Enhancement, mapping, and validation of viral vectors for primate optogenetics

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PROJECT SUMMARY Mapping the visual and visuomotor circuits of the brain using opsins and other actuators to target and control neurons is a central goal of modern neuroscience. Actuators have become key to studying neuronal circuits, modeling brain disorders, and developing new therapies. Neural actuator applications to research in rodents and other small animals have achieved great success. In primates, however, these approaches have yet to be transformative. The main problem is that viral vectors are required to deliver actuator genes, but both viral transduction and gene expression have been unreliable across, and even within, primate labs. Efficacy is hindered by the complex innate and adaptive retaliatory immune response in primates, and even when the approach does work, cell-type specificity is lacking. The overall purpose of this project is to incorporate recent advances in virology, gene therapy, and immunology to maximize viral transduction, maintain chronic gene expression, and gain cell-type specificity through retrograde transportation of viruses in visual circuits of the macaque brain. Throughout the project, optogenetics is the actuator-mediated intervention and the visual- oculomotor system is the testbed. We focus on two viruses that provide retrograde transport: retrograde adeno- associated virus-2 (rAAV2-retro) and fusion glycoprotein-E pseudotyped lentiviral vector (NeuRet). Each will deliver genes encoding the Red-activatable Channelrhodopsin (ReaChR). Our team, spanning Duke, NYU, and UNC-Chapel Hill, has extensive expertise in vector technology and macaque neurobiology. Aim 1 is dedicated towards the maturation of pharmacological regimens that modify both arms of the primate's immune system to enhance viral transduction and promote long-term constitutive expression of opsin transgenes. Aim 2 will establish comprehensive expression maps of retrogradely transduced neurons. This mapping is a critical step toward providing cell- and circuit-level specificity and supplies a means for physiologically identifying neurons, through phototagging, based on their anatomical connectivity. Aim 3 will use phototagging paired with projection targeting to identify and neurophysiologically characterize neurons contributing to specific circuits within the visual and visuomotor circuitry of the macaque brain. In combination, this work will enhance the efficacy of viral vectors for neuroscientific research of the macaque visual and visuomotor system, provide both anatomical and functional validation of the developed protocols, and provide new insights into the functional role these specific circuits serve in vision and visuomotor behaviors. Finally, this project will provide fundamental insights for improving human gene therapies that depend on viral delivery of therapeutic genes.