AAV5-miHTT lowers huntingtin mRNA and protein without off-target effects in patient-derived neuronal cultures and astrocytes

Abstract Huntington disease (HD) is a fatal neurodegenerative genetic disorder, thought to reflect a toxic gain of function in huntingtin (Htt) protein. Adeno-associated viral vector serotype 5 (AAV5)- microRNA targeting huntingtin (miHTT) is a HD gene-therapy candidate that efficiently lowers HTT using RNA interference (RNAi). This study analyzed the efficacy and potential for off-target effects with AAV5-miHTT in neuronal- and astrocyte-cell cultures differentiated from induced-pluripotent stem cells (iPSC) from two individuals with HD (HD71 and HD180). One-time AAV5-miHTT treatment significantly reduced human HTT mRNA by 57% and Htt protein by 68% in neurons. Small RNA sequencing showed that mature miHTT was processed correctly without off-target passenger strand. No cellular microRNAs were dysregulated, indicating that endogenous-RNAi machinery was unaffected by miHTT overexpression. Quantitative polymerase-chain reaction validation of in-silico predicted off-target transcripts, next-generation sequencing, and pathway analysis confirmed absence of dysregulated genes due to sequence homology or seed-sequence activity of miHTT. Minor effects on gene expression were observed in both AAV5-miHTT and AAV5-green-fluorescent protein-treated samples, suggesting that they were due to viral transduction rather than miHTT. This study confirms the efficacy of AAV5-miHTT in HD patient iPSC-derived-neuronal cultures and lack of off-target effects in gene expression and regulation in neuronal cells and astrocytes.