Recombinant adenovirus expressing adeno-associated virus cap and rep proteins supports production of high-titer recombinant adeno-associated virus.
It has been difficult to produce a chimeric vector containing both Ad and AAV rep and cap, and to grow such chimeric vectors in 293 cells. By recombination in vitro in a bacterial host, we were able to produce recombinant plasmid AdAAV (pAdAAVrep-cap), which could be used to generate recombinant AdAAV (rAdAAVrep-cap) after transfection into 293 cells. A recombinant adenovirus, rAdAAVGFP, in which the green fluorescent protein (GFP) gene is flanked by the AAV terminal repeats cloned into the E1-deleted site of Ad was also generated. Co-infection of rAdAAVrep-cap together with rAdAAVGFP into 293 cells resulted in production of high titers of rAAV expressing GFP. It was noted that the titer of rAdAAVrep-cap was lower than the titer of control AdCMVLacZ. The lower titer of rAdAAvrep-cap was associated with expression of Rep protein. Non-homologous recombination occurs after high passage and results in deletions within the AAV rep genes. These results indicate that (1) rAdAAVrep-cap can be produced; (2) rAdAAVrep-cap + rAdAAVGFP is a convenient and efficient way to transfect 293 cells to grow high titer rAAV; and (3) frozen stock is required to avoid propagation of rep-deleted pAdAAVrep-cap.
Zhang
H G
HG
The University of Alabama at Birmingham, Department of Medicine, Division of Clinical Immunology and Rheumatology, Birmingham, AL 35294, USA.
Wang
Y M
YM
Xie
J F
JF
Liang
X
X
Hsu
H C
HC
Zhang
X
X
Douglas
J
J
Curiel
D T
DT
Mountz
J D
JD
D000256:Adenoviridae / Q000235:genetics*; D001483:Base Sequence; D018929:Cell Culture Techniques; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D000229:Dependovirus / Q000235:genetics* / Q000378:metabolism / Q000502:physiology; D017353:Gene Deletion; D005822:Genetic Vectors; D006801:Humans; D008969:Molecular Sequence Data; D010957:Plasmids / Q000235:genetics; D011995:Recombination, Genetic; D014764:Viral Proteins / Q000235:genetics / Q000378:metabolism*; D014779:Virus Replication / Q000235:genetics
D004268:DNA-Binding Proteins; D014764:Viral Proteins; C068734:rep proteins, Adeno-associated virus 2