Exploratory Safety Profile of EDIT-101, a First-in-Human in vivo CRISPR Gene Editing Therapy for CEP290-related Retinal Degeneration

Abstract

Purpose : Leber congenital amaurosis type 10 is a retinal degenerative condition that causes severe visual impairment. To restore vision, we developed EDIT-101, an adeno-associated virus type 5 encoding Staphylococcus aureus Cas9 (SaCas9) expressed under the photoreceptor-specific GRK1 promoter and two guide RNAs designed to excise the disease-causing CEP290 mutation, c.2991+1655A>G (CEP290-IVS26). Safety and tolerability of EDIT-101 is being evaluated in the ongoing BRILLIANCE (NCT03872479) trial, an open-label, Phase 1/2 single ascending dose study enrolling adults and children. Viral shedding in body fluids of EDIT-101 treated subjects is a key exploratory safety endpoint in the trial. To monitor this key safety assessment, a SaCas9-based real-time quantitative polymerase chain reaction (qPCR) assay was developed and validated.

Methods : We developed a qPCR assay to quantify EDIT-101 vector genomes in blood and tears by targeting the SaCas9 transgene. Assay validation was performed to establish linearity, reproducibility, limits of detection, biological matrix effects, and acceptance criteria for EDIT-101 detection in samples collected throughout the BRILLIANCE trial.

Results : The validated assay for blood and tears has a lower and upper limit of quantification (LLOQ & ULOQ) of 25 to 1x10⁷ double stranded EDIT-101 vector genomes, respectively, and a lower limit of detection (LLOD) of 10 vector genomes. No matrix effect or amplification above the LLOD was observed in the absence of EDIT-101. The assay is highly reproducible and specific with an efficiency of 92% to 103% and a R² ≥ 0.998.
EDIT-101 vector genomes were only detected in a small number of clinical samples. The highest levels were observed the day after treatment, then rapidly dropped below the LLOQ. Vector genomes were only identified in tears collected from inside the lower eyelid of the treated, but not the untreated eye. Of the blood and tear samples tested, fewer blood samples contained detectable vector genomes. Levels of vector shed were minimal compared to the initial dose, indicating low risk to the patient or environment.

Conclusions : We developed a SaCas9-specific qPCR assay to evaluate viral shedding in the BRILLIANCE trial. Our data suggests observed EDIT-101 shedding is transient and at low levels of detection, with no risk of systemic viral persistence.

This abstract was presented at the 2022 ARVO Annual Meeting, held in Denver, CO, May 1-4, 2022, and virtually.