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

David M. Markusic, Roland W. Herzog, George V. Aslanidi, Brad E. Hoffman, Baozheng Li, Mengxin Li, Giridhara R. Jayandharan, Chen Ling, Irene Zolotukhin, Wenqin Ma, Sergei Zolotukhin, Arun Srivastava, Li Zhong

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110 Scopus citations


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.

Original languageEnglish (US)
Pages (from-to)2048-2056
Number of pages9
JournalMolecular Therapy
Issue number12
StatePublished - Dec 2010
Externally publishedYes

Bibliographical note

Funding 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.


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