Novel AAV gene therapy for treating CNS neuropathy of MPS IIIC

PROJECT SUMMARY MPS IIIC is a devastating lysosomal storage disease (LSD) for which there is no definitive treatment. To address this unmet need, this proposed project is to develop an effective gene therapy (GT) product for treating MPS IIIC using AAV9 vector to target the root cause. Notably, the neuropathologies in MPS IIIC are global, involving the entire nervous system, and the defective HGSNAT is a trans-lysosomal-membrane protein, which, unlike the majority of lysosomal enzymes, cannot be secreted and taken-up by neighboring cells. Therefore, optimal therapeutic benefit requires targeting as many cells as possible, which can be achieved by high-dose vector delivery, such as in the approved Zolgensma product for SMA.25 In comparison, studies have demonstrated that gene therapy for MPS disorders involving a secretable protein required significantly lower vector doses. The high vector dose has been a major challenge in translation of rAAV gene therapy due to the scale-up capacity of vector production for human application, as well as potential toxicity. All cells are known to continuously release extracellular vesicles (EVs) and communicate by exchanging large molecules via EV traffic. The demonstrated properties of EVs in intercellular interactions offer a potential tool to facilitate bystander effect in AAV GT, which may ease the high vector dose requirement and reduce the potential risk of toxicity. We hypothesize that incorporation of EV-mRNA packaging signals in GT vectors will engender by-stander effects for a normally non-secreted protein via the EV-mRNA cargo. Our preliminary studies support our hypothesis, showing that incorporation of a 25nt EV-mRNA packaging zip code (ZC) sequence in a rAAV- hHGSNATEV vector mediates EV-hHGSNAT-mRNA packaging. Importantly, the EVs were shown to transport the hHGSNAT-mRNA into non-transduced human MPS IIIC cells, in which the hHGSNAT-mRNA was translated into functional rHGSNAT protein, thus leading to the cross-correction of the lysosomal storage pathology in the recipient cells. These data lead to the further hypothesis that this EV-facilitated by-stander effect has great potential of achieving the optimal therapeutic benefits without having to transduce every cell due to cross- correction. Notably, for enhanced transduction efficiency, we have also constructed a self-complementary (sc) vector, scAAV9-hHGSNATEV. This Phase I STTR proposal is to further develop the proposed rAAV9- hHGSNATEV GT product for treating MPS IIIC towards clinical application and commercialization.