Methods for AAV vector reapplication

Abstract

This invention relates to combinations of recombinant AAVpo1 vector and recombinant AAV9, AAVrh10 or AAVrh74 vectors for gene therapy of muscle and / or nervous system disorders in individuals in need.

Description

Technical Field

[0001] This invention relates to the field of gene therapy. Specifically, this invention relates to a method for re-administering adeno-associated virus (AAV) vectors using a combination of recombinant porcine AAV serotype 1 (AAVpo1) vectors and recombinant AAV serotype 9, rh74, or rh10 (AAV9, AAVrh74, AAVrh10) vectors, and its application in gene therapy for diseases, particularly muscle and / or nervous system disorders, and more particularly neuromuscular diseases, such as hereditary neuromuscular diseases. Background Technology

[0002] Recombinant adeno-associated virus (rAAV or AAV) vectors are widely used for in vivo gene transfer, and clinical trials using AAV vectors to treat a variety of diseases are currently underway.

[0003] AAV is a non-pathogenic virus belonging to the genus Dependent Virus in the family Parvoviridae. AAV is a non-enveloped virus, composed of a capsid approximately 25 nm in diameter and 4.7 kb of single-stranded DNA. Its genome carries two genes, rep and cap, flanked by palindromic sequence regions called inverted terminal repeats (ITRs), which serve as the origin of viral replication and packaging signals. The cap gene encodes three structural proteins that constitute the AAV capsid: VP1, VP2, and VP3. VP1, VP2, and VP3 share the same C-terminus, which is the entirety of VP3. Using AAV2 as a reference, VP1 has 735 amino acids (GenBank YP_680426); VP2 (598 amino acids) begins at threonine 138 (T138); and VP3 (533 amino acids) begins at methionine 203 (M203). The rep gene encodes four proteins required for viral replication: Rep78, Rep68, Rep52, and Rep40. The recombinant AAV vector packages an rAAV genome flanked by an ITR, in which a therapeutic gene expression cassette replaces the AAV protein coding sequence.

[0004] Tissue specificity is determined by the capsid serotype. Commonly used AAV vector serotypes isolated from humans (AAV2, 3, 5, 6) and non-human primates (AAV1, 4, 7-11) can transduce specific organs more effectively than other serotypes. For example, AAV6, AAV8, AAV9, and AAVrh74 are used for muscle tissue, and AAV2, AAV9, AAVrh10, AAVcy.10, AAV-PHP.B, AAV-PHP.EB, and F-branch AAVHSCs (such as AAVHSC7, AAVHSC15, and AAVHSC17) are used for neural tissue.

[0005] Immunogenicity of AAV vectors is a major limiting factor for AAV vector gene transfer. On the one hand, pre-existing immunity exists in commonly used AAV vector serotypes isolated from humans and non-human primates; for example, the seropositivity rate of AAV2 in humans is as high as 80% (Fu et al., Hum Gene Ther Clin Dev., 2017 Dec;28(4):187-196; Stanford et al., Res Pract Thromb Haemost., 2019, 3: 261-267). On the other hand, the high levels of neutralizing antibodies generated after administration of commonly used AAV vector serotypes hinder the possibility of vector re-administration.

[0006] In the presence of anti-AAV antibodies, various methods have been explored for AAV vector re-administration, such as plasma exchange before the second vector injection or treatment with IgG cleavage endopeptidase (IdeS). However, this approach is complex.

[0007] Recombinant AAV vectors have been constructed using the capsids of different porcine AAVs (AAVpo1, po2.1, po4-6). After systemic administration in mice, AAVpo1 was reported to exhibit strong transgenic expression in all major skeletal muscle types, but poor transduction in other tissues, even showing complete off-target effects in the liver. (Bello et al., GeneTherapy, 2009, 16, 1320-1328) . doi: 10.1038 / gt.2009.82; Bello et al., Sci Rep.,2014, 4, 6644, doi: 10.1038 / srep06644; Tulalamba et al., Gene Therapy, 2019, doi.org / 10.1038 / s41434-019-0106-3; WO 2009 / 030025). Peptide-modified AAVpo1 (AAVpo1.A1) can be further transduced into the central nervous system (brain and spinal cord) and achieve transgenic expression levels in different muscle groups and the central nervous system, at least comparable to, or even better than, the AAV9 vector, while avoiding liver targeting (WO 2021 / 219762).

[0008] There is still a need in the field for methods for re-administering adeno-associated virus (AAV) vectors. Summary of the Invention

[0009] The inventors' research confirmed that, contrary to previous publications (Bello et al., Tulalamba et al., cited above; WO 2009 / 030025), there is cross-neutralization between porcine AAV serotypes and some currently isolated AAV vector serotypes from humans and non-human primates. Furthermore, AAVpo1 can be neutralized by human immunoglobulins, but the neutralizing titer of this porcine AAV serotype is lower than that of existing AAV serotypes. Despite these phenomena, the inventors demonstrated that, with a reasonable combination of AAVpo1 and existing AAV vector serotypes, vector re-administration is still possible after the initial AAV vector administration.

[0010] The embodiments of this application demonstrate that antibodies generated after injection of AAV8, AAV9, AAVrh74, or AAVrh10 vectors cannot neutralize AAVpo1 (see...). Figure 1 and Figure 3 Therefore, after the initial injection of AAV8, AAV9, AAVrh74, or AAVrh10 vectors, AAVpo1 vector can be administered (see [link to relevant documentation]). Figure 6 and Figure 7 Conversely, when AAV8, AAV9, or AAVrh10 vectors are administered after the initial injection of the AAVpo1 vector, the presence of neutralizing antibodies renders skeletal muscle transduction levels undetectable, and even before the second injection, the levels of neutralizing antibodies against AAV8 or AAVrh10 are very low (see [link to relevant documentation]). Figure 2 , Figure 4 , Figure 6 , Figure 7 ).

[0011] This invention relates to a combination of recombinant porcine adeno-associated virus serotype 1 (AAVpo1) vector and recombinant adeno-associated virus 9, rh10 or rh74 vector (AAV9, AAVrh10, AAVrh74) for gene therapy of diseases; preferably, the AAV9, AAVrh10 or AAVrh74 vector is administered at an initial time point and the AAVpo1 vector is administered at a subsequent time point.

[0012] In some embodiments, the AAVpo1, AAV9, AAVrh10, and AAVrh74 serotypes are selected from the group consisting of: AAV capsids comprising a sequence having at least 95% identity with any one of SEQ ID NO: 1 and 3-5; and hybrids or peptide-modified derivatives thereof having at least 90% identity with any one of SEQ ID NO: 1 and 3-5.

[0013] In some preferred embodiments, the peptide-modified AAV serotype comprises a peptide containing at least an RGD sequence, preferably a 7-peptide containing at least an RGD sequence. In some preferred embodiments, the peptide-modified AAV serotype comprises peptides selected from the group consisting of: SEQ ID NO: 6-60 and 77-82; preferably, SEQ ID NO: 6, 15, 33-60 and 77-82; more preferably, SEQ ID NO: 6, 15, 36, 44, 50, 56, 58, 60, 77 and 79.

[0014] In some preferred embodiments, the AAVpo1 serotype has at least 95% sequence identity with SEQ ID NO: 61 and comprises peptide A1 (SEQ ID NO: 15). In some preferred embodiments, the AAV9 serotype has at least 95% sequence identity with SEQ ID NO: 3 and comprises peptides selected from the group consisting of SEQ ID NO: 6-60 and 77-82, preferably selected from the group consisting of SEQ ID NO: 6, 15, 33-60 and 77-82, more preferably SEQ ID NO: 6, 15, 36, 44, 50, 56, 58, 60, 77 and 79. In some preferred embodiments, the AAV9 serotype is a heterozygote AAV9rh74, which has at least 95% sequence identity with SEQ ID NO: 62; preferably, it comprises peptide P1 (SEQ ID NO: 6) or any one of SEQ ID NO: 77-82; more preferably, it has at least 95% identity with SEQ ID NO: 64 or 65 and comprises peptide P1 (SEQ ID NO: 6), or has at least 95% identity with SEQ ID NO: 83 and comprises peptide SEQ ID NO: 77.

[0015] In some embodiments, the recombinant AAVpo1 vector and the recombinant AAV9, AAVrh74, or AAVrh10 vector encode the target transgene for therapeutic purposes. In some specific embodiments, the transgene encoded by the recombinant AAVpo1 vector is the same as the transgene encoded by the recombinant AAV9, AAVrh10, or AAVrh74 vector. In other specific embodiments, the transgene encoded by the recombinant AAVpo1 vector is different from the transgene encoded by the recombinant AAV9, AAVrh10, or AAVrh74 vector.

[0016] In some implementations, the therapeutically intended transgenic material is selected from the group consisting of: (i) Therapeutic genes; (ii) a gene encoding a therapeutic protein or peptide, said therapeutic protein or peptide being, for example, a therapeutic antibody or antibody fragment and a genome editing enzyme; and (iii) A gene encoding a therapeutic RNA, such as interfering RNA, guide RNA for genome editing, and antisense RNA capable of exon skipping.

[0017] In some specific embodiments, the AAV serotype used for initial administration and the recombinant AAV serotype used for subsequent administration are selected from: (i) the natural AAV9 capsid of SEQ ID NO: 3 and the AAVpo capsid modified with peptide A1 of SEQ ID NO: 61; (ii) the AAVrh10 capsid, particularly the natural AAVrh10 capsid of SEQ ID NO: 4 and the AAVpo1 capsid modified with peptide A1 of SEQ ID NO: 61; (iii) the peptide-modified AAV9 capsid comprising peptides selected from SEQ ID NO: 33-60 and the AAVpo1 capsid modified with peptide A1 of SEQ ID NO: 61; and (iv) the AAVrh74 capsid, particularly the natural AAVrh74 capsid of SEQ ID NO: 5 and the AAVpo1 capsid modified with peptide A1 of SEQ ID NO: 61.

[0018] In some embodiments, the combination is administered via a systemic route, preferably an intravascular route; more preferably an intravenous route.

[0019] In some embodiments, the combination according to this disclosure is used for gene therapy of muscle and / or nervous system diseases or conditions.

[0020] In some implementations, the target cells for gene therapy are in the nervous system (central and / or peripheral nerves), muscles (cardiac, smooth, and / or skeletal muscles), or combinations thereof.

[0021] In some embodiments, the disease is selected from the group consisting of: neurological diseases, muscle diseases, and combinations thereof. Preferably, the disease is a neuromuscular disease affecting the nervous system, and more preferably, a hereditary neuromuscular disease affecting the nervous system. In some preferred embodiments, the hereditary neuromuscular disease is selected from the group consisting of: (i) myopathy, such as congenital myopathy, myasthenia gravis, metabolic myopathy, distal myopathy, muscular dystrophy with or without cardiomyopathy; and (ii) spinal muscular atrophy (SMA) and motor neuron disease; preferably SMN1 gene-associated spinal muscular atrophy, spinal muscular atrophy with progressive myoclonic epilepsy (SMA-PME), spinal bulbar muscular atrophy (SBMA), and amyotrophic lateral sclerosis (ALS).

[0022] In some preferred embodiments, the target genes for gene therapy are selected from the group consisting of: DMD, DYSF, FKRP, α-muscle Polysaccharide (SGCA), β-caryogamic acid (SGCB), γ-caryogamic acid (SGCG), calpain 3 (CAPN3) , Anoctamin 5 (ANO5), MTM1, DNM2, BIN1, GAA, AGL, ColQ, DOK7, SMN1, ASAH1, MCEP2, and AR genes .

[0023] The present invention also relates to products comprising a recombinant AAVpo1 vector and a recombinant AAV9, AAVrh10 or AAVrh74 vector, as a combination formulation for sequential use in the treatment of a condition according to the present disclosure. Detailed Implementation

[0024] This invention relates to a combination of recombinant porcine adeno-associated virus serotype 1 (AAVpo1) vector and recombinant adeno-associated virus serotype 9, rh10, or rh74 (AAV9, AAVrh10, AAVrh74) vector for gene therapy of diseases in individuals in need. Preferably, the AAV9, AAVrh10, or AAVrh74 vector is administered at an initial time point, and the AAVpo1 vector is administered at a subsequent time point.

[0025] As used in this article, the term "AAV carrier" refers to AAV carrier particles.

[0026] As used herein, the term "recombinant porcine AAV serotype 1 (AAVpo1) vector" or AAVpo1 vector refers to an AAV vector containing porcine AAV serotype 1 capsid proteins. The term "recombinant AAV serotype 9, rh10, rh74 (AAV9, AAVrh10, AAVrh74) vector" or AAV9, AAVrh10, AAVrh74 vector refers to an AAV vector containing AAV serotype 9, rh10, or rh74 capsid proteins.

[0027] As used herein, “AAV serotype” refers to the AAV capsid serotype. AAV serotypes 9, rh10, and rh74 refer to the capsids of AAV9, AAVrh10, and AAVrh74, respectively. AAVpo1 serotype refers to the AAVpo1 capsid. AAV serotypes include natural AAV (i.e., wild-type AAV) and artificial AAV serotypes, such as variants and hybrid capsids derived from natural AAV serotypes, as well as peptide-modified AAV serotypes derived from natural, variant, or hybrid AAV serotypes. An AAV serotype refers to a functional AAV capsid capable of transducing and expressing transgenes in target tissue or organ cells.

[0028] As used in this article, AAV capsid refers to AAV, VP1, VP2 and / or VP3 proteins.

[0029] As used herein, "target tissue or organ" refers to a single tissue or organ or multiple tissues or organs that can be targeted by a combination of the recombinant AAVpo1 serotype vector and the recombinant AAV9, AAVrh10, or AAVrh74 serotype vector according to this disclosure. Gene therapy targets the target tissue or organ, i.e., by sequentially administering combinations of AAV vectors according to this disclosure to treat disease.

[0030] As used in this article, "gene therapy for a disease or condition" means "used to treat a disease or condition through gene therapy" or "used in gene therapy for a disease or condition".

[0031] As used herein, "disease" or "symptom" refers to a disease in which gene therapy can be performed using combinations of AAV vectors according to this disclosure. Diseases specifically include those related to gene mutations, and are therefore suitable for AAV gene therapy.

[0032] Unless explicitly stated in the context, the articles “a / an” and “the” both cover plural references. Therefore, “a / an,” “one or more,” or “at least one” are used interchangeably in this text; unless otherwise specified, “or” means “and / or.”

[0033] AAVpo1 (GenBank accession number FJ688147, accession date July 24, 2016) contains a portion of the viral genome sequence (2977 bp) containing the Cap gene located at positions 780-2930: VP1 CDS at positions 780-2930; VP2 CDS at positions 1188-2930; and VP3 CDS at positions 1329-2930. The sequence of the AAVpo1 capsid protein (VP1) has GenBank accession number ACN42940.1 (accession date July 24, 2016), or SEQ ID NO:1. The amino acid sequence corresponding to the AAV9 capsid protein has GenBank accession number AY530579.1, or protein ID number AAS99264.1 (accession date June 24, 2004, SEQ ID NO:3). The AAVrh10 capsid (GenBank accession number AY243015.1 or protein ID AAO88201.1, accession date May 14, 2003) corresponds to the amino acid sequence SEQ ID NO:4. The AAVrh74 capsid corresponds to the amino acid sequence SEQ ID NO:5.

[0034] The AAVpo1, AAV9, AAVrh74, or AAVrh10 serotypes include the aforementioned natural (wild-type) serotypes (SEQ ID NO: 1 and 3-5), as well as any synthetic serotypes, including any variants, hybrids, and / or peptide-modified AAV capsids derived from said serotypes. This invention covers the use of AAVpo1, AAV9, AAVrh74, or AAVrh10 capsid sequences (SEQ ID NO: 1 and 3-5) having at least 95%, 96%, 97%, 98%, or 99% identity. The present invention also includes the use of hybrids and / or peptide-modified AAV capsids derived from the AAVpo1, AAV9, AAVrh74, or AAVrh10 capsids or serotypes, wherein the hybrids and / or peptide-modified AAV capsids have at least 90% identity with the aforementioned AAVpo1, AAV9, AAVrh74, or AAVrh10 capsid sequences (SEQ ID NO: 1 and 3-5). The hybrids and / or peptide-modified AAV capsids have 91%, 92%, 93%, 94%, 95%, or higher (96%, 97%, 98%, or 99%) identity with the aforementioned AAVpo1, AAV9, AAVrh74, or AAVrh10 capsid sequences (SEQ ID NO: 1 and 3-5). The peptide-modified AAV capsid according to the present invention: (i) is derived from an initial AAV capsid (without peptides) having at least 95%, 96%, 97%, 98%, or 99% identity with the above-described AAVpo1, AAV9, AAVrh74, or AAVrh10 capsid sequences (SEQ ID NO: 1 and 3-5); and (ii) has at least 90% identity with the above-described AAVpo1, AAV9, AAVrh74, or AAVrh10 capsid sequences (SEQ ID NO: 1 and 3-5). The hybrid AAV capsid according to the invention: (i) is derived from at least two different AAV serotypes, including at least one AAVpo1, AAV9, AAVrh74, or AAVrh10 capsid or serotype, having at least 95%, 96%, 97%, 98%, or 99% identity with the aforementioned AAVpo1, AAV9, AAVrh74, or AAVrh10 capsid sequence (SEQ ID NO: 1 and 3-5); and (ii) has at least 90% sequence identity with the aforementioned at least one AAVpo1, AAV9, AAVrh74, or AAVrh10 capsid sequence (SEQ ID NO: 1 and 3-5). The artificial serotype according to the invention has serum-neutralizing properties similar to the natural serotype from which it is derived. This means that the artificial serotype can be neutralized by AAV antibodies, and the antibody titer is similar to that of the natural serotype. Preferably, the difference in dilution factor between similar antibody titers is less than 2-fold; preferably less than 3-fold.3.16 times (10. 0.5 A 100-fold dilution is called a semi-log dilution or semi-log dilution.

[0035] The term "identity" refers to the sequence similarity between two polypeptide molecules or two nucleic acid molecules. Two molecules are identical at that position when a position in the compared sequences is occupied by the same base or the same amino acid residue. The percentage of identity between two sequences is equal to the number of matching positions shared by the two sequences divided by the total number of positions compared, multiplied by 100. Typically, when aligning two sequences, the maximum identity is obtained through comparison. Identity can be calculated using alignments such as the GCG (Genetics Computer Group, Program Manual for the GCG Package, Version 7, Madison, Wisconsin) pileup program, or any sequence alignment algorithm such as BLAST, FASTA, or CLUSTALW.

[0036] In some implementations, AAVpo1, AAV9, AAVrh10, and AAVrh74 serotypes (natural, variant, and hybrid capsids) are modified with peptides, specifically peptides that enhance transduction efficiency in target tissue or organ cells. Libraries of AAV capsid variants displaying short random peptides on the surface of various AAV serotypes have been constructed to screen gene therapy vectors that alter cell specificity and / or transduction efficiency (see Büning et al., Molecular Therapy: Methods & Clinical Development, 2019, 12, 248 for a review). Existing technologies disclose various peptides (targeting peptides) obtained through library screening that can target AAV vectors to target organs (Michelfelder et al., PLoS ONE, 2009, 4, e5122; Kienle EC (Dissertation for the degree of Doctor of natural Sciences, Combined Faculties for the Natural Sciences and for Mathematics of the Ruperto-Carola University of Heidelberg, Germany, 2014); WO 2019 / 207132; Börner et al.(Molecular Therapy, April 2020, 28, 1017-1032; Weinmann et al., Nature Communications, 2020, 11, 5432; Tabebordbar et al., Cell, 2021, 184, 4919-4938). Ai Vu Hong et al., Nature Communications, 2024, 15, 7965- and WO2023 / 237748 disclose peptides targeting integrin αVβ6 that enhance the myophilicity of AAV vectors. The targeting peptides typically consist of sequences of up to 30 amino acids. The peptide may consist of 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 amino acids. In some embodiments, the peptide consists of up to 15 amino acids, preferably 3-10 amino acids, and particularly 7 or 10 amino acids.

[0037] Non-limiting examples of targeting peptides include the peptides disclosed below: Börner et al. (Tables S2, S3), Weinmann et al., Tabebordbar et al.; Ai Vu Hong et al. and WO 2023 / 237748: P1 (RGDLGLS), P2 (CDCRGDCFC), P3 (RGDAVGV), P4 (NDVRSAN), P5 (NDVRAVS), P6 (CNHRYMQMC), P7 (SPGARAF), P8 (DGPWRKM), P9 (FGQKASS), A1 (MPLGAAG), A2 (NYSRGVD), A3 (GSVPRLG), A4 (PVVPRPA), A5 (GERAKPA), A6 (NEARVRE), D1 (NSSRDLG), D2 (MVNNFEW), T1 (SEGLKNL), T2 (PSVPRPP), K1 (SLRSPPS), K2 (NFTRLSA), L1 (GDVGPPG), L2 (NDVRPER), L4 (NRVEEKL), L5 (TTSVRPA), L6 (HLHGRPA), Kera2 (PRGDLAP), RGDLRVS, 1C (RGDLSTP), 1B (RGDLNQY), 1A (RGDLTTP), 1F (RGDATEL), 1D (RGDQLYH), RGDVAAK, 1E (RGDTMSK), 1G (RGDMINT), 1H (RGDLNDS), RGDTMNY, 2A (GPGRGDQTTL), 2B (AEGRGDQYTR), 2C (ATGRGDLGQA), 2D (AVARGDQGLI), 2E (NISRGDQGYQ), 2F (APARGDQGSQ), 3A (RGDYVGL), 3B (RGDYSGL), 3C (RGDYSSV), 3D (RGDYREL), 3E (RGDHGVL), 3F (RGDHASW), 4A (SNSRGDYNSL), 4B (STVRGDYTS), 4C (QERRGDYTSM), 4D (ASTRGDHGVL), 4E (ENRRGDFNNT);And RGDLXXL / I, where XX is any pair of amino acids, which can bind with high affinity to the integrin heterodimer αVβ6 (ITGAV-B6), particularly 4um9 (RGDLGRL), 4um9_modified (RGDLGEL), 5ffo (RGDLATI), 5ffo_modified (RGDLAEL), 5nem (RGDLQVL), and 5nem_modified (RGDLAEI), corresponding to SEQ ID NO: 6-60 and 77-82.

[0038] It has been reported that AAV capsids containing peptides with RGD motifs can improve gene delivery efficiency in muscles after systemic administration. Peptides containing RGD motifs are known to bind to a variety of different cell surface integrins. Peptides containing the RGD motif have been disclosed in Tabebordbar et al., Cell, 2021, 184, 4919-4938, Ai Vu Hong et al., and WO 2023 / 237748; and include: RGDLSTP (1C), RGDLNQY (1B), RGDLTTP (1A), RGDATEL (1F), RGDQLYH (1D), RGDVAAK, RGDTMSK (1E), RGDMINT (1G), RGDLNDS, RGDTMNY, 2A, 2B, 2C, 2D, 2E, 2F, 3A, 3B, 3C, 3D, 3E, 3F, 4A, 4B, 4C, 4D, 4E, RGDLGRL (4um9), RGDLGEL (4um9_modified), RGDLATI (5ffo), RGDLAEL (5ffo_modified), RGDLQVL (5nem), and RGDLAEI. (5nem_modified). The AAV capsids with the best muscle transduction efficiency in mice, non-human primates, and / or human primary myotubes contain peptides with RGDL motifs: RGDLGLS or P1 (AAVMYO or AAV9P1), RGDLTTP (MyoAAV 1A), GPGRGDQTTL (MyoAAV 2A), SNSRGDYNSL (MyoAAV 4A), ENRRGDFNNT (MyoAAV 4E), SAQRGDYVGL (MyoAAV 3A), QERRGDYTSM (MyoAAV 4C) with insertion into variable region VIII (WO 2019 / 207132; Weinmann et al., Nature Communications, 2020, 11, 5432; Tabebordbar et al., Cell, 2021, 184, 4919-4938); or RGDLGRL (4um9), RGDLGEL (4um9_modified), RGDLATI (5ffo), RGDLAEL (5ffo_modified), RGDLQVL (5nem), and RGDLAEI (5nem_modified) are inserted into the variable region IV (Ai VuHong et al. and WO 2023 / 237748).In some preferred embodiments, the AAVpo1, AAV9, AAVrh10, and AAVrh74 serotypes are modified with peptides selected from the group consisting of: P1 (RGDLGLS), A1 (MPLGAAG), MyoAAV peptides (SEQ ID NO: 33-60) and SEQ ID NO: 77-82; particularly SEQ ID NO: 36 (MyoAAV 1A peptide), SEQ ID NO: 44 (MyoAAV 2A peptide), SEQ ID NO: 50 (MyoAAV 3A peptide), SEQ ID NO: 56 (MyoAAV 4A peptide), SEQ ID NO: 58 (MyoAAV 4C peptide), SEQ ID NO: 60 (MyoAAV 4E peptide), SEQ ID NO: 77 (4um9 peptide) and SEQ ID NO: 79 (5ffo peptide).

[0039] In some embodiments, the AAVpo1 serotype is natural AAVpo1; variant AAVpo1, heterozygous AAVpo1, or peptide-modified derivatives thereof, preferably natural AAVpo1 or peptide-modified AAVpo1. In some specific embodiments, the AAVpo1 serotype (natural, heterozygous, mutant capsid) has at least 95% identity with SEQ ID NO: 1. In some specific embodiments, the peptide comprises or consists of sequences selected from the group consisting of SEQ ID NO: 6-60 and 77-82, preferably SEQ ID NO: 15, 33-60, and 77-82. In some more specific embodiments, the peptide comprises or consists of the sequence MPLGAAG (peptide A1 or SEQ ID NO: 15). In some specific embodiments, the AAVpo1 capsid has at least 90% identity with SEQ ID NO: 1, preferably at least 95% identity with SEQ ID NO: 1, and comprises a peptide selected from SEQ ID NO: 15, 33-60, 77-82, preferably SEQ ID NO: 15. In some preferred embodiments, the peptide-modified AAVpo1 capsid protein comprises a sequence selected from the group consisting of SEQ ID NO: 61, and sequences having at least 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 61, and such sequences comprise the peptide of SEQ ID NO: 15 or a fragment thereof corresponding to the VP2 or VP3 capsid protein. SEQ ID NO: 61 corresponds to the AAVpo1 capsid modified with the A1 peptide, named AAVpo1.A1, disclosed in WO2021 / 219762. VP2 corresponds to the amino acid sequence from K136 to the terminal of SEQ ID NO: 61. VP3 corresponds to the amino acid sequence from M184 to the end of SEQ ID NO: 61. In some preferred embodiments, the peptide-modified AAVpo1 capsid protein comprises SEQ ID NO: 61, or a fragment thereof corresponding to the VP2 or VP3 capsid protein.

[0040] In some embodiments, the AAV9 serotype is natural (wild-type) AAV9 or peptide-modified AAV9 comprising peptides selected from SEQ ID NO: 3-60, 77-82; preferably P1 peptide, A1 peptide, MyoAAV peptide selected from SEQ ID NO: 33-60, or RGDLXXL / I peptide selected from SEQ ID NO: 77-82, particularly SEQ ID NO: 36, 44, 50, 56, 58, 60, 77, and 79. The P1-modified AAV9 capsid (AAV9P1 or AAVMYO) is disclosed in WO 2019 / 207132 and corresponds to the amino acid sequence SEQ ID NO: 63.

[0041] In some embodiments, the AAV9 serotype is a heterozygous AAV9 or a peptide-modified derivative thereof. In specific embodiments, the peptide comprises or consists of sequences selected from or composed of sequences from the group consisting of SEQ ID NO: 6-60 and 77-82, preferably SEQ ID NO: 6, 15, 33-60 and 77-82. In specific embodiments, the AAV9 serotype is a heterozygous AAV9.rh74, a P1-modified AAV9.rh74, or an AAV9.rh74 modified with RGDLXXL / I peptide 4um9, 4um9_modified, 5ffo, 5ffo_modified, 5nem or 5nem_modified. RGDLXXL / I peptide-modified AAV9.rh74 is preferably modified with peptide 4um9. The AAV9.rh74 capsid is disclosed in WO 2019 / 193119, corresponding to the amino acid sequence SEQ ID NO: 62. P1-modified AAV9rh74 capsids are disclosed in WO 2022 / 053630 and WO 2020 / 200499, corresponding to SEQ ID NO: 64 and SEQ ID NO: 65, respectively. AAV9rh74 modified with RGDLXXL / I peptides 4um9, 4um9_modified, 5ffo, 5ffo_modified, 5nem, or 5nem_modified is disclosed in WO 2023 / 237748, corresponding to SEQ ID NO: 83-88, respectively. SEQ ID NO: 83 is LICA1. In some embodiments, the AAV9 serotype is an AAV9 variant or a peptide-modified derivative thereof. In specific embodiments, the peptide comprises or consists of sequences selected from or composed of sequences from the group consisting of SEQ ID NO: 6-60 and 77-82, preferably SEQ ID NO: 6, 15, 33-60, and 77-82. AAV9 variants specifically include: AAV-PHP, such as AAV-PHP.A, AAV-PHP.B, AAV-PHP.eB, AAV-PHP.S; AAV9BI; AAV9LD; AAVS1 and AAVS10 (El Andari et al., Science Advances, 2022, 8, 1-21; WO2019207132A1). Peptide-modified AAV9 variants specifically include AAVMYO2 (SEQ ID NO: 66) and AAVMYO3 (SEQ ID NO: 67), corresponding to P1-modified AAVS1 and AAVS10 capsids, respectively (El Andari et al.; WO2019207132A1).

[0042] In some specific embodiments, the AAV9 serotype has at least 90% identity with SEQ ID NO: 3, preferably at least 95% identity with SEQ ID NO: 3. In some specific embodiments, the AAV9 serotype has at least 90% identity with SEQ ID NO: 3, preferably at least 95% identity with SEQ ID NO: 3 and comprises a peptide comprising or consisting of sequences selected from the group consisting of: SEQ ID NO: 6-60 and 77-82, preferably SEQ ID NO: 6, 15, 33-60, 77-82, more preferably peptide P1 (SEQ ID NO: 6); further preferably comprising a sequence having at least 95% identity with any one of SEQ ID NO: 63, 66 or 67, comprising the P1 peptide (SEQ ID NO: 6); even more preferably comprising any one of SEQ ID NO: 63, 66 or 67.

[0043] In some specific embodiments, the AAV9 serotype is a heterozygous AAV9rh74, preferably having at least 95% identity with SEQ ID NO:62, and / or comprising a peptide comprising or consisting of a sequence selected from the group consisting of: SEQ ID NO: 6-60 and 77-82, preferably SEQ ID NO: 6, 15, 33-60 and 77-82; more preferably peptide P1 (SEQ ID NO:6) or RGDLXXL / I peptide selected from 4um9, 4um9_modified, 5ffo, 5ffo_modified, 5nem and 5nem_modified, preferably peptide 4um9 (SEQ ID NO: 77). In some preferred embodiments, the heterozygous AAV9rh74 serotype comprises a sequence having at least 95% identity with SEQ ID NO: 62, or a sequence having at least 95% identity with SEQ ID NO: 64 or 65, comprising the peptide P1; more preferably, comprising any one of SEQ ID NO: 62, 64 or 65. In other preferred embodiments, the heterozygous AAV9rh74 serotype comprises a sequence having at least 95% identity with SEQ ID NO: 62, or a sequence having at least 95% identity with SEQ ID NO: 83-88, comprising the RGDLXXL / I peptide selected from 4um9, 4um9_modified, 5ffo, 5ffo_modified, 5nem and 5nem_modified, respectively; more preferably, comprising any one of SEQ ID NO: 83-88; even more preferably SEQ ID NO: 83.

[0044] In some embodiments, the AAVrh10 serotype is natural AAVrh10; variant AAVrh10; hybrid AAVrh10; or a peptide-modified derivative thereof. In specific embodiments, the AAVrh10 serotype (natural, hybrid, variant capsid) comprises a peptide selected from SEQ ID NO: 6-60 and 77-82, preferably a P1 peptide, an A1 peptide, or a MyoAAV peptide selected from SEQ ID NO: 33-60, and an RGDLXXL / I peptide selected from SEQ ID NO: 77-82, particularly SEQ ID NO: 36, 44, 50, 56, 58, 60, 77, and 79. In some specific embodiments, the AAVrh10 serotype has at least 95% identity with SEQ ID NO: 4. In some specific embodiments, the AAVrh10 serotype has at least 90% identity with SEQ ID NO: 4, preferably at least 95% identity with SEQ ID NO: 4 and contains P1 peptide (SEQ ID NO: 6), or MyoAAV peptide selected from SEQ ID NO: 33-60, or RGDLXXL / I peptide selected from SEQ ID NO: 77-82, particularly SEQ ID NO: 36, 44, 50, 56, 58, 60, 77 and 79.

[0045] In some embodiments, the AAVrh74 serotype is natural AAVrh74, variant AAVrh74, hybrid AAVrh74, or a peptide-modified derivative thereof. In a particular embodiment, the AAVrh10 serotype (natural, hybrid, or variant capsid) comprises a peptide selected from SEQ ID NO: 6-60 and 77-82, preferably P1 peptide, A1 peptide, or MyoAAV peptide selected from SEQ ID NO: 33-60, or RGDLXXL / I peptide selected from SEQ ID NO: 77-82, particularly SEQ ID NO: 36, 44, 50, 56, 58, 60, 77, and 79. In some particular embodiments, the AAVrh74 serotype is the aforementioned hybrid AAVrh74 or a peptide-modified derivative thereof. In some particular embodiments, the AAVrh74 serotype has at least 95% identity with SEQ ID NO: 5. In some specific embodiments, the AAVrh74 serotype has at least 90% identity with SEQ ID NO: 5, preferably at least 95% identity with SEQ ID NO: 5 and contains a P1 peptide (SEQ ID NO: 6), or a MyoAAV peptide selected from SEQ ID NO: 33-60, or an RGDLXXL / I peptide selected from SEQ ID NO: 77-82, particularly SEQ ID NO: 36, 44, 50, 56, 58, 60, 77, and 79; more preferably, it contains SEQ ID NO: 6 or SEQ ID NO: 77. In some specific embodiments, the AAVrh74 serotype is a heterozygous AAVrh74, preferably having at least 95% identity with SEQ ID NO: 62, or at least 95% identity with SEQ ID NO: 64 or 65 and containing a P1 peptide (SEQ ID NO: 6); more preferably, it contains a sequence selected from SEQ ID NO: 62, 64, or 65. In some other preferred embodiments, the hybrid AAV9rh74 comprises a sequence having at least 95% identity with SEQ ID NO: 62, or a sequence having at least 95% identity with SEQ ID NO: 83-88 and comprising the RGDLXXL / I peptide, which is selected from 4um9, 4um9_modified, 5ffo, 5ffo_modified, 5nem, and 5nem_modified, respectively; more preferably, it comprises any one of SEQ ID NO: 83-88; even more preferably, it comprises SEQ ID NO: 83.

[0046] In some embodiments, the rAAV combinations for initial and subsequent administration respectively comprise: (i) a natural AAV9 capsid and a peptide-modified AAVpo1 capsid containing peptide A1 (AAVpo1A1); (ii) an AAVrh10 capsid, particularly a natural AAVrh10 capsid and a peptide-modified AAVpo1 capsid containing peptide A1 (AAVpo1A1); (iii) a peptide-modified AAV9 capsid containing MyoAAV peptides selected from SEQ ID NO: 33-60 and a peptide-modified AAVpo1 capsid containing peptide A1 (AAVpo1A1); (iv) (v) AAVrh74 capsids, particularly natural AAVrh74 capsids and peptide-modified AAVpo1 capsids containing peptide A1 (AAVpo1A1); (v) peptide-modified AAV9 capsids containing peptide P1 (AAV9P1 or AAVMYO) and peptide-modified AAVpo1 capsids containing peptide A1 (AAVpo1A1); (vi) peptide-modified AAV9 variant capsids containing peptide P1, named AAVS1P1 (AAVMYO2) and peptide-modified AAVpo1 capsids containing peptide A1 (AAVpo1A1); (vii) peptide-modified AAV9 variant capsids containing peptide P1, named AAVS10P1 (AAVMYO3) and peptide-modified AAVpo1 capsids containing peptide A1 (AAVpo1A1); (viii) hybrid AAV9rh74 capsids, including peptide-modified hybrid AAV9rh74 capsids, particularly containing peptide P1. (AAV9rh74P1) or peptide 4um9 (LICA1), and a peptide-modified AAVpo1 capsid containing peptide A1 (AAVpo1A1). Preferably, the rAAV combination for initial and subsequent administration comprises: (i) a natural AAV9 capsid and a peptide-modified AAVpo1 capsid containing peptide A1 (AAVpo1A1); (ii) an AAVrh10 capsid, particularly a natural AAVrh10 capsid and a peptide-modified AAVpo1 capsid containing peptide A1 (AAVpo1A1); (iii) a peptide-modified AAV9 capsid comprising the MyoAAV peptide selected from SEQ ID NO: 33-60, and a peptide-modified AAVpo1 capsid containing peptide A1 (AAVpo1A1); (iv) an AAVrh74 capsid, particularly a natural AAVrh74 capsid and a peptide-modified AAVpo1 capsid containing peptide A1 (AAVpo1A1).

[0047] In some specific embodiments, the present invention also relates to a recombinant porcine adeno-associated virus serotype 1 (AAVpo1) vector for gene therapy of diseases according to the present disclosure, wherein the AAVpo1 vector is administered to patients with natural immunity to AAV9, AAVrh10, and / or AAVrh74 vectors according to the present disclosure. As used herein, a patient with natural immunity to AAV9, AAVrh10, and / or AAVrh74 vectors is a patient who has pre-existing neutralizing antibodies against AAV9, AAVrh10, or AAVrh74 due to natural infection with adeno-associated virus (AAV). The presence and / or level of neutralizing antibodies against AAV9, AAVrh10, or AAVrh74 in the patient's plasma or serum can be determined using appropriate neutralization assays known in the art and disclosed in this application. Meliani et al., Hum. Gene Ther. Methods, 2015, 26, 45-53 discloses an example of a neutralization assay.

[0048] The genome of the AAV vectors used in combination according to this disclosure can be a single-stranded genome or a self-complementary double-stranded genome (McCarty et al, Gene Therapy, 2003, Dec., 10(26), 2112-2118). Self-complementary vectors are generated by deleting terminal splitting sites (trs) from one of the terminal repeat sequences of the AAV. These modified vectors (whose replicated genome is half the length of the wild-type AAV genome) readily package DNA dimers. The AAV genome is flanked by ITRs. In certain embodiments, the AAV vector is a pseudotype vector, i.e., the genome and capsid are derived from different serotypes of AAV. In some embodiments, the vector contains the AAV2 rep protein and / or the AAV2 ITR.

[0049] The AAV vectors for combined use according to this disclosure are prepared using standard methods well-known in the art for preparing AAV vectors (see Apone-Ubillus for a review). et al.(Applied Microbiology and Biotechnology, 2018, 102: 1045-1054). In short, after co-transfection with an expression plasmid expressing AAV Rep and capsid proteins, and a plasmid containing a recombinant AAV vector genome (the recombinant AAV vector genome contains the target gene inserted into the expression cassette, flanked by AAV ITRs), the rAAV vector genome can be packaged into AAV capsid particles in the presence of sufficient helper functions. Cells are cultured for a sufficient time to produce AAV vector particles, then the cells are collected, lysed, and the AAV vector particles are purified by standard purification methods such as affinity chromatography or iodixanol or cesium chloride density gradient ultracentrifugation.

[0050] The AAV vector particles typically package a therapeutic target gene (i.e., a transgene). "Therapeutic target gene," "therapeutic target gene," "target gene," or "heterologous target gene" refers to a therapeutic gene or a gene encoding a therapeutic protein, peptide, or RNA, or a combination thereof.

[0051] In some embodiments, the transgene encoded by the recombinant AAVpo1 vector is the same as the transgene encoded by the recombinant AAV9, AAVrh10, or AAVrh74 vector. In other embodiments, the transgene encoded by the recombinant AAVpo1 vector is different from the transgene encoded by the recombinant AAV9, AAVrh10, or AAVrh74 vector. This invention covers various vector systems, such as dual-vector systems, wherein the vector combination encodes overlapping segments of the transgene, which form a complete transgene upon co-delivery of the vector combination into a cell.

[0052] A target gene is any nucleic acid sequence that can modify a target gene or target cellular pathway in a target tissue or organ cell. For example, the gene can modify the expression, sequence, or regulatory pattern of a target gene or cellular pathway.

[0053] In some specific implementations, the target tissue / organ includes muscles and / or the nervous system.

[0054] As used herein, the term "muscle" refers to cardiac muscle (heart), smooth muscle, and skeletal muscle. The term "myocellular" refers to myocytes, myotubes, myoblasts, and / or satellite cells.

[0055] As used herein, the term "nervous system" refers to both the central nervous system (CNS) and the peripheral nervous system (PNS). The term "central nervous system or CNS" refers to the brain, spinal cord, retina, cochlea, optic nerve, and / or olfactory nerve. As used herein, the term "CNS cell" refers to CNS cells comprising neurons and glial cells (oligodendrocytes, astrocytes, ependymal cells, microglia). PNS refers to nerves and ganglia outside the brain and spinal cord.

[0056] Depending on the type of disease, the target tissue or organ may primarily include the nervous system (CNS and / or PNS), primarily include muscles, or primarily include both muscles and the nervous system.

[0057] In some embodiments, the target gene is a functional version of a gene or a fragment thereof. The functional version of the gene includes a wild-type gene, a variant gene, such as a variant gene belonging to the same gene family, or a truncated version that at least partially retains the function of the protein it encodes. The functional version of the gene can be used as an adjunct to gene therapy to replace a defective or nonfunctional gene in a patient's body. For example, functional versions of dystrophin genes include various mini-dystrophin and micro-dystrophin gene constructs known in the art, as well as other functional versions of dystrophin genes. In other embodiments, the target gene is a gene that inactivates a dominant allele that causes an autosomal dominant genetic disease. Gene fragments can be used as recombination templates in combination with genome editing enzymes.

[0058] Alternatively, the target gene may encode a target protein for a specific application (e.g., an antibody or antibody fragment, a gene-editing enzyme) or RNA. In some embodiments, the protein is a therapeutic protein, comprising a therapeutic antibody or antibody fragment, or a gene-editing enzyme. In some embodiments, the RNA is therapeutic RNA.

[0059] In some implementations, the target gene sequence is optimized for expression in the treated individual, preferably a human individual. Sequence optimization may include various alterations to the nucleic acid sequence, including codon optimization, increasing GC content, reducing the number of CpG islands, reducing the number of variable open reading frames (ARFs), and / or reducing the number of splice donor and splice acceptor sites.

[0060] The target gene is a functional gene capable of producing encoded proteins, peptides, or RNA in disease target cells. In some embodiments, the target gene is a human gene. The AAV vector contains the target gene in a form expressible in target organ cells. Specifically, the target gene is operatively linked to an appropriate regulatory sequence to express the transgene in the target cells, tissues, or organs of an individual. Such sequences known in the art particularly include promoters, and regulatory sequences capable of further controlling transgene expression, such as, but not limited to, enhancers, terminators, introns, silencers, particularly tissue-specific silencers, and microRNAs. The target gene is operatively linked to a broad-spectrum, tissue-specific, or inducible promoter that functions in the target organ cells. In some specific embodiments, the target gene is operatively linked to at least two promoters, at least one of which is a target cell-specific or inducible promoter that functions in the target tissue or organ cells. In some specific implementations, the target gene is operatively linked to at least two promoters, one of which is a target cell-specific or inducible promoter that functions in the cells of a target tissue or organ, and the other is a target-specific or inducible promoter that functions in the cells of a different target tissue or organ. The target gene may be inserted into an expression cassette that also contains other regulatory sequences as described above.

[0061] Examples of ubiquitous promoters include the CAG promoter, phosphoglycerate kinase 1 (PGK) promoter, cytomegalovirus enhancer / promoter (CMV), SV40 early promoter, Rous sarcoma virus retrovirus (RSV) LTR promoter, dihydrofolate reductase promoter, β-actin promoter, and EF1 promoter.

[0062] Muscle-specific promoters include, but are not limited to, desin (Des) promoter, creatine kinase MCK promoter, α-myosin heavy chain α-MHC promoter, myosin light chain 2 (MLC-2) promoter, cardiac troponin C (cTnC) promoter, human skeletal muscle actin (HSA) promoter, or synthetic muscle promoters such as SpC5-12 promoter, CK6 promoter, or MHCK7 promoter.

[0063] Promoters for expression in the nervous system, such as the CNS, include promoters that drive broad-spectrum expression and promoters that drive neuronal expression. Representative promoters driving broad-spectrum expression include, but are not limited to: CAG promoters (including the cytomegalovirus enhancer / chicken β-actin promoter, the first exon and first intron of the chicken β-actin gene, and the splice acceptor of the rabbit β-globin gene); PGK (phosphoglycerate kinase 1) promoter; β-actin promoter; EF1α promoter; and CMV promoter. Representative promoters driving neuronal expression include, but are not limited to, the calcitonin gene-related peptide (CGRP) promoter, a known motor neuron-derived factor. Other neuron-selective promoters include promoters for choline acetyltransferase (ChAT), neuron-specific enolase (NSE), synaptic proteins, Hb9, and ubiquitous promoters including neuronal restriction silencing elements (NRSE). Representative promoters driving glial cell-selective expression include the promoter for the glial fibrillary acidic protein gene (GFAP).

[0064] For expression in muscle cells (skeletal muscle and cardiomyocytes), the target gene is advantageously controlled by the desmin promoter, particularly the human desmin promoter (Raguz et al., Dev. Biol., 1998, 201, 26-42; Paulin D&Li Z, Exp. Cell. Res., 2004, Nov 15;301(1):1-7) or the MHCK7 promoter. For skeletal muscle expression, the target gene is advantageously controlled by the desmin promoter, particularly the human desmin promoter, and also contains the miR208a target sequence that inhibits expression in cardiomyocytes (i.e., in the heart, Roudault et al., Circulation, 2013, 128, 1094-104. doi: 10.1161 / CIRCULATIONAHA.113.001340).

[0065] RNA can advantageously complement target DNA or RNA sequences or bind to target proteins. For example, RNA can be interfering RNA, such as shRNA; microRNA; guide RNA (gRNA) used in combination with Cas enzymes or similar enzymes for genome editing; or exon-jugating antisense RNA, such as modified small RNA (snRNA) or long non-coding RNA. Interfering RNA or microRNA can be used to regulate the expression of target genes associated with muscle and / or neurological diseases. A complex of guide RNA for genome editing with Cas enzymes or similar enzymes can be used to modify target genes, particularly correcting the sequence of mutated / defective genes associated with diseases (especially muscle and / or neurological disorders), or modifying the expression of target genes. Exon-jugating antisense RNA is particularly used to correct reading frames and restore the expression of defective genes with interrupted reading frames. In some embodiments, the NA is a therapeutic RNA.

[0066] The genome editing enzyme according to the present invention refers to any enzyme or enzyme complex capable of modifying a target gene or a target cellular pathway. For example, a genome editing enzyme can modify the expression, sequence, or regulation of a target gene or cellular pathway. Advantageously, genome editing enzymes are engineered nucleases, such as, but not limited to, macronucleases, zinc finger nucleases (ZFNs), transcription activation-like effector nucleases (TALENs), Cas enzymes of the CRISPR-Cas system, and similar enzymes. Genome editing enzymes, particularly engineered nucleases, such as Cas enzymes or similar enzymes, can be functional nucleases that can generate double-strand breaks (DSBs) or single-strand DNA breaks (nicking enzymes such as Cas9 (D10A)) at target genomic sites and are used for site-specific genome editing applications, including but not limited to: gene correction, gene substitution, gene insertion, gene knockout, mutagenesis, chromosomal translocation, chromosomal deletion, etc. In site-specific genome editing applications, genome editing enzymes, particularly engineered nucleases such as Cas enzymes or similar enzymes, can be used in conjunction with homologous recombination (HR) templates or templates (also known as DNA donor templates) that modify target genomic sites through homologous recombination induced by double-strand breaks (DSBs). Specifically, HR templates can introduce target transgenes into target genomic sites or repair mutations at target genomic sites, preferably in abnormal or deleted genes causing muscle and / or nervous system diseases. Genome editing enzymes, such as Cas enzymes and similar enzymes, can be DNA base editors, such as cytosine base editors and adenine base editors, or prime editors. Base editors can introduce all four types of transformation mutations, while prime editors extend the scope of donor-free precise DNA editing to produce not only all transformation and transversion mutations but also small insertion and deletion mutations. Overall, DNA base editing and prime editing tools can precisely replace nucleotides in a programmable manner without the need for a donor template. Alternatively, genome editing enzymes (such as Cas enzymes or similar enzymes) can be engineered to lack nuclease activity and then used as DNA-binding proteins for various genome engineering operations, such as, but not limited to, transcriptional activation, transcriptional repression, epigenome modification, genome imaging, and DNA or RNA pull-down.

[0067] The AAV vector combinations disclosed herein are used for gene therapy of diseases, particularly for targeted gene therapy of target tissues or organs.

[0068] The combination of AAV vector serotypes according to this disclosure is preferably used in the form of a single pharmaceutical composition comprising a therapeutically effective amount of AAV vector particles of each serotype, preferably packaged as AAV vector particles of a therapeutically targeted gene according to this disclosure.

[0069] As used herein, "gene therapy" refers to a treatment method that delivers a target nucleic acid into a patient's cells to treat a disease. Nucleic acid delivery is typically achieved using a delivery vector (also called a vector). The combination of rAAV vector particles according to this disclosure can be used to introduce genes into patient cells.

[0070] As used herein, “cell therapy” refers to the process of delivering cells stably transduced according to the rAAV vector particle assembly of the present disclosure to an individual in need by any suitable means (e.g., intravenous injection (infusion) or injection into a target tissue (implantation or transplantation)). In a particular embodiment, cell therapy includes collecting cells from said individual, transducing the individual’s cells according to the rAAV vector particle assembly of the present disclosure, and administering the stably transduced cells back into the patient. As used herein, “cell” means isolated cells, natural or artificial cell aggregates, bioartificial cell scaffolds, and bioartificial organs or tissues.

[0071] Gene therapy can be performed through gene transfer, gene editing, exon skipping, RNA interference, trans-splicing, or any other gene modification that encodes or regulates intracellular sequences, including those in the cell nucleus, mitochondria, or as symbiotic nucleic acids, such as, but not limited to, viral sequences contained within the cell.

[0072] The two main types of gene therapy are as follows: - Therapies designed to provide functional replacement genes for defective / abnormal genes: replacement or additive gene therapy; - Therapies aimed at gene or genome editing: In this case, the goal is to provide cells with the necessary tools to correct the sequence of defective / abnormal genes or modify their expression or regulation, thereby enabling the functional expression of genes or inhibiting (inactivating) abnormal genes: This is gene editing therapy.

[0073] In add-on gene therapy, the target gene is a functional version of a defective or mutated gene in the patient (e.g., a gene associated with a genetic disease). In this case, the target gene will restore the expression of the functional gene.

[0074] Gene or genome editing involves the application of one or more target genes, such as: (i) Genes encoding the therapeutic RNA mentioned above, such as interfering RNAs, like shRNA or miRNA; guide RNAs (gRNAs) used in combination with Cas enzymes or similar enzymes; or antisense RNAs capable of exon jumping, such as modified small suitable RNAs (snRNAs); and (ii) Genes encoding genome editing enzymes as defined above, such as engineered nucleases like macronucleases, zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), Cas enzymes, or similar enzymes; or combinations of such genes, and functional gene fragments as defined above that can be used as a recombination template.

[0075] Gene therapy can be used to treat a variety of hereditary (genotypic) or acquired diseases or conditions that affect the structure or function of target tissues or organs. These diseases can be caused by trauma, infection, degenerative changes, structural or metabolic defects, tumors, inflammatory or autoimmune diseases, stroke, or other causes.

[0076] In some specific implementations, the disease affects one or more of the following target tissues or organs: muscles, including skeletal muscle, smooth muscle, or cardiac muscle; the nervous system, including the brain or spinal cord; the disease may affect muscles, including skeletal muscle or cardiac muscle; the nervous system, including the brain or spinal cord; or both.

[0077] In some specific embodiments, the disease is a myopathy, such as skeletal myopathy and / or cardiomyopathy, preferably a hereditary myopathy.

[0078] In some specific embodiments, the disease is a neurological disorder (neural cell disease), preferably a hereditary neurological disorder. The neurological disorder may affect the CNS and / or PNS, such as the brain, spinal cord, or both. CNS diseases include, for example, Alzheimer's disease, Parkinson's disease, frontotemporal dementia, Huntington's disease, etc.

[0079] In some specific embodiments, the disease is a neuromuscular disorder, preferably a hereditary neuromuscular disorder. Neuromuscular disease or disorder (NMD) is a very broad term encompassing a range of conditions that impair muscle function, whether direct (lesions of voluntary muscles) or indirect, involving the peripheral nervous system or neuromuscular junctions. Neuromuscular diseases are a broad group of conditions involving damage or dysfunction of peripheral nerves, muscles, or neuromuscular junctions. The site of damage can be located in the cell body (i.e., amyotrophic lateral sclerosis [ALS] or sensory ganglion disease), axon (i.e., peripheral axonal neuropathy or brachial plexus neuropathy), Schwann cells (i.e., chronic inflammatory demyelinating polyradiculoneuropathy), neuromuscular junction (i.e., myasthenia gravis or Lambert-Eaton myasthenia gravis syndrome), muscle (i.e., inflammatory myopathy or muscular dystrophy), or any combination of these sites. Some neuromuscular diseases are also associated with central nervous system disorders, such as ALS or spinal muscular atrophy.

[0080] The table below lists the mutated genes in hereditary neurological disorders that can be targeted for treatment using gene therapy based on the AAV vector combination of the present invention: Hereditary neurological disorders

[0081] Other examples of mutated genes for targeted hereditary neurological disorders that can be treated using the AAV vector combination gene therapy of the present invention include genes causing spinal muscular atrophy (SMA) and motor neuron disease; hereditary motor and sensory neuropathy; and genes for hereditary paraplegia and hereditary ataxia, listed in the table below. In some specific embodiments, the neurological disorders are selected from the group consisting of spinal muscular atrophy (SMA). SMN1, ASAH1 Genes), amyotrophic lateral sclerosis (ALS) SOD1, ALS2 SETX, FUS, ANG, TARDBP, FIG4, OPTN etc.); hereditary spastic paraplegia (etc.) SPAST (SPG4), SPG7 Other SPG Genes such as SPG11, SPG20 and SPG21 Specially for SPAST (SPG4) and SPG7 ) and 4B1 type Charcot-Marie-Tuss disease ( MTMR2 In some preferred embodiments, the gene is selected from the group consisting of: SMN1, ASAH1, DNM2, MTMR2 and SPAST Genes. In some other preferred embodiments, the genes are selected from the group consisting of: SOD1, ALS2, SETX, FUS, ANG, TARDBP, FIG4 and OPTN .

[0082] Examples of hereditary myopathy mutations that can be targeted for gene therapy using the AAV vector combination of the present invention are listed in the table below: Muscular dystrophy

[0083] Congenital muscular dystrophy

[0084] Congenital myopathy

[0085] Distal myopathy

[0086] Other myopathy

[0087] Myotonia syndrome

[0088] Ion channel myopathy

[0089] Malignant hyperthermia

[0090] Metabolic myopathy

[0091] Hereditary cardiomyopathy

[0092] Congenital myasthenia gravis

[0093] Spinal muscular atrophy (SMA) and motor neuron disease

[0094] Hereditary motor and sensory neuropathy

[0095] Hereditary paraplegia

[0096] Other neuromuscular disorders

[0097] Hereditary ataxia

[0098] Any of the genes in the table above can be used as a target site for alternative gene therapy, where the target gene is the functional version of the defective / mutated gene.

[0099] Alternatively, the genes mentioned above can serve as targets for gene editing. Gene editing is used to correct mutated gene sequences or modify the expression or regulation of defective / abnormal genes, enabling muscle cells to express functional genes. In such cases, the target gene is selected from genes encoding therapeutic RNA, such as interfering RNA, guide RNA for gene editing, or exon-jugating antisense RNA, where these therapeutic RNAs target the pathogenic genes listed above. Tools such as CRISPR / Cas9 can be used in this editing process.

[0100] Therefore, through gene editing or gene substitution, a modified version of the gene can be provided in the target cells of affected patients, particularly muscle cells and / or nervous system (PNS and / or CNS) cells, which could contribute to the effective treatment of the disease.

[0101] In some implementations, the target gene for gene therapy (additional gene therapy or gene editing) is the pathogenic gene for the neurological disorders, muscle disorders or neuromuscular disorders disclosed herein.

[0102] Diseases for which gene therapy using AAV vector serotype combinations according to this disclosure specifically include: muscular dystrophy, congenital muscular dystrophy, congenital myopathy, distal myopathy, other myopathy, myotonic syndrome, ion channel myopathy, malignant hyperthermia, metabolic myopathy, hereditary cardiomyopathy, congenital myasthenia gravis, myasthenia gravis, spinal muscular atrophy and motor neuron disease, hereditary paraplegia, hereditary ataxia, hereditary motor sensory neuropathy and other neuromuscular diseases; said diseases can be addressed by gene therapy targeting the diseases listed in the table. The diseases can be classified into the following categories: (i) Myopathy: including hereditary cardiomyopathy, metabolic myopathy, other myopathy, distal myopathy, muscular dystrophy and congenital myopathy; muscular dystrophy includes Duchenne muscular dystrophy; congenital myopathy includes myotubular myopathy and central nucleus myopathy; (ii) Spinal muscular atrophy (SMA) and motor neuron disease include: amyotrophic lateral sclerosis (ALS), progressive bulbar palsy (PBP), pseudobulbar palsy, progressive muscular atrophy (PMA), primary lateral sclerosis (PLS), various types of spinal muscular atrophy (SMA), such as spinal muscular atrophy with progressive myoclonic epilepsy (SMA-PME), SMN1-associated spinal muscular atrophy, spinal bulbar muscular atrophy (SBMA) and unilateral muscular atrophy (MMA) and some rare subtypes of ALS; (iii) Myotonic syndrome, including myotonic dystrophy type 1 ( DMPK (gene) and type 2 myotonic dystrophy ( CNBP (iv) Congenital myasthenia gravis syndrome; (v) Hereditary motor and sensory neuropathy; (vi) Hereditary paraplegia and hereditary ataxia.

[0103] In some preferred embodiments, the hereditary neuromuscular disease is selected from the group consisting of: (i) myopathy, such as congenital myopathy, myasthenia gravis, metabolic myopathy, distal myopathy, muscular dystrophy with or without cardiomyopathy; (ii) spinal muscular atrophy (SMA) and motor neuron disease; preferably SMN1-associated spinal muscular atrophy, spinal muscular atrophy with progressive myoclonic epilepsy (SMA-PME), spinal bulbar muscular atrophy (SBMA) and amyotrophic lateral sclerosis (ALS).

[0104] Hereditary neuromuscular diseases that can be treated with AAV serotype combination therapy according to this disclosure include, for example: - Amyotrophic lateral sclerosis is caused by DMD X-linked myopathy spectrum caused by pathogenic gene variants, the gene encoding dystrophin. Muscular dystrophy includes Duchenne muscular dystrophy (DMD), Benedict's muscular dystrophy (BMD), and DMD-associated dilated cardiomyopathy.

[0105] Limb-girdle muscular dystrophy (LGMD) is a group of diseases with clinical manifestations similar to DMD, but can be caused by autosomal recessive or autosomal dominant inheritance, and can affect both males and females. LGMD is caused by mutations in genes encoding sarcoglycan and other proteins associated with muscle cell membranes, which interact with dystrophin. The term LGMD1 refers to a dominant (autosomal dominant) genotype, while LGMD2 is an autosomal recessive genotype. More than 50 pathogenic loci have been reported (LGMD1A-LGMD1G, LGMD2A-LGMD2W). Calcinopathy (LGMD2A) is caused by… CAPN3 Gene mutations cause LGMD, and more than 450 pathogenic agents have been described to date. Genes associated with the LGMD phenotype include: anoctamin 5 ( ANO5 ), vascular extracardiac matrix proteins ( BVES ), Caloprotein 3 ( CAPN3 ), caveolin 3 ( CAV3 ), CDP-L-fucose phosphate A ( CRPPA ), muscular dystrophy proteoglycan 1 ( DAG1 ), desmin ( DES ), DNAJ heat shock protein family ( Hsp40 Homeotype B subfamily 6 ( DNAJB6 ), muscle repair protein ( DYSF ), fukutin-related protein ( FKRP ), fukutin ( FKT GDP-mannose phosphorylase B ( GMPPB ), heterogeneous nucleoribonucleoprotein D-like ( HNRNPDL ), containing LIM zinc finger domain 2 ( LIMS2 ), lain A:C ( LMNA ), actin MYOT ), lectin protein ( PLEC ), protein O-glucose transferase 1 ( PLOGLUT1 ), protein O-linked mannose β1,2-N-acetylglucosamine transferase 1 (β1,2-)( POMGNT1 ), protein O-mannose kinase ( POMK), protein O-mannose transferase 1 ( POMT1 ), protein O-mannose transferase 2 ( POMT2 Sarcoglycan α ( SGCA Sarcoglycan β ( SGCB Sarcoglycan δ( SGCD Sarcoglycan γ ( SGCG ), myone-cap ( TCAP ), transporter 3 ( TNPO3 ), torsin 1A interacting protein ( TOR1AIP1 ), transporter particle complex 11 ( TRAPPC11 Triple motif protein 32 (TRIM32) and titin (TTN) are the main pathogenic genes leading to the LGMD phenotype. CAPN3, DYSF, FKRP and ANO5 (Babi Ramesh Reddy Nallamilli et al., Annals of Clinical and Translational Neurology, 2018, 5, 1574-1587).

[0106] Emory-Dreyfus muscular dystrophy (EDMD) is caused by a defect in one of the following genes: EMD (encoding nuclear membrane protein) FHL1 Gene, LMNA Genes (encoding lamins A and C).

[0107] Nesprin-1 and Nesprin-2-related muscular dystrophy are respectively caused by... SYNE1 , SYNE2 Genetic defects cause LUMA-related muscular dystrophy. TMEM43 LAP1B-related muscular dystrophy is caused by a genetic defect. TOR1AIP1 Caused by genetic defects.

[0108] - Type 1 facioscapulohumeral muscular dystrophy (FSHD1A), such as... DUX4 It is associated with gene (shortening of the D4Z4 macrosatellite repeat sequence in the subtelomere region of chromosome 4q35) or FRG1 gene defects; type 2 facial-scapular-humeral muscular dystrophy (FSHD1B) is caused by SMCHD1 Caused by genetic defects.

[0109] - Abnormalities in the muscle repair protein (Dysferlin) are also involved in a variety of neurological disorders, including multiple sclerosis (Hochmeister et al., J. Neuropathol. Exp. Neurol., 2006 Sep;65(9):855-65); Alzheimer's disease (Galvin et al., Acta Neuropathol., 2006 Dec;112(6):665-71) and choreiform dyskinesia (Takahashi T, et al., Mov. Disord., 2006, Sep;21(9):1513-5).

[0110] - Spinal muscular atrophy is a disease of the survival motor neuron 1 ( SMN1 Hereditary diseases caused by gene mutations, characterized by weakness and atrophy of the muscles used for movement. ASAH1 Gene mutations can lead to SMA-PME (spinal muscular atrophy with progressive myoclonic epilepsy). - Central nucleus myopathy: including X-linked actin fibromyopathy ( MTM1 ) and autosomal dominant central karyotype myopathy ( DNM2, BIN1 (etc.), as shown in the table above. X-linked actin fibromyalgia is a disease caused by the actinin gene (etc.). MTM1 This is a genetic disease caused by a mutation that primarily affects the muscles used for movement (skeletal muscles) and occurs almost exclusively in males. The disease is characterized by muscle weakness (myopathy) and decreased muscle tone (hypotonia).

[0111] - Myosinemia is a genetic disorder caused by mutations in the myosin (TTN) gene. It has been reported to have both dominant and recessive forms. TTN Mutations can cause widespread myocardial and skeletal muscle disorders. Dominant myosinopathy includes hereditary myopathy of early-onset respiratory failure (HMERF) caused by exon 344 mutations and late-onset tibial muscular dystrophy (TMD). Recessive myosinopathy includes limb girdle muscular dystrophy type 2J, early-onset distal myosinopathy in adolescents or adults, Emory-Dreyfus myopathy without cardiomyopathy, and congenital myopathy with or without heart disease.

[0112] Pompe disease is caused by acid α-glucosidase ( GAA Pompe disease is a hereditary condition caused by gene mutations. Mutations in the GAA gene can hinder the effective breakdown of glycogen by acid α-glucosidase, leading to the accumulation of this sugar in lysosomes at toxic levels. This accumulation can damage organs and tissues throughout the body, especially muscles, thus triggering progressively worsening clinical manifestations and symptoms of Pompe disease.

[0113] Type III glycogen storage disease GSD3) is an autosomal recessive metabolic disorder caused by Amylo-α-1,6-glucosidase and 4-α-glucosidase, which encode glycogen debranching enzymes. AGL This is caused by homozygous or compound heterozygous mutations in the GSD gene, leading to the accumulation of abnormal glycogen (with shorter outer chains) in the body. Clinically, GSD3 patients often present with hepatomegaly, hypoglycemia, and growth retardation in infancy or early childhood. Type IIIa patients experience mild muscle weakness in childhood, but this can worsen in adulthood; some patients may develop cardiomyopathy.

[0114] Glycogen storage disease type IV (GSD IV) is an extremely rare autosomal recessive disorder caused by a mutation in the GBE1 gene, which encodes glycogen branching enzyme (GBE). GSD IV accounts for approximately 3% of all glycogen storage diseases. The phenotype of GSD IV is broad, ranging from neonatal death to mild adult onset, and is accompanied by varying degrees of involvement of the liver, muscles, nervous system, skin, and heart.

[0115] - Genome-wide association studies confirm BIN1 The locus is a major regulator of genetic risk in Alzheimer's disease (AD) (Voskobiynyk et al., eLife doi: 10.7554 / eLife.57354; July 13, 2020). Hereditary spastic paraplegia (HSP) is a group of rare inherited neurological diseases with extremely high clinical and genetic heterogeneity; the core symptom of all HSPs is spasticity of the lower limbs caused by upper motor neuron involvement. Genes contributing to HSP include at least 79 species. SPG Gene. SPG7 and SPAST Gene mutations are a common cause of hereditary spastic paraplegia (HSP) (Review, Lallemant-Dudek P). et al. Fac. Rev., 2021, Mar 10;10:27).

[0116] MECP2 (methyl CpG-binding protein 2) is essential for the normal function of nerve cells. It is a key recognition protein for DNA methylation. Its methyl CpG-binding domain (MBD) recognizes and binds to the 5-methylcytosine region. MECP2 The gene is X-linked and regulated by X chromosome inactivation. MECP2 Gene mutations are the cause of the vast majority of Rett syndrome cases. Rett syndrome is a progressive neurodevelopmental disorder and one of the most common causes of intellectual disability in women. At least 53 pathogenic mutations have been identified to date.

[0117] Androgen receptor (AR)-related diseases include androgen insensitivity syndrome (partial or complete) and X-linked spinal-bulbar muscular atrophy type 1 (SMAX1 or SBMA). SBMA is an X-linked, adult-onset neuromuscular disease caused by abnormal amplification of the polyglutamine (polyQ) fragment of the androgen receptor (AR) protein. AR isoform 2 is a naturally occurring variant that encodes a truncated AR lacking the polyQ binding region. Delivery of this isoform using rAAV9 restores the abnormal transcriptional activity of the polyQ AR and improves the disease phenotype in SBMA mice (Lim et al., Science Advances, 2021, 7, 34).

[0118] In some specific implementations, the target gene for gene therapy (additional gene therapy or gene editing) is selected from the following group of neuromuscular disease-related genes: Duchenne muscular dystrophy and Becker muscular dystrophy (DMD genes); ligamentous muscular dystrophy (LGMD) genes; and so on. CAPN3, DYSF, FKRP, ANO5, DNAJB6 Isogenes, and SGCA, SGCB, SGCG Other genes); spinal muscular atrophy ( SMN1, ASAH1 Genetic) and progressive muscular atrophy ( SOD1, ALS2, SETX, FUS, ANG, TARDBP, FIG4, OPTN etc.); myotubular myopathy ( MTM1 Gene); Central karyotype myopathy ( MTM1, DNM2, BIN1 Genes); Nimarin myopathy ( NEB, ACTA1, KLHL40, KLHL41, KBTBD13, LMOD3, TNNT1, TNNT3, TPM2, TPM3, CFL2, MYPN Gene); Selenin N-related myopathy ( SEPN1 Genes); Congenital myasthenia gravis ( ColQ, CHRNE, RAPSN, DOK7, MUSK, CHAT, AGRN Genes); Pompa disease ( GAA Gene); Type III glycogen storage disease ( GSD3 (AGL gene); Myotonic myopathy type 1 ( DMPK (gene) and type 2 ( CNBP / ZNF9 Genes); Hereditary paraplegia ( SPAST (SPG4), SPG7 Other SPG genes, such as SPG11 SPG20 and SPG21 ,in particular SPAST (SPG4) and SPG7 Charcot-Marie-Tooth disease type 4B1 ( MTMR2 Rett syndrome MCEP2 And spinal cord and brainstem muscle atrophy, X-linked type 1 (…). SMAX1 or SBMA ()( AR Gene).

[0119] In some preferred embodiments, the target gene is selected from the group consisting of: DMD, CAPN3, DYSF, FKRP, SGCA, SGCB, SGCG, ANO5, MTM1, DNM2, BIN1, GAA, AGL, ColQ, DOK7, SMN1, MECP2, AR and ASAH1Gene.

[0120] In this invention, a therapeutically effective dose refers to a dose sufficient to reverse, alleviate, or inhibit the progression of the condition or symptom referred to in the term, or to reverse, alleviate, or inhibit the progression of one or more symptoms of the condition or symptom. The terms "effective amount" or "effective dose" are defined as a dose sufficient to achieve, or at least partially achieve, the intended effect.

[0121] Determining and adjusting the effective dose depends on a variety of factors, such as the composition used, route of administration, individual patient characteristics (such as sex, age and weight), concomitant medications, and other factors that a medical professional can identify.

[0122] In various embodiments of the present invention, the pharmaceutical composition comprises a pharmaceutically acceptable carrier and / or medium.

[0123] "Pharmaceutically acceptable carriers" refer to media that, when administered appropriately to mammals, especially humans, will not produce serious adverse reactions, allergic reactions, or other harmful effects. Pharmaceutically acceptable carriers / excipients are non-toxic solid, semi-solid, or liquid fillers, diluents, encapsulating materials, or various formulation additives.

[0124] Preferably, the pharmaceutical composition comprises a pharmaceutically acceptable carrier suitable for injection. In particular, these carriers may be isotonic, sterile saline solutions (monosodium or disodium phosphate, sodium chloride, potassium chloride, calcium chloride or magnesium chloride, or mixtures of such salts), or in a dry form, particularly lyophilized compositions, which, when sterile water or saline is added as appropriate, form an injectable solution.

[0125] Suitable drug formulations for injection include sterile aqueous solutions or suspensions. These solutions or suspensions may contain additives compatible with the viral vector and that do not impede the entry of viral vector particles into target cells. In all cases, the formulation must be sterile and must have sufficient fluidity for easy aspiration with a syringe. It must be stable under production and storage conditions and protected against contamination by microorganisms such as bacteria and fungi. Examples of suitable solutions include buffers, such as phosphate-buffered saline (PBS) or lactated Ringer's solution.

[0126] This invention also provides a method for treating muscle and / or nervous system disorders, comprising: - Administer a therapeutically effective dose of AAV9, AAVrh10, or AAVrh74 vector to the patient at the initial time point, and then administer a therapeutically effective dose of AAVpo1 vector to the patient at subsequent time points.

[0127] The present invention also provides the use of the pharmaceutical composition according to the present disclosure in the preparation of a medicament for treating muscle or nervous system disorders, particularly muscle or CNS disorders according to the present disclosure; preferably muscle and nervous system disorders, particularly muscle and CNS disorders according to the present disclosure.

[0128] The present invention also provides a product comprising AAVpo1 carrier and AAV9, AAVrh10, and AAVrh74 carriers, as a combination formulation for sequential use in the treatment of muscular and / or neurological disorders.

[0129] As described herein, the terms "patient" or "individual" include humans and other mammals receiving preventative or therapeutic treatment. Preferably, the patient or individual according to the invention is a human.

[0130] In some embodiments, the individual has been previously tested and shown to have no or only low levels of neutralizing antibodies against the AAV serotype used for initial administration. Neutralizing antibodies against the AAV serotype can be detected by standard AAV neutralization assays well known in the art, which have been disclosed in the embodiments of this application. In some specific embodiments, the low level of neutralizing antibodies in the neutralization assays disclosed in the embodiments of this application has a titer of less than or equal to 1:10.

[0131] As used herein, “treatment” means the application or administration of a therapeutic agent or combination of therapeutic agents to a patient suffering from a disease (particularly a muscle disease), or the application or administration of said therapeutic agent to an isolated tissue or cell line taken from the patient, in order to cure, repair, alleviate, reduce, alter, correct, improve, or affect the disease or any of its symptoms. Specifically, the term “treatment” means the reduction or relief of at least one adverse clinical symptom associated with the disease.

[0132] As used in this article, the term "treatment" also refers to the preventative application of a therapeutic agent.

[0133] The pharmaceutical compositions of the present invention are typically administered according to a known procedure at an effective dose and time that induces a therapeutic effect in the patient. The pharmaceutical compositions may be administered via any convenient route, such as, but not limited to, infusion or bolus injection, or absorption through the epithelium or mucosa (e.g., oral mucosa, rectal mucosa, and intestinal mucosa).

[0134] As used herein, the term "systemic administration" refers to the route by which a substance (carrier) is administered into the circulatory system, including enteric or parenteral administration. Parenteral administration includes injection, infusion, implantation, and others.

[0135] The administration can be systemic, local, or a combination of systemic and local administration; systemic administration includes parenteral and oral administration, and local administration includes local and regional administration. Systemic administration is preferably parenteral, such as subcutaneous (SC), intramuscular (IM), intravascular, such as intravenous (IV), intra-arterial or coronary; intraperitoneal (IP); intradermal (ID), epidural, or other routes. Local administration is preferably intracerebral, intraventricular, cerebrospinal fluid, and / or intrathecal administration. The route of administration can be, for example, injection or perfusion. In some preferred embodiments, the route of administration is parenteral, preferably intravascular, such as intravenous (IV), arterial or coronary, intramuscular or subcutaneous; more preferably intravascular, such as intravenous (IV) or arterial; and even more preferably intravenous (IV). In other preferred embodiments, the administration is intracerebral, intraventricular, cerebrospinal fluid, and / or intrathecal, which can be performed alone or in combination with parenteral administration, preferably intravascular, intramuscular, or subcutaneous; more preferably intravascular, such as intravenous (IV) or intra-arterial, and even more preferably intravenous (IV). In some other preferred embodiments, administration is parenteral, preferably intravascular alone, or in combination with intracerebral, ventricular, cerebrospinal fluid, and / or intrathecal administration. Initial and subsequent time-point administrations may be administered via the same or different routes.

[0136] In some implementations, the initial time point and subsequent time points of application of the carrier combination are spaced weeks, months or years apart.

[0137] In some embodiments, the vector used for initial administration and the vector used for subsequent time points encode the same transgene, and the subsequent time points are characterized by a decrease in the transgene expression level or therapeutic effect of the AAV vector administered at the initial time point. According to this embodiment, sequential administration of a combination of AAV vector serotypes via a systemic route can produce sustained high levels of transgene expression in target organs compared to the vector used for initial administration.

[0138] In some other embodiments, the vector used for initial administration and the vector used for subsequent time point administration encode different transgenes, and the combination of different serotype AAV vectors administered sequentially via a systemic route can express two different transgenes in the target organ compared to the vector used for initial administration.

[0139] Vector transduction and transgene expression were determined by sequential systemic administration of AAV vector serotype combinations in animal models (such as mouse models well known in the art and disclosed in the embodiments of this application). Administration of the vector used for initial administration was used for comparison. Vector transduction could be determined by measuring the vector genome copy number in each diploid genome, using standard assays well known in the art, such as real-time PCR assays disclosed in the embodiments of this application. Transgene expression could be determined by measuring mRNA or protein levels, using standard assays well known in the art, such as quantitative RT-PCR assays and quantitative Western blot analysis disclosed in the embodiments of this application.

[0140] Unless otherwise stated, the present invention will be carried out using conventional techniques in the art. These techniques have been described in detail in the literature.

[0141] The present invention will now be described with reference to the accompanying drawings and through the following embodiments, which are not limiting: Attached Figure Description

[0142] Figure 1 Neutralizing antibody titers against rAAVpo1 in mice injected with different rAAV serotypes. A. Mice injected with AAV8, AAV9, AAVrh10, AAVpo1A1, and AAV9P1. B. Mice injected with AAVrh74.

[0143] Figure 2 Neutralizing antibody titers against different rAAV serotypes in mice injected with rAAVpo1.

[0144] Figure 3 Neutralizing antibody titers against rAAVpol in rats injected with different rAAV serotypes.

[0145] Figure 4 Neutralizing antibody titers against different rAAV serotypes in rats injected with rAAVpo1.

[0146] Figure 5 Serological detection rates of AAV8, AAV9, AAVpo1 (=AAVpo1WT), and AAVpo1.A1 in the serum of 50 human donors. A. Serological detection rate percentage. B. Serological positivity rate percentage.

[0147] Figure 6The biodistribution of the vector in skeletal muscle was analyzed in rats injected with AAV8, AAV9, or AAVrh10 carrying the rat Mtm1 gene, followed by administration of the AAVpo1.A1-luciferase gene; or rats injected with AAVpo1.A1 carrying the rat Mtm1 gene, followed by administration of the AAV8-, AAV9-, or AAVrh10-luciferase gene; and a control group. A. Vector copy number in the tibialis anterior muscle. B. Vector copy number in the diaphragm muscle.

[0148] Figure 7 The study included rats injected with AAV8, AAV9, or AAVrh10 carrying the rat Mtm1 gene, followed by administration of AAVpo1.A1-luciferase, or rats injected with AAVpo1.A1 carrying the rat Mtm1 gene, followed by administration of AAV8-, AAV9-, or AAVrh10-luciferase, and a control group. The luciferase activity in skeletal muscle was measured. A. Luciferase activity in the tibialis anterior muscle, expressed as relative luminescent units (RLUs) and normalized to µg protein. B. Luciferase activity in the diaphragm muscle, expressed as relative luminescent units (RLUs) and normalized to µg protein.

[0149] Example Example 1 Materials and Methods Neutralizing antibody detection was performed as described above (Meliani et al., Hum. Gene Ther. Methods, 2015, 26, 45-53). The 2V6.11 cell line (accession number CVCL_6355) can be induced to express the human adenovirus E4 ORF 634kDa tumor protein, previously described by Mohammadi et al. (Nucleic Acids Res., 2004, 32, 2652-2659). This cell line was chosen because of its highest AAV transduction efficiency, as described by Meliani et al. The recombinant AAV vector serotype expresses a luciferase reporter gene under the control of the CMV promoter. In short, on day 1, 2 × 10⁻⁶ cells were transduced into the recombinant AAV vector. 4Two V6.11 cells were seeded in 96-well plates and cultured for 24 hours in the presence of ponasterone A (Life Technologies, Carlsbad, USA). Each AAV serotype was used with a multiplicity of infection (MOI) of 200, resulting in transduction efficiencies of 10,000–1,000,000 relative optical units (RLUs) depending on the serotype. Recombinant AAV-CMV-luciferase (AAV-CMV-Luc) was diluted with serum-free DMEM medium (Life Technologies, Carlsbad, USA) and mixed with semi-logarithmic serial dilutions (1 / 1 to 1 / 3160) of the serum samples, followed by incubation at 37°C for 1 hour. The initial dilution of the samples could be adjusted after injection. Subsequently, the serum-vector mixture was added to cells cultured in DMEM medium containing 10% FCS at 37°C and 5% CO2. Two replicates were set for each dilution. 24 hours later, cells were lysed using the Bright Lite system (Promega, Madison, USA) and analyzed using a chemiluminescence analyzer (ENSPIRE). TM Luciferase activity was measured on a microscope (Perkin Elmer, Waltham, USA). Transduction efficiency was expressed as relative light units per second (RLU). Neutralization titer was defined as the highest serum dilution that inhibited AAV transduction by ≥50% compared to a serum-free control group (100% transduction).

[0150] result 1. Neutralization of rAAVpo1 using mouse serum injected with various human / non-human primate (NHP) serotypes of rAAV. Three-week-old mice (n=3-10 per group) were intravenously injected with AAV vectors (AAVpo1.A1, AAV8, AAV9, AAVrh10) expressing transgenes under the control of the Desmin or CAG promoter at a dose of 2×10⁻⁶. 13 vg / kg. Four 7-week-old mice were intravenously injected with the AAV9P1 vector at a dose of 5 × 10⁻⁶ g / kg. 13 vg / kg. AAVpo1.A1 is the A1-modified wild-type AAVpo1, previously described in WO2021 / 219762. AAVpo1.A1 represents AAVpo1. AAV9P1 has been previously described in Weinmann et al., Nat Commun. 2020 Oct 28;11(1):5432. doi: 10.1038 / s41467-020-19230-w and WO2019 / 207132. Three-week-old mice were intravenously injected (n=4 per group) with the AAVrh74 vector at a dose of 10.12 vg / kg or 5×10 12 vg / kg. AAV8 was used as a control. Mice serum was collected 6–24 weeks after injection, and the titers of neutralizing antibodies against AAVpo1.A1 and the injected serotype were measured.

[0151] Mice injected with AAV9 and AAVrh10 produced neutralizing antibodies against the injected serum types, but not against AAVpo1.A1. Figure 1 A). Injection of AAVrh74 yielded similar results ( Figure 1 B); These results for AAVrh74 are in line with expectations, as its capsid sequence shares 99% identity with that of AAVrh10. These results suggest that AAVpo1 vectors, such as AAVpo1.A1, can be subsequently administered after initial administration of AAV9, AAVrh10, or AAVrh74 vectors.

[0152] 2. The effect of mouse serum injected with rAAVpo1 on neutralizing rAAV from various human / non-human serotypes. Five 3-week-old mice were intravenously injected with the AAVpo1.A1 vector expressing MTM1 under the control of the desmin promoter at a dose of 2 × 10⁻⁶. 13 vg / kg. Serum was collected 12 weeks after injection, and the titers of neutralizing antibodies against AAV8, AAV9, AAV5, wild-type AAVpo1 (AAVpo1WT), and AAVpo1.A1 were determined.

[0153] Mice injected with AAVpo1.A1 produced high levels of neutralizing antibodies against the injected serotype (AAVpo1.A1) and AAVpo1WT (titers ranging from 1:316 to 1:3160). Neutralizing titers against AAV9 and AAV5 were low, ranging from 1:3.16 to 1:31.6 (only 2 / 5 of mice achieved AAV9 titers of 1:100 and 1:316). In contrast, neutralizing titers against AAV8 were significantly lower (undetectable in 3 / 5 of mice, and 1:1 in 2 / 5 of mice). Figure 2 ).

[0154] 3. Neutralizing rAAVpo1 with rat serum injected with rAAV serotypes from various human / non-human primates. Three-week-old rats (n=5-15 per group) were intravenously injected with AAV vectors (AAVpo1.A1, AAV8, AAV9, AAVrh10) expressing transgenes under the control of the desmin promoter at a dose of 2 × 10⁻⁶. 13 vg / kg. Serum was collected 3-6 weeks after injection, and the titers of neutralizing antibodies against AAVpo1.A1 and the injected serotype were measured.

[0155] Rats injected with AAV9 and AAVrh10 produced neutralizing antibodies against the injected serum types, but produced almost no neutralizing antibodies against AAVpo1.A1 or at low titers (undetectable in 11-12 / 15 rats; titer of 1:1 in 3-4 / 15 rats); Figure 3 These results indicate that AAVpo1 vectors (such as AAVpo1.A1 vectors) can be used as a follow-up treatment after initial administration of AAV9 or AAVrh10 vectors.

[0156] 4. Neutralization of rAAV from various human / non-human serotypes using rat serum injected with rAAVpo1. Five 3-week-old rats were intravenously injected with AAV vectors (AAVpo1.A1, AAV8, AAV9, and AAVrh10) expressing MTM1 under the control of the desmin promoter at a dose of 2 × 10⁻⁶. 13 vg / kg. Serum was collected 3 weeks after injection, and the titers of neutralizing antibodies against AAV8, AAV9, AAVrh10, and AAVpo1.A1 were measured.

[0157] Rats injected with AAVpo1.A1 produced high levels of neutralizing antibodies (titers greater than 1:1000) against the injected serotype (AAVpo1.A1). Neutralizing antibody titers against AAV9 were lower, at 1:100. In contrast, neutralizing antibody titers against AAV8 and AAVrh10 were significantly lower (for AAV8: titers were 1:1 in 3 / 5 rats, and 1:3.16 and 1:10 in 2 / 5 rats, respectively; for AAVrh10: undetectable in 2 / 5 rats, and 1:1, 1:3.16, and 1:10 in 3 / 5 rats). Figure 4 ).

[0158] 5. Neutralize rAAV from porcine AAV serotype 1 and various human or non-human primate serotypes with mixed human immunoglobulins. Neutralizing antibody detection was performed using normal human immunoglobulin (TEGELINE, 50 mg / mL). The neutralizing antibody titers for AAVpo1 (AAVpo1WT and AAVpo1.A1) ranged from 1:10 to 1:31.6, while the titers for AAV8 and AAV9 were approximately 1:316. These results indicate the presence of neutralizing antibodies against AAVpo1 in human serum.

[0159] 6. Human AAV seropositivity rate Serum samples from 50 randomly selected human blood donors were analyzed to determine the serological detection rates of AAV8, AAV9, and AAVpo1 (AAVpo1.A1 or AAVpo1WT). The serological detection rates of AAVpo1 (AAVpo1.A1 and AAVpo1WT) were significantly lower than those of AAV8 and AAV9. Figure 5 A). All AAVpo1 (AAVpo1WT or AAVpo1.A1) positive donors were also AAV8 and AAV9 positive. However, the neutralizing antibody titer against AAVpo1 (AAVpo1WT or AAVpo1.A1) was lower than that against AAV8 and AAV9. Figure 5 B).

[0160] Table 1. AAV9 and AAVpo1.A1 neutralizing antibody titers in human donors.

[0161] Approximately 60% of AAV9-positive patients are AAVpo1-negative (60.4% AAV9-positive / AAVpo1A1-negative (Table 1); 57.6% AAV9-positive / AAVpo1WT-negative), therefore AAVpo1 (AAVpo1WT or AAVpo1A1) can be administered. Furthermore, if a neutralizing antibody titer of 1:10 or lower is used as the positivity threshold, then 14 out of 16 AAV9-positive patients can be injected with AAVpo1.A1 (Table 1).

[0162] Example 2 Three-week-old Sprague-Dawley rats were intravenously injected with an AAV vector expressing rat Mtm1 cDNA at a dose of 2 × 10⁻⁶. 13 vg / kg; 6 weeks later, administer intravenously the AAV vector expressing the luciferase gene at a dose of 1×10 13 vg / kg. Rats were euthanized 6 weeks (15 weeks old) after the second injection. Blood was collected every 3 weeks, and blood was also collected before sacrifice for immunoassay. Muscle and organs were collected and flash-frozen in liquid nitrogen for biochemical analysis.

[0163] Materials and Methods Vector genome copy number analysis Samples were placed in cell lysis buffer (QIAGEN) and homogenized for 40 seconds at 5 m / s using a Bead Mill 24 homogenizer (FisherBrand). Total genomic DNA was extracted using the Gentra Puregen Blood Kit (QIAGEN) according to the manufacturer's instructions. The total gDNA concentration was determined using a Nanodrop 8000 (ThermoScientific) spectrophotometer. 32 ng of total DNA was used to quantify the copy number of vectors within the diploid genome using TaqMan real-time PCR on a LightCycler 480 quantitative PCR instrument (Roche). Using the titin gene as an internal control, the following primers and probes were used for normalization: 5'-gTCCCCTgCgTATCTgCTATg-3' (forward; SEQ ID NO: 68), 5'-AgAgAggTAgTATTgAAAACgAgCg-3' (reverse; SEQ ID NO: 69), and 5'-TCCgCAgCTCTAgTggAAgAACCACC-3' (probe; SEQ ID NO: 70). The primers used for vector genome (Mtm1 transgene) amplification were: 5'-TGGATGGATGGGCGATTTAC-3' (forward; SEQ ID NO: 71), 5'-GCGCTGATTGACGAAACTTATC-3' (reverse; SEQ ID NO: 72), and 5'-TTGAAGAGTACAGAAGGCAGGGCC-3' (probe; SEQ ID NO: 73), and the primers used for luciferase transgene amplification were: 5'-GTGTTGGGCGCGTTATTTATC-3' (forward: SEQ ID NO: 74), 5'-TAGGCTGCGAAATGTTCATACT-3' (reverse: SEQ ID NO: 75), and 5'-TTGCGCCCGCGAACGACATTTATA-3' (probe: SEQ ID NO: 76).

[0164] Luciferase activity in tissues Samples were placed in a mixture of PBS buffer and protease inhibitor (Roche) and homogenized for 30 seconds at 4 m / s using a Bead Mill 24 homogenizer (FisherBrand). 50 µL of lysis buffer was taken, and 100 µL of detection buffer (1 mM DTT, 25 mM Tris / base, 1 mM EDTA, 15% glycerol, 8 mM MgCl2, 2 mM ATP (Roche)) and 100 µL of 167 µM D-luciferin (Interchim) were added. Luciferase activity was then measured on a luminescence analyzer (ENSPIRE™, Perkin Elmer, Waltham, USA), expressed as relative light units (RLU) / second. The luciferase signal was normalized to the total protein content as determined by the BCA protein assay (ThermoFisherScientific).

[0165] result Based on quantitative analysis of the vector genome in the tibialis anterior muscle and diaphragm ( Figure 6 ), in the first time with 2×10 13 After administering AAV8, AAV9, and AAVrh10 vectors at a dose of vg / kg, at a dose of 1×10 13 Administering the AAVpo1A1 vector at a dose of vg / kg effectively transduced skeletal muscle, with efficacy comparable to a single dose of AAVpo1A1. In contrast, a second administration of AAVpo1A1 was neutralized by antibodies produced during the first injection, thus preventing the second vector from transducing skeletal muscle. Similarly, neutralizing antibodies against AAVpo1A1 were detected in the serum of one of the rats injected with both AAV8 and AAVpo1A1, 6 weeks after AAV8 injection, rendering the second-administered vector ineffective in transducing skeletal muscle.

[0166] By measuring luciferase activity in the tibialis anterior muscle and diaphragm ( Figure 7 The expression of the luciferase gene carried by the second-injection vector was quantified. The luminescence level was correlated with the vector copy number: after the initial administration of AAV8, AAV9, or AAVrh10 vectors, injection of AAVpo1A1 resulted in luciferase expression levels similar to those achieved with a single dose of the vector.

[0167] These data indicate that AAVpo1 vectors (such as AAVpo1.A1 vectors) can be used after prior injection of AAV8, AAV9, and AAVrh10. This also suggests that AAVrh74 can be used in combination with AAVpo1 vectors, as AAVpo1.A1 is not neutralized by antibodies generated after AAVrh74 injection. Figure 1 B).

[0168] In contrast, after injecting AAVpo1.A1 first, 1×10 13 At a dose of vg / kg of AAV8 or AAVrh10, skeletal muscle transduction levels could not be detected prior to the second injection due to the presence of neutralizing antibodies (even at low levels). (See [link to injection]) Figure 4 and Figure 6 ).

Claims

1. A combination of a recombinant porcine adeno-associated virus serotype 1 (AAVpo1) vector and a recombinant adeno-associated virus serotype 9, rh10, or rh74 (AAV9, AAVrh10, AAVrh74) vector for gene therapy of muscle and / or nervous system diseases in individuals in need, wherein the AAV9, AAVrh10, or AAVrh74 vector is administered at an initial time point and the AAVpo1 vector is administered at a subsequent time point.

2. The combination for use according to claim 1, wherein the AAVpo1, AAV9, AAVrh10 or AAVrh74 serotype is selected from the group consisting of: AAV capsids comprising a sequence having at least 95% identity with any one of SEQ ID NO: 1 and 3-5; and hybrids or peptide-modified derivatives thereof having at least 90% identity with any one of SEQ ID NO: 1 and 3-5.

3. The combination for use according to claim 2, wherein the peptide-modified AAV serotype comprises a peptide containing the sequence RGD, preferably a 7-mer peptide containing the sequence RGD.

4. The combination for use according to claim 2 or 3, wherein the peptide-modified AAV serotype comprises peptides selected from the group consisting of: SEQ ID NO: 6-60 and 77-82; preferably SEQ ID NO: 6, 15, 33-60 and 77-82; more preferably SEQ ID NO: 6, 15, 36, 44, 50, 56, 58, 60, 77 and 79.

5. The combination for its use according to any one of claims 1-4, wherein: - The AAVpo1 serotype has at least 95% identity with SEQ ID NO: 61 and contains peptide A1 of SEQ ID NO: 15; - The AAV9 serotype has at least 95% identity with SEQ ID NO: 3 and contains peptides selected from the group consisting of SEQ ID NO: 6-60 and 77-82, preferably selected from the group consisting of SEQ ID NO: 6, 15, 33-60 and 77-82, more preferably SEQ ID NO: 6, 15, 36, 44, 50, 56, 58, 60, 77 and 79; - The AAV9 serotype is a heterozygote AAV9rh74, which has at least 95% identity with SEQ ID NO: 62; preferably, it contains peptide P1 of SEQ ID NO: 6 or any one of SEQ ID NO: 77-82; more preferably, it has at least 95% identity with SEQ ID NO: 64 or 65 and contains peptide P1 of SEQ ID NO: 6, or has at least 95% identity with SEQ ID NO: 83 and contains peptide SEQ ID NO:

77.

6. The combination for use according to any one of claims 1-5, wherein the recombinant AAVpo1 vector and the recombinant AAV9, AAVrh74 or AAVrh10 vector encode a therapeutic transgene.

7. The combination for use according to claim 6, wherein the transgene encoded by the recombinant AAVpo1 vector is the same as the transgene encoded by the recombinant AAV9, AAVrh74 or AAVrh10 vector.

8. The combination for use according to claim 6, wherein the transgene encoded by the recombinant AAVpo1 vector is different from the transgene encoded by the recombinant AAV9, AAVrh10 or AAVrh74 vector.

9. The combination for use according to any one of claims 6-8, wherein the therapeutically intended transgenic material is selected from the group consisting of: (i) Therapeutic genes; (ii) a gene encoding a therapeutic protein or peptide, said therapeutic protein or peptide being, for example, a therapeutic antibody or antibody fragment and a genome editing enzyme; and (iii) A gene encoding a therapeutic RNA, such as interfering RNA, guide RNA for genome editing, and antisense RNA capable of exon skipping.

10. The combination for use according to any one of claims 1-9, wherein the recombinant AAV serotype for initial administration and the recombinant AAV serotype for subsequent administration are selected from: (i) the natural AAV9 capsid of SEQ ID NO: 3 and the AAVpo1 capsid modified with peptide A1 of SEQ ID NO: 61; (ii) the AAVrh10 capsid, particularly the natural AAVrh10 capsid of SEQ ID NO: 4 and the AAVpo1 capsid modified with peptide A1 of SEQ ID NO: 61; (iii) the peptide-modified AAV9 capsid comprising peptides selected from SEQ ID NO: 33-60 and the AAVpo1 capsid modified with peptide A1 of SEQ ID NO: 61; (iv) the AAVrh74 capsid, particularly the natural AAVrh74 capsid of SEQ ID NO: 5 and the AAVpo1 capsid modified with peptide A1 of SEQ ID NO:

61.

11. The combination for use according to any one of claims 1-10, wherein it is administered via a systemic route, preferably an intravascular route, and even more preferably an intravenous route.

12. The combination for use according to any one of claims 1-11, wherein the disease is selected from the group consisting of: nervous system diseases, muscle diseases, and combinations thereof.

13. The combination for use according to claim 12, wherein the disease is a neuromuscular disease.

14. The combination for use according to any one of claims 1-13, wherein the disease is a hereditary disease.

15. The combination for use according to claim 14, wherein the target gene for gene therapy of the hereditary disease is selected from the group consisting of: DMD, DYSF, FKRP, MTM1, SGCA, SGCB, SGCG, CAPN3, ANO5, DNM2, BIN1, GAA, AGL, ColQ, DOK7, SMN1, ASAH1, MCEP2 and AR Gene.

16. A product comprising a recombinant AAVpo1 vector and a recombinant AAV9, AAVrh10 or AAVrh74 vector, as a combination formulation for sequential use in the treatment of muscular and / or neurological disorders.

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