Humoral Immune Response to AAV

Tissues from rhesus monkeys were screened by PCR for the presence of sequences homologous to known adeno-associated virus (AAV) serotypes 1-6. DNA spanning entire rep-cap ORFs from two novel AAVs, called AAV7 and AAV8, were isolated. Sequence comparisons among these and previously described AAVs revealed the greatest divergence in capsid proteins. AAV7 and AAV8 were not neutralized by heterologous antisera raised to the other serotypes. Neutralizing antibodies to AAV7 and AAV8 were rare in human serum and, when present, were low in activity. Vectors formed with capsids from AAV7 and AAV8 were generated by using rep and inverted terminal repeats (ITRs) from AAV2 and were compared with similarly constructed vectors made from capsids of AAV1, AAV2, and AAV5. Murine models of skeletal muscle and liver-directed gene transfer were used to evaluate relative vector performance. AAV7 vectors demonstrated efficiencies of transgene expression in skeletal muscle equivalent to that observed with AAV1, the most efficient known serotype for this application. In liver, transgene expression was 10- to 100-fold higher with AAV8 than observed with other serotypes. This improved efficiency correlated with increased persistence of vector DNA and higher number of transduced hepatocytes. The efficiency of AAV8 vector for liver-directed gene transfer of factor IX was not impacted by preimmunization with the other AAV serotypes. Vectors based on these novel, nonhuman primate AAVs should be considered for human gene therapy because of low reactivity to antibodies directed to human AAVs and because gene transfer efficiency in muscle was similar to that obtained with the best known serotype, whereas, in liver, gene transfer was substantially higher than previously described. Show more

The potential for using Adeno-associated virus (AAV) as a vector for human gene therapy has stimulated interest in the Dependovirus genus. Serologic data suggest that AAV infections are prevalent in humans, although analyses of viruses and viral sequences from clinical samples are extremely limited. Molecular techniques were used in this study to successfully detect endogenous AAV sequences in 18% of all human tissues screened, with the liver and bone marrow being the most predominant sites. Sequence characterization of rescued AAV DNAs indicated a diverse array of molecular forms which segregate into clades whose members share functional and serologic similarities. One of the most predominant human clades is a hybrid of two previously described AAV serotypes, while another clade was found in humans and several species of nonhuman primates, suggesting a cross-species transmission of this virus. These data provide important information regarding the biology of parvoviruses in humans and their use as gene therapy vectors. Show more

Adeno-associated viral vectors are highly safe and efficient gene delivery vehicles. However, numerous challenges in vector design remain, including neutralizing antibody responses, tissue transport and infection of resistant cell types. Changes must be made to the viral capsid to overcome these problems; however, very often insufficient information is available for rational design of improvements. We therefore applied a directed evolution approach involving the generation of large mutant capsid libraries and selection of adeno-associated virus (AAV) 2 variants with enhanced properties. High-throughput selection processes were designed to isolate mutants within the library with altered affinities for heparin or the ability to evade antibody neutralization and deliver genes more efficiently than wild-type capsid in the presence of anti-AAV serum. This approach, which can be extended to additional gene delivery challenges and serotypes, directs viral evolution to generate 'designer' gene delivery vectors with specified, enhanced properties. Show more