Abstract
Huntington’s disease (HD) is caused by a trinucleotide expansion in exon 1 of the Huntingtin gene, which leads to neuronal dysfunction and eventually cell death. While the full function of the Huntingtin (HTT) protein has not been completely elucidated, it is thought to play a role in vesicle transport, gene transcription, and the regulation of RNA trafficking. This makes the prospect of allele-specific reduction important, as it is not known what the consequences of total knockdown of the Huntingtin protein could be in the long term. Our project aimed to use single nucleotide polymorphisms (SNPs) found in the patient population to reduce the expression of only the mutant HTT with a modified CRISPR system in HD patient-derived cells. We first characterized disease-associated single nucleotide polymorphisms (SNPs) in patient-derived primary cells that could be used as therapeutic target in the HD patient population. We identified heterozygous SNPs near regulatory regions of the Huntingtin promoter in HD patient iPSCs that can be targeted by customized guide RNAs. It was important that these SNPs were heterozygous to discriminate the mutant from the healthy allele. Validation of these targeting SNPs was done through a Kompetitive Allele Specific PCR (KASP) genotyping panel that we designed. This allowed genotyping patient cells in an efficient and cost-effective manner. We identified several previously uncharacterized heterozygous targets, which allowed for the design of allele-specific guide RNAs containing the SNP proximal to the PAM site, which is needed for the Cas9 protein to recognize and bind to DNA. To accomplish this goal we cloned a new Cas9 variant, dxCas(3.7) tethered to repressive effector domains into a novel vector via Gibson cloning (dxiCas9). dxCas(3.7) has been shown to have a broader variety of PAM sites and higher binding specificity, which is necessary to target the mutant-allele associated SNPs in the HTT promoter. These SNPs were previously unable to be targetable as they were not proximal to a canonical NGG PAM site that is recognized by spCas9, making the cloning of this plasmid essential. We created four novel dxiCas9 plasmids that were fused with either no effector, KRAB, DNMT3a, or a combination of DNMT3a and KRAB tethered to the N- and C-terminus of the Cas9 protein, respectively. The various dxiCas and gRNA plasmids were introduced to an HD patient-derived fibroblast line through electroporation as an initial proof-of-concept study. After delivery of our gRNAs and different dxiCas proteins, we performed qRT-PCRs to assess knockdown of total Huntingtin RNA as a screen for gRNA selection to move forward with. Two gRNAs that were targeted to rs3856973 did not show any knockdown. We did see substantial knockdown of total HTT using our gRNA targeted to rs762855 when paired with dxiCas-KRAB. Interestingly, downregulation was not observed when targeting rs762855 when paired with DNMT3a-dxiCas or the DNMT-dxiCas-KRAB fusion. While there is a wide breadth of research being conducted in the HD field, there is still a large unmet therapeutic need. If we are able to complete a sustained knockdown of the HTT protein via an epigenetic mechanism, it could potentially allow for a one-time treatment. This research also looks into the validity of the new dxCas(3.7) as a gene editing technology along with the effectiveness of a dual repressor domain fusion as a means for knockdown. In addition, this work helps to further advance the field of targeted CRISPR/dCas9-mediated gene silencing and allow its application in many other diseases and epigenetic studies.