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

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.

Highlights

Huntington disease (HD) is a fatal neurodegenerative genetic disorder that affects motor function and leads to behavioral symptoms and cognitive decline in adulthood, resulting in total physical and mental deterioration over a 12- to 15-year period.[1,2] HD is caused by the expansion of CAG trinucleotides in exon 1 of a multifunctional gene coding for the huntingtin (Htt) protein.[2]
These data indicate that human induced pluripotent stem cells (iPSCs)-derived neuronal cultures from HD patients can be effectively transduced by associated viral vector serotype 5 (AAV5)-microRNA targeting huntingtin (miHTT), which results in significant lowering of human HTT mRNA and human Htt protein
Machinery Having demonstrated the efficacy of AAV5-miHTT, we investigated the processing of the therapeutic miHTT in human cells

Summary

Introduction

Huntington disease (HD) is a fatal neurodegenerative genetic disorder that affects motor function and leads to behavioral symptoms and cognitive decline in adulthood, resulting in total physical and mental deterioration over a 12- to 15-year period.[1,2] HD is caused by the expansion of CAG trinucleotides in exon 1 of a multifunctional gene coding for the huntingtin (Htt) protein.[2]. To lower HTT, we developed an adeno-associated viral vector serotype 5 (AAV5) that delivers a microRNA (miRNA) that targets human HTT mRNA (AAV5-miHTT). AAV5-miHTT is a one-time gene therapy candidate for HD based on the lowering of mutant HTT expression using RNAi.[4,5,6,7,8] The expression cassette for AAV5miHTT consists of the cytomegalovirus immediate-early enhancer fused to chicken b-actin (CAG) promoter, the pri-miHTT expression cassette engineered in the pri-miR-451 backbone, and the human growth hormone poly(A) signal.[4] The AAV5-miHTT cassette is flanked by two intact noncoding inverted terminal repeats (ITRs) that originate from the wild-type AAV2 viral genome. Minipig, and nonhuman primate studies, AAV5-miHTT has demonstrated preferential vector distribution and miRNA expression in the striatum, the primary site of neurodegeneration in HD, and in the sensorimotor cortex, affected during disease progression.[6,8] This expression of therapeutic miHTT in key brain areas relating to HD results in lowering of HTT mRNA and mutant Htt protein, reduction in mutant Htt aggregates, and improvements in neuronal dysfunction, behavior, and survival in different small and large animal disease models.[5,6,7,8]

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