Affiliation:
1. Departments of Pediatrics and Genetics, School of Medicine, Stanford University, 300 Pasteur Drive, Stanford, California 94305
Abstract
ABSTRACT
We and others have recently reported highly efficient liver gene transfer with adeno-associated virus 8 (AAV-8) pseudotypes, i.e., AAV-2 genomes packaged into AAV-8 capsids. Here we studied whether liver transduction could be further enhanced by using viral DNA packaging sequences (inverted terminal repeats [ITRs]) derived from AAV genotypes other than 2. To this end, we generated two sets of vector constructs carrying expression cassettes embedding a
gfp
gene or the human factor IX (
hfIX
) gene flanked by ITRs from AAV genotypes 1 through 6. Initial in vitro analyses of
gfp
vector DNA replication, encapsidation, and cell transduction revealed a surprisingly high degree of interchangeability among the six genotypes. For subsequent in vivo studies, we cross-packaged the six
hfIX
variants into AAV-8 and infused mice via the portal vein with doses of 5 × 10
10
to 1.8 × 10
12
particles. Notably, all vectors expressed comparably high plasma hFIX levels within a dose cohort over the following 6 months, concurrent with the finding of equivalent vector DNA copy numbers per cell. Partial hepatectomies resulted in ∼80% drops of hFIX levels and vector DNA copy numbers in all groups, indicating genotype-independent persistence of predominantly episomal vector DNA. Southern blot analyses of total liver DNA in fact confirmed the presence of identical and mostly nonintegrated molecular vector forms for all genotypes. We conclude that, unlike serotypes, AAV genotypes are not critical for efficient hepatocyte transduction and can be freely substituted. This corroborates our current model for AAV vector persistence in the liver and provides useful information for the future design and application of recombinant AAV.
Publisher
American Society for Microbiology
Subject
Virology,Insect Science,Immunology,Microbiology
Cited by
102 articles.
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