Herpes simplex virus type 1 thymidine kinase sequence fused to β-galactosidase gene increases levels of β-galactosidase activity per genome of both first generation and high-capacity adenoviral vectors in vitro and in vivo

PUNTEL; MONDKAR; MUHAMMAD; LIU; SCIASCIA; XIONG; KROEGER; CZER; PALMER; NG; CASTRO; LOWENSTEIN

Seattle, Washington, USA

Congreso; American Society of Gene Therapy; 2007

Herpes simplex virus type 1 thymidine kinase sequence fused to β-galactosidase gene increases levels of β-galactosidase activity per genome of both first generation and high-capacity adenoviral vectors in vitro and in vivoM. Puntel1, S. Mondkar1, A.K.M. Muhammad1, C. Liu1, S. Sciascia1, W. Xiong1, K.M. Kroeger1, P. Czer1, D. Palmer2, P. Ng2, M. G. Castro1 and P. R. Lowenstein1.1Board of Governors Gene Therapeutics Research Institute, Cedars-Sinai Medical Center, and Departments of Medicine, and Molecular and Medical Pharmacology, University of California Los Angeles, Los Angeles, CA90048, USA. 2Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, TX 77030, USA. Email: lowensteinp@cshs.orgViral vector-associated toxicity, a limiting factor in clinical gene therapy trials, is dose-dependent. Enhanced transgene expression per viral vector genome will reduce the therapeutic vector dose needed, and increase the safety and efficacy of gene therapy. Increases in transgene expression have been achieved by: (i) use of stronger promoters (i.e. viral vs. cellular promoters), and (ii) through the use of viral cis-acting elements that increase mRNA half life. We previously found that expression of HSV1-TK from first generation adenoviral vectors in the brain was higher and longer lived than expression of -galactosidase, both expressed from the hCMV promoter. Thus, we wished to determine if the HSV1-TK sequence could confer higher expression levels onto a different transgene. This was compared to WPRE, a cis-acting post-transcriptional regulatory element commonly used to enhance transgene expression. First generation adenoviral vectors containing HSV1-TK sequences fused to β-galactosidase showed 2.5-10 fold enhanced levels of -galactosidase activity per genome in rodent, dog, primate, and human cell lines; WPRE enhanced -galactosidase activity per genome from 2.8-13 fold. First generation adenoviruses, however, remain sensitive to anti-adenoviral immune responses, while high capacity adenoviral vectors provide stable expression even in the presence of anti-adenoviral immune responses. We thus determined the activity of HSV1-TK in high capacity adenovirus (HC-Ad) vectors. In HC-Ad, HSV1-TK enhanced -galactosidase levels from 2-18-fold; while WPRE did not increase -galactosidase activity. Our results demonstrate that HSV1-TK sequence could be used to enhance levels of transgene expression per vector genome from both first generation and HC-Ad vectors.Funded by NINDS 1RO1 NS 42893.01, U54 NS045309-01, 1R21 NS047298-01; and Bram & Elaine Goldsmith Chair in Gene Therapeutics to P.R.L.; NINDS 1R01 NS44556.01, 1 R21 NS054143-01 and NIDDK 1 RO3 TW006273-01 to MGC; The Linda Tallen & David Paul Kane Foundation and the Board of Governors at CSMS.