OA21365A

OA21365A - Synergistic effect of SMN1 and MIR-23A in treating spinal muscular atrophy.

Info

Publication number: OA21365A
Application number: OA1202300302
Authority: OA
Inventors: Dmitry Valentinovich MOROZOV; Dmitriy Aleksandrovich MADERA; Pavel Mikhailovich GERSHOVICH; Anna Sergeevna VESELOVA; Aleksei Sergeevich SIUTKIN
Original assignee: Joint Stock Company ‘‘Biocad’’
Priority date: 2021-01-29
Filing date: 2022-01-28
Publication date: 2024-05-10

Abstract

The present application relates to the fields of biotechnology, virology, genetics, and molecular biology. More specifically, the present invention relates to an isolated nucleic acid for producing a gene therapy viral product, said isolated nucleic acid comprising a nucleic acid that encodes the SMN1 protein having the amino acid sequence of SEQ ID NO: 1, and a nucleic acid that encodes the microRNA miR-23a, an expression cassette and a vector based thereon, as well as an AAV9based recombinant virus for expressing the SMN1 gene in target cells, a pharmaceutical composition that includes said recombinant virus, and various uses of the above recombinant virus and the above composition.

Description

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

FIELD OF THE INVENTION

The présent application relates to the fields of biotechnology, virology, genetics, and molecular biology. More specifically, the présent invention relates to an isolated nucleic acid for producing a gene therapy viral product, said isolated nucleic acid comprising a nucleic acid that encodes the SMN1 protein (survival motor neuron protein) having the amino acid sequence of SEQ ID NO: 1, and a nucleic acid that encodes ,the microRNA miR-23a, to an expression cassette and a vector based thereon, as well as to an AAV9 (adeno-associated virus serotype 9)-based recombinant virus for expressing the SMN1 gene in target cells, to a pharmaceutical composition that includes said recombinant virus, and to various uses of the above recombinant virus and the above composition.

BACKGROUND OF THE INVENTION

Spinal muscular atrophy (SMA) relates tp the group of neuromuscular diseases and is characterized by predominantly autosomal récessive inheritance pattern. Currently, the term SMA typically refers to the most common form of the disease that develops as a resuit of mutations and (or) délétions in the SMN1 (survival motor neuron 1) gene located on the long arm of chromosome 5 (5qll.2-ql3.3) (Lefebvre et al., Cell (1995) 80:155-165). The disease is accompanied by motor neuron degeneration in the ventral (anterior) hom of the spinal cord, which leads to hypotonia of the proximal muscles responsible for gross motor skills, for example, crawling, walking, neck control, and also to swallowing disorder and breathing disorder (Sumner C.J., NeuroRx (2006) 3:235-245). Consequently, SMA patients présent with increased tendencies for respiratory distress and superaddition of intercurrent diseases.

Gene therapy is a promising approaeh to treating spinal muscular atrophy.

Adeno-associated virus (AAV) vectors are considered effective in CNS gene therapy because they hâve suitable toxicity and immunogenicity profiles, they may be used in nerve cell transduction, and they are able to médiate long-term expression in the CNS.

Adeno-associated virus (AAV) is a small (20 nm), independent replication-defective, nonenveloped virus. Many different AAV serotypes hâve been described in human and primates. The adeno-associated virus genome is composed of (+ or -) single-stranded DNA (ssDNA) being about 4,700 nucléotides long. At the ends of a genomic DNA molécule, there are accommodated terminal inverted repeats (ITRs). The genome comprises two open reading frames (ORFs), Rep and Cap, comprising several alternative reading frames encoding various protein products. The ¹ 1 rep products are essential for AAV réplication, whereas three capsid proteins (VP1, VP2, and VP3), along with other alternative products, are encoded by the Cap gene. VP1, VP2, and VP3 are présent at 1:1:10 ratio to form an icosahedral capsid (Xie Q. et al. The atomic structure of adenoassociated virus (AAV-2), a vector for human gene therapy. Proc Natl Acad Sci USA, 2002; 99:10405-10410). During recombinant AAV (rAAV) vector production, an expression cassette flanked by ITR is packaged into an AAV capsid. The genes required for AAV réplication are not included in the cassette. Recombinant AAV is considered one of the safest and most widely used viral vectors for in vivo gene transfer. Vectors can infect cells of multiple tissue types to provide strong and sustained transgene expression. They are also non-pathogenic, and hâve a low immunogenicity profile (High KA et al., rAAV human trial expérience Methods Mol Biol. 2011 ; 807:429-57).

International applications WO2017106354 (Al), W02010129021 (Al), W02015060722 (Al), WO2017066579 (Al), WO2015158749 (A2) disclose various variants of AAV comprising the SMN1 gene and uses thereof. ,

Description of the invention

The authors of the invention hâve developed an isolated nucleic acid for producing a gene therapy viral product, said isolated nucleic acid comprising a nucleic acid that encodes the SMN1 protein having the amino acid sequence of SEQ ID NO: 1, and a nucleic acid that encodes the microRNA miR-23a, an expression cassette and a vector based thereon, as well as an AAV9-based recombinant virus for expressing the SMN1 gene in target cells, a pharmaceutical composition that includes said recombinant virus, and various uses of the above recombinant virus and the above composition. The authors of the invention surprisingly found that miR-23a enhances the functional effect of SMN1 in vitro and revealed the synergistic effect of SMN1 and miR-23a in treating SMA in an animal model of the disease

Brief description of the invention

Tn one aspect, the présent invention relates to an isolated nucleic acid for producing a gene therapy viral product, said isolated nucleic acid, comprising a nucleic acid that encodes the SMN1 protein (survival motor neuron protein) having the amino acid sequence of SEQ ID NO: 1, and a nucleic acid that encodes the microRNA miR-23a.

Tn some embodiments, the isolated nucleic acid that encodes the SMN1 protein having the amino acid sequence of SEQ ID NO: 1 includes the nucléotide sequence of SEQ ID NO: 2.

Tn some embodiments, the isolated nucleic acid includes the microRNA miR-23a that has the nucléotide sequence of SEQ ID NO: 3.

Tn some embodiments, the isolated nucleic acid includes a nucleic acid encoding the microRNA miR-23a that includes the nucléotide sequence of SEQ ID NO: 4.

In some embodiments, the isolated nucleic acid includes the following éléments in the 5'end to 3'-end direction:

a nucleic acid that encodes the SMN1 protein having the amino acid sequence of SEQ ID NO: 1, and a nucleic acid that encodes the microRNA miR-23a.

In one aspect, the présent invention relates to an expression cassette comprising any of the above nucleic acids.

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

a left-hand (first) ITR (inverted terminal repeats);

a CMV (cytomégalovirus) enhancer;

a CMV (cytomégalovirus) promoter;

an intron of the hBGl gene (hemoglobin subunit gamma 1 gene);

a nucleic acid that encodes the SMN1 protein;

an hGHl polyadenylation signal (human growth hormone gene polyadenylation signal);

an SV40 promoter (simian virus 40 promoter);

a nucleic acid that encodes the microRNA miR-23a;

an SV40 polyadenylation signal (simian virus 40 polyadenylation signal), and a right-hand (second) ITR.

Tn some embodiments, the expression cassette includes a nucleic acid with SEQ ID NO: 6.

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

Tn one aspect, the présent invention relates to an AAV9 (adeno-associated virus serotype 9)-based recombinant virus for the expression of the SMN1 gene in target cells, which includes a capsid and any of the above expression cassettes.

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

Tn some embodiments, the AAV9-based recombinant virus includes the AAV9 protein VP 1 having the amino acid sequence of SEQ ID NO: 7.

Tn some embodiments, the AAV9-based recombinant virus includes the AAV9 protein VP 1 having the amino acid sequence of SEQ ID NO: 7 with one or more point mutations.

Tn some embodiments, the AAV9-based recombinant virus includes the AAV9 protein VP 1 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 éléments in the 5'-end to 3'-end direction:

Y.

a left-hand (first) ITR (inverted terminal repeats);

a CMV (cytomégalovirus) enhancer;

a CMV (cytomégalovirus) promoter;

an intron of the hBGl gene (hemoglobin subunit gamma 1 gene);

a nucleic acid that encodes the SMN1 protein;

an SV40 promoter (simian virus 40 promoter);

a nucleic acid that encodes the microRNA miR-23a;

an SV40 polyadenylation signal (simian virus 40 polyadenylation signal), and a right-hand (second) ITR. ।

In some embodiments, the AAV9-based recombinant virus includes the AAV9 protein VP1 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 with SEQ ID NO: 6.

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

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

Tn one aspect, the présent invention relates to the use of any of the above AAV9-based recombinant viruses or the above composition for survival of a subject that has spinal muscular atrophy and/or that does not hâve fully functional copies of the SMN1 gene.

Tn one aspect, the présent invention relates to the use of any of the above AAV9-based recombinant viruses or the above composition for providing the SMN1 protein to a subject that has spinal muscular atrophy and/or that does not hâve fully functional copies of the SMN1 gene.

Tn one aspect, the présent invention relates to the use of any of the above AAV9-based recombinant viruses or the above composition for treating spinal muscular atrophy in a subject that has spinal muscular atrophy.

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

Tn one aspect, the présent invention relates to a method for providing the SMN protein to a subject having spinal muscular atrophy, said method comprising administering a therapeutically >4 effective amount of any of the above AAV9-based recombinant viruses or the above composition into the cells of the subject in need thereof.

In one aspect, the présent invention relates to a method for delivering the SMN1 gene to the target cells of a subject having spinal muscular atrophy, said method comprising administering any of the above AAV9-based recombinant viruses or the above composition into the cells of the subject.

In one aspect, the présent invention relates to a method for treating spinal muscular atrophy in a subject, said method comprising administering a therapeutically effective amount of any of the above AAV9-based recombinant viruses or the above composition into a subject having spinal muscular atrophy.

Brief description of drawings

Figure 1 is a graph showing SMN1 expression at mRNA level following SMN1 knockdown using siRNA and transduction by AAV9-GFP, AAV9-GFP-miR-23a, AAV9-SMN1 and AAV9SMNl-miR-23a viruses. U-87 cells were transfected with 20 nmol/1 siRNA spécifie for SMN1 (or control siRNA), thereafter they were transduced by the viruses AAV9-GFP, AAV9-GFP-miR23a, AAV9-SMN1 and AAV9-SMNl-miR-23a at MOI = 400,000. 120 h following transduction, the copy number of the SMN1 gene in each sample was determined by quantitative PCR (n = 3). The copy number of the GAPDH household gene was also determined. Ail obtained levels for SMN1 were normalized to the amounts of copies of the GAPDH gene in each sample. Provided are data on normalized mean copy number of SMN1, showing standard déviation.

Figure 2 is a graph showing SMN expression at protein level following SMN1 knockdown using siRNA and transduction by the viruses AAV9-GFP, AAV9-GFP-miR-23a, AAV9-SMN1 and AAV9-SMNl-miR-23a. U-87 cells were transfected with 20 nmol/1 siRNA spécifie for SMN1 (or control siRNA), thereafter they were transduced by the viruses AAV9-GFP, AAV9-GFP-miR23a, AAV9-SMN1 and AAV9-SMNl-miR-23a at MOI = 400,000. 120 h following transduction, the amount of SMN protein in each sample was determined using ELISA (n = 3). Ail resulting amounts of SMN protein (in pg) were normalized against the total amount of protein in each sample (in pg). Provided are data on normalized mean amount of SMN protein, showing standard déviation.

Figure 3 is a graph showing miR-23a expression following SMN1 knockdown using siRNA and transduction by the viruses AAV9-GFP, AAV9-GFP-miR-23a, AAV9-SMN1 and AAV9SMNl-miR-23a. U-87 cells were transfected with 20 nmol/1 siRNA spécifie for SMN1 (or control siRNA), thereafter they were transduced by the viruses AAV9-GFP, AAV9-GFP-miR-23a, AAV9-SMN1 and AAV9-SMNl-miR-23a at MOI = 400,000. 120 h following transduction, the amount oÎmiR-23a against the copy number of the GAPDH gene in each sample was determined 5 by quantitative PCR (n = 3), using the ΔΔϋτ method. Provided are data on normalized mean amount of miR-23a, showing standard déviation. The relative amount of miR-23a in nontransfected and non-transduced Controls was taken as 100%.

Figure 4 is a graph showing Senataxin expression following SMN1 knockdown using siRNA and transduction by the viruses AAV9-GFP, AAV9-GFP-miR-23a, AAV9-SMN1 and AAV9-SMNl-miR-23a. U-87 cells were transfected with 20 nmol/1 siRNA spécifie for wild-type SMN1 (or control siRNA), thereafter they were transduced by the viruses AAV9-GFP, AAV9GFP-miR-23a, AAV9-SMN1 and AAV9-SMNl-miR-23a at MOI = 400,000. 120 h following transduction, the amount of Senataxin against that of Vinculin protein in each sample was determined using Western blot. Provided is a graph showing normalized expression of Senataxin against Vinculin in experimental samples with indication of standard déviations (n = 3). The relative amount of Senataxin in non-transfected and non-transduced Controls was taken as 100%.

For Figures 1 to 4:

siNEG is a négative control of siRNA, siSMNl is siRNA for SMN1.

Figure 5 is a graph showing the survival curves for SMA model mice injected with the viruses AAV9-SMN1 and AAV9-SMNl-miR-23a. Survival curves for placebo-injected animais and wild-type control mice (from the same litter as the experimental ones) are also shown.

Définitions and general methods ,

Unless defined otherwise herein, ail technical and scientific terms used in connection with the présent invention will hâve the same meaning as is commonly understood by those skilled in the art.

Furthermore, unless otherwise required by context, singular terms shall include plural terms, and the plural terms shall include the singular terms. Typically, the présent classification and methods of cell culture, molecular biology, immunology, microbiology, genetics, analytical chemistry, organic synthesis chemistry, medical and pharmaceutical chemistry, as well as hybridization and chemistry of protein and nucleic acids described herein are well known by those skilled and widely used in the art. Enzyme reactions and purification methods are performed according to the manufacturer's guidelines, as is common in the art, or as described herein.

“Isolated” means altered or removed from the natural State. For example, a nucleic acid or a peptide naturally présent in an animal is not “isolated”, but the same nucleic acid or peptide partially or completely separated from the coexisting materials of its natural state is isolated . An isolated nucleic acid or protein can exist in substantially purified form, or can exist in a non-native environment such as, for example, a genetically modified cell.

The terms “naturally occurring,” “native,” or “wild-type” are used to describe an object that can be found in nature as distinct from being artificially produced. For example, a protein or nucléotide sequence présent in an organism (including a virus), which can be isolated from a source in nature and that has not been intentionally modified by a person in the laboratory, is naturally occurring.

The tenu “genome” refers to the complété genetic material of an organism.

As used in the présent description and claims that follow, unless otherwise dictated by the context, the words include and comprise, or variations thereof such as having, includes, including, comprises, or comprising, will be understood to imply the inclusion of a stated integer or group of integers but not the exclusion of any other integer or group of integers.

Protein (Peptide)

As used in the présent description, the terms peptide, “polypeptide” and “protein” are used interchangeably, and they refer to a compound consisting of amino acid residues that are covalently linked by peptide bonds. A protein or peptide must contain at least two amino acids, and no limitation is placed on the maximum number of amino acids that can comprise a protein's or peptide's sequence. Polypeptides include any peptide or protein comprising two or more amino acids joined to each other by peptide bonds. As used in the présent description, the term refers to both short chains, which also commonly are referred to in the art, for example, as peptides, oligopeptides and oligomers, and to longer chains, which generally are referred to in the art as proteins, of which there are many types. “Polypeptides” include, inter alia, for example, biologically active fragments, substantially homologous polypeptides, oligopeptides, homodimers, heterodimers, variants of polypeptides, modified polypeptides, dérivatives, analogs, fusion proteins. The polypeptides include natural peptides, recombinant peptides, synthetic peptides, or a combination thereof.

Nucleic acid molécules

The terms nucleic acid, nucleic sequence, nucleic acid sequence, polynucleotide, oligonucleotide, polynucleotide sequence and nucléotide sequence, used interchangeably in the présent description, mean a précisé sequence of nucléotides, modified or not, determining a fragment or a région of a nucleic acid, containing unnatural nucléotides or not, and being either a double-strand DNA or RNA, a single-strand DNA or RNA, or transcription products of said DNAs.

As used in the présent description, polynucleotides include, as non-limiting examples, ail nucleic acid sequences which are obtained by any means available in the art, including, as nonlimiting examples, recombinant means, i.e. the cloning of nucleic acid sequences from a recombinant library or a cell genome, using ordinary cloning technology and PCR and the like, and by synthetic means.

It should also be included here that the présent invention does not relate to nucléotide sequences in their naturel chromosomal environment, i.e. in a naturel State. The sequences of the présent invention hâve been isolated and/or purified, i.e., they were sampled directly or indirectly, for example by copying, their environment having been at least partially modifïed. Thus, isolated nucleic acids obtained by recombinant genetics, by means, for example, of host cells, or obtained by Chemical synthesis should also be mentioned here.

Unless otherwise indicated, the term nucléotide sequence encompasses its complément. Thus, a nucleic acid having a particular sequence should be understood as one which encompasses the complementary strand thereof with the complementary sequence thereof.

Adeno-associatedvirus (AAV)

Viruses of the Parvoviridae family are small DNA-containing animal viruses. The Parvoviridae family may be divided into two subfamilies: the Parvovirinae, which members infect vertebrates, and the Densovirinae, which members infect insects. By 2006, there hâve been 11 serotypes of adeno-associated virus described (Mori, S. ET AL., 2004, Two novel adenoassociated viruses from cynomolgus monkey: pseudotyping characterization of capsid protein, Virology, T. 330 (2): 375-83). Ail of the known serotypes can infect cells from multiple tissue types. Tissue specificity is determined by the capsid protein serotype; therefore, the adenoassociated virus-based vectors are constructed by assigning the desired serotype. Further information on parvoviruses and other members of the Parvoviridae is described in the literature (Kenneth L Bems, Parvoviridae: The Viruses and Their Réplication, Chapter 69 in Fields Virology (3d Ed. 1996)).

The genomic organization of ail known AAV serotypes is very similar. The genome of AAV is a linear, single-stranded DNA molécule that is less than about 5000 nucléotides (nt) in length: Inverted terminal repeats (ITRs) flank the unique coding nucléotide sequences of réplication of non-structural proteins (Rep) and structural proteins (Cap). The Cap gene encodes the VP proteins (VP1, VP2, and VP3) which form the capsid. The terminal 145 nucléotides are self-complementary and are organized such that an energetically stable intramolecular duplex forming a T-shaped hairpin may be formed. Such hairpin structures fonction as an origin for virus DNA réplication, serving as primers for the cellular DNA polymerase complex. Following wildtype AAV (wtAAV) infection in mammalian cells, the Rep genes (e.g. Rep78 and Rep52) are expressed using the P5 promoter and the P19 promoter, respectively, and the both Rep proteins hâve a certain fonction in the réplication of the viral genome. A splicing event in the Rep open reading frame (Rep ORF) results in the expression of actually four Rep proteins (e.g. Rep78,

Rep68, Rep52, and Rep40). However, it has been shown that the unspliced mRNA encoding Rep78 and Rep52 proteins is sufficient for AAV vector production in mammalian cells.

Vector

The term vector as used herein means a nucleic acid molécule capable of transporting another nucleic acid to which it has been linked. Furthermore, the term vector herein refers to a viral particle capable of transporting a nucleic âcid.

As used in the présent description, the term “expression” is defïned as the transcription and/or translation of a particular nucléotide sequence driven by its promoter.

Use

Gene therapy is the insertion of genes into subject's cells and/or tissues to treat a disease, typically hereditary diseases, in which a defective mutant allele is replaced with a functional one.

“Treat”, “treating” and “treatment” refer to a method of alleviating or abrogating a biologicâl disorder and/or at least one of its attendant symptoms. As used herein, to alleviate a disease, disorder or condition means reducing the severity and/or occurrence frequency of the symptoms of a disease, disorder, or condition. Further, references herein to “treatment” include references to curative, palliative and prophylactic treatment.

Tn one aspect, the subject of treatment, or patient, is a mammal, preferably a human subject. Said subject may be either male or female, of any âge.

The term disorder means any condition that would benefit from treatment with the compound of the présent invention.

“Disease” is a state of health of a subject where the subject cannot maintain homeostasis, and where if the disease is not ameliorated then the subject's health continues to deteriorate.

The ternis “subject,” “patient,” “individual,” and the like are used interchangeably in the présent description, and they refer to any animal which is amenable to the methods described in the présent description. In certain non-limiting embodiments, the subject, patient or individual is a human. Said subject may be either male or female, of any âge.

Therapeutically effective amount or effective amount refers to that amount of the therapeutic agent being administered which will relieve to some extent one or more of the symptoms of the disease being treated.

Detailed description of the invention

Nucleic acid

In one aspect, the présent invention relaies to an isolated nucleic acid for producing a gene therapy viral product, said isolated nucleic acid comprising a nucleic acid that encodes the SMN1 protein (survival motor neuron protein) having the amino acid sequence MAMSSGGSGGGVPEQEDSVLFRRGTGQSDDSDIWDDTALIKAYDKAVASFKHALKNG

DICETSGKPKTTPKRKPAKKNKSQKKNTAASLQQWKVGDKCSAIWSEDGCIYPATIASI DFKRETCVWYTGYGNREEQNLSDLLSPICEVANNIEQNAQENENESQVSTDESENSRSP GNKSDNIKPKSAPWNSFLPPPPPMPGPRLGPGKPGLKFNGPPPPPPPPPPHLLSCWLPPFPS GPPIIPPPPPICPDSLDDADALGSMLISWYMSGYHTGYYMGFRQNQKEGRCSHSLN (SEQ ID NO: 1), and a nucleic acid that encodes the microRNA miR-23a;

Tn some embodiments, the above nucleic acid is used for producing a gene therapy viral product that is an expression vector of the invention or an AAV9-based recombinant virus of the invention.

An isolated nucleic acid molécule is one which is identified and separated from at least one nucleic acid molecule-impurity, which the former is typically bound to in the natural source of nuclease nucleic acid. An isolated nucleic acid molécule is different from the form or set in which it is found under natural conditions. Thus, an isolated nucleic acid molécule is different from a nucleic acid molécule that exists in cells under natural conditions.

In some embodiments, the isolated nucleic acid is DNA.

As would be appreciated by those skilled in the art, because of the redundancy of the genetic code, a variety of different DNA sequences can encode the SMN1 protein having the amino acid sequence of SEQ ID NO: 1. It is well within the skill of a person trained in the art to create these alternative DNA sequences encoding the same amino acid sequences. Such variant DNA sequences are within the scope of the présent invention.

Tn some embodiments, the isolated nucleic acid that encodes the SMN1 protein having the amino acid sequence of SEQ ID NO: 1 includes the nucléotide sequence

ATGGCCATGAGCAGCGGCGGCAGCGGCGGCGGCGTGCCTGAGCAAGAGGAC AGCGTGCTGTTCAGAAGAGGCACCGGCCAGAGCGACGACAGCGACATCTGGGACGA CACCGCCCTGATCAAGGCCTACGACAAGGCCGTGGCCAGCTTCAAGCACGCCCTGA AGAACGGCGACATCTGCGAGACCAGCGGCAAGCCCAAGACCACCCCCAAGAGAAA GCCCGCCAAGAAGAACAAGAGCCAGAAGAAGAACACCGCCGCCAGCCTGCAGCAG TGGAAGGTGGGCGACAAGTGCAGCGCCATCTGGAGCGAGGACGGCTGCATCTACCC CGCCACCATCGCCAGCATCGACTTCAAGAGAGAGACCTGCGTGGTGGTGTACACCG GCTACGGCAACAGAGAGGAGCAGAACCTGAGCGACCTGCTGAGCCCCATCTGCGAG GTGGCCAACAACATCGAGCAGAACGCCCAAGAGAACGAGAACGAGAGCCAAGTGA GCACCGACGAGAGCGAGAACAGCAGAAGCCCCGGCAACAAGAGCGACAACATCAA GCCCAAGAGCGCCCCCTGGAACAGCTTCCTGCCCCCTCCCCCCCCTATGCCCGGCCC TAGAÇTGGGCCCTGGCAAGCCTGGCCTGAAGTTCAACGGCCCCCCCCCCCCTCCTCC TCCTCCTCCTCCTCACCTGCTGAGCTGCTGGCTGCCCCCCTTCCCCAGCGGCCCTCCT ATCATCCCTCCTCCCCCCCCCATCTGCCCCGACAGCCTGGACGACGCCGACGCCCTG

GGCAGCATGCTGATCAGCTGGTACATGAGCGGCTACCACACCGGCTACTACATGGG CTTCAGACAGAACCAGAAGGAGGGCCGGTGCAGCCACAGCCTGAAC (SEQ ID NO: 2).

Tn some embodiments, the isolated nucleic acid includes the microRNA miR-23a that has the nucléotide sequence

GGCCGGCUGGGGUUCCUGGGGAUGGGAUUUGCUUCCUGUCACAAAUCACAUUGCC AGGGAUUUCCAACCGACC (SEQ ID NO: 3).

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

CATGCAAGTTGCTGTAGCCTCCTTGTCCCGCATGGGCCCTCTAGGTATCTCTG cctctccagtcctggggctggaacgGagggcacagctaggctccagctccccgtgtg GTGGCTCCTGCATATGAGAAAAGAGCTTCCCTGTGATCAAAGGAAGCATCTGGGGA CCTGGAGGGGAGGTGTCCCCAAATCTCATTACCTCCTTTGCTCTCTCTCTCTTTCTCC CCTCCAGGTGCCAGCCTCTGGCCCCGCCCGGTGCCCCCCTCACCCCTGTGCCACGGC CGGCTGGGGTTCCTGGGGATGGGATTTGCTTCCTGTCACAAATCACATTGCCAGGGA TTTCCAACCGACCCTGAGCTCTGCCACCGAGGATGCTGCCCGGGGACGGGGTGGCA GAGAGGCCCCGAAGCCTGTGCCTGGCCTGAGGAGCAGGGCTTAGCTGCTTGTGAGC AGGGTCCACACCAAGTCGTGTTCACAGTGGCTAAGTTCCGCCCCCCAGGCCCTCACC TCCTCTGGCCTTGCCGCCTGTCCCCTGCTGCCGCCTGTCTGCCTGCCATCCTGCTGCC TGGCCTCCCTGGGCTCTGCCTCCCGTGCCTACTGAGCTGAAACACAGTTGGTTTGTG TACACTGGCTCAGTTCAGCAGGAACA (SEQ ID NO: 4).

The above nucleic acid, which encodes the microRNA miR-23a, having the nucléotide sequence SEQ ID NO: 4 is a nucleic acid before processing.

Following processing, the nucleic acid, which encodes the microRNA miR-23a, has the nucléotide sequence

GGCCGGCTGGGGTTCCTGGGGATGGGATTTGCTTCCTGTCACAAATCACATTGCCAG GGATTTCCAACCGACC (SEQ ID NO:5).

Tn some embodiments, the isolated nucleic acid includes the following éléments in the 5'end to 3'-end direction: ¹ a nucleic acid that encodes the SMN1 protein having the amino acid sequence of SEQ ID NO: 1, and a nucleic acid that encodes the microRNA miR-23a.

Expression cassette. Expression vector.

The term expression cassette, as used herein, refers in particular to a DNA fragment that is capable, in an appropriate setting, of triggering 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, inter alia, capable of engaging cellular mechanisms to transcribe the polynucleotide encoding the polypeptide of interest into RNA that is then typically further processed and eventually translated into the polypeptide of interest. The expression cassette may be contained in an expression vector. '

The term cassette which expresses or expression cassette, as used herein, refers in particular to a DNA fragment that is capable, in an appropriate setting, of triggering 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, inter alia, capable of engaging cellular mechanisms to transcribe the polynucleotide encoding the polypeptide of interest into RNA that is then typically further processed and eventually translated into the polypeptide of interest. The expression cassette may be contained in an expression vector.

The expression cassette of the présent invention comprises a promoter as an element. The term promoter as used herein refers in particular to a DNA element that promûtes the transcription of a polynucleotide to which the promoter is operably linked. The promoter may further form part of a promoter/enhancer element. Although the physical boundaries between the promoter and enhancer éléments are not always clear, the term promoter typically refers to a site on the nucleic acid molécule to which ah RNA polymerase and/or any associated factors binds and at which transcription is initiated. Enhancers potentiate promoter activity temporally as well as spatially. Many promoters are known in the art to be 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 derepression. The both classes are suitable for protein expression. Promoters that are used for high-level production of polypeptides in eukaryotic cells and, in particular, in mammalian cells, should be strong and preferably 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, it is not herein necessary to describe them in detail. In accordance with the idea of the présent invention, it is préférable to use the cytomégalovirus (CMV) promotet. A promoter or promoter/enhancer derived from the immédiate early (IE) région of human cytomégalovirus (hCMV) is particularly suitable as a promoter in the expression cassette of the présent invention. Human cytomégalovirus (hCMV) immédiate early (IE) région and functional expression-inducing and/or funcfional expression-enhancing fragments derived therefrom, for example, hâve been disclosed in EP0173177 and EP0323997, and are well known in the art. Thus, several fragments of the hCMV immédiate early (IE) région may 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 présent invention.

Tn some embodiments, the expression cassette includes the following éléments in the 5'end to 3'-end direction:

a left-hand (first) ITR (inverted terminal repeats);

a CMV (cytomégalovirus) enhancer;

a CMV (cytomégalovirus) promoter;

an intron of the hBGl gene (hemoglobin subunit gamma 1 gene);

a nucleic acid that encodes the SMN1 protein;

an SV40 promoter (simian virus 40 promoter);

a nucleic acid that encodes the microRNA miR-23a;

Tn some embodiments, the left-hand (first) ITR (inverted terminal repeats) has the following nucleic acid sequence:

cctgcaggcagctgcgcgctcgctcgctcactgaggccgcccgggcgtcgggcgacctttggtcgcccggcctcagtgagcgagcgag cgcgcagagagggagtggccaactccatcactaggggttcct (SEQ ID NO: 10).

In some embodiments, the CMV (cytomégalovirus) enhancer has the following nucleic acid sequence:

cgttacataacttacggtaaaiggcccgcctggctgaccgcccaacgacccccgcccattgacgtcaataatgacgtatgttcccatagtaac gCcaatagggactttccattgacgtcaatgggtggagtatttacggtaaactgcccacttggcagtacatcaagtgtatcatatgccaagtac gccccctattgacgtcaatgacggtaaatggcccgcctggcattatgcccagtacatgaccttatgggactttcctacttggcagtacatctac gtattagtcatcgctattaccatg (SEQ ID NO: 11).

Tn some embodiments, the CMV (cytomégalovirus) promoter has the following nucleic acid sequence:

gtgatgcggttttggcagtacatcaatgggcgtggatagcggtttgactcacggggatttccaagtctccaccccattgacgtcaatgggagt ttgttttgGcaccaaaatcaacgggactttccaaaatgtcgtaacaactccgccccattgacgcaaatgggcggtaggcgtgtacggtggga ggtctatataagcagagct (SEQ ID NO: 12).

In some embodiments, the intron of the hBGl (hemoglobin subunit gamma 1) gene has the following nucleic acid sequence:

cgaatcccggccgggaacggtgcattggaacgcggattccccgtgccaagagtgacgtaagtaccgcctatagagtctataggcccacaa aaaatgctttcttcttttaatatacttttttgtttatcttatttctaatactttccctaatctctttctttcagggcaataatgatacaatgtatcatgcctcttt gcaccattctaaagaataacagtgalaatttctgggttaaggcaatagcaatatttctgcatataaatatttctgcatataaattgtaactgatgtaa gaggtttcatattgctaatagcagctacaatccagctaccattctgcttttattttatggttgggataaggctggattattctgagtccaagctagg 13 cccttttgctaatcatgttcatacctcttatcttcctcccacagctcctgggcaacgtgctggtctgtgtgctggcccatcactttggcaaagaatt gggat (SEQ ID NO: 13).

In some embodiments, the hGHl (human growth hormone 1 gene) polyadenylation signal has the following nucleic acid sequence:

acgggtggcatccctgtgacccctccccagtgcctctcctggccctggaagttgccactccagtgcccaccagccttgtcctaataaaattaa gttgcatcattttgtctgactaggtgtccttctataatattatggggtggaggggggtggtatggagcaaggggcaagttgggaagacaacct gtagggcctgcggggtctattgggaaccaagctggagtgcagtggcacaatcttggctcactgcaatctccgcctcctgggttcaagcgatt ctcctgcctcagcctcccgagttgttgggattccaggcatgcatgaccaggctcagctaatttttgtttttttggtagagacggggtttcaccata ttggccaggctggtctccaactcctaatctcaggtgatctacccaccttggcctcccaaattgctgggattacaggcgtgaaccactgctccct tccctgtcctt (SEQ ID NO: 14).

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

TGCATCTCAATTAGTCAGCAACCATAGtcccgcccctaactccgcccatcccgcccctaactccgcccagttccg cccattctccgccccatcgctgactaattttttttatttatgcagaggccgaggccgcctcggcctctgagctattccagaagtagtgaggagg cttttttggaggcctaggcttttgcaaaaagct (SEQ ID NO: 15).

Tn some embodiments, the SV40 polyadenylation signal (simian virus 40 polyadenylation signal) has the . following nucleic acid sequence: aataaaatatctttattttcattacatctgtgtgttggttttttgtgtgaatcgatagtactaacatacgctctccatcaaaacaaaacgaaacaaaac aaactagcaaaataggctgtccccagtgcaaGTGCAGGTGCCAGAACATTTCTCT (SEQ ID NO: 16).

In some embodiments, the right-hand (second) ITR has the following nucleic acid sequence:

aggaacccctagtgatggagttggccactccctctctgcgcgctcgctcgctcactgaggccgggcgaccaaaggtcgcccgacgcccg ggctttgcccgggcggcctcagtgagcgagcgagcgcgcagctgcctgcagg (SEQ ID NO: 17).

Tn some embodiments, the expression cassette includes a nucleic acid with the nucléotide sequence cctgcaggcagctgcgcgctcgctcgctcactgaggccgcccgggcgtcgggcgacctttggtcgcccggcctcagtgagcgagcgag cgcgcagagagggagtggcçaactccatcactaggggttcctgcggccgcacgcgtctagttattaatagtaatcaattacggggtcattag ttcatagcccatatatggagttccgcgttacataacttacggtaaatggcccgcctggctgaccgcccaacgacccccgcccattgacgtca ataatgacgtatgttcccatagtaacgCcaatagggactttccattgacgtcaatgggtggagtatttacggtaaactgcccacttggcagtac atcaagtgtatcatatgccaagtacgccccctattgacgtcaatgacggtaaatggcccgcctggcattatgcccagtacatgaccttatggg actttcctacttggcagtacatctacgtattagtcatcgctattaccatggtgatgcggttttggcagtacatcaatgggcgtggatagcggtttg actcacggggatttccaagtctccaccccattgacgtcaatgggagtttgttttgGcaccaaaatcaacgggactttccaaaatgtcgtaaca actccgccccattgacgcaaatgggcggtaggcgtgtacggtgggaggtctatataagcagagctcgtttagtgaaccgtcagatcgcctg gagacgccatccacgctgttttgacctccatagaagacaccgggaccgatccagcctccgcggattcgaatcccggccgggaacggtgc attggaacgcggattccccgtgccaagagtgacgtaagtaccgcctatagagtctataggcccacaaaaaatgctttcttcttttaatatactttt ttgtttatcttatttctaatactttccctaatctctttctttcagggcaataatgatacaatgtatcatgcctctttgcaccattctaaagaataacagtg ataatttctgggttaaggcaatagcaatatttctgcatataaatatttctgcatataaattgtaactgatgtaagaggtttcatattgctaatagcagc tacaatccagctaccattctgcttttattttatggttgggataaggctggattattctgagtccaagctaggcccttttgctaatcatgttcatacctc ttatcttcctcccacagctcctgggcaacgtgctggtctgtgtgctggcccatcactttggcaaagaattgggattcgaacatCGATTGT AATTCATGAGCCACCATGGCCATGAGCAGCGGCGGCAGCGGCGGCGGCGTGCCTGA GCAAGAGGACAGCGTGCTGTTCAGAAGAGGCACCGGCCAGAGCGACGACAGCGAC ATCTGGGACGACACCGCCCTGATCAAGGCCTACGACAAGGCCGTGGCCAGCTTCAA GCACGCCCTGAAGAACGGCGACATCTGCGAGACCAGCGGCAAGCCCAAGACCACCC CCAAGAGAAAGCCCGCCAAGAAGAACAAGAGCCAGAAGAAGAACACCGCCGCCAG CCTGCAGCAGTGGAAGGTGGGCGACAAGTGCAGCGCCATCTGGAGCGAGGACGGCT GCATCTACCCCGCCACCATCGCCAGCATCGACTTCAAGAGAGAGACCTGCGTGGTG GTGTACACCGGCTACGGCAACAGAGAGGAGCAGAACCTGAGCGACCTGCTGAGCCC CATCTGCGAGGTGGCCAACAACATCGAGCAGAACGCCCAAGAGAACGAGAACGAG AGCCAAGTGAGCACCGACGAGAGCGAGAACAGCAGAAGCCCCGGCAACAAGAGCG ACAACATCAAGCCCAAGAGCGCCCCCTGGAACAGCTTCCTGCCCCCTCCCCCCCCTA TGCCCGGCCCTAGACTGGGCCCTGGCAAGCCTGGCCTGAAGTTCAACGGCCCCCCCC CCCCTCCTCCTCCTCCTCCTCCTCACCTGCTGAGCTGCTGGCTGCCCCCCTTCCCCAG CGGCCCTCCTATCATCCCTCCTCCCCCCCCCATCTGCCCCGACAGCCTGGACGACGC CGACGCCCTGGGCAGCATGCTGATCAGCTGGTACATGAGCGGCTACCACACCGGCT ACTACATGGGCTTCAGACAGAACCAGAAGGAGGGCCGGTGCAGCCACAGCCTGAAC TGATctagagtcgacctgcagaagcttgcctcgagcagcgctgctcgagagatctacgggtggcatccctgtgacccctccccagtgc ctctcctggccctggaagttgccactccagtgcccaccagccttgtcctaataaaattaagttgcatcattttgtctgactaggtgtccttctataa tattatggggtggaggggggtggtatggagcaaggggcaagttgggaagacaacctgtagggcctgcggggtctattgggaaccaagct ggagtgcagtggcacaatcttggctcactgcaatctccgcctcctgggttcaagcgattctcctgcctcagcctcccgagttgttgggattcca ggcatgcatgaccaggctcagctaatttttgtttttttggtagagacggggtttcaccatattggccaggctggtctccaactcctaatctcaggt gatctacccaccttggcctcccaaattgctgggattacaggcgtgaaccactgctcccttccctgtccttctgattttgtaggtaaccacTAG AGAAATGTTCTGGCACCTGCACttgcactggggacagcctattttgctagtttgttttgtttcgttttgttttgatggagagcg tatgttagtactatcgattcacacaaaaaaccaacacacagatgtaatgaaaataaagatattttattgcggccgcTGTTCCTGCTGA ACTGAGCCAGTGTACACAAACCAACTGTGTTTCAGCTCAGTAGGCACGGGAGGCAG AGCCCAGGGAGGCCAGGCAGCAGGATGGCAGGCAGACAGGCGGCAGCAGGGGACA GGCGGCAAGGCCAGAGGAGGTGAGGGCCTGGGGGGCGGAACTTAGCCACTGTGAA CACGACTTGGTGTGGACCCTGCTCACAAGCAGCTAAGCCCTGCTCCTCAGGCCAGGC ACAGGCTTCGGGGCCTCTCTGCCACCCCGTCCCCGGGCAGCATCCTCGGTGGCAGAG CTCAGGGTCGGTTGGAAATCCCTGGCAATGTGATTTGTGACAGGAAGCAAATCCCAT CCCCAGGAACCCCAGCCGGCCGTGGCACAGGGGTGAGGGGGGCACCGGGCGGGGC

CAGAGGCTGGCACCTGGAGGGGAGAAAGAGAGAGAGAGCAAAGGAGGTAATGAGA TTTGGGGACACCTCCCCTCCAGGTCCCCAGATGCTTCCTTTGATCACAGGGAAGCTC TTTTCTCATATGCAGGAGCCACCACACGGGGAGCTGGAGCCTAGCTGTGCCCTCCGT TCCAGCCCCAGGACTGGAGAGGCAGAGATACCTAGAGGGCCCATGCGGGACAAGG AGGCTACAGCAACTTGCATGgccgcagctttttgcaaaagcctaggcctccaaaaaagcctcctcactacttctggaata gctcagaggccgaggcggcctcggcctctgcataaataaaaaaaattagtcagcgatggggcggagaatgggcggaactgggcggagt taggggcgggatgggcggagttaggggcgggaCTATGGTTGCTGACTAATTGAGATGCAgggccgctccaag tacctcccgtaccttaagtgcggaccgagcggccgcaggaacccctagtgatggagttggccactccctctctgcgcgctcgctcgctcac tgaggccgggcgaccaaaggtcgcccgacgcccgggctttgcccgggcggcctcagtgagcgagcgagcgcgcagctgcctgcagg (SEQ ID NO: 6).

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

In some embodiments, a vector is a viral vector, wherein additional DNA segments may be ligated into the viral genome.

In some embodiments, vectors are capable of autonomous réplication in a host cell into which they are introduced (e.g. bacterial vectors having a bacterial origin site of réplication and episomal mammalian vectors). In further embodiments, vectors (e.g. non-episomal mammalian vectors) may be integrated into the genome of a host cell upon introduction into a host cell, and thereby are replicated along with the host gene. Moreover, certain 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 vectors include plasmids, retroviruses, adenoviruses, adeno-associated viruses (AAVs), plant viruses, such as cauliflower mosaic virus, tobacco mosaic virus, cosmids, YACs, EBV derived episomes, and the like. DNA molécules may be ligated into a vector such that transcriptional and translational control sequences within the vector serve their intended fonction of regulating the transcription and translation of the DNA. An expression vector and expression control sequences may be chosen to be compatible with the expression host cell used. DNA molécules may be introduced into the expression vector by standard methods (e.g. ligation of complementary restriction sites, or blunt end ligation if no restriction sites are présent).

The recombinant expression vector may also encode a leader peptide (or a signal peptide) that facilitâtes the sécrétion of the protein of interest from a host cell. The gene of 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 other leader peptide (that is, a non-immunoglobulin protein leader peptide).

In addition to the SMN1 and microRNA miR-23a genes according to the présent invention, the recombinant expression of the vectors accofding to the présent invention may carry regulatory sequences that control the expression of the SMN1 gene and microRNA miR-23a gene in a host cell. It will be understood by those skilled in the art that the design of an expression vector, including the sélection of regulatory sequences, may dépend on such factors as the choice of a host cell to be transformed, the level of expression of a desired protein, and so forth. Preferred control sequences for an expression host cell in mammals include viral éléments that ensure high levels of protein expression in mammalian cells, such as promoters and/or enhancers derived from a rétro viral LTR, cytomégalovirus (CMV) (such as a CMV promoter/enhancer), simian virus 40 (SV40) (such as a SV40 promoter/enhancer), adenovirus, (e.g. the major late promoter adenovirus (AdMLP)), polyomavirus and strong mammalian promoters such as native immunoglobulin promoter or actin promoter.

The term control sequences refers to DNA sequences necessary for the expression of an operably linked coding sequence in a particular host organism. The control sequences that are suitable for prokaryotes are, for example, a promoter, optionally an operator sequence and a ribosome binding site. Eukaryotic cells are known to include promoters, polyadenylation signais, and enhancers.

As used in the présent description, the term “promoter” or “transcription 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 with respect to the direction of reading relative to the direction of transcription from the transcription initiation site of the coding sequence, and is structurally identified by the presence of a binding site for DNA-dependent RNA polymerase, transcription initiation sites and any other DNA sequences, including, but not limited to, transcription factor binding sites, repressor and activator protein binding sites, and any other sequences of nucléotides known to one of skill in the art that directly or indirectly regulate the level of transcription with said promoter. A “constitutive” promoter is a promoter that is active in most tissues under typical physiological and developmental conditions. An “inducible” promoter is a promoter that is physiologically or developmentally regulated, e.g. under the influence of a Chemical inducer. A “tissue spécifie” promoter is only active in spécifie types of tissues or cells.

The tenus “enhancers” or “enhancer” as used herein may refer to a DNA sequence that is located adjacent to the DNA sequence that encodes a recombinant product. Enhancer éléments are typically located in a 5' direction from a promoter element or can be located downstream of or within a coding DNA sequence (e.g. a DNA sequence transcribed or translated into a recombinant product or products). Hence, an enhancer element may be located 100 base pairs, 200 base pairs, or 300 or more base pairs upstream of a DNA sequence that encodes a recombinant product, or downstream of said sequence. Enhancer éléments may increase the amount of a recombinant product being expressed from a DNA sequence above the level of expression associated with a single promoter element. Multiple enhancer éléments are readily available to those of ordinary skill in the art.

The term expression control sequence as used in the présent description refers to polynucleotide sequences that are necessary to effect 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 signais such as splicing and polyadenylation signais; sequences that stabilize cytoplasmic mRNA; sequences that enhance translation efficiency (i.e., Kozak consensus sequence); sequences that enhance protein stability; and when desired, sequences that enhance protein sécrétion. The nature of such control sequences differs depending upon the host organism; in prokaryotes, such control sequences generally include the promoter of ribosome binding site, and transcription termination sequences; in eukaryotes, typically, such control sequences include promoters and transcription termination sequences. The term control sequences includes at least ail components, the presence of which is essential for expression and processing, and can also include additional components, the presence of which is advantageous, for example, leader sequences and fusion partner sequences. .

Tn one embodiment of the présent invention, expression vector relates to a vector comprising one or more polynucleotide sequences of interest, genes of interest, or transgenes that are flanked by parvoviral sequences or inverted terminal repeat (ITR) sequences.

Neither the cassette nor the vector of the invention comprises nucléotide sequences of genes encoding non-structural proteins (Rep) and structural proteins (Cap) of the adeno-associated virus.

AAV9 (adeno-associated virus serotype 9)-based recombinant virus

The term AAV-based recombinant virus (or AAV-based virus-like particle, or AAV recombinant virus strain, or AAV recombinant vector, or rAAV vector) as used in this description refers to the above expression cassette (or the above expression vector), which is enclosed within the AAV capsid.

The Cap gene, among other alternative products, encodes 3 capsid proteins (VP1, VP2, and VP3). VP1, VP2, and VP3 are présent at 1:1:10 ratio to form 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 starts from a single promoter, p40. The molecular weights of the corresponding proteins (VP1, VP2 n VP3) are 87, 72, and 62 kDa, respectively. Ail of the three proteins are translated from a single mRNA. Following transcription, pre-mRNA may be spliced in two different manners, where either longer or shorter intron is excised to form mRNAs of various nucléotide lengths.

During the production of the AAV (rAAV)-based recombinant virus, an expression cassette flanked by ITR is packaged into an AAV capsid. The genes required for AAV réplication, as mentioned above, are not included in the cassette.

The expression cassette DNA is packaged into a viral capsid in the form of a single stranded DNA molécule (ssDNA) being approximately 3000 nucléotides long. Once a cell is infected with the virus, the single-stranded DNA is converted to the form of double-stranded DNA (dsDNA). The dsDNA can only be used by the cell's proteins, which transcribe the présent gene or genes into RNA.

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

Tn some embodiments, the AAV9-based recombinant virus has a capsid that includes the AAV9 protein VP1 having the following amino acid sequence MAADGYLPDWLEDNLSEGIREWWALKPGAPQPKANQQHQDNARGLVLPGYKYLGPG NGLDKGEPVNAADAAALEHDKAYDQQLKAGDNPYLKYNHADAEFQERLKEDTSFGG NLGRAVFQAKKRLLEPLGLVEEAAKTAPGKKRPVEQSPQEPDSSAGIGKSGAQPAKKRL NFGQTGDTESVPDPQPIGEPPAAPSGVGSLTMASGGGAPVADNNEGADGVGSSSGNWH CDSQWLGDRVITTSTRTWALPTYNNHLYKQISNSTSGGSSNDNAYFGYSTPWGYFDFN RFHCHFSPRDWQRLINNNWGFRPKRLNFKLFNIQVKEVTDNNGVKTIANNLTSTVQVFT DSDYQLPYVLGSAHEGCLPPFPADVFMIPQYGYLTLNDGSQAVGRSSFYCLEYFPSQML RTGNNFQFSYEFENVPFHSSYAHSQSLDRLMNPLIDQYLYYLSKTINGSGQNQQTLKFSV AGPSNMAVQGRNYIPGPSYRQQRVSTTVTQNNNSEFAWPGASSWALNGRNSLMNPGP AMASHKEGEDRFFPLSGSLIFGKQGTGRDNVDADKVMITNEEEIKTTNPVATESYGQVA TNHQSAQAQAQTGWVQNQGILPGMVWQDRDVYLQGPIWAKIPHTDGNFHPSPLMGGF GMKHPPPQILIKNTPVPADPPTAFNKDKLNSFITQYSTGQVSVEIEWELQKENSKRWNPE IQYTSNYYKSNNVEFAVNTEGVYSEPRPIGTRYLTRNL (SEQ ID NO: 7).

O?

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

In some embodiments, the AAV9-based recombinant virus has a capsid that includes the AAV9 protein VP2. ¹

Jh some embodiments, the AAV9-based recombinant virus has a capsid that includes the AAV9 protein VP2 having the following amino acid sequence: TAPGKKRPVEQSPQEPDSSAGIGKSGAQPAKKRLNFGQTGDTESVPDPQPIGEPPAAPSG VGSLTMASGGGAPVADNNEGADGVGSSSGNWHCDSQWLGDRVITTSTRTWALPTYNN HLYKQISNSTSGGSSNDNAYFGYSTPWGYFDFNRFHCHFSPRDWQRLINNNWGFRPKRL NFKLFNIQVKEVTDNNGVKTIANNLTSTVQVFTDSDYQLPYVLGSAHEGCLPPFPADVF MIPQYGYLTLNDGSQAVGRSSFYCLEYFPSQMLRTGNNFQFSYEFENVPFHSSYAHSQS LDRLMNPLIDQYLYYLSKTINGSGQNQQTLKFSVAGPSNMAVQGRNYIPGPSYRQQRVS TTVTQNNNSEFAWPGASSWALNGRNSLMNPGPAMASHKEGEDRFFPLSGSLIFGKQGT GRDNVDADKVMIINEEEIKTTNPVATESYGQVATNHQSAQAQAQTGWVQNQGILPGM VWQDRDVYLQGPIWAKJPHTDGNFHPSPLMGGFGMKHPPPQILIKNTPVPADPPTAFNK DKLNSFITQYSTGQVSVEIEWELQKENSKRWNPEIQYTSNYYKSNNVEFAVNTEGVYSE PRPIGTRYLTRNL (SEQ ID NO: 8).

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

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

In some embodiments, the AAV9-based recombinant virus has a capsid that includes the AAV9 protein VP3 having the following amino acid sequence MASGGGAPVADNNEGADGVGSSSGNWHCDSQWLGDRVITTSTRTWALPTYNNHLYK QISNSTSGGSSNDNAYFGYSTPWGYFDFNRFHCHFSPRDWQRLINNNWGFRPKRLNFKL FNIQVKEVTDNNGVKTIANNLTSTVQVFTDSDYQLPYVLGSAHEGCLPPFPADVFMIPQ YGYLTLNDGSQAVGRSSFYCLEYFPSQMLRTGNNFQFSYEFENVPFHSSYAHSQSLDRL MNPLIDQYLYYLSKTINGSGQNQQTLKFSVAGPSNMAVQGRNYIPGPSYRQQRVSTTVT QNNNSEFAWPGASSWALNGRNSLMNPGPAMASHKEGEDRFFPLSGSLIFGKQGTGRDN VDADKVMITNEEEIKTTNPVATESYGQVATNHQSAQAQAQTGWVQNQGILPGMVWQD RDVYLQGPIWAKIPHTDGNFHPSPLMGGFGMKHPPPQILIKNTPVPADPPTAFNKDKLNS FITQYSTGQVSVEIEWELQKENSKRWNPEIQYTSNYYKSNNVEFAVNTEGVYSEPRPIGT RYLTRNL (SEQ ID NO: 9).

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

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

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

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

The phrase more point mutations refers to two, three, four, five, six, seven, eight, nine, or ten point substitutions.

Particularly preferred embodiments include substitutions (mutations) that are conservative in nature, i.e. substitutions that take place within a family of amino acids that are joined in their side chains. In particular, amino acids are typically divided into four families: (1) acidic amino acids are aspartate and glutamate; (2) basic amino acids are lysine, arginine, histidine; (3) nonpolar amino acids are alanine, valine, leucine, isoleucine, proline, phenylalanine, méthionine, tryptophan, and (4) uncharged pblar amino acids are glycine, asparagine, glutamine, cysteine, serine, threonine, tyrosine. Phenylalanine, tryptophan, and tyrosine are sometimes classified as aromatic amino acids. For example, it is reasonably predictable that an isolated substitution of leucine for isoleucine or valine, an aspartate for a glutamate, a threonine for a serine, or a similar conservative substitution of an amino acid for a structurally related amino acid, will not hâve a major effect on the biological activity. For example, the polypeptide of interest may include up to about 5-10 conservative or non-conservative amino acid substitutions, so long as the desired fonction of the molécule remains intact.

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

Conservative substitutions are shown in Table A under preferred substitutions.

Table A
Original residue	Exemplary substitutions	Preferred substitutions
Ala(A)	Val; Leu; Ile	Val

'21

Arg(R)	Lys; Gin; Asn	Lys
Asn(N)	Gin; His; Asp, Lys; Arg	Gin
Asp (D)	Glu; Asn	Glu
Cys (C)	Ser; Ala	Ser
Gln(Q)	Asn; Glu	Asn
Glu (E)	Asp; Gin	Asp
Gly(G)	Ala	Ala
His (H)	Asn; Gin; Lys; Arg	Arg
Ile (I)	Leu; Val; Met; Ala; Phe; Norleucine	Leu
Leu (L)	Norleucine; Ile; Val; Met; Ala; Phe	Ile
Lys (K)	Arg; Gin; Asn	Arg
Met (M)	Leu; Phe; Ile ¹	Leu
Phe(F)	Trp; Leu; Val; Ile; Ala; Tyr	Tyr
Pro (P)	Ala	Ala
Ser(S)	Thr	Thr
Thr (T)	Val; Ser	Ser
Trp(W)	Tyr; Phe	Tyr
Tyr(Y)	Trp; Phe; Thr; Ser	Phe
Val (V)	Ile; Leu; Met; Phe; Ala; Norleucine	Leu

Tn some embodiments, the AAV9-based recombinant virus has a capsid that includes the AAV9 protein VP1 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 éléments in the 5'-end to 3'-end direction:

a left-hand (first) ITR (inverted terminal repeats);

a CMV (cytomégalovirus) enhancer;

a CMV (cytomégalovirus) promoter;

an intron of the hBGl gene (hemoglobin subunit gamma 1 gene);

a nucleic acid that encodes the SMN1 protein;

an SV40 promoter (simian virus 40 promoter);

a nucleic acid that encodes the microRNA miR-23a;

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

a left-hand (first) ITR (inverted terminal repeats);

a CMV (cytomégalovirus) enhancer;

a CMV (cytomégalovirus) promoter;

an intron of the hBGl gene (hemoglobin subunit gamma 1 gene);

a nucleic acid that encodes the SMN1 protein;

an SV40 promoter (simian virus 40 promoter);

a nucleic acid that encodes the microRNA miR-23a;

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

a left-hand (first) ITR (inverted terminal repeats);

a CMV (cytomégalovirus) enhancer;

a CMV (cytomégalovirus) promoter;

an intron of the hBGl gene (hemoglobin subunit gamma 1 gene);

a nucleic acid that encodes the SMN 1 protein;

an SV40 promoter (simian virus 40 promoter);

a nucleic acid that encodes the microRNA miR-23a;

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

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

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

Pharmaceutical composition

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

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

Tn particular embodiments, the présent invention relates to a pharmaceutical composition comprising the AAV9-based recombinant virus of the invention in a pharmaceutically acceptable carrier or in other pharmaceutical agents, adjuvants, diluents, etc. For injection, the carrier will typically be a liquid carrier. For other methods of administration, the carrier may be either solid or liquid, such as stérile pyrogen-free water or stérile pyrogen-free phosphate-buffered saline solution. For inhalation administration, the carrier is respirable, and preferably is in a solid or liquid particulate form. As an injection medium, it is preferred to use water that contains the additives that are common for injection solutions, such as stabilizing agents, salts or saline, and/or buffers.

Pharmaceutical composition means a composition comprising the above AAV9-based recombinant virus of the invention and at least one of components selected from the group consisting of pharmaceutically acceptable and pharmacologically compatible excipients, such as fillers, solvents, diluents, carriers, auxiliary, distributing agents, delivery agents, preservatives, stabilizers, emulsifiers, suspending agents, thickeners, prolonged delivery controllers, the choice and proportions of which dépend on the type and route of administration and dosage. Pharmaceutical compositions of the présent invention and methods of préparation thereof will be undoubtedly apparent to those skilled in the art. The pharmaceutical compositions should preferably be manufactured in compliance with the GMP (Good Manufacturing Practice) requirements. The composition may comprise a'buffer composition, tonicity agents, stabilizers and solubilizers.

Pharmaceutically acceptable means a material that does not hâve biological or other négative side effects, for example, the material can be administered to a subject without causing any undesirable biological effects. Thus, such pharmaceutical compositions may be used, for example, in transfection of a cell ex vivo or in administration in vivo of the AAV9-based recombinant virus of the invention directly to a subject.

The term excipient is used herein to describe any ingrédient other than the above ingrédients of the invention. These are substances of inorganic or organic nature which are used in the pharmaceutical manufacturing in order to give drug products the necessary physicochemical properties.

Stabilizer refers to an excipient or a mixture of two or more excipients that provide the physical and/or Chemical stability of the active agent.

The term buffer, buffer composition, buffering agent refers to a solution, which is capable of resisting changes in pH by the action of its acid-base conjugate components, which allows the rAAV9 vector product to resist changes in pH. Generally, the pharmaceutical composition preferably has a pH in the range from 4.0 to 8.0. Examples of buffers used include, but are not limited to, acetate, phosphate, citrate, histidine, succinate, etc. buffer solutions.

The pharmaceutical composition is stable if the active agent retains physical stability and/or Chemical stability and/or biological activity thereof during the specified shelf life at storage température, for example, of 2-8 °C. Preferably, the active agent retains both physical and Chemical stability, as well as biological activity. Storage period is adjusted based on the results of stability test in accelerated or naturel aging conditions.

A pharmaceutical composition of the invention can be manufactured, packaged, or widely sold in the form of a single unit dose or a plurality of single unit doses in the form of a ready formulation. The term single unit dose as used herein refers to discrète quantity of a pharmaceutical composition containing a predetermined quantity of an active ingrédient. The quantity of the active ingrédient typically equals the dose of the active ingrédient to be administered in a subject, or a convenient portion of such dose, for example, half or a third of such dose.

Use

The authors of the invention surprisingly found that miR-23a enhances the functional effect of SMN1 in vitro and revealed the synergistic effect of SMN1 and miR-23a in treating SMA in an animal model of the disease. .

In one aspect, the présent invention relates to the use of any of the above AAV9-based recombinant viruses or the above composition for survival of a subject that has spinal muscular atrophy and/or that does not hâve folly functioilal copies of the SMN1 gene.

In one aspect, the présent invention relates to the use of any of the above AAV9-based recombinant viruses or the above composition for providing the SMN1 protein to a subject that has spinal muscular atrophy and/or that does not hâve fully functional copies of the SMN1 gene.

In one aspect, the présent invention relates to the use of any of the above AAV9-based recombinant viruses or the above composition for treating spinal muscular atrophy in a subject that has spinal muscular atrophy.

In one aspect, the présent invention relates to a method for modulating motor fonction in a subject having a motor neuron disorder, said method comprising administering a therapeutically effective amount of any of the above AAV9-based recombinant viruses or the above composition into the cells of the subject.

In one aspect, the ptesent invention relates to a method for providing the SMN protein to a subject having spinal muscular atrophy, said method comprising administering a therapeutically effective amount of any of the above AAV9-baSed recombinant viruses or the above composition into the cells of the subject in need thereof.

In one aspect, the présent invention relates to a method for delivering the SMN1 gene to the target cells of a subject having spinal muscular atrophy, saidmethod comprising administering any of the above AAV9-based recombinant viruses or the above composition into the cells of the subject.

In one aspect, the présent invention relates to a method for treating spinal muscular atrophy in a subject, said method cc>mprising administering a therapeutically effective amount of any of the above AAV9-based recombinant viruses or the above composition into a subject having spinal muscular atrophy.

The lack of folly fonctional copies of the SMN1 gene refers to inactivating mutations or délétions in ail copies of the SMN1 gene in the genome, which resuit in the loss or defect of the fonction of the SMN1 gene.

Subject refers to any animal that is amenable to the techniques provided in the présent description. In certain non-limiting embodiments, the subject is a human. Said subject may be either male or female, of any âge.

A subject in need of delivering the SMN1 gene to target cells, or a subject in need of being provided with the SMN1 protein, or a subject in need of delivering the SMN1 gene to target cells refers to a subject who has spinal muscular atrophy or a subject who has inactivating mutations or délétions in the SMN1 gene that lead to loss or defect in the fonction of the SMN1 gene.

Exemplary modes of administration include topical application, intranasal, inhalation, transmucosal, transdermal, enterai (e.g. oral, rectal), parentéral (e.g. intravenous, subcutaneous, intradermal, intramuscular) administrations, as well as direct tissue or organ injections.

Injectables canbe prepared in conventional forms, either as liquid solutions or suspensions, solid forms suitable for the préparation of solution or suspensions in liquid prior to injection, or as émulsions. Altematively, one may administer the above AAV9-based recombinant virus of the présent invention in a local rather than systemic manner, for example in a depot or sustainedrelease formulation.

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

Dosages of the above AAV9-based recombinant virus of the invention will dépend on the mode of administration, the particular viral vector, and they can be determined in a routine manner. Exemplary doses for achieving therapeutic effects are viral titers of at least about ΙΟ⁵, ΙΟ⁶, ΙΟ⁷, ΙΟ⁸, ΙΟ⁹, ΙΟ¹⁰, ΙΟ¹¹, ΙΟ¹², ΙΟ¹³, ΙΟ¹⁴, ΙΟ¹⁵,10¹⁶ transducing units or more, preferably about 10⁹ to 10¹⁵ transducing units, yet more preferably 10¹⁴ transducing units per kilogram.

The cell for administering the above AAV9-based recombinant viras of the invention may be a cell of any type, including but not limited to motor neurons or other tissues of the nervous System, épithélial cells (e.g. skin, respiratory and gut épithélial cells), hepatic cells, muscle cells, pancreatic cells (including islet cells), hepatic cells, spleen cells, fibroblasts, endothélial cells, and the like.

The above AAV9-based recombinant viras of the invention is not used to modify the genetic integrity of human germ line cells.

Examples

The following examples are provided for better understanding of the invention. These examples are for purposes of illustration only and are not to be constraed as limiting the scope of the invention in any manner.

Ail publications, patents, and patent applications cited in this spécification are incorporated herein by reference. Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, it will be readily apparent to those of ordinary skill in the art in light of the teachings of this invention that certain changes and modifications may be made thereto without departing from the spirit or scope of the appended embodiments.

Materials and general methods

Recombinant DNA techniques

Standard methods were used to manipulate DNA as described in Sambrook, J. et al, Molecular cloning: A laboratory manual; Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 1989. The molecular biological reagents were used according to the manufacturer protocols. Briefly, plasmid DNA was produced for further manipulation in E. coli cells grown under sélective antibiotic pressure so that the plasmids were not lost in the cell population. We isolated the plasmid DNA from cells using commercial kits, measured the concentration, and used it for cloning by restriction endonuclease treatment or PCR amplification. The DNA fragments were ligated to each other using ligases and transformed into bacterial cells for the sélection of clones and further production. Ail resulting genetic constructs were confirmed by restriction patterns and complété Sanger sequencing.

Gene synthesis

Desired gene segments were prepared from oligonucleotides made by Chemical synthesis. Gene segments of 300 to 1000 bp long, which were flanked by unique restriction sites, were collected by renaturing oligonucleotides on top of each other, followed by PCR amplification from border primers. As a resuit, a mixture of fragments was produced, including the desired one. The fragments were cloned at restriction sites into intermediate vectors, following which the DNA sequences of the subcloned fragments were confirmed by DNA sequencing.

DNA sequence détermination

DNA sequences were determined by Sanger sequencing. DNA and protein sequences were analyzed and sequence data was processed in SnapGene Viewer 4.2 or higher for sequence création, mapping, analysis, annotation and illustration.

Culturing cell cultures

The experiments used HEK293 (Human Embryonic Kidney clone 293) and U-87 MG (human glioblastoma) cell lines. The cells were cultured under standard conditions at 37 °C and 5%CO₂, on a DMEM complété culture medium supplemented with 10% FBS and an antibiotic. To culture HSMCs, the culture plastic was pre-coated with collagen (Gibco). Cells were subcultured upon reaching 80-90% confluence. Cell viability was assessed using either Trypan Blue stain and a hemocytometer or PI stain and flow cytometry.

siRNA cell transfection

A commercial siRNA spécifie for the ,SMN1 gene was ordered from the manufacturer (ThermoFisher Scientific) along with a non-specific control.

Cell lines were inoculated the day before transfection into 6-well plates such that they reached 70-80% confluence by the time of transfection. Transfection was performed using commercial lipofection kits according to the manufacturées protocol. 24 h following transfection, transduction was performed (see below). 120 h following transduction, the cells were treated with trypsin solutions or similar, removed from the substrate, washed in a phosphate buffer, and collected for further analysis of expression of target genes and proteins. Enzyme immunoassay (ELISA) was performed to control the level of SMN protein knockdown.

Ail samples were analyzed with 3 experimental replicates.

Gene expression analysis

Expression o/SMNl at the mRNA level and expression of miR-23a was measured using quantitative PCR. Briefly, we used primers and a sample that are spécifie for the nucléotide sequence encoding the SMN1 protein. For miR-23a, we used a commercial kit for quantitative PCR spécifie for processed miR-23a (Thermo Fisher Scientific). Primers and a sample spécifie for the GAPDH household gene were used to control the initial RNA levels. Calibration curves were plotted for each set of primers and samples using a known copy number of linearized plasmid DNA comprising the amplified sequence of the corresponding gene. Expression was analyzed by detennining, using the calibration curves, the copy number of SMN1 and GAPDH in each sample, following which we normalized the copy number of SMN1 to 10,000 copies of GAPDH. The resulting values were compared for different samples within the same experiment.

Détermination of SMN protein expression

Thermo Fisher Scientific kit was used to détermine the amount of total protein. Cellular précipitâtes were lysed, the resulting lysâtes were introduced into microplate wells together with standard dilutions of BSA at a known concentration. A working reagent was added to the samples, and the samples were incubated. At the end of incubation, we measured absorption at a wavelength of 562 nm on a microplate reader. Concentration of total protein in test samples was calculated according to BSA standard curve. Next, the lysâtes were diluted to a total protein concentration of 10 pg/ml.

Content of SMN protein in cells was assessed by enzyme immunoassay (ELISA) using a commercial Abcam kit according to the manufacturées instructions. Microplate wells coated with anti-SMN antibodies were loaded with test samples and standards at known concentrations.

Following incubation and washing, SMN-specific polyclonal antibodies for détection were added to each well, the samples were incubated and washed. Secondary antibodies conjugated with horseradish peroxidase were added, and the samples were incubated. Excess of reagents was washed off and TMB substrate was added. Following a short-term incubation, the enzymatic reaction was stopped with stop reagent. Optical density of the yellow-colored product was measured by spectrophotometry at a wavelength of 450 nm. Amount of SMN in test samples was determined using the calibration plot of optical density versus concentration of SMN in the standards. ₁

Détermination of expression of SMN target, Senataxin protein (functional test)

Senataxin protein is a polyfimctional enzyme involved in the resolution of DNA-RNA structures called R-loops, which occur during transcription in case of absent or underexpressed SMN protein. The literature has reported a direct corrélation between SMN and Senataxin expression levels, and indirect activation of Senataxin is one of the fonctions of SMN. In this regard, change in Senataxin expression was used as a functional test for SMN.

Senataxin expression level was determined by Western blot. Briefly, cells were lysed with RIPA buffer supplemented with protease inhibitor cocktail following experimental exposure (transfection, transduction, incubation), thereby obtaining protein lysate samples. The samples were applied onto a 10% polyacrylamide denaturing gel, thereafter the proteins were transferred onto PVDF membrane.

Membrane was incubated for 1 hour in 5% bovine sérum albumin (BSA) solution in TBST buffer, thereafter the BSA solution was replaçed with 1% BSA solution in TBS-T supplemented with primary antibodies spécifie for Senataxin. After 2 hours of incubation, the membrane was washed 3 times with 1% BSA solution in TBS-T and secondary antibody solution in TBS-T supplemented with 1% BSA was added. We used secondary antibodies conjugated with enzyme horseradish peroxidase.

Secondary antibodies were washed 3 times with 1% BSA solution in TBS-T, thereafter the protein signal was detected at the membrane using a chemiluminescence substrate and a gel imaging System. Resulting images were saved in digital format and the signal intensity was analyzed using the software of the gel imaging System.

The protein signal of the vinculin household gene was used as a control for normalization. Staining was carried out in a similar fashion..

Assembly and purification of viral particles of AAV recombinant vectors

To assemble AAV particles containing the SMN1 gene or GFP control gene, we used HEK293 packaging cells, into which 3 plasmids were transfected using polyethylenimine as folio ws:

1. A plasmid comprising the AAV genome having a transgene expression cassette (SNM1, GFP, SMN1 + miR-23a or GFP + miR-23a);

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

3. A plasmid for expression of Ad5 (adenovirus serotype 5) genes that are 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 methods. The titer of the viral particles was determined by quantitative PCR with primers and a sample that were spécifie for the région of the recombinant viral genome and expressed as the copy number of viral genomes per 1 ml.

Transduction of cell cultures

U-87 cell line was inoculated similarly to transfection experiments, transfection with siRNA was carried out, following which the prôduct with viral particles was added and, after 120 h, the cells were analyzed. Transduction efficiency was estimated by measuring the percentage of GFP+ cells.

The cultures being used were pre-tested with the check of the transduction efficiency. Briefly, the AAV9-GFP viral product was transduced into the cell fines in different ratios of cells and viral particles. The ratio of viral particle number to cell number is referred to as multiplicity of infection (MOI). The MOI of the AAV9-GFP virus ranged from 50,000 to 1,000,000. As a resuit, the optimal MOI of 400,000 was chosen for the U-87 line. Further U-87 transduction works were carried out at this MOI for ail viruses.

Following transduction, gene and protein expression was analyzed as described above.

Ail samples were analyzed with 3 experimental replicates.

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

Viral particles AAV9-SMN1, AAV9-GFP, AAV9-SMNl-miR-23a and AAV9-GFP-miR23a were used for injection into SMA model mice. These mice do not express the mouse Smn gene, but hâve in the genome thereof one copy of the human SMN2 gene and one copy of the human SMN1 gene with exon 7 missing (8ΜΝ1Δ7). Without intervention or with a placebo injection, such mice are bom, but poorly gain weight after birth and die after an average of 21 days.

Mice of the model fine were genotyped on day 1 following birth, thereafter mice that contained no copy of the Smn gene were injected systemically (in the tail vein) with either a placebo (a solution that does not contain viral particles, but contains a buffer for dilution thereof), or one of the viruses at a dose of 3.2xl0¹⁴ vg/kg of body weight. Thereafter, the animais were kept under standard conditions and weighed daily, and survival curves were also plotted. 90 days 31 following injection, survived animais were sacrificed for tissue analysis and the experiment was terminated.

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

SMN1 gene sequence was produced by amplification with spécifie primers with cDNA synthesized based on total RNA of HEK293 cells, or assembled from a sériés of oligonucleotides (see above). During the amplification process, the Kozak sequence and Clal restriction site were added from the 5’-end of the gene, and the Xbal restriction site was added from the 3’-end. The sequence of the SMN1 gene was thereafter cloned by the restriction-ligase method at the Clal and Xbal sites into a commercial construct pAAV-GFP Control plasmid (VPK-402) from CellBiolab (USA), with substitution of the GFP gene with SMN1, thereby producing the pAAV-SMNl construct.

An additional miR-23a expression cassette was inserted into the plasmids pAAV-GFP and pAAV-SMNl that were produced previously. The additional miR-23a expression cassette consists of a promoter, a gene of interest (miR-23a, produced using PCR from genomic DNA of Huh7 cell line), and a polyadenylation signal. Target vectors were produced by linearizing at the Pmll site the récipient vectors (pAAV-GFP, pAAV-SMNl) followed by incorporation of the expression cassette with miR-23a at the cohesive ends.

The final vector contains ail the necessary éléments for expression and assembly of the gene as part of the recombinant AAV genome:

1. ITRs at the ends of the sequence that is encapsidated into a viral capsid;

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

3. If any, miR-23a expression cassette (promoter, miR-30-based microRNA cassette encoding miR-23a forward and reverse strands, polyadenylation signal);

4. The bacterial réplication origin and antibiotic résistance gene to produce plasmid DNA in bacterial cells.

Example 2. Creating of viral products expressing SMN1 and miR-23a

Plasmids pAAV-SMNl, pAAV-GFP, pAAV-SMNl-miR-23a, pAAV-GFP-miR-23a along with other plasmids required for producing recombinant AAV viral particles (see above) were used for the AAV production bioprocess. The serotype used was wild-type AAV9 or that having various mutations, fri ail cases, the properties of viral particles were compared only as long as the serotype used and capsid mutations, if any, were identical. Ail serotypes based on AAV9, either that of wild type or with mutations, are hereinafter referred to as AAV9 without specifying mutations.

Bioprocess resulted in recombinant viral particles designated as AAV9-SMN1, AAV9GFP, AAV9-SMNl-miR-23a, AAV9-GFP-miR-23a. Following determining the titers of viral particles, ail 3 products having the same MOI of 400,000 were used to transduce permissive cells, U-87, pre-transfected (24 hours before) with siRNA against SMN1 or siRNA having a non-specific sequence. Further analysis was performed only as long as the GFP transduction efficiency was at least 70%.

Following successful transduction, the cells were removed from the substrate, washed in a phosphate buffer, and the expression of SMN1 was analyzed at the mRNA and SMN protein levels as described above. It was shown that when transducing with viruses AAV9-SMN1 and AAV9SMNl-miR-23a, SMN1 expression exceeded the endogenous mRNA level (Table 1, Fig. 1) and reconstituted to the endogenous level (observed in the control without SMN1 -spécifie siRNA) at protein level (Table 2, Fig. 2).

Table 1.

Sample name	Mean mRNA copy number normalized to GAPDH copy number	Standard déviation
No siRNA, no virus	0.00275	0.00021
siNeg	0.00168	0.00009
siSMNl	. 0.0002	0.00002
siNeg+ AAV9-GFP	0.00203	0.00033
siSMNl + AAV9-GFP	0.00039	0.00006
siNeg + AAV9-GFP-miR-23a	0.00185	0.00028
siSMNl + AAV9-GFP-miR-23a	, 0.00039	0.00001
siNeg+ AAV9-SMN1	0.10363	0.01045
siSMNl+ AAV9-SMN1	0.33121	0.01495
siNeg + AAV9-SMNl-miR-23a	0.24863	0.01715
siSMNl + AAV9-SMNl-miR-23a	0.34258	________0.0705

Table 2.

Sample name	Mean SMN amount (pg) normalized to total protein (pg)	Standard déviation
No siRNA, no virus	83.37	8.58
siNeg	73.43	5.53
siSMNl	5.45	1.36
siNeg+ AAV9-GFP	67.95	12.41
siSMNl+ AAV9-GFP	. 5.4	1.02
siNeg + AAV9-GFP-miR-23a	71.82	3.16
siSMNl + AAV9-GFP-miR-23a	4.8	1.23
siNeg + AAV9-SMN1	' 116.4	1.85
siSMNl+ AAV9-SMN1	69.15	5.71

siNeg + AAV9-SMNl-miR-23a siSMNl + AAV9-SMNl-miR-23a

99.95

91.13

7.1

4.93

Also, we determined miR-23a expression level in samples post-transduction. A signifïcant excess of miR-23a expression was shown in samples transduced with viruses AAV9-SMNl-miR23a, AAV9-GFP-miR-23a. In other samples, miR-23a expression did not differ from endogenous one. Transfection of siRNA against SMN1 did not affect miR-23a expression (Table 3, Fig. 3).

Table 3.

Sample name	Mean normalized amount of miR-23a (%)	Standard déviation
No siRNA, no virus	100	. 12
siNeg	95	10
siSMNl	113	13
siNeg+ AAV9-GFP	124	12.41
siSMNl+ AAV9-GFP	107	11
siNeg + AAV9-GFP-miR-23a	843	33
siSMNl + AAV9-GFP-miR-23a	768	26
siNeg+ AAV9-SMN1	116.4	16
siSMNl+ AAV9-SMN1	77	8
siNeg + AAV9-SMNl-miR-23a	803	15
siSMNl + AAV9-SMNl-miR-23a	920	19

It should be noted that changes in SMN1 expression with knockdown or reconstitution using the viral product did not affect miR-23a expression. Also, miR-23a overexpression had no effect on SMN1 expression.

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

The above design of experiment of SMN1 knockdown and reconstitution of expression thereof using transduction with viral particles was used to evaluate the functional coopération of SMN1 and miR-23a in vitro. Among the many fonctions of SMN1, the protein is responsible for correct resolution of DNA-RNA duplexes that occur during transcription. Further, the main protein involved in resolution of duplexes is Senataxin, which, according to the literature, is indirectly activated by SMN. Thus, activation of Senataxin may be considered a functional test of SMN activity in cells.

Senataxin expression level in samples was determined by Western blot. It was found that with knockdown of SMN expression, Senataxin expression was decreased by about 2 times, and with reconstitution of SMN expression with virus AAV9-SMN1, it reconstituted to endogenous level. We observed a trend towards a slight increase in Senataxin expression in the case of SMN knockdown during transduction with control virus AAV9-GFP-miR-23a, but it was not 34 statistically significant. It is important to note that in case of transduction with virus AAV9-SMN1miR-23a, Senataxin expression was not only reconstituted to the endogenous level against the background of endogenous SMN knockdown, but also increased statistically significantly by 1.52 times (Table 4, Fig. 4). ' .. .

Table 4.

Sample name	Mean normalized amount of Senataxin (%)	Standard déviation
No siRNA, no virus	100	15
siNeg	109	17
siSMNl	46	8
siNeg+ AAV9-GFP	115	11
siSMNl + AAV9-GFP	53	12
siNeg + AAV9-GFP-miR-23a	98	14
siSMNl + AAV9-GFP-miR-23a	73	19
siNeg + AAV9-SMN1	124	21
siSMNl + AAV9-SM NI	110	13
siNeg + AAV9-SMNl-miR-23a	196	17
siSMNl + AAV9-SMNl-miR-23a	168	7

This confirms the synergistic effect of SMN and miR-23a on Senataxin expression, exceeding the effect of both miR-23a and SMN separately on the régulation of the gene.

Example 4. Synergistic effect of SMN1 and miR-23a in treating animais of SMA mouse model

Viral products AAV9-SMN1 and AAV9-SMNl-miR-23a were injected into the tail vein of SMA model mice on day 1 following birth at a dose of 3.6x10¹⁴ vg/kg. We used a solution without viral particles (placebo) and a group of wild-type sibling mice that did not hâve the SMA phenotype due to their expression of Smn. Next, survival fonctions of mice in ail groups were built. Upon reaching the point where it was possible to détermine the médian survival times for ail groups, the médians were calçulated.

The SMA phenotype was most corrected in the group injected with the virus AAV9SMNl-miR-23a. Médian survival time for the group was 55 days. This resuit was significantly different from the group injected with the virus AAV9-SMN1, where the médian survival time was 21 days. For placebo-injected mice, the médian was 16 days following birth (Fig. 5). This resuit shows the synergistic effect of SMN1 and miR-23a in treating SMA in an animal model of the disease.

Claims

1. An isolated nucleic acid for producing a gene therapy viral product, said isolated nucleic acid comprising a nucleic acid that encodes the SMN1 protein (survival motor neüron protein) having the amino acid sequence of SEQ ID NO: 1, and a nucleic acid that encodes the microRNA miR-23a.

2. The isolated nucleic acid according to claim 1, wherein the nucleic acid that encodes the SMN1 protein having the amino acid sequence of SEQ ID NO: 1 comprises the nucléotide sequence of SEQ ID NO: 2.

3. The isolated nucleic acid according to claim 1, wherein the microRNA miR-23a has the nucléotide sequence of SEQ ID NO: 3.

4. The isolated nucleic acid according to claim 1, wherein the nucleic acid that encodes the microRNA miR-23a comprises the nucléotide sequence of SEQ ID NO: 4.

5. An expression cassette that comprises the nucleic acid according to any one of claims 1 to 4.

fa.

The expression cassette according to claim 5, said expression cassette comprising the following éléments in the 5'-end to 3'-end direction:

a left-hand (first) ITR (inverted terminal repeats);

a CMV (cytomégalovirus) enhancer;

a CMV (cytomégalovirus) promoter;

an intron of the hBGl gene (hemoglobin subunit gamma 1 gene);

a nucleic acid that encodes the SMN1 protein;

an SV40 promoter (simian virus 40 promoter);

a nucleic acid that encodes the microRNA miR-23a;

7. The expression cassette according to claim 6, said expression cassette comprising a nucleic acid with SEQ ID NO: 6.

8. An expression vector that comprises the nucleic acid according to any one of claims 1 to 4 or the cassette according to ahy one of claims 5 to 7.

9. An AAV9 (adeno-associated virus serotype 9)-based recombinant virus for the expression of the SMN1 gene in target cells, said AAV9-based recombinant virus comprising a capsid and the expression cassette according to any one of claims 5 to 7.

10. The AAV9-based recombinant virus according to claim 9, wherein the capsid comprises the AAV9 protein VP1.

11. The AAV9-based recombinant virus according to claim 10, wherein the capsid comprises the AAV9 protein VP1 having the amino acid sequence of SEQ ID NO: 7.

12. The AAV9-based recombinant virus according to claim 10, wherein the capsid includes the AAV9 protein VP1 havihg the amino acid sequence of SEQ ID NO: 7 with one or more point mutations.

13. The AAV9-based recombinant virus according to any one of claims 9 to 12, wherein the capsid comprises the AAV9 protein VP1 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 éléments in the 5'-end to 3'-end direction:

a left-hand (first) ITR (inverted terminal repeats);

a CMV (cytomégalovirus) enhancer;

a CMV (cytomégalovirus) promoter;

an intron of the hBGl gene (hemoglobin subunit gamma 1 gene);

a nucleic acid that encodes the SMN1 protein;

an SV40 promoter (simian virus 40 promoter);

a nucleic acid that encodes the microRNA miR-23a;

14. The AAV9-based recombinant virus according to claim 10, wherein the capsid comprises the AAV9 protein VP1 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 with SEQ ID NO: 6.

15. A pharmaceutical composition for delivering the SMN1 gene to target cells, said pharmaceutical composition comprising the AAV9-based recombinant virus according to claims 9 to 14 in combination with one or more pharmaceutically acceptable excipients.

16. Use of the AAV9-based recombinant virus according to claims 9 to 14 or the composition according to claim 15 for delivering the SMN1 gene to target cells.

17. Use of the AAV9-based recombinant virus according to claims 9 to 14 or the composition according to claim 15 for survival of a subject that has spinal muscular atrophy and/or that does not hâve fully functional copies of the SMN1 gene.

18. Use of the AAV9-based recombinant virus according to claims 9 to 14 or the composition according to claim 15 for providing the SMN1 protein to a subject that has spinal muscular atrophy and/or that does not hâve fully functional copies of the SMN1 gene.

19. Use of the AAV9-based recombinant virus according to claims 9 to 14 or the composition according to claim 15 for treating spinal muscular atrophy in a subject that has spinal muscular atrophy.

20. A method for modulating motor function in a subject having a motor neuron disorder, said method comprising administering a therapeutically effective amount of the AAV9-based recombinant virus according to claims 9 to 14 or the composition according to claim 15 into the cells of the subject.

21. A method for providing the SMN protein to a subject having spinal muscular atrophy, said method comprising administering a therapeutically effective amount of the AAV9-based recombinant virus according to claims 9 to 14 or the composition according to claim 15 into the cells of the subject in need thereof.

22. A method for delivering the SMN1 gene to the target cells of a subject having spinal muscular atrophy, said method comprising administering the AAV9-based recombinant virus according to claims 9 to 14 or the composition according to claim 15 into the cells of the subject.

23. A method for treating spinal muscular atrophy in a subject, said method comprising administering a therapeutically effective amount of the AAV9-based recombinant virus according to claims 9 to 14 or the composition according to claim 15 to a subject having spinal muscular atrophy.