High-efficiency transduction and correction of murine hemophilia B using AAV2 vectors devoid of multiple surface-exposed tyrosines.
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.
Markusic
David M
DM
Division of Cellular and Molecular Therapy, Department of Pediatrics, University of Florida College of Medicine, Gainesville, Florida 32611, USA.
Herzog
Roland W
RW
Aslanidi
George V
GV
Hoffman
Brad E
BE
Li
Baozheng
B
Li
Mengxin
M
Jayandharan
Giridhara R
GR
Ling
Chen
C
Zolotukhin
Irene
I
Ma
Wenqin
W
Zolotukhin
Sergei
S
Srivastava
Arun
A
Zhong
Li
L
D000818:Animals; D000229:Dependovirus / Q000235:genetics*; D015316:Genetic Therapy; D005822:Genetic Vectors / Q000235:genetics; D006367:HeLa Cells; D002836:Hemophilia B / Q000235:genetics* / Q000628:therapy*; D022781:Hepatocytes / Q000378:metabolism; D006801:Humans; D051379:Mice; D008810:Mice, Inbred C57BL; D010957:Plasmids; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics; D014161:Transduction, Genetic; D014443:Tyrosine / Q000737:chemistry
D011994:Recombinant Proteins; D014443:Tyrosine