ASHG Abstract Presented by Camelle Catamura, University of California, Berkeley.
The following is a significant abstract released on Friday, November 3, during the Annual American Association of Human Genetics meeting (ASHG 2023), being held in Washington, DC, November 1-5.
In the ASHG abstract, Catamura and her colleagues wrote the following:
“Repeat expansion diseases that follow a dominant negative inheritance pattern, such as Huntington’s disease, present a great challenge for gene therapies. The most appealing therapeutic strategy requires allele-specific silencing of the disease allele while leaving the wild-type allele intact. Because some amount of repeat sequence is present in both alleles, selective CRISPR targeting of the mutant allele is profoundly challenging.”
“Here, we present a general framework for allele-specific suppression of repeat expansion genes and show its efficacy in suppressing the gene responsible for Huntington’s disease, HTT. Our solution is to engineer a poison exon into only the disease allele: an alternative exon that, when included in an mRNA, will introduce an early stop codon and trigger nonsense-mediated mRNA decay (NMD). mRNA expressed from the edited disease allele would be degraded, while mRNA from the wild-type allele would be unchanged.”
“To target only the disease allele, we take advantage of benign, heterozygous SNPs within introns of the disease gene. CRISPR guides specific to the SNP allele that is in phase with the pathogenic repeat could recruit an RNA-guided deaminase base editor to introduce nucleotide substitutions within a narrow window on only the disease allele.”
“Using a deep learning model of splicing based on thousands of bases of sequence context, we screen in silico for positions where targeting a base editor could create sequences that form new splice sites. Importantly, the novel exon need not be near the actual disease mutation; any highly heterozygous SNP in an intron could potentially serve this purpose.”
“We applied this model to screen SNPs within introns of HTT and found positions where editing adjacent to the SNP was predicted to give rise to new poison exons. We validated these targets at the endogenous HTT locus in human cell culture, showing that the edits created new exons spliced into HTT mRNAs with over 75% inclusion. The resulting mRNAs were degraded by NMD, substantially reducing HTT expression.”
“We conclude that editing of just a few carefully chosen nucleotides in a gene can be sufficient to create a new exon, making it a feasible approach for allele-specific mRNA depletion. Our approach combines the power of CRISPR editing with deep learning models of RNA sequence to engineer poison exons with therapeutic potential for a disease that, at present, has no viable treatments.”