US5753500A
Expired / Lapsed
US
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Helper-free stocks of recombinant adeno-associated virus vectors
Assignee: Princeton University
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Filing: 1995-04-03
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Granted: 1998-05-19
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Expires: 2015-04-03
Abstract
Abstract The present invention relates to a method for producing helper-free stocks of recombinant adeno-associated virus (rAAV) which can be used to efficiently and stably transduce foreign genes into host cells or organisms. The method comprises the cotransfection of eukaryotic cells with rAAV and with helper AAV DNA in the presence of helper virus (e.g. adenovirus or herpesvirus) such that the helper AAV DNA is not associated with virion formation. The crux of the invention lies in the inability of the helper AAV DNA to recombine with rAAV vector, thereby preventing the generation of wild-type virus. In a specific embodiment of the invention, the vector comprises a recombinant AAV genome containing only the terminal regions of the AAV chromosome bracketing a non-viral gene, and the helper AAV DNA comprises a recombinant AAV genome containing that part of the AAV genome which is not present in the vector, and in which the AAV terminal regions are replaced by adenovirus sequences. In a further embodiment of the invention, cell lines are created which incorporate helper AAV DNA which can directly produce substantially pure recombinant AAV virus. The pure stocks of recombinant AAV produced according to the invention provide an AAV viral expression vector system with increased yield of recombinant virus, improved efficiency, higher definition, and greater safety than presently used systems.
Inventors
- 👤 Thomas E. Shenk
- 👤 Richard Jude Samulski
- 👤 Long-Sheng Chang
Bibliographic Details
Application Number
—
Jurisdiction
US
Data Source
google_patents
Last Synchronized
2026-11-03 13:01
CPC Classifications
C; C12; C12N; C12N15/00; C12N15/09; C12N15/63; C12N15/79; C12N15/85; C12N15/86; C12N2750/00; C12N2750/00011; C12N2750/14011; C12N2750/14111; C12N2750/14141; C12N2750/14143
Claims Analysis
Total Claims: 17
Claim 1
Independent Claim
39 words
A stock of recombinant adeno-associated virus free of adeno-associated helper virus comprising a packaged recombinant adeno-associated virus vector containing a heterologous gene of interest but lacking adeno-associated virus genes required for replication or packaging of said adeno-associated virus vector.
Claim 2
Dependent Claim
39 words
A stock of recombinant adeno-associated virus free of adeno-associated helper virus comprising a packaged recombinant adeno-associated virus vector containing a heterologous gene of interest but lacking adeno-associated virus genes required for replication or packaging of said adeno-associated virus vector.
Claim 3
Dependent Claim
24 words
The helper-free stock of recombinant adeno-associated virus of claim 1 wherein the virus is pAAV/neo, deposited with the ATCC and assigned accession number 68065.
Claim 4
Dependent Claim
143 words
A stock of recombinant adeno-associated virus free of adeno-associated helper virus, produced using a method comprising: a) cotransfecting cells permissive for adeno-associated virus replication, in the presence of helper virus infection with i) a recombinant adeno-associated virus vector which contains a portion of foreign DNA sequence and which can be incorporated into an infectious virion and ii) a recombinant helper adeno-associated virus DNA which provides viral functions sufficient for the replication and packaging into infectious virions of said recombinant adeno-associated virus vector, and which comprises a nucleotide sequence not found in wild-type adeno-associated virus which promotes expression of adeno-associated genes which results in the viral functions provided, but which lacks adeno-associated virus terminal repeat sequence and shares no adeno-associated virus sequences with said vector, and which cannot itself be incorporated into an infectious virion in said cotransfected cells; and b) collecting virions produced.
Claim 5
Dependent Claim
143 words
A stock of recombinant adeno-associated virus free of adeno-associated helper virus, produced using a method comprising: a) cotransfecting cells permissive for adeno-associated virus replication, in the presence of helper virus infection with i) a recombinant adeno-associated virus vector which contains a portion of foreign DNA sequence and which can be incorporated into an infectious virion and ii) a recombinant helper adeno-associated virus DNA which provides viral functions sufficient for the replication and packaging into infectious virions of said recombinant adeno-associated virus vector, and which comprises a nucleotide sequence not found in wild-type adeno-associated virus which promotes expression of adeno-associated genes which results in the viral functions provided, but which lacks adeno-associated virus terminal repeat sequence and shares no adeno-associated virus sequences with said vector, and which cannot itself be incorporated into an infectious virion in said cotransfected cells; and b) collecting virions produced.
Claim 6
Dependent Claim
124 words
A stock of recombinant adeno-associated virus free of adeno-associated helper virus, produced using a method comprising: a) cotransfecting cells permissive for adeno-associated virus replication, in the presence of helper-virus infection with i) a recombinant adeno-associated virus vector which contains a portion of foreign DNA sequence and which can be incorporated into an infectious virion and ii) a recombinant helper adeno-associated virus DNA which provides viral functions sufficient for the replication and packaging into infectious virions of said recombinant adeno-associated virus vector, and which comprises adenovirus terminal DNA sequence but which lacks adeno-associated virus terminal repeat sequence and shares no adeno-associated virus sequences with said vector, and which cannot itself be incorporated into an infectious virion in said cotransfected cells; and b) collecting virions produced.
Claim 7
Dependent Claim
124 words
A stock of recombinant adeno-associated virus free of adeno-associated helper virus, produced using a method comprising: a) cotransfecting cells permissive for adeno-associated virus replication, in the presence of helper-virus infection with i) a recombinant adeno-associated virus vector which contains a portion of foreign DNA sequence and which can be incorporated into an infectious virion and ii) a recombinant helper adeno-associated virus DNA which provides viral functions sufficient for the replication and packaging into infectious virions of said recombinant adeno-associated virus vector, and which comprises adenovirus terminal DNA sequence but which lacks adeno-associated virus terminal repeat sequence and shares no adeno-associated virus sequences with said vector, and which cannot itself be incorporated into an infectious virion in said cotransfected cells; and b) collecting virions produced.
Claim 8
Dependent Claim
31 words
The helper-free stock of recombinant adeno-associated virus of claim 3 or 4 in which the helper virus is selected from the group consisting of herpesvirus, cytomegalovirus, Epstein-Barr virus and vaccinia virus.
Claim 9
Dependent Claim
29 words
The helper-free stock of recombinant adeno-associated virus of claim 3 or 4 in which the recombinant adeno-associated vector comprises a transcriptional promoter not present in the wild-type adeno-associated virus.
Claim 10
Dependent Claim
30 words
The helper-free stock of recombinant adeno-associated virus of claim 3 or 4 in which the recombinant adeno-associated virus vector comprises pAAV/neo, deposited with the ATCC and assigned accession number 68065.
Claim 11
Dependent Claim
32 words
The helper-free stock of recombinant adeno-associated virus of claim 3 or 4 in which the recombinant helper adeno-associated virus DNA is plasmid AAV/Ad, deposited with the ATCC and assigned accession number 68066.
Claim 12
Dependent Claim
120 words
A stock of recombinant adeno-associated virus free of adeno-associated helper virus, produced using a method comprising: (a) providing a host cell infected with helper virus and permissive for adeno-associated virus replication, which host cell provides in trans adeno-associated virus functions sufficient for adeno-associated virus replication and packaging; (b) providing a recombinant adeno-associated virus vector containing a heterologous gene of interest but lacking: (i) adeno-associated virus genes required for replication and packaging; and (ii) sequences homologous to adeno-associated virus sequences present in the host cell; (c) transfecting the host cell of (a) with the recombinant adeno-associated virus vector of (b), so that the recombinant adeno-associated virus vector is replicated and packaged into virions; and (d) collecting the virions produced in (c).
Claim 13
Dependent Claim
120 words
A stock of recombinant adeno-associated virus free of adeno-associated helper virus, produced using a method comprising: (a) providing a host cell infected with helper virus and permissive for adeno-associated virus replication, which host cell provides in trans adeno-associated virus functions sufficient for adeno-associated virus replication and packaging; (b) providing a recombinant adeno-associated virus vector containing a heterologous gene of interest but lacking: (i) adeno-associated virus genes required for replication and packaging; and (ii) sequences homologous to adeno-associated virus sequences present in the host cell; (c) transfecting the host cell of (a) with the recombinant adeno-associated virus vector of (b), so that the recombinant adeno-associated virus vector is replicated and packaged into virions; and (d) collecting the virions produced in (c).
Claim 14
Dependent Claim
29 words
The helper-free stock of recombinant adeno-associated virus of claim 9 in which the helper virus is selected from the group consisting of herpesvirus, cytomegalovirus, Epstein-Barr virus and vaccinia virus.
Claim 15
Dependent Claim
27 words
The helper-free stock of recombinant adeno-associated virus of claim 9 in which the recombinant adeno-associated vector comprises a transcriptional promoter not present in the wild-type adeno-associated virus.
Claim 16
Dependent Claim
27 words
The helper-free stock of recombinant adeno-associated virus of claim 9 in which the recombinant adeno-associated vector comprises pAAV/neo deposited with the ATCC and assigned accession number 68065.
Claim 17
Dependent Claim
28 words
The helper-free stock of recombinant adeno-associated virus of claim 9 in which the recombinant adeno-associated helper DNA comprises pAAV/Ad, deposited with the ATCC and assigned accession number 68066.
References Cited (Backward Citations)
US4797368A
Cited
US5436146A
Cited
USPTO Annuity & Maintenance Windows
| Annuity Stage | Window Opens | Payment Deadline | Surcharge Period Until | Estimated Fee | Status |
|---|---|---|---|---|---|
| 3.5 Years (Year 4) | 2001-05-18 | 2001-11-17 | 2002-05-19 | $2000.00 | Window Closed / Lapsed |
| 7.5 Years (Year 8) | 2005-05-18 | 2005-11-17 | 2006-05-19 | $3760.00 | Window Closed / Lapsed |
| 11.5 Years (Year 12) | 2009-05-18 | 2009-11-17 | 2010-05-19 | $7700.00 | Window Closed / Lapsed |