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

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.

Absence of a desmopressin response after therapeutic expression of factor VIII in hemophilia A dogs with liver-directed neonatal gene therapy

“…Stable and therapeutic levels of FVIII have been achieved in HA mice (reviewed in refs. [5][6][7][8][9] by transduction of liver with gamma retroviral vectors (RVs) [Ͼ20% of normal FVIII (10)], adenoviral vectors [Ͼ50% of normal (11)(12)(13)(14)(15)], lentiviral vectors [Ͼ5% of normal (16,17)], hydrodynamic injection of plasmid DNA [Ͼ300% of normal (18)], and adeno-associated virus vectors [2-100% of normal (19)(20)(21)(22)(23)]. Ex vivo gene therapy of endothelial cells (24) or hematopoietic stem cells (25) has also resulted in Ͼ50% of normal FVIII activity.…”

Prolonged and inducible transgene expression in the liver using gutless adenovirus: A potential therapy for liver cancer

“…11,14 -16 In addition, the elimination of all the adenoviral genes leaves room to allocate large expression cassettes, and therefore gutless adenoviruses are also designed as high-capacity adenoviral vectors. 11,14 -16 For correction of metabolic and/or hereditary diseases, different authors have used gutless vectors with nonregulable promoters showing long-term expression of transgenes such as ␣1-antitrypsin, 17,18 leptin, 19 apolipoprotein E, 20 and factor VIII and IX [21][22][23][24] at therapeutic levels without apparent hepatic toxicity.…”

Developing a HER3 Vaccine to Prevent Resistance to Endocrine Therapy

“…A key aspect of these Ad5 vectors is that expression of Ad late genes is greatly reduced (65,69,70). For example, production of the capsid fiber proteins could be detected in vivo for [E1-] Ad5 vectors, while fiber expression was ablated from [E1-, E2b-] Ad5 vector vaccines (67,68,71 (74,75). These data suggest that the lack of Ad5 gene expression renders infected cells invisible to anti-Ad activity and permits infected cells to produce the transgene for extended periods of time.…”

“…Overcoming the problem of pre-existing anti-vector immunity has been a subject of intense investigation. (65)(66)(67)(68). A key aspect of these Ad5 vectors is that expression of Ad late genes is greatly reduced (65,69,70).…”