Gene therapy-corrected autologous hepatocyte-like cells from induced pluripotent stem cells for the treatment of pediatric single enzyme disorders

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.

The long-term objective of this multidisciplinary program is to develop a gene-corrected stem cell-derived liver cell 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 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 cells from patients with UCDs into stem cells followed by adding a corrected copy of the gene and then developing these cells into functional liver cells or hepatocytes. These human cells will then be transplanted into a mice that have 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 an affected patient's induced pluripotent stem cells for neonatal and juvenile liver cell treatment. In the first and second years 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 three cell lines and also a control or normal cell line. We have characterized of these stem cells by gene studies, determined their gene mutations, and have demonstrated that they can turn into all three germ layers when injected into immune-deficient mice. We have prepared adequate stocks of these fibroblasts and induced pluripotent stem cells and stored them 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 to try to avoid activating any gene that may lead to cancer in these cells ,and inserting the gene at a site that could be used for all of the urea cycle disorders and for other disorders of the liver such as maple syrup urine disease and alpha1-antitrypsin. We have been able to shown by looking at the DNA data that the gene is inserted at the exact location that we have chosen, thus successfully targeting this "safe" site for gene integration. We have also ben examining the level of for gene expression of the urea cycle disorder gene that is being corrected in our gene-corrected human patient disease-specific cell lines. We have demonstrated expression of the gene which is as high as 40% of the level of expression in fetal liver cells. We have begun interventions on the mouse model, first by delivering injections to ensure that the mice can tolerate procedures under anesthesia and not have complications afterwards. These procedures are now established such that testing will soon begin of different doses of cells to the mouse model of this urea cycle disorder.