Next generation of adeno-associated virus 2 vectors: point mutations in tyrosines lead to high-efficiency transduction at lower doses

Abstract

Recombinant adeno-associated virus 2 (AAV2) vectors are in use in several Phase I/II clinical trials, but relatively large vector doses are needed to achieve therapeutic benefits. Large vector doses also trigger an immune response as a significant fraction of the vectors fails to traffic efficiently to the nucleus and is targeted for degradation by the host cell proteasome machinery. We have reported that epidermal growth factor receptor protein tyrosine kinase (EGFR-PTK) signaling negatively affects transduction by AAV2 vectors by impairing nuclear transport of the vectors. We have also observed that EGFR-PTK can phosphorylate AAV2 capsids at tyrosine residues. Tyrosine-phosphorylated AAV2 vectors enter cells efficiently but fail to transduce effectively, in part because of ubiquitination of AAV capsids followed by proteasome-mediated degradation. We reasoned that mutations of the surface-exposed tyrosine residues might allow the vectors to evade phosphorylation and subsequent ubiquitination and, thus, prevent proteasome-mediated degradation. Here, we document that site-directed mutagenesis of surface-exposed tyrosine residues leads to production of vectors that transduce HeLa cells approximately 10-fold more efficiently in vitro and murine hepatocytes nearly 30-fold more efficiently in vivo at a log lower vector dose. Therapeutic levels of human Factor IX (F.IX) are also produced at an approximately 10-fold reduced vector dose. The increased transduction efficiency of tyrosine-mutant vectors is due to lack of capsid ubiquitination and improved intracellular trafficking to the nucleus. These studies have led to the development of AAV vectors that are capable of high-efficiency transduction at lower doses, which has important implications in their use in human gene therapy.

Fig. 1.

Site-directed mutational analyses of surface-exposed tyrosine residues on AAV2 capsid proteins and generation of recombinant scAAV2-EGFP vectors. (A) A surface representation of AAV2 VP3 shown in different colors for icosahedral symmetry related monomers (salmon, reference; green, twofold; bluish purple, threefold; and wheat, fivefold). The position of the seven surface-exposed tyrosine residues on the AAV2 capsid surface, Y252, Y272, Y444, Y500, Y700, Y704, and Y730, are indicated by the arrows and colored blue, cyan, red, green, pink, orange, and yellow, respectively, on all of the VP3 monomers. The icosahedral twofold axis is shown with the filled oval, the threefold axis is shown in the red triangle, and the fivefold axis is shown in the filled pentagon. A viral asymmetric unit is shown in the open triangle. Site-directed mutations of these seven 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 after transduction with tyrosine-mutant capsid scAAV2-EGFP vectors. (Magnification, ×100.) (C) Quantitation of the transduction efficiency in HeLa cells. *, P < 0.01 vs. WT scAAV2-EGFP.

Fig. 2.

AAV2-mediated transduction of hepatocytes from normal C57BL/6 mice injected via tail vein with tyrosine-mutant capsid scAAV2-EGFP vectors. (A) Transgene expression was detected by fluorescence microscopy 2 weeks after injection of 1 × 10¹⁰ viral particles per animal via the tail vein (n = 2 per experimental group). (Magnification, ×50.) (B) Quantitation of the transduction efficiency in hepatocytes in C57BL/6 mice. *, P < 0.01 vs. WT scAAV2-EGFP.

Fig. 3.

Comparative analyses of AAV2-mediated transduction efficiency in HeLa C12 cells with or without coinfection with adenovirus and treatment with proteasome- or EGFR-PTK-inhibitors after transduction with tyrosine-mutant capsid scAAV2-EGFP vectors. (A) Cells were mock-infected or infected with adenovirus after transduction with the WT, Y444F, or Y730F scAAV2-EGFP vectors. (Magnification, 100×.) (B) Quantitation of the transduction efficiency in HeLa C12 cells. (C) Cells were mock-treated or treated with Tyr-23 or MG132, after transduction with the WT or Y730F scAAV2-EGFP vectors. (Magnification, ×100.) (D) Quantitation of the transduction efficiency. *, P < 0.05 vs. control.

Fig. 4.

Western blot analyses of ubiquitinated AAV2 capsid proteins in HeLa cells after transduction with tyrosine-mutant scAAV2-EGFP vectors. WCLs prepared from cells (untreated or treated with MG132) after mock-infection (lanes 1 and 2), or infected with the WT (lanes 3 and 4), Y730F (lanes 5 and 6), or Y444F (lanes 7 and 8) scAAV2-EGFP vectors were immunoprecipitated with anti-AAV2 capsid antibody A20 followed by Western blot analyses with anti-Ub monoclonal antibody P4D1.

Fig. 5.

Southern blot analyses of intracellular trafficking of the WT and tyrosine-mutant scAAV2-EGFP vectors and cytoplasmic (C) and nuclear (N) distribution of AAV2 genomes. HeLa cells were mock-infected (lanes 1 and 2) or infected with the WT (lanes 3 and 4), Y730F (lanes 5 and 6), or Y444F (lanes 7 and 8) scAAV2-EGFP vectors. (A) Nuclear and cytoplasmic fractions were obtained 18 h after infection. Low-M_r DNA samples were isolated and electrophoresed on 1% agarose gels followed by Southern blot hybridization, using a ³²P-labeled EGFP DNA probe. (B) Quantitation of relative amounts of viral genomes. These results are representative of two independent experiments.

Fig. 6.

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 was determined as a function of time after injection of 1 × 10¹¹ viral particles/animal in BALB/c (A), and C3H/HeJ (B) mice via tail vein (tv), and 1 × 10¹⁰ viral particles/animal in C57BL/6 mice via tail vein (tv) (C), or portal vein (pv) (D). Fold increase of hF.IX peak levels of Y730F vectors compared with the WT capsid vectors is indicated for each panel. Data are mean ± SD (n = 4 per experimental group).