Using the CRISPR-Cas9 gene-editing tool, researchers created a pig model of phenylketonuria (PKU) that better mimics the disorder’s signs and symptoms in humans, compared with classic mouse models, a study reported.
This new pig model will allow researchers to further investigate PKU’s physical impact on people with the rare disease. It also will support a more accurate assessment of new PKU therapies, researchers said.
The study, “A porcine model of phenylketonuria generated by CRISPR/Cas9 genome editing,” was published in the journal JCI Insight.
The classic preclinical animal model for PKU therapeutic development is the Pahenu2 mouse. It carries a mutation in the PAH gene, leading to elevated phenylalanine levels and low levels of tyrosine, as seen in people with the condition.
However, despite increased phenylalanine levels, ranging from above-normal to high, many of the neurological features observed in humans are not found in these mice. Indeed, these mice display only moderately impaired behavior. The limitations are most likely due to developmental and anatomical differences between the mouse and human brain. But they restrict the model’s usefulness.
Furthermore, in advance of clinical trials testing new PKU treatments, the U.S. Food and Drug Administration has asked that neurocognitive benefit be demonstrated, not just phenylalanine reduction. As such, there is an urgent need for a PKU animal model that better mimics the human disease.
Now, researchers based at the University of Pittsburgh School of Medicine used CRISPR-Cas9 gene-editing technology to create a PKU model in minipigs (Sus scrofa). These minipigs have a body and brain size, anatomy, physiology, metabolism, and genome sequence that more closely resembles humans than those of mice.
Minipigs are widely used for pharmaceutical and preclinical toxicity studies as well as for behavioral and MRI studies.
To generate PKU pigs, CRISPR-Cas9 reagents with instructions for creating deletions in the PAH gene were injected into pig zygotes. These zygotes are at the earliest stage of development following fertilization, before embryo formation.
After allowing them to grow for a few days, these embryos were tested for PAH mutations and transferred into a surrogate female pig (sow). The sow gave birth to a single founding litter of PKU swine. One pig was a full PKU model, while one other was only a carrier of the mutations.
At five days of age, the PKU pig had excess blood levels of phenylalanine and a phenylalanine-to-tyrosine ratio of 10.5, consistent with classical PKU. Phenylalanine metabolites also were found in excess in the urine. In contrast, the carrier PKU pig had normal phenylalanine and tyrosine levels and very little urinary metabolites.
The PKU pig had lighter patches of skin (hypopigmentation), which is common in untreated PKU patients. Compared with the PKU carrier, the early growth of the PKU pig was impaired due to chronic phenylalanine toxicity during pre- and postnatal development.
Giving the PKU pig a phenylalanine-restricted diet, as would be done in human PKU patients, cut its phenylalanine levels in half after four days. Those levels kept decreasing until reaching therapeutic levels by day 14. When phenylalanine was added back into the diet, phenylalanine levels increased.
Brain MRI scans performed at seven months of age showed that the PKU pig had reduced volumes in various regions of the brain, including the grey and white matter. Additionally, the PKU pig showed a volume reduction in the cerebellum — the brain region responsible for coordinating movements — and an increased volume in brain cavities known as ventricles, consistent with a loss of brain tissue.
Despite very high levels of phenylalanine and changes in brain composition, the PKU pig showed no apparent signs of neurological impairment. It responded to external stimulation, was able to walk, and did not experience seizures. Cognitively, the affected pig was the same as the PKU carrier, and there were no noticeable differences during phenylalanine-restricted dietary treatment.
These results were an “unexpected finding because the prolonged exposure of extreme [high phenylalanine] associated with classical PKU results in debilitating neurological and neurobehavioral clinical phenotypes,” the researchers wrote.
Although CRISPR/Cas9 genome editing is highly specific, there may be off-target editing of genes with similar DNA sequences, the researchers said. While several single mutations were detected, there was no sign of genome editing across predicted off-target sites.
Finally, whole-genome sequencing confirmed that targeting the PAH gene in these pigs was both efficient and accurate.
The researchers plan to conduct a post-mortem (after death) analysis on the PKU pig compared to the carrier littermate, as well as long-term MRI, neurological, behavioral, and cognitive studies on early treated and untreated animals from future generations.
“In conclusion, we have established a large-animal preclinical model of PKU to investigate pathophysiology and to assess new therapeutic interventions,” the investigators wrote.