Developing a Synthetic Adeno-Associated Virus (AAV) for Engineering Safer Gene Therapies

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PROJECT SUMMARY Medicine is currently undergoing a revolution, where viable gene therapies are being developed for multiple disorders, including diseases of the central nervous system (CNS). One of the obstacles that limits the use of gene therapy is the availability of safe and effective vectors for widespread delivery of genes. Due to its stable transgene expression, broad tropism, and modest immunogenicity, recombinant adeno-associated virus (rAAV) is the most widely used viral vector for human gene therapy. Almost 200 rAAV therapies have been completed or are currently in clinical trials, including two FDA-approved therapies for genetic diseases of the CNS. However, evidence is mounting that rAAV-based gene therapies are not without toxicity or significant risk, with several rAAV-related deaths and numerous adverse outcomes reported during the past three years alone. In a recent trial for Sanfilippo syndrome, 1 patient died and others demonstrated concerning MRI changes at rAAV injection sites within the brain, halting the study. Other rAAV trials have reported serious adverse effects ranging from thrombocytopenia to acute kidney failure to cardio-pulmonary insufficiency. While some of these adverse effects are thought to be caused by immune reactions to the AAV capsid or transgene, increasing evidence indicates that the rAAV genome, which contains two 145-base pair DNA segments named inverted terminal repeats (ITRs), is a major source of rAAV toxicity. While conducting fundamental experiments on learning and memory, we discovered that rAAV was toxic to dividing neural progenitor cells (NPCs) and immature neurons, completely ablating adult neurogenesis in the mouse hippocampus. Consistent with previous work, these experiments indicate that the AAV ITRs appear to be sufficient and necessary for this toxicity. Embarking on a new research direction, we will utilize our complimentary expertise in neuroscience, stem cell biology, and engineering to develop new methods for rAAV production and create the first rAAVs with engineered ITRs that are safer for human gene therapy. These new therapies will be particularly important in the treatment of neurodevelopmental and other diseases in children who have active proliferation of stem/progenitor cells, which are exquisitely sensitive to rAAV toxicity. In the current proposal we aim to: Aim 1. Determine which components of the ITR DNA sequence are required for toxicity in NPCs in vivo. Aim 2. Develop a cell-free synthetic rAAVs capable of packaging genomes with mutant ITRs. Aim 3. Engineer an rAAV that will rescue loss of function in a murine model of Rett syndrome while demonstrating less toxicity than conventional rAAVs.