Optimization of AAV gene therapy through the unbiased, high-throughput screening of novel ITR domains in the mouse brain

Adeno-associated viral (AAV) vectors have become the gold standard for delivery of genetic cargo the central nervous system. Remarkable efficacy and strong safety profiles have led to FDA approval of multiple AAV gene therapies for genetic disorders validating this platform as a key leader in the space. However, AAV gene therapies are still limited in certain aspects as it pertains to broadly treating and advancing to clinical trials for brain disorders. One of the key factors that determines clinical efficacy and success is related to transduction efficiency, biodistribution, and efficient transgene expression. Revolutionary progress has been made on the first two fronts through the identification of naturally occurring serotypes with CNS tropism and through capsid evolution to increase blood-brain barrier permeability and distribution, however increasing transgene expression has been limited to studies of promoter optimization. The critical barrier for transgene expression following AAV transduction is mediated through the single strand DNA virus undergoing double-strand DNA synthesis allowing for mRNA to be transcribed. This process is mediated by the only non-optimized, wild-type sequence within the AAV process, the inverted terminal repeats (ITR). This proposal aims to modernize and engineer better transgene expression through the identification and screening of a large library of ITR domains in the mouse brain. These library screens will allow for the high throughput identification of optimal ITR domains that outperform the natural ITR found in AAV plasmids and at least equivalent if not improved performance when compared to self-complementary ITR. Additionally, this proposal will explore novel mechanisms in which to enhance AAV production through increased particle secretion into the extracellular space. Through this high-risk/high-reward project we aim to provide the foundational information needed to improve upon specific aspects of AAV gene therapy effectiveness and translational capabilities. While the primary goal is to increase the efficiency in which a single strand DNA virus can efficiently form the double- strand substrate needed for transcription, this system may also enable and unlock additional benefits as it relates to AAV gene therapy such as reduced innate immune response, shorter timeframe to express therapeutic transgene therefore shorting the treatment window in which an organism could receive therapeutic rescue, improved packaging and manufacturing metrics, and the ability to lower the vector genome dose needed to achieve therapeutic efficacy thereby making these treatments safer for the patient population. Taken together, this initial screen and optimization of ITR may have an outsized impact on AAV gene therapy for those suffering from genetic neurological conditions. Project Number: 1R21NS142879-01A1 | Fiscal Year: 2026 | NIH Institute/Center: National Institute of Neurological Disorders and Stroke (NINDS) | Principal Investigator: Kyle Fink (+1 co-PI) | Institution: UNIVERSITY OF CALIFORNIA AT DAVIS, DAVIS, CA | Award Amount: $442,750 | Activity Code: R21 | Study Section: Therapeutic Approaches to Genetic Diseases Study Section[TAG] View on NIH RePORTER: https://reporter.nih.gov/project-details/11305097