WO2026020002A1

WO2026020002A1 - Enhancing neuronal resistance to neurodegeneration

Info

Publication number: WO2026020002A1
Application number: PCT/US2025/038051
Authority: WO
Inventors: Hoa Quang My NGUYEN; Esteban O. MAZZONI
Original assignee: New York University NYU
Current assignee: New York University NYU
Priority date: 2024-07-17
Filing date: 2025-07-17
Publication date: 2026-01-22
Anticipated expiration: 2027-01-17

Abstract

The application relates to methods of enhancing neuronal resistance to disease characterized by TAR DNA-binding protein 43 (TDP-43) mislocalization or dysfunction, e.g., a neurodegenerative disease.

Description

ENHANCING NEURONAL RESISTANCE TO NEURODEGENERATION

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH

[0001] This invention was made with government support under R21 AG067174 awarded by the National Institutes of Health. The government has certain rights in the invention.

CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims priority to U.S. Provisional Application No. 63/672,452, filed July 17, 2024, the contents of which is incorporated by reference herein in its entirety.

SEQUENCE LISTING

[0003] The instant application contains a Sequence Listing which has been submitted electronically in XML format and is hereby incorporated by reference in its entirety. Said XML copy, created on July 9, 2025, is named 243735_000444_SL.xml and is 982,474 bytes in size.

FIELD OF THE INVENTION

[0004] The application relates to methods of enhancing neuronal resistance to disease characterized by TAR DNA-binding protein 43 (TDP-43) mislocalization or dysfunction, e.g., a neurodegenerative disease.

BACKGROUND OF THE INVENTION

[0005] Neurodegenerative diseases are age-related conditions characterized by the gradual loss of specific neuronal types of neurons while sparing others similar to the ones dying.

Neurodegenerative disorders such as Frontotemporal Lobar Degeneration (FTLD), Limbic- predominant Age-Related TDP-43 Encephalopathy (LATE), Frontotemporal Dementia (FTD), and Amyotrophic Lateral Sclerosis (ALS) are characterized by the progressive loss of neurons and function. A key pathological feature shared by these conditions is the abnormal reduction of TDP-43 protein in the nucleus and/or accumulation in aggregates. TDP-43, normally involved in RNA processing, becomes pathologically altered through phosphorylation, ubiquitination, and cleavage, leading to its aggregation in neurons and glial cells. This accumulation disrupts cellular functions and particularly splicing regulation, contributing to the neurodegenerative processes underlying these diseases. Frontotemporal dementia (FTD) and Amyotrophic Lateral Sclerosis (ALS) are closely related neurodegenerative diseases with shared clinical manifestations, molecular changes, and genetic mutations. Thus, patients lie on an FTD/ ALS continuum with cognitive and motor manifestations. Eventually, all FTD/ ALS patients with reduced mobility develop difficulties in swallowing due to tongue weakness due to the degeneration of hypoglossal motor neurons. However, they communicate with eye-tracking devices because the motor neurons that innervate the eye muscles are relatively resistant to degeneration. Thus, differential neuronal sensitivity to FTD/ ALS is a paradoxical case to understand why some neurons degenerate while others resist.

[0006] Understanding why some neuronal types are more sensitive to neurodegeneration than others remains a challenging and understudied problem, hindering comprehension of these diseases and potential therapeutic strategies.

SUMMARY OF THE INVENTION

[0007] As specified in the Background section above, there is a need to understand how different neuronal types respond to the stress associated with neurodegenerative diseases such as FTD/ ALS and to identify therapeutic strategies for treating neurodegenerative diseases. The present application addresses these and other needs.

[0008] In one aspect, provided herein is a method of treating a disease characterized by TAR DNA-binding protein 43 (TDP-43) mislocalization or dysfunction in a subject in need thereof, the method comprising providing to cells or tissues or organs of the subject having TDP-43 mislocalization or dysfunction a wild-type protein selected from neuro-oncological ventral antigen 1 (NOVAI), embryonic lethality and abnormal visual like protein 1 (ELAVL1), ELAVL2, ELAVL3, ELAVL4, RNA binding fox-1 homolog 2 (RBFOX2), matrin 3 (MATR3), Heterogeneous nuclear ribonucleoprotein Al (HNRNPA1), splicing factor proline- and glutamine-rich (SFPQ), Fused in Sarcoma (FUS) or a functional fragment thereof, or any combination thereof, or one or more nucleic acid molecules encoding the said wild-type protein or the functional fragment thereof.

[0009] In some embodiments, the disease is a neurodegenerative disease. [0010] In some embodiments, the neurodegenerative disease is Amyotrophic Lateral Sclerosis (ALS), Frontotemporal Lobar Degeneration (FTLD), Frontotemporal dementia (FTD), Limbic-predominant Age-Related TDP-43 Encephalopathy (LATE), Chronic Traumatic Encephalopathy (CTE), or Alzheimer’s Disease (AD).

[0011] In some embodiments, providing said protein(s) or nucleic acid molecules(s) involves administering a gene therapy to the subject.

[0012] In some embodiments, the nucleic acid molecule is contained within a viral vector which is administered to the subject.

[0013] In some embodiments, the viral vector is a lentiviral vector, an adenoviral vector, or an adeno-associated viral (AAV) vector.

[0014] In some embodiments, the viral vector is an adeno-associated viral (AAV) vector.

[0015] In some embodiments, the AAV vector has tropism for neural tissue or neurons.

[0016] In some embodiments, the adeno-associated viral vector is AAV9 or AAV-B1.

[0017] In some embodiments, the viral vector is administered to the subject intracranially, systemically, or intrathecally.

[0018] In some embodiments, the intracranial or intrathecal administration of the viral vector results in the appearance of the viral vector in the cerebrospinal fluid of the subject.

[0019] In some embodiments, the nucleic acid molecule is operably linked to a promoter.

[0020] In some embodiments, the promoter is an inducible promoter or a constitutive promoter.

[0021] In some embodiments, the promoter is a tissue-specific promoter or a cell-specific promoter.

[0022] In some embodiments, the promoter is a neural tissue-specific promoter or a neuronspecific promoter.

[0023] In some embodiments, the promoter is a synapsin promoter.

[0024] In some embodiments, the promoter is a human ubiquitin C (hUBC) promoter; a chicken [Lactin promoter, CMV enhancer, and rabbit [3-globin splice acceptor (pCAG) promoter; a human cytomegalovirus (HCMV) promoter; a mouse phosphoglycerate kinase (mPGK) promoter; or a homeobox gene (Hb9) promoter.

[0025] In some embodiments, the wild-type protein is NOVAI and/or ELAVL2.

[0026] In some embodiments, the NOVAl is NOVAl P51513-4. [0027] In some embodiments, the cells are neurons.

[0028] In some embodiments, the neurons are motor neurons.

[0029] In some embodiments, the motor neurons are hypoglossal motor neurons and/or oculomotor motor neurons.

[0030] In some embodiments, the subject is a mammal.

[0031] In some embodiments, the mammal is a human.

[0032] In one aspect, provided herein is a method of treating a disease characterized by TAR DNA-binding protein 43 (TDP-43) mislocalization or dysfunction in a subject in need thereof, the method comprising providing to cells or tissues or organs of the subject having TDP-43 mislocalization or dysfunction a means for restoring misregulated splicing events to wild-type splicing events of one or more of NOVAI, ELAVL1, ELAVL2, ELAVL3, ELAVL4, RBFOX2, MATR3, HNRNPA1, SFPQ and FUS.

[0033] In some embodiments, the restoring misregulated splicing events to wild-type splicing events comprises skipping of an abnormal exon and/or cryptic exons and/or favoring inclusion of a normal exon.

[0034] In some embodiments, the disease is a neurodegenerative disease.

[0035] In some embodiments, the neurodegenerative disease is Amyotrophic Lateral Sclerosis (ALS), Frontotemporal Lobar Degeneration (FTLD), Limbic-predominant Age-Related TDP-43 Encephalopathy (LATE), Chronic Traumatic Encephalopathy (CTE), Frontotemporal dementia (FTD), or Alzheimer's Disease (AD).

[0036] In some embodiments, the means for restoring misregulated splicing events to wildtype splicing events:

(i) enhance expression and/or function of one or more splicing factors and/or splicing regulators, and/or

(ii) decrease expression and/or function of one or more mislocalized or dysfunctional splicing factors and/or splicing regulators.

[0037] In some embodiments, the means for restoring misregulated splicing events to wildtype splicing events is administered intrathecally.

[0038] In some embodiments, the means for restoring misregulated splicing events to wildtype splicing events comprises one or more splicing modifiers and/or antisense oligonucleotide(s) (ASO(s)). [0039] In some embodiments, the one or more ASO(s) is(are) splice- switching ASO(s) and/or restore normal splicing.

[0040] In some embodiments, the one or more ASO(s) is(are) targeted to pre-mRNA of one or more of NO VAI, ELAVL1, ELAVL2, ELAVL3, ELAVL4, RBFOX2, MATR3, HNRNPA1, SFPQ and FUS.

[0041] In some embodiments, the one or more ASO(s) is(are) targeted to one or more exons of one or more pre-mRNA of NO VAI, ELAVL1, ELAVL2, ELAVL3, ELAVL4, RBFOX2, MATR3, HNRNPA1, SFPQ and FUS.

[0042] In some embodiments, the one or more ASO(s) is(are) targeted to pre-mRNA of NOVAI and/or EL A VL2.

[0043] In some embodiments, the one or more ASO(s) is(are) targeted to: a) exon 8 of NOVAI pre-mRNA; b) exon 6 of ELAVL1 pre-mRNA; c) exon 6 of ELAVL2 pre-mRNA; d) exon 10 and 11 of ELAVL4 pre-mRNA; e) exon 3 of RBFOX2 pre-mRNA; f) exon 15 of MATR3 pre-mRNA; g) exon 6 of HNRNPA1 pre-mRNA; h) exon 9 of SFPQ pre-mRNA; i) exon 4 of ELAVL3 pre-mRNA; and/or j) exon 3 and exon 7 of FUS pre-mRNA.

[0044] In some embodiments, the one or more ASO(s) binds to a pre-mRNA sequence flanking a mis-spliced exon of one or more of NOVAI, ELAVL1, ELAVL2, ELAVL3, ELAVL4, RBFOX2, MATR3, HNRNPA1, SFPQ and FUS.

[0045] In some embodiments, the one or more ASO(s) is(are) optimized by tiling relevant splice junctions of one or more pre-mRNA of NOVAI, ELAVL1, ELAVL2, ELAVL3, ELAVL4, RBFOX2, MATR3, HNRNPA1, SFPQ and FUS.

[0046] In some embodiments, the NOVAl is NOVAl P51513-4.

[0047] In some embodiments, the cells are neurons.

[0048] In some embodiments, the neurons are motor neurons. [0049] In some embodiments, the motor neurons are hypoglossal motor neurons and/or oculomotor motor neurons.

[0050] In some embodiments, the subject is a mammal.

[0051] In some embodiments, the mammal is a human.

[0052] In some embodiments, the method further comprises identifying the subject as having a disease characterized by TAR DNA-binding protein 43 (TDP-43) mislocalization or dysfunction.

[0053] In some embodiments, the identifying comprises determining level(s) of (i) one or more clinical biomarkers generated by splicing defects in a sample isolated from the subject, and/or (ii) neopeptides and/or neoantigens, and/or antibodies and/or T cells recognizing neopeptides and/or neoantigens, in a sample isolated from the subject.

[0054] In some embodiments, the one or more clinical biomarkers comprise peptide inclusions comprising mis-spliced proteins.

[0055] In some embodiments, the mis-spliced proteins comprise one or more of NOVAI, ELAVL1, ELAVL2, ELAVL3, ELAVL4, RBFOX2, MATR3, HNRNPA1, SFPQ and FUS. [0056] In some embodiments, the neopeptides and/or neoantigens comprise mis-spliced proteins.

BRIEF DESCRIPTION OF THE DRAWINGS

[0057] Figures 1A-1G show generating and validating inducible Neurog2-Isll-Phox2a (NIP) and Neurog2-Isll-Lhx3 (NIL) induced pluripotent stem cell (iPSC) lines. (Figure 1A) Schematic for line generation, selection, and screening. iPSCs are transfected by pubic transposase with NIP or NIL expression cassettes on day 0. From day 0 to day 5, blasticidin selection removes cells without a NIP or NIL insertion. Clones are picked and expanded. NIP and NIL cassette expression is then induced for 2 days with doxycycline. Clones with low Islet-1 (Isl 1) induced expression are eliminated and the rest are differentiated for 10 days. Clones with low vesicular acetylcholine transporter (VAChT) expression are eliminated and those with the highest expression kept as new NIP or NIL inducible iPSC lines. (Figure IB) Schematic for differentiation protocol (Figure 1C) Representative images of p3-tubulin, microtubule associated protein 2 (MAP2), and VAChT-tdTomato in day 10 NIP iCrMNs and NIL iSpMNs. Scale bar = 100 pm (Figure ID) Principal Component Analysis (PCA) of iPSC NIP, iPSC NIL, iNeurog2/l, hypoglossal motor neuron (hMN) VAChT-tdT day 10 (circle), hMN VAChT-tdT day 30 (triangle), oMN VAChT-tdT day 10 (circle), oculomotor motor neuron (oMN) VAChT-tdT day 30 (triangle), DESeq2 normalized mRNA expression. (Figure IE) Volcano plot of genes differentially expressed in hMN day 10 (log2 fold change < -1.5, -logioP > 0.05) and oMN day 10 (log2 fold change > 1.5, -logioP > 0.05). (Figure IF) Heatmap showing the expression levels of selected iPSC, neuron, hMN, and oMN marker genes for 5 different cell types: iPSC, induced NIL on day 10 and 30, induced NIP on day 10 and 30. Gene expression values were normalized using z-score transformation across samples for each gene. Cell types are grouped along the columns, and marker genes are grouped along rows. (Figure 1G) Principle Component Analysis (PCA) of single cells from hMN day 10, day 30, and oMN day 10, day 30 normalized mRNA expression.

[0058] Figures 2A-2K depict cell type specific splicing changes after n-carbobenzoxy-1- leucyl-l-leucyl-l-leucinal (MG132, also known as Z-Leu-Leu-Leu-al) induced TDP43 nuclear depletion in hMNs and oMNs. (Figure 2A) Experimental outline: oMNs and hMNs are differentiated from day 0 to day 10 and treated with 1.0 pM (oMN) or 1.5 pM (hMN) MG132 on day 10 and collected 24 hours later for RNA-seq followed by differential splicing analysis using LeafCutter. (Figure 2B) LeafCutter analysis of differentially spliced intron clusters in stathmin-2 (STMN2) in hMNs and oMNs. Black boxes represent boxes. Change in percent spliced in (APSI). Dark grey lines represent positive APSI, light gray lines represent negative APSI.

(Figure 2C) Box plot of STMN2 DESeq2 normalized count in MG132 treated and dimethyl sulfoxide (DMSO) control hMNs and oMNs (n = 4 replicates, Welch Two sample t-test).

(Figure 2D) Volcano plot showing genes differentially expressed between DMSO control and MG132 treatment in hMN and oMN (|log2 fold change) > 0, -logioP > 0.05). Selected genes are highlighted: STMN2, potassium voltage-gated channel subfamily Q member 2 (KCNQ2), Unc- 13 homolog A (UNC I 3 A). (Figure 2E) Pie chart showing the proportion of different splicing events in MG132 treated hMNs and oMNs compared to control. Splicing events are defined by Leafcutter and include Annotated, Cryptic 5 ’, Cryptic 3 ’, Novel annotated pair, and Two cryptic sites. (Figure 2F) Total number of splicing defects in MG132 treated hMNs (1440 genes) and oMNs (1098 genes) versus control (Figure 2G) MG132 versus DMSO differentially spliced genes (DSGs) specific to hMNs (666 genes), oMNs (324 genes), and shared (774 genes).

(Figures 2H-2J) Gene ontology (GO) enriched molecular functions for shared (Figure 2H) hMN-specific (Figure 21) and oMN-specific (Figure 2J) DMSO vs MG132 DSGs. (Figure 2K) Sensitive hMNs enter a downward spiral of proteotoxic stress and disrupted splicing leading to degeneration.

[0059] Figures 3A-3H show TDP-43 binding in hMNs and oMNs. (Figure 3A) RNA immunoprecipitation sequencing (RIP-seq) experimental outline for TDP-43 in hMNs versus oMNs. (Figure 3B) TDP-43 RIP-seq BigWig coverage reads for UNC13A and STMN2 in hMNs and oMNs. (Figure 3C) TDP-43 binding motif in hMNs and oMNs. (Figure 3D) Total number of TDP-43 bound genes in hMNs (10189 genes) and oMNs (10673 genes). (Figure 3E) TDP-43 bound genes specific to hMNs (1408 genes), oMNs (1964 genes), and shared (8709 genes). (Figure 3F) Proportion of cell type mis-spliced genes that are bound by TDP-43 in hMNs (64%, 425 genes), and oMNs (65%, 212 genes). (Figures 3G-3H) Gene ontology (GO) enriched molecular functions for hMN specific (Figure 3G) and oMN specific (Figure 3H) TDP-43 bound mis-spliced genes.

[0060] Figures 4A-4K show cell type specific splicing changes over time in TDP43^M337X and C9orf72 HRE oMNs and hMNs. (Figure 4A) Experimental outline: mutant hMNs and oMNs are differentiated to day 10 and day 30 and collected for RNA-seq followed by differential splicing analysis using LeafCutter. (Figure 4B) Box plot of STMN2 DESeq2 normalized count in hMNs day 10 and day 30, oMNs day 10 and day 30 (n = 4 replicates, Welch Two sample t-test). ns = not significant, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. (Figure 4C) Total number of splicing defects in TDP43^M337V hMNs (284 genes) and oMNs (202 genes) day 10 compared to wild-type (Figure 4D) TDP43^M337V versus wild-type differentially spliced genes (DSGs) specific to day 10 hMNs (210 genes), oMNs (128 genes), and shared (74 genes). (Figure 4E) Pie chart showing the proportion of different splicing events in day 10 TDP43^M337 hMNs and oMNs compared to wild-type. (Figure 4F) Gene ontology (GO) enriched molecular functions for shared day 10 hMN-specific TDP43^M337V versus wild-type mis-spliced genes. (Figure 4G) Total number of splicing defects C9orf72 HRE hMNs (821 genes) and oMNs (513 genes) day 30 compared to wild-type (Figure 4H) C9orf72 HRE versus wild-type differentially spliced genes (DSGs) specific to day 30 hMNs (506 genes), oMNs (198 genes), and shared (315 genes).

(Figure 41) Pie chart showing the proportion of different splicing events in day 30 C9orf72 HRE hMNs and oMNs compared to wild-type. (Figure 4J) Gene ontology (GO) enriched molecular functions for shared day 30 hMN-specific C9orf72 HRE versus wild-type mis-spliced genes. (Figure 4K) Proportion of shared mis-spliced genes in MG132 treated versus mutant hMNs and oMNs.

[0061] Figures 5A-5F show spinal cord postmortem dissection splicing changes in ALS patients versus control cohort. (Figure 5A) Pie chart showing the proportion of different splicing events in ALS spinal cord compared to control. (Figure 5B) Gene ontology (GO) enriched molecular functions for ALS spinal cord versus control mis-spliced genes. (Figure 5C) Proportion of splicing defects in vivo spinal cords overlapping with splicing defects in vitro MG132 treated hMNs (17%), TDP43^M337V hMN day 10 (3%), TDP43^M337V hMN day 30 (10%), C9orf72 HRE hMN day 30 (8%). (Figure 5D) Proportion of splicing defects in vitro MG132 treated hMNs (70%), TDP43^M337V hMN day 10 (78%), TDP43^M337V hMN day 30 (72%), C9orf72 HRE hMN day 30 (73%) overlapping with in vivo spinal cords. (Figure 5E) Gene ontology (GO) enriched molecular functions for shared mis-spliced genes between in vivo spinal cords and in vitro hMNs. Selected RNA binding GO terms. (Figure 5F) Heatmap showing normalized mRNA expression log2fold change between ALS versus control spinal cord and MG132 treated versus DMSO control for selected splicing regulators.

[0062] Figures 6A-6B show enhancing neuronal resistance to neurodegeneration by rescuing the neurodegenerative cascade. (Figure 6A) Illustration of the splicing cascade in healthy neurons. TDP-43 regulates the splicing and mRNA levels of essential splicing regulators, ensuring a healthy neuronal transcriptome. (Figure 6B) During TDP-43 dysfunction, the levels and activity of these splicing regulators decrease, exacerbating the splicing errors caused by TDP-43 malfunction. Reintroducing these critical splicing regulators, as described in panel (Figure 6A), helps mitigate splicing defects and restores the health of the neuronal transcriptome.

[0063] Figures 7A-7J show splicing events that can be corrected by the antisense oligonucleotides of the present invention. The arrows in the figures indicate the exons that are mis-regulated. These mis-regulated exons can be corrected by, e.g., the ASOs of the present disclosure.

[0064] Figures 8A-8D show a critical role for NOVAI in sensitive neurons. (Figure 8A) scRNAseq: N0VA1 expression in hMNs (Days in culture). (Figure 8B) N0VA1 splicing defects induced by TDP-43 mutations. (Figure 8C) NOVAI splicing defects induced by MG- 132. (Figure 8D) NOVAI mRNA levels were reduced by MG-132 stress and a TDP-43 mutation.

DETAILED DESCRIPTION

[0065] As described in the Background section above, there is a need to understand how sensitive (hypoglossal-like) and resistant (oculomotor-like) motor neurons and cortical neurons respond to the stress associated with a disease characterized by TDP-43 mislocalization or dysfunction such as, but not limited to, neurodegenerative diseases. Exemplary neurodegenerative diseases include Alzheimer’s disease (AD), Parkinson’s disease, amyotrophic lateral sclerosis (ALS), Frontotemporal Lobar Degeneration (FTLD), Limbic-predominant Age- Related TDP-43 Encephalopathy (LATE), Frontotemporal Dementia (FTD), Chronic Traumatic Encephalopathy (CTE), Huntington’s disease, spinobulbar muscular atrophy, dentatorubral pallidoluysian atrophy, Kennedy disease, and spinocerebellar ataxias. Additionally, there is a need to improve the neuronal response to neurodegenerative stress.

[0066] Similar neuronal types exhibit contrasting responses to neurodegeneration. Dr. Stephen Hawking is an atypical case, yet a well-known example. He experienced severe speech and swallowing difficulties caused by hypoglossal motor neuron (hMN) loss but communicated using eye-tracking devices thanks to resilient oculomotor motor neurons (oMNs).

[0067] Splicing defects are a key cell-autonomous neuronal contributor to neurodegeneration. The studies described herein illustrate correction of splicing as a therapeutic strategy for treating neurodegenerative disorders. TDP-43 is prone to aggregation, forming seeds to recruit more TDP-43¹⁰⁶, and even low levels of TDP-43 overexpression cause neurodegeneration in mice. [0068] The invention of the present disclosure illustrates rescuing specific splicing defects downstream of TDP-43 as a therapeutic intervention. The inventors hypothesized that rescuing defects in splicing factors affected by TDP-43 depletion could improve a significant fraction of the neuronal transcriptome affected by TDP-43 depletion. To regulate splicing in neurodegeneration, splicing regulators and/or splicing factors downstream of TDP-43 are overexpressed. An overexpression of splicing regulators and/or splicing factors can be achieved by cDNA expression delivered by viral vectors or restoring their splicing by alternative spliceswitching oligonucleotides (ASOs). The splicing regulators and/or splicing factors include, without limitation, neuro-oncological ventral antigen 1 (N0VA1), embryonic lethality and abnormal visual like protein 1 (ELAVL1), ELAVL2, ELAVL3, ELAVL4, RNA binding fox-1 homolog 2 (RBFOX2), matrin 3 (MATR3), heterogeneous nuclear ribonucleoprotein Al (HNRNPA1), splicing factor proline- and glutamine-rich (SFPQ), fused in sarcoma (FUS) or functional fragments thereof, or any combination thereof, or one or more nucleic acid molecules encoding any of the aforementioned splicing regulators and/or splicing factors. In some embodiments, the N0VA1 is NOVAl P51513-4.

Definitions

[0069] To facilitate an understanding of the principles and features of the various embodiments of the invention, various illustrative embodiments are explained below. Although exemplary embodiments of the invention are explained in detail, it is to be understood that other embodiments are contemplated. Accordingly, it is not intended that the invention is limited in its scope to the details of construction and arrangement of components set forth in the following description or examples. The invention is capable of other embodiments and of being practiced or carried out in various ways. Also, in describing the exemplary embodiments, specific terminology will be resorted to for the sake of clarity.

[0070] Unless otherwise defined herein, scientific and technical terms used in connection with the present invention shall have the meanings that are commonly understood by those of ordinary skill in the art. Further, unless otherwise required by context, singular terms shall include pluralities and plural terms shall include the singular. Generally, nomenclatures used in connection with, and techniques of, cell and tissue culture, molecular biology, immunology, microbiology, genetics and protein and nucleic acid chemistry and hybridization described herein are those well-known and commonly used in the art.

[0071] The term “polynucleotide” or “nucleotide sequence” mean a series of nucleotide bases (also called “nucleotides”) in DNA and RNA and mean any chain of two or more nucleotides. A nucleotide sequence typically carries genetic information, including the information used by cellular machinery to make proteins and enzymes. The term “polynucleotide” or “nucleotide sequence” as referred to herein means a polymeric form of nucleotides of at least 10 bases in length, either ribonucleotides or deoxyribonucleotides or a modified form of either type of nucleotide. These terms include double or single stranded genomic and cDNA, RNA, any synthetic and genetically manipulated polynucleotide, and both sense and anti-sense polynucleotide. This includes single- and double-stranded molecules, i.e., DNA-DNA, DNA- RNA, and RNA-RNA hybrids.

[0072] The term “isolated polynucleotide” as used herein means a polynucleotide of genomic, cDNA, or synthetic origin or some combination thereof, which by virtue of its origin or source of derivation, the “isolated polynucleotide” has one to three of the following: (1) is not associated with all or a portion of a polynucleotides with which the “isolated polynucleotide” is found in nature, (2) is operably linked to a polynucleotide to which it is not linked in nature, or (3) does not occur in nature as part of a larger sequence. “Operably linked” sequences include both expression control sequences that are contiguous with the gene of interest and expression control sequences that act in trans or at a distance to control the gene of interest.

[0073] The term “gene” as used herein means a DNA sequence that codes for a particular noncoding (untranslated) RNA or a sequence of amino acids, which comprise all or part of one or more proteins or enzymes and may include regulatory (non-transcribed) DNA sequences, such as promoter sequences, which determine for example the conditions under which the gene is expressed.

[0074] The term “oligonucleotide” as used herein, refers to a nucleic acid, generally of at least 8, preferably no more than 100 nucleotides, which is hybridizable to a genomic DNA molecule, a cDNA molecule, or an RNA molecule. Oligonucleotides can be labeled, e.g., with ³²P- nucleotides or nucleotides to which a label, such as biotin, has been covalently conjugated. In one embodiment, a labeled oligonucleotide can be used as a probe to detect the presence of a nucleic acid. In another embodiment, oligonucleotides can be used as PCR primers, either for cloning or for detection of a specific nucleic acid. In a further embodiment, an oligonucleotide of the invention can be used as an antisense oligonucleotide to inhibit a function or expression of a nucleic acid molecule. Generally, oligonucleotides are prepared synthetically, for example without limitation, on a nucleic acid synthesizer. Accordingly, oligonucleotides can be prepared with non-naturally occurring phosphoester analog bonds, such as thioester bonds, etc.

[0075] The terms “express” and “expression” mean allowing or causing the information in a gene or DNA sequence to become manifest, for example, producing a non-coding (untranslated) RNA or a protein by activating the cellular functions involved in transcription and translation of a corresponding gene or DNA sequence. A DNA sequence is expressed in or by a cell to form an “expression product” such as RNA or a protein. The expression product itself, e.g., the resulting RNA or protein, may also be said to be “expressed” by the cell.

[0076] The term “expression control sequence” as used herein means polynucleotide sequences that are necessary to effect the expression and/or processing of coding sequences to which they are ligated. Expression control sequences include, without limitation, appropriate transcription initiation, termination, promoter and enhancer sequences; efficient RNA processing signals such as splicing and polyadenylation signals; sequences that stabilize cytoplasmic mRNA; sequences that enhance translation efficiency (e.g., Kozak consensus sequence); sequences that enhance protein stability; and when desired, sequences that enhance protein secretion. The nature of such control sequences differs depending upon the host organism; in prokaryotes, such control sequences generally, can include promoter, ribosomal binding site, and transcription termination sequences; in eukaryotes, generally, such control sequences can include promoters and transcription termination sequence.

[0077] The term “control sequences” is intended to include, at a minimum, all components whose presence is essential for expression and/or processing and can also include additional components whose presence is advantageous, for example, leader sequences and fusion partner sequences.

[0078] A sequence “encoding” an expression product, such as an RNA, polypeptide, protein, or enzyme, is a minimum nucleotide sequence that, when expressed, results in the production of that RNA, polypeptide, protein, or enzyme.

[0079] The term “antisense” nucleic acid molecule or oligonucleotide as used herein refers to a single stranded (ss) nucleic acid molecule, which may be DNA, RNA, a DNA-RNA chimera, or a derivative thereof, which, upon hybridizing under physiological conditions with complementary bases in an RNA or DNA molecule of interest, inhibits or activates (in the case of “activating antisense oligonucleotides”) the expression of the corresponding gene by modulating, e.g., RNA transcription, RNA processing such as without limitation splicing, RNA transport, mRNA translation, or RNA stability.

[0080] The term “vector,” as used herein, means a vehicle capable of transporting a nucleic acid into a host cell. Vectors include plasmids, cosmids, phages, viruses, etc. Vectors may further comprise selectable markers. In some embodiments, the vector is a plasmid, e.g., a circular double stranded DNA loop into which additional DNA segments may be ligated. In some embodiments, the vector is a viral vector, and additional DNA segments may be ligated into the viral genome. In some embodiments, the vectors are capable of autonomous replication in a host cell into which they are introduced (e.g., bacterial vectors having a bacterial origin of replication and episomal mammalian vectors). In other embodiments, the vectors (e.g., non-episomal mammalian vectors) can be integrated into the genome of a host cell upon introduction into the host cell, and thereby are replicated along with the host genome. Moreover, certain vectors are capable of directing the expression of genes to which they are operatively linked. Such vectors are referred to herein as “recombinant expression vectors” (or simply, “expression vectors”). [0081] The term “promoter” as used herein is defined as a DNA sequence recognized by the synthetic machinery of the cell, or introduced synthetic machinery, required to initiate the specific transcription of a polynucleotide sequence. As used herein, the term “regulatory sequence” means a nucleic acid sequence which can regulate expression of a gene product operably linked to the regulatory sequence. In some instances, this sequence may be the core promoter sequence and in other instances, this sequence may also include an enhancer sequence and other regulatory elements which are required for expression of the gene product. The promoter or regulatory sequence may, for example, be one which expresses the gene product in a tissue specific manner.

[0082] The term “recombinant host cell” (or simply “host cell”), as used herein, means a cell into which an exogenous nucleic acid and/or recombinant vector has been introduced. It should be understood that “recombinant host cell” and “host cell” mean not only the particular subject cell but also the progeny of such a cell. Because certain modifications may occur in succeeding generations due to either mutation or environmental influences, such progeny may not, in fact, be identical to the parent cell, but are still included within the scope of the term “host cell” as used herein.

[0083] The term “percent sequence identity” means a ratio, expressed as a percent of the number of identical residues over the total number of residues compared. Sequence identity for nucleic acid sequences may be analyzed over a stretch of at least about nine nucleotides, usually at least about 18 nucleotides, more usually at least about 24 nucleotides, typically at least about 28 nucleotides, more typically at least about 32 nucleotides, and preferably at least about 36, 48, or more nucleotides. There are a number of different algorithms known in the art which can be used to measure nucleotide sequence identity. For instance, polynucleotide sequences can be compared using FASTA, Gap, or Bestfit, which are programs in Wisconsin Package Version 10.0, Genetics Computer Group (GCG), Madison, Wis. FASTA, which includes, e.g., the programs FASTA2 and FASTA3, provides alignments and percent sequence identity of the regions of the best overlap between the query and search sequences (Pearson, Methods Enzymol. 183:63-98 (1990); Pearson, Methods Mol. Biol. 132: 185-219 (2000); Pearson, Methods Enzymol. 266:227-258 (1996); Pearson, J. Mol. Biol. 276:71-84 (1998); herein incorporated by reference). Unless otherwise specified, default parameters for a particular program or algorithm are used. For instance, percent sequence identity between nucleic acid sequences can be determined using FASTA with its default parameters (a word size of 6 and the NOPAM factor for the scoring matrix) or using Gap with its default parameters as provided in GCG Version 6.1, herein incorporated by reference. A reference to a nucleotide sequence encompasses its complement unless otherwise specified. Thus, a reference to a nucleic acid having a particular sequence should be understood to encompass its complementary strand, with its complementary sequence. Sequence identity for polypeptides, is typically measured using sequence analysis software. Protein analysis software matches sequences using measures of similarity assigned to various substitutions, deletions and other modifications, including conservative amino acid substitutions. For instance, GCG contains programs such as “Gap” and “Bestfit” which can be used with default parameters, as specified with the programs, to determine sequence homology or sequence identity between closely related polypeptides, such as homologous polypeptides from different species of organisms or between a wild-type protein and a mutein thereof. See, e.g., GCG Version 6.1. Polypeptide sequences also can be compared using FASTA using default or recommended parameters, see GCG Version 6.1. (University of Wisconsin Wis.) FASTA (e.g., FASTA2 and FASTA3) provides alignments and percent sequence identity of the regions of the best overlap between the query and search sequences (Pearson, Methods Enzymol. 183:63-98 (1990); Pearson, Methods Mol. Biol. 132: 185-219 (2000)). Another preferred algorithm when comparing a sequence of the invention to a database containing a large number of sequences from different organisms is the computer program BLAST, especially blastp or tblastn, using default parameters, as supplied with the programs. See, e.g., Altschul etal., J. Mol. Biol. 215:403-410 (1990); Altschul et al., Nucleic Acids Res. 25:3389-402 (1997).

[0084] The length of polypeptide sequences compared for homology will generally be at least about 16 amino acid residues, usually at least about 20 residues, more usually at least about 24 residues, typically at least about 28 residues, and preferably more than about 35 residues. When searching a database containing sequences from a large number of different organisms, it is preferable to compare amino acid sequences.

[0085] The term “substantial similarity” or “substantial sequence similarity”, when referring to a nucleic acid or fragment thereof, means that when optimally aligned with appropriate nucleotide insertions or deletions with another nucleic acid (or its complementary strand), there is nucleotide sequence identity in at least about 85%, preferably at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of the nucleotide bases, as measured by any well- known algorithm of sequence identity, such as FASTA, BLAST, or Gap, as discussed above. As applied to polypeptides, the term “substantial identity” means that two peptide sequences, when optimally aligned, such as by the programs GAP or BESTFIT using default gap weights, as supplied with the programs, share at least 70%, 75%, 80%, or 85% sequence identity, preferably at least 90%, 91%, 92%, 93%, 94% 95%, 96%, 97%, 98%, or 99% sequence identity. In certain embodiments, residue positions that are not identical differ by conservative amino acid substitutions.

[0086] The terms “treat” or “treatment” of a state, disease, disorder, or condition, or the like, include: (1) preventing, delaying, or reducing the incidence and/or likelihood of the appearance of at least one clinical or sub-clinical symptom of the state, disease, disorder, or condition developing in a subject that may be afflicted with or predisposed to the state, disease, disorder, or condition but does not yet experience or display clinical or subclinical symptoms of the state, disease, disorder, or condition; or (2) inhibiting the state, disease, disorder, or condition, e.g., arresting, reducing, or delaying the development of the state, disease, disorder, or condition; or a relapse thereof (in case of maintenance treatment) or at least one clinical or sub-clinical symptom thereof; or (3) relieving the state, disease, disorder, or condition, e.g., by causing regression of the state, disease, disorder, or condition or at least one of its clinical or sub-clinical symptoms. The benefit to a subject to be treated is either statistically significant or at least perceptible to the patient or to the physician.

[0087] As used herein, the term “isolated” means that the material being referred to has been removed from the environment in which it is naturally found and is characterized to a sufficient degree to establish that it is present in a particular sample. Such characterization can be achieved by any standard technique, such as, e.g., sequencing, hybridization, immunoassay, functional assay, expression, size determination, or the like. Thus, a biological material can be “isolated” if it is free of cellular components, i.e., components of the cells in which the material is found or produced in nature. A nucleic acid molecule excised from the chromosome that it is naturally a part of is considered to be isolated. Such a nucleic acid molecule may or may not remain joined to regulatory, or non-regulatory, or non-coding regions, or to other regions located upstream or downstream of the gene when found in the chromosome. Nucleic acid molecules that have been spliced into vectors such as plasmids, cosmids, artificial chromosomes, phages, and the like are considered isolated. In a particular embodiment, a splicing regulator-encoding nucleic acid spliced into a recombinant vector, and/or transformed into a host cell, is considered to be “isolated”. An isolated material may or may not be “purified”. The term “purified” as used herein refers to a material (e.g, a nucleic acid molecule or a protein) that has been isolated under conditions that detectably reduce or eliminate the presence of other contaminating materials. Contaminants may or may not include native materials from which the purified material has been obtained. A purified material preferably contains less than about 90%, less than about 75%, less than about 50%, less than about 25%, less than about 10%, less than about 5%, or less than about 2% by weight of other components with which it was originally associated.

[0088] Methods for purification are well-known in the art. For example, nucleic acids or polynucleotide molecules can be purified by precipitation, chromatography (e.g.. by affinity chromatography, preparative solid phase chromatography, oligonucleotide hybridization, and triple helix chromatography), ultracentrifugation, and other means. Polypeptides can be purified by various methods including, without limitation, preparative disc-gel electrophoresis, isoelectric focusing, HPLC, reverse-phase HPLC, gel filtration, affinity chromatography, ion exchange and partition chromatography, precipitation and salting-out chromatography, extraction, and countercurrent distribution. Cells can be purified by various techniques, including centrifugation, matrix separation (e.g, nylon wool separation), panning and other immunoselection techniques, depletion (e.g., complement depletion of contaminating cells), and cell sorting (e.g., fluorescence activated cell sorting (FACS)). Other purification methods are possible.

[0089] The term “substantially pure” indicates the highest degree of purity that can be achieved using conventional purification techniques currently known in the art. In the context of analytical testing of the material, “substantially free” means that contaminants, if present, are below the limits of detection using current techniques, or are detected at levels that are low enough to be acceptable for use in the relevant art, for example, no more than about 2-5% (w/w). Accordingly, with respect to the purified material, the term “substantially pure” or “substantially free” means that the purified material being referred to is present in a composition where it represents 95% (w/w) or more of the weight of that composition. Purity can be evaluated by chromatography, gel electrophoresis, immunoassay, composition analysis, biological assay, or any other appropriate method known in the art.

[0090] As used herein, the term “therapeutically effective” applied to dose or amount refers to that quantity of a compound or pharmaceutical composition that is sufficient to result in a desired activity upon administration to a subject in need thereof. Within the context of the present invention, the term “therapeutically effective” refers to that quantity of a compound or pharmaceutical composition that is sufficient to reduce or eliminate at least one symptom of a neurodegenerative disorder. Note that when a combination of active ingredients is administered the effective amount of the combination may or may not include amounts of each ingredient that would have been effective if administered individually.

[0091] As used herein, the phrase “pharmaceutically acceptable” refers to molecular entities and compositions that are generally regarded as physiologically tolerable.

[0092] As used herein, the term “combination” of a compound or a composition, and at least a second pharmaceutically active ingredient means at least two, but any desired combination of compound or composition can be delivered simultaneously or sequentially.

[0093] The terms “patient”, “individual”, “subject”, and “animal” are used interchangeably herein and refer to mammals, including, without limitation, human and veterinary animals (e.g., cats, dogs, cows, horses, goats, sheep, pigs, etc.) and experimental animal models (e.g., rodents such as mice and rats, rabbits, and non-human primates). In a preferred embodiment, the subject is a human.

[0094] As used herein, the term “healthy subject” refers to a subject that is without known disorder (e.g., a neurodegenerative disorder) by using conventional diagnostic methods. In certain embodiments, a healthy subject is a subject without a known first degree relative with a neurodegenerative disorder. In certain embodiments, a matched healthy subject is matched by age, gender, and/or ethnicity.

[0095] The term “carrier” refers to a diluent, adjuvant, excipient, or vehicle with which the compound is administered. Such pharmaceutical carriers can be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable, or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like. Water or aqueous solution saline solutions and aqueous dextrose and glycerol solutions are preferably employed as carriers, particularly for injectable solutions. Alternatively, the carrier can be a solid dosage form carrier, including but not limited to one or more of a binder (for compressed pills), a glidant, an encapsulating agent, a flavorant, and a colorant. Suitable pharmaceutical carriers are described in “Remington’s Pharmaceutical Sciences” by E.W. Martin.

[0096] The term “about” or “approximately” means within a statistically meaningful range of a value. Such a range can be within an order of magnitude, preferably within 50%, more preferably within 20%, still more preferably within 10%, and even more preferably within 5% of a given value or range. The allowable variation encompassed by the term “about” or “approximately” depends on the particular system under study, and can be readily appreciated by one of ordinary skill in the art.

[0097] The terms “a,” “an,” and “the” do not denote a limitation of quantity, but rather denote the presence of “at least one” of the referenced item.

[0098] The practice of the present invention employs, unless otherwise indicated, conventional techniques of statistical analysis, molecular biology (including recombinant techniques), microbiology, cell biology, and biochemistry, which are within the skill of the art. Such tools and techniques are described in detail in e.g., Sambrook et al. (2001) Molecular Cloning: A Laboratory Manual. 3^rd ed. Cold Spring Harbor Laboratory Press: Cold Spring Harbor, New York; Ausubel et al. eds. (2005) Current Protocols in Molecular Biology. John Wiley and Sons, Inc.: Hoboken, NJ; Bonifacino et al. eds. (2005) Current Protocols in Cell Biology. John Wiley and Sons, Inc.: Hoboken, NJ; Coligan etal. eds. (2005) Current Protocols in Immunology, John Wiley and Sons, Inc.: Hoboken, NJ; Coico et al. eds. (2005) Current Protocols in Microbiology, John Wiley and Sons, Inc.: Hoboken, NJ; Coligan et al. eds. (2005) Current Protocols in Protein Science, John Wiley and Sons, Inc.: Hoboken, NJ; Enna et al. eds. (2005) Current Protocols in Pharmacology, John Wiley and Sons, Inc.: Hoboken, NJ; Oligonucleotide Synthesis (Gait ed. 1984); Nucleic Acid Hybridization (Hames and Higgins eds. 1985); Transcription And Translation (Hames and Higgins eds. 1984); Animal Cell Culture (Freshney ed. 1986);

Immobilized Cells And Enzymes (IRL Press, 1986); B. Perbal, A Practical Guide To Molecular Cloning (1984); and Ausubel et al. eds. Additional techniques are explained, e.g., in U.S. Patent No. 7,912,698 and U.S. Patent Appl. Pub. Nos. 2011/0202322 and 2011/0307437.

[0099] Also, in describing the exemplary embodiments, terminology will be resorted to for the sake of clarity. It is intended that each term contemplates its broadest meaning as understood by those skilled in the art and includes all technical equivalents that operate in a similar manner to accomplish a similar purpose.

[00100] It is also to be understood that the mention of one or more method steps does not preclude the presence of additional method steps or intervening method steps between those steps expressly identified. Similarly, it is also to be understood that the mention of one or more components in a composition does not preclude the presence of additional components than those expressly identified.

[00101] The materials described hereinafter as making up the various elements of the present invention are intended to be illustrative and not restrictive. Many suitable materials that would perform the same or a similar function as the materials described herein are intended to be embraced within the scope of the invention. Such other materials not described herein can include, but are not limited to, materials that are developed after the time of the development of the invention, for example. Any dimensions listed in the various drawings are for illustrative purposes only and are not intended to be limiting. Other dimensions and proportions are contemplated and intended to be included within the scope of the invention.

Methods of the invention

[00102] Provided herein are methods of treating a disease characterized by TAR DNA-binding protein 43 (TDP-43) mislocalization or dysfunction in a subject. In some embodiments, the disease is a disease or condition characterized by TDP-43 nuclear depletion. In some embodiments, the disease is a neurodegenerative disease. In certain embodiments, the neurodegenerative disease is a neurodegenerative disease characterized by TDP-43 mislocalization or dysfunction in a subject. In certain embodiments, the neurodegenerative disease is Alzheimer’s disease (AD), Parkinson’s disease, amyotrophic lateral sclerosis (ALS), Frontotemporal Lobar Degeneration (FTLD), Limbic-predominant Age-Related TDP-43 Encephalopathy (LATE), Frontotemporal Dementia (FTD), Chronic Traumatic Encephalopathy (CTE), Huntington’s disease, spinobulbar muscular atrophy, dentatorubral pallidoluysian atrophy, Kennedy disease, and spinocerebellar ataxias.

[001031 TDP-43 is a mainly nuclear RNA binding protein that regulates RNA, including mRNA stability, transcriptional regulation, stress granule formation, microRNA processing, and alternative splicing ⁹- ¹⁰. TDP-43 nuclear depletion leads to FTD/ALS characteristic splicing defects such as those in Stathmin-2 (STMN2) and unc-13 homolog A (UNC13A) ⁿ’¹³. TAR DNA binding protein (TARDBP) (which codes for TDP-43) mutations imply a direct causal relationship between FTD/ALS mutations and the well-characterized splicing defects¹³’¹⁵. The disease-triggering mechanism for chromosome 9 open reading frame 72 (C9orf72) has not been conclusively established. It has been hypothesized to range from loss of function to toxic repeat- associated non-ATG-dependent dipeptide repeat proteins ¹⁶. However, C9orf72 mutant patient neurons contain TDP-43 inclusions and the associated nuclear depletion ¹⁷. Thus, TDP-43 nuclear depletion is likely a downstream bottleneck and a significant factor in C9orf72-initiated FTD/ALS. Moreover, 57% of Alzheimer’s disease cases contain TDP-43 aggregates ¹⁸. Thus, how some neurons control TDP-43 localization and its effect on the neuronal transcriptome may be important to understanding FTD/ALS and other neurodegenerative diseases illustrated herein the present disclosure.

[00104] Splicing defects caused by TDP-43 dysfunction can contribute to the progressive neuronal loss. Rescuing specific splicing defects downstream of TDP-43 can be a promising therapeutic intervention. Correcting splicing defects with the proposed splicing regulations is a more holistic and comprehensive way to help prevent or slow down neurodegeneration by ensuring that neurons can produce the correct set of proteins needed for their survival and function.

[00105] In one aspect provided herein is a method of treating a disease characterized by TDP-43 mislocalization or dysfunction in a subject comprising providing to the cells or tissues or organs of the subject having TDP-43 mislocalization or dysfunction one or more splicing regulators and/or splicing factors. In some embodiments, the method of treating a disease characterized by TDP-43 mislocalization or dysfunction in a subject comprises administering to the cells or tissues or organs of the subject having TDP-43 mislocalization or dysfunction one or more splicing regulators and/or splicing factors. [00106] In some embodiments, one or more splicing regulators and/or splicing factors are administered via a gene therapy. In some embodiments, one or more splicing regulators and/or splicing factors are administered via a vector. In some embodiments, the method of treating a disease characterized by TDP-43 mislocalization or dysfunction in a subject comprises restoring expression and/or function of the one or more splicing regulators and/or splicing factors.

[00107] In some embodiments, the method of treating a disease characterized by TDP-43 mislocalization or dysfunction in a subject comprises providing a means for restoring the misregulated splicing events to wild-type splicing events of one or more splicing regulators and/or splicing factors in the cells or tissues or organs of the subject having TDP-43 mislocalization or dysfunction. In some embodiments, the restoring misregulated splicing events to wild-type splicing events involves skipping of abnormal exons and/or cryptic exons and/or favoring inclusion of normal exons. In some embodiments, the means for restoring the misregulated splicing events to wild-type splicing events of one or more splicing regulators and/or splicing factors (i) enhances expression and/or function of one or more splicing factors and/or splicing regulators, and/or (ii) decreases expression and/or function of one or more mislocalized or dysfunctional splicing factors and/or splicing regulators.

[00108] In some embodiments, the method of treating a disease characterized by TDP-43 mislocalization or dysfunction in a subject comprises enhancing expression and/or function of a correctly spliced form of one or more splicing regulators and/or splicing factors in the cells or tissues or organs of the subject having TDP-43 mislocalization or dysfunction. In some embodiments, the method of treating a disease characterized by TDP-43 mislocalization or dysfunction in a subject comprises decreasing expression and/or function of one or more incorrectly spliced or mis-spliced splicing regulators and/or splicing factors in the cells of the subject having TDP-43 mislocalization or dysfunction.

[00109] In some embodiments, the one or more splicing regulators and/or splicing factors are restored. In some embodiments, the one or more splicing regulators and/or splicing factors are correctly spliced. In some embodiments, the one or more splicing regulators and/or splicing factors are the mis-spliced splicing regulators and/or splicing factors that are generated by mislocalized or dysfunctional TDP-43. In some embodiments, the one or more splicing regulators and/or splicing factors are the mis-spliced splicing regulators and/or splicing factors that are generated by TDP-43 depletion. In some embodiments, the one or more splicing regulators and/or splicing factors are the mis-spliced splicing regulators and/or splicing factors that are generated by TDP-43 -induced splicing defect(s). In some embodiments, the one or more splicing factors are hypoglossal motor neuron (hMN)-specific splicing factors. In some embodiments, the one or more splicing factors are neuron-specific splicing factors. In some embodiments, the one or more splicing factors are general splicing factors. In some embodiments, the one or more splicing factors are ocular motor neuron (oMN)-specific splicing factors. In some embodiments, the one or more splicing regulators and/or splicing factors are selected from neuro-oncological ventral antigen 1 (N0VA1), embryonic lethality and abnormal visual like protein 1 (ELAVL1), ELAVL2, ELAVL3, ELAVE4, RNA binding fox-1 homolog 2 (RBF0X2), matrin 3 (MATR3), heterogeneous nuclear ribonucleoprotein Al (HNRNPA1), splicing factor proline- and glutamine-rich (SFPQ), and fused in Sarcoma (FUS), or a functional fragment thereof, or any combination thereof, or one or more nucleic acid molecules encoding any of the aforementioned splicing regulators and/or splicing factors.

[00110] The term “one or more” as used in the present application includes, but is not limited to, two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, or all. In some embodiments, the two or more splicing regulators and/or splicing factors are selected from NOVAI, ELAVL1, ELAVL2, ELAVL3, ELAVL4, RBFOX2, MATR3, HNRNPA1, SFPQ, and FUS, or a functional fragment thereof, or one or more nucleic acid molecules encoding any of the said splicing regulators and/or splicing factors. In some embodiments, the three or more splicing regulators and/or splicing factors are selected from NO VAI, ELAVL1, ELAVL2, ELAVL3, ELAVL4, RBFOX2, MATR3, HNRNPA1, SFPQ, and FUS, or a functional fragment thereof, or one or more nucleic acid molecules encoding any of the said splicing regulators and/or splicing factors. In some embodiments, the four or more splicing regulators and/or splicing factors are selected from NOVAI, ELAVL1, ELAVL2, ELAVL3, ELAVL4, RBFOX2, MATR3, HNRNPA1, SFPQ, and FUS, or a functional fragment thereof, or one or more nucleic acid molecules encoding any of the said splicing regulators and/or splicing factors. In some embodiments, the five or more splicing regulators and/or splicing factors are selected from NOVAI, ELAVL1, ELAVL2, ELAVL3, ELAVL4, RBFOX2, MATR3, HNRNPA1, SFPQ, and FUS, or a functional fragment thereof, or one or more nucleic acid molecules encoding any of the said splicing regulators and/or splicing factors. In some embodiments, the six or more splicing regulators and/or splicing factors are selected from NO VAI, ELAVL1, ELAVL2, ELAVL3, ELAVL4, RBFOX2, MATR3, HNRNPA1, SFPQ, and FUS, or a functional fragment thereof, or one or more nucleic acid molecules encoding any of the said splicing regulators and/or splicing factors. In some embodiments, the seven or more splicing regulators and/or splicing factors are selected from N0VA1, ELAVL1, ELAVL2, ELAVL3, ELAVL4, RBFOX2, MATR3, HNRNPA1, SFPQ, and FUS, or a functional fragment thereof, or one or more nucleic acid molecules encoding any of the said splicing regulators and/or splicing factors. In some embodiments, the splicing regulators and/or splicing factors are NO VAI, ELAVL1, ELAVL2, ELAVL3, ELAVL4, RBF0X2, MATR3, HNRNPA1, SFPQ, and FUS, or a functional fragment thereof, or nucleic acid molecules encoding said splicing regulators and/or splicing factors.

[00111] In some embodiments, the method of treating a disease characterized by TDP-43 mislocalization or dysfunction in a subject comprises providing to cells or tissues or organs of the subject having TDP-43 mislocalization or dysfunction a wild-type protein selected from NO VAI, ELAVL1, ELAVL2, ELAVL3, ELAVL4, RBFOX2, MATR3, HNRNPA1, SFPQ, and FUS, or a functional fragment thereof, or any combination thereof, or one or more nucleic acid molecules encoding any of the aforementioned wild-type proteins or functional fragments thereof. In some embodiments, the method of treating a disease characterized by TDP-43 mislocalization or dysfunction in a subject comprises administering to cells or tissues or organs of the subject having TDP-43 mislocalization or dysfunction a wild-type protein selected from NOVAI, ELAVL1, ELAVL2, ELAVL3, ELAVL4, RBFOX2, MATR3, HNRNPA1, SFPQ, and FUS, or a functional fragment thereof, or any combination thereof, or one or more nucleic acid molecules encoding any of the aforementioned wild-type proteins or functional fragments thereof. In some embodiments, the method of treating a disease characterized by TDP-43 mislocalization or dysfunction in a subject comprises restoring the misregulated splicing of pre- mRNA of a wild-type protein selected from NOVAI, ELAVL1, ELAVL2, ELAVL3, ELAVL4, RBFOX2, MATR3, HNRNPA1, SFPQ, and FUS, or a functional fragment thereof, or any combination thereof to normal. In some embodiments, the method of treating a disease characterized by TDP-43 mislocalization or dysfunction in a subject comprises enhancing expression and/or function of one or more of NOVAI, ELAVL1, ELAVL2, ELAVL3, ELAVL4, RBFOX2, MATR3, HNRNPA1, SFPQ, and FUS, or a functional fragment thereof, in the cells or tissues or organs of the subject having TDP-43 mislocalization or dysfunction. In some embodiments, the method of treating a disease characterized by TDP-43 mislocalization or dysfunction in a subject comprises enhancing expression and/or function of one or more of a correctly spliced N0VA1, ELAVL1, ELAVL2, ELAVL3, ELAVL4, RBF0X2, MATR3, HNRNPA1, SFPQ, and FUS, or a functional fragment thereof. In some embodiments, the method of treating a disease characterized by TDP-43 mislocalization or dysfunction in a subject comprises decreasing expression and/or function of one or more of an incorrectly spliced or misspliced N0VA1, ELAVL1, ELAVL2, ELAVL3, ELAVL4, RBF0X2, MATR3, HNRNPA1, SFPQ, and FUS, or a functional fragment thereof.

[00112] In certain embodiments of the methods, uses, and compositions of the present application, the wild-type protein is NOVAI and/or ELAVL2. In some embodiments of the methods, uses, and compositions of the present application, the NOVAI is NOVAI P51513-4. [00113] In some embodiments, one or more splicing regulators and/or splicing factors are provided to cells or tissues or organs of the subject by a gene therapy. In some embodiments, providing said protein(s) or nucleic acid molecules(s) involves administering a gene therapy to the subject. In some embodiments, one or more splicing regulators and/or splicing factors are delivered to the cells via a vector as described herein. In some embodiments, the vector comprises one or more genes encoding one or more splicing regulators and/or splicing factors. In some embodiments, the vector comprises one or more genes encoding one or more of NOVAI, ELAVL1, ELAVL2, ELAVL3, ELAVL4, RBFOX2, MATR3, HNRNPA1, SFPQ, and FUS, or a functional fragment thereof.

[00114] In one aspect provided herein is a method of treating a disease characterized by TDP-43 mislocalization or dysfunction in a subject comprises providing to cells or tissues or organs of the subject having TDP-43 mislocalization or dysfunction a means for restoring misregulated splicing events to wild-type splicing events of one or more of NOVAI, ELAVL1, ELAVL2, ELAVL3, ELAVL4, RBFOX2, MATR3, HNRNPA1, SFPQ, and FUS. In some embodiments, the method of treating a disease characterized by TDP-43 mislocalization or dysfunction in a subject comprises providing to cells or tissues or organs of the subject having TDP-43 mislocalization or dysfunction a means for enhancing expression and/or function of one or more of NOVAI, ELAVL1, ELAVL2, ELAVL3, ELAVL4, RBFOX2, MATR3, HNRNPA1, SFPQ, and FUS, or a functional fragment thereof, e.g. correctly spliced NOVAI, ELAVL1, ELAVL2, ELAVL3, ELAVL4, RBFOX2, MATR3, HNRNPA1, SFPQ, and/or FUS. In some embodiments, the method of treating a disease characterized by TDP-43 mislocalization or dysfunction in a subject comprises providing to cells or tissues or organs of the subject having TDP-43 mislocalization or dysfunction a means for decreasing expression and/or function of one or more of a mislocalized or dysfunctional N0VA1, ELAVL1, ELAVL2, ELAVL3, ELAVL4, RBF0X2, MATR3, HNRNPA1, SFPQ, and FUS, or a functional fragment thereof, e.g., incorrectly spliced or mis-spliced N0VA1, ELAVL1, ELAVL2, ELAVL3, ELAVL4, RBF0X2, MATR3, HNRNPA1, SFPQ, and/or FUS.

[00115] In some embodiments, the means for restoring misregulated splicing events to wildtype splicing events or enhancing expression and/or function of one or more of N0VA1, ELAVL1, ELAVL2, ELAVL3, ELAVL4, RBFOX2, MATR3, HNRNPA1, SFPQ, and FUS, or a functional fragment thereof, is selected from a small molecule, a protein, a protein-drug conjugate, a nucleotide molecule, a gene editing system, an engineered cell system, and any combination thereof.

[00116] In some embodiments, the means for restoring misregulated splicing events to wildtype splicing events or enhancing expression and/or function of one or more of N0VA1, ELAVL1, ELAVL2, ELAVE3, ELAVE4, RBFOX2, MATR3, HNRNPA1, SFPQ, and FUS, or a functional fragment thereof is a protein selected from a peptide; an antibody or antigen-binding fragment thereof; a monobody; engineered, low-density -lipoprotein-receptor-derived, A domain (LDLR-A) (e.g., Avimers™); a designed ankyrin repeat protein (DARPin) lipocalin (e.g., anticalins); an affibody; engineered, Protein-A-derived, Z domain (Affibodies™) CTLD3 (e.g., Tetranectin); C-type lectin-like domain scaffolds; Sac7d-derived polypeptides (e.g., Nanoffitins® or affitins); engineered, tenascin-derived, tenascin type III domain (e.g., Centyrin™), thioredoxin (e.g., peptide aptamer); KAEBITOR®; the P-sandwich (e.g., iMab); gamma-B crystallin-derived scaffold or engineered, ubiquitin-derived scaffold (e.g., Affilins); engineered, protease inhibitor- derived, Kunitz domain (e.g., EETI-II/AGRP, BPTI/LACI-D1/ITI-D2); engineered antibody mimics; miniproteins; engineered, Fyn-derived, SH2 domain (e.g., Fynomers®); genetically manipulated counterparts of the foregoing that retains its binding functionality, and any combination thereof.

[00117] In some embodiments, the means for restoring misregulated splicing events to wildtype splicing events or enhancing expression and/or function of one or more of N0VA1, ELAVL1, ELAVL2, ELAVL3, ELAVL4, RBFOX2, MATR3, HNRNPA1, SFPQ, and FUS, or a functional fragment thereof is an antibody or antigen-binding fragment thereof selected from an intact antibody, an antigen-binding (Fab) fragment, an Fab’ fragment, an (Fab’)2 fragment, an Fd, an Fv, a dAb, a single domain fragment or single monomeric variable antibody domain, a single-chain Diabody (scDb), a single-chain variable fragment (scFv), a VH domain, a nanobody, a Bi-specific T-cell engager (BiTE), a bispecific killer cell engager (BiKE), a bispecific macrophage engager (BiME), a CrossMab, a tri-specific binding partner, and any combination thereof.

[00118] In some embodiments, the means for restoring misregulated splicing events to wildtype splicing events or enhancing expression and/or function of one or more of NOVAI, ELAVL1, ELAVL2, ELAVL3, ELAVL4, RBFOX2, MATR3, HNRNPA1, SFPQ, and FUS, or a functional fragment thereof is a nucleotide molecule selected from an antisense oligonucleotide, a micro RNA (miRNA), a small interfering RNA (siRNA), a short hairpin RNA (shRNA), a single guide RNA (sgRNA), and any combination thereof.

[00119] In some embodiments, the means for restoring misregulated splicing events to wildtype splicing events or enhancing expression and/or function is a gene editing system comprising a CRISPR-associated protein (Cas) nuclease, a zinc finger nuclease (ZFN), a transcription activator-like effector nuclease (TALEN), a meganuclease, any endo- or exo-nuclease, variants thereof, fragments thereof, or any combination thereof.

[00120] In some embodiments, the means for restoring misregulated splicing events to wildtype splicing events or enhancing expression and/or function is a CAR modified cell selected from a CAR-modified T cell (CAR-T cell), a CAR-modified natural killer (NK) cell (CAR-NK cell), or a CAR-macrophage (CAR-M).

[00121] In some embodiments, the means for restoring misregulated splicing events to wildtype splicing events of one or more of N0VA1, ELAVL1, ELAVL2, ELAVL3, ELAVL4, RBFOX2, MATR3, HNRNPA1, SFPQ, and FUS, or a functional fragment thereof, comprises one or more splicing modifiers and/or antisense oligonucleotides (ASOs). In some embodiments, the one or more ASOs are splice-switching ASOs.

[00122] The splicing modifiers can be a small molecule, a protein, a protein-drug conjugate, a nucleotide molecule, a gene editing system, an engineered cell system, and any combination thereof. [00123] In some embodiments, the gene therapy or the means for restoring misregulated splicing events to wild-type splicing events or enhancing expression and/or function of one or more of N0VA1, ELAVL1, ELAVL2, ELAVL3, ELAVL4, RBFOX2, MATR3, HNRNPA1, SFPQ, and FUS, or a functional fragment thereof can reduce stress associated with neurodegenerative diseases.

[00124] In some embodiments, the means for restoring misregulated splicing events to wildtype splicing events (i) enhance expression and/or function of one or more splicing factors and/or splicing regulators (e.g., N0VA1, ELAVL1, ELAVL2, ELAVL3, ELAVL4, RBFOX2, MATR3, HNRNPA1, SFPQ, and FUS, or a functional fragment thereof) , and/or (ii) decrease expression and/or function of one or more mislocalized or dysfunctional splicing factors and/or splicing regulators (e.g., N0VA1, ELAVL1, ELAVL2, ELAVL3, ELAVL4, RBFOX2, MATR3, HNRNPA1, SFPQ, and FUS, or a functional fragment thereof). In some embodiments, the means for restoring misregulated splicing events to wild-type splicing events (i) enhance expression and/or function of one or more correctly spliced splicing factors and/or splicing regulators, and/or (ii) decrease expression and/or function of one or more mis-spliced splicing factors and/or splicing regulators.

Vectors

[00125] In one aspect, the present disclosure provides a means for providing one or more splicing regulators and/or splicing factors to the cells or tissues or organs of the subject. In some embodiments, the means for providing one or more splicing regulators and/or splicing factors to the cells or tissues or organs of the subject is a gene therapy.

[00126] In some embodiments, one or more splicing regulators and/or splicing factors are provided to cells or tissues or organs of the subject via a vector.

[00127] In some embodiments, one or more splicing regulators and/or splicing factors are antisense oligonucleotides provided to cells or tissues or organs of the subject via a vector. The present invention further provides compositions and constructs for cloning and expressing any of the polynucleotide molecules encoding one or more of NOVAI, ELAVL1, ELAVL2, ELAVL3, ELAVL4, RBFOX2, MATR3, HNRNPA1, SFPQ, and FUS, or a functional fragment thereof, including cloning vectors, expression vectors, transformed host cells comprising any of said vectors, and novel strains or cell lines derived therefrom. In one embodiment, the present invention provides a recombinant vector comprising a polynucleotide molecule having a nucleotide sequence encoding a wild-type protein selected from N0VA1, ELAVL1, ELAVL2, ELAVL3, ELAVL4, RBFOX2, MATR3, HNRNPA1, SFPQ, and FUS, or a functional fragment thereof, or any combination thereof.

[00128] Recombinant vectors of the present invention, particularly expression vectors, are constructed so that the coding sequence for the polynucleotide molecule of the present invention is in operative association with, or operably linked to, one or more regulatory elements necessary for transcription of the coding sequence to produce a wild-type protein (e.g., a correctly spliced protein) selected from N0VA1, ELAVL1, ELAVL2, ELAVL3, ELAVL4, RBFOX2, MATR3, HNRNPA1, SFPQ, and FUS, or a functional fragment thereof, or any combination thereof. As used herein, the term “regulatory element” includes, but is not limited to, nucleotide sequences that encode inducible and non-inducible promoters, enhancers, operators, and other elements known in the art that serve to drive and/or regulate expression of polynucleotide coding sequences.

[00129] Methods are known in the art for constructing recombinant vectors containing coding sequences in operative association with appropriate regulatory elements (such as coding sequences operably linked to a promoter), and these can be used to practice the present invention. These methods include in vitro recombinant techniques, synthetic techniques, and in vivo genetic recombination. See, e.g., the techniques described in Ausubel et al., 1989; Sambrook etal., 1989; Saiki e/ a/., 1988; Reyes et al., 2001; Wu et al., 1989; U.S. Pat. Nos. 4,683,202; 6,335,184; and 6,027,923.

[00130] A variety of expression vectors are known in the art that can be utilized to express a polynucleotide molecule of the present invention, including recombinant bacteriophage DNA, plasmid DNA, and cosmid DNA expression vectors containing the coding sequences. Typical prokaryotic expression vector plasmids that can be engineered to contain a polynucleotide molecule of the present invention include pUC8, pUC9, pBR322, and pBR329 (Biorad Laboratories, Richmond, Calif), pPL and pKK223 (Pharmacia, Piscataway, N.J ), pQE50 (Qiagen, Chatsworth, Calif), and pGEM-T EASY (Promega, Madison, Wis.), pcDNA6.2/V5- DEST and pcDNA3.2/V5DEST (Invitrogen, Carlsbad, Calif.) among many others. Typical eukaryotic expression vectors that can be engineered to contain a polynucleotide molecule of the present invention include an ecdysone-inducible mammalian expression system (Invitrogen, Carlsbad, Calif.), cytomegalovirus promoter-enhancer-based systems (Promega, Madison, Wis.; Stratagene, La Jolla, Calif.; Invitrogen), and baculovirus-based expression systems (Promega), among many others.

[00131] The vector can specifically infect (i.e., exhibit tropism to) a certain tissue type, e.g., neural tissue, or a specific cell type, e.g., motor neurons.

[00132] The regulatory elements of these and other vectors can vary in their strength and specificities. Depending on the host/vector system utilized, any of a number of suitable transcription elements can be used. For instance, when cloning in mammalian cell systems, promoters isolated from the genome of mammalian cells, e.g., mouse metallothionein promoter, or from viruses that grow in these cells, e.g., vaccinia virus 7.5 K promoter or Maloney murine sarcoma virus long terminal repeat, can be used. Promoters obtained by recombinant DNA or synthetic techniques can also be used to provide for transcription of the inserted sequence. In addition, expression from certain promoters can be elevated in the presence of inducers, e.g., zinc and cadmium ions for metallothionein promoters. Non-limiting examples of transcriptional regulatory regions or promoters include for bacteria, the P-galactosidase (P-gal) promoter, the T7 promoter, the TAC promoter, trp and lac promoters, trp-lac fusion promoters, etc:, for yeast, glycolytic enzyme promoters, such as ADH-I and -II promoters, GPK promoter, PGI promoter, TRP promoter, etc:, and for mammalian cells, SV40 early and late promoters, and adenovirus major late promoters, among others.

[00133] In some embodiments, the nucleic acid molecule encoding the wild-type protein (such as splicing factor and/or splicing regulator) is operably linked to a promoter. The promoter can be an inducible promoter or a constitutive promoter. In some embodiments, the promoter is a tissue-specific promoter or a cell-specific promoter. In some embodiments, the promoter is a neural tissue-specific promoter or a neuron-specific promoter. In some embodiments, the promoter is a synapsin promoter. Non-limiting examples of promoters include a human ubiquitin C (hUBC) promoter, a CMV early enhancer/chicken P-actin promoter and a rabbit P-globin splice acceptor (pCAG) promoter, a human cytomegalovirus (HCMV) promoter, a mouse phosphoglycerate kinase (mPGK) promoter, or a homeobox gene (Hb9) promoter.

[00134] Expression vectors can also be constructed that will express a fusion/chimeric RNA comprising one or more of NO VAI, ELAVL1, ELAVL2, ELAVL3, ELAVL4, RBFOX2, MATR3, HNRNPA1, SFPQ, and FUS, or a functional fragment thereof, or a fragment thereof (e.g, a correctly spliced NO VAI , ELAVL1 , ELAVL2, ELAVL3, ELAVL4, RBFOX2, MATR3, HNRNPA1, SFPQ, and/or FUS). Such fusion/chimeric RNA can be used, e.g., to study the functional and/or structural properties of the one or more of N0VA1, ELAVL1, ELAVL2, ELAVL3, ELAVL4, RBF0X2, MATR3, HNRNPA1, SFPQ, and FUS, or a functional fragment thereof, or to aid in the identification or purification, or to improve the stability, of a recombinantly-expressed one or more of N0VA1, ELAVL1, ELAVL2, ELAVL3, ELAVL4, RBF0X2, MATR3, HNRNPA1, SFPQ, and FUS, or a functional fragment thereof, .

[00135] Expression vectors of the present invention also include various expression vectors for expression of one or more of N0VA1, ELAVL1, ELAVL2, ELAVL3, ELAVL4, RBFOX2, MATR3, HNRNPA1, SFPQ, and FUS, or a functional fragment thereof, e.g., correctly spliced NO VAI, ELAVL1, ELAVL2, ELAVL3, ELAVL4, RBFOX2, MATR3, HNRNPA1, SFPQ, and/or FUS (as well as any other molecules interacting with one or more of N0VA1, ELAVL1, ELAVL2, ELAVL3, ELAVL4, RBFOX2, MATR3, HNRNPA1, SFPQ, and FUS, or a functional fragment thereof ). Possible fusion protein expression vectors include, but are not limited to, vectors incorporating sequences that encode P-galactosidase and trpE fusions, maltose-binding protein (MBP) fusions, glutathione-S-transferase (GST) fusions, polyhistidine fusions (e.g., Hise (SEQ ID NO: 3)), V5, HA, and myc.

[00136] Methods known in the art can be used to construct expression vectors encoding these and other fusion proteins. In a specific embodiment, the present invention provides expression vectors for production of fusion proteins of one or more of NOVAI, ELAVL1, ELAVL2, ELAVL3, ELAVL4, RBFOX2, MATR3, HNRNPA1, SFPQ, and FUS, or a functional fragment thereof (e. ., a correctly spliced NO VAI, ELAVL1, ELAVL2, ELAVL3, ELAVL4, RBFOX2, MATR3, HNRNPA1, SFPQ, and/or FUS), e.g., to assist their purification or detection. In nonlimiting embodiments, e.g., a fusion protein of one or more of NOVAI, ELAVL1, ELAVL2, ELAVL3, ELAVL4, RBFOX2, MATR3, HNRNPA1, SFPQ, and FUS, or a functional fragment thereof, can be purified using amylose resin; a fusion protein of one or more of N0VA1, ELAVL1, ELAVL2, ELAVL3, ELAVL4, RBFOX2, MATR3, HNRNPA1, SFPQ, and FUS, or a functional fragment thereof, can be purified using glutathione-agarose beads; and a fusion protein of one or more of NO VAI, ELAVL1, ELAVL2, ELAVL3, ELAVL4, RBFOX2, MATR3, HNRNPA1, SFPQ, and FUS, or a functional fragment thereof, can be purified using divalent nickel resin. Alternatively, antibodies against a carrier protein or peptide can be used for affinity chrom tography purification of the fusion protein. In one embodiment, a nucleotide sequence coding for the FLAG™ epitope tag (International Biotechnologies Inc.), which is a hydrophilic marker peptide, can be inserted by standard techniques into the expression vector at a point corresponding, e.g., to the amino or carboxyl terminus of the one or more of N0VA1, ELAVL1, ELAVL2, ELAVL3, ELAVL4, RBF0X2, MATR3, HNRNPA1, SFPQ, and FUS, or a functional fragment thereof, . The expressed one or more of N0VA1, ELAVL1, ELAVL2, ELAVL3, ELAVL4, RBF0X2, MATR3, HNRNPA1, SFPQ, and FUS, or a functional fragment thereof, protein-FLAG™ epitope fusion product can then be detected and affinity -purified using commercially available anti-FLAG™ antibodies. The expression vector can also be engineered to contain polylinker sequences that encode specific protease cleavage sites so that the expressed one or more of NO VAI, ELAVL1, ELAVL2, ELAVL3, ELAVL4, RBF0X2, MATR3, HNRNPA1, SFPQ, and FUS, or a functional fragment thereof, can be released from a carrier region or fusion partner by treatment with a specific protease. For example, the fusion protein vector can include a nucleotide sequence encoding a thrombin or factor Xa cleavage site, among others.

[00137] To aid in the selection of host cells transformed or transfected with a recombinant vector of the present invention, the vector can be engineered to further comprise a coding sequence for a reporter gene product or other selectable marker. Such a coding sequence is preferably in operative association with the regulatory elements, as described above. Reporter genes that are useful in practicing the invention are known in the art, and include, but are not limited to, those encoding chloramphenicol acetyltransferase (CAT), green fluorescent protein (GFP), firefly luciferase, and human growth hormone (hGH), among others. Nucleotide sequences encoding selectable markers are known in the art and include, but are not limited to, those that encode gene products conferring resistance to antibiotics or anti-metabolites, or that supply an auxotrophic requirement. Examples of such sequences include those that encode thymidine kinase activity or resistance to methotrexate, penicillin, ampicillin, kanamycin, chloramphenicol, tetracycline, zeocin, pyrimethamine, aminoglycosides, hygromycin, blasticidine, or neomycin, among others. Other examples include positive and negative selection markers, for example, glutamate synthase genes, HSV-TK, HSV-TK derivatives for ganciclovir selection, or bacterial purine nucleoside phosphorylase gene for 6-methylpurine selection (Gadi et al., 7 Gene Ther. 1738-1743 (2000)). A nucleic acid sequence encoding a selection marker or the cloning site may be upstream or downstream of a nucleic acid sequence encoding a polypeptide of interest or cloning site.

[00138] Non-limiting examples of additional vectors that can be used in accordance with the present disclosure include Moloney murine leukemia viruses (MLV), Moloney murine leukemia viruses pseudotyped with vesicular stomatitis virus G protein (MLV-VSV-G), murine stem cell viruses (MSCV), lentiviruses, lentiviruses pseudotyped with vesicular stomatitis virus G protein (LV-VSV-G), adenoviruses (e.g., Ad5, Ad41), adeno-associated viruses (AAVs such as AAV1, AAV2, AAV5, AAV6, AAV8, AAV9, AAV10, AAV-B1), and variants and derivatives thereof. Additional suitable vectors include those described in Buckinx and Timmermans, Histochem Cell Biol (2016) 146:709-720, which is incorporated herein by reference in its entirety.

[00139] In some embodiments, the virus comprising a gene encoding one or more of NOVAI, ELAVL1, ELAVL2, ELAVL3, ELAVL4, RBFOX2, MATR3, HNRNPA1, SFPQ, and FUS, or a functional fragment thereof (e.g., a correctly spliced N0VA1, ELAVL1, ELAVL2, ELAVL3, ELAVL4, RBFOX2, MATR3, HNRNPA1, SFPQ, and/or FUS), is a viral vector. In some embodiments, the viral vector is a lentiviral vector, an adenoviral vector, or an adeno-associated viral vector.

[00140] In some embodiments, the adeno-associated viral vector exhibits tropism to neural tissue or to neurons, e.g., motor neurons. In some embodiments, the adeno-associated viral vector exhibits tropism to human motor neurons, e.g., hypoglossal motor neurons or ocular motor neurons. In some embodiments, the adeno-associated viral vector is AAV9 or AAV-B1. In one embodiment, the AAV vector is AAV9. In one embodiment, the AAV vector is AAV-B1. [00141] Numerous techniques are known in the art for the introduction of foreign genes into cells and may be used to construct the recombinant cells for purposes of carrying out the described methods, in accordance with the various embodiments described and exemplified herein. The technique used should provide for the stable transfer of the heterologous gene sequence to the host cell, such that the heterologous gene sequence is heritable and expressible by the cell progeny, and so that the necessary development and physiological functions of the recipient cells are not disrupted. Techniques which may be used include, but are not limited to, chromosome transfer (e.g., cell fusion, chromosome mediated gene transfer, micro cell mediated gene transfer), physical methods (e.g., transfection, spheroplast fusion, microinjection, electroporation, liposome carrier), viral vector transfer (e.g., recombinant DNA viruses, recombinant RNA viruses) and the like (described in Cline, 29 Pharmac. Ther. 69-92 (1985)). Calcium phosphate precipitation and polyethylene glycol (PEG)-induced fusion of bacterial protoplasts with mammalian cells may also be used to transform cells.

Vectors as a gene therapy

[00142] In one aspect, the present disclosure provides a gene therapy for treating a disease characterized by TDP-43 mislocalization or dysfunction in a subject. In some embodiments, the gene therapy is a vector-based gene therapy. Non-limiting examples of vector that can be used as therapeutics include a lentiviral vector, an adenoviral vector, and an adeno-associated viral vector. In some embodiments, the vector that can be used as therapeutics is a viral vector. In some embodiments, the viral vector is an adeno-associated viral vector.

[00143] Adeno-associated viral vectors (AAVs) and recombinant adeno-associated virus (rAAV) vectors are well known delivery vehicles that can be constructed and used to deliver a nucleic acid molecule to cells, as described in Shi et al., “Therapeutic Expression of an AntiDeath Receptor-5 Single-Chain Fixed Variable Region Prevents Tumor Growth in Mice,” Cancer Res. 66: 11946-53 (2006); Fukuchi et al., “Anti-A0 Single-Chain Antibody Delivery via Adeno- Associated Virus for Treatment of Alzheimer’s Disease,” Neurobiol. Dis. 23:502-511 (2006); Chatteijee et al., “Dual-Target Inhibition of HIV-1 In Vitro by Means of an Adeno- Associated Virus Antisense Vector,” Science 258: 1485-1488 (1992); Ponnazhagan et al., “Suppression of Human Alpha-globin Gene Expression Mediated by the Recombinant Adeno- associated Virus 2- based Antisense Vectors,” J Exp. Med. 179:733-738 (1994), which are hereby incorporated by reference in their entirety. In vivo use of these vehicles is described in Flotte et al., “Stable In Vivo Expression of the Cystic Fibrosis Transmembrane Conductance Regulator With an Adeno- Associated Virus Vector,” Proc. Nat’l. Acad. Sci. 90:10613-10617 (1993), which is hereby incorporated by reference in its entirety.

[00144] Recombinant adeno-associated virus (rAAV) vectors provide the ability to stably transduce and express genes with very long-term (many years) duration in skeletal muscle, and depending on the AAV vector serotype and its modification, to do so with high muscle-tropism and selectivity whether using local intramuscular injection or systemic routes of delivery (Phillips et al., “Systemic Gene Transfer to Skeletal Muscle Using Reengineered AAV Vectors,” Methods Mol. Biol. 709:141-51 (2011) and Muraine et al., “Transduction Efficiency of Adeno- Associated Virus Serotypes After Local Injection in Mouse and Human Skeletal Muscle,” Hum. Gene Ther. 31(3-4):233-240 (2020), which are hereby incorporated by reference in their entirety). Moreover, for certain AAV serotypes and engineered variants, particularly AAV8 and its engineered variants, studies in mice have been shown to be predictive of human skeletal muscle transduction and gene expression, as found in clinical trials for skeletal muscle transmission and expression (Phillips et al., “Systemic Gene Transfer to Skeletal Muscle Using Reengineered AAV Vectors,” Methods Mol. Biol. 709: 141-51 (2011) and Muraine et al., “Transduction Efficiency of Adeno- Associated Virus Serotypes After Local Injection in Mouse and Human Skeletal Muscle,” Hum. Gene Ther. 31(3-4) :233 -240 (2020), which are hereby incorporated by reference in their entirety).

[00145] In certain embodiments, a single-stranded AAV (ssAAV) can be used. In certain embodiments, a self-complementary vector, e.g., scAAV, can be used (see, e.g., Wu, 2007, Human Gene Therapy, 18(2): 171-82, McCarty et al, 2001, Gene Therapy, Vol. 8, Number 16, Pages 1248-1254; and U.S. Patent Nos. 6,596,535; 7,125,717; and 7,456,683, each of which is incorporated herein by reference in its entirety). Self-complementary vectors may include a mutant ITR sequence.

[00146] In additional embodiments, rAAV particles comprise a pseudotyped rAAV particle. In some embodiments, the pseudotyped rAAV particle comprises (a) a nucleic acid vector comprising AAV ITRs and (b) a capsid comprised of capsid proteins derived from AAVx (e.g., AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV2i8, AAV2.5, AAVrh.8, AAVrh.10, AAVrh.43, AAVrh.74, AAVhu.37, AAVAAV.hu31, AAVhu.32. or AAV-B1), in particular AAV9 or AAV-B1. In some embodiments, rAAV particles comprise a pseudotyped rAAV particle containing AAV8 capsid protein. In some embodiments, the pseudotyped rAAV8 particle is an rAAV2/8 pseudotyped particle. In some embodiments, rAAV particles comprise a pseudotyped rAAV particle containing AAV9 capsid protein, or In some embodiments, rAAV particles comprise a pseudotyped rAAV particle containing AAV-B1 capsid protein. Methods for producing and using pseudotyped rAAV particles are known in the art (see, e.g., Duan et al., “Enhancement of Muscle Gene Delivery with Pseudotyped Adeno-Associated Virus Type 5 Correlates with Myoblast Differentiation,” J. Virol. 75(16):7662-7671 (2001); Halbert et al., “Repeat Transduction in the Mouse Lung by Using Adeno-Associated Virus Vectors with Different Serotypes,” J. Virol. 74(3): 1524-1532 (2000); Zolotukhin et al., “Production and Purification of Serotype 1, 2, and 5 Recombinant Adeno-Associated Viral Vectors,” Methods 28(2): 158-167 (2002); and Auricchio et al., “Exchange of Surface Proteins Impacts on Viral Vector Cellular Specificity and Transduction Characteristics: the Retina as a Model,” Hum. Molec. Genet. 10:3075-3081 (2001), which are hereby incorporated by reference in their entirety).

[00147] The AAV vector described herein may comprise a sequence isolated or derived from an AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV- Bl, or any combination thereof. In some embodiments, the AAV or rAAV particles comprise an AAV capsid protein chimeric of AAV9 or AAV-B1 capsid protein and one or more AAV capsid proteins from an AAV serotype selected from AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV2i8, AAV2.5, AAVrh.8, AAVrh.10, AAVrh.43, AAVrh.74, AAVhu.37, AAVAAV.hu31, AAV-B1, or AAVhu.32.

[00148] In some embodiments, the AAV vector is a recombinant vector.

[00149] In one embodiment, the AAV vector is AAV8. AAV8 derived from macaques is very poorly immunogenic, resulting in long-term expression of the encoded transgene (for many years), and efficiently transduce skeletal muscle with high tropism and selectivity in both human and mouse (Phillips et al., “Systemic Gene Transfer to Skeletal Muscle Using Reengineered AAV Vectors,” Methods Mol. Biol. 709: 141-51 (2011); Muraine etal, “Transduction Efficiency of Adeno-Associated Virus Serotypes After Local Injection in Mouse and Human Skeletal Muscle,” Hum. Gene Ther. 31(3-4):233-240 (2020); Blankinship et al, “Efficient Transduction of Skeletal Muscle Using Vectors Based on Adeno-associated Virus Serotype 6,” Mo/. Ther.

10(4):671 -8 (2004); and Gregorevic et al, “Viral Vectors for Gene Transfer to Striated Muscle,” Curr. Opin. Mol. Ther. 6(5):491 -8 (2004), which are hereby incorporated by reference in their entirety). AAV8 shows essentially no liver tropism, is largely specific for skeletal fibers and satellite cells, and has been shown to transduce skeletal muscles throughout the body (Wang et al, “Construction and Analysis of Compact Muscle-specific Promoters for AAV Vectors,” Gene Ther. 15(22): 1489-99 (2008), which is hereby incorporated by reference in its entirety).

[00150] In some embodiments, the adeno-associated viral vector exhibits tropism to neural tissue or to neurons, e.g., motor neurons. In some embodiments, the adeno-associated viral vector exhibits tropism to human motor neurons, e.g., hypoglossal motor neurons or ocular motor neurons. In some embodiments, the adeno-associated viral vector is AAV9 or AAV-B1. In one embodiment, the AAV vector is AAV9. In one embodiment, the AAV vector is AAV-B1. [00151] In some embodiments, the viral vector comprises a promoter operably linked to the nucleic acid encoding the wild-type protein. The promoter can be an inducible promoter or a constitutive promoter. In some embodiments, the promoter is a tissue-specific promoter or a cellspecific promoter. In some embodiments, the promoter is a neural tissue-specific promoter or a neuron-specific promoter. Non-limiting examples of promoters include a human ubiquitin C (hUBC) promoter, a CMV early enhancer/chicken -actin promoter and a rabbit (3-globin splice acceptor (pCAG) promoter, a human cytomegalovirus (HCMV) promoter, a mouse phosphoglycerate kinase (mPGK) promoter, or a homeobox gene (Hb9) promoter.

[00152] Another aspect of the present disclosure involves making molecules disclosed herein. In some embodiments, a molecule according to the disclosure is made by providing a nucleotide comprising the nucleic acid sequence encoding any of the capsid protein molecules; and using a packaging cell system to prepare corresponding rAAV particles with capsid coats made up of the capsid protein. In some embodiments, the nucleic acid encodes a sequence having at least 60%, 70%, 80%, 85%, 90%, or 95%, including 96%, 97%, 98%, 99%, or 99.9%, identity to the sequence of the AAV9 capsid protein, while retaining (or substantially retaining) biological function of the AAV9 capsid protein. In some embodiments, the nucleic acid encodes a sequence having at least 60%, 70%, 80%, 85%, 90%, or 95%, including 96%, 97%, 98%, 99%, or 99.9%, identity to the sequence of the AAV-B1 capsid protein, while retaining (or substantially retaining) biological function of the AAV-B1 capsid protein.

[00153] The capsid protein, coat, and rAAV particles may be produced by techniques known in the art. In some embodiments, the viral genome comprises at least one inverted terminal repeat (ITR) to allow packaging into a vector. In some embodiments, the viral genome further comprises a cap gene and/or a rep gene for expression and splicing of the cap gene. In embodiments, the cap and rep genes are provided by a packaging cell and not present in the viral genome.

[00154] In some embodiments, the nucleic acid encoding the engineered capsid protein is cloned into an AAV Rep-Cap plasmid in place of the existing capsid gene. When introduced together into host cells, this plasmid helps package an rAAV genome into the engineered capsid protein as the capsid coat. Packaging cells can be any cell type possessing the genes necessary to promote AAV genome replication, capsid assembly, and packaging. [00155] Numerous cell culture-based systems are known in the art for production of rAAV particles, any of which can be used to practice a method disclosed herein. The cell culture-based systems include transfection, stable cell line production, and infectious hybrid virus production systems which include, but are not limited to, adenovirus-AAV hybrids, herpesvirus- AAV hybrids and baculovirus-AAV hybrids. rAAV production cultures for the production of rAAV virus particles require: (1) suitable host cells, including, for example, but are not limited to, human-derived cell lines, mammalian cell lines, or insect-derived cell lines; (2) suitable helper virus function, provided by wild type or mutant adenovirus (such as temperature-sensitive adenovirus), herpes virus, baculovirus, or a plasmid construct providing helper functions; (3) AAV rep and cap genes and gene products; (4) a transgene (such as a therapeutic transgene) flanked by AAV ITR sequences and optionally regulatory elements; and (5) suitable media and media components (nutrients) to support cell growth/survival and rAAV production.

[00156] Non-limiting examples of host cells include A549, WEHI, 10T1/2, BHK, MDCK, COS1, COS7, BSC1, BSC40, BMT10, VERO, W138, HeLa, HEK293 and their derivatives (HEK293T cells, HEK293F cells), Saos, C2C12, L, HT1080, HepG2, primary fibroblast, hepatocyte, myoblast cells, CHO cells or CHO-derived cells, or insect-derived cell lines such as SF-9 (e.g. in the case of baculovirus production systems). See Aponte-Ubillus etal., “Molecular Design for Recombinant Adeno-Associated Virus (rAAV) Vector Production,” Appl. Microbiol. Biotechnol. 102: 1045-1054 (2018), which is incorporated by reference herein in its entirety for manufacturing techniques.

[00157] In some embodiments, provided herein is a method of producing rAAV particles, comprising (a) providing a cell culture comprising a cell; (b) introducing into the cell one or more baculovirus vectors encoding at least one of: i. an rAAV genome to be packaged, ii. an AAV rep protein sufficient for packaging, and iii. an AAV cap protein sufficient for packaging; (c) adding to the cell culture sufficient nutrients and maintaining the cell culture under conditions that allow production of the rAAV particles. In some embodiments, the method comprises using a first baculovirus vector encoding the rep and cap genes and a second baculovirus vector encoding the rAAV genome. In some embodiments, the method comprises using a baculovirus encoding the rAAV genome and a cell expressing the rep and cap genes. In some embodiments, the method comprises using a baculovirus vector encoding the rep and cap genes and the rAAV genome. In some embodiments, the cell is an insect cell. In some embodiments, the insect cell is an Sf-9 cell. In some embodiments, the insect cell is an Sf-9 cell comprising one or more stably integrated heterologous polynucleotide encoding the rep and cap genes.

[00158] In some embodiments, a method disclosed herein uses a baculovirus production system. In some embodiments the baculovirus production system uses a first baculovirus encoding the rep and cap genes and a second baculovirus encoding the rAAV genome. In some embodiments the baculovirus production system uses a baculovirus encoding the rAAV genome and a host cell expressing the rep and cap genes. In some embodiments, the baculovirus production system uses a baculovirus encoding the rep and cap genes and the rAAV genome. In some embodiments, the baculovirus production system uses insect cells, such as Sf-9 cells. A skilled artisan is aware of the numerous methods by which AAV rep and cap genes, AAV helper genes (e.g., adenovirus Ela gene, Elb gene, E4 gene, E2a gene, and VA gene), and rAAV genomes (comprising one or more genes of interest flanked by ITRs) can be introduced into cells to produce or package rAAV. The phrase “adenovirus helper functions” refers to a number of viral helper genes expressed in a cell (as RNA or protein) such that the AAV grows efficiently in the cell. The skilled artisan understands that helper viruses, including adenovirus and herpes simplex virus (HSV), promote AAV replication and certain genes have been identified that provide the essential functions, e.g., the helper may induce changes to the cellular environment that facilitate such AAV gene expression and replication. In some embodiments of a method disclosed herein, AAV rep and cap genes, helper genes, and rAAV genomes are introduced into cells by transfection of one or more plasmid vectors encoding the AAV rep and cap genes, helper genes, and rAAV genome. In some embodiments of a method disclosed herein, AAV rep and cap genes, helper genes, and rAAV genomes can be introduced into cells by transduction with viral vectors, for example, rHSV vectors encoding the AAV rep and cap genes, helper genes, and rAAV genome. In some embodiments of a method disclosed herein, one or more of AAV rep and cap genes, helper genes, and rAAV genomes are introduced into the cells by transduction with an rHSV vector. In some embodiments, the rHSV vector encodes the AAV rep and cap genes. In some embodiments, the rHSV vector encodes the helper genes. In some embodiments, the rHSV vector encodes the rAAV genome. In some embodiments, the rHSV vector encodes the AAV rep and cap genes. In some embodiments, the rHSV vector encodes the helper genes and the rAAV genome. In some embodiments, the rHSV vector encodes the helper genes and the AAV rep and cap genes. [00159] In one aspect, provided herein is a method of producing rAAV particles, comprising (a) providing a cell culture comprising a host cell; (b) introducing into the cell one or more rHSV vectors encoding at least one of: i. an rAAV genome to be packaged, ii. helper functions necessary for packaging the rAAV particles, iii. an AAV rep protein sufficient for packaging, and iv. an AAV cap protein sufficient for packaging; (c) adding to the cell culture sufficient nutrients and maintaining the cell culture under conditions that allow production of the rAAV particles. In some embodiments, the rHSV vector encodes the AAV rep and cap genes. In some embodiments, the rHSV vector encodes helper functions. In some embodiments, the rHSV vector comprises one or more endogenous genes that encode helper functions. In some embodiments, the rHSV vector comprises one or more heterogeneous genes that encode helper functions. In some embodiments, the rHSV vector encodes the rAAV genome. In some embodiments, the rHSV vector encodes the AAV rep and cap genes. In some embodiments, the rHSV vector encodes helper functions and the rAAV genome. In some embodiments, the rHSV vector encodes helper functions and the AAV rep and cap genes. In some embodiments, the cell comprises one or more stably integrated heterologous polynucleotide encoding the rep and cap genes.

[00160] In one aspect, provided herein is a method of producing rAAV particles, comprising (a) providing a cell culture comprising a mammalian cell; (b) introducing into the cell one or more polynucleotides encoding at least one of: i. an rAAV genome to be packaged, ii. helper functions necessary for packaging the rAAV particles, iii. an AAV rep protein sufficient for packaging, and iv. an AAV cap protein sufficient for packaging; (c) adding to the cell culture sufficient nutrients and maintaining the cell culture under conditions that allow production of the rAAV particles. In some embodiments, the helper functions are encoded by adenovirus genes. In some embodiments, the mammalian cell comprises one or more stably integrated heterologous polynucleotide encoding the rep and cap genes.

[00161] Molecular biology techniques to develop plasmid or viral vectors encoding the AAV rep and cap genes, helper genes, and/or rAAV genome are commonly known in the art. In some embodiments, AAV rep and cap genes are encoded by one plasmid vector. In some embodiments, AAV helper genes (e.g., adenovirus Ela gene, Elb gene, E4 gene, E2a gene, and VA gene) are encoded by one plasmid vector. In some embodiments, the E1A gene or Elb gene is stably expressed by the host cell, and the remaining AAV helper genes are introduced into the cell by transfection by one viral vector. In some embodiments, the El a gene and Elb gene are stably expressed by the host cell, and the E4 gene, E2a gene, and VA gene are introduced into the cell by transfection by one plasmid vector. In some embodiments, one or more helper genes are stably expressed by the host cell, and one or more helper genes are introduced into the cell by transfection by one plasmid vector. In some embodiments, the helper genes are stably expressed by the host cell. In some embodiments, AAV rep and cap genes are encoded by one viral vector. In some embodiments, AAV helper genes (e.g., adenovirus Ela gene, Elb gene, E4 gene, E2a gene, and VA gene) are encoded by one viral vector. In some embodiments, the Ela gene or Elb gene is stably expressed by the host cell, and the remaining AAV helper genes are introduced into the cell by transfection by one viral vector. In some embodiments, the Ela gene and Elb gene are stably expressed by the host cell, and the E4 gene, E2a gene, and VA gene are introduced into the cell by transfection by one viral vector. In some embodiments, one or more helper genes are stably expressed by the host cell, and one or more helper genes are introduced into the cell by transfection by one viral vector. In some embodiments, the AAV rep and cap genes, the adenovirus helper functions necessary for packaging, and the rAAV genome to be packaged are introduced to the cells by transfection with one or more polynucleotides, e.g., vectors. In some embodiments, a method disclosed herein comprises transfecting the cells with a mixture of three polynucleotides: one encoding the cap and rep genes, one encoding adenovirus helper functions necessary for packaging (e.g., adenovirus Ela gene, Elb gene, E4 gene, E2a gene, and VA gene), and one encoding the rAAV genome to be packaged. In some embodiments, the AAV cap gene is an AAV8 cap gene. In some embodiments, the AAV cap gene is an AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV2i8, AAV2.5, AAVrh.8, AAVrh.10, AAVrh.43, AAVrh.74, AAVhu.37, AAVAAV.hu31, AAV-B1, or AAVhu.32 cap gene. In some embodiments, the vector encoding the rAAV genome to be packaged comprises a gene of interest flanked by AAV ITRs.

[00162] Any combination of vectors can be used to introduce AAV rep and cap genes, AAV helper genes, and rAAV genome to a cell in which rAAV particles are to be produced or packaged. In some embodiments of a method disclosed herein, a first plasmid vector encoding an rAAV genome comprising a gene of interest flanked by AAV inverted terminal repeats (ITRs), a second vector encoding AAV rep and cap genes, and a third vector encoding helper genes can be used. In some embodiments, a mixture of the three vectors is co-transfected into a cell. In some embodiments, a combination of transfection and infection is used by using both plasmid vectors as well as viral vectors. The gene of interest includes genes of one or more of N0VA1, ELAVL1, ELAVL2, ELAVL3, ELAVE4, RBF0X2, MATR3, HNRNPA1, SFPQ, and FUS, or a functional fragment thereof (e.g., a correctly spliced N0VA1, ELAVL1, ELAVL2, ELAVL3, ELAVL4, RBF0X2, MATR3, HNRNPA1, SFPQ, and/or FUS).

[00163] In some embodiments, one or more of rep and cap genes, and AAV helper genes are constitutively expressed by the cells and does not need to be transfected or transduced into the cells. In some embodiments, the cell constitutively expresses rep and/or cap genes. In some embodiments, the cell constitutively expresses one or more AAV helper genes. In some embodiments, the cell constitutively expresses El a. In some embodiments, the cell comprises a stable transgene encoding the rAAV genome.

[00164] In some embodiments, AAV rep, cap, and helper genes (e.g., Ela gene, Elb gene, E4 gene, E2a gene, or VA gene) can be of any AAV serotype. In some embodiments, AAV rep and cap genes to produce a rAAV particle are from different serotypes. For example, the rep gene is from AAV2 whereas the cap gene is from AAV8.

[00165] In some embodiments, the rep gene is from AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV2i8, AAV2.5, AAVrh.8, AAVrh.10, AAVrh.43, AAVrh.74, AAVhu.37, AAVAAV.hu31, AAV-B1, or AAVhu.32or other AAV serotypes (e.g., a hybrid serotype harboring sequences from more than one serotype). In other embodiments, the rep and the cap genes are from the same serotype. In still other embodiments, the rep and the cap genes are from the same serotype, and the rep gene comprises at least one modified protein domain or modified promoter domain. In certain embodiments, the at least one modified domain comprises a nucleotide sequence of a serotype that is different from the capsid serotype. The modified domain within the rep gene may be a hybrid nucleotide sequence consisting of fragments different serotypes.

[00166] Hybrid rep genes provide improved packaging efficiency of rAAV particles, including packaging of a viral genome comprising a therapeutic protein transgene (such as NOVAI, ELAVL1, ELAVL2, ELAVL3, ELAVL4, RBFOX2, MATR3, HNRNPA1, SFPQ, and FUS, or a functional fragment thereof, e.g., a correctly spliced NOVAI, ELAVL1, ELAVL2, ELAVL3, ELAVL4, RBFOX2, MATR3, HNRNPA1, SFPQ, and/or FUS ) greater than 4 kb, greater than 4.1 kb, greater than 4.2 kB, greater than 4.3 kb, greater than 4.4 kB, greater than 4.5 kb, or greater than 4.6 kb. AAV rep genes consist of nucleic acid sequences that encode the non- structural proteins needed for replication and production of virus. Transcription of the rep gene initiates from the p5 or pl9 promoters to produce two large (Rep78 and Rep68) and two small (Rep52 and Rep40) nonstructural Rep proteins, respectively. Additionally, Rep78/68 domain contains a DNA-binding domain that recognizes specific ITR sequences within the ITR. All four Rep proteins have common helicase and ATPase domains that function in genome replication and/or encapsidation (Maurer and Weitzman, “Adeno-Associated Virus Genome Interactions Important for Vector Production and Transduction,” Hum. Gene Ther. 31 (9- 10) : 499-511 (2020), which is hereby incorporated by reference in its entirety). Transcription of the cap gene initiates from a p40 promoter, which sequence is within the C- terminus of the rep gene, and it has been suggested that other elements in the rep gene may induce p40 promoter activity. The p40 promoter domain includes transcription factor binding elements EFl A, MLTF, and ATF, Fos/Jun binding elements (AP-1), Sp 1 -like elements (Spl and GGT), and the TATA element (Pereira and Muzyczka, “The Adeno-Associated Virus Type 2 p40 Promoter Requires a Proximal Spl Interaction and a pl9 CArG-like Element to Facilitate Rep Transactivation,” J. Virol. 71(6): 4300-4309 (1997), which is hereby incorporated by reference in its entirety). In some embodiments, the rep gene comprises a modified p40 promoter. In some embodiments, the p40 promoter is modified at any one or more of the EF1A binding element, MLTF binding element, ATF binding element, Fos/Jun binding elements (AP-1), Sp 1 -like elements (Spl or GGT), or the TATA element. In other embodiments, the rep gene is of serotype 1, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, rh8, rhlO, rh20, rh39, rh.74, RHM4- 1, or hu37, and the portion or element of the p40 promoter domain is modified to serotype 2. In some embodiments, the rep gene is of serotype 8 or 9, and the portion or element of the p40 promoter domain is modified to serotype 2.

[00167] ITRs contain A and A’ complimentary sequences, B and B’ complimentary sequences, and C and C’ complimentary sequences; and the D sequence is contiguous with the ssDNA genome. The complimentary sequences of the ITRs form hairpin structures by selfannealing (Bems, KI., “The Unusual Properties of the AAV Inverted Terminal Repeat,” Hum. Gene Ther. 31 (9-10): 518-523 (2020), which is hereby incorporated by reference in its entirety). The D sequence contains a Rep Binding Element (RBE) and a terminal resolution site (TRS), which together constitute the AAV origin of replication. The ITRs are also required as packaging signals for genome encapsidation following replication. In some embodiments, the ITR sequences and the cap genes are from the same serotype, except that one or more of the A and A’ complimentary sequences, B and B’ complimentary sequences, C and C’ complimentary sequences, or the D sequence may be modified to contain sequences from a different serotype than the capsid. In some embodiments, the modified ITR sequences are from the same serotype as the rep gene. In other embodiments, the ITR sequences and the cap genes are from different serotypes, except that one or more of the ITR sequences selected from A and A’ complimentary sequences, B and B’ complimentary sequences, C and C’ complimentary sequences, or the D sequence are from the same serotype as the capsid (cap gene), and one or more of the ITR sequences are from the same serotype as the rep gene. In some embodiments, the rep and the cap genes are from the same serotype, and the rep gene comprises a modified Rep78 domain, DNA binding domain, endonuclease domain, ATPase domain, helicase domain, p5 promoter domain, Rep68 domain, p5 promoter domain, Rep52 domain, p!9 promoter domain, Rep40 domain or p40 promoter domain. In other embodiments, the rep and the cap genes are from the same serotype, and the rep gene comprises at least one protein domain or promoter domain from a different serotype. In one embodiment, an rAAV comprises a transgene flanked by AAV2 ITR sequences, an AAV8 cap, and a hybrid AAV2/8 rep. In another embodiment, the AAV2/8 rep comprises serotype 8 rep except for the p40 promoter domain or a portion thereof is from serotype 2 rep. In other embodiments, the AAV2/8 rep comprises serotype 2 rep except for the p40 promoter domain or a portion thereof is from serotype 8 rep. In some embodiments, more than two serotypes may be utilized to construct a hybrid rep/cap plasmid. The composition of the present application may further comprise one or more targeting elements. Suitable targeting elements include, without limitation, agents such as saponins or cationic polyamides (see, e.g., U.S. Patent Nos. 5,739,118 and 5,837,533, which are hereby incorporated by reference in their entirety); microparticles, microcapsules, liposomes, or other vesicles; lipids; cell-surface receptors; transfecting agents; peptides (e.g., one known to enter the nucleus); or ligands (such as one subject to receptor-mediated endocytosis). Suitable means for using such targeting elements include, without limitation: microparticle bombardment; coating the polynucleotide with lipids, cell- surface receptors, or transfecting agents; encapsulation of the polynucleotide in liposomes, microparticles, or microcapsules; administration of the polynucleotide linked to a peptide which is known to enter the nucleus; or administration of the polynucleotide linked to a ligand subject to receptor-mediated endocytosis (see, e.g., Wu et al, “Receptor-Mediated in vitro Gene Transformation by a Soluble DNA Carrier System,” J. Biol. Chem. 262:4429-4432 (1987), which is hereby incorporated by reference in its entirety), which can be used to target cell types specifically expressing the receptors. Alternatively, a polynucleotide-ligand complex can be formed allowing the polynucleotide to be targeted for cell specific uptake and expression in vivo by targeting a specific receptor (see, e.g., PCT Application Publication Nos. WO 92/06180, WO 92/22635, WO 92/203167, WO 93/14188, and WO 93/20221, which are hereby incorporated by reference in their entirety).

[00168] Any suitable method known in the art may be used for transfecting a cell may be used to produce rAAV particles according to a method disclosed herein. In some embodiments, a method disclosed herein comprises transfecting a cell using a chemical-based transfection method. In some embodiments, the chemical-based transfection method uses calcium phosphate, highly branched organic compounds (dendrimers), cationic polymers (e.g., DEAE dextran or polyethylenimine (PEI)), lipofection. In some embodiments, the chemicalbased transfection method uses cationic polymers (e.g., DEAE dextran or polyethylenimine (PEI)). In some embodiments, the chemi cal -based transfection method uses polyethylenimine (PEI). In some embodiments, the chemical-based transfection method uses DEAE dextran. In some embodiments, the chemi cal -based transfection method uses calcium phosphate.

[00169] Provided are methods of testing of the infectivity of a recombinant vector disclosed herein, for example AAV or rAAV particles. Vector copy numbers may be assessed using polymerase chain reaction techniques and level of therapeutic protein expression may be tested by measuring levels of therapeutic protein (such as NOVAI, ELAVL1, ELAVL2, ELAVL3, ELAVL4, RBFOX2, MATR3, HNRNPA1, SFPQ, and FUS, or a functional fragment thereof, e.g., a correctly spliced N0VA1, ELAVL1, ELAVL2, ELAVL3, ELAVL4, RBFOX2, MATR3, HNRNPA1, SFPQ, and/or FUS) mRNA in the cells.

[00170] In some embodiments, provided herein is a composition comprising an adeno- associated viral (AAV) vector as described herein. In some embodiments, the composition of the present application further comprises a buffer solution.

[00171] In some embodiments, the composition further includes a transfection reagent. The transfection reagent may be a positively charged transfection reagent. Suitable transfection reagents are well known in the art and include, e.g., Lipofectamine® RNAiMAX (Invitrogen™), Lipofectamine® 2000 (Invitrogen™), Lipofectamine® 3000 (Invitrogen™), Tnvivofectamine™ 3.0 (Invitrogen™), Lipofectamine™ MessengerMAX™ (Invitrogen™), Lipofectin™ (Invitrogen™), siLentFet™ (Bio-Rad), DharmaFECT™ (Dharmacon), HiPerFect (Qiagen), TransIT-X2® (Minis), jetMESSENGER® (Polyplus), Trans-Hi™, JetPEI® (Polyplus), and ViaFect™ (Promega).

[00172] In some embodiments, the composition is an aqueous composition. Aqueous compositions of the present application comprise an effective amount of the vector, dissolved, or dispersed in a pharmaceutically acceptable carrier or aqueous medium.

[00173] In some embodiments, provided herein is a pharmaceutical composition comprising an adeno-associated viral (AAV) vector described herein and a pharmaceutically acceptable carrier. [00174] The vector(s) (/.<?., adeno-associated viral (AAV) vector and/or lentiviral vectors disclosed herein) and/or pharmaceutical composition(s) disclosed herein can be formulated according to any available conventional method. Examples of preferred dosage forms include a tablet, a powder, a subtle granule, a granule, a coated tablet, a capsule, a syrup, a troche, an inhalant, a suppository, an injectable, an ointment, an ophthalmic ointment, an eye drop, a nasal drop, an ear drop, a cataplasm, a lotion and the like. In the formulation, generally used additives such as a diluent, a binder, a disintegrant, a lubricant, a colorant, a flavoring agent, and if necessary, a stabilizer, an emulsifier, an absorption enhancer, a surfactant, a pH adjuster, an antiseptic, an antioxidant, and the like can be used.

[00175] In some embodiments, the method of the present disclosure involves contacting cell or tissues or organs having TDP-43 mislocalization or dysfunction with an adeno-associated viral (AAV) vector or a composition described herein under conditions effective to express one or more splicing factors and/or splicing regulators, in the cells or tissues or organs to increase the level of one or more splicing factors and/or splicing regulators, and/or to restore compromised one or more splicing factors and/or splicing regulators.

[00176] In some embodiments, the method of the present disclosure involves contacting cell or tissues or organs having TDP-43 mislocalization or dysfunction with an adeno-associated viral (AAV) vector or a composition described herein under conditions effective to express one or more of N0VA1, ELAVL1, ELAVL2, ELAVL3, ELAVL4, RBFOX2, MATR3, HNRNPA1, SFPQ, and FUS, or a functional fragment thereof, in the cells or tissues or organs to increase the level of one or more of NOVAI, ELAVL1, ELAVL2, ELAVL3, ELAVL4, RBFOX2, MATR3, HNRNPA I , SFPQ, and FUS., (e.g., a correctly spliced NO VAI , ELAVL1, ELAVL2, ELAVL3, ELAVL4, RBF0X2, MATR3, HNRNPAI, SFPQ, and/or FUS) or a functional fragment thereof, and/or to restore compromised, mislocalized, and/or dysfunctional (e.g., mis-spliced or incorrectly spliced) one or more of N0VA1, ELAVL1, ELAVL2, ELAVL3, ELAVL4, RBF0X2, MATR3, HNRNPAI, SFPQ, and FUS

[00177] Suitable cells for use according to the methods of the present application include, without limitation, mammalian cells such as rodent (mouse or rat) cells, cat cells, dog cells, rabbit cells, horse cells, sheep cells, pig cells, cow cells, and non-human primate cells. In some embodiments the cells are human cells.

[00178] In some embodiments, the method further involves culturing the cells ex vivo under conditions effective to express one or more splicing factors and/or splicing regulators. In some embodiments, the method further involves culturing the cells ex vivo under conditions effective to express one or more of NOVAI, ELAVL1, ELAVL2, ELAVL3, ELAVL4, RBFOX2, MATR3, HNRNPAI, SFPQ, and FUS, or a functional fragment thereof, . In some embodiments, the one or more of NOVAI, ELAVL1, ELAVL2, ELAVL3, ELAVL4, RBFOX2, MATR3, HNRNPAI, SFPQ, and FUS is correctly spliced. In some embodiments, the one or more of NOVAI, ELAVL1, ELAVL2, ELAVL3, ELAVL4, RBFOX2, MATR3, HNRNPAI, SFPQ, and FUS is restored. In some embodiments, the one or more of NOVAI, ELAVL1, ELAVL2, ELAVL3, ELAVL4, RBFOX2, MATR3, HNRNPAI, SFPQ, and FUS is incorrectly spliced or mis-spliced. In some embodiments, the method is carried out in vivo.

[00179] Contacting, according to the methods of the present application, may be carried out by intracranial administration, systemic administration, or intrathecal administration. In some embodiments, the vectors as described herein are administered to the subject via intracranial administration, systemic administration, or intrathecal administration. Contacting, according to the methods of the present application, may also be carried out by oral administration, by topical administration, by transdermal administration, by parenteral administration, by subcutaneous administration, by intravenous administration, by intramuscular administration, by intraperitoneal administration, by intranasal instillation administration, by intracavitary or by intravesical instillation, by intraocular administration, by intraarterial administration, by intralesional administration, or by application to mucous membranes. In some embodiments, the contacting is carried out by intramuscular administration, by intracranial administration, intravenous administration, by intrathecal administration, by subcutaneous administration, by oral administration, or by intraperitoneal administration to a subject.

Antisense oligonucleotides

[00180] In one aspect, the present disclosure provides an antisense oligonucleotide targeting one or more splicing factors and/or splicing regulators. In some embodiments, the antisense oligonucleotide targets one or more of NOVAI, ELAVL1, ELAVL2, ELAVL3, ELAVL4, RBFOX2, MATR3, HNRNPA1, SFPQ, and FUS

[00181] The targeting of one or more splicing factors and/or splicing regulators may include, but is not limited to, targeting pre-mRNA of one or more exons of NOVAI, ELAVL1, ELAVL2, ELAVL3, ELAVL4, RBFOX2, MATR3, HNRNPA1, SFPQ, and FUS. In some embodiments, the antisense oligonucleotide targets pre-mRNA of one or more exons of NOVA 1, ELAVL1, ELAVL2, ELAVL3, ELAVL4, RBFOX2, MATR3, HNRNPA1, SFPQ, and FUS. In some embodiments, the antisense oligonucleotide binds to a pre-mRNA sequence flanking a misspliced exon of one or more of NO VAI, ELAVL1, ELAVL2, ELAVL3, ELAVL4, RBFOX2, MATR3, HNRNPA1, SFPQ, and FUS

[00182] Non-limiting examples of exons include exon 8 of NOVAI pre-mRNA, exon 6 of ELAVL1 pre-mRNA, exon 6 of ELAVL2 pre-mRNA, exon 4 of ELAVL3 pre-mRNA, exon 10 and 11 ofELAVL4 pre-mRNA, exon 3 ofRBFOX2 pre-mRNA, exon 15 ofMATR3 pre- mRNA, exon 6 of HNRNPA1 pre-mRNA, exon 9 of SFPQ pre-mRNA, exon 3 and 7 of FUS pre-mRNA, and any combinations thereof.

[00183] Non-limiting examples of exons include exon 8 (chrl4:26, 472, 320-26, 472, 391) of NOVAI pre-mRNA, exon 6 (chrl9:7, 967, 565-7, 967, 790) of ELAVL1 pre-mRNA, exon 6 (chr9:23, 762, 163-23,762,249) ofELAVL2 pre-mRNA, exon 4 (chrl9: 11,458,457-11,458,611) of ELAVL3 pre-mRNA, exon 10 (chrl :50, 193,765-50, 193,918) and 11 (chrl :50,195,561- 50,195,786) of ELAVL4 pre-mRNA, exon 3 (chr22:35, 938, 847-35, 938, 897) of RBFOX2 pre- mRNA, exon 15 (chr5: 139,325,440-139,325,662) of MATR3 pre-mRNA, exon 6

(chrl2:54, 283, 079-54, 283, 234) of HNRNPA1 pre-mRNA, exon 9 (chrl : chrl:35, 177,989- 35,178,036) of SFPQ pre-mRNA, exon 3 (chrl6:31, 183, 858-31, 183, 999) and 7

(chrl6:31, 186, 802-31, 186, 836) of FUS pre-mRNA, and any combinations thereof. [00184] In some embodiments, antisense oligonucleotides are 100% complementary to the target sequence, or may include one or more mismatches, e.g., to improve selective targeting of allele containing the disease-associated modifications or mis-splices exons, if a heteroduplex formed between the antisense oligonucleotide and target sequence is sufficiently stable to withstand the action of cellular nucleases and other modes of degradation which may occur in vivo. In some embodiments, antisense oligonucleotides may have about or at least about 70% sequence complementarity, e.g., 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence complementarity, between the oligonucleotide and the target sequence. If antisense oligonucleotides include mismatches, they are typically less destabilizing toward the end regions of the hybrid duplex than in the middle. The number of mismatches may depend on the percentage of G:C base pairs in the duplex, the length of the oligonucleotide, and the position of the mismatch(es) in the duplex, according to well understood principles of duplex stability.

[00185] In some embodiments, the antisense oligonucleotides are complementary to splice sites or splicing regulatory elements of one or more of NOVAI, ELAVL1, ELAVL2, ELAVL3, ELAVL4, RBFOX2, MATR3, HNRNPA1, SFPQ, and FUS. The splicing regulatory elements comprise exonic splicing enhancers (ESEs).

[00186] In some embodiments, the antisense oligonucleotides are optimized by tiling relevant splice junctions of one or more pre-mRNA of NOVAI, ELAVL1, ELAVL2, ELAVL3, ELAVL4, RBFOX2, MATR3, HNRNPA1, SFPQ, and FUS. The splice sites (5’ or 3’ ends of exons) and/or splicing regulatory elements (enhancers or silencers) can be tiled to saturation. The tiling may include designing a series of overlapping ASOs that span the entire exon-intron boundary, and/or splicing regulatory elements. There is a 5-10 nucleotide overlap between consecutive ASOs. Each ASO is specific to the target sequence and may not have significant off- target binding in the genome.

[00187] In some embodiments, the antisense oligonucleotides can interfere with mRNA maturation. An mRNA maturation can be inhibited by hindering the 5’ cap formation. In some embodiments, the antisense oligonucleotides disrupt the addition of the 5’ cap. The disruption of the addition of the 5’ cap may destabilize the mRNA and diminish its translation efficiency. In some embodiments, the antisense oligonucleotides are capable to influence pre-mRNA splicing events. The antisense oligonucleotides that are capable to influence pre-mRNA splicing events may also termed as splice-switching oligonucleotides. An antisense oligonucleotide hybridizes with a pre-mRNA sequence and thereby alters splice site recognition. The alteration of the splice site recognition may lead to inclusion or exclusion of exons during mRNA processing. This changes in splicing patterns may produce an altered mRNA isoform (e.g., an altered mRNA with the exclusion of targeted one or more exons). The hybridization of the antisense oligonucleotide and pre-mRNA sequence affects resulting protein products or triggering mRNA degradation. [00188] In some embodiments, the antisense oligonucleotides physically obstruct ribosomal activity by binding to a target mRNA sequence. In some embodiments, the antisense oligonucleotides bind near translation initiation sites or along coding regions of the target mRNA sequence. In some embodiments, the antisense oligonucleotides obstruct physically ribosomal activity by inducing RNase-H endonuclease cleavage of a target RNA. The RNase-H endonuclease cleavage of a target RNA can be induced by inhibiting 5’ cap formation or by altering splicing. The obstruction of ribosomal activity by binding to a specific site on the target mRNA sequence can create steric hindrance which affects ribosome binding or progression of the target mRNA sequence and thereby reducing protein synthesis efficiency of the target mRNA sequence.

[00189] The term “antisense” broadly includes RNA-RNA interactions, RNA-DNA interactions, and RNase-H mediated arrest. Antisense oligonucleotides can be encoded by a recombinant gene for expression in a cell (see, e.g., U.S. Pat. Nos. 5,814,500 and 5,811,234), or alternatively they can be prepared synthetically (see, e.g., U.S. Pat. No. 5,780,607). According to the present disclosure, the expression(s) and/or function(s) of one or more of NOVAI, ELAVL1, ELAVL2, ELAVL3, ELAVL4, RBFOX2, MATR3, HNRNPA1, SFPQ, and FUS may be identified, modulated, and studied using antisense oligonucleotides described herein. Such ASOs can be derived based on one or more of NOVAI, ELAVL1, ELAVL2, ELAVL3, ELAVL4, RBFOX2, MATR3, HNRNPA1, SFPQ, and FUS nucleic acid molecules. Furthermore, as disclosed herein, due to their ability to modulate expression of one or more of N0VA1, ELAVL1, ELAVL2, ELAVL3, ELAVL4, RBFOX2, MATR3, HNRNPA1, SFPQ, and FUS, N0VA1-, ELAVL1-, ELAVL2-, ELAVL3-, ELAVL4-, RBFOX2-, MATR3-, HNRNPA1-, SFPQ-, and FUS- specific antisense oligonucleotides may be useful as therapeutics to treat a disease characterized by TDP-43 mislocalization or dysfunction in a subject. The disease characterized by TDP-43 mislocalization or dysfunction includes a neurodegenerative disease. [00190] A modulation of expression (e.g, an activation or enhancement of expression) of one or more ofNOVAl, ELAVL1, ELAVL2, ELAVL3, ELAVL4, RBFOX2, MATR3, HNRNPA1, SFPQ, and FUS can be achieved by antisense oligonucleotides described herein. Antisense oligonucleotides of the invention comprise from about 6 to about 200 nucleotides but are typically about 13 to about 50 nucleotides in length. Such “activating” antisense oligonucleotides may function by targeting pre-mRNA of exon(s), binding to a pre-mRNA sequence flanking a mis-spliced exon, and/or by optimizing by tiling relevant splice junction(s) of one or more pre- mRNA ofNOVAl, ELAVL1, ELAVL2, ELAVL3, ELAVL4, RBFOX2, MATR3, HNRNPA1, SFPQ, and FUS. Exemplary antisense oligonucleotides are typically 18-22 nucleotides long, with a GC content between 40-60%. The splice sites (5’ or 3’ ends of exons) and/or splicing regulatory elements (enhancers or silencers) can be tiled to saturation.

[00191] The antisense oligonucleotides of the invention comprise sequences complementary to at least a portion of the corresponding splicing factor or splicing factor-encoding nucleic acid. The antisense oligonucleotides of the invention comprise sequences complementary to at least a portion of the corresponding splicing regulator or splicing regulator-encoding nucleic acid. The antisense oligonucleotides of the invention comprise sequences complementary to at least a portion of the corresponding wild-type protein-encoding nucleic acid. The wild-type protein according to the present invention is selected from NOVAI, ELAVL1, ELAVL2, ELAVL3, ELAVL4, RBFOX2, MATR3, HNRNPA1, SFPQ, and FUS, or any combinations thereof. The antisense oligonucleotides of the invention comprise sequences complementary to a functional fragment of one or more ofNOVAl, ELAVL1, ELAVL2, ELAVL3, ELAVL4, RBFOX2, MATR3, HNRNPA1, SFPQ, and FUS. However, 100% sequence complementarity is not required so long as formation of a stable duplex (for single stranded antisense oligonucleotides) or triplex (for double stranded antisense oligonucleotides) can be achieved.

[00192] In some embodiments, ASOs include oligonucleotides that contain phosphorothioates, aminoalkylphosphotriesters, phosphotriesters, chiral phosphorothioates, phosphorodithi oates, methyl and other alkyl phosphonates optionally comprising 3’ alkylene phosphonates and chiral phosphonates, phosphoramidates optionally comprising 3 ’-amino phosphoramidate and aminoalkylphosphoramidates, phosphinates, thionoalkylphosphonates, thionophosphoramidates, thionoalkylphosphotriesters, boranophosphates having normal 3’-5’ linkages, 2’-5’ linked analogs of these, those having inverted polarity wherein the adjacent pairs of nucleoside units are linked 3’-5’ to 5’-3’ or 2’-5’ to 5’-2’, short chain alkyl or cycloalkyl intersugar linkages, or short chain heteroatomic or heterocyclic intersugar linkages. Some antisense oligonucleotides contain with CH₂— NH— O— CH₂, CH₂— N(CH₃)— O— CH₂, CH₂— O— N(CH₃)— CH₂, CH₂— N(CH₃) — N(CH₃) — CH₂ and O — N(CH₃) — CH₂ — CH₂ backbones (where phosphodiester is O — PO₂ — O — CH₂). U.S. Pat. No. 5,677,437 describes heteroaromatic oligonucleoside linkages. Nitrogen linkers or groups containing nitrogen can also be used to prepare oligonucleotide mimics (U.S. Pat. Nos. 5,792,844 and 5,783,682). U.S. Pat. No. 5,637,684 describes phosphoramidate and phosphorothioamidate oligomeric compounds.

[00193] “Phosphorothioates” (also known as S-oligos) are a variant of normal DNA with one of the nonbridging oxygens replaced by a sulfur. The sulfurization of the internucleotide bond reduces the action of endo-and exo- nucleases including 3’ to 5’ and 5’ to 3’ DNA Polymerase I (Pol I) exonuclease, serum nucleases, nucleases SI and PI, RNases, and snake venom phosphodiesterase. Phosphorothioates can be made by two principal routes: (i) by the method of sulfurizing phosphite triesters with either 3H-1, 2-bensodithiol-3-one 1, 1 -dioxide (BDTD) or tetraethylthiuram disulfide (TETD) (see, e.g., Iyer et al., J. Org. Chem. 55, 4693-4699, 1990), or (ii). by the action of a solution of elemental sulfur in carbon disulfide on a hydrogen phosphonate. The former methods avoid the problem of elemental sulfur’s insolubility in most organic solvents and the toxicity of carbon disulfide. The BDTD and TETD methods also yield higher purity phosphorothioates.

[00194] In some embodiments, ASOs are oligonucleotides having morpholino backbone structures (U.S. Pat. No. 5,034,506) (/.e., an oligonucleotide in which the bases are linked to 6- membered morpholine rings, which are connected to other morpholine-linked bases via nonionic phosphorodiamidate intersubunit linkages). Morpholino oligonucleotides are highly resistant to nucleases and have good targeting predictability, high in-cell efficacy, and high sequence specificity (U.S. Pat. No. 5,034,506; Summerton, Biochim. Biophys. Acta 1999; 1489: 141-158; Summerton and Weller, Antisense Nucleic Acid Drug Dev. 1997; 7: 187-195; Arora et al., J. Pharmacol. Exp. Ther. 2000; 292:921-928; Qin et al., Antisense Nucleic Acid Drug Dev. 2000; 10: 11-16; Heasman et al., Dev. Biol. 2000; 222: 124-134; Nasevicius and Ekker, Nat. Genet. 2000; 26:216-220). [00195] In some embodiments, ASOs are oligonucleotides having a gapmer.

[00196] In some embodiments, a “Gapmer” of an oligonucleotide is 10-50 nucleosides in length. For example, a gapmer may be 10-15, 10-40, 10-50, 10-45, 20-40, 10-35, 10-30, 15-20, 10-25, 10-20, 15-40, 15-35, 30-40, 15-30, 15-25, 20-35, 20-30, 20-25, 25-40, 25-35, 25-30, 30- 35, or 35-40 nucleosides in length. In some embodiments, a gapmer 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, or 40 nucleosides in length. In some embodiments, a gapmer is about 16 to about 20 nucleosides in length. In some embodiments, a gapmer is 16 nucleotides in length. In some embodiments, a gapmer is 17 nucleotides in length. In some embodiments, a gapmer is 18 nucleotides in length. In some embodiments, a gapmer is 19 nucleotides in length. In some embodiments, a gapmer is 20 nucleotides in length.

[00197] An oligonucleotide having a “Gapmer” is an oligonucleotide comprising an internal region containing a plurality of nucleosides that support RNase-H cleavage positioned between external regions containing one or more nucleosides, wherein the nucleosides comprising the internal region are chemically distinct from the nucleoside or nucleosides comprising the external regions. The internal region can be referred to as the “gap” and the external regions can be referred to as the “wings.” In some embodiments, a gapmer may have a 3-10-3 configuration or a 5-10-5 configuration. In some embodiments, a gapmer may have 5’ and 3’ wings each having 2-6 nucleotides and a gap having 7-12 nucleotides.

[00198] In some embodiments, the gap region of the gapmer polynucleotide contains modified nucleotides for efficient RNase-H action in addition to DNA nucleotides, such as acyclic nucleotides, C4’ -substituted nucleotides, and arabino-configured nucleotides. In some embodiments, one or both flanking regions each independently comprise one or more phosphorothioate internucleoside linkages (e.g., phosphorothioate intemucleoside linkages or other linkages) between at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, or at least 10, or more nucleotides. In some embodiments, the gap region and two flanking regions each independently comprise modified internucleoside linkages (e.g, phosphorothioate intemucleoside linkages or other linkages) between at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, or at least 10, or more nucleotides. In some embodiments, the gap region comprises one or more unmodified intemucleosides. In some embodiments, each intemucleotide linkage in the gap segment comprises a phosphorothioate linkage. In some embodiments, each intemucleotide linkage in the gapmer comprises a phosphorothioate linkage. In some embodiments, each internucleotide linkage in the 5’ or 3’ wing region comprises a phosphorothioate linkage. [00199] An oligonucleotide having a “Gapmer” commonly has the formula 5’-X-Y-Z-3’, wherein X and Z are flanking regions around a gap region Y. In some embodiments, flanking region X of formula 5’-X-Y-Z-3’ is also called 5’ wing region X, flanking sequence X, X region, or 5’ wing segment. In some embodiments, flanking region Z of formula 5’-X-Y-Z-3’ is also called 3’ wing region Z, flanking sequence Z, Z region, or 3’ wing segment. In some embodiments, gap region Y of formula 5’-X-Y-Z-3’ is also called gap segment, Y segment, Y region, gap-segment Y, or gap region. In some embodiments, each nucleoside in the gap region Y is a 2’ -deoxyribonucleoside., In some embodiments, each nucleoside in neither the 5’ wing region X or the 3’ wing region Z comprises any 2’ -deoxyribonucleosides.

[00200] In some embodiments, the gap region in a gapmer is 5-20 nucleosides in length. For example, the gap region Y may be 5-10, 10-20, 5-20, 5-15, 10-15, or 15-20 nucleosides in length. In some embodiments, the gap region is 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleosides in length. In some embodiments, one or more nucleosides in the gap region Y is a 2’-deoxyribonucleoside. In some embodiments, every nucleotide in the gap region is a deoxyribonucleoside. In some embodiments, one or more of the nucleosides in the gap region is a modified nucleoside (e. ., a 2’ modified nucleoside as described herein). In some embodiments, one or more cytosines in the gap region Y are 5-methyl-cytosines. In some embodiments, every cytosine in a gapmer is a 5-methyl-cytosine. In some embodiments, every cytosine in the gap region Y is a 5-methyl-cytosine.

[00201] In some embodiments, the Y region comprises a contiguous stretch of nucleotides, e.g., a region of 5 or more DNA nucleotides. In some embodiments, the Y region can recruit an RNase including, but is not limited to, Rnase-H. In some embodiments, the gapmer can bind to a target nucleic acid such that an Rnase is recruited to cleave the target nucleic acid (e.g., m-RNA). In some embodiments, the Y region may be flanked both 5’ and 3’ by regions X and Z which contain high-affinity modified nucleosides, e.g., 1-10 high-affinity modified nucleosides. Exemplary high affinity modified nucleosides include, but are not limited to, 2’ -modified nucleosides (e.g., 2’-M0E, 2’0-Me, 2’-F) and 2’-4’ bicyclic nucleosides (e.g., methylene (LNA), (S)-constrained ethyl (cEt), ethylene (ENA)). [00202] “2’ O-Me” containing oligonucleotide molecules carry a methyl group at the 2’-OH residues of the ribose molecule. 2’-O-Me-RNAs show the same (or similar) behavior as DNA, except for 2’-O-Me-RNAs are protected against nuclease degradation. 2’-O-Me-RNAs can be combined with phosphothioate oligonucleotides (PTOs) for further stabilization. 2’ -O-Me oligonucleotides (phosphodiester or phosphothioate) can be synthesized according to techniques known in the art (see, e.g., Yoo et al., Nucleic Acids Res. 32:2008-16, 2004).

[00203] In some embodiments, the flanking sequences X and Z can be of 1-5 nucleotides, 1-10 nucleotides, 1-20 nucleotides, or 1-30 nucleotides, in length. The flanking sequences X and Z can be of similar length or of dissimilar lengths. In some embodiments, the flanking sequences X and Z are each 3 nucleotides in length. In some embodiments, the flanking sequences X and Z are each 5 nucleotides in length. In some embodiments, the flanking sequences X and Z are each 7 nucleotides in length. In some embodiments, the gap-segment Y can be a nucleotide sequence of 5-10 nucleotides, 5-20 nucleotides, or 5-30 nucleotides in length. In some embodiments, the gap segment is 10 nucleotides in length. In some embodiments, the gap segment is 20 nucleotides in length. In some embodiments, the gap segment is 30 nucleotides in length.

[00204] In some embodiments, the 5’ wing region and the 3’ wing region of a gapmer are independently 1-20 nucleosides (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleosides) long. For example, the 5’ wing region and the 3’ wing region of the gapmer may be independently 1-3, 1-2, 1- 20, 1-15, 2-5, 2-7, 1-10, 1-7, 1-5, 10-20, 10-15, 3-5, 3-7, 5-20, 5-15, 5-10, or 15-20 nucleosides long. In some embodiments, the 5’ wing region and the 3’ wing region of a gapmer are of different lengths. In some embodiments, the 5’ wing region and the 3’ wing region of the gapmer are of the same length. In some embodiments, the 5’ wing region is shorter than the 3’ wing region of the gapmer. In some embodiments, the 5’ wing region is longer than the 3’ wing region of a gapmer.

[00205] In some embodiments, one or more nucleosides in the 5’ wing region and/or the 3’ wing region of a gapmer are modified nucleotides. In some embodiments, the modified nucleotide may be a 2’- modified nucleoside, e.g., a non-bicyclic 2’ -modified nucleoside or 2’ -4’ bicyclic nucleoside. In some embodiments, the nucleoside can be a 2’-4’ bicyclic nucleoside (e.g., cEt, LNA, or ENA) or a non-bicyclic 2’-modified nucleoside (e.g., 2’-fluoro (2’-F), 2’-O- methyl (2’-O-Me), 2’-O-dimethylaminopropyl (2’-O-DMAP), 2’-O-dimethylaminoethyl (2’-O- DMAOE), 2’-O-methoxy ethyl (2’-M0E), 2’-O-dimethylaminoethyloxyethyl (2’-O-DMAEOE), 2 ’-O-ami nopropyl (2’-0-AP), or 2’-O-N-methylacetamido (2’-0-NMA)). In some embodiments, every nucleotide in a wing region is a modified nucleotide. In some embodiments, every nucleotide in a wing region is a 2’ -MOE, cET, or LNA nucleotide.

[00206] In some embodiments, a gapmer described herein comprises one or more modified nucleoside linkages in each of the X, Y, and Z regions. In some embodiments, each internucleoside linkage comprises phosphorothioate linkage. In some embodiments, each of the X, Y, and Z regions independently comprises a combination of phosphodiester linkages and phosphorothioate linkages. In some embodiments, each intemucleoside linkage in the gap region Y is a phosphorothioate linkage, the 5’ wing region X comprises a combination of phosphorothioate linkages and phosphodiester linkages, and the 3’ wing region Z comprises a combination of phosphorothioate linkages and phosphodiester linkages.

[00207] In some embodiments, ASOs include, without limitation, peptide nucleic acids (PNAs). PNAs are analogs of DNA in which the backbone is structurally homomorphous with a deoxyribose backbone, consisting of N-(2-aminoethyl) glycine units to which pyrimidine or purine bases are linked. PNAs which contain natural pyrimidine and purine bases hybridize to complementary oligonucleotides obeying Watson-Crick base-pairing rules, and mimic DNA in terms of base pair recognition (Egholm, Buchardt et al. 1993). The backbone of PNAs is formed by peptide bonds, making them well-suited for antisense applications. The backbone is uncharged, resulting in PNA/RNA or PNA/DNA duplexes that exhibit greater than normal thermal stability. PNAs may not be recognized by nucleases or proteases.

[00208] PNAs are capable of sequence-specific binding in a helix form to DNA or RNA. The non-limiting characteristics of PNAs include a destabilizing effect caused by single-base mismatch, a high binding affinity to complementary DNA or RNA, hybridization with DNA or RNA independent of salt concentration, resistance to nucleases and proteases, and triplex formation with homopurine DNA. One of the known PNAs is Bts PNA monomer (Bts; benzothiazole-2-sulfonyl group). The PNA oligomerization using Bts PNA monomers is composed of repetitive cycles of deprotection, coupling, and capping. PNAs can be produced synthetically using any technique known in the art. See, e.g., U.S. Pat. Nos. 5,539,082;

5,714,331; and 5,719,262 for the preparation of PNAs. See also U.S. Pat. Nos. 5,539,082; 5,714,331; 5,719,262; 6,969,766; 7,211,668; 7,022,851; 7,125,994; 7,145,006; and 7,179,896. PNA compounds can be found in Nielsen et al., Science, 254: 1497-1500, 1991 . Each of the foregoing is incorporated by reference in its entirety.

[00209] In some embodiments, the PNA backbone or the phosphodiester backbone of the oligonucleotide may be replaced with a polyamide backbone, the bases being bound directly or indirectly to the aza nitrogen atoms of the polyamide backbone (Nielsen et al., Science 1991; 254: 1497). Other ASOs may contain substituted sugar moieties comprising one of the following at the 2’ position: OH, SH, SCH3, F, OCN, O(CH2)_nNH2, or O(CH2)_nCH3, where n is from 1 to about 10; Ci to C10 lower alkyl, substituted lower alkyl, alkaryl, or aralkyl; Cl; Br; CN; CF3; OCF3; 0— S— , or N-alkyl; O-, S-, or N-alkenyl; SOCH3; SO2CH3; ONO₂; NO₂; N₃; NH₂; heterocycloalkyl; heterocycloalkaryl; aminoalkylamino; polyalkylamino; substituted sialyl; a fluorescein moiety; an RNA cleaving group; a reporter group; an intercalator; a group for improving the pharmacokinetic properties of an oligonucleotide; or a group for improving the pharmacodynamic properties of an oligonucleotide, and other substituents having similar properties.

[00210] ASOs may also have sugar mimetics such as cyclobutyls or other carbocyclics in place of the pentofuranosyl group. Nucleotide units having nucleosides other than adenosine, cytidine, guanosine, thymidine, and uridine may be used, such as inosine.

[00211] The sugar moieties may include 2’-ribose substitutions (e.g., 2’-O-methyl, 2’-O- methoxyethyl, a 2’-deoxy, 2’-fluoro, 2’-O-aminopropyl, 2’-O-dimethylaminoethyl, 2’-O- dimethylaminopropyl, 2’-O-dimethylaminoethyloxyethyl, and/or 2’-O-N-methylacetamido), creation of bridged nucleic acids (e.g., locked nucleic acid (LNA), 2’,4’-constrained 2’-O-ethyl bridged nucleic acid, and/or 2’-( ,4’-C-ethylene bridged nucleic acid), and/or creation of a phosphorodiamidate morpholino oligonucleotide (i.e., the five-membered ribose sugar is replaced by a six-membered morpholine ring). In some embodiments, each nucleotide of the ASO molecule can be a modified nucleotide e.g., a 2’-modified nucleotide).

[00212] In other embodiments, LNA can be used (reviewed in, e.g., Jepsen and Wengel, Curr. Opin. Drug Discov. Devel. 2004; 7: 188-194; Crinelli et al., Curr. Drug Targets 2004; 5:745- 752). LNA are nucleic acid analog(s) with a 2’-O, 4’-C methylene bridge. LNA enhances backbone preorganization and base stacking to increase hybridization and thermal stability. This bridge restricts the flexibility of the ribofuranose ring and locks the structure into a rigid C3-endo conformation, conferring enhanced hybridization performance and exceptional biostability. LNA allows the use of very short oligonucleotides (less than 10 bp) for efficient hybridization in vivo. [00213] The various structures of LNAs can be found, e.g, in Wengel, et al., Chemical Communications (1998) 455; Tetrahedron (1998) 54:3607, and Accounts of Chem. Research (1999) 32:301); Bioorganic Medicinal Chemistry (2008) 16:9230, and Obika, etal., Tetrahedron Letters (1997) 38:8735; (1998) 39:5401. Antisense oligonucleotides described herein may incorporate one or more LNAs or the antisense oligonucleotides may be entirely composed of LNAs. Methods for the synthesis of individual LNA nucleoside subunits and their incorporation into oligonucleotides are described, e.g., in U.S. Pat. Nos 6,794,499, 6,670,461, 7,034,133, 7,053,207, 7,060,809, 7,084,125, 7,569,575, and 7,572,582, each of which is incorporated by reference in its entirety. Generally, intersubunit linkers include phosphodiester and phosphorothioate moi eties. In some cases, non-phosphorous containing linkers may be employed. In some embodiments, an antisense oligonucleotide comprises an LNA containing compound, wherein each LNA subunit is separated by a DNA subunit. Certain antisense oligonucleotides are composed of alternating DNA and LNA subunits, wherein the intersubunit linker is phosphorothioate.

[00214] In some embodiments, antisense oligonucleotides comprise nucleotide sequences that are fragments of the nucleotide sequences of any of the splicing factors- and/or splicing regulators-related antisense oligonucleotides described herein. Such fragments comprise at least about 5%, at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, or at least about 99% of the contiguous nucleotide sequence of the antisense oligonucleotides described herein, or the complements of such oligonucleotide sequences.

[00215] Such fragments comprise at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 30, at least 35, or more consecutive nucleotides of any of the antisense oligonucleotides described herein, or the complements of such oligonucleotide sequences.

[00216] Such fragments can be used for a variety of purposes including, e.g., to produce a portion of one or more splicing factors and/or splicing regulators in an appropriate expression system, to identify functional or structural domains of one or more splicing factors and/or splicing regulators, to use it to enhance expression or function of one or more splicing factors and/or splicing regulators, e.g., a correctly spliced splicing factor and/or splicing regulator. [00217] In addition to the nucleotide sequences of any of the one or more splicing factors- and/or splicing regulators- related antisense oligonucleotides described herein, polynucleotide molecules of the present invention can further comprise, or alternatively may consist of, nucleotide sequences selected from those sequences that naturally flank one or more splicing factors- and/or splicing regulators- encoding nucleotide sequence in the chromosome, including regulatory sequences.

[00218] The present invention further provides an oligonucleotide molecule that hybridizes to a polynucleotide molecule of the present invention, or that hybridizes to a polynucleotide molecule having a nucleotide sequence that is the complement of a nucleotide sequence of a polynucleotide molecule of the present invention. Such an oligonucleotide molecule: (i) is about 10 nucleotides to about 200 nucleotides in length or from about 15 to about 100 nucleotides in length, or about 20 to about 50 nucleotides in length, and (ii) hybridizes to one or more of the polynucleotide molecules of the present invention under highly stringent conditions (e.g., washing in 6*SSC/0.5% sodium pyrophosphate at about 37°C. for about 14-base oligos, at about 48°C; for about 17-base oligos, at about 55°C; for about 20-base oligos, at about 60°C; and for about 23-base oligos).

[00219] An antisense oligonucleotide may comprise a short nucleotide sequence which is complementary or substantially complementary to a target nucleotide sequence in a pre-mRNA molecule, mRNA molecule, or heterogeneous nuclear RNA (hnRNA). An antisense sequence can form a stable double stranded hybrid with the target nucleotide sequence in the RNA molecule under physiological conditions depending on the degree of complementarity (or substantial complementarity) of the antisense sequence. Antisense oligonucleotides can be synthetic and chemically modified. Without wishing to be bound by theory, “complementarity” of nucleic acids can mean that a nucleotide sequence in one strand of nucleic acid, e.g., due to orientation of its nucleobase groups, forms hydrogen bonds with another sequence on an opposing nucleic acid strand. The complementary bases in DNA are generally A paired with T and C paired with G. In RNA, the complementary bases are generally C with paired with G and U paired with A. Complementarity can be perfect or substantial/sufficient. Perfect complementarity between two nucleic acids means that the two nucleic acids can form a duplex in which every base within the duplex is bonded to a complementary base by Watson-Crick pairing. “Substantial” or “sufficient” complementarity means that a sequence in one strand is not completely and/or perfectly complementary to a sequence in an opposing strand but that sufficient bonding takes place between bases on the two strands to form a stable hybrid complex in set of hybridization conditions (e.g., temperature and/or salt concentration). Such conditions can be determined by, e.g., empirical determination of Tm (melting temperature) by employing routine methods in the art or by using the sequences and standard mathematical calculations to predict the Tm of hybridized strands. Tm can include the temperature at which a population of hybridization complexes formed between two nucleic acid strands are 50% denatured (z.e., a population of double-stranded nucleic acid molecules becomes half dissociated into single strands). At a temperature below the Tm, formation of a hybridization complex can be favored, while at a temperature above the Tm, melting or separation of the strands in the hybridization complex can be favored. ASO can be synthetic and chemically modified.

[00220] Nucleobases can be conventional bases (A, G, C, T, U), analogs thereof (e. , modified uridines such as 5 -methoxyuridine, pseudouridine, or N1 -methylpseudouridine, or others); inosine; derivatives of purines or pyrimidines (e.g., N4-methyl deoxyguanosine, deaza- or azapurines, deaza- or aza-pyrimidines, pyrimidine bases with substituent groups at the 5 or 6 position (e.g., 5-methylcytosine), purine bases with a substituent at the 2, 6, or 8 positions, 2- amino-6- methylaminopurine, 6-O-methylguanine, 4-thio-pyrimidines, 4-amino-pyrimidines, 4- dimethylhydrazine-pyrimidines, and 4-O-alkyl-pyrimidines. Nucleic acids can include one or more “abasic” residues where the backbone includes no nitrogenous base for position(s) of the polymer.

[00221] In some embodiments, the nucleobase modifications may include a pyrimidine methylation, such as a 5-methylcytidine or a 5-methyluridine, an abasic nucleotide, or an inverted abasic residues.

[00222] The antisense oligonucleotides can be DNA or RNA or chimeric mixtures, or derivatives or modified versions thereof, and can be single-stranded or double-stranded. The antisense oligonucleotides can be modified at the base moiety, sugar moiety, or phosphate backbone, or a combination thereof. For example, a splicing factor- and/or a splicing regulatorspecific antisense oligonucleotide can comprise at least one modified base moiety selected from a group including but not limited to -fluorouracil, 5-bromouracil, 5-chlorouracil, 5-iodouracil, hypoxanthine, xantine, 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-methyl adenine, 2-methylguanine, 3 -methylcytosine, 5 -methyl cytosine, N6-adenine, 7-methylguanine, 5-methylaminomethyluracil, 5-methoxyaminomethyl-2-thiouracil, beta-D-mannosylqueosine, 5-methoxycarboxymethyluracil, 5 -methoxyuracil, 2-methylthio-N6- isopentenyl adenine, uracil-5-oxyacetic acid (v), pseudouracil, queosine, 2-thiocytosine, 5- methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil, uracil-5-oxyacetic acid methylester, uracil-5-oxyacetic acid (v), 5-methyl-2-thiouracil, 3-(3-amino-3-N-2- carboxypropyl) uracil, (acp3)w, and 2,6-diaminopurine.

[00223] In another embodiment, the splicing factor- and/or splicing regulator- specific antisense oligonucleotide comprises at least one modified sugar moiety, e.g., a sugar moiety selected from arabinose, 2-fluoroarabinose, xylulose, and hexose.

[00224] In yet another embodiment, the splicing factor- and/or splicing regulator- specific antisense oligonucleotide comprises at least one modified phosphate backbone selected from a phosphorothioate, a phosphorodi thioate, a phosphoramidothioate, a phosphoramidate, a phosphordiamidate, a methylphosphonate, an alkyl phosphotri ester, and a formacetal or analog thereof. In some embodiment, the present invention provides phosphorothioate antisense oligonucleotides (e.g, 6NOVA1 and anti-6^NOVA1 phosphothioate modified at 3’ and 5’ ends to increase their stability) and chimeras between methylphosphonate and phosphodiester oligonucleotides. These oligonucleotides appear to provide good in vivo activity due to solubility, nuclease resistance, good cellular uptake, ability to activate RNase-H, and high sequence selectivity.

[00225] The antisense oligonucleotide can include other appending groups such as peptides, or agents facilitating transport across the cell membrane (see, e.g, Letsinger et al., Proc. Natl. Acad. Sci. USA 1989; 86:6553-6556; Lemaitre et al., Proc. Natl. Acad. Sci. USA 1987; 84:648- 652; PCT Publication No. WO 88/09810) or blood-brain barrier (see, e.g., PCT Publication No. WO 89/10134), hybridization-triggered cleavage agents (see, e.g., Krol et al., BioTechniques 1988; 6:958-976), intercalating agents (see, e.g., Zon, Pharm. Res. 1988; 5:539-549), etc.

[00226] In another embodiment, the antisense oligonucleotide can include a-anomeric oligonucleotides. An a-anomeric oligonucleotide forms specific double-stranded hybrids with complementary RNA in which, contrary to the usual P-units, the strands run parallel to each other (Gautier etal., Nucl. Acids Res. 1987; 15:6625-6641).

[00227] In one embodiment, the antisense oligonucleotide molecule of the present invention is 100% complementary over its entire length to a portion of at least one of the polynucleotide molecules of splicing factors and/or splicing regulators of the present invention. In another embodiment, an oligonucleotide molecule of the present invention is greater than 90% complementary over its entire length to a portion of at least one of the polynucleotide molecules of the splicing factors and/or splicing regulators of the present invention.

[00228] In one embodiment, an antisense oligonucleotide molecule of the present invention is 100% complementary over its entire length to a portion of at least one of the polynucleotide molecules of NO VAI, ELAVL1, ELAVL2, ELAVL3, ELAVL4, RBFOX2, MATR3, HNRNPA1, SFPQ, and FUS. In another embodiment, an oligonucleotide molecule of the present invention is greater than 90% complementary over its entire length to a portion of at least one of the polynucleotide molecules of N0VA1, ELAVL1, ELAVL2, ELAVL3, ELAVL4, RBFOX2, MATR3, HNRNPA1, SFPQ, and FUS.

[00229] Antisense oligonucleotide molecules of the present invention can be labeled, e.g., with radioactive labels e.g., y³²P), biotin, fluorescent labels, etc. In one embodiment, a labeled antisense oligonucleotide molecule can be used as a probe to detect the presence of a nucleic acid molecule encoding one or more of NOVAI, ELAVL1, ELAVL2, ELAVL3, ELAVL4, RBFOX2, MATR3, HNRNPA1, SFPQ, and FUS.

[00230] The antisense oligonucleotide molecules of the invention are capable of modulating function and/or expression of one or more splicing factors and/or splicing regulators of the invention. In some embodiments, antisense oligonucleotide molecules of the invention are capable of producing one or more splicing factors and/or splicing regulators that are correctly spliced. In some embodiments, antisense oligonucleotide molecules of the invention are capable of restoring misregulated splicing events to wild-type splicing events of one or more splicing factors and/or splicing regulators. The antisense oligonucleotide molecules of the invention are capable of modulating function and/or expression of one or more of NO VAI, ELAVL1, ELAVL2, ELAVL3, ELAVL4, RBFOX2, MATR3, HNRNPA1, SFPQ, and FUS. Generally, antisense oligonucleotide molecules are prepared synthetically, preferably on a nucleic acid synthesizer, and may be prepared with non-naturally occurring phosphoester analog bonds, such as thioester bonds, where appropriate.

[00231] Antisense oligonucleotides of the invention may be chemically synthesized, for example using appropriately protected ribonucleoside phosphoramidites and a conventional DNA/RNA synthesizer. Antisense nucleic acid oligonucleotides of the invention can also be produced intracellularly by transcription from an exogenous sequence. For example, a vector can be introduced in vivo such that it is taken up by a cell within which the vector or a portion thereof is transcribed to produce an antisense RNA. Such a vector can remain episomal or become chromosomally integrated, so long as it can be transcribed to produce the desired antisense RNA. Such vectors can be constructed by recombinant DNA technology methods standard in the art. Vectors can be plasmid, viral, or others known in the art, used for replication and expression in mammalian cells. In another embodiment, “naked” antisense nucleic acids can be delivered to adherent cells via “scrape delivery”, whereby the antisense oligonucleotide is added to a culture of adherent cells in a culture vessel, the cells are scraped from the walls of the culture vessel, and the scraped cells are transferred to another plate where they are allowed to re-adhere. Scraping the cells from the culture vessel walls serves to pull adhesion plaques from the cell membrane, generating small holes that allow the antisense oligonucleotides to enter the cytosol.

Screening methods of the antisense oligonucleotides

[00232] In one embodiment, the present invention provides a method for identifying candidate antisense oligonucleotides useful for modulating an expression and/or function of one or more splicing factors and/or splicing regulators, said method comprising: (a) contacting a first cell with an antisense oligonucleotide for a time period sufficient to allow the cell to respond to said contact with the test compound; (b) determining in the cell prepared in step (a) the expression and/or function of one or more splicing factors and/or splicing regulators; and (c) comparing the expression and/or function of one or more splicing factors and/or splicing regulators determined in step (b) to the expression and/or function of the one or more splicing factors and/or splicing regulators in a second (control) cell that has not been contacted with the antisense oligonucleotide of the present invention; wherein a detectable change in the function and/or expression of the one or more splicing factors and/or splicing regulators in the first cell in response to contact with the antisense oligonucleotide compared to the function and/or expression of the one or more splicing factors and/or splicing regulators in the second cell that has not been contacted with the antisense oligonucleotide, indicates that the antisense oligonucleotide modulates the function and/or expression of the one or more splicing factors and/or splicing regulators and is a candidate compound.

[00233] In one embodiment, the present invention provides a method for identifying candidate antisense oligonucleotides useful for modulating a function and/or expression of one or more wild-type proteins selected from NOVAI, ELAVL1, ELAVL2, ELAVL3, ELAVL4, RBFOX2, MATR3, HNRNPA1, SFPQ, and FUS, or any combinations thereof, said method comprising: (a) contacting a first cell or tissue or organ with an antisense oligonucleotide for a time period sufficient to allow the cell or tissue or organ to respond to said contact with the test compound; (b) determining in the cell or tissue or organ prepared in step (a) the expression and/or function of the wild-type protein selected from NOVAI, ELAVL1, ELAVL2, ELAVL3, ELAVL4, RBFOX2, MATR3, HNRNPA1, SFPQ, and FUS, or any combination thereof; and (c) comparing the expression and/or function of the wild-type protein selected from NOVAI, ELAVL1, ELAVL2, ELAVL3, ELAVL4, RBFOX2, MATR3, HNRNPA1, SFPQ, and FUS, or any combination thereof determined in step (b) to the expression and/or function of the wild-type protein selected from NO VAI, ELAVL1, ELAVL2, ELAVL3, ELAVL4, RBFOX2, MATR3, HNRNPA1, SFPQ, and FUS, or any combination thereof in a second (control) cell or tissue or organ that has not been contacted with the antisense oligonucleotide of the present invention; wherein a detectable change in the function and/or expression of the wild-type protein selected from N0VA1, ELAVL1, ELAVL2, ELAVL3, ELAVL4, RBFOX2, MATR3, HNRNPA1, SFPQ, and FUS, or any combination thereof in the first cell or tissue or organ in response to contact with the antisense oligonucleotide compared to the function and/or expression of the wild-type protein selected from N0VA1, ELAVL1, ELAVL2, ELAVL3, ELAVL4, RBFOX2, MATR3, HNRNPA1, SFPQ, and FUS, or any combination thereof in the second cell or tissue or organ that has not been contacted with the antisense oligonucleotide, indicates that the antisense oligonucleotide modulates the function and/or expression of the wild-type protein selected from NOVAI, ELAVL1, ELAVL2, ELAVL3, ELAVL4, RBFOX2, MATR3, HNRNPA1, SFPQ, and FUS, or any combination thereof and is a candidate compound.

[00234] A function and/or expression of the wild-type protein selected from N0VA1, ELAVL1, ELAVL2, ELAVL3, ELAVL4, RBFOX2, MATR3, HNRNPA1, SFPQ, and FUS, or any combinations thereof assayed according to this method can be any function, e.g., activation of the expression of the wild-type protein selected from N0VA1, ELAVL1, ELAVL2, ELAVL3, ELAVL4, RBF0X2, MATR3, HNRNPA1, SFPQ, and FUS, or any combination thereof; formation of the wild-type protein selected from N0VA1, ELAVL1, ELAVL2, ELAVL3, ELAVL4, RBF0X2, MATR3, HNRNPA1, SFPQ, and FUS, or any combination thereof; restoration of misregulated splicing events to wild-type splicing events of the wild-type protein selected from N0VA1, ELAVL1, ELAVL2, ELAVL3, ELAVL4, RBF0X2, MATR3, HNRNPA1, SFPQ and FUS or any combination thereof; correction of the splicing defects of the pre mRNA of the wild-type protein selected from N0VA1, ELAVL1, ELAVL2, ELAVL3, ELAVL4, RBF0X2, MATR3, HNRNPA1, SFPQ, and FUS, or any combination thereof; inhibition of the production of aberrant proteins generated by the TDP-43 induced splicing defects, wherein the protein is selected from N0VA1, ELAVL1, ELAVL2, ELAVL3, ELAVL4, RBF0X2, MATR3, HNRNPA1, SFPQ, and FUS, or any combination thereof; reduction of stress associated with neurodegenerative diseases, etc.

[00235] The splicing events that can be corrected by the antisense oligonucleotides of the present invention comprises correcting mis-splicing of one or more of exons include exon 8 of NOVAI pre-mRNA, exon 6 of ELAVL1 pre-mRNA, exon 6 of ELAVL2 pre-mRNA, exon 4 of ELAVL3 pre-mRNA, exon 10 and 11 ofELAVL4 pre-mRNA, exon 3 ofRBFOX2 pre-mRNA, exon 15 of MATR3 pre-mRNA, exon 6 of HNRNPA1 pre-mRNA, exon 9 of SFPQ pre-mRNA, exon 3 and 7 of FUS pre-mRNA, and any combinations thereof.

[00236] The splicing events that can be corrected by the antisense oligonucleotides of the present invention comprises correcting mis-splicing of one or more of exon 8 (chrl4:26,472,320- 26,472,391) of NOVAI pre-mRNA, exon 6 (chrl 9:7,967,565-7,967,790) of ELAVL1 pre- mRNA, exon 6 (chr9: 23, 762, 163-23,762,249) of ELAVL2 pre-mRNA, exon 10

(chrl :50, 193, 765-50, 193, 918) and 11 (chrl :50, 195,561-50, 195,786) ofELAVL4 pre-mRNA, exon 4 (chrl9: 11,458,457-11,458,611) of ELAVL3 pre-mRNA, exon 3 (chr22:35,938,847- 35,938,897) ofRBFOX2 pre-mRNA, exon 15 (chr5: 139,325,440-139,325,662) ofMATR3 pre- mRNA, exon 6 (chrl2:54, 283, 079-54, 283, 234) of HNRNPA1 pre-mRNA, exon 9 (chrl : chrl :35, 177, 989-35, 178, 036) of SFPQ pre-mRNA, and exon 3 (chrl6:31, 183, 858-31, 183, 999) and 7 (chrl6:31, 186, 802-31, 186, 836) ofFUS [00237] In another embodiment, the present invention provides a method for identifying an antisense oligonucleotide capable of binding to an RNA molecule encoding a eukaryotic wildtype protein selected from N0VA1, ELAVL1, ELAVL2, ELAVL3, ELAVL4, RBFOX2, MATR3, HNRNPA1, SFPQ, and FUS, or any combination thereof, said method comprising: (a) contacting the RNA molecule (such as pre-mRNA or mRNA) encoding a wild-type protein selected from N0VA1, ELAVL1, ELAVL2, ELAVL3, ELAVL4, RBFOX2, MATR3, HNRNPA1, SFPQ, and FUS, or any combination thereof with an antisense oligonucleotide under conditions that permit binding of the antisense oligonucleotide to the RNA molecule (such as pre-mRNA or mRNA) encoding a wild-type protein selected from NOVAI, ELAVL1, ELAVL2, ELAVL3, ELAVL4, RBFOX2, MATR3, HNRNPA1, SFPQ, and FUS, or any combination thereof; and (b) detecting binding of the antisense oligonucleotide to the RNA molecule (such as pre-mRNA or mRNA) encoding a wild-type protein selected from NOVAI, ELAVL1, ELAVL2, ELAVL3, ELAVL4, RBFOX2, MATR3, HNRNPA1, SFPQ, and FUS, or any combination thereof. The binding of the antisense oligonucleotide to the RNA molecule (such as pre-mRNA or mRNA) encoding a wild-type protein selected from NOVAI, ELAVL1, ELAVL2, ELAVL3, ELAVL4, RBFOX2, MATR3, HNRNPA1, SFPQ, and FUS, or any combination thereof can be detected, e.g., by detecting the expression of the wild-type protein selected from NOVAI, ELAVL1, ELAVL2, ELAVL3, ELAVL4, RBFOX2, MATR3, HNRNPA1, SFPQ, and FUS, or any combination thereof (using, e.g., immunochemistry), or cell thermotolerance (using, e.g., MTS cell viability assays).

[00238] In another embodiment, the present invention provides a method for identifying an antisense oligonucleotide capable of binding to a DNA molecule encoding a eukaryotic wildtype protein selected from NOVAI, ELAVL1, ELAVL2, ELAVL3, ELAVL4, RBFOX2, MATR3, HNRNPA1, SFPQ, and FUS, or any combination thereof, said method comprising: (a) contacting the DNA molecule encoding a wild-type protein selected from NOVAI, ELAVL1, ELAVL2, ELAVL3, ELAVL4, RBFOX2, MATR3, HNRNPA1, SFPQ, and FUS, or any combination thereof with an antisense oligonucleotide under conditions that permit binding of the antisense oligonucleotide to the DNA molecule encoding a wild-type protein selected from NO VAI, ELAVL1, ELAVL2, ELAVL3, ELAVL4, RBFOX2, MATR3, HNRNPA1, SFPQ, and FUS, or any combination thereof; and (b) detecting binding of the antisense oligonucleotide to the DNA molecule encoding a wild-type protein selected from NOVAI, ELAVL1, ELAVL2, ELAVL3, ELAVL4, RBF0X2, MATR3, HNRNPA1, SFPQ, and FUS, or any combination thereof. The binding of the antisense oligonucleotide to the DNA molecule encoding a wild-type protein selected from NO VAI, ELAVL1, ELAVL2, ELAVL3, ELAVL4, RBF0X2, MATR3, HNRNPA1, SFPQ, and FUS, or any combination thereof can be detected, e.g., by detecting the expression of the wild-type protein selected from N0VA1, ELAVL1, ELAVL2, ELAVL3, ELAVL4, RBF0X2, MATR3, HNRNPA1, SFPQ, and FUS, or any combination thereof (using, e.g., immunochemistry), or cell thermotolerance (using, e.g., MTS cell viability assays). [00239] In one embodiment of this screening method, both antisense oligonucleotide treated cells and control cells can be subjected to stress (e.g., stress associated with neurodegen erative diseases). In this embodiment, the antisense oligonucleotide can be added after cells had been subjected to stress, or after a preconditioning stress but before the lethal stress, or before cells had been subjected to stress.

[00240] The above-identified screening methods can be used to identify a candidate compound that can be used to treat a condition, a disorder, or a disease that can be treated by modulating a function of a eukaryotic wild-type protein selected from N0VA1, ELAVL1, ELAVL2, ELAVL3, ELAVL4, RBFOX2, MATR3, HNRNPA1, SFPQ, and FUS, or any combination thereof. Such conditions, disorders, or diseases include conditions, disorders, or diseases characterized by TDP-43 mislocalization or dysfunction such as neurodegeneration and related disorders.

Use of the antisense oligonucleotides to treat a disease characterized by TDP-43 mislocalization or dysfunction

[00241] As described herein the present disclosure, the means for restoring misregulated splicing events to wild-type splicing events of one or more of N0VA1, ELAVL1, ELAVL2, ELAVL3, ELAVL4, RBFOX2, MATR3, HNRNPA1, SFPQ, and FUS of the invention such as, e.g., antisense oligonucleotides, can be used to enhance expression and/or function of one or more of N0VA1, ELAVL1, ELAVL2, ELAVL3, ELAVL4, RBFOX2, MATR3, HNRNPA1, SFPQ, and FUS e.g, a correctly spliced N0VA1, ELAVL1, ELAVL2, ELAVL3, ELAVL4, RBFOX2, MATR3, HNRNPA1, SFPQ, and/or FUS), and in this way provide a basis for developing novel therapeutics to treat a disease characterized by TDP-43 mislocalization or dysfunction. Alternatively or additionally, the means for restoring misregulated splicing events to wild-type splicing events of one or more of N0VA1 , ELAVL1 , ELAVL2, ELAVL3, ELAVL4, RBF0X2, MATR3, HNRNPA1, SFPQ, and FUS of the invention such as, e.g., antisense oligonucleotides, can be used to decrease expression and/or function of one or more of a mislocalized or dysfunctional N0VA1, ELAVL1, ELAVL2, ELAVL3, ELAVL4, RBF0X2, MATR3, HNRNPA1, SFPQ, and FUS (e.g., a incorrectly spliced or mis-spliced N0VA1, ELAVL1, ELAVL2, ELAVL3, ELAVL4, RBF0X2, MATR3, HNRNPA1, SFPQ, and/or FUS), and in this way provide a basis for developing novel therapeutics to treat a disease characterized by TDP-43 mislocalization or dysfunction.

[00242] To develop effective therapeutics to treat a disease characterized by TDP-43 mislocalization or dysfunction based on the splicing factor- and/or splicing regulator- specific antisense oligonucleotides, candidate antisense oligonucleotides molecules can be first tested in vitro, e.g., by testing their ability to prevent/decrease neuronal cell death (e.g., upon expression of polyglutamine-expanded proteins such as mutant huntingtin) or cardiomyocyte cell death (e.g., induced by hydrogen peroxide; see Zou et al., Circulation 2003; 108:3024-3030). Therapeutics which produce the best effect in in vitro assays can be further tested in vivo in various animal models of neurodegenerative diseases. In these models, the activity of the novel therapeutics in inhibiting neurodegenerative disorders can be determined using various methods known in the art, including without limitation, determination of electrical activity of neural tissue or myocardium, ECG, determination of reduction in neuronal or cardiomyocyte cell death, etc. [00243] Splicing factor- and/or splicing regulator- specific antisense oligonucleotides which show the strongest effect in vitro and in animal models can be further optimized (e.g., by increasing their resistance to nucleases, increasing the efficiency of their targeting to cells, increasing their sequence specificity (e.g., by introducing phosphothioate or morpholino modifications or using LNA, and reducing the size) making them even more potent in inhibition of various signs of neurodegeneration and activation of the one or more splicing factors and/or splicing regulators expression.

[00244] In conjunction with the therapeutics of the invention (e.g., the splicing factors- and/or splicing regulators-specific antisense oligonucleotides), the present invention also provides a method for inhibiting a neurodegenerative disorder in a mammal comprising administering said therapeutics to the mammal. In a specific embodiment, the mammal is human. [00245] The novel therapeutics of the invention can be used in the prophylaxis as well as in the therapeutic treatment of various neurodegenerative diseases. Non-limiting examples of neurodegenerative diseases include Alzheimer’s disease (AD), Parkinson’s disease, amyotrophic lateral sclerosis (ALS), Frontotemporal Lobar Degeneration (FTLD), Limbic-predominant Age- Related TDP-43 Encephalopathy (LATE), Frontotemporal Dementia (FTD), Chronic Traumatic Encephalopathy (CTE), Huntington’s disease, spinobulbar muscular atrophy, dentatorubral pallidoluysian atrophy, Kennedy disease, and spinocerebellar ataxias.

[00246] The novel therapeutics of the present invention can be used in conjunction with existing treatments such as pharmacotherapy (e.g., but not limited to, therapy using cholinesterase inhibitors or NMD A receptor antagonists for Alzheimer’s disease or therapy using ropinirole for ALS).

[00247] In some embodiments, provided herein is a composition comprising an ASO as described herein. In some embodiments, the composition of the present application further comprises a carrier, an excipient, and/or a buffer solution.

[00248] In some embodiments, the ASO as described herein can be delivered to the cells via a vector or other nucleic acid. The vector can be any vector as described in the present application. The vector can be any vector which contains any nucleic acid molecule as described in the present application. In some embodiments, provided herein is a composition comprising a vector or nucleic acid comprising an ASO as described herein, optionally further comprising a carrier, an excipient, and/or a buffer solution.

[00249] In some embodiments, the composition further includes a transfection reagent capable of introducing an ASO as described herein to the cells. The transfection reagent may be a positively charged transfection reagent. Suitable transfection reagents are well known in the art and include, e.g., Lipofectamine® RNAiMAX (Invitrogen™), Lipofectamine® 2000 (Invitrogen™), Lipofectamine® 3000 (Invitrogen™), Invivofectamine™ 3.0 (Invitrogen™), Lipofectamine™ MessengerMAX™ (Invitrogen™), Lipofectin™ (Invitrogen™), siLentFet™ (Bio-Rad), DharmaFECT™ (Dharmacon), HiPerFect (Qiagen), TransIT-X2® (Minis), jetMESSENGER® (Polyplus), Trans-Hi™, JetPEI® (Polyplus), and ViaFect™ (Promega).

[00250] In some embodiments, the composition is an aqueous composition. Aqueous compositions of the present application comprise an effective amount of the ASO as described herein, dissolved, or dispersed in a pharmaceutically acceptable carrier or aqueous medium. [00251] In some embodiments, provided herein is a pharmaceutical composition comprising an ASO described herein and a pharmaceutically acceptable carrier, excipient, and/or buffer. In some embodiments, provided herein is a pharmaceutical composition comprising a vector or nucleic acid comprising an ASO described herein and a pharmaceutically acceptable carrier, excipient, and/or buffer.

[00252] The ASOs as disclosed herein and/or pharmaceutical composition(s) disclosed herein can be formulated according to any available conventional method. Examples of preferred dosage forms include, but are not limited to, a tablet, a powder, a subtle granule, a granule, a coated tablet, a capsule, a syrup, a troche, an inhalant, a suppository, an injectable, an ointment, an ophthalmic ointment, an eye drop, a nasal drop, an ear drop, a cataplasm, a lotion and the like. In the formulation, generally used additives such as a diluent, a binder, a disintegrant, a lubricant, a colorant, a flavoring agent, and if necessary, a stabilizer, an emulsifier, an absorption enhancer, a surfactant, a pH adjuster, an antiseptic, an antioxidant, and the like can be used.

[00253] In some embodiments, the ASOs as disclosed herein and/or pharmaceutical composition(s) disclosed herein can be administered to the subject by intracranial administration, systemic administration, or intrathecal administration. Other methods of administration include oral administration, topical administration, transdermal administration, parenteral administration, subcutaneous administration, intravenous administration, intramuscular administration, intraperitoneal administration, intranasal instillation administration, intracavitary or intravesical instillation, intraocular administration, intraarterial administration, intralesional administration to a subject, or application to mucous membranes of a subject. In some embodiments, the administration includes intramuscular administration, intracranial administration, intravenous administration, intrathecal administration, subcutaneous administration, oral administration, or intraperitoneal administration to a subject.

Cells

[00254] In some embodiments, the cell is a cell of the subject having TDP-43 mislocalization or dysfunction. In some embodiments, the cells are neurons. In some embodiments, the neurons are motor neurons. In some embodiments, the motor neurons are hypoglossal motor neurons (hMN) and/or oculomotor motor neurons (oMN). Methods of diagnosing a disease characterized by TDP-43 mislocalization or dysfunction [00255] There is a considerable effort in the field to define the set of peptides generated by cryptic exon inclusions regulated directly by TDP-43 as biomarkers. There is a much richer source of clinical biomarkers generated by splicing defects in response to the neurodegenerative splicing cascade as described herein. For example, these include peptide inclusions in response to N0VA1 and the other splicing factors and/or splicing regulators in sensitive motor neurons. Thus, understanding the critical splicing regulators in the “neurodegenerative splicing cascade” as described in the present disclosure increases the number of possible epitopes that should be evaluated as biomarkers of the neurodegenerative diseases such as Alzheimer’s disease (AD), Parkinson’s disease, amyotrophic lateral sclerosis (ALS), Frontotemporal Lobar Degeneration (FTLD), Limbic-predominant Age-Related TDP-43 Encephalopathy (LATE), Frontotemporal Dementia (FTD), Chronic Traumatic Encephalopathy (CTE), Huntington’s disease, spinobulbar muscular atrophy, dentatorubral pallidoluysian atrophy, Kennedy disease, and spinocerebellar ataxias. This strategy is compatible with current biomarker technologies. Enhancing the biomarker repertoire as described herein can improve diagnostic accuracy, aid in early detection, and provide a more comprehensive disease profiling.

[00256] In one aspect, provided herein is a method of identifying the subject having a disease characterized by TDP-43 mislocalization or dysfunction.

[00257] In some embodiments, the method of identifying the subject having a disease characterized by TDP-43 mislocalization or dysfunction comprises determining level(s) of one or more proteins that are generated by TDP-43 -induced splicing defects, and/or (ii) neopeptides and/or neoantigens, and/or antibodies and/or T cells recognizing neopeptides and/or neoantigens, in a sample isolated from the subject. In some embodiments, the identifying comprises determining level(s) of one or more clinical biomarkers generated by splicing defects, and/or (ii) neopeptides and/or neoantigens, and/or antibodies and/or T cells recognizing neopeptides and/or neoantigens, in a sample isolated from the subject. In some embodiments, the method of identifying the subject having a disease characterized by TDP-43 mislocalization or dysfunction comprises determining level(s) of one or more proteins that are generated by splicing defects, and/or (ii) neopeptides and/or neoantigens, and/or antibodies and/or T cells recognizing neopeptides and/or neoantigens, in a sample isolated from the subject in response to the neurodegenerative splicing cascade. [00258] In some embodiments, the subject having a disease characterized by TDP-43 mislocalization or dysfunction has higher level(s) of the one or more proteins that are generated by splicing defects and/or (ii) neopeptides neoantigens, and/or antibodies and/or T cells recognizing neopeptides and/or neoantigens relative to a subject who does not have the disease. The one or more proteins that are generated by splicing defects may also be termed as clinical biomarkers.

[00259] In some embodiments, the clinical biomarkers comprise peptide inclusions. In some embodiments, the peptide inclusions comprise mis-spliced proteins. Non-limiting examples of mis-spliced proteins include N0VA1, ELAVL1, ELAVL2, ELAVL3, ELAVL4, RBFOX2, MATR3, HNRNPA1, SFPQ, and FUS or any combinations thereof.

Dosage and administration regimens

[00260] Provided herein is a method of treating or preventing a disease characterized by TDP-

43 mislocalization or dysfunction in a subject in need thereof, comprising administering to the subject an effective amount of a vector encoding one or more splicing factors and/or splicing regulators.

[00261] Exemplary vectors include, but are not limited to, vectors encoding a protein selected from one or more of NO VAI, ELAVL1, ELAVL2, ELAVL3, ELAVL4, RBFOX2, MATR3, HNRNPAl, SFPQ, and FUS, or a functional fragment thereof (e.g., a correctly spliced NOVAI, ELAVL1, ELAVL2, ELAVL3, ELAVL4, RBFOX2, MATR3, HNRNPAl, SFPQ, and/or FUS), or any combinations thereof.

[00262] In some embodiments, the administration of the viral vector encoding one or more splicing factors and/or splicing regulators results in the appearance of the viral vector in the cerebrospinal fluid of the subject.

[00263] Provided herein is a method of treating or preventing a disease characterized by TDP-

43 mislocalization or dysfunction in a subject in need thereof, comprising administering to the subject an effective amount of a means for restoring misregulated splicing events to wild-type splicing events or enhancing expression and/or function of one or more of NOVAI, ELAVL1, ELAVL2, ELAVL3, ELAVL4, RBFOX2, MATR3, HNRNPAl, SFPQ, and FUS. In some embodiments, the antisense oligonucleotide of the present invention is administered for restoring misregulated splicing events to wild-type splicing events of one or more of N0VA1, ELAVL1, ELAVL2, ELAVL3, ELAVL4, RBF0X2, MATR3, HNRNPA1, SFPQ, and FUS.

[00264] In some embodiments, the means for restoring misregulated splicing events to wildtype splicing events or enhancing expression and/or function of one or more of N0VA1, ELAVL1, ELAVL2, ELAVL3, ELAVL4, RBFOX2, MATR3, HNRNPA1, SFPQ, and FUS refers to a therapeutic agent capable of detectably enhancing the expression of and/or the function of the one or more of NO VAI, ELAVL1, ELAVL2, ELAVL3, ELAVL4, RBFOX2, MATR3, HNRNPA1, SFPQ, and FUS (e.g, a correctly spliced N0VA1, ELAVL1, ELAVL2, ELAVL3, ELAVL4, RBFOX2, MATR3, HNRNPA1, SFPQ, and/or FUS), compared to a control without treatment with the means for restoring misregulated splicing events to wild-type splicing events described herein. The enhanced expression of and/or activity level of the one or more of N0VA1, ELAVL1, ELAVL2, ELAVL3, ELAVL4, RBFOX2, MATR3, HNRNPA1, SFPQ, and FUS can be 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or higher than that in a control without treatment with the means for restoring misregulated splicing events to wild-type splicing events described herein. In certain aspects, the enhanced expression is 1.5-fold, 2-fold, 2.5-fold, 3-fold, 3.5-fold, 4-fold, 4.5-fold, 5- fold, 5.5-fold, 6-fold, 7-fold, 8-fold, 9-fold, or 10-fold, or more in comparison to a control.

[00265] In some embodiments, the means for restoring misregulated splicing events to wildtype splicing events or enhancing expression and/or function of one or more of N0VA1, ELAVL1, ELAVL2, ELAVL3, ELAVL4, RBFOX2, MATR3, HNRNPA1, SFPQ, and FUS refers to a therapeutic agent capable of detectably decreasing the expression of and/or the function of the one or more of NO VAI, ELAVL1, ELAVL2, ELAVL3, ELAVL4, RBFOX2, MATR3, HNRNPA1, SFPQ, and FUS (e.g., an incorrectly spliced or mis-spliced N0VA1, ELAVL1, ELAVL2, ELAVL3, ELAVL4, RBFOX2, MATR3, HNRNPA1, SFPQ, and/or FUS), compared to a control without treatment with the means for restoring misregulated splicing events to wild-type splicing events described herein. The decreased expression of and/or activity level of the one or more of N0VA1, ELAVL1, ELAVL2, ELAVL3, ELAVL4, RBFOX2, MATR3, HNRNPA1, SFPQ, and FUS can be 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or lower than that in a control without treatment with the means for restoring misregulated splicing events to wild-type splicing events described herein. In certain aspects, the decreased expression is 1.5-fold, 2-fold, 2.5-fold, 3-fold, 3.5-fold, 4-fold, 4.5-fold, 5-fold, 5.5-fold, 6-fold, 7-fold, 8-fold, 9-fold, or 10-fold, or more in comparison to a control.

[00266] In some embodiments, the means for restoring misregulated splicing events to wildtype splicing events described herein can enhance expression of splicing factors and/or splicing regulators (e.g., by partially or totally binding, partially or totally blocking RNA processing of the mis-spliced or incorrectly spliced splicing factors and/or splicing regulators by decreasing, preventing, or delaying activation of the mis-spliced or incorrectly spliced splicing factors and/or splicing regulators; or inactivating, desensitizing, or down-regulating gene expression, signal transduction, or enzymatic activity of the mis-spliced or incorrectly spliced splicing factors and/or splicing regulators).

[00267] In some embodiments, the method comprises administering to the subject an effective amount of (a) a vector encoding one or more splicing factors and/or splicing regulators (e.g., a correctly spliced splicing factor and/or splicing regulator) or (b) an antisense oligonucleotide that can bind to the mis-spliced splicing factors and/or splicing regulators, and a therapy selected from an immunotherapy, a gene therapy, and/or an RNA interference (RNAi) therapy. Nonlimiting examples of immunotherapies include an antibody or an antigen-binding fragment, an oligonucleotide, a peptide, and an aptamer. Non-limiting examples of RNAi therapies include a miRNA, an shRNA, and an siRNA. Non-limiting examples of gene therapies include a vectorbased therapy, a site-directed nuclease-based therapy (such as a CRISPR-associated protein (Cas) nuclease, a ZFN, a TALEN, a meganuclease, any endo- or exo-nuclease, variants thereof, fragments thereof, or any combination thereof), and/or a CAR-based therapy (a CAR-T cell, a CAR-NK cell, or CAR-M). Any therapeutics that are known to be effective in neurodegenerative disorders can be combined with the vector or antisense oligonucleotides of the present disclosure for therapeutic purposes.

[00268] In certain embodiments, an amount of (a) a vector encoding one or more splicing factors and/or splicing regulators (e.g., a correctly spliced splicing factor and/or splicing regulator) or (b) an antisense oligonucleotide that can bind to the mis-spliced splicing factors and/or splicing regulators and/or a therapy selected from an immunotherapy, a gene therapy, and/or an RNAi therapy, or pharmaceutical composition thereof, which is administered to a subject as described herein is a therapeutically effective amount. [00269] The phrase “therapeutically effective amount” as used herein, means an amount sufficient to achieve the desired effect for which it is administered. In some embodiments, the therapeutically effective amount of the antisense oligonucleotides includes an amount sufficient to restore misregulated splicing events to wild-type splicing events of one or more of N0VA1, ELAVL1, ELAVL2, ELAVL3, ELAVL4, RBFOX2, MATR3, HNRNPA1, SFPQ and FUS. The subject can be from any suitable species, such as mammalian or eukaryotic subjects (e.g, human subject or non-human mammalian subject). A mammal can be, but is not limited to, a non-human mammal, a hamster, a rodent, a mouse, a human, or a rat. Non-human mammals include, but are not limited to, non-human primates, for example, monkeys and apes. The term “non-human” means excluding humans.

[00270] In some embodiments, the subject is human. The human can be a patient. In some embodiments, the subject is a veterinary animal or an experimental model.

[00271] In certain embodiments, the method of treating disease is in a patient that has received no prior treatment. In certain embodiments, the method of treating disease is in a patient that has received a prior treatment with therapeutic agent(s), (e.g., an immunotherapy, a gene therapy, and/or an RNAi therapy). In certain embodiments, the patient has developed an acquired resistance to the previous treatment of one or more immunotherapies, gene therapies, and/or RNAi therapies. In further embodiments, the patient has developed bypass resistance to the previous treatment of one or more immunotherapies, gene therapies, and/or RNAi therapies. [00272] In some embodiments, the (a) vector encoding one or more splicing factors and/or splicing regulators (e.g., a correctly spliced splicing factor and/or splicing regulator) or (b) antisense oligonucleotide that can bind to the mis-spliced splicing factors and/or splicing regulators, and/or therapy selected from an immunotherapy, a gene therapy, and/or an RNAi therapy, or pharmaceutical composition thereof, is administered as a weight-based dose to a subject.

[00273] In some embodiments, the (a) vector encoding one or more splicing factors and/or splicing regulators (e.g., a correctly spliced splicing factor and/or splicing regulator) or (b) antisense oligonucleotide that can bind to the mis-spliced splicing factors and/or splicing regulators and/or therapy selected from an immunotherapy, a gene therapy, and/or an RNAi therapy, or pharmaceutical composition thereof, is administered as a fixed dose to a subject. [00274] A “weight-based dose” (e.g., a dose measured in mg/kg) as used herein, is a dose of the (a) vector encoding one or more splicing factors and/or splicing regulators or (b) antisense oligonucleotide that can bind to the mis-spliced splicing factors and/or splicing regulators and/or the therapy selected from an immunotherapy, a gene therapy, and/or an RNAi therapy that will vary depending on the subject’s weight.

[00275] In other embodiments, the (a) vector encoding one or more splicing factors and/or splicing regulators (e.g., a correctly spliced splicing factor and/or splicing regulator) or (b) antisense oligonucleotide that can bind to the mis-spliced splicing factors and/or splicing regulators and/or the therapy selected from an immunotherapy, a gene therapy, and/or an RNAi therapy is administered as a fixed dose.

[00276] A “fixed dose” (e.g., a dose measured in mg) means that one dose of the (a) vector encoding one or more splicing factors and/or splicing regulators or (b) antisense oligonucleotide that can bind to the mis-spliced splicing factors and/or splicing regulators and/or the therapy selected from an immunotherapy, a gene therapy, and/or an RNAi therapy is used consistently for all subjects regardless of any subject-specific factors, e.g., weight.

[00277] In certain embodiments, the (a) vector encoding one or more splicing factors and/or splicing regulators (e.g., a correctly spliced splicing factor and/or splicing regulator) or (b) antisense oligonucleotide that can bind to the mis-spliced splicing factors and/or splicing regulators and/or the therapy selected from an immunotherapy, a gene therapy, and/or an RNAi therapy is administered to a subject at a dosing frequency of about five times a week, about four times a week, about twice a week, about once a week, about once every two weeks, about once every three weeks, about once a month, about once every five weeks, about once every six weeks, about once every seven weeks, about once every two months, about once every three months, or less frequently so long as an effective therapeutic response is achieved. In certain embodiments, the (a) vector encoding one or more splicing factors and/or splicing regulators or (b) antisense oligonucleotide that can bind to the mis-spliced splicing factors and/or splicing regulators and/or the therapy selected from an immunotherapy, a gene therapy, and/or an RNAi therapy can be administered to a subject at a dosing frequency of about six times a year, about four times a year, about twice a year, about once a year, about once every two years, about once every three years, about once every four years, about once every five years, about once every six years, about once every eight years, about once a decade, about once every twelve years, about once every fifteen years, or less frequently so long as an effective therapeutic response is achieved.

[00278] Dosage ranges and frequencies of administration of (a) vectors encoding one or more splicing factors and/or splicing regulators (e.g., a correctly spliced splicing factor and/or splicing regulator) or (b) antisense oligonucleotides that can bind to the mis-spliced splicing factors and/or splicing regulators and/or therapies selected from immunotherapies, gene therapies, and/or RNAi therapies as described herein can vary depending on the parameters of a specific subject, the route of administration, the type of medical condition, and/or the severity of the medical condition. A more accurate dose may depend on the subject in which it is administered, e.g., a lower dose may be used if the subject in which it is administered is juvenile and a higher dose may be used if the subject is adult. In some embodiments, a more accurate dose may depend on the subject weight.

[00279] In certain embodiments, multiple doses of (a) a vector encoding one or more splicing factors and/or splicing regulators (e.g., a correctly spliced splicing factor and/or splicing regulator) or (b) an antisense oligonucleotide that can bind to the mis-spliced splicing factors and/or splicing regulators and/or a therapy selected from an immunotherapy, a gene therapy, and/or an RNAi therapy are administered over a defined time course.

[00280] In some embodiments, the (a) vector encoding one or more splicing factors and/or splicing regulators (e.g., a correctly spliced splicing factor and/or splicing regulator) or (b) antisense oligonucleotide that can bind to the mis-spliced splicing factors and/or splicing regulators and/or the therapy selected from an immunotherapy, a gene therapy, and/or an RNAi therapy may be administered following a repeated dosing regimen wherein the (a) vector encoding one or more splicing factors and/or splicing regulators (e.g., a correctly spliced splicing factor and/or splicing regulator) or (b) antisense oligonucleotide that can bind to the mis-spliced splicing factors and/or splicing regulators and/or the therapy selected from an immunotherapy, a gene therapy, and/or an RNAi therapy may be administered an initial time (e.g., as an initial dose) and then may be re-administered at any amount for any number of subsequent times thereafter over the time course of treatment. For example, the (a) vector encoding one or more splicing factors and/or splicing regulators (e.g., a correctly spliced splicing factor and/or splicing regulator) or (b) antisense oligonucleotide that can bind to the mis-spliced splicing factors and/or splicing regulators and/or the therapy selected from an immunotherapy, a gene therapy, and/or an RNAi therapy may be re-administered one time, two times, three times, four times, five times, six times, seven times, eight times, nine times, ten times, fifteen times, twenty times, or more, over the time course of treatment which may occur over any amount of time (e. ., days, weeks, or years). In certain embodiments, the (a) vector encoding one or more splicing factors and/or splicing regulators (e.g., a correctly spliced splicing factor and/or splicing regulator) or (b) antisense oligonucleotide that can bind to the mis-spliced splicing factors and/or splicing regulators and/or the therapy selected from an immunotherapy, a gene therapy, and/or an RNAi therapy which is/are administered first in a repeat dosing regimen may comprise the same (a) vector encoding one or more splicing factors and/or splicing regulators (e.g., a correctly spliced splicing factor and/or splicing regulator) or (b) antisense oligonucleotide that can bind to the misspliced splicing factors and/or splicing regulators which is/are re-administered second or thereafter in the regimen, for any number of subsequent times thereafter. In certain embodiments, the (a) vector encoding one or more splicing factors and/or splicing regulators (e.g., a correctly spliced splicing factor and/or splicing regulator) or (b) antisense oligonucleotide that can bind to the mis-spliced splicing factors and/or splicing regulators and/or the therapy selected from an immunotherapy, a gene therapy, and/or an RNAi therapy which is/are administered first in a repeat dosing regimen may comprise a different therapy selected from an immunotherapy, a gene therapy, and/or an RNAi therapy than is/are re-administered second or thereafter in the regimen, for any number of subsequent times thereafter.

[00281] In certain embodiments, the (a) vector encoding one or more splicing factors and/or splicing regulators (e.g., a correctly spliced splicing factor and/or splicing regulator) or (b) antisense oligonucleotide that can bind to the mis-spliced splicing factors and/or splicing regulators and/or the therapy selected from an immunotherapy, a gene therapy, and/or an RNAi therapy can be administered using a stepwise dosing regimen. Stepwise dosing can refer to dividing dosing of the same (a) vector encoding one or more splicing factors and/or splicing regulators (e.g., a correctly spliced splicing factor and/or splicing regulator) or (b) antisense oligonucleotide that can bind to the mis-spliced splicing factors and/or splicing regulators over multiple administrations. In certain embodiments, the dosing of the (a) vector encoding one or more splicing factors and/or splicing regulators (e.g., a correctly spliced splicing factor and/or splicing regulator) or (b) antisense oligonucleotide that can bind to the mis-spliced splicing factors and/or splicing regulators is/are broken up one time, two times, three times, four times, five times, six times, seven times, eight times, nine times, ten times, fifteen times, twenty times, or more, over the time course of the treatment which can occur over any period of time (e.g., over any number of days, weeks, or years). In certain embodiments, when a stepwise dose regimen is used in the administration of the (a) vector encoding one or more splicing factors and/or splicing regulators (e.g., a correctly spliced splicing factor and/or splicing regulator) or (b) antisense oligonucleotide that can bind to the mis-spliced splicing factors and/or splicing regulators, the regimen may encompass an increase or decrease in dosage levels with each administration of the (a) vector encoding one or more splicing factors and/or splicing regulators (e.g., a correctly spliced splicing factor and/or splicing regulator) or (b) antisense oligonucleotide that can bind to the mis-spliced splicing factors and/or splicing regulators.

[00282] In some embodiments, the (a) vector encoding one or more splicing factors and/or splicing regulators (e.g., a correctly spliced splicing factor and/or splicing regulator) or (b) antisense oligonucleotide that can bind to the mis-spliced splicing factors and/or splicing regulators and/or the therapy selected from an immunotherapy, a gene therapy, and/or an RNAi therapy are administered via intratumoral, intravenous, intradermal, intraperitoneal, subcutaneous, intramuscular delivery, inhalation, oral delivery, lipid nanoparticle-based delivery (LNP), cellular delivery, viral and/or non-viral delivery, or gene editing, or as a cargo in a cell, or any combination thereof.

Non-limiting embodiments of the invention

1. A method of treating a disease characterized by TAR DNA-binding protein 43 (TDP-43) mislocalization or dysfunction in a subject in need thereof, the method comprising: providing to cells or tissues or organs of the subject having TDP-43 mislocalization or dysfunction a wild-type protein selected from neuro-oncological ventral antigen 1 (N0VA1), embryonic lethality and abnormal visual like protein 1 (ELAVL1), ELAVL2, ELAVL3, ELAVL4, RNA binding fox-1 homolog 2 (RBFOX2), matrin 3 (MATR3), Heterogeneous nuclear ribonucleoprotein Al (HNRNPA1), splicing factor proline- and glutamine-rich (SFPQ), Fused in Sarcoma (FUS) or a functional fragment thereof, or any combination thereof, or one or more nucleic acid molecules encoding the said wild-type protein or the functional fragment thereof.

2. The method of embodiment 1, wherein the disease is a neurodegenerative disease. 3. The method of embodiment 2, wherein the neurodegenerative disease is Amyotrophic Lateral Sclerosis (ALS), Frontotemporal Lobar Degeneration (FTLD)Frontotemporal dementia (FTD), Limbic-predominant Age-Related TDP-43 Encephalopathy (LATE), Chronic Traumatic Encephalopathy (CTE), or Alzheimer’s Disease (AD).

4. The method of any one of embodiments 1-3, wherein providing said protein(s) or nucleic acid molecules(s) involves administering a gene therapy to the subject.

5. The method of any one of embodiments 1-4, wherein the nucleic acid molecule is contained within a viral vector which is administered to the subject.

6. The method of embodiment 5, wherein the viral vector is a lentiviral vector, an adenoviral vector, or an adeno-associated viral (AAV) vector.

7. The method of embodiment 6, wherein the viral vector is an adeno-associated viral (AAV) vector.

8. The method of embodiment 7, wherein the AAV vector has tropism for neural tissue or neurons.

9. The method of embodiment 7, wherein the adeno-associated viral vector is AAV9 or AAV- Bl.

10. The method of any one of embodiments 4-9, wherein the viral vector is administered to the subject intracranially, systemically, or intrathecally.

11. The method of embodiment 10, wherein the intracranial or intrathecal administration of the viral vector results in the appearance of the viral vector in the cerebrospinal fluid of the subject.

12. The method of any one of embodiments 4-11, wherein the nucleic acid molecule is operably linked to a promoter.

13. The method of embodiment 12, wherein the promoter is an inducible promoter or a constitutive promoter.

14. The method of embodiment 12 or embodiment 13, wherein the promoter is a tissue-specific promoter or a cell-specific promoter.

15. The method of embodiment 14, wherein the promoter is a neural tissue-specific promoter or a neuron-specific promoter.

16. The method of embodiment 15, wherein the promoter is a synapsin promoter.

17. The method of embodiment 12, wherein the promoter is a human ubiquitin C (hUBC) promoter; a chicken P-actin promoter, CMV enhancer, and rabbit P-globin splice acceptor (pCAG) promoter; a human cytomegalovirus (HCMV) promoter; a mouse phosphoglycerate kinase (mPGK) promoter; or a homeobox gene (Hb9) promoter.

18. The method of any one of embodiments 1-17, wherein the wild-type protein is N0VA1 and/or ELAVL2.

19. The method of embodiment 18, wherein the N0VA1 is N0VA1 P51513-4.

20. The method of any one of embodiments 1-19, wherein the cells are neurons.

21. The method of embodiment 20, wherein the neurons are motor neurons.

22. The method of embodiment 21, wherein the motor neurons are hypoglossal motor neurons and/or oculomotor motor neurons.

23. The method of any one of embodiments 1-22, wherein the subject is a mammal.

24. The method of embodiment 23, wherein the mammal is a human.

25. A method of treating a disease characterized by TAR DNA-binding protein 43 (TDP-43) mislocalization or dysfunction in a subject in need thereof, the method comprising: providing to cells or tissues or organs of the subject having TDP-43 mislocalization or dysfunction a means for restoring misregulated splicing events to wild-type splicing events of one or more of NOVAI, ELAVL1, ELAVL2, ELAVL3, ELAVL4, RBFOX2, MATR3, HNRNPA1, SFPQ and FUS.

26. The method of embodiment 25, wherein restoring misregulated splicing events to wild-type splicing events comprises skipping of an abnormal exon and/or cryptic exons and/or favoring inclusion of a normal exon.

27. The method of embodiment 25 or embodiment 26, wherein the disease is a neurodegenerative disease.

28. The method of embodiment 27, wherein the neurodegenerative disease is Amyotrophic Lateral Sclerosis (ALS), Frontotemporal Lobar Degeneration (FTLD), Limbic-predominant Age- Related TDP-43 Encephalopathy (LATE), Chronic Traumatic Encephalopathy

(CTE), Frontotemporal dementia (FTD), or Alzheimer's Disease (AD).

29. The method of any one of embodiments 25-28, wherein the means for restoring misregulated splicing events to wild-type splicing events:

(i) enhance expression and/or function of one or more splicing factors and/or splicing regulators, and/or (ii) decrease expression and/or function of one or more mislocalized or dysfunctional splicing factors and/or splicing regulators.

30. The method of any one of embodiments 25-29, wherein the means for restoring misregulated splicing events to wild-type splicing events is administered intrathecally.

31. The method of any one of embodiments 25-30, wherein the means for restoring misregulated splicing events to wild-type splicing events comprises one or more splicing modifiers and/or antisense oligonucleotide(s) (ASO(s)).

32. The method of embodiment 31, wherein the one or more ASO(s) is(are) splice-switching ASO(s) and/or restore normal splicing.

33. The method of embodiment 31 or embodiment 32, wherein the one or more ASO(s) is(are) targeted to pre-mRNA of one or more of NOVAI, ELAVL1, ELAVL2, ELAVL3, ELAVL4, RBFOX2, MATR3, HNRNPA1, SFPQ and FUS.

34. The method of embodiment 33, wherein the one or more ASO(s) is(are) targeted to one or more exons of one or more pre-mRNA of NO VAI, ELAVL1, ELAVL2, ELAVL3, ELAVL4, RBFOX2, MATR3, HNRNPA1, SFPQ and FUS.

35. The method of embodiment 33 or embodiment 34, wherein the one or more ASO(s) is(are) targeted to pre-mRNA of NOVA 1 and/or ELAVL2.

36. The method of embodiment 33 or embodiment 34, wherein the one or more ASO(s) is(are) targeted to: a) exon 8 of NOVAI pre-mRNA; b) exon 6 of ELAVL1 pre-mRNA; c) exon 6 of ELAVL2 pre-mRNA; d) exon 10 and 11 of ELAVL4 pre-mRNA; e) exon 3 of RBFOX2 pre-mRNA; f) exon 15 of MATR3 pre-mRNA; g) exon 6 of HNRNPA1 pre-mRNA; h) exon 9 of SFPQ pre-mRNA; i) exon 4 of ELAVL3 pre-mRNA; and/or j) exon 3 and exon 7 of FUS pre-mRNA. 37. The method of embodiment 33, wherein the one or more ASO(s) binds to a pre-mRNA sequence flanking a mis-spliced exon of one or more of N0VA1, ELAVL1, ELAVL2, ELAVL3, ELAVL4, RBF0X2, MATR3, HNRNPA1, SFPQ and FUS.

38. The method of any one of embodiments 29-35, wherein the one or more ASO(s) is(are) optimized by tiling relevant splice junctions of one or more pre-mRNA of N0VA1, ELAVL1, ELAVL2, ELAVL3, ELAVL4, RBFOX2, MATR3, HNRNPA1, SFPQ and FUS.

39. The method of any one of embodiments 25-38, wherein the N0VA1 is N0VA1 P51513-4.

40. The method of any one of embodiments 25-39, wherein the cells are neurons.

41. The method of embodiment 40, wherein the neurons are motor neurons.

42. The method of embodiment 41, wherein the motor neurons are hypoglossal motor neurons and/or oculomotor motor neurons.

43. The method of any one of embodiments 25-42, wherein the subject is a mammal.

44. The method of embodiment 43, wherein the mammal is a human.

45. The method of any one of embodiments 1-44, further comprising: identifying the subject as having a disease characterized by TAR DNA-binding protein

43 (TDP-43) mislocalization or dysfunction.

46. The method of embodiment 45, wherein the identifying comprises determining level(s) of

(i) one or more clinical biomarkers generated by splicing defects in a sample isolated from the subject, and/or

(ii) neopeptides and/or neoantigens, and/or antibodies and/or T cells recognizing neopeptides and/or neoantigens, in a sample isolated from the subject.

47. The method of embodiment 46, wherein the one or more clinical biomarkers comprise peptide inclusions comprising mis-spliced proteins.

48. The method of embodiment 47, wherein the mis-spliced proteins comprise one or more of NO VAI, ELAVL1, ELAVL2, ELAVL3, ELAVL4, RBFOX2, MATR3, HNRNPA1, SFPQ and FUS.

49. The method of embodiment 46, wherein the neopeptides and/or neoantigens comprise mis- spliced proteins. EXAMPLES

[00283] The following examples are provided to further describe some of the embodiments disclosed herein. The examples are intended to illustrate, not to limit, the disclosed embodiments.

Example 1. Development of a new human pluripotent stem cell differentiation system to enable decreased heterogeneity in differentiated cell populations

[00284] FTD and ALS are closely related diseases, sharing clinical, pathological, and genetic traits. Cortical neurons are affected in pure FTD, causing cognitive and behavioral impairment. Spinal motor neurons (SpMNs) are among the first populations to deteriorate in pure ALS, leading to muscle denervation, paralysis, and eventually death. It is common for patients to present mixed FTD and ALS symptoms. For example, ~ 50% of ALS patients develop FTD neuropsychological deficits h The accumulation of insoluble protein aggregates is a prevalent feature in neurodegenerative diseases. TDP-43 cytoplasmic aggregation is typical for FTD/ ALS: in 50% FTD and 90% ALS patients ^2-4 Additionally, ALS and FTD share common genetic causes. A GGGGCC (G4C2) hexanucleotide repeat expansion in the first intron of C9orf72 is responsible for most familial FTD and ALS cases and some sporadic cases ⁵’⁶. In sum, ALS and FTD lay clinically, pathologically, and mechanistically on a continuum of neurodegenerative disorders with overlapping clinical symptoms ⁷’ ⁸. Collectively, they are referred to as FTD/ ALS throughout the application in agreement with the pathophysiological overlap and the difficulty separating neuronal molecular events in TDP-43 protein cytoplasmic accumulation, nuclear depletion, and splicing defects.

[00285] Differential motor neuron (MN) survival FTD/ ALS patients is a paradoxical case of differential sensitivity to neurodegeneration. All FTD/ ALS cases with muscle weaknesses eventually develop bulbar symptoms and difficulties in speech and swallowing. Moreover, bulbar ALS onset tends to be very aggressive. The degeneration of hypoglossal MNs (hMNs) is a main contributor to bulbar symptoms. On the other hand, MNs responsible for controlling eye movements (oMNs: oculomotor, trochlear, and abducens nerves, which are types of cranial motor neurons) remain unaffected until the late stages of the disease. As a result, patients who lose the ability to speak can still communicate through eye-tracking devices that rely on eye movements¹⁹’ ²⁰. It has been proposed that oMNs resist excitotoxicity, but this feature does not explain the reduced protein accumulation and splicing defects ^{21, 22}. Thus, there may be additional factors that contribute to their differential sensitivity.

[00286] A key hurdle to addressing this knowledge gap is acquiring large quantities of human neurons that display differential sensitivity to FTD/ALS stressors, such as TDP-43 nuclear depletion or C9orf72 and TARDBP mutations. A robust protocol was developed to differentiate human induced pluripotent stem cells (iPSCs) from healthy and FTD/ALS patients into hMN- and oMN-like neurons to overcome this obstacle. These neurons recapitulate important differences in their response to FTD/ALS stress. With this cellular model, undescribed differences were identified that could explain their relative sensitivity and produce opportunities to improve neuronal health upon TDP-43 nuclear depletion: resistant neurons have redundant protection while hMNs enter an accelerated splicing neurodegenerative downward spiral (Figure 2K).

[00287] It was proposed that resistant neurons utilize two nested mechanisms: retain more nuclear TDP-43 and reduce splicing defects even after TDP-43 nuclear depletion. In contrast, the sensitive neurons experience a splicing neurodegenerative downward spiral: TDP-43 reduction induces more splicing defects in sensitive neurons, including hMN-specific splicing factors that enhance the initial splicing defects. The invention disclosed herein challenges this model and may rescue the degenerative cascade in sensitive neurons. Herein, cell-autonomous neuronal differences and methods to enhance neuronal resistance to TDP-43 perturbations have been described.

[00288] Non-limiting aspects of the invention described herein include:

1. Studying fundamental cellular responses affected in most FTD/ALS patients rather than a single mutation. '. TDP-43 cytoplasmic accumulation and concomitant nuclear depletion are shared features across familial FTD/ALS and sporadic cases ²³. Rather than focusing on a single gene, how different neurons react to TDP-43 perturbations, a feature in most FTD/ALS patients, was explored. Additionally, the relationship between TDP-43 and C9orf72 (the most common FTD/ALS mutation ²⁴) mutations across neuronal types was investigated.

2. Comparing different neuron types of the same genotype under the same stress. Due to technical limitations in differentiating human pluripotent stem cells, most FTD/ALS studies examine one neuronal type. Brachial motor neurons for ALS or cortical neurons (CN) for FTD are the typical neuronal classes for studies differentiating human induced pluripotent stem cells (iPSCs), including consortia with extensive iPSC collections ²⁵'³⁰. The absence of comparative studies using different neuronal types from the same patient-derived genotype can restrict the scope of analysis, making it challenging to differentiate between events that have significant consequences for neuronal survival or death and those that do not. Herein, inter-neuronal comparisons are made between these motor neurons and CN commonly used in the field to model FTD. Additionally, TARDBP mutations are uncommon in FTD, while C9orf72 repeat expansion is common across FTD/ASL. How CN and hMN respond to these mutations are studied as described herein.

3. The development of an efficient and robust human iPSC differentiation protocol that recapitulates key neuronal differences. '. Various methodologies exist for differentiating pluripotent stem cells from neurons ^{26, 31, 32}. Morphogen-based and organoid methods can be used for studying developmental transitions and tissue organization, as has been done for gene regulatory networks and epigenetic studies ³³'³⁷. However, these methods produce heterogeneous cell populations that may limit the study of cell-intrinsic properties and impose tremendous variability across iPSC lines, introducing extra complexity in comparative analyses ³⁸. A human pluripotent stem cell differentiation system is developed to address this limitation and generate clinically relevant cell types ^{39, 40}. This technology may rely on the power of direct programming by transcription factor combinations to generate hMN and oMN, as transcription factors control progressive acquisition of terminal cell fate (differentiating from pluripotent cells to neuronal precursor cells, motor neuron progenitor cells, and finally to motor neurons). The differentiation strategy described in the present invention is robust, reproducible, accessible, and scalable and complements the CN differentiation by Neurogenin2.

[00289] Neuronal type autonomous and non-cell autonomous factors contribute to the neuronal type’s vulnerability and resilience ⁴². Early studies with conditional mutant mice revealed a strong motor neuron cell-autonomous component initiating the FTD/ALS neurodegenerative process ^{43, 44}. Thus, the invention described herein focuses on the cell-autonomous neuronal response in one of the aspects. However, mouse models make isolating the strictly cell- autonomous mechanisms contributing to different neuronal sensitivity in FTD/ALS challenging. Additionally, mouse-human interspecies differences downstream of TDP-43 relocalization require human cells to model the disease ^{12, 15}. Therefore, pluripotent stem cell-derived spinal motor neurons were critical to discovering pathological TDP-43 splicing defects such as STMN2 (which does not happen in mice) and UNCI 3a ^{11-13, 45, 46}. Moreover, the drug ropinirole, which is entering phase l/2a of clinical trials for ALS, is based on in vitro differentiated spinal motor neurons ^{47, 48}. Therefore, pluripotent stem cell differentiation into disease-related neurons has become a critical platform for understanding disease mechanisms and developing new therapeutic approaches ⁴⁹'^;’¹.

[00290] Direct programming via the forced expression of transcription factors (TFs) overcomes limitations associated with other differentiation approaches, such as the production of heterogeneous cell populations and differences across stem cell lines ^{32, 52}'⁵⁴. CN fate can be induced in human pluripotent stem cells by the activity of the Neurogenin 2 (Neurog2) transcription factor ⁵³’ ⁵⁵'⁵⁷. Neurog2 -programmed neurons are models for neurodegenerative diseases (including TDP-43 splicing) ^{13, 38, 59}, neuronal plasticity ⁶⁰ , and schizophrenia ^{61, 62}. Discovering neuronal-type specific mechanisms that contribute to differential resistance or sensitivity requires studying different neuronal types. Thus, a method was developed to enhance the Neurog2 approach, establishing an efficient and reliable method for differentiating FTD/ALS sensitive and resistant motor neurons.

[00291] A protocol to differentiate mouse pluripotent stem cells into hMNs (FTD/ALS sensitive) and oMNs (FTD/ALS resistant) was developed (Figures 1A and IB). A non-limiting aspect of this approach was combining Neurog2 with pairs of synergistic transcription factors required for their embryonic differentiation, namely Islet- 1 and LIM homeobox protein 3 (Isll- Lhx3) for hMNs and Islet-1 and Paired like homeobox 2A (Isll-Phox2a) for oMNs ^{37, 63}. These neuronal differentiations are termed as Neurog2-Isll-Lhx3 (NIL) and Neurog2-Isl 1 - Phox2a(NIP)-directed differentiation in the present disclosure (Figures 1A and IB). These neurons may be electrically active, express the appropriate fate markers, and send axons through the correct track when implanted into a developing spinal cord ⁶³. The in vzTro-derived neurons revealed that an enhanced proteostasis capacity protects oMNs from generating protein aggregates ^{40, 64}. This behavior was corroborated with primary neurons in the Superoxide dismutase 1 (SOD1) mouse model, a transgenic model expressing a mutant form of the human SoDl gene ⁴⁰. Thus, direct hMN and oMN programming generate neurons that model cell- autonomous differences and responses observed in vivo.

[00292] A protocol for differentiating oMNs and hMNs from human iPSCs was recently developed. This updated method employs adherent cultures, removing the variability associated with floating embryoid body formation. Furthermore, the rostral -caudal identity of the resulting neurons was modulated through precise retinoic acid concentrations. On day 10, -90% of the cells in the dish were postmitotic hMNs or oMNs (Figure 1C). The iPSC-derived hMNs or oMNs expressed neuronal markers 03 -tubulin (TUBB3) and microtubule associated protein 2 (MAP2) and the motor neuron markers vesicular acetylcholine transporter (VAChT) and choline O-acetyltransferase (CHAT). RNA sequencing (RNA-seq) and immunocytochemistry analysis revealed the oMNs expressed the paired-like homeobox 2B (Phox2b), T-box transcription factor 20 (Tbx20), and NK6 homeobox 1 (Nkx6.1) fate markers (-80% of the neurons). hMNs expressed Motor neuron and pancreas homeobox 1 (MNX1, Hb9), Homeobox protein Hox-B4 (Hoxb4, which control MN diversity), and Teashirt zinc finger homeobox 1 (Tshzl) required factor for their in vivo differentiation ⁶³ (markers as depicted in Figures 1C, IE, and IF). The neurons are different from the commonly used iNeurongl/2 neurons (Figure ID). oMNs and hMNs for two groups of non-overlapping cell fates, thus the described process produces two distinct neuronal populations (Figure 1G). Consistent with findings in mouse models, human oMNs are more resistant to chemically induced proteotoxic stress than hMNs ³⁹. Notably, a recent study using the hMN transcription factor differentiation protocol demonstrated that programmed hMNs recapitulate splicing defects seen in sporadic ALS postmortem samples with TDP-43 pathologies, while other iPCS-derived neurons do not ⁶⁶. Thus, the invention described herein establishes a rapid and efficient human iPSC differentiation protocol that models cell- autonomous splicing defects seen in FTD/ALS.

[00293] Leveraging the iPSC differentiation system described herein, a multi-tiered system that protects oMNs and exacerbates hMN degeneration was enabled. According to the invention described herein this model is challenged and to obtain a novel intervention step to reduce neuronal splicing defects resulting from FTD/ALS stress. Overall, the model as described herein may generate a blueprint for the molecular, cellular, and systemic mechanisms that give rise to neurodegeneration in specific neuron types, thus opening a path to develop therapeutics.

Elucidation o f control TDP-43 perturbations by resistant neurons

[00294] oMNs resist the FTD/ALS neurodegenerative stress stemming from TARDBP mutations and C9orf72 repeat expansion. The precise cell-autonomous mechanisms responsible for this resistance remain poorly understood. Thus, there is a need to understand the nested mechanisms that protect oMNs. Based on previous observations of differential proteostasis capacity in mouse neurons ^{40, 64} the hypothesis that retaining TDP-43 in the nucleus in response to FTD/ALS mutations is the first line of defense for human FTD/ALS-resistant motor neurons was tested. Secondly, the data suggests an undescribed subsequent tier of resistance far less studied: oMNs reduce splicing defects, even under strong TDP-43 perturbations. To test this hypothesis, the effect on splicing of lowering nuclear TDP-43 in oMNs, versus CNs and hMNs is measured.

Effect of different neuronal types on nuclear TDP-43 depletion and depletion rates [00295] A postmortem analysis of ALS patients revealed that 100% of dual FTD/ALS diagnosed patients had hypoglossal TDP-43 accumulation ⁶⁷. On the other hand, only a few oMNs accumulate TDP-43 at advanced stages ⁶⁸. The postmortem data cannot distinguish between cell-autonomous versus non-cell-autonomous mechanisms responsible for hMNs versus oMN differences. The studies of mouse neurons proposed a higher oMNs proteostasis capacity that allows them to prevent protein aggregates ⁴⁰. It was hypothesized that retaining nuclear TDP-43 is the first layer in the cell-autonomous components that enables human oMNs to resist FTD/ALS. This hypothesis is tested in human iPSC-derived neurons carrying TARDBP and C9orf72 mutations.

Methods:

[00296] hiPSC line selection'. To enhance comparisons, experiments are conducted using two orthogonal genetic approaches: 1) patient-derived lines representing patient genotypes and 2) an isogenic background with engineered mutations isolating the mutation effect from the overall background effects.

1) The following patient-derived iPCS lines were obtained: two lines with 800 G4C2 repeats at C9orf72: CSOBUUiALS (female) and CS7VCZiALS (male), two TARDBP mutant lines: CS8EDMiALS-nxx (female; G384R mutation), CSIUWUiALS-nxx (Male; N390D mutation), and two control CS3GFTiCTR (male) and CS6EFYiCTR-nxx (female). All these lines were carefully selected to match different parameters, including stem cell reprogramming method (episomal plasmid), somatic cell origin (PBMC), sex (female/male), ancestry (Caucasian), and age range (60-65). Both sexes are analyzed because of the described iPSC differentiation sex signature ²⁵. As representation and inclusion apply to disease modeling, more genetic backgrounds are included as iPSC lines become available. 2) Isogenic iPSC lines with FTD/ALS mutations. The “standard” KOLF2.1 J iPSC line selected by the iPSC Neurodegenerative Disease Initiative (iNDI) project ⁴¹ and its M337V TDP-43 mutant derivative is used. Additionally, two KOLF2.1J derivatives with 200 G4C2 engineered repeats at the C9orf72 locus recapitulating FDT/ALS mutations were obtained. The iNDI project is generating a line with 800 repeats that is requested after verification. Although it cannot be known where these genotypes would lie in the FTD/ALS spectrum, they may be important reductionist tools because they can isolate the effect of the mutation from the overall genetic background.

[00297] Neuronal differentiation'. Each iPSC line is differentiated into the three neuronal types using transcription factor direct cell programming ^{39, 40}. A single “enhanced” piggyBac transposon is used, which contains the doxycycline reverse transactivator (rtTA) ⁶⁹ , and the following inducible transcription factors: CN) Neurog2 (iN), hMN) Neurog2+Isl 1 +Lhx3 (iNIL) and oMN) Neurog2+Isll+Phox2a (iNIP) (Figure 1A). Three clones demonstrating similar Neurog2 induction levels for each genotype are picked and used for biological replicates to avoid biases introduced by studying a single clone.

[00298] For differentiation, iPSCs were plated on Matrigel in a neuron differentiation medium with 3 pM doxycycline to induce Neurog2, NIL, or NIP cassette expression on day 0 (Figure IB and ³⁹). The CN generation by Neurog2 expression is well established ^{52, 57} Briefly, Neurog2 expression combined with Wingless-related integration site (WNT) inhibition (rostralizing conditions) generates CNs. Caudalizing conditions were established by retinoic acid (RA) concentrations to complement TF combinations: NIP+0.01 pM for oMN, and NIL+1 pM for hMN. Adarotene is added from day 6 onward to eliminate active dividing cells without the toxic effects of Arabinose operon regulatory protein (AraC) and Floxuridine (FUdR) ⁷⁰. To maintain a healthy culture beyond day 10, the media is replaced every 4 days with the following neurotropic factors: Brain-derived neurotrophic factor (BDNF)/ Ciliary neurotrophic factor (CNTF)/ Glial cell line-derived neurotrophic factor (GDNF)/Vitamin C. The GENtonik maturation cocktail is added to accelerate maturation ⁷¹.Thus, an entirely adherent protocol requiring only media changes produces oMNs and hMNs at high efficiency and complements the well-described CN protocol.

[00299] The control and the M337V TDP-43 KOLF2.1J lines (Figure 1C) were differentiated. Additionally, 3 clones of the patient-derived C9orf72 mutant CS7VCZiALS line were differentiated with high reproducibility across different clones (Figure ID). All proposed genotype alternatives were surveyed, and all differentiated well, eliminating concerns about differentiating cells . Using the methods described herein, neurons may be maintained without glia support for more than 45 days.

[00300] TDP-43 quantification'. TDP-43 nuclear retention across neuronal types and genotypes were compared. The longest neurons were cultivated for without any sign of cell death: day 10 (young neurons), day 30 (matured neurons), and day 45 are used. Staining for TDP-43 and its phosphorylated form (pTDP-43) that accumulates in pathological aggregates in FTD/ALS is conducted ²’ ⁴. Delineation is performed for the nucleus with DNA (4’,6-diamidino-2- phenylindole, (DAP I)) and anti-lamin B antibody to quantify TDP-43 protein in the nucleus and cytoplasm ⁿ’ ¹². The anti-lamin B antibody staining can also reveal possible nuclear lamina distortions observed during TDP-43 mislocalization in FTD/ALS ⁷³.

[00301] The TDP-43^M337V mutation was reported in a Japanese cohort with bulbar ALS; thus, strong hypoglossal degeneration with TDP-43 aggregates ⁷⁴ TDP-43 nuclear levels were measured in oMNs and hMNs derived from a TDP-43^M337 mutant iPSC line. In agreement with postmortem data, oMNs retain more nuclear TDP-43 and have reduced TDP-43 condensates compared to hMNs ³⁹.

[00302] Investigating all three neuron types and genotypes provides significant insights into the first line of cell-autonomous response of CN, oMN, and hMN to FTD/ALS mutations.

Maintaining TDP-43 in the nucleus can be the first line of defense that allows oMNs to remain less sensitive to TDP-43 perturbations induced by TARDBP and C9orf72 mutations. C9orf72 mutations are common across the FTD/ALS spectrum, and TARDBP mutations are rare in pure FTD ⁸. While hMNs are sensitive to both mutations, CN could be more sensitive to C9orf72 than TARDBP. Finally, CN could show an intermediate phenotype between these two motor neurons based on their differential sensitivity.

Effect of TDP-43 and C9orf72 mutations on splicing defects across sensitive and resistant neurons

[00303] Splicing defects are found across ALS/FTD TDP-43 and C9orf72 mutations, including UNCI 3a and STMN2, reproduced in in vitro differentiated neurons ^{l 2}- ^{4 46} However, no comprehensive comparisons exist of the splicing defects induced by TARDBP and C9orf72 mutations between resistant and sensitive neuronal types. Without this knowledge, it is challenging to pinpoint causative mRNA changes directly associated with their neurodegenerative response in sensitive neurons. The inventors of the present invention hypothesized that the neuronal-type transcriptomes are differentially affected by TARDBP (direct effect on TDP-43) and by C9orf72 mutations (indirect), allowing oMNs to maintain a robust splicing pattern in response to FTD/ALS stress. To test this, control, TARDBP, and C9orf72-induced splicing defects in young and mature CN, oMNs, and hMNs are compared. Methods:

[00304] iPCS cell lines and differentialion. As described above.

[00305] Transcriptome and splice variant analysis'. To understand the relationship between the TARDBP and C9orf72 and splicing defects, deep bulk RNA-seq is performed. RNA is collected from young Day 10 neurons and mature Day 30 to obtain a temporal progression that matches the preliminary TDP-43 nuclear depletion . This time series can be enhanced if TDP-43 nuclear differences become exacerbated with time. cDNA is generated with the RiboMinus™ Eukaryote Kit for RNA-Seq to avoid the 3’ bias of polyA-based amplification kits that limit splicing analysis. The state-of-the-art Genome Technology Center at NYU Grossman School of Medicine is used for deep paired-end 300bp Illumina reads (a regular service by the NYU facility and an appropriate format for transcriptome analysis). The External RNA Controls Consortium (ERCC) RNA spike-in is added to normalize read counts across experiments.

[00306] First, DESeq2 is used to analyze differentially expressed genes between neuronal types, genotypes, and time points ⁷⁵. Subsequently, differential splicing analysis is performed using LeafCutter⁸¹ (Figure 2A). LeafCutter was chosen because it is agnostic to gene annotations and thus ideal for identifying cryptic intron (non-annotated) retention typically induced in FTD/ALS ii, 13, 45, 82 Complementarity, splicing is analyzed with MAJIQ and its updated v2 package that shares the unconstrained splicing annotation with LeafCutter ⁸³- ⁸⁴

[00307] Nuclear depletion of TDP-43 causes splicing defects in spinal motor neurons. The mutation-induced depletion of nuclear TDP-43 was found to lead to splicing defects in these neurons, including the RNA-binding fox-1 homolog 2 (RBFOX2) and Matrin 3 (MATR3) RNA binding factors suspected to play a role in FTD/ALS ⁸⁵. A proof of principle experiment was completed in a TARDBP mutant background and observed evident splicing defects even in young hMNs. Additionally, a preliminary splicing analysis was performed for young C9orf72 hMNs. hMNs mis-splice several RNA binding proteins, including the known MATR3 mRNA ⁸⁶. Importantly, the initial splicing defects were found in Neuro-oncological ventral antigen 1 (NO VAI).

[00308] A time series is established across three neuronal types with C9orf72 and TARDBP mutations. Deteriorating splicing over time is anticipated, consistent with TDP-43 mislocalization. The collective findings enable the comparison of TDP-43 nuclear depletion and splicing defects across neuronal types. Similar TDP-43 nuclear depletion may lead to varying splicing defects in different neuronal types, explaining their resistance. This preliminary data suggests that oMNs may maintain a more normal splicing landscape even with mislocalized TDP-43. TARDBP mutations may impact hMNs more than CN due to their higher sensitivity. Thus, the disentanglement of TDP-43 mislocalization and splicing defects stemming from FTD/ALS mutations across human neuronal types is important for understanding where these three neurons part ways to produce differential outcomes in response to FTD/ALS stress.

Effects of TDP-43 nuclear depletion on various neuron types

[00309] Changes in TDP-43 nuclear depletion rates can only partially account for oMN resistance, as some oMNs contain TDP-43 aggregates ⁸⁷. Therefore, additional, less obvious resistance mechanisms were explored that enable oMNs to resist neurodegeneration. TDP-43 depletion in the nucleus results in the incorporation of cryptic exons or introns in hundreds of mRNAs, often disrupting their translation and promoting nonsense-mediated decay ¹⁵‘ ^{59, 88}. Hence, the transcriptome of different neuron types may exhibit varying sensitivity to TDP-43 alterations. Thus, oMNs may exhibit resistance at multiple levels: controlling TDP-43 accumulation in response to FTD/ALS mutations and reducing splicing complications even when TDP-43 depletes from the nucleus. Since the neuronal repose to FTD/ALS mutations includes TDP-43 localization and possible splicing differences, TDP-43 ’s direct effects are isolated by experimentally reducing its nuclear localization in CNs, oMNs, and hMNs.

Methods.

[00310] iPCS cell line and differentiation'. Control genotypes as described above Control genotypes and C9orf72 mutations are used, and TARDBP mutations are omitted since TDP-43 levels are reduced.

[00311] TDP-43 nuclear depletion: TDP-43 nuclear depletion recapitulates neuron splicing defects. Two complementary approaches are taken: a) Proteasome inhibitor and b) TDP-43 knockdown (KD). a) Transient, non-lethal stress induces cytoplasmic TDP-43 aggregates in neurons ⁸⁹. Acute proteasome activity reduction mimics some aspects of cellular aging and induces TDP-43 cytoplasmic accumulation and nuclear depletion ^90-94 The proteasome inhibitor MG-132 induces splicing defects in iPSC-derived motor neurons seen in ALS patients ¹². To model proteotoxic stress and acute TDP-43 nuclear depletion, MG-132 concentrations were titrated: 24 hours of 1.0 and 1.5 pM MG-132 produce similar TDP-43 nuclear depletion in oMNs and hMNs (Figure 2A)³⁹. b) MG-132 cellular stress reduces nuclear TDP-43. Thus, a targeted TDP-43 depletion is performed with an established small interfering RNA (siRNA)-based method ⁴⁶. Differentiated CN, hMNs, and oMNs are transfected with SMARTpool ON-TARGETplus siRNA targeting TDP-43 or scramble (control) siRNA pool at 100 nM. The reduction is verified with quantitative polymerase chain reaction (qPCR), western blot, and immunocytochemistry to measure cell-to- cell variation 96 h after the initial transfection, as described ⁴⁶. The siRNA-based strategy was tested and obtained a 50% reduction of TDP-43 mRNA, as reported ⁴⁶'

[00312] Transcriptome splice variant analysis'. As described above.

[00313] Splicing differences were analyzed in oMNs and hMNs under MG-132-induced TDP- 43 mislocalization. While this was done in a single time point and with control neurons (no FDT/ALS mutations), clear UNC13a and STMN2 splicing defects were observed. Additionally, hMNs exhibited more splicing defects even with similar TDP-43 mislocalization levels (Figure 2G). These findings support the hypothesis that oMNs and hMNs respond differently under similar TDP-43 nuclear depletion and stress.

[00314] Preliminary data suggest that oMNs exhibit resistance to neurodegeneration at two redundant levels: maintaining nuclear TDP-43 and reducing splicing differences even after TDP- re nuclear depletion.

[00315] oMNs may have a better proteostasis capacity that allows them to reduce the accumulation of insoluble protein aggregates. Enhanced protein degradation protects mouse oMNs from accumulating insoluble SOD1 and p63 aggregates in vitro and in vivo ^{40 64}. Preliminary data with TARDBP mutants suggests these results expand to human oMNs with FTD/ALS mutations. Thus, the first layer of defense of oMN is controlling proteostasis; which seems to be conserved across species and supports the recent findings in mouse cells ⁶⁴ [00316] The second resistance introduces a novel mechanism (illustrated herein). The initial observation that resistant neurons display fewer splicing defects even when nuclear TDP-43 protein levels were experimentally manipulated (Figures 2D, 2E, 2F, and 2G) opened a new area of investigation: how does the neuronal type-specific transcriptome respond to TDP-43 mislocalization? This question is addressed under acute TDP-43 perturbations and moderate and sustained stress by TARDBP (direct) and C9orf72 (indirect) mutations (Figures 4C, 4D, 4E, 4G, 4H, and 41). The results show disentangle direct TDP-43 targets from the product of chronic stress caused by mutations in different neurons. Comparisons with similar neuron types but with varying sensitivities to FTD/ALS stress may be novel lines of investigation and can reveal the multi-tiered cell-autonomous mechanism that, at least in part, can explain why neurons respond differently to the FDT/ALS stress.

[00317] While shown to be effective in oMNs and hMNs, TDP-43 knockdown using siRNA could be less efficient in CNs. If so, two tested alternative approaches can be used: antisense oligonucleotides (ASOs) targeting TARDBP (TDP-43) RNAs ¹¹ or CRISPR interference (CRISPi) to decrease TARDBP transcription, which has been extensively used in iPSC-derived neurons ⁹⁵. To facilitate execution, a strict quality control and record-keeping system has been established for all cell lines, neuronal identity, and library preparation.

Defining the neurode generative splicing cascade in sensitive neurons

[00318] Prior experiments focused on the initial protein and RNA responses to TDP-43 perturbations, presenting the first two layers that discriminate between resistant and sensitive neurons. Here, new layers to the multi-tiered system are introduced: Preliminary analysis suggests the presence of an undescribed amplification mechanism in sensitive neurons (Figure 6B). First, how the neuronal type and FTD/ALS genotypes influence the direct TDP-43 regulome is described, explaining differential outcomes to its nuclear depletion. Secondly, measurements of the contributions of the hMN-specific splicing regulator NO VAI to the observed splicing abnormalities in FTD/ALS-sensitive neurons are made. Finally, testing if restoring hMN-affected splicing factors mitigates TDP-43 -induced splicing defects is performed. Association of DP-43 splicing factor with different mRNAs in CN, hMNs, and oMNs

[00319] Studies in mouse, human, and rat neurons reveal that TDP-43 binds to approximately one-third of all mRNAs ^{7 9}’ ⁹⁶’ ⁹⁷. The most prominently affected transcripts in TDP-43 perturbations are long mRNAs, typically enriched in neurons. However, how TDP-43 interacts with the resident transcriptome of sensitive and resistant neurons has been less explored. Additionally, the effect of FTD/ALS mutations in the neuronal type-specific TDP-43 regulome is understudied.

[00320] The TDP-43 nuclear depletion data in hMNs and oMNs demonstrate that common and neuronal type-specific transcripts are differentially spliced between these two neurons (Figures 2E, 2F, and 2G). This observation presents the following questions: 1) how many splicing defect differences between oMNs and hMNs can be attributed to the fact that TDP-43 associates with different mRNAs in different neuronal types? 2) how do FTD/ALS mutations affect this regulation? To address these, TDP-43 associated mRNAs is characterized in control, TDP-43, and C9orf72 CNs, oMNs, and hMNs to measure the effect of neuronal type in combination with FTD/ALS mutations in the TDP-43 regulome.

Methods:

[00321] iPCS cell line and differentiation. As described above. These experiments are initially performed on day 10 and 30 to consolidate data. Following the results from TDP-43 mislocalization and nuclear depletion phenotypes, this timeline are expanded to reach Day 45.

[00322] TDP-43 associated mRNAs'. To identify TDP-43 protein-associated mRNAs, the updated TDP-43 RNA immunoprecipitation sequencing (RIP-seq) protocol is used. Briefly, single-cell suspensions are fixed in 0.3% methanol-free formaldehyde for 30 min at 4 °C to link transcripts and proteins. After RIPA buffer lysis, TDP-43+RNA complexes are pulled down with the immunoprecipitation (IP)-validated antibody TDP-43: A303-223A, Bethyl Laboratories ⁸³. After washing and elution, RNA-seq is performed from the input and TDP-43 pull-downs. The ERCC RNA spike-in is added to normalize read counts across experiments.

[00323] The TDP-43 RIP-seq protocol was established. The TDP-43 RIP-seq identifies the canonical TDP-43 binding sites on the STMN2 and Uncl3a transcripts in control and C9orf72 hMN and oMNs (Figure 3B). This data establishes technical feasibility, but additionally biological replicates to analyze differential TDP-43 binding are needed.

[00324] These results have a straightforward yet impactful interpretation. TDP-43 -associated mRNAs in CN, oMNs, and hMNs are compared to test the hypothesis that the TDP-43 protein associates and regulates different mRNA species in each cell type, thus explaining some of the differential response. If confirmed, TDP-43 protein is sensitive to the resident neuronal type transcriptome in two ways. 1) Commonly expressed genes could associate differently with TDP- 43, possibly explaining some of the different sensitivity to perturbations in FTD/ALS, with potential implications in future efforts to understand what controls differences in TDP-43- associated RNAs. 2) TDP-43 associates with differentially expressed mRNAs. This result suggests inherent differences as a secondary effect of the transcriptome of each neuron type. N0VA1 is a critical component of the splicing cascade that renders hMNs sensitive to TDP-43 perturbations.

[00325] The preliminary splicing data across neuronal types, mutations and MG- 132 provided a surprising observation. Genes mis-spliced in hMNs only are enriched in splicing factors. This effect is not minor; the first seven enriched Gene Ontology (GO) terms are associated with splicing or RNA biology (Figures 21, 3G, and 4F). Surprisingly, several genes are highly expressed in hMNs, including N0VA1. However, some candidates like RBFOX2 are not differentially affected in these neurons and thus may be unlikely to explain the differential response. Thus, the hMN-specific splicing factors seem very sensitive to FTD/ALS stress. [00326] NOVA are central nervous system-specific RNA-binding proteins. While Neuro- oncological ventral antigen 2 (N0VA2) has higher expression in the dorsal spinal cord, NOVAI is highly expressed in the ventral spinal cord, including motor neurons ¹⁰°. N0VA1 mutant mice die postnatally with severe motor deficits primarily due to motor neuron defects ¹⁰¹. Furthermore, N0VA1 protein is reduced in ALS postmortem tissue⁸⁵. N0VA1 and TDP-43 share ~ 60% of their targets, suggesting they can compound their effect when both are compromised ⁸⁵. Finally, the preliminary data supports a critical role for NOVAI in sensitive neurons: a) N0VA1 is highly expressed in hMN (single-cell RNA-seq revealed most hMN express NOVAI as they mature Figure 8A) and not in oMNs; b) NOVAI levels decrease in TARDBP mutant hMNs (Figure 8B) and C9orf72 hMNs; c) NOVAI splicing defects in C9orf72 and TARDBP mutations, and after TDP-43 nuclear reduction with MG- 132 (Figures 8C and 8D); d) From the preliminary RIP experiment NO VAI was detected as a direct TDP-43 target in hMNs. This data suggests an hMN amplification system initiated by TDP-43 mislocalization, including the splicing regulator NOVAI. Hindering the understanding of the NOVAI regulome in disease, there is no systematic characterization of NOVAI targets in control and FTD/ALS human hMNs. Thus, quantification of the NOVA 1 -dependent regulome is important in both control and FDT/ALS mutant hMNs as a potential mediator of differential neuronal vulnerability to neurodegeneration. Methods.

[00327] iPCS cell line and differentiation. NOVAI ’ s role in control and FTD/ALS hMNs is investigated. The control, TARDBP, and C9orf72 mutant iPSCs are differentiated into hMN as previously described.

[00328] NOVAI reduction'. N0VA1 is reduced, repeating the successful TDP-43 siRNA knockdown with probes targeting NOVAI. Reduction is verified by qPCR, western blot, and immunocytochemistry to measure cell-to-cell variation.

[00329] Transcriptome splice variant analysis'. The expression and splicing changes are described with Deseq2 and LeafCutter. Days 10 and 30 will mark the start, as explained above. The timeline will be extended after the initial experiments.

[00330] NOVAl-associated mRNAs'. NOVAI is directly identified using the validated antibody for NOVA1-RNA interactions (abl83034, Abeam) ⁸⁵. The protocol that was successfully used for TDP-43 is followed (Figure 3A).

[00331] These experiments highlight the importance of the neuronal type-specific splicing regulator NOVAI in maintaining the hMN transcriptome in control and FTD/ALS mutant neurons. The TDP-43/NOVA1 splicing hierarchy is resolved by comparing these results with those for TDP-43, identifying common and unique mRNA targets for each protein. Thus, the expected splicing defects that depend strictly on NOVAI in control, TDP-43, and C9orf72 mutant hMNs are quantified. Because they share targets with TDP-43, a large fraction of NOVAI targets is possibly affected by FTD/ALS mutations. Thus, TDP-43 reduction and the consequent NOVAI reduction are “double hit” for commonly regulated RNAs. The preliminary data supports a model that places NOVAI as a mediation of the hMN-specific splicing stress. ELAV-like RNA binding proteins (ELAVL) 2 and 3 are also possible mediators. ELAVL proteins were implicated in neurodegeneration ¹⁰²- ¹⁰³. ELAVL3 exhibits nuclear depletion in ALS patients and is enriched in hMNs ¹⁰⁴

Restoring splicing factors levels to improve the sensitive neuron transcriptome after TDP-43 nuclear depletion.

[00332] Splicing defects are a key cell-autonomous neuronal contributor to neurodegeneration. Correcting splicing has been proposed as a possible therapeutic venue. TDP-43 is prone to aggregation, forming seeds to recruit more TDP-43 ^{105, 106}, and even low levels of TDP-43 overexpression cause neurodegeneration in mice ¹⁰⁷. Because TDP-43 expression is unviable, rescuing specific splicing defects downstream of TDP-43 emerged as a possible therapeutic intervention. Based on previous observations, it is hypothesized that rescuing defects in splicing factors affected by TDP-43 depletion can improve a significant fraction of the neuronal transcriptome affected by TDP-43 depletion.

[00333] A self-positive feedback loop maintains high N0VA1. The exon 4 of N0VA1 mRNA contains five YCAY motifs bound by NOVAI protein. NOVAI binding is necessary and sufficient for exon 4 exclusion contributing to N0VA1 high levels ¹⁰⁸. Therefore, increasing the N0VA1 protein can restore splicing defects, including NOVAI, amplifying the intervention. Because N0VA1 and TDP-43 share targets, NOVAI might rescue defects induced by TDP-43 nuclear depletion. Thus, quantification of NOVAI sufficiency to mitigate TDP-43 -induced splicing and mRNA levels in control and FTD/ALS mutant hMNs is performed. Additionally, this approach can serve as a proof of principle to rescue degenerative phenotypes by restoring upstream splicing factors affected by TDP-43 nuclear reduction.

Methods:

[00334] iPCS cell line, differentiation and transcriptome splice variant analysis'. iPSCs are differentiated into hMNs. To quantify the rescue, initially the hMN splicing and expression are compared across different samples: control, TDP-43 reduction, and TDP-43 reduction+NOVAl. [00335] NOVAI rescue in TDP-43 reduction. Control hMNs express “canonical” N0VA1 isoform P51513-4, thus rescue is performed with this isoform that excludes the exon 4 incorporated in TDP-43 perturbations, beta-galactosidase (b-Gal, control) or NOVAI cDNA and constitutively expressed green fluorescent protein (GFP) are virally delivered in Day 10 young hMNs.

[00336] Transcriptome splice variant analysis'. GFP cells (expressing the control b-Gal or N0VA1) are sorted. The expression and splicing changes are described with Deseq2 and LeafCutter. Days 10 and 30 are the starting point. The timeline can be extended after the initial experiments.

[00337] Comparing the splicing defects induced by TDP-43 and N0VA1 establishes a hierarchy of splicing control started by TDP-43 nuclear depletion. The RNA binding studies discriminate between parallel or sequential mechanisms. Overall, the contribution of the sensitive neuronspecific N0VA1 to the TDP-43 phenotype is explored. Finally, the rescue induced by forced NOVAI expression after TDP-43 reduction is quantified. This can serve as proof of principle for downstream interventions to control the neurodegenerative process.

[00338] The other splicing factors affected can be added in hMNs after TDP-43 reduction: N0VA2 (although aNOVAl paralog, it regulates a different set of mRNAs in neurons ¹⁰⁹), ELAVL2 (identified in the screen for hMN-specific splicing factors) and ELAVL4 (with potential involvement in FTD as expressed above). Splice-switching ASOs targeting splicing factors can be developed as described in the present disclosure to maximize the intervention reach.

[00339] Well-established protocols are proposed, supported by preliminary data or proof of concept. N0VA1 mutant iPCS cells can be generated and differentiated into hMNs. NOVAI is not expressed in pluripotent stem cells and thus should not affect their maintenance. However, the neurons will differentiate with low N0VA1 levels, therefore not mimicking a postmitotic neuronal reduction expected in FTD/ALS. As the preliminary data shows, RNA is not degraded for RIP-seq to increase sensitivity at the expense of base resolution. If base pair resolution is required, PAR-CLIP (photoactivatable ribonucleoside-enhanced crosslinking and immunoprecipitation) can be employed to discriminate TDP-43 versus N0VA1 RNA binding ¹¹⁰- ⁿⁱ. PAR-CLIP is significantly more complex but offers greater accuracy in determining protein- RNA interactions. The use of single-cell (sc) RNA-seq was assessed as an alternative to bulk RNA-seq. , scRNA-seq can be used if significant cell-to-cell variability is encountered as measured by immunocytochemistry in the knockdown experiments.

[00340] Drawing inspiration from FTD/ALS patients who retain eye movement despite motor neuron degeneration, a cell-autonomous mechanism that sets sensitive neurons on a degenerative downward spiral is described herein. The initial failure to maintain adequate levels of TDP-43 triggers splicing defects. The magnitude of splicing defects is greater in sensitive neurons, even at similar nuclear TDP-43 levels. Splicing factors are among those affected in sensitive neurons, implying the pathological phenotype. Conversely, the multi-tiered cascade opens the possibility of interventions at many levels, individually or in combination.

[00341] Using a human pluripotent stem cell differentiation strategy described herein, the problem is approached with an innovative comparison to shine light from a new angle. Finally, the proposed model could be the blueprint for future studies introducing more neurodegenerative genotypes and other neuronal types, such as basal forebrain cholinergic neurons affected in Alzheimer’s disease.

Rigor, reproducibility, and validation'.

[00342] All cell lines, DNA constructs, and mutations are routinely sequenced. Phenotypes are analyzed in at least three independently generated cell lines or derived clones. The analysis is blinded to a second investigator for phenotypic analysis. Sequencing files are coded and handed blind to a collaborator bioinformatician. Three biological repeats of RNA-seq and RIP-seq experiments are performed to measure reproducibility following the Encyclopedia of DNA Elements (ENCODE) guidelines ^{113, 114}. If outliers or underpowered studies are identified, two additional replicates are added. As expressed before, iPCS for both sexes are introduced to account for the reported sex difference in in vitro differentiated motor neurons and diverse genetic backgrounds can be added when possible.

Example 2. Dissecting differential neuronal sensitivity to neurodegeneration.

[00343] The death of specific neuronal types characterizes each neurodegenerative disease. Identifying the cellular responses that influence the susceptibility of specific neuronal populations to neurodegenerative stress is critical to understanding the disease and developing effective therapeutic strategies.

[00344] FTD and ALS are closely related neurodegenerative diseases. Therefore, patients lie on an FTD/ ALS spectrum with shared neuropathological and molecular markers. While most cases are sporadic, TDP-43 and C9orf72 mutations are common in familial FTD/ ALS cases.

Regardless of the cause, most FTD/ ALS patients develop TDP-43 cytoplasmic accumulation and nuclear reduction, resulting in characteristic TDP-43 -induced splicing defects.

[00345] Not all neurons are equally affected in FTD/ ALS. At later disease stages, FTD/ ALS patients with compromised mobility have severe difficulties swallowing and speaking, in part due to hypoglossal motor neuron (hMN) degeneration. However, patients communicate with eye-tracking devices because motor neurons controlling eye movement (the oculomotor motor neurons: oMNs) remain relatively unaffected. In addition, there is neuronal sensitivity across the FTD/ ALS spectrum mutations. Cortical neurons (CNs) tend to be more sensitive to C9orf72 than TDP-43 mutations than hMNs. The reasons for these differential sensitivities are unknown. The present invention described herein identifies the cellular responses that affect oMN, hMN, and CN susceptibility to TDP-43 perturbations and TDP-43 and C9orf72 mutations.

[00346] To study the differential sensitivity to FTD/ALS stress, a novel protocol was established to differentiate a high efficiency and maintain human oMNs and hMNs from patients and control iPSCs. This protocol complements the well-established CN differentiation. hMNs and oMNs were found to respond differently to FTD/ALS stress recapitulating in vivo phenotypes. Resistant neurons have redundant mechanisms to cope with TDP-43 nuclear depletion, but sensitive neurons enter a neurodegenerative downward spiral.

[00347] A multi-tiered model (Table 1) is described herein. Resistant neurons maintain nuclear TDP-43 (Tier 1) and have reducing splicing defects in response to TDP-34 perturbations splicing defects in sensitive neurons (Tier 2-3). On the other hand, sensitive hMNs have increased defects in splicing factors, including N0VA1, that amplify the phenotype (Tier 4-5).

Table 1. Overview of the multitiered mechanisms to understand the cell-autonomous differences in response to FTD/ALS stress.

Dissecting how resistant neurons control TDP-43 perturbations.

[00348] The initial defense mechanism employed by FTD/ALS-resistant oMNs is the retention of TDP-43 in the nucleus is tested (Tier 1). TDP-43 nuclear retention is measured in CNs, oMNs, and hMNs with TDP-43 and C9orf72 mutation. Additionally, the data as described herein suggests a novel mechanism: resistant neurons mitigate splicing defects even under significant TDP-43 perturbations (Tier 2). To test this hypothesis, nuclear TDP-43 is reduced and the splicing defects in these three neuronal types is measured.

Defining the neurodegenerative splicing cascade in sensitive neurons.

[00349] The analysis indicates the presence of an undescribed amplification mechanism in hMNs, intensifying initial splicing defects that lead to degeneration. It was hypothesized that the TDP-43 -dependent regulome differs in resistant and sensitive neurons (Tier 3). Specifically, TDP-43 depletion affects hMN’s splicing factors that further enhance splicing. Thus, NOVAl ’s contribution to maintaining healthy hMN splicing and mRNA content is quantified (Tier 4) and the extent to which restoring N0VA1 levels reverses splicing defects in hMNs caused by reduced TDP-43 (Tier 5) is measured.

[00350] A novel cell differentiation platform was developed to study how neuronal types are differentially sensitive to degeneration.

[00351] The sensitive hMNs embark on a neurodegenerative cascade are exacerbating their initial vulnerability. The non-limiting illustrations of the present disclosure and their results may enhance the understanding of differential neuronal sensitivity and pave the way for innovative therapeutic strategies for FTD/ALS, including suppressing its effects by restoring compromised splicing factors in sensitive neurons.

Example 3. Dissecting differential neuronal sensitivity to neurodegeneration.

[00352] Neurodegenerative diseases are age-related conditions characterized by several common features including the gradual loss of specific types of neurons, genetic mutations e.g., 90% sporadic and 10% familial in ALS), protein aggregation, and abnormal cellular functions (e.g., mis-splicing). Understanding why some neuronal types are more sensitive to neurodegeneration than others remains a challenging and understudied problem, possibly hindering comprehension of these diseases and potential therapeutic strategies. For example, FTD/ALS patients experience extensive motor neuron degeneration and become paralyzed. However, they can communicate with eye-tracking devices because the motor neurons that innervate the eye are relatively resistant to degeneration.

[00353] A comprehensive protocol was developed to investigate neuron-autonomous mechanisms that affect neuronal resistance. Initial investigations using the invention and methods described herein have unveiled a novel multitiered response mechanism that differentiates FTD/ALS resistant from sensitive neurons.

[00354] The first-tier concerns retaining nuclear TDP-43 as a defense mechanism in resilient motor neurons. Remarkably, the depletion of nuclear TDP-43 results in fewer splicing defects within these resilient neurons, marking it the second tier of defense. The last two tiers amply the sensitivity. A distinct TDP-43 regulome in sensitive neurons was found compared to susceptible counterparts. This disparity triggers an amplification cascade in sensitive neurons that involves other splicing factors.

[00355] The invention as described herein evaluated a cell-autonomous mechanism that sets sensitive neurons on a degenerative downward spiral. The initial failure to maintain adequate levels of TDP-43 triggers splicing defects in critical splicing factors, culminating in an amplification of the pathological phenotype. The insights gained from the non-limiting illustrations of the present disclosure have the potential to pave the way for innovative therapeutic strategies aimed at addressing neurodegenerative diseases, such as FTD and ALS.

EXAMPLE 4. Rescuing splicing defects in TDP-43 pathologies

[00356] Splicing defects are a non-limiting aspect of the cell-autonomous neuronal contribution to neurodegeneration. Correcting splicing has been proposed as a possible therapeutic venue. TDP-43 is prone to aggregation, forming seeds to recruit more TDP-43-and even low levels of TDP-43 overexpression cause neurodegeneration in mice. Because TDP-43 expression is unviable, rescuing specific splicing defects downstream of TDP-43 emerged as a possible therapeutic intervention. It was hypothesized that rescuing defects in splicing factors affected by TDP-43 depletion can improve a significant fraction of the neuronal transcriptome affected by TDP-43 depletion.

[00357] The following splicing regulators are overexpressed, to determine the therapeutic potential of splicing regulation in neurodegeneration: NOVAI P51513-4, NOVA2, ELAVL1, ELAVL2, ELAVL3, ELAVL4, RBFOX2 and MATR3 (Figure 6B). Overexpression is achieved by cDNA expression delivered by viral vectors or restoring their splicing by Splice-switching oligonucleotides (SSOs).

[00358] cDNA expression in neurons. To express these proteins in neurons, the full-length cDNAs (mature mRNA) from neurons is expanded and cloned into the following viral vectors: Lentivirus: pcDNA3.1 (+) IRES GFP ¹³² Adeno-associated viral: AAVretro-CAG-GFP, and AAV9-CAG-tdTomato, where GFP replaces tdTomato.

NOVAI :

MMAAAPIQQNGTHTGVPIDLDPPDSRKRPLEAPPEAGSTKRTNTGEDGQYFLKVLIPSY AAGSIIGKGGQTIVQLQKETGATIKLSKSKDFYPGTTERVCLIQGTVEALNAVHGFIAEKI REMPQNVAKTEPVSILQPQTTVNPDRIKQTLPSSPTTTKSSPSDPMTTSRANQVKIIVPNS

TAGLIIGKGGATVKAVMEQSGAWVQLSQKPDGINLQERVVTVSGEPEQNRKAVELIIQK

IQEDPQSGSCLNISYANVTGPVANSNPTGSPYANTAEVLPTAAAAAGLLGHANLAGVAA

FPAVLSGFTGNDLVAITSALNTLASYGYNLNTLGLGLSQAAATGALAAAAASANPAAA

AANLLATYASEASASGSTAGGTAGTFALGSLAAATAATNGYFGAASPLAASAILGTEKS

TDGSKDVVEIAVPENLVGAILGKGGKTLVEYQELTGARIQISKKGEFVPGTRNRKVTITG

TPAATQAAQYLITQRITYEQGVRAANPQKVG (SEQ ID NO: 1)

RBFOX2:

MQNEPLTPGYHGFPARDSQGNQEPTTTPDAMVQPFTTIPFPPPPQNGIPTEYGVPHTQDY

AGQTGEHNLTLYGSTQAHGEQSSNSPSTQNGSLTTEGGAQTDGQQSQTQSSENSESKST

PKRLHVSNIPFRFRDPDLRQMFGQFGKILDVEIIFNERGSKGFGFVTFENSADADRAREKL

HGTWEGRKIEVNNATARVMTNKKMVTPYANGWKLSPVVGAVYGPELYAASSFQAD

VSLGNDAAVPLSGRGGINTYIPLISLPLVPGFPYPTAATTAAAFRGAHLRGRGRTVYGAV

RAVPPTAIPAYPGVVYQDGFYGADLYGGYAAYRYAQPATATAATAAAAAAAAYSDGY

GRVYTADPYHALAPAASYGVGAVASLYRGGYSRFAPY (SEQ ID NO: 2)

Table 2. Exemplary sequences of splicing factors and splicing regulators Example 5. Development of AAV vector encoding splicing factors and/or splicing regulators

[003591 The cDNAs of NOVAI, ELAVL1, ELAVL2, ELAVL3, ELAVL4, RBFOX2, MATR3, HNRNPA1, SFPQ, and/or FUS are cloned in an AAV backbone.

[00360] Strong promoters such as, without limitation, hUBC, pCAGs, hCMV, and mPGK, the synapsin (hSYN), and the motor neuron promoter Hb9 can be used to rescue gene expression in neurons.

[00361] AAV9 and AAV-B1 display neuronal tropism and can mediate stable, long-term expression with a single administration.

[00362] The purified AAV particles are delivered to neurons via intracranially, systemic, or intrathecal injection. This method allows for direct access to the cerebrospinal fluid and efficient transduction of neurons.

[00363] Splicing defects are a key contributor to the neurodegenerative process that involves TDP-43 dysfunction. The re-expression of critical splicing regulators, including NOVAI, ELAVL1, ELAVL2, ELAVL3, ELAVL4, RBFOX2, MATR3, HNRNPA1, SFPQ, and FUS either individually or in combination, would help mitigate the effects of TDP-43 -induced splicing defects. This approach is expected to improve the neuronal transcriptome and enhance neuronal survival in response to TDP-43 dysfunction associated with FTD, AL), Parkinson’s disease (PD), and other such diseases.

Example 6. Construction of antisense oligonucleotides

[00364] The ASOs to bind to the pre-mRNA sequence flanking the mis-spliced exons are designed. The ASOs designed as described in the present disclosure are complementary to the splice sites or splicing regulatory elements, such as exonic splicing enhancers (ESEs). The relevant splice junctions are tiled to the ASOs to restore and select the most efficient ASOs. [00365] ASOs are transfected and their impact on splicing in human neurons derived from ALS or FTD patients, or with induced TDP-43 dysfunction are assessed.

[00366] Successful correction of splicing is demonstrated by RT-PCR and long-read mRNA sequencing, showing the presence of the correctly spliced mRNA. This indicates that the ASO is effectively targeting the mis-spliced exon and restoring normal splicing. [00367] Correct splicing should lead to the production of normal, functional protein. The protein levels are measured using western blotting or immunocytochemistry to confirm that the ASO treatment restores protein expression.

Example 7. Enhancing Neuronal Resistance to Neurodegeneration

Neurodegenerative diseases involve the death of specific types of neurons.

[00368] To develop effective treatments, an understanding of how different neurons respond to stress caused by these diseases may be useful. In ALS, patients gradually lose their ability to move but can still control their eye movements because the MNs that control the eyes resist the disease until its late stages. Currently, research often treats motor neurons as a monolithic group, without comparing different types of neurons to see why some are more resistant to stress. This approach may have a major drawback as it may limit an understanding of the specific events that lead to neuron degeneration and limits the development of targeted therapies. The inventors suggest a new therapeutic idea that may address this gap in research.

There are two innovations described herein:

[00369] 1) Leveraging natural differences in MN subtypes to understand and treat ALS. The inventors’ research is based in part on the observation that hMNs degenerate in ALS patients, while oMNs resist until late disease stages. This difference is conserved across familial and sporadic ALS, thus a general phenomenon. To investigate the different responses of these sensitive and resistant motor neurons to ALS, the inventors developed a human hMN and oMN differentiation platform from iPSCs, as discussed herein. This comparison allowed identification of ALS-specific responses in motor neurons that are sensitive to the disease.

[00370] The “neurodegenerative splicing cascade” (Figure 6A): TDP-43 nuclear depletion and splicing defects are a characteristic of ALS. Yet, it is not known why MNs suffer more than other neurons from TDP-43 dysfunction. The inventors found that sensitive hMNs exhibited six times more splicing defects in genes related to RNA splicing compared to oMNs with the same TDP-43 loss of nuclear TDP-43, as shown herein. The inventors also found a set of genes that are responsible for maintaining splicing health in hMNs and are directly controlled by TDP-43. Thus, TDP-43 dysfunction causes their mRNA downregulation that amplifies defects in cryptic and alternative splice isoforms. These defects can be termed a “neurodegenerative splicing cascade” that the inventors aim to restore and make sensitive MNs more similar to oMNs. This strategy utilizes differences in neuronal responses to TDP-43 dysfunction to enhance the survival of motor neurons.

[00371] 2) Correcting the “neurodegenerative splicing cascade” (Figure 6B). The inventors propose a novel approach to rescuing MNs affected by neurodegenerative stress, validated with data from ALS patient post-mortem tissues. Current strategies involve the challenging task of restoring nuclear TDP-43 with its technical and biological complications. Alternatively, some strategies aim to fix specific downstream splicing events like STMN2 that, by definition, improve a single cellular property. Moreover, it is challenging and inefficient to restore the many splicing downstream splicing defects one at a time.

[00372] The inventors propose targeting the “neurodegenerative splicing cascade” by rescuing the function of upstream splicing regulators to improve all aspects of neuronal physiology affected by TDP-43. Some important players in this response that the inventors propose to rescue and restore splicing defects in sensitive neurons to make them more like resistant MNs include NO VAI, ELAVL1, ELAVL2, ELAVL3, ELAVL4, RBFOX2, MATR3, HNRNPA1, SFPQ, and FUS. The inventors propose to re-express these splicing regulators in MNs to enhance the MNs’ overall response to TDP-43 dysfunction in ALS.

[00373] The proposed therapeutic idea has a testable hypothesis: re-expressing TDP-43 controlled splicing regulators that maintain a healthy transcriptome can reduce splicing defects and improve the survival of MNs with ALS mutations and/or ALS-related stressors (. The inventors propose to identify the minimal set of splicing factors that are sufficient to rescue splicing defects and survival of motor neurons carrying ALS-associated mutations and TDP-43 dysfunction. The identified target genes can be immediately targeted for gene therapy or splicing correction using gene therapy, such as viral vectors (e.g., AAVs) expressing wild-type splicing regulators and/or splicing factors, or splice-switching ASOs.

[00374] MN survival is a paradoxical case of differential sensitivity to neurodegeneration, yet the reasons for differential sensitivity between MN types are unknown. ALS cases with muscle weakness eventually develop bulbar symptoms and difficulties in speech and swallowing due to the degeneration of hMNs. On the other hand, ALS patients maintain eye movement because the oMNs remain unaffected until the late stages of the diseasel. It has been proposed that oMNs resist excitotoxicity, but this feature does not explain the reduced protein accumulation and splicing defects observed in ALS patient oMNs 2. Thus, additional factors may contribute to their differential sensitivity, and the reasons for these differential sensitivities are unknown. [00375] The inventors developed a protocol to differentiate human pluripotent stem cells into oMN- and hMN-like neurons to understand the differential sensitivity. An important step was combining Neurog2 with pairs of synergistic transcription factors (TFs) required for their embryonic differentiation, namely Neurog2-Isll-Lhx3 (the NIL combination) for MNs expressing marker genes associated with rostral spinal MN identity and Neurog2-Isll-Phox2a (the NIP combination) for generating rostral cranial MNs, containing oMNs 3. Both hMN and oMN neurons are electrically active, express the appropriate fate markers, and send axons through the correct track when implanted into a developing spinal cord 3. These hPSC-derived neurons revealed that an enhanced proteostasis capacity protects oMNs from generating superoxide dismutase 1 (SOD1) and sequestosome 1 (p62) protein aggregates. 4. NIL and NIP - induced neurons expressed neuronal and MN markers at day 10 and maintain them until day 30. NIL-induced MNs expressed MNX1 (Homeobox gene 9; Hbx9), Homeobox B4 (Hoxb4), and Teashirt Zinc Finger Homeobox 1 (Tshzl), which are required for hMN in vivo differentiation. The inventors have recently collected single-cell RNA-seq data, and the two MN fates form tight, non-overlapping clusters. Thus, the inventors call them hMN-induced and oMN-induced (hiMN and oiMN).

[00376] The inventors have identified a set of splicing regulators affected in sensitive MNs (Figure 6A). Studies in mouse, human, and rat neurons revealed that one regulator, TDP-43, binds to approximately one-third of all neuronal mRNAs 5. However, these studies do not explain why some neurons die while others do not. Therefore, the inventors leveraged the comparative analysis described herein to identify potential targets that could explain and rescue sensitive MNs.

[00377] First, the inventors examined all MN splicing defects in response to the FTD/ALS mutations TARDBP, C9orf72, and ALS-relevant proteotoxic stress. Sensitive hMNs exhibited six times more splicing defects in genes related to RNA biology than oMNs, indicating a potential amplification of splicing defects in sensitive neurons. Second, the inventors selected direct TDP-43-bound RNAs in MNs based on the RNA immunoprecipitation sequencing (RIP- seq) data discussed herein. These genes shared a common signature: sensitive hMNs had six times more genes coding for RNA binding and splicing regulators than oMNs. Third, the inventors compared this list of defects with splicing defects observed in postmortem spinal cord data from patients and concentrated on genes common to both sets. Splicing defects can produce novel proteins and/or induce mRNA decay. Fourth, the inventors focused on genes where the mRNA is reduced in response to splicing changes as a proxy for reduced function.

[00378] This approach enabled the inventors to identify specific genes whose splicing is directly regulated by TDP-43 and whose mRNA levels are reduced in sensitive MNs in ALS patients. These genes participated in the same biological process and are direct targets of TDP- 43. Genes having a known function in maintaining neuronal health can be prioritized for further study. The gene therapy target validation genes are:

N0VA1 P51513-4, ELAVL1, ELAVL2, ELAVL3, ELAVL4, RBFOX2, MATR3, HNRNPA1, SFPQ, and FUS. ^{6 3}.

[00379] These genes can be important critical components of the “neurodegeneration cascade” that work with TDP-43 to maintain a healthy transcriptome.

[00380] Without wishing to be bound by theory, it is suggested that the re-expression of these splicing regulators can mitigate the effects of TDP-43 -induced splicing defects, improve the MN transcriptome, and enhance MN survival in response to ALS mutations (Figure 6B).

Research Strategy

[00381] MN Differentiation: Control, TARDBP, and C9orf72 mutant iPSCs are differentiated into MN using two protocols: a) the inventors’ previously successful hMN differentiation protocol by TF induction (NIL) 14; and/or b) an efficient small molecule guided MN differentiation protocol where cells transition through all natural progenitor stages 15.

[00382] Rescuing the Expression of Splicing Regulators: The hMN-expressed cDNA of the genes described above and P-Gal (control) is cloned into in a viral backbone with constitutively expressed GFP. These vectors are delivered individually and in combination on Day 10, young hMNs. These vectors are expressed in pools and the ones with the most significant effect are isolated.

[00383] Transcriptome Splice: The ability of the splicing regulators to rescue normal hMN splicing in response to TARDBP & C9orf72 mutations, and ALS-relevant proteotoxic stress is measured. [00384] Survival: The ability of the selected splicing regulators to improve the survival of control, TARDBP, and C9orf72 mutant MNs under normal conditions and in response to ALS- relevant proteotoxic stress is measured.

[00385] Correction of neuronal subtype-specific splicing defects represents a possible therapeutic approach for restoring a healthy transcriptome to ALS patients. TDP-43 is prone to aggregation, forming seeds to recruit more TDP-43 16. Even low levels of TDP-43 overexpression cause neurodegeneration in mice 17. Because simply supplying additional TDP- 43 expression is not a viable option, rescuing specific splicing defects downstream of TDP-43 is a possible therapeutic intervention.

[00386] As discussed herein, this approach includes restoring the splicing defects immediately downstream of TDP-43 in MNs. Without wishing to be bound by theory, it is suggested that rescuing defects in splicing factors affected by TDP-43 depletion can improve a significant fraction of the neuronal transcriptome affected by TDP-43 depletion. This can assist in understanding MN transcriptome regulation and identifying therapeutic targets for ALS. The inventors have identified and selected certain splicing regulators for study as discussed herein. Because the downregulation of these splicing regulators is the product of splicing defects, the relevant gene's expression can be restored by "traditional" cDNA expression and/or spliceswitching ASOs. TDP-43 aggregates in around 90% of ALS patients; thus, the proposed strategy applies to most ALS cases.

Biomarkers

[00387] There is a considerable effort in the field to define the set of peptides generated by cryptic exon inclusions regulated directly by TDP-43 as biomarkers. The inventors suggest that there is a much richer source of clinical biomarkers generated by splicing defects in response to the neurodegenerative splicing cascade. For example, these include peptide inclusions in response to N0VA1 and other splicing regulators in sensitive motor neurons. Thus, the inventors propose that understanding the critical splicing regulators in the “neurodegenerative splicing cascade” can increase the number of possible epitopes that can be evaluated as being ALS biomarkers, which can improve diagnostic accuracy, aid in early detection, and provide a more comprehensive disease profiling. References

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* * *

[00388] The present invention is not to be limited in scope by the specific embodiments described herein. Indeed, various modifications of the invention in addition to those described herein will become apparent to those skilled in the art from the foregoing description. Such modifications are intended to fall within the scope of the appended claims.

[00389] All patents, applications, publications, test methods, literature, and other materials cited herein are hereby incorporated by reference in their entirety as if physically present in this specification.

Claims

2. The method of claim 1, wherein the disease is a neurodegenerative disease.

3. The method of claim 2, wherein the neurodegenerative disease is Amyotrophic Lateral Sclerosis (ALS), Frontotemporal Lobar Degeneration (FTLD) Frontotemporal dementia (FTD), Limbic-predominant Age-Related TDP-43 Encephalopathy (LATE), Chronic Traumatic Encephalopathy (CTE), or Alzheimer’s Disease (AD).

4. The method of any one of claims 1-3, wherein providing said protein(s) or nucleic acid molecules(s) involves administering a gene therapy to the subject.

5. The method of any one of claims 1-4, wherein the nucleic acid molecule is contained within a viral vector which is administered to the subject.

6. The method of claim 5, wherein the viral vector is a lentiviral vector, an adenoviral vector, or an adeno-associated viral (AAV) vector.

7. The method of claim 6, wherein the viral vector is an adeno-associated viral (AAV) vector.

8. The method of claim 7, wherein the AAV vector has tropism for neural tissue or neurons.

9. The method of claim 7, wherein the adeno-associated viral vector is AAV9 or AAV-B 1.

10. The method of any one of claims 4-9, wherein the viral vector is administered to the subject intracranially, systemically, or intrathecally.

11. The method of claim 10, wherein the intracranial or intrathecal administration of the viral vector results in the appearance of the viral vector in the cerebrospinal fluid of the subject.

12. The method of any one of claims 4-11, wherein the nucleic acid molecule is operably linked to a promoter.

13. The method of claim 12, wherein the promoter is an inducible promoter or a constitutive promoter.

14. The method of claim 12 or claim 13, wherein the promoter is a tissue-specific promoter or a cell-specific promoter.

15. The method of claim 14, wherein the promoter is a neural tissue-specific promoter or a neuron-specific promoter.

16. The method of claim 15, wherein the promoter is a synapsin promoter.

17. The method of claim 12, wherein the promoter is a human ubiquitin C (hUBC) promoter; a chicken P-actin promoter, CMV enhancer, and rabbit P-globin splice acceptor (pCAG) promoter; a human cytomegalovirus (HCMV) promoter; a mouse phosphoglycerate kinase (mPGK) promoter; or a homeobox gene (Hb9) promoter.

18. The method of any one of claims 1-17, wherein the wild-type protein is NOVAI and/or ELAVL2.

19. The method of claim 18, wherein the NOVAI is NOVAI P51513-4.

20. The method of any one of claims 1-19, wherein the cells are neurons.

21. The method of claim 20, wherein the neurons are motor neurons.

22. The method of claim 21, wherein the motor neurons are hypoglossal motor neurons and/or oculomotor motor neurons.

23. The method of any one of claims 1-22, wherein the subject is a mammal.

24. The method of claim 23, wherein the mammal is a human.

25. A method of treating a disease characterized by TAR DNA-binding protein 43 (TDP-43) mislocalization or dysfunction in a subject in need thereof, the method comprising: providing to cells or tissues or organs of the subject having TDP-43 mislocalization or dysfunction a means for restoring misregulated splicing events to wild-type splicing events of one or more of NO VAI, ELAVL1, ELAVL2, ELAVL3, ELAVL4, RBFOX2, MATR3, HNRNPA1, SFPQ and FUS.

26. The method of claim 25, wherein restoring misregulated splicing events to wild-type splicing events comprises skipping of an abnormal exon and/or cryptic exons and/or favoring inclusion of a normal exon.

27. The method of claim 25 or claim 26, wherein the disease is a neurodegenerative disease.

28. The method of claim 27, wherein the neurodegenerative disease is Amyotrophic Lateral Sclerosis (ALS), Frontotemporal Lobar Degeneration (FTLD), Limbic-predominant Age-Related TDP-43 Encephalopathy (LATE), Chronic Traumatic Encephalopathy (CTE), Frontotemporal dementia (FTD), or Alzheimer's Disease (AD).

29. The method of any one of claims 25-28, wherein the means for restoring misregulated splicing events to wild-type splicing events:

30. The method of any one of claims 25-29, wherein the means for restoring misregulated splicing events to wild-type splicing events is administered intrathecally.

31. The method of any one of claims 25-30, wherein the means for restoring misregulated splicing events to wild-type splicing events comprises one or more splicing modifiers and/or antisense oligonucleotide(s) (ASO(s)).

32. The method of claim 31, wherein the one or more ASO(s) is(are) splice-switching ASO(s) and/or restore normal splicing.

33. The method of claim 31 or claim 32, wherein the one or more ASO(s) is(are) targeted to pre- mRNA of one or more of NOVAI, ELAVL1, ELAVL2, ELAVL3, ELAVL4, RBFOX2, MATR3, HNRNPA1, SFPQ and FUS.

34. The method of claim 33, wherein the one or more ASO(s) is(are) targeted to one or more exons of one or more pre-mRNA of NO VAI, ELAVL1, ELAVL2, ELAVL3, ELAVL4, RBFOX2, MATR3, HNRNPA1, SFPQ and FUS.

35. The method of claim 33 or claim 34, wherein the one or more ASO(s) is(are) targeted to pre- mRNA of NO VAI and/or ELAVL2.

36. The method of claim 33 or claim 34, wherein the one or more ASO(s) is(are) targeted to: a) exon 8 of NOVAI pre-mRNA; b) exon 6 of ELAVL1 pre-mRNA; c) exon 6 of ELAVL2 pre-mRNA; d) exon 10 and 11 ofELAVL4 pre-mRNA; e) exon 3 of RBFOX2 pre-mRNA; f) exon 15 ofMATR3 pre-mRNA; g) exon 6 of HNRNPA1 pre-mRNA; h) exon 9 of SFPQ pre-mRNA; i) exon 4 of ELAVL3 pre-mRNA; and/or j) exon 3 and exon 7 of FUS pre-mRNA.

37. The method of claim 33, wherein the one or more ASO(s) binds to a pre-mRNA sequence flanking a mis-spliced exon of one or more of N0VA1, ELAVL1, ELAVL2, ELAVL3, ELAVL4, RBF0X2, MATR3, HNRNPA1, SFPQ and FUS.

38. The method of any one of claims 29-35, wherein the one or more ASO(s) is(are) optimized by tiling relevant splice junctions of one or more pre-mRNA of N0VA1, ELAVL1, ELAVL2, ELAVL3, ELAVL4, RBFOX2, MATR3, HNRNPA1, SFPQ and FUS.

39. The method of any one of claims 25-38, wherein the N0VA1 is N0VA1 P51513-4.

40. The method of any one of claims 25-39, wherein the cells are neurons.

41. The method of claim 40, wherein the neurons are motor neurons.

42. The method of claim 41, wherein the motor neurons are hypoglossal motor neurons and/or oculomotor motor neurons.

43. The method of any one of claims 25-42, wherein the subject is a mammal.

44. The method of claim 43, wherein the mammal is a human.

45. The method of any one of claims 1-44, further comprising: identifying the subject as having a disease characterized by TAR DNA-binding protein

43 (TDP-43) mislocalization or dysfunction.

46. The method of claim 45, wherein the identifying comprises determining level(s) of

47. The method of claim 46, wherein the one or more clinical biomarkers comprise peptide inclusions comprising mis-spliced proteins.

48. The method of claim 47, wherein the mis-spliced proteins comprise one or more of NOVAI, ELAVL1, ELAVL2, ELAVL3, ELAVL4, RBFOX2, MATR3, HNRNPA1, SFPQ and FUS.

49. The method of claim 46, wherein the neopeptides and/or neoantigens comprise mis-spliced proteins.