High-efficiency transduction and correction of murine hemophilia B using AAV2 vectors devoid of multiple surface-exposed tyrosines

Abstract

Elimination of specific surface-exposed single tyrosine (Y) residues substantially improves hepatic gene transfer with adeno-associated virus type 2 (AAV2) vectors. Here, combinations of mutations in the seven potentially relevant Y residues were evaluated for further augmentation of transduction efficiency. These mutant capsids packaged viral genomes to similar titers and retained infectivity. A triple-mutant (Y444+500+730F) vector consistently had the highest level of in vivo gene transfer to murine hepatocytes, approximately threefold more efficient than the best single-mutants, and ~30-80-fold higher compared with the wild-type (WT) AAV2 capsids. Improvement of gene transfer was similar for both single-stranded AAV (ssAAV) and self-complementary AAV (scAAV) vectors, indicating that these effects are independent of viral second-strand DNA synthesis. Furthermore, Y730F and triple-mutant vectors provided a long-term therapeutic and tolerogenic expression of human factor IX (hF.IX) in hemophilia B (HB) mice after administration of a vector dose that only results in subtherapeutic and transient expression with WT AAV2 encapsidated vectors. In summary, introduction of multiple tyrosine-mutations into the AAV2 capsid results in vectors that yield at least 30-fold improvement of transgene expression, thereby lowering the required therapeutic dose and potentially vector-related immunogenicity. Such vectors should be attractive for treatment of hemophilia and other genetic diseases.

Figure 1

AAV2-mediated transgene expression in (a,b) HeLa cells and in (c,d) H2.35 murine hepatocytes following transduction with single and multiple surface-exposed tyrosine-mutant capsid scAAV2-EGFP vectors. (a,c) Transgene expression was detected by fluorescence microscopy 48 hours postinfection with (a) 500 or (c) 2,000 vgs/cell. Original magnification ×100. (b) Quantitative analyses of AAV2 transduction efficiency in HeLa cells. (d) Quantitative analyses of AAV2 transduction efficiency in H2.35 cells. Images from five visual fields were analyzed quantitatively by ImageJ analysis software. Transgene expression was assessed as total area of green fluorescence (pixel²) per visual field (mean ± SD). One-way ANOVA and Dunnett's multiple comparison test was used to determine which multiple tyrosine-mutants provided significantly higher transgene expression (P < 0.001) compared to Y730F. ANOVA, analysis of variance; AAV2, adeno-associated virus type 2; EGFP, enhanced green fluorescent protein; scAAV2, self-complementary AAV2; vgs, vector genomes.

Figure 2

Hepatic gene transfer in C57BL/6 mice with single and multiple tyrosine-mutant capsid scAAV2-EGFP vectors. (a) Transgene expression was detected by fluorescence microscopy 4 weeks postinjection of 1 × 10⁹ vgs/animal. Original magnification ×50. (b) Quantitative analyses of EGFP expression (see legend to Figure 1 for details). EGFP, enhanced green fluorescent protein; scAAV2, self-complementary adeno-associated virus type 2; vgs, vector genomes.

Figure 3

Time course of transgene expression in livers of C57BL/6 mice transduced with tyrosine-mutant capsid scAAV2-EGFP and ssAAV2-EGFP vectors. (a,b) Transgene expression was detected by fluorescence microscopy 2–26 weeks postinjection of 1 × 10⁹ vgs/animal of scAAV2-EGFP vector (original magnification ×50) and quantitatively analyzed as described in the legend to Figure 1. *P < 0.01 versus WT scAAV2-EGFP. (c,d) Transgene expression 2–17 weeks postinjection of 1 × 10¹⁰ vgs/animal of ssAAV2-EGFP vector. *P < 0.01 versus WT ssAAV2-EGFP. EGFP, enhanced green fluorescent protein; scAAV2, self-complementary adeno-associated virus type 2; vgs, vector genomes; WT, wild type.

Figure 4

Systemic expression and humoral immune response to hF.IX following peripheral vein delivery of WT AAV2, Y730F, and triple-mutant ssAAV2-ApoE/hAAT-hF.IX vector. (a) hF.IX expression (mean ± SD) in HB mice 4 weeks following injection of 2 × 10¹¹ vgs of WT (n = 3), Y730F (n = 4), or triple-mutant (n = 4) AAV2 vectors via the tail vein. (b) hF.IX expression (mean ± SD) in C3H/HeJ/F9^‐/‐ mice following injection of 2 × 10¹¹ vgs/animal of WT, Y730F, or triple-mutant AAV2 vectors via the tail vein. (c) Coagulation times (aPTT in seconds, mean ± SD) (b,c) Y730F-injected mice were challenged at indicated time point (dotted arrow) with hF.IX in CFA. Triple-mutant transduced mice were challenged at indicated time point with repeated administration of hF.IX (solid arrow and line). (d) Antibody titers (IgG 1 mean ± SD) against hF.IX as a function of time after WT, Y730F, or triple-mutant AAV2 vector administration. Note that one mouse injected with the triple-mutant AAV2 vector formed a transient low-titer antibody. (e) Inhibitors to hF.IX as measured by Bethesda assay. AAV2 injected mice challenged at indicated time point with either hF.IX/CFA (n = 4, dashed arrow) or with repeated hF.IX (n = 3, dashed line). Y730F-injected mice were challenged at indicated time point dotted with hF.IX/CFA (dotted arrow). Triple-mutant transduced mice were challenged with repeated hF.IX (solid arrow and line). (f) Bethesda titers in naive (i.e., nontransduced) HB mice challenged with hF.IX/CFA or with repeated hF.IX administration (one i.p. injection followed by three weekly i.v. injections). AAV2, adeno-associated virus type 2; ApoE, apolipoprotein E; aPTT, activated partial thromboplastin times; BU, Bethesda unit; CFA, complete Freund's adjuvant; HB, hemophilia B; hF.IX, human factor IX; i.p., intraperitoneal; i.v., intravascular; scAAV2, self-complementary AAV2.

Figure 5

Representative immunofluorescence staining of hF.IX (red) in liver cross-sections after gene transfer to C3H/HeJ/F9^‐/‐ mice with (a) WT AAV2 3 months, (b) Y730F AAV2 7 months, or (c) triple-mutant AAV2 5 months following gene transfer. Original magnification: ×200. AAV2, adeno-associated virus type 2; hF.IX, human factor IX; WT, wild-type.