Methods for treating oxidative phosphatidylcholine-related disorders

Abstract

Methods for treating TDP-43 aggregation-related diseases and disorders (e.g., ALS) using antibodies that specifically bind to OxPC or polynucleotides encoding antibodies that specifically bind to OxPC are provided herein.

Description

[Technical Field]

[0001] Methods for treating TDP-43 aggregation-related diseases and disorders (e.g., ALS) using antibodies that specifically bind to OxPC or polynucleotides encoding antibodies that specifically bind to OxPC are provided herein.

[0002] Related applications This application claims the benefit of U.S. Provisional Patent Application No. 63 / 498,932, filed April 28, 2023, the contents of which are incorporated herein by reference in their entirety.

[0003] Sequence List The electronically submitted sequence listing XML (name: 209266_SL.xml; size: 14,693 bytes; created April 17, 2024) is incorporated herein by reference in its entirety. [Background technology]

[0004] Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease that causes progressive loss of motor neurons, ultimately leading to paralysis and premature death. ALS is a multifactorial disease characterized by protein aggregation, neuroinflammation, mitochondrial dysfunction, axonal damage, and neuromuscular junction (NMJ) damage. The majority of patients diagnosed with ALS die within 3 to 5 years, often due to respiratory failure. Currently, there is no cure for ALS, and most treatments rely on symptom management.

[0005] Misfolded TAR DNA-binding protein-43 (TDP-43) has been associated with 97% of sporadic ALS cases, as well as the pathology of hereditary forms of ALS. In ALS, misfolded TDP-43 interferes with the translation of mitochondrial proteins at the neuromuscular junction (NMJ), leading to mitochondrial dysfunction. In addition to TDP-43 pathology, a major metabolic abnormality in ALS motor neurons is a dramatic increase in glycerophospholipids that induces the formation of oxidized phosphatidylcholine (OxPC), a type of oxidized phospholipid (OxPL) that is neurotoxic. OxPC is involved in multiple diseases, including multiple sclerosis and ALS, and therefore, targeting OxPC is a possible novel therapeutic approach to treat these diseases. [Overview of the project] [Problems that the invention aims to solve]

[0006] Therefore, there is a need in this field for ALS treatments that effectively reduce or eliminate symptoms. [Means for solving the problem]

[0007] A method is provided herein for inhibiting OxPL-induced neurotoxicity, inhibiting TDP-43 aggregate-induced neurotoxicity, preventing or reducing TDP-43 aggregates, and / or treating a disease or disorder associated with elevated levels of oxidized phosphatidylcholine (OxPC) in subjects requiring such treatment, by administering to a subject an antibody or antigen-binding fragment thereof that specifically binds to OxPC, or a polynucleotide encoding an antibody or antigen-binding fragment thereof that specifically binds to OxPC. The method disclosed herein is particularly advantageous because it prevents and / or reduces TDP-43 aggregates and prevents OxPC-induced neurotoxicity in subjects having a condition associated with TDP-43 aggregates (e.g., ALS).

[0008] In some embodiments, methods for inhibiting oxidized phospholipid (OxPL)-induced neurotoxicity in a subject requiring such inhibition are provided herein, comprising the steps of administering to a subject a therapeutically effective amount of (a) an antibody or antigen-binding fragment thereof that specifically binds to OxPC; or (b) a polynucleotide encoding an antibody or antigen-binding fragment thereof that specifically binds to OxPC.

[0009] In some embodiments, a method for inhibiting neurotoxicity induced by TDP-43 aggregates in a subject requiring such inhibition is provided herein, comprising the step of administering to a subject a therapeutically effective amount of (a) an antibody or antigen-binding fragment thereof that specifically binds to OxPC; or (b) a polynucleotide encoding an antibody or antigen-binding fragment thereof that specifically binds to OxPC.

[0010] In some embodiments, a method for preventing or reducing TDP-43 aggregates in a subject suffering from a condition associated with TDP-43 aggregate formation is provided herein, comprising the step of administering to a subject a therapeutically effective amount of (a) an antibody or antigen-binding fragment thereof that specifically binds to OxPC; or (b) a polynucleotide encoding an antibody or antigen-binding fragment thereof that specifically binds to OxPC.

[0011] In the embodiments, the subject has amyotrophic lateral sclerosis (ALS), Alzheimer's disease, motor neuron disease, Parkinson's disease, or frontotemporal lobar degeneration. In the embodiments, the subject has sporadic ALS (sALS).

[0012] In some embodiments, methods for treating a disease or disorder associated with elevated levels of oxidized phosphatidylcholine (OxPC) in a subject requiring treatment are provided herein, comprising the step of administering to a subject a therapeutically effective amount of (a) an antibody or antigen-binding fragment thereof that specifically binds to OxPC; or (b) a polynucleotide encoding an antibody or antigen-binding fragment thereof that specifically binds to OxPC.

[0013] In an embodiment, the disease or disorder is amyotrophic lateral sclerosis (ALS), Alzheimer's disease, motor neuron disease, Parkinson's disease, or frontotemporal lobe degeneration. In an embodiment, the disease or disorder is sporadic ALS (sALS).

[0014] In an embodiment, the antibody or antigen-binding fragment comprises a heavy chain variable region comprising complementarity-determining regions CDRH1, CDRH2, and CDRH3 of the amino acid sequence of the heavy chain variable region shown in SEQ ID NO: 7, and a light chain variable region comprising complementarity-determining regions CDRL1, CDRL2, and CDRL3 of the amino acid sequence of the light chain variable region shown in SEQ ID NO: 8.

[0015] In an embodiment, the antibody or antigen-binding fragment comprises CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and CDRL3, each comprising the amino acid sequence shown in SEQ ID NOs: 1, 2, 3, 4, 5, and 6.

[0016] In an embodiment, the antibody or its antigen-binding fragment comprises a heavy chain variable region comprising an amino acid sequence having at least 80% identity to SEQ ID NO: 7. In an embodiment, the antibody or its antigen-binding fragment comprises a light chain variable region comprising an amino acid sequence having at least 80% identity to SEQ ID NO: 8.

[0017] In an embodiment, the antibody or its antigen-binding fragment comprises a heavy chain variable region comprising an amino acid sequence having at least 80% identity to the amino acid sequence shown in SEQ ID NO: 7, and a light chain variable region comprising an amino acid sequence having at least 80% identity to the amino acid sequence shown in SEQ ID NO: 8.

[0018] In an embodiment, the antibody or its antigen-binding fragment is a humanized antibody.

[0019] In an embodiment, the antibody or its antigen-binding fragment is a single-chain variable fragment (scFv).

[0020] In the embodiment, scFv includes a peptide linker between the heavy chain variable region and the light chain variable region. In the embodiment, the peptide linker includes the amino acid sequence GGGGSGGGGSGGGGS (SEQ ID NO: 9).

[0021] In the embodiment, the polynucleotide is contained within the vector. In the embodiment, the vector is a viral vector.

[0022] In the embodiment, the viral vector is selected from the group consisting of adeno-associated viruses (AAV), adenoviruses, retroviruses, orthomyxoviruses, paramyxoviruses, papovaviruses, picornaviruses, lentiviruses, herpes simplex viruses, vaccinia viruses, poxviruses, and alphaviruses.

[0023] In the embodiment, the vector is an AAV vector contained within recombinant AAV (rAAV), where rAAV includes an AAV capsid containing an AAV capsid protein and an rAAV genome.

[0024] In this embodiment, the capsid protein is one of the following capsid proteins: clade A, clade B, clade C, clade D, clade E, clade F, clade G, clade H, clade I, AAVgo.1, AAV3, AAV4, AAV10, AAV11, AAV12, rh.32, rh32.33, rh.33, rh.34, BAAV, or AAV5, or a genetically modified variant thereof.

[0025] In one embodiment, the capsid protein contains an amino acid sequence having at least 95% identity with amino acids 193-725 of SEQ ID NO: 11. In another embodiment, the capsid protein contains an amino acid sequence having at least 99% identity with amino acids 193-725 of SEQ ID NO: 11. In yet another embodiment, the capsid protein contains the amino acid sequence of amino acids 193-725 of SEQ ID NO: 11.

[0026] In one embodiment, the capsid protein contains an amino acid sequence having at least 95% identity with amino acids 138-725 of SEQ ID NO: 11. In another embodiment, the capsid protein contains an amino acid sequence having at least 99% identity with amino acids 138-725 of SEQ ID NO: 11. In yet another embodiment, the capsid protein contains the amino acid sequence from amino acids 138-725 of SEQ ID NO: 11.

[0027] In one embodiment, the capsid protein contains an amino acid sequence having at least 95% identity with SEQ ID NO: 10 or 11. In another embodiment, the capsid protein contains an amino acid sequence having at least 99% identity with SEQ ID NO: 10 or 11. In yet another embodiment, the capsid protein contains the amino acids of SEQ ID NO: 10 or 11.

[0028] In this embodiment, the rAAV genome further includes a CBh promoter containing the nucleic acid sequence shown in SEQ ID NO: 12.

[0029] In this embodiment, the rAAV genome further comprises an SV40 polyA tail containing the nucleic acid sequence shown in SEQ ID NO: 13.

[0030] In the embodiment, rAAV is administered to the subject intravenously, intraperitoneally, subcutaneously, intramuscularly, intrathecally, or intradermally.

[0031] In this embodiment, the method neutralizes the OxPC activity in the subject.

[0032] In this embodiment, the method prevents or reduces TDP-43 aggregates.

[0033] In the embodiment, the method reduces the expression of one or more ALS-related genes. In the embodiment, the one or more ALS-related genes are apoE, COL4A1, CTSS, DAB2, TIMP1, or any combination thereof.

[0034] In the embodiment, the method reduces the expression of one or more ALS-related genes. In the embodiment, the one or more ALS-related genes are C9orf72, GRM3, SYP, GRIN2B, CHRNA7, MYD88, ITPR2, GRN, VEGFA, FKBP5, or any combination thereof.

[0035] In the embodiments, the method neutralizes neurotoxicity mediated by OxPC. In the embodiments, the method increases the electrical firing of neurons. In the embodiments, the method increases neurite outgrowth on neurons.

[0036] In the embodiment, the vector targets cortical neurons, spinal neurons, and / or astrocytes.

[0037] In this embodiment, the subject is a human subject.

[0038] In some embodiments, antibodies or antigen-binding fragments thereof that specifically bind to OxPC, or nucleic acid sequences encoding antibodies or antigen-binding fragments thereof that specifically bind to OxPC, are provided herein for use in inhibiting neurotoxicity induced by TDP-43 aggregates in subjects requiring such use, and the treatment is carried out according to any one of the methods disclosed herein.

[0039] In some embodiments, antibodies or antigen-binding fragments thereof that specifically bind to oxidized phospholipids (OxPL) or nucleic acid sequences encoding antibodies or antigen-binding fragments thereof that specifically bind to oxidized phospholipids (OxPL) are provided herein for use in inhibiting neurotoxicity induced by oxidized phospholipids (OxPL) in subjects requiring such treatment, and the treatment is carried out according to any one of the methods disclosed herein.

[0040] In some embodiments, antibodies or antigen-binding fragments thereof that specifically bind to OxPC, or nucleic acid sequences encoding antibodies or antigen-binding fragments thereof that specifically bind to OxPC, are provided herein for use in preventing or reducing TDP-43 aggregates in subjects requiring such use, and the treatment is carried out according to any one of the methods disclosed herein.

[0041] In some embodiments, antibodies or antigen-binding fragments thereof that specifically bind to OxPC, or nucleic acid sequences encoding antibodies or antigen-binding fragments thereof that specifically bind to OxPC, are provided herein for use in the manufacture of pharmaceuticals for inhibiting neurotoxicity induced by TDP-43 aggregates in subjects requiring such treatment, and the treatment is carried out according to any one of the methods disclosed herein.

[0042] In some embodiments, antibodies or antigen-binding fragments thereof that specifically bind to oxidized phospholipids (OxPL) or nucleic acid sequences encoding antibodies or antigen-binding fragments thereof that specifically bind to oxidized phospholipids (OxPL) are provided herein for use in the manufacture of pharmaceuticals for inhibiting neurotoxicity induced by oxidized phospholipids (OxPL) in subjects requiring such treatment, and the treatment is carried out according to any one of the methods disclosed herein.

[0043] In some embodiments, antibodies or antigen-binding fragments thereof that specifically bind to OxPC, or nucleic acid sequences encoding antibodies or antigen-binding fragments thereof that specifically bind to OxPC, are provided herein for use in the manufacture of pharmaceuticals for preventing or reducing TDP-43 aggregates in subjects requiring such treatment, and the treatment is carried out according to any one of the methods disclosed herein.

[0044] In some embodiments, the use of an antibody or antigen-binding fragment thereof that specifically binds to OxPC, or a nucleic acid sequence encoding an antibody or antigen-binding fragment thereof that specifically binds to OxPC, is provided herein for inhibiting neurotoxicity induced by TDP-43 aggregates in subjects requiring such treatment, and the treatment is carried out according to any one of the methods disclosed herein.

[0045] In some embodiments, the use of an antibody or antigen-binding fragment thereof that specifically binds to oxidized phospholipids (OxPL) or a nucleic acid sequence encoding an antibody or antigen-binding fragment thereof that specifically binds to oxidized phospholipids (OxPL) is provided herein for inhibiting neurotoxicity induced by OxPL in subjects requiring such treatment, and the treatment is carried out according to any one of the methods disclosed herein.

[0046] In some embodiments, the use of an antibody or antigen-binding fragment thereof that specifically binds to OxPC, or a nucleic acid sequence encoding an antibody or antigen-binding fragment thereof that specifically binds to OxPC, for preventing or reducing TDP-43 aggregates in subjects requiring such treatment, wherein the treatment is carried out according to any one of the methods disclosed herein. [Brief explanation of the drawing]

[0047] [Figure 1] This graph shows OxPC expression in SOD1G93AALS motor neurons and healthy motor neurons (%OxPC+ cells). Values ​​are expressed as mean (healthy=1.000; SOD1G93A=1.239) ± SEM and normalized to healthy neurons; ***p=0.0008. Unpaired two-tailed t-test; 3 independent experiments, N=2-4 replicates each. [Figure 2A]This graph shows the effect of OxPC (PONPC) treatment on neurite outgrowth in healthy motor neurons. Values ​​are expressed as mean ± SEM and normalized to the 25 μM PSPC condition. Two-way ANOVA with Tukey post-hoc test (F-statistic = 25.67; p = 0.0368) (p < 0.05); n = 3 independent experiments, 1-4 replicates. [Figure 2B-C] Figure 2B is a graph showing the elevated apoE profile (% positive cells and mean intensity) in SOD1G93AALS motor neurons compared to healthy motor neurons. Values ​​are expressed as mean ± SEM and normalized to healthy neurons; *p=0.0241, ****p<0.0001. Unpaired two-tailed t-test; two independent experiments, n=2 replicates each. Figure 2C is a graph showing the elevated apoE profile in SOD1G93AALS motor neurons treated with 24 hours of OxPC treatment + 6 hours of washing or 24 hours of OxPC treatment + 24 hours of washing. Scale bar = 20 μM (apoE, FITC). [Figure 2D-E] Figure 2D is a graph showing increased TDP-43 aggregation in healthy motor neurons treated with OxPC or TDP-43M337VALS motor neurons treated with OxPC. Figure 2E is a graph showing pTDP-43 levels in healthy motor neurons treated with OxPC. [Figure 3A-B] Figure 3A is a graph showing the differential directional properties of AAV-mE06 across human-derived neurons and astrocytes (GFP(FITC), green fluorescent protein). Scale bar = 300 μM. Figure 3B shows the expression of mE06-scFv transcript after AAV transduction at various MOIs in motor neurons and astrocytes. The scatter plot shows the mean gene expression, normalized to HPrt1 and compared to the non-transduction condition (background). N=3 (Hprt1, hypoxanthine phosphoribosyltransferase 1). [Figure 3C]This figure shows mE06-scFv protein expression in motor neurons and astrocytes after AAV transduction. Both cell types were transduced with AAV-mE06-scFv at various MOIs. The scatter plot shows the mean gene expression. The maximum signal (OD450) using 4× dilution was used to estimate the scFv concentration. N=3. [Figure 4A-B] Figure 4A is a graph showing TDP-43 aggregation in healthy motor neurons and TDP-43M337VALS motor neurons transduced with AAV5.2-mE06 and treated with OxPC. Figure 4B is a graph showing the number of axons in SOD1G93AALS motor neurons transduced with AAV5.2-mE06 and treated with OxPC. Values ​​are expressed as mean ± SEM and normalized to 25 μM PSPC conditions. The number of axons was counted in the distal compartment according to the ImageJ plugin and Tuj-1 used as a neuronal marker. N=2 experiments. [Figure 4C] This graph shows the electrical bursts in healthy motor neurons and TDP-43M337VALS motor neurons transduced using AAV5.2-mE06 and treated with OxPC. Values ​​are expressed as mean ± SEM and normalized for untransduced neurons treated with OxPC (PONPC). N=4 experiments. [Figure 5A-B] Figure 5A is a graph showing the motor dysfunction score in a sALS-CSF mouse model transduced using AAV5.2-mE06. Figure 5B is a graph showing the normalized grip strength relative to baseline in a sALS-CSF mouse model transduced using AAV5.2-mE06. [Figure 5C] This graph shows the number of choline acetyltransferase-positive (ChAT+) motor neurons in sALS-CSF mouse models transduced using AAV5.2-mE06 and treated with OxPC. Motor impairment scores, normalized grip strength scores, and immunostaining intensity were analyzed using one-way ANOVA with Bonferroni post-hoc analysis. N=3-6 mice per experiment. [Figure 6A-B] Figures 6A–G are a series of graphs showing the concentrations of various OxPC molecular species in ng / mL in plasma from wild-type, PBS-treated SOD1G93A transgenic mice, and SOD1G93A transgenic mice treated with AAV5.2-mE06. Differences between OxPC concentrations were compared using an unpaired t-test with Welch correction (unequal standard deviation). [Figure 6C-D] Figures 6A–G are a series of graphs showing the concentrations of various OxPC molecular species in ng / mL in plasma from wild-type, PBS-treated SOD1G93A transgenic mice, and SOD1G93A transgenic mice treated with AAV5.2-mE06. Differences between OxPC concentrations were compared using an unpaired t-test with Welch correction (unequal standard deviation). [Figure 6E-F] Figures 6A–G are a series of graphs showing the concentrations of various OxPC molecular species in ng / mL in plasma from wild-type, PBS-treated SOD1G93A transgenic mice, and SOD1G93A transgenic mice treated with AAV5.2-mE06. Differences between OxPC concentrations were compared using an unpaired t-test with Welch correction (unequal standard deviation). [Figure 6G] Figures 6A–G are a series of graphs showing the concentrations of various OxPC molecular species in ng / mL in plasma from wild-type, PBS-treated SOD1G93A transgenic mice, and SOD1G93A transgenic mice treated with AAV5.2-mE06. Differences between OxPC concentrations were compared using an unpaired t-test with Welch correction (unequal standard deviation). [Modes for carrying out the invention]

[0048] This specification provides a method for inhibiting neurotoxicity induced by oxidized phospholipids (OxPL), inhibiting neurotoxicity induced by TDP-43 aggregates, preventing or reducing TDP-43 aggregates, and / or treating diseases or disorders associated with elevated levels of oxidized phosphatidylcholine (OxPC) in subjects requiring such treatment, by administering to a subject an antibody or antigen-binding fragment thereof that specifically binds to OxPC, or a polynucleotide encoding an antibody or antigen-binding fragment thereof that specifically binds to OxPC. The method disclosed herein is particularly advantageous because it prevents and / or reduces TDP-43 aggregates and prevents OxPC-induced neurotoxicity in subjects having a condition associated with TDP-43 aggregates (e.g., ALS).

[0049] I. Definition As used herein, the terms “antibody” and “antibodies” include full-length antibodies, antigen-binding fragments of full-length antibodies, and molecules containing antibody CDR, VH region, and / or VL region. Examples of antibodies include, but are not limited to, monoclonal antibodies, recombinant antibodies, monospecific antibodies, multispecific antibodies (including bispecific antibodies), human antibodies, humanized antibodies, chimeric antibodies, immunoglobulins, synthetic antibodies, tetrameric antibodies containing two heavy chain molecules and two light chain molecules, antibody light chain monomers, antibody heavy chain monomers, antibody light chain dimers, antibody heavy chain dimers, antibody light chain-antibody heavy chain pairs, intrabodies, heteroconjugate antibodies, antibody-drug conjugates, single-domain antibodies, monovalent antibodies, single-chain antibodies or single-chain Fv(scFv), camelized antibodies, aphibodies, F(ab')2 fragments, disulfide-linked Fv(sdFv), anti-idiotype (anti-Id) antibodies (e.g., anti-anti-Id antibodies), variable domains (VNARs) of novel antigen receptors, antigen-binding (Fab) fragments, monobodies, DARPin, VHH antibodies, and any of the antigen-binding fragments listed above. Antibodies may be of any type of immunoglobulin molecule (e.g., IgG, IgE, IgM, IgD, IgA, or IgY), any class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, or IgA2), or any subclass (e.g., IgG2a or IgG2b).

[0050] As used herein, the term “CDR” or “complementarity-determining region” means a discontinuous antigen-binding site found within the variable regions of heavy and light chain polypeptides. These specific regions have been described, for example, by Kabat et al., J. Biol. Chem. 252, 6609-6616 (1977) and Kabat et al., Sequences of proteins of immunological interest. (1991), by Chothia et al., J. Mol. Biol. 196:901-917 (1987), and by MacCallum et al., J. Mol. Biol. 262:732-745 (1996), all of which are incorporated herein by reference in their entirety, in which the definition includes duplication or subsets of amino acid residues when compared to one another. In embodiments, "CDR" is the CDR as defined by MacCallum et al., J. Mol. Biol. 262:732-745 (1996) and Martin A. "Protein Sequence and Structure Analysis of Antibody Variable Domains," in Antibody Engineering, Kontermann and Dubel, eds., Chapter 31, pp. 422-439, Springer-Verlag, Berlin (2001). In embodiments, the term "CDR" is the CDR as defined by Kabat et al., J. Biol. Chem. 252, 6609-6616 (1977) and Kabat et al., Sequences of proteins of immunological interest. (1991). In embodiments, the heavy chain CDR and light chain CDR of an antibody are defined using different conventions. In the embodiment, the heavy chain CDR and / or light chain CDR are defined by performing a structural analysis of the antibody and identifying residues in the variable region that are expected to come into contact with the epitope region of the target molecule.CDRH1, CDRH2, and CDRH3 represent heavy-chain CDRs, while CDRL1, CDRL2, and CDRL3 represent light-chain CDRs.

[0051] As used herein, the terms “variable region” and “variable domain” are interchangeable and common in the art. A variable region is a portion of an antibody, typically a portion of the light or heavy chain, that differs significantly in sequence between antibodies and is used in the binding and specificity of a particular antibody to a particular antigen. It is typically the amino-terminal portion of the mature heavy chain (approximately 110–120 amino acids or 110–125 amino acids) and the mature light chain (approximately 90–115 amino acids). Sequence variability is concentrated in a region called the complementarity-determining region (CDR), and more highly conserved regions within the variable region are called the framework region (FR). Without intending to adhere to any particular mechanism or theory, the CDRs of the light and heavy chains are considered to be primarily responsible for the antibody’s interaction with the antigen and its specificity. In certain embodiments, the variable region is a human variable region. In certain embodiments, the variable region includes rodent or mouse CDRs and human framework regions (FRs). In the embodiment, the variable region is a primate (e.g., non-human primate) variable region. In the embodiment, the variable region includes a rodent or mouse CDR and a primate (e.g., non-human primate) framework region (FR).

[0052] As used herein, the terms "VH" and "VL" refer to the antibody heavy chain and light chain variable regions, respectively, as described in Kabat et al., (1991) Sequences of proteins of immunological interest (NIH Publication No. 91-3242, Bethesda), which are incorporated herein by reference in their entirety.

[0053] As used herein, the term “constant region” is common in the art. The constant region is an antibody moiety, such as the carboxyl-terminal portion of the light and / or heavy chain, which does not directly contribute to the antibody’s binding to an antigen but can exhibit various effector functions, such as interactions with Fc receptors (e.g., Fcγ receptors).

[0054] As used herein, the term “heavy chain” may mean any of the different types, e.g., alpha (α), delta (δ), epsilon (ε), gamma (γ), and mu (μ), based on the amino acid sequence of the constant region that gives rise to the IgA, IgD, IgE, IgG, and IgM classes of the antibody, including subclasses of IgG such as IgG1, IgG2, IgG3, and IgG4, respectively.

[0055] As used herein, the term “light chain,” when used in reference to an antibody, may mean either a different type, for example, kappa (κ) or lambda (λ), based on the amino acid sequence of the constant region. Light chain amino acid sequences are well known in the art. In embodiments, the light chain is a human light chain.

[0056] As used herein, the term "AAV" is a standard abbreviation for adeno-associated virus.

[0057] As used herein, the terms “recombinant adeno-associated virus” or “rAAV” mean an AAV containing a genome lacking functional rep and cap genes.

[0058] As used herein, the term “cap gene” means the nucleic acid sequence encoding the capsid protein. With respect to AAV, the capsid protein may be VP1, VP2, or VP3. The VP1, VP2, and / or VP3 capsid proteins assemble into a capsid surrounding the rAAV genome.

[0059] As used herein, the term “rep gene” means a nucleic acid sequence that encodes a non-structural protein (e.g., rep78, rep68, rep52, and rep40) required for the replication and production of AAV.

[0060] As used herein, the term “rAAV genome” means a nucleic acid molecule (e.g., DNA and / or RNA) containing the rAAV genome sequence. Those skilled in the art will understand that if the rAAV genome contains a transgene (e.g., an antibody), the rAAV genome may be oriented in a sense or antisense direction relative to the transcription direction of the transgene.

[0061] As used herein, “isolated polynucleotide” means a polynucleotide isolated from one or more nucleic acid molecules present in a natural source of polynucleotides.

[0062] As used herein, "percent identity" between two nucleotide sequences or two amino acid sequences is calculated by multiplying the number of matches between the aligned sequence pair by 100 and dividing by the length of the aligned region, including internal gaps. Identity scoring counts only perfect matches and does not consider the degree of amino acid similarity to each other. Where a sequence is described herein as being of a certain percentage identity with respect to a reference sequence, the percentage identity with respect to the reference sequence is determined over the entire length of the reference sequence.

[0063] As used herein, the term "subject" may refer to either a human or a non-human animal. In some embodiments, the subject is a non-human mammal. In some embodiments, the subject is a human.

[0064] As used herein, the term “effective dose” in the context of administering AAV to a subject means the amount of AAV that achieves the desired prophylactic or therapeutic effect.

[0065] As used herein, the terms “about” or “approximately” refer to measurable values ​​such as dosages and include variations of ±20%, ±10%, ±5%, ±1%, or ±0.1% of a given value or range that is appropriate for performing the methods disclosed herein.

[0066] II. Antioxidant phosphatidylcholine (OxPC) antibodies Antibodies that specifically bind to OxPC (i.e., anti-OxPC antibodies) that are useful in the methods described herein include, but are not limited to, those listed below.

[0067] In the embodiment, the antibody includes a heavy chain variable region (VH) containing the CDRH1, CDRH2, and CDRH3 amino acid sequences of the VH amino acid sequence shown in SEQ ID NO: 7. In the embodiment, the antibody includes a heavy chain variable region (VH) containing the CDRH1, CDRH2, and CDRH3 amino acid sequences of the VH amino acid sequence shown in SEQ ID NO: 7 and a light chain variable region (VL) containing the CDRL1, CDRL2, and CDRL3 amino acid sequences of the VL amino acid sequence shown in SEQ ID NO: 8.

[0068] In the embodiment, the antibody contains the CDRH1, CDRH2, and CDRH3 amino acid sequences shown in SEQ ID NOs: 1, 2, and 3, respectively. In the embodiment, the antibody contains the CDRL1, CDRL2, and CDRL3 amino acid sequences shown in SEQ ID NOs: 4, 5, and 6, respectively. In the embodiment, the antibody contains the CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and CDRL3 amino acid sequences shown in SEQ ID NOs: 1, 2, 3, 4, 5, and 6, respectively.

[0069] In the embodiment, the antibody comprises VH containing the amino acid sequence shown in SEQ ID NO: 7. In the embodiment, the antibody comprises VL containing the amino acid sequence shown in SEQ ID NO: 8. In the embodiment, the antibody comprises VH containing an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence shown in SEQ ID NO: 7; and VL containing an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence shown in SEQ ID NO: 8.

[0070] In this embodiment, the antibody comprises VH containing the amino acid sequence shown in SEQ ID NO: 7 and VL containing the amino acid sequence shown in SEQ ID NO: 8.

[0071] In the embodiments, the antibody includes a heavy chain constant region selected from the group consisting of human IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. In the embodiments, the heavy chain constant region is IgG1. In the embodiments, the heavy chain constant region is IgG2. In the embodiments, the antibody includes a human κ light chain constant region or a human λ light chain constant region.

[0072] In the embodiment, the antibody is scFv. In the embodiment, the antibody is mouse E06 (mE06), and its amino acid sequence is provided in Table 1 below.

[0073] [Table 1] TIFF2026516795000003.tif43165

[0074] In the embodiment, scFv includes a peptide linker between the heavy chain variable region and the light chain variable region. In the embodiment, the peptide linker includes the amino acid sequence GGGGSGGGGSGGGGS (SEQ ID NO: 9).

[0075] III. Polynucleotides and Vectors Polynucleotides and vectors encoding antibodies or antigen-binding fragments thereof (i.e., anti-OxPC antibodies) that are useful in the methods described herein include, but are not limited to, those listed below.

[0076] In some embodiments, a method for inhibiting oxidized phospholipid (OxPL)-induced neurotoxicity in a subject requiring such inhibition is provided herein, comprising the step of administering to a subject a nucleic acid sequence encoding an antibody or an antigen-binding fragment thereof that specifically binds to a therapeutically effective amount of OxPC.

[0077] In some embodiments, a method for inhibiting neurotoxicity induced by TDP-43 aggregates in a subject requiring such inhibition is provided herein, comprising the step of administering to a subject a therapeutically effective amount of (a) an antibody or antigen-binding fragment thereof that specifically binds to OxPC; or (b) a polynucleotide encoding an antibody or antigen-binding fragment thereof that specifically binds to OxPC.

[0078] In some embodiments, a method for preventing or reducing TDP-43 aggregates in a subject suffering from a condition associated with TDP-43 aggregate formation is provided herein, comprising the step of administering to a subject a therapeutically effective amount of (a) an antibody or antigen-binding fragment thereof that specifically binds to OxPC; or (b) a polynucleotide encoding an antibody or antigen-binding fragment thereof that specifically binds to OxPC.

[0079] In some embodiments, methods for treating a disease or disorder associated with elevated levels of oxidized phosphatidylcholine (OxPC) in a subject requiring treatment are provided herein, comprising the step of administering to a subject a therapeutically effective amount of (a) an antibody or antigen-binding fragment thereof that specifically binds to OxPC; or (b) a polynucleotide encoding an antibody or antigen-binding fragment thereof that specifically binds to OxPC.

[0080] In this embodiment, the polynucleotide is contained within the vector.

[0081] In the embodiments, the vector is a nonviral vector. Examples of nonviral vectors include, but are not limited to, plasmid DNA, transposons, episomal plasmids, minicircles, ministrings, and oligonucleotides (e.g., mRNA, naked DNA). In the embodiments, the nonviral vector is a transposon-based vector. In the embodiments, the nonviral vector is a PiggyBac-based vector or a Sleeping Beauty-based vector.

[0082] In the embodiments, the vector is a viral vector. The viral vector may or may not be able to replicate. The viral vector may be integrating or non-integrating. Numerous virus-based systems have been developed for gene transfer into mammalian cells, and suitable viral vectors can be selected by those skilled in the art. Exemplary viral vectors include, but are not limited to, adenovirus vectors (e.g., adenovirus type 5), adeno-associated virus (AAV) vectors (e.g., AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9), retrovirus vectors (e.g., MMSV, MSCV), lentivirus vectors (e.g., HIV-1, HIV-2), gamma-retrovirus vectors, herpesvirus vectors (e.g., HSV1, HSV2), alphavirus vectors (e.g., SFV, SIN, VEE, M1), flaviviruses (e.g., Kunzin, West Nile, dengue virus), rhabdovirus vectors (e.g., rabies virus, VSV), measles virus vectors, Newcastle disease virus vectors, poxvirus vectors, and picornavirus vectors (e.g., coxsackievirus). In the embodiment, the viral vector is selected from the group consisting of adeno-associated viruses (AAV), adenoviruses, retroviruses, orthomyxoviruses, paramyxoviruses, papovaviruses, picornaviruses, lentiviruses, herpes simplex viruses, vaccinia viruses, poxviruses, and alphaviruses.

[0083] In the embodiment, the vector targets cortical neurons, spinal neurons, and / or astrocytes.

[0084] In the embodiment, the vector is an AAV vector contained within recombinant AAV (rAAV), where rAAV includes an AAV capsid containing an AAV capsid protein and an rAAV genome.

[0085] In this embodiment, the capsid protein is one of the following capsid proteins: clade A, clade B, clade C, clade D, clade E, clade F, clade G, clade H, clade I, AAVgo.1, AAV3, AAV4, AAV10, AAV11, AAV12, rh.32, rh32.33, rh.33, rh.34, BAAV, or AAV5, or a genetically modified variant thereof.

[0086] In the embodiments, the capsid protein is a genetically modified mutant capsid protein containing amino acid sequences derived from at least two different AAV capsid proteins. In the embodiments, the capsid protein contains amino acid sequences derived from AAV2 and AAV5 capsid proteins. In the embodiments, the capsid protein contains the VP1 amino acid sequence derived from AAV2 and the VP2 and VP3 amino acid sequences derived from AAV5.

[0087] In one embodiment, the capsid protein contains an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 98% identity with amino acids 193-725 of SEQ ID NO: 11. In another embodiment, the capsid protein contains an amino acid sequence having at least 99% identity with amino acids 193-725 of SEQ ID NO: 11. In yet another embodiment, the capsid protein contains the amino acid sequence of amino acids 193-725 of SEQ ID NO: 11.

[0088] In one embodiment, the capsid protein contains an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 98% identity with amino acids 138-725 of SEQ ID NO: 11. In another embodiment, the capsid protein contains an amino acid sequence having at least 99% identity with amino acids 138-725 of SEQ ID NO: 11. In yet another embodiment, the capsid protein contains the amino acid sequence of amino acids 138-725 of SEQ ID NO: 11.

[0089] In one embodiment, the capsid protein contains an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 98% identity with SEQ ID NO: 10 or 11. In another embodiment, the capsid protein contains an amino acid sequence having at least 99% identity with SEQ ID NO: 10 or 11. In yet another embodiment, the capsid protein contains the amino acids of SEQ ID NO: 10 or 11.

[0090] In one embodiment, the rAAV genome further comprises a CBh promoter containing a nucleic acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 98% identity with SEQ ID NO: 12.

[0091] TIFF2026516795000004.tif66165TIFF2026516795000005.tif35166

[0092] In the embodiment, the rAAV genome further comprises an SV40 polyA tail containing a nucleic acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 98% identity with SEQ ID NO: 13.

[0093] TIFF2026516795000006.tif23165

[0094] In the embodiment, the capsid protein comprises peptides that target cortical neurons, spinal neurons, and / or astrocytes.

[0095] In the embodiment, rAAV comprises an AAV capsid containing an AAV capsid protein, and an rAAV genome containing a nucleic acid encoding an antibody or an antigen-binding fragment thereof that specifically binds to OxPC.

[0096] In embodiments, the rAAV genome further comprises a 5' inverted end repeat (5'ITR) nucleotide sequence and a 3' inverted end repeat (3'ITR) nucleotide sequence. Any ITR sequence or variant thereof derived from any AAV serotype can be used in the rAAV genome disclosed herein. The 5' and 3'ITRs may be derived from the same serotype of AAV or from different serotypes of AAV.

[0097] Exemplary capsid protein sequences are disclosed in Table 2 below.

[0098] [Table 2] TIFF2026516795000008.tif95166

[0099] IV. Treatment method In some embodiments, methods for inhibiting oxidized phospholipid (OxPL)-induced neurotoxicity in a subject requiring such inhibition are provided herein, comprising the steps of administering to a subject a therapeutically effective amount of (a) an antibody or antigen-binding fragment thereof that specifically binds to OxPC; or (b) a polynucleotide encoding an antibody or antigen-binding fragment thereof that specifically binds to OxPC.

[0100] In some embodiments, a method for inhibiting neurotoxicity induced by TDP-43 aggregates in a subject requiring such inhibition is provided herein, comprising the step of administering to a subject a therapeutically effective amount of (a) an antibody or antigen-binding fragment thereof that specifically binds to OxPC; or (b) a polynucleotide encoding an antibody or antigen-binding fragment thereof that specifically binds to OxPC.

[0101] In some embodiments, a method for preventing or reducing TDP-43 aggregates in a subject suffering from a condition associated with TDP-43 aggregate formation is provided herein, comprising the step of administering to a subject a therapeutically effective amount of (a) an antibody or antigen-binding fragment thereof that specifically binds to OxPC; or (b) a polynucleotide encoding an antibody or antigen-binding fragment thereof that specifically binds to OxPC.

[0102] In the embodiments, the subject has amyotrophic lateral sclerosis (ALS), Alzheimer's disease, motor neuron disease, Parkinson's disease, or frontotemporal lobar degeneration. In the embodiments, the subject has sporadic ALS (sALS).

[0103] In some embodiments, methods for treating a disease or disorder associated with elevated levels of oxidized phosphatidylcholine (OxPC) in a subject requiring treatment are provided herein, comprising the step of administering to a subject a therapeutically effective amount of (a) an antibody or antigen-binding fragment thereof that specifically binds to OxPC; or (b) a polynucleotide encoding an antibody or antigen-binding fragment thereof that specifically binds to OxPC.

[0104] In the embodiments, the disease or disorder is amyotrophic lateral sclerosis (ALS), Alzheimer's disease, motor neuron disease, Parkinson's disease, or frontotemporal lobar degeneration. In the embodiments, the disease or disorder is sporadic ALS (sALS).

[0105] In the embodiment, rAAV is administered to the subject intravenously, intraperitoneally, subcutaneously, intramuscularly, intrathecally, or intradermally.

[0106] In this embodiment, the method neutralizes the OxPC activity in the subject.

[0107] In the embodiments, the method prevents or reduces TDP-43 aggregates. In the embodiments, the method results in a reduction of approximately 1, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100% of TDP-43 aggregates in the subject.

[0108] In the embodiments, the method reduces the expression of one or more ALS-related genes. In the embodiments, the one or more ALS-related genes are apoE, COL4A1, CTSS, DAB2, TIMP1, or any combination thereof. In the embodiments, the method results in a reduction of approximately 1, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100% of the expression of one or more ALS-related genes in the subject.

[0109] In the embodiments, the method increases the expression of one or more ALS-related genes. In the embodiments, the one or more ALS-related genes are C9orf72, GRM3, SYP, GRIN2B, CHRNA7, MYD88, ITPR2, GRN, VEGFA, FKBP5, or any combination thereof. In the embodiments, the method results in an increase of approximately 1, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100% in the expression of one or more ALS-related genes in the subject.

[0110] In the embodiments, the method neutralizes neurotoxicity mediated by OxPC. In the embodiments, the method increases the electrical firing of neurons. In the embodiments, the method increases neurite outgrowth on neurons.

[0111] In the embodiment, the vector targets cortical neurons, spinal neurons, and / or astrocytes.

[0112] In this embodiment, the subject is a human subject.

[0113] In embodiments, the method further includes the step of administering an additional therapeutic agent.

[0114] In some embodiments, antibodies or antigen-binding fragments thereof that specifically bind to OxPC, or polynucleotides encoding antibodies or antigen-binding fragments thereof that specifically bind to OxPC, are provided herein for use in inhibiting neurotoxicity induced by TDP-43 aggregates in subjects requiring such use, and the treatment is carried out according to any one of the methods disclosed herein.

[0115] In some embodiments, antibodies or antigen-binding fragments thereof that specifically bind to oxidized phospholipids (OxPL) or polynucleotides encoding antibodies or antigen-binding fragments thereof that specifically bind to oxidized phospholipids (OxPL) are provided herein for use in inhibiting neurotoxicity induced by oxidized phospholipids in subjects requiring such treatment, and the treatment is carried out according to any one of the methods disclosed herein.

[0116] In some embodiments, antibodies or antigen-binding fragments thereof that specifically bind to OxPC, or polynucleotides encoding antibodies or antigen-binding fragments thereof that specifically bind to OxPC, are provided herein for use in preventing or reducing TDP-43 aggregates in subjects requiring such use, and the treatment is carried out according to any one of the methods disclosed herein.

[0117] In some embodiments, antibodies or antigen-binding fragments thereof that specifically bind to OxPC, or polynucleotides encoding antibodies or antigen-binding fragments thereof that specifically bind to OxPC, are provided herein for use in the manufacture of pharmaceuticals for inhibiting neurotoxicity induced by TDP-43 aggregates in subjects requiring such treatment, and the treatment is carried out according to any one of the methods disclosed herein.

[0118] In some embodiments, antibodies or antigen-binding fragments thereof that specifically bind to oxidized phospholipids (OxPL) or polynucleotides encoding antibodies or antigen-binding fragments thereof that specifically bind to oxidized phospholipids (OxPL) are provided herein for use in the manufacture of pharmaceuticals for inhibiting neurotoxicity induced by oxidized phospholipids (OxPL) in subjects requiring such treatment, and the treatment is carried out according to any one of the methods disclosed herein.

[0119] In some embodiments, antibodies or antigen-binding fragments thereof that specifically bind to OxPC, or polynucleotides encoding antibodies or antigen-binding fragments thereof that specifically bind to OxPC, are provided herein for use in the manufacture of pharmaceuticals for preventing or reducing TDP-43 aggregates in subjects requiring such treatment, and the treatment is carried out according to any one of the methods disclosed herein.

[0120] In some embodiments, the use of an antibody or antigen-binding fragment thereof that specifically binds to OxPC, or a polynucleotide encoding an antibody or antigen-binding fragment thereof that specifically binds to OxPC, is provided herein for inhibiting neurotoxicity induced by TDP-43 aggregates in subjects requiring such treatment, and the treatment is carried out according to any one of the methods disclosed herein.

[0121] In some embodiments, the use of an antibody or antigen-binding fragment thereof that specifically binds to oxidized phospholipids (OxPL) or a polynucleotide encoding an antibody or antigen-binding fragment thereof that specifically binds to oxidized phospholipids (OxPL) is provided herein for the purpose of inhibiting neurotoxicity induced by oxidized phospholipids in subjects requiring such treatment, and the treatment is carried out according to any one of the methods disclosed herein.

[0122] In some embodiments, the use of an antibody or antigen-binding fragment thereof that specifically binds to OxPC, or a polynucleotide encoding an antibody or antigen-binding fragment thereof that specifically binds to OxPC, for preventing or reducing TDP-43 aggregates in subjects requiring such treatment, wherein the treatment is carried out according to any one of the methods disclosed herein. [Examples]

[0123] Example 1: OxPC induces ALS-like transcriptome signatures in motor neurons. Familial ALS (fALS) accounts for 5-10% of ALS diagnoses, and cell lines carrying fALS mutations can be used to study the ALS mechanism in vitro. Therefore, two well-characterized ALS motor neuron cell lines (TDP-43) are available. M337V and SOD1 G93A The OxPC profile and transcriptome in ALS were evaluated by comparing them with healthy motor neurons treated with OxPC.

[0124] (a) OxPC Profile To evaluate the OxPC profile, cells were washed with PBS, permeabilized with 0.5% saponin (47036-50G-F, Sigma) for 15 minutes, washed three times with PBS, and incubated with blocking solution (5% goat serum [31873, Thermo Fisher Scientific]) at room temperature for 45 minutes. Subsequently, cells were incubated overnight at 4°C or at room temperature for 1 hour with mouse anti-phosphatidylcholine antibody (Absolute Antibody) diluted in blocking solution. After three washes in 1% goat serum, cells were incubated at room temperature for 1 hour in the dark with secondary antibody diluted in blocking solution, followed by two washes with PBS and incubated at room temperature for 10 minutes in the dark with Hoechst (1:10000 in PBS). After one wash with PBS, cells were stored at 4°C until imaging. The results from this analysis compared SOD1 levels in healthy motor neurons. G93A This showed an increase in the number of OxPC-positive cells in ALS motor neurons (Figure 1).

[0125] (b) Transcriptome analysis Next, to identify the molecular mechanism by which SOD1 is activated in the presence of OxPC, G93A Transcriptome analysis of ALS motor neurons and healthy motor neurons was performed using NanoString technology.

[0126] 1-Palmitoyl-2-(9-oxo-nonanoyl)-sn-glycero-3-phosphocholine (PONPC) (870605P-1MG, Avanti Polar Lipids), one of the main OxPC molecular species in rat, pig, and human brains, was used as the OxPC treatment in this analysis. First, PONPC was dissolved in 100% ethanol (EtOH), heated to 35°C, and then sonicated (37 kHz) for 30–60 seconds. The PONPC solution was diluted to 2× final concentration in complete motor neuron maintenance medium. Motor neurons were treated with 25–100 μM PONPC and incubated for 24 hours, or incubated for 24 hours followed by washing for 24 hours. 0.3% EtOH was used as a vehicle control. Motor neuron samples were prepared for NanoString analysis according to the NanoString Gene Expression CodeSet RNA Hybridization Protocol (MAN-10056-05). Two pre-designed NanoString neuroscience panels were selected for gene expression analysis: (a) neuropathology and (b) neuroinflammation. Quality control and expression changes were analyzed using nSolver 4.0 analysis software.

[0127] Of the 770 transcripts included in each panel, exposure of healthy motor neurons to OxPC resulted in the differential expression of 174 genes (neuropathology) and 161 genes (neuroinflammation). G93A OxPC exposure in ALS motor neurons resulted in differential expression of 137 genes (neuroinflammation). Of all transcripts analyzed in healthy motor neurons, approximately 25% showed a response to OxPC exposure. A similar profile was observed in SOD1. G93AObserved in the same analysis performed on ALS motor neurons. Gene ontology (GO) analysis of genes differentially expressed after OxPC treatment in healthy motor neurons revealed that the most prominent pathways were response to stimulus, programmed cell death, apoptosis, regulation of molecular function, transcriptome reorganization, and protein phosphorylation. When exposed to OxPC, both healthy motor neurons and SOD1 G93A Approximately 50% overlap was found between differentially expressed genes in both healthy and SOD1

[0128] TDP-43 M337V and SOD1 G93A ALS motor neurons. Transcriptome analysis of ALS motor neurons was performed to find common gene expression signatures between both cell lines. TDP-43 M337V and SOD1 G93A Similarities were observed in each panel between the transcriptomes (NP = 35.08%; NI = 34.35%). Considerable transcriptome overlap was also found between healthy motor neurons exposed to OxPC and SOD1 G93A ALS motor neurons (NP = 39.69%; NI = 50.31%), and to a lesser extent, between healthy motor neurons and TDP-43 M337V motor neurons (NP = 28.48%; NI = 50.31%). Of all differentially expressed transcripts, 31.03% (NP) and 22.58% (NI) were common among the three types of motor neuron cell lines (healthy, TDP-43 M337V , and SOD1 G93A ).

[0129] Data mining analysis was performed using the transcriptome data described above, as well as two independent ALS transcriptome datasets: (1) ALSoD (Abel et al. 2012) and (2) postmortem spinal cord tissue from patients with sporadic ALS (D'Erchia et al. 2017). The analysis revealed that 20% of transcripts previously associated with ALS altered expression after OxPC treatment of healthy motor neurons. These transcripts are listed in Table 3, along with a brief description of their function.

[0130] [Table 3] TIFF2026516795000010.tif188168

[0131] Example 2: OxPC induces phenotypic changes in ALS motor neurons, including disruption of neural networks and TDP-43 aggregation. Fully differentiated health and TDP-43 M337V ALS motor neurons were cultured and matured, then exposed to PONPC(OxPC). Subsequently, neurite outgrowth, lipid peroxidation characteristics, phenotypic changes including TDP-43 aggregation and phosphorylation, and apoE profiles were evaluated.

[0132] Healthy motor neurons and TDP-43 M337V ALS motor neurons were treated for 24 hours with either 25 μM PONPC or 1-palmitoyl-2-stearoyl-sn-glycero-3-phosphocholine (PSPC) control. Mean neurite outgrowth was quantified by running a neurite tracing protocol on CellReporterXpress software, and the percentage of positive cells and the average intensity of all cells were quantified by running a cell scoring protocol. Exposure of healthy motor neurons to OxPC (25 μM) resulted in a dramatic decrease in neurite outgrowth (Figure 2A). Mitochondrial dysfunction is an important marker of oxidative stress. However, healthy motor neurons or TDP-43 M337VNo differences in mitochondrial profiles were observed in any of the ALS motor neurons after OxPC exposure.

[0133] In the CNS, apoE is involved in lipid redistribution to supply cholesterol and phospholipids required for its biological processes to various cell types. When body fluids from ALS patients were analyzed, a preferential OxPL content was found for apoE in cerebrospinal fluid (CSF), but not in plasma. To further confirm the association between apoE, OxPC, and ALS, apoE expression was evaluated in ALS motor neurons treated with OxPC and healthy motor neurons. Cells were washed with PBS, permeabilized with 0.1% Triton X-100 for 15 minutes at room temperature, washed three times with PBS, and incubated with blocking solution (3% BSA [422371X, VWR]) for 30 minutes at room temperature. Cells were incubated with diluted mouse anti-apoE antibody (NovusBio) in blocking solution overnight at 4°C or for 1 hour at room temperature. Cells were washed three times with PBS and then incubated with diluted secondary antibody in blocking solution in the dark for 1 hour at room temperature. The cells were washed twice with PBS and incubated at room temperature for 10 minutes in the dark with Hoechst (H3570, Invitrogen) diluted 1:10000 in PBS. Increased apoE expression was observed in SOD1 treated with OxPC. G93A It was detected in ALS motor neurons (Figure 2B) and healthy motor neurons (Figure 2C).

[0134] TDP-43 aggregation and phosphorylation are important features in ALS pathology. Healthy motor neurons and TDP-43 M337VALS motor neurons were treated with OxPC and analyzed for TDP-43 aggregation. TDP-43 aggregates were analyzed using the HTRF kit (TDP-43 Aggregation Kit, cisbio) according to the manufacturer's instructions. Increased TDP-43 aggregation was observed 24 hours after OxPC treatment in both healthy and ALS motor neurons (Figure 2D). Phospho-TDP-43 (pTDP-43) was analyzed by IHC using rabbit anti-pTDP-43 antibody (Proteintech) according to the method described above for ApoE detection. The results showed a trend toward increased pTDP-43 expression in healthy motor neurons treated with OxPC under similar conditions (Figure 2E), but this effect was not statistically significant.

[0135] Next, we will discuss the functional outcomes of OxPC, and the health and TDP-43 levels of those exposed to OxPC. M337VALS motor neurons were evaluated by determining their electrical activity profiles. Multielectrode array (MEA) activity was monitored on days 7, 16, 20, 21, and 22 after cell seeding using Maestro Pro (Axion Biosystems). Electrical activity was recorded according to the following settings: nerve spike detection (shape), 12.5 kHz (sampling frequency), and 200 Hz to 3 kHz (filter). Data analysis included the following settings: 0 to 180 seconds (analysis start and end), 5 spk / min (active electrode reference), 11 kOhm (minimum target resistance), simultaneous artifact electrode burst removal setting - Poisson Surprise algorithm, 5 (minimum surprise), network burst setting - envelope algorithm, 1.25 (threshold coefficient), 100 ms (minimum IBI), 75% (burst inclusion): 35% (minimum electrode). Exclusion criteria were applied to wells exhibiting poor cell adhesion / viability, fewer than four active electrodes, and / or a weighted mean firing rate (wMFR) less than 1.5 Hz. The assay was performed using four biological replicates. Prior to OxPC treatment on day 21, ALS motor neurons consistently showed lower MEA activity compared to healthy motor neurons at different time points and analyzed criteria. A slight decrease in wMFR was observed in healthy motor neurons after OxPC treatment, but not in ALS motor neurons. OxPC exposure induced reduced burst activity in both healthy and ALS motor neurons.

[0136] Example 3: Vectorized anti-OxPC scFv is expressed in the CNS and targets ALS-associated cell types. The limited permeability of the blood-brain barrier (BBB) ​​is a major obstacle to the use of antibody-based therapies for CNS diseases. To overcome BBB impairment, we developed vectorized anti-OxPC scFv(E06) as an antibody therapeutic strategy for neutralizing OxPC in CNS cell types (e.g., motor neurons, astrocytes). AAV5.2 was selected as the AAV capsid for this strategy because it is known to target cells within the CNS. Briefly, vectorized mouse E06-scFv(AAV5.2-CBh-mE06) and humanized E06-scFv(AAV5.2-CBh-hE06), as well as AAV5.2 carrying sequences against the control AAV5.2-CAG-GFP, were generated in a baculovirus expression system. The resulting recombinant AAVs are referred to herein as "AAV5.2-mE06," "AAV5.2-hE06," and "AAV5.2-GFP."

[0137] To test the transduction efficiency of recombinant AAV, human iPSC-derived neurons and astrocytes were subjected to 10 fertilization in culture medium 7-10 days after seeding. 5 , 10 6 , and 10 7 AAV5.2-mE06 or AAV5.2-GFP was transduced at the specified MOI. Half of the culture medium was removed from the wells, and then the AAV mixture was added. The same volume of fresh medium was added to the cells 4 hours after transduction.

[0138] To determine the ability of AAV5.2 to transduce CNS cell types, AAV5.2-GFP was evaluated first. ALS-related cell types such as astrocytes and co-cultures of astrocytes and neurons showed high transduction rates (60–80%, Figure 3A). In motor neurons, approximately 40–60% of the cells were successfully transduced by AAV5.2-GFP.

[0139] Transduction and expression were correlated in a dose-dependent manner, as measured by mE06 mRNA (Figure 3B) and protein (Figure 3C). In summary, AAV5.2-mE06 is highly efficient in transducing and expressing mE06 in motor neurons and astrocytes.

[0140] Example 4: Vectorized anti-OxPC scFv normalizes OxPC-induced pathways and expression changes. The ability of AAV5.2-mE06 to neutralize the effects of OxPC was demonstrated in (i) healthy motor neurons exposed to OxPC and (ii) SOD1 transduced by AAV5.2-mE06. G93A ALS motor neurons were evaluated by NanoString transcriptome assay (as described in Example 1 above). Transcriptome analysis showed that transduction by AAV5.2-mE06 was associated with important biological processes, including the regulation of cell death, apoptosis, protein metabolism, molecular function, and response to endogenous stimuli. In contrast, processes such as aging and protein phosphorylation were associated with SOD1 transduction by AAV5.2-mE06. G93A Improvement was observed only in ALS motor neurons.

[0141] In most cases, treatment with AAV5.2-mE06 reversed the transcriptome changes induced by OxPC treatment in healthy neurons. Approximately 40% of all OxPC-sensitive transcripts showed the same improvement (≥0.1x change) after transduction with AAV5.2-mE06 (see Table 4 below). A similar effect was observed in SOD1 not treated with OxPC. G93A This was observed after AAV5.2-mE06 transduction of ALS motor neurons (Table 2).

[0142] [Table 4]

[0143] Example 5: Vectorized anti-OxPC scFv provides protection against OxPC-mediated neurotoxicity. The ability of OxPC to induce TDP-43 aggregation in both healthy and ALS motor neurons was described above. We evaluated whether this phenotype could be prevented using AAV5.2-mE06 or AAV5.2-hE06 transduced before treatment. Notably, both AAV5.2-mE06 and AAV5.2-hE06 were able to completely resolve the TDP-43 aggregation induced by OxPC exposure (Figure 4A).

[0144] Axonal degeneration is a crucial mechanism in motor neuron diseases. Therefore, the effects of OxPC toxicity on nerve projections and axonal health are considered in relation to SOD1 G93A Evaluation was performed on ALS motor neurons. These neurons were chosen due to the established axonal dysfunction associated with this genotype. SOD1 was treated with OxPC using the OMEGA-NMJ chip (eNUVIO). G93A A significant reduction in the number of projection axons in ALS motor neurons (approximately 73%) was observed (Figure 4B). Transduction with AAV5.2-mE06 prior to OxPC treatment substantially reduced this toxicity to the level of the untreated state (approximately 80%).

[0145] Next, health and TDP-43 M337V The ability of AAV5.2-hE06 to prevent the effects of OxPC on ALS motor neurons was evaluated by determining its electrical activity profile. In short, 125,000 healthy and 25,000 TDP-43 M337V ALS motor neurons / astroglial cells were seeded in a co-culture setting on a CytoView 96-well plate (M768-tMEA-96B, Axion Biosystems) with an MOI of 10. 6Cells were transduced using AAV5.2-hE06 and then treated with OxPC. Multielectrode array (MEA) activity was evaluated as in Example 2. The assay was performed using four biological replicates. A decrease in burst count was observed in cells treated only with OxPC, but this decrease was prevented in cells transduced with AAV5.2-hE06 prior to OxPC treatment (Figure 4C).

[0146] Example 6: Vectorized anti-OxPC scFv provides protection against motor dysfunction and ALS pathology transmission in an in vivo mouse model. Previous studies have reported the neurotoxic properties of CSF derived from ALS patients and demonstrated the transmission of disease pathology to animals injected with CSF from ALS patients (Wong et al., Brain Commun. 2022 Aug 22;4(4):fcac207. doi: 10.1093). We investigated the ability of AAV5.2-mE06 to prevent the transmission of ALS pathology in mice injected with CSF from ALS patients (sALS-CSF mouse model). The study was conducted at the Tisch MS Research Center of New York (Tisch Center). Briefly, CSF from sporadic ALS patients and control CSF were obtained from the Tisch Center's CSF bank. Informed consent in accordance with institutional ethics committee approval and the Declaration of Helsinki was obtained prior to CSF ​​collection. Samples were collected using sterile techniques, either by lumbar puncture or aspiration from the access port of a surgically implanted pump. The CSF samples were centrifuged at 200×g for 15 minutes to remove cells, and microscopic observation confirmed the absence of red blood cell contamination. Subsequently, they were stored as aliquots at -80°C.

[0147] Adult female C57BL / 6J mice (8-12 weeks old) purchased from Jackson Laboratory (Bar Harbor, ME) were used in all in vivo experiments. All procedures were approved by the Institutional Animal Experimentation Committee of Mispro Biotech Services (New York). Prior to surgery, mice were anesthetized with a cocktail of ketamine (110 mg / kg) and xylazine (10 mg / kg) and subcutaneously injected with 0.1 mg / kg buprenorphine, 2.5 mg / kg baytril, and 1 mL of 0.9% saline. Laminectomy was performed at the C4 and C5 cervical vertebral levels to expose the underlying spinal cord. A 32-gauge Hamilton syringe was inserted subdurally, and 5 μL of saline / AAV5.2-mE06 or 3 μL of saline / CSF as a control was slowly injected into the subarachnoid space. AAV5.2-mE06, 4.3e 11 Eight-week-old mice were intrathecally infused with a genome copy (gc) / mouse dose, followed by saline / CSF infusion four weeks later (12-week-old mice). A minimum of three mice were infused per individual patient CSF sample. Mice were assigned to different treatment groups in a randomized manner.

[0148] (a) Motor impairment score test After intrathecal delivery of CSF, all mice underwent motor tests 1 day post-infusion (DPI). Forelimb reaching, grasping, and tail relaxation were evaluated on a 3-point scale. Mice were held by their tails over a cage bar and asked to extend their forelimbs to grasp the bar for five trials. Mice showing no motor impairment were given a score of 0. Impairment in either reaching or grasping was given a score of 1. Specifically, uncertain reaching was considered a reaching impairment, and weak grasping strength or forelimb clenching was scored as a grasping impairment. Tail relaxation was also given a score of 1. All motor tests were performed blindly for each treatment group. Mice pre-treated with saline prior to sALS-CSF infusion showed increased motor impairment, while mice pre-treated with AAV5.2-mE06 developed less severe motor impairment (Figure 5A).

[0149] (b) Gripping strength test Mice were accustomed to a grip strength meter (TSE Systems) for three days prior to surgery. Each mouse was given one minute to explore the grip strength meter, then held by the tail, and gripped the bar with both forelimbs for five consecutive trials. After a 30-second rest period, the mice underwent five more gripping trials and were then returned to their home cages. Baseline grip strength was measured again one day before surgery and at 1 DPI. Average grip strength was calculated from the five trials. Normalized grip strength values ​​were calculated by dividing the average grip strength on the post-injection test day by the average baseline grip strength. A decrease in grip strength was observed in mice treated with physiological saline prior to injection with sALS-CSF, but this was prevented in mice pre-treated with AAV5.2-mE06 (Figure 5B).

[0150] (c) Motor Neuron Histology Tissue recovery: Mice were sacrificed using an overdose of ketamine (300 mg / kg) and xylazine (30 mg / kg), and transcardially perfused with phosphate-buffered saline (PBS) followed by 4% paraformaldehyde in 0.1 M PBS, pH 7.4. The spinal cord and brain were dissected, fixed overnight in 4% paraformaldehyde, and then placed overnight in 30% sucrose for cryoprotection.

[0151] The cervical spinal cord was severed 0.5 cm rostrally and 0.5 cm caudally from the injection site, and a 1 cm section of thoracic spinal cord was also recovered. Subsequently, the 1 cm fragment was embedded in Tissue Tek® (VWR International, PA) and frozen. The spinal cord was thinly sectioned into 20 μm thick sagittal sections using a cryostat (Leica), and then slide-mounted onto Histobond® slides (VWR International, PA). As described in more detail below, the anatomical orientation of the tissue sections, as well as the order and position of mounting them on the slides, were kept constant to facilitate unbiased histological comparison. The brain was thinly sectioned into 30 μm coronal sections, and the suspension sections were stored in 0.01% sodium azide in PBS.

[0152] Immunofluorescence staining: Immunostaining was performed on a series of spinal cord sections spaced 100 μm apart across the entire cervical or thoracic spinal cord. Spinal cord sections or slides containing cells, or suspended brain sections, were washed three times in 0.1% triton X-100 in PBS (PBS / T), followed by incubation in 10% normal goat serum (NGS) or normal donkey serum (NDS) in PBS / T for 1 hour at room temperature. The primary antibody, goat anti-ChAT (Millipore, 1:100), was diluted in 10% NGS or NDS in PBS / T and incubated overnight at 4°C. After incubation, slides or sections were rinsed three times in PBS and incubated for 1.5 hours at room temperature in a 1:750 dilution of appropriate Alexa-Fluor secondary antibody (Invitrogen) in 10% NGS or NDS in PBS / T. Slides or sections were rinsed three times in PBS, followed by counter-staining with 1:2500 DAPI (Invitrogen) in PBS for 5 minutes. After two final washes in PBS, the suspended brain sections were placed on slides and mounted using Fluoromount (Sigma).

[0153] Histological analysis: Images were acquired at 20x magnification using a Zeiss Axio Imager. Acquisition parameters and exposure times were kept constant for each antibody stain. To ensure unbiased comparisons between experimental groups, spinal cord images were acquired from similar tissue section numbers on the slide, and matching anatomical regions were verified by the experimenter. Motor neuron count and immunostaining intensity were quantified using ImageJ software. For cell count, three images were quantified for each mouse to calculate the average number of motor neurons. Fluorescence intensity was measured as the average density value in the area of ​​interest. Both imaging and quantification were performed by blinded experimenters for each treatment group. A decrease in motor neuron count was observed in mice treated with saline prior to sALS-CSF injection, but this was prevented in mice pre-treated with AAV5.2-mE06 (Figure 5C).

[0154] Example 7: Vectorized anti-OxPC scFv is SOD1 G93A Normalizing OxPC concentration in an ALS mouse model We investigated the ability of AAV5.2-mE06 to normalize the levels of various OxPC molecular species in a mouse model of ALS pathology.

[0155] SOD1 G93A Transgenic (TG) and wild-type (WT) mice were given PBS / 0.001% Pluronic or 1 × 10⁶ mice at 45 days of age. 12 Either gc / mouse AAV5.2-mE06 was administered intrathecally. Subsets from each dose group were sacrificed at 70 and 90 days of age and sampled. The remaining mice from each dose group were sacrificed at the humane endpoint (survival) or at 160 days of age. 24 mice were treated with PBS and SOD1. G93A Mice and 15 AAV5.2-mE06 treated SOD1 G93A Plasma was collected from mice and used for OxPC analysis.

[0156] To measure OxPC molecular species, lipids were extracted from 50 μL of mouse plasma according to the Folch method. The supernatant was evaporated to dryness and degraded with methanol. The degraded samples were analyzed using an Agilent 1290 HPLC system equipped with a binary pump, multisampler, and column thermostat with a Zorbax Eclipse plus C-18, 50 × 2.1 mm, 1.8 μm, 40°C column. The gradient consisted of 32–97% B for 21 minutes with a stop time of 27 minutes. The flow rate was set to 0.4 mL / min and the injection volume was 5 μL. HPLC was combined with an Agilent 6470 triple quadrupole mass spectrometer (Agilent Technologies, Santa Clara, USA) using optimized electrospray ionization source parameters. Analysis was performed using multiple reaction monitoring according to Solati et al., Front. Med. 1;8:716944 (2021), doi: 10.3389 / fmed.2021.716944. Analytes were identified by characteristic mass transitions and retention times. For quantification, external POVPC and SOVPC standards (Cayman Chemical, Ann Arbor, MI) were used, and concentrations were expressed in ng / mL units.

[0157] Of the 22 different OxPC molecular species previously identified by Solati et al. (see above), 19 are WT and TG SOD1 G93A Detected in mice. Of the measured OxPC molecular species, seven were identified using AAV5.2-mE06 for SOD1 G93A The normalized OxPC concentrations during the treatment of mice are shown (Figures 6A to 6G).

[0158] conclusion These results demonstrate that, upon binding to OxPC, anti-OxPC scFv neutralizes the neurotoxic and neuroinflammatory mechanisms of OxPC, alters gene expression patterns in an ALS mouse model, and restores in vivo OxPC levels, as evidenced by the prevention of neurotoxicity induced by TDP-43 aggregates and OxPC. Therefore, anti-OxPC scFv, particularly vectorized anti-OxPC scFv, represents a potential strategy for treating ALS.

[0159] The present invention is not limited in scope by the specific embodiments described herein. In fact, various modifications of the present invention will be apparent to those skilled in the art from the foregoing description and the accompanying drawings. Such modifications are intended to fall within the scope of the accompanying claims.

[0160] All references cited herein (e.g., publications or patents or patent applications) are incorporated herein by reference in whole and for all purposes to the same extent that each individual reference (e.g., publications or patents or patent applications) is specifically and individually indicated to be incorporated herein by reference in whole and for all purposes. Other embodiments fall within the scope of the following claims.

Claims

1. Therapeutic effective dose: (a) an antibody or antigen-binding fragment thereof that specifically binds to oxidized phosphatidylcholine (OxPC); or (b) A polynucleotide encoding an antibody or antigen-binding fragment thereof that specifically binds to OxPC. A method for inhibiting oxidized phospholipid (OxPL)-induced neurotoxicity in a subject requiring it, comprising the step of administering to the subject.

2. Therapeutic effective dose: (a) an antibody or antigen-binding fragment thereof that specifically binds to OxPC; or (b) A polynucleotide encoding an antibody or antigen-binding fragment thereof that specifically binds to OxPC. A method for inhibiting TDP-43 aggregate-induced neurotoxicity in a subject requiring it, comprising the step of administering to the subject.

3. Therapeutic effective dose: (a) an antibody or antigen-binding fragment thereof that specifically binds to OxPC; or (b) A polynucleotide encoding an antibody or antigen-binding fragment thereof that specifically binds to OxPC. A method for preventing or reducing TDP-43 aggregates in a subject suffering from a condition associated with TDP-43 aggregate formation, comprising the step of administering to the subject.

4. The method according to any one of claims 1 to 3, wherein the subject has amyotrophic lateral sclerosis (ALS), Alzheimer's disease, motor neuron disease, Parkinson's disease, or frontotemporal lobar degeneration.

5. The method according to any one of claims 1 to 4, wherein the subject has sporadic ALS (sALS).

6. A method for treating a disease or disorder mediated by oxidized phosphatidylcholine (OxPC) in a subject requiring treatment, comprising the step of administering to a subject a polynucleotide encoding an antibody or an antigen-binding fragment thereof that specifically binds to a therapeutically effective amount of OxPC.

7. The method according to claim 6, wherein the disease or disorder is amyotrophic lateral sclerosis (ALS), Alzheimer's disease, motor neuron disease, Parkinson's disease, or frontotemporal lobar degeneration.

8. The method according to claim 6 or 7, wherein the disease or disorder is sporadic ALS (sALS).

9. The method according to any one of claims 1 to 8, wherein the antibody or antigen-binding fragment comprises a heavy chain variable region including the complementarity-determining regions CDRH1, CDRH2, and CDRH3 of the heavy chain variable region amino acid sequence shown in SEQ ID NO: 7, and a light chain variable region including the complementarity-determining regions CDRL1, CDRL2, and CDRL3 of the light chain variable region amino acid sequence shown in SEQ ID NO:

8.

10. The method according to any one of claims 1 to 9, wherein the antibody or antigen-binding fragment comprises CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and CDRL3, each comprising the amino acid sequences shown in SEQ ID NOs: 1, 2, 3, 4, 5, and 6, respectively.

11. The method according to any one of claims 1 to 10, wherein the antibody or its antigen-binding fragment includes a heavy chain variable region having an amino acid sequence having at least 80% identity with SEQ ID NO:

7.

12. The method according to any one of claims 1 to 11, wherein the antibody or its antigen-binding fragment includes a light chain variable region having an amino acid sequence having at least 80% identity with SEQ ID NO:

8.

13. The method according to any one of claims 1 to 12, wherein the antibody or its antigen-binding fragment comprises a heavy chain variable region having at least 80% identity with the amino acid sequence shown in SEQ ID NO: 7 and a light chain variable region having at least 80% identity with the amino acid sequence shown in SEQ ID NO:

8.

14. The method according to any one of claims 1 to 13, wherein the antibody or its antigen-binding fragment is a humanized antibody.

15. The method according to any one of claims 1 to 14, wherein the antibody or its antigen-binding fragment is a single-chain variable fragment (scFv).

16. The method according to claim 15, wherein the scFv includes a peptide linker between the heavy chain variable region and the light chain variable region.

17. The method according to claim 16, wherein the peptide linker comprises the amino acid sequence GGGGSGGGGSGGGGS (SEQ ID NO: 9).

18. The method according to any one of claims 1 to 17, wherein the vector contains a polynucleotide.

19. The method according to claim 18, wherein the vector is a viral vector.

20. The method according to claim 19, wherein the viral vector is selected from the group consisting of adeno-associated virus (AAV), adenovirus, retrovirus, orthomyxovirus, paramyxovirus, papovavirus, picornavirus, lentivirus, herpes simplex virus, vaccinia virus, poxvirus, and alphavirus.

21. The method according to any one of claims 18 to 20, wherein the vector is an AAV vector contained within a recombinant AAV (rAAV), and the rAAV comprises an AAV capsid containing an AAV capsid protein; and an rAAV genome.

22. The method according to claim 21, wherein the capsid protein is one of the following: clade A, clade B, clade C, clade D, clade E, clade F, clade G, clade H, clade I, AAVgo.1, AAV3, AAV4, AAV10, AAV11, AAV12, rh.32, rh32.33, rh.33, rh.34, BAAV, or AAV5 capsid proteins, or genetically modified variants thereof.

23. The method according to claim 21 or 22, wherein the capsid protein comprises an amino acid sequence having at least 95% identity with amino acids 193-725 of SEQ ID NO:

11.

24. The method according to any one of claims 21 to 23, wherein the capsid protein comprises an amino acid sequence having at least 99% identity with amino acids 193 to 725 of SEQ ID NO:

11.

25. The method according to any one of claims 21 to 24, wherein the capsid protein comprises the amino acid sequence of amino acids 193 to 725 of SEQ ID NO:

11.

26. The method according to any one of claims 21 to 25, wherein the capsid protein comprises an amino acid sequence having at least 95% identity with amino acids 138 to 725 of SEQ ID NO:

11.

27. The method according to any one of claims 21 to 26, wherein the capsid protein comprises an amino acid sequence having at least 99% identity with amino acids 138 to 725 of SEQ ID NO:

11.

28. The method according to any one of claims 21 to 27, wherein the capsid protein comprises the amino acid sequence of amino acids 138 to 725 of SEQ ID NO:

11.

29. The method according to any one of claims 21 to 28, wherein the capsid protein comprises an amino acid sequence having at least 95% identity with SEQ ID NO: 10 or 11.

30. The method according to any one of claims 21 to 29, wherein the capsid protein comprises an amino acid sequence having at least 99% identity with SEQ ID NO: 10 or 11.

31. The method according to any one of claims 21 to 30, wherein the capsid protein comprises the amino acids of SEQ ID NO: 10 or 11.

32. The method according to any one of claims 21 to 31, wherein the rAAV genome further comprises a CBh promoter containing the nucleic acid sequence shown in SEQ ID NO:

12.

33. The method according to any one of claims 21 to 32, wherein the rAAV genome further comprises an SV40 polyA tail containing the nucleic acid sequence shown in SEQ ID NO:

13.

34. The method according to any one of claims 1 to 33, wherein an antibody, polynucleotide, or rAAV is administered to a subject intravenously, intraperitoneally, subcutaneously, intramuscularly, intrathecally, or intradermally.

35. The method according to any one of claims 1 to 34, for neutralizing OxPC in a test subject.

36. The method according to any one of claims 1 to 35 for preventing or reducing TDP-43 aggregates.

37. The method according to any one of claims 1 to 36, which reduces the expression of one or more ALS-related genes.

38. The method according to claim 37, wherein one or more ALS-related genes are apoE, COL4A1, CTSS, DAB2, TIMP1, or any combination thereof.

39. The method according to any one of claims 1 to 38, which increases the expression of one or more ALS-related genes.

40. The method according to claim 39, wherein one or more ALS-related genes are C9orf72, GRM3, SYP, GRIN2B, CHRNA7, MYD88, ITPR2, GRN, VEGFA, FKBP5, or any combination thereof.

41. The method according to any one of claims 1 to 40, for neutralizing neurotoxicity mediated by OxPC.

42. The method according to any one of claims 1 to 41, for increasing the electrical firing of neurons.

43. The method according to any one of claims 1 to 42, which increases neurite outgrowth on a neuron.

44. The method according to any one of claims 21 to 43, wherein the vector targets cortical neurons, spinal neurons, and / or astrocytes.

45. The method according to any one of claims 1 to 44, wherein the subject is a human subject.

46. An antibody or antigen-binding fragment thereof that specifically binds to OxPC, or a nucleic acid sequence encoding an antibody or antigen-binding fragment thereof that specifically binds to OxPC, for use in inhibiting neurotoxicity induced by TDP-43 aggregates in subjects requiring such use, wherein the treatment is carried out according to the method of any one of claims 1 to 45.

47. An antibody or antigen-binding fragment thereof that specifically binds to oxidized phospholipids (OxPL) or a polynucleotide encoding an antibody or antigen-binding fragment thereof that specifically binds to oxidized phospholipids, for use in inhibiting neurotoxicity induced by oxidized phospholipids (OxPL) in subjects requiring such use, wherein the treatment is carried out according to the method of any one of claims 1 to 45.

48. An antibody or antigen-binding fragment thereof that specifically binds to OxPC, or a polynucleotide encoding an antibody or antigen-binding fragment thereof that specifically binds to OxPC, for use in preventing or reducing TDP-43 aggregates in subjects requiring such use, wherein the treatment is carried out according to the method of any one of claims 1 to 45.

49. An antibody or antigen-binding fragment thereof that specifically binds to OxPC, or a polynucleotide encoding an antibody or antigen-binding fragment thereof that specifically binds to OxPC, for use in the manufacture of a pharmaceutical for inhibiting neurotoxicity induced by TDP-43 aggregates in a subject requiring such use, wherein the treatment is carried out according to the method of any one of claims 1 to 45.

50. An antibody or antigen-binding fragment thereof that specifically binds to OxPC, or a polynucleotide encoding an antibody or antigen-binding fragment thereof that specifically binds to OxPC, for use in the manufacture of a pharmaceutical for inhibiting neurotoxicity induced by oxidized phospholipids (OxPL) in a subject requiring such use, wherein the treatment is carried out according to the method of any one of claims 1 to 45.

51. An antibody or antigen-binding fragment thereof that specifically binds to OxPC, or a polynucleotide encoding an antibody or antigen-binding fragment thereof that specifically binds to OxPC, for use in the manufacture of a pharmaceutical for preventing or reducing TDP-43 aggregates in a subject requiring such use, wherein the treatment is carried out according to the method of any one of claims 1 to 45.

52. The use of an antibody or antigen-binding fragment thereof that specifically binds to OxPC, or a polynucleotide encoding an antibody or antigen-binding fragment thereof that specifically binds to OxPC, for the purpose of inhibiting neurotoxicity induced by TDP-43 aggregates in a subject requiring such use, wherein the treatment is carried out according to the method of any one of claims 1 to 45.

53. The use of an antibody or antigen-binding fragment thereof that specifically binds to oxidized phospholipids (OxPL) or a polynucleotide encoding an antibody or antigen-binding fragment thereof that specifically binds to oxidized phospholipids, for the purpose of inhibiting neurotoxicity induced by oxidized phospholipids (OxPL) in a subject requiring such use, wherein the treatment is carried out according to the method of any one of claims 1 to 45.

54. The use of an antibody or antigen-binding fragment thereof that specifically binds to OxPC, or a nucleic acid sequence encoding an antibody or antigen-binding fragment thereof that specifically binds to OxPC, for preventing or reducing TDP-43 aggregates in a subject requiring such use, wherein the treatment is carried out according to the method of any one of claims 1 to 45.

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