AAV vector carrying hepatocyte growth factor gene optimized for intrathecal administration

Abstract

The present invention relates to an AAV (adeno-associated virus) vector into which a hepatocyte growth factor gene optimized for intrathecal administration has been introduced. The AAV vector of the present invention and the AAV particles produced thereby are excellent in productivity of AAV particles containing the hepatocyte growth factor gene, and are also highly efficient in expressing hepatocyte growth factor when administered intrathecally to a mammal. Therefore, when the AAV vector and particles of the present invention are used for intrathecal administration, they can be usefully used for the prevention or treatment of associated diseases by inducing expression of the hepatocyte growth factor protein.

Description

[Technical field]

[0001] This patent application claims priority to Korean Patent Application No. 10-2022-0052369, filed with the Korean Intellectual Property Office on April 27, 2022, the disclosure of which is incorporated herein by reference.

[0002] The present invention relates to an adeno-associated virus (AAV) vector carrying a hepatocyte growth factor gene that is optimized for intrathecal administration. [Background technology]

[0003] Gene therapy is a method to treat genetic and incurable diseases by transferring therapeutic genes into the patient's cells using recombinant gene technology, causing genetic changes in the target cells or expressing specific proteins. The substance that transfers the gene into the body is called a carrier or vector, and vectors are broadly classified into viral vectors and non-viral vectors. Representative viral vectors include retroviruses and adenoviruses, and non-viral vectors include naked DNA and liposomes.

[0004] The present inventors have previously produced adeno-associated viruses (AAVs) carrying the hepatocyte growth factor gene for gene therapy using hepatocyte growth factor (Lee et al., BBRC 517:452-457, 2019; Lee et al., Acta Neuropathologica Communications 7:14, 2019). However, conventional AAVs have low productivity and are limited by a very low protein expression level upon intrathecal administration. Thus, the need for the development of new AAV vectors with improved productivity and optimized for intrathecal administration to improve protein expression levels has been raised.

[0005] Throughout this specification, a number of papers and patent documents are referred to and citations are provided, the disclosures of which are incorporated herein by reference in their entirety in order to more clearly describe the state of the art to which the present invention pertains and the content of the present invention. Summary of the Invention [Problem to be solved by the invention]

[0006] The present inventors have made extensive research efforts to develop a novel AAV (adeno-associated virus) vector with improved productivity and protein expression levels. As a result, the present inventors have confirmed that the productivity of AAV particles is greatly improved when a specific promoter, AAV serotype, and polyadenylation sequence are combined. The present inventors have also found that the protein expression level is greatly improved when the AAV particles are administered intrathecally, and have completed the present invention.

[0007] Therefore, an object of the present invention is to provide an AAV vector, an AAV particle, and a pharmaceutical composition comprising the same, into which a hepatocyte growth factor gene optimized for intrathecal administration has been introduced.

[0008] Other objects and advantages of the present invention will become more apparent from the following detailed description of the invention, the claims and the drawings. [Means for solving the problem]

[0009] According to one aspect of the invention, the invention provides an AAV (adeno-associated virus) vector comprising a nucleic acid molecule comprising:

[0010] (a) a CMV (Cytomegalovirus) promoter sequence or an EF-1alpha (Elongation Factor 1-alpha) promoter sequence; and

[0011] (b) a polynucleotide sequence encoding a hepatocyte growth factor (HGF), operably linked to the promoter sequence.

[0012] The present inventors have made extensive research efforts to develop a novel AAV (adeno-associated virus) vector with improved productivity and protein expression levels. As a result, the present inventors have confirmed that the productivity of AAV particles is greatly improved when a specific promoter, AAV serotype, and polyadenylation sequence are combined. The present inventors have also found that the protein expression level is greatly improved when the AAV particles are administered intrathecally.

[0013] As used herein, the term "vector" refers to a non-chromosomal nucleic acid that contains an intact replicon such that it can be replicated when placed in a cell by a transformation process. The vector may be viral or non-viral. Viral vectors include, but are not limited to, retroviruses, adenoviruses, adeno-associated viruses (AAV), herpesviruses, baculoviruses, modified baculoviruses, papoviruses, and the like. Non-viral vectors for delivering nucleic acids include the use of naked DNA; DNA complexed with cationic lipids, alone or in combination with cationic polymers; anionic and cationic liposomes; DNA-protein complexes and particles, including DNA condensed with heterologous polylysine, oligopeptides of defined length, and cationic polymers such as polyethyleneimine, in some cases contained in liposomes; and ternary complexes including viruses and polylysine-DNA.

[0014] A "viral vector" is defined as a recombinantly produced virus or virus particle that contains a polynucleotide that is delivered to a host cell in vivo, ex vivo, or in vitro. Examples of viral vectors include retroviral vectors, AAV vectors, lentiviral vectors, adenoviral vectors, alphavirus vectors, and the like. Alphavirus vectors, such as Semliki Forest virus-based vectors and Sindbis virus-based vectors, have also been developed for use in gene therapy and immunotherapy (see Schlesinger and Dubensky (1999) Curr. Opin. Biotechnol. 5:434-439 and Ying, et al. (1999) Nat. Med. 5(7):823-827).

[0015] The vector may include a nucleic acid sequence that allows replication in a host cell, such as an origin of replication. The vector may lack a nucleic acid sequence that allows replication in a host cell. The vector may also include a gene of interest, one or more selectable marker genes, other genetic elements known in the art, or any other suitable insert.

[0016] In one embodiment of the present invention, "adeno-associated virus" or "AAV" belongs to the family Parvoviridae and the genus Dependoparvovirus. Multiple serotypes of this virus are known to be suitable for gene transfer. At least eleven sequentially numbered AAV serotypes are known in the art: AAV1, AAV2, AAV4, AAV5, AAV6, AAV8, AAV9, AAV10, AAV11, AAV12, and AAV13.

[0017] In one embodiment of the invention, the AAV serotypes include the following variant serotypes: AAV-DJ.AAV-DJ / 8, AAV-PHP.Eb, AAV-PHP.S, and AAV rh10.

[0018] As used herein, the term "AAV vector" refers to a vector that contains one or more polynucleotides of interest (or transgenes) flanked by inverted terminal repeat (ITR) sequences. An AAV vector is a vector that encodes and expresses viral DNA replication (rep) and encapsidation (cap) gene products that can be replicated and packaged into infectious viral particles when present in a transfected host cell.

[0019] An "AAV virion" or "AAV virus particle" or "AAV vector particle" refers to a viral particle composed of at least one AAV capsid protein and an encapsidated polynucleotide AAV vector.

[0020] The "AAV" is a replication-deficient parvovirus whose single-stranded DNA genome is approximately 4.7 kb in length and contains two nucleotide inverted terminal repeat (ITR) sequences. There are multiple serotypes of AAV. The nucleotide sequences of the genomes of the above-mentioned AAV serotypes are known in the art. For example, the complete genome of AAV-1 is provided under GenBank Accession No. NC_002077; the complete genome of AAV-2 is provided under GenBank Accession No. NC_001401 and Srivastava et al., J. Virol., 45:555-564 (1983); the complete genome of AAV-3 is provided under GenBank Accession No. NC_1829; the complete genome of AAV-4 is provided under GenBank Accession No. NC_001829; the AAV-5 genome is provided under GenBank Accession No. AF085716; the complete genome of AAV-6 is provided under GenBank Accession No. NC_001862; at least portions of the AAV-7 and AAV-8 genomes are provided under GenBank Accession Nos. AX753246 and AX753249, respectively; and the AAV-9 genome is provided by Gao et al. al., J. Virol., 78:6381-6388 (2004); the AAV-10 genome is provided in Mol. Ther., 13(1):67-76 (2006); and the AAV-11 genome is provided in Virology, 330(2):375-383 (2004).

[0021] In one embodiment of the present invention, the genome of the AAV may be a self-complementary genome. In this case, Cis-acting sequences that direct viral DNA replication (rep), encapsidation (cap) / packaging, and host cell chromosomal integration are contained within the AAV ITR sequences. The Rep protein has multiple enzymatic properties that ultimately contribute to the replication of the viral genome. The Cap gene is expressed from the p40 promoter and encodes three capsid proteins VP1, VP2, and VP3. Alternatively, to generate an AAV vector, the rep and cap proteins may be provided in trans.

[0022] The AAV may be packaged using a viral packaging system, such as a retrovirus, adenovirus, herpesvirus, or baculovirus packaging system.

[0023] In one embodiment of the invention, packaging is accomplished by using a helper virus or helper plasmid and a cell line that contains elements and sequences (E4, E2a, VA, etc.) that facilitate the transfer of genetic material into the cell.

[0024] The helper plasmid may, for example, contain at least one helper DNA sequence derived from a replication-incompetent viral genome that encodes in trans all the virion proteins necessary to package replication-incompetent AAV and to produce virion proteins capable of packaging high-titer replication-incompetent AAV without producing replication-incompetent AAV.

[0025] During the packaging process, helper plasmids and plasmids encoding AAV viral proteins are transiently co-transfected into mammalian cells capable of producing virus, such as human embryonic kidney cells, e.g., 293 cells (ATCC No. CRL1573, ATCC, Rockville, Md.), to produce high titer recombinant retrovirus-containing supernatants.

[0026] As used herein, the term "promoter" refers to any sequence that regulates the expression of a coding sequence, e.g., a gene. Promoters can be, for example, constitutive, inducible, repressible, or tissue-specific. A "promoter" is a region of a polynucleotide sequence responsible for controlling the initiation and rate of transcription.

[0027] In one embodiment of the present invention, the promoter includes, but is not limited to, a Rous sarcoma virus (RSV) LTR promoter (optionally including an RSV enhancer), a cytomegalovirus (CMV) promoter, an SV40 promoter, a dihydroborate reductase promoter, a β-actin promoter, a phosphoglycerol kinase (PGK) promoter, a U6 promoter, or an EF-1alpha (Elongation Factor 1-alpha) promoter.

[0028] In a specific embodiment of the present invention, the promoter is a CMV promoter or an EF-1alpha promoter.

[0029] As used herein, the term "nucleic acid molecule" is intended to comprehensively include DNA (gDNA and cDNA) and RNA molecules, and the nucleotides that are the basic building blocks of nucleic acid molecules include not only natural nucleotides but also analogues in which the sugar or base moiety has been modified (Scheit, Nucleotide Analogs, John Wiley, New York (1980); Uhlman and Peyman, Chemical Reviews, 90:543-584 (1990)).

[0030] It will be obvious to those skilled in the art that the nucleotide sequence encoding the hepatocyte growth factor of the present invention is not limited to any particular nucleotide sequence, but may be any nucleotide sequence encoding the amino acid sequence constituting the hepatocyte growth factor or a variant thereof.

[0031] This is because even if a mutation occurs in a nucleotide sequence, when the mutated nucleotide sequence is expressed as a protein, it may not result in a change in the protein sequence. This is called codon degeneracy. Thus, the nucleotide sequence includes nucleotide sequences that contain functionally equivalent codons or codons that code for the same amino acid (e.g., due to codon degeneracy, there are six codons for arginine or serine), or codons that code for biologically equivalent amino acids.

[0032] Considering the above-mentioned mutations having biological equivalent activity, the nucleic acid molecule of the present invention encoding the amino acid sequence constituting the hepatocyte growth factor is interpreted as including a sequence showing substantial identity thereto. The above-mentioned substantial identity means at least 60% or more homology (e.g., 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, or 69%), more specifically 70% or more homology (e.g., 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, 101%, 102%, 103%, 104%, 105%, 106%, 107%, 108%, 109%, 110%, 111%, 112%, 113%, 114%, 115%, 116%, 117%, 118%, 119%, 120%, 121%, 122%, 123%, 124%, 125%, 126%, 127%, 128%, 129%, 130%, 131%, 132%, 133%, 134%, 135%, 136%, 137%, 138%, 139%, 140%, 141%, 142%, 143%, 144%, 145%, 146%, 147%, 148%, 149%, 150%, 151%, 152%, 153%, , 77%, 78% or 79%), even more specifically 80% or more homology (e.g., 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88% or 89%), even more specifically 90% or more homology (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%), and most specifically 95% or more homology (e.g., 95%, 96%, 97%, 98% or 99%). All integers from 60% to 100% inclusive and decimals therebetween are included within the scope of the present invention in relation to % homology.

[0033] Alignment methods for sequence comparison are well known in the art. Various methods and algorithms for alignment are disclosed in Smith and Waterman, Adv. Appl. Math. 2:482 (1981); Needleman and Wunsch, J. Mol. Bio. 48:443 (1970); Pearson and Lipman, Methods in Mol. Biol. 24:307-31 (1988); Higgins and Sharp, Gene 73:237-44 (1988); Higgins and Sharp, CABIOS 5:151-3 (1989); Corpet et al., Nuc. Acids Res. 16:10881-90 (1988); Huang et al., Comp. Appl. BioSci. 8:155-65 (1992) and Pearson et al., Meth. Mol. Biol. 24:307-31 (1994). The NCBI Basic Local Alignment Search Tool (BLAST) (Altschul et al., J. Mol. Biol. 215:403-10 (1990)) is available at NCBI (National Center for Biological Information) and can be used in conjunction with sequence analysis programs such as blastp, blastn, blastx, tblastn, and tblastx on the Internet. BLAST can be accessed from the BLAST page on the ncbi website. Methods for sequence homology comparison using this program can be found on the BLAST help page on the ncbi website.

[0034] In one embodiment of the present invention, the AAV vector of the present invention may include a polynucleotide sequence encoding any polypeptide having another function, instead of the polynucleotide sequence encoding the above-mentioned hepatocyte growth factor.

[0035] As used herein, the term "operatively linked" refers to the functional association of a nucleic acid expression control sequence (e.g., a promoter, a signal sequence, or an array of transcription factor binding sites) with another nucleic acid sequence, whereby the control sequence controls the transcription and / or translation of the other nucleic acid sequence.

[0036] In one embodiment of the present invention, the hepatocyte growth factor is a hepatocyte growth factor isoform (HGF isoform). As used herein, the term "HGF isoform" refers to an HGF polypeptide having an amino acid sequence that is at least 80%, for example, 80%, 85%, 90%, or 95%, identical to the amino acid sequence of HGF naturally occurring in animals, including all allelic variants. For example, HGF isoform includes the normal form or wild type of HGF, and various variants of HGF (for example, splicing variants and deletion variants).

[0037] In one embodiment of the present invention, the hepatocyte growth factor isoform is full-length hepatocyte growth factor (flHGF), deleted variant HGF (dHGF), or a combination thereof, but is not limited thereto.

[0038] As used herein, the term "flHGF" refers to the amino acid sequence of amino acids 1-728 of the amino acid sequence of an animal HGF, in one embodiment, the amino acid sequence of amino acids 1-728 of the amino acid sequence of a mammalian HGF, and in another embodiment, the amino acid sequence of amino acids 1-728 of the amino acid sequence of a human HGF.

[0039] As used herein, the term "dHGF" refers to a truncated variant of an animal HGF gene, according to one embodiment, an HGF protein produced by alternative splicing of a mammalian HGF gene. According to another embodiment of the present invention, the dHGF of the present invention is a 723 amino acid human HGF in which five amino acids (F, L, P, S, and S) are deleted from the full-length HGF sequence in the first Kringle domain of the alpha chain.

[0040] In one embodiment of the present invention, HGF isoforms may be encoded by a hybrid HGF gene that simultaneously expresses two or more different isoforms, for example, flHGF and dHGF.

[0041] In one embodiment of the present invention, the hybrid HGF gene comprises a sequence corresponding to exons 1 to 4 of the human HGF gene; a sequence of intron 4 or a fragment thereof of the human HGF gene; and a sequence corresponding to exons 5 to 18 of the human HGF gene.

[0042] In a specific embodiment of the present invention, the full-length hepatocyte growth factor comprises the amino acid sequence of SEQ ID NO: 1, and the truncated mutant hepatocyte growth factor comprises the amino acid sequence of SEQ ID NO: 2, but is not limited thereto.

[0043] In one embodiment of the present invention, the hybrid HGF gene including intron 4 is 7113 bp long and comprises the nucleotide sequence of SEQ ID NO:3.

[0044] In another embodiment of the invention, the hybrid HGF gene may optionally contain a fragment of intron 4 between exon 4 and exon 5 of the HGF cDNA.

[0045] In one embodiment of the present invention, the polynucleotide sequence of a gene to be inserted into the AAV vector of the present invention may be modified for stable expression of a polypeptide encoded by the polynucleotide sequence, for example, by modifying the polynucleotide sequence to include only a portion of an intron sequence instead of the entire intron sequence of the inserted gene, thereby shortening the length of the inserted polynucleotide sequence.

[0046] In a more specific embodiment of the present invention, the hybrid HGF gene comprises a nucleotide sequence selected from the group consisting of the nucleotide sequences of SEQ ID NO: 5 to SEQ ID NO: 9 and SEQ ID NO: 19. The nucleotide sequences of SEQ ID NO: 5 to SEQ ID NO: 8 represent HGF-X7-d1 to HGF-X7-d4 genes in which the length of the nucleotide sequence of HGF-X7 shown in SEQ ID NO: 4 has been shortened to include only a portion of the intron 4 sequence of the HGF gene.

[0047] The "HGF isoforms" and "hybrid HGF genes" (eg, HGF-X7) of the present invention have been reported in WO2003 / 078568, the disclosure of which is incorporated by reference in its entirety into this specification.

[0048] In one embodiment of the present invention, the polynucleotide sequence encoding the hepatocyte growth factor or its variant is codon optimized.

[0049] In a specific embodiment of the present invention, the codon-optimized polynucleotide sequence encoding hepatocyte growth factor or a variant thereof comprises the nucleotide sequence of SEQ ID NO:9.

[0050] In one embodiment of the present invention, the polynucleotide sequence encoding the hepatocyte growth factor or a variant thereof may be at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to a nucleotide sequence selected from the group consisting of the nucleotide sequences of SEQ ID NO: 4 to SEQ ID NO: 9, or SEQ ID NO: 19, but is not limited thereto.

[0051] In one embodiment of the present invention, the AAV vector is an AAV vector whose serotype is AAV1, AAV2, AAV4, AAV5, AAV6, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV-DJ.AAV-DJ / 8, AAV-PHP.Eb, AAV-PHP.S, or AAV rh10, but is not limited thereto.

[0052] In one embodiment of the present invention, the vector is an AAV vector whose serotype is AAV1 or AAV9.

[0053] In one embodiment of the present invention, the CMV promoter sequence may include a nucleotide sequence of SEQ ID NO: 10 or 11, or may include a sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the nucleotide sequence of SEQ ID NO: 10 or 11.

[0054] In one embodiment of the present invention, the CMV promoter sequence may include a fragment of the entire CMV promoter sequence. The fragment includes a functional minimal sequence of the CMV promoter sequence. For example, the CMV promoter sequence may include the nucleotide sequence of SEQ ID NO: 10, and may further include a CMV promoter sequence at the 5' end or 3' end of the fragment.

[0055] In one embodiment of the present invention, the EF-1alpha promoter sequence may comprise a nucleotide sequence of SEQ ID NO: 12 or may comprise a sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the nucleotide sequence of SEQ ID NO: 12. All integers from 90% to 100% and decimals therebetween are included within the scope of the present invention in relation to percent identity.

[0056] In one embodiment of the present invention, the nucleic acid molecule further comprises a posttranscriptional regulatory element (PRE).

[0057] In a specific embodiment of the present invention, the posttranscriptional regulatory element includes, but is not limited to, the Woodchuck hepatitis virus posttranscriptional regulatory element (WPRE).

[0058] In one embodiment of the present invention, the nucleic acid molecule further comprises a polyadenylation sequence.

[0059] In one embodiment of the present invention, the polyadenylation sequence includes, but is not limited to, a human growth hormone (hGH) polyadenylation sequence, a bovine growth hormone (bGH) polyadenylation sequence, an SV40 polyadenylation sequence, or a combination thereof.

[0060] In specific embodiments of the invention, the human growth hormone (hGH) polyadenylation sequence may comprise the nucleotide sequence of SEQ ID NO: 13 or SEQ ID NO: 14, or may comprise a sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the nucleotide sequence of SEQ ID NO: 13 or SEQ ID NO: 14. All integers from 90% to 100% and decimals therebetween are included within the scope of the invention in relation to the % identity.

[0061] In one embodiment of the present invention, the human growth hormone (hGH) polyadenylation sequence may comprise a fragment of the entire hGH sequence. The fragment comprises a functional minimal sequence of the hGH polyadenylation sequence. For example, the hGH polyadenylation sequence may comprise the nucleotide sequence of SEQ ID NO: 13, and may further comprise the hGH polyadenylation sequence at the 5'-end or 3'-end of the fragment.

[0062] In a specific embodiment of the invention, the bovine growth hormone (bGH) polyadenylation sequence may comprise the nucleotide sequence of SEQ ID NO: 15 or SEQ ID NO: 16, or may comprise a sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the nucleotide sequence of SEQ ID NO: 15 or SEQ ID NO: 16. All integers from 90% to 100% and decimals therebetween are included within the scope of the invention in relation to the % identity.

[0063] In one embodiment of the present invention, the bovine growth hormone (bGH) polyadenylation sequence may comprise a fragment of the entire bGH polyadenylation sequence. The fragment comprises a functional minimal sequence of the bGH polyadenylation sequence. For example, the bGH polyadenylation sequence may comprise the nucleotide sequence of SEQ ID NO: 15, and may further comprise the bGH polyadenylation sequence at the 5' end or 3' end of the fragment.

[0064] In specific embodiments of the invention, the SV40 polyadenylation sequence may comprise the nucleotide sequence of SEQ ID NO: 17 or SEQ ID NO: 18, or may comprise a sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the nucleotide sequence of SEQ ID NO: 17 or SEQ ID NO: 18. All integers from 90% to 100% and decimals therebetween are included within the scope of the invention in relation to the percent identity.

[0065] In one embodiment of the present invention, the SV40 polyadenylation sequence may comprise a fragment of the entire SV40 polyadenylation sequence. The fragment comprises a functional minimal sequence of the SV40 polyadenylation sequence. For example, the SV40 polyadenylation sequence may comprise the nucleotide sequence of SEQ ID NO: 17, and may further comprise the SV40 polyadenylation sequence at the 5' end or 3' end of the fragment.

[0066] In one embodiment of the present invention, the nucleic acid molecule comprises two AAV inverted terminal repeat sequences (ITR sequences) on either side of the nucleic acid sequence.

[0067] According to another aspect of the invention, there is provided an isolated host cell comprising the AAV vector.

[0068] Any host cell known in the art can be used as a host cell capable of stably and continuously cloning and expressing the AAV vector of the present invention. For example, suitable eukaryotic host cells for the vector include, but are not limited to, yeast (Saccharomyce cerevisiae), insect cells, monkey kidney cells 7 (COS7), NSO cells, SP2 / 0, Chinese hamster ovary (CHO) cells, W138, baby hamster kidney (BHK) cells, MDCK, myeloma cell lines, HuT78 cells, and HEK-293 cells.

[0069] As used herein, the terms "transformed," "transduced," or "transfected" refer to the process by which exogenous nucleic acid is transferred or introduced into a host cell. A "transformed," "transduced," or "transfected" cell is a cell that has been transformed, transduced, or transfected with exogenous nucleic acid, including the cell itself and its progeny by subculture.

[0070] When the host cell is a eukaryotic cell, the vector of the present invention can be delivered into the host cell by microinjection (Capecchi, MR, Cell, 22:479 (1980)), calcium phosphate precipitation (Graham, FL et al., Virology, 52:456 (1973)), electroporation (Neumann, E. et al., EMBO J., 1:841 (1982)), liposome-mediated transfection (Wong, TK et al., Gene, 10:87 (1980)), DEAE-dextran treatment (Gopal, Mol. Cell Biol., 5:1188-1190 (1985)), gene bombardment (Yang et al., Proc. Natl. Acad. Sci., 87:9568-9572 (1990)), or the like.

[0071] In the present invention, the vector injected into the host cell can express proteins necessary for the production of AAV viral particles in the host cell, and AAV viral particles are obtained through the action of the expressed proteins and a combination of these proteins.

[0072] The culture is generally carried out under aerobic conditions such as shaking culture or rotation by a rotor. The culture temperature is preferably in the range of 10 to 40°C, and the culture time is generally 5 hours to 7 days. The pH of the medium is preferably maintained in the range of 3.0 to 9.0 during the culture. The pH of the medium can be adjusted with inorganic or organic acids, alkaline solutions, urea, calcium carbonate, ammonia, etc. During the culture, antibiotics such as ampicillin, streptomycin, chloramphenicol, kanamycin, and tetracycline can be added for the maintenance and expression of the recombinant vector, if necessary. When culturing a host cell transformed with a recombinant expression vector having an inducible promoter, an appropriate inducer may be added to the medium if necessary. For example, when the expression vector contains a lac promoter, IPTG (isopropyl-beta-D-thiogalactopyranoside) can be added to the medium, and when it contains a trp promoter, indoleacrylic acid can be added to the medium.

[0073] According to yet another aspect, the present invention provides an AAV particle comprising the AAV vector described above.

[0074] In one embodiment of the invention, the AAV particles are for intrathecal administration.

[0075] The AAV particles according to one embodiment of the present invention are effective in expressing a transformed gene of interest (GOI) as a protein when administered intrathecally.

[0076] According to yet another aspect of the present invention, there is provided a pharmaceutical composition for preventing or treating amyotrophic lateral sclerosis (ALS) or diabetic peripheral neuropathy (DPN), comprising the AAV particles.

[0077] In one embodiment of the present invention, the diabetic peripheral neuropathy is polyneuropathy or focal neuropathy.

[0078] Since the pharmaceutical composition of the present invention uses the above-mentioned AAV vector or AAV particle of the present invention as an active ingredient, the content common to both will be omitted in order to avoid overly complicating this specification.

[0079] The pharma- ceutically acceptable carriers contained in the pharmaceutical composition of the present invention are those commonly used in formulation, including, but not limited to, lactose, dextrose, sucrose, sorbitol, mannitol, starch, gum acacia, calcium phosphate, alginate, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methylcellulose, methyl hydroxybenzoate, propyl hydroxybenzoate, talc, magnesium stearate, and mineral oil. In addition to the above ingredients, the pharmaceutical composition of the present invention may further include lubricants, wetting agents, sweeteners, flavoring agents, emulsifiers, suspending agents, preservatives, etc. Suitable pharma- ceutically acceptable carriers and formulations are described in detail in Remington's Pharmaceutical Sciences (19th ed., 1995).

[0080] The pharmaceutical composition of the present invention can be administered orally or parenterally, for example, intravenously, intrathecally, intraspinally, intracerebrally, intraventricularly, intraventricularly, intracranially, subcutaneously, intramuscularly, intraperitoneally, intrasternally, topically, intranasally, intrapulmonaryly, and intrarectally. Preferably, it may be administered intrathecally, but is not limited thereto.

[0081] The appropriate dosage of the pharmaceutical composition of the present invention varies depending on factors such as the formulation method, administration method, age, weight, sex, pathological condition, diet, administration time, administration route, excretion rate and reaction sensitivity of the patient, and an ordinary physician skilled in the art can easily determine and prescribe an effective dosage for the desired treatment or prevention.

[0082] According to a preferred embodiment of the present invention, the daily dose of the pharmaceutical composition of the present invention is 1×10 9 ~1×10 12 As used herein, the term "pharmaceutical effective amount" refers to an amount sufficient to prevent or treat the above-mentioned diseases.

[0083] The term "prophylaxis" as used herein means the prevention or protective treatment of a disease or disease state. The term "treatment" as used herein means the reduction, suppression, mitigation or eradication of a disease state.

[0084] The pharmaceutical composition of the present invention may be prepared in a unit dose form or in a multi-dose container by formulating it with pharma- ceutically acceptable carriers and / or excipients in a manner that can be easily carried out by a person having ordinary skill in the art to which the present invention pertains. In this case, the dosage form may be in the form of a solution, suspension or emulsion in an oil or aqueous medium, or in the form of an extract, powder, suppository, powder, granule, tablet or capsule, and may further include a dispersant or stabilizer.

[0085] According to yet another aspect of the present invention, there is provided a method for preventing or treating amyotrophic lateral sclerosis (ALS) or diabetic peripheral neuropathy (DPN), comprising administering the above-mentioned pharmaceutical composition to a subject in need of treatment.

[0086] In one embodiment of the present invention, the pharmaceutical composition comprises, as an active ingredient, the AAV vector described above, an isolated host cell containing the AAV vector described above, or an AAV particle containing the AAV vector described above.

[0087] The subject of the present invention is a mammal, such as a human, chimpanzee, orangutan, non-human primate, dog, cat, horse, hamster, mouse, rat, gerbil, pig, sheep, cow, and most particularly, but not limited to, a human.

[0088] The method for preventing or treating amyotrophic lateral sclerosis (ALS) or diabetic peripheral neuropathy (DPN) of the present invention includes a step of administering a pharmaceutical composition which is one embodiment of the present invention, and therefore, the description of the overlapping content will be omitted in order to avoid excessive duplication in this specification. Effect of the Invention

[0089] The present invention provides an AAV vector or AAV particle carrying a hepatocyte growth factor gene optimized for intrathecal administration, and a pharmaceutical composition comprising the same for preventing or treating amyotrophic lateral sclerosis (ALS) or diabetic peripheral neuropathy (DPN).

[0090] The AAV vector and AAV particle of the present invention are excellent in productivity of AAV particles containing a hepatocyte growth factor gene, and are also highly efficient in expressing hepatocyte growth factor when administered intrathecally to a mammal. Therefore, when the AAV vector and particle of the present invention are used for intrathecal administration, they can be usefully used for the prevention or treatment of associated diseases by inducing expression of the hepatocyte growth factor protein. [Brief description of the drawings]

[0091] [Figure 1] FIG. 1 shows the configuration of the AAV vector candidates shown in Table 1 of the present invention, based on the types of promoter, GOI, and polyadenylation sequence.

[0092] [Diagram 2] FIG. 1 shows a comparison of hHGF expression levels according to the configurations of the AAV vector candidate group in Table 1 of the present invention.

[0093] [Diagram 3] FIG. 1 shows a comparison of the productivity of AAV particles produced by the configurations of the AAV vector candidate group in Table 1 of the present invention.

[0094] [Figure 4] FIG. 1 shows a comparison of hHGF expression levels after AAV particles prepared using the configurations of the AAV vector candidate group in Table 2 of the present invention and administered into the spinal cavity of mice. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0095] The present invention will be described in more detail below using examples. It will be apparent to those skilled in the art that these examples are merely intended to more specifically explain the present invention, and that the scope of the present invention is not limited to these examples according to the gist of the present invention. EXAMPLES

[0096] Throughout this specification, "%" used to indicate the concentration of a particular substance is (wt / wt)% for solid / solid, (wt / vol)% for solid / liquid, and (vol / vol)% for liquid / liquid, unless otherwise specified.

[0097] Example 1: Preparation of candidate plasmids and comparison of expression efficiency (in vitro)

[0098] The present inventors constructed six types of plasmids containing different promoters and poly A tails by cloning as described below.

[0099] (1) A backbone sequence (sequence number 20) including ITR(L) and ITR(R) was obtained by synthesis.

[0100] (2) As promoters, the CMV promoter (SEQ ID NO: 11) and the EF-1alpha S series promoter (SEQ ID NO: 12) were synthesized so as to contain MluI and ClaI restriction enzyme sequences, respectively, and inserted into the backbone.

[0101] (3) The HGF-X7-d4 sequence of SEQ ID NO:8 was inserted using the ClaI and SalI restriction enzyme sequences.

[0102] (4) Candidate Poly(A) sequences, hGH poly A tail (SEQ ID NO: 14), bGH poly A tail (SEQ ID NO: 16), and SV40 poly A (SEQ ID NO: 18), were synthesized to contain SalI and RsrII restriction enzyme sequences, respectively, and inserted into the backbone.

[0103] The structures of the six plasmids are shown in Table 1 and Figure 1. VM202 (pCK-HGF-X7) was used as a positive control.

[0104] [Table 1]

[0105] FIG. 1 is a schematic diagram showing the configuration of the AAV vector candidate group shown in Table 1 of the present invention, based on the types of promoter, GOI, and polyadenylation sequence.

[0106] HEK293 cells were cultured in DMEM + 10% FBS medium. One day before transfection, 1 × 10 5The cells were seeded at 1000 cells / well. The next day, 0.5 ug each of NC (negative control), VM202 (positive control), and plasmids 1 to 6 were added, and transfection was performed using PEI. After 72 hours, the supernatant was collected and subjected to human HGF ELISA (R&D, DHG00), and the results are shown in Figure 2. Figure 2 shows a comparison of the hHGF expression levels according to the composition of the AAV vector candidate group listed in Table 1 of the present invention.

[0107] As shown in Figure 2, we first confirmed that hHGF was well expressed in VM202, the positive control group. Plasmids 1 to 3, which have a CMV-series promoter, expressed relatively high levels of hHGF, and plasmid 3, which contains SV40 polyA, expressed hHGF at a lower level than plasmids 1 and 2. Plasmids 4 to 6, which contain the EF1 alpha S-series promoter, showed a relatively significantly low hHGF expression rate.

[0108] Example 2: Comparison of AAV productivity of candidate plasmids (in vitro)

[0109] The present inventors prepared AAV vectors using the six plasmids in Example 1. First, HEK293 cells were seeded in a T225 flask, and three plasmids (pAAV-RC, pAAV-helper, and pAAV-HGF) were co-transfected using PEI. After 72 hours, the cells were harvested and AAV was purified, followed by titration of AAV. The results are shown in FIG. 3. FIG. 3 is a diagram showing a comparison of the productivity of AAV particles produced by the composition of the AAV vector candidate group in Table 1 of the present invention.

[0110] As shown in Figure 3, plasmids 1 and 3, which contained CMV-series promoters, showed the lowest levels of AAV productivity, and among them, plasmid 2, which had bGH polyA, showed the highest AAV productivity among the group containing CMV-series promoters.

[0111] On the other hand, plasmids 4 to 6, which contain EF1 alpha series promoters, showed relatively high levels of AAV productivity compared to plasmids containing CMV series promoters, and in particular, plasmid 5 showed approximately 20-fold increased AAV production compared to plasmid 1.

[0112] Example 3: Comparison of hHGF expression rates of candidate plasmids (in vivo)

[0113] The inventors produced AAV1 or AAV9 using plasmids 1, 4, and 5 prepared in previous examples, and plasmid 11, which is a plasmid in which the expression gene in plasmid 5 was replaced with codon-optimized HGFX7d4, and administered these to mice to compare the expression levels of hHGF protein (flHGF) under in vivo conditions.

[0114] [Table 2]

[0115] AAV (1 × 10 10vg / head) was administered intrathecally to C57BL / 6 mice, and 7 days later, the lumbar spinal cord and dosal root ganglia (DRG) were harvested. Proteins were extracted from the harvested tissue samples using RIPA buffer, and the total protein amount was quantified using a BCA protein assay kit (Thermo, Cat#23225). The expression levels of hHGF protein were then compared using a Human HGF Quantikine ELISA kit (R&D, DHG00). 50uL of lysate from the spinal cord and dorsal root ganglia (DRG) were added per well and mixed well to react with the antibody. A standard curve was drawn using the Human HGF standard in the ELISA kit, and the amount of HGF in the final sample was derived using this curve. The results are shown in Figure 4. FIG. 4 is a graph showing a comparison of the levels of hHGF expression after AAV particles produced using the compositions of the AAV vector candidate group shown in Table 2 of the present invention were administered into the spinal cavity of mice.

[0116] As shown in Figure 4, contrary to our expectations, AAVs with EF1 alpha promoters expressed higher levels of hHGF overall than AAVs with CMV promoters. This is significant because it is a contradictory result to the in vitro results. In particular, the HGF-X7-d4 codon optimized candidate with EF1 alpha promoter expressed significantly higher levels of hHGF. Quantitative comparison showed that AAV_#11_codon_opti showed approximately 16-fold higher expression levels in the spinal cord and approximately 5-fold higher expression levels in the dorsal ganglion (DRG) than AAV_#1.

[0117] Based on the test results, plasmid 11, which had high AAV productivity and high in vivo hHGF expression, was selected as the final candidate.

[0118] Although certain parts of the present invention have been described in detail above, it will be apparent to those skilled in the art that such specific descriptions are merely preferred embodiments and do not limit the scope of the present invention.

Claims

1. An AAV (adeno-associated virus) vector comprising a nucleic acid molecule comprising: (a) a CMV (Cytomegalovirus) promoter sequence or an EF-1alpha (Elongation Factor 1-alpha) promoter sequence; and (b) a polynucleotide sequence encoding hepatocyte growth factor (HGF), operably linked to the promoter sequence.

2. The AAV vector of claim 1 , wherein the hepatocyte growth factor is a hepatocyte growth factor isoform (HGF isoform).

3. The AAV vector according to claim 2, wherein the hepatocyte growth factor variant is full-length hepatocyte growth factor (flHGF), deleted variant HGF (dHGF), or a combination thereof.

4. The AAV vector of claim 3, wherein the full-length hepatocyte growth factor comprises the amino acid sequence of SEQ ID NO:1, and the deleted mutant hepatocyte growth factor comprises the amino acid sequence of SEQ ID NO:

2.

5. The AAV vector of claim 1, wherein the polynucleotide sequence encoding the hepatocyte growth factor is at least 95% identical to a nucleotide sequence selected from the group consisting of the nucleotide sequences of SEQ ID NO:4 to SEQ ID NO:9 and SEQ ID NO:

19.

6. The AAV vector of claim 1 , wherein the polynucleotide sequence encoding the hepatocyte growth factor comprises the nucleotide sequence of SEQ ID NO:8 or SEQ ID NO:

9.

7. The AAV vector according to claim 1, wherein the vector is an AAV vector having a serotype of AAV1, AAV2, AAV4, AAV5, AAV6, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV-DJ.AAV-DJ / 8, AAV-PHP.Eb, AAV-PHP.S, or AAV rhlO.

8. The AAV vector according to claim 1 , wherein the vector is an AAV vector whose serotype is AAV1 or AAV9.

9. The AAV vector of claim 1 , wherein the CMV promoter sequence comprises the nucleotide sequence of SEQ ID NO: 10 or SEQ ID NO:

11.

10. The AAV vector according to claim 1, wherein the EF-1alpha promoter sequence comprises the nucleotide sequence of SEQ ID NO:

12.

11. The AAV vector of claim 1 , wherein the nucleic acid molecule further comprises a polyadenylation sequence.

12. 12. The AAV vector of claim 11, wherein the polyadenylation sequence comprises an SV40 polyadenylation sequence, a human growth hormone (hGH) polyadenylation sequence, a bovine growth hormone (bGH) polyadenylation sequence, or a combination thereof.

13. The AAV vector of claim 1, wherein the nucleic acid molecule comprises two AAV inverted terminal repeat sequences (ITR repeat sequences) on either side of the nucleic acid sequence.

14. An isolated host cell comprising an AAV vector according to any one of claims 1 to 13.

15. An AAV particle comprising the AAV vector described in any one of claims 1 to 13.

16. The AAV particle of claim 15, wherein the AAV particle is for intrathecal administration.

17. A pharmaceutical composition for preventing or treating amyotrophic lateral sclerosis (ALS) or diabetic peripheral neuropathy (DPN), comprising the AAV particles according to claim 15.

18. The pharmaceutical composition according to claim 17, wherein the diabetic peripheral neuropathy is polyneuropathy or focal peripheral neuropathy.

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