PROJECT SUMMARY A heterozygous hexanucleotide (GGGGCC) repeat expansion in a single allele of the C9orf72 gene is the most frequent known genetic cause of frontotemporal dementia (FTD) and amyotrophic lateral sclerosis (ALS), two fatal and irreversible neurodegenerative diseases. Given that there are no effective treatments for FTD (an Alzheimer’s-related dementia), novel therapeutic strategies are urgently needed. Targeting the C9orf72 gene itself by CRISPR/Cas9 genome editing may provide a curative intervention. We have established a novel dual sgRNA strategy, which can excise the C9orf72 repeat region while preserving all exons in patient-derived induced pluripotent stem cells (iPSCs). The excision eliminated pathological hallmarks of C9-FTD and preserved normal C9orf72 protein levels in neurons derived from these iPSCs. Given sequence conservation in this region, this editing strategy applies to all patients, regardless of genetic background or mutation size. We have also engineered a novel silica nanocapsule (SNC) platform that can cross the blood-brain barrier (BBB) after intravenous (IV) injection and deliver CRISPR genome editors to the whole brain of healthy animals with intact BBBs. The SNCs possess many desirable properties including the ability to deliver diverse biologics, high delivery efficiency, versatile surface chemistry for ligand conjugation, small particle sizes, excellent in vivo stability, good biocompatibility, and scalable production. Combining our unique C9orf72 editing approach with our innovative SNC capable of brain-wide systemic delivery of genome editors, we aim to develop a safe and efficiency genome editing therapy to treat C9-FTD. In Aim 1, we will maximize efficiency of our gRNA strategy by identifying sgRNA pairs that excise the C9orf72 repeat region with high efficiency and no side effects in post-mitotic patient-derived neurons in vitro. In Aim 2, we will optimize the SNC formulation and the injection regimen for maximal delivery to the brain in Ai14 reporter and C9-FTD mouse models, maximizing delivery efficacy. In Aim 3, we will determine the long-term biosafety and therapeutic efficacy of excising the C9orf72 repeat region via IV delivery of SNC in the C9-FTD mouse model. With the successful completion of the proposed IND-enabling studies, we will have optimized a candidate therapeutic that targets the C9orf72 mutation, and reaches the highest therapeutic efficacy in human neurons in vitro and in a C9-FTD mouse model in vivo. The optimal therapeutic will then be ready for scale-up, manufacturing/CMC development and IND-enabling safety studies in nonhuman primate and rat. We will have additionally addressed a number of open questions in the field, including whether editing efficiencies in post- mitotic neurons differ from mitotic cells, how to deliver CRISPR/Cas9 with multiple sgRNAs widely throughout the mouse brain and whether it is possible to reverse or arrest clinical phenotypes in symptomatic mice. Our goal is to accelerate genome editing for neurodegenerative diseases toward the clinic.

PROJECT NARRATIVE The Alzheimer’s-related disease frontotemporal dementia (FTD) and amyotrophic lateral sclerosis (ALS) are two fatal and incurable neurodegenerative diseases linked by a shared genetic cause – a heterozygous hexanucleotide (GGGGCC) repeat expansion in a single allele of the C9orf72 gene. The goal of this work is to develop our novel CRSIPR genome editing platform to remove the C9orf72 mutant repeat region in human cells and mice. Our platform combines a novel dual gRNA approach to editing the mutant C9orf72 gene that preserves normal gene function paired with a first-in-class engineered silica nanocapsule (SNC) that can cross the blood brain barrier and widely distribute throughout the brain. We will evaluate our strategy to remove the repeat expansion in patient-derived induced neurons, determine optimal dosage of SNCs for highest editing rates in mouse brain, and determine the therapeutic effect and biosafety of our platform in the mouse model of C9-FTD.