Year 1

The long-term objective of this multidisciplinary program is to develop a gene-corrected induced pluripotent stem cell-derived hepatocyte transplantation approach for clinical trials in children to replace the deficiency of single-enzyme defects in urea cycle disorders (UCDs) and other single-enzyme deficiencies that affect the liver. At present, liver transplantation for UCDs replaces a liver that is normal architecturally and in all other aspects except for a single enzyme. It is believed that establishing enzyme function to 10% or less in many of these disorders may result in a cure.
We are performing studies reprogramming of skin fibroblasts from human patients with UCDs into induced pluripotent stem cells followed by a gene addition approach and subsequent differentiation into functional hepatocytes. These human cells will be transplanted into a mouse model with a urea cycle disorder to demonstrate proof-of-principle enzyme replacement, define cell dose to replace enzyme activity to low normal levels, and characterize cell behavior after transplantation. These studies will serve as a preclinical proof of concept for a potential development candidate for a currently unmet need by using corrected and differentiated derivatives of a patient’s induced pluripotent stem cells for neonatal and juvenile hepatic regenerative medicine.
In the first year of this award, we have obtained three human skin samples/fibroblasts from patients with a urea cycle disorder. We have gone through the regulatory process at our institution and completed all approvals related to stem cells. We have been developing induced pluripotent stem cells from the first two lines and also a control line. We require characterization of these stem cells by gene studies and by demonstrating that they can turn into all three germ layers; some of these studies have been completed and others are in process. We have prepared adequate stocks of these fibroblasts and induced pluripotent stem cells for these ongoing studies. We have been developing what we believe will be a safe approach to adding a gene; in this way supplying the correct copy of the abnormal gene to treat the disorder. We are developing this as a universal method of gene addition. That is, one that could be used for all of the urea cycle disorders and for other disorders of the liver such as maple syrup urine disease, alpha1-antitrypsin, and others. Our data suggests that have been able to successfully target this “safe” site for gene integration; further studies are in progress to prove this finding. We have also examined for gene expression of the urea cycle disorder gene that is being corrected in both our gene-corrected control cell line and in our first gene-corrected human patient disease-specific cell line. Both have demonstrated expression of the gene. We are now working to demonstrate that the gene is present at the correct location and that it is functional in that it is producing protein to correct this disorder.