Blood Disorders

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

Role of intracytoplasmic pattern recognition receptors in HSC engraftment

Funding Type: 
Basic Biology V
Grant Number: 
RB5-07379
ICOC Funds Committed: 
$615 639
Disease Focus: 
Blood Disorders
oldStatus: 
Closed
Public Abstract: 
The research performed through this project is very important for the fields of solid organ and bone marrow transplantation because it focuses on a potential new target to increase engraftment of stem cells. Currently, patients that receive stem cell transplants from a non-identical donor must take medications to suppress their immune system; otherwise the stem cells will be rejected. Stem cell trials have been extended to solid organ transplantation, where it has been shown that kidney transplants can be managed with little or no immunosuppressive medications when stem cells are given to the patient at the time of transplantation. In many cases though the stem cells are rejected and the patient must resume toxic medications. Our laboratory has been very interested in understanding ways to prevent the rejection of stem cells and has focused on a phylogenetically conserved group of cellular receptors called pattern recognition receptors. This project is focused on understanding how to prevent rejection of stem cells through modifications of these receptors. We hope to identify novel targets to prevent the rejection of stem cells in order to decrease the occurrence of graft versus host disease after bone marrow transplantation and also improve the opportunities for long-term transplant survival without the use of toxic immunosuppressive medications.
Statement of Benefit to California: 
The research we will undertake will benefit the State of California and its residents in two major ways. First it promises to define a novel targets to prevent rejection of stem cells that are transplanted into their new host. This is very important because rejection of hematopoietic stem cells is a major impediment to successful efforts at both bone marrow and solid organ transplantation. Patients needed life-saving solid organ transplants and patients that receive bone marrow transplants from donors that are not perfectly matched to them reject their grafts unless they take powerful medications to suppress their immune system. This project is focused on finding a way to help prevent the rejection of these grafts without the need for immunosuppressive medications. The second way the project will benefit the State of California is to provide new employment opportunities within the State at a large University that conducts biomedical research. This project will not only directly support the employment of three California citizens devoted to biomedical research, but the work it generates will support California-based biomedical science companies, California University personal and other local companies that employ California citizens that produce the reagents and the supplies used in the proposed studies.

Self-renewal and senescence in iPS cells derived from patients with a stem cell disease

Funding Type: 
Basic Biology II
Grant Number: 
RB2-01497
ICOC Funds Committed: 
$1 430 908
Disease Focus: 
Blood Disorders
Pediatrics
Stem Cell Use: 
iPS Cell
Cell Line Generation: 
iPS Cell
oldStatus: 
Active
Public Abstract: 
The discovery of induced pluripotent stem (iPS) cell technology promises to revolutionize our understanding of human disease and to allow the development of new cellular therapies for regenerative medicine applications. The ability to reprogram a patient's fibroblasts to iPS cells creates the opportunity to expand human cells with a specific genetic defect and to study that defect in a defined cell population, either to understand the basic biology of the disease or to study potential therapeutics. Furthermore, the genetic defects in iPS cells can be repaired and the iPS cells used as a source for cellular therapies after differentiation to specific cell lineages. Although tremendous strides have been made in recent years in treating human disease, replacing damaged tissue remains almost completely beyond our grasp. Harnessing human iPS stem cells for this purpose will open completely new areas of regenerative medicine. However, a limited understanding of iPS cell self-renewal and differentiation is a major roadblock in realizing this long-term goal. One shared characteristic of iPS cells and adult stem cells that reside in many of our tissues is the ability to self-renew. Self-renewal is the ability of a stem cell to divide and give rise to a daughter cell that is undifferentiated and capable of giving rise to all the same lineages as the parent stem cell. Senescence pathways – pathways that cause dividing cells to permanently stop dividing – represents a significant barrier in the reprogramming process to engineer new iPS cells. Understanding how iPS cells self-renew is critical for determining how to maintain these cells, how to differentiate them toward specific tissue lineages and how to expand more committed stem cells or progenitor cells in cell culture. In this proposal, we investigate the molecular mechanism of self-renewal and senescence in human iPS cells using skin cells isolated from patients with a defect in the enzyme telomerase. Telomerase is an enzyme complex expressed in embryonic stem cells, some tissue stem cells and in almost all human cancers. Most differentiated cells lack telomerase expression. Telomerase adds DNA repeats to structures at the ends of our chromosomes, termed telomeres. Telomeres are very important in protecting chromosome ends and in preventing chromosome ends from breaking down or sticking to other ends inappropriately. By maintaining telomeres, telomerase supports the ability of stem cells to divide a large number of times. People with telomerase mutations develop a stem cell disease – dykeratosis congenita. In this disease, patients have defects in skin, blood and lung – tissues that depend on tissue stem cell function to maintain these organs during life. We will reprogram skin cells from dyskeratosis patients to understand how senescence responses limit iPS cell self-renewal and differentiation to specific cell lineages.
Statement of Benefit to California: 
This proposal will benefit California and its citizen in two general ways. First, I have recruited new scientists to California from Texas and from Brazil to work on this proposal. These are new taxpayers and consumers, which will benefit local businesses. They would have been less likely to come to California in the absence of the CIRM program and its strong emphasis on human stem cell biology. Second, this novel grant will generate new intellectual property in the form of patents. These patents may in fact be licensed to California companies or be used to support the formation of new start-up companies. The growth of such companies has historically fueled much of the profound growth in California. The future of California is linked to new technologies in the stem cell, biotechnology and other technology.
Progress Report: 
  • Over the past year, we have analyzed five induced pluripotent stem (iPS) cell lines engineered from different individuals with a genetic stem cell disease. Dyskeratosis congenita is a rare disease affecting stem cells in multiple tissues. Patients with dyskeratosis congenita develop life-threatening bone marrow failure and pulmonary fibrosis, and are highly prone to cancers. In addition, they develop defects in skin, nails and many other organs. Dyskeratosis congenita is caused by mutations in an enzyme - telomerase - that is particularly important in stem cells. Telomerase elongates telomeres, caps that protect chromosome ends. If telomerase is defective, telomeres shorten and loss of the protective cap at telomeres can cause serious problems in stem cells. It has been very difficult to study this disease because isolating stem cells from dyskeratosis congenita patients is challenging. To overcome this problem, we engineered iPS cells from five patients. This is a way to change skin cells into cells that closely resemble embryonic stem cells - stem cells that can give rise to all tissues within the body. We studied these iPS cells from dyskeratosis congenita patients and found that the type of effects on telomerase were very specific and depended on the specific gene that is mutated in the patient. For example, mutations in TERT, the catalytic protein in the telomerase complex, resulted in a 50% reduction in telomerase activity in the patient's iPS cells. In contrast, mutations in the protein dyskerin, seen in the X-linked form of the disease, reduced telomerase activity by a much greater amount - 90% compared to controls. Mutations in another telomerase protein, TCAB1, left telomerase activity unaffected, but made the enzyme mislocalize within the nucleus. We studied how telomeres elongated with reprogramming of skin cells to iPSCs for each patient. Normal cells from healthy people show significant elongation of telomeres during the making of iPSCs, because telomerase is reactivated during this process. For TERT-mutant patients, elongation still happened, but elongation was significantly blunted. For dyskerin-mutant iPS cells and TCAB1-mutant iPS cells, elongation was completely blocked by the mutations and instead, telomeres shortened during this process and with passage in culture. Importantly, the much more severe telomere defect in dyskerin-mutant and TCAB1-mutant cells corresponds closely with the severity of the disease in the patients themselves. Our data show that iPS cells are a very accurate system for studying dyskeratosis congenita and revealed for the first time that the severity of the disease correlates with the severity of the telomerase defect in stem cells. These findings create new opportunities to study stem cell diseases in cell culture and to develop therapies that could specifically reverse the disease defect.
  • Over the past year, we have generated and analyzed new induced pluripotent stem (iPS) cell lines engineered from different individuals with a genetic stem cell disease. Dyskeratosis congenita is a rare disease affecting stem cells in multiple tissues. Patients with dyskeratosis congenita develop life-threatening bone marrow failure and pulmonary fibrosis, and are highly prone to cancers. In addition, they develop defects in skin, nails and many other organs. Dyskeratosis congenita is caused by mutations in an enzyme - telomerase - that is particularly important in stem cells. Telomerase elongates telomeres, caps that protect chromosome ends. If telomerase is defective, telomeres shorten and loss of the protective cap at telomeres can cause serious problems in stem cells. It has been very difficult to study this disease because isolating stem cells from dyskeratosis congenita patients is challenging. To overcome this problem, we engineered iPS cells from dyskeratosis congenita patients. This is a way to change skin cells into cells that closely resemble embryonic stem cells - stem cells that can give rise to all tissues within the body. We studied these iPS cells from dyskeratosis congenita patients and found that the type of effects on telomerase were very specific and depended on the specific gene that is mutated in the patient. Normal cells from healthy people show significant elongation of telomeres during the making of iPSCs, because telomerase is reactivated during this process. In iPS cells from patients with dyskeratosis congenita by contrast, telomere elongation during reprogramming is compromised. These findings create new opportunities to study stem cell diseases in cell culture and to develop therapies that could specifically reverse the disease defect.
  • Over the past year, we have generated and analyzed new induced pluripotent stem (iPS) cell lines engineered from individuals with a genetic stem cell disease. Dyskeratosis congenita is a rare disease affecting stem cells in multiple tissues. Patients with dyskeratosis congenita develop life-threatening bone marrow failure and pulmonary fibrosis, and are highly prone to cancers. In addition, they develop defects in skin, nails and many other organs. Dyskeratosis congenita is caused by mutations in an enzyme - telomerase - that is particularly important in stem cells. Telomerase elongates telomeres, caps that protect chromosome ends. If telomerase is defective, telomeres shorten and loss of the protective cap at telomeres can cause serious problems in stem cells. It has been very difficult to study this disease because isolating stem cells from dyskeratosis congenita patients is challenging. To overcome this problem, we engineered iPS cells from dyskeratosis congenita patients. This is a way to change skin cells into cells that closely resemble embryonic stem cells - stem cells that can give rise to all tissues within the body. We studied these iPS cells from dyskeratosis congenita patients and found that the type of effects on telomerase were very specific and depended on the specific gene that is mutated in the patient. Normal cells from healthy people show significant elongation of telomeres during the making of iPSCs, because telomerase is reactivated during this process. In iPS cells from patients with dyskeratosis congenita by contrast, telomere elongation during reprogramming is compromised. These findings create new opportunities to study stem cell diseases in cell culture and to develop therapies that could specifically reverse the disease defect.a

Development of Induced Pluripotent Stem Cells for Modeling Human Disease

Funding Type: 
New Cell Lines
Grant Number: 
RL1-00649
ICOC Funds Committed: 
$1 737 720
Disease Focus: 
Amyotrophic Lateral Sclerosis
Neurological Disorders
Autism
Blood Disorders
Rett's Syndrome
Neurological Disorders
Pediatrics
Stem Cell Use: 
iPS Cell
Cell Line Generation: 
iPS Cell
oldStatus: 
Closed
Public Abstract: 
Human embryonic stem cells (hESC) hold great promise in regenerative medicine and cell replacement therapies because of their unique ability to self-renew and their developmental potential to form all cell lineages in the body. Traditional techniques for generating hESC rely on surplus IVF embryos and are incompatible with the generation of genetically diverse, patient or disease specific stem cells. Recently, it was reported that adult human skin cells could be induced to revert back to earlier stages of development and exhibit properties of authentic hES cells. The exact method for “reprogramming” has not been optimized but currently involves putting multiple genes into skin cells and then exposing the cells to specific chemical environments tailored to hES cell growth. While these cells appear to have similar developmental potential as hES cells, they are not derived from human embryos. To distinguish these reprogrammed cells from the embryonic sourced hES cells, they are termed induced pluripotent stem (iPS) cells. Validating and optimizing the reprogramming method would prove very useful for the generation of individual cell lines from many different patients to study the nature and complexity of disease. In addition, the problems of immune rejection for future therapeutic applications of this work will be greatly relieved by being able to generate reprogrammed cells from individual patients. We have initiated a series of studies to reprogram human and mouse fibroblasts to iPS cells using the genes that have already been suggested. While induction of these genes in various combinations have been reported to reprogram human cells, we plan to optimize conditions for generating iPS cells using methods that can control the level of the “reprogramming” genes, and also can be used to excise the inducing genes once reprogramming is complete; thus avoiding unanticipated effects on the iPS cells. Once we have optimized the methods of inducing human iPS cells from human fibroblasts, we will make iPS cells from patients with 2 different neurological diseases. We will then coax these iPS cells into specific types of neurons using methods pioneered and established in our lab to explore the biological processes that lead to these neurological diseases. Once we generate these cell based models of neural diseases, we can use these cells to screen for drugs that block the progress, or reverse the detrimental effects of neural degeneration. Additionally, we will use the reprogramming technique to study models of human blood and liver disease. In these cases, genetically healthy skin cells will be reprogrammed to iPS cells, followed by introduction of the deficient gene and then coaxed to differentiate into therapeutic cell types to be used in transplantation studies in animal models of these diseases. The ability of the reprogrammed cell types to rescue the disease state will serve as a proof of principle for therapeutic grafting in
Statement of Benefit to California: 
It has been close to a decade since the culture of human embryonic stem (hES) cells was first established. To this day there are still a fairly limited number of stem cell lines that are available for study due in part to historic federal funding restrictions and the challenges associated with deriving hES cell lines from human female egg cells or discarded embryos. In this proposal we aim to advance the revolutionary new reprogramming technique for generating new stem cell lines from adult cells, thus avoiding the technical and ethical challenges associated with the use of human eggs or embryos, and creating the tools and environment to generate the much needed next generation of human stem cell lines. Stem cells offer a great potential to treat a vast array of diseases that affect the citizens of our state. The establishment of these reprogramming techniques will enable the development of cellular models of human disease via the creation of new cell lines with genetic predisposition for specific diseases. Our proposal aims to establish cellular models of two specific neurological diseases, as well as developing methods for studying blood and liver disorders that can be alleviated by stem cell therapies. California has thrived as a state with a diverse population, but the stem cell lines currently available represent a very limited genetic diversity. In order to understand the variation in response to therapeutics, we need to generate cell lines that match the rich genetic diversity of our state. The generation of disease-specific and genetically diverse stem cell lines will represent great potential not only for CA health care patients but also for our state’s pharmaceutical and biotechnology industries in terms of improved models for drug discovery and toxicological testing. California is a strong leader in clinical research developments. To maintain this position we need to be able to create stem cell lines that are specific to individual patients to overcome the challenges of immune rejection and create safe and effective transplantation therapies. Our proposal advances the very technology needed to address these issues. As a further benefit to California stem cell researchers, we will be making available the new stem cell lines created by our work.
Progress Report: 
  • Public Summary for: CIRM New Cell Line Project - Progress Report.
  • Our research team has been working over the last year on developing new human stem cell lines that are specifically useful for studying human diseases and developing new therapeutic strategies. Human embryonic stem (hES) cells were first established in 1998 and in the past decade have been shown to be capable to differentiating to a vast array of different cell types. This full developmental potential is termed pluripotency. Until recently these were the only established human cell type that could be robustly grown in the laboratory setting and still maintain full pluripotent developmental potential. In November of 1997, a new type of human pluripotent cell was created. By turning on a set of 4 genes, researchers succeeded in reprogramming human skin cells back into a cell type that appeared to have very similar properties and potential as the hES cell. These new stem cells are called induced pluripotent stem (iPS) cells in order to keep the name distinct from their embryonic derived counterpart. One of the scientific limitations of hES cells is the impracticality of generating patient or disease specific stem cell lines. This opportunity now becomes theoretically practical with the advent of human iPS cell line generation. We report here on significant progress demonstrating the practicality of generating disease-linked cellular models of human diseases.
  • We have identified 2 specific human neurological diseases that have a known, or strongly suggested genetic component, and have set about to generate disease-linked iPS cell lines. We have obtained skin cell samples from patients with these neurological diseases and have successfully reprogrammed them back to iPS cells. These disease-linked pluripotent stem cells have been carefully characterized and we have demonstrated that they do indeed behave very similar to existing hES cells and also to the genetically healthy control iPS cell lines that we have generated. Therefore the disease phenotype is not detrimental to reprogramming or proliferation as a stem cell. Furthermore, we have succeeded in coaxing these disease-linked iPS cells to turn into specific types of human neurons, the very cells that are suspected to be involved in the neurological disorders. We now have established a viable model for studying human neural disorders in the laboratory, and have already observed some potentially important functional differences between the disease-linked and control iPS generated neurons. In the coming year we will be evaluating the differences between the disease-linked and control neurons and investigating potential therapeutic approaches to stop or reverse the defects.
  • We have also been working on developing new methods for generating iPS cells that will make them more useful in clinical or pre-clinical settings where it is important that the original set of 4 genes used to reprogram the skin cells are removed once they have become iPS cells. Significant progress has been made in this regard and will be completed in the coming year. Looking forward we will also be applying this approach to generate human disease-linked iPS cells for specific hematological (blood) related disorders. The derivation of iPS-based models of hematological disorders will allow us develop gene therapy approaches to correct the disease causing defects and establish proof of principle for therapeutic approaches.
  • This research project is focused on developing new human stem cell lines that are specifically useful for studying human diseases and developing new therapeutic strategies. Human embryonic stem (hES) cells were first established in 1998 and in the past decade have been shown to be capable of differentiating to a vast array of different cell types. This full developmental potential is termed "pluripotency." Until recently these were the only established human cell types that could be robustly grown in the laboratory setting and still maintain full pluripotent developmental potential. In November 1997 a new type of human pluripotent cell was created. By turning on a set of 4 genes, researchers succeeded in reprogramming human skin cells back into a cell type that appeared to have very similar properties and potential as the hES cell. These new stem cells are called induced pluripotent stem (iPS) cells in order to keep the name distinct from their embryonic derived counterpart. One of the scientific limitations of hES cells is the impracticality of generating patient or disease specific stem cell lines. This opportunity now becomes theoretically practical with the advent of human iPS cell line generation. We report here on significant progress demonstrating the practicality of generating disease-linked cellular models of human diseases.
  • We have identified 2 specific human neurological diseases that have known, or strongly suggested, genetic components and have set about to generate disease-linked iPS cell lines. We have obtained skin cell samples from patients with these neurological diseases and have successfully reprogrammed them back to iPS cells. These disease-linked pluripotent stem cells have been carefully characterized and we have demonstrated that they do indeed behave very similar to existing hES cells and also to the genetically healthy control iPS cell lines that we have generated. Therefore, the disease phenotype is not detrimental to reprogramming or proliferation as a stem cell. Furthermore, we have succeeded in coaxing these disease-linked iPS cells to turn into specific types of human neurons, the very cells that are suspected to be involved in the neurological disorders. We now have established a viable model for studying human neural disorders in the laboratory, and have already observed some potentially important functional differences between the disease-linked and control iPS-generated neurons. Importantly, we have found defects in the function of disease-linked neurons that can be corrected in part following specific drug treatments. This discovery demonstrates the potential utility to use this method of modeling human diseases in the laboratory as a tool for understanding the detailed pathways, which might contribute to the development of the disease state and, importantly, as a target for screening potential therapeutic compounds that might be used to block or slow the progress of human neural disorders. In the coming year we will finalize our efforts on this project.
  • We have also succeeded in developing an improved method for the delivery of the reprogramming genes into the patient cells in order to become iPS cells. This method allows the reprogramming genes to be removed thus mitigating the potential for unwanted and potentially detrimental reactivation of these reprogramming genes subsequent to the iPS cell state. We have begun work using this new reprogramming methodology to generate iPS cell lines that are specifically linked to diseases of the blood and immune system. The new methodology appears to be working well and we anticipate completing the generation and characterization of these new disease-linked stem cell lines within the next year of this project.
  • This research project has been focused on developing new human stem cell lines that are specifically useful for studying human diseases and developing new therapeutic strategies. Human embryonic stem (hES) cells were first established in 1998 and in the past decade have been shown to be capable to differentiating of a vast array of different cell types. This full developmental potential is termed "pluripotency". Until recently these were the only established human cell type that could be robustly grown in the laboratory setting and still maintain full pluripotent developmental potential. In November of 2007, a new type of human pluripotent cell was created. By turning on a set of 4 genes, researchers succeeded in reprogramming human skin cells back into a cell type that appears to have very similar properties and potential as the hES cell. These new stem cells are called induced pluripotent stem (iPS) cells in order to keep the name distinct from their embryonic derived counterpart. One of the scientific limitations of hES cells is the impracticality of generating patient or disease specific stem cell lines. This opportunity now becomes theoretically practical with the advent of human iPS cell line generation. We report here on significant progress demonstrating the practicality of generating disease-linked cellular models of human diseases.
  • We have identified 2 specific human neurological diseases, Rett’s Syndrome and Schizophrenia that have a known, or strongly suggested genetic components, and have set about to generate disease-linked iPS cell lines. We have obtained skin cell samples from patients with these neurological diseases and have successfully reprogrammed them back to iPS cells. These disease-linked pluripotent stem cells have been carefully characterized and we have demonstrated that they do indeed behave very similar to existing hES cells and also to the healthy control iPS cell lines that we have generated. Therefore, the disease phenotype is not detrimental to reprogramming or proliferation as a stem cell. Furthermore, we have succeeded in coaxing these disease-linked iPS cells to turn into specific types of functional human neurons, the very cells that are suspected to be involved in the neurological disorders. We now have established a viable model for studying human neural disorders in the laboratory, and have already observed some potentially important functional differences between the disease-linked and control iPS generated neurons. Importantly, we have found defects in the function of disease-linked neurons that can be corrected in part following specific drug treatments. This discovery demonstrates the potential utility to use this method of modeling human diseases in the laboratory as a tool for understanding the detailed pathways that might contribute to the development of the disease state and importantly as a target for screening potential therapeutic compounds that might be used to block or slow the progress of human neural disorders.
  • We have also succeeded in developing an improved method for the delivery of the reprogramming genes into the patient cells in order to become iPS cells. This method combines all the of the reprogramming genes into a single cassette, and also allows the reprogramming genes to be removed thus mitigating the potential for unwanted and potentially detrimental reactivation of these reprogramming genes subsequent to the iPS cell state. We have demonstrated the success of this new reprogramming methodology to generate iPS cell lines that are specifically linked to a disease of the immune system. In addition to creating a panel of disease-linked iPS cell lines that are free of the externally introduced reprogramming transgenes, we have shown progress in achieving correction of the DNA mutation that leads to the disease state. Our extended research on these new disease specific iPS cell lines has shown utility for creating in vitro models of human neural disorders, and potential for genetically corrected patient specific iPS cell lines that could be used for cell based transplantation therapies.

Curing Hematological Diseases

Funding Type: 
Early Translational I
Grant Number: 
TR1-01273
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.

Prospective isolation of hESC-derived hematopoietic and cardiomyocyte stem cells

Funding Type: 
Comprehensive Grant
Grant Number: 
RC1-00354
ICOC Funds Committed: 
$2 636 900
Disease Focus: 
Blood Disorders
Heart Disease
Immune Disease
Stem Cell Use: 
Embryonic Stem Cell
oldStatus: 
Closed
Public Abstract: 
The capacity of human embryonic stem cells (hESCs) to perpetuate themselves indefinitely in culture and to differentiate to all cell types of the body has lead to numerous studies that aim to isolate therapeutically relevant cells for the benefit of patients, and also to study how genetic diseases develop. However, hESCs can cause tumors called teratomas when placed in the body and therefore, we need to separate potentially beneficial cells from hazardous hESCs. Thus, potential therapeutics cannot advance until the development of methodologies that eliminate undifferentiated cells and enrich tissue stem cells. In our proposal we hope to define the cell surface markers that are differentially expressed by committed hESC-derived stem cells and others that are expressed by teratogenic hESCs. To do this we will carry out a large screen of cell subsets that form during differentiation using a collection of unique reagents called monoclonal antibodies, many already obtained or made by us, to define the cell-surface markers that are expressed by teratogenic cells and others that detect valuable tissue stem cells. This collection, after filing for IP protection, would be available for CIRM investigators in California. We were the first to isolate mouse and human adult blood-forming stem cells, human brain stem cells, and mouse muscle stem cells, all by antibody mediated cell-sorting approaches. Antibody mediated identification of cell subsets that arise during early hESC differentiation will allow separation and characterization of defined subpopulations; we would isolate cells that are committed to the earliest lineage known to form multiple cell types in the body including bone, blood, heart and muscle. These cells would be induced to differentiate further to the blood forming and heart muscle forming lineages. Enriched, and eventually purified hESC-derived blood-forming stem cells and heart muscle stem cells will be tested for their potential capacity to engraft and improve function in animal models. Blood stem cells will be transplanted into immunodeficient mice to test their capacity to give rise to all blood cell types; and heart muscle stem cells will be transferred to mouse hearts that had an artificial coronary artery blockage, a model for heart attack damage. Finally, we will test the capacity of blood stem cell transplantation to induce transplantation tolerance towards heart muscle stem cells from the same donor cell line. Transplantation tolerance in this case means that the heart cells would be accepted as ‘self’ by the mouse that had it’s unrelated donor immune system replaced wholly or in part by blood forming stem cells from the same hESC line that gave rise to the transplantable heart stem cells, and therefore would not be rejected by it’s own immune system. This procedure would allow transplantation of beneficial tissues such as heart, insulin-producing cells, etc., without the use of immunosuppressive drugs.
Statement of Benefit to California: 
The principle objective of this proposal is to develop reagents which, in combinations, can identify and isolate tissue-regenerating stem cells derived from hESC lines. The undifferentiated hESCs are dangerous for transplantation into humans, as they cause tumors. We propose to prepare reagents that identify and can be used to delete or prospectively isolate these tumor-causing undifferentiated hESCs. HESC-derived tissue stem cells have the potential to regenerate damaged tissues and organs, and don’t cause tumors. We propose to develop reagents that can be used to identify and prospectively isolate pure human blood-forming stem cells derived from hESCs, and separately other reagents that can be used to identify and prospectively isolate pure heart-forming stem or progenitor cells. These “decontaminated” hESC-derived tissue stem cells may eventually be used to treat human tissue degenerative diseases. These reagents could also be used to isolate the same cells from somatic cell nuclear transfer (SCNT)-derived pluripotent stem cell lines from patients with genetic diseases. This procedure would enable us to analyze the effects of the genetic abnormalities on blood stem and progenitor cells in patients with genetic blood and immune system disorders, and on heart stem and progenitor cells in patients with heart disorders. The antibodies and stem cells (hESCs, tissue regenerating, etc) that will be isolated from patients with specific diseases will be invaluable tools that can be used to create model(s) for understanding the diseases and their progression. In addition, the antibodies and the stem cells generated in these studies are entities that could be patented or protected by copyright, forming an intellectual property portfolio shared by the state and the state institutions wherein the research was carried out. The funds generated from the licensing of these technologies will help pay back the state, will help support increasing faculty and staff (many of whom bring in other, out of state funds for their research), and could be used to ameliorate the costs of clinical trials. Only California businesses are likely to be able to license these antibodies and cells, to develop them into diagnostic and therapeutic entities; such businesses are the heart of the CIRM strategy to enhance the California economy. Most importantly, however, is that this research will lead to tissue stem cell therapies. Such therapies will address chronic diseases that cause considerable disability and misery, currently have no cure, and therefore lead to huge medical expenses. Because tissue stem cells renew themselves for life, stem cell therapies are one-time therapies with curative intent. We expect that California hospitals and health care entities will be first in line for trials and therapies, and for CIRM to negotiate discounts on such therapies for California taxpayers, thus California will benefit both economically and with advanced novel medical care.
Progress Report: 
  • The objectives of our proposal are the isolations of blood-forming and heart-forming stem cells from human embryonic stem cell (hESCs) cultures, and the generation of monoclonal antibodies (mAbs) that eliminate residual teratogenic cells from transplantable populations of differentiated hESCs. For isolation of progenitors, we hypothesized that precursors derived from hESCs could be identified and isolated using mAbs that label unique combinations of lineage-specific cell surface molecules. We used hundreds of defined mAbs, generated hundreds of novel anti-hESC mAbs, and used these to isolate and characterize dozens of hESC-derived populations. We discovered four precursor types from early stages of differentiating cells, each expressing genes indicative of commitment to either embryonic or extraembryonic tissues. Together, these progenitors are candidates to give rise to meso-endodermal lineages (heart, blood, pancreas, etc), and yolk sac, umbilical cord and placental tissues, respectively. Importantly, we have found that cells of the meso-endodermal population give rise to beating cardiomyocytes. We are currently enriching cardiomyocyte precursors from this population using cardiac-specific genetic markers, and are assaying the putative progenitors using electrophysiological assays and by transplantation into animal hearts (a test for restoration of heart function). In addition, we established in vitro conditions that effectively promote hESC-differentiation towards the hematopoietic (blood) lineages and isolated populations that resemble hematopoietic stem cells (HSCs) in both surface phenotype as well as lineage potentials, as determined by assays in vitro. We have generated hESC-lines that express the anti-apoptotic gene BCL2, and have found that these cells produce significantly greater amounts of hematopoietic and cardiac cells, because of their increased survival during culturing and sorting. We are currently isolating hematopoietic precursors from BCL2-hESCs and will test their ability to engraft in immunodeficient mice, to examine the capacity of hESC-derived HSCs to regenerate the blood system. Finally, we have utilized the novel mAbs that we prepared against undifferentiated hESCs, to deplete residual teratogenic cells from differentiated cultures that were transplanted into animal models. We discovered that following depletion teratoma rarely formed, and we expect to determine a final cocktail of mAbs for removal of teratogenic cells from transplantation products this year.
  • The main objective of our proposal is to isolate therapeutic stem cells and progenitors from human embryonic stem cells (hESCs) that give rise to blood and heart cells. Our approach involves isolation of differentiated precursor subset of cells using monoclonal antibodies (mAbs) and cell sorting instruments, and subsequent characterization of their respective hematopoietic and cardiomyogenic potential in culture as well as following engraftment into mouse models of disease. In addition, we aim to develop mAbs that specifically bind to undifferentiated hESCs for removal of residual teratoma-initiating cells from therapeutic cell preparations, to ensure transplantation safety.
  • We have made substantial advancement towards achieving these goals. First, we discovered that the initial differentiation of hESCs occurs through only 4-5 different progenitor types, of which one is destined to give rise to heart lineages. We purified this population using three novel cell surface markers, and found a significant enrichment of cardiomyocyte clones in colony formation assays that we developed. This subset also expressed particularly high levels of cardiac genes and was receptive to further differentiation into beating cardiomyocytes or vascular endothelial cells. When transplanted into immunodeficient mice these progenitors differentiated into ventricular myocytes and vascular endothelial cells. In the coming year we will perform transplantation experiments to evaluate whether they improve the functional outcome of heart infarction in hearts of mice. Second, we have optimized cell culture conditions and cell surface markers to sort hematopoietic progenitors derived from hESCs. We have also begun to transplant these populations into immunodeficient mouse recipients to identify blood-reconstituting hematopoietic populations. Third, we identified 5 commercial and 1 custom mAbs that are specific to human pluripotent cells (hESCs and induced pluripotent cells). We are currently testing the capacity of combinations of 3 pluripotency surface markers to remove all teratoma-initiating cells from transplanted differentiated cell populations. In summary, we expect provide functional validation of the blood and heart precursor populations that we identified from hESCs by the end term of this grant.
  • The main objective of our proposal is to isolate therapeutic stem and progenitor cells derived from human embryonic stem cells (hESCs) that can give rise to blood and heart cells. Our approach involves developing differentiation protocols to drive hematopoietic (blood) and cardiac (heart) development of hESCs, then to identify and isolate stem/progenitor cells using monoclonal antibodies (mAbs) specific to surface markers expressed on blood and heart stem/progenitor cells, and finally to characterize their functional properties in vitro and in vivo. In addition, we sought to develop mAbs that specifically bind to undifferentiated hESCs for removal of residual teratoma (tumor)-initiating cells from therapeutic preparations, to ensure transplantation safety.
  • We have made substantial progress toward achieving these goals. First, we discovered that the initial differentiation of hESCs occurs through only 4-5 different progenitor types, of which one is destined to give rise to heart lineages. We purified this population using four novel cell surface markers (ROR2, PDGFRα, KDR, and CD13), and found a significant enrichment of cardiomyocyte clones in colony formation assays that we developed. This subset also expressed particularly high levels of cardiac genes and was receptive to further differentiation into beating cardiomyocytes or vascular endothelial cells. When transplanted into immunodeficient mice these progenitors differentiated into ventricular myocytes and vascular endothelial cells. We have also successfully developed a human fetal heart xenograft model to test hESC-derived cardiomyocyte stem/progenitor cells in human heart tissue for engraftment and function.
  • Second, we have optimized cell culture conditions and cell surface markers to sort hematopoietic progenitors derived from hESCs. In doing so, we have mapped the earliest stages of hematopoietic specification and commitment from a bipotent hematoendothelial precursor. Our culture conditions drive robust hematopoietic differentiation in vitro but these hESC-derived hematopoietic progenitors do not achieve hematopoietic engraftment when transplanted in mouse models. Furthermore, we overexpressed the anti-apoptotic protein BCL2 in hESCs, and discovered a significant improvement in viability upon single cell sorting, embryoid body formation, and in cultures lacking serum replacement. Moving forward, we feel the survival advantages exhibited by this BCL2-expressing hESC line will improve our chances of engrafting hESC-derived hematopoietic stem/progenitor cells.
  • Third, we identified a cocktail of 5 commercial and 1 novel mAbs that enable specific identification of human pluripotent cells (hESCs and induced pluripotent cells). We have found combinations of 3 pluripotency surface markers that can remove all teratoma-initiating cells from differentiated hESC and induced pluripotent stem cell (iPSC) populations prior to transplant. While these combinations can vary depending on the differentiation culture, we have generated a simple, easy-to-follow protocol to remove all teratogenic cells from large-scale differentiation cultures.
  • In summary, we accomplished most of the goals stated in our original proposal. We successfully achieved cardiac engraftment of an hESC-derived cardiomyocyte progenitor using a novel human heart model of engraftment. While we unfortunately did not attain hematopoietic engraftment of hESC-derived cells, we are exploring a strategy to address this. Our research has led to four manuscripts: one on the protective effects of BCL2 expression on hESC viability and pluripotency (published in PNAS, 2011), another describing markers of pluripotency and their use in depleting teratogenic potential in differentiated PSCs (accepted for publication in Nature Biotechnology), and two submitted manuscripts, one describing a novel xenograft assay to test PSC-derived cardiomyocytes for functional engraftment and the other describing the earliest fate decisions downstream of a PSC.

Differentiation of Human Hematopoietic Stem Cells into iNKT Cells

Funding Type: 
Basic Biology V
Grant Number: 
RB5-07089
ICOC Funds Committed: 
$614 400
Disease Focus: 
Blood Disorders
oldStatus: 
Active
Public Abstract: 
Blood stem cells living in the bone marrow of adult humans give rise to all of the cells in our blood, including the red blood cells that carry oxygen to supply our body, and the white blood cells such as T and B lymphocytes that fight infections and keep us healthy. Among the T lymphocytes there is a small population called invariant natural killer T (iNKT) cells. Despite their low frequency in humans (~0.001-1% in blood), iNKT cells have the remarkable capacity to mount immediate and potent responses when stimulated, and have been suggested to play important roles in regulating multiple human diseases including infections, allergies, cancer, and autoimmunity (such as Type I diabetes and multiple sclerosis). However, successful clinical interventions with iNKT cells have been greatly hindered by our limited knowledge on how these cells are produced by blood stem cells, largely due to the lack of tools to track these cells in humans. We therefore propose a novel model system to overcome this research bottleneck by transplanting human blood stem cells into a mouse and genetically programming these cells to develop into iNKT cells. This “humanized” mouse model will allow us to directly track the differentiation of human blood stem cells into iNKT cells in a living animal. From this study, we will address some critical unanswered questions for iNKT cell development, and shed light on developing stem-cell based iNKT cell therapies.
Statement of Benefit to California: 
Allergies, cancer and autoimmunity are leading health hazards in California. These diseases affect millions of Californians, impairing their life quality and creating huge economic burdens for the State of California. This proposal intends to study invariant natural killer (iNKT) T cells, a special population of T lymphocytes that have been suggested to play important roles in regulating these diseases. To date, clinical applications of iNKT cells have been greatly limited by their low frequency in humans and their high variability between individuals (~0.001-1% in blood). Thus, an improved understanding of how these cells are naturally generated is important for their use clinically. Like all other cells in blood, iNKT cells are descendants of the blood stem cells that live in the bone marrow of adult humans. Our goal is to study how human blood stem cells give rise to iNKT cells. If successful, our results can be exploited to develop stem cell-based iNKT cell therapies to treat allergies, cancer and autoimmunity, and therefore may benefit the millions of Californians currently suffering from these diseases. In addition, the knowledge and reagents generated from this proposed study will be shared freely with non-profit and academic organizations in California, and any new intellectual property derived from this study will be developed under the guidance of CIRM to benefit the State of California.

A Treatment For Beta-thalassemia via High-Efficiency Targeted Genome Editing of Hematopoietic Stem Cells

Funding Type: 
Strategic Partnership II
Grant Number: 
SP2-06902
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.

Development of a cell and gene based therapy for hemophilia

Funding Type: 
Early Translational IV
Grant Number: 
TR4-06809
ICOC Funds Committed: 
$2 322 440
Disease Focus: 
Blood Disorders
Liver Disease
Pediatrics
Stem Cell Use: 
iPS Cell
Cell Line Generation: 
iPS Cell
oldStatus: 
Closed
Public Abstract: 
Hemophilia B is a bleeding disorder caused by the lack of FIX in the plasma and affects 1/30,000 males. Patients suffer from recurrent bleeds in soft tissues leading to physical disability in addition to life threatening bleeds. Current treatment (based on FIX infusion) is transient and plagued by increased risk for blood-borne infections (HCV, HIV), high costs and limited availability. This has fueled a search for gene/cell therapy based alternatives. Being the natural site of FIX synthesis, the liver is expected to provide immune-tolerance and easy circulatory access. Liver transplantation is a successful, long-term therapeutic option but is limited by scarcity of donor livers and chronic immunosuppression; making iPSC-based cell therapy an attractive prospect. As part of this project, we plan to generate iPSCs from hemophilic patients that will then be genetically corrected by inserting DNA capable of making FIX. After validation for correction, we will then differentiate these iPSCs into liver cells that can be transplanted into our mouse model of hemophilia that is capable of accepting human hepatocytes and allowing their proliferation. These mice exhibit disease symptoms similar to human patients and we propose that by injecting our corrected liver cells they will exhibit normal clotting as measured by various biochemical and physiological assays. If successful, this will provide a long-term cure for hemophilia and other liver diseases.
Statement of Benefit to California: 
Generation of iPSCs from adult cells unlocked the potential of tissue engineering, replacement and cell transplant therapies to cure a host of debilitating diseases without the ethical concerns of working with embryos or the practical problems of immune-rejection. We aim to develop a POC for a novel cell- and gene-therapy based approach towards the treatment of hemophilia B. In addition to the obvious and direct benefit to the affected patients and families by providing a potential long-term cure; the successful development of our proposal will serve as a POC for moving other iPSC-based therapies to the clinic. Our proposal also has the potential to treat a host of other hepatic diseases like alpha-1-antitrypsin deficiency, Wilson’s disease, hereditary hypercholesterolemia, etc. These diseases have devastating effects on the patients in addition to the huge financial drain on the State in terms of the healthcare costs. There is a pressing need to find effective solutions to such chronic health problems in the current socio-economic climate. The work proposed here seeks to redress this by developing cures for diseases that, if left untreated, require substantial, prolonged medical expenditures and cause increased suffering to patients. Being global leaders in these technologies, we are ideally suited to this task, which will establish the state of California at the forefront of medical breakthroughs and strengthen its biomedical/biotechnology industries.

Beta-Globin Gene Correction of Sickle Cell Disease in Hematopoietic Stem Cells

Funding Type: 
Early Translational IV
Grant Number: 
TR4-06823
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.

A Phase 1/2, Open Label Study Evaluating the Safety and Efficacy of Gene Therapy in Subjects with β-Thalassemia by Transplantation of Autologous Hematopoietic Stem Cells [REDACTED]

Funding Type: 
Strategic Partnership I
Grant Number: 
SP1-06477
Investigator: 
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.

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