Several Log Increase in Therapeutic Transgene Delivery by Distinct Adeno-Associated Viral Serotype Vectors

Abstract

We previously demonstrated that rAAV vectors carrying human and canine factor IX (FIX) cDNA can infect, stably persist, and secrete functional human and canine FIX following direct intramuscular injection. In an attempt to improve FIX protein secretion for eventual therapeutic use, we set out to determine if alteration of the AAV capsid would affect skeletal muscle transduction and factor IX secretion. Two reasons to pursue this question were (1) the persistence of high-titer neutralizing antibody (NAB) to AAV2 and (2) our previous study that supported a restricted tropism of muscle fiber types to AAV2 transduction. Using an identical CMV/canine factor IX (cFIX) expression cassette, we cross-packaged this genome into virions generated from each of the five AAV serotypes. In a dose-response assay, equivalent amounts of rAAV/cFIX serotypes were tested in vitro and in vivo. In tissue culture cells, FIX antigen levels secreted into the supernatant varied depending on the AAV serotype used; type 2 transduced maximally, with serotypes 3, 1, 5, and 4, respectively, expressing lower levels. However, when the same viruses were tested in vivo using immunodeficient NOD/SCID animals, we obtained surprisingly different results. While the time to onset of detectable serum levels appeared the same for all serotypes, types 1, 3, and 5 produced 100- to 1000-fold more cFIX than type 2. In fact, 12 weeks after transduction, type 1 continued to express levels of cFIX on average at 80 microg/ml followed by type 5 (6.52 microg/ml), type 3 (3.27 microg/ml), type 4 (258 ng/ml), and finally type 2 (90 ng/ml). Coagulant activity of cFIX as measured by aPTT supported the circulating levels measured by ELISA demonstrating the secreted protein was functional, and RT-PCR of injected tissue correlated with the serotype-specific transduction data. In summary, we found significant differences in cFIX expression upon introducing various rAAV serotypes into mouse muscle. These data have direct bearing on the design of AAV gene therapy clinical trials for hemophilia and should also extend to most therapeutic transgenes.

Adeno-associated vectors: Are we there yet?

“…Recent papers by Sarkar et al 1 and Wang et al 2 suggest that sustained therapeutic levels of coagation factors FVIII and FIX, respectively, were achieved in murine knockout and canine deficient animals. These results stem from previous observations 3,4 and the isolation of new adeno-associated virus (AAV) serotypes in primates. 5 The recent ASGT meeting in St Louis saw notable abstracts listing the use of AAV serotypes, as well as the unveiling of new serotypes (AAV10).…”

Long-term correction of murine glycogen storage disease type Ia by recombinant adeno-associated virus-1-mediated gene transfer

“…[22][23][24] Studies have shown that the capsid gene of the AAV determines the properties of the viral particles including their tissue tropism and antigenic properties. 28 The different efficiencies in transducing the Figure 3 Histological analysis of the liver and kidney from the AAV1-G6Pase-a-2X infused G6Pase-a À/À mice.…”

“…[22][23][24] These are gaining popularity not only because of their increased transduction efficiency but also because they circumvent the pre-existing immunity to the most common AAV2 serotype that is prevalent in over 70% of humans. In this study, we show that neonatal infusion of G6Pase-a À/À mice with either a rAAV serotype 1 vector carrying G6Pase-a (AAV1-G6Pase-a) or a rAAV serotype 8 vector carrying G6Pase-a (AAV8-G6Pase-a) effectively delivers the G6Pase-a transgene to the liver and markedly improves the survival of the mice.…”

Chromatographic purification of recombinant adenoviral and adeno-associated viral vectors: methods and implications

“…81 rAAV produced from serotypes 1 and 5 transduce skeletal muscle much more efficiently than AAV of serotype 2. 82,83 Discovery of new AAV serotypes could allow further improvements in cell transduction efficiencies as well as broaden the range of somatic host cell types. Screening human and non-human primate tissues has revealed over 40 novel AAV variants, most of which have been derived via genomic recombination at the capsid protein regions between different parental serotypes.…”