Abstract C240: Modification on the capsid of recombinant adeno-associated virus vectors (rAAV) leads to high-efficiency transduction of human monocyte-derived dendritic cells (moDCs).

Abstract

Abstract Genetically modified dendritic cell (DC) has been extensively studied and numerous phase I and II clinical trials evaluating the efficacy of DC-based vaccine have been initiated. Among different methods for gene delivery, vectors based on a human parvovirus, the adeno-associated virus (AAV), have attracted much attention mainly because of the non-pathogenic nature of this replication deficient virus, and its ability to mediate long-term, sustained therapeutic gene expression. Successful transduction of different subsets of DC by different commonly used serotypes of AAV was demonstrated in recent studies and the potential advantage of an AAV-based antitumor vaccine was discussed. However, further improvement of gene transfer by rAAV to DC in terms of specificity and transduction efficiency should be made to have a significant impact in the choice of vector used as an antitumor vaccine. Serine/threonine protein kinases, such as p-38 MAPK, are involved in a wide variety of cellular processes such as differentiation, transcription regulation and development of many cell types including immune cells. Such kinases can also negatively regulate efficiency of rAAV-mediated gene transfer by phosphorylating serine or threonine surface exposed residues on viral capsid and target virus for proteasome-mediated degradation. In present studies we show that pre-treatment of cells with selective p-38 MAPK inhibitors prior to AAV infection significantly increased the transduction by AAV2 vectors. We hypothesized that prevention of phosphorylation of the surface-exposed serine residues might allow the vectors to evade phosphorylation and subsequent ubiquitination and, thus, prevent proteasomal degradation. The AAV2 capsid contains 50 serine residues in the VP3 region, of which 15 residues (S261, S264, S267, S276, S384, S458, S468, S492, S498, S578, S658, S662, S668, S707, S721) are surface-exposed. Each of the fifteen serine (S) residues was substituted with valine (V), and each S-V mutant vector was evaluated for transduction efficiency in moDC. We identified that single serine residue (S662) substitution to valine leads to increased transduction efficiency of moDC up to 6 times. We next evaluated the possibility whether S662V-AAV vectors could be used for generating antigen-specific cytotoxic T-cells (CTLs). To this end, S662V-AAV2 vectors encoding a truncated human telomerase (hTERT) gene were generated, and used to stimulate CTLs against a human immortalised myelogenous leukaemia cell line, K562. Our data show that S662V-AAV2-hTERT-transduced DCs resulted in rapid (within one week), specific T-cell clone proliferation and generation of robust CTLs. A killing curve was generated and specific cell lysis was determined by fluorescence-activated cell sorting (FACS) analysis of live/dead cell ratios. These results suggest that high-efficiency transduction of moDCs by capsid-modified AAV vectors is feasible, which supports the potential utility of these vectors for future human DC-based vaccine studies. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr C240.