Blood Disorders

Coding Dimension ID: 
278
Coding Dimension path name: 
Blood Disorders
Funding Type: 
Strategic Partnership II
Grant Number: 
SP2-06902
Investigator: 
Type: 
PI
ICOC Funds Committed: 
$6 374 150
Disease Focus: 
Blood Disorders
Pediatrics
Stem Cell Use: 
Adult Stem Cell
oldStatus: 
Active
Public Abstract: 

β-thalassemia is a genetic disease caused by diverse mutations of the β-globin gene that lead to profoundly reduced red blood cell (RBC) development. The unmet medical need in transfusion-dependent β-thalassemia is significant, with life expectancy of only ~30-50 years despite standard of care treatment of chronic blood transfusions and iron chelation therapy. Cardiomyopathy due to iron overload is the major cause of mortality, but iron-overload induced multiorgan dysfunction, blood-borne infections, and other disease complications impose a significant physical, psychosocial and economic impact on patients and families. An allogeneic bone marrow transplant (BMT) is curative. However, this therapy is limited due to the scarcity of HLA-matched related donors (<20%) combined with the significant risk of graft-versus-host disease (GvHD) after successful transplantation of allogeneic cells.

During infancy, gamma-globin-containing fetal hemoglobin protects β-thalassemia patients from developing disease symptoms until gamma globin is replaced by adult-type β-globin chains. The proposed therapeutic intervention combines the benefits of re-activating the gamma globin gene with the curative potential of BMT, but without the toxicities associated with acute and chronic immunosuppression and GvHD. We hypothesize that harvesting hematopoietic stem and progenitor cells (HSPCs) from a patient with β-thalassemia, using genome editing to permanently re-activate the gamma globin gene, and returning these edited HSPCs to the patient could provide transfusion independence or greatly reduce the need for chronic blood transfusions, thus decreasing the morbidity and mortality associated with iron overload. The use of a patient’s own cells avoids the need for acute and chronic immunosuppression, as there would be no risk of GvHD. Moreover, due to the self-renewing capacity of HSPCs, we anticipate a lifelong correction of this severe monogenic disease.

Statement of Benefit to California: 

Our proposed treatment for transfusion dependent β-thalassemia will benefit patients in the state by offering them a significant improvement over current standard of care. β-thalassemia is a genetic disease caused by diverse mutations of the β-globin gene that lead to profoundly reduced red blood cell (RBC) development and survival resulting in the need for chronic lifelong blood transfusions, iron chelation therapy, and important pathological sequelae (e.g., endocrinopathies, cardiomyopathies, multiorgan dysfunction, bloodborne infections, and psychosocial/economic impact). Incidence is estimated at 1 in 100,000 in the US, but is more common in the state of California (incidence estimated at 1 in 55,000 births) due to immigration patterns within the State. While there are estimated to be about 1,000-2,000 β-thalassemia patients in the US, one of our proposed clinical trial sites has the largest thalassemia program in the Western United States, with a population approaching 300 patients. Thus, the state of California stands to benefit disproportionately compared to other states from our proposed treatment for transfusion dependent β-thalassemia.

An allogeneic bone marrow transplant (BMT) is curative for β-thalassemia, but limited by the scarcity of HLA-matched related donors (<20%) combined with the significant risk of graft-versus-host disease (GvHD) after successful transplantation of allogeneic cells. Our approach is to genetically engineer the patient’s own stem cells and thus (i) solve the logistical challenge of finding an appropriate donor, as the patient now becomes his/her own donor; and (ii) make use of autologous cells abrogating the risk of GvHD and need for acute and chronic immunosuppression.

Our approach offers a compelling pharmacoeconomic benefit to the State of California and its citizens. A lifetime of chronic blood transfusions and iron chelation therapy leads to a significant cost burden; despite this, the prognosis for a transfusion dependent β-thalassemia patient is still dire, with life expectancy of only ~30-50 years. Our proposed one-time treatment aims to reduce or eliminate the need for costly chronic blood transfusions and iron chelation therapy, while potentially improving the clinical benefit to patients, including the morbidity and mortality associated with transfusion-induced iron overload.

Progress Report: 
  • Summary of progress
  • Our CIRM-funded effort aims to develop a treatment for beta-thalassemia. Beta-thalassemia is an inherited genetic disorder that is caused by mutations (changes in the DNA) in a gene called beta-globin. This gene produces a protein that forms hemoglobin in red blood cells that carry oxygen to through the body. In an individual with beta-thalassemia, beta-globin is not produced (or is made in dramatically reduced quantities), and so the person does not make enough healthy red blood cells. The treatment, which is essential for life in these patients, is repeated blood transfusions (typically once a month or more frequently). The transfusion of blood this frequently results in a dangerous condition called “iron overload,” which must be treated with costly drugs. In general, the quality of life of many people with beta-thalassemia is poor.
  • At present, there is only one cure, and that is to carry out a bone marrow transplant. This involves taking special cells from a healthy person called “hematopoietic stem cells” that give rise to blood cells for the whole of a person's life, and giving them to the patient so that they that they are now able to make healthy red blood cells for their lifetime. However, the cell donor must be an immunologic match to the patient and for many people with beta-thalassemia, such donors are not available.
  • Our approach to treating beta-thalassemia aims to genetically engineer a person’s hematopoietic stem cells (change the DNA inside the cell) to allow them to make healthy red blood cells using a technology that we have developed called "zinc finger nucleases,” or ZFNs. We plan to obtain stem cells from a beta-thalassemia patient, genetically engineer them by transiently exposing them to ZFNs, and then transplant them back into the same individual, making the patient their own donor. The genetic engineering is designed to replicate a situation observed in certain people with beta-thalassemia who have milder symptoms than others. Such patients have a much higher than average level of a “backup” form of beta-globin, called fetal globin, in their blood.
  • All people make fetal globin while in utero and after birth, but in infancy the levels of fetal globin decrease and the child begins to make adult beta-globin. It is at this stage that the symptoms of beta-thalassemia become evident. However, if person with beta-thalassemia has high level of fetal globin, they will be spared the severe effects of the disease.
  • We know that certain individuals who have an elevated level of fetal globin do so because they have a less active form of a gene called BCL11A that normally shuts down the production of fetal globin during infancy. Making use of this observation, our approach is to knock out BCL11A in a patient’s own stem cells, transplant them back into the patient to allow the production of fetal hemoglobin and, as a consequence, increase production of healthy red blood cells.
  • In order to test drugs in humans investigators must consult with the US Federal Drug Administration (FDA) and ultimately submit data about the investigational drug to various regulatory bodies including the FDA as part of Investigational New Drug (IND) application. This past year, we held a meeting with the Center for Biologics Evaluation and Research of the FDA known as a “pre-IND” and received useful guidance on issues that we should address in preparing the IND filing. We also presented our program to the Recombinant DNA Advisory Committee of the NIH (RAC); our proposed preclinical safety assessment program and plan for the phase I clinical trial received unanimous approval from the RAC.
  • Our work this year focused on two major deliverables that are necessary to achieve the goal of beginning a clinical trial of our approach. The first one relates to our ability to purify and efficiently genetically engineer a sufficient quantity of stem cells from a patient with beta-thalassemia. Working with healthy volunteers, and in a setting that is identical to the one we plan to use during our clinical trial, we have been able to consistently obtain sufficient quantities of hematopoietic stem cells to treat an individual with beta-thalassemia, and attain high levels of targeted genetic engineering in those cells.
  • As part of a preclinical safety assessment program, we have initiated and completed a series of studies to determine whether the genetic engineering we perform has any unforeseen untoward consequences in the cell. When we have completed this effort, we aim to file the IND application with the FDA before the end of the year and, pending FDA acceptance, initiate the phase 1 clinical trial in 2015.
Funding Type: 
Early Translational IV
Grant Number: 
TR4-06823
Investigator: 
Type: 
PI
ICOC Funds Committed: 
$1 815 308
Disease Focus: 
Blood Disorders
Pediatrics
Stem Cell Use: 
Adult Stem Cell
Cell Line Generation: 
Adult Stem Cell
oldStatus: 
Active
Public Abstract: 

Disorders affecting the blood, including Sickle Cell Disease (SCD), are the most common genetic disorders in the world. SCD causes significant suffering and early death, despite major improvements in medical management and advances in understanding the complex disease-related biology. A bone marrow transplant (BMT) can greatly benefit patients with SCD, by providing a life-long source of normal red blood cells. However, BMT is limited by the availability of suitable donors and immune complications, especially for the more than 80% of patients who lack a matched sibling donor. An alternative treatment approach for SCD is to isolate some of the patient’s own bone marrow and then use gene therapy methods to correct the sickle gene defect in the blood stem cells before transplanting them back into the patient. The gene-corrected stem cells could make normal blood cells for the life of the patient, essentially eliminating the SCD. Such an approach would avoid the complications typically associated with transplants from non-matched donors. We will define the optimal techniques to correct the sickle gene mutation in the bone marrow stem cells to develop as a therapy for patients with SCD.

Statement of Benefit to California: 

Development of methods for regenerative medicine using stem cells will have widespread applications to improve the health and to provide novel, effective therapies for millions of Californians and tens of millions of people worldwide. Many severe medical conditions can be cured or improved by transplantation of blood-forming hematopoietic stem cells (HSC), including genetic diseases of blood cells, such as sickle cell disease and inborn errors of metabolism, cancer and leukemia, and HIV/AIDS. Precise genetic engineering of stem cells to repair inherited mutation may be the best way to correct genetic defects affecting the mature cells they produce. This project will advance methods to precisely repair the genetic defect that underlies sickle cell disease in hematopoietic stem cells, which can then be transplanted to ameliorate the disease. These advances will have direct and immediate applications to enhance current medical therapies of sickle cell disease and will more broadly help to advance the capacities for regenerative medicine. 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 people of the State of California.

Progress Report: 
  • Sickle-cell disease (SCD) is characterized by a single point mutation in the seventh codon of the beta-globin gene. Site-specific correction of the sickle mutation in adult bone marrow hematopoietic stem cells (HSCs) would allow for permanent production of normal red blood cells. Site-specific correction can be achieved using proteins called zinc-finger nucleases (ZFNs) which recognize and bind the region of the genome surrounding the sickle mutation. The ZFNs are able to create a break in the DNA which the cells repair using existing repair machinery. If, at the time of repair, a homologous donor template containing the corrective base is present, the cells' repair machinery can use this template and the resulting cell genome will contain the wild-type base instead of the sickle mutation. By doing this in hematopoietic setm cells, the cell is permanently corrected and each red blood cell (RBC) derived from this corrected stem cell will produce normal, non-sickle RBCs. In this report, we show efficient targeted cleavage by the ZFNs at the beta-globin locus with minimal off-target modification. In addition, we compare two different homologous donor templates (an integrase-defective lentiviral vector [IDLV] and a single-stranded DNA oligonucleotide [oligo]) to determine the optimal donor template. In both wild-type as well as sickle cell disease patient CD34+ HSCs, we are able to deliver the ZFN and donor templates and specifically correct the genome at rates of up to 30%. When these cells are differentiated into RBCs in vitro, we demonstrate that they are not altered in their differentiation capacity and are able to produce wild-type hemoglobin at high levels (35% of all hemoglobins) by HPLC. These results provide a strong basis for moving forward with this work as we begin our efforts to increase the number of treated cells to achieve clinical levels of corrected cells as well as characterize the ability of these cells to engraft a murine model in vivo. The progress made in this year is an exciting step towards a clinical therapy and potential treatment for sickle cell disease.
Funding Type: 
Strategic Partnership I
Grant Number: 
SP1-06477
Investigator: 
Institution: 
Type: 
PI
ICOC Funds Committed: 
$9 363 335
Disease Focus: 
Blood Disorders
Stem Cell Use: 
Adult Stem Cell
Cell Line Generation: 
Adult Stem Cell
oldStatus: 
Closed
Public Abstract: 

[REDACTED] plans to carry out a Phase 1/2 study to evaluate the safety and efficacy of [REDACTED] for the treatment of β-Thalassemia Major(BTM). [REDACTED] consists of autologous patient hematopoietic stem cells(HSC) that have been genetically modified ex vivo with a lentiviral vector that encodes a therapeutic form of the β-globin gene. [REDACTED] is administered through autologous hematopoietic cell transplant(HCT), with the goal of restoring normal levels of hemoglobin and red blood cell(RBC) production in BTM patients who are dependent on RBC transfusions for survival.

Because they cannot produce functional hemoglobin, BTM patients require lifelong RBC transfusions that cause widespread organ damage from iron overload. While hemosiderosis can be mitigated with chelation therapy, poor compliance, efficacy and tolerability remain key challenges, and a majority BTM patients die in their 3rd-5th decade. The only cure for BTM is allogeneic HCT, which carries a significant risk of mortality and morbidity from immune-incompatibility between the donor and recipient, and is hampered by the limited availability of HLA matched sibling donors.

By stably inserting functional copies of β-globin into the genome of a patient’s own HSC, treatment with [REDACTED] promises to be a one-time transformative therapy for BTM. The β-globin gene in the [REDACTED] vector carries a single codon mutation [REDACTED] that allows for quantitative monitoring of therapeutic globin production but that does not alter oxygen carrying capacity. Treatment with an earlier version of the vector has been shown to correct β-thalassemia in mice [REDACTED]. In a clinical trial [REDACTED], 3 BTM patients were treated–one of whom became transfusion independent 1 year after treatment and remains so 4 years later.

Given the prevalence of patients with a common BTM genotype in California, [REDACTED] plans to open at least 2, and up to 4, clinical sites in California. Development activities are on track to initiate the trial in 1H 2013, and to complete the trial with 2 years of follow-up within the award window. [REDACTED] has completed a pre-IND meeting with the FDA and successfully manufactured a GMP lot of [REDACTED] vector that is available for clinical use. The Company expects to complete all IND enabling activities by Q4 2012.

In the last year, the company has made scientific advances that have allowed for a significant improvement in the efficiency of HSC genetic modification that will be help ensure clinical efficacy in BTM. Moreover, through collaborations with contract manufacturers, [REDACTED] is now producing large scale GMP lots of vector, and is on track to qualify a GMP cell processing facility with commercial capabilities prior to study initiation. [REDACTED].

Statement of Benefit to California: 

The company expects to spend a major component of its financial resources conducting business within the state of California during the period of this CIRM award. Specifically: 1) we will have at least two clinical sites in California, and more likely up to 4 sites, 2) our viral vector manufacturing will occur in California, 3) our cell processing will occur in California, 4) we will hire several consultants and full-time employees within California to support the program. Overall, several million dollars will be spent employing the services of people, academic institutions, and other companies within the state of California.

Moreover, the disease we aim to treat occurs at a substantially greater rate of in California than other parts of the United States. As such, it is a significant public health concern, for which our therapy could provide a dramatically improved outcome and significant reduction in the lifetime cost of treatment, along with increased productivity. Due to the prevalence of the disease in California, if brought to the market, the pharmacoeconomic and social benefit of our therapy will accrue disproportionately to the state of California.

Funding Type: 
New Faculty Physician Scientist
Grant Number: 
RN3-06532
Investigator: 
ICOC Funds Committed: 
$2 661 742
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.

Progress Report: 
  • Our group works on developing methods for successful transplantation of blood stem cells to treat fetuses with genetic disorders such as sickle cell disease or thalassemia. In this grant, we are using novel stem cells that will differentiate into blood-forming cells and other techniques to improve the “engraftment” of these cells. This year, we focused on using a new technique that creates “space” in the bone marrow of the recipient using an antibody (ACK2) to deplete the host’s blood stem cells. In a mouse model, we showed that this antibody is very effective is improving the engraftment of transplanted blood stem cells. In fact, the treatment is more effective in the fetal environment than the adult. These findings were recently published and we are planning to use this strategy in the monkey model as a step toward clinical applications. We are also working on transplanting human blood stem cells into immunodeficient mouse fetuses to understand whether different sources of stem cells vary in their ability to make blood cells in this setting.
  • The goal of our grant is to optimize the strategy of in utero transplantation of hematopoietic stem cells, with the ultimate goal of treating fetuses with congenital stem cell disorders. Our project includes transplantation of HSC into both mice and non-human primates. This year, we have continued our work with in utero transplantation of human HSCs into the fetuses of an immunodeficient mouse strain. We have observed engraftment of the cells and differentiation into multiple blood lineages, including T cells, B cells, and regulatory T cells. We are working with other HSC types, such as those derived from iPS cells, to determine whether they can engraft in these mice as well. We are also testing different routes of administration of these cells, including into the placenta, which is a site of hematopoiesis. These experiments are designed with the goal of translating these discoveries to treat fetuses with genetic disorders such as thalassemia or sickle cell disease.
Funding Type: 
New Faculty Physician Scientist
Grant Number: 
RN3-06479
Investigator: 
ICOC Funds Committed: 
$3 084 000
Disease Focus: 
Blood Disorders
Blood Cancer
Cancer
Stem Cell Use: 
iPS Cell
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.

Progress Report: 
  • We aim to understand how blood stem cells develop from blood vessels during development. We are also interested in learning whether the blood-making program can be turned back on in blood vessel cells for blood production outside the human body. During the past year we have been able to extract and culture blood vessel cells that once had blood making capacity. We have also started experiments that will help uncover the regulation of the blood making program. In addition, we have developed tools to help the process of understanding whether iPS technology can "turn back time" in mature blood vessels and turn on the blood making program.
  • We aim to understand how blood stem cells develop from blood vessels during development. We are also interested in learning whether the blood-making program can be turned back on in blood vessel cells for bloodproduction outside the human body. During the past year we have made progress in understanding early human hematopoiesis such that we have designed new tools that may enable us to try and generate hematopoietic cells in culture. We have also gained ground in refining our screening strategy that we hope to adapt for finding new regulators of blood development that can be used for culturing hematopoietic stem cells.
Funding Type: 
Tools and Technologies II
Grant Number: 
RT2-02060
Investigator: 
Institution: 
Type: 
PI
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.
  • The main focus of our program is to improve methods to generate pure populations of tissue-specific stem cells that form only heart tissue or blood. Such tissue-specific stem cells are necessary for developing safe and effective therapies. If injected into patients with heart damage, heart-forming stem cells might be used to regenerate healthy heart tissue. Blood-forming stem cells are capable of regenerating the blood-forming system after cancer therapy and replacing a defective blood forming-system. We showed that blood-forming stem cells from a given donor induce in the recipient permanent transplant tolerance of all organs, tissues, or other tissue stem cells from the same donor. Therefore, having a single biological source of each of the different types of stem cells (e.g., blood and heart) would revolutionize regenerative medicine.
  • Our projects involve generating tissue-specific stem cells from pluripotent stem cells (PSCs), the latter of which are stem cells that can form all tissues of the body. PSCs (which include embryonic stem cells and induced pluripotent stem cells) can turn into all types of more specialized cells in a process known as “differentiation.” Because PSCs can be grown to very large numbers, differentiating PSCs into tissue-specific stem cells could lead to banks of defined tissue stem cells for transplantation into patients—the ultimate reason to conduct PSC research.
  • However, current methods to differentiate PSCs often generate mixtures of various cell types that are unsafe for injection into patients. Therefore, generating a pure population of a desired cell type from PSC is pivotal for regenerative medicine—purity is a key concern for cell therapy as it is with medications.
  • We have invented technologies to purify desired types of cells from complex cell populations, allowing us to precisely isolate a pure population of tissue-specific stem cells from differentiating PSCs for cell therapy. For instance, in our work on heart-forming cells, we developed labels for cells that progressively give rise to heart cells. We used these labeled cells to clarify the natural, stepwise, differentiation process that leads from PSCs to heart-forming stem cells, and finally to different cells within the heart. Exploiting these technologies to isolate desired cell types, we have completed the first step of turning human PSCs into heart-forming stem cells. In the laboratory, when we transplanted these heart-forming stem cells into a human heart, they integrated with the surrounding tissue and participated in correctly timed beating. In the future we hope to deliver heart-forming stem cells into the damaged heart to regenerate healthy tissue.
  • We have also attempted to turn PSCs into blood-forming stem cells by understanding the complex process of blood formation in the early embryo. As mentioned above, if blood-forming stem cells are transplanted into patients, they create in the recipient an immune system that will tolerate (i.e., not reject) other tissues and types of tissue stem cells (e.g., for skin or heart) from the same donor. Thus, turning PSCs into blood-forming stem cells will provide the basis for all regenerative medicine, whereby the blood-forming stem cells and the needed other tissue stem cells can be generated from the same pluripotent cell line and be transplanted together.
  • In parallel studies to those above, we have turned PSCs into liver-forming stem cells. In the embryo, the liver emerges from a cell type known as endoderm, whereas the blood and heart emerge from a different cell type known as mesoderm. We learned that PSCs could only be steered to form endoderm (and subsequently, liver) by diverting them away from the path that leads to mesoderm. Through this approach, we could turn human PSCs into endoderm cells (at >99% purity) and then into liver-forming stem cells that, when injected into the mouse liver, gave rise to human liver cells. This could be of therapeutic importance for human patients with liver damage.
  • Finally, we have developed methods to deplete PSCs from mixtures of cells differentiated from PSCs, because residual PSCs in these mixtures can form tumors (known as teratomas). These methods should increase the safety of PSC-derived tissue stem cell therapy.
  • In summary, we have developed methods to turn PSCs to tissue-specific stem cells that exclusively develop into only heart, blood cells, or liver cells. This work has 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.
Funding Type: 
Research Leadership
Grant Number: 
LA1-08014
Investigator: 
Name: 
Type: 
PI
ICOC Funds Committed: 
$5 174 715
Disease Focus: 
Blood Disorders
Stem Cell Use: 
Adult Stem Cell
Public Abstract: 
Bone marrow and peripheral blood transplantation utilizing blood stem cells can provide curative treatment for patients with cancers and non-cancerous diseases of the blood and immune systems. Such treatments can be curative because the stem cells contained within the bone marrow or peripheral blood of healthy donors are capable of replacing the entirety of the patient’s blood system and providing a new immune system which can eradicate the patient’s cancer cells. The application of blood stem cell transplantation could be applied to a much larger population of patients if methods could be developed to expand blood stem cells in vitro or in vivo. This would be particularly beneficial for the broadened application of human cord blood transplantation for the many patients who lack an immune-matched sibling or unrelated donor. Furthermore, a method to expand human blood stem cells in vivo could be highly beneficial for the thousands of patients with cancer who require toxic chemotherapy which frequently results in decreased blood counts, infections and bleeding complications. A systemic treatment (i.e. a shot) which could cause blood stem cells to grow and produce more blood cells in patients could markedly improve patient’s outcomes after they receive such chemotherapy in the curative treatment of cancer. However, the development of treatments capable of inducing human blood stem cells to grow in the body has been very slow, in part due to a lack of understanding of the processes which govern blood stem cell growth in general. In my laboratory, we have developed mouse genetic models which allow us to discover new proteins produced in the bone marrow (the “soil” where blood stem cells reside) which make blood stem cells grow. We have recently discovered that 2 proteins are secreted by blood vessels within the bone marrow and cause blood stem cells to grow rapidly following damage with radiation. We are currently in the process of developing one of these into a growth factor that we can deliver to patients via injection as a means to cause their blood stem cells to grow after cord blood transplantation or following chemotherapy treatment for cancer. In this proposal, we will utilize our unique mouse models to discover the additional growth factors that make blood stem cells grow and we will perform pre-clinical studies to test whether these newly discovered growth factors can cause human blood stem cells to grow in vitro and in vivo. This proposal has the potential to generate new understanding of how human stem cells grow in vivo and to facilitate the development of new therapies which can regenerate human blood stem cells and the blood system as a whole in patients.
Statement of Benefit to California: 
My research program has both basic science and pre-clinical components which I believe will benefit California in several important ways: First, my basic research program will contribute new fundamental knowledge in stem cell biology which will benefit students, fellows and faculty. My research will also synergize with other campus laboratories and other centers in California and will lead to collaborations and accelerated translation of these discoveries for regenerative medicine. Second, my research program has the potential to directly benefit patients in California. We have already discovered two niche-derived proteins which promote hematopoietic stem cell regeneration in vivo and are focusing substantial efforts now to develop these proteins as therapeutics for Phase I clinical trials. For example, we are developing one of the HSC regenerative factors which we discovered for a Phase I clinical trial to test its efficacy as a systemic therapy to accelerate cord blood engraftment and hematologic recovery in adult cord blood transplant patients. This has literal potential benefit for patients since approximately 10% of cord blood transplant patients die from complications of graft failure or delayed hematologic recovery. In addition, patients with cancer who receive myelosuppressive chemotherapy can potentially benefit from systemic administration of [REDACTED] or other HSC regenerative factors that we discover to accelerate hematologic recovery after chemotherapy. If we are able to show that administration of such regenerative factors can accelerate hematologic recovery in patients after chemotherapy, then remission rates for cancer patients may increase via more effective delivery of curative chemotherapy on time and to completion. Third, my research will provide new intellectual property. These inventions from my laboratory will be available for licensure to biotech or pharmaceutical companies in California. I have experience with licensing inventions from my laboratory to biotech companies and am eager to see my future inventions licensed to accelerate development for regenerative medicine. Fourth, my research program will provide new jobs and professional opportunities. At present, my research program provides partial or complete funding for more than 30 employees internally and more than 30 employees at our partner institutions in academia and biotechnology. I will also bring substantial federal research funding with me to California and will be hiring new fellows, technicians and faculty promptly upon my arrival. Taken together, I am hopeful that my research program will have a major benefit for the scientific community of California, for patients who may benefit from treatments we are developing, for the biotechnology community via the development of new intellectual property and for the larger economy via the creation of many new jobs. I sincerely look forward to the opportunity to bring my program to California.
Progress Report: 
  • During the past year, we have made substantial progress in the discovery and translation of niche-derived growth factors which stimulate hematopoietic stem cell regeneration. In one aim, we interrogated the activity of bone marrow niche cells which are responsible for the generation of bone cells which reside adjacent to hematopoietic stem cells. We discovered that deletion of cell death proteins from bone forming niche cells caused the protection of hematopoietic stem cells in mice following radiation exposure. We subsequently screened the bone forming cells for secreted proteins and discovered a soluble protein which is produced by bone forming cells and was strongly associated with the regeneration of hematopoietic stem cells. Further, treatment of hematopoietic stem cells with this bone-derived factor rescued hematopoietic stem cells from radiation-induced depletion in vitro. Systemic treatment of mice following lethal dose irradiation with this bone-derived factor potently accelerated the regeneration of hematoopoietic stem cells in vivo and markedly increased the survival of irradiated mice compared to controls. In complementary studies, we discovered a novel transmembrane receptor which is highly expressed on mouse and human hematopoietic stem cells and have shown that genetic deletion of this receptor significantly increased hematopoietic stem cell repopulating capacity following transplantation into recipient mice. This led to examination of the structure-activity-relationships of candidate small molecules with the transmembrane receptor. Based on this analysis, we functionally screened several candidate modulators of this receptor for activity against hematopoietic stem cells in culture. One candidate small molecule caused a significant expansion of hematopoietic stem cells in culture. Subsequently, we irradiated wild type mice with a lethal dose of irradiation and demonstrated that systemic administration of this candidate receptor modulator caused a significant acceleration in the recovery of hematopoietic stem cells in vivo, as well as the marked increase in survival of irradiated mice compared to controls. These 2 complementary studies provide the foundation for the development and translation of these hematopoietic regenerative growth factors for therapeutic purposes in the coming year.
Funding Type: 
Research Leadership
Grant Number: 
LA1-06917
Investigator: 
Institution: 
Type: 
PI
ICOC Funds Committed: 
$6 152 065
Disease Focus: 
Blood Disorders
Stem Cell Use: 
iPS Cell
oldStatus: 
Closed
Public Abstract: 

The development of induced pluripotent stem cell (iPSC) technology may be the most important advance in stem cell biology for the future of medicine. This technology allows one to generate a patient’s own pluripotent stem cells (PSCs) from skin or blood cells. iPSCs can then be reprogrammed to multiply and produce high quality mature cells for cell therapy. Because iPSCs
are derived from a patient's own cells, therapies that use them will not stimulate unwanted immune reactions or necessitate lifelong immunosuppression. If organs can be generated from iPSCs, many patients with organ failure awaiting transplants will be helped. The goal of this project is to further develop iPSC technology to bring about personalized regenerative medicine for treating intractable diseases such as cancers, viral infections, genetic blood disorders, and organ failure. Specifically, we would like to establish three major core programs for generating from iPSCs: personalized immune cells; an unlimited supply of blood stem
cells; and functional organs.

First, we will generate iPSC-derived immune cells that kill viruses and cancer cells. Current immunotherapy uses immune cells that are exhausted (have limited ability to function and proliferate) after they multiply in a test tube. To supply active nonexhausted immune cells, iPSCs will be generated from a patient’s immune cells that target tumor cells and infections and then redifferentiated to mature immune cells with the same targets.

Second, we aim to develop iPSC technology to generate blood stem cells that replenish all blood cells throughout life. Harvesting blood stem cells from a leukemia patient for transplantation back to the patient after chemotherapy and radiation has been challenging because few blood stem cells can be harvested and may be contaminated with cancer cells. Alternatively, transplanting blood stem cells from cord blood or another person requires genetic matching to prevent immune reactions. However, generating blood stem cells from a patient’s iPSCs may avoid contamination with cancer cells, immune reactions, and
the need to find a matched donor. Furthermore, we aim to generate iPSCs from a patient with a genetic blood disease, correct the genetic defect in the iPSCs, and generate from these corrected iPSCs healthy blood stem cells that may be curative when transplanted back into the patient.

Lastly, we will try to generate from iPSCs not just mature cells, but organs for transplantation, to potentially address the tremendous shortage of donated organs. In a preliminary study, we generated preclinical models that could not develop pancreases. When we injected stem cells into these models, they developed functional pancreases derived from the injected cells and survived to adulthood. We hope that within 10 years, we will be able to provide a needed organ to a patient by growing it from the patient’s own PSCs in a compatible animal.

Statement of Benefit to California: 

Cancer is the second leading cause of death, accounting for 24% of all deaths in the U.S. Nearly 55,000 people will die of the disease--about 150 people each day or one of every four deaths in California. In 2012, nearly 144,800 Californians will be diagnosed with cancer. We need effective treatment to cure cancer.

End-stage organ failure is another difficult disease to treat. Transplantation of kidneys, liver, heart, lungs, pancreas, and small intestine has become an accepted treatment for organ failure. In California, more than 21,000 people are on the waiting lists at transplant centers. However, one in three of these people will die waiting for transplants because of the shortage of donated
organs. While end-stage renal failure patients can survive for decades with hemodialysis treatment, they suffer from high morbidity and mortality. In addition, the high medical costs for increasing numbers of dialysis patients is a social issue. We need to find a way to increase organs that can be used for transplantation. In our proposed projects, we aim to use iPSC technology and recent discoveries to develop new methods for treating cancers,
viral infections, and organ failure. More specifically, we will pursue our recent discoveries using iPSCs to: (1) multiply person’s T cells that specifically target cancers and viral infections; (2) generate normal blood-forming stem cells that can be transplanted back into a patient to correct a blood disease (3) regenerate tissues and organs from a patient’s cells for transplantation back into that patient.

These projects are likely to benefit the state of California in several ways. Many of the methods, cells, and reagents generated by this research will be patentable, forming an intellectual property portfolio shared by the state and the institutions where the research is performed. 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. Most importantly, this research will set the platform for stem cell-based therapies. Because tissue stem cells are capable of lifelong
self-renewal, these therapies have the potential to provide a single, curative treatment. Such therapies will address chronic diseases that have no cure and cause considerable disability, leading to substantial medical expenses and loss of work. We expect that California hospitals and health care entities will be first in line for trials and therapies. Thus, California will benefit economically and the project will help advance novel medical care.

Progress Report: 
  • Adoptive immunotherapy with functional T cells is a potentially effective therapeutic strategy against various types of cancers and viral infections. A major challenge however lies with the “exhaustion” (loss of cytotoxic and proliferative capacities) of antigen-specific T cells during expansion in culture. For an effective adoptive immunotherapy, what we need is not the "exhausted" T cells, but large number of "young and active" CD8+ T cells that can kill tumors or virus infected cells efficiently. To address this issue, we generated induced pluripotent stem cells (iPSCs) from EBV-specific CD8+ T cells from an EBV-infected patient. We then redifferentiated these iPSCs into CD8+ T cells or we like to call them “rejuvenated” T cells since they are newly generated and highly proleferative. These rejT cells possessed antigen-specific killing activity and exhibited TCR gene rearrangement patterns identical to those of the original T cell clone from the patient. In order to confirm in vivo efficacy of these rejT cell, we innoculated EBV-induced tumors into immunodeficient mice and after confimation of tumor growth, we injected these rejT cells. Results indicated that these rejT cells eliminated tumors more efficiently than the original EBV-specific CD8+ T cells, thus confirming in vivo efficacy of these T cells.
  • Another aspect we worked on is generation of a functional organ in livestock animals. In the past, we have demonstrated generation of rat pancreas in mouse utilising a method called "blastocyst complementation". In ancillary work, we successfully generated exogenous-pig pancreata using the same principle. Whilst these studies prepared us to examine the feasibility of generating human PSC-derived pancreata in pancreatogenesis-disabled pigs, some ethical issues on making such “admix chimeras” have yet to be solved. A part of the concern comes from the possibility that human iPSC-derived cells contribute to neural or germ cells in chimeric animals. To overcome this issue, we attempted to restrict differentiation of PSC-derived cells into endodermal organs by introducing a gene that is important for the development of internal organs. When the expression of this gene was induced after transfer of embryo to foster mother, differentiation of ES-derived cells were directed toward interenal organs avoiding contribution of those cells in germ cells, skin and nervous systems. We termed this type of organ generation as "Targeted organ generation" and this should, in principle, reduce the ethical concern when making human-livestock chimeras.
  • In addition, we found that the inhibition of nuclear translocation of a molecule called b-CATENIN enhances conversion of mouse EpiSCs (non-chimera forming) to naive-like PSCs (chimera forming). Since most human ES/iPSCs are considered EpiSCs and non-chimera forming, the finding is of importance for the generation of human organs in ivestock animals.
Funding Type: 
Early Translational I
Grant Number: 
TR1-01273
Investigator: 
Type: 
PI
ICOC Funds Committed: 
$6 649 347
Disease Focus: 
Blood Disorders
Immune Disease
Stem Cell Use: 
iPS Cell
Cell Line Generation: 
iPS Cell
oldStatus: 
Closed
Public Abstract: 

The primary aim of this project is to develop treatments for incurable diseases of the blood and immune system. X-linked Severe Combined Immunodeficiency (X-SCID) and Fanconi anemia (FA) are two blood diseases where mutations in a single gene results in the disease. XSCID, more commonly known as the “bubble boy” disease, is characterized by a complete failure of the immune system, and typically results in early childhood fatality. The most common treatment for X-SCID is bone marrow transplant using a matched sibling donor. Unfortunately, the lack of suitable donors limits the application of this treatment. In 2000, the first gene therapy "success" resulted in X-SCID patients with a functional immune system. These trials were stopped when it was discovered that several patients in one trial had developed lymphoma, a blood related cancer resulting from unintended consequences of the therapy. FA is a disease where the stability of the genome is compromised and results in premature cell death and lethal anemia. Gene therapy trials for such patients have been largely unsuccessful due to the inability to culture the cells long enough for the correction of the gene. Like XSCID there is a shortage of suitable bone marrow donors for patients, thus development of treatments via other methods is warranted.

From this study and others we have learned 1) gene therapy can work to cure certain diseases, 2) adequate safeguards must be developed to prevent unintended cancer formation, and 3) we need better sources of matched cells and tissues to avoid the problems of rejection.

Our proposal will be using one of the most exciting new developments in regenerative medicine, that is the ability to reprogram a patient’s skin, or even hair follicle back to an induced pluripotent stem (iPS) cell, which is similar to embryonic stem cells, without involving embryo destruction. The iPS cell is a good candidate for repair of the specific genetic defects that cause diseases like X-SCID and FA. The reprogrammed, genetically corrected cells are a perfect match for transplantation therapy since they come from the patient. At this stage the corrected cells will be augmented with additional safety factors that work to avoid the downstream potential for cancer. These safe and genetically corrected cells will then be coaxed back into the cells that form the blood and immune systems and used for transplant therapy.

In this work we will be using mouse models that mimic the human diseases of X-SCID and FA and are amenable to treatment with human hematopoietic stem cells. We will be working with human patient and disease-specific cells to demonstrate the feasibility and evaluate the safety in a pre-clinical setting to advance these pioneering new techniques that combine the latest developments in regenerative medicine and gene therapy. Our proposed work will also benefit the successful stem cell based therapies for many other diseases like Parkinson’s and diabetes.

Statement of Benefit to California: 

The idea that embryonic stem cells (ES cells) have the ability to differentiate into a variety of cell types, tissues, and organs, opens the possibility of tissue engineering, replacement, and cell transplant therapies to cure diseases ranging from Parkinson’s, Alzheimer’s, diabetes, blood disorders and a host of other debilitating disorders. Rarely comes along a new technology that has the potential to make such a major impact on human health. Recently researchers have discovered methods to reprogram adult fibroblasts and skin cells back into a cell referred to as induced pluripotent stem cell (iPS) that appears to be indistinguishable from the pluripotent ES cell. This is accomplished without the need for embryo destruction and offers great potential to alleviate the problems of immune rejection in cell or tissue transplantation by allowing a patient’s own cells to be reprogrammed, expanded then used in therapeutic applications. The principle aim of this proposal is to develop new technologies that can be used to treat two specific devastating hematological disorders X-linked Severe Combined Immunodeficiency (X-SCID) and Fanconi Anemia (FA). Both are rare genetic diseases, and both have devastating effects on the immune and blood systems.

The successful development of therapies for these diseases will have an obvious and direct effect on the patients and their families affected by these diseases. From a broader perspective, the establishment of these regenerative medicine techniques has the potential to treat a vast array of disease like Parkinson’s, Alzheimer’s, diabetes and other blood disorders like thalassemia, Sickle cell anemia, and hemophilia. These diseases all have devastating effects on the patients afflicted, but they also place a tremendous burden on the State in terms of health care cost. Ever more, we need to spend state resources wisely and finding ways to reduce the continually increasing cost of long-term medical care is critical. The work proposed here seeks to do just that by creating outright cures for diseases that if left untreated require substantial and prolonged medical expenditures and incredible suffering for the patients and their families. In other regards keeping the state of California at the forefront of medical breakthroughs and strengthening our biomedical and biotechnology industries. We are a leading force in these fields, not only across the nation but also worldwide.

Progress Report: 
  • The primary aim of this project is to develop treatments for incurable diseases of the blood and immune system. X-linked Severe Combined Immunodeficiency (X-SCID) and Fanconi anemia (FA) are two blood diseases where mutations in a single gene results in the disease. XSCID, more commonly known as the “bubble boy” disease, is characterized by a complete failure of the immune system, and typically results in early childhood fatality. The most common treatment for X-SCID is bone marrow transplant using a matched sibling donor. Unfortunately, the lack of suitable donors limits the application of this treatment. In 2000, the first gene therapy "success" resulted in X-SCID patients with a functional immune system. These trials were stopped when it was discovered that several patients in one trial had developed lymphoma, a blood related cancer resulting from unintended consequences of the therapy. FA is a disease where the stability of the genome is compromised and results in premature cell death and lethal anemia. Gene therapy trials for such patients have been largely unsuccessful due to the inability to culture the cells long enough for the correction of the gene. Like XSCID there is a shortage of suitable bone marrow donors for patients, thus development of treatments via other methods is warranted.
  • From this study and others we have learned: 1) gene therapy can work to cure certain diseases, 2) adequate safeguards must be developed to prevent unintended cancer formation, and 3) we need better sources of matched cells and tissues to avoid the problems of rejection.
  • We proposed to reprogram a patient’s skin, or even hair follicle back to an induced pluripotent stem (iPS) cell, which is similar to embryonic stem cells, without involving embryo destruction. The iPS cell is a good candidate for repair of the specific genetic defects that cause diseases like X-SCID and FA. We have reprogrammed many patients cells to generate iPS. More importantly, we have gotten early hints of success in making hematopoietic stem cells and other blood cells from them. We have also started to make iPS cells from both X-SCID patients.
  • The primary aim of this project is to develop treatments for incurable diseases of the blood and immune system. X-linked Severe Combined Immunodeficiency (X-SCID) and Fanconi anemia (FA) are two blood diseases where mutations in a single gene results in the disease. XSCID, more commonly known as the “bubble boy” disease, is characterized by a complete failure of the immune system, and typically results in early childhood fatality. The most common treatment for X-SCID is bone marrow transplant using a matched sibling donor. Unfortunately, the lack of suitable donors limits the application of this treatment. In 2000, the first gene therapy "success" resulted in X-SCID patients with a functional immune system. These trials were stopped when it was discovered that several patients in one trial had developed lymphoma, a blood related cancer resulting from unintended consequences of the therapy. FA is a disease where the stability of a patients genome is compromised and results in premature cell death and lethal anemia. Gene therapy trials for such patients have been largely unsuccessful due to the inability to culture the affected cells long enough for the correction of the gene. Like XSCID there is a shortage of suitable bone marrow donors for patients, thus development of treatments via other methods is warranted.
  • From this study and others we have learned: 1) gene therapy can work to cure certain diseases, 2) adequate safeguards must be developed to prevent unintended cancer formation, and 3) we need better sources of matched cells and tissues to avoid the problems of rejection.
  • Our approach starts with a patient’s skin, hair follicle or other easily accessible adult cell/tissue sample and employs a newly developed and robust technique to safely reprogram these cells back to an induced pluripotent stem (iPS) cell fate, which is similar to that of embryonic stem cells in potential, but is patient specific thereby avoiding downstream problems of immune rejection. The iPS cell is a good candidate for repair of the specific genetic defects that cause diseases like X-SCID and FA. We have successfully reprogrammed cells from human patients of each of these diseases to generate iPS cell lines. We are employing the latest technology to perform genetic correction of these cells. In parallel we are advancing the state-of-the-art in developing reliable methods to direct the differentiation of these disease corrected stem cells into the appropriate therapeutic cell types capable of reconstituting the blood and immune systems and thereby effecting cures for these hematological diseases.
  • The primary aim of this project is to develop treatments for incurable diseases of the blood and immune system. X-linked Severe Combined Immunodeficiency (X-SCID) and Fanconi anemia (FA) are two blood diseases where mutations in a single gene results in the disease. XSCID, more commonly known as the “bubble boy” disease, is characterized by a complete failure of the immune system, and typically results in early childhood fatality. The most common treatment for X-SCID is bone marrow transplant using a matched sibling donor. Unfortunately, the lack of suitable donors limits the application of this treatment. In 2000, the first gene therapy "success" resulted in X-SCID patients with a functional immune system. These trials were stopped when it was discovered that several patients in one trial had developed lymphoma, a blood related cancer resulting from unintended consequences of the therapy. FA is a disease where the stability of a patients genome is compromised and results in premature cell death and lethal anemia. Gene therapy trials for such patients have been largely unsuccessful due to the inability to culture the affected cells long enough for the correction of the gene. Like XSCID, there is a shortage of suitable bone marrow donors for patients, thus development of treatments via other methods is warranted.
  • From this study and others we have learned: 1) gene therapy can work to cure certain diseases, 2) adequate safeguards must be developed to prevent unintended cancer formation, and 3) we need better sources of matched cells and tissues to avoid the problems of rejection.
  • Our approach starts with a patient’s skin, hair follicle or other easily accessible adult cell/tissue sample and employs a newly developed and robust technique to safely reprogram these cells back to an induced pluripotent stem (iPS) cell fate, which is similar to that of embryonic stem cells in potential, but is patient specific thereby avoiding downstream problems of immune rejection. The iPS cell is a good candidate for repair of the specific genetic defects that cause diseases like X-SCID and FA. We have successfully reprogrammed cells from human patients of each of these diseases to generate iPS cell lines. We are employing the latest technology to perform genetic correction of these cells. In parallel we are advancing the state-of-the-art in developing reliable methods to direct the differentiation of these disease corrected stem cells into the appropriate therapeutic cell types capable of reconstituting the blood and immune systems and thereby effecting cures for these hematological diseases.
  • This project is focused on developing treatments for incurable diseases of the blood and immune system. X-linked Severe Combined Immunodeficiency (X-SCID) and Fanconi anemia (FA) are two blood diseases where mutations in a single gene results in the disease. XSCID, more commonly known as the “bubble boy” disease, is characterized by a complete failure of the immune system, and typically results in early childhood fatality. The most common treatment for X-SCID is bone marrow transplant using a matched sibling donor. Unfortunately, the lack of suitable donors limits the application of this treatment. In 2000, the first gene therapy "success" resulted in X-SCID patients with a functional immune system. These trials were stopped when it was discovered that several patients in one trial had developed lymphoma, a blood related cancer resulting from unintended consequences of the therapy. FA is a disease where the stability of a patients genome is compromised and results in premature cell death and lethal anemia. Gene therapy trials for such patients have been largely unsuccessful due to the inability to culture the affected cells long enough for the correction of the gene. Like XSCID, there is a shortage of suitable bone marrow donors for patients, thus development of treatments via other methods is warranted. From this study and others we have learned: 1) gene therapy can work to cure certain diseases, 2) adequate safeguards must be developed to prevent unintended cancer formation, and 3) we need better sources of matched cells and tissues to avoid the problems of rejection.
  • Our approach starts with a patient’s skin, hair follicle or other easily accessible adult cell/tissue sample and employs newly developed and robust techniques to safely reprogram these cells back to an induced pluripotent stem (iPS) cell fate, which is similar to that of embryonic stem cells in potential, but is patient specific thereby avoiding downstream problems of immune rejection. The iPS cell is a good candidate for repair of the specific genetic defects that cause diseases like X-SCID and FA. To date, we have successfully reprogrammed cells from human patients of each of these diseases to generate iPS cell lines. We have also had success employing the latest technology to perform genetic correction of these cells, effectively repairing the DNA mutations that cause the diseases. In parallel we are advancing the state-of-the-art in developing reliable methods to direct the differentiation of these disease corrected stem cells into the appropriate therapeutic cell types capable of reconstituting the blood and immune systems and thereby effecting cures for these hematological diseases.
Funding Type: 
Transplantation Immunology
Grant Number: 
RM1-01733
Investigator: 
Institution: 
Type: 
PI
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.

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