Development of Novel Recombinant AAV Vectors and Strategies for the Potential Gene Therapy of Hemophilia

Abstract

Recombinant vectors based on a non-pathogenic human parvovirus, the adeno-associated virus (AAV), have gained attention as a potentially safe and useful alternative to the more commonly used retroviral and adenoviral vectors. AAV vectors are currently in use in Phase I/II clinical trials for gene therapy of a number of diseases such as cystic fibrosis, α-1 antitrypsin deficiency, muscular dystrophy, Batten's disease, and Parkinson's disease, and have shown efficacy in patients with Leber's congenital amaurosis, and hemophilia B. For patients with hemophilia B, however, relatively large vector doses are needed to achieve therapeutic benefits. Large vector doses also trigger an immune response as significant fraction of the vectors fails to traffic efficiently to the nucleus, and is targeted for degradation by the host cell proteasome machinery. With a better understanding of the various steps in the life cycle of AAV vectors, strategies leading to the development of novel AAV vectors that are capable of high-efficiency transduction at lower doses are needed. In this review, we summarize our strategies to develop novel AAV vectors for the potential gene therapy of both hemophilia B and hemophilia A, based on our recent studies on the basic molecular biology of AAV. These strategies, including the development of novel AAV vectors by site-directed mutagenesis of critical surface-exposed tyrosine residues on AAV2 capsids to circumvent the ubiquitination step and the use of different AAV serotypes and self-complementary (sc) AAV2 vectors, and their use as helper vectors to circumvent the obstacles of second-strand DNA synthesis of single-stranded (ss) AAV, should dramatically accelerate the progress towards the potential gene therapy of both hemophilia A and hemophilia B.

Figure 1

Novel tyrosine-mutant AAV2 vectors. (A) The position of the 7 surface-exposed tyrosine residues on the AAV2 capsid surface, Y252, Y272, Y444, Y500, Y700, Y704, and Y730, are indicated by the arrows. Site-directed mutations of these tyrosine residues to phenylalanine residues (tyrosine-phenylalanine, Y-F) were performed and tyrosine-mutant capsid scAAV2-EGFP vectors were generated. (B) AAV2-mediated transgene expression in HeLa cells following transduction with tyrosine-mutant scAAV2-EGFP vectors. (C) Quantitative analyses of the transduction efficiency*P<0.01 vs. WT scAAV2-EGFP. (D) AAV2-mediated transduction of hepatocytes from normal C57BL/6 mice injected via tail vein with tyrosine-mutant capsid scAAV2-EGFP vectors. (E) Quantitative analyses of AAV2 transduction efficiency. *P < 0.01 vs WT scAAV2-EGFP. (F) Comparative analyses of the WT or Y730F ssAAV2-ApoE/hAAT-hF.IX vector-mediated transduction efficiency in hepatocytes in mice in vivo. Human F.IX (hF.IX) expression in plasma as a function of time after injection of 1×10¹¹ viral particles/animal in BALB/c and C3H/HeJ mice via tail vein (tv), and 1×10¹⁰ viral particles/animal in C57BL/6 mice via tail vein (tv) or portal vein (pv). Fold-increase of hF.IX peak levels of Y730F vectors compared to the WT capsid vectors is indicated for each panel [Proc. Natl. Acad. Sci., USA, 105: 7827–7832, 2008].

Figure 2

F.IX^−/− C3H/HeJ mice (n=4/vector) were injected into the tail vein with 2×10¹¹ vgs of AAV-ApoE/hAAT-hF.IX packaged into WT AAV-2 (dashed line), or AAV2-Y730F (solid line) or AAV2-Y730+500+444F (dotted line) capsids. Resulting systemic hF.IX expression (A) and coagulation times (aPTT) (B) are shown. Each line represents average SD. Horizontal lines in A mark the range of aPTTs for normal mouse plasma (25–35 sec) or untreated hemophilia B mouse plasma (>60 sec). Y-F vector-treated mice were challenged with subcutaneous hF.IX/CFA (arrow) or weekly intravenous hF.IX (arrow + line). C–E. Immunostain for hF.IX expressing hepatocytes 3–7 months after gene transfer with WT (C), Y730F (D), or Y730+500+444F (E) capsid vectors. Average percent of hF.IX positive hepatocytes are indicated [Mol. Ther., 18: 2048–2056, 2010].

Figure 3

Comparative analyses of the ssAAV2-ApoE/hAAT-hF.IX vector-mediated transduction efficiency in hepatocytes with and without co-administration of scAAV8-PP5 helper-viruses in C57BL/6 mice in vivo. (A) Human F.IX (hF.IX) expression in plasma was determined as a function of time after injection of 1×10¹⁰ or 5×10¹⁰ vgs of each vector/animal. Data are mean ± SEM (n=4 per experimental group). (B) Representative liver sections obtained 10 weeks following injection of ssAAV2-hAAT-hF.IX vectors (AAV2-F.IX) with or without scAAV8-TTR-PP5 helper-virus (PP5). Sections were immunofluorescently stained for hF.IX and the ranges of percent positive hepatocytes for each group are shown in each panel. Original magnification: 200x [Hum. Gene Ther., 21: 271–283, 2010].