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

Funding Type: 
Early Translational IV
Grant Number: 
Award Value: 
Disease Focus: 
Liver Disease
Genetic Disorder
Stem Cell Use: 
iPS Cell
Cell Line Generation: 
iPS Cell
Public Abstract: 

Liver transplantation (LT) has been used to treat a variety of liver diseases. Within hours after birth, neonates can present with disorders of the urea cycle (UCDs), the critical metabolic liver pathway needed to detoxify waste nitrogen from the diet and cellular turnover. The overall incidence of UCDs is estimated to be 1 in 8200 births. An increase in ammonia concentrations is particularly toxic to the central nervous system (CNS), causing brain edema, with multiple episodes affecting survival and often resulting in mental retardation and cerebral palsy. While LT is recommended in neonatal-onset patients with these single-enzyme defects, organ availability is a major limitation and transplantation requires lifelong immunosuppression. In addition, transplant morbidity and mortality are not inconsequential: 1-year survival is about 91.9%, and 5-year survival in children transplanted at less than 5 kg is 74%. Transplantation of genetically corrected embryonic stem cell-derived hepatocytes from the affected patients themselves is a potential way to replace LT for the treatment of metabolic liver disease and will likely not require immunosuppression; importantly, the supply will be limitless, allowing early transplantation before CNS injury. We propose to explore this approach by using a new and robust liver repopulation UCD mouse to advance this therapy: treatment of single-enzyme liver defects with patient-derived and genetically corrected stem cell-induced liver cells.

Statement of Benefit to California: 

Unfortunately there is a substantial wait for people in California who need a liver transplant, resulting in many who develop significant disability or die while waiting. While many are adults with chronic disease, some are children with metabolic disorders including urea cycle disorders (UCDs).

UCDs are caused by mutations resulting in enzyme deficiencies responsible for removing waste nitrogen, which as ammonia can cause irreversible brain damage, coma and/or death. Newborns can become catastrophically ill within 36-48 hours after birth. These and other inborn errors represent a substantial cause of brain damage and death among newborns and infants and because many cases remain undiagnosed, or infants with the disorders die without a definitive diagnosis, the exact incidence is unknown and likely underestimated.

Present treatment is dietary for most which is onerous & incomplete; definitive therapy is liver transplantation which is challenging in infants who have greater rates of complications and morbidity. In this proposal we will develop genetically-corrected hepatocyte-like cells from induced pluripotent stem cells from patients with arginase deficiency, a UCD. These will be tested for the ability to correct the disorder in a unique UCD liver repopulation animal model. The advantage of the proposed methodology over current therapy is that genetically-corrected cells will be limitless and will require no major surgery or immunosuppression and its short- & long-term risks.

Progress Report: 

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.