AAV: Analysis of virus quality and DNA integration into the human genome

Abstract

Gene therapy approaches can be divided into two fractions: Targeting dividing cell-types, i.e. stem cells, requires an integrating gene delivery vehicle, while non-dividing cells can be modified without integration of the transgene. Here, we investigate wild-type adeno-associated virus (wtAAV) for the first and recombinant AAV (rAAV) for the second task. The combination of stem cells and gene therapy is promising for the future treatment of a variety of human diseases. Genetic modification of stem cells is commonly achieved through the use of retroviral vectors, which integrate the transgene stably and efficiently, but bear the risk of insertional mutagenesis. wtAAV possesses the unique ability to integrate its genome in a site-specific manner into the cellular DNA without imposing deleterious effects on the host. By investigating the following four questions about the biology of wtAAV integration into human cells, we address the validity of this virus as a gene delivery vehicle for dividing cell-types: (1) What is the integration efficiency of wtAAV into unselected human cells? (2) What is the molecular organization of the provirus? (3) What is the genetic organization surrounding the provirus? (4) What is the proportion of site-specific versus random integration? To date, rAAV vectors are successfully used to achieve gene delivery to nondividing cells. However, the two main concerns are low efficacy and the induction of a host immune response. It is likely that rAAV preparations of maximum quality may be able to increase efficacy and avoid a host immune response. In this dissertation the quality of viral preparations for rAAV was established and compared to that of wtAAV. Surprisingly – and unprecedented for a human virus – a near-perfect quality of wtAAV as compared to rAAV was determined. We, therefore propose comparing rAAV preparations to wtAAV as a tool to evaluate improved methods of the production for rAAV