394 - A New Knockout Rabbit Model for Sepiapterin Reductase Exhibits Spontaneous Motor Deficits in the Newborn

Poster Number: 394
Publication Number: 394.236

Zhongjie Shi, Wayne State University School of Medicine, Detroit, MI, United States; Kehuan Luo, Children's Hospital of Michigan, Detroit, MI, United States; Charlie Trice, Wayne State University School of Medicine, Detroit, MI, United States; Jie Xu, University of Michigan Medical School, Ann Arbor, MI, United States; Sidhartha Tan, Children's Hospital of Michigan, Wayne State University and Central Michigan University, Detroit, MI, United States

Background: Tetrahydrobiopterin (BH4) is an essential cofactor for many neurotransmitter producing enzymes in the brain. Congenital BH4 deficiency in humans caused by mutations in sepiapterin reductase (SPR) often mimic cerebral palsy. BH4 treatment congenital BH4 deficiency decreases motor deficits in humans. In our rabbit model, preventive BH4 therapy reduces motor deficits following antenatal hypoxia-ischemia.

Objective: Our objective was to develop a SPR knockout (KO) rabbit with help of CRISPR/Cas9 technology, with the overall goal to investigate the role of BH4 in motor development.

Design/Methods: SPR sequences were analyzed, and a single guide RNA (sgRNA) designed and validated. Next, Cas9 mRNA and sgRNA were microinjected to pronuclear stage rabbit embryos, followed by embryo transfer to synchronized recipients. Two founder animals were confirmed germline transmission and produced F1 generation heterozygous SPR KO kits (HTZ). Interbreeding of F1 HTZ KO pairs successfully produced F2 generation HTZ and homozygous SPR KO (HOM) rabbits. A breeding program was implemented. Body weight of newborn kits were documented, and a neurobehavioral battery of tests were evaluated in a blind manner

Results: In kits of normal delivery, there was no difference in weight between WT, HTZ and HOM at P1 indicating that intrauterine growth was normal after SPR KO. There was also no difference in weight gain from P1 to P5 and to P11 (WT n=27, HTZ n=22, HOM n=5). Neurobehavioral tests at P1 showed all WT newborn kits to be normal appearing. In the HTZ group, there was one severe and one mild kit based on dystonia and locomotor scores with an abnormality rate of 2/22= 9%. In the HOM group there were three mildly affected kits, with an incidence rate of 3/5 = 60%. There was one dystonia manifestation in the HTZ and HOM groups each. The only mildly hypertonic kit was in the HOM group. Looking at the individual score of each element of the neurobehavioral battery, there were no differences in between HTZ and WT. However, there were significant differences in eleven of the variables of the neurobehavioral battery (α error < 0.05) and five variables between HOM and WT, using α error ( < 0.0028) with Bonferroni correction for multiple comparisons.Conclusion(s): Establishment of a knockout SPR rabbit model will allow further investigation of the role of BH4 in motor deficits. Even without hypoxia-ischemia, there are spontaneous motor deficits in some HOM kits.
Neurobehavioral differences between HOM (worse) and WT kits, all significant, p < 0.0028).