Optimizing AAV-Mediated Gene Delivery in the Brain: Blocking TLR9 Minimizes the Negative Impacts of AAV on Neuronal Morphology

Abstract

Recombinant adeno-associated viruses (rAAVs) have transformed the fields of basic neuroscience and human gene therapy. Although rAAVs are considered relatively non-immunogenic, AAV elicits an immune response in the brain, including upregulation of immune proteins known to negatively regulate dendritic complexity and synaptic transmission. This suggests that AAV could alter the structure and function of neurons, a previously unexplored possibility. To quantify AAV-induced changes in neuronal morphology, DiOlistic labeling, Sholl analysis, and Strahler analysis were performed on layer 4-6 pyramidal cells in mouse primary somatosensory cortex. We found that AAV simplifies dendritic morphology, irrespective of AAV serotype, promoter, transgene, or production facility. We demonstrated that AAV induces a broad spatial reduction in dendritic complexity by 21 days post injection, which is associated with a decrease in cortical cell density. By corroborating electrophysiology recordings with Western blot quantification, we determined that AAV changes synaptic transmission by altering the ratio of Ca2+-permeable to Ca2+-impermeable AMPA receptors. To determine if the toll-like receptor (TLR9), which detects viral DNA, is required for AAV detection in the brain, TLR9 was inhibited. We found that systemically blocking TLR9 prevents AAV-induced dendritic simplification. In this thesis, for the first time, we characterize the negative impacts of AAV on neuronal morphology. We propose to block TLR9 as a solution to minimize these unintended consequences of AAV in basic neuroscience research and to develop safer and more effective gene therapies.