Recombinant adenovirus expressing adeno-associated virus cap and rep proteins supports production of high-titer recombinant adeno-associated virus

H. G. Zhang, Y. M. Wang, J. F. Xie, X. Liang, H. C. Hsu, X. Zhang, J. Douglas, D. T. Curiel, J. D. Mountz

Research output: Contribution to journalArticlepeer-review

24 Scopus citations

Abstract

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 AdCM-VLacZ. 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.

Original languageEnglish
Pages (from-to)704-712
Number of pages9
JournalGene therapy
Volume8
Issue number9
DOIs
StatePublished - 2001

Keywords

  • AdAAV
  • Adeno-associated virus
  • Adenovirus
  • Cap
  • Chimeric vector
  • Rep

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