JP2002528087A - Improved AAV vector production - Google Patents

Abstract

  (57) [Summary]   The present invention relates to genetically engineered viral vectors, particularly adeno-associated virus (AAV) vectors, for use in gene therapy. The present invention is provided with a nucleic acid encoding at least one gene product that provides an AAV helper function that is substantially free of a helper virus such as an adenovirus and is capable of producing AAV without producing a related helper virus. , High titers of recombinant adeno-associated virus (AAV) packing cells.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001] The present invention relates to the field of genetically engineered viral vectors for gene therapy, and more particularly to adeno-associated virus (AAV) vectors.

[0002] Adeno-associated viruses are non-pathogenic human parvoviruses (Berns, 1
990a, Berns, 1990b). This virus replicates as a single-stranded DNA of approximately 4.6 kb. The plus and minus strands are packaged and infectious. Efficient replication of AAV requires co-infection of cells with a helper virus. Viruses that have been identified to support AAV include adenovirus, herpes simplex virus (HSV), cytomegalovirus (CMV)
And pseudorabies virus (Berns, 1996). In the absence of the helper virus, no substantial replication of AAV is observed. Therefore, AAV is also classified as a dependent virus. In the absence of a helper virus, the AAV genome can be integrated into the host genome. Wild-type virus is the first
Strong preference (70%) at the integration site (q13.3-q ter) of the long arm of chromosome 9
(Kotin et al., 1990, Samulski, 199).
3, Samulski et al. 1991). After integration, viral gene expression is undetectable. The integrated provirus is replicated as a normal part of the host cell genome during division of the transfected cells, resulting in daughter cells. This stage of the virus life cycle is known as the latent stage. This incubation stage is stable, but can hinder helper virus infection of the transgene. After infection with the helper virus, AAV is excised from the host genome and begins to replicate. During the early stages of this lysis cycle, the rep gene is expressed. After about 12-16 hours, the capsid proteins VP1, VP2, and VP3 are produced in detectable amounts, and the replicated viral DNA is packaged in virions. A schematic diagram of the AAV genome and its genes is shown in FIG. Virions accumulate in the nucleus of cells and are released when lysed as a result of AAV and helper virus accumulation (Bern
s, 1990a, Berns, 1990b). Thus, six serotypes of primates have so far been characterized (Berns et al., 19).
94, Ruttlege et al. , 1998). The AAV genome contains two genes, rep and cap (FIG. 1). Three promoters (P5, P19
, And P40) have four Rep proteins (Rep78, Rep68, Re40).
p52, and Rep40) and three capsid proteins (VP1, VP
2, and drives the synthesis of mRNA encoding VP3). The AAV genome
Flanking both ends of a 145 bp sequence called the inverted terminal repeat (ITR), which is thought to contain all cis-acting sequences necessary for viral integration, replication and capsid formation (Lusby et al. , 1980, Samuls
ki et al. , 1989).

[0003] During a productive infection, the P5 promoter is first activated, directing the production of Rep 78 and Rep 68. These proteins contain the viral gene AVV
Essential for replication and trans regulation. REp52 and Rep40 are expressed from the P19 promoter and are thought to be involved in the packaging of the AAV genome (Chejanovsky and Carter, 1989, Smit).
and Kotin, 1998). Capsid proteins VP1, VP2,
And VP3 are produced from a 2.6 kb transcript of the AAV P40 promoter, which is spliced into two 2.3 kb mRNAs by using the same splice donor and two different splice acceptor sites. Splice acceptor sites are located at both ends of the VP1 transcription initiation signal. VP1 is
Translated from a messenger using a splice acceptor directly before the VP1 translation initiation codon. VP2 and VP3 are VP1 ATG acceptor 3
Translated from messenger mRNA spliced to Proteins VP2 and VP3 are either ACG translation initiation (Vp2) or downstream ATG (VP3)
Translated from this messenger by use of Since all three coding regions are in frame, the capsid proteins share a large domain with identical amino acid sequences. VP3 is completely contained in VP1 and VP2, the latter two containing additional amino-terminal sequences. Similarly, VP1 contains the complete VP2 protein but contains additional N-terminal sequences. All three capsid proteins terminate at the same position (Ruffing et al., 19).
94). AAV capsids are 20-40 nm in diameter (Berns a
nd Bohensky, 1987, Srivastava et al. , 1
983), about 5% VP1, 5% VP2, and 90% VP3. This ratio is
Relative abundance of alternatively spliced messengers and AC of VP2
It is believed to reflect a reduction in translation initiation efficiency at the G initiation codon.

[0004] Adeno-associated virus vectors can be made by replacing the rep and cap genes of wild-type AAV with sequences of interest. For the production of recombinant viruses, the related helper virus that has infected human cells is re-expressed by different means.
It is necessary to supply the p gene and the cap gene. This is routinely done by transfection of a so-called packaging plasmid that confers an AAV packaging function, containing the AAV rep and cap genes but lacking the AAV ITR. Recombinant AAV is typically produced by co-transfection of a helper virus-infected cell with a plasmid containing the recombinant AAV and a packaging plasmid. Recombinant virus is typically recovered from this culture 48-72 hours after cell transfection. Recombinant AAV produced by this method can be produced at high titers and essentially free of wild-type AAV (Allen et al., 1997, Samulski et.
al. , 1989). Cells are also associatedly infected with a helper virus (usually an adenovirus) so that this helper virus is also produced (Cla
rk et al. , 1997, Flotte et al. , 1993, He
rzog et al. , 1997, Monahan et al. , 1998
, Snyder et al. , 1997a). AVV replication and packaging can also be performed in vitro using cell free systems (Hong et a
l. , 1992, Hong et al. , 1994, Ni et al. , 1
994, Zhou and Muzyczka, 1998, Ward et a.
l. , 1998).

[0005] The presence of helper virus in rAAV preparations is undesirable. Related helper viruses are potential pathogens and are unacceptable for clinical use, even for small amounts of helper virus contamination of recombinant AVV preparations. Several methods are used to remove helper virus from recombinant AAV preparations. For adenovirus,
These include differences in density and temperature sensitivity. AAV particles are 1.41-1
. Adenovirus whereas a density of 45 g / cm ³ is 2-5 g / cm ^3, the helper virus is most commonly used is the density of 1.33 g / cm ^3. This difference is used to separate the two viruses by density gradient centrifugation (Clark eet al., 1997; Herzog et al.
, 1997). Differences in temperature sensitivity can also be used to remove contaminated adenovirus. Adeno-associated virus particles are more resistant to heat treatment than adenovirus particles. Routinely, the recombinant AAV preparation is incubated at 56 ° C. for 1 hour. Recombinant AAV is resistant to this treatment, whereas adenovirus (helper virus) is not (Flotte et al., 1993, M
onahan eet al. , 1998, Snyder et al. , 19
97a). Although these methods are sufficient to remove most helper viruses, they are not ideal for recombinant AAV clinical applications. For one reason, clinical applications require the production of large amounts of recombinant AAV. This means that large quantities of helper virus are also produced, after which the rAAV preparation must be completely removed. Furthermore, it is difficult to confirm the absence of a helper virus.

The present invention relates to the production, production, and purification of genetically engineered viral vectors designed to transfer and express a gene of interest into mammalian cells. The present invention provides methods for producing high titers of recombinant adeno-associated virus (AAV) vectors that are essentially free of helper viruses such as adenovirus. Some viruses can provide helper functions for AAV. Adenovirus helper functions are constantly being best characterized. In adenovirus, four regions have been identified that require fully permissive AAV infection. These are the E1, E2a, E4orf6, and VA regions. In El, genes from the Ela and Elb regions are important. The role of these genes has been discovered and characterized studies have been reviewed (Carter, 1990).
. HSV can also function as a helper virus for AAV. So far, the identified HSV gene with helper virus function has been
Contains the P8 and IPC4 genes (viral DNA polymerase and possibly viral helicase) (Berns, 1996). The present invention provides an adeno-associated virus (AAV) packaging cell provided with a nucleic acid encoding a gene product that provides an AAV helper function capable of producing a recombinant AAV without producing an associated helper virus.

More particularly, the present invention relates to methods, cell lines, recombinant adenoviral vectors, and recombinant DNA molecules that are particularly suitable for the large scale production of high titers of recombinant AAV strains free of replicable adenovirus. I will provide a. In normal cells, AAV replication and packaging are undetectable. However, low levels of replication and packaging can be induced in the absence of helper virus function. AA on a small scale
Several methods have been published for inducing proliferative replication of V. These include treatment of cells with cytostatic drugs or UV irradiation (Yacobson et a.
l. , 1989), but are not suitable for large-scale production of high titer recombinant AAV strains that do not contain a replicable helper virus. The present invention provides a structurally better solution to completely avoid helper virus production during recombinant AAV production. Prevention of helper virus production eliminates the need for laborious purification and subsequent confirmation and testing of the preparation. The present invention relates to AAV
Methods are provided for eliminating the production of helper virus during AAV production by eliminating the helper virus required for replication.

In recent years, improved methods for producing recombinant AAV have received much attention. Various methods of expression of the AAV genes, rep and cap, have been found to improve the yield of recombinant AAV over standard methods (Allen et al., 1997).
Conway et al. , 1997, Li et al. , 1997, V
incent et al. , 1997). In addition, helper virus function is under investigation and methods have been found to improve both the quality and yield of recombinant AVV preparations (Ferrari et al., 1997; Ferrari et a).
l. , 1996, Xiao et al. , 1998b). In one aspect of the invention, the adenovirus E2A gene and the further required helper function are expressed, and the further required helper function is the same as the E2A helper function already present in the packaging cell that induces RCA formation. Provide packaging cells that do not carry overlapping sequences. Preferably, the E-2A gene is an adenovirus ts1
Derived from 25. In another embodiment of the invention, the adenovirus E1 region and the additional required helper function are expressed, and the additional required helper function is a sequence that overlaps the E1 helper function already present in the packaging cell that induces RCA formation. Provide packaging cells that are not retained. In one particular aspect of the invention,
Packaging cells include PER cell lines. The PER cell line is a fully characterized plasmid containing the human adenovirus 5E1 region (WO 97/0032).
It can be produced from normal human embryonic retinoblasts (HER) immortalized in 6). PER cells are particularly useful for preventing the formation of RCA in combination with a novel E1-deleted adenovirus vector that does not carry sequences overlapping the E1 region present in PER cells (WO 97/00326). In one aspect of the invention, the PER cells are provided with additional necessary helper virus functions by insertion of an E1-deficient adenovirus that does not contain a sequence that overlaps with the E1 sequence already present in the PER cells that induces RCA formation. . In another aspect of the invention, the PER cells contain a plasmid DNA containing a gene encoding a helper virus function (this plasmid does not contain a sequence that overlaps with the E1 sequence already present in the PER cells that induces RCA formation). To provide additional necessary helper virus functions. Examples of such PER cells include Per. C6 is Center fo
r Applied Microbiology Research (CAMR
) Has been deposited under accession number 9602940 ECACC. The most commonly used cell lines for rAAV production are HeLa and 293. Although these cell lines are widely used, there are some disadvantages. H
Since eLa cells are derived from human cancer, they carry one or more oncogenes in their DNA. It is believed that some chromosomal DNA will be co-packaged with the viral vector produced for these cells and will be present in the patient's target cells. Compared to HeLa cells, 293 cells have the advantage that they are not derived from human cancer. However, 293 cells are stably transfected with some adenovirus sequences and consequently express the El gene (Gr
aham et al. , 1977a). This E1 gene expression was
(Herzog et al., 1997, Snyder).
et al. , 1997b, Zhou et al. , 1998). However, 2
The 93 cell line has drawbacks. Not only does the E1 region stably integrate into cellular DNA. From the left half of the adenovirus genome, it is known that the cell line carries at least the adenovirus 5 sequence 1-4344, including the left ITR, the packaging signal, the El gene, and the gene encoding protein IX (Lo
uis et al. , 1997). Not only the presence of the E1 sequence alone, but leaving significant regions at both ends that overlap with the most commonly used E1-deleted adenovirus vectors or deletion mutants (such as d1312 (Snyder et al., 1997b)). ing. The overlapping region is sufficient for homologous recombination between the most commonly used E1-deleted adenovirus vector and the adenovirus 5 sequence in 293. By such homologous recombination, replicable adenovirus (RCA) (H
ehir et al. , 1996). Especially in large scale preparations, the presence of RCA in E1-deleted adenovirus vector strains is problematic (Imler et al., 1996, Lochmuller eet).
al. , 1994). (Ferrari et al., 1996) and WO
No. 96/40240 discloses an XbaI-digested Dba from adenovirus d1309 to obtain an adenovirus helper function for the production of recombinant AAV.
Includes transfection of 293 cells with 35,000 bp DNA isolated from NA. This technique is not ideal because this XbaI fragment significantly overlaps with the adenovirus sequence in 293, which allows for the accidental production of a replicable adenovirus. Another disadvantage is that d1309 has DNA inserted into the E3 region. A subtle adjustment of this technology has produced adenovirus helper plasmids that lack adenovirus genes while retaining helper virus function for recombinant AAV production (WO97 / 17458, Ferra).
ri et al. , 1997, Li et al. , 1997, Xiao e
t al. , 1998a). The use of these adenovirus late gene deletion helper plasmids to avoid RCA is generally limited to 293 cells.
As described above, this cell line has several disadvantages, and one further disadvantage of the 293 cell line is that it expresses only the E1 region, thus requiring additional helper functions for efficient large-scale production of recombinant AAV. And that this function must be supplied separately. Moreover, culturing 293 cells is considered difficult.

[0009] In another embodiment of the present invention, there is provided a stable packaging cell expressing adenovirus regions E1 and E2a, for example from adenovirus ts125. In this preferred embodiment of the invention, the functional expression of E2a can be adjusted to optimize the yield of recombinant AAV. Further helper functions required
Obtained in the form of an E1, E2a-deleted adenovirus or in the form of a plasmid DNA containing a gene encoding a helper virus function, the helper adenovirus vector or plasmid DNA is used in the packaging cells of the present invention to induce RCA formation. Does not contain sequences that overlap with existing helper virus functions. In this preferred embodiment, the E2a helper function may be additionally provided to the packaging cell if the method does not transfer a sequence that overlaps the E1 region already present in the packaging cell that induces RCA formation. it can.

[0010] In a preferred embodiment of the invention, adenovirus late gene expression is either interposed in the transcript of the late gene or the removal of one or more coding genes from DNA encoding further necessary helper functions. Essentially suppresses.

In another aspect of the invention, the cells of the invention are grown in large numbers for production to produce recombinant A
The AV is recovered and purified. For the production of recombinant AAV, the cells are treated with recombinant AAV.
Supply DNA, genes including AAV rep and cap genes, and DNA including helper virus functions. In a preferred embodiment of the present invention, A
The rep and cap genes of the AV are physically linked to the plasmid DNA such that they are present in exactly the same molecule, further obtaining the necessary helper functions. The cells can be supplied with the DNA required for the production of recombinant AAV just before the start of recombinant AAV production, in which case for each production the cells will have to
NA needs to be supplied. The method can be any method suitable for transfecting or infecting a large number of cells with DNA. In a particularly preferred embodiment of the invention, the DNA required for recombinant AAV production is poly (2- (dimethylamino) ethyl-10-4-aminobutyl) phosphazene or other poly (organo)
Transfect PER cells with phosphazene. Alternatively, recombinant A
Part of the DNA required for AV production can be stably integrated into PER cell chromosomal DNA.

In another aspect of the present invention, a recombinant AAV, a cell of the present invention, is produced using a packaging cell of the present invention grown in suspension culture using a fully defined serum-free medium. Let it.

In one embodiment of the present invention, there is provided a method of producing a packaging cell comprising all the helper functions required for the AAV replication cycle, wherein the helper functions do not include duplication that induces the formation of a replicable helper virus. . In a preferred embodiment of the invention, the packaging cells are stably transformed with an adenovirus E1 region that does not further overlap with the required helper functions. In a particularly preferred embodiment of the present invention,
The packaging cells have been stably transformed with the E1 and E2a regions.
In this particularly preferred embodiment of the invention, the E2a function can be switched on and off freely according to the signal. In a preferred embodiment of the present invention,
Since the E2a gene is derived from the adenovirus mutant H5ts125, the signal switches with temperature. In another particularly preferred embodiment of the invention, the packaging cells are stably transformed with the adenovirus 5E1 region, the E2a gene, and the adenovirus 5VA region (Martinez et al., 1989) and / or the adenovirus 5E4orf6 gene. I do. In this particularly preferred embodiment, the adenovirus 5VA region and / or adenovirus 5E
Regulates the transcriptional activity of the 4orf6 gene. This means that the transcription activity can be freely switched on or off according to the signal.

As used herein, the term “further required helper function” also refers to a recombinant gene in which the encoding gene is not stably integrated into the DNA of the recombinant AAV-producing cell or where further expression is desired. Refers to a helper virus function that enables efficient (large-scale) production of AAV. Such additional necessary helper functions can be performed using any viral or non-viral method (poly (organo)) capable of introducing foreign genetic material into mammalian cells.
Phosphazene, polyethyleneimine, calcium phosphate precipitation, electroporation, recombinant, lipid or liposome-mediated gene transfer, including, but not limited to).

In one embodiment of the present invention, there is provided a packaging cell requiring only additional AAV packaging functions and a recombinant AAV vector for recombinant AAV production. The packaging cells contain stably integrated adenovirus DNA
Includes and provides the necessary adenovirus helper functions of the origin. In one aspect of the invention, the helper function is provided by a stably integrated E1 region. In another aspect of the invention, the helper function is provided by a stably integrated E1 region and a stably integrated E2a gene. In this particular embodiment of the invention, E
The 2a function can be switched on or off freely according to the signal. In a preferred embodiment of the invention, the signal switches with temperature, since the E2a gene is derived from the adenovirus mutant H5ts125. In another particularly preferred embodiment of the present invention, the packaging cell comprises the adenovirus 5E1 region, the E2a gene, and the adenovirus 5VA region (Martinez et.
al. , 1989) or the adenovirus 5E4orf6 gene or both. In this particularly preferred embodiment, the adenovirus 5VA
Regulates the transcriptional activity of the region and / or the adenovirus 5E4orf6 gene, which means that the transcriptional activity can be switched on or off freely according to the signal.

[0016] The present invention provides a cell culture comprising the cells of the present invention. Large scale production of recombinant vectors for human gene therapy requires easy and scalable culture methods for producer cell lines, and suspension cultures (ie, non- Serum media) or other large scale cultures (such as bioreactor cultures) are preferred. Several systems have been devised for propagation into large numbers of mammalian cells. These include, but are not limited to, rolling bottle cultures, cell cubes, and bioreactors. Each of these systems has advantages and disadvantages. Bioreactors in which cells grow in suspension are the easiest to normalize and scale up to larger scales. However, one disadvantage is that cells in suspension are not easily transfected. Many different cell cultures are developed to support optimal growth of many different cells. Most of these media are supplemented with bovine serum based on a modification of Dulbecco's Modified Eagle's Medium (DMEM). The present inventors adapted the cells of the present invention to a suspension medium using a defined serum-free medium. Serum-free medium is
It has the advantage of being completely clear, as it does not depend on natural serum sources which can vary in the quality and presence of the foreign factor. These serum-free media contain alternatives to essential components for cell growth in serum.

The present invention also provides methods of producing recombinant adeno-associated viruses, including the use of the cells or cell cultures of the invention, and the use of these adeno-associated virus vectors in gene therapy.

Although the invention has been described on the basis of AAV-2, other AAV serotypes (1 and 3-5) or serotypes not yet discovered can also be applied for the same purpose. Is evident. Dependent viruses common to other species can also be used for the same purpose, for example, infecting human cells with canine adeno-associated virus. In addition, human AAV replicates in as many mammalian cells as the species-specific adenovirus is present, and each species-specific adenovirus can be used to generate dependent virus from other species by the cells and methods of the invention. Can be produced. Non-limiting examples of non-primate dependent viruses include birds,
It is an adeno-associated virus of dogs and cattle (Berns, 1996). For example, it will be apparent to one of skill in the art that adenoviruses 1-4, 6-51, or other human or animal adenoviruses can be engineered for the same purpose if the functions of the gene products are similar. A similar AAV helper function is obtained, but with a different virus (
Gene products derived from, but not limited to, HSV, CMV, and pseudorabies virus), from other natural sources, or produced in synthetic form, for the same purpose. Can be.

The present invention is further described in the experimental part and drawings herein, which do not limit the invention.

(Experimental part) Materials and methods. DNA construct. Packaging plasmid pIM4
5 (7.3 kb) contains the AAV-2 rep and cap genes (M
cCarty et al. , 1991). It was a gift from Dr. Zolotukhin. pACV-βgal (8.3 kb) is AAV
-A plasmid containing the CMV-LacZ expression cassette between the ITRs, A. It was a gift from Dr. Kleinschmidt. Plasmid pIG. E1A. E1B regulates Ad5 under the transcriptional control of the human PGK promoter.
Comprising the Ela and Elb genes (nucleotides 459 to 3510 of Ad5);
This is described in WO / 97/00326. Plasmid pE2a is also known as plasmid pcDNA3wtE2A, described below. Plasmid pE4or
f6 was generated by insertion of a 929 bp fragment encoding the Ad5 E4orf6 protein into the BamHI site of pCMV / neo (Hinds
et al. , 1990).

PBr / Ad. Bam-rITR (ECACC deposit p97082122). I
To facilitate blunt-end cloning of TR sequences, wild-type human adenovirus type 5 (Ad5) DNA was treated with Klenow enzyme in the presence of excess dNTPs. After inactivation of the Klenow enzyme and phenol / chloroform extraction and ethanol precipitation, the DNA was digested with BamHI. This DNA preparation was used without further purification in a ligation reaction using vector DNA derived from pBR322 prepared as follows: pBR322 DNA was used for EcoRV and BamH
I, digested with TSAP enzyme (Life Technologies), dephosphorylated and purified on LMP agarose gel (SeaPlaque GTG). Competent E. After transformation into E. coli DH5a (Life Tech.) and analysis of ampicillin resistant colonies, one clone showing the expected digestion pattern was selected for inserts from the BamHI site in Ad5 to the right ITR. Cloning border in right ITR (cloning b
order) revealed that most of the 3′G residue of the ITR was missing, and the rest of the ITR was found to be valid.

PBr / Ad. Cla-Bam (ECACC Accession No. P97082117).
Wild type adeno type 5 DNA was digested with ClaI and BamHI and 20.6 kb
Fragments were isolated from the gel by electroelution. pBR322 was digested with the same enzymes and purified from agarose gel by Geneclean. Both fragments were ligated and transformed into competent DH5α. The resulting clone pBr / Ad. Cla-Bam was analyzed by restriction enzyme digestion and 91
It was found to contain an insert with 9-21566 bp of adenovirus sequence.

PBr / Ad. AflII-Bam (ECACC accession number P97082114)
). Clone pBr / Ad. Cla-Bam was replaced with EcoRI (in pBR322)
And partially digested with AflII. After heat denaturation of AflII at 65 ° C. for 20 minutes, the ends of the fragment were filled with Klenow enzyme. The DNA is then transferred to Pac
Blunt -ended double -stranded oligo linker containing an I site (5'-AATTGTC TTAAT
TAA CCGCTTAA-3 ′). This linker is used for the following 2
Oligonucleotides: 5'-AATTGTCTTAATTAACCGC-3
'And 5'-AATTGCGGTTAATTAAGAC-3' were made by annealing and blunting with Klenow enzyme. The ligated DNA is precipitated and the buffer exchanged, which is digested with excess PacI enzyme to remove oligo concatemers. A 22016 bp partial fragment containing the sequence from 3534 to 215666 bp and the vector sequence was ligated to LMP agarose (Seaa).
Plaque), religated and transformed into competent DH5α. One clone, containing a PacI site and found to retain a large adenofragment, was selected and the 5 'end was sequenced to determine the P.
The correct insertion of the acI linker was evaluated.

PBr / Ad. Bam-rITRpac # 2 (ECACC accession number P9708)
2120) and pBr / Ad. Bam-rITR # 8 (ECACC deposit number P
97082121). Clone pBr / Ad. I of Ad5 in Bam-rITR
About 190 nucleotides between the ClaI site and the start of the ITR sequence in the pBR322 backbone were removed to allow insertion of a PacI site near the TR. This was performed as follows. pBr / Ad. Bam-rITR to Cl
digested with aI and nuclease B for various times (2, 5, 10, and 15 minutes)
treated with all3l. After removal of the nucleotides, reactions were performed individually on pBR322 DNA (also digested at the ClaI site) using the same buffer and conditions. The Bal31 enzyme was inactivated by incubation at 75 ° C. for 10 minutes, the DNA precipitated and resuspended in a smaller volume of TE buffer. The DNA was further treated with T4 DNA polymerase in the presence of excess dNTP to check for blunt ends. PBR322 DNA with SalI (control)
After digestion, satisfactory degradation in samples treated for 10 or 15 minutes (approximately 1
50 bp). PBr / Ad. Treated for 10 or 15 minutes. Ba
The m-rITR sample was ligated to the blunt-ended PacI linker described above (see pBr / Ad.AflII-Bam). The ligation was purified by precipitation, digested with excess PacI, and separated from the linker on an LMP agarose gel. After replication, the DNA was transformed into competent DH5α and colonies were analyzed. Ten clones were selected that showed deletions of approximately the desired length and were selected for T-track sequencing (T7 sequencing kit, Pharmacia Biote).
ch)). A clone was found with a PacI linker inserted just downstream of the rITR. After digestion with PacI, clone # 2 bound to the ITR is 28 bp and clone # 8 is 27 bp.

PWE / Ad. AflII-rITR (ECACC Deposit No. P9708211)
6). The larger Ad5 insert was cloned using the cosmid vector pWE15 (Clontech). First, a linker containing a unique PacI site was inserted into the EcoRI site of pWE15 to allow pWE15. Pac was produced. For this reason, pBr / Ad. The double-stranded PacI oligo described in AflII-Bam but additionally having its EcoRI overhang was used. The following fragments were then isolated from the agarose gel by electroelution: PacI digested pWE15. Pac, PacI and BamHI digested pBr /
Ad. AflII-Bam, and BamHI and PacI digested pBr / A
d. Bam-rITR # 2. These fragments were ligated together and packaged using λ phage packaging extraction (Stratagene) according to the manufacturer's protocol. After infection with host bacteria, colonies were grown on plates and analyzed for the presence of the complete insert. pWE / Ad. Af
The II-rITR contains the entire adenovirus type 5 sequence from 3534 (AflII site) to the base containing the right ITR (lacking most 3'G residues).

PWE / Ad. Δ5 '. Construct pWE / Ad. Δ5 ′ is the two adenovirus ITRs and the entire adenovirus sequence between 3510 and 35938 bp (ie, the complete adenovirus genome except for El and the packaging signal)
It is an example of the replication molecule of the present invention containing pWE / Ad. Δ5 ′ was generated on a cosmid vector background from three fragments. First, the 5'ITR from Ad was converted to the following primer: ITR-EPH: 5'-CGG-AAT-TCT-TAATTA-AGT-TA
A-CAT-CAT-CAA-TAA-TAT-ACC-3 'and ITR-PIX: 5'-ACG-GCG-CGC-CTT-AAG-CCA-C
GC-CCA-CAC-ATT-TCA-GTA-CGT-ACT-AGT-C
Amplification was performed using TA-CGT-CAC-CCG-CCC-CGT-TCC-3 '. The resulting PCR fragment was digested with EcoRI and AscI and digested with the same enzymes, vector pNEB193 (New England Biol).
abs). The resulting construct is called pNEB / ITR-pIX. By sequencing, the expected sequence of GenBank (accession number M732)
60 / M29978) (i.e., excess G residues
PIX promoters (3511-35) except for those found just upstream of the II site.
Ad5 sequence in the left ITR (Ads from 1 to 103) linked to the Ad5 sequence of No. 38
(5 sequences). This ITR-pIX fragment was
Ec isolated with oRI and AflII and containing the Ad5 sequence 3539-21567
ligated into the oRI-AflII vector. The latter fragment is called pBr
/ Ad. Obtained by digestion of Cla-Bam with EcoRI and partial digestion with AflII. The resulting clone is called pAd / LITR (Δ5 ′)-BamHI. The final construct pWE / Ad. The cosmid vector pWE15. PAd of Pac (supra) digested with PacI / BamHI
/ LITR (Δ5 ′)-pB digested with BamHI and PacI / BamHI
r / Ad. Bam-rITR. pac # 2 (supra).

PWE / Ad. AflII-rITRΔE2A. This cosmid contains pWE / Ad. Except for the deletion of the E2A coding sequence. It is essentially identical to AflII-rITR (ECACC accession number P97082116). pWE / Ad. AflII-r
Deletion of the E2A coding sequence from the ITR (ECACC accession number P97082116) was performed as follows. Adenovirus sequences flanking the E2A coding region on the left and right sides were compared to Expand PCR according to the manufacturer's protocol.
Plasmid pB in a PCR reaction using the system (Boehringer)
R / Ad. Sal. Amplified from rITR (ECACC deposit number P97082119). The following primers were used. Right flanking sequence (corresponding to Ad5 nucleotides 24033-25180): ΔE2A. SnaBI: 5'-GGC. GTA. CGT. AGC. CCT. GT
C. GAA. AG-3 ′ ΔE2A. DBP-start: 5'-CCA. ATG. CAT . TCG. AA
G. FIG. TAC. TTC. CTT. CTC. CTA. TAG. GC-3 '. Replicated DNA fragments were digested with SnaBI and NsiI (NsiI was generated in primer ΔE2A.DBP-start (underlined)). Left flanking sequence (corresponding to Ad5 nucleotides 21557-22442): ΔE2A. DBP-stop: 5'-CCA. ATG. CAT . ACG. GCG
. CAG. ACG. G-3 ′ ΔE2A. BamHI: 5'-GAG. GTG. GAT. CCC. ATG. GA
C. GAG-3 '. Replicated DNA fragments were digested with BamHI and NsiI (NsiI was generated in primer ΔE2A.DBP-stop (underlined)). The digested DNA fragment was then digested with SnaI / BamHI digested pBr / Ad. S
al-rITR and ligated into pBR. Ad. Sal-rITRΔE2A was obtained. By sequencing, plasmid pBR. Ad. Correct replacement of the DBP coding sequence with a unique NsiI site in Sal-rITRΔE2A was confirmed.

Next, the cosmid pWE / Ad. AflII-rITRΔE2A was prepared. Plasmid pBR. Ad. Sal-rITRΔE2A was purified from BamHI and Spe
I digested. 3.9 Kb with E2A coding region replaced with a unique NsiI site
Was isolated. pWE / Ad. AflII-rITR cosmid
Digested with BamHI and SpeI. BamHI / S containing E2A coding sequence
A 35 Kb fragment with the peI fragment removed was isolated. Fragments were ligated and packaged with λ phage according to the manufacturer's protocol, and the cosmid pWE / Ad. AflII-rITRΔE2A
I got

PVA. pVA (3.7 kb) is the adenovirus type 5 VAI and VAI
PUC119 plasmid containing the I region (nucleotides 10555-11075). The adenovirus type 5 VA gene was ligated with the primer 5′-ACGCGTC.
GACCCTCTGGCCGGTCAGGGCGGCGCAA-3 'and 5'-
ACGCGGATCCCGCATCTGCCGGCAGCACCGGATGC-3
And cloned by PCR for isolated DNA from wild-type adenovirus type 5. Expand long Tem according to manufacturer's instructions
PCR was performed using the plate ^™ PCR kit (Boehrnger).
The resulting fragment present in the primers was digested with SalI and BamHI and ligated into SalI, BamHI digested pUC119.

Cell culture. 293 cells (Graham et al., 1977b) and H
eLa cells (Cancer Res., 12, 264, 1952)
Dulbecco's modified Eagle's medium containing heat-denatured fetal bovine serum (DMEM, Life
(Technologies Breda, The Netherlands) at 37 ° C., 10% CO ₂ . PER. 10% adherent culture of C6 cells
37 ° C. in DMEM medium supplemented with fetal calf serum and MgCl ₂ (10 mM)
It was grown in 10% CO _2. PER. Suspension cultures of C6 cells were treated with Ex-Cell ^™ 525 (hereinafter Ex) supplemented with 1 × L-glutamine (GIBCO BRL).
-Called Cell ^TM ) at 37 ° C, 10% CO ₂ in 6-well dishes (Grein
er, Alphen aan de Rijn, The Netherlands
s) or Erlenmeyer tissue culture flasks (Cornin
g) by any of the 100 RPM shaking cultures.

Transfection. Transfection of monolayer culture. HeLa and 293 cells were transfected using a calcium phosphate transfection system (Life technologies, Almere) according to the manufacturer's instructions. PER. Monolayers of C6 cells were plated according to the manufacturer's instructions, LipofectAMINE ^™ (Life technologies, B).
reda).

Transfection of suspension culture. PER. In logarithmic growth phase. C6 cells were collected by centrifugation (3000 g, retention time 5 minutes). Cells are 2 × 10 ⁶ cells / m
resuspended in the transfection mixture (described below) at a concentration of 1
It was incubated for 3 hours at O _2. Unless otherwise specified, transfection
Performed with continuous shaking (100 RPM). DMRIE-C ^TM (Life tec
For transfection with H. gnologies, Breda), the transfection mixture was according to the manufacturer's instructions. Made in DMEM. After incubation for 3 hours at the transfection mixture, the cells were harvested by centrifugation (3000 g, retention time of 5 minutes), and resuspended to a final concentration of 10 ⁶ cells / ml in fresh Ex-Cell ^TM medium. FuGENE ^TM 6 (Boehri
transfection was performed using a transfection mixture made in Ex-Cell ^™ medium according to the manufacturer's instructions. After incubation for 3 hours with transfection mixture, the cells were diluted to a final concentration of 10 ⁶ cells / ml in Ex-Cell ^TM medium. The transfection mixture was mixed with poly (2- (dimethylamino) ethyl-10
It was produced as follows using (-4-aminobutyl) phosphazene (PPZ).
A stock solution of PPZ (2.4 mg / ml) was made by dissolution of the solid compound in Hepes (5 mM, pH = 7.3). The chemical formula of PPZ is

Embedded image It is.

The transfection mixture was made by adding the indicated amount of PPZ to 500 μl of Ex-Cell ^™ medium. This solution was mixed with an equal volume of Ex-Cell ^™ containing the indicated amount of DNA. The mixture was incubated for 1 hour and used to generate 2 × 10 ⁶ PER. The pellet of C6 cells was resuspended. Cells were incubated for transfection mixture for 3 hours, and diluted to a final concentration of 10 ⁶ cells / ml in Ex-Cell ^TM medium. Transfected cells were harvested 48 hours later and analyzed for β-galactosidase activity.

Β-galactosidase activity assay. Cells can be transformed into β-galacto in two different ways.
Stained for tosidase activity. Histochemical analysis and identification of the number of infectious units
The following procedure was used. Cells were purified from PBS (NPBI, Emmer-Compa).
wash, twice with 0.2% glutaraldehyde (Sigma, Zwi)
jndrecht, The Netherlands) in PBS for 10 minutes
Fixed. The cells are washed twice with PBS, and X-Gal solution (0.1 M phosphate buffer)
(PH 7.4) in 2 mM MgCl₂・ 6H₂O, 5 mM K₂Fe (CN) ₆ , 5 mM K4Fe (CN)₆・ 3H₂O and 40 mg / ml X-Gal
(5-bromo-4-chloro-3-indolyl-b-galactopyranoside, Mol
ecolubes Europe, Leiden, The Neth
erlands)). After overnight staining at 37 ° C., blue cells were removed by light microscopy.
Counting was performed using a mirror (Olympus CK2-TR). PER. For suspension culture of C6
For the determination of β-galactosidase activity in the manufacturer's protocol
Therefore, FluoReporterTM LacZ / Galactosidas
e Quantitation kit (Molecular Probes,
Leiden, The Netherlands). Β of each sample
-Galactosidase activity in 10 μl of cell suspension β-galactosidase
By comparing the enzyme activity with a serial dilution of purified β-galactosidase of known concentration
evaluated.

[0035] Recombinant AAV titration. HeLa cells were seeded at 4 × 10 ⁴ cells / cm ² . The medium was replaced the following day with fresh medium containing serial dilutions of rAAV and adenovirus ts149 (20 pfu / cell). After 4 hours, the medium was replaced with fresh medium, and the cells were incubated at 37 ° C., 10% CO ₂ for 24 hours, followed by β-galactosidase staining. The titer of the recombinant AAV strain was calculated by counting the number of blue cells from the highest dilution at which blue cells were obtained and multiplying this number by the dilution factor.

[0036] Recombinant AAV production on adherent cells. Cells were seeded the next day to reach 70% confluency. The cells were then transformed into pACV-βgal and pIM45 (1: 4
w / w) and adenovirus helper virus ts14
9 (moi = 20), E1-deleted adenovirus helper virus IG. A
d. MLP. Luc (Vincent et sl., 1996) or transfected with adenovirus helper plasmid DNA. In the latter case, the total number of adenovirus helper gene plasmids is pACV-βgal
And 1.5 times (w / w) the total number of pIM45 DNA and pIM45 DNA. When more than one adenovirus helper virus was used, equal amounts (w / w) of different adenovirus helper plasmids were used. Adhesive PER. C6tsE2A. c5-
9 with PER.9 except for some modifications. Performed as described for C6 cell line. Cell lines were grown at 39 ° C., 10% CO ₂ . Prior to transfection, cells were seeded at 39 ° C., 10% CO ₂ and 70% confluence the next day. The cells were then cultured for 1 day at 32 ° C., 10% CO ₂ . Then, PER. Cells were transfected at 37 ° C., 10% CO ₂ as described for the C6 strain. Recombinant AAV was recovered 48 hours after transfection. Cells were scraped from the culture medium and subjected to three freeze-thaw cycles. Cell debris was centrifuged (2000 RPM, retention time 10 minutes). Adenovirus ts1
When 49 or E1-deleted adenovirus vectors were used, the supernatant was heat denatured at 56 ° C. for 1 hour. When using an adenovirus DNA fragment to supplement adenovirus production, the supernatant was not heat denatured. Filter all supernatants (0.
45 μM, Millipore) and stored at −20 ° C.

Example 1 Production of a production cell line for producing a recombinant adenovirus vector lacking the early region 1 and the early region 2A. Described herein is the generation of a cell line for the production of a recombinant adenovirus vector lacking the early region 1 (E1) and the early region 2A (E2A). The producer cell line is
Complement the E1 and E2A deletions from the recombinant adenovirus vector in trans with constitutive expression of the E1 and E2A genes, respectively. Pre-established Ad
5-E1 transformed human embryonic retinoblast cell line PER. C6 (WO 97/00326)
And Ad5 transformed human embryonic kidney cell line 293 (Graham et al., 1).
977b) was further provided with an E2A expression cassette.

The adenovirus E2A gene has a high affinity of 72 kDa for single-stranded DNA.
Encodes a DNA binding protein (DBP). Because of this feature, constitutive expression of DBP is cytotoxic. The ts125E2A mutant encodes a DBP that replaces Pro → Ser at amino acid 413 (Vliet van der e)
t al. , 1975). This mutation results in ts125E2 encoding DBP.
A is fully active at a permissive temperature of 32 ° C, but ssD at a nonpermissive temperature of 39 ° C.
Does not bind to NA. This makes it possible to generate cell lines that express E2A constitutively, which are non-functional and non-toxic at the non-permissive temperature of 39 ° C., but become functional after the temperature change to the permissive temperature of 32 ° C. .

A. Generation of plasmids expressing wild type E2A or temperature sensitive ts125E2A. pcDNA3wtE2A. The complete wild-type early region 2A (E2A) coding region was purified from plasmid pBR / Ad. Using primers DBPpcr1 and DBPpcr2 using the Expand ^™ Long Template PCR system according to standard protocols from the supplier (Boehringer Mannheim). Bam-rITR (ECACC accession number P97082122)
). PCR was performed using Biometra Trio Thermoblo.
The following program was performed using ck: 1 cycle at 94 ° C. for 2 minutes, 94 cycles
1 cycle at 10 ° C. for 10 seconds, + 51 ° C. for 30 seconds, + 68 ° C. for 2 minutes, and 10 cycles at 94 ° C.
10 cycles of + 58 ° C for 30 seconds, 68 ° C for 2 minutes, and 94 ° C for 10 seconds
Extension at 58 ° C. for 30 seconds + 68 ° C. for 2 minutes and 10 seconds per cycle
One cycle of 5 minutes at 68 ° C. Primer DBPpcr1: CGG
GAT CC G CCA CCA TGG CCA GTC GGG AAG
AGG AG (5 ′ → 3 ′) is a unique BamHI restriction site (underlined), Koza
Includes the 5 'of the k sequence (italics) and the start codon of the E2A coding sequence. Primer DBPpcr2: CG G AAT TCT TAA AAA TCA AAG
GGG TTC TGC CGC (5 ′ → 3 ′) contains a unique EcoRI restriction site (underlined), 3 ′ of the stop codon of the E2A coding sequence. Bold letters refer to sequences from the E2A coding region. The PCR fragment was digested with BamHI / EcoRI and cloned into BamHI / EcoRI digested pcDNA3 (Invitrogen) to obtain pcDNA3wtE2A.

[0040] pcDNA3tsE2A. The complete ts125E2A coding sequence was converted to the temperature-sensitive adenovirus mutant H5ts125 (Ensinger and Ginsb).
erg, 1972, Vliet van der et al. , 1975). The procedure of PCR amplification is the same as the procedure of amplification of wtE2A. The PCR fragment was digested with BamHI / EcoRI and
Cloning into mHI / EcoRI digested pcDNA3 (Invitrogen) yielded pcDNA3tsE2A. The coding sequences of wtE2A and tsE2A were confirmed by sequencing.

B. Growth characteristics of producer cells for production of recombinant adenovirus vectors cultured at 32 ° C, 37 ° C, and 39 ° C. PER. C6 cells were incubated with 10% fetal bovine serum (FBS, GIBCO BRL) and 10 mM in any of 32 ° C., 37 ° C., or 39 ° C. in 10% CO ₂ .
Dulbecco's modified Eagle medium supplemented with M MgCl ₂ (DMEM, GIBC
OBRL). On day 0, a total of 1 × 10 ⁶ PER. 25 C6 cells
cm ² tissue culture flasks (Nunc) were seeded and cells were cultured at either 32 ° C, 37 ° C, or 39 ° C. Cells were counted on days 1-8. FIG. 3 shows PER. 3 shows the comparison between the growth rate and final cell density of C6 cell culture at 37 ° C. PER. The growth rate and final density of C6 cultures were slightly reduced compared to those at 37 ° C or 39 ° C. No significant cell death was observed at any of the incubation temperatures. Therefore, PER. C6 cells are t
It works fairly well at both 32 ° C, the allowable temperature for s125E2A, and 39 ° C, the non-permissive temperature.

C. PER.E2A expression vector Transfection of C6 and 293; colony formation and production of cell lines 2 × 10 ⁶ PER. C6 cells were grown in a 6 cm tissue culture dish (Gre in DME with 10% FBS and 10 mM MgCl _2).
inner) and incubated at 37 ° C., 10% CO ₂ . The next day, 39
℃ and except to transfection against 10% CO ₂ cells in based on the standard protocol of the supplier (GIBCO BRL), Lipofec
3 μl per tissue culture dish using tAMINE PLUS® reagent kit
g, 5 μg, or 8 μg of pcDNA3, pcDNA3wtE2A or pc
Cells were transfected with DNAtsE2A plasmid DNA. After transfection, cells were kept constant at 39 ° C., the non-permissive temperature of ts125E2A. After 3 days, 10% FBS, 10 mM MgCl ₂ , and 0.25 m
Cells were placed in DME supplemented with g / l G418 (GIBCO BRL) and the first G418 resistant colonies appeared 10 days after transfection. As shown in Table 1, the total number of colonies obtained after pcDNA3 transfection (
There was a significant difference between the total number of colonies obtained after transfection of pcDNA3tsE2A (〜200 colonies) or pcDNA3tsE2A (〜100 colonies) and the total number of colonies obtained after transfection of pcDNA3wtE2A (only 4 colonies). These results indicate that the toxicity of constitutively expressed E2A indicates that E2A (t
It has been shown that a temperature-sensitive mutant of s125E2A) can be overcome by culturing the cells at a non-permissive temperature of 39 ° C.

From each transfection, several colonies were pipetted off the cells from the tissue culture dish. Subsequently, the detached cells were placed in 24-well tissue culture dishes (Greiner), 10% FBS, 10 mM MgCl ₂ , and 0
. The cells were cultured in DME supplemented with 25 mg / l G418 at 39 ° C. and 10% CO ₂ . As shown in Table 1, 10 pcDNA3 transfected colonies
0% (4/4) and 82% (37/45) of the pcDNA3tsE2A transfected colonies established a stable cell line (the remaining 8 pcDNA3t).
sE2A transfection colonies grew slowly and were discarded). In contrast, only one pcDNA3wtE2A transfection colony could be established. The other three died shortly after collection.

Next, the E2A expression levels of the different cell lines were determined by Western blotting. The cell line is seeded in a 6-well tissue culture dish, and the subconfluent culture is
(NPBI), washed twice and dissolved in RIPA (1% NP-40, 0.5% sodium deoxycholate, and PBS containing 0.1% SDS, further 1 mM
Phenylmethylsulfonyl fluoride and 0.1 mg / ml trypsin inhibitor). After 15 minutes incubation on ice, the lysate was removed by centrifugation. Protein concentration was determined by the Bio-Rad protein assay based on the standard protocol of the supplier (BioRad). Equal amounts of whole cell extracts were fractionated by SDS-PAGE on a 10% gel. The protein was transferred to an Immobilon-P membrane (Millipore) and the αDBP monoclonal antibody B6 (Reich et al., 1983).
) And incubated. The secondary antibody was a horseradish peroxidase-conjugated goat anti-mouse antibody (BioRad). Western blotting and antibody incubations were performed according to the protocol provided by Millipore. Antibody conjugate was visualized by the ECL detection system based on the manufacturer's (Amersham) protocol. FIG. 3 shows that pcDNA3
Show that all cell lines derived from the tsE2A transfection express the 72 kDa EZA protein (top panel, lanes 4-14; middle panel,
Lanes 1 to 13; lower panel, lanes 1 to 12). In contrast, pcDNA3
Only cell lines derived from wtE2A transfection failed to express E2A protein (lane 2). E2A protein was not detected in extracts from the pcDNA3 transfection-derived cell line (lane 1) and is used as a negative control. PER. Extracts of C6 cells were transiently transfected with pcDNA3wtE2A (lane 3) and used as a positive control for Western blotting. These data confirm that constitutive expression of wtE2A is toxic to cells and that this toxicity can be avoided by using a ts125 mutant of E2A.

PER. In contrast to C6 cells, culturing 293 cells at 39 ° C is problematic. Therefore, pcDNA3, pcDNA3wtE2A, or pcDN
Transfection of 293 cells with any of A3tsE2A was performed using DBP
Under CO ₂ at 37 ° C., the semi-permissive temperature of ts125E2A, which encodes
The day before transfection, 293 cells were treated with 10% FBS and 10 mM Mg.
Cells were seeded at a density of 3.6 × 10 ⁵ cells per 6 cm tissue culture dish (Greiner) supplemented with cl2. Five hours before the start of transfection, cells were given fresh medium. Cells were transformed with pcDNA3 by the calcium phosphate transfection system based on standard protocols from the supplier (GIBCO BRL).
Transfected with 7.2 μg of either pcDNA3wtE2A or pcDNA3tsE2A plasmid DNA. Two days after transfection, cells were plated in selective medium, ie, 10% FBS, 10 mM MgCl ₂ ,
And 0.25 mg / l of G418 in DMEM. The first colonies appeared 12 days after transfection. As shown in Table 2, pcDN
The total number of colonies obtained after transfection of A3 (（100 colonies) and the total number of colonies obtained after transfection of pcDNA3tsE2A (〜25 colonies) were obtained from the total number of colonies obtained after transfection of pcDNA3wtE2A (only 2 colonies). Was also significantly higher. These results indicate that constitutively expressed ESA is toxic to cells and that this toxicity is
It again shows that this can be avoided by using s125E2A. Furthermore, this is true for PER. This indicates that it is not unique to C6 cells, but generally applies to eukaryotic cells (eg, 293 cells).

D. PER.ts expressing constitutively ts125E2A. Ad5. By C6 cells. d
Complementation of E2A deletion in l802 Adenovirus Ad5. dl802 is an Ad5-derived vector in which the main part of the E2A coding region has been deleted, and does not produce a functional DBP (Rice and
Klessing, 1985). Ad5. dl802 is a PER. constitutively expressing ts125E2A. It was used to test the E2A trans complementation activity of C6 cells. Parent PER. C6 cells or PER. C6tsE2A clone 3-9
At 39 ° C. in DME supplemented with 10% FBS and 10 mM MgCl _2.
Cultured in 25 cm ² flask with 10% CO ₂ , multiplicity of infection (moi) 5
Ad5. Either dl802 infection or membrane mock infection was performed. continue,
Infected cells were cultured at 32 ° C. and cells were screened for expression of cytopathic effect (CPE) by determining changes in cell morphology and detachment of cells from flasks. Table 3 shows that PER. Ad5. Infected with C6tsE2A clone 3-9. This shows that complete CPE expression occurred in dl802. These data indicate that PER. Expressing constitutively expressing ts125E2A. C6
Cells were allowed to reach Ad5. It shows that E2A deletion of the dl802 vector is complemented with trans.

E. PER. Serum-free suspension culture of C6tsE2A cell line. Large-scale production of recombinant adenovirus vectors for human gene therapy allows for assay and scale-up of production cell lines, preferably suspension cultures, in media lacking any human or animal constituents Requires a culture method. For that purpose, some PER. The C6tsE2A clone was cultured in suspension.
For example, cell line PER. C6tsE2Ac5-9 (denoted as c5-9) at 39 ° C.
In 10% CO ₂ is 10% FBS and 10 mM MgCl ₂ were cultured in 175cm ² flasks (Nunc) containing DME was added. At subconfluence (70-80% confluence), cells are washed with PBS (NPBI) and the medium is washed with 1 × L-glutamine (GI
It was replaced with 25 ml of serum-free suspension medium Ex-cell (registered trademark) 525 (JRH) (hereinafter referred to as Ex-cell (registered trademark)) to which BCO BRL) was added. Two days later, the cells were detached from the flask by tapping the cells, and the cells were removed for 10 minutes for 10 minutes.
Centrifuge at 00 rpm. The cell pellet is mixed with 5 ml of Ex-Cell®.
And 0.5 ml of the cell suspension was transferred to an 80 cm ² tissue culture flask (Nunc) with 12 ml of fresh Ex-cell®. Two days later, cells were harvested (all cells in suspension) and counted on a Burker cell counter. The cells were then transferred to 125 ml tissue culture Erlenmeyer (Corn) at a density of 3 × 10 ⁵ cells per ml in a total volume of 20 ml Ex-cell®.
ing). The cells were further cultured in an orbital shaker (GFL) at 39 rpm at 39 ° C., 10% CO ₂ . On days 1-6, cells were cultivated with Burker
The cells were counted with a cell counter. FIG. 4 shows the average growth curves obtained from eight cultures. P
ER. C6tsE2Ac5-9 works well in serum-free suspension culture. A maximum cell density of about 2 × 10 ⁶ cells / ml was achieved within 5 days of culture.

Example 2 PER cells as cells producing recombinant AAV. PER cells are derived from human retinal cells. The retina is known for its ability to sustain AAV replication. Therefore, we have identified that PER cell cells
Check if AV production is allowed. LipofectAMINE (registered trademark)
Using PER. C6 cells were transformed with the packaging plasmid pIM45, rAAV-
Transfected with the vector pACV-βgal (ratio 10: 1 w / w),
Adenovirus ts149 was infected. Recombinant AAV was isolated two days later and titrated on adenovirus infected HeLa cells. PER. PAC produced in C6 cells
V-βgal is 2 × 10 ⁷ infectious units (IU) per ml or 2 per cell.
It had a titer of 0 IU. Virus yields per cell obtained with this system were reported in the 293 cell line with the packaging plasmid pIM45 (
Vincent et al. , 1997) or better. At the same time, the pattern and level of the expressed E1 protein
It was analyzed whether it was sufficient for AAV production. To address this issue, PER. C6 cells were transformed with the packaging plasmid pIM45, rA
Transfection with the AV-vector pACV-βgal (ratio 10: 1 w / w) was performed and the E1-deleted adenovirus vector IG. Ad. MLP. Luc (Vinc
ent et al. , 1996). E1 deleted adenovirus vector IG. Ad. MLP. The yield of rAVV using Luc was 2 × 10 ⁷ AI
U / ml, similar to adenovirus ts149. This result is
R. Both patterns and levels of E1 expression in C6 have been shown to enable sufficient rAAV production.

PER. Production by recombinant AAV on C6 in the absence of viral vector.
Recombinant AA using adenovirus DNA as helper rather than virus
Production of V has been reported (WO 96/40240, WO 97/17458).
, (Ferrari et al., 1997; Ferrari et a.
l. , 1996; laser iridotomy et al. , 19
97; Xiao et al. , 1998a)). This study was performed only on the 293 cell line. The present inventors have proposed PER. We also wanted to determine if the C6 cell line could be used to produce recombinant AAV. To this end, we have constructed two constructs, pWE / Ad., Which contain all adenovirus genes except the E1 region. D5 'and pWE / Ad. AflII-rITR was tested. As already mentioned, the construct pWE / Ad. AflII-rITR contains all adenovirus genes except the E1 region, but has a deletion in the promoter of the protein IX gene. On the other hand, pWE / Ad. D5 'contains the left ITR and the full length protein IX promoter. Recombinant AAV could be produced with both adenovirus DNA fragments. The yield of recombinant AAV was different for both constructs. pWE / Ad. The yield of recombinant AAV using AflII-rITR was pWE
/ Ad. It was significantly higher than that using D5 '(Table 4). One reason for the difference in yield is due to differences in the expression of related proteins from the two different plasmids. Another reason is that expression of adenovirus 5 protein IX negatively affects recombinant AAV production.

PER. Minimum requirements for recombinant AAV production of C6. PER. To determine the minimum requirements for recombinant AAV production on C6 cells, plasmid clones of adenovirus genes known to adversely affect AAV production were obtained (ie, E2a, E4orf6, and VA). ). Therefore, all adenovirus genes known to affect AAV production are PER. Since it can be used immediately with the E1 region already existing in C6,
PER. Their effect on recombinant AAV production on C6 was tested.
In contrast to HeLa cells, PER. C6 cells contain pIM45 and pACV-β
A small but detectable amount of recombinant AAV was produced when transfected with gal. Efficient production of recombinant AAV required transfection of another helper function-encoding gene. Transfection of all three expression cassettes or pE2 and pVA yielded the highest amounts of recombinant AAV (Table 4). Transfection with pE2a alone or with pE4orf6 resulted in ± 10% recombinant AAV yield. Transfection of E4 alone or VA alone did not produce significant amounts of AAV (Table 4). PER. To which no special adenovirus helper gene was added. C6t
Production on sE2A yielded a titer of 1.3 × 10 ³ . By adding pEorf6 and pVA alone or in combination with pE2A, recombinant AAV
Increased in yield. The highest yields were obtained with construct pWE / Ad. aflII-rIT
Obtained using R.

Large Scale Production of Recombinant AAV Several systems have been devised for growing mammalian cells in large quantities. They include, but are not limited to, rolling bottle cultures, cell cubes and bioreactors. Each of these systems has advantages and disadvantages.
Bioreactors in which cells grow in suspension are the easiest to standardize and scale up even larger. However, one disadvantage is that cells in suspension are not easily transfected. Many different cell culture media have been developed to support optimal growth of a wide variety of different cells. Most of these media are based on modified Dulbecco's Modified Eagle's Medium (DMEM) and are supplemented with bovine serum. The present inventors have conducted PER. C6 cells were adapted. Serum-free culture has the advantage that it can be completely defined because it varies in quality and does not depend on naturally occurring serum containing foreign agents. The serum-free medium contains an additive that substitutes for an essential component for cell growth in serum. We found that the cells were more DMEM than if they were cultured in serum-free Ex-Cell® medium.
It was observed that more transfection reagents, especially liposomes, functioned better when cultured with (not shown). However, PER. Transfection of C6 cells was performed using the non-liposomal reagent FuGEN
E.RTM. 6 or by using the liposome: DNA complex (D
MRIE-C) and contact with Ex-Cell® media (Table 5). Another transfection reagent is poly (organo) phosphazene. Little is known about the ability of cells to be transfected with these reagents. To investigate the use of the above reagents for transfection of PER cells in suspension, we determined that a certain amount of D
Increasing amount of compound poly (2- (dimethylamino) ethyl) -10-4- with NA
Transfection with (aminobutyl) phosphazene (PPZ) was performed. Cells were exposed to the transfection mixture for 3 hours before diluting the medium. Transfection of suspension cells as determined by X-Gal staining and fluorometric analysis depends on the amount of PPZ added (Tables 6 and 7).
5% X-Gal with 320 μg PPZ (Table 6) and 160 μg PPZ (Table 7)
Positive cells were reached. The higher PPZ in the transfection mixture was related to massive cell loss.

Next, we assessed the potential of PER cells for large-scale production of recombinant DNA. The mass production potential was first determined by the adhesive PER. C6 cells were evaluated.
DMEM + 10% FCS for 10 170 cm ² (Greiner) dishes
2 × 10 ⁷ PER. C6 was seeded. Cells were purified with pACV-βgal
, PIM45, and pWE / Ad. AflII-rITR (each 2: 8:
30 μg). Virus was collected after 48 hours using the following protocol. The medium is removed from the cells and the cells are scraped and collected.
ml / dish in fresh DMEM. The cell suspension was freeze-thawed twice, followed by 37
Incubated with DNaseI (100 μg / ml) for 30 minutes at ° C. The suspension was subjected to two additional freeze-thaw cycles, after which cell debris was removed by centrifugation (3,000 rpm, 10 minutes). The supernatant was incubated with 13.3 ml of saturated (NH ₄ ) 2 SO ₄ (4 ° C., 10 minutes). SW2
The precipitate was removed by centrifugation at 10,000 rpm (4 ° C., 15 minutes) using a 7.1 rotor. An additional 26.6 ml of saturated (NH ₄ ) ₂ SO ₄ was added and the supernatant was incubated and further incubated at 4 ° C. for 20 minutes. The virus was pelleted by centrifugation at 12,000 rpm (4 ° C., 30 minutes) using a SW27.1 rotor. Pellet 5 ml PBS (NPBI)
And resuspended in two tubes (Quick-Seal Ultra-Clear tu).
be) (Beckman Instruments, Mijdrecht,
The Netherlands). Add virus suspension to an equal volume of O
ptiPrep (Nycomed Pharma As, Oslo, Nor
way). The sealed tube was rotated at an angle of 80 degrees (
20 min, 10 rpm). The virus uses a Vti80 rotor (Beckman
Instruments, density gradient centrifugation (3 hours, 71,000 rpm)
). Fractions were collected (200 μl per fraction) and titrated for the presence of recombinant AAV. Positive fractions are pooled (± 400-600 μl) and
Diluted with BS (NPBI), followed by Centriplus 100 (Centriplu)
s 100) and Centricon 100 (Am)
icon and Capelle A / D Ijssel). Titration The concentrated fractions were found to have 8.5 × 10 ⁹ titer of infectious units per 1 ml.

[0053]

[Table 1]

[0054]

[Table 2]

[0055]

[Table 3]

[0056]

[Table 4]

[0057]

[Table 5]

[0058]

[Table 6]

[0059]

[Table 7]

[Brief description of the drawings]

FIG. 1 shows the structure of wtAAV and the structure of the genome.

FIG. C6 cells were seeded at a density of 1 × 10 ⁶ cells per 25 cm ³ tissue culture flask and cultured at either 32 ° C., 37 ° C., or 39 ° C. At the indicated times, cells were counted on a Burker cell counter. PER. C6 is 32
It grows well at any of C, 37C, and 39C.

FIG. 3: pcDNA3 (upper panel, lane 1), pcDNA3wtE2A (
Upper panel, lane 2), pcDNA3tsE2A (upper panel, lane 4)
-14, middle panel, lanes 1-13, and lower panel, lanes 1-12
). C6 or pcDNA3tsE
2A (upper panel, lane 3). C6
FIG. 2 shows a Western blot using 35 mg of a whole cell extract derived from a cell line prepared from cells. Blots were run with an antibody specific for the E2A gene product (B6a
DBP) and visualized using the ECL detection system. All PER. C6ts
The E2A cell line expresses the tsE2A-encoded temperature-sensitive DBP protein.

FIG. 4. Cell line expressing tsE2A PER. C6tsE2A. c5-9, serum-free E
The cells were cultured in a suspension of x-cell ^™ . At the indicated times, cells were counted on a Burker cell counter. The results of eight independent cultures are shown. PER. C6tsE
2A grows well in suspension in serum-free Ex-cell ^™ medium.

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI Theme coat ゛ (Reference) C12R 1:91) (C12N 7/00 (C12N 5/10 C12R 1:93) C12R 1:91) C12N 5 / 00 B (C12N 7/00 15/00 A C12R 1:93) (81) Designated country EP (AT, BE, CH, CY, DE, DK, ES, FI, FR, GB, GR, IE, IT , LU, MC, NL, PT, SE), OA (BF, BJ, CF, CG, CI, CM, GA, GN, GW, ML, MR, NE, SN, TD, TG), AP (GH, GM) , KE, LS, MW, SD, SL, SZ, TZ, UG, ZW), EA (AM, AZ, BY, KG, KZ, MD, RU, TJ, TM), AE, AL, AM, AT, A , AZ, BA, BB, BG, BR, BY, CA, CH, CN, CR, CU, CZ, DE, DK, DM, EE, ES, FI, GB, GD, GE, GH, GM, HR, HU, ID, IL, IN, IS, JP, KE, KG, KP, KR, KZ, LC, LK, LR, LS, LT, LU, LV, MA, MD, MG, MK, MN, MW, MX , NO, NZ, PL, PT, RO, RU, SD, SE, SG, SI, SK, SL, TJ, TM, TR, TT, TZ, UA, UG, US, UZ, VN, YU, ZA, ZW F term (reference) 4B024 AA01 CA04 DA02 EA02 EA06 GA13 GA18 HA12 HA17 4B065 AA93X AA95Y AB01 AC20 BA02 BA25 BB32 BD01 BD14 CA44

Claims (16)

[Claims] 1. An adeno-associated virus (adeno-associated virus) provided with a nucleic acid encoding at least one gene product that provides an AAV helper function capable of producing a recombinant adeno-associated virus (AAV) without producing an associated helper virus. AAV) Packaging cells. 2. The cell of claim 1, wherein said nucleic acid encodes an adenovirus gene product. 3. The cell of claim 2, wherein said gene product comprises adenovirus E1 or a functional fragment thereof. 4. The cell of claim 2, wherein said gene product comprises adenovirus E2A or a functional fragment thereof, wherein said E2A is preferably derived from adenovirus ts125. 5. The cell according to claim 1, further comprising at least one further nucleic acid encoding a required helper function. 6. The gene product encodes adenovirus E1 or a functional fragment thereof, wherein at least one of the additional nucleic acids encodes adenovirus E2A or a functional fragment thereof, wherein the E2A is adenovirus ts12.
The cell according to claim 5, which is preferably derived from No. 5. 7. The cell of claim 6, further comprising a nucleic acid encoding adenovirus E4orf6 or a functional fragment thereof. 8. The method according to claim 1, which is derived from human embryonic retinoblasts.
The cell according to Item. 9. Center for Applied Microbiolo
gy research (CAMR) with accession number 9602940
PER. Deposited at ECACC. 9. The cell according to claim 8, comprising a C6 cell or a cell derived therefrom. 10. The cosmid pWE / Ad. AflII-rITR (CAMR E
The cell of any one of claims 1 to 9, comprising CACC deposit number p97082116). 11. The cosmid pWE / Ad. The cell according to any one of claims 1 to 10, comprising AflII-rITRΔE2A. 12. The cell according to claim 1, comprising the plasmid pcDNA3wtE2A. A cell culture method comprising the cell according to any one of claims 1 to 12. 14. The cell culture method of claim 13, comprising a medium lacking any human or animal components. 15. The cell culture method according to claim 13 or 14, comprising a suspension cell culture or other mass culture. 16. A recombinant adeno-associated virus using the cell according to any one of claims 1 to 12 or the cell culture method according to any one of claims 13 to 15. A method for producing