Cell sheets for gene transfer

Abstract

The present inventors have completed the present invention by developing cell sheets as a local gene delivery platform to solve the problems associated with systemic administration of gene delivery systems. [Solution] The present invention relates to a cell sheet for gene delivery. Unlike cell sheets used in existing tissue regeneration applications, the cell sheet of the present invention can be used as a local gene delivery system. In particular, when a virus is used as a gene delivery system, the virus can be propagated within the cell sheet, allowing the virus to act locally at the treatment site. This makes the cell sheet excellent for preventing or treating cancer, preventing cancer recurrence or metastasis, and particularly for treating multifocal tumors, even with a significantly reduced viral dose compared to systemic or intratumoral administration of the virus.

Description

[Technical field]

[0001] The present invention relates to a cell sheet for gene transfer. [Background technology]

[0002] Despite the rapid development of cancer therapeutics, cancer remains one of the diseases with a high mortality rate worldwide. The main cancer treatment methods currently used clinically are surgery, radiation therapy, anticancer drug therapy, or a combination of these, which removes as many cancer cells as possible from the patient. However, these treatments have the problem that they are only effective in relatively early cancers when the cancer cells are completely removed without metastasis.

[0003] In particular, hepatocellular carcinoma (HCC) is known to be the fifth most common cancer in the world and the third most common cause of cancer-related deaths. Chronic liver diseases such as hepatitis B and hepatitis C virus infections and resulting liver cirrhosis account for 80-90% of all liver cancer cases. One of the core characteristics of HCC is that it can develop multifocal tumor lesions (multifocal / multinucleated / intracerebrovascular) simultaneously, and this multifocal hepatocyte contributes to high recurrence rates, drug-related characteristics, and morbidity. Importantly, long-term cohort studies have shown a strong amount of correlation between liver cirrhosis and multifocal / multifocal HCC. Chronic viral infection is closely associated with repeated hepatocyte necrosis, followed by regeneration. Such accelerated cell cycles may be associated with the accumulation of genetic errors in the liver that cause tumors in many liver sites, and these patients can be classified as multifocal HCC.

[0004] The current standard treatment for HCC includes selective internal radiation therapy and systemic therapy with chemotherapy agents. However, these treatment modalities lack cancer specificity and are highly toxic to the liver, which causes serious problems for many HCC patients who show liver dysfunction, making it difficult to administer sufficient amounts of drugs to eradicate the tumor. For the reasons mentioned above, surgical resection remains the preferred treatment, but for a variety of reasons such as multiple sclerosis and cirrhosis, most patients with hepatocellular carcinoma (<70%) cannot undergo resection [El-Serag,HB,et al.,Diagnosis and treatment of hepatocellular carcinoma.Gastroenterology,2008.134(6):p.1752-63;Belghiti,J.and R.Kianmanesh,Surgical treatment of hepatocellular carcinoma.HPB(Oxford),2005.7(1):p.42-9;Ziser,A.,et al.,Morbidity and mortality in cirrhotic patients undergoing anesthesia and surgery.Anesthesiology,1999.90(1):p.42-53].

[0005] Even with curative resection, the high recurrence rate of patients remains a significant challenge in the treatment of hepatocellular carcinoma.

[0006] Tumor-killing therapies that have demonstrated potent and cancer-specific cell-killing efficacy in various clinical trials may be promising candidates to overcome the targeted cell toxicity associated with small molecule chemotherapy.

[0007] Among the various oncolytic vectors, adenoviruses (Ad) have several beneficial properties, such as easy production at high titers and high levels of transgene expression without the risk of inducing insertional mutagenesis, making them advantageous for oncogene therapy. Despite promising preclinical results, several obstacles, such as inadequate delivery and insufficient levels of therapeutic gene delivery or viral replication, must be overcome to elicit optimal antitumor efficacy in clinical trials. Importantly, safety issues and limited efficacy of systemically administered virions mean that intratumoral inoculation of virions, presented as the preferred route of administration in clinical trials, may not be possible in the case of multifocal tumors. Currently, there is a lack of effective and standardized protocols to simultaneously treat various tumors with oncolytic viruses. Although systemic administration could circumvent these limitations with different standard therapies, the strong immunogenicity of oncolytic viruses and the high pre-existing immunity of patients to Ad make such an approach impractical in the clinic.

[0008] To overcome the inherent limitations of standard treatment regimens and tumor-killing therapies for multifocal HCC, we investigated cell sheets as a promising candidate to improve the efficacy of tumor-killing Ad in multifocal HCC.

[0009] Traditionally, cell sheets have been used in tissue engineering primarily to replace or restore the function of damaged tissues. The main advantages of cell sheets are their biocompatibility, durable engraftment induction ability, and reduced risk of adverse inflammatory reactions compared to synthetic antibodies. However, no studies have been reported using such cell sheets for gene transfer. Summary of the Invention [Problem to be solved by the invention]

[0010] The present inventors have completed the present invention by developing a cell sheet as a local gene delivery platform to solve the problems associated with systemic administration of gene delivery systems. [Means for solving the problem]

[0011] The present invention relates to Contains two or more cell layers, one or more of the cell layers is introduced with a gene delivery system; A cell sheet is provided.

[0012] The present invention also provides a method for producing a method for manufacturing a semiconductor device comprising the steps of: A temperature-responsive culture dish containing a temperature-sensitive polymer is forming a cell sheet comprising two or more cell layers; introducing a gene delivery system into one or more of the cell layers; Separating the cell sheet from the temperature-responsive culture dish. The present invention provides a method for producing a cell sheet, comprising:

[0013] The present invention also provides a method for producing a method for manufacturing a semiconductor device comprising the steps of: A gene therapy agent comprising the cell sheet is provided.

[0014] The present invention also provides a method for producing a method for manufacturing a semiconductor device comprising the steps of: The present invention provides a method for preventing recurrence of cancer or a method for treating cancer, which comprises transplanting the cell sheet into a site of a cancer removal surgery or a site requiring cancer treatment. Effect of the Invention

[0015] The cell sheet according to the present invention can be used as a local gene delivery system, unlike cell sheets used in existing tissue regeneration applications. In particular, when a virus is used as a gene delivery system, the virus can be proliferated within the cell sheet and acts locally only at the treatment site, making it excellent for preventing or treating cancer, preventing cancer recurrence or metastasis, and particularly for treating multifocal tumors, even with a significantly reduced virus dosage compared to systemic or intratumoral administration of the virus. [Brief description of the drawings]

[0016] Figure 1 shows the appearance of oAd-DCN / CFCS isolated on a temperature-responsive culture dish (a), histological analysis of oAd-DCN / CFCS by hematoxylin and eosin staining (b), and immunostaining detection of Ad E1A protein in PBS-treated control CFCSs (c) and oAd-DCN / CFCSs (d) [Scale bars: 1 cm (a) and 2 μm (b-d)]. Figure 2 shows the virus production and therapeutic gene expression profiles of oAd-DCN / CFCS [(a) Virus production of oAd-DCN infected with CFCS. CFCSs were infected with naked oAd-DCN at an MOI of 0.5. CFCSs and supernatants were harvested at 4, 12, 24, 48, 72, and 96 h postinfection, and then viral genome copies were measured by real-time quantitative PCR. (b) DCN expression in oAd-DCN / CFCSs. Representative western blot of DCN using cell lysates and supernatants harvested 48 h postinfection with oAd-DCN at 1 MOI data shown as mean ± SD. *P<0.05, **P<0.01]. Figure 3 shows the degradation profile and evaluation of viral persistence of oAd-DCN / CFCSs in vivo after intrahepatic transplantation. (a) Degradation profile of cell sheet. CFCSs were made of fibroblasts and firefly luciferase-expressing cancer cells (CFCSs / Fluc). Subsequently, CFCSs / Fluc were infected with oAd-DCN and then transplanted into the left hepatic lobe of nude mice bearing orthotopic HCC tumors. (b) Evaluation of viral persistence after transplantation of oAd-DCN / CFCS. CFCSs were infected with firefly luciferase-expressing oAd-DCN (oAd-DCN / Fluc / CFCSs) and then transplanted onto the surface of the left hepatic lobe of tumor-bearing mice. Bioluminescence imaging was monitored daily after transplantation. Figure 4 shows the histological results of multifocal hepatocellular carcinoma (HCC) [cross-sections of multifocal hepatocellular carcinoma tissues were obtained 21 days after tumor cell injection in the PBS-treated group and stained with hematoxylin and eosin. Original magnification: ×40, white dotted line: tumor]. Figure 5 shows the strong antitumor efficacy of oAd-DCN / CFCS. Figure 5a shows the antitumor efficacy of oAd-DCN / CFCS in a Hep3B orthotopic tumor model [orthotopic liver tumors were 1 × 10 6 The tumor was established by injecting 1000 cells of Hep3B expressing firefly luciferase. Immediately after cell injection, the site of cell injection was either sprayed with 3 × 10 PBS or sprayed with 1000 μg of PBS. 8 VP was implanted into oAd-DCN (spray)-treated or PBS-treated CFCs or oAd-DCN / CFCS (n=6). Six mice were injected with Hep3B cells and then injected with 3 × 10 8 VP was injected systemically. Tumor growth was monitored 2, 5, 7, 14 and 21 days after treatment. Figure 5b shows the bioluminescence signals from HCC from each treatment group after background subtraction [data shown as mean ± standard deviation; *P<0.05]. Figure 6 shows the histological analysis of tumor tissues from mice treated with PBS, oAd-DCN intravascular injection, oAd-DCN intraperitoneal injection, CFCS alone, or oAd-DCN / CFCS [cross sections of treated multifocal hepatocellular carcinoma were obtained 14 days after treatment with PBS, oAd-DCN intravascular injection, oAd-DCN intraperitoneal injection, CFCS alone, or oAd-DCN / CFCS. Original magnification: ×50, black dotted line: tumor]. FIG. 7 illustrates the process of forming an oAd-DCN / CFCS. FIG. 8 shows the biological activity of the adenovirus replicated from the cell sheet loaded with the tumor-selective killing adenovirus. FIG. 9 shows the results of confirming whether or not tumors recurred after tumor removal surgery and then attaching a cell sheet carrying a tumor-selective killing adenovirus. FIG. 10 shows the formation of a cell sheet loaded with vaccinia virus. FIG. 11 shows the results of confirming the vaccinia virus replication ability of the cell sheet loaded with vaccinia virus. FIG. 12 shows the results of confirming vaccinia virus cell killing in a cell sheet loaded with vaccinia virus. FIG. 13 shows the results of confirming cell viability of cancer cells irradiated with radiation. FIG. 14 shows the results of confirming the adenovirus replicative ability in a cell sheet containing irradiated cancer cells. BEST MODE FOR CARRYING OUT THEINVENTION

[0017] The present invention relates to Contains two or more cell layers, one or more of the cell layers is introduced with a gene delivery system; A cell sheet is provided.

[0018] In the present invention, the cell sheet is used for local gene delivery, i.e., it serves as a kind of carrier that can locally deliver a gene delivery system to a body site where gene therapy is required.

[0019] The cell sheet preferably has mechanical properties suitable for handling by the user during the production and implantation processes.

[0020] For this purpose, the cell sheet includes one of the cell layers that acts as a support.

[0021] In one embodiment, the cell sheet comprises a cell layer comprising somatic cells as a support.

[0022] The cell layer containing somatic cells serves as a support for the cell layer into which the gene delivery system is introduced and can provide an environment similar to that in the body, allowing the attachment, proliferation, differentiation and culture of a variety of cells.

[0023] Any somatic cell suitable for performing such a function may be used, and the type of the somatic cell is not particularly limited. Specifically, the somatic cell may be one or more selected from the group consisting of fibroblasts, chondrocytes, epithelial cells, myoepithelial cells, dermal cells, epithelial keratinocytes, Schwann cells, glial cells, osteoblasts, cardiomyocytes, megakaratyocytes, adipocytes, stem cells (e.g., mesenchymal stem cells), and cancer cells, but is not limited thereto.

[0024] Any gene delivery system known to be useful in gene therapy can be used in the present invention.

[0025] For example, the gene delivery system of the present invention may be in the form of: (i) a naked recombinant DNA molecule, (ii) a plasmid, (iii) a viral vector, and (iv) a liposome or niosome encapsulating said naked recombinant DNA molecule or plasmid.

[0026] All gene delivery systems used in conventional gene therapy can be applied to the cell sheet of the present invention, and preferably, plasmids, adenoviruses (Lockett LJ, et al., Clin. Cancer Res. 3: 2075-2080 (1997)), adeno-associated viruses (Adeno-associated viruses: AAV, Lashford LS., et al., Gene Therapy Technologies, Applications and Regulations Ed. A. Meager, 1999), retroviruses (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 viruses (Chamber R., et al., Proc. Natl. Acad. Sci USA 92:1411-1415(1995)), vaccinia virus (Puhlmann M. et al., Human Gene Therapy 10:649-657(1999)), liposomes (Methods in Molecular Biology, Vol 199, S.C. Basu and M. Basu (Eds.), Human Press 2002) or niosomes may be used.

[0027] i. Adenovirus Adenoviruses are widely used as gene transfer vectors due to their medium genome size, ease of manipulation, high titer, wide range of target cells, and excellent infectivity. Both ends of the genome contain 100-200 bp ITRs (inverted terminal repeats), which are cis elements essential for DNA replication and packaging. The E1 region (E1A and E1B) of the genome encodes proteins that regulate transcription and host cell gene transcription. The E2 region (E2A and E2B) encodes proteins involved in viral DNA replication.

[0028] Among the adenovirus vectors currently developed, many are replication-incompetent adenoviruses lacking the E1 region. Meanwhile, the E3 region is removed from conventional adenovirus vectors to provide a site for inserting foreign genes (Thimmappaya, B. et al., Cell, 31:543-551 (1982); and Riordan, J. R. et al., Science, 245:1066-1073 (1989)). Thus, the nucleotide sequence of interest to be delivered into the cell is preferably inserted into the deleted E1 region (E1A region and / or E1B region, preferably the E1B region) or the E3 region, more preferably into the deleted E1 region. The term "deletion" as used herein in connection with the viral genome sequence includes not only complete deletion of the sequence, but also partial deletion.

[0029] Adenovirus has 42 different serotypes and subgroups AF. Among them, adenovirus type 2 and type 5 belonging to subgroup C are the most preferred starting materials for obtaining the adenovirus vector of the present invention. The biochemical and genetic information for adenovirus type 2 and type 5 is well known.

[0030] The foreign gene delivered by adenovirus is replicated in an episomal manner, which results in very low genetic toxicity to host cells, and therefore gene therapy using the adenovirus gene delivery system of the present invention is considered to be very safe.

[0031] ii. Retroviruses Retroviruses are widely used as gene transfer vectors because they can insert their own genes into the host genome, deliver large amounts of foreign genetic material, and infect a wide spectrum of cells.

[0032] To construct a retroviral vector, the nucleotide sequence of interest to be delivered into a cell is inserted into the retroviral genome in place of the retroviral sequence to produce a replication-incompetent virus. To produce virions, a packaging cell line is constructed that contains the gag, pol and env genes but lacks the LTR (long terminal repeat) and Ψ sequence (Mann et al., Cell, 33:153-159 (1983)). When a recombinant plasmid containing the nucleotide sequence of interest to be delivered, the LTR and the Ψ sequence is introduced into the cell line, the Ψ sequence allows the production of RNA transcripts of the recombinant plasmid, which are packaged into viruses and excreted 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 and concentrated for use in the gene delivery system.

[0033] Gene transfer using a second-generation retroviral vector has been reported. Kasahara et al. Science, 266:1373-1376 (1994) produced a mutant of Moloney Murine Leukemia Virus (MMLV) in which an EPO (erythropoietin) sequence was inserted into the envelope site to produce a chimeric protein with new binding properties. The gene transfer system of the present invention can also be produced by such a construction strategy of a second-generation retroviral vector.

[0034] iii. AAV vectors Adeno-associated virus (AAV) is suitable for the gene delivery system of the present invention because it can infect non-dividing cells and has the ability to infect a wide variety of cell types. Detailed information on the production and use of AAV vectors is disclosed in detail in U.S. Patent Nos. 5,139,941 and 4,797,368.

[0035] Studies regarding AAV as a gene delivery system are disclosed in LaFace et al, Viology, 162:483-486 (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).

[0036] Typically, AAV virus is produced by cotransformation of a plasmid containing a gene sequence of interest (the nucleotide sequence of interest to be delivered into a cell) flanked by two AAV terminal repeats (McLaughlin et al., J. Virol., 62:1963-1973 (1988); and Samulski et al., J. Virol., 63:3822-3828 (1989)) and an expression plasmid containing the wild-type AAV coding sequence lacking the terminal repeats (McCarty et al., J. Virol., 65:2936-2945 (1991)).

[0037] iv. Other viral vectors Other viral vectors can also be used in the gene delivery system of the present invention. Vaccinia 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)), lentivirus (Wang G. et al., J. Clin. Invest. 104(11):R55-62(1999)) or herpes simplex virus (Chamber Vectors derived from the method described by R., et al., Proc. Natl. Acad. Sci USA 92:1411-1415 (1995) can also be used as a delivery system capable of delivering a desired nucleotide sequence into cells.

[0038] Other viral vectors include reovirus, poxvirus, Semliki Forest virus and Measles virus.

[0039] v. Liposomes Liposomes are formed automatically by phospholipids dispersed in an aqueous phase. Examples of successful delivery of foreign DNA molecules into cells using liposomes are disclosed in Nicolau and Sene, Biochim. Biophys. Acta, 721:185-190 (1982) and Nicolau et al., Methods Enzymol., 149:157-176 (1987). On the other hand, the reagent most frequently used for transformation of animal cells using liposomes is Lipofectamine (Gibco BRL). Liposomes encapsulating the target nucleotide sequence to be delivered interact with cells through mechanisms such as endocytosis, adsorption to the cell surface, or fusion with the plasma membrane, and deliver the target nucleotide sequence into the cells.

[0040] The method of introducing a gene delivery system into one or more cell layers is carried out by contacting the gene delivery system with cells that form the cell layer.

[0041] In the present invention, when the gene delivery system is based on a viral vector, the contacting step is carried out by a viral infection method known in the art. Infection of host cells with a viral vector is described in the above cited references.

[0042] In the present invention, when the gene delivery system is a naked recombinant DNA molecule or a plasmid, the gene delivery system may be a recombinant DNA molecule or a plasmid, which ... Genes can be transferred into cells by methods such as the DEAE-dextran treatment method (Gopal, Mol. Cell Biol., 5:1188-1190 (1985)), and gene bombardment (Yang et al., Proc. Natl. Acad. Sci., 87:9568-9572 (1990)), and can be used to introduce genes into cells.

[0043] In one embodiment of the present invention, the cell sheet according to the present invention is used as a localized delivery platform capable of viral replication. The tumor-killing virus covers the target tumor site, and expresses a specific gene (e.g., decorin, etc.) that is advantageous for tumor elimination using the tumor-killing virus, allowing effective replication of the tumor-killing virus and therapeutic gene. In addition, the cell sheet allows active replication of virions in both the cell sheet and tumor tissue until the cell sheet is completely degraded in the body, thereby allowing long-term maintenance of the tumor-killing virus in the tumor tissue.

[0044] This allows the cell sheet to elicit a stronger antitumor effect than existing oncolytic viruses administered intratumorally, and effectively prevents the formation of multifocal carcinomas. Furthermore, cell sheet-mediated delivery of oncolytic viruses is localized to the tumor site, preventing nonspecific release of virions into normal tissues. Administration of oncolytic viruses loaded onto cell sheets can prolong, amplify and enhance the therapeutic efficacy of the virus while improving the safety profile by simultaneously enhancing intratumoral localization and preventing nonspecific release of the virus.

[0045] In one embodiment of the present invention, the gene delivery system may be a recombinant adenovirus.

[0046] As various advantages of recombinant adenovirus as a gene transfer vector emerge, its frequency of use in cancer gene therapy is steadily increasing. In particular, when trying to treat cancer with gene therapy agents, it has the advantage that long-term and sustained expression of therapeutic genes is not required. In addition, the host immune response induced by the virus used as a vector is not a major problem or can even act as an advantage, so recombinant adenovirus is in the spotlight as a gene transfer vehicle for cancer therapy.

[0047] The recombinant adenovirus may be a replication-incompetent adenovirus or an oncolytic adenovirus.

[0048] Replication-incompetent adenoviruses are engineered by inserting a therapeutic gene in place of the E1 gene (all or part) essential for adenovirus replication, so that the adenovirus cannot replicate in the cells into which it is introduced.

[0049] Oncolytic adenovirus is an adenovirus with a partially deleted E1B 55kDa gene, and can grow only in cells with functionally inactivated p53. In cancer cells where the function of p53 is suppressed, the virus grows actively, whereas in normal cells, the virus grows inhibited. Therefore, oncolytic adenovirus can selectively kill only cancer cells without affecting normal cells, which is particularly advantageous in the treatment of cancer.

[0050] In one embodiment of the invention, the recombinant adenovirus has an inactivated E1B 19kDa gene, an E1B 55kDa gene or an E1B 19kDa / E1B 55kDa gene, preferably an inactivated E1B 19kDa and E1B 55kDa gene.

[0051] As used herein, the term "inactivated" in relation to a gene means that the transcription and / or decoding of the gene is not performed normally, and the normal protein function encoded by the gene is not expressed. For example, an inactivated E1B 19kDa gene is a gene in which a mutation (substitution, addition, partial deletion, or total deletion) occurs in the gene, and the gene does not produce active E1B 19kDa protein. When the E1B 19kDa gene is absent, the cell killing ability can be increased, and when the E1B 55kDa gene is absent, the gene has tumor cell specificity (see Korean Patent Application No. 2002-0023760).

[0052] According to one embodiment of the present invention, the recombinant adenovirus of the present invention can contain an active E1A gene. The recombinant adenovirus containing the E1A gene will have the property of being replicable. According to a more preferred embodiment of the present invention, the recombinant adenovirus of the present invention contains an inactivated E1B 19kDa / E1B 55kDa gene and an active E1A gene. According to one embodiment of the present invention, the recombinant adenovirus of the present invention is one in which the E1B 19kDa / E1B 55kDa gene is deleted, the recombinant adenovirus contains an active E1A gene, and a decorin-encoding nucleotide sequence is inserted into the deleted E1 region.

[0053] In one embodiment of the present invention, The cell layer into which the gene delivery system is introduced can include cancer cells or stem cells.

[0054] When using a tumor-killing adenovirus as a gene delivery system, the cell layer into which the gene delivery system is introduced may contain cancer cells. As previously explained, the tumor-killing adenovirus grows only in cancer cells, and its replication is suppressed in normal cells other than the cell layer containing cancer cells. Therefore, even if the cell sheet is cultured for a long period of time, there is no need to worry about the deterioration of mechanical properties due to the death of the cell layer that acts as a support. In addition, since the cancer cells themselves are killed by the tumor-killing adenovirus after the cell sheet is transplanted into the body, there is no need to worry about the cancer cells remaining in the body.

[0055] Even if a gene delivery system other than tumor-killing adenovirus is used, the cancer cells can be irradiated to lose their own replicative ability, so there is no problem in using the gene delivery system in the cell layer into which the cell sheet is to be introduced. In one embodiment of the present invention, the cell sheet includes a cell layer containing cancer cells into which a gene delivery system has been introduced, and the cancer cells may be irradiated.

[0056] Specific examples of the cancer cells include glioblastoma, laryngeal cancer, pancreatic cancer, lung cancer, non-small cell lung cancer, colon cancer, bone cancer, pancreatic cancer, skin cancer, head or neck cancer, ovarian cancer, uterine cancer, rectal cancer, stomach cancer, anal cancer, colon cancer, breast cancer, fallopian tube cancer, endometrial cancer, cervical cancer, vaginal cancer, vulvar cancer, Hodgkin's disease, esophageal cancer, small intestine cancer, endocrine gland cancer, thyroid cancer, parathyroid cancer, adrenal gland cancer, soft tissue sarcoma, urethral cancer, penile cancer, prostate cancer, chronic or acute leukemia, lymphocytic lymphoma, bladder cancer, kidney or ureter cancer, renal cell carcinoma, renal pelvic carcinoma, central nervous system cancer, and the like. The cancer cells may be derived from one or more cancers selected from the group consisting of central nervous system (CNS) tumors, primary central nervous system lymphomas, spinal cord tumors, hepatocellular carcinomas, bronchial carcinomas, nasopharyngeal carcinomas, brain stem gliomas, and pituitary adenomas, but are not limited thereto.

[0057] In another embodiment, the cell layer into which the gene delivery system is introduced may include stem cells. Stem cells have tumor targeting ability, and therefore the cell sheet of the present invention may be particularly advantageous when used for cancer patients. However, since it is generally known that stem cells have a low infection rate with adenovirus, it may be preferable to use a recombinant adenovirus with improved stem cell introduction ability. Although not limited thereto, when the cell layer into which the gene delivery system is introduced includes stem cells, it may be preferable to use a recombinant adenovirus with improved stem cell introduction ability, which includes a fiber knob of serotype 35. In the case of a recombinant adenovirus with a fiber knob of serotype 35, it has a remarkably excellent introduction efficiency into mesenchymal stem cells, and has the characteristic that it can be well introduced even at a low virus concentration.

[0058] Without being limited thereto, in one embodiment of the present invention, the recombinant adenovirus of the present invention has an inactivated E1B 19kDa gene, an E1B 55kDa gene or an E1B 19kDa / E1B 55kDa gene, preferably has an inactivated E1B 19kDa and E1B 55kDa genes.

[0059] As used herein, the term "inactivated" in relation to a gene means that the transcription and / or decoding of the gene is not performed normally, and the normal protein function encoded by the gene is not expressed. For example, an inactivated E1B 19kDa gene is a gene in which a mutation (substitution, addition, partial deletion, or total deletion) occurs in the gene, and the gene does not produce active E1B 19kDa protein. When the E1B 19kDa gene is absent, the cell killing ability can be increased, and when the E1B 55kDa gene is absent, the gene has tumor cell specificity (see Korean Patent Application No. 2002-0023760).

[0060] According to a preferred embodiment of the present invention, the recombinant adenovirus of the present invention comprises an active E1A gene. The recombinant adenovirus comprising the E1A gene has the property of being replicable. According to a more preferred embodiment of the present invention, the recombinant adenovirus of the present invention comprises an inactivated E1B 19kDa / E1B 55kDa gene and an active E1A gene. According to a most preferred embodiment of the present invention, the recombinant adenovirus of the present invention is deleted of the E1B 19kDa / E1B 55kDa gene, comprises an active E1A gene, and a decorin-encoding nucleotide sequence is inserted into the deleted E1 region.

[0061] The recombinant adenovirus used in the present invention contains a promoter operable in animal cells, preferably mammalian cells. Promoters suitable for the present invention include promoters derived from mammalian viruses and promoters derived from the genome of mammalian cells, such as the CMV (Cytomegalovirus) promoter, the U6 promoter and the H1 promoter, the MLV (Murine Leukemia Virus) LTR (Long terminal repeat) promoter, the adenovirus early promoter, the adenovirus late promoter, the vaccinia virus 7.5K promoter, the SV40 promoter, the HSV tk promoter, the RSV promoter, the EF1 promoter, the metallothionine promoter, the β-actin promoter, the human IL-2 gene promoter, the human IFN gene promoter, the human IL-4 gene promoter, the human lymphotoxin gene promoter, the human GM-CSF gene promoter, an inducible promoter, a cancer cell-specific promoter (e.g., the TERT promoter, the modified TERT promoter, the PSA promoter, the PSMA promoter, the CEA promoter, the Survivin promoter, the E2F promoter, the modified E2F promoter, the AFP promoter, the modified AFP promoter, the E2F-AFP hybrid promoter, and the E2F-TERT promoter). Examples of promoters include, but are not limited to, a human phosphoglycerate kinase (PGK) promoter, a mouse phosphoglycerate kinase (PGK) promoter, and a hybrid promoter, and a tissue-specific promoter (e.g., albumin promoter). Most preferred is a CMV promoter. In an expression construct for expressing a transgene, a polyadenylation sequence is preferably linked downstream of the transgene.The polyadenylation sequence may be a bovine growth hormone terminator (Gimmi, ER, et al., Nucleic Acids Res. 17:6983-6998 (1989)), a polyadenylation sequence derived from SV40 (Schek, N, et al., Mol. Cell Biol. 12:5386-5393 (1992)), HIV-1 polyA (Klasens, BIF, et al., Nucleic Acids Res. 26:1870-1876 (1998)), β-globin polyA (Gil, A., et al., Cell 49:399-406 (1987)), HSV TK polyA (Cole, CN and TP Stacy, Mol. Cell. Biol. 5:2104-2113 (1985)), or polyoma virus polyA (Batt, DB and Examples of such antibodies include, but are not limited to, those disclosed in GG Carmichael, Mol. Cell. Biol. 15:4783-4790 (1995)).

[0062] The recombinant adenovirus of the present invention may further comprise an antibiotic resistance gene and a reporter gene (e.g., GFP (green fluorescence protein), luciferase and β-glucuronidase) as a selection marker. The antibiotic resistance gene includes antibiotic resistance genes commonly used in the art, such as ampicillin, gentamicin, carbenicillin, chloramphenicol, streptomycin, kanamycin, geneticin, neomycin and tetracycline resistance genes, preferably the neomycin resistance gene.

[0063] The viral vector (e.g., recombinant adenovirus) loaded into the cell sheet according to the present invention is preferably loaded at an amount of 0.1 to 500 MOI (Multiplicity of infection). More preferably, it may be loaded at 0.1 to 200 MOI, 0.1 to 100 MOI, 0.1 to 50 MOI, 0.1 to 10 MOI, 0.1 to 5 MOI, 0.5 to 200 MOI, 0.5 to 100 MOI, 0.5 to 50 MOI, 0.5 to 10 MOI, or 0.5 to 5 MOI. This is the tumor-killing viral dose of 1×10 used in existing tumor treatment methods. 10 VP~5×10 10 Since it can grow in cancer cells even when used in a significantly smaller amount than VP, the amount of virus carried can be significantly reduced compared to existing tumor treatments using tumor-killing viruses, significantly reducing the burden on doctors.

[0064] The present invention also provides a method for producing a method for manufacturing a semiconductor device comprising the steps of: A temperature-responsive culture dish containing a temperature-sensitive polymer is forming a cell sheet comprising two or more cell layers; introducing a gene delivery system into one or more of the cell layers; Separating the cell sheet from the temperature-responsive culture dish. The present invention provides a method for producing a cell sheet comprising the steps of:

[0065] All of the contents described above in relation to the cell sheet can be directly or mutatis mutandis applied to the method for producing the cell sheet.

[0066] The method for producing a cell sheet according to the present invention may include, but is not limited to, the following steps: culturing somatic cells on a temperature-responsive culture dish containing a temperature-sensitive polymer to form a first cell layer containing somatic cells; a step of culturing cancer cells on the first cell layer to form a second cell layer containing the cancer cells, thereby producing a cell sheet; inoculating said second layer of cells with an oncolytic virus; and Separating the cell sheet from the temperature responsive culture dish.

[0067] To specifically explain the above example, First, a silicon ring is placed on a temperature-responsive culture dish containing a temperature-sensitive polymer, and somatic cells that act as a support are dispensed onto the inside of the silicon ring and cultured in a thermostatic device to form a first cell layer containing somatic cells.

[0068] The temperature-sensitive culture dish allows cells to attach to the surface above the lower critical solution temperature (LCST) to form a cell sheet, and below the lower critical solution temperature, the polymer swells and cells can be collected in sheet form.

[0069] The temperature-sensitive polymer may be at least one selected from the group consisting of poly(N-isopropylacrylamide), poly(N-vinylcaprolactam), polycaprolactone (PCL) and polylactate-co-glycolate (PLGA), but any polymer having temperature sensitivity may be used without limitation.

[0070] Next, cancer cells are dispensed onto the first cell layer and cultured in a thermostatic device to form a second cell layer containing the cancer cells, thereby producing a cell sheet.

[0071] In particular, the method may further include a step of irradiating the second cell layer with radiation in order to eliminate the possibility of carcinogenesis caused by cancer cells constituting the cell sheet.

[0072] The second cell layer is then inoculated with an oncolytic virus to load the cell sheet with the virus.

[0073] In this case, the oncolytic virus is inoculated at an amount of 0.1 to 500 MOI (more preferably 0.1 to 200 MOI, 0.1 to 100 MOI, 0.1 to 50 MOI, 0.1 to 10 MOI, 0.1 to 5 MOI, 0.5 to 200 MOI, 0.5 to 100 MOI, 0.5 to 50 MOI, 0.5 to 10 MOI, or 0.5 to 5 MOI) 12 hours to 1 day after the formation of the cell sheet. Only when the cells are inoculated at a certain time interval, can the cells form a sheet, and the amount of virus carried per number of cells can be calculated. By inoculating such an oncolytic virus, the cancer cells naturally disappear after a certain period (12 hours to 7 days after inoculation). In addition, the oncolytic virus can be amplified by the cancer cells, and the cancer cells can be treated by administering a small amount.

[0074] Finally, the cell sheet is detached from the temperature-responsive culture dish.

[0075] The temperature-sensitive culture dish has a polymer that swells below the lower critical temperature of the surface, allowing the cell sheet to be detached from the culture dish. In this case, it is preferable to separate the cell sheet from the culture dish 6 hours to 1 day after the virus inoculation, since the cell sheet can be peeled off in the form of a firm cell sheet without being decomposed by the replication of the carried virus.

[0076] The present invention also provides a gene therapy agent comprising the cell sheet of the present invention as an active ingredient.

[0077] The term "gene therapy agent" as used herein means a cell or a pharmaceutical product in which genetic material or a carrier into which genetic material has been transferred is administered to a subject for the purpose of treating a disease, etc. It also means a pharmaceutical product used for the purpose of treating or preventing a genetic defect by injecting a normal gene or a gene having a therapeutic effect into a damaged gene of a subject.

[0078] Pharmaceutically acceptable carriers that can be used as gene therapy agents are sterile and biocompatible, and can be used by mixing one or more of saline, sterile water, Ringer's solution, buffered saline, albumin injection solution, dextrose solution, maltodextrin solution, glycerol, ethanol, and other common additives such as antioxidants, buffers, bacteriostatic agents, etc., as necessary. In addition, diluents, dispersants, surfactants, binders, and lubricants can be added to formulate the agent into an injectable dosage form such as an aqueous solution, suspension, emulsion, etc., pills, capsules, granules, or tablets, and target organ-specific antibodies or other ligands can be bound to the carriers to specifically act on the target organs.

[0079] Preferably, the gene therapy agent of the present invention is used for the prevention or treatment of cancer, or for preventing the recurrence or metastasis of cancer.

[0080] The cancers include multifocal hepatocellular carcinoma, glioma, glioblastoma, laryngeal cancer, pancreatic cancer, lung cancer, non-small cell lung cancer, colon cancer, bone cancer, pancreatic cancer, skin cancer, head or neck cancer, ovarian cancer, uterine cancer, rectal cancer, stomach cancer, anal cancer, colon cancer, breast cancer, fallopian tube cancer, endometrial cancer, cervical cancer, vaginal cancer, vulvar cancer, Hodgkin's disease, esophageal cancer, small intestine cancer, endocrine gland cancer, thyroid cancer, parathyroid cancer, adrenal gland cancer, soft tissue sarcoma, urethral cancer, penile cancer, prostate cancer, chronic or acute leukemia, lymphocytic lymphoma, bladder cancer, kidney or ureter cancer, renal cell carcinoma, renal pelvic carcinoma, central nervous system cancer, The tumor may be one or more selected from the group consisting of primary central nervous system (CNS) tumor, primary central nervous system lymphoma, spinal cord tumor, liver cancer, bronchial cancer, nasopharyngeal cancer, brain stem glioma, and pituitary adenoma.

[0081] By administering the cell sheet of the present invention to the surgical site where the cancer (tumor) was removed or to the site where the cancer developed, it is possible to treat the cancer or prevent the cancer metastasis or recurrence. The preferred dosage of the cell therapy agent of the present invention varies depending on the condition and weight of the individual, the degree of the disease, the drug form, the route and duration of administration, and can be appropriately selected by those skilled in the art. The agent can be administered once a day or in several divided doses, and the dosage does not limit the scope of the present invention in any aspect.

[0082] The term "prevention" as used in the present invention refers to any action of suppressing cancer (tumor) or delaying the onset of the disease by administering the cell sheet according to the present invention.

[0083] The term "treatment" as used in the present invention refers to any action that improves or favorably modifies symptoms of cancer (tumor) by administration of the cell sheet according to the present invention.

[0084] The term "metastasis" as used herein means a state in which a malignant tumor spreads to other tissues distant from the organ where the tumor originated.

[0085] The term "recurrence" as used herein means the re-growth of the same tumor after it has disappeared due to surgical removal or radiation exposure, the proliferation of remaining tumor cells, or the complete disappearance of tumor cells followed by the development of a new tumor.

[0086] The present invention also includes a method for preventing cancer recurrence, comprising the step of transplanting the cell sheet of the present invention into a site of a cancer removal surgery in a subject.

[0087] The present invention also includes a method for treating cancer, comprising transplanting the cell sheet of the present invention into a site in a subject that requires cancer treatment.

[0088] In the present invention, the term "subject" refers to a subject requiring treatment for a disease, and more specifically refers to mammals such as humans or non-human primates, rodents (rats, mice, guinea pigs, etc.), dogs, cats, horses, cows, sheep, pigs, goats, camels, and antelopes.

[0089] The cell sheet according to the present invention will disappear naturally after a certain period of time (6 to 10 days) after transplantation, as the cancer cell layer will be destroyed by viral replication.

[0090] The method for transplanting the cell sheet of the present invention into a target site may include, but is not limited to, the following steps: The culture medium is removed from the virus-infected cell sheet formed in a temperature-responsive culture dish, and the cell sheet is washed with PBS. The membrane is left on the cell sheet, the temperature is lowered, and the cell sheet is peeled off, resulting in a cell sheet with the cell sheet still attached to the membrane. The membrane with the cell sheet attached is placed on the target (lesion) site, and the membrane is carefully peeled off, resulting in the cell sheet adhering to the target site. By removing the membrane, the cell sheet can be transplanted to the target site. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0091] In the following, preferred examples are presented to aid in understanding the present invention. However, the following examples are merely provided to facilitate understanding of the present invention, and the contents of the present invention are not limited to the following examples.

[0092] [Example] Experimental Method Cell lines and cell culture All cell lines were cultured in Dulbecco's modified Eagle's medium (DMEM, Gibco BRL, Grand Island, NY USA) supplemented with 10% fetal bovine serum (FBS, Gibco BRL) and penicillin-streptomycin (100 IU / mL, Gibco BRL).

[0093] HEK293 (a human embryonic kidney cell line expressing the Ad E1 region), A549 (a lung cancer cell line), Hep3B (a hepatocellular carcinoma cell line), U343 (a glioblastoma cell line), and NIH3T3 (a fibroblast cell line) cell lines were purchased from the American Type Culture Collection (ATCC, Manassas, VA).

[0094] All cell lines were maintained at 37° C. in a humidified atmosphere of 5% CO 2 .

[0095] Construction of oAd-DCN (decorin-expressing tumor-killing Ad) pCA14 / DCN was cut with Bgl II restriction enzyme to obtain a DCN expression cassette, which was then ligated to pdE1sp1B(p)-HRE-mTERT-Rd19 cut with BamHI restriction enzyme to produce the pdE1sp1B(p)-HRE-mTERT-Rd19 / DCN E1 shuttle vector.

[0096] The above vector was treated with XmnI restriction enzyme to make it single-stranded, and dl324-k35, a vector in which the adenovirus knob was replaced with that of Ad35, was treated with BstBI restriction enzyme to make it single-stranded, and the two vectors were simultaneously transformed into E. coli BJ5183 to induce homologous recombination, thereby constructing a DCN-expressing oAd vector (HmT-Rd19-k35 / DCN, oAd-DCN).

[0097] Ad-loaded cancer cell / fibroblast cell sheets (oAd / CFCSs) A cell sheet (CFCS) consisting of a first cell layer of fibroblasts and a second cell layer of cancer cells was fabricated using a temperature-responsive culture dish (TRCD; UpCell; NUNC, Tokyo, Japan).

[0098] To prepare a bilayer cell sheet, NIH3T3 cells (3 × 10 5) were placed in the hollow inner layer of a silicon ring (radius 1.8 cm) in a 35 mm TRCD and cultured at 37 °C. After 48 hours of culture, U343 cells (3 × 10 5 ) was added to the silicon ring-defined area to form the second layer of the cell sheet and cultured for 24 h. After replacing the medium of each dish with fresh DMEM containing 5% FBS, the cells were infected with decorin-expressing tumor-killing Ad (oAd-DCN) at a multiplicity of infection (MOI) of 0.5 or 5. The Ad-DCN-infected cancer cell / fibroblast cell sheets (oAd-DCN / CFCSs) were detached from the dishes 4 h after infection by lowering the culture temperature to room temperature for 30 min.

[0099] histology oAd-DCN CFCSs were generated as described above using 5 MOI of oAd-DCN, and control CFCSs were generated by treating cell sheets with phosphate-buffered saline (PBS).

[0100] Tumor-killed Ad-loaded or PBS-treated CFCs were harvested 12 hours after treatment by lowering the culture temperature to room temperature for 30 minutes and fixing with 4% paraformaldehyde for 24 hours. Fixed samples were embedded in paraffin, cut into 5um sections, and the paraffin was removed for hematoxylin and eosin (H&E) staining.

[0101] Virus production analysis To evaluate viral production of tumor-killing Ad in cell sheets, CFCs were plated in 12-well plates and infected with oAd-DCN at an MOI of 0.5. Four hours after infection, wells were washed with PBS and replaced with fresh DMEM containing 5% FBS.

[0102] At 4, 12, 24, 48, 72, and 96 h postinfection, the supernatants and whole cell sheets were collected and the numbers of Ad particles were assessed by real-time quantitative PCR (Q-PCR, TaqMan PCR detection, Applied Biosystems, CA, USA).

[0103] Western blotting analysis To evaluate the level of Ad-mediated DCN expression and tumor killing by oAd-DCN / CFCSs, sheets were infected with oAd-DCN at an MOI of 1 and cultured for 48 h. Subsequently, sheets were homogenized in ice-cold RIPA buffer (Elipis Biotech, Taejeon, Korea) containing a protease inhibitor cocktail (Sigma, MO, USA), and the resulting homogenate was centrifuged at 13,200 rpm for 10 min.

[0104] Total protein concentrations were measured by BCA protein assay (Pierce, Rockford, IL, USA), and equivalent protein amounts (200 μg per sample) were loaded onto sodium dodecyl sulfate-polyacrylamide gels for electrophoresis. Proteins were transferred to polyvinylidene fluoride membranes and incubated with goat anti-DCN antibody (Ab, R&D Systems, MN, USA) or rabbit anti-β-actin antibody (Cell Signaling Technology, Beverly, MA, USA).

[0105] Membranes were incubated with secondary Abs, horseradish peroxidase-conjugated mouse anti-goat IgG Ab or goat anti-rabbit IgG (Cell Signaling), and immunoreactive bands were visualized via enhanced chemiluminescence (Amersham Pharmacia Biotech, Uppsala, Sweden).

[0106] The expression levels of DCN were semi-quantitatively analyzed using ImageJ software (National Institutes of Health, Bethesda, MD, USA).

[0107] MTT analysis To evaluate viral replication-mediated degradation of CFCs in vitro, CFCs were plated in 48-well plates and then infected with oAd-DCN at an MOI of 5.

[0108] At 4, 12, 24, 48, 72, and 96 hours after infection, the medium was removed and the CFCSs were treated with 500 μL of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT, Sigma, MO, USA). Subsequently, the plates were read on a microplate reader at 540 nm. The absorbance from the PBS-treated group was set as 100% viability.

[0109] In vivo degradation profile and viral persistence of oAd-DCN / CFCS To evaluate the degradation of CFCSs and viral persistence in vivo, two different types of cell sheets were fabricated.

[0110] The degradation profile of CFCSs was evaluated using CFCSs composed of fibroblasts and firefly luciferase (fluc)-expressing cancer cells (CFCSs / fluc), followed by infection with oAd-DCN at an MOI of 5 to generate oAd-DCN / CFCSs / fluc.

[0111] To evaluate the viral persistence of the cell sheets, fibroblasts and cancer cells that do not express the fluc gene were infected with firefly luciferase-expressing oAd-DCN (oAd-DCN-fluc) at an MOI of 5 to generate CFCs. oAd-DCN-fluc / CFCSs, oAd-DCN / CFCSs / fluc, and oAd-DCN-fluc / CFCS were all transplanted into the largest liver lobe of HCC-bearing mice 4 hours after infection. On days 2, 4, 6, 8, and 10 after transplantation, the mice were anesthetized in a chamber filled with 2% isoflurane in oxygen and intraperitoneally injected with D-luciferin (150 mg / kg, Caliper, Hopkinton, MA) to evaluate the degradation of oAd-DCN / CFCS / fluc and the persistence of tumor-killing Ad of oAd-DCN-fluc / CFCS through an IVIS imaging system (Xenogen, Alameda, CA, USA).

[0112] In vivo antitumor efficacy To evaluate the antitumor effect of oAd-DCN, CFCSs and oAd-DCN / CFCSs were transplanted into the largest lobe of the liver in 6- to 7-week-old athymic nude mice (OrientBioInc., Seongnam, Korea) in an orthotopic HCC xenograft model at 1 × 10 6 Fluc-expressing Hep3B cells (Hep3B / fluc) were injected.

[0113] Immediately after tumor cell injection, the hepatic injection site was treated with trypsin for 5 min, and then implanted with CFCS groups (CFCS or oAd-DCN / CFCS) or injected with PBS or oAd-DCN (5 MOI). Finally, one of the treatment groups was systemically administered with oAd-DCN via the tail vein (same amount of virus administered as the other oncolytic Ad-containing groups).

[0114] Two days after cell injection, mice were anesthetized in a chamber filled with 2% isoflurane in oxygen and injected intraperitoneally with D-luciferin (150 mg / kg; Caliper, Hopkinton, MA) to confirm successful engraftment of Hep3B / fluc cells via an IVIS imaging system (Xenogen).

[0115] In vivo bioluminescence signal intensity was obtained in photons per second (photons / second [p / s]) at the body site of interest (tumor) on days 2, 5, 7, 14, and 21 after treatment.

[0116] Histological and immunohistochemical analyses Hep3B-HCC tumor tissues were harvested 21 days after HCC cell injection, fixed in 10% formalin, embedded in paraffin, and cut at 5 μm sections. Sections were stained with H&E and examined under a light microscope. To detect Ad particles in tumor tissues, tumor sections were immunostained with rabbit anti-Ad E1A polyclonal antibody (Santa Cruz Biotechnology).

[0117] Tumor sections were also immunostained with a proliferating cell nuclear antigen (PCNA)-specific antibody (Dako, Glostrup, Denmark) or terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) to assess tumor cell proliferation or induction of cell death after treatment, followed by treatment with horseradish peroxidase-conjugated goat anti-rat IgG (BD Biosciences Pharmingen) or horseradish peroxidase-conjugated goat anti-mouse IgG (Southern Biotech, Birmingham, AL, USA) as secondary antibodies.

[0118] Diaminobenzidine / hydrogen peroxidase (DAKO) was used as the chromogenic substrate. All slides were counterstained with Mayer's hematoxylin.

[0119] statistical analysis Data are presented as mean ± standard deviation (SD). Statistical significance was determined by two-tailed Student T-test or One-way Anova test (SPSS 13.0 software, SPSS, Chicago, IL). P values ​​less than 0.05 were considered statistically significant.

[0120] Confirmation of cell viability of irradiated cancer cells After irradiating U343 cell line with various intensities of radiation (1, 5, 10, 15, 30, 50 Gy), the irradiated U343 cell line was seeded into a 96-well plate and MTT assay was performed up to day 9 to confirm the cell viability of the irradiated cancer cells.

[0121] Confirmation of viral replication ability from cell sheets using irradiated cancer cells A cell sheet (CFCS) consisting of a first cell layer of fibroblasts and a second cell layer of cancer cells was fabricated using a temperature-responsive culture dish (TRCD; UpCell; NUNC, Tokyo, Japan).

[0122] To prepare a bilayer cell sheet, NIH3T3 cells (3 × 10 5 ) were placed in the hollow inner layer of a silicon ring (radius 1.8 cm) in a 35 mm TRCD and cultured at 37°C. After 48 hours of culture, U343 cells that had not been irradiated (control group) or U343 cells that had been irradiated at 5 Gy (3 × 10 5 ) was added to the area limited by the silicon ring to form the second layer of the cell sheet, which was then cultured for 24 hours. After replacing the medium in each dish with fresh DMEM containing 5% FBS, the cells were infected with DCN-expressing tumor-killing Ad (oAd-DCN) at a multiplicity of infection (MOI) of 0.5. After that, the medium was replaced with fresh medium to remove non-infectious viruses at 4 hours, and the medium and cells were harvested at 4, 24, and 48 hours, and the presence of viruses was confirmed by Q-PCR.

[0123] Confirmation of biological activity and tumor development after transplanting cell sheets into the tumor removal surgery (target) site As shown in Figure 7, NIH3T3 cells were dispensed into a temperature-responsive culture dish to form the first layer, and three days later, U343 cell line was dispensed to form the second layer. After 48 hours, the cells were infected with a tumor-selective killing adenovirus, and after 24 hours, the temperature of the temperature-responsive culture dish was lowered to 20°C and the cell sheet was peeled off.

[0124] The culture medium was removed from the virus-infected cell sheet formed in the temperature-responsive culture dish, and the cell sheet was washed three times with 1X PBS. The membrane was placed on the cell sheet and the temperature was lowered to 20°C to obtain a membrane with the cell sheet attached.

[0125] The tumor was surgically removed from a mouse model bearing a subcutaneous H460 lung cancer tumor, and the membrane with the cell sheet attached was placed on the tumor removal site. The membrane was then carefully peeled off, and the cell sheet was attached to the tumor removal site. The surgical site was then sutured, and the presence or absence of tumor recurrence was observed for up to 30 days.

[0126] Biological activity analysis of virus produced in cell sheets To evaluate the biological activity of tumor-killing adenovirus replicated in the cell sheets, CFCs were placed in 12-well plates and infected with oAd-DCN at 0.5 MOI. Four hours after infection, the wells were washed with 1XPBS and replaced with fresh DMEM containing 5% FBS. The supernatants and whole cell sheets were collected at 4, 12, 24, 48, and 72 hours after infection.

[0127] A549 cells were cultured in a 96-well plate at 1 × 10 4 The cells were seeded at 1000 μL / well, and 25, 50, and 100 μL of each collected sample was applied to the cells. After 48 hours, an MTT assay was performed to confirm the cancer cell killing ability of the virus produced in the cell sheet.

[0128] Production of cell sheets loaded with viruses To prepare a cell sheet for replicating or transmitting vaccinia virus or adenovirus, a silicon ring with an inner diameter of 1.8 mm was placed on a temperature-responsive culture dish (TRCD), and NIH3T3 cell line was inoculated at 3 × 10 5 After 48 hours, 3 × 10 U343 cells were 5 The cells were then dispensed to produce cell sheets.

[0129] After 24 hours, the cell sheets were infected with vaccinia virus or adenovirus. After a certain period of time, the culture medium and silicon ring of the infected cell sheets were removed from the temperature-responsive culture dish, and the cells were washed three times with 1XPBS. Then, 2mL of fresh 1XPBS was added and the cells were peeled off at 20℃ to confirm the formation of the cell sheet.

[0130] Experimental Results oAd-DCN / CFCS generation and characteristic clarification To fabricate CFCS permissive for Ad replication, we used bilayer cell sheets composed of a human brain glioblastoma cell line (U343) and a mouse fibroblast cell line (NIH3T3).

[0131] CFCs were generated in temperature-responsive culture dishes (TRCDs) and infected with oAd-DCN at an MOI of 5 to generate tumor-killing Ad-infected CFCs (oAd-DCN / CFCSs). 12 h after infection, oAd-DCN / CFCSs were separated from TRCDs by lowering the plate temperature to room temperature for 30 min. As shown in Figure 1a, circular sheets with uniform sizes were easily separated from TRCDs. H&E staining of the sheets showed that cancer cells and normal fibroblast components were present in the sheet layer (Figure 1b).

[0132] Importantly, Ad E1A staining of the cell sheets showed widespread distribution of oncolytic Ad visible as red spots (Fig. 1d). In contrast, PBS-treated control sheets had no detectable spots (Fig. 1c). It is noteworthy that PBS- and oncolytic Ad-loaded CFCs showed similar structure and morphology at 12 h postinfection. These results also indicated that oncolytic Ad could be loaded into cell sheets and would not harm the overall structural integrity of the cell sheets.

[0133] Viral replication and gene expression profile in oAd-DCN / CFCS To assess whether the cancer cell layer of the sheets was permissive for tumor-killing Ad infection, viral replication and therapeutic gene expression profiles were analyzed by Q-PCR and Western blotting, respectively.

[0134] As shown in Figure 2a, the oncolytic Ad replicated effectively in the cell sheets up to 72 hours after infection, demonstrating that the cell sheets were permissive for oncolytic Ad replication (at 4 hours, the amount of virus infected in the cell sheets was detected, but because a low MOI was used, the value was below the detection limit of Q-PCR and was not analyzed).

[0135] At 96 h after treatment, a slight decrease in the amount of Ad was observed due to the tumor-killing activity of oAd-DCN, and a significant disintegration of the cell sheet was induced at 96 h after treatment. These results demonstrated that oAd-DCN could effectively generate DCN in oAd-DCN / CFCS by Western blot analysis (Figure 2b).

[0136] These results also demonstrated that cell sheets were susceptible to oncolytic Ad infection and, ultimately, that the sheets could function as a transduction scaffold permissive for viral replication and therapeutic gene expression.

[0137] Degradation and viral persistence of oAd-DCN / CFCSs in vivo To monitor and visualize the degradation of cell sheets and the persistence of loaded tumor-killing Ad in CFCSs, HCC xenograft mice were implanted with oAd-DCN-loaded fluc-expressing CFCSs (oAd-DCN / CFCSs / Fluc) (Fig. 3a) or CFCSs (without reporter gene) infected with fluc-expressing oAd-DCN (oAd-DCN-Fluc / CFCSs) (Fig. 4b).

[0138] As shown in Figure 3a, the luciferase signal of the cell sheets decreased in a time-dependent manner and completely disappeared by 10 days after implantation.

[0139] In addition, the mice were sacrificed 10 days after transplantation, and it was confirmed with the naked eye that all of the transplanted CFCS had been completely degraded in the liver.

[0140] Similarly, the signal of the tumor-killing Ad virus also decreased in a time-dependent manner and showed a similar time-dependent decrease in luciferase expression pattern as the 10-day cell sheets (FIG. 3b).

[0141] Potent antitumor efficacy of oAd-DCN / CFCS against multifocal hepatocellular carcinoma Orthotopic tumor models have emerged as one of the important cancer research models due to their clinical relevance. Because multifocality of HCC is a major challenge toward successful treatment in the clinic, a multifocal hepatocellular orthotopic tumor model was established by multiple injections of luciferase-expressing Hep3B cells into the largest lobe of the liver.

[0142] As shown in Figure 4 , HCC lesions were detected in the livers of PBS-treated mice, confirming that orthotopic multifocal HCC was successfully established.

[0143] As shown in FIG. 5a, oAd-DCN / CFCS treatment showed significantly higher antitumor activity than any other treatment group at 21 days after treatment, and the tumor-specific activity was significantly higher than that of PBS (7.2×10 8 ±4.5×10 8 ), PBS-treated CFCs (1.7 × 10 8 ±9.3×10 7 ), or oAd-DCN(sprayed)(1.2×10 8 ±1.3×10 8 ) showed 20.4-fold, 4.7-fold, and 3.5-fold greater tumor growth inhibition than IFN-γ-α, ...β, or IFN-γ-α, respectively (FIG. 5b).

[0144] Moreover, all oAd-DCN / CFCSs-treated mice did not exhibit multifocal hepatocellular carcinoma in the liver, suggesting that CFCS-mediated delivery of tumor-killing Ad can prevent the formation of multifocal tumors (Fig. 6 ).

[0145] These results also suggest that oAd-DCN loaded onto cell sheets could be effectively delivered to tumor tissues to elicit potent antitumor efficacy and prevent the formation of multifocal HCC.

[0146] Histological analysis of oAd-DCN / CFCS for multifocal hepatocellular carcinoma To further investigate the therapeutic effect, tumor tissues were collected 2 weeks after treatment with each group (PBS, intravascular injection of oAd-DCN, intraperitoneal injection of oAd-DCN, CFCS alone, or oAd-DCN / CFCS) and analyzed through histological and immunohistological analysis.

[0147] As shown in FIG. 6, H&E staining demonstrated large multifocal areas of proliferating tumor cells in PBS-treated, oAd-DCN intravascularly injected, oAd-DCN intraperitoneally injected, CFCSs alone-treated tissues or macrophages.

[0148] Small areas of tumor cells and multiple tumors were not observed with oAd-DCN / CFCS. Taken together, this demonstrates that the use of CFCS for local delivery of oAd improves antitumor efficacy and prevents the development of multifocal tumors.

[0149] Confirmation of biological activity and tumor development after transplanting cell sheets into the tumor removal surgery (target) site FIG. 8 shows the biological activity of the adenovirus replicated from a cell sheet loaded with a tumor-selective killing adenovirus. It was confirmed that the later the sample, which contained a larger amount of replicated virus, had an increased cell-killing ability, proving that the adenovirus replicated from the cell sheet had biological activity.

[0150] FIG. 9 shows the results of examining whether or not tumor recurrence occurred after tumor removal surgery and then attaching a cell sheet loaded with a tumor-selective killing adenovirus. In the control group in which no cell sheet was attached after tumor removal surgery, tumor recurrence was observed 30 days after surgery, whereas in the experimental group in which a cell sheet was attached and sutured, no tumor recurrence occurred.

[0151] Confirmation of cell sheet formation infected with vaccinia virus It was confirmed that cell sheets were well formed regardless of whether or not the cells were infected with a virus.

[0152] Figure 10 shows a photograph of a cell sheet 6 hours after infection with vaccinia virus at an MOI of 0.5 on a temperature-responsive culture dish (a), a photograph of the cell sheet after the silicone ring has been removed and the cell sheet has been washed three times with 1X PBS in order to detach it from the temperature-responsive culture dish (b), a photograph of the cell sheet not infected with the virus after the temperature-responsive culture dish has been reacted for 15 minutes at 20°C and the cell sheet has been detached (c), and a photograph of the cell sheet infected with vaccinia virus at an MOI of 0.5 (d).

[0153] Confirmation of vaccinia virus replication in cell sheets To evaluate the replication ability of vaccinia virus in the cancer cell layer of the cell sheet, a cell sheet consisting of a first cell layer of fibroblasts and a second cell layer of cancer cells was formed as shown in Figure 11a, and infected with vaccinia virus at an MOI of 0.1. After 4 hours, all non-infectious vaccinia virus was removed by washing, and vaccinia virus was confirmed in the cell culture medium and cell sheet via Q-PCR at 24, 48, and 72 hours.

[0154] As shown in FIG. 11b, vaccinia virus replicated efficiently within the cell sheets over time up to 72 hours post-infection, demonstrating that the cell sheets were permissive for vaccinia virus replication.

[0155] Confirmation of cell death in cell sheets caused by vaccinia virus To confirm cell death in the cell sheet due to virus infection, as shown in Figure 11a, a cell sheet was formed consisting of a first cell layer of fibroblasts and a second cell layer of cancer cells, and infected with vaccinia virus at an MOI of 2 or 5. After that, 7-AAD dye, which can stain dead cells red, was added to confirm cell death over time.

[0156] As can be seen from Figure 12a, the number of dead cells increased over time in the cell sheets infected with vaccinia virus at an MOI of 2 or 5, and the red area in the photograph was quantified (Figure 12b).

[0157] Taking the results of Figures 11 and 12 together, it was demonstrated that the cell sheet can be infected not only by adenovirus but also by vaccinia virus, enabling the virus to replicate, and that the cell sheet is biodegradable due to viral replication.

[0158] Confirmation of cell viability of irradiated cancer cells The cell viability of U343 cells irradiated with various levels of radiation (1, 5, 10, 15, 30, 50 Gy) was confirmed by MTT assay. U343 cell lines irradiated with 1 Gy divided at a similar rate to non-irradiated cells (control group), and when irradiated with 5 Gy, the cell division rate was slower than the control group, and cells began to die after the 7th day. In the case of U343 cells irradiated with 10-50 Gy, cell division hardly occurred and all cells died by the 6th day (Figure 13).

[0159] Confirmation of adenovirus replication in cell sheets containing irradiated cancer cells To evaluate the viral replication ability in the irradiated cancer cell layer of the cell sheet, a cell sheet consisting of a first fibroblast cell layer and a second cancer cell layer was formed as shown in Figure 11a, and infected with adenovirus at 0.5 MOI. After 4 hours, all non-infectious adenovirus was removed by washing, and adenovirus was confirmed from the cell culture medium and cell sheet by Q-PCR at 24, 48, and 72 hours. As a result, it was confirmed that adenovirus replication occurred even in irradiated cancer cells (Figure 14).

[0160] discussion Multiple hepatocellular carcinoma (HCC), which frequently occurs in the liver due to chronic stress, is highly mutated and covers much of the liver, making it difficult to perform curative resection surgery in many patients.

[0161] Even patients who undergo surgical resection have a poor prognosis, with a 5-year survival rate of 50%, due to a high recurrence rate.

[0162] Also, the extensive liver damage in this patient precludes systemic administration of adequate amounts of chemotherapy agents, as these drugs cause severe hepatotoxicity.

[0163] To address such limitations of traditional HCC treatment options, we generated biodegradable and Ad replication-permissive cell sheets to efficiently deliver tumor-killing Ad to the multifocal orthotopically localized sites of HCC.

[0164] The permissive nature of the cell sheet delivery platform for viral replication means that other common therapeutic routes (many local and systemic administration of oncolytic Ad) require 1–5 × 10 10 This allows for the effective treatment of multifocal hepatocellular carcinoma with a relatively low viral dose compared to VP (which is required for chemotherapy).

[0165] At comparable viral doses, oc-DCN / CFCS treatment led to long-term tumor-killing Ad release, demonstrating superior tumor growth inhibition and prevention of multiple hepatocellular carcinoma formation than systemic or local administration of naked oAd-DCN (Figure 6).

[0166] One explanation for this is that viral replication mediated cell lysis and many virions released from the CFCS reaching the tumor are able to infect large areas of multifocal tumor sites.

[0167] Ultimately, maintaining infectivity of therapeutic viruses for extended periods of time remains a major obstacle in clinical trials. There are several other local delivery platforms such as hydrogels, patches and intratumoral injections currently being developed to improve the localization and therapeutic efficacy of therapeutic agents [Pesonen, S., L. Kangasniemi, and A. Hemminki, Oncolytic adenoviruses for the treatment of human cancer: focus on translational and clinical data. Mol Pharm, 2011.8(1):p.12-28; Wang, C., et al., Enhanced Cancer Immunotherapy by Microneedle Patch-Assisted Delivery of Anti-PD1 Antibody. Nano Lett, 2016.16(4):p.2334-40; Kasala, D., et al., Evolving lessons on nanomaterial-coated viral vectors for local and systemic gene therapy. Nanomedicine(Lond), 2016.11(13):p.1689-713]. One of the main problems with such platforms is that they often use synthetic components such as polymers, liposomes, and nanoparticles, and the decomposition products can induce inflammation and other side effects. However, unlike other existing local delivery platforms, the cell sheet (e.g., oAd-DCN / CFCS) loaded with the tumor-killing virus of the present invention is excellent in biocompatibility and degradability because it does not contain any synthetic components. One of the major concerns and obstacles for the CFCS approach in the clinic is the use of a cancer cell layer that can generate other tumors and thus support viral replication. To address this concern, irradiated cancer cells were used to generate CFCS, which induced the eradication of the cancer cell layer after initial transplantation and supported viral replication (Figure 13). In addition, irradiation enhanced viral replication in the cell sheet layer (Figure 14).

[0168] These results indicate that DNA damage induced by radiation and adjuvant radiation therapy can enhance the replication of tumor-killing Ad, and that accelerating DNA repair can further enhance the replication of episomal adenoviral DNA.

[0169] Moreover, irradiated cancer cells are currently being evaluated as promising candidates for cancer vaccines because they can provide tumor-associated antigens to the host immune system to recognize and induce tumor-specific immune responses, potentially representing a promising immunotherapy platform for the co-delivery of tumor vaccines and tumor-killing Ads.

[0170] Potential immunological modulation of CFCS-mediated delivery of tumor-killing Ads (oncolytic Ads) is currently being evaluated in future studies.

[0171] conclusion Some embodiments of the present invention combine oncolytic viruses with cell sheets to create an efficient delivery system for multifocal tumor treatment, which exhibits efficient amplification and sustained release, and prevents nonspecific excretion of oAds by permissive cell sheets.

[0172] In conclusion, some embodiments of the present invention demonstrate that the use of an oncolytic virus / cell sheet system can maximize the therapeutic effect of oncolytic viruses by overcoming the limitations of existing cancer gene therapy methods.

[0173] The above description of the present invention is for illustrative purposes only, and those skilled in the art will understand that the present invention can be easily modified into other specific forms without changing the technical concept or essential features of the present invention. Therefore, the above-described embodiments should be understood as illustrative and not restrictive in all respects. In particular, the embodiments described herein only exemplify the use of the present invention for some viruses, and the present invention can be modified to other virus systems.

Claims

1. a cell layer comprising somatic cells as a support; a cell layer comprising mesenchymal stem cells and into which a tumor-killing adenovirus has been introduced; The somatic cells are at least one selected from the group consisting of fibroblasts, chondrocytes, epithelial cells, myoepithelial cells, dermal cells, epithelial keratinocytes, Schwann cells, glial cells, osteoblasts, cardiomyocytes, megakaratocytes, and adipocytes; A cell sheet for preventing or treating cancer, or for preventing cancer recurrence or metastasis.

2. The cell sheet according to claim 1 for transplantation at a site where cancer has been formed or at a site where cancer has been removed.

3. The cell sheet according to claim 1 , wherein the tumor-killing adenovirus is loaded in an amount of 0.1 to 500 MOI (Multiplicity of infection).

4. A temperature-responsive culture dish containing a temperature-sensitive polymer is forming a cell layer containing somatic cells as a support; forming a cell layer containing mesenchymal stem cells on the cell layer; Introducing a tumor-killing adenovirus into a cell layer containing mesenchymal stem cells; Separating the cell sheet from the temperature responsive culture dish; The somatic cells are at least one selected from the group consisting of fibroblasts, chondrocytes, epithelial cells, myoepithelial cells, dermal cells, epithelial keratinocytes, Schwann cells, glial cells, osteoblasts, cardiomyocytes, megakaratocytes, and adipocytes; A method for producing the cell sheet according to claim 1 for preventing or treating cancer, or for preventing cancer recurrence or metastasis.

5. 5. The method of claim 4, wherein the temperature sensitive polymer is at least one selected from the group consisting of poly(N-isopropylacrylamide), poly(N-vinylcaprolactam), polycaprolactone (PCL) and polylactate-co-glycolate (PLGA).

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