KR20090092536A - Composition comprising recombinant adenovirus and liposome with enhanced gene transfer - Google Patents

Abstract

A composition for improving gene transfer containing a recombinant adenovirus and DOTAP (1,2-dioleoyl-3-trimethylammonium propane) is provided to increase the gene transfer efficiency of recombinant adenovirus which expresses interleukin-12 mutant and have the excellent anticancer effect. A composition for enhancing gene transfer comprises a recombinant adenovirus and DOTAP(1,2-dioleoyl-3-trimethylammonium propane). The recombinant adenovirus expresses cytokine, chemokine, antigen, tumor suppressor gene, suicide gene, anti-angiogenic factor, prodrug activating gene or immunostimulatory gene.

Description

Composition comprising recombinant adenovirus and liposome with enhanced gene transfer}

The present invention relates to a composition for enhancing gene delivery comprising recombinant adenovirus and DOTAP (1,2-dioleoyl-3-trimethylammonium propane) and to a method for enhancing gene delivery efficiency of adenovirus using the composition. In particular, the composition of the present invention has an excellent effect to enhance the gene transfer efficiency in cancer cells of recombinant adenovirus expressing the interleukin 12 variant gene, thereby exhibiting a high anticancer effect. Thus, the present invention also relates to a composition for anticancer comprising a recombinant adenovirus expressing a human interleukin 12 variant gene and DOTAP.

Gene therapy is a new way to treat a variety of diseases, such as disease or cancer, and the basic concept of gene therapy is to substitute a functioning gene for a gene that is mutated and malfunctions. Treating damaged cells by expressing a treatable gene in the target cell. At this time, the most essential condition for gene therapy is the development of a system that delivers genes accurately to target cells with high efficiency.

Such a gene carrier which is widely used for gene delivery and gene therapy is adenovirus (adenovirus). This is because adenovirus is easily infected with various kinds of cells regardless of whether the cells are divided or not, and thus can efficiently transfer desired genes, easily produce high titer virus, and can be stored by freeze drying. (I. Kovesdi, Curr Opin Biotechnol, 8 (5): 583-589, 1997). The size of the transfer gene is also as large as 8kb. In addition, unlike the retrovirus that binds to the nucleus of the target cell, since it exists outside the nucleus, it has excellent advantages in terms of safety.

Adenovirus is infected through the COxsackievirus and adenovirus receptor (CAR), the receptor for the target cell. In general, most cells express enough CAR, whereas immune cells such as muscle cells, dendritic cells, melanoma and Some of the cancer cells, such as rarely express the CAR, there is a limit to the gene transfer to these cells using adenovirus. In addition, most humans are already infected with adenovirus, and since the antibody (neutralizing Ab) is formed, the transmission effect is low. Therefore, overcoming these problems is essential for gene therapy using adenovirus vectors.

Various approaches have been studied to overcome the limitations of gene delivery of adenoviruses that depend on such CAR receptors.

First, there is a method to improve the gene transfer efficiency by processing high titer virus. However, when a large amount of adenovirus is used, a problem may occur that causes the toxicity of the adenovirus itself or the virus is lost due to a strong immune response of the host. Second, there is a method of increasing the type of cells that adenovirus can infect by modifying the fiber portion that binds to the cell receptor when the adenovirus is infected. For example, a study that complements the drawbacks of CAR-dependent adenoviruses by introducing RGD motifs targeting integrins, known as the second receptors of adenoviruses, at the carboxyl terminal sites or HI loops of adenovirus fibers. (Wickham, TJJ Virol., 71: 8221-8229, 1997; Kransnykh, V. et al. J. Virol., 72: 1884-1852, 1998). However, these methods also did not provide a significant effect on improving low delivery efficiency in vivo delivery, and efforts to develop a method for improving delivery efficiency continue.

Under these circumstances, the present inventors have made diligent efforts to develop a method for improving intracellular delivery efficiency of adenoviruses for use in gene therapy. As a result, the present inventors have improved the delivery efficiency of adenovirus vectors using DOTAP, a type of liposome. The present invention was completed by confirming the improvement.

One object of the present invention is to provide a composition for enhancing gene delivery comprising recombinant adenovirus and DOTAP.

Another object of the present invention is to provide a method of enhancing the efficiency of intracellular gene transfer of adenovirus by treating the recombinant adenovirus and DOTAP together.

Another object of the present invention is to provide an anticancer composition comprising a recombinant adenovirus and DOTAP expressing a mouse or human interleukin-12 variant gene.

In order to achieve the above object, as one aspect the present invention relates to a composition for enhancing gene delivery comprising recombinant adenovirus and DOTAP.

As used herein, the term “recombinant adenovirus” refers to any adenovirus vector comprising exogenous DNA inserted into its genome encoding a polypeptide. Adenoviruses used in the recombinant adenoviruses included in the compositions of the present invention may be of various types, such as Type 1, Type 2, Type 3, Type 4, Type 5, and the like.

In addition, it includes both recombinant adenovirus replicable and non-replicable in the composition of the present invention. Adenoviruses are known that the E1A gene is essential for replication, and thus the replicable recombinant adenoviruses used in the recombinant adenoviruses included in the compositions of the present invention include the E1A gene, whereas the non-replicating recombinant adenoviruses The E1A gene is mutated so that it cannot be deleted or replicated. The replicable recombinant adenoviruses included in the compositions of the present invention can kill tumor cells with high efficiency by introducing them into tumor cells at high transduction rates and then replicating them. Expression of the therapeutic gene after introduction into the target cell at the transduction rate can treat various diseases including tumors with improved efficacy.

In the present invention, the exogenous DNA inserted into the recombinant adenovirus and delivered into the cell is not limited, but the expression of the CAR receptor is low so that the expression of the recombinant adenovirus is low (eg, cancer tissue) using the composition of the present invention. For the purpose of the present invention to enhance gene transfer efficiency, preferably cytokines, chemokines, antigens, tumor suppressor genes, suicide genes, antiangiogenic factors Genes related to cancer gene therapy, such as anti-angiogenic factor, prodrug activating gene and immunostimulatory gene, are preferred. Most preferably the interleukin 12 or interleukin 12 variant genes are preferred.

In a specific embodiment of the present invention, a recombinant adenovirus expressing IL-12N220L, an interleukin-12 (IL-12) variant gene, was used. IL-12N220L was originally developed by the present inventors and is a gene mutated Asn-220 amino acid, which is the N-glycosylation portion of the murine IL-12p40 subunit. Expression of this gene is known to increase the expression of active IL-12p70 and decrease the secretion of IL-12p40. Optimal cell mediation when immunized with HCV E2 gene in small animal model mice Immune Responses In particular, it has been reported that such immune responses are continuously induced after a long period of time (National Patent No. 10-0399728; Ha et al., Nat. Biotechnol. 20: 381-6, 2002. And the contents of the paper are incorporated herein by reference).

IL-12 is a heteropolymer of p70 consisting of two covalently bonded subunits, p35 and p40. Thus, the IL-12 variant gene used in the present invention specifically comprises (1) the mouse IL-12p35 subunit gene and (2) the codon encoding Asn-220 of the mouse IL-12p40 subunit gene. Gene construct consisting of an IL-12p40 subunit gene substituted with a codon encoding (-220).

In addition, the inventors of the present invention have previously conducted a study (National Patent No. 10-0399728; Ha et al., Nat. Biotechnol. 20: 381-6, 2002) to confirm that Asn-220 of the mouse IL-12p40 subunit is leucine ( Leu-220) as well as non-glycosylated amino acids, such as non-polarized or branched non-polar side-chain amino acid has been demonstrated to have the same immunopotentiating effect, IL-12 variant gene of the present invention Codons encoding Asn-220 of (1) the mouse IL-12p35 subunit gene and (2) the mouse IL-12p40 subunit gene are leucine (Leu-220), glutamine (Gln-220) or isoleucine (Ile) And a IL-12 variant gene, which is a gene construct consisting of an IL-12p40 subunit gene substituted with a codon encoding -220). In addition, the IL-12 variant gene preferably comprises an internal ribosome entry site (IRES) between the subunit genes (1) and (2).

In addition, the amino acid sequence of the mouse and human IL-12p40 shows a very high homology. In particular, Asn-220 of human IL-12p40 corresponding to Asn-220 of mouse IL-12p40 is located in amino acid sequence 222, and the surrounding amino acid sequence is It is known to be very similar. In addition, the inventors have conducted, through prior studies, codons encoding Asn-222 of (1) the human IL-12p35 subunit gene and (2) the human IL-12p40 subunit gene, leucine (Leu-222) and glutamine (Gln). -222) or IL-12 variant gene, a gene construct consisting of an IL-12p40 subunit gene substituted with a codon encoding isoleucine (Ile-222), has similar immunopotentiating effects to IL-12 variant genes in mice. Proved to have. Therefore, the present invention may also include the IL-12 variant gene of such a human. In addition, the IL-12 variant gene preferably comprises an internal ribosome entry site (IRES) between the subunit genes (1) and (2).

The present invention also includes DOTAP, which is one of the cationic liposomes to enhance the intracellular gene transfer efficiency of recombinant adenoviruses.

As used herein, the term "liposome" refers to a closed structure in which an external lipid surrounding an internal water-soluble space forms a multilayer, and cationic liposomes and anionic liposomes, depending on their ionicity. In the present invention, when using DOTAP, one of the cationic liposomes, the gene delivery efficiency of the recombinant adenovirus was found to be particularly excellent.

As used herein, the term “DOTAP” refers to a transfection lipid comprising one monovalent cationic trimethylammonium head group and two unsaturated hydrocarbon chains as one of the cationic liposomes. )to be. DOTAP is itself a nonviral vector, known as a gene carrier that complexes with plasmid DNA and delivers DNA into cells. However, in the present invention, DOTAP is used as a kind of gene therapy aid, which enhances the gene transfer efficiency of the recombinant adenovirus, which is a gene carrier.

The composition of the present invention comprising recombinant adenovirus and DOTAP in which exogenous DNA is inserted can be used for the treatment of various genetic diseases depending on the type of exogenous DNA.

Introduction of foreign nucleic acids with transporters at in vitro or in vivo can cure several defects in cells due to lack of expression or overexpression of genes, or to modify cellular proteins and their expression. Can be. The delivery of nucleic acids to cells with the adenovirus and DOTAP provided herein can be used to treat animals suffering from diseases or disorders resulting from abnormal or rarely present, or inappropriately low or high protein expression. Such diseases and disorders include, but are not limited to, various cancers and genetic defect diseases.

In the present invention, the following method demonstrated the superiority of the composition of the present invention.

Specifically, in the embodiment of the present invention, the improvement of the delivery efficiency of the adenovirus vector using DOTAP was tested in in vitro cancer cells. When adenovirus vectors expressing the fluorescent labeling factor GFP (Green Fluorescent Protein) were treated with DOTAP, it was confirmed that improved gene transfer efficiency was obtained when adenovirus alone was treated (FIGS. 1, 2, 3 and 4).

In addition, in the present invention, the improvement of the delivery efficiency of adenovirus vectors was tested in cancer tissues using DOTAP. Adenovirus vectors expressing IL-12N220L, which show anticancer effects, were injected into rat cancer tissues, such as DOTAP, and showed improved gene transfer efficiency compared to treatment with adenovirus alone in serum and cancer tissues after 24 or 48 hours. Could be (FIGS. 5 and 6).

In addition, in the present invention, it was tested whether the anticancer effect was enhanced by improving the delivery efficiency of adenovirus vectors using DOTAP. Treatment of cancer cells in vitro with an adenovirus vector expressing IL-12N220L, which exhibits anti-cancer effects, in an optimal concentration of DOTAP, followed by subcutaneous injection of rats, resulted in less severe cancer progression than adenovirus treatment alone. Improved survival was confirmed (FIG. 7 and FIG. 8).

In the present invention, the mechanism by which DOTAP enhances the gene transfer efficiency of recombinant adenovirus is not clearly known. However, the cationic DOTAP reduces the repulsive force between the adenovirus and the cell membrane negative, and serves as a bridge between the two. It is thought that this will increase the infection rate of the virus.

As another aspect, the present invention relates to a composition for anticancer comprising a recombinant adenovirus expressing the interleukin 12 variant gene and DOTAP. Specifically, the IL-12 variant gene is (1) the mouse or human IL-12p35 subunit gene and (2) the Asn- of the codon or human IL-12p40 subunit encoding Asn-220 of the mouse IL-12p40 subunit. The codon encoding 222 is a gene construct consisting of an IL-12p40 subunit gene substituted with leucine, glutamine or isoleucine.

As described above, in the present invention, the composition of the present invention has a particularly excellent effect in enhancing the gene transfer efficiency in cancer cells of recombinant adenovirus expressing the interleukin 12 variant gene, thereby demonstrating high anticancer effect. .

The anticancer composition of the present invention may further comprise a pharmaceutically usable carrier.

As used herein, the term " pharmaceutically acceptable carrier " refers to a medium that is generally available by participating in the administration of adenoviruses and liposomes to animals, including humans. Pharmaceutically available carriers can be prepared according to a variety of factors well known to those skilled in the art, including, for example, the particular bioactive agent used, its concentration, stability and intended bioavailability, diseases and disorders to be treated with adenoviruses. Or factors such as, but not limited to, conditions, subjects to be treated, age, size and general condition, routes used to administer the composition, such as nasal, oral, ocular, topical, transdermal and muscular conditions. Pharmaceutically available carriers which are generally used for the administration of physiologically active substances other than the oral route of administration include aqueous solutions comprising D5W, dextrose and physiological salts within 5% of the volume. Pharmaceutically available carriers may also include additional ingredients that can enhance the stability of the active ingredients such as preservatives and antioxidants.

The route of administration of the pharmaceutical composition of the present invention may be any suitable route of administration. In addition, the dosage of the pharmaceutical composition of the present invention is administered at a dose effective for the intended treatment. The therapeutically effective amount required to treat or inhibit the progress of a particular medical disease can be readily determined by one skilled in the art using preclinical and clinical studies known in the medical arts. As used herein, the term “therapeutically effective amount” refers to the amount of active ingredient that elicits a biological or medical response in a particular tissue, system, and animal or human, as desired by a clinician or investigator.

As another aspect, the present invention relates to a method for enhancing the efficiency of intracellular gene transfer of adenovirus by treating the recombinant adenovirus and DOTAP together.

As described above, the delivery efficiency of the adenovirus vector can be greatly improved through the method using the DOTAP of the present invention. In addition, the gene delivery method of the present invention can be sufficiently extended to be applied to genes in general as well as culture cells, that is, gene transfer to animal cells, animal tissues and animals.

As described above, the composition comprising the recombinant adenovirus and DOTAP of the present invention can deliver the desired substance, ie, nucleic acid, better to various animal cells than the conventional adenovirus, and also significantly reduce the cytotoxicity, so that gene therapy It can be usefully used for effectively transporting a gene of interest to a cell.

1 is a graph showing the improvement of adenovirus vector delivery efficiency by various concentrations of DOTAP in mouse melanoma cells B16F10.

2 is a graph showing the improvement of adenovirus vector delivery efficiency by various concentrations of DOTAP in human melanoma cells A375.

3 is a cell diagram showing fluorescence showing improved gene transfer efficiency of adenovirus vectors containing fluorescent genes when optimal concentrations of DOTAP were used in B16F10, a mouse melanoma cell, and A375, a human melanoma cell.

4 is a graph showing the improvement of gene transfer efficiency according to the amount of adenovirus vector when the optimal concentration of DOTAP is used in mouse melanoma cells B16F10.

FIG. 5 shows that adenovirus vectors alone are administered in 24 hours and 48 hours after administration of adenovirus vectors expressing genes expressing DOTAP and anti-cancer effects into cancer tissues formed by subcutaneous injection of melanoma cells B16F10. It is a graph compared with the hour.

FIG. 6 shows adenoviruses for improving gene transfer in cancer tissues 24 and 48 hours after administration of adenovirus vectors expressing DOTAP and genes exhibiting anticancer effects into cancer tissues formed by subcutaneous injection of mouse melanoma cells B16F10. It is a graph compared with the vector administration alone.

FIG. 7 shows in vitro treatment of mouse melanoma B16F10 cells with a mixture of adenovirus vectors expressing IL-12N220L and DOTAP for 6 hours after anti-cancer effect, and then injected these cells subcutaneously into mice 24 hours later. It is a graph showing the expression level of the gene IL-12N220L introduced in the serum.

FIG. 8 shows in vitro treatment of mouse melanoma B16F10 cells with a mixture of adenovirus vectors expressing IL-12N220L and DOTAP which exhibit anticancer effects, followed by injection of these cells subcutaneously into rats. It is a graph showing the progress and survival rate of the rat.

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, and the scope of the present invention is not limited by these examples in accordance with the gist of the present invention to those skilled in the art. Will be self explanatory.

Example  One: DOTAP Using Adenovirus  In vitro Delivery Efficiency Enhancement Effect of Vector

To test whether DOTAP can improve the delivery efficiency of adenovirus vectors in vitro, rAd / EGFP, an adenovirus vector expressing the fluorescent labeling factor, Green Fluorescent Protein (GFP), was used. First, rAd / EGFP was mixed with 5, 10, 20, and 40 µg / ml of DOTAP, and left at room temperature for 30 minutes, and then infected with mouse melanoma cells B16F10 and human melanoma cells A375 for 2 hours. At this time, the experiment was independently performed with rAd.EGFO without DOTAP as a control, and the amount of adenovirus used for infection was 200 MOI (multiplicity of infection) for B16F10 and 10 MOI for A375. After 2 hours, the virus mixture was removed, changed into culture medium, incubated in a CO2 incubator for 2 days, and then infected cells were removed with trypsin to measure intracellular GFP levels by flow cytometry.

As a result, GFP gene transfer efficiency improved in proportion to the DOTAP concentration. In particular, it was confirmed that the gene transfer effect of B16F10 was increased by 5 times and A375 by 3.5 times as compared with the adenovirus treatment alone at 10 ㎍ / ㎖ showing the optimum improvement effect (Fig. 1 to 3). In addition, when the amount of rAd / EGFP at 10 ~ 500 MOI at the optimal DOTAP concentration (10 ㎍ / ㎖), it was confirmed that the gene transfer efficiency was significantly improved in all amounts of adenovirus (Fig. 4).

Example  2: DOTAP Using Adenovirus  Effect of Vector Increasing Efficiency in Cancer Cells

To test whether DOTAP can improve the delivery efficiency of adenovirus vectors in vivo, rAd / IL-12N220L, an adenovirus vector expressing the immunomodulatory substance IL-12N220L, was used. First, 3 × 10 ⁸ pfu (plaque forming unit) rAd / IL-12N220L was mixed with 5, 10 and 20 μg / ml of DOTAP, left at room temperature for 30 minutes, and then B16F10 cancer solidified subcutaneously at 5-10 mm. Serum was taken 24 and 48 hours after injection into the tissue, and cancer tissue was collected 48 hours later and the amount of IL-12 expressed in them was quantified by IL-12 p70 ELISA (Enzyme-Linked ImmunoSorbent Assay) method.

As a result, it was confirmed that IL-12 expression was increased in serum and cancer tissue in proportion to the amount of DOTAP used. In particular, when the optimal concentration of 10 ㎍ / ㎖ DOTAP was used, it was confirmed that the gene delivery effect increased by 2 times in serum after 24 hours, 5 times in cancer tissue after 48 hours (Fig. 5 and 6).

Example  3: DOTAP Using Adenovirus  Promote anticancer effect of vector

The improved delivery of DOTAP's superior adenovirus vectors identified in vitro and in cancer tissues was tested to improve the anticancer effects of gene therapy. To this end, rAd / IL-12N220L, an adenovirus vector expressing IL-12N220L, an anti-tumor effect previously reported, was used. At this time, the experiment was independently performed with rAd / mock having only adenovirus vectors without a transfer gene as a control. First, 100 MOI of rAd / IL-12N220L was mixed with 10 μg / ml of DOTAP, left at room temperature for 30 minutes, and then infected with a mouse melanoma cell B16F10 cell line for 6 hours. Infected cells were removed with trypsin and injected subcutaneously in mice, and 24 hours later, the expression level of IL-12 in serum was measured by ELISA.

As a result, no IL-12 was found in the control group treated with rAd / mock, and 40 times more IL-12 was detected in the serum group treated with DOTAP compared to the group treated with adenovirus vectors alone. (FIG. 7). In addition, treatment with DOTAP was able to significantly reduce the cancer progression, and the survival rate on day 56 was also 42.8%, which was significantly improved than 21.4% of the group treated with adenovirus alone (FIG. 8).

Claims (11)

A composition for enhancing gene delivery, comprising recombinant adenovirus and DOTAP (1,2-dioleoyl-3-trimethylammonium propane). The method of claim 1, wherein the recombinant adenovirus is cytokine (cytokine), chemokine (chemokine), antigens, tumor suppressor gene (suicide gene), suicide gene (anti-angiogenic factor) And a recombinant adenovirus expressing a therapeutic gene selected from the group consisting of prodrug activating genes and immunostimulatory genes. 3. The composition of claim 2, wherein said cytokine is an interleukin-12 (IL-12) or interleukin-12 variant gene. The codon according to claim 3, wherein the IL-12 variant gene comprises (1) a mouse IL-12p35 subunit gene and (2) a codon leucine (Leu-220) encoding Asn-220 of a mouse IL-12p40 subunit gene, A composition that is a gene construct consisting of an IL-12p40 subunit gene substituted with a codon encoding glutamine (Gln-220) or isoleucine (Ile-220). 5. The composition of claim 4, wherein said IL-12 variant gene is a gene construct comprising an internal ribosome entry site (IRS) between subunit genes (1) and (2). The codon according to claim 3, wherein the IL-12 variant gene comprises (1) a human IL-12p35 subunit gene and (2) a codon leucine (Leu-222) encoding Asn-222 of a human IL-12p40 subunit gene, A composition that is a gene construct consisting of an IL-12p40 subunit gene substituted with a codon encoding glutamine (Gln-222) or isoleucine (Ile-222). 7. The composition of claim 6, wherein said IL-12 variant gene is a gene construct comprising an internal ribosome entry site (IRS) between subunit genes (1) and (2). A method for treating intracellular gene transfer efficiency of recombinant adenovirus by treating the recombinant adenovirus and DOTAP together. (1) a mouse or human IL-12p35 subunit gene and (2) a codon encoding Asn-220 of a mouse IL-12p40 subunit or a codon encoding Asn-222 of a human IL-12p40 subunit leucine, An anticancer composition comprising a recombinant adenovirus expressing an IL-12 variant gene consisting of an IL-12p40 subunit gene substituted with glutamine or isoleucine, and DOTAP. 10. The composition of claim 9, wherein said IL-12 variant gene is a gene construct comprising an internal ribosome entry site (IRS) between subunit genes (1) and (2). 10. The composition of claim 9, further comprising the pharmaceutically acceptable carrier.