Methods and compositions for treating neurologic disorders

Abstract

Methods and compositions described herein provide a solution for treating Huntington's disease and other neurodegenerative diseases by administering a recombinant polypeptide, such as a wild-type Huntingtin (HTT) fragment (e.g., SEQ ID NO:4), Brahma-related gene 1 (BRG1), Brahma (BRM), PNKP, Matrin 3 (MATRN3), or functional variants thereof, to a subject having, at risk of developing, or suspected of having a neurodegenerative disease characterized by persistent DNA double-strand breaks and impaired RNA processing. The polypeptides, delivered via mRNA, circular RNA, or AAV vectors, restore transcription-coupled non-homologous end-joining (TC-NHEJ) activity and RNA processing, ameliorating disease progression in neuronal and non-neuronal brain cells.

Description

METHODS AND COMPOSITIONS FOR TREATING NEUROLOGIC DISORDERSPRIORITY PARAGRAPH

[0001] This Application claims priority to US Provisional Patent Application serial number 63 / 680,946 filed August 8, 2024, which is incorporated herein by reference in its entirety.STATEMENT REGARDING FEDERALLY FUNDED RESEARCH

[0002] This invention was made with government support under NS130830 awarded by the National Institutes of Health. The government has certain rights in the invention.REFERENCE TO SEQUENCE LISTING

[0003] A sequence listing required by 37 CFR 1.821-1.825 is being submitted electronically with this application. The sequence listing is incorporated herein by reference. The sequence listing that is contained in the file named "UTMBP0420" which is 144 KB (as measured in Microsoft Windows®) and was created on 8 / 8 / 2025.BACKGROUND

[0004] Huntingtin is a protein encoded by the HTT gene in humans and is primarily expressed in the brain. It plays a crucial role in neuronal function, particularly in embryonic development, maintaining adult brain health, and regulating transcription-coupled DNA repair, transcription and RNA processing. The HTT gene contains a region where a trinucleotide repeat (CAG) can expand, leading to a pathological condition known as Huntington's disease (HD) when the repeat number is typically 36 or more CAG repeats. In HD, the expanded CAG repeats result in an abnormal and mutant form of the huntingtin protein (mHTT), which forms toxic aggregates within neurons, impairs DNA repair, transcription and RNA processing, and ultimately leads to neuronal dysfunction and neurodegeneration. Symptoms of Huntington's disease typically manifest in midadulthood and include involuntary movements (chorea), cognitive decline, and psychiatric symptoms.

[0005] Research on huntingtin protein and into Huntington's disease (HD) aims to understand the protein's normal function and how its altered (mutant) form contributes to neurodegeneration. This knowledge is crucial for developing treatments that can potentially modify or halt the progression of Huntington's disease.

[0006] There remains a need for additional treatments for neurodegenerative disorders such as Huntington’s disease and related neurological disorders, including Alzheimer's disease (AD), Parkinson's disease (PD), Frontotemporal dementia (FTD) and amyotrophic lateral sclerosis (ALS).SUMMARY

[0007] As a solution to the problem of Huntington’s disease and other neurodegenerative diseases, the inventors describe a treatment using the administration of a wild-type Huntingtin (HTT) protein, an N-terminal HTT fragment (e.g., SEQ ID NO:4), Brahma-related gene 1 (BRG1), Brahma (BRM), or Matrin 3 (MATRN3), or functional variants thereof, to a subject having, at risk of developing, or suspected of having a neurodegenerative disease characterized by the persistence of DNA double-strand breaks (DSBs) and impaired RNA processing.

[0008] The persistence of DNA double-strand breaks (DSBs) is an existential threat to postmitotic cells like neurons. Flawed DNA repair resulting in accumulation of DSBs is implicated as the underlying pathological mechanism of several neurodegenerative diseases, including Huntington’ s disease (HD). It was recently shown that huntingtin (HTT) assembles a transcription- coupled DNA repair complex responsible for repairing DNA lesions during transcription, while mutant HTT (mHTT) lacks this capacity resulting in accumulated DNA damage and HD. The Inventors report that HTT plays a much broader role in DNA repair, by organizing a transcription- coupled non-homologous end-joining (TC-NHEJ) complex with Brahma-related gene 1 (BRG1) ATPase subunit of SWI / SNF-like chromatin remodeler that resolves DSBs in transcribed regions to maintain genome integrity of the coding regions of genome. The presence of mutant HTT (mHTT) in TC-NHEJ complex compromises repair activity, resulting in DSB accumulation in HD. Functional studies show that restoring TC-NHEJ in the Drosophila HD model dramatically improves genome integrity, motor defects, and lifespan. These results indicate that unlike mHTT, the HTT-assembled TC-NHEJ complex maintains neuronal health by facilitating DSB repair.

[0009] Certain embodiments are directed to methods for treating a neurodegenerative disorder associated with defective DNA repair comprising administering a recombinant polypeptide or an expression vector encoding the same, for example one or more polypeptides (in the various combinations and permutations) having an amino acid identity of at least 90, 95, 98, or 100% to SEQ ID NO:2, 4, 6, 34, 36, 38, or functional variants thereof to a subject having, at risk ofdeveloping, or suspected of having a neurodegenerative disorder and / or a defect in DNA repair, e.g., TC-NHEJ facilitated repair.

[0010] Certain embodiments are directed to recombinant protein compositions comprising a recombinant polypeptide having an amino acid sequence that is at least 90, 95, 98, or 100% identical to 2, 4, 6, 34, 36, 38, or functional variants thereof, wherein said polypeptide is expressed in a host cell for the treatment of a neurodegenerative disorder. In certain aspects, the recombinant polypeptide or an expression vector encoding the recombinant polypeptide is formulated for delivery to cells of the central or peripheral nervous system.

[0011] Certain embodiments are directed to methods for treating a neurodegenerative disorder comprising administering to a subject in need thereof an effective amount of a recombinant protein composition described herein (e g., HTT or HTT fragment, PNKP polypeptide, BRG1, BRM, and / or MATRN3, or a functional variant thereof).

[0012] Other embodiments are directed to the use of a recombinant protein composition described herein for the manufacture of a medicament for the treatment of a neurodegenerative disorder associated with defective DNA repair and aberrant transcription.

[0013] Certain embodiments include pharmaceutical compositions comprising one or more recombinant protein(s) described herein or one or more expression vector encoding one or more recombinant protein(s) described herein, and a pharmaceutically acceptable carrier or excipient.

[0014] Other embodiments are directed to recombinant vector(s) comprising a nucleic acid sequence encoding one or more recombinant protein(s) described herein (e g., HTT or HTT fragment, PNKP polypeptide, BRG1, BRM, and / or MATRN3, or a functional variant thereof), wherein said vector is capable of expressing said recombinant protein in a host cell (e.g., neuron or other central or peripheral nervous system cells) or cell-free system.

[0015] Certain embodiments are directed to methods for ameliorating defective DNA repair comprising administering a recombinant HTT fragment having an amino acid identity of at least 90, 95, 98, or 100% to SEQ ID NO:4 or a functional variant thereof to a neuron and / or nonneuronal brain cell (e.g., glial cells or astrocytes) or other cell in the peripheral or central nervous system. The method can further comprise administering a polypeptide or fragment thereof having an amino acid identity of at least 90, 95, 98, or 100% to SEQ ID NO:2, 6, 34, 36, 38 or a functional variant thereof.

[0016] Certain embodiments are directed to methods of facilitating DNA repair comprising administering a recombinant HTT fragment (including functional variants) having an amino acid identity of at least 90, 95, 98, or 100% to SEQ ID NO:4 to a cell or subject in need thereof.

[0017] Definitions - For the purposes of this specification and the appended claims, the following terms shall have the meanings set forth below unless the context requires otherwise:

[0018] Functional Variant: A polypeptide or nucleic acid sequence that retains at least 80% of the biological activity (e.g., DNA repair activity, RNA processing activity, or transcription- coupled non-homologous end-joining (TC-NHEJ) function) of the reference sequence, such as SEQ ID NO:4 (N-terminal Huntingtin fragment), SEQ ID NO:2 (full-length Huntingtin), SEQ ID NO:34 (BRG1), SEQ ID NO:36 (BRM), or SEQ ID NO:38 (MATRN3). A functional variant may include amino acid or nucleotide substitutions, deletions, or insertions that do not substantially impair the protein’s ability to assemble or participate in the TC-NHEJ complex or regulate RNA processing, as assessed by assays such as western blotting, filter trap assay, or mitochondrial function analysis described herein.

[0019] Transcription-Coupled Non-Homologous End-Joining (TC-NHEJ): A multi -protein complex comprising wild-type Huntingtin (HTT), Brahma-related gene 1 (BRG1), Brahma (BRM), MATRIN 3 (MATRN3), and associated proteins (e.g., PNKP, TDP-43, FUS, hNRNPK) that coordinately and concomitantly facilitates the flawless repair of DNA double-strand breaks (DSBs) in transcriptionally active regions of the neuronal genome and regulates RNA processing and transcription. The TC-NHEJ complex maintains genome integrity and neuronal health, as impaired by mutant HTT (mHTT) in neurodegenerative disorders like Huntington’s disease.

[0020] Recombinant Polypeptide: A protein produced through recombinant DNA technology, including wild-type Huntingtin (HTT, SEQ ID NO:2), an N-terminal truncated HTT fragment (SEQ ID NO:4), Brahma-related gene 1 (BRG1, SEQ ID NO:34), Brahma (BRM, SEQ IDNO:36), MATRIN 3 (MATRN3, SEQ ID NO:38), or PNKP (SEQ ID NO:6), or their functional variants, expressed in a host cell (e.g., neuronal cell, glial cell, or cell-free system) for therapeutic administration.

[0021] Expression Vector: A nucleic acid construct designed to express a recombinant polypeptide in a target cell, including linear mRNA, circular RNA (cirRNA), self-amplifying RNA (saRNA), or adeno-associated viral (AAV) vectors. The mRNA or cirRNA vectors may include5'-UTR (SEQ ID NO: 125) and 3'-UTR (SEQ ID NO: 126) sequences to enhance stability and translation efficiency.

[0022] Neurodegenerative and Neurological Disorder: A condition characterized by progressive neuronal dysfunction, loss, or injury, including but not limited to dementia (e.g., Alzheimer’s disease, Frontotemporal dementia, Lewy body dementia, vascular dementia, Creutzfeldt-Jakob disease, progressive supranuclear palsy, corticobasal degeneration, normal pressure hydrocephalus, HIV-associated dementia), Huntington’s disease, Parkinson’s disease (PD), amyotrophic lateral sclerosis (ALS), Hereditary Spinocerebellar Ataxias (SCAs), multiple sclerosis (MS), stroke, and traumatic brain injury (TBI), wherein such conditions are associated with defective DNA repair, impaired RNA processing, or acute neurological damage. The specification provides support for negative limitations in the claim, as one or more neurodegenerative conditions can be explicitly excluded.

[0023] Mutant Huntingtin (mHTT): An abnormal form of the Huntingtin protein encoded by the HTT gene with expanded CAG repeats (typically 36 or more), resulting in the accumulation of toxic aggregates, impaired TC-NHEJ activity, and disrupted RNA processing in the brain cells, leading to neuronal dysfunction and finally to neurodegeneration in Huntington’s disease.

[0024] Lipid Nanoparticle (LNP): A delivery system comprising ionizable lipids (e.g., ALC- 0315, SM-102, MC3, C12-200), DSPC, cholesterol, and PEG-lipids (e.g., DMG-PEG2000, C14- PEG2000) formulated to encapsulate mRNA, cirRNA, or saRNA for delivery to CNS cells, with optional peptide ligands (e.g., transferrin or ApoE-binding motifs) to enhance neuronal uptake.

[0025] RNA Processing: The cellular processes involving the modification, splicing, or transport of RNA molecules, regulated by the TC-NHEJ complex through interactions with RNA- binding proteins such as hnRNP K, TDP-43 and FUS, which are disrupted in neurodegenerative disorders like Huntington’s disease.

[0026] Central Nervous System (CNS) Delivery: The administration of therapeutic agents (e.g., recombinant polypeptides, expression vectors) to cells of the brain or spinal cord via routes such as intrathecal (IT), intracerebroventricular (ICV), or intranasal (IN) injection, designed to cross biological barriers and target neurons or non-neuronal brain cells.

[0027] “Substantially similar” with respect to nucleic acid sequences or amino acid sequences, means at least about 65% identity between two or more sequences. Preferably, the term refers to at least about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, to 99%.Such identity can be determined using algorithms known in the art, such as the mBLAST algorithm.

[0028] The terms “treating” or “treatment” refer to any success or indicia of success in the attenuation or amelioration of an injury, pathology or condition, including any objective or subjective parameter such as abatement, remission, diminishing of symptoms or making the injury, pathology, or condition more tolerable to the patient, slowing in the rate of degeneration or decline, making the final point of degeneration less debilitating, improving a subject's physical or mental well-being, or prolonging the length of survival. The treatment or amelioration of symptoms can be based on objective or subjective parameters, including the results of a physical examination, neurological examination, and / or psychiatric evaluations.

[0029] “Effective amount” and “therapeutically effective amount” are used interchangeably herein and refer to an amount of a therapeutic polypeptide or functional fragment thereof, as described herein, effective to achieve a particular biological or therapeutic result such as, but not limited to, the biological or therapeutic results disclosed herein.

[0030] Other objects, features and advantages of the present invention will become apparent from the following detailed description. It should be understood, however, that the detailed description and the specific examples, while indicating specific embodiments of the invention, are given by way of illustration only, since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description. Each embodiment described herein is understood to be embodiments of the invention that are applicable to all aspects of the invention. It is contemplated that any embodiment discussed herein can be implemented with respect to any method or composition of the invention, and vice versa.DESCRIPTION OF THE DRAWINGS

[0031] The following drawings form part of the present specification and are included to further demonstrate certain aspects of the present invention. The invention may be better understood by reference to one or more of these drawings in combination with the detailed description of the specification embodiments presented herein.

[0032] FIG. 1A-1P: Characterization of the HTT-Assembled TC-NHEJ Complex. (A) Western blotting of HTT immunocomplexes (ICs) from nuclear extracts (NEs) of 3-month-oldwild-type C 57BL / 6 mouse brains, immunoprecipitated (IP’d) with anti-HTT antibody (MAB2170, Millipore- Sigma). ICs show BRG1, DNA-PKcs, Ku70, PNKP, XRCC4, LIG IV, POLR2A, CSB, and TFIIS. Lane 1: Molecular weight marker; Lane 2: Input; Lane 3: IgG control; Lane 4: HTT IP. (B) Western blotting of mHTT ICs from NEs of 3-month-old zQ175 HD transgenic mouse cortex, IP’d with anti-HTT antibody, showing similar NHEJ factors as in (A). Lane 1: Marker; Lane 2: Input; Lane 3: IgG control; Lane 4: mHTT IP. (C-H) Proximity ligation assay (PLA) in SH-SY5Y cells assessing HTT interactions with Ku70 (C-D), XRCC4 (E-F), and PNKP (G-H) before and after etoposide (ET) treatment. Red fluorescence indicates interactions (arrows in panels 3-4). Nuclei stained with DAPI. Relative PLA signals (D, F, H) show increased interactions post-ET (mean± SD, ****p<0.001). (I) Chromatin immunoprecipitation (ChIP) in SH-SY5Y cells showing HTT-genome interactions under stress-free conditions, quantified by qPCR (mean ± SEM, ****p<0.001). (I) Neutral comet assay in SH-SY5Y cells pre- and post-bleomycin (BL) treatment, showing increased DSBs (comet tails, arrows) post-BL. (K) ChIP in SH-SY5Y cells pre- and post-BL treatment, showing 2- to 3-fold increased HTT-genome interaction post-DSB induction (mean ± SEM, ****p<0.001). (L-P) ChIP -re-ChIP in wild-type C57BL / 6 mouse cortex, sequentially IP’d with anti-HTT and anti-Ku70 (L), anti-XRCC4 (N), anti -LIG IV (O), or anti- PNKP (P) antibodies. Quantitative real time PCR (qRT-PCR) of Neurodi and Tubb3 genomic loci confirms co-occupancy (mean ± SEM, ****p<0.001). (M) Reciprocal ChlP-re-ChIP with anti- Ku70 antibody followed by anti-HTT antibody, further confirming co-occupancy.

[0033] FIG. 2A-2E: HTT-Mediated DSB Repair. (A) Western blotting of NEs from control, HTT-KD (HHT -knocked down), and HTT-OE (HTT-overexpressing) SH-SY5Y cells, detecting HTT, Ku70, Ku80, PNKP, XRCC4, and -actin (loading control). Lane 1 : Marker; Lane 2: Control; Lane 3: HTT-KD; Lane 4: HTT-OE. (B) Immunostaining of HTT-KD and control SH- SY5Y cells with anti-yI I2AX antibody (9718, Cell Signaling), showing DSBs (red puncta, arrows) in HTT-KD cells. Nuclei stained with DAPI. (C-D) LA-QPCR of genomic DNA from control, HTT-KD, and HTT-OE SH-SY5Y cells, amplifying NEURODI, NEUROG1, BDNF, BCL2L2, and ENO2y. HTT-KD shows reduced amplification; HTT-OE shows increased amplification (mean ± SD, ****p<0.001). Lane 10: 1-kb ladder. (E) Chromosome aberration analysis in control and HTT-KD SH-SY5Y cells post-2 Gray (2 Gy) irradiation, showing increased aberrations in HTT-KD cells (mean ± SD, ****p<0.001).

[0034] FIG. 3A-3D: HTT Facilitates recruitments of various NHEJ Factors (A) Ligation- mediated PCR post-CRISPR-CAS9 gRNA delivery to chromosome 17 (Chrl7A, Chrl7B) in SH- SY5Y cells, measuring uncut DNA. (B) ChIP post-CRISPR-CAS9 DSB induction at Chrl7A, showing reduced NHEJ factor (Ku70, Ku80, 53BP1, PNKP) recruitment in HTT-KD cells (mean ± SD, **p<0.05, ***p<0.01, ****p<0.001). (C-D) LA-QPCR of genomic DNA from control, HTT-KD, and HTT-OE SH-SY5Y cells post-BL treatment (300 ng / mL, 30 min) and 10-min recovery, showing reduced DSBs in HTT-OE cells (mean ± SD, ***p<0.01, ****p<0.001).

[0035] FIG. 4A-4G: mHTT Impairs TC-NHEJ in Transcriptionally Active Regions. (A) ChIP showing HTT recruitment to transcriptionally active (Chr-1 A, Chr-17A) vs. inactive (Chr-IB, Chr- 17B) loci pre- and post-DSB induction in SH-SY5Y cells (mean ± SD, ****p<0.001). (B-C) ChIP of POLR2A at DSB sites (Chrl7A, Chr-IA) in control and HTT-KD cells, showing reduced recruitment in HTT-KD cells (mean ± SD, ****p<0.001). (D-E) Immunostaining of wild-type and zQ175 mouse cortex (12 weeks) with anti-53BPl antibody (2675, Cell Signaling), showing DSBs (arrows) in zQ175 brains. (F-G) LA-QPCR of transcriptionally active genes (Grinl, Grial, Rab3a, Tgoln2, Napg) in zQ175 vs. control mouse cortex, showing reduced amplification in zQ175 (mean ± SD, ****p<0.001).

[0036] FIG. 5A-5I: wtHTT and PNKP Enhance DNA Repair in HD Models. (A-B) LA-QPCR of genomic DNA from STHdhQ7, STHdhQl l l, and STHdhQl 1 l+wtHTT-Q19 cells, showing reduced DNA damage in wtHTT-Q19 cells (mean ± SD, ****p<0.001). (C) MTT assay showing improved cell viability in STHdhQl 1 l+wtHTT-Q19 cells (mean ± SD, ****p<0.001). (D-E) LA- QPCR of STHdhQ7, STHdhQl 11, and STHdhQl 11+PNKP cells, showing reduced DNA damage with PNKP (mean ± SD, ****p<0.001). (F-I) LA-QPCR of -tubulin locus in wild-type, UAS- 128QhttFL, and UAS-128QhttFL+wtHTT-Q16 or +PNKP Drosophila, showing reduced DNA damage with wtHTT or PNKP (mean ± SD, ****p<0.001).

[0037] FIG. 6A-6G: DNA Repair Improves HD Phenotypes in Drosophila model of HD. (A- D) Climbing assays at 10, 15, 20, and 25 days post-eclosion in wild-type, UAS-128QhttFL, and UAS-128QhttFL+wtHTT-Q16 or +PNKP Drosophila, showing improved motor function (mean ± SD, *p<0.05, **p<0.01, ***p<0.001, ****p<0.001). (E) Lifespan analysis showing extended longevity in UAS-128QhttFL+wtHTT-Q16 or+PNKP flies (mean± SD, **p<0.01, ****p<0.001). (F-G) RNAseq analysis of gene expression in UAS-128QhttFL vs. wild-type, and UAS-128QhttFL+wtHTT-Q16 or +PNKP, showing normalized expression of neurodegeneration- related genes (color-coded variance, ****p<0.0001, ***p<0.001).

[0038] FIG. 7A-7F: DSB Persistence in HD. (A-B) Immunostaining of HD (Q50, Q53) and control (Q20, Q18) primary neurons with anti-53BPl antibody, showing DSBs in HD (arrows). (C-D) Confocal imaging of HD patient and control brain sections (basal ganglia) with anti-53BPl antibody, showing DSBs in HD (arrows). (E-F) LA-QPCR of NEURODI, BDNF, and ENOLASE in HD patient and control caudate, showing reduced amplification in HD (mean ± SD, ****p<0.001).

[0039] FIG. 8A-8C: Transcriptional Normalization in HD Drosophila (A-B) RNAseq analysis of UAS-128QhttFL vs. wild-type, and UAS-128QhttFL+wtHTT-Q16 or +PNKP, showing normalized expression of neurodegeneration-related genes (color-coded variance). (C) Diagram of wtHTT and BRG1 assembling TC-NHEJ complex for DSB repair, and mHTT’s disruption leading to persistent DSBs and neurotoxicity.

[0040] FIG. 9A-9M: Restoring mtDNA Repair in Drosophila model of HD. (A-B) PNKP 3'- phosphatase activity in ME from STHdhQ7 (lane 3), STHdhQl l l (lane 4), and STHdhQl l l+wtHTT (lane 2), with substrate (blue arrowhead) and phosphate release (red arrowhead). Lane 1 : No extract. (B) Relative activity (mean ± SD, ****p<0.001). (C-D) LA- QPCR of DNA from STHdhQ7 (lanes 1-3), STHdhQl l l (lanes 4-6), and STHdhQl l l+wtHTT (lanes 7-9), amplifying 10.6 kb and 0.2 kb mtDNA (arrowheads). Lane 10: 1-kb ladder. (D) Relative amplification (mean ± SD, ****p<0.001). (E) JC-10 mitochondrial membrane potential in STHdhQ7, STHdhQl l l, and STHdhQl l l+wtHTT cells (mean ± SD, ****p<0.001). (F-G) LA- QPCR of Drosophila mtDNA from wild-type (lanes 1-3), UAS-128QhttFL (lanes 4-6), and UAS- 128QhttFL+wtHTT (lanes 7-9), amplifying 14.2 kb and 0.15 kb segments (arrowheads). Lane 10: 1-kb ladder. (G) Relative amplification (mean ± SD, ****p<0.001, n=25). (H) Western blotting of proteins from wild-type (lane 2), PNKP-OE1 (lane 3), and PNKP-OE2 (lane 4) Drosophila heads, detecting PNKP; Lane 1 : Marker (kDa). (I- J) LA-QPCR of Drosophila mtDNA from wild-type (lanes 1-3), UAS-128QhttFL (lanes 4-6), and UAS-128QhttFL+PNKP (lanes 7-9), amplifying 14.2 kb and 0.15 kb segments (arrowheads). Lane 10: 1-kb ladder. (J) Relative amplification (mean ± SD, ****p<0.001, n=25). (K-L) Climbing assays in wild-type, UAS-128QhttFL, and UAS- 128QhttFL+wtHTT (K) or +PNKP (L) Drosophila, showing improved motor function (*p<0.001,#p<0.01). (M) Diagram of mHTT impairing mtDNA repair complex (HTT, POLRMT, TFAM, POLGA, CSB, TFB1M / TFB2M, PNKP), leading to mtDNA damage and neurotoxicity.

[0041] FIG. 10A-10E: TDP-43 in TC-NHEJ Complex. (A) Volcano plot of TDP-43- interacting proteins in SH-SY5Y nuclear fraction, IP’d with anti-TDP-43 or IgG antibodies, analyzed by mass spectrometry. Red points indicate significant interactions with DNA repair, chromatin remodeling, RNA processing, and transcription proteins. (B) STRING network of TDP- 43-interacting proteins, showing interactions with DNA repair and transcription proteins. (C-E) Western blotting of HTT (C), TDP-43 (D), and BRG1 (E) ICs from benzonase-treated NE of 3- month-old C57BL / 6 mouse brain (n=6), IP’d with anti-HTT (MAB2170, Millipore-Sigma), anti- TDP-43 (Cl 5410266, Diagenode), or anti-BRGl (NB 100-2594, Novus) antibodies, detecting BRG1, TDP-43, ATXN2, POLR2A, FUS, and TC-NHEJ factors (DNA-PKcs, PNKP, LIG IV, CSB). Lane 1 : Marker; Lane 2: Input; Lane 3: IgG IP; Lane 4: Specific IP.

[0042] FIG. 11 A-l IE: TDP-43 Co-Occupancy in TC-NHEJ Complex. (A-E) ChlP-re-ChIP in wild-type C57BL / 6 mouse brain (n=6 for A-B, n=3 for D-E), sequentially IP’d with anti-HTT (MAB2170, Millipore-Sigma) and anti-TDP-43 (C15410266, Diagenode) (A), anti-TDP-43 and anti-HTT (B), anti-TDP-43 and anti-PNKP (MBP-1-A7257, Novus) (C), anti-HTT and anti- ATXN2 (SC-515602, Santa Cruz) (D), or anti-TDP-43 and anti-ATXN2 (E). qPCR of Neurodi, Neurogl, Neurod2 loci confirms co-occupancy (mean ± SEM, *p<0.001).

[0043] FIG. 12A-12B : TDP-43 Interactions with TC-NHEJ Components. (A) Western blotting of input from SH-SY5Y cells co-transfected with p-Myc-TDP -43 and p-FLAG-HTT-Q19, -BRG1, -BRM, -ATXN3-Q23, -PNKP, -FUS, -TBP-Q13, or -ATXN2-Q27, detecting Myc-TDP-43 and FLAG-tagged proteins. Lane 1 : Marker (kDa). (B) Western blotting of Myc-TDP-43 ICs from above cells, IP’d with anti-Myc antibody (SC-40, Santa Cruz), detecting FLAG-tagged proteins.

[0044] FIG. 13A-13G: HTT Interactions with TC-NHEJ Components. (A-B) Western blotting of input (A) and Myc-HTT-Q19 ICs (B) from SH-SY5Y cells co-transfected with p-Myc-HTT- Q19 and p-FLAG-BRGl, -BRM, -ATXN3, -TDP-43, -FUS, -PNKP, -TBP-Q13, -POLR2A, or - ATXN2-Q27, detecting Myc-HTT and FLAG-tagged proteins. Lane 1 : Marker (kDa). (C-D) Western blotting of input (C) and Myc-TDP-43 ICs (D) from SH-SY5Y cells co-transfected with p-Myc-TDP-43 and p-FLAG-NT-HTT-Q 19, -Q24, -Q40, -Q46, -Q79, -QI 09, detecting Myc- TDP-43 and FLAG-NT-HTT. Lane 1 : Marker (kDa). (E) Diagram of TDP-43 domains: NTD (1- 106), RRM1 / RRM2 (106-262), CTD (274-414), and combinations. (F-G) Western blotting ofinput (F) and FLAG-HTT-Q19 ICs (G) from SH-SY5Y cells co-transfected with p-FLAG-HTT- Q19 and p-Myc-TDP-43, -NTD, -RRMs, -CTD, -(NTD+RRMs), or -(RRMs+CTD), detecting FLAG-HTT and Myc-tagged TDP-43 domains (arrows).

[0045] FIG. 14A-14E: ATXN2 Interactions with TC-NHEJ Components. (A-B) Western blotting of input (A) and Myc-ATXN2-Q27 ICs (B) from SH-SY5Y cells co-transfected with p- Myc-ATXN2-Q27 and p-FLAG-HTT-Q19, -BRG1, -BRM, -ATXN3-Q23, -PNKP, -TDP-43, - FUS, or -TBP-Q13, detecting Myc-ATXN2 and FLAG-tagged proteins. Lane 1 : Marker (kDa). (C-D) Western blotting of input (C) and Myc-ATXN2 ICs (D) from SH-SY5Y cells co-transfected with p-FLAG-TDP-43 and p-Myc-ATXN2-Q23, -NT-ATXN2-Q23, or -CT-ATXN2, detecting FLAG- TDP-43 and Myc-tagged ATXN2. Lane 1: Marker (kDa). (E-F) Western blotting of input (E) and FLAG-TDP-43 ICs (F) from SH-SY5Y cells co-transfected with p-FLAG-TDP-43 and p- Myc-ATXN2-Q23, -Q27, -Q33, or -Q52, detecting FLAG-TDP-43 and Myc-ATXN2. Lane 1 : Marker (kDa).

[0046] FIG. 15: ATXN2 Depletion Reduces NHEJ. NHEJ efficacy in control and ATXN2-KD SH-SY5Y cells, showing reduced NHEJ frequency at gene-rich (Chr-1 A) and gene-poor (Chr-IB) sites (mean ± SD, **p<0.05, ****p<0.0001).

[0047] FIG. 16A-16G: Regulation of TDP-43 Recruitment. (A-G) ChIP in control, BRG1-KD, ATXN2-KD, or HTT-KD SH-SY5Y cells post-CRISPR-CAS9 (Chrl7A) or I-Scel (ChrlA) DSB induction, assessing TDP-43 (A, C, G), HTT (B, D), or BRG1 (E, F) recruitment. Reduced recruitment in KD cells (mean ± SD, **p<0.005, ***p<0.001, ****p<0.0001, NS).

[0048] FIG. 17A-17B: ATXN2 Depletion Increases DNA Damage. (A-B) LA-QPCR of DNA from control (lanes 1-3) and ATXN2-KD (lanes 4-6) SH-SY5Y cells, amplifying 8-10 kb segments of transcriptionally active (BCL2L2, NEURODI, BDNF) and inactive (MYH4, MYH6) genes (arrowheads). Lane 7: 1-kb ladder. (B) Relative damage, higher in active regions (mean ± SD, ***p<0.001, ****p<0.0001).

[0049] FIG. 18A-18C: ATXN2 Depletion Downregulates NHEJ Proteins. (A-B) Western blotting of proteins from control and ATXN2-KD SH-SY5Y cells, detecting HTT, Ku70, Ku80, PNKP, XRCC4, DNA-PKcs, ATM, PARP1, TDP-43; P-actin as control. (B) Relative levels, showing reduced DNA-PKcs and ATM (mean ± SD, ***p<0.001, ****p<0.0001, NS). (C) qRT- PCR of NHEJ protein mRNA in control and ATXN2-KD cells (mean ± SD, NS).

[0050] FIG. 19A-19C: TDP-43 Depletion Downregulates NHEJ Proteins (A-B) Western blotting of proteins from control and TDP-43-KD SH-SY5Y cells, detecting ATXN2, DNA-PKcs, ATM, HTT, Ku70, Ku80, PNKP; [Lactin as control. (B) Relative levels, showing reduced DNA- PKcs and ATM (mean ± SD, ***p<0.001, ****p<0.0001, NS). (C) qRT-PCR of NHEJ protein mRNA in control and TDP-43-KD cells (mean ± SD, NS).

[0051] FIG. 20A-20C: Mutant Tdp-43 Downregulates NHEJ Proteins. (A-B) Western blotting of proteins from control and Tdp-432FL mouse cortex, detecting HTT, DNA-PKcs, ATM, Ku70, Ku80, PNKP, XRCC4, LIG IV; (3-actin as control. (B) Relative levels, showing reduced DNA- PKcs, ATM, HTT (mean ± SD, ***p<0.001, ****p<0.0001, NS). (C) qRT-PCR of NHEJ protein mRNA in control and Tdp-432FL cortex (mean ± SD, NS).

[0052] FIG. 21A-21C: Tdp-432FL Induces DNA Damage. (A-B) LA-QPCR of DNA from Tdp-432FL (lanes 4-6) and control (lanes 1-3) mouse cortex, amplifying 6-8 kb transcriptionally active genes (Bdnf, Bcl212, Arc, GluRl, GluNl, Rab3a, Napg). Lane 7: 1-kb ladder. (B) Relative amplification, reduced in active genes (mean ± SD, ****p<0.0001). (C) LA-QPCR of transcriptionally inactive genes (Myodl, Myogl, Myh2) in Tdp-432FL and control cortex, showing minimal damage (mean ± SD, NS).

[0053] FIG. 22A-22D: PolyQ Expansion and TDP-43 Localization. (A-D) Immunostaining of zQ175 (A) and control (B) mouse brain, and HD patient (C) and control (D) brain sections with anti-TDP-43 antibody, analyzed by confocal microscopy. Cytosolic TDP-43 in zQ175 and HD (arrows); nuclear TDP-43 in controls (arrows). Nuclei stained with DAPI.

[0054] FIG. 23: Mechanism of TDP-43 in TC-NHEJ Diagram of TDP-43 as a regulatory component of the HTT-assembled TC-NHEJ complex, interacting with HTT, BRG1, ATXN2, PNKP, POLR2A, Ku70, Ku80, DNA-PKcs, XRCC4, LIG IV, Artemis, CSA, CSB to repair DSBs. Nuclear exclusion of TDP-43 increases PNKP SUMOylation, reduces DSB repair, and leads to DNA damage, ATM / DNA-PK / p53 activation, and neurotoxicity in ALS / FTLD.DESCRIPTION

[0055] The following discussion is directed to various embodiments of the invention. The term “invention” is not intended to refer to any particular embodiment or otherwise limit the scope of the disclosure. Although one or more of these embodiments may be preferred, the embodiments disclosed should not be interpreted, or otherwise used, as limiting the scope of the disclosure,including the claims. In addition, one skilled in the art will understand that the following description has broad application, and the discussion of any embodiment is meant only to be an example of that embodiment and not intended to imply that the scope of the disclosure, including the claims, is limited to that embodiment.

[0056] Neurons are metabolically highly active and thus generate excessive reactive oxygen species (ROS) as byproducts of mitochondrial oxidation. These damaging free radicals (ROS) that escape cellular antioxidant defense systems induce oxidative DNA damage in both the nuclear and mitochondrial genomes. In healthy neurons, this oxidative DNA damage is rapidly repaired by a plethora of DNA repair systems to strictly maintain genomic integrity and ensure the health and normal function of neurons. A repertoire of DNA repair systems plays a pivotal role in neurons to maintain genome integrity and prevent premature neuronal loss, which would be catastrophic due to the postmitotic nature of most neurons, which largely survive for the entire lifespan of the organism. Therefore, a defective or impaired DNA repair system can lead to persistent damage, and accumulation of these lesions can impede neuronal gene expression and concurrently activate pro-degenerative DNA damage-response (DDR) p53 signaling, driving neuronal dysfunction and premature loss in many neurodegenerative diseases.

[0057] Among several different DNA lesions that occur in the neuronal genome, DNA doublestrand breaks (DSBs) pose an existential threat to neurons. Impaired DNA repair mechanism(s) resulting in cumulative accumulation of DSBs in the neuronal genome are implicated in eliciting neurotoxicity in many neurodegenerative diseases. However, how DNA repair mechanism(s) become dysfunctional, and specifically whether dysfunctional DNA repair mechanisms cause early neurotoxicity in these slowly progressing neurological diseases, remains unknown. We have discovered that the huntingtin protein (HTT) acts as a structural scaffold to assemble a multifactorial transcription-coupled non-homologous end-joining (TC-NHEJ) DNA repair complex with RNA polymerase, transcription factors and various DNA repair proteins, and that this novel macromolecular complex repairs DNA lesions in the neuronal genome during transcription to maintain genome integrity. Conversely, mutant HTT impairs the activity of the TC-NHEJ complex, resulting in defective DNA repair, ultimately leading to DNA lesion accumulation, neurotoxicity, and neurodegeneration in HD.

[0058] HTT assembles a transcription-coupled DNA repair complex that repairs DNA lesions during transcription, while mutant HTT (mHTT) impairs its activity, resulting in damageaccumulation in HD (PMID: 30994454). In a major advance, we have further discovered that HTT plays a much broader role in DNA repair and genome maintenance by organizing the transcription- coupled non-homologous end-joining (TC-NHEJ) complex with Brahma-related gene 1 (BRG1), an ATPase subunit of the SWI / SNF-like chromatin remodeler. This complex repairs double-strand breaks (DSBs) in the transcriptionally active neuronal genome to maintain genome integrity, ensuring that the encoded proteins remain functional and unmutated. We find that the presence of mutant HTT in the TC-NHEJ complex significantly compromises its activity, resulting in DNA lesion accumulation, and transcriptional dysregulation and early neurotoxicity in HD. We have demonstrated that expression of exogenous wildtype HTT or the DNA repair enzyme PNKP in a Drosophila model of HD dramatically improves the genome integrity, rescues motor defects, and extends the lifespan of the mutant flies. Moreover, restoring genome integrity reverses the transcriptional landscape in the Drosophila model of HD, indicating that the HTT-assembled TC- NHEJ complex maintains neuronal health by facilitating DSB repair, which is compromised by mutant HTT carrying extended polyglutamine tracts.

[0059] In another major advance, we have discovered that TDP-43 and FUS, proteins mechanistically linked to neurodegenerative diseases such as ALS, FTLD, and AD, are integral regulatory components of the HTT-assembled TC-NHEJ repair complex in neurons. We find that TDP-43 and FUS positively regulate the activity of the TC-NHEJ complex in the nervous system. Additionally, we report that ATXN2, whose glutamine expansion causes spinocerebellar ataxia type 2, is also a key component of the neuronal TC-NHEJ complex. We have discovered that TDP- 43 and / or FUS directly interact with the N-terminal regions of HTT and ATXN2 that carry polyglutamine sequences. These protein interactions are critical for the nuclear retention and function of TDP-43 and for maintaining TC-NHEJ activity, efficacy of DNA repair, and genome integrity.

[0060] We find that longer polyglutamine lengths in HTT and / or ATXN2 facilitate the expulsion of TDP-43 from the TC-NHEJ complex and the nucleus, resulting in the inactivation of the TC-NHEJ complex. This leads to DNA repair deficiency and persistent accumulation of DNA lesions at early stages of ALS, FTLD, and AD. The persistence and cumulative accumulation of DNA lesions in the neuronal genome at early stages cause neurotoxicity and premature neuronal death. Our discovery explains the genetic elements that facilitate nuclear expulsion of TDP-43 and / or FUS and why their nuclear depletion adversely impacts DNA repair, resulting in DNAdamage accumulation, predominantly within the transcriptionally active genome. This leads to chronic activation of pro-apoptotic p53 signaling in the FTLD / ALS nervous system, which could give rise to overlapping phenotypes in these insidious diseases.

[0061] Mass spectrometry and co-immunoprecipitation (co-IP) analyses of nuclear extracts from wildtype C57BL / 6 mouse brain cortex identified Ku70, Ku80, PNKP, DNA-PKcs, MATRN3, BRG1, TDP-43, ATXN2, FUS, and Cockayne Syndrome protein B (CSB) in HTT immunocomplexes, confirming their roles in the TC-NHEJ complex. Reverse co-IP experiments using anti-TDP-43 and anti-BRGl antibodies further validated the presence of HTT, MATRN3, ATXN2, FUS, POLR2A, and NHEJ factors, with the absence of APE1 ensuring specificity. Sequential chromatin immunoprecipitation (ChlP-re-ChIP) demonstrated co-occupancy of HTT, TDP-43, ATXN2, and PNKP on transcriptionally active genomic regions (e.g., Neurodi, Neurogl, Tubb3) in mouse brain. Additionally, co-IP studies in SH-SY5Y cells showed that TDP-43 interacts with HTT’s N-terminal domain and RNA-recognition motifs (RRM1 + RRM2), while ATXN2 interacts with TDP-43 via its N-terminal polyQ region, with interactions persisting across normal and pathogenic polyQ lengths (e.g., Q23, Q27, Q33, and Q52).

[0062] ATXN2 plays a critical role in TC-NHEJ-mediated DNA repair, as demonstrated by experiments showing that its depletion in SH-SY5Y cells reduces NHEJ activity by approximately 70%, with minimal impact on homologous recombination (20% reduction), indicating specificity for TC-NHEJ. ChIP analyses revealed that ATXN2 regulates the recruitment of TDP-43, BRG1, and HTT to double-strand break (DSB) sites in transcriptionally active genomic regions, with significantly impaired recruitment in ATXN2-knockdown (KD) cells. ATXN2 depletion also reduced DNA-PKcs and ATM protein levels by over 80% without affecting their mRNA levels, suggesting a translational regulatory role. Long-amplicon quantitative PCR (LA-QPCR) analysis showed reduced amplification efficiency of transcriptionally active genes (e.g., BCL2L2, NEURODI, BDNF) in ATXN2-KD cells compared to controls, indicating increased DNA damage, while transcriptionally inactive regions (e.g., MYH4, MYH6) showed minimal damage. These findings underscore ATXN2’s importance in maintaining genome integrity through TC- NHEJ, making it a potential therapeutic target for neurodegenerative diseases.

[0063] The loss of TDP-43 nuclear function significantly impairs TC-NHEJ, contributing to DNA damage accumulation in neurodegenerative diseases. In SH-SY5Y cells, TDP-43 depletion via shRNA reduced DNA-PKcs, ATM, and HTT protein levels without altering their mRNAlevels, indicating a translational block. In the TDP-432FL mouse model, where TDP-43 nuclear function is abrogated by phenylalanine-to-leucine mutations at positions 147 and 149, steady-state levels of DNA-PKcs, ATM, and PARP1 were severely reduced, while PNKP, Ku70, Ku80, XRCC4, and LIG IV levels remained unchanged. LA-QPCR analysis of TDP-432FL mouse cortex revealed significantly lower amplification efficiencies for transcriptionally active genes (e.g., Bdnf, Arc, GluRl) compared to controls, indicating higher DNA damage, whereas transcriptionally inactive genes (e.g., Myodl, Myogl) showed marginal damage. Immunostaining of brain sections from zQ175 HD mice and HD patients confirmed TDP-43 nuclear exclusion and cytoplasmic accumulation, correlating with impaired TC-NHEJ and increased DSBs in gene-rich regions, as detected by anti-yH2AX antibodies. These data highlight TDP-43 ’s critical role in TC- NHEJ and its dysregulation in HD pathology.

[0064] We found that increasing expression of wildtype HTT in a Drosophila model of HD dramatically improves TC-NHEJ activity, restores genome integrity, enhances motor function, and increases longevity of mutant flies. Importantly, we also find that ectopic expression of either wildtype HTT or PNKP facilitates nuclear inclusion of TDP-43. These data suggest that increasing PNKP levels or the N-terminal fragment of wildtype HTT in HD, using delivery methods such as AAV-mediated delivery, can be developed as a powerful therapeutic strategy to halt or delay neurodegeneration and neuronal dysfunction in HD and related diseases.

[0065] Our data suggest that the polyglutamine lengths in HTT and / or ATXN2 regulate the nucleocytoplasmic distribution of TDP-43 and FUS; longer polyglutamine lengths facilitate nuclear exclusion of TDP-43 / FUS, triggering neurotoxicity. The lengths of CAG repeats encoding polyglutamine in HTT and / or ATXN2 may serve as a genetic signature predisposing individuals to disease development.I. Huntington’s disease and Similar Neurodegenerative Disorders

[0066] Huntington’s disease (HD), a dominantly inherited fatal neurodegenerative disease, is caused by the expansion of a CAG trinucleotide repeat tract, encoding a polyglutamine (polyQ) stretch, within the N-terminal region of HTT. Over the past two decades, strong evidence has shown that mutant HTT (mHTT) carrying an extended polyQ sequence acquires a toxic gain of function, and numerous studies suggest that mHTT disrupts mitochondrial function, contributing to HD pathology. For instance, the association of mHTT with the outer mitochondrial membranehas been proposed to impair protein transport across the mitochondrial membrane, and its interaction with dynamin-related protein- 1 (DRP1) has been proposed to cause abnormal mitochondrial quality control. Furthermore, mitochondrial DNA (mtDNA) damage / lesions are commonly observed in HD and are implicated in perturbing energy homeostasis, thus causing mitochondrial dysfunction. These data point to a significant contribution of mitochondrial dysfunction to the progression of HD.

[0067] Consistent with the postulated role of wildtype HTT in facilitating the assembly of multiprotein complexes, we recently demonstrated that wildtype HTT assembles a macromolecular transcription-coupled DNA repair (TCR) complex with RNA polymerase II subunit A (POLR2A), ataxin-3, and essential DNA repair enzymes, including DNA ligase 3 (Lig3) and polynucleotide kinase 3 '-phosphatase (PNKP), in the nucleus. Using various mouse and cellbased models, we found that this novel HTT-assembled multiprotein complex helps maintain genome integrity and neuronal function by resolving DNA damage / lesions that occur during routine transcription and neuronal activities. Additionally, the presence of mHTT in the nuclear TCR complex impairs its DNA repair activity, resulting in DNA damage accumulation in HD and various HD models. While this complex is critical for stimulating DNA repair and maintaining nuclear genome integrity, it is unknown whether a similar repair complex may also be present in mitochondria to resolve mtDNA damage.

[0068] The mitochondrial genome is relentlessly exposed to reactive oxygen species (ROS) due to its proximity to the site of ROS generation - the electron transport chain (ETC) complex. Notably, actively transcribed mtDNA lacks nucleosomes, making the mitochondrial genome vulnerable to ROS-induced damage / lesions. For these reasons, a range of DNA repair mechanisms have evolved to resolve mtDNA lesions and maintain mtDNA integrity. Base excision repair (BER) is the primary mechanism for repairing oxidized mtDNA bases, but there is some evidence supporting nucleotide excision repair (NER) within mitochondria. Knockdown of wildtype HTT in murine embryonic stem cells was found to disrupt the structural and functional integrity of mitochondria, emphasizing its importance in maintaining mitochondrial function. Furthermore, previous studies have implicated a role for mHTT in mitochondrial degeneration and dysfunction. While these data suggest that wildtype HTT may play a critical role in maintaining mitochondrial function, the precise mechanisms remain poorly understood.A. Huntingtin and Related Polypeptides

[0069] The function of the huntingtin protein (HTT) is enigmatic in that the native protein provides neuroprotection, while mutant HTT (mHTT), carrying an expanded polyglutamine stretch, triggers neurotoxicity in Huntington’s disease (HD). HTT assembles a DNA repair complex in mitochondria with mitochondrial RNA polymerase, mitochondrial transcription factors, and PNKP, which resolves DNA lesions to preserve mitochondrial genome integrity and function. Pathogenic mHTT reduces the activity of this mitochondrial DNA repair complex, resulting in persistent DNA lesions and impaired mitochondrial function in HD. Restoring the activity of this repair complex in a Drosophila model of HD dramatically improves mitochondrial genome integrity and motor deficits.

[0070] Huntingtin (HTT) is a large 3, 114-amino-acid protein that functions as a powerful and versatile neuroprotective factor in vitro and in vivo. The neuroprotective roles of HTT were demonstrated by findings that native wildtype HTT (wtHTT) protects cells against pro-death stimuli, whereas its depletion increases their vulnerability to the same stimuli. Consistent with these observations, transgenic expression of wildtype HTT in mouse brain confers neuroprotection against excitotoxic and ischemic injury, while its brain-specific inactivation at postnatal stages triggers neurodegeneration and motor deficits. Although the mechanism by which wildtype HTT confers neuroprotection is unclear, recent studies indicate that its role in protecting mitochondrial function may be relevant.

[0071] One example of a human full-length HTT nucleotide sequence of SEQ ID NO: 1 (GenBank accession NM 002111). SEQ ID NO: 1 encodes the human full-length HTT protein sequence having the amino acid sequence of SEQ ID NO:2.

[0072] In certain embodiments a recombinant HTT polypeptide having a modified HTT sequence of the N-terminal fragment of human HTT is encoded, for example, by SEQ ID NO:3. SEQ ID NO:3 encoding the N-terminal fragment of HTT having the amino acids of SEQ ID NO:4.

[0073] One example of a nucleic acid sequence encoding human PNKP (NM_007254) has the nucleic acid sequence of SEQ ID NO:5. SEQ ID NO:5 encoding a human PNKP protein (NP 009185) having the amino acid sequence of SEQ ID NO:6.

[0074] One example of a nucleic acid encoding Brahma-related gene 1 (BRG1) has the nucleic acid sequence of SEQ ID NO:33. SEQ ID NO:33 encoding a BRG1 protein having an amino acid sequence of SEQ ID NO:34.

[0075] One example of a nucleic acid encoding Brahma (BRM) has the nucleic acid sequence of SEQ ID NO:35. SEQ ID NO:35 encoding a BRM protein having an amino acid sequence of SEQ ID NO:36.

[0076] One example of a nucleic acid encoding Matrin 3 (MATRN3) has the nucleic acid sequence of SEQ ID NO:37. SEQ ID NO:37 encoding a BRG1 protein having an amino acid sequence of SEQ ID NO: 38.

[0077] In certain aspects a polynucleotide can include a variant of SEQ ID NO: 1, 3, 5, 33, 35, or 37, having 1, 5, 10, 15, 20, or more nucleotide substitutions. The polynucleotide can comprise a continuous polynucleotide segment that is at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100 percent identical to SEQ ID NO: 1, 3, 5, 33, 35, or 37. In certain aspects a polypeptide of the invention can be a variant of SEQ ID NO:2, 4, 6, 34, 36, or 38 having 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more amino acid substitutions, or amino acid truncations. A polypeptide can comprise a continuous polypeptide segment that is at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% identical to SEQ ID NO: 2, 4, 6, 34, 36, or 38.B. Formulation and Administration

[0078] In the field of neuroscience and neuropharmacology, the direct and targeted delivery of peptides or nucleic acids encoding peptides to neurons in vivo presents a challenge due to the blood-brain barrier (BBB), and cellular targeting is often required. Peptides hold immense therapeutic potential for treating neurological disorders and conditions, offering precise modulation of neuronal activity and signaling pathways. Achieving effective delivery to neurons in vivo can harness the full therapeutic benefits of peptides.

[0079] The present invention discloses methods and compositions for the targeted administration of peptides to neurons in vivo, overcoming the limitations posed by the BBB and ensuring selective uptake by neuronal cells. The methods encompass novel peptide formulations, delivery systems, and administration protocols designed to facilitate efficient penetration of peptides or expression vectors encoding the peptides across the BBB and their subsequent binding and internalization into neurons.

[0080] In terms of functional equivalents, it is well understood by those skilled in the art that a “biologically functional equivalent” protein or polynucleotide has a limit to the number of changes that may be made within a defined portion of the molecule while retaining an acceptablelevel of equivalent biological activity. Biologically functional equivalents or variants are thus defined herein as proteins (or polynucleotides) in which selected amino acids (or nucleotides) may be substituted. In certain aspects, a polypeptide (e.g., HTT or PNKP) is 80, 85, 90, 92, 94, 96, 98, or 100% identical to the wildtype form of the polypeptide. In certain aspects, polypeptide(s) that are 80, 85, 90, 92, 94, 96, 98, or 100% identical to SEQ ID NO:2, 4, 6, 34, 36, or 38 are used, or nucleic acids encoding the same. A combination / permutation of any 2, 3, 4, 5, or 6 of SEQ ID NO:2, 4, 6, 34, 36, or 38, or variants thereof can be used in various embodiments.

[0081] In general, the shorter the length of the molecule, the fewer changes can be made while retaining function. Longer domains may tolerate an intermediate number of changes. The full- length protein will have the greatest tolerance for a larger number of changes. However, it must be appreciated that certain molecules or domains highly dependent on their structure may tolerate little or no modification. The function of a polypeptide can be determined using various assays known to detect the activity of the polypeptide of interest.

[0082] Some amino acid substitutions are generally based on the relative similarity of the amino acid side-chain substituents, for example, their hydrophobicity, hydrophilicity, charge, size, and / or the like. An analysis of the size, shape, and / or type of the amino acid side-chain substituents reveals that arginine, lysine, and histidine are all positively charged residues; that alanine, glycine, and serine are all of similar size; and that phenylalanine, tryptophan, and tyrosine all have a generally similar shape. Therefore, based on these considerations, arginine, lysine, and histidine; alanine, glycine, and serine; and phenylalanine, tryptophan, and tyrosine are defined herein as biologically functional equivalents.

[0083] To effect more quantitative changes, the hydropathic index of amino acids may be considered. Each amino acid has been assigned a hydropathic index based on its hydrophobicity and charge characteristics: isoleucine (+4.5); valine (+4.2); leucine (+3.8); phenylalanine (+2.8); cysteine / cystine (+2.5); methionine (+1.9); alanine (+1.8); glycine (-0.4); threonine (_0.7); serine (-0.8); tryptophan (-0.9); tyrosine (-1.3); proline (-1.6); histidine (-3.2); glutamate (-3.5); glutamine (_3.5); aspartate (-3.5); asparagine (_3.5); lysine (-3.9); and arginine (_4.5).

[0084] The importance of the hydropathic amino acid index in conferring interactive biological function on a protein is generally understood in the art (Kyte & Doolittle, 1982, incorporated herein by reference). It is known that certain amino acids may be substituted for others with a similar hydropathic index or score while retaining similar biological activity. In making changes based onthe hydropathic index, the substitution of amino acids whose hydropathic indices are within ±2 is preferred, those within ±1 are particularly preferred, and those within ±0.5 are even more particularly preferred.

[0085] It is also understood in the art that the substitution of like amino acids can be made effectively based on hydrophilicity. As detailed in U.S. Patent 4,554,101, the following hydrophilicity values have been assigned to amino acid residues: arginine (+3.0); lysine (+3.0); aspartate (+3.0+1); glutamate (+3.0+1); serine (+0.3); asparagine (+0.2); glutamine (+0.2); glycine (0); threonine (-0.4); proline (-0.5+1); alanine (_0.5); histidine (_0.5); cysteine (_1.0); methionine (-1.3); valine (-1.5); leucine (-1.8); isoleucine (-1.8); tyrosine (-2.3); phenylalanine (-2.5); tryptophan (-3.4). In making changes based on similar hydrophilicity values, the substitution of amino acids whose hydrophilicity values are within ±2 is preferred, those within ±1 are particularly preferred, and those within ±0.5 are even more particularly preferred.1. Peptide Formulations

[0086] The invention includes various peptide / polypeptide formulations optimized for stability, solubility, and neuronal uptake. These formulations may incorporate modifications such as liposomal encapsulation, conjugation with BBB -penetrating moieties, or chemical modifications to enhance peptide / polypeptide stability and bioavailability in neuronal environments. In certain aspects a delivered peptide can have the amino acid sequence of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6 (or a variant thereof), SEQ ID NO:34 (or a variant thereof), SEQ ID NO:36 (or a variant thereof), SEQ ID NO:38 (or a variant thereof) or a combination of 2 or more of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6 (or variants thereof), SEQ ID NO:34 (or a variant thereof), SEQ ID NO:36 (or a variant thereof), or SEQ ID NO:38 (or a variant thereof).2. Delivery Systems:

[0087] Delivery systems can be designed to facilitate the passage of peptide(s) or nucleic acid(s) encoding the peptide(s) across the BBB and their subsequent targeting to neurons. Examples include nanocarriers, lipid-based vesicles, polymeric nanoparticles, and viral vectors engineered to deliver peptides specifically to neuronal cells.3. Administration Protocols:

[0088] The invention can use specific protocols for administering peptide or expression vector formulations to achieve optimal bioavailability and therapeutic efficacy. These protocols mayinvolve intravenous, intranasal, intrathecal, or direct injection routes, with adjustments made for dose, frequency, and duration of administration based on the therapeutic application and patient requirements. To enhance CNS delivery, additional methods were explored beyond IT and ICV injections. Intravenous administration of LNPs decorated with brain-specific ligands, such as transferrin receptor-binding peptides or ApoE mimetics, was tested to facilitate blood-brain barrier (BBB) penetration. These LNPs, formulated as described herein, achieved a 3 -fold increase in striatal uptake in wild-type mice compared to non-targeted LNPs, as measured by ex vivo IVIS imaging at 24 hours post-injection. Additionally, focused ultrasound with microbubbles was investigated to transiently disrupt the BBB, enabling systemic delivery of mRNA-LNPs to the cortex and hippocampus. Biodistribution studies showed detectable HTT expression (SEQ ID NO:4) in neurons 48 hours post-treatment, supporting the feasibility of these methods for clinical translation.4. Targeting Mechanisms:

[0089] Mechanisms for specific targeting of peptides to distinct neuronal populations are disclosed, utilizing peptide sequences or ligands that recognize and bind to receptors or transporters uniquely expressed on neuronal cell surfaces. Such targeting ensures selective delivery of therapeutic peptides to desired regions of the brain or spinal cord.5. Therapeutic Applications:

[0090] Therapeutic applications of peptide or expression vector administration to neurons are provided for the treatment of neurodegenerative diseases.

[0091] The invention provides for the targeted administration of peptides or expression vectors to neurons in vivo. The disclosed methods, formulations, and delivery systems represent an effective approach to modulating neuronal function for therapeutic purposes.II. Nucleic Acid Compositions

[0092] The term “nucleic acid vector” refers to a carrier nucleic acid molecule into which a nucleic acid sequence can be inserted for introduction into a cell where it can be replicated, transcribed, and / or translated (i.e., expressed). A nucleic acid sequence can be “exogenous,” meaning it is foreign to the cell into which the vector is introduced, or “endogenous” to the cell but located in a position where it is not ordinarily found. In certain aspects, an exogenous vector can encode an endogenous nucleic acid. Nucleic acid vectors include plasmids, viral genomes, andother expression vectors (e.g., bacteriophage, animal viruses, and plant viruses). Given the current disclosure, one skilled in the art would be well equipped to construct a vector through standard recombinant techniques.

[0093] The term “expression vector” refers to any genetic construct comprising a nucleic acid coding for an RNA that is subsequently transcribed. In some cases, RNA molecules are translated into a protein, polypeptide, or peptide. Expression vectors can contain “control sequences” that govern transcription and / or translation of an operably linked coding sequence in a particular host cell. In addition to control sequences for transcription and translation, vectors and expression vectors may contain nucleic acid sequences serving other functions. These sequences may be modified, given that multiple codons can encode the same amino acid, while still encoding the same or a similar protein or polypeptide. Codon optimization can also be undertaken based on the expression host organism.III. Delivery Vectors

[0094] In certain aspects, components are delivered to the central nervous system, brain, or other organs or tissues using nucleic acids that encode or express such components. Viral and non- viral delivery vectors can be used in the methods described herein (e.g., Syner-III). The terms “nucleic acids,” “nucleic acid molecules,” “nucleic acid sequences,” “nucleotide sequences,” and “nucleotide molecules” are used interchangeably herein and, unless otherwise specified, refer to a polymer of deoxyribonucleic acids, including cDNA, DNA, or RNA.A. Viral delivery

[0095] The ability of certain viruses to infect cells or enter cells via receptor-mediated endocytosis and express virally encoded genes makes them attractive candidates for transferring foreign nucleic acids into cells (e.g., mammalian cells). Viruses may thus be utilized to encode and express peptides or polypeptides. Non-limiting examples of viral vectors that may be used to deliver nucleic acids include adenoviral vectors, AAV vectors, retroviral vectors, and the like.B. Lipid-Mediated Transfection.

[0096] In a further embodiment, a nucleic acid may be entrapped in a lipid particle, such as a liposome. Liposomes are vesicular structures characterized by a phospholipid bilayer membrane and an inner aqueous medium. Multilamellar liposomes have multiple lipid layers separated byaqueous medium. They form spontaneously when phospholipids are suspended in an excess of aqueous solution (Ghosh and Bachhawat, In: Liver Diseases, Targeted Diagnosis and Therapy Using Specific Receptors and Ligands, Wu et al. (Eds.), Marcel Dekker, NY, 87-104, 1991).C. Receptor Mediated Transfection

[0097] A nucleic acid may be delivered to a target cell via receptor-mediated delivery vehicles. These take advantage of the selective uptake of macromolecules by receptor-mediated endocytosis occurring in a target cell. Given the cell type-specific distribution of various receptors, this delivery method adds another degree of specificity to the present invention.IV. Examples

[0098] The following examples as well as the figures are included to demonstrate preferred embodiments of the invention. It should be appreciated by those of skill in the art that the techniques disclosed in the examples or figures represent techniques discovered by the inventors to function well in the practice of the invention and thus can be considered to constitute preferred modes for its practice. However, those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments which are disclosed and still obtain a like or similar result without departing from the spirit and scope of the invention.EXAMP E 1 - HUNTTNGTIN, IN ASSOCIATION WITH BRAHMA-RELATED GENE- 1 ASSEMBLES A TRANSCRIPTION-COUPLED NON-HOMOLOGOUS END-JOINING COMPLEX IN NEURON.

[0099] The inventors have discovered and characterized a novel transcription-coupled nonhom ologous end-joining (TC-NHEJ) complex essential for neuronal DNA repair, organized by HTT) in association with the chromatin remodeling protein BRG1. Co-immunoprecipitation (coIP) of HTT from nuclear extracts followed by mass spectrometry and western blot analysis revealed the presence of BRG1 and core NHEJ factors, including Ku70, Ku80, DNA-PKcs, XRCC4, and LIG IV (FIG. 1 A). While mHTT also forms a structurally similar complex (FIG. IB), its function is impaired. The physiological relevance of these interactions was confirmed in situ using proximity ligation assays (PLAs), which showed a significant increase in association between HTT and NHEJ factors following DNA damage (FIG. 1C-1H). Furthermore, sequential chromatin immunoprecipitation (ChIP -re-ChIP) assays demonstrated that HTT and key NHEJcomponents like Ku70 and PNKP co-occupy the same genomic loci, confirming the assembly of a functional repair complex on DNA (FIG. 1L-1P).

[0100] The function of this HTT-assembled TC-NHEJ complex is critical for maintaining neuronal genomic integrity. Modulating HTT levels directly impacts DNA repair capacity. RNAi- mediated knockdown of HTT (HTT-KD) resulted in a significant accumulation of spontaneous DNA double-strand breaks (DSBs), evidenced by increased yH2AX foci, higher rates of chromosome aberration, and reduced amplification efficiency in long-amplicon QPCR (LA- QPCR) assays (FIG. 2). Conversely, overexpression of wild-type HTT (HTT-OE) reduced baseline DNA damage and conferred resistance to genotoxic stress by accelerating the repair of induced DSBs (FIG. 3C, 3D). These results establish that HTT is a key factor that facilitates DSB repair in neurons.

[0101] Mechanistically, HTT appears to function as a scaffolding protein that orchestrates the recruitment of the TC-NHEJ repair machinery to sites of DNA damage. This activity is targeted predominantly to transcriptionally active, gene-rich regions of the genome (FIG. 4A). In HTT- deficient cells, the recruitment of essential NHEJ factors to CRISPR-induced DSBs was markedly diminished (FIG. 3B). The pathology of Huntington’s disease arises from the functional impairment of this process by mHTT, which leads to the persistent accumulation of DSBs specifically within these critical, actively transcribed genomic regions. This accumulation of unrepaired damage was confirmed in both zQ175 HD mouse models and post-mortem human HD brain tissue (FIG. 4D-4G, FIG. 7).

[0102] Crucially, the DNA repair deficiency and its pathological consequences in HD models are reversible. Providing a functional component of the TC-NHEJ complex rescued the disease phenotype in both cellular and animal models. In a striatal cell line expressing mHTT, transgenic expression of either wtHTT or the downstream repair enzyme PNKP dramatically reduced genomic DNA damage and improved cell viability (FIG. 5A-5E). This therapeutic effect was validated in vivo using a Drosophila model of HD, where expression of wtHTT or PNKP mitigated DNA damage, reversed motor deficits, and significantly extended lifespan (FIG. 5F-5I, FIG. 6). Restoring this repair pathway also normalized the transcriptional dysregulation associated with the disease (FIG. 8A, 8B), confirming that the HTT-assembled TC-NHEJ complex is a potent therapeutic target for Huntington's and related neurodegenerative diseases.EXAMPLE 2 HUNTINGTIN AINTAINS MITOCHONDRIAL GENOME INTEGRIT Y AND FUNCTION

[0103] In addition to its role in nuclear DNA repair, the inventors have determined that wtHTT is a key scaffolding protein for a novel transcription-coupled mtDNA repair complex. This complex was shown to contain essential components of the mitochondrial transcription and repair machinery, including mitochondrial RNA polymerase (POLRMT), DNA polymerase gamma (POLGA), mitochondrial transcription factor TFAM, and the DNA repair enzyme PNKP. Coimmunoprecipitation and proximity ligation assays confirmed that these proteins form a complex within the mitochondria of neuronal cells, and chromatin immunoprecipitation assays demonstrated that this complex is recruited to mtDNA, particularly in response to oxidative damage. This establishes a new, critical function for wtHTT in maintaining mitochondrial genome integrity.

[0104] This mitochondrial repair function is pathologically impaired in HD. In various models, from immortalized striatal cells to patient-derived iPSC neurons and transgenic mice, the presence of mutant HTT (mHTT) was found to significantly reduce the enzymatic activity of PNKP within the complex. This functional impairment leads to a persistent accumulation of mtDNA damage, which correlates with compromised mitochondrial health and the selective vulnerability of neuronal populations in HD.

[0105] Crucially, these deficits are reversible and represent a viable therapeutic target. As demonstrated in a Drosophila model of HD (FIG. 9), correcting the repair deficiency by introducing either exogenous wtHTT or PNKP not only restored mtDNA integrity but also led to a significant rescue of the progressive motor defects characteristic of the disease. These findings provide a direct link between the molecular mechanism of mtDNA repair, mitochondrial health, and neuronal function, strongly supporting the conclusion that restoring this pathway is a promising therapeutic strategy for HD.EXAMPLE 3 MUTANT ATXN2 FACILITATES TDP-43 NUCLEAR EXPULSION, IMPEDES TRANSLATION OF DNA-PKCS AND ATM TO IMPAIR DNA REPAIR.A. Methods

[0106] Construction ofplasmidDNA Plasmids pBacMam2-DiEx-LIC-C-flag_huntingtin_full- length_Q19 (Addgene plasmid # 111741; URL n2t.net / addgene: 111741; RRID: Addgene l 11741), pBacMam2-DiEx-LIC-C-flag_huntingtin_full-length_Q24 (Addgene plasmid # 111742; URL n2t.net / addgene Addgene _111742), pBacMam2-DiEx-LIC-C-flag_huntingtin_full_length_Q30, (Addgene plasmid # 111744; URL n2t.net / addgene: 111744; RRID: Addgene_l 11744), pBacMam2-DiEx-LIC-C-flag_huntingtin_full_length_Q36 (Addgene plasmid # 111745; URL n2t.net / addgene: 111745; RRID: Addgene 111745), pBacMam2-DiEx- LIC-C-flag_huntingtin_full_length_Q42 (Addgene plasmid # 111746; URL n2t.net / addgene: 111746; RRID: Addgene l 11746), pBacMam2-DiEx-LIC-C-flag_huntingtin_full_length_Q48 (Addgene plasmid # 111747; URL n2t.net / addgene: 111747; RRID: Addgene_l 11747), pBacMam2-DiEx-LIC-C-Flag_huntingtin_full-length_Q79 (Addgene plasmid # 111729; URL n2t.net / addgene: 111729; RRID: Addgene_l 11729), pBacMam2-DiEx-LIC-C- flag_huntingtin_full-length_Q109, (Addgene plasmid # 111730; URL n2t.net / addgene: 111730; RRID: Addgene l 11730), pBacMam2-DiEx-LIC-C-C-flag_huntingtin_full-length_Q145(Addgene plasmid # 111731; URL n2t.net / addgene: 111731; RRID: Addgene_l 11731 were kind gifts from Cheryl Arrowsmith. The full-length huntingtin (HTT) cDNA was PCR-amplified from these plasmids using appropriate primers, and the PCR products were sub-cloned into plasmid pcDNA3.1 (Invitrogen, USA) using appropriate linkers to construct plasmids p-FLAG-HTT-Q19, P-FLAG-HTT-Q24, p-FLAG-HTT-Q30, p-FLAG-HTT-Q36, p-FLAG-HTT-Q42, p-FLAG-HTT- Q48, p-FLAG-HTT-Q79, p-FLAG-HTT-Q 109 and p-FLAG-HTT-Q145, expressing full-length FLAG-tagged HTT-Q19, HTT-Q24, HTT-Q30, HTT-Q36, HTT-Q42, HTT-Q48, HTT-Q79, HTT-Q109 and HTT-Q145 respectively. The N-terminal truncated fragments (Exon 1) of HTT carrying various lengths of polyQ sequences (Q19, Q24, Q30, Q36, Q42, Q48, Q79, Q109 or QI 45) were PCR amplified using appropriate primers from the parent plasmids and the PCR products were sub-cloned into plasmid p-CMV-(DYKDDDDK)-N (Takara, USA) to construct recombinant plasmid p-FLAG-NT-HTT-Q19, p-FLAG-NT-HTT-Q24, p-FLAG-NT-HTT-Q30, P-FLAG-NT-HTT-Q36, p-FLAG-NT-HTT-Q42, p-FLAG-NT-HTT-Q48, p-FLAG-NT-HTT- Q79, p-FLAG-NT-HTT-Q109 and p-FLAG-NT-HTT-Q145 respectively. Plasmid expressing DNA Ligase IV (LIG IV), Artemis, and ATXN2 cDNA were purchased from Origene, USA. The cDNA sequences of LIG IV, Artemis, or ATXN2 were PCR-amplified from the parent plasmids using appropriate primers, and the PCR products were sub-cloned into plasmid p-CMV- (DYKDDDDK)-N (Takara, USA) to construct plasmids p-FLAG-LIG IV, p-FL AG- Artemis, and p-FLAG-ATXN2-Q27, expressing FLAG-tagged ligase IV, Artemis, or ATXN2 respectively. Plasmid pWZL-Neo-Myr Flag PNKP was a gift from Willium Hahn and Jean Zhao (Addgene plasmid # 20594; http: / / n2t.nct / addgene:20594; RRID: Addgene_20594). PNKP cDNA sequenceswere PCR amplified from this plasmid using appropriate primers and the purified PCR fragment was subcloned into plasmid pCMV-Myc-N or pCMV-(DYKDDDDK)-N (Takara, USA) to construct p-FLAG-PNKP or p-Myc-PNKP.

[0107] Plasmids pEGFP-Cl-FLAG-Ku70 (Addgene plasmid # 46957; URL n2t.net / addgene: 46957; RRID: addgene_46957), pEGFP-Cl-FLAG-Ku80 (Addgene plasmid # 46958; URL n2t.net / addgene: 46958; RRID: Addgen e_46958) and pEGFP-Cl-FLAG-XRCC4 (Addgene plasmid # 46959; URL n2t.net / addgene:46959; RRID: Addgene_46959) were gift from Steve Jackson. Plasmid pCMV-PARPl-3xFlag-WT was a gift from Thomas Muir (Addgene plasmid # 111575; URL n2t.net / addgene: 111575; RRID: addgene 11575). Plasmid pcDNA3.1(+)-Flag- His-ATM was a gift from Michael Kastan (Addgene plasmid # 31985; URL n2t.net / addgene: 31985; RRID: addgene_31985). The plasmid hu-DNA-PKcs was a gift from Katheryn Meek (Addgene plasmid # 83317). The full-length cDNA sequences of Ku70, Ku80, DNA-PKcs XRCC4, and PARP1 were PCR amplified with appropriate primers and the PCR fragments were sub-cloned into plasmid pCMV-(DYKDDDDK)-N (Takara, USA) to construct recombinant plasmids expressing FLAG-tagged proteins. The sequence integrity of the clones was verified by sequencing, and expression of the proteins / protein fragments were checked using western blotting with respective antibodies. Plasmid encoding human TDP-43 and FUS were purchased from Origene, USA and the full-length TDP-43 or FUS cDNA were PCR-amplified using appropriate primers, and the PCR products were subcloned into either plasmid pCMV-Myc-N or pCMV- (DYKDDDDK)-N (Takara, USA) to construct p-Myc-TDP-43, p-Myc-FUS, p-FLAG-TDP-43 and p-FLAG-FUS, expressing Myc-tagged TDP-43 and FUS, and FLAG-tagged TDP-43 and FUS respectively. Various functional domains of TDP-43 were PCR-amplified using appropriate primers and the PCR products were sub-cloned into pCMV-Myc-N (Takara, USA) to construct plasmids expressing various functional domains of TDP-43 as Myc-tagged peptides. The plasmid pCMV5-BRGl-Flag was a gift from Joan Massague (Addgene plasmid # 19143; URL n2t.net / addgene: 19143; PRID: Addgene_19143). The full-length human BRG1 cDNA sequences from the plasmid pCMV5-BRGl-Flag were PCR-amplified with appropriate primers, and the PCR product was sub-cloned into either plasmid pCMV-Myc-N or pCMV-(DYKDDDDK)-N plasmids (Takara, USA) to construct plasmid pMyc-BRGl or p-FLAG-BRGl, expressing Myc-tagged and FLAG-tagged BRG1 respectively. Various domains of BRG1 were PCR amplified with appropriate primers and the PCR products were subcloned into pCMV-Myc-N (Takara, USA) toconstruct plasmids expressing various functional domains of BRG1 as Myc-tagged peptides. All the recombinant plasmid DNA were isolated and purified using Qiagen Plasmid Maxi Kit (Cat # 12163; Qiagen).

[0108] Cell culture, and Transfection. Human neuroblastoma SH-SY5Y cells were purchased from ATCC (Cat # CRL-2266) and cultured in DMEM containing 15% FBS, 1% penicillin / streptomycin in CO2 incubator at 37 oC. Purified plasmid DNA encoding the human micro-RNA-adapted shRNA sequences targeting TDP-43, ATXN2, HTT, or BRG1 (Horizon Discovery, USA) were transfected into SH-SY5Y cells, and the transfected cells were selected for puromycin resistance. The knockdown efficiencies in the shRNA-expressing SH-SY5Y cells were assessed by western blotting using appropriate antibodies. Plasmids expressing the full-length FLAG-tagged wildtype and mutant HTT cDNA (p-FLAG-wtHTT-Q19, p-FLAG-wtHTT-Q24, p- FLAG-mHTT-Q79, p-FLAG-mHTT-Q109 and p-FLAG-mHTT-Q145) were linearized and transfected into SH-SY5Y cells with Lipofectamine 2000 reagent (Invitrogen, USA), and the positive transfected cells were selected for G418 resistance, and the stable cells expressing the wtHTT or mHTT were cultured in DMEM containing 15% FBS in CO2 incubator, and transgene expression was assessed by western blot using an anti-FLAG or anti-HTT antibodies.

[0109] Cells were transfected with various plasmid DNA constructs using Lipofectamine 2000 reagent (Invitrogen, USA). Human embryonic kidney 293 (HEK293) cells were purchased from Coriell Institute, USA (Cat # CRL-1573) and cultured in EMEM, containing 10% FBS, 1% penicillin-streptomycin. HEK293 cells were co-transfected with plasmids p-Myc-TDP-43 and p- FLAG-Ku70, p-FLAG-Ku80, p-FLAG-DNA-PKcs, p-FLAG-XRCC4, p-FLAG-Ligase IV, p- FLAG-PNKP, p-FLAG-PARPl or p-FL AG- Artemis using Lipofectamine 2000 (Invitrogen, USA). Similarly, HEK293 cells were co-transfected with plasmids p-Myc-TDP-43 and p-FLAG- HTT, p-FLAG-ATXN3-Q23, p-FLAG-ATXN2-Q27, p-FLAG-PNKP, p-FLAG-BRGl, p-FLAG- TBP-Q13, p-FLAG-POLR2A, or p-FLAG-FUS using Lipofectamine 2000 (Invitrogen, USA). Plasmids expressing Myc-tagged TDP-43 (p-Myc-TDP-43) and expressing the N-terminal truncated fragments (Exon 1) of HTT (p-FLAG-NT-HTT-[CAG]n carrying various lengths of polyQ sequences (Q15, Q19, Q30, Q36, Q42, Q48, Q79, Q109 or Q145) were transiently transfected into HEK293 cells and cells were cultured in MEM medium (Invitrogen, USA). All the cell lines used in this study were authenticated by short tandem repeat analysis in the UTMB Molecular Genomics Core. We routinely tested mycoplasma contaminations in all our cell linesusing GeM Mycoplasma Detection Kit (SIGMA, Cat # MP0025) and cells were found to be free from mycoplasma contamination.

[0110] Preparation of nuclear and cytosolic protein extracts for western blotting analysis. Cell pellets or mouse brain tissues were homogenized, and total protein was isolated using a total protein extraction kit (Millipore, USA). The cytosolic and nuclear fractions were isolated from cells / tissue extract using a NE-PER protein extraction kit (Cat# 78833; Fisher Thermo Scientific, USA). Briefly, 5 x 106 cells were ground with a glass homogenizer in an ice bath for 20 to 22 strokes. Cytoplasmic and nuclear fractions were separated by differential centrifugation (600 x g, 10 min, 4°C and 10,000 x g, 10 min, 4°C). The supernatant (cytosolic fraction) and pellet (nuclear fraction) were collected. The nuclear and cytosolic pellets were lysed to yield the final cytosolic and nuclear lysates, and the extracted proteins were analyzed by western blotting as described previously.

[0111] Antibodies for the western blot analysis. Western blots were performed according to the standard procedure, and each experiment was performed a minimum of three times to ensure reproducible and statistically significant results. The rabbit and mouse antibodies for TDP-43 were from Diagenode (Cat # Cl 5410266) and Novus Biologicals (Cat # H00023435-M01) respectively. The antibodies for APE1 (Cat # 4128), Ku80 (Cat # 2753), 53BP1 (Cat # 4937), phospho-53BPl (Cat # 2675), p-ATM (Cat # 4526), rabbit polyclonal Ab for BRG1 (Cat # 3508), mouse monoclonal Ab for BRG1 (Cat # 52251), rabbit monoclonal Ab for ATM (Cat # 2873), and rabbit monoclonal Ab for DNA ligase IV (Cat # 14649) were from Cell Signaling Technology (USA); anti-p-ATM-S1981 rabbit monoclonal (Cat # ab81292) and anti-p-DNA-PKcs (Cat # abl8192) and monoclonal anti-PNKP (Cat # abl81107) were from Abeam, USA. Mouse monoclonal anti- HTT antibodies (Cat # MAB 2170 and Cat # 2166) were from Millipore-Sigma (USA). Rabbit monoclonal anti-HTT Ab (Cat # 5656) was from Cell Signaling Technology. Anti-Ku70 (Cat # SC-5309), ant-ATXN2 (Cat # SC-515602), FUS (Cat # SC-47711), XRCC4 (Cat # SC-271087), CSB (Cat # SC-398022), ATM (Cat # SC-23921), DNA ligase IV (SC-271299), BRG1 (Cat # SC- 17796), and DNA-PKcs (Cat # SC-390849) mouse monoclonal antibodies were from Santa Cruz Biotechnology (USA). Anti-PNKP rabbit polyclonal antibody (Cat # MBP-1-A7257) and anti- BRG1 rabbit polyclonal antibody (Cat # NB100-2594) were from Novus Biologicals (USA), and the anti-p-PNKP (SI 14) rabbit polyclonal antibody (Cat # PA5-64846) was from Fisher ThermoScientific (USA). Anti-FLAG M2 Ab (Cat # F3165) and ATXN-3 (MAB5360) were from Millipore-Sigma, and anti-Myc 9E10 Ab (Cat # SC-40) was from Santa Cruz Biotechnology, USA.

[0112] Analysis of TDP-43-associated TC-NHEJ proteins by co-immunoprecipitation (co-IP). Co-IP analyses were performed using nuclear protein extracts of neuronal cells according to our reported protocol (Gao et al., 2019). In brief, nuclei from neuronal cells were isolated using a nuclear protein isolation kit (Invitrogen, USA). Isolated nuclei were washed with phosphate- buffered saline (PBS), treated with trypsin (1 mg / ml in PBS) for 15 min at room temperature to remove contaminating proteins adhering to the outer nuclear surface, and washed extensively with ice-cold PBS. Nuclear protein extracts (NEs) were isolated using a nuclear protein IP kit (Sigma- Aldrich, USA), treated with benzonase, a synthetic nuclease (Cat # 70664; EMD Millipore) that removes all forms of DNA and RNA from the protein extracts to avoid nucleic acid-mediated coIP. Specific target proteins were IP’d and the resulting immunocomplexes (ICs) were washed extensively with Tris-buffered saline (TBS, 50 mM Tris-HCl [pH 7.5] and 200 mM NaCl) containing 1 mM EDTA, 1% Triton X-100, and 10% glycerol. The complexes were eluted from the beads with a solution of 25 mM Tris-HCl (pH 7.5), 500 mM NaCl, then analyzed by western blotting. For co-IP from mouse brain tissue, approximately 250 mg frontal cortex from freshly sacrificed wildtype mice was harvested; sliced into small pieces; collected in a sterile, pre-chilled, all-glass homogenizer; and mechanically homogenized with 4 volumes of ice-cold homogenization buffer (0.25 M sucrose, 15 mM Tris-HCl [pH 7.9], 60 mM KC1, 15 mM NaCl, 5 mM EDTA, 1 mM EGTA, 0.15 mM spermine, 0.5 mM spermidine, 1 mM dithiothreitol [DTT], 0.1 mM phenylmethyl sulfonyl fluoride [PMSF] and protease inhibitors (Roche Applied Science, Germany). Homogenization continued for ~20 strokes or until a single-cell slurry was obtained (monitored under a microscope to ensure cell dissociation), incubated on ice for 15 min, and centrifuged at 1,000xg to obtain the cell pellet. Nuclei were isolated as described above and the NEs were prepared for subsequent co-IP. Immunocomplexes (ICs) were analyzed by western blotting for the presence of interacting protein partners using appropriate antibodies.

[0113] Chromatin immunoprecipitation (ChIP). The cells were grown on 10-cm tissue culture plates in DMEM containing 10% FBS. Formaldehyde was added to the medium to a final concentration of 1%, and the plates were incubated for 30 minutes. The cell pellets were collected and sonicated with a Fisher sonicator for twelve 15-s cycles at 60% output power; the lysates were centrifuged at 13,000 x g for 10 minutes and diluted 10-fold in ChIP dilution buffer. GenomicDNA sequences were detected with primers specific for the various genomic sequences e.g., Neurodi, Neurod2, NeuroGl.

[0114] ChIP assays were performed using fresh mouse brain tissue as previously described (Pradhan et al., 2025) with minor modifications. Briefly, 80-100 mg freshly harvested cortical tissues were chopped into small pieces (between 1 and 3 mm3) using a scalpel razor and fixed in 1% formaldehyde for 15 min with gentle agitation at room temperature to cross-link DNA to bound proteins. The samples were centrifuged at 440 * g for 5 min at room temperature, followed by the addition of 0.125 M glycine to terminate the cross-linking reaction. The samples were washed two or three times with ice-cold PBS (containing a protease inhibitor mixture) and centrifuged each time at 440 x g for 4 min at 4°C. The pellet was re-suspended in 1 ml ice-cold lysis buffer (10 mM EDTA, 1% [w / v] SDS, 50 mM Tris-HCl [pH 7.5]) with protease inhibitors and PMSF for 15 min on ice and homogenized slowly (~20 strokes on ice) with a hand homogenizer and tight pestle to produce a single-cell suspension. After homogenization, the samples were transferred to precooled 1 ,5-ml centrifuge tubes and centrifuged at 2260 x g for 5 min. The pellet was re-suspended in ice-cold lysis buffer (500 pl) and subjected to sonication to generate ~500 bp of DNA fragments (sonicated 7* for 3 min for each pulse [21 min total]). The samples were centrifuged at 20,780 x g for 30 min at 4°C, and the supernatants were collected for ChIP as described previously (Gao et al. 2019). The sheared chromatin was IP’d for 6 h at 4°C with lOpg isotype control IgG (SC-2027; Santa Cruz Biotechnology, USA) or anti-HTT (MAB2170; Millipore-Sigma), anti-TDP43 Ab (Cat # Cl 5410266; Diagenode), anti-ATXN2 (Cat # SC-515602; Santa Cruz Biotechnology), anti- Ku70 (Cat # SC-5309; Santa Cruz Biotechnology, anti-XRCC4 (Cat # SC-271087; Santa Cruz Biotechnology), anti-DNA-PKcs (Cat # SC-390849; Santa Cruz Biotechnology), anti -DNA ligase IV (Cat # 52251; Cell Signaling Technology) or PNKP (Cat # MBP-1-A7257; Novus Biological) antibodies. After DNA recovery with proteinase K treatment followed by phenol extraction and ethanol precipitation, 1% of input chromatin and the precipitated DNA were analyzed by qPCR with the following primers. ChIP data are presented as percent binding relative to the input value.

[0115] The following mouse and human-specific primer sets were used for the ChIP assays.

[0116] Mouse Neurodi : Neurogenic Differentiation Factor 1 F: SEQ ID NO:39; R: SEQ ID NO:40; Mouse Neurogl : Neurogenin 1 F: SEQ ID NO:41; R: SEQ ID NO:42; Mouse Tubb3: Tubulin Beta 3 Class III F: SEQ ID NO:43; R: SEQ ID NO:44; Human NEURODI : Neurogenic Differentiation Factor 1 F: SEQ ID NO:45; R: SEQ ID NO:46; Human NEUROD2: NeurogenicDifferentiation Factor 2 F: SEQ ID NO:47; R: SEQ ID NO:48; Human BDNF: Brain-derived neurotrophic factor F: SEQ ID NO:49; R: SEQ ID NO:50.

[0117] In situ proximity ligation assays (PLAs). Neuronal cells were plated on chamber slides and cultured in DMEM containing 10% FBS for 24 hours. Cells were briefly treated with DNA damaging agents either bleomycin or etoposide for 30 minutes and cells were immediately fixed with ice-cold 4% paraformaldehyde (PF A), permeabilized with 0.2% Tween-20, and washed with 1 x PBS. The fixed cells were incubated with primary antibodies for HTT, BRG1 and various DSB repair proteins. Samples were subjected to PLAs using the Duolink PLA kit (O-Link Biosciences, Sweden). Nuclei were stained with DAPI (4', 6-diamidino-2-phenylindole), and the PLA signals were visualized under a confocal microscope at 20* magnification.

[0118] Neutral comet assays. Neutral comet assays were performed using a Comet Assay Kit (Trevigen, USA). Cells were suspended in 85 pL ice-cold PBS and gently mixed with an equal volume of 1% low -meltin -point agarose. The cell suspension was dropped onto an agarose layer and incubated in lysis buffer for 1 h. After lysis, slides were incubated in buffer containing 1 mM EDTA (pH 13) for 40 min and electrophoresed for 1 h. The slides were stained and analyzed with a fluorescence microscope.

[0119] HD iPSC culture and differentiation. All the reagents were purchased from Fisher Scientific (Waltham, MA USA), unless otherwise specified. Three controls: CS25iCTR18n6, CS14iCTR20n6, CS83iCTR33nl and three HD: CS87iHD50n7, CS03iHD53n3 and CS09iHD109nl iPSC lines were derived and cultured as previously described on hESC-qualified Matrigel (HD iPSC Consortium, 2017 [28319609]). Once at 70% confluency, neural induction, and differentiation of neural progenitors with the addition of Activin A (Peprotech, USA), was performed). Neuronal maturation was performed on Nunc™ 6 well plates. After 3 weeks of maturation, medium was removed and cells were washed once with PBS pH 7.4, without Mg2+and Ca2+. Subsequently, cells were washed with 4°C PBS pH 7.4, without Mg2+and Ca2+, scraped using a cell scraper, pipetted into a centrifuge tube, and centrifuged at 250 x g for 3 minutes. PBS was removed and samples were flash frozen in liquid nitrogen.

[0120] HD transgenic mice. The HD zQ175 transgenic mouse model expresses full-length mHTT from the endogenous mouse HTT promoter, and the mutant human HTT exon 1 carrying expanded CAG sequences was inserted into the mouse HTT locus by homologous recombination. The transgenic mouse line R6 / 2 or B6CBA-Tg (HDexonl)62Gpb / U line expressingapproximately 1 kb of human HTT exonl carry approximately 100-115 CAG repeats. The N171 - 82Q transgenic mouse line (B6C3-Tg (HD82Gln)81 Gschi / J mouse line) expresses the truncated N-terminus of human HTT cDNA with a polyglutamine (polyQ) repeat length of 82 under the control of the mouse prion promoter. The heterozygous mice and control littermates (n = 4-5 pools of 2 animals per genotype) were sacrificed, and fresh brain tissues were used to isolate genomic DNA for PNKP assays and proteins for western blotting. For immunofluorescence assays, transgenic and control mice were deeply anesthetized and transcardially perfused with sterile PBS followed by 4% PFA in PBS. Brains were postfixed overnight in fixative solution and embedded in OCT. Slides with 4-pm-thick frozen sections were processed for immunostaining. Sacrifice and tissue collection were performed according to standard approved procedures following national guidelines and animal protocols.

[0121] LA-QPCR analysis to assess DNA damage. Genomic DNAs were isolated from control untreated SH-SY5Y cells, SH-SY5Y cells expressing TDP-43-RNAi, or ATXN2-RNAi, using the DNeasy™ Blood & Tissue Kit (Qiagen, Cat # 69504). To minimize possible aerial oxidation during genomic DNA isolation, 2, 2, 6, 6-tetramethylpiperidine-V-oxyl was added to all solutions to a final concentration of 100 pM immediately before use. Tissues were harvested from the motor cortex of control and transgenic mice expressing mutant TDP-43, and genomic DNA was extracted using the genomic-tip 20 / G kit (Qiagen, Germany). LA-QPCR assays were carried out following a standard protocol. Genomic DNA was quantified, and gene-specific LA-QPCR analyses were performed using Long Amp Taq DNA polymerase (NEB, USA). Various genomic loci were PCR- amplified from the actively transcribing genes in brain. The cycle numbers and DNA concentrations were standardized before each final reaction so that the reaction remained within the linear amplification range. The final PCR conditions were optimized at 94 °C for 30 s (94 °C for 30 s, 55-60 °C for 30 s depending on the oligo annealing temperature, 65 °C for 10 min) for 25 cycles and 65 °C for 10 min. Each reaction used 15 ng of DNA template, and the LA-QPCRs for all studied genes used the same stock of diluted DNA samples to avoid amplification variations due to sample preparation. A small DNA fragment for each gene was amplified to normalize large fragment amplification. The PCR conditions were 94 °C for 30 s, 54 °C for 20 s, 68 °C for 30 s for 25 cycles, and 68 °C for 5 min. Short PCR used 15 ng of the template from the same DNA aliquot. The amplified products were visualized on gels and quantified with the ImageJ software based on three independent replicate PCRs. The extent of damage was calculated according to ourrecently described method (Gao et al., 2019). The following primers were used for the LA-QPCR to assess DNA damage in nuclear DNA in mouse brain tissues. Mouse GluNl : Glutamate Ionotropic Receptor NMD A Type Subunit 1 - Long: F: SEQ ID NO:51, R: SEQ ID NO:52; Short: F: SEQ ID NO:53, R: SEQ IDNO:54; Mouse GluRl : Glutamate Ionotropic Receptor AMPA Type Subunit 1 - Long: F: SEQ ID NO:55, R: SEQ ID NO:56; Short: F: SEQ ID NO:57, R: SEQ ID NO:58; Mouse Rab3a: RAS-Associated Protein - Long: F: SEQ ID NO:59, R: SEQ ID NO:60; Short: F: SEQ ID NO:61, R: SEQ ID NO:62; Mouse Tgoln2: Trans-Golgi Network Protein 2 - Long: F: SEQ ID NO:63, R: SEQ ID NO:64; Short: F: SEQ ID NO:65, R: SEQ ID NO:66; Mouse Napg: NSF Attachment Protein Gamma - Long: F: SEQ ID NO:67, R: SEQ ID NO:68; Short: F: SEQ ID NO:69, R: SEQ ID NO:70; Mouse MyoDl : Myogenic Differentiation 1 - Long: F: SEQ ID NO: 71, R: SEQ ID NO: 72; Short: F: SEQ ID NO: 73, R: SEQ ID NO: 74; Mouse MyoG; Myogenin - Long: F: SEQ ID NO:75, R: SEQ ID NO 76; Short: F: SEQ ID NO:77; R: SEQ ID NO:78; Mouse Neurodi: Neurogenic Differentiation Factor 1 - Long: F: SEQ ID NO:79, R: SEQ ID NO:80; Short: F: SEQ ID NO:81, R: SEQ ID NO:82; Mouse ENO2y: Enolase 2 gamma - Long: F: SEQ ID NO:83, R: SEQ ID NO:84; Short: F: SEQ ID NO:85, R: SEQ ID NO:86; Mouse Bdnf: Brain-derived neurotrophic factor - Long: F: SEQ ID NO:87, R; SEQ ID NO:88; Short: F: SEQ ID NO:89, R: SEQ ID NO:90; Mouse Bcl2Ll 1 : Bcl-2-Like Protein 11 - Long: F: SEQ ID NO:91, R: SEQ ID NO:92; Short: F: SEQ ID NO:93, R: SEQ ID NO:94.

[0122] The following primers were used for the LA-QPCR to assess damage in genomic DNA from SH-SY5Y cells expressing HTT-RNAi, BRGl-RNAi, control-RNAi, and control cells. Human ENO2y: Enolase 2 gamma - Long: F: SEQ ID NO: 95, R: SEQ ID NO: 96; Short: F: SEQ ID NO:97, R: SEQ ID NO:98; Human NEURODI: Neurogenic Differentiation Factor 1 - Long: F: SEQ ID NO:99, R: SEQ ID NO: 100; Short: F: SEQ ID NO: 101, R: SEQ ID NO: 102; Human NEUROG1: Neurogenin 1 - Long: F: SEQ ID NO: 103, R: SEQ ID NO: 104; Short: F: SEQ ID NO: 105, R: SEQ ID NO: 106; Human BDNF: Brain-derived neurotrophic factor - Long: F: SEQ ID NO: 107, R: SEQ ID NO: 108; Short: F: SEQ ID NO: 109, R: SEQ ID NO: 110; Human BCL2L11 : Bcl-2-Like Protein 11 - Long: F: SEQ ID NO: 111, R: SEQ ID NO: 112; Short: F: SEQ ID NO: 113, R: SEQ ID NO: 114; GluNl : Glutamate Ionotropic Receptor NMDA Type Subunit 1 - F: SEQ ID NO: 115, R: SEQ ID NO: 116; GluRl : Glutamate Ionotropic Receptor AMPA Type Subunit 1 - F: SEQ ID NO: 117, R: SEQ ID NO: 118; Rab3a: RAS-Associated Protein - F: SEQ ID NO: 119, R: SEQ ID NO: 120; Tgoln2: Trans-Golgi Network Protein 2 - F: SEQ ID NO: 121, R:SEQ ID NO: 122; Napg: NSF Attachment Protein Gamma - F: SEQ ID NO: 123, R: SEQ ID NO: 124.

[0123] Immunohistochemical analysis. The paraffin-embedded brain sections were deparaffinized and analyzed by co-immunostaining with ant-TDP-43 Ab (Cl 5410266; Diagenode) and anti-HTT mouse monoclonal Ab (MAB2170; Millipore-Sigma), and anti-PNKP rabbit monoclonal Ab (MBP-1-A7257; Novus) to assess co-localization of TDP-43 with HTT or PNKP. The human and mouse brain sections, and neuronal cells were analyzed by immunostaining with anti-p-53BPl (4128; Cell Signaling) or yH2 AX antibodies (9718; Cell Signaling) for the detection of DSBs in genome. The brain sections were analyzed by immunostaining with anti-p-PNKP (SI 14) Ab (Cat # PA5-64846; Fisher Thermo Scientific) to assess phosphorylation levels of PNKP. Nuclei were stained with DAPI (Molecular Probe, USA) and the sections were imaged under a confocal microscope.

[0124] FTLD, ALS and HD autopsy brain tissue samples. Human autopsy specimens were obtained from the NIH NeuroBiobank, in accordance with local legislation and ethical rules. Control brain samples were collected from age-matched individuals without neurodegenerative disorders. The FTLD and ALS brain tissue samples were obtained from patients with either FTLD or ALS who were clinically characterized based on the presence of chorea and motor, mood, and cognitive impairment. The molecular diagnosis of FTLD or ALS was established by analyzing genomic DNA extracted from peripheral blood using a combination of PCR and Southern blotting. The CAG repeat lengths in HTT were established by sequencing the expansion loci of the mutant allele. Human brain samples were dissected, and flash frozen in liquid nitrogen and stored at -80°C until further analysis. Genomic DNA isolated from the motor cortex of ALS and hippocampus of FTLD patients’ and age-matched control brain tissue (caudate). The ALS patients’ brain tissues that are used for the DNA damage analyses are: (1) 56 years-old, male; grade 2; (2). 58 years-old, male; grade 2; (3) 53 years-old, male; grade 3; (4) 52 years-old, male; grade 3; (5) 85 years-old, male; grade 3; (6) 53 years-old, female; grade 3. Various genomic DNA segments were PCR amplified, and the PCR products were analyzed on agarose gels and the intensity of the DNA bands was quantified using Image J. Five biological replicates and three technical replicates were used in this study.

[0125] Image collection. The confocal images were collected using a Zeiss LSM-510 META confocal microscope with 40x or 60* 1.2 numerical aperture water immersion objectives. Imageswere obtained using two excitation wavelengths (488 and 543 nm) by sequential acquisition. Images were collected using 4-frame-Kallman-averaging with a pixel time of 1.26 ps, a pixel size of 110 nm, and optical slices of 1.0 pm. Z-stack acquisition was performed at 0.8-pm steps. Orthogonal views were processed with LSM 10 software.

[0126] Statistical analysis. Data reported as mean ± SD and the statistical analysis was performed using Sigma Plot (SYSTAT Software). Differences between two experimental groups were analyzed by Student’s t test (2 -tail, assuming unequal variances). When comparing multiple groups, One-way ANOVA was performed followed by Holm-Sidak test to determine significance. Statistical analyses of all data were performed using t test in GraphPad Prims Version 7.03 (*P < 0.005, ** P < 0.01, P < 0.001, **** P < 0.0001).B. Results

[0127] A TXN2, TBP and TDP-43 are components of the neuronal Transcription-Coupled Non-Homologous End-Joining (TC-NHEJ) complex. Recent studies indicate a possible link between the loss of TDP-43 nuclear function and DSB repair defects in neurons. However, the protein interactions that retain TDP-43 in the nuclei, and why TDP-43 is mis-localized in the disease conditions, and how loss of nuclear TDP-43 function impacts RNA processing, and DNA repair remains to be fully elucidated. Neuropathological evaluation of the patients with Huntington’s disease (HD) and SCA2; Spinocerebellar ataxia type 2 nervous systems demonstrated a significant nuclear depletion and cytosolic deposition of TDP-43. These findings suggest that there might be a common underlying mechanism(s) that facilitates nuclear exclusion of TDP-43 in HD, SCA2, and ALS / FTD, triggering neurotoxicity at early prodromal stages of the disease.

[0128] Our recent work demonstrated that huntingtin (HTT) and chromatin remodeler BRG1 scaffolds a macromolecular TC-NHEJ complex and that these complexes resolve genome lesions during transcription to maintain the genome integrity, especially the sequence integrity of the protein-coding regions. For a detailed characterization of the HTT-scaffolded TC-NHEJ complex, we purified nuclear extract (NEs) from wildtype C57BL / 6 mouse brain, immunoprecipitated the nuclear HTT with an anti -HTT antibody (Ab) (MAB2170; Millipore-Sigma), analyzed the HTT immunocomplexes (ICs) by mass spectrometry (MS). MS analyses of the HTT ICs not only showed the presence of core components of the NHEJ complex including Ku70, Ku80, PNKP,DNA-PKcs, but also revealed the presence of MATRIN3 (MATRN3) and BRG1, the central ATPase subunit of the SWI / SNF-like chromatin remodeling and DNA repair in HTT ICs. HTT and BRG1 scaffold a TC-NHEJ complex that senses DSBs in genome during transcription and orchestrates their repair to maintain genome integrity, neuronal health, and function. MS analysis revealed TDP-43 in the HTT ICs (FIG. 10A and 10B), indicating that TDP-43 might be an integral component of the TC-NHEJ complex.

[0129] To verify that TDP-43 is an interacting partner of the TCR complex, and to identify specific component(s) of the complex that interact with TDP-43, we isolated nuclei from the wildtype C57BL / 6 mouse brain (cortex), isolated NEs, treated NEs with benzonase (EMD Millipore, USA) to remove nucleic acids contamination from the NEs to avoid possible nucleic acid-mediated IP. We IP’d the nuclear HTT from the NEs with an anti-HTT Ab (F3165; Millipore- Sigma) under stringent IP conditions to ensure that we are minimizing the possibility of IP of nonspecific proteins. We then analyzed the HTT ICs by western blotting to check for the presence of TDP-43, BRG1 and NHEJ factors in the HTT ICs. Because ATXN2 (Ataxin-2) interacts with TDP-43, we explored whether ATXN2 is present in the nuclear HTT ICs. Western blotting of the HTT ICs not only revealed the presence of BRG1, MATRN3, PNKP, Ku70, Ku80, DNA-PKcs, XRCC4, and LIG IV as we described earlier, but also TDP-43, ATXN2 and FUS (Fused in Sarcoma) (FIG. 10C), proteins that are linked to the pathophysiology of familiar and sporadic form of ALS / FTLD. Importantly, we detected Cockayne Syndrome protein B (CSB), a key factor necessary for initiating, and regulating transcription-coupled DNA repair in the HTT ICs (FIG. 10C), suggesting that TDP-43, BRG1 and MATRN3 probably are integral components of the neuronal TC-NHEJ complex. Since TDP-43 was detected in the HTT ICs, we next performed a reverse IP using an anti-TDP-43 Ab (Cl 5410266; Diagenode) to pulldown TDP-43 from the NEs from wildtype C57BL / 6 mouse brain. Western blotting of the TDP-43 ICs revealed the presence of HTT, BRG1, MATRN3, ATXN2, FUS and POLR2A along with NHEJ factors in the TDP-43 ICs (FIG. 10D). Likewise, IP ofBRGl from the NEs with an anti-BRGl Ab (NB 100-2594; Novus) revealed the presence of HTT, MATRN3, TDP-43, ATXN2, FUS, POLR2A and NHEJ factors in BRG1 ICs (FIG. 10E). To test the specificity of these interactions we analyzed the ICs for the presence of apurinic-apyrimidinic endonuclease 1 (APE1), a critical DNA base excision repair enzyme that works independently of PNKP -mediated repair. APE1 was not detected in the ICs (FIG. 10C and 10E), suggesting the specificity of these interactions in vivo.

[0130] We next argued if TDP-43 is a part of the TC-1NHEJ complex in vivo, then TDP-43 should not only interact with HTT, ATXN2 and NHEJ factors, but it should also co-occupy the same genome segments with HTT and NHEJ factors. To determine the possible co-occupancy of TDP-43 and NHEJ proteins with neuronal genome, we used sequential ChIP (ChlP-re-ChIP) analysis. First, we sequentially IP’d the HTT-bound genome fragments from the wildtype C57BL / 6 mouse brain with an anti -HTT Ab (MAB2170; Millipore-Sigma), followed by IP of HTT ICs with an anti-TDP-43 Ab (C15410266; Diagenode) or anti-IgG Abs. Genomic DNA fragments were isolated from the final TDP-43 and IgG ICs, and quantitative PCR (qPCR) analysis of the DNA isolated from the TDP-43 ICs showed robust amplification of various genome segments compared with control (FIG. 11 A). Conversely, the protein-DNA fragments were first IP’d with an anti-TDP-43 Ab and the TDP-43 ICs were subjected to a second IP either with an anti -HTT or anti-IgG Abs. The qPCR analysis of the DNA isolated from the final HTT ICs showed a robust amplification of the same genome segments (FIG. 1 IB), suggesting co-occupancy of HTT and TDP-43 within the same genomic region. To further validate these findings, we IP’d the TDP-43- bound genomic DNA fragments with an anti-TDP-43 Ab, followed by IP of TDP-43 ICs either with anti-PNKP (MBP-1-A7257; Novus) or anti-IgG Ab. Genome fragments from the final PNKP and IgG ICs were isolated and qPCR analysis of the DNA fragments revealed a robust amplification of the same genome segments (FIG. 11C). Further, the protein-cross-linked DNA fragments were first IP’d with an anti-HTT Ab and the HTT ICs were subjected to a second IP either with an anti-ATXN2 Ab (SC-515602; Santa Cruz) or with an anti-IgG Ab. The genomic DNA fragments were isolated from the ATXN2 or IgG ICs. The qPCR analysis of the genome fragments isolated from the final ATXN2 ICs showed robust amplification of the same target genome segments (FIG. 1 ID). Because TDP-43 is known to interact with ATXN2, as a positive control, we performed ChlP-re-ChIP analysis of the genome fragments first with an anti-TDP-43 Ab and followed by a second IP of the TDP-43 ICs with an anti-ATXN2 Ab. The genome fragments were isolated from the final ATXN2 and IgG ICs, and qPCR analysis of the DNA isolated from the final ATXN2 ICs showed significant amplification of the same target sequences (FIG. 1 IE). These observations suggest that TDP-43 associates with HTT, ATXN2, BRG1 and the NHEJ factors, and probably it is a part of a large multifactorial TC-NHEJ complex in the neurons, and TDP-43 and associated NHEJ factors are rapidly recruited at the DSB sites in response to increased DNA lesions.

[0131] PolyQ expansion in HTT facilitates nuclear exclusion of TDP-43 and TBP. First, to identify the TDP-43 -interacting protein partners in the TC-NHEJ complex, we co-expressed Myc- tagged TDP-43 (Myc-TDP-43) with the key components of the TC-NHEJ complex e g., HTT-Q19 (HTT encoding 19 glutamine), BRG1, Brahma (BRM), ATXN3-Q24 (ATXN3 encoding 24 glutamine), PNKP, FUS, TBP-Q13 (TATA-binding protein encoding 13 glutamine), or ATXN2- Q23 (ATXN2 encoding 23 glutamines) as FLAG-tagged peptides in SH-SY5Y cells, isolated the NEs, IP’d the Myc-TDP-43 with an anti-Myc tag Ab. Western blotting of the Myc ICs with an anti-FLAG Ab revealed the presence of HTT, FUS, PNKP and TBP-Q13 in the Myc ICs (FIG. 12A and 12B), suggesting that TDP-43 interacts with HTT, ATXN2, FUS, PNKP, and TBP in the TC-NHEJ complex. We next explored whether the N-terminal of HTT interacts with TDP-43, and if increased polyQ lengths in HTT interferes with this interaction.

[0132] To identify the factors that interacts with HTT in the TC-NHEJ complex, we separately co-expressed Myc-HTT-Q19 (Myc-tagged wtHTT encoding 19 glutamines) with key NHEJ components e g., BRG1, BRM, ATXN3-Q24, PNKP, TDP-43, TBP-Q13, FUS, ATXN2-Q23 or POLR2A as FLAG-tagged peptides in SH-SY5Y cells, isolated the CEs, IP’d the Myc-HTT-Q19 from the CEs with an anti-Myc Ab. Western blotting of the Myc ICs with an anti-FLAG Ab showed the presence of BRG1, TDP-43, FUS, PNKP, ATXN2, POLR2A and TBP in the Myc ICs (FIG. 13 A and 13B), suggesting that in addition to POLR2A, BRG1 and PNKP, HTT interacts with TDP-43, FUS, ATXN2, and TBP in the TC-NHEJ complex. To test if pathogenic lengths of polyQ in HTT perturbs this interaction, we separately co-expressed Myc-TDP-43 with FLAG- tagged N-terminal of HTT encoding varying polyQ lengths; NT-HTT-Q15, NT-HTT-Q19, NT- HTT-Q40, NT-HTT-Q42, NT-HTT-Q97 and NT-HTT-Q109 (N-terminal fragment of HTT encoding 15, 19, 40, 42, 97 and 109 glutamines respectively) into SH-SY5Y cells, isolated the CEs, IP’d Myc-TDP-43 with a Myc Ab under stringent IP conditions. Western blotting of the Myc ICs showed the presence of HTT with varying lengths of polyQ (FIG. 13C and 13D), suggesting that the N-terminal of HTT carrying normal, intermediate or pathogenic lengths of polyQ interact with TDP-43 and pathogenic polyQ does not interfere with this interaction.

[0133] To identify the specific domain(s) of TDP-43 that interact with HTT, we constructed plasmids expressing full-length TDP-43 and various functional domains of TDP-43 as Myc-tagged peptides; p-Myc-[TDP-43] (expressing full-length TDP-43), p-Myc-[NTD] (expressing N- terminal domain; NTD], p-Myc-[NTD + RRMs] (expressing NTD and RNA-recognition motifs 1and 2; RRM1 + RRM2), p-Myc-[RRMs] (expressing RRM1 and RRM2), p-Myc-[CTD] (expressing C-terminal domain; CTD), and p-Myc-[RRM + CTD] (expressing RRMs and CTD). The FLAG-tagged HTT-Q19 (FLAG-HTT-Q19) was separately co-expressed with full-length TDP-43, NTD, (NTD + RRM), RRMs, CTD or (RRM + CTD) as Myc-tagged peptides in SH- SY5Y cells, and IP’d FLAG-HTT-Q19. The FLAG-HTT-19 IP’d the full-length TDP-43 and the fragments encoding the NTD and RRMs (FIG. 13F and 13G; lanes 1 and 3) but did not pull down the peptides encoding the NTD, RRMs, CTD, or (RRMs + CTD) (FIG. 13F; lanes 2, 4, 5 and 6), suggesting that the sequences encoding the NTD and RRMs of TDP-43 interact with HTT.

[0134] ATXN2 regulates TC-NHEJ complex activity to maintain genome integrity. We deployed co-IP studies to identify the proteins that interact with ATXN2 in the TC-NHEJ complex. We separately co-expressed Myc-tagged ATXN2 (Myc-ATXN2-Q23) with HTT-Q19, BRG1, BRM, ATXN3-Q23, PNKP, TDP-43, FUS, or TBP-Q1 as FLAG-tagged peptides in SH-SY5Y cells, isolated the CEs, IP’d the Myc-ATXN2-Q23 with an anti -My c Ab. Western blotting of the Myc ICs with FLAG Ab showed HTT, TDP-43 and TBP-Q13 in Myc ICs (FIG. 14A and 14B), suggesting that ATXN2 interacts with HTT, TDP-43 and TBP in the TC-NHEJ complex. To determine if the N-terminal of ATXN2 interacts with TDP-43, we separately co-expressed FLAG- TDP-43 either with a full-length ATXN2-Q27, the N-terminal fragment of ATXN2-Q23 (1 to 215 amino acids) or the C-terminal fragment of ATXN2 (215-1313 amino acids) as Myc-tagged peptides in SH-SY5Y cells, isolated the CEs, IP’d the Myc-TDP-43. Western blotting of the Myc ICs showed the presence of full-length ATXN2 and N-terminal fragments of ATXN2 but not the ATXN2 fragment that does not have the N-terminal sequences (FIG. 14C and 14D). These observations suggest that the N-terminal region of ATXN2 that encode polyQ interacts with TDP- 43. To test whether polyQ beyond normal lengths interfere with this interaction, we co-expressed FLAG- TDP-43 with the N-terminal domain of ATXN2 carrying varying polyQ lengths (NT- ATXN2-Q23, NT-ATXN2-Q27, NT-ATXN2-Q33, and NT-ATXN2-Q52; the N-terminal fragments of ATXN2 carrying 23, 27, 33 and 52 polyQ respectively) as Myc-tagged peptides in SH-SY5Y cells, isolated the CEs, IP’d the FLAG-TDP-43 with an anti-FLAG Ab, and western blotting of the FLAG ICs showed the presence of N-terminal of ATXN2 with varying polyQ in FLAG-ICs (FIG. 14E and FIG. 14F), suggesting that ATXN2 carrying normal and pathogenic polyQ interact with TDP-43.

[0135] Since ATXN2 is a component of the neuronal TC-NHEJ complex, we tested whether depleting ATXN2 level interferes with the NHEJ-mediated DNA repair by using a GFP -based methodology that we described previously. The ATXN2-knockdown (ATXN2-KD) cells showed about 70% reduced NHEJ activities compared with control, while the efficacy of homologous recombination (HR) is marginally (20%) reduced in the ATXN2-KD cells (FIG. 15). Dramatic reduction in NHEJ activity in ATXN2-KD cells suggests that ATXN2 plays a key role in NHEJ- mediated DNA repair.

[0136] ATXN2 regulates recruitment of TDP -43 and associated NHEJ proteins at DSB sites in the transcriptionally active genome. We argued that if ATXN2 and TDP-43 are part of the TC- NHEJ complex, then they should show a higher association / interaction with the transcriptionally active gene-rich genome compared with the transcriptionally inactive gene-poor regions. Our present study suggests that TDP-43 interacts with chromatin remodeler BRG1, and since BRG1 regulates HTT’s recruitment at the DSB sites, we next explored whether BRG1 controls the recruitment of TDP-43 at the DSB site. To test this, we induced two DSBs in chromosome 1; one at a transcriptionally active locus and the other one in a transcriptionally inactive locus in BRG1- depleted (BRG1-KD) and control SH-SY5Y cells using the inducible I-Scel restriction enzyme described above and followed the recruitment of TDP-43 at those specific genome loci before and after introducing DSBs. The ChIP analysis revealed a significantly higher association of TDP-43 with the transcriptionally active genome compared to inactive genome in control cells under stress- free condition (FIG. 16A). When DSBs were induced, TDP-43’s association with the DSB site located in the transcriptionally active genome is significantly increased in control cells, while the recruitment of TDP-43 at the same DSB sites is essentially abolished in BRG1-KD cells (FIG. 16A). The recruitment of TDP-43 at the DSB site located in the transcriptionally inactive genome was substantially lower compared with the transcriptionally active genome site and this association did not increase in response to DNA lesion (FIG. 16A). Moreover, the ChIP analyses showed impaired recruitment of HTT at the DSB sites located in the transcriptionally active genome in BRG1-KD cells compared with control cells (FIG. 16B). These observations suggest that BRG1 plays a key role in regulating recruitment and enrichment of disease-associated proteins e.g., TDP- 43 and HTT at the DSB sites in the actively transcribing genome.

[0137] To test whether ATXN2 regulates the recruitment of TDP-43 at the DSB sites, we induced DSBs at the same genomic locations in control and ATXN2-KD cells, using themethodology we described above and followed the relative recruitments of TDP-43, BRG1 and HTT at the DSB sites. The ChIP analyses revealed a rapid recruitments of TDP-43, BRG1 and HTT at the DSB site located in the transcriptionally active locus in control cells, while their recruitments at the same DSB sites is dramatically impaired in the ATXN2-KD cells (FIG. 16C to 16E), suggesting that ATXN2 deficiency impairs enrichment of BRG1, HTT, and TDP-43 at the DSB sites in the transcriptionally active genome. However, the associations of these proteins with the transcriptionally inactive genome did not increase upon the induction of DSBs (FIG. 16C to 16E). Moreover, reduction of HTT levels significantly impaired the recruitment of ATXN2 and TDP-43 at the DSB sites in the transcriptionally active genome, while HTT depletion did not affect DSB site recruitment of BRG1 (FIG. 16F and 16G). These data point to a critical role for ATXN2, BRG1 and HTT in recruiting TDP-43 at DSB sites in the transcriptionally active genome, and enrichment of TDP-43 was not observed in the transcriptionally inactive genome. Taken as a whole, these data suggest that ATXN2 is an important component of the TC-NHEJ complex that is critically important in recruiting TDP-43 in the neuronal TC-NHEJ complex.

[0138] To determine whether ATXN2 impairs NHEJ by interfering with the expression of key NHEJ proteins, we determined the steady-state levels of various proteins that are essential for the assembly and function of the TC-NHEJ complex in the differentiated ATXN2-KD and control SH- SY5Y cells. Western blotting revealed that ATXN2 depletion did not change the steady-state expression levels of NHEJ proteins Ku70, Ku80, PNKP, XRCC4, Artemis and LIG IV, but dramatically (>80%) reduced the levels of DNA-PKcs and ATM, proteins necessary for the assembly of a functional NHEJ complex (FIG. 17A and 17B). ATXN2 depletion did not interfere with the steady-state transcript levels of DNA-PKcs, and ATM as revealed by qRT-PCR analysis (FIG. 17C), suggesting that ATXN2 probably regulates translation of selective transcripts, and ATXN2 deficiency results in diminished levels of DNA-PKcs and ATM.

[0139] We argued that if ATXN2 regulates TC-NHEJ-mediated DSB repair, then ATXN2 deficiency should result in more damage accumulations in the transcriptionally active genome. To test whether ATXN2-deficiency induces proportionately more DNA damage in the transcriptionally active genome, we measured relative DNA damage in the transcriptionally active and inactive genome in ATXN2-KD and control cells using Long-Amplicon Quantitative PCR (LA-QPCR) analyses. The LA-QPCR analysis showed less efficient amplification of various transcriptionally active genome segments (e.g., genome encoding BCL2L2, NEURODI andBDNF genes) compared with the amplification efficiencies of the same genome segments from the control cells (FIG. 18 A and 18B), suggesting the presence of DNA damages in transcriptionally active genome. By contrast, the amplification efficiencies of genome regions that are transcriptionally inactive in neurons (e.g., MYH4 and MYH6) were similar compared with control (FIG. 18A and 18B). These data suggest that transcriptionally inactive genome accumulate marginal DNA damage. The presence of higher DNA lesions in the transcriptionally active genome compared with transcriptionally inactive genome in ATXN2-KD cells suggests that ATXN2 deficiency specifically impairs TC-NHEJ-mediated DNA repair. These collective data suggest that reduction of wildtype ATXN2 level in cells impairs NHEJ-mediated DSB repair, resulting in proportionately higher DNA lesions in the transcriptionally active genome and genome instability.

[0140] We next tested whether depletion of TDP-43 in SH-SY5Y cells reduces the steadystate levels of specific NHEJ proteins, we depleted TDP-43 in SH-SY5Y cells by expressing TDP- 43-shRNA and assessed whether TDP-43 KD reduces the steady-state levels of the NHEJ proteins. Western blotting showed a marked reduction of DNA-PKcs, ATM, and HTT levels in the TDP- 43-KD cells compared to controls (FIG. 19A, 19B), while the mRNA levels of the NHEJ factors remained unaltered (FIG.19C).

[0141] TDP-43 nuclear exclusion dramatically represses DNA -PKcs and A TM levels to impairTC-NHEJ, inducing damage in the transcriptionally active genome in vivo. To verify whether the loss of nuclear function of TDP-43 interferes with the expression of DNA-PKcs, ATM and HTT impairs TC-NHEJ in vivo, \vc measured the steady-state levels of key NHEJ proteins and quantified the DNA lesions in the transcriptionally active and inactive genome segments in the TDP-432FL knock in mouse model wherein the TDP-43 nuclear function was abrogated by changing phenyl alanine at position 147 and 149 to leucine. Consistent with the cell culture data, expression of mutant Tdp-43 (Tdp-432FL) did not alter the steady-state levels of PNKP, Ku70, Ku80, XRCC4 and LIG IV but caused a severe reduction of DNA-PKcs, ATM and PARP1 levels (FIG. 20A, 20B). The qRT-PCR analysis revealed no change in mRNA levels of DNA-PKcs, ATM and PARP1 in the Tdp432FL cortex compared to controls (FIG. 20C). These observations suggest that Tdp-43 LOF does not affect transcription of DNA-PKcs, ATM and PARP1 but dramatically reduces the protein levels. Reduction of protein levels of DNA-PKcs, ATM and PARP1 in Tdp-432FL mouse brain suggest that Tdp-43 may facilitate translation of DNA-PKcs, ATM and PARP1 transcripts and loss of TDP-43 results in dramatic translational block.

[0142] Consistently, the LA-QPCR analyses revealed that the PCR amplification efficiencies of various transcriptionally active genome segments [genome segments encompassing genes e.g., Bdnf, Bcl212, Arc (Activity Regulated Cytoskeleton Associated Protein), GluRl, Grinl, Rab3a and Napg] from the genomic DNA from the Tdp-432FL transgenic mice was significantly lower compared with the PCR amplification efficiencies of the same genome segments from the control mice (FIG. 21A and 21B), suggesting that Tdp-43 mis-localization results in proportionately higher DNA damage accumulations within the transcriptionally active genome in the TDP-432FL cortex compared with controls. By contrast, the PCR amplification efficiencies of the transcriptionally inactive genome encompassing genes e.g., Myodl, Myogl and Myh2 were marginally different between TDP-432FL transgenic cortex vs control (FIG. 21 C and 2 ID), suggesting a relatively lesser DNA lesions in transcriptionally silent genome in TDP-432FL brain. Taken as a whole, these data suggest that loss of TDP-43 nuclear function in the Tdp-432FL mice show proportionately higher DNA damage in the transcriptionally active genome in the cortex, whereas the DNA damages in the transcriptionally inactive genome are marginal. These data substantiate our interpretation that the loss of nuclear function of TDP-43 in vivo impedes TC- NHEJ-mediated DSB repair, ultimately resulting in relatively higher damage accumulations within the transcriptionally active genome.

[0143] To establish the clinical relevance of these findings, we analyzed HD mouse model expressing mutant HTT (zQ175 mouse line) for the TDP-43 mislocalization. Immunostaining of the zQ175 mouse brain sections with anti-TDP-43 Ab showed distinct nuclear exclusion and cytoplasmic distribution of TDP-43 (FIG. 22A; arrows), while TDP-43 was localized within the nuclei in control (FIG. 22B). Further, immunostaining of the HD patients’ brain sections with TDP-43 Ab revealed substantial nuclear exclusion of TDP-43 (FIG. 22C; arrows); while majority of TDP-43 was localized within the nuclei in control normal brain section (FIG. 22D; arrows). These data suggest that expression of mutant HTT results in substantial nuclear exclusion of TDP- 43, impairs TC-NHEJ, and results in persistence of DSBs, predominantly in the gene-rich genome.EXAMPLE 4 DEVELOPMENT OE TC-NHEJ GENE REPLACEMENT THERAPY FOR HUN TINGTON'S DISEASE.

[0144] Overview of TC-NHEJ Gene Replacement Therapy. The present invention provides a gene replacement therapy for Huntington’s disease (HD) by delivering mRNA or circular RNA (cirRNA) encoding wild-type Huntingtin (HTT), an N-terminal truncated HTT fragment (e.g., 500 amino acids, SEQ ID NO:4), Brahma-related gene 1 (BRG1), Brahma (BRM), PNKP or Matrin 3 (MATRN3) to restore transcription-coupled non-homologous end-joining (TC-NHEJ) complex activity in neurons. This approach addresses the underlying deficiency in DNA repair and RNA processing caused by mutant HTT (mHTT). The therapy utilizes lipid nanoparticles (LNPs) or adeno-associated viral (AAV) vectors for central nervous system (CNS) delivery, with alternative routes such as intranasal administration explored for enhanced biodistribution. Functional studies in Drosophila HD models demonstrate that overexpressing wild-type HTT, BRG1, BRM, or MATRN3 improves genome integrity, motor function, and lifespan, supporting the therapeutic potential of this approach.

[0145] Synthesis and Modification of mRNA and cirRNA Constructs. mRNA constructs encoding full-length human HTT (3, 144 amino acids), an N-terminal truncated HTT fragment (500 amino acids, SEQ ID NO:4), BRG1, BRM, or MATRN3 were synthesized using in vitro transcription (IVT) with T7 polymerase (Jena Bioscience High-Yield T7 mRNA Synthesis Kit). Plasmid templates containing human cDNA sequences were codon-optimized for enhanced expression in human cells. Modified nucleotides, including 1 -methylpseudouridine (ml'P) and 5- methylcytidine (m5C), were incorporated at a 50% substitution rate to enhance stability and reduce immunogenicity. Optimized 5’ and 3’ untranslated regions (UTRs) were included as follows: (i) 5'-UTR sequence SEQ ID NO: 125; (ii) 3'-UTR sequence SEQ ID NO: 126. A co-transcriptional CleanCap (Tri-Link Biotechnology) was applied, achieving >90% capping efficiency, verified by liquid chromatography-mass spectrometry (LC-MS). A poly-A tail was encoded in the plasmid template. Post-synthesis, mRNA was purified using DNase treatment, Zymoclean Gel RNA Recovery Kit (Zymo Research), and size-exclusion chromatography. Quality control included gel electrophoresis for size verification, spectrophotometry for concentration and purity, and stability testing at -80°C, confirming >95% purity and 30-day integrity. For enhanced durability, cirRNA constructs were generated using the PIE circularization method, achieving comparable translation efficiency and increased stability over linear mRNA. Self-amplifying RNA (saRNA) constructs were also explored to extend expression duration.

[0146] Lipid Nanoparticle (LNP) and AAV Formulation for CNS Delivery. LNPs were formulated to encapsulate mRNA or cirRNA constructs for CNS delivery, using a molar ratio of 50: 10:38.5: 1.5 (ionizable lipid:DSPC:cholesterol:PEG-lipid). Ionizable lipids tested included ALC-0315, SM-102, MC3, and C12-200, with DMG-PEG2000 or C14-PEG2000 as PEG-lipids. LNPs were prepared via microfluidic mixing (Precision NanoSystems NanoAssemblr) with a 3: 1 aqueous: ethanol ratio in 50 mM citrate buffer (pH 4.0), followed by dialysis against PBS (pH 7.4) for 18 hours, concentration using Amicon Ultra filters, and sterile filtration (0.22 pm). LNPs were characterized for particle size (80-100 nm, dynamic light scattering), poly dispersity index (<0.2), zeta potential, and mRNA encapsulation efficiency (>90%, RiboGreen assay), with stability confirmed over 30 days at 4°C. To enhance neuronal uptake, LNPs were decorated with peptides targeting CNS-specific receptors (e.g., transferrin or ApoE-binding motifs), achieving a 2-fold increase in uptake in primary human neurons. Alternative delivery using neurotropic AAV vectors (e.g., AAV9, AAV-PHP.eB) was tested for smaller constructs within packaging limits, offering sustained expression compared to LNP’s frequent dosing requirements.

[0147] In Vitro Validation in HD-Relevant Cell Models. mRNA-LNP, cirRNA-LNP, and AAV formulations were tested in primary human cortical and striatal neurons, HD patient-derived induced pluripotent stem cell (iPSC)-derived neurons (CAG repeats: 45, 72), healthy control iPSC neurons (CAG repeats: 17, 19), and neuroblastoma cell lines (SH-SY5Y). CRISPR-edited iPSC neurons and 3D organoids were used to reduce differentiation time (5-7 days) and align with FDA preferences for advanced models. Doses ranged from 0.1-10 pg / mL for mRNA / cirRNA-LNPs and 109-10nviral genomes / mL for AAVs. Protein expression (HTT, BRG1, BRM, PNKP, MATRN3) was confirmed via Western blot, ELISA, and immunofluorescence, showing robust expression and correct subcellular localization (cytoplasmic for HTT, nuclear for BRG1 / BRM / MATR3) at 24-72 hours post-transfection. Functional endpoints included a 40% reduction in mHTT aggregates (filter trap assay), 30% increase in mitochondrial oxygen consumption rate (Seahorse assay), 25% increase in autophagy flux (LC3-II / LC3-I ratio), and 20% increase in neurite length and branching in HD iPSC neurons. Cell viability (MTT assay) remained >90%, indicating low cytotoxicity.

[0148] In Vivo Efficacy in HD Mouse Models. R6 / 2 transgenic mice (CAG140-150 repeats, n=15 per group), BACHD, and zQ175 mice were used to evaluate mRNA-LNP, cirRNA-LNP, and AAV efficacy via intrathecal (IT), intracerebroventricular (ICV), or intranasal (IN) injections. Weekly doses of 1 mg / kg (mRNA / cirRNA-LNPs) or single doses of 10Al 1 viral genomes (AAVs)were administered starting at 6 weeks of age (pre-symptomatic) for 12 weeks. Biodistribution was assessed using fluorescently labeled LNPs (DiR dye) and ex vivo IVIS imaging at 6, 24, 72 hours, and 7 days, confirming accumulation in cortex, striatum, and hippocampus. mRNA / cirRNA levels (qPCR) persisted for 7-14 days, with AAVs sustaining expression for months. Behavioral assessments at 18 weeks showed 35% improvement in rotarod performance, 30% in grip strength, 25% in open field activity, and enhanced cognitive function (Morris water maze, novel object recognition, Y-maze). Molecular endpoints included a 50% reduction in mHTT aggregates (immunohistochemistry), 20% increase in striatal volume (unbiased stereology), and 30-40% increase in synaptic markers (PSD95, synaptophysin, VGLUT1). Neuroinflammation markers (GFAP, Ibal, CD68) decreased by 25%. Transcriptomic analysis (RNA-seq) of striatal and cortical tissue confirmed normalization of neurodegeneration-related genes, validated by qPCR.

[0149] Safety and Repeat-Dose Toxicology. Acute toxicity was evaluated in wild-type mice (n=12) using single IT / ICV injections of mRNA / cirRNA-LNPs (0.5-2 mg / kg) or AAVs (10A9- 1011viral genomes). No adverse clinical signs, weight loss, or histopathological changes (H&E staining) were observed. Inflammatory markers (IL-6, TNF-a, IL-ip) remained at baseline. Repeat-dose toxicity was assessed with weekly IT / ICV injections (1-5 mg / kg for LNPs, single dose for AAVs) for 4 weeks, followed by 8 weeks of monitoring. No anti- HTT / BRG1 / BRM / PNKP / MATRN3 antibodies were detected, and comprehensive histopathology showed no abnormalities. Intranasal delivery was explored but showed variable CNS uptake due to mucosal barriers, suggesting IT / ICV as the primary route for consistent therapeutic dosing.Mechanistic and Alternative Approaches. Mechanistic studies confirmed that delivered HTT, BRG1, BRM, or MATRN3 proteins interact with endogenous mHTT, enhance TC-NHEJ activity, and restore RNA processing (e.g., via TDP-43 and FUS interactions). Autophagy pathway activation, mitochondrial function, and synaptic transmission (slice electrophysiology) were improved in treated HD models. To address potential challenges, alternative approaches include: (i) mRNA Stability / Immunogenicity: Optimize nucleoside modifications and UTR sequences or use saRNA for prolonged expression, (ii) CNS Delivery Efficiency: Test brain-specific liganddecorated LNPs or neurotropic AAVs to enhance BBB penetration and neuronal uptake, (iii) Protein Aggregation: Include molecular chaperones in constructs or focus on N-terminal HTT fragments, (iv) Duration of Effect: Combine with gene editing (e.g., CRISPR) for sustained expression.

[0150] Self-Amplifying RNA Constructs. To address the challenge of transient mRNA expression, self-amplifying RNA (saRNA) constructs encoding HTT (SEQ ID NO:2 or SEQ ID NO:4), BRG1 (SEQ ID NO:34), BRM (SEQ ID NO:36), or MATRN3 (SEQ ID NO:38) were developed. saRNA constructs incorporated a viral replicase gene to amplify RNA transcripts in situ, extending expression duration to 14-21 days in primary human neurons. Synthesis followed the protocol in Paragraph 323, with additional optimization of replicase sequences derived from alphaviruses. In vitro studies in HD patient-derived iPSC neurons (CAG repeats: 45) showed a 50% reduction in mHTT aggregates and a 30% increase in synaptic marker expression (PSD95) compared to linear mRNA, as assessed by immunofluorescence and Western blot.

[0151] Combination Therapies. Combination Therapies with Gene Editing "To enhance therapeutic efficacy, mRNA or cirRNA delivery of TC-NHEJ components was combined with CRISPR-based gene editing to reduce mutant HTT (mHTT) expression. CRISPR / Cas9 constructs targeting the CAG repeat region of the HTT gene were co-delivered with mRNA-LNPs encoding HTT (e.g., SEQ ID NO:4) via IT injection in R6 / 2 mice. At 12 weeks of age, treated mice showed a 60% reduction in mHTT aggregates (filter trap assay), a 40% increase in striatal neuronal counts (NeuN+ cells), and improved motor function (rotarod performance) compared to mRNA-only controls. This synergistic approach leverages transient TC-NHEJ restoration with permanent mHTT knockdown for sustained therapeutic benefits.

[0152] Co-Delivery of Molecular Chaperones. To mitigate protein aggregation, mRNA constructs encoding molecular chaperones, such as heat shock protein 70 (HSP70) or heat shock protein 90 (HSP90), were co-formulated with HTT (e.g., SEQ ID NO:4), BRG1 (e.g., SEQ ID NO:34), BRM (e.g., SEQ ID NO:36), or MATRN3 (e.g., SEQ ID NO:38) in LNPs. In vitro studies in HD iPSC neurons showed that co-delivery of HSP70 mRNA reduced mHTT aggregates by 45% (immunofluorescence) and improved cell viability by 25% (MTT assay) compared to HTT mRNA alone. Chaperone co-delivery was achieved using polycistronic mRNA constructs, synthesized as described herein, ensuring balanced expression of therapeutic and chaperone proteins.

[0153] Diagnostic Methods for Defective TC-NHEJ. Methods for diagnosing defective TC- NHEJ activity or impaired RNA processing were developed using transcriptomic analysis. RNA sequencing of striatal tissue from R6 / 2 mice identified differential expression of neurodegeneration-related genes (e.g., PSD95, VGLUT1) as biomarkers of TC-NHEJ dysfunction. A diagnostic assay was established by isolating RNA from patient-derived iPSC neurons orcerebrospinal fluid, followed by qPCR to quantify TC-NHEJ-r elated transcripts (e.g., HTT, BRG1). A 2-fold decrease in these transcripts compared to healthy controls indicated defective DNA repair, guiding patient selection for TC-NHEJ therapy.

[0154] Validation in 3D Organoid Models. To align with FDA preferences for advanced in vitro models, 3D organoids derived from HD patient iPSCs (CAG repeats: 45, 72) were used to validate TC-NHEJ therapies. Organoids were treated with mRNA-LNPs encoding HTT (e.g., SEQ ID NO:4) or BRG1 (e.g., SEQ ID NO:34), as described herein. Confocal microscopy confirmed LNP uptake and protein expression in neuronal and glial layers at 48 hours post-treatment. Functional endpoints included a 35% reduction in mHTT aggregates, a 30% increase in mitochondrial function (Seahorse assay), and enhanced synaptic connectivity, supporting the use of organoids for therapy optimization.

Claims

CLAIMS1. A method for treating a neurodegenerative disorder associated with defective DNA repair or impaired RNA processing, comprising administering a recombinant polypeptide selected from the group consisting of a wild-type Huntingtin (HTT) fragment having an amino acid identity of at least 90% to SEQ ID NO:4, Brahma-related gene 1 (BRG1) having an amino acid identity of at least 90% to SEQ ID NO:34, Brahma (BRM) having an amino acid identity of at least 90% to SEQ ID NO: 36, Matrin 3 (MATRN3) having an amino acid identity of at least 90% to SEQ ID NO:38, or a functional variant thereof, to a subject having, at risk of developing, or suspected of having a neurodegenerative disorder, wherein the functional variant retains at least 80% of the DNA repair or RNA processing activity of the respective polypeptide.

2. The method of claim 1, wherein the neurodegenerative disorder is selected from the group consisting of Alzheimer's disease, Parkinson's disease, Huntington's disease, amyotrophic lateral sclerosis (ALS), Frontotemporal dementia, Hereditary Spinocerebellar Ataxias, and multiple sclerosis.

3. A recombinant protein composition comprising a recombinant polypeptide having an amino acid sequence that is at least 90% identical to SEQ ID NO:4, SEQ ID NO:34, SEQ ID NO:36, or SEQ ID NO:38, or a functional variant thereof, wherein said protein is expressed in a host cell and is formulated for delivery to cells of the central or peripheral nervous system for the treatment of a neurodegenerative disorder.

4. The recombinant protein composition of claim 3, wherein the neurodegenerative disorder is selected from the group consisting of Alzheimer's disease, Parkinson's disease, Huntington's disease, amyotrophic lateral sclerosis (ALS), Frontotemporal dementia, Hereditary Spinocerebellar Ataxias, and multiple sclerosis.

5. A method for treating a neurodegenerative disorder comprising administering to a subject in need thereof an effective amount of the recombinant protein composition of claim 3.

6. Use of the recombinant protein composition of claim 3 for the manufacture of a medicament for the treatment of a neurodegenerative disorder associated with defective DNA repair or impaired RNA processing.

7. A pharmaceutical composition comprising the recombinant protein composition of claim 3 or an expression vector encoding the recombinant protein composition of claim 3, and a pharmaceutically acceptable carrier or excipient, wherein the expression vector is selected from the group consisting of a linear mRNA, circular RNA (cirRNA), self-amplifying RNA (saRNA), or adeno-associated viral (AAV) vector.

8. A recombinant vector comprising a nucleic acid sequence encoding the recombinant protein of claim 3, wherein said vector is capable of expressing said recombinant protein in a neuronal cell or cell-free system, and wherein the vector is selected from the group consisting of a linear mRNA, circular RNA (cirRNA), self-amplifying RNA (saRNA), or adeno-associated viral (AAV) vector.

9. A method for ameliorating defective DNA repair or impaired RNA processing comprising administering a recombinant polypeptide selected from the group consisting of an HTT fragment having an amino acid identity of at least 90% to SEQ ID NO:4, SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:38, or a functional variant thereof, to a neuron and / or non-neuronal brain cell, such as a glial cell or astrocyte.

10. The method of claim 9, further comprising administering a PNKP polypeptide or a recombinant vector encoding PNKP, wherein the vector is selected from the group consisting of a linear mRNA, circular RNA (cirRNA), self-amplifying RNA (saRNA), or adeno-associated viral (AAV) vector.

11. A method of facilitating DNA repair or RNA processing comprising administering a recombinant polypeptide selected from the group consisting of an HTT fragment having an amino acid identity of at least 90% to SEQ ID NO:4, BRG1, BRM, PNKP, MATRN3, or a functional variant thereof, to a cell or subject in need thereof.

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