Facilitate by-stander effects by EV-mRNA cargo in AAV gene therapy for MPS IIIC

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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 a novel effective gene therapy product for treating MPS IIIC using AAV vector to target the root cause. Notably, the neuropathologies in MPS IIIC are global, involving the entire nervous system, and 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 benefits requires targeting as many cells as possible, which can be achieved by high-dose vector delivery, such as recently approved Zolgensma 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 doses has been a major challenge in translation of AAV gene therapy due to the scale-up capacity of vector production for human application. All cells are known to continuously release extracellular vesicles (EVs) and communicate by exchanging large molecules via EV traffic. The demonstrated properties of EV intercellular interaction offer a potential tool to ease this challenge to translation of rAAV gene therapy. We hypothesize that incorporation of EV-mRNA packaging signals in gene therapy vectors will engender by-stander effects for normally non-secreted protein via the mRNA-containing EVs. To test this hypothesis, we will construct rAAV vectors that express hHGSNAT and contain different putative EV packaging signals, and identify effective signals for EV-mRNA loading. The goal is to develop an effective rAAV-hHGSNAT gene delivery approach for treating MPS IIIC. Data generated may also provide powerful tools for developing gene therapy to treat other diseases involving non- secreted transgene products. We believe this project is ideally suited to the R21 high risk/high impact funding mechanism.