Development of a cell and gene based therapy for hemophilia

Hemophilia B is a bleeding disorder caused by the lack of FIX in the plasma and affects 1 in 30,000 males. Patients suffer from recurrent bleeds in soft tissues leading to physical disability in addition to life threatening bleeds. Current treatment (based on FIX infusion) is transient and plagued by increased risk for blood-borne infections (HCV, HIV), high costs and limited availability. This has fueled a search for gene/cell therapy based alternatives. Gene therapy with viruses is beset with problems of safety and increased immunogenicity. Being the natural site of FIX synthesis, the liver is expected to provide immune-tolerance and easy circulatory access. Liver transplantation is a successful, long-term therapeutic option but is limited by scarcity of donor livers and chronic immunosuppression, making iPSC-based cell therapy an attractive prospect. As part of this project, we will generate iPSCs from hemophilic patients that will be genetically corrected by inserting FIX coding DNA. After correction, we will differentiate these iPSCs into liver cells which will then be transplanted into our mouse model of hemophilia that can accept human hepatocytes and allow their proliferation. These mice exhibit disease symptoms similar to human patients and we propose that by injecting our corrected liver cells they will exhibit normal clotting as measured by various biochemical and physiological assays. If successful, this will provide a long-term cure for hemophilia and will serve as a proof-of-concept for the treatment of other liver diseases. With this long term aim, during the first year of the project, we have procured two hemophilic patient samples and two control samples from our collaborators. We have successfully generated iPSCs with no long-term genomic changes. We are currently working towards identifying the mutations in the patients that were responsible for the disease. Our efforts are presently directed towards correcting the mutations in the patient derived iPSCs so that they can now produce a functional FIX protein.

Reporting Period:

Year 2

Hemophilia B is a bleeding disorder caused by the lack of FIX in the plasma & affects 1/30,000 males. Patients suffer from recurrent bleeds in soft tissues leading to physical disability in addition to life threatening bleeds. Current treatment (based on FIX infusion) is transient & plagued by increased risk for blood-borne infections (HCV, HIV), high costs & limited availability. This has fueled a search for gene/cell therapy based alternatives. Gene therapy with viruses is beset with problems of safety & increased immunogenicity. Being the natural site of FIX synthesis, the liver is expected to provide immune-tolerance & easy circulatory access. Liver transplantation is a successful, long-term therapeutic option but is limited by scarcity of donor livers & chronic immunosuppression; making iPSC-based cell therapy an attractive prospect. As part of this project, we plan to generate iPSCs from hemophilic patients that will then be genetically corrected by inserting DNA coding for FIX. After validation for correction, we will then differentiate these iPSCs into liver cells that can be transplanted into our mouse model of hemophilia that is capable of accepting human hepatocytes and allowing their proliferation. These mice exhibit disease symptoms similar to human patients & we propose that by injecting our corrected liver cells they will exhibit normal clotting as measured by various biochemical & physiological assays. If successful, this will provide a long-term cure for hemophilia & other liver diseases. So far, we have generated multiple stem cell lines from two hemophilia B patients through cells in their peripheral blood. We have further sequenced the coding regions of the FIX gene in these patients to identify the mutations responsible for the disease. We have corrected the responsible single nucleotide change in two stem cell lines for one patient and are working towards correcting the other patient. For the other patient, we are currently planning to use a universal correction strategy that will bypass the need to sequence and identify the mutations each time. This will allow the integration of a functional and corrected F9 gene sequence under its native regulatory sequence. We have also generated a new mouse model for the disease that is deficient in 4 genes, making it permissive to the transplantation and expansion of human hepatocytes in its liver. We demonstrate the hemophilic nature of this new mouse strain through prolonged clotting time, absence of circulating protein and other biochemical assays. This hemophilic mousewill allow us to screen for symptoms of disease alleviation upon administration of patient derived hepatocytes. We are currently optimizing our differentiation protocol and conducting preliminary studies where these in vitro differentiated cells will be transplanted in our mouse model.

Reporting Period:

Year 3

In the last three years of this grant, we established the feasibility of hepatocyte transplantation as a therapeutic approach for hemophilia B. We transplanted cadaveric human hepatocytes into our quadruple knock-out hemophilic mice and found that these human hepatocytes could engraft and expand in the mouse liver for up to a year. The transplanted animals also showed dramatic improvement in their clotting phenotype attesting to the feasibility of this option. Our objective here was to extend cell therapy to patient iPSC-derived hepatocytes to mitigate the immune risks and the dire need for transplantable livers.

Pursuing this direction, we procured blood samples from 2 hemophilia B patients (HB001 and HB002) and derived iPSCs from them. We established their pluripotency and genomic stability and sequenced the FIX to identify the disease causing mutations in each case, then used the CRISPR/Cas9 technology to correct the mutation and to restore functionality of the FIX gene/protein. We developed 2 different approaches for this: one where we only changed the defective nucleotide and another where we inserted the full length FIX cDNA under control of the endogenous promoter bypassing the need to sequence every patient. We differentiated these autologous iPSC lines into “hepatocyte like cells/HLCs” using our in-house differentiation protocol. We demonstrated correction of the genetic defect through multiple approaches whereby expression of FIX is restored in these HLCs in culture. Finally we transplanted these in vitro differentiated HLCs into our mouse model and will be testing for their in vivo presence.

Grant Application Details

Application Title:

Development of a cell and gene based therapy for hemophilia

Public Abstract:

Hemophilia B is a bleeding disorder caused by the lack of FIX in the plasma and affects 1/30,000 males. Patients suffer from recurrent bleeds in soft tissues leading to physical disability in addition to life threatening bleeds. Current treatment (based on FIX infusion) is transient and plagued by increased risk for blood-borne infections (HCV, HIV), high costs and limited availability. This has fueled a search for gene/cell therapy based alternatives. Being the natural site of FIX synthesis, the liver is expected to provide immune-tolerance and easy circulatory access. Liver transplantation is a successful, long-term therapeutic option but is limited by scarcity of donor livers and chronic immunosuppression; making iPSC-based cell therapy an attractive prospect. As part of this project, we plan to generate iPSCs from hemophilic patients that will then be genetically corrected by inserting DNA capable of making FIX. After validation for correction, we will then differentiate these iPSCs into liver cells that can be transplanted into our mouse model of hemophilia that is capable of accepting human hepatocytes and allowing their proliferation. These mice exhibit disease symptoms similar to human patients and we propose that by injecting our corrected liver cells they will exhibit normal clotting as measured by various biochemical and physiological assays. If successful, this will provide a long-term cure for hemophilia and other liver diseases.

Statement of Benefit to California:

Generation of iPSCs from adult cells unlocked the potential of tissue engineering, replacement and cell transplant therapies to cure a host of debilitating diseases without the ethical concerns of working with embryos or the practical problems of immune-rejection. We aim to develop a POC for a novel cell- and gene-therapy based approach towards the treatment of hemophilia B. In addition to the obvious and direct benefit to the affected patients and families by providing a potential long-term cure; the successful development of our proposal will serve as a POC for moving other iPSC-based therapies to the clinic. Our proposal also has the potential to treat a host of other hepatic diseases like alpha-1-antitrypsin deficiency, Wilson’s disease, hereditary hypercholesterolemia, etc. These diseases have devastating effects on the patients in addition to the huge financial drain on the State in terms of the healthcare costs. There is a pressing need to find effective solutions to such chronic health problems in the current socio-economic climate. The work proposed here seeks to redress this by developing cures for diseases that, if left untreated, require substantial, prolonged medical expenditures and cause increased suffering to patients. Being global leaders in these technologies, we are ideally suited to this task, which will establish the state of California at the forefront of medical breakthroughs and strengthen its biomedical/biotechnology industries.

Publications

Proc Natl Acad Sci U S A (2017): Systemic delivery of factor IX messenger RNA for protein replacement therapy. (PubMed: 28202722)