Genetically blocking HPD via CRISPR-Cas9 protects against lethal liver injury in a pig model of tyrosinemia type I

Abstract

Hereditary tyrosinemia type I (HT1) results from the loss of fumarylacetoacetate hydrolase (FAH) activity and can lead to lethal liver injury (LLI). Therapeutic options for HT1 remain limited. The FAH À/À pig, a well-characterized animal model of HT1, represents a promising candidate for testing novel therapeutic approaches to treat this condition. Here, we report an improved single-step method to establish a biallelic (FAH À/À ) mutant porcine model using CRISPR-Cas9 and cytoplasmic microinjection. We also tested the feasibility of rescuing HT1 pigs through inactivating the 4-hydroxyphenylpyruvic acid dioxygenase (HPD) gene, which functions upstream of the pathogenic pathway, rather than by directly correcting the diseasecausing gene as occurs with traditional gene therapy. Direct intracytoplasmic delivery of CRISPR-Cas9 targeting HPD before intrauterine death reprogrammed the tyrosine metabolism pathway and protected pigs against FAH deficiency-induced LLI. Characterization of the F1 generation revealed consistent liver-protective features that were germline transmissible. Furthermore, HPD ablation ameliorated oxidative stress and inflammatory responses and restored the gene profile relating to liver metabolism homeostasis. Collectively, this study not only provided a novel large animal model for exploring the pathogenesis of HT1, but also demonstrated that CRISPR-Cas9-mediated HPD ablation alleviated LLI in HT1 pigs and represents a potential therapeutic option for the treatment of HT1.

Integrated analyses reveal evolutionarily conserved and specific injury response genes in dorsal root ganglion

“…Cldn4 belonging to the tight junction protein, has been shown to protect against acute lung injury 61 . We found some genes related with metabolism that showed rat-preference, including iron homeostasis ( Hamp , Rasd1 / Dexras1 ), tetrahydrobiopterin production ( Spr , Dhfr ) 49 , tyrosine ( Hpd 62 ), tryptophan ( Tph2 ), and calcium homeostasis ( Atp2b4 /PMCA4 56 ). Hamp , induced by inflammation stimulus, has been validated up-regulation in rat DRG upon ScNI and could be transported into regenerating axons upon ScNI and effect neuroprotective by reducing iron in rat primary cortical neuron against hemin and iron-mediated neurotoxicity 50 , 63 .…”

In vivodissection of the mouse tyrosine catabolic pathway with CRISPR-Cas9 identifies modifier genes affecting hereditary tyrosinemia type 1

“…For example, while Fah -/- Hpd -/- and Fah -/- Hgd -/- animals survived without NTBC, Fah -/- Gstz1 -/- mice displayed an even more severe phenotype than Fah -/- mice 30, 37, 38 ( Figure 1A ). Accordingly, both short hairpin RNA (shRNA)-mediated knockdown and CRISPR-Cas9- directed inactivation of Hpd in the liver of Fah -/- animals enabled the survival after NTBC withdrawal, suggesting that metabolically blocking the pathway in the liver is sufficient for a systemic therapeutic effect 39–42 . Still, the impact of liver-specific inactivation of Hgd and Gstz1 via in vivo genome editing in the HT-I mouse model has yet to be described.…”

Leverage of nuclease-deficient CasX for preventing pathological angiogenesis

“… 1 This CRISPR technology originated from a natural bacterial defense mechanism against phage infection and plasmid transfer 2 and is capable of manipulating nearly any genomic sequence specified by a small RNA-guided Cas to work, 2 including correction of disease-causing mutations. 3 , 4 , 5 , 6 CRISPR/Cas-mediated genome engineering is expected to become an important tool for the treatment or cure of a variety of human diseases, including but not limited to tumors, neurodegenerative diseases, sickle cell anemia, hereditary diseases, viral infection, immune system disease, and vascular disease. 7 , 8 , 9 , 10 , 11 , 12 , 13 , 14 …”