KR20090055392A - Compositions for enhancing gene delivery efficiency of gene delivery systems and for preserving viruses - Google Patents

Abstract

A composition for improving the efficiency of gene delivery to an animal cell of gene delivery system containing an aprotic polar solvent is provided to preserve a virus containing a capsid protein for a short and long time. A composition for improving the efficiency of gene delivery to an animal cell of gene delivery system comprises a gene delivery system and an aprotic polar solvent. The aprotic polar solvent is DMSO(dimethyl sulfoxide), DMF(dimethyl formamide), N-methylpyrolidone, N-methylmorpholine, gamma-butyrolactone, acetonitrile, tetrahydrofuran, and hexamethyl phosphoric triamide. The gene delivery system is a plasmid, recombinant adenovirus, adeno-associated viruses(AAV), retrovirus, lentivirus, herpes simplex virus, liposome or noisome. A pharmaceutical anti-tumor composition comprises therapeutically effective dose of adenovirus, aprotic polar solvent, and pharmaceutically allowable carrier. A composition for preserving a virus containing a capsid protein comprises the aprotic polar solvent. The virus covered with the capside protein is the adenovirus, adeno-associated virus(AAV), retrovirus, lentivirus, or herpes simplex virus.

Description

Compositions for Enhancing Gene Delivery Efficiency of Gene Delivery Systems and for Preserving Viruses

The present invention relates to a technique for increasing the gene transfer efficiency of a gene carrier, and more particularly, to a composition for improving gene transfer efficiency and a gene delivery composition for animal cells of the gene carrier.

The mechanism by which adenovirus infects and enters into a cell is first performed by the coupling between the coxsackievirus and adenovirus receptor (CAR) expressed on the surface of the target cell and the fiber knob of the adenovirus. As the integrin, the secondary receptor of adenovirus, is revealed, receptor-mediated endocytosis occurs due to the secondary binding of Fentonbase protein and integrin, in addition to the primary binding of adenovirus fiber knob to CAR. . Subsequently, endosomes are formed by endocytosis, and the adenovirus penetrates into the cytoplasm by the acidic pH of the endosomes. After binding to the nuclear pores, the DNA of the adenovirus moves into the nucleus.

The expression of CAR has been reported to be low in brain cancer cell lines, head and neck cancers, melanoma and bladder cancer.In the case of cell expressions with low CAR expression, intracellular infection of adenovirus is very low and intracellular CAR expression is increased. The result shows that the infection rate of adenovirus increases when poor CAR expression acts as a barrier to adenovirus infection.

Throughout this specification, many papers and patent documents are referenced and their citations are indicated. The disclosures of cited papers and patent documents are incorporated herein by reference in their entirety, and the level of the technical field to which the present invention belongs and the contents of the present invention are more clearly explained.

The present inventors have tried to develop a method of increasing the gene delivery efficiency into animal cells of a gene delivery system, and as a result, when aprotic polar solvent is used together with the gene delivery system, the gene delivery efficiency is improved. By confirming that it can greatly increase, the present invention has been completed.

Accordingly, it is an object of the present invention to provide a composition for improving gene transfer efficiency of animal genes to animal cells.

Another object of the present invention to provide a composition for gene delivery.

Another object of the present invention is to provide a pharmaceutical anti-tumor composition.

Another object of the present invention is to provide a composition for virus conservation.

Other objects and advantages of the present invention will become apparent from the following detailed description, claims and drawings.

According to an aspect of the present invention, the present invention provides a composition for improving gene delivery efficiency of an animal carrier comprising an aprotic polar solvent as an active ingredient.

According to another aspect of the present invention, the present invention provides a kit comprising: (a) a gene carrier; And (b) provides a composition for gene delivery comprising an aprotic polar solvent.

The present inventors have sought to develop a method for increasing the gene delivery efficiency of a gene delivery system into animal cells. As a result, it was confirmed that the use of an aprotic polar solvent in combination with a gene carrier can greatly increase gene transfer efficiency.

The aprotic polar solvent used in the present invention is not particularly limited and may be used in the present invention as long as it exhibits an aprotic polarity. That is, polar solvents having no acidic hydrogen atoms can be used in the present invention.

According to a preferred embodiment of the present invention, the aprotic polar solvent used in the present invention is dimethyl sulfoxide (DMSO), dimethyl formamide (DMF), N-methylpyrrolidone, N-methylmorpholine, γ-butyrolactone , Acetonitrile, tetrahydrofuran or hexamethylphosphoric triamide, most preferably DMSO.

DMSO is an aprotic polar solvent as shown in the formula of C ₂ H ₆ OS. DMSO is a chemically stable solvent with high boiling point (189 ° C), high flash point (95 ° C) and low toxicity, and is easy to use. In addition, DMSO is a by-product produced during the pulp process, and is characterized by not being decomposed by microorganisms in water or in soil for a long time. DMSO is an aprotic compound consisting of a hydrophilic sulfoxide group and two hydrophobic methyl groups and can dissolve a polar or nonpolar compound.

Gene delivery vehicles that can improve gene delivery efficiency by the present invention is not particularly limited. Preferably, the gene carrier used in the present invention is a plasmid, adenovirus (Lockett LJ, et al., Clin . Cancer Res . 3: 2075-2080 (1997)), Adeno-associated viruses: AAV, Lashford LS., Et al., Gene Therapy Technologies , Applications and Regulations Ed. A. Meager, 1999), retroviral (Gunzburg WH, et al., Retroviral vectors. Gene Therapy Technologies, Applications and Regulations Ed. A. Meager, 1999), lentiviruses (Wang G. et al., J. Clin . Invest . 104 (11): R55-62 (1999)), herpes simplex virus (Chamber R., et al., Proc . Natl. Acad. Sci USA 92: 1411-1415 (1995)), basinian virus (Puhlmann M. et al., Human Gene Therapy 10: 649-657 (1999)), liposomes (Methods in Molecular Biology, Vol 199, SC Basu and M. Basu (Eds.), Human Press 2002) or niosomes.

Most preferably, the gene carrier in which the gene transfer efficiency can be improved by the present invention is a recombinant adenovirus.

In the case of recombinant adenoviruses, they are infected into cells via a CAR (coxsackievirus and adenovirus receptor). However, since the expression of CAR is low in brain cancer cells, head and neck cancer cells, melanoma cells, and bladder cancer cells, it is a barrier in gene therapy by recombinant adenovirus. In addition, even in cells with high CAR expression, increasing the infection rate of recombinant adenovirus is very helpful for gene therapy.

The present invention makes it possible to overcome these barriers to gene therapy. For example, the use of aprotic polar solvents allows the introduction of genes into cells using adenoviruses CAR-independently.

Detailed description of examples of gene carriers that can be used in the present invention are as follows:

Iii. Adenovirus

Adenoviruses are widely used as gene transfer vectors because of their medium genome size, ease of manipulation, high titers, wide range of target cells and excellent infectivity. Both ends of the genome contain 100-200 bp of Inverted Terminal Repeat (ITR), which is an essential cis element for DNA replication and packaging. The genome El region (E1A and E1B) encodes proteins that regulate transcription and transcription of host cell genes. The E2 regions (E2A and E2B) encode proteins that are involved in viral DNA replication.

Among the adenovirus vectors currently developed, non-replicating adenoviruses lacking the E1 region are widely used. On the other hand, the E3 region is removed from conventional adenovirus vectors to provide a site for insertion of foreign genes (Thimmappaya, B. et al., Cell , 31: 543-551 (1982); and Riordan, JR et al., Science , 245: 1066-1073 (1989). Therefore, the foreign gene is preferably inserted into the deleted E1 region (E1A region and / or E1B region, preferably E1B region) or E3 region, and more preferably inserted into the deleted E3 region. On the other hand, the target nucleotide sequence to be carried into the cell is preferably inserted into the deleted E1 region (E1A region and / or E1B region, preferably E1B region) or E3 region, more preferably inserted into the deleted E1 region do. The insertion sequences can also be inserted into the deleted E4 region. As used herein, the term "deletion" as used in connection with a viral genome sequence has the meaning including not only a complete deletion of the sequence, but also a partial deletion.

According to the most preferred embodiment of the present invention, the adenovirus gene delivery system of the present invention has a “promoter-purpose nucleotide sequence-poly A sequence” structure, wherein the “promoter-purpose nucleotide sequence-poly A sequence” is deleted. It is inserted into an E1 region (E1A region and / or E1B region, preferably E1B region) or E3 region, preferably the deleted E1 region. In addition, the first target nucleotide and the second target nucleotide are linked by an internal ribosome entry site (IRES) such as “promoter-first target nucleotide sequence-poly A sequence-IRES-second target nucleotide sequence-poly A sequence”. It can also be expressed by a bicistronic expression system.

In addition, because adenovirus can pack up to about 105% of the wild-type genome, about 2 kb can be additionally packaged (Ghosh-Choudhury et al., EMBO J. , 6: 1733-1739 (1987)). Thus, the above-described foreign sequence inserted into the adenovirus may additionally bind to the genome of the adenovirus.

Adenoviruses have 42 different serotypes and subgroups of A-F. Of these, adenovirus type 5 belonging to subgroup C is the most preferred starting material for obtaining the adenovirus vector of the present invention. Biochemical and genetic information for adenovirus type 5 is well known.

Foreign genes carried by adenoviruses replicate in the same way as episomes, with very low genetic toxicity to host cells. Therefore, gene therapy using the adenovirus gene delivery system of the present invention is considered to be very safe.

Ii. Retrovirus

Retroviruses are widely used as gene transfer vectors because they insert their genes into the host genome, carry large amounts of foreign genetic material, and have a broad spectrum of cells that can infect them.

To construct the retroviral vector, the target nucleotide sequence to be delivered is inserted into the retroviral genome instead of the sequence of the retrovirus to produce a nonreplicating virus. To produce virion, a packaging cell line comprising the gag, pol and env genes but no LTR (long terminal repeat) and Ψ sequences is constructed (Mann et al., Cell , 33: 153-159 (1983)). When a recombinant plasmid comprising the target nucleotide sequence, LTR and Ψ sequence to be transported is introduced into the cell line, the Ψ sequence enables the production of RNA transcripts of the recombinant plasmid, which are packaged into a virus and the virus is Discharged into the medium (Nicolas and Rubinstein "Retroviral vectors," In: Vectors: A survey of molecular cloning vectors and their uses , Rodriguez and Denhardt (eds.), Stoneham: Butterworth, 494-513 (1988)). The medium containing the recombinant retrovirus is collected, concentrated and used as a gene delivery system.

Gene delivery using second generation retroviral vectors has been announced. Kasahara et al. Science , 266: 1373-1376 (1994)) prepared a variant of the Moloney murine leuchemia virus, in which an erythropoietin (EPO) sequence was inserted into the envelope site to produce a chimeric protein with new binding properties. The gene delivery system of the present invention can also be prepared according to this second generation retroviral vector construction strategy.

Iii. AAV vector

Adeno-associated virus (AAV) is suitable as the gene delivery system of the present invention because it can infect non-dividing cells and has the ability to infect various kinds of cells. Details of the preparation and use of AAV vectors are disclosed in detail in US Pat. Nos. 5,139,941 and 4,797,368.

Studies on AAV as a gene delivery system are described in LaFace et al, Viology , 162: 483486 (1988), Zhou et al., Exp . Hematol . (NY), 21: 928-933 (1993), Walsh et al, J. Clin . Invest . 94: 1440-1448 (1994) and Flotte et al., Gene Therapy , 2: 29-37 (1995). Recently, AAV vectors have undergone clinical I as a therapeutic agent for cystic fibrosis.

Typically, AAV viruses are plasmids containing the gene sequence of interest, with two AAV terminal repeats flanked (McLaughlin et al., J. Virol . , 62: 1963-1973 (1988); and Samulski et al. , J. Virol . , 63: 3822-3828 (1989)) and expression plasmids containing wild type AAV coding sequences without terminal repeats (McCarty et al., J. Virol . , 65: 2936-2945 (1991)). Prepared by cotransformation.

Iii. Different virus vector

Other viral vectors can also be used in the gene delivery system of the present invention. Basinian virus (Puhlmann M. et al., Human Gene Therapy 10: 649-657 (1999); Ridgeway, "Mammalian expression vectors," In: Vectors : A survey of molecular cloning vectors and their uses . Rodriguez and Denhardt, eds. Stoneham: Butterworth, 467-492 (1988); Baichwal and Sugden, "Vectors for gene transfer derived from animal DNA viruses: Transient and stable expression of transferred genes," In: Kucherlapati R, ed. Gene transfer . New York: Plenum Press, 117-148 (1986) and Coupar et al., Gene , 68: 1-10 (1988)), lentiviruses (Wang G. et al., J. Clin . Invest . 104 (11) : R55-62 (1999)) or vectors derived from the herpes simplex virus (Chamber R., et al., Proc . Natl . Acad . Sci USA 92: 1411-1415 (1995)). Nucleotide sequences can be used as a delivery system capable of delivering intracellularly.

Iii. Liposomes

Liposomes are automatically formed by phospholipids dispersed in the aqueous phase. Examples of successfully delivering foreign DNA molecules into liposomes into cells include Nicolau and Sene, Biochim . Biophys . Acta , 721: 185-190 (1982) and Nicolau et al., Methods Enzymol . 149: 157-176 (1987). On the other hand, Lipofectamine (Gibco BRL) is the most used reagent for the transformation of animal cells using liposomes. Liposomes containing the target nucleotide sequence to be transported carry the target nucleotide sequence to be transported into the cell by interacting with the cell through mechanisms such as endocytosis, adsorption to the cell surface, or fusion with a plasma cell membrane.

The target nucleotide sequence that can be carried intracellularly by the gene carrier in the present invention may be any nucleotide sequence, for example, a cancer therapeutic gene that induces death of cancer cells and ultimately degrades tumors, including tumor suppressor genes and immune regulatory genes. [E.g. cytokine genes, chemokine genes and costimulatory factors (cofactors necessary for T cell activity such as B7.1 and B7.2)], antigenic genes, suicide genes, cytotoxic genes, cell proliferation inhibition Genes, pro-apoptotic genes and anti-angiogenic genes are included.

Suicide genes are nucleic acid sequences that express substances that cause cells to be killed by external factors or cause toxic conditions to cells. Well known such suicide genes are the thymidine kinase (TK) genes (US Pat. Nos. 5,631,236 and 5,601,818). Cells expressing the TK gene product are susceptible to selective killing by administration of gancyclovir. Tumor suppressor genes refer to genes encoding polypeptides that inhibit tumor formation. Tumor suppressor genes are naturally occurring genes in mammals, and it is believed that deletion or inactivation of these genes is an essential prerequisite for tumor development. Examples of tumor suppressor genes include APC, DPC4, NF-1, NF-2, MTS1, WT1, BRCA1, BRCA2, VHL, p53, Rb, MMAC-1, MMSC-2, retinoblastoma genes (Lee et al. Nature , 329: 642 (1987)), adenomatous polyposis coli protein (US Pat. No. 5,783,666), a throat disease suppressor gene located on chromosome 3p21.3 (Cheng et al. Proc . Nat . Acad . Sci . , 95: 3042-3047 (1998)), members of the INK4 family of tumor suppressor genes, including the missing colon tumor (DCC) gene, MTS1, CDK4, VHL, p110Rb, p16 and p21, and therapeutically effective fragments thereof (Eg, p56Rb, p94Rb, etc.). Those skilled in the art will appreciate that all other known anti-tumor genes besides the genes exemplified above may be used in the present invention.

As used herein, the term “antigenic gene” refers to a nucleotide sequence that is expressed in a target cell to produce cell surface antigenic proteins that can be recognized by the immune system. Examples of such antigenic genes include carcinoembryonic antigen (CEA) and prostate specific antigen (PSA), α-feto protein (AFP), p53 (WO 94/02167). To facilitate recognition of the immune system, the antigenic gene can be linked to an MHC type I antigen.

As used herein, the term "cytotoxic gene" refers to a nucleotide sequence that is expressed in a cell and exhibits a toxic effect. Examples of such cytotoxic genes include nucleotide sequences encoding Pseudomonas exotoxin, lysine toxin, diphtheria toxin, and the like.

As used herein, the term "cytostatic gene" refers to a nucleotide sequence that is expressed intracellularly and stops the cell cycle during the cell cycle. Examples of such cell proliferation inhibitory genes include p21, retinoblastoma gene, E2F-Rb fusion protein gene, genes encoding cyclin-dependent kinase inhibitors (eg, p16, p15, p18 and p19), growth stop specific homeo Growth arrest specific homeobox (GAX) genes (WO 97/16459 and WO 96/30385), and the like.

In addition, many therapeutic genes that can be usefully used to treat various diseases are also carried by the system of the present invention. For example, cytokines (eg interferon-alpha, -beta, -delta and -gamma), interleukins (eg IL-1, IL-2, IL-4, IL-6, IL-7, IL-10 , Genes encoding IL-12, IL-19 and IL-20) and colony stimulating factors (eg, GM-CSF and G-CSF), chemokine groups (monocyte chemotactic protein 1 (MCP-1), monocyte chemotaxis) Protein 2 (MCP-2), monocyte chemotactic protein 3 (MCP-3), monocyte chemotactic protein 4 (MCP-4), macrophage inflammatory protein 1α (MIP-1α), macrophage inflammatory protein 1β (MIP-1 β) , Macrophage inflammatory protein 1γ (MIP-1γ), macrophage inflammatory protein 3α (MIP-3α), macrophage inflammatory protein 3β (MIP-3β), chemokine (ELC), macrophage inflammatory protein 4 (MIP-4), macrophage inflammatory protein 5 (MIP-5), LD78β, RANTES, SIS-epsilon (p500), thymus activation-regulated chemokine (TARC), eotaxin, I-309, human protein HCC-1 / NCC-2, human protein HCC-3, Mouse protein C10, etc.). Also included are genes expressing tissue plasminogen activator (tPA) or urokinase and LAL generating genes that provide sustained thrombotic effects to prevent cholesterol hyperemia. In addition, many polynucleotides are known for treating viral, malignant and inflammatory diseases and conditions such as cystic fibrosis, adenosine deaminase deficiency, and AIDS.

As used herein, the term “pro-apoptotic gene” refers to a nucleotide sequence that is expressed and induces programmed cell death. Examples of such pro-apoptotic genes include p53, adenovirus E3-11.6K (derived from Ad2 and Ad5) or adenovirus E3-10.5K (derived from Ad), adenovirus E4 gene, Fas ligand, TNF-, TRAIL, p53 pathway gene and gene encoding caspase are included.

As used herein, the term "anti-angiogenic gene" refers to a nucleotide sequence that is expressed and releases an anti-angiogenic factor extracellularly. Anti-angiogenic factors include angiostatin, inhibitors of vascular endothelial growth factor (VEGF) such as Tie 2 (PNAS, 1998, 95,8795-800), endostatin and the like.

In addition, the relaxin gene or decorin gene, which has been found to be suitable for the treatment of adenovirus genes by the present inventors, is also a gene that can be carried intracellularly by a gene carrier.

Nucleotide sequences of the purpose described above can be obtained from DNA sequence databanks such as GenBank or EMBL.

According to the present invention, gene carriers can be introduced into various animal cells with increased efficiency. For example, the present invention can be applied to mammalian (human, bovine, pig, mouse, rat, goat, hamster, sheep and horse) or avian cells. In addition, the present invention can be applied to cells of various states or types. For example, the present invention can be applied to normal cells, cancer cells, tumor cells, primary-cultured cells and stem cells to introduce gene carriers at increased efficiency.

According to an aspect of the present invention, the present invention provides a composition for improving gene delivery efficiency of an animal carrier comprising an aprotic polar solvent as an active ingredient.

According to another aspect of the present invention, the present invention provides a kit comprising: (a) a gene carrier; And (b) provides a composition for gene delivery comprising an aprotic polar solvent.

According to another aspect of the present invention, the present invention provides a gene delivery method comprising the step of contacting the above-described gene delivery composition of the present invention to a biosample comprising a cell.

In the present invention, when the gene delivery system is constructed based on the viral vector, the contacting step is performed according to a virus infection method known in the art. Infection of host cells with viral vectors is described in the references cited above.

According to another aspect of the invention, the invention provides a pharmaceutical composition comprising (a) a therapeutically effective amount of adenovirus; (b) aprotic polar solvents; And (c) provides a pharmaceutical anti-tumor composition comprising a pharmaceutically acceptable carrier.

In addition to the adenovirus used as an active ingredient in the pharmaceutical composition of the present invention, the aprotic polar solvent used to increase the intracellular infection rate of the adenovirus is preferably DMSO (dimethyl sulfoxide), DMF (dimethyl formamide), or N. -Methylpyrrolidone, N-methylmorpholine, γ-butyrolactone, acetonitrile, tetrahydrofuran or hexamethylphosphoric triamide, most preferably DMSO.

Adenoviruses used in the pharmaceutical compositions of the present invention include any adenovirus used for gene therapy.

The genome of an adenovirus used in the present invention includes an inverted terminal repeat (ITR) nucleotide sequence and a target nucleotide sequence.

The pharmaceutical anti-tumor composition of the present invention increases the gene transfer efficiency of conventional recombinant adenoviruses, greatly increasing the efficacy of cancer treatment by adenoviruses.

Because only a small part of the adenovirus genome is known to be required in cis (Tooza, J. Molecular biology of DNA Tumor viruses , 2nd ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY (1981)), anenoviruses are capable of carrying large quantities of foreign DNA molecules, especially when using certain cell lines such as 293. In this aspect, in the adenovirus used in the present invention, sequences of adenoviruses other than the gene of interest to be carried include at least an ITR sequence.

The gene of interest is preferably inserted into the E1 region (E1A region and / or E1B region, preferably E1B region) or E3 region, more preferably in the E3 region. On the other hand, other foreign nucleotide sequences (e.g., cytokines, immune-suppressive factors, suicide genes and tumor suppressor genes) may additionally be included in the adenovirus, which is an E1 region (E1A region and / or E1B region, preferably It is preferably inserted into an E1B region or an E3 region, more preferably in an E1 region (E1A region and / or E1B region, preferably E1B region). In addition, the insertion sequences may be inserted in the E4 region.

In addition, because adenovirus can pack up to about 105% of the wild-type genome, about 2 kb can be additionally packaged. Thus, the above-described foreign sequence inserted into the adenovirus may additionally bind to the genome of the adenovirus.

In a preferred embodiment of the invention, the recombinant adenovirus of the invention has an inactivated E1B 19 gene, E1B 55 gene or E1B 19 / E1B 55 gene. As used herein, the term “inactivation” as used in connection with a gene means that the transcription and / or translation of the gene is not performed normally, so that the function of the normal protein encoded by the gene does not appear. For example, an inactivated E1B 19 gene is a gene in which a mutation (substitution, addition, partial deletion or total deletion) occurs in that gene, resulting in no active E1B 19 kDa protein. In the case of lacking E1B 19, it is possible to increase apoptosis, and in the absence of E1B 55 gene, it has tumor cell specificity (see Korean Patent Application No. 10-2002-0023760).

According to a preferred embodiment of the present invention, the adenovirus used in the present invention comprises an active E1A gene. Recombinant adenoviruses comprising the E1A gene will have replicable properties. According to a more preferred embodiment of the present invention, the adenovirus used in the present invention comprises an inactivated E1B 19 / E1B 55 gene and an active E1A gene. According to the most preferred embodiment of the present invention, the adenovirus used in the present invention comprises an inactivated E1B 19 / E1B 55 gene and an active E1A gene, and the nucleotide sequence of interest is inserted into the deleted E3 region.

In addition, the adenovirus used in the present invention has an “ITR-E1A-ΔE1B-promoter-target gene-poly A sequence” structure, wherein the “promoter-target gene-poly A sequence” is inserted into the deleted E3 region. will be.

As exemplified in the Examples below, administration of adenoviruses in combination with aprotic polar solvents, especially DMSO, greatly improves gene transfer efficiency by adenoviruses and does not depend on CAR expression. This dramatic increase in gene transfer efficiency results in greatly increased antitumor effects by adenoviruses. This improvement in anti-frontal effects is seen in not only replicable recombinant adenoviruses, but also in non-replicable recombinant adenoviruses.

In order to induce an effective anti-tumor effect using adenovirus, it is necessary to induce an effective cell killing effect through faster virus proliferation and proliferation into adjacent cells compared to rapidly growing cancer cells. In addition, in order to successfully perform cancer gene therapy using adenoviruses, a method for improving safety with high therapeutic effect should be developed. The co-administration method of the aprotic polar solvent developed in the present invention greatly increases the intracellular infection rate of adenovirus, thereby significantly increasing the antitumor effect. As a result, the virus dose required for cancer treatment can be reduced, greatly reducing in vivo toxicity and immune response by the virus.

As the adenovirus included in the composition of the present invention exhibits killing efficacy against various tumor cells, as described above, the pharmaceutical composition of the present invention can be used for various diseases or diseases related to the tumor, such as gastric cancer, lung cancer, breast cancer, and ovarian cancer. , Liver cancer, bronchial cancer, nasopharyngeal cancer, laryngeal cancer, pancreatic cancer, bladder cancer, colon cancer and cervical cancer and the like. As used herein, the term “treatment” means (i) prevention of tumor cell formation; (Ii) inhibiting a disease or condition associated with a tumor following removal of tumor cells; And (iii) alleviation of a disease or disorder associated with a tumor following removal of tumor cells. Thus, the term “therapeutically effective amount” herein means an amount sufficient to achieve the above pharmacological effect.

Pharmaceutically acceptable carriers included in the compositions of the present invention are those commonly used in the formulation, lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia rubber, calcium phosphate, alginate, gelatin, calcium silicate , Microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methyl cellulose, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, mineral oil, and the like, but are not limited thereto. no. In addition to the above components, the pharmaceutical composition of the present invention may further include a lubricant, a humectant, a sweetener, a flavoring agent, an emulsifier, a suspending agent, a preservative, and the like.

The pharmaceutical composition of the present invention is preferably parenteral, and may be administered using, for example, intravenous administration, intraperitoneal administration, intramuscular administration, subcutaneous administration, or topical administration. Intraperitoneal administration in ovarian cancer and portal cancer in liver can be administered by injection method, in the case of breast cancer by injection directly into tumor mass, and in the case of colon cancer by direct enema Can be administered by injection, in the case of bladder cancer can be administered by injection directly into the catheter.

Suitable dosages of the pharmaceutical compositions of the invention vary depending on factors such as the formulation method, mode of administration, age, weight, sex of the patient, degree of disease symptom, food, time of administration, route of administration, rate of excretion and response to reaction. In general, the skilled practitioner can readily determine and prescribe a dosage effective for the desired treatment. In general, the pharmaceutical compositions of the present invention comprise 1 × 10 ⁵ -1 × 10 ¹⁵ pfu / ml of recombinant adenovirus and typically are injected 1 × 10 ¹⁰ pfu once every two days for two weeks.

The pharmaceutical compositions of the present invention are prepared in unit dosage form by being formulated with pharmaceutically acceptable carriers and / or excipients, according to methods which may be readily practiced by those skilled in the art. Or may be prepared by incorporating into a multi-dose container. In this case, the formulation may be in the form of a solution, suspension or emulsion in an oil or an aqueous medium, or may be in the form of extracts, powders, granules, tablets or capsules, and may further include a dispersant or stabilizer.

The pharmaceutical composition of the present invention may be used as a single therapy, but may also be used in combination with other conventional chemotherapy or radiation therapy, and when the combination therapy is performed, cancer treatment may be more effectively performed. Chemotherapeutic agents that can be used with the compositions of the present invention are cisplatin, carboplatin, procarbazine, mechlorethamine, cyclophosphamide, phospho Ifosfamide, melphalan, chlorambucil, bisulfan, nitrosourea, diactinomycin, daunorubicin, doxorubicin , Bleomycin, plicomycin, mitomycin, etoposide, tamoxifen, taxol, transplatinum, 5-fluoro 5-urauracil, vincristin, vinblastin, methotrexate and the like. Radiation therapy that can be used with the composition of the present invention is X-ray irradiation, γ-ray irradiation, and the like.

According to another aspect of the present invention, the present invention provides a virus preservation composition having a capsid protein comprising an aprotic polar solvent as an active ingredient.

The inventors have found that aprotic polar solvents exert very good efficacy in conserving viruses surrounded by genetic material by capsid proteins.

As used herein, the term “capsid” refers to the protein shell of a virus. Capsids consist of several oligomeric subunits of protein and surround the genetic material of the virus. Capsids can be divided according to structure, and most viruses have capsids of helical or icosahedral structures (eg, adenoviruses).

According to a preferred embodiment of the present invention, the aprotic polar solvent is dimethyl sulfoxide (DMSO), dimethyl formamide (DMF), N-methylpyrrolidone, N-methylmorpholine, γ-butyrolactone, acetonitrile, Tetrahydrofuran or hexamethylphosphoric triamide, most preferably DMSO.

According to a preferred embodiment of the invention, the virus surrounded by the capsid protein which can be preserved by the composition of the invention is adenovirus, Adeno-associated viruses (AVA), retroviruses, lentiviruses, herpes Simplex virus or basinian virus, most preferably adenovirus.

The features and advantages of the present invention are summarized as follows:

(Iii) The present invention greatly improves the gene transfer efficiency of the gene carrier to animal cells by using an aprotic polar solvent.

(Ii) Although a lot of lipofectamine is commonly used in the art to introduce genes into cells, the aprotic polar solvent of the present invention shows a much better improvement in gene delivery efficiency than lipofectamine.

(Iii) For gene carriers used for gene therapy, the efficiency of gene delivery into cells is very important for achieving the desired effect. If gene transfer efficiency is a problem, the present invention can significantly improve this problem.

(Iii) The composition of the present invention comprising an aprotic polar solvent (particularly DMSO) exhibits excellent properties in terms of safety since it hardly causes cell damage, as demonstrated in the examples below.

(Iii) In addition, the composition of the present invention comprising an aprotic polar solvent has excellent short-term and long-term preservation against viruses having capsid proteins.

Hereinafter, the present invention will be described in more detail with reference to Examples. These examples are only for illustrating the present invention in more detail, it will be apparent to those skilled in the art that the scope of the present invention is not limited by these examples in accordance with the gist of the present invention. .

Example

Experimental Materials and Methods

Target cell line and cell culture

Human liver cancer cell lines (Huh7 and Hep1), brain cancer cell lines (U87MG, U251N and U343), lung cancer cell lines (H1299, H460 and A549), breast cancer cell lines (MDA-MB-435, MDA-MB-231 and MCF-7), head and neck Cancer cell lines such as cancer cell lines (FaDu, 1YB, QLL1 and CAL27), skin cancer cell lines (A431), prostate cancer cell lines (DU145) and human normal cell lines (CBHEL and IMR90) were used. In addition, human bone tumor cell lines SJSA and U20S and human fibrocarcinoma cell line HT1080 were used.

Mouse cell lines (B16F1, B16F10 and B16BL6), lung cancer cell lines (CMT-64), rectal cancer cell lines (CMT-93), breast cancer cell lines (EF43-fgf) and fibroblast-like Mus dunni cell lines were used. Hamster ovary cell line (Lec2) and rat cell line (YPEN) were used. All of these cell lines were purchased from the American Type Culture Collection (ATCC), Manassas, UA, USA, and all cell lines except FADU cell lines (MEM medium containing 10% fetal bovine serum) contained 5% or 10% fetal bovine serum (AAAA). ) DMEM (AAAA) medium containing the antibiotic penicillin / streptomycin (Invitrogen) as a culture medium was incubated in a 37 ℃ incubator in the presence of 5% CO ₂ . Lipofectamine was purchased from Invitrogen (Carlsbad, CA, USA) and dimethylsulfoxide (DMSO) was purchased from Sigma (St. Louis, MO, USA).

2. X-gal staining

To verify gene transfer efficiency by lipofectamine or DMSO, X-gal staining was performed using a virus with a LacZ marker gene. After dispensing 3 x 10 ⁵ cells into 6-wells of various cell lines, LacZ virus alone (dl / LacZ, see WO 2006/075819), lipofectamine / Plus (2: 6) combination or DMSO combination Two days later, X-gal staining was performed. Cells were fixed in fixative (1% formaldehyde, 0.2% glutaraldehyde in H ₂ O) for 5 minutes, washed twice with PBS and then X-Gal (5-bromo-4-chloro-3-indoly-bD galactopyanoside: The reaction of β-galactosidase was observed by reaction at 37 ° C. for 12 hours with a dye solution containing Life Technologies, Rokuill, MD).

3. Cell Lesion (CPE) Analysis

To verify the effect of lipofectamine and DMSO on the replication of adenovirus, the CPEs resulting from the replication of the virus were compared. The cancer cells were dispensed into 24-well plates with 2-5 × 10 ⁴ cells, and then cloned Ad-ΔB7 virus developed by the inventors (Korean Patent No. 0764122) at 1, 10, 100 or 1000 MOI. (multiplicity of infection) virus alone, in combination with lipofectamine, in combination with 2% DMSO or 5% DMSO. When any of these viruses completely killed the infected cells at the titer of 1 MOI, all the media was removed and the remaining cells were fixed and stained with 0.5% crystal violet (50% methanol) and analyzed. The unit% indicating the concentration of DMSO throughout this specification represents v / v%.

4. Production Comparison of Adenoviruses

To compare virus production by increased gene transfer efficiency when co-administered with lipofectamine or DMSO, 4 x 10 ⁴ A549, MDA-MB-231, Hep1 or FaDu cell lines were dispensed in 24-well plates. After 24 hours, Ad-ΔB7 virus was co-administered with virus alone, lipofectamine, or 1% DMSO, and A549 infected 0.5 MOI, 5 MOI for MDA-MB-231 and Hep1 cell lines, and 10 MOI for FaDu cell line. After infection, A549, MDA-MB-231, and Hep1 recovered cells and medium, respectively, after 48 hours in the FaDu cell line. Infected cells were released by repeated freezing and thawing three times, and the titer of virus in the medium and cells was calculated by performing a limiting titeration assay in HEK293 cell line.

5. FACS Analysis

Cell lines of A549, MDA-MB-231, or MDA-MB-435 in 6-wells were tested to determine whether the expression of CAR and integrin, which are important receptors for intracellular adenovirus, was increased when treated with lipofectamine or DMSO. Cell lines were obtained 4 and 24 hours after lipofectamine, 2% or 5% DMSO after x 10 ⁵ aliquots, and flow cytometry was performed. Each cell line was obtained and washed twice with PBS and then FITC bound anti-integrin α _V β ₃ (mouse anti-human integrin α _V β ₃ monoclonal antibody, clone name: LM 609, Chemicon, AAAAa) , Anti-integrin α _V β ₅ (mouse anti-human integrin α _V β ₅ monoclonal antibody, clone name: P1F6, Chemicon) antibody was added and reacted at 4 ℃ for 45 minutes, washed twice with PBS and 2% paraform After fixation with aldehyde, flow cytometry was performed. In case of CAR, mouse anti-human CAR purified antibody (RmcB, DiNonA) was added as a primary antibody and reacted at 4 ° C. for 45 minutes, washed twice with PBS, followed by PE-bound goat anti-mouse IgG immunoglobulin (cat number: 80014F, DiNonA) was added. After reacting them at 4 ° C. for 30 minutes, each cell was washed twice with PBS, fixed with 2% paraformaldehyde, and then subjected to flow cytometry.

6.Verification of stability and usefulness of adenovirus proteins by DMSO

DMSO is known as a solvent that induces degeneration of the internal keratin of human skin. Based on this fact, it was verified whether DMSO induced degeneration of the structural protein of adenovirus. As a negative control group, 4% sucrose solution, which is a general preservation solution of adenovirus, was used. dl / LacZ virus (see WO 2006/075819) was dissolved in 4% sucrose solution, sucrose solution containing 20% DMSO or sucrose solution containing 40% DMSO, and then left at 0 ° C. for 6 hours. Virus alone, 1% or 2% DMSO containing 1483 cells, head and neck cancer cell lines were infected and then X-gal staining 48 hours later. DMSO has also been reported to prevent protein accumulation by refolding mis-folded proteins as chemical chaperones. To verify whether adenovirus aggregation was inhibited by DMSO, dl / LacZ adenovirus dissolved in a preservative containing 4% sucrose and 20% DMSO was stored at room temperature for 1, 3, 5 and 7 days. After 8 days X-gal staining was performed.

7. Verification of short-term or long-term adenovirus preservation capacity using DMSO

DMSO is known to be a very useful reagent as a cryopreservative solution of cells. Based on this fact, it was verified whether DMSO could be used to preserve adenovirus for short or long term. As a negative control, 4% sucrose solution, which is a general preservation solution of adenovirus, was used. dl / LacZ virus was dissolved in 4% sucrose preservative containing 4% sucrose preservative and 20% DMSO, then stored in -80 ° C rapid freezer for 30 minutes or 24 hours before X-gal staining. . In addition, to verify long-term adenovirus preservation, the same batch of dl / LacZ virus was dissolved in 4% sucrose preservative or 4% sucrose preservative containing 20% DMSO and stored at -80 ° C monthly. After months, all were thawed for X-gal staining.

8. Analysis of Cell Changes by Electron Microscopy

Human lung cancer cell line H460 was dispensed in a 25T flask 1 × 10 ⁶ and the next day cells were obtained using a scraper and centrifuged and washed twice with PBS. Each cell was treated with virus alone, in combination with lipofectamine / Plus (2: 6) or in combination with 2% DMSO, 5 minutes, 1 hour after washing the cells twice with PBS, followed by 2% glutaraldehyde. Fixed. After fixing for 1 hour in 1% O _S O ₄ dissolved in 0.1 M cacodylate-HCl (pH 7.4) was observed by electron microscopy.

9. H & E Dyeing

In order to verify the gene transfer efficiency by intratumoral DMSO, 1 x 10 ⁶ EF43-fgf cell line was injected into the abdominal wall of BALB / C mice 6-8 weeks old (Central Laboratory Animal, Korea). The 60-80 mm 3 dl / LacZ virus was injected three times intratumorally at 1 × 10 ⁸ PFU (plaque forming unit) at two-day intervals. Three days after the last virus injection, the tumors were extracted and subjected to X-gal staining. In addition, when the tumor was grown to 60-80 뒤 after forming tumors of the FaDu cell line, human head and neck cancer cell lines, Ad-ΔB7 virus was injected three times with 1 x 10 ⁸ PFU, and tumors were extracted three days after the last virus administration. H & E staining was performed (hemotoxyline-eosine staining).

10. In vivo anti-tumor effect verification

FaDu cells, a human head and neck cancer cell line, were cultured in a single layer in MEM (Invitrogen) medium containing 10% fetal bovine serum, treated with 0.25% trypsin-EDTA (Invitrogen), and washed with Hanks' Balanced salt solution (HBSS). It was suspended later and injected subcutaneously into the abdominal wall of the mouse. About 8-10 days after tumor cell transplantation, when the tumor grows about 60-80 ㎣, Ad-ΔB7 adenovirus can be treated with virus alone, lipofectamine / Plus (2: 6) at a dose of 1 X 10 ⁸ PFU. Co-administration or co-administration with 5% DMSO was administered intratumorally five times every two days. As a negative control group, experiments were performed in the group administered with lipofectamine alone and the group administered with DMSO alone. The volume of the tumor was measured using a caliper to measure the long and short axis of the tumor and was calculated using the following formula: volume of the tumor = (short mm) ² x long axis mm x 0.523

Experiment result

1. Verification of gene transfer efficiency by DMSO co-administration in human and mouse cell lines

To verify the gene transfer efficiency of adenovirus by DMSO, LacZ analysis was performed using several human cell lines. As a marker gene, dl / LacZ, a non-replicating adenovirus expressing LacZ, was infected by MOI and then stained with X-gal. As can be seen in Figure 1, adenovirus-infected cell lines with low cell infection rate of FaDu, 1YB, QLL1, CAL27, A431, MDA-MB-435, MCF-7, CBHEL and IMR90 were infected with adenovirus alone Increased gene delivery efficiency was observed when co-administered with lipofectamine or DMSO. In particular, cell lines such as FaDu, 1YB, A431, CBHEL, and IMR90 showed a greater increase in gene transfer efficiency when adenovirus was administered with DMSO than when lipofectamine and adenovirus were administered. there was. In addition, gene transfer efficiency was slightly increased in 2% DMSO compared to 1% DMSO.

On the other hand, in the case of U87MG, U251N, U343, H1299, DU145, MDA-MB-231, Huh7, Hep1, H460, and A549 cell lines with high adenovirus infection rates, adenoviruses were used in combination with lipofectamine and adenovirus. It was observed that the gene transfer efficiency was reduced rather than the infection alone (Fig. 2). In contrast, when adenovirus was administered in combination with DMSO, it was observed that the gene transfer efficiency was similar to or slightly increased when the virus was administered alone. In particular, in the case of a combination of adenovirus and DMSO in the U87MG, DU145, MDA-MB-231, Hep1 and A549 cell lines, the gene delivery efficiency was increased compared to the virus alone. In addition, it could be observed that in combination with DMSO at 2% compared with the case of 1% DMSO co-induced increased gene transfer efficiency.

In addition, as a result of verifying the gene transfer efficiency in mouse and rat cell lines, as shown in Figure 3, the expression of CAR is very low B16-F1 and B16-F10 mouse melanoma cell lines with low adenovirus infection rate Showed that the expression efficiency of LacZ in combination with lipofectamine or DMSO and adenovirus was significantly increased compared to the case of infection with adenovirus alone, especially LacZ in combination with DMSO and adenovirus. Gene expression efficiency was confirmed to increase significantly compared to the case of co-administration of lipofectamine and adenovirus (Fig. 3). In addition, in the CMT-64, CMT-93, YPEN, E43-fgf mouse, and rat cell lines with a relatively high infection rate of adenoviruses, the gene transfer efficiency of a combination of DMSO and adenoviruses infected with adenoviruses alone. While similar or slightly increased, the expression of LacZ was decreased when co-administered with lipofectamine.

2. Verification of cell killing ability of replicable adenovirus by co-administration of lipofectamine and DMSO

In order to investigate the effect of lipofectamine or DMSO on the replication of adenovirus when adenovirus is administered with lipofectamine or DMSO, the degree of apoptosis due to the replication of adenovirus using a replicable adenovirus was measured. Observed by analysis. As shown in FIG. 4, when Ad-ΔB7 replication-capable adenovirus was administered alone or in combination with lipofectamine or 2% DMSO and 5% DMSO to various types of human cell lines with low adenovirus infection rate In addition, it was observed that the cell killing ability increased more in combination with DMSO than administration of the replicable virus alone. In particular, MCF-7, SJSA, and HT1080 cell lines showed higher cell killing ability when combined with lipofectamine or DMSO than Ad-ΔB7 replicable virus alone, and 5% DMSO in combination. In the case of administration, it was observed that induction of better cell killing ability compared with the case of co-administration of lipofectamine. In the case of FADU, A431, U20S, CBHEL, and IMR90 cell lines, the combination of lipofectamine and the apoptotic cell showed similar apoptosis, but DMSO co-administration resulted in increased cell death. The killing ability could be observed. In other words, the cell killing ability of the replicable adenovirus co-administered with DMSO was improved in the cell lines with low CAR expression compared with the virus alone, and the cell killing ability was increased compared with the co-administration of lipofectamine. I could confirm it.

On the other hand, in the case of U87MG, U343, DU145, MDA-MB-231, Hep1, H460, and A549 cell lines, which have high expression of CAR and high infection rate of adenovirus, lipofectamine was used as in the LacZ analysis. It was confirmed that the cell killing ability in the case of co-administration with Ad-ΔB7 replicable virus decreased compared with the case in which only virus was administered alone (FIG. 5). However, when the virus was co-administered with DMSO, the cell killing capacity was not decreased compared with the virus alone. Especially, the cell killing ability was increased in DU145, H460, and A549 cell lines. In addition, in the case of mouse and rat cell lines, it was confirmed that the cell killing capacity when the replicable adenovirus was coadministered with DMSO was increased compared with the virus alone administration (FIG. 6). In particular, in the CMT-64 cell line, apoptotic adenovirus was co-administered with lipofectamine, but the cell killing capacity was decreased compared with the virus alone. Was not observed.

3. Verification of Changes in Adenovirus Production by DMSO Co-administration

To investigate whether adenovirus can increase cell infectivity by a combination with DMSO and thus increase adenovirus production, Ad-ΔB7, a replicable adenovirus, was applied to MDA-MB-231 and A549 cell lines, respectively. After 48 hours of treatment with MOI and 0.5 MOI, both infected cells and cultures were recovered to calculate virus titers. As can be seen in Figure 7, in all MDA-MB-231 and A549 cell line, when the virus is co-administered with lipofectamine, while the virus production is significantly reduced compared to the virus alone, in combination with DMSO When administered, virus production did not decrease. In particular, in the case of the A549 cell line, Ad-ΔB7 adenovirus co-administered with DMSO was found to increase about four times as compared with the virus alone.

4. Observation of CAR and Integrin Expression in Lipofectamine or DMSO Co-administration

To determine whether the enhanced gene transfer efficiency and cell killing ability when adenovirus was administered with DMSO was due to increased expression of CAR and integrin, which are important receptors in the path of infection of adenovirus, the expression levels of CAR and integrin were examined. Validation was performed by FACS analysis. MDA-MB-231, MDA-MB-435, A549 cell lines were treated with lipofectamine, 2% DMSO, or 5% DMSO for 4 hours and 24 hours before obtaining cells to obtain CAR, α _ν β ₃ , or The change in expression of α _v β ₅ was observed. As shown in Table 1, it was confirmed that the expression level of α _ν β ₃ or α _ν β ₅ integrin did not change when lipofectamine or DMSO was administered in all cell lines used in the experiment. No significant change in expression level was observed.

MDA-MB-231 cell line Item Reaction time Badge alone Lipofectamine 2% DMSO 5% DMSO α _ν β ₃ 4 hr 6.20 6.45 6.45 6.24 24 hr 5.78 6.86 6.41 6.30 α _ν β ₅ 4 hr 5.24 5.68 6.13 6.23 24 hr 6.30 6.43 3.94 4.04 CAR 4 hr 18.52 20.36 17.73 17.29 24 hr 27.12 32.76 32.23 35.93

MDA-MB-435 cell line Item Reaction time Badge alone Lipofectamine 2% DMSO 5% DMSO α _ν β ₃ 4 hr 3.86 3.97 4.05 4.33 24 hr 4.87 5.08 4.89 5.18 α _ν β ₅ 4 hr 2.90 2.71 2.99 3.00 24 hr 2.39 2.48 2.38 2.58 CAR 4 hr 3.27 7.21 3.66 3.89 24 hr 3.20 5.69 3.43 4.95

A549 cell line Item Reaction time Badge alone Lipofectamine 2% DMSO 5% DMSO α _ν β ₃ 4 hr 6.35 7.60 6.74 6.4 24 hr 6.66 8.17 6.79 6.80 α _ν β ₅ 4 hr 19.30 18.88 18.19 17.31 24 hr 15.46 16.60 15.22 14.86 CAR 4 hr 22.70 25.33 23.88 26.06 24 hr 42.24 45.59 46.70 47.09

5. Verification of structural stability change of adenovirus by DMSO

Capsid, the structural protein of adenoviruses, regularly surrounds the DNA genome and consists of three major proteins: fiber, fentonbase, and hexon. In particular, a fiber that plays an important role in intracellular infection of adenovirus has a knob portion that binds to a CAR receptor expressed on the cell surface, and the knob is composed of a triple structure of two β-sheets. DMSO is a solvent used to induce protein denaturation and is known to induce denaturation of the internal keratin of human skin from the α-helix structure to the β-sheet structure. Based on this fact, it was verified whether DMSO induced degeneration of the structural protein of adenovirus. After adenovirus was dissolved in 20% or 40% DMSO at the final concentration and incubated at 0 ° C. for 6 hours, the expression level of LacZ was observed. As shown in Figure 8, the expression of LacZ in adenovirus dissolved in adenovirus storage buffer (10 mM Tris, 2 mM MgCl ₂ ) or virus dissolved in 20%, 40% DMSO was not significantly different. In all cases, the increase in LacZ expression was observed when DMSO was added during cell infection compared to the case where only adenovirus was infected. It was also observed that LacZ gene transfer efficiency increased when the virus was infected with the final concentration of DMSO at 2%, compared to when the virus was infected with the final concentration of DMSO at 1%.

In addition, DMSO has been reported to be similar to the role of chaperone protein as a chemical chaperone, preventing the aggregation of intracellular proteins and refolding mis-folded proteins. Against this background, dl / LacZ adenoviruses were stored in storage buffer or storage buffer with 20% DMSO to freeze at -80 ° C to determine whether they could effectively inhibit the accumulation of adenovirus. After standing at room temperature for 1, 3, 5 and 7 days, the degree of adenovirus accumulation was observed by LacZ analysis. As can be seen in Figure 9, the adenovirus preserved in the storage buffer was observed that the expression of LacZ is sharply inhibited, especially in the virus left for 24 hours at room temperature it can be observed that the expression of LacZ is greatly reduced. there was. However, in the case of the virus preserved in the storage buffer containing 20% DMSO, it was confirmed that the expression of LacZ was not inhibited at all compared to the control virus, and the expression of LacZ was maintained even when left at room temperature for 7 days. . These results confirm that DMSO inhibits adenovirus accumulation and increases the structural stability of adenovirus proteins.

6. Verification of the use of DMSO as a preservative of adenovirus

DMSO is a cryopreservation solution of various cells that functions to protect cells from excessive dehydration during freezing and to inhibit intracellular ice crystal formation. In order to examine the adenovirus's preservation ability as it is excellent as a cell cryopreservative solution, dl / LacZ adenovirus was added to a storage buffer containing 4% sucrose or 20% DMSO, and then rapidly frozen at -80 ° C. After 30 minutes and 24 hours of thawing, LacZ expression was observed. As can be seen in FIG. 10, the activity of the preserved adenovirus in the storage buffer containing 20% DMSO was not decreased at all compared to the adenovirus stored in the storage buffer containing 4% sucrose used as a positive control. 1483 LacZ expression was observed in head and neck cancer cells.

In addition, to determine the long-term preservation of adenovirus by DMSO, adenovirus was added at a titer of 3.21 × 10 ¹² VP in storage buffer containing sucrose or storage buffer containing 20% DMSO. Adenoviruses were stored at −80 ° C. for months, 4 months, 5 months, and 6 months, followed by freeze thawing and LacZ analysis. As can be seen in FIG. 11, it was observed that the adenoviruses stored in the storage buffer containing 20% DMSO showed a similar or slightly increased expression of LacZ compared to the adenoviruses stored in the storage buffer.

Based on the above results, DMSO can be used for long-term preservation of adenovirus, and it can be confirmed that there is an advantage that can increase gene delivery efficiency more than the current 4% sucrose preservative.

7. Verification of cell type change by DMSO

In order to precisely observe the morphological changes of the cells by DMSO treatment, the change of the cell surface was observed using a scanning electron microscope (SEM). The H460 human lung cancer cell line was dosed with dl / LacZ adenovirus in combination with lipofectamine or 2% DMSO at a titer of 50 MOI and observed for 5 minutes or 1 hour using a scanning electron microscope. As shown in FIG. 12A, when only dl / LacZ adenovirus was administered alone, the cell surface or morphology of the H460 cells without adenovirus treatment was similar, and the cell surface or morphology was hardly changed by virus infection. Could. On the other hand, in the case of lipofectamine-treated cells, there were many changes in the cell surface, and in particular, most of the cell surface proteins observed in the negative control group were removed. In addition, it was also observed that holes were drilled on the cell surface. However, when 2% DMSO was treated in the cells, proteins adhered to the cell surface were also observed, and no holes were punctured in the cell membrane as in the case of lipofectamine. In addition, observation of the morphological changes of these cells by transmission electron microscopy (TEM) revealed that the cell membranes remained intact in the case of H460 cells not treated with adenovirus, a negative control, and cells treated with adenovirus alone. On the other hand, the cell membranes co-administered with lipofectamine were observed to be damaged and no part of the cell membranes were falling off (FIG. 12B). On the contrary, when 2% DMSO was coadministered with adenovirus, cell membrane damage was not observed, and these results were confirmed to be in agreement with the results of scanning electron microscopy (SEM).

8. Verification of in vivo gene delivery enhancement effect by DMSO using tumor model

EF43-fgf mouse breast cancer model was used to observe whether the adenovirus and DMSO co-administration increased gene transfer efficiency. When EF43-fgf tumors were formed in about 6 weeks old male BALB / C mice and grown to about 100-120 mm ³ , dl / LacZ virus alone, with lipofectamine or 5% DMSO, was administered every other day. Three doses were combined and mouse tumors were extracted 3 days after the last virus administration and subjected to X-gal staining. As shown in FIG. 13, it was observed that the amount of LacZ expression increased when co-administered with lipofectamine or 5% of DMSO compared to the case of virus alone, especially when co-administered with 5% DMSO In the case, LacZ expression was induced in a wider tumor tissue, and gene delivery efficiency of adenovirus was significantly increased than that of co-administration with lipofectamine.

In addition, the antitumor effect of tumor selective killing adenovirus could be increased by increasing gene transfer efficiency of adenovirus following DMSO co-administration. Adenovirus, a replicable adenovirus, Ad-ΔB7 (1 × 10 ⁸ PFU), alone or in combination with lipofectamine or 5% DMSO, was administered three times every two days to increase the gene transfer efficiency of adenoviruses. It was observed whether the antitumor effect was increased. As can be seen in Figure 14a, when the negative control of either lipofectamine or DMSO alone, tumors grew rapidly, and at 27 days, respectively, 2001.66㎣, 2080.55㎣, and about 1078.28㎣ when the virus alone was administered. The tumor grew to a degree. In contrast, when DMSO and virus were administered in combination, the tumor size was approximately 574.86 mm 3, indicating that tumor growth was significantly inhibited. However, when co-administered with lipofectamine and the virus, the tumor growth was significantly increased, compared with the virus alone, and the tumor size was increased to 1903.03㎣. H & E staining was performed to more closely observe changes in the tumor tissue. As shown in FIG. 14B, when Ad-ΔB7 adenovirus was administered alone, the tumor was actively growing, and tumor tissues administered with 5% DMSO and adenovirus were markedly marked in tumor size. Not only did they decrease, but a lot of cell necrosis occurred in the tumor. However, when co-administered with lipofectamine and virus, not only tumor size was reduced but also tumor growth was actively observed that cancer cells were actively proliferating in tumor tissues.

Having described the specific part of the present invention in detail, it is apparent to those skilled in the art that the specific technology is merely a preferred embodiment, and the scope of the present invention is not limited thereto. Thus, the substantial scope of the present invention will be defined by the appended claims and equivalents thereof.

Figure 1a is a photograph showing the results of X-gal staining to investigate the gene transfer efficiency of the combination of DMSO (dimethyl sulfoxide) and adenovirus in FaDu, 1YB and QLL1 head and neck cancer cell lines. "Ad" represents dl-Lac Z adenovirus.

Figure 1b is X-gal investigating the gene transfer efficiency of the combination of DMSO and adenovirus in the head and neck cancer cell line CAL27, skin cancer cell line A431, breast cancer cell lines MDA-MB-435 and MCF-7, and normal fibroblasts CBHEL and IMR90 The photo shows the staining result.

Figure 2a is a photograph showing the results of X-gal staining to investigate the gene transfer efficiency of the combination of DMSO and adenovirus in brain cancer cell lines U87MG, U251N and U343.

Figure 2b is a photograph showing the X-gal staining results of the gene delivery efficiency of the combination of DMSO and adenovirus in lung cancer cell line H1299, prostate cancer cell line DU145, breast cancer cell line MDA-MB-231, and liver cancer cell lines Huh7 and Hep1 .

Figure 2c is a photograph showing the results of X-gal staining to investigate the gene transfer efficiency according to the co-administration of DMSO and adenovirus in lung cancer cell lines H460 and A549.

Figure 3 is a mouse cell line melanoma cell line (B16F1, B16F10), lung cancer cell line (CMT-64), rectal cancer cell line (CMT-93) and breast cancer cell line (EF43-fgf), hamster ovary cell line (Lec2) and rat cell line (YPEN) ) Shows the results of X-gal staining to investigate the gene transfer efficiency of DMSO and adenovirus in combination.

Figure 4 shows the results of cell lesion analysis to confirm the increase in cell killing ability according to the co-administration of DMSO and adenovirus. Human cancer cell lines MCF-7, SJSA, HT1080, FADU, A431 and U20S, human normal fibroblasts CBHEL and IMR90 were used.

Figure 5 shows the results of cell lesion analysis to confirm the increase in cell killing ability according to the co-administration of DMSO and adenovirus in a cell line with a relatively high expression of CAR.

Figure 6 shows the results of cell lesion analysis to confirm the increase in cell killing ability according to the combination of DMSO and adenovirus in mouse and rat cell lines.

Figure 7 is a graph showing the increase in production of Ad-ΔB7 adenovirus following the co-administration of DMSO and adenovirus in human cancer cell lines. V.O, administration of adenovirus alone; Combination administration of Lipo, lipofectamine and adenovirus; 1% DM: Combination of 1% DMSO with Adenovirus.

8 is an X-gal staining result of analyzing the effect on the structural stability of adenovirus by DMSO.

Figure 9a is the result of X-gal staining analyzing whether the aggregation (aggregation) of adenovirus is inhibited by DMSO. CNTL, dl / LacZ adenovirus was used as a control. The storage buffer consisted of 10 mM Tris, pH 8.0, 2 mM MgCl ₂ and 4% sucrose.

Figure 9b is the result of X-gal staining analyzing whether the accumulation of adenovirus is inhibited by DMSO.

10 shows X-gal staining results of short-term preservation of adenovirus by DMSO.

11A and 11B show X-gal staining results for analyzing long-term preservation of adenovirus by DMSO.

12a is a scanning electron microscope (SEM) photograph of morphological changes following DMSO treatment.

12b is a transmission electron microscope (TEM) image of morphological changes observed with DMSO treatment.

Figure 13 is a photograph of the result of H & E staining (hemotoxyline-eosine staining) for the tissue of the mouse transplanted (xenotransplantation) EF43-fgf breast cancer cell line. When treated with 5% DMSO, it can be seen that the efficiency of gene transfer in vivo is increased.

14A and 14B are graphs showing an increase in antitumor efficacy when 5% DMSO and adenovirus were co-administered in mice transplanted with FaDu head and neck cancer cell lines (xenotransplantation). 14B is a photograph of H & E staining result of tumor tissue.

Claims (10)

A composition for improving gene transfer efficiency of an animal carrier comprising an aprotic polar solvent as an active ingredient. (a) gene carriers; And (b) an aprotic polar solvent. The aprotic polar solvent according to claim 1 or 2, wherein the aprotic polar solvent is dimethyl sulfoxide (DMSO), dimethyl formamide (DMF), N-methylpyrrolidone, N-methylmorpholine, γ-butyrolactone, acetonitrile. , Tetrahydrofuran or hexamethylphosphoric triamide. 4. The composition of claim 3, wherein said aprotic polar solvent is DMSO. The method of claim 1 or 2, wherein the gene carrier is a plasmid, recombinant adenovirus, Adeno-associated viruses (AVA), retrovirus, lentivirus, herpes simplex virus, basinia virus, liposomes, or Niosomes, characterized in that the composition. (a) a therapeutically effective amount of adenovirus; (b) aprotic polar solvents; And (c) a pharmaceutically acceptable carrier. A virus preservation composition having a capsid protein comprising an aprotic polar solvent as an active ingredient. The method of claim 6 or 7, wherein the aprotic polar solvent is dimethyl sulfoxide (DMSO), dimethyl formamide (DMF), N-methylpyrrolidone, N-methylmorpholine, γ-butyrolactone, acetonitrile , Tetrahydrofuran or hexamethylphosphoric triamide. 9. The composition of claim 8, wherein said aprotic polar solvent is DMSO. The method of claim 7, wherein the virus surrounded by the capsid protein is adenovirus, Adeno-associated viruses (AVA), retrovirus, lentivirus, herpes simplex virus or basinian virus Composition.

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