In vitro and in vivo modeling of gene therapy vector stability when challenged by natural virus infection in human hepatocytes - PROJECT SUMMARY Gene therapies based on adeno-associated virus (AAV) vectors have been a revolutionary medical advancement in treating human genetic diseases. With a single dose, AAV-mediated gene therapies can confer long-term correction or abatement of disease for the lifetime of the patient. Recombinant (r)AAVs that are used for the transfer of therapeutic genes are considered safe, partly because these vectors are removed of all viral genes. The only viral elements that are retained are the inverted terminal repeat (ITR) sequences that are at both the ends of the vector genome. However, this feature may make them vulnerable to natural viral infections from wildtype (wt)AAV and helper viruses, such as adenoviruses (AdV), herpesviruses, or papillomaviruses. It is speculated that these challenges may destabilize the therapeutic vector genome or may amplify them in a process called “mobilization”. Unfortunately, models for these events have yet to be explored, and the extent to which these processes can alter the abundance or structure of the vector genomes are unknown. Understanding the stability of rAAV genomes in treated tissues under these naturally occurring and unavoidable circumstances is critically important for the gene therapy field. This project proposal aims to define if and how natural virus infections that can act to alter the composition of AAV-based gene therapy in human hepatocytes, a prevalent gene therapy target for rAAV-based treatments. The objectives are to develop novel methods to characterize the structure of vector genomes as they transition from linear species, to extrachromosomal episomes, and to vector genomes that have integrated into the host cell genome. The bases of this work leverages expertise in long-read sequencing technologies, bioinformatics, and vectorology to address these decades-long unanswered questions. The grant proposal is divided into two main aims: • Aim 1. Track changes in vector genome abundance and episomal configurations within transduced primary human hepatocytes following wtAAV and wtAdV infection. A relevant in vitro model will be developed to track the kinetics of transgene expression and to assess the stability of rAAV genomes in transduced human hepatocytes following wtAAV and wtAdV infection over time. • Aim 2. Assessing the ability for wtAAV and wtAdV infection to destabilize rAAV episomes in humanized mouse livers. Whether wtAAV and wtAdV infection will act to destabilize or mobilize rAAV genomes in human hepatocytes, or whether they will act to drive integration of vectors into the host cell genome will be tested in the Fah/Rag2/Il2rgc-deficient (FRG)-humanized liver mouse model.
