RNA-Targeted Gene Therapy for Huntington’s Disease Using Single Nucleotide Polymorphisms

Huntington’s Disease (HD) is an autosomal dominant neurodegenerative disorder caused by a CAG repeat expansion in exon 1 of the Huntingtin (HTT) gene. Expanded CAG repeats lead to a larger polyglutamine tract in the mutant Huntingtin (mHTT) protein, which forms aggregates in the brain and contributes to overall disease progression. Lowering overall Huntingtin protein levels can lower the mHTT protein and provide a therapeutic benefit for HD patients, but may also affect the normal HTT (wtHTT) protein, whose loss is not well understood and may lead to unintended long-term consequences. Thus, a targeted approach that leaves wtHTT protein unaffected is considered to be the ideal strategy to treat HD. Although several therapies have been developed to achieve this goal, they have failed in clinical trials for various reasons. Here, we evaluate the use of Artificial Site-specific Endonucleases (ASREs) to target mHTT RNA in order to develop a novel targeted gene therapy for HD. ASREs consist of two functional domains: a) a programmable RNA-binding Pumilio-F (PUF) domain; and b) an RNA endonuclease (PIN), which cleaves the RNA at the site of PUF binding. Previous research has shown that ASREs can be designed to bind and destroy targeted RNA transcripts by recognizing specific 8-ribonucleotide sequences. Extensive genomic analysis has revealed that mHTT alleles are frequently linked with single nucleotide polymorphisms (SNPs), providing unique targets for allele-selective therapies. We examined the hypothesis that SNP-specific ASREs can be designed to selectively target mHTT transcripts for degradation. However, the PUF domain’s ability to distinguish transcripts based on a single nucleotide difference has not been evaluated in mammalian cells. To assess binding ability of the PUF domain to its target RNA, we optimized RNA Immunoprecipitation (RIP) and qPCR assays by successfully detecting RNA bound to an endogenous protein. These results allow for future characterization of the SNP-targeting capabilities of ASRE technology and will help validate its application in developing a novel protein-based RNA-targeted therapy for HD.