WO1999061640A2 - Aav derived vector - Google Patents
Aav derived vector Download PDFInfo
- Publication number
- WO1999061640A2 WO1999061640A2 PCT/GB1999/001633 GB9901633W WO9961640A2 WO 1999061640 A2 WO1999061640 A2 WO 1999061640A2 GB 9901633 W GB9901633 W GB 9901633W WO 9961640 A2 WO9961640 A2 WO 9961640A2
- Authority
- WO
- WIPO (PCT)
- Prior art keywords
- adeno
- gene
- producer cell
- viral
- associated viral
- Prior art date
Links
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
- C12N15/86—Viral vectors
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2750/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssDNA viruses
- C12N2750/00011—Details
- C12N2750/14011—Parvoviridae
- C12N2750/14111—Dependovirus, e.g. adenoassociated viruses
- C12N2750/14141—Use of virus, viral particle or viral elements as a vector
- C12N2750/14143—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector
Definitions
- the present invention relates to an improved adeno-associated viral vector, to a method of transfecting target cells, an adeno-associated viral producer cell and a method for the production of an adeno-associated viral vector.
- Adeno-associated virus is a vector which has shown promise in virally-mediated gene therapy protocols.
- AAV is a small, 20-22nm, replication defective, non-pathogenic parvo virus.
- Six serotypes of AAV are currently known (types 1 to 6) , of which type 2 is the most extensively studied.
- AAV type 2 has a single stranded DNA genome of 4.7kB which encodes both structral proteins through a cap gene and non-structural proteins through a rep gene.
- the virus has a pair of 146bp inverted terminal repeats (ITRs) which are the only els acting elements in AAV required for replication and packaging.
- ITRs inverted terminal repeats
- the present invention aims to increase the level of transgene expression from an adeno-associated viral vector.
- the present invention provides an adeno-associated viral vector containing a transgene and encapsidating a protein with adenovirus E4 ORF6 activity.
- the protein is preferably an adenovirus E4 ORF6 protein such as is obtainable from adenovirus .
- the present invention provides the adeno- associated viral vector for use as a medicament, especially in gene therapy. Also provided is a method for transfecting target cells, which comprises contacting target cells with the adeno-associated viral vector under conditions to achieve adeno-associated viral infection.
- E4 ORF6 needs to be present as a polypeptide to facilitate conversion of the AAV single stranded genome to the double stranded replicative form.
- the present invention provides an adeno- associated viral producer cell containing a gene encoding a protein with E4 ORF6 activity, which gene is under the control of an inducible promoter.
- E4 0RF6 constitutive expression of E4 0RF6 in the AAV producer cell tends to have an adverse effect on the cell. This was unexpected. However, by putting the gene under control of an inducible promoter the producer cell is able to tolerate the presence of the protein.
- Any inducible promoter may be used, such as the MMTV-LTR (Mouse Mammary Tumour Virus-Long Terminal Repeat) promoter, which is inducible by dexamethasone .
- the inducer may be added to the medium in which the cells grow or, alternatively, may be produced upon infection of the cell by AAV or helper virus for AAV.
- an AAV inducible promoter was used; the P5 promoter. In this case, upon infection with both adeno-virus and AAV, the P5 promoter is induced so as to induce the E4 ORF6 protein.
- the gene comprises a drug-resistance marker, such as an antibiotic resistance marker which enables the cells to be grown in medium containing the relevant drug or antibiotic.
- the selectable marker may be a cell surface marker such as a receptor which can be specifically targeted by a complimentary molecule such as an antibody thereto.
- One such cell surface marker is nerve growth factor (NGF) .
- the gene encoding adenovirus E4 ORF6 protein may be conveniently introduced into the producer cell on a plasmid.
- the E4 ORF6 gene may be readily obtained from an adenovirus such as adenovirus type 2, which is deposited as ATCC VR846, the sequence of which is published by EMBL at locus HACG. See also Ohman et al in Virology 194 (1) , 50-58, 1993 where cloning of E4 ORF is reported. From the EMBL sequence the E4 region spans nucleotides 32801-35609 and E4 ORF6 from Ohman et al spans nucleotides 33193-34115 .
- the protein with E4 ORF6 activity has a nuclear localisation sequence which is typically situated outside of the functional part of the E4 ORF6 protein, typically at or near the N- or C-terminal sequence of the protein.
- the nuclear localisation sequence confers on the protein an important property.
- the AAV producer cell which incorporates the gene encoding the protein with E4 ORF6 activity may be any producer cell commonly used to produce AAV vectors, such as a human 293 producer cell (ATCC CRL 1573) .
- the AAV will be rep " cap " and so either a second transfection step involving the introduction of rep and cap on, for example, a plasmid would need to be undertaken or the producer cells would have to be made rep + cap + .
- the producer cell is a stable cell containing rep and cap, both associated with a selectable marker as described above.
- transfection or infection by AAV DNA (rep " cap " ) requires no second plasmid transfection step.
- the producer cell comprises a rep-containing region which comprises a rep gene associated with a marker gene for rep, and a cap-containing region which comprises a cap gene associated with a marker gene for cap.
- the rep-containing and cap-containing regions are preferably sufficiently spaced apart to prevent their simultaneous recombination with adeno-associated virus. This confers a further advantage on the viral producer cells .
- the AAV DNA would necessarily contain inverted terminal repeats and so a problem arises in producing AAV vectors because there is a low but significant frequency of homologous recombination.
- the AAV producer cells contain a rep gene contiguous with a cap gene, homologous recombination events result in contaminating amounts of wild-type AAV being formed. This problem is avoided where the cap and rep genes are spaced sufficiently apart to prevent such simultaneous co- recombination of rep and cap.
- the rep gene and/or the cap gene is plasmid-borne so as to avoid contaminating homologous recombination. More preferably, both rep and cap genes are plasmid-borne.
- the viral producer cell is formed from a multicopy plasmid which carries a rep gene and cap gene. In this embodiment it is preferable for the plasmid to have an origin of replication.
- the producer cell is preferably capable of producing a replication initiator for the origin of replication of the plasmid. In this way, multiple copies of the plasmid are made in the cell.
- Various combinations of replication initiator and origin of replication are known. A particularly useful combination is where the cell is a COS cell and the origin of replication is SV40 ori .
- COS cells produce T antigen as a replication initiator for SV40 ori.
- Another example of such a combination is EBNA1 as a replication initiator for oriP.
- EBNA1 and oriP are derived from the Epstein Barr virus which is capable of infecting a variety of cells, including human cell lines.
- the rep gene encodes a rep mRNA which has reduced efficiency of translation as compared with wild-type rep mRNA. More preferably, the rep mRNA of the viral producer cells has a mutant initiation codon which reduces the efficiency of translation. Wild-type initiation codons are AUG whereas mutant initiation codons may be UUG, CUG, or GUG, preferably ACG.
- the adeno-associated viral producer cell comprises at least one rep gene and at least two cap genes, wherein the number of cap genes exceeds the number of rep genes . This may be achieved by locating a rep gene on one plasmid and locating a cap gene on a multicopy plasmid.
- the present invention provides a method for the production of an adeno-associated viral vector, which comprises introducing into the viral producer cell DNA comprising a transgene flanked by adeno-associated virus inverted terminal repeats, allowing formation of adeno- associated viral vectors in the viral producer cell, and isolating the adeno-associated viral vectors.
- the DNA may be introduced into the producer cell by transfection, for example as part of a suitable plasmid, in the presence of an adeno- associated helper virus, or by infection as part of a suitable virus.
- the DNA may be integrated into the genome of an adeno- associated helper virus which infects the producer cell or the DNA may comprise the genome of an adeno-associated virus which is infected into the producer cell in the presence of an adeno-associated helper virus.
- the helper virus comprises an adenovirus, such as adenovirus type 5.
- This example compares level of transgene expression in target cells using AAV type 2 viruses from producer cell lines containing E4 0RF6 with and without an NLS .
- a stable cell line was made by lipofecting COS 7 cells (ATCC CRL 1651) with the E4 ORF6 containing plasmid containing a hygromycin resistance cassette.
- Adenovirus E40RF6 cDNA was a very kind gift from Gorkan Akusvardi.
- the cDNA was excised from pBR322 using BamHI and inserted into pBK-CMV (Stratagene) to give pCMV-ORF6.
- the ORF6 cDNA was then subcloned into the hygromycin resistant expression plasmid p3"SS (Stratagene) by digestion with Hind III/XbaI and subsequent ligation to Hind III/XbaI digested p3"SS. This gave p3"SS/ORF6.
- NLS nuclear localisation signal
- Hygromycin resistant stable COS-7 cell transfectant lines were created using both the above plasmids to give lines termed COS/ORF6/NLS and COS/ORF6.
- rAAV Three different aliquots of rAAV were prepared, one prepared using the C0S/0RF6/NLS cell line, one using COS/ORF6 and one using unmodified parental COS cells.
- Helper and Vector Plasmids pUC 18 was modified by the insertion of a pair of linkers:- 5' CAT CGA TGG CCA GAT CTG ATA TCG ATG 3 ' and 5 ' CAT GGT AGC TAC CGG TCT AGA CTA TAG CTA CCT AG 3 ' between the Kpn I /Bam HI sites to give an extended multiple cloning site with additional Cla I, Bal I, Bgl II, and Eco RV sites. Wild type
- AAV was obtained from the ATCC. After digest with Bgl II, the resulting AAV fragment was cloned into the modified pUC18 to give pUC/AAV.
- Dra III and Nco I Digestion of pUC/AAV with Dra III and Nco I removes the two genes rep and cap, essentially leaving the ITRs of the wild type virus . Blunting the Dra III/Nco I ends with T4 DNA polymerase allows ligation of the ⁇ -galactosidase cDNA plasmid (Promega) to yield pUC/AAV/ ⁇ -gal (vector plasmid) .
- the ITR helper plasmid was obtained by sequential removal of the ITRs from pUC/AAV. The 5' ITR was removed by digestion with Eco RV and SnaBI and religating the DNA. This plasmid was then digested with Bal I to remove the 3 ' ITR and religated to give pUC/rep/cap. (helper plasmid) .
- AAV ⁇ - galactosidase AAV ⁇ -gal
- helper plasmid 3:1 ratio of helper to vector plasmid
- the COS cells were washed twice with HBSS and after the final aspiration the DNA/lipofectin mixture was layered onto the cells with fresh Opti-MEM to ensure the cells were adequately covered.
- the COS cells were then incubated for 5 hours at 37°C and 5% C0 2 , after which the serum free medium was replaced with fresh DMEM containing 10% FCS and adenovirus type 5 at a multiplicity of infection (MOI) of 5. Incubation was then continued until full cytopathic effect of the adenovirus was seen, typically after 48 to 72 hours.
- the infected cells were scraped off and collected together with the supernatant. Trypsin was added to a final concentration of 0.02% and deoxycholate to a final concentration of 0.5%.
- the sample was then incubated at 37°C for 2 hours and then subjected to three cycles of freeze- thawing to release any virus remaining within the cells. Following the final freeze cycle, the sample was incubated at 56°C for 1 hour to inactivate the heat-labile adenovirus. The sample was spun at 2100 xG for 10 minutes to pellit the cellular debris and the supernatant containing the recombinant AAV was decanted into a separate 50ml tube (Falcon) .
- the concentrated viral preparation was desalted using a Pharmacia Biotech pD-10 Sephadex desalt column. The desalted viral preparation was then further concentrated using a Centriprep 30 concentrator to give a final volume of approximately 500 ⁇ l.
- the viral sample Prior to infecting cells, the viral sample was sterilised free of bacteria using a 0.2 ⁇ m filter (Sartorius - Centrisart C4). The viral concentrate was then either analysed immediately, used to infect target cells or stored at -70°C.
- Viral titre was quantitated using a dot-blot assay as follows. Hybond-N nylon membrane (Amersham-Life Science) was prewetted in 6x SSC, and then inserted in the dot blot apparatus (Bio- Rad) .
- the reference control DNA and the recombinant AAV preparation were diluted in lOx SSC to the following ratios, neat, 1:2, 1:4, 1:8, 1:16, 1:32, 1:64, 1:128, 1:256, 1:512, 1:1024 and 1:2048 and immediately prior to loading, the samples were denatured by heating at 100°C for 5 minutes. The samples were then loaded onto the nylon membrane and sucked through under negative pressure. The nylon filter was then removed from the dot blot apparatus and dried by incubation for 2 hours at 80°C. After drying both sides of the filter were UV irradiated (Stratagene UV Stratalinker) .
- Prehybridisation buffer (24 mis 20x SSC, 8 mis 50x Denharts, 4mls 10% SDS, 80 ⁇ l (lOng/ml) salmon sperm DNA) , was warmed to 60°C and added to the filter present in a hybridisation bottle. Prehybridisation was allowed to continue for 4 hours at 65°C. After this time, the prehybridisation buffer was then discarded and replaced with hybridisation buffer (9 mis 20x SSC, 3mls 50x Denharts, 1.5mls 10% SDS, 300 ⁇ l EDTA 0.5M, 30 ⁇ l salmon sperm DNA (lOng/ml) , 16.2ml H 2 0) containing a radiolabelled probe.
- the probe used was the CMV promoter present in both the viral preparation and the reference plasmid.
- the CMV promoter probe was radiolabelled using the rediprime DNA labelling system (Amersham Life Science) following the manufacturers instructions. After the labelling reaction had completed, unincorporated radio- label was removed using a Qiagen desalt column following the manufacturers instructions.
- the probe Prior to the addition of the probe to the hybridisation mix and the filter, the probe was denatured by heating to 100°C for 5 minutes. Hybridisation was allowed to proceed for 18 hours at 65°C. Post hybridisation, the filter was washed twice in 2x SSC and 0.1% SDS at 37°C for 10 minutes, followed by two washes in 2x SSC and 0.1% SDS at 65°C for 10 minutes.
- ⁇ -galactosidase activity was assayed in the virally infected cells as follows. The medium was carefully aspirated from the infected cells and the cells were then washed twice with PBS buffer (137mM NaCl, 2.7mM KCl, 4.3mM Na 2 HP0 4 , 1.47mM KH 2 P0 4 , pH 7.1) . After washing 400 ⁇ l of Reporter Lysis Buffer (Promega) was added to each cell population, the cells were incubated with the lysis buffer for 15 minutes at room temperature. The cells were then carefully scraped off from each well and transferred to separate microcentrifuge tubes. Each tube was vortexed for 15 seconds and centrifuged at 13,000rpm for 2 minutes. After this the supernatants were transferred to fresh microcentrifuge tubes.
- PBS buffer 137mM NaCl, 2.7mM KCl, 4.3mM Na 2 HP0 4 , 1.47mM KH 2 P0 4 , pH 7.1
- Target cells were assessed as being positive after incubation with ONPG as described above .
- This Example demonstrates construction of a COS Cell Line capable of improving AAV-type 2 titre.
- This cell line is suitable for use in the present invention as a vehicle for a gene encoding E40RF6.
- a cell line containing multiple copies of the AAV cap gene was created as follows.
- pUC 18 was modified by the insertion of a pair of linkers:- 5' CAT CGA TGG CCA GAT CTG ATA TCG ATG 3 ' and 5 ' CAT GGT AGC TAC CGG TCT AGA CTA TAG CTA CCT AG 3 ' between the Kpn I/Bam HI sites to give an extended multiple cloning site with additional Cla I, Bal I, Bgl II, and Eco RV sites. Wild type
- AAV was obtained from the ATCC. After digest with Bgl II, the resulting AAV fragment was cloned into the modified pUC18 to give pUC/AAV.
- the AAV cap gene was excised from pUC/AAV by digestion with SnaBI and HinD III and ligated into HinD III/ Sma I digested pBK-CMV (Stratagene) to yield pCMV-cap.
- a COS cell stable transfectant cell line was created by introducing 2 ⁇ g pCMV-cap DNA into exponentially growing COS cells using Lipofectin following the manufacturers' instructions. This gave COS/cap.
- p3 ' SS (Stratagene) was digested with Pfl MI and Nsi I to release a 1.8kbp Hygromycin resistance cassette. This was blunted with T4 DNA polymerase.
- the AUG initiation codon of rep68/78 in pUC/AAV/Neo was mutated by standard techniques to give an ACG codon. This was verified by sequencing.
- This construct was digested with Apa I and Nde I to remove the cap gene and the ends blunted with T4 DNA polymerase.
- the hygromycin cassette was ligated to the mutated rep gene cassette to give pUC/ACGrep/Hygro.
- COS cell stable transfectant cell line was created by introducing 2 ⁇ g pUC/ACGrep/Hygro DNA into exponentially growing COS/cap cells using Lipofectin following the manufacturers' instructions. This gave COS/rep/cap.
- COS/rep/cap or COS/cap or parental COS cells were grown to 80% confluency in 92mm tissue culture plates (Nunclon) in DMEM supplemented with 10% FCS.
- 4.6 ⁇ g of AAV ⁇ -galactosidase (AAV ⁇ -gal) and in the case of parental COS cells, 9.4 ⁇ g of rep/cap plamid were addded to a 17 x 120mm conical polystyrene sterile tube (Falcon) , together with lOO ⁇ l of serum free medium (Opti- MEM - Life Sciences) .
- 56 ⁇ l of lipofectin was added to a second polystyrene tube together with another lOO ⁇ l of Opti-MEM.
- the DNA/lipofectin mixture was mixed gently and incubated for 15 minutes at room temperature.
- the cells were washed twice with HBSS and after the final aspiration the DNA/lipofectin mixture was layered onto the cells with fresh Opti-MEM to ensure the cells were adequately covered.
- the cells were then incubated for 5 hours at 37°C and 5% C0 2 , after which the serum free medium was replaced with fresh DMEM containing 10% FCS and adenovirus type 5 at a multiplicity of infection (MOI) of 5. Incubation was then continued until full cytopathic effect of the adenovirus was seen, typically after 48 to 72 hours.
- MOI multiplicity of infection
- the infected cells were scraped off and collected together with the supernatant. Trypsin was added to a final concentration of 0.02% and deoxycholate to a final concentration of 0.5%. The sample was then incubated at 37°C for 2 hours and then subjected to three cycles of freeze-thawing to release any virus remaining within the cells. Following the final freeze cycle, the sample was incubated at 56°C for 1 hour to inactivate the heat-labile adenovirus. The sample was spun at 2100 xG for 10 minutes to pellet the cellular debris and the supernatant containing the recombinant AAV was decanted into a separate 50ml tube (Falcon) .
- Falcon 50ml tube
- the concentrated viral preparation was desalted using a Pharmacia Biotech pD-10 Sephadex desalt column. The desalted viral preparation was then further concentrated using a Centriprep 30 concentrator to give a final volume of approximately 500 ⁇ l.
- the viral sample Prior to infecting cells, the viral sample was sterilised free of bacteria using a 0.2 ⁇ m filter (Sartorius - Centrisart C4). The viral concentrate was then either analysed immediately, used to infect target cells or stored at -70°C.
- Viral titre was quantitated using a dot-blot assay as follows. Hybond-N nylon membrane (Amersham-Life Science) was prewetted in 6x SSC, and then inserted in the dot blot apparatus (Bio- Rad) .
- the reference control DNA and the recombinant AAV preparation were diluted in lOx SSC to the following ratios, neat, 1:2, 1:4, 1:8, 1:16, 1:32, 1:64, 1:128, 1:256, 1:512, 1:1024 and 1:2048 and immediately prior to loading, the samples were denatured by heating at 100°C for 5 minutes. The samples were then loaded onto the nylon membrane and sucked through under negative pressure. The nylon filter was then removed from the dot blot apparatus and dried by incubation for 2 hours at 80°C. After drying both sides of the filter were UV irradiated (Stratagene UV Stratalinker) .
- Prehybridisation buffer (24 mis 20x SSC, 8 mis 50x Denharts, 4mls 10% SDS, 80 ⁇ l (lOng/ml) salmon sperm DNA) , was warmed to 60°C and added to the filter present in a hybridisation bottle. Prehybridisation was allowed to continue for 4 hours at 65°C. After this time, the prehybridisation buffer was then discarded and replaced with hybridisation buffer (9 mis 20x SSC, 3mls 50x Denharts, 1.5mls 10% SDS, 300 ⁇ l EDTA 0.5M, 30 ⁇ l salmon sperm DNA (lOng/ml) , 16.2ml H 2 0) containing a radiolabelled probe.
- the probe used was the CMV promoter present in both the viral preparation and the reference plasmid.
- the CMV promoter probe was radiolabelled using the rediprime DNA labelling system (Amersham Life Science) following the manufacturers' instructions. After the labelling reaction had completed, unincorporated radio-label was removed using a Qiagen desalt column following the manufacturers instructions.
- the probe Prior to the addition of the probe to the hybridisation mix and the filter, the probe was denatured by heating to 100°C for 5 minutes. Hybridisation was allowed to proceed for 18 hours at 65°C. Post hybridisation, the filter was washed twice in 2x SSC and 0.1% SDS at 37°C for 10 minutes, followed by two washes in 2x SSC and 0.1% SDS at 65°C for 10 minutes.
- Titres of virus, as determined by the viral dot-blot, obtained from the COS rep/cap cells were higher than those obtained using parental cells and the two plasmid system. No virus was produced from the COS/cap cells .
- the results are set out in Table 2.
- All titres refer to the number of genomes obtained per ml. of virus as assessed by dot-blot hybridisation.
- a gene encoding E40RF6 may be incorporated into this improved COS cell line, as described above.
Landscapes
- Life Sciences & Earth Sciences (AREA)
- Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Engineering & Computer Science (AREA)
- Zoology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Biomedical Technology (AREA)
- Organic Chemistry (AREA)
- Biotechnology (AREA)
- General Engineering & Computer Science (AREA)
- Chemical & Material Sciences (AREA)
- Wood Science & Technology (AREA)
- Microbiology (AREA)
- Physics & Mathematics (AREA)
- Plant Pathology (AREA)
- Virology (AREA)
- Molecular Biology (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- Biophysics (AREA)
- Medicines Containing Material From Animals Or Micro-Organisms (AREA)
- Peptides Or Proteins (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
An adeno-associated viral vector containing a transgene and encapsidating a protein with adenovirus E4 ORF6 activity.
Description
AAV DERIVED VECTOR
Field of the Invention
The present invention relates to an improved adeno-associated viral vector, to a method of transfecting target cells, an adeno-associated viral producer cell and a method for the production of an adeno-associated viral vector.
Background of the Invention
Adeno-associated virus (AAV) is a vector which has shown promise in virally-mediated gene therapy protocols. AAV is a small, 20-22nm, replication defective, non-pathogenic parvo virus. Six serotypes of AAV are currently known (types 1 to 6) , of which type 2 is the most extensively studied. AAV type 2 has a single stranded DNA genome of 4.7kB which encodes both structral proteins through a cap gene and non-structural proteins through a rep gene. The virus has a pair of 146bp inverted terminal repeats (ITRs) which are the only els acting elements in AAV required for replication and packaging.
In J.Virol.70 (5) :3772-3234, 1996, Ferrari et al have proposed that the conversion of the AAV single stranded genome to the double stranded form is a rate-limiting step for efficient transduction by recombinant AAV vectors. In J.Virol. 70 (1) :520-532 , 1996, Fischer et al proposed that transduction with recombinant AAV for gene therapy is limited by leading- strand synthesis. The authors showed that the addition of adenovirus increased the levels of expression of an AAV transgene. Studies using various adenovirus mutants showed that a polypeptide encoded by the adenovirus E4 region open reading frame 6 (E4 ORF6) was responsible although the precise nature of the polypeptide is unknown.
Summary of the Invention
The present invention aims to increase the level of transgene expression from an adeno-associated viral vector.
Accordingly, in a first aspect, the present invention provides an adeno-associated viral vector containing a transgene and encapsidating a protein with adenovirus E4 ORF6 activity. The protein is preferably an adenovirus E4 ORF6 protein such as is obtainable from adenovirus .
In a further aspect, the present invention provides the adeno- associated viral vector for use as a medicament, especially in gene therapy. Also provided is a method for transfecting target cells, which comprises contacting target cells with the adeno-associated viral vector under conditions to achieve adeno-associated viral infection.
It has been found that by engineering an appropriate adeno- associated viral producer cell it is possible to incorporate into an AAV vector both the transgene and the protein with E4 ORF6 activity. The level of expression of the transgene in target cells is found to be usefully increased. Without wishing to be bound by theory, it is thought E4 ORF6 needs to be present as a polypeptide to facilitate conversion of the AAV single stranded genome to the double stranded replicative form.
In a further aspect, the present invention provides an adeno- associated viral producer cell containing a gene encoding a protein with E4 ORF6 activity, which gene is under the control of an inducible promoter.
It has been found that constitutive expression of E4 0RF6 in the AAV producer cell tends to have an adverse effect on the cell. This was unexpected. However, by putting the gene
under control of an inducible promoter the producer cell is able to tolerate the presence of the protein. Any inducible promoter may be used, such as the MMTV-LTR (Mouse Mammary Tumour Virus-Long Terminal Repeat) promoter, which is inducible by dexamethasone . The inducer may be added to the medium in which the cells grow or, alternatively, may be produced upon infection of the cell by AAV or helper virus for AAV. In the examples below, an AAV inducible promoter was used; the P5 promoter. In this case, upon infection with both adeno-virus and AAV, the P5 promoter is induced so as to induce the E4 ORF6 protein.
In order to maintain the gene encoding the protein with E4 ORF6 activity in the viral producer cell it is advantageous to associate the gene with a marker gene which encodes a selectable marker so as to enable producer cells containing the gene to be readily identified and maintained. Typically, the marker comprises a drug-resistance marker, such as an antibiotic resistance marker which enables the cells to be grown in medium containing the relevant drug or antibiotic. Alternatively, the selectable marker may be a cell surface marker such as a receptor which can be specifically targeted by a complimentary molecule such as an antibody thereto. One such cell surface marker is nerve growth factor (NGF) .
The gene encoding adenovirus E4 ORF6 protein may be conveniently introduced into the producer cell on a plasmid. The E4 ORF6 gene may be readily obtained from an adenovirus such as adenovirus type 2, which is deposited as ATCC VR846, the sequence of which is published by EMBL at locus HACG. See also Ohman et al in Virology 194 (1) , 50-58, 1993 where cloning of E4 ORF is reported. From the EMBL sequence the E4 region spans nucleotides 32801-35609 and E4 ORF6 from Ohman et al spans nucleotides 33193-34115 .
Advantageously, the protein with E4 ORF6 activity has a nuclear localisation sequence which is typically situated outside of the functional part of the E4 ORF6 protein, typically at or near the N- or C-terminal sequence of the protein. The nuclear localisation sequence confers on the protein an important property. Once the protein is produced in the producer cell it is transported by the cell to the nucleus. It is in the nucleus where AAV capsid assembly occurs and it is thought that localisation of the protein in the region of the nucleus facilitates encapsidation of the protein by AAVs .
The AAV producer cell which incorporates the gene encoding the protein with E4 ORF6 activity may be any producer cell commonly used to produce AAV vectors, such as a human 293 producer cell (ATCC CRL 1573) . Typically, the AAV will be rep" cap" and so either a second transfection step involving the introduction of rep and cap on, for example, a plasmid would need to be undertaken or the producer cells would have to be made rep+ cap+. In one embodiment, the producer cell is a stable cell containing rep and cap, both associated with a selectable marker as described above. In this embodiment, transfection or infection by AAV DNA (rep" cap") requires no second plasmid transfection step. In a further embodiment, the producer cell comprises a rep-containing region which comprises a rep gene associated with a marker gene for rep, and a cap-containing region which comprises a cap gene associated with a marker gene for cap.
The rep-containing and cap-containing regions are preferably sufficiently spaced apart to prevent their simultaneous recombination with adeno-associated virus. This confers a further advantage on the viral producer cells . The AAV DNA would necessarily contain inverted terminal repeats and so a
problem arises in producing AAV vectors because there is a low but significant frequency of homologous recombination. Where the AAV producer cells contain a rep gene contiguous with a cap gene, homologous recombination events result in contaminating amounts of wild-type AAV being formed. This problem is avoided where the cap and rep genes are spaced sufficiently apart to prevent such simultaneous co- recombination of rep and cap.
Preferably, the rep gene and/or the cap gene is plasmid-borne so as to avoid contaminating homologous recombination. More preferably, both rep and cap genes are plasmid-borne. In an advantageous embodiment, the viral producer cell is formed from a multicopy plasmid which carries a rep gene and cap gene. In this embodiment it is preferable for the plasmid to have an origin of replication. The producer cell is preferably capable of producing a replication initiator for the origin of replication of the plasmid. In this way, multiple copies of the plasmid are made in the cell. Various combinations of replication initiator and origin of replication are known. A particularly useful combination is where the cell is a COS cell and the origin of replication is SV40 ori . COS cells produce T antigen as a replication initiator for SV40 ori. Another example of such a combination is EBNA1 as a replication initiator for oriP. EBNA1 and oriP are derived from the Epstein Barr virus which is capable of infecting a variety of cells, including human cell lines.
It is also thought that rep has a role in down-regulating cap transcription. Therefore, by reducing rep translation efficiency an increase in the AAV vector titre in the producer cell can be achieved. Accordingly, in a preferred embodiment, the rep gene encodes a rep mRNA which has reduced efficiency of translation as compared with wild-type rep mRNA.
More preferably, the rep mRNA of the viral producer cells has a mutant initiation codon which reduces the efficiency of translation. Wild-type initiation codons are AUG whereas mutant initiation codons may be UUG, CUG, or GUG, preferably ACG.
Particularly preferably, as described in copending PCT patent application (PWF 91911) filed on 21st May 1999, claiming priority from UK patent application number 9811172.7, the adeno-associated viral producer cell comprises at least one rep gene and at least two cap genes, wherein the number of cap genes exceeds the number of rep genes . This may be achieved by locating a rep gene on one plasmid and locating a cap gene on a multicopy plasmid.
It has been found that by making multiple copies of the cap gene in the viral producer cell and by reducing the translation efficiency of the rep gene product, an increased yield of adeno-associated viral vectors is achieved.
In a further aspect, the present invention provides a method for the production of an adeno-associated viral vector, which comprises introducing into the viral producer cell DNA comprising a transgene flanked by adeno-associated virus inverted terminal repeats, allowing formation of adeno- associated viral vectors in the viral producer cell, and isolating the adeno-associated viral vectors. The DNA may be introduced into the producer cell by transfection, for example as part of a suitable plasmid, in the presence of an adeno- associated helper virus, or by infection as part of a suitable virus. The DNA may be integrated into the genome of an adeno- associated helper virus which infects the producer cell or the DNA may comprise the genome of an adeno-associated virus which is infected into the producer cell in the presence of an
adeno-associated helper virus. Typically, the helper virus comprises an adenovirus, such as adenovirus type 5.
Detailed Description of the Invention
The invention will now be described in further detail, by way of example only, with reference to the following experimental work .
EXAMPLE 1
This example compares level of transgene expression in target cells using AAV type 2 viruses from producer cell lines containing E4 0RF6 with and without an NLS .
E4 ORF6 Vectors and Cell Lines
A stable cell line was made by lipofecting COS 7 cells (ATCC CRL 1651) with the E4 ORF6 containing plasmid containing a hygromycin resistance cassette.
Adenovirus E40RF6 cDNA was a very kind gift from Gorkan Akusvardi. The cDNA was excised from pBR322 using BamHI and inserted into pBK-CMV (Stratagene) to give pCMV-ORF6. The ORF6 cDNA was then subcloned into the hygromycin resistant expression plasmid p3"SS (Stratagene) by digestion with Hind III/XbaI and subsequent ligation to Hind III/XbaI digested p3"SS. This gave p3"SS/ORF6. Addition of a nuclear localisation signal (NLS) was achieved by sequential rounds of insertional mutagenesis using standard techniques to add, in frame, the amino acid sequence Pro-Lys-Lys-Lys-Arg-Lys-Ala. (NLS first described by Kalderon D. et al . A short amino acid sequence able to specify nuclear location. Cell 39 (3 ) .499-509 , 1984 and Kalderon D. et al . Sequence requirements for nuclear location of simian virus 40 large-T antigen. Nature. 311(5981) :33-8, 1984) .
The DNA sequence was verified by sequencing. This gave p3 "SS/ORF6/NLS . An additional plasmid was constructed which did not contain the NLS called p3 SS/ORF6.
Hygromycin resistant stable COS-7 cell transfectant lines were created using both the above plasmids to give lines termed COS/ORF6/NLS and COS/ORF6.
Three different aliquots of rAAV were prepared, one prepared using the C0S/0RF6/NLS cell line, one using COS/ORF6 and one using unmodified parental COS cells.
Construction of Helper and Vector Plasmids pUC 18 was modified by the insertion of a pair of linkers:- 5' CAT CGA TGG CCA GAT CTG ATA TCG ATG 3 ' and 5 ' CAT GGT AGC TAC CGG TCT AGA CTA TAG CTA CCT AG 3 ' between the Kpn I /Bam HI sites to give an extended multiple cloning site with additional Cla I, Bal I, Bgl II, and Eco RV sites. Wild type
AAV was obtained from the ATCC. After digest with Bgl II, the resulting AAV fragment was cloned into the modified pUC18 to give pUC/AAV.
Digestion of pUC/AAV with Dra III and Nco I removes the two genes rep and cap, essentially leaving the ITRs of the wild type virus . Blunting the Dra III/Nco I ends with T4 DNA polymerase allows ligation of the β-galactosidase cDNA plasmid (Promega) to yield pUC/AAV/β-gal (vector plasmid) .
The ITR helper plasmid was obtained by sequential removal of the ITRs from pUC/AAV. The 5' ITR was removed by digestion with Eco RV and SnaBI and religating the DNA. This plasmid was
then digested with Bal I to remove the 3 ' ITR and religated to give pUC/rep/cap. (helper plasmid) .
Production of Recombinant AAV
Production of recombinant AAV requires a two plasmid system, with the rep and cap gene functions being supplied in trans by the AAV helper plasmid and the vector plasmid containing the expression cassette between the two ITR's. COS cells were grown to 80% confluency in 92mm tissue culture plates (Nunclon) in DMEM supplemented with 10% FCS . 4.6μg of AAV β- galactosidase (AAV β-gal) and 9.4μg of helper plasmid (3:1 ratio of helper to vector plasmid) were addded to a 17 x 120mm conical polystyrene sterile tube (Falcon) , together with lOOμl of serum free medium (Opti-MEM - Life Sciences) . 56μl of lipofectin was added to a second polystyrene tube together with another lOOμl of Opti-MEM. These tubes were individually mixed and then the contents of the second tube were added to the first. The DNA/lipofectin mixture was mixed gently and incubated for 15 minutes at room temperature . The COS cells were washed twice with HBSS and after the final aspiration the DNA/lipofectin mixture was layered onto the cells with fresh Opti-MEM to ensure the cells were adequately covered. The COS cells were then incubated for 5 hours at 37°C and 5% C02, after which the serum free medium was replaced with fresh DMEM containing 10% FCS and adenovirus type 5 at a multiplicity of infection (MOI) of 5. Incubation was then continued until full cytopathic effect of the adenovirus was seen, typically after 48 to 72 hours. The infected cells were scraped off and collected together with the supernatant. Trypsin was added to a final concentration of 0.02% and deoxycholate to a final concentration of 0.5%. The sample was then incubated at 37°C for 2 hours and then subjected to three cycles of freeze- thawing to release any virus remaining within the cells. Following the final freeze cycle, the sample was incubated at
56°C for 1 hour to inactivate the heat-labile adenovirus. The sample was spun at 2100 xG for 10 minutes to pellit the cellular debris and the supernatant containing the recombinant AAV was decanted into a separate 50ml tube (Falcon) .
Purification of Recombinant AAV Usincr Cellufine Affinity Column
Sulfonated cellulose (Cellulofine) was packed into the column (Pharmacia Biotech XK50) and equilibrated with PBS (pH 7.8). The supernatant containing recombinant AAV was then applied to the column, which was then washed with PBS (pH 7.8) until the absorbance of the elutate at 280nm had returned to the baseline level. The AAV bound to the cellulofine was eluted with lOmM phosphate buffer containing 1.0M NaCl . 3ml fractions were collected, and the aliquots containing eluted AAV were identified by the increase in absorbance detected at 280nm. These fractions were then pooled and concentrated using a Centriprep 50 concentrator, to a final volume of approximately 2.5mls. The concentrated viral preparation was desalted using a Pharmacia Biotech pD-10 Sephadex desalt column. The desalted viral preparation was then further concentrated using a Centriprep 30 concentrator to give a final volume of approximately 500μl. Prior to infecting cells, the viral sample was sterilised free of bacteria using a 0.2μm filter (Sartorius - Centrisart C4). The viral concentrate was then either analysed immediately, used to infect target cells or stored at -70°C.
Dot Blot Hybridisation
Viral titre was quantitated using a dot-blot assay as follows. Hybond-N nylon membrane (Amersham-Life Science) was prewetted in 6x SSC, and then inserted in the dot blot apparatus (Bio- Rad) . The reference control DNA and the recombinant AAV preparation were diluted in lOx SSC to the following ratios,
neat, 1:2, 1:4, 1:8, 1:16, 1:32, 1:64, 1:128, 1:256, 1:512, 1:1024 and 1:2048 and immediately prior to loading, the samples were denatured by heating at 100°C for 5 minutes. The samples were then loaded onto the nylon membrane and sucked through under negative pressure. The nylon filter was then removed from the dot blot apparatus and dried by incubation for 2 hours at 80°C. After drying both sides of the filter were UV irradiated (Stratagene UV Stratalinker) .
Prehybridisation buffer (24 mis 20x SSC, 8 mis 50x Denharts, 4mls 10% SDS, 80μl (lOng/ml) salmon sperm DNA) , was warmed to 60°C and added to the filter present in a hybridisation bottle. Prehybridisation was allowed to continue for 4 hours at 65°C. After this time, the prehybridisation buffer was then discarded and replaced with hybridisation buffer (9 mis 20x SSC, 3mls 50x Denharts, 1.5mls 10% SDS, 300μl EDTA 0.5M, 30μl salmon sperm DNA (lOng/ml) , 16.2ml H20) containing a radiolabelled probe. The probe used was the CMV promoter present in both the viral preparation and the reference plasmid. The CMV promoter probe was radiolabelled using the rediprime DNA labelling system (Amersham Life Science) following the manufacturers instructions. After the labelling reaction had completed, unincorporated radio- label was removed using a Qiagen desalt column following the manufacturers instructions. Prior to the addition of the probe to the hybridisation mix and the filter, the probe was denatured by heating to 100°C for 5 minutes. Hybridisation was allowed to proceed for 18 hours at 65°C. Post hybridisation, the filter was washed twice in 2x SSC and 0.1% SDS at 37°C for 10 minutes, followed by two washes in 2x SSC and 0.1% SDS at 65°C for 10 minutes. After washing the filter was wrapped in Saran wrap, and placed next to X-Ray film (Fuji) and exposed for 3 hours at -70°C. The exposed film was then developed.
Viral titre was assessed by comparing the relative signal strengths of the test and reference standards.
Detection of β-Galactosidase Activity in Transfected Cells β-galactosidase activity was assayed in the virally infected cells as follows. The medium was carefully aspirated from the infected cells and the cells were then washed twice with PBS buffer (137mM NaCl, 2.7mM KCl, 4.3mM Na2HP04, 1.47mM KH2P04, pH 7.1) . After washing 400μl of Reporter Lysis Buffer (Promega) was added to each cell population, the cells were incubated with the lysis buffer for 15 minutes at room temperature. The cells were then carefully scraped off from each well and transferred to separate microcentrifuge tubes. Each tube was vortexed for 15 seconds and centrifuged at 13,000rpm for 2 minutes. After this the supernatants were transferred to fresh microcentrifuge tubes.
In a flat bottomed, 96 well plate, serial dilutions of each cell extract, to a maximum volume of 25μl were diluted with lx Reporter Lysis Buffer to give a final volume of 50μl. A 50μl aliquot of 2x Assay Buffer (120mM Na2HP04, 80mM NaH2P04, 2mM MgCl2, lOOmM β-mercaptoethanol and 1.33mg/ml ONPG) was then added to each sample which was then mixed, the plate was then covered and incubated at 37°C for 30 minutes. The reaction was stopped by the addition of 150μl of IM sodium carbonate to each well. The absorbance of the samples was read at 405nm using a plate reader (Anthos reader 2001) .
RESULTS
Cells infected with the viruses prepared from the COS/ORF6/NLS cell line produced a higher titre of β-gal activity as compared to cells infected with virus prepared from the other two cell lines using the same titre of virus as determined by the viral dot -blot.
The results are set out in Table 1 below.
TABLE ONE
Method of Viral producer cell Percentage target virus line . cells positive for production. β-galactosidase .
Dual COS cells 45% transfection
Dual COS cells 55% transfection Containing E4 ORF6 minus nuclear localisation signal
Dual COS cells 85% transfection Containing E4 ORF6 plus nuclear localisation signal
Target cells were assessed as being positive after incubation with ONPG as described above .
EXAMPLE 2
This Example demonstrates construction of a COS Cell Line capable of improving AAV-type 2 titre. This cell line is suitable for use in the present invention as a vehicle for a gene encoding E40RF6.
Improved COS Cell Line Construction
A cell line containing multiple copies of the AAV cap gene was created as follows.
pUC 18 was modified by the insertion of a pair of linkers:- 5' CAT CGA TGG CCA GAT CTG ATA TCG ATG 3 ' and 5 ' CAT GGT AGC TAC CGG TCT AGA CTA TAG CTA CCT AG 3 ' between the Kpn I/Bam HI
sites to give an extended multiple cloning site with additional Cla I, Bal I, Bgl II, and Eco RV sites. Wild type
AAV was obtained from the ATCC. After digest with Bgl II, the resulting AAV fragment was cloned into the modified pUC18 to give pUC/AAV.
The AAV cap gene was excised from pUC/AAV by digestion with SnaBI and HinD III and ligated into HinD III/ Sma I digested pBK-CMV (Stratagene) to yield pCMV-cap. A COS cell stable transfectant cell line was created by introducing 2μg pCMV-cap DNA into exponentially growing COS cells using Lipofectin following the manufacturers' instructions. This gave COS/cap.
p3 ' SS (Stratagene) was digested with Pfl MI and Nsi I to release a 1.8kbp Hygromycin resistance cassette. This was blunted with T4 DNA polymerase. The AUG initiation codon of rep68/78 in pUC/AAV/Neo was mutated by standard techniques to give an ACG codon. This was verified by sequencing. This construct was digested with Apa I and Nde I to remove the cap gene and the ends blunted with T4 DNA polymerase. The hygromycin cassette was ligated to the mutated rep gene cassette to give pUC/ACGrep/Hygro.
A COS cell stable transfectant cell line was created by introducing 2μg pUC/ACGrep/Hygro DNA into exponentially growing COS/cap cells using Lipofectin following the manufacturers' instructions. This gave COS/rep/cap.
Production of Recombinant AAV
COS/rep/cap or COS/cap or parental COS cells were grown to 80% confluency in 92mm tissue culture plates (Nunclon) in DMEM supplemented with 10% FCS. 4.6μg of AAV β-galactosidase (AAV β-gal) and in the case of parental COS cells, 9.4μg of rep/cap plamid were addded to a 17 x 120mm conical polystyrene sterile
tube (Falcon) , together with lOOμl of serum free medium (Opti- MEM - Life Sciences) . 56μl of lipofectin was added to a second polystyrene tube together with another lOOμl of Opti-MEM. These tubes were individually mixed and then the contents of the second tube were added to the first. The DNA/lipofectin mixture was mixed gently and incubated for 15 minutes at room temperature. The cells were washed twice with HBSS and after the final aspiration the DNA/lipofectin mixture was layered onto the cells with fresh Opti-MEM to ensure the cells were adequately covered. The cells were then incubated for 5 hours at 37°C and 5% C02, after which the serum free medium was replaced with fresh DMEM containing 10% FCS and adenovirus type 5 at a multiplicity of infection (MOI) of 5. Incubation was then continued until full cytopathic effect of the adenovirus was seen, typically after 48 to 72 hours. The infected cells were scraped off and collected together with the supernatant. Trypsin was added to a final concentration of 0.02% and deoxycholate to a final concentration of 0.5%. The sample was then incubated at 37°C for 2 hours and then subjected to three cycles of freeze-thawing to release any virus remaining within the cells. Following the final freeze cycle, the sample was incubated at 56°C for 1 hour to inactivate the heat-labile adenovirus. The sample was spun at 2100 xG for 10 minutes to pellet the cellular debris and the supernatant containing the recombinant AAV was decanted into a separate 50ml tube (Falcon) .
Purification of Recombinant AAV Using Cellufine Affinity Column
Sulfonated cellulose (Cellulofine) was packed into the column (Pharmacia Biotech XK50) and equilibrated with PBS (pH 7.8). The supernatant containing recombinant AAV was then applied to the column, which was then washed with PBS (pH 7.8) until the absorbance of the elutate at 280nm had returned to the
baseline level. The AAV bound to the cellulofine was eluted with lOmM phosphate buffer containing 1. OM NaCl . 3ml fractions were collected, and the aliquots containing eluted AAV were identified by the increase in absorbance detected at 280nm. These fractions were then pooled and concentrated using a Centriprep 50 concentrator, to a final volume of approximately 2.5mls. The concentrated viral preparation was desalted using a Pharmacia Biotech pD-10 Sephadex desalt column. The desalted viral preparation was then further concentrated using a Centriprep 30 concentrator to give a final volume of approximately 500μl. Prior to infecting cells, the viral sample was sterilised free of bacteria using a 0.2μm filter (Sartorius - Centrisart C4). The viral concentrate was then either analysed immediately, used to infect target cells or stored at -70°C.
Dot Blot Hybridisation
Viral titre was quantitated using a dot-blot assay as follows. Hybond-N nylon membrane (Amersham-Life Science) was prewetted in 6x SSC, and then inserted in the dot blot apparatus (Bio- Rad) . The reference control DNA and the recombinant AAV preparation were diluted in lOx SSC to the following ratios, neat, 1:2, 1:4, 1:8, 1:16, 1:32, 1:64, 1:128, 1:256, 1:512, 1:1024 and 1:2048 and immediately prior to loading, the samples were denatured by heating at 100°C for 5 minutes. The samples were then loaded onto the nylon membrane and sucked through under negative pressure. The nylon filter was then removed from the dot blot apparatus and dried by incubation for 2 hours at 80°C. After drying both sides of the filter were UV irradiated (Stratagene UV Stratalinker) .
Prehybridisation buffer (24 mis 20x SSC, 8 mis 50x Denharts, 4mls 10% SDS, 80μl (lOng/ml) salmon sperm DNA) , was warmed to 60°C and added to the filter present in a hybridisation
bottle. Prehybridisation was allowed to continue for 4 hours at 65°C. After this time, the prehybridisation buffer was then discarded and replaced with hybridisation buffer (9 mis 20x SSC, 3mls 50x Denharts, 1.5mls 10% SDS, 300μl EDTA 0.5M, 30μl salmon sperm DNA (lOng/ml) , 16.2ml H20) containing a radiolabelled probe. The probe used was the CMV promoter present in both the viral preparation and the reference plasmid. The CMV promoter probe was radiolabelled using the rediprime DNA labelling system (Amersham Life Science) following the manufacturers' instructions. After the labelling reaction had completed, unincorporated radio-label was removed using a Qiagen desalt column following the manufacturers instructions. Prior to the addition of the probe to the hybridisation mix and the filter, the probe was denatured by heating to 100°C for 5 minutes. Hybridisation was allowed to proceed for 18 hours at 65°C. Post hybridisation, the filter was washed twice in 2x SSC and 0.1% SDS at 37°C for 10 minutes, followed by two washes in 2x SSC and 0.1% SDS at 65°C for 10 minutes. After washing the filter was wrapped in Saran wrap, and placed next to X-Ray film (Fuji) and exposed for 3 hours at -70°C. The exposed film was then developed. Viral titre was assessed by comparing the relative signal strengths of the test and reference standards.
RESULTS .
Titres of virus, as determined by the viral dot-blot, obtained from the COS rep/cap cells were higher than those obtained using parental cells and the two plasmid system. No virus was produced from the COS/cap cells . The results are set out in Table 2.
Cell type Transfection Vector/Help Titre of AAV method. er ratio observed
293 cells Dual plasmid 3 :1 1.5 x 108
COS cells Dual plasmid 3 :1 1 x 107
Improved
Helper COS Single plasmid N/A 1 x 109
All titres refer to the number of genomes obtained per ml. of virus as assessed by dot-blot hybridisation.
A gene encoding E40RF6 may be incorporated into this improved COS cell line, as described above.
Claims
1. An adeno-associated viral vector containing a transgene and encapsidating a protein with adenovirus E4 ORF6 activity.
2. An adeno-associated viral vector according to claim 1, wherein the protein is an adenovirus E4 ORF6 protein.
3. An adeno-associated viral vector according to claim 1 or claim 2, for use in gene therapy.
4. A method for transfecting target cells, which comprises contacting target cells with an adeno-associated viral vector according to claim 1 or claim 2 under conditions to achieve adeno-associated viral infection.
5. An adeno-associated viral producer cell containing a gene encoding a protein with an adenovirus E4 ORF6 activity, which gene is under the control of an inducible promoter.
6. An adeno-associated viral producer cell according to claim 5, wherein the gene encodes adenovirus E4 0RF6 protein.
7. An adeno-associated viral producer cell according to claim 6, wherein the gene is associated with a selectable marker gene .
8. An adeno-associated viral producer cell according to claim 7, wherein the marker gene encodes a drug resistance marker or a cell surface marker.
9. An adeno-associated viral producer cell according to any one of claims 5 to 8 , wherein the gene is plasmid-borne.
10. An adeno-associated viral producer cell according to any one of claims 5 to 9, wherein the protein has a nuclear localisation sequence.
11. An adeno-associated viral producer cell according to any one of claims 5 to 10, which further comprises a rep- containing region which comprises a rep gene associated with a second marker gene, and a cap-containing region which comprises a cap . gene associated with a third marker gene, the rep-containing and cap-containing regions being sufficiently spaced apart to prevent their sumultaneous recombination with adeno-associated virus.
12. A method for the production of an adeno-associated viral vector, which comprises introducing into a viral producer cell according to any one of claims 5 to 11, DNA comprising a transgene flanked by adeno-associated virus inverted terminal repeats, allowing formation of adeno-associated viral vectors in the viral producer cell, and isolating the adeno-associated viral vectors .
13. A method according to claim 12, wherein the DNA is transfected into the viral producer cell in the presence of an adeno-associated helper virus.
14. A method according to claim 12, wherein the DNA is integrated into the genome of an adeno-associated helper virus which is infected into the viral producer cell.
15. A method according to claim 12, wherein the DNA comprises the genome of an adeno-associated virus which is infected into the viral producer cell in the presence of an adeno-associated helper virus.
16. A method according to any one of claims 13 to 15, wherein the helper virus is an adenovirus.
Applications Claiming Priority (2)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
GBGB9811171.9A GB9811171D0 (en) | 1998-05-22 | 1998-05-22 | Viral vector |
GB9811171.9 | 1998-05-22 |
Publications (2)
Publication Number | Publication Date |
---|---|
WO1999061640A2 true WO1999061640A2 (en) | 1999-12-02 |
WO1999061640A3 WO1999061640A3 (en) | 2000-01-27 |
Family
ID=10832634
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
PCT/GB1999/001633 WO1999061640A2 (en) | 1998-05-22 | 1999-05-21 | Aav derived vector |
Country Status (2)
Country | Link |
---|---|
GB (1) | GB9811171D0 (en) |
WO (1) | WO1999061640A2 (en) |
Cited By (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
DE10056210A1 (en) * | 2000-11-13 | 2002-05-29 | Arimedes Biotechnology Gmbh | Viral expression system |
WO2003104467A1 (en) * | 2002-04-25 | 2003-12-18 | Crucell Holland B.V. | Means and methods for the production of adenovirus vectors |
US8912142B2 (en) | 2009-04-01 | 2014-12-16 | The Medical Research, Infrastructure and Health Services Fund of the Tel Aviv Medical Center | Method of regulating proliferation and differentiation of keratinocyes |
Citations (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO1996000587A1 (en) * | 1994-06-30 | 1996-01-11 | University Of Pittsburgh | Aav capsid vehicles for molecular transfer |
WO1996022378A1 (en) * | 1995-01-20 | 1996-07-25 | Rhone-Poulenc Rorer S.A. | Cells for the production of recombinant adenoviruses |
WO1996039530A2 (en) * | 1995-06-05 | 1996-12-12 | The Trustees Of The University Of Pennsylvania | Recombinant adenovirus and adeno-associated virus, cell lines, and methods of production and use thereof |
WO1997009441A2 (en) * | 1995-09-08 | 1997-03-13 | Genzyme Corporation | Improved aav vectors for gene therapy |
-
1998
- 1998-05-22 GB GBGB9811171.9A patent/GB9811171D0/en not_active Ceased
-
1999
- 1999-05-21 WO PCT/GB1999/001633 patent/WO1999061640A2/en active Application Filing
Patent Citations (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO1996000587A1 (en) * | 1994-06-30 | 1996-01-11 | University Of Pittsburgh | Aav capsid vehicles for molecular transfer |
WO1996022378A1 (en) * | 1995-01-20 | 1996-07-25 | Rhone-Poulenc Rorer S.A. | Cells for the production of recombinant adenoviruses |
WO1996039530A2 (en) * | 1995-06-05 | 1996-12-12 | The Trustees Of The University Of Pennsylvania | Recombinant adenovirus and adeno-associated virus, cell lines, and methods of production and use thereof |
WO1997009441A2 (en) * | 1995-09-08 | 1997-03-13 | Genzyme Corporation | Improved aav vectors for gene therapy |
Non-Patent Citations (3)
Title |
---|
FERRARI F. K. ET AL.: "SECOND-STRAND SYNTHESIS IS A RATE-LIMITING STEP FOR EFFICIENT TRANSDUCTION BY RECOMBINANT ADENO-ASSOCIATED VIRUS VECTORS" JOURNAL OF VIROLOGY,US,THE AMERICAN SOCIETY FOR MICROBIOLOGY, vol. 70, no. 5, page 3227-3234 XP000570467 ISSN: 0022-538X cited in the application * |
FISHER K. J. ET AL.: "TRANSDUCTION WITH RECOMBINANT ADENO-ASSOCIATED VIRUS FOR GENE THERAPY IS LIMITED BY LEADING-STRAND SYNTHESIS" JOURNAL OF VIROLOGY,US,THE AMERICAN SOCIETY FOR MICROBIOLOGY, vol. 70, no. 1, page 520-532 XP000570468 ISSN: 0022-538X cited in the application * |
LEPPARD K. N.: "E4 gene function in adenovirus, adenovirus vector and adenovirus infections" JOURNAL OF GENERAL VIROLOGY,GB,SOCIETY FOR GENERAL MICROBIOLOGY, READING, vol. 78, page 2131-2138 XP002086863 ISSN: 0022-1317 * |
Cited By (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
DE10056210A1 (en) * | 2000-11-13 | 2002-05-29 | Arimedes Biotechnology Gmbh | Viral expression system |
WO2003104467A1 (en) * | 2002-04-25 | 2003-12-18 | Crucell Holland B.V. | Means and methods for the production of adenovirus vectors |
EA010828B1 (en) * | 2002-04-25 | 2008-12-30 | Круселл Холланд Б.В. | Recombinant adenovirus vectors and methods for the production thereof and use |
US8912142B2 (en) | 2009-04-01 | 2014-12-16 | The Medical Research, Infrastructure and Health Services Fund of the Tel Aviv Medical Center | Method of regulating proliferation and differentiation of keratinocyes |
Also Published As
Publication number | Publication date |
---|---|
WO1999061640A3 (en) | 2000-01-27 |
GB9811171D0 (en) | 1998-07-22 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
US8409842B2 (en) | Recombinant adeno-associated virus production | |
US6943019B2 (en) | Methods and vector constructs useful for production of recombinant AAV | |
JP4573437B2 (en) | Adeno-associated virus serotype 1 nucleic acid sequence, vector and host cell containing the same | |
US8637255B2 (en) | Adeno-associated virus serotype I nucleic acid sequences, vectors and host cells containing same | |
AU699973B2 (en) | Recombinant viral vector system | |
Collaco et al. | A helper virus-free packaging system for recombinant adeno-associated virus vectors | |
US20040087026A1 (en) | Host cells for packing a recombinant adeno-associated virus (raav), method for the production and use thereof | |
CA2359748A1 (en) | Method of producing a recombinant adeno-associated virus, suitable means for producing the same and use thereof for producing a medicament | |
US20110201088A1 (en) | Production of rAAV in Vero Cells Using Particular Adenovirus Helpers | |
CZ230697A3 (en) | Cells for producing recombinant adenoviruses | |
WO1996036364A9 (en) | Recombinant viral vector system | |
US20020064878A1 (en) | Compositions and methods for recombinant adeno-associated virus production | |
JP2001500015A (en) | Method for producing inducible recombinant adeno-associated virus using T7 polymerase | |
US20070202587A1 (en) | Recombinant AAV production in mammalian cells | |
CA2397532A1 (en) | Recombinant aav packaging systems | |
NZ562881A (en) | High titer recombinant AAV production | |
Shull et al. | Genomic clones of bovine parvovirus: construction and effect of deletions and terminal sequence inversions on infectivity | |
US20040014031A1 (en) | Inducible highly productive rAAV packaging cell-lines | |
US20070275449A1 (en) | Method for Large-Scale Production, Isolation, Purification and the Uses of Multi-Type Recombinant Adeno-Associated Virus Vectors | |
WO1999061640A2 (en) | Aav derived vector | |
EP1845163A2 (en) | Adeno-associated virus serotype l nucleic acid sequences, vetors and host cells containing same | |
JP2024525121A (en) | A system for high-level rAAV production | |
US7037723B1 (en) | Recombinant herpes viruses for preparing recombinant adeno-associated viruses | |
WO1999061641A1 (en) | Aav-vector production | |
WO2003084977A1 (en) | Gene expression control system and its use in recombinant virus packaging cell lines |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
AK | Designated states |
Kind code of ref document: A2 Designated state(s): JP US |
|
AL | Designated countries for regional patents |
Kind code of ref document: A2 Designated state(s): AT BE CH CY DE DK ES FI FR GB GR IE IT LU MC NL PT SE |
|
121 | Ep: the epo has been informed by wipo that ep was designated in this application | ||
AK | Designated states |
Kind code of ref document: A3 Designated state(s): JP US |
|
AL | Designated countries for regional patents |
Kind code of ref document: A3 Designated state(s): AT BE CH CY DE DK ES FI FR GB GR IE IT LU MC NL PT SE |
|
DFPE | Request for preliminary examination filed prior to expiration of 19th month from priority date (pct application filed before 20040101) | ||
122 | Ep: pct application non-entry in european phase |