SRA

Huntington's disease (HD) is an incurable neurodegenerative disorder caused by genetic expansion of a CAG repeat sequence in one allele of the huntingtin (HTT) gene. Reducing expression of the mutant HTT (mutHTT) protein has remained a clear therapeutic goal but reduction of wild-type HTT (wtHTT) is undesirable as it compromises gene function and potential therapeutic efficacy. One promising allele- selective approach involves targeting the CAG repeat expansion with steric binding small RNAs bearing central mismatches. However, successful genetic encoding requires consistent placement of mismatches to the target within the small RNA guide sequence, which involves 5' processing precision by cellular enzymes. Here, we used small RNA sequencing to monitor processing precision of a limited set of CAG repeat- targeted small RNAs expressed from multiple scaffold contexts. Small RNA sequencing identified expression constructs with high guide strand 5' processing precision that also conferred promising allele-selective inhibition of mutHTT. However, mRNA-seq revealed varying degrees of transcriptome-wide off-target effects, including certain CAG repeat-containing mRNAs. These results support the continued investigation and optimization of genetically encoded repeat-targeted small RNAs for allele-selective HD gene therapy and underscore the value of sequencing methods to balance specificity with allele selectivity during the design and selection process. Overall design: HD fibroblasts (GM04281) were obtained from Coriell Institute and used as the cellular context for this mRNA-Seq experiment. Patient-derived HD fibroblast cells were transduced with small RNA expressing lentiviral particles. RNA was harvested by trizol methods and mRNA was isolated by poly-A magnetic bead selection. The sequencing library was prepared by cDNA-PCR methods using an Oxford Nanopore Technologies kit and sequenced on a PromethION 2 Solo (ONT). Sequence analysis was performed using DESeq2 to identify transcriptome changes between mock, shMCHR, and shHD2.4 control samples against shHD33-full mimic, shHD33-full nb, and shHD33-full UAC as HTT allele selective treatments. DESeq2 result tables, expression profile plots and gene counts were generated and used for interpretation of data in R Studio running the DESeq2 analysis program.