences are also intended to have arbitrary nucleotide chemistry.

[0031] Also disclosed herein are ASOs according to any one of SEQ ID NOs. 555-571 or SEQ ID NOs. 579 or 580. In these sequences, U and T are interchangeable. These sequences are also intended to have any nucleotide chemistry.

[0032] Also disclosed herein are methods for modulating UNC13A splicing in a subject, comprising administering a therapeutically effective amount of a first or second embodiment of ASO, a third embodiment of guide RNA, a fourth embodiment of a viral vector, or a fifth embodiment of a pharmaceutical composition to a subject requiring such treatment. For example, an ASO, guide RNA, viral vector, or pharmaceutical composition can be used to prevent and / or restore the function of the UNC13A protein. For example, an ASO, guide RNA, viral vector, or pharmaceutical composition can be used to modulate splicing so that the UNC13A latent exon is not incorporated into the UNC13A mRNA.

[0033] Also disclosed herein are isolated latent exon sequences of UNC13A and its flanking region, corresponding to SEQ ID NO: 1. SEQ ID NO: 1 corresponds to a premRNA sequence to which ASO can bind. Also disclosed herein are isolated latent exon sequences corresponding to SEQ ID NO: 2 (short variant) or SEQ ID NO: 3 (long variant). The latent exon sequences of UNC13A and its flanking region may be used to explore TDP-43 pathology and / or UNC13A dysfunction, and its role in disease.

[0034] The inventors have discovered a previously unreported latent exon in UNC13A, the gene encoding the important synaptic protein UNC13A. This novel latent exon is found to cause UNC13A downregulation at both the transcriptional and protein levels. Notably, the latent exon was specifically detected in postmortem brain regions of patients with TDP-43 protein disorders, including both ALS and FTD, and was found to overlap with disease-associated variant rs12973192, which has been previously identified in multiple genome-wide association studies linked to ALS / FTLD risk and disease invasiveness. While the latent exon was found to be absent in UNC13A mRNA at normal TDP-43 levels, the latent exon was included in both the presence and absence of the risk SNP when TDP-43 levels were depleted. Risk SNPs have been found to induce high levels of latent exons when TDP-43 loss occurs in both cellular and postmortem brain tissue. This suggests that the increased level of these latent exons directly contributes to the disease, and therefore inhibiting their inclusion in mRNA may be therapeutically beneficial.

[0035] The UNC13A latent exon is associated with TDP pathology and disease invasiveness, and therefore represents a novel therapeutic target for such diseases. Accordingly, we have developed novel therapeutic agents that can target UNC13A in the latent exon and modulate UNC13A splicing. Using the ASO, guide RNA, and viral vectors disclosed herein, it is possible to prevent the incorporation of the UNC13A latent exon into mature mRNA and thus prevent the loss of UNC13A translation protein so that fully functional UNC13A is produced. Using the ASO, splice regulatory elements (SREs) including motifs within the UNC13A latent exon, UNC13A latent exon splice sites (e.g., splice 5' donor or 3' acceptor sites), branching points, or RNA-binding protein sites associated with splice enhancers and UNC13A latent exon can be targeted. Using the ASO, guide RNA, and viral vectors disclosed herein, it is possible to mask key elements that splice the latent exon. The ASOs, guide RNAs, and viral vectors disclosed herein can prevent the splicing mechanism from recognizing latent exons and incorporating them into UNC13A mRNA. The ASOs, guide RNAs, and viral vectors disclosed herein can bind to UNC13A latent exons and their intron flanking regions and can therefore be used to explore TDP-43 pathology and / or UNC13A dysfunction, as well as its role in disease.

[0036] The following disclosures are described with reference to the drawings. [Brief explanation of the drawing]

[0037] [Figure 1]Figure 1(B) shows a schematic diagram illustrating how the ASO of this disclosure can modulate UNC13A splicing, a) an abnormal splicing event in which a novel UNC13A latent exon is incorporated into mRNA, with the upper part of Figure 1(A) showing premRNA and the lower part showing mature mRNA, and b) the ASO can target the latent exon to modulate splicing and prevent its incorporation into mature mRNA, with the upper part of Figure 1(B) showing premRNA and the lower part showing mature mRNA. [Figure 2] Differential splicing analysis of TDP knockdown cells and control cells revealed that the UNC13A transcript may contain a novel latent cassette exon not previously reported in TDP-43 depleted cells. [Figure 3] This study shows that the UNC13A latent event induced by TDP-43 knockdown is close to two polymorphisms (SNPs) previously linked to both ALS and FTD: rs12973192 and rs12608932. One of the polymorphisms, rs12973192, places 16 bp within the latent event, while the other, rs12608932, is located 534 bp downstream of the 3' splice site of the latent event. Exons 20 and 21 are also shown. [Figure 4] This study demonstrates that rs12973192 is a key SNP driving alterations in UNC13A latent splicing associated with the risk of invasive disease progression. The effects of rs12973192 (exon SNP) and rs12608932 (intron SNP) on UNC13A were examined using a minigene with four possible combinations of SNP alleles. The results show that the presence of the risk allele in rs12973192 can, by itself, drive increased UNC13A CE splicing when TDP-43 is depleted (see the boxes drawn on the gel, where a high molecular weight band is present with the 2× risk and REHI combination). [Figure 5] This study demonstrates that expression of the UNC13A latent exon was found across multiple datasets from TDP-43 knockdown in neuron-like cells. [Figure 6] The efficiency of TARDBP depletion shows a strong correlation with the amount of UNC13A latent exon present, and in the sample, the greatest reduction in TARDBP RNA is observed compared to the control with the greatest inclusion of UNC13A latent exon. [Figure 7] This paper demonstrates qPCR verification of latent events in SH-SY5Y TDP-43 KD cells. [Figure 8] This paper demonstrates how TDP-43 loss leads to a reduction in UNC13A at the transcriptional level. [Figure 9] This shows how TDP-43 loss leads to a reduction in UNC13A at the protein level (A-D). [Figure 10] This study demonstrates that the UNC13A latent exon is present only in the brains of ALS and FTLD patients with TDP-43 pathology. [Figure 11] This study demonstrates that the UNC13A latent exon is present in FTLD brains with TDP-43 pathology in the cerebellum, frontal cortex, and temporal cortex. [Figure 12] This study demonstrates that the UNC13A latent exon is present in the brains of ALS and FTLD patients with TDP-43 pathology in the cerebellum, cervical spinal cord, lumbar spinal cord, motor cortex, temporal cortex, and thoracic spinal cord. [Figure 13] This shows the correlation between the UNC13A latent exon and the STMN2 latent region. [Figure 14A] This paper shows the results of experiments transfecting SK-N-DZ cells with numerous different ASOs, and the number of precisely spliced ​​fractions when treated with confirmed antisense polynucleotides. All cells were treated with doxycycline except for NT (untreated). All ASOs had LNA-modified bases except for the OMe_ sequence. Error bars indicate the standard deviation of three copies. *=adjusted p<0.05, **=adjusted p<0.01, ***=adjusted p<0.001, ****=adjusted p<0.0001; Tukey method; significance calculated against Dox. [Figure 14B] This is a continuation of Figure 14A. [Figure 15]An example of RT-PCR results showing splicing patterns with and without the UNC13A latent exon is presented. The bottom band originates from precisely spliced ​​samples. As expected, it is abundant in NT (untreated samples) but rare in Dox-treated samples. 21nt_Don4 (SEQ ID NO: 560) rescues the level of this precisely spliced ​​band, while the scrambled control does not. [Figure 16] Figure 14A shows the qPCR validation of TDP-43 knockdown in the sample. Error bars indicate the standard deviation. The TDP-43 Ct value was normalized to the GAPDH Ct value to explain the variation in cDNA amount. The average TDP-43 abundance in untreated cells was normalized to 1. [Figure 17] A schematic diagram showing the binding site and degree of rescue for an example of ASO is shown. The shading of the bars indicates the strength of the rescue. The number of bars represents the nucleotide length of the ASO. [Figure 18-1]This study demonstrates that TDP-43 depletion in neurons leads to changes in the splicing of synaptic genes UNC13A and UNC13B. (A) Differential splicing using MAJIQ, (B) Differential expression of CRISPRi TDP-43 depleted (N=3) iPSC-derived cortical-like i3Neuron with control (N=4). Each point indicates a splice junction (A) or gene (B). (C) Representative sashimi plot showing the inclusion of a latent exon (CE) between exons 20 and 21 of UNC13A by TDP-43 knockdown (KD). (D, F) Schematic showing intron retention (IR, lower schematic, orange), TDP-43 binding region and two ALS / FTLD-related SNPs. (E) Representative sashimi plot of UNC13B showing the inclusion of a frameshift exon (fsE) by TDP-43 KD. (G) LocusZoom plot of the UNC13A locus using the latest ALS GWAS17. Read SNP rs12973192 is plotted as a diamond shape in 1000 European genomes, with other SNPs colored due to linkage disequilibrium with rs12973192. (H) BaseScope detection of UNC13A CE in i3Neurons with TDP-43, neurite (TUBB3), and nucleus co-stained, control (upper) and TDP-43 KD (lower). (I) Representative images of RT-PCR products using iPSC-derived neurons prepared from the independent iPSC strain NCRM5, with untargeted control sgRNA (sgTARDBP-), intermediate TDP-43 KD (sgTARDBP+), or stronger TDP-43 KD (sgTARDBP++). (J) Quantification of (I) plotted mean ± SEM = sgControl (n=6), sgTARDBP + (n=5), sgTARDBP ++ (n=6). One-way ANOVA for multiple comparisons. Significance levels are reported as * (p<0.05), ** (p<0.01), *** (p<0.001), **** (p<0.0001). Error bars show the mean standard error. (K) Schematic diagram of the quantified nanopore long reads.(L) Percentage of targeted UNC13A long reads having a TDP-43 regulatory splice event containing either CE or IR, or both, in TDP-43 KD SH-SY5Y cells. [Figure 18-2] This is a continuation of Figure 18-1. [Figure 18-3] This is a continuation of Figure 18-2. [Figure 18-4] This is a continuation of Figure 18-3. [Figure 19-1] This study demonstrates that UNC13A and UNC13B are downregulated after TDP-43 knockdown due to the production of NMD-sensitive transcripts. (A) Ribosome profiling of TDP-43 knockdown i3Neuron shows a reduction in ribosome occupancy in STMN2, UNC13A, and UNC13B transcripts. (B) Mass spectrometry-based proteomics analysis shows a dose-dependent reduction in the protein abundance of UNC13A and UNC13B in i3Neuron due to TDP-43 knockdown. Two-sample t-test. (C) Quantification of TDP-43, UNC13A, and UNC13B proteins and RNAs in SH-SY5Y with varying levels of DOX-induced TDP-43 knockdown. (D) Transcriptional expression induced by CHX treatment suggests that UNC13A and UNC13B, rather than STMN2, are sensitive to nonsense-mediated decay. HNRNPL (heteronuclear ribonucleoprotein L) is a positive control. Shaded bars indicate that a separate experiment was conducted for UNC13B. Single-sample t-test. Significance levels are reported as *(p<0.05), **(p<0.01), ***(p<0.001), and ****(p<0.0001). Error bars show the mean standard error. [Figure 19-2] This is a continuation of Figure 19-1. [Figure 20-1]This study demonstrates that UNC13A CE is highly expressed in the tissues of ALS / FTLD patients and correlates with known markers of TDP-43 loss of function. (A) Expression of UNC13A and STMN2 CE from a published dataset of prefrontal cortical neuronal nuclei from ALS / FTLD patients, sorted according to TDP-4327 expression. (B) UNC13A CE expression in bulk RNA-seq from the NYGC ALS Consortium, normalized to library size of disease and tissue samples. ALS cases were stratified by mutation status, and FTLD cases were stratified by pathological subtype. (C) CE expression across all ALS / FTLD-TDP cases in tissue, with the number of tissue samples in parentheses. (D) BaseScope detection of UNC13A CE (focus) in prefrontal cortical samples from FTLD-TDP (n=9) rather than controls (n=5) or FTLD-tau (n=4), with background-corrected intergroup focus frequency quantification. Scale bar is 10 μm. Error bars represent the mean standard error. (E) Correlation between UNC13A and STMN2 CE PSI in the ALS / FTLD-TDP cortex of patients with at least 30 splice reads at the CE locus. (B, C) Wilcoxon test. Significance levels are reported as * (p<0.05), ** (p<0.01), *** (p<0.001), and **** (p<0.0001). [Figure 20-2] This is a continuation of Figure 20-1. [Figure 20-3] This is a continuation of Figure 20-2. [Figure 20-4] This is a continuation of Figure 20-3. [Figure 21-1]This study demonstrates that the UNC13A ALS / FTD risk variant enhances UNC13A CE splicing in patients and in vitro by altering TDP-43 premRNA binding. (A) Ratio of UNC13A / STMN2 CE PSI, split by genotype of UNC13A risk allele. (B) Proprietary cDNA from targeted RNA-seq in 10 CE SNP heterozygous FTLD-TDP patients. p-values ​​for one-sided binomial test. FTD1, 5, and 7 are C9 or f72 hexanucleotide repeat carriers. (C) Diagram of the UNC13A minigene containing exon 20, intron 20, and exon 21, as well as combinations of UNC13A alleles. (D) Representative image of RT-PCR product of UNC13A minigene in SH-SY5Y ± TDP-43 KD. (E) Quantification of (D) plotted mean ± SE. The results of independent t-tests comparing each variant with a co-transfected healthy minigene (N=3). (F) TDP-43 iCLIP of SH-SY5Y containing 2R and 2H minigenes, with the top panel showing the mean crosslink density and the middle panel showing the mean density change for 2R-2H (rolling window = 20 nt, unit = 1,000 crosslinks). The bottom diagram shows the predicted TDP-43 binding footprint (UGNNUG motif). (G) Mean change in E value (measure of binding enrichment) in the protein for heptamers containing risk / healthy CE SNP alleles, for TDP-43. (H) Binding affinity between TDP-43 and 14-nt RNA containing healthy or risk sequences of CE (n=4) or introns (n=3), as measured by ITC. Two-sample t-test. (I) Representative image of RT-PCR product from UNC13A minigene with the mutant UGNNUG TDP-43 binding motif shown in (F). (J) The data is plotted on mean ± SE, and the results of (I), where N=3, are quantified. The analysis is performed in the same manner as in D and E, and the significance levels are reported as * (p<0.05), ** (p<0.01), *** (p<0.001), and **** (p<0.0001). [Figure 21-2] This is a continuation of Figure 21-1. [Figure 21-3] This is a continuation of Figure 21-2. [Figure 21-4]This is a continuation of Figure 21-3. [Figure 22-1] Further evidence is presented of missplicing of UNC13A after TDP-43 knockdown in neuronal cell lines. (A, B) RNA-seq traces from IGV70 of representative samples from i3Neuron control (top) and TARDBP KD (bottom) show intron retention in UNC13A(A) (mean 4.50 ± 1.50 increased IR in KD) and UNC13B (mean 1.86 ± 0.63 increased IR in KD), (B) overlapping with the published TDP-43 iCLIP peak 26, (C) histogram showing the number of basecope latent foci per nucleus in WTC11-derived i3Neuron control and TDP-43 KD (gray), independent t-test with p<0.0001. (D, E) RNA levels of TARDBP and UNC13A in WTC11-derived (D) and NCRM-5-derived i3Neuron (D), representing untargeted control sgRNA (sgTARDBP -), moderate TDP-43 KD (sgTARDBP +), or high TDP-43 KD (sgTARDBP ++). (F) Representative images of RT-PCR products of UNC13A CE. (G) Quantification of (F) plotted with mean ± SEM = sgControl (n=6), sgTARDBP + (n=6), sgTARDBP ++ (n=6). One-way ANOVA with multiple comparisons. (H~K) Expression of TDP-43 regulatory splicing of UNC13A (H, I) and UNC13B (J, K) in control and TDP-43 KD using publicly available neuron datasets. Intron retention (IR) (I, K), and CE and fsE PSI (H, J) are the relative gene expression levels of TARDBP in 11,25 of the (L) neuron datasets that significantly increase after TDP-43 depletion in most experiments using the Wilcoxon test. RNA counts normalized to the control are shown. The numbers represent the log2 factor change calculated by DESeq2. Significance is shown as the DESeq2 adjusted p-value. Significance levels are reported as * (p<0.05), ** (p<0.01), *** (p<0.001), and **** (p<0.0001). [Figure 22-2] This is a continuation of Figure 22-1. [Figure 22-3] This is a continuation of Figure 22-2. [Figure 22-4] This is a continuation of Figure 22-3. [Figure 23-1] This paper demonstrates the verification of UNC13A and UNC13B missplicing after TDP-43 KD in multiple neuronal cell lines. (A) Sanger sequencing of latent bands in both SH-SY5Y and SK-N-DZ cells confirms CE splice junctions. (B, C) Crosslink density of UNC13A(chr19)(B) and UNC13B(chr9)(C) genomic loci from novel iCLIPs in endogenous TDP-43 in SH-SHY5Y cells. The crosslink density of both genes peaked at CE / fsE (dotted line on the left) and retained introns (dotted line on the right). Coordination is shown at hg38 percent for all targeted UNC13A long reads in SH-SY5Y cells containing either no CE or IR, both, or retention of latent exons or introns, as determined by Sanger sequencing. Most reads in both the control and TDP-43 KD cells did not contain either event, and while the IR event was present in the control, CE was detected only in TDP-43 KD. (Bottom) Targeted nanopore sequencing reveals that UNC13A CE and IR events occur almost independently in vitro. (A) Percentage of all targeted UNC13A long reads in SHSY5Y cells containing neither CE nor IR, both, or either CE or IR. Most reads in both the control and TDP-43 KD cells did not contain either event, and while the IR event was present in the control, CE was detected only in TDP-43 KD. [Figure 23-2] This is a continuation of Figure 23-1. [Figure 24-1]This study demonstrates that the reduction in UNC13A and UNC13B after TDP-43 knockdown correlates with TDP-43 levels and is caused by nonsense-mediated decay. Relative gene expression levels of UNC13A(A) and UNC13B(B) after TDP-43 knockdown in neuronal cell lines 11 and 25. RNA counts normalized to the control are shown. Numbers indicate log-fold changes calculated by DESeq2. Significance is shown as adjusted p-values ​​by DESeq2. (C, D) RT-qPCR analysis shows that TDP-43, UNC13A, and UNC13B gene expression is reduced by TARDBP shRNA knockdown in both SH-SY5Y and SK-N-DZ human cell lines. Graphs show mean ± SE, N=6, 1-sample t-test. (E) 5' ends of 29nt reads relative to annotated start codons from a representative ribosome profiling dataset (TDP-43 KD replication B). As expected, the inventors detected strong read enrichment in the annotated coding sequences with strong 3-nucleotide periodicity and upstream untranslated regions. (F) Protein levels of UNC13A, UNC13B, and TDP-43 with varying levels of DOX-inducible TDP-43 knockdown in SH-SY5Y cells, as measured by Western blot. Tubulin is used as an endogenous control, N=3. (G) Quantification of RT-PCR products from transcripts containing UNC13A CE, UNC13A intron retention, UNC13B fsE, and UNC13B intron retention with varying levels of DOX-inducible TDP-43 knockdown in SH-SY5Y cells. Error bars show mean standard error, N=3. (H) UPF1 siRNA knockdown resulted in rescue of transcripts of hnRNPL (positive control), UNC13A, and UNC13B, but not STMN2. Error bars show mean standard error, N=4, 1-sample t-test. (I) UNC13A CE-containing transcript PSI increases after UPF1 knockdown in i3Neuron. Error bars show mean standard error, N=6. (J) RT-PCR products from UNC13A in a setting of moderate TDP-43 knockdown with either DMSO (control) or CHX (NMD inhibitor) addition.(K) Quantification of (J) plotted on mean ± SE, N=4. Significance levels are reported as *(p<0.05), **(p<0.01), ***(p<0.001), and ****(p<0.0001). [Figure 24-2] This is a continuation of Figure 24-1. [Figure 24-3] This is a continuation of Figure 24-2. [Figure 24-4] This is a continuation of Figure 24-3. [Figure 24-5] This is a continuation of Figure 24-4. [Figure 24-6] This is a continuation of Figure 24-5. [Figure 25-1] The differences in technical factors between samples in which UNC13A CE was detected and those in which it was not differed between cortical and spinal cord samples. (A) Detection rates of UNC13A CE in tissues by RNA sequencing platform and read length. When sequencing was performed mechanically using 125 bp compared to 100 bp, UNC13A CE was more likely to be detected in the cervical and motor cortex. (B) There were no significant differences in total RNA-seq library size (log10 scale). (C) RNA integrity score (RIN) was significantly lower in the motor and temporal cortex in samples in which UNC13A was detected. (D) Cell type degradation revealed that samples in which UNC13A CE was detected had a high proportion of neurons in the cervical and lumbar spinal cord, while samples in which UNC13A CE was detected had a low proportion of neurons in the frontal, temporal, and motor cortex, and samples in which UNC13A CE was detected had a high proportion of astrocytes in the motor and temporal cortex. Astrocy. - Astrocyte, Endothi. - Endothelium, Microgl - Microglia, Neur. - Neuron, Oligiodendr. - Oligodendrocyte (Oligodendrycyte). The p-values ​​shown are from Fisher's exact test (A) or Wilcoxon test (B-D). The number of tissue samples is indicated in parentheses below. [Figure 25-2] This is a continuation of Figure 25-1. [Figure 25-3] This is a continuation of Figure 25-2. [Figure 26] This study demonstrates that targeted long reads in the FTLD prefrontal cortex indicate that UNC13A CE and IR occur independently in vivo. (A) Percentage of targeted UNC13A long reads having TDP-43 regulatory splice events containing either CE or IR, or both, in four of the FTLD prefrontal cortex segments. (B) Percentage of all targeted UNC13A long reads in (A) that contain neither CE nor IR, both CE and IR, or either CE or IR. [Figure 27] This study demonstrates the expression of TDP-43-regulated UNC13A and UNC13B introns in human neural tissue from NYGC tissue samples. The IR ratios for UNC13A exons 31-32 (A) and UNC13B exons 21-22 (B) in NYGC tissue samples are shown. UNC13A IR was lower in ALS-TDP cases than in controls in the cervical spinal cord, prefrontal cortex, and motor cortex, and higher in FTLD-TDP cases than in controls in the prefrontal cortex and temporal cortex. This is thought to reflect the difference in the effect of cell type composition in disease states. Wilcoxon test results are reported with significance levels as *(p<0.05), **(p<0.01), ***(p<0.001), and ****(p<0.0001). [Figure 28] This study demonstrates that STMN2 CE PSI correlates with TDP-43 regulatory potential in the NYGC RNA-seq dataset. (A, B) Previously described CEs in RAP1GAP and PFKP regulated by TDP-439-11 correlate with STMN2 CE, suggesting that STMN2 CE PSI may act as a read of TDP-43 function. Only samples with at least 30 splice reads at each CE locus are included in the correlation. Spearman correlation. [Figure 29-1]This study demonstrates that the UNC13A risk allele increases UNC13A CE expression after TDP-43 depletion by altering TDP-43 binding affinity in UNC13A CE-containing introns. (A) UNC13A CE PSI by genotype (Wilcoxon test) (B) Effect of CE or intron SNPs on the correlation between STMN2 and UNC13A CE PSI in ALS / FTD cortex of samples with at least 30 junction reads at the CE locus. Spearman correlation. (C) Raw tape station gel images of UNC13A CE products of 2H and 2R minigenes, and quantification of PCR products (n=3), two-way ANOVA (D) Raw tape station gel image corresponding to Figure 4E. Either the control (upper column) or the mutant minigene (lower column) was amplified using two sets of primers. Left panel: Single transfection was performed to ensure primer specificity. Right panel: Three copies of double transfection. (E) Fractional changes in the iCLIP peak of 2R relative to 2H minigene (mean and 75% confidence interval shown). Peaks within 50nt of each SNP are highlighted. (F) Mean crosslink density around exon (upper) and intron (lower) SNPs of 2H and 2R minigenes relative to the 5' end of the minigene (error bars = standard deviation, dashed lines indicate SNP location). (G, H) Individual TDP-43 E scores for CE(G) and intron(H) heptamers in data30. (I) Mean change in E value (measure of binding enrichment) in protein for heptamers containing risk / healthy intron SNP alleles, with TDP-43 shown. Significance levels are reported as *(p<0.05), **(p<0.01), ***(p<0.001), ****(p<0.0001). [Figure 29-2] This is a continuation of Figure 29-1. [Figure 29-3] This is a continuation of Figure 29-2. [Figure 30]Figures A-D show the binding of TDP-43 to SNP-containing intron RNA. ITC measurement of the interaction between TDP-43 and 14-nt RNA containing healthy sequences of CE SNPs (A, B) and intron SNPs (C, D). Representative datasets have been reported, and raw data (A, C) and integrated heat plots (B, D) are used. Circles represent integrated heat plots, and curves represent optimality. (E) Raw tape station gel image corresponding to Figure 4J. In each experiment, two RT-PCRs were performed using different primer sets to amplify either the control minigene (upper column, minigene 2H) or the mutant minigene (lower column). Left: Single transfection to ensure primer specificity to either the control or mutant minigene. Right: Three replications of double transfection with control minigene 2H and either mutant minigene. [Figure 31-1] (A) Expression of splice junction reads supporting UNC13A CE in tissues and disease subtypes. Junction count normalized to library size in millions (junctions per million). The long novel acceptor junction is expressed in all disease subtypes of the cerebellum. (B) Example of RNA-seq trace from IGV showing the UNC13A cerebellar exon sharing the long novel acceptor junction as UNC13A CE. [Figure 31-2] This is a continuation of Figure 31-1. [Figure 32] This demonstrates ASO processing targeting short and long latent acceptors. [Figure 33] This document shows SHSY5Y cells treated with three concentrations of 21nt LNA donor ASO as described herein. [Modes for carrying out the invention]

[0038] I. Compositions of the Disclosure The studies described herein relate to compositions and methods for suppressing or preventing the inclusion of latent exons into UNC13A mRNA. The inclusion of latent exons into UNC13A mRNA can result in cleaved transcripts and proteins.

[0039] UNC13A expression can be restored by suppressing the latent splicing form of UNC13A that occurs when TDP-43 is sequestered or its functionality is reduced, for example by blocking the emergence or accumulation of the latent form and converting or restoring it to functional UNC13A RNA (e.g., by administration of ASO or antisense oligonucleotides). In addition, the studies described herein relate to compositions and methods for increasing UNC13A protein synthesis, i.e., increasing UNC13A protein expression.

[0040] Also provided in this disclosure are compositions and formulations comprising ASO or antisense polynucleotides as described herein.

[0041] In some embodiments, the composition is formulated for administration via a specific route, such as intravenous injection, intramuscular injection, or subcutaneous injection.

[0042] In another aspect of this disclosure, methods of using the antisense polynucleotides, compositions, and formulations described herein are provided. In some embodiments, the antisense polynucleotides, compositions, and formulations of this disclosure may be used as tools to control gene expression, including but not limited to transcriptional control, splicing control, translational control, and post-translational control. In some embodiments, the antisense polynucleotides, compositions, and formulations of this disclosure may be therapeutic agents for treating and preventing diseases.

[0043] Some aspects of this disclosure provide naked antisense polynucleotides. Other aspects of this disclosure provide antisense polynucleotides encapsulated or formulated with a carrier, such as a lipid-containing carrier.

[0044] Antisense polynucleotides (ASO compounds) In certain embodiments, the herein provides oligomeric compounds comprising oligonucleotides or polynucleotides consisting of linked nucleosides. As used herein, the terms “oligonucleotide” and “polynucleotide” may be used interchangeably. These may also be referred to as ASOs throughout this disclosure.

[0045] ASOs or polynucleotides may be unmodified (RNA or DNA) or modified. Modified ASOs or antisense polynucleotides contain at least one modification relative to unmodified RNA or DNA. That is, a modified polynucleotide contains at least one modified nucleoside (including a modified sugar moiety and / or a modified nucleic acid base), and / or at least one modified nucleoside linkage.

[0046] The antisense polynucleotide (ASO compound) of the present invention may contain a nucleotide sequence of 13 to 30 nucleotides, the nucleotide sequence having a sequence complementary to SEQ ID NO: 1 (i.e., the 13 to 30 nucleotide sequence is complementary to a portion of the SEQ ID NO: 1 sequence having the same length as the nucleotide sequence). SEQ ID NO: 1 corresponds to a portion of the premRNA UNC13A sequence and includes the UNC13A latent exon + intron flanking region.

[0047] ASO can prevent abnormal splicing of UNC13A premRNA, thereby preventing the inclusion of UNC13A latent exon into mature UNC13A mRNA. In one embodiment, ASO prevents splicing mechanisms (e.g., protein splicing factors) from recognizing UNC13A latent exon. In one embodiment, ASO can prevent the inclusion of UNC13A latent exon into mature UNC13A mRNA.

[0048] In one embodiment, ASO can bind to the UNC13A latent exon and / or its intron flanking region (i.e., within UNC13A premRNA). In one embodiment, the flanking region corresponds to 844 bp upstream or 303 bp downstream of the latent exon sequence. In one embodiment, the UNC13A latent exon and its intron region correspond to positions chr19:17,641,557~17642844. In one embodiment, ASO can bind to the UNC13A latent exon. In one embodiment, the UNC13A latent exon corresponds to positions chr19:17642414~17,642,541. In one embodiment, the UNC13A latent exon corresponds to SEQ ID NO: 2. In one embodiment, ASO contains a nucleotide sequence complementary to SEQ ID NO: 2. In one embodiment, the UNC13A latent exon is a long variant of the UNC13A latent exon corresponding to positions chr19:17642414~17642591. In one embodiment, the UNC13A latent exon is a long variant of the latent exon corresponding to SEQ ID NO: 3. In one embodiment, ASO contains a nucleotide sequence complementary to SEQ ID NO: 3.

[0049] In one embodiment, the ASO can bind to splice sites of the UNC13A latent exon (i.e., and its flanking region). Targeting splice sites makes them less available for splicing. In one embodiment, the splice sites correspond to positions chr19:17642414, chr19:17,642,541, or chr19:17642591. In one embodiment, the splice site may be a 5' splice site (i.e., a donor splice site) or a 3' splice site (i.e., an acceptor splice site). In some embodiments, the splice site is a 3' splice site (acceptor site), which is either a long acceptor site (i.e., corresponding to position chr19:17642414) or a short acceptor site (i.e., corresponding to position chr19:17,642,541). In some embodiments, the splice site is the 5' splice site (i.e., the donor site corresponding to position chr19:17642591).

[0050] In some embodiments, the ASO sequence includes a sequence complementary to the splice sites described herein (for example, splice donor sites, i.e., those corresponding to phosphodiester bonds between chr19:17,642,413~17,642,414, or splice acceptor sites, i.e., those corresponding to phosphodiester bonds between chr19:17,642,541~17,642,542 or chr19:17,642,591~17,642,592).

[0051] In some embodiments, the ASO includes sequences complementary to sequence numbers 547-551 (i.e., sequences complementary to the splice donor site). In some embodiments, the ASO may include one or more of sequence numbers 270-352.

[0052] In some embodiments, the ASO includes sequences complementary to sequence numbers 552-554 (i.e., sequences complementary to the splice acceptor site).

[0053] In some embodiments, the ASO may include one or more of sequence numbers 105 to 189.

[0054] In some embodiments, the ASO includes a sequence complementary to the short acceptor site.

[0055] In some embodiments, the ASO includes a sequence containing one or more of the sequence numbers 155-180 or 160-175.

[0056] In some embodiments, the ASO includes a sequence complementary to the long acceptor region.

[0057] In some embodiments, the ASO includes a sequence containing one or more of the sequence numbers 105-127 or 110-122.

[0058] The ASO capable of binding to the splice site of the UNC13A latent exon may have a sequence complementary to the target sequence including the splice site (e.g., a donor or acceptor splice site) and its flanking region. In some embodiments, the flanking region may be at least 100 nucleotides (nt) upstream or downstream of the splice site (e.g., a donor or acceptor splice site), or at least 90 nucleotides, at least 80 nucleotides, at least 70 nucleotides, at least 60 nucleotides, at least 50 nucleotides, at least 40 nucleotides, at least 30 nucleotides, at least 25 nucleotides, at least 20 nucleotides, at least 15 nucleotides, at least 10 nucleotides, or at least 5 nucleotides upstream or downstream of the splice site.

[0059] In some embodiments, the ASO can be directly bonded to the splice site.

[0060] In one embodiment, the ASO can bind to the bifurcation of the UNC13A latent exon. Targeting the bifurcation reduces the efficiency of splicing. In one embodiment, the bifurcation corresponds to position chr19:17642800. In one embodiment, the ASO sequence includes a sequence complementary to the bifurcation described herein.

[0061] In one embodiment, ASO can bind to a splice regulatory element (SRE) associated with UNC13A to modulate the splicing of the UNC13A latent exon. Targeting the splice regulatory element restricts the binding of RNA-binding proteins that enhance the inclusion of the latent exon into mature UNC13A mRNA.

[0062] In one embodiment, the SRE can be determined in silico. In one embodiment, the SRE is a splice enhancer and the ASO can bind to the splice enhancer. In one embodiment, the SRE is a TDP-43 binding site and the ASO can bind to the TDP-43 binding site to regulate the splicing of the UNC13A latent exon. In one embodiment, the SRE is a latent exon and the ASO can bind to the latent exon. In one embodiment, the SRE is a SNP in the intron flanking region of the CE. The ASO may be able to bind to a portion of the sequence in UNC13A premRNA transcribed from a sequence containing the intron SNP. The intron SNP is rs12608932. In one embodiment, the SRE is a SNP of the CE (e.g., a latent exon SNP). The ASO may be able to bind to a portion of the sequence in UNC13A premRNA transcribed from a sequence containing the CE SNP (e.g., a latent exon SNP). The CE SNP is rs12973192. In some embodiments, the ASO can bind to a sequence containing both the CE SNP rs12973192 and the donor splice site; in other words, the ASO is complementary to both the CE SNP and the donor splice site.

[0063] In one embodiment, ASO blocks the interaction between a specific protein and UNC13A premRNA, for example, a splicing factor. In one embodiment, binding of ASO to UNC13A premRNA prevents deletion of fully translated UNC13A protein (i.e., ASO corrects the reduction in UNC13A protein levels).

[0064] In one embodiment, ASO prevents the inclusion of latent exons into UNC13A mRNA. In one embodiment, ASO prevents deletion of UNC13A translation protein and / or restores its function.

[0065] UNC13A and latent exons According to this disclosure, Sequence ID 1 refers to a target sequence of the UNC13A latent exon and its intron flanking region. As otherwise described herein, Sequence ID 1 of a target sequence includes sequences having a secondary allele (e.g., a risk variant of an SNP) or a primary allele at rs12973192, and therefore also includes sequences in which G is replaced by C at rs12973192 and / or U is replaced by G at rs12608932. Thus, while Sequence ID 1 as described herein is demonstrated by G at rs12973192 (position 415 of Sequence ID 1) and U at rs12608932 (position 965 of Sequence ID 1), Sequence ID 1 may include either G or C at position 415 and / or either U or G at position 965. Coordination refers to the hg38 assembly.

[0066] Sequence IDs 2 and 3 disclosed herein are short and long sequences of the UNC13A latent exon. As otherwise described herein, Sequence IDs 2 and 3 include sequences with and without an SNP at rs12973192, and therefore also include sequences in which G is replaced by C at rs12973192. Thus, Sequence IDs 2 and 3 disclosed herein are demonstrated by G at rs12973192 (i.e., corresponding to positions 112 and 162 of Sequence IDs 2 and 3, respectively), although Sequence IDs 2 and 3 may contain G or C at positions 112 and 162, respectively.

[0067] The UNC13A latent exon, as defined herein, refers to a latent exon sequence that is abnormally present in UNC13A mRNA due to TDP pathology in some cases. The UNC13A latent exon may correspond to SEQ ID NO: 2 (short UNC13A latent variant) at position chr19:17642414~17,642,541, or to SEQ ID NO: 3 (long UNC13A latent variant) at position chr19:17642414~17642591. The UNC13A latent exon is located between exons 20 and 21 of UNC13A. SEQ ID NO: 1 corresponds to the UNC13A latent exon and its flanking region in the UNC13A preRNA.

[0068] As defined herein, key elements involved in the splicing of latent exons include a) branching points, b) splicing sites including i) 3' splice sites (otherwise referred to as splice acceptor sites), ii) 5' splice sites (otherwise referred to as splice donor sites), and c) splicing regulatory elements (SREs). As defined herein, splice sites refer to sites or sequences where splicing occurs. A branching point, as defined herein, refers to an "A" nucleotide upstream of a splice acceptor, whose loss can often be compensated for by another adjacent "A". A splicing regulatory element (SRE), as referenced herein, is a site or sequence to which an RNA-binding protein binds and facilitates the splicing event. SREs, as defined herein, include portions of a transcribed UNC13A premRNA sequence containing splice enhancers, TDP-43 binding sites and RNA-binding protein sites, and / or SNPs (e.g., risk SNPs-rs12973192 and / or rs12608932).

[0069] Complementarity In one embodiment, an antisense polynucleotide (e.g., ASO) has at least 95% sequence complementarity to SEQ ID NO: 1, or at least 95.5%, or at least 96%, or at least 96.5%, or at least 97%, or at least 97.5%, or at least 98%, or at least 98.5%, or at least 99%, or at least 99.5%, or at least 100% complementarity to SEQ ID NO: 1.

[0070] In one embodiment, an antisense polynucleotide (e.g., ASO) has at least 95% sequence complementarity to SEQ ID NO: 2, or at least 95.5%, or at least 96%, or at least 96.5%, or at least 97%, or at least 97.5%, or at least 98%, or at least 98.5%, or at least 99%, or at least 99.5%, or at least 100% complementarity to SEQ ID NO: 2.

[0071] In one embodiment, an antisense polynucleotide (e.g., ASO) has at least 95% sequence complementarity to SEQ ID NO: 3, or at least 95.5%, or at least 96%, or at least 96.5%, or at least 97%, or at least 97.5%, or at least 98%, or at least 98.5%, or at least 99%, or at least 99.5%, or at least 100% complementarity to SEQ ID NO: 3.

[0072] Complementarity with SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3 refers to the fact that an antisense polynucleotide (e.g., ASO) is complementary to a part of SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3.

[0073] It is possible to introduce mismatched bases without eliminating activity. For example, Gautschi et al (J. Natl. Cancer Inst. 93:463-471, March 2001) demonstrated that an oligonucleotide with 100% complementarity to bcl-2 mRNA and three mismatches to bcl-xL mRNA has the ability to reduce the expression of both bcl-2 and bcl-xL in vitro and in vivo.

[0074] In certain embodiments, the antisense polynucleotide or antisense oligonucleotide (ASO) is complementary to the target nucleic acid over its entire length. In certain embodiments, the antisense polynucleotide or antisense oligonucleotide (ASO) is 99%, 95%, 90%, 85%, or 80% complementary to the target nucleic acid. In certain embodiments, the oligonucleotide is at least 80% complementary to the target nucleic acid over its entire length and includes a portion that is 100% or completely complementary to the target nucleic acid. In certain embodiments, the completely complementary portion is the length of 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleic acid bases.

[0075] In certain embodiments, the antisense polynucleotide or antisense oligonucleotide (ASO) contains one or more mismatched nucleic acid bases relative to the target nucleic acid. In certain embodiments, the mismatch is located at position 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 from the 5' end of the oligonucleotide.

[0076] In some embodiments, the nucleic acid-based compositions described herein, including the ASO described herein, contain oligonucleotides or polynucleotides that are at least 80% complementary to a region of the target transcript. This region of the target transcript to which the nucleic acid-based composition hybridizes or binds is referred to as the “target sequence” or “target site.”

[0077] The term "complementary to" means that hybridization is possible under physiological conditions, or in some cases, under strict conditions regarding hybridization temperature and salt concentration. It should be understood that thymidine (T) in any given DNA sequence can be replaced by uridine (U) in the corresponding RNA transcript, and that this difference does not change the understanding of the term "complementarity."

[0078] The nucleic acid-based compositions described (e.g., antisense polynucleotides or antisense oligonucleotides (ASOs)) may share at least 80% sequence identity, at least 90% sequence identity, at least 95% sequence identity, at least 98% sequence identity, at least 99% sequence identity, or 100% sequence identity with the reverse complement of the target sequence. Thus, the reverse complement of the described nucleic acid-based compositions has a high degree of sequence identity with the target sequence. The target sequence may have the same length as the nucleic acid-based composition, i.e., the same number of nucleotides, or the target sequence may have a similar length compared to the length of the nucleic acid-based composition, i.e., a similar length in the range of 1 nucleotide or less, 2 nucleotides or less, 3 nucleotides or less, 4 nucleotides or less, or 5 nucleotides or less. The nucleic acid-based compositions may hybridize with all or part of the target sequence, or may intermittently hybridize with the targeted sequence. In some embodiments, the target sequence can hybridize with all or part of the nucleic acid-based composition described herein, or the target sequence can hybridize intermittently with the nucleic acid-based composition.

[0079] In some embodiments, the target sequence contains at least 8 nucleotides. Thus, the target sequence may be 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides in length. In some cases, the target sequence is longer than 30 nucleotides. In some embodiments, the target sequence is between 6 and 18 nucleotides in length.

[0080] In some embodiments, the target sequence has a length of 7 to 18 nucleotides. In some embodiments, the target sequence has a length of 8 to 18 nucleotides. In some embodiments, the target sequence has a length of 9 to 18 nucleotides. In some embodiments, the target sequence has a length of 10 to 18 nucleotides. In some embodiments, the target sequence has a length of 11 to 18 nucleotides. In some embodiments, the target sequence has a length of 12 to 18 nucleotides. In some embodiments, the target sequence has a length of 13 to 18 nucleotides. In some embodiments, the target sequence has a length of 14 to 18 nucleotides. In some embodiments, the target sequence has a length of approximately 14 nucleotides. In some embodiments, the target sequence has a length of approximately 15 nucleotides. In some embodiments, the target sequence has a length of approximately 16 nucleotides.

[0081] For example, in a particular embodiment, the nucleic acid-based composition described (e.g., antisense polynucleotide or antisense oligonucleotide (ASO)) is 12-13, 12-14, 12-15, 12-16, 12-17, 12-18, 12-19, 12-20, 12-21, 12-22, 12-23, 12-24, 12-25, 12-26, 12-27, 12-28, 12-29, 12-30, 13-14, 13-15, 13-16, 13-17, 13-18, 13-19, 13-20 pieces, 13~21 pieces, 13~22 pieces, 13~23 pieces, 13~24 pieces, 13~25 pieces, 13~26 pieces, 13~27 pieces, 13~28 pieces, 13~2 9 pieces, 13~30 pieces, 14~15 pieces, 14~16 pieces, 14~17 pieces, 14~18 pieces, 14~19 pieces, 14~20 pieces, 14~21 pieces, 14~2 2 pieces, 14~23 pieces, 14~24 pieces, 14~25 pieces, 14~26 pieces, 14~27 pieces, 14~28 pieces, 14~29 pieces, 14~30 pieces, 15~ 16 pieces, 15~17 pieces, 15~18 pieces, 15~19 pieces, 15~20 pieces, 15~21 pieces, 15~22 pieces, 15~23 pieces, 15~24 pieces, 15~ 25 pieces, 15~26 pieces, 15~27 pieces, 15~28 pieces, 15~29 pieces, 15~30 pieces, 16~17 pieces, 16~18 pieces, 16~19 pieces, 16 ~20 pieces, 16~21 pieces, 16~22 pieces, 16~23 pieces, 16~24 pieces, 16~25 pieces, 16~26 pieces, 16~27 pieces, 16~28 pieces, 16 ~29 pieces, 16~30 pieces, 17~18 pieces, 17~19 pieces, 17~20 pieces, 17~21 pieces, 17~22 pieces, 17~23 pieces, 17~24 pieces, 1 7~25 pieces, 17~26 pieces, 17~27 pieces, 17~28 pieces, 17~29 pieces, 17~30 pieces, 18~19 pieces, 18~20 pieces, 18~21 pieces, 1 8~22 pieces, 18~23 pieces, 18~24 pieces, 18~25 pieces, 18~26 pieces, 18~27 pieces, 18~28 pieces, 18~29 pieces, 18~30 pieces, 19~20 pieces, 19~21 pieces, 19~22 pieces, 19~23 pieces, 19~24 pieces, 19~25 pieces, 19~26 pieces, 19~29 pieces, 19~28 pieces, 19~29 pieces, 19~30 pieces, 20~21 pieces, 20~22 pieces, 20~23 pieces, 20~24 pieces, 20~25 pieces, 20~26 pieces, 20~27 pieces, 20~28 pieces, 20~29 pieces, 20~30 pieces, 21~22 pieces, 21~23 pieces, 21~24 pieces, 21~25 pieces, 21~26 pieces, 21~27 pieces,21~28 pieces, 21~29 pieces, 21~30 pieces, 22~23 pieces, 22~24 pieces, 22~25 pieces, 22~26 pieces, 22~27 pieces, 22~28 pieces, 22~29 pieces, 2 2~30 pieces, 23~24 pieces, 23~25 pieces, 23~26 pieces, 23~27 pieces, 23~28 pieces, 23~29 pieces, 23~30 pieces, 24~25 pieces, 24~26 pieces, 24~ It consists of 27, 24-28, 24-29, 24-30, 25-26, 25-27, 25-28, 25-29, 25-30, 26-27, 26-28, 26-29, 26-30, 27-28, 27-29, 27-30, 28-29, 28-30, or 29-30 linked nucleosides.

[0082] The ASOs described herein may have or contain lengths of 13 to 30 nucleotides. In one embodiment, the ASO may essentially consist of 13 to 30 nucleotides complementary to SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3.

[0083] In one embodiment, the ASO has at least 13 nucleotides, or at least 14 nucleotides, or at least 15 nucleotides, or at least 16 nucleotides, or at least 17 nucleotides, 18 nucleotides, or at least 19 nucleotides, or at least 20 nucleotides, or at least 21 nucleotides, or at least 22 nucleotides, or at least 23 nucleotides, or at least 24 nucleotides. In one embodiment, the ASO has less than 30 nucleotides, or less than 29 nucleotides, or less than 28 nucleotides, or less than 27 nucleotides, or less than 26 nucleotides, or less than 25 nucleotides, or less than 24 nucleotides, or less than 23 nucleotides, or less than 22 nucleotides, or less than 21 nucleotides, or less than 20 nucleotides. In one embodiment, ASO has 15-30 nucleotides, or 16-30 nucleotides, or 17-30 nucleotides, or 17-28 nucleotides, or 18-30 nucleotides, or 17-28 nucleotides, or 18-28 nucleotides, or 19-26 nucleotides, or 19-25 nucleotides, or 20-25 nucleotides, or 20-24 nucleotides, or 20-23 nucleotides, or 20-22 nucleotides, or 21-24 nucleotides, or 21-23 nucleotides, or 22-24 nucleotides. In one embodiment, ASO may have 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides.

[0084] In one embodiment, the ASO may have 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides complementary to SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3. In one embodiment, the ASO has a length of 13 to 30 nucleotides, and the ASO contains 13 to 30 nucleotides complementary to SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3.

[0085] In some embodiments, the ASO may include a nucleotide overhang (i.e., in the context of hybridizing to a target). The nucleotide overhang is part of the ASO that is not complementary to the target sequence (e.g., SEQ ID NO: 1). In some embodiments, the overhang may contain 1 to 3 nucleotides. In some embodiments, the overhang is located at the 3' end. In one embodiment, the ASO may not contain a nucleotide overhang, and for example, the entire ASO may be complementary to SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3.

[0086] array The ASO of the present invention comprises a nucleotide sequence of about 13 to about 30 nucleotides, the nucleotide sequence having sequence complementarity to SEQ ID NO: 1. As described in other parts of this specification, SEQ ID NO: 1 of the target sequence is also intended to include sequences in which one or more SNPs are located within the latent exons and / or flanking regions of rs12973192 and / or rs12608932. Coordination refers to the hg38 assembly.

[0087] In some embodiments, the ASO consists of a nucleotide sequence of 13 to 30 nucleotides, and the nucleotide sequence is sequence-complementary to SEQ ID NO: 1.

[0088] In one embodiment of the first and second aspects, the ASO can bind to the UNC13A latent exon or its flanking region. In one embodiment, the ASO (i.e., at least a portion of the ASO) includes a 13-nucleotide sequence corresponding to one or more of sequence numbers 4 to 546.

[0089] In one embodiment, the ASO (i.e., at least a portion of the ASO) includes at least 13 nucleotide sequences corresponding to one or more of sequence numbers 4-104. These ASOs can target branching points to reduce splicing efficiency.

[0090] In one embodiment, the ASO (i.e., at least a portion of the ASO) includes at least one 13-nucleotide sequence corresponding to one or more of the sequence numbers 105-189 or 270-352. These sequences correspond to sequences that target the UNC13A latent exon splice site (i.e., the acceptor and donor splice sites, respectively).

[0091] In one embodiment, the ASO (i.e., at least a portion of the ASO) includes at least one 13-nucleotide sequence corresponding to one or more of sequence numbers 105-189. These correspond to sequences that target the UNC13A latent exon 3' splice site (i.e., acceptor site). In some embodiments, the ASO includes at least one 13-nucleotide sequence corresponding to sequence numbers 150-185, more preferably sequence numbers 160-175. In one embodiment, the ASO (i.e., at least a portion of the ASO) includes at least one 13-nucleotide sequence corresponding to one or more of sequence numbers 270-352. These correspond to sequences that target the UNC13A latent exon 5' splice site. In some embodiments, the ASO includes at least one 13-nucleotide sequence corresponding to one or more of the following: SEQ ID NOs: 270-375, more preferably 275-340, more preferably 280-330, more preferably 285-325, more preferably 290-320, and more preferably 295-324.

[0092] In one embodiment, the ASO (i.e., at least a portion of the ASO) includes at least one 13-nucleotide sequence corresponding to one or more of the sequence numbers 190-269, 353-426, 427-474, or 475-546. These correspond to sequences that target the UNC13A splice control element.

[0093] In one embodiment, the SRE is a TDP-43 binding site. The ASO (i.e., at least a portion of the ASO) may contain a 13-nucleotide sequence corresponding to one or more of sequence numbers 427-474. In one embodiment, the SRE is an enhancer. The ASO may contain at least one 13-nucleotide sequence corresponding to one or more of sequence numbers 475-546.

[0094] In one embodiment, the SRE is located within a latent exon. In one embodiment, the ASO can bind to the UNC13A latent exon. In one embodiment, the UNC13A latent exon corresponds to SEQ ID NO: 2 or SEQ ID NO: 3. As described in other parts of this specification, the sequences of SEQ ID NOs: 2 and 3 are also intended to encompass a latent SNP within the latent exon of rs12973192. In one embodiment, the ASO is complementary to SEQ ID NO: 2 or SEQ ID NO: 3. In one embodiment, the ASO (i.e., or at least a portion thereof) includes at least one 13-nucleotide sequence corresponding to one or more of SEQ ID NOs: 190-269. These correspond to sequences that target a latent exon. Targeting a latent exon prevents the splicing mechanism from recognizing the CE.

[0095] In one embodiment, the SRE is an intronic SNP. In one embodiment, the ASO (i.e., at least a portion of the ASO) contains at least one 13-nucleotide sequence corresponding to one or more of sequence numbers 353-426. These correspond to sequences that target premRNA transcribed from sequences containing the intronic SNP. The intronic SNP is disease-linked. The intronic SNP is rs12608932.

[0096] In some embodiments, the ASO (i.e., at least a portion of the ASO) contains 13 nucleotides and can precisely correspond to one or more sequences of SEQ ID NOs. 4-546. In some embodiments, the ASO contains one or more additional nucleotides (1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, or 17 additional nucleotides) that flank one of the sequences of SEQ ID NOs. 4-546, for example, the ASO has 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides, respectively. The additional nucleotides may be located at either the 5' and / or 3' end of the 13-nucleotide sequence corresponding to SEQ ID NOs. 4-546.

[0097] In one embodiment, the ASO is longer than 13 nucleotides and contains two or more sequences complementary to SEQ ID NO: 1. In one embodiment, the ASO may contain 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, or 18 overlapping consecutive sequences selected from, for example, SEQ ID NOs: 4-104, 105-189, 190-269, 270-352, 353-426, or SEQ ID NOs: 427-474 or 475-546, where consecutive means the number of SEQ ID NOs. For example, an ASO containing 20 nucleotides may contain 8 consecutive sequences selected from SEQ ID NOs: 4-104 (for example, the ASO may contain SEQ ID NOs: 4, 5, 6, 7, 8, 9, 10, and 11). In some embodiments, the ASO includes sequence number n, sequence number n+1, possibly sequence number n+2, possibly sequence number n+3, possibly sequence number n+4, possibly sequence number n+5, possibly sequence number n+6, possibly sequence number n+7, possibly sequence number n+8, possibly sequence number n+9, possibly sequence number n+10, possibly sequence number n+11, possibly sequence number n+12, possibly sequence number n+13, possibly sequence number n+14, possibly sequence number n+15, possibly sequence number n+16, possibly sequence number n+17, and possibly sequence number n+18, where n is the sequence number.

[0098] In some embodiments, the ASO includes a nucleotide sequence that is sequence-complementary to one or more of sequence numbers 547-551 or one or more of sequence numbers 552-554.

[0099] qualification Modifications can be introduced into the polynucleotides (e.g., ASOs) described herein. The polynucleotides (e.g., ASOs) of this disclosure may contain one, two, three or more modifications. The modifications may be a variety of specific modifications. In some embodiments, modifications may be located in various regions and fragments of the polynucleotides of this disclosure, including but not limited to coding regions, untranslated regions, flanking regions and / or terminal or tail regions.

[0100] Modification, compared to unmodified polynucleotides (e.g., ASOs), makes nucleic acid molecules more resistant to degradation within cells and / or more stable within cells when introduced into cells. Modification can also increase the biological function of nucleic acid molecules compared to unmodified polynucleotides, such as binding to RBP or other polynucleotides (e.g., ASOs).

[0101] Modifications can be structural and / or chemical modifications. Chemical modifications can include nucleic acid base modifications and / or sugar modifications, as well as nucleotide and / or nucleoside modifications, including nucleoside linkage modifications (i.e., nucleoside linkages, e.g., linked phosphates, phosphodiester linkages, and phosphodiester skeletons). Structural modifications can include secondary and tertiary structural modifications.

[0102] The modifications described herein may involve the modification of ribonucleic acid (RNA) to deoxyribonucleic acid (DNA), threose nucleic acid (TNA), glycol nucleic acid (DNA), peptide nucleic acid (PNA), locked nucleic acid (LNA), or hybrids thereof.

[0103] In some embodiments, one, two, or more (depending on the context) nucleoside or nucleotide modifications can be incorporated into the polynucleotides (e.g., ASOs) of the Disclosure. Wherever used herein, “nucleoside” is defined as a compound containing a sugar molecule (e.g., pentose or ribose) or a derivative thereof in combination with an organic base (e.g., purine or pyrimidine) or a derivative thereof (also referred to herein as “nucleic acid base”). The five major / standard nucleic acid bases: adenine (A), cytosine (C), guanine (G), thymine (T), and uracil (U) are the basic units of nucleic acid molecules. Adenine and guanine are referred to as purine bases and have a fused ring skeleton structure derived from purine, while uracil and thymine, derived from pyrimidine, are referred to as pyrimidine bases. Where used herein, “nucleotide” is defined as a nucleoside containing a phosphate group or other skeletal linkages (internucleoside linkages).

[0104] In some embodiments, a polynucleotide (e.g., an ASO or antisense polynucleotide) includes at least one modification described herein. In other embodiments, a polynucleotide (e.g., an ASO) includes two, three, four or more (depending on the case) chemical modifications described herein. The modifications may be a combination of nucleic acid bases (purines and / or pyrimidines), sugars, and skeletal (internucleoside) linkage modifications. The modifications may be located on one or more nucleotides of the polynucleotide. In some embodiments, all nucleotides of the polynucleotide (e.g., an ASO) are chemically modified. In some embodiments, all nucleotides of a biologically functional nucleic acid sequence are chemically modified.

[0105] The polynucleotides (e.g., ASOs) of this disclosure contain modified nucleotides in amounts ranging from about 1% to about 100% (relative to the overall nucleotide content or relative to one or more types of nucleotides, i.e., one or more of A, G, T / U, or C), or any intermediate percentage (e.g., 1% to 20%, 1% to 25%, 1% to 50%, 1% to 60%, 1% to 70%, 1% to 80%, 1% to 90%, 1% to 95%, 10% to 20%, 10% to 25%, 10% to 50%, 10% to 60%, 10% to 70%, 10% to 80%, 10% to 90%, 10% to 10% It can be contained in amounts of 95%, 10%~100%, 20%~25%, 20%~50%, 20%~60%, 20%~70%, 20%~80%, 20%~90%, 20%~95%, 20%~100%, 50%~60%, 50%~70%, 50%~80%, 50%~90%, 50%~95%, 50%~100%, 70%~80%, 70%~90%, 70%~95%, 70%~100%, 80%~90%, 80%~95%, 80%~100%, 85%~95%, 85%~100%, 90%~95%, 90%~100%, and 95%~100%).

[0106] In some embodiments, polynucleotides (e.g., ASOs) are modified by at least 50%, for example, at least 50% of the nucleotides. In some embodiments, polynucleotides (e.g., ASOs) are modified by at least 75%, for example, at least 75% of the nucleotides. Since each nucleotide (sugar, base, and phosphate moiety, e.g., linkage) can be modified, it should be understood that any modification to any part of a nucleotide or nucleoside constitutes a modification.

[0107] In some embodiments, a polynucleotide (e.g., ASO) is modified by at least 10% of only one component of the nucleotide, such component being a linkage between nucleic acid bases, sugars, or nucleosides. For example, the modification may be carried out on at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% of the nucleic acid bases, sugars, or linkages of the polynucleotide (e.g., ASO) described herein.

[0108] Polynucleotides (e.g., ASOs) can be designed as patterned arrays of sugars, nucleic acid bases, or ligation modifications. In some embodiments, polynucleotides (e.g., ASOs) may include modifications to maximize stability.

[0109] Modification: sugar Modifications of modified nucleosides and nucleotides can be present in sugar subunits. In some embodiments, polynucleotides (e.g., ASOs) contain at least one sugar modification. Generally, RNA contains ribose, which is a sugar subunit and is a five-membered ring with oxygen.

[0110] For example, the 2'-hydroxyl group (OH) can be modified or replaced with a number of different substituents. Exemplary substitutions at the 2'OH position include H; halo; and optionally, substituted C. 1~6 Alkyl; optionally substituted C1-6 alkoxy; optionally substituted C 6~10 Aryloxy; C may be substituted. 3~8 Cycloalkyl; optionally substituted C 3~8 Cycloalkoxy; optionally substituted C 6~10 Aryloxy; C may be substituted. 6~10 Aryl-C 1~6 Alkoxy; C may be substituted. 1~12(Heterocyclyl)oxy; sugar (e.g., ribose, pentose or any as described herein); polyethylene glycol (PEG) -O(CH2CH2O)nCH2CH2OR (where R is H or optionally substituted alkyl and n is an integer from 0 to 20 (e.g., 0 to 4, 0 to 8, 0 to 10, 0 to 16, 1 to 4, 1 to 8, 1 to 10, 1 to 16, 1 to 20, 2 to 4, 2 to 8, 2 to 10, 2 to 16, 2 to 20, 4 to 8, 4 to 10, 4 to 16 and 4 to 20)); "locked" nucleic acid (LNA) (where the 2'-hydroxyl is connected to the 4'-carbon of the same ribose sugar by a C 1~6 alkylene or C 1~6 heteroalkylene bridge, and exemplary bridges include methylene, propylene, ether or amino bridges; aminoalkyl; aminoalkoxy; amino; and amino acids, but are not limited thereto.

[0111] Other exemplary sugar modifications include replacement of oxygen (O) in ribose (e.g., with S, Se, or alkylene, e.g., methylene or ethylene); addition of a double bond (e.g., to replace ribose with cyclopentenyl or cyclohexenyl); ring contraction of ribose (e.g., to form a 4-membered ring of cyclobutane or oxetane); ring expansion of ribose (e.g., a 6- or 7-membered ring having additional carbon or heteroatoms, e.g., anhydrohexitol, altritol, mannitol, cyclohexanyl, cyclohexenyl and morpholino also having a phosphoramidate backbone); polycyclic forms (e.g., tricyclic); and "unlocked" forms, e.g., glycol nucleic acid (GNA) (e.g., R-GNA or S-GNA, where ribose is replaced by a glycol unit linked by a phosphodiester bond), threose nucleic acid (TNA, where ribose is replaced by α-L-threofuranosyl-(3'→2')), and peptide nucleic acid (PNA, where the 2-amino-ethyl-glycine linkage replaces the ribose and phosphodiester backbone).

[0112] The sugar subunit may also contain one or more carbon atoms having the opposite stereochemical configuration to the corresponding carbon atoms of ribose. Thus, the antisense polynucleotides (e.g., ASOs) described herein may include nucleotides containing, for example, arabinose as the sugar.

[0113] In some embodiments, at least one of the 2' positions (OH in RNA or H in DNA) of the nucleotide sugars of a polynucleotide (e.g., ASO) is substituted with -O-methoxyethyl and is referred to as 2'-OMe. In some embodiments, at least one of the 2' positions (OH in RNA or H in DNA) of the nucleotide sugars of a polynucleotide (e.g., ASO) is substituted with -F- and is referred to as 2'-F. In some embodiments, the sugar modification may be one or more locked nucleic acids (LNAs). In some embodiments, the polynucleotide (e.g., ASO) may be a fully modified 2'-MOE sugar.

[0114] In certain embodiments, the modified sugar moiety is a non-bicyclic modified sugar moiety. In certain embodiments, the modified sugar moiety is a bicyclic or tricyclic sugar moiety. In certain embodiments, the modified sugar moiety is a sugar substitute. Such a sugar substitute may contain one or more substituents corresponding to other types of modified sugar moieties. In certain embodiments, the modified sugar moiety is a non-bicyclic modified sugar moiety comprising a furanosyl ring having one or more substituents (none of which bridge two atoms of the furanosyl ring to form a bicyclic structure). Such non-bridged substituents may be at any position of the furanosyl ring and include, but are not limited to, substituents at the 2', 4', and / or 5' positions. In certain embodiments, one or more non-bridged substituents of the non-bicyclic modified sugar moiety are branched. Suitable 2' substituents for the non-bicyclic modified sugar moiety include, but are not limited to, 2'-F, 2'-OCH3 ("OMe" or "O-methyl"), 2'-O(CH2)2OCH3 ("MOE" or "O-methoxyethyl"), 2'-ON-alkylacetamides, such as 2'-ON-methylacetamide ("NMA"), 2'-ON-dimethylacetamide, 2'-ON-ethylacetamide, or 2'-ON-propylacetamide. See, for example, U.S. Patent No. 6,147,200, Prakash et al., 2003, Org. Lett., 5, 403-406.

[0115] In a particular embodiment, the 2' substituent is halo, allyl, amino, azide, SH, CN, OCN, CF, OCF3, O-C1~C 10 Alkyl, O-C1~C10 substituted alkoxy, O-C1~C 10 Alkyl, O-C1~C 10A substituted alkyl, S-alkyl, N(Rm)-alkyl, O-alkenyl, S-alkenyl, N(Rm)-alkenyl, O-alkynyl, S-alkynyl, N(Rm)-alkynyl, O-alkylenyl-O-alkyl, alkynyl, alkaryl, aralkyl, O-alkaryl, O-aralkyl, O(CH2)2SCH3, O(CH2)2ON(Rm)(Rn) or OCH2C(=O)-N(Rm)(Rn) (wherein each Rm and Rn is independently H, an amino protecting group, or a substituted or unsubstituted C1-C10 alkyl), as well as a 2' substituent as described in Cook et al. U.S. Patent No. 6,531,584, Cook et al. U.S. Patent No. 5,859,221 and Cook et al. U.S. Patent No. 6,005,087. Certain embodiments of these 2' substituents may be further substituted with one or more substituents independently selected from hydroxyl, amino, alkoxy, carboxy, benzyl, phenyl, nitro(NO2), thiol, thioalkoxy, thioalkyl, halogen, alkyl, aryl, alkenyl, and alkynyl. Suitable 4' substituents for the non-bicyclic modified sugar moiety include, but are not limited to, alkoxy (e.g., methoxy), alkyl, and those described in Manoharan et al. International Publication 2015 / 106128. Suitable 5' substituents for the non-bicyclic modified sugar moiety include, but are not limited to, 5'-methyl(R or S), 5'-vinyl, and 5'-methoxy. In certain embodiments, the non-bicyclic modified sugar moiety comprises two or more non-crosslinked sugar substituents, for example, a 2'-F-5'-methyl sugar moiety and a modified sugar moiety, as well as modified nucleosides described in International Publication 2008 / 101157 by Migawa et al. and U.S. Patent Application 2013 / 0203836 by Rajeev et al.

[0116] In certain embodiments, the 2'-substituted non-bicyclic modified nucleoside comprises a sugar moiety containing a non-crosslinked 2' substituent selected from F, NH2, N3, OCF3OCH3, O(CH2)3NH2, CH2CH=CH2, OCH2CH=CH2, OCH2CH2OCH3, O(CH2)2SCH3, O(CH2)2ON(Rm)(Rn), O(CH2), ON(CH3)2, O(CH2)2O(CH2)2N(CH3)2, and N-substituted acetamides (OCH2C(=O)-N(Rm)(Rn)) (where each Rm and Rn is independently H, an amino protecting group, or a substituted or unsubstituted C1-C10 alkyl, e.g., OCH2C(=O)-N(H)CH3 ("NMA")).

[0117] In certain embodiments, the 2'-substituted non-bicyclic modified nucleosides are F, OCF3OCH3, OCH2CH2OCH3, O(CH2)2SCH3, O(CH2)2ON(CH3)2, O(CH2)2O(CH2)2N(CH3)2, and OCH2C(=O)-N(H)C H3 The sugar moiety comprises a non-crosslinked 2' substituent selected from ("NMA"). In certain embodiments, the 2'-substituted non-bicyclic modified nucleoside comprises a sugar moiety comprising a non-crosslinked 2' substituent selected from F, OCH, OCH2CH2OCH3 and OCH2C(=O)-N(H)CH3.

[0118] Certain modified sugar moieties include substituents that bridge two atoms of a furanosyl ring to form a second ring, resulting in a bicyclic sugar moiety. In certain such embodiments, the bicyclic sugar moiety includes a bridge between the 4' and 2' furanose ring atoms. Examples of such 4'-to-2' crosslinked sugar substituents include 4'-CH2-2', 4'-(CH2)2-2', 4'-(CH2)3-2', 4'-CH2-O-2' ("LNA"), 4'-CH2-S-2', 4'-(CH2)2-O-2' ("ENA"), 4'-CH(CH3)-O-2' (referred to as "restricted ethyl" or "cEt"), 4'-CH2-O-CH2-2', 4'-CH2-N(R)-2', 4'-CH(CH2OCH3)-O-2' ("restricted MOE" or "cMOE") and their analogues (e.g., U.S. Patent No. 7,399,845 by Seth et al., U.S. Patent No. 7,569,686 by Bhat et al., U.S. Patent No. 7,569,686 by Swayze et al.) See Japanese Patent No. 7,741,457 and U.S. Patent No. 8,022,193 by Swayze et al.), 4'-C(CH3)(CH3)-O-2' and its analogues (see, for example, U.S. Patent No. 8,278,283 by Seth et al.), 4'-CH2-N(OCH)-2' and its analogues (see, for example, U.S. Patent No. 8,278,425 by Prakash et al.), 4'-CH2-ON(CH3)-2' (see, for example, U.S. Patent No. 7,696,345 by Allerson et al. and U.S. Patent No. 8,124,745 by Allerson et al.), 4'-CH2-C(H)(CH3)-2' (see, for example, Zhou et al., J. Examples include, but are not limited to, Org. Chem., 2009, 74, 118-134, 4'-CH2-C(=CH2)-2' and its analogues (see, for example, U.S. Patent No. 8,278,426 by Seth et al.), 4,-C(RaRb)-N(R)-0-2', 4'-C(RaRb)-ON(R)-2', 4'-CH2-0-N(R)-2' and 4'-CH2-N(R)-0-2' (where R, Ra, and Rb are independently H, protecting groups, or C1-C12 alkyl groups) (see, for example, U.S. Patent No. 7,427,672 by Imanishi et al.).

[0119] In a particular embodiment, such a 4'-to-2' crosslink independently comprises 1 to 4 linking groups independently selected from -[C(Ra)(R)]n-, -[C(Ra)(Rb)]nO-, -C(Ra)=C(Rb)-, -C(Ra)=N-, -C(=NRa)-, -C(=O)-, -C(=S)-, -O-, -Si(Ra)2-, -S(=O)x- and -N(Ra)-, where x is 0, 1 or 2, and n is 1, 2, 3 or 4, and each Ra and Rb independently comprises H, a protecting group, hydroxyl, C1-C12 alkyl, substituted C1-C12 alkyl, C2-C12 alkenyl, substituted C2-C12 alkenyl, C2-C12 alkynyl, substituted C2-C12 alkynyl, C5-C20 aryl, substituted C5-C20 aryl The group consists of a heterocyclic group, a substituted heterocyclic group, a heteroaryl group, a substituted heteroaryl group, a C5-C7 alicyclic group, a substituted C5-C7 alicyclic group, a halogen, OJi, NJIJ2, SJi, N3, COOJi, an acyl (C(=O)-H), a substituted acyl, CN, a sulfonyl (S(=O)2-Ji), or a sulfoxyl (S(=O)-Ji), where each Ji and J2 is independently H, a C1-C12 alicyclic group. These are lucyl, substituted C1-C12 alkyl, C2-C12 alkenyl, substituted C2-C12 alkenyl, C2-C12 alkynyl, substituted C2-C12 alkynyl, C5-C20 aryl, substituted C5-C20 aryl, acyl (C(=O)-H), substituted acyl, heterocyclic group, substituted heterocyclic group, C1-C12 aminoalkyl, substituted C1-C12 aminoalkyl, or protecting group.

[0120] The additional bicyclic sugar moieties are known in the art, for example, Freier et al., Nucleic Acids Research, 1997, 25(22), 4429-4443; Albaek et al., J. Org. Chem., 2006, 71, 7731-7740; Singh et al., Chem. Commun., 1998, 4, 455-456; Koshkin et al., Tetrahedron, 1998, 54, 3607-3630; Kumar et al., Bioorg. Med. Chem. Lett., 1998, 8, 2219-2222; Singh et al., Org. Chem., 1998, 63, 10035-10039; Srivastava et al., J. Am. Chem. Soc., 2007, 129, 8362~8379; Wengel et al., U.S. Patent No. 7,053,207; Imanishi et al., U.S. Patent No. 6,268,490; Imanishi et al., U.S. Patent No. 6,770,748; Imanishi et al., U.S. Reissue Patent No. 44,779; Wengel et al., U.S. Patent No. 6,794,499; Wengel et al., U.S. Patent No. 6,670,461; Wengel et al., U.S. Patent No. 7,034,133; Wengel et al., U.S. Patent No. 8,080,644; Wengel et al., U.S. Patent No. 8,034,909; Wengel et al., U.S. Patent No. 8,153,365; Wengel et al., U.S. Patent No. 7,572,582; Ramasamy et al., U.S. Patent No. 6,525,191; Torsten et al., International Publication No. 2004 / 106356; Wengel et al., International Publication No. 1999 / 014226; Seth et al., International Publication No. 2007 / 134181; Seth et al., U.S. Patent No. 7,547,684; Seth et al., U.S. Patent No. 7,666,854; Seth et al., U.S. Patent No. 8,088,746; Seth et al., U.S. Patent No. 7,750,131; Seth et al., U.S. Patent No. 8,030,467; Seth et al., U.S. Patent No. 8,268,980; Seth et al., U.S. Patent No. 8,546,556; Seth et al., U.S. Patent No. 8,530,640; Migawa et al., U.S. Patent No. 9,012,421; Sethet et al., U.S. Patent No. 8,501,805; See also U.S. Patent Publication No. 2008 / 0039618 by Allerson et al. and U.S. Patent Publication No. 2015 / 0191727 by Migawa et al.

[0121] In certain embodiments, the bicyclic sugar moiety and the nucleoside incorporating such a bicyclic sugar moiety are further defined by their isomer configuration. For example, an LNA nucleoside (as described herein) may have an α-L configuration or a bD configuration.

[0122] LNA (β-D configuration), α-L-LNA (α-L configuration), cross-linked = 4'-CH2-O-2', cross-linked = 4'-CH2-O-2'. α-L-methyleneoxy(4'-CH2-O-2') or α-L-LNA bicyclic nucleosides are incorporated into oligonucleotides that exhibit antisense activity (Frieden et al., Nucleic Acids Research, 2003, 21, 6365-6372). Here, the general description of a bicyclic nucleoside includes both isomer configurations. Where the position of a particular bicyclic nucleoside (e.g., LNA or cEt) is identified in the exemplary embodiments herein, unless otherwise specified, these are the bD configuration.

[0123] In a particular embodiment, the modified sugar moiety comprises one or more non-crosslinked sugar substituents and one or more crosslinked sugar substituents (e.g., 5'-substituted and 4'-2'-crosslinked sugars).

[0124] In certain embodiments, the modified sugar moiety is a sugar substitute. In certain such embodiments, the oxygen atom of the sugar moiety is replaced with, for example, a sulfur, carbon, or nitrogen atom. In certain such embodiments, such a modified sugar moiety also includes the crosslinked and / or non-crosslinked substituents described herein. For example, certain sugar substitutes include a 4' sulfur atom and substitutions at the 2' position (see, for example, U.S. Patent No. 7,875,733 and U.S. Patent No. 7,939,677 by Bhat et al.) and / or at the 5' position.

[0125] In certain embodiments, the sugar substitute comprises a ring having more than five atoms. For example, in certain embodiments, the sugar substitute comprises a six-membered tetrahydropyran ("THP"). Such tetrahydropyrans may be further modified or substituted. Nucleosides containing such modified tetrahydropyrans include, but are not limited to, hexitol nucleic acids ("HNA"), anitol nucleic acids ("ANA"), mannitol nucleic acids ("MNA") (see, e.g., Leumann, CJ. Bioorg. & Med. Chem. 2002, 10, 841-854), fluoroHNA ("F-HNA," e.g., Swayze et al., U.S. Patent No. 8,088,904; Swayze et al., U.S. Patent No. 8,440,803; Swayze et al., U.S. Patent No. 8,796,437; and Swayze et al., U.S. Patent No. 9,005,906; F-HNA may also be referred to as F-THP or 3'-fluorotetrahydropyran), and nucleosides containing additional modified THP compounds.

[0126] In certain embodiments, the sugar substitute comprises a ring having more than five atoms and two or more heteroatoms. For example, their use in nucleosides and oligonucleotides containing a morpholino sugar moiety has been reported (see, e.g., Braasch et al., Biochemistry, 2002, 41, 4503-4510, and U.S. Patents 5,698,685, 5,166,315, 5,185,444, and 5,034,506 by Summerton et al.). As used herein, the term “morpholino” means a sugar substitute having the following structure:

[0127] In certain embodiments, morpholino can be modified, for example, by adding or altering various substituents from the morpholino structure described above. Such sugar substitutes are referred to herein as “modified morpholino.”

[0128] In certain embodiments, the sugar substitute includes an acyclic moiety. Examples of nucleosides and oligonucleotides containing such acyclic sugar substitutes include, but are not limited to, peptide nucleic acids ("PNA"), acyclic butyl nucleic acids (see, e.g., Kumar et al., Org. Biomol. Chem., 2013, 11, 5853-5865), and nucleosides and oligonucleotides described in International Publication No. 2011 / 133876 by Manoharan et al.

[0129] Many other bicyclic and tricyclic sugars and sugar substitute ring systems that can be used as modified nucleosides are known in the art.

[0130] Modification: Nucleoside In some embodiments, the ASO comprises an unmodified nucleoside, such as adenine, guanine, uracil, thymine, or cytosine.

[0131] In one embodiment, ASO may include modified variants of nucleosides (i.e., provided that Watson-Crick base pairing of the base nucleoside is unaffected). In one embodiment, ASO may include modified A, modified C, modified G, or modified U. In one embodiment, modified ASO may include, but is not limited to, substitution with modified C, e.g., 5-methylcytosine or 5-hydroxymethylcytosine, modified U, e.g., 5-methyluridine or thymine, or modified A, e.g., N6-methyladenine. In one embodiment, ASO may include unmodified and a mixture of v-nucleosides. While unmodified nucleosides are shown in SEQ ID NOs. 4-546 described herein, the present invention also encompasses modified variants of nucleosides.

[0132] In certain embodiments, the oligonucleotide comprises one or more nucleosides containing unmodified nucleic acid bases. In certain embodiments, the modified oligonucleotide comprises one or more nucleosides containing modified nucleic acid bases. In certain embodiments, the modified oligonucleotide comprises one or more nucleosides that do not contain nucleic acid bases and are referred to as debased nucleosides.

[0133] The polynucleotides of this disclosure (e.g., ASOs) may include nucleoside modifications. One or more atoms of the pyrimidine nucleic acid bases may be replaced or substituted with, for example, optionally substituted aminos, optionally substituted thiols, optionally substituted alkyls (e.g., methyl or ethyl), optionally substituted or halo (chloro or fluoro) atoms or groups.

[0134] As a non-limiting example, all uracil nucleosides of the polynucleotides (e.g., ASOs) of this disclosure are modified. The modifications may be identical or different. In some embodiments, all guanine nucleosides of the polynucleotides (e.g., ASOs) of this disclosure are modified. The modifications may be identical or different. In some embodiments, all guanine nucleosides of the polynucleotides (e.g., ASOs) of this disclosure are modified. The modifications may be identical or different. In some embodiments, all cytosine nucleosides of the polynucleotides (e.g., ASOs) of this disclosure are modified. The modifications may be identical or different. In some embodiments, all adenine nucleosides of the polynucleotides (e.g., ASOs) of this disclosure are modified. The modifications may be identical or different.

[0135] Modification: Nucleic acid bases Modified nucleosides and nucleotides may contain modified nucleic acid bases. Examples of RNA nucleic acid bases include, but are not limited to, adenine (A), guanine (G), cytosine (C), and uracil (U). Examples of DNA nucleic acid bases include, but are not limited to, adenine (A), guanine (G), cytosine (C), and thymine (T).

[0136] In some embodiments, the modified nucleic acid base is modified uracil (U).

[0137] Exemplary nucleic acid bases and nucleosides having modified uracil include pseudouridine (ψ), pyridine-4-onribonucleoside, 5-aza-uridine, 6-aza-uridine, 2-thio-5-aza-uridine, and 2-thiouridine (s 2 U), 4-thio-uridine (s 4 U), 4-thiopsoiduridine, 2-thiopsoiduridine, 5-hydroxyuridine (ho 5 U), 5-aminoallyl-uridine, 5-halo-uridine (eg), 5-iodo-uridine (I 5 U) or 5-bromo-uridine (br 5 U)), 3-methyluridine(m 3 U), 5-methoxyuridine (mo 5 U), Uridine 5-oxyacetic acid (cmo 5 U), Uridine 5-oxyacetate methyl ester (mcmo 5 U), 5-carboxymethyl-uridine (cm 5 U), 1-carboxymethyl-psoidouridine, 5-carboxyhydroxymethyl-uridine (chm 5 U), 5-carboxyhydroxymethyl-uridinemethyl ester (mchm 5 U), 5-methoxycarbonylmethyluridine (mcm 5 U), 5-methoxycarbonylmethyl-2-thiouridine (mcm 5 s 2 U), 5-aminomethyl-2-thiouridine (nm 5 s 2U), 5-methylaminomethyluridine (mnm 5 U), 5-methylaminomethyl-2-thiouridine (mnm 5 s 2 U), 5-methylaminomethyl-2-seleno-uridine (mnm 5 se 2 U), 5-Carbamoylmethyluridine (ncm 5 U), 5-carboxymethylaminomethyluridine (cmnm 5 U), 5-carboxymethylaminomethyl-2-thiouridine (cmnm 5 s 2 U), 5-propynyl-uridine, 1-propynyl-psoidouridine, 5-taurinomethyl-uridine (τm 5 U), 1-taurinomethyl-psoidouridine, 5-taurinomethyl-2-thiouridine (τm 5 s 2 U), 1-taurinomethyl-4-thiopsoiduridine, 5-methyluridine (m 5 U, i.e., having the nucleic acid base deoxythymine), 1-methylpsoiduridine (m 1 ψ), 5-methyl-2-thiouridine (m 5 s 2 U), pseudouracil (ψ), 1-methyl-4-thio-psoidouridine (m 1 s 4 ψ), 4-thio-1-methyl-psoidouridine, 3-methyl-psoidouridine (m 3 ψ), 2-thio-1-methyl-psoidouridine, 1-methyl-1-deaz-psoidouridine, 2-thio-1-methyl-1-deaz-psoidouridine, dihydrouridine(D), dihydropsoidouridine, 5,6-dihydrouridine, 5-methyl-dihydrouridine(m 5 D) 2-thio-dihydrouridine, 2-thio-dihydropsoiduridine, 2-methoxy-uridine, 2-methoxy-4-thiouridine, 4-methoxy-psoiduridine, 4-methoxy-2-thiopsoiduridine, N1-methylpsoiduridine (also known as 1-methylpsoiduridine (m 1ψ)), 3-(3-amino-3-carboxypropyl)uridine (acp 3 U), 1-methyl-3-(3-amino-3-carboxypropyl)psoidouridine (acp 3 ψ), 5-(isopentenylaminomethyl)uridine (inm 5 U), 5-(isopentenylaminomethyl)-2-thiouridine(inm 5 s 2 U), α-thio-uridine, 2'-O-methyl-uridine (Um), 5,2'-O-dimethyl-uridine (m 5 Um), 2'-O-methylpsoidouridine(ψm), 2-thio-2'-O-methyluridine(s 2 Um), 5-methoxycarbonylmethyl-2'-O-methyluridine (mcm 5 Um), 5-Carbamoylmethyl-2'-O-methyluridine (ncm 5 Um), 5-carboxymethylaminomethyl-2'-O-methyluridine (cmnm 5 Um), 3,2'-O-dimethyluridine (m 3 Um), 5-(isopentenylaminomethyl)-2'-O-methyluridine(inm 5 Examples include Um), 1-thiouridine, deoxythymidine, 2'-F-alar-uridine, 2'-F-uridine, 2'-OH-alar-uridine, 5-(2-carbomethoxyvinyl)uridine, and 5-[3-(1-E-propenylamino)uridine.

[0138] In some embodiments, the modified nucleic acid base is modified cytosine (C).

[0139] Exemplary nucleic acid bases and nucleosides having modified cytosine include 5-aza-cytidine, 6-aza-cytidine, pseudoisocytidine, and 3-methylcytidine (m 3 C), N4-acetylcytidine (ac 4 C), 5-formyl-cytidine (f 5 C), N4-methylcytidine (m 4 C), 5-methylcytidine (m 5C), 5-halo-cytidine (e.g., 5-iodocytidine), 5-hydroxymethylcytidine (hm 5 C) 1-methyl-psoidisocytidine, pyrrolo-cytidine, pyrrolo-psoidisocytidine, 2-thio-cytidine (s 2 C) 2-thio-5-methylcytidine, 4-thio-psoidisocytidine, 4-thio-1-methyl-psoidisocytidine, 4-thio-1-methyl-1-deaz-psoidisocytidine, 1-methyl-1-deaz-psoidisocytidine, zebralin, 5-aza-zebralin, 5-methyl-zebralin, 5-aza-2-thio-zebralin, 2-thio-zebralin, 2-methoxycytidine, 2-methoxy-5-methylcytidine, 4-methoxy-psoidisocytidine, 4-methoxy-1-methyl-psoidisocytidine, lysidine (k 2 C), α-thiocytidine, 2'-O-methylcytidine (Cm), 5,2'-O-dimethylcytidine (m 5 Cm), N4-acetyl-2'-O-methylcytidine (ac 4 Cm), N4,2'-O-dimethylcytidine (m 4 Cm), 5-formyl-2'-O-methylcytidine (f 5 Cm), N4,N4,2'-O-trimethylcytidine (m 4 Examples include 2Cm), 1-thiocytidine, 2'-F-alacytidine, 2'-F-cytidine, and 2'-OH-alacytidine.

[0140] In some embodiments, the modified nucleic acid base is modified adenine (A).

[0141] Exemplary nucleobases and nucleosides having modified adenine include 2 - amino - purine, 2,6 - diamino purine, 2 - amino - 6 - halo - purine (e.g., 2 - amino - 6 - chloro - purine), 6 - halo - purine (e.g., 6 - chloro - purine), 2 - amino - 6 - methyl - purine, 8 - azido - adenosine, 7 - deaza - adenine, 7 - deaza - 8 - aza - adenine, 7 - deaza - 2 - amino - purine, 7 - deaza - 8 - aza - 2 - amino - purine, 7 - deaza - 2,6 - diamino purine, 7 - deaza - 8 - aza - 2,6 - diamino purine, 1 - methyl - adenosine (m 1 A), 2 - methyl - adenosine (m 2 A), N6 - methyl - adenosine (m 6 A), 2 - methylthio - N6 - methyl - adenosine (ms 2 m 6 A), N6 - isopentenyl - adenosine (i 6 A), 2 - methylthio - N6 - isopentenyl - adenosine (ms 2 i 6 A), N6 - (cis - hydroxyisopentenyl) adenosine (io 6 A), 2 - methylthio - N6 - (cis - hydroxyisopentenyl) adenosine (ms 2 io 6 A), N6 - glycylcarbamoyl - adenosine (g 6 A), N6 - threonylcarbamoyl - adenosine (t 6 A), N6 - methyl - N6 - threonylcarbamoyl - adenosine (m 6 t 6 A), 2 - methylthio - N6 - threonylcarbamoyl - adenosine (ms 2 g 6 A), N6,N6 - dimethyl - adenosine (m 6 2A), N6 - hydroxynorvalylcarbamoyl - adenosine (hn 6 A), 2 - methylthio - N6 - hydroxynorvalylcarbamoyl - adenosine (ms 2 hn 6 A), N6 - acetyl - adenosine (ac 6A) 7-methyl-adenine, 2-methylthio-adenine, 2-methoxy-adenine, α-thio-adenosine, 2'-O-methyl-adenosine (Am), N6,2'-O-dimethyl-adenosine (m 6 Am), N6,N6,2'-O-trimethyl-adenosine (m 6 2Am), 1,2'-O-dimethyl-adenosine (m 1 Examples include Am), 2'-O-ribosyladenosine (phosphate) (Ar(p)), 2-amino-N6-methyl-purine, 1-thio-adenosine, 8-azido-adenosine, 2'-F-ala-adenosine, 2'-F-adenosine, 2'-OH-ala-adenosine, and N6-(19-amino-pentaoxanonadecyl)-adenosine.

[0142] In some embodiments, the modified nucleic acid base is modified guanine (G).

[0143] Examples of nucleic acid bases and nucleosides having modified guanine include inosine(I) and 1-methyl-inosine(m). 1 I) Wyosin (imG), Methyl Wyosin (mimG), 4-Dimethyl Wyosin (imG-14), Iso Wyosin (imG2), Wybutosin (yW), Peroxywybutosin (o2yW), Hydroxywybutosin (OHyW), Unmodified Hydroxywybutosin (OHyW*), 7-Deaza-Guanosine, Quosin (Q), EpoxyQuosin (oQ), Galactosyl Quosin (galQ), Mannosyl Quosin (manQ), 7-Cyano-7-Deaza-Guanosine (preQ0), 7-Aminomethyl-7-Deaza-Guanosine (preQ1), Alkaeosin (G + ), 7-deaza-8-aza-guanosine, 6-thio-guanosine, 6-thio-7-deaza-guanosine, 6-thio-7-deaza-8-aza-guanosine, 7-methyl-guanosine (m 7 G), 6-thio-7-methyl-guanosine, 7-methyl-inosine, 6-methoxy-guanosine, 1-methyl-guanosine (m 1 G), N2-methyl-guanosine (m 2G), N2,N2-dimethyl-guanosine (m 2 2G), N2,7-dimethyl-guanosine (m 2,7 G), N2, N2,7-dimethyl-guanosine (m 2,2,7 G), 8-oxo-guanosine, 7-methyl-8-oxo-guanosine, 1-methyl-6-thio-guanosine, N2-methyl-6-thio-guanosine, N2,N2-dimethyl-6-thio-guanosine, α-thio-guanosine, 2'-O-methyl-guanosine (Gm), N2-methyl-2'-O-methyl-guanosine (m 2 Gm), N2,N2-dimethyl-2'-O-methyl-guanosine (m 2 2Gm), 1-methyl-2'-O-methyl-guanosine (m 1 Gm), N2,7-dimethyl-2'-O-methyl-guanosine (m 2,7 Gm), 2'-O-methyl-inosine (Im), 1,2'-O-dimethyl-inosine (m 1 Examples include Im) and 2'-O-ribosylguanosine (phosphate) (Gr(p)).

[0144] In some embodiments, the nucleic acid salt of the nucleotide may be independently selected from purines, pyrimidines, purines, or pyrimidine analogs. Nucleic acid bases and / or analogs include pyrazolo[3,4-d]pyrimidine, 5-methylcytosine (5-me-C), 5-hydroxymethylcytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-methyl and other alkyl derivatives of adenine and guanine, 2-propyl and other alkyl derivatives of adenine and guanine, 2-thiouracil, 2-thiothymine and 2-thiocytosine, 5-propynyluracil and cytosine, 6-azouracil, cytosine and thymine, 5-uracil (pseudouracil), 4-thiouracil, 8-halo (e.g., 8-bromo), 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl and other 8-substituted adenine and guanine, 5-halo, especially 5-bromo, 5-triph Examples include, but are not limited to, adenines, cytosines and other 5-substituted uracils and cytosines, 7-methylguanine and 7-methyladenine, 8-azaguanine and 8-azaadenine, deazaguanine, 7-deazaguanine, 3-deazaguanine, deazaadenine, 7-deazaadenine, 3-deazaadenine, pyrazolo[3,4-d]pyrimidine, imidazo[1,5-a]1,3,5-triazinon, 9-deazapurine, imidazo[4,5-d]pyrazine, thiazolo[4,5-d]pyrimidine, pyrazine-2-one, 1,2,4-triazine, pyridazine, and 1,3,5-triazine, which can be independently selected from natural and synthetic derivatives of adenines, cytosines, guanine, thymine, uracil, and bases.

[0145] In certain embodiments, the modified nucleic acid bases are selected individually or in combination from 5-substituted pyrimidines, 6-azapyrimidines, alkyl or alkynyl-substituted pyrimidines, alkyl-substituted purines, and N-2, N-6, and O-6-substituted purines. In certain embodiments, the modified nucleic acid bases are 2-aminopropyladenine, 5-hydroxymethylcytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-N-methylguanine, 6-N-methyladenine, 2-propyladenine, 2-thiouracil, 2-thiothymine and 2-thiocytosine, 5-propynyl(-C≡C-CH)uracil, 5-propynylcytosine, 6-azouracil, 6-azocytosine, 6-azocymine, 5-ribosyluracil (pseudouracil), 4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl, 8-az, and others Selected from 8-substituted purines, 5-halos, particularly 5-bromo, 5-trifluoromethyl, 5-halouracil and 5-halocytosine, 7-methylguanine, 7-methyladenine, 2-F-adenine, 2-aminoadenine, 7-deazaguanine, 7-deazaadenine, 3-deazaguanine, 3-deazaadenine, 6-N-benzoyladenine, 2-N-isobutyrylguanine, 4-N-benzoylcytosine, 4-N-benzoyluracil, 5-methyl4-N-benzoylcytosine, 5-methyl4-N-benzoyluracil, universal bases, hydrophobic bases, random bases, size-expanding bases, and fluorinated bases. Further modified nucleic acid bases include tricyclic pyrimidines, such as 1,3-diazaphenoxadin-2-one, 1,3-diazaphenothiazine-2-one, and 9-(2-aminoethoxy)-1,3-diazaphenoxadin-2-one (G-clamp). Modified nucleic acid bases may also include those in which a purine or pyrimidine base is replaced by another heterocycle, such as 7-deazaadenine, 7-deazaguanosine, 2-aminopyridine, and 2-pyridone.Further nucleic acid bases include those disclosed in U.S. Patent No. 3,687,808 by Merigan et al., The Concise Encyclopedia Of Polymer Science And Engineering, Kroschwitz, JI, Ed., John Wiley & Sons, 1990, 858-859; Englisch et al., Angewandte Chemie, International Edition, 1991, 30, 613; Sanghvi, YS, Chapter 15, Antisense Research and Applications, Crooke, ST and Lebleu, B., Eds., CRC Press, 1993, 273-288; and Chapters 6 and 15, Antisense Drug Technology, Crooke ST, Ed., CRC Press, 2008, 163-166 and 442-443.

[0146] The publications teaching the preparation of the modified nucleic acid bases shown above, as well as specific other modified nucleic acid bases, include, but are not limited to, U.S. Patent Application No. 2003 / 0158403 by Manoharan et al.; U.S. Patent Application No. 2003 / 0175906 by Manoharan et al.; U.S. Patent No. 4,845,205 by Dinh et al.; U.S. Patent No. 5,130,302 by Spielvogel et al.; U.S. Patent No. 5,134,066 by Rogers et al.; U.S. Patent No. 5,175,273 by Bischofberger et al.; U.S. Patent No. 5,367,066 by Urdea et al.; U.S. Patent No. 5,432,272 by Benner et al.; U.S. Patent No. 5,434,257 by Matteucci et al.; U.S. Patent No. 5,457,187 by Gmeiner et al.; U.S. Patent No. 5,459,255 by Cook et al.; Froehler et al., U.S. Patent No. 5,484,908; Matteucci et al., U.S. Patent No. 5,502,177; Hawkins et al., U.S. Patent No. 5,525,711; Haralambidis et al., U.S. Patent No. 5,552,540; Cook et al., U.S. Patent No. 5,587,469; Froehler et al., U.S. Patent No. 5,594,121; Switzer et al., U.S. Patent No. 5,596,091; Cook et al., U.S. Patent No. 5,614,617; Froehler et al., U.S. Patent No. 5,645,985; Cook et al., U.S. Patent No. 5,681,941; Cook et al., U.S. Patent No. 5,811,534; Cook et al., U.S. Patent No. 5,750,692; Cook et al., U.S. Patent No. 5,948,903; Cook et al., U.S. Patent No. 5,587,470; Cook et al., U.S. Patent No. 5,457,191; Examples include U.S. Patent No. 5,763,588 by Matteucci et al.; U.S. Patent No. 5,830,653 by Froehler et al.; U.S. Patent No. 5,808,027 by Cook et al.; U.S. Patent No. 6,166,199 by Cook et al.; and U.S. Patent No. 6,005,096 by Matteucci et al.

[0147] Modification: Internucleoside linking In certain embodiments, the nucleosides of modified oligonucleotides may be linked using any internucleoside linkages. Two main classes of nucleoside linkages are defined by the presence or absence of a phosphorus atom. Representative phosphorus-containing internucleoside linkages include, but are not limited to, phosphodiesters containing a phosphodiester linkage P(O2)=O (also referred to as unmodified or naturally occurring linkages); phosphotriesters; methylphosphonates; methoxypropylphosphonates ("MOP"); phosphoramidates; mesylphosphoramidates; phosphorothioates (P(O2)=S); and phosphorodithioates (HS-P=S). Representative phosphorus-free internucleoside linking groups include, but are not limited to, methylenemethylimino (-CH2-N(CH)-0-CH2-); thiodiesters; thionocarbamates (-OC(=O)(NH)-S-); siloxanes (-O-SiH2-O-); and N,N'-dimethylhydrazine (-CH2-N(CH3)-N(CH3)-). Compared to naturally occurring phosphate linkages, modified internucleoside linkages can be used to alter, typically increase, the nuclease resistance of oligonucleotides. In certain embodiments, internucleoside linkages having chiral atoms can be prepared as racemic mixtures or separate enantiomers. Methods for preparing phosphorus-containing and phosphorus-free internucleoside linkages are well known to those skilled in the art.

[0148] Representative nucleoside linkages having a chiral center include, but are not limited to, alkylphosphonates and phosphorothioates. Modified oligonucleotides containing nucleoside linkages having a chiral center can be prepared as a group of modified oligonucleotides containing stereodisordered nucleoside linkages, or in particular as a group of modified oligonucleotides containing phosphorothioate nucleoside linkages in a specific stereochemical configuration. In a particular embodiment, the group of modified oligonucleotides contains phosphorothioate nucleoside linkages in which all of the phosphorothioate nucleoside linkages are stereodisordered. Such modified oligonucleotides can be produced using a synthetic method that results in a random selection of the stereochemical configuration of each phosphorothioate nucleoside linkage. In any case, as will be well understood by those skilled in the art, each individual phosphorothioate of each individual oligonucleotide molecule has a defined stereochemical configuration. In a particular embodiment, the group of modified oligonucleotides is concentrated with modified oligonucleotides containing one or more phosphorothioate nucleoside linkages in a particular stereochemical configuration, in particular independently selected. In a particular embodiment, a specific phosphorothioate nucleoside linkage in a particular configuration is present in at least 65% of the molecules in the population. In a particular embodiment, a specific phosphorothioate nucleoside linkage in a particular configuration is present in at least 70% of the molecules in the population. In a particular embodiment, a specific phosphorothioate nucleoside linkage in a particular configuration is present in at least 80% of the molecules in the population. In a particular embodiment, a specific phosphorothioate nucleoside linkage in a particular configuration is present in at least 90% of the molecules in the population. In a particular embodiment, a specific phosphorothioate nucleoside linkage in a particular configuration is present in at least 99% of the molecules in the population. Such chiral enrichments of modified oligonucleotides can be produced using synthetic methods known in the art, for example, the methods described by Oka et al., JACS, 2003, 125, 8307, Wan et al., Nuc. Acid. Res., 2014, 42, 13456, and International Publication No. 2017 / 015555.In a particular embodiment, the population of modified oligonucleotides is enriched with modified oligonucleotides having at least one designated phosphorothioate in the (Sp) configuration. In a particular embodiment, the population of modified oligonucleotides is enriched with modified oligonucleotides having at least one phosphorothioate in the (Rp) configuration.

[0149] Unless otherwise specified, the chiral nucleoside linkages of the modified oligonucleotides described herein may be stereodisordered or, in particular, have a specific stereochemical configuration.

[0150] Examples of neutral nucleoside linkages include, but are not limited to, phosphotriesters, methylphosphonates, MMI (3'-CH2-N(CH3)-O-5'), amide-3 (3'-CH2-C(=O)-N(H)-5'), amide-4 (3'-CH2-N(H)-C(=O)-5'), formacetal (3'-O-CH2-O-5'), methoxypropyl, and thioformacetal (3'-S-CH2-O-5'). Further neutral nucleoside linkages include nonionic linkages containing siloxane (dialkylsiloxane) carboxylic acid esters, carboxamides, sulfides, sulfonic acid esters, and amides (see, for example, *Carbohydrate Modifications in Antisense Research*, YS Sanghvi and PD Cook, Eds., ACS Symposium Series 580; Chapters 3 and 4, 40-65). Further neutral nucleoside linkages include nonionic linkages containing mixed N, O, S, and CH2 component moieties.

[0151] In certain embodiments, the modified nucleoside linkages are any of those described in International Publication No. 2021 / 030778, which is incorporated herein by reference.

[0152] Modification: Skeletal phosphate group The skeletal phosphate group can be modified by replacing one or more oxygen atoms with different substituents. Furthermore, modified nucleosides and nucleotides may include large-scale replacement of the unmodified phosphate moiety by other internucleoside linkages as described herein. Examples of modified phosphate groups include, but are not limited to, phosphorothioates, methylphosphonates, phosphoroselenates, boranophosphates, boranophosphate esters, hydrogen phosphonates, phosphoramides, phosphorodiamidates, alkyl or arylphosphonates, and phosphotryesters. In phosphorodithioates, both unlinked oxygen atoms are replaced by sulfur. Phosphate linkers can also be modified by replacing the linked oxygen atoms with nitrogen (bridged phosphoramide), sulfur (bridged phosphorothioate), and carbon (bridged methylenephosphonate).

[0153] The α-thio-substituted phosphate moiety is provided to confer stability to RNA and DNA polynucleotides via non-natural phosphorothioate backbone linking. Phosphothioate-linked DNA and RNA exhibit increased nuclease resistance and subsequently a longer half-life in the cellular environment. Phosphothioate-linked polynucleotide molecules are also expected to reduce the innate immune response through weak binding / activation of cellular innate immune molecules.

[0154] In some embodiments, the polynucleotides of the Disclosure (e.g., ASOs) include modifications such as at least one phosphorothioate linkage, methylphosphonate linkages between nucleotides, 5'-(E)-vinylphosphonate (5'-EP), and phosphate mimetic compounds.

[0155] The ASOs defined herein may have any suitable backbone (i.e., any suitable nucleoside linkage and / or any suitable nucleoside, where the nucleoside may have any suitable sugar and / or any suitable nucleic acid base). In one embodiment, the ASOs defined herein are resistant to RNase H cleavage. In one embodiment, the AO may be a sterically barrierped ASO. In one embodiment, if the ASO binds to an important element or target sequence, it may be resistant to cleavage and / or an inadequate substrate for RNase H. In one embodiment, the ASO does not cause degradation of UNC13A premRNA or mRNA.

[0156] In one embodiment, the backbone is formed from RNA (alternating phosphate and ribose), LNA (locked nucleic acid), tcDNA (tricycloDNA), cEt (restricted ethyl crosslinked nucleic acid), ENA (ethylene crosslinked nucleic acid), HNA (hexitol nucleic acid), TNA (threose nucleic acid), PMO (phosphodiamide morpholino oligomer) PMO, PNA (peptide nucleic acid), 2-OMe-RNA, 2'-O-methoxyethyl (MOE) nucleic acid or 2-O-(2-methylcarbamoyl (MCE)) nucleic acid, or any combination thereof. The ASO is a portion of the DNA nucleotide, for example, at least 10%, or at least 20%, or at least 30%, or at least 40%, or at least 50%, or at least 60%, or 70% of the DNA nucleotide, i.e., LNA, tcDNA, cET, ENA, The ASO further comprises combinations with HNA, TNA, PMO, PNA, 2'-OMe-RNA, 2'-O-methoxyethyl (MOE) nucleic acid, or MCE nucleotides. The skeleton may consist entirely of one nucleotide or a mixture of one or more nucleotides. In one embodiment, the ASO has a crosslinked nucleic acid (i.e., locked or restricted) skeleton, such as LNA, cET, or ENA skeletons. These skeletons may have good stability, a high binding constant to RNA, and / or resistance to RNase H cleavage. In some embodiments, the ASO contains phosphate (i.e., phosphodiester) nucleotide linkages. In some embodiments, the ASO contains phosphorodiamidate nucleotide linkages. In other embodiments, the ASO contains phosphorothioate nucleotide linkages (i.e., known as also known as PS-ASO).

[0157] In some embodiments, ASO comprises LNA, i.e., LNA and DNA, and more specifically, phosphorothioate LNA and DNA. In some examples disclosed herein, ASO comprises LNA or 2-OMe-RNA.

[0158] In some examples, the ASO contains only a portion of the crosslinked nucleic acid, e.g., LNA. In some examples, the ASO contains about 20-60% crosslinked nucleic acid (e.g., LNA), or preferably about 30-50% crosslinked nucleic acid (e.g., LNA). In some embodiments, the ASO having an LNA backbone contains crosslinked nucleic acid (e.g., LNA bases) in combination with DNA bases. In some examples, the ASO contains at least 20% crosslinked nucleic acid (e.g., LNA), or at least 30% or at least 40% crosslinked nucleic acid (e.g., LNA). In some examples, the ASO contains less than 100% crosslinked nucleic acid (e.g., LNA), or less than 90%, or less than 80%, or less than 70%, or less than 60%, or less than 50% crosslinked nucleic acid (e.g., LNA). In some embodiments, the ASO contains crosslinked nucleic acid (e.g., LNA) every three or more nucleotides, or every two or more nucleotides.

[0159] In some cases, ASO contains 2-OMe-RNA. In some cases, ASO contains 100% 2-OMe-RNA, more specifically 100% phosphorothioate-modified 2-OMe-RNA. In some cases, ASO contains only a portion of 2-OMe-RNA.

[0160] In one embodiment, the ASO has an RNA backbone. The ASO sequences / parts of ASO sequences provided in the sequence listings herein have an RNA backbone, but any other suitable backbone may be used, provided that the nucleoside sequences are the same (as described in other parts of this specification, in the ASOs disclosed herein, the "U" and "T" nucleosides, e.g., uracil or thymine, can be used interchangeably. Thus, in any of the ASO sequences described herein (e.g., including SEQ ID NOs. 4 to 546), "U" can be replaced with "T").

[0161] Modification: Motif Different sugar modifications, nucleic acid base modifications, and / or internucleoside linkages (e.g., skeletal structures) can be introduced at various positions of the polynucleotides (e.g., ASOs) described herein. Those skilled in the art will understand that nucleotide analogs or other modifications can be placed at any position of the polynucleotide (e.g., ASO) such that the function of the polynucleotide (e.g., ASO) is not substantially diminished.

[0162] Modification: Sugar motif In certain embodiments, the oligonucleotide comprises one or more types of modified and unmodified sugar moieties arranged in a definite pattern or sugar motif along the oligonucleotide or a portion thereof. In certain cases, such sugar motifs include, but are not limited to, any of the sugar modifications discussed herein.

[0163] Certain modified oligonucleotides have a gapmer motif, which is defined by two outer regions or "wings" and a central or inner region or "gap". The three regions of the gapmer motif (5' wing, gap, and 3' wing) form a contiguous sequence of nucleosides, where at least a portion of the sugar moiety of each wing's nucleoside is different from at least a portion of the sugar moiety of the gap's nucleoside. Specifically, at least the sugar moiety of each wing's nucleoside closest to the gap (the nucleoside of the 5' wing closest to 3' and the nucleoside of the 3' wing closest to 5') is different from the sugar moiety of the adjacent gap nucleotide, thus defining the wing-gap boundary (i.e., the wing / gap junction). In certain embodiments, the sugar moieties within the gap are identical to each other. In certain embodiments, the gap contains one or more nucleosides having sugar moieties different from the sugar moieties of one or more other nucleosides within the gap. In certain embodiments, the sugar moieties of the two wings are identical to each other (symmetric gapmer). In certain embodiments, the sugar motif of the 5' wing is different from the sugar motif of the 3' wing (asymmetric gapmer). In certain embodiments, the modified oligonucleotide of the present invention is not a gapmer.

[0164] In certain embodiments, the gapmer wing contains 1 to 6 nucleosides. In certain embodiments, each nucleoside in each wing of the gapmer contains a modified sugar moiety. In certain embodiments, at least 1, at least 2, at least 3, at least 4, at least 5, or at least 6 nucleosides in each wing of the gapmer contain a modified sugar moiety.

[0165] In a particular embodiment, the gap of the gapmer contains 7 to 12 nucleosides. In a particular embodiment, each nucleoside in the gap of the gapmer contains a 2'-deoxyribosyl sugar moiety. In a particular embodiment, at least one nucleoside in the gap of the gapmer contains a modified sugar moiety, and the remaining nucleosides each contain a 2'-deoxyribosyl sugar moiety.

[0166] Here, the lengths (number of nucleosides) of the three regions of the gapmer can be given using the notation [number of nucleosides in the 5' wing]-[number of nucleosides in the gap]-[number of nucleosides in the 3' wing]. Thus, a 5-10-5 gapmer consists of 5-linked nucleosides in each wing and 10-linked nucleosides in the gap. If a specific modification follows such naming, that modification is a modification in each sugar moiety of each wing, and the gap nucleoside contains a 2'-deoxyribosyl sugar moiety. Thus, a 5-10-5 MOE gapmer consists of a 5-linked 2'-MOE nucleoside in the 5' wing, a 10-linked 2'-deoxyribosyl nucleoside in the gap, and a 5-linked 2'-MOE nucleoside in the 3' wing.

[0167] In certain embodiments, each nucleoside of a modified oligonucleotide or a portion thereof comprises a 2'-substituted sugar moiety, a bicyclic sugar moiety, a sugar substitute, or a 2'-deoxyribosyl sugar moiety. In certain embodiments, the 2'-substituted sugar moiety is selected from a 2'-MOE sugar moiety, a 2'-NMA sugar moiety, a 2'-OMe sugar moiety, and a 2'-F sugar moiety. In certain embodiments, the bicyclic sugar moiety is selected from a cEt sugar moiety and a LNA sugar moiety. In certain embodiments, the sugar substitute is selected from morpholino, modified morpholino, PNA, THP, and F-HNA.

[0168] In certain embodiments, the modified oligonucleotide comprises at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, or at least 20 nucleosides, including the modified sugar moiety. In certain embodiments, the modified sugar moiety is independently selected from 2'-substituted sugar moieties, bicyclic sugar moieties, or sugar substitutes. In certain embodiments, the 2'-substituted sugar moiety is selected from 2'-MOE sugar moieties, 2'-NMA sugar moieties, 2'-OMe sugar moieties, and 2'-F sugar moieties. In certain embodiments, the bicyclic sugar moiety is selected from cEt sugar moieties and LNA sugar moieties. In certain embodiments, the sugar substitute is selected from morpholino, modified morpholino, THP, and F-HNA.

[0169] In certain embodiments, each nucleoside of a modified oligonucleotide contains a modified sugar moiety ("fully modified oligonucleotide"). In certain embodiments, each nucleoside of a fully modified oligonucleotide contains a 2'-substituted sugar moiety, a bicyclic sugar moiety, or a sugar substitute. In certain embodiments, the 2'-substituted sugar moiety is selected from the 2'-MOE sugar moiety, the 2'-NMA sugar moiety, the 2'-OMe sugar moiety, and the 2'-F sugar moiety. In certain embodiments, the bicyclic sugar moiety is selected from the cEt sugar moiety and the LNA sugar moiety. In certain embodiments, the sugar substitute is selected from morpholino, modified morpholino, THP, and F-HNA. In certain embodiments, each nucleoside of a fully modified oligonucleotide contains the same modified sugar moiety ("uniformly modified sugar moiety"). In certain embodiments, the uniformly modified sugar motif has a length of 7 to 20 nucleosides. In certain embodiments, each nucleoside of the uniformly modified sugar motif contains a 2'-substituted sugar moiety, a bicyclic sugar moiety, or a sugar substitute. In certain embodiments, the 2'-substituted sugar moiety is selected from the 2'-MOE sugar moiety, the 2'-NMA sugar moiety, the 2'-OMe sugar moiety, and the 2'-F sugar moiety. In certain embodiments, the bicyclic sugar moiety is selected from the cEt sugar moiety and the LNA sugar moiety. In certain embodiments, the sugar substitute is selected from morpholino, modified morpholino, THP, and F-HNA.

[0170] In certain embodiments, the modified oligonucleotide has a sugar motif comprising at least one, at least two, at least three, or at least 42'-deoxyribonucleosides, otherwise it is fully modified. In certain embodiments, the modified oligonucleotide having at least one fully modified sugar motif may also contain one or fewer, two or fewer, three or fewer, or 42'-deoxyribonucleosides. In certain embodiments, the modified oligonucleotide having at least one fully modified sugar motif may also contain exactly one, exactly two, exactly three, or exactly 42'-deoxyribonucleosides. In certain embodiments, the modified oligonucleotide contains more than 42'-deoxyribonucleosides, provided that it does not contain a region comprising four or more consecutive 2'-deoxyribonucleosides.

[0171] Modification: Nucleic acid base motif In certain embodiments, the ASO or oligonucleotide comprises modified and / or unmodified nucleic acid bases arranged in a definite pattern or motif along the ASO or oligonucleotide or a portion thereof. In certain embodiments, each nucleic acid base is modified. In certain embodiments, none of the nucleic acid bases are modified. In certain embodiments, each purine or pyrimidine is modified. In certain embodiments, each adenine is modified. In certain embodiments, each guanine is modified. In certain embodiments, each thymine is modified. In certain embodiments, each uracil is modified. In certain embodiments, each cytosine is modified. In certain embodiments, some or all of the cytosine nucleic acid bases of the modified oligonucleotide are 5-methylcytosine. In certain embodiments, all cytosine nucleic acid bases are 5-methylcytosine, and all other nucleic acids of the modified oligonucleotide are unmodified nucleic acid bases.

[0172] In certain embodiments, the modified ASO or oligonucleotide comprises a block of modified nucleic acid bases. In certain such embodiments, the block is located at the 3' end of the oligonucleotide. In certain embodiments, the block is located within three nucleosides at the 3' end of the oligonucleotide. In certain embodiments, the block is located at the 5' end of the oligonucleotide. In certain embodiments, the block is located within three nucleosides at the 5' end of the oligonucleotide. In certain embodiments, the oligonucleotide having a gapmer motif comprises a nucleoside containing the modified nucleic acid base. In certain such embodiments, one nucleoside containing the modified nucleic acid base is located in the central gap of the oligonucleotide having a gapmer motif. In certain such embodiments, the sugar moiety of the nucleoside is a 2'-deoxyribosyl sugar moiety. In certain embodiments, the modified nucleic acid base is selected from 2-thiopyrimidine and 5-propympyrimidine.

[0173] Modification: Internucleoside linking motif In certain embodiments, the ASO or oligonucleotide includes modified and / or unmodified nucleoside linkages arranged in a defined pattern or motif along the oligonucleotide or a portion thereof. In certain embodiments, each nucleoside linkage group is a phosphodiester nucleoside linkage. In certain embodiments, each nucleoside linkage group of the modified oligonucleotide is a phosphorothioate nucleoside linkage. In certain embodiments, each nucleoside linkage of the modified oligonucleotide is independently selected from phosphorothioate nucleoside linkages and phosphodiester nucleoside linkages. In certain embodiments, each phosphorothioate nucleoside linkage is independently selected from stereodisordered phosphorothioate, (Sp)phosphorothioate, and (Rp)phosphorothioate. In certain embodiments, the sugar motif of the modified oligonucleotide is a gapmer, and all nucleoside linkages within the gap are modified. In certain such embodiments, some or all of the nucleoside linkages of the wings are unmodified phosphodiester nucleoside linkages. In certain embodiments, the terminal nucleoside linkages are modified. In certain embodiments, the sugar motif of the modified oligonucleotide is a gapmer, and the nucleoside linkage motif contains at least one phosphodiester nucleoside linkage in at least one wing, at least one phosphodiester nucleoside linkage is not a terminal nucleoside linkage, and the remaining nucleoside linkages are phosphorothioate nucleoside linkages. In certain such embodiments, all phosphorothioate nucleoside linkages are sterically disordered. In certain embodiments, all phosphorothioate nucleoside linkages of the wings are (SP)phosphorothioate, and the gap contains at least one Sp,Sp,Rp motif. In certain embodiments, the population of modified oligonucleotides is enriched with modified oligonucleotides containing such nucleoside linkage motifs.

[0174] In a particular embodiment, the modified oligonucleotide comprises at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, or at least 19 phosphodiester nucleoside linkages. In a particular embodiment, the modified oligonucleotide comprises at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, or at least 19 phosphothioate nucleoside linkages. In a particular embodiment, the modified oligonucleotide comprises at least one, at least two, at least three, at least four, or at least five phosphodiester nucleoside linkages, the remainder of which are phosphorothioate nucleoside linkages.

[0175] Conjugate In certain embodiments, the ASO or oligonucleotide is covalently bonded to one or more conjugate groups. In certain embodiments, the conjugate groups modify one or more properties of the conjugated oligonucleotide, including but not limited to pharmacodynamics, pharmacokinetics, stability, binding, absorption, tissue distribution, cell distribution, cell uptake, charge, and clearance. In certain embodiments, the conjugate groups impart new properties to the conjugated oligonucleotide, such as being fluorophores or reporter groups that enable the detection of the oligonucleotide. Certain conjugate groups or conjugate moieties have been previously described, for example, the cholesterol moiety (Letsinger et al., Proc. Natl. Acad. Sci. USA, 1989, 86, 6553-6556), cholic acid (Manoharan et al., Bioorg. Med. Chem. Lett., 1994, 4, 1053-1060), thioethers, for example, hexyl-S-tritylthiol (Manoharan et al., Ann. NY Acad. Sci., 1992, 660, 306-309; Manoharan et al., Bioorg. Med. Chem. Lett., 1993, 3, 2765-2770), thiocholesterol (Oberhauser et al., Nucl. Acids Res., 1992, 20, 533~538), aliphatic chains, e.g., dodecane-diol or undecyl residues (Saison-Behmoaras et al., EMBOJ., 1991, 10, 1111~1118; Kabanov et al., FEBS Lett., 1990, 259, 327~330; Svinarchuk et al., Biochimie, 1993, 75, 49~54), phospholipids, e.g., dihexadecyl-rac-glycerol or triethyl-ammonium 1,2-di-0-hexadecyl-rac-glycerol-3-H-phosphonate (Manoharan et al., Tetrahedron Lett., 1995, 36, 3651~3654; Shea et al., Nucl. Acids Res., 1990, 18, 3777~3783), polyamine or polyethylene glycol chain (Manoharan et al., Nucleosides & Nucleotides, 1995, 14, 969~973), or adamantane acetate palmityl moiety (Mishra et al., Biochim. Biophys. Acta, 1995, 1264, 229~237), octadecylamine or hexylamino-carbonyl-oxycholesterol moiety (Crooke et al., . / . Pharmacol. Exp. Ther., 1996, 277, 923~937), tocopherol group (Nishina et al., Molecular Therapy Nucleic Acids, 2015, 4, e220; and Nishina et al., Molecular This refers to Therapy, 2008, 16, 734-740, or the GalNAc cluster (e.g., International Publication No. 2014 / 179620).

[0176] The conjugate portion may include, but is not limited to, intervening substances, reporter molecules, polyamines, polyamides, peptides, carbohydrates, vitamin portions, polyethylene glycol, thioethers, polyethers, cholesterol, thiocholesterol, cholic acid portions, folic acid, lipids, lipophilic groups, phospholipids, biotin, phenazine, phenanthridine, anthraquinone, adamantane, acridine, fluorescein, rhodamine, coumarin, fluorophores, and pigments.

[0177] In certain embodiments, the conjugate portion includes an active drug substance, such as aspirin, warfarin, phenylbutazone, ibuprofen, suprofen, fenbufen, ketoprofen, (S')-(+)-pranoprofen (pranoprofcn), capprofen, dansyl sarcosine, 2,3,5-triiodobenzoic acid, fingolimod, flufenamic acid, folinic acid, benzothiadiazide, chlorothiazide, diazepine, indomethacin, barbiturates, cephalosporins, sulfonamides, antidiabetic drugs, antibacterial agents, or antibiotics.

[0178] Conjugate Linker The conjugate moiety is bound to the oligonucleotide via a conjugate linker. In certain oligomeric compounds, the conjugate linker is a single chemical bond (i.e., the conjugate moiety is directly bound to the oligonucleotide via a single bond). In certain oligomeric compounds, the conjugate moiety is bound to the oligonucleotide via a more complex conjugate linker, which includes one or more conjugate linker moieties that constitute the conjugate linker. In certain embodiments, the conjugate linker includes a chain structure, e.g., a hydrocarbyl chain, or a repeating unit, e.g., an oligomer of ethylene glycol, a nucleoside, or an amino acid unit.

[0179] In certain embodiments, the conjugate linker comprises one or more groups selected from alkyl, amino, oxo, amide, disulfide, polyethylene glycol, ether, thioether, and hydroxylamino. In certain such embodiments, the conjugate linker comprises groups selected from alkyl, amino, oxo, amide, and ether groups. In certain embodiments, the conjugate linker comprises groups selected from alkyl and amide groups. In certain embodiments, the conjugate linker comprises groups selected from alkyl and ether groups. In certain embodiments, the conjugate linker comprises at least one phosphite moiety. In certain embodiments, the conjugate linker comprises at least one phosphate group. In certain embodiments, the conjugate linker comprises at least one neutral linking group.

[0180] In certain embodiments, conjugate linkers including the conjugate linker described above are known to those skilled in the art to be useful for conjugating a conjugate group to a bifunctional linkage, for example, a parent compound, such as an oligonucleotide provided herein. Generally, a bifunctional linkage includes at least two functional groups. One of the functional groups is selected to bond to a specific site on the parent compound, and the other is selected to bond to a conjugate group. Examples of functional groups used in a bifunctional linkage include, but are not limited to, electrophiles that react with nucleophiles, and nucleophiles that react with electrophiles. In certain embodiments, the bifunctional linkage includes one or more groups selected from amines, hydroxyls, carboxylic acids, thiols, alkyls, alkenyls, and alkynyls.

[0181] Examples of conjugate linkers include, but are not limited to, pyrrolidine, 8-amino-3,6-dioxaoctanoic acid (ADO), succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC), and 6-aminohexanoic acid (AHEX or AHA). Other examples of conjugate linkers include, but are not limited to, substituted or unsubstituted C1-C10 alkyls, substituted or unsubstituted C2-C10 alkenyls, or substituted or unsubstituted C2-C10 alkynyls. A non-limiting list of preferred substituents includes hydroxyl, amino, alkoxy, carboxy, benzyl, phenyl, nitro, thiol, thioalkoxy, halogen, alkyl, aryl, alkenyl, and alkynyl.

[0182] In certain embodiments, the conjugate linker contains 1 to 10 linker nucleosides. In certain embodiments, the conjugate linker contains 2 to 5 linker nucleosides. In certain embodiments, the conjugate linker contains exactly 3 linker nucleosides. In certain embodiments, the conjugate linker contains a TCA motif. In certain embodiments, such linker nucleosides are modified nucleosides. In certain embodiments, such linker nucleosides contain a modified sugar moiety. In certain embodiments, the linker nucleosides are unmodified. In certain embodiments, the linker nucleosides contain optionally protected heterocyclic bases selected from purines, substituted purines, pyrimidines, or substituted pyrimidines. In certain embodiments, the cleavable portion is a nucleoside selected from uracil, thymine, cytosine, 4-N-benzoylcytosine, 5-methylcytosine, 4-N-benzoyl-5-methylcytosine, adenine, 6-N-benzoyladenine, guanine, and 2-N-isobutyrylguanine. Typically, it is desirable for the linker nucleoside to be cleaved from the oligomer compound after reaching the target tissue. Thus, the linker nucleosides are typically linked to each other and to the rest of the oligomer compound via cleavable bonds. In certain embodiments, such cleavable bonds are phosphodiester bonds.

[0183] In this specification, linker nucleosides are not considered part of the oligonucleotide. Therefore, in embodiments in which an oligomeric compound comprises an oligonucleotide consisting of a specific number or range of linked nucleosides and / or having a specific percentage of complementarity to a reference nucleic acid, and the oligomeric compound also comprises a conjugate group comprising a conjugate linker containing a linker nucleoside, these linker nucleosides are not included in the length of the oligonucleotide and are not used to determine the percentage of complementarity of the oligonucleotide to the reference nucleic acid. For example, an oligomeric compound may contain (1) a modified oligonucleotide consisting of 8 to 30 nucleosides, and (2) a conjugate group comprising 1 to 10 linker nucleosides consecutively with the nucleosides of the modified oligonucleotide. The total number of nucleosides consecutively linked to such an oligomeric compound may exceed 30. Alternatively, the oligomeric compound may contain a modified oligonucleotide consisting of 8 to 30 nucleosides and may not contain a conjugate group. The total number of nucleosides continuously linked to such oligomeric compounds is 30 or less. Unless otherwise specified, the conjugate linker contains 10 or fewer linker nucleosides. In certain embodiments, the conjugate linker contains 5 or fewer linker nucleosides. In certain embodiments, the conjugate linker contains 3 or fewer linker nucleosides. In certain embodiments, the conjugate linker contains 2 or fewer linker nucleosides. In certain embodiments, the conjugate linker contains 1 or fewer linker nucleosides.

[0184] In certain embodiments, it is desirable that the conjugate group be cleaved from the oligonucleotide. For example, in certain circumstances, an oligomeric compound containing a particular conjugate moiety is better taken up by a particular cell type, but it is desirable that, upon uptake of the oligomeric compound, the conjugate group be cleaved so as to release the unconjugated or parent oligonucleotide. Thus, a particular conjugate linker may contain one or more cleavable moieties. In certain embodiments, the cleavable moiety is a cleavable bond. In certain embodiments, the cleavable moiety is a group of atoms containing at least one cleavable bond. In certain embodiments, the cleavable moiety contains a group of atoms having one, two, three, four, or more than four cleavable bonds. In certain embodiments, the cleavable moiety is selectively cleaved within a cell or intracellular compartment, such as a lysosome. In certain embodiments, the cleavable moiety is selectively cleaved by an endogenous enzyme, such as a nuclease.

[0185] In certain embodiments, the cleavable bond is selected from amides, esters, ethers, one or both phosphodiesters, phosphate esters, carbamates, or disulfides. In certain embodiments, the cleavable bond is one or both phosphodiesters. In certain embodiments, the cleavable moiety comprises a phosphate or a phosphodiester. In certain embodiments, the cleavable moiety is a phosphate linkage between an oligonucleotide and a conjugate moiety or conjugate group.

[0186] In certain embodiments, the cleavable moiety comprises or consists of one or more linker nucleosides. In certain such embodiments, one or more linker nucleosides are linked to each other and / or to the rest of the oligomeric compound via cleavable bonds. In certain embodiments, such cleavable bonds are unmodified phosphodiester bonds. In certain embodiments, the cleavable moiety is a 2'-deoxyribonucleoside that is linked to either the 3' or 5' terminal nucleoside of the oligonucleotide via phosphate nucleoside linkages and is covalently linked to the conjugated linker or the rest of the conjugated moiety via phosphate or phosphorothioate nucleoside linkages.

[0187] In a particular such embodiment, the cleavable portion is 2'-deoxyadenosine.

[0188] Preferred ASO Also disclosed herein are ASOs comprising 17 to 24 nucleotides that can bind to UNC13A splice sites or their flanking regions (i.e., to modulate UNC13A latent exon splicing). In one embodiment, the ASO can bind to UNC13A splice donor sites or their flanking regions to modulate UNC13A latent splicing (for example, it can bind to one or more of SEQ ID NOs: 547, 548, 549, or 551 and / or is substantially complementary). In some embodiments, the ASO is a) at least SEQ ID NOs: 295, 297, 299, 300 and 301, or b) at least SEQ ID NOs: 296, 298, 299, 300 and 301, or c) at least SEQ ID NOs: 297, 299 and 300, 301 and 302, or d) at least SEQ ID NOs: 298, 299, 300, 301 and 302, or e) at least SEQ ID NOs: 299, 300, 301, 302 and 303, or f) at least SEQ ID NOs: 300, 301, 302, 303 and 304, or g) at least SEQ ID NOs: 301, 302 and 303, 304 and 305, or h i) at least SEQ ID NOs: 302, 303, 304, 305 and 306, or i) at least SEQ ID NOs: 303, 304, 305, 306 and 307, or j) at least SEQ ID NOs: 304, 305, 306, 307 and 308, k) at least SEQ ID NOs: 305, 306, 307, 308 and 309, or l) at least SEQ ID NOs: 306, 307, 308, 309 and 310, or m) at least SEQ ID NOs: 307, 308, 309, 310 and 311, or n) at least SEQ ID NOs: 308, 309, 310, 311 and 312, or o) at least SEQ ID NOs: 312 and 313,SEQ ID NOs: 314, 315 and 316, or p) at least SEQ ID NOs: 313, 314, 315, 316 and 317, or q) at least SEQ ID NOs: 314, 315, 316, 317 and 318, or r) at least SEQ ID NOs: 315, 316, 317, 318 and 319, or s) at least SEQ ID NOs: 316, 317, 318, 319 and sequence number 320, or t) at least SEQ ID NO: 317, SEQ ID NO: 318, SEQ ID NO: 319, SEQ ID NO: 320 and SEQ ID NO: 321, or u) at least SEQ ID NO: 318, SEQ ID NO: 319, SEQ ID NO: 320, SEQ ID NO: 321 and SEQ ID NO: 322 and SEQ ID NO: 323, or w) at least SEQ ID NO: 320, SEQ ID NO: 321, SEQ ID NO: 322, SEQ ID NO: 323 and SEQ ID NO: 324, or a combination thereof. In one embodiment, the ASO can be coupled to a splice acceptor site or its flanking region, more specifically, to a short acceptor site (for example, it can be coupled to or substantially complementary to SEQ ID NO: 552 or SEQ ID NO: 554), and the ASO can be coupled to at least aa) SEQ ID NO: 159, SEQ ID NO: 160, SEQ ID NO: 161, SEQ ID NO: 162 and SEQ ID NO: 163, or bb) SEQ ID NO: 160, SEQ ID NO: 161, SEQ ID NO: 162, SEQ ID NO: 163 and SEQ ID NO: 164 and SEQ ID NO: 165, or dd) SEQ ID NO: 162, SEQ ID NO: 163, SEQ ID NO: 164, SEQ ID NO: 165, and SEQ ID NO: 166, or ee) SEQ ID NO: 163, SEQ ID NO: 164, SEQ ID NO: 165, SEQ ID NO: 166, SEQ ID NO: 167, and SEQ ID NO: 168, or ff) SEQ ID NO: 164, SEQ ID NO: 165, SEQ ID NO: 166, SEQ ID NO: 167, SEQ ID NO: 168, and SEQ ID NO: 169, or hh) SEQ ID NO: 166, SEQ ID NO: 167, SEQ ID NO: 168, SEQ ID NO: 169, and SEQ ID NO: 170, or ii) SEQ ID NO: 167, SEQ ID NO: 168, SEQ ID NO: 169, SEQ ID NO: 170, and SEQ ID NO: 171, or jj) SEQ ID NO: 168,Includes SEQ ID NOs: 169, 170, 171, and 172, or kk) SEQ ID NOs: 169, 170, 171, 172, and 173, or ll) SEQ ID NOs: 169, 170, 171, 172, and 173, or mm) SEQ ID NOs: 170, 171, 172, 173, and 174, or nn) SEQ ID NOs: 171, 172, 173, 174, and 175, or oo) SEQ ID NOs: 172, 173, 174, 175, and 176, or pp) SEQ ID NOs: 173, 174, 175, 176, and 177, or combinations thereof. In one embodiment, the ASO can be bound to a splice acceptor site, more specifically, to a long acceptor site (for example, it can be bound to or substantially complementary to SEQ ID NO: 552 or SEQ ID NO: 553), and the ASO can be at least aaa) SEQ ID NO: 105, SEQ ID NO: 106, SEQ ID NO: 107, SEQ ID NO: 108 and SEQ ID NO: 109, or bbb) SEQ ID NO: 106, SEQ ID NO: 107, SEQ ID NO: 108, SEQ ID NO: 109 and SEQ ID NO: 110 and SEQ ID NO: 110, or ccc) SEQ ID NO: 107, SEQ ID NO: 108, SEQ ID NO: 109, SEQ ID NO: 110 and SEQ ID NO: 111 and SEQ ID NO: 112, or eee) SEQ ID NO: 109, SEQ ID NO: 110, SEQ ID NO: 111, SEQ ID NOs: 112 and 113, or fff) SEQ ID NOs: 110, 111, 112, 113 and 114, or ggg) SEQ ID NOs: 111, 112, 113, 114 and 115, or hhh) SEQ ID NOs: 112, 113, 114, 115 and 116, or iii) SEQ ID NOs: 113, 114, 115, 116 and 117, or jjj) SEQ ID NOs: 114, 115, 116, 117 and 118, or kkk) SEQ ID NOs: 115, 116, 117, 118 and 119, or lll) SEQ ID NOs: 116, 117, 118,This includes sequence numbers 119 and 120, or mmm) sequence numbers 117, 118, 119, 120 and 121, or nnnn) sequence numbers 118, 119, 120, 121 and 122, or ooo) sequence numbers 119, 120, 121, 122 and 123, or ppp) sequence numbers 120, 121, 122, 123 and 124, or qqq) sequence numbers 120, 121, 122, 123 and 124, or rrr) sequence numbers 121, 122, 123, 124 and 125, or combinations thereof. In a preferred embodiment, the ASO comprises crosslinked nucleic acid, preferably 30-50% crosslinked nucleic acid, and preferably the crosslinked nucleic acid is LNA. In a preferred embodiment, the ASO comprises phosphothioate linkage.

[0189] Also disclosed herein are ASOs comprising 20 to 24 nucleotides that can bind to the UNC13A splice site or its flanking region. In one embodiment, the ASO can bind to the donor splice site or its flanking region (for example, it can bind to or is substantially complementary to one or more of SEQ ID NOs: 547, 548, 549, 550, or 551). In one embodiment, the ASO includes SEQ ID NOs: 295, 297, 299, 300, 301, 302, 303, 304, optionally SEQ ID NOs: 305 (i.e., a 21-nucleotide ASO), possibly SEQ ID NOs: 306 (i.e., a 22-nucleotide ASO), possibly SEQ ID NOs: 307 (i.e., a 23-nucleotide ASO), and possibly SEQ ID NOs: 308 (i.e., a 24-nucleotide ASO). In one embodiment, the ASO includes SEQ ID NOs: 296, 298, 299, 300, 301, 302, 303, 304, optionally SEQ ID NOs: 305 (i.e., 21-nucleotide ASO), possibly SEQ ID NOs: 306 (i.e., 22-nucleotide ASO), possibly SEQ ID NOs: 307 (i.e., 23-nucleotide ASO), and possibly SEQ ID NOs: 308 (i.e., 24-nucleotide ASO). In one embodiment, the ASO includes SEQ ID NOs: 297, 299, 300, 301, 302, 303, 304, 305, optionally SEQ ID NOs: 306 (i.e., 21-nucleotide ASO), possibly SEQ ID NOs: 307 (i.e., 22-nucleotide ASO), possibly SEQ ID NOs: 308 (i.e., 23-nucleotide ASO), and possibly SEQ ID NOs: 309 (i.e., 24-nucleotide ASO).In one embodiment, the ASO includes SEQ ID NOs: 298, 299, 300, 301, 302, 303, 304, 305, optionally SEQ ID NOs: 306 (i.e., 21-nucleotide ASO), possibly SEQ ID NOs: 307 (i.e., 22-nucleotide ASO), possibly SEQ ID NOs: 308 (i.e., 23-nucleotide ASO), and possibly SEQ ID NOs: 309 (i.e., 24-nucleotide ASO). In one embodiment, the ASO includes SEQ ID NOs: 299, 300, 301, 302, 303, 304, 305, 306, optionally SEQ ID NOs: 307 (i.e., 21-nucleotide ASO), possibly SEQ ID NOs: 308 (i.e., 22-nucleotide ASO), possibly SEQ ID NOs: 309 (i.e., 23-nucleotide ASO), and possibly SEQ ID NOs: 310 (i.e., 24-nucleotide ASO). In one embodiment, the ASO includes SEQ ID NO: 300, SEQ ID NO: 301, SEQ ID NO: 302, SEQ ID NO: 303, SEQ ID NO: 304, SEQ ID NO: 305, SEQ ID NO: 306, SEQ ID NO: 307, optionally SEQ ID NO: 308 (i.e., 21-nucleotide ASO), possibly SEQ ID NO: 309 (i.e., 22-nucleotide ASO), possibly SEQ ID NO: 310 (i.e., 23-nucleotide ASO), and possibly SEQ ID NO: 311 (i.e., 24-nucleotide ASO). In one embodiment, the ASO includes SEQ ID NO: 301, SEQ ID NO: 302, SEQ ID NO: 303, SEQ ID NO: 304, SEQ ID NO: 305, SEQ ID NO: 306, SEQ ID NO: 307, SEQ ID NO: 308, optionally SEQ ID NO: 309 (i.e., 21-nucleotide ASO), possibly SEQ ID NO: 310 (i.e., 22-nucleotide ASO), possibly SEQ ID NO: 311 (i.e., 23-nucleotide ASO), and possibly SEQ ID NO: 312 (i.e., 24-nucleotide ASO).In one embodiment, the ASO includes SEQ ID NOs: 302, 303, 304, 305, 306, 307, 308, 309, possibly SEQ ID NOs: 310 (i.e., 21-nucleotide ASO), possibly SEQ ID NOs: 311 (i.e., 22-nucleotide ASO), possibly SEQ ID NOs: 312 (i.e., 23-nucleotide ASO), and possibly SEQ ID NOs: 313 (i.e., 24-nucleotide ASO). In one embodiment, the ASO includes SEQ ID NOs: 303, 304, 305, 306, 307, 308, 309, 310, possibly SEQ ID NOs: 311 (i.e., 21-nucleotide ASO), possibly SEQ ID NOs: 312 (i.e., 22-nucleotide ASO), possibly SEQ ID NOs: 313 (i.e., 23-nucleotide ASO), and possibly SEQ ID NOs: 314 (i.e., 24-nucleotide ASO). In one embodiment, the ASO includes SEQ ID NOs: 304, 305, 306, 307, 308, 309, 310, 311, optionally SEQ ID NOs: 312 (i.e., 21-nucleotide ASO), possibly SEQ ID NOs: 313 (i.e., 22-nucleotide ASO), possibly SEQ ID NOs: 314 (i.e., 23-nucleotide ASO), and possibly SEQ ID NOs: 315 (i.e., 24-nucleotide ASO). In one embodiment, the ASO includes SEQ ID NOs: 305, 306, 307, 308, 309, 310, 311, 312, optionally SEQ ID NOs: 313 (i.e., 21-nucleotide ASO), possibly SEQ ID NOs: 314 (i.e., 22-nucleotide ASO), possibly SEQ ID NOs: 315 (i.e., 23-nucleotide ASO), and possibly SEQ ID NOs: 316 (i.e., 24-nucleotide ASO).In one embodiment, the ASO includes SEQ ID NOs: 306, 307, 308, 309, 310, 311, 312, 313, optionally SEQ ID NOs: 314 (i.e., 21-nucleotide ASO), possibly SEQ ID NOs: 315 (i.e., 22-nucleotide ASO), possibly SEQ ID NOs: 316 (i.e., 23-nucleotide ASO), and possibly SEQ ID NOs: 317 (i.e., 24-nucleotide ASO). In one embodiment, the ASO includes SEQ ID NOs: 307, 308, 309, 310, 311, 312, 313, 314, optionally SEQ ID NOs: 315 (i.e., 21-nucleotide ASO), possibly SEQ ID NOs: 316 (i.e., 22-nucleotide ASO), possibly SEQ ID NOs: 317 (i.e., 23-nucleotide ASO), and possibly SEQ ID NOs: 318 (i.e., 24-nucleotide ASO). In one embodiment, the ASO includes SEQ ID NOs. 308, 309, 310, 311, 312, 313, 314, 315, optionally SEQ ID NOs. 316 (i.e., 21-nucleotide ASO), possibly SEQ ID NOs. 317 (i.e., 22-nucleotide ASO), possibly SEQ ID NOs. 318 (i.e., 23-nucleotide ASO), and possibly SEQ ID NOs. 319 (i.e., 24-nucleotide ASO). In one embodiment, the ASO includes SEQ ID NOs. 309, 310, 311, 312, 313, 314, 315, 316, optionally SEQ ID NOs. 317 (i.e., 21-nucleotide ASO), possibly SEQ ID NOs. 318 (i.e., 22-nucleotide ASO), possibly SEQ ID NOs. 319 (i.e., 23-nucleotide ASO), and possibly SEQ ID NOs. 320 (i.e., 24-nucleotide ASO).In one embodiment, the ASO includes SEQ ID NOs: 310, 311, 312, 313, 314, 315, 316, 317, optionally SEQ ID NOs: 318 (i.e., 21-nucleotide ASO), possibly SEQ ID NOs: 319 (i.e., 22-nucleotide ASO), possibly SEQ ID NOs: 320 (i.e., 23-nucleotide ASO), and possibly SEQ ID NOs: 321 (i.e., 24-nucleotide ASO). In one embodiment, the ASO includes SEQ ID NOs: 311, 312, 313, 314, 315, 316, 317, 318, optionally SEQ ID NOs: 319 (i.e., 21-nucleotide ASO), possibly SEQ ID NOs: 320 (i.e., 22-nucleotide ASO), possibly SEQ ID NOs: 321 (i.e., 23-nucleotide ASO), and possibly SEQ ID NOs: 322 (i.e., 24-nucleotide ASO). In one embodiment, the ASO includes SEQ ID NOs: 312, 313, 314, 315, 316, 317, 318, 319, possibly SEQ ID NOs: 320 (i.e., 21-nucleotide ASO), possibly SEQ ID NOs: 321 (i.e., 22-nucleotide ASO), possibly SEQ ID NOs: 322 (i.e., 23-nucleotide ASO), and possibly SEQ ID NOs: 323 (i.e., 24-nucleotide ASO). In one embodiment, the ASO includes SEQ ID NOs: 313, 314, 315, 316, 317, 318, 319, 320, possibly SEQ ID NOs: 321 (i.e., 21-nucleotide ASO), possibly SEQ ID NOs: 322 (i.e., 22-nucleotide ASO), possibly SEQ ID NOs: 323 (i.e., 23-nucleotide ASO), and possibly SEQ ID NOs: 324 (i.e., 24-nucleotide ASO).In one embodiment, the ASO includes SEQ ID NOs: 314, 315, 316, 317, 318, 319, 320, 321, optionally SEQ ID NOs: 322 (i.e., 21-nucleotide ASO), possibly SEQ ID NOs: 323 (i.e., 22-nucleotide ASO), possibly SEQ ID NOs: 324 (i.e., 23-nucleotide ASO), and possibly SEQ ID NOs: 325 (i.e., 24-nucleotide ASO). In one embodiment, the ASO includes SEQ ID NOs: 315, 316, 317, 318, 319, 320, 321, 322, optionally SEQ ID NOs: 323 (i.e., 21-nucleotide ASO), possibly SEQ ID NOs: 324 (i.e., 22-nucleotide ASO), possibly SEQ ID NOs: 325 (i.e., 23-nucleotide ASO), and possibly SEQ ID NOs: 326 (i.e., 24-nucleotide ASO). In one embodiment, the ASO includes SEQ ID NOs: 316, 317, 318, 319, 320, 321, 322, 323, optionally SEQ ID NOs: 324 (i.e., 21-nucleotide ASO), possibly SEQ ID NOs: 325 (i.e., 22-nucleotide ASO), possibly SEQ ID NOs: 326 (i.e., 23-nucleotide ASO), and possibly SEQ ID NOs: 327 (i.e., 24-nucleotide ASO). In a preferred embodiment, the ASO includes a crosslinked nucleic acid, preferably 30-50% crosslinked nucleic acid, and preferably the crosslinked nucleic acid is LNA. In a preferred embodiment, the ASO includes a phosphothioate linkage.

[0190] Also disclosed herein are ASOs comprising 20 to 24 nucleotides that can bind to a UNC13A splice site, where the ASO is an acceptor splice site (for example, it can bind to SEQ ID NO: 552). In one embodiment, the ASO includes SEQ ID NO: 159, SEQ ID NO: 160, SEQ ID NO: 161, SEQ ID NO: 162, SEQ ID NO: 163, SEQ ID NO: 164, SEQ ID NO: 165, SEQ ID NO: 166, optionally SEQ ID NO: 167 (i.e., a 21-nucleotide ASO), possibly SEQ ID NO: 168 (i.e., a 22-nucleotide ASO), possibly SEQ ID NO: 169 (i.e., a 23-nucleotide ASO), and possibly SEQ ID NO: 170 (i.e., a 24-nucleotide ASO). In one embodiment, the ASO includes SEQ ID NOs: 160, 161, 162, 163, 164, 165, 166, 167, optionally SEQ ID NOs: 168 (i.e., 21-nucleotide ASO), possibly SEQ ID NOs: 169 (i.e., 22-nucleotide ASO), possibly SEQ ID NOs: 170 (i.e., 23-nucleotide ASO), and possibly SEQ ID NOs: 171 (i.e., 24-nucleotide ASO). In one embodiment, the ASO includes SEQ ID NOs: 161, 162, 163, 164, 165, 166, 167, 168, optionally SEQ ID NOs: 169 (i.e., 21-nucleotide ASO), possibly SEQ ID NOs: 170 (i.e., 22-nucleotide ASO), possibly SEQ ID NOs: 171 (i.e., 23-nucleotide ASO), and possibly SEQ ID NOs: 172 (i.e., 24-nucleotide ASO). In one embodiment, the ASO includes SEQ ID NOs: 161, 162, 163, 164, 165, 166, 167, 168, optionally SEQ ID NOs: 169 (i.e., 21-nucleotide ASO), possibly SEQ ID NOs: 170 (i.e., 22-nucleotide ASO), possibly SEQ ID NOs: 171 (i.e., 23-nucleotide ASO), and possibly SEQ ID NOs: 172 (i.e., 24-nucleotide ASO).In one embodiment, the ASO includes SEQ ID NOs: 162, 163, 164, 165, 166, 167, 168, 169, possibly SEQ ID NOs: 170 (i.e., 21-nucleotide ASO), possibly SEQ ID NOs: 171 (i.e., 22-nucleotide ASO), possibly SEQ ID NOs: 172 (i.e., 23-nucleotide ASO), and possibly SEQ ID NOs: 173 (i.e., 24-nucleotide ASO). In one embodiment, the ASO includes SEQ ID NOs: 163, 164, 165, 166, 167, 168, 169, 170, possibly SEQ ID NOs: 171 (i.e., 21-nucleotide ASO), possibly SEQ ID NOs: 172 (i.e., 22-nucleotide ASO), possibly SEQ ID NOs: 173 (i.e., 23-nucleotide ASO), and possibly SEQ ID NOs: 174 (i.e., 24-nucleotide ASO). In one embodiment, the ASO includes SEQ ID NOs: 164, 165, 166, 167, 168, 169, 170, 171, optionally SEQ ID NOs: 172 (i.e., 21-nucleotide ASO), possibly SEQ ID NOs: 173 (i.e., 22-nucleotide ASO), possibly SEQ ID NOs: 174 (i.e., 23-nucleotide ASO), and possibly SEQ ID NOs: 175 (i.e., 24-nucleotide ASO). In one embodiment, the ASO includes SEQ ID NOs: 165, 166, 167, 168, 169, 170, 171, 172, optionally SEQ ID NOs: 173 (i.e., 21-nucleotide ASO), possibly SEQ ID NOs: 174 (i.e., 22-nucleotide ASO), possibly SEQ ID NOs: 175 (i.e., 23-nucleotide ASO), and possibly SEQ ID NOs: 176 (i.e., 24-nucleotide ASO).In one embodiment, the ASO includes SEQ ID NOs: 166, 167, 168, 169, 170, 171, 172, 173, optionally SEQ ID NOs: 174 (i.e., 21-nucleotide ASO), possibly SEQ ID NOs: 175 (i.e., 22-nucleotide ASO), possibly SEQ ID NOs: 176 (i.e., 23-nucleotide ASO), and possibly SEQ ID NOs: 177 (i.e., 24-nucleotide ASO). In one embodiment, the ASO includes SEQ ID NOs: 167, 168, 169, 170, 171, 172, 173, 174, optionally SEQ ID NOs: 175 (i.e., 21-nucleotide ASO), possibly SEQ ID NOs: 176 (i.e., 22-nucleotide ASO), possibly SEQ ID NOs: 177 (i.e., 23-nucleotide ASO), and possibly SEQ ID NOs: 178 (i.e., 24-nucleotide ASO). In one embodiment, the ASO includes SEQ ID NOs: 168, 169, 170, 171, 172, 173, 174, 175, optionally SEQ ID NOs: 176 (i.e., 21-nucleotide ASO), possibly SEQ ID NOs: 177 (i.e., 22-nucleotide ASO), possibly SEQ ID NOs: 178 (i.e., 23-nucleotide ASO), and possibly SEQ ID NOs: 179 (i.e., 24-nucleotide ASO). In one embodiment, the ASO includes SEQ ID NOs: 169, 170, 171, 172, 173, 174, 175, 176, optionally SEQ ID NOs: 177 (i.e., 21-nucleotide ASO), possibly SEQ ID NOs: 178 (i.e., 22-nucleotide ASO), possibly SEQ ID NOs: 179 (i.e., 23-nucleotide ASO), and possibly SEQ ID NOs: 180 (i.e., 24-nucleotide ASO). In a preferred embodiment, the ASO comprises a crosslinked nucleic acid, preferably 30-50% crosslinked nucleic acid, and preferably the crosslinked nucleic acid is LNA. In a preferred embodiment, the ASO comprises a phosphothioate linkage.

[0191] In a preferred embodiment, the ASO can bind to the UNC13A donor splice site, and the ASO contains 21 nucleotides. In one embodiment, the ASO includes SEQ ID NOs: 295, 297, 299, 300, 301, 302, 303, 304, and 305. In one embodiment, the ASO includes SEQ ID NOs: 296, 298, 299, 300, 301, 302, 303, 304, and 305. In one embodiment, the ASO includes SEQ ID NOs: 297, 299, 300, 301, 302, 303, 304, 305, and 306. In one embodiment, the ASO includes SEQ ID NOs: 298, 299, 300, 301, 302, 303, 304, 305, and 306. In one embodiment, ASO includes SEQ ID NOs: 299, 300, 301, 302, 303, 304, 305, 306, and 307. In one embodiment, ASO includes SEQ ID NOs: 300, 301, 302, 303, 304, 305, 306, 307, and 308. In one embodiment, ASO includes SEQ ID NOs: 301, 302, 303, 304, 305, 306, 307, 308, and 309. In one embodiment, ASO includes SEQ ID NOs: 302, 303, 304, 305, 306, 307, 308, 309, and 310. In one embodiment, ASO includes SEQ ID NOs: 303, 304, 305, 306, 307, 308, 309, 310, and 311. In one embodiment, ASO includes SEQ ID NOs: 304, 305, 306, 307, 308, 309, 310, 311, and 312.In one embodiment, ASO includes SEQ ID NOs: 305, 306, 307, 308, 309, 310, 311, 312, and 313. In one embodiment, ASO includes SEQ ID NOs: 306, 307, 308, 309, 310, 311, 312, 313, and 314. In one embodiment, ASO includes SEQ ID NOs: 307, 308, 309, 310, 311, 312, 313, 314, and 315. In one embodiment, ASO includes SEQ ID NOs: 308, 309, 310, 311, 312, 313, 314, 315, and 316. In one embodiment, ASO includes SEQ ID NOs: 309, 310, 311, 312, 313, 314, 315, 316, and 317. In one embodiment, ASO includes SEQ ID NOs: 310, 311, 312, 313, 314, 315, 316, 317, and 318. In one embodiment, ASO includes SEQ ID NOs: 311, 312, 313, 314, 315, 316, 317, 318, and 319. In one embodiment, ASO includes SEQ ID NOs: 312, 313, 314, 315, 316, 317, 318, 319, and 320. In one embodiment, ASO includes sequence numbers 313, 314, 315, 316, 317, 318, 319, 320, and 321. In one embodiment, ASO includes sequence numbers 314, 315, 316, 317, 318, 319, 320, 321, and 322. In one embodiment, ASO includes sequence numbers 315, 316, 317, 318, 319, 320, 321, 322, and 323.In one embodiment, ASO includes SEQ ID NOs: 316, 317, 318, 319, 320, 321, 322, 323, and 324. In a preferred embodiment, ASO includes crosslinked nucleic acid, preferably 30-50% crosslinked nucleic acid, and preferably the crosslinked nucleic acid is LNA. In a preferred embodiment, ASO includes phosphothioate linkage.

[0192] Also disclosed herein are pharmaceutical compositions comprising a first ASO selected from the above and a second ASO different from the first ASO selected from the above. In some embodiments, the pharmaceutical composition comprises a first ASO capable of binding to the UNC13A donor splice site or its flanking region, and a second ASO capable of binding to the UNC13A acceptor splice site or its flanking region. In some embodiments, the pharmaceutical composition comprises a first ASO capable of binding to the long acceptor splice site or its flanking region of UNC13A, and a second ASO capable of binding to the short acceptor splice site or its flanking region. In some embodiments, the pharmaceutical composition comprises a first ASO capable of binding to the risk (i.e., secondary) allele of a latent exon SNP, and a second ASO capable of binding to the main allele of a latent exon SNP.

[0193] As disclosed above, the flanking region may consist of fewer than 100, more preferably fewer than 75, more preferably fewer than 50, more preferably fewer than 25, more preferably fewer than 20, and more preferably fewer than 10 nucleotides upstream or downstream of the splice site. As disclosed above, “can bind” means “complementary” or “substantially complementary.”

[0194] Production and / or synthesis of antisense polynucleotides The polynucleotides described herein (e.g., ASOs) may be synthesized using any method known in the art or the methods described herein.

[0195] evaluation Purification of antisense polynucleotides (e.g., ASOs) as described herein includes, but is not limited to, cleanup, quality assurance, and quality control of the polynucleotides. Cleanup can be carried out by methods known in the art, such as, but is not limited to, AGEN-COURT® beads (Beckman Coulter Genomics, Danvers, Mass.), Poly-T beads, LNA® oligo-T capture probes (EX-IQON® Inc., Vedbaek, Denmark), or HPLC-based purification methods, such as strong anion exchange HPLC, weak anion exchange HPLC, reverse-phase HPLC (RP-HPLC), and hydrophobic interaction HPLC (HIC-HPLC). The term “purified” as used in relation to polynucleotides, such as “purified polynucleotides,” means that they have been separated from at least one contaminant. As used herein, “contaminant” is any substance that makes another unsuitable, impure, or inferior. Therefore, purified polynucleotides (e.g., DNA and RNA) exist in a different form or configuration than those found in nature, or in a different form or configuration than those that existed before being subjected to processing or purification methods.

[0196] In some embodiments, antisense polynucleotides (e.g., ASOs) may be quantified using methods such as, but not limited to, ultraviolet-visible spectroscopy (UV / Vis). An unspecified example of a UV / Vis spectrometer is the NANODROP® spectrometer (ThermoFisher, Waltham, Mass.). The quantified polynucleotides can be analyzed to determine if polynucleotide degradation has occurred in order to determine if the polynucleotides are of an appropriate size.

[0197] The degradation of antisense polynucleotides (e.g., ASOs) can be investigated by methods such as, but not limited to, agarose gel electrophoresis; HPLC-based purification methods such as strong anion exchange HPLC, weak anion exchange HPLC, reverse-phase HPLC (RP-HPLC), and hydrophobic interaction HPLC (HIC-HPLC); liquid chromatography-mass spectrometry (LCMS); capillary electrophoresis (CE); and capillary gel electrophoresis (CGE).

[0198] II. Pharmaceutical Compositions Provided by this disclosure are compositions, for example, pharmaceutical compositions comprising at least one antisense polynucleotide (e.g., ASO) as described herein. Compositions comprising antisense polynucleotides (e.g., ASO) as described herein may be formulated for administration to specific target cells, target tissues or target organs and / or subjects.

[0199] The pharmaceutical compositions of the present invention may contain one or more polynucleotides described herein (e.g., ASO, guide RNA, vectorization constructs), or two or more, three or more, or four or more polynucleotides described herein (e.g., ASO, guide RNA, vectorization constructs).

[0200] In some embodiments, the pharmaceutical composition comprises two or more, three or more, or four or more ASOs as described herein. In some embodiments, the pharmaceutical composition comprises two or more ASOs that can be bound to different portions of SEQ ID NO: 1 (i.e., UNC13A latent exon or its intron flanking region).

[0201] In some embodiments, the pharmaceutical composition comprises one or more ASOs capable of binding to the UNC13A donor splice site and one or more ASOs capable of binding to the UNC13A acceptor splice site. In some embodiments, the pharmaceutical composition comprises two or more ASOs capable of binding to the UNC13A donor site.

[0202] In some embodiments, the pharmaceutical composition comprises two or more ASOs capable of binding to the UNC13A acceptor site, where one or more ASOs can bind to a short acceptor site and one or more ASOs can bind to a long acceptor site. In some embodiments, the pharmaceutical composition comprises a first ASO capable of binding to the risk (i.e., sub) allele of the CE SNP (e.g., substantially complementary) and a second ASO capable of binding to the main allele of the CE SNP (e.g., substantially complementary). This ensures that enhanced binding is obtained for both alleles.

[0203] In some embodiments, the pharmaceutical composition comprises two or more, three or more, or four or more guide RNAs as described herein. In some embodiments, the pharmaceutical composition comprises two or more, three or more, or four or more viral vectors as described herein. In some embodiments, the pharmaceutical composition may include a combination of one or more ASOs, one or more guide RNAs, and / or one or more viral vectors as described herein.

[0204] Antisense polynucleotide (e.g., ASO) compositions may also be formulated to be delivered directly to organs or tissues by any of several methods in the art, including, but not limited to, direct sedimentation or immersion by catheter, gel, powder, ointment, cream, lotion, and / or infusion, or by the use of a substrate such as fibers or biodegradable materials coated or impregnated with the composition.

[0205] formulation Pharmaceutical formulations may additionally contain pharmaceutically acceptable excipients, which, as used herein, may be water, any solvent including dispersion media and diluents, or other liquid vehicles, dispersion or suspension aids, surfactants, isotonic agents, solubilizers, isotonic agents, pH adjusters, buffers, thickeners or emulsifiers, preservatives, etc., to suit a particular desired dosage form.

[0206] Techniques for preparing various excipients and compositions for formulation pharmaceutical compositions are publicly known in the art (the entirety of which is incorporated herein by reference, Remington: The Science and Practice of Pharmacy, 21 st See Edition, AR Gennaro, Lippincott, Williams & Wilkins, Baltimore, MD, 2006. The use of conventional excipient media may be intended to be within the scope of this disclosure, except where any conventional excipient media may be incompatible with the substance or derivative, for example, producing any undesirable biological effect, or otherwise interacting in a detrimental manner with any other component of the pharmaceutical composition.

[0207] Formulations of the pharmaceutical compositions described herein may be prepared by any known or later developed method in the field of pharmacology. Generally, such preparation methods include the steps of associating an active ingredient (e.g., antisense polynucleotide) with excipients and / or one or more other auxiliary components, and then, if necessary and / or desirable, dividing, shaping and / or packaging the product into desired single or multi-dose units.

[0208] The pharmaceutical compositions described herein are prepared, packaged and / or sold in bulk as single unit doses and / or multiple single unit doses. As used herein, “unit dose” is an isolated volume of a pharmaceutical composition containing a predetermined amount of the active ingredient. The amount of the active ingredient is generally equal to the dose of the active ingredient administered to a subject and / or a convenient fraction of such dose, for example, half or one-third of such dose.

[0209] The relative amounts of the active ingredient, pharmaceutically acceptable excipients and / or any additional ingredients in the pharmaceutical compositions according to the present disclosure may vary depending on the nature, size and / or condition of the subject to be treated, and further depending on the route by which the composition is administered. By way of example, the composition can contain from 0.1% to 100%, for example, from 0.5 to 50%, from 1 to 30%, from 5 to 80%, from 10 to 50%, from 20 to 90%, at least 70% (w / w), or at least 80% (w / w) of the active ingredient.

[0210] In some embodiments, the formulations described herein can contain at least one antisense polynucleotide (e.g., ASO). In some embodiments, the formulation can contain at least 1, 2, 3, 4 or 5 antisense polynucleotides (e.g., ASO) having different sequences. In one embodiment, the formulation contains at least 2 antisense polynucleotides (e.g., ASO). In one embodiment, the formulation contains at least 3 antisense polynucleotides (e.g., ASO). In another embodiment, the formulation contains at least 4 antisense polynucleotides (e.g., ASO). In yet another embodiment, the formulation contains at least 5 antisense polynucleotides (e.g., ASO).

[0211] The pharmaceutical compositions and formulations of the present disclosure can be formulated with one or more excipients to increase the stability of the antisense polynucleotide (e.g., ASO), increase cell permeability, enable sustained, controlled or delayed release, alter the biodistribution (e.g., target a nucleic acid vaccine composition to a particular tissue or cell type), increase the in vivo translation of the encoded protein, and / or alter the in vivo release of the encoded protein.

[0212] In addition to traditional excipients, the excipients of the present disclosure include, but are not limited to, lipids, lipidoids, liposomes, lipid nanoparticles, polymers, lipoplexes, core-shell nanoparticles, peptides, proteins, nucleic acid molecules, cells, organelles, explants, nanoparticle mimics, and combinations thereof.

[0213] In vivo delivery of nucleic acids can be affected by many parameters including, but not limited to, the composition of the formulation, the nature of the particles, the loading degree, the ratio of polynucleotide (e.g., ASO) to lipid / lipidoid, the nature of the polynucleotide (e.g., ASO), such as sequence content, single-stranded or double-stranded, linear or circular, length and modification, particle size and charge, and the route of administration.

[0214] The present disclosure contemplates formulations and uses in the delivery of at least one antisense polynucleotide (e.g., ASO) composition and at least one pharmaceutically acceptable carrier. Complexes, micelles, liposomes or particles containing any suitable lipid and lipidoid can be prepared, and thus effective delivery of the antisense polynucleotide composition can occur after injection of the formulation by various means including, but not limited to, those described herein via local and / or systemic administration routes.

[0215] Lipids and lipidoids In some embodiments, the antisense polynucleotides (ASO compounds) and compositions of the present disclosure can be formulated using one or more lipids and / or lipidoids. As used herein, the term "lipidoid" refers to any material having the characteristics of a lipid. Lipidoids are lipid-like structures containing multiple secondary and tertiary amine functionalities, which confer extremely efficient interactions with nucleic acid molecules.

[0216] The synthesis of lipids and lipidoids has been extensively studied, and formulations containing lipids and lipidoids are particularly suitable for nucleic acid delivery. The use of lipids and lipidoids for the formulation and effective delivery of double-stranded small RNA (siRNA), single-stranded mRNA, and gene therapies has been described in mice and non-human primates (e.g., Lvins et al., 2010); Akinc et al., Nat Biotechnol. 2008 26:561~569; Love et al., Proc Natl Acad Sci US A. 2010, 107:1864~1869; Siegwart et al., Proc Natl Acad Sci US A. 2011, 108:12996~3001; Leuschner et al., Nat Biotechnol. 2011, 29:1005~1010; Roberts et al., Methods Mol. Biol., 2016, 1364:2991~310; Ball et al., Nato. Lett. 2018, 18(6):3814~3822; Lokras et al., Methods Mol. Biol., 2021, 2282:137~157; Schrom et al., Mol. Ther. Nucleic Acids, 2017, 7:350~365; all of these contents are incorporated herein by reference in their entirety).

[0217] Lipids and lipidoids can be cationic lipids and lipidoids. Cationic lipids are typically characterized by a positively charged head followed by a hydrophobic tail of various compositions, with the head and tail connected by a linker, such as an ether, ester, or amide. While we do not wish to be bound by any theory, these cationic head groups neutralize the anionic charge of the nucleic acid they transport.

[0218] In some embodiments, ionizable cationic lipids can be used in the formulation.

[0219] In some embodiments, the lipids may be anionic lipids.

[0220] In some embodiments, lipids and lipidoids may be neutral lipids.

[0221] In some embodiments, ionizable lipids, such as Dlin-MC3-DMA(MC3), Dlin-KC2-DMA(KC2), and cKK-E12, may be used as the packaged cyclic nucleic acid molecule.

[0222] Non-limiting examples of lipidoids for formulation include "98N12-5" disclosed in Akinc et al., Mol Ther. 2009, 17:872-879; and "C12-200" disclosed in Love et al., Proc Natl Acad Sci US A. 2010, 107:1864-1869, and Liu and Huang, Molecular Therapy. 2010, 669-670.

[0223] Polymers and polymer nanoparticles (NP) In some embodiments, the polynucleotide (e.g., ASO) compositions of the present disclosure may be formulated using polymers containing one or more polymers or nanoparticles (NPs). In some embodiments, the polymers may be biocompatible and biodegradable.

[0224] The physicochemical properties of polymers (e.g., composition, molecular weight, and polydispersity) can be modified to achieve formulations specialized for nucleic acid delivery. Polymers may be naturally derived or synthetic. In some embodiments, the polymers used in this disclosure are processed to reduce and / or inhibit the binding of unwanted substances, such as bacteria, to the polymer surface. The polymers may be processed by methods known and / or described in the art and / or in PCT Patent Application International Publication No. 2012150467 (the contents of which are incorporated herein by reference in their entirety).

[0225] Many polymer techniques have demonstrated effectiveness for in vivo delivery of nucleic acids into the cytoplasm (reviewed in de Fougerolles' Hum Gene Ther. 2008, 19:125-132). Techniques using dynamic polyconjugates have been shown to effectively deliver siRNA in vivo in mice and to arrest the expression of endogenous target mRNA in hepatocytes. In this technique, the multicomponent polymer system includes a membrane-active polymer in which nucleic acids are covalently linked via disulfide bonds, and both PEG (charge masking) and N-acetylgalactosamine (hepatocyte targeting) groups are linked via pH-sensitive binding. Upon binding to hepatocytes and entering endosomes, the polymer complex is disintegrated in a low pH environment, exposing the polymer's positive charge, leading to endosomal escape and cytoplasmic release of siRNA from the polymer. Replacing the N-acetylgalactosamine group with a mannose group can alter the targeting of sinusoidal endothelial and Kupffer cells (Rozema et al., Proc Natl Acad Sci US A. 2007, 104:12982~12887). Another method of formulating siRNA using cyclodextrin-containing polycationic nanoparticles has demonstrated targeted arrest of the EWS-FLI1 gene product in Ewing sarcoma tumor cells (Hu-Lieskovan et al., Cancer Res. 2005, 65:8984~8982); the contents of each of these are incorporated herein by reference in their entirety. Both of these delivery strategies incorporate reasonable methods using polymers for both targeted delivery and endosomal escape mechanisms.

[0226] In some embodiments, the antisense polynucleotides (e.g., ASOs) of this disclosure may be formulated using naturally derived polymers, structural proteins, and polysaccharides, such as cationic collagen derivatives and chitosan. Cationic collagen proteins have been used for nucleic acid delivery to articular cartilage and bone for regenerative medicine and metastatic tumor treatment (Capito et al., Gene Ther., 2007, 14:721-732; Curtin et al., Adv. Healthc. Mater., 2015, 4:223-227). Chitosan, a linear cationic polysaccharide, is produced by the deacetylation of chitin (poly-d-glucosamine), is non-toxic even at high concentrations, and can be formulated into polyplexes. Non-limiting examples of chitosan-based formulations include the outer portion of a positively charged chitosan core and a negatively charged substrate (U.S. Patent Publication No. 20120258176; this is incorporated herein by reference in its entirety). Chitosan includes, but is not limited to, N-trimethyl chitosan, mono-N-carboxymethyl chitosan (MCC), N-palmitoyl chitosan (NPCS), EDTA-chitosan, low molecular weight chitosan, chitosan derivatives, or combinations thereof.

[0227] Cyclodextrins (CDs) are another family of naturally derived carbohydrate-based polymers with desirable physicochemical properties. α-, β-, or γ-CD types can be used in combination with other cationic polymers to deliver nucleic acids, for example, to the liver and metastatic tumors.

[0228] The antisense polynucleotides (e.g., ASOs) of this disclosure can be formulated using synthetic polymers, which allows for the controlled incorporation of diverse chemistry and provides flexibility and more options for polynucleotide formulations. Various synthetic strategies exist in the art to control polymerization reactions and thus the properties of the resulting polymers. Examples of methods include controlled free radical polymerization, e.g., reversible addition-fragmentation-chain transfer (RAFT) polymerization and atom transfer radical polymerization (ATRP) (Boyer et al., Chem. Rev., 2009, 109:5402-5436). Polymers formulated with the polynucleotide (e.g., ASO) compositions of this disclosure can be synthesized by methods described in PCT Patent Application International Publication No. 2012082574 or No. 2012068187 (the contents of which are incorporated herein by reference in their entirety, respectively).

[0229] In some embodiments, cationic groups can be incorporated into polymers to formulate nucleic acid molecules. While we do not wish to be bound by any theory, cationic groups can assist in the loading of negatively charged nucleic acid cargo, and once the loaded polynucleotides are delivered to cells, they can facilitate interaction with negatively charged glycoproteins in the cell membrane.

[0230] In some embodiments, the antisense polynucleotide (e.g., ASO) composition of the present disclosure comprises at least one polymer compound, for example, but not limited to, polyethene, polyethylene glycol (PEG), poly(l-lysine) (PLL), PEG grafted onto PLL, cationic lipopolymer, biodegradable cationic lipopolymer, polyethyleneimine (PEI), crosslinked branched poly(alkyleneimine), polyamine derivative, modified poloxamer, biodegradable polymer, elastic biodegradable polymer, biodegradable block polymer, biodegradable random copolymer, biodegradable polyester copolymer, biodegradable polyester block copolymer, biodegradable polyester block random copolymer, multiblock copolymer, linear biodegradable copolymer, poly[α-(4-aminobutyl)-L-glycolic acid)( This may include PAGA, biodegradable crosslinked cationic multiblock copolymers, polycarbonates, polyanhydrides, polyhydroxy acids, polypropylfumerates, polycaprolactones, polyamides, polyacetals, polyethers, polyesters, poly(orthoesters), polycyanoacrylates, polyvinyl alcohols, polyurethanes, polyphosphazenes, polyacrylates, polymethacrylates, polycyanoacrylates, polyureas, polystyrenes, polyamines, polylysine, poly(ethyleneimine), poly(serine esters), poly(L-lactide-co-L-lysine), poly(4-hydroxy-L-proline esters), acrylic polymers, amine-containing polymers, dextran polymers, dextran polymer derivatives, or combinations thereof.

[0231] In some embodiments, the synthetic polymers are biodegradable. Synthetic biodegradable polymers can be produced by assembling low molecular weight monomers into a polymer via bioreversible linkages, such as sulfide or ester bonds. Examples of synthetic biodegradable polymers include, but are not limited to, poly(lactic acid) (PLA), poly(glycolic acid) (PGA), poly(lactic acid-co-glycolic acid) (PLGA), poly(beta-amino)esters (PBAE), and poly(amine-co-esters) (PACE).

[0232] Biodegradable polymers have been previously used to protect nucleic acids from degradation and have been shown to result in sustained release of payloads in vivo (Rozema et al., Proc Natl Acad Sci U S A. 2007, 104:12982-12887; Sullivan et al., Expert Opin Drug Deliv. 2010, 7:1433-1446; Convertine et al., Biomacromolecules. 2010, Oct 1; Chu et al., Acc Chem Res. 2012, Jan 13; Manganiello et al., Biomaterials. 2012, 33:2301-2309; Benoit et al., Biomacromolecules. 2011, 12:2708-2714; Singha et al., Nucleic Acid Ther. 2011, 2:133-147; de Fougerolles Hum Gene Ther. 2008, 19:125-132; Schaffert and Wagner, Gene Ther. 2008, 16:1131-1138; Chaturvedi et al., Expert Opin Drug Deliv. 2011, 8:1455-1468; Davis, Mol Pharm. 2009, 6:659-668; Davis, Nature, 2010, 464:1067-1070; the contents of which are hereby incorporated by reference in their entirety).

[0233] The biodegradable polymer can be a polymer containing a polyethyleneimine group as described in U.S. Patent No. 7,700,542. The polymer can be a biodegradable cationic lipopolymer prepared by the methods described in U.S. Patent No. 6,696,038, and U.S. Patent Publications Nos. 20030073619 and 20040142474 (the contents of which are hereby incorporated by reference in their entirety).

[0234] The antisense polynucleotides (e.g., ASOs) of this disclosure may be formulated on polymer carriers using different polymer-containing nanoparticles. For example, therapeutic polymer nanoparticles may be identified by the method described in U.S. Patent Publication No. 20120140790 (which is incorporated herein by reference in its entirety). In some embodiments, the therapeutic nanoparticles may include a polymer matrix. As an example of non-limiting examples, nanoparticles may include two or more polymers and diblock copolymers, such as, but are not limited to, polyethylene, polycarbonate, polyanhydride, polyhydroxy acid, polypropylfumerate, polycaprolactone, polyamide, polyacetal, polyether, polyester, poly(orthoester), polycyanoacrylate, polyvinyl alcohol, polyurethane, polyphosphazene, polyacrylate, polymethacrylate, polycyanoacrylate, polyurea, polystyrene, polyamine, polylysine, poly(ethyleneimine), poly(serine ester), poly(L-lactide-co-L-lysine), poly(4-hydroxy-L-proline ester), or combinations thereof. The polymers include those described in PCT Patent Applications International Publication Nos. 2011115862, 2012082574, and 2012068187, and U.S. Patent Application Publication No. 20120283427; the polymer of formula Z described in International Publication No. 2011115862; and the polymers of formulas Z, Z', or Z'' described in PCT Patent Applications International Publication Nos. 2012082574 and 2012068187, and U.S. Patent Application Publication No. 2012028342, the contents of which are incorporated herein by reference in their entirety.

[0235] As a non-limiting example, the antisense polynucleotide (e.g., ASO) compositions of this disclosure may be formulated in therapeutic nanoparticles comprising PLGA-PEG block copolymers (see U.S. Patent Publication No. 20120004293 and U.S. Patent No. 8,236,330), or PEG-PLA diblock copolymers or PEG-PLGA copolymers (see U.S. Patent No. 8,246,968 and PCT Patent International Publication No. 2012166923), or multiblock copolymers as described in U.S. Patent Nos. 8,263,665 and 8,287,910, the contents of which are incorporated herein by reference in whole.

[0236] In some embodiments, the block copolymers may include those of formulas I, II, III, IV, V, VI, and VII of PCT Patent Application International Publication 2015017519 (the contents of which are incorporated herein by reference in their entirety). The block copolymers may be contained in polyionic composites comprising nonpolymeric micelles and block copolymers. (See, for example, U.S. Patent Application Publication 20120076836, the contents of which are incorporated herein by reference in their entirety).

[0237] In some embodiments, the antisense polynucleotide (e.g., ASO) compositions of the Disclosure may be formulated using acrylic polymers, including but not limited to acrylic acid, methacrylic acid, acrylic acid and methacrylic acid copolymers, methyl methacrylate copolymers, ethoxyethyl methacrylate, cyanoethyl methacrylate, aminoalkyl methacrylate copolymers, poly(acrylic acid), poly(methacrylic acid), polycyanoacrylate, and combinations thereof. In other embodiments, the antisense polynucleotide (e.g., ASO) compositions of the Disclosure may be formulated using amine-containing polymers, for example, but not limited to polylysine, polyethyleneimine, poly(amideamine) dendrimers, and poly(beta-aminoesters) (see, for example, U.S. Patent No. 8,287,849, the entirety of which is incorporated herein by reference).

[0238] In some embodiments, the nanoparticles may contain at least one biodegradable polyester that may contain polycationic side chains. Biodegradable polyesters include, but are not limited to, poly(serine esters), poly(L-lactide-co-L-lysine), poly(4-hydroxy-L-proline esters), and combinations thereof. Biodegradable polyesters may include PEG conjugations to form PEGylated polymers. In other embodiments, the polynucleotide (e.g., ASO) compositions of this disclosure may be formulated with at least one crosslinkable polyester. Crosslinkable polyesters include those known in the art and those described in U.S. Patent Application Publication No. 20120269761, which is incorporated herein by reference in its entirety.

[0239] In some embodiments, polymer formulations comprising the polynucleotides (e.g., ASOs) of the present disclosure may include branched-chain PEG molecules described in or prepared by the method described in PCT Patent Application International Publication No. 20180126084. The branched-chain PEGs that may be used in the formulations described in No. 20180126084 may have formulas I, II, III, IV, V, and VI, the contents of which are incorporated herein by reference in their entirety.

[0240] In some embodiments, antisense polynucleotides (e.g., ASOs) are covalently bonded to a carrier molecule. In one example, the ASO is covalently bonded to a carbohydrate, protein, small molecule (e.g., α-tocopherol), peptide (e.g., cell-permeable peptide), antibody, lipid (e.g., cholesterol), or polymer (e.g., PEG). In one embodiment, the ASO is not covalently bonded to a carrier molecule.

[0241] In some embodiments, polymer nanoparticles formulating the antisense polynucleotides of the Disclosure (e.g., ASOs) may include a conjugation of at least one targeting ligand. The targeting ligand may be any ligand known in the art, for example, but not limited to monoclonal antibodies, peptides, and nucleic acids (e.g., aptamers). In other embodiments, polymer nanoparticles may selectively target cells, tissues, and / or organs through the expression of different ligands (e.g., folic acid, transferrin, and N-acetylgalactosamine (GalNAc)).

[0242] In some embodiments, polymer nanoparticles used to formulate the antisense polynucleotide (e.g., ASO) compositions of the Disclosure may enable sustained or delayed release of the polynucleotide composition. The modified release profile of the antisense polynucleotide composition can result in long-term control. In some embodiments, sustained-release polymer formulations may include, but are not limited to, PLGA microspheres, ethylene vinyl acetate (EVA), poloxamers, and fibrinogen polymers. For example, an antisense polynucleotide composition may be formulated in PLGA microspheres by preparing PLGA microspheres with adjustable release rates (days and weeks), encapsulating the polynucleotide (e.g., ASO) composition in the PLGA microspheres, and maintaining the integrity of the polynucleotide during the encapsulation process.

[0243] In some embodiments, the antisense polynucleotide (e.g., ASO) compositions of the present disclosure can be formulated for controlled release in polymer formulations containing copoly(lactic / glycol) acid (see, for example, U.S. Patent No. 4,675,189, Kent et al.) or a block copolymer of lactic acid and PEG, which can be administered by subcutaneous or intramuscular injection to achieve a controlled-release depot formulation.

[0244] As a non-limiting example, the antisense polynucleotides (e.g., ASOs) of this disclosure may be formulated as polymer formulations comprising a polymer compound of PEG-PLL as described in U.S. Patent No. 6,177,274, or formulations comprising a PLGA-PEG-PLG block copolymer as described in U.S. Patent No. 6,004,573, or as dry formulations or liquid formulations that may be anhydrous as described in U.S. Patent Application Publications 20090042829 and 20090042825, the contents of which are incorporated herein by reference in their entirety.

[0245] Liposomes The polynucleotide(s) and polynucleotide (e.g., ASO(s) and ASO) compositions of this disclosure can be formulated using one or more liposomes. As used herein, the term “liposome” refers to an artificially prepared vesicle, mainly composed of one or more lipid bilayers, that can be used as a delivery vehicle. Liposomes may be of different sizes, and are not limited to, for example, multilayer vesicles (MLVs) which may have a diameter of several hundred nanometers and contain a series of concentric bilayers separated by a narrow aqueous compartment; small unicellular vesicles (SUVs) which may have a diameter of less than 50 nm; and large monolayer vesicles (LUVs) which may have a diameter between 50 and 500 nm.

[0246] Liposome design may include, but is not limited to, opsonins or ligands that improve liposome binding to unhealthy tissue or activate events such as endocytosis, for example. Liposomes can contain low or high pH to improve the delivery of pharmaceutical formulations. The formulation of liposomes may depend on physicochemical characteristics, such as, for example, the encapsulated pharmaceutical formulation and liposome components, the properties of the medium in which the lipid vehicle is dispersed, the effective concentration and potential toxicity of the encapsulated substance, any additional processes involved in the application and / or delivery of the vesicles, the optimized vesicle size for the intended use, polydispersibility and shelf life, and the batch-to-batch reproducibility and possibility of large-scale production of safe and efficient liposome products.

[0247] Liposomes can be lipid-based liposomes, polymer-based liposomes, or hybrids. Liposomes can be cationic liposomes or neutral liposomes. Cationic liposomes have been used to deliver siRNA to various cell types (e.g., U.S. Patent Application Publication 2004 / 0204377).

[0248] In some embodiments, the material may contain glycosylated lipids as disclosed in Bally et al. U.S. Patent No. 5,595,756 (the contents of which are incorporated herein by reference in their entirety). The lipids are gangliosides and cerebrosides and may be present in amounts of about 10 moles.

[0249] In some embodiments, liposomes are formed by the self-assembly of dissolved lipid molecules and / or polymers. The polynucleotides of the Disclosure (e.g., ASOs) can be passively encapsulated in a lipid bilayer via liposome preparation, and can be encapsulated in the aqueous core of the liposome or in the aqueous phase between the bilayers using, for example, passive loading methods, such as reverse-phase evaporation, dehydration-rehydration methods, or active loading involving a pH gradient of the liposome membrane (Szoka and Papahadjopoulos, PNAS, 1978;9:4194~4198; Shew and Deamer, Biochim. Biophy Acta., 1985;1:1~8; and Mayer et al., Biochim. Biophy Acta., 1986;1:123~126).

[0250] Liposome formulations may be affected by, but are not limited to, the selection of cationic lipid components, the degree of saturation of cationic lipids, the nature of PEGylation, the proportion of all components, and biophysical parameters such as size. In one example by Semple et al. (Semple et al., Nature Biotech. 2010, 28:172-176; this content is incorporated herein by reference in its entirety), the liposome formulation consisted of 57.1% cationic lipids, 7.1% dipalmitoylphosphatidylcholine, 34.3% cholesterol, and 1.4% PEG-c-DMA.

[0251] In some embodiments, liposomes can be targeted to surface-bound ligands that can recognize and bind to target cells. Targeted liposomes can increase the delivery and accumulation of liposomes and inclusive polynucleotides (e.g., ASOs) in desired tissues and organs. Examples of surface-targeting ligands include immunoglobulins (Ig) and their fragments, peptides, and aptamers.

[0252] In some embodiments, the surface of liposomes may be coated with an inert, biocompatible polymer, such as PEG. The polymer coating forms a protective layer on the liposome surface, slowing opsonin recognition of liposomes and thereby increasing in vivo circulation of liposomes.

[0253] In some embodiments, the polynucleotides (e.g., ASOs) and pharmaceutical compositions comprising polynucleotides (e.g., ASOs) described herein are not limited to liposomes, such as 1,2-dioleyloxy-N,N-dimethylaminopropane (DODMA) liposomes, Marina Biotech's (Bothell, WA) DiLa2 liposomes, and SMARTICLES® / NOV340 (Marina Biotech). Biotech (Bothell), 1,2-dilinoleyloxy-3-dimethylaminopropane (DLin-DMA), 2,2-dilinoleyl-4-(2-dimethylaminoethyl)-[1,3]-dioxolane (DLin-KC2-DMA), MC3 (U.S. Patent No. 20100324120; this content is incorporated herein by reference in its entirety), neutral DOPC (1,2-dioleoyl-sn-glycero-3-phosphocholine)-based liposomes (e.g., siRNA delivery to ovarian cancer (Landen et al., Cancer Biology & Therapy 2006, 5(12): 1708~1713); this content is incorporated herein by reference in its entirety), hyaluronan-coated liposomes (Quiet Therapeutics, Israel), and small molecule drugs, e.g., Janssen Examples include those formed from liposomes capable of delivering DOXIL®, a registered trademark of Biotech, Inc. (Horsham, PA).

[0254] In some embodiments, the polynucleotides (e.g., ASOs) and pharmaceutical compositions comprising polynucleotides (e.g., ASOs) described herein may include, but are not limited to, those formed from the synthesis of stabilized plasmid lipid particles (SPLPs) or stabilized nucleic acid lipid particles (SNALPs), which have been previously described and shown to be suitable for oligonucleotide delivery in vitro and in vivo (the entire content of which is incorporated herein by reference: Wheeler et al., Gene Therapy. 1999, 6:271~281; Zhang et al., Gene Therapy. 1999, 6:1438~1447; Jeffs et al., Pharm Res. 2005, 22:362~372; Mo, Nat Biotechnol. 2005, 2:1002~1007; Zimmermann et al., Nature. 2006, 441:111~114; Heyes et al., J Contr Rel. See also 2005, 107:276~287; Semple et al., Nature Biotech. 2010, 28:172~176; Judge et al., J Clin Invest. 2009, 119:661~673; deFougerolles Hum Gene Ther. 2008, 19:125~132). The original manufacturing method by Wheeler et al. was a detergent dialysis method, which was later improved by Jeffs et al. and is called the spontaneous vesicle formation method. The liposome formulation may consist of 3 to 4 lipid components in addition to the nucleic acid vaccine composition. As an unspecified example, liposomes may contain, but are not limited to, 55% cholesterol, 20% disteroylphosphatidylcholine (DSPC), 10% PEG-S-DSG, and 15% 1,2-dioleyloxy-N,N-dimethylaminopropane (DODMA), as described by Jeffs et al.In another example, a particular liposomal formulation may contain, but is not limited to, 48% cholesterol, 20% DSPC, 2% PEG-c-DMA, and 30% cationic lipids, as described by Heyes et al., where the cationic lipids may be 1,2-distearyloxy-N,N-dimethylaminopropane (DSDMA), DODMA, DLin-DMA, or 1,2-dilinolenyloxy-3-dimethylaminopropane (DLenDMA). In another example, the nucleic acid-lipid particles may include cationic lipids comprising about 50 mol% to about 85 mol% of the total lipids present in the particles, non-cationic lipids comprising about 13 mol% to about 49.5 mol% of the total lipids present in the particles, and conjugate lipids that inhibit particle aggregation comprising about 0.5 mol% to about 2 mol% of the total lipids present in the particles, as described in International Publication No. 2009127060 by Maclachlan et al. (the entirety of which is incorporated herein by reference). In another example, the nucleic acid-lipid particles may be any nucleic acid-lipid particles disclosed in U.S. Patent Application No. 2006008910 by Maclachlan et al. (the entirety of which is incorporated herein by reference). In a non-limiting example, the nucleic acid-lipid particles may include cationic lipids of formula I, non-cationic lipids, and conjugate lipids that inhibit particle aggregation.

[0255] In some embodiments, the polynucleotide (e.g., ASO) compositions of this disclosure may be formulated in liposomes containing cationic lipids. The liposomes may have a molar ratio (N:P ratio) of nitrogen atoms in the cationic lipid to phosphate in the nucleic acid vaccine composition between 1:1 and 20:1, as described in PCT Patent Application International Publication No. 2013006825 (the contents of which are incorporated herein by reference in their entirety). In some embodiments, the liposomes may have an N:P ratio greater than 20:1 or less than 1:1.

[0256] In some embodiments, polynucleotide (e.g., ASO) compositions may be formulated in any amphoteric liposome, as disclosed in Panzner's PCT Patent Application International Publication No. 2008043575 and Essler et al.'s U.S. Patent No. 8,580,297 (Marina Biotech) (the contents of which are incorporated herein by reference in their entirety). The amphoteric liposomes may contain a mixture of lipids comprising a mixture of cationic amphiphilic substances, anionic amphiphilic substances, and optionally one or more neutral amphiphilic substances. The amphoteric liposomes may contain amphoteric compounds based on amphiphilic molecules, where the head group of the amphiphilic molecule is substituted with one or more amphoteric groups. In some embodiments, the pharmaceutical compositions may be formulated in amphoteric lipids comprising one or more amphoteric groups having an isoelectric point between 4 and 9, as disclosed in Essler et al.'s U.S. Patent Application Publication No. 20140227345 (Marina Biotech) (the contents of which are incorporated herein by reference in their entirety). In some embodiments, the pharmaceutical composition can be formulated in amphoteric liposomes comprising at least one amphiphilic cationic lipid, at least one amphiphilic anionic lipid and at least one neutral lipid, as disclosed in U.S. Patent No. 7,780,983 (Novosom) by Panzner et al. (the contents of which are incorporated herein by reference in their entirety), or in liposomes comprising at least one amphiphilic lipid having both positive and negative charges and at least one neutral lipid, wherein the liposomes are stable at pH 4.2 to pH 7.5.

[0257] In some embodiments, polynucleotide (e.g., ASO) compositions may be formulated in liposomes containing sterol derivatives, as disclosed in Panzner's U.S. Patent No. 7,312,206 (Novosom) (the contents of which are incorporated herein by reference in their entirety).

[0258] In some embodiments, the pharmaceutical composition may be formulated in liposomes containing a serum-stable lipid mixture, as taught in Panzner et al., U.S. Patent Application Publication No. 20110076322 (the contents of which are incorporated herein by reference in their entirety), which can encapsulate the nucleic acid vaccine composition of the present disclosure. The lipid mixture contains phosphatidylcholine and phosphatidylethanolamine in a ratio ranging from about 0.5 to about 8. The lipid mixture may also contain pH-sensitive anionic and cationic amphiphilic substances such that the mixture is amphoteric, negatively charged or neutral at pH 7.4 and positively charged at pH 4. The drug / lipid ratio can be adjusted to target the liposomes to specific organs or other sites of the body. In some embodiments, the liposomes loaded with the nucleic acid vaccine composition of the present disclosure as cargo are prepared by the method disclosed in Panzner et al., U.S. Patent Application Publication No. 20120021042 (the contents of which are incorporated herein by reference in their entirety). The method comprises the steps of mixing an aqueous solution of a polyvalent anionic activator (e.g., a polynucleotide) with an alcohol solution of one or more amphiphilic substances, and buffering the mixture to an acidic pH, wherein one or more amphiphilic substances are sensitive to forming amphoteric liposomes at an acidic pH, thereby forming amphoteric liposomes in the suspension encapsulating the activator.

[0259] Lipoplex The compositions of this disclosure can be formulated using one or more lipoplexes. In some embodiments, antisense polynucleotide (e.g., ASO) compositions can be formulated as lipoplexes, for example, but not limited to, the ATUPLEX® system, the DACC system, the DBTC system and other RNA-lipoplex technologies of Silence Therapeutics (London, United Kingdom), STEMFECT® of STEMGENT® (Cambridge, MA), and polyethyleneimine (PEI).

[0260] In some embodiments, lipoplex-formulated RNA (RNA-LPX) can be produced by complexing RNA with liposomes containing / composed of the cationic lipid DOTMA and the helper lipid DOPE (Kranz et al., Nature, 2016;534(7607):396~401). RNA-lipoplexes may be formed for intravenous injection and possess pharmaceutical and physiological characteristics that enable selective targeting of antisense polynucleotides (e.g., ASOs) toward target cells, tissues, and / or organs. RNA-lipoplex products may be formed in the self-assembly process of RNA and dedicated cationic (positively charged) liposomes, including a topological transfer from liposomes to dense RNA-lipoplex nanoparticles having a specific internal molecular structure. The in vitro and in vivo efficacy of lipoplex formulations is evaluated by individually investigating the effects of parameters on biological activity, such as particle charge, size, molecular structure, lipid composition, and phase state. The ratio of cationic lipids to RNA is expressed as a charge ratio and determines the particle characteristics and target selectivity. RNA-lipoplex formulations can be formed using either an excess of positive (cationic liposomes) or negative (RNA) charge. Positive or negative charge lipoplexes can affect the delivery target. Therefore, variations in liposome characteristics used in lipoplex assembly and biological activity can be further controlled. In some embodiments, lipoplex formation can be achieved in the presence of various monovalent and divalent ions, peptides, and buffers.

[0261] Lipid nanoparticles (LNPs) In some embodiments, the antisense polynucleotides (e.g., ASOs) and compositions of this disclosure may be formulated with lipid nanoparticles (LNPs).

[0262] Generally, LNPs can be characterized as small solid or semi-solid particles having an outer lipid layer with a hydrophilic outer surface exposed to a non-LNP environment, an inner void that may be aqueous (vesicle-like) or non-aqueous (micelle-like), and at least one hydrophobic intermembrane void. The LNP membrane may be layered or non-layered and may consist of one, two, three, four, five or more layers.

[0263] The LNPs for formulating the antisense polynucleotides (e.g., ASOs) of this disclosure may have a diameter of 10 to 1000 nm. Nanoparticles are 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, 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, 305, 310, 3 15, 320, 325, 330, 335, 340, 345, 350, 355, 360, 365, 370, 375, 380, 385, 390, 395, 400, 405, 410, 415, 420, 425, 430, 435, 440, 445, 450, 455, 460, 465, 470, 475, 480, 485, 490, 495, 500, 505, 510, 515, 520, 525, 530, 535, 540, 545, 550, 555, 560, 565, 570, 575, 580, 585, 590, 595, 600, 60 5, 610, 615, 620, 625, 630, 635, 640, 645, 650, 655, 660, 665, 670, 675, 680, 685, 690, 695, 700, 705, 710, 715, 720, 725, 730, 735, 740, 745, 750, 755, 760, 765, 770, 775, 780, 785, 790, 795, 800, 805, 810, 815, 820, 825, 830, 835, 840, 845, 850, 855, 860, 865, 870, 875, 880, 885, 890, 895 , 900, 905, 910, 915, 920, 925, 930, 935, 940, 945, 950, 955, 960, 965, 970, 975, 980, 985, 990, 995 or 1000 nm, or possibly greater than 100 nm, greater than 150 nm, greater than 200 nm, greater than 250 nm, greater than 300 nm, greater than 350 nm, greater than 400 nm, greater than 450 nm, greater than 500 nm, greater than 550 nm, greater than 600 nm, greater than 650 nm, greater than 700 nm, greater than 750 nm, greater than 800 nm, greater than 850 nm, greater than 900 nm, 950 nm or greater than 1000 nm.

[0264] In some embodiments, lipid nanoparticles for formulating the antisense polynucleotide (e.g., ASO) of the Disclosure may have diameters of approximately 1 to approximately 100 nm, for example, but not limited to approximately 1 nm to approximately 10 nm, approximately 1 nm to approximately 20 nm, approximately 1 nm to approximately 30 nm, approximately 1 nm to approximately 40 nm, approximately 1 nm to approximately 50 nm, approximately 1 nm to approximately 60 nm, approximately 1 nm to approximately 70 nm, approximately 1 nm to approximately 80 nm, approximately 1 nm to approximately 90 nm, approximately 5 nm to approximately 100 nm, approximately 5 nm to approximately 10 nm, approximately 5 nm to approximately 20 nm, approximately 5 nm to approximately 30 nm, approximately 5 nm to approximately 40 nm, approximately 5 nm to approximately 50 nm, approximately 5 nm to approximately 60 nm, approximately 5 nm to approximately 70 nm, approximately 5 nm to approximately 80 nm, approximately 5 nm to approximately 90 nm and / or approximately 5 nm to approximately 100 nm.

[0265] In some embodiments, the lipid nanoparticles used to formulate the antisense polynucleotide (e.g., ASO) of the Disclosure may be approximately 10 to approximately 100 nm in size, for example, not limited to approximately 10 nm to approximately 20 nm, approximately 10 nm to approximately 30 nm, approximately 10 nm to approximately 40 nm, approximately 10 nm to approximately 50 nm, approximately 10 nm to approximately 60 nm, approximately 10 nm to approximately 70 nm, approximately 10 nm to approximately 80 nm, approximately 10 nm to approximately 90 nm, approximately 20 nm to approximately 30 nm, approximately 20 nm to approximately 40 nm, approximately 20 nm to approximately 50 nm, approximately 20 nm to approximately 60 nm, and approximately 20 nm. ~about 70nm, about 20nm to about 80nm, about 20nm to about 90nm, about 20nm to about 100nm, about 30nm to about 40nm, about 30nm to about 50nm, about 30nm to about 60nm, about 30nm to about 70nm, about 30nm to about 80nm, about 30nm to about 9 0nm, about 30nm to about 100nm, about 40nm to about 50nm, about 40nm to about 60nm, about 40nm to about 70nm, about 40nm to about 80nm, about 40nm to about 90nm, about 40nm to about 100nm, about 50nm to about 60nm, about 50nm to about 70nm It can have a diameter of approximately 50nm to 80nm, approximately 50nm to 90nm, approximately 50nm to 100nm, approximately 60nm to 70nm, approximately 60nm to 80nm, approximately 60nm to 90nm, approximately 60nm to 100nm, approximately 70nm to 80nm, approximately 70nm to 90nm, approximately 70nm to 100nm, approximately 80nm to 90nm, approximately 80nm to 100nm and / or approximately 90nm to 100nm.

[0266] In some embodiments, the lipid nanoparticles used to formulate the antisense polynucleotide (e.g., ASO) of the Disclosure may have a diameter of about 10 to about 500 nm.

[0267] In some embodiments, the LNPs used to formulate the antisense polynucleotides (e.g., ASOs) of this disclosure are less than 0.1 μm to 100 μm, for example, less than 0.1 μm, less than 1.0 μm, less than 5 μm, less than 10 μm, less than 15 μm, less than 20 μm, less than 25 μm, less than 30 μm, less than 35 μm, less than 40 μm, less than 50 μm, less than 55 μm, less than 60 μm, less than 65 μm, less than 70 μm, less than 75 μm, less than 80 μm, less than 85 μm, less than 90 μm, less than 95 μm, less than 100 μm, less than 125 μm, less than 150 μm, less than 175 μm, less than 200 μm, and 225 μm. These are small LNPs with diameters of less than μm, less than 250 μm, less than 275 μm, less than 300 μm, less than 325 μm, less than 350 μm, less than 375 μm, less than 400 μm, less than 425 μm, less than 450 μm, less than 475 μm, less than 500 μm, less than 525 μm, less than 550 μm, less than 575 μm, less than 600 μm, less than 625 μm, less than 650 μm, less than 675 μm, less than 700 μm, less than 725 μm, less than 750 μm, less than 775 μm, less than 800 μm, less than 825 μm, less than 850 μm, less than 875 μm, less than 900 μm, less than 925 μm, less than 950 μm, and less than 975 μm.

[0268] Useful LNPs as used herein are known in the art and generally include cholesterol (which aids stability and promotes membrane fusion), helper lipids (e.g., phospholipids that provide structure to the LNP bilayer and can also aid in endosomal escape), polyethylene glycol (PEG) derivatives (which reduce LNP aggregation, "shield" LNPs from nonspecific endocytosis by immune cells, and reduce opsonization and reticuloendothelial clearance by serum proteins), and ionizable lipids (which complex with negatively charged RNA and enhance endosomal escape), which form an LNP-forming composition. The components of the LNP may be selected based on the desired target, tropism, cargo (e.g., antisense polynucleotides), size, or other desired features or properties. The ionizable lipids, helper lipids, cholesterol, and PEG substantially influence the efficacy of the lipid nanoparticles and can be optimized for a given application and route of administration.

[0269] Generally, the antisense polynucleotide (e.g., ASO) compositions of this disclosure can be formulated using LNPs in the inner space, in the intermembrane space, on the outer surface, or any combination thereof.

[0270] The LNPs for formulating antisense polynucleotides (e.g., ASOs) of this disclosure comprise at least one cationic lipid. The cationic lipid can be selected from any lipid known in the art, such as DLin-MC3-DMA, DLin-DMA, C12-200, and DLin-KC2-DMA, but is not limited to these.

[0271] In some embodiments, cationic lipids that can be used in the formulations of the present disclosure are PCT Patent Applications International Publication Nos. 2012040184, 2011153120, 2011149733, 2011090965, 2011043913, 2011022460, 2012061259, 2012054365, 2012044638, and 2010080. Cationic lipids may be selected from, but are not limited to, those described in Patent No. 724, No. 201021865 and No. 2008103276, U.S. Patent No. 7,893,302, No. 7,404,969 and No. 8,283,333, and U.S. Patent Publication No. 20100036115 and No. 20120202871 (the contents of which are incorporated herein by reference in their entirety, respectively). Cationic lipids may also be selected from, but are not limited to, formula A as described in PCT Patent Applications International Publications Nos. 2012040184, 2011153120, 2011149733, 2011090965, 2011043913, 2011022460, 2012061259, 2012054365 and 2012044638 (each of which is incorporated herein by reference in its entirety). Alternatively, the cationic lipid may be selected from, but is not limited to, formulas CLI-CLXXIX of PCT Patent Application International No. 2008103276, formula CLI-CLXXIX of U.S. Patent No. 7,893,302, formula CLI-CLXXXXII of U.S. Patent No. 7,404,969, and formulas I-VI of U.S. Patent Application Publication No. 20100036115 (each of which is incorporated herein by reference in its entirety). The cationic lipid may also be a polyvalent cationic lipid disclosed in U.S. Patent No. 7,223,887 (each of which is incorporated herein by reference in its entirety), which has a positively charged head group containing two quaternary amine groups and a hydrophobic moiety containing four hydrocarbon chains.The cationic lipid may also be biodegradable, such as the biodegradable lipid disclosed in Maier et al.'s U.S. Patent Application Publication No. 20130195920 (the contents of which are incorporated herein by reference in their entirety), having one or more biodegradable groups located on the lipid portion of the cationic lipid. In some embodiments, the cationic lipid may also be the cationic lipid disclosed in Manoharan et al.'s U.S. Patent Application Nos. 20130156845 and 20130129785, Wasan et al.'s International Publication No. 2012047656, Chen et al.'s International Publication No. 2010144740, Ansell et al.'s International Publication No. 2013086322, and Manoharan et al.'s International Publication No. 2012016184 (the contents of which are incorporated herein by reference in their entirety, respectively).

[0272] Non-limiting examples of cationic lipids include (20Z,23Z)-N,N-dimethylnonacosa-20,23-dien-10-amine, (17Z,20Z)-N,N-dimethylhexacosa-17,20-dien-9-amine, (1Z,19Z)-N5N-dimethylpentacosa-16,19-dien-8-amine, (13Z,16Z)-N,N-dimethyldocosa-13,16-dien-5-amine, (12Z,15Z)-N,N-dimethylhenicosa-12,15-dien-4-amine, (14Z,17Z)-N,N-dimethyltricosa-14,17-dien-6-amine, and (15Z,18Z)-N,N-dimethyltetracosa-15,18-dien-7-amine. N, (18Z,21Z)-N,N-dimethylheptacosa-18,21-diene-10-amine, (15Z,18Z)-N,N-dimethyltetracosa-15,18-diene-5-amine, (14Z,17Z)-N,N-dimethyltricosa-14,17-diene-4-amine, (19Z,22Z)-N,N-dimethyloctacosa-19,22-diene-9-amine, (18Z,21 (Z)-N,N-dimethylheptacosa-18,21-diene-8-amine, (17Z,20Z)-N,N-dimethylhexacosa-17,20-diene-7-amine, (16Z,19Z)-N,N-dimethylpentacosa-16,19-diene-6-amine, (22Z,25Z)-N,N-dimethylhenthriaconta-22,25-diene-10-amine, (21Z (24Z)-N,N-dimethyltriaconta-21,24-dien-9-amine, (18Z)-N,N-dimethylheptacosa-18-en-10-amine, (17Z)-N,N-dimethylhexacosa-17-en-9-amine, (19Z,22Z)-N,N-dimethyloctacosa-19,22-dien-7-amine, N,N-dimethylheptacosa-10-amine, (20Z,23Z)-N-ethyl-N-methylnonacosa-20,23-dien-10-amine, 1-[(11Z,14Z)-l-nonylicosa-11,14-dien-l-yl]pyrrolidine, (20Z)-N,N-dimethylheptacosa-20-en-l O-amine, (15Z)-N,N-dimethyleptacosa-15-ene-l O-amine, (14Z)-N,N-dimethylnonacosa-14-ene-l O-amine, (17Z)-N,N-dimethylnonacosa-17-en-10-amine, (24Z)-N,N-dimethyltritriaconta-24-en-10-amine, (20Z)-N,N-dimethylnonacosa-20-en-1 0-amine, (22Z)-N,N-dimethylhenthriaconta-22-ene-10-amine, (16Z)-N,N-dimethylpentacosa-16-ene-8-amine, (12Z,15Z)-N,N-dimethyl-2-nonylhenicosa-12,15-dien-1-amine, (13Z,16Z)-N,N-dimethyl-3-nonyldocosa-13,16-dien-1-amine, N,N-dimethyl-1-[(1S,2R)-2-octylcyclopropyl]eptadecane-8-amine, 1-[(1S,2R)-2-hexylcyclopropyl]-N,N-dimethylnonadecane-10-amine, N,N-dimethyl-1-[(1S ,2R)-2-octylcyclopropyl]nonadecane-10-amine, N,N-dimethyl-21-[(1S,2R)-2-octylcyclopropyl]henicosane-10-amine, N,N-dimethyl-1-[(1S,2S)-2-{[(1S,2R)-2-pentylcisiopropyl]methyl}cyclopropyl]nonadecane-10-amine, N,N-dimethyl-1-[(1S,2R)-2-octylcyclopropyl]hexadecane-8-amine, N,N-dimethyl-[(1S,2R)-2-undecycyclopropyl]tetradecane-5-amine, N,N-dimethyl-3-{7-[(1S,2R)-2-octylcyclopropyl]heptyl}dodecane-1-amine, 1-[(1R,2S)-2-heptylcyclopropyl]-N,N-dimethyloctadecane-9-amine, 1-[(1S,2R)-2-decylcyclopropyl]-N,N-dimethylpentadecane-6-amine, N,N-dimethyl-l-[(lS,2R)-2-octylcyclopropyl]pentadecane-8-amine, RN,N-dimethyl-1-[(9Z,12Z)-octadeca-9,12-diene-1-yloxy]-3-(octyloxy)propane-2-amine, SN,N-dimethyl-1-[(9Z,12Z)-octadeca-9,12-diene-1-yloxy]-3-(octyloxy)propane-2-amine, 1-{2-[(9Z,12Z)-octadeca-9,12-diene-1-yloxy]-1-[(octyloxy)methyl]ethyl}pyrrolidine, (2S)-N,N-dimethyl-1-[(9Z,12Z)-octadeca-9,12-diene-1-yloxy]-3-[(5Z)-octa-5-ene-1-yloxy]propan-2-amine, 1-{2-[(9Z,12Z)-octadeca-9,12-diene-1-yloxy]-1-[(octyloxy)methyl]ethyl}azetidine, (2S)-1-(hexyloxy)-N,N-dimethyl-3-[(9Z,12Z)- Octadeca-9,12-diene-1-yloxy]propan-2-amine, (2S)-1-(heptyloxy)-N,N-dimethyl-3-[(9Z,12Z)-octadeca-9,12-diene-1-yloxy]propan-2-amine, N,N-dimethyl-1-(nonyloxy)-3-[(9Z,12Z)-octadeca-9,12-diene-1-yloxy]propan-2-amine, N,N-dimethyl-1-[(9Z)-octadeca-9-en-1-yloxy]-3-(octyloxy)propan-2-amine; (2S)-N,N-dimethyl-1-[(6Z,9Z,12Z)-octadeca-6,9,12-triene-1-yloxy]-3-(octyloxy)propan-2-amine, (2S)-1-[(11Z,14Z)-icosa-11,14-diene-1-yloxy]-N,N-dimethyl-3-(pentyloxy)propan-2-amine, (2S)-1-(hexyloxy)-3-[(11Z,14Z)-icosa-11,14-diene-1-yloxy]-N,N-dimethylpropan-2-amine, 1-[(11Z,14Z)-icosa-11,14-diene-1-yloxy]-N,N -Dimethi-1-3-(octyloxy)propan-2-amine, 1-[(13Z,16Z)-docosa-13,16-diene-1-yloxy]-N,N-dimethyl-3-(octyloxy)propan-2-amine, (2S)-1-[(13Z,16Z)-docosa-13,16-diene-1-yloxy]-3-(hexyloxy)-N,N-dimethylpropan-2-amine, (2S)-1-[(13Z)-docosa-13-en-1-yloxy]-3-(hexyloxy)-N,N-dimethylpropan-2-amine, 1-[(13Z)-docosa-13-en-1-yloxy]-N,N-dimethyl-3-(octyloxy)propan-2-amine, 1-[(9Z)-hexadeca-9-en-1-yloxy]-N,N-dimethyl-3-(octyloxy)propan-2-amine, (2R)-N,N-dimethyl-H(1-methoyloctyl)oxy]-3-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]propan-2-amine, (2R)-1-[(3,7-dimethyloctyl)oxy]-N,N-dimethyl-3-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]propan-2 -amines, N,N-dimethyl-1-(octyloxy)-3-({8-[(1S,2S)-2-{[(1R,2R)-2-pentylcyclopropyl]methyl}cyclopropyl]octyl}oxy)propan-2-amine, N,N-dimethyl-1-{[8-(2-octylcyclopropyl)octyl]oxy}-3-(octyloxy)propan-2-amine, and (1IE,20Z,23Z)-N,N-dimethylnonacosa-1,20,2-triene-10-amine, or pharmaceutically acceptable salts or stereoisomers thereof can be selected.

[0273] In some embodiments, LNPs for formulating antisense polynucleotide (e.g., ASO) compositions may include a plurality of cationic lipids, e.g., first and second cationic lipids described in Hope et al., U.S. Patent Application Publication 20130017223 (which is incorporated herein by reference in its entirety). The first cationic lipid may be selected based on a first property, and the second cationic lipid may be selected based on a second property. The first and second properties may be complementary. In some embodiments, the LNP may comprise one or more first cationic lipids and one or more second cationic lipids, where the lipid particles comprise a solid core described in Cullis et al., U.S. Patent Application Publication 20120276209 (which is incorporated herein by reference in its entirety).

[0274] In some embodiments, LNP may contain one or more ionizable lipids, such as C12-200, DLin-KC2-DMA and / or HGT5001, helper lipids, structural lipids, PEG-modified lipids, MC3, DLinDMA, DLinkC2DMA, cKK-El2, ICE, HGT5000, DODAC, DDAB, DMRIE, DOSPA, DOGS, DODAP, DODMA, DMDMA, DODAC, DLenDMA, DMRIE, ClinDMA, CpLinDMA, DMOBA, DOcarbDAP, DLinDAP, DLincarbDAP, DLinCDAP, KLin-K-DMA, DLin-K-XTC2-DMA, HGT4003, and combinations thereof.

[0275] In some embodiments, cationic lipids can be synthesized by methods known in the art and / or described in PCT Patent Applications International Publications 2012040184, 2011153120, 2011149733, 2011090965, 2011043913, 2011022460, 2012061259, 2012054365, 2012044638, 2010080724 and 201021865 (each of which is incorporated herein by reference in its entirety).

[0276] In some embodiments, the nanoparticles described herein may include at least one cationic polymer described herein and / or known in the art.

[0277] LNPs for formulating antisense polynucleotides (e.g., ASOs) according to this disclosure may contain at least one helper lipid. The helper lipid in the LNP contributes to the stability and delivery efficiency of the LNP due to its cationic properties and / or can mitigate toxicity. In some embodiments, the helper lipid is a lipid having a conical geometry, such as dioleoylphosphatidylethanolamine (DOPE). In some embodiments, the helper lipid is a cylindrical lipid, such as phosphatidylcholine.

[0278] In some embodiments, the LNPs for formulating the antisense polynucleotides (e.g., ASOs) of this disclosure may include, but are not limited to, phospholipids such as DDPC, DLPC, DMPC, DPPC, DSPC, DOPC, POPC, and DEPC; DMPG, DPPG, DSPG, and POPG; DMPA, DPPA, and DSPA; DMPE, DPPE, DSPE, and DOPE; DOPS, as well as synthetic phospholipids and polyglycerol-bound phospholipids (PG phospholipids). The phospholipids may be selected based on the route of administration; for example, DPPC, POPC, and POPG may be used for the LNPs in injection, and DOPC, POPC, and DDPC may be used for the LNPs in lung delivery. In some embodiments, the phospholipids may be purified lipids from natural sources.

[0279] In some embodiments, the LNP for formulating the antisense polynucleotide (e.g., ASO) of the present disclosure may include one or more neutral helper lipids, such as dioleoylphosphoethanolamine (DOPE), prostaglandins, eicosanoids, glycerides, glycosylated diacylglycerols, oxygenated fatty acids, NAGly, and PAHSA.

[0280] The LNPs for formulating antisense polynucleotides (e.g., ASOs) of this disclosure include cholesterol, naturally occurring cholesterol analogs or synthetic cholesterol-like compounds and cholesterol derivatives. In some embodiments, the naturally occurring cholesterol analogs may be selected from those provided by Patel et al. (Nature Communications, 2020;983:doi.org / 10.1038 / s41467-020-14527-2) (the contents of which are incorporated herein by reference in their entirety). In some embodiments, the LNP includes one or more cholesterol derivatives, e.g., PtdChol.

[0281] The LNPs for formulating antisense polynucleotides (e.g., ASOs) of this disclosure include at least PEGylated compounds, such as PEG polymers and PEGylated lipids.

[0282] In some embodiments, the antisense polynucleotide (e.g., ASO) compositions of the present disclosure may contain at least one PEGylated lipid as described in PCT Patent Application International Publication No. 2012099755 (the contents of which are incorporated herein by reference in their entirety).

[0283] In some embodiments, the ratio of PEG in lipid nanoparticle (LNP) formulations can be increased or decreased, and / or the carbon chain length of PEG lipids can be modified to C14-C18 to alter the pharmacokinetics and / or biodistribution of LNP formulations.

[0284] In some embodiments, the LNP for formulating the antisense polynucleotide (e.g., ASO) of the Disclosure comprises PEG-c-DOMG. In some embodiments, PEG-c-DOMG may be replaced with a PEG lipid, for example, but not limited to PEG-DSG (1,2-distearoyl-sn-glycerol, methoxypolyethylene glycol), PEG-DPG (1,2-dipalmitoyl-sn-glycerol, methoxypolyethylene glycol), or PEG-DMG 2000 (1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000]). In non-limiting examples, the LNP formulation may contain PEG-DMG 2000, DLin-DMA, DSPC, and cholesterol. As another non-limiting example, an LNP preparation may contain PEG-DMG 2000, DLin-DMA, DSPC, and cholesterol in a molar ratio of 2:40:10:48 (see, for example, Geall et al., PNAS, 2012, 109(36):14604-14609, the whole of which is incorporated herein by reference).

[0285] In some embodiments, the LNP formulations described herein may include a polycationic composition. For example, the polycationic composition may be selected from formulas 1 to 60 of U.S. Patent Application Publication No. 20050222064 (the contents of which are incorporated herein by reference in their entirety). LNP formulations comprising a polycationic composition may be used for the in vivo and / or in vitro delivery of the nucleic acid vaccine compositions described herein.

[0286] Lipid nanoparticle formulations can be improved by replacing cationic lipids with biodegradable cationic lipids, known as rapidly eliminated lipid nanoparticles (reLNPs). Ionizable cationic lipids, such as but not limited to DLinDMA, DLin-KC2-DMA, and DLin-MC3-DMA, have been shown to accumulate in plasma and tissues over time and can be a potential source of toxicity. The rapid metabolism of rapidly eliminated lipids can improve the tolerability and therapeutic index of lipid nanoparticles. The inclusion of enzymatically degradable ester linkages can improve the degradation and metabolic profile of the cationic components while simultaneously maintaining the activity of the reLNP formulation. Ester linkages may be internally positioned within the lipid chain or terminally positioned at the ends of the lipid chain. Internal ester linkages can replace any carbon in the lipid chain.

[0287] In some embodiments, the LNPs for formulating the antisense polynucleotides (e.g., ASOs) of this disclosure may include cleavable lipids, such as those described in PCT Patent Application International Publication No. 2012170889 (the contents of which are incorporated herein by reference in their entirety).

[0288] In some embodiments, the LNP for formulating the antisense polynucleotide (e.g., ASO) compositions of this disclosure may include a conjugate lipid. In non-limiting examples, the conjugate lipid may have a formula, for example, that described in Lin et al., U.S. Patent Application Publication No. 20120264810 (the contents of which are incorporated herein by reference in their entirety). The conjugate lipid may form lipid particles further comprising cationic lipids, neutral lipids, and lipids that can reduce aggregation.

[0289] In some embodiments, the LNP for formulating the antisense polynucleotides (e.g., ASOs) of this disclosure may include a mixture of a cationic compound and a neutral lipid. In non-limiting examples, the cationic compound may be formula (I) as disclosed in Ansell et al., PCT Patent Application International Publication No. 1999010390 (the contents of which are incorporated herein by reference in their entirety), and the neutral lipid may be selected from the group consisting of diacylphosphatidylcholine, diacylphosphatidylethanolamine, ceramide, and sphingomyelin.

[0290] In some embodiments, the LNP formulations described herein may additionally include permeable enhancer molecules. Non-limiting permeable enhancer molecules are described in U.S. Patent Application Publication No. 20050222064, which is incorporated herein by reference in its entirety.

[0291] In some embodiments, LNPs for formulating the antisense polynucleotide (e.g., ASO) compositions of the present disclosure may be encapsulated in any polymer known in the art that can form a gel when injected into a subject. In another non-limiting example, lipid nanoparticles may be encapsulated in a polymer matrix that may be biodegradable.

[0292] In some embodiments, LNPs for formulating the antisense polynucleotide (e.g., ASO) compositions of this disclosure can be encapsulated with a lipid formulation to form stable nucleic acid lipid particles (SNALPs), for example, as described in U.S. Patent No. 8,546,554 by de Fougerolles et al. (the contents of which are incorporated herein by reference in their entirety). In one non-limiting example, the SNALP comprises 40% 2,2-dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane (lipid A), 10% dioleoylphosphatidylcholine (DSPC), 40% cholesterol, and 10% polyethylene glycol (PEG)-C-DOMG (mol percent), with a particle size of 63.0 ± 20 nm and a nucleic acid / lipid ratio of 0.027.

[0293] In some embodiments, the LNP for formulating the antisense polynucleotide (e.g., ASO) compositions of this disclosure may include endosomal membrane destabilizers, as disclosed in U.S. Patent No. 7,189,70 by Lam et al. (the contents of which are incorporated herein by reference in their entirety). In a non-limiting example, the endosomal membrane destabilizer may be Ca 2+ It could be an ion.

[0294] In some embodiments, the LNPs for formulating the antisense polynucleotide (e.g., ASO) compositions of this disclosure may include charged lipids or aminolipids. As used herein, the term “charged lipid” is intended to include lipids having one or two fatty acyl or fatty alkyl chains and a quaternary amino head group. Quaternary amines carry a permanent positive charge. The head group may optionally include an ionizable group, e.g., a primary, secondary, or tertiary amine that can be prontated at physiological pH. The presence of a quaternary amine may alter the pKa of the ionizable group compared to the pKa of the group in structurally similar compounds lacking a quaternary amine (e.g., the quaternary amine is replaced by a tertiary amine). In non-limiting examples, the charged lipids used in any of the formulations described herein may be any of the charged lipids described in Manoharan et al. European Patent No. EP2509636 (the contents of which are incorporated herein by reference in their entirety). In some embodiments, charged lipids are referred to as “aminolipids.” In an unspecified example, the aminolipid may be any aminolipid described in Hope et al.'s U.S. Patent Application Publication No. 20110256175 (the contents of which are incorporated herein by reference in their entirety). For example, the aminolipid may have structures (II) disclosed in Tables 3-7 of Hope, e.g., DLin-K-C2-DMA, DLin-K2-DMA, DLin-K6-DMA, etc. In another unspecified example, the aminolipid may be any aminolipid described in Hope et al.'s U.S. Patent Application No. 20110117125 (the contents of which are incorporated herein by reference in their entirety), e.g., a lipid of structure (I) such as DLin-K-DMA, DLin-C-DAP, DLin-DAC, DLin-MA, DLin-S-DMA, etc. In another non-limiting example, the aminolipid may have structures (I), (II), (III), or (IV) as described in Manoharan et al.'s PCT Patent Application International Publication No. 2009132131 (the contents of which are incorporated herein by reference in their entirety), or 4-(R)-DLin-K-DMA(VI), 4-(S)-DLin-K-DMA(V).

[0295] In some embodiments, the LNP for formulating the antisense polynucleotide (e.g., ASO) compositions of the present disclosure may include, for example, a reverse head-based lipid formulated with a zwitterionic lipid containing a head group, wherein the positive charge is located near the acyl chain region and the negative charge is located at the distal end of the head group, and the lipid has structure (A) or structure (I) as described, for example, in Leung et al. PCT Patent Application International Publication No. 2011056682 (the contents of which are incorporated herein by reference in their entirety).

[0296] In some embodiments, the ratio (mass / mass ratio) of the lipid components of the LNP to the nucleic acid (e.g., the ratio of lipids to polynucleotides (e.g., ASOs) composition) is in the range of approximately 1:1 to approximately 50:1, approximately 1:1 to approximately 25:1, approximately 3:1 to approximately 15:1, approximately 4:1 to approximately 10:1, approximately 5:1 to approximately 9:1, or approximately 6:1 to approximately 9:1, or 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 11:1, 12:1, 13:1 , 14:1, 15:1, 16:1, 17:1, 18:1, 19:1, 20:1, 21:1, 22:1, 23:1, 24:1, 25:1, 26:1, 27:1, 28:1, 29:1, 30:1, 31:1, 32:1, 33:1, 34:1, 35:1, 36:1, 37:1, 38:1, 38:1, 39:1, 40:1, 41:1, 42:1, 43:1, 44:1, 45:1, 46:1, 47:1, 48:1, 49:1 or 50:1.

[0297] In some embodiments, LNP formulations can be formulated by the methods described in International Publication No. 2011127255 or WO2008103276. In non-limiting examples, the antisense polynucleotide (e.g., ASO) compositions of this disclosure may be encapsulated with any of the lipid nanoparticle (LNP) formulations described in International Publication No. 2011127255 and / or WO2008103276 (the contents of which are each incorporated herein by reference in their entirety).

[0298] As a non-limiting example, LNPs may be lipid nanoparticles as described in PCT Patent Application International Publication No. 2012170930 (the contents of which are incorporated herein by reference in their entirety). As another non-limiting example, lipid formulations may include cationic lipids of formula A, neutral lipids, sterols, and PEG or PEG-modified lipids as disclosed in Akinc et al. U.S. Patent Application Publication No. 20120101148 (the contents of which are incorporated herein by reference in their entirety).

[0299] As another non-limiting example, LNPs may be nanoparticles delivered by parenteral routes, as described in U.S. Patent Application Publication No. 20120207845 (the contents of which are incorporated herein by reference in their entirety).

[0300] In some embodiments, an antisense polynucleotide (e.g., ASO) composition can be encapsulated in rapidly excluded lipid nanoparticles, which can then be encapsulated with polymers, hydrogels, and / or surgical sealants described herein and / or known in the art. Non-limiting examples of polymers, hydrogels, or surgical sealants include PLGA, ethylene vinyl acetate (EVAc), poloxamer, GELSITE® (Nanotherapeutics, Inc. Alachua, FL), HYLENEX® (Halozyme Therapeutics, San Diego, CA), surgical sealants such as fibrinogen polymer (Ethicon Inc. Cornelia, GA), TISSELL® (Baxter International, Inc., Deerfield, IL), PEG-based sealants, and COSEAL® (Baxter International, Inc., Deerfield, IL).

[0301] In some embodiments, antisense polynucleotide (e.g., ASO) compositions may be formulated with solid lipid nanoparticles (SLNs). The solid lipid nanoparticles (SLNs) may be spherical, having an average diameter between 10 and 1000 nm or between 100 and 1000 nm. The SLNs have a solid lipid core matrix capable of solubilizing lipophilic molecules and may be stabilized with surfactants and / or emulsifiers. The lipid nanoparticles may be self-assembling lipid polymer nanoparticles (see Zhang et al., ACS Nano, 2008, 2(8):1696-1702, the entire content of which is incorporated herein by reference).

[0302] In some embodiments, the LNPs for formulating the antisense polynucleotide (e.g., ASO) compositions of this disclosure may include targeted lipids having a targeted moiety, for example, the targeted moiety disclosed in Manoharan et al., U.S. Patent Application Publication 20130202652 (the contents of which are incorporated herein by reference in their entirety). Non-limiting examples include the targeted moiety of Formula I of Manoharan et al., U.S. Patent Application Publication 20130202652, which may be selected so as to be advantageous for the lipid to be localized to a desired organ, tissue, cell, cell type or subtype, or organelle. Non-limiting targeted moieties intended in this disclosure include transferrin, anisamide, RGD peptide, prostate-specific membrane antigen (PSMA), fucose, antibody, or aptamer.

[0303] Micelle In some embodiments, antisense polynucleotide (e.g., ASO) compositions may be formulated in micelles. The term “micelle,” as used herein, refers to any water-soluble aggregate spontaneously and reversibly formed from an amphiphilic compound or ion. The size of the micelles may be small, less than 10 nm, or less than 9 nm, or less than 8 nm, or less than 7 nm, or less than 6 nm.

[0304] In some embodiments, antisense polynucleotide (e.g., ASO) compositions may be formulated in reverse micelles. Reverse micelles are microemulsions comprising a dispersion of water-in-oil nanodroplets. A reverse micelle may be defined as a system in which water forms the inner phase and the hydrophobic tails of lipids form the continuous phase. Reverse micelles may also contain phospholipids or sphingolipids. Reverse micelles containing oil, a surfactant, an auxiliary surfactant, and an aqueous phase are also characterized as water-in-oil microemulsions. In non-limiting examples, reverse micelles may be prepared using the method described in U.S. Patent No. 8,877,237 (the contents of which are incorporated herein by reference in their entirety). Reverse micelles may be formulated for the absorption of antisense polynucleotides delivered to mucous membranes, e.g., the oral, nasal, and / or rectal mucosa.

[0305] In some embodiments, the antisense polynucleotide composition may be formulated in micelles using a temperature-sensitive polymer. The micelles can be coated with glucose, which allows for meaningful delivery of the antisense polynucleotide composition to the brain. The temperature-sensitive copolymer comprises a cationic block (e.g., a cationic amino acid polymer block) and a temperature-sensitive block (e.g., polyethylene glycol). In non-limiting examples, the temperature-sensitive polymer may be prepared by the method described in PCT International Publication No. 2016186204 (the contents of which are incorporated herein by reference in their entirety).

[0306] exosomes and ECVs Exosomes and extracellular vesicles (ECVs) are small membrane vesicles of intracellular origin that are released into the extracellular environment after fusion of the polysodeoxycholic body with the cell membrane. Exosomes and ECVs can transport large molecules suitable for nucleic acid delivery vehicles (e.g., Raposo, 2013, J Cell Biol, 200:373 and Validi, 2007, Nat Cell Biol, 9:654). Exosomes can be as small as 10-200 nm, such as 10-150 nm, 20-180 nm, 40-120 nm, or 10 nm, 20 nm, 30 nm, 40 nm, 50 nm, 60 nm, 70 nm, 80 nm, 100 nm, 120 nm, 140 nm, 160 nm, 180 nm, and 200 nm. In some embodiments, the exosomes and ECVs may be large in size, ranging from 100 to 1000 nm or 100 to 600 nm.

[0307] In some embodiments, antisense polynucleotide (e.g., ASO) compositions may be formulated using extracellular vesicles (ECVs) and / or exosomes. Exosomes may be produced using exosome-producing cells. In non-limiting examples, antisense polynucleotide-exosome formulations may be produced using methods known in the art and / or described in PCT International Publication No. 2017054085 (the contents of which are incorporated herein by reference in their entirety).

[0308] In some embodiments, antisense polynucleotide (e.g., ASO) compositions can be formulated into exosomes by transferring the antisense polynucleotide (e.g., ASO construct) to exosome-producing cells using any method known in the art, for example, but not limited to, electroporation, transfection using a transfection agent, such as lipofection, heat shock, and transformation using viral infection. Non-limiting examples include the preparation of polynucleotide-exosome formulations using the methods described in U.S. Patent No. 10,695,443 (the contents of which are incorporated herein by reference in their entirety).

[0309] In some embodiments, the antisense polynucleotide (e.g., ASO) compositions of the Disclosure may be formulated using a targeted and modular exosome loading system. The targeted exosome may contain a fusion protein comprising an RNA-binding domain and an exosome-targeting domain. The RNA-binding domain of the fusion protein may be bound to the antisense polynucleotide (e.g., ASO) of the Disclosure so that the antisense polynucleotide is packaged inside the exosome. The exosome-targeting domain may include the exosome-targeting domain of a lysosome-associated protein (e.g., LAMP and LIMP).

[0310] Other nanoparticles and delivery agents In some embodiments, the antisense polynucleotide (e.g., ASO) compositions of this disclosure may be complexed with a cationic amphiphilic substance in an oil-in-water (o / w) emulsion, for example, as described in European Patent Publication No. 2298358 by Satishchandran et al. (the entire contents of which are incorporated herein by reference). The cationic amphiphilic substance may be a cationic lipid, modified or unmodified spermine, bupivacaine, or benzalkonium chloride, and the oil may be a vegetable oil or an animal oil. In a non-limiting example, at least 10% of the nucleic acid cationic amphiphilic substance complex may be present in the oily phase of the oil-in-water emulsion (see, for example, the complex described in European Patent Publication No. 2298358 by Satishchandran et al., the entire contents of which are incorporated herein by reference).

[0311] The antisense polynucleotide (e.g., ASO) compositions of this disclosure may be formulated with carbohydrate nanoparticles comprising a carbohydrate carrier. Examples of carbohydrate carriers that are not limited to these include, but are not limited to, anhydrous-modified phytoglycogen or glycogen-type materials, phytoglycogen octanyl succinate, phytoglycogen betadextrin, and anhydrous-modified phytoglycogen betadextrin. (See, for example, PCT Patent Application International Publication 2012109121, the entirety of which is incorporated herein by reference).

[0312] The antisense polynucleotide (e.g., ASO) compositions of the present disclosure may be encapsulated on synthetic nanocarriers, linked to synthetic nanocarriers, and / or associated with synthetic nanocarriers. The synthetic nanocarriers include PCT Patent Application International Publications No. 2010005740, 2010030763, 201213501, 2012149252, 2012149255, 2012149259, 2012149265, 2012149268, 2012149282, 2012149301, and 2012 Examples include, but are not limited to, those described in Patent Nos. 149393, 2012149405, 2012149411, 2012149454, and 2013019669, as well as U.S. Patent Application Publications 20110262491, 20100104645, 20100087337, and 20120244222. Synthetic nanocarriers may be formulated using methods known in the art and / or described herein, for example, by methods described in PCT International Patent Applications Nos. 2010005740, 2010030763 and 201213501, and U.S. Patent Applications Nos. 20110262491, 20100104645, 20100087337 and 2012024422. Synthetic nanocarrier formulations may be lyophilized by methods described in PCT International Patent Application No. 2011072218 and U.S. Patent No. 8,211,473 (the contents of which are incorporated herein by reference in their entirety).

[0313] In some embodiments, the synthetic nanocarrier may contain a reactive group that releases the antisense polynucleotide (e.g., ASO) composition of the Disclosure (see International Publication No. 20120952552 and U.S. Patent Application Publication No. 20120171229, the entire contents of which are incorporated herein by reference). The synthetic nanocarrier may be formulated to release the antisense polynucleotide (e.g., ASO) composition after a specific pH and / or a desired time interval, for example, 24 hours and / or at a pH of 4.5 (see International Publication Nos. 2010138193 and 2010138194 and U.S. Patent Application Publication Nos. 20110020388 and 20110027217, the entire contents of which are incorporated herein by reference).

[0314] In some embodiments, synthetic nanocarriers may be formulated for targeted release, control, and / or sustained release of antisense polynucleotide (e.g., ASO) compositions of the Disclosure. In non-limiting examples, synthetic nanocarriers for sustained release may be formulated by methods known in the Art, described herein, and / or PCT Patent Application International Publication 2010138192 and U.S. Patent Application Publication 20100303850 (the contents of which are incorporated herein by reference in their entirety).

[0315] In some embodiments, the antisense polynucleotide (e.g., ASO) compositions of this disclosure may be formulated as modular compositions, for example, those described in U.S. Patent No. 8,575,123 by Manoharan et al. (the contents of which are incorporated herein by reference in their entirety). The modular compositions may include at least one endosomal soluble component and at least one targeted ligand.

[0316] In some embodiments, the antisense polynucleotide (e.g., ASO) compositions of this disclosure may be formulated in formulated lipid particles (FLip) as disclosed in U.S. Patent No. 8,148,344 by Akinc et al. (the contents of which are incorporated herein by reference in their entirety).

[0317] In some embodiments, the antisense polynucleotide (e.g., ASO) compositions of this disclosure can be fully encapsulated in lipid particles as disclosed in Maurer et al., U.S. Patent Application Publication No. 20120276207 (the contents of which are incorporated herein by reference in their entirety). The lipid particles may include a lipid composition comprising a cationic lipid having formula A, or a pre-forming lipid vesicle, and an activator and an stabilizer that forms a mixture.

[0318] In some embodiments, the antisense polynucleotide (e.g., ASO) compositions of this disclosure may be formulated as neutral liposome formulations, for example, as disclosed in U.S. Patent Application Publication No. 20120244207 by Fitzgerald et al. (the contents of which are incorporated herein by reference in their entirety). The term “neutral liposome formulation” refers to a liposome formulation having a near-neutral or neutral surface charge at a physiological pH (e.g., about 7.0).

[0319] In some embodiments, the antisense polynucleotide (e.g., ASO) compositions of the present disclosure can be formulated on a lipid bilayer carrier, where the antisense polynucleotide (e.g., ASO) composition can be combined with a lipid detergent mixture comprising an anti-aggregating agent, a lipid mixture of cationic lipids and membrane-fusion lipids, and a detergent. The nucleic acid lipid detergent mixture is dialyzed with a buffer salt solution to remove the detergent, and the nucleic acid is encapsulated on the lipid bilayer carrier to provide a lipid bilayer nucleic acid composition (see, for example, Cullis et al., PCT Patent Application International Publication No. 1999018933, whose entire contents are incorporated herein by reference).

[0320] In some embodiments, formulations comprising the antisense polynucleotide (e.g., ASO) compositions described herein may also be constructed or modified to be passively or actively directed in vivo to different cell types, including but not limited to immune cells, endothelial cells, antigen-presenting cells, and leukocytes (Akinc et al., Mol Ther. 2010, 18:1357~1364; Song et al., Nat Biotechnol. 2005, 23:709~717; Judge et al., J Clin Invest. 2009, 119:661~673; Kaufmann et al., Microvasc Res, 2010, 80:286~293; Santel et al., Gene Ther 2006, 13:1222~1234; Santel et al., Gene Ther, 2006, 13:1360~1370; Gutbier et al., Pulm Pharmacol. Ther. 2010, 23:334-344; Basha et al., Mol. Ther. 2011, 19:2186-2200; Fenske and Cullis, Expert Opin Drug Deliv. 2008, 5:25-44; Peer et al., Science. 2008, 319:627-630; Peer and Lieberman, Gene Ther. 2011, 18:1127-1133; (These contents are incorporated herein by reference in their entirety).

[0321] Examples of passive targeting of hepatocytes with pharmaceutical formulations include DLin-DMA, DLin-KC2-DMA, and DLin-MC3-DMA-based lipid formulations, which have been shown to bind to apolipoprotein E and promote the binding and uptake of these formulations into hepatocytes in vivo (Akinc et al., Mol Ther. 2010, 18:1357~1364). LNPs can also be coated with cell-specific ligands for selective targeting.Examples of ligands include folic acid, transferrin, N-acetylgalactosamine (GalNAc), and antibodies, as well as / or fragment targeting techniques (Kolhatkar et al., Curr Drug Discov Technol. 2011, 8:197~206; Musacchio and Torchilin, Front Biosci. 2011, 16:1388~1412; Yu et al., Mol Membr Biol. 2010, 27:286~298; Patil et al., Crit Rev Ther Drug Carrier Syst. 2008, 25:1~61; Benoit et al., Biomacromolecules. 2011, 12:2708~2714; Zhao et al., Expert Opin Drug Deliv. 2008, 5:309~319; Akinc et al., Mol Ther. 2010, 18:1357~1364; Srinivasan et al., Methods Mol Biol. 2012, 820:105~116; Ben~Arie et al., Methods Mol Biol. 2012, 757:497~507; Peer J Control Release. 2010, 20:63~68; Peer et al., Proc Natl Acad Sci US A. 2007, 104:4095~4100; Kim et al., Methods Mol Biol. 2011, 721:339~353; Subramanya et al., Mol Ther. 2010, 18:2028~2037; Song et al., Nat Biotechnol. 2005, 23:709~717; Peer et al., Science. 2008, 319:627-630; Peer and Lieberman, Gene Ther. 2011, 18:1127-1133; the contents of these works are incorporated herein by reference in their entirety.

[0322] In some embodiments, formulations comprising the antisense polynucleotide (e.g., ASO) compositions described herein are disease target specific.

[0323] In some embodiments, formulations comprising the polynucleotide (e.g., ASO) compositions described herein may also be constructed or modified to suit different routes of administration, e.g., parenteral (intravenous, intramuscular, or subcutaneous), oral, rectal, intraocular, and / or topical administration. Non-limiting examples include formulations described herein which may be optimized for oral administration by comprising at least one cationic biopolymer, e.g., chitosan or a derivative thereof (see U.S. Patent Application Publication No. 20120282343, the entirety of which is incorporated herein by reference).

[0324] In some embodiments, the antisense polynucleotide (e.g., ASO) compositions of this disclosure may be formulated for controlled release and / or targeted delivery. As used herein, “controlled release” refers to a pharmaceutical composition or compound release profile that matches a specific release pattern affecting therapeutic outcomes. As used herein, “sustained release” refers to a pharmaceutical composition or compound that matches a release rate over a specific period of time. This period may be, but is not limited to, hours, days, weeks, months, or years. In some embodiments, the antisense polynucleotide (e.g., ASO) compositions may be encapsulated with delivery agents described herein and / or known in the art for controlled release and / or targeted delivery. As used herein, the term “encapsulation” means sealing, surrounding, or wrapping. In relation to the formulation of the compositions of this disclosure, encapsulation may be substantial, complete, or partial. The term “substantially encapsulated” means that at least 50, 60, 70, 80, 85, 90, 95, 96, 97, 98, 99, 99.9, more than 99.9, or more than 99.999% of the pharmaceutical composition of the Disclosure may be sealed, surrounded, or enclosed within the delivery agent. “Partially encapsulated” means that less than 10, 10, 20, 30, 40, or 50 or less of the pharmaceutical composition or compound of the Disclosure may be sealed, surrounded, or enclosed within the delivery agent. Advantageously, encapsulation may be determined by measuring the elution or activity of the pharmaceutical composition of the Disclosure using fluorescence and / or electron microscopy. For example, at least 1, 5, 10, 20, 30, 40, 50, 60, 70, 80, 85, 90, 95, 96, 97, 98, 99, 99.9, 99.99, or more than 99.99% of the pharmaceutical composition of the present disclosure is encapsulated with a delivery agent.

[0325] In some embodiments, formulations comprising an antisense polynucleotide (e.g., ASO) composition for controlled release and / or targeted delivery may also include at least one controlled release coating. Examples of controlled release coatings include, but are not limited to, OPADRY®, polyvinylpyrrolidone / vinyl acetate copolymer, polyvinylpyrrolidone, hydroxypropyl methylcellulose, hydroxypropylcellulose, hydroxyethylcellulose, EUDRAGIT RL®, EUDRAGIT RS®, and cellulose derivatives, such as aqueous dispersions of ethylcellulose (AQUACOAT® and SURELEASE®). In some embodiments, controlled release and / or targeted delivery formulations may include at least one biodegradable polyester that may contain polycationic side chains.

[0326] In some embodiments, formulations comprising an antisense polynucleotide (e.g., ASO) composition may be formulated with sustained-release nanoparticles containing polymers described in PCT Patent Application International Publication No. 2010075072 and U.S. Patent Applications Publications No. 20100216804, 20110217377, and 20120201859 (the contents of which are incorporated herein by reference in their entirety).

[0327] Excipients and adjuvants In some embodiments, the pharmaceutical formulation may additionally contain pharmaceutically acceptable excipients, which, as used herein, may be any solvent, dispersion medium, diluent or other liquid vehicle, dispersion or suspension aid, surfactant, isosmolarity agent, thickener or emulsifier, preservative, solid binder, lubricant, flavoring agent, stabilizer, antioxidant, osmotic pressure regulator, pH regulator, etc., to suit a particular desired dosage form. Various excipients for formulating pharmaceutical compositions and techniques for preparing compositions are known in the art (see Remington: The Science and Practice of Pharmacy, 21" Edition, AR Gennaro, (Lippincott, Williams & Wilkins, Baltimore, Md, 2006), which is incorporated herein by reference in whole). The use of conventional excipient media is intended and may be intended to be within the scope of this disclosure, except where any conventional excipient media may be incompatible with a substance or derivative, for example, producing any undesirable biological effect, or otherwise interacting in a detrimental manner with any other component of the pharmaceutical composition.

[0328] In some embodiments, pharmaceutically acceptable excipients may have a purity of at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%. In some embodiments, the excipients are approved for use in humans and animals. In some embodiments, the excipients may be approved by the U.S. Food and Drug Administration. In some embodiments, the excipients may be pharmaceutical grade. In some embodiments, the excipients may meet the standards of the United States Pharmacopeia (USP), European Pharmacopoeia (EP), British Pharmacopoeia, and / or International Pharmacopoeia. Pharmaceutically acceptable excipients used in the manufacture of pharmaceutical compositions include, but are not limited to, inert diluents, dispersants and / or granulators, surfactants and / or emulsifiers, disintegrants, binders, preservatives, buffers, lubricants, and / or oils. Such excipients may, as they may be, be included in the pharmaceutical composition. The composition may also contain excipients, such as cocoa butter and suppository waxes, colorants, coatings, sweeteners, flavorings, and / or fragrances.

[0329] Examples of diluents include, but are not limited to, calcium carbonate, sodium carbonate, calcium phosphate, dicalcium phosphate, calcium sulfate, calcium hydrogen phosphate, sodium phosphate, lactose, sucrose, cellulose, microcrystalline cellulose, kaolin, mannitol, sorbitol, inositol, sodium chloride, dried starch, corn starch, powdered sugar, and / or combinations thereof.

[0330] Examples of granulating and / or dispersing agents include, but are not limited to, potato starch, corn starch, tapioca starch, sodium starch glycolate, clay, alginic acid, guar gum, citrus pulp, agar, bentonite, cellulose and wood products, natural sponges, cation exchange resins, calcium carbonate, silicates, sodium carbonate, cross-linked polyvinylpyrrolidone (crospovidone), sodium carboxymethyl starch (sodium starch glycolate), carboxymethylcellulose, cross-linked sodium carboxymethylcellulose (croscamellose), methylcellulose, pregelatinized starch (starch 1500), microcrystalline starch, water-insoluble starch, calcium carboxymethylcellulose, magnesium aluminum silicate (VEEGUM®), sodium lauryl sulfate, quaternary ammonium compounds, and / or combinations thereof.

[0331] Exemplary surfactants and / or emulsifiers include natural emulsifiers (e.g., acacia, agar, alginic acid, sodium alginate, tragacanth, condorucus, cholesterol, xanthan gum, pectin, gelatin, egg yolk, casein, lanolin, cholesterol, wax, and lecithin), colloidal clays (e.g., bentonite (aluminum silicate) and VEEGUM® (magnesium aluminum silicate)), long-chain amino acid derivatives, high molecular weight alcohols (e.g., stearyl alcohol, cetyl alcohol, oleyl alcohol, triacetin monostearate, ethylene glycol distearate, glyceryl monostearate, and propylene glycol monostearate, polyvinyl alcohol), carbomers (e.g., carboxypolymethylene, polyacrylic acid, acrylic acid polymers, and carboninyl polymers), carrageenan, cellulose derivatives (e.g., sodium carboxymethylcellulose, powdered cellulose, hydroxymethylcellulose, hydroxypropylcellulose, hydroxypropylmethylcellulose, methylcellulose), sorbitan fatty acid esters (e.g., polyoxy Ethylene sorbitan monolaurate (TWEEN® 20), polyoxyethylene sorbitan (TWEEN® 60), polyoxyethylene sorbitan monooleate (TWEEN® 80), sorbitan monopalmitate (SPAN® 40), sorbitan monostearate (SPAN® 60), sorbitan tristearate (SPAN® 65), glyceryl monooleate, sorbitan monooleate (SPAN® 80), polyoxyethylene ester (e.g., polyoxyethylene) Lenmonostearate (MYRJ® 45), polyoxyethylene hydrogenated castor oil, polyethoxylated castor oil, polyoxymethylene stearate and SOLUTOL®), sucrose fatty acid ester, polyethylene glycol fatty acid ester (e.g., CREMOPHOR®), polyoxyethylene ether (e.g., polyoxyethylene lauryl ether (BRIJ® 30)), poly(vinyl-pyrrolidone), diethylene glycol monolaurate, triethanolamine oleate, sodium oleate,Potassium oleate, ethyl oleate, oleic acid, ethyl laurylate, sodium lauryl sulfate, PLUORINC® F68, POLOXAMER® 188, cetrimonium bromide, cetylpyridinium chloride, benzalkonium chloride, sodium docusate, etc., and / or combinations thereof, are examples, but are not limited to these.

[0332] Exemplary preservatives include, but are not limited to, antioxidants, chelating agents, antimicrobial preservatives, antifungal preservatives, alcohol preservatives, acidic preservatives, and / or other preservatives. Oxidation is a potential degradation pathway for mRNA, particularly for liquid mRNA formulations. Antioxidants can be added to formulations to prevent oxidation. Exemplary antioxidants include, but are not limited to, alpha-tocopherol, ascorbic acid, ascorbyl palmitate, benzyl alcohol, butylated hydroxyanisole, EDTA, m-cresol, methionine, butylated hydroxytoluene, monothioglycerol, potassium metabisulfite, propionic acid, propyl gallate, sodium ascorbate, sodium bisulfite, sodium metabisulfite, thioglycerol, and / or sodium sulfite. Examples of chelating agents include ethylenediaminetetraacetic acid (EDTA), citrate monohydrate, disodium edetate, dipotassium edetate, edetate, fumaric acid, malic acid, phosphoric acid, sodium edetate, tartaric acid, and / or trisodium edetate. Examples of antimicrobial preservatives include, but are not limited to, benzalkonium chloride, benzethonium chloride, benzyl alcohol, bronopol, cetrimide, cetylpyridinium chloride, chlorhexidine, chlorobutanol, chlorocresol, chloroxylenol, cresol, ethyl alcohol, glycerin, hexetidine, imidourea, phenol, phenoxyethanol, phenylethyl alcohol, phenylmercury nitrate, propylene glycol, and / or thimerosal. Examples of antifungal preservatives include, but are not limited to, butylparaben, methylparaben, ethylparaben, propylparaben, benzoic acid, hydroxybenzoic acid, potassium benzoate, potassium sorbate, sodium benzoate, sodium propionate, and / or sorbic acid. Examples of alcohol preservatives include, but are not limited to, ethanol, polyethylene glycol, phenol, phenolic compounds, bisphenol, chlorobutanol, hydroxybenzoate, and / or phenylethyl alcohol.Examples of acidic preservatives include, but are not limited to, vitamin A, vitamin C, vitamin E, beta-carotene, citric acid, acetic acid, dehydroacetic acid, ascorbic acid, sorbic acid, and / or phytic acid. Other preservatives include, but are not limited to, tocopherol, tocopherol acetate, deteroxime mesylate, cetrimide, butylated hydroxyanisole (BHA), butylated hydroxytoluene (hydroxytoluene d) (BHT), ethylenediamine, sodium lauryl sulfate (SLS), sodium lauryl ether sulfate (SLES), sodium bisulfite, sodium metabisulfite, potassium sulfite, potassium metabisulfite, GLYDANT PLUS®, PHENONIP®, methylparaben, GERMALL® 115, GERMABEN®, NEOLONE®, KATHON®, and / or EUXYL®.

[0333] Exemplary binders include, but are not limited to, starches (e.g., corn starch and starch paste), gelatin, sugars (e.g., sucrose, glucose, dextrose, dextrin, molasses, lactose, lactitol, mannitol), amino acids (e.g., glycine), natural and synthetic rubbers (e.g., acacia, sodium alginate, Irish moss extract, panwal gum, gatchigum, Isapol bark mucus, carboxymethylcellulose, methylcellulose, ethylcellulose, hydroxyethylcellulose, hydroxypropylcellulose, hydroxypropylmethylcellulose, microcrystalline cellulose, cellulose acetate, poly(vinylpyrrolidone), aluminum magnesium silicate (VEEGUM®), and Larchia labogalactan), alginates, polyethylene oxide, polyethylene glycol, inorganic calcium salts, silicic acid, polymethacrylate, waxes, water, alcohols, and / or combinations thereof.

[0334] In some embodiments, the pH of the pharmaceutical solution is maintained between pH 5 and pH 8 to improve stability. Exemplary buffers for controlling pH include, but are not limited to, sodium phosphate, sodium citrate, sodium succinate, histidine (or histidine-HCl), sodium carbonate, and / or sodium malate. In other embodiments, the exemplary buffers listed above may be used with additional monovalent counterions (potassium is an example, but is not limited to potassium). Divalent cations may also be used as buffer counterions, but these are undesirable due to complex formation and / or mRNA degradation.

[0335] Exemplary buffers include, but are not limited to, citrate buffer, acetate buffer, phosphate buffer, ammonium chloride, calcium carbonate, calcium chloride, calcium citrate, calcium glubionate, calcium gluceptate, calcium gluconate, D-gluconic acid, calcium glycerophosphate, calcium lactate, propanoic acid, calcium levulinate, pentanoic acid, dicalcium phosphate, phosphoric acid, tricalcium phosphate, calcium hydroxide phosphate, potassium acetate, potassium chloride, potassium gluconate, potassium mixture, dipotassium phosphate, monopotassium phosphate, potassium phosphate mixture, sodium acetate, sodium bicarbonate, sodium chloride, sodium citrate, sodium lactate, disodium phosphate, monosodium phosphate, sodium phosphate mixture, tromethamine, magnesium hydroxide, aluminum hydroxide, alginic acid, pyrogen-removed water, isotonic saline, Ringer's solution, ethyl alcohol, and / or combinations thereof.

[0336] Examples of lubricants include, but are not limited to, magnesium stearate, calcium stearate, stearic acid, silica, talc, malt, glyceryl behenate, hydrogenated vegetable oil, polyethylene glycol, sodium benzoate, sodium acetate, sodium chloride, leucine, magnesium lauryl sulfate, sodium lauryl sulfate, and / or combinations thereof.

[0337] Examples of oils include almond oil, apricot kernel oil, avocado oil, babassu oil, bergamot oil, blackcurrant oil, borage oil, juniper oil, chamomile oil, canola oil, carauwae oil, carnauba oil, castor oil, cinnamon oil, cocoa butter, coconut oil, cod liver oil, coffee bean oil, corn oil, cottonseed oil, emu oil, eucalyptus oil, evening primrose oil, fish oil, linseed oil, geraniol oil, melon oil, grape seed oil, hazelnut oil, hyssop oil, isopropyl myristate, jojoba oil, kukui nut oil, lavandin oil, lavender oil, lemon oil, litsea cubeba oil, and macadamia oil. Examples of oils that may be used include, but are not limited to, nut oils, mallow oil, mango seed oil, meadowfoam seed oil, mink oil, nutmeg oil, olive oil, orange oil, orange roughy oil, palm oil, palm kernel oil, peach kernel oil, peanut oil, poppy oil, pumpkin seed oil, rapeseed oil, rice bran oil, rosemary oil, safflower oil, sandalwood oil, susquamum oil, savory oil, sea buckthorn oil, sesame oil, shea butter, silicone oil, soybean oil, sunflower oil, tea plant oil, thistle oil, camellia oil, vetiver oil, walnut oil, and wheat germ oil. Examples of oils include, but are not limited to, butyl stearate, glyceryl tricaprylate, glyceryl tricaprate, cyclomethicone, diethyl sebacate, dimethicone 360, isopropyl myristate, mineral oil, octyldodecanol, oleyl alcohol, silicone oils, and / or combinations thereof.

[0338] Excipients, such as cocoa butter and suppository wax, colorants, coatings, sweeteners, flavorings, and / or flavorings, may be present in the composition at the discretion of the compounder.

[0339] Examples of additives include the addition of physiologically compatible buffers (e.g., trimethylamine hydrochloride), chelating agents (e.g., DTPA or DTPA-bisamide), or calcium chelate complexes (e.g., calcium DTPA, CaNaDTPA-bisamide), or optionally, calcium or sodium salts (e.g., calcium chloride, calcium ascorbate, calcium gluconate, or calcium lactate). In addition, antioxidants and suspending agents may be used.

[0340] In some embodiments, the polynucleotides of this disclosure encoding an antigen (e.g., antisense polynucleotides) may be formulated as nucleic acid vaccines with adjuvants. Adjuvants can be used to enhance the immunogenicity of nucleic acid vaccines, modify the immune response, reduce the amount of nucleic acid vaccine required for immunization, reduce the frequency of necessary additional or "booster" immunizations, or produce an improved immune response in weakened or immunocompromised immune systems or in elderly individuals. Concomitant administration of adjuvants may be by any method known in the art or described herein, for example, intravenous (IV), intramuscular (IM), subcutaneous (SC), or intradermal (ID).

[0341] Adjuvants may be selected by those skilled in the art for use with nucleic acid vaccines. Adjuvants may be natural or synthetic. Adjuvants may also be organic or inorganic. In some embodiments, adjuvants used with polynucleotide vaccines include certain classes of adjuvants, for example, but not limited to, carbohydrates, microorganisms, mineral salts (e.g., aluminum hydroxide, aluminum phosphate gel or calcium phosphate gel), emulsions (e.g., oil emulsions, surfactant-based emulsions, purified saponins and oil-in-water emulsions), inactive vehicles, granular adjuvants (e.g., monolayer liposome vehicles, e.g., visomes, or saponin-lipid structured complexes, e.g., polylactide co-glycerides (PLGs)), microbial derivatives, endogenous human immunomodulators, and tension-active compounds. A list of adjuvants that may be used with the nucleic acid vaccines described herein can be found in the web-based vaccine adjuvant database Vaxjo (see, for example, Sayers et al., Journal of Biomedicine and Biotechnology; 2012; 2012:831486, which is incorporated herein by reference in its entirety).

[0342] Other exemplary adjuvants include interferon, TNF-alpha, TNF-beta, chemokines (e.g., CCL21, eotaxin, HMGB1, SA100-8 alpha, GCSF, GMCSF, granulosin, lactoferrin, ovalbumin, CD40L, CD28 agonist, PD1, soluble PD1, PDL1, PDL2), or interleukins (e.g., IL1, IL2, IL4, IL6, IL7, IL10, IL12, IL13, IL15, IL17, IL18, IL21, and IL23), Abisco-100 vaccine adjuvant, adamantylamide dipeptide vaccine adjuvant, Adjumer®, AF03, albumin heparin microparticle vaccine adjuvant, and Algal. Glucan, Algammulin, Alhydrogel, Aluminum Hydroxide Vaccine Adjuvant, Aluminum Phosphate Vaccine Adjuvant, Aluminum Potassium Sulfate Adjuvant, Aluminum Vaccine Adjuvant, Amorphous Aluminum Hydroxyphosphate Sulfate Adjuvant, Arlacel A, AS0, AS04, AS03, AS-2 Vaccine Adjuvant, Avridine®, B7-2 Vaccine Adjuvant, Bay R1005, Bordetella Pertussis Constituent Vaccine Adjuvant, Bupivacaine Vaccine Adjuvant, Calcium Phosphate Gel, Calcium Phosphate Vaccine Adjuvant, Cationic Liposome Vaccine Adjuvant, Cationic Liposome DNA Complex JVRS-100, Cholera Toxin, Cholera Toxin B Subunit, Corynebacterium-derived P40 Vaccine Adjuvant, CpG DNA vaccine adjuvant, CRL1OO5, CTA1-DD gene fusion protein, DDA adjuvant, DHEA vaccine adjuvant, DL-PGL (polyester poly(DL-lactide-co-glycolide)) vaccine adjuvant, DOC / Alum complex, Escherichia coli (E. coli)coli) heat-unstable toxin, Etx B subunit adjuvant, flagellin, Freund's complete adjuvant, Freund's incomplete adjuvant, gamma inulin, Gerbu adjuvant, GM-CSF, GMDP, imiquimod, immunoliposomes containing antibodies against costimulatory molecules, ISCOM(s) (trademark), ISCOMA-TRIX (registered trademark), dead Corynebacterium parvum vaccine adjuvant, lipopolysaccharide, liposomes, loxoribine, LTK63 vaccine mutation adjuvant, LTK72 vaccine adjuvant, LTR192G vaccine adjuvant, Matrix-S, MF59, Montanide incomplete Seppic adjuvant, Montanide ISA 51, Montanide ISA 720 adjuvant, MPL-SE vaccine adjuvant, MPL(trademark) adjuvant, MTP-PE liposome, Muramylide, Muramyl dipeptide adjuvant, Murapalmitine, D-Murapalmitine, NAGO, nanoemulsion vaccine adjuvant, nonionic surfactant vesicle, non-toxic variant E112K of cholera toxin mCT-E112K, PMMA, Poly(LC), polygene vaccine adjuvant, protein cocreat, QS-21, Quil-A vaccine adjuvant, RC529 vaccine adjuvant, recombinant hlfN-gamma / interferon-g, Rehydragel EV, Rehydragel Examples of adjuvants include, but are not limited to, HPA, reciquimod, Ribi vaccine adjuvant, SAF-1, saponin vaccine adjuvant, Sclavo peptide, Sendai proteoliposome, Sendai-containing lipid matrix, Specol, SPT (antigen preparation), squalene-based adjuvant, stearyl tyrosine, Theramide®, threonyl muramyl dipeptide (TMDP), Titer-Max gold adjuvant, Ty particle vaccine adjuvant, and VSA-3 adjuvant.

[0343] Viral vector The present invention also provides a viral vector for encoding or delivering the antisense polynucleotide (e.g., ASO) of the present invention. The viral vector may include or encode the ASO and / or guide RNA described herein. In one embodiment, the ASO has an RNA backbone. The viral vector can be used to deliver the ASO and / or guide RNA to target cells. The viral vector can improve the delivery of the ASO and / or guide RNA to target cells (e.g., nerve cells).

[0344] In some embodiments, the viral vector includes a sequence that expresses nucleic acids comprising the ASO and / or guide RNA described herein.

[0345] Viral vectors can be designed to deliver and / or encode ASO or guide RNA sequences using standard practices known in the art.

[0346] The viral vector may include an expression cassette, which encodes a transcript comprising an ASO or guide RNA. In some embodiments, the viral vector includes one or more regions comprising a sequence encoding the ASO or guide RNA as described herein, and a reverse-ended repeat (ITR) sequence that flanks the ASO or guide RNA sequence. In some embodiments, the sequence is operably ligated to a promoter. In some embodiments, the promoter is a tissue-specific (e.g., CNS-specific) promoter.

[0347] In one embodiment, the viral vector is a retrovirus, lentivirus, adenovirus (AV) or adeno-associated virus (AAV), or herpes simplex virus. The viral vector may be derived from any suitable serotype or subgroup. The viral vector may be a human viral vector or a non-human viral vector.

[0348] III. Route of administration, dosage, and delivery This disclosure encompasses the delivery of one or more antisense polynucleotide (e.g., ASO) compositions for any therapeutic, prophylactic, pharmaceutical, diagnostic, or research use by any suitable route, taking into account the potential for advances in the chemistry of drug delivery.

[0349] delivery This disclosure provides a method for delivering an antisense polynucleotide (ASO) to cells, wherein the ASO modulates the splicing of UNC13A to prevent the inclusion of latent exons into UNC13A RNA. This can prevent deletion of UNC13A translational protein and / or functional UNC13A protein. In one embodiment, this is an in vitro method. In one embodiment, the cells are central nervous system cells. In one embodiment, the cells are nerve cells. In some embodiments, the ASO is delivered to the cells by a vector, for example, a viral vector described herein. The method may be an in vitro or in vivo method. The in vitro method can be used to explore or modulate UNC13A function. The in vivo method can be used to treat neurodegenerative disorders described in other parts of this specification.

[0350] Antisense polynucleotides (e.g., ASOs) and compositions comprising antisense polynucleotides (e.g., ASOs) as disclosed herein may be loaded onto a vehicle, e.g., a formulation component as discussed herein, for administration to target cells, tissues, and / or organs. The formulation may contain modified and / or unmodified antisense polynucleotide (e.g., ASO) compositions. The formulation may further include, but is not limited to, cell penetration agents, pharmaceutically acceptable carriers, delivery agents, bio-erosive or biocompatible polymers, solvents, and sustained-release delivery depots. Formulated antisense polynucleotide (e.g., ASO) compositions may be delivered to cells using administration routes known in the art and described herein.

[0351] Delivery may also be naked. Antisense polynucleotides (e.g., ASOs) and compositions may be delivered naked to cells. As used herein, “naked” refers to the delivery of an antisense polynucleotide (e.g., ASO) composition that does not contain any transfection-promoting agents. For example, an antisense polynucleotide (e.g., ASO) composition delivered to cells may not contain any modifications. Naked antisense polynucleotide (e.g., ASO) compositions may be delivered to cells using administration routes known in the art and described herein.

[0352] Route of administration When used as a pharmaceutical or in the treatment methods described herein, antisense polynucleotides (e.g., ASOs) or pharmaceutical compositions may be administered to a subject by any preferred method of administration, such as injection.

[0353] The antisense polynucleotide (e.g., ASO) compositions of this disclosure may be administered via any route that produces therapeutically effective results. These include, but are not limited to, enteral, gastrointestinal, epidural, oral, percutaneous, epidural (on the dura mater), intracerebral (in the cerebrum), lateral ventricle (inside the ventricle), cutaneous (application on the skin), intradermal (inside the skin itself), subcutaneous (under the skin), nasal administration (inside the nose), intravenous (inside the vein), intraarterial (inside the artery), intramuscular (inside the muscle), intracardiac (inside the heart), intraosseous injection (inside the bone marrow), intrasacral (inside the spinal canal), intraperitoneal (injection into the peritoneum), intravesical injection, intravitreous (inside the eye), intracavitary injection (at the base of the penis), vaginal administration, intrauterine, extraamniotic administration, percutaneous (diffusion through untreated skin for systemic distribution), transmucosal (diffusion through the mucous membrane), inhalation (through the nose), sublingual, sublabial, enema, eye drops (on the conjunctiva), or ear drops. In certain embodiments, the composition may be administered in a manner that allows it to cross the blood-brain barrier, the blood barrier, or other epithelial barriers.

[0354] Dosage The pharmaceutical compositions described herein may be formulated for the dosage forms and routes of administration described herein.

[0355] Polynucleotides and / or pharmaceutical compositions may be administered to a subject in single or multiple doses. In one embodiment, multiple doses include two, three, or four or more doses. In one embodiment, the ASO, guide RNA, viral vector, or pharmaceutical composition is administered to the subject at regular intervals, for example, weekly, bi-weekly, monthly, every two months, every three months, every four months, every five months, every six months, or annually. In some embodiments, a first set of doses (e.g., two, three, four, five, six, seven, eight, or ten doses) is administered monthly, with further doses administered at less frequent intervals (e.g., every three months, every six months, or annually).

[0356] IV.How to use This disclosure provides antisense polynucleotides (e.g., ASOs), compositions comprising the antisense polynucleotides of this disclosure, and methods for using the formulations.

[0357] Gene expression can be controlled at multiple levels using polynucleotides, compositions and formulations comprising the polynucleotides of this disclosure. Some aspects of this disclosure provide a method for controlling gene expression in cells, comprising administering antisense polynucleotides (e.g., ASO compounds), compositions and formulations comprising the polynucleotides described herein to cells.

[0358] In some embodiments, gene expression is controlled at the transcriptional level, the post-transcriptional level, the translational level, or the post-translational level.

[0359] therapeutic use Antisense polynucleotides (e.g., ASOs), compositions and formulations comprising the polynucleotides of this disclosure can be used as therapeutic agents for the treatment of diseases. The therapeutic use of antisense polynucleotides (e.g., ASOs), compositions and formulations comprising antisense polynucleotides (ASOs) of this disclosure can provide protection against disease by engaging in the regulation of endogenously present RNA.

[0360] In some embodiments, the therapeutic use of antisense polynucleotides, compositions and formulations comprising antisense polynucleotides of this disclosure (e.g., ASOs) may involve the administration of polynucleotides manipulated and produced in vitro. The synthetic antisense polynucleotides (e.g., ASOs) described herein may be delivered to target cells to function therapeutically.

[0361] The polynucleotides (e.g., ASOs) or pharmaceutical compositions described herein may be intended for or used as pharmaceuticals, for example, in therapy.

[0362] Also disclosed herein are methods for treating neurodegenerative disorders, comprising administering a therapeutically effective amount of an ASO of the first or second embodiment, a guide RNA of the third embodiment, a viral vector of the fourth embodiment, or a pharmaceutical composition of the fifth embodiment to a subject in need thereof.

[0363] Also disclosed herein are methods for treating conditions associated with TDP-43 pathology, comprising administering a therapeutically effective amount of a first or second embodiment of ASO, a third embodiment of guide RNA, a fourth embodiment of a viral vector, or a fifth embodiment of a pharmaceutical composition to a subject in need thereof.

[0364] The loss of function of the UNC13A protein can be prevented and / or restored using the ASO, guide RNA, viral vector, or pharmaceutical composition described herein. For example, splicing can be modified using the ASO, guide RNA, viral vector, or pharmaceutical composition so that the UNC13A latent exon is not incorporated into the UNC13A mRNA.

[0365] In one embodiment, the pharmaceutical is intended for use in or used to modulate the splicing of UNC13A premRNA. In one embodiment, the pharmaceutical is intended for use in or used to prevent the inclusion of UNC13A latent exon into UNC13A mature RNA so that the function of the UNC13A protein is restored. In one embodiment, the pharmaceutical is intended for use in or used in methods for treating diseases associated with TDP-443 pathology.

[0366] The ASOs, guide RNAs, viral vectors, or pharmaceutical compositions described herein are intended for use in or used in methods for treating neurodegenerative disorders. In one embodiment, the neurodegenerative disorder is associated with a reduction in nuclear TDP-43. In one embodiment, the neurodegenerative disorder is caused by nuclear-cytoplasmic mislocalization of TDP-43. In one embodiment, the neurodegenerative disorder is associated with TDP-43 pathology (e.g., pathological TDP-43).

[0367] In one embodiment, the treatment method first includes diagnosing a subject having a neurodegenerative disorder associated with TDP-43 pathology prior to the treatment method. In one embodiment, this is determined using a biomarker of TDP-43 pathology. In one embodiment, this may be determined genetically, for example, by gene mutation. In one embodiment, TDP-43 pathology associated with ALS may be determined if FUS and SOD1 mutations are not found in the subject. In one embodiment, TDP pathology associated with FTD may be determined if C9orf72 or PGRN mutations are not found in the subject. In one embodiment, a biomarker of TDP-43 pathology may be mutant TDP-43. In some embodiments, TDP-43 pathology may be determined by TDP-43 phosphorylation. In some embodiments, TDP-43 pathology may be determined by the expression of the STMN2 latent exon.

[0368] In one embodiment, the treatment method first includes determining whether the subject has SNP variants related to rs12973192 and / or rs12608932 before the treatment method is performed. This can be determined by genomics.

[0369] In one embodiment, the neurodegenerative disorder may be selected from ALS, frontotemporal dementia, Alzheimer's disease, Parkinson's disease, FOSMNN (facial onset sensory and motor neuron disorder), Perry syndrome, or any combination thereof.

[0370] In one embodiment, the neurodegenerative disorder is ALS (amyotrophic lateral sclerosis). ALS is a chronic and fatal form of motor neuron disease (MND), otherwise it may be called MNS, Charcot disease, or Lou Gehrig's disease. In some embodiments, ALS may be familial ALS or sporadic (idiopathic) ALS. Familial ALS (FALS) is a family-related form of ALS and accounts for about 10% of ALS cases. Sporadic ALS is non-familial ALS. In one embodiment, ALS may not be ALS-FUS and ALS-SOD1, which are the genetically defined forms of ALS. The ASOs, guide RNAs, viral vectors, or pharmaceutical compositions for use or treatment methods described herein may restore one or more symptoms associated with ALS. Symptoms of ALS may include fasciculations (muscle spasms), muscle cramps, stiff and rigid muscles (spasticity), muscle weakness, slurred nasal voice, and difficulty chewing or swallowing. ALS leads to progressive deterioration of muscle function and ultimately, in many cases, death due to respiratory failure.

[0371] In one embodiment, the neurodegenerative disorder is frontotemporal dementia (FTD). Frontotemporal dementia is a type of dementia affecting the frontal and temporal lobes of the brain. The ASOs, guide RNAs, viral vectors, or pharmaceutical compositions for use or treatment methods described herein can restore one or more symptoms associated with FTD. Symptoms of FTD may include personality and behavioral changes, language problems, problems with mental abilities, memory problems, and physical problems (e.g., motor difficulties). FTD can be characterized by frontotemporal lobar degeneration (FTLD). FTLD may also be FTLD-TDP, which is FTLD associated with TDP-43 pathology. This can be characterized by ubiquitin and TDP-43 positive, tau negative, and FUS negative inclusions. FTLD-TDP may be type A, type B, type C, or type D. Type A is a type of FTLD-TDP that shows small neurites and neuronal cytoplasmic inclusions in the superior (surface) cortical layer. Rod-shaped intranuclear inclusions are also observed, but their number is relatively small. Type B is a type of FTLD-TDP that shows neuronal and glial cytoplasmic inclusions in both the superior (surface) and inferior (deep) cortical layers and lower motor neurons. Intranuclear inclusions may be absent or present in relatively small numbers. Type B may be associated with ALS and C9ORF92 mutations. Type C is a type of FTLD-TDP that presents long neuronal profiles found in the surface cortical layers. Neuronal cytoplasmic inclusions, intranuclear inclusions, or glial cytoplasmic inclusions may be relatively few or absent. FTLD-TDP is often associated with semantic dementia. Type D is a type of FTLD-TDP that shows neuronal intranuclear inclusions and degenerated neurites. Inclusions may be absent in the granule cell layer of the hippocampus. Type D may be associated with VCP mutations. In one embodiment, FTLD may not be of the FTLD-FUS or FTLD-tau type.

[0372] In one embodiment, the neurodegenerative disorder is Alzheimer's disease. Alzheimer's disease is a chronic neurodegenerative disease that begins slowly and gradually worsens over time, and is a major cause of dementia. The ASOs, guide RNAs, viral vectors, or pharmaceutical compositions for use or treatment methods described herein can restore one or more symptoms associated with Alzheimer's disease. Symptoms of Alzheimer's include memory problems, confusion and disorientation, speech and language problems, motor problems, personality changes, and combinations thereof. Alzheimer's disease may be associated with TDP pathology.

[0373] In one embodiment, the neurodegenerative disorder is Parkinson's disease. Parkinson's disease is a progressive neurological disorder affecting movement. The symptoms of Parkinson's disease usually begin gradually and worsen over time. The ASOs, guide RNAs, viral vectors, or pharmaceutical compositions for use or treatment methods described herein can restore one or more symptoms associated with Parkinson's disease. Symptoms may include tremor, bradykinesia (slow movement), and muscle rigidity. Parkinson's disease may be associated with TDP pathology.

[0374] In one embodiment, the neurodegenerative disorder is FOSMN (facial-onset sensory and motor neuron disorder). FOSMN is a rare, slowly progressive motor neuron disorder. The ASOs, guide RNAs, viral vectors, or pharmaceutical compositions for use or treatment described herein can restore one or more symptoms associated with FOSMN. Symptoms include loss of sensation and motor function in the face (e.g., facial tingling or numbness), which may further extend to loss of sensation and motor function in the scalp, neck, upper trunk, and arms. FOSMN may be associated with TDP pathology.

[0375] In one embodiment, the neurodegenerative disorder is Perry syndrome. Perry syndrome is a progressive brain disease. The ASOs, guide RNAs, viral vectors, or pharmaceutical compositions for use or treatment methods described herein can restore one or more symptoms associated with Perry syndrome. These symptoms include parkinsonism (patterns of movement abnormalities), mental changes, weight loss, and hypoventilation. Perry syndrome may be associated with TDP pathology.

[0376] In one embodiment, the neurodegenerative disorder is associated with hereditary motor neuropathy. Hereditary motor neuropathy may be associated with TDP-43. In one embodiment, hereditary motor neuropathy may be hereditary motor sensory neuropathy (HMSN), otherwise it may also be known as Charcot-Marie-Tooth (CMT) disease or peroneal muscle atrophy (PMA). The ASOs, guide RNAs, viral vectors, or pharmaceutical compositions for use or treatment methods described herein can restore one or more symptoms associated with HMSN. Symptoms include weakness of the feet, ankles, legs, or hands; and pathologies of clumsy movements and numbness of the feet, arms, and hands.

[0377] The ASO, guide RNA, viral vector, or pharmaceutical composition may be administered to any suitable subject when used as a pharmaceutical or in the treatment methods described herein. In a preferred embodiment, the subject is human. In one embodiment, the subject has SNP variants associated with rs12973192 and / or rs12608932. The human subject is of any suitable age, for example, infants (under 1 year old), children (under 18 years old), including adolescents (10 to 18 years old), or adults (over 18 years old), including elderly subjects (over 65 years old).

[0378] Adjusting splicing Disclosed herein are methods for modulating UNC13A splicing, comprising administering a therapeutically effective amount of a first or second embodiment of ASO, a third embodiment of guide RNA, a fourth embodiment of a viral vector, or a fifth embodiment of a pharmaceutical composition to cells or a subject requiring such treatment. For example, an ASO, guide RNA, viral vector, or pharmaceutical composition can be used to prevent loss of function of the UNC13A protein and / or restore its function. For example, an ASO, guide RNA, viral vector, or pharmaceutical composition can be used to modulate splicing so that the UNC13A latent exon is not incorporated into the UNC13A mRNA.

[0379] CRISPR system: gene therapy The present invention also provides a guide RNA containing the ASO and Cas nuclease scaffold sequences of the present invention. In one embodiment, the ASO has an RNA backbone. The guide RNA can be used in a CRISPR / Cas system to regulate UNC13A splicing. In a preferred embodiment, the guide RNA is a single guide RNA (sgRNA) containing both the ASO and the scaffold sequence. In one embodiment, the sgRNA contains the scaffold sequence upstream of the ASO.

[0380] Guide RNAs can be designed to include these sequences using standard practices known in the art. In this regard, the ASO functions as the crRNA, i.e., the portion of the guide RNA complementary to the nucleic acid target, while the scaffold sequence is the tracer RNA, i.e., the portion of the guide RNA that serves as a binding scaffold for the Cas nuclease.

[0381] The scaffold sequence includes a binding sequence for a Cas nuclease for use in the CRISPR / Cas system. Any suitable scaffold sequence specific to the Cas nuclease can be selected. The Cas nuclease may be any suitable Cas nuclease capable of binding to RNA, and may be dead or inactive (e.g., a dCas nuclease). In one embodiment, the Cas nuclease is a Cas13 nuclease. The Cas13 nuclease may be any suitable subtype. In one embodiment, the Cas13 nuclease is a 13a or 13d subtype.

[0382] ASO and / or guide RNA (i.e., a part of the CRISPR / dCas system) are further used to mask key elements of CE splicing (splice donor and acceptor sites), as well as / or splice regulatory elements (SREs). These therapeutics prevent the splicing mechanism from recognizing CE, thereby preventing the incorporation of latent exons into mature UNC13A RNA.

[0383] This disclosure also provides a Cas system comprising the guide RNA and dCas nuclease described herein. The Cas nuclease may be one of those described above.

[0384] Using the guide RNAs and associated Cas systems described herein, key elements of latent exon splicing, including splice sites (splice donors and acceptors) and / or splice control elements, can be masked.

[0385] VI.Definition To facilitate understanding of the present invention, numerous terms and phrases are defined below.

[0386] When used herein, the term “approximately” is understood to mean within the range of tolerances typically used in the art, e.g., within two standard deviations of the mean. “Approximately” can be understood to mean within 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, 0.05%, or 0.01% of the stated value. When the term “approximately” refers to a numerical value or range, it means that the numerical value or range referred to is an approximation within the range of experimental variability (or statistical experimental error), and therefore the numerical value or range may vary. Typical experimental variability may stem, for example, from the changes and adjustments required when scaling up from laboratory experiments and manufacturing settings to larger scales.

[0387] Analogue: As used herein, the term “analogue” is intended to include polypeptide variants that differ by one or more amino acid variations, e.g., substitution, addition or deletion of amino acid residues, but still retain one or more properties of the parent or starting polypeptide.

[0388] Animals: As used herein, the term “animal” refers to any member of the animal kingdom. In some embodiments, “animal” refers to a human at any developmental stage. In some embodiments, “animal” refers to a non-human animal at any developmental stage. In certain embodiments, a non-human animal is a mammal (e.g., rodents, mice, rats, rabbits, monkeys, dogs, cats, sheep, cattle, primates, or pigs). In some embodiments, animals include, but are not limited to, mammals, birds, reptiles, amphibians, fish, and helminths. In some embodiments, an animal is a genetically modified animal, a genetically engineered animal, or a clone.

[0389] Antisense Polynucleotides: As used herein, “antisense polynucleotides” or “antisense oligonucleotides” (ASOs) have the usual meaning in the art and refer to synthetic single-stranded nucleosides joined by nucleoside linkages. Nucleosides may be linked by phosphate-based linkages, phosphodiesters, phosphoramidates or phosphorothioate linkages, or combinations thereof and similar as described herein. ASOs are used in the art as therapeutic agents, for example, to target mRNA. Typically, they bind complementaryally to a region of the nucleotide sequence of pre-messenger ribonucleic acid (pre-mRNA) or mature mRNA via Watson-Crick base pairing (“antisense”), modulating mRNA function or splicing. As used herein, “can bind” refers to any sequence that is sufficiently complementary to the target sequence or key element and forms an association with the target sequence or its element. In most cases, ASOs are of synthetic origin, and therefore the term is intended to exclude any naturally occurring or transcription RNA products in this context.

[0390] The ASOs defined herein bind to UNC13A premRNA. The ASO sequences described herein are synthetic oligonucleotides that are isolated and identified from any genomic or transcriptome sequence. Nevertheless, ASOs may form a portion of another longer synthetic sequence (e.g., the guide RNA disclosed herein).

[0391] Associated: As used herein, the terms “associated,” “conjugated,” “linked,” “bonded,” and “anchored” mean, when used in reference to two or more parts, that these parts physically associate or connect with each other, either directly or through one or more additional parts acting as linkers, to form a sufficiently stable structure, thereby remaining physically associated under the conditions in which the structure is used, e.g., physiological conditions. “Association” does not necessarily have to be strictly via direct covalent chemical bonds. Ionic or hydrogen bonding, or hybridization-based connectivity, may also suggest sufficient stability for the “associated” entities to remain physically associated.

[0392] Co-administered: As used herein, the terms “co-administered” or “to administer concurrently” mean administering the first construct or system with one or more additional constructs or systems, or other therapeutic agents or components, within a time interval sufficient to enhance the effects of the first construct or system, or other therapeutic agents or components.

[0393] Complementarity: As used herein, the term “complementarity” refers to Watson-Crick base pairing in RNA, for example, here where A binds with U (or a modified variant thereof) and C binds with G (or a modified variant thereof). The complementary sequence strands are referred to as sense and antisense, the sense strand being the post-transcriptional premRNA, and the antisense sequence (e.g., ASO or therapeutic agent) being complementary to the sense sequence. In the ASOs disclosed herein, the “U” and “T” nucleosides, e.g., uracil or thymine, may be used interchangeably. Thus, in any of SEQ ID NOs. 4-546, “U” can be replaced with “T”. Because there is a bond between the two nucleic acid base compounds, complementarity does not need to be 100% or “perfect”. The complementarity may be 30%, 40%, 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, or any integer between 30% and 100%. The complementarity may be relative to the entire length of the polynucleotide or antisense polynucleotide, or to the target sequence. Such complementarity may be any degree from 1% to 100% of the entire length of the antisense polynucleotide or target, or any length such as 30%, 40%, 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% of the whole.

[0394] Compound: When used in the specification, the terms “compound” or “construct” are intended to include all stereoisomers, geometric isomers, tautomers, and isotopes of the construct being described.

[0395] The compounds or constructs described herein may be asymmetric (e.g., having one or more stereocenters). Unless otherwise specified, all stereoisomers, e.g., enantiomers and diastereomers, are intended. Compounds of this disclosure containing asymmetrically substituted carbon atoms may be isolated in optically active or racemic forms. Methods for preparing optically active forms from optically active starting materials are known in the art, e.g., by reparation or stereoselective synthesis of racemic mixtures. Many geometric isomers, such as olefins and C=N double bonds, may also be present in the compounds described herein, and all such stable isomers are intended in this disclosure. Cis and trans geometric isomers of the compounds of this disclosure are described and may be isolated as mixtures of isomers or as separate isomers. Compounds or constructs of this disclosure also include tautomers. Tautomers arise from the exchange of a single bond for an adjacent double bond and the accompanying transfer of a proton. Tautomers include prototrophic tautomers, which are isomer protonated states with the same empirical formula and total charge. Examples of prototrophic tautomers include ketone-enol pairs, amide-imido acid pairs, lactam-lactim pairs, amide-imido acid pairs, enamine-imine pairs, and cyclic forms in which protons can occupy two or more positions in the heterocyclic system, such as 1H- and 3H-imidazoles, 1H-, 2H- and 4H-1,2,4-triazoles, 1H- and 2H-isoindoles, and 1H- and 2H-pyrazoles. The tautomer forms may be in equilibrium or may be sterically locked into one form by appropriate substituents. The compounds or constructs of this disclosure also include all isotopes of the atoms appearing in the intermediate or final compound. "Isotope" refers to an atom having the same number of atoms but different mass numbers resulting from different numbers of neutrons in the nucleus. For example, isotopes of hydrogen include tritium and deutherium. The compounds or constructs and salts of this disclosure can be p...

Claims

1. An antisense oligonucleotide (ASO) comprising 17-24 nucleotides capable of binding to the UNC13A cryptic splice site or its flanking region and modulating UNC13A splicing.

2. The ASO of claim 1, comprising a cross-linked nucleic acid.

3. The ASO of claim 1 or 2, comprising an LNA.

4. An ASO described in any one of claims 1 to 3, comprising 30 to 50% LNA.

5. An ASO described in any one of claims 1 to 4, comprising phosphorothioate linkages.

6. An ASO described in any one of claims 1 to 5, capable of directly binding to an UNC13A cryptic splice site.

7. An ASO described in any one of claims 1 to 6, capable of binding to the UNC13A donor splice site.

8. The ASO of claim 7, which is complementary to SEQ ID NO: 547 or SEQ ID NO: 548, more preferably SEQ ID NO: 549 or SEQ ID NO: 550, and even more preferably SEQ ID NO:

551.

9. a) at least SEQ ID NO:295, SEQ ID NO:297, SEQ ID NO:299, SEQ ID NO:300 and SEQ ID NO:301, or b) at least SEQ ID NO:296, SEQ ID NO:298, SEQ ID NO:299, SEQ ID NO:300 and SEQ ID NO:301, or c) at least SEQ ID NO:297, SEQ ID NO:299, SEQ ID NO:300, SEQ ID NO:301 and SEQ ID NO:302, or d) at least SEQ ID NO:298, SEQ ID NO:299, SEQ ID NO:300, SEQ ID NO:301 and SEQ ID NO:302, or e) at least SEQ ID NO:299, SEQ ID NO:300, SEQ ID NO:301, SEQ ID NO:302 and SEQ ID NO:303, or or f) at least SEQ ID NO:300, SEQ ID NO:301, SEQ ID NO:302, SEQ ID NO:303 and SEQ ID NO:304, or g) at least SEQ ID NO:301, SEQ ID NO:302 and SEQ ID NO:303, SEQ ID NO:304 and SEQ ID NO:305, or h) at least SEQ ID NO:302, SEQ ID NO:303, SEQ ID NO:304, SEQ ID NO:305 and SEQ ID NO:306, or i) at least SEQ ID NO:303, SEQ ID NO:304, SEQ ID NO:305, SEQ ID NO:306 and SEQ ID NO:307, or j) at least SEQ ID NO:304, SEQ ID NO:305, SEQ ID NO:306, SEQ ID NO:307 and SEQ ID NO:

308. k) at least SEQ ID NO:305, SEQ ID NO:306, SEQ ID NO:307, SEQ ID NO:308 and SEQ ID NO:309, or l) at least SEQ ID NO:306, SEQ ID NO:307, SEQ ID NO:308, SEQ ID NO:309 and SEQ ID NO:310, or m) at least SEQ ID NO:307, SEQ ID NO:308, SEQ ID NO:309, SEQ ID NO:310 and SEQ ID NO:311, or n) at least SEQ ID NO:308, SEQ ID NO:309, SEQ ID NO:310, SEQ ID NO:311 and SEQ ID NO:312, or o) at least SEQ ID NO:312, SEQ ID NO:313, SEQ ID NO:314, SEQ ID NO:315 and SEQ ID NO:316, or p) at least SEQ ID NO:313, SEQ ID NO:314, SEQ ID NO:315, SEQ ID NO:316 and SEQ ID NO:317, or q) at least SEQ ID NO:314, SEQ ID NO:315, SEQ ID NO:316, SEQ ID NO:317 and SEQ ID NO:318, or r) at least SEQ ID NO:315, SEQ ID NO:316, SEQ ID NO:317, SEQ ID NO:318 and SEQ ID NO:319, or s) at least SEQ ID NO:316, SEQ ID NO:317, SEQ ID NO:318, SEQ ID NO:319 and SEQ ID NO:320, or t) at least SEQ ID NO:317, SEQ ID NO:318, SEQ ID NO:319, SEQ ID NO:320 and SEQ ID NO:321,or u) at least SEQ ID NO:318, SEQ ID NO:319, SEQ ID NO:320, SEQ ID NO:321 and SEQ ID NO:322, or v) at least SEQ ID NO:319, SEQ ID NO:320, SEQ ID NO:321, SEQ ID NO:322 and SEQ ID NO:323, or w) at least SEQ ID NO:320, SEQ ID NO:321, SEQ ID NO:322, SEQ ID NO:323 and SEQ ID NO:324, or a combination thereof.

10. comprises 20-24 nucleotides capable of binding to the UNC13A cryptic splice site; and i. comprising SEQ ID NO:295, SEQ ID NO:297, SEQ ID NO:299, SEQ ID NO:300, SEQ ID NO:301, SEQ ID NO:302, SEQ ID NO:303, SEQ ID NO:304, optionally SEQ ID NO:305 (i.e., a 21 nucleotide ASO), optionally SEQ ID NO:306 (i.e., a 22 nucleotide ASO), optionally SEQ ID NO:307 (i.e., a 23 nucleotide ASO), and optionally SEQ ID NO:308 (i.e., a 24 nucleotide ASO); or ii. comprising SEQ ID NO:296, SEQ ID NO:298, SEQ ID NO:299, SEQ ID NO:300, SEQ ID NO:301, SEQ ID NO:302, SEQ ID NO:303, SEQ ID NO:304, optionally SEQ ID NO:305 (i.e. a 21 nucleotide ASO), optionally SEQ ID NO:306 (i.e. a 22 nucleotide ASO), optionally SEQ ID NO:307 (i.e. a 23 nucleotide ASO), and optionally SEQ ID NO:308 (i.e. a 24 nucleotide ASO); or iii. comprising SEQ ID NO:297, SEQ ID NO:299, SEQ ID NO:300, SEQ ID NO:301, SEQ ID NO:302, SEQ ID NO:303, SEQ ID NO:304, SEQ ID NO:305, optionally SEQ ID NO:306 (i.e. a 21 nucleotide ASO), further optionally SEQ ID NO:307 (i.e. a 22 nucleotide ASO), further optionally SEQ ID NO:308 (i.e. a 23 nucleotide ASO), and further optionally SEQ ID NO:309 (i.e. a 24 nucleotide ASO); or iv. comprising SEQ ID NO:298, SEQ ID NO:299, SEQ ID NO:300, SEQ ID NO:301, SEQ ID NO:302, SEQ ID NO:303, SEQ ID NO:304, SEQ ID NO:305, optionally SEQ ID NO:306 (i.e. a 21 nucleotide ASO), further optionally SEQ ID NO:307 (i.e. a 22 nucleotide ASO), further optionally SEQ ID NO:308 (i.e. a 23 nucleotide ASO), and further optionally SEQ ID NO:309 (i.e. a 24 nucleotide ASO); or v. comprising SEQ ID NO:299, SEQ ID NO:300, SEQ ID NO:301, SEQ ID NO:302, SEQ ID NO:303, SEQ ID NO:304, SEQ ID NO:305, SEQ ID NO:306, optionally SEQ ID NO:307 (i.e., a 21 nucleotide ASO), optionally SEQ ID NO:308 (i.e., a 22 nucleotide ASO), optionally SEQ ID NO:309 (i.e., a 23 nucleotide ASO), and optionally SEQ ID NO:310 (i.e., a 24 nucleotide ASO); or vi. comprising SEQ ID NO:300, SEQ ID NO:301, SEQ ID NO:302, SEQ ID NO:303, SEQ ID NO:304, SEQ ID NO:305, SEQ ID NO:306, SEQ ID NO:307, SEQ ID NO:308, optionally SEQ ID NO:309 (i.e. a 21 nucleotide ASO), further optionally SEQ ID NO:309 (i.e. a 22 nucleotide ASO), further optionally SEQ ID NO:310 (i.e. a 23 nucleotide ASO), and further optionally SEQ ID NO:311 (i.e. a 24 nucleotide ASO); or vii. comprising SEQ ID NO:300, SEQ ID NO:301, SEQ ID NO:302, SEQ ID NO:303, SEQ ID NO:304, SEQ ID NO:305, SEQ ID NO:306, SEQ ID NO:307, optionally SEQ ID NO:308 (i.e. a 21 nucleotide ASO), optionally SEQ ID NO:310 (i.e. a 22 nucleotide ASO), optionally SEQ ID NO:311 (i.e. a 23 nucleotide ASO), and optionally SEQ ID NO:312 (i.e. a 24 nucleotide ASO); or viii. comprising SEQ ID NO:302, SEQ ID NO:303, SEQ ID NO:304, SEQ ID NO:305, SEQ ID NO:306, SEQ ID NO:307, SEQ ID NO:308, SEQ ID NO:309, optionally SEQ ID NO:310 (i.e. a 21 nucleotide ASO), further optionally SEQ ID NO:311 (i.e. a 22 nucleotide ASO), further optionally SEQ ID NO:312 (i.e. a 23 nucleotide ASO), and further optionally SEQ ID NO:313 (i.e. a 24 nucleotide ASO); or ix. comprising SEQ ID NO:303, SEQ ID NO:304, SEQ ID NO:305, SEQ ID NO:306, SEQ ID NO:307, SEQ ID NO:308, SEQ ID NO:309, SEQ ID NO:310, optionally SEQ ID NO:311 (i.e., a 21 nucleotide ASO), optionally SEQ ID NO:312 (i.e., a 22 nucleotide ASO), optionally SEQ ID NO:313 (i.e., a 23 nucleotide ASO), and optionally SEQ ID NO:314 (i.e., a 24 nucleotide ASO); or x. comprising SEQ ID NO:304, SEQ ID NO:305, SEQ ID NO:306, SEQ ID NO:307, SEQ ID NO:308, SEQ ID NO:309, SEQ ID NO:310, SEQ ID NO:311, optionally SEQ ID NO:312 (i.e., a 21 nucleotide ASO), optionally SEQ ID NO:313 (i.e., a 22 nucleotide ASO), optionally SEQ ID NO:314 (i.e., a 23 nucleotide ASO), and optionally SEQ ID NO:315 (i.e., a 24 nucleotide ASO); or xi. comprising SEQ ID NO:305, SEQ ID NO:306, SEQ ID NO:307, SEQ ID NO:308, SEQ ID NO:309, SEQ ID NO:310, SEQ ID NO:311, SEQ ID NO:312, optionally SEQ ID NO:313 (i.e., a 21 nucleotide ASO), optionally SEQ ID NO:314 (i.e., a 22 nucleotide ASO), optionally SEQ ID NO:315 (i.e., a 23 nucleotide ASO), and optionally SEQ ID NO:316 (i.e., a 24 nucleotide ASO); or xii. comprising SEQ ID NO:306, SEQ ID NO:307, SEQ ID NO:308, SEQ ID NO:309, SEQ ID NO:310, SEQ ID NO:311, SEQ ID NO:312, SEQ ID NO:313, optionally SEQ ID NO:314 (i.e. a 21 nucleotide ASO), optionally SEQ ID NO:315 (i.e. a 22 nucleotide ASO), optionally SEQ ID NO:316 (i.e. a 23 nucleotide ASO), and optionally SEQ ID NO:317 (i.e. a 24 nucleotide ASO); or xiii. comprising SEQ ID NO:307, SEQ ID NO:308, SEQ ID NO:309, SEQ ID NO:310, SEQ ID NO:311, SEQ ID NO:312, SEQ ID NO:313, SEQ ID NO:314, optionally SEQ ID NO:315 (i.e., a 21 nucleotide ASO), optionally SEQ ID NO:316 (i.e., a 22 nucleotide ASO), optionally SEQ ID NO:317 (i.e., a 23 nucleotide ASO), and optionally SEQ ID NO:318 (i.e., a 24 nucleotide ASO); or xiv. comprising SEQ ID NO:308, SEQ ID NO:309, SEQ ID NO:310, SEQ ID NO:311, SEQ ID NO:312, SEQ ID NO:313, SEQ ID NO:314, SEQ ID NO:315, optionally SEQ ID NO:316 (i.e., a 21 nucleotide ASO), optionally SEQ ID NO:317 (i.e., a 22 nucleotide ASO), optionally SEQ ID NO:318 (i.e., a 23 nucleotide ASO), and optionally SEQ ID NO:319 (i.e., a 24 nucleotide ASO); or xv. comprising SEQ ID NO:309, SEQ ID NO:310, SEQ ID NO:311, SEQ ID NO:312, SEQ ID NO:313, SEQ ID NO:314, SEQ ID NO:315, SEQ ID NO:316, optionally SEQ ID NO:317 (i.e., a 21 nucleotide ASO), optionally SEQ ID NO:318 (i.e., a 22 nucleotide ASO), optionally SEQ ID NO:319 (i.e., a 23 nucleotide ASO), and optionally SEQ ID NO:320 (i.e., a 24 nucleotide ASO); or xvi. comprising SEQ ID NO:310, SEQ ID NO:311, SEQ ID NO:312, SEQ ID NO:313, SEQ ID NO:314, SEQ ID NO:315, SEQ ID NO:316, SEQ ID NO:317, optionally SEQ ID NO:318 (i.e. a 21 nucleotide ASO), optionally SEQ ID NO:319 (i.e. a 22 nucleotide ASO), optionally SEQ ID NO:320 (i.e. a 23 nucleotide ASO), and optionally SEQ ID NO:321 (i.e. a 24 nucleotide ASO); or xvii. comprising SEQ ID NO:311, SEQ ID NO:312, SEQ ID NO:313, SEQ ID NO:314, SEQ ID NO:315, SEQ ID NO:316, SEQ ID NO:317, SEQ ID NO:318, optionally SEQ ID NO:319 (i.e. a 21 nucleotide ASO), optionally SEQ ID NO:320 (i.e. a 22 nucleotide ASO), optionally SEQ ID NO:321 (i.e. a 23 nucleotide ASO), and optionally SEQ ID NO:322 (i.e. a 24 nucleotide ASO); or xviii. comprising SEQ ID NO:312, SEQ ID NO:313, SEQ ID NO:314, SEQ ID NO:315, SEQ ID NO:316, SEQ ID NO:317, SEQ ID NO:318, SEQ ID NO:319, optionally SEQ ID NO:320 (i.e. a 21 nucleotide ASO), optionally SEQ ID NO:321 (i.e. a 22 nucleotide ASO), optionally SEQ ID NO:322 (i.e. a 23 nucleotide ASO), and optionally SEQ ID NO:323 (i.e. a 24 nucleotide ASO); or xix. comprising SEQ ID NO:313, SEQ ID NO:314, SEQ ID NO:315, SEQ ID NO:316, SEQ ID NO:317, SEQ ID NO:318, SEQ ID NO:319, SEQ ID NO:320, optionally SEQ ID NO:321 (i.e., a 21 nucleotide ASO), optionally SEQ ID NO:322 (i.e., a 22 nucleotide ASO), optionally SEQ ID NO:323 (i.e., a 23 nucleotide ASO), and optionally SEQ ID NO:324 (i.e., a 24 nucleotide ASO); or xx. comprising SEQ ID NO:314, SEQ ID NO:315, SEQ ID NO:316, SEQ ID NO:317, SEQ ID NO:318, SEQ ID NO:319, SEQ ID NO:320, SEQ ID NO:321, optionally SEQ ID NO:322 (i.e., a 21 nucleotide ASO), optionally SEQ ID NO:323 (i.e., a 22 nucleotide ASO), optionally SEQ ID NO:324 (i.e., a 23 nucleotide ASO), and optionally SEQ ID NO:325 (i.e., a 24 nucleotide ASO); or xxi. comprising SEQ ID NO:315, SEQ ID NO:316, SEQ ID NO:317, SEQ ID NO:318, SEQ ID NO:319, SEQ ID NO:320, SEQ ID NO:321, SEQ ID NO:322, optionally SEQ ID NO:323 (i.e., a 21 nucleotide ASO), optionally SEQ ID NO:324 (i.e., a 22 nucleotide ASO), optionally SEQ ID NO:325 (i.e., a 23 nucleotide ASO), and optionally SEQ ID NO:326 (i.e., a 24 nucleotide ASO); or xxii. comprising SEQ ID NO:316, SEQ ID NO:317, SEQ ID NO:318, SEQ ID NO:319, SEQ ID NO:320, SEQ ID NO:321, SEQ ID NO:322, SEQ ID NO:323, optionally SEQ ID NO:324 (i.e., a 21 nucleotide ASO), optionally SEQ ID NO:325 (i.e., a 22 nucleotide ASO), optionally SEQ ID NO:326 (i.e., a 23 nucleotide ASO), and optionally SEQ ID NO:327 (i.e., a 24 nucleotide ASO); An ASO described in any one of claims 1 to 7.

11. An ASO described in any one of claims 1 to 6, capable of binding to the UNC13A acceptor splice site.

12. The ASO of claim 11, which is complementary to sequence number 552.

13. The ASO of claim 11 or 12, wherein the acceptor splice site is a short acceptor site.

14. or bb) SEQ ID NO:160, SEQ ID NO:161, SEQ ID NO:162, SEQ ID NO:163 and SEQ ID NO:164, or cc) SEQ ID NO:161, SEQ ID NO:162, SEQ ID NO:163, SEQ ID NO:164 and SEQ ID NO:165, or dd) SEQ ID NO:162, SEQ ID NO:163, SEQ ID NO:164, SEQ ID NO:165 and SEQ ID NO:166, or ee) SEQ ID NO:163, SEQ ID NO:164, SEQ ID NO:165, SEQ ID NO:166 and SEQ ID NO:167, or ff) SEQ ID NO:164, SEQ ID NO:165, SEQ ID NO:166, SEQ ID NO:167 and SEQ ID NO:168, or gg) SEQ ID NO:165, SEQ ID NO:166, SEQ ID NO:167, SEQ ID NO:168 and SEQ ID NO:169, or hh) SEQ ID NO:166, SEQ ID NO:167, SEQ ID NO:168, SEQ ID NO:169 and SEQ ID NO:170, or ii) SEQ ID NO:167, SEQ ID NO:1 68, SEQ ID NO:169, SEQ ID NO:170, and SEQ ID NO:171, or jj) SEQ ID NO:168, SEQ ID NO:169, SEQ ID NO:170, SEQ ID NO:171, and SEQ ID NO:172, or kk) SEQ ID NO:169, SEQ ID NO:170, SEQ ID NO:171, SEQ ID NO:172, and SEQ ID NO:173, or ll) SEQ ID NO:169, SEQ ID NO:170, SEQ ID NO:171, SEQ ID NO:172, and SEQ ID NO:173, or mm) SEQ ID NO:170, SEQ ID NO:171, SEQ ID NO:172, SEQ ID NO:173, and SEQ ID NO:174, or nn) SEQ ID NO:171, SEQ ID NO:172, SEQ ID NO:173, SEQ ID NO:174, and SEQ ID NO:175, or oo) SEQ ID NO:172, SEQ ID NO:173, SEQ ID NO:174, SEQ ID NO:175, and SEQ ID NO:176, or pp) SEQ ID NO:173, SEQ ID NO:174, SEQ ID NO:175, SEQ ID NO:176, and SEQ ID NO:177, or a combination thereof.

15. The ASO of claim 11 or 12, wherein the acceptor site is a long acceptor site.

16. at least aaa) SEQ ID NO:105, SEQ ID NO:106, SEQ ID NO:107, SEQ ID NO:108 and SEQ ID NO:109, or bbb) SEQ ID NO:106, SEQ ID NO:107, SEQ ID NO:108, SEQ ID NO:109 and SEQ ID NO:110, or ccc) SEQ ID NO:107, SEQ ID NO:108, SEQ ID NO:109, SEQ ID NO:110 and SEQ ID NO:111, or ddd) SEQ ID NO:108, SEQ ID NO:109, SEQ ID NO:110, SEQ ID NO:111 and SEQ ID NO:112, or eee) SEQ ID NO:109, SEQ ID NO:110, SEQ ID NO:11 1, SEQ ID NO:112 and SEQ ID NO:113, or fff) SEQ ID NO:110, SEQ ID NO:111, SEQ ID NO:112, SEQ ID NO:113 and SEQ ID NO:114, or ggg) SEQ ID NO:111, SEQ ID NO:112, SEQ ID NO:113, SEQ ID NO:114 and SEQ ID NO:115, or hhh) SEQ ID NO:112, SEQ ID NO:113, SEQ ID NO:114, SEQ ID NO:115 and SEQ ID NO:116, or iii) SEQ ID NO:113, SEQ ID NO:114, SEQ ID NO:115, SEQ ID NO:116 and SEQ ID NO:117, or jjj) SEQ ID NO:114 , SEQ ID NO:115, SEQ ID NO:116, SEQ ID NO:117, and SEQ ID NO:118, or kkk) SEQ ID NO:115, SEQ ID NO:116, SEQ ID NO:117, SEQ ID NO:118 and SEQ ID NO:119, or lll) SEQ ID NO:116, SEQ ID NO:117, SEQ ID NO:118, SEQ ID NO:119 and SEQ ID NO:120, or mmm) SEQ ID NO:117, SEQ ID NO:118, SEQ ID NO:119, SEQ ID NO:120 and SEQ ID NO:121, or nnn) SEQ ID NO:118, SEQ ID NO:119, SEQ ID NO:120, SEQ ID NO:121 and SEQ ID NO: 22, or ooo) SEQ ID NO:119, SEQ ID NO:120, SEQ ID NO:121, SEQ ID NO:122 and SEQ ID NO:123, or ppp) SEQ ID NO:120, SEQ ID NO:121, SEQ ID NO:122, SEQ ID NO:123 and SEQ ID NO:124, or qqq) SEQ ID NO:120, SEQ ID NO:121, SEQ ID NO:122, SEQ ID NO:123 and SEQ ID NO:124, or rrr) SEQ ID NO:121, SEQ ID NO:122, SEQ ID NO:123, SEQ ID NO:124 and SEQ ID NO:125, or a combination thereof.

17. The ASO of any one of claims 1 to 6, which is an ASO of SEQ ID NO:555, SEQ ID NO:556, SEQ ID NO:557, SEQ ID NO:558, SEQ ID NO:559, SEQ ID NO:560, SEQ ID NO:561, SEQ ID NO:562, SEQ ID NO:563, SEQ ID NO:564, SEQ ID NO:565, SEQ ID NO:569, SEQ ID NO:570, SEQ ID NO:571, SEQ ID NO:579, or SEQ ID NO:

580.

18. A pharmaceutical composition comprising one or more ASOs described in any one of claims 1 to 17.

19. The pharmaceutical composition of claim 18, comprising a first ASO capable of binding to the UNC13A donor splice site or its flanking region, and a second ASO capable of binding to the UNC13A acceptor splice site or its flanking region.

20. The pharmaceutical composition of claim 18, comprising a first ASO capable of binding to the short acceptor splice site or its flanking region of UNC13A, and a second ASO capable of binding to the long acceptor splice site or its flanking region of UNC13A.

21. The pharmaceutical composition of claim 18, comprising a first ASO capable of binding to a minor allele of a cryptic exonic SNP, and a second ASO capable of binding to a major allele of a cryptic exonic SNP.

22. An ASO according to any one of claims 1 to 17 or a pharmaceutical composition according to any one of claims 18 to 21 for use as a medicament.

23. An ASO according to any one of claims 1 to 17 or a pharmaceutical composition according to any one of claims 18 to 21 for use in a method for treating a neurodegenerative disorder.

24. 1. A method for reducing expression of an UNC13A cryptic exon splice variant of UNC13A mature mRNA in a cell, comprising: reducing expression of an UNC13A cryptic exon splice variant of UNC13A mature mRNA in the cell by contacting the cell with an antisense oligonucleotide; The method, wherein the oligonucleotide is complementary to at least 8 consecutive nucleotides of SEQ ID NO:

1.

25. 25. The method of claim 24, wherein the antisense oligonucleotide is complementary to at least 8 consecutive nucleotides of SEQ ID NO:2 including the cryptic exon splice site in SEQ ID NO:

2.

26. 26. The method of claim 25, wherein the cryptic exon splice site is a donor splice site.

27. 26. The method of claim 25, wherein the cryptic exon splice site is an acceptor splice site.

28. 25. The method of claim 24, wherein the antisense oligonucleotide is complementary to at least 8 consecutive nucleotides of SEQ ID NO:3 including the cryptic exon splice site in SEQ ID NO:

3.

29. 29. The method of claim 28, wherein the cryptic exon splice site is a donor splice site.

30. 29. The method of claim 28, wherein the cryptic exon splice site is an acceptor splice site.

31. 25. The method of claim 24, wherein the antisense oligonucleotide is complementary to at least 8 consecutive nucleotides of any one of SEQ ID NOs: 547-551.

32. 25. The method of claim 24, wherein the antisense oligonucleotide is complementary to at least 8 consecutive nucleotides of any one of SEQ ID NOs: 552-554.

33. 25. The method of claim 24, wherein the antisense oligonucleotide is 13 to 30 nucleotides in length.

34. The method of claim 24, wherein the antisense oligonucleotide is 17 to 24 nucleotides in length.

35. 25. The method of claim 24, which results in increased expression of UNC13A mature mRNA.

36. 1. A method for regulating splicing of UNC13A in a cell, comprising: modulating splicing of UNC13A in a cell by contacting the cell with an antisense oligonucleotide; The method, wherein the antisense oligonucleotide is complementary to at least 8 consecutive nucleotides of SEQ ID NO:

1.

37. 37. The method of claim 36, which results in reduced expression of the UNC13A cryptic exon splice variant of UNC13A mature mRNA in a cell.

38. 37. The method of claim 36, which results in increased expression of UNC13A mature mRNA in the cell.

39. 37. The method of claim 36, wherein the antisense oligonucleotide is complementary to at least 8 consecutive nucleotides of SEQ ID NO:2 including a cryptic exon splice site in SEQ ID NO:

2.

40. 40. The method of claim 39, wherein the cryptic exon splice site is a donor splice site.

41. 37. The method of claim 36, wherein the antisense oligonucleotide is complementary to at least 8 consecutive nucleotides of any one of SEQ ID NOs: 547-551.

42. 40. The method of claim 39, wherein the cryptic exon splice site is an acceptor splice site.

43. 37. The method of claim 36, wherein the antisense oligonucleotide is complementary to at least 8 consecutive nucleotides of any one of SEQ ID NOs: 552-554.

44. 37. The method of claim 36, wherein the antisense oligonucleotide is complementary to at least 8 consecutive nucleotides of SEQ ID NO:3 including a cryptic exon splice site in SEQ ID NO:

3.

45. 37. The method of claim 36, wherein the antisense oligonucleotide is 13 to 30 nucleotides in length.

46. 37. The method of claim 36, wherein the antisense oligonucleotide is 17 to 24 nucleotides in length.

47. 1. A method for reducing expression of an UNC13A cryptic exon splice variant of UNC13A mature mRNA in a cell, comprising: reducing expression of an UNC13A cryptic exon splice variant of UNC13A mature mRNA in the cell by contacting the cell with an antisense oligonucleotide; The method, wherein the oligonucleotide is complementary to at least 8 consecutive nucleotides of any one of SEQ ID NOs: 547-554.

48. 48. The method of claim 47, wherein the antisense oligonucleotide is complementary to at least 8 consecutive nucleotides of any one of SEQ ID NOs: 547-551.

49. 48. The method of claim 47, wherein the antisense oligonucleotide is complementary to at least 8 consecutive nucleotides of any one of SEQ ID NOs: 552-554.

50. 48. The method of claim 47, wherein the antisense oligonucleotide is 13 to 30 nucleotides in length.

51. 48. The method of claim 47, wherein the antisense oligonucleotide is 17 to 24 nucleotides in length.