AAV5-miHTT Lowers Huntingtin mRNA and Protein without Off-Target Effects in Patient-Derived Neuronal Cultures and Astrocytes

Sonay Keskin; Cynthia C Brouwers; Marina Sogorb-Gonzalez; Raygene Martier; Josse A Depla; Astrid Vallès; Sander J van Deventer; Pavlina Konstantinova; Melvin M Evers

doi:10.1016/j.omtm.2019.09.010

AAV5-miHTT Lowers Huntingtin mRNA and Protein without Off-Target Effects in Patient-Derived Neuronal Cultures and Astrocytes

Mol Ther Methods Clin Dev. 2019 Oct 4:15:275-284. doi: 10.1016/j.omtm.2019.09.010. eCollection 2019 Dec 13.

Authors

Sonay Keskin¹, Cynthia C Brouwers¹, Marina Sogorb-Gonzalez^{1

2}, Raygene Martier^{1

2}, Josse A Depla^{1

3}, Astrid Vallès¹, Sander J van Deventer^{1

2}, Pavlina Konstantinova¹, Melvin M Evers¹

Affiliations

¹ Department of Research & Development, uniQure biopharma, B.V., Amsterdam, the Netherlands.
² Department of Gastroenterology and Hepatology, Leiden University Medical Center, Leiden, the Netherlands.
³ Department of Medical Microbiology - Clinical Virology, Amsterdam Medical Center, Amsterdam, the Netherlands.

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 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 (iPSCs) 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. qPCR 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-GFP-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.

Keywords: AAV5; AAV5-miHTT; Huntingtin; Huntington disease; RNA-seq; gene therapy; iPSC; microRNA; off-target; patient-derived neuronal cultures.