RU2812279C2 - RECOMBINANT ADEN-ASSOCIATED VIRUS, WHICH IS HYBRID OF AAV9 AND AAVrh74 SEROTYPES, WITH REDUCED TROPISM FOR LIVER TISSUE - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: recombinant adeno-associated virus (AAV) capsid protein is described that is a hybrid of the capsid proteins of AAV serotype 9 (AAV9) and AAV serotype 74 (AAVrh74), comprising a sequence having at least 85% identity with the following SEQ ID NO: 3, and has a reduced tropism for liver tissue compared to the capsid proteins of the original serotypes AAV9 and AAVrh74. The following is presented: a polynucleotide encoding the capsid protein of a recombinant hybrid AAV in an expressed form. Also, the following is presented: an AAV-based vector particle for expression of the desired gene in muscle cells containing a nucleotide sequence corresponding to the capsid protein of the recombinant hybrid AAV.

EFFECT: invention expands the arsenal of agents for genetic therapy.

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Description

Field of technology to which the invention relates

This invention relates to the capsid of a recombinant adeno-associated virus (AAV), which is a hybrid of the capsid protein of AAV serotype 9 (AAV9) and the capsid protein of AAV serotype rh74 (AAVrh74), having tropism for liver tissue, reduced compared to that of the original capsid proteins AAV9 and AAVrh74. This invention also relates to vector particles based on AAV hybrid serotypes carrying the desired gene, and their use in gene therapy, in particular for the treatment of genetically determined neuromuscular diseases.

State of the art

Recombinant adeno-associated virus (rAAV) vectors are widely used for in vivo gene transfer. rAAV-based vectors are nonenveloped particles consisting of a 20 nm diameter capsid and 4.7 kilobase (kb) single-stranded DNA. The genome has two genes, rep and cap, flanked by two palindromic regions called inverted terminal repeats (ITRs). The cap gene encodes three structural proteins, designated VP1, VP2, and VP3, that form the AAV capsid. The VP1, VP2 and VP3 proteins have the same C-terminal region, the sequence of which makes up the entire VP3 protein. In adeno-associated virus serotype 2 (AAV2), which can be taken as typical, the VP1 protein consists of 735 amino acid residues (GenBank YP_680426); The sequence of VP2 includes 598 amino acid residues, starting with threonine at position 138 (T138), while VP3 has 533 residues, its sequence begins with methionine at position 203 (M203). AAV serotypes are determined by their capsid. There are several different serotypes, each with its own tissue specificity. Therefore, the choice of serotype depends on which tissue is to be targeted. Skeletal muscle cells and liver cells are infected and transduced with vectors based on different AAV serotypes, such as AAV8, AAV9 and AAV-rh74.

Chimeric or hybrid AAV serotypes were created by exchanging fragments of nucleotide sequences encoding capsid proteins between different naturally occurring AAV serotypes in order to increase transduction efficiency or enhance the tropism of the virus to desired tissues or cell types.

Hybrid AAV capsids were prepared by combining structural domains of AAV capsids of serotype 8 and serotypes isolated from primate brain. The resulting hybrid AAV serotypes are capable of transduction into human and mouse retinal tissue, with transduction efficiency no greater than that of AAV2 and AAV5 based vectors (Charbel Issa et al., PLOS ONE, 2013, 8, e60361). However, one of these hybrid AAV serotypes exhibited increased transduction efficiency in adipose tissue compared with AAV1, AAV8 and AAV9 (Liu et al., Molecular Therapy, 2014, 1, 8, doi:10.1038/mtm).

Publication WO 2015/191508 describes recombinant hybrid AAV capsids obtained by exchanging variable regions of AAV capsids isolated from various species (humans, primates, birds, snakes, cattle), in particular AAV capsids with tropism for central nervous cells systems to create chimeric capsids specific to the tissue of the central nervous system.

WO 2017/096164 describes recombinant AAV capsids that are hybrids of AAV capsids of serotypes 1, 2, 3b, 6 and 8, which have increased tropism for human skeletal muscle tissue.

However, all naturally occurring adeno-associated virus serotypes tested to date tend to accumulate in the liver. This creates difficulties, in particular when introducing AAV-based vectors into the body systemically. First, the transferred gene, which should be expressed in muscle cells, can cause harmful effects in the liver. Secondly, due to the fact that the AAV-based vector gets to the liver, there will be less of it in the skeletal muscles than necessary. Accordingly, you will have to increase its dosage. And this increases hepatotoxicity and the costs of using the vector.

Differences in gene expression in different tissues are achieved through tissue-specific promoters and regulatory mechanisms involving small non-coding RNAs (microRNAs). But these regulatory mechanisms do not exclude the possibility that after systemic administration, the genome of an AAV-based vector will express itself in organs other than its intended target, for example, it will be expressed in the liver.

Mutations affecting the essential amino acid arginine residues at positions 585 or 588 (R585, R588) of the capsid protein have been shown to weaken heparin/heparin sulfate binding and reduce the liver tropism of AAV2-based vectors (Asokan et al., Nat. Biotechnol., 2010, 28, 79-82). However, this approach is only suitable for serotypes AAV2 and AAV6, in which tropism for the liver is determined by the binding of basic residues of the capsid protein to heparin.

Thus, there is an urgent need for new adeno-associated virus vectors that have much lower tropism for liver tissue than for muscle tissue. New vectors are also needed that can effectively infect muscle cells but do not penetrate liver or brain cells.

Disclosure of the Invention

The authors of this invention have created new hybrid serotypes of adeno-associated virus by combining two serotypes that effectively penetrate muscle and liver tissue cells. namely AAV9 and AAV-rh74. Two new hybrid AAV serotypes were created by exchanging variable regions of the cap gene between serotypes AAV9 and AAVrh74 (see Fig. 1A and 1B). It turned out that the hybrid serotype of the adeno-associated virus lacks the tropism for liver tissue characteristic of the original serotypes AAV9 and AAVrh74 (see Fig. 4C and 4D). At the same time, for the hybrid AAV serotype, a higher vector titer and a higher efficiency of gene transduction in skeletal and cardiac muscle cells were observed.

New hybrid AAV serotypes are valuable for gene therapy of neuromuscular disorders, including genetically determined diseases, autoimmune diseases, neurodegenerative diseases and cancers.

Thus, this invention covers recombinant adeno-associated viruses with capsids hybridized between AAV9 and AAVrh74, having reduced tropism for liver tissue; AAV-based vector particles containing recombinant adeno-associated virus hybrid capsids; compositions containing vector particles based on AAV hybrid serotypes, as well as methods for producing and using said vector particles based on AAV hybrid serotypes and compositions including them, in particular use in gene therapy.

Carrying out the invention

Fusion protein of the capsid of a recombinant adeno-associated virus

In one aspect, this invention relates to a recombinant adeno-associated virus (AAV) capsid protein that is a hybrid between the capsid proteins of AAV serotypes 9 (AAV9) and 74 (AAVrh74), wherein the recombinant AAV capsid fusion protein has a reduced tropism for liver tissue compared to with capsid proteins of the original serotypes AAV9 and AAVrh74.

As used herein, the term “tropism” refers to the specificity of infection of a certain cell type or certain tissues by viral particles containing the AAV capsid protein.

AAV capsid tropism for a particular cell type or particular tissue can be determined by measuring, by standard analytical methods known in the art (e.g., described in the Examples section of this application), the ability of AAV vector particles containing an AAV capsid fusion protein to infect cells of a certain type or certain tissues or carry out transduction in them.

As used herein, the term “liver tissue tropism”/“hepatotropism” refers to tropism for liver tissue and liver cells, including hepatocytes.

According to the present invention, the tropism of the AAV capsid fusion protein for liver tissue is at least 20%, 30%, 40%, 50% or more, preferably at least 50%, 60% 70%, 80%, 90% or 99% lower than the tropism for liver tissue of the capsid proteins of the original serotypes AAV9 or AAVrh74.

According to the present invention, the AAV capsid fusion protein has tropism for muscle tissues and muscle cells.

Muscle tissues include, but are not limited to, cardiac muscle tissue and skeletal muscle tissue.

As used herein, the term “muscle cells” refers to myocytes, myotubes, myoblasts and/or satellite cells.

In some embodiments of the present invention, the tropism of the AAV fusion capsid protein for muscle tissue is similar to that of the capsid proteins of the parent serotypes AAV9 and/or AAVrh74. Preferably, the muscle tropism of the AAV fusion capsid protein is at least 50%, 60%, 70%, 80%, 90%, 99% or more of the muscle tropism of the capsid proteins of the parent serotypes AAV9 and/or AAVrh74.

In some embodiments of the present invention, the AAV fusion capsid protein is a VP1, VP2, or VP3 protein fusion.

In some embodiments of the present invention, the AAV fusion capsid protein has tropism for at least musculoskeletal tissue. In some preferred embodiments of the present invention, the AAV fusion capsid protein has tropism for both skeletal muscle tissue and cardiac muscle tissue. An example of such a hybrid protein is the AAV hybrid capsid having the amino acid sequence shown as SEQ ID NO: 3 (in the Examples section this protein is referred to as “Hybrid Cap9-rh7”). This type of AAV capsid can be used to treat cardiac and skeletal muscle disorders.

The AAV capsid fusion protein of this invention can be derived from any sequence of the AAV9 and AAVrh74 capsid proteins; these sequences are well known in the art; they are available in public amino acid sequence databases. For example, the AAV9 capsid protein corresponds to the sequences presented in the GenBank database with the following identifiers: AY530579.1; SEQ ID NO: 123 (as per publication WO 2005/033321); SEQ ID NO: 1 (as per publication WO 2012/112832); AAV9 capsid variants in which one or more of the natural amino acid residues at positions 271 (D), 446 (Y) and 470 (N) are replaced by other amino acid residues, as described in WO 2012/112832; AAV9 capsid variants with amino acid substitutions K143R, T251A, S499A, S669A and S490A, as described in US patent No. 2014/0162319. The following sequences correspond to the AAVrh74 capsid protein: SEQ ID NO: 1 according to publication WO 2015/013313; SEQ ID NO: 6 according to WO 2006/110689; SEQ ID NO: 1 according to WO 2013/123503; SEQ ID NO: 4 according to WO 2013/158879; variants with substitutions of amino acid residues K137R, K333R, K550R, K552R, K569R, K691R, K695R, K709R and combinations of these variants.

In some embodiments of the present invention, the AAV capsid fusion protein of the present invention is derived from the AAV9 capsid protein having the amino acid sequence of SEQ ID NO: 1 (GenBank AY530579.1) and the AAVrh74 capsid protein having the amino acid sequence of SEQ ID NO: 2.

In some embodiments of the present invention, the AAV capsid fusion protein of the present invention is obtained by replacing a variable region in the capsid protein sequence of AAV9 or AAVrh74 with the corresponding variable region of the capsid protein sequence of an adeno-associated virus of a different serotype,

wherein the AAV9 capsid variable region corresponds to a stretch of the amino acid sequence SEQ ID NO: 1 of the AAV9 capsid (reference sequence) starting from any of positions 331 to 493 to any of positions 556 to 736 or a fragment, consisting of at least 10, 15, 20, 25, 30, 35, 40, 45, 50, 55 or 60 consecutive amino acid residues from the region between the 493rd and 556th positions of the sequence SEQ ID NO: 1 AAV9 capsid; And

the variable region of the AAVrh74 capsid corresponds to a segment of the amino acid sequence SEQ ID NO: 2 of the AAVrh74 capsid (reference sequence) starting from any of positions 332 to 495 and to any of positions 558 to 738 or a fragment consisting of at least 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, or 60 consecutive amino acid residues from the region between positions 495 and 558 of SEQ ID NO: 2 AAVrh74 capsids.

The present invention includes AAV capsid fusion proteins that are derivatives of any of the amino acid sequences of the capsid proteins AAV9 and AAVrh74, obtained by replacing the variable region in the capsid sequence of AAV9 or AAVrh74 with the corresponding variable region of the capsid sequence of an adeno-associated virus of another serotype, as described above. In this invention, the variable region is defined using the capsid sequences SEQ ID NO: 1 AAV9 and SEQ ID NO: 2 AAVrh74 as reference. After comparing any other AAV9 capsid sequence with SEQ ID NO: 1 or any other AAVrh74 capsid sequence with SEQ ID NO: 2 by standard protein amino acid sequence alignment methods using computer programs well known in the art BLAST, FASTA, CLUSTALW, etc. . One skilled in the art can easily determine the corresponding variable region sequence positions in other AAV9 or AAVrh74 capsid sequences.

In some preferred embodiments of the present invention, the AAV fusion capsid protein of the present invention is formed by replacing the variable region corresponding to the sequence 449 to 609 of the capsid sequence SEQ ID NO: 1 AAV9 or 450 to 611 of the capsid sequence sequence SEQ ID NO: 2 AAVrh74, to the corresponding variable region of the capsid of a virus of a different serotype.

In some embodiments of the present invention, the AAV capsid fusion protein comprises an amino acid sequence selected from the group consisting of the sequences SEQ ID NO: 3 and SEQ ID NO: 4; sequences having at least 85%, 90%, 95%, 97%, 98% or 99% identity with said sequences, and a fragment thereof corresponding to the VP2 or VP3 capsid protein. The VP2 protein corresponds to a portion of the sequence SEQ ID NO: 3 or 4 from amino acid residue T138 to its end. The VP3 protein corresponds to a portion of the sequence SEQ ID NO: 3 from amino acid residue M203 to its end or sequence SEQ ID NO: 4 from amino acid residue M204 to its end.

The sequence of SEQ ID NO: 3 is derived from the AAV9 capsid protein having the amino acid sequence of SEQ ID NO: 1 by replacing the AAV9 variable region (positions 449 to 609 of SEQ ID NO: 1) with the AAVrh74 capsid protein variable region ( positions 450 to 611 of SEQ ID NO: 2); in the Examples section the corresponding hybrid is called Hybrid Cap9-rh74. The VP2 protein corresponds to a portion of the sequence SEQ ID NO: 3 from amino acid residue T138 to its end. The VP3 protein corresponds to a portion of the sequence SEQ ID NO: 3 from amino acid residue M203 to its end.

The sequence of SEQ ID NO: 4 is derived from the AAVrh74 capsid protein having the amino acid sequence of SEQ ID NO: 2 by replacing the AAVrh74 variable region (positions 450 to 611 of SEQ ID NO: 2) with the AAV9 capsid protein variable region ( positions 449 to 609 of SEQ ID NO: 1); in the Examples section the corresponding hybrid is called Hybrid Caprh74-9. The VP2 protein corresponds to a portion of the sequence SEQ ID NO: 4 from amino acid residue T138 to its end. The VP3 protein corresponds to a portion of the sequence SEQ ID NO: 4 from amino acid residue M204 to its end.

In some preferred embodiments of the present invention, the AAV fusion capsid protein of the present invention is derived from the AAV9 capsid protein by replacing the AAV9 variable region with the corresponding variable region of the AAVrh74 capsid sequence as described above; preferably, the AAV fusion capsid protein comprises replacing the variable region corresponding to the region from positions 449 to 609 of the capsid sequence of SEQ ID NO: 1 of AAV9 with a variable region corresponding to the region from positions 450 to 611 of the capsid sequence of SEQ ID NO: 2 AAVrh74. Preferably, said hybrid capsid protein comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 3 and sequences having at least 85%, 90%, 95%, 97%, 98%, or 99 identity to said sequence %; more preferably, said AAV fusion protein contains the sequence SEQ ID NO: 3.

The term “identity” as used herein refers to the similarity of the amino acid sequences of two polypeptides (or the nucleotide sequences of two nucleic acids). When two sequences being compared have the same amino acid residues (nucleotides) at a given position, the sequences being compared are said to be identical at that position. The degree of identity of two sequences is the ratio of the number of positions at which these sequences are identical to the number of positions being compared, expressed as a percentage. Typically comparisons are made by aligning sequences to establish maximum identity. The degree of identity is calculated by alignment using, for example, the GCG software package (Genetics Computer Group, Program Manual for the GCG Package, Version 7; Madison, Wis.) or any other sequence comparison algorithm, such as BLAST, FASTA or CLUSTALW.

In some embodiments of the present invention, the AAV fusion capsid protein of the present invention produces a large number of recombinant AAV vector particles. Preferably, the titer of the recombinant AAV hybrid capsid vector is or exceeds 10 ¹¹ viral genomes per ml (vg/ml). The high efficiency of production of recombinant AAV vector particles is very valuable for use in gene therapy.

In some embodiments of the present invention, the AAV fusion capsid protein of the present invention also includes additional modifications, for example, increasing the targeting of AAV-based vectors to skeletal muscle tissue or cardiac muscle. A non-limiting example of such modifications is the addition of antopleurin-B to the N-terminus of the AAV capsid protein VP2 (Finet et al., Virology, 2018, 513, 43-51). Another example is the insertion of a peptide into a region exposed on the surface of the capsid, in particular near position 588 according to the numbering of amino acid residues in the sequence SEQ ID NO: 1. Examples of such peptides (not limiting) are described in the work of Michelfelder et al. PLoS ONE, 2009, 4, e5122. The insertion is preferably made in the region between positions 587 and 592 according to the numbering of amino acid residues in the sequence SEQ ID NO: 1. When inserting a peptide, it is possible (but not required) to delete some or all amino acid residues in the specified region. The insertion peptide sequence preferably consists of no more than 20 amino acid residues and may contain certain sequences of no more than five amino acid residues located at its N- and/or C-terminus, for example GQSG (SEQ ID NO: 35) and AQAA (SEQ ID NO: 36) at the N- and C-terminus of the peptide, respectively.

In some embodiments of the present invention, the insert peptide contains or is (ie consists of) a sequence selected from the group consisting of SEQ ID NOs: 12 to 34. Preferably, the insert peptide has the sequence GQSG at its N-terminus (SEQ ID NO: 35 ), and at the C-terminus there is the sequence AQAA (SEQ ID NO: 36). Preferably, the inserted peptide replaces all amino acid residues from position 587 to position 592 of SEQ ID NO: 1 of the AAV capsid protein. Preferably, the inserted peptide increases targeting to cardiac muscle tissue and in some cases to skeletal muscle tissue. In some preferred embodiments of the present invention, the AAV fusion capsid protein modified by the insertion of a peptide comprises or is comprised of a sequence selected from the group consisting of the sequence SEQ ID NO: 9 and sequences having a degree of identity to that sequence of at least 85%, 90%, 95%, 97%, 98% or 99%; more preferably, the fusion capsid protein contains the sequence of SEQ ID NO:9. The sequence of SEQ ID NO: 9 is derived from the Cap9-rh74 fusion protein having the amino acid sequence of SEQ ID NO: 3 by insertion of a peptide having the sequence of SEQ ID NO: 12. Also included within the scope of this invention are the chimeric AAV capsid proteins VP1 and VP2, derived from the AAV9/rh74 fusion capsid protein VP3 of the present invention, wherein the VP1-specific N-terminal region and/or the VP2-specific N-terminal region belong to natural or artificial AAV serotypes other than AAV9 and AAVrh74.

In some embodiments of the present invention, the chimeric AAV VP1 capsid protein comprises:

(i) a VP1-specific N-terminal region having a sequence taken from a natural or artificial AAV serotype other than AAV9 and AAVrh74,

(ii) a VP2-specific N-terminal region having a sequence taken from AAV9, AAVrh74, or natural or artificial AAV serotypes other than AAV9 and AAVrh74, and

(iii) A C-terminal region of VP3 having a VP3 fusion protein sequence of the present invention.

In some embodiments of the present invention, the chimeric AAV VP2 capsid proteins comprise

(i) a VP2-specific N-terminal region having a sequence taken from a natural or artificial AAV serotype other than AAV9 and AAVrh74, and

(ii) A C-terminal region of VP3 having a VP3 fusion protein sequence of the present invention.

Polynucleotide, vector and their use for obtaining an AAV-based vector

Another aspect of the present invention is a polynucleotide encoding a recombinant AAV capsid fusion protein in an expressible form. Said polynucleotide may be RNA, DNA, or synthetic or semi-synthetic nucleic acid.

In some embodiments of the present invention, the polynucleotide is a cap AAV9/rh74 fusion gene encoding the VP1, VP2 and VP3 fusion capsid proteins of the present invention. In some preferred embodiments of the present invention, said polynucleotide comprises the sequence SEQ ID NO: 5 (encoding an AAV fusion capsid protein having the sequence SEQ ID NO: 3) or the sequence SEQ ID NO: 7 (encoding an AAV fusion capsid protein having the sequence SEQ ID NO: 4).

In some embodiments of the present invention, said polynucleotide is a chimeric cap gene that encodes the VP3 AAV9/rh74 fusion capsid protein of the present invention and the VP1 chimeric capsid protein, and optionally the VP2 chimeric capsid protein, wherein the VP1-specific N-terminal region and , optionally, the VP2-specific N-terminal region is from a natural or artificial AAV serotype other than AAV9 and AAVrh74. Such a chimeric cap gene can be produced by any suitable method using the nucleotide sequence encoding the AAV9/rh74 fusion capsid protein VP3 of the present invention in combination with heterologous sequences taken from selected AAV serotypes other than those indicated or from non-adjacent regions genomes of the specified serotypes, or from viruses other than AAV, or from non-viral sources.

In some embodiments of the present invention, the polynucleotide also encodes adeno-associated virus replicase (Rep AAV) in expressible form, preferably Rep AAV2.

The polynucleotide of this invention is preferably inserted into a recombinant vector; Recombinant vectors include, but are not limited to, linear or circular DNA or RNA composed of chromosomal or non-chromosomal, synthetic or semi-synthetic nucleic acids, such as viral vectors, plasmids or RNA vectors.

There are many known vectors into which the desired nucleotide sequences can be inserted in order to introduce them into eukaryotic host cells and ensure their preservation there; the choice of a suitable vector is determined by its intended use (for example, for replication of the desired nucleotide sequence, or for its expression, or for persistence as an extrachromosomal element, or for integration into the chromosomal DNA of the host cell), as well as the nature of the host cell.

In some embodiments of the present invention, the vector is a plasmid.

The present invention uses a recombinant expression vector comprising elements necessary for the expression of an AAV fusion capsid protein, as well as, if necessary, an AAV replicase. Typically, each coding sequence (for the fusion capsid protein and for the AAV replicase) is included in a separate expression cassette, either in the same vector or in different ones. Each expression cassette includes an open reading frame (ORF) coding sequence operably linked to regulatory sequences that direct expression of the corresponding protein in the cell producing AAV virions, such as a specific promoter, promoter/enhancer, start codon (ATG), stop codon, and transcription termination signal. Alternatively, the fusion capsid protein and AAV replicase are expressed from a single expression cassette using an internal ribosome entry site (IRES) that is inserted between two coding sequences, or a viral 2A peptide. Additionally, nucleotide sequences encoding the fusion capsid protein and AAV replicase are preferably optimized for expression in specific AAV-producing cells, particularly human cells.

The vector of the present invention, preferably a plasmid, can be used to produce an AAV fusion vector containing an AAV fusion capsid protein using standard AAV production methods known in the art (Review in Aponte-Ubillus et al., Applied Microbiology and Biotechnology, 2018, 102: 1045-1054).

After cotransfection, the cells are incubated for a time sufficient to allow generation of AAV vector particles to occur; the cells are then harvested, lysed, and the vector particles are purified using standard purification methods, such as cesium chloride density gradient centrifugation.

AAV-based particles, pharmaceutical composition and therapeutic use

In another aspect, the present invention provides AAV-based particles containing a fusion recombinant AAV capsid protein of the present invention. These particles may contain fusion capsid proteins VP1, VP2 and VP3, encoded by the fusion cap gene of this invention. Alternatively, or in addition, these particles contain chimeric capsid proteins VP1 and VP2 and a fusion protein VP3 encoded by the chimeric cap gene of the present invention.

In some embodiments of the present invention, the AAV-based particles are mosaic in the sense that they also contain another AAV capsid protein of a natural or artificial serotype other than AAV9 and AAVrh74, and these AAV-based mosaic particles have reduced tropism for liver tissue by compared with that of AAV9 and AAVrh74. The artificial AAV serotype may be (not limited to) a chimeric AAV capsid, a recombinant AAV capsid, or a humanized AAV capsid. Such an artificial capsid can be prepared by any suitable method, using a selected AAV sequence (for example, a fragment of the VP1 capsid protein sequence) in combination with heterologous sequences taken from AAVs of various selected serotypes, with regions of the genome (not adjacent to the given sequence) of the same AAV serotype, from a non-viral source of AAV sequences, or from a non-viral source.

Preferably, the AAV-based particles of this invention are vector particles. The AAV vector genome is a single-stranded nucleic acid or double-stranded self-complementary (McCarty et al, Gene Therapy, 2003, Dec., 10(26), 2112-2118).

Self-complementary vectors are generated by deleting the terminal attachment site (TRS) in one of the terminal repeats of AAV. Vectors modified in this way, in which the replicating genome is half as long as the wild-type AAV genome, tend to form DNA dimers. The AAV genome is flanked by inverted terminal repeats (ITRs). In some embodiments of the present invention, the AAV-based vector is pseudotyped, that is, its genome and capsid are derived from AAVs of different serotypes. In some preferred embodiments of the present invention, the genome of the pseudotyped vector is derived from AAV2.

In some preferred embodiments of the present invention, the AAV vector particles carry the gene of interest.

The AAV-based particles of the present invention can be produced using the methods for generating recombinant AAV-based vector particles of the present invention.

The phrase “desired/desired gene” means a gene that is valuable for a particular application, such as diagnostics, reporter gene insertion, modification, treatment and genome editing (the listed herein is not limiting).

For example, the desired gene may be a therapeutic gene, a reporter gene, or a genome editing enzyme gene.

The phrases “therapeutic target gene”, “therapeutic target gene” or “heterologous target gene” mean a gene capable of producing a therapeutic effect, or a gene encoding a protein, peptide or RNA having a therapeutic effect.

A target gene is any nucleotide sequence that can affect a target gene or cellular mechanism to be affected, particularly in muscle cells. For example, the gene modifies the expression, sequence, or regulation of a target gene or cellular machinery to be affected. In some embodiments of the present invention, the gene of interest is a functional variant of a gene or fragment thereof. Functional variants of the gene may be a wild type gene, a variant of the gene (for example, a variant from the same gene family or other variants), a truncated gene, the encoded protein of which retains the same functionality at least partially. A functional gene variant may be useful in gene therapy to replace or complement a gene that is nonfunctional or defective in a patient. In other embodiments of the present invention, the gene of interest is a gene that inactivates a dominant allele causing an autosomal dominant genetic disorder. The gene fragment may be useful as a template for recombination when used in combination with a genome editing enzyme.

Or the gene of interest is a gene that encodes a protein for a specific application (for example, an antibody, an antibody fragment, or an enzyme used for genome editing) or RNA. In some embodiments of the present invention, the protein encoded by the gene of interest is a protein that produces a therapeutic effect, including therapeutic antibodies or genome editing enzymes. In some embodiments of the present invention, the RNA encoded by the gene of interest has therapeutic value. The desired gene is a functional gene that can ensure the formation of the protein, peptide or RNA it encodes in target cells for a given disease, in particular in muscle cells. The AAV-based vector of the present invention contains the desired gene in a form expressed in muscle cells, including cardiac and skeletal muscle cells. In particular, the desired gene is operably linked to universal, tissue-specific or inducible promoters capable of acting in muscle cells. The desired gene can be inserted into an expression cassette that also contains polyA sequences.

The RNA encoded by the desired gene is preferably complementary to the target DNA or RNA sequence or binds to the target protein. For example, the RNA encoded by the gene of interest is interfering RNA, such as short hairpin RNA (shRNA); microRNA; small RNA, which acts as a template for genome editing (gRNA) and is used in combination with the Cas enzyme or a similar protein that serves for genome editing; antisense RNA capable of skipping damaged exons, such as modified small nuclear RNA (snRNA) or long non-coding RNA. Interfering RNAs or microRNAs can be used to regulate the expression of a target gene involved in muscle disease. An RNA genome editing template in combination with a Cas enzyme or similar genome editing protein can be used to modify the sequence of a target gene, particularly to correct the sequence of a mutant or defective gene or to influence the expression of a target gene involved in a disease , in particular neuromuscular diseases. Antisense RNA, capable of skipping damaged exons, can be used, in particular, to correct the reading frame and restore the expression of a defective gene with a disrupted reading frame. In some embodiments of the present invention, the RNA of interest is RNA having therapeutic value.

Genome editing enzymes of the present invention are any enzymes or enzyme complexes capable of altering a target gene or influencing the cellular machinery being affected, particularly in muscle cells. For example, a genome editing enzyme may affect the expression, sequence, or regulation of a target gene or cellular machinery. Preferably, the genome editing enzyme is an engineered nuclease, such as a meganuclease, a zinc finger nuclease (ZEN), a transcription activator-like effector nuclease (TALEN), an enzyme encoded by the cas gene from the CRISPR/Cas system (CRISP for short). palindromic repeats arranged in groups evenly spaced from each other) or other similar enzyme (the listed here is not limiting). A genome editing enzyme, particularly an engineered nuclease, such as a protein encoded by the cas gene or the like, may be a functional nuclease that creates DNA double strand breaks (DSBs) at a target locus; such enzymes are used in site-specific genome editing applications, including gene correction, gene replacement, gene knockout, gene knockin, mutagenesis, chromosomal translocations, chromosomal deletions, etc. (The items listed here are not limiting). In site-specific genome editing applications, a genome editing enzyme, particularly an engineered nuclease, such as the enzyme encoded by the cas gene and similar proteins, can be used in combination with a homologous recombination template (HR template, also called donor DNA). matrix), resulting in a change in the target locus through homologous recombination caused by a double DNA break at this locus. In particular, with the help of an HR matrix it is possible to introduce the desired gene (transgene) into a target locus of the genome or to carry out repair at the site of a mutation in the target locus, preferably in an abnormal or defective gene causing a neuromuscular disease. Alternatively, a genome editing enzyme, such as the enzyme encoded by the cas gene and similar proteins, is engineered such that it loses nuclease activity and is used as a DNA binding protein for various genome engineering applications, such as transcriptional activation, silencing transcription, epigenomic modifications, visualization of genomic data, co-precipitation of RNA or DNA, etc.

In another aspect, the present invention provides a pharmaceutical composition comprising a therapeutically effective amount of AAV particles comprising a fusion recombinant AAV capsid protein of the present invention, preferably AAV vector particles carrying the desired therapeutic gene.

In some embodiments of the present invention, the proposed pharmaceutical composition is intended for use as a medicament, in particular in gene therapy. The scope of this invention includes the use of the proposed pharmaceutical composition as a medicinal product, in particular for the treatment of diseases by gene therapy.

Gene therapy is carried out by gene transfer, gene editing, damaged exon skipping, RNA interference, trans-splicing or any other genetic changes to coding or regulatory nucleotide sequences in the cell, including nucleotide sequences found in the nucleus, mitochondria or those belonging to commensals, e.g. viral nucleotide sequences contained in the cell.

The main types of gene therapy are as follows:

- therapy, the purpose of which is to ensure the replacement of an abnormal or defective gene with a functional gene; such therapy is called replacement or complementary therapy;

- therapy, the purpose of which is gene or genome editing; in this case, the task is to provide the cell with the necessary means of correcting the sequences or changing the expression or regulation of the abnormal or defective gene in this way. so that a functional gene is expressed or an abnormal gene is suppressed (inactivated); in these cases they talk about gene-editing therapy.

In complementary gene therapy, the desired gene is a functional version of a gene that is mutant or defective in the patient, as, for example, in some genetically determined diseases. In such cases, the desired gene is expressed to produce a functional product of that gene.

In gene editing therapy, one or more desired genes are used to edit a gene or genome, for example:

(i) a gene encoding RNA that may have a therapeutic effect (see above), for example, interfering RNAs such as small hairpin RNAs (shRNAs), small genome editing RNAs (gRNAs) and used in combination with the Cas enzyme or similar protein, antisense RNAs capable of skipping damaged RNA exons, such as modified small nuclear RNAs (snRNAs); And

(ii) a gene encoding a genome editing enzyme (see above), e.g. an engineered nuclease, e.g. meganuclease, zinc finger nuclease (ZEN), transcription activator-like effector nuclease (TALEN), enzyme encoded by the cas gene , or other similar enzyme, or a combination of such genes; and also a fragment of a functional gene variant for use as a template for recombination, as described above.

Gene therapy is used to treat a variety of diseases, including genetic diseases such as neuromuscular genetic disorders, cancers, neurodegenerative diseases, and autoimmune diseases (not limited to).

In some embodiments of the present invention, gene therapy is used to treat diseases affecting muscle tissue, particularly skeletal muscle tissue and/or cardiac muscle, such as neuromuscular genetic disorders, cardiomyopathies, rhabdomyosarcomas, polymyositis, dermatomyositis, juvenile polymyositis, etc. (listed herein) is not restrictive).

Examples of mutant genes playing a role in neuromuscular genetic disorders that can be treated by gene therapy using the pharmaceutical composition of the present invention are listed in the tables below.

Muscular dystrophies

Gene Protein DMD Dystrophin EMD Emerin FHL1 FHL1 protein (four and a half protein 1, contains four LIM domains and one zinc finger) LMNA Lamin A/C SYNE1 Nesprin 1 (spectrin repeat-containing nuclear envelope protein 1) SYNE2 Nesprin 2 (spectrin repeat-containing nuclear envelope protein 2) TMEM43 Transmembrane protein 43 TOR1AIP1 Torsin A-interacting protein 1 DUX4 Paired homeodomain transcription factor transcription activator 1 SMCHD1 Chromosome structural support protein containing a hinge domain, 1 PTRF Polymerase I and transcript releasing factor MYOT Myotilin CAV3 Kaveolin 3 DNAJB6 Heat shock protein homologue 40, subfamily B, member 6 DES Desmin TNPO3 Transportin 3 HNRNPDL D-protein-like protein of the heterogeneous nuclear ribonucleoprotein family CAPN3 Kalpain 3 DYSF Dysferlin SGCG γ-sarcoglycan SGCA α-sarcoglycan SGCB β-sarcoglycan SGCD δ-sarcoglycan TCAP Telefonin TRIM32 Tripartite motif (TRIM) containing protein, 32 FKRP Fukutin-related protein TTN Titin POMT1 Protein O-mannosyltransferase 1 ANO5 Anoctamin 5 FKTN Fukutin POMT2 Protein O-mannosyltransferase 2 POMGNT1 β-1,2-N-acetylglucosaminyltransferase 1 protein-O-linked mannose PLEC Plectin TRAPPC11 Particle transport protein complex 11 GMPPB GDP-mannose pyrophosphorylase B DAG1 Dystroglycan 1 DPM3 Dolichyl phosphate mannosyltransferase polypeptide 3 ISPD Protein containing an isoprenoid synthase domain VCP Valosin-containing protein LIMS2 LIM domain and senescent cell antigen-like domain 2 GAA Acid α-glucosidase

Congenital muscular dystrophies

Gene Protein LAMA2 Laminin _α2 chain in merosin COL6A1 α1 chain of type VI collagen COL6A2 α2 chain of type VI collagen COL6A3 α3 chain of collagen type VI SEPN1 Selenoprotein N1 FHL1 FHL1 protein (four and a half protein 1, contains four LIM domains and one zinc finger) ITGA7 α7 integrin precursor DNM2 Dynamin 2 TCAP Telefonin LMNA Lamin A/C FKTN Fukutin POMT1 Protein O-mannosyltransferase 1 POMT2 Protein O-mannosyltransferase 2 FKRP Fukutin-related protein POMGNT1 β-1,2-N-acetylglucosaminyltransferase 1 protein-O-linked mannose ISPD Protein containing an isoprenoid synthase domain POMGNT2 β-1,2-N-acetylglucosaminyltransferase 2 protein-O-linked mannose B3GNT1 UDP-N-acetylglucosamine:β-galactosidase β-1,3-N-acetylglucosaminyl-transferase 1 GMPPB GDP-mannose phosphorylase B (mannose-1-phosphate guanylyltransferase (GDP)) LARGE Glycosyltransferase-like protein DPM1 Dolichyl phosphate mannosyltransferase polypeptide 1 (catalytic subunit) DPM2 Dolichyl phosphate-mannosyltransferase polypeptide 2 (regulatory subunit) ALG13 UDP-N-acetylglucosaminyltransferase B3GALNT2 β-1,3-N-acetylgalactosaminyltransferase 2 TMEM5 Transmembrane protein 5 POMK Protein O-mannose kinase CHKB Choline kinase β ACTA1 α-Skeletal muscle actin TRAPPC11 Particle transport protein complex 11

Congenital myopathies

Gene Protein TPM3 Tropomyosin 3 NEB Nebulin ACTA1 α-Skeletal muscle actin TPM2 Tropomyosin 2 (β) TNNT1 Slow troponin T KBTBD13 Kelch repeat and BTB domain (POZ) protein, 13 CFL2 Cofilin 2 (muscular) KLHL40 Protein is a member of the Kelch family of proteins 40 KLHL41 Protein is a member of the Kelch family of proteins 41 LMOD3 Leiomodin 3 (fetal) SEPN1 Selenoprotein N1 RYR1 Ryanodine receptor 1 (skeletal muscle) MYH7 Cardiac β-myosin heavy chain 7 MTM1 Myotubularin DNM2 Dynamin 2 BIN1 Amphiphysin TTN titin SPEG The protein is a product of the SPEG complex locus MEGF10 Transmembrane protein with EGF-like domains 10 MYH2 Skeletal muscle myosin heavy chain 2 MYBPC3 Cardiac myosin binding protein C CNTN1 Kontaktin-1 TRIM32 Protein containing tripartite motif, 32 PTPLA Protein – member A of the tyrosine phosphatase family (3R-hydroxyacyl-coenzyme A dehydratase) CACNA1S α1S-subunit of voltage-gated calcium channel type L

Distal myopathies

Gene Protein DYSF Dysferlin TTN Titin GNE UDP-N-acetylglucosamine-2-epimerase/N-acetylmannosamine kinase MYH7 Cardiac β-myosin heavy chain 7 MATR3 Matrin 3 TIA1 RNA-binding protein associated with cytoplasmic granules of cytolytic cells MYOT Myotilin NEB Nebulin CAV3 Kaveolin 3 LDB3 LIM domain binding protein 3 ANO5 Anoctamin 5 DNM2 Dynamin 2 KLHL9 Protein homologous to Kelch cup proteins, 9 FLNC Filamin C, γ (actin binding protein 280) VCP Valosin-containing protein

Other myopathies

Gene Protein ISCU Homolog of the carrier protein of the iron-sulfur cluster self-assembly apparatus in Escherichia coli MSTN Myostatin FHL1 FHL1 protein (four and a half protein 1, contains four LIM domains and one zinc finger) BAG3 Athanogen associated with BCL2, 3 ACVR1 Activin A receptor type 2 kinase II MYOT Myotilin FLNC Filamin C, γ (actin binding protein 280) LDB3 LIM domain binding protein 3 LAMP2 Lysosome-associated membrane protein precursor 2 VCP Valosin-containing protein CAV3 Kaveolin 3 SEPN1 Selenoprotein N1 CRYAB α-Crystallin B DES Desmin VMA21 Vacuolar homolog of H ⁺ -ATPase (Saccharomyces cerevisiae) PLEC Plectin PABPN1 Nuclear poly(A) binding protein 1 TTN Titin RYR1 Ryanodine receptor 1 (skeletal muscle) CLN3 Neuronal waxy lipofuscinosis (Batten disease) associated protein 3 (battenin) TRIM54 Protein containing tripartite motif, 54 TRIM63 Tripartite motif 63-containing protein (E3 ubiquitin ligase)

Myotonic syndromes

Gene Protein DMPK Protein kinase associated with myotonic dystrophy CNPB Cellular nucleotide binding protein CLCN1 Chloride ion channel 1 (skeletal muscle) associated with autosomal dominant Thomsen's disease CAV3 Kaveolin 3 HSPG2 Perlecan ATP2A1 ATPase that transports Ca ²⁺ ions in fast-twitch muscles, 1

Muscle diseases associated with ion channels

Gene Protein CLCN1 Chloride ion channel 1 (skeletal muscle) associated with autosomal dominant Thomsen's disease SCN4A α-subunit of voltage-gated sodium channel type IV SCN5A α-subunit of voltage-gated sodium channel type V CACNA1S α1S-subunit of voltage-gated calcium channel type L CACNA1A α1A-Subunit of the voltage-gated calcium channel P/Q type KCNE3 Voltage-gated potassium channel protein, Isk-type family member 3 KCNA1 Voltage-gated potassium channel protein, member of shaker channel-related subfamily 1 KCNJ18 Inward rectifying potassium channel 2.6 (Kir2.6) KCNJ2 Inward rectifying potassium channel J2 KCNH2 Voltage-gated potassium channel protein member 2 of subfamily H KCNQ1 Voltage-gated potassium channel protein member 1 of KQT subfamily KCNE2 Voltage-gated potassium channel protein, Isk-type family member 2 KCNE1 Voltage-gated potassium channel protein, Isk-type family member 1

Malignant hyperthermia

Gene Protein RYR1 Ryanodine receptor 1 (skeletal muscle) CACNA1S α1S-subunit of voltage-gated calcium channel type L

Metabolic myopathies

Gene Protein GAA Acid α-1,4-glucosidase (preproprotein) AGL Glycogen debranching protein (amylo-1,6-glucosidase + 4-α-glucanotransferase) GBE1 Glycogen (1,4-α-glucan-)-branching enzyme 1 associated with Andersen's disease (glycogenosis type IV) P.Y.G.M. Glycogen phosphorylase PFKM Phosphofructokinase (muscle) PHKA1 Phosphorylase B kinase α-subunit PGM1 Phosphoglucomutase 1 GYG1 Glycogenin 1 GYS1 Glycogen synthase 3, glycogen synthase 1 (muscle) PRKAG2 Non-catalytic γ2 subunit of AMP-activated protein kinase RBCK1 RanBP and C3HC4-type zinc finger-containing protein 1 (heme-oxidized iron regulatory protein 2 (IRP2) ubiquitin ligase 1) PGK1 Phosphoglycerate kinase 1 PGAM2 Phosphoglycerate mutase 2 (muscle) LDHA Lactate dehydrogenase A ENO3 Tissue-specific β-enolase 3 (muscle) CPT2 Carnitine palmitoyltransferase II SLC22A5 Carnitine transport protein (member 5 of the SLC22 organic cation transport protein family) SLC25A20 Carnitine acylcarnitine translocase ETFA α-Subunit of electron transfer flavoprotein ETFB β-Subunit of electron transfer flavoprotein ETFDH Electron transfer flavoprotein dehydrogenase (ETF dehydrogenase) ACADVL Acyl-coenzyme A dehydrogenase (very long-chain fatty acid substrates) ABHD5 α/β-hydrolase domain-containing protein, 5 PNPLA2 Adipocyte triglyceride lipase (desnutrin) LPIN1 Lipin 1 (phosphatidate phosphatase 1) PNPLA8 Protein containing phospholipase patatin-like domain, 8

Hereditary cardiomyopathies

Gene Protein MYH6 Myosin heavy chain 6 MYH7 Myosin heavy β-chain 7 (cardiac muscle) TNNT2 Troponin T2 (cardiac) TPM1 α-Tropomyosin 1 MYBPC3 Cardiac myosin binding protein C PRKAG2 Non-catalytic γ2 subunit of AMP-activated protein kinase TNNI3 Troponin I (cardiac) MYL3 Myosin light chain 3 TTN Titin MYL2 Myosin light chain 2 ACTC1 α-actin precursor (cardiac muscle) CSRP3 Cysteine-glycine-rich protein 3 (LIM family cardiac protein) TNNC1 Slow troponin C VCL Vinculin MYLK2 Myosin light chain kinase 2 CAV3 Kaveolin 3 MYOZ2 Myozenin 2, or calsarcin 1 (Z-disc protein) JPH2 Dzhanktofilin-2 PLN Phospholamban NEXN Nexilin (F-actin binding protein) ANKRD1 Ankyrin repeat domain-containing protein 1 (cardiac muscle) ACTN2 α-Actinin 2 NDUFAF1 NADH-ubiquinone-oxidoreductase subcomplex 1α TSFM Mitochondrial translation elongation factor Ts gene AARS2 Mitochondrial alanyl-tRNA synthetase 2 MRPL3 Mitochondrial ribosomal protein L3 COX15 Protein homologous to the assembly protein of the yeast cytochrome c oxidase complex MTO1 Mitochondrial tRNA-binding translation optimization factor 1 (yeast) MRPL44 Mitochondrial ribosomal protein L44 LMNA Lamin A/C LDB3 LIM domain binding protein 3 SCN5A α-subunit of voltage-gated sodium channel type V DES Desmin EYA4 Transcription factor, member 4 of the EYA (“eyes absent”) protein family 4 SGCD δ-Sarcoglycan TCAP Telefonin ABCC9 ATP-binding cassette transporter, member 9 of subfamily C of the cassette transporter family TMPO Inner nuclear membrane protein 2 PSEN2 Presenelin 2 CRYAB αB-Crystallin FKTN Fukutin TAZ Tafatzin DMD Dystrophin LAMA4 α-Laminin 4 ILK Integrin-linked kinase MYPN Myopalladin RBM20 RNA binding motif 20 SYNE1 Nesprin 1 (spectrin repeat-containing nuclear envelope protein 1) MURC Muscle double helix protein DOLK Dolichol kinase GATAD1 Protein is a member of the GATA family of transcription factors containing a zinc finger domain, 1 SDHA A subunit of the succinate dehydrogenase complex (flavoprotein) GAA Acid α-glucosidase (pre-protein) DTNA α-Dystrobrevin FLNA α-Filamin A (actin binding protein 280) TGFB3 Transforming growth factor β3 RYR2 Ryanodine receptor 2 TMEM43 Transmembrane protein 43 DSP Desmoplakin PKP2 Plakophilin 2 DSG2 Desmoglein 2 DSC2 Desmocollin 2 JUP Plakoglobin (γ-catenin) CASQ2 Calsequestrin 2 (heart muscle) KCNQ1 Voltage-gated potassium channel protein, member 1 of KQT-type subfamily KCNH2 Voltage-gated potassium channel protein, member 2 of KQT-type subfamily ANK2 Ankirin 2 KCNE1 Voltage-gated potassium channel protein, Isk-type family member 1 KCNE2 Voltage-gated potassium channel protein, Isk-type family member 2 KCNJ2 Inward rectifying potassium channel J2 CACNA1C α 1C subunit of voltage-gated calcium channel type L SCN4B β – Subunit of voltage-gated sodium channel type IV AKAP9 Protein A kinase anchor 9 (PRKA) (YOTIAO) SNTA1 α1-Syntrophin KCNJ5 Inward rectifying potassium channel, subfamily J member 5 NPPA Natriuretic peptide A precursor KCNA5 Voltage-gated potassium channel protein, member of the shaker channel-related subfamily 5 GJA5 Connexin 40 SCN1B β-Subunit of voltage-gated sodium channel type I SCN2B β-Subunit of voltage-gated sodium channel type II NUP155 Nucleoporin 155 kDa GPD1L Glycerol-3-phosphate dehydrogenase 1 peptides CACNB2 β2 subunit of voltage-gated calcium channel KCNE3 Voltage-gated potassium channel protein, Isk-type family member 3 SCN3B β-Subunit of voltage-gated sodium channel type III HCN4 Cyclic nucleotide-dependent sodium channel, activated by hyperpolarization, 4

Congenital myasthenic syndromes

Gene Protein CHRNA1 α-Subunit 1 of the nicotinic acetylcholine receptor CHRNB1 Nicotinic acetylcholine receptor β-subunit 1 (muscle) CHRN Nicotinic acetylcholine receptor δ-subunit CHRNE ε-Subunit of the nicotinic acetylcholine receptor RAPSN Rapsin CHAT Choline acetyltransferase isoform COLQ Collagen-like acetylcholinesterase anchor polypeptide MUSK Skeletal muscle tyrosine kinase receptor DOK7 Docking protein 7 AGRN Agrin GFPT1 Glutamine fructose 6-phosphate transaminase 1 DPAGT1 Dolichyl phosphate (UDP-N-acetylglucosamine) N-acetylglucosamine phosphate transferase 1 LAMB2 β2 – Laminin chain (laminin S) SCN4A α-subunit of voltage-gated sodium channel type IV CHRNG γ-Nicotinic acetylcholine receptor subunit PLEC Plectin ALG2 α -1,3/1,6-mannosyltransferase ALG14 UDP-N-acetylglucosamine transferase SYT2 Synaptotagmin II PREPL Prolyl endopeptidase-like protein

Motor neuron diseases

Gene Protein SMN1 Motor neuron survival factor 1 (encoded by the telomeric gene variant) IGHMBP2 Immunoglobulin μ-chain binding helicase, 2 PLEKHG5 Pleckstrin homologous domain-containing protein, G family member 5 (with RhoGef domain) HSPB8 Heat shock protein β8 (27 kDa) HSPB1 Heat shock protein β1 (27 kDa) HSPB3 Heat shock protein β3 (27 kDa) AARS Alanyl-tRNA synthetase GARS Glycyl-tRNA synthetase BSCL2 Seiping REEP1 Expression enhancer protein 1 (auxiliary receptor protein 1) SLC5A7 Carnitine transport protein (member 7 of the SLC5 family of organic cation transport proteins) DCTN1 Dynactin 1 UBA1 Ubiquitin-activating enzyme 1 ATP7A α-Polypeptide of Cu ³⁺ transfer ATPase DNAJB2 DnaJ heat shock protein homologous protein (Hsp40), member 2 of subfamily B TRPV4 Temperature-sensitive ion channel from the TRP (fast receptor potential) family, member of subfamily 4 DYNC1H1 Cytoplasmic dynein heavy chain 1 BICD2 Homologue 2 of the protein encoded by the bicaudal mutation gene, D Drosophila FBXO38 F-box domain-containing protein 38 ASAH1 N-acylsphingosine amidohydrolase (acid ceramidase) 1 VAPB Membrane protein associated with vesicles B and C EXOSC8 Exosome component 8 SOD1 Soluble superoxide dismutase 1 ALS2 Alsin SETX Senataxin FUS Hybrid protein resulting from the t(12;16) translocation in malignant liposarcoma ANG Angiogenin TARDBP Protein that binds DNA containing a TAR (trans-activation response) element FIG4 Sac domain-containing inositol phosphatase 3 OPTN Optineurin ATXN2 Ataxin 2 VCP Valosin-containing protein UBQLN2 Ubiquilin 2 SIGMAR1 Non-opioid intracellular receptor ς 1 CHMP2B Charged multivesicular body protein 2B PFN1 Profilin 1 MATR3 Matrin 3 NEFH Heavy neurofilament protein PRPH Perinephrine C9orf72 Protein encoded by chromosome 9 open reading frame 72 CHCHD10 Protein containing CHCH domain (double helix-helix-double helix) 10 SQSTM1 Sequestosome 1 AR Androgen receptor GLE1 Homologue of yeast nuclear pore protein mediating RNA egress, 1 ERBB3 Homologue of the v-erbB oncogene of avian erythroblastoma virus 3 PIP5K1C Phosphatidylinositol 4-phosphate 5-kinase type γ I EXOSC3 Exosome component 3 VRK1 Vaccinia-associated protein kinase 1 SLC52A3 Riboflavin transport protein (member 3 of the SLC52 organic cation transport protein family) SLC52A2 Riboflavin transport protein (member 2 of the SLC52 organic cation transport protein family) HEXB Hexosaminidase B

Hereditary motor and sensory neuropathies

Gene Protein PMP22 Peripheral nerve myelin protein 22 MPZ Myelin protein null LITAF Transcription factor for lipopolysaccharide-inducible tumor necrosis factor EGR2 Early response protein 2 NEFL Light neurofilament protein (68 kDa) HOXD10 Homeobox domain-encoded protein D10 ARHGEF10 Rho domain-containing guanine nucleotide exchange factor 10 FBLN5 Fibulin 5 (intercellular matrix) DNM2 Dynamin 2 YARS Tyrosyl-tRNA synthetase INF2 Inverted formin 2 GNB4 β-Subunit of G protein 4 (guanine nucleotide binding protein) GDAP1 Ganglioside-inducible cell differentiation-associated protein 1 MTMR2 Myotubularin-binding phosphatase 2 SBF2 SET domain binding factor 2 SBF1 SET domain binding factor 1 SH3TC2 Protein KIAA1985 NDRG1 Protein product of the gene controlled by the N-myc gene, 1 PRX Periaxin HK1 Hexokinase 1 FGD4 Frabin, which binds actin microfilaments FIG4 Sac domain-containing inositol phosphatase 3 SURF1 Mitochondrial membrane protein encoded by the surfeit locus 1 GJB1 Connexin 32 (gap junction protein β1, molecular weight 32 kDa) AIFM1 Mitochondrion-associated apoptosis induction factor 1 PRPS1 Phosphoribosylpyrophosphate synthetase 1 PDK3 Pyruvate dehydrogenase kinase isoenzyme 3 KIF1B Protein 1B of the kinesin family MFN2 Mitofusin 2 RAB7A Late endosome marker RAB7 from the RAS oncogene family TRPV4 Thermosensitive ion channel of the TRP family, member 4 of subfamily V GARS Glycyl-tRNA synthetase HSPB1 Heat shock protein 1 (27 kDa) HSPB8 Heat shock protein 8 (27 kDa) AARS Alanyl-tRNA synthetase DYNC1H1 Cytoplasmic dynein heavy chain 1 LRSAM1 Membrane metalloendopeptidase containing leucine-rich repeats and SAM domain, 1 DHTKD1 Protein containing dehydrogenase E1 and transkelotase domains, 1 TRIM2 Protein containing tripartite motif, 2 TFG Receptor tyrosine kinase (TRK) fusion protein MARS Methionyl-tRNA synthetase KIF5A Protein 5A from the kinesin family LMNA Lamin A/C MED25 Transmitter complex subunit 25 DNAJB2 DnaJ heat shock protein homologous protein (Hsp40), member 2 of subfamily B HINT1 Nucleotide binding protein containing a histidine triad motif, 1 KARS Lysyl tRNA synthetase PLEKHG5 Pleckstrin homologous domain-containing protein, G family member 5 (with RhoGef domain) COX6A1 Cytochrome c oxidase subunit VIa polypeptide 1 IGHMBP2 Immunoglobulin μ-chain binding helicase, 2 SPTLC1 Serine palmitoyltransferase subunit 1 SPTLC2 Long chain 2 serine palmitoyltransferase ATL1 Atlastin 1 KIF1A Protein 1A from the kinesin family WNK1 Protein kinase WNK (lysine poor) 1 IKBKAP κ light chain gene enhancer inhibitor associated with the kinase complex of B cells NGF Nerve growth factor β-polypeptide DNMT1 DNA(cytosine-5)-methyltransferase 1 SLC12A6 Potassium and chloride ion cotransporter KCC3 GJB3 Connexin 31 (gap junction β3 protein, molecular weight 31 kDa) Sept-09 Septin 9 GAN Gigaxonin CTDP1 Carboxy-terminal domain (CTD) phosphatase subunit 1 VRK1 Vaccinia-associated protein kinase 1

Hereditary paraplegia

Gene Protein ATL1 Atlastin 1 SPAST Spastin NIPA1 Protein is the product of a gene that is not imprinted in Prader-Willi syndrome and Angelman syndrome, 1 KIAA0196 Strumpellin KIF5A Protein 5A from the kinesin family RTN2 Reticulon 2 HSPD1 Heat shock protein 1 (molecular weight 60 kDa) (chaperonin) BSCL2 Seiping REEP1 Expression enhancer protein 1 (auxiliary receptor protein 1) ZFYVE27 Protrudin SLC33A1 Acetyl coenzyme A transporter, member 33 of the SLC family of organic ion transport proteins CYP7B1 Cytochrome P450 family 7 subfamily B polypeptide 1 SPG7 Paraplegin SPG11 Spataxin ZFYVE26 Spastisin ERLIN2 Endoplasmic reticulum lipid raft-associated protein, 2 SPG20 Spartina SPG21 Maspardine B4GALNT1 β-1,4-N-acetyl-galactoaminyltransferase 1 DDHD1 DDHD domain-containing protein 1 KIF1A Protein 1A from the kinesin family FA2H Fatty acid hydrolase 2 PNPLA6 Protein containing phospholipase patatin-like domain, 6 C19orf12 Protein encoded by open reading frame 12 of chromosome 19 GJC2 Gap junction protein γ2 (molecular weight 47 kDa) NT5C2 Cytosolic 5'-nucleotidase II GBA2 β-Glucosidase 2, involved in the metabolism of bile acids AP4B1 β1 subunit of adapter protein complex 4 AP5Z1 ζ1 subunit of adapter protein complex 5 (LOC9907) TECPR2 Tectonin-β-propeller repeat-containing protein, 2 AP4M1 μ1 subunit of adapter protein complex 4 AP4E1 ε1 subunit of adapter protein complex 4 AP4S1 ς1 subunit of adapter protein complex 4 DDHD2 DDHD domain-containing protein 2 C12orf65 Protein encoded by open reading frame 65 of chromosome 12 CYP2U1 Cytochrome P450 family 2 subfamily U polypeptide 1 ARL6IP1 ADP-ribosylation factor-like protein 6.1 interacting protein AMPD2 Adenosine monophosphate deaminase 2 ENTPD1 Ectonucleoside triphosphate diphosphohydrolase 1 ALDH3A2 Aldehyde dehydrogenase 3A2 ALS2 Alsin L1CAM L1 neuronal cell adhesion molecule PLP1 Proteolipid protein 1 MTPAP Mitochondrial poly(A) polymerase AFG3L2 AFG3-like protein-2 (paraplegin-like ATP-dependent protease (Saccharomyces cerevisiae) 1 SACS Saksin

Other neuromuscular disorders

Gene Protein TOR1A Torsin A SGCE ε-Sarcoglycan IKBKAP κ light chain gene enhancer inhibitor associated with the kinase complex of B cells TTR Transthyretin (prealbumin associated with type I amyloidosis) KIF21A Protein 21A from the kinesin family PHOX2A Aristaless-related homeodomain-containing protein, paired type, 2A TUBB3 β-Tubulin 3 TPM2 β-Tropomyosin 2 MYH3 Myosin heavy chain 3 of embryonic skeletal muscle TNNI2 Troponin I type 2 TNNT3 Skeletal muscle troponin T3 SYNE1 Nesprin 1 (spectrin repeat-containing nuclear envelope protein 1) MYH8 Skeletal muscle myosin heavy chain 8 in the perinatal period POLG Mitochondrial DNA polymerase γ SLC25A4 Mitochondrial adenine nucleotide transfer protein C10orf2 Protein encoded by open reading frame 2 of chromosome 10 POLG2 Accessory subunit of mitochondrial DNA polymerase RRM2B Small subunit (M2B) of ribonucleotide reductase, induced by pP53 protein) TK2 Mitochondrial thymidine kinase 2 SUCLA2 β-Subunit of ATP-specific (ADP-forming) succinate-coenzyme A ligase (succinate thiokinase) OPA1 Dynamin-like GTPase associated with optic atrophy STIM1 Transmembrane endoplasmic reticulum calcium sensor 1 of the STIM family ORAI1 Calcium release activator modulator 1 PUS1 Pseudouridylate synthase 1 CHCHD10 Protein containing CHCH domain (double helix-helix-double helix) 10 CASQ1 Calsequestrin 1 fast twitch skeletal muscle YARS2 Mitochondrial tyrosyl-tRNA synthetase 2

Any of the genes listed in the tables above can serve as a target for gene replacement therapy, with the desired gene being a functional variant of the defective or mutant gene.

Or the genes listed above can be used as objects of gene editing. Gene editing is used to correct the nucleotide sequences of mutant genes or to change the expression or regulation of defective/abnormal genes, resulting in expression of a functional gene in target cells, such as muscle cells. In such cases, the desired gene is selected from genes encoding RNAs that have therapeutic effects, such as interfering RNAs, genome editing template RNAs (gRNAs), and antisense RNAs capable of skipping damaged exons; The RNA targets with a therapeutic effect are the genes listed above. Tools such as the CRISPR/Cas9 system are used for gene editing.

In some embodiments of the present invention, the target of gene therapy (complementary gene therapy or gene editing) is a gene that causes one of the muscular dystrophies listed above, in particular Duchenne muscular dystrophy (DMD; DMD, BMD genes); limb-girdle (damage to the pelvic and shoulder girdle) muscular dystrophy (LGMD) (CAPN3 gene and other genes); Facioscapulohumeral muscular dystrophy type 1 (FSHD1A) (DUX4 or FRG1 gene) and type 2 (FSHD1B) (SMCHD1 gene) and titinopathies (TTN gene).

In some embodiments of the present invention, the pharmaceutical composition provided is for use in the treatment of muscle diseases (e.g., myopathies) or muscle lesions, in particular genetically determined neuromuscular disorders without liver involvement, for example, muscular dystrophies, congenital muscular dystrophies, congenital myopathies, distal myopathies, other myopathies, myotonic syndromes, diseases affecting ion channels, malignant hyperthermia, metabolic myopathies, hereditary cardiomyopathies, congenital myasthenic syndromes, motor neuron diseases, hereditary paraplegia, hereditary motor and sensory neuropathies and other neuromuscular disorders.

Muscular dystrophies include, but are not limited to:

- dystrophinopathies - various muscle diseases linked to the X chromosome, which are caused by pathological variants of the DMD gene, encoding the dystrophin protein; dystrophinopathies include Duchenne muscular dystrophy (DMD), Becker muscular dystrophy (BMD) and dilated cardiomyopathy associated with the DMD gene;

- limb girdle muscular dystrophy (LGMD); disorders of this group are similar in clinical picture to Duchenne muscular dystrophy, but occur in people of both sexes as a result of autosomal recessive and autosomal dominant inheritance. Limb-girdle muscular dystrophies are caused by mutations in genes encoding muscle cell membrane-bound sarcoglycans and other proteins that interact with dystrophin. The designation LGMD1 refers to diseases inherited in an autosomal dominant manner, and LGMD2 refers to diseases inherited in an autosomal recessive manner. Pathogenic gene variants have been reported at more than 50 loci (LGMD1A - LGMD1H; LGMD2A - LGMD2Y);

- calpainopathy (LGMD2A), which is caused by mutations in the CAPN3 gene, and more than 450 of its pathogenic variants have been described;

- Emery-Dreyfus muscular dystrophy (EDMD), which is caused by defects in one of the genes, including the EMD gene encoding emerin, the FHL1 gene and the LMNA gene encoding lamins A and C);

- muscular dystrophy associated with nesprin-1 and nesprin-2, which is caused by defects in the SYNE1 and SYNE2 genes, respectively;

- LUMA protein-associated muscular dystrophy, which is caused by defects in the TMEM43 gene;

- muscular dystrophy associated with the LAP1B protein, which is caused by defects in the TOR1AIP1 gene; And

- Facioscapulohumeral muscular dystrophy type 1 (FSHD1A), associated with defects in the DUX4 gene (reduction in the number of D4Z4 macrosatellite repeats in the subtelomeric region of chromosome 4q35) or the FRG1 gene;

Facioscapulohumeral muscular dystrophy type 2 (FSHD1B), which is caused by defects in the SMCHD1 gene.

A specific example of gene editing would be the treatment of limb-girdle muscular dystrophy type 2A (LGMD2), which is caused by mutations in the CAPN3 gene, which encodes calpain-3. Another example is treatment by correcting mutations in the DMD or TNT genes.

Thus, through gene editing or gene replacement therapy, the presence of a normal variant of the gene, the defect of which causes the disease, is ensured in the patient’s muscle cells, which contributes to the effective treatment of the disease. Using the same principle, it is possible to treat the above genetically determined muscle diseases by replacing or editing the corresponding genes.

Replacement or complementary gene therapy can be used to treat cancers, particularly rhabdomyosarcomas. In cancer, the gene of interest may be a gene involved in the regulation of the cell cycle, or metabolism, or the migration of tumor cells, or the induction of tumor cell death. For example, expression of the gene encoding inducible caspase-9 in muscle cells activates the cell death mechanism, which can be used preferentially in combination therapy to induce a long-lasting antitumor immune response.

Gene editing can be used to change gene expression in muscle cells in autoimmune conditions or cancers, or to disrupt the life cycle of viruses in these cells. In such cases, the gene of interest is preferably selected from genes encoding genome editing template RNA (gRNA), site-specific endonuclease genes (TALEN nucleases, meganucleases, zinc finger nucleases, Cas nucleases), DNA templates and interfering RNAs, e.g. short hairpin RNAs (shRNAs) and microRNAs. Tools such as the CRISPR/Cas9 system are used for these purposes.

In some embodiments of the present invention, gene therapy is used to treat diseases affecting other tissues by expressing genes that produce a therapeutic effect in muscle tissue. This avoids the expression of a gene that produces a therapeutic effect in the liver, particularly in patients with an underlying liver disorder such as hepatitis. In such cases, the therapeutic gene preferably encodes a protein, peptide or antibody that has a therapeutic effect and is released from muscle cells into the bloodstream, which delivers the protein, peptide or antibody to other target tissues, such as the liver. Examples of genes that provide a therapeutic effect include, but are not limited to those listed herein, genes encoding coagulation factors VIII and IX, as well as acid α-glucosidase.

The pharmaceutical composition of this invention containing vector particles based on adeno-associated virus with reduced tropism for liver tissue can be administered to patients with concomitant liver disease, such as hepatitis, including viral or toxic hepatitis.

As used herein, the term “therapeutically effective amount” refers to a dose sufficient to reverse, attenuate, or suppress the progression of the disorder or condition being treated, or to reverse, attenuate, or suppress the progression of one or more symptoms of the disorder or condition being treated.

The effective dose is determined and selected by a medical professional depending on a number of factors, including the specific composition used, the route of administration and data of the individual, such as gender, age, body weight, medications taken, etc.

In various embodiments of this invention, the proposed pharmaceutical composition contains a pharmaceutically acceptable carrier and/or carrier medium.

The term “pharmaceutically acceptable carrier” as used herein refers to carriers that do not cause adverse, allergic or other adverse reactions when administered to a mammal, especially a human, as appropriate. A pharmaceutically acceptable carrier or excipient is a non-toxic solid, semi-solid or liquid excipient, diluent, enveloping material or composition having some auxiliary function.

Preferably, the pharmaceutical composition of this invention contains a carrier medium/carrier that is pharmaceutically acceptable for the injectable drug. Such a medium/carrier may be, in particular, an isotonic sterile saline solution (mono- or dibasic sodium phosphate, sodium chloride, potassium or magnesium and similar salts or mixtures of these salts) or an anhydrous, in particular lyophilized composition, which, when added (as appropriate ) sterile water or saline solution becomes a solution suitable for administration by injection.

Dosage forms suitable for administration by injection include sterile aqueous solutions or suspensions. Such a solution or suspension may contain additives that are compatible with viral vectors and do not interfere with the penetration of vector particles into target cells. In any case, the dosage form of this invention must be sterile and liquid to such an extent that it can be easily administered by syringe. The dosage form of this invention must be stable under the conditions of manufacture and storage and protected from contamination by microorganisms, such as bacteria and fungi. An example of a solution suitable for this invention is a buffer solution, such as phosphate buffered saline (PBS) or lactated Ringer's solution (Hartmann's solution).

The present invention also provides a method of treating diseases affecting muscle tissue, in particular skeletal muscle and/or cardiac muscle, comprising administering to the patient a therapeutically effective amount of the pharmaceutical composition described above.

The present invention also provides a method for treating diseases by expressing genes that provide a therapeutic effect, comprising administering to the patient a therapeutically effective amount of the pharmaceutical composition described above.

As used herein, the terms “patient,” “patient,” or “individual” refer to mammals. Preferably, the patient/patient/individual of this invention is a human.

In the context of this invention, the term “treatment” is used herein to mean “reversing, reducing, or suppressing the progression of the disorder or condition to which the term is used,” or “reversing, reducing, or suppressing the progression of one or more symptoms of the disorder or condition in which the term is used.” in relation to which this term is used."

The pharmaceutical composition of this invention is administered to an individual, generally by known methods, in a dosage and regimen effective to produce a therapeutic effect in that individual.

Administration of the pharmaceutical composition of this invention to an individual may be parenteral, oral, local or local-regional. Parenteral administration is preferably by injection or perfusion, such as subcutaneous (SC), intramuscular (IM), intravascular (eg, intravenous (IV)), intraperitoneal (IP), intradermal (ID), etc. Administration of the pharmaceutical composition of the present invention is preferred. produces a systemic effect throughout the body, that is, in all muscles of the individual, including the diaphragm and heart muscle. Preferably, administration of the pharmaceutical composition of this invention is systemic, more preferably parenteral.

The practice of this invention assumes (unless otherwise indicated) the use of conventional methods and technologies within the competence of a person skilled in the art. Such methods and technologies are fully described in the literature.

Below, this invention is disclosed using non-limiting examples, illustrated by the figures attached to the present description.

Description of illustrations

Fig. 1. Scheme of the formation of the cap gene (encoding the Cap protein) of a new AAV serotype, which is a hybrid of the AAV9 and AAVrh74 serotypes.

A. The cap (VP1 protein) genes of serotypes AAV9 and AAVrh74 are depicted; the sequence of the variable region is highlighted. The N- and C-terminal regions of the variable region of the corresponding proteins are the sequences of SEQ ID NO: 37 and SEQ ID NO: 38 in serotype AAV9 and SEQ ID NO: 39 and SEQ ID NO: 40 in serotype AAVrh74. B. Hybrid genes of cap serotypes AAV9-rh74 and AAVrh74-9 (VP1 protein).

Fig. 2. Formation of virions of new AAV serotypes, which are hybrids of the AAV9 and AAVrh74 serotypes.

HEK293T cells produced virions of hybrid serotypes (AAV9-rh74 and AAVrh74-9) and control serotypes (AAV9 and AAVrh74). The number of viral genomes was determined by real-time polymerase chain reaction with 5' tag release (TaqMan). Error bars represent standard error of the mean (SEM).

Fig. 3. Scheme of searching for target tissues (studying the biodistribution of the substance) Vg – viral genome (vg).

Fig. 4. Quantitative determination of transgene expression in muscles and organs after systemic administration of AAV hybrid serotypes.

We determined the expression of luciferase in skeletal muscles (A and B) and organs (C and D) in mice that were injected with virions of hybrid serotypes AAV9-rh74 and AAVrh74-9 and control AAV9 and AAVrh74 at two different doses: low = 2 E10 vg/individual (A and C), high = 1 E11 vg/individual (B and D). Error bars represent standard error of the mean (SEM). TA - tibialis anterior; Pso – psoas muscle; Qua – quadriceps muscle; Dia – diaphragm; RLU – relative luminescence units.

Examples

Example 1. Design and production of vectors based on hybrid serotypes of recombinant adeno-associated virus (rAAV) with hybrid capsids

AAV9-rh74 and rh74-AAV9

1. Materials and methods

Construction of plasmids for new AAV serotypes

To construct a plasmid containing nucleotide sequences encoding the Rep AAV2 replicase and the hybrid capsid protein Cap 9-rh74, a fragment of 1029 nucleotides in length was synthesized containing a highly variable region of the sequence encoding the capsid protein Cap AAV-rh74, flanked by fragments of the sequence encoding Cap AAV9, and restriction sites BsiWI at the 5' end and Eco47III at the 3' end (GeneWiz). Using the indicated restriction sites, the resulting fragment was inserted into the plasmid pAAV2-9 containing the nucleotide sequences encoding the AAV2 Rep and AAV9 Cap proteins to replace the corresponding AAV9 Cap sequence.

To construct a plasmid containing nucleotide sequences encoding Rep AAV2 replicase and the hybrid capsid protein Cap rh74-9, a fragment of 2611 nucleotides in length was synthesized containing a highly variable region of the sequence encoding the capsid protein Cap AAV-9, flanked by the rest of the sequence Cap AAV_rh74, part of the sequence , encoding Rep AAV2, and restriction sites HindIII at the 5' end and PmeI at the 3' end (GeneWiz). Using the indicated restriction sites, the resulting fragment was inserted into the plasmid pAAV2-9 containing the nucleotide sequences encoding the AAV2 Rep and AAV9 Cap proteins to replace the entire AAV9 Cap sequence.

Receiving AAV

In this study, two protocols were used, corresponding to two scales of virus production. Under small-scale virus production conditions, adherent HEK293 cells were cultured in a 6-well plate in DMEM containing 10% fetal bovine serum (FBS). Under conditions for large-scale virus production, HEK293 cells were cultured in suspension in 250 ml of serum-free medium. Cells were transfected with three plasmids: i) a transgenic plasmid containing AAV2 inverted terminal repeats (ITRs) flanking an expression cassette that encoded firefly luciferase; ii) an auxiliary plasmid pXX6 containing adenoviral nucleotide sequences necessary for the formation of AAV; iii) a plasmid containing the genes encoding the AAV Rep and Cap proteins, which determine the AAV serotype. Two days after transfection, cells were lysed to release AAV viral particles.

The lysate containing viral particles was purified: first, it was centrifuged twice in a cesium chloride density gradient, then dialysis or affinity chromatography was performed. The content of viral genomes was determined by real-time 5' tag release polymerase chain reaction (TaqMan) using primers and probes corresponding to inverted terminal repeats (ITRs) in the AAV vector genome (Rohr et al., J. Virol. Methods , 2002, 106, 81–88).

2. Results

Construction of new serotypes

The amino acid sequences of the VP1 protein (encoded by the Cap genes) of AAV9 (SEQ ID NO: 1) and AAV-rh74 (SEQ ID NO: 2) were aligned using the BLASTP program. Highly variable regions were identified located between positions 449 and 609 of the AAV9 Cap polypeptide chain and between positions 450 and 611 of the AAV-rh74 Cap polypeptide chain (see Fig. 1A). Two new cap genes were constructed (sequences SEQ ID NO: 5 and SEQ ID NO: 7) by replacing the highly variable region of one of these two serotypes with the corresponding region of the second serotype and vice versa (see Fig. 1B). each of the resulting two hybrid cap genes was inserted into a plasmid containing a nucleotide sequence encoding the AAV2 Rep protein, which ensured the formation of recombinant AAV viral particles. The new serotypes were given the following names: the serotype that had most of the AAV9 Cap protein sequence and the AAV-rh74 highly variable region is “AAV Hybrid 9-rh74” (SEQ ID NO: 3); the serotype that had most of the Cap sequence of AAV-rh74 and the highly variable region of AAV9 is the “AAV rh74-9 Hybrid” (SEQ ID NO: 4).

Generation of new hybrid AAV serotypes

AAV viral particles of new hybrid serotypes and control ones were obtained in two versions: on a small scale (2 ml) in a 6-well plate and on a larger scale (250 ml) in a suspension of cultured cells. As shown in Figure 2, new hybrid serotypes are produced in yields suitable for gene transfer applications

Example 2. Study of the biodistribution of vector particles based on hybrid serotypes with AAV9-rh74 and AAVrh74-9 capsids

1. Materials and methods

In vivo experiments

AAV-based vector particles were administered to male B6Albino mice at 1 month of age; administration was carried out by injection into the tail vein. We took two dosages: low - 2 E10 (2x10 ¹⁰ ) viral genomes (vg) per 1 individual and high - 1 E11 (10 ¹¹ ) vg / individual. 15 days after vector administration, mice were anesthetized (ketamine + xylazine) and given intraperitoneal luciferin, after which luciferase activity was determined by measuring luminescence using an IVIS Lumina in vivo imaging system (manufactured by Perkin Elmer). 30 days after injection of the vector, mice were sacrificed, samples of skeletal muscles and internal organs were taken and frozen in liquid nitrogen.

Molecular analysis

Tissue samples were homogenized in cell lysis buffer [Tris (base) 25 mM, magnesium chloride ( _MgCl2 ) 8 mM, dithiothrietol (DTT) 1 mM, ethylenediaminetetraacetate (EDTA) 1 mM, glycerol 15%, Triton X-100 0, 2%], to which a mixture of protease inhibitors Protease Inhibitor Cocktail (manufactured by Roche) was added. Luciferase expression was determined in sample lysates using an EnSpire Multimode Plate Reader (manufactured by Roche), in cell buffer solution [Tris (base) 25 mM, magnesium chloride (MgCl ₂ ) 8 mM, dithiothriethol (DTT) 1 mM, ethylenediaminetetraacetate (EDTA) 1 mM, glycerol 15%, adenosine triphosphate (ATP) 2 mM], to which luciferin (83 μM) was added immediately before use. The total protein content of the samples was also determined using the Pierce BCA protein assay kit (manufactured by Thermo Fisher). The measurement results (luminescence intensity) were normalized by the total protein content.

To determine the content of viral genomes in the samples (VCN - vector copy number), DNA was isolated from them using the NucleoSpin Tissue kit (manufactured by Macherey-Nagel). For real-time polymerase chain reaction, 100 ng of DNA was collected and followed the same protocol as described above for determining the titer of AAV-based vectors. For genomic control, the Mex5 exon of the titin gene was amplified in the same experiment.

2.Results

To assess the biodistribution of new serotypes, hybrid AAV and control vectors containing an expression cassette with a luciferase reporter gene under the control of the CMV promoter, which is considered universal, were obtained. These vectors were administered to mice systemically by injection in two dosages: low - 2 E10 (2x10 ¹⁰ ) viral genomes (vg) per 1 individual and high - 1 E11 (10 ¹¹ ) vg / individual. 15 days after administration, the distribution of vectors throughout the body was visualized, and after 30 days, samples were taken from various skeletal muscles and internal organs (see Fig. 3).

The intensity of luciferase expression in the samples was determined and the data were normalized to the total protein content in these samples.

In muscle, the AAV 9-74 hybrid gave good levels of transgene expression in all muscles tested, including skeletal and cardiac muscle, similar to that of AAVrh74 (see FIGS. 4A to 4D).

It turned out that in the liver for both hybrid vectors the level of transgene expression is significantly reduced compared to its high level in the case of the AAV9 or AAV-rh74 vectors taken as control (see Fig. 4C - 4D).

Two new AAV serotypes were created by combining serotypes AAV9 and AAV-rh74, which efficiently infect both skeletal muscle and liver. And the hybrid serotype vectors of this invention provided gene transfer in skeletal muscles, but did not provide effective transduction in the liver.

Example 3. Vector based on the hybrid serotype AAV9-rh74, encoding a capsid protein with a peptide modification

Peptides were modified in the amino acid sequence of the Cap9-rh74 hybrid protein in order to increase the tropism of the vector based on the hybrid AAV 9-rh74 serotype to muscle tissue. Modification of the AAV capsid was carried out according to the work of Kienle E.C. (Ph.D. thesis, 2014, Joint Faculty of Science and Mathematics, Ruperto_Carol University Heidelberg, Germany), using the peptide described in Michelfelder et al. (PLoS ONE, 2009, 4, e5122). Briefly, we did the following. The hexapeptide QQNAAP (SEQ ID NO: 41), present in the VP1 protein of the 9-rh74 hybrid capsid (positions 587 to 592 of SEQ ID NO: 3) as a result of mutation was converted into the octapeptide GQSGAQAA (SEQ ID NO: 42) and the P1 peptide (RGDLGLS; SEQ ID NO: 12) was inserted between the glycine residue at position 4 and the alanine residue at position 5. The hybrid capsid protein Cap9-rh74, modified with the P1 peptide, has the amino acid sequence SEQ ID NO: 9 (it corresponds to the nucleotide sequence SEQ ID NO: 10). Vectors were prepared by triple transfection of HEK293 cells cultured in suspension and purified by affinity chromatography as in Example 1. Vectors were administered to mice, which were sacrificed one month after this injection, and tissue samples were collected for biodistribution determination as in Example 2. Expected effect This modification is an increase in the amount of vector in muscle tissue.

Claims (14)

1. A recombinant adeno-associated virus (AAV) capsid protein that is a hybrid of the capsid proteins of AAV serotype 9 (AAV9) and AAV serotype 74 (AAVrh74), comprising a sequence having at least 85% identity to SEQ ID NO: 3 and having reduced tropism to liver tissue compared to the capsid proteins of the parent serotypes AAV9 and AAVrh74. 2. The recombinant hybrid AAV capsid protein according to claim 1, which has a tropism for muscle tissue similar to that of the original capsid proteins AAV9 and/or AAVrh74. 3. Capsid protein of recombinant hybrid AAV according to claim 1 or 2, wherein said hybrid is formed by replacing the variable region located between positions 449 and 609 of the capsid sequence AAV9 SEQ ID NO: 1 with the corresponding variable region located between positions 450 and 611 of the capsid sequence AAVrh74 SEQ ID NO: 2. 4. Recombinant hybrid AAV capsid protein according to any one of claims. 1-3, comprising a sequence having at least 90%, 95%, 97%, 98% or 99% identity with the specified sequence of SEQ ID NO: 3. 5. The recombinant hybrid AAV capsid protein according to claim 4, which includes the sequence of SEQ ID NO: 3. 6. Recombinant hybrid AAV capsid protein according to any one of claims. 1-5, which is a fusion protein of VP1, VP2 or VP3. 7. A polynucleotide encoding the capsid protein of a recombinant hybrid AAV according to any one of claims. 1-6 in expressed form. 8. Vector particle based on AAV, for expression of the desired gene in muscle cells, containing a nucleotide sequence corresponding to the capsid protein of the recombinant hybrid AAV according to any one of paragraphs. 1-6. 9. The AAV vector particle of claim 8, which further comprises at least one AAV capsid protein of a natural or artificial AAV serotype other than AAV9 and AAVrh74. 10. The AAV-based vector particle of claim 8 or 9, wherein the desired gene is selected from the group consisting of (i) genes that provide a therapeutic effect; (ii) genes encoding proteins or peptides that provide therapeutic effects, such as antibodies or antibody fragments, or enzymes for genome editing; And (iii) genes encoding RNAs that provide therapeutic effects, such as interfering RNAs, small RNAs that act as templates for genome editing (gRNAs), and antisense RNAs that can skip damaged exons.