ALLELE-SELECTIVE RNA-FOCUSED THERAPEUTIC APPROACHES FOR REPEAT EXPANSION DISORDERS

Author

Mansi Anil ParasrampuriaFollow

Date of Award

12-1-2024

Degree Name

Doctor of Philosophy

Department

Molecular Biology, Microbiology and Biochemistry

First Advisor

Gagnon, Keith T.

Abstract

Tandem repeats, or repeat expansion diseases (RED), are polymorphic nucleotide sequences distributed throughout the human genome. More than fifty neurological disorders are attributed to tandem repeats, which are an essential cause of human neurological disorders. These short repeat expansions come in various sizes, from dinucleotides to longer, and can be found in coding and non-coding sequences of different genes, resulting in diverse groups of diseases. The general mechanism of the tandem repeat expansion disease pathologies associated with bidirectional transcription, intranuclear RNA foci, and repeat-associated non-AUG (RAN) translation are main highlights for the introduction chapter. Our primary focus on REDs is on molecular mechanisms driving disease pathogenesis, which vary depending on repeat size, location, and the phenomena of clinical anticipation. Additionally, we discuss the emerging therapeutic approaches for several known repeat expansion diseases and their potential clinical applications. Throughout the chapter, our primary focus is on the molecular mechanisms and RNA biology of mainly two REDs, Huntington's disease and C9ORF72-associated Frontotemporal dementia and Amyotrophic lateral Sclerosis. In the later chapters, we study the allele-selective potential RNA-centric therapeutic approaches for the mutant HTT gene in Huntington’s disease and the mutant C9ORF72 gene for FTD/ALS.

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 the 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.

The following two chapters describe the two potential RNA-focused therapeutics for C9FTD/ALS, an incurable neurological disorder. Amyotrophic Lateral Sclerosis (ALS) is a progressive neurological disease characterized by degeneration of motor neurons in the brain and spinal cord. The leading genetic cause of ALS is the hexanucleotide repeat expansion in chromosome 9 open reading frame 72 (C9ORF72). The molecular pathology cascade is primarily driven by the production of repeat expansion RNAs from that locus, which are subsequently translated into poly-dipeptide repeat (poly-DPR) proteins. Gaps in the underlying mechanism persist, which can aid in identifying promising therapeutic approaches. Selective and safe reduction of transcription across the mutant C9ORF72 locus could reduce repeat RNA and poly-DPR production and potentially offer a therapeutic window. Our first RNA focused approach is the low or moderate inhibition of guanosine triphosphate (GTP) and cytidine triphosphate (CTP) synthesis via targeting purine and pyrimidine pathways can serve as a treatment for C9ORF72-mediated amyotrophic lateral sclerosis (C9-FTD/ALS) by achieving the safe and selective reduction of the (G4C2)n repeat expansion RNA transcription. Pharmacological or genetic inhibition of enzymes involved in GTP and CTP biosynthesis represent successful antiviral therapies and treatments for organ transplant rejection. Our in-vitro screen gave us some potential small molecule inhibitors with specific doses that induce low toxicity on expanded and non-expanded reporter cells. We demonstrated that small molecule inhibitors targeting GTP and CTP depletion by targeting purine and pyrimidine biosynthesis reduced both sense and antisense repeat expansion RNA, and selectively preserved C9ORF72 protein at low and safe doses in patient-derived neural stem cells. We also genetically depleted the enzymes involved in the GTP and CTP biosynthesis pathway, which selectively reduced G4C2 repeat expansion RNA expression in our cell-based experiments, strengthening our proof of concept. Taken together, the findings provide a novel approach that ensures safety in assessing its efficacy and molecular mechanisms of action in cellular models of C9FTD/ALS.

In the second RNA focused approach, we propose utilizing CRISPRoff, a highly precise and effective CRISPR-based dCas9-methyltransferase fusion system, to target the methylation of the C9ORF72 promoter and repeat expansion sequence. CRISPRoff enables precise CpG methylation for targeted and long-term gene silencing with minimal off-target effects and does not induce DNA cleavage. We investigated the impact of CRISPRoff in C9FTD/ALS models and patient-derived induced pluripotent stem cells (iPSCs). Our results demonstrated that transiently transfection with CRISPRoff and specific sgRNAs targeting the TetON promoter and CCCCGG repeat expansion in model cells led to allele-selective methylation, substantially reducing mCherry reporter expression and poly-(GP) in expanded poly-(GP)88-mCherry reporter cells. Additionally, in patient-iPSCs, transient targeting of the C9ORF72 promoter and repeat expansion resulted in a notable and significant decrease in repeat RNA. Nanopore sequencing confirmed the increased methylation at the repeat expansion sequences for reporter and patient-iPSCs cells.

These findings suggest that multiple methylation events are occurring, and inducing silencing and targeting the repeat sequence could be selectively applied to the expanded allele. Given the relative rarity of G4C2 repeat tracts in the human genome, CRISPRoff offers a promising safety profile for C9FTD/ALS therapy. This two study not only provides a novel therapeutic approach for slowing down the progression of C9FTD/ALS but also highlights the potential of targeting nucleoside/nucleotide depletion and using CRISPRoff for epigenetic regulation of repeat expansion loci in multiple repeat expansion disorders. Taken all together, allele-selective RNA focused therapeutics provides therapeutic approaches for Huntington and C9FTD/ALS disease.