TY - JOUR
T1 - AAV5-miHTT Lowers Huntingtin mRNA and Protein without Off-Target Effects in Patient-Derived Neuronal Cultures and Astrocytes
AU - Keskin, Sonay
AU - Brouwers, Cynthia C.
AU - Sogorb-Gonzalez, Marina
AU - Martier, Raygene
AU - Depla, Josse A.
AU - Vallès, Astrid
AU - van Deventer, Sander J.
AU - Konstantinova, Pavlina
AU - Evers, Melvin M.
PY - 2019
Y1 - 2019
N2 - 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.
AB - 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.
UR - https://www.scopus.com/inward/record.uri?partnerID=HzOxMe3b&scp=85074442435&origin=inward
U2 - https://doi.org/10.1016/j.omtm.2019.09.010
DO - https://doi.org/10.1016/j.omtm.2019.09.010
M3 - Article
C2 - 31737741
SN - 2329-0501
VL - 15
SP - 275
EP - 284
JO - Molecular therapy. Methods & clinical development
JF - Molecular therapy. Methods & clinical development
ER -