Michael P. Seiler

Helper‐dependent adenoviral gene therapy mediates long‐term correction of the clotting defect in the canine hemophilia A model

Summary.  Background: Adenoviral vector‐mediated gene therapy might have potential for long‐term correction of the monogenic disease hemophilia A. Objective: In this study, we tested the efficacy of administering a helper‐dependent adenoviral vector (HDV) designed for maximal liver‐restricted canine factor VIII (cFVIII) expression on three out‐bred hemophilia A dogs. Methods: Three FVIII‐deficient animals from the University of North Carolina colony were injected with 1 × 1012 (Dog A), and 3 × 1012 (Dog B and C) vp kg−1 helper‐dependent adenoviral vector, and we performed systematic analysis of toxicity, persistence of therapeutic gene expression, and molecular analysis of gene transfer. Results: We observed acute dose‐dependent elevation in liver enzymes and thrombocytopenia after injection, although both were transient and resolved within 2 weeks. The whole blood clotting time (WBCT), plasma FVIII concentration, FVIII activity, and activated partial thromboplastin time in all animals improved significantly after treatment, and two animals receiving a higher dose reached near normal WBCT with low‐level FVIII activity until terminal sacrifice at 3 months, and 2 years. Importantly, the treated dogs suffered no bleeding events after injection. Moreover, we observed persistent vector‐specific DNA and RNA in liver tissue collected from one high‐dose animal at days 18 and 79, and could not detect the formation of inhibitory antibodies. Conclusion: Although vector‐associated toxicity remains an obstacle, a single injection of HDV led to long‐term transgene expression and vector persistence in two FVIII‐deficient animals with conversion of their severe phenotype to a moderate one.

627. Calcium Phopspate Precipitation of Adenovirus Enhances Gene Delivery into Human Dendritic Cells

Top of pageAbstract Mature dendritic cells (DC) are potent antigen presenting cells (APC) that have been used in vaccine studies as well as adoptive immunotherapy protocols. Adenovirus (Ad) vectors are frequently used to genetically modify DC; however gene transfer with recombinant adenovirus (serotype 5) into human DC is inefficient since DC do not express the cognate Ad5 receptor. Calcium phosphate (CaPi) precipitation of Ad has been shown to enhance gene transfer into murine DC and the aim of this study was to evaluate if CaPi precipitation enhances the gene delivery into human DC without interfering with their ability to activate antigen-specific T cells. Immature, monocyte derived DC were transduced with Ad5 or Ad5:CaPi complexes and gene expression was determined using GFP as a marker gene. CaPi precipitation of Ad5 increased DC gene transfer on average 35-fold when compared to unmodified Ad5. Ad5:CaPi complexes mediated gene delivery was similar in direct comparison to the optimal Ad for DC gene transfer (Ad5f35). Moreover, CaPi also increased the transduction efficiency of Ad5f35 at least 2-fold. CaPi transduction of immature DC did not reduce cell viability or their ability to be matured as judged by expression of CD83, and DR. To determine if Ad:CaPi treated DC can reactivate antigen-specific T cells, DC were incubated with an Ad5f35 vector encoding the subdominant Epstein Barr virus (EBV) antigens, latent membrane proteins (LMP) 2, which is expressed in EBV-positive malignancies. CaPi precipitation increased the expression of LMP2 in human DC 5|[ndash]|10 fold compared to virus alone, resulting in enhanced activation of LMP2-specific T cells in vitro. In conclusion, CaPi complexes increased gene transfer into human DC with both Ad5 and Ad5f35 vectors without changing their phenotype or their ability to activate antigen-specific T cells. Ad5:CaPi mediated transduction resulted in similar transduction as Ad5f35 vectors, which allows the use of existing Ad5 vector technology to genetically modify human DC.