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 (Y444500730F) 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.
Bibliographical noteFunding Information:
This research was supported in part by Public Health Service grants P01 HL-078810 (Project 3) and R01 AI/HL-51390 from the National Institutes of Health (to R.W.H.); R01 EB-002073, R01 HL-065770, HL-076901, and P01 DK-058327 (Project 1) from the National Institutes of Health (to A.S.); and Grant No. 8187368876 from the Roche Foundation for Anemia Research, a research grant from the Fanconi Anemia Research Fund, Inc., and an Institutional Research Grant No. IRG-01-188-04 from the American Cancer Society (to L.Z.). G.R.J. was supported in part by an “Overseas Associate Fellowship-2006” from the Department of Biotechnology, Government of India, and D.M.M. is supported by a Ruth L. Kirschstein National Research Service Award F32 HL-096281 from the National Institutes of Health.