CAG-targeting artificial miRNA with reduced off-target risk for efficient lowering of pathogenic polyglutamine proteins.

Huntington's disease (HD) is the best-known example of a neurodegenerative disorder caused by the expansion of a glutamine-encoding CAG repeat in the causative gene. Growing evidence indicates that somatic CAG expansions play a key role in disease progression, providing a strong rationale for therapeutic strategies directly targeting the repeat tract. However, achieving sufficient efficacy while maintaining allele selectivity and minimizing off-target effects remains a major challenge. Here, we developed allele-selective, CAG-targeting artificial microRNA (amiRNA) molecules that exhibit significantly reduced off-target risk. This was achieved by introducing specific substitutions at selected positions within the guide strand. These molecules effectively downregulated polyglutamine (polyQ) proteins in cellular models of HD, spinocerebellar ataxias types 1 and 3, and dentatorubral pallidoluysian atrophy. The most promising candidate, amiR136-13A, reduced mutant huntingtin levels in different brain regions of the HD mouse model and did not induce toxicity up to 28 weeks following a single administration of an AAV5 vector. Transcriptomic profiling of human HD neural stem cells treated with amiR136-13A revealed minor changes in gene expression. Moreover, amiR136-13A reduced the level of HTT1a, a short pathogenic isoform of huntingtin. Collectively, these findings identify amiR136-13A as a potent, selective, and safe therapeutic candidate for HD and potentially other polyQ disorders.