US5646034A
Expired / Lapsed
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Increasing rAAV titer
Assignee: University of California
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Filing: 1995-06-07
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Granted: 1997-07-08
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Expires: 2015-06-07
Abstract
Abstract Methods, kits and compositions for increasing the titer of rAAV vectors are provided.
Inventors
- 👤 Michael Mamounas
- 👤 Flossie Wong-Staal
- 👤 Mark Leavitt
- 👤 Mang Yu
Bibliographic Details
Application Number
—
Jurisdiction
US
Data Source
google_patents
Last Synchronized
2026-11-03 10:41
CPC Classifications
C; C12; C12N; C12N15/00; C12N15/09; C12N15/63; C12N15/79; C12N15/85; C12N15/86; A; A61; A61K; A61K47/00; A61K47/50; A61K47/51; A61K47/62; A61K47/64; A61K47/645; A61K47/69; A61K47/6901; C12N15/87; A61K48/00; C12N2750/00; C12N2750/00011; C12N2750/14011; C12N2750/14111; C12N2750/14141; C12N2750/14143
Claims Analysis
Total Claims: 34
Claim 1
Independent Claim
89 words
A method for producing high titers of recombinant adeno-associated virus (AAV) vector comprising the steps of: (i) binding a recombinant encapsidatable rAAV nucleic acid and a recombinant AAV helper nucleic acid, wherein the AAV helper nucleic acid comprises sequences necessary for rAAV replication and encapsidation, to an AAV helper virus, thereby producing a bound AAV helper virus; (ii) contacting a cell with the bound AAV helper virus; and (iii) culturing the cell under conditions which permit replication and encapsidation of the rAAV nucleic acid, thereby producing an rAAV vector.
Claim 2
Independent Claim
89 words
A method for producing high titers of recombinant adeno-associated virus (AAV) vector comprising the steps of: (i) binding a recombinant encapsidatable rAAV nucleic acid and a recombinant AAV helper nucleic acid, wherein the AAV helper nucleic acid comprises sequences necessary for rAAV replication and encapsidation, to an AAV helper virus, thereby producing a bound AAV helper virus; (ii) contacting a cell with the bound AAV helper virus; and (iii) culturing the cell under conditions which permit replication and encapsidation of the rAAV nucleic acid, thereby producing an rAAV vector.
Claim 3
Dependent Claim
13 words
The method of claim 1, wherein the AAV helper virus is an adenovirus.
Claim 4
Dependent Claim
14 words
The method of claim 1, wherein the AAV helper nucleic acid is replication defective.
Claim 5
Dependent Claim
18 words
The method of claim 1, wherein the AAV helper nucleic acid comprises adenovirus ITR sequences and AAV sequences.
Claim 6
Dependent Claim
13 words
The method of claim 1, wherein the AAV helper nucleic acid is Ad8.
Claim 7
Dependent Claim
13 words
The method of claim 1, wherein the AAV helper virus is replication defective.
Claim 8
Dependent Claim
12 words
The method of claim 1, wherein the cell is a human cell.
Claim 9
Dependent Claim
17 words
The method of claim 1, wherein step (ii) further comprises simultaneously infecting the cell with an adenovirus.
Claim 10
Dependent Claim
19 words
The method of claim 1, wherein the rAAV nucleic acid comprises AAV ITR sequences and heterologous nucleic acid sequences.
Claim 11
Dependent Claim
20 words
The method of claim 1, wherein the rAAV nucleic acid comprises AAV ITR sequences and heterologous anti-HIV nucleic acid sequences.
Claim 12
Dependent Claim
21 words
The method of claim 1, wherein the rAAV nucleic acid comprises AAV ITR sequences and heterologous anti-HIV ribozyme nucleic acid sequences.
Claim 13
Dependent Claim
18 words
The method of claim 1, wherein the method further includes the step of isolating the recombinant rAAV vector.
Claim 14
Dependent Claim
21 words
The method of claim 1, wherein the bound AAV helper virus of step (i) further comprises a nucleic acid binding molecule.
Claim 15
Dependent Claim
19 words
The method of claim 1, wherein the bound AAV helper virus of step (i) further comprises poly-1-lysine and transferrin-poly-1-lysine.
Claim 16
Dependent Claim
19 words
The method of claim 1, wherein the rAAV nucleic acid and the AAV helper nucleic acid are not homologous.
Claim 17
Dependent Claim
14 words
The method of claim 13, wherein the nucleic acid binding molecule is a polycation.
Claim 18
Dependent Claim
13 words
The method of claim 13, wherein the nucleic acid binding molecule is poly-1-lysine.
Claim 19
Independent Claim
94 words
A method of replicating an rAAV vector through receptor mediated endocytosis, comprising: (i) binding an rAAV nucleic acid and an AAV helper nucleic acid to a receptor-binding ligand, thereby forming an AAV nucleic acid-receptor binding ligand complex, wherein the receptor-binding ligand, upon binding a cell membrane receptor causes endocytosis of the receptor and the receptor binding ligand; (ii) contacting a cell with the rAAV nucleic acid-AAV helper nucleic acid-receptor binding ligand complex; and, (iii) incubating the cell under conditions which permit replication and encapsidation of the rAAV nucleic acid, thereby creating an rAAV vector.
Claim 20
Independent Claim
94 words
A method of replicating an rAAV vector through receptor mediated endocytosis, comprising: (i) binding an rAAV nucleic acid and an AAV helper nucleic acid to a receptor-binding ligand, thereby forming an AAV nucleic acid-receptor binding ligand complex, wherein the receptor-binding ligand, upon binding a cell membrane receptor causes endocytosis of the receptor and the receptor binding ligand; (ii) contacting a cell with the rAAV nucleic acid-AAV helper nucleic acid-receptor binding ligand complex; and, (iii) incubating the cell under conditions which permit replication and encapsidation of the rAAV nucleic acid, thereby creating an rAAV vector.
Claim 21
Dependent Claim
15 words
The method of claim 18, wherein the receptor binding ligand is an AAV helper virus.
Claim 22
Dependent Claim
21 words
The method of claim 18, wherein the receptor binding ligand is selected from the group consisting of adenovirus and herpes virus.
Claim 23
Dependent Claim
19 words
The method of claim 18, wherein the AAV nucleic acid-receptor binding ligand complex further comprises an AAV helper virus.
Claim 24
Dependent Claim
18 words
The method of claim 18, wherein the cell of step (ii) is infected with an AAV helper virus.
Claim 25
Dependent Claim
40 words
The method of claim 18, wherein the cell of step (ii) is infected with an AAV helper virus at the same time as it is contacted with the rAAV nucleic acid-AAV helper nucleic acid-receptor binding ligand complex of step (i).
Claim 26
Independent Claim
119 words
A method of transducing a cell with a target nucleic acid, comprising: (i) binding a recombinant rAAV nucleic acid and a recombinant AAV helper nucleic acid to an AAV helper virus, thereby producing a bound AAV helper virus, wherein the AAV helper nucleic acid comprises sequences necessary for rAAV replication and encapsidation, and the rAAV nucleic acid comprises a target nucleic acid sequence; (ii) contacting a cell with the bound helper virus; and (iii) culturing the cell under conditions which permit replication and encapsidation of the rAAV nucleic acid, thereby producing an rAAV vector; (iv) isolating the rAAV vector, thereby producing an isolated rAAV vector; and (v) contacting a cell with the isolated rAAV vector, thereby transducing the cell.
Claim 27
Independent Claim
119 words
A method of transducing a cell with a target nucleic acid, comprising: (i) binding a recombinant rAAV nucleic acid and a recombinant AAV helper nucleic acid to an AAV helper virus, thereby producing a bound AAV helper virus, wherein the AAV helper nucleic acid comprises sequences necessary for rAAV replication and encapsidation, and the rAAV nucleic acid comprises a target nucleic acid sequence; (ii) contacting a cell with the bound helper virus; and (iii) culturing the cell under conditions which permit replication and encapsidation of the rAAV nucleic acid, thereby producing an rAAV vector; (iv) isolating the rAAV vector, thereby producing an isolated rAAV vector; and (v) contacting a cell with the isolated rAAV vector, thereby transducing the cell.
Claim 28
Dependent Claim
12 words
The method of claim 24, wherein the cell is a human cell.
Claim 29
Dependent Claim
13 words
The method of claim 24, wherein the rAAV helper virus is an adenovirus.
Claim 30
Dependent Claim
20 words
A composition, comprising an AAV helper virus, nucleic acid binding molecule, rAAV nucleic acid and an AAV helper nucleic acid.
Claim 31
Dependent Claim
20 words
A composition, comprising an AAV helper virus, nucleic acid binding molecule, rAAV nucleic acid and an AAV helper nucleic acid.
Claim 32
Dependent Claim
8 words
The composition of claim 27, further comprising transferrin.
Claim 33
Dependent Claim
13 words
The composition of claim 27, wherein the nucleic acid binding molecule is poly-1-lysine.
Claim 34
Dependent Claim
13 words
The composition of claim 27, wherein the AAV helper virus is an adenovirus.
References Cited (Backward Citations)
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| Annuity Stage | Window Opens | Payment Deadline | Surcharge Period Until | Estimated Fee | Status |
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| 3.5 Years (Year 4) | 2000-07-07 | 2001-01-06 | 2001-07-08 | $2000.00 | Window Closed / Lapsed |
| 7.5 Years (Year 8) | 2004-07-07 | 2005-01-06 | 2005-07-08 | $3760.00 | Window Closed / Lapsed |
| 11.5 Years (Year 12) | 2008-07-07 | 2009-01-06 | 2009-07-08 | $7700.00 | Window Closed / Lapsed |