RU2839898C2 - Synergetic action of smn1 and mir-23a in treating spinal muscular atrophy - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: invention relates to biotechnology, specifically to gene therapeutic preparations based on SMN1 gene, and can be used in medicine in therapy of spinal muscular atrophy (SMA). Disclosed is an isolated nucleic acid for producing a gene therapeutic viral preparation for SMN1 gene expression in mammalian target cells, which contains in direction from 5'-end to 3'-terminal nucleic acid coding SMN1 protein, and nucleic acid coding microRNA miR-23a. Invention also discloses an expression cassette and a vector containing said nucleic acid, as well as a recombinant virus based on AAV9 (adeno-associated virus of serotype 9).

EFFECT: invention provides enhanced functional action of SMN1 in vitro when using SMN1 gene in combination with miR-23a as a part of a gene therapeutic agent, which can be used in treating SMA.

12 cl, 5 dwg, 5 tbl, 4 ex

Description

AREA OF TECHNOLOGY

The present application relates to the field of biotechnology, virology, genetics and molecular biology. More specifically, the present invention relates to an isolated nucleic acid for producing a gene therapeutic viral preparation, which comprises a nucleic acid that encodes a SMN1 protein (survival motor neuron protein) with the amino acid sequence of SEQ ID NO: 1, and a nucleic acid that encodes a miR-23a microRNA, an expression cassette and a vector based on it, as well as a recombinant virus based on AAV9 (adeno-associated virus serotype 9) for expressing the SMN1 gene in target cells, a pharmaceutical composition that includes this recombinant virus, and various uses of the above-mentioned recombinant virus and the above-mentioned composition.

LEVEL OF TECHNOLOGY

Spinal muscular atrophy (CMA/SMA) is a group of neuromuscular diseases and is characterized predominantly by an autosomal recessive type of inheritance. Today, the term SMA is generally understood to mean the most common form of the disease, which develops as a result of mutations and/or deletions in the SMN1 gene (survival motor neuron 1), located on the long arm of chromosome 5 (5q11.2-q 13.3) (Lefebvre et al., Cell (1995) 80:155-165). The disease is accompanied by degeneration of motor neurons in the ventral (anterior) horn of the spinal cord and/or brainstem, which leads to hypotonia of the proximal muscle groups responsible for basic motor skills, such as crawling, walking, holding the head, as well as to impaired swallowing and breathing (Sumner C.J., NeuroRx (2006) 3:235-245). Thus, patients with SMA have a high risk of developing respiratory failure and the addition of intercurrent diseases.

Gene therapy is a promising treatment for spinal muscular atrophy.

Adeno-associated viral (AAV) vectors are considered effective for CNS gene therapy because they have a favorable toxicity and immunogenicity profile, can be used in neural cell transduction, and are able to mediate long-term expression in the CNS.

Adeno-associated virus (AAV) is a small (20 nm), non-enveloped, non-spontaneous virus. Many different AAV serotypes have been described in humans and primates. The adeno-associated virus genome contains (+ or -) single-stranded DNA (ssDNA) approximately 4.7 thousand nucleotides long. At the ends of the genomic DNA molecule are inverted terminal repeats (ITRs). The genome contains two open reading frames (ORFs): Rep and Cap, which contain several alternative reading frames encoding different protein products. The Rep products are essential for AAV replication, and the Cap gene, in addition to other alternative products, encodes three capsid proteins (VP1, VP2, and VP3). The VP1, VP2, and VP3 proteins are present in a 1:1:10 ratio, forming an icosahedral capsid (Xie Q. et al. The atomic structure of adeno-associated virus (AAV-2), a vector for human gene therapy. Proc Natl Acad Sci USA, 2002; 99:10405-10410). In the formation of a recombinant AAV vector (rAAV), an expression cassette flanked by an ITR is packaged into the AAV capsid. The genes required for AAV replication are not included in the cassette. Recombinant AAV is considered the safest and one of the most widely used viral vectors for in vivo gene transfer. Vectors can infect cells from a variety of tissue types, providing potent and robust transgene expression. They are also non-pathogenic and have a low immunogenicity profile (High KA et al., “rAAV human trial experience” Methods Mol Biol. 2011; 807:429-57).

International applications WO 2017106354 (A1), WO 2010129021 (A1), WO 2015060722 (A1), WO 2017066579 (A1), WO 2015158749 (A2) describe various AAV variants with the SMN1 gene and their use.

Description of the invention

The inventors have developed an isolated nucleic acid for obtaining a gene therapy viral preparation, which contains a nucleic acid encoding the SMN1 protein with the amino acid sequence SEQ ID NO: 1, and a nucleic acid encoding the miR-23a microRNA, an expression cassette and a vector based on it, as well as a recombinant virus based on AAV9 for expressing the SMN1 gene in target cells, a pharmaceutical composition that includes this recombinant virus, and various options for using the above-mentioned recombinant virus and the above-mentioned composition. The inventors have unexpectedly found that miR-23a enhances the functional effect of SMN1 in vitro and have established a synergistic effect of SMN1 and miR-23a in the treatment of SMA in an animal model of this disease.

Brief description of the invention

In one aspect, the present invention relates to an isolated nucleic acid for producing a gene therapy viral preparation, which comprises a nucleic acid that encodes a SMN1 protein (survival motor neuron protein) with the amino acid sequence of SEQ ID NO: 1, and a nucleic acid that encodes a miR-23a microRNA.

In some embodiments, an isolated nucleic acid that encodes an SMN1 protein with the amino acid sequence of SEQ ID NO: 1 comprises the nucleotide sequence of SEQ ID NO: 2.

In some embodiments, the isolated nucleic acid comprises miR-23a microRNA, which has the nucleotide sequence of SEQ ID NO: 3.

In some embodiments, the isolated nucleic acid comprises a nucleic acid encoding a miR-23a microRNA that comprises the nucleotide sequence of SEQ ID NO: 4.

In some embodiments, the isolated nucleic acid comprises the following elements in the 5' to 3' direction:

a nucleic acid that encodes the SMN1 protein with the amino acid sequence of SEQ ID NO: 1, and

nucleic acid that encodes the microRNA miR-23a.

In one aspect, the present invention relates to an expression cassette that comprises any of the above nucleic acids.

In some embodiments, the expression cassette includes the following elements in the 5' to 3' direction:

left (first) ITR (inverted terminal repeats);

CMV (cytomegalovirus) enhancer;

CMV (cytomegalovirus) promoter;

intron of the hBG1 gene (hemoglobin gamma-1 subunit gene);

nucleic acid that encodes the SMN1 protein;

hGH1 polyadenylation signal (human growth hormone gene polyadenylation signal);

SV40 promoter (simian virus 40 promoter); nucleic acid that encodes the microRNA miR-23a;

SV40 polyadenylation signal (simian virus 40 polyadenylation signal) and right (second) ITR.

In some embodiments, the expression cassette comprises a nucleic acid of SEQ ID NO: 6.

In one aspect, the present invention relates to an expression vector that comprises any of the above nucleic acids or any of the above cassettes.

In one aspect, the present invention relates to a recombinant AAV9 (adeno-associated virus 9)-based virus for expressing the SMN1 gene in target cells, which comprises a capsid and any of the above cassettes.

In some embodiments, the recombinant AAV9-based virus includes the AAV9 VP1 protein.

In some embodiments, the recombinant AAV9-based virus comprises an AAV9 VP1 protein having the amino acid sequence of SEQ ID NO: 7.

In some embodiments, the recombinant AAV9-based virus comprises an AAV9 VP1 protein having the amino acid sequence of SEQ ID NO: 7 with one or more point mutations.

In some embodiments, the recombinant AAV9-based virus comprises an AAV9 VP1 protein having the amino acid sequence of SEQ ID NO: 7 or the amino acid sequence of SEQ ID NO: 7 with one or more point mutations, and the expression cassette comprises the following elements in the 5'-to-3'-direction:

left (first) ITR (inverted terminal repeats);

CMV (cytomegalovirus) enhancer;

CMV (cytomegalovirus) promoter;

intron of the hBG1 gene (hemoglobin gamma-1 subunit gene);

nucleic acid that encodes the SMN 1 protein;

hGH1 polyadenylation signal (human growth hormone gene polyadenylation signal);

SV40 promoter (simian virus 40 promoter);

nucleic acid that encodes the microRNA miR-23a;

SV40 polyadenylation signal (simian virus 40 polyadenylation signal) and right (second) ITR.

In some embodiments, the recombinant AAV9-based virus comprises an AAV9 VP1 protein having the amino acid sequence of SEQ ID NO: 7 or the amino acid sequence of SEQ ID NO: 7 with one or more point mutations, and the expression cassette comprises a nucleic acid of SEQ ID NO: 6.

In one aspect, the present invention relates to a pharmaceutical composition for delivering an SMN1 gene to target cells, which comprises any of the above-mentioned recombinant AAV9-based viruses in combination with one or more pharmaceutically acceptable excipients.

In one aspect, the present invention relates to the use of any of the above-mentioned recombinant AAV9-based viruses or the above-mentioned composition for delivering the SMN1 gene to target cells.

In one aspect, the present invention relates to the use of any of the above-mentioned recombinant AAV9-based viruses or the above-mentioned composition for the survival of a subject who has spinal muscular atrophy and/or lacks fully functional copies of the SMN1 gene.

In one aspect, the present invention relates to the use of any of the above-mentioned recombinant AAV9-based viruses or the above-mentioned composition for providing SMN1 protein to a subject who has spinal muscular atrophy and/or lacks fully functional copies of the SMN1 gene.

In one aspect, the present invention relates to the use of any of the above-mentioned recombinant AAV9-based viruses or the above-mentioned composition for the treatment of spinal muscular atrophy in a subject having spinal muscular atrophy.

In one aspect, the present invention relates to a method of modulating motor function in a subject with a motor neuron disorder, comprising administering a therapeutically effective amount of any of the above-mentioned recombinant AAV9-based viruses or the above-mentioned composition to the cells of the subject.

In one aspect, the present invention relates to a method of providing SMN protein to a subject with spinal muscular atrophy, comprising administering a therapeutically effective amount of any of the above-mentioned recombinant AAV9-based viruses or the above-mentioned composition to cells of the subject in need thereof.

In one aspect, the present invention relates to a method of delivering an SMN1 gene to target cells of a subject with spinal muscular atrophy, comprising administering any of the above-mentioned recombinant AAV9-based viruses or the above-mentioned composition to the cells of the subject.

In one aspect, the present invention relates to a method of treating spinal muscular atrophy in a subject, which comprises administering a therapeutically effective amount of any of the above-mentioned recombinant AAV9-based viruses or the above-mentioned composition to a subject with spinal muscular atrophy.

Brief description of the drawings

Fig. 1 is a graph showing the expression of SMN1 at the mRNA level after SMN1 knockdown by siRNA and transduction with AAV9-GFP, AAV9-GFP-miR-23a, AAV9-SMN1, and AAV9-SMN1-miR-23a viruses. U-87 cells were transfected with 20 nmol/L SMN 1-specific siRNA (or control siRNA) and then transduced with AAV9-GFP, AAV9-GFP-miR-23a, AAV9-SMN1, and AAV9-SMN1-miR-23a viruses at an MOI of 400,000. At 120 h post-transduction, the copy number of SMN1 gene in each sample was determined by qPCR (n=3). The copy number of housekeeping gene GAPDH was also determined. All SMN1 counts were normalized to the GAPDHv gene copy numbers in each sample. Data are presented as normalized mean SMN1 copy numbers with standard deviation.

Fig. 2 is a graph showing the expression of SMN at the protein level after SMNJ knockdown by siRNA and transduction with AAV9-GFP, AAV9-GFP-miR-23a, AAV9-SMN1, and AAV9-SMN1-miR-23a viruses. U-87 cells were transfected with 20 nmol/L SMN1-specific siRNA (or control siRNA) and then transduced with AAV9-GFP, AAV9-GFP-miR-23a, AAV9-SMN1, and AAV9-SMN1-miR-23a viruses at an MOI of 400,000. At 120 h post-transduction, the amount of SMN protein in each sample was determined by ELISA (n=3). All SMN protein amounts (in pg) were normalized to the total protein amount in each sample (in μg). The data are presented as normalized mean SMN protein amounts, with standard deviations.

Fig. 3 is a graph showing the expression of miR-23a after SMN1 knockdown by siRNA and transduction with AAV9-GFP, AAV9-GFP-miR-23a, AAV9-SMN1, and AAV9-SMN1-miR-23a viruses. U-87 cells were transfected with 20 nmol/L SMN1-specific siRNA (or control siRNA) and then transduced with AAV9-GFP, AAV9-GFP-miR-23a, AAV9-SMN1, and AAV9-SMN1-miR-23a viruses at an MOI of 400,000. At 120 h post-transduction, the amount of miR-23a relative to the number of GAPDH gene copies in each sample was determined by qPCR (n=3) using the ΔΔC _T method. The data are presented as normalized mean miR-23a amounts with standard deviation. The relative amount of miR-23a in the untransfected and untransduced controls is taken as 100%.

Fig. 4 is a graph showing the expression of Senataxin after SMN1 knockdown by siRNA and transduction with AAV9-GFP, AAV9-GFP-miR-23a, AAV9-SMN1, and AAV9-SMN1-miR-23a viruses. U-87 cells were transfected with 20 nmol/L siRNA specific for wild-type SMN1 (or control siRNA) and then transduced with AAV9-GFP, AAV9-GFP-miR-23a, AAV9-SMN1, and AAV9-SMN1-miR-23a viruses at an MOI of 400,000. At 120 h post-transduction, the amount of Senataxin relative to the amount of Vinculin protein in each sample was determined by Western blot. The graph of normalized expression of Senataxin relative to Vinculin in experimental samples with standard deviations (n=3) is presented. The relative amount of Senataxin in the untransfected and untransduced control is taken as 100%.

For figures 1-4:

siNEG is a negative control siRNA,

siSMN1 is siRNA for SMN1.

Fig. 5 is a graph showing the survival curves of SMA mice injected with AAV9-SMN1 and AAV9-SMN1-miR-23a viruses. Survival curves of placebo-injected animals and wild-type control mice (from the same litter as the experimental mice) are also shown.

Definitions and General Methods

Unless otherwise defined herein, scientific and technical terms used in connection with the present invention shall have the meanings that are commonly understood by those skilled in the art.

In addition, unless the context otherwise requires, singular terms include plural terms, and plural terms include singular terms. Generally, the classification and methods of cell culture, molecular biology, immunology, microbiology, genetics, analytical chemistry, organic synthetic chemistry, medicinal and pharmaceutical chemistry, as well as hybridization and protein and nucleic acid chemistry described herein are well known to those skilled in the art and are widely used in the art. Enzymatic reactions and purification methods are carried out in accordance with the manufacturer's instructions, as is customary in the art, or as described herein.

"Isolated" means altered or removed from its natural state. For example, a nucleic acid or peptide naturally present in an animal is not "isolated," but the same nucleic acid or peptide partially or completely separated from the materials accompanying it in its natural state is "isolated." An isolated nucleic acid or protein may exist in a substantially purified form or may exist in a non-natural environment, such as, for example, a genetically modified cell.

The terms "naturally occurring," "native," or "wild-type" are used to describe something that can be found in nature as distinct from something produced artificially. For example, a protein or nucleotide sequence that is present in an organism (including a virus), that can be isolated from its source in nature, and that has not been intentionally modified by a technician in a laboratory, is naturally occurring.

The term "genome" refers to the complete genetic material of an organism.

In the present description and in the following claims, unless the context otherwise requires, the words “include” and “comprise” or variations thereof such as “having,” “includes,” “including,” “comprises,” or “containing,” are to be understood as including the stated whole or group of wholes, but not excluding any other whole or group of wholes.

Protein (Peptide)

As used herein, the terms "peptide", "polypeptide" and "protein" are used interchangeably and refer to a compound consisting of amino acid residues covalently linked by peptide bonds. A protein or peptide must contain at least two amino acids, and there is no limit to the maximum number of amino acids that a protein or peptide sequence may contain. Polypeptides include any peptide or protein containing two or more amino acids linked to each other by peptide bonds. As used herein, the term refers to both short chains, also commonly referred to in the art as, for example, peptides, oligopeptides and oligomers, and longer chains, commonly referred to in the art as proteins, of which there are many types. "Polypeptides" include, but are not limited to, for example, biologically active fragments, substantially homologous polypeptides, oligopeptides, homodimers, heterodimers, polypeptide variants, modified polypeptides, derivatives, analogs, fusion proteins. Polypeptides include natural peptides, recombinant peptides, synthetic peptides, or a combination thereof.

Nucleic acid molecules

The terms "nucleic acid", "nucleic sequence" or "nucleic acid sequence", "polynucleotide", "oligonucleotide", "polynucleotide sequence" and "nucleotide sequence", which are used interchangeably in this specification, denote a distinct sequence of nucleotides, modified or unmodified, defining a fragment or section of nucleic acid, containing or not containing non-natural nucleotides and being either double-stranded DNA or RNA, or single-stranded DNA or RNA, or transcription products of said DNA.

As used herein, polynucleotides include, but are not limited to, all nucleic acid sequences obtained by any methods available in the art, including, but not limited to, recombinant methods, i.e., cloning nucleic acid sequences from a recombinant library or cell genome, using conventional cloning and PCR technology, etc., and synthetic methods.

It should also be mentioned here that the present invention does not relate to nucleotide sequences in their natural chromosomal environment, i.e. in their natural state. The sequences of the present invention have been isolated and/or purified, i.e. taken directly or indirectly, for example by copying, whereby their environment has been at least partially modified. Thus, isolated nucleic acids obtained by genetic recombination, for example with the help of host cells, or obtained by chemical synthesis should also be understood here.

The term nucleotide sequence includes its complement unless otherwise specified. Thus, a nucleic acid having a certain sequence should be understood as including its complementary strand with its complementary sequence.

Adeno-associated virus (AAV)

Viruses of the Parvoviridae family are small DNA-containing animal viruses. The Parvoviridae family can be divided into two subfamilies: Parvovirinae, which infect vertebrates, and Densovirinae, which infect insects. By 2006, 11 serotypes of adeno-associated virus had been described (Mori, S. ET AL., 2004, “Two novel adeno-associated viruses from cynomolgus monkey: pseudotyping characterization of capsid protein,” Virology, T. 330 (2): 375-83). All known serotypes can infect cells of many tissue types. Tissue specificity is determined by the serotype of the capsid proteins, so adeno-associated virus-based vectors are constructed by specifying the required serotype. Additional information on parvoviruses and other members of the Parvoviridae is described in the literature (Kenneth I. Berns, “Parvoviridae: The Viruses and Their Replication,” Chapter 69 in Fields Virology (3d Ed. 1996)).

The genomic organization of all known AAV serotypes is very similar. The AAV genome is a linear, single-stranded DNA molecule that is less than approximately 5,000 nucleotides (nt) in length. Inverted terminal repeats (1TR) flank unique nucleotide coding sequences for the replication nonstructural proteins (Rep) and structural proteins (Cap). The Cap gene encodes the VP proteins (VP1, VP2, and VP3) that form the capsid. The terminal 145 nt are self-complementary and are organized in such a way that an energetically stable intramolecular duplex can be formed, forming a T-shaped hairpin structure. These hairpin structures function as origins of viral DNA replication, serving as primers for the cellular DNA polymerase complex. Following infection of mammalian cells with wild-type AAV (wtAAV), Rep genes (e.g., Rep78 and Rep52) are expressed through the P5 promoter and P19 promoter, respectively, and both Rep proteins have a defined function in viral genome replication. Splicing within the Rep open reading frame (Rep ORF) results in the expression of virtually four Rep proteins (e.g., Rep78, Rep68, Rep52, and Rep40). However, unspliced mRNA encoding the Rep78 and Rep52 proteins has been shown to be sufficient for AAV vector production in mammalian cells.

Vector

The term "vector" as used herein means a nucleic acid molecule capable of transporting another nucleic acid to which it is linked. Additionally, the term "vector" as used herein means a viral particle capable of transporting a nucleic acid.

As used herein, the term "expression" is defined as the transcription and/or translation of a particular nucleotide sequence driven by its promoter.

Application

"Gene therapy" is the insertion of genes into a subject's cells and/or tissues to treat a disease, usually a hereditary disease, whereby a defective mutant allele is replaced or supplemented with a functional allele.

"Treat," "treatment," and "therapy" refer to a method of alleviating or eliminating a biological disorder and/or at least one of its associated symptoms. As used herein, the term "alleviate" a disease, disorder, or condition means to reduce the severity and/or frequency of the symptoms of the disease, disorder, or condition. Additionally, references herein to "treatment" include references to curative, palliative, and prophylactic therapy.

In one aspect, the subject of treatment or patient is a mammal, preferably a human subject. The said subject may be male or female of any age.

The term “disorder” means any condition that can be improved by treatment according to the present invention.

"Disease" is a state of health of a subject where the subject cannot maintain homeostasis, and where, unless the disease is alleviated, the subject's health continues to deteriorate.

The term "subject", "patient", "individual" and the like are used interchangeably herein and refer to any animal that is amenable to the methods described herein. In certain non-limiting embodiments, the subject, patient or individual is a human. The aforementioned subject may be male or female of any age.

A "therapeutically effective amount" or "effective amount" is the amount of a therapeutic agent administered that will relieve to some degree one or more symptoms of the disease being treated.

Detailed description of the invention

Nucleic acid

In one aspect, the present invention relates to an isolated nucleic acid for producing a gene therapy viral preparation, which comprises a nucleic acid that encodes a SMN1 (survival motor neuron protein) protein with an amino acid sequence and a nucleic acid that encodes the microRNA miR-23a.

In some embodiments, the above nucleic acid is used to produce a gene therapy viral preparation, which is an expression vector according to the invention or a recombinant virus based on AAV9 according to the invention.

An "isolated" nucleic acid molecule is a nucleic acid molecule that is identified and separated from at least one contaminant nucleic acid molecule with which it is normally associated in a natural source of the nuclease nucleic acid. An isolated nucleic acid molecule is different from the form or composition in which it is naturally found. Thus, an isolated nucleic acid molecule is different from a nucleic acid molecule that naturally exists in cells.

In some embodiments, the isolated nucleic acid is DNA.

It will be obvious to those skilled in the art that the SMN1 protein with the amino acid sequence of SEQ ID NO: 1 can be encoded by a wide variety of different DNA sequences, given the degeneracy of the genetic code. It is well known to those skilled in the art to obtain such alternative DNA sequences encoding the same amino acid sequences. Such variant DNA sequences are within the scope of the present invention.

In some embodiments, an isolated nucleic acid that encodes an SMN1 protein with the amino acid sequence of SEQ ID NO: 1 comprises a nucleotide sequence

In some embodiments, the isolated nucleic acid comprises the miR-23a microRNA, which has the nucleotide sequence

In some embodiments, the isolated nucleic acid comprises a nucleic acid encoding the miR-23a microRNA that comprises the nucleotide sequence

The above nucleic acid encoding the miR-23a microRNA with the nucleotide sequence of SEQ ID NO: 4 is a nucleic acid before processing.

After processing, the nucleic acid that encodes the miR-23a microRNA has the nucleotide sequence

In some embodiments, the isolated nucleic acid comprises the following elements in the 5' to 3' direction:

a nucleic acid that encodes the SMN1 protein with the amino acid sequence of SEQ ID NO: 1, and

nucleic acid that encodes the microRNA miR-23a.

Expression cassette. Expression vector.

In one aspect, the present invention relates to an expression cassette that comprises any of the above nucleic acids.

The term "expression cassette" as used herein refers in particular to a fragment of DNA that is capable, under appropriate circumstances, of driving the expression of a polynucleotide encoding a polypeptide of interest that is included in said expression cassette. When introduced into a host cell, the expression cassette is, among other things, capable of engaging the cellular machinery to transcribe the polynucleotide encoding the polypeptide of interest into RNA, which is then typically further processed and finally translated into the polypeptide of interest. The expression cassette may be contained in an expression vector.

The term "expression cassette" or "expression cassette" as used herein, in particular, refers to a fragment of DNA that is capable, under appropriate circumstances, of driving the expression of a polynucleotide encoding a polypeptide of interest that is included in said expression cassette. When introduced into a host cell, the expression cassette is, among other things, capable of engaging the cellular machinery to transcribe the polynucleotide encoding the polypeptide of interest into RNA, which is then typically further processed and finally translated into the polypeptide of interest. The expression cassette may be contained in an expression vector.

The expression cassette of the present invention comprises a promoter element. The term "promoter" as used herein particularly refers to a DNA element that promotes transcription of a polynucleotide to which the promoter is operably linked. A promoter may also form part of a "promoter/enhancer" element. Although the physical boundaries between the "promoter" and "enhancer" elements are not always clear, the term "promoter" generally refers to a site on a nucleic acid molecule to which RNA polymerase and/or associated factors bind and from which transcription is initiated. Enhancers enhance promoter activity temporally as well as spatially. A variety of promoters are known in the art that are transcriptionally active in a wide range of cell types. Promoters can be divided into two classes: those that function constitutively and those that are regulated by induction or release of repression. Both classes are useful for protein expression. Promoters which are used for the production of high levels of polypeptides in eukaryotic cells and in particular in mammalian cells should be strong and preferably should be active in a wide range of cell types. Strong constitutive promoters which are capable of driving expression in many cell types are well known in the art and therefore do not need to be described in detail herein. According to the teaching of the present invention, it is preferred to use a cytomegalovirus (CMV) promoter. A promoter or promoter/enhancer derived from the immediate early (IE) region of human cytomegalovirus (hCMV) is particularly suitable as a promoter in the expression cassette of the present invention. The immediate early (IE) region of human cytomegalovirus (hCMV) and functional expression driving fragments and/or functional expression enhancing fragments derived therefrom are, for example, described in EP 0 173 177 and EP 0 323 997 and are also well known in the art. Thus, several fragments of the immediate early (IE) region of hCMV can be used as a promoter and/or promoter/enhancer. According to one embodiment of the invention, the human CMV promoter is used in the expression cassette of the present invention.

In some embodiments, the expression cassette includes the following elements in the 5' to 3' direction:

left (first) ITR (inverted terminal repeats);

CMV (cytomegalovirus) enhancer;

CMV (cytomegalovirus) promoter;

intron of the hBG1 gene (hemoglobin gamma-1 subunit gene);

nucleic acid that encodes the SMN1 protein;

hGH1 polyadenylation signal (human growth hormone gene polyadenylation signal);

SV40 promoter (simian virus 40 promoter);

nucleic acid that encodes the microRNA miR-23a;

SV40 polyadenylation signal (simian virus 40 polyadenylation signal) and right (second) ITR.

In some variants, the left (first) ITR (inverted terminal repeats) has the following nucleic acid sequence:

In some CMV (cytomegalovirus) variants, the enhancer has the following nucleic acid sequence:

In some CMV (cytomegalovirus) variants, the promoter has the following nucleic acid sequence:

In some variants, the intron of the hBG1 gene (hemoglobin gamma-1 subunit gene) has the following nucleic acid sequence:

In some embodiments, the hGH1 polyadenylation signal (the human growth hormone gene polyadenylation signal) has the following nucleic acid sequence:

In some SV40 variants, the promoter (simian virus 40 promoter) has the following nucleic acid sequence:

In some embodiments, the SV40 polyadenylation signal (simian virus 40 polyadenylation signal) has the following nucleic acid sequence:

In some variants, the right (second) ITR has the following nucleic acid sequence:

In some embodiments, the expression cassette comprises a nucleic acid having a nucleotide sequence

In one aspect, the present invention relates to an expression vector that comprises any of the above nucleic acids or any of the above cassettes.

In some embodiments of the invention, the vector is a plasmid, i.e., a circular double-stranded piece of DNA into which additional DNA segments can be ligated.

In some embodiments of the invention, the vector is a viral vector in which additional DNA segments can be ligated into the viral genome.

In some embodiments, vectors are capable of autonomous replication in a host cell into which they are introduced (e.g., bacterial vectors having a bacterial origin of replication and mammalian episomal vectors). In other embodiments, vectors (e.g., non-episomal mammalian vectors) can be integrated into the host cell genome upon introduction into the host cell, and thus replicate along with the host gene. Moreover, some vectors are capable of directing the expression of genes to which they are operably linked. Such vectors are referred to herein as "recombinant expression vectors" (or simply "expression vectors" ("expression vector" or "expression vector")).

Expression vectors include plasmids, retroviruses, adenoviruses, adeno-associated viruses (AAV), plant viruses such as cauliflower mosaic virus, tobacco mosaic viruses, cosmids, YAC, EBV derived episomes, and the like. DNA molecules can be ligated into a vector such that transcription and translation control sequences in the vector perform the intended function of regulating transcription and translation of DNA. The expression vector and expression control sequences can be selected to be compatible with the expression host cell used. DNA molecules can be introduced into the expression vector by standard methods (e.g., ligation of complementary restriction sites or blunt end ligation if restriction sites are not present).

The recombinant expression vector may also encode a leader peptide (or signal peptide) that facilitates production of the protein of interest by the host cell. The gene for the protein of interest may be cloned into the vector such that the signal peptide is linked in-frame to the amino terminus of the protein of interest. The leader peptide (or signal peptide) may be an immunoglobulin leader peptide or a different leader peptide (i.e., a leader peptide of a protein that is not immunoglobulin in nature).

In addition to the SMN1 and miR-23a microRNA genes of the present invention, the recombinant expression vectors of the present invention may carry regulatory sequences that control the expression of the SMN1 gene and the miR-23a microRNA gene in a host cell. Those skilled in the art will appreciate that the design of the expression vector, including the choice of regulatory sequences, may depend on factors such as the selection of the host cell for transformation, the level of expression of the desired protein, etc. Preferred regulatory sequences for a mammalian expression host cell include viral elements that provide high level expression of proteins in mammalian cells, such as promoters and/or enhancers derived from a retroviral LTR, cytomegalovirus (CMV) (e.g., the CMV promoter/enhancer), simian virus 40 (SV40) (e.g., the SV40 promoter/enhancer), adenovirus (e.g., the adenovirus large late promoter (AdMLP)), polyoma virus, as well as strong mammalian promoters such as the native immunoglobulin promoter or the actin promoter.

The expression "control sequences" refers to DNA sequences required for the expression of a functionally linked coding sequence in a given host organism. Suitable control sequences for prokaryotes are, for example, a promoter, optionally an operator, and a ribosome binding site. Promoters, polyadenylation signals, and enhancers are known to be present in eukaryotic cells.

As used herein, the term "promoter" or "transcriptional regulatory sequence" or "regulatory sequence" refers to a nucleic acid fragment that controls the transcription of one or more coding sequences and that is located upstream of the transcription initiation site of the coding sequence and that is structurally identifiable by the presence of a binding site for DNA-dependent RNA polymerase, transcription initiation sites, and other DNA sequences, including, but not limited to, transcription factor binding sites, repressor and activator protein binding sites, and any other nucleotide sequences known to those skilled in the art that directly or indirectly regulate the level of transcription from the promoter. A "constitutive" promoter is one that is active in most tissues under normal physiological and developmental conditions. An "inducible" promoter is one that is subject to physiological or developmental regulation, such as by exposure to a chemical inducer. A "tissue-specific" promoter is active only in specific tissue or cell types.

The terms "enhancers" or "enhancer" as used herein may refer to a DNA sequence that is located adjacent to a DNA sequence encoding a recombinant product. Enhancer elements are typically located 5' of a promoter element or may be located downstream of or within a coding DNA sequence (e.g., a DNA sequence transcribed or translated into a recombinant product or products). Thus, an enhancer element may be located 100 base pairs, 200 base pairs, or 300 base pairs or more upstream of or after a DNA sequence encoding a recombinant product. Enhancer elements may increase the amount of recombinant product expressed from a DNA sequence beyond that resulting from a single promoter element. A variety of enhancer elements are available to those skilled in the art.

The term "expression control sequence" as used herein refers to polynucleotide sequences that are necessary to affect the expression and processing of coding sequences to which they are ligated. Expression control sequences include appropriate transcription initiation, termination, promoter, and enhancer sequences; efficient RNA processing signals such as splicing and polyadenylation signals; sequences that stabilize cytoplasmic mRNA; sequences that enhance translation efficiency (i.e., the Kozak consensus sequence); sequences that enhance protein stability; and, optionally, sequences that enhance protein secretion. The nature of such control sequences varies depending on the host organism; in prokaryotes, such control sequences typically include a promoter, a ribosome binding site, and transcription termination sequences; in eukaryotes, such control sequences typically include promoters and transcription termination sequences. The term "control sequences" includes, at a minimum, all components whose presence is essential for expression and processing, and may also include additional components whose presence is beneficial, such as leader sequences and fusion sequences.

In one embodiment of the present invention, an "expression vector" refers to a vector comprising one or more polynucleotide sequences of interest, genes of interest, or "transgenes" that are flanked by parvovirus or inverted terminal repeat (ITR) sequences.

Neither the cassette nor the vector according to the invention contains nucleotide sequences of genes encoding non-structural proteins (Rep) and structural proteins (Cap) of the adeno-associated virus.

Recombinant virus based on AAV9 (adeno-associated virus 9 serotype)

In one aspect, the present invention relates to a recombinant AAV9 (adeno-associated virus 9)-based virus for expressing the SMN1 gene in target cells, which comprises a capsid and any of the above cassettes.

The term "recombinant AAV-based virus" (or "AAV-based virus-like particle", or "recombinant AAV viral strain", or "recombinant AAV vector", or "rAAV vector") as used herein refers to the above expression cassette (or the above expression vector), which is contained within the AAV capsid.

The Cap gene, in addition to other alternative products, encodes three capsid proteins (VP1, VP2, and VP3). The VP1, VP2, and VP3 proteins are in a 1:1:10 ratio, forming an icosahedral capsid (Xie Q. et al. The atomic structure of adeno-associated virus (AAV-2), a vector for human gene therapy. Proc Natl Acad Sci USA, 2002; 99:10405-10410). Transcription of these genes begins from a single promoter, p40. The molecular weight of the corresponding proteins (VP1, VP2, and VP3) is 87, 72, and 62 kDa, respectively. All three proteins are translated from a single mRNA. After transcription, the pre-mRNA can be spliced in two different ways, with a longer or shorter intron being excised and mRNAs of different nucleotide lengths being formed.

In the formation of a recombinant AAV-based virus (rAAV), an expression cassette flanked by an ITR is packaged into the AAV capsid. The genes required for AAV replication, as noted above, are not included in the cassette.

The expression cassette DNA is packaged into the viral capsid as a single-stranded DNA (ssDNA) molecule approximately 3,000 nucleotides long. Once a cell is infected with the virus, the single-stranded DNA is converted to a double-stranded DNA (dsDNA) form. Only dsDNA can be used by the cell's proteins, which transcribe the gene or genes it contains into RNA.

In some embodiments, the recombinant AAV9-based virus has a capsid that includes the AAV9 VP1 protein.

In some embodiments, the recombinant AAV9-based virus has a capsid that includes the AAV9 VP1 protein having the following amino acid sequence:

In some embodiments, the recombinant AAV9-based virus has a capsid that includes an AAV9 VP1 protein having the amino acid sequence of SEQ ID NO: 7 with one or more point mutations.

In some embodiments, the recombinant AAV9-based virus has a capsid that includes the AAV9 VP2 protein.

In some embodiments, the recombinant AAV9-based virus has a capsid that includes the AAV9 VP2 protein, which has the following amino acid sequence:

In some embodiments, the recombinant AAV9-based virus has a capsid that includes an AAV9 VP2 protein having the amino acid sequence of SEQ ID NO: 8 with one or more point mutations.

In some embodiments, the recombinant AAV9-based virus has a capsid that includes the AAV9 VP3 protein.

In some embodiments, the recombinant AAV9-based virus has a capsid that includes the AAV9 VP3 protein, which has the following amino acid sequence:

In some embodiments, the recombinant AAV9-based virus has a capsid that includes an AAV9 VP3 protein having the amino acid sequence of SEQ ID NO: 9 with one or more point mutations.

In some embodiments, the recombinant AAV9-based virus has a capsid that includes the AAV9 VP1, VP2, and VP3 proteins.

In some embodiments, the recombinant AAV9-based virus has a capsid that includes proteins VP1 with the amino acid sequence of SEQ ID NO: 7, VP2 with the amino acid sequence of SEQ ID NO: 8, and VP3 with the amino acid sequence of SEQ ID NO: 9.

In some embodiments, the recombinant AAV9-based virus has a capsid that includes VP1 proteins with the amino acid sequence of SEQ ID NO: 7 with one or more point mutations, VP2 with the amino acid sequence of SEQ ID NO: 8 with one or more point mutations, and VP3 with the amino acid sequence of SEQ ID NO: 9 with one or more point mutations.

By "multiple point mutations" we mean two, three, four, five, six, seven, eight, nine, or ten point substitutions.

Particularly preferred variants include substitutions (mutations) that are conservative in nature, i.e., those substitutions that occur within a family of amino acids that are grouped together by their side chains. In particular, amino acids are usually divided into four families: (1) acidic - aspartate and glutamate; (2) basic - lysine, arginine, histidine; (3) nonpolar - alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan; and (4) uncharged polar - glycine, asparagine, glutamine, cysteine, threonine series, tyrosine. Phenylalanine, tryptophan, and tyrosine are sometimes classified as aromatic amino acids. For example, it is reasonably predictable that a selected substitution of leucine for isoleucine or valine, aspartate for glutamate, threonine for serine, or a similar conservative substitution of an amino acid for a structurally related amino acid will not have an important effect on biological activity. For example, a polypeptide of interest may include up to about 5-10 conservative or non-conservative amino acid substitutions, provided that the desired function of the molecule remains intact.

A variant of point mutations in the sequences of the AAV9 VP1, VP2 or VP3 proteins using amino acid substitutions is a replacement of at least one amino acid residue in the AAV9 VP1, VP2 or VP3 protein with another amino acid residue.

Conservative substitutions are shown in Table A under the heading "preferred substitutions."

In some embodiments, the recombinant AAV9-based virus has a capsid that includes an AAV9 VP1 protein having the amino acid sequence of SEQ ID NO: 7 or the amino acid sequence of SEQ ID NO: 7 with one or more point mutations, and the expression cassette includes the following elements in the 5'-to-3'-direction:

left (first) ITR (inverted terminal repeats);

CMV (cytomegalovirus) enhancer;

CMV (cytomegalovirus) promoter;

intron of the hBG1 gene (hemoglobin gamma-1 subunit gene);

nucleic acid that encodes the SMN1 protein;

hGH1 polyadenylation signal (human growth hormone gene polyadenylation signal);

SV40 promoter (simian virus 40 promoter);

nucleic acid that encodes the microRNA miR-23a;

SV40 polyadenylation signal (simian virus 40 polyadenylation signal) and right (second) ITR.

In some embodiments, the recombinant AAV9-based virus has a capsid that includes VP1 proteins with the amino acid sequence of SEQ ID NO: 7, VP2 proteins with the amino acid sequence of SEQ ID NO: 8, and VP3 proteins with the amino acid sequence of SEQ ID NO: 9, and the expression cassette includes the following elements in the 5'-to-3'-direction:

left (first) ITR (inverted terminal repeats);

CMV (cytomegalovirus) enhancer;

CMV (cytomegalovirus) promoter;

intron of the hBG1 gene (hemoglobin gamma-1 subunit gene);

nucleic acid that encodes the SMN1 protein;

hGH1 polyadenylation signal (human growth hormone gene polyadenylation signal);

SV40 promoter (simian virus 40 promoter);

nucleic acid that encodes the microRNA miR-23a;

SV40 polyadenylation signal (simian virus 40 polyadenylation signal) and right (second) ITR.

In some embodiments, the recombinant AAV9-based virus has a capsid that includes VP1 proteins with the amino acid sequence of SEQ ID NO: 7 with one or more point mutations, VP2 with the amino acid sequence of SEQ ID NO: 8 with one or more point mutations, and VP3 with the amino acid sequence of SEQ ID NO: 9 with one or more point mutations, and the expression cassette includes the following elements in the direction from the 5' end to the 3' end:

left (first) ITR (inverted terminal repeats);

CMV (cytomegalovirus) enhancer;

CMV (cytomegalovirus) promoter;

intron of the hBG1 gene (hemoglobin gamma-1 subunit gene);

nucleic acid that encodes the SMN 1 protein;

hGH1 polyadenylation signal (human growth hormone gene polyadenylation signal);

SV40 promoter (simian virus 40 promoter);

nucleic acid that encodes the microRNA miR-23a;

SV40 polyadenylation signal (simian virus 40 polyadenylation signal) and right (second) 1TR.

In some embodiments, the recombinant AAV9-based virus has a capsid that includes an AAV9 VP1 protein having the amino acid sequence of SEQ ID NO: 7 or the amino acid sequence of SEQ ID NO: 7 with one or more point mutations, and the expression cassette includes a nucleic acid of SEQ ID NO: 6.

In some embodiments, the recombinant AAV9-based virus has a capsid that includes VP1 proteins with the amino acid sequence of SEQ ID NO: 7, VP2 with the amino acid sequence of SEQ ID NO: 8, and VP3 with the amino acid sequence of SEQ ID NO: 9, and the expression cassette includes a nucleic acid with SEQ ID NO: 6.

In some embodiments, the recombinant AAV9-based virus has a capsid that includes VP1 proteins with the amino acid sequence of SEQ ID NO: 7 with one or more point mutations, VP2 with the amino acid sequence of SEQ ID NO: 8 with one or more point mutations, and VP3 with the amino acid sequence of SEQ ID NO: 9 with one or more point mutations, and the expression cassette includes a nucleic acid with SEQ ID NO: 6.

Pharmaceutical composition

In one aspect, the present invention relates to a pharmaceutical composition for delivering an SMN1 gene to target cells, which comprises any of the above-mentioned recombinant AAV9-based viruses in combination with one or more pharmaceutically acceptable excipients.

The active substance in the above composition is in an effective amount, for example, in a biologically effective amount.

In particular embodiments, the present invention relates to a pharmaceutical composition comprising a recombinant AAV9-based virus of the invention in a pharmaceutically acceptable carrier or in other pharmaceutical agents, adjuvants, diluents, etc. The carrier for injection is usually liquid. The carrier for other modes of administration can be either solid or liquid, such as sterile pyrogen-free water or sterile pyrogen-free phosphate-buffered saline. For administration by inhalation, the carrier is respirable and is preferably in solid or liquid dispersed form. As an injection medium, it is preferable to use water containing additives conventional for injection solutions, such as stabilizing agents, salts or saline solutions and/or buffers.

"Pharmaceutical composition" means a composition comprising the above-mentioned recombinant AAV9-based virus of the invention and at least one component selected from the group consisting of pharmaceutically acceptable and pharmacologically compatible excipients such as fillers, solvents, diluents, carriers, auxiliary, distributing, delivery vehicles, preservatives, stabilizers, emulsifiers, suspending agents, thickeners, regulators of prolonged delivery, the choice and ratio of which depends on the nature and route of administration and dosage. The pharmaceutical compositions of the present invention and the methods for their manufacture will be undoubtedly obvious to those skilled in the art. The manufacture of pharmaceutical compositions should preferably comply with the requirements of GMP (good manufacturing practice). The composition may include a buffer composition, tonic agents, stabilizers and solubilizers.

A "pharmaceutically acceptable" material is one that does not have biological or other contraindications, e.g., the material can be administered to a subject without any undesirable biological effects. Thus, such pharmaceutical compositions can be used, for example, to transfect a cell ex vivo or to administer in vivo a recombinant AAV9-based virus of the invention directly to a subject.

The term "excipient" or "auxiliary substance" is used in this document to describe any component different from those previously described in this invention. These are substances of inorganic or organic origin used in the process of production, manufacturing of medicinal products to impart the necessary physicochemical properties to them.

A "stabilizer" is an excipient or a mixture of two or more excipients that ensures the physical and/or chemical stability of the active agent.

The term "buffer", "buffer composition", "buffering agent" means a solution capable of maintaining a pH value due to the interaction of acidic and alkaline components included in its composition, which enables the rAAV9-based vector preparation to exhibit resistance to pH changes. In general, the pH values of the pharmaceutical composition from 4.0 to 8.0 are preferable. For example, acetate, phosphate, citrate, histidine, succinate, etc. buffer solutions can be used as buffer agents, but are not limited to them.

A pharmaceutical composition is "stable" if the active agent retains its physical stability and/or chemical stability and/or biological activity during the stated shelf life at a storage temperature of, for example, 2-8°C. Preferably, the active agent retains both physical and chemical stability, as well as biological activity. The shelf life is selected based on the results of stability studies under accelerated and natural storage.

The pharmaceutical composition of the present invention may be manufactured, packaged, or commercially sold in a single unit dose or multiple single unit doses in a finished dosage form. As used herein, the term "unit dose" means a discrete quantity of the pharmaceutical composition containing a predetermined amount of the active ingredient. The quantity of the active ingredient is typically equal to the dosage of the active ingredient to be administered to the subject, or a convenient fraction of such a dosage, such as one-half or one-third of such a dosage.

Application

The authors unexpectedly found that miR-23a enhances the functional effect of SMN1 in vitro and established a synergistic effect of SMN1 and miR-23a in the treatment of SMA in an animal model of the disease.

In one aspect, the present invention relates to the use of any of the above-mentioned recombinant AAV9-based viruses or the above-mentioned composition for delivering the SMN1 gene to target cells.

In one aspect, the present invention relates to the use of any of the above-mentioned recombinant AAV9-based viruses or the above-mentioned composition for the survival of a subject who has spinal muscular atrophy and/or lacks fully functional copies of the SMN1 gene.

In one aspect, the present invention relates to the use of any of the above-mentioned recombinant AAV9-based viruses or the above-mentioned composition for providing SMN1 protein to a subject who has spinal muscular atrophy and/or lacks fully functional copies of the SMN1 gene.

In one aspect, the present invention relates to the use of any of the above-mentioned recombinant AAV9-based viruses or the above-mentioned composition for the treatment of spinal muscular atrophy in a subject having spinal muscular atrophy.

In one aspect, the present invention relates to a method of modulating motor function in a subject with a motor neuron disorder, comprising administering a therapeutically effective amount of any of the above-mentioned recombinant AAV9-based viruses or the above-mentioned composition to the cells of the subject.

In one aspect, the present invention relates to a method of providing SMN protein to a subject with spinal muscular atrophy, comprising administering a therapeutically effective amount of any of the above-mentioned recombinant AAV9-based viruses or the above-mentioned composition to cells of the subject in need thereof.

In one aspect, the present invention relates to a method of delivering an SMN1 gene to target cells of a subject with spinal muscular atrophy, comprising administering any of the above-mentioned recombinant AAV9-based viruses or the above-mentioned composition to the cells of the subject.

In one aspect, the present invention relates to a method of treating spinal muscular atrophy in a subject, which comprises administering a therapeutically effective amount of any of the above-mentioned recombinant AAV9-based viruses or the above-mentioned composition to a subject with spinal muscular atrophy.

Absence of fully functional copies of the SMN1 gene refers to inactivating mutations or deletions in all copies of the SMN1 gene in the genome that result in a loss or defect in the function of the SMN1 gene.

By subject is meant any animal that is susceptible to the methods described herein. In certain non-limiting embodiments, the subject is a human. The aforementioned subject may be male or female of any age.

A subject in need of delivery of the SMN1 gene to target cells, or a subject in need of providing SMN1 protein, or a subject in need of delivery of the SMN1 gene to target cells, means a subject who has spinal muscular atrophy or a subject who has inactivating mutations or deletions in the SMN1 gene that result in a loss or defect in the function of the SMN 1 gene.

Examples of routes of administration include topical, intranasal, inhalational, transmucosal, transdermal, enteral (e.g., oral, rectal), parenteral (e.g., intravenous, subcutaneous, intradermal, intramuscular) administration, and injection directly into tissue or into an organ.

Injectable preparations can be prepared in conventional dosage forms: as liquid solutions or suspensions, solid forms suitable for preparing solutions or suspensions in liquid prior to injection, or as emulsions. Alternatively, the above-mentioned recombinant AAV9-based virus of the present invention can be administered locally rather than systemically, for example, as a depot or in a sustained-release composition.

The recombinant AAV9-based virus is administered to the body in an effective amount. The recombinant AAV9-based virus is preferably administered to the body in a biologically effective amount. A "biologically effective" amount of a recombinant virus is an amount that is sufficient to cause infection (or transduction) and expression of a nucleic acid sequence in a cell. If the virus is administered to a cell in vivo (e.g., the virus is administered to a subject as described below), a "biologically effective" amount of the viral vector is an amount that is sufficient to cause transduction and expression of the nucleic acid sequence in the target cell.

The dosages of the above recombinant AAV9-based virus of the present invention will depend on the route of administration, the specific viral vector, and can be determined by routine methods. Exemplary dosages for achieving a therapeutic effect are viral titers of at least about 10 ⁵ , 10 ⁶ , 10 ⁷ , 10 ⁸ , 10 ⁹ , 10 ¹⁰ , 10 ¹¹ , 10 ¹² , 10 ¹³ , ^{10 14} , 10 ¹⁵ , 10 ¹⁶ transducing units or more, preferably about 10 ⁹ to 10 ¹⁵ transducing units, even more preferably 10 ¹⁴ transducing units per kilogram.

The cell for administering the above-mentioned recombinant AAV9-based virus of the invention may be any type of cell, including, but not limited to, motor neurons or other nervous system tissues, epithelial cells (e.g., epithelial cells of the skin, respiratory tract and intestine), liver cells, muscle cells, spleen cells, fibroblasts, endothelial cells and the like.

The above-mentioned recombinant AAV9-based virus according to the invention is not used to modify the genetic integrity of human germline cells.

Examples

For a better understanding of the invention, the following examples are given. These examples are given for illustrative purposes only and should not be construed as limiting the scope of the invention in any way.

All publications, patents and patent applications mentioned in this specification are incorporated herein by reference. Although the above invention has been described in some detail by way of illustration and example for the purpose of avoiding ambiguity, it will be readily apparent to those skilled in the art, based on the teachings disclosed in this invention, that certain changes and modifications may be made without departing from the spirit and scope of the accompanying embodiments of the invention.

Materials and general methods

Recombinant DNA methods

DNA manipulations followed standard procedures described in Sambrook, J., et al., Molecular cloning: A laboratory manual; Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 1989. Molecular biology reagents were used according to the manufacturers' instructions. Briefly, plasmid DNA for further manipulations was produced in E. coli cells grown under selective pressure with antibiotics to ensure that plasmids were not lost in the cell population. Plasmid DNA was isolated from the cells using commercial kits, measured for concentration, and used for cloning by restriction endonuclease digestion or PCR amplification. DNA fragments were ligated together using ligases and transformed into bacterial cells for clone selection and further development. All resulting genetic constructs were confirmed by restriction patterns and complete Sanger sequencing.

Gene synthesis

The desired gene segments were obtained from oligonucleotides created by chemical synthesis. Gene segments of 300 to 1000 bp in length, flanked by unique restriction sites, were assembled by annealing the oligonucleotides on each other, followed by PCR amplification from the outer primers. The result was a mixture of fragments, including the desired one. The fragments were cloned into intermediate vectors by restriction sites, after which the DNA sequences of the subcloned fragments were confirmed by DNA sequencing.

Determination of DNA sequences

DNA sequences were determined by Sanger sequencing. DNA and protein sequence analysis and sequence data processing were performed using SnapGene 4.2 and higher for sequence generation, mapping, analysis, annotation, and illustration.

Cultivation of cell cultures

The HEK293 (Human Embryonic Kidney clone 293) and U-87 MG (human glioblastoma) cell lines were used in the experiments. The cells were cultured under standard conditions at 37°C and 5% CO ₂ , in a complete DMEM nutrient medium supplemented with 10% FBS and antibiotic. For HSMC cultivation, the culture plastic was pre-coated with collagen (Gibco). Cell passage was performed upon reaching 80-90% confluency. Cell viability was assessed using Trypan Blue staining and a Goryaev chamber or using PI staining and flow cytometry.

Transfection of cells with siRNA

Commercial siRNA specific to the SMN1 gene along with non-specific control was ordered from the manufacturer (ThermoFisher Scientific).

Cell lines were seeded in 6-well plates the day before transfection so that they reached 70-80% confluency at the time of transfection. Transfection was performed using commercial lipofection kits according to the manufacturer's protocol. Transduction was performed 24 h after transfection (see below). At 120 h after transduction, cells were treated with trypsin solutions or similar, removed from the substrate, washed in phosphate buffer and collected for further analysis of target gene and protein expression. Enzyme-linked immunosorbent assay (ELISA) was used to monitor the level of SMN protein knockdown.

All samples were placed in three experimental replicates.

Gene expression analysis

SMN1 mRNA expression as well as miR-23a expression were verified by quantitative PCR. Briefly, primers and probe specific for the nucleotide sequence encoding SMN 1 protein were used. For miR-23a, a commercial miR-23a-processed specific qPCR kit (Thermo Fisher Scientific) was used. Primers and probe specific for the housekeeping gene GAPDH were used to control the amount of input RNA. Calibration curves were constructed for each primer and probe set using known copy numbers of linearized plasmid DNA containing the amplified sequence of the respective gene. Expression analysis was performed by determining the copy numbers of SMN1 and GAPDH in each sample from the calibration curves and then normalizing the copy number of SMN1 per 10,000 copies of GAPDH. The obtained values were compared with each other for different samples within the same experiment.

Determination of SMN protein expression

The Thermo Fisher Scientific kit was used to determine the amount of total protein. Cell pellets were lysed, the resulting lysates were added to the wells of a microplate along with standard dilutions of BSA with a known concentration. The working reagent was added to the samples and they were incubated. At the end of the incubation, the absorption was measured at a wavelength of 562 nm on a microplate reader. The concentration of total protein in the studied samples was calculated using the curve of the standard BSA sample. Then, the lysates were diluted to a total protein concentration of 10 m kg/ml.

The SMN protein content in the cells was assessed by enzyme-linked immunosorbent assay (ELISA) using a commercial Abcam kit according to the manufacturer's instructions. The test samples and standards with known concentrations were added to the wells of a microplate coated with antibodies to SMN. After incubation and washing, SMN-specific polyclonal antibodies for detection were added to each well, incubated, and washed. Secondary antibodies conjugated with horseradish peroxidase were added and incubated. Excess reagents were washed off and TMB substrate was added. After a short incubation, the enzymatic reaction was stopped with a stop reagent. The optical density of the yellow-colored product was measured spectrophotometrically at a wavelength of 450 nm. The amount of SMN in the test samples was determined using a calibration graph of the dependence of optical density on the SMN concentration in the standards.

Determination of expression of the SMN target protein Senataxin (functional test)

Senataxin protein is a multifunctional enzyme involved in the resolution of DNA-RNA structures called R-loops that arise during transcription in the absence or insufficient expression of SMN protein. A direct correlation between SMN and Senataxin expression levels has been shown in the literature, and indirect activation of Senataxin is one of the functions of SMN. In this regard, changes in Senataxin expression have been used as a functional test for SMN.

Senataxin expression levels were determined using Western blot. Briefly, cells were lysed with RIPA buffer supplemented with protease inhibitor cocktail after experimental treatments (transfection, transduction, incubation) to obtain protein lysate samples. Samples were loaded onto 10% polyacrylamide denaturing gels, and proteins were then transferred to PVDF membrane.

The membrane was incubated for 1 hour in a solution of 5% bovine serum albumin (BSA) in TBS-T buffer, after which the BSA solution was replaced with a solution of 1% BSA in TBS-T with the addition of primary antibodies specific for Senataxin. After 2 hours of incubation, the membrane was washed 3 times with a solution of 1% BSA in TBS-T and a solution of secondary antibodies in TBS-T with 1% BSA was added. Secondary antibodies conjugated with the enzyme horseradish peroxidase were used.

The secondary antibodies were washed 3 times with 1% BSA in TBS-T, and the protein signal on the membrane was visualized using a chemiluminescence substrate and a gel imaging system. The resulting images were saved in digital format, and the signal intensity was analyzed using the gel imaging system software.

The protein signal of the housekeeping gene vinculin was used as a control for normalization. Staining for it was carried out similarly.

Assembly and purification of viral particles of recombinant AAV vectors

To assemble AAV viral particles containing the SMNJ gene or the GFP control gene, HEK293 packaging cells were used, into which the following plasmids were transfected using polyethyleneimine 3:

1. A plasmid containing the AAV genome with a cassette for transgene expression (SNMJ, GFP, SMN1+miR-23a or GFP+miR-23a);

2. Plasmid for expression of the AAV9 serotype Cap gene and the AAV2 serotype Rep gene. Each gene encodes several protein products using alternative reading frames;

3. Plasmid for expression of adenovirus Ad5 genes required for assembly and packaging of AAV capsids.

After 72 hours, the cells were lysed and the viral particles were purified and concentrated using filtration and chromatography. The titer of viral particles was determined using quantitative PCR with primers and probe specific to a region of the recombinant viral genome and was expressed as the number of copies of viral genomes per 1 ml.

Transduction of cell cultures

The U-87 cell line was seeded similarly to the transfection experiments, transfected with siRNA, then the preparation with viral particles was added and the cells were analyzed after 120 h. Transduction efficiency was calculated by analyzing the percentage of GFP + cells.

Experiments to test transduction efficiency were performed in advance for the cultures used. Briefly, the AAV9-GFP virus preparation was transduced into cell lines in different ratios of cells and virus particles. The ratio of the number of virus particles to cells is called the multiplicity of infection (MOI). The MOI of the AAV9-GFP virus varied from 50,000 to 1,000,000. As a result, an optimal MOI of 400,000 was selected for the U-87 line. Further work with U-87 transduction was carried out with this MOI for all viruses.

After transduction, gene and protein expression analysis was performed as described above.

All samples were set up in three experimental replicates.

Injection of viral preparations into a mouse model of spinal muscular atrophy (SMA)

AAV9-SMN1, AAV9-GFP, AAV9-SMN1-miR-23a, and AAV9-GFP-miR-23a viral particles were injected into SMA model mice. These mice do not express the mouse Smn gene, but have one copy of the human SMN2 gene and one copy of the human SMN1 gene with exon 7 missing (SMN1Δ7) in their genome. Without intervention or with placebo injection, these mice are born, but gain weight poorly after birth and die on average after 21 days.

Mice of the model line were genotyped on the first day after birth, after which mice lacking any copy of the Smn gene were injected systemically (into the tail vein) with either a placebo (a solution containing no viral particles but containing a buffer for their dilution) or one of the viruses at a dose of 3.2×10 ¹⁴ vg/kg of weight. After this, the animals were kept under standard conditions and their weight was measured daily, and survival curves were plotted. Ninety days after the injection, surviving animals were killed for tissue analysis and the experiment was terminated.

Example 1. Assembly of genetic constructs carrying a recombinant AAV genome and encoding the SMN1, GFP and miR-23a genes

The SMN1 gene sequence was obtained by amplification with specific primers from cDNA synthesized on the basis of total RNA of HEK293 cells or assembled from a series of oligonucleotides (see above). During the amplification process, the Kozak sequence and the ClaI restriction site were added to the 5'-end of the gene, and the XbaI restriction site was added to the 3'-end. After that, the SMN1 gene sequence was cloned by the restriction enzyme-ligase method at the ClaI and XbaI sites into the commercial construct pAAV-GFP Control plasmid (VPK-402) from CellBiolab (USA), with the GFP gene replaced by SMN1, resulting in the pAAV-SMN1 construct.

An additional cassette for miR-23a expression was inserted into the previously obtained plasmids pAAV-GFP and pAAV-SMN1. The additional cassette for miR-23a expression consists of a promoter, a gene of interest (miR-23a, obtained using PCR with genomic DNA of the Huh7 cell line) and a polyadenylation signal. Target vectors were obtained by linearization at the PmlI site of recipient vectors (pAAV-GFP, pAAV-SMN1) and subsequent insertion of the expression cassette with miR-23a at the sticky ends.

The final vector contains all the necessary elements for gene expression and assembly into the recombinant AAV genome:

1. Terminal ITR repeats at the ends of the sequence that is encapsidated into the viral capsid;

2. Cassette for expression of the target gene (promoter, enhancer, intron, Kozak sequence, transgene, polyadenylation site);

3. If available, miR-23a expression cassette (promoter, miR-30-based microRNA cassette encoding the forward and reverse chains of miR-23a, polyadenylation signal);

4. Origin of bacterial replication and antibiotic resistance gene for the production of plasmid DNA in bacterial cells.

Example 2. Creation of viral preparations expressing SMN1 and miR-23a

Plasmids pAAV-SMN1, pAAV-GFP, pAAV-SMN1-miR-23a, pAAV-GFP-miR-23a along with other plasmids required for the production of recombinant AAV viral particles (see above) were used for the bioprocess of AAV production. The AAV9 serotype, wild-type or with various mutations, was used as a serotype. In all cases, the properties of the viral particles were compared only in the case of identity of the serotype used and the capsid mutations, if present. All serotypes based on AAV9, wild-type or with mutations, are hereinafter referred to as AAV9 without specifying the mutations.

As a result of the bioprocess, recombinant viral particles designated as AAV9-SMN1, AAV9-GFP, AAV9-SMN1-miR-23a, AAV9-GFP-miR-23a were obtained. After determination of viral particle titers, all 3 preparations with the same MOI equal to 400 thousand were used for transduction of permissive cells - U-87, pre-transfected (24 h before) with siRNA against SMN1 or siRNA with a non-specific sequence. Further analysis was performed only if the efficiency of GFP transduction was at least 70%.

After successful transduction, cells were removed from the substrate, washed in phosphate buffer, and SMN1 expression was analyzed at the SMN mRNA and protein levels as described above. It was shown that upon transduction with AAV9-SMN1 and AAV9-SMN1-miR-23a viruses, SMN1 expression exceeded the endogenous level for mRNA (Table 1, Fig. 1) and was restored to the endogenous level (observed in the control without siRNA specific for SMN1) at the protein level (Table 2, Fig. 2).

The expression level of miR-23a in the samples after transduction was also determined. A significant increase in miR-23a expression was shown in the samples transduced with AAV9-SMN1-miR-23a and AAV9-GFP-miR-23a viruses. In the remaining samples, miR-23a expression did not differ from endogenous. Transfection of siRNA against SMN1 did not affect miR-23a expression (Table 3, Fig. 3).

Notably, altering SMNJ expression upon knockdown or rescue with the viral drug did not affect miR-23a expression. Also, overexpression of miR-23a did not affect SMN1 expression.

Example 3. Functional assessment of SMN1 and miR-23a in vitro after transduction

The above-described experimental design of SMN1 knockdown and expression restoration using viral particle transduction was used to assess the functional cooperation of SMN1 and miR-23a in vitro. Among the many functions of SMN1, this protein is responsible for the correct resolution of DNA-RNA duplexes that arise during transcription. The main protein involved in duplex resolution is Senataxin, which, according to the literature, is indirectly activated by SMN. Thus, Senataxin activation can be considered a functional test of SMN activity in cells.

The expression level of Senataxin in the samples was determined using the Western blot method. It was found that upon knockdown of SMN expression, Senataxin expression also decreased by about 2 times, and upon restoration of SMN expression by the AAV9-SMN1 virus, it returned to the endogenous level. Upon transduction with the control virus AAV9-GFP-miR-23a, a tendency towards a slight increase in Senataxin expression was also observed in the case of SMN knockdown, but it was not statistically significant. It is important to note that upon transduction with the AAV9-SMN1-miR-23a virus, Senataxin expression was not only restored to the endogenous level against the background of endogenous SMN knockdown, but also increased statistically significantly by 1.5-2 times (Table 4, Fig. 4).

This indicates the presence of a synergistic effect of SMN and miR-23a on Senataxin expression, which exceeds the effect of either miR-23a or SMN individually on the regulation of this gene.

Example 4. Synergistic effect of SMN1 and miR-23a in the treatment of mouse models of SMA

Viral preparations AAV9-SMN1 and AAV9-SMN1-miR-23a were injected into the tail vein of SMA model mice on the first day after birth at a dose of 3.6×10 ¹⁴ vg/kg. A solution without viral particles (placebo) and a group of wild-type sibling mice that did not have the SMA phenotype due to their expression of Smn were used as controls. Then, the survival functions of mice in all groups were constructed. When it was possible to determine the medians of survival time for all groups, the medians were calculated.

The SMA phenotype was corrected to the greatest extent in the AAV9-SMN1-miR-23a-injected group. The median survival time for the group was 55 days. This result was significantly different from the AAV9-SMN1-injected group, where the median survival time was 21 days. For placebo-injected mice, the median was 16 days after birth (Fig. 5). This result indicates the presence of a synergistic effect of SMNJ and miR-23a in the treatment of SMA in an animal model of this disease.

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Sequence listing

<110> LLC "ANABION"

<120> Synergistic effect of SMN1 and miR-23a in treating spinal muscular atrophy

<150>RU2021102051

<151> 01/29/2021

<160> 17

<170> BiSSAP 1.3.6

<210> 1

<211> 294

<212> PRT

<213>Natural Sequence

<220>

<223> Amino acid sequence of SMN1 protein

<400> 1

Met Ala Met Ser Ser Gly Gly Ser Gly Gly Gly Val Pro Glu Gln Glu

1 5 10 15

Asp Ser Val Leu Phe Arg Arg Gly Thr Gly Gln Ser Asp Asp Ser Asp

20 25 30

Ile Trp Asp Asp Thr Ala Leu Ile Lys Ala Tyr Asp Lys Ala Val Ala

35 40 45

Ser Phe Lys His Ala Leu Lys Asn Gly Asp Ile Cys Glu Thr Ser Gly

50 55 60

Lys Pro Lys Thr Thr Pro Lys Arg Lys Pro Ala Lys Lys Asn Lys Ser

65 70 75 80

Gln Lys Lys Asn Thr Ala Ala Ser Leu Gln Gln Trp Lys Val Gly Asp

85 90 95

Lys Cys Ser Ala Ile Trp Ser Glu Asp Gly Cys Ile Tyr Pro Ala Thr

100 105 110

Ile Ala Ser Ile Asp Phe Lys Arg Glu Thr Cys Val Val Val Tyr Thr

115 120 125

Gly Tyr Gly Asn Arg Glu Glu Gln Asn Leu Ser Asp Leu Leu Ser Pro

130 135 140

Ile Cys Glu Val Ala Asn Asn Ile Glu Gln Asn Ala Gln Glu Asn Glu

145 150 155 160

Asn Glu Ser Gln Val Ser Thr Asp Glu Ser Glu Asn Ser Arg Ser Pro

165 170 175

Gly Asn Lys Ser Asp Asn Ile Lys Pro Lys Ser Ala Pro Trp Asn Ser

180 185 190

Phe Leu Pro Pro Pro Pro Met Pro Gly Pro Arg Leu Gly Pro Gly

195 200 205

Lys Pro Gly Leu Lys Phe Asn Gly Pro Pro Pro Pro Pro Pro Pro Pro

210 215 220

Pro Pro His Leu Leu Ser Cys Trp Leu Pro Pro Phe Pro Ser Gly Pro

225 230 235 240

Pro Ile Ile Pro Pro Pro Pro Pro Ile Cys Pro Asp Ser Leu Asp Asp

245 250 255

Ala Asp Ala Leu Gly Ser Met Leu Ile Ser Trp Tyr Met Ser Gly Tyr

260 265 270

His Thr Gly Tyr Tyr Met Gly Phe Arg Gln Asn Gln Lys Glu Gly Arg

275 280 285

Cys Ser His Ser Leu Asn

290

<210> 2

<211> 882

<212> DNA

<213> Artificial Sequence

<220>

<223> Nucleic acid sequence encoding SMN1 protein

<400> 2

atggccatga gcagcggcgg cagcggcggc ggcgtgcctg agcaagagga cagcgtgctg 60

ttcagaagag gcaccggcca gagcgacgac agcgacatct gggacgacac cgccctgatc 120

aaggcctacg acaaggccgt ggccagcttc aagcacgccc tgaagaacgg cgacatctgc 180

gagaccagcg gcaagcccaa gaccaccccc aagagaaagc ccgccaagaa gaacaagagc 240

cagaagaaga acaccgccgc cagcctgcag cagtggaagg tgggcgacaa gtgcagcgcc 300

atctggagcg aggacggctg catctacccc gccaccatcg ccagcatcga cttcaagaga 360

gagacctgcg tggtggtgta caccggctac ggcaacagag aggagcagaa cctgagcgac 420

ctgctgagcc ccatctgcga ggtggccaac aacatcgagc agaacgccca agagaacgag 480

aacgagagcc aagtgagcac cgacgagagc gagaacagca gaagccccgg caacaagagc 540

gacaacatca agcccaagag cgccccctgg aacagcttcc tgccccctcc cccccctatg 600

cccggccta gactgggccc tggcaagcct ggcctgaagt tcaacggccc ccccccccct 660

cctcctcctc ctcctcctca cctgctgagc tgctggctgc cccccttccc cagcggccct 720

cctatcatcc ctcctccccc ccccatctgc cccgacagcc tggacgacgc cgacgccctg 780

ggcagcatgc tgatcagctg gtacatgagc ggctaccaca ccggctacta catgggcttc 840

agacagaacc agaaggagg ccggtgcagc cacagcctga ac 882

<210> 3

<211> 73

<212> RNA

<213> Artificial Sequence

<220>

<223> microRNA miR-23a

<400> 3

ggccggcugg gguuccuggg gaugggauuu gcuuccuguc acaaaucaca uugccaggga 60

uuuccaaccg acc 73

<210> 4

<211> 648

<212> DNA

<213> Artificial Sequence

<220>

<223> Nucleic acid sequence encoding microRNA miR-23a (prior to processing)

<400> 4

catgcaagtt gctgtagcct ccttgtcccg catgggccct ctaggtatct ctgcctctcc 60

agtcctgggg ctggaacgga gggcacagct aggctccagc tccccgtgtg gtggctcctg 120

catatgagaa aagagcttcc ctgtgatcaa aggaagcatc tggggacctg gaggggaggt 180

gtccccaaat ctcattacct cctttgctct ctctctcttt ctcccctcca ggtgccagcc 240

tctggccccg cccggtgccc ccctcacccc tgtgccacgg ccggctgggg ttcctgggga 300

tgggatttgc ttcctgtcac aaatcacatt gccagggatt tccaaccgac cctgagctct 360

gccaccgagg atgctgcccg gggacggggt ggcagagagg ccccgaagcc tgtgcctggc 420

ctgaggagca gggcttagct gcttgtgagc agggtccaca ccaagtcgtg ttcacagtgg 480

ctaagttccg ccccccaggc cctcacctcc tctggccttg ccgcctgtcc cctgctgccg 540

cctgtctgcc tgccatcctg ctgcctggcc tccctgggct ctgcctcccg tgcctactga 600

gctgaaacac agttggtttg tgtacactgg ctcagttcag caggaaca 648

<210> 5

<211> 73

<212> DNA

<213> Artificial Sequence

<220>

<223> Nucleic acid sequence encoding microRNA miR-23a (following processing)

<400> 5

ggccggctgg ggttcctggg gatgggattt gcttcctgtc acaaatcaca ttgccaggga 60

tttccaaccg acc 73

<210> 6

<211> 3972

<212> DNA

<213> Artificial Sequence

<220>

<223> Nucleic acid sequence of the expression cassette (complete)

<400> 6

cctgcaggca gctgcgcgct cgctcgctca ctgaggccgc ccgggcgtcg ggcgaccttt 60

ggtcgcccgg cctcagtgag cgagcgagcg cgcagagagg gagtggccaa ctccatcact 120

aggggttcct gcggccgcac gcgtctagtt attaatagta atcaattacg gggtcattag 180

ttcatagccc atatatggag ttccgcgtta cataacttac ggtaaatggc ccgcctggct 240

gaccgcccaa cgacccccgc ccattgacgt caataatgac gtatgttccc atagtaacgc 300

caatagggac tttccattga cgtcaatggg tggagtattt acggtaaact gcccacttgg 360

cagtacatca agtgtatcat atgccaagta cgccccctat tgacgtcaat gacggtaaat 420

ggcccgcctg gcattatgcc cagtacatga ccttatggga ctttcctact tggcagtaca 480

tctacgtatt agtcatcgct attaccatgg tgatgcggtt ttggcagtac atcaatgggc 540

gtggatagcg gtttgactca cggggatttc caagtctcca ccccattgac gtcaatggga 600

gtttgttttg gcaccaaaat caacgggact ttccaaaatg tcgtaacaac tccgccccat 660

tgacgcaaat gggcggtagg cgtgtacggt gggaggtcta tataagcaga gctcgtttag 720

tgaaccgtca gatcgcctgg agacgccatc cacgctgttt tgacctccat agaagacacc 780

gggaccgatc cagcctccgc ggattcgaat cccggccggg aacggtgcat tggaacgcgg 840

attccccgtg ccaagagtga cgtaagtacc gcctatagag tctataggcc cacaaaaaat 900

gctttcttct tttaatatac ttttttgttt atcttatttc taatactttc cctaatctct 960

ttctttcagg gcaataatga tacaatgtat catgcctctt tgcaccattc taaagaataa 1020

cagtgataat ttctgggtta aggcaatagc aatatttctg catataaata tttctgcata 1080

taaattgtaa ctgatgtaag aggtttcata ttgctaatag cagctacaat ccagctacca 1140

ttctgctttt attttatggt tgggataagg ctggattatt ctgagtccaa gctaggccct 1200

tttgctaatc atgttcatac ctcttatctt cctcccacag ctcctgggca acgtgctggt 1260

ctgtgtgctg gcccatcact ttggcaaaga attgggattc gaacatcgat tgtaattcat 1320

gagccaccat ggccatgagc agcggcggca gcggcggcgg cgtgcctgag caagaggaca 1380

gcgtgctgtt cagaagaggc accggccaga gcgacgacag cgacatctgg gacgacaccg 1440

ccctgatcaa ggcctacgac aaggccgtgg ccagcttcaa gcacgccctg aagaacggcg 1500

acatctgcga gaccagcggc aagcccaaga ccacccccaa gagaaagccc gccaagaaga 1560

acaagagcca gaagaagaac accgccgcca gcctgcagca gtggaaggtg ggcgacaagt 1620

gcagcgccat ctggagcgag gacggctgca tctaccccgc caccatcgcc agcatcgact 1680

tcaagagaga gacctgcgtg gtggtgtaca ccggctacgg caacagagag gagcagaacc 1740

tgagcgacct gctgagcccc atctgcgagg tggccaacaa catcgagcag aacgcccaag 1800

agaacgagaa cgagagccaa gtgagcaccg acgagagcga gaacagcaga agccccggca 1860

acaagagcga caacatcaag cccaagagcg ccccctggaa cagcttcctg ccccctcccc 1920

cccctatgcc cggccctaga ctgggccctg gcaagcctgg cctgaagttc aacggccccc 1980

ccccccctcc tcctcctcct cctcctcacc tgctgagctg ctggctgccc cccttcccca 2040

gcggccctcc tatcatccct cctccccccc ccatctgccc cgacagcctg gacgacgccg 2100

acgccctggg cagcatgctg atcagctggt acatgagcgg ctaccacacc ggctactaca 2160

tgggcttcag acagaaccag aaggaggcc ggtgcagcca cagcctgaac tgatctagag 2220

tcgacctgca gaagcttgcc tcgagcagcg ctgctcgaga gatctacggg tggcatccct 2280

gtgacccctc cccagtgcct ctcctggccc tggaagttgc cactccagtg cccaccagcc 2340

ttgtcctaat aaaattaagt tgcatcattt tgtctgacta ggtgtccttc tataatatta 2400

tggggtggag gggggtggta tggagcaagg ggcaagttgg gaagacaacc tgtagggcct 2460

gcggggtcta ttgggaacca agctggagtg cagtggcaca atcttggctc actgcaatct 2520

ccgcctcctg ggttcaagcg attctcctgc ctcagcctcc cgagttgttg ggattccagg 2580

catgcatgac caggctcagc taatttttgt ttttttggta gagacggggt ttcaccatat 2640

tggccaggct ggtctccaac tcctaatctc aggtgatcta cccaccttgg cctcccaaat 2700

tgctgggatt acaggcgtga accactgctc ccttccctgt ccttctgatt ttgtaggtaa 2760

ccactagaga aatgttctgg cacctgcact tgcactgggg acagcctatt ttgctagttt 2820

gttttgtttc gttttgtttt gatggagagc gtatgttagt actatcgatt cacacaaaaa 2880

accaacacac agatgtaatg aaaataaaga tattttattg cggccgctgt tcctgctgaa 2940

ctgagccagt gtacacaaac caactgtgtt tcagctcagt aggcacggga ggcagagccc 3000

agggaggcca ggcagcagga tggcaggcag acaggcggca gcaggggaca ggcggcaagg 3060

ccagaggagg tgagggcctg gggggcggaa cttagccact gtgaacacga cttggtgtgg 3120

accctgctca caagcagcta agccctgctc ctcaggccag gcacaggctt cggggcctct 3180

ctgccacccc gtccccgggc agcatcctcg gtggcagagc tcagggtcgg ttggaaatcc 3240

ctggcaatgt gatttgtgac aggaagcaaa tcccatcccc aggaacccca gccggccgtg 3300

gcacaggggt gaggggggca ccgggcgggg ccagaggctg gcacctggag gggagaaaga 3360

gagagagagc aaaggaggta atgagatttg gggacacctc ccctccaggt ccccagatgc 3420

ttcctttgat cacagggaag ctcttttctc atatgcagga gccaccacac ggggagctgg 3480

agcctagctg tgccctccgt tccagcccca ggactggaga ggcagagata cctagagggc 3540

ccatgcggga caaggaggct acagcaactt gcatggccgc agctttttgc aaaagcctag 3600

gcctccaaaa aagcctcctc actacttctg gaatagctca gaggccgagg cggcctcggc 3660

ctctgcataa ataaaaaaaa ttagtcagcg atggggcgga gaatgggcgg aactgggcgg 3720

agttaggggc gggatgggcg gagttagggg cgggactatg gttgctgact aattgagatg 3780

cagggccgct ccaagtacct cccgtacctt aagtgcggac cgagcggccg caggaacccc 3840

tagtgatgga gttggccact ccctctctgc gcgctcgctc gctcactgag gccgggcgac 3900

caaaggtcgc ccgacgcccg ggctttgccc gggcggcctc agtgagcgag cgagcgcgca 3960

gctgcctgca gg 3972

<210> 7

<211> 736

<212> PRT

<213>Natural Sequence

<220>

<223> Amino acid sequence of AAV9 protein VP1

<400> 7

Met Ala Ala Asp Gly Tyr Leu Pro Asp Trp Leu Glu Asp Asn Leu Ser

1 5 10 15

Glu Gly Ile Arg Glu Trp Trp Ala Leu Lys Pro Gly Ala Pro Gln Pro

20 25 30

Lys Ala Asn Gln Gln His Gln Asp Asn Ala Arg Gly Leu Val Leu Pro

35 40 45

Gly Tyr Lys Tyr Leu Gly Pro Gly Asn Gly Leu Asp Lys Gly Glu Pro

50 55 60

Val Asn Ala Ala Asp Ala Ala Ala Leu Glu His Asp Lys Ala Tyr Asp

65 70 75 80

Gln Gln Leu Lys Ala Gly Asp Asn Pro Tyr Leu Lys Tyr Asn His Ala

85 90 95

Asp Ala Glu Phe Gln Glu Arg Leu Lys Glu Asp Thr Ser Phe Gly Gly

100 105 110

Asn Leu Gly Arg Ala Val Phe Gln Ala Lys Lys Arg Leu Leu Glu Pro

115 120 125

Leu Gly Leu Val Glu Glu Ala Ala Lys Thr Ala Pro Gly Lys Lys Arg

130 135 140

Pro Val Glu Gln Ser Pro Gln Glu Pro Asp Ser Ser Ala Gly Ile Gly

145 150 155 160

Lys Ser Gly Ala Gln Pro Ala Lys Lys Arg Leu Asn Phe Gly Gln Thr

165 170 175

Gly Asp Thr Glu Ser Val Pro Asp Pro Gln Pro Ile Gly Glu Pro Pro

180 185 190

Ala Ala Pro Ser Gly Val Gly Ser Leu Thr Met Ala Ser Gly Gly Gly

195 200 205

Ala Pro Val Ala Asp Asn Asn Glu Gly Ala Asp Gly Val Gly Ser Ser

210 215 220

Ser Gly Asn Trp His Cys Asp Ser Gln Trp Leu Gly Asp Arg Val Ile

225 230 235 240

Thr Thr Ser Thr Arg Thr Trp Ala Leu Pro Thr Tyr Asn Asn His Leu

245 250 255

Tyr Lys Gln Ile Ser Asn Ser Thr Ser Gly Gly Ser Ser Asn Asp Asn

260 265 270

Ala Tyr Phe Gly Tyr Ser Thr Pro Trp Gly Tyr Phe Asp Phe Asn Arg

275 280 285

Phe His Cys His Phe Ser Pro Arg Asp Trp Gln Arg Leu Ile Asn Asn

290 295 300

Asn Trp Gly Phe Arg Pro Lys Arg Leu Asn Phe Lys Leu Phe Asn Ile

305 310 315 320

Gln Val Lys Glu Val Thr Asp Asn Asn Gly Val Lys Thr Ile Ala Asn

325 330 335

Asn Leu Thr Ser Thr Val Gln Val Phe Thr Asp Ser Asp Tyr Gln Leu

340 345 350

Pro Tyr Val Leu Gly Ser Ala His Glu Gly Cys Leu Pro Pro Phe Pro

355 360 365

Ala Asp Val Phe Met Ile Pro Gln Tyr Gly Tyr Leu Thr Leu Asn Asp

370 375 380

Gly Ser Gln Ala Val Gly Arg Ser Ser Phe Tyr Cys Leu Glu Tyr Phe

385 390 395 400

Pro Ser Gln Met Leu Arg Thr Gly Asn Asn Phe Gln Phe Ser Tyr Glu

405 410 415

Phe Glu Asn Val Pro Phe His Ser Ser Tyr Ala His Ser Gln Ser Leu

420 425 430

Asp Arg Leu Met Asn Pro Leu Ile Asp Gln Tyr Leu Tyr Tyr Leu Ser

435 440 445

Lys Thr Ile Asn Gly Ser Gly Gln Asn Gln Gln Thr Leu Lys Phe Ser

450 455 460

Val Ala Gly Pro Ser Asn Met Ala Val Gln Gly Arg Asn Tyr Ile Pro

465 470 475 480

Gly Pro Ser Tyr Arg Gln Gln Arg Val Ser Thr Thr Val Thr Gln Asn

485 490 495

Asn Asn Ser Glu Phe Ala Trp Pro Gly Ala Ser Ser Trp Ala Leu Asn

500 505 510

Gly Arg Asn Ser Leu Met Asn Pro Gly Pro Ala Met Ala Ser His Lys

515 520 525

Glu Gly Glu Asp Arg Phe Phe Pro Leu Ser Gly Ser Leu Ile Phe Gly

530 535 540

Lys Gln Gly Thr Gly Arg Asp Asn Val Asp Ala Asp Lys Val Met Ile

545 550 555 560

Thr Asn Glu Glu Glu Ile Lys Thr Thr Asn Pro Val Ala Thr Glu Ser

565 570 575

Tyr Gly Gln Val Ala Thr Asn His Gln Ser Ala Gln Ala Gln Ala Gln

580 585 590

Thr Gly Trp Val Gln Asn Gln Gly Ile Leu Pro Gly Met Val Trp Gln

595 600 605

Asp Arg Asp Val Tyr Leu Gln Gly Pro Ile Trp Ala Lys Ile Pro His

610 615 620

Thr Asp Gly Asn Phe His Pro Ser Pro Leu Met Gly Gly Phe Gly Met

625 630 635 640

Lys His Pro Pro Pro Gln Ile Leu Ile Lys Asn Thr Pro Val Pro Ala

645 650 655

Asp Pro Pro Thr Ala Phe Asn Lys Asp Lys Leu Asn Ser Phe Ile Thr

660 665 670

Gln Tyr Ser Thr Gly Gln Val Ser Val Glu Ile Glu Trp Glu Leu Gln

675 680 685

Lys Glu Asn Ser Lys Arg Trp Asn Pro Glu Ile Gln Tyr Thr Ser Asn

690 695 700

Tyr Tyr Lys Ser Asn Asn Val Glu Phe Ala Val Asn Thr Glu Gly Val

705 710 715 720

Tyr Ser Glu Pro Arg Pro Ile Gly Thr Arg Tyr Leu Thr Arg Asn Leu

725 730 735

<210> 8

<211> 599

<212> PRT

<213>Natural Sequence

<220>

<223> Amino acid sequence of AAV9 protein VP2

<400> 8

Thr Ala Pro Gly Lys Lys Arg Pro Val Glu Gln Ser Pro Gln Glu Pro

1 5 10 15

Asp Ser Ser Ala Gly Ile Gly Lys Ser Gly Ala Gln Pro Ala Lys Lys

20 25 30

Arg Leu Asn Phe Gly Gln Thr Gly Asp Thr Glu Ser Val Pro Asp Pro

35 40 45

Gln Pro Ile Gly Glu Pro Pro Ala Ala Pro Ser Gly Val Gly Ser Leu

50 55 60

Thr Met Ala Ser Gly Gly Gly Ala Pro Val Ala Asp Asn Asn Glu Gly

65 70 75 80

Ala Asp Gly Val Gly Ser Ser Ser Gly Asn Trp His Cys Asp Ser Gln

85 90 95

Trp Leu Gly Asp Arg Val Ile Thr Thr Ser Thr Arg Thr Trp Ala Leu

100 105 110

Pro Thr Tyr Asn Asn His Leu Tyr Lys Gln Ile Ser Asn Ser Thr Ser

115 120 125

Gly Gly Ser Ser Asn Asp Asn Ala Tyr Phe Gly Tyr Ser Thr Pro Trp

130 135 140

Gly Tyr Phe Asp Phe Asn Arg Phe His Cys His Phe Ser Pro Arg Asp

145 150 155 160

Trp Gln Arg Leu Ile Asn Asn Asn Trp Gly Phe Arg Pro Lys Arg Leu

165 170 175

Asn Phe Lys Leu Phe Asn Ile Gln Val Lys Glu Val Thr Asp Asn Asn

180 185 190

Gly Val Lys Thr Ile Ala Asn Asn Leu Thr Ser Thr Val Gln Val Phe

195 200 205

Thr Asp Ser Asp Tyr Gln Leu Pro Tyr Val Leu Gly Ser Ala His Glu

210 215 220

Gly Cys Leu Pro Pro Phe Pro Ala Asp Val Phe Met Ile Pro Gln Tyr

225 230 235 240

Gly Tyr Leu Thr Leu Asn Asp Gly Ser Gln Ala Val Gly Arg Ser Ser

245 250 255

Phe Tyr Cys Leu Glu Tyr Phe Pro Ser Gln Met Leu Arg Thr Gly Asn

260 265 270

Asn Phe Gln Phe Ser Tyr Glu Phe Glu Asn Val Pro Phe His Ser Ser

275 280 285

Tyr Ala His Ser Gln Ser Leu Asp Arg Leu Met Asn Pro Leu Ile Asp

290 295 300

Gln Tyr Leu Tyr Tyr Leu Ser Lys Thr Ile Asn Gly Ser Gly Gln Asn

305 310 315 320

Gln Gln Thr Leu Lys Phe Ser Val Ala Gly Pro Ser Asn Met Ala Val

325 330 335

Gln Gly Arg Asn Tyr Ile Pro Gly Pro Ser Tyr Arg Gln Gln Arg Val

340 345 350

Ser Thr Thr Val Thr Gln Asn Asn Asn Ser Glu Phe Ala Trp Pro Gly

355 360 365

Ala Ser Ser Trp Ala Leu Asn Gly Arg Asn Ser Leu Met Asn Pro Gly

370 375 380

Pro Ala Met Ala Ser His Lys Glu Gly Glu Asp Arg Phe Phe Pro Leu

385 390 395 400

Ser Gly Ser Leu Ile Phe Gly Lys Gln Gly Thr Gly Arg Asp Asn Val

405 410 415

Asp Ala Asp Lys Val Met Ile Thr Asn Glu Glu Glu Ile Lys Thr Thr

420 425 430

Asn Pro Val Ala Thr Glu Ser Tyr Gly Gln Val Ala Thr Asn His Gln

435 440 445

Ser Ala Gln Ala Gln Ala Gln Thr Gly Trp Val Gln Asn Gln Gly Ile

450 455 460

Leu Pro Gly Met Val Trp Gln Asp Arg Asp Val Tyr Leu Gln Gly Pro

465 470 475 480

Ile Trp Ala Lys Ile Pro His Thr Asp Gly Asn Phe His Pro Ser Pro

485 490 495

Leu Met Gly Gly Phe Gly Met Lys His Pro Pro Pro Gln Ile Leu Ile

500 505 510

Lys Asn Thr Pro Val Pro Ala Asp Pro Pro Thr Ala Phe Asn Lys Asp

515 520 525

Lys Leu Asn Ser Phe Ile Thr Gln Tyr Ser Thr Gly Gln Val Ser Val

530 535 540

Glu Ile Glu Trp Glu Leu Gln Lys Glu Asn Ser Lys Arg Trp Asn Pro

545 550 555 560

Glu Ile Gln Tyr Thr Ser Asn Tyr Tyr Lys Ser Asn Asn Val Glu Phe

565 570 575

Ala Val Asn Thr Glu Gly Val Tyr Ser Glu Pro Arg Pro Ile Gly Thr

580 585 590

Arg Tyr Leu Thr Arg Asn Leu

595

<210> 9

<211> 534

<212> PRT

<213>Natural Sequence

<220>

<223> Amino acid sequence of AAV9 protein VP3

<400> 9

Met Ala Ser Gly Gly Gly Ala Pro Val Ala Asp Asn Asn Glu Gly Ala

1 5 10 15

Asp Gly Val Gly Ser Ser Ser Gly Asn Trp His Cys Asp Ser Gln Trp

20 25 30

Leu Gly Asp Arg Val Ile Thr Thr Ser Thr Arg Thr Trp Ala Leu Pro

35 40 45

Thr Tyr Asn Asn His Leu Tyr Lys Gln Ile Ser Asn Ser Thr Ser Gly

50 55 60

Gly Ser Ser Asn Asp Asn Ala Tyr Phe Gly Tyr Ser Thr Pro Trp Gly

65 70 75 80

Tyr Phe Asp Phe Asn Arg Phe His Cys His Phe Ser Pro Arg Asp Trp

85 90 95

Gln Arg Leu Ile Asn Asn Asn Trp Gly Phe Arg Pro Lys Arg Leu Asn

100 105 110

Phe Lys Leu Phe Asn Ile Gln Val Lys Glu Val Thr Asp Asn Asn Gly

115 120 125

Val Lys Thr Ile Ala Asn Asn Leu Thr Ser Thr Val Gln Val Phe Thr

130 135 140

Asp Ser Asp Tyr Gln Leu Pro Tyr Val Leu Gly Ser Ala His Glu Gly

145 150 155 160

Cys Leu Pro Pro Phe Pro Ala Asp Val Phe Met Ile Pro Gln Tyr Gly

165 170 175

Tyr Leu Thr Leu Asn Asp Gly Ser Gln Ala Val Gly Arg Ser Ser Phe

180 185 190

Tyr Cys Leu Glu Tyr Phe Pro Ser Gln Met Leu Arg Thr Gly Asn Asn

195 200 205

Phe Gln Phe Ser Tyr Glu Phe Glu Asn Val Pro Phe His Ser Ser Tyr

210 215 220

Ala His Ser Gln Ser Leu Asp Arg Leu Met Asn Pro Leu Ile Asp Gln

225 230 235 240

Tyr Leu Tyr Tyr Leu Ser Lys Thr Ile Asn Gly Ser Gly Gln Asn Gln

245 250 255

Gln Thr Leu Lys Phe Ser Val Ala Gly Pro Ser Asn Met Ala Val Gln

260 265 270

Gly Arg Asn Tyr Ile Pro Gly Pro Ser Tyr Arg Gln Gln Arg Val Ser

275 280 285

Thr Thr Val Thr Gln Asn Asn Asn Ser Glu Phe Ala Trp Pro Gly Ala

290 295 300

Ser Ser Trp Ala Leu Asn Gly Arg Asn Ser Leu Met Asn Pro Gly Pro

305 310 315 320

Ala Met Ala Ser His Lys Glu Gly Glu Asp Arg Phe Phe Pro Leu Ser

325 330 335

Gly Ser Leu Ile Phe Gly Lys Gln Gly Thr Gly Arg Asp Asn Val Asp

340 345 350

Ala Asp Lys Val Met Ile Thr Asn Glu Glu Glu Ile Lys Thr Thr Asn

355 360 365

Pro Val Ala Thr Glu Ser Tyr Gly Gln Val Ala Thr Asn His Gln Ser

370 375 380

Ala Gln Ala Gln Ala Gln Thr Gly Trp Val Gln Asn Gln Gly Ile Leu

385 390 395 400

Pro Gly Met Val Trp Gln Asp Arg Asp Val Tyr Leu Gln Gly Pro Ile

405 410 415

Trp Ala Lys Ile Pro His Thr Asp Gly Asn Phe His Pro Ser Pro Leu

420 425 430

Met Gly Gly Phe Gly Met Lys His Pro Pro Pro Gln Ile Leu Ile Lys

435 440 445

Asn Thr Pro Val Pro Ala Asp Pro Pro Thr Ala Phe Asn Lys Asp Lys

450 455 460

Leu Asn Ser Phe Ile Thr Gln Tyr Ser Thr Gly Gln Val Ser Val Glu

465 470 475 480

Ile Glu Trp Glu Leu Gln Lys Glu Asn Ser Lys Arg Trp Asn Pro Glu

485 490 495

Ile Gln Tyr Thr Ser Asn Tyr Tyr Lys Ser Asn Asn Val Glu Phe Ala

500 505 510

Val Asn Thr Glu Gly Val Tyr Ser Glu Pro Arg Pro Ile Gly Thr Arg

515 520 525

Tyr Leu Thr Arg Asn Leu

530

<210> 10

<211> 130

<212> DNA

<213>Natural Sequence

<220>

<223> left-hand (first) ITR (inverted terminal repeats)

<400> 10

cctgcaggca gctgcgcgct cgctcgctca ctgaggccgc ccgggcgtcg ggcgaccttt 60

ggtcgcccgg cctcagtgag cgagcgagcg cgcagagagg gagtggccaa ctccatcact 120

aggggttcct 130

<210> 11

<211> 304

<212> DNA

<213>Natural Sequence

<220>

<223> CMV (Cytomegalovirus) enhancer

<400> 11

cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc cccgcccatt 60

gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc attgacgtca 120

atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgt atcatatgcc 180

aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt atgcccagta 240

catgacctta tgggactttc ctacttggca gtacatctac gtattagtca tcgctattac 300

catg 304

<210> 12

<211> 204

<212> DNA

<213>Natural Sequence

<220>

<223> CMV (Cytomegalovirus) promoter

<400> 12

gtgatgcggt tttggcagta catcaatggg cgtggatagc ggtttgactc acggggattt 60

ccaagtctcc accccattga cgtcaatggg agtttgtttt ggcaccaaaa tcaacgggac 120

tttccaaaat gtcgtaacaa ctccgcccca ttgacgcaaa tgggcggtag gcgtgtacgg 180

tgggaggtct atataagcag agct 204

<210> 13

<211> 493

<212> DNA

<213>Natural Sequence

<220>

<223> intron of hBG1 gene (hemoglobin subunit gamma 1)

<400> 13

cgaatcccgg ccgggaacgg tgcattggaa cgcggattcc ccgtgccaag agtgacgtaa 60

gtaccgccta tagagtctat aggcccacaa aaaatgcttt cttcttttaa tatacttttt 120

tgtttatctt atttctaata ctttccctaa tctctttctt tcagggcaat aatgatacaa 180

tgtatcatgc ctctttgcac cattctaaag aataacagtg ataatttctg ggttaaggca 240

atagcaatat ttctgcatat aaatatttct gcatataaat tgtaactgat gtaagaggtt 300

tcatattgct aatagcagct acaatccagc taccattctg cttttatttt atggttggga 360

taaggctgga ttattctgag tccaagctag gcccttttgc taatcatgtt catacctctt 420

atcttcctcc cacagctcct gggcaacgtg ctggtctgtg tgctggccca tcactttggc 480

aaagaattgg gat 493

<210> 14

<211> 479

<212> DNA

<213>Natural Sequence

<220>

<223> polyadenylation signal of hGH1

(polyadenylation signal (poly(A)) of human growth hormone)

<400> 14

acgggtggca tccctgtgac ccctccccag tgcctctcct ggccctggaa gttgccactc 60

cagtgcccac cagccttgtc ctaataaaat taagttgcat cattttgtct gactaggtgt 120

ccttctataa tattatgggg tggagggggg tggtatggag caaggggcaa gttgggaaga 180

caacctgtag ggcctgcggg gtctattggg aaccaagctg gagtgcagtg gcacaatctt 240

ggctcactgc aatctccgcc tcctgggttc aagcgattct cctgcctcag cctcccgagt 300

tgttgggatt ccaggcatgc atgaccaggc tcagctaatt tttgtttttt tggtagagac 360

ggggtttcac catattggcc aggctggtct ccaactccta atctcaggtg atctacccac 420

cttggcctcc caaattgctg ggattacagg cgtgaaccac tgctcccttc cctgtcctt 479

<210> 15

<211> 202

<212> DNA

<213>Natural Sequence

<220>

<223> SV40 promoter (simian virus 40 promoter)

<400> 15

tgcatctcaa ttagtcagca accatagtcc cgcccctaac tccgcccatc ccgcccctaa 60

ctccgcccag ttccgcccat tctccgcccc atcgctgact aatttttttt atttatgcag 120

aggccgaggc cgcctcggcc tctgagctat tccagaagta gtgaggaggc ttttttggag 180

gcctaggctt ttgcaaaaag ct 202

<210> 16

<211> 154

<212> DNA

<213>Natural Sequence

<220>

<223> SV40 polyadenylation signal (simian virus 40 polyadenylation signal)

<400> 16

aataaaatat ctttattttc attacatctg tgtgttggtt ttttgtgtga atcgatagta 60

ctaacatacg ctctccatca aaacaaaacg aaacaaaaca aactagcaaa ataggctgtc 120

cccagtgcaa gtgcaggtgc cagaacattt ctct 154

<210> 17

<211> 141

<212> DNA

<213>Natural Sequence

<220>

<223> right-hand (second) ITR

<400> 17

aggaacccct agtgatggag ttggccactc cctctctgcg cgctcgctcg ctcactgagg 60

ccgggcgacc aaaggtcgcc cgacgcccgg gctttgcccg ggcggcctca gtgagcgagc 120

gagcgcgcag ctgcctgcag g 141

<---

Claims (34)

1. An isolated nucleic acid for producing a gene therapy viral preparation for expressing the SMN1 gene in target mammalian cells, which comprises, in the direction from the 5'-end to the 3'-end, a nucleic acid that encodes the SMN1 protein (motor neuron survival protein) with the amino acid sequence of SEQ ID NO: 1, and a nucleic acid that encodes the miR-23a microRNA with the nucleotide sequence of SEQ ID NO: 3. 2. The isolated nucleic acid of claim 1, wherein the nucleic acid that encodes the SMN1 protein with the amino acid sequence of SEQ ID NO: 1 comprises the nucleotide sequence of SEQ ID NO: 2. 3. The isolated nucleic acid of claim 1, wherein the nucleic acid that encodes the miR-23a microRNA comprises the nucleotide sequence of SEQ ID NO: 4. 4. An expression cassette that comprises a nucleic acid according to any one of claims 1-3. 5. An expression cassette according to claim 4, comprising the following elements in the direction from the 5'-end to the 3'-end: left (first) ITR (inverted terminal repeats); CMV (cytomegalovirus) enhancer; CMV (cytomegalovirus) promoter; intron of the hBG1 gene (hemoglobin gamma-1 subunit gene); nucleic acid that encodes the SMN1 protein; hGH1 polyadenylation signal (human growth hormone gene polyadenylation signal); SV40 promoter (simian virus 40 promoter); nucleic acid that encodes the microRNA miR-23a; SV40 polyadenylation signal (simian virus 40 polyadenylation signal) and right (second) ITR. 6. The expression cassette of claim 5, which comprises a nucleic acid with SEQ ID NO: 6. 7. An expression vector that comprises a nucleic acid according to any one of claims 1-3 or a cassette according to any one of claims 4-6. 8. A recombinant virus based on AAV9 (adeno-associated virus 9) for expression of the SMN1 gene in target mammalian cells, which includes 1) AAV9 capsid and 2) a nucleic acid according to any one of claims 1-3 or an expression cassette according to any one of claims 4-6. 9. A recombinant virus based on AAV9 according to claim 8, wherein the capsid comprises the VP1 protein of AAV9. 10. A recombinant AAV9-based virus according to claim 9, wherein the capsid comprises the AAV9 VP1 protein having the amino acid sequence SEQ ID NO: 7. 11. A recombinant AAV9-based virus according to claims 8-10, wherein the capsid comprises the AAV9 VP1 protein having the amino acid sequence SEQ ID NO: 7, and the expression cassette comprises the following elements in the direction from the 5'-end to the 3'-end: left (first) ITR (inverted terminal repeats); CMV (cytomegalovirus) enhancer; CMV (cytomegalovirus) promoter; intron of the hBG1 gene (hemoglobin gamma-1 subunit gene); nucleic acid that encodes the SMN1 protein; hGH1 polyadenylation signal (human growth hormone gene polyadenylation signal); SV40 promoter (simian virus 40 promoter); nucleic acid that encodes the microRNA miR-23a; SV40 polyadenylation signal (simian virus 40 polyadenylation signal) and right (second) ITR. 12. A recombinant AAV9-based virus according to claim 8, wherein the capsid comprises the AAV9 VP1 protein having the amino acid sequence of SEQ ID NO: 7, and the expression cassette comprises the nucleic acid with SEQ ID NO: 6.