RT Journal Article
SR Electronic
T1 Circumventing Recombination Events Encountered with Production of a Clinical-Grade Adenoviral Vector with a Double-Expression Cassette
JF Molecular Pharmacology
JO Mol Pharmacol
FD American Society for Pharmacology and Experimental Therapeutics
SP 1488
OP 1493
DO 10.1124/mol.106.025619
VO 70
IS 5
A1 Natalya Belousova
A1 Raymond Harris
A1 Kurt Zinn
A1 Mary Ann Rhodes-Selser
A1 Alexander Kotov
A1 Olga Kotova
A1 Minghui Wang
A1 Rosemarie Aurigemma
A1 Zeng B. Zhu
A1 David T. Curiel
A1 Ronald D. Alvarez
YR 2006
UL http://molpharm.aspetjournals.org/content/70/5/1488.abstract
AB Delivery of multiple exogenous genes into target cells is important for a broad range of gene therapy applications, including combined therapeutic gene expression and noninvasive imaging. Previous studies ( Mol Ther:-231, 2001 ) have described the adenoviral vector RGDTKSSTR with a double-expression cassette that encodes herpes simplex virus thymidine kinase (HSVtk) for molecular chemotherapy and human somatostatin receptor subtype-2 (hSSTR2) for indirect imaging. In this vector, both genes are inserted in place of the E1 region of the adenoviral genome and expressed independently from two cytomegalovirus (CMV) promoters. During production of clinical-grade RGDTKSSTR, we found that the CMV promoters and simian virus 40 (SV40) poly(A) regions located in both expression cassettes provoked homologous recombination and deletion of one of the cassettes. To resolve this problem, we designed a strategy for substituting the duplicate promoters and poly(A) regions. We placed the hSSTR2 gene in the new Ad5.SSTR/TK.RGD vector under the control of a CMV promoter with a bovine growth hormone poly(A) region, whereas the SV40 promoter, enhancer, and poly(A) signal controlled HSVtk expression. This use of different regulatory sequences allowed independent expression of both transgenes from a single adenoviral vector and circumvented the recombination problem. Reconstruction of the vector with a double-expression cassette enables its use in human clinical trials. The American Society for Pharmacology and Experimental Therapeutics