Recombinant adeno-associated virus (rAAV) vector genome structure and regulation in vivo, biodistribution, AAV capsid structure in solution, and vector manufacturing and testing technology.
Recombinant adeno-associated viral (rAAV) vectors are capable of mediating long-term gene
expression following administration to a variety of tissues. In rodent skeletal muscle,
the vector genomes persist in the nucleus in concatemeric episomal forms. In non-human
primates, rAAV vectors integrate inefficiently into the chromosomes of myocytes and reside
predominantly as episomal monomeric and concatemeric circles. A goal of the lab is to understand how episomal rAAV vector genomes
and vector gene expression persist long-term in vivo.
We have shown that the episomal rAAV genomes assimilate into
chromatin with a typical nucleosomal pattern. The persistence of the vector
genomes and gene expression for years in quiescent tissues suggests that a
bona fide chromatin structure is
important for episomal maintenance and transgene expression.
A second goal of the lab is to determine the smallest dose of a rAAV vector injected intramuscularlly that is able to be detected in blood, and to discern the relationship between injected dose and longevity in blood. The tests we are developing involve the collection of blood and the analysis of DNA by PCR and qPCR assays. Validation of all of the steps involved (blood collection, DNA extraction, transport, and PCR analysis) is necessary. Data generated in the non-human primate is the basis for developing a legally defensible commercial qPCR assay that will eventually be used to test athletes. Given that gene transfer technology encompasses a variety of vectors, transgenes, expression cassettes, routes of administration, as well as injection formulations, vector biodistribution can vary widely, thus an outcome of our work will be to better define assays that can be utilized to elucidate vector distribution for legitimate gene therapy applications.
A third goal is to understand the structure of the AAV capsid in solution using molecular methods. Proteolytic mapping of AAV provides a useful tool for gaining a better understanding of dynamic structural changes in the capsid that must occur as it performs its various functions during the virus life cycle. For example, different proteolytic signatures are seen between empty and full (DNA-containing) capsids, indicating that DNA packaging can change the capsid surface. Additionally, we have demonstrated that proteolytic mapping can be used as a diagnostic tool to distinguish among different AAV serotypes.
In addition, the lab is developing and optimizing production, purification, and testing methods for rAAV vectors.
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