Abstract
Huntington's disease is a fatal neurodegenerative disorder caused by an inherited mutation in the huntingtin (HTT) gene comprising an expanded cytosine-adenine-guanine (CAG) trinucleotide repeat sequence that results in a pathogenic huntingtin protein. Adeno-associated viral (AAV) gene therapy containing a primary artificial microRNA (pri-amiRNA) specifically targeting HTT messenger RNA (mRNA) has the potential to provide long-lasting therapeutic benefit, through durable reduction of mutant HTT expression after a single administration. The efficiency and precision of processing of the pri-amiRNA precursor to the mature guide (G) strand by transduced cells are critical for specific and potent HTT mRNA lowering. The selection of the optimized pri-amiRNA comprised a series of in vitro studies followed by in vivo studies in small and then large mammals. Our studies demonstrate the predictivity of certain cell culture systems and rodent models for nonhuman primates with respect to some, but not all key features of pri-amiRNA processing. In addition, our results show that the processing of pri-amiRNAs to the mature guide strand can differ greatly across different scaffolds and sequences while providing the same levels of target lowering. Importantly, our data demonstrate that there is a combinatorial effect of guide and passenger (P) strand sequences, together with the scaffold, on pri-amiRNA processing, with different guide and passenger strand sequences within the same scaffold dramatically altering pri-amiRNA processing. Taken together, our results highlight the importance of optimizing not only target lowering but also the efficiency and precision of pri-amiRNA processing in vitro, in rodents and in large mammals to identify the most potent and selective AAV gene therapy that harnesses the endogenous microRNA (miRNA) biogenesis pathway for target lowering without perturbing the endogenous cellular miRNA profile. The optimized pri-amiRNA was selected with this focus on efficiency and precision of pri-amiRNA processing in addition to its pharmacological activity on HTT mRNA lowering and general tolerability in vivo.