AAV-based anti-ga therapy

Abstract

Aspects of the present disclosure relate to compositions and methods for diagnosing and / or treating certain neurodegenerative diseases, e.g., diseases associated with repeat-associated non-ATG (RAN) translation proteins, such as amyotrophic lateral sclerosis (ALS). In some embodiments, the present disclosure relates to recombinant adeno-associated viruses (rAAV) expressing antibodies and antigen-binding fragments thereof that bind to poly(GA) RAN proteins. In some embodiments, the present disclosure relates to methods of treating RAN protein-associated diseases by administering rAAV to a subject in need thereof.

Description

[0001] Related applications

[0002] This application claims the benefit of U.S. Provisional Application No. 63 / 608,667, filed December 11, 2023; U.S. Provisional Application No. 63 / 554,852, filed February 16, 2024; and U.S. Provisional Application No. 63 / 645,222, filed May 10, 2024, pursuant to 35 USC §119(e), the entire contents of which are incorporated herein by reference.

[0003] Reference to the electronic sequence list

[0004] The contents of the electronic serial number (U120270132WO00-SEQ-KZM.xml; size: 91,478 bytes; creation date: December 5, 2024) are incorporated herein by reference in their entirety.

[0005] Government support

[0006] This invention was completed with the support of the U.S. government, grant number HT9425-23-1-0287, granted by the U.S. Army Medical Research Acquisition Activity. The U.S. government holds certain rights to this invention. Background Technology

[0007] Microsatellite repeat amplification is known to cause more than sixty neurodegenerative disorders. Many of these disorders share common molecular features including the accumulation of RNA foci containing both sense and antisense amplified transcripts, and the accumulation of proteins resulting from repeat-associated non-AUG (RAN) translation. RAN translation can occur across a wide range of repeat lengths, from premutated lengths (approximately 20–40 repeats) to fully amplified lengths (greater than 11,000 repeats). Growing evidence suggests that RAN proteins are toxic and contribute to a growing number of diseases and conditions, including but not limited to amyotrophic lateral sclerosis (ALS), frontotemporal dementia (FTD), myotonic dystrophy type 1 (DM1), myotonic dystrophy type 2 (DM2), Huntington's disease (HD), Alzheimer's disease (AD), and fragile X-tremor ataxia syndrome (FXTAS). Summary of the Invention

[0008] This disclosure relates to compositions and methods for diagnosing and / or treating certain neurodegenerative diseases, such as those associated with repetitive non-ATG (RAN) translational proteins, such as amyotrophic lateral sclerosis (ALS). Part of this disclosure is based on viral vectors, such as recombinant adeno-associated virus (rAAV) vectors and rAAV particles encoding antibodies (e.g., monoclonal antibodies) that bind to polyglycine-alanine (poly(GA)) RAN proteins and their antigen-binding fragments (e.g., single-chain variable fragments, scFv). In some embodiments, this disclosure relates to methods for reducing the aggregation of RAN proteins (e.g., poly(GA) RAN proteins) in a subject by administering an rAAV vector or rAAV particles to a subject. In some embodiments, this disclosure relates to methods for treating RAN protein-related diseases by administering rAAV to a subject in need of it.

[0009] Therefore, in some aspects, this disclosure provides a recombinant adeno-associated virus (rAAV) vector comprising a nucleic acid sequence encoding an anti-poly(GA) RAN protein antibody or an antigen-binding fragment thereof, the sequence being flanked by adeno-associated virus (AAV) inverted terminal repeat (ITR) sequences.

[0010] In some implementations, AAV ITR is AAV2 ITR.

[0011] In some embodiments, the anti-poly(GA) RAN protein antibody or its antigen-binding fragment comprises a heavy chain variable region (VH), which includes: a CDR1 region comprising or consisting of the amino acid sequence shown in SEQ ID NO:1; a CDR2 region comprising or consisting of the amino acid sequence shown in SEQ ID NO:2; and / or a CDR3 region comprising or consisting of the amino acid sequence shown in SEQ ID NO:3.

[0012] In some embodiments, the anti-poly(GA) RAN protein antibody or antigen-binding fragment includes a light chain variable region (VL) comprising: a CDR1 region comprising or consisting of the amino acid sequence shown in SEQ ID NO:4; a CDR2 region comprising or consisting of the amino acid sequence shown in SEQ ID NO:5; and / or a CDR3 region comprising or consisting of the amino acid sequence shown in SEQ ID NO:6.

[0013] In some embodiments, the anti-poly(GA) RAN protein antibody or antigen-binding fragment comprises a variable heavy chain, which comprises or consists of the amino acid sequence shown in SEQ ID NO:7.

[0014] In some embodiments, the anti-poly(GA) RAN protein antibody or antigen-binding fragment comprises a variable light chain that contains or consists of the amino acid sequence shown in SEQ ID NO:12.

[0015] In some embodiments, the anti-poly(GA) RAN protein antibody or antigen-binding fragment comprises a heavy chain containing or consisting of the amino acid sequence shown in SEQ ID NO:8 or 10.

[0016] In some embodiments, the anti-poly(GA) RAN protein antibody or antigen-binding fragment comprises a light chain containing or consisting of the amino acid sequence shown in SEQ ID NO:13 or 15.

[0017] In some embodiments, the anti-poly(GA) RAN protein antibody or antigen-binding fragment includes a heavy chain variable region (VH), which comprises: a CDR1 region comprising or consisting of the amino acid sequence shown in SEQ ID NO:20; a CDR2 region comprising or consisting of the amino acid sequence shown in SEQ ID NO:21; and / or a CDR3 region comprising or consisting of the amino acid sequence shown in SEQ ID NO:22.

[0018] In some embodiments, the anti-poly(GA) RAN protein antibody or antigen-binding fragment includes a light chain variable region (VL) comprising: a CDR1 region comprising or consisting of the amino acid sequence shown in SEQ ID NO:23; a CDR2 region comprising or consisting of the amino acid sequence shown in SEQ ID NO:24; and / or a CDR3 region comprising or consisting of the amino acid sequence shown in SEQ ID NO:25.

[0019] In some embodiments, the anti-poly(GA) RAN protein antibody or antigen-binding fragment comprises a variable heavy chain containing the amino acid sequence shown in SEQ ID NO:26 or consisting of the amino acid sequence shown in SEQ ID NO:26.

[0020] In some embodiments, the anti-poly(GA) RAN protein antibody or antigen-binding fragment comprises a variable light chain containing the amino acid sequence shown in SEQ ID NO:31 or composed of the amino acid sequence shown in SEQ ID NO:31.

[0021] In some embodiments, the anti-poly(GA) RAN protein antibody or antigen-binding fragment comprises a heavy chain containing or consisting of the amino acid sequence shown in SEQ ID NO:27 or 29.

[0022] In some embodiments, the anti-poly(GA) RAN protein antibody or its antigen-binding fragment comprises a light chain containing or consisting of the amino acid sequence shown in SEQ ID NO:32 or 34.

[0023] In some embodiments, the anti-poly(GA) RAN protein antigen-binding fragment comprises a single-chain variable fragment (scFv) containing a heavy chain variable region comprising: a CDR1 region comprising or consisting of the amino acid sequence shown in SEQ ID NO:1; a CDR2 region comprising or consisting of the amino acid sequence shown in SEQ ID NO:2; and / or a CDR3 region comprising or consisting of the amino acid sequence shown in SEQ ID NO:3.

[0024] In some embodiments, the anti-poly(GA) RAN protein antigen-binding fragment comprises a single-chain variable fragment (scFv) containing a light chain variable region comprising: a CDR1 region comprising or consisting of the amino acid sequence shown in SEQ ID NO:4; a CDR2 region comprising or consisting of the amino acid sequence shown in SEQ ID NO:5; and / or a CDR3 region comprising or consisting of the amino acid sequence shown in SEQ ID NO:6.

[0025] In some embodiments, the scFv comprises a variable heavy chain that contains or consists of the amino acid sequence shown in SEQ ID NO:17.

[0026] In some embodiments, the scFv comprises a variable light chain that contains or consists of the amino acid sequence shown in SEQ ID NO:18.

[0027] In some embodiments, the scFv includes a linker molecule connecting the heavy chain variable region and the light chain variable region. In some embodiments, the linker molecule includes a polyGS linker.

[0028] In some embodiments, the antibody or its antigen-binding fragment further comprises a signal peptide.

[0029] In some embodiments, the rAAV vector comprises the sequence shown in any one of SEQ ID NO:36-40.

[0030] In some embodiments, the rAAV vector comprises a sequence that is at least 75% identical to the sequence shown in any one of SEQ ID NO:43-46.

[0031] In some aspects, this disclosure provides a recombinant adeno-associated virus (rAAV) comprising: the rAAV vector described herein; and one or more adeno-associated virus (AAV) capsid proteins.

[0032] In some aspects, this disclosure provides a method for reducing poly(GA) RAN protein aggregation in a subject, the method comprising administering the rAAV described herein to the subject.

[0033] In some embodiments, one or more AAV capsid proteins comprise the AAV9 capsid protein.

[0034] In some embodiments, one or more AAV capsid proteins comprise AAV1 capsid protein.

[0035] In some embodiments, one or more AAV capsid proteins comprise a VP1 protein containing an ERDRTRG peptide (e.g., as shown in SEQ ID NO:49). In some embodiments, the VP1 protein comprises the amino acid sequence shown in SEQ ID NO:48.

[0036] In some aspects, this disclosure provides a composition comprising the rAAV carrier or rAAV described herein, and a pharmaceutically acceptable carrier or buffer.

[0037] In some aspects, this disclosure provides a method for expressing an antibody or antigen-binding fragment in a subject, the method comprising administering the rAAV described herein to the subject.

[0038] In some implementations, the subject is a mammal. In some implementations, the subject is a human.

[0039] In some implementations, the subject expresses one or more RAN proteins. In some implementations, the subject expresses a poly(GA) RAN protein.

[0040] In some implementations, the subjects have or are suspected of having Alzheimer's disease, amyotrophic lateral sclerosis (ALS), or frontotemporal dementia.

[0041] In some implementations, the subject has or is suspected of having ALS.

[0042] In some aspects, this disclosure provides a method for treating a subject suffering from ALS, the method comprising administering the rAAV described herein to the subject.

[0043] In some implementations, the subject is a human being.

[0044] Brief description of the attached figures

[0045] Figure 1 Representative immunohistochemical (IHC) data are shown, indicating that the anti-poly(GA)-IgG1 antibody expressed by rAAV reduced the number of poly(GA) RAN protein aggregates in the brain tissue of a C9orf72 mouse model of ALS (C9 BAC).

[0046] Figure 2Representative data are shown, indicating that the anti-poly(GA)-IgG1 antibody expressed by rAAV reduced the number of poly(GA) RAN protein aggregates in a C9orf72 mouse model of ALS. The leftmost inset shows a histogram representation of the number (N) of poly(GA) aggregates per nucleus, the middle inset shows a histogram representation of the total area of ​​poly(GA) aggregates, and the rightmost inset shows a histogram representation of the average size of poly(GA) aggregates. In each histogram, the first column shows the results for control mice, the second column shows the results for PBS-treated C9 BAC mice, the third column shows the results for C9 BAC mice treated with rAAV9 containing a transgene encoding anti-poly(GA)-IgG1 A, the fourth column shows the results for C9 BAC mice treated with rAAV9 containing a transgene encoding anti-poly(GA)-ScFv, the fifth column shows the results for C9 BAC mice treated with rAAV9 containing a transgene encoding anti-poly(GA)-IgG1 B, and the sixth column shows the results for C9 BAC mice treated with rAAV9 containing a transgene encoding anti-poly(GA)-IgG2 antibody. N=5 animals per group. One-way ANOVA was used, with Sidak multiple comparison tests performed. Data represent mean ± SEM. * <0.05.

[0047] Figure 3 Representative microscopic data are shown, indicating that anti-poly(GA) antibodies administered using AAV-based delivery co-localize with poly(GA) RAN protein aggregates in brain tissue of a C9BAC mouse model.

[0048] Figure 4 Representative data are shown, indicating that AAV9-based anti-poly(GA) antibody delivery reduced GP levels in the frontal cortex of a C9 BAC mouse model. N per group > Five animals. In some embodiments, the anti-poly(GA) antibodies described herein reduce GP aggregates because they improve protein homeostasis in cells.

[0049] Figures 5A-5B Representative data show that delivery of AAV9-based anti-poly(GA) antibodies reduces neuroinflammation in the motor cortex of C9 BAC mice. Figure 5A Immunohistochemical results are shown, displaying GFAP staining (neuroinflammatory marker) in motor cortical tissues from untreated (NT) control mice, PBS-treated C9 BAC mice, and C9 BAC mice treated with rAAV9 containing a transgenic antibody encoding anti-poly(GA). Figure 5B show Figure 5AThe data shown is quantitative. N per group. > Five animals.

[0050] Figure 6 Representative data show that the novel monoclonal α-GA antibody recognizes GA aggregates in the brains of C9 BAC mice and in human ALS cell line models (human patient-derived cells (iMN)).

[0051] Figures 7A-7B Representative data are shown, indicating that the α-GA recombinant antibody was expressed and secreted. Figure 7A Western blot analysis of cell protein lysates from HEK293T cells expressing FLAG-labeled anti-poly(GA)-ScFv or unlabeled anti-poly(GA)-IgG1 A, anti-poly(GA)-IgG1 B or anti-poly(GA)-IgG2 antibodies. Figure 7B Fluorescence microscopy analysis of the supernatant shows that the supernatant contains substances that recognize GFP-GA overexpression. 60 The primary antibody against GA aggregates in HEK293T (anti-poly(GA) antibody (FLAG-labeled anti-poly(GA)-ScFv or unlabeled anti-poly(GA)-IgG1 A, anti-poly(GA)-IgG1 B or anti-poly(GA)-IgG2 antibody)).

[0052] Figure 8 Representative data from immunohistochemical analysis are shown, indicating that the recombinant α-GA antibody recognizes GA aggregates in the brains of C9 mice.

[0053] Figures 9A-9B Representative data were shown, indicating that the recombinant α-GA antibody reduced GFP-GA in HEK293T cells. 60 . Figure 9A The expression of α-GA recombinant antibody and GFP-GA showed that... 60 Western blot analysis of protein lysates obtained from cells. Figure 9B Display as shown Figure 9A The total GFP-GA level was quantified after treatment with the recombinant α-GA antibody.

[0054] Figures 10A-10B Representative data obtained from immunofluorescence analysis are shown, indicating that the recombinant α-GA antibody reduces the number and size of GA aggregates. Figure 10A The expression of α-GA recombinant antibody and GFP-GA showed that... 60 Fluorescence microscopy analysis of protein lysates obtained from cells. Figure 10B Display as shown Figure 10AThe figure shows the quantification of the number of aggregates after treatment with recombinant α-GA antibody. The treatments shown from top to bottom in the legend correspond to the treatments shown from left to right on the x-axis of the histogram.

[0055] Figure 11A-11D The development of an ERDR-IgG-GFP isotype control that can be delivered using recombinant AAV is demonstrated. Figure 11A A schematic diagram is shown of recombinant α-GA immunoglobulin and nucleic acids containing transgenes encoding the recombinant α-GA immunoglobulin. Figure 11B A schematic diagram showing the α-GA single-stranded variable fragment (scFv) and the nucleic acid containing the transgene encoding the α-GA scFv. Figure 11C A schematic diagram showing the α-GFP isotype control and the nucleic acid containing the transgene encoding the α-GFP isotype control. Figure 11D This shows representative data from fluorescence microscopy analysis of a supernatant containing an α-GFP isotype control that had been in contact with GFP-transfected cells.

[0056] Figure 12 This demonstrates a non-limiting example of a efficacy study strategy using AAV1-ERDR-IgG C9-BAC mice.

[0057] Figure 13 Representative data show that AAV1-ERDR-TFP is widely delivered to ependymal cells and motor cortical cells after ICV injection.

[0058] Figures 14A-14B Representative data obtained from the suspension test in injected mouse subjects are shown. Figure 14A This displays the suspension line scores of 16-week-old injected mice, indicating the number of times they reached either end of the line and the number of falls. The left inset represents the score for the number of times an animal reached the edge of the device. The middle and right insets both represent the score for the number of times an animal fell from the device. In the middle inset, each animal starts with a score of 10, deducting points based on the number of falls. In the right inset, each animal starts with a score of 0, adding points based on the number of falls. Figure 14B This shows a photograph taken during the suspension analysis of injected mouse subjects.

[0059] Figure 15A-15F Representative data obtained from open field analysis of injected mouse subjects are shown. Figure 15A Open field analysis of C9orf72 positive mice showed a variable mobility phenotype compared to non-transgenic littermates. Figure 15B This shows representative data (movement parameters) from open field analysis in treated and untreated C9orf72 BAC mice at 23 weeks of age. Figure 15CThis shows representative data (stereotyped behavior, resting time, and exploratory behavior) from open field analysis of 23-week-old treated and untreated C9orf72 BAC mice. Figure 15D The data show that treatment B rescued 11 / 12 open field abnormalities in C9-BAC mice compared to the mediator treatment. Figure 15E The data show representative results, indicating that the C9-BAC mice treated in group B had no difference in open field behavior compared to the NT animals. Figure 15F Representative data show that treatment A moderately improved four stereotyped combing / exploration parameters in C9-BAC mice, but did not improve movement parameters. Figure 15B-15C The treatment groups shown from left to right along the x-axis are: C9 uninjected, C9 medium, C9 Apricot, C9 Blue, NT medium, and NT uninjected. For Figure 15D-15F The gray boxes define regions of significant difference (p≤0.05) found in the antibody treatment cohorts compared to the C9- or NT-mediated controls, n≥5 / group. Data represent mean ± SEM. *<0.05, **<0.01, ***<0.001, ****<0.0001.

[0060] Figure 16 Representative data are shown, indicating that treatment B improved the survival rate of C9-BAC mice compared to treatment A and the vector treatment. Kaplan-Meier survival curves from Bonferroni analysis of multiple comparisons between treatment groups at 23 weeks of age are presented. The analysis showed that C9 mice treated with IgG B had a significantly increased survival rate compared to the C9-vector and C9 IgGA treatment cohorts.

[0061] Figure 17 Representative data are presented, showing that treatment A reduces GA aggregates in the posterior cingulate cortex (RSC) of C9-BAC mice. Compared to C9-mediated animals, C9-IgG-B treatment resulted in reduced GA RAN aggregates in the RSC of mice.

[0062] Figure 18 Representative data obtained from the analysis of the distribution of recombinant α-GA antibodies in brain and spinal cord tissues after AAV-based delivery are shown.

[0063] Figures 19A-19B Representative data show that AAV-based delivery of recombinant α-GA antibody reduces GA aggregates in the frontal cortex of treated C9-BAC mice. Figure 19A Data obtained from immunohistochemical analysis show that the recombinant α-GA antibody recognizes GA aggregates in the frontal cortex of treated C9-BAC mice. Figure 19B show Figure 19A Quantitative analysis of data.

[0064] Figures 20A-20B Representative data show that delivery of AAV-based recombinant α-GA antibody increases the survival of motor neurons in the brains of treated C9-BAC mice. Figure 20A The data obtained from the microscopic analysis of the processed C9-BAC motor neurons are shown. Figure 20B The treatment showed that the brains of C9-BAC mice were positive for choline acetyltransferase (ChAt). + Data obtained from motor neuron analysis.

[0065] Figures 21A-21B Representative data obtained from microscopic analysis of the brains of treated C9-BAC mice are shown, indicating that the recombinant α-GA antibody based on AAV delivery was retained in the CA2 region.

[0066] Figures 22A-22B Representative data obtained from microscopic analysis of the brains of treated C9-BAC mice are shown, demonstrating the biodistribution of AAV-based recombinant α-GA antibodies in the mouse brain.

[0067] Figures 23A-23B AAV-GA antibody treatment showed a reduction in brain damage in C9 female mice ( Figure 23A ) and organoids derived from C9orf72 ALS / FTD ( Figure 23B GA level in ).

[0068] Figures 24A-24B AAV-GA antibody showed that it improved the suspension line performance. Figure 24A and reduced GA levels in the brains of male C9-BAC mice. Figure 24B ). Detailed Implementation

[0069] This disclosure relates to compositions and methods for diagnosing and / or treating certain neurodegenerative diseases, such as those associated with repetitive non-ATG (RAN) translational proteins, like ALS. Part of this disclosure is based on rAAV vectors and rAAV particles encoding antibodies (such as monoclonal antibodies) that bind to polyglycine-alanine (poly(GA)) RAN proteins and their antigen-binding fragments (such as single-chain variable fragments, scFv). In some embodiments, this disclosure relates to methods for reducing the aggregation of RAN proteins (such as poly(GA) RAN proteins) in a subject by administering the rAAV vector or rAAV particles to the subject. In some embodiments, this disclosure relates to methods for treating RAN protein-related diseases by administering rAAV to a subject who requires it.

[0070] Recombinant AAV (rAAV)

[0071] This disclosure relates to certain viral vectors, such as recombinant adeno-associated virus (rAAV) vectors, and rAAV particles (also referred to as "rAAV") containing such vectors, which express transgenes encoding antibodies against RAN proteins (such as anti-poly(GA) RAN protein antibodies or antigen-binding fragments thereof). In some embodiments, rAAV is administered to a subject (such as a subject suffering from a disease characterized by RAN protein translation and accumulation, such as a human subject in which rAAV is administered to treat a disease or condition associated with RAN protein translation and accumulation), and the expression of the antibody or its antigen-binding fragment reduces RAN protein translation and RAN protein accumulation in the subject.

[0072] In some embodiments, the recombinant rAAV particle comprises a nucleic acid vector, such as a single-stranded (ss) or self-complementary (sc) recombinant AAV vector. In some embodiments, the rAAV vector comprises a transgene encoding an antibody against the poly(GA) RAN protein described herein or an antigen-binding fragment thereof (such as scFv), and one or more regions flanking the expression construct containing inverted terminal repeat (ITR) sequences (such as wild-type ITR sequences or engineered ITR sequences). In some embodiments, the rAAV vector is encapsulated with a viral capsid. In some embodiments, the transgene is operatively linked to a promoter, such as a constitutive promoter or an inducible promoter. In some embodiments, the promoter is a tissue-specific (such as CNS-specific) promoter.

[0073] Therefore, in some embodiments, the rAAV particle comprises a viral capsid and a nucleic acid vector (such as an rAAV vector) encapsulated therein. In some embodiments, the viral capsid comprises 60 capsid protein subunits, including VP1, VP2, and VP3. In some embodiments, the VP1, VP2, and VP3 subunits are present in the capsid in a ratio of approximately 1:1:10.

[0074] The ITR sequence of the nucleic acid or nucleic acid vector described herein may be derived from any AAV serotype (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10), or from more than one serotype. In some embodiments of the nucleic acid or nucleic acid vector provided herein, the ITR sequence is derived from AAV2. ITR sequences and plasmids containing ITR sequences are known in the art and are commercially available (see products and services offered by Vector Biolabs (Philadelphia, PA), Cellbiolabs (San Diego, CA), Agilent Technologies (Santa Clara, CA), and Addgene (Cambridge, MA); and Gene delivery to skeletal muscle results in sustained expression and systemic delivery of a therapeutic protein. Kessler PD, Podsakoff GM, Chen X, McQuiston SA, Colosi PC, Matelis LA, Kurtzman GJ, Byrne BJ. Proc Natl AcadSci US A. 1996 Nov 26;93(24):14082-7; and Curtis A. Machida. Methods in Molecular Medicine™. Viral Vectors for Gene Therapy Methods and Protocols.10.1385 / 1-59259-304-6:201 © Humana Press Inc. 2003. Chapter 10. Targeted Integration by Adeno-Associated Virus. Matthew D. Weitzman, Samuel M. Young Jr., Toni Cathomen and Richard Jude Samulski; U.S. Patent Nos. 5,139,941 and 5,962,313, the entire latter of which are incorporated herein by reference. An exemplary AAV2 ITR sequence is shown below.

[0075] TTGGCCACTCCCTCTCTGCGCGCTCGCTCGCTCACTGAGGCCGGGCGACCAAAGGTCGCCCGACGCCCGGGCTTTGCCCGGGCGGCCTCAGTGAGCGAGCGAGCGCGCAGAGAGGGAGTGGCCAACTCCATCACTAGGGGTTCCT (SEQ ID NO: 42)

[0076] In some embodiments, the size of the expression construct is no more than 7 kilobases, no more than 6 kilobases, no more than 5 kilobases, no more than 4 kilobases, or no more than 3 kilobases. In some embodiments, the size of the expression construct is between 4 kilobases and 7 kilobases.

[0077] In some embodiments, the rAAV particles comprise the nucleic acids described herein (such as vectors or their recombinant genomes or transgenes). In some embodiments, the rAAV particles comprise at least 75% identical nucleic acids to any one of SEQ ID NO:43-46. For example, in some embodiments, the rAAV particles comprise at least 75% identical nucleic acids to any one of the sequences shown in SEQ ID NO:43-46, such that the nucleic acid contains an ITR and / or regulatory sequence different from the sequence, and / or wherein the nucleic acid contains one or more codons different from the sequence (e.g., wherein the nucleic acid is codon-optimized). In some embodiments, the rAAV particles comprise 75-80%, 80-85%, 85-90%, 90-95%, 96%, 97%, 98%, or 99% identical nucleic acids to any one of SEQ ID NO:43-46. In some embodiments, the rAAV particles comprise the nucleic acids or portions thereof shown in any one of SEQ ID NO:43-46, such as ITRs, transgenes, regulatory sequences, etc., or any combination thereof. In some embodiments, the anti-GA antibody described herein is encoded by a nucleotide sequence contained in any one of SEQ ID NO:43-46. Non-limiting embodiments of the nucleic acids disclosed herein are shown in Table 1 below.

[0078] Table 1. Representative vectors used for anti-GA antibody expression

[0079]

[0080]

[0081]

[0082]

[0083]

[0084]

[0085]

[0086]

[0087]

[0088] The capsid of rAAV particles can be any AAV serotype (such as AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, or AAV10), including any derivatives (including non-naturally occurring variants of the serotype) or pseudotypes. In some embodiments, the AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, or AAV10 capsid proteins are mammalian AAV serotypes (mammal AAV1, mammalian AAV2, mammalian AAV3, mammalian AAV4, mammalian AAV5, mammalian AAV6, mammalian AAV7, mammalian AAV8, mammalian AAV9, or mammalian AAV10, respectively). In some embodiments, the AAV capsid protein is the AAV9 capsid protein. In some embodiments, the AAV capsid protein is the AAV1 capsid protein.

[0089] In some implementations, rAAV particles are pseudotyped rAAV particles. Non-limiting examples of rAAV pseudotypes include mammalian AAV2 / 1, mammalian AAV2 / 5, mammalian AAV2 / 6, mammalian AAV2 / 8, mammalian AAV2 / 9, mammalian AAV3 / 1, mammalian AAV3 / 5, mammalian AAV3 / 8, and mammalian AAV3 / 9, where the slash indicates that the rAAV genome of one serotype is packaged in the capsid of a different serotype (e.g., an rAAV genome containing AAV2 ITR packaged in the capsid of AAV5 is AAV2 / 5).

[0090] In some embodiments, the rAAV particles comprise hybrid or mutant mammalian AAV capsid protein derivatives, such as AAVrh.10, AAVrh.74, AAVhu.14, AAV3a / 3b, AAVrh32.33, AAV-HSC15, AAV-HSC17, AAVhu.37, AAVrh.8, CHt-P6, AAV2.5, AAV6.2, AAV2i8, AAV-HSC15 / 17, AAVM41, AAV9.45, AAV6(Y445F / Y731F), AAV2.5T, AAV-HAE1 / 2, and AAV clone. 32 / 83, AAVShHIO, AAV2(Y->F), AAV8(Y733F), AAV2.15, AAV2.4, AAVM41, AAV2(pen taYF), AAV2-BCDG(T491V+K556R), AAV5-M2, AAV5(Y719F), AAV6(T492V+S663V), AA V6(T492V+Y705F+Y731F), AAV6(S551V+S663V), AAV8-C&G(T494V), AAV8-M3, AAV8( Y733F), AAV8(T494V+Y733F), AAV8(Y275F+Y447F+Y733F), AAV9-PHP.B or AAVr3.45.

[0091] In some embodiments, the VP1 protein of the AAV capsid comprises the ERDTRG peptide (SEQ ID NO:49). The VP1 protein comprising the ERDTRG (SEQ ID NO:49) peptide can be any AAV serotype (such as AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, or AAV10). In some embodiments, the VP1 variant protein is the AAV9 serotype. In some embodiments, the VP1 variant protein is the AAV1 serotype. In some embodiments, the VP1 protein of the AAV1 serotype comprises the amino acid sequence shown in SEQ ID NO:48 below.

[0092] have ERDRTRG (SEQ ID NO:49) AAV1 capsid peptide with peptide insertion

[0093] MAADGYLPDWLEDNLSEGIREWWDLKPGAPKPKANQQKQDDGRGLVLPGYKYLGPFNGLDKGEPVNAADAAALEHDKAYDQQLKAGDNPYLRYNHADAEFQERLQEDTSFGGNLGRAVFQAKKRVLEPLGLVEEGAKTAPGKKRPVEQSPQEPDSSSGIGKTGQQPAKKRLNFGQTGDSESVPDPQPLGEPPATPAAVGPTTMASGGGAPMADNNEGADGVGNASGNWHCDSTWLGDRVITTSTRTWALPTYNNHLYKQISSASTGASNDNHYFGYSTPWGYFDFNRFHCHFSPRDWQRLINNNWGFRPKRLNFKLFNIQVKEVTTNDGVTTIANNLTSTVQVFSDSEYQLPYVLGSAHQGCLPPFPADVFMIPQYGYLTLNNGSQAVGRSSFYCLEYFPSQMLRTGNNFTFSYTFEEVPFHSSYAHSQSLDRLMNPLIDQYLYYLNRTQNQSGSAQNKDLLFSRGSPAGMSVQPKNWLPGPCYRQQRVSKTKTDNNNSNFTWTGASKYNLNGRESIINPGTAMASHKDDEDKFFPMSGVMIFGKESAGASNTALDNVMITDEEEIKATNPVATERFGTVAVNFQSSSTDSSA ERDRTRG ASPATGDVHAMGALPGMVWQDRDVYLQGPIWAKIPHTDGHFHPSPLMGGFGLKNPPPQILIKNTPVPANPPAEFSATKFASFITQYSTGQVSVEIEWELQKENSKRWNPEVQYTSNYAKSANVDFTVDNNGLYTEPRPIGTRYLTRPL*(SEQ ID NO: 48)

[0094] Other AAV serotypes and derivatives / pseudotypes, as well as methods for producing such derivatives / pseudotypes, are known in the art (see Mol Ther. 20 April 2012 (4):699-708. doi: 10.1038 / mt.2011.287. Epub 24 January 2012. The AAV vector toolkit: poised at the clinical crossroads. Asokan A1, Schaffer DV, Samulski RJ.).

[0095] Typically, rAAV particles are generated in host cells upon contact with helper and packaging nucleic acids that support AAV replication. These functions include, but are not limited to, activation of AAV gene transcription, stage-specific AAV mRNA splicing, AAV DNA replication, synthesis of capsid expression products, and AAV capsid assembly.

[0096] Helper nucleic acids typically comprise the E1 gene, E2A gene, E4 gene, VA gene, or any combination thereof. In some embodiments, helper nucleic acids are provided by contacting host cells with a vector (e.g., a plasmid) containing the helper nucleic acid. In some embodiments, helper nucleic acids are provided by contacting host cells with a helper virus. In some embodiments, the helper virus is an adenovirus (e.g., human adenovirus 2, human adenovirus 5, or human adenovirus 12) or a recombinant herpes simplex virus containing an AAV helper gene. Preferably, the AAV helper nucleic acid supports efficient rAAV particle production without producing any detectable wild-type AAV particles (e.g., AAV particles containing functional rep and capsid protein genes). Helper nucleic acids and methods for preparing said nucleic acids have been previously described and are commercially available (see, for example, the following helper nucleic acids and references to public disclosures related to rAAV production, incorporated by reference): pDM, pDG, pDP1rs, pDP2rs, pDP3rs, pDP4rs, pDP5rs, pDP6rs, pDG(R484E / R585E) and pDP8.ape plasmids from Plasmid Factory (Bielefeld, Germany); other products and services from Vector Biolabs (Philadelphia, PA), Cellbiolabs (San Diego, CA), Agilent Technologies (Santa Clara, CA) and Addgene (Cambridge, MA); pxx6; Grimm et al. (1998), Novel Tools for Production and Purification of Recombinant Adeno associated Virus Vectors, Human Gene Therapy, Vol. 9, 2745-2760; Kern, A. et al. (2003), Identification of a Heparin-Binding Motif on Adeno-Associated Virus Type 2Capsids, Journal of Virology, Vol. 77, 11072-11081; Grimm et al. (2003), HelperVirus-Free, Optically Controllable, and Two-Plasmid-Based Production ofAdeno-associated Virus Vectors of Serotypes 1 to 6, Molecular Therapy, Vol.7, 839-850; Kronenberg et al. (2005), A Conformational Change in the Adeno-Associated Virus Type 2 Capsid Leads to the Exposure of Hidden VP1 N Termini, Journal of Virology, Vol. 79, 5296-5303; and Moullier, P. and Snyder, RO (2008), International efforts for recombinant adeno associated viral vector reference standards, Molecular Therapy, Vol. 16, 1185-1188). .

[0097] Packaging nucleic acids typically provide the nucleotide sequences upon which AAV replication depends, including sequences encoding the AAV rep protein and the AAV capsid protein. In some embodiments, the rAAV described herein is generated using a packaging nucleic acid containing a nucleotide sequence encoding the AAV capsid protein of a mammalian AAV serotype. In some embodiments, the rAAV described herein is generated using a packaging nucleic acid containing the nucleotide sequence GAGAGGGATCGGACTAGGGGT (SEQ ID NO:50) located in the sequence encoding the AAV capsid protein. In some embodiments, the packaging nucleic acid contains the nucleotide sequence GAGAGGGATCGGACTAGGGGT (SEQ ID NO:50) described herein. ERDRTRG The rAAV of the VP1 variant protein of the (SEQ ID NO:49) peptide is generated using a packaging nucleic acid containing the nucleotide sequence shown in SEQ ID NO:47. In some embodiments, the nucleotide sequence encoding the ERDRTRG (SEQ ID NO:49) peptide is codon-optimized.

[0098] Encoding has ERDRTRG (SEQ ID NO:49) Nucleic acid sequence of AAV1 capsid protein with peptide insertion.

[0099] GAGAGGGATCGGACTAGGGGT GCTAGCCCTGCCACTGGTGACGTGCATGCTATGGGTGCCTTACCTGGCATGGTGTGGCAAGATAGAGACGTGTACCTGCAGGGTCCCATTTGGGCCAAAATTCCTCACACAGATGGACACTTTCACCCGTCTCCTCTTATGGGCGGCTTTGGACTCAAGAACCCGCCTCCTCAGATCCTCATCAAAAACACGCCTGTTCCTGCGAATCCTCCGGCGGAGTTTTCAGCTACAAAGTTTGCTTCATTCATCACCCAATACTCCACAGGACAAGTGAGTGTGGAAATTGAATGGGAGCTGCAGAAAGAAAACAGCAAGCGCTGGAATCCCGAAGTGCAGTACACATCCAATTATGCAAAATCTGCCAACGTTGATTTTACTGTGGACAACAATGGACTTTATACTGAGCCTCGCCCCATTGGCACCCGTTACCTTACCCGTCCCCTGTAA (SEQ ID NO: 47)

[0100] In some embodiments, components cultured in host cells to package the rAAV genome in a capsid may be trans-provided to the host cells. In some embodiments, rAAV particles may be produced using a triple transfection method (see, for example, U.S. Patent No. 6,001,650, the disclosure of which relating to a triple transfection method for rAAV production is incorporated herein by reference). Typically, rAAV particles are produced by transfecting host cells with an AAV vector to be packaged into rAAV particles (see, for example, Table 1) and at least one nucleic acid containing an AAV helper gene and / or packaging gene. In some embodiments, two nucleic acids are used, including a helper nucleic acid and a packaging nucleic acid. In some embodiments, any one or more components used for rAAV particle production (such as an AAV vector, AAV rep sequence, AAV cap sequence, and / or helper nucleic acid) may be provided by host cells engineered to stably contain said one or more components. Such host cells will typically contain said one or more components under the control of an inducible promoter or a constitutive promoter. Examples of suitable inducible and constitutive promoters are provided. Methods for constructing nucleic acids or their rAAV particles have been described (see, for example, Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Press, Cold Spring Harbor, NY, the disclosure relating to nucleic acid engineering is incorporated herein by reference, see also K. Fisher et al., J. Virol., 70:520-532 (1993) and U.S. Patent No. 5,478,745, the disclosure relating to methods for producing rAAV is incorporated herein by reference).

[0101] RAN protein

[0102] "RAN proteins (repetition-associated non-ATG translational proteins)" are polypeptides that translate from bidirectionally transcribed, positive or negative RNA sequences via repeat amplification mutations in the absence of an AUG start codon. RAN protein coding sequences can be found at multiple loci in the genome, including but not limited to open reading frame 72 (C9orf72) on chromosome 9, open reading frame 80 (C2orf80) on chromosome 2, LRP8, CASP8, CRNDE, EXOC6B, SV2B, PPML1, ADARB2, GREB1, and MSMO1. The characterization of proteins associated with C9orf72 is currently unclear, but they are known to be abundant in neurons, particularly in the cerebral cortex and motor neurons. C9orf72 proteins are thought to be located at the presynaptic terminal. C9orf72 proteins may influence RNA transcription, translation, and intracellular localization. The C9orf72 gene contains a GGGGCC repeat. This hexanucleotide repeat appears in a variable number of repeats, with fewer repeats not associated with any pathology.

[0103] Typically, RAN proteins contain amplified repeats of single, di, tri, or quad-amino acids (such as tetra-amino acids), referred to as polyamino acid repeats. For example, “AAAAAAAAAAAAAAAAAAAAAAA” (SEQ ID NO: 51) (polyalanine), “LLLLLLLLLLLLLLLLLLLL” (SEQ ID NO: 52) (polyleucine), “SSSSSSSSSSSSSSSSSSSS” (SEQ ID NO: 53) (polyserine), or “CCCCCCCCCCCCCCCCCCCC” (SEQ ID NO: 54) (polycysteine) are polyamino acid repeats, each with a length of 20 amino acid residues. Examples of diamino acid RAN proteins include GPGPGPGPGPGPGPGPGPGPGPGP (SEQ ID NO: 55) (polyGP), GAGAGAGAGAGAGAGAGAGAGA (SEQ ID NO: 56) (polyGA), GRGRGRGRGRGRGRGRGRGR (SEQ ID NO: 57) (polyGR), PAPAPAPAPAPAPAPAPAPAPA (SEQ ID NO: 58) (polyPA), and PRPRPRPRPRPRPRPRPRPR (SEQ ID NO: 59) (polyPR). Examples of tetraamino acid repeats include LPACLPACLPAC (SEQ ID NO: 60) (e.g., polyLPAC) and QAGRQAGRQAGR (SEQ ID NO: 61) (e.g., polyQAGR). RAN proteins may have polyamino acid repeats of at least 20, at least 25, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, or at least 200 amino acid residues. In some embodiments, the RAN protein has polyamino acid repeats of more than 200 amino acid residues (e.g., 500, 1000, 5000, 10,000, etc.).

[0104] Typically, RAN proteins are translated from aberrant repetitive amplifications of DNA (such as TCT repeats, hexanucleotide repeats such as GGGGCC, etc.). This disclosure is partly based on the identification of microsatellite repeats in subjects with certain RAN protein-related diseases characterized by the expression of one or more (e.g., 2, 3, 4, 5, or more) RAN proteins, such as poly(glycine-alanine) [poly(GA)]. In some embodiments, the disease status of subjects with or suspected of having RAN protein-related diseases is classified by the number and / or type of microsatellite repeats present in the subject (e.g., in the subject's genome or in the subject's genes).

[0105] "Subjects who have or are suspected of having a disease (such as a neurological disease) related to RAN protein expression, translation and / or accumulation" generally refers to subjects who exhibit signs and symptoms of one or more neurodegenerative diseases, including but not limited to memory deficits (such as short-term memory loss), confusion, deficits in executive functions (such as attention, planning, flexibility, abstract thinking, etc.), loss of speech, degeneration or loss of motor skills, or subjects who have or are identified as having one or more gene mutations related to RAN protein expression, translation and / or accumulation.

[0106] "Subjects with or suspected of having amyotrophic lateral sclerosis (ALS)" can be subjects exhibiting one or more signs and symptoms of ALS, including but not limited to memory impairment (such as short-term memory loss), confusion, impairment of executive functions (such as attention, planning, flexibility, abstract thinking, etc.), loss of speech, decline or loss of motor skills, or subjects who have or are identified as having one or more gene mutations associated with ALS (e.g., mutations in specific genes, including C9orf72). In some embodiments, the subject has been diagnosed with ALS by a medical professional. "Subjects with or suspected of having Alzheimer's disease (AD)" can be subjects exhibiting one or more signs and symptoms of AD, including but not limited to memory impairment (such as short-term memory loss), confusion, impairment of executive functions (such as attention, planning, flexibility, abstract thinking, etc.), loss of speech, decline or loss of motor skills, or subjects who have or are identified as having one or more gene mutations associated with AD (e.g., mutations in specific genes). In some embodiments, the subject has been diagnosed with AD by a medical professional. "Subjects with or suspected of having frontotemporal dementia (FTD)" can be subjects exhibiting one or more signs and symptoms of FTD, including but not limited to memory deficits (such as short-term memory loss), confusion, deficits in executive functions (such as attention, planning, flexibility, abstract thinking, etc.), loss of speech, regression or loss of motor skills, or subjects who have or are identified as having one or more gene mutations (e.g., mutations in specific genes) associated with FTD. In some embodiments, the subject has been diagnosed with ALS by a medical professional. The subject can be a mammal (such as a human, mouse, rat, dog, cat, or pig). In some embodiments, the subject is a non-human animal, such as a mouse, rat, guinea pig, cat, dog, horse, camel, etc. In some embodiments, the subject is a human.

[0107] In some embodiments, subjects with fewer than 10 repeat sequences do not exhibit signs or symptoms of RAN protein-related disease characterized by RAN protein translation. In some embodiments, subjects with between 10 and 40 repeats may or may not exhibit one or more signs or symptoms of RAN protein-related disease characterized by RAN protein translation. In some embodiments, subjects with more than 40 trinucleotide repeats exhibit one or more signs or symptoms of RAN protein-related disease characterized by RAN protein translation. In some cases, subjects are identified as having RAN protein-related disease characterized by a large number (>100) of repeats. Microsatellite repeat sequences encoding RAN proteins are generally known. In some embodiments, the RAN protein-related disease is Alzheimer's disease.

[0108] In some implementations, subjects with or suspected of having RAN protein-related diseases possess one or more microsatellite repeat sequences encoding poly(GA) RAN proteins. Examples of microsatellite repeat sequences encoding poly(GA) proteins include GGGGCC.

[0109] In some aspects, this disclosure relates to the discovery of length-dependent aggregation patterns of RAN proteins (such as poly(GA)). For example, RAN proteins with polyamino acid repeats of length >20, >48, or >80 residues aggregate differently in the brains of subjects. Generally, the differential aggregation characteristics of RAN proteins of different lengths can be used to detect RAN proteins in biological samples. Longer RAN proteins are present at higher levels in biological samples (such as blood, serum, or CSF). In some embodiments, RAN proteins with polyamino acid repeats of length >40, >50, >60, >70, or >80 amino acid residues can be detected in biological samples.

[0110] Monoclonal antibodies

[0111] Various aspects of this disclosure relate to antibodies and antigen-binding fragments that specifically bind to RAN proteins, and methods for preparing and using said antibodies and antigen-binding fragments. In some embodiments, the antibody or antigen-binding fragment specifically binds to poly(glycine-alanine) [poly(GA)] RAN proteins. In some embodiments, the antibody directly binds to poly(GA) repeat regions (e.g., binding to the GAGAGA (SEQ ID NO:62) motif). In some embodiments, the antibody binds to non-repeatable regions of RAN proteins (e.g., poly(GA) RAN proteins), such as the unique C-terminal amino acid sequence of the RAN protein.

[0112] As used herein, antibody broadly refers to an immunoglobulin molecule or any functional mutant, variant, or derivative thereof. Desired functional mutants, variants, and their derivatives, as well as antigen-binding fragments, retain the essential epitope binding characteristics of an Ig molecule. An antibody is capable of specifically binding to a target through at least one antigen recognition site located in the variable region of an immunoglobulin molecule. Typically, a complete or full-length antibody consists of two heavy chains and two light chains. Each heavy chain contains a heavy chain variable region (VH) and first, second, and third constant regions (CH1, CH2, and CH3). Each light chain contains a light chain variable region (VL) and a constant region (CL). The VH and VL regions can be further subdivided into hypervariable regions called complementarity-determining regions (CDRs), interspersed with more conserved regions called framework regions (FRs). The CDR components on the heavy chain are called CDRH1, CDRH2, and CDRH3, while the CDR components on the light chain are called CDRL1, CDRL2, and CDRL3.

[0113] CDR usually refers to Kabat CDR, as described in Sequences of Proteins of Immunological Interest, US Department of Health and Human Services (1991), eds. Kabat et al. Another standard for characterizing antigen-binding sites is the hypervariable loop described by Chothia. See Chothia, D. et al. (1992) J. Mol. Biol. 227:799-817; and Tomlinson et al. (1995) EMBO J. 14:4628-4638. Yet another standard is the AbM definition used by the Oxford Molecular AbM antibody modeling software. See Protein Sequence and Structure Analysis of Antibody Variable Domains. In: Antibody Engineering Lab Manual (Ed.: Duebel, S., and Kontermann, R., Springer-Verlag, Heidelberg). The implementation described in the Kabat CDR can be implemented alternatively using similar descriptive relations regarding the Chothia high-variable ring or the AbM defined ring, or any combination of these methods.

[0114] Each VH and VL consists of three CDRs and four FRs, arranged in the following order from the amino terminus to the carboxyl terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. Full-length antibodies can be of any class, such as IgD, IgE, IgG, IgA, or IgM (or their subclasses), and the antibody does not need to be of any specific class. Immunoglobulins are classified into different classes based on the amino acid sequence of their heavy chain constant domains. There are five major classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, and several of these classes can be further subdivided into subclasses (isotypes), such as IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. The heavy chain constant domains corresponding to different classes of immunoglobulins are called α, δ, ε, γ, and μ, respectively. The subunit structures and three-dimensional conformations of different classes of immunoglobulins are well known.

[0115] The term "antigen-binding fragment" refers to any antibody derivative that is shorter than full length and capable of specifically binding to a target. Preferably, the antigen-binding fragments provided herein retain the ability to specifically bind to RAN proteins. Antigen-binding fragments may contain a heavy chain variable region (VH), a light chain variable region (VL), or both. VH and VL typically each contain three complementarity-determining regions, CDR1, CDR2, and CDR3.

[0116] Examples of antigen-binding fragments include, but are not limited to, Fab, Fab', F(ab')2, scFv, Fv, dsFv, biantibodies, affinity molecules, and Fd fragments. Antigen-binding fragments can be generated by any suitable method. For example, antigen-binding fragments can be generated by enzymatic or chemical fragmentation of an intact antibody, or by recombination of a gene encoding a partial antibody sequence. Alternatively, antigen-binding fragments can be generated entirely or partially synthetically. Antigen-binding fragments may optionally be single-chain antibody fragments. Alternatively, the fragment may comprise multiple chains linked together by, for example, disulfide bonds. Antigen-binding fragments may also optionally be multi-molecular complexes. Functional antigen-binding fragments will typically contain at least about 50 amino acids, and more typically will contain at least about 200 amino acids.

[0117] A single-chain Fv (scFv) is a recombinant antigen-binding fragment composed of only the light chain variable region (VL) and the heavy chain variable region (VH) covalently linked together by a polypeptide linker. Both VL and VH can be NH2-terminal domains. The polypeptide linker can have variable length and composition, as long as the two variable domains are bridged without significant steric hindrance. Typically, the linker mainly consists of a fragment of glycine and serine residues, interspersed with some glutamic acid or lysine residues to increase solubility. ScFv is encompassed within the term "antigen-binding fragment".

[0118] Biantibodies are dimeric scFvs. The components of biantibodies typically have shorter peptide linkers than most scFvs, and they exhibit a preference for binding in a dimeric form (see Holliger, P., et al. (1993) Proc. Natl. Acad. Sci. USA 90:6444-6448; Poljak, RJ, et al. (1994) Structure 2: 1121-1123). Biantibodies are also encompassed within the term "antigen-binding fragment".

[0119] Fv fragments are antigen-binding fragments consisting of a VH and a VL domain held together by non-covalent interactions. Although the two domains, VL and VH, of an Fv fragment can be encoded by separate genes, they can be linked together using recombinant methods via synthetic linkers, enabling them to be produced as single-chain proteins, where the VL and VH regions pair to form a monovalent molecule (called a single-chain Fv (scFv); see, for example, Bird et al. (1988) Science 242:423-426; and Huston et al. (1988) Proc. Natl. Acad. Sci. USA 85:5879-5883). Such single-chain antibodies are also intended to be included within the term "antigen-binding fragment" of antibodies. The term dsFv is used herein to refer to Fvs with engineered intermolecular disulfide bonds to stabilize the VH-VL pair. dsFv is also encompassed within the term "antigen-binding fragment."

[0120] The F(ab')2 fragment is essentially equivalent to the antigen-binding fragment obtained by digesting immunoglobulins (usually IgG) with pepsin at pH 4.0–4.5. This fragment can be recombined. F(ab')2 is also included within the term "antigen-binding fragment".

[0121] A Fab fragment is essentially equivalent to an antigen-binding fragment obtained by reducing the disulfide bonds connecting the two heavy chain segments in the F(ab')2 segment. Fab' fragments can be recombined. Fab' is also encompassed within the term "antigen-binding fragment".

[0122] Fab fragments are essentially equivalent to antigen-binding fragments obtained by digesting immunoglobulins (usually IgG) with papain. Fab fragments can be recombined. The heavy chain segment of a Fab fragment is the Fd segment. Fab fragments are also included within the term "antigen-binding fragment".

[0123] Affinities are small proteins consisting of triple-helical bundles that act as antigen-binding molecules (such as antibody mimics). Typically, affinities are about 58 amino acids in length and have a molar mass of about 6 kDa. Affinity molecules with unique binding properties are obtained by randomizing 13 amino acids located in the two α-helices involved in the binding activity of the parent protein domain. Specific affinity molecules binding to the desired target protein can be isolated from pools (libraries) containing billions of different variants using methods such as phage display. Affinities are also encompassed within the term "antigen-binding fragment."

[0124] The term "human antibody" refers to an antibody having variable and constant regions substantially corresponding to or derived from antibodies obtained from human subjects, such as those encoded by human germline immunoglobulin sequences or variants thereof. Human antibodies may contain one or more amino acid residues not encoded by human germline immunoglobulins (e.g., mutations introduced through random or site-directed mutagenesis in vitro or through somatic mutations in vivo). Such mutations may be present in one or more CDRs (especially CDR3) or in one or more frame regions. In some embodiments, a human antibody may have at least one, two, three, four, five, or more positions replaced by amino acid residues not encoded by human germline immunoglobulin sequences. However, the term "human antibody" as used herein is not intended to include antibodies in which a CDR sequence derived from another mammalian species (such as a mouse) has been grafted onto a human frame sequence.

[0125] As used herein, the term “recombinant human antibody” is intended to include all human antibodies prepared, expressed, created, or isolated by recombinant means, such as antibodies expressed using recombinant expression vectors transfected into host cells, antibodies isolated from recombinant human antibody libraries (Hoogenboom HR, (1997) TIB Tech. 15:62-70; Azzazy H., and Highsmith WE, (2002) Clin. Biochem. 35:425-445; Gavilondo JV, and Larrick J. W. (2002) BioTechniques 29: 128-145; Hoogenboom H., and Chames P. (2000) Immunology Today 21:371-378), and antibodies isolated from animals transgenic with human immunoglobulin genes (such as mice) (see Taylor, LD, et al. (1992) Nucl. Acids Res.). 20:6287-6295; Kellermann S-A., and Green LL (2002) Current Opinion in Biotechnology 13:593-597; Little M. et al. (2000) Immunology Today 21:364-370), or antibodies prepared, expressed, created, or isolated by any other means involving splicing human immunoglobulin gene sequences into other DNA sequences. Such recombinant human antibodies have variable and constant regions as defined above. However, in some embodiments, such recombinant human antibodies may be mutagenized in vitro (or in vivo somatic cell mutagenized when using transgenic animals with human Ig sequences), and therefore the amino acid sequences of the VH and VL regions of the recombinant antibody may be sequences that, although derived from and related to human germline VH and VL sequences, may not naturally exist in an in vivo human antibody germline library.

[0126] In some embodiments, the antibody or antigen-binding fragment comprises a heavy chain containing the amino acid sequence shown in any one of SEQ ID NO: 8, 10, 27, and 29. In some embodiments, the anti-RAN antibody and antigen-binding fragment of this disclosure comprises a light chain containing the amino acid sequence shown in any one of SEQ ID NO: 13, 15, 32, and 34.

[0127] In some embodiments, the anti-RAN antibody or antigen-binding fragment may or may not include a frame region of the antibody, such as a frame region amino acid sequence. In some embodiments, the anti-RAN antibody is a mouse antibody. In some embodiments, the anti-RAN antibody is a chimeric or humanized antibody.

[0128] In some embodiments, the antibody or antigen-binding fragment comprises a VH sequence as shown in any one of SEQ ID NO:7, 17, and 26. In some embodiments, the antibody or antigen-binding fragment comprises a VL sequence as shown in any one of SEQ ID NO:12, 18, or 31.

[0129] In some embodiments, the antibody or antigen-binding fragment comprises a VH sequence as shown in SEQ ID NO:7 and a VL sequence as shown in SEQ ID NO:12.

[0130] In some embodiments, the antibody or antigen-binding fragment comprises a VH sequence as shown in SEQ ID NO:17 and a VL sequence as shown in SEQ ID NO:18.

[0131] In some embodiments, the antibody or antigen-binding fragment comprises a VH sequence as shown in SEQ ID NO:26 and a VL sequence as shown in SEQ ID NO:31.

[0132] In some embodiments, the antibody or antigen-binding fragment comprises six complementarity-determining regions (CDRs): CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and CDRL3, wherein CDRH1 comprises the sequence shown in SEQ ID NO:1, CDRH2 comprises the sequence shown in SEQ ID NO:2, CDRH3 comprises the sequence shown in SEQ ID NO:3, CDRL1 comprises the sequence shown in SEQ ID NO:4, CDRL2 comprises the sequence shown in SEQ ID NO:5, and CDRL3 comprises the sequence shown in SEQ ID NO:6.

[0133] In some embodiments, the antibody or antigen-binding fragment comprises six complementarity-determining regions (CDRs): CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and CDRL3, wherein CDRH1 comprises the sequence shown in SEQ ID NO:20, CDRH2 comprises the sequence shown in SEQ ID NO:21, CDRH3 comprises the sequence shown in SEQ ID NO:22, CDRL1 comprises the sequence shown in SEQ ID NO:23, CDRL2 comprises the sequence shown in SEQ ID NO:24, and CDRL3 comprises the sequence shown in SEQ ID NO:25.

[0134] It should be understood that, in some embodiments, this disclosure considers variants (like homologs) of the amino acid and nucleic acid sequences of the antibody heavy chain variable region and light chain variable region. "Homology" refers to the percentage identity between two polynucleotides or two polypeptide moieties. When referring to a nucleic acid or fragment thereof, the term "substantial homology" means that, when optimally aligned with another nucleic acid (or its complementary strand) with appropriate insertions or deletions of nucleotides, there is nucleotide sequence identity in about 90% to 100% of the aligned sequences. For example, in some embodiments, nucleic acid sequences sharing substantial homology have sequence identity of at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%. When referring to a polypeptide or fragment thereof, the term "substantial homology" means that, when optimally aligned with another polypeptide with appropriate insertions or deletions, there is amino acid sequence identity in about 90% to 100% of the aligned sequences. The term "highly conserved" means at least 80% identity, preferably at least 90% identity, and more preferably more than 97% identity. For example, in some implementations, highly conserved proteins share at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity. In some cases, high conservatism may refer to 100% identity. Identity can be readily determined by those skilled in the art, for example, by using algorithms and computer programs known to them.

[0135] In some embodiments, the RAN antibody of this disclosure can bind to RAN proteins with high affinity, such as Kd less than 10. -7 M, 10 -8 M, 10 -9 M, 10 -10 M, 10 -11 M or lower. For example, anti-RAN antibodies or antigen-binding fragments can bind RAN proteins with an affinity between 5 pM and 500 nM, such as between 50 pM and 100 nM, or between 500 pM and 50 nM. This disclosure also includes antibodies or antigen-binding fragments that compete with any antibody described herein for binding to RAN proteins and have an affinity of 50 nM or lower (e.g., 20 nM or lower, 10 nM or lower, 500 pM or lower, 50 pM or lower, or 5 pM or lower). The affinity and binding kinetics of anti-RAN protein antibodies can be tested using any method known in the art, including but not limited to biosensor technologies such as OCTET or BIACORE.

[0136] In some embodiments, the anti-RAN antibody of this disclosure includes the VH, VL, and CDR amino acid sequences shown in Table 2 below.

[0137] Table 2: Representative sequences of developed anti-poly(GA) monoclonal antibodies

[0138]

[0139] In some embodiments, antibody clone 27B11 binds to poly(GA). In some embodiments, clone 27B11 is an IgG1 antibody. In some embodiments, clone 27B11 is an IgG2 antibody. In some embodiments, clone 27B11 is scFv. In some embodiments, antibody clone 23H2 binds to poly(GA). In some embodiments, antibody clone 23H2 is an IgG1 antibody. In some embodiments, antibody clone 23H2 is an IgG2 antibody.

[0140] Anti-RAN antibodies can be used to treat or as adjunctive therapy for one or more symptoms of diseases associated with the RAN protein. In some embodiments, diseases associated with the RAN protein are selected from the group consisting of: amyotrophic lateral sclerosis (ALS), Alzheimer's disease (AD), and frontotemporal dementia. In some embodiments, the neurological disease associated with the RAN protein is Alzheimer's disease (AD).

[0141] In some embodiments, anti-RAN antibodies can be used to treat or as adjunctive therapy for one or more symptoms of RAN protein-related diseases, for example by administering a therapeutically effective amount of one or more anti-RAN antibodies to a subject diagnosed with one or more symptoms of RAN protein-related diseases (such as early Alzheimer's disease) or at risk of developing RAN protein-related diseases (such as based on one or more assays described in this application).

[0142] As used herein, “treating” a disease (such as a disease associated with poly(GA) RAN translation, such as Alzheimer’s disease) means reducing the frequency or severity of at least one sign or symptom of the disease or condition experienced by the subject. The compositions described above or elsewhere herein are generally administered to the subject in an effective amount, i.e., an amount capable of producing the desired outcome. The desired outcome will depend on the active agent administered. An acceptable therapeutic amount of an anti-RAN protein antibody may be an amount capable of treating a disease (such as Alzheimer’s disease) by reducing the expression and / or aggregation of RAN proteins and / or the presence or number of RNA foci containing RAN protein-encoding microsatellite repeat sequences. As is well known in the medical and veterinary fields, the dosage for any subject depends on a variety of factors, including the subject’s body size, body surface area, age, the specific composition to be administered, the active ingredient in the composition, the time and route of administration, general health condition, and any other medications administered concurrently.

[0143] In some embodiments, one or more anti-RAN antibodies or antigen-binding fragments disclosed herein are administered to a subject, wherein the subject has been characterized as having a RAN protein-related disease by detecting at least one RAN protein in a biological sample obtained from the subject.

[0144] Production of anti-RAN antibodies

[0145] Typically, polyclonal antibodies are produced by inoculating suitable mammals (such as mice, rabbits, or goats). An antigen is injected into the mammal. This induces B lymphocytes to produce IgG immunoglobulins specific to that antigen. This polyclonal IgG is then purified from the mammalian serum. Monoclonal antibodies are typically produced from a single cell line (such as a hybridoma cell line). In some embodiments, anti-RAN antibodies are purified (e.g., isolated from serum). In some embodiments, the antigen contains a poly(GA)RAN protein repeat sequence.

[0146] Several methods can be used to obtain anti-RAN antibodies. For example, antibodies can be generated using recombinant DNA methods. Monoclonal antibodies can also be produced by generating hybridomas (see, for example, Kohler and Milstein (1975) Nature, 256:495-499), according to known methods. Hybridomas formed in this manner are then screened using standard methods, such as enzyme-linked immunosorbent assays (ELISA; such as RCA-based ELISA or rtPCR-based ELISA) and surface plasmon resonance (such as OCTET or BIACORE) assays, to identify one or more hybridomas that produce antibodies that specifically bind to a particular antigen. Any form of the specific antigen (such as RAN protein) can be used as an immunogen, such as a recombinant antigen, a naturally occurring form, any variant or fragment thereof. An exemplary method for preparing antibodies involves screening protein expression libraries, such as phage or ribosome display libraries, that express the antibody or fragments thereof (such as scFv). Phage display is described, for example, in Ladner et al., U.S. Patent No. 5,223,409; Smith (1985) Science 228:1315-1317; Clackson et al. (1991) Nature, 352:624-628; Marks et al. (1991) J. Mol. Biol., 222:581-597; WO 92 / 18619; WO 91 / 17271; WO 92 / 20791; WO 92 / 15679; WO 93 / 01288; WO 92 / 01047; WO 92 / 09690; and WO 90 / 02809.

[0147] In another embodiment, monoclonal antibodies are obtained from non-human animals and then modified using recombinant DNA techniques known in the art, such as to prepare chimeric antibodies. Various methods for preparing chimeric antibodies have been described. See Morrison et al., Proc. Natl. Acad. Sci. USA 81:6851, 1985; Takeda et al., Nature 314:452, 1985; Cabilly et al., U.S. Patent No. 4,816,567; Boss et al., U.S. Patent No. 4,816,397; Tanaguchi et al., European Patent Publication EP171496; European Patent Publication 0173494; and British Patent GB 2177096B.

[0148] Antibodies can also be humanized using methods known in the art. For example, monoclonal antibodies with desired binding specificity are commercially humanized (Scotgene, Scotland; and Oxford Molecular, Palo Alto, Calif.). Fully humanized antibodies, such as those expressed in transgenic animals, are within the scope of this invention (see Green et al. (1994) Nature Genetics 7, 13; and U.S. Patents 5,545,806 and 5,569,825). For other antibody production techniques, see Antibodies: A Laboratory Manual, Second Edition. Edited by Edward A. Greenfield, Dana-Farber Cancer Institute, ©2014. This disclosure is not necessarily limited to any particular source, method of production, or other special characteristics of the antibody.

[0149] Some aspects of this disclosure relate to isolated cells (such as host cells) transformed with polynucleotides or vectors. In some embodiments, the vector is an rAAV vector. In some embodiments, host cells are transduced or infected with rAAV particles containing an rAAV vector encoding an anti-poly(GA) antibody.

[0150] The host cell can be a prokaryotic or eukaryotic cell. The polynucleotides or vectors present in the host cell may be integrated into the host cell genome or retained extrachromosomally. The host cell can be any prokaryotic or eukaryotic cell, such as bacterial, insect, fungal, plant, animal, or human cells. In some embodiments, fungal cells are, for example, cells of the genus *Saccharomyces*, particularly *Saccharomyces cerevisiae*. The term "prokaryotic" includes all bacteria that can be transformed or transfected with DNA or RNA molecules to express antibodies or corresponding immunoglobulin chains. Prokaryotic hosts can include Gram-negative and Gram-positive bacteria, such as, for example, *Escherichia coli*, *Salmonella typhimurium*, *Serratia marcescens*, and *Bacillus subtilis*. The term "eukaryotic" includes yeast, higher plant, insect, and vertebrate cells, such as mammalian cells, such as NSO and CHO cells. Depending on the host used in the recombinant production process, the antibody or immunoglobulin chain encoded by the polynucleotide may or may not be glycosylated. The antibody or corresponding immunoglobulin chain may also contain a starting methionine amino acid residue.

[0151] In some embodiments, once the vector has been integrated into a suitable host, the host can be maintained under conditions suitable for high-level expression of the nucleotide sequence, and subsequently, if necessary, the immunoglobulin light chain, heavy chain, light / heavy chain dimer, or intact antibody, antigen-binding fragment, or other immunoglobulin forms can be collected and purified; see Beychok, Cells of Immunoglobulin Synthesis, Academic Press, NY, (1979). Thus, the introduction of polynucleotides or vectors into cells leads to the subsequent production of antibodies or antigen-binding fragments. Furthermore, transgenic animals (preferably mammals) containing the aforementioned host cells can be used for large-scale production of antibodies or antibody fragments.

[0152] The transformed host cells can be grown in a fermenter and cultured according to techniques known in the art to achieve optimal cell growth. Once expressed, the complete antibody, its dimer, individual light and heavy chains, other immunoglobulin forms, or antigen-binding fragments can be purified according to standard procedures in the art, including ammonium sulfate precipitation, affinity chromatography, column chromatography, gel electrophoresis, etc.; see Scopes, "Protein Purification", Springer Verlag, NY (1982). The antibody or antigen-binding fragment can then be isolated from the growth medium, cell lysate, or cell membrane fraction. For example, the isolation and purification of antibodies or antigen-binding fragments expressed by microorganisms can be performed by any conventional means, such as preparative chromatographic separation and immunological separation, such as those involving the use of monoclonal or polyclonal antibodies targeting, for example, the constant region of the antibody.

[0153] This disclosure relates to hybridomas, which provide an indefinite source of monoclonal antibodies. As used herein, “hybridoma cells” refers to immortalized cells derived from the fusion of B lymphoblasts with myeloma fusion companion cells. To prepare monoclonal antibody-producing cells (such as hybridoma cells), individual animals (such as mice) with confirmed antibody titers are selected, and their spleens or lymph nodes are removed 2 to 5 days after the last immunization. The antibody-producing cells contained therein are then fused with myeloma cells to prepare the desired monoclonal antibody-producing hybridoma. The antibody titer in the antiserum can be measured, for example, by reacting a labeled protein (described below) with the antiserum and then measuring the activity of the labeling agent bound to the antibody. Cell fusion can be performed according to known methods, such as those described by Kochler and Milstein (Nature 256:495 (1975)). Polyethylene glycol (PEG) or Sendai virus (HVJ) can be used as fusion promoters, for example.

[0154] Examples of myeloma cells include NS-1, P3U1, SP2 / 0, AP-1, etc. The ratio of antibody-producing cells (spleen cells) to myeloma cells is preferably from about 1:1 to about 20:1. PEG (preferably PEG 1000-PEG 6000) is preferably added at a concentration of about 10% to about 80%. Cell fusion can be effectively achieved by incubating the mixture of the two cell types at about 20°C to about 40°C (preferably about 30°C to about 37°C) for about 1 minute to 10 minutes.

[0155] Several methods can be used to screen hybridomas that produce antibodies (such as tumor antigens or autoantibodies against the present invention). For example, the supernatant of the hybridoma is added to a solid phase (such as a microplate), where antibodies are adsorbed directly or together with a carrier, and then radioactive material or enzyme-labeled anti-immunoglobulin antibodies (anti-mouse immunoglobulin antibodies are used if mouse cells are used in cell fusion) or protein A is added to detect monoclonal antibodies against proteins bound to the solid phase. Alternatively, the supernatant of the hybridoma is added to a solid phase adsorbed with anti-immunoglobulin antibodies or protein A, and then radioactive material or enzyme-labeled protein is added to detect monoclonal antibodies against proteins bound to the solid phase.

[0156] The selection of monoclonal antibodies can be performed according to any known method or its modifications. Typically, animal cell culture media supplemented with HAT (hypoxanthine, aminopterin, thymidine) are used. Any selection and growth medium can be used as long as the hybridoma can grow. For example, RPMI 1640 medium containing 1% to 20% (preferably 10% to 20%) fetal bovine serum, GIT medium containing 1% to 10% fetal bovine serum, serum-free medium for hybridoma culture (SFM-101, Nissui Seiyaku), etc., can be used. Culture is typically carried out at 20°C to 40°C (preferably 37°C) in approximately 5% CO2 gas for approximately 5 days to 3 weeks (preferably 1 to 2 weeks). The antibody titer in the hybridoma culture supernatant can be measured using the same method as described above for the antiprotein antibody titer in antiserum.

[0157] As an alternative to obtaining immunoglobulins directly from hybridoma cultures, immortalized hybridoma cells can be used as a source of rearranged heavy and light chain loci for subsequent expression and / or gene manipulation. The rearranged antibody genes can be reverse transcribed from suitable mRNA to produce cDNA. If desired, the heavy chain constant region can be exchanged for a different isotype of constant region or completely eliminated. Variable regions can be linked to encode single-stranded Fv regions. Multiple Fv regions can be linked to confer binding capability to more than one target, or a chimeric combination of heavy and light chains can be employed. Any suitable method can be used to clone antibody variable regions and generate recombinant antibodies.

[0158] In some embodiments, suitable nucleic acids encoding the variable regions of the heavy and / or light chains are obtained and inserted into an expression vector that can be transfected into standard recombinant host cells. A variety of such host cells can be used. In some embodiments, mammalian host cells may facilitate efficient processing and production. Typical mammalian cell lines used for this purpose include CHO cells, 293 cells, or NSO cells. Production of antibodies or antigen-binding fragments can be carried out by culturing modified recombinant hosts under culture conditions suitable for host cell growth and expression of the encoding sequence. Antibody or antigen-binding fragments can be recovered by isolating them from the culture. The expression system can be designed to contain a signal peptide such that the resulting antibody is secreted into the culture medium; however, intracellular production is also possible.

[0159] This disclosure also includes a polynucleotide encoding at least one variable region of an immunoglobulin chain of the antibody described herein. In some embodiments, the variable region encoded by the polynucleotide includes at least one complementarity-determining region (CDR) of the VH and / or VL of the variable region of an antibody generated from any of the hybridomas described above.

[0160] The polynucleotide encoding an antibody or antigen-binding fragment can be such as DNA, cDNA, RNA, or synthetically produced DNA or RNA, or a chimeric nucleic acid molecule resulting from recombination containing any of these polynucleotides (alone or in combination). In some embodiments, the polynucleotide is part of a vector. Such vectors may contain other genes, such as marker genes, which allow selection of the vector in suitable host cells and under suitable conditions.

[0161] In some embodiments, the polynucleotide is operatively linked to an expression control sequence that allows expression in prokaryotic or eukaryotic cells. Expression of the polynucleotide involves transcribing it into translatable mRNA. Regulatory elements ensuring expression in eukaryotic cells (preferably mammalian cells) are well known to those skilled in the art. These may include regulatory sequences that promote transcription initiation and optionally poly-A signals that promote transcription termination and transcript stabilization. Other regulatory elements may include transcription and translation enhancers, and / or naturally associated or heterologous promoter regions. Possible regulatory elements allowing expression in prokaryotic host cells include promoters such as PL, Lac, Trp, or Tac in *E. coli*, and examples of regulatory elements allowing expression in eukaryotic host cells are the AOX1 or GAL1 promoter in yeast, or CMV promoters, SV40 promoters, RSV promoters (Rouse sarcoma virus), CMV enhancers, SV40 enhancers, or globin introns in mammalian and other animal cells.

[0162] In addition to elements responsible for transcription initiation, such regulatory elements may also include transcription termination signals, such as SV40-poly-A sites or tk-poly-A sites downstream of the polynucleotide. Furthermore, depending on the expression system used, a leader sequence capable of guiding the polypeptide into a cellular compartment or secreting it into a culture medium may be added to the coding sequence of the polynucleotide, and is well known in the art. The leader sequence is assembled with the translation, initiation, and termination sequences in appropriate phases, and preferably, is a leader sequence capable of guiding the translated protein or a portion thereof to secrete into, for example, an extracellular culture medium. Optionally, a heteropolynucleotide sequence encoding a fusion protein may be used, containing a C- or N-terminal identifying peptide conferring desired properties, such as for stabilization or simplified purification of the expressed recombinant product.

[0163] In some embodiments, the polynucleotide encoding at least the light chain and / or heavy chain variable domains may encode the variable domains of two immunoglobulin chains or only one chain. Similarly, the polynucleotides may be expressed under the control of the same promoter or separately. Furthermore, some aspects relate to vectors, particularly plasmids, granules, viruses, and phages commonly used in genetic engineering, which contain polynucleotides encoding the variable domains of immunoglobulin chains encoding antibody or antigen-binding fragments; optionally combined with polynucleotides encoding the variable domains of another immunoglobulin chain encoding antibodies.

[0164] In some embodiments, the expression control sequence is provided as a eukaryotic promoter system in a vector capable of transforming or transfecting eukaryotic host cells, but a prokaryotic host control sequence may also be used. Expression vectors derived from viruses such as retroviruses, vaccinia viruses, adeno-associated viruses, herpesviruses, or bovine papillomaviruses can be used to deliver polynucleotides or vectors to target cell populations (e.g., engineered cells to express antibodies or antigen-binding fragments). A variety of suitable methods can be used to construct recombinant viral vectors. In some embodiments, the polynucleotide and vector can be reconstructed into liposomes for delivery to target cells. Vectors containing the polynucleotide (e.g., heavy chain and / or light chain variable domains of an immunoglobulin chain coding sequence and an expression control sequence) can be transferred into host cells using suitable methods that vary depending on the type of cell host.

[0165] Pharmaceutical Composition

[0166] In some aspects, this disclosure relates to pharmaceutical compositions comprising an rAAV carrier or rAAV (such as rAAV encoding an anti-RAN antibody or antigen-binding fragment). In some embodiments, the composition comprises an rAAV carrier or rAAV particles, and a pharmaceutically acceptable carrier. As used herein, the term "pharmaceutically acceptable carrier" is intended to include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic agents, and absorption delayers compatible with drug administration. The use of such media and reagents for pharmaceutically active substances is well known in the art. The use of any conventional media or reagent in a composition is contemplated unless it is incompatible with the active compound. Additional active compounds may also be incorporated into the composition. Pharmaceutical compositions may be prepared as described below. The active ingredient may be mixed or compounded with any conventional pharmaceutically acceptable carrier or excipient. The composition may be sterile.

[0167] Typically, pharmaceutical compositions are formulated to deliver an effective amount of an agent (such as rAAV encoding an anti-RAN antibody). Generally, the “effective amount” of an active agent refers to an amount sufficient to elicit the desired biological response (such as improvement of one or more symptoms of ALS). The effective amount of an active agent can vary depending on factors such as the desired biological endpoint, the pharmacokinetics of the compound, the disease being treated (such as ALS, amplification disorders), the route of administration, and the patient.

[0168] A composition is referred to as a “pharmaceutically acceptable carrier” if its administration is tolerated by the recipient patient. Sterile phosphate-buffered saline is an example of a pharmaceutically acceptable carrier. Other suitable carriers are well known in the art. See, for example, REMINGTON'S PHARMACEUTICAL SCIENCES, 18th Ed. (1990).

[0169] Those skilled in the art will understand that any conventionally used administration method, medium, or carrier that is inert to the active agent can be used to prepare and administer the pharmaceutical compositions of this disclosure. Exemplary descriptions of such methods, media, and carriers can be found, for example, in Remington's Pharmaceutical Sciences, 4th ed. (1970), the disclosure of which is incorporated herein by reference. Those skilled in the art, upon understanding the principles of this disclosure, will be able to readily determine suitable media, excipients, and carriers, or to combine the active ingredient therewith, to form the pharmaceutical compositions of this disclosure.

[0170] An effective amount (also called a therapeutically effective amount) of a compound (such as rAAV encoding an anti-RAN antibody) is an amount sufficient to alleviate at least one adverse reaction of a RAN protein-related disease (such as memory loss, cognitive impairment, coordination loss, speech disorder, etc.). In some embodiments, the RAN protein-related neurological disease is selected from the group consisting of amyotrophic lateral sclerosis (ALS), frontotemporal dementia, and Alzheimer's disease. In a specific embodiment, the RAN protein-related neurological disease is ALS. The therapeutically effective amount contained in the pharmaceutical composition depends in each case on several factors, such as the type of patient to be treated, body size and condition, the intended administration method, the patient's ability to be admitted to the intended dosage form, etc. Typically, the amount of active agent contained in each dosage form is provided from about 0.1 to about 250 mg / kg, preferably from about 0.1 to about 100 mg / kg. Those skilled in the art will be able to determine the appropriate therapeutically effective amount empirically. In some embodiments, the effective amount of rAAV contained in the pharmaceutical composition is at least 1 × 10⁻⁶ mg / kg. 5 A vector genome (vg), such as in which a drug composition is administered to a subject, and the effective amount of rAAV in the drug composition is 1 × 10⁻⁶ per kilogram (kg) of the subject's body weight. 5 per vg (vg / kg). For example, in some implementations, rAAV is approximately 1 × 10 6 vg / kg up to 10 10 The subject was given a dose of vg / kg or higher (e.g., 2 × 10⁻⁶). 6 vg / kg, 3 × 10 6 vg / kg, 4 × 10 6 vg / kg, 5 × 10 6 vg / kg, 6 × 10 6 vg / kg, 7 × 10 6 vg / kg, 8×10 6 vg / kg, 9×10 6 vg / kg, 1 x 10 7 vg / kg, 2 x 107 vg / kg, 3 x 10 7 vg / kg, 4 x 10 7 vg / kg, 5 x 10 7 vg / kg, 6 x 10 7 vg / kg, 7 x 10 7 vg / kg, 8 x 10 7 vg / kg, 9 x 10 7 vg / kg, 1 x10 8 vg / kg, 2 x 10 8 vg / kg, 3 x 10 8 vg / kg, 4 x 10 8 vg / kg, 5 x 10 8 vg / kg, 6 x 10 8 vg / kg, 7 x 10 8 vg / kg, 8 x 10 8 vg / kg, 9 x 10 8 , 1 x 10 9 vg / kg, 2 x 10 9 vg / kg, 3 x 10 9 vg / kg, 4 x 10 9 vg / kg, 5 x 10 9 vg / kg, 6 x 10 9 vg / kg, 7 x 10 9 vg / kg, 8 x 10 9 vg / kg, 9 x10 9 vg / kg, vg / kg, 1 x 10 10 vg / kg, 2 x 10 10 vg / kg, 3 x 10 10 vg / kg, 4 x 10 10 vg / kg, 5 x10 10 vg / kg, 6 x 10 10 vg / kg, 7 x 10 10 vg / kg, 8 x 10 10 vg / kg, 9 x 10 10 vg / kg or higher). In some embodiments, the rAAV is administered at about 1×10 6 vg / kg to 1×10 14 vg / kg, 1×10 8 vg / kg to 1×1013 vg / kg or 1×10 9 vg / kg up to 1×10 12 The subjects were given a dose of vg / kg.

[0171] Based on the teachings provided herein, by selecting from various active compounds and weighing factors such as potency, relative bioavailability, patient weight, severity of adverse side effects, and chosen administration method, an effective prophylactic or therapeutic treatment regimen that does not cause substantial toxicity but is fully effective in treating a specific subject can be devised. The effective amount for any particular application may vary depending on factors such as the disease or condition being treated, the specific therapeutic agent administered, the subject's body size, or the severity of the disease or condition. Those skilled in the art can determine the effective amount of a particular nucleic acid and / or other therapeutic agent empirically without excessive experimentation.

[0172] The term pharmaceutically acceptable carrier refers to one or more compatible solid or liquid fillers, diluents, or encapsulating substances suitable for administration to humans or other vertebrates. The term carrier also refers to an organic or inorganic (natural or synthetic) component combined with the active ingredient to facilitate application. The components of the pharmaceutical composition are also miscible with and with the compounds of this disclosure in a manner that does not materially impair the desired therapeutic effect.

[0173] The pharmaceutical composition may also contain a suitable solid or gel phase carrier or excipient. Examples of such carriers or excipients include, but are not limited to, calcium carbonate, calcium phosphate, various sugars, starch, cellulose derivatives, gelatin, and polymers such as polyethylene glycol.

[0174] Suitable liquid or solid pharmaceutical formulations include, for example, for inhalation, microencapsulation, encochleated, coated on microscopic gold particles, contained in liposomes, nebulized, aerosolized, pellets for implantation into the skin, or aqueous or saline solutions dried onto sharp objects for skin penetration. Pharmaceutical compositions also include granules, powders, tablets, coated tablets, (micro)capsules, suppositories, syrups, emulsions, suspensions, creams, drops, or formulations with sustained release of the active compound, conventionally prepared using excipients and additives and / or adjuvants as described above, such as disintegrants, binders, coating agents, swelling agents, lubricants, flavoring agents, sweeteners, or solubilizers. Pharmaceutical compositions are suitable for a variety of drug delivery systems. For a brief overview of drug delivery methods, see Langer R (1990) Science 249:1527-1533, which is incorporated herein by reference.

[0175] The compound may be given itself (in its pure form) or in the form of a pharmaceutically acceptable salt. When used in pharmaceuticals, the salt should be pharmaceutically acceptable, but non-pharmaceutically acceptable salts may be conveniently used to prepare their pharmaceutically acceptable salts. Such salts include, but are not limited to, those prepared from the following acids: hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, maleic acid, acetic acid, salicylic acid, p-toluenesulfonic acid, tartaric acid, citric acid, methanesulfonic acid, formic acid, malonic acid, succinic acid, naphthalene-2-sulfonic acid, and benzenesulfonic acid. Furthermore, such salts may be prepared as alkali metal or alkaline earth metal salts, such as sodium, potassium, or calcium salts of carboxylic acid groups.

[0176] The composition can be conveniently presented in unit dosage form and can be prepared by any method well known in the pharmaceutical industry. All methods involve the step of combining the compound with a carrier constituting one or more excipients. Typically, the composition is prepared by uniformly and tightly binding the compound with a liquid carrier, a finely dispersed solid carrier, or both, and then shaping the product if desired. Liquid dosage units are vials or ampoules. Solid dosage units are tablets, capsules, and suppositories.

[0177] For medical purposes

[0178] Therapeutic agents can be delivered by any suitable means known in the art.

[0179] This disclosure relates to delivering a therapeutically effective amount of a therapeutic agent to a subject. In some embodiments, a therapeutically effective amount is an amount that effectively reduces repeat expression or RAN protein translation in the subject. In some embodiments, a therapeutically effective amount is an amount that effectively reduces RNA transcription that produces RAN proteins in the subject. In some embodiments, a therapeutically effective amount is an amount that effectively reduces RAN protein translation in the subject. In some embodiments, a therapeutically effective amount is an amount that effectively treats a disease associated with repeat expression or RAN protein translation. “Reduced” repeat expression or RAN protein translation refers to a decrease in the amount or level of repeat expression or RAN protein translation in the subject after administration of the therapeutic agent (and relative to the amount or level in the subject before administration).

[0180] Therefore, aspects of this disclosure further relate to rAAV carriers, rAAV, and pharmaceutical compositions (such as for treating subjects with or suspected of having a disease related to RAN protein translation or RAN protein accumulation) and the use of rAAV carriers and rAAV in the preparation of pharmaceuticals (such as pharmaceuticals for treating subjects with or suspected of having a disease related to RAN protein translation or RAN protein accumulation).

[0181] In some embodiments, an effective amount is an amount that effectively reduces RAN protein levels by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or at least 98% (e.g., RAN protein levels relative to RAN protein levels in cells or subjects that have not received a therapeutic agent).

[0182] The pharmaceutical compositions described herein can be prepared by any method known in the field of pharmacology. Typically, such preparation methods involve combining the compound described herein (i.e., the “active ingredient”) with a carrier or excipient and / or one or more other auxiliary ingredients, and then, if desired and / or expected, shaping and / or packaging the product into desired single-dose or multi-dose units.

[0183] Pharmaceutical compositions may be prepared, packaged, and / or sold in bulk, as a single unit dose, and / or as multiple unit doses. A “unit dose” is a discrete amount of a pharmaceutical composition containing a predetermined amount of an active ingredient. The amount of active ingredient is generally equal to the dose of active ingredient to be given to a subject and / or a convenient fraction of that dose, such as half or one-third of the dose.

[0184] The relative amounts of the active ingredient, pharmaceutically acceptable excipients, and / or any other components in the pharmaceutical compositions described herein will vary depending on the identity, body size, and / or condition of the treated subject, and further depending on the route of administration of the composition. The composition may contain 0.1% to 100% (w / w) of the active ingredient.

[0185] The therapeutic agents described herein are typically formulated in dosage units to facilitate administration and dosage uniformity. However, it should be understood that the total daily dosage of the compositions described herein will be determined by a physician within reasonable medical judgment. The specific therapeutically effective dose level for any particular subject or organism will depend on a variety of factors, including the severity of the disease and condition being treated; the activity of the specific active ingredient used; the specific composition used; the subject's age, weight, general health condition, sex, and diet; the time of administration, route of administration, and excretion rate of the specific active ingredient used; the duration of treatment; drugs used in combination with or coincidentally used with the specific active ingredient used; and similar factors well known in the medical field.

[0186] Therapeutic agents can be administered via any route, including enterally (e.g., orally), parenterally, intravenously, intramuscularly, intraarterially, intramedullaryly, intrathecally, intracerebrally, transdermally, intradermally, rectally, intravaginally, intraperitoneally, topically (e.g., powder, ointment, cream, and / or drops), mucosally, nasally, buccally, sublingually; via endotracheal instillation, bronchial instillation, and / or inhalation; and / or as oral sprays, nasal sprays, and / or aerosols. Specific routes of administration considered include oral administration, intravenous administration (e.g., systemic intravenous injection), administration through areas supplied by blood and / or lymph, and / or direct administration to the affected site. Generally, the most appropriate route of administration will depend on a number of factors, including the nature of the agent (e.g., its stability in the gastrointestinal environment) and / or the condition of the subject (e.g., whether the subject can tolerate oral administration).

[0187] In some embodiments, administration of the rAAV described herein to subjects with or suspected of having RAN protein-related diseases (such as Alzheimer's disease, amyotrophic lateral sclerosis (ALS), or frontotemporal dementia) can reduce the expression and / or aggregation of RAN proteins (such as poly(GA)RAN protein) in one or more central nervous system tissues (such as the cortex, frontal cortex, motor cortex, posterior cingulate cortex, and / or hippocampus). In some embodiments, administration of the rAAV described herein can reduce the expression and / or aggregation of RAN proteins (such as poly(GA)RAN protein) in the cortex, frontal cortex, motor cortex, posterior cingulate cortex, and / or hippocampus of subjects. In some embodiments, administration of the rAAV described herein can increase motor neuron survival in subjects (such as subjects characterized as having or suspected of having RAN protein-related diseases such as Alzheimer's disease, amyotrophic lateral sclerosis (ALS), or frontotemporal dementia). In some embodiments, administration of the rAAV described herein can increase Fc receptor levels in subjects, such as in one or more central nervous system tissues (e.g., the cortex, frontal cortex, motor cortex, posterior cingulate cortex, and / or hippocampus). In some embodiments, administration of the rAAV described herein can increase Fc receptor levels in subjects who express RAN proteins such as poly(GA) RAN proteins (e.g., subjects characterized as having or suspected of having RAN protein-related diseases such as Alzheimer's disease, amyotrophic lateral sclerosis (ALS), or frontotemporal dementia).

[0188] The exact therapeutic dose required to achieve an effective amount will vary from subject to subject, depending on factors such as species, age, general condition, severity of side effects or illness, characteristics of the specific compound, and administration method. An effective amount may be contained in a single dose (e.g., a single oral dose) or multiple doses (e.g., multiple oral doses). In some embodiments, when multiple doses are administered to a subject or applied to a biological sample, tissue, or cell, any two doses in the multiple doses contain different or substantially the same amounts of the compound described herein. In some embodiments, when multiple doses are administered to a subject or applied to a biological sample, tissue, or cell, the frequency of administering multiple doses or applying multiple doses to the biological sample, tissue, or cell may be three doses per day, two doses per day, one dose per day, one dose every other day, one dose every three days, one dose per week, one dose every two weeks, one dose every three weeks, or one dose every four weeks. In some embodiments, the frequency of administering multiple doses or applying multiple doses to a biological sample, tissue, or cell is one dose per day. In some embodiments, the frequency of administering multiple doses or applying multiple doses to a subject is two doses per day. In some embodiments, the frequency of administering multiple doses to a subject or applying multiple doses to a biological sample, tissue, or cell is three doses per day. In some embodiments, when administering multiple doses to a subject or applying them to a biological sample, tissue, or cell, the duration between the first and last doses of the multiple doses is one day, two days, four days, one week, two weeks, three weeks, one month, two months, three months, four months, six months, eight months, nine months, one year, two years, three years, four years, five years, seven years, ten years, fifteen years, twenty years, or the lifetime of the subject, tissue, or cell. In some embodiments, the duration between the first and last doses of the multiple doses is three months, six months, or one year. In some embodiments, the duration between the first and last doses of the multiple doses is the lifetime of the subject, tissue, or cell.

[0189] In some implementations, the central nervous system (CNS) of the subject in need is treated for a disease related to RAN protein expression. As used herein, “central nervous system (CNS)” refers to all cells and tissues of the subject’s brain and spinal cord, including but not limited to neurons, glial cells, astrocytes, cerebrospinal fluid, etc. Methods of administering the therapeutic agent to the subject’s CNS include direct injection into the brain (e.g., intracerebral injection, intraventricular injection, intraparenchymal injection, etc.), direct injection into the subject’s spinal cord (e.g., intrathecal injection, lumbar injection, etc.), or any combination thereof.

[0190] In some embodiments, the treatments described herein are administered systemically to the subject, for example, via intravenous injection. In some embodiments, the systemically administered therapeutic molecules may be modified to improve molecule delivery to the subject's CNS. Examples of modifications to improve CNS delivery of the therapeutic molecules include, but are not limited to, co-administration or conjugation with blood-brain barrier targets (such as transferrin, melanin-transferrin, low-density lipoprotein (LDL), vascular peptides, RVG peptides, etc., as disclosed in Georgieva et al. Pharmaceuticals 6(4):557-583 (2014)), co-administration with BBB disruptors (such as bradykinin), and physical disruption of the BBB prior to administration (such as by MRI-guided focused ultrasound).

[0191] The following examples are intended to illustrate the benefits of the invention and describe specific implementations, but are not intended to demonstrate the full scope of the invention. Therefore, it should be understood that the examples are not intended to limit the scope of the invention.

[0192] Example

[0193] This example describes the expression and testing of an AAV-based monoclonal antibody that binds to poly(GA) RAN protein in a C9orf72 mutant mouse model of amyotrophic lateral sclerosis (ALS) (C9 BAC). The ALS C9orf72 mouse model (C9 BAC) was treated with either a control (PBS) or a transgenic recombinant AAV9 virus containing a recombinant α-poly(GA) antibody (IgG1 A, ScFv, IgG1 B, or IgG2) at 30–37, 37–44, or 44–50 weeks of age prior to immunohistochemical analysis. Figure 1 Representative immunohistochemical (IHC) data showed that the anti-poly(GA)-IgG1 antibody expressed by rAAV reduced the number of poly(GA) RAN protein aggregates in the C9BAC of ALS. These observations were further confirmed by quantification of GA aggregates using one-way ANOVA and Sidak multiple comparison tests. Figure 2 Representative data showed that the anti-poly(GA)-IgG1 antibody expressed by rAAV reduced the number of poly(GA) RAN protein aggregates in a C9orf72 mouse model of ALS. Further analysis was performed to confirm that the anti-poly(GA)-IgG1 antibody targets poly(GA) RAN protein aggregates. Figure 3 Representative microscopic data show that the AAV-based anti-poly(GA) antibody co-localizes with poly(GA)RAN protein aggregates in the C9 BAC mouse model.

[0194] Figure 4This shows representative data obtained from a mesoscale detection (MSD) immunoassay performed in the frontal cortex of treated animals using monoclonal mouse α-GP antibody (capture) and rabbit polyclonal α-GP antibody (detection). These results indicate that AAV9-based anti-poly(GA) antibody delivery reduces total GP levels in a C9 BAC mouse model through a mechanism that may involve improving protein homeostasis.

[0195] Figures 5A-5B The data presented represent the results, showing that delivery of AAV9-based anti-poly(GA) antibodies reduced neuroinflammation in the motor cortex of C9 BAC mice. Figure 5A Immunohistochemistry revealed GFAP staining as a marker of neuroinflammatory activity in brain tissue obtained from PBS-treated NT mice and PBS- or antibody-treated C9 animals. Figure 5B show Figure 5A The data shown is quantitative.

[0196] Figure 6 Representative data show that the novel α-GA antibody recognizes GA aggregates in C9-BAC mouse brain and human patient-derived cells (iMN). HEK293 cells were engineered to contain nucleic acids encoding the α-GA antibody. The culture medium was changed 24 hours post-transfection, and protein lysates and supernatants were obtained 48 hours post-transfection. These samples were subsequently used for Western blotting and fluorescence microscopy analysis to validate the expression levels of ScFv-FLAG, IgG1-A, IgG1-B, and IgG2. Figures 7A-7B The data show representative figures indicating that the α-GA recombinant antibody was expressed and secreted. Figure 7A Western blot analysis of cell protein lysates collected from HEK293 cells. Figure 7B Fluorescence microscopy analysis of supernatant containing primary anti-α-GA antibody collected from HEK293 cells.

[0197] Figure 8 The data show representative findings indicating that the recombinant antibody recognizes GA aggregates in the brains of C9 mice.

[0198] HEK293 cells were engineered to contain nucleic acids encoding α-GA antibodies and GFP-GA antibodies. 60 Nucleic acids were extracted. The culture medium was changed 24 hours after transfection, and protein lysates and supernatants were obtained 48 hours after transfection. These samples were then used for Western blotting and fluorescence microscopy analysis. Figures 9A-9B Representative data show that the recombinant α-GA antibody reduced GFP-GA in HEK293T cells. 60 . Figure 9A The expression of α-GA recombinant antibody and GFP-GA showed that... 60Western blot analysis of protein lysates obtained from cells. Figure 9B show Figure 9A The data shown is quantitative. Figures 10A-10B The data show that the recombinant α-GA antibody reduces the number and size of GA aggregates. Figure 10A The expression of α-GA recombinant antibody and GFP-GA showed that... 60 Fluorescence microscopy analysis of protein lysates obtained from cells. Figure 10B show Figure 10B The data shown is quantitative.

[0199] Figure 11A A schematic diagram showing recombinant α-GA immunoglobulin and nucleic acids containing transgenes encoding the recombinant α-GA immunoglobulin. Figure 11B A schematic diagram showing the α-GA single-stranded variable fragment (scFv) and the nucleic acid containing the transgene encoding the α-GA scFv is presented. A recombinant AAV-antibody targeting green fluorescent protein was developed as an isotype control for in vivo experiments, which will allow for the evaluation of the effects of non-GA-targeting versus GA-targeting antibody treatments. Figure 11C-11D The development of a recombinant AAV AAV1 ERDR-IgG-GFP isotype control was demonstrated. Figure 11C A schematic diagram showing the α-GFP isotype control and the nucleic acid containing the transgene encoding the α-GFP isotype control. Figure 11D This displays representative data from fluorescence microscopy analysis of the α-GFP isotype control. The α-GFP isotype control was delivered by rAAV containing the AAV1 serotype VP1 capsid protein and an ERDRTRG (SEQ ID NO:49) peptide insert. The VP1 protein is encoded by the nucleic acid of SEQ ID NO:43 and contains the amino acid sequence of SEQ ID NO:44.

[0200] Figure 12 This is a non-limiting example of a efficacy study strategy using AAV1-ERDR-IgG C9-BAC mice.

[0201] Figure 13 The data show representative figures demonstrating the extensive delivery of AAV1-ERDR-TFP following ICV injection.

[0202] Figures 14A-14B This shows representative data obtained from the suspension test in injected mouse subjects. Figure 14A This displays the suspension score of the injected mouse subjects. Figure 14B The image shows photographs taken during the suspension analysis of injected mouse subjects. Further results of the suspension performance analysis were shown... Figures 24A-24B The results showed that AAV-GA antibody improved the suspension line performance ( Figure 24Aand reduced GA levels in the brains of male C9-BAC mice. Figure 24B ).

[0203] Figure 15A-15F This shows representative data obtained from open field analysis of injected mouse subjects. Figure 15A Open field analysis of C9orf72 positive mice shows a variable mobility phenotype. Figure 15B This shows representative data (movement parameters) from open field analysis of treated and untreated C9orf72 BAC mice. Figure 15C This shows representative data (stereotyped and exploratory behaviors) from open field analysis of treated and untreated C9orf72 BAC mice. Figure 15D The data show that treatment B rescued 11 / 12 open field abnormalities in C9-BAC mice compared to the mediator treatment. Figure 15E The data show representativeness, indicating that the C9-BAC mice treated in group B did not show any difference in open field behavior compared to NT animals. Figure 15F The data show that treatment A moderately improved four stereotyped combing / exploration parameters in C9-BAC mice, but did not improve movement parameters.

[0204] Figure 16 The data show representative findings indicating that treatment B improved the survival rate of C9-BAC mice compared to treatment A and the vector treatment.

[0205] Figure 17 The data shown are representative and demonstrate that treatment A reduces GA aggregates.

[0206] Brain and spinal cord tissues were harvested from mouse subjects who had never received treatment or administration of AAV containing nucleic acids encoding recombinant control antibodies or AAV containing nucleic acids encoding recombinant α-GA IgG1. Lysates generated from the tissue samples were diluted and assayed to measure α-GA IgG1 levels. Figure 18 The data shown are representative of the recombinant α-GA antibody distribution analysis, indicating that delivery of AAV-based α-GA antibodies to mouse subjects resulted in antibody presence in brain and spinal cord tissues.

[0207] Figures 19A-19B Representative data show that AAV-based delivery of recombinant α-GA antibody reduces GA aggregates in the frontal cortex of C9-BAC mice. Furthermore, Figures 20A-20B The data show representative findings indicating that delivery of AAV-based recombinant α-GA antibody increases motor neuron survival in the brains of C9-BAC mice.

[0208] Further microscopic analysis confirmed that the α-GA antibody was delivered to the brains of treated mouse subjects. Figures 21A-21BRepresentative data obtained from microscopic analysis of the brains of C9-BAC mice show that recombinant α-GA antibody is retained in the CA2 region after AAV-based delivery. Figures 22A-22B The images show representative data obtained from microscopic analysis of the C9-BAC mouse brain, demonstrating the delivery of the AAV-based recombinant α-GA antibody in the mouse brain.

[0209] also, Figures 23A-23B AAV-GA antibody treatment showed a reduction in brain damage in C9 female mice ( Figure 23A ) and organoids derived from C9orf72ALS / FTD ( Figure 23B GA level in ).

[0210] Representative sequence

[0211] > Anti-polyGA antibody clone 27B11 heavy chain CDR1 amino acid sequence (SEQ ID NO:1)

[0212] GFAFSNYG

[0213] > Anti-polyGA antibody clone 27B11 heavy chain CDR2 amino acid sequence (SEQ ID NO:2)

[0214] INSDGDST

[0215] > Anti-polyGA antibody clone 27B11 heavy chain CDR3 amino acid sequence (SEQ ID NO:3)

[0216] ARVGGNYDFAMDY

[0217] > Anti-polyGA antibody clone 27B11 light chain CDR1 amino acid sequence (SEQ ID NO:4)

[0218] QSLLNSRTRKNY

[0219] > Anti-polyGA antibody clone 27B11 light chain CDR2 amino acid sequence (SEQ ID NO:5)

[0220] WTS

[0221] > Anti-polyGA antibody clone 27B11 light chain CDR3 amino acid sequence (SEQ ID NO:6)

[0222] KQSYNNPWT

[0223] > Anti-polyGA antibody clone 27B11 heavy chain variable region amino acid sequence (SEQ ID NO:7)

[0224] EVQLQESGGGSVQPGGSLKLSCAASGFAFSNYGMSWVRQTPDKRLELVTTINSDGDSTFYPDSVKGRFTISRDNAKNALYLQMSSLKSDDTAMYYCARVGGNYDFAMDYWGQGTSVIVSS

[0225] > Amino acid sequence of heavy chain IgG1 of anti-polyGA antibody clone 27B11 (SEQ ID NO: 8)

[0226] EVQLQESGGGSVQPGGSLKLSCAASGFAFSNYGMSWVRQTPDKRLELVTTINSDGDSTFYPDSVKGRFTISRDNAKNALYLQMSSLKSDDTAMYYCARVGGNYDFAMDYWGQGTSVIVSSAKTTPPSVYPLAPGSAAQTNSMVTLGCLVKGYFPEPVTVTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVPSSTWPSETVTCNVAHPASSTKVDKKIVPRDCGCKPCICTVPEVSSVFIFPPKPKDVLTITLTPKVTCVVVDISKDDPEVQFSWFVDDVEVHTAQTQPREEQFNSTFRSVSELPIMHQDWLNGKEFKCRVNSAAFPAPIEKTISKTKGRPKAPQVYTIPPPKEQMAKDKVSLTCMITDFFPEDITVEWQWNGQPAENYKNTQPIMDTDGSYFVYSKLNVQKSNWEAGNTFTCSVLHEGLHNHHTEKSLSHSPGK

[0227] > Amino acid sequence of heavy chain IgG1 of anti-polyGA antibody clone 27B11 (comprising a signal peptide) (SEQ ID NO: 9)

[0228] MDWTWRVFCLLAVAPGAHSEVQLQESGGGSVQPGGSLKLSCAASGFAFSNYGMSWVRQTPDKRLELVTTINSDGDSTFYPDSVKGRFTISRDNAKNALYLQMSSLKSDDTAMYYCARVGGNYDFAMDYWGQGTSVIVSSAKTTPPSVYPLAPGSAAQTNSMVTLGCLVKGYFPEPVTVTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVPSSTWPSETVTCNVAHPASSTKVDKKIVPRDCGCKPCICTVPEVSSVFIFPPKPKDVLTITLTPKVTCVVVDISKDDPEVQFSWFVDDVEVHTAQTQPREEQFNSTFRSVSELPIMHQDWLNGKEFKCRVNSAAFPAPIEKTISKTKGRPKAPQVYTIPPPKEQMAKDKVSLTCMITDFFPEDITVEWQWNGQPAENYKNTQPIMDTDGSYFVYSKLNVQKSNWEAGNTFTCSVLHEGLHNHHTEKSLSHSPGK

[0229] > Amino acid sequence of anti-polyGA antibody clone 27B11 heavy chain IgG2 (SEQ ID NO: 10)

[0230] EVQLQESGGGSVQPGGSLKLSCAASGFAFSNYGMSWVRQTPDKRLELVTTINSDGDSTFYPDSVKGRFTISRDNAKNALYLQMSSLKSDDTAMYYCARVGGNYDFAMDYWGQGTSVIVSSAKTTAPSVYPLAPVCGDTTGSSVTLGCLVKGYFPEPVTLTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVTSSTWPSQSITCNVAHPASSTKVDKKIEPRGPTIKPCPPCKCPAPNLLGGPSVFIFPPKIKDVLMISLSPIVTCVVVDVSEDDPDVQISWFVNNVEVHTAQTQTHREDYNSTLRVVSALPIQHQDWMSGKEFKCKVNNKDLPAPIERTISKPKGSVRAPQVYVLPPPEEEMTKKQVTLTCMVTDFMPEDIYVEWTNNGKTELNYKNTEPVLDSDGSYFMYSKLRVEKKNWVERNSYSCSVVHEGLHNHHTTKSFSRTPGK

[0231] > Amino acid sequence of heavy chain IgG2 of anti-polyGA antibody clone 27B11 (including signal peptide) (SEQ ID NO: 11)

[0232] MKCSWVIFFLMAVVIGINSEVQLQESGGGSVQPGGSLKLSCAASGFAFSNYGMSWVRQTPDKRLELVTTINSDGDSTFYPDSVKGRFTISRDNAKNALYLQMSSLKSDDTAMYYCARVGGNYDFAMDYWGQGTSVIVSSAKTTAPSVYPLAPVCGDTTGSSVTLGCLVKGYFPEPVTLTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVTSSTWPSQSITCNVAHPASSTKVDKKIEPRGPTIKPCPPCKCPAPNLLGGPSVFIFPPKIKDVLMISLSPIVTCVVVDVSEDDPDVQISWFVNNVEVHTAQTQTHREDYNSTLRVVSALPIQHQDWMSGKEFKCKVNNKDLPAPIERTISKPKGSVRAPQVYVLPPPEEEMTKKQVTLTCMVTDFMPEDIYVEWTNNGKTELNYKNTEPVLDSDGSYFMYSKLRVEKKNWVERNSYSCSVVHEGLHNHHTTKSFSRTPGK

[0233] > Amino acid sequence of the light chain variable region of anti-polyGA antibody clone 27B11 (SEQ ID NO: 12)

[0234] DIVMSQFPSSLAVSAGDKVTMSCKSSQSLLNSRTRKNYLAWYQQKPGQSPKLLIYWTSTRESGVPDRFTGSRSGTDFTLTISSVQAEDLAVYYCKQSYNNPWTFGGGTKLEIK

[0235] > Amino acid sequence of the light chain IgG1 of anti-polyGA antibody clone 27B11 (SEQ ID NO: 13)

[0236] DIVMSQFPSSLAVSAGDKVTMSCKSSQSLLNSRTRKNYLAWYQQKPGQSPKLLIYWTSTRESGVPDRFTGSRSGTDFTLTISSVQAEDLAVYYCKQSYNNPWTFGGGTKLEIKRADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC

[0237] > Anti-polyGA antibody clone 27B11 light chain IgG1 amino acid sequence (including signal peptide) (SEQ ID NO:14)

[0238] MKLPVRLLVLMFWIPASSSDIVMSQFPSSLAVSAGDKVTMSCKSSQSLLNSRTRKNYLAWYQQKPGQSPKLLIYWTSTRESGVPDRFTGSRSGTDFTLTISSVQAEDLAVYYCKQSYNN PWTTFGGGTKLEIKRADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC

[0239] > Anti-polyGA antibody clone 27B11 light chain IgG2 amino acid sequence (SEQ ID NO:15)

[0240] DIVMSQFPSSLAVSAGDKVTMSCKSSQSLLNSRTRKNYLAWYQQKPGQSPKLLIYWTSTRESGVPDRFTGSRSGTDFTLTISSVQAEDLAVYYCKQSYNNPWTFGGGTKL EIKRADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC

[0241] > Anti-polyGA antibody clone 27B11 light chain IgG2 amino acid sequence (including signal peptide) (SEQ ID NO:16)

[0242] MKLPVRLLVLMFWIPASSSDIVMSQFPSSLAVSAGDKVTMSCKSSQSLLNSRTRKNYLAWYQQKPGQSPKLLIYWTSTRESGVPDRFTGSRSGTDFTLTISSVQAEDLAVYYCKQSYNN PWTTFGGGTKLEIKRADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC

[0243] Anti-polyGA antibody clone 27B11 scFv heavy chain (SEQ ID NO:17)

[0244] EVQLQESGGGSVQPGGSLKLSCAASGFAFSNYGMSWVRQTPDKRLELVTTINSDGDSTFYPDSVKGRFTISRDNAKNALYLQMSSLKSDDTAMYYCARVGGNYDFAMDYWGQGTSVIVSS

[0245] Anti-polyGA antibody clone 27B11 scFv light chain (SEQ ID NO:18)

[0246] DIVMSQFPSSLAVSAGDKVTMSCKSSQSLLNSRTRKNYLAWYQQKPGQSPKLLIYWTSTRESGVPDRFTGSRSGTDFTLTISSVQAEDLAVYYCKQSYNNPWTFGGGTKLEIK

[0247] The complete sequence of anti-polyGA antibody clone 27B11 scFv (including signal peptide and polyGA linker) (SEQ ID NO:19)

[0248] MDWTWRVFCLLAVAPGAHSEVQLQESGGGSVQPGGSLKLSCAASGFAFSNYGMSWVRQTPDKRLELVTTINSDGDSTFYPDSVKGRFTISRDNAKNALYLQMSSLKSDDTAMYYCARVGGNYDFAMDYWGQGT SVIVSSGGGGSGGGGSGGGGSDIVMSQFPSSLAVSAGDKVTMSCKSSQSLLNSRTRKNYLAWYQQKPGQSPKLLIYWTSTRESGVPDRFTGSRSGTDFTLTISSVQAEDLAVYYCKQSYNNPWTFGGGTKLEIK

[0249] > Anti-polyGA antibody clone 23H2 heavy chain CDR1 amino acid sequence (SEQ ID NO:20)

[0250] GFTFSSHG

[0251] > Anti-polyGA antibody clone 23H2 heavy chain CDR2 amino acid sequence (SEQ ID NO:21)

[0252] INSNGGST

[0253] > Anti-polyGA antibody clone 23H2 heavy chain CDR3 amino acid sequence (SEQ ID NO:22)

[0254] ARVGDNDDFAMGY

[0255] > Anti-polyGA antibody clone 23H2 light chain CDR1 amino acid sequence (SEQ ID NO:23)

[0256] QSLFNSRTRKNY

[0257] > Anti-polyGA antibody clone 23H2 light chain CDR2 amino acid sequence (SEQ ID NO:24)

[0258] WTS

[0259] > Anti-polyGA antibody clone 23H2 light chain CDR3 amino acid sequence (SEQ ID NO:25)

[0260] KQSYNNPWT

[0261] > Anti-polyGA antibody clone 23H2 heavy chain variable region amino acid sequence (SEQ ID NO:26)

[0262] EVQLQESGGGSVQPGGALQLSCAASGFTFSSHGMSWVRQTPDKRLEMVATINSNGGSTYYPDSVKGRFIISRDNAKNTLYLQMSSLKSEDTAMYYCARVGDNDDFAMGYWGQGTSVTVSS

[0263] > Anti-polyGA antibody clone 23H2 heavy chain IgG1 amino acid sequence (SEQ ID NO:27)

[0264] EVQLQESGGGSVQPGGALQLSCAASGFTFSSHGMSWVRQTPDKRLEMVATINSNGGSTYYPDSVKGRFIISRDNAKNTLYLQMSSLKSEDTAMYYCARVGDNDDFAMGYWGQGTSVTVSSAKTTPPSVYPLAPGSAAQTNSMVTLGCLVKGYFPEPVTVTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVPSSTWPSETVTCNVAHPASSTKVDKKIVPRDCGCKPCICTVPEVSSVFIFPPKPKDVLTITLTPKVTCVVVDISKDDPEVQFSWFVDDVEVHTAQTQPREEQFNSTFRSVSELPIMHQDWLNGKEFKCRVNSAAFPAPIEKTISKTKGRPKAPQVYTIPPPKEQMAKDKVSLTCMITDFFPEDITVEWQWNGQPAENYKNTQPIMDTDGSYFVYSKLNVQKSNWEAGNTFTCSVLHEGLHNHHTEKSLSHSPGK

[0265] > Amino acid sequence of the heavy chain IgG1 of anti-polyGA antibody clone 23H2 (including signal peptide) (SEQ ID NO: 28)

[0266] MDWTWRVFCLLAVAPGAHSEVQLQESGGGSVQPGGALQLSCAASGFTFSSHGMSWVRQTPDKRLEMVATINSNGGSTYYPDSVKGRFIISRDNAKNTLYLQMSSLKSEDTAMYYCARVGDNDDFAMGYWGQGTSVTVSSAKTTPPSVYPLAPGSAAQTNSMVTLGCLVKGYFPEPVTVTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVPSSTWPSETVTCNVAHPASSTKVDKKIVPRDCGCKPCICTVPEVSSVFIFPPKPKDVLTITLTPKVTCVVVDISKDDPEVQFSWFVDDVEVHTAQTQPREEQFNSTFRSVSELPIMHQDWLNGKEFKCRVNSAAFPAPIEKTISKTKGRPKAPQVYTIPPPKEQMAKDKVSLTCMITDFFPEDITVEWQWNGQPAENYKNTQPIMDTDGSYFVYSKLNVQKSNWEAGNTFTCSVLHEGLHNHHTEKSLSHSPGK

[0267] > Amino acid sequence of anti-polyGA antibody clone 23H2 heavy chain IgG2 (SEQ ID NO: 29)

[0268] EVQLQESGGGSVQPGGALQLSCAASGFTFSSHGMSWVRQTPDKRLEMVATINSNGGSTYYPDSVKGRFIISRDNAKNTLYLQMSSLKSEDTAMYYCARVGDNDDFAMGYWGQGTSVTVSSAKTTAPSVYPLAPVCGDTTGSSVTLGCLVKGYFPEPVTLTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVTSSTWPSQSITCNVAHPASSTKVDKKIEPRGPTIKPCPPCKCPAPNLLGGPSVFIFPPKIKDVLMISLSPIVTCVVVDVSEDDPDVQISWFVNNVEVHTAQTQTHREDYNSTLRVVSALPIQHQDWMSGKEFKCKVNNKDLPAPIERTISKPKGSVRAPQVYVLPPPEEEMTKKQVTLTCMVTDFMPEDIYVEWTNNGKTELNYKNTEPVLDSDGSYFMYSKLRVEKKNWVERNSYSCSVVHEGLHNHHTTKSFSRTPGK

[0269] > Amino acid sequence of the heavy chain IgG2 of anti-polyGA antibody clone 23H2 (including signal peptide) (SEQ ID NO: 30)

[0270] MDWTWRVFCLLAVAPGAHSEVQLQESGGGSVQPGGALQLSCAASGFTFSSHGMSWVRQTPDKRLEMVATINSNGGSTYYPDSVKGRFIISRDNAKNTLYLQMSSLKSEDTAMYYCARVGDNDDFAMGYWGQGTSVTVSSAKTTAPSVYPLAPVCGDTTGSSVTLGCLVKGYFPEPVTLTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVTSSTWPSQSITCNVAHPASSTKVDKKIEPRGPTIKPCPPCKCPAPNLLGGPSVFIFPPKIKDVLMISLSPIVTCVVVDVSEDDPDVQISWFVNNVEVHTAQTQTHREDYNSTLRVVSALPIQHQDWMSGKEFKCKVNNKDLPAPIERTISKPKGSVRAPQVYVLPPPEEEMTKKQVTLTCMVTDFMPEDIYVEWTNNGKTELNYKNTEPVLDSDGSYFMYSKLRVEKKNWVERNSYSCSVVHEGLHNHHTTKSFSRTPGK

[0271] > Amino acid sequence of the variable region of the light chain of anti-polyGA antibody clone 23H2 (SEQ ID NO: 31)

[0272] DIVMSQSPSSLAVSEGEKVTLTCKSSQSLFNSRTRKNYLAWYQQKPGQPPKLLIYWTSTRESGVPDRFTGSGYGTDFTLTISSVQAEDLAVYYCKQSYNNPWTFGGGTKLEIK

[0273] > IgG1 light chain amino acid sequence of anti-polyGA antibody clone 23H2 (SEQ ID NO: 32)

[0274] DIVMSQSPSSLAVSEGEKVTLTCKSSQSLFNSRTRKNYLAWYQQKPGQPPKLLIYWTSTRESGVPDRFTGSGYGTDFTLTISSVQAEDLAVYYCKQSYNNPWTFGGGTKLEIKRADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC

[0275] > Anti-polyGA antibody clone 23H2 light chain IgG1 amino acid sequence (including signal peptide) (SEQ ID NO:33)

[0276] MKLPVRLLLVLMFWIPASSSDIVMSQSPSSLAVSEGEKVTLTCKSSQSLFNSRTRKNYLAWYQQKPGQPPKLLIYWTSTRESGVPDRFTGSGYGTDFTLTISSVQAEDLAVYYCKQSYNN PWTTFGGGTKLEIKRADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC

[0277] > Anti-polyGA antibody clone 23H2 light chain IgG2 amino acid sequence (SEQ ID NO:34)

[0278] DIVMSQSPSSLAVSEGEKVTLTCKSSQSLFNSRTRKNYLAWYQQKPGQPPKLLIYWTSTRESGVPDRFTGSGYGTDFTLTISSVQAEDLAVYYCKQSYNNPWTFGGGTKL EIKRADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC

[0279] > Anti-polyGA antibody clone 23H2 light chain IgG2 amino acid sequence (including signal peptide) (SEQ ID NO:35)

[0280] MKLPVRLLLVLMFWIPASSSDIVMSQSPSSLAVSEGEKVTLTCKSSQSLFNSRTRKNYLAWYQQKPGQPPKLLIYWTSTRESGVPDRFTGSGYGTDFTLTISSVQAEDLAVYYCKQSYNN PWTTFGGGTKLEIKRADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC

[0281] > Anti-polyGA antibody clone 27B11 IgG1 nucleic acid sequence (containing signal peptide and bGH poly(A) signal) (SEQ ID NO:36)

[0282]

[0283] > Anti-polyGA antibody clone 27B11 IgG2 nucleic acid sequence (containing signal peptide and bGH poly(A) signal) (SEQ ID NO:37)

[0284]

[0285] > Anti-polyGA antibody clone 27B11 ScFv nucleic acid sequence (containing signal peptide, FLAG and bGH poly(A) signal) (SEQ ID NO:38)

[0286]

[0287] > Anti-polyGA antibody clone 23H2 IgG1 nucleic acid sequence (containing signal peptide and bGH poly(A) signal) (SEQ ID NO:39)

[0288]

[0289] > Anti-polyGA antibody clone 23H2 IgG2 nucleic acid sequence (containing signal peptide and bGH poly(A) signal) (SEQ ID NO:40)

[0290]

[0291] > F2A self-cleaving peptide (SEQ ID NO:41)

[0292] VKQTLNFDLLKLAGDVESNPGP

[0293] Equivalent scheme

[0294] Although several inventive embodiments have been described and illustrated herein, those skilled in the art will readily conceive of various other means and / or structures for performing the functions described herein and / or obtaining the results and / or one or more advantages described herein, and each such variation and / or modification is considered to be within the scope of the inventive embodiments described herein. More generally, those skilled in the art will readily understand that all parameters, dimensions, materials, and configurations described herein are intended to be exemplary, and actual parameters, dimensions, materials, and / or configurations will depend on a particular application or application using the teachings of this invention. Those skilled in the art will recognize or be able to determine many equivalents of the specific inventive embodiments described herein using only conventional experiments. Therefore, it should be understood that the foregoing embodiments are presented by way of example only, and embodiments of the invention may be practiced in ways different from the specific descriptions and claims within the scope of the appended claims and their equivalents. The inventive embodiments of this disclosure pertain to each individual feature, system, article of manufacture, material, kit, and / or method described herein. Furthermore, any combination of two or more such features, systems, articles of manufacture, materials, kits, and / or methods is included within the scope of this disclosure if such features, systems, articles of manufacture, materials, kits, and / or methods do not contradict each other.

[0295] All definitions defined and used herein should be understood to take precedence over dictionary definitions, definitions in incorporated documents by reference, and / or the general meaning of the defined terms.

[0296] All references, patents and patent applications disclosed herein are incorporated herein by reference and refer to the subject matter cited, and in some cases may cover the entire contents of the documents.

[0297] Unless explicitly indicated to the contrary, the non-limiting articles “a” and “an” used in the specification and claims shall be understood to mean “at least one”.

[0298] The phrase “and / or” as used in the specification and claims should be understood to mean “any one or both” of the elements so combined, i.e., elements that coexist in some cases and exist separately in others. Multiple elements listed with “and / or” should be interpreted in the same way, i.e., “one or more” of the elements so combined. Other elements may optionally be present, whether related to or unrelated to the specifically designated element, in addition to the element specifically specified in the “and / or” clause. Therefore, as a non-limiting example, when used in conjunction with open-ended language such as “comprising,” reference to “A and / or B” may in one embodiment refer only to A (optionally including elements other than B); in another embodiment refer only to B (optionally including elements other than A); in yet another embodiment refer to both A and B (optionally including other elements); and so on.

[0299] As used in the specification and claims, “or” should be understood to have the same meaning as “and / or” as defined above. For example, when separating items in a list, “or” or “and / or” should be interpreted as inclusive, meaning that it includes at least one, but also includes more than one, multiple or list elements, and optionally includes other unlisted items. Only when explicitly indicating the opposite, such as “only one” or “exactly one,” or when used in the claims, “consisting of” will refer to the inclusion of exactly one of multiple or list elements. In general, the term “or” as used herein is interpreted as indicating an exclusive alternative (i.e., “one or the other but not both”) only when placed after exclusive terms such as “either,” “one of,” “only one,” or “exactly one.” “Substantially consisting of” when used in the claims should have its ordinary meaning in the field of patent law.

[0300] As used in the specification and claims, the phrase "at least one" referring to a list of one or more elements should be understood to mean at least one element selected from any one or more elements in the list, but not necessarily including at least one of each element specifically listed in the list, and does not exclude any combination of elements in the list. This definition also allows for the optional presence of elements other than those specifically specified in the list referred to by the phrase "at least one," whether related to or unrelated to the specifically specified element. Thus, as a non-limiting example, "at least one of A and B" (or equivalently "at least one of A or B," or equivalently "at least one of A and / or B") in one embodiment may refer to at least one (optionally including more than one) A, with no B (and optionally including elements other than B); in another embodiment, it may refer to at least one (optionally including more than one) B, with no A (and optionally including elements other than A); in yet another embodiment, it may refer to at least one (optionally including more than one) A and at least one (optionally including more than one) B (and optionally including other elements); and so on.

[0301] It should also be understood that, unless expressly indicated otherwise, in any method claimed herein that includes more than one step or action, the order of the steps or actions of the method is not necessarily limited to the order of the steps or actions of the method described herein.

[0302] In the claims and the foregoing description, all transitional phrases such as “comprising,” “including,” “carrying,” “having,” “containing,” “involving,” “holding,” “consisting of,” etc., shall be understood as open-ended, meaning including but not limited to. Only the transitional phrases “consisting of” and “substantially consisting of” shall be closed or semi-closed transitional phrases, respectively, as described in Section 2111.03 of the U.S. Patent Examination Procedure Manual. It should be understood that embodiments described herein using open-ended transitional phrases (such as “comprising”) are also considered as alternative embodiments “consisting of” and “substantially consisting of” the feature described in that open-ended transitional phrase. For example, if the disclosure describes “a composition comprising A and B,” the disclosure also considers the alternative embodiments “a composition comprising A and B” and “a composition substantially comprising A and B.”

Claims

1. A recombinant adeno-associated virus (rAAV) vector comprising a nucleic acid sequence encoding an antibody against a poly(GA) repeat-associated non-ATG (RAN) protein or an antigen-binding fragment thereof, wherein the nucleic acid sequence is flanked by inverted terminal repeat (ITR) sequences of adeno-associated virus (AAV).

2. The rAAV vector according to claim 1, wherein the anti-poly(GA)RAN protein antibody or its antigen-binding fragment comprises a heavy chain variable region (VH), said VH comprising: (i) Complementarity-determining region (CDR) 1 (CDR1) region, which contains or is composed of the amino acid sequence shown in SEQ ID NO:1; (ii) a CDR2 region comprising or consisting of the amino acid sequence shown in SEQ ID NO:2; and / or (iii) CDR3 region, which contains or consists of the amino acid sequence shown in SEQ ID NO:

3.

3. The rAAV vector according to claim 1 or 2, wherein the anti-poly(GA) RAN protein antibody or its antigen-binding fragment comprises a light chain variable region (VL), said VL comprising: (i) Complementarity-determining region (CDR) 1 (CDR1) region, which contains or is composed of the amino acid sequence shown in SEQ ID NO:4; (ii) a CDR2 region comprising or consisting of the amino acid sequence shown in SEQ ID NO:5; and / or (iii) CDR3 region, which contains or consists of the amino acid sequence shown in SEQ ID NO:

6.

4. The rAAV vector according to any one of claims 1 to 3, wherein the anti-poly(GA) RAN protein antibody or antigen-binding fragment comprises a variable heavy chain, said variable heavy chain comprising or consisting of the amino acid sequence shown in SEQ ID NO:

7.

5. The rAAV vector according to any one of claims 1 to 4, wherein the anti-poly(GA) RAN protein antibody or antigen-binding fragment comprises a variable light chain, said variable light chain comprising or consisting of the amino acid sequence shown in SEQ ID NO:

12.

6. The rAAV vector according to any one of claims 1 to 5, wherein the anti-poly(GA) RAN protein antibody or antigen-binding fragment comprises a heavy chain, said heavy chain comprising or consisting of the amino acid sequence shown in SEQ ID NO: 8 or 10.

7. The rAAV vector according to any one of claims 1 to 6, wherein the anti-poly(GA) RAN protein antibody or antigen-binding fragment comprises a light chain, said light chain comprising or consisting of the amino acid sequence shown in SEQ ID NO: 13 or 15.

8. The rAAV vector of claim 1, wherein the anti-poly(GA) RAN protein antibody or antigen-binding fragment comprises a heavy chain variable region (VH), said VH comprising: (i) Complementarity-determining region (CDR) 1 (CDR1) region, which contains or is composed of the amino acid sequence shown in SEQ ID NO:20; (ii) a CDR2 region comprising or consisting of the amino acid sequence shown in SEQ ID NO:21; and / or (iii) CDR3 region, which contains or consists of the amino acid sequence shown in SEQ ID NO:

22.

9. The rAAV vector according to claim 1 or 8, wherein the anti-poly(GA) RAN protein antibody or antigen-binding fragment comprises a light chain variable region (VL), said VL comprising: (i) Complementarity-determining region (CDR) 1 (CDR1) region, which contains or is composed of the amino acid sequence shown in SEQ ID NO:23; (ii) a CDR2 region comprising or consisting of the amino acid sequence shown in SEQ ID NO:24; and / or (iii) CDR3 region, which contains or consists of the amino acid sequence shown in SEQ ID NO:

25.

10. The rAAV vector according to claim 8 or 9, wherein the anti-poly(GA) RAN protein antibody or antigen-binding fragment comprises a variable heavy chain, said variable heavy chain comprising or consisting of the amino acid sequence shown in SEQ ID NO:

26.

11. The rAAV vector according to any one of claims 8 to 10, wherein the anti-poly(GA) RAN protein antibody or antigen-binding fragment comprises a variable light chain, said variable light chain comprising or consisting of the amino acid sequence shown in SEQ ID NO:

31.

12. The rAAV vector according to any one of claims 8 to 11, wherein the anti-poly(GA) RAN protein antibody or antigen-binding fragment comprises a heavy chain, said heavy chain comprising or consisting of the amino acid sequence shown in SEQ ID NO: 27 or 29.

13. The rAAV vector according to any one of claims 8 to 12, wherein the anti-poly(GA) RAN protein antibody or antigen-binding fragment comprises a light chain, said light chain comprising or consisting of the amino acid sequence shown in SEQ ID NO: 32 or 34.

14. The rAAV vector of claim 1, wherein the anti-poly(GA) RAN protein antigen-binding fragment comprises a single-chain variable fragment (scFv), said scFv comprising a heavy chain variable region, said heavy chain variable region comprising: (i) Complementarity-determining region (CDR) 1 (CDR1) region, which contains or is composed of the amino acid sequence shown in SEQ ID NO:1; (ii) a CDR2 region comprising or consisting of the amino acid sequence shown in SEQ ID NO:2; and / or (iii) CDR3 region, which contains or consists of the amino acid sequence shown in SEQ ID NO:

3.

15. The rAAV vector according to claim 1 or 14, wherein the anti-poly(GA) RAN protein antigen-binding fragment comprises a single-chain variable fragment (scFv), said scFv comprising a light chain variable region, said light chain variable region comprising: (i) Complementarity-determining region (CDR) 1 (CDR1) region, which contains or is composed of the amino acid sequence shown in SEQ ID NO:4; (ii) a CDR2 region comprising or consisting of the amino acid sequence shown in SEQ ID NO:5; and / or (iii) CDR3 region, which contains or consists of the amino acid sequence shown in SEQ ID NO:

6.

16. The rAAV vector according to claim 14 or 15, wherein the scFv comprises a variable heavy chain, the variable heavy chain comprising or consisting of the amino acid sequence shown in SEQ ID NO:

17.

17. The rAAV vector according to any one of claims 14 to 16, wherein the scFv comprises a variable light chain, the variable light chain comprising or consisting of the amino acid sequence shown in SEQ ID NO:

18.

18. The rAAV vector according to any one of claims 15 to 17, wherein the scFv comprises a linker molecule connecting the heavy chain variable region and the light chain variable region, optionally wherein the linker molecule comprises a polyGS linker.

19. The rAAV vector according to any one of claims 1 to 18, wherein the antibody or its antigen-binding fragment further comprises a signal peptide.

20. The rAAV vector according to any one of claims 1 to 19, comprising the sequence shown in any one of SEQ ID NO:36-40.

21. The rAAV vector according to any one of claims 1 to 20, comprising at least 75% identical sequences to those shown in any one of SEQ ID NO:43-46.

22. The rAAV vector according to any one of claims 1 to 21, wherein the rAAV comprises the sequence shown in any one of SEQ ID NO:43-46.

23. A recombinant adeno-associated virus (rAAV) comprising: (i) the rAAV carrier according to any one of claims 1 to 22; and (ii) One or more adeno-associated virus (AAV) capsid proteins.

24. The rAAV of claim 23, wherein one or more AAV capsid proteins comprise AAV9 capsid protein.

25. The rAAV of claim 23, wherein one or more AAV capsid proteins comprise AAV1 capsid protein.

26. The rAAV according to any one of claims 23 to 25, wherein one or more AAV capsid proteins comprise VP1 protein, said VP1 protein comprising the ERDRTRG peptide as shown in SEQ ID NO:

49.

27. The rAAV of claim 26, wherein the VP1 protein comprises the amino acid sequence of SEQ ID NO:

48.

28. A composition comprising the rAAV carrier of any one of claims 1 to 22 or the rAAV of any one of claims 23 to 27, and a pharmaceutically acceptable carrier or buffer.

29. A method for expressing an antibody or antigen-binding fragment in a subject, the method comprising administering rAAV according to any one of claims 23 to 27 to the subject.

30. The method of claim 29, wherein the subject is a mammal, optionally wherein the subject is a human.

31. The method of claim 29 or 30, wherein the subject expresses one or more RAN proteins.

32. The method according to any one of claims 29 to 31, wherein the subject expresses poly(GA) RAN protein.

33. The method according to any one of claims 29 to 32, wherein the subject has or is suspected of having Alzheimer's disease, amyotrophic lateral sclerosis (ALS), or frontotemporal dementia.

34. The method of claim 33, wherein the subject has or is suspected of having ALS.

35. A method for reducing poly(GA) RAN protein aggregation in a subject, the method comprising administering rAAV according to any one of claims 23 to 27 to the subject.

36. The method of claim 35, wherein the subject is a mammal, optionally wherein the subject is a human.

37. The method of claim 35 or 36, wherein the subject expresses one or more RAN proteins.

38. The method according to any one of claims 35 to 37, wherein the subject expresses poly(GA) RAN protein.

39. The method according to any one of claims 35 to 38, wherein the subject has or is suspected of having Alzheimer's disease, amyotrophic lateral sclerosis (ALS), or frontotemporal dementia.

40. The method of claim 39, wherein the subject has or is suspected of having ALS.

41. A method for treating a subject suffering from amyotrophic lateral sclerosis (ALS), the method comprising administering rAAV according to any one of claims 23 to 27 to the subject.

42. The method of claim 41, wherein the subject is a human being.

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