JP2005269997A - Method for producing adenovirus vector - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method for producing an adenovirus vector in high efficiency. <P>SOLUTION: The method for producing the adenovirus vector containing a capsid protein obtained by packaging an adenovirus genome indispensable in packaging of the adenovirus, and a genome containing a transduction gene cassette following the adenovirus genome comprises a step of cotransfecting (1) a vector DNA comprising the adenovirus genome required for the packaging and amplification, and the transduction gene cassette, and (2) a helper adenovirus DNA containing the adenovirus genome containing a 5' ligand protein (TPC), a cassette of 5' inverse terminal repeat (ITR) of the adenovirus, a 5' loxP site, an excisable packaging signal cassette, a 3' loxP site, an E2 region except the deleted whole or part of an E1 region, an E4 region and a late gene, a 3' ITR cassette of the adenovirus, and TPC of 3', with a mammal cell, and a step of subcultivating the cotransfected product by repeating rescue and propagation. <P>COPYRIGHT: (C)2006,JPO&NCIPI

Description

  The present invention relates to a method for producing an adenovirus vector used for gene therapy and the like.

In adenovirus (Ad), a double-stranded linear DNA having a size of about 36 kb and a protein encoded by itself are covalently bonded to both ends. Since this virus is excellent in gene transfer efficiency, research as a gene for therapeutic gene transfer has been intensively conducted.
The human adenovirus genome consists of a 5 ′ inverted terminal repeat (ITR), a packaging signal (ψ), a region E1, consisting of early regions E1A and E1B, region E2, region E3, region E4, late regions L1-L5, and Includes 3'ITR. Regions E1 and E4 contain regulatory proteins, region E2 encodes a protein required for replication, and L region encodes a viral structural protein.

  When adenovirus is left in the wild, human cells are damaged by infection and propagation of the virus. Therefore, in order to use for gene therapy, recombination of the gene is necessary so that the virus does not grow. As a result of examination of adenovirus gene recombination, Patent Document 1 reports a method for deleting all or part of the E1 gene from the wild adenovirus genome, and Patent Document 2 discloses the region E1a-E1b. However, adenoviral genomes have been reported that include genomes required for replication or packaging. Such a method of deleting a specific gene of wild adenovirus and then introducing a target gene is called a first generation method. Although this method succeeded in making the vector incompletely replicated, animal experiments have shown that it involves the development of inflammation at the target site of the vector.

  In order to overcome the problems of the first generation adenovirus vector, a helper virus system in which the adenovirus region is removed from the vector and is provided by the helper virus in trans, as described in Patent Document 3, for example. Was proposed (second generation method). This vector requires the supplementation of viral regulatory and structural proteins supplied in trans for packaging and rescue.

  The helper virus system, which is the second generation method, uses a helper virus. In order to eliminate the influence of this helper virus, an attempt has been made to use a helper virus having a genome from which the E1 gene cassette and / or packaging cassette has been removed. For example, Patent Document 4 proposes a method in which a packaging cassette part of a helper virus is sandwiched between loxPs, and the packaging cassette part is deleted with an enzyme of Cre cells. Patent Document 5 discloses a technique using a packaging cassette having little homology with the wild type. The helper dependent vector using this helper virus was considered promising for gene therapy, but the replication of the helper virus and the plasmid containing the transgene competes for vector replication and gene recombination, so the yield is high. It turns out that there is a drawback of low.

  As a method for improving the yield of adenovirus vector, as described in Non-patent Document 1, the entire viral genome inserted with cosmid is efficiently propagated using adenovirus DNA-terminal protein gene. A method is disclosed. The method disclosed in this document relates to a method for propagating a first generation viral vector, and nothing is disclosed about application to the second generation.

JP 10-507928 gazette Japanese National Patent Publication No. 11-506111 Japanese National Patent Publication No. 11-503910 Special Table 2000-514312 Special table 2002-535986 gazette SANAE MIYAKE et al., Proc. Natl. Acad. Sci. USA. Vol. 93, pp. 1320-1324, February 1996

  An object of the present invention is to provide a method for producing an adenoviral vector with high efficiency.

  As a result of intensive studies to solve the above-mentioned problems, the present inventors have obtained an adenovirus vector by cotransfecting a mammalian cell with a vector DNA containing a transgene cassette and a helper adenovirus genome having TPC. The inventors have found that it can be produced efficiently and have completed the present invention.

  That is, according to the present invention, (1) a vector DNA comprising an adenovirus genome necessary for packaging and amplification and a transgene cassette, and (2) a 5 ′ ligand protein (TPC), an adenovirus 5 ′ reverse end Contains the E2 region, E4 region, and late genes except that the repeat cassette (ITR), 5'loxP site, excisable packaging signal cassette, 3'loxP site, all or part of the E1 region are deleted A step of co-transfecting a mammalian cell with an adenoviral genome and a helper adenoviral DNA containing an adenoviral 3 ′ ITR cassette and a 3 ′ TPC, and a step of repeatedly subculturing rescue and propagation Adenovirus essential for adenovirus packaging, including Sugenomu a manufacturing method of an adenoviral vector comprising a capsid protein genome is being packaged therein comprising a transgene cassette followed is provided.

  According to another aspect of the present invention, (1) a vector DNA comprising an adenovirus genome necessary for packaging and amplification and a transgene cassette, (2) a 5 ′ ligand protein (TPC), an adenovirus 5 ′ Includes an inverted terminal repeat cassette (ITR), a packaging signal cassette and an adenoviral genome containing the E2 region, E4 region, and late genes, except that all or part of the E1 gene is deleted, and a 3 ′ ITR. Adenovirus genome and subsequent transgene cassette essential for adenovirus packaging, including co-transfection of helper adenovirus DNA into mammalian cells, and repeated subculture of rescue and propagation A cap containing a genome containing A method for producing an adenoviral vector containing a sid protein is provided.

Preferably, the mammalian cell is Cre293.
Preferably, the transgene cassette is a gene cassette for the treatment of muscular dystrophy.
Preferably, cotransfection is performed by a method using calcium phosphate or a lipofection method.

  According to the present invention, it has become possible to provide a method for producing an adenovirus vector with high efficiency. The adenoviral vector produced by the method of the present invention can be used for gene therapy, antibody production (for example, vaccine), and research for testing gene regulation in vivo or in vitro.

Embodiments of the present invention will be described below.
An outline of a conventional method and a method for producing an adenoviral vector according to the method of the present invention is shown in FIG. The production of an adenoviral vector involves two steps, rescue and propagation. In the conventional method (transfection and infection method), HDAdv (helper-dependent adenoviral vector) is first rescued by transfection of vector DNA into Cre-293 cells followed by helper virus infection. The rescued HDAdv is then propagated by repeated co-infection of HDAdv and helper virus. On the other hand, in the method of the present invention (co-transfection method), HDAdv is rescued by co-transfecting vector DNA and helper virus DNA-TPC into Cre-293 cells. The rescued HDAdv is propagated as in the conventional method. After the helper virus DNA is transferred by infection or transfection, the packaging signal (Ψ) is removed by Cre-recombinase. Vector DNA containing a transgene (such as a therapeutic gene) is packaged in a viral capsid produced from helper virus DNA. Helper virus DNA is not packaged because the packaging signal is removed. In FIG. 1, vector DNA and helper virus DNA are indicated by a black rectangle and a white rectangle.

Hereafter, the component of this invention is demonstrated concretely.
1. Adenovirus Adenoviruses have no envelope, are icosahedral (having 20 equilateral triangular surfaces and 12 vertices), and are approximately 65-80 nm in diameter. Proteins called fibers protrude from each vertex. Normally, a Faber protein is composed of three identical polypeptide chains, but the length varies depending on the serotype. Adenovirus types 2 and 5 used for the production of adenovirus vectors are well characterized. The adenovirus used in the present invention can be obtained from any adenovirus. Preferably, human adenovirus serotypes, more preferably human adenovirus serotypes 1 to 47, more preferably group C serotypes (eg, serotypes 1, 2, 5, and 6) can be used. A preferred group C serotype is human adenovirus serotype 2 or 5, more preferably serotype 5.

2. The protein coat (capsid) of capsid adenovirus is composed of 252 subunits (capsomer), of which 240 are hexons and 12 are pentons. Each penton contains a penton base on the capsid surface and a fiber protein protruding from the base. For example, the Ad2 penton base protein has been found to be an 8 × 9 nm cyclic complex composed of 5 identical protein subunits each consisting of 571 amino acids. Adenovirus protein IX is also present on the surface of the viral capsid.

3. Adenovirus gene sequence Human adenoviruses consist of a linear, approximately 36 kb, double-stranded DNA genome. This is divided into 100 crossing units (mu), each of which is 360 bp in length. This DNA contains short inverted terminal repeats (ITRs) at each end of the genome that are required for viral DNA replication. The gene product is organized into early (E1 to E4) and late (L1 to L5) regions based on expression before or after initiation of viral DNA synthesis [eg, Horwitz, Vorology, 2nd edition, edited by B. N. Fields, Raven Press, Ltd., New York (1990)].

4). Vector DNA
Vector DNA is composed of an adenovirus genome and a transgene cassette necessary for packaging and amplification. Vector DNA refers to a viral vector that contains cis elements necessary for adenoviral amplification and packaging, but lacks the trans elements required for adenoviral replication and packaging. The cis elements necessary for virus production are adenovirus 3 ′ ITR, packaging signal, and adenovirus 5 ′ ITR. The transelements required for virus production are the proteins encoded by E1, E2, and E4, and the L adenovirus region. Preferably, the vector DNA lacks a nucleic acid encoding an adenoviral protein. In the present invention, vector DNA is used as a vector for gene delivery to cells in vivo. Helper virus elements useful for facilitating production and isolation of vector DNA include a low homology excisable packaging signal and a replacement E3 region.

5. Helper wade adenovirus "Helper adenovirus" refers to a virus that expresses one or more proteins necessary for viral production of vector DNA. Preferably, the helper adenovirus comprises an adenovirus 3 ′ ITR, an adenovirus packaging signal, an adenovirus 5 ′ ITR and encodes adenovirus E2 and E4 and L region proteins. Transelements not obtained by helper viruses can be obtained by other means such as cells. For example, 293 cells can be used with a helper virus that does not express such proteins to supply the E1 protein required in trans.

6). ITR
An ITR is an inverted terminal repeat that includes a 5 ′ ITR (base pairs 1-103 of the adenovirus genome); and a packaging / enhancer domain (base pairs 194-358 of the adenovirus genome). . The vector DNA used in the present invention includes all the packaging / enhancer domains, including up to base pairs 194-455 of the adenovirus genome.

7). Packaging signal For example, the adenovirus serotype 5 wild-type packaging signal is formed by at least 7 functional units called A repeats present between nt230 and nt380 of the genome. The A element has the consensus sequence TTTGN8CG (Schmid et al, 1997, J. Virol. 71, 3375-3384).

  The recombinase recognition sequence is recombined when acting based on the recombinase that recognizes the sequence, the nucleic acid is cut out, and the intervention of the nucleic acid is repeated. The recognition sequences must be sufficiently separated (at least about 180 bp) so that the nucleic acid segment to be recombined is properly positioned. Preferably, the recombinase recognition sequence is loxP or frt.

  The loxP recognition site may be present 3 ′ and 5 ′ of the packaging signal that allows excision by Cre recombinase. Cells such as 293cre cells that stably express Cre recombinase can efficiently remove nucleic acids present between loxP recognition sites. (Parks, et al., Proc. Natl. Acad. Sci. USA, 93, 13565-13570, 1996 and Parks, et al, J. Virology, 71, 4, 3293-3298, 1997). In the present invention, Cre-293 cells (described in Virology, 309 (2003) 330-338, Cre / loxp-mediated adenovirus type 5 packaging signal excision demonstrates that core element VI is sufficient for virus packaging) can be used. The first loxP site adjacent to the packaging signal is removed by homologous recombination within the packaging signal.

  For example, if the first loxP site adjacent to the packaging signal is removed by homologous recombination within the packaging signal, the resulting helper virus will not be able to excise the packaging signal, so Avoid selection.

8). Transgenes In the present invention, transgenes that are delivered and expressed by adenovirus include, for example, enzymes, blood derivatives, hormones, lymphokines such as interleukins and interferons, coagulation factors, growth factors, neurotransmitters, tumor suppressors, Examples include, but are not limited to, apolipoproteins, antigens or antibodies, and genes encoding other biologically active proteins. Specific examples of transgenes include the cystic fibrosis transmembrane regulator (CFTR), dystrophin, glucocerebrosidase, tumor necrosis factor p53, p21, herpes simplex thymidine kinase and ganciclovir, retinoblastoma (Rb), and adenosine deaminase (ADA), cytosine deaminase, erythropoietin, ornithine transcarbamylase, interleukin-2, α1-antitrypsin, thrombopoietin and the like, but are not limited thereto. Transgenes encoding antisense molecules or ribozymes can also be used. Vector DNA may include one or more transgenes under the control of one or more regulatory elements.

9. TPC
TPC is involved in the replication, rescue and integration of the adenovirus genome.

10. Specific examples of host cells the host cells are 293 cells, 911 cells, and PERC. 6 cells (International Publication WO97 / 00326), A549 cells, HER3 cells (human embryo retinoblasts transformed with Ad12), or vK-20 cells. Appropriate cells can be constructed to obtain useful proteins such as viral proteins or recombinases. Preferably, a complementary human embryonic kidney (HEK) 293 cell line (a human kidney cell line containing a functional adenovirus E1a gene that provides a trans-acting E1a protein) can be used.

11. A number of adenoviruses are available from Genbank, including adenovirus gene type Ad5. As the sequence of adenovirus, 41 types of identified human types may be used, or adenovirus known to infect other animals may be used. Adenovirus strains can be obtained from the American Type Culture Collection (Rockville, Maryland), other commercial products can be used, and other research sources You can also get it from

12 Preparation of vector DNA and helper adenoviruses Vector DNA and helper adenovirus genomes are prepared using techniques well known in the art (eg, Current Protocols in Molecular Biology, John Wiley, 1987-1988 and Sambrook, et al., Molecular Cloning, A laboratory Manual, 2nd edition, Cold Spring Harbor Laboratory Press, 1989, etc.) and the like, and the method described in the examples of this specification or a method analogous thereto.

13. Cotransfection In the present invention, mammalian cells are cotransfected with vector DNA and a helper adenovirus genome having TPC. Cotransfection can be performed by a method using calcium phosphate or a lipofection method.

14 Rescue and propagation
After the cytopathic effect (CPE) appears, the cells can be recovered, freeze-thawed, and then purified using cesium chloride centrifugation to recover the virus. Alternatively, virus purification can be performed using chromatographic methods, such as those shown in PCT / US96 / 13872.

15. The gene / protein (capsid) complex rescued adenoviral genome is packaged into a preformed protein capsid and, after cell degradation, released as an infectious virion that packages the adenoviral DNA genome. The adenoviral vector produced by the method of the present invention is replication-defective, i.e., is not capable of producing a productive infection in the host cell.

16. Measurement of titer The titer of an adenovirus vector produced by the method of the present invention can be measured using a marker or the like possessed by the vector. For example, if the marker gene LacZ is inserted, measure the titer by destroying the rescued / propagated cells, infecting some of them with COS7 cells, staining lacZ, and counting blue cells Can do. Endpoint dilution with an antibody against the adenoviral hexon protein may be used to quantify virus production or infection efficiency against target cells.

17. Transgene Expression When the adenoviral vector produced by the method of the present invention encodes a marker or other transgene, assays for examining gene expression include, for example, Northern blot, S1 analysis or reverse transcription-polymerase chain reaction. Examples include measurement of mRNA of a transgene by (RT-PCR). The presence of the protein encoded by the transgene may be detected by Western blot, immunoprecipitation, immunocytochemistry, or other methods known to those skilled in the art.

18. Composition for Delivery The adenovirus vector produced by the method of the present invention may be formed into a complex using a cationic amphiphile. Specific examples of the cationic amphiphile include DOTMA (Felgner et al., Proc. Natl. Acad. Sci. USA 84: 7413-7417, 1987); (N- [1- (2,3-dioleyl). Oxy) propyl] -N, N, N-trimethylammonium chloride); DOGS (dioctadecylamidoglycylspermine) (Behr et al., Proc. Natl. Acad. Sci. USA 86: 6982-6986, 1989); DMRIE (1,2-Dimyrostyloxypropyl-3-dimethyl-hydroxyethylammonium bromide) (Felgner et al., J. Biol. Chem. 269: 2550-2561, 1994); and CD-chol (3B [N- Cationic lipids such as, but not limited to, N ′, N′-dimethylaminoethane) -carbamoyl] cholesterol) (US Pat. No. 5,283,185).

  In addition, a composition containing an adenovirus vector is formulated with one or more cationic amphiphiles together with a neutral coexisting lipid (such as dioleoylphosphatidylethanolamine (DOPE)) into a cell. It may be easier to deliver. Specific examples of other coexisting lipids that may be used in these complexes include diphytanoyl phosphatidylethanolamine, lyso-phosphatidylethanolamine, other phosphatidylethanolamines, phosphatidylcholine, lyso-phosphatidylcholine, and cholesterol. However, it is not limited to these.

19. Administration Method When the adenoviral vector produced by the method of the present invention is used for gene therapy, the target site is not particularly limited, and for example, it can target neurons, hepatocytes, epithelial cells and the like. The administration route of the adenoviral vector produced by the method of the present invention may be administered directly to the target organ, tissue or site, or intranasal, intravenous, intramuscular, subcutaneous, intradermal, oral and Other parenteral routes of administration may be used. If desired, two or more routes of administration can be combined.

20. In the formulation of a complex containing a cationic amphiphile with a dose adenoviral vector, 10 ⁷ to 10 ¹⁰ of a preferred range of infectious units of virus is 10 ⁴ to 10 ⁶ per virus particle. Can be mixed with a range of cationic amphiphiles. The therapeutically effective human dose of the adenoviral vector produced by the method of the present invention is, for example, an adenoviral vector concentration of about 1 × 10 ⁷ to 1 × 10 ¹⁰ bfu / ml (bfu represents a blue forming unit). About 20 ml to about 50 ml of physiological saline solution containing can be administered.
The following examples further illustrate the present invention, but the present invention is not limited to the examples.

Example 1: Helper virus production
A 42 Kb cosmid pAdex1cw (assigned by Dr. Izumi Saito, Institute of Medical Science, The University of Tokyo) was cleaved with the restriction enzyme SalI, and a 4.2 Kb pAdexSal1self containing the left end of adenovirus was prepared by self-ligation. A synthetic oligo loxP sequence having a linker ligated to each site was inserted into the restriction enzyme AfLIII recognition site at nucleotide 143 (nt143) and the restriction enzyme ClaI recognition site of nt 454 from the left end of the virus. The plasmid generated by this manipulation was named pAdex-ASw-lox-sal. From this plasmid, the left end portion of adenovirus having two loxP sites was recovered by digestion with restriction enzymes EcoRI and SwaI. A cosmid pAdex-Asw-lox was prepared by ligating this fragment and a fragment of a 24 Kb adenovirus produced by digestion of EcoRI and SwaI with pAdex1cw into pAdex1cw cleaved with EcoRI. As a result, loxP was inserted into the pAdex1cw adenovirus at the left end of the packaging signal.

  In addition, the following operations were performed for the purpose of marking a helper virus and for increasing the density difference of HDAdv by making the helper virus genome as large as possible. In order to enlarge the helper virus genome, a 3.7 KbBspHI fragment of λ phage (nt 890-nt 4650 of λ phage) was blunt-ligated to the HindIII site of pGL3-Control (Promega). From this plasmid, a 6.2 Kb DNA fragment containing a luciferase expression cassette and a 3.7 Kbλ fragment was recovered by digestion with restriction enzymes MluI and SalI. This fragment was blunt ligated to the SwaI site of cosmid pAdex-Asw-lox. This produced cosmid pAdex-Asw-lox-lucλ.

  The virus was prepared by digesting the left end with EcoT22I and co-transfection with 0.5 μg of adenovirus genome covalently bound with terminal protein at the right end and 14.5 μg of the above cosmid pAdex-Asw-lox-lucλ1 into HEK293 cells.

Example 2: Establishment of Cre-293 cells
Cre was amplified from AxCANCre (TaKaRa Biomedicals) using PCR to remove the nuclear translocation signal. This was blunt ligated to the SmaI site of pCI-neo (Promega), and the resulting plasmid was designated as pCI-neo-Cre. This plasmid was linearized with BglII and introduced into HEK293 cells. Thereafter, the cells were maintained in DMEM / 10% FBS containing 400 μg / ml G418 (Sigma), and the resulting colonies were picked up and further grown in a selective medium.
The contents of Example 1 and Example 2 are described in virology, 309 (2003) 330-338, Cre / loxp-mediated adenovirus type 5 packaging signal excision demonstrates that core element VI is sufficient for virus packaging.

Example 3: Preparation of vector DNA
pBluescript KS + (STRATAGENE) was digested with KpnI and PstI, and the stump was branded and self-ligated. We digested with SacI and BamHI, branded the stumps and self-ligated to eliminate unnecessary multiple cloning sites. PacI-NotI-KpnI-SwaI-SpeI-PmeI-XhoI-NotI-PacI synthetic oligos were inserted into the remaining SmaI site. This plasmid was designated as pPNKSSPXNP.

  The ITR + packaging signal ψ was recovered by digesting pAdexSal1self with BamHI and SwaI. This was inserted into the XhoI site. ITR was also recovered by digesting pAdexSal1self with BamHI and BsrGI and inserting it into the KpnI site. The mouse Emx2 gene K2 clone (transferred from Dr. Aizawa) pEmx2K2 was used as the stuffer DNA. The SpeI / SwaI fragment (2.6 Kb) of pEmx2K2 of pEmx2K2 was inserted between SwaI and SpeI. This was designated as pPN2.6NP. Further, a 7.4 Kb SpeI fragment was inserted into the SpeI site of pPN2.6NP to obtain pPN10NP.

  The lacZ gene expressed from the RSV promoter was used as a marker. First, pRc / RSV (Invitrogen) was digested with SpeI and XbaI, followed by branding and self-ligation, leaving only HindIII as a cloning site and eliminating unnecessary sites. The LacZ gene was inserted into the remaining HindIII site. This expression plasmid was digested with NruI and NaeI to recover the LacZ expression unit that moves under the RSV promoter. This was inserted into the previous SPN site of pPN10NP to obtain pPN10LacZ.

A dystrophin expression unit for expressing Dystrophin cDNA with cytomegarovirus early gene enhancer and chicken β-actin promoter (CAG promoter) was further inserted. First, pBluescriptKS + was digested with XbaI and EcoRV, self-ligated after branding. Next, the polyA signal of enhancer / promoter / β-globin was extracted by digestion of SalI and HindIII from pCAGGS (assigned by Dr. Junichi Miyazaki and Dr. Kenichi Yamamura) and inserted into the above plasmids SalI and HindIII to make pBluescript CAG. . A mouse full-length dystrophin cDNA (transferred from Dr. Cremens) recovered from NotC from pCCL-DMD was inserted into this EcoRI site. The dystrophin expression unit was recovered by digestion with SalI and NotI and inserted into the PmeI site of pPN10LacZ. This produced pRSVLacZ-dys.
In preparing HDAdv, RSVlacZ-dys was cut with NotI and used for transfection.

Example 4: Recovery of TPC-binding helper adenovirus DNA
Recovery of TPC-linked helper adenovirus DNA was performed according to the method described in Miyake, PNAS Vol93 pp.1320-1324 Feb. 1996, Efficient generation of recombinant adenoviruses using adenovirus DNA-terminal protein complex and a cosmid bearing the full-length genome. It went as follows.
10 ⁷ HEK293 cells were infected with HEK293 cell plaque-purified helper virus. After about 1 week, cytopathic effect (CPE) was confirmed in all cells, and the culture supernatant and cells were collected. Freeze-thaw is performed to release virus particles from the cells, which are then infected with 10 ⁸ HEK293 and amplified as before. Next, 5 × 10 ⁹ HEK293 cells are infected to cause CPE, and only the cells are collected by centrifugation. Suspend it in 0.1 M Tris buffer and add 1/10 vol 5% sodium deoxycholate to disrupt the cells. Add 1.8 ml of CsCl-saturated TE to 3.1 ml of virus suspension and purify the virus in a density gradient by centrifugation at 35.000 rpm over night using Beckman Sw50.1 Rotor. After centrifugation, the virus band is recovered and once again subjected to density gradient purification under the same conditions, and the virus band is recovered to obtain a purified virus solution.

Add an equal volume of 8M guanidine hydrochloride to this purified virus solution, and then fill with 50g CsCl / 50ml 4M guanidine hydrochloride solution to fill the Beckman NVT65 rotor dedicated centrifuge tube, and centrifuge overnight at 55.000 rpm. After the density gradient is created, fractionation is performed. Add ethidium bromide to a portion of each fraction and irradiate with UV to confirm the DNA fraction.
Fractions containing DNA are collected and dialyzed against TE. After dialysis, OD260 is measured using a portion, the concentration is calculated in the same manner as normal DNA, and stored frozen at -80 ° C.

Example 5: Rescue method of HDAdv
14 μg vector DNA cut with NotI and 1 μg helper virus DNA-TPC are co-transfected into Cre-293 of a 60 mm cell culture dish by the calcium phosphate method. After transfection with Overnight, wash the crystals with PBS (-) and continue culturing in a new medium. Many cells develop CPE in about 4 days. At this point, collect all media and cells, freeze / thaw 3 times, and disrupt the cells.

  COS7 cells in a 35 mm cell culture dish are infected with 1/20 of the medium containing HDAdv. Cells are stained X-gal after 2 days. The results were captured with a microscope, LacZ positive cells were counted macroscopically, the number of HDAdv was calculated, and the titer of HDAdv was expressed as Blue forming unit (BFU).

Example 6: Propagation of HDAdv At the time of propagation, a portion of each rescue / propagation was used to infect COS7 cells for 2 days, the titer of HDAdv was determined by X-gal staining, and the same number of Cre- 293 was prepared and used for the next propagation. In other words, in propagation, one Cre-293 cell was infected with 1-2BFU of HDAdv for about 2 hours, and then helper Asw was infected with moi3-5. When the culture was continued and CPE was confirmed in all cells in about 2-3 days, the medium and cells were collected. It was frozen / thawed 3 times to disrupt the cells.

  The measurement results of Example 5 and Example 6 are shown in FIGS. FIG. 2A shows the structure of vector DNA. Vector DNA includes mouse full-length dystrophin cDNA under the control of CAG promoter / enhancer, bacterial β-galactosidase ORF driven by RSV promoter / enhancer, part of mouse Emx-2 gene (stuffer DNA), adenovirus type 5 packaging It contains a signal (Ψ) and two ITRs. In FIG. 2B, HDAdv was quantified on COS7 cells in a 6-well culture plate using 1/20 of the culture supernatant and cell lysate. Cells were infected for 2 days before X-gal staining. Even number numbers were transfected with exogenous Cre expression plasmids along with vector DNA, while odd numbers were not transfected. FIG. 2C shows the production amount of HDAdv LacZ-dys under each rescue condition. The numbers correspond to those in FIG.

FIG. 3 shows the amplification of HDAdv through a series of propagations.
White circle: HDAdv rescued by the helper virus DNA-TPC cotransfection method of the present invention was amplified by sequentially co-infecting Cre-293 cells with HDAdv and helper virus.
Black diamond shape: Rescue HDAdv was amplified by conventional transfection and infection methods.
The number of infected Cre-293 in each propagation increased in steps as HDAdv production increased.

FIG. 1 shows an outline of a conventional method and a method for producing an adenovirus vector according to the method of the present invention. FIG. 2 shows the rescue efficiency of the helper-dependent adenoviral vector under each condition. FIG. 3 shows the amplification of HDAdv through a series of propagations.

Claims (5)

(1) a vector DNA consisting of an adenovirus genome and a transgene cassette necessary for packaging and amplification, (2) a 5 ′ ligand protein (TPC), an adenovirus 5 ′ inverted terminal repeat cassette (ITR), An adenovirus genome comprising an E2 region, an E4 region and a late gene except that the 5'loxP site, excisable packaging signal cassette, 3'loxP site, all or part of the E1 region are deleted, and an adenovirus An adenovirus package comprising co-transfecting a mammalian cell with a helper adenovirus DNA comprising a 3 ′ ITR cassette of 3 ′ and a TPC of 3 ′, and repeated subculture of rescue and propagation Essential adenovirus genome and subsequent guidance Method for producing an adenoviral vector comprising a capsid protein that genome comprising a gene cassette comprising packaged therein. (1) a vector DNA consisting of an adenovirus genome and a transgene cassette necessary for packaging and amplification, (2) a 5 ′ ligand protein (TPC), an adenovirus 5 ′ inverted terminal repeat cassette (ITR), A helper adenoviral DNA containing a packaging signal cassette and an adenoviral genome containing the E2 region, E4 region and late gene except that all or part of the E1 gene is deleted, and a 3 ′ ITR; The adenovirus genome essential for adenovirus packaging, followed by the genome containing the transgene cassette, including the step of co-transfecting and the step of repeated subculture of rescue and propagation. Adenovirus containing capsid protein A method for producing a spvector. The method for producing an adenoviral vector according to claim 1 or 2, wherein the mammalian cell is Cre293. The method for producing an adenovirus vector according to any one of claims 1 to 3, wherein the transgene cassette is a gene cassette for muscular dystrophy treatment. The method for producing an adenovirus vector according to any one of claims 1 to 4, wherein the cotransfection is performed by a method using calcium phosphate or a lipofection method.