Compositions and methods for modulating SPTLC1

Abstract

Aspects of the present disclosure relate to compositions and methods for modulating the translation of one or more RNA transcripts (e.g., mRNA transcripts) in a cell or subject. The present disclosure is based, in part, on isolated nucleic acids that bind to an mRNA transcript of a gene involved in L-serine biosynthesis, such as serine palmitoyltransferase long chain base subunit 1 (SPTLC1). In some embodiments, the compositions of the present disclosure are useful for the treatment of diseases or conditions associated with dysregulation of L-serine biosynthesis, such as retinal diseases (e.g., MacTel2 and age-related macular degeneration (AMD), such as AMD with geographic atrophy), HSAN1 (e.g., HSAN1 (a)), diabetic retinopathy, peripheral neuropathy (e.g., neuropathy), and the treatment of diseases or conditions associated with dysregulation of L-serine biosynthesis, such as retinal diseases (e.g., MacTel2 and age-related macular degeneration (AMD), such as AMD with geographic atrophy). ), neurodegenerative disorders (e.g., Parkinson's disease, Friedel's ataxia, motor neuron disease, Alzheimer's disease, amyotrophic lateral sclerosis (ALS), etc.), spasmodic paraplegia or psychiatric disorders (e.g., neurodegenerative disorders, neurodegenerative disorders, etc. ), resistant depression, refractory depression, schizophrenia, and / or refractory schizophrenia.

Description

[0001] Related Applications

[0002] This application claims the benefit under 35 U.S.C. 119(e) of U.S. Provisional Application No. 63 / 489,247, filed March 9, 2023, U.S. Provisional Application No. 63 / 492,595, filed March 28, 2023, U.S. Provisional Application No. 63 / 507,959, filed June 13, 2023, U.S. Provisional Application No. 63 / 512,292, filed July 7, 2023, U.S. Provisional Application No. 63 / 580,819, filed September 6, 2023, U.S. Provisional Application No. 63 / 588,141, filed October 5, 2023, U.S. Provisional Application No. 63 / 611,369, filed December 18, 2023, U.S. Provisional Application No. 63 / 650,766, filed February 7, 2024, and U.S. Provisional Application No. 63 / 556,381, filed February 21, 2024, each of which is incorporated by reference in its entirety.

[0003] Reference to Electronic Sequence Listing

[0004] The contents of the electronic sequence listing (L090770037WO00-SEQ-KZM.xml; size: 267,715 bytes; and date created: March 6, 2024) is incorporated by reference herein in its entirety. BACKGROUND

[0005] L-serine is important for many biological processes in the central nervous system (CNS), such as lipid biosynthesis and production of D-serine. In the CNS, L-serine biosynthesis occurs in astrocytes. Mutations in genes associated with L-serine biosynthesis and lipid (e.g., sphingolipid) biosynthesis are associated with certain diseases and disorders of the CNS, such as macular telangiectasia type 2 (MacTel2) and hereditary sensory neuropathy type 1 (HSAN1). SUMMARY

[0006] Aspects of the present disclosure relate to isolated nucleic acids that bind to mRNA transcripts of genes involved in L-serine biosynthesis, such as serine palmitoyltransferase long chain base subunit 1 (SPTLC1). In some embodiments, compositions of the present disclosure are used to treat diseases or disorders associated with dysregulation of L-serine biosynthesis, such as retinal diseases (e.g., MacTel2 and age-related macular degeneration (AMD), such as AMD with geographic atrophy), HSAN1 (e.g., HSAN1(a)), diabetic retinopathy, peripheral neuropathy (e.g., motor neuropathy, sensory neuropathy, autonomic neuropathy, combined neuropathy, inherited or acquired peripheral neuropathy, such as diabetic neuropathy, chemotherapy-induced neuropathy, or other forms of toxic peripheral neuropathy, etc.), neurodegeneration (e.g., Parkinson’s disease, Friedreich’s ataxia, motor neuron disease, Alzheimer’s disease, amyotrophic lateral sclerosis (ALS), etc.), spastic paraplegia, or psychiatric disorders (e.g., resistant depression, treatment resistant depression, schizophrenia, and / or treatment resistant schizophrenia). The present disclosure is based, in part, on compositions and methods for modulating the level, transcription, splicing, and / or translation of one or more RNA transcripts (e.g., mRNA transcripts) in a cell or subject.

[0007] Accordingly, in some aspects, the present disclosure provides an isolated nucleic acid comprising a region complementary to a human SPTLC1 mRNA transcript and that, when bound to the mRNA transcript, reduces the level, transcription, splicing, and / or translation of functional SPTLC1 protein from the mRNA transcript. In some embodiments, the isolated nucleic acid comprises a nucleic acid sequence that is at least 60% (e.g., 60-70%, 70-80%, 80-90%, 90-95%, 95-99%, or 100%) identical to any one of the nucleic acid sequences set forth in SEQ ID NOs: 1-299.

[0008] In some embodiments, the isolated nucleic acid comprises an RNA. In some embodiments, the isolated nucleic acid is an antisense oligonucleotide (ASO).

[0009] In some embodiments, the isolated nucleic acid comprises or consists of 10 to 40 nucleotides. In some embodiments, the isolated nucleic acid comprises or consists of 18 to 25 nucleotides.

[0010] In some embodiments, the isolated nucleic acid comprises one or more chemical modifications. In some embodiments, the one or more chemical modifications comprise one or more nucleoside modifications and / or one or more sugar-phosphate backbone modifications. In some embodiments, the one or more nucleoside modifications comprise a 2’-O-methyl (2’-OMe) modification, a 2’-O-fluoro modification, or a locked nucleic acid (LNA) modification. In some embodiments, the one or more sugar-phosphate backbone modifications comprise a phosphorothioate backbone modification. In some embodiments, the isolated nucleic acid is fully chemically modified (e.g., contains a fully modified sugar-phosphate backbone, and all nucleotides of the isolated nucleic acid are chemically modified).

[0011] In some embodiments, the isolated nucleic acid comprises one or more deoxynucleotides. In some embodiments, the isolated nucleic acid is a gapmer.

[0012] In some embodiments, the region of complementarity is located in an untranslated region of the SPLTC1 mRNA transcript. In some embodiments, the untranslated region comprises a 5’ UTR, an intron, or a 3’ UTR of the SPLTC1 mRNA transcript.

[0013] In some embodiments, the region of complementarity is located in a protein-coding region of the SPLTC1 mRNA transcript.

[0014] In some embodiments, the region of complementarity is located at an intron-exon boundary of the SPLTC1 mRNA transcript (e.g., the region of complementarity spans the intron-exon boundary such that the isolated nucleic acid simultaneously hybridizes to both the intron and the exon).

[0015] In some embodiments, the region of complementarity comprises at least 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 contiguous nucleotides of the sequence set forth in SEQ ID NO: 300.

[0016] In some embodiments, the isolated nucleic acid comprises a nucleotide sequence set forth in any one of the nucleotide sequences set forth in Table 1.

[0017] In some aspects, the disclosure provides a method for increasing L-serine biosynthesis in a cell or a subject, the method comprising administering to a subject in need thereof an isolated nucleic acid as described herein.

[0018] In some embodiments, the subject is characterized as having low serine.

[0019] In some embodiments, the subject comprises one or more mutations in a gene associated with L-serine biosynthesis. In some embodiments, the gene is SPTLC1.

[0020] In some embodiments, the cell or subject is a human cell or human subject.

[0021] In some embodiments, the subject has or is suspected of having a disease or disorder associated with dysregulation of L-serine metabolism. In some embodiments, the disease or disorder is a retinal disease (e.g., MacTel2 and age-related macular degeneration (AMD), such as AMD with geographic atrophy), HSAN1 (e.g., HSAN1(a)), diabetic retinopathy, peripheral neuropathy (e.g., motor neuropathy, sensory neuropathy, autonomic neuropathy, combined neuropathy, inherited or acquired peripheral neuropathy, such as diabetic neuropathy, chemotherapy-induced neuropathy, or other forms of toxic peripheral neuropathy, etc.), neurodegeneration (e.g., Parkinson’s disease, Friedreich’s ataxia, motor neuron disease, Alzheimer’s disease, amyotrophic lateral sclerosis (ALS), etc.), spastic paraplegia, or a psychiatric disorder (e.g., treatment-resistant depression, treatment-resistant depression, schizophrenia, and / or treatment-resistant schizophrenia).

[0022] In some embodiments, the administration is systemic administration. In some embodiments, the systemic administration comprises intravenous injection.

[0023] In some embodiments, the administration comprises direct administration to a target tissue of the subject. In some embodiments, the direct administration comprises direct injection to the central nervous system (CNS) of the subject. In some embodiments, the direct administration comprises direct injection to the peripheral nervous system (PNS) of the subject. In some embodiments, the direct administration comprises direct administration to the eye of the subject (e.g., via intraocular injection, surface injection, etc.).

[0024] In some embodiments, the administration comprises placing the subject in a head-low feet-high position (Trendelenburg position) during the administration.

[0025] In some aspects, the disclosure provides a method for reducing deoxy-sphingolipid (deoxy-SL) biosynthesis in a cell or subject, the method comprising administering to a subject in need thereof an isolated nucleic acid as described herein.

[0026] In some embodiments, the subject is characterized as having low serine.

[0027] In some embodiments, the subject comprises one or more mutations in a gene associated with L-serine biosynthesis. In some embodiments, the gene is SPTLC1.

[0028] In some embodiments, the cell or subject is a human cell or human subject.

[0029] In some embodiments, the subject has or is suspected of having a disease or disorder associated with dysregulation of L-serine metabolism. In some embodiments, the disease or disorder is a retinal disease (e.g., MacTel2 and age-related macular degeneration (AMD), such as AMD with geographic atrophy), HSAN1 (e.g., HSAN1(a)), diabetic retinopathy, peripheral neuropathy (e.g., motor neuropathy, sensory neuropathy, autonomic neuropathy, combined neuropathy, inherited or acquired peripheral neuropathy, such as diabetic neuropathy, chemotherapy-induced neuropathy, or other forms of toxic peripheral neuropathy, etc.), neurodegeneration (e.g., Parkinson’s disease, Friedreich’s ataxia, motor neuron disease, Alzheimer’s disease, amyotrophic lateral sclerosis (ALS), etc.), spastic paraplegia, or a psychiatric disorder (e.g., treatment-resistant depression, treatment-refractory depression, schizophrenia, and / or treatment-refractory schizophrenia).

[0030] In some embodiments, the administration is systemic administration. In some embodiments, the systemic administration comprises intravenous injection.

[0031] In some embodiments, the administration comprises direct administration to a target tissue of the subject. In some embodiments, the direct administration comprises direct injection to the central nervous system (CNS) of the subject. In some embodiments, the direct administration comprises direct injection to the peripheral nervous system (PNS) of the subject. In some embodiments, the direct administration comprises direct administration to the eye of the subject (e.g., via intraocular injection, surface injection, etc.).

[0032] In some embodiments, the administration comprises placing the subject in a head-low feet-high position (Trendelenburg position) during the administration.

[0033] In some aspects, the disclosure provides a method for preventing or treating a disease or disorder associated with dysregulation of L-serine biosynthesis in a subject in need thereof, the method comprising administering to the subject an isolated nucleic acid as described herein.

[0034] In some embodiments, the subject is a human.

[0035] In some embodiments, the disease or disorder is a retinal disease (e.g., MacTel2 and age-related macular degeneration (AMD), such as AMD with geographic atrophy), HSAN1 (e.g., HSAN1(a)), diabetic retinopathy, peripheral neuropathy (e.g., motor neuropathy, sensory neuropathy, autonomic neuropathy, combined neuropathy, inherited or acquired peripheral neuropathy, such as diabetic neuropathy, chemotherapy-induced neuropathy, or other forms of toxic peripheral neuropathy, etc.), neurodegeneration (e.g., Parkinson’s disease, Friedreich’s ataxia, motor neuron disease, Alzheimer’s disease, amyotrophic lateral sclerosis (ALS), etc.), spastic paraplegia, or a psychiatric disorder (e.g., treatment-resistant depression, treatment-refractory depression, schizophrenia, and / or treatment-refractory schizophrenia).

[0036] In some embodiments, the administering comprises direct administration to a target tissue of the subject. In some embodiments, the direct administration comprises direct injection to the central nervous system (CNS) of the subject. In some embodiments, the direct administration comprises direct injection to the peripheral nervous system (PNS) of the subject. In some embodiments, the direct administration comprises direct administration to the eye of the subject (e.g., via intraocular injection, surface injection, etc.). BRIEF DESCRIPTION OF DRAWINGS

[0037] Figure 1 A schematic depicting the modulation of translation of an RNA (e.g., mRNA, such as a mature mRNA or a pre-mRNA) by an antisense oligonucleotide (ASO) is shown. Constituent “A” represents an ASO that binds to the 5’ untranslated region (5’ UTR) of the RNA. Constituent “B” represents an ASO that binds to an intron of the RNA. Constituent “C” represents an ASO that binds to a splice boundary (e.g., splice junction) between an exon and an intron of the RNA. Constituent “D” represents an ASO that binds to an exon (e.g., protein-coding region) of the RNA. Constituent “E” represents an ASO alone that binds to the 3’ UTR of the RNA or in combination with a trans -regulator. Constituent “F” represents a “gapmer” ASO that binds to an exon (e.g., protein-coding region) of the RNA and mediates RNase H decay. Constituent “G” represents a “gapmer” ASO that binds to the 3’ UTR of the RNA and mediates RNase H decay (alone or with a trans -regulator). In some embodiments, an ASO that binds to the RNA results in translation of a truncated protein that has a dominant negative effect on the wild-type full-length protein.

[0038] Figures 2A-2C Representative data from an expression profiling analysis for human serine palmitoyltransferase long chain base subunit 1 (SPTLC1) is shown. Figure 2ABulk tissue gene expression is shown for human SPTLC1 ; data indicate that SPTLC1 mRNA is ubiquitously expressed. Figure 2B A schematic diagram depicting the exons and introns present in the SPTLC1 gene (e.g., NCBI Reference Sequence NM_006415.4) is shown. Figure 2C Representative data from an exon expression analysis of human SPTLC1 splice variants in CNS tissues is shown.

[0039] Figures 3A-3E Representative in vitro data for ASOs targeting SPTLC1 RNA are shown. Figure 3A A schematic of SPTLC1 RNA is shown, with exons indicated by boxes and introns indicated by broken lines. Based on the location of the target region on the SPTLC1 RNA for the ASOs, 216 designed ASOs are indicated (gray and dark gray rectangles; Table 1). The most potent ASOs in each chemical are colored dark gray. The region of SPTLC1 amplified in the qPCR reaction is indicated by the rectangularly labeled “qPCR amplicon.” Figure 3B SPTLC1 RNA levels following ASO treatment are shown. Normalized SPTLC1 RNA levels in U-118 MG cells as measured by RT-qPCR 48 hours after transfection with non-targeting control ASO (gray), siRNA targeting SPTLC1 (gray), or ASOs targeting SPTLC1 at 5 nM and 20 nM doses are presented (Chemical 1: indicated by a set of light and dark hatched bars to the right of the water and siRNA control; Chemical 2: indicated by a set of light and dark hatched bars in the distance to the right). SPTLC1 expression was normalized to HPRT1 expression and presented as a percentage of the untransfected control. The mean between biological replicates (N=2) is presented; error bars are standard deviations. Figure 3C SPTLC1 RNA downregulation by selected ASOs is shown. Normalized SPTLC1 RNA levels in U-118 MG cells as measured by RT-qPCR 48 hours after transfection with 20 nM non-targeting control ASO (gray) or 23 ASOs that showed >50% reduction in SPTLC1 RNA levels at a 20 nM dose are presented (Chemical 1: indicated by a set of light and dark hatched bars to the right of the water and siRNA control; Chemical 2: indicated by a set of light and dark hatched bars in the distance to the right). SPTLC1 expression was normalized to HPRT1 expression and presented as a percentage of the untransfected control. The mean of biological replicates (N=2) is presented; error bars are standard deviations. Figure 3DRepresentative data from an analysis of SPTLC1 RNA downregulation following administration of selected ASOs to U-251 MG cells at the indicated half maximal effective concentration (EC50) is shown. SPTLC1 RNA in U-251 MG cells was measured by RT-qPCR 48 hours after transfection with the indicated ASO and normalized to HPRT1 expression. Figure 3E Representative results from an analysis of U-251MC cells following administration of selected ASOs are shown. The left panel shows the level of SPTLC1 RNA in ASO treated U-251 MG cells relative to the level of SPTLC1 RNA in U-251 MG cells treated with negative control. The right panel shows the level of HPRT1 RNA in ASO treated U-251 MG cells relative to the level of HPRT1 RNA in U-251 MG cells treated with negative control. RNA expression levels were measured 48 hours after transfection with the indicated ASO at a dose of 40 nM, 20 nM, 10 nM, 5 nM, 2.5 nM, 1.25 nM, 0.625 nM, or 0.3125 nM. In some embodiments, the ASO shown includes an ASO comprising the nucleotide sequence of SEQ ID NO: 22, the gapmer structure, and the chemical modifications as shown in column A and column C of row 23 of Table 1; an ASO comprising the nucleotide sequence of SEQ ID NO: 147, the gapmer structure, and the chemical modifications as shown in column A and column C of row 148 of Table 1 (or also referred to herein as “SPTLC1 ASO 2”); an ASO comprising the nucleotide sequence of SEQ ID NO: 152, the gapmer structure, and the chemical modifications as shown in column A and column C of row 153 of Table 1; an ASO comprising the nucleotide sequence of SEQ ID NO: 162, the gapmer structure, and the chemical modifications as shown in column A and column C of row 163 of Table 1 (or also referred to herein as “SPTLC1 ASO 1”); an ASO comprising the nucleotide sequence of SEQ ID NO: 193, the gapmer structure, and the chemical modifications as shown in column A and column C of row 194 of Table 1; an ASO comprising the nucleotide sequence of SEQ ID NO: 221, the gapmer structure, and the chemical modifications as shown in column A and column C of row 222 of Table 1; and an ASO comprising the nucleotide sequence of SEQ ID NO: 254, the gapmer structure, and the chemical modifications as shown in column A and column C of row 255 of Table 1. Figures 3D-3E

[0040] Figures 4A-4D High consistency of SPTLC1 knockdown in different cell types is shown. Figure 4A ​Representative results obtained when 61 SPTLC1 -targeting ASOs were tested in U-251 MG cells and the knockdown was compared to the knockdown previously observed in U-118 MG cells are shown. U-251 MG cells were reverse transfected with 5 nM dose or 20 nM dose of ASO (Chemical 1 (skipmer): light and dark shaded circles and triangles, respectively; Chemical 2 (gapmer): light and dark shaded circles and triangles, respectively) and SPTLC1 expression was measured by RT-qPCR at 48 hours. The ASO-targeting sequence of SPTLC1 is either conserved in cynomolgus and / or mouse, or not conserved in cynomolgus and mouse, as indicated. The effect of the ASOs tested on U-251 MG cells and U-118 MG cells was determined to be highly correlated (R = 0.86). Figure 4B Representative results obtained when ASO comprising the nucleotide sequence of SEQ ID NO: 162, the gapmer structure, and the chemical modifications as shown in column A and column C of line 163 of Table 1 (or also referred to herein as “SPTLC1 ASO 1”) or a non-targeting ASO were transfected in COS-7 cells at different concentrations are shown. SPTLC1 mRNA levels were measured by RT-qPCR 48 hours post transfection and presented as percent of vehicle control. Mean values are presented; error bars are standard deviations of N = 2 biological replicates. X-axis is a Log10 scale. Figure 4C Representative results obtained when water, a non-targeting ASO, an ASO comprising the nucleotide sequence of SEQ ID NO: 147, the gapmer structure, and the chemical modifications as shown in column A and column C of line 148 of Table 1 (or also referred to herein as “SPTLC1 ASO 2”), or an ASO comprising the nucleotide sequence of SEQ ID NO: 162, the gapmer structure, and the chemical modifications as shown in column A and column C of line 163 of Table 1 (or also referred to herein as “SPTLC1 ASO 1”) were transfected in COS-7 cells at different concentrations (2.5 nM, 5 nM, 10 nM, and 20 nM, shown from left to right in the bar graph above each treatment group). SPTLC1 mRNA levels were measured by RT-qPCR 48 hours post transfection and presented as percent of vehicle (water) control. Mean values are presented; error bars are standard deviations of N = 2 biological replicates. X-axis is a Log10 scale. Figure 4DRepresentative results showing human iPSC-derived spinal cord motor neurons (left panel) and human iPSC-derived spinal cord motor neurons (right panel) treated with ASO comprising the nucleotide sequence of SEQ ID NO: 162, gapmer structure, and chemical modifications as shown in column A and column C of row 163 of Table 1 (or also referred to herein as“SPTLC1 ASO 1”) via gymnotic delivery for 5 days. SPTLC1 mRNA levels were measured using RT-qPCR. In human iPSC-derived spinal cord motor neurons (left panel), concentration-dependent potency was observed with a low micromolar EC50 of 0.8 µM. The left panel shows the mean + / - SD, with the x-axis on a Log10 scale. N=2 wells. In human iPSC-derived spinal cord motor neurons (right panel), concentration-dependent potency was observed with a low micromolar EC50 of 0.1 µM. The right panel shows the mean + / - SD, with the x-axis on a Log10 scale. N=3 wells.

[0041] Figures 5A-5B Dose response of SPTLC1 -targeting ASO in U-251 MG cells is shown. ASO was applied in vitro to U-251 cells at 8 doses: 40 nM, 20 nM, 10 nM, 5 nM, 2.5 nM, 1.25 nM, 0.625 nM, and 0.3125 nM. U-251 MG cells were treated in 96-well plates by reverse transfection and SPTLC1 expression was assayed after 48 hours by RT-qPCR. ASO treatment was normalized to control-transfected cells. The mean between biological replicates (N=2) is presented; error bars are standard deviations. Figure 5ARepresentative expression data from assays for SPTLC1 mRNA levels following in vitro administration of 6 different ASOs to U-251 MG cells are shown. The chemistry of the ASO assayed is indicated (Chemistry 1 (skipmer): dark gray; Chemistry 2 (gapmer): light gray). 0% knockdown and 50% knockdown are indicated by dotted lines (black and gray, respectively). The ASOs shown include: an ASO comprising the nucleotide sequence, gapmer structure, and chemical modifications of SEQ ID NO: 46 as shown in column A and column C of row 47 of Table 1; an ASO comprising the nucleotide sequence, gapmer structure, and chemical modifications of SEQ ID NO: 147 as shown in column A and column C of row 148 of Table 1 (or also referred to herein as “SPTLC1 ASO 2”); an ASO comprising the nucleotide sequence, gapmer structure, and chemical modifications of SEQ ID NO: 195 as shown in column A and column C of row 196 of Table 1; an ASO comprising the nucleotide sequence, gapmer structure, and chemical modifications of SEQ ID NO: 233 as shown in column A and column C of row 234 of Table 1; an ASO comprising the nucleotide sequence, skipmer structure, and chemical modifications of SEQ ID NO: 245 as shown in column A and column C of row 246 of Table 1; and an ASO comprising the nucleotide sequence, gapmer structure, and chemical modifications of SEQ ID NO: 248 as shown in column A and column C of row 249 of Table 1. Figure 5B Representative expression data from assays for SPTLC1 mRNA levels 48 hours following in vitro administration of two ASOs (an ASO comprising the nucleotide sequence, gapmer structure, and chemical modifications of SEQ ID NO: 162 as shown in column A and column C of row 163 of Table 1 (or also referred to herein as “SPTLC1 ASO 1”) and an ASO comprising the nucleotide sequence, gapmer structure, and chemical modifications of SEQ ID NO: 147 as shown in column A and column C of row 148 of Table 1 (or also referred to herein as “SPTLC1 ASO 2”)) to U-251 MG cells are shown. 0% knockdown is indicated by a black dotted line, 20% knockdown is indicated by a black dashed line, and 50% knockdown is indicated by a gray dotted line.

[0042] Figures 6A-6D Representative in vitro data for ASOs targeting SPTLC1 RNA in induced pluripotent stem cell (iPSC)-derived neurons are shown. Figure 6ASPTLC1 RNA levels in iPSC-derived neurons after ASO treatment are shown. iPSC-derived neurons were treated with 0 µM ASO (PBS control) or a range of 0.3 µM to 10 µM ASO. 50% knockdown of SPTLC1 expression is indicated by the dashed line. Figure 6B SPTLC1 RNA levels in iPSC-derived neurons after ASO treatment are shown. iPSC-derived neurons were treated with 0 µM ASO (PBS control) or a range of 0.3 µM to 10 µM ASO. 50% knockdown of SPTLC1 expression is indicated by the dashed line. Figure 6A HPRT1 RNA levels in iPSC-derived neurons after ASO treatment in Example 2 are shown. The effect of ASOs targeting SPTLC1 (up to 1 µM ASO) on HPRT1 expression was generally attenuated, with the maximum reduction in HPRT1 expression observed at the 10 µM ASO dose. Figure 6C A comparison between the SPTLC1 knockdown observed in iPSC-derived neurons treated with 3 µM ASO and the SPTLC1 knockdown observed in U251-MG cells treated with 20 µM ASO is shown. The effect of each dose on SPTLC1 expression in each respective cell type was determined to be highly correlated (r = 0.73). Figure 6D Representative data from an analysis of WDR47 and ZFHX3 expression in human iPSC-derived cortical neurons treated with an ASO comprising the nucleotide sequence of SEQ ID NO: 162, gapmer structure, and chemical modifications as shown in column A and column C of line 163 of Table 1 (or also referred to herein as “SPTLC1 ASO 1”) is shown. Mean values are presented; error bars are standard deviations for N = 3 biological replicates. The X-axis is on a Log10 scale.

[0043] Figure 7 Representative data for SPTLC1 mRNA levels in iPSC-derived neurons comprising a clinical HSAN1 mutation is shown. “WT” indicates wild-type human iPSCs. “Het KO” indicates iPSCs that are heterozygous for a knockout of the PTLC1 gene. “Het KI C133W” and “Het KI” indicate iPSCs that are heterozygous for a knock-in of a SPTCL1 allele encoding the C133W mutation seen in HSAN1 patients. “ASO 1” indicates treatment with an ASO comprising the nucleotide sequence of SEQ ID NO: 162, gapmer structure, and chemical modifications as shown in column A and column C of line 163 of Table 1 (or also referred to herein as “SPTLC1 ASO 1”). “Mycosporine.” indicates human iPSCs treated with the non-selective SPT inhibitor mycosporine. : p<0.05, : p<0.01, p < 0.001 (by one-way ANOVA followed by Dunnett’s test for post-hoc comparisons for Het C133W KI versus treatment or different SPTLC1 genotypes).

[0044] Figures 8A-8K Representative data on the effects of nucleotide sequence, gapmer structure, and chemically modified SPTLC1ASO1 on toxic lipids in iPSC-derived neurons containing clinical HSAN1 mutations, as shown in columns A and C of row 163 of Table 1. Figure 8A The sphingolipid biosynthesis pathway is shown, with non-limiting examples of toxic deoxysphingolipids and sphingolipids associated with neurological diseases shown in boxes. Figure 8B Increased production of toxic 1-deoxysphingoid bases was observed in human iPSCs derived from HSAN1 patients with the SPTC1 C133W mutation. Figure 8C The production of 1-deoxydihydrosphingosine was shown in human iPSC-derived neurons. Figure 8D This study demonstrates the production of deoxysphingosine in human iPSC-derived neurons. Figure 8E This study demonstrates the production of dihydrosphingosine in human iPSC-derived neurons. Figure 8F This demonstrates the production of sphingosine in human iPSC-derived neurons. Figure 8G Showing Figures 8C-8F The data is represented by a heatmap. "DSB" indicates deoxysphingosine bases. "DHSph" indicates dihydrosphingosine. "Deoxy-DHSph" indicates 1-deoxydihydrosphingosine. "Sph" indicates sphingosine. "Deoxy-Sph" indicates 1-deoxysphingosine. Figure 8H This study demonstrates the production of 1-deoxyceramide in human iPSC-derived neurons. Figure 8I This study demonstrates the production of ceramides in human iPSC-derived neurons. Figure 8J Showing Figures 8H-8I Heatmap representation of the data. "Deoxy-Cer" indicates 1-deoxyceramide. "Cer" indicates ceramide. For Figures 8C-8J“WT” indicates wild-type human iPSCs. “Het KO” indicates heterozygous iPSCs for SPTLC1 gene knockout. “Het KI C133W” and “Het KI” indicate heterozygous iPSCs for knock-in of an SPTCL1 allele encoding the C133W mutation seen in HSAN1 patients. “ASO 1” indicates treatment with SPTLC1 ASO1 comprising the nucleotide sequence of SEQ ID NO: 162, gapmer structure, and chemical modifications as shown in column A and column C of row 163 of Table 1. “Lead myr.” indicates human iPSCs treated with the nonselective SPT inhibitor myriocin. Figure 8K Representative data showing the modulation of sphingolipid levels by SPTLC1 ASO 1 in human iPSC-derived cortical neurons is shown. Human iPSC-derived cortical neurons were treated with SPTLC1 ASO 1 via gymnotic delivery for 5 days. SPTLC1 mRNA levels were examined using RT-qPCR, and sphingolipid profiles were analyzed using HPLC-MS / MS and normalized to total protein input. SPTLC1 ASO 1 treatment decreased SPTLC1 expression as well as total ceramide levels. For Figure 8C , 8E -8F and 8H-8I: : p < 0.05, : p < 0.01, : p < 0.001 (by one-way ANOVA followed by Dunnett’s test for post-hoc comparisons for Het C133W KI vs. treatment or different SPTLC1 genotypes).

[0045] Figures 9A-9D Representative data showing the in vivo reduction of SPTLC1 mRNA levels in mouse brain is shown. Figure 9ARelative SPTLC1 mRNA levels in hippocampal tissue of mouse subjects seven days after a series of three weekly ICV injections of vehicle (artificial CSF), 3 ug of polyoxin, 200 ug (100 ug + 100 ug) or 300 ug (100 ug + 100 ug + 100 ug) dose of non-specific ASO, or 300 ug (100 ug + 100 ug + 100 ug) dose of SPTLC1 ASO 1 or SPTLC1 ASO 2 are shown. Figure 9B Relative SPTLC1 mRNA levels in cortical tissue of mouse subjects seven days after a series of three weekly ICV injections of vehicle (artificial CSF), 3 ug of polyoxin, 200 ug (100 ug + 100 ug) or 300 ug (100 ug + 100 ug + 100 ug) dose of non-specific ASO, or 300 ug (100 ug + 100 ug + 100 ug) dose of SPTLC1 ASO 1 or SPTLC1 ASO 2 are shown. Figure 9C Relative SPTLC1 mRNA levels in hippocampal tissue of mouse subjects seven days after a series of three weekly ICV injections of vehicle (artificial CSF) or 300 ug (100 ug + 100 ug + 100 ug) of ASO by catheter are shown. Figure 9D Relative SPTLC1 mRNA levels in cortical tissue of mouse subjects seven days after a series of three weekly ICV injections of vehicle (artificial CSF) or 300 ug (100 ug + 100 ug + 100 ug) of ASO by catheter are shown. “SPTLC1 ASO 1” comprises the nucleotide sequence of SEQ ID NO: 162, the gapmer structure, and the chemical modifications as shown in column A and column C of line 163 of Table 1. “SPTLC1 ASO 2” comprises the nucleotide sequence of SEQ ID NO: 147, the gapmer structure, and the chemical modifications as shown in column A and column C of line 148 of Table 1.

[0046] Figures 10A-10B SPTLC1 ASO 1 pharmacokinetics (ASO levels) in cortical and hippocampal tissue samples obtained from injected mouse subjects are shown. Figure 10ASPTLC1 ASO 1 pharmacokinetics in cortical and hippocampal tissue samples obtained from mouse subjects that received a single intracerebroventricular (ICV) injection of a control agent (vehicle) or a 300 pg dose (three 100 pg injections administered 7 days apart) of SPTLC1 ASO 1. SPTLC1 ASO 1 levels were measured in cortical tissue samples collected from mouse subjects one week after injection. For Figure 10B SPTLC1 ASO 1 pharmacokinetics in cortical and hippocampal tissue samples obtained from mouse subjects that received a single intracerebroventricular (ICV) injection of a control agent (vehicle) or a 300 pg dose (three 100 pg injections administered 7 days apart) of SPTLC1 ASO 1. SPTLC1 ASO 1 levels were measured in cortical tissue samples collected from mouse subjects one week after injection. For Figures 10A-10B “SPTLC1 ASO 1” comprises the nucleotide sequence, gapmer structure, and chemical modifications of SEQ ID NO: 162 as shown in column A and column C of row 163 of Table 1. “SPTLC1 ASO 2” comprises the nucleotide sequence, gapmer structure, and chemical modifications of SEQ ID NO: 147 as shown in column A and column C of row 148 of Table 1. “Vehicle” refers to artificial CSF (aCSF). “pg / g” refers to micrograms of SPTLC1 ASO 1 per gram of tissue sample. Each point on the graph represents a respective mouse subject. Bars represent mean + / - standard deviation.

[0047] Figures 11A-11M Representative data for SPTLC1 ASO 1 pharmacokinetics (ASO levels), pharmacodynamics (SPTLC1 mRNA expression), and toxicity sphingolipid levels in tissue samples obtained from injected non-human primate subjects. “SPTLC1 ASO 1” comprises the nucleotide sequence, gapmer structure, and chemical modifications of SEQ ID NO: 162 as shown in column A and column C of row 163 of Table 1. For Figures 11B-11J Each point on the graph represents a respective non-human primate subject. For Figures 11D-11K“Assay 1” refers to samples analyzed with an RT-qPCR probe that binds SPTLC1 mRNA at a sequence located near the SPTLC1 ASO 1 binding site, and “Assay 2” refers to samples analyzed with an RT-qPCR probe that binds indiscriminately to all SPTLC1 mRNA isoforms, including an alternative shorter transcript that lacks a domain critical to SPTLC1 activity and is not targeted by SPTLC1 ASO 1. Figure 11A A non-limiting example of a study design is shown, in which a series of two intrathecal (IT) injections (at Day 1 and Day 14) of a 40 mg (20 mg + 20 mg) dose of vehicle (artificial CSF) or SPTLC1 ASO 1 are administered to non-human primate subjects. N = 2-3 non-human primate animal subjects. Figure 11B SPTLC1 ASO 1 levels in frontal cortex (“cortex”), hippocampus, and lumbar spinal cord tissue samples obtained from non-human primate subjects who underwent injections of SPTLC1 ASO 1 according to Figure 11A SPTLC1 ASO 1 levels in frontal cortex (“cortex”), hippocampus, and lumbar spinal cord tissue samples obtained from non-human primate subjects who underwent injections of SPTLC1 ASO 1 according to Figure 11C SPTLC1 ASO 1 levels in frontal cortex (“cortex”), hippocampus, and lumbar spinal cord tissue samples obtained from non-human primate subjects who underwent injections of SPTLC1 ASO 1 according to Figure 11A SPTLC1 ASO 1 levels in frontal cortex (“cortex”), hippocampus, and lumbar spinal cord tissue samples obtained from non-human primate subjects who underwent injections of SPTLC1 ASO 1 according to Figure 11D RT-qPCR analysis data for SPTLC1 mRNA expression in frontal cortex tissue samples obtained from non-human primate subjects who underwent injections of SPTLC1 ASO 1 according to Figure 11A RT-qPCR analysis data for SPTLC1 mRNA expression in frontal cortex tissue samples obtained from non-human primate subjects who underwent injections of SPTLC1 ASO 1 according to : p < 0.05 for treatment vs. vehicle by unpaired T-test. Bars represent mean + / - standard deviation. Figure 11E RT-qPCR analysis data obtained using two different SPTLC1 probes (corresponding to Assay 2 and Assay 1 in Figure 11D RT-qPCR analysis data obtained using two different SPTLC1 probes (corresponding to Assay 2 and Assay 1 in Figure 11ASPTLC1 mRNA expression in frontal cortex tissue of non-human primate subjects harvested after intrathecal injection of vehicle (artificial CSF), 20 mg (10 mg + 10 mg) or 40 mg (20 mg + 20 mg) dose of non-specific ASO or 40 mg (20 mg + 20 mg) dose of SPTLC1 ASO 1. SPTLC1 mRNA expression was normalized to PGK1 mRNA expression and shown as a percentage of the vehicle group. Bars indicate mean + / - standard deviation. Figure 11F A comparison of data obtained from RT-qPCR analysis of SPTLC1 mRNA expression in sensory cortex tissue of non-human primate subjects who underwent administration according to Figure 11A A comparison of data obtained from RT-qPCR analysis of SPTLC1 mRNA expression in sensory cortex tissue of non-human primate subjects who underwent administration according to Figure 11E A comparison of data obtained from RT-qPCR analysis of SPTLC1 mRNA expression in sensory cortex tissue of non-human primate subjects who underwent administration according to : p<0.05 treatment vs. vehicle by unpaired T-test. Bars indicate mean + / - standard deviation of the mean. Figure 11G A comparison of data obtained from RT-qPCR analysis of SPTLC1 mRNA expression in sensory cortex tissue of non-human primate subjects who underwent administration according to Figure 11A SPTLC1 mRNA expression in sensory cortex tissue of non-human primate subjects harvested after intrathecal injection of vehicle (artificial CSF), 20 mg (10 mg + 10 mg) or 40 mg (20 mg + 20 mg) dose of non-specific ASO or 40 mg (20 mg + 20 mg) dose of SPTLC1 ASO 1. SPTLC1 mRNA expression was normalized to PGK1 mRNA expression and shown as a percentage of the vehicle group. Bars indicate mean + / - standard deviation. N=2-3 / group. Figure 11H A comparison of data obtained from RT-qPCR analysis of SPTLC1 mRNA expression in sensory cortex tissue of non-human primate subjects who underwent administration according to Figure 11G A comparison of data obtained from RT-qPCR analysis of SPTLC1 mRNA expression in sensory cortex tissue of non-human primate subjects who underwent administration according to : p<0.01. Figure 11I A comparison of data obtained from RT-qPCR analysis of SPTLC1 mRNA expression in sensory cortex tissue of non-human primate subjects who underwent administration according to Figure 11A A comparison of data obtained from RT-qPCR analysis of SPTLC1 mRNA expression in sensory cortex tissue of non-human primate subjects who underwent administration according to Figure 11G A comparison of data obtained from RT-qPCR analysis of SPTLC1 mRNA expression in sensory cortex tissue of non-human primate subjects who underwent administration according to : p<0.05 treatment vs. vehicle by unpaired T-test. Bars indicate mean + / - standard deviation. Figure 11JIt shows that by experiencing according to Figure 11A RT-qPCR analysis of SPTLC1 mRNA expression in the sensory cortex tissue of non-human primate subjects was obtained. Figure 11I Comparison of data from the previous section. SPTLC1 mRNA expression was normalized to PGK1 mRNA expression and displayed as a percentage of the mediator group. The unpaired t-test showed that the p-value for treatment versus the mediator was <0.05. The bar indicates the standard error of the mean plus or minus the mean. Figure 11K The pharmacokinetics of SPTLC1 ASO1 in tissue samples from the frontal cortex (left panel) and sensory cortex (right panel) are compared. RT-qPCR analysis was performed using assay 1. The unpaired t-test showed that the p-value for treatment versus the mediator was <0.05. The bar indicates the standard error of the mean plus or minus the mean. Figure 11L Showing Figures 11D-11K The data is presented in a table. The average knockdown level is shown as a percentage difference from the media group. The average knockdown level was analyzed at a 95% confidence interval (“CI”) (lower and upper limits are shown in parentheses). The p-values ​​related to the average knockdown level determined from the confidence interval analysis are shown in scientific notation. The “IDT” probe and the “Thermo” probe correspond to… Figure 11D , 11E Dark (Measurement 2) and light (Measurement 1) shaded bars in 11G. Figure 11M Liquid chromatography-tandem mass spectrometry analysis of toxic sphingolipid levels in lumbar spinal cord samples obtained from non-human primate subjects who received an IT injection of 40 mg (20 mg + 20 mg) SPTLC1 ASO 1 is presented. Each spot represents one animal. Bars show the mean plus / minus the standard error of the mean. N = 3 / group. The unpaired t-test showed that the treatment versus the medium was p<0.05.

[0048] Figure 12A schematic depicting SPTCL1 ASO 1 is shown, comprising the nucleotide sequence of SEQ ID NO: 162, gapmer structure, and chemical modifications as shown in column A and column C of line 163 of Table 1, which targets a sequence present in the 3’ untranslated region (UTR) of SPTLC1 transcripts encoding the full-length canonical isoform of SPTLC1. Shorter transcripts encoding minor isoforms of SPTLC1 lack the sequence targeted by SPTLC1 ASO 1. This shorter isoform also lacks the catalytic domain that is critical for SPTLC1 activity, while retaining the SPTLC1 inhibition and dimerization domains. Also shown are the regions of the SPTLC1 transcript detected by the RT-qPCR probes used in Assay 1 and Assay 2 (see, e.g., Figures 11D-11K ).

[0049] Figures 13A-13J Representative immunostimulatory effects of SPTLC1 ASOs on human peripheral blood mononuclear cells (huPBMCs) harvested from healthy donors are shown. huPBMCs were either left untreated, treated with cytokine / chemokine response control agents (XD-01024, CL097, R837, ODN2216, ODN2006, ODN2395, TL8-506, poly (l:c), or XD-00366) for 24 hours, or treated with SPTLC1 ASOs at concentrations of 1 µM, 3 µM, or 10 µM for 24 hours (indicated on the x-axis). Cytokine / chemokine levels (indicated by the y-axis) were then analyzed using the MSD-U-Plex platform. “SPTLC1 ASO 1” comprises the nucleotide sequence of SEQ ID NO: 162, gapmer structure, and chemical modifications as shown in column A and column C of line 163 of Table 1 (right panel). “SPTLC1 ASO 2” comprises the nucleotide sequence of SEQ ID NO: 147, gapmer structure, and chemical modifications as shown in column A and column C of line 148 of Table 1 (left panel). Graphs show mean + / - standard error. Each point represents an individual donor. N = 4 donors (2 male and 2 female). Figure 13A Analysis of IFN-a2a levels is shown. Figure 13B Analysis of IFN-b levels is shown. Figure 13C Analysis of IL-1B levels is shown. Figure 13D Analysis of IL-6 levels is shown. Figure 13E Analysis of IL-10 levels is shown. Figure 13F Analysis of IP-10 levels is shown. Figure 13G Analysis of MCP-1 levels is shown. Figure 13H Analysis of MIP-1a levels is shown. Figure 13IAnalysis of MIP-1b levels is shown. Figure 13B Analysis of TNF-a levels is shown.

[0050] Figure 14 A non-limiting example of a study design is shown in which a series of four intrathecal injections of a 95 mg (30 mg + 25 mg + 20 mg + 20 mg) dose of either vehicle (artificial CSF) or ASO were administered to non-human primate subjects. Each intrathecal injection was performed two weeks apart (Day 0, 14, 28, and 42).

[0051] Figure 15 shows representative data from analysis of pharmacokinetics (ASO levels), pharmacodynamics (SPTLC1 mRNA expression), and toxic sphingolipid levels in tissue samples obtained from injected non-human primate subjects that received a series of four intrathecal (IT) injections comprising SPTLC1 ASO 1 having the nucleotide sequence, gapmer structure, and chemical modifications of SEQ ID NO: 162 as shown in columns A and C of row 163 of Table 1. As shown, the non-human primate subjects received a 95 mg (30 mg + 25 mg + 20 mg + 20 mg) dose of SPTLC1 ASO 1 via intrathecal injection. The indicated samples were obtained two weeks after the last injection of ASO (Day 56). Figure 14 Figure 16 shows representative data from analysis of pharmacokinetics (ASO levels), pharmacodynamics (SPTLC1 mRNA expression), and toxic sphingolipid levels in tissue samples obtained from injected non-human primate subjects that received a series of four intrathecal (IT) injections comprising SPTLC1 ASO 1 having the nucleotide sequence, gapmer structure, and chemical modifications of SEQ ID NO: 162 as shown in columns A and C of row 163 of Table 1. As shown, the non-human primate subjects received a 95 mg (30 mg + 25 mg + 20 mg + 20 mg) dose of SPTLC1 ASO 1 via intrathecal injection. The indicated samples were obtained two weeks after the last injection of ASO (Day 56). Figure 15A Figure 17 shows ASO levels in prefrontal cortex, hippocampus, and lumbar spinal cord samples obtained from injected non-human primate subjects and assessed by liquid chromatography-tandem mass spectrometry (LC-MS / MS). Each point represents one sample obtained from a different non-human primate subject. N=3 for each of the indicated sample groups. Bars show the mean + / - standard error of the mean. Figure 15B Figure 18 shows ASO levels in dorsal root ganglion (DRG), hippocampus, lumbar spinal cord, motor cortex, prefrontal cortex, and temporal cortex samples obtained from injected non-human primate subjects and assessed by LC-MS / MS. Each point represents one sample obtained from a different non-human primate subject. N=2-3 for each of the indicated sample groups. Bars show the mean + / - standard error of the mean. Figure 15C Figure 19 shows RT-qPCR analysis data for SPTLC1 mRNA expression in prefrontal cortex tissue samples obtained from injected non-human primate subjects. Bars show the mean + / - standard error of the mean. Figure 15D Figure 20 shows liquid chromatography-tandem mass spectrometry analysis of toxic sphingolipid levels in cerebrospinal fluid (CSF) samples obtained from non-human primate subjects. Bars show the mean + / - standard error of the mean. N=3 / group. (+) by paired t-test, p < 0.1 for day 56 vs. baseline for animals treated with SPTLC1 ASO 1. DETAILED DESCRIPTION

[0052] Aspects of the present disclosure relate to compositions and methods for modulating the level, transcription, splicing, and / or translation of one or more RNA transcripts (e.g., mRNA transcripts) in a cell or subject. The present disclosure is based in part on isolated nucleic acids that bind to mRNA transcripts of genes involved in L-serine biosynthesis or lipid (e.g., sphingolipid) biosynthesis, such as serine palmitoyltransferase long chain base subunit 1 (SPTLC1). In some embodiments, the compositions of the present disclosure can be used to treat diseases or disorders associated with dysregulation of L-serine biosynthesis or lipid (e.g., sphingolipid) biosynthesis, such as retinal diseases (e.g., MacTel2 and age-related macular degeneration (AMD), such as AMD with geographic atrophy), HSAN1 (e.g., HSAN1 (a)), diabetic retinopathy, peripheral neuropathy (e.g., motor neuropathy, sensory neuropathy, autonomic neuropathy, combined neuropathy, inherited or acquired peripheral neuropathy, such as diabetic neuropathy, chemotherapy-induced neuropathy, or other forms of toxic peripheral neuropathy, etc.), neurodegeneration (e.g., Parkinson’s disease, Friedreich’s ataxia, motor neuron disease, Alzheimer’s disease, amyotrophic lateral sclerosis (ALS), etc.), spastic paraplegia, or psychiatric disorders (e.g., resistant depression, treatment-resistant depression, schizophrenia, and / or treatment-resistant schizophrenia).

[0053] L-serine and lipid biosynthesis

[0054] Serine is a non-essential amino acid that is important for a variety of biochemical processes. In some cases, the typical role of serine is related to its function in metabolism. In some cases, serine is important for the biosynthesis of proteins, the amino acids glycine and cysteine, and purines and pyrimidines. Once incorporated into a protein, the side chain chemistry of serine can enable different roles, including but not limited to, acting as a nucleophile during protease cleavage, acting as a site for phosphorylation by protein kinases, and as an acceptor residue for O-linked protein glycosylation.

[0055] In the mammalian central nervous system (CNS), L-serine is used for the production of lipids such as sphingolipids, which are involved in signaling functions at the cell membranes of brain cells. Sphingolipid production from serine in the brain typically occurs in astrocytes. In some cases, dietary L-serine can be used directly by cells for sphingolipid synthesis. In other cases, serine is synthesized from glucose. The biosynthesis of L-serine involves several enzymatic steps, including, for example, the conversion of 3-phosphoglycerate, which is derived from glucose, to 3-phosphohydroxypyruvate by phosphoglycerate dehydrogenase (PHGDH). Ultimately, 3-phosphoglycerate is used as a substrate for a series of enzymatic reactions to produce L-serine.

[0056] Exogenous dietary and glucose-derived serine is used to produce sphingolipids by the condensation of L-serine with palmitoyl-CoA by serine palmitoyltransferase (SPT). One subunit of SPT, in humans, is provided by the SPTLC1 gene, which subunit implements a self-regulatory role in the enzymatic activity of SPT. Autosomal dominant mutations of SPTLC1 lead to atypical production of sphingolipids and are associated with hereditary sensory neuropathies (HSAN1 and HSAN1(A)) caused by the condensation of palmitoyl-CoA with alanine to form cytotoxic deoxysphingolipids. HSAN1 leads to peripheral neuropathy (e.g., motor neuropathy, sensory neuropathy, autonomic neuropathy, combined neuropathy, inherited or acquired peripheral neuropathy, such as diabetic neuropathy, chemotherapy-induced neuropathy, or other forms of toxic peripheral neuropathy, etc.), in some cases, manifesting as severe loss of sensation to temperature, pressure, and pain. In some cases, long-term cases of HSAN1 lead to painless injuries, chronic skin ulcers, bone destruction, bone infection, digit amputations, and motor dysfunction.

[0057] Aspects of the present disclosure relate to the inventors’ recognition that certain nucleic acid molecules, e.g., isolated nucleic acids, such as RNA processing modulators (e.g., antisense oligonucleotides), can be used to modulate the transcription and / or translation of certain mRNAs encoded by genes associated with L-serine biosynthesis.

[0058] A“gene associated with L-serine biosynthesis” refers to a gene encoding a gene product (e.g., mRNA, protein, etc.) that is genetically, biochemically, or functionally associated with the production of L-serine in a cell or subject. A“gene associated with lipid biosynthesis” refers to a gene encoding a gene product (e.g., mRNA, protein, etc.) that is genetically, biochemically, or functionally associated with the production of a lipid in a cell or subject. In some embodiments, the gene associated with lipid biosynthesis is serine palmitoyltransferase long chain base subunit 1 (SPTLC1). In some embodiments, the lipid biosynthesis is sphingolipid biosynthesis or deoxy-sphingolipid (d-SL) biosynthesis.

[0059] In some embodiments, the gene associated with lipid biosynthesis encodes an mRNA encoding a subunit of an SPT protein, such as serine palmitoyltransferase long chain base subunit 1 (SPTLC1). In humans, SPTLC1 is encoded by the SPTLC1 gene, which is located on chromosome 9 (e.g., by Ensembl ID NO: ENSG00000090054, chromosome 9: 92,000,087-92,115,413 antisense strand). In some embodiments, SPTCL1 encodes a peptide represented by NCBI Reference Sequence NP_006406.1. In some embodiments, the SPTCL1 gene encodes an mRNA comprising the sequence set forth in NCBI Reference Sequence NM_006415.4. In some embodiments, the mRNA is encoded by the SPTCL1 gene comprising the sequence set forth below:

[0060] NM_006415.4

[0061]

[0062] The skilled artisan recognizes that when referring to the sequence of a gene encoding an mRNA, the sequence of the mRNA is identical to the sequence of the referenced gene except that each instance of "T" is replaced with "U".

[0063] In some embodiments, the SPTLC1 gene (or mRNA encoded by the SPTLC1 gene) comprises one or more nucleotide substitutions, one or more nucleotide insertions, and / or one or more nucleotide deletions relative to the wild-type SPTLC1 gene (or mRNA encoded by the wild-type SPTLC1 gene), and can be referred to as a "mutant" SPTLC1 gene or SPTLC1 variant. The number of nucleotide substitutions, nucleotide insertions, and / or nucleotide deletions in the PHGDH or SPTLC1 variant can vary. In some embodiments, the SPTLC1 variant comprises 1 to 20, 5 to 10, 2 to 15, 10 to 30, or 20 to 100 nucleotide substitutions, nucleotide insertions, and / or nucleotide deletions relative to the wild-type SPTLC1 gene (or mRNA encoded by the wild-type SPTLC1 gene). In some embodiments, the one or more nucleotide substitutions, one or more nucleotide insertions, and / or one or more nucleotide deletions result in an amino acid substitution in the protein encoded by the SPTLC1 variant. In some embodiments, the one or more nucleotide substitutions, one or more nucleotide insertions, and / or one or more nucleotide deletions result in a nonsense mutation (e.g., insertion of a premature stop codon) in the mRNA encoded by the SPTLC1 variant. In some embodiments, the SPTLC1 variant protein forms part of an SPT protein complex that preferentially uses alanine instead of serine for the production of 3-keto-sphingosine.

[0064] In some embodiments, the one or more nucleotide substitutions, one or more nucleotide insertions, and / or one or more nucleotide deletions result in a frameshift mutation in the SPTLC1 variant relative to the wild-type SPTLC1 gene. In some embodiments, the mutation or mutations present in the SPTLC1 variant result in the production of one or more splice variants of the SPTLC1 mRNA. A "splice variant" can refer to an mRNA that results from one or more mutations in the DNA sequence that occur at the boundaries (splice sites) of exons and introns of a gene. Splice site mutations typically disrupt RNA splicing and result in the loss of an exon or inclusion of an intron and an altered protein coding sequence (e.g., a "splice variant").

[0065] Aspects of the present disclosure relate to isolated nucleic acids, e.g., RNA processing modulators (e.g., ASOs), that bind to one or more target regions of an mRNA encoded by a gene associated with L-serine biosynthesis. In some embodiments, the isolated nucleic acids bind to multiple or more splice variants of the SPTLC1 gene (e.g., human SPTLC1 splice variants). In some embodiments, the isolated nucleic acids described by the present disclosure bind to a region of a SPTLC1 splice variant (e.g., an mRNA encoded by a SPTLC1 variant) selected from an untranslated region (UTR). In some embodiments, the UTR is a 5’ UTR. In some embodiments, the UTR is a 3’ UTR. In some embodiments, the UTR is an intron. In some embodiments, the isolated nucleic acids described by the present disclosure bind to an intron-exon boundary of a SPTLC1 splice variant (e.g., an mRNA encoded by a SPTLC1 variant). An intron-exon boundary refers to a contiguous sequence of nucleotides that includes a portion of an intron and a portion of an exon that are adjacent to each other in an mRNA transcript. In some embodiments, the isolated nucleic acids (e.g., antisense oligonucleotides) bind to an mRNA expressed from a particular allele of PHGDH (e.g., bind to the target mRNA in an allele-specific manner).

[0066] Isolated nucleic acids

[0067] In some embodiments of the present disclosure, the nucleic acid is an isolated nucleic acid. In some cases, the nucleic acid is alternatively referred to as an oligonucleotide. In some embodiments, the isolated nucleic acid comprises DNA (e.g., deoxyribonucleotides). In some embodiments, the isolated nucleic acid comprises RNA (e.g., ribonucleotides). In some embodiments, the isolated nucleic acid comprises both DNA (e.g., deoxyribonucleotides) and RNA (e.g., ribonucleotides), such as an isolated nucleic acid comprising a gapmer structure comprising a region of deoxyribonucleotides flanked by regions of ribonucleotides. The isolated nucleic acid can be single-stranded or double-stranded. In some embodiments, the isolated nucleic acid is an RNA oligonucleotide. In some embodiments, the isolated nucleic acid is a single-stranded RNA oligonucleotide (which can also be referred to as a single-stranded RNA polynucleotide).

[0068] As used herein, the term “isolated” means artificially produced. Artificial production of an isolated nucleic acid can be achieved, for example, by in vitro amplification by polymerase chain reaction (PCR), recombinant cloning, or chemical synthesis. Methods of synthesizing isolated nucleic acids (e.g., RNA) are known in the art, for example, as described in Soukchareun et al. Preparation and characterization of antisense oligonucleotide-peptide hybrids containing viral fusion peptides. Bioconjug Chem. 1995 Jan-Feb;6(1):43-53. doi: 10.1021 / bc00031a004. PMID: 7711103.

[0069] The length of an isolated nucleic acid can vary. In some embodiments, an isolated nucleic acid (e.g., single-stranded RNA) is 10, 15, 20, 25, 30, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or up to 120 nucleotides in length. In some embodiments, an isolated nucleic acid is between about 1 and 100, 2 and 30, 5 and 20, 10 and 40, or 20 and 80 nucleotides in length. In some embodiments, an isolated nucleic acid is about 10 to 50 nucleotides in length. In some embodiments, an isolated nucleic acid is 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 nucleotides in length. In some embodiments, an isolated nucleic acid is greater than 50 nucleotides in length (e.g., 60, 70, 80, 90, 100, etc. nucleotides in length). In some embodiments, an isolated nucleic acid is no more than 200 nucleotides in length.

[0070] In some embodiments, an isolated nucleic acid of the present disclosure comprises an antisense oligonucleotide comprising a sequence set forth in any one of SEQ ID NOs: 1-299 (provided in column A of Table 1). In some embodiments, an isolated nucleic acid of the present disclosure comprises an antisense oligonucleotide comprising at least 15 nucleotides (e.g., at least 15, at least 16, at least 17, at least 18, at least 19, or at least 20 nucleotides) of any one of the sequences set forth in SEQ ID NOs: 1-299 (provided in column A of Table 1).

[0071] In some embodiments of the application, the isolated nucleic acid is modified (e.g., comprises one or more modifications, e.g., chemical modifications, such as those in Column C of Table 1). A modified nucleic acid can refer to an oligonucleotide that has been structurally altered in a non-natural way (e.g., a way that does not occur in nature). Nucleic acid modifications can be used to impart specific functional characteristics to the nucleic acid relative to unmodified nucleic acids. In some embodiments, the modification of the isolated nucleic acid facilitates binding of the isolated nucleic acid to a target molecule or increases the stability of the isolated nucleic acid (e.g., renders the isolated nucleic acid resistant to enzymatic degradation).

[0072] In some embodiments, the one or more modifications are 1 to 50 modifications, 2 to 20, 5 to 30, 10 to 40, or 15 to 50 modifications. In some embodiments, the isolated nucleic acid comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 modifications. In some embodiments, the isolated nucleic acid comprises more than 50 modifications (e.g., 60 modifications, 70 modifications, 80 modifications, 90 modifications, 100 modifications, etc.). In some embodiments, the isolated nucleic acid comprises more than 50 modifications (e.g., up to 60 modifications, 70 modifications, 80 modifications, 90 modifications, or 100 modifications, etc.). In some embodiments, the isolated nucleic acid comprises a chemical modification on every nucleotide and every sugar-phosphate backbone linkage. Such a modified isolated nucleic acid can be referred to as a “fully modified” isolated nucleic acid. In some embodiments, less than all of the nucleotides of the isolated nucleic acid are modified.

[0073] Chemical modifications can include modifications of the nucleobases or nucleotides, and / or modifications of the sugar-phosphate backbone (e.g., modifications of one or more sugar-phosphate backbone linkages).

[0074] In some embodiments, the isolated nucleic acid of the present disclosure comprises one or more chemical modifications listed in Column C of Table 1.

[0075] In some embodiments, the isolated nucleic acid comprises one or more modifications to the 5'-carbon atom (e.g., the 5'-carbon atom of the sugar) and / or one or more modifications to the 5-carbon of the nucleobase. Examples of modifications include, but are not limited to, 5-(2-amino)propyl uridine, 5-bromouridine, 5-propynyl uridine, 5-propenyl uridine, 5-carboxymethylaminomethyl-2-thioxyuracil, and 5-carboxymethylaminomethyluracil. In other embodiments, the nucleic acid modification targets the 6-carbon atom of the nucleobase. In some embodiments, the isolated nucleic acid comprises one or more modifications to the 6-carbon atom (e.g., the 6-carbon atom of the sugar), such as 6-(2-amino)propyl uridine. In some embodiments, the isolated nucleic acid comprises one or more modifications to the 8-carbon atom (e.g., the 8-carbon atom of the nucleobase). Examples of 8 modifications include, but are not limited to, 8-bromoguanosine, 8-chloroguanosine, and 8-fluoroguanosine.

[0076] In some embodiments, the isolated nucleic acid comprises one or more modifications to the 2' carbon of the sugar group. Examples of modified sugar groups include, but are not limited to, D-ribose, 2'-O-alkyl (including 2'-O-methyl and 2'-O-ethyl), i.e., 2'-alkoxy, 2'-amino, 2'-S-alkyl, 2'-halo (including 2'-fluoro), 2'-2-O-methoxyethoxy, 2'-allyl, 2'-propargyl, 2'-propyl, ethynyl, vinyl, propenyl, and cyano, among others. In some embodiments, the modified sugar moiety comprises a hexose and is incorporated into an oligonucleotide as described in Augustyns, K., et al., Nucl. Acids. Res. 18:4711 (1992). Other examples of 2' modifications include, but are not limited to, substitution of the bound OH group with H, OR, R, F, Cl, Br, I, SH, SR, NH, NHR, NR, COOR, or OR, wherein R is a substituted or unsubstituted aliphatic group. Other 2' modifications are found in the art. As used herein, the term "aliphatic" includes saturated and unsaturated straight chain (i.e., unbranched), branched, acyclic, cyclic, or polycyclic aliphatic hydrocarbons, which are optionally substituted with one or more functional groups. As will be appreciated by one of ordinary skill in the art, "aliphatic" is intended herein to include, but not be limited to, alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, and cycloalkynyl moieties.

[0077] In some embodiments, the isolated nucleic acid modification is a sugar-phosphate backbone modification. One example of a phosphate group modification is the replacement of an oxygen atom with a sulfur atom. In other embodiments, the backbone of the nucleic acid is modified. Examples of backbone modifications include, but are not limited to, phosphorothioate, borano-phosphate, alkylphosphonate nucleic acids, peptide nucleic acids, and morpholinos. For example, morpholino backbones are described by Corey and Abrams Genome Biol. 2001; 2(5): reviews1015.1 - reviews1015.3.

[0078] Other examples of modified bases include N4,N4-ethanocytosine, 7-deazaxanthine, 7- deazaguanosine, 8-oxo-N6-methyladenine, 4-acetylcytosine, dihydrouracil, inosine, N6- isopentenyladenine, 1-methyladenine, 1-methylpseudouracil, 1-methylguanine, 1- methylinosine, 2,2-dimethylguanine, 2-methyladenine, 2-methylguanine, 3-methylcytosine, 5- methylcytosine, N6-methyladenine, 7-methylguanine, 2-methylthio-N6-isopentenyladenine, pseudouracil, 5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 2-thiocytosine, and 2,6- diaminopurine. Other examples of nucleic acid modifications are described by, e.g., Eckstein, Antisense Nucleic Acid Drug Dev. 2000 Apr. 10(2): 117-21, Rusckowski et al. Antisense Nucleic Acid Drug Dev. 2000 Oct. 10(5):333-45, Stein, Antisense Nucleic Acid Drug Dev. 2001 Oct. 11(5): 317-25, Vorobjev et al. Antisense Nucleic Acid Drug Dev. 2001 Apr. 11(2):77-85, Duffy. BMC Bio. 2020 Sep. 2(8): 112, and U.S. Patent No. US5684143.

[0079] Additional modifications of the isolated nucleic acid (e.g., ASO) are described by Duffy et al. BMC Biology volume 18, Article number: 112 (2020), which is incorporated by reference in its entirety.

[0080] In some embodiments, the isolated nucleic acid of the present disclosure comprises a nucleic acid sequence from column A of Table 1 and one or more chemical modifications (or combinations of chemical modifications) from column C of Table 1, optionally wherein the column A and column C are from the same row of Table 1.

[0081] RNA processing modulator

[0082] Aspects of the present disclosure relate to compositions (e.g., isolated nucleic acids, reagents, etc.) that modulate a mRNA encoded by a gene associated with L-serine biosynthesis or lipid (e.g., sphingolipid) biosynthesis. In some embodiments, the gene associated with lipid biosynthesis is SPTLC1 (e.g., the human SPTLC1 gene). In some embodiments, the composition comprises an RNA processing modulator. As used herein, an “RNA processing modulator” or “RPM” refers to an agent that binds to a target mRNA (e.g., a mRNA encoded by a gene associated with L-serine biosynthesis, such as SPTLC1, or a gene product, such as a protein encoded by the mRNA) and upregulates, downregulates, or otherwise alters the function or activity of the target mRNA by affecting the level, transcription, splicing, and / or translation of the mRNA. The RNA processing modulator can be an isolated nucleic acid or an ASO as described herein. In some embodiments, the RNA processing modulator is an isolated nucleic acid that affects the splicing, transcription, or translation of a target mRNA (e.g., a mRNA encoded by a SPTLC1 gene). In some embodiments, the RNA processing modulator is an antisense oligonucleotide that affects the transcription, level, splicing, and / or translation of a target mRNA (e.g., a mRNA encoded by a SPTLC1 gene). In some embodiments, the mRNA (e.g., a target mRNA, such as a mRNA encoded by a SPTLC1 gene) is a pre-mRNA (e.g., an RNA that has been transcribed from a gene, such as a SPTLC1 gene, but has not yet been processed (e.g., by splicing) to remove introns). In some embodiments, the mRNA is a mature mRNA that has been processed (e.g., a mRNA transcribed from a SPTLC1 gene and subjected to processing).

[0083] In some embodiments, the RNA processing modulator upregulates the transcription, level, splicing, and / or translation of the target mRNA. Upregulation of transcription or translation can include binding to a regulatory region (e.g., an untranslated region, such as a 5’ UTR or 3’ UTR) of the target mRNA, and reducing non-productive splicing or translation initiation from alternative start codons present in the target mRNA, for example, by sterically blocking a non-productive splicing site or alternative start codon (e.g., an “upstream alternative start codon” located in the 5’ UTR of the target mRNA), or causing a mRNA frameshift (e.g., a splice variant) that results in translation of a protein variant from the target mRNA that lacks one or more inhibitory domains.

[0084] The amount of upregulation of transcription or translation mediated by the RNA processing modulator can vary. In some embodiments, the RNA processing modulator increases transcription or translation of a target mRNA transcript by 1-fold to 100-fold, 2-fold to 10-fold, 5-fold to 20-fold, 10-fold to 30-fold, 20-fold to 50-fold, or 25-fold to 100-fold, or any value therebetween, relative to the cell or subject prior to administration of the RPM, or relative to a control cell or subject. In some embodiments, the RNA processing modulator increases transcription or translation of a target mRNA transcript by more than 100-fold, e.g., at least 200-fold, 400-fold, 500-fold, or 1000-fold. In some embodiments, the RNA processing modulator increases transcription, levels, splicing, and / or translation of a target mRNA transcript by no more than 1000-fold. In some embodiments, the upregulation of levels, transcription, splicing, and / or translation of a target mRNA can be used to increase expression of a desired (e.g., wild-type) allele that encodes the target mRNA. In some embodiments, the desired allele encodes an SPTLC1 subunit that, when included in an SPT complex, preferentially uses serine over alanine for 3-keto-sphingosine production.

[0085] In some embodiments, the RNA processing modulator downregulates transcription and / or translation of a target mRNA. Downregulation of transcription or translation can include binding to a regulatory region (e.g., untranslated region, such as a 5’ UTR or 3’ UTR) of the target mRNA and blocking (e.g., by steric blocking of the transcriptional start site) transcription of the target mRNA, binding to the mRNA and subsequently initiating RNAse H-mediated degradation (e.g., in the context of a “gapmer” RNA processing modulator), or causing a frameshift (e.g., a splice variant) in the mRNA that results in translation of a protein variant from the target mRNA that is inactive or has reduced function or activity (e.g., enzymatic activity, ability to interact with other proteins to form protein complexes, etc.). In some embodiments, the resulting protein variant is a dominant negative protein variant. In some embodiments, the downregulation of levels, transcription, splicing, and / or translation of a target mRNA can be used to increase expression of an undesired (e.g., mutant, or disease-associated) allele that encodes the target mRNA. In some embodiments, the undesired allele of SPTLC1 encodes an SPTLC1 subunit that, when included in an SPT complex, preferentially uses alanine over serine for 3-keto-sphingosine production.

[0086] The amount of downregulation of transcription or translation mediated by the RNA processing modulator can vary. In some embodiments, the RNA processing modulator reduces transcription or translation of a target mRNA transcript by 1-fold to 100-fold, 2-fold to 10-fold, 5-fold to 20-fold, 10-fold to 30-fold, 20-fold to 50-fold, or 25-fold to 100-fold, or any value therebetween. In some embodiments, the RNA processing modulator increases transcription or translation of a target mRNA transcript by more than 100-fold, such as at least 200-fold, 400-fold, 500-fold, or 1000-fold. In some embodiments, the RNA processing modulator reduces transcription, levels, splicing, and / or translation of a target mRNA transcript by more than 100-fold, such as at least 200-fold, 400-fold, 500-fold, or 1000-fold. In some embodiments, the RNA processing modulator reduces transcription, levels, splicing, and / or translation of a target mRNA transcript by no more than 1000-fold.

[0087] The RNA processing modulator can alter the number and / or characteristics of splice variants of a target mRNA. In some embodiments, the RNA processing modulator increases the number of different splice variants of an mRNA or the ratio between different splice variants of an mRNA (relative to native transcription or translation of the target mRNA). In some embodiments, the RNA processing modulator decreases the number of different splice variants of an mRNA or the ratio between different splice variants of an mRNA (relative to native transcription or translation of the target mRNA). In some embodiments, contacting a target mRNA with an RNA processing modulator results in 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more splice variants of the target mRNA being transcribed and / or translated. In some embodiments, contacting a target mRNA with an mRNA processing modulator results in a single splice variant of the target mRNA being transcribed and / or translated.

[0088] The location of binding of the RNA processing modulator can vary. In some embodiments, the RNA processing modulator affects splicing of a target mRNA. For example, the RNA processing modulator can bind to a target mRNA at a splice junction (e.g., a location spanning an intron-exon boundary) and mediate skipping of one or more exons in the mRNA transcript. In some embodiments, skipping of one or more exons in the target mRNA results in production of a truncated protein variant of the protein encoded by the target mRNA. In another example, the RNA processing modulator can bind to a target mRNA at a splice junction and mediate alternative splicing, in which an intron is translated and produces a protein variant of the target gene. In some embodiments, the RNA processing modulator binds to a target mRNA at a location comprising a coding sequence (e.g., a protein-coding sequence or exon).

[0089] In some embodiments, the RNA processing modulator comprises an agent selected from the group consisting of a nucleic acid, a peptide (including a polypeptide), and a small molecule. Examples of small molecule RNA processing inhibitors include, but are not limited to, translational read-through inducing drugs (TRIDs), such as certain aminoglycosides, non-aminoglycoside antibiotics (e.g., negamycin), ataluren (PTC124), and amlexanox. Examples of peptides include, but are not limited to, activator proteins (e.g., transcription factors), repressor proteins (e.g., inducible cAMP early repressor (ICER), bZIP repressors, SP1 repressors, certain histone deacetylases, etc.), antibodies, and the like. Examples of nucleic acids include, but are not limited to, suppressor tRNAs, dsRNAs, siRNAs, microRNAs (miRNAs), artificial miRNAs (ami-RNAs), aptamers, and antisense oligonucleotides. In some embodiments, the RNA processing modulator comprises an antisense oligonucleotide (ASO).

[0090] As used herein, the term “antisense nucleic acid” or “ASO” refers to a single-stranded nucleic acid that has sequence complementarity to a target sequence and is specifically hybridizable to a nucleic acid having the target sequence, e.g., under stringent conditions. An antisense nucleic acid is specifically hybridizable when its binding to a target nucleic acid is sufficient to create a complementary base pairing between the antisense nucleic acid and the target nucleic acid, and there is a sufficient degree of complementarity to reduce or avoid non-specific binding of the antisense nucleic acid to non-target nucleic acids under conditions in which specific binding is desired, e.g., under physiological conditions in the case of in vivo assays or therapeutic treatment, and under conditions in which assays are performed in the case of in vitro assays, under the conditions in which the assay is performed. In some embodiments, the ASO is chemically synthesized. The ASO can be a DNA polynucleotide, an RNA polynucleotide, or a DNA / RNA polynucleotide (e.g., an ASO comprising a gapmer structure comprising a region of deoxyribonucleotides flanked by regions comprising ribonucleotides).

[0091] Complementarity refers to the capacity of two nucleotides to pair precisely. For example, if the nucleotide at a certain position of an antisense nucleic acid is capable of hydrogen bonding with the nucleotide at the corresponding position of a target nucleic acid (e.g., a target RNA), then the antisense nucleic acid and the target nucleic acid are considered to be complementary to one another at that position. An antisense nucleic acid and a target nucleic acid are complementary to one another when a sufficient number of corresponding positions in each molecule are occupied by nucleotides which can hydrogen bond with one another. Thus, “complementary” is a term that is used to indicate a sufficient degree of complementarity or precise pairing that enables stable and specific binding between an antisense nucleic acid and a target nucleic acid. However, it is understood that 100% complementarity is not required. For example, in some embodiments, an antisense nucleic acid (e.g., an oligonucleotide) can be at least 80% complementary to a contiguous nucleotide of a target nucleic acid (e.g., a target nucleic acid comprising an mRNA sequence encoded by SEQ ID NO: 300) (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% complementary to a contiguous nucleotide of a target nucleic acid (e.g., a target nucleic acid comprising an mRNA sequence encoded by SEQ ID NO: 300)).

[0092] Sequence identity, including determining the sequence complementarity of nucleic acid sequences, can be determined by sequence comparison and alignment algorithms known in the art. To determine the percent identity of two nucleic acid sequences, the sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced in the first sequence or second sequence for optimal alignment). The nucleotides at corresponding positions are then compared. When a position in the first sequence is occupied by the same residue as the corresponding position in the second sequence, then the molecules are identical at that position. In some embodiments, the percent identity between two sequences is a function of the number of identical positions shared by the sequences (e.g., % homology = the number of identical positions / total number of positions x 100), optionally penalizing the number and / or length of gaps introduced.

[0093] In some embodiments, an antisense oligonucleotide has a length in the range of 5 to 40 nucleotides, 5 to 30 nucleotides, 10 to 30 nucleotides, 10 to 25 nucleotides, or 15 to 25 nucleotides. In some embodiments of the disclosure, an antisense oligonucleotide comprises a length of 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 nucleotides.

[0094] In some embodiments, the antisense nucleic acid comprises a region of complementarity that is perfectly complementary to a portion of the target nucleic acid (e.g., 100% of the nucleotides of the ASO hybridize to nucleotides of the target RNA, such as a target mRNA (e.g., an mRNA sequence encoded by SEQ ID NO: 300)). However, it will be appreciated that in some embodiments, the antisense nucleic acid comprises less than 100% sequence complementarity to the target nucleic acid (e.g., 50%, 60%, 70%, 80%, 90%, 95%, or 99% of the nucleotides of the ASO hybridize to nucleotides of the target RNA, such as a target mRNA (e.g., an mRNA sequence encoded by SEQ ID NO: 300)). Furthermore, to minimize the likelihood of off-target effects, the antisense nucleic acid can be designed to ensure that it does not have sequences (e.g., 5 or more contiguous nucleotides) that are complementary to off-target nucleic acids (e.g., mRNAs that are not transcribed from the SPTLC1 gene).

[0095] In some embodiments, the antisense oligonucleotide comprises a region that is complementary to an mRNA encoded by (e.g., transcribed from) the SPTLC1 gene. In some embodiments, the antisense oligonucleotide comprises a region that is complementary to a pre-mRNA sequence encoded by the human SPTLC1 gene, e.g., (e.g., encoded by ENSG00000090054, chromosome 9: 92,000,087-92,115,413 antisense strand). In some embodiments, the complementary region of the antisense nucleic acid hybridizes to at least 6, e.g., at least 7, at least 8, at least 9, at least 10, at least 15, or more contiguous nucleotides of the target nucleic acid, e.g., a pre-mRNA encoded by ENSG00000090054, chromosome 9: 92,000,087-92,115,413 antisense strand. In some embodiments, the antisense oligonucleotide comprises a region that is complementary to at least 6, e.g., at least 7, at least 8, at least 9, at least 10, at least 15, or more contiguous nucleotides of an intron encoded by ENSG00000090054, chromosome 9: 92,000,087-92,115,413 antisense strand. Those skilled in the art recognize that the plus strand of such nucleic acids encoding pre-mRNA transcripts or mature mRNA transcripts can also be targeted.

[0096] In some embodiments, the antisense oligonucleotide comprises a region of complementarity to an mRNA encoded by (e.g., transcribed from) the SPTLC1 gene. In some embodiments, the antisense nucleic acid oligonucleotide comprises a region of complementarity to an mRNA encoded by a sequence as set forth in SEQ ID NO: 300. In some embodiments, the region of complementarity of the antisense nucleic acid hybridizes to at least 6, e.g., at least 7, at least 8, at least 9, at least 10, at least 15, or more, consecutive nucleotides of a target nucleic acid, e.g., an mRNA encoded by a sequence as set forth in SEQ ID NO: 300. In some embodiments, the antisense oligonucleotide comprises a region of complementarity to a 5' UTR, a 3' UTR, an exon sequence, a splice donor sequence, a splice acceptor sequence, or a lariat branch point encoded by the human SPTLC1 gene. In some embodiments, the oligonucleotide binds to an mRNA expressed from a particular allele of SPTLC1 (e.g., binds to the target mRNA in an allele-specific manner).

[0097] In some embodiments, the antisense oligonucleotide comprises a complementary region that is complementary to 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 contiguous nucleotides of SEQ ID NO: 300. In some embodiments, the antisense oligonucleotide comprising a region having complementarity to an mRNA transcript encoded by SEQ ID NO: 300 comprises at least 60% sequence identity (e.g., 60-70%, 70-80%, 80-90%, 90-95%, or greater than 95% sequence identity) to the nucleic acid sequence set forth in any of SEQ ID NOs: 1-299 as described in Column A of Table 1. In some embodiments, the antisense oligonucleotide comprises a sequence of 10 or more contiguous nucleotides (e.g., 10, 11, 12, 13, 14, 15, 16, 17, 18 or more contiguous nucleotides) of any of the sequences set forth in SEQ ID NOs: 1-299 as described in Column A of Table 1. In some embodiments, the antisense oligonucleotide comprises the nucleic acid sequence set forth in any of SEQ ID NOs: 1-299 as described in Column A of Table 1. In some embodiments, the antisense oligonucleotide comprises a nucleotide sequence having one or more mismatches (e.g., one or more bases that are not complementary to the nucleotide at a given position of the target mRNA) relative to an mRNA transcript encoded by the sequence set forth in SEQ ID NO: 300. In some embodiments, the antisense oligonucleotide comprises a sequence having 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 mismatches relative to an mRNA transcript encoded by the sequence set forth in SEQ ID NO: 300. In some embodiments, the antisense oligonucleotide comprising one or more mismatches relative to an mRNA transcript encoded by SEQ ID NO: 300 comprises at least 60% sequence identity (e.g., 60-70%, 70-80%, 80-90%, 90-95%, or greater than 95% sequence identity) to a sequence of 10 or more contiguous nucleotides of any of the sequences set forth in SEQ ID NOs: 1-299 as described in Column A of Table 1.In some embodiments, the sequence of 10 or more contiguous nucleotides (e.g., 10, 11, 12, 13, 14, 15, 16, 17, 18, or more contiguous nucleotides) of the sequence of any one of SEQ ID NOs: 1-299 of the antisense oligonucleotide comprising at least 60% sequence identity differs at one or more nucleotide positions (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more nucleotide positions comprising a substitution, insertion, or deletion) relative to the sequence of 10 or more contiguous nucleotides of any one of SEQ ID NOs: 1-299 as described in column A of Table 1. In some embodiments, the antisense oligonucleotide comprising one or more mismatches relative to the mRNA transcript encoded by SEQ ID NO: 300 comprises at least 60% sequence identity (e.g., 60-70%, 70-80%, 80-90%, 90-95%, or greater than 95% sequence identity) to the nucleic acid sequence set forth in any one of SEQ ID NOs: 1-299. In some embodiments, the antisense oligonucleotide comprising at least 60% sequence identity to the nucleic acid sequence set forth in any one of SEQ ID NOs: 1-299 differs at one or more nucleotide positions (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more nucleotide positions comprising a substitution, insertion, or deletion) relative to the nucleic acid sequence set forth in any one of SEQ ID NOs: 1-299.

[0098] In some embodiments, the RNA processing modulators (e.g., antisense oligonucleotides) are provided in a homogeneous preparation, e.g., wherein at least 85%, at least 90%, at least 95%, or at least 99% of the RNA processing modulators (e.g., antisense oligonucleotides) are identical. For example, in some embodiments, a homogeneous preparation of antisense oligonucleotides is provided, wherein at least 85%, at least 90%, at least 95%, or at least 99% of the oligonucleotides in the preparation are 10 to 25 nucleotides in length and comprise a region of complementarity that is complementary to at least 6 contiguous nucleotides of an mRNA transcript encoded by an SPTLC1 gene (e.g., an SPTLC1 gene comprising the nucleic acid sequence set forth in SEQ ID NO: 300). In some embodiments, the RNA processing modulators (e.g., antisense oligonucleotides) are provided in a homogeneous preparation, e.g., the homogeneous preparation comprises at least 2, 3, 4, 5, 6, 7, 8, 9, 10, or more different RNA processing modulators (e.g., antisense oligonucleotides each targeting a different sequence of the SPTLC1 mRNA transcript).

[0099] The RNA processing modulators of the disclosure (e.g., antisense oligonucleotides) can be modified to achieve one or more desired properties, such as, for example, improved cellular uptake, improved stability, reduced immunogenicity, improved potency, improved target hybridization, sensitivity to RNase cleavage, and the like. In some embodiments, the antisense nucleic acid is modified such that when present in a cell containing the SPTLC1 gene, it is capable of hybridizing to RNA transcribed from the SPTLC1 gene without inducing cleavage of the RNA by an RNase. In some embodiments, the antisense nucleic acid is modified such that when present in a cell containing the SPTLC1 gene, it is capable of hybridizing to RNA transcribed from the SPTLC1 gene and inducing cleavage of the RNA by an RNase.

[0100] The RNA processing modulators (e.g., antisense oligonucleotides, e.g., a nucleic acid sequence set forth in any one of SEQ ID NOs: 1-299 as described in Column A of Table 1) can be modified at the base moiety, the sugar moiety, and / or the phosphate backbone. Thus, the RNA processing modulators (e.g., antisense oligonucleotides) can have one or more modified nucleotides (e.g., nucleotide analogs) and / or one or more backbone modifications (e.g., modified internucleotide linkages). The RNA processing modulators (e.g., antisense oligonucleotides) can have a combination of modified and unmodified nucleotides. The RNA processing modulators (e.g., antisense oligonucleotides) can also have a combination of modified and unmodified internucleotide linkages. The RNA processing modulators (e.g., antisense oligonucleotides) can comprise one or more chemical modifications (or combinations of chemical modifications) from Column C of Table 1. In some embodiments, the RNA processing modulators comprise a nucleic acid sequence from Column A of Table 1 and one or more chemical modifications (or combinations of chemical modifications) from Column C of Table 1, wherein Column A and Column C are from the same row of Table 1.

[0101] In some embodiments, the one or more modifications are 1 to 50 modifications, 2 to 20, 5 to 30, 10 to 40, or 15 to 50 modifications. In some embodiments, the RNA processing modulator (e.g., antisense oligonucleotide) comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 modifications. In some embodiments, the RNA processing modulator (e.g., antisense oligonucleotide) comprises more than 50 modifications (e.g., 60 modifications, 70 modifications, 80 modifications, 90 modifications, 100 modifications, etc.). In some embodiments, the RNA processing modulator (e.g., antisense oligonucleotide) comprises a chemical modification on each nucleotide and each sugar-phosphate backbone linkage. Such a modified RNA processing modulator (e.g., antisense oligonucleotide) can be referred to as a “fully modified” RNA processing modulator (e.g., antisense oligonucleotide). In some embodiments, the fully modified antisense oligonucleotide comprises the nucleic acid sequence of any one of SEQ ID NOs: 1-299. In some embodiments, not all of the nucleotides of the antisense oligonucleotide are modified.

[0102] RNA processing modulators (e.g., antisense oligonucleotides) can include ribonucleotides, deoxyribonucleotides, and combinations thereof (e.g., RNA processing modulators comprising gapmer structures). Examples of nucleotides that can be used in modifications of antisense nucleic acids include, for example, 5-fluorouracil, 5-bromouracil, 5-chlorouracil, 5-iodouracil, hypoxanthine, xanthine, 4-acetylcytosine, 5-(carboxyhydroxylmethyl) uracil, 5-carboxymethylaminomethyl-2-thiouridine, 5-carboxymethylaminomethyluracil, dihydrouracil, beta-D-galactosylqueosine, inosine, N6-isopentenyladenine, 1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine, 2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-adenine, 7-methylguanine, 5-methylaminomethyluracil, 5-methoxyaminomethyl-2-thiouracil, beta-D-mannosylqueosine, 5'-methoxycarboxymethyluracil, 5-methoxyuracil, 2-methylthio-N6-isopentenyladenine, uracil-5-oxyacetic acid (v), wybutoxosine, pseudouracil, queosine, 2-thiocytosine, 5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil, uracil-5-oxyacetic acid methyl ester, uracil-5-oxyacetic acid (v), 5-methyl-2-thiouracil, 3-(3-amino-3-N-2-carboxypropyl) uracil, and 2,6-diaminopurine.

[0103] In some embodiments, the modified nucleotide is a 2’-modified nucleotide. For example, the 2’-modified nucleotide can be a 2’-deoxy, 2’-fluoro, 2’-O-methyl, 2’-O-methoxyethyl, 2’-amino, and 2’-aminoalkoxy modified nucleotide. In some embodiments, the 2’-modified nucleotide comprises a 2’-O-4’-C methylene bridge, such as a locked nucleic acid (LNA) nucleotide. In some embodiments of the 2’-modified nucleotide, the 2’-hydroxyl group is linked to the 3’ or 4’ carbon atom of the sugar ring, thereby forming a bicyclic sugar moiety. In such embodiments, the linkage can be a methylene (-CH2-) group bridging the 2’ oxygen atom and the 3’ or 4’ carbon atom, wherein n is 1 or 2. In some embodiments, the linkage comprises a cEt modification (e.g., -CH3substitutes a hydrogen in the methylene group of the bridge). n In some embodiments, the 2’-modified nucleotide comprises a 2’-O-4’-C methylene bridge, such as a locked nucleic acid (LNA) nucleotide. In some embodiments of the 2’-modified nucleotide, the 2’-hydroxyl group is linked to the 3’ or 4’ carbon atom of the sugar ring, thereby forming a bicyclic sugar moiety. In such embodiments, the linkage can be a methylene (-CH2-) group bridging the 2’ oxygen atom and the 3’ or 4’ carbon atom, wherein n is 1 or 2. In some embodiments, the linkage comprises a cEt modification (e.g., -CH3substitutes a hydrogen in the methylene group of the bridge).

[0104] RNA processing modulators (e.g., antisense oligonucleotides) can include combinations of LNA nucleotides and unmodified nucleotides. Antisense nucleic acids can include combinations of LNA and RNA nucleotides. Antisense nucleic acids can include combinations of LNA and DNA nucleotides. Further preferred oligonucleotide modifications include locked nucleic acids (LNAs), in which the 2'-hydroxyl group is linked to the 3' or 4' carbon of the sugar ring, thereby forming a bicyclic sugar moiety.

[0105] RNA processing modulators (e.g., antisense oligonucleotides) can also include nucleobase-modified nucleotides, e.g., nucleotides containing non-naturally occurring nucleobases rather than naturally occurring nucleobases. For example, the bases can be modified to block the activity of adenosine deaminases. Examples of modified nucleobases include, but are not limited to, uridine and / or cytidine modified at the 5-position, e.g., 5-(2-amino)propyl uridine, 5-bromo uridine; adenosine and / or guanosine modified at position 8, e.g., 8-bromo guanosine; deazanucleotides, e.g., 7-deaza-adenosine; O-alkylated nucleotides and N-alkylated nucleotides, e.g., N6-methyladenosine are suitable. It is noted that the above modifications can be combined.

[0106] Within the antisense nucleic acids (e.g., antisense oligonucleotides) of the present disclosure, as few as one and as many as all of the nucleotides are modified. In some embodiments, the modified RNA processing modulators (e.g., antisense oligonucleotides) will contain as few modified nucleotides as necessary to achieve a desired level of in vivo stability and / or bioaccessibility or other desired property.

[0107] Certain antisense oligonucleotides can include non-ionic DNA analogs such as alkyl- and aryl-phosphonates in which the charged, non-bridging oxygen is replaced by an alkyl or aryl group, phosphodiester and alkylphosphotriester in which the charged oxygen moiety is alkylated. Nucleic acids containing diols such as tetraethyleneglycol or hexaethyleneglycol at either or both ends have also been shown to have significant resistance to nuclease degradation and can be used herein. In some embodiments, the antisense nucleic acids can include at least one lipophilic substituted nucleotide analog and / or pyrimidine-purine dinucleotides.

[0108] In some embodiments, the RNA processing modulator (e.g., antisense oligonucleotide) can have one or two accessible 5' ends. It is possible to produce modified oligonucleotides having two such 5' ends, for example, by 3'-3' linkage of two oligonucleotides to generate an oligonucleotide having one or two accessible 5' ends. The 3'-3' linkage can be phosphodiester, phosphorothioate, or any other modified internucleoside bridge. Additionally, the 3'-3' linked oligonucleotide can be made using additional spacers such as triethylene glycol phosphate or tetraethylene glycol phosphate moieties, where the linkage between the 3' terminal nucleosides is not phosphodiester, phosphorothioate, or other modified bridge. In some embodiments, a triazole ring is used.

[0109] The phosphodiester internucleotide linkages of the RNA processing modulator (e.g., antisense oligonucleotide) can be replaced with modified linkages. The modified linkages can be selected from, for example, phosphorothioate, phosphorodithioate, NR1R2-phosphoramidate, borano-phosphates, alpha-hydroxybenzyl phosphonate, phosphate-(Ci-C2i)-O-alkyl ester, phosphate-[(C6-Ci2)aryl-(Ci-C2i)-O-alkyl] ester, (Ci-C8)alkyl phosphonate, and / or (C6-Ci2)aryl phosphonate bridges, and (C7-Ci2)-alpha-hydroxymethyl-aryl.

[0110] The phosphate backbone of the RNA processing modulator (e.g., antisense oligonucleotide) can be modified to generate a peptide nucleic acid molecule. As used herein, the term "peptide nucleic acid" or "PNA" refers to a nucleic acid mimic, e.g., a DNA mimic, in which the deoxyribose phosphate backbone is replaced with a pseudopeptide backbone and the four natural nucleobases are retained. The neutral backbone of PNA has been shown to allow for specific hybridization to DNA and RNA under low ionic strength conditions. For example, synthesis of PNA oligomers can be performed using standard solid phase peptide synthesis protocols.

[0111] The RNA processing modulator (e.g., antisense oligonucleotide) can also be formulated as a morpholino oligonucleotide. In this embodiment, the nucleoside portion of each subunit of the oligonucleotide agent of the oligonucleotide is converted to a morpholino moiety. The morpholino can also be modified, e.g., as a peptide-conjugated morpholino, etc.

[0112] Aspects of the disclosure relate to RNA processing modulators (e.g., antisense oligonucleotides) comprising a "gapmer" structure. A "gapmer" refers to an oligonucleotide comprising the following formula X n1 -(Y) n2 -(X) n3antisense oligonucleotide having a gapmer structure, wherein (X) is a ribonucleotide (e.g., an RNA base), (Y) is a deoxyribonucleotide (e.g., a DNA base), and wherein each of n1, n2, and n3 is an integer ranging from 1 to 50 (including all integers therebetween). In some embodiments, the antisense oligonucleotide having a gapmer structure binds to (e.g., hybridizes with) a target mRNA (e.g., an mRNA encoded by the SPTLC1 gene) and induces degradation of the target mRNA mediated by ribonuclease H1 (RNAseH1). Gapmer antisense oligonucleotides are known in the art, for example, as described by Kasuya et al. Sci Rep. 2016; 6: 30377.

[0113] The number of DNA bases in the gapmer can vary. In some embodiments, the gapmer comprises 1 to 10 DNA bases (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 DNA bases). In some embodiments, the gapmer comprises 2 to 6 DNA bases (e.g., 2, 3, 4, 5, or 6 DNA bases). The DNA bases of the gapmer antisense oligonucleotide can be located at the 5’ end of the ASO (e.g., within 1, 2, 3, 4, 5, etc. nucleotides of the 5’ terminal nucleotide of the ASO), at the 3’ end of the ASO (e.g., within 1, 2, 3, 4, 5, etc. nucleotides of the 3’ terminal nucleotide of the ASO), or in the middle of the ASO (e.g., with an equal number of RNA bases flanking the DNA bases).

[0114] In other embodiments, the RNA processing modulator (e.g., antisense oligonucleotide) can be linked to a functional group such as a peptide (e.g., for targeting a host cell receptor in vivo) or an agent that facilitates transport across a cell membrane or the blood-brain barrier. For example, the oligonucleotide agents of the disclosure can also be modified with a chemical moiety (e.g., cholesterol) that improves the in vivo pharmacological properties of the RNA processing modulator. In some embodiments, the functional group comprises a peptide, a small molecule, a sugar, a lipid, a nucleic acid, or any combination of the foregoing.

[0115] The sequences and chemical modifications of representative RNA processing modulators (e.g., antisense oligonucleotides) targeting SPTLC1 (e.g., an mRNA encoded by the SPTLC1 gene, such as a pre-mRNA or a mature mRNA) are shown in columns A and C, respectively, of Table 1.

[0116] Table 1: Representative RPMs targeting SPTLC1

[0117]

[0118]

[0119]

[0120]

[0121]

[0122]

[0123]

[0124]

[0125]

[0126]

[0127] In some embodiments, the RNA processing modulator (e.g., antisense oligonucleotide) comprises at least 18 contiguous nucleotides of any one of the nucleic acid sequences set forth in SEQ ID NOs: 1-299 (see column A of Table 1). In some embodiments, the RNA processing modulator consists of 18 contiguous nucleotides of any one of the nucleic acid sequences set forth in column A of Table 1 (labeled as “18-mer” in column B of the same row in Table 1). In some embodiments, the RNA processing modulator comprises 18 contiguous nucleotides of any one of the nucleic acid sequences set forth in column A of Table 1 (labeled as “18-mer” in column B of the same row in Table 1), and comprises one additional nucleotide (at the 5’ end or the 3’ end) or two additional nucleotides (both at the 5’ end, both at the 3’ end, or one at the 5’ end and the other at the 3’ end) that are complementary to a target sequence in the SPTLC1 mRNA that hybridizes to the 18 contiguous nucleotides of the nucleic acid sequence selected from column A of Table 1. In some embodiments, the RNA processing modulator consists of 19 contiguous nucleotides of any one of the nucleic acid sequences set forth in column A of Table 1 (labeled as “20-mer” in column B of the same row in Table 1), and comprises one additional nucleotide at the 5’ end or the 3’ end that are complementary to a target sequence in the SPTLC1 mRNA that hybridizes to the 20 contiguous nucleotides of the nucleic acid sequence selected from column A of Table 1. In some embodiments, the RNA processing modulator comprises or consists of 20 contiguous nucleotides of any one of the nucleic acid sequences set forth in column A of Table 1 (labeled as “20-mer” in column B of the same row in Table 1). In some embodiments, the RNA processing modulator comprises 20 contiguous nucleotides of any one of the nucleic acid sequences set forth in column A of Table 1 (labeled as “20-mer” in column B of the same row in Table 1), and comprises one or more additional nucleotides at the 5’ end, at the 3’ end, or at both the 5’ end and the 3’ end that are complementary to a target sequence in the SPTLC1 mRNA that hybridizes to the 20 contiguous nucleotides of the nucleic acid sequence selected from column A of Table 1. In some embodiments, the RNA processing modulator comprising at least 18 contiguous nucleotides of any one of the nucleic acid sequences set forth in SEQ ID NOs: 1-299 (e.g., an ASO comprising 18 nucleotides, 19 nucleotides, or 20 contiguous nucleotides of any one of the nucleic acid sequences set forth in column A of Table 1, or consisting thereof) comprises one or more chemical modifications as set forth in any one of column B of Table 1.In some embodiments, an RNA processing modulator comprising at least 18 contiguous nucleotides of any one of the nucleic acid sequences set forth in SEQ ID NOs: 1-299 (e.g., an ASO comprising or consisting of 18 nucleotides, 19 nucleotides, or 20 contiguous nucleotides of any of the nucleic acid sequences set forth in column A of Table 1) comprises a chemical modification pattern as set forth in any of the rows in column B of Table 1. In some embodiments, the at least 18 contiguous nucleotides comprised in the RNA processing modulator are set forth in the nucleic acid sequence of SEQ ID NO: 22. In some embodiments, the at least 18 contiguous nucleotides comprised in the RNA processing modulator are set forth in the nucleic acid sequence of SEQ ID NO: 46. In some embodiments, the at least 18 contiguous nucleotides comprised in the RNA processing modulator are set forth in the nucleic acid sequence of SEQ ID NO: 147. In some embodiments, the at least 18 contiguous nucleotides comprised in the RNA processing modulator are set forth in the nucleic acid sequence of SEQ ID NO: 152. In some embodiments, the at least 18 contiguous nucleotides comprised in the RNA processing modulator are set forth in the nucleic acid sequence of SEQ ID NO: 162. In some embodiments, the at least 18 contiguous nucleotides comprised in the RNA processing modulator are set forth in the nucleic acid sequence of SEQ ID NO: 193. In some embodiments, the at least 18 contiguous nucleotides comprised in the RNA processing modulator are set forth in the nucleic acid sequence of SEQ ID NO: 195. In some embodiments, the at least 18 contiguous nucleotides comprised in the RNA processing modulator are set forth in the nucleic acid sequence of SEQ ID NO: 221. In some embodiments, the at least 18 contiguous nucleotides comprised in the RNA processing modulator are set forth in the nucleic acid sequence of SEQ ID NO: 233. In some embodiments, the at least 18 contiguous nucleotides comprised in the RNA processing modulator are set forth in the nucleic acid sequence of SEQ ID NO: 248. In some embodiments, the at least 18 contiguous nucleotides comprised in the RNA processing modulator are set forth in the nucleic acid sequence of SEQ ID NO: 254. In some embodiments, the RNA processing modulator comprising at least 18 contiguous nucleotides of any one of the nucleic acid sequences set forth in SEQ ID NOs: 1-299, or a composition thereof, reduces the level of SPTLC1 mRNA (e.g., mature mRNA or pre-mRNA) and / or SPTLC1 protein in a cell or one or more tissues (such as a cell or one or more tissues of a subject (e.g., cerebrospinal fluid, plasma, and / or brain tissue)) by 50% or more (e.g., 50-60%, 60-70%, 70-80%, 80-90%, 90-95%, or 95-100%) when the RNA processing modulator or the composition thereof is administered to the subject in an effective amount.In some embodiments, when the RNA processing modulator or composition thereof is administered to the subject in an effective amount, the RNA processing modulator comprising at least 18 contiguous nucleotides of any of the nucleic acid sequences set forth in SEQ ID NOs: 1-299 reduces the level of one or more lipids (e.g., toxic lipids, such as toxic deoxysphingolipids) in the cell or one or more tissues (such as the cell or one or more tissues of the subject (e.g., cerebrospinal fluid, plasma, and / or brain tissue)) by 50% or more (e.g., 50-60%, 60-70%, 70-80%, 80-90%, 90-95%, or 95-100%). In some embodiments, when the RNA processing modulator or composition thereof is administered to the subject in an effective amount, the RNA processing modulator comprising at least 18 contiguous nucleotides of any of the nucleic acid sequences set forth in SEQ ID NO: 22, 46, 147, 152, 162, 193, 195, 221, 233, 248, or 254 reduces the level of SPTLC1 mRNA (e.g., mature mRNA or pre-mRNA) and / or SPTLC1 protein in the cell or one or more tissues (such as the cell or one or more tissues of the subject (e.g., cerebrospinal fluid, plasma, and / or brain tissue)) by 50% or more (e.g., 50-60%, 60-70%, 70-80%, 80-90%, 90-95%, or 95-100%). In some embodiments, when the RNA processing modulator or composition thereof is administered to the subject in an effective amount, the RNA processing modulator comprising at least 18 contiguous nucleotides of any of the nucleic acid sequences set forth in SEQ ID NO: 22, 46, 147, 152, 162, 193, 195, 221, 233, 248, or 254 reduces the level of one or more lipids (e.g., toxic lipids, such as toxic deoxysphingolipids) in the cell or one or more tissues (such as the cell or one or more tissues of the subject (e.g., cerebrospinal fluid, plasma, and / or brain tissue)) by 50% or more (e.g., 50-60%, 60-70%, 70-80%, 80-90%, 90-95%, or 95-100%).

[0128] In some embodiments, the RNA processing modulator comprises or consists of 18 contiguous nucleotides of any one of the nucleic acid sequences set forth in SEQ ID NOs: 1-299 (see column A of Table 1), or comprises or consists of 19 contiguous nucleotides thereof, or comprises or consists of 20 contiguous nucleotides thereof, wherein one or more positions comprising a “T” residue is substituted with a “U” residue. In some embodiments, the RNA processing modulator comprises or consists of 18 contiguous nucleotides of any one of the nucleic acid sequences set forth in SEQ ID NOs: 1-299 (see column A of Table 1), or comprises or consists of 19 contiguous nucleotides thereof, or comprises or consists of 20 contiguous nucleotides thereof, wherein each position comprising a “T” residue is substituted with a “U” residue. In some embodiments, the RNA processing modulator comprising at least 18 contiguous nucleotides of any one of the nucleic acid sequences set forth in SEQ ID NOs: 1-299 (e.g., an ASO comprising or consisting of 18 nucleotides, 19 nucleotides, or 20 contiguous nucleotides of any one of the nucleic acid sequences set forth in column A of Table 1), wherein one or more positions comprising a “T” residue is substituted with a “U” residue, and wherein the RNA processing modulator comprises one or more chemical modifications as set forth in any row of column B of Table 1. In some embodiments, the RNA processing modulator comprising at least 18 contiguous nucleotides of any one of the nucleic acid sequences set forth in SEQ ID NOs: 1-299 (e.g., an ASO comprising or consisting of 18 nucleotides, 19 nucleotides, or 20 contiguous nucleotides of any one of the nucleic acid sequences set forth in column A of Table 1), wherein one or more positions comprising a “T” residue is substituted with a “U” residue, and wherein the RNA processing modulator comprises a pattern of chemical modifications as set forth in any row of column B of Table 1. In some embodiments, one or more positions in the RNA processing modulator comprising a “U” residue comprises a uracil-containing base or a chemically modified uracil-containing base described herein and a deoxyribose or a chemically modified deoxyribose described herein. In some embodiments, the at least 18 contiguous nucleotides comprised in the RNA processing modulator is as set forth in the nucleic acid sequence of SEQ ID NO: 22, wherein one or more positions comprising a “T” residue in SEQ ID NO: 22 (e.g., each position comprising a “T” residue in SEQ ID NO: 22) is substituted with a “U” residue. In some embodiments, the at least 18 contiguous nucleotides comprised in the RNA processing modulator is as set forth in the nucleic acid sequence of SEQ ID NO: 46, wherein one or more positions comprising a “T” residue in SEQ ID NO: 46 (e.g., each position comprising a “T” residue in SEQ ID NO: 46) is substituted with a “U” residue.In some embodiments, the at least 18 contiguous nucleotides comprised in the RNA processing modulator are as set forth in the nucleic acid sequence of SEQ ID NO: 147, wherein one or more positions in SEQ ID NO: 147 comprising a “T” residue (e.g., every position in SEQ ID NO: 147 comprising a “T” residue) is / are substituted to a “U” residue. In some embodiments, the at least 18 contiguous nucleotides comprised in the RNA processing modulator are as set forth in the nucleic acid sequence of SEQ ID NO: 152, wherein one or more positions in SEQ ID NO: 152 comprising a “T” residue (e.g., every position in SEQ ID NO: 152 comprising a “T” residue) is / are substituted to a “U” residue. In some embodiments, the at least 18 contiguous nucleotides comprised in the RNA processing modulator are as set forth in the nucleic acid sequence of SEQ ID NO: 162, wherein one or more positions in SEQ ID NO: 162 comprising a “T” residue (e.g., every position in SEQ ID NO: 162 comprising a “T” residue) is / are substituted to a “U” residue. In some embodiments, the at least 18 contiguous nucleotides comprised in the RNA processing modulator are as set forth in the nucleic acid sequence of SEQ ID NO: 193, wherein one or more positions in SEQ ID NO: 193 comprising a “T” residue (e.g., every position in SEQ ID NO: 193 comprising a “T” residue) is / are substituted to a “U” residue. In some embodiments, the at least 18 contiguous nucleotides comprised in the RNA processing modulator are as set forth in the nucleic acid sequence of SEQ ID NO: 195, wherein one or more positions in SEQ ID NO: 195 comprising a “T” residue (e.g., every position in SEQ ID NO: 195 comprising a “T” residue) is / are substituted to a “U” residue. In some embodiments, the at least 18 contiguous nucleotides comprised in the RNA processing modulator are as set forth in the nucleic acid sequence of SEQ ID NO: 221, wherein one or more positions in SEQ ID NO: 221 comprising a “T” residue (e.g., every position in SEQ ID NO: 221 comprising a “T” residue) is / are substituted to a “U” residue. In some embodiments, the at least 18 contiguous nucleotides comprised in the RNA processing modulator are as set forth in the nucleic acid sequence of SEQ ID NO: 233, wherein one or more positions in SEQ ID NO: 233 comprising a “T” residue (e.g., every position in SEQ ID NO: 233 comprising a “T” residue) is / are substituted to a “U” residue.In some embodiments, the at least 18 contiguous nucleotides comprised in the RNA processing modulator are as set forth in the nucleic acid sequence of SEQ ID NO: 248, wherein one or more positions in SEQ ID NO: 248 comprising a “T” residue (e.g., every position in SEQ ID NO: 248 comprising a “T” residue) is / are substituted with a “U” residue. In some embodiments, the at least 18 contiguous nucleotides comprised in the RNA processing modulator are as set forth in the nucleic acid sequence of SEQ ID NO: 254, wherein one or more positions in SEQ ID NO: 254 comprising a “T” residue (e.g., every position in SEQ ID NO: 254 comprising a “T” residue) is / are substituted with a “U” residue.

[0129] In some embodiments, the RNA processing modulator (e.g., antisense oligonucleotide) comprises a gapmer structure, wherein a region of 10 deoxyribonucleotides is flanked by regions each comprising 4 ribonucleotides (for a total of 8 ribonucleotides and 10 deoxyribonucleotides). In some embodiments, the 1, 2, 3, or 4 ribonucleotides comprised in each region flanking the region of 10 deoxyribonucleotides comprise a 2’-O-methoxyethyl (-OCH2CH2OCH3 (2’ MOE)) modification. In some embodiments, the 1, 2, 3, or 4 ribonucleotides comprised in each region flanking the region of 10 deoxyribonucleotides are connected by phosphorothioate linkages or phosphodiester linkages. In some embodiments, 1-10 deoxyriboses (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 deoxyriboses) comprised in the region of 10 deoxyribonucleotides are connected by phosphorothioate linkages. In some embodiments, one or more riboses (e.g., 2, 3, or 4 riboses) comprised in each region flanking the region of 10 deoxyribonucleotides are connected by phosphorothioate linkages. In some embodiments, 16 of the 18 positions are connected by phosphorothioate linkages. In some embodiments, 16 of the 18 positions are connected by phosphorothioate linkages, wherein the second position is connected to the third position (relative to the 5’ end) by a phosphodiester linkage, and the sixteenth position is connected to the seventeenth position (relative to the 5’ end) by a phosphodiester linkage. In some embodiments, the RNA processing modulator (e.g., antisense oligonucleotide) comprising a gapmer structure comprises or consists of 18 contiguous nucleotides of any one of the nucleic acid sequences set forth in SEQ ID NOs: 1-299 (see column A of Table 1). In some embodiments, the RNA processing modulator (e.g., antisense oligonucleotide) comprising a gapmer structure comprises or consists of 18 contiguous nucleotides of the nucleic acid sequence of SEQ ID NO: 22. In some embodiments, the RNA processing modulator (e.g., antisense oligonucleotide) comprising a gapmer structure comprises or consists of 18 contiguous nucleotides of the nucleic acid sequence of SEQ ID NO: 46. In some embodiments, the RNA processing modulator (e.g., antisense oligonucleotide) comprising a gapmer structure comprises or consists of 18 contiguous nucleotides of the nucleic acid sequence of SEQ ID NO: 147. In some embodiments, the RNA processing modulator (e.g., antisense oligonucleotide) comprising a gapmer structure comprises or consists of 18 contiguous nucleotides of the nucleic acid sequence of SEQ ID NO: 152. In some embodiments, the RNA processing modulator (e.g., antisense oligonucleotide) comprising a gapmer structure comprises or consists of 18 contiguous nucleotides of the nucleic acid sequence of SEQ ID NO: 162.In some embodiments, the RNA processing modulator (e.g., antisense oligonucleotide) comprising a gapmer structure comprises or consists of 18 contiguous nucleotides of the nucleic acid sequence of SEQ ID NO: 193. In some embodiments, the RNA processing modulator (e.g., antisense oligonucleotide) comprising a gapmer structure comprises or consists of 18 contiguous nucleotides of the nucleic acid sequence of SEQ ID NO: 195. In some embodiments, the RNA processing modulator (e.g., antisense oligonucleotide) comprising a gapmer structure comprises or consists of 18 contiguous nucleotides of the nucleic acid sequence of SEQ ID NO: 221. In some embodiments, the RNA processing modulator (e.g., antisense oligonucleotide) comprising a gapmer structure comprises or consists of 18 contiguous nucleotides of the nucleic acid sequence of SEQ ID NO: 233. In some embodiments, the RNA processing modulator (e.g., antisense oligonucleotide) comprising a gapmer structure comprises or consists of 18 contiguous nucleotides of the nucleic acid sequence of SEQ ID NO: 248. In some embodiments, the RNA processing modulator (e.g., antisense oligonucleotide) comprising a gapmer structure comprises or consists of 18 contiguous nucleotides of the nucleic acid sequence of SEQ ID NO: 254. In some embodiments, the RNA processing modulator comprising a gapmer structure reduces the level of SPTLC1 mRNA (e.g., mature mRNA or pre-mRNA) and / or SPTLC1 protein in a cell or one or more tissues (such as a cell or one or more tissues of a subject (e.g., cerebrospinal fluid, plasma, and / or brain tissue)) by 50% or more (e.g., 50-60%, 60-70%, 70-80%, 80-90%, 90-95%, or 95-100%) when the RNA processing modulator or a composition thereof is administered to a subject in an effective amount. In some embodiments, the RNA processing modulator comprising a gapmer structure reduces the level of one or more lipids (e.g., toxic lipids such as toxic deoxysphingolipids) in a cell or one or more tissues (such as a cell or one or more tissues of a subject (e.g., cerebrospinal fluid, plasma, and / or brain tissue)) by 50% or more (e.g., 50-60%, 60-70%, 70-80%, 80-90%, 90-95%, or 95-100%) when the RNA processing modulator or a composition thereof is administered to a subject in an effective amount.

[0130] In some embodiments, the RNA processing modulator (e.g., antisense oligonucleotide) comprises a gapmer structure in which a region of 10 deoxyribonucleotides is flanked by regions each comprising 5 ribonucleotides (for a total of 10 ribonucleotides and 10 deoxyribonucleotides). In some embodiments, 1, 2, 3, 4, or 5 ribonucleotides in each of the regions flanking the region of 10 deoxyribonucleotides comprise a 2’-O-methoxyethyl (-OCH2CH2OCH3 (2’ MOE)) modification. In some embodiments, the 1, 2, 3, 4, or 5 ribonucleotides comprised in each of the regions flanking the region of 10 deoxyribonucleotides are connected by phosphorothioate linkages or phosphodiester linkages. In some embodiments, 1-10 deoxyribos (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 deoxyribos) comprised in the region of 10 deoxyribonucleotides are connected by phosphorothioate linkages. In some embodiments, one or more ribos (e.g., 2, 3, 4, or 5 ribos) comprised in each of the regions flanking the region of 10 deoxyribonucleotides are connected by phosphorothioate linkages. In some embodiments, 16 of the 20 positions are connected by phosphorothioate linkages. In some embodiments, 16 of the 20 positions are connected by phosphorothioate linkages, wherein the second and third positions (relative to the 5’ terminal end), the third and fourth positions (relative to the 5’ terminal end), the seventeenth and eighteenth positions (relative to the 5’ terminal end), and the eighteenth and nineteenth positions (relative to the 5’ terminal end) are each connected by phosphodiester linkages. In some embodiments, 18 of the 20 positions are connected by phosphorothioate linkages. In some embodiments, 18 of the 20 positions are connected by phosphorothioate linkages, wherein the second and third positions (relative to the 5’ terminal end) are connected by phosphodiester linkages and the seventeenth and eighteenth positions (relative to the 5’ terminal end) are connected by phosphodiester linkages. In some embodiments, the RNA processing modulator (e.g., antisense oligonucleotide) comprising a gapmer structure comprises or consists of 20 contiguous nucleotides of any one of the nucleic acid sequences set forth in SEQ ID NOs: 1-299 (see column A of Table 1). In some embodiments, the RNA processing modulator (e.g., antisense oligonucleotide) comprising a gapmer structure comprises or consists of 20 contiguous nucleotides of the nucleic acid sequence of SEQ ID NO: 46. In some embodiments, the RNA processing modulator (e.g., antisense oligonucleotide) comprising a gapmer structure comprises or consists of 20 contiguous nucleotides of the nucleic acid sequence of SEQ ID NO: 162.In some embodiments, the RNA processing modulator (e.g., antisense oligonucleotide) comprising a gapmer structure comprises or consists of 20 contiguous nucleotides of the nucleic acid sequence of SEQ ID NO: 193. In some embodiments, the RNA processing modulator (e.g., antisense oligonucleotide) comprising a gapmer structure comprises or consists of 20 contiguous nucleotides of the nucleic acid sequence of SEQ ID NO: 195. In some embodiments, the RNA processing modulator (e.g., antisense oligonucleotide) comprising a gapmer structure comprises or consists of 20 contiguous nucleotides of the nucleic acid sequence of SEQ ID NO: 233. In some embodiments, the RNA processing modulator (e.g., antisense oligonucleotide) comprising a gapmer structure comprises or consists of 20 contiguous nucleotides of the nucleic acid sequence of SEQ ID NO: 248. In some embodiments, the RNA processing modulator (e.g., antisense oligonucleotide) comprising a gapmer structure comprises or consists of 20 contiguous nucleotides of the nucleic acid sequence of SEQ ID NO: 254. In some embodiments, the RNA processing modulator comprising a gapmer structure reduces the level of SPTLC1 mRNA (e.g., mature mRNA or pre-mRNA) and / or SPTLC1 protein in a cell or one or more tissues (such as a cell or one or more tissues of a subject (e.g., cerebrospinal fluid, plasma, and / or brain tissue)) by 50% or more (e.g., 50-60%, 60-70%, 70-80%, 80-90%, 90-95%, or 95-100%) when the RNA processing modulator or a composition thereof is administered to a subject in an effective amount. In some embodiments, the RNA processing modulator comprising a gapmer structure reduces the level of one or more lipids (e.g., toxic lipids such as toxic deoxysphingolipids) in a cell or one or more tissues (such as a cell or one or more tissues of a subject (e.g., cerebrospinal fluid, plasma, and / or brain tissue)) by 50% or more (e.g., 50-60%, 60-70%, 70-80%, 80-90%, 90-95%, or 95-100%) when the RNA processing modulator or a composition thereof is administered to a subject in an effective amount.

[0131] In some embodiments, the isolated nucleic acid (e.g., RNA processing modulator, such as an antisense oligonucleotide) comprises the nucleic acid sequence set forth in SEQ ID NO: 1 and the following modification pattern: fully PS; 2’MOE; 20-mer. In some embodiments, the isolated nucleic acid (e.g., RNA processing modulator, such as an antisense oligonucleotide) comprises the nucleic acid sequence set forth in SEQ ID NO: 2 and the following modification pattern: fully PS; 2’MOE; 20-mer. In some embodiments, the isolated nucleic acid (e.g., RNA processing modulator, such as an antisense oligonucleotide) comprises the nucleic acid sequence set forth in SEQ ID NO: 3 and the following modification pattern: fully PS; 2’MOE; 18-mer. In some embodiments, the isolated nucleic acid (e.g., RNA processing modulator, such as an antisense oligonucleotide) comprises the nucleic acid sequence set forth in SEQ ID NO: 4 and the following modification pattern: fully PS; 2’MOE; 18-mer. In some embodiments, the isolated nucleic acid (e.g., RNA processing modulator, such as an antisense oligonucleotide) comprises the nucleic acid sequence set forth in SEQ ID NO: 5 and the following modification pattern: fully PS; 2’MOE; 20-mer; 5-10-5, PO from the 2ndposition from the 5’ end, PO from the 3rdposition from the 3’ end. In some embodiments, the isolated nucleic acid (e.g., RNA processing modulator, such as an antisense oligonucleotide) comprises the nucleic acid sequence set forth in SEQ ID NO: 6 and the following modification pattern: fully PS; 2’MOE; 20-mer. In some embodiments, the isolated nucleic acid (e.g., RNA processing modulator, such as an antisense oligonucleotide) comprises the nucleic acid sequence set forth in SEQ ID NO: 7 and the following modification pattern: fully PS; 2’MOE; 18-mer. In some embodiments, the isolated nucleic acid (e.g., RNA processing modulator, such as an antisense oligonucleotide) comprises the nucleic acid sequence set forth in SEQ ID NO: 8 and the following modification pattern: fully PS; 2’MOE; 20-mer. In some embodiments, the isolated nucleic acid (e.g., RNA processing modulator, such as an antisense oligonucleotide) comprises the nucleic acid sequence set forth in SEQ ID NO: 9 and the following modification pattern: fully PS; 2’MOE; 18-mer. In some embodiments, the isolated nucleic acid (e.g., RNA processing modulator, such as an antisense oligonucleotide) comprises the nucleic acid sequence set forth in SEQ ID NO: 10 and the following modification pattern: fully PS; 2’MOE; 20-mer; 5-10-5. In some embodiments, the isolated nucleic acid (e.g., RNA processing modulator, such as an antisense oligonucleotide) comprises the nucleic acid sequence set forth in SEQ ID NO: 11 and the following modification pattern: fully PS; 2’MOE; 20-mer.In some embodiments, the isolated nucleic acid (e.g., RNA processing modulator, such as an antisense oligonucleotide) comprises the nucleic acid sequence set forth in SEQ ID NO: 12 and the following modification pattern: fully PS; 2’MOE; 18-mer. In some embodiments, the isolated nucleic acid (e.g., RNA processing modulator, such as an antisense oligonucleotide) comprises the nucleic acid sequence set forth in SEQ ID NO: 13 and the following modification pattern: fully PS; 2’MOE; 18-mer. In some embodiments, the isolated nucleic acid (e.g., RNA processing modulator, such as an antisense oligonucleotide) comprises the nucleic acid sequence set forth in SEQ ID NO: 14 and the following modification pattern: fully PS; 2’MOE; 20-mer. In some embodiments, the isolated nucleic acid (e.g., RNA processing modulator, such as an antisense oligonucleotide) comprises the nucleic acid sequence set forth in SEQ ID NO: 15 and the following modification pattern: fully PS; 2’MOE; 20-mer. In some embodiments, the isolated nucleic acid (e.g., RNA processing modulator, such as an antisense oligonucleotide) comprises the nucleic acid sequence set forth in SEQ ID NO: 16 and the following modification pattern: fully PS; 2’MOE; 18-mer. In some embodiments, the isolated nucleic acid (e.g., RNA processing modulator, such as an antisense oligonucleotide) comprises the nucleic acid sequence set forth in SEQ ID NO: 17 and the following modification pattern: fully PS; 2’MOE; 18-mer. In some embodiments, the isolated nucleic acid (e.g., RNA processing modulator, such as an antisense oligonucleotide) comprises the nucleic acid sequence set forth in SEQ ID NO: 18 and the following modification pattern: fully PS; 2’MOE; 18-mer. In some embodiments, the isolated nucleic acid (e.g., RNA processing modulator, such as an antisense oligonucleotide) comprises the nucleic acid sequence set forth in SEQ ID NO: 19 and the following modification pattern: fully PS; 2’MOE; 18-mer; 4-10-4. In some embodiments, the isolated nucleic acid (e.g., RNA processing modulator, such as an antisense oligonucleotide) comprises the nucleic acid sequence set forth in SEQ ID NO: 20 and the following modification pattern: fully PS; 2’MOE; 20-mer. In some embodiments, the isolated nucleic acid (e.g., RNA processing modulator, such as an antisense oligonucleotide) comprises the nucleic acid sequence set forth in SEQ ID NO: 21 and the following modification pattern: fully PS; 2’MOE; 18-mer. In some embodiments, the isolated nucleic acid (e.g., RNA processing modulator, such as an antisense oligonucleotide) comprises the nucleic acid sequence set forth in SEQ ID NO: 22 and the following modification pattern: fully PS; 2’MOE; 18-mer; 4-10-4; PO from the 2nd position from the 5’ end, PO from the 3rd position from the 3’ end.In some embodiments, the isolated nucleic acid (e.g., RNA processing modulator, such as an antisense oligonucleotide) comprises the nucleic acid sequence set forth in SEQ ID NO: 23 and the following modification pattern: all PS; 2’MOE; 20-mer. In some embodiments, the isolated nucleic acid (e.g., RNA processing modulator, such as an antisense oligonucleotide) comprises the nucleic acid sequence set forth in SEQ ID NO: 24 and the following modification pattern: all PS; 2’MOE; 18-mer. In some embodiments, the isolated nucleic acid (e.g., RNA processing modulator, such as an antisense oligonucleotide) comprises the nucleic acid sequence set forth in SEQ ID NO: 25 and the following modification pattern: all PS; 2’MOE; 20-mer. In some embodiments, the isolated nucleic acid (e.g., RNA processing modulator, such as an antisense oligonucleotide) comprises the nucleic acid sequence set forth in SEQ ID NO: 26 and the following modification pattern: all PS; 2’MOE; 18-mer. In some embodiments, the isolated nucleic acid (e.g., RNA processing modulator, such as an antisense oligonucleotide) comprises the nucleic acid sequence set forth in SEQ ID NO: 27 and the following modification pattern: all PS; 2’MOE; 18-mer. In some embodiments, the isolated nucleic acid (e.g., RNA processing modulator, such as an antisense oligonucleotide) comprises the nucleic acid sequence set forth in SEQ ID NO: 28 and the following modification pattern: all PS; 2’MOE; 18-mer; 4-10-4. In some embodiments, the isolated nucleic acid (e.g., RNA processing modulator, such as an antisense oligonucleotide) comprises the nucleic acid sequence set forth in SEQ ID NO: 29 and the following modification pattern: all PS; 2’MOE; 20-mer. In some embodiments, the isolated nucleic acid (e.g., RNA processing modulator, such as an antisense oligonucleotide) comprises the nucleic acid sequence set forth in SEQ ID NO: 30 and the following modification pattern: all PS; 2’MOE; 20-mer. In some embodiments, the isolated nucleic acid (e.g., RNA processing modulator, such as an antisense oligonucleotide) comprises the nucleic acid sequence set forth in SEQ ID NO: 31 and the following modification pattern: all PS; 2’MOE; 20-mer; 5-10-5. In some embodiments, the isolated nucleic acid (e.g., RNA processing modulator, such as an antisense oligonucleotide) comprises the nucleic acid sequence set forth in SEQ ID NO: 32 and the following modification pattern: all PS; 2’MOE; 20-mer. In some embodiments, the isolated nucleic acid (e.g., RNA processing modulator, such as an antisense oligonucleotide) comprises the nucleic acid sequence set forth in SEQ ID NO: 33 and the following modification pattern: all PS; 2’MOE; 18-mer; 4-10-4.In some embodiments, the isolated nucleic acid (e.g., RNA processing modulator, such as an antisense oligonucleotide) comprises the nucleic acid sequence set forth in SEQ ID NO: 34 and the following modification pattern: all PS; 2’MOE; 18-mer; 4-10-4. In some embodiments, the isolated nucleic acid (e.g., RNA processing modulator, such as an antisense oligonucleotide) comprises the nucleic acid sequence set forth in SEQ ID NO: 35 and the following modification pattern: all PS; 2’MOE; 20-mer. In some embodiments, the isolated nucleic acid (e.g., RNA processing modulator, such as an antisense oligonucleotide) comprises the nucleic acid sequence set forth in SEQ ID NO: 36 and the following modification pattern: all PS; 2’MOE; 18-mer. In some embodiments, the isolated nucleic acid (e.g., RNA processing modulator, such as an antisense oligonucleotide) comprises the nucleic acid sequence set forth in SEQ ID NO: 37 and the following modification pattern: all PS; 2’MOE; 20-mer. In some embodiments, the isolated nucleic acid (e.g., RNA processing modulator, such as an antisense oligonucleotide) comprises the nucleic acid sequence set forth in SEQ ID NO: 38 and the following modification pattern: all PS; 2’MOE; 18-mer. In some embodiments, the isolated nucleic acid (e.g., RNA processing modulator, such as an antisense oligonucleotide) comprises the nucleic acid sequence set forth in SEQ ID NO: 39 and the following modification pattern: all PS; 2’MOE; 20-mer; 5-10-5, PO from the 2nd position from the 5’ end, PO from the 3rd position from the 3’ end. In some embodiments, the isolated nucleic acid (e.g., RNA processing modulator, such as an antisense oligonucleotide) comprises the nucleic acid sequence set forth in SEQ ID NO: 40 and the following modification pattern: all PS; 2’MOE; 20-mer. In some embodiments, the isolated nucleic acid (e.g., RNA processing modulator, such as an antisense oligonucleotide) comprises the nucleic acid sequence set forth in SEQ ID NO: 41 and the following modification pattern: all PS; 2’MOE; 20-mer. In some embodiments, the isolated nucleic acid (e.g., RNA processing modulator, such as an antisense oligonucleotide) comprises the nucleic acid sequence set forth in SEQ ID NO: 42 and the following modification pattern: all PS; 2’MOE; 18-mer. In some embodiments, the isolated nucleic acid (e.g., RNA processing modulator, such as an antisense oligonucleotide) comprises the nucleic acid sequence set forth in SEQ ID NO: 43 and the following modification pattern: all PS; 2’MOE; 18-mer. In some embodiments, the isolated nucleic acid (e.g., RNA processing modulator, such as an antisense oligonucleotide) comprises the nucleic acid sequence set forth in SEQ ID NO: 44 and the following modification pattern: all PS; 2’MOE; 18-mer.In some embodiments, the isolated nucleic acid (e.g., RNA processing modulator, such as an antisense oligonucleotide) comprises the nucleic acid sequence set forth in SEQ ID NO: 45 and the following modification pattern: all PS; 2’MOE; 18-mer. In some embodiments, the isolated nucleic acid (e.g., RNA processing modulator, such as an antisense oligonucleotide) comprises the nucleic acid sequence set forth in SEQ ID NO: 46 and the following modification pattern: all PS; 2’MOE; 20-mer; 5-10-5, PO from the 2nd position from the 5’-end, PO from the 3rd position from the 3’-end. In some embodiments, the isolated nucleic acid (e.g., RNA processing modulator, such as an antisense oligonucleotide) comprises the nucleic acid sequence set forth in SEQ ID NO: 47 and the following modification pattern: all PS; 2’MOE; 18-mer. In some embodiments, the isolated nucleic acid (e.g., RNA processing modulator, such as an antisense oligonucleotide) comprises the nucleic acid sequence set forth in SEQ ID NO: 48 and the following modification pattern: all PS; 2’MOE; 18-mer. In some embodiments, the isolated nucleic acid (e.g., RNA processing modulator, such as an antisense oligonucleotide) comprises the nucleic acid sequence set forth in SEQ ID NO: 49 and the following modification pattern: all PS; 2’MOE; 20-mer. In some embodiments, the isolated nucleic acid (e.g., RNA processing modulator, such as an antisense oligonucleotide) comprises the nucleic acid sequence set forth in SEQ ID NO: 50 and the following modification pattern: all PS; 2’MOE; 20-mer. In some embodiments, the isolated nucleic acid (e.g., RNA processing modulator, such as an antisense oligonucleotide) comprises the nucleic acid sequence set forth in SEQ ID NO: 51 and the following modification pattern: all PS; 2’MOE; 20-mer; 5-10-5. In some embodiments, the isolated nucleic acid (e.g., RNA processing modulator, such as an antisense oligonucleotide) comprises the nucleic acid sequence set forth in SEQ ID NO: 52 and the following modification pattern: all PS; 2’MOE; 20-mer; 5-10-5, PO from the 2nd position from the 5’-end, PO from the 3rd position from the 3’-end. In some embodiments, the isolated nucleic acid (e.g., RNA processing modulator, such as an antisense oligonucleotide) comprises the nucleic acid sequence set forth in SEQ ID NO: 53 and the following modification pattern: all PS; 2’MOE; 20-mer. In some embodiments, the isolated nucleic acid (e.g., RNA processing modulator, such as an antisense oligonucleotide) comprises the nucleic acid sequence set forth in SEQ ID NO: 54 and the following modification pattern: all PS; 2’MOE; 18-mer; 4-10-4. In some embodiments, the isolated nucleic acid (e.g., RNA processing modulator, such as an antisense oligonucleotide) comprises the nucleic acid sequence set forth in SEQ ID NO: 55 and the following modification pattern: all PS; 2’MOE; 20-mer.In some embodiments, the isolated nucleic acid (e.g., RNA processing modulator, such as an antisense oligonucleotide) comprises the nucleic acid sequence set forth in SEQ ID NO: 56 and the following modification pattern: all PS; 2’MOE; 18-mer. In some embodiments, the isolated nucleic acid (e.g., RNA processing modulator, such as an antisense oligonucleotide) comprises the nucleic acid sequence set forth in SEQ ID NO: 57 and the following modification pattern: all PS; 2’MOE; 18-mer. In some embodiments, the isolated nucleic acid (e.g., RNA processing modulator, such as an antisense oligonucleotide) comprises the nucleic acid sequence set forth in SEQ ID NO: 58 and the following modification pattern: all PS; 2’MOE; 20-mer. In some embodiments, the isolated nucleic acid (e.g., RNA processing modulator, such as an antisense oligonucleotide) comprises the nucleic acid sequence set forth in SEQ ID NO: 59 and the following modification pattern: all PS; 2’MOE; 18-mer. In some embodiments, the isolated nucleic acid (e.g., RNA processing modulator, such as an antisense oligonucleotide) comprises the nucleic acid sequence set forth in SEQ ID NO: 60 and the following modification pattern: all PS; 2’MOE; 20-mer. In some embodiments, the isolated nucleic acid (e.g., RNA processing modulator, such as an antisense oligonucleotide) comprises the nucleic acid sequence set forth in SEQ ID NO: 61 and the following modification pattern: all PS; 2’MOE; 18-mer. In some embodiments, the isolated nucleic acid (e.g., RNA processing modulator, such as an antisense oligonucleotide) comprises the nucleic acid sequence set forth in SEQ ID NO: 62 and the following modification pattern: all PS; 2’MOE; 18-mer; 4-10-4. In some embodiments, the isolated nucleic acid (e.g., RNA processing modulator, such as an antisense oligonucleotide) comprises the nucleic acid sequence set forth in SEQ ID NO: 63 and the following modification pattern: all PS; 2’MOE; 20-mer. In some embodiments, the isolated nucleic acid (e.g., RNA processing modulator, such as an antisense oligonucleotide) comprises the nucleic acid sequence set forth in SEQ ID NO: 64 and the following modification pattern: all PS; 2’MOE; 18-mer. In some embodiments, the isolated nucleic acid (e.g., RNA processing modulator, such as an antisense oligonucleotide) comprises the nucleic acid sequence set forth in SEQ ID NO: 65 and the following modification pattern: all PS; 2’MOE; 20-mer. In some embodiments, the isolated nucleic acid (e.g., RNA processing modulator, such as an antisense oligonucleotide) comprises the nucleic acid sequence set forth in SEQ ID NO: 66 and the following modification pattern: all PS; 2’MOE; 20-mer. In some embodiments, the isolated nucleic acid (e.g., RNA processing modulator, such as an antisense oligonucleotide) comprises the nucleic acid sequence set forth in SEQ ID NO: 67 and the following modification pattern: all PS; 2’MOE; 20-mer.In some embodiments, the isolated nucleic acid (e.g., RNA processing modulator, such as an antisense oligonucleotide) comprises the nucleic acid sequence set forth in SEQ ID NO: 68 and the following modification pattern: all PS; 2’OMe; 20-mer. In some embodiments, the isolated nucleic acid (e.g., RNA processing modulator, such as an antisense oligonucleotide) comprises the nucleic acid sequence set forth in SEQ ID NO: 69 and the following modification pattern: all PS; 2’OMe; 20-mer. In some embodiments, the isolated nucleic acid (e.g., RNA processing modulator, such as an antisense oligonucleotide) comprises the nucleic acid sequence set forth in SEQ ID NO: 70 and the following modification pattern: all PS; 2’OMe; 18-mer. In some embodiments, the isolated nucleic acid (e.g., RNA processing modulator, such as an antisense oligonucleotide) comprises the nucleic acid sequence set forth in SEQ ID NO: 71 and the following modification pattern: all PS; 2’OMe; 20-mer; 5-10-5. In some embodiments, the isolated nucleic acid (e.g., RNA processing modulator, such as an antisense oligonucleotide) comprises the nucleic acid sequence set forth in SEQ ID NO: 72 and the following modification pattern: all PS; 2’OMe; 20-mer. In some embodiments, the isolated nucleic acid (e.g., RNA processing modulator, such as an antisense oligonucleotide) comprises the nucleic acid sequence set forth in SEQ ID NO: 73 and the following modification pattern: all PS; 2’OMe; 20-mer. In some embodiments, the isolated nucleic acid (e.g., RNA processing modulator, such as an antisense oligonucleotide) comprises the nucleic acid sequence set forth in SEQ ID NO: 74 and the following modification pattern: all PS; 2’OMe; 20-mer. In some embodiments, the isolated nucleic acid (e.g., RNA processing modulator, such as an antisense oligonucleotide) comprises the nucleic acid sequence set forth in SEQ ID NO: 75 and the following modification pattern: all PS; 2’OMe; 20-mer. In some embodiments, the isolated nucleic acid (e.g., RNA processing modulator, such as an antisense oligonucleotide) comprises the nucleic acid sequence set forth in SEQ ID NO: 76 and the following modification pattern: all PS; 2’OMe; 18-mer. In some embodiments, the isolated nucleic acid (e.g., RNA processing modulator, such as an antisense oligonucleotide) comprises the nucleic acid sequence set forth in SEQ ID NO: 77 and the following modification pattern: all PS; 2’OMe; 20-mer. In some embodiments, the isolated nucleic acid (e.g., RNA processing modulator, such as an antisense oligonucleotide) comprises the nucleic acid sequence set forth in SEQ ID NO: 78 and the following modification pattern: all PS; 2’OMe; 18-mer; 4-10-4, PO from the 2nd position from the 5’ end, PO from the 3rd position from the 3’ end.In some embodiments, the isolated nucleic acid (e.g., RNA processing modulator, such as an antisense oligonucleotide) comprises the nucleic acid sequence set forth in SEQ ID NO: 79 and the following modification pattern: all PS; 2’MOE; 18-mer. In some embodiments, the isolated nucleic acid (e.g., RNA processing modulator, such as an antisense oligonucleotide) comprises the nucleic acid sequence set forth in SEQ ID NO: 80 and the following modification pattern: all PS; 2’MOE; 18-mer. In some embodiments, the isolated nucleic acid (e.g., RNA processing modulator, such as an antisense oligonucleotide) comprises the nucleic acid sequence set forth in SEQ ID NO: 81 and the following modification pattern: all PS; 2’MOE; 20-mer; 5-10-5. In some embodiments, the isolated nucleic acid (e.g., RNA processing modulator, such as an antisense oligonucleotide) comprises the nucleic acid sequence set forth in SEQ ID NO: 82 and the following modification pattern: all PS; 2’MOE; 18-mer. In some embodiments, the isolated nucleic acid (e.g., RNA processing modulator, such as an antisense oligonucleotide) comprises the nucleic acid sequence set forth in SEQ ID NO: 83 and the following modification pattern: all PS; 2’MOE; 20-mer. In some embodiments, the isolated nucleic acid (e.g., RNA processing modulator, such as an antisense oligonucleotide) comprises the nucleic acid sequence set forth in SEQ ID NO: 84 and the following modification pattern: all PS; 2’MOE; 20-mer. In some embodiments, the isolated nucleic acid (e.g., RNA processing modulator, such as an antisense oligonucleotide) comprises the nucleic acid sequence set forth in SEQ ID NO: 85 and the following modification pattern: all PS; 2’MOE; 18-mer. In some embodiments, the isolated nucleic acid (e.g., RNA processing modulator, such as an antisense oligonucleotide) comprises the nucleic acid sequence set forth in SEQ ID NO: 86 and the following modification pattern: all PS; 2’MOE; 18-mer. In some embodiments, the isolated nucleic acid (e.g., RNA processing modulator, such as an antisense oligonucleotide) comprises the nucleic acid sequence set forth in SEQ ID NO: 87 and the following modification pattern: all PS; 2’MOE; 20-mer. In some embodiments, the isolated nucleic acid (e.g., RNA processing modulator, such as an antisense oligonucleotide) comprises the nucleic acid sequence set forth in SEQ ID NO: 88 and the following modification pattern: all PS; 2’MOE; 20-mer. In some embodiments, the isolated nucleic acid (e.g., RNA processing modulator, such as an antisense oligonucleotide) comprises the nucleic acid sequence set forth in SEQ ID NO: 89 and the following modification pattern: all PS; 2’MOE; 18-mer. In some embodiments, the isolated nucleic acid (e.g., RNA processing modulator, such as an antisense oligonucleotide) comprises the nucleic acid sequence set forth in SEQ ID NO: 90 and the following modification pattern: all PS; 2’MOE; 18-mer; 4-10-4.In some embodiments, the isolated nucleic acid (e.g., RNA processing modulator, such as an antisense oligonucleotide) comprises the nucleic acid sequence set forth in SEQ ID NO: 91 and the following modification pattern: fully PS; 2’MOE; 20-mer. In some embodiments, the isolated nucleic acid (e.g., RNA processing modulator, such as an antisense oligonucleotide) comprises the nucleic acid sequence set forth in SEQ ID NO: 92 and the following modification pattern: fully PS; 2’MOE; 20-mer. In some embodiments, the isolated nucleic acid (e.g., RNA processing modulator, such as an antisense oligonucleotide) comprises the nucleic acid sequence set forth in SEQ ID NO: 93 and the following modification pattern: fully PS; 2’MOE; 18-mer. In some embodiments, the isolated nucleic acid (e.g., RNA processing modulator, such as an antisense oligonucleotide) comprises the nucleic acid sequence set forth in SEQ ID NO: 94 and the following modification pattern: fully PS; 2’MOE; 20-mer. In some embodiments, the isolated nucleic acid (e.g., RNA processing modulator, such as an antisense oligonucleotide) comprises the nucleic acid sequence set forth in SEQ ID NO: 95 and the following modification pattern: fully PS; 2’MOE; 20-mer. In some embodiments, the isolated nucleic acid (e.g., RNA processing modulator, such as an antisense oligonucleotide) comprises the nucleic acid sequence set forth in SEQ ID NO: 96 and the following modification pattern: fully PS; 2’MOE; 20-mer. In some embodiments, the isolated nucleic acid (e.g., RNA processing modulator, such as an antisense oligonucleotide) comprises the nucleic acid sequence set forth in SEQ ID NO: 97 and the following modification pattern: fully PS; 2’MOE; 20-mer. In some embodiments, the isolated nucleic acid (e.g., RNA processing modulator, such as an antisense oligonucleotide) comprises the nucleic acid sequence set forth in SEQ ID NO: 98 and the following modification pattern: fully PS; 2’MOE; 18-mer. In some embodiments, the isolated nucleic acid (e.g., RNA processing modulator, such as an antisense oligonucleotide) comprises the nucleic acid sequence set forth in SEQ ID NO: 99 and the following modification pattern: fully PS; 2’MOE; 18-mer. In some embodiments, the isolated nucleic acid (e.g., RNA processing modulator, such as an antisense oligonucleotide) comprises the nucleic acid sequence set forth in SEQ ID NO: 100 and the following modification pattern: fully PS; 2’MOE; 18-mer. In some embodiments, the isolated nucleic acid (e.g., RNA processing modulator, such as an antisense oligonucleotide) comprises the nucleic acid sequence set forth in SEQ ID NO: 101 and the following modification pattern: fully PS; 2’MOE; 18-mer. In some embodiments, the isolated nucleic acid (e.g., RNA processing modulator, such as an antisense oligonucleotide) comprises the nucleic acid sequence set forth in SEQ ID NO: 102 and the following modification pattern: fully PS; 2’MOE; 18-mer.In some embodiments, the isolated nucleic acid (e.g., RNA processing modulator, such as an antisense oligonucleotide) comprises the nucleic acid sequence set forth in SEQ ID NO: 103 and the following modification pattern: all PS; 2’MOE; 20-mer. In some embodiments, the isolated nucleic acid (e.g., RNA processing modulator, such as an antisense oligonucleotide) comprises the nucleic acid sequence set forth in SEQ ID NO: 104 and the following modification pattern: all PS; 2’MOE; 20-mer. In some embodiments, the isolated nucleic acid (e.g., RNA processing modulator, such as an antisense oligonucleotide) comprises the nucleic acid sequence set forth in SEQ ID NO: 105 and the following modification pattern: all PS; 2’MOE; 20-mer; 5-10-5. In some embodiments, the isolated nucleic acid (e.g., RNA processing modulator, such as an antisense oligonucleotide) comprises the nucleic acid sequence set forth in SEQ ID NO: 106 and the following modification pattern: all PS; 2’MOE; 20-mer. In some embodiments, the isolated nucleic acid (e.g., RNA processing modulator, such as an antisense oligonucleotide) comprises the nucleic acid sequence set forth in SEQ ID NO: 107 and the following modification pattern: all PS; 2’MOE; 20-mer. In some embodiments, the isolated nucleic acid (e.g., RNA processing modulator, such as an antisense oligonucleotide) comprises the nucleic acid sequence set forth in SEQ ID NO: 108 and the following modification pattern: all PS; 2’MOE; 18-mer; 4-10-4. In some embodiments, the isolated nucleic acid (e.g., RNA processing modulator, such as an antisense oligonucleotide) comprises the nucleic acid sequence set forth in SEQ ID NO: 109 and the following modification pattern: all PS; 2’MOE; 18-mer. In some embodiments, the isolated nucleic acid (e.g., RNA processing modulator, such as an antisense oligonucleotide) comprises the nucleic acid sequence set forth in SEQ ID NO: 110 and the following modification pattern: all PS; 2’MOE; 20-mer. In some embodiments, the isolated nucleic acid (e.g., RNA processing modulator, such as an antisense oligonucleotide) comprises the nucleic acid sequence set forth in SEQ ID NO: 111 and the following modification pattern: all PS; 2’MOE; 18-mer; 4-10-4. In some embodiments, the isolated nucleic acid (e.g., RNA processing modulator, such as an antisense oligonucleotide) comprises the nucleic acid sequence set forth in SEQ ID NO: 112 and the following modification pattern: all PS; 2’MOE; 18-mer; 4-10-4, PO from the 2nd position from the 5’ end, PO from the 3rd position from the 3’ end. In some embodiments, the isolated nucleic acid (e.g., RNA processing modulator, such as an antisense oligonucleotide) comprises the nucleic acid sequence set forth in SEQ ID NO: 113 and the following modification pattern: all PS; 2’MOE; 18-mer.In some embodiments, the isolated nucleic acid (e.g., RNA processing modulator, such as an antisense oligonucleotide) comprises the nucleic acid sequence set forth in SEQ ID NO: 114 and the following modification pattern: all PS; 2’MOE; 20-mer. In some embodiments, the isolated nucleic acid (e.g., RNA processing modulator, such as an antisense oligonucleotide) comprises the nucleic acid sequence set forth in SEQ ID NO: 115 and the following modification pattern: all PS; 2’MOE; 18-mer. In some embodiments, the isolated nucleic acid (e.g., RNA processing modulator, such as an antisense oligonucleotide) comprises the nucleic acid sequence set forth in SEQ ID NO: 116 and the following modification pattern: all PS; 2’MOE; 18-mer; 4-10-4. In some embodiments, the isolated nucleic acid (e.g., RNA processing modulator, such as an antisense oligonucleotide) comprises the nucleic acid sequence set forth in SEQ ID NO: 117 and the following modification pattern: all PS; 2’MOE; 18-mer. In some embodiments, the isolated nucleic acid (e.g., RNA processing modulator, such as an antisense oligonucleotide) comprises the nucleic acid sequence set forth in SEQ ID NO: 118 and the following modification pattern: all PS; 2’MOE; 20-mer; 5-10-5. In some embodiments, the isolated nucleic acid (e.g., RNA processing modulator, such as an antisense oligonucleotide) comprises the nucleic acid sequence set forth in SEQ ID NO: 119 and the following modification pattern: all PS; 2’MOE; 18-mer. In some embodiments, the isolated nucleic acid (e.g., RNA processing modulator, such as an antisense oligonucleotide) comprises the nucleic acid sequence set forth in SEQ ID NO: 120 and the following modification pattern: all PS; 2’MOE; 20-mer. In some embodiments, the isolated nucleic acid (e.g., RNA processing modulator, such as an antisense oligonucleotide) comprises the nucleic acid sequence set forth in SEQ ID NO: 121 and the following modification pattern: all PS; 2’MOE; 18-mer. In some embodiments, the isolated nucleic acid (e.g., RNA processing modulator, such as an antisense oligonucleotide) comprises the nucleic acid sequence set forth in SEQ ID NO: 122 and the following modification pattern: all PS; 2’MOE; 20-mer. In some embodiments, the isolated nucleic acid (e.g., RNA processing modulator, such as an antisense oligonucleotide) comprises the nucleic acid sequence set forth in SEQ ID NO: 123 and the following modification pattern: all PS; 2’MOE; 18-mer. In some embodiments, the isolated nucleic acid (e.g., RNA processing modulator, such as an antisense oligonucleotide) comprises the nucleic acid sequence set forth in SEQ ID NO: 124 and the following modification pattern: all PS; 2’MOE; 20-mer.In some embodiments, the isolated nucleic acid (e.g., RNA processing modulator, such as an antisense oligonucleotide) comprises the nucleic acid sequence set forth in SEQ ID NO: 125 and the following modification pattern: all PS; 2’MOE; 20-mer; 5-10-5, PO from the 2nd position from the 5’-end, PO from the 3rd position from the 3’-end. In some embodiments, the isolated nucleic acid (e.g., RNA processing modulator, such as an antisense oligonucleotide) comprises the nucleic acid sequence set forth in SEQ ID NO: 126 and the following modification pattern: all PS; 2’MOE; 20-mer. In some embodiments, the isolated nucleic acid (e.g., RNA processing modulator, such as an antisense oligonucleotide) comprises the nucleic acid sequence set forth in SEQ ID NO: 127 and the following modification pattern: all PS; 2’MOE; 18-mer. In some embodiments, the isolated nucleic acid (e.g., RNA processing modulator, such as an antisense oligonucleotide) comprises the nucleic acid sequence set forth in SEQ ID NO: 128 and the following modification pattern: all PS; 2’MOE; 18-mer; 4-10-4. In some embodiments, the isolated nucleic acid (e.g., RNA processing modulator, such as an antisense oligonucleotide) comprises the nucleic acid sequence set forth in SEQ ID NO: 129 and the following modification pattern: all PS; 2’MOE; 20-mer. In some embodiments, the isolated nucleic acid (e.g., RNA processing modulator, such as an antisense oligonucleotide) comprises the nucleic acid sequence set forth in SEQ ID NO: 130 and the following modification pattern: all PS; 2’MOE; 20-mer. In some embodiments, the isolated nucleic acid (e.g., RNA processing modulator, such as an antisense oligonucleotide) comprises the nucleic acid sequence set forth in SEQ ID NO: 131 and the following modification pattern: all PS; 2’MOE; 18-mer. In some embodiments, the isolated nucleic acid (e.g., RNA processing modulator, such as an antisense oligonucleotide) comprises the nucleic acid sequence set forth in SEQ ID NO: 132 and the following modification pattern: all PS; 2’MOE; 20-mer. In some embodiments, the isolated nucleic acid (e.g., RNA processing modulator, such as an antisense oligonucleotide) comprises the nucleic acid sequence set forth in SEQ ID NO: 133 and the following modification pattern: all PS; 2’MOE; 18-mer. In some embodiments, the isolated nucleic acid (e.g., RNA processing modulator, such as an antisense oligonucleotide) comprises the nucleic acid sequence set forth in SEQ ID NO: 134 and the following modification pattern: all PS; 2’MOE; 18-mer; 4-10-4. In some embodiments, the isolated nucleic acid (e.g., RNA processing modulator, such as an antisense oligonucleotide) comprises the nucleic acid sequence set forth in SEQ ID NO: 135 and the following modification pattern: all PS; 2’MOE; 20-mer.In some embodiments, the isolated nucleic acid (e.g., RNA processing modulator, such as an antisense oligonucleotide) comprises the nucleic acid sequence set forth in SEQ ID NO: 136 and the following modification pattern: all PS; 2’MOE; 20-mer. In some embodiments, the isolated nucleic acid (e.g., RNA processing modulator, such as an antisense oligonucleotide) comprises the nucleic acid sequence set forth in SEQ ID NO: 137 and the following modification pattern: all PS; 2’MOE; 20-mer. In some embodiments, the isolated nucleic acid (e.g., RNA processing modulator, such as an antisense oligonucleotide) comprises the nucleic acid sequence set forth in SEQ ID NO: 138 and the following modification pattern: all PS; 2’MOE; 20-mer. In some embodiments, the isolated nucleic acid (e.g., RNA processing modulator, such as an antisense oligonucleotide) comprises the nucleic acid sequence set forth in SEQ ID NO: 139 and the following modification pattern: all PS; 2’MOE; 18-mer. In some embodiments, the isolated nucleic acid (e.g., RNA processing modulator, such as an antisense oligonucleotide) comprises the nucleic acid sequence set forth in SEQ ID NO: 140 and the following modification pattern: all PS; 2’MOE; 20-mer. In some embodiments, the isolated nucleic acid (e.g., RNA processing modulator, such as an antisense oligonucleotide) comprises the nucleic acid sequence set forth in SEQ ID NO: 141 and the following modification pattern: all PS; 2’MOE; 20-mer. In some embodiments, the isolated nucleic acid (e.g., RNA processing modulator, such as an antisense oligonucleotide) comprises the nucleic acid sequence set forth in SEQ ID NO: 142 and the following modification pattern: all PS; 2’MOE; 18-mer; 4-10-4. In some embodiments, the isolated nucleic acid (e.g., RNA processing modulator, such as an antisense oligonucleotide) comprises the nucleic acid sequence set forth in SEQ ID NO: 143 and the following modification pattern: all PS; 2’MOE; 18-mer. In some embodiments, the isolated nucleic acid (e.g., RNA processing modulator, such as an antisense oligonucleotide) comprises the nucleic acid sequence set forth in SEQ ID NO: 144 and the following modification pattern: all PS; 2’MOE; 18-mer. In some embodiments, the isolated nucleic acid (e.g., RNA processing modulator, such as an antisense oligonucleotide) comprises the nucleic acid sequence set forth in SEQ ID NO: 145 and the following modification pattern: all PS; 2’MOE; 20-mer. In some embodiments, the isolated nucleic acid (e.g., RNA processing modulator, such as an antisense oligonucleotide) comprises the nucleic acid sequence set forth in SEQ ID NO: 146 and the following modification pattern: all PS; 2’MOE; 20-mer.In some embodiments, an isolated nucleic acid (e.g., an RNA processing modulator, such as an antisense oligonucleotide) comprises the nucleic acid sequence set forth in SEQ ID NO: 147 and the following modification pattern: all PS; 2’MOE; 18-mer; 4-10-4, PO from the 2nd position from the 5’-end, PO from the 3rd position from the 3’-end. In some embodiments, an isolated nucleic acid (e.g., an RNA processing modulator, such as an antisense oligonucleotide) comprises the nucleic acid sequence set forth in SEQ ID NO: 148 and the following modification pattern: all PS; 2’MOE; 18-mer. In some embodiments, an isolated nucleic acid (e.g., an RNA processing modulator, such as an antisense oligonucleotide) comprises the nucleic acid sequence set forth in SEQ ID NO: 149 and the following modification pattern: all PS; 2’MOE; 18-mer; 4-10-4. In some embodiments, an isolated nucleic acid (e.g., an RNA processing modulator, such as an antisense oligonucleotide) comprises the nucleic acid sequence set forth in SEQ ID NO: 150 and the following modification pattern: all PS; 2’MOE; 20-mer. In some embodiments, an isolated nucleic acid (e.g., an RNA processing modulator, such as an antisense oligonucleotide) comprises the nucleic acid sequence set forth in SEQ ID NO: 151 and the following modification pattern: all PS; 2’MOE; 20-mer; 5-10-5. In some embodiments, an isolated nucleic acid (e.g., an RNA processing modulator, such as an antisense oligonucleotide) comprises the nucleic acid sequence set forth in SEQ ID NO: 152 and the following modification pattern: all PS; 2’MOE; 18-mer; 4-10-4; PO from the 2nd position from the 5’-end, PO from the 3rd position from the 3’-end. In some embodiments, an isolated nucleic acid (e.g., an RNA processing modulator, such as an antisense oligonucleotide) comprises the nucleic acid sequence set forth in SEQ ID NO: 153 and the following modification pattern: all PS; 2’MOE; 20-mer; 5-10-5. In some embodiments, an isolated nucleic acid (e.g., an RNA processing modulator, such as an antisense oligonucleotide) comprises the nucleic acid sequence set forth in SEQ ID NO: 154 and the following modification pattern: all PS; 2’MOE; 18-mer; 4-10-4. In some embodiments, an isolated nucleic acid (e.g., an RNA processing modulator, such as an antisense oligonucleotide) comprises the nucleic acid sequence set forth in SEQ ID NO: 155 and the following modification pattern: all PS; 2’MOE; 20-mer. In some embodiments, an isolated nucleic acid (e.g., an RNA processing modulator, such as an antisense oligonucleotide) comprises the nucleic acid sequence set forth in SEQ ID NO: 156 and the following modification pattern: all PS; 2’MOE; 20-mer.In some embodiments, the isolated nucleic acid (e.g., RNA processing modulator, such as an antisense oligonucleotide) comprises the nucleic acid sequence set forth in SEQ ID NO: 157 and the following modification pattern: all PS; 2’OMe; 20-mer. In some embodiments, the isolated nucleic acid (e.g., RNA processing modulator, such as an antisense oligonucleotide) comprises the nucleic acid sequence set forth in SEQ ID NO: 158 and the following modification pattern: all PS; 2’OMe; 20-mer. In some embodiments, the isolated nucleic acid (e.g., RNA processing modulator, such as an antisense oligonucleotide) comprises the nucleic acid sequence set forth in SEQ ID NO: 159 and the following modification pattern: all PS; 2’OMe; 20-mer. In some embodiments, the isolated nucleic acid (e.g., RNA processing modulator, such as an antisense oligonucleotide) comprises the nucleic acid sequence set forth in SEQ ID NO: 160 and the following modification pattern: all PS; 2’OMe; 18-mer. In some embodiments, the isolated nucleic acid (e.g., RNA processing modulator, such as an antisense oligonucleotide) comprises the nucleic acid sequence set forth in SEQ ID NO: 161 and the following modification pattern: all PS; 2’OMe; 18-mer. In some embodiments, the isolated nucleic acid (e.g., RNA processing modulator, such as an antisense oligonucleotide) comprises the nucleic acid sequence set forth in SEQ ID NO: 162 and the following modification pattern: all PS; 2’OMe; 20-mer; 5-10-5; 4 PO from the 2nd base from the 5’, 3rd base from the 5’, 3rd base from the 3’, 4th base from the 3’ after. In some embodiments, the isolated nucleic acid (e.g., RNA processing modulator, such as an antisense oligonucleotide) comprises the nucleic acid sequence set forth in SEQ ID NO: 163 and the following modification pattern: all PS; 2’OMe; 18-mer. In some embodiments, the isolated nucleic acid (e.g., RNA processing modulator, such as an antisense oligonucleotide) comprises the nucleic acid sequence set forth in SEQ ID NO: 164 and the following modification pattern: all PS; 2’OMe; 20-mer. In some embodiments, the isolated nucleic acid (e.g., RNA processing modulator, such as an antisense oligonucleotide) comprises the nucleic acid sequence set forth in SEQ ID NO: 165 and the following modification pattern: all PS; 2’OMe; 18-mer. In some embodiments, the isolated nucleic acid (e.g., RNA processing modulator, such as an antisense oligonucleotide) comprises the nucleic acid sequence set forth in SEQ ID NO: 166 and the following modification pattern: all PS; 2’OMe; 18-mer. In some embodiments, the isolated nucleic acid (e.g., RNA processing modulator, such as an antisense oligonucleotide) comprises the nucleic acid sequence set forth in SEQ ID NO: 167 and the following modification pattern: all PS; 2’OMe; 18-mer.In some embodiments, the isolated nucleic acid (e.g., RNA processing modulator, such as an antisense oligonucleotide) comprises the nucleic acid sequence set forth in SEQ ID NO: 168 and the following modification pattern: all PS; 2’MOE; 18-mer. In some embodiments, the isolated nucleic acid (e.g., RNA processing modulator, such as an antisense oligonucleotide) comprises the nucleic acid sequence set forth in SEQ ID NO: 169 and the following modification pattern: all PS; 2’MOE; 20-mer; 5-10-5. In some embodiments, the isolated nucleic acid (e.g., RNA processing modulator, such as an antisense oligonucleotide) comprises the nucleic acid sequence set forth in SEQ ID NO: 170 and the following modification pattern: all PS; 2’MOE; 18-mer. In some embodiments, the isolated nucleic acid (e.g., RNA processing modulator, such as an antisense oligonucleotide) comprises the nucleic acid sequence set forth in SEQ ID NO: 171 and the following modification pattern: all PS; 2’MOE; 18-mer. In some embodiments, the isolated nucleic acid (e.g., RNA processing modulator, such as an antisense oligonucleotide) comprises the nucleic acid sequence set forth in SEQ ID NO: 172 and the following modification pattern: all PS; 2’MOE; 18-mer. In some embodiments, the isolated nucleic acid (e.g., RNA processing modulator, such as an antisense oligonucleotide) comprises the nucleic acid sequence set forth in SEQ ID NO: 173 and the following modification pattern: all PS; 2’MOE; 18-mer. In some embodiments, the isolated nucleic acid (e.g., RNA processing modulator, such as an antisense oligonucleotide) comprises the nucleic acid sequence set forth in SEQ ID NO: 174 and the following modification pattern: all PS; 2’MOE; 20-mer. In some embodiments, the isolated nucleic acid (e.g., RNA processing modulator, such as an antisense oligonucleotide) comprises the nucleic acid sequence set forth in SEQ ID NO: 175 and the following modification pattern: all PS; 2’MOE; 18-mer; 4-10-4. In some embodiments, the isolated nucleic acid (e.g., RNA processing modulator, such as an antisense oligonucleotide) comprises the nucleic acid sequence set forth in SEQ ID NO: 176 and the following modification pattern: all PS; 2’MOE; 18-mer. In some embodiments, the isolated nucleic acid (e.g., RNA processing modulator, such as an antisense oligonucleotide) comprises the nucleic acid sequence set forth in SEQ ID NO: 177 and the following modification pattern: all PS; 2’MOE; 18-mer; 4-10-4. In some embodiments, the isolated nucleic acid (e.g., RNA processing modulator, such as an antisense oligonucleotide) comprises the nucleic acid sequence set forth in SEQ ID NO: 178 and the following modification pattern: all PS; 2’MOE; 18-mer; 4-10-4.In some embodiments, the isolated nucleic acid (e.g., RNA processing modulator, such as an antisense oligonucleotide) comprises the nucleic acid sequence set forth in SEQ ID NO: 179 and the following modification pattern: all PS; 2’MOE; 20-mer. In some embodiments, the isolated nucleic acid (e.g., RNA processing modulator, such as an antisense oligonucleotide) comprises the nucleic acid sequence set forth in SEQ ID NO: 180 and the following modification pattern: all PS; 2’MOE; 18-mer. In some embodiments, the isolated nucleic acid (e.g., RNA processing modulator, such as an antisense oligonucleotide) comprises the nucleic acid sequence set forth in SEQ ID NO: 181 and the following modification pattern: all PS; 2’MOE; 20-mer. In some embodiments, the isolated nucleic acid (e.g., RNA processing modulator, such as an antisense oligonucleotide) comprises the nucleic acid sequence set forth in SEQ ID NO: 182 and the following modification pattern: all PS; 2’MOE; 18-mer; 4-10-4. In some embodiments, the isolated nucleic acid (e.g., RNA processing modulator, such as an antisense oligonucleotide) comprises the nucleic acid sequence set forth in SEQ ID NO: 183 and the following modification pattern: all PS; 2’MOE; 18-mer. In some embodiments, the isolated nucleic acid (e.g., RNA processing modulator, such as an antisense oligonucleotide) comprises the nucleic acid sequence set forth in SEQ ID NO: 184 and the following modification pattern: all PS; 2’MOE; 18-mer. In some embodiments, the isolated nucleic acid (e.g., RNA processing modulator, such as an antisense oligonucleotide) comprises the nucleic acid sequence set forth in SEQ ID NO: 185 and the following modification pattern: all PS; 2’MOE; 18-mer. In some embodiments, the isolated nucleic acid (e.g., RNA processing modulator, such as an antisense oligonucleotide) comprises the nucleic acid sequence set forth in SEQ ID NO: 186 and the following modification pattern: all PS; 2’MOE; 20-mer. In some embodiments, the isolated nucleic acid (e.g., RNA processing modulator, such as an antisense oligonucleotide) comprises the nucleic acid sequence set forth in SEQ ID NO: 187 and the following modification pattern: all PS; 2’MOE; 20-mer. In some embodiments, the isolated nucleic acid (e.g., RNA processing modulator, such as an antisense oligonucleotide) comprises the nucleic acid sequence set forth in SEQ ID NO: 188 and the following modification pattern: all PS; 2’MOE; 18-mer. In some embodiments, the isolated nucleic acid (e.g., RNA processing modulator, such as an antisense oligonucleotide) comprises the nucleic acid sequence set forth in SEQ ID NO: 189 and the following modification pattern: all PS; 2’MOE; 18-mer.In some embodiments, the isolated nucleic acid (e.g., RNA processing modulator, such as an antisense oligonucleotide) comprises the nucleic acid sequence set forth in SEQ ID NO: 190 and the following modification pattern: all PS; 2’MOE; 20-mer; 5-10-5. In some embodiments, the isolated nucleic acid (e.g., RNA processing modulator, such as an antisense oligonucleotide) comprises the nucleic acid sequence set forth in SEQ ID NO: 191 and the following modification pattern: all PS; 2’MOE; 20-mer; 5-10-5, PO from the 2nd position from the 5’-end, PO from the 3rd position from the 3’-end. In some embodiments, the isolated nucleic acid (e.g., RNA processing modulator, such as an antisense oligonucleotide) comprises the nucleic acid sequence set forth in SEQ ID NO: 192 and the following modification pattern: all PS; 2’MOE; 18-mer; 4-10-4. In some embodiments, the isolated nucleic acid (e.g., RNA processing modulator, such as an antisense oligonucleotide) comprises the nucleic acid sequence set forth in SEQ ID NO: 193 and the following modification pattern: all PS; 2’MOE; 20-mer; 5-10-5; PO from the 2nd position from the 5’-end, PO from the 3rd position from the 3’-end. In some embodiments, the isolated nucleic acid (e.g., RNA processing modulator, such as an antisense oligonucleotide) comprises the nucleic acid sequence set forth in SEQ ID NO: 194 and the following modification pattern: all PS; 2’MOE; 18-mer. In some embodiments, the isolated nucleic acid (e.g., RNA processing modulator, such as an antisense oligonucleotide) comprises the nucleic acid sequence set forth in SEQ ID NO: 195 and the following modification pattern: all PS; 2’MOE; 20-mer; 5-10-5; PO from the 2nd position from the 5’-end, PO from the 3rd position from the 3’-end. In some embodiments, the isolated nucleic acid (e.g., RNA processing modulator, such as an antisense oligonucleotide) comprises the nucleic acid sequence set forth in SEQ ID NO: 196 and the following modification pattern: all PS; 2’MOE; 18-mer. In some embodiments, the isolated nucleic acid (e.g., RNA processing modulator, such as an antisense oligonucleotide) comprises the nucleic acid sequence set forth in SEQ ID NO: 197 and the following modification pattern: all PS; 2’MOE; 20-mer. In some embodiments, the isolated nucleic acid (e.g., RNA processing modulator, such as an antisense oligonucleotide) comprises the nucleic acid sequence set forth in SEQ ID NO: 198 and the following modification pattern: all PS; 2’MOE; 18-mer. In some embodiments, the isolated nucleic acid (e.g., RNA processing modulator, such as an antisense oligonucleotide) comprises the nucleic acid sequence set forth in SEQ ID NO: 199 and the following modification pattern: all PS; 2’MOE; 18-mer.In some embodiments, the isolated nucleic acid (e.g., RNA processing modulator, such as an antisense oligonucleotide) comprises the nucleic acid sequence set forth in SEQ ID NO: 200 and the following modification pattern: all PS; 2’MOE; 20-mer. In some embodiments, the isolated nucleic acid (e.g., RNA processing modulator, such as an antisense oligonucleotide) comprises the nucleic acid sequence set forth in SEQ ID NO: 201 and the following modification pattern: all PS; 2’MOE; 20-mer. In some embodiments, the isolated nucleic acid (e.g., RNA processing modulator, such as an antisense oligonucleotide) comprises the nucleic acid sequence set forth in SEQ ID NO: 202 and the following modification pattern: all PS; 2’MOE; 20-mer. In some embodiments, the isolated nucleic acid (e.g., RNA processing modulator, such as an antisense oligonucleotide) comprises the nucleic acid sequence set forth in SEQ ID NO: 203 and the following modification pattern: all PS; 2’MOE; 18-mer. In some embodiments, the isolated nucleic acid (e.g., RNA processing modulator, such as an antisense oligonucleotide) comprises the nucleic acid sequence set forth in SEQ ID NO: 204 and the following modification pattern: all PS; 2’MOE; 18-mer. In some embodiments, the isolated nucleic acid (e.g., RNA processing modulator, such as an antisense oligonucleotide) comprises the nucleic acid sequence set forth in SEQ ID NO: 205 and the following modification pattern: all PS; 2’MOE; 18-mer. In some embodiments, the isolated nucleic acid (e.g., RNA processing modulator, such as an antisense oligonucleotide) comprises the nucleic acid sequence set forth in SEQ ID NO: 206 and the following modification pattern: all PS; 2’MOE; 20-mer. In some embodiments, the isolated nucleic acid (e.g., RNA processing modulator, such as an antisense oligonucleotide) comprises the nucleic acid sequence set forth in SEQ ID NO: 207 and the following modification pattern: all PS; 2’MOE; 18-mer; 4-10-4. In some embodiments, the isolated nucleic acid (e.g., RNA processing modulator, such as an antisense oligonucleotide) comprises the nucleic acid sequence set forth in SEQ ID NO: 208 and the following modification pattern: all PS; 2’MOE; 18-mer. In some embodiments, the isolated nucleic acid (e.g., RNA processing modulator, such as an antisense oligonucleotide) comprises the nucleic acid sequence set forth in SEQ ID NO: 209 and the following modification pattern: all PS; 2’MOE; 18-mer. In some embodiments, the isolated nucleic acid (e.g., RNA processing modulator, such as an antisense oligonucleotide) comprises the nucleic acid sequence set forth in SEQ ID NO: 210 and the following modification pattern: all PS; 2’MOE; 20-mer. In some embodiments, the isolated nucleic acid (e.g., RNA processing modulator, such as an antisense oligonucleotide) comprises the nucleic acid sequence set forth in SEQ ID NO: 211 and the following modification pattern: all PS; 2’MOE; 18-mer.In some embodiments, the isolated nucleic acid (e.g., RNA processing modulator, such as an antisense oligonucleotide) comprises the nucleic acid sequence set forth in SEQ ID NO: 212 and the following modification pattern: all PS; 2’MOE; 20-mer. In some embodiments, the isolated nucleic acid (e.g., RNA processing modulator, such as an antisense oligonucleotide) comprises the nucleic acid sequence set forth in SEQ ID NO: 213 and the following modification pattern: all PS; 2’MOE; 20-mer. In some embodiments, the isolated nucleic acid (e.g., RNA processing modulator, such as an antisense oligonucleotide) comprises the nucleic acid sequence set forth in SEQ ID NO: 214 and the following modification pattern: all PS; 2’MOE; 18-mer; 4-10-4. In some embodiments, the isolated nucleic acid (e.g., RNA processing modulator, such as an antisense oligonucleotide) comprises the nucleic acid sequence set forth in SEQ ID NO: 215 and the following modification pattern: all PS; 2’MOE; 20-mer. In some embodiments, the isolated nucleic acid (e.g., RNA processing modulator, such as an antisense oligonucleotide) comprises the nucleic acid sequence set forth in SEQ ID NO: 216 and the following modification pattern: all PS; 2’MOE; 18-mer. In some embodiments, the isolated nucleic acid (e.g., RNA processing modulator, such as an antisense oligonucleotide) comprises the nucleic acid sequence set forth in SEQ ID NO: 217 and the following modification pattern: all PS; 2’MOE; 20-mer. In some embodiments, the isolated nucleic acid (e.g., RNA processing modulator, such as an antisense oligonucleotide) comprises the nucleic acid sequence set forth in SEQ ID NO: 218 and the following modification pattern: all PS; 2’MOE; 18-mer. In some embodiments, the isolated nucleic acid (e.g., RNA processing modulator, such as an antisense oligonucleotide) comprises the nucleic acid sequence set forth in SEQ ID NO: 219 and the following modification pattern: all PS; 2’MOE; 20-mer; 5-10-5. In some embodiments, the isolated nucleic acid (e.g., RNA processing modulator, such as an antisense oligonucleotide) comprises the nucleic acid sequence set forth in SEQ ID NO: 220 and the following modification pattern: all PS; 2’MOE; 20-mer. In some embodiments, the isolated nucleic acid (e.g., RNA processing modulator, such as an antisense oligonucleotide) comprises the nucleic acid sequence set forth in SEQ ID NO: 221 and the following modification pattern: all PS; 2’MOE; 18-mer; 4-10-4; PO from the 2nd position from the 5’ end, PO from the 3rd position from the 3’ end. In some embodiments, the isolated nucleic acid (e.g., RNA processing modulator, such as an antisense oligonucleotide) comprises the nucleic acid sequence set forth in SEQ ID NO: 222 and the following modification pattern: all PS; 2’MOE; 18-mer; 4-10-4.In some embodiments, the isolated nucleic acid (e.g., RNA processing modulator, such as an antisense oligonucleotide) comprises the nucleic acid sequence set forth in SEQ ID NO: 223 and the following modification pattern: all PS; 2’MOE; 18-mer. In some embodiments, the isolated nucleic acid (e.g., RNA processing modulator, such as an antisense oligonucleotide) comprises the nucleic acid sequence set forth in SEQ ID NO: 224 and the following modification pattern: all PS; 2’MOE; 18-mer. In some embodiments, the isolated nucleic acid (e.g., RNA processing modulator, such as an antisense oligonucleotide) comprises the nucleic acid sequence set forth in SEQ ID NO: 225 and the following modification pattern: all PS; 2’MOE; 18-mer. In some embodiments, the isolated nucleic acid (e.g., RNA processing modulator, such as an antisense oligonucleotide) comprises the nucleic acid sequence set forth in SEQ ID NO: 226 and the following modification pattern: all PS; 2’MOE; 20-mer; 5-10-5. In some embodiments, the isolated nucleic acid (e.g., RNA processing modulator, such as an antisense oligonucleotide) comprises the nucleic acid sequence set forth in SEQ ID NO: 227 and the following modification pattern: all PS; 2’MOE; 20-mer; 5-10-5, PO from the 2nd position from the 5’ end, PO from the 3rd position from the 3’ end. In some embodiments, the isolated nucleic acid (e.g., RNA processing modulator, such as an antisense oligonucleotide) comprises the nucleic acid sequence set forth in SEQ ID NO: 228 and the following modification pattern: all PS; 2’MOE; 18-mer. In some embodiments, the isolated nucleic acid (e.g., RNA processing modulator, such as an antisense oligonucleotide) comprises the nucleic acid sequence set forth in SEQ ID NO: 229 and the following modification pattern: all PS; 2’MOE; 18-mer. In some embodiments, the isolated nucleic acid (e.g., RNA processing modulator, such as an antisense oligonucleotide) comprises the nucleic acid sequence set forth in SEQ ID NO: 230 and the following modification pattern: all PS; 2’MOE; 18-mer; 4-10-4. In some embodiments, the isolated nucleic acid (e.g., RNA processing modulator, such as an antisense oligonucleotide) comprises the nucleic acid sequence set forth in SEQ ID NO: 231 and the following modification pattern: all PS; 2’MOE; 20-mer. In some embodiments, the isolated nucleic acid (e.g., RNA processing modulator, such as an antisense oligonucleotide) comprises the nucleic acid sequence set forth in SEQ ID NO: 232 and the following modification pattern: all PS; 2’MOE; 20-mer. In some embodiments, the isolated nucleic acid (e.g., RNA processing modulator, such as an antisense oligonucleotide) comprises the nucleic acid sequence set forth in SEQ ID NO: 233 and the following modification pattern: all PS; 2’MOE; 20-mer; 5-10-5, PO from the 2nd position from the 5’ end, PO from the 3rd position from the 3’ end.In some embodiments, the isolated nucleic acid (e.g., RNA processing modulator, such as an antisense oligonucleotide) comprises the nucleic acid sequence set forth in SEQ ID NO: 234 and the following modification pattern: all PS; 2' MOE; 20-mer. In some embodiments, the isolated nucleic acid (e.g., RNA processing modulator, such as an antisense oligonucleotide) comprises the nucleic acid sequence set forth in SEQ ID NO: 235 and the following modification pattern: all PS; 2' MOE; 20-mer. In some embodiments, the isolated nucleic acid (e.g., RNA processing modulator, such as an antisense oligonucleotide) comprises the nucleic acid sequence set forth in SEQ ID NO: 236 and the following modification pattern: all PS; 2' MOE; 18-mer. In some embodiments, the isolated nucleic acid (e.g., RNA processing modulator, such as an antisense oligonucleotide) comprises the nucleic acid sequence set forth in SEQ ID NO: 237 and the following modification pattern: all PS; 2' MOE; 18-mer. In some embodiments, the isolated nucleic acid (e.g., RNA processing modulator, such as an antisense oligonucleotide) comprises the nucleic acid sequence set forth in SEQ ID NO: 238 and the following modification pattern: all PS; 2' MOE; 18-mer. In some embodiments, the isolated nucleic acid (e.g., RNA processing modulator, such as an antisense oligonucleotide) comprises the nucleic acid sequence set forth in SEQ ID NO: 239 and the following modification pattern: all PS; 2' MOE; 18-mer. In some embodiments, the isolated nucleic acid (e.g., RNA processing modulator, such as an antisense oligonucleotide) comprises the nucleic acid sequence set forth in SEQ ID NO: 240 and the following modification pattern: all PS; 2' MOE; 20-mer. In some embodiments, the isolated nucleic acid (e.g., RNA processing modulator, such as an antisense oligonucleotide) comprises the nucleic acid sequence set forth in SEQ ID NO: 241 and the following modification pattern: all PS; 2' MOE; 20-mer. In some embodiments, the isolated nucleic acid (e.g., RNA processing modulator, such as an antisense oligonucleotide) comprises the nucleic acid sequence set forth in SEQ ID NO: 242 and the following modification pattern: all PS; 2' MOE; 20-mer. In some embodiments, the isolated nucleic acid (e.g., RNA processing modulator, such as an antisense oligonucleotide) comprises the nucleic acid sequence set forth in SEQ ID NO: 243 and the following modification pattern: all PS; 2' MOE; 20-mer; 5-10-5. In some embodiments, the isolated nucleic acid (e.g., RNA processing modulator, such as an antisense oligonucleotide) comprises the nucleic acid sequence set forth in SEQ ID NO: 244 and the following modification pattern: all PS; 2' MOE; 20-mer; 5-10-5, PO from the 2nd position from the 5' end, PO from the 3rd position from the 3' end.In some embodiments, the isolated nucleic acid (e.g., RNA processing modulator, such as an antisense oligonucleotide) comprises the nucleic acid sequence set forth in SEQ ID NO: 245 and the following modification pattern: all PS; 2’MOE; 20-mer. In some embodiments, the isolated nucleic acid (e.g., RNA processing modulator, such as an antisense oligonucleotide) comprises the nucleic acid sequence set forth in SEQ ID NO: 246 and the following modification pattern: all PS; 2’MOE; 20-mer. In some embodiments, the isolated nucleic acid (e.g., RNA processing modulator, such as an antisense oligonucleotide) comprises the nucleic acid sequence set forth in SEQ ID NO: 247 and the following modification pattern: all PS; 2’MOE; 20-mer. In some embodiments, the isolated nucleic acid (e.g., RNA processing modulator, such as an antisense oligonucleotide) comprises the nucleic acid sequence set forth in SEQ ID NO: 248 and the following modification pattern: all PS; 2’MOE; 20-mer; 5-10-5, PO from the 2ndposition from the 5’ end, PO from the 3rdposition from the 3’ end. In some embodiments, the isolated nucleic acid (e.g., RNA processing modulator, such as an antisense oligonucleotide) comprises the nucleic acid sequence set forth in SEQ ID NO: 249 and the following modification pattern: all PS; 2’MOE; 20-mer. In some embodiments, the isolated nucleic acid (e.g., RNA processing modulator, such as an antisense oligonucleotide) comprises the nucleic acid sequence set forth in SEQ ID NO: 250 and the following modification pattern: all PS; 2’MOE; 20-mer. In some embodiments, the isolated nucleic acid (e.g., RNA processing modulator, such as an antisense oligonucleotide) comprises the nucleic acid sequence set forth in SEQ ID NO: 251 and the following modification pattern: all PS; 2’MOE; 18-mer; 4-10-4. In some embodiments, the isolated nucleic acid (e.g., RNA processing modulator, such as an antisense oligonucleotide) comprises the nucleic acid sequence set forth in SEQ ID NO: 252 and the following modification pattern: all PS; 2’MOE; 20-mer; 5-10-5. In some embodiments, the isolated nucleic acid (e.g., RNA processing modulator, such as an antisense oligonucleotide) comprises the nucleic acid sequence set forth in SEQ ID NO: 253 and the following modification pattern: all PS; 2’MOE; 20-mer. In some embodiments, the isolated nucleic acid (e.g., RNA processing modulator, such as an antisense oligonucleotide) comprises the nucleic acid sequence set forth in SEQ ID NO: 254 and the following modification pattern: all PS; 2’MOE; 20-mer; 5-10-5; PO from the 2ndposition from the 5’ end, PO from the 3rdposition from the 3’ end. In some embodiments, the isolated nucleic acid (e.g., RNA processing modulator, such as an antisense oligonucleotide) comprises the nucleic acid sequence set forth in SEQ ID NO: 255 and the following modification pattern: all PS; 2’MOE; 18-mer.In some embodiments, the isolated nucleic acid (e.g., RNA processing modulator, such as an antisense oligonucleotide) comprises the nucleic acid sequence set forth in SEQ ID NO: 256 and the following modification pattern: fully PS; 2’ MOE; 20-mer. In some embodiments, the isolated nucleic acid (e.g., RNA processing modulator, such as an antisense oligonucleotide) comprises the nucleic acid sequence set forth in SEQ ID NO: 257 and the following modification pattern: fully PS; 2’ MOE; 20-mer; 5-10-5. In some embodiments, the isolated nucleic acid (e.g., RNA processing modulator, such as an antisense oligonucleotide) comprises the nucleic acid sequence set forth in SEQ ID NO: 258 and the following modification pattern: fully PS; 2’ MOE; 20-mer. In some embodiments, the isolated nucleic acid (e.g., RNA processing modulator, such as an antisense oligonucleotide) comprises the nucleic acid sequence set forth in SEQ ID NO: 259 and the following modification pattern: fully PS; 2’ MOE; 20-mer. In some embodiments, the isolated nucleic acid (e.g., RNA processing modulator, such as an antisense oligonucleotide) comprises the nucleic acid sequence set forth in SEQ ID NO: 260 and the following modification pattern: fully PS; 2’ MOE; 18-mer; 4-10-4. In some embodiments, the isolated nucleic acid (e.g., RNA processing modulator, such as an antisense oligonucleotide) comprises the nucleic acid sequence set forth in SEQ ID NO: 261 and the following modification pattern: fully PS; 2’ MOE; 18-mer. In some embodiments, the isolated nucleic acid (e.g., RNA processing modulator, such as an antisense oligonucleotide) comprises the nucleic acid sequence set forth in SEQ ID NO: 262 and the following modification pattern: 20-mer; Gapmer; 5-10-5, PO from the 2nd base from the 5’ end, PO from the 3rd base from the 3’ end; fully PS; 2’ MOE. In some embodiments, the isolated nucleic acid (e.g., RNA processing modulator, such as an antisense oligonucleotide) comprises the nucleic acid sequence set forth in SEQ ID NO: 263 and the following modification pattern: 20-mer; Gapmer; 5-10-5, PO from the 2nd base from the 5’ end, PO from the 3rd base from the 3’ end; fully PS; 2’ MOE. In some embodiments, the isolated nucleic acid (e.g., RNA processing modulator, such as an antisense oligonucleotide) comprises the nucleic acid sequence set forth in SEQ ID NO: 264 and the following modification pattern: 20-mer; Gapmer; 5-10-5, PO from the 2nd base from the 5’ end, PO from the 3rd base from the 3’ end; fully PS; 2’ MOE.In some embodiments, the isolated nucleic acid (e.g., RNA processing modulator, such as an antisense oligonucleotide) comprises the nucleic acid sequence set forth in SEQ ID NO: 265 and the following modification pattern: 20-mer; Gapmer; 5-10-5, PO from the 2nd base from the 5' end, PO from the 3rd base from the 3' end; Fully PS; 2' MOE. In some embodiments, the isolated nucleic acid (e.g., RNA processing modulator, such as an antisense oligonucleotide) comprises the nucleic acid sequence set forth in SEQ ID NO: 266 and the following modification pattern: 20-mer; Gapmer; 5-10-5, PO from the 2nd base from the 5' end, PO from the 3rd base from the 3' end; Fully PS; 2' MOE. In some embodiments, the isolated nucleic acid (e.g., RNA processing modulator, such as an antisense oligonucleotide) comprises the nucleic acid sequence set forth in SEQ ID NO: 267 and the following modification pattern: 20-mer; Gapmer; 5-10-5, PO from the 2nd base from the 5' end, PO from the 3rd base from the 3' end; Fully PS; 2' MOE. In some embodiments, the isolated nucleic acid (e.g., RNA processing modulator, such as an antisense oligonucleotide) comprises the nucleic acid sequence set forth in SEQ ID NO: 268 and the following modification pattern: 20-mer; Gapmer; 5-10-5, PO from the 2nd base from the 5' end, PO from the 3rd base from the 3' end; Fully PS; 2' MOE. In some embodiments, the isolated nucleic acid (e.g., RNA processing modulator, such as an antisense oligonucleotide) comprises the nucleic acid sequence set forth in SEQ ID NO: 269 and the following modification pattern: 20-mer; Gapmer; 5-10-5, PO from the 2nd base from the 5' end, PO from the 3rd base from the 3' end; Fully PS; 2' MOE. In some embodiments, the isolated nucleic acid (e.g., RNA processing modulator, such as an antisense oligonucleotide) comprises the nucleic acid sequence set forth in SEQ ID NO: 270 and the following modification pattern: 20-mer; Gapmer; 5-10-5, PO from the 2nd base from the 5' end, PO from the 3rd base from the 3' end; Fully PS; 2' MOE. In some embodiments, the isolated nucleic acid (e.g., RNA processing modulator, such as an antisense oligonucleotide) comprises the nucleic acid sequence set forth in SEQ ID NO: 271 and the following modification pattern: 20-mer; Gapmer; 5-10-5, PO from the 2nd base from the 5' end, PO from the 3rd base from the 3' end; Fully PS; 2' MOE.In some embodiments, the isolated nucleic acid (e.g., RNA processing modulator, such as an antisense oligonucleotide) comprises the nucleic acid sequence set forth in SEQ ID NO: 272 and the following modification pattern: 20mer; Gapmer; 5-10-5, PO from the 2nd base from the 5' end, PO from the 3rd base from the 3' end; Fully PS; 2' MOE. In some embodiments, the isolated nucleic acid (e.g., RNA processing modulator, such as an antisense oligonucleotide) comprises the nucleic acid sequence set forth in SEQ ID NO: 273 and the following modification pattern: 20mer; Gapmer; 5-10-5, PO from the 2nd base from the 5' end, PO from the 3rd base from the 3' end; Fully PS; 2' MOE. In some embodiments, the isolated nucleic acid (e.g., RNA processing modulator, such as an antisense oligonucleotide) comprises the nucleic acid sequence set forth in SEQ ID NO: 274 and the following modification pattern: 20mer; Gapmer; 5-10-5, PO from the 2nd base from the 5' end, PO from the 3rd base from the 3' end; Fully PS; 2' MOE. In some embodiments, the isolated nucleic acid (e.g., RNA processing modulator, such as an antisense oligonucleotide) comprises the nucleic acid sequence set forth in SEQ ID NO: 275 and the following modification pattern: 20mer; Gapmer; 5-10-5, PO from the 2nd base from the 5' end, PO from the 3rd base from the 3' end; Fully PS; 2' MOE. In some embodiments, the isolated nucleic acid (e.g., RNA processing modulator, such as an antisense oligonucleotide) comprises the nucleic acid sequence set forth in SEQ ID NO: 276 and the following modification pattern: 20mer; Gapmer; 5-10-5, PO from the 2nd base from the 5' end, PO from the 3rd base from the 3' end; Fully PS; 2' MOE. In some embodiments, the isolated nucleic acid (e.g., RNA processing modulator, such as an antisense oligonucleotide) comprises the nucleic acid sequence set forth in SEQ ID NO: 277 and the following modification pattern: 20mer; Gapmer; 5-10-5, PO from the 2nd base from the 5' end, PO from the 3rd base from the 3' end; Fully PS; 2' MOE. In some embodiments, the isolated nucleic acid (e.g., RNA processing modulator, such as an antisense oligonucleotide) comprises the nucleic acid sequence set forth in SEQ ID NO: 278 and the following modification pattern: 20mer; Gapmer; 5-10-5, PO from the 2nd base from the 5' end, PO from the 3rd base from the 3' end; Fully PS; 2' MOE.In some embodiments, the isolated nucleic acid (e.g., RNA processing modulator, such as an antisense oligonucleotide) comprises the nucleic acid sequence set forth in SEQ ID NO: 279 and the following modification pattern: 20mer; Gapmer; 5-10-5, PO from the 2nd base from the 5' end, PO from the 3rd base from the 3' end; Fully PS; 2' MOE. In some embodiments, the isolated nucleic acid (e.g., RNA processing modulator, such as an antisense oligonucleotide) comprises the nucleic acid sequence set forth in SEQ ID NO: 280 and the following modification pattern: 20mer; Gapmer; 5-10-5, PO from the 2nd base from the 5' end, PO from the 3rd base from the 3' end; Fully PS; 2' MOE. In some embodiments, the isolated nucleic acid (e.g., RNA processing modulator, such as an antisense oligonucleotide) comprises the nucleic acid sequence set forth in SEQ ID NO: 281 and the following modification pattern: 20mer; Gapmer; 5-10-5, PO from the 2nd base from the 5' end, PO from the 3rd base from the 3' end; Fully PS; 2' MOE. In some embodiments, the isolated nucleic acid (e.g., RNA processing modulator, such as an antisense oligonucleotide) comprises the nucleic acid sequence set forth in SEQ ID NO: 282 and the following modification pattern: 20mer; Gapmer; 5-10-5, PO from the 2nd base from the 5' end, PO from the 3rd base from the 3' end; Fully PS; 2' MOE. In some embodiments, the isolated nucleic acid (e.g., RNA processing modulator, such as an antisense oligonucleotide) comprises the nucleic acid sequence set forth in SEQ ID NO: 283 and the following modification pattern: 20mer; Gapmer; 5-10-5, PO from the 2nd base from the 5' end, PO from the 3rd base from the 3' end; Fully PS; 2' MOE. In some embodiments, the isolated nucleic acid (e.g., RNA processing modulator, such as an antisense oligonucleotide) comprises the nucleic acid sequence set forth in SEQ ID NO: 284 and the following modification pattern: 20mer; Gapmer; 5-10-5, PO from the 2nd base from the 5' end, PO from the 3rd base from the 3' end; Fully PS; 2' MOE. In some embodiments, the isolated nucleic acid (e.g., RNA processing modulator, such as an antisense oligonucleotide) comprises the nucleic acid sequence set forth in SEQ ID NO: 285 and the following modification pattern: 20mer; Gapmer; 5-10-5, PO from the 2nd base from the 5' end, PO from the 3rd base from the 3' end; Fully PS; 2' MOE.In some embodiments, the isolated nucleic acid (e.g., RNA processing modulator, such as an antisense oligonucleotide) comprises the nucleic acid sequence set forth in SEQ ID NO: 286 and the following modification pattern: 20-mer; Gapmer; 5-10-5, PO from the 2nd base from the 5' end, PO from the 3rd base from the 3' end; Fully PS; 2' MOE. In some embodiments, the isolated nucleic acid (e.g., RNA processing modulator, such as an antisense oligonucleotide) comprises the nucleic acid sequence set forth in SEQ ID NO: 287 and the following modification pattern: 20-mer; Gapmer; 5-10-5, PO from the 2nd base from the 5' end, PO from the 3rd base from the 3' end; Fully PS; 2' MOE. In some embodiments, the isolated nucleic acid (e.g., RNA processing modulator, such as an antisense oligonucleotide) comprises the nucleic acid sequence set forth in SEQ ID NO: 288 and the following modification pattern: 20-mer; Gapmer; 5-10-5, PO from the 2nd base from the 5' end, PO from the 3rd base from the 3' end; Fully PS; 2' MOE. In some embodiments, the isolated nucleic acid (e.g., RNA processing modulator, such as an antisense oligonucleotide) comprises the nucleic acid sequence set forth in SEQ ID NO: 289 and the following modification pattern: 20-mer; Gapmer; 5-10-5, PO from the 2nd base from the 5' end, PO from the 3rd base from the 3' end; Fully PS; 2' MOE. In some embodiments, the isolated nucleic acid (e.g., RNA processing modulator, such as an antisense oligonucleotide) comprises the nucleic acid sequence set forth in SEQ ID NO: 290 and the following modification pattern: 20-mer; Gapmer; 5-10-5, PO from the 2nd base from the 5' end, PO from the 3rd base from the 3' end; Fully PS; 2' MOE. In some embodiments, the isolated nucleic acid (e.g., RNA processing modulator, such as an antisense oligonucleotide) comprises the nucleic acid sequence set forth in SEQ ID NO: 291 and the following modification pattern: 20-mer; Gapmer; 5-10-5, PO from the 2nd base from the 5' end, PO from the 3rd base from the 3' end; Fully PS; 2' MOE. In some embodiments, the isolated nucleic acid (e.g., RNA processing modulator, such as an antisense oligonucleotide) comprises the nucleic acid sequence set forth in SEQ ID NO: 292 and the following modification pattern: 20-mer; Gapmer; 5-10-5, PO from the 2nd base from the 5' end, PO from the 3rd base from the 3' end; Fully PS; 2' MOE.In some embodiments, the isolated nucleic acid (e.g., RNA processing modulator, such as an antisense oligonucleotide) comprises the nucleic acid sequence set forth in SEQ ID NO: 293 and the following modification pattern: 20mer; Gapmer; 5-10-5, PO from the 2nd base from the 5' end, PO from the 3rd base from the 3' end; Fully PS; 2' MOE. In some embodiments, the isolated nucleic acid (e.g., RNA processing modulator, such as an antisense oligonucleotide) comprises the nucleic acid sequence set forth in SEQ ID NO: 294 and the following modification pattern: 20mer; Gapmer; 5-10-5, PO from the 2nd base from the 5' end, PO from the 3rd base from the 3' end; Fully PS; 2' MOE. In some embodiments, the isolated nucleic acid (e.g., RNA processing modulator, such as an antisense oligonucleotide) comprises the nucleic acid sequence set forth in SEQ ID NO: 295 and the following modification pattern: 20mer; Gapmer; 5-10-5, PO from the 2nd base from the 5' end, PO from the 3rd base from the 3' end; Fully PS; 2' MOE. In some embodiments, the isolated nucleic acid (e.g., RNA processing modulator, such as an antisense oligonucleotide) comprises the nucleic acid sequence set forth in SEQ ID NO: 296 and the following modification pattern: 20mer; Gapmer; 5-10-5, PO from the 2nd base from the 5' end, PO from the 3rd base from the 3' end; Fully PS; 2' MOE. In some embodiments, the isolated nucleic acid (e.g., RNA processing modulator, such as an antisense oligonucleotide) comprises the nucleic acid sequence set forth in SEQ ID NO: 297 and the following modification pattern: 20mer; Gapmer; 5-10-5, PO from the 2nd base from the 5' end, PO from the 3rd base from the 3' end; Fully PS; 2' MOE. In some embodiments, the isolated nucleic acid (e.g., RNA processing modulator, such as an antisense oligonucleotide) comprises the nucleic acid sequence set forth in SEQ ID NO: 298 and the following modification pattern: 20mer; Gapmer; 5-10-5, PO from the 2nd base from the 5' end, PO from the 3rd base from the 3' end; Fully PS; 2' MOE. In some embodiments, the isolated nucleic acid (e.g., RNA processing modulator, such as an antisense oligonucleotide) comprises the nucleic acid sequence set forth in SEQ ID NO: 299 and the following modification pattern: 20mer; Gapmer; 5-10-5, PO from the 2nd base from the 5' end, PO from the 3rd base from the 3' end; Fully PS; 2' MOE.In any of the foregoing embodiments: positions comprising a "T" residue comprise a thymine (T) nitrogenous base bonded to the 1' carbon of a ribose or deoxyribose sugar; an isolated nucleic acid comprising a gapmer structure is indicated by a structure denoted as "(X)-(Y)-(X)", wherein "(X)" refers to a region comprising 'X' number of nucleotide positions having ribose, and which is flanked by a region, denoted as "(Y)", comprising 'Y' number of nucleotide positions having deoxyribose; an isolated nucleic acid comprising a skipmer structure (or also referred to herein as a skipper) is indicated by the absence of the notation as "(X)-(Y)-(X)", and each nucleotide position comprises ribose; "fully PS" refers to each nucleotide position connected by an internucleotide linkage comprising a phosphorothioate modification, unless otherwise indicated, except that the 3' carbon of the ribose comprises a phosphodiester group (PO); "2' MOE" refers to the 2' carbon of a ribose sugar bonded to an oxygen atom which is bonded to a methoxyethyl group; "#mer" refers to the number of nucleotide positions; and "PO" refers to a nucleotide position in which the 3' carbon of the ribose comprises a phosphodiester bond connecting the nucleotide position to the nucleotide position immediately adjacent in the 3' direction, wherein a nucleotide position comprising a PO group at the 3' carbon of the ribose (e.g., a position referred to as the 2nd or 3rd position from the 5' end or 3' end) is indicated by the position of the PO group, which can be referred to as "positioned at" or "following".

[0132] In some embodiments, the RPM (e.g., antisense oligonucleotide) comprises a structure set forth in Formula (I):

[0133] Formula (I)

[0134] Pharmaceutical compositions

[0135] In some embodiments of the disclosure, RNA processing modulators (e.g., antisense oligonucleotides) are assembled into compositions for therapeutic purposes. In some embodiments, compositions are designed to enhance the therapeutic effect of the RNA processing modulators, for example, by increasing biocompatibility, targeting the RNA processing modulators to a site of interest in the body, decreasing in vivo clearance of the isolated nucleic acid (e.g., antisense oligonucleotide), increasing in vivo stability of the isolated nucleic acid (e.g., antisense oligonucleotide), increasing uptake of the isolated nucleic acid (e.g., antisense oligonucleotide) into a target cell, or amplifying the in vivo expected effect of the isolated nucleic acid (e.g., antisense oligonucleotide).

[0136] In some embodiments, the RNA processing modulator (e.g., antisense oligonucleotide) is provided in combination with a pharmaceutically acceptable carrier. By "pharmaceutically acceptable carrier" is meant a pharmaceutically acceptable material, composition or carrier, such as a liquid or solid filler, stabilizer, dispersing agent, suspending agent, diluent, excipient, thickening agent, solvent or encapsulating material, involved in carrying or transporting the compounds useful within the application into or around the patient's body so that the compounds can perform their intended function. Additional ingredients which can be included in the pharmaceutical compositions used in the practice of the application are known in the art and described, for example, in Remington's Pharmaceutical Sciences (Genaro, Ed., Mack Publishing Co., 1985, Easton, PA), which is incorporated herein by reference.

[0137] In some embodiments, the composition comprising an RNA processing modulator (e.g., an antisense oligonucleotide) comprises a sterile artificial cerebrospinal fluid (CSF). In some embodiments, the artificial CSF comprises a pH of about 6.8 ± 7.8. In some embodiments, the pH is 6.9 to 7.7, 7.0 to 7.6, or 7.1 to 7.5. In some embodiments, the artificial CSF comprises a sodium phosphate buffer. In some embodiments, the concentration of the sodium phosphate buffer is 0.5 mM to 2.0 mM, such as 0.5-0.75 mM, 0.75-1.0 mM, 1.25-1.5 mM, 1.5-1.75 mM, or 1.75-2.0 mM. In some embodiments, the sodium phosphate buffer comprises a pH of about 6.90, 6.95, 7.00, 7.05, 7.10, 7.15, 7.20, 7.25, or 7.30. In some embodiments, the artificial CSF comprises sodium chloride. In some embodiments, the concentration of the sodium chloride is 50 mM to 300 mM, such as 75 mM to 250 mM, 100 mM to 225 mM, or 125 mM to 200 mM. In some embodiments, the artificial CSF comprises potassium chloride. In some embodiments, the concentration of the potassium chloride is 0.5 mM to 5 mM, such as 1 mM to 5 mM, or 2 mM to 5 mM. In some embodiments, the artificial CSF comprises calcium chloride dihydrate. In some embodiments, the concentration of the calcium chloride dihydrate is 1.0 mM to 2.0 mM, such as 1.1 mM to 1.9 mM, 1.2 mM to 1.8 mM, or 1.3 mM to 1.7 mM. In some embodiments, the artificial CSF comprises magnesium chloride hexahydrate. In some embodiments, the concentration of the magnesium chloride hexahydrate is 0.1 mM to 1.5 mM, such as 0.3 mM to 1.2 mM, 0.4 mM to 1.1 mM, or 0.5 mM to 1.0 mM. In some embodiments, the artificial CSF is a sterile aqueous solution comprising any combination of sodium phosphate buffer, sodium chloride, potassium chloride, calcium chloride dihydrate, and magnesium chloride hexahydrate.

[0138] In some embodiments, the composition comprising an RNA processing modulator (e.g., an antisense oligonucleotide) comprises a total volume of about 5 mL to about 30 mL. In some embodiments, the total volume is about 10 mL, 15 mL, 20 mL, or about 25 mL. In some embodiments, the total volume of the composition is contained in a vial, such as a glass vial. In some embodiments, the composition is prepared under sterile processing using sterile filtration and filling.

[0139] The present disclosure also provides compositions comprising an RNA processing modulator (e.g., an antisense oligonucleotide) at a concentration of about 1 mg / mL to 20 mg / mL. In some embodiments, the concentration of the RNA processing modulator (e.g., antisense oligonucleotide) in the composition is 1 mg / mL to 15 mg / mL, 3 mg / mL to 15 mg / mL, or 5 mg / mL to 15 mg / mL. In some embodiments, the concentration of the RNA processing modulator (e.g., antisense oligonucleotide) in the composition is greater than 5 mg / mL and less than 10 mg / mL.

[0140] Methods and medical uses

[0141] Aspects of the present disclosure relate to methods of modulating the transcription, splicing, translation, function, or activity of a gene associated with L-serine biosynthesis or lipid biosynthesis (e.g., sphingolipid biosynthesis) in a cell or subject. Also provided are RNA processing modulators (e.g., antisense oligonucleotides) described herein for use as a medicament. The RNA processing modulators (e.g., antisense oligonucleotides) can be used in methods of modulating, for example, the transcription, translation, function, or activity of a gene associated with L-serine biosynthesis or lipid biosynthesis (e.g., sphingolipid biosynthesis) in a cell or subject. In some embodiments, the method comprises administering to the cell or subject a composition comprising one or more RNA processing modulators (e.g., 1, 2, 3, 4, 5, or more RNA processing modulators, e.g., 1, 2, 3, 4, 5, or more antisense oligonucleotides). In some embodiments, the administration of the composition (e.g., RNA processing modulator) results in an alteration of lipid (e.g., sphingolipid) biosynthesis in the cell or subject. The cell is in vitro, in vivo, or ex vivo.

[0142] For example, in some embodiments, the administration of an RNA processing modulator (e.g., antisense oligonucleotide) targeting SPTLC1 mRNA results in an increase in the production of sphingolipids (which utilize L-serine as a precursor molecule) in the cell or subject. For example, in some embodiments, the administration of an RNA processing modulator (e.g., antisense oligonucleotide) targeting SPTLC1 mRNA results in an increase in the production of deoxy-sphingolipids (which result from mutations in certain genes involved in L-serine biosynthesis and / or lipid biosynthesis, e.g., SPTLC1) in the cell or subject. The present disclosure is based, in part, on the recognition that, in some embodiments, contacting a cell or subject with an RNA processing modulator that decreases the level, transcription, splicing, translation, function, or activity of SPTLC1 protein results in decreased deoxy-sphingolipid production in the subject.

[0143] Accordingly, in some aspects, the present disclosure provides methods for increasing sphingolipid biosynthesis in a cell or subject, the method comprising administering to a subject in need thereof an isolated nucleic acid as described herein. In some embodiments, the isolated nucleic acid comprises an antisense oligonucleotide comprising a sequence set forth in any one of SEQ ID NOs: 1-299 (provided in Column A of Table 1, optionally which comprises one or more modifications in Column C of Table 1, and optionally wherein the sequence in Column A and the chemistry in Column C are provided in the same row of Table 1).

[0144] Generally, it is desirable to increase sphingolipid levels (or decrease deoxysphingolipids) in certain subjects, e.g., subjects having low serine, e.g., subjects having retinal disease (e.g., MacTel2 and age-related macular degeneration (AMD), such as AMD with geographic atrophy), HSAN1 (e.g., HSAN1(a)), diabetic retinopathy, peripheral neuropathy (e.g., motor neuropathy, sensory neuropathy, autonomic neuropathy, combined neuropathy, inherited or acquired peripheral neuropathy, such as diabetic neuropathy, chemotherapy-induced neuropathy, or other forms of toxic peripheral neuropathy, etc.), neurodegeneration (e.g., Parkinson’s disease, Friedreich’s ataxia, motor neuron disease, Alzheimer’s disease, amyotrophic lateral sclerosis (ALS), etc.), spastic paraplegia, or psychiatric disorders (e.g., resistant depression, refractory depression, schizophrenia, and / or refractory schizophrenia). However, in some embodiments, it is understood that the present disclosure provides methods for decreasing sphingolipid biosynthesis (or increasing deoxo-sphingolipid biosynthesis) in a cell or subject, the method comprising administering to a subject in need thereof an isolated nucleic acid as described herein. In some embodiments, the isolated nucleic acid comprises an antisense oligonucleotide comprising a sequence set forth in any one of SEQ ID NOs: 1-299 (provided in Column A of Table 1, optionally which comprises one or more modifications in Column C of Table 1, and optionally wherein the sequence in Column A and the chemistry in Column C are provided in the same row of Table 1).

[0145] In some aspects, the RNA processing modulators described herein (e.g., antisense oligonucleotides) can be used to treat a disease or disorder associated with dysregulation of L-serine biosynthesis. Accordingly, provided herein are RNA processing modulators described herein (e.g., antisense oligonucleotides) for use in a method of treating a disease or disorder associated with dysregulation of L-serine biosynthesis. A disease or disorder associated with dysregulation of L-serine biosynthesis refers to a disease or disorder in which a subject (e.g., a patient) 1) is characterized as having low serine, and / or 2) has one or more mutations in one or more genes associated with L-serine biosynthesis, and / or 3) has one or more mutations in one or more genes involved in metabolic pathways that utilize serine (e.g., sphingolipid biosynthesis, which is dependent on SPTLC1). A disease or disorder associated with dysregulation of L-serine biosynthesis refers to a disease or disorder in which a subject (e.g., a patient) is characterized as having one or more mutations in one or more genes associated with lipid (e.g., sphingolipid) biosynthesis (e.g., SPTLC1), or as having low serine that leads to increased production of toxic deoxysphingolipids.

[0146] Methods of measuring serine levels in a subject are known in the art, e.g., as described by Méneret et al. Arch Neurol. 2012; 69(7):908-911. In some embodiments, the L-serine level of a subject is determined by measuring the concentration of L-serine in a biological sample (e.g., a blood sample, a serum sample, a cerebrospinal fluid (CSF) sample, etc.) obtained from the subject. In some embodiments, a subject having “low serine” refers to a subject having a plasma serine level of less than 0.66 mg / dL and / or a cerebrospinal fluid (CSF) serine level of less than 0.21 mg / dL.

[0147] Examples of genes associated with L-serine biosynthesis include PHGDH, phosphoserine transaminase (PSTAT1), and phosphoserine phosphatase (PSPH). In some embodiments, a subject having a disease or disorder associated with dysregulation of L-serine biosynthesis comprises one or more mutations in a gene selected from PHGDH, PSTAT1, and PSPH. In some embodiments, the subject has one or more mutations in the PHGDH gene. In some embodiments, a subject having one or more mutations in the PHGDH gene has MacTel2 (or is at risk of developing MacTel2). Methods of detecting mutations in a subject’s genes are known in the art and include, e.g., DNA sequencing, RNA sequencing, microarray analysis, etc.

[0148] In some embodiments, the subject has a disease or disorder associated with dysregulation of lipid (e.g., sphingolipid) biosynthesis and comprises one or more mutations in one or more genes involved in metabolic pathways that utilize serine (e.g., L-serine or D-serine). For example, L-serine is used to produce sphingolipids through condensation of L-serine with palmitoyl-CoA by serine palmitoyltransferase (SPT). In humans, one subunit of SPT is provided by the SPTLC1 gene, which subunit implements a self-regulatory role in the enzymatic activity of SPT. Autosomal dominant mutations in SPTLC1 lead to atypical production of sphingolipids and are associated with hereditary sensory neuropathy (HSAN1) (including HSAN1(a)) caused by condensation of palmitoyl-CoA with alanine to form cytotoxic deoxysphingolipids. HSAN1 leads to peripheral neuropathy (e.g., motor neuropathy, sensory neuropathy, autonomic neuropathy, combined neuropathy, inherited or acquired peripheral neuropathy, such as diabetic neuropathy, chemotherapy-induced neuropathy, or other forms of toxic peripheral neuropathy, etc.), in some cases, manifesting as severe loss of sensation to temperature, pressure, and pain. In some cases, long-term cases of HSAN1 lead to painless injuries, chronic skin ulcers, bone destruction, bone infection, finger / toe amputations, and motor dysfunction. In some embodiments, subjects having one or more mutations in genes encoding SPT subunits can benefit from administration of RNA processing modulators that increase transcription, translation, function, or activity of SPTLC1. Without wishing to be bound by any particular theory, increased levels or activity of SPTLC1 can lead to restoration of appropriate sphingolipid:deoxysphingolipid levels in the subject. Measurement of sphingolipid and deoxysphingolipid levels in a subject is known, e.g., as described by Johnson et al. Nature Communications volume 11, Article number: 2471 (2020), Weiss et al. Hum Mol Genet. 2021 Feb 25;29(24):3945-3953. doi: 10.1093 / hmg / ddaa248, and Clark et al. (2020) FEBS Lett, 594:3579-3582.

[0149] Deoxysphingolipid production due to reduced SPT activity and subsequent metabolic shift to alanine utilization over L-serine is observed in other diseases and disorders. In some cases, diabetic neuropathy caused by type 2 diabetes is associated with excess production of deoxysphingolipids. In some cases, amyotrophic lateral sclerosis (ALS) is associated with excess production of deoxysphingolipids.

[0150] Accordingly, in some aspects, the present disclosure provides methods for treating a disease or disorder associated with dysregulation of lipid (e.g., sphingolipid) biosynthesis, the method comprising administering an isolated nucleic acid as described herein to a subject in need thereof. Also provided are RNA processing modulators (e.g., antisense oligonucleotides) described by the present disclosure for use in a method of treating a disease or disorder associated with dysregulation of lipid (e.g., sphingolipid) biosynthesis. The method can comprise administering an isolated nucleic acid as described herein to a subject in need thereof. In some embodiments, the isolated nucleic acid comprises an antisense oligonucleotide comprising a sequence set forth in any one of SEQ ID NOs: 1-299 (provided in column A of Table 1, optionally which comprises one or more modifications in column C of Table 1, and optionally wherein the sequence in column A and the chemistry in column C are provided in the same row of Table 1). In some embodiments, the disease is selected from retinal diseases (e.g., MacTel2 and age-related macular degeneration (AMD), such as AMD with geographic atrophy), HSAN1 (e.g., HSAN1(a)), diabetic retinopathy, peripheral neuropathy (e.g., motor neuropathy, sensory neuropathy, autonomic neuropathy, combined neuropathy, inherited or acquired peripheral neuropathy, such as diabetic neuropathy, chemotherapy-induced neuropathy, or other forms of toxic peripheral neuropathy, etc.), neurodegeneration (e.g., Parkinson’s disease, Friedreich’s ataxia, motor neuron disease, Alzheimer’s disease, amyotrophic lateral sclerosis (ALS), etc.), spastic paraplegia, or psychiatric disorders (e.g., treatment-resistant depression, treatment-refractory depression, schizophrenia, and / or treatment-refractory schizophrenia).

[0151] As used herein, “treat” or “treating” means preventing or delaying the onset of a disease, reducing or preventing the development of symptoms associated with a disease, reducing the severity of a disease, and / or preventing the worsening of symptoms associated with a disease. Thus, in some aspects, the present disclosure provides methods for treating a subject having or suspected of having a disease caused by dysregulated L-serine biosynthesis or dysregulated lipid biosynthesis (e.g., sphingolipid biosynthesis). The treatment of a subject involves administering a composition (e.g., an RNA processing modulator, such as an antisense oligonucleotide) as described herein to the subject.

[0152] As used herein, the term “treatment” means the application or administration of a composition (e.g., an RNA processing modulator, such as an antisense oligonucleotide as described herein) to a subject having a disease or disorder associated with low levels of serine or with dysregulation of L-serine biosynthesis or with dysregulation of lipid biosynthesis, with the purpose to heal, relieve, alleviate, lessen, change, remedy, improve, or affect symptoms of a disorder associated with dysregulation of L-serine biosynthesis, a disease, or a predisposition toward the disease.

[0153] Alleviating a disease associated with L-serine biosynthesis dysregulation includes delaying the development or progression of the disease, or reducing the severity of the disease. Alleviating a disease does not necessarily require a curative outcome. As used herein, "delaying" the development of a disease (such as a disease associated with L-serine biosynthesis dysregulation or a disease associated with lipid biosynthesis dysregulation) means to defer, hinder, slow, retard, stabilize, and / or postpone the progression of the disease. This delay can be of varying lengths, depending on the history of the disease and / or the individual being treated. A method that "delays" or alleviates the development of a disease or delays the onset of a disease is a method that reduces the probability of developing one or more symptoms of the disease within a given time frame and / or reduces the extent of symptoms within a given time frame when compared to not using the method. Such comparisons are typically based on clinical studies using a large number of subjects sufficient to give statistically significant results.

[0154] "Development" or "progression" of a disease means the initial presentation and / or subsequent progression of the disease. The development of a disease can be detected and assessed using standard clinical techniques well known in the art. However, development also refers to progression that can not be detectable. For purposes of the present disclosure, development or progression refers to the biological process of symptoms. "Development" includes occurrence, recurrence, and onset. As used herein, "onset" or "occurrence" of a disease associated with low serine levels and / or L-serine biosynthesis dysregulation and / or lipid biosynthesis dysregulation.

[0155] A subject can be a human, mouse, rat, pig, dog, cat, or non-human primate. In some embodiments, a subject has or is suspected of having a disease or disorder associated with low serine levels and / or L-serine biosynthesis dysregulation and / or lipid biosynthesis dysregulation. In some embodiments, a subject having a disease or disorder associated with low serine levels and / or L-serine biosynthesis dysregulation and / or lipid biosynthesis dysregulation comprises at least one SPTLC1 allele having a mutation, such as a loss-of-function mutation, or a mutation that alters the substrate specificity of the SPT protein comprising a mutant SPTLC1 subunit to have an alanine preference over serine. In some embodiments, the SPTLC1 allele having a mutation comprises a frameshift mutation, a splice site mutation, a missense mutation, a truncation mutation, or a nonsense mutation. A subject can have two SPTLC1 alleles with the same mutation (homozygous state) or two SPTLC1 alleles with different mutations (compound heterozygous state).

[0156] The optimal process for administration or delivery of the RNA processing modulators (e.g., antisense oligonucleotides) of the present disclosure can vary depending on the desired outcome and / or the subject to be treated. As used herein, “administration” refers to contacting a cell with the RNA processing modulator and can be performed in vitro or in vivo. The compositions (e.g., pharmaceutical compositions) provided herein can be administered by a variety of routes, including, but not limited to, by oral administration, intravenous administration (e.g., systemic intravenous injection / administration), administration to the brain and / or spinal cord, intracerebral injection, intraventricular injection, intracisternal injection, intraparenchymal injection, intrathecal injection, and any combination of the foregoing. In some embodiments, administration includes administration to the cerebrospinal fluid and / or direct administration to the affected site (e.g., a target tissue, such as an ocular tissue, a central nervous system (CNS) tissue, or a peripheral nervous system (PNS) tissue).

[0157] Generally, the most appropriate route of administration will depend on a variety of factors, including the nature of the agent (e.g., its stability in the gastrointestinal environment) and / or the condition of the subject (e.g., whether the subject can tolerate oral administration, injection, etc.). In certain embodiments, the compounds or pharmaceutical compositions described herein are suitable for topical administration to the eye of a subject. In some embodiments, administration (e.g., injection) of the compounds or pharmaceutical compositions is performed on a patient in a head-low feet-high position. In some embodiments, the composition is administered to the subject by only one route of administration. In some embodiments, multiple routes of administration (e.g., serially or simultaneously) can be utilized for administering the composition to the subject.

[0158] In some embodiments, it can be desirable to deliver the RNA processing modulators (e.g., antisense oligonucleotides) of the disclosure to the CNS of a subject. By “CNS” is meant all cells and tissues of the vertebrate brain and spinal cord. Thus, this term includes, but is not limited to, neuronal cells, neuroglial cells, astrocytes, cerebrospinal fluid (CSF), interstitial space, bone, cartilage, etc. The RNA processing modulators (e.g., antisense oligonucleotides) of the disclosure can be delivered directly to the CNS or brain by using neurosurgical techniques known in the art, such as by stereotactic injection, with a needle, catheter, or related device, into, for example, the ventricular region as well as the striatum (e.g., the caudate or putamen of the striatum), the spinal cord, and neuromuscular junctions or cerebellar lobes (see, e.g., Stein et al., J Virol 73:3424-3429, 1999; Davidson et al., PNAS 97:3428-3432, 2000; Davidson et al., Nat. Genet. 3:219-223, 1993; and Alisky and Davidson, Hum. Gene Ther. 11:2315-2329, 2000). In some embodiments, the RNA processing modulators (e.g., antisense oligonucleotides) of the disclosure are administered by intravenous injection. In some embodiments, the RNA processing modulators (e.g., antisense oligonucleotides) of the disclosure are administered by intracerebral injection. In some embodiments, the RNA processing modulators (e.g., antisense oligonucleotides) of the disclosure are administered by intracerebroventricular injection. In some embodiments, the RNA processing modulators (e.g., antisense oligonucleotides) of the disclosure are administered by intrathecal injection. In some embodiments, the RNA processing modulators (e.g., antisense oligonucleotides) of the disclosure are administered by intralumbar injection. In some embodiments, the RNA processing modulators (e.g., antisense oligonucleotides) of the disclosure are administered by intrastriatal injection. In some embodiments, the RNA processing modulators (e.g., antisense oligonucleotides) of the disclosure are delivered by intracranial injection. In some embodiments, the RNA processing modulators (e.g., antisense oligonucleotides) of the disclosure are delivered by cisterna magna injection. In some embodiments, the RNA processing modulators (e.g., antisense oligonucleotides) of the disclosure are delivered by lateral cerebroventricular injection. One of skill in the art will also recognize that the foregoing routes of administration can be combined in a single subject (e.g., a subject can be administered the RNA processing modulators (e.g., antisense oligonucleotides) of the disclosure using a combination of two or more of the foregoing techniques).

[0159] In some embodiments, administration of the RNA processing modulator (e.g., antisense oligonucleotide) of the present disclosure results in delivery of the RNA processing modulator (e.g., antisense oligonucleotide) to ocular tissue. Delivery of the RNA processing modulator (e.g., antisense oligonucleotide) to a mammalian subject can be by, for example, intraocular injection, subretinal injection, topical administration (e.g., eye drops), or by injection into the eye of the mammalian subject to ocular tissue (e.g., intravitreal injection). As used herein, “ocular tissue” refers to any tissue derived from or contained in the eye. Non-limiting examples of ocular tissue include neurons, retina (e.g., photoreceptor cells), sclera, choroid, retina, vitreous, macula, fovea, optic disc, lens, pupil, iris, aqueous fluid, cornea (e.g., keratocytes, corneal endothelial cells, corneal basal cells, corneal wing cells, and corneal squamous cells), conjunctiva ciliary body, and optic nerve. The retina is located at the back of the eye and contains photoreceptor cells. These photoreceptor cells (e.g., rod cells, cone cells) confer vision by distinguishing color as well as contrast in the visual field.

[0160] In some embodiments, an effective amount (e.g., an amount sufficient to increase transcription, translation, function, or activity of a target mRNA) is administered to a subject. In some embodiments, an effective amount of the RNA processing modulator (e.g., antisense oligonucleotide) is an amount sufficient to increase transcription, translation, function, or activity of a target mRNA. In some embodiments, an effective amount of the RNA processing modulator (e.g., antisense oligonucleotide) is an amount sufficient to decrease transcription, translation, function, or activity of a target mRNA. Effective amounts will depend primarily on factors such as the species, age, weight, health, and the tissue to be targeted of the subject, and thus can vary from animal to animal and tissue to tissue.

[0161] In some embodiments, an effective amount (e.g., an amount sufficient to increase transcription, translation, function, or activity of a target mRNA or an amount sufficient to decrease transcription, translation, function, or activity of a target mRNA) is 1 ng - 100 mg. In some embodiments, an effective amount (e.g., an amount sufficient to increase transcription, translation, function, or activity of a target mRNA or an amount sufficient to decrease transcription, translation, function, or activity of a target mRNA) is 1 - 1000 ng. In some embodiments, an effective amount (e.g., an amount sufficient to increase transcription, translation, function, or activity of a target mRNA or an amount sufficient to decrease transcription, translation, function, or activity of a target mRNA) is 1 - 10, 10 - 50, 50 - 100, 100 - 200, 200 - 300, 300 - 500, 500 - 750, or 750 - 1000 ng. In some embodiments, an effective amount (e.g., an amount sufficient to increase transcription, translation, function, or activity of a target mRNA or an amount sufficient to decrease transcription, translation, function, or activity of a target mRNA) is 0.1 pg - 100.0 pg. In some embodiments, an effective amount (e.g., an amount sufficient to increase transcription, translation, function, or activity of a target mRNA or an amount sufficient to decrease transcription, translation, function, or activity of a target mRNA) is 0.1 - 1.0, 1.0 - 5.0, 5.0 - 20.0, 20.0 - 50.0, or 50.0 - 100.0 pg. In some embodiments, an effective amount (e.g., an amount sufficient to increase transcription, translation, function, or activity of a target mRNA or an amount sufficient to decrease transcription, translation, function, or activity of a target mRNA) is 1 pg - 1000 pg. In some embodiments, an effective amount (e.g., an amount sufficient to increase transcription, translation, function, or activity of a target mRNA) is 100 - 250, 250 - 500, 500 - 750, or 750 - 1000 pg. In some embodiments, an effective amount (e.g., an amount sufficient to increase transcription, translation, function, or activity of a target mRNA or an amount sufficient to decrease transcription, translation, function, or activity of a target mRNA) is 0.1 - 1.0, 1.0 - 5.0, 5.0 - 20.0, 20.0 - 50.0, or 50.0 - 100.0 mg. In some embodiments, an effective dose is administered from a composition containing an RNA processing modulator (e.g., an antisense oligonucleotide) at a concentration of about 1 mg / mL to 20 mg / mL. In some embodiments, the concentration of the RNA processing modulator (e.g., an antisense oligonucleotide) in the composition is 1 mg / mL to 15 mg / mL, 3 mg / mL to 15 mg / mL, or 5 mg / mL to 15 mg / mL. In some embodiments, the concentration of the RNA processing modulator (e.g., an antisense oligonucleotide) in the composition is greater than 5 mg / mL and less than 10 mg / mL.

[0162] In some embodiments, the effective amount (e.g., an amount sufficient to increase transcription, translation, function, or activity of a target mRNA or an amount sufficient to decrease transcription, translation, function, or activity of a target mRNA) is 0.1-1.0, 1.0-20.0, 20.0-50.0, 50.0-200.0, or 200.0-500.0 mg. In some embodiments, an effective dose is administered from a composition containing an RNA processing modulator (e.g., an antisense oligonucleotide) at a concentration of about 1 mg / mL to 50 mg / mL. In some embodiments, the concentration of the RNA processing modulator (e.g., an antisense oligonucleotide) in the composition is 1 mg / mL to 45 mg / mL, 10 mg / mL to 45 mg / mL, or 15 mg / mL to 45 mg / mL.

[0163] During the course of treatment, administration of the composition can be altered or adjusted accordingly. For example, expression of a protein encoded by a nucleic acid targeted by the isolated nucleic acid of the pharmaceutical composition can be monitored to give information on the method of use of the composition. For example, expression information can be obtained by measuring changes in the level of a protein or RNA product of the target nucleic acid. Alternatively, sequencing analysis of the target nucleic acid can be used to determine whether expression changes include alterations in the structure or sequence of the protein or RNA product of the target nucleic acid sequence.

[0164] The amount of the composition will vary depending on a number of factors such as, but not limited to, the clinical characteristics of the subject (e.g., disease severity, rate of disease progression, physical characteristics, etc.) and the mode of administration. Thus, in certain instances, the composition can be administered to a single subject one or more than one time. In certain instances, the composition can be administered to the same subject by different modes or routes at different times during the course of treatment. In some embodiments, the composition is administered to the subject more than one time. In some embodiments, the composition is administered to the subject two, three, four, or more times. In some embodiments, the composition is administered to the subject less than twelve times over a 12 month period. In some embodiments, the time between each administration of the composition is days, weeks, or months. In some embodiments, the time between each administration is 1-2 weeks, 2-4 weeks, or 1-4 months. In some embodiments, the time between each administration of the composition is the same. In other embodiments, the time between each administration of the composition can be different. In some embodiments, the time between the first administration of the composition and the second administration of the composition is 1-2 weeks. In some embodiments, the time between the second administration of the composition and the third administration of the composition is 1-3 months.

[0165] Examples

[0166] Example 1: RNA Processing Modulators (RPMs)

[0167] This example describes the use of RNA processing modulators (RPMs) to modulate the translation of one or more mRNA transcripts in a cell or subject. RPMs act by binding to a target-specific mRNA sequence and altering (e.g., upregulating or downregulating) protein translation of the mRNA sequence.

[0168] In some embodiments, the RPM is an antisense oligonucleotide (ASO). Antisense oligonucleotides (ASOs) typically range in length from about 10 to 30 nucleotides and can comprise a non-natural sugar-phosphate backbone (e.g., phosphorodiamidate morpholino backbone, phosphorothioate backbone, etc.) and / or one or more modified sugar moieties (e.g., 2’-O-methoxyethylribose (2’-O-MOE) modification, etc.).

[0169] In some embodiments, the RPM (e.g., ASO) targets a structural element of the mRNA transcript, such as an untranslated region (UTR), to modulate expression of the target (e.g., the target gene encoding the mRNA transcript) by increasing or decreasing the transcription and / or translation of the protein encoded by the mRNA transcript (or alternatively referred to as up- or down-regulating expression). In another example, the RPM (e.g., ASO) can target a regulatory region of the UTR region (and thus interfere with protein binding, such as ribosomal protein binding) to up- or down-regulate expression of the target. Alternatively, the RPM (e.g., ASO) can target a splice site (e.g., a splice acceptor site or a splice donor site in the UTR region or one or more nucleotide positions thereof) to up- or down-regulate expression of the target (and thus generate a new protein variant). Other examples of structural elements that can be targeted by the RPM (e.g., ASO) include, but are not limited to: an intronic regulatory site, an exonic regulatory site, an exon-intron boundary, an antisense binding site of the target mRNA transcript, a long non-coding RNA (LncRNA) binding site of the target gene, and a retained exon of a canonical mRNA.

[0170] Non-limiting examples of ASOs targeting various structural elements of mRNA are provided in Figure 1The compositions are shown in FIG. 1. Composition "A" represents an ASO that binds to the 5' untranslated region (5' UTR) of the RNA. Composition "B" represents an ASO that binds to an intron of the RNA. Composition "C" represents an ASO that binds to the splice boundary (e.g., splice junction) between an exon and an intron of the RNA. Composition "D" represents an ASO that binds to an exon (e.g., protein coding region) of the RNA. Composition "E" represents a combination of ASOs that bind to the 3' UTR of the RNA (alone or with a trans- regulator). Composition "F" represents a "gapmer" ASO that binds to an exon (e.g., protein coding region) of the RNA and mediates RNase H decay. Composition "G" represents a "gapmer" ASO that binds to the 3' UTR of the RNA and mediates RNase H decay (alone or with a trans-regulator). In some embodiments, the ASO that binds to the RNA results in the translation of a truncated protein that has a dominant negative effect on the wild-type full-length protein.

[0171] Example 2: L-Serine and Lipid Biosynthesis

[0172] This example describes diseases and disorders associated with L-serine biosynthesis, particularly diseases and disorders associated with dysregulation of L-serine biosynthesis in the central nervous system (CNS).

[0173] L-serine is generally classified as a non-essential amino acid. However, in the CNS, an external supply of L-serine is required for the synthesis of sphingolipids and phosphatidylserine (PS) and for the survival of neurons. In astrocytes, L-serine is generally synthesized from metabolized glucose by several enzymes, including phosphoglycerate dehydrogenase (PHGDH). L-serine then interacts with serine palmitoyltransferase (SPT) to produce certain lipids (e.g., sphingolipids) that are exported to neurons for cell membrane biosynthesis. L-serine also interacts with serine racemase to produce D-serine, which is exported to neurons. Dysregulation of L-serine biosynthesis (e.g., caused by mutations in PHGDH or SPTLC1, a gene encoding a SPT subunit) has been shown to result in the accumulation of toxic deoxy-sphingolipids (deoxy-SLs), which can cause inflammation and is further associated with certain CNS diseases and disorders.

[0174] Extensive lipidomic profiling is useful in pointing to biomarkers associated with dysregulation of L-serine synthesis and can provide a means for detecting disease states associated with problems with L-serine biosynthesis.

[0175] Recent data suggest that PHGDH loss-of-function mutations (e.g., haploinsufficiency) and variants in the context of macular capillary telangiectasia type 2 (MacTel2) are an “allelic series” that links reduced enzyme levels / activity to disease impact. Notably, even MacTel2 patients without severe PHGDH mutations have been observed to have low L-serine levels. These low serine levels have been observed to lead to high deoxy-SL through the established SPT pathway. In short, in the presence of low serine levels, the SPT enzymes are left to utilize alanine instead of serine to make complex sphingolipids (SLs), which leads to the production of deoxy-SLs. In some embodiments, administration of an RNA processing modulator (RPM) described herein (e.g., an antisense oligonucleotide) leads to translation of an SPTLC1 protein isoform that prefers serine over alanine, even in the presence of low serine in the subject.

[0176] Interestingly, SPT mutations have also been observed to cause MacTel2. For example, dominant mutations in the SPT genes SPTLC1 or SPTLC2 have been observed to cause preferential use of alanine and also to cause rare familial MacTel2. Low serine has also been associated with oxidative stress. In this regard, serine biosynthesis is an important antioxidant in the macula of the eye, and reduced PHGDH function leads to oxidative stress and reduced glutathione. In addition, SPT mutations have also been observed to cause hereditary sensory neuropathy type 1 (HSAN1), including HSAN1(a), and a subset of these HSAN1 patients have both diseases. Certain gain-of-function mutations in SPTLC1, such as SPTLC1 C133W, reduce substrate selectivity and lead to the production of deoxy-sphingolipids. In HSAN1 patients, multiple toxic deoxy-SLs are elevated, such as 1-deoxy sphingodihydrol, 1-deoxysphingol, and 1-deoxynervonate.

[0177] Rare mutations in SPT have been observed to cause early onset ALS and are associated with other neurodegenerative diseases such as Parkinson’s disease and Alzheimer’s disease. Interestingly, SPTLC1 mutations associated with ALS are different from those seen in MacTel2 or HSAN1. SPTLC1 variants associated with ALS are associated with reduced levels of SPTLC1 and ceramides in plasma, cerebrospinal fluid, and spinal cord tissue. Elevated ceramide levels are also observed in tissues such as plasma and cerebrospinal fluid of Parkinson’s disease and Alzheimer’s disease subjects, as well as in other models of these diseases (e.g., patient cells) harboring mutations in SPTLC1 and other SPT subunits. In Alzheimer’s disease patients, plasma ceramide levels are positively correlated with baseline neurofilament light chain (Nfl) plasma levels, and also positively correlated with longitudinal changes in Nfl. Similar to observations made in models of Parkinson’s disease and Alzheimer’s disease, lipid analysis has shown accumulation of ceramides in the general ALS population, which includes ceramides in patient plasma samples. Non-limiting examples of SPTLC1 mutations associated with ALS include SPTLC1 A20S, SPTLC1 Y23F, SPTLC1 L38R, SPTLC1 L39 deletion, SPTLC1 F40 deletion + S41 deletion, SPTLC1 S331F, SPTLC1 S331Y, SPTLC1 exon 2 deletion, and SPTLC1 rs75431233. Different mutations in the SPTLC1 subunit, which increase the canonical activity of the enzyme and lead to over-accumulation of sphingolipids, result in a monogenic form of ALS. In some embodiments, administration of an RNA processing modulator as described herein to a subject having ALS or Alzheimer’s disease results in a decrease in Nfl plasma levels in the subject (e.g., relative to Nfl plasma levels in the subject prior to administration).

[0178] Adult-onset diabetes has also been observed to result in decreased serine levels, which can be due to excessive liver uptake.

[0179] Thus, accumulation of toxic deoxy-SL is observed in several disease indications, and elevated non-deoxy-SL (e.g., dihydroceramides, ceramides, and sphingosine) can play an important role in the pathophysiology of neurodegenerative diseases such as Parkinson’s disease, Alzheimer’s disease, and ALS.

[0180] Dysregulated deoxy-SL production is associated with other conditions. For example, in contrast to the role of dysregulated L-serine synthesis in the development of MacTel2 and HSAN1, upregulation of toxic deoxy-SL can be a therapeutic approach for cancer. For example, inhibition of PHGDH increased the sensitivity of primary cultures of muller glia from cadaveric eyes to oxidative stress conditions. This suggests that increased deoxy-SL is cytotoxic and makes cells sensitive to other stresses. Inhibition of SPT function alone was associated with reduced markers of atherosclerosis.

[0181] Existing drugs that inhibit SPT (e.g., globomycin and reportedly cycloserine) can not be specific enough to mediate therapeutic effects, and globomycin can have some toxicity associated with its use.

[0182] Example 3: Regulation of L-serine and lipid biosynthesis

[0183] In some embodiments, an isolated nucleic acid described herein (e.g., an RNA processing modulator, such as an antisense oligonucleotide) is capable of modulating the level of one or more non-deoxy-SLs (e.g., dihydrosphingosine, sphingosine, and / or ceramide) and / or one or more deoxy-SLs (e.g., deoxydihydrosphingosine and / or deoxyceramide) in a cell, such as a cell of a subject (e.g., a human subject). In some embodiments, modulating the level of one or more non-deoxy-SLs and / or one or more deoxy-SLs comprises using an isolated nucleic acid described herein to modulate the expression of a gene associated with L-serine biosynthesis and / or lipid biosynthesis. In some embodiments, modulating the expression of a gene associated with L-serine biosynthesis and / or lipid biosynthesis comprises using an isolated nucleic acid described herein (e.g., an RNA processing modulator, such as an antisense oligonucleotide) to increase or decrease the transcription and / or translation of mRNA encoded by the gene.

[0184] In some embodiments, the gene associated with L-serine biosynthesis and / or lipid biosynthesis is a mutant SPTLC1 gene. In some embodiments, the mutant SPTLC1 gene comprises one or more nucleotide substitutions, one or more nucleotide insertions, and / or one or more nucleotide deletions relative to a wild-type SPTLC1 gene (or an mRNA encoded by a wild-type SPTLC1 gene, such as a gene comprising the sequence set forth in SEQ ID NO: 300). In some embodiments, the mutant SPTLC1 gene encodes SPTLC1 C133W. In some embodiments, the mutant SPTLC1 gene encodes SPTLC1 A20S. In some embodiments, the mutant SPTLC1 gene encodes SPTLC1 Y23F. In some embodiments, the mutant SPTLC1 gene encodes SPTLC1 L38R. In some embodiments, the mutant SPTLC1 gene encodes SPTLC1 L39 deletion. In some embodiments, the mutant SPTLC1 gene encodes SPTLC1 F40 deletion + S41 deletion. In some embodiments, the mutant SPTLC1 gene encodes SPTLC1 S331F. In some embodiments, the mutant SPTLC1 gene encodes SPTLC1 S331Y. In some embodiments, the mutant SPTLC1 gene comprises SPTLC1 exon 2 deletion. In some embodiments, the mutant SPTLC1 gene comprises SPTLC1 rs75431233.

[0185] In some embodiments, the mutant SPTLC1 gene is associated with a disease or disorder described herein, including but not limited to, a retinal disease (e.g., MacTel2 and age-related macular degeneration (AMD), such as AMD with geographic atrophy), HSAN1 (e.g., HSAN1 (a)), diabetic retinopathy, peripheral neuropathy (e.g., motor neuropathy, sensory neuropathy, autonomic neuropathy, combined neuropathy, inherited or acquired peripheral neuropathy, such as diabetic neuropathy, chemotherapy-induced neuropathy, or other forms of toxic peripheral neuropathy, etc.), neurodegeneration (e.g., Parkinson’s disease, Friedreich’s ataxia, motor neuron disease, Alzheimer’s disease, amyotrophic lateral sclerosis (ALS), etc.), spastic paraplegia, or a psychiatric disorder (e.g., treatment-resistant depression, treatment-resistant depression, schizophrenia, and / or treatment-resistant schizophrenia). In some embodiments, the mutant SPTLC1 gene is associated with Parkinson’s disease, Alzheimer’s disease, ALS, or HSAN1.

[0186] In some embodiments, the mutant SPTLC1 gene comprises one or more nucleotide substitutions, one or more nucleotide insertions, and / or one or more nucleotide deletions relative to the wild-type SPTLC1 gene (or mRNA encoded by the wild-type SPTLC1 gene) that are associated with HSAN1 in the subject. In subjects having a mutated SPTLC1 gene, HSAN-1 can cause peripheral neuropathy (e.g., motor neuropathy, sensory neuropathy, autonomic neuropathy, combined neuropathy, inherited or acquired peripheral neuropathy, such as diabetic neuropathy, chemotherapy-induced neuropathy, or other forms of toxic peripheral neuropathy, etc.), which in some cases manifests as a severe loss of sensation to temperature, pressure, and pain. In some cases, long-term cases of HSAN1 result in painless injuries, chronic skin ulcers, bone destruction, bone infection, finger / toe amputations, and motor dysfunction. In some embodiments, the mutant SPTLC1 gene encoding SPTLC1 C133W is associated with HSAN1 in the subject.

[0187] In some embodiments, the mutant SPTLC1 gene comprises one or more nucleotide substitutions, one or more nucleotide insertions, and / or one or more nucleotide deletions relative to the wild-type SPTLC1 gene (or mRNA encoded by the wild-type SPTLC1 gene) that are associated with ALS in the subject. Amyotrophic lateral sclerosis (ALS) is a fatal, rapidly progressive neurodegenerative disease. In subjects with a mutated SPTLC1 gene, the degeneration of upper and lower motor neurons leads to spasticity, weakness, muscle atrophy, and ultimately death due to respiratory muscle involvement. Most cases present with weakness of the limbs, but up to one-third can begin with bulbar symptoms. Cognitive and behavioral symptoms can include pseudobulbar affect and behavioral-variant frontotemporal dementia. In some embodiments, a mutant SPTLC1 gene encoding SPTLC1 C133W is associated with ALS in the subject. In some embodiments, a mutant SPTLC1 gene encoding SPTLC1 A20S is associated with ALS in the subject. In some embodiments, a mutant SPTLC1 gene encoding SPTLC1 Y23F is associated with ALS in the subject. In some embodiments, a mutant SPTLC1 gene encoding SPTLC1 L38R is associated with ALS in the subject. In some embodiments, a mutant SPTLC1 gene encoding SPTLC1 L39 deletion is associated with ALS in the subject. In some embodiments, a mutant SPTLC1 gene encoding SPTLC1 F40 deletion + S41 deletion is associated with ALS in the subject. In some embodiments, a mutant SPTLC1 gene encoding SPTLC1 S331F is associated with ALS in the subject. In some embodiments, a mutant SPTLC1 gene encoding SPTLC1 S331Y is associated with ALS in the subject. In some embodiments, a mutant SPTLC1 gene comprising a SPTLC1 exon 2 deletion is associated with ALS in the subject. In some embodiments, a mutant SPTLC1 gene comprising SPTLC1 rs75431233 is associated with ALS in the subject.

[0188] In some embodiments, an isolated nucleic acid described herein (e.g., an RNA processing modulator, such as an antisense oligonucleotide) is administered to a subject (e.g., a mammalian subject, such as a human subject) having or suspected of having one or more mutations in the SPTLC1 gene. In some embodiments, the subject has or is suspected of having a disease or disorder described herein. In some embodiments, the disease or disorder is Parkinson’s disease, Alzheimer’s disease, ALS, or HSAN1. In some embodiments, administration of an isolated nucleic acid described herein (e.g., an RNA processing modulator, such as an antisense oligonucleotide) is capable of modulating one or more non-deoxy-SLs (e.g., dihydro sphingosine, sphingosine, and / or ceramide) and / or one or more deoxy-SLs (e.g., deoxydihydro sphingosine and / or deoxyceramide) in a subject (e.g., a mammal, such as a human subject). In some embodiments, administration of an isolated nucleic acid to a subject modulates the level of one or more non-deoxy-SLs (e.g., dihydro sphingosine, sphingosine, and / or ceramide) and / or one or more deoxy-SLs (e.g., deoxydihydro sphingosine and / or deoxyceramide) in a biological sample obtained from the subject. In some embodiments, the biological sample is cerebrospinal fluid (CSF), plasma, or spinal cord tissue.

[0189] Example 4: ASOs targeting SPTLC1

[0190] This example describes the design of RPMs (e.g., ASOs) targeting human SPTLC1. In the context of a disease associated with dysregulation of L-serine biosynthesis, it is desirable to reduce the protein level of SPTLC1 (e.g., mutant SPTLC1), for example, by reducing the translation of SPTLC1 mRNA or reducing the activity or function of SPT. In some embodiments, the ASO is designed to target a region of the SPTLC1 mRNA that will result in reduced translation of SPTLC1 protein or SPT and / or reduced activity of SPTLC1 protein or SPT.

[0191] Expression profiling was performed on human serine palmitoyltransferase long chain base subunit 1 (SPTLC1) (e.g., NCBI Reference Sequence NM_006415.4). Figure 2A Bulk tissue gene expression of human SPTLC1 was shown. The data indicates that SPTLC1 mRNA is ubiquitously expressed. The types and number of SPTLC1 mRNA splice variants were investigated Figure 2B ). The data indicates that eight isoforms of SPTLC1 protein are encoded by SPTLC1 splice variants. Figure 2CRepresentative data from exon expression analysis of human SPTLC1 splice variants in CNS tissue is shown. The data indicates that full-length SPTLC1 is the most frequently expressed isoform in brain tissue. Table 1 shows representative RPM (e.g., antisense oligonucleotide) sequences targeting mRNA encoded by SPTLC1.

[0192] Example 5: In vitro screening of ASOs

[0193] Cell lines (e.g., U-118 MG human glioblastoma cells) are cultured and maintained using appropriate media (e.g., Dulbecco’s Modified Eagle Medium containing 10% fetal bovine serum). At the appropriate time, several methods are used to generate in vitro models of MacTel2 and HSAN1. For example, cell lines can be engineered to stably express mutant forms of SPTLC1. Cells harboring mutations in SPTLC1 are verified by sequencing. At the appropriate time, serine-deficient media is optionally used to expand the phenotype of cells with dysregulated L-serine biosynthesis.

[0194] Screening of ASOs targeting SPTLC1 RNA (Table 1, Figure 3A ) was performed in 96-well plates by seeding approximately 20,000 U-118 MG cells per well and treating with ASOs at concentrations of 5 nM and 20 nM using a Lipofectamine protocol. Each concentration was transfected in 2 independent wells for biological replicates. Two different ASO chemistries were assayed against SPTLC1 RNA. Short interfering RNA (siRNA) was used as a positive control for SPTLC1 expression measurement. Non-targeting ASO sequences with matching chemistry and length were used as negative controls in addition to wells treated with PBS or water. Treated cells were incubated at 37°C in a cell incubator for 48 hours prior to isolation of total RNA for measurement of gene expression. Total RNA was isolated and converted (reverse transcribed) to cDNA, then Taqman gene expression assays were used to quantify SPTLC1 gene modulation (e.g., TaqMan™ Fast Advanced Cells-to-CT™ Kit (ThermoFisher #A35378)). qPCR reactions were multiplexed with probes targeting SPTLC1 Figure 3A ; the amplified region of SPTLC1 is indicated by the rectangle labeled “qPCR amplicon”) and the human housekeeping gene hypoxanthine guanine phosphoribosyltransferase 1 (HPRT1). Cycle threshold (Ct) was generated for both SPTLC1 and HPRT1 and used for analysis.

[0195] Using Ct method to analyze SPTLC1 gene expression levels. For each sample, SPTLC1 gene expression levels provided as cycle threshold (Ct) values were normalized to the housekeeping gene HPRT1 (2 -(SPTLC1 Ct – HPRT1 Ct) ). SPTLC1 expression for each sample relative to control was then calculated based on the average of the untransfected control wells treated with PBS or water within each plate and displayed as a percentage ((sample / control average) 100). The resulting values for all treatment groups are shown in Figure 3B .

[0196] Five (5) SPTLC1 -targeting ASOs tested resulted in greater than 50% reduction in SPTLC1 RNA expression at a 5 nM dose and achieved knockdown comparable to the siRNA positive control. An additional seventeen (17) ASOs resulted in greater than 50% reduction in SPTLC1 RNA expression at a 20 nM dose. SPTLC1 -targeting ASOs for each tested chemistry successfully resulted in greater than 50% reduction in SPTLC1 RNA expression at a 20 nM dose. The effects of the 23 most potent ASOs tested are shown in Figure 3C .

[0197] U-251 MG cells were transfected with ASOs at a dose of 40 nM, 20 nM, 10 nM, 5 nM, 2.5 nM, 1.25 nM, 0.625 nM, or 0.3125 nM. Forty-eight hours post-transfection, the effect of ASO administration on SPTLC1 RNA and housekeeping control (HPRT1) was analyzed using RT-qPCR. The SPTLC1 -targeting ASOs tested showed concentration-dependent knockdown of SPTLC1 mRNA. An EC50 = 3.25 nM ( Figures 3C-3E ) was observed following administration of an ASO comprising the nucleotide sequence, gapmer structure, and chemical modifications of SEQ ID NO: 193 as shown in Table 1, line 194, columns A and C. An EC50 = 5.95 nM ( Figures 3C-3E ) was observed following administration of an ASO comprising the nucleotide sequence, gapmer structure, and chemical modifications of SEQ ID NO: 147 as shown in Table 1, line 148, columns A and C (or also referred to herein as “SPTLC1 ASO2”). An EC50 = 1.83 nM ( Figures 3C-3E ) was observed following administration of an ASO comprising the nucleotide sequence, gapmer structure, and chemical modifications of SEQ ID NO: 162 as shown in Table 1, line 163, columns A and C (or also referred to herein as “SPTLC1 ASO1”).

[0198] The effects of these ASOs on L-serine, SL, and deoxy-SL levels can be further tested by methods well known in the art. For example, chemically extracted SL and deoxy-SL can be measured using liquid chromatography-mass spectrometry (LCMS). ASO efficacy can be determined by comparing levels of L-serine, SL, and deoxy-SL between treated and untreated cells. Alternatively, L-serine biosynthesis can be measured by metabolic labeling experiments in intact cells or lysed cells using reagents such as radioactive, fluorescent, and bioorthogonal labels. Alternatively, SL and deoxy-SL can be measured by flow cytometry platforms after gating on cells that were not exposed to fluorescent antibodies (where intact cells are exposed to fluorescent antibodies that bind SL and deoxy-SL prior to flow cytometry analysis).

[0199] To further characterize ASO-dependent changes in L-serine biosynthesis and / or lipid biosynthesis, cell toxicity can be measured to understand the physiological impact of changes in deoxy-SL levels. Cell viability can be measured by generating survival curves by manually counting trypan blue-stained cells after ASO treatment. Alternatively, cells can be stained with propidium iodide and then analyzed by flow cytometry to measure cell death.

[0200] Example 6: In vitro dose response of ASOs targeting SPTLC1

[0201] To further characterize ASOs targeting SPTLC1, selected ASOs (e.g., from Table 1) were assayed with a second cell line, U251-MG cells. Based on high endogenous levels of SPTLC1 expression, U251-MG cells were selected for further study. To assess efficacy of ASOs targeting SPTLC1 in U251-MG cells, 61 ASOs (e.g., from Table 1) were selected for further study and tested at a 5 nM dose or a 20 nM dose. The 61 ASOs included ASOs with gapmer chemistry or skipmer chemistry. U251-MG cells were reverse transfected with 5 nM or 20 nM ASO using Lipofectamine 2000 protocol, 2 biological replicates per transfection condition, and plated at 1.5 x 105cells per well in a 96-well plate. Control cells were reverse transfected with water and a non-targeting control. After incubation at 37 °C for 48 hours, gene expression levels were analyzed by RT-qPCR as previously described with 3 technical replicates. The effects of ASOs targeting SPTLC1 on SPTLC1 gene expression were compared to the corresponding conditions previously tested in U118-MG cells. It was observed that the ASOs tested effectively inhibited SPTLC1 expression in both cell types 4 Example 6: In vitro dose response of ASOs targeting SPTLC1 Figure 4AFurthermore, the degree of knockout was determined to be highly correlated between the two cell types (R=0.86). These results demonstrate the high reproducibility of ASO effectiveness in different cell lines and suggest that these ASOs will be effective in multiple cell types. Further analysis showed that the ASO containing the nucleotide sequence, gapmer structure, and chemical modifications of SEQ ID NO: 162 as shown in columns A and C of row 163 of Table 1 (or referred to herein as “SPTLC1 ASO 1”) resulted in targeted knockdown of SPTLC1 in non-human primate cells and human-derived iPSCs. Figures 4B-4D The nucleotide sequence, gapmer structure, and chemically modified ASO (or referred to herein as “SPTLC1 ASO 2”) containing the SEQ ID NO:147 shown in columns A and C of row 148 of Table 1 resulted in powerful knockout of both SPTLC1 in non-human primate cells and human-derived iPSCs, and Sptlc1 in mouse cells. Figure 4C ).

[0202] The efficacy of selected ASOs in U251-MG cells was further determined by testing ASOs at a range of concentrations. Six different ASOs (e.g., from Table 1) were selected for further investigation and tested at concentrations of 40 nM, 20 nM, 10 nM, 5 nM, 2.5 nM, 1.25 nM, 0.625 nM, and 0.3125 nM. As before, U251-MG cells were reverse transfected with up to 40 nM ASO using the Lipofectamine 2000 protocol, with two biological replicates per transfection condition, at a density of 1.5 × 10⁶ cells per well. 4 Cells were seeded in 96-well plates. Control cells were reverse transfected with water and a non-targeted control. After incubation at 37°C for 48 hours, SPTLC1 gene expression levels were analyzed by RT-qPCR as previously described, with three technical replicates. All assayed ASOs were observed to inhibit SPTLC1 expression in U251-MG cells, achieving >50% knockdown between 1.25 nM and 40 nM, with most ASOs achieving >50% knockdown around 10 nM. Figure 5A and 5B ).

[0203] Example 7: In vitro dose-response of ASO targeting SPTLC1 in glutamatergic neurons

[0204] To further determine the efficacy of ASOs targeting SPTLC1 on SPTLC1 gene expression levels in a range of cell types, a panel of 9 ASOs (e.g., as described in Table 1) was assayed in induced pluripotent stem cell (iPSC)-derived glutamatergic neurons. Briefly, iPSC-derived neurons were cultured and transfected with 0 µM, 0.3 µM, 1 µM, 3 µM, or 10 µM ASO and incubated at 37°C for 5 days. Control cells were treated with non-targeting control in PBS. Following incubation for 5 days, SPTLC1 gene expression levels were analyzed by RT-qPCR as previously described. All assayed ASOs, except one, achieved >50% knockdown at 1 µM or around 1 µM, with several achieving >50% knockdown at 0.3 µM or around 0.3 µM ( Figure 6A ). The ASOs targeting SPTLC1 generally had little to no effect on expression of the HPRT1 housekeeping gene at most doses, however a significant effect on HPRT1 expression was generally observed at 10 µM, indicating that the highest dose can be toxic to iPSC-derived neurons ( Figure 6B ). Importantly, it was observed that SPTLC1 knockdown at the 3 µM dose was highly correlated between iPSC-derived neurons and U251-MG cells previously tested at a 20 nM dose ( Figure 6C ).

[0205] To assess the ability of an ASO comprising the nucleotide sequence of SEQ ID NO: 162, gapmer structure, and chemical modifications as shown in column A and column C of line 163 of Table 1 (or referred to herein as “SPTLC1 ASO 1”) to enter neurons and reduce SPTLC1 in a relevant cell type, hiPSC-derived spinal cord motor neurons were treated with PBS or an ASO comprising the nucleotide sequence of SEQ ID NO: 162, gapmer structure, and chemical modifications as shown in column A and column C of line 163 of Table 1 (or referred to herein as “SPTLC1 ASO 1”) (0.08 µM, 0.31 µM, 1.25 µM, 5 µM, 20 µM) via gymnotic delivery. Five days after treatment, SPTLC1 mRNA levels were quantified by RT-qPCR and normalized to the housekeeping gene HPRT1. Treatment with an ASO comprising the nucleotide sequence of SEQ ID NO: 162, gapmer structure, and chemical modifications as shown in column A and column C of line 163 of Table 1 (or also referred to herein as “SPTLC1 ASO 1”) resulted in a concentration-dependent decrease in SPTLC1 mRNA levels, with an EC50 of less than 1 µM ( Figure 6D). To confirm the effects observed in hiPSC-derived motor neurons in a second relevant cell type, hiPSC-derived cortical neurons were treated with PBS or ASOs comprising the nucleotide sequence of SEQ ID NO: 162, the gapmer structure, and chemical modifications as indicated in column A and C of line 163 of Table 1 (or also referred to herein as “SPTLC1 ASO 1”) via gymnotic delivery (0.08 µM, 0.31 µM, 1.25 µM, 5 µM, 20 µM). After 5 days of treatment, SPTLC1 mRNA levels were quantified by RT-qPCR and normalized to the housekeeping gene HPRT1. Treatment with ASOs comprising the nucleotide sequence of SEQ ID NO: 162, the gapmer structure, and chemical modifications as indicated in column A and C of line 163 of Table 1 (or also referred to herein as “SPTLC1 ASO 1”) resulted in a concentration-dependent reduction of SPTLC1 mRNA levels with an EC50 of 0.1 µM.

[0206] Computer off-target analysis identified two genes, WDR47 and ZFHX3, with two mismatches to the ASO comprising the nucleotide sequence, gapmer structure, and chemical modifications of SEQ ID NO: 162 as shown in column A and C of line 163 of Table 1 (or also referred to herein as “SPTLC1 ASO 1”). Because both genes are expressed in the CNS, and because the primary impact of the ASO comprising the nucleotide sequence, gapmer structure, and chemical modifications of SEQ ID NO: 162 as shown in column A and C of line 163 of Table 1 (or also referred to herein as “SPTLC1 ASO 1”) would occur in the CNS following intrathecal administration, the impact of the ASO comprising the nucleotide sequence, gapmer structure, and chemical modifications of SEQ ID NO: 162 as shown in column A and C of line 163 of Table 1 (or also referred to herein as “SPTLC1 ASO 1”) on these two genes was evaluated in vitro in neuronal cell types. hiPSC-derived cortical neurons were treated with PBS or the ASO comprising the nucleotide sequence, gapmer structure, and chemical modifications of SEQ ID NO: 162 as shown in column A and C of line 163 of Table 1 (or also referred to herein as “SPTLC1 ASO 1”) (0.08 µM, 0.31 µM, 1.25 µM, 5 µM, 20 µM) via gymnotic delivery. After 5 days of treatment, WDR47 and ZFHX3 mRNA levels were quantified by RT-qPCR and normalized to the housekeeping gene HPRT1. The ASO comprising the nucleotide sequence, gapmer structure, and chemical modifications of SEQ ID NO: 162 as shown in column A and C of line 163 of Table 1 (or also referred to herein as “SPTLC1 ASO 1”) was 89-fold more potent at reducing SPTLC1 (EC50= 0.1 µM) than for WDR47 (EC50= 8.9 µM) and 273-fold more potent for ZFHX3 (EC50= 27.3 µM). At the EC50 of SPTLC1, WDR47 and ZFHX3 mRNA levels were reduced by less than 20% Figure 6D

[0207] These results further demonstrate the efficacy of ASOs targeting SPTLC1 on SPTLC1 gene expression levels in a range of cell types, including non-cancer cell types.

[0208] Example 8: In vivo ASO methods

[0209] ​Animals are maintained in a consistent light and dark cycle and allowed to acclimate for at least five days prior to experimentation. Animals are fed regularly at a fixed time, frequency, and amount each day. ASOs targeting SPTLC1 are administered to animals via infusion. When multiple ASO infusions are performed, administration of ASOs is performed at the same time each day to minimize metabolic changes due to circadian rhythms. ASO infusions are provided directly to the affected region or into the cerebrospinal fluid (CSF). Animals can be placed in a head-low feet-high position during and after infusion to aid in distribution of the ASO into the tissues of the animal (e.g., CNS tissues). ASOs are dissolved in an appropriate buffer and sterilized prior to infusion. Following infusion, animals are maintained for a predetermined period of time prior to analysis. In some cases, animals are fed a diet containing radioactive glucose to determine the extent of L-serine incorporation into SL. To analyze the effects of ASO treatment, animals are anesthetized and tissues are harvested. Harvested tissue samples are snap-frozen in an appropriate extraction buffer. In appropriate cases, blood samples are isolated and mixed with buffer for preservation purposes. Harvested tissue samples are cryosectioned and used for immunohistochemical analysis. Blood samples are used to measure blood serine levels.

[0210] Example 9: Toxic lipid analysis of iPSC-derived neurons

[0211] Human iPSC-derived neuronal lines were engineered to contain gain-of-function mutations in SPTLC1 to characterize the effect of SPTLC1 mRNA knockdown on toxic lipid production in vitro. The PGP1 human iPSC line containing wild-type SPTLC1 was used to engineer two lines, where the “Het SPTLC1 KO” line was engineered to contain a heterozygous knock-out of SPTLC1 and the “Het SPTLC1C133W KI” line was engineered from the Het SPTLC1 KO to contain a heterozygous knock-in of a SPTLC1 gain-of-function (GOF) allele encoding the C133W mutation associated with HSAN1. Engineered human iSPC lines were verified by karyotype analysis and sequencing.

[0212] RT-qPCR analysis indicated that Het SPTLC1 KO exhibited a 64% reduction in SPTLC1 mRNA. Het SPTLC1 C133 KI had mRNA levels comparable to WT control. Treatment with a non-selective serine palmitoyltransferase (SPT) inhibitor control (myriocin) had no effect on SPTLC1 mRNA levels. However, when treated with an ASO comprising the nucleotide sequence of SEQ ID NO: 162, gapmer structure, and chemical modifications as shown in column A and column C of row 163 of Table 1 (or also referred to herein as“SPTLC1 ASO 1”), Het SPTLC1 C133 KI exhibited a 56% knockdown of SPTLC1 mRNA levels relative to untreated control.

[0213] Further analysis was used to assess production of toxic lipids due to SPTLC1 mutation and ASO treatment. Multiple lipid levels were assayed, including toxic deoxy-sphingolipids (e.g., 1-deoxy-dihydro sphingosine, 1-deoxysphingosine, and 1-deoxy ceramide) that are elevated in diseases like HSAN1, and non-deoxy lipids (e.g., dihydro sphingosine, sphingosine, and ceramide) that are elevated in diseases like ALS Figure 8A ). The lipid production assay was first validated in undifferentiated iPSCs derived from a HSAN1 patient that contains SPTLC1 encoding a C133W mutation. Analysis of the human HSAN1 patient line showed an increase in production of toxic 1-deoxysphingosine bases Figure 8B

[0214] To assess toxic lipid production in engineered iPSC-derived neurons, single cells were plated for an appropriate amount of time prior to treatment with 30 nM of an ASO comprising the nucleotide sequence of SEQ ID NO: 162, gapmer structure, and chemical modifications as shown in column A and column C of row 163 of Table 1 (or also referred to herein as“SPTLC1 ASO 1”) or 20 uM of myriocin. After 72 hours, cells were harvested and assayed for toxic lipid production. A total of two replicates were performed in all treatments.

[0215] ​Very low to undetectable levels of toxic deoxy sphingoid bases (DSBs) were detected in wild-type and Het SPTLC1 KO iPSCs. Het SPTLC1 KO iPSCs exhibited a 40% and 55% reduction in dihydrosphingosine and sphingosine levels, respectively, relative to wild-type iPSCs. Het SPTLC1 C133W KI iPSCs exhibited elevated levels of toxic DSBs relative to wild-type iPSCs. Treatment of Het SPTLC1 C133W KI iPSCs with the positive control, globomycin, reduced both sphingosine bases and toxic deoxy sphingoid bases by over 90%. When SPTLC1 ASO 1 was administered, 1-deoxydihydrosphingosine levels were reduced by approximately about 20% compared to Het SPTLC1 C133W KI iPSCs. Additionally, SPTLC1 ASO 1 treatment reduced dihydrosphingosine and sphingosine levels by 48% and 63%, respectively, which is comparable to the levels observed in Het SPTLC1 KO iPSCs Figures 8C-8G

[0216] When ceramide and deoxyceramide levels were measured, a 54% increase in 1-deoxyceramide was observed in Het SPTLC1 C133W KI iPSCs compared to wild-type and Het SPTLC1 KO iPSCs. A 34% increase in ceramide was observed in Het SPTLC1 C133W KI iPSCs compared to wild-type iPSCs. Treatment of Het SPTLC1 C133W KI iPSCs with the positive control, globomycin, resulted in a 98% reduction in 1-deoxyceramide and ceramide levels. When SPTLC1 ASO 1 was administered, 1-deoxyceramide and ceramide levels were reduced by 21% and 29%, respectively Figures 8H-8J

[0217] These results indicate that SPTLC1 knockout results in reduced sphingolipid production, while the SPTLC1 C133W mutation results in elevated production of toxic deoxy sphingolipids. Furthermore, these results indicate that treatment with SPTLC1 ASO 1 or globomycin modulates sphingolipid production and reduces the levels of toxic deoxy sphingosine bases.

[0218] ​​To confirm that the lipid effects observed in the hiPSC disease model studies were applicable to neurons, a small study was performed in wild-type hiPSC-derived cortical neurons. The main focus of the sphingolipid readout was total ceramide levels. hiPSC-derived cortical neurons in multiple wells of 6 plates were treated via gymnotic delivery with PBS or SPTLC1 ASO 1 (5 mM). After 5 days of treatment, the wells from each treatment group were pooled together for mRNA and targeted lipid analysis. SPTLC1 mRNA levels were measured by RT-qPCR, and SPTLC1 ASO 1 treatment resulted in a -90% reduction in SPTLC1 levels Figure 8K ). The sphingolipid profile was analyzed using LC / MS / MS and normalized to total protein input. STPCL1 ASO 1 treatment reduced total ceramide levels by >70% Figure 8K ), recapitulating the results observed in the hiPSC disease models. This further demonstrates that SPTLC1 ASO 1 is effective at reducing SPTLC1 in neurons and has a corresponding effect on reducing elevated sphingolipid levels in different diseases.

[0219] Example 10: In vivo knockdown of SPTLC1 mRNA in mouse subjects

[0220] This example describes in vivo knockdown of SPTLC1 mRNA using the ASOs described herein. Briefly, C57BL / 6 mice were administered a single ICV injection or repeated ICV injections of ASO and mRNA levels were quantified in brain tissue (samples harvested from left cortex 1 and left hippocampus). Frozen tissue was lysed and homogenized in RLT buffer using beads (MP Biomedical) and RNA extraction was performed using RNeasy mini kit (Qiagen) in a QIAcube workstation (Qiagen). RNA concentration was evaluated using a nanodrop spectrometer (ThermoFisher) and integrity using a 2100 Bioanalyzer LabChip (Agilent). 500 ng of RNA was reverse transcribed using SuperScript IV VILO Master Mix ezDNase (Invitrogen). qPCR was performed in a Quantstudio thermocycler (Applied Biosystems) with TaqMan Fast Advance Master Mix (Invitrogen) for 2 independent Taqman (VIC) assays for SPTLC1 (IDT TF Mm01339824_g1, TFMm00447346_m1). Pgkl and PPIA levels were measured using Taqman (FAM) assays (Mm00435617_m1, Thermofischer) for normalization using the Ct method.

[0221] Figures 9A-9D Representative data showing in vivo reduction of SPTLC1 mRNA levels in the brain of C57BL / 6 mice is shown. Figure 9A Representative data showing relative SPTLC1 mRNA levels in hippocampal tissue of mouse subjects 3 weeks after a single bilateral stereotactic ICV injection of vehicle (artificial CSF), 3 ug of polyoxin, 200 ug or 300 ug dose of non-specific ASO, or 300 ug dose of ASO comprising the nucleotide sequence, gapmer structure, and chemical modifications of SEQ ID NO: 162 as shown in column A and column C of row 163 of Table 1 (or also referred to herein as “SPTLC1 ASO 1”) or comprising the nucleotide sequence and chemical modifications of SEQ ID NO: 147 as shown in column A and column C of row 148 of Table 1 (or also referred to herein as “SPTLC1 ASO 2”) is shown. Figure 9B ​Relative SPTLC1 mRNA levels in the cortex tissue of mouse subjects 3 weeks after a single bilateral stereotactic ICV injection of vehicle (artificial CSF), 3 ug of multicalyxin, 200 ug or 300 ug dose of non-specific ASO, or 300 ug dose of SPTLC1 ASO 1 or SPTLC1 ASO 2 are shown. Figure 9C Relative SPTLC1 mRNA levels in the hippocampus tissue of mouse subjects one week after the last of three ICV injections (1 week apart) of vehicle (artificial CSF) or 100 ug + 100 ug + 100 ug ASO via a catheter are shown. Figure 9D Relative SPTLC1 mRNA levels in the cortex tissue of mouse subjects one week after the last of three ICV injections (1 week apart) of vehicle (artificial CSF) or 100 ug + 100 ug + 100 ug ASO via a catheter are shown. “SPTLC1 ASO 1” corresponds to an ASO comprising the nucleotide sequence, gapmer structure, and chemical modifications of SEQ ID NO: 162 as shown in column A and column C of line 163 of Table 1. “SPTLC1 ASO 2” corresponds to an ASO comprising the nucleotide sequence, gapmer structure, and chemical modifications of SEQ ID NO: 147 as shown in column A and column C of line 148 of Table 1. Values for 2 technical replicates were averaged and shown as percent of vehicle-control group. Statistical analysis was performed using a linear model, comparing treatment groups to aCSF group, and adjusting for RNA isolation batch : p < 0.05, : p < 0.01, : p < 0.001. Figures 9A-9D Representative data showing in vivo reduction of SPTLC1 mRNA levels in mouse brain is shown. Figure 9A Relative SPTLC1 mRNA levels in the hippocampus tissue of mouse subjects seven days after a series of three ICV injections of vehicle (artificial CSF), 3 ug of multicalyxin, 200 ug (100 ug + 100 ug) or 300 ug (100 ug + 100 ug + 100 ug) dose of non-specific ASO, or 300 ug (100 ug + 100 ug + 100 ug) dose of SPTLC1 ASO 1 or SPTLC1 ASO 2 once per week are shown. Figure 9BRelative SPTLC1 mRNA levels in the cortex tissue of mouse subjects seven days after a series of three weekly ICV injections of vehicle (artificial CSF), 3 ug of globomycin, 200 ug (100 ug + 100 ug) or 300 ug (100 ug + 100 ug + 100 ug) dose of non-specific ASO, or 300 ug (100 ug + 100 ug + 100 ug) dose of SPTLC1 ASO 1 or SPTLC1 ASO 2 are shown. Figure 9C Relative SPTLC1 mRNA levels in the cortex tissue of mouse subjects seven days after a series of three weekly ICV injections of vehicle (artificial CSF), 3 ug of globomycin, 200 ug (100 ug + 100 ug) or 300 ug (100 ug + 100 ug + 100 ug) dose of non-specific ASO, or 300 ug (100 ug + 100 ug + 100 ug) dose of SPTLC1 ASO 1 or SPTLC1 ASO 2 are shown. Figure 9D Relative SPTLC1 mRNA levels in the cortex tissue of mouse subjects seven days after a series of three weekly ICV injections of vehicle (artificial CSF), 3 ug of globomycin, 200 ug (100 ug + 100 ug) or 300 ug (100 ug + 100 ug + 100 ug) dose of non-specific ASO, or 300 ug (100 ug + 100 ug + 100 ug) dose of SPTLC1 ASO 1 or SPTLC1 ASO 2 are shown.

[0222] In the repeat ICV injection study, some animals exhibited mild acute in-life observations after administration of globomycin, SPTLC1 ASO 1 or SPTLC1 ASO 2, which largely resolved within 1-2 hours. No major impact on life was observed in animals receiving administration of globomycin or SPTLC1 ASO.

[0223] Repeat ICV injection of SPTLC1 ASO 2 resulted in 19-22% knockdown of SPTLC1 mRNA in the cortex and hippocampus, respectively. Figures 9C-9D SPTLC1 ASO 2 exhibited cross-reactivity to SPTLC1 mRNA in mice and non-human primates. SPTLC1 ASO 1 resulted in significant SPTLC1 knockdown in the cortex in one assay. Figure 9D No significant impact on SPTLC1 mRNA was observed in control-treated subjects administered vehicle, globomycin or non-specific ASO.

[0224] Further analysis determined pharmacokinetics (ASO levels) as a result of SPTLC1 ASO 1 administration via ICV injection into the cortex of mouse subjects. Briefly, mouse subjects received a single ICV injection of control agent (artificial CSF; non-specific ASO at a 10 pg dose; or SPTLC1 ASO 2 at a 10 pg dose) as described herein or SPTLC1 ASO 1 at a 10 pg dose. Mouse subjects were sacrificed two weeks after ICV injection. SPTLC1 ASO 1 levels were measured using hybridization ELISA analysis of cortical tissue samples collected from the same side (ipsilateral) of the cortex as the single ICV injection was performed. No SPTLC1 ASO 1 levels were detected in cortical tissue samples obtained from mouse subjects that received a single ICV injection of artificial CSF, non-specific ASO, or SPTLC1 ASO 2. The sum of all SPTLC1 ASO 1 level measurements from cortical tissue samples obtained from mouse subjects that received a single ICV injection of non-specific ASO was not significant. When analyzing cortical samples obtained from mouse subjects that received a single ICV injection of SPTLC1 ASO 1, high SPTLC1 ASO 1 levels were detected Figure 10A

[0225] PK of ASO 1 exposure was evaluated in mouse ipsilateral cortex and hippocampus by hybridization enzyme-linked immunosorbent assay (hELISA) using custom capture and detection probes. Male C57BL / 6 mice (N=5 / group, 8-9 weeks old) were administered 100 pL of vehicle (aCSF) or 100 pg of SPTLC1 ASO 1 once per week via unilateral ICV for three weeks. One week after the last dose, animals were sacrificed and pharmacokinetics (PK) of mouse ipsilateral cortex and hippocampus were assessed. hELISA data showed an average exposure of 34.8 pg / g (16.9-45 pg / g) in the cortex and an average exposure of 53.3 pg / g (31.7-102 pg / g) in the hippocampus, indicating ASO 1 exposure in the CNS Figure 10B

[0226] Example 11: In vivo ASO administration to non-human primates

[0227] ​​This example describes the in vivo administration of SPTLC1 ASOs to cynomolgus monkey (Macaca fascicularis) subjects (also referred to as “non-human primate subjects”). Briefly, non-human primate subjects received two intrathecal (IT) injections of either vehicle (artificial CSF) or an ASO comprising the nucleotide sequence of SEQ ID NO: 162, gapmer structure, and chemical modifications as shown in column A and column C of row 163 of Table 1 (or also referred to herein as “SPTLC1 ASO 1”). Each group (vehicle-injected and ASO-injected) consisted of two male subjects and one female subject, and each round of IT injection was performed two weeks apart (day 1 and day 14). In the two rounds of ASO IT injection, SPTLC1 ASO 1 was administered at a dose of 20 mg to result in a total dose of 40 mg of SPTLC1 ASO 1 administered over a 28-day period. At day 28 (two weeks after the last intrathecal injection), the non-human primate subjects were sacrificed and samples of cerebrospinal fluid and brain (frontal cortex, sensory cortex, and hippocampus), lumbar spinal cord, dorsal root ganglion, kidney, liver, spleen, heart, stomach, and gonadal tissue were collected. Additionally, serum and plasma were collected 3 days prior to day 1 and 3 days prior to day 14 ( Figure 11A ). Liquid chromatography-mass spectrometry analysis was used to measure SPTLC1 ASO 1 pharmacokinetics (ASO levels), and RT-qPCR was used to measure SPTLC1 ASO 1 pharmacodynamics (SPTLC1 mRNA expression). SPTLC1 ASO 1 concentrations were then quantified in frontal cortex, hippocampus, and lumbar spinal cord tissue samples to determine SPTLC1 ASO 1 levels as a result of administration by IT injection.

[0228] Table 2. SPTLC1 ASO 1 levels in non-human primate subject tissue samples

[0229]

[0230] Subjects receiving SPTLC1 ASO 1 treatment comprising an ASO having the nucleotide sequence of SEQ ID NO: 162 as shown in column A and column C of row 163 of Table 1, respectively ), and chemical modifications

[0231] ​​SPTLC1 ASO 1 was not detected in tissue samples obtained from non-human primate subjects who received two IT injections with the vector. No in-life safety signals were observed in non-human primate subjects who received two IT injections with either the vector or SPTLC1 ASO 1. The average concentration of SPTLC1 ASO 1 detected in tissue samples obtained from non-human primate subjects who received two IT injections with an ASO targeting SPTLC1 mRNA was approximately 14–55 µg / g tissue. Figure 11B (and Table 2). In addition, the pharmacokinetics of ASO obtained from tissue samples obtained from non-human primate subjects administered via IT injection met or exceeded the pharmacokinetic data recorded in previous non-human primate ASO studies (see, for example, Malat1 ASO administration in Jafar-Nejad et al. (2021). NucleicAcids Research. 49 (2): 657–673).

[0232] Further analysis showed that two IT injections of SPTLC1 ASO1 in frontal cortex and sensory cortex tissue samples obtained from non-human primate subjects resulted in significant SPTLC1 mRNA knockdown (approximately 30%-40%). Figures 11D-11K Knockdown of SPTLC1 mRNA using a medium (artificial CSF) or nonspecific ASO injection had no significant effect. Figures 11D-11L Furthermore, no changes in SPTLC1 mRNA expression were observed when RT-qPCR analysis was performed using probes that indiscriminately bound to all SPTLC1 mRNA isoforms (including shorter alternative transcripts lacking domains crucial for SPTLC1 activity and not targeted by SPTLC1 ASO1). Figures 11D-11L Determination 2 in the middle; see also Figure 12 However, when SPTLC1 mRNA expression was analyzed by RT-qPCR using a probe that binds to SPTLC1 mRNA at a sequence located near the SPTLC1 ASO1 binding site, approximately 30%-40% knockdown of SPTLC1 mRNA was observed. Figures 11D-11L Determination 1 in the middle; see also the determination 1 in the middle; Figure 12). This indicates that SPTLC1 ASO 1 specifically reduced the expression of SPTLC1 encoded by SPTLC1 canonical transcripts in the frontal cortex and sensory cortex of non-human primates after repeated injections with high doses of SPTLC1 ASO 1. Liquid chromatography-tandem mass spectrometry analysis of lumbar spinal cord samples indicated a reduction of toxic sphingolipid levels in the samples, which also showed high levels of bio-distribution of SPTLC1 ASO 1 and reduced levels of SPTLC1 mRNA Figure 11M

[0233] A series of four IT injections of ASO was evaluated in relation to pharmacokinetic effects in additional non-human primate subjects. Three male non-human primate subjects were administered either vehicle (artificial CSF) or a series of four IT injections of 95 mg (30 mg + 25 mg + 20 mg + 20 mg) doses of an ASO comprising the nucleotide sequence of SEQ ID NO: 162, the gapmer structure, and chemical modifications as shown in columns A and C in row 163 of Table 1 (or also referred to herein as “SPTLC1 ASO 1”). For all non-human primate subjects, each round of IT injections was performed two weeks apart (days 0, 14, 28, and 42). Samples of cerebrospinal fluid as well as brain (frontal cortex, sensory cortex, and hippocampus), lumbar spinal cord, dorsal root ganglion, kidney, liver, spleen, heart, stomach, and gonadal tissues were collected two weeks after the last IT injection Figure 14 ). Liquid chromatography-tandem mass spectrometry (LC-MS / MS) analysis was used to measure ASO pharmacokinetics (ASO levels) in tissue samples obtained from the injected non-human primates and toxic sphingolipid levels in cerebrospinal fluid. ASO concentrations in tissue samples were quantified to determine SPTLC1 ASO 1 levels as a result of the IT injections Figures 15A-15B ). Toxic sphingolipid levels in cerebrospinal fluid samples were quantified to determine the effect of SPTLC1 ASO 1 administration via IT injections Figure 15D ). At the same time, SPTLC1 mRNA levels were analyzed by RT-qPCR to correlate pharmacokinetic effects and reduction of ceramide levels in cerebrospinal fluid with pharmacodynamic kinetics effects of SPTLC1 ASO 1 administration Figure 15C ). In SPTLC1 ASO 1 treated subjects, an average 32% reduction of SPTLC1 mRNA levels was observed in all treated animals, and an average 44% reduction of SPTLC1 mRNA levels in 2 animals with drug exposure Figures 15B-15C ), and a 45% reduction of ceramide levels in CSF compared to baseline Figure 15D

[0234] Example 12: In vitro immunostimulatory effects of ASOs​​

[0235] This example describes an analysis of the immunostimulatory effects of SPTLC1 ASOs in human peripheral blood mononuclear cells (huPBMCs).

[0236] All ASOs were prepared using in vivo mass scale material in a manner consistent with the analyses performed in animal subjects described above. huPBMCs were harvested from healthy donors and either left untreated, treated with cytokine / chemokine response controls, or treated with ASOs comprising the nucleotide sequence, gapmer structure, and chemical modifications of SEQ ID NO: 162 as shown in column A and column C of line 163 of Table 1 (or also referred to herein as “SPTLC1 ASO 1”) or comprising the nucleotide sequence, gapmer structure, and chemical modifications of SEQ ID NO: 147 as shown in column A and column C of line 148 of Table 1 (or also referred to herein as “SPTLC1 ASO 2”) at a concentration of 1 µM, 3 µM, or 10 µM for 24 hours.

[0237] Cytokine / chemokine response controls included: XD-01024, which is a cholesterol-conjugated ApoB siRNA, which has a TLR7 / 8 agonist effect; CL097, which is a water-soluble derivative of the imidazoquinoline compound R848, which is a TLR7 / 8 ligand; Imiquimod (R837), which is an immune response modifier, which has potent antiviral activity and also induces cytokine production and activates TLR7; ODN2216, which is a 20-mer oligonucleotide containing an unmethylated CpG, and has a TLR9 agonist effect; ODN2006, which is biased for TLR9, is one or more CpG, class B; ODN2395, TLR9, CpG with palindromic motif, class C; TL8-506, which is a benzazepine with a TLR8 agonist effect; LMW poly (l:c), which has a TLR3 agonist effect; and XD-00366, which is a 25-mer double-stranded, unmodified, blunt-ended LacZ RNA duplex, which has a TLR7 / 8 agonist effect. (benzoazepine) with a TLR8 agonist effect; LMW poly (l:c), which has a TLR3 agonist effect; and XD-00366, which is a 25-mer double-stranded, unmodified, blunt-ended LacZ RNA duplex, which has a TLR7 / 8 agonist effect.

[0238] Following treatment of huPBMCs, levels of IFN-a2a, IFN-b, IL-1B, IL-6, IL-10, IP-10, MCP-1, MIP-1a, MIP-1b, and TNF-a were measured using the MSD-U-Plex platform. Representative data from these analyses are shown in Figures 13A-13JNegative control cells exhibited minimal or undetectable increases in chemokine / cytokine levels following treatment. As expected, treatment with the TLR agonist positive control resulted in increased chemokine / cytokine levels. Immune responses to SPTLC1 ASO 1 or SPTLC1 ASO 2 were minimal or undetectable when compared to cells samples treated under negative or positive control conditions Figures 13A-13J

[0239] equivalents

[0240] While several inventive embodiments have been described and illustrated herein, those of ordinary skill in the art will readily envision a variety of other means and / or structures for performing the functions and / or obtaining the results and / or one or more of the advantages described herein, and each of the foregoing are deemed a equivalent to those described herein. More generally, those skilled in the art will readily appreciate that all parameters, dimensions, materials, and configurations described herein are meant to be exemplary and that the actual parameters, dimensions, materials, and / or configurations will depend upon the specific application or applications for which a inventive embodiment is used. Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific inventive embodiments described herein. It is, therefore, to be understood that the foregoing embodiments are presented by way of example only and that, within the scope of the appended claims and equivalents thereto, inventive embodiments can be practiced otherwise than as specifically described and claimed. Inventive embodiments of the present disclosure are directed to each individual feature, system, article, material, kit, and / or method described herein. In addition, any combination of two or more such features, systems, articles, materials, kits, and / or methods, if such features, systems, articles, materials, kits, and / or methods are not mutually inconsistent, is

[0241] All definitions, as defined and used herein, should be understood to control over dictionary definitions, definitions in documents incorporated by reference, and / or ordinary meanings of the defined terms.

[0242] All references, patents and patent applications disclosed herein are incorporated by reference with respect to the subject matter for which each is cited, which can cover, in some cases, the entirety of the document.

[0243] The indefinite articles "a" and "an," as used herein in the specification and in the claims, unless clearly indicated to the contrary, should be understood to mean "at least one."

[0244] ​The phrase “and / or,” as used herein in the specification and in claims, should be understood to mean “either or both of” the elements so conjoined, i.e., elements conjoined with “and / or” are to be construed in the fashion that permits claims to use a single criterion for embarrassed elements followed by “and / or” in a conjunctive fashion. Multiple elements listed with “and / or” should be construed in the same fashion, i.e., “one or more” of the elements so conjoined. Other elements can optionally be present other than the elements specifically identified by the “and / or” clause, whether related or unrelated to those elements specifically identified. Thus, as a non-limiting example, a reference to “A and / or B” when used in this specification is a reference to A only (optionally including elements other than B); to B only (optionally including elements other than A) or to both A and B (optionally including elements other than A or B).

[0245] As used herein in the specification and in the claims, “or” should be understood to have the same meaning as “and / or” as defined above. For example, when used in the context of listing items, “or” or “and / or” shall be interpreted as the inclusive, i.e., the collection of items, one or more of which can be present, also includes more than one, and optionally, additional unlisted items. Only terms that are clearly expressed as the exclusive alternative, such as “only one of’ or “exactly one of,” or, when used in the claims, “consisting of,” will refer to the collection of items that includes exactly one of each named element, but no others. In general, the term “or” as used herein, unless limited by an exclusive -use term such as “either,” “one of,” “only one of,” or “exactly one of,” shall be interpreted to mean an inclusive “or” (i.e., “one or the other, but not both”).

[0246] As used in the specification and the claims, the phrase “at least one,” with respect to a list of one or more elements, should be understood to mean at least one of the elements, but not necessarily including at least one of each and every element specifically listed within the list of elements and not excluding any combinations of elements in the list of elements. This definition also allows that the

[0247] It should also be understood that, unless clearly indicated to the contrary, in any methods claimed herein that include more than one step or act, the order of the steps or acts of the method is not necessarily limited to the order in which the steps or acts are recited.

[0248] In the claims, as well as in the specification above, all transitional phrases such as “comprising,” “including,” “carrying,” “having,” “containing,” “involving,” “holding,” “composed of,” and the like are to be understood to be open-ended, i.e., to mean including but not limited to. Only the transitional phrases “consisting of’ and “consisting essentially of’ shall be closed or semi-closed transitional phrases, as set forth in the United States Patent Office Manual of Patent Examining Procedures, Section 2111.03. It should be understood that in alternative embodiments, the embodiments described herein that use open-ended transitional phrases such as “comprising” can also consist of or consist essentially of, the elements a transitional phrase describes. For example, if the present disclosure describes a composition “comprising A and B,” then the present disclosure also contemplates alternative embodiments consisting of or consisting essentially of a composition of A and B.

Claims

1. An isolated nucleic acid comprising a region having complementarity to a human SPTLC1 mRNA transcript, having at least 60% identity to the nucleic acid sequence set forth in any one of SEQ ID NOs: 1-299, and when bound to the mRNA transcript, reduces translation of a functional SPTLC1 protein encoded by the mRNA transcript.

2. The isolated nucleic acid of claim 1, wherein the isolated nucleic acid comprises RNA.

3. The isolated nucleic acid of claim 1 or 2, wherein the isolated nucleic acid is an antisense oligonucleotide.

4. The isolated nucleic acid of any one of claims 1 to 3, comprising or consisting of 10 to 40 nucleotides.

5. The isolated nucleic acid of claim 4, wherein the isolated nucleic acid comprises or consists of 18 to 25 nucleotides.

6. The isolated nucleic acid of any one of claims 1 to 5, wherein the isolated nucleic acid comprises one or more chemical modifications.

7. The isolated nucleic acid of claim 6, wherein the one or more chemical modifications comprise one or more nucleoside modifications and / or one or more sugar-phosphate backbone modifications.

8. The isolated nucleic acid of claim 7, wherein the one or more nucleoside modifications comprise a 2’-O-methyl (2’-OMe) modification, a 2’-O-MOE modification, a 2’-O-fluoro modification, or a locked nucleic acid (LNA) modification.

9. The isolated nucleic acid of claim 7 or 8, wherein the one or more sugar-phosphate backbone modifications comprise a phosphorothioate backbone modification.

10. The isolated nucleic acid of any one of claims 1 to 9, wherein the isolated nucleic acid is fully chemically modified.

11. The isolated nucleic acid of any one of claims 1 to 10, wherein the isolated nucleic acid comprises one or more deoxyribonucleic acids, optionally wherein the isolated nucleic acid is a gapmer.

12. The isolated nucleic acid of any one of claims 1 to 11, wherein the region having complementarity is located in an untranslated region (UTR) of the SPTLC1 mRNA transcript.

13. The isolated nucleic acid of claim 12, wherein the untranslated region comprises a 5’ UTR, an intron, or a 3’ UTR of the SPTLC1 mRNA transcript.

14. The isolated nucleic acid of any one of claims 1 to 11, wherein the region having complementarity is located in a protein-coding region of the SPTLC1 mRNA transcript.

15. The isolated nucleic acid of any one of claims 1 to 11, wherein the region having complementarity is located at an intron-exon boundary of the SPTLC1 mRNA transcript.

16. The isolated nucleic acid of any one of claims 1 to 15, wherein the region having complementarity comprises at least 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 contiguous nucleotides of the sequence set forth in SEQ ID NO:

300.

17. The isolated nucleic acid of any one of claims 1 to 16, comprising a nucleotide sequence set forth in any one of the nucleotide sequences set forth in Table 1.

18. A composition comprising the isolated nucleic acid of any one of claims 1 to 17, and a pharmaceutically acceptable excipient.

19. A method for increasing lipid biosynthesis in a cell or a subject, the method comprising administering to a cell or a subject in need thereof the isolated nucleic acid of any one of claims 1 to 17 or the composition of claim 18.

20. The method of claim 19, wherein the subject is characterized as having low serine.

21. The method of claim 19 or 20, wherein the subject comprises one or more mutations in a gene associated with lipid biosynthesis, optionally wherein the gene is SPTLC1.

22. The method of any one of claims 19 to 21, wherein the cell is a human cell, optionally wherein the cell is in a subject.

23. The method of any one of claims 19 to 22, wherein the subject is a human subject.

24. The method of any one of claims 19 to 23, wherein the subject has or is suspected of having a disease or disorder associated with dysregulation of L-serine biosynthesis.

25. The method of claim 24, wherein the disease or disorder is a retinal disease (e.g., MacTel2 and age-related macular degeneration (AMD), such as AMD with geographic atrophy), HSAN1 (e.g., HSAN1(a)), diabetic retinopathy, peripheral neuropathy (e.g., motor neuropathy, sensory neuropathy, autonomic neuropathy, combined neuropathy, inherited or acquired peripheral neuropathy, such as diabetic neuropathy, chemotherapy-induced neuropathy, or other forms of toxic peripheral neuropathy, etc.), neurodegeneration (e.g., Parkinson’s disease, Friedreich’s ataxia, motor neuron disease, Alzheimer’s disease, amyotrophic lateral sclerosis (ALS), etc.), spastic paraplegia, or a psychiatric disorder (e.g., treatment-resistant depression, treatment-refractory depression, schizophrenia, and / or treatment-refractory schizophrenia).

26. The method of any one of claims 19 to 25, wherein the administration is systemic administration, optionally wherein the systemic administration comprises intravenous injection.

27. The method of any one of claims 19 to 25, wherein the administration comprises direct administration to a target tissue of the subject, optionally wherein the direct administration comprises direct injection to the central nervous system (CNS), direct injection to the peripheral nervous system (PNS), or direct administration to the eye.

28. The method of claim 27, wherein the administration comprises having the subject in a head-low-feet-high position during the administration.

29. A method for decreasing deoxy-sphingolipid (deoxy-SL) biosynthesis in a cell or a subject, the method comprising administering to a cell or a subject in need thereof the isolated nucleic acid of any one of claims 1 to 17 or the composition of claim 18.

30. The method of claim 29, wherein the subject is characterized as having low serine.

31. The method of claim 29 or 30, wherein the subject comprises one or more mutations in a gene associated with L-serine biosynthesis, optionally wherein the gene is SPTLC1.

32. The method of any one of claims 29 to 31, wherein the cell or subject is a human cell or human subject.

33. The method of any one of claims 29 to 32, wherein the subject has or is suspected of having a disease or disorder associated with dysregulation of L-serine metabolism.

34. The method of claim 33, wherein the disease or disorder is a retinal disease (e.g., MacTel2 and age-related macular degeneration (AMD), such as AMD with geographic atrophy), HSAN1 (e.g., HSAN1(a)), diabetic retinopathy, peripheral neuropathy (e.g., motor neuropathy, sensory neuropathy, autonomic neuropathy, combined neuropathy, inherited or acquired peripheral neuropathy, such as diabetic neuropathy, chemotherapy-induced neuropathy, or other forms of toxic peripheral neuropathy, etc.), neurodegeneration (e.g., Parkinson’s disease, Friedreich’s ataxia, motor neuron disease, Alzheimer’s disease, amyotrophic lateral sclerosis (ALS), etc.), spastic paraplegia, or a psychiatric disorder (e.g., resistant depression, refractory depression, schizophrenia, and / or refractory schizophrenia).

35. The method of any one of claims 29 to 34, wherein the administering is systemic administration, optionally wherein the systemic administration comprises intravenous injection.

36. The method of any one of claims 29 to 34, wherein the administering comprises direct administration to the central nervous system (CNS), optionally wherein the direct administration comprises direct injection to the CNS.

37. The method of claim 36, wherein the administering comprises positioning the subject in a head-low feet-high position during the administering.

38. A method for preventing or treating a disease and disorder associated with dysregulation of L-serine biosynthesis in a subject in need thereof, the method comprising administering to a subject in need thereof the isolated nucleic acid of any one of claims 1 to 17 or the composition of claim 18.

39. The method of claim 38, wherein the subject is a human.

40. The method of claim 38 or 39, wherein the disease or disorder is a retinal disease (e.g., MacTel2 and age-related macular degeneration (AMD), such as AMD with geographic atrophy), HSAN1 (e.g., HSAN1(a)), diabetic retinopathy, peripheral neuropathy (e.g., motor neuropathy, sensory neuropathy, autonomic neuropathy, combined neuropathy, inherited or acquired peripheral neuropathy, such as diabetic neuropathy, chemotherapy-induced neuropathy, or other forms of toxic peripheral neuropathy, etc.), neurodegeneration (e.g., Parkinson’s disease, Friedreich’s ataxia, motor neuron disease, Alzheimer’s disease, amyotrophic lateral sclerosis (ALS), etc.), spastic paraplegia, or a psychiatric disorder (e.g., treatment-resistant depression, treatment-resistant depression, schizophrenia, and / or treatment-resistant schizophrenia).

41. The method of any one of claims 38-40, wherein the administering comprises direct administration to a target tissue of the subject, optionally wherein the direct administration comprises direct injection to the central nervous system (CNS), direct injection to the peripheral nervous system (PNS), or direct administration to the eye.

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