Method of gene delivery using wildtype adeno associated viral (AAV) vectors with insertions

• US 6,153,436 A
• Filed: 01/09/1998
• Issued: 11/28/2000
• Est. Priority Date: 01/10/1997
• Status: Expired due to Fees

• Current Assignee: The Board of Trustees of the University of Arkansas (University of Arkansas System)
• Original Assignee: The Board of Trustees of the University of Arkansas (University of Arkansas System)
• Inventors: Li, Han, Bishop, Brian, Quirk, J. Gerald, Hermonat, Paul L.
• Primary Examiner(s): REYNOLDS, DEBORAH J
• Application Number:
  US09/004,877
• Time in Patent Office: 1,054 Days
• Field of Search: 435/440, 435/320.1, 435/235.1, 435/455, 435/456, 514/44, 424/93.2
• US Class Current: 435/456
• CPC Class Codes: A61K 48/00 Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
• CPC Class Codes: C12N 15/86 Viral vectors
• CPC Class Codes: C12N 2750/14143 viral genome or elements thereof as genetic vector

Abstract

The present invention provides a method of human gene therapy using AAV vectors with essentially wildtype phenotype. Genes of 900 bases or less can be inserted into wildtype AAV and still allow the resulting vector to have a largely wildtype phenotype. For example, several antisense genes could be inserted and still allow packaging. Such wildtype vectors have several advantages. First, high titers of such vectors is easy to accomplish. Second, the vectors, since they include the Rep78 gene, integrate specifically into human chromosome 19. Third, such vectors, being wildtype, spread after their initial introduction. Another method for use of large wildtype AAV genomes is as complementors for fully defective AAV vectors. Such complementors can be delivered by virus infection and, be introduced easily into 100% of the cells used to produce virus. Viral infection is superior to synthetic techniques for introducing DNA into tissue culture producer cells. When large essentially wildtype AAV complementor virus are used in conjunction with AAV vector virus allowing for the introduction of both vectors into all cells, high titers of recombinant AAV virus is achieved.

Claims

1. A method of delivering DNA to a target cell, in vitro, using adeno-associated viral vectors, comprising the step of:

administering an adeno-associated viral vector to said cell, wherein said viral vector retain all adeno-associated viral genomic sequences, wherein said viral vector contains a DNA insert of a size up to about 1000 nucleotide base pairs and wherein said insert is placed after the last coding region of said virus and prior to the right ITR of said virus.

2. The method of claim 1, wherein said DNA insert is less than 900 nucleotide base pairs.

3. The method of claim 1, wherein said DNA insert is inserted into a non-essential portion of the viral vector, wherein non-essential portions do not harbor cis or trans sequences that are required for viral replication or transcription.

4. The method of claim 1, wherein said viral vector integrates into chromosome 19 of said cell.

5. A method of complementing a defective adeno-associated viral vector, comprising the step of:

administering a defective adeno-associated viral vector to a cell, in vitro; and

administering a non-defective adeno-associated viral vector to said cell, said non-defective viral vector retaining all adeno-associated viral genomic sequences, wherein said non-defective viral vector contains a DNA insert of a size up to about 1000 nucleotide base pairs and wherein said non-defective viral vector is capable of generating virus particles of the defective adeno-associated viral vector.