Severe combined immunodeficiency caused by mutations in the IL2RG gene on the x-chromosome (SCID-X1 or "bubble boy disease") is a devastating genetic disease that results in boys not being able to form an immune system. If they are exposed to the environment for even a short period of time they can get infections that a normal immune system would eliminate without problems but instead can be lethal. While in the past the only treatment for this disease was to keep the boys protected from the environment by being isolated in a bubble, hence its colloquial name, now we treat SCID-X1 with allogeneic bone marrow transplantation (allo-BMT). In allo-BMT the defective immune system of the patient is replaced by the functional immune system of the donor. Allo-BMT now saves the life of 70-95% of patients depending on where the donor immune system comes from and how sick the patient is before receiving the transplant. There remain, however, significant limitations to allo-BMT. These include that in some patients the new immune system is still not as good as a normal immune system, thus keeping the patient at risk for lethal infections, and toxicity from the new immune system causing a reaction in which the donor immune system sees the patient as "foreign" and attacks the tissues causing graft vs host disease. In rare patients, however, a single stem or progenitor cell that gives rise to the immune system will have a spontaneous mutation that reverts the disease causing DNA sequence back into a non-disease causing sequence thereby correcting the gene. The goal of this program is to develop a specific gene correction procedure that could be applied to almost every patient with SCID-X1 rather than to it naturally occur in an extremely rare lucky few.
Towards this end we have developed a system in which we make a specific break in the IL2RG gene. This break activates the cell to repair the break and we can take advantage of the cell fixing the break to insert a good copy of the gene at the site of the break. In this way, we utilize the cell's own repair machinery to fix the gene. We have shown that we can do gene repair in human blood stem and progenitor cells from anyone and create corrected cells thousands of corrected stem and progenitor cells rather than just a single cell rarely occurs naturally. We have shown that these modified cells can create blood cells, including immune cells. The goal of this specific project is to further improve the gene correction system by optimizing the different components, to assure that the gene correction system is safe and does not cause deleterious effects in the blood stem and progenitor cells, to scale the process up to a size that would be needed to treat a patient and to perform the regulatory tasks that are needed to bring what would be a first-in-human gene correction approach to patients.
SCID-X1 is a rare disease that only affects a handful of patients in the state of California each year. Finding a genetic cure based on gene correction, therefore, might seem not to be of great benefit to the state of California or its citizens, This would be a mistaken impression for several reasons. For the handful of patient's and families that are affected that are affected every year, dealing with the disease will be among the most challenging life events they will ever face and finding a gene correction cure would be of tremendous, life-changing benefit to them. Moreover, t's significance far outstrips its incidence because of its notoriety as the "bubble boy disease" and the recognition that it is a seminal proof-of-concept genetic disease. That is, if one can figure out how to genetically correct stem cells to cure SCID-X1 then that provides the foundation for a strategy to genetically correct stem cells that cause a multitude of other genetic diseases. That is, a pipeline for gene correction for all children with genetic diseases in California will be started. As succinctly summarized by the head of research and development of a large international pharmaceutical company "One will get you a hundred."
While the medical benefits of first curing SCID-X1 and then curing other genetic diseases is clear, the financial ramifications of turning chronic lifelong genetic diseases that directly cost society sometimes millions of dollars per patient per lifetime and indirectly cost society even more into acute diseases that can be cured with one procedure are enormous.
Finally, California attracts the best and the brightest from all over the world because it is known as a place where transformative, innovative, and impactful discoveries are made and supported. When we are successful with this definitive and innovative approach to curing a genetic disease, it will continue to re-affirm the seminal importance of California and its citizens in making the world a better place.
As the largest provider of bone marrow cell transplants in California, and the second largest in the nation, our institution has great expertise and an excellent record of safety in the delivery of stem cell treatments. We now propose to create the Alpha Clinic for Cell Therapy and Innovation (ACT-I) in which new, state-of-the-art, stem cell treatments for cancer and devastating blood-related diseases will be conducted and evaluated. As these experimental therapies prove to be effective, and become routine practice, our ACT-I Program will serve as the clinical center for delivery of these treatments. ACT-I will be an integral part of our Hematologic Malignancy and Stem Cell Transplantation Institute, placing it in the center of our institutional strengths, expertise, infrastructure and investment over the next decade. To move quickly once the CIRM award is made, ACT-I can be launched within our institution’s Day Hospital, a brand new, outpatient blood stem cell transplantation center opened in late 2013 with California Department of Health approval for 24 hour a day operation. This will ensure that ACT-I will have all the clinical and regulatory expertise, trained personnel, state-of-the-art facilities and other infrastructure in place to conduct first-in-human clinical trials and to deliver future, stem cell-based therapies for cancer and blood-related diseases, including AIDS. When our new Ambulatory Treatment Center is complete in 2018, it will double our capacity for patient visits and allow for expansion of the ACT-I pipeline of new stem cell products in a state-of-the-art facility.
Beyond our campus, we operate satellite clinics covering an area that includes urban, suburban and rural sites. More than 17.7 million people live in this area, and represent some of the greatest racial and ethnic diversity seen in any part of the country. Our ACT-I is prepared to serve a significant, diverse and underserved portion of the population of California.
CLINICAL TRIALS. Our proposal has two lead clinical trials that will be the first to be tested in ACT-I. One will deliver transplants of blood stem cells that have been modified to treat patients suffering from AIDS and lymphoma. The second will use neural stem cells to deliver drugs directly to cancer cells hiding in the brain. These studies represent some of the new and exciting biomedical technologies being developed at our institution. In addition to the two lead trials, we have several additional clinical studies poised to use and be tested in this special facility for clinical trials. In summary, ACT-I is well prepared to accommodate the long list of clinical trials and begin to fulfill the promise of providing new stem cell therapies for the citizens of California.
California’s citizens voted for the California Stem Cell Research and Cures Act to support the development of stem cell-based therapies that treat incurable diseases and relieve human suffering. To achieve this goal, we propose to establish an Alpha Clinic for Cellular Therapies and Innovation (ACT-I) as an integral part of our Hematological Malignancies and Stem Cell Transplantation Institute, and serve as the clinical center for the testing and delivery of new, cutting-edge, cellular treatments for cancer and other blood-related diseases. Our institution is uniquely well-suited to serve as a national leader in the study and delivery of stem cell therapeutics because we are the largest provider of stem cell transplants in California, and the second largest in the country. According to national benchmarking data, our Hematopoietic Cell Transplantation program is the only program in the nation to have achieved survival outcomes above expectation for each of the past nine years. This program currently offers financially sustainable, research-driven clinical care for patients with cancer, HIV and other life-threatening diseases. CIRM funding will allow the ACT-I clinic to ramp up quickly, drawing upon institutionally established protocols, personnel and infrastructure to conduct first-in-human clinical trials for assessment of efficacy. As CIRM funding winds down, ACT-I will have institutional support to offer proven cellular therapeutics to patients. The lead studies at the forefront of the ACT-I pipeline of clinical trials focus on treatments for HIV-1 infection and brain tumors, two devastating and incurable conditions. These first trials are closely followed by a robust queue of other stem cell therapeutics for leukemia, lymphoma, prostate cancer, brain cancers and thalassemia.
Our long list of proposed treatments addresses diseases that have a major impact on the lives of Californians. Thalassemia is found in up to 1 in 2,200 children born in California; prostate cancer affects 211,300 men, and HIV-1 infection occurs in 111,000 of our citizens. From 2008 to 2010, 6,705 Californians were diagnosed with brain cancers, 4,580 of whom died. In considering hematological malignancies during this same period, 2,800 patients were diagnosed with Hodgkin lymphoma (416 died), 20,351 with non-Hodgkin lymphoma (6,241 died), 13,358 with leukemia (6,961 died), 3,900 with acute myelogenous leukemia (2,972 died), 2,129 with acute lymphoblastic leukemia (648 died) and 4,198 with chronic lymphocytic leukemia (1,271 died). Standard of care fails in many cases; mortality rates for patients with hematological malignancies range from 25% to 76%. Successful stem cell therapeutics hold the promise to reduce disease-related mortality while improving disease-related survival and quality of life for the citizens of California, and for those affected by these diseases worldwide.
Tens of thousands of patients need bone marrow transplants (BMT) every year, some for bone marrow (BM) cancers and some for inherited diseases such as sickle cell anemia and thalassemia, but many lack a BM donor. African Americans, Asian Americans, and people of Hispanic descent are more likely than others to lack a stem cell donor.
BMTs provide hematopoietic (blood) stem and progenitor cells (HS/PCs) that replace the patient’s diseased BM with healthy BM. The new BM provides all the circulating blood cells throughout life.
Many BMTs use HS/PCs that do not come from the BM. One such ‘other’ source is umbilical cord blood (UCB). UCB HS/PCs have many advantages over other HS/PC sources (i.e., BM or peripheral blood). For example, we can easily obtain UCB HS/PCs without any risk to the donor, and we can keep the cells stored in freezers to be available when a patient needs them. However, most UCB samples contain too few HS/PCs to be used to treat people.
Expanding the number of HS/PCs in UCB samples will increase the number of clinically usable UCB samples, offering new hope for thousands of patients who currently lack a donor. We previously screened >120,000 compounds for their ability to expand UCB HS/PCS, and identified a short list of lead candidates. This grant will fund the next step in our effort to develop a novel, clinically-useful UCB HS/PC expansion protocol. Successful completion of this proposal will result in life-saving treatment for thousands of patients.
Our proposal seeks to establish a novel method to expand umbilical cord blood hematopoietic stem/progenitor cells (HS/PCs) to make bone marrow transplants (BMTs) available to thousands of patients who currently lack a stem cell donor. The benefits to California are wide-ranging:
• Grow California’s skilled workforce and create jobs: This project will train scientists in stem cell research and technology, and our success will attract more talent from outside California.
• Increase innovation: This proposal is highly translational, with a goal to move rapidly from bench to bedside. However, our research will also provide basic insights into stem cell biology that can be applied by other scientists to help patients more broadly.
• Enhancing the medical treatment of California residents: Compounds that expand UBC HS/PCs have the potential to improve clinical benefit and reduce health care costs by increasing the success rate of stem-cell transplants. Given California’s diverse ethnic population, we have many patients who need a BMT yet lack a donor, so our residents will directly benefit from our success.
• Attracting venture capital and commercialization: We aim to develop technology that will be highly attractive to the biotechnology industry. We have identified GE as a partner to commercialize our reagents and processes. Furthermore, commercially viable compounds will attract venture capital to fund cell therapies and create new biotech jobs for the California economy.
The overall goal of this proposal is to develop new methods and technologies to improve our ability to engineer hematopoietic stem cells. These are the adult stem cells found in the bone marrow that give rise to all of the components of the blood and immune systems. Being able to engineer these cells provides potential treatments for diseases of the blood including genetic diseases, such as sickle cell disease or severe immune deficiencies, as well as serious infections such as HIV/AIDS. We work with a new class of genetic engineering tools called targeted nucleases that have the potential to make genetic engineering of stem cells much more precise and therefore safer. In addition, we are exploring methods to deliver these reagents directly to the stem cells in the body, without the currently necessary steps of first removing the cells and performing the genetic engineering in a lab. Such capabilities would greatly improve the safety of human gene therapy, as well as facilitate its practical implementation. HIV/AIDS is our disease of focus, and we will use these techniques to develop new treatments that go beyond the current use of targeted nucleases in patients, where HIV’s co-receptor gene, called CCR5, is being disrupted. Our goal is to develop a next-generation of anti-HIV therapies and we expect that the techniques we develop will be broadly applicable to other disease of the blood and immune systems where stem cell therapies could be of benefit.
HIV/AIDS is a major social, economic and health burden to California and its citizens. The numbers are sobering: California has 14% of all US cases of HIV, second only to New York, with 220,543 cases reported through June 2014, including 98,161 deaths. With the advent of improved antiretroviral drugs, mortality has significantly decreased, but so has the length of time people need to take the drugs, and the economic burden to the state is revealed by the cost of drugs representing 85% of all AIDS-related costs. Both federal and state laws require that the AIDS Drug Assistance Program be the payer of last resort for these medications, and its budget is underwritten by the General Fund. Beyond the fiscal concerns, patients live with the potential for developing side effects to the drugs or drug-resistant virus, and accessing these life-long drug regimens is a daily struggle for many. Consequently, the development of stem cell based therapies for HIV brings the potential of one-shot and long-lasting treatments that could arm a patient’s own immune system with the capability to suppress HIV in the absence of drugs. Such an outcome would provide economic returns over the long-run by reducing spending on drugs, as well as improving the quality of life for individuals with HIV/AIDS. Beyond HIV, the development of technologies to improve the efficiency, safety and implementation of hematopoietic stem cell therapies will benefit other diseases where such cells could be curative.
A goal of stem-cell therapy is to transplant into a patient “tissue-specific” stem cells, which can regenerate a particular type of healthy tissue (e.g., heart or blood cells). A major obstacle to this goal is obtaining tissue-specific stem cells that (1) are available in sufficient numbers; and (2) will not be rejected by the recipient. One approach to these challenges is to generate tissue-specific stem cells in the lab from “pluripotent” stem cells, which can produce all types of tissue-specific stem cells. The rationale is that pluripotent stem cells that will be tolerated are easier to directly obtain than tissue-specific stem cells that will be tolerated. Furthermore, descendants of a tolerated pluripotent stem cell will also be tolerated and can be produced abundantly.
The goal of the proposed project is to develop techniques for generating transplantable blood-forming stem cells from pluripotent stem cells. In pursuit of this goal, we will study how blood-forming stem cells arise during development. We will also test new methods--less toxic than current chemotherapy and radiation--for preparing recipients for transplantation of blood-forming stem cells.
Additional benefit: Successful transplantation of blood-forming stem cells allows the recipient to tolerate other tissue or organ transplants from the same donor. Thus, transplanted blood-forming stem cells could allow people to receive organs that they may otherwise reject, without taking immune-suppressing drugs.
We aim to generate from stem cells that can produce all tissues of the body those stem cells that specifically form blood. We will also test new methods--less toxic than current chemotherapy and radiation--for pretreatment before transplantation of blood-forming stem cells. A large number of patients in California could benefit from advances in this field, primarily those with diseases affecting the production of blood and immune cells: leukemia, lymphoma, thalassemia, certain types of anemia, immune deficiency diseases, autoimmune diseases (e.g., lupus), etc. For leukemia and lymphoma alone, in 2014 in California, there will be an estimated 12,060 newly diagnosed cases, 103,400 existing cases, and 4,620 deaths (per the California Cancer Registry). The cost of these blood cancers are difficult to estimate but they account for 6% of cancers in women and 9% in men in California, where the estimated cost of cancer per year is $28.3 billion.
The reagents generated in these studies can be patented, forming an intellectual property portfolio shared by the state. The funds generated from the licensing of these technologies will provide revenue for the state, help increase hiring of faculty and staff (many of whom will bring in other, out-of-state funds to support their research) and could reduce the costs of related clinical trials. Only California businesses are likely to be able to license these reagents and to develop them into diagnostic and therapeutic entities.
Our goal is to develop tools that address major bottlenecks that have prevented the generation of blood forming stem cells in culture for therapeutic use. To help overcome these bottlenecks, we will generate a suite of human embryonic stem cell reporter lines that can be used to monitor key milestones in blood stem cell development. These lines will serve as tools to identify factor combinations to improve the in vitro differentiation of hESCs to functional blood stem cells. Once individual lines have been validated, lines that contain multiple fluorescent reporters will be generated, and a multi factor screen will be performed to optimize conditions that induce these blood stem cell regulators. To track the location and quantity of transplanted cells in recipient small animal model, we will generate hESC lines with in vivo reporter system that combines bioluminescent or PET imaging, and serum-based assay. Our in vivo tracking tools will be broadly relevant and not restricted to studying the in vivo biology of blood forming cells. These tools will help translate the promise of stem cells to cell based therapies to treat human disease.
This project will help improve California economy as many of the vendors used for reagents and supplies are located in California. This project will also help create and maintain jobs for skilled personnel and helps train post-doctoral fellows who will become the next generation of stem cell scientists. The long-term goal of this project is to improve in vitro differentiation protocols to create transplantable blood forming stem cells for therapeutic use. If we, or others who will use our reporter lines generated in this study, achieve this goal, there will be new, theoretically unlimited sources of HLA-matched or patient specific blood stem cells that can be used for treating many serious blood diseases, including leukemias and inherited immunodeficiencies or anemias. Availability of patient specific blood stem cells for transplantation would be a major benefit in California, as there is currently limited availability of suitable bone marrow donors for individuals from mixed ethnic backgrounds.
The research performed through this project is very important for the fields of solid organ and bone marrow transplantation because it focuses on a potential new target to increase engraftment of stem cells. Currently, patients that receive stem cell transplants from a non-identical donor must take medications to suppress their immune system; otherwise the stem cells will be rejected.
Stem cell trials have been extended to solid organ transplantation, where it has been shown that kidney transplants can be managed with little or no immunosuppressive medications when stem cells are given to the patient at the time of transplantation. In many cases though the stem cells are rejected and the patient must resume toxic medications.
Our laboratory has been very interested in understanding ways to prevent the rejection of stem cells and has focused on a phylogenetically conserved group of cellular receptors called pattern recognition receptors. This project is focused on understanding how to prevent rejection of stem cells through modifications of these receptors. We hope to identify novel targets to prevent the rejection of stem cells in order to decrease the occurrence of graft versus host disease after bone marrow transplantation and also improve the opportunities for long-term transplant survival without the use of toxic immunosuppressive medications.
The research we will undertake will benefit the State of California and its residents in two major ways. First it promises to define a novel targets to prevent rejection of stem cells that are transplanted into their new host. This is very important because rejection of hematopoietic stem cells is a major impediment to successful efforts at both bone marrow and solid organ transplantation. Patients needed life-saving solid organ transplants and patients that receive bone marrow transplants from donors that are not perfectly matched to them reject their grafts unless they take powerful medications to suppress their immune system. This project is focused on finding a way to help prevent the rejection of these grafts without the need for immunosuppressive medications.
The second way the project will benefit the State of California is to provide new employment opportunities within the State at a large University that conducts biomedical research. This project will not only directly support the employment of three California citizens devoted to biomedical research, but the work it generates will support California-based biomedical science companies, California University personal and other local companies that employ California citizens that produce the reagents and the supplies used in the proposed studies.
- The research performed through this project is very important for the fields of solid organ and bone marrow transplantation because it focuses on a potential new target to increase engraftment of stem cells. Currently, patients that receive stem cell transplants from a non-identical donor must take medications to suppress their immune system; otherwise the stem cells will be rejected.
- In our first year of this project we were able to develop a reliable model to delete specific receptors which directly influence stem cell engraftment in genetically different hosts. We have also found that deletion of defined receptors greatly improves stem cell engraftment for up to 20 weeks after injection in a murine model of bone marrow transplantation.
- The plan for the next reporting period is to continue to focus our studies on characterizing the lodging and fate of the engrafted stem cells in long-lasting chimeric animals and to look into ways to improve the opportunities for long-term transplant survival without the use of toxic immunosuppressive medications.
Blood stem cells living in the bone marrow of adult humans give rise to all of the cells in our blood, including the red blood cells that carry oxygen to supply our body, and the white blood cells such as T and B lymphocytes that fight infections and keep us healthy. Among the T lymphocytes there is a small population called invariant natural killer T (iNKT) cells. Despite their low frequency in humans (~0.001-1% in blood), iNKT cells have the remarkable capacity to mount immediate and potent responses when stimulated, and have been suggested to play important roles in regulating multiple human diseases including infections, allergies, cancer, and autoimmunity (such as Type I diabetes and multiple sclerosis). However, successful clinical interventions with iNKT cells have been greatly hindered by our limited knowledge on how these cells are produced by blood stem cells, largely due to the lack of tools to track these cells in humans. We therefore propose a novel model system to overcome this research bottleneck by transplanting human blood stem cells into a mouse and genetically programming these cells to develop into iNKT cells. This “humanized” mouse model will allow us to directly track the differentiation of human blood stem cells into iNKT cells in a living animal. From this study, we will address some critical unanswered questions for iNKT cell development, and shed light on developing stem-cell based iNKT cell therapies.
Allergies, cancer and autoimmunity are leading health hazards in California. These diseases affect millions of Californians, impairing their life quality and creating huge economic burdens for the State of California. This proposal intends to study invariant natural killer (iNKT) T cells, a special population of T lymphocytes that have been suggested to play important roles in regulating these diseases. To date, clinical applications of iNKT cells have been greatly limited by their low frequency in humans and their high variability between individuals (~0.001-1% in blood). Thus, an improved understanding of how these cells are naturally generated is important for their use clinically. Like all other cells in blood, iNKT cells are descendants of the blood stem cells that live in the bone marrow of adult humans. Our goal is to study how human blood stem cells give rise to iNKT cells. If successful, our results can be exploited to develop stem cell-based iNKT cell therapies to treat allergies, cancer and autoimmunity, and therefore may benefit the millions of Californians currently suffering from these diseases. In addition, the knowledge and reagents generated from this proposed study will be shared freely with non-profit and academic organizations in California, and any new intellectual property derived from this study will be developed under the guidance of CIRM to benefit the State of California.
- Despite their small numbers (~0.001-1% in blood), invariant natural killer T (iNKT) cells in humans have been suggested to play important roles regulating multiple diseases including infections, allergies, cancer and autoimmunity. Like all other immune cells, iNKT cells are derived from the blood stem cells living in the bone marrow of adult humans. Successful clinical interventions with iNKT cells have been greatly hindered by our limited knowledge on how these cells are produced by blood stem cells, largely due to the lack of tools and track these cells in humans. Our project proposes to overcome this research bottleneck by transplanting human blood stem cells into a mouse and genetically engineer these cells to develop into human iNKT cells. This “humanized” mouse model will allow us to directly track the differentiation of human blood stem cells into iNKT cells in a living animal. In this reporting period, we have demonstrated the feasibility of this model system, and have successfully generated stem cell-engineered human iNKT cells. In the coming year, we plan to use this established model system to address some critical unanswered questions for iNKT cell development, and explore the therapeutic potential of stem-cell based iNKT cell therapies.
β-thalassemia is a genetic disease caused by diverse mutations of the β-globin gene that lead to profoundly reduced red blood cell (RBC) development. The unmet medical need in transfusion-dependent β-thalassemia is significant, with life expectancy of only ~30-50 years despite standard of care treatment of chronic blood transfusions and iron chelation therapy. Cardiomyopathy due to iron overload is the major cause of mortality, but iron-overload induced multiorgan dysfunction, blood-borne infections, and other disease complications impose a significant physical, psychosocial and economic impact on patients and families. An allogeneic bone marrow transplant (BMT) is curative. However, this therapy is limited due to the scarcity of HLA-matched related donors (<20%) combined with the significant risk of graft-versus-host disease (GvHD) after successful transplantation of allogeneic cells.
During infancy, gamma-globin-containing fetal hemoglobin protects β-thalassemia patients from developing disease symptoms until gamma globin is replaced by adult-type β-globin chains. The proposed therapeutic intervention combines the benefits of re-activating the gamma globin gene with the curative potential of BMT, but without the toxicities associated with acute and chronic immunosuppression and GvHD. We hypothesize that harvesting hematopoietic stem and progenitor cells (HSPCs) from a patient with β-thalassemia, using genome editing to permanently re-activate the gamma globin gene, and returning these edited HSPCs to the patient could provide transfusion independence or greatly reduce the need for chronic blood transfusions, thus decreasing the morbidity and mortality associated with iron overload. The use of a patient’s own cells avoids the need for acute and chronic immunosuppression, as there would be no risk of GvHD. Moreover, due to the self-renewing capacity of HSPCs, we anticipate a lifelong correction of this severe monogenic disease.
Our proposed treatment for transfusion dependent β-thalassemia will benefit patients in the state by offering them a significant improvement over current standard of care. β-thalassemia is a genetic disease caused by diverse mutations of the β-globin gene that lead to profoundly reduced red blood cell (RBC) development and survival resulting in the need for chronic lifelong blood transfusions, iron chelation therapy, and important pathological sequelae (e.g., endocrinopathies, cardiomyopathies, multiorgan dysfunction, bloodborne infections, and psychosocial/economic impact). Incidence is estimated at 1 in 100,000 in the US, but is more common in the state of California (incidence estimated at 1 in 55,000 births) due to immigration patterns within the State. While there are estimated to be about 1,000-2,000 β-thalassemia patients in the US, one of our proposed clinical trial sites has the largest thalassemia program in the Western United States, with a population approaching 300 patients. Thus, the state of California stands to benefit disproportionately compared to other states from our proposed treatment for transfusion dependent β-thalassemia.
An allogeneic bone marrow transplant (BMT) is curative for β-thalassemia, but limited by the scarcity of HLA-matched related donors (<20%) combined with the significant risk of graft-versus-host disease (GvHD) after successful transplantation of allogeneic cells. Our approach is to genetically engineer the patient’s own stem cells and thus (i) solve the logistical challenge of finding an appropriate donor, as the patient now becomes his/her own donor; and (ii) make use of autologous cells abrogating the risk of GvHD and need for acute and chronic immunosuppression.
Our approach offers a compelling pharmacoeconomic benefit to the State of California and its citizens. A lifetime of chronic blood transfusions and iron chelation therapy leads to a significant cost burden; despite this, the prognosis for a transfusion dependent β-thalassemia patient is still dire, with life expectancy of only ~30-50 years. Our proposed one-time treatment aims to reduce or eliminate the need for costly chronic blood transfusions and iron chelation therapy, while potentially improving the clinical benefit to patients, including the morbidity and mortality associated with transfusion-induced iron overload.
- Summary of progress
- Our CIRM-funded effort aims to develop a treatment for beta-thalassemia. Beta-thalassemia is an inherited genetic disorder that is caused by mutations (changes in the DNA) in a gene called beta-globin. This gene produces a protein that forms hemoglobin in red blood cells that carry oxygen to through the body. In an individual with beta-thalassemia, beta-globin is not produced (or is made in dramatically reduced quantities), and so the person does not make enough healthy red blood cells. The treatment, which is essential for life in these patients, is repeated blood transfusions (typically once a month or more frequently). The transfusion of blood this frequently results in a dangerous condition called “iron overload,” which must be treated with costly drugs. In general, the quality of life of many people with beta-thalassemia is poor.
- At present, there is only one cure, and that is to carry out a bone marrow transplant. This involves taking special cells from a healthy person called “hematopoietic stem cells” that give rise to blood cells for the whole of a person's life, and giving them to the patient so that they that they are now able to make healthy red blood cells for their lifetime. However, the cell donor must be an immunologic match to the patient and for many people with beta-thalassemia, such donors are not available.
- Our approach to treating beta-thalassemia aims to genetically engineer a person’s hematopoietic stem cells (change the DNA inside the cell) to allow them to make healthy red blood cells using a technology that we have developed called "zinc finger nucleases,” or ZFNs. We plan to obtain stem cells from a beta-thalassemia patient, genetically engineer them by transiently exposing them to ZFNs, and then transplant them back into the same individual, making the patient their own donor. The genetic engineering is designed to replicate a situation observed in certain people with beta-thalassemia who have milder symptoms than others. Such patients have a much higher than average level of a “backup” form of beta-globin, called fetal globin, in their blood.
- All people make fetal globin while in utero and after birth, but in infancy the levels of fetal globin decrease and the child begins to make adult beta-globin. It is at this stage that the symptoms of beta-thalassemia become evident. However, if person with beta-thalassemia has high level of fetal globin, they will be spared the severe effects of the disease.
- We know that certain individuals who have an elevated level of fetal globin do so because they have a less active form of a gene called BCL11A that normally shuts down the production of fetal globin during infancy. Making use of this observation, our approach is to knock out BCL11A in a patient’s own stem cells, transplant them back into the patient to allow the production of fetal hemoglobin and, as a consequence, increase production of healthy red blood cells.
- In order to test drugs in humans investigators must consult with the US Federal Drug Administration (FDA) and ultimately submit data about the investigational drug to various regulatory bodies including the FDA as part of Investigational New Drug (IND) application. This past year, we held a meeting with the Center for Biologics Evaluation and Research of the FDA known as a “pre-IND” and received useful guidance on issues that we should address in preparing the IND filing. We also presented our program to the Recombinant DNA Advisory Committee of the NIH (RAC); our proposed preclinical safety assessment program and plan for the phase I clinical trial received unanimous approval from the RAC.
- Our work this year focused on two major deliverables that are necessary to achieve the goal of beginning a clinical trial of our approach. The first one relates to our ability to purify and efficiently genetically engineer a sufficient quantity of stem cells from a patient with beta-thalassemia. Working with healthy volunteers, and in a setting that is identical to the one we plan to use during our clinical trial, we have been able to consistently obtain sufficient quantities of hematopoietic stem cells to treat an individual with beta-thalassemia, and attain high levels of targeted genetic engineering in those cells.
- As part of a preclinical safety assessment program, we have initiated and completed a series of studies to determine whether the genetic engineering we perform has any unforeseen untoward consequences in the cell. When we have completed this effort, we aim to file the IND application with the FDA before the end of the year and, pending FDA acceptance, initiate the phase 1 clinical trial in 2015.
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.
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.
- 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.