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This overall goal of this pilot investigation of phenylketonuria (PKU) being done in collaboration with Randall S. Prather, PhD, and Kevin D. Wells, PhD, at the University of Missouri, is to develop a model of PKU using CRISPR-Cas9 technology for biochemical and neurobiological characterization and future development of novel therapeutic approaches.
In this project the miniature pig enables us to develop a better, clinically relevant animal model PKU to understand the biomedical bases and to develop therapeutic approaches, especially for mental retardation, neurological, and neuropsychological features. Using bioinformatics and phylogenetic comparison to human, we initially assembled the entire pig Pah gene, encoding a 452 amino acid enzyme, and confirmed high expression of PAH in pig liver and kidney. Furthermore, we have successfully targeted deletions and inversions of the Pah gene using a CRISPR/Cas9 RNA-guided nuclease approach. Our studies over the first eight months of the NPKUA funding period have utilized our in vitro SCH model system and successfully optimized the genome editing reagents and mutation-detection assays for the pig Pah locus. We transfected CRISPR gRNA/Cas9 vectors into two cell lines and used a deletion-PCR assay with primers located in introns 5 and 6 outside the targeted region on genomic DNA. The intensity of the deletion-breakpoint band with each of the four pairs of gRNAs gives an estimate of the relative efficiency of editing, suggesting that the optimal gRNA reagents were the combination of gRNAs 5-1 + 6-2. DNA sequencing confirmed the presence of locus-specific ~ 1,216-bp deletions in both cell types, clearly generated by repair of two DSBs by NHEJ since the breakpoints have minor heterogeneity. In addition, inversion-PCR assays demonstrated the successful detection of specific bands for the two inversion-breakpoints in each cell line transfected with pairs of CRISPR gRNA/Cas9 vectors. Thus, our strategy for genome editing at the pig Pah locus efficiently induces both deletions and inversions at high frequency.
The PKU alleles that we detect in pig models may be deletion or inversion alleles, but in each case exon 6 is lost from the Pah transcriptional unit with the end result being a null mutation with loss of exon 6 as well as a frame-shift after histidine residue #170 and premature termination of protein translation. Working with our Missouri collaborators, the CRISPR gRNAs have been shown to function in vivo in pig pre-implantation embryos and a pregnancy has been obtained from embryo transfers of genome-edited embryos (~35 percent modified alleles). Once we identify minipigs having the PKU genotype, these can be used for breeding and to characterize the biochemical and neurobiological phenotype of pigs with PKU. A porcine PKU-model will allow an understanding of the pathophysiology of PKU, and in the future also for maternal PKU syndrome (MPKUS), including mental retardation, abnormal behavior and neurological features, and will provide a clinically accurate animal model for therapeutic testing for PKU.
Investigators have submitted a competitive renewal grant application to National PKU Alliance for a further year of funding, in which the goal is to perform initial biochemical and neurobiological characterization of a minipig model of PKU developed using CRISPR-Cas9 technology, as well as a pilot trial of hepatocyte transplantation to rescue the disease phenotype.
The team also intends to apply for a for a three-year National Institutes of Health research grant for a collaborative project in which the goal is to characterize minipig models of PKU and MPKUS produced using CRISPR-Cas9 technology, for biochemical and neurobiological phenotypes, to identify biological and therapeutic markers, and to initiate therapeutic approaches assessing diet and for future testing of enzyme replacement or gene therapy.
National PKU Alliance
Robert D. Nicholls, PhD
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