PROJECT SUMMARY Inborn errors of metabolism (IEMs) are rare, devasting disorders arising from pathogenic variants in genes encoding enzymes of key biochemical pathways. The liver plays an important role in the pathogenesis of over 150 IEMs, often failing to metabolize a toxic metabolite that can injure secondary organs, such as brain. Liver transplantation is employed in some IEMs; however, its utility is limited by scarcity of donors and lifelong risk of post-transplant complications. To address the unmet medical need of IEM patients, we aim to develop a master protocol for the rapid development of personalized prime-editing therapies for severe, rare hepatic IEMs. We envision a platform regulatory framework where IND-enabling studies for a “leader” hepatic IEM prime-editing therapy also support programs for varied “follower” indications. The leader and follower therapies will differ only in patient-variant-specific guide RNA sequences. For our leader indication, we have selected phenylketonuria (PKU). In PKU, an autosomal recessive disorder, impaired phenylalanine (Phe) catabolism in the liver induces neurotoxic Phe accumulation. In our proof-of-concept studies, a single dose of an AAV8-based, R408W prime- editing therapy completely and durably normalized Phe levels in humanized PKU R408W mice. We will leverage our PKU R408W IND-enabling studies to accelerate development for our follower indications: ultra-rare, severe, neonatal urea cycle disorders (UCDs) and organic acidemias (OAs) that affect ≈1:20,000 births. Neonates with UCDs or OAs present with severe toxic accumulation of ammonia and/or organic acids. While liver transplantation can improve metabolic control, patients must grow to an appropriate size to receive a transplant. During this wait, most patients experience irreversible neurologic damage and episodes of life- threatening metabolic decompensation. We seek to develop just-in-time, personalized, liver-directed prime- editing therapies addressing the pathogenic variants identified through newborn screening at our large referral center. A platform regulatory approach is essential to develop therapies in time to meaningfully improve outcomes in this patient cohort who typically suffer significant early morbidity and mortality. Our primary objectives are to (1) establish a therapeutic platform, comprising a single prime editor and AAV delivery system, for numerous hepatic IEMs, and (2) streamline approvals through regulatory innovation, informed by stakeholder input and disseminated to the scientific community.

PROJECT NARRATIVE Inherited disorders of metabolism, including phenylketonuria (PKU), urea cycle disorders (UCDs), and organic acidemias (OAs), often cause debilitating and even life-threatening medical problems as early as birth. We seek to establish an unprecedented platform to efficiently create personalized gene-editing therapies for patients with these diseases, using a CRISPR technology called prime editing to directly and permanently correct each patient’s disease-causing gene variant in the liver. This “one-and-done” treatment strategy could substantially improve the long-term health and survival of these patients who currently have severely limited treatment options.