Adeno-associated virus (AAV) has in vitro properties that may be useful in gene therapy. These are the specificities of AAV integration in the human genome in the lack of pathogenicity of the AAV vector. However, early vector production methods did not generate large amounts of high titer recombinant AAV uncontaminated by adenovirus, replication competent AAV, or cellular toxins. Since such contaminants may effect the activity and/or persistence of recombinant AAV, as well as its potential for eliciting acute, humoral, and/or cellular immunological responses, comprehensive in vivo evaluation will require uncontaminated vector. We evaluated in vivo high titer, recombinant AAV lacking detectable contamination with adenovirus, replication competent AAV, or cellular toxins. Virus was produced by Targeted Genetics, Inc. Analysis of purity indicated no contaminating material at the limits of test sensitivity: < 10 infectious adenoviruses / 10e10 DRP (DNase-Resistant Particles), < 2.5 x 10e5 Adenoviral genomes / 10e12 DRP, < 1 replication competent AAV / 10e10 DRP, and < 0.003 endotoxin units / 10e10 DRP. Viral titer was 1.4 x 10e12 DRP per ml. 1 x 10e11 DRP of AAVBgal or control vehicle was administered to mice intravenously or by catheter-mediated, manometry-guided retrograde biliary infusion. Animals were evaluated 28 days later for (1) the pattern and extent of gene transfer and recombinant protein expression using both immunohistochemistry and quantitative DNA analysis; and (2) the presence of any serological or histological evidence of pathology. Immunohistochemical evidence of gene transfer and recombinant protein expression was detected in both cholangiocytes and hepatocytes 28 days following retrograde biliary administration of recombinant AAVBgal.
