Blood Disorders

Coding Dimension ID: 
278
Coding Dimension path name: 
Blood Disorders

In Utero Embryonic Stem Cell Transplantation to Treat Congenital Anomalies

Funding Type: 
New Faculty Physician Scientist
Grant Number: 
RN3-06532
ICOC Funds Committed: 
$2 836 742
Disease Focus: 
Blood Disorders
Pediatrics
Stem Cell Use: 
Embryonic Stem Cell
oldStatus: 
Active
Public Abstract: 
Many fetuses with congenital blood stem cell disorders such as sickle cell disease or thalassemia are prenatally diagnosed early enough in pregnancy to be treated with stem cell transplantation. The main benefit to treating these diseases before birth is that the immature fetal immune system may accept transplanted cells without needing to use immunosuppressant drugs to prevent rejection. Moreover, transplanting stem cells into the fetus—in which many stem cell types are actively multiplying and migrating—can promote similar growth and differentiation of the transplanted cells. Although this strategy works well in animal models, when applied clinically, the number of surviving cells in the blood (“engraftment”) has been too low to achieve a reliable cure. Our lab studies ways to improve engraftment, with the long-term goal of applying these strategies to treat fetuses with congenital blood disorders. In this application, we will use novel embryonic stem cells that may be better suited to differentiate into blood cells in the fetal environment. We will also test various approaches to improve the survival advantage of these stem cells in fetal organs that make blood cells. Finally, we will study the fetal immune system to determine how fetuses become tolerant to the transplanted cells. The experiments in this proposal will give us important information to design clinical trials to treat fetuses with common, currently incurable stem cell disorders.
Statement of Benefit to California: 
The long-term goal of our project is to develop safe and effective ways to perform prenatal stem cell transplantation to treat fetuses with congenital blood disorders, such as thalassemia and hemoglobin disorders. These diseases affect many California citizens. For example, hemoglobin disorders are so common that they are routinely screened for at birth (and prenatal screening is performed if there is a family history). Thalassemias are found more commonly in persons of Mediterranean or Asian descent and are therefore prevalent in our state’s population. Prenatal screening is routinely offered, especially to patients with a family history or those with an ethnic predisposition. Fetal stem cell transplantation would also benefit children with sickle cell disease, 2000 of which are born each year in the United States, and inborn errors of metabolism, which occur in 1 in 4000 births. Thus, once we develop reliable techniques to treat these disorders before birth, there will be an enormous potential to make a difference. Fetal surgery was pioneered in California and is performed only in select centers across the country. Therefore, once we have developed safe and effective therapies for fetuses with stem cell disorders, we also expect increased referrals of such patients to California. The convergence of our expertise in fetal therapies with those in stem cell biology carries great promise for finally realizing the promise of fetal stem cell transplantation.

Human endothelial reprogramming for hematopoietic stem cell therapy.

Funding Type: 
New Faculty Physician Scientist
Grant Number: 
RN3-06479
ICOC Funds Committed: 
$3 259 000
Disease Focus: 
Blood Disorders
Blood Cancer
Cancer
Stem Cell Use: 
Directly Reprogrammed Cell
Cell Line Generation: 
Directly Reprogrammed Cell
oldStatus: 
Active
Public Abstract: 
The current roadblocks to hematopoietic stem cell (HSC) therapies include the rarity of matched donors for bone marrow transplant, engraftment failures, common shortages of donated blood, and the inability to expand HSCs ex vivo in large numbers. These major obstacles would cease to exist if an extensive, bankable, inexhaustible, and patient-matched supply of blood were available. The recent validation of hemogenic endothelium (blood vessel cells lining the vessel wall give rise to blood stem cells) has introduced new possibilities in hematopoietic stem cell therapy. As the phenomenon of hemogenic endothelium only occurs during embryonic development, we aim to understand the requirements for the process and to re-engineer mature human endothelium (blood vessels) into once again producing blood stem cells (HSCs). The approach of re-engineering tissue specific de-differentiation will accelerate the pace of discovery and translation to human disease. Engineering endothelium into large-scale hematopoietic factories can provide substantial numbers of pure hematopoietic stem cells for clinical use. Higher numbers of cells, and the ability to grow cells from matched donors (or the patients themselves) will increase engraftment and decrease rejection of bone marrow transplantation. In addition, the ability to program mature lineage restricted cells into more primitive versions of the same cell lineage will capitalize on cell renewal properties while minimizing malignancy risk.
Statement of Benefit to California: 
Bone marrow transplantation saves the lives of millions with leukemia and other diseases including genetic or immunologic blood disorders. California has over 15 centers serving the population for bone marrow transplantation. While bone marrow transplantation can be seen as a standard to which all stem cell therapies should aspire, there still remains the difficulty of finding matched donors, complications such as graft versus host disease, and the recurrence of malignancy. While cord blood has provided another donor source of stem cells and improved engraftment, it still requires pooling from multiple donors for sufficient cell numbers to be transplanted, which may increase transplant risk. By understanding how to reprogram blood vessels (such as those in the umbilical cord) for production of blood stem cells (as it once did during human development), it could eventually be possible to bank umbilical cord vessels to provide a patient matched reproducible supply of pure blood stem cells for the entire life of the patient. Higher numbers of cells, and the ability to grow cells from matched donors (or the patients themselves) will increase engraftment and decrease rejection of bone marrow transplantation. In addition, the proposed work will introduce a new approach to engineering human cells. The ability to turn back the clock to near mature cell specific stages without going all the way back to early embryonic stem cell stages will reduce the risk of malignancy.

Antibody tools to deplete or isolate teratogenic, cardiac, and blood stem cells from hESCs

Funding Type: 
Tools and Technologies II
Grant Number: 
RT2-02060
ICOC Funds Committed: 
$1 869 487
Disease Focus: 
Blood Disorders
Heart Disease
Liver Disease
Stem Cell Use: 
Embryonic Stem Cell
iPS Cell
oldStatus: 
Active
Public Abstract: 
Purity is as important for cell-based therapies as it is for treatments based on small-molecule drugs or biologics. Pluripotent stem cells possess two properties: they are capable of self regeneration and they can differentiate to all different tissue types (i.e. muscle, brain, heart, etc.). Despite the promise of pluripotent stem cells as a tool for regenerative medicine, these cells cannot be directly transplanted into patients. In their undifferentiated state they harbor the potential to develop into tumors. Thus, tissue-specific stem cells as they exist in the body or as derived from pluripotent cells are the true targets of stem cell-based therapeutic research, and the cell types most likely to be used clinically. Existing protocols for the generation of these target cells involve large scale differentiation cultures of pluripotent cells that often produce a mixture of different cell types, only a small fraction of which may possess therapeutic potential. Furthermore, there remains the real danger that a small number of these cells remains undifferentiated and retains tumor-forming potential. The ideal pluripotent stem cell-based therapeutic would be a pure population of tissue specific stem cells, devoid of impurities such as undifferentiated or aberrantly-differentiated cells. We propose to develop antibody-based tools and protocols to purify therapeutic stem cells from heterogeneous cultures. We offer two general strategies to achieve this goal. The first is to develop antibodies and protocols to identify undifferentiated tumor-forming cells and remove them from cultures. The second strategy is to develop antibodies that can identify and isolate heart stem cells, and blood-forming stem cells capable of engraftment from cultures of pluripotent stem cells. The biological underpinning of our approach is that each cell type can be identified by a signature surface marker expression profile. Antibodies that are specific to cell surface markers can be used to identify and isolate stem cells using flow cytometry. We can detect and isolate rare tissue stem cells by using combinations of antibodies that correspond to the surface marker signature for the given tissue stem cell. We can then functionally characterize the potential of these cells for use in regenerative medicine. Our proposal aims to speed the clinical application of therapies derived from pluripotent cell products by reducing the risk of transplanting the wrong cell type - whether it is a tumor-causing residual pluripotent cell or a cell that is not native to the site of transplant - into patients. Antibodies, which exhibit exquisitely high sensitivity and specificity to target cellular populations, are the cornerstone of our proposal. The antibodies (and other technologies and reagents) identified and generated as a result of our experiments will greatly increase the safety of pluripotent stem cell-derived cellular therapies.
Statement of Benefit to California: 
Starting with human embryonic stem cells (hESC), which are capable of generating all cell types in the body, we aim to identify and isolate two tissue-specific stem cells – those that can make the heart and the blood – and remove cells that could cause tumors. Heart disease remains the leading cause of mortality and morbidity in the West. In California, 70,000 people die annually from cardiovascular diseases, and the cost exceeded $48 billion in 2006. Despite major advancement in treatments for patients with heart failure, which is mainly due to cellular loss upon myocardial injury, the mortality rate remains high. Similarly, diseases of the blood-forming system, e.g. leukemias, remain a major health problem in our state. hESC and induced pluripotent stem cells (collectively, pluripotent stem cells, or PSC) could provide an attractive therapeutic option to treat patients with damaged or defective organs. PCS can differentiate into, and may represent a major source of regenerating, cells for these organs. However, the major issues that delay the clinical translation of PSC derivatives include lack of purification technologies for heart- or blood-specific stem cells from PSC cultures and persistence of pluripotent cells that develop into teratomas. We propose to develop reagents that can prospectively identify and isolate heart and blood stem cells, and to test their functional benefit upon engraftment in mice. We will develop reagents to identify and remove residual PSC, which give rise to teratomas. These reagents will enable us to purify patient-specific stem cells, which lack cancer-initiating potential, to replenish defective or damaged tissue. The reagents generated in these studies can be patented forming an intellectual property portfolio shared by the state and the institutions where the research is carried out. The funds generated from the licensing of these technologies will provide revenue for the state, will help increase hiring of faculty and staff (many of whom will bring in other, out-of-state funds to support their research) and could be used to ameliorate the costs of clinical trials – the final step in translation of basic science research to clinical use. Only California businesses are likely to be able to license these reagents and to develop them into diagnostic and therapeutic entities; such businesses are at the heart of the CIRM strategy to enhance the California economy. Most importantly, this research will set the platform for future stem cell-based therapies. Because tissue stem cells are capable of lifelong self-renewal, stem cell therapies have the potential to be a single, curative treatment. Such therapies will address chronic diseases with no cure that cause considerable disability, leading to substantial medical expense. We expect that California hospitals and health care entities will be first in line for trials and therapies. Thus, California will benefit economically and it will help advance novel medical care.
Progress Report: 
  • Our program is focused on improving methods that can be used to purify stem cells so that they can be used safely and effectively for therapy. A significant limitation in translating laboratory discoveries into clinical practice remains our inability to separate specific stem cells that generate one type of desired tissue from a mixture of ‘pluripotent’ stem cells, which generate various types of tissue. An ideal transplant would then consist of only tissue-specific stem cells that retain a robust regenerative potential. Pluripotent cells, on the other hand, pose the risk, when transplanted, of generating normal tissue in the wrong location, abnormal tissue, or cancer. Thus, we have concentrated our efforts to devise strategies to either make pluripotent cells develop into desired tissue-specific stem cells or to separate these desired cells from a mixture of many types of cells.
  • Our approach to separating tissue-specific stem cells from mixed cultures is based on the theory that every type of cell has a very specific set of molecules on its surface that can act as a signature. Once this signature is known, antibodies (molecules that specifically bind to these surface markers) can be used to tag all the cells of a desired type and remove them from a mixed population. To improve stem cell therapy, our aim is to identify the signature markers on: (1) the stem cells that are pluripotent or especially likely to generate tumors; and (2) the tissue-specific stem cells. By then developing antibodies to the pluripotent or tumor-causing cells, we can exclude them from a group of cells to be transplanted. By developing antibodies to the tissue-specific stem cells, we can remove them from a mixture to select them for transplantation. For the second approach, we are particularly interested in targeting stem cells that develop into heart (cardiac) tissue and cells that develop into mature blood cells. As we develop ways to isolate the desired cells, we test them by transplanting them into animals and observing how they grow.
  • Thus, the first goal of our program is to develop tools to isolate pluripotent stem cells, especially those that can generate tumors in transplant recipients. To this end, we tested an antibody aimed at a pluripotent cell marker (stage-specific embryonic antigen-5 [SSEA-5]) that we previously identified. We transplanted into animals a population of stem cells that either had the SSEA-5-expressing cells removed or did not have them removed. The animals that received the transplants lacking the SSEA-5-expressing cells developed smaller and fewer teratomas (tumors consisting of an abnormal mixture of many tissues). Approaching the problem from another angle, we analyzed teratomas in animals that had received stem cell transplants. We found SSEA-5 on a small group of cells we believe to be responsible for generating the entire tumor.
  • The second goal of the program is to develop methods to selectively culture cardiac stem cells or isolate them from mixed cultures. Thus, in the last year we tested procedures for culturing pluripotent stem cells under conditions that cause them to develop into cardiac stem cells. We also tested a combination of four markers that we hypothesized would tag cardiac stem cells for separation. When these cells were grown under the proper conditions, they began to ‘beat’ and had electrical activity similar to that seen in normal heart cells. When we transplanted the cells with the four markers into mice with normal or damaged hearts, they seemed to develop into mature heart cells. However, these (human) cells did not integrate with the native (mouse) heart cells, perhaps because of the species difference. So we varied the approach and transplanted the human heart stem cells into human heart tissue that had been previously implanted in mice. In this case, we found some evidence that the transplanted cells differentiated into mature heart cells and integrated with the surrounding human cells.
  • The third goal of our project is to culture stem cells that give rise only to blood cells and test them for transplantation. In the past year, we developed a new procedure for treating cultures of pluripotent stem cells so that they differentiate into specific stem cells that generate blood cells and blood vessels. We are now working to refine our understanding and methods so that we end up with a culture of differentiated stem cells that generates only blood cells and not vessels.
  • In summary, we have discovered markers and tested combinations of antibodies for these markers that may select unwanted cells for removal or wanted cells for inclusion in stem cell transplants. We have also begun to develop techniques for taking a group of stem cells that can generate many tissue types, and growing them under conditions that cause them to develop into tissue-specific stem cells that can be used safely for transplantation.
  • Our program is focused on improving methods to purify blood-forming and heart-forming stem cells so that they can be used safely and effectively for therapy. Current methods to identify and isolate blood-forming stem cells from bone marrow and blood are efficient. In addition, we found that if blood-forming stem cells are transplanted, they create in the recipient an immune system that will tolerate (i.e., not reject) organs, tissues, or other types of tissue stem cells (e.g. skin, brain, or heart) from the same donor. Many living or recently deceased donors often cannot contribute these stem cells, so we need, in the future, a single biological source of each of the different types of stem cells (e.g., blood and heart) to change the entire field of regenerative medicine. The ultimate reason to fund embryonic stem cell and other pluripotent stem cell research is to create safe banks of predefined pluripotent cells. Protocols to differentiate these cells to the appropriate blood-forming stem cells could then be used to induce tolerance of other tissue stem cells from the same embryonic stem cell line. However, existing protocols to differentiation stem cells often lead to pluripotent cells (cells that generate multiple types of tissue), which pose a risk of generating normal tissue in the wrong location, abnormal tissue, or cancers called teratomas. To address these problems, we have concentrated our efforts to devise strategies to (a) make pluripotent cells develop into desired tissue-specific stem cells, and (b) to separate these desired cells from all other cells, including teratoma-causing cells. In the first funding period of this grant, we succeeded in raising antibodies that identify and eliminate teratoma-causing cells.
  • In the past year, we identified surface markers of cells that can only give rise to heart tissue. First we studied the genes that were activated in these cells, further confirming that they would likely give rise to heart tissue. Using antibodies against these surface markers, we purified heart stem cells to a higher concentration than has been achieved by other purification methods. We showed that these heart stem cells can be transplanted such that they integrate into the human heart, but not mouse heart, and participate in strong and correctly timed beating.
  • In the embryo, a group of early stem cells in the developing heart give rise to (a) heart cells; (b) cells lining the inner walls of blood vessels; and (c) muscle cells surrounding blood vessels. We identified cell surface markers that could be used to separate each of these subsets from each other and from their common stem cell parents. Finally, we determined that a specific chemical in the body, fibroblast growth factor, increased the growth of a group of pluripotent stem cells that give rise to more specific stem cells that produce either blood cells or the lining of blood vessels. This chemical also prevented blood-forming stem cells from developing into specific blood cells.
  • In the very early embryo, pluripotent cells separate into three distinct categories called ‘germ layers’: the endoderm, mesoderm, and ectoderm. Each of these germ layers later gives rise to certain organs. Our studies of the precursors of mesoderm (the layer that generates the heart, blood vessels, blood, etc.) led us by exclusion to develop techniques to direct ES cell differentiation towards endoderm (the layer that gives rise to liver, pancreas, intestinal lining, etc.). A graduate student before performed most of this work before he joined in our effort to find ways to make functional blood forming stem cells from ES cells. He had identified a group of proteins that we could use to sequentially direct embryonic stem cells to develop almost exclusively into endoderm, then subsets of digestive tract cells, and finally liver stem cells. These liver stem cells derived from embryonic stem cells integrated into mouse livers and showed signs of normal liver tissue function (e.g., secretion of albumin, a major protein in the blood). Using the guidelines of the protocols that generated these liver stem cells, we have now turned our attention back to our goal of generating from mesoderm the predecessors of blood-forming stem cells, and, ultimately, blood-forming stem cells.
  • In summary, we have continued to discover signals that cause pluripotent stem cells (which can generate many types of tissue) to become tissue-specific stem cells that exclusively develop into only heart, blood cells, blood vessel lining cells, cells that line certain sections of the digestive tract, or liver cells. This work has also involved determining the distinguishing molecules on the surface of various cells that allow them to be isolated and nearly purified. These results bring us closer to being able to purify a desired type of stem cell to be transplanted safely to generate only a single type of tissue.

Purified allogeneic hematopoietic stem cells as a platform for tolerance induction

Funding Type: 
Transplantation Immunology
Grant Number: 
RM1-01733
ICOC Funds Committed: 
$1 403 557
Disease Focus: 
Blood Disorders
Immune Disease
Muscular Dystrophy
Stem Cell Use: 
Adult Stem Cell
oldStatus: 
Active
Public Abstract: 
Blood and immune cells originate and mature in the bone marrow. Bone marrow cells are mixtures of blood cells at different stages of development, and include rare populations of blood-forming stem cells. These stem cells are the only cells capable of generating the blood system for the life of an individual. Bone marrow transplants (BMT) have been performed > 50 years, to replace a diseased patient’s blood system with that of a donor. Unfortunately, BMT have associated dangers which make the procedure high risk. Major risks include a syndrome called graft-versus-host disease (GvHD) which results when the donor’s mature blood cells attack the organs of the host, and toxicity from the treatments (radiation and chemotherapy) required to permit the donor cells to take in the recipient. These risk factor limit the use of BMT to only immediate life-threatening diseases. If made safer, BMT could cure many other debilitating diseases. In addition to being curative of blood cancers and non-malignant blood diseases (such as sickle-cell anemia), these transplants can cure autoimmune diseases, such as juvenile (type I) diabetes and multiple sclerosis. In addition, simultaneous BMT with organ transplants induces “tolerance” to the new organ, meaning the recipient will not reject the graft because the new blood system provides continuous proteins to re-train the recipient immune system not to attack it. This establishment of tolerance eliminates the need for drugs that suppress the immune system. In efforts to make BMT safer, our research has focused on isolating the blood stem cells away from the other bone marrow cells because transplants of pure stem cells do not cause GvHD. We developed the methods to purify the blood stem cells from mouse and human blood forming sources and showed in mice that transplants of blood stem cells can cure autoimmune disease and induce tolerance to solid organ transplants. However, this technology has not been tested in human clinical trials because safer methods must be developed that permit the stem cells to engraft in recipients. Our studies in mice show that we can replace the toxic drugs and radiation used to prepare recipients for BMT with non-toxic proteins that target the cells responsible for rejection of blood stem cells. The goal of this study is to translate this technology from mice to patient clinical trials. If successful, the studies will open the door to the use of blood stem cell transplants to the many thousands of patients who could benefit from this approach. The science behind achieving blood stem cell engraftment by the methods we propose look toward the future when blood stem cells and other tissues will be developed from pluripotent stem cells (ES, NT and iPS). We envision that the blood stem cells will induce tolerance to tissues derived from the same pluripotent stem cell line, in the same way that adult blood stem cells induce tolerance to organs from the same living donor.
Statement of Benefit to California: 
The science and the preclinical pathway to induce human immune tolerance in patients with degenerative diseases so that new blood and tissue stem cells can regenerate their lost tissues: For stem cell biology to launch the era of regenerative medicine, stem cells capable of robust and specific regeneration upon transplantation must be found, and methods for safe patient administration must be developed. In the cases where cell donation cannot come from the host, immune responses will reject the donor stem cells. Successful transplants of blood-forming stem cells (HSC) leads to elimination immune cells that reject organ grafts from donors. While bone marrow or cord blood transplants contain immune cells called T cells that will attack the host in a potentially lethal graft against host immune reaction, purified HSC do not do this. Pluripotent stem cells (ES, NT, iPS) can make all cell types in the body and provide a shortcut to find tissue and organ stem cells. Just as co-transplants of adult HSC prevent rejection of organs from the same donor, co-transplants of HSC derived from pluripotent cells should protect tissues derived from the same pluripotent line. Attack by a patient's blood system against one’s own organs cause the syndromes of autoimmune disease including juvenile diabetes, multiple sclerosis, and lupus. Transplanted HSC from donor mice genetically resistant to these diseases end the autoimmune attack permanently. We have in mice, substituted minimally toxic antibodies for toxic chemoradiotherapy to prepare the host for HSC transplants. Now it is time to take these advances to humans, with human immune cell and HSC-targeting antibodies. Long-term potential benefits to the state of California and its residents: The justification for Proposition 71 was to establish in California centers of research not funded adequately in the areas of stem cell biology and regenerative medicine. This research, if successful, is the platform for the application of stem cell biology to regenerative medicine. The costs for long-term immune suppression to patients who receive organ transplants are enormous, both in terms of quality of life, even survival, and healthcare resources. Add to that the lifetime costs of insulin to treat juvenile diabetes, with the inevitable premature diseases of compromised blood vessels and organs, and the shortened lifespan of patients. Add to that the costs to lives and the healthcare system of lupus, of multiple sclerosis, of other autoimmune diseases like juvenile and adult rheumatoid arthritis and scleroderma, and of muscular dystrophy, to mention a few, and the value to Californians and people everywhere is obvious. If our studies are successful, and if the clinical trials were first done in California, our citizens will have the first chance at successful treatment. Further, if these studies are successful - new antibodies, if produced by CIRM funds, will generate royalties which eventually will return to the state.
Progress Report: 
  • The successful transplantation of blood forming stem cells from one person to another can alter the recipient immune system in profound ways. The transplanted blood forming cells can condition the recipient to accept organs from the original stem cell donor without the need for drugs to suppress their immune system; and such transplantations can be curative of autoimmune diseases such as childhood diabetes and multiple sclerosis. Modification of the immune system in these ways is called immune tolerance induction.
  • Unfortunately, the current practice of blood stem cell transplantation is associated with serious risks, including risk of death in 10-20% of recipients. It has been a long-standing goal of investigators in this field to make transplantations safer so that patients that must undergo this procedure have better outcomes, and so that patients who need an organ graft or that suffer from an autoimmune disorder can be effectively treated by this powerful form of cellular therapy. The major objectives of our proposal are to achieve this goal by developing methods to prepare patients to accept blood forming stem cell grafts with reagents that specifically target cell populations in recipients that constitute the barriers to engraftment, and to transplant only purified blood forming stem cells thereby avoiding the potentially lethal complication call graft-vs-host disease.
  • The proposal has four Specific Aims. Aims 1 and 2 focus on development of biologic agents that specifically target recipient barrier cells. Aims 3 and 4 propose to test the reagents and approaches developed in the first two aims in mouse models to induce tolerance to co-transplanted tissues and to cure animals with Type 1 diabetes mellitus or multiple sclerosis. These aims have not changed in this reporting period.
  • One parameter of success in this project is the development of one or more biologic reagents that can replace toxic radiation and chemotherapy that can be used in human clinical trials by the end of the third year of funding (Aim 2). In this regard, significant progress has been made in the last year. A reagent critical to the success of donor blood forming stem cell engraftment is one that targets and eliminates the stem cells that already reside in the recipients. Recipient blood stem cells block the ability of donor stem cells to take. In our prior mouse studies we determined that a protein (antibody) that specifically targets a molecule on the surface of blood forming stem cells called CD117 is capable of eliminating recipient blood stem cells thus opening up special niches and allowing donor stem cells to engraft. This antibody was highly effective in permitting engraftment of purified donor blood stem cells in mice that lack a functional immune system. In this application we proposed to develop and test reagents that could target and eliminate human blood forming stem cells by targeting human CD117. This year we have identified and tested such an antibody which is manufactured by a third party. This anti-CD117 antibody has been evaluated in early clinical trials for an indication separate from our proposed use and appears to be non-toxic. In mice that we generated to house a human blood system, the antibody was capable eliminating the human blood forming stem cells. We plan to pursue the use of this reagent in a clinical trial as a non-toxic way to prepare children with a disease called severe combined immunodeficiency (SCID) for transplantation. Without a transplant children with SCID will die. The use of the anti-CD117 antibody and transplantation of purified blood forming stem cells has the potential to significantly reduce the complications of such transplants and improve the outcomes for these patients. The trial will be the first step to using this form of targeted therapy and serve as a pioneering study for all indications for which a blood forming stem cell transplant is needed, including the induction of immune tolerance.
  • The transplantation of blood forming stem cells from one individual to another can alter the recipient immune system in profound ways. Transplanted blood forming cells can condition the recipient to accept organs from the original stem cell donor without the need for drugs to suppress their immune system. Such transplantations can also be curative of autoimmune diseases such as childhood diabetes and multiple sclerosis. Modification of the immune system in these ways is called immune tolerance induction.
  • The major goal of this project is to enable the use of blood forming stem cell transplantation for the purpose of immune tolerance induction without unwanted side effects. The current practice of blood stem cell transplantation is associated with serious risks, including risk of death in 10-20% of recipients due to complications of transplant conditioning and graft-versus-host disease. We aim to abolish or reduce the risks of these transplantations so that this curative form of stem cell therapy can safely treat patients who need an organ graft or who suffer from an autoimmune disorder. To achieve our goals, we proposed the development of methods to prepare patients to accept blood forming stem cell grafts with reagents that specifically target recipient cell populations that constitute the barriers to engraftment, and to transplant only purified blood forming stem cells, thereby avoiding graft-versus-host disease.
  • The proposal has four Specific Aims. Aims 1 and 2 focus on development of biologic agents that specifically target recipient barrier cells. Aims 3 and 4 propose testing the reagents and approaches developed in the first two aims in mouse models to induce tolerance to co-transplanted tissues and to cure animals with muscular dystrophy, Type 1 diabetes mellitus and multiple sclerosis. These aims have not changed in this reporting period.
  • In this reporting period, significant progress has been made in the first three aims. In prior years we identified a biologic reagent that has the potential to replace toxic radiation and chemotherapy. Radiation and chemotherapy are used in transplantation to eliminate the blood forming stem cells of recipients because recipient stem cells block the ability of donor cells to take. The novel reagent we have studied is a protein, called a monoclonal antibody, which differs from radiation and chemotherapy because it specifically targets and eliminates recipient blood stem cells. This antibody reagent recognizes a molecule on the surface of blood stem cells called CD117. In years 1 and 2 we began testing of an anti-human CD117 (anti-hCD117) antibody in mice. Mice were engrafted with human blood cells and we showed that this antibody safely and specifically eliminated the human blood forming cells. These studies were proof-of-concept that the antibody is appropriate for use in human clinical trials.
  • This last year we were awarded a CIRM Disease Team grant to move the testing of this anti-hCD117 from the experimental phase in mice to a clinical trial for the treatment of children with a disease call severe combined immunodeficiency (SCID), also known as the “bubble boy” disease. Children with SCID are missing certain types of white blood cells (lymphocytes) so they cannot defend themselves from infections. Without a transplant, children with SCID will die. The use of the anti-CD117 antibody and transplantation of purified blood forming stem cells has the potential to significantly reduce the complications of such transplants and improve the outcomes for these patients. The use of the anti-CD117 antibody and transplantation of purified blood forming stem cells has the potential to significantly reduce the complications of such transplants and improve the outcomes for these patients. The trial will be the first step to using this form of targeted therapy and serve as a pioneering study for all indications for which a blood forming stem cell transplant is needed, including the induction of immune tolerance.
  • In the last year we have moved forward with the purification of skeletal muscle stem cells based upon labeling and sorting of primitive muscle cells that express an array of molecules on the cell surface. We have also transplanted a special strain of mice (mdx) that are a model for muscular dystrophy with blood forming stem cells from normal mouse donors. In the coming year we will perform simultaneous transplants of blood forming stem cells and skeletal muscle stem cells from normal donor mice into the mdx mice. We will determine if the blood stem cells permit the long-term survival of the muscle stem cells in recipients transplanted across histocompatibility barriers. Our ultimate goal is to achieve long-term recovery of muscle cell function in the recipients of these co-transplantations.
  • The transplantation of blood forming stem cells from one individual to another is widely used to treat patients with otherwise incurable cancers. Because such transplantations alter the recipient immune system in profound ways there are many other applications for this powerful form of therapy. The studies proposed in this grant focused on the use of blood stem cell transplantation for the purpose of immune tolerance induction. Tolerance induction in this setting means that transplantation of blood stem cells trains the body of a recipient to accept organs from same stem cell donor without the need for drugs to suppress their immune system. Blood stem transplantations can also reverse aberrant immune responses in individuals with autoimmune diseases such as childhood diabetes and multiple sclerosis.
  • In this project we sought to develop new ways to perform blood stem cell transplants to make the procedure safer and therefore more widely useable for a broad spectrum of patients. Transplants can be dangerous and sometimes fatal. Serious complications are caused by the toxic chemotherapy or radiation which are used to permit stem cells to engraft, and by a syndrome called graft-versus-host disease. Our research has aimed to replace the toxic treatments by testing novel reagents that more specifically target and eliminate the cells in recipients that constitute the barriers to stem cell engraftment. Furthermore, we perform transplantations of purified blood forming stem cells, and thus are able to avoid the problem of graft-versus-host disease which is caused by non-stem cell “passenger” immune cells in the donor grafts.
  • The proposal has four Specific Aims. Aims 1 and 2 focus on development of biologic agents that specifically target recipient barrier cells. Aims 3 and 4 propose testing the reagents and approaches developed in the first two aims in mouse models to induce tolerance to co-transplanted tissues and to cure animals with muscular dystrophy, Type 1 diabetes mellitus and multiple sclerosis. These aims have not changed in this reporting period.
  • Our prior reports highlighted our progress in Aim 2, which is now complete. Aim 2 focused on the identification and testing of an antibody directed against a molecule called CD117 present on surface of human blood stem cells. We demonstrated that this antibody can safely target and eliminate human blood stem cells in mice that had been previously engrafted with human cells. Based upon these studies we were awarded a CIRM Disease Team Grant, which will test this anti-human CD117 antibody in a clinical trial for the treatment of children with severe combined immune deficiency (SCID), also known as the “bubble boy” disease. Children with SCID are missing certain types of white blood cells (lymphocytes) so they cannot defend themselves from infections. Without a transplant, SCID patients usually die before the age of two. Our proposed clinical study has the potential to significantly improve the success of transplants for these patients. This clinical trial will be a first to test a reagent that specifically targets recipient stem cells to clear niche space and allow replacement therapy by healthy donor stem cells.
  • In the last year we have continued to make significant progress on Aims 1, 3 and 4. Aim 1 proposed to study how to improve blood stem cell engraftment using novel agents in mice that have intact immune systems. The anti-CD117 antibody discussed above works well in recipients that lack lymphocytes but not recipients with normal immune function. We have tested the anti-CD117 antibody in mice that lack more defined lymphocyte subsets to narrow down which lymphocyte type must be neutralized or eliminated. We have also tested novel reagents that inhibit the activity of specific immune cells and observed a stronger effect of the anti-CD117 antibody when co-administered with these reagents. For Aims 3 and 4, we have successfully achieved our goal of performing blood stem cell transplants that result in the stable mixing of blood cells between donor and recipients (called partial chimerism). For Aim 3, recipients are from a specialized mouse strain that models muscular dystrophy (MDX mice). We have transplanted purified skeletal muscle stem cells (SMSC) and observed engraftment of SMSC in MDX mice injected with genetically-matched SMSC. The next step is to test if co-transplants of blood stem cells plus SMSC from genetically mismatched donors will permanently engraft and expand in MDX recipients. For Aim 4, two mouse models are studied: (1) NOD mice which model childhood diabetes, and (2) mice that develop multiple sclerosis. We can successfully block the progression of disease in these animals with blood stem cell transplants. Our next steps are to apply the therapies developed in Aim 1 to these disease models. In the post-award period we will continue to carry out studies testing the novel approaches developed here in models of tolerance induction.

Inactivating NK cell reactivity to facilitate transplantation of stem cell derived tissue

Funding Type: 
Transplantation Immunology
Grant Number: 
RM1-01730
ICOC Funds Committed: 
$958 808
Disease Focus: 
Blood Disorders
Stem Cell Use: 
Adult Stem Cell
Cell Line Generation: 
Adult Stem Cell
oldStatus: 
Closed
Public Abstract: 
One of the great promises of stem cell research is that it will one day be possible to prepare replacement cells or organs from stem cells such as embryonic stem cells, which can be transplanted to patients to substitute for diseased or defective patient tissues or organs. Unfortunately, the immune system reacts against, and rejects, transplanted tissues that are not perfectly matched with the recipient. A promising approach around this problem is a two step procedure, in which a patient is first transplanted with blood stem cells of a specific type, and later with replacement tissues or cells derived from the same embryonic stem cells as the blood stem cells. If the blood cell transplant is successful, the patient’s blood cells will forever after be composed of a mixture of their own blood cells and the donor blood cells (“chimerism”). It is known that blood cell chimerism induces the recipient to be accept diverse types of grafts of the same source as the blood cells. Thus, the blood cell graft prepares the recipient to accept other types of grafts derived from the same stem cells. Unfortunately, blood stem cell grafts are themselves subject to a specific type of immune rejection, mediated by natural killer (NK) cells. Hence, successful application of the two step procedure requires the development of methods to prevent NK cells from rejecting blood cell grafts. We have developed evidence that NK cells can be induced to become tolerant of mismatched blood cell grafts. We propose studies to develop a general procedure to induce tolerance of a recipient’s NK cells to mismatched blood cell grafts. Using an experimental model, we will test whether the procedure facilitates transplantation of blood cells derived from embryonic stem cells, the generation of blood cell chimerism, and the subsequent transplantation of other tissues in a two step procedure.
Statement of Benefit to California: 
The proposed research is designed to provide novel methods to facilitate therapeutic transplantation of stem cell derived cells and tissues to patient’s suffering from numerous disorders and diseases. Such approaches will ultimately benefit millions of Californians suffering from diabetes, heart disease, neurodegenerative diseases, etc. Breakthroughs in stem cell research in California will also generate new industries, reducing joblessness and bolstering the California economy.
Progress Report: 
  • Our plan is to find ways to facilitate transplants of stem cell-derived cells to genetically different recipients. We propose to inactivate the rejection capability of natural killer cells, a white blood cell type that can reject transplanted cells. To explore this we started with a mouse model. Previously we generated evidence that there are one or more cell types in normal mice can inactivate the rejection capacity of natural killer cells. Our first aim is to identify that cell type to see it can be injected into mice to inactivate the natural killer cells. In the last year we have generated evidence that the relevant cell type is a non-blood cell type. We will now test various non-blood cell types to see which ones have this capability. The hope is that once the cell type has been identified, it could be generated from stem cells and injected into patients to facilitate transplants of other cell types derived from the same stem cells.
  • Our plan is to find ways to facilitate transplants of stem cell-derived cells to genetically different recipients. We propose to inactivate the rejection capability of natural killer cells, a white blood cell type that can reject transplanted cells. To explore this we started with a mouse model. Previously we generated evidence that there are one or more cell types in normal mice can inactivate the rejection capacity of natural killer cells. Our first aim is to identify that cell type to see it can be injected into mice to inactivate the natural killer cells. In the last year we discovered that the relevant cells include both blood cell types and non blood cell types. We showed however, that tolerance induced by non-blood cell types induces a more stable type of tolerance than that induced by blood cell types. We went on to develop a system in live mice to test subtypes of cells that can induce tolerance. Using this system, we could show that a heterogeneous mixture of blood cell types could induce tolerance. The system is suitable for testing specific blood cell, or non blood cell, types for their capacity to induce tolerance. We will undertake those studies in the coming months. The hope is that once the cell type has been identified, it could be generated from stem cells and injected into patients to facilitate transplants of other cell types derived from the same stem cells.
  • Stem cell therapy involves transfer of stem cells to patients. Transferring stem cells from a donor to a patient holds particular promise, because the stem cells may be reliably modified to rectify a specific defect and restore a particular function. This approach is limited by the patient’s immune response, which may reject the foreign transplant. Immune suppressive drugs can be used to prevent rejection of the stem cell, but these drugs leave patients sensitive to infections. We aim to find new, less debilitating methods to facilitate transplantation of foreign stem cell derived tissues. One approach is to establish a state of immune tolerance in the patient, by transplanting blood cells to generate blood cell mixing, called chimerism— that the patient tolerates. Once that is successful, stem cells of other tissues will also be tolerated, as long as they are from the same donor as the donor blood cells. Our efforts have focused on enabling patients to accept foreign blood cells—the first step in this approach. Natural killer cells are immune cells that are known to reject foreign blood cells, when the donor cells are mismatched at genes that control tissue rejection. We have shown that natural killer cells can be rapidly converted to a tolerant state when exposed to specific foreign cells from a donor. Subsequently, they will ignore transplants from the same donor. These findings suggested we could develop methods to readily prevent rejection of foreign blood cells by natural killer cells, but we also learned that this tolerance is fragile: when infections occurred, the tolerance could be reversed and the donor cells rejected. If this occurred after stem cell therapy, the donor stem cells would be rejected, abrogating the benefit of stem cell therapy. However, we also learned that this outcome did not occur in all circumstances. We learned that the fragile state of tolerance occurred when natural killer cells were exposed to foreign blood cells, but a distinct or deeper state of tolerance occurred when NK cells were exposed to other types of foreign cells, not of the blood cell lineages. In that case, tolerance was much less fragile and was sustained even when infections occurred. Our research is geared to identifying the key cell types that induce a deeper and less fragile form of tolerance of natural killer cells, in order to improve the effectiveness of therapeutic stem cell therapy.

Maternal and Fetal Immune Responses to In Utero Hematopoietic Stem Cell Transplantation

Funding Type: 
Transplantation Immunology
Grant Number: 
RM1-01718
ICOC Funds Committed: 
$1 324 229
Disease Focus: 
Pediatrics
Immune Disease
Blood Disorders
oldStatus: 
Active
Public Abstract: 
The immune system is the body’s defense system against disease and can recognize foreign cells. Because of this, stem cells and organs that are transplanted from one person to another are usually “rejected” by the immune system, forcing doctors to use powerful immune suppressive drugs with severe side effects. This natural defense system will therefore limit our ability to use stem cell therapies until we develop better solutions to prevent rejection (“induce tolerance”). We are developing a unique solution to this problem: if we transplant cells in utero, before the immune system is fully developed, we can educate the fetus to tolerate the foreign cells and avoid rejection without using any drugs. This strategy could be useful for many inherited stem cell disorders such as sickle cell disease, thalassemias, and muscular dystrophy. In addition, if tolerance to a particular donor is established, it may be used to transplant an organ (eg. kidney) from the same donor for other congenital anomalies. Many of these diseases can be diagnosed early in pregnancy and the surgical expertise for performing the transplants safely already exists. Although this strategy has been successful in animal models, cells transplanted in utero have mostly been rejected and we have been doing research to improve these results. Our lab has recently made the important discovery that the mother’s immune system is also responsible for rejection: we believe that cells from the mother help the immature fetal immune system develop faster and facilitate rejection of the transplanted cells. In this proposal, we will study this idea in both an animal model and in patients who have fetal surgery for other diseases. We will examine whether surgery leads to changes in the mother and fetus which prompt rejection of the transplanted cells. The strategy of treating stem cell disorders in utero to avoid rejection has a high likelihood of success and our team is uniquely qualified to perform a clinical trial of in utero stem cell transplantation once we have evidence of safety and efficacy in animal models. The experiments in this proposal will give us important information to design innovative treatments for common, currently incurable stem cell disorders.
Statement of Benefit to California: 
The long term goal of our team is to develop strategies for safe and effective stem cell transplants to cure fetuses with congenital stem cell disorders. Many common diseases can be diagnosed early in pregnancy and may potentially be treated with in utero stem cell transplantation. For example, blood stem cells may be used to treat sickle cell disease and thalassemias. Muscle stem cells may be used to treat muscular dystrophies and liver stem cells may be used to treat metabolic disorders. Furthermore, transplantation of blood stem cells may be used to develop tolerance to a particular donor so that organs can be transplanted without immunosupression. Recent progress in our understanding of immune interactions between the mother and fetus has brought us closer to realizing this goal. Congenital stem cell disorders are common and affect many patients in California. For example, hemoglobin disorders are so common that they are routinely screened in all babies (and prenatal diagnosis can be done if there is a family history): each year, 2000 children are born with sickle cell disease in the United States, 150 children in California alone (www.scdfc.org). Thalassemias are found more commonly in persons of Mediterranean or Asian descent and are therefore prevalent in our state’s population. Muscular dystrophy affects 1/3500 births and currently has no cure while inborn errors of metabolism affect 1/4000. Given that more than 500,000 children are born each year in California, the potential to make a difference is enormous. Furthermore, our studies will improve our knowledge about the uniquely tolerant state of the fetus and may allow us to then design treatments to improve tolerance in adult patients. in utero surgery was born in California and is performed in only select centers in the country. Therefore, once we have developed safe and effective therapies for stem cell disorders, we also expect increased referrals of such patients to California. The convergence of our expertise in fetal therapies with those in stem cell biology carries great promise for finally realizing the promise of in utero stem cell transplantation.
Progress Report: 
  • We are working on developing better treatments for patients with genetic stem cell disorders. Our strategy is to treat fetuses before birth with stem cell transplantation in order to induce tolerance to the foreign transplanted cells and avoid immunosuppression. We have noted that the mother’s immune system is involved in rejecting the cells that are transplanted into the fetus and are now studying how the fetal immune system responds to the transplant. This year, we learned that the fetal immune system becomes aware of the transplanted cells as early as 2 weeks after the transplant. However, T cells that would react to the transplant are also deleted, which is one way that the fetus learns to tolerate the foreign cells. We are also analyzing immune development in human patients who undergo fetal surgery. In our analysis of human patients, we learned that open fetal surgery increases the amount of maternal cells that have trafficked into the fetus. We are now studying whether the fetus becomes sensitized or tolerant to these maternal cells after surgery.
  • We are working on developing better treatments for patients with genetic stem cell disorders. Our strategy is to treat fetuses before birth with stem cell transplantation in order to induce tolerance to the foreign transplanted cells and avoid immunosuppression. We have noted that the mother’s immune system is involved in rejecting the cells that are transplanted into the fetus. We are now studying how the fetal immune system responds to the transplant. This year, we learned that, although the fetal immune system becomes tolerant to the transplanted cells by deleting T cells, it does not become tolerant by making regulatory T cells, which would be a more robust mechanism of tolerance. Therefore, the strategy of adding in more regulatory T cells may boost tolerance. We are also analyzing immune development in human patients who undergo fetal surgery. We have refined our assays to include the most relevant pathway by which maternal T cells recognize the foreign fetus and have found that, in addition to maternal T cells recognizing the fetus, fetal T cells are also capable of recognizing the mother. We are now understanding whether this recognition is enhanced after fetal surgery, which would indicate sensitization and possible rejection.
  • Our lab studies in utero hematopoietic stem cell transplantation as a way novel strategy to treat fetuses with congenital stem cell disorders. This method can potentially allow us to transplant genetically foreign stem cells without rejection by the immune system. In our previous experiments, we have determined that the mother's immune system can be a barrier to success but the fetal immune system does not reject the transplanted cells. In these experiments, we first used a mouse model and performed a detailed analysis of the fetal host immune response to transplantation to understand why rejection does not occur. We also analyzed human maternal and cord blood samples to understand human fetal immune maturation, to determine whether clinical applications will involve any immune response from the fetus or the mother. Our results are an exciting preclinical platform for considering in utero transplantation for fetuses with disorders such as hemoglobinopathies.

Role of Innate Immunity in hematopoeitic stem cell-mediated allograft tolerance

Funding Type: 
Transplantation Immunology
Grant Number: 
RM1-01709
ICOC Funds Committed: 
$1 746 684
Disease Focus: 
Blood Disorders
Stem Cell Use: 
Adult Stem Cell
oldStatus: 
Active
Public Abstract: 
The research proposed in this project has very high potential to identify new medications to boost the natural ability of stem cells to prevent rejection of transplanted organs. This is a very important goal, because patients that receive a life-saving transplanted organ must take toxic medications that increase their risk for cancer and serious infections. Experimental clinical trials have recently shown that stem cells given to patients at the same time as they receive their transplanted organ can engraft in the patient and prevent rejection of the transplanted organ, without the need to take immunosuppressive medications. The problem though is that the stem cells don't last forever; they are eventually rejected by the patient's own immune system. A promising target to prevent rejection of stem cells in patients is a group of primitive molecules that are receptors on stem cells, as well as many other cells in the body. These primitive receptors are called innate immune receptors and they provide the trigger for activation of a cascade of mechanisms that lead to rejection of the stem cells. If the trigger is not pulled, then the stem cells will not be rejected. Therefore, our proposal focuses on how to block activation of the rejection cascade so that stem cells are able to engraft in the patient and prevent rejection of transplanted organs, without the life-long use of toxic medications. We have extensive experience studying innate immune receptors and transplantation and therefore are poised to make significant advances in our understanding of how stem cells are rejected by signals that depend on innate immune receptors. Furthermore, once we identify which innate immune receptors are relevant, targeted rationale blockade of these receptors can be proposed.
Statement of Benefit to California: 
The proposed research will benefit the State of California and its residents by providing important knowledge about new ways to prevent rejection of transplanted organs. Currently, patients with transplanted organs must take life-long toxic medications to prevent rejection of their organs. This proposal will help develop ways to avoid the use of these toxic medications, while allowing life-saving organ transplants to survive in their new host. The use of stem cells in recipients of solid organ transplants is the first new breakthrough in decades for transplantation and therefore it is very important to try to optimize the use of stem cells to allow the survival of transplanted organs without toxic immunosuppressive medications.
Progress Report: 
  • Recent studies conducted first in animals and subsequently confirmed in humans have shown that tolerance to solid organ transplants can be achieved using donor-derived hematopoietic stem cells (HSCs). HSCs can induce tolerance by embedding in the recipient’s thymus. Once in the thymus they cause the deletion or inhibition of recipient cells that would otherwise cause rejection of a transplanted organ from the same donor. The coexistence of donor and host hematopoietic cells is called mixed chimerism and as long as the donor cells remain in the host, an allograft from the donor can be accepted without the need for immunosuppression.
  • Although many studies have shown that mixed chimerism can be obtained, donor tissue can still reject because the host responses to engrafted organs are not completely suppressed. Therefore, before HSC strategies can be widely used, additional refinements are needed to prevent activation of host responses. A logical approach, based on recent new information about the early activation events, involves targeting primitive receptors that are initial triggers of adaptive immunity – pattern recognition receptors (PRRs).
  • Pattern recognition receptors have recently been linked to activation of HSCs because it is known that HSCs undergo massive expansion and migration in inflammation. Two families of PRRs have been identified in HSCs - toll-like receptors (TLRs) and NOD-like receptors (NLRs). TLRs reside on cell membranes and NLRs are found within the cells HSCs. TLR/NLR-induced expansion and differentiation of HSCs results in their differentiation into activated cells that trigger rejection of donor cells (i.e., chimeric donor cells that would otherwise 'tolerize' host T cells are rejected). The end result of the PRR-induced activation of HSCs is loss of mixed chimerism and graft rejection. We have already shown that targeted blockade of specific PRRs can prevent ischemia-mediated tissue injury, inflammatory responses to the tissue injury, and also prolong survival of highly immunogenic allografts.
  • The overall objective of our project is to identify novel potential drug targets that promote HSC-mediated tolerance to transplanted solid organs. The idea is that signals mediated through PRRs interfere with HSC-mediated mixed chimerism and tolerance induction. We proposed to test our hypothesis in three interrelated aims. The first aim focused on testing the role of PRRs in the induction of tolerance. The second aim focused on the role of donor cells in the induction of host T cell unresponsiveness. The third aim focused on the role of HSC-mediated mixed chimerism on donor graft survival.
  • The first year of funding has already led to some important initial findings that are setting the stage for our understanding the role of hematopoietic stem cell induced tolerance. We believe that many, unavoidable, signals are activated during the course of HSC harvest and transplantation and that some of these signals reduce the ability of the transplanted HSCs to engraft in the host. Our initial findings suggest that if some of these signals are blocked, HSC engraftment, and transplant tolerance, can be enhanced. We are currently testing our initial exciting findings and progressing on the second and third aims of the study.
  • During the past funding period several significant advances were made towards each of the three aims of the proposal. We found that innate immune receptors were critically important to engraftment of hematopoietic stem cells and we have begun to understand how engraftment is enhanced in the absence of some of these receptors. We also discovered important aspects of the biology of the KO cells and how they might confer better engraftment. Our ongoing studies are focused on the mechanistic factors that lead to enhanced hematopoietic stem cell engraftment in our model.
  • Significant progress was made in the three aims of this project. Most important was the finding that we could markedly improve engraftment of foreign hematopoietic stem cells by removing certain receptors of the innate immune system from the donor stem cells. We have pursued an understanding of how cells without these innate immune receptors can be better at engraftment. It appears that T cells lacking these receptors are less able to proliferate in response to the foreign antigens.

Improving microenvironments to promote hematopoietic stem cell development from human embryonic stem cells

Funding Type: 
SEED Grant
Grant Number: 
RS1-00420
ICOC Funds Committed: 
$577 037
Disease Focus: 
Blood Disorders
Stem Cell Use: 
Embryonic Stem Cell
oldStatus: 
Closed
Public Abstract: 
Hematopoietic stem cells (HSC) have been used successfully to cure various life-threatening blood diseases. Yet, matching HSCs are not available for every patient. Human embryonic stem cells (hESC) may provide an unlimited source of HSCs for therapeutic use. However, hESC derived hematopoietic cells do not develop properly in those culture conditions that are currently used, and thereby their function is impaired. Hematopoietic cells that are derived from human ES cells lack the ability to self-renew, which is a prerequisite for the ability to generate blood cells for the individual’s lifetime. HSCs can only develop and function normally if they receive correct signal from their microenvironment, the stem cell niche. The goal of our proposal is take advantage of our knowledge of development of hematopoietic stem cells during embryogenesis, and mimic the environments where HSCs normally develop to provide the cues for proper HSC development in culture. We will attempt to mimic physiological HSC niches by deriving stroma lines from human placentas, which we have shown to be an important site for HSC development. We will further modify these lines by adding regulatory molecules that are known to aid HSC self-renewal, or inhibit molecules that might promote premature differentiation. Alternatively, we will use placental villi as a niche where ES cell derived hematopoietic cells could develop during culture. Subsequently, hESC derived cells are tested in animal models where human hematopoietic tissues have been implanted to provide an optimal environment for human HSCs to function. These studies are expected to shed light on the mechanisms that enable human HSCs to establish and maintain self-renewal ability and multipotency, and improve the differentiation of hESCs towards functional HSCs, which could be used to treat leukemias, other cancers, and inherited disease of the blood and immune system. To ensure hESC lines derived in different conditions respond in a similar way to these developmental cues, non-federally approved lines have to be used in this study, and thus governmental funding is not attainable for this project {REDACTED}.
Statement of Benefit to California: 
We aim to develop hematopoietic stem cells (HSC) from human ES cells (hESC) for ultimate theraoutic use for blood diseases. Only up to 50% of the patients that could be cured by HSC transplantation are able to receive this treatment, as matching donors are not available for every patient. If functional HSCs could be generated from hESCs, patients in California that suffer from leukemias or other acquired or inherited diseases of the blood and immune system could be treated. We aim to develop novel approaches to differentiate HSCs from hESCs by mimicking the physiological niches where human HSCs normally develop. Through these studies, we aim to understand what the critical properties in HSC microenvironment are that signal for HSCs to preserve their functionality. Identification of the regulatory cues that alter HSC fates between self-renewal and differentiation might also lead to innovative discoveries that could be developed into biotechnological or pharmaceutical products in California, thereby improving the industry and economy in California.
Progress Report: 
  • Our goal has been to improve the microenvironment where human embryonic stem cells (hESC) differentiate in order to generate functional hematopoietic stem/progenitor cells (HS/PC) in culture, with the ultimate goal to use these HS/PCs for the treatment of leukemias and other blood diseases. We have tested various human and mouse stroma lines for their ability to support expansion of multipotential human HS/PCs as well as hematopoietic specification from hESCs. So far mouse mesenchymal stem cells (MSC) have proven to provide the best supportive ability for human hematopoiesis. By combining embryoid body differentiation and co-culture on mouse MSC stroma, we have succesfully generated HS/PCs that phenotypically resemble bona fide human HSCs (CD34+CD38-CD90+CD45+). However, so far their differentiation ability has been biased toward myeloerythroid cells, with poor ability to generate B-cells in culture. Based on microarray data that we obtained from a related project supported by the CIRM New Faculty Award, we have identified molecular programs that are defective in hES derived HS/PCs. Future efforts will be directed in modifying the culture microenvironment as well as the cell intrinsic regulatory machinery in hES derived HS/PCs in order to improve their differentiation and self-renewal potential.
  • Our goal has been to improve the microenvironment where human embryonic stem cells (hESC) differentiate in order to generate functional hematopoietic stem/progenitor cells (HS/PC) in culture, with the ultimate goal to use these HS/PCs for the treatment of leukemias and other blood diseases. We have optimized a two step differentiation protocol that combines embryoid body differentiation and subsequent stroma co-culture to generate HS/PCs that exhibit the same phenotype as HSCs obtained from human hematopoietic tissues (CD34+CD38-CD90+CD45+). However, our findings indicate that the hESC derived HS/PCs have restricted developmental potential as compared to fetal liver or cord blood derived HS/PCs, and they senesce prematurely in culture, and are unable to generate B-cells . Our functional and molecular studies suggest that hES-derived HS/PCs resemble closely lineage-restricted progenitors found early in development in human hematopoietic tissues. Our recent studies have focused on exploring the possibility that another precursor that develops in the embryoid bodies could have lymphoid potential when placed in an appropriate microenvironment. Our preliminary data suggests that development of T-lymphocytes from hESCs in vitro may be feasible. Our future work will continue to focus on generating fully functional HSCs by improving the in vitro microenvironment where HS/PCs develop, and/or programming HSC transcriptional program using inducible lentiviral vectors.

Generation of long-term cultures of human hematopoietic multipotent progenitors from embryonic stem cells

Funding Type: 
SEED Grant
Grant Number: 
RS1-00280
ICOC Funds Committed: 
$538 211
Disease Focus: 
Blood Disorders
Stem Cell Use: 
Embryonic Stem Cell
oldStatus: 
Closed
Public Abstract: 
For many therapeutic reasons it is important to have available large numbers of blood cells. However, it is difficult to generate large numbers of specialized blood cells that have the ability to neutralize autoimmunity and response to tumor cell growth. In this study we would develop a technique that would allow the production of large numbers of different types of blood cells from human embryonic stem cells. For example, a subset of white blood cells, called dendrititc cells, is currently manipulated in the laboratory in a manner that allows them to attack cancer cells. The same cells also are altered in the laboratory to counter-act the development of autoimmune diseases. A problem with these experiments is that it is difficult to isolate large numbers of these cells, since they are relatively rare. With the technology that is described in this grant application we would be able to generate large numbers of such cells in the laboratory using as a starting point, human embryonic stem cells.
Statement of Benefit to California: 
In this study we would develop an approach that would allow the production of large numbers of different types of blood cells from human embryonic stem cells. For example, a subset of white blood cells, called dendrititc cells, is currently manipulated in the laboratory in a manner that allows them to attack cancer cells. The same cells also are altered in the laboratory to counter-act the development of autoimmune diseases. A problem with these experiments is that it is difficult to isolate large numbers of these cells, since they are relatively rare. With the technology that is described in this grant application we would be able to generate large numbers of such cells in the laboratory using as a starting point, human embryonic stem cells. The approach is novel and straightforward and could be applied immediately once it has been established.
Progress Report: 
  • A prominent subset of white blood cells, named CD4 helper T cells, are critical in modulating the immune response against viral and bacterial pathogens. During HIV infection, the CD4 compartment is selectively reduced, suppressing the activity and response of cytolytic CD8 T cells, needed to abolish cells infected with the virus. Pharmaceutical therapies have been developed but they are not consistently effective and multidrug resistant viral strains are increasingly prevalent. Similarly, in vitro manipulated human dendritic cells are now being explored to tolerize against autoimmune disease or to stimulate antitumor responses. However, the number of dendritic cells that can be isolated form patients using current technologies is small. Consequently, different approaches need to be developed to enhance T cell reconstitution. In principle, multipotent hematopoietic progenitors could be derived from hESCs without long-term in vitro culture. A drawback is that the number of human hematopoietic progenitors derived from human ES cell cultures is limited using current culture conditions. Consequently, a subset of studies involving in vitro manipulated human cells would be difficult to perform. The transduction of human progenitor cells can be achieved using a tissue culture system in which human cord blood progenitors are differentiated in the presence of stromal cells that express the Notch ligand DL-1 towards the T cell lineage. However, the efficiency by which human progenitor cells differentiate into the T lineage cells is low. In the original application we proposed to develop a novel strategy that would permit the generation of large numbers of human T cell progenitors (up to 109) from human hematopoietic stem cells. To accomplish this objective we would target a critical regulator of early hematopoieisis, named E2A. Indeed during the two years period funded by CIRM we have demonstrated that murine hematopoietic progenitors that overexpress an inhibitor of E2A, named Id2, can be grown indefinitely in culture without losing their ability to generate many different types of white blood cells in the laboratory. This strategy is unconventional since it would permit the growth and isolation of large numbers of T cell progenitors, which has not been achieved so far by conventional culture conditions. We will continue these studies and use the same strategy to establish a long-term culture of human hematopoietic progenitor cells. If successful the approach would enable clinicians to reconstitute the hematopoietic compartments of patients carrying invading pathogens, including HIV infected patients, with large numbers of T cells that either express either a wild-type TCR repertoire or TCRs with specificities directed against invading pathogens. I expect this to succeed since we have already achieved this objective using murine progenitors as demonstrated during the past two years using funds obtained form the CIRM.

Stem Cell Gene Therapy for Sickle Cell Disease

Funding Type: 
Disease Team Research I
Grant Number: 
DR1-01452
ICOC Funds Committed: 
$9 212 365
Disease Focus: 
Blood Disorders
Pediatrics
Stem Cell Use: 
Adult Stem Cell
Cell Line Generation: 
Adult Stem Cell
oldStatus: 
Active
Public Abstract: 
Sickle cell disease (SCD), which results from an inherited mutation in the hemoglobin gene that causes red blood cells to "sickle" under conditions of low oxygen, occurs with a frequency of 1/500 African-Americans, and is also common in Hispanic-Americans, who comprise up to 5% of SCD patients in California. The median survival based on 1991 national data was 42 years for males and 48 years for females. More recent data indicate that the median survival for Southern California patients with SCD is only 36 years, suggesting that serious problems exist regarding access to optimal medical care in this community. By twenty years of age, about 15% of children with SCD suffer major strokes and by 40 years of age, almost half of the patients have had central nervous system damage leading to significant cognitive dysfunction. These patients suffer recurrent damage to lungs and kidneys as well as severe chronic pain that impacts on quality of life. While current medical therapies for SCD can make an important difference in short-term effects, the progressive deterioration in organ function results in compromised quality of life and early deaths in ethnic populations who are generally adversely affected by health care disparity. Transplantation of bone marrow from a healthy donor as a source of new adult blood-forming ("hematopoietic") stem cells can benefit patients with SCD, by providing a source for life-long production of normal red blood cells. However, bone marrow transplant is limited by the availability of well-matched donors and the problems that arise from immune reactions between the cells of the donor and the patient. Thus, despite major improvements in clinical care of SCD patients, SCD continues to be a major cause of illness and early death. The stem cell therapy approach to be developed by this Disease Team will be used to treat patients with SCD by transplanting them with their own bone marrow adult hematopoietic stem cells that are genetically corrected by adding a hemoglobin gene that blocks sickling of the red blood cells. This approach has the potential to permanently cure this debilitating and common illness with significantly less toxicity than with a bone marrow transplant from another person. A clinical trial using stem cell gene therapy for patients with SCD will be developed to be performed by this Team. This multi-disciplinary Disease Team combines world-leading experts in stem cell gene therapy, clinical bone marrow transplantation and the care of patients with sickle cell disease. Successful use of stem cell gene therapy for sickle cell disease has the potential to provide a more effective and safe treatment for this disease to a larger proportion of affected patients.
Statement of Benefit to California: 
Development of methods for regenerative medicine using genetically-corrected human stem cells will result in novel, effective therapies that improve the health for millions of Californians and tens of millions of people world-wide. Sickle cell disease is an inherited disease of the red blood cells that results from a specific gene mutation. Sickle cell disease disproportionately afflicts poor minority patients in the State of California, causing severe morbidity, early mortality and high medical costs. We will develop a clinical trial to evaluate a novel treatment for patients with sickle cell disease, using their own adult blood-forming stem cells, after correcting the hemoglobin gene defect. Successful treatment of sickle cell disease using adult blood forming “hematopoietic” stem cells corrected with gene therapy may provide a clinically beneficial way to treat sickle cell disease with greater safety and wider availability than current options. The clinical trial to be developed will treat sickle cell disease patients from across the state of California through the network of institutions incorporated into this Disease Team. All scientific findings and biomedical materials produced from our studies will be publicly available to non-profit and academic organizations in California, and any intellectual property developed by this Project will be developed under the guidelines of CIRM to benefit the State of California.
Progress Report: 
  • The clinical complications of sickle cell disease are due to the inherited abnormality of the oxygen-carrying hemoglobin protein in red blood cells (RBC). The RBC are made from stem cells in the bone marrow and transplantation of stem cells from the bone marrow of a healthy donor to someone with sickle cell disease (SCD) can lead to significant improvements in their health. However, most people do not have a matched sibling donor, and transplants from unrelated donors have higher risks for complications, mainly due to immune reactions between the donor and the recipient.
  • The goal of this project is to bring to the clinic a trial of treating patients with SCD by transplanting them with their own bone marrow stem cells that have been modified in the lab by adding the gene for a version of human beta-globin that will act to inhibit sickling of the patient’s RBC (“anti-sickling” gene). This approach may provide a way to improve the health of people with SCD, with advantages over clinical treatments using transplantation of bone marrow stem cells from another person.
  • The major Year 1 Milestone was to demonstrate the feasibility of this approach, i.e. that the clinical cell product, the subject’s bone marrow stem cells modified with the anti-sickling gene, can be produced suitably for clinical transplantation and that enough of the anti-sickling hemoglobin is made to reverse sickling of RBC made from the gene-modified stem cells.
  • Studies done by the Laboratory component of our Disease Team showed that the gene transfer lentiviral vector we developed to insert the anti-sickling gene into bone marrow stem cells met pre-set technical criteria for: the amount of vector that can be made, its efficiency to insert the anti-sickling gene into human bone marrow stem cells, the levels of anti-sickling beta-globin protein made by the vector in RBC made from bone marrow stem cells, and the absence of adverse effects on the stem cells or their ability to make new RBC. These successful results allow advancement to the major lab focus for Years 2-3, pre-clinical efficacy and safety studies to support an IND application.
  • The Clinical/Regulatory component of our Disease team established the proposed network of California clinical hematology sites to obtain bone marrow samples from volunteer donors with SCD for laboratory research studies on cell product development (UCLA, CHLA and CHRCO). We put into place the necessary IRB-approved protocols to collect bone marrow samples at these sites to use for the laboratory research at UCLA and USC. This network obtained its first BM sample from a SCD donor on 3/18/2010 and a total of 15 over the year. These patient-derived samples have been truly essential to the advancement of the laboratory work because bone marrow from SCD patients is needed for studies to measure expression of the anti-sickling gene and improvement in RBC sickling.
  • The Clinical Regulatory component has also produced a complete first draft of the clinical trial protocol, which defines which specific people with SCD would be eligible for participation in this study, and the exact approach of the clinical study, including how the patients will be evaluated before the procedure, the details of the bone marrow harvest, stem cell processing and transplant processes, and how the effects of the procedure will be assessed. This protocol was conceived with input from the Team of physicians and scientists with expertise in clinical and experimental hematology, bone marrow transplantation, transfusion medicine, gene therapy and cell processing laboratory methods, regulatory affairs, and biostatistics.
  • These efforts provided sufficient laboratory data and definition of the clinical approach that we could have a pre-pre-IND exchange with the FDA (on 09/30/10). This interaction provided us the opportunity to receive initial guidance for three key areas that would comprise the IND application: the draft clinical protocol, the methods to make and characterize the gene-modified stem cell product for transplant, and the planned pre-clinical safety studies. The meeting was encouraging and informative.
  • In Year 2, our laboratory work will focus on determining the functional effects of inserting the anti-sickling gene into bone marrow stem cells from SCD donors on sickling of the RBC. We will begin to define the laboratory test methods that would be used to measure the results in the clinical trial (% of stem and blood cells with the gene, the amounts of anti-sickling beta-globin made, and the effects on RBC sickling). We will continue to design the studies to formally test vector safety (Toxicology study). The major goal is to advance to a pre-IND meeting with the FDA which should provide further guidance to finalize the design of the pre-clinical toxicology study and the clinical trial design. We will then be ready to implement the toxicology study and begin regulatory reviews of the protocol by local and federal authorities.
  • The clinical complications of sickle cell disease are due to the inherited abnormality of the oxygen-carrying hemoglobin protein in red blood cells (RBC). The RBC are made from stem cells in the bone marrow and transplantation of stem cells from the bone marrow of a healthy donor to someone with sickle cell disease (SCD) can lead to significant improvements in their health. However, most people do not have a matched sibling donor, and transplants from unrelated donors have higher risks for complications, mainly due to immune reactions between the donor and the recipient.
  • The goal of this project is to bring to the clinical trial of treating patients with SCD by transplanting them with their own bone marrow stem cells that have been modified in the laboratory by adding the gene for a version of human beta-globin that will act to inhibit sickling of the patient’s RBC (“anti-sickling” gene). This approach may provide a way to improve the health of people with SCD, with advantages over clinical treatments using transplantation of bone marrow stem cells from another person.
  • In the first 2 years of this project we were able to demonstrate the feasibility of this approach, i.e. that the clinical cell product, the subject’s bone marrow stem cells modified with the anti-sickling gene, can be produced suitably for clinical transplantation and that enough of the anti-sickling hemoglobin is made to reverse sickling of RBC made from the gene-modified stem cells.
  • Studies done by the Laboratory component of our Disease Team showed that the gene transfer lentiviral vector we developed to insert the anti-sickling gene into bone marrow stem cells met pre-set technical criteria for: the amount of vector that can be made, its efficiency to insert the anti-sickling gene into human bone marrow stem cells, the levels of anti-sickling beta-globin protein made by the vector in RBC, and the absence of adverse effects on the stem cells or their ability to make new RBC. These successful results allow advancement to the major lab focus for Year 3, safety studies to support an IND application.
  • The Clinical/Regulatory component of our Disease team established the proposed network of California clinical hematology sites to obtain bone marrow samples from volunteer donors with SCD for laboratory research studies on cell product development (UCLA, CHLA and CHRCO). We put into place the necessary IRB-approved protocols to collect bone marrow samples at these sites to use for the laboratory research at UCLA and USC. This network obtained its first BM sample from a SCD donor on 3/18/2010 and a total of 29 over 2 years. These patient-derived samples have been truly essential to the advancement of the laboratory work because bone marrow from SCD patients is needed for studies to measure expression of the anti-sickling gene and improvement in RBC sickling.
  • The Clinical Regulatory component has also produced a complete first draft of the clinical trial protocol, which defines which specific people with SCD would be eligible for participation in this study, and the exact approach of the clinical study, including how the patients will be evaluated before the procedure, the details of the bone marrow harvest, stem cell processing and transplant processes, and how the effects of the procedure will be assessed. This protocol was conceived with input from the Team of physicians and scientists with expertise in clinical and experimental hematology, bone marrow transplantation, transfusion medicine, gene therapy and cell processing laboratory methods, regulatory affairs, and biostatistics.
  • These efforts provided sufficient laboratory data and definition of the clinical approach that we could have a pre-IND meeting with the FDA (on 08/22/11). This interaction provided us the opportunity to receive guidance for three key areas that would comprise the IND application: the draft clinical protocol, the methods to make and characterize the gene-modified stem cell product for transplant, and the planned pre-clinical safety studies. The meeting was encouraging and informative.
  • In Year 3, our laboratory work will focus on performing pre-clinical safety studies (Toxicology study), qualifying end point assays and finalizing stem cell processing.
  • The clinical complications of sickle cell disease are due to the inherited abnormality of the oxygen-carrying hemoglobin protein in red blood cells (RBC). The RBC are made from stem cells in the bone marrow and transplantation of stem cells from the bone marrow of a healthy donor to someone with sickle cell disease (SCD) can lead to significant improvements in their health. However, most people do not have a matched sibling donor, and transplants from unrelated donors have higher risks for complications, mainly due to immune reactions between the donor and the recipient.
  • The goal of this project is to develop a clinical trial to treat patients with SCD by transplanting them with their own bone marrow stem cells that have been modified in the laboratory by adding the gene for a version of human beta-globin that will act to inhibit sickling of the patient’s RBC (“anti-sickling” gene). This approach may provide a way to improve the health of people with SCD, with advantages over clinical treatments using transplantation of bone marrow stem cells from another person.
  • In the first 2 years of this project we demonstrated the feasibility of this approach, i.e. that the clinical cell product, the subject’s bone marrow stem cells modified with the anti-sickling gene, can be produced suitably for clinical transplantation and that enough of the anti-sickling hemoglobin is made to reverse sickling of RBC made from the gene-modified stem cells. The Clinical/Regulatory component of our Disease Team established the proposed network of California clinical hematology sites to obtain bone marrow samples from volunteer donors with SCD for laboratory research studies on cell product development (UCLA, CHLA and CHRCO). We put into place the necessary IRB-approved protocols to collect bone marrow samples at these sites to use for the laboratory research at UCLA and USC. This network obtained its first BM sample from a SCD donor on 3/18/2010 and a total of 45 over 3 years. These patient-derived samples have been truly essential to the advancement of the laboratory work because bone marrow from SCD patients is needed for studies to measure expression of the anti-sickling gene and improvement in RBC sickling. The Clinical Regulatory component has also produced the clinical trial protocol, which defines which specific people with SCD would be eligible for participation in this study, and the exact approach of the clinical study, including how the patients will be evaluated before the procedure, the details of the bone marrow harvest, stem cell processing and transplant processes, and how the effects of the procedure will be assessed. This protocol was conceived with input from the Team of physicians and scientists with expertise in clinical and experimental hematology, bone marrow transplantation, transfusion medicine, gene therapy and cell processing laboratory methods, regulatory affairs, and biostatistics.
  • During the third year the Clinical Gene Therapy Laboratory component of the Team has demonstrated the feasibility of the stem cell processing procedure. Mimicking the future clinical scenario, the Lab was able to isolate stem cells from a largescale bone marrow harvest, insert the anti-sickling gene in adequate amount and recover the needed amount of stem cells that would be transplanted into the patient. The Clinical/Regulatory component of our Disease Team is focusing on validating all the assays that will be used during the clinical trial i.e. to characterize the final cell product and also the end-point assays to analyze the efficacy of this approach in patients. Another major focus during the third year has been safety and toxicology studies in a murine model of bone marrow transplant; the studies are still ongoing and will be completed in the next year. These successful results allow advancement to support an IND application in year 4.
  • CIRM DR1-01452 - Stem Cell Gene Therapy for Sickle Cell Disease
  • Scientific Progress in Year 4
  • The clinical complications of sickle cell disease are due to the inherited abnormality of the oxygen-carrying hemoglobin protein in red blood cells (RBC). The RBC are made from stem cells in the bone marrow and transplantation of stem cells from the bone marrow of a healthy donor to someone with sickle cell disease (SCD) can lead to significant improvements in their health. However, most people do not have a matched sibling donor, and transplants from unrelated donors have higher risks for complications, mainly due to immune reactions between the donor and the recipient.
  • The goal of this project is to develop a clinical trial to treat patients with SCD by transplanting them with their own bone marrow stem cells that have been modified in the laboratory by adding the gene for a version of human beta-globin that will act to inhibit sickling of the patient’s RBC (“anti-sickling” gene). This approach may provide a way to improve the health of people with SCD, with advantages over clinical treatments using transplantation of bone marrow stem cells from another person.
  • In the first 2 years of this project, we demonstrated the feasibility of this approach, i.e. that the clinical cell product, the subject’s bone marrow stem cells modified with the anti-sickling gene, can be produced suitably for clinical transplantation and that enough of the anti-sickling hemoglobin is made to reverse sickling of RBC made from the gene-modified stem cells. The Clinical/Regulatory component of our Disease Team established the proposed network of California clinical hematology sites to obtain bone marrow samples from volunteer donors with SCD for laboratory research studies on cell product development (UCLA, CHLA and CHRCO). We put into place the necessary IRB-approved protocols to collect bone marrow samples at these sites to use for the laboratory research at UCLA and USC. This network obtained its first BM sample from a SCD donor on 3/18/2010 and a total of 56 over 4 years. These patient-derived samples have been truly essential to the advancement of the laboratory work because bone marrow from SCD patients is needed for studies to measure expression of the anti-sickling gene and improvement in RBC sickling. The Clinical Regulatory component has also produced the clinical trial protocol, which defines which specific people with SCD would be eligible for participation in this study, and the exact approach of the clinical study, including how the patients will be evaluated before the procedure, the details of the bone marrow harvest, stem cell processing and transplant processes, and how the effects of the procedure will be assessed. This protocol was conceived with input from the Team of physicians and scientists with expertise in clinical and experimental hematology, bone marrow transplantation, transfusion medicine, gene therapy and cell processing laboratory methods, regulatory affairs, and biostatistics. It has now been approved by the UCLA Institutional Review Board and the Institutional Scientific Protocol review Committee, as well as the NIH Recombinant DNA Advisory Committee.
  • During the last 2 years the Clinical Gene Therapy Laboratory component of the Team has demonstrated the feasibility of the stem cell processing procedure. Mimicking the future clinical scenario, the Lab was able to isolate stem cells from a large scale bone marrow harvest, insert the anti-sickling gene in adequate amount and recover the needed amount of stem cells that would be transplanted into the patient. The Clinical/Regulatory component of our Disease Team validated all the assays that will be used during the clinical trial i.e. to characterize the final cell product and also the end-point assays to analyze the efficacy of this approach in patients. Another major focus during the third and fourth year has been safety and toxicology studies in a murine model of bone marrow transplant; these successful results allow advancement to support an IND application in the second quarter of 2014, with a goal of opening the trial in the third quarter of the year.

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