Construction of adeno-associated virus packaging plasmids and cells that directly select for AAV helper functions.
Recombinant adeno-associated virus type 2 (rAAV) has promise for use as a gene therapy vector. Potential problems in the production of rAAV stocks are both the limited amount of recombinant virus that is produced by traditional methods and the possibility of wild-type replication competent adeno-associated virus (wtAAV) contamination. The presence of these contaminants is largely dependent upon the helper plasmid used. Whilst wtAAV is not a pathogen, the presence of these contaminants is undesirable as they may affect experiments concerning the biology of rAAV. Additionally as protocols using rAAV with altered tropism are becoming more prevalent, it is important that no recombination be permitted that may cause the creation of a replication competent AAV with modified (targeting) capsids. Many experimental protocols require the generation of large amounts of high titre rAAV stocks. We describe the production of several AAV helper plasmids and cell lines designed to achieve this goal. These plasmids possess split AAV rep and cap genes to eliminate the production of wtAAV and they possess a selection mechanism which is operatively linked to expression from the AAV cap gene. This allows positive selection of those cells expressing the highest level of the structural capsid proteins and therefore those cells which yield the highest amount of rAAV.
Department of Haematology, Royal Free and University College Medical School, Royal Free Campus, Rowland Hill Street, Hampstead, London NW3 2PF, UK.
Alistair
A
Whiteway
Wale
W
Deru
H Grant
HG
Prentice
Robert
R
Anderson
D015704:CD4 Antigens / Q000235:genetics / Q000378:metabolism; D036022:Capsid Proteins / Q000235:genetics / Q000378:metabolism; D002460:Cell Line; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism; D000229:Dependovirus / Q000235:genetics*; D005822:Genetic Vectors; D006367:HeLa Cells; D006378:Helper Viruses / Q000235:genetics*; D006801:Humans; D010957:Plasmids / Q000235:genetics*; D014176:Protein Biosynthesis; D018741:Replication Origin; D014162:Transfection; D014764:Viral Proteins / Q000235:genetics / Q000378:metabolism; D019065:Virus Assembly
D015704:CD4 Antigens; D036022:Capsid Proteins; D004268:DNA-Binding Proteins; D014764:Viral Proteins; C068734:rep proteins, Adeno-associated virus 2