In vivo Genome Editing for Hereditary Tyrosinemia Type I Using a Compact, Hyper-accurate Cas9 from Neisseria meningitidis

Univ Of Massachusetts Med Sch Worcester, Worcester MA

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Abstract

Project Summary Hereditary tyrosinemia type I (HT-I) is an infantile metabolic disorder caused by loss of function mutations in the gene that makes fumarylacetoacetate hydrolase (FAH), which catalyzes the last step of tyrosine degradation. As a result, toxic intermediates accumulate in liver tissue, leading to severe liver damage. Fortunately, HT-I can be managed with (i) the medication nitisinone (i.e., NTBC, short for its chemical name) to inhibit an early step of tyrosine degradation, (ii) a strict protein diet, and (iii) careful clinical monitoring. However, management of HT-I with NTBC is expensive, restrictive, and lifelong, and NTBC causes side effects or developmental abnormalities in some children. Gene editing approaches that block early steps in tyrosine catabolism or that correct the FAH gene might improve the health and quality of life of children with HT-I by eliminating the need for NTBC or dietary restriction. The goal of this project is to develop efficient, accurate, and safe CRISPR gene editing approaches for HT- I treatment. In this approach, a nuclease (Cas9) and guide RNA (gRNA) target a specific DNA sequence and create a double-stranded break. Imprecise repair of a DNA break typically results in a small insertion or deletion and is used to inactivate a target gene. Precise repair of a DNA break by homologous recombination is typically used to introduce a defined change or to repair a mistake in a target gene. However, available Cas9 nucleases are too large for efficient therapeutic delivery and they frequently cleave off-target sites in the genome, introducing undesirable mutations. This project will take advantage of a recently discovered compact, hyper-accurate Cas9 nuclease derived from Neisseria meningitidis (Nme2Cas9). Nme2Cas9 targeting requires a short sequence motif whose flexible consensus allows for the selection of more target sites than are available to other Cas9 nucleases. The high- fidelity of Nme2Cas9 significantly reduces off-target editing. Nme2Cas9 is small enough that its gene can be co-delivered with gRNA in a convenient all-in-one adeno-associated virus (AAV) vector. Our preliminary studies show that Nme2Cas9 delivered by AAV is functional in vivo. In Aim 1, HT-1 mice will be treated with all-in-one AAV Nme2Cas9 vectors designed to knock out genes (Hpd and Hgd) that act early in the tyrosine degradation pathway and thus block the accumulation of toxic metabolites. In Aim 2, HT-1 mice will be treated with an all-in-one AAV Nme2Cas9 vector that includes a 500- bp DNA donor to correct the Fah gene and thus restore FAH enzyme activity. Vectors will be packaged into AAV8 to target the liver. In both strategies, edited cells gain a growth advantage and repopulate the liver, rescuing the lethal phenotype of HT-1 mice. These studies will advance the utility of CRISPR for in vivo functional genomic studies and inform the future development of one-time Cas9-based therapies to improve the quality of life of children with HT-I.