Investigating Mechanisms of Viral Impairment of Neurogenesis Using Recombinant AAV

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PROJECT SUMMARY There is growing evidence that viral infections of the central nervous system (CNS) contribute to chronic brain disease. During development, multiple viruses including cytomegalovirus, herpes simplex virus, rubella virus, human immunodeficiency virus, and Zika virus annually cause thousands of cases of microcephaly — small head size resulting from impaired neurogenesis within the cerebral cortex. The sequalae of these viruses later in neurodevelopment and adulthood are less understood, but here they also disrupt neurogenesis and have been implicated in disease. These viruses share a common ability to eliminate neural progenitor cells (NPCs) in the developing and adult brain. However, the complex biology of these viruses has precluded our ability to identify a precise mechanism by which these infectious agents ablate neurogenesis. We recently discovered that the widely used recombinant adeno-associated virus (rAAV) rapidly kills dividing NPCs and early post-mitotic neurons in the adult murine dentate gyrus (DG) in a dose-dependent manner. Unlike the other viruses described above, rAAV is replication defective and is not known to cause significant pathology. This has resulted in its wide use as a vector in both experimental biology and human gene therapy. However, evidence is mounting that rAAV-based gene therapies are not without significant risk, with at least 7 rAAV-related deaths and numerous adverse outcomes reported in pediatric rAAV trials during the past three years alone. While some of these adverse effects are thought to be caused by immune reactions to the 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. Our preliminary experiments indicate that rAAV ITRs bind to and deplete Parp1, a first responder in cellular DNA damage response (DDR) within the nucleus. Moreover, rAAV toxicity mimics pharmacological inhibition of Parp1, inducing cell cycle arrest and cell death, and can be partially reversed by Parp1 activation. We aim to capitalize on these findings to identify the cellular pathways that mediate ITR-induced toxicity and discern whether therapeutic targets within these pathways are shared among viruses that cause microcephaly.