Abstract
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.
Footnotes
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This research was funded with federal funds from the National Cancer Institute, National Institutes of Health, under contract N01-CO12400.
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ABBREVIATIONS: CAR, coxsackie-adenovirus receptor; HSVtk, herpes simplex virus thymidine kinase; hSSTR2, human somatostatin receptor subtype-2; CMV, cytomegalovirus; SV40, simian virus 40; RGD-4C, arginine-glycine-aspartic motif; GCV, ganciclovir; HEK, human embryonic kidney; DMEM, Dulbecco's modified Eagle's medium; PCR, polymerase chain reaction; MOI, multiplicity of infection; pfu, plaque-forming units; bp, base pair(s).
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↵1 Current affiliation: Department of Experimental Diagnostic Imaging, The University of Texas M.D. Anderson Cancer Center, Houston, Texas.
- Received April 13, 2006.
- Accepted August 8, 2006.
- The American Society for Pharmacology and Experimental Therapeutics
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