WO1996028560A1

WO1996028560A1 - Novel cell strains

Info

Publication number: WO1996028560A1
Application number: PCT/JP1996/000656
Authority: WO
Inventors: Takashi Shimada; Katsuhiko Akiyama; Hidekazu Kuma; Yousuke Suzuki
Original assignee: Hisamitsu Pharmaceutical Co., Inc.
Priority date: 1995-03-16
Filing date: 1996-03-15
Publication date: 1996-09-19
Also published as: AU4954896A

Abstract

A recombinant adeno-associated virus helper plasmid in which the ITR sequence on the genome 5' side and the ITR sequence on the genome 3' side of the wild type adeno-associated virus have been replaced by the ITR sequence of the Adenovirus; recombinant adeno-associated virus vector-producing cells having the above-mentioned recombinant adeno-associated virus helper plasmid stably integrated into the genome thereof via transfection of animal cells with the helper plasmid; and a process for producing the recombinant adeno-associated virus vector with the use of these recombinant adeno-associated virus vector-producing cells as the packaging cells.

Description

Specification

New cell line technology

The present invention relates to a recombinant adeno-associated virus vector (hereinafter, recombinant AV vector), a method for producing the same, and a cell producing the same. Background art

With the rapid development of genetic engineering, various molecular biological techniques have been developed. Along with that, 著しい remarkable progress has been made in the analysis of gene information and the elucidation of gene functions, and many attempts have been made to transfer the results obtained to actual treatment sites. One of the most advanced fields is the gene therapy field. In addition to discovering and decoding the causative genes of various hereditary diseases, methods for introducing those genes into cells by physical and chemical techniques have been developed. It has evolved before clinical applications are implemented.

遗 As for the clinical application of gene therapy, since the first clinical trial of gene therapy in the United States in 1989, clinical trials have already started in Italy, the Netherlands, France, the United Kingdom, and China. In the United States, in particular, by July 1994, 54 gene therapy protocols had been approved by the NIH Recombinant DNA Committee (RAC), and congenital immunodeficiencies (adenosine deminase deficiency) and familial Gene therapy has been attempted for genetic diseases such as resterolemia and cystic fibrosis and various cancers such as gloma and melanoma. Recently, many basic studies on gene therapy for AIDS have been made.

Gene therapy is classified into germ cell gene therapy (Germ line Cell Gene Therapy) and somatic cell gene therapy (Somatic Cell Gene Therapy) according to the type of cells (target cells) to which the gene is to be introduced. It has been. Also, additional gene therapy (Augmentation Gene Therapy) that adds a new (normal) gene while leaving the abnormal (cause) gene intact y) and Replacement gene therapy (Replacement gene gene therapy) in which the abnormal gene is replaced with a normal gene. At present, however, additional genes for somatic cells are subject to ethical and technical constraints. Only treatment is given. Furthermore, as a method of gene therapy, an autotransplantation method in which target cells are first taken out of a patient, the target gene is introduced, and then the cells are returned to the patient's body (ex Vivo) (Gene therapy) is currently being conducted, but in the future, a method of directly administering genes to patients (in vivo gene therapy) is also being studied. One of the major technical issues in clinical application of gene therapy as described above is how to introduce foreign genes efficiently and safely into target cells. In the early 1980s, application of physical techniques such as microinjection was attempted, but gene transfer efficiency was low and stable transfer was not possible. Did not connect. Later, a recombinant virus (virus vector) was developed as a vector to efficiently introduce foreign genes into target cells, and for the first time clinical application of gene therapy became possible. There are several types of virus vectors, as shown below. In the current gene therapy, the most widely used virus vector is mouse leukemia ゥ inores (MoMLV: Moloneyurine Leukemia V). irus) is a retrovirus vector that utilizes the advantages of the propagation of this virus. Retroviruses are enveloped RNA viruses that enter the cell by binding their envelope proteins to receptors on the host cell. After invasion, single-stranded viral RNA is converted to double-stranded DNA by reverse transcriptase and is randomly but stably integrated into infected cell genomic DNA. However, in order to be integrated, the cells must be dividing and proliferating (DG Miller, eta 1., Molecular and Ce11u1arBiology, 10.8.4439, 1990). The integrated retrovirus gene is called a provirus. RNA is transcribed from the provirus, and viral proteins are synthesized. To those proteins and viral RNA New virus particles are created. In this case, the retrovirus gene is recombined into a foreign gene to form a retrovirus vector (AD Mi 11 er, Current To picsin Microbiolo gy a nd Immu no lo gy, 158.1, 1992). There has been a great deal of research on retroviral vectors, especially MoMLV vectors, and many improvements have been made in their safety, and no major problems have occurred so far. However, in MoML V vectors, the integration of target cells into the genomic DNA is random, and long-term terminal repeats (LTRs), which are part of the viral gene, have a promotion activity for gene expression. Properties are known. Therefore, although there has been no report so far, it is possible to completely deny the possibility that random integration of a foreign gene could result in the activation of an oncogene that is present in the vicinity of the gene and causing the target cell to become cancerous. It is impossible, and the development of safer vectors is strongly desired. Furthermore, the most practical problem with MoMLV vectors is the inability to transfer genes into non-dividing cells. Therefore, gene repair of nerve cells, which is a problem in many congenital metabolic disorders, cannot be performed. In addition, gene transfer efficiency of hematopoietic stem cells, hepatocytes, muscle cells, etc., which are the target cells for gene therapy, is usually low in the stationary phase. Cells taken out of the body have been treated to promote division in order to increase the efficiency of gene transfer.However, it is considered difficult to transfer genes to these cells in vivo. There is a need to develop vectors that can efficiently transfer genes to non-dividing cells.

Herpesvirus vectors are expected to be vectors that can transfer foreign genes into nerve cells (TD Pa1e11a, eta1..Mo and Ce11.Biol., 8). , 457, 1988) Due to its high power and cytotoxicity, and the virus itself has a very large genome size of 150 kb, development has not progressed at present.

The HIV vector (human immunodeficiency virus) was developed as a vector that allows the introduction of specific genes into CD4-positive T lymphocytes due to the host characteristics of the virus itself (T. Shimada, eta 1 .. J. C li n. Inves t. 88. 1043, 1991). Since lymphocytes are important target cells for gene therapy such as congenital immunodeficiency disease, AIDS, and 瘙, the usefulness of HIV vectors is expected to be high. The biggest drawback of HIV vectors is the potential for contamination with wild-type strains, which, if resolved, could potentially be used for in vivo gene therapy by intravascular administration.

Adenovirus vectors have recently received the most attention because they allow the transfer of genes into non-dividing cells and can easily concentrate vectors to about 10 10. Recent studies have shown that this adenovirus vector can transfer genes into airway epithelial cells, hepatocytes, muscle cells, etc. at a high rate in vitro (LD Lavrero, et al., Hum. Gene Therapy, 1, 241, 1990, B. Qu antin. Proc. Natl. Acad. Sci. USA. 89, 2581, 1992). On the other hand, this vector has the essential property that, when a foreign * gene is introduced into cells, it will not be incorporated into genomic DNA. The effect of the introduction of messenger is lost in Rikitsuki. For this reason, gene transfer must be repeated frequently, which causes problems such as an increase in physical and mental burden on patients and a decrease in gene transfer efficiency due to the appearance of anti-adenovirus antibodies. In addition, clinical trials of transbronchially administering an adenoviral vector to the lung for the treatment of cystic fibrosis are currently underway, but this is thought to be due to the immunogenicity and cytotoxicity of adenoviral particles. It is said that an inflammatory reaction occurs.

On the other hand, the adeno-associated virus (AAV) vector has no foreign gene integrated into the target cell genome, DNA, and has no pathogenicity or cytotoxicity (N. Muzyczk a. Current T.). opicsin Microbiolo gy and Immu nolo gy. 158, 97, 1992). In addition, packaging into viral particles-ITR (Inverted Terminal Repeat) required for gene integration into genomic DNA has no promoter activity for gene expression. Setting allows for the on-off of gene expression and the use of tissue-specific promoters, Since it has a wide host range and can respond to various target cell diseases, it is expected to be a new viral vector replacing the MoMLV vector. It has also been discovered that wild-type AAV integrates at a specific position S on chromosome 19 (MS uwa d0 goand RG Roeder, Proc. Atl. Acad. Sci. USA. 82. 4394 1985), which has attracted attention as a vector that can target gene integration sites.

At present, AAV vectors are prepared at the time of use by co-transfection of a helper plasmid and a vector plasmid (R. M. Kotin, Human Gene Therapy, 5, 793. 1994). The transfection method typified by the calcium phosphate method has the following limitations: (1) The efficiency of gene transfer into cells is limited, and it is not possible to obtain a high-titer viral vector required in a clinical setting. Difficult. (2) When transfection is performed by dividing into several mouths, the transfection efficiency between the lots varies, stabilizing the virus vector with a constant titer. ③ It is difficult to prepare a large amount of vector at a time due to the complicated operation of transfection, ④ A large amount of AA

To prepare a V vector, there are several major problems: it is necessary to prepare a correspondingly large amount of plasmid for Transfection X-rays. As a means to solve these problems, packaging cell lines in which helper plasmid and / or packaging plasmid are integrated into the genomic DNA of virus vector-producing cells have been devised. This cell line is inherited by daughter cells during cell division because the DNA is stably integrated into the genomic DNA. The replacement virus can be obtained stably. Generally, in order to establish these packaging cell lines, a helper plasmid and / or a packaging plasmid carrying a drug resistance gene such as a neomycin resistance gene or a hygromycin resistance gene are produced, and a virus vector is produced. Transfection of cells to be performed. Most of the genes introduced into the cells are not integrated into the genomic DNA at this time—they are occasionally present in the cells and then disappear, but the genes can be integrated into the genomic DNA with very low probability . In order to selectively obtain only the latter cells, trans A long-term culture tie is performed in the presence of the drug corresponding to the drug-resistant gene that has been subjected to the functioning to obtain a packaging cell line. It is important to select a clone with a higher copy number in the packaging cell genome DNA to obtain a higher titer of the viral vector. By these methods, PA 317 (AD Miller, eta 1 .. Sowat. Cell Mo 1. Genet. 12. 175, 1986) and Ψ2 (R. eta 1., Cell, 33. 153, 1983), etc., and various packaging cell lines have been established and used for vector preparation in actual gene therapy. On the other hand, the establishment of a packaging cell line in the AAV vector has been tried but has not yet been achieved, and its establishment is strongly desired.

The AAV vector is expected to be a new viral vector that replaces the MoMLV vector that has been used in the field of gene therapy, but its preparation method has not been completed, and at present, helper plasmid and vector plasmid are used at the time of use. It is prepared by transfusion. However, in this method, the transfection efficiency is not constant each time, so that the titer of the vector fluctuates and stable supply cannot be performed.The transfection operation is complicated, so that a large amount of vector is prepared. There is a major problem in that a large amount of AAV vector cannot be prepared, and a large amount of plasmid vector must be prepared to match it. For the MoMLV vector, a packaging cell line has been established in which helper plasmid and vector plasmid have been stably integrated into genomic DNA, but the establishment of a packaging cell line for the AAV vector has not yet been achieved. Absent. Disclosure of the invention

The present invention has been made in view of the circumstances described above, and has as its object to provide a method for stably preparing an AAV vector in a large amount.

The present inventors have conducted intensive studies to achieve the above object, and as a result, a packaging cell for producing a novel recombinant adeno-associated virus (AAV) vector and a plasmid for establishing the packaging cell. Vector and the plasmid And succeeded in developing a method for establishing a packaging cell using a recombinant vector and a method for producing a recombinant AAV vector using the packaging cell, thereby completing the present invention.

That is, the present invention provides a recombinant adeno-associated virus HELPER plasmid in which the ITR on the 5 ′ side of the genome of the wild-type adeno-associated virus and the ITR sequence on the 3 ′ side of the genome are replaced by the ITR sequence of the adenovirus. .

The present invention also relates to a transfection of the above-mentioned recombinant adeno-associated virus helper plasmid into animal cells, wherein the recombinant adeno-associated virus helper plasmid is stably integrated into the cell genome. An adeno-associated virus vector producing cell is provided.

The present invention also relates to a wild-type adeno-associated virus, wherein the genomic sequence of the wild-type adeno-associated virus between the 5 ′ ITR and the 3 ′ ITR sequence of the wild-type adeno-associated virus is replaced by an exogenous gene sequence containing a promoter and a poly-A signal. Provided a recombinant adeno-associated virus vector plasmid.

The present invention further comprises transfecting the above-mentioned recombinant adeno-associated virus vector plasmid into the above-mentioned recombinant adeno-associated virus vector-producing cell, and stably transforming the recombinant adeno-associated virus vector plasmid into the recombinant adeno-associated virus vector. Provided is a cell producing a recombinant adeno-associated virus vector producing an exogenous gene, which is integrated into the genome of the cell producing an associated virus vector.

The present invention further provides a recombinant adeno-associated virus vector or an exogenous gene-encapsulated cell by infecting the above-mentioned recombinant adeno-associated virus vector-producing cell or exogenous transgene-encapsulated recombinant adeno-associated virus vector-producing cell with an adenovirus. Methods for propagating the production of an incoming recombinant adeno-associated virus vector are provided.

The present invention further provides a recombinant adeno-associated virus vector or a foreign adeno-associated virus vector by freezing and thawing the recombinant adeno-associated virus vector-producing cells or the exogenous gene-encapsulated recombinant adeno-associated virus vector-producing cells grown by the above method. Provided is a method for recovering a sex-gene-encapsulated recombinant adeno-associated virus vector. The present invention further provides an exogenous gene-encapsulated recombinant adeno-associated virus vector obtained by the above recovery method. BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is a schematic diagram showing the structure of helper plasmid pAAVZAd.

FIG. 2 is a schematic diagram showing the structure of plasmid psub 201 into which the entire genome sequence of the wild-type adeno-associated virus has been incorporated.

FIG. 3 is a schematic diagram showing the structure of the recombinant vector plasmid pNAV.

FIG. 4 is a schematic diagram showing the positions of primers in the rep gene used for PCR.

Figure 5 is a diagram (electrophoresis photograph) showing the results of Southern printing.

FIG. 6 shows the position in the cp gene of the probe used for Southern blotting. FIG. 7 is a diagram (photograph of electrophoresis) showing the results of Southern printing.

FIG. 8 is a diagram (photograph showing the morphology of an organism) showing the effect of the pNAV packaging cells prepared in Example 4.

FIG. 9 is a diagram (photograph showing the morphology of an organism) showing the effect of the pNAV packaging cells prepared in Example 5. BEST MODE FOR CARRYING OUT THE INVENTION

The configuration and preferred embodiments of the present invention will be described in detail below.

First, the recombinant adeno-associated virus helper plasmid in which the ITR sequence at the 5 'end of the wild-type adeno-associated virus and the ITR sequence at the 3' end of the genome of the wild-type adeno-associated virus shown in Fig. 1 have been replaced by the ITR sequence of the adenovirus. Is constructed by a known method. Here, the ITR (inverted termina 1 repeat) of the wild-type adeno-associated virus is a sequence of 145 bases at both ends of the genomic DNA of the adeno-associated virus, which has a T-shaped hairpin structure and replicates the virus. R. eta 1 .. Proc. Nat, Ac ad. Sci. USA. 79: 2077-2081. 1982 ). The ITR of adeno-associated virus has no promoter activity and is distinguished from the ITR of adenovirus having promoter activity in this regard. It is. In the present invention, a recombinant adeno-associated virus is constructed by replacing the ITR on the 5′-side of the adeno-associated virus and the ITR on the 3′-side of the genome with the ITR sequence of the adenovirus.

The ITRE sequence encoded by the helper plasmid was used to replace an animal cell-derived or virus-derived gene sequence with promoter activity, preferably an adenovirus-derived ITR sequence. It is easier to establish packaging cells described later, and more preferably, those derived from adenovirus type 8 are more likely to establish packaging cells.

The recombinant adeno-associated virus helper plasmid of the present invention has a promoter activity by introducing an ITR derived from adenovirus, and thus produces the virus shell itself without the coexistence of adenovirus. can do.

The helper plasmid thus obtained is used together with a plasmid encoding a drug resistance gene such as a neomycin resistance gene or a hygromycin resistance gene or the like (hereinafter abbreviated as drug resistance plasmid) together with a known method. Cotransfection of animal cells (for example, calcium phosphate method). The animal cell used herein is preferably a cell line that can be infected with adenovirus and adeno-associated virus. In addition, to ensure that all cell lines that have acquired drug resistance have also undergone transformation with helper brasmid, the amount of each brasmid used in cotransfection must be in molar ratios. It is preferable that the drug resistance plasmid is about 1 with respect to the helper plasmid 10. By culturing this transfectant for a long time in the presence of the drug corresponding to the drug-resistant plasmid and selecting only the drug-resistant strain, the recombinant adeno-associated virus helper plasmid is stably introduced into the cells. An integrated recombinant adeno-associated virus vector producing cell is obtained, which can be used as a packaging cell.

The packaging cells are very useful for producing a recombinant adeno-associated virus vector. As a specific example of such a packaging cell, September 1, 1994, HA-10NMS, 1-3-1, Higashi, Tsukuba, Ibaraki, Japan As FE RM P—1 450 0 Those deposited in Japan are listed. This deposit has been transferred to the International Deposit under the accession number F ERM BP-5459.

Next, a novel recombinant adeno-associated virus vector can be produced using the above-described cells producing the recombinant adeno-associated virus vector (packaging cells).

First, the coding region encoded between the ITR sequence at the 5 'end and the ITR sequence at the 3' end of the plasmid of the plasmid encoding the wild-type adeno-associated virus represented by psub201 was deleted. To construct a plasmid (vector plasmid) into which any foreign gene is inserted. That is, the genomic sequence of the wild-type adeno-associated virus between the ITR sequence at the 5 'end of the genome and the ITR sequence at the 3' end of the wild-type adeno-associated virus is replaced by an exogenous gene sequence containing a promoter and a poly-A signal. The recombinant adeno-associated virus vector plasmid is constructed by a known method. As the promoter, a promoter of human simple virus-derived thymidine kinase is preferable. In addition, drug resistance genes such as the neomycin resistance gene can be incorporated for selection.

The thus obtained adeno-associated virus vector plasmid (vector plasmid) is then transfected to the above-mentioned adeno-associated virus vector producing cell (packaging cell). After culturing for several days, the desired recombinant adeno-associated virus vector plasmid is transformed into the recombinant adenovirus by a known method (Samu1 ski R. J., eta 1., J. Viol. 63 3822 1989). An exogenous gene-encapsulated adeno-associated virus vector-producing cell integrated into the genome of the associated virus vector-producing cell can be obtained. According to this method, the plasmid vector transfected when preparing the adeno-associated virus vector is only the vector plasmid, and no helper plasmid is required. The titer of the resulting recombinant adeno-associated virus vector is the same as that of cotransfection of helper plasmid and vector blasmid, since helper plasmid has already been integrated into all cells. Higher than the price. In addition, by optionally changing the number of cells used, a recombinant adeno-associated virus vector can be produced in a necessary amount. On the other hand, the above-mentioned packaging cells are also used to re-produce the produced recombinant AAV vector. The packaging cells cultured on an arbitrary scale are infected with a recombinant AAV vector, cultured for several days, and then a recombinant AAV vector is obtained by a known method.

When producing very large amounts of recombinant AAV vectors, or when producing frequently used recombinant AAV vectors, helper plasmids and vector plasmids can be used to reduce the complexity of vector plasmid transfection. A packaging cell in which the cells are integrated together is used. This packaging cell can be established by the same method as described above using the helper plasmid and an arbitrary vector plasmid.

Further, by infecting the above-mentioned recombinant adeno-associated virus vector-producing cells or exogenous gene-encapsulated recombinant adeno-associated virus vector-producing cells with an adenovirus, a recombinant adeno-associated virus vector or an exogenous gene-encapsulated recombinant adeno-associated virus is produced. Viral vector production can be amplified.

In addition, since the adeno-associated virus vector is in the nuclear membrane of the cell, it can be translocated by freezing and thawing to obtain a high-titer vector. That is, the above-mentioned recombinant adeno-associated virus vector-producing cell or exogenous gene-encapsulated recombinant adeno-associated virus vector-producing cell is frozen and thawed to obtain a recombinant adeno-associated virus vector or exogenous gene-encapsulated recombinant adeno-associated virus. The vector can be recovered. Such freezing and thawing techniques are known to those skilled in the art.

As described in detail above, the present invention provides a method for stably preparing an AAV vector in a large amount. The method of the present invention has a very wide range of applications, and its industrial use can be expected. Example

Hereinafter, the present invention will be described in more detail with reference to examples, but the present invention is not limited to the following examples.

Example 1: Construction of Plasmid All the operations related to the construction of the brassamide were performed using a general gene manipulation method. As shown in FIG. 1, the helper plasmid pAAVZ Ad contains the ITR sequence on the 5 ′ side of the plasmid (psub 201, FIG. 2) incorporating the entire genome sequence of wild-type adeno-associated virus (hereinafter, wild-type AAV), and It was constructed by replacing the 3 'ITR sequence with the ITR sequence of type 8 adenovirus. Recombinant vector plasmid pNAV (Fig. 3) is a human simple virus-derived thymidine kinase derived from the wild-type AAV genomic sequence between the 5 'and 3' ITR sequences of psub201. And a neomycin resistance gene. Example 2: Transfection of helper plasmid into cells and selection of transfectants by drug

The transfection method was performed by the calcium phosphate method. Sterile purified water and an aqueous solution of calcium chloride were added to 10 g of pAAV / Ad obtained in Example 1 and 1 g of plasmid LH / B encoding a hygromycin-resistant gene to make a total volume of 0.5 ml. did. This mixture was added dropwise to 0.5 ml of an HBSP buffer solution with shaking, and allowed to stand at room temperature for 30 minutes to obtain a co-precipitate of calcium brasmidolate. 9 cm Dish between about 70% Konfuruen 4:00 in culture medium of Bok H e L a cells cultured in the state of the above coprecipitate was added to C_〇 ₂ Inkyubeta within one Incubation after sucrose emissions of fresh The medium was replaced with a fresh culture solution and incubated for another 2 days.

Next, in order to select only those transfectants in which the recombinant DNA was retained in the genomic DNA of the cells, hygromycin was added to the culture solution to a final concentration of 500 s / m 1, and 10 Incubated for days. The surviving cell populations (colony) were separated one by one, and further incubation was performed in a fresh culture solution to obtain a hygromycin-resistant cell line. Example 3: Gene analysis by PCR method and Southern printing method

Helper is inserted into the genomic DNA of the hygromycin-resistant strain obtained in Example 2. The stable integration of the recombinant gene derived from one plasmid was confirmed by the PCR method and the Southern printing method. Extraction of genomic DNA from the cells was carried out by a known method (B1 in N. and DW Stafford, Nucl e.c.

The rep gene contained in the helper plasmid was detected by the PCR method. The primer was set at the position shown in Fig. 4 so that a 655 bp gene fragment was amplified if the repII gene was included in the sample. As shown in FIG. 5, a 655 bp band derived from the rep gene was observed in c clone 8. In addition, genomic DNA of cell lines that were positive for the rep gene by PCR was further analyzed by Southern blotting. The probe used was a part of the cap gene radiolabeled with ³² P (Fig. 6). Genomic DNA extracted from cells is digested with the restriction enzyme XbaI, and if the helper plasmid is integrated into the genomic DNA without causing rare-rangement, it is emitted around 4.3 kb. Activity was detected. Fig. 7 shows the results. It was found that the genomic DNA of clone 8 in which the rep 解 gene was positive showed that the gene derived from the helper plasmid was stably integrated into the genomic DNA sequence without causing rearrangement. This cell line was named AAVZH e La. Example 4: Transfection of vector plasmid into established packaging cells and preparation of recombinant AAV vector

Using the packaging cells (AAVZHeLa) established in Example 3, preparation of a recombinant AAV vector was attempted. The packaging cells were cultured in a 9 cm culture dish until 70% confluent. Using Bekutapu Rasumi de pNAV 10 g constructed in Example 1, Example by a method similar to the method shown in 2 to prepare plasmid dollies phosphate calcium method coprecipitate, _c 4 of this was added to the culture dishes after standing time C0 ₂ Inkyubeta in one, and further incubated for 2 days and replaced with fresh medium. After washing twice with PBS (-), the cells were detached from the culture dish with a mixture of tribcine and EDTA, and replated on a new 9 cm culture dish. After confirming that the cells have adhered to the culture dish, neomycin is a related substance in the culture solution.

G418 was added to a final concentration of 1000 / ml. C0 ₂ Inkyu after standing for 10 days in beta in one, surviving cell population (colony) and one by one separated further continued fin incubation in fresh culture medium, packaging of producing p NA V vector Cells (pNAV packaging cells) were obtained.

Preparation of the pNAV vector followed the recombinant AAV vector preparation method of Samulski et al. Example 5: Width of Recombinant AAV Vector Using Established Packaging Cells The width of the recombinant AAV vector using the packaging cells was examined. _{C The} packaging cells established in Example 3 were cultured in a 4 cm culture dish. were seeded (4 ¹⁰⁴ cells / dish), recombinant AAV vectors prepared by the method of example 4 (PNAV vector titer ^{lxl 0 5 cfu / ml) 100} 1 together with DMEM3m 1 containing 10% FC S Was added. After incubation release C 0 ₂ incubator for 2 days, PBS (-) was washed with and re-seeded in culture dishes peeling to 9 cm cells by trypsin one EDTA mixture. After confirming that the cells adhered to the culture dish, G418 was added to the culture dish to a final concentration of 1000 g / m1. After standing 10 days C_〇 ₂ incubator base in one coater, the packaging that forms produce a p NAV vector continued for an additional fin incubation in fresh culture medium and one by one separated surviving cell population (colony) Cells (pNAV packaging cells) were obtained. The pNAV vector was prepared according to a known method for preparing a recombinant AAV vector, as in Example 4. Example 6: Assay of prepared recombinant AAV vector

The assay of the obtained virus solution was performed by the bioassay method using 3T3 cells shown below. Seeded 4x 10 ⁵ pieces of 3 T 3 cells in culture dishes 4 cm, and allowed to stand in Ichi晚C0 ₂ incubator. After washing the cells twice with PBS (-), 3 ml DMEM containing 1001 virus solution and 10% FCS was added. At this time, as a negative control, He La cells without transfection were used. For the group to which the supernatant was added, the following examination was performed in the same manner. The cells were incubated in a CO ₂ incubator for 2 days, washed with PBS (1), detached with Tribcine-EDTA, and replated on a 9 cm culture dish. After confirming that the cells were contact wear culture dish, G418 were cultured at a final concentration of 500 u / m 1 and so as to the added _c 10 days C_〇 ₂ incubator to stain the colonies by crystal bio column Bok.

As shown in FIG. 8, in the negative control group, no colonies showing resistance to G418 were found, whereas the pNAV packaging cells prepared in Example 4 were shown in Example 4. Many colonies were observed in the group treated with the known virus method and applying the obtained virus solution to 3T3 cells. This result proves that a recombinant AAV vector can be prepared by transfection of vector plasmid with AAV / HeLa.

FIG. 9 shows an assay of the virus solution prepared by the method shown in Example 5. While no colonies were observed in the negative control group, many colonies were observed in the group in which the virus solution obtained from pNAV packaging cells was allowed to act on 3T3 cells. This result demonstrates that the same recombinant AAV vector can be newly prepared by infecting AAV / HeLa with the recombinant AAV vector.

Claims

The scope of the claims

1. Recombinant adeno-associated virus helper plasmid in which the ITR sequence at the 5 'end of the wild-type adeno-associated virus and the ITR sequence at the 3' end of the genome have been replaced by the ITR sequence of the adenovirus.

2. A recombinant adeno-associated virus vector in which the recombinant adeno-associated virus helper plasmid of claim 1 is transfused into animal cells, and the recombinant adeno-associated virus helper plasmid is stably integrated into the cell genome. Producing cells.

3. The genomic sequence of the wild-type adeno-associated virus between the 5 ′ ITR sequence and the 3 ′ ITR sequence of the wild-type adeno-associated virus is replaced by an exogenous gene sequence containing a promoter and Bol A signal. Recombinant adeno-associated virus vector plasmid.

4. The above recombinant adeno-associated virus vector plasmid is transfected into the above-mentioned recombinant adeno-associated virus vector-producing cells, and the recombinant adeno-associated virus vector plasmid is stably transfected with the recombinant adeno-associated virus vector. An exogenous gene-encapsulated recombinant adeno-associated virus vector-producing cell integrated into the genome of a vector-producing cell.

5. Recombinant adeno-associated virus vector or exogenous gene by infecting the recombinant adeno-associated virus vector producing cell of claim 3 or the exogenous gene-encapsulated recombinant adeno-associated virus vector producing cell of claim 4 with adenovirus. A method of propagating the production of an encapsulated recombinant adeno-associated virus vector.

6. Freezing and thawing the recombinant adeno-associated virus vector-producing cell or the exogenous gene-encapsulated recombinant adeno-associated virus vector-produced cell grown by the method of claim 5 to thereby obtain the recombinant adeno-associated virus vector or exogenous cell. A method for recovering a gene-encapsulated recombinant adeno-associated virus vector.

7. A foreign vector-encapsulated recombinant adeno-associated virus vector obtained by the method of claim 6.