I06 Efficacy and safety of AAV5-MIHTT in hd patient-derived neurons

Background We are developing an HTT lowering approach based on adeno-associated serotype 5 virus carrying an expression cassette of an engineered therapeutic microRNA (AAV5-miHTT). Main efficacy and safety concerns with microRNA gene therapy approaches would be long-term expression and possible off-target activity of the microRNA. As off-target activity could be both species and cell type-specific, off-target analyses were done in a cell system that as closely as possible resembles the human patient situation: neuronal cultures derived from HD patient induced pluripotent stem (iPS) cells. Aims Investigate efficacy and safety of AAV5-miHTT in HD patient-derived neurons. Methods iPS-derived neurons originating from two HD patients were transduced with AAV5-miHTT and AAV5-GFP. HTT mRNA and protein lowering were assessed, and the miHTT processing pattern determined by small RNA sequencing. In silico off-target analysis was performed, followed by unbiased next-generation sequencing (NGS). Results AAV5-miHTT was able to efficiently transduce HD patient iPS-derived neurons, resulting in a significant mutant huntingtin protein lowering of 76.8%. Small RNA sequencing showed that miHTT was processed correctly, without any passenger strand, limiting potential off-target activity. No other endogenous microRNAs were dysregulated, indicating that the endogenous RNA interference machinery is not saturated due to miHTT overexpression. qPCR validation of in silico predicted off-target transcripts, NGS, and pathway analysis confirmed the absence of dysregulated genes due to sequence homology or seed sequence activity of miHTT. Only an effect was observed in both miHTT and GFP treated samples, due to the viral transduction. Conclusions AAV5-miHTT efficiently lowers mutant huntingtin, without any off-target effects, in HD patient iPS-derived neurons.