Funding opportunities

Developmental candidate: hESC derived therapies for neonatal urea cycle defects

Funding Type: 
Early Translational I
Grant Number: 
TR1-01217
Funds requested: 
$3 010 555
Funding Recommendations: 
Not recommended
Grant approved: 
No
Public Abstract: 
For severe liver diseases, transplantation is the only effective therapy. In California 750-800 liver transplants are performed annually, saving many lives but at a staggering cost: $300,000 for the first year plus $30,000 per patient per year, totaling over $440 million every year. Liver transplantation is limited by the supply of donor organs, especially livers suited to pediatric transplant. Urea cycle disorders (UCD) are severe diseases of newborns in which ammonia is not correctly metabolized in the liver. Ammonia accumulation rapidly leads to nerve damage and often death. Liver transplantation is currently the only cure. Because the livers of UCD patients are normal in all other functions, thus stem cell therapy could be a successful, minimally invasive, UCD treatment. There recently has been importance progress in developing stem cell therapy for cellular therapies for liver disease. Stem cell therapy for liver disease can be viewed as a platform technology. When the basic strategy fro engrafting new cells in the liver is achieved in a defined group of high-risk UCD patients, the resulting understanding would pave the way for much broader applications. We propose to generate mouse models of UCD from immunocompromised mice and use these disease models to demonstrate that human embryonic stem cells, developed in the lab into committed liver progenitor cells, can engraft into intact livers and function as liver cells. Implantation of committed progenitor cells will greatly reduce the risk of cancer (teratoma formation). To minimize transplant rejection we will build a bank of progenitor cells from different human embryonic stem cells lines developed by members of our team at [REDACTED], and assess tissue matching with normal human lymphocytes. We will use advanced imaging, developed by our team, to noninvasively track the engraftment of the human progenitor cells into the mouse liver. If embryonic stem cell-based cell therapies perform as anticipated in these dire diseases, they could be directed toward many conditions not necessarily perceived as liver disease. Hemophilia A and B are two examples of genetic diseases stemming from defective proteins produced by the liver that greatly affect quality of life. The liver is the site of LDL processing and a genetic mutation in the LDL receptor results in familial hypercholesterolemia, a disorder leading to early heart disease in 1 of 600 individuals. Repopulating patients’ diseased livers with stem cells carrying normal genes could cure these diseases. In the future, modifying the genes of stem cells before engrafting them into the liver could treat an even broader spectrum of liver disease, including hepatitis C. Furthermore, the technologies developed for monitoring the relevant events following stem cell implant in patient models could direct development of additional stem cell therapies.
Statement of Benefit to California: 
Our plan addresses important unmet medical needs for liver replacement surgery with new research to develop stem cell therapy that will be rapidly translated into patient treatment. For many severe liver diseases, transplantation is the only effective therapy. In California 750-800 liver transplants are performed annually-a great advance saving many lives but at a staggering cost: $300,000 for the first year plus $30,000 per patient each year for medications, totaling over $440 million every year. Liver transplantation is, however, limited by the supply of donor organs. Of the 20,000 patients awaiting surgery approximately 3,000 will die before a suitable liver becomes available. Other patients will not be viable candidates for transplant surgery. Long-term immunosuppression exacerbates other diseases and reduces life expectancy. This is especially true for pediatric liver transplant recipients, in which normal development is affected and long-term morbidities accumulate. Clearly there is a need for new therapies that are less invasive and more available and cost-effective. Rapid advances in stem cell shows true progress toward cellular therapies for liver disease. Stem cell therapy for liver disease can be viewed as a platform technology. When the basic strategy fro engrafting new cells in the liver is achieved in a defined group of high-risk patients the resulting understanding would pave the way for much broader applications. Infants with lethal genetic disorders of liver metabolism, called urea cycle defects, form one such patient population. They cannot properly metabolize dietary protein and suffer severe or lethal neurological damage. The affected child’s liver is otherwise normal, thus research indicates that partial liver replacement with cells derived from normal human embryonic stem cells (hESC) would be a viable therapy, if scientific hurdles can be cleared and the treatment shown to be safe and effective. If hESC-based cell therapies perform as anticipated in these dire diseases, they could be directed toward many conditions not necessarily perceived as liver disease. Hemophilia A and B are two examples of genetic diseases stemming from defective proteins produced by the liver that greatly affect quality of life. The liver is the site of LDL (bad cholesterol) processing and a genetic mutation in the LDL receptor results in familial hypercholesterolemia, a disorder leading to early heart disease in 1 of 600 individuals. Repopulating patients’ diseased livers with stem cells carrying normal genes could cure these diseases. In the future, modifying the genes of stem cells before engrafting them into the liver could treat an even broader spectrum of liver disease, including hepatitis C. Furthermore, the technologies developed for monitoring the relevant events following stem cell implant in patient models could direct development of additional stem cell therapies.
Review Summary: 
This proposal is focused on the development of a stem cell therapy to treat urea cycle disorders (UCDs). UCDs are severe diseases affecting newborns in which ammonia is not properly metabolized by the liver, leading to hyperammonemia, neurologic complications and eventual death. The applicant proposes to first engineer mouse and human embryonic stem cells (mESCs and hESCs) to express fluorescent reporter genes to allow the differentiation and selection of endodermal precursor cells (EPs). These EPs are capable of hepatocyte differentiation and engraftment in vivo and will be tested for therapeutic efficacy in ornithine transcarbamylase (OTC) deficient mice, a model of a common UCD. In addition, the applicant proposes to monitor immune responses to transplanted EPs, establish a hESC bank genetically labeled for endoderm, and develop strategies for HLA-matching of donors and patients. Finally, the applicant plans to develop methods for tracking transplanted EPs using MRI and multimodal imaging. Reviewers agreed that this proposal addresses an unmet medical need. UCDs are devastating diseases that have an early-onset, are lethal and largely untreatable except by organ transplant. Reviewers felt that the rationale for the proposal was well justified but that its potential impact was severely limited by issues of feasibility. Reviewers also cautioned that potential findings might not be easily translated to the clinic. Specifically, one reviewer discussed the applicant’s plan to generate hESC and mESC lines expressing fluorescent reporter genes to aid differentiation and selection of EPs. The reviewer noted that this work alone is extremely ambitious and could easily require the duration of the funding period. Furthermore, this reviewer did not consider the (gene manipulation/gene therapy) strategy to be translational, stating that it will be essential to use unmodified ESC derivatives in human patients in the first-in-man clinical studies. The reviewer suggested taking advantage of published protocols for directed differentiation of ESCs along the hepatocyte lineage and employing surface markers (e.g. Flk1, Cxcl4, etc.) for selection, as an alternative approach. Reviewers described the proposal as ambitious and diverse but underdeveloped. They raised a number of concerns about the research design and feasibility. One reviewer noted that the basis for establishing correction of the OTC deficiency in the mouse model is not ammonia levels as noted, but measurements of orotic acid levels in the urine and correction of the animal’s sparse-fur phenotype. While the measurement of orotic acid levels is appropriate, a complete investigation should include measurements of blood ammonia levels, OTC activity in the recipient organ and the presence of donor cells (via OTC immunostaining, OTC RNA expression and donor-specific DNA). This reviewer also noted that the applicant expects that transplanted EPs will proliferate such that 20-30% of the liver is repopulated by donor cells following a single injection of cells but provides no meaningful discussion or preliminary data to support this hypothesis. The reviewer commented that it is frequently necessary to induce proliferation and expansion of transplanted cells to arrive at levels of repopulation that are meaningful for the correction of metabolic liver diseases. Another reviewer noted that the experiments proposed in Aim 3 were designed with considerable thought and appear to fit best with the expertise of the applicant. However, this reviewer felt that, like earlier aims, Aim 3 represents an extremely ambitious project on its own, requiring development of a large number of stage-specific reporter lines and challenging assays using human lymphocyte transfer models in NOD/SCID mice. Another reviewer raised concerns about the imaging experiments described in Aim 4, noting a lack of detail about the MRI experiments (i.e. information about instrumentation, resolution, pulse sequences and time points). In addition, the section describing multimodal optical imaging was cut off due to page limitations. The reviewers raised several issues with the applicant’s use and interpretation of the literature. One reviewer did not feel that that the hepatocyte transplant literature was adequately referenced in the proposal. This reviewer noted that direct injections into the liver parenchyma are not the usual method of delivery for transplanted hepatocytes and are reserved for emergency situations where alternative routes, such as the portal vein or spleen, are unavailable. The applicant does not discuss or provide rationale for their choice of transplant site of delivery, which is not a clinically relevant approach. Furthermore, studies describing hepatocyte transplants into human OTC patients have been published but were not cited in the proposal. Another reviewer pointed out that the publication cited for information about imaging ferritin-labeled hepatocytes, suggests that this technique may be toxic to these cells. The reviewers agreed that the applicant is well qualified to carry out the proposed research and will be assisted by a number of expert investigators. However they commented that the project would benefit from additional expertise in the areas of directed differentiation of hESCs and in vivo imaging. Reviewers agreed that the applicant’s available resources and research environment are excellent. Overall, reviewers found this proposal to be overly ambitious and raised significant doubts about its feasibility, design and potential for translation to the clinic.
Conflicts: 

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