Sequencing-Guided Design of Genetically Encoded Small RNAs Targeting CAG Repeats for Selective Inhibition of Mutant Huntingtin [I]. Sequencing-Guided Design of Genetically Encoded Small RNAs Targeting CAG Repeats for Selective Inhibition of Mutant Huntingtin [I]

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: To identify processing of designed repeat shRNA/miRNAs in mammalian cell tissues, HEK 293T mammalian cells were transfected with repeat shRNA/miRNA encoding lentiviral plasmids. Sequence analysis was performed using pre-existing RNA seq analysis methods as well as in house excel spreadsheets for reconstructing read constructs. ImageJ software was used to evaluate transfection efficiency between transfected samples in order to normalize read abundance levels. Expression profiling using sRNAtoolbox and further analysis was used to evaluate transfected small RNAs and endogenous miRNA expression levels.