US5843742A
Expired / Lapsed
US
⏱️ 0.0 years remaining
Adeno-associated derived vector systems for gene delivery and integration into target cells
Assignee: Avigen Inc
•
Filing: 1995-09-08
•
Granted: 1998-12-01
•
Expires: 2015-09-08
Abstract
Abstract A novel nucleotide sequence integration and targeting system is described. The system employs adeno-associated virus (AAV) derived vectors which include a selected nucleotide sequence bounded by AAV inverted terminal repeats (ITRs). An AAV rep coding region is also provided, enabling the targeted integration of the selected nucleotide sequence into the genome of a suitable target cell. The nucleotide sequence integration system of the present invention can deliver and integrate large segments of DNA into the genome of target cells. Further, the subject integration system provides the advantage of site-specific integration of the selected nucleotide sequences in a target cell genome, thereby avoiding insertional mutagenesis events experienced with prior systems.
Inventors
- 👤 Georges Natsoulis
- 👤 Gary Kurtzman
Bibliographic Details
Application Number
—
Jurisdiction
US
Data Source
google_patents
Last Synchronized
2026-11-09 22:55
CPC Classifications
C; C12; C12N; C12N15/00; C12N15/09; C12N15/63; C12N15/79; C12N15/85; C12N15/86; C07; C07K; C07K14/00; C07K14/435; C07K14/745; C07K14/755; C12N2750/00; C12N2750/00011; C12N2750/14011; C12N2750/14111; C12N2750/14141; C12N2750/14143
Claims Analysis
Total Claims: 44
Claim 1
Independent Claim
144 words
A method of integrating a selected nucleotide sequence into the genome of a mammalian cell, comprising: (a) providing: (i) a mammalian cell; (ii) a first virion-free nucleic acid construct comprising a nucleotide sequence flanked by a 5' and a 3' adeno-associated virus inverted terminal repeat; and (iii) a second virion-free nucleic acid construct having a rep coding region operably linked to control elements which direct the transcription and translation of the rep coding region in said mammalian cell; (b) introducing, in any order, said first and second virion-free nucleic acid constructs into said mammalian cell; and (c) expressing the rep coding region of said second nucleic acid construct, thereby producing an amount of rep expression product under conditions such that an increased frequency of integration of said nucleotide sequence of said first nucleic acid construct into the genome of said mammalian cell is achieved.
Claim 2
Independent Claim
144 words
A method of integrating a selected nucleotide sequence into the genome of a mammalian cell, comprising: (a) providing: (i) a mammalian cell; (ii) a first virion-free nucleic acid construct comprising a nucleotide sequence flanked by a 5' and a 3' adeno-associated virus inverted terminal repeat; and (iii) a second virion-free nucleic acid construct having a rep coding region operably linked to control elements which direct the transcription and translation of the rep coding region in said mammalian cell; (b) introducing, in any order, said first and second virion-free nucleic acid constructs into said mammalian cell; and (c) expressing the rep coding region of said second nucleic acid construct, thereby producing an amount of rep expression product under conditions such that an increased frequency of integration of said nucleotide sequence of said first nucleic acid construct into the genome of said mammalian cell is achieved.
Claim 3
Dependent Claim
22 words
The method of claim 1, wherein said inverted terminal repeats in said first nucleic acid construct were excised from said adeno-associated virus.
Claim 4
Dependent Claim
23 words
The method of claim 1, wherein said first nucleic acid construct and said second nucleic acid construct are present on the same vector.
Claim 5
Dependent Claim
22 words
The method of claim 1, wherein said first nucleic acid construct and said second nucleic acid construct are present on different vectors.
Claim 6
Dependent Claim
25 words
The method of claim 1, wherein said nucleotide sequence of said first nucleic acid construct is integrated into chromosome 19 q of said mammalian cell.
Claim 7
Dependent Claim
21 words
The method of claim 1, wherein said nucleotide sequence of said first nucleic acid construct is more than 5,000 base pairs.
Claim 8
Dependent Claim
18 words
The method of claim 1, wherein said nucleotide sequence of said first nucleic acid construct encodes a polypeptide.
Claim 9
Dependent Claim
16 words
The method of claim 2, wherein said first nucleic acid construct is contained in a plasmid.
Claim 10
Dependent Claim
14 words
The method of claim 8, wherein said mammalian cell is transfected with said plasmid.
Claim 11
Dependent Claim
21 words
The method of claim 6, wherein said nucleotide sequence of said first nucleic acid construct is more than 10,000 base pairs.
Claim 12
Dependent Claim
18 words
The method of claim 7, wherein said polypeptide encodes at least a portion of the factor VIII gene.
Claim 13
Independent Claim
131 words
A method of integrating a selected nucleotide sequence into the genome of a mammalian cell, comprising: (a) providing: (i) a mammalian cell; (ii) a first virion-free nucleic acid construct comprising a nucleotide sequence flanked by a 5' and a 3' adeno-associated virus inverted terminal repeat, and (iii) an amount of rep expression product which increases the frequency of integration of said nucleotide sequence of said first nucleic acid construct; (b) introducing said first nucleic acid construct and said rep expression product, in any order, into said mammalian cell; and (c) integrating said nucleotide sequence of said first nucleic acid construct into said mammalian cell, under conditions such that an increased frequency of integration of said nucleotide sequence of said first virion-free nucleic acid construct is achieved by said rep expression product.
Claim 14
Independent Claim
131 words
A method of integrating a selected nucleotide sequence into the genome of a mammalian cell, comprising: (a) providing: (i) a mammalian cell; (ii) a first virion-free nucleic acid construct comprising a nucleotide sequence flanked by a 5' and a 3' adeno-associated virus inverted terminal repeat, and (iii) an amount of rep expression product which increases the frequency of integration of said nucleotide sequence of said first nucleic acid construct; (b) introducing said first nucleic acid construct and said rep expression product, in any order, into said mammalian cell; and (c) integrating said nucleotide sequence of said first nucleic acid construct into said mammalian cell, under conditions such that an increased frequency of integration of said nucleotide sequence of said first virion-free nucleic acid construct is achieved by said rep expression product.
Claim 15
Dependent Claim
22 words
The method of claim 12, wherein said inverted terminal repeats in said first nucleic acid construct were excised from said adeno-associated virus.
Claim 16
Dependent Claim
14 words
The method of claim 12, wherein the mammalian cell is transfected with said plasmid.
Claim 17
Dependent Claim
25 words
The method of claim 12, wherein said nucleotide sequence of said first nucleic acid construct is integrated into chromosome 19 q of said mammalian cell.
Claim 18
Dependent Claim
21 words
The method of claim 12, wherein said nucleotide sequence of said first nucleic acid construct is more than 5,000 base pairs.
Claim 19
Dependent Claim
12 words
The method of claim 12, wherein said nucleotide sequence encodes a polypeptide.
Claim 20
Dependent Claim
16 words
The method of claim 13, wherein said first nucleic acid construct is contained in a plasmid.
Claim 21
Dependent Claim
21 words
The method of claim 16, wherein said nucleotide sequence of said first nucleic acid construct is more than 10,000 base pairs.
Claim 22
Dependent Claim
103 words
A mammalian cell, comprising: (a) a virion-free first nucleic acid construct comprising a nucleotide sequence flanked by a 5' and a 3' adeno-associated virus inverted terminal repeat; and (b) a virion-free second nucleic acid construct having a rep coding region operably linked to control elements which direct the transcription and translation of the rep coding region in said mammalian cell to produce an amount of rep expression product to increase the frequency of integration of said nucleotide sequence of said first nucleic acid construct and said nucleotide sequence of said first nucleic acid construct is integrated into the genome of said mammalian cell.
Claim 23
Dependent Claim
103 words
A mammalian cell, comprising: (a) a virion-free first nucleic acid construct comprising a nucleotide sequence flanked by a 5' and a 3' adeno-associated virus inverted terminal repeat; and (b) a virion-free second nucleic acid construct having a rep coding region operably linked to control elements which direct the transcription and translation of the rep coding region in said mammalian cell to produce an amount of rep expression product to increase the frequency of integration of said nucleotide sequence of said first nucleic acid construct and said nucleotide sequence of said first nucleic acid construct is integrated into the genome of said mammalian cell.
Claim 24
Dependent Claim
23 words
The mammalian cell of claim 20, wherein said inverted terminal repeats in said first nucleic acid construct were excised from said adeno-associated virus.
Claim 25
Dependent Claim
24 words
The mammalian cell of claim 20, wherein said first nucleic acid construct and said second nucleic acid construct are present on the same vector.
Claim 26
Dependent Claim
23 words
The mammalian cell of claim 20, wherein said first nucleic acid construct and said second nucleic acid construct are present on different vectors.
Claim 27
Dependent Claim
26 words
The mammalian cell of claim 20, wherein said nucleotide sequence of said first nucleic acid construct is integrated into chromosome 19 q of said mammalian cell.
Claim 28
Dependent Claim
22 words
The mammalian cell of claim 20, wherein said nucleotide sequence of said first nucleic acid construct is more than 5,000 base pairs.
Claim 29
Dependent Claim
19 words
The mammalian cell of claim 20, wherein said nucleotide sequence of said first nucleic acid construct encodes a polypeptide.
Claim 30
Dependent Claim
18 words
The method of claim 20, wherein said polypeptide encodes at least a portion of the factor VIII gene.
Claim 31
Dependent Claim
17 words
The mammalian cell of claim 21, wherein said first nucleic acid construct is contained in a plasmid.
Claim 32
Dependent Claim
15 words
The mammalian cell of claim 28, wherein said plasmid is transfected into said mammalian cell.
Claim 33
Dependent Claim
22 words
The mammalian cell of claim 25, wherein said nucleotide sequence of said first nucleic acid construct is more than 10,000 base pairs.
Claim 34
Dependent Claim
19 words
The mammalian cell of claim 26, wherein said polypeptide encodes at least a portion of the factor VIII gene.
Claim 35
Dependent Claim
55 words
A mammalian cell, comprising: (a) a nucleic acid construct comprising a nucleotide sequence flanked by a 5' and a 3' adeno-associated virus inverted terminal repeat; and (b) an amount of rep expression product which increases the frequency of integration of said nucleotide sequence of said nucleic acid construct into the genome of said mammalian cell.
Claim 36
Dependent Claim
55 words
A mammalian cell, comprising: (a) a nucleic acid construct comprising a nucleotide sequence flanked by a 5' and a 3' adeno-associated virus inverted terminal repeat; and (b) an amount of rep expression product which increases the frequency of integration of said nucleotide sequence of said nucleic acid construct into the genome of said mammalian cell.
Claim 37
Dependent Claim
22 words
The mammalian cell of claim 32, wherein said inverted terminal repeats in said nucleic acid construct were excised from said adeno-associated virus.
Claim 38
Dependent Claim
16 words
The mammalian cell of claim 32, wherein said nucleic acid construct is contained in a plasmid.
Claim 39
Dependent Claim
15 words
The mammalian cell of claim 32, wherein said plasmid is transfected into said mammalian cell.
Claim 40
Dependent Claim
25 words
The mammalian cell of claim 32, wherein said nucleotide sequence of said nucleic acid construct is integrated into chromosome 19 q of said mammalian cell.
Claim 41
Dependent Claim
21 words
The mammalian cell of claim 32, wherein said nucleotide sequence of said nucleic acid construct is more than 5,000 base pairs.
Claim 42
Dependent Claim
19 words
The mammalian cell of claim 32, wherein said nucleotide sequence of said first nucleic acid construct encodes a polypeptide.
Claim 43
Dependent Claim
21 words
The mammalian cell of claim 37, wherein said nucleotide sequence of said nucleic acid construct is more than 10,000 base pairs.
Claim 44
Dependent Claim
19 words
The mammalian cell of claim 38, wherein said polypeptide encodes at least a portion of the factor VIII gene.
References Cited (Backward Citations)
US4722848A
Cited
US4965199A
Cited
US5139941A
Cited
US5219740A
Cited
WO1988009809A2
Cited
WO1989003429A1
Cited
US5399346A
Cited
US5436146A
Cited
US5225347A
Cited
WO1991012882A1
Cited
WO1992001070A1
Cited
WO1992003545A1
Cited
US5173414A
Cited
WO1993003769A1
Cited
EP0592836A1
Cited
WO1996013598A2
Cited
WO1996015777A1
Cited
USPTO Annuity & Maintenance Windows
| Annuity Stage | Window Opens | Payment Deadline | Surcharge Period Until | Estimated Fee | Status |
|---|---|---|---|---|---|
| 3.5 Years (Year 4) | 2001-11-30 | 2002-06-01 | 2002-12-01 | $2000.00 | Window Closed / Lapsed |
| 7.5 Years (Year 8) | 2005-11-30 | 2006-06-01 | 2006-12-01 | $3760.00 | Window Closed / Lapsed |
| 11.5 Years (Year 12) | 2009-11-30 | 2010-06-01 | 2010-12-01 | $7700.00 | Window Closed / Lapsed |