EPCO-23. MOLECULAR CHARACTERIZATION OF CRISPROFF EPIGENETIC EDITOR ACTIVITY REVEALS MUTANT ALLELE-SPECIFIC <i>TERT</i> REPRESSION IN GLIOBLASTOMA

Abstract Telomerase reactivation via mutations in the telomerase reverse transcriptase promoter (TERTp) is a recurrent feature of glioblastoma, enabling replicative immortality in ~80% of patient cases and presents a critical oncogenic event attractive for therapeutic targeting. TERTp hotspot mutations in glioblastoma generate de novo transcription factor binding sites that recruit the transcription factor GABP, driving aberrant TERT expression. CRISPRoff is an engineered CRISPR/Cas9 fusion protein that can potently and heritably silence cis-regulatory elements such as promoters and enhancers through combined action of DNA and histone methylation. Here, we use CRISPRoff to perform targeted silencing of the TERTp in three patient-derived primary glioblastoma models harboring TERTp mutations. We delivered mRNA encoding CRISPRoff and TERTp-targeting sgRNAs using optimized lipid nanoparticles (LNPs) into glioblastoma primary cultures and organoids. Epigenetic and transcriptional phenotypes were profiled using RT-qPCR, RNA-seq, targeted bisulfite DNA sequencing, long read native DNA sequencing using Oxford Nanopore, ChIP-qPCR, and CUT&amp;TAG. A single transient CRISPRoff delivery was sufficient to stably silence TERT expression by &amp;gt;95% in primary glioblastoma cultures, leading to induction of apoptosis over time and complete replicative senescence in clonal populations. The CpG island encompassing TERTp exhibited ~5-fold increases in cytosine DNA methylation and long read Nanopore sequencing revealed that increased 5mC DNA methylation was specific to the mutant allele of TERTp. Analysis of 5hmC revealed enrichment of this modification upstream of the -124C&amp;gt;T promoter mutation, which was lost upon CRISPRoff activity. ChIP-qPCR revealed a 68% decrease in GABPA occupancy at the TERT promoter. Analysis of H3K9me3 enrichment revealed deposition of heterochromatin within the CpG island of TERTp, and this enrichment was among the most statistically significant increased H3K9me3 peaks genome-wide. In sum, the molecular characterization of CRISPRoff activity against TERTp revealed the ability to achieve mutant allele-specific reprogramming of epigenetic state that could be harnessed for epigenetic therapeutics.