Gene therapy for prostate cancer: toxicological profile of four HSV-tk transducing adenoviral vectors regulated by different promoters.
Adenoviral vector delivery of the Herpes simplex virus thymidine kinase (HSV-tk) gene in combination with the prodrug ganciclovir (GCV) has been tested in phase I clinical trials for prostate cancer and found to exhibit a satisfactory toxicity profile. We have developed additional adenoviral vectors with differing promoters to optimize the expression profile and in the present study evaluate the potential systemic toxicity of these vectors. Four recombinant adenoviral vectors that express the HSV-tk gene were generated using three different promoters: CMV (leftward orientation); RSV (both rightward and leftward orientation); and the mouse caveolin-1 (cav-1) promoter (leftward orientation). Efficacy was determined in vitro by cytotoxicity assays in a mouse prostate cancer cell line, RM-9, and in vivo by treating orthotopic tumors. Potential toxicity was evaluated from liver histology and apoptotic cell counts and enzyme levels in the serum following intravenous adenoviral vector injection. Although there were differences in HSV-tk expression at the protein level among the four vectors there were no significant differences in in-vitro cytotoxicity studies with GCV or in vivo in tumor growth suppression of an orthotopic mouse prostate cancer model in GCV treated mice. Intravenous delivery of high doses of all adenoviral vectors lead to abnormalities in liver function as measured by specific serum markers and histological evaluation of liver tissue and increased levels of apoptosis in the liver. These abnormalities were most prevalent with the vector containing the CMV promoter and the rightward oriented RSV promoter. They were least prevalent in the vector regulated by the cav-1 promoter. Upregulation of specific chemokines, MIP-2 and MIP-1beta was correlated with apoptotic counts. Our results demonstrate that comprehensive toxicological analysis of adenoviral vectors provides internally consistent information that can differentiate vectors with comparable efficacy based on toxicity. In these studies vectors with the cav-1 promoter-driven and leftward RSV-driven HSV-tk gene demonstrated minimal toxicities with cytotoxic effectiveness comparable to more toxic vectors. Our studies further suggest that promoter selection can influence the toxic effects of an adenoviral gene therapy vector.
Ebara
S
S
Scott Department of Urology, Baylor College of Medicine, Houston, Texas 77030, USA.
Shimura
S
S
Nasu
Y
Y
Kaku
H
H
Kumon
H
H
Yang
G
G
Wang
J
J
Timme
T L
TL
Aguilar-Cordova
E
E
Thompson
T C
TC
D000230:Adenocarcinoma / Q000628:therapy*; D000256:Adenoviridae / Q000235:genetics*; D000818:Animals; D017209:Apoptosis; D001358:Avian Sarcoma Viruses / Q000235:genetics*; D051242:Caveolin 1; D022461:Caveolins / Q000235:genetics*; D054407:Chemokine CCL4; D018946:Chemokine CCL5 / Q000096:biosynthesis / Q000235:genetics; D054357:Chemokine CXCL10; D054426:Chemokine CXCL2; D018925:Chemokines / Q000096:biosynthesis* / Q000235:genetics; D019743:Chemokines, CXC / Q000096:biosynthesis / Q000235:genetics; D003587:Cytomegalovirus / Q000235:genetics*; D003673:Defective Viruses / Q000235:genetics*; D015870:Gene Expression; D015967:Gene Expression Regulation, Viral; D005813:Genes, Synthetic; D005814:Genes, Viral; D015316:Genetic Therapy; D005822:Genetic Vectors / Q000235:genetics / Q000627:therapeutic use / Q000633:toxicity*; D006524:Hepatitis, Viral, Animal / Q000209:etiology*; D007275:Injections, Intravenous; D008111:Liver Function Tests; D019402:Macrophage Inflammatory Proteins / Q000096:biosynthesis / Q000235:genetics; D008297:Male; D051379:Mice; D008810:Mice, Inbred C57BL; D015846:Monokines / Q000096:biosynthesis / Q000235:genetics; D011401:Promoter Regions, Genetic; D011471:Prostatic Neoplasms / Q000628:therapy*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D018139:Simplexvirus / Q000201:enzymology / Q000235:genetics*; D013937:Thymidine Kinase / Q000037:antagonists & inhibitors / Q000235:genetics*; D014407:Tumor Cells, Cultured / Q000473:pathology / Q000821:virology; D014764:Viral Proteins / Q000037:antagonists & inhibitors / Q000235:genetics*
C495541:Cav1 protein, mouse; D051242:Caveolin 1; D022461:Caveolins; D054407:Chemokine CCL4; D018946:Chemokine CCL5; D054357:Chemokine CXCL10; D054426:Chemokine CXCL2; D018925:Chemokines; D019743:Chemokines, CXC; D019402:Macrophage Inflammatory Proteins; D015846:Monokines; D014764:Viral Proteins; D013937:Thymidine Kinase