Use of synthetic AAV capsids for gene therapy of muscle and central nervous system disorders

Abstract

The present invention relates to the use of recombinant porcine adeno-associated virus (AAV) vectors comprising peptide-modified porcine AAV serotype 1 (AAVpo1) capsids in the gene therapy of muscle and / or central nervous system (CNS) disorders, particularly neuromuscular diseases such as genetic neuromuscular diseases.

Description

Technical Field

[0001] The present invention relates to the use of a recombinant porcine adeno-associated virus (AAV) vector containing a peptide-modified porcine AAV serotype 1 (AAVpo1) capsid in the gene therapy of muscle and / or central nervous system (CNS) disorders, particularly neuromuscular diseases such as genetic neuromuscular diseases.

Background Art

[0002] Recombinant adeno-associated virus (rAAV or AAV) vectors are widely used for in vivo gene transfer, and clinical trials using AAV vectors are currently being conducted for the treatment of a number of diseases.

[0003] The AAV vector is a non-enveloped vector composed of a 20-nm diameter capsid and 4.7 kb of single-stranded DNA. The genome carries two palindromic regions called inverted terminal repeats (ITRs), and two adjacent genes, rep and cap. The cap gene encodes three structural proteins, VP1, VP2, and VP3, that make up the AAV capsid. VP1, VP2, and VP3 share the same C-terminus, which is the entirety of VP3. Using AAV2 as a reference, VP1 has a 735-amino acid sequence (GenBank YP_680426); VP2 (598 amino acids) starts at threonine 138 (T138), and VP3 (533 amino acids) starts at methionine 203 (M203).

[0004] Tissue specificity is determined by the capsid serotype, and commonly used AAV serotypes isolated from humans (AAV2, 3, 5, 6) and non-human primates (AAV1, 4, 7-11) can transduce specific organs more efficiently than others. For example, AAV6, AAV8, AAV9, and AAV-rh74 in muscle tissue, and AAV2, AAV9, AAVrh10, AAVcy.10, AAV-PHP.B, AAV-PHP.EB, and clade F AAVHSC, such as AAVHSC7, AAVHSC15, and AAVHSC17 in neural tissue.

[0005] However, all commonly used, naturally occurring AAV serotypes and their variants validated to date tend to accumulate in the liver. This poses a problem, particularly when AAV vectors are administered via systemic routes. Firstly, transgenes intended for expression in muscle may be toxic to the liver. Secondly, AAV vectors that enter the liver reduce the amount of vector available for muscle or nerve tissue. Consequently, higher doses of AAV vector are required. This increases the potential for inducing hepatotoxicity and the cost of vector production.

[0006] Additionally, pre-existing immunity to AAV is a weakness in commonly used AAV vector serotypes isolated from humans and non-human primates, particularly AAV2 and other serotypes, which have a seroprevalent of up to 80% of the population (Fu et al., Hum Gene Ther Clin Dev., December 2017; 28(4): pp. 187-196; Stanford et al., Res Pract Thromb Haemost., 2019, 3: pp. 261-267).

[0007] Recombinant AAV vectors were generated using capsids derived from various porcine AAVs (AAVpo1, po2.1, po4 to 6). Strong transgene expression in all major types of skeletal muscle was reported for AAVpo1, combined with poor transduction in other tissues, including complete detargeting from the liver, after systemic administration in mice. AAVpo2.1 was also detargeted from the liver after peripheral administration, while AAVpo4 and AAVpo6 efficiently transduced all major organs sampled, including the brain. Porcine AAV vectors were not cross-neutralized by antiserum produced against all other commonly used AAVs or by pooled human Ig (Bello et al., Gene Therapy, 2009, 16, pp. 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; Puppo et al., PLOS ONE, 2013, 8, e59025; WO 2009 / 030025). Overall, this makes recombinant porcine AAV vectors, particularly AAVpo1 and AAVpo2.1 detargeted from the liver, attractive vectors for human gene therapy of muscle diseases. However, while the transgene expression levels achieved in muscle using recombinant porcine AAV vectors were high, they were only half to one-third of those achieved with AAV9, which is often considered the absolute standard for muscle-directed gene therapy. Additionally, the transduction efficiencies reported in the brain using AAVpo1 and AAVpo2.1 were low.

[0008] A library of AAV capsid variants displaying short peptides on the surface of various AAV serotypes was generated to screen gene therapy vectors with altered cell specificity and / or transduction efficiency (Borner et al., Molecular Therapy, April 2020, 28, pp. 1017-1032; 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 2018 / 189244)). Peptide-modified AAV1, 7-9, rh10, and DJ capsids effective in in vitro transduction of human T cell lines, primary human macrophages, hepatocytes, and astrocytes have been reported. Peptides sharing the motif NXXRXXX (SEQ ID NO: 12) were disclosed as even more effective for transduction of multiple cell types in vitro in the context of multiple AAV serotypes.

[0009] The ability to efficiently, selectively, and safely transduce various muscle groups and the central nervous system using AAV vectors delivered throughout the body is beneficial for gene therapy of many human diseases. [Prior art documents] [Patent Documents]

[0010] [Patent Document 1] WO 2009 / 030025 [Patent Document 2] WO 2018 / 189244 [Non-patent literature]

[0011] [Non-Patent Document 1] Fu, Hum Gene Ther Clin Dev., December 2017; 28(4): 187-196 [Non-licensed Document 2] Stanford, Res Pract Thromb Haemost, 2019, 3: 261-267° [Non-licensed Document 3] Bello, Gene Therapy, 2009, 16, pp. 1320-1328. doi: 10.1038 / gt.2009.82 [Non-licensed Document 4] Bello, Sci Rep., 2014, 4, 6644, DOI: 10.1038 / srep06644 [Non-licensed Document 5] Tulalamba, Gene Therapy, 2019, doi.org / 10.1038 / s41434-019-0106-3 [Non-licensed Document 6] Puppo, PLOS ONE, 2013, 8, e59025 [Non-licensed Document 7] Borner, Molecular Therapy, April 2020, 28, pp. 1017-1032 [Non-licensed Document 8] 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 [Non-licensed Document 9] Girod, Nat. Med., 1999, 5, pages 1052~1056 [Non-licensed Document 10] Grifman, Molecular Therapy, 2001, 3, pages 964~975 [Non-licensed Document 11] Rabinowitz, Virology, 1999, pages 265, 274~285 [Non-licensed Document 12] Wu, J. Virol., 2000, 74, pp. 8635-8647. [Non-licensed Document 13] McCarty, Gene Therapy, December 10, 2003 (26), pages 2112~2118 [Non-licensed Document 14] Aponte-Ubillus, Applied Microbiology and Biotechnology, 2018, 102: pages 1045~1054 [Non-licensed Document 15] Raguz, Dev. Biol., 1998, 201, pp. 26-42 [Non-licensed Document 16] Paulin D & Li Z, Exp. Cell. Res., November 15, 2004; 301(1): 1-7 [Non-licensed Document 17] Roudault, Circulation, 2013, pp. 128, 1094-104. doi: 10.1161 / CIRCULATIONAHA.113.001340 [Non-licensed Document 18] Babi Ramesh Reddy Nallamilliら, Annals of Clinical and Translational Neurology, 2018, 5, pages 1574~1587 [Non-licensed Document 19] Hochmeister, J. Neuropathol. Exp. Neurol., September 2006; 65(9): 855-65) [Non-licensed Document 20] Galvin, Acta Neuropathol, December 2006; 112(6): 665-71 [Non-licensed Document 21] Takahashi Tら、Mov. Disord.、September 2006;21(9):1513~5 pages [Non-licensed Document 22] Voskobiynyk et al., eLife doi: 10.7554 / eLife.57354; July 13, 2020 [Non-Patent Document 23] Lallemant-Dudek P. et al., Fac. Rev., March 10, 2021; 10:27 [Non-Patent Document 24] Buj-Bello et al., PNAS, 2002, 99, pp. 15060 - 5. doi:10.1073 / pnas.212498399 [Non-Patent Document 25] Al-Qusairi et al., PNAS, 2009, 106, pp. 18763 - 8. doi:10.1073 / pnas.0900705106 [Non-Patent Document 26] Meyer et al., Molecular Therapy, 2015, 23. doi: 10.1038 / mt.2014.210 [Summary of the Invention] [Means for Solving the Problems]

[0012] The inventors used a recombinant porcine adeno-associated virus (AAV) vector containing a peptide-modified capsid protein derived from porcine AAV serotype 1 (AAVpo1) to deliver a target therapeutic gene by systemic administration in mice. Some of the best AAV vector serotypes commonly used for muscle (AAV8, AAV9) or CNS (AAV9, AAVrh10) transduction were simultaneously tested for comparison. The inventors were surprised to find that the peptide-modified AAVpo1 vector achieved advantageous transgene expression levels in various muscle groups and the central nervous system (brain and spinal cord) that were at least equivalent to, if not superior to, those of the AAV9 vector, while simultaneously being detargeted from the liver. Furthermore, this porcine AAV vector is expected not to be neutralized by pre-existing antibodies against common AAVs (human and non-human primate AAVs). For these reasons, the use of such peptide-modified AAVpo1 vectors represents a potentially efficient, selective, and safer therapeutic approach for gene therapy of muscle and / or CNS disorders, particularly neuromuscular diseases such as genetic neuromuscular disorders. In some embodiments, peptide-modified AAVpo1 vectors are used to target nervous system cells or nervous system and muscle cells to treat nervous system and neuromuscular diseases, particularly genetic nervous system and genetic neuromuscular diseases.

[0013] Accordingly, one aspect of the present invention relates to a recombinant porcine adeno-associated virus (AAV) vector comprising a peptide-modified capsid protein derived from porcine AAV serotype 1 for gene therapy of muscle and central nervous system (CNS) disorders; in particular, nervous system disorders and neuromuscular disorders affecting the nervous system, such as CNS disorders affecting the CNS and neuromuscular disorders.

[0014] In some embodiments, recombinant porcine AAV vectors for use according to the present invention are characterized by a combination of hepatic detargeting after systemic administration, particularly intravenous administration, and transgene expression levels in various muscle groups, as well as in the brain and spinal cord, which are at least equivalent, if not superior, to those of the AAV9 vector.

[0015] In some embodiments, the peptide-modified capsid protein comprises at least one peptide containing the sequence MPLGAAG (SEQ ID NO: 2) or a variant containing one or two amino acid mutations (insertions, deletions, or substitutions) in the sequence, preferably one or two amino acid substitutions in the sequence. In some preferred embodiments, the peptide comprises the sequence GMPLGAAGA (SEQ ID NO: 3) or a variant containing up to four (1, 2, 3, or 4) amino acid mutations (insertions, deletions, or substitutions) in the sequence, preferably one or two amino acid deletions or substitutions in the sequence, advantageously the deletions being at the N and / or C-terminus. In some preferred embodiments, the sequence SEQ ID NO: 2 or 3 or a variant thereof has up to five amino acids adjacent to its N and / or C-terminus, for example, GQR and GAA at their N and C-terminuses. In some even more preferred embodiments, the peptide comprises or consists of the sequence GQRGMPLGAAGAQAA (SEQ ID NO: 4).

[0016] In some embodiments, the peptide is inserted between residues N567 and S568 or between residues N569 and T570 of the capsid protein; the position is determined by alignment with SEQ ID NO: 1. Preferably, the peptide is inserted between positions N567 and S568, replacing all residues at positions 565-567 and 568-570, or the peptide is inserted between position N569 and T570, replacing all residues at positions 567-569 and 570-572; the position is determined by alignment with SEQ ID NO: 1.

[0017] In some preferred embodiments, the peptide-modified AAVpo1 capsid protein includes the sequence of SEQ ID NO: 5, and a sequence selected from the group consisting of a sequence having at least 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 5, including the peptide according to this disclosure, and a fragment thereof corresponding to a VP2 or VP3 capsid protein.

[0018] In some embodiments, recombinant porcine AAV vectors are vector particles that package the target gene for therapeutic purposes.

[0019] In some preferred embodiments, the gene of interest is operably linked to a promoter that is functional in neurons and / or glial cells.

[0020] In some preferred embodiments, the gene of interest for therapeutic purposes is: (i) Therapeutic genes; (ii) Genes encoding therapeutic proteins or peptides such as therapeutic antibodies or antibody fragments, and genome editing enzymes; and (iii) Genes encoding therapeutic RNA such as interfering RNA, guide RNA for genome editing, and antisense RNA that can be exon-skipped. It is selected from the group consisting of the following.

[0021] In some embodiments, the disease is a neuromuscular disease, preferably a genetic neuromuscular disease. Preferably, the disease is a neuromuscular disease affecting the nervous system, preferably a genetic neuromuscular disease affecting the nervous system.

[0022] In some embodiments, hereditary neuromuscular disorders are selected from the group including: (i) myopathy, such as hereditary cardiomyopathy, metabolic myopathy, other myopathy, distal myopathy, muscular dystrophy and congenital myopathy; and (ii) spinal muscular atrophy (SMA) and motor neuron disease; preferably congenital myopathy and muscular dystrophy, and spinal muscular atrophy (SMA) and motor neuron disease.

[0023] In some embodiments, the gene of interest for therapeutic purposes is a functional version of a gene causing a genetic neuromuscular disorder selected from the group including Duchenne muscular dystrophy, Becker muscular dystrophy, limb-girdle muscular dystrophy, myotonic dystrophy, myotubular myopathy, central nucleus myopathy, nemaline myopathy, selenoprotein N-related myopathy, Pompe disease, glycogen storage disease III, spinal muscular atrophy, and amyotrophic lateral sclerosis, or a therapeutic RNA that targets the said gene causing the disease.

[0024] In some embodiments, the genes causing genetic neuromuscular disorders are selected from the group including the following genes: DMD, CAPN3, DYSF, FKRP, ANO5, MTM1, DNM2, BIN1, ACTA1, KLHL40, KLHL41, KBTBD13, TPM3, TPM2, TNNT1, CFL2, LMOD3, SEPN1, GAA, AGL, SMN1, and ASAH1.

[0025] In some preferred embodiments, hereditary neuromuscular disorders are selected from the group including: (i) myopathy, such as muscular dystrophy including congenital muscular dystrophy; (ii) spinal muscular atrophy (SMA) and motor neuron disorders; (iii) myotonic syndromes, particularly type 1 and type 2 myotonic dystrophy; (iv) hereditary sensorimotor neuropathy; (v) hereditary paraplegia and hereditary ataxia; (vi) congenital myasthenic syndrome; preferably congenital myasthenic syndrome, muscular dystrophy including congenital muscular dystrophy; and spinal muscular atrophy (SMA) and motor neuron disorders.

[0026] In some preferred embodiments, the genes of interest for therapeutic purposes include: Duchenne muscular dystrophy and Becker muscular dystrophy (DMD gene); limb-girdle muscular dystrophy (DYSF, FKRP gene); type 1 (DMPK gene) and type 2 (CNBP / ZNF9 gene) myotonic dystrophy, central nuclear myopathy (DNM2, BIN1 gene), Pompe disease (GAA gene); glycogen storage disease III (AGL gene); spinal muscular atrophy (SMN1, ASAH1 gene); amyotrophic lateral sclerosis (SOD1, ALS2, SETX, FUS, ANG, TARDBP, FIG4, OPTN, etc.); hereditary paraplegia (SPAST A functional version of a gene causing a genetic neuromuscular disorder affecting the nervous system, selected from the group including (SPG4), SPG7 and other SPG genes (e.g., SPG11, SPG20 and SPG21), Charcot-Marie-Tooth, type 4B1 (MTMR2), and congenital myasthenic syndrome (CHAT, AGRN), or therapeutic RNA that targets such disease-causing genes.

[0027] In some more preferred embodiments, the genes causing genetic neuromuscular disorders affecting the nervous system are selected from the group including the DMD, DYSF, FKRP, DNM2, BIN1, GAA, AGL, SMN1, and ASAH1 genes.

[0028] In other more preferred embodiments, the genes causing genetic neuromuscular disorders affecting the nervous system are selected from the group including: FKTN, POMT1, POMT2, POMGNT1, POMGNT2, LMNA, ISPD, GMPPB, LARGE, LAMA2, TRIM32, and B3GALNT2 genes.

[0029] In some embodiments, the recombinant porcine AAV vector is intended for use in gene therapy for spinal muscular atrophy, and the vector comprises a peptide-modified capsid protein containing a sequence having at least 95%, 96%, 97%, 98%, or 99% identity to the sequence of SEQ ID NO: 5 or any one of the peptides SEQ ID NOs from 2 to 4, and the vector further packages a human SMN1 gene operably linked to a promoter that is functional in neurons and / or glial cells.

[0030] In some embodiments, the recombinant porcine AAV vector according to this disclosure is administered systematically, preferably intravenously.

[0031] In some embodiments, the recombinant porcine AAV vectors according to this disclosure are used in methods for treating neuromuscular diseases. [Modes for carrying out the invention]

[0032] Peptide-modified AAVpo1 vector This invention relates to a recombinant adeno-associated virus vector containing peptide-modified porcine AAV serotype 1 capsid protein for use in gene therapy for muscle and nervous system disorders, such as muscle and central nervous system (CNS) disorders. Recombinant adeno-associated virus vectors containing peptide-modified porcine AAV serotype 1 capsid protein can be used in gene therapy for diseases affecting only the nervous system (PNS and / or CNS) or both the nervous system and muscles. These include, in particular, CNS diseases and neuromuscular diseases.

[0033] Porcine AAV serotype 1 (AAVpo1) vectors comprising peptide-modified capsid proteins (or peptide-modified AAVpo1 vectors) for use according to the present invention combine detargeting from off-target organs, particularly the liver, with high transgene expression levels in target organs (i.e., the nervous system, e.g., CNS; or the muscle and nervous system, e.g., muscle and CNS).

[0034] As used herein, the term “detargeting” refers to a reduction of vector transduction and transgene expression in off-target organs to a minimum level, preferably as close as possible to the detection limit. Peptide-modified AAVpo1 vectors according to this disclosure that are detargeted at the transduction level favorably contain at least one-tenth the number of vector genome copies per diploid genome compared to AAV8 and AAV9 vectors after systemic administration at the same dose. Peptide-modified AAVpo1 vectors according to this disclosure that are detargeted at the transgene expression level favorably contain vector-derived protein levels lower than the endogenous levels of the protein when using a vector expressing a human transgene.

[0035] As used herein, the term “muscle” refers to cardiac muscle (i.e., heart) and skeletal muscle.

[0036] As used herein, the term "muscle cell" refers to muscle cells, myotubes, myoblasts, and / or satellite cells.

[0037] As used herein, the term “nervous system” refers to both the central (CNS) and peripheral (PNS) nervous systems.

[0038] As used herein, the term “central nervous system or CNS” means the brain, spinal cord, retina, cochlea, optic nerve, and / or olfactory nerve and epithelium. As used herein, the term “CNS cells” means any cells of the CNS, including neurons and glial cells (oligodendrocytes, astrocytes, ependymal cells, microglia).

[0039] As used herein, PNS refers to nerves and ganglia outside the brain and spinal cord.

[0040] As used herein, the term "systemic administration" refers to the route of administration of a substance (vector) into the circulatory system, including enteral or parenteral administration. Parenteral administration includes injection, infusion, and implantation.

[0041] As used herein, the term "AAV vector" refers to an AAV vector particle.

[0042] As used herein, the terms “porcine AAV vector” or “AAVpo1 vector” refer to an AAV vector containing porcine AAV serotype 1 capsid protein.

[0043] As used herein, the term AAV serotype includes natural and artificial AAV serotypes, such as variants and hybrid capsids derived from natural AAV serotypes. An AAV serotype refers to a functional AAV capsid capable of transducing a target organ and expressing a transgene in the target organ.

[0044] As used herein, “for use in gene therapy for muscular and nervous system disorders, such as muscular and central nervous system (CNS) disorders” means “for use in gene therapy for the treatment of muscular and nervous system disorders, such as muscular and central nervous system (CNS) disorders” or “for use in gene therapy for the treatment of muscular and nervous system disorders, such as muscular and central nervous system (CNS) disorders.”

[0045] As used herein, "or" means "and / or".

[0046] Neuromuscular disorders (NMDs) are a very broad term encompassing a wide range of conditions that impair muscle function, whether directly affecting voluntary muscles or indirectly affecting the peripheral nervous system or neuromuscular junctions. Neuromuscular diseases broadly define a group of disorders involving damage or dysfunction of peripheral nerves, muscles, or neuromuscular junctions. The site of damage may be in the cell body (i.e., amyotrophic lateral sclerosis [ALS] or sensory ganglion disorders), axons (i.e., axonal peripheral neuropathy or brachial plexus disorders), Schwann cells (i.e., chronic inflammatory demyelinating polyradiculomyelopathy), neuromuscular junctions (i.e., myasthenia gravis or Lambert-Eaton myasthenia gravis syndrome), muscles (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.

[0047] As used herein, “neuromuscular disease or disorder affecting the nervous system” means a neuromuscular disease including nervous system injury. A neuromuscular disease may further include, for example, muscle injury as a result of primary nervous system injury.

[0048] In some embodiments, the peptide-modified AAVpo1 vector for use according to the present invention, after systemic administration, is simultaneously detargeted from the liver while resulting in high transgene expression levels in target organs (i.e., the nervous system, e.g., CNS; or the muscle and nervous system, e.g., muscle and CNS). Transduction (vector copy number) in the nervous system, e.g., CNS; or the muscle and nervous system, e.g., muscle and CNS) is advantageously increased using the peptide-modified AAVpo1 vector compared to a control AAVpo1 vector containing an unmodified capsid. Transgene expression levels in the muscle and nervous system, e.g., muscle and CNS, using the peptide-modified AAVpo1 vector are preferably at least 2-fold, preferably 3, 4, or 5-fold or more, in muscle, particularly skeletal muscle, and the central nervous system, compared to a control AAVpo1 vector containing an unmodified capsid. In various muscle types and in the nervous system, for example, in various muscle types and in the CNS, the transgene expression levels achieved using the peptide-modified AAVpo1 vector are preferably at least the same as (more than two-thirds; i.e., comparable) those of the AAV8, AAV9, and AAVrh10 vectors.

[0049] Vector transduction and transgene expression are well known in the art and are determined by systemic administration of peptide-modified AAVpo1 vectors in animal models, such as the mouse models disclosed in the examples of this application. AAVpo1 vectors commonly used for muscle transduction, including unmodified capsids and the best AAV vector serotypes (AAV2, AAV8, AAV9, AAVrh10, etc.), are advantageously used for comparison. Vector transduction can be determined by measuring the vector genome copy number per diploid genome by standard assays well known in the art, such as the real-time PCR assay disclosed in the examples of this application. Transgene expression is measured at the mRNA or protein level by standard assays well known in the art, such as the quantitative RT-PCR assay and quantitative Western blot analysis disclosed in the examples of this application.

[0050] In some embodiments, the peptide-modified AAVpo1 vector for use according to the present invention is detargeted from the liver and at least one other non-target organ, such as the spleen.

[0051] In some embodiments, the peptide-modified AAVpo1 vector for use according to the present invention favorably yields high transgene expression levels in various muscle groups, preferably including the major muscle groups, after systemic administration, particularly intravenous administration. The major skeletal muscle groups that make up the upper body of a human are the abdomen, pectoralis, deltoid, trapezius, latissimus dorsi, erector spinae, biceps, triceps, and diaphragm. The major skeletal muscle groups of the lower body of a human are the quadriceps, flexor thigh, gastrocnemius, soleus, and gluteal muscles. The muscles of the anterior part of the lower leg are the tibialis anterior, extensor digitorum longus, extensor hallucis longus, peroneus longus, peroneus brevis, and peroneus tertius. The ability of the peptide-modified AAVpo1 vector to deliver high transgene expression levels in various muscle groups after systemic administration is illustrated in the example of this application (Figure 5), which shows high transgene expression levels in the tibia (TA), extensor digitorum longus (EDL), quadriceps (Qua), gastrocnemius (Ga), soleus (Sol), triceps, biceps, and diaphragm of mice intravenously injected with the peptide-modified AAVpo1 vector.

[0052] In some embodiments, the peptide-modified AAVpo1 vector for use according to the present invention is characterized by a combination of hepatic detargeting after systemic administration, particularly intravenous administration, and transgene expression levels in various muscle groups, as well as in the brain and spinal cord, which are at least equivalent, if not superior, to those of the AAV9 vector.

[0053] AAVpo1 (GenBank accession number FJ688147, accessed July 24, 2016) contains the Cap gene at positions 780 to 2930 of the partial viral genome sequence (2977 bp): VP1 CDS is at positions 780 to 2930; VP2 CDS is at positions 1188 to 2930; and VP3 CDS is at positions 1329 to 2930. The AAVpo1 capsid protein (VP1) has the sequence of GenBank accession number ACN42940.1, accessed July 24, 2016, or Sequence ID No. 1. Examples of hybrid vectors include vectors containing the AAVpo1 capsid and the AAV2 rep protein and / or AAV2 ITR. AAVpo1 serotypes include the natural AAVpo1 serotypes listed above and any artificial variants or hybrids derived from said serotypes. The present invention encompasses the use of peptide-modified AAVpo1 vectors derived from AAV capsid sequences having at least 95%, 96%, 97%, 98%, or 99% identity to the AAVpo1 capsid sequences listed above.

[0054] In some embodiments, the peptide-modified AAVpo1 capsid protein is derived from an AAV capsid sequence having at least 95%, 96%, 97%, 98%, or 99% identity to the sequence of SEQ ID NO: 1.

[0055] The term "identity" refers to the sequence similarity between two polypeptide molecules or two nucleic acid molecules. If one position in both sequences being compared is occupied by the same base or amino acid residue, then the molecules are identical at that position. The percentage of identity between two sequences is calculated by dividing the number of matching positions shared by the two sequences by the number of positions being compared and multiplying by 100. Generally, comparisons are performed when the two sequences are aligned to yield the greatest possible identity. Identity can be calculated, for example, by alignment using the GCG (Genetics Computer Group, Program Manual for the GCG Package, Version 7, Madison, Wisconsin) pile-up program, or any sequence comparison algorithm, such as BLAST, FASTA, or CLUSTALW.

[0056] The peptide is preferably up to 30 amino acids. In some preferred embodiments, the peptide is up to 25, 20, or 15 amino acids (i.e., 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, or 15 amino acids).

[0057] In some embodiments, the peptide, preferably up to 30 amino acids, comprises or consists of the sequence MPLGAAG (SEQ ID NO: 2) or a variant containing one or two amino acid mutations (insertions, deletions, or substitutions) in the sequence, preferably one or two amino acid substitutions. In some preferred embodiments, the peptide comprises or consists of the sequence GMPLGAAGA (SEQ ID NO: 3) or a variant containing up to four (1, 2, 3, or 4) amino acid mutations (insertions, deletions, or substitutions) in the sequence, preferably one or two amino acid deletions or substitutions; the deletions are preferably located at the N and / or C terminus. In some preferred embodiments, the sequence of SEQ ID NO: 2 or 3 or its variant as defined above has up to five (1, 2, 3, 4, or 5) or more amino acids at its N and / or C terminus, for example, GQR and QAA at their N and C terminus. Alternatively, the adjacent sequences may contain or consist of alanine (A) residues. In some more preferred embodiments, the peptide comprises or consists of the sequence GQRGMPLGAAGAQAA (SEQ ID NO: 4).

[0058] The peptide-modified AAVpo1 capsid protein contains at least one copy of the peptide inserted into the AAVpo1 capsid protein. Depending on the insertion site, the peptide may be inserted into VP1, VP1 and VP2, or VP1, VP2 and VP3. The peptide-modified AAVpo1 capsid protein may contain up to five copies of the peptide, preferably one copy of the peptide.

[0059] The peptide-modified AAVpo1 capsid protein according to the present invention contains one or more peptides inserted into a site exposed on the surface of the AAV capsid. Sites on the AAV capsid that are exposed on the capsid surface and allow peptide insertion, i.e., do not affect the assembly and packaging of the viral capsid, are well known in the art and include, for example, AAV capsid surface loops or antigenic loops (Girod et al., Nat. Med., 1999, pp. 5, 1052-1056; Grifman et al., Molecular Therapy, 2001, pp. 3, 964-975); other sites are disclosed in Rabinowitz et al., Virology, 1999, pp. 265, 274-285; Wu et al., J. Virol., 2000, pp. 74, 8635-8647.

[0060] In particular, at least one peptide is inserted at positions N567, S568, N569, and T570 of the capsid protein, according to the numbering in SEQ ID NO: 1, preferably between positions N567 and S568 or between positions N569 and T570. The insertion of the peptide may or may not result in the deletion of some residues preceding and / or following the peptide insertion site, preferably one to three (1, 2, or 3) of said residues. In some embodiments, the peptide is inserted between positions N567 and S568, replacing all residues at positions 565-567 and 568-570. In some other embodiments, the peptide is inserted between positions N569 and T570, replacing all residues at positions 567-569 and 570-572. The position is indicated by reference to the AAVpo1 capsid protein of SEQ ID NO: 1; those skilled in the art can easily find the corresponding position in another AAVpo1 capsid protein sequence after alignment using SEQ ID NO: 1.

[0061] In some preferred embodiments, the peptide-modified AAVpo1 capsid protein includes a sequence selected from the group consisting of the sequence of SEQ ID NO: 5 and a sequence having at least 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 5 containing the peptide according to this disclosure, and a fragment thereof corresponding to a VP2 or VP3 capsid protein. VP2 corresponds to the amino acid sequence of SEQ ID NO: 5 from K136 to the terminal. VP3 corresponds to the amino acid sequence of SEQ ID NO: 5 from M184 to the terminal. In some preferred embodiments, the peptide-modified AAVpo1 capsid protein includes the sequence of SEQ ID NO: 5 or a fragment thereof corresponding to a VP2 or VP3 capsid protein.

[0062] The present invention also includes AAVpo1 VP1 and VP2 chimeric capsid proteins derived from the peptide-modified AAVpo1 VP3 capsid protein according to the present disclosure, wherein the VP1-specific N-terminal region and / or VP2-specific N-terminal region are derived from another natural or artificial AAV serotype, preferably another AAVpo serotype selected from known AAVpo serotypes, particularly the AAVpo2.1 serotype. The present invention further includes mosaic peptide-modified AAVpo1 vectors, wherein the vector particles further comprise another AAV capsid protein derived from another natural or artificial AAV serotype, preferably another AAVpo serotype selected from known AAVpo serotypes, particularly the AAVpo2.1 serotype according to the present disclosure.

[0063] The genome of the peptide-modified AAVpo1 vector may be either single-stranded or self-complementary double-stranded (McCarty et al., Gene Therapy, December 2003, 10(26), pp. 2112-2118). Self-complementary vectors are generated by deleting a terminal resolution site (trs) from one of the AAV terminal repeats. These modified vectors, whose replicated genome is half the length of the wild-type AAV genome, tend to package DNA dimers. The AAV genome is adjacent to the ITR. In specific embodiments, the AAV vector is a pseudotype vector, i.e., its genome and capsid are derived from different serotypes of AAV. In some preferred embodiments, the genome of the pseudotype vector is derived from AAV2.

[0064] The peptide-modified AAVpo1 vector for use according to this disclosure is produced by standard methods for producing AAV vectors that are well known in the art (review in Aponte-Ubillus et al., Applied Microbiology and Biotechnology, 2018, 102: pp. 1045-1054). Briefly, after co-transfection using an expression plasmid for the AAV Rep and capsid protein, and a plasmid containing a recombinant AAV vector genome with the gene of interest inserted adjacent to the AAV ITR in the expression cassette, in the presence of sufficient helper function to enable packaging of the rAAV vector genome into AAV capsid particles, the cells are incubated for a sufficient time to enable the production of AAV vector particles, the cells are then collected and lysed, and the AAV vector particles are purified by standard purification methods such as affinity chromatography or ultracentrifugation with an iodixanol or cesium chloride density gradient.

[0065] Peptide-modified AAVpo1 vector particles typically package genes of therapeutic interest. “Genes of therapeutic interest,” “genes of therapeutic interest,” “target gene,” or “target heterogene” refers to genes encoding therapeutic genes or therapeutic proteins, peptides, or RNA. These therapeutic genes may be used in combination with genome editing enzymes.

[0066] The gene of interest is any nucleic acid sequence that can alter a target gene or target cellular pathway in cells of a target organ (i.e., the nervous system, e.g., CNS; or muscle and / or nervous system, e.g., muscle and CNS). Depending on the type of disease, the target organ may essentially include the nervous system, e.g., CNS, or may further include muscle. In some specific embodiments, the target organ includes at least the nervous system, e.g., CNS. In some preferred embodiments, the target organ includes the nervous system and muscle, e.g., CNS and muscle. For example, the gene can alter the expression, sequence, or control of a target gene or cellular pathway. In some embodiments, the gene of interest is a functional version of a gene or a fragment thereof. Examples of functional versions of the gene include wild-type genes, variant genes such as variants belonging to the same family that at least partially preserve the function of the encoded protein, or truncated versions. Functional versions of genes are useful for substitution or additive gene therapy, such as replacing a gene that is deficient or non-functional in a patient. In other embodiments, the gene of interest is a gene that inactivates a dominant allele causing an autosomal dominant genetic disorder. Gene fragments are useful as recombinant templates for use in combination with genome editing enzymes.

[0067] Alternatively, the gene of interest may encode a protein of interest for a specific use (e.g., an antibody or antibody fragment, a genome editing enzyme) or RNA. In some embodiments, the protein is a therapeutic protein containing a therapeutic antibody or antibody fragment, or a genome editing enzyme. In some embodiments, the RNA is therapeutic RNA.

[0068] In some embodiments, the sequence of the gene of interest is optimized for expression in the individual being treated, preferably a human individual. Sequence optimization may involve a number of changes in the nucleic acid sequence, including codon optimization, increased GC content, decreased number of CpG islands, decreased number of alternative translation regions (ARFs), and / or decreased number of splice donor and splice acceptor sites.

[0069] The gene of interest is a functional gene capable of producing an encoded protein, peptide, or RNA in disease target cells, particularly muscle cells and cells of the nervous system (CNS and / or PNS). Depending on the type of disease, the target cells may essentially include nervous system cells such as CNS cells, and may further include muscle cells. In some specific embodiments, the disease target cells include at least nervous system cells such as CNS cells. In some preferred embodiments, the disease target cells include nervous system cells and muscle cells, e.g., CNS cells and muscle cells. In some embodiments, the gene of interest is a human gene. The peptide-modified AAVpo1 vector contains the gene of interest in a form expressible in cells of the target organ (i.e., the nervous system, e.g., CNS; or muscle and / or nervous system, e.g., muscle and / or CNS). In some specific embodiments, the gene of interest is in a form expressible in at least nervous system cells, e.g., CNS cells. In some preferred embodiments, the gene of interest is in a form expressible in nervous system cells and muscle cells, e.g., CNS cells and muscle cells. In particular, the gene of interest is operably ligated to a regulatory sequence appropriate for the expression of the transgene in target cells, tissues, or organs of an individual. Such sequences, well known in the art, include, in particular, promoters, and more not limited to, enhancers, terminators, introns, silencers, and especially tissue-specific silencers, which are regulatory sequences that can further regulate the expression of the transgene, as well as microRNAs. The gene of interest is operably ligated to a ubiquitous, tissue-specific, or inducible promoter that is functional in cells of a target organ (i.e., muscle and / or nervous system, e.g., muscle and / or CNS). In some specific embodiments, the target organ includes at least the nervous system, e.g., CNS. In some preferred embodiments, the target organ includes the nervous system and muscle, e.g., CNS and muscle. In some specific embodiments, the gene of interest is operably ligated to a ubiquitous, tissue-specific, or inducible promoter that is functional in nervous system cells, e.g., neurons and / or glial cells; or in nervous system cells, e.g., neurons and / or glial cells and muscle cells.In some specific embodiments, the gene of interest is operably ligated to at least two promoters, at least one of which is a neuron and / or glial cell-specific or inducible promoter that is functional in neurons and / or glial cells. In some specific embodiments, the gene of interest is operably ligated to at least two promoters, one of which is a neuron and / or glial cell-specific or inducible promoter that is functional in neurons and / or glial cells, and the other is a muscle-specific or inducible promoter that is functional in muscle cells.

[0070] The target gene may be inserted into an expression cassette further comprising the additional regulatory sequences disclosed above. Examples of ubiquitous promoters include the CAG promoter, phosphoglycerate kinase 1 (PGK) promoter, cytomegalovirus enhancer / promoter (CMV), SV40 initial promoter, retroviral Roussarcoma virus (RSV) LTR promoter, didihydrofolate reductase promoter, β-actin promoter, and EF1 promoter.

[0071] Examples of muscle-specific promoters, without limitation, include the desmin (Des) promoter, muscle creatine kinase (MCK) promoter, CK6 promoter, alpha-myosin heavy chain (alpha-MHC) promoter, myosin light chain 2 (MLC-2) promoter, cardiac troponin C (cTnC) promoter, synthetic muscle-specific SpC5-12 promoter, and human skeletal actin (HSA) promoter.

[0072] Promoter codes for the nervous system, such as CNS expression, include promoters that drive ubiquitous expression and promoters that drive expression in neurons. Representative promoters that drive ubiquitous expression, non-limitingly, include: CAG promoter (cytomegalovirus enhancer / chicken beta-actin promoter, including the first exon and first intron of the chicken beta-actin gene and the splice receptor of the rabbit beta-globin gene); PGK (phosphoglycerate kinase 1) promoter; β-actin promoter; EF1a promoter; CMV promoter. Representative promoters that drive expression in neurons, non-limitingly, include calcitonin gene-related peptide (CGRP) and promoters of known motor neuron-derived factors. Other neuron-selective promoters include ubiquitous promoters, including choline acetyltransferase (ChAT), neuron-specific enolase (NSE), synapsin, Hb9 promoters, and neuron-specific silencer element (NRSE) promoters. A representative promoter that drives selective expression in glial cells is the promoter of the glial filament acid protein gene (GFAP).

[0073] For expression in muscle cells (skeletal and cardiomyocytes), the gene of interest is favorably regulated by the desmin promoter, particularly the human desmin promoter (Raguz et al., Dev. Biol., 1998, pp. 201, 26-42; Paulin D & Li Z, Exp. Cell. Res., November 15, 2004; 301(1): pp. 1-7). For expression in skeletal muscle cells, the gene of interest is favorably regulated by the desmin promoter, particularly the human desmin promoter, and further includes the miR208a target sequence that suppresses expression in cardiomyocytes (i.e., in the heart; Roudault et al., Circulation, 2013, pp. 128, 1094-104. doi: 10.1161 / CIRCULATIONAHA.113.001340).

[0074] RNA is advantageously complementary to the target DNA or RNA sequence, or binds to the target protein. For example, RNA can be interfering RNA, such as shRNA, microRNA, guide RNA (gRNA) for use in combination with Cas enzymes or similar enzymes for genome editing, exon-skipping antisense RNA, such as modified nuclear small RNA (snRNA), or long non-coding RNA. Interfering RNA or microRNA may be used to control the expression of target genes involved in muscular or nervous system diseases, such as muscular or CNS diseases. In some embodiments, the disease is a nervous system disease, such as a CNS disease. In these embodiments, the target gene is located in nervous system cells, such as CNS and / or PNS cells, particularly neurons and / or glial cells. In some other embodiments, the disease is a nervous system and muscular disease, such as CNS and muscular diseases. In other embodiments, the target gene is located in at least nervous system cells, e.g., CNS and / or PNS cells, particularly neurons and / or glial cells; the target gene may be located in essentially nervous system cells, e.g., CNS and / or PNS cells, particularly neurons and / or glial cells; or it may be located in nervous system cells and muscle cells, e.g., CNS and / or PNS cells, particularly neurons and / or glial cells, as well as muscle cells. Guide RNA in a complex containing a Cas enzyme or a similar enzyme for genome editing may be used to alter the sequence of the target gene, in particular to correct the sequence of a mutated / deficient gene, or to alter the expression of a target gene involved in a disease, particularly a muscle or nervous system disorder, e.g., a muscle or central nervous system (CNS) disorder. Exon-skipping antisense RNA is used in particular to correct the reading frame and restore the expression of a deficient gene whose reading frame has been disrupted. In some embodiments, the RNA is therapeutic RNA.

[0075] The genome editing enzymes according to the present invention are any enzymes or enzyme complexes capable of altering a target gene or target cellular pathway, particularly in muscle cells and / or nervous system cells, e.g., muscle cells and / or CNS cells. In some embodiments, the target gene is located in at least nervous system cells, e.g., CNS and / or PNS cells, particularly including neurons and / or glial cells; the target gene may be located in essentially nervous system cells, e.g., CNS and / or PNS cells, particularly including neurons and / or glial cells; or it may be located in nervous system cells and muscle cells, e.g., CNS and / or PNS cells, particularly including neurons and / or glial cells, as well as muscle cells. In some specific embodiments, the target gene is located in nervous system cells, e.g., CNS and / or PNS cells, particularly including neurons and / or glial cells. In some other specific embodiments, the target gene may be located in nervous system cells and muscle cells, e.g., CNS and / or PNS cells, particularly including neurons and / or glial cells, as well as muscle cells. For example, the genome editing enzyme can alter the expression, sequence, or control of a target gene or cellular pathway. Genome editing enzymes are, advantageously, non-limiting engineered nucleases such as meganucleases, zinc finger nucleases (ZFNs), transcription activator-like effector-based nucleases (TALENs), Cas enzymes derived from clustered and regularly arranged palindromic sequence repeats (CRISPR)-Cas systems, and similar enzymes. Genome editing enzymes, particularly engineered nucleases such as Cas enzymes and similar enzymes, can be functional nucleases used for site-directed genome editing applications, including, but not limited to, gene modification, gene substitution, gene knock-in, gene knock-out, mutation introduction, chromosome translocation, and chromosome deletion, by inducing double-strand breaks (DSBs) or single-strand breaks (nickase, e.g., Cas9(D10A)) at target genomic loci. For site-directed genome editing applications, engineered nucleases such as Cas enzymes and similar enzymes can be used in combination with homologous recombination (HR) matrices or templates (also known as DNA donor templates) that modify target genomic loci by double-strand break (DSB)-induced homologous recombination.In particular, the HR template can introduce the desired transgene into a target genomic locus, or, preferably, repair mutations in the target genomic locus in abnormal or deficient genes causing muscle or central nervous system (CNS) disorders. In some embodiments, the disease is a nervous system disorder, e.g., a CNS disorder. In some other embodiments, the disease is a nervous system and muscle disorder, e.g., a CNS and muscle disorder. Alternatively, genome editing enzymes such as Cas enzymes and similar enzymes may be engineered to be nuclease-deficient and can be used as DNA-binding proteins for various genome engineering purposes, including but not limited to: transcriptional activation, transcriptional repression, epigenetic modification, genome imaging, and DNA or RNA pulldown.

[0076] In some embodiments, peptide-modified AAVpo1 vector particles packaging the target gene for therapeutic purposes target skeletal muscle cells and / or neurons.

[0077] An example of a preferred vector for use according to the present invention is an AAVpo1 vector comprising a peptide-modified capsid protein comprising a sequence having at least 95%, 96%, 97%, 98%, or 99% identity to the sequence of SEQ ID NO: 5 or any one of the peptides SEQ ID NOs from 2 to 4, wherein the vector further packages a gene of interest for therapeutic purposes operably linked to a desmin promoter, preferably a human desmin promoter, and ultimately an operable linker to a miR208a target sequence. This first vector is useful for systemic, particularly intravascular, administration of the gene of interest, to express only in muscle (skeleton and heart; expression cassette without the miR208a target sequence) or skeletal muscle (expression cassette with the miR208a target sequence), but not in the liver.

[0078] Another example of a preferred vector for use according to the present invention is an AAVpo1 vector comprising a peptide-modified capsid protein comprising a sequence having at least 95%, 96%, 97%, 98%, or 99% identity to the sequence comprising the sequence of SEQ ID NO: 5 or any one of the peptides SEQ ID NOs: 2 to 4, wherein the vector further packages a gene of interest for therapeutic purposes operably linked to a CAG promoter, preferably further comprising a human betaglobin polyadenylation signal. This second vector is useful for systemic, particularly intravascular, administration of the gene of interest to prevent its expression in the liver, but in the muscles, including the heart, and in the nervous system, for example, in the muscles, including the heart, and in the CNS.

[0079] Another preferred vector for use according to the present invention is an AAVpo1 vector comprising a peptide-modified capsid protein comprising a sequence having at least 95%, 96%, 97%, 98%, or 99% identity to the sequence of SEQ ID NO: 5 or any one of the peptides SEQ ID NOs: 2 to 4, wherein the vector further packages a gene of interest for therapeutic purposes operably linked to a promoter functional in neurons and / or glial cells. The promoter may be a tissue-specific promoter such as a ubiquitous promoter, e.g., CAG, or an inductive promoter functional in neurons and / or glial cells. In some specific embodiments, the promoter is a neuron and / or glial cell-specific or inductive promoter functional in neurons and / or glial cells. This third vector is useful for expressing the gene of interest in the nervous system but not in the liver after systemic, particularly intravascular, administration.

[0080] Another example of a preferred vector for use according to the present invention is an AAVpo1 vector comprising a peptide-modified capsid protein comprising a sequence having at least 95%, 96%, 97%, 98%, or 99% identity to the sequence comprising the sequence of SEQ ID NO: 5 or any one of the peptides SEQ ID NOs: 2 to 4, wherein the vector further packages a gene of interest for therapeutic purposes operably linked to a promoter or combination of promoters that is functional in muscle cells and in neurons and / or glial cells. This fourth vector is useful for systemic, particularly after intravascular administration, to express the gene of interest in the muscle, including the heart, and in the nervous system, for example, in the muscle, including the heart, and in the CNS, without expressing it in the liver. The promoter may be a ubiquitous promoter, such as CAG, a tissue-specific promoter or an inducible promoter, or a combination of promoters comprising a first promoter functional in muscle cells and a second promoter functional in neurons and / or glial cells. In some specific embodiments, the gene of interest is operably linked to at least two promoters, one of which is a neuron and / or glial cell-specific or inducible promoter functional in neurons and / or glial cells, and the other is a muscle-specific or inducible promoter functional in muscle cells.

[0081] Gene therapy for muscle and nervous system disorders, such as muscle and CNS disorders. The peptide-modified AAVpo1 vectors according to this disclosure are used in gene therapy for muscle and / or nervous system diseases or disorders, such as muscle and / or CNS diseases or disorders. In some embodiments, the peptide-modified AAVpo1 vectors according to this disclosure are used in gene therapy for diseases affecting the nervous system, e.g., the CNS, where the disease may essentially affect the nervous system, e.g., the CNS, or affect the nervous system and muscles, e.g., the CNS and muscles. For example, a disease may initially affect the nervous system, and primary damage to the nervous system may result in secondary damage to the muscles. In some specific embodiments, the peptide-modified AAVpo1 vectors according to this disclosure are used in gene therapy for nervous system diseases, particularly CNS diseases. In some other specific embodiments, the peptide-modified AAVpo1 vectors according to this disclosure are used in gene therapy for nervous system and muscle diseases, e.g., CNS and muscle diseases, particularly neuromuscular diseases affecting at least the nervous system (CNS and / or PNS).

[0082] The peptide-modified AAVpo1 vectors according to this disclosure are preferably used in the form of peptide-modified AAVpo1 vector particles, preferably in the form of a pharmaceutical composition comprising a therapeutically effective amount of peptide-modified AAVpo1 vector particles packaging the therapeutic gene of interest according to this disclosure.

[0083] Gene therapy may be carried out by gene transfer, gene editing, exon skipping, RNA interference, trans-splicing, or any other genetic modification of any coding or regulatory sequence in cells, including, but not limited to, symbiotic nucleic acids such as viral sequences contained in the nucleus, midchondria, or cells.

[0084] The two main types of gene therapy are as follows: - Therapies aimed at providing functional replacement genes for deficient / abnormal genes: This is replacement or additive gene therapy; - Therapies aimed at gene or genome editing: In such cases, the goal is to provide cells with the tools necessary to modify the sequence or alter the expression or control of a deletion / abnormal gene so that a functional gene is expressed or an abnormal gene is suppressed (inactivated): This is gene editing therapy.

[0085] In additive gene therapy, the target gene may be a functional version of a gene that is missing or mutated in the patient, for example, in the case of a genetic disorder. In this case, the target gene restores the expression of the functional gene.

[0086] Gene or genome editing is: (i) Therapeutic RNA as defined above, e.g., interfering RNA such as shRNA or microRNA, guide RNA (gRNA) for use in combination with Cas enzyme or similar enzyme, or antisense RNA that can be exon-skipped, e.g., genes encoding modified nuclear small RNA (snRNA); and (ii) genes encoding genome editing enzymes as defined above, for example, meganucleases, zinc finger nucleases (ZFNs), transcription activator-like effector-based nucleases (TALENs), Cas enzymes or similar enzymes, or combinations of such genes, and fragments of functional versions of genes for use as recombinant templates as defined above. Use one or more target genes, such as those mentioned above.

[0087] Gene therapy is used to treat a variety of hereditary or acquired diseases or disorders affecting the structure or function of the muscle and / or nervous system, including, for example, the skeleton or cardiac muscle, brain or spinal cord. Diseases may result from trauma, infection, degeneration, structural or metabolic defects, tumors, autoimmune disorders, stroke, etc. In some embodiments, gene therapy is used to treat hereditary or acquired diseases or disorders affecting the structure or function of at least the nervous system (PNS and / or CNS), particularly the CNS including the brain and / or spinal cord. Diseases essentially affect the nervous system (PNS and / or CNS), particularly the CNS including the brain and / or spinal cord, or affect the muscle including the skeleton and / or cardiac muscle. In some specific embodiments, the disease is a nervous system disease, particularly a CNS disease and / or PNS disease; a CNS disease may affect the brain and / or spinal cord. In some other specific embodiments, the disease is a disease of the nervous system (PNS and / or CNS) and the muscles, e.g., a disease of the CNS and muscles; the disease affects the nervous system, e.g., the brain and / or spinal cord, and further affects the muscles, e.g., the skeleton and / or myocardium. As used herein, diseases of the nervous system and muscles include diseases resulting from secondary muscle complications or injuries, particularly of the nervous system, particularly of the CNS, as a result of primary complications or injuries. Thus, diseases of the nervous system and muscles disclosed herein are different from muscle diseases, which are diseases characterized by primary muscle injury or complications.

[0088] In some embodiments, gene therapy is used to treat neurological disorders, particularly CNS disorders, and especially genetic neurological disorders. Examples of CNS disorders include Alzheimer's disease, Parkinson's disease, and frontotemporal dementia.

[0089] Examples of mutated genes in genetic neurological disorders that can be targeted by gene therapy using the pharmaceutical composition of the present invention are listed in the table below:

[0090] Genetic neurological disorders

[0091] [Table 1]

[0092] Other examples of mutated genes in hereditary neurological disorders that can be targeted by gene therapy using the pharmaceutical compositions of the present invention are the genes causing spinal muscular atrophy (SMA) and motor neuron diseases; hereditary sensorimotor neuropathy; hereditary paraplegia and hereditary ataxia; as listed in the table below. In some specific embodiments, the neurological disorders are selected from the group consisting of: spinal muscular atrophy (SMN1, ASAH1 genes); amyotrophic lateral sclerosis (SOD1, ALS2, SETX, FUS, ANG, TARDBP, FIG4, OPTN, etc.); hereditary paraplegia (SPAST (SPG4), SPG7 and other SPG genes, e.g., SPG11, SPG20 and SPG21; in particular SPAST (SPG4) and SPG7) and Charcot-Marie-Tooth, type 4B1 (MTMR2). In some preferred embodiments, the genes are selected from the group consisting of: SMN1, ASAH1, DNM2, MTMR2 and SPAST genes. In some other preferred embodiments, the gene is selected from the group consisting of SOD1, ALS2, SETX, FUS, ANG, TARDBP, FIG4, and OPTN.

[0093] In some embodiments, gene therapy is used to treat neuromuscular diseases in humans, particularly genetic neuromuscular disorders. Examples of mutated genes in genetic neuromuscular disorders, including genetic muscle disorders that can be targeted by gene therapy using the pharmaceutical compositions of the present invention, are listed in the following table:

[0094] Muscular dystrophy

[0095] [Table 2]

[0096] Congenital muscular dystrophy

[0097] [Table 3]

[0098] Congenital myopathy

[0099] [Table 4]

[0100] Distal myopathy

[0101] [Table 5]

[0102] Other myopathies

[0103] [Table 6]

[0104] myotonic syndrome

[0105] [Table 7]

[0106] Ion channel muscle disease

[0107] [Table 8]

[0108] Malignant hyperthermia

[0109] [Table 9]

[0110] Metabolic myopathy

[0111] [Table 10]

[0112] Hereditary cardiomyopathy

[0113] [Table 11A]

[0114] [Table 11B]

[0115] Congenital myasthenic syndrome

[0116] [Table 12]

[0117] Spinal muscular atrophy (SMA) and motor neuron diseases

[0118] [Table 13A]

[0119] [Table 13B]

[0120] Hereditary motor sensory neuropathy

[0121] [Table 14A]

[0122] [Table 14B]

[0123] Hereditary paraplegia

[0124] [Table 15A]

[0125] [Table 15B]

[0126] Other neuromuscular disorders

[0127] [Table 16]

[0128] Hereditary ataxia

[0129] [Table 17A]

[0130] [Table 17B]

[0131] Any of the genes listed above can be targeted in gene replacement therapy, where the gene of interest is a functional version of the deleted or mutated gene.

[0132] Alternatively, the genes listed above can be used as targets for gene editing. Gene editing is used to modify the sequence of mutated genes so that functional genes are expressed in muscle cells, or to alter the expression or control of deletion / abnormal genes. In such cases, the target gene is selected from those encoding therapeutic RNA, such as interfering RNA, guide RNA for genome editing, and antisense RNA that can be exon-skipped, where the therapeutic RNA targets the genes in the table above. Tools such as CRISPR / Cas9 can be used for this purpose.

[0133] Therefore, gene editing or gene substitution can deliver the correct version of this gene to the muscle cells and / or nervous system (PNS and / or CNS) cells of affected patients, particularly to the muscle cells and CNS cells of affected patients, which can contribute to an effective treatment of this disease.

[0134] In some embodiments, the target gene for gene therapy (additional gene therapy or gene editing) is a gene that causes one of the neuromuscular diseases selected from the group including: (i) myopathy, e.g., hereditary cardiomyopathy, metabolic myopathy, other myopathy, distal myopathy, muscular dystrophy and congenital myopathy; (ii) spinal muscular atrophy (SMA) and motor neuron disease; (iii) myotonic syndrome, in particular type 1 and type 2 myotonic dystrophy; congenital myasthenic syndrome; hereditary sensorimotor neuropathy; hereditary paraplegia and hereditary ataxia, in particular congenital myopathy and muscular dystrophy and spinal muscular atrophy (SMA) and motor neuron disease.

[0135] In some specific embodiments, the target genes for gene therapy (additional gene therapy or gene editing) are those listed above, preferably Duchenne muscular dystrophy and Becker muscular dystrophy (DMD genes), limb-girdle muscular dystrophy (LGMD) (e.g., CAPN3, DYSF, FKRP, ANO5 genes), spinal muscular atrophy (SMN1, ASAH1 genes) and amyotrophic lateral sclerosis (SOD1, ALS2, SETX, FUS, ANG, TARDBP, FIG4, OPTN genes), myotubular myopathy (MTM1 gene), and central nuclear myopathy (MTM1, DNM2, BIN1 gene). It is a gene that causes one of the neuromuscular diseases selected from a group that includes hereditary paraplegia (SPAST) and Charcot-Marie-Tooth, type 4B1 (MTMR2), nemaline myopathy (ACTA1, KLHL40, KLHL41, KBTBD13 genes), selenoprotein N-related myopathy (SEPN1 gene), congenital myasthenia gravis (ColQ, CHRNE, RAPSN, DOK7, MUSK genes), Pompe disease (GAA gene), glycogen storage disease III (GSD3) (AGL gene), type 1 (DMPK gene) and type 2 (CNBP / ZNF9 gene) myotonic dystrophy; hereditary paraplegia (SPAST) and Charcot-Marie-Tooth, type 4B1 (MTMR2). In some more preferred embodiments, the target gene is selected from the group consisting of the following genes: DMD, CAPN3, DYSF, FKRP, ANO5, MTM1, DNM2, BIN1, ACTA1, KLHL40, KLHL41, KBTBD13, TPM3, TPM2, TNNT1, CFL2, LMOD3, SEPN1, GAA, AGL, SMN1, and ASAH1.

[0136] In some preferred embodiments, the target gene for gene therapy (additional gene therapy or gene editing) is one of the genes listed above, preferably: (i) myopathy, e.g., muscular dystrophy including congenital muscular dystrophy; (ii) spinal muscular atrophy (SMA) and motor neuron disease; (iii) myotonic syndrome, particularly type 1 and type 2 myotonic dystrophy; (iv) hereditary sensorimotor neuropathy; (v) hereditary paraplegia and hereditary ataxia; (vi) congenital myasthenic syndrome, particularly a gene causing one of at least one neuromuscular disease affecting the nervous system, selected from the group including muscular dystrophy including congenital muscular dystrophy, congenital myasthenic syndrome, and spinal muscular atrophy (SMA) and motor neuron disease.

[0137] In some more preferred embodiments, the target gene for gene therapy is a gene that causes myopathy affecting at least the nervous system, such as a muscular dystrophy including congenital muscular dystrophy affecting at least the nervous system, and is selected from the group consisting of FKTN, POMT1, POMT2, POMGNT1, POMGNT2, LMNA, ISPD, GMPPB, LARGE, LAMA2, TRIM32, and B3GALNT2.

[0138] In some other, more preferred embodiments, the target gene for gene therapy is a gene that causes at least a nervous system-affecting myopathy, a congenital myasthenic syndrome, such as congenital myasthenia gravis, and is selected from the genes that cause congenital myasthenic syndrome listed in the table above.

[0139] In some other more preferred embodiments, the target genes for gene therapy (additional gene therapy or gene editing) are listed above, preferably Duchenne muscular dystrophy and Becker muscular dystrophy (DMD genes), limb-girdle muscular dystrophy (LGMD) (DYSF, FKRP), spinal muscular atrophy (SMN1, ASAH1 genes) and amyotrophic lateral sclerosis (SOD1, ALS2, SETX, FUS, ANG, TARDBP, FIG4, OPTN, etc.), central nuclear myopathy (DNM2, BIN1 genes), Pompe disease (GAA gene), and glycogen It is a gene that causes one of at least one neuromuscular disorders affecting the nervous system, selected from the group including disease III (GSD3) (AGL gene), type 1 (DMPK gene), and type 2 (CNBP / ZNF9 gene) myotonic dystrophy; hereditary paraplegia (SPAST (SPG4), SPG7, and other SPG genes, e.g., SPG11, SPG20, and SPG21; especially SPAST (SPG4) and SPG7); Charcot-Marie-Tooth, type 4B1 (MTMR2); and congenital myasthenic syndromes, e.g., congenital myasthenia gravis (CHAT, AGRN gene).

[0140] In some more preferred embodiments, the target gene is selected from the group consisting of the DMD, DYSF, FKRP, DNM2, BIN1, GAA, AGL, SMN1, and ASAH1 genes.

[0141] In some preferred embodiments, the peptide-modified AAVpo1 vector according to this disclosure is used to target motor neurons to treat motor neuron diseases. The target gene may be any one of the genes involved in spinal muscular atrophy (SMA) and motor neuron diseases listed in the table above. Motor neuron diseases include amyotrophic lateral sclerosis (ALS), progressive bulbar palsy (PBP), pseudobulbar palsy, progressive muscular atrophy (PMA), primary lateral sclerosis (PLS), spinal muscular atrophy (SMA), and monomelic atrophy (MMA), as well as several even rarer variants similar to ALS.

[0142] Dystrophin disorders are a spectrum of X-linked muscle diseases caused by pathogenic variants in the DMD gene, which encodes the protein dystrophin. Dystrophin disorders include Duchenne muscular dystrophy (DMD), Becker muscular dystrophy (BMD), and DMD-associated dilated cardiomyopathy.

[0143] Limb-girdle muscular dystrophy (LGMD) is a group of disorders that clinically resemble DMD but occur in both sexes as a result of autosomal recessive and autosomal dominant inheritance. Limb-girdle dystrophy is caused by mutations in genes encoding sarcoglycans and other proteins related to muscle cell membranes that interact with dystrophin. In terminology, LGMD1 refers to the type of gene that exhibits dominant inheritance (autosomal dominant), while LGMD2 refers to the type that exhibits autosomal recessive inheritance. Pathogenic variants have been reported at more than 50 loci (LGMD1A to LGMD1G; LGMD2A to LGMD2W). Calpainopathy (LGMD2A) is caused by mutations in the CAPN3 gene, which has more than 450 described pathogenic variants.Genes contributing to the LGMD phenotype include: anoctamin 5 (ANO5), blood vessel epicardial substance (BVES), calpain 3 (CAPN3), caveolin 3 (CAV3), CDP-L-ribitol pyrophosphorylase A (CRPPA), dystroglycan 1 (DAG1), desmin (DES), DNAJB6 (DNAJB6), dysferrin (DYSF), fukutin-related protein (FKRP), fukutin (FKT), GDP-mannose pyrophosphorylase B (GMPPB), heteronuclear ribonucleoprotein D-like (HNRNPDL), LIM zinc finger domain-containing 2 (LIMS2), and lain. Examples include A:C (LMNA), myotilin (MYOT), plectin (PLEC), protein O-glucose transferase 1 (PLOGLUT1), protein O-linked mannose N-acetylglucosamine transferase 1 (beta-1,2-) (POMGNT1), protein O-mannose kinase (POMK), protein O-mannosyl transferase 1 (POMT1), protein O-mannosyl transferase 2 (POMT2), sarcoglycan alpha (SGCA), sarcoglycan beta (SGCB), sarcoglycan delta (SGCD), sarcoglycan gamma (SGCG), titin-cap (TCAP), transportin 3 (TNPO3), tosin 1A interacting protein (TOR1AIP1), transport protein particle complex 11 (TRAPPC11), tri-element motif-containing 32 (TRIM 32), and titin (TTN). The main genes contributing to the LGMD phenotype include CAPN3, DYSF, FKRP, and ANO5 (Babi Ramesh Reddy Nallamilli et al., Annals of Clinical and Translational Neurology, 2018, 5, pp. 1574-1587).

[0144] Disferrin is involved in neurological disorders including multiple sclerosis (Hochmeister et al., J. Neuropathol. Exp. Neurol., September 2006; 65(9): pp. 855-865), Alzheimer's disease (Galvin et al., Acta Neuropathol., December 2006; 112(6): pp. 665-671), and chorea (Takahashi T et al., Mov. Disord., September 2006; 21(9): pp. 1513-155).

[0145] Spinal muscular atrophy is a genetic disorder caused by mutations in the Survival Motor Neuron 1 (SMN1) gene, characterized by weakness and wasting (atrophy) of the muscles used for movement. Mutations in the ASAH1 gene result in SMA-PME (Spinal Muscular Atrophy with Progressive Myoclonus Epilepsy).

[0146] X-linked myotubular myopathy is a genetic disorder caused by mutations in the myotubularin (MTM1) gene, affecting the muscles used for movement (skeletal muscles), and occurring almost exclusively in males. This condition is characterized by muscle weakness (myopathy) and decreased muscle tone (hypotonia).

[0147] Pompe disease is a genetic disorder caused by mutations in the acid alpha-glucosidase (GAA) gene. Mutations in the GAA gene prevent acid alpha-glucosidase from effectively breaking down glycogen, causing this sugar to increase to toxic levels in lysosomes. This increase damages organs and tissues throughout the body, especially muscles, leading to the progressive signs and symptoms of Pompe disease.

[0148] Glycogen storage disease (GSD3) is an autosomal recessive metabolic disorder caused by homozygous or compound heterozygous mutations in the amylo-alpha-1,6-glucosidase,4-alpha-glucanotransferase (AGL) gene, which encodes glycogen debranching enzyme and is associated with the accumulation of abnormal glycogen with a short outer chain. Clinically, patients with GSD3 present with hepatomegaly, hypoglycemia, and developmental delay in infancy or early childhood. Muscle weakness in those with GSD3a is mild in childhood but becomes more severe in adulthood; some patients develop cardiomyopathy.

[0149] Genome-wide association studies have identified the BIN1 locus as 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 genetic neurological disorders characterized by broad clinical and genetic heterogeneity. Lower limb spasticity with initial motor neuron complications is a core symptom of all HSPs. The genes that cause HSP include at least 79 SPG genes. Mutations in SPG7 and SPAST are common causes of hereditary spastic paraplegia (HSP) (Lallemant-Dudek P. et al., Fac. Rev., March 10, 2021; 10:27 summary).

[0150] A non-limiting example of a vector for use in gene therapy for myotubular myopathy is an AAVpo1 vector comprising a peptide-modified capsid protein containing a sequence having at least 95%, 96%, 97%, 98%, or 99% identity to the sequence of SEQ ID NO: 5 or any one of SEQ ID NOs from 2 to 4, wherein the vector further packages the human MTM1 gene operably linked to a human desmin promoter and an operable linker to the miR208a target sequence. This vector is useful for expressing the target gene in skeletal muscle without expression in the liver after systemic administration, such as intravascular injection.

[0151] Another non-limiting example of a vector for use in gene therapy for spinal muscular atrophy is an AAVpo1 vector comprising a peptide-modified capsid protein containing a sequence having at least 95%, 96%, 97%, 98%, or 99% identity to the sequence of SEQ ID NO: 5 or any one of the peptides SEQ ID NOs from 2 to 4, wherein the vector packages a human SMN1 gene operably linked to a CAG promoter and preferably further comprising a human betaglobin polyadenylation signal. This vector is useful for expressing the gene of interest in the muscle, including the heart, and the nervous system, particularly in the muscle, including the heart, and the CNS, without expression in the liver, after systemic administration such as intravascular injection.

[0152] The pharmaceutical composition of the present invention, comprising peptide-modified AAVpo1 vector particles with reduced liver-specification, can be administered to patients who simultaneously have liver degeneration, such as fibrosis, non-alcoholic fatty liver disease, non-alcoholic steatohepatitis, viral or toxic hepatitis, or who have underlying genetic disorders that induce liver degeneration.

[0153] In the context of the present invention, a therapeutically effective dose means a dose sufficient to reverse, reduce or inhibit the progression of the disorder or condition to which such terms apply, or to reverse, reduce or inhibit the progression of one or more symptoms of the disorder or condition to which such terms apply.

[0154] The effective dose is determined and adjusted according to factors such as the composition used, the route of administration, the physical characteristics of the individual being studied, such as sex, age, and weight, concomitant drug therapies, and other factors recognized by those skilled in the medical field.

[0155] In various embodiments of the present invention, the pharmaceutical composition comprises a pharmaceutically acceptable carrier and / or vehicle.

[0156] A "pharmaceutically acceptable carrier" refers to a vehicle that does not cause allergic or other adverse effects when appropriately administered to mammals, particularly humans. A pharmaceutically acceptable carrier or excipient refers to a non-toxic solid, semi-fluid, or liquid extender, diluent, encapsulating material, or any type of formulation aid.

[0157] Preferably, the pharmaceutical composition contains a pharmaceutically acceptable vehicle for an injectable formulation. These may be, in particular, isotonic, sterile, physiological saline (such as monosodium or disodium phosphate, sodium chloride, potassium chloride, calcium chloride or magnesium chloride, or mixtures of such salts), or optionally, a dried, especially lyophilized, composition that can be used to form an injectable solution by adding sterile water or physiological saline.

[0158] Suitable pharmaceutical forms for injectable use include sterile aqueous solutions or suspensions. The solution or suspension may contain additives that are compatible with the viral vector and do not hinder the entry of viral vector particles into target cells. In all cases, the formulation must be sterile and fluid enough to pass through a syringe easily. It must be stable under manufacturing and storage conditions and stored in a manner resistant to contamination by microorganisms, such as bacteria and fungi. Examples of suitable solutions are buffer solutions, such as phosphate-buffered saline (PBS) or Ringer's lactate.

[0159] The present invention also provides a method for treating muscle or nervous system disorders, in particular muscle or CNS disorders according to the present disclosure, comprising administering a therapeutically effective amount of the pharmaceutical composition described above to a patient. More preferably, the present invention provides a method for treating muscle and nervous system disorders, in particular muscle and CNS disorders according to the present disclosure.

[0160] The present invention also provides the use of pharmaceutical compositions according to the present disclosure for pharmaceutical preparations for treating muscular or nervous system disorders, particularly muscular or CNS disorders according to the present disclosure; preferably muscular and nervous system disorders, particularly muscular and CNS disorders according to the present disclosure.

[0161] As used herein, the terms “patient” or “individual” mean a mammal. Preferably, the patient or individual according to the present invention is a human.

[0162] In the context of the present invention, the terms “to treat” or “to treat” as used herein mean to reverse, mitigate or suppress the progression of the disorder or condition to which the term applies, or to reverse, mitigate or suppress one or more symptoms of the disorder or condition to which such term applies.

[0163] The pharmaceutical composition of the present invention is generally administered to patients in a sufficient dose and for a sufficient duration to induce a therapeutic effect, according to known procedures.

[0164] Administration may be systemic, topical, or systemic in combination with topical administration. Systemic administration is preferably parenteral, e.g., subcutaneous (SC), intramuscular (IM), intravascular, e.g., intravenous (IV) or intraarterial; intraperitoneal (IP); intradermal (ID) or other. Topical administration is preferably intracerebral, intraventricular, intracisional, and / or subarachnoid. Administration may be, for example, by injection or perfusion. In some preferred embodiments, administration is parenteral, preferably intravascular, e.g., intravenous (IV) or intraarterial. In some other preferred embodiments, administration is intracerebral, intraventricular, intracisional, and / or subarachnoid, alone or in combination with parenteral administration, preferably intravascular. In some other preferred embodiments, administration is parenteral, preferably intravascular, alone or in combination with intracerebral, intraventricular, intracisional, and / or subarachnoid.

[0165] The implementation of this invention will, unless otherwise indicated, utilize the prior art, which is within the scope of the art of the art. Such art is fully described in the literature.

[0166] The present invention is illustrated with reference to the attached figures using the following, non-limiting embodiments. [Brief explanation of the drawing]

[0167] [Figure 1] This figure shows the body weight over time of Mtm1-KO mice treated with various AAV vectors expressing hMTM1: AAVpo1 (KO-AAVpo1), AAVpo1A1 (KO-AAVpo1A1), AAV8 (KO-AAV8), AAV9 (KO-AAV9), and AAVrh10 (KO-AAVrh10). Untreated wild-type (WT-PBS) and Mtm1-KO (KO-PBS) mice were used as control subjects. [Figure 2] This figure shows the muscle weights of Mtm1-KO mice treated with various AAV vectors expressing hMTM1. AAVpo1 (KO + AAVpo1), AAVpo1A1 (KO + AAVpo1A1), AAV8 (KO + AAV8), AAV9 (KO + AAV9), AAVrh10 (KO + AAVrh10). Untreated wild-type (WT + PBS) and Mtm1-KO (KO + PBS) mice are used as controls. TA: Tibialis anterior, EDL: Extensor digitorum longus, Qua: Quadriceps, Ga: Gastrocnemius, Sol: Soleus, Triceps, Biceps, Diaphragm, Heart. Statistical analysis was performed using Tukey's post-hoc multiple comparison test, following one-way ANOVA (* P<0.05 vs KO + AAV8; ** P<0.01 vs KO + AAV; *** P<0.001 vs KO + AAV8; $ P<0.05 vs KO + AAV9; $$ P<0.01 vs KO + AAV9; $$$ P<0.001 vs KO + AAV9). [Figure 3]This figure shows the vector copy number (VCN) in the muscle of Mtm1-KO mice treated with various AAV vectors expressing hMTM1. AAVpo1 (KO + AAVpo1), AAVpo1A1 (KO + AAVpo1A1), AAV8 (KO + AAV8), AAV9 (KO + AAV9), AAVrh10 (KO + AAVrh10). Untreated wild-type mice (WT-PBS) are used as a control. TA: Tibialis anterior, EDL: Extensor digitorum longus, Qua: Quadriceps, Ga: Gastrocnemius, Sol: Soleus, Triceps, Biceps, Diaphragm, Heart. Statistical analysis was performed using Tukey's post-hoc multiple comparison test, following a one-way ANOVA (* P<0.05 vs KO + AAV8; ** P<0.01 vs KO + AAV; *** P<0.001 vs KO + AAV8; $ P<0.05 vs KO + AAV9; $$ P<0.01 vs KO + AAV9). [Figure 4] This figure shows the vector copy number (VCN) in organs of Mtm1-KO mice treated with various AAV vectors expressing hMTM1: AAVpo1 (KO + AAVpo1), AAVpo1A1 (KO + AAVpo1A1), AAV8 (KO + AAV8), AAV9 (KO + AAV9), and AAVrh10 (KO + AAVrh10). Statistical analysis was performed using Tukey's post-hoc multiple comparison test following one-way ANOVA (* P<0.05 vs KO + AAV8; ** P<0.01 vs KO + AAV; *** P<0.001 vs KO + AAV8; $ P<0.05 vs KO + AAV9; $$ P<0.01 vs KO + AAV9; $$$ P<0.001 vs KO + AAV9). [Figure 5]This figure shows the hMTM1 mRNA levels in the muscle of Mtm1-KO mice treated with various AAV vectors expressing hMTM1. AAVpo1 (KO + AAVpo1), AAVpo1A1 (KO + AAVpo1A1), AAV8 (KO + AAV8), AAV9 (KO + AAV9), AAVrh10 (KO + AAVrh10). MTM1 mRNA levels are expressed in comparison to expression in KO + AAV8. TA: tibia EDL: Extensor digitorum longus Qua: quadriceps Ga: gastrocnemius Sol: soleus triceps biceps diaphragm heart. Statistical analysis was performed using Tukey's post-hoc multiple comparison test, following one-way ANOVA (* P<0.05 vs KO + AAV8; ** P<0.01 vs KO + AAV; *** P<0.001 vs KO + AAV8; $ P<0.05 vs KO + AAV9; $$ P<0.01 vs KO + AAV9; $$$ P<0.001 vs KO + AAV9). [Figure 6] This figure shows the hMTM1 mRNA levels in organs of Mtm1-KO mice treated with various AAV vectors expressing MTM1. AAVpo1 (KO + AAVpo1), AAVpo1A1 (KO + AAVpo1A1), AAV8 (KO + AAV8), AAV9 (KO + AAV9), AAVrh10 (KO + AAVrh10). hMTM1 mRNA levels are expressed in comparison to expression in KO + AAV8. Statistical analysis was performed using Tukey's post-hoc multiple comparison test following one-way ANOVA (* P<0.05 vs KO + AAV8; ** P<0.01 vs KO + AAV; *** P<0.001 vs KO + AAV8; $ P<0.05 vs KO + AAV9; $$$ P<0.001 vs KO + AAV9). [Figure 7]This figure shows the levels of hMTM1 protein in the muscle of Mtm1-KO mice treated with various AAV vectors expressing hMTM1. AAVpo1 (KO + AAVpo1), AAVpo1A1 (KO + AAVpo1A1), AAV8 (KO + AAV8), AAV9 (KO + AAV9), AAVrh10 (KO + AAVrh10). Untreated wild-type (WT + PBS) and Mtm1-KO (KO + PBS) mice are used as controls. GAPDH is used as an internal control. [Figure 8] This figure shows the immunolocalization of SMN protein in spinal cord neurons of C57BL / 6 mice injected with the AAVpo1A1-SMN1 vector at 5 x 10¹³ vg / kg. SMN protein is fused to an HA-tag and detected using an anti-HA antibody. Neurons were labeled using an anti-NeuN antibody. Arrows indicate motor neurons expressing HA-SMN. Scale bars = 200 μm (left) or 50 μm (right). [Examples]

[0168] Materials and methods The porcine-derived AAVpo1A1 capsid (the nucleotide sequence encoding the protein of SEQ ID NO: 5, which includes the peptide of SEQ ID NO: 4, which replaces all residues at positions 567-569 and 570-572 of the AAVpo1 capsid protein of SEQ ID NO: 1, SEQ ID NO: 13) was compared with serotypes 8, 9, rh10, and po1 (Bello et al., Gene Therapy, 2009, pp. 16, 1320-1328. doi: 10.1038 / gt.2009.821) in constitutive knockout of the myotubularin gene (Mtm1 KO mouse line), as previously described (Buj-Bello et al., PNAS, 2002, pp. 99, 15060-1505. doi:10.1073 / pnas.212498399; Al-Qusairi et al., PNAS, 2009, 106). pp. 18763-18768. doi:10.1073 / pnas.0900705106). The vector was entirely produced using HEK 293 cells via triple transfection and contained cassettes expressing the target sequences of human MTM1 and miR208a under the regulation of the human desmin promoter (1kb) (Raguz et al., Dev. Biol., 1998, pp. 201, 26-42; Paulin D & Li Z, Exp. Cell. Res., November 15, 2004; 301(1):1-7; Roudault et al., Circulation, 2013, pp. 128, 1094-104. doi: 10.1161 / CIRCULATIONAHA.113.001340). AAVpo1A1 and AAV9 capsids were also evaluated in C57BL / 6 mice using cassettes expressing human SMN fused to the HA tag sequence under the control of a ubiquitous CAG promoter (Meyer et al., Molecular Therapy, 2015, 23. doi: 10.1038 / mt.2014.210).

[0169] 2x10 for each vector expressing MTM1 13A single dose of vg / kg was administered intravenously to 3-week-old mutant mice, tissue was collected, and frozen in nitrogen 4 weeks after injection. PBS was injected as a control to Mtm1-KO and wild-type male littermates. C57BL / 6 mice were given either AAV9 or AAVpo1A1 vector (8x10⁻¹⁰ 12 The patient received a dose of vg / kg at 4 weeks of age, and tissue samples were collected 3 weeks later.

[0170] The number of vector genomes per diploid genome from 32 ng of total DNA was quantified by Taqman real-time PCR using a LightCycler480 thermocycler (Roche). The titin gene was used for standardization with primers and probes: 5'-AAAACGAGCAGTGACGTGAGC-3' (forward; SEQ ID NO: 6), 5'-TTCAGTCATGCTGCTAGCGC-3' (reverse; SEQ ID NO: 7), and 5'-TGCACGGAAGCGTCTCGTCTCAGTC-3' (probe; SEQ ID NO: 8). The primers used for vector genome (MTM1) amplification were: 5'-TTGGTTGTCCAGTTTGGAGTCTACT-3' (forward; SEQ ID NO: 9), 5'-CCGTCACTGCAATGCACAAG-3' (reverse; SEQ ID NO: 10), and 5'-ATATCAAGCTCGTTTTGAC-3' (probe; SEQ ID NO: 11). The primers used for vector genome (SMN1) amplification were: 5'-CAGTGCAGGCTGCCTATCAG-3' (forward; SEQ ID NO: 15), 5'-TGTGGGCCAGGGCATTAG-3' (reverse; SEQ ID NO: 16), and 5'-AAGTGGTGGCTGGTGTG-3' (probe; SEQ ID NO: 17). Other primers used for vector genome (SMN1) amplification were: 5'-GCTGCCTCCATTTCCTTCTG-3' (forward; SEQ ID NO: 18), 5'-ACATACTTCCCAAAGCATCAGCAT-3' (reverse; SEQ ID NO: 19), and 5'-CACCACCTCCCATATGTCCAGATTCTCTTG-3' (probe; SEQ ID NO: 20).

[0171] The level of MTM1 transcript was quantified from 350 ng of total RNA by reverse transcription using the RevertAid H Minus Reverse Transcriptase Kit (Thermo Scientific). Next, the amount of cDNA was amplified by qPCR using a LightCycler480 thermocycler (Roche). The RPLP0 gene was used for standardization with primers and probes: 5'-CTCTGGAGAAACTGCTGCCT-3' (forward; SEQ ID NO: 21), 5'-CTGCACATCACTCAGAATTTCAA-3' (reverse; SEQ ID NO: 22), and 5'-AGGACCTCACTGAGATTCGGGATATGC-3' (probe; SEQ ID NO: 23).

[0172] Proteins were extracted and analyzed by NuPAGE 4-12% Bis-Tris gel electrophoresis and Western blotting. Membranes were searched using a polyclonal antibody against human myotubularin (Abnova). A mouse monoclonal antibody specific to GAPDH (Merck Millopore) was used as an internal control. Detection was performed using secondary antibodies (donkey anti-goat 800 or goat anti-mouse 680 (Invitrogen)) and the Odyssey infrared imaging system (LI-COR Biotechnology Inc.).

[0173] For immunostaining of vector-derived HA-SMN, 5x10 C57BL / 6 mice were used. 13A single dose of vg / kg AAVpo1A1 vector was injected, and euthanasia was performed 4 weeks later by intraperitoneal anesthetic injection (10 mg / kg xylazine, 100 mg / kg ketamine), followed by intracardiac perfusion with PBS, then 4% paraformaldehyde (PFA). Tissue was isolated and post-fixed by incubation in 4% PFA. Next, the spinal cord was incubated in PBS-sucrose solution (30%). Serial coronal cryostat sections of the lumbar spinal cord were treated with mouse IgG blocking using mouse-on-mouse IgG blocking solution (Invitrogen), then with anti-HA tag (hSMN) staining using rabbit anti-HA primary antibody (Sigma-Aldrich), and anti-NeuN staining using mouse anti-NeuN primary antibody (Sigma-Aldrich). Detection was performed using fluorescent conjugate secondary antibodies (goat anti-rabbit Alexa Fluor 488 and goat anti-mouse Alexa Fluor 594 (Invitrogen)). The sections were mounted using FluoroMount-G medium + DAPI, and images were captured using axioscan Z1 (Zeiss).

[0174] result AAV vectors expressing MTM1 (AAVpo1, AAVpo1A1, AAV8, AAV9, AAVrh10) were administered to Mtm1-KO mice at 3 weeks of age in 2x10⁶ doses. 13 The drug was administered intravenously at vg / kg. From two weeks after injection, the body weight of treated KO and WT mice was similar, while the body weight of untreated KO mice began to decrease after 6 weeks of age (Figure 1). Skeletal muscles such as the tibialis anterior (TA), quadriceps (Qua), gastrocnemius (Ga), and triceps (Tri) of mutant mice from the AAVpo1- and AAVpo1A1- treated groups were heavier than those of mice treated with AAV8 (Figure 2).

[0175] Vector genome quantification in skeletal muscle (Figures 3 and 4) showed that the AAVpo1A1 vector efficiently transduced most skeletal muscle, similar to the AAV8 vector. Interestingly, the AAVpo1A1 vector produced lower levels of transduction in organs such as the heart, liver, spleen, kidneys, lungs, and brain.

[0176] The expression of the MTM1 transgene was analyzed by RT-qPCR in various muscles and organs (Figures 5 and 6). The AAVpo1A1 vector resulted in higher MTM1 transcript levels in all skeletal muscles compared to AAV8, despite similar transduction levels, and achieved transgene expression levels comparable to those of the AAV9 vector. Furthermore, administration of the AAVpo1A1 vector detargeted transgene expression in organs such as the liver and spleen, at even lower MTM1 transcript levels than those observed after AAV8 vector delivery. Transgene expression levels were higher in central nervous system regions such as the cortex and spinal cord in AAVpo1A1-treated mice compared to the AAV8 group, and even higher in the spinal cord than in AAV9-treated mice.

[0177] MTM1 protein expression was analyzed in various muscles (gastrocnemius, triceps, and diaphragm) by immunoblotting (Figure 7). Administration of AAVpo1A1 vector resulted in higher MTM1 protein levels in the skeletal muscle of mutant mice compared to AAV8 and AAVpo1 vectors.

[0178] SMN1-expressing AAVpo1A1 and AAV9 vectors were administered to C57BL / 6 mice at 4 weeks of age in 8x10⁶ units. 12 The vector was administered intravenously at vg / kg. Several muscle and organ samples were collected after 3 weeks. The AAVpo1A1 vector transduced all skeletal muscle and heart to similar levels in WT mice. As previously observed in Mtm1-KO mice, administration of the AAVpo1A1 vector resulted in low transduction of the liver.

[0179] Transgene expression was analyzed by RT-qPCR, and the results showed that SMN1 transcript levels were similar in skeletal muscle after transduction with AAVpo1A1 and AAV9 vectors. AAVpo1A1-derived SMN1 mRNA levels were lower in the heart, liver, spleen, and kidneys compared to AAV9. In the central nervous system, AAVpo1A1-derived SMN1 transcript was present at slightly higher levels in all analyzed regions (cortex, cerebellum, and spinal cord) than in the spinal cord.

[0180] To evaluate the cellular locality of SMN in the spinal cord, immunofluorescence staining using anti-HA antibody and anti-NeuN antibody was performed in 5x10⁻¹⁰⁻¹ 13 The procedure was performed 4 weeks after injection of the AAVpo1A1-SMN1 vector at vg / kg. As shown in Figure 8, HA-SMN was expressed in neurons, particularly large motor neurons located in the anterior horn of the spinal cord. The majority of motor neurons (mean 80%, range 72% to 94%, mouse n=4) were transduced using AAVpo1A1 and expressed the transgene.

[0181] Overall, this demonstrates improved strength and tissue specificity of the AAVpo1A1 vector for muscle- and / or CNS-directed gene transfer, as it favorably combines high transgene expression levels in skeletal muscle, brain, and spinal cord with detargeted transgene expression in other organs such as the liver and spleen.

Claims

1. An agent for delivering a target gene to a nervous system cell, comprising a recombinant porcine AAV vector containing a peptide-modified capsid protein of porcine adeno-associated virus (AAV) serotype 1, wherein the peptide-modified capsid protein contains the sequence of SEQ ID NO: 5, or a sequence having at least 95% identity with SEQ ID NO: 5, and the peptide from SEQ ID NO: 2 to 4 is inserted between residues N567 and S568 or between residues N569 and T570 of the capsid protein, the position of which is determined by alignment with SEQ ID NO:

1.

2. The agent according to claim 1, wherein the peptide replaces all residues at positions 565-567 and 568-570 or all residues at positions 567-569 and 570-572, and the positions are determined by alignment with SEQ ID NO:

1.

3. The agent according to claim 1 or 2, wherein the peptide-modified capsid protein contains a sequence having at least 96%, 97%, 98%, or 99% identity with SEQ ID NO:

5.

4. The agent according to any one of claims 1 to 3, wherein the AAV vector is a vector particle packaging a gene of interest for therapeutic purposes.

5. The agent according to claim 4, wherein the gene of interest is operably linked to a promoter that is functional in neurons and / or glial cells.

6. The target gene for treatment, (i) Therapeutic genes; (ii) A gene encoding a therapeutic protein or peptide; or (iii) Genes encoding therapeutic RNA The agent according to claim 4 or 5, selected from the group consisting of the following.

7. The agent according to claim 6, wherein the therapeutic protein or peptide is selected from a therapeutic antibody or antibody fragment and a genome editing enzyme.

8. The agent according to claim 6, wherein the therapeutic RNA is selected from interfering RNA, guide RNA for genome editing, and antisense RNA capable of exon skipping.

9. A pharmaceutical agent for gene therapy of neurological disorders, comprising the agent according to any one of claims 1 to 8.

10. A pharmaceutical agent for gene therapy of a neuromuscular disease affecting the nervous system, comprising the agent according to any one of claims 1 to 8, wherein the disease is a genetic neuromuscular disease affecting the nervous system, and the disease is selected from the group consisting of (i) spinal muscular atrophy and motor neuron disease, (ii) hereditary motor sensory neuropathy, (iii) hereditary paraplegia and hereditary ataxia, and (iv) congenital myasthenic syndrome.

11. The pharmaceutical product according to claim 10, wherein the gene of interest for treatment is a functional version of a gene causing a genetic neuromuscular disorder affecting the nervous system, selected from the group consisting of spinal muscular atrophy (SMN1, ASAH1 gene); amyotrophic lateral sclerosis (SOD1, ALS2, SETX, FUS, ANG, TARDBP, FIG4, OPTN gene); hereditary paraplegia (SPAST, SPG7 gene); Charcot-Marie-Tooth, type 4B1 (MTMR2 gene); and congenital myasthenic syndrome (CHAT, AGRN gene), or a therapeutic RNA that targets the gene causing the disease.

12. The pharmaceutical product according to claim 11, wherein the gene causing a genetic neuromuscular disorder affecting the nervous system is selected from the group consisting of the SMN1 and ASAH1 genes.

13. The pharmaceutically acceptable drug according to claim 11 or 12, wherein the vector further packages a human SMN1 gene operably linked to a promoter that is functional in neurons and / or glial cells, for use in gene therapy for spinal muscular atrophy.

14. An agent according to any one of claims 1 to 8, or a pharmaceutical agent according to any one of claims 9 to 13, administered by an intravascular, intracerebral, intraventricular, intracisional, and / or subarachnoid route, or a combination thereof.

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