Establishment and characterization of Lesch-Nyhan syndrome rabbit model.

ABSTRACT

Lesch-Nyhan syndrome (LNS) is a congenital defect disease that results in defective purine metabolism. It is caused by pathogenic variants of the HPRT gene. Its clinical symptoms mainly include high uric acid levels, gout, and kidney stones and damage. The mechanism of LNS has not been fully elucidated, and no cure exists. Animal models have always played an important role in exploring causative mechanisms and new therapies. This study combined CRISPR/Cas9 and microinjection to knock out the HPRT gene to create an LNS rabbit model. A sgRNA targeting exon 3 of HPRT gene was designed. Subsequently, Cas9 mRNA and sgRNA were injected into rabbit zygotes, and injected embryos were transferred to the uterus. The genotype and phenotype of rabbits were analyzed after birth. Four infant rabbits (named R1, R2, R3 and R4), which showed varying levels of gene modification, were born. The gene-editing efficiency was 100%. No wild-type sequences at the target HPRT gene were detected in R4 rabbit. Next, 6-thioguanine drug testing confirmed that HPRT enzymatic activity was deficient in R4 infant rabbit. HE staining revealed kidney abnormalities in all infant rabbits. Overall, an sgRNA capable of knocking out the HPRT gene in rabbits was successfully designed, and HPRT gene-modified rabbits were successfully constructed by using CRISPR/Cas9 technology and microinjection. This study provides a new nonrodent animal model for studying LNS syndrome.