A method for enhancing gene expression using AAV vectors

Abstract

To provide a composition for gene transfer comprising a recombinant virus vector, where the composition for gene transfer improves the efficiency of gene transfer using a virus vector.SOLUTION: Specifically, the present invention provides a composition for gene transfer which comprises a recombinant virus vector comprising a gene of interest for expression and a sugar at a concentration of at least 40 mM. The virus vector is preferably an adeno-associated virus vector. By using the composition of the present invention, the efficiency of gene transfer using a virus vector can be improved by about 50 times or more.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to a gene transfer composition comprising a recombinant viral vector. More specifically, the present invention relates to a gene transfer composition comprising a recombinant adeno-associated virus (rAAV) vector and a sugar. [Background technology]

[0002] In recent years, viral vectors have been used to introduce foreign genes into cells. Examples of viral vectors used in mammalian cells include adenoviruses, adeno-associated viruses, retroviruses (such as lentiviruses), and herpesviruses. These viral vectors are also being used and studied as viral vectors for disease treatment (Non-Patent Document 1).

[0003] Gene vectors utilizing adeno-associated virus (AAV) can introduce genes into cells such as nerve cells, hepatocytes (hepatocytes), retinal cells, muscle cells, cardiomyocytes, vascular cells, and adipocytes, and allow for long-term expression. Therefore, they are increasingly being used clinically as gene therapy vectors for conditions such as Parkinson's disease, hemophilia, and retinitis pigmentosa (Patent Documents 1-4, Non-Patent Documents 2-4). Furthermore, they are frequently used as vectors for gene introduction of sgRNA and CAS9 proteins in gene editing, and for gene introduction of photosensitive proteins such as channelrhodopsin in optogenetics (Non-Patent Documents 5, 6). Modifications such as genome structure alteration, mutation introduction into capsid proteins, and promoter substitution are also performed to improve introduction efficiency and gene expression (Non-Patent Documents 7, 8, 9). However, in many cases, effective gene expression requires, for example, 10 4 Infection with rAAV vectors of more than one vector genome (abbreviated as vg, synonymous with copy number) is required, which is often inefficient and necessitates the purification of large quantities of vectors. [Prior art documents] [Patent Documents]

[0004] [License 1] International Publication No. 2003 / 018821 [License 2] International Publication No. 2003 / 053476 [License 3] International Publication No. 2007 / 001010 [License 4] International Publication No. 2012 / 057363 [Non-licensed literature]

[0005] [Non-licensed Document 1] Kenjiro Tani, "2 Treatments for Orphans in Japan", Orphan Medicine MOOK 30, pp. 38-49, published June 20, 2016. [Non-licensed Document 2] Dunber CE et al.: Science. 359: eaan4672, 2018 [Non-licensed Document 3] Shinichi Muramatsu: "Treatment of パーキンソン disease with dandruff" Japanese Clinical 75: 146-150, 2017 [Non-licensed Document 4] Hastie E, Samulski RJ: Hum Gene Ther 26:257-265, 2015 [Non-licensed Document 5] Ohmori T et al.: Sci Rep. 7:4159. 2017 [Non-licensed Document 6] Prakash R et al.: Nat Methods. 9:1171-1179, 2012 [Non-licensed Document 7] McCarty DM, et al.: Gene Ther. 8:1248-1254, 2001 [Non-licensed Document 8] Ling C, et al.: Hum Gene Ther Methods. 27:143-149, 2016 [Non-licensed Document 9] Chen SJ, et al.: Hum Gene Ther Methods. 24:270-278, 2013 Summary of the Invention Problems to be Solved by the Invention

[0006] Therefore, when using an rAAV vector as a recombinant viral vector, it is desired to provide a composition and means for more efficiently achieving the expression of a target gene. Means for Solving the Problems

[0007] The inventors of the present invention, through various trials and errors, found that in a gene transfer composition containing an rAAV vector as a recombinant viral vector, by including a higher concentration of sugar, a recombinant viral vector-containing composition that can be more efficiently transfected into various target cells (for example, hepatocytes, neurons, etc.) was found, and the present invention was completed.

[0008] That is, the present invention provides a gene transfer composition containing a recombinant viral vector, sugar, and an aqueous medium, etc. Specifically, the following gene transfer compositions, kits, and methods are provided. [1] A gene transfer composition comprising a recombinant viral vector containing a gene for the expression of interest, at least 40 mM of sugar, and an aqueous medium. [2] The composition according to [1] above, wherein the recombinant viral vector is selected from the group consisting of an adeno-associated virus (AAV) vector, an adenovirus vector, a gamma-retrovirus vector, and a lentivirus vector. [[ID=**27**]] [3] The composition according to [1] or [2] above, wherein the recombinant viral vector is derived from AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAVrh10, AAVrh39, AAVrh43, AAV-B1, AAV-PHP.B, or AAV-PHP.eB. [4] The composition according to any one of [1] to [3] above, wherein the recombinant viral vector comprises a capsid containing a protein having an amino acid sequence described in SEQ ID NOs: 1 to 4, or an amino acid sequence having approximately 90% or more identity with the amino acid sequences described in SEQ ID NOs: 1 to 4. [5] The composition according to any one of the above [1] to [4], wherein the composition contains sugar at a concentration of 0.04 to 2 M. [6] The composition according to any one of the above [1] to [5], wherein the sugar comprises a monosaccharide, a disaccharide, or a combination thereof. [7] The composition according to any one of [1] to [6] above, wherein the monosaccharide comprises at least one selected from glucose, galactose, fructose, mannose, and combinations thereof. [8] The composition according to any one of the above [1] to [7], wherein the monosaccharide contains 60% or more of the D-isomer. [9] The composition according to any one of the above [1] to [8], wherein the disaccharide comprises at least one selected from the group consisting of sucrose, trehalose, maltose, lactose, and combinations thereof.

[10] The composition according to any one of the above [1] to [9], wherein the aqueous medium is water for injection.

[11] The composition described in any one of the above [1] to

[10] , which is a pharmaceutical product in the form of an injection or infusion.

[12] A composition according to any one of the above [1] to

[11] , which is a drug used exovivo or in vitro.

[13] The composition according to any one of the above [1] to

[12] , wherein the composition is frozen for storage and thawed before use.

[14] A kit for preparing the composition according to any one of the above [1] to

[13] , comprising a recombinant viral vector and a sugar.

[15] A method for gene transfer comprising the steps of (a) providing a composition comprising a recombinant virus and a sugar at a concentration of 40 mM or more, and (b) contacting cultured cells with the composition in a culture medium containing the sugar at a final concentration of 10 to 250 mM.

[16] A method for gene transfer comprising: (a) providing a gene transfer composition containing a recombinant virus and containing or not containing sugar; (b) contacting the gene transfer composition with cultured cells; and (c) culturing the cells in a medium containing 10 to 250 mM sugar immediately after step (b) to 72 hours later.

[17] The method according to

[15] or

[16] above, wherein the cultured cells are cells taken from a living organism or established cells.

[18] A gene transfer composition according to any one of the above [1] to

[13] for use in a surgery to isolate an organ, tissue or part thereof that is to be gene-transferred. [Effects of the Invention]

[0009] The gene transfer composition according to the present invention, which includes a recombinant viral vector, sugar, and aqueous medium, enables efficient gene transfer into various target cells (e.g., hepatocytes, nerve cells, etc.). Specifically, it was confirmed that using the gene transfer composition according to the present invention, which includes an AAV vector as the recombinant viral vector, a high concentration of sugar, and an aqueous medium, results in an expression enhancement effect of approximately 50 times or more compared to the control. [Brief explanation of the drawing]

[0010] [Figure 1A] Figure 1A shows the results of in vitro rAAV infection with and without sucrose. GFP expression in cells was measured one day after infection. [Figure 1B] Figure 1B shows fluorescence microscope images of the cells from the experiment shown in Figure 1A. [Figure 2A] Figure 2A shows the results of in vitro infection with rAAV by adding sucrose, mannitol, or glucose. [Figure 2B] Figure 2B shows fluorescence microscope images of the cells from the experiment shown in Figure 2A. [Figure 3] Figure 3 shows the results of in vitro infection with rAAV after sucrose supplementation. [Figure 4A]Figure 4A shows the results of in vitro infection immediately after mixing the virus solution with sucrose, 10 minutes later, and 1 hour later. [Figure 4B] Figure 4B shows the results one day after in vitro infection with a mixture of virus solution and sucrose, either immediately or one hour later. [Figure 4C] Figure 4C shows the results when infection with a sucrose-free virus solution was performed for 3 hours, followed by replacement with a culture medium containing sucrose. [Figure 5A] Figure 5A shows the results of in vitro rAAV infection using D-glucose or L-glucose. [Figure 5B] Figure 5B shows the results of the experiment in Figure 5A one day, two days, and three days later. [Figure 6] Figure 6 shows the results of in vitro rAAV infection using various concentrations of sucrose. [Figure 7] Figure 7 shows the results of in vitro rAAV infection using various concentrations of glucose. [Figure 8A] Figure 8A shows the results of in vitro rAAV infection using various concentrations of sucrose, trehalose, maltose, and lactose, respectively. [Figure 8B] Figure 8B shows fluorescence microscopy images of cells taken 3 days after using a sugar concentration of 150 mM as shown in Figure 8A. [Figure 8C] Figure 8C shows the results of the experiment in Figure 8A one day, two days, three days, and six days later. [Figure 9A] Figure 9A shows the results of in vitro rAAV infection using galactose, fructose, mannose, and glucose, respectively. [Figure 9B] Figure 9B shows fluorescence microscopy images of cells taken 3 days after using a sugar concentration of 200 mM as shown in Figure 9A. [Modes for carrying out the invention]

[0011] The present invention provides a gene transfer composition comprising a recombinant viral vector containing a gene for expression of a target, a sugar at a concentration of 40 mM or higher, and an aqueous medium. The recombinant viral vector may, but is not limited to, adeno-associated virus (AAV) vectors, adenovirus vectors, gamma-retrovirus vectors, and lentiviral vectors.

[0012] 1. Regarding the viral vector used in the composition of the present invention The recombinant viral vector used in the gene transfer composition of the present invention (hereinafter also referred to as "the composition of the present invention") may include a known viral vector that infects eukaryotic cells, preferably mammalian cells. Examples of viral vectors that can be used in the compositions of the present invention include, but are not limited to, adeno-associated virus vectors, adenovirus vectors, retrovirus vectors (including gamma-retrovirus vectors and lentivirus vectors), herpesvirus vectors, poxvirus vectors, vaccinia virus vectors, Sendai virus vectors, and Borna virus vectors. Preferably, the recombinant viral vector used in the present invention is an adeno-associated virus vector or an adenovirus vector, and more preferably an adeno-associated virus vector.

[0013] 1.1. Adeno-associated virus vectors Natural adeno-associated viruses (AAVs) are characterized by being non-pathogenic viruses and by the long-term presence of the viral genome contained within the adeno-associated virus vector in the episomes of the nucleus of infected host cells. These characteristics are utilized to create various recombinant viral vectors and deliver desired genes for gene therapy (see, for example, WO2003 / 018821, WO2003 / 053476, WO2007 / 001010, and the Japanese Journal of Pharmaceutical Sciences 126(11)1021-1028).

[0014] There are numerous isolates (types) of known adeno-associated viruses (AAVs). Adeno-associated viruses are known to have specificity or targeting properties for specific organs. For example, adeno-associated viruses that show a tendency to target the liver include types 2, 3 (including 3A and 3B), and 8 (e.g., WO 2012 / 057363, WO 2008 / 124724). Adeno-associated viruses that show a tendency to target nerve cells include types 1, 2, 9, rh10, rh39, rh43, B-1, PHP.B, and PHP.eB (e.g., references 10-13 below). However, the adeno-associated viruses used as therapeutic viral vectors in the present invention are not limited to these isolates, and it is also possible to use isolates or variants known in the field. Reference 10. Sorrentino NC, et al.: Mol Ther. 24:276-286, 2016. Reference 11. Choudhury SR, et al.: Mol Ther. 24:1247-1257, 2016. Reference 12. Chan KY, et al.: Nat Neurosci. 20:1172-1179, 2017. Reference 13. Iida A, et al.: Biomed Res Int. 2013:974-819, 2013.

[0015] The wild-type AAV genome is a single-stranded DNA molecule with a total length of approximately 5 kb, and is either a sense strand or an antisense strand. Generally, the AAV genome has inverted terminal repeat (ITR) sequences of approximately 145 nucleotides at both the 5' and 3' ends of the genome. These ITRs are known to have diverse functions, including functioning as the origin of AAV genome replication and as a packaging signal for this genome within the viral particle (e.g., Journal of Pharmaceutical Sciences 126(11)1021-1028). The internal region of the wild-type AAV genome, flanked by the ITRs (hereinafter referred to as the internal region), contains AAV replication (rep) genes and capsid (cap) genes. These rep and cap genes encode Rep, a protein involved in viral replication, and capsid proteins (e.g., at least one of VP1, VP2, and VP3) that form the capsomere, the outer shell of the icosahedron structure, respectively. For further details, see, for example, Human Gene Therapy, 13, pp.345-354, 2002; Neuronal Development 45, pp.92-103, 2001; Experimental Medicine 20, pp.1296-1300, 2002; Pharmaceutical Journal 126(11)1021-1028; Hum Gene Ther, 16, 541-550, 2005.

[0016] The rAAV vectors used in this invention are derived from, but are not limited to, natural adeno-associated viruses type 1 (AAV1), type 2 (AAV2), type 3 (AAV3a / AAV3b), type 4 (AAV4), type 5 (AAV5), type 6 (AAV6), type 7 (AAV7), type 8 (AAV8), type 9 (AAV9), and rhAAV10 (rhAAV10: Hu, C et al., Molecular Therapy vol.22, no.10, Oct.2014, 1792-1802). The nucleotide sequences of these adeno-associated virus genomes are publicly known, and their respective nucleotide sequences can be referenced using GenBank registry numbers: AF063497.1 (AAV1), AF043303 (AAV2), NC_001729 (AAV3), U48704 (AAV3A), AF028705.1 (AAV3B), NC_001829.1 (AAV4), NC_006152.1 (AAV5), AF028704.1 (AAV6), NC_006260.1 (AAV7), NC_006261.1 (AAV8), AY530579.1 (AAV9), and AY631965 (AAV10). In the present invention, the protein contained in the recombinant viral vector has an amino acid sequence that is at least 80%, preferably 85% or more, about 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, 99.1% or more, 99.2% or more, 99.3% or more, 99.4% or more, 99.5% or more, 99.6% or more, 99.7% or more, 99.8% or more, or 99.9% or more identical to the amino acid sequence described in SEQ ID NOs: 1 to 4, and is a protein that forms a viral vector or forms the capsid (outer shell) of a viral vector under physiological conditions.

[0017] 1.2. Adenovirus vectors Gene therapy vectors using adenoviruses (hereinafter also called recombinant adenovirus vectors) can be said to be the most widely used vectors in clinical trials of pharmaceuticals to date (Japanese Patent Publication No. 08-501686, Japanese Patent Publication No. 10-506542, Non-Patent Literature 1, References 14, 15 below). Furthermore, as a practical application, a recombinant adenovirus vector incorporating the human tumor suppressor gene p53 was the first in the world to be approved as a gene therapy drug in 2003 and is sold under the trade name "Gendicin". Recombinant adenovirus vectors are known to have a wide host range (target animal species, cell tumors, etc.). In addition, since the adenovirus gene exists independently in the cell nucleus of the target cell without being integrated into the chromosome, this characteristic allows recombinant adenovirus vectors to transiently express the target gene. Specifically, when a recombinant adenovirus vector is administered to a living organism, the expression of the target gene can usually disappear within 1 to 2 weeks. One drawback of recombinant adenovirus vectors is their high immunogenicity in living organisms. Reference 14. Yokoda RT, et al.: Biomedicines. 6:33, 2018. Reference 15. Zhang C and Zhou D: Hum Vaccin Immunother. 12:2064-2074, 2016.

[0018] The adenovirus genome is a linear double-stranded DNA molecule of approximately 36kb, and it encodes more than 30 genes, including early and late structural genes necessary for viral replication. Furthermore, the adenovirus's outer shell has an exposed capsid, which is made up of capsid protein, similar to the outer shell of adeno-associated viruses.

[0019] The early genes of adenoviruses are divided into four regions in the adenovirus genome (E1-E4: E indicates early). These contain six transcription units and have their own promoters. The late genes (L1-L5: L indicates late) partially overlap with the early transcription units and are transcribed mostly from the major late promoter (MLP).

[0020] For further details on recombinant viral vectors using adenoviruses, see, for example, references 14 and 15 mentioned above.

[0021] 1.3. Retroviral vectors As used herein, retroviral vectors are intended to include gamma retroviral vectors (or oncoviral vectors) and lentiviral vectors. Examples of gamma retroviral vectors include those derived from Moloney's mouse leukemia virus (MoMLV). Examples of lentiviral vectors include those derived from human immunodeficiency virus type 1 (HIV 1) and simian immunodeficiency virus (SIV).

[0022] Retroviral vectors can permanently express a target polynucleotide by efficiently incorporating it into the chromosomes of target cells. However, because these retroviral vectors originate from viruses that cause diseases such as leukemia and immunodeficiency syndromes, there are concerns that they may also cause malignant neoplasms. Examples of genes that can be introduced include those of 9kb or less (Doi et al., Virus, Vol. 65, No. 1, pp. 27-36, 2015; Vargas JE, et al.: J Transl Med. 14:288, 2016; Poletti V and Mavilio F: Mol Ther Methods Clin Dev. 8:31-41, 2017).

[0023] Retroviruses possess an envelope protein and envelope membrane as their outer shell. Furthermore, retroviruses often have RNA as their genome, and the genes encoded by this genome include gag / pol genes that encode precursor proteins for viral structural proteins, proteases, reverse transcriptases, and integrases, as well as env genes that encode envelope glycoproteins. The viral genome is flanked at both ends by long terminal repeats (LTRs), which contain enhancers, promoters, and polyadenylation signals.

[0024] For further information on the characteristics and properties of retroviral vectors, please refer to the following literature, for example: Mann et al., Cell 33:153-159 (1983); Cone and Mulligan, Proc. Natl.Acad.Sci.USA 81: 6349-6353 (1984); Vargas JE, et al.: J Transl Med. 14:288, 2016; Poletti V and Mavilio F: Mol Ther Methods Clin Dev. 8:31-41, 2017.

[0025] 1.4. Regarding the gene to be expressed The recombinant viral vector used in the present invention may contain a target expression gene (polynucleotide) for introduction into target cells within the recombinant viral genome contained within the viral vector. Such a target expression gene to be introduced may include, but is not limited to, a gene containing a functional sequence itself, a sequence whose transcript is functional, a sequence that is translated into a protein and functions, and a combination of these sequences. In other words, the target expression gene contained in the recombinant viral vector used in the present invention is not particularly limited as long as it can be loaded into the recombinant viral vector, taking into account gene size and other factors.

[0026] Examples of target expression genes included in the recombinant viral vector used in the present invention include sequences that function on their own, such as promoter sequences, telomere sequences, polyadenylation signal sequences, and target protein binding sequences.

[0027] Examples of sequences in which the transcripts used in the recombinant vectors of the present invention function include, but are not limited to, antisense molecules, ribozymes, interfering RNA (iRNA), and microRNA (miRNA), which are polynucleotides used to alter (e.g., disrupt or reduce) the function of a target endogenous gene, or polynucleotides used to alter (e.g., reduce) the expression level of an endogenous protein. Methods for preparing and using double-stranded RNA (dsRNA, siRNA, shRNA, or miRNA) are well known from numerous publications (see Japanese Patent Publication No. 2002-516062; U.S. Publication No. 2002 / 086356A; Nature Genetics, 24(2), 180-183, 2000 Feb., etc.).

[0028] Examples of sequences for protein translation included in the recombinant vector used in the present invention include sequences encoding proteins such as growth factors, trophic factors, cytokines, antigens, antibodies, tumor suppressors, metabolic enzymes, suicide genes, receptors, transporters, growth inhibitors, genome editing and repair methods, and combinations of these sequences. Furthermore, an expression cassette containing a known reporter gene such as green fluorescent protein (GFP) may also be used. The recombinant viral vector used in the present invention may include multiple vectors having the gene for expression described above. Furthermore, the recombinant viral vector used in the present invention may also contain multiple genes for expression within a single vector.

[0029] Examples of target genes to be expressed by the recombinant viral vector of the present invention include, but are not limited to, hepatocyte growth factor (HGF), nerve growth factor (NGF), hemoglobin, interleukin-1, interleukin-2, interleukin-3, interleukin-4, interleukin-5, interleukin-6, interleukin-7, interleukin-8, interleukin-9, interleukin-10, interleukin-11, GM-CSF, G-CSF, M-CSF, human growth factor, insulin, factor VIII, factor IX, tPA, LDL receptor, tumor necrosis factor (TNF), PDGF, EGF, NGF, IL-1ra, EPO, β-globin, and enzymes for cleaving specific sites on the genome (e.g., TALEN, CRISPR, and ZFN). The target polynucleotides to be introduced by the recombinant viral vector of the present invention may be heterologous or homologous to the target cells, tissues, or patient. The promoter sequence usable in this invention is not particularly limited as long as it can express the gene for the purpose described above within the target cell. For example, in the present invention, general-purpose mammalian-derived promoters or virus-derived promoters can be used. Examples of such promoters include, but are not limited to, the PGK promoter, EF1-α promoter, β-globin promoter, CMV promoter, SV40 promoter, MMLV-LTR promoter, and HIV-LTR promoter. Furthermore, in the present invention, a promoter specific to the target cell can be used. When the target cell is a cell of the nervous system, examples include, but are not limited to, synapsin I promoter sequences, myelin basic protein promoter sequences, neuron-specific enolase promoter sequences, calcium / calmodulin-dependent protein kinase II (CMKII) promoter sequences, tubulin αI promoter sequences, platelet-derived growth factor β chain promoter sequences, glial fibrillary acidic protein (GFAP) promoter sequences, L7 promoter sequences (cerebellar Purkinje cell-specific promoter), glial fibrillary acidic protein (hGfa2) promoter sequences, glutamate receptor delta 2 promoter (cerebellar Purkinje cell-specific promoter) sequences, and glutamate decarboxylase (GAD65 / GAD67) promoter sequences. In the present invention, when the target cells are hepatocytes, examples of promoters include, but are not limited to, the ApoE promoter, antitrypsin promoter, cKit promoter, promoters for liver-specific transcription factors (HNF-1, HNF-2, HNF-3, HNF-6, C / ERP, DBP), albumin promoter, thyroxine-binding globulin (TBG) promoter, and synthetic promoters (such as the HCRhAAT promoter, as described in WO2018 / 131551, etc.). These promoter sequences can be appropriately modified for use in the present invention, as long as they retain promoter function in target cells.

[0030] 1.5. Target Diseases Examples of therapeutic methods using the composition of the present invention as a pharmaceutical include gene therapy, cancer virus therapy, and viral immunotherapy. In gene therapy, research is being conducted on congenital genetic disorders and diseases caused by age-related functional decline. In cancer virus therapy, viruses that target cancer cells and induce lysis, apoptosis, etc., are being studied. In viral immunotherapy, for example, treatment methods that use viruses to induce an immune response advantageous for treating the target disease, such as CART therapy, are being studied.

[0031] Examples of diseases targeted by viral vector therapy include congenital genetic disorders such as adenosine aminase deficiency (ADA-SCID), X-linked severe combined immunodeficiency (X-SCID), chronic granulomatous disease (CGD), β-thalassemia, Leber congenital amaurosis (LCA), and adrenoleukodystrophy (ALD). Examples of cancers targeted by cancer viral therapy include lung cancer, kidney cancer, prostate cancer, esophageal cancer, brain tumors, and melanoma. Other diseases being studied include obstructive arteriosclerosis, angina pectoris, myocardial infarction, Alzheimer's disease, Parkinson's disease, ALS, HIV, hepatitis, age-related macular degeneration, diabetes mellitus, and rheumatoid arthritis (e.g., Non-Patent Literature 1, Non-Patent Literature 2, Piguet F et al.: Hum Gene Ther. 28:988-1003, 2017).

[0032] 2.sugar The composition of the present invention is characterized by containing sugar together with the recombinant viral vector described above. In this specification, "sugar" may also be referred to as "saccharides". The sugars usable in the compositions of the present invention are sugars known to be usable in the pharmaceutical field. Preferably, the sugars used in the present invention are those usable in injectable or infusion preparations. Such sugars include monosaccharides (including trioses, tetrose, pentoses, and hexoses) and disaccharides. The sugars used in the compositions of the present invention are preferably erythrose, threose, ribose, arabinose, xylose, lyxose, allose, altrose, glucose, mannose, growth, idose, galactose, talose, erythrolose, ribulose, xylulose, psicose, fructose, sorbose, tagatose, sucrose, trehalose, maltose, lactose, and combinations thereof, but are not limited thereto.

[0033] The sugars used in the compositions of the present invention may include stereoisomers. For example, the structure of each sugar (monosaccharide) contained in the compositions of the present invention may be the D-isomer, the L-isomer, or a mixture thereof. Preferably, about 60% or more, 70% or more, 80% or more, 90% or more, or 95% or more of the monosaccharides contained in the compositions of the present invention may be the D-isomer.

[0034] The monosaccharides used in the compositions of the present invention may have a cyclic or linear structure. Examples of cyclic structures include the pyranose type, which is a six-membered ring, and the furanose type, which is a five-membered ring. These ring structures have either an α-form or a β-form depending on the position of the hydroxyl group bonded to the carbon atom at position 1 (C1). The monosaccharides used in the compositions of the present invention may be α-forms, β-forms, or mixtures thereof. In this specification, unless otherwise specified, the ring structure of a monosaccharide includes α-forms, β-forms, and combinations thereof.

[0035] Furthermore, the compositions of the present invention may contain known sugar alcohols that can be used as pharmaceuticals. Preferred sugar alcohols include, but are not limited to, erythritol, xylitol, sorbitol, mannitol, and combinations thereof.

[0036] The compositions of the present invention may contain known sugar modifiers that can be used as pharmaceuticals. Modifications to obtain such sugar modifiers include, but are not limited to, known modifications applied to pharmaceuticals, such as acetylation, N-acetylglucosamine, N-acetylgalactosamine, sialylation, glucuronization, iduronization, phosphorylation, sulfation, and ribosylation. Preferred sugar modifications used in the compositions of the present invention include, but are not limited to, N-acetylglucosamine, N-acetylgalactosamine, sialylation, glucuronization, iduronization, and combinations thereof.

[0037] The composition of the present invention may be in the form of an aqueous solution containing at least 40 mM of sugar. This concentration of 40 mM corresponds to an aqueous solution of approximately 0.7% for monosaccharides and approximately 1.4% for disaccharides. For example, the sugar concentrations in the composition of the present invention are, at a lower limit, 40 mM glucose, 80 mM sucrose, 40 mM trehalose, 40 mM maltose, 40 mM lactose, or higher, and at an upper limit, the saturated aqueous concentration of the monosaccharide or disaccharide contained in the composition, such as 240 mM glucose, 200 mM sucrose, 200 mM trehalose, 200 mM maltose, 200 mM lactose, or lower (provided that the lower limit < upper limit). For example, the composition of the present invention may contain monosaccharides or disaccharides at concentrations of 200 mM glucose, 160 mM sucrose, 160 mM trehalose, 160 mM maltose, and 160 mM lactose. The sugars mentioned above may be combinations of two or more sugars. For example, combinations of glucose and sucrose, sucrose and maltose, glucose, sucrose and maltose, or sucrose and maltose. In this case, the sugar concentrations can be set to concentrations suitable for the desired gene transfer, such as the lower and upper limits of the concentration of each sugar, or the lower and upper limits of the total concentration of each sugar for one type of sugar.

[0038] Generally, in basal culture media used for eukaryotic cells, especially mammalian cells, the sugar concentration is 1 to 4.5 g / L. This concentration corresponds to approximately 6 to 25 mM in glucose equivalent. When using the composition of the present invention, in ex vivo or in vitro gene transfer, the final sugar concentrations can be, for example, 40 to 240 mM glucose, 80 to 200 mM sucrose, 40 to 200 mM trehalose, 40 to 200 mM maltose, and 40 to 200 mM lactose. These sugars may be a combination of two or more sugars as described above. With respect to the compositions of the present invention, the final sugar concentration in the case of ex vivo or in vitro gene transfer can be appropriately calculated depending on the amount of culture medium used.

[0039] Regarding blood glucose levels in living organisms, in typical humans, the lower limit is 80-100 mg / dL (e.g., between meals or during fasting), and the upper limit is 150-160 mg / dL (e.g., after a meal). Combining these, blood glucose levels are considered to be within a narrow range of 5.0-8.5 mM. When using the composition of the present invention, the final concentration of each sugar can be adjusted to 10 mM or less in vivo. These sugars may be a combination of two or more sugars as described above. With respect to the composition of the present invention, the final glucose concentration in the case of in vivo gene transfer can be calculated by considering the weight, water content, blood volume, volume of the patient to be administered the gene, blood, tissue fluid (e.g., cerebrospinal fluid), etc. In such calculations, known amounts can be used, for example, in the case of a human adult weighing 60 kg, such as approximately 60% water content and approximately 8% (approximately 5 L) of blood volume. Depending on the circumstances, surgical procedures can be used to increase the local concentrations of the recombinant viral vector and sugar in the gene transfer composition of the present invention. For example, a specific organ, tissue, or part thereof to be gene-transferred can be isolated from the body and temporarily exposed to a higher concentration of sugar. More specifically, the blood flow, lymph, cerebrospinal fluid, etc., of the site to be gene-transferred (e.g., organ, tissue, or part thereof) can be temporarily stopped (isolated) using clamps, etc., and replaced, perfused, or flushed with a pharmaceutically usable aqueous medium (such as physiological saline), and then the composition of the present invention can be administered to the flushed site. A specific example of such a surgical procedure is known to be a method in which portal vein blood in the liver is temporarily flushed with physiological saline and then the AAV vector is administered there (Mimuro et al., Molecular Therapy vol.21 no.2 feb. 2013, 318-323). In this case, the final sugar concentration can be, for example, 50-100 mM, 60-90 mM, 70-80 mM, etc., but is not limited to these. Furthermore, when the composition of the present invention is used for intrathecal administration, a final concentration higher than the blood concentration may be used, taking into account the volume of cerebrospinal fluid (for example, about 150 mL in adults). In this case, the final glucose concentration may be, but is not limited to, concentrations such as 50-100 mM, 60-90 mM, or 70-80 mM.

[0040] The sugars contained in the composition of the present invention may be combined with a recombinant viral vector at a desired concentration during a period from immediately before use (usually 5 minutes or less) to one day before use, for example, 10 minutes, 20 minutes, 30 minutes, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 12 hours (one night before), or one day before use. When the composition of the present invention is prepared in this manner before use, the sugar concentrations in the composition of the present invention may be prepared to include, for example, glucose at concentrations of 40-240 mM, sucrose at 80-200 mM, trehalose at 40-200 mM, maltose at 40-200 mM, and lactose at 40-200 mM. These concentrations may be used in amounts that take into account the final concentrations described above. Furthermore, the composition of the present invention may be provided in the form of a concentrate and diluted before use (for example, immediately before use (5 minutes or less), 10 minutes before, 20 minutes before, 30 minutes before, etc.). Alternatively, the composition of the present invention may be frozen and stored in a state containing the above-mentioned sugar concentration. When frozen and stored, it may be in the form of a concentrate or not. When frozen and stored, the composition of the present invention can preferably be stored for a period of one month or less, two months, three months, six months, one year, two years, five years, ten years or more. Thereafter, it is thawed before use (for example, immediately before use (five minutes or less), ten minutes before, twenty minutes before, thirty minutes before, or overnight) and optionally diluted to the desired concentration.

[0041] The step of bringing target cells or tissues into contact with the composition of the present invention can also be performed temporarily. For example, contact between target cells, tissues, or organs and the composition of the present invention can be performed at culture temperature (or a temperature lower than the culture temperature, such as ambient temperature) for a certain period of time, for example, from 30 minutes to several days (7 days), more specifically, for periods such as 1 hour, 1.5 hours, 2 hours, 2.5 hours, 3 hours, 6 hours, 12 hours (overnight), 18 hours, 24 hours (1 day), or 2 days. After that, the target cells, tissues, or organs may be replaced with fresh culture medium that does not contain the composition of the present invention, or the target cells, tissues, or organs may be repeatedly brought into contact with the composition of the present invention. In this operation, the sugars used and their final concentrations may be the above-mentioned final concentrations, for example, glucose at 40-240 mM, sucrose at 80-200 mM, trehalose at 40-200 mM, maltose at 40-200 mM, and lactose at 40-200 mM in ex vivo or in vitro (e.g., final concentration in culture medium), or a monosaccharide or disaccharide at a concentration of 10 mM in vivo (e.g., in blood). The composition of the present invention allows for a shorter administration period than conventionally used in in vivo administration, thereby reducing the burden on the patient.

[0042] 3. Other features of the composition of the present invention 3.1. Preparation at the time of use The composition of the present invention contains a predetermined concentration of sugar. When actually used as a pharmaceutical composition, it is conceivable that the amount of sugar administered may need to be limited or reduced depending on the patient's health condition. In such cases, the composition of the present invention can be prepared immediately before use.

[0043] In one embodiment, the composition of the present invention may be provided in the form of a solution containing a predetermined concentration of sugar. In another embodiment, the composition of the present invention may be provided in a form that is prepared at the time of use. Such a form may be, for example, a kit comprising a vial or ampoule containing a recombinant viral vector and a vial or ampoule containing sugar. The vector and sugar in these vials or ampoules may be in the form of an aqueous solution or in a dried form, such as by freeze-drying. Furthermore, the kit according to the present invention may further include a solution (an aqueous medium such as water for injection) for dissolving the recombinant viral vector and sugar. Furthermore, the kit according to the present invention is packaged together with instructions provided by the manufacturer.

[0044] When the composition of the present invention is prepared in advance of use, it can be prepared between immediately before use (5 minutes or less) and 12 hours before use (1 night before), for example, 10 minutes, 20 minutes, 30 minutes, 1 hour, 2 hours, 3 hours, 6 hours, and 12 hours before use (1 night before). Furthermore, the composition of the present invention may be provided in the form of a concentrate and diluted before use (for example, between immediately before use (5 minutes or less) and 12 hours before use (one night before), for example, 10 minutes, 20 minutes, 30 minutes before, etc.). Alternatively, the composition of the present invention may be frozen and stored for a period of time, for example, one month or less, two months, three months, six months, one year, two years, five years, ten years or more, with or without the above-mentioned sugar concentration, and may be thawed before use (for example, immediately before use (five minutes or less), ten minutes before, twenty minutes before, thirty minutes before, etc.) and optionally diluted to a composition containing the desired final sugar concentration. The temperature used for preparation as described above may be below the culture temperature, for example, ambient temperature (room temperature), or it may be low enough that the solution of the composition of the present invention does not freeze.

[0045] 3.2. Use in vivo, exo vivo, or in vitro The composition of the present invention can be brought into contact with cultured cells. Examples of such cells include, but are not limited to, established cultured cells and primary cells collected from living organisms. Cells into which the target gene has been introduced using the composition of the present invention can be used for therapeutic, research, and other purposes.

[0046] The compositions of the present invention may be used in vitro or ex vivo, or in vivo. Preferably, the compositions of the present invention are pharmaceutical compositions and may be used when culturing cells to be extracted from a living organism and returned to the living organism, or may be administered directly to a living organism. The compositions of the present invention may also be in the form of reagents, for example, comprising a recombinant viral vector having a reporter gene.

[0047] The compositions of the present invention can be administered into the body (in vivo) as pharmaceutical compositions. For example, adeno-associated viruses have the advantage of being targeted to various target tissues depending on the isolate (type). In this case, a composition prepared in advance to the desired sugar concentration can be administered to the body using dosage forms such as injections or infusions. Furthermore, the compositions of the present invention can also be administered via known routes of administration, such as peripheral administration, intramuscular administration, portal vein administration, intrathecal administration, and intracerebral administration. Furthermore, the composition of the present invention can be used in surgery to isolate an organ, tissue, or part thereof that is to be gene-transmitted. In such surgery, in order to increase the local concentration of the recombinant viral vector and sugar in the composition of the present invention, the blood flow, lymph, cerebrospinal fluid, etc. of the site to be introduced (e.g., organ, tissue, or part of an organ or tissue) is temporarily isolated using clamps, etc., and replaced, perfused, or flushed with a pharmaceutically usable aqueous medium (such as physiological saline). Subsequently, the gene-transmission composition of the present invention can be administered to the isolated site. As a specific example of this, a method is known in which portal vein blood in the liver is temporarily flushed with physiological saline and then the AAV vector is administered thereto (Mimuro et al., Molecular Therapy vol. 21 no. 2 feb. 2013, 318-323).

[0048] 3.3 Dosage Forms of Pharmaceutical Compositions When the composition of the present invention is used as a pharmaceutical composition, it can be administered, for example, orally, parenterally (intravenously), via cerebrospinal fluid, muscle, oral mucosa, rectally, vaginally, transdermally, nasally, or by inhalation, but parenteral administration is preferred. Intravenous administration is even more preferred. The active ingredient of the pharmaceutical composition of the present invention may be formulated alone or in combination, but it can also be provided in the form of a formulation by incorporating a pharmaceutically acceptable carrier or formulation additive. In this case, the active ingredient of the present invention can be contained in the composition in an amount of, for example, 0.1 to 20% by weight, preferably 1 to 5% by weight.

[0049] The active ingredient of the pharmaceutical composition of the present invention may be formulated alone or in combination, but it can also be provided in the form of a formulation by incorporating a pharmaceutically acceptable carrier or formulation additive. In this case, the active ingredient of the present invention can be contained in the composition in an amount such as 0.1 to 20% by weight, preferably 1 to 5% by weight, in the formulation. Examples of pharmaceutically acceptable carriers or additives include diluents, solubilizers, solubilizers, isotonic agents, pH adjusters, stabilizers, dyes, etc.

[0050] Suitable formulations for parenteral administration include, for example, injections, infusions, and suppositories. For parenteral administration, the recombinant viral vector, sugar, and other components used in the pharmaceutical composition of the present invention can be dissolved in an aqueous medium (purified water, sterile purified water, sterile water for injection, physiological saline, etc.), sesame oil, or peanut oil, or in an aqueous solution of propylene glycol. The aqueous solution should be buffered as needed (preferably to pH 8 or higher), and the liquid diluent must first be made isotonic. For example, physiological saline can be used as such a liquid diluent. The prepared aqueous solution is suitable for intravenous injection, while the oily solution is suitable for intra-articular, intramuscular, and subcutaneous injection. All of these solutions can be easily manufactured under sterile conditions using standard pharmaceutical techniques well known to those skilled in the art.

[0051] Examples of formulations suitable for oral administration include liquids or syrups. For oral administration, in addition to lactose, high molecular weight polyethylene glycol can be used. When preparing an aqueous suspension and / or elixir for oral administration, the active ingredient can be used in combination with various sweeteners or flavorings, colorants or dyes, and if necessary, emulsifiers and / or suspending agents can also be used, along with water, ethanol, propylene glycol, glycerin, and diluents combining these.

[0052] The dosage of the pharmaceutical composition according to the present invention is not particularly limited, and an appropriate dosage can be selected according to various conditions such as the type of disease, the patient's age and symptoms, the route of administration, the purpose of treatment, and the presence or absence of concomitant drugs. The dosage of the pharmaceutical composition according to the present invention is, for example, 1 to 5000 mg per day for an adult (e.g., weighing 60 kg), preferably 10 to 1000 mg, but is not limited to these. These daily doses may be administered in two to four divided doses. For example, when using vg (vector genome) as the dosage unit, for example, 10 per kg of body weight 9 ~10 14 vg, preferably 10 10 ~10 13 vg, more preferably 10 10 ~10 12 Dosages within the VG range can be selected, but are not limited to these. Furthermore, the units and amounts may be changed depending on the virus used. Any commonly used and known dosage can be used in the compositions of this invention.

[0053] 3.4. Reagents and Kits of the Present Invention The composition of the present invention may be in the form of a reagent used in cultured cells ex vivo or in vitro. In this case, the composition of the present invention may be used as an aqueous medium, such as the above-mentioned aqueous medium such as water for injection or physiological saline, as well as a basal medium or a concentrated medium. Furthermore, the recombinant viral vector of the present invention may contain a known reporter gene such as GFP. Such a marker can be used, for example, to select gene-transformed cells ex vivo or in vitro, or to verify the transduction state in vivo.

[0054] The compositions of the present invention may be provided in the form of a kit for preparing the compositions. In such a kit (also known as the kit of the present invention), the recombinant viral vector used in the present invention may include, for example, an empty vector DNA, a helper virus, or helper virus DNA for packaging within the recombinant viral vector. More specifically, for example, in the case of an rAAV vector, the first polynucleotide is DNA into which the user incorporates the gene (polynucleotide) of interest for expression, and the second polynucleotide is DNA encoding a helper virus (e.g., AdV helper). The kit of the present invention may also further include manufacturer's instructions describing the procedure for the user to prepare a recombinant viral vector using the kit of the present invention.

[0055] 4. Other terms used in this specification The meanings of each term used herein are as follows. Unless otherwise specified, terms are intended to refer to the meanings that are ordinarily understood by those skilled in the art.

[0056] Where used herein, unless otherwise specified, the terms “viral vector,” “viral virion,” and “viral particle” are interchangeable. Furthermore, “viral vector” refers to a genetically modified vector unless otherwise explicitly stated.

[0057] As used herein, the term “polynucleotide” is interchangeable with “nucleic acid,” “gene,” or “nucleic acid molecule,” and refers to a polymer of nucleotides. As used herein, the term “nucleotide sequence” is interchangeable with “nucleic acid sequence” or “base sequence,” and refers to a sequence of deoxyribonucleotides (abbreviated as A, G, C, and T).

[0058] The "viral genome" according to the present invention may be in the form of DNA (e.g., cDNA or genomic DNA) or RNA (e.g., mRNA), unless otherwise specified. The viral genome used herein may be double-stranded or single-stranded DNA or RNA. The single-stranded DNA or RNA may be a coding strand (i.e., sense strand) or a non-coding strand (i.e., antisense strand).

[0059] Unless otherwise specified, when describing the genetic arrangement of promoters, target genes, polyadenylation signals, etc., encoded by the rAAV genome, the description refers to the sense strand itself if the rAAV genome is a sense strand, or to its complementary strand if it is an antisense strand. Furthermore, the recombination symbol "r" may be omitted in this specification where it is clear from the context.

[0060] In this specification, "protein" and "polypeptide" are used interchangeably, and polymers of amino acids are intended. Polypeptides used herein follow the convention of peptide notation, with the left end being the N-terminus (amino terminus) and the right end being the C-terminus (carboxyl terminus). The partial peptides of the polypeptide of the present invention (which may be abbreviated as "partial peptides of the present invention" in this specification) are the partial peptides of the polypeptide of the present invention described above, preferably having properties similar to those of the polypeptide of the present invention described above.

[0061] In this specification, the term "plasmid" means various known genetic elements, such as plasmids, phages, transposons, cosmids, chromosomes, etc. Plasmids can replicate in a specific host and transport gene sequences between cells. In this specification, plasmids comprise various known nucleotides (DNA, RNA, PNA, and mixtures thereof) and may be single-stranded or double-stranded, but preferably double-stranded. For example, in this specification, the term "rAAV vector plasmid" is intended to include a double-stranded genome formed by the rAAV vector genome and its complementary strand, unless otherwise specified. Plasmids used in the present invention may be linear or circular.

[0062] In this specification, terms not specifically defined are intended to mean what is ordinarily understood by those skilled in the art. [Examples]

[0063] The present invention will be described in more detail below with reference to examples, but the scope of the present invention is not limited to the following examples.

[0064] A. Materials and Methods (1) GFP-expressing AAV vector Three types of AAV vectors (AAV3B, yf2AAV9, and AAVGT4) were used. yf2AAV9 has tyrosine (Y) at positions 446 and 731 of the AAV9 envelope protein VP1 replaced with phenylalanine (F) (iida et al., BioMed Research International, Vol. 2013, Article ID 974819). AAVGT4 has serine (S) at position 587 of VP1 of AAV3B replaced with alanine (A) (SEQ ID NO: 4). All AAV vectors have an expression cassette consisting of a cytomegalovirus promoter (CMV) promoter, green fluorescent protein (AcGFP) cDNA, and SV40 poly(A) inserted between the inverted terminal repeats (ITRs) of the known AAV3A. AAV3A: GenBank Accession # U48704 (The amino acid sequence of the VP1 protein is shown in SEQ ID NO: 1.) AAV3B: GenBank Accession #AF028705.1 (The amino acid sequence of the VP1 protein is shown in SEQ ID NO: 2.) AAV9: GenBank Accession # AY530579.1 yf2AAV9: (The amino acid sequence of the VP1 protein is shown in SEQ ID NO: 3.) AAVGT4: (The amino acid sequence of the VP1 protein is shown in SEQ ID NO: 4.) (b) Cell culture 1) HEK 293 cells 5×10 4 / well of HEK293 cells were seeded and cultured at 5% CO2, 37 °C using 10% fetal calf serum (FCS)-DMEM / F12 medium. 2) HepG2 cells 5×10 4 / well of HepG2 cells were seeded and cultured at 5% CO2, 37 °C using 10% FCS DMEM low glucose medium (Thermo Scientific). (c) Sugars Monosaccharides: glucose, galactose, fructose, mannose Disaccharides: sucrose, trehalose, maltose, lactose They were added to the medium to reach the following final concentrations. Glucose: 0 - 200 mM[[ID=3,6]] Other monosaccharides: 100 mM, 200 mM Sucrose: 0 - 187 mM Other disaccharides: 100 mM, 125 mM, 150 mM (d) Infection with viral vectors AAV3B-CMV-AcGFP expressing GFP described in (a) was added to the cultured cells in (b) at 6×10 7 ~5×108 vg / well, or yf2AAV9-CMV-AcGFP 2×10 9 VG / well was added and the cells were cultured for 3-6 days. (e) Evaluation of GFP expression The fluorescence intensity of GFP was measured and compared using a plate reader (Biotech Japan). Representative field images were also captured using a fluorescence microscope (Olympus IX83).

[0065] Experimental Results and Discussion Experiment 1: Effect of sucrose on AAV infection and expression HEK293 cells 5×10 4 Cells / well were seeded into a 96-well optical bottom plate (ThermoFisher), and the following day, AAV3B-CMV-AcGFP 2×10 8 vg / well, or yf2AAV9-CMV-AcGFP 2×10 9 vg / well was added to the culture medium. Subsequently, sucrose (Wako) was added to achieve a final concentration of 125 mM in the medium. Specifically, a 1.25 M sucrose solution dissolved in the medium was added to make up 1 / 10 of the total volume of the medium. For the control (sucrose-free), the same amount of medium as the sucrose solution was added. After incubation for 3 days at 37°C in a 5% CO2 incubator, the fluorescence intensity of GFP was measured and compared using a plate reader (Biotech Japan) (Figure 1A). In addition, representative field images were taken using a fluorescence microscope Olympus IX83 (Figure 1B). The results of this experiment showed that the addition of sucrose (represented by (+)) increased GFP expression by 15.4 times for AAV3B and 19.6 times for yf2AAV9. [Table 1]

[0066] Experiment 2: Presence or absence of increased expression levels from sucrose, mannitol, glucose, and NaCl. HEK293 cells 5×10 4Cells / well were seeded into a 96-well Optical Bottom Plate, and the following day, AAV3B-CMV-AcGFP 6×10 7 vg / well was added to the culture medium. Subsequently, 125 mM sucrose was added, along with glucose (Sigma), mannitol (Nippon Pharmaceutical), or NaCl (Wako) at concentrations that resulted in approximately the same osmotic pressure. The final concentrations were 125.0 mM, 132.3 mM, 132.3 mM, and 71.5 mM, respectively. An equal volume of culture medium was added to each solution to serve as a control. After incubation for 3 days at 37°C in a 5% CO2 incubator, the fluorescence intensity of GFP was measured and compared using a plate reader (Figure 2A). In addition, representative field images were taken using an Olympus IX83 fluorescence microscope (Figure 2B). The results of this experiment showed that the addition of sucrose, glucose, and mannitol increased expression by 11-fold, 8-fold, and 2-fold, respectively. However, the addition of NaCl resulted in a drastic decrease in expression. [Table 2]

[0067] Experiment 3: Effect of increased expression in HepG2 cells HepG2 cells 5×10 4 Cells / well were seeded into a 96-well optical bottom plate. The following day, AAVGT4-CMV-AcGFP 5×10 8 vg / well, or AAV3B-CMV-AcGFP 5×10 8 vg / well was added to the culture medium. Then, sucrose was added to the medium until the final concentration in the medium was 125 mM. For the control (sucrose-free), the same amount of medium as the sucrose solution was added. After incubation for 3 days at 37°C in a 5% CO2 incubator, the fluorescence intensity of GFP was measured and compared using a plate reader. The results of this experiment showed that in HepG2 cells, a human liver cancer cell line, the addition of sucrose increased the expression of AAV3B by 16.1 times and AAVGT4 by 19.1 times. [Table 3]

[0068] Experiment 4: Sucrose expression enhancement effect by various addition methods HEK293 cells 5×10 4 Cells / well were seeded in a 96-well Optical Bottom Plate, and the following day, AAV vector and sucrose were administered using various methods. All samples were cultured for 3 days at 37°C in a 5% CO2 incubator, after which the fluorescence intensity of GFP was measured and compared using a plate reader. All AAV vectors used were AAV3B-CMV-AcGFP 1×10⁶. 8 The concentration was set to vg / well, and the final concentration of sucrose was 125 mM. As the first method, sucrose was added immediately after AAV vector administration (less than 5 minutes), immediately after sucrose was added to the cells beforehand (less than 5 minutes), 10 minutes later, or 1 hour later before administering the AAV vector. For the control group (sucrose-free), the same amount of culture medium as the sucrose solution was added. In the sucrose-added group, a 10-fold increase in expression was observed with either method (Figure 4A). [Table 4A] As a second method, the AAV vector and sucrose were mixed beforehand, and immediately after mixing (less than 5 minutes) and after standing at room temperature for 1 hour, the amount of AAV3B-CMV-AcGFP was 1 × 10⁻⁶. 8 Cells were administered vg / well with sucrose at a final concentration of 125 mM. Sucrose(-) refers to a solution prepared by mixing the AAV vector with an equal volume of sucrose solution and allowing it to stand at room temperature for 1 hour. GPF expression levels were measured after 3 days in the same manner as above. The results of this experiment showed that even when the AAV vector was mixed with sucrose before administration, a 12-fold or greater enhancement of expression was obtained in both immediate and 1-hour administration (Figure 4B). [Table 4B] As a third method, AAV3B-CMV-AcGFP vector 1×10 8Immediately after administration of vg / well (within 5 minutes), sucrose(+) or (-) medium was added, and 3 hours later, the entire volume was replaced with 125 mM sucrose(+) or (-) medium. GPF expression levels were measured 3 days later in the same manner as above. These experiments showed that sucrose was present immediately after AAV administration, resulting in a 4.9-fold enhancement of expression. However, even when sucrose was added 3 hours later, when AAV infection was considered complete, a 2.8-fold enhancement was still observed (Figure 4C). [Table 4C]

[0069] Experiment 5: Expression enhancement effect of D-glucose and L-glucose L-glucose (Sigma), which does not exist in nature and is not utilized by cells (not incorporated into glycolysis), or normally present D-glucose, was mixed with AAV3B-CMV-AcGFP and left to stand at room temperature for 1 hour. After this, the amount of AAV increased to 1 × 10⁻⁶. 8 Cells were administered vg / well, with each glucose concentration reaching a final concentration of 132 mM. After culturing for 1, 2, and 3 days in a 37°C, 5% CO2 incubator, the fluorescence intensity of GFP was measured and compared using a plate reader. The cells used were HEK293 cells, 5 × 10⁶. 4 The cells / well were used the day after sowing in a 96-well optical bottom plate. The results of this experiment showed that, one day after administration, D-glucose addition resulted in approximately a 17-fold increase in expression, while L-glucose addition also resulted in an 11-fold increase. This suggests that the reason for this is not that the mixed sugars activated cellular metabolism, but rather that the sugars were involved in the AAV vector infection itself (Figure 5A). [Table 5A] On the other hand, the difference in expression intensity between the D and L forms (D / L) was 1.5 times after 1 day, but widened to 2.1 times after 3 days, suggesting that sugar may also enhance expression by increasing cell activity (Figure 5B). [Table 5B]

[0070] Experiment 6: Differences in expression enhancement effects depending on sucrose concentration in the culture medium. AAV3B-CMV-AcGFP was pre-mixed with sucrose at various concentrations, allowed to stand at room temperature for 1 hour, and then administered to cells. The final dose of the AAV vector was 1 × 10⁻⁶. 8 The final sucrose concentration was 80 mM to 160 mM, based on the 1g / well ratio. For sucrose-free cells, the sucrose solution was mixed with an equal volume of culture medium. As a control, to prevent cytotoxicity from the buffer during sucrose dilution, the medium was changed 3 hours after administration with a sucrose solution of the same concentration prepared with culture medium. After culturing for 3 days at 37°C in a 5% CO2 incubator, the GFP fluorescence intensity was measured and compared using a plate reader. The cells used were HEK293 cells, 5 × 10⁶. 4 The cells / well were used the day after sowing in a 96-well optical bottom plate. The results of this experiment showed that a sucrose concentration of 80 mM in the culture medium resulted in a more than 5-fold enhancement. The effect increased in a concentration-dependent manner, with high enhancements of approximately 30-fold observed at 145, 150, and 160 mM. The optimal concentration after 3 days was 150 mM (Figure 6). Furthermore, when the sucrose concentration was increased to 187 mM, cells were damaged and detached, and GFP intensity decreased drastically to about 20% (data not shown). [Table 6]

[0071] Experiment 7: Differences in expression enhancement effects depending on glucose concentration in the culture medium. AAV3B-CMV-AcGFP was pre-mixed with glucose at various concentrations, allowed to stand at room temperature for 1 hour, and then administered to cells. The final AAV dose was 1 × 10⁻⁶. 8The final glucose concentration was 40 mM to 200 mM, based on vg / well. For glucose-free solutions, an equal amount of culture medium was mixed with the glucose solution. To prevent cytotoxicity due to buffer during glucose dilution, the culture medium was changed 3 hours after administration with a glucose solution of the same concentration prepared in the culture medium. After culturing for 3 days at 37°C in a 5% CO2 incubator, the fluorescence intensity of GFP was measured and compared using a plate reader. The cells used were HEK293 cells, 5 × 10⁶. 4 The cells / well were used the day after sowing in a 96-well optical bottom plate. The results of this experiment showed a more than threefold enhancement at a glucose concentration of 80 mM in the culture medium. This enhancement effect increased in a concentration-dependent manner, and a nearly 60-fold enhancement was obtained at 200 mM (Figure 7). [Table 7]

[0072] Experiment 8: Effect of disaccharides on enhancing AAV expression HEK293 cells 5×10 4 The day after seeding cells / wells in a 96-well Optical Bottom Plate, the medium was changed with a culture medium containing four disaccharides: sucrose, trehalose, maltose, and lactose (all Sigma except sucrose), at concentrations of 100, 125, and 150 mM. Then, AAV3B-CMV-AcGFP 1×10⁶ was added. 8 Vg / well was administered. The control group was given the same amount of culture medium as the glucose solution. After incubation at 37°C in a 5% CO2 incubator for 1, 2, 3, and 6 days, the fluorescence intensity of GFP was measured and compared using a plate reader (Figure 8A). In addition, representative field images were taken using an Olympus IX83 fluorescence microscope (Figure 8B). The results of this experiment showed that, three days after administration, all sugars exhibited a concentration-dependent enhancement effect of 2 to 6 times. [Table 8-1] When compared at a sugar concentration of 150 mM, lactose showed the highest effect, but lactose has extremely low solubility compared to other sugars and may not be suitable for practical use. After 6 days, expression was stronger with maltose, and there were differences in how expression enhancement manifested depending on the sugar (Figure 8C). [Table 8-2]

[0073] Experiment 9: Enhancement effect of monosaccharides on AAV expression HEK293 cells 5×10 4 The day after seeding cells / well into a 96-well Optical Bottom Plate, AAV3B-CMV-AcGFP was mixed with two concentrations of monosaccharides, glucose, galactose, fructose, and mannose (all Sigma), left to stand at room temperature for 1 hour, and then administered to the cells. The final AAV dose was 1 × 10⁻⁶. 8 The sugar concentrations were 100 mM and 200 mM at a dose of vg / well. For the control, the same amount of culture medium was mixed with the sugar solution. After incubation for 3 days at 37°C in a 5% CO2 incubator, the fluorescence intensity of GFP was measured and compared using a plate reader (Figure 9A). In addition, representative field images were taken using an Olympus IX83 fluorescence microscope 3 days after administration (Figure 9B). The results of this experiment showed that among the monosaccharides glucose, galactose, fructose, and mannose, glucose showed an overwhelmingly high enhancement effect, with an effect of 120 times at 200 mM. The effects of fructose and mannose were similar to those of sucrose. Galactose had a lower effect than the other sugars. [Table 9] [Industrial applicability]

[0074] The composition of the present invention can significantly improve the efficiency of gene transfer using recombinant viral vectors. The composition of the present invention is expected to be used as a pharmaceutical for gene therapy. [Sequence Listing Free Text]

[0075] Sequence ID 1: Amino acid sequence of the AAV3A VP1 protein Sequence ID 2: Amino acid sequence of AAV3B VP1 protein Sequence ID 3: Amino acid sequence of yf2AAV9 VP1 protein (Y446F / Y731F mutant) Sequence ID 4: Amino acid sequence of AAV GT4 VP1 protein

Claims

1. A gene transfer composition for use in intrathecal administration, comprising a recombinant adeno-associated virus (AAV) vector containing a gene for expression of a target, at least 40 mM sugar, and an aqueous medium, The aforementioned composition is a gene transfer composition administered so that the final glucose concentration during intrathecal administration is 50 to 100 mM.

2. The composition according to claim 1, wherein the recombinant AAV vector is derived from AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAVrh10, AAVrh39, AAVrh43, AAV-B1, AAV-PHP.B, or AAV-PHP.eB.

3. The composition according to claim 1 or 2, wherein the recombinant AAV vector comprises a capsid containing a protein having an amino acid sequence described in SEQ ID NOs: 1 to 4, or an amino acid sequence having approximately 90% or more identity with the amino acid sequences described in SEQ ID NOs: 1 to 4.

4. The composition according to any one of claims 1 to 3, wherein the composition contains sugar at a concentration of 0.04 to 2 M.

5. The composition according to any one of claims 1 to 4, wherein the sugar comprises glucose, galactose, fructose, mannose, sucrose, trehalose, maltose, lactose, or a combination thereof.

6. The composition according to any one of claims 1 to 5, wherein the aqueous medium is water for injection.

7. The composition according to any one of claims 1 to 6, which is a pharmaceutical product in the form of an injection or infusion.

8. The composition according to any one of claims 1 to 7, wherein the composition is frozen for storage and thawed before use.

9. A kit for preparing, immediately after use, the composition according to any one of claims 1 to 8, comprising a recombinant adeno-associated virus (AAV) vector and a sugar.

10. (a) A step of providing a composition comprising a recombinant adeno-associated virus (AAV) vector and a sugar at a concentration of 40 mM or higher, and (b) A step of contacting cultured cells with the AAV vector in the composition in a culture medium containing sugar at a final concentration of 40 to 250 mM. A method for exo-vivo or in vitro gene transfer, including the above.

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