Sustained Human Factor VIII Expression in Hemophilia A Mice Following Systemic Delivery of a Gutless Adenoviral Vector

doi:doi:10.1006/mthe.2001.0510

Molecular Therapy (2002) 5, 63–73; doi: 10.1006/mthe.2001.0510

Sustained Human Factor VIII Expression in Hemophilia A Mice Following Systemic Delivery of a Gutless Adenoviral Vector

P. Seshidhar Reddy¹, Kiran Sakhuja¹, Shanthi Ganesh¹, Lijuan Yang¹, Dawn Kayda¹, Terrence Brann¹, Scott Pattison¹, Doug Golightly¹, Neeraja Idamakanti¹, Anne Pinkstaff¹, Michele Kaloss¹, Catherine Barjot², Jeffrey S. Chamberlain², Michael Kaleko¹ and Sheila Connelly¹

¹Genetic Therapy, Inc. (A Novartis Company), 9 West Watkins Mill Road, Gaithersburg, Maryland 20878, USA
²Department of Neurology, University of Washington School of Medicine, Seattle, Washington 98195-7720, USA

Correspondence: Sheila Connelly, Fax: (301) 258-4680. E-mail: sheila.connelly@pharma.novartis.com.

Received 24 August 2001; Accepted 19 November 2001.

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Abstract

Gutless adenoviral vectors are devoid of all viral coding regions and display reduced cytotoxicity, diminished immunogenicity, and an increased coding capacity compared with early generation vectors. Using hemophilia A, a deficiency in clotting factor VIII (FVIII), as a model disease, we generated and evaluated a gutless vector encoding human FVIII. The FVIII gutless vector grew to high titer and was reproducibly scaled-up from vector seed lots. Extensive viral DNA analyses revealed no rearrangements of the vector genome. A quantitative PCR assay demonstrated helper virus contamination levels of <2%, with the best preparation containing 0.3% helper virus. We compared the gutless vector with an E1/E2a/E3-deficient (Av3) early generation vector encoding an identical FVIII expression cassette following intravenous administration to hemophilia A mice. Gutless vector-treated mice displayed 10-fold higher FVIII expression levels that were sustained for at least 9 months. In contrast, mice treated with the Av3 vector displayed FVIII levels below the limit of sensitivity of the assay at 3 months. Assessment of hepatotoxicity by measuring the serum levels of liver enzymes demonstrated that the gutless vector was significantly less toxic than the Av3 vector at time points later than 7 days. At the highest dose used, both vectors caused a transient 10-fold increase in liver enzymes 1 day after vector administration, suggesting that this increase was caused by direct toxicity of the input capsid proteins. These data demonstrate that the gutless vector displayed increased duration and levels of FVIII expression, and was significantly less toxic than an analogous early generation vector.