Neurological Disorders

Coding Dimension ID: 
303
Coding Dimension path name: 
Neurological Disorders

A hESc-based Development Candidate for Huntington's Disease

Funding Type: 
Early Translational II
Grant Number: 
TR2-01841
ICOC Funds Committed: 
$4 045 253
Disease Focus: 
Huntington's Disease
Neurological Disorders
Stem Cell Use: 
Embryonic Stem Cell
Cell Line Generation: 
Embryonic Stem Cell
oldStatus: 
Active
Public Abstract: 
Huntington’s disease (HD) is a devastating degenerative brain disease with a 1 in 10,000 prevalence that inevitably leads to death. These numbers do not fully reflect the large societal and familial cost of HD, which requires extensive caregiving. HD has no effective treatment or cure and symptoms unstoppably progress for 15-20 years, with onset typically striking in midlife. Because HD is genetically dominant, the disease has a 50% chance of being inherited by the children of patients. Symptoms of the disease include uncontrolled movements, difficulties in carrying out daily tasks or continuing employment, and severe psychiatric manifestations including depression. Current treatments only address some symptoms and do not change the course of the disease, therefore a completely unmet medical need exists. Human embryonic stem cells (hESCs) offer a possible long-term treatment approach that could relieve the tremendous suffering experienced by patients and their families. HD is the 3rd most prevalent neurodegenerative disease, but because it is entirely genetic and the mutation known, a diagnosis can be made with certainty and clinical applications of hESCs may provide insights into treating brain diseases that are not caused by a single, known mutation. Trials in mice where protective factors were directly delivered to the brains of HD mice have been effective, suggesting that delivery of these factors by hESCs may help patients. Transplantation of fetal brain tissue in HD patients suggests that replacing neurons that are lost may also be effective. The ability to differentiate hESCs into neuronal populations offers a powerful and sustainable alternative for cell replacement. Further, hESCs offer an opportunity to create cell models in which to identify earlier markers of disease onset and progression and for drug development. We have assembled a multidisciplinary team of investigators and consultants who will integrate basic and translational research with the goal of generating a lead developmental candidate having disease modifying activity with sufficient promise to initiate IND-enabling activities for HD clinical trials. The collaborative research team is comprised of investigators from multiple California institutions and has been assembled to maximize leverage of existing resources and expertise within the HD and stem cell fields.
Statement of Benefit to California: 
The disability and loss of earning power and personal freedom resulting from Huntington's disease (HD) is devastating and creates a financial burden for California. Individuals are struck in the prime of life, at a point when they are their most productive and have their highest earning potential. As the disease progresses, individuals require institutional care at great financial cost. Therapies using human embryonic stem cells (hESCs) have the potential to change the lives of hundreds of individuals and their families, which brings the human cost into the thousands. For the potential of hESCs in HD to be realized, a very forward-thinking team effort will allow highly experienced investigators in HD, stem cell research and clinical trials to come together and identify a lead development candidate for treatment of HD. This early translation grant will allow for a comprehensive and systematic evaluation of hESC-derived cell lines to identify a candidate and develop a candidate line into a viable treatment option. HD is the 3rd most prevalent neurodegenerative disease, but because it is entirely genetic and the mutation known, a diagnosis can be made with certainty and clinical applications of hESCs may provide insights into treating brain diseases that are not caused by a single, known mutation. We have assembled a strong team of California-based investigators to carry out the proposed studies. Anticipated benefits to the citizens of California include: 1) development of new human stem cell-based treatments for HD with application to other neurodegenerative diseases such as Alzheimer's and Parkinson's diseases that affect thousands of individuals in California; 2) improved methods for following the course of the disease in order to treat HD as early as possible before symptoms are manifest; 3) transfer of new technologies and intellectual property to the public realm with resulting IP revenues coming into the state with possible creation of new biotechnology spin-off companies; and 4) reductions in extensive care-giving and medical costs. It is anticipated that the return to the State in terms of revenue, health benefits for its Citizens and job creation will be significant.
Progress Report: 
  • Huntington’s disease (HD) is a devastating degenerative brain disease with a 1 in 10,000 prevalence that inevitably leads to death. Because HD is genetically dominant, the disease has a 50% chance of being inherited by the children of patients. Symptoms of the disease include uncontrolled movements, difficulties in carrying out daily tasks or continuing employment, and severe psychiatric manifestations including depression. Current treatments only address some symptoms and do not change the course of the disease, therefore a completely unmet medical need exists. Human embryonic stem cells (hESCs) offer a possible long-term treatment approach that could relieve the tremendous suffering experienced by patients and their families. Because HD is entirely genetic and the mutation known, a diagnosis can be made with certainty and clinical applications of hESCs may provide insights into treating brain diseases that are not caused by a single, known mutation. The ability to differentiate hESCs into neuronal populations offers a powerful and sustainable treatment opportunity. We have established the multidisciplinary team of investigators and consultants to integrate basic and translational research with the goal of generating a lead developmental candidate having disease modifying activity with sufficient promise to initiate IND-enabling activities for HD clinical trials.
  • In preliminary experiments, the transplantation of mouse neural stem cells, which survived in the brain for the four week period of the trial, provided protective effects in delaying disease progression in an HD mouse model and increased production of protective molecules in the brains of these mice. In the first year, the team has developed and established methods to differentiate hESCs into neural, neuronal and astrocyte precursors to be used for transplantation and has determined the correct cells to use that can be developed for future clinical development of these cells. In initial studies during this year, transplantation of neural stem cells (NSCs) provided both neurological and behavioral benefit to a HD mouse model. In addition, neuroprotective molecules were increased. Three immunosuppression regimens were tested to optimize methods for next stage preclinical trials. Finally, breeding of the three different HD mouse models has been initiated. Taken as a whole, progress supports the feasibility of the CIRM-funded studies to transplant differentiated hESCs into HD mice for preclinical development with the ultimate goal of initiating IND-enabling activities for HD clinical trials.
  • Huntington’s disease (HD) is a devastating degenerative brain disease with a 1 in 10,000 prevalence that inevitably leads to death. Because HD is genetically dominant, the disease has a 50% chance of being inherited by the children of patients. Symptoms of the disease include uncontrolled movements, difficulties in carrying out daily tasks or continuing employment, and severe psychiatric manifestations including depression. Current treatments only address some symptoms and do not change the course of the disease, therefore a completely unmet medical need exists. Human embryonic stem cells (hESCs) offer a possible long-term treatment approach that could relieve the tremendous suffering experienced by patients and their families. Because HD is entirely genetic and the mutation known, a diagnosis can be made with certainty and clinical applications of hESCs may provide insights into treating brain diseases that are not caused by a single, known mutation. The ability to differentiate hESCs into neuronal populations offers a powerful and sustainable treatment opportunity. We have established the multidisciplinary team of investigators and consultants to integrate basic and translational research with the goal of generating a lead developmental candidate having disease modifying activity with sufficient promise to initiate IND-enabling activities for HD clinical trials.
  • We previously performed transplantation of human neural stem cells into an HD mouse model and found that a subset of cells survived in the brain for the four week period of the trial, providing protective effects in delaying disease progression. In the past year, we have increased production and characterization of human neural stem cells (hNSCs) into neuronal (hNPC) and astrocyte (hAPC) precursors to be used for transplantation and optimized methods for shipping and implantation. Immunosuppression regimens were improved to optimize cell survival of implanted cells in HD mice. Transplantation of both human NSCs and NPCs are neuroprotective to HD mice and transplantation of hAPCs is in progress. Once completed, the cell giving the greatest protective benefit will be transplanted into mice that display slower progression over a longer time frame to validate and optimize approach for subsequent human application. All three HD mouse models have been bred and are ready for stem cell transplants. Taken as a whole, progress supports the feasibility of the CIRM-funded studies to transplant differentiated hESC-derived cell types into HD mice for preclinical development with the ultimate goal of identifying a lead candidate cell type and initiating IND-enabling activities for HD clinical trials.
  • Huntington’s disease (HD) is a devastating degenerative brain disease with a 1 in 10,000 prevalence that inevitably leads to death. Because HD is genetically dominant, the disease has a 50% chance of being inherited by the children of patients. Symptoms of the disease include uncontrolled movements, difficulties in carrying out daily tasks or continuing employment, and severe psychiatric manifestations including depression. Current treatments only address some symptoms and do not change the course of the disease, therefore a completely unmet medical need exists. Human embryonic stem cells (hESCs) offer a possible long-term treatment approach that could relieve the tremendous suffering experienced by patients and their families. Because HD is entirely genetic and the mutation known, a diagnosis can be made with certainty and clinical applications of hESCs may provide insights into treating brain diseases that are not caused by a single, known mutation. The ability to differentiate hESCs into neuronal populations offers a powerful and sustainable treatment opportunity. We have established the multidisciplinary team of investigators and consultants to integrate basic and translational research with the goal of generating a lead developmental candidate having disease modifying activity with sufficient promise to initiate Investigational New Drug (IND) enabling activities for HD clinical trials.
  • We have completed several rounds of transplantation of human neural stem cells into an HD mouse model and found that the cells survived in the brain for the four-week period of the trial, provided protective effects in delaying disease progression and increased production of protective molecules in the brains of these mice. In the last year the team differentiated hESCs into neural, neuronal and astrocyte precursors and performed transplantation studies to determine the best cell candidate to use and develop for future clinical work. We determined that the human neural stem cells produce the most robust effect. We have now selected a GMP grade hNSC line that will be carried forward for further testing in both rapidly progressing and slower progressing HD mice, as well as in mouse preclinical dosing studies. Taken as a whole, progress supports the feasibility of the CIRM-funded studies to transplant differentiated hESCs into HD mice for preclinical development with the ultimate goal on initiating IND-enabling activities for HD clinical trials.

A Phase I/IIa Dose Escalation Safety Study of [REDACTED] in Patients with Cervical Sensorimotor Complete Spinal Cord Injury

Funding Type: 
Strategic Partnership III Track A
Grant Number: 
SP3A-07552
ICOC Funds Committed: 
$14 323 318
Disease Focus: 
Spinal Cord Injury
Neurological Disorders
Stem Cell Use: 
Embryonic Stem Cell
oldStatus: 
Active
Public Abstract: 
The proposed project is designed to assess the safety and preliminary activity of escalating doses of human embryonic stem cell derived oligodendrocyte progenitor cells (OPCs) for the treatment of spinal cord injury. OPCs have two important functions: they produce factors which stimulate the survival and growth of nerve cells after injury, and they mature in the spinal cord to produce myelin, the insulation which enables electrical signals to be conducted within the spinal cord. Clinical testing of this product initiated in 2010 after extensive safety and efficacy testing in more than 20 nonclinical studies. Initial clinical safety testing was conducted in five subjects with neurologically complete thoracic injuries. No safety concerns have been observed after following these five subjects for more than two years. The current project proposes to extend testing to subjects with neurologically complete cervical injuries, the intended population for further clinical development, and the population considered most likely to benefit from the therapy. Initial safety testing will be performed in three subjects at a low dose level, with subsequent groups of five subjects at higher doses bracketing the range believed most likely to result in functional improvements. Subjects will be monitored both for evidence of safety issues and for signs of neurological improvement using a variety of neurological, imaging and laboratory assessments. By completion of the project, we expect to have accumulated sufficient safety and dosing data to support initiation of an expanded efficacy study of a single selected dose in the intended clinical target population.
Statement of Benefit to California: 
The proposed project has the potential to benefit the state of California by improving medical outcomes for California residents with spinal cord injuries (SCIs), building on California’s leadership position in the field of stem cell research, and creating high quality biotechnology jobs for Californians. Over 12,000 Americans suffer an SCI each year, and approximately 1.3 million people in the United States are estimated to be living with a spinal cord injury. Although specific estimates for the state of California are not available, the majority of SCI result from motor vehicle accidents, falls, acts of violence, and recreational sporting activities, all of which are common in California. Thus, the annual incidence of SCI in California is likely equal to or higher than the 1,400 cases predicted by a purely population-based distribution of the nationwide incidence. The medical, societal and economic burden of SCI is extraordinarily high. Traumatic SCI most commonly impacts individuals in their 20s and 30s, resulting in a high-level of permanent disability in young and previously healthy individuals. At one year post injury, only 11.8% of SCI patients are employed, and fewer than 35% are employed even at more than twenty years post-injury (NSCISC Spinal Cord Injury Facts and Figures 2013). Life expectancies of SCI patients are significantly below those of similar aged patients with no SCI. Additionally, many patients require help with activities of daily living such as feeding and bathing. As a result, the lifetime cost of care for SCI patients are enormous; a recent paper (Cao et al 2009) estimated lifetime costs of care for a patient obtaining a cervical SCI (the population to be enrolled in this study) at age 25 at $4.2 million. Even partial correction of any of the debilitating consequences of SCI could enhance activities of daily living, increase employment, and decrease reliance on attendant and medical care, resulting in substantial improvements in both quality of life and cost of care for SCI patients. California has a history of leadership both in biotechnology and in stem cell research. The product described in this application was invented in California, and has already undergone safety testing in five patients in a clinical study initiated by a California corporation. The applicant, who has licensed this product from its original developer and recruited many of the employees responsible for its previous development, currently employs 17 full-time employees at its California headquarters, with plans to significantly increase in size over the coming years. The successful performance of the proposed project would enable significant additional jobs creation in preparation for pivotal trials and product registration.

Role of the NMD RNA Decay Pathway in Maintaining the Stem-Like State

Funding Type: 
Basic Biology IV
Grant Number: 
RB4-06345
ICOC Funds Committed: 
$1 360 450
Disease Focus: 
Neurological Disorders
Stem Cell Use: 
Embryonic Stem Cell
Cell Line Generation: 
iPS Cell
oldStatus: 
Active
Public Abstract: 
A subset of intellectual disability cases in humans are caused by mutations in an X-linked gene essential for a quality control mechanism called nonsense-mediated RNA decay (NMD). Patients with mutations in this gene—UPF3B—commonly have not only ID, but also schizophrenia, autism, and attention-deficit/hyperactivity disorder. Thus, the study of UPF3B and NMD may provide insight into a wide spectrum of cognitive and psychological disorders. To examine how mutations in UPF3B can cause mental defects, we will generate and characterize induced-pluripotent stem cells from intellectual disability patients with mutations in the UPF3B gene. In addition to having a role in neural development, our recent evidence suggests that NMD is important for maintaining the identity of ES cells and progenitor cells. How does NMD do this? While NMD is a quality control mechanism, it is also a well characterized biochemical pathway that serves to rapidly degrade specific subsets of normal messenger ribonucleic acids (mRNAs), the transiently produced copies of our genetic material: deoxyribonucleic acid (DNA). We have obtained evidence that NMD preferentially degrades mRNAs that interfere with the stem cell program (i.e., NMD promotes the decay mRNAs encoding proteins that promote differentiation and inhibit cell proliferation). In this proposal, we will identify the target mRNAs of NMD in stem and progenitor cells and directly address the role of NMD in maintaining the stem-like state.
Statement of Benefit to California: 
iPS cells provide a means to elucidate the mechanisms underlying diseases that afflict a growing number of Californians. Our proposed project concerns making and testing iPS cells from patients with mutations in the UPF3B gene, all of whom have intellectual disabilities. In addition, many of these patients have autism, attention-deficit disorders, and schizophrenia. By using iPS cells to identify the cellular and molecular defects in these patients, we have the potential to ultimately ameliorate the symptoms of many of these patients. This is important, as over 1.6 million people in California have serious mental illness. Moreover, a large proportion of patients with UPF3B mutations have autism, a disorder that has undergone an alarming 12-fold increase in California between 1987 and 2007. The public mental health facilities in California are inadequate to meet the needs of people with mental health disorders. Furthermore, what is provided is expensive: $4.4 billion was spent on public mental health agency services in California in 2006. Mental health problems also exert a heavy burden on California’s criminal justice system. In 2006, over 11,000 children and 40,000 adults with mental health disorders were incarcerated in California’s juvenile justice system. Our research is also directed towards understanding fundamental mechanisms by which all stem cells are maintained, which has the potential to also impact non-psychiatric disorders suffered by Californians.
Progress Report: 
  • A key quality of stem cells is their ability to switch from a proliferative cell state in which they reproduce themselves to a differentiated cell state that ultimately allows them to carry out the functions of a fully mature cell. Most research on the nature of this switch has focused on the role of proteins that determine whether the genetic material—DNA—generates a copy of it itself in the form of messenger RNA, a process called transcription. In stem cells, such proteins—which are called transcription factors—activate the production of messenger RNAs encoding proteins that promote the proliferative and undifferentiated cell state. They also increase the production of messenger mRNAs that encode inhibitors of differentiation and cell proliferation. The level and profile of such transcription factors are altered in response to signals that trigger stem cells to differentiate. For example, transcription factors that promote the undifferentiated cell state are decreased in level and transcription factors that drive differentiation down a particular lineage are increased in level. While this transcription factor-centric view of stem cells explains some aspects of stem cell biology, it is, in of itself, insufficient to explain many of their behaviors, including both their ability to maintain the stem-like state and to differentiate. We hypothesize that a molecular pathway that complements transcription-base mechanisms in controlling stem cell maintenance vs. differentiation decisions is an RNA decay pathway called nonsense-mediated RNA decay (NMD). Messenger RNA decay is as important as transcription in determining the level of messenger RNA. Signals that trigger increased decay of a given messenger RNA leads to decreased levels of its encoded protein, while signals that trigger the opposite response increase the level of the encoded protein. Our project revolves around two main ideas. First, that NMD promotes the stem-like state by preferentially degrading messenger RNAs that encode differentiation-promoting proteins and proliferation inhibitor proteins. Second, that NMD must be downregulated in magnitude to allow stem cells to differentiate. During the progress period, we obtained substantial evidence for both of these hypotheses. With regard to the first hypothesis, we have used genome-wide approaches to identify hundreds of messenger RNAs that are regulated by NMD in both in vivo (in mice) and in vitro (in cell lines). To determine which of these messenger mRNAs are directly degraded by NMD, we have used a variety of approaches. This work has revealed that NMD preferentially degrades messenger RNAs encoding neural differentiation inhibitors and proliferation inhibitors in neural stem cells. In contrast, very few messenger RNAs encoding pro-stem cell proteins or pro-proliferation proteins are degraded by NMD. Together this provides support for our hypothesis that NMD promotes the stem-like state by shifting the proportion of messenger RNAs in a cell towards promoting an undifferentiated, proliferative cell state. With regard to the second hypothesis, we have found that many proteins that are directly involved in the NMD pathway are downregulated upon differentiation of stem and progenitor cells. Not only are NMD proteins reduced in level, but we find that the magnitude of NMD itself is reduced. We have used a variety of molecular techniques to determine whether this NMD downregulatory response has a role in neural differentiation and found that NMD downreglation is both necessary and sufficient for this event. Such experiments have also revealed particular messenger mRNAs degraded by NMD that are crucial for the NMD downregulatory response to promote neural differentiation. Our research has implications for intellectual disability cases in humans caused by mutations in an X-linked gene essential for NMD. Patients with mutations in this gene—UPF3B—not only have intellectual disability, but also schizophrenia, autism, and attention-deficit/hyperactivity disorder. Thus, the study of NMD may provide insight into a wide spectrum of cognitive and psychological disorders. We are currently in the process of generating induced-pluripotent stem (iPS) cells from intellectual disability patients with mutations in the UPF3B gene towards this goal.
  • A key quality of stem cells is their ability to switch from a proliferative cell state in which they reproduce themselves to a differentiated cell state that ultimately allows them to carry out the functions of a fully mature cell. Most research on the nature of this switch has focused on the role of proteins that determine whether the genetic material—DNA—generates a copy of it itself in the form of messenger RNA, a process called transcription. In stem cells, such proteins—which are called transcription factors—activate the production of messenger RNAs encoding proteins that promote the proliferative and undifferentiated cell state. They also increase the production of messenger mRNAs that encode inhibitors of differentiation and cell proliferation. The level and profile of such transcription factors are altered in response to signals that trigger stem cells to differentiate. For example, transcription factors that promote the undifferentiated cell state are decreased in level and transcription factors that drive differentiation down a particular lineage are increased in level. While this transcription factor-centric view of stem cells explains some aspects of stem cell biology, it is, in of itself, insufficient to explain many of their behaviors, including both their ability to maintain the stem-like state and to differentiate. We hypothesize that a molecular pathway that complements transcription-base mechanisms in controlling stem cell maintenance vs. differentiation decisions is an RNA decay pathway called nonsense-mediated RNA decay (NMD). Messenger RNA decay is as important as transcription in determining the level of messenger RNA. Signals that trigger increased decay of a given messenger RNA leads to decreased levels of its encoded protein, while signals that trigger the opposite response increase the level of the encoded protein. Our project revolves around two main ideas. First, that NMD promotes the stem-like state by preferentially degrading messenger RNAs that encode differentiation-promoting proteins and proliferation inhibitor proteins. Second, that NMD must be downregulated in magnitude to allow stem cells to differentiate. During the progress period, we obtained substantial evidence for both of these hypotheses. With regard to the first hypothesis, we have used genome-wide approaches to identify hundreds of messenger RNAs that are regulated by NMD in both in vivo (in mice) and in vitro (in cell lines). To determine which of these messenger mRNAs are directly degraded by NMD, we have used a variety of approaches. This work has revealed that NMD preferentially degrades messenger RNAs encoding neural differentiation inhibitors and proliferation inhibitors in neural stem cells. In contrast, very few messenger RNAs encoding pro-stem cell proteins or pro-proliferation proteins are degraded by NMD. Together this provides support for our hypothesis that NMD promotes the stem-like state by shifting the proportion of messenger RNAs in a cell towards promoting an undifferentiated, proliferative cell state. During the progress period, we have obtained considerable evidence that this hypothesis not only applies to mouse stem cells but also human embryonic stem cells. With regard to the second hypothesis, we have found that many proteins that are directly involved in the NMD pathway are downregulated upon differentiation of stem and progenitor cells. Not only are NMD proteins reduced in level, but we find that the magnitude of NMD itself is reduced. We have used a variety of molecular techniques to determine whether this NMD downregulatory response has a role in neural differentiation and found that NMD downreglation is both necessary and sufficient for this event. Such experiments have also revealed particular messenger mRNAs degraded by NMD that are crucial for the NMD downregulatory response to promote neural differentiation. During the progress period, we obtained both experimental and genome-wide data that this applies to human embryonic stem cells. Our research has implications for intellectual disability cases in humans caused by mutations in an X-linked gene essential for NMD. Patients with mutations in this gene—UPF3B—not only have intellectual disability, but also schizophrenia, autism, and attention-deficit/hyperactivity disorder. Thus, the study of NMD may provide insight into a wide spectrum of cognitive and psychological disorders. We are currently in the process of generating and characterizing induced-pluripotent stem (iPS) cells from intellectual disability patients with mutations in the UPF3B gene towards this goal.

Mechanism and Utility of Direct Neuronal Conversion with a MicroRNA-Chromatin Switch

Funding Type: 
Basic Biology IV
Grant Number: 
RB4-05886
ICOC Funds Committed: 
$1 392 426
Disease Focus: 
Neurological Disorders
Stem Cell Use: 
Directly Reprogrammed Cell
oldStatus: 
Active
Public Abstract: 
Many human diseases and injuries that affect the brain and nervous system could potentially be treated by either introducing healthy neurons or persuading the cells that normally provide supporting functions to become functioning neurons. A number of barriers must be traversed to bring these goals to practical therapies. Recently our laboratory and others have found ways of converting different human cell types to functioning neurons. Surprisingly, two routes for the production of neurons have been discovered. Our preliminary evidence indicates that these two routes are likely to work together and therefore more effective ways of producing neurons can likely be provided by understanding these two routes, which is one aim of this application. Another barrier to effective treatment of human neurologic diseases has been the inability to develop good models of human neurologic disease due to inability to sample tissues from patients with these diseases. Hence we will understand ways of making neurons from blood cells and other cells, which can be easily obtained from patients with little or no risk. Our third goal will be to understand how different types of neurons can be produced from patient cells. We would also like to understand the barriers and check points that keep one type of cell from becoming another another type of cell. Understanding these mysterious processes could help provide new sources of human cells for replacement therapies and disease models.
Statement of Benefit to California: 
The state of California and its citizens are likely to benefit from the work described in this proposal by the development of more accurate models for the testing of drugs and new means of treatment of human neurologic diseases. Presently these diseases are among the most common afflicting Californians, as well as others and will become more common in an aging population. Common and devastating diseases such as Alzheimer’s, Schizophrenia, Parkinson's Disease, and others lack facile cell culture models that allow one to probe the basis of the disease and to test therapies safely and without risk to the patient. Our work is already providing these models, but we hope to make even better ones by understanding the fundamental processes that allow one cell type (such as a skin cell or blood cell) to be converted to human neurons, where the disease process can be investigated. In the past the inability to make neurons from patients with specific diseases has been a major roadblock to treatment. In the future the studies described here might be able to provide healthy neurons to replace ones loss through disease or injury.
Progress Report: 
  • During the past year, our laboratory has investigated the way that human skin cells can be changed to neurons. To do this, we have used a natural switch that occurs as embryonic cells decide to become neurons. We have found that this process proceeds in a highly ordered series of stages that involve first a resetting of fundamental cell biologic processes characteristic of neurons. This is followed by activation of genes encoding proteins that allow different types of neurons to interact and develop communication between one another. This finding surprised us since we expected to find changes in transcription factors, which instruct the formation of neurons. Instead, we find that the natural switching mechanism in neurons first regulates cell-to-cell communication.
  • We are exploring the way that normal human skin and other types of cells can be converted to neurons. We have found that there are at least two fundamental genetic pathways of doing this that are influenced by different genes and may therefore represent a fertile ground for developing new methods for converting cells of different types to neurons. This could perhaps be useful for replacing neurons from other cell types in states where neurons are damaged or lost such as a variety of neurodegenerative diseases.

Common molecular mechanisms in neurodegenerative diseases using patient based iPSC neurons

Funding Type: 
Basic Biology IV
Grant Number: 
RB4-06079
ICOC Funds Committed: 
$1 506 420
Disease Focus: 
Huntington's Disease
Neurological Disorders
Parkinson's Disease
Stem Cell Use: 
iPS Cell
Cell Line Generation: 
iPS Cell
oldStatus: 
Active
Public Abstract: 
A major medical problem in CA is the growing population of individuals with neurodegenerative diseases, including Parkinson’s (PD) and Huntington’s (HD) disease. These diseases affect millions of people, sometimes during the prime of their lives, and lead to total incapacitation and ultimately death. No treatment blocks the progression of neurodegeneration. We propose to conduct fundamental studies to understand the basic common disease mechanisms of neurodegenerative disorders to begin to develop effective treatments for these diseases. Our work will target human stem cells made from cells from patients with HD and PD that are developed into the very cells that degenerate in these diseases, striatal neurons and dopamine neurons, respectively. We will use a highly integrated approach with innovative molecular analysis of gene networks that change the states of proteins in these diseases and state-of-the-art imaging technology to visualize living neurons in a culture dish to assess cause and effect relationships between biochemical changes in the cells and their gradual death. Importantly, we will test whether drugs effective in animal model systems are also effective in blocking the disease mechanisms in the human HD and PD neurons. These human preclinical studies could rapidly lead to clinical testing, since some of the drugs have already been examined extensively in humans in the past for treating other disorders and are safe.
Statement of Benefit to California: 
Neurodegenerative diseases, such as Parkinson’s (PD) and Huntington’s disease (HD), are devastating to patients and families and place a major financial burden on California. No treatments effectively block progression of any neurodegenerative disease. A forward-thinking team effort will allow highly experienced investigators in neurodegenerative disease and stem cell research to investigate common basic mechanisms that cause these diseases. Most important is the translational impact of our studies. We will use neurons and astrocytes derived from patient induced pluripotent stem cells to identify novel targets and discover disease-modifying drugs to block the degenerative process. These can be quickly transitioned to testing in preclinical and clinical trials to treat HD and other neurodegenerative diseases. We are building on an existing strong team of California-based investigators to complete the studies. Future benefits to California citizens include: 1) discovery and development of new HD treatments with application to other diseases, such as PD, that affect thousands of Californians, 2) transfer of new technologies and intellectual property to the public realm with resulting IP revenues to the state with possible creation of new biotechnology spin-off companies, and 3) reductions in extensive care-giving and medical costs. We anticipate the return to the State in terms of revenue, health benefits for its Citizens and job creation will be significant.
Progress Report: 
  • The goal of our study is to identify common mechanisms that cause the degeneration of neurons and lead to most neurodegenerative disorders. Our work focuses on the protein homeostasis pathways that are disrupted in many forms of neurodegeneration, including Huntington’s disease (HD) and Parkinson’s disease (PD). In this first reporting period we have made great progress in developing novel methods to probe the autophagy pathway in single cells. This pathway is involved in the turnover of misfolded proteins and dysfunction organelles. Using our novel autophagy assays, we have preliminary data that indicate that the autophagy pathway in neurons from HD patients is modulated compared to healthy controls. We have also begun validating small molecules that activate the autophagy pathway and we are now moving these inducers into human neurons from HD patients to see if they reduce toxicity or other disease related phenotypes. Using pathway analysis we have also identified specific genes within the proteostasis network that are modulated in HD. We are now testing whether modulating these genes in human neurons from HD patients can lead to a reduction in neurodegeneration. In the final part of this study we are investigating whether neurodegenerative diseases, such as HD and PD, share changes in similar genes or pathways, specifically those involved in protein homeostasis. We have now established a human neuron model for PD and have used it to identify potential targets that modulate the disease phenotype via changes in proteostasis. Using the assays, autophagy drugs and pathway analysis described above, we hope to identify overlapping targets that could potentially rescue disease associated phenotypes in both HD and PD.
  • The goal of our study is to identify common mechanisms that cause the degeneration of neurons and lead to most neurodegenerative disorders. Our work focuses on the protein homeostasis pathways that are disrupted in many forms of neurodegeneration, including Huntington’s disease (HD) and Parkinson’s disease (PD). In this reporting period we have made good progress in both developing new assays and novel autophagy compounds and identifying potential genetic targets that could lead to novel therapeutic strategies for patients with HD and PD. We have developed methods to measure the degradation rates of proteins involved in causing neurodegeneration and the decay rates of mitochondria that are disrupted during the progression of these diseases. We are now investigating if and how these degradation rates differ in cells from patients with HD. We have developed novel compounds that can activate the autophagy pathway which is critical for degrading the toxic proteins that cause neurodegeneration. We are now testing if these compounds can increase the survival of neurons derived from iPSCs from patients with HD. Using pathway analysis we have also identified specific genes within the proteostasis network that are modulated in HD. Specifically we have identified deubiquitinating enzymes as modulators of HD induced toxicity and autophagy modulation, potentially indicating that importance of the autophagy pathway in the disease progression. We are also using RNAseq analysis to investigate if neurons derived from iPSCs from PD patients exhibit differences in the genes expressed in the proteostasis network. If we identify key genes we will use our established human neuron model for PD to validate whether these genes modulate the disease phenotype via changes in proteostasis. Ultimately we hope to identify overlapping targets that could potentially rescue disease associated phenotypes in both HD and PD.

Functional Neural Relay Formation by Human Neural Stem Cell Grafting in Spinal Cord Injury

Funding Type: 
Early Translational III
Grant Number: 
TR3-05628
ICOC Funds Committed: 
$4 699 569
Disease Focus: 
Spinal Cord Injury
Neurological Disorders
Stem Cell Use: 
Embryonic Stem Cell
oldStatus: 
Active
Public Abstract: 
We aim to develop a novel stem cell treatment for spinal cord injury (SCI) that is substantially more potent than previous stem cell treatments. By combining grafts of neural stem cells with scaffolds placed in injury sites, we have been able to optimize graft survival and filling of the injury site. Grafted cells extend long distance connections with the injured spinal cord above and below the lesion, while the host spinal cord also sends inputs to the neural stem cell implants. As a result, new functional relays are formed across the lesion site. These result in substantially greater functional improvement than previously reported in animal studies of stem cell treatment. Work proposed in this grant will identify the optimal human neural stem cells for preclinical development. Furthermore, in an unprecedented step in spinal cord injury research, we will test this treatment in appropriate preclinical models of SCI to provide the greatest degree of validation for human translation. Successful findings could lead to clinical trials of the most potent neural stem cell approach to date.
Statement of Benefit to California: 
Spinal cord injury (SCI) affects approximately 1.2 million people in the United States, and there are more than 11,000 new injuries per year. A large number of spinal cord injured individuals live in California, generating annual State costs in the billions of dollars. This research will examine a novel stem cell treatment for SCI that could result in functional improvement, greater independence and improved life styles for injured individuals. Results of animal testing of this approach to date demonstrate far greater functional benefits than previous stem cell therapies. We will generate neural stem cells from GMP-compatible human embryonic stem cells, then test them in the most clinically relevant animal models of SCI. These studies will be performed as a multi-center collaborative effort with several academic institutions throughout California. In addition, we will leverage expertise and resources currently in use for another CIRM-funded project for ALS, thereby conserving State resources. If successful, these studies will form the basis for clinical trials in a disease of great unmet medical need, spinal cord injury. Moreover, the development of this therapy would reduce costs for clinical care while bringing novel biomedical resources to the State.
Progress Report: 
  • In the first 12 months of this project we have made important progress in the following areas:
  • 1) Identified the lead embryonic stem cell type for potential use in a translational clinical program.
  • 2) Replicated the finding that implants of ES-derived neural progenitor cells from this lead cell type extend axons out from the spinal cord lesion site in very high numbers and over very long distances.
  • 3) Begun efforts to scale this work to larger animal models of spinal cord injury.
  • Very good progress has been made in the last year on this project. We are attempting to address a great unmet medical need to develop effective therapies for human spinal cord injury (SCI). We aim to develop and optimize a pluripotent neural stem cell line for grafting to sites of spinal cord injury, and develop this line for clinical translation. Unlike other programs of stem cell therapy for SCI, we are transplanting neural stem cells directly into the injury site, in high numbers, and we observe very extensive growth of axons both into and out of the graft. The amount of axon growth in this model is substantially greater than that observed with other approaches to the injured spinal cord, including approaches currently in clinical trials. Accordingly, we believe that our approach provides a substantially greater opportunity to improve outcomes after SCI.
  • In the last year, we have identified a lead stem cell line for potential human translation, and validated its ability to engraft to the injured spinal cord. We have observed that human neural stem cells, grafted into mice and rats, exhibit a human time frame for maturation and growth: cells require at least one year to develop and mature. This knowledge is very important for planning human clinical trials.
  • Remaining work will characterize the long term safety and efficacy of these cells in rodent and large animal models of SCI.

Modeling disease in human embryonic stem cells using new genetic tools

Funding Type: 
Basic Biology IV
Grant Number: 
RB4-05855
ICOC Funds Committed: 
$1 387 800
Disease Focus: 
Neuropathy
Neurological Disorders
Stem Cell Use: 
Embryonic Stem Cell
oldStatus: 
Active
Public Abstract: 
The use of stem cells or stem cell-derived cells to treat disease is one important goal of stem cell research. A second, important use for stem cells is the creation of cellular models of human development and disease, critical for uncovering the molecular roots of illness and testing new drugs. However, a major limitation in achieving these goals is the difficulty in manipulating human stem cells. Existing means of generating genetically modified stem cells are not ideal, as they do not preserve the normal gene regulation, are inefficient, and do not permit removal of foreign genes. We have developed a method of genetically modifying mouse embryonic stem cells that is more efficient than traditional methods. We are adapting this approach for use with human embryonic stem cells, so that these cells can be better understood and harnessed for modeling, or even treating, human diseases. We will use this approach to create a human stem cell model of Charcot-Marie-Tooth (CMT) disease, an inherited neuropathy. How gene dysfunction leads to nerve defects in CMT is not clear, and there is no cure or specific therapy for this neurological disease. Thus, we will use our genetic tools to investigate how gene function is disrupted to cause CMT. By developing these tools and using them to gain understanding of CMT, we will illustrate how this system can be used to gain insight into other important diseases.
Statement of Benefit to California: 
Although human stem cells hold the potential to generate new understanding about human biology and new approaches to important diseases, the inability to efficiently and specifically modify stem cells currently limits the pace of research. Also, there is presently no safe means of changing genes compatible with the use of the stem cells in therapies. We are developing new genetic tools to allow for the tractable manipulation of human stem cells. By accelerating diverse other stem cell research projects, these tools will enhance the scientific and economic development of California. We will use these tools to create cellular models of Charcot-Marie-Tooth (CMT), a neurological disease with no cure that affects about 15,000 Californians. This model will facilitate understanding of the etiology of CMT, and may lead to insights that can be used to develop specific therapies. Beyond gaining insight into CMT, the ability to engineer specific genetic changes in human stem cells will be useful for many applications, including the creation of replacement cells for personalized therapies, reporter lines for stem cell-based drug screens, and models of other diseases. Thus, our research will assist the endeavors of the stem cell community in both the public and private arenas, contributing to economic growth and new product development. This project will also train students and postdoctoral scholars in human stem cell biology, who will contribute to the economic capacity of California.
Progress Report: 
  • An important use for stem cells is the creation of cellular models of human development and disease, critical for uncovering the molecular roots of illness and testing new drugs. However, a major limitation in achieving these goals is the difficulty in manipulating human stem cells. We have developed a method of genetically modifying mouse embryonic stem cells that is more efficient than traditional methods. During the first year of this project, we adapted this approach for use with human embryonic stem cells. We have also created gene trap mutations in a diversity of human embryonic stem cell genes that can be used to better harness human embryonic stem cells for modeling, or even treating, human diseases.
  • An important use for stem cells is the creation of cellular models of human development and disease, critical for uncovering the molecular roots of illness and testing new drugs. However, a major limitation in achieving these goals is the difficulty in manipulating human stem cells. We have developed a method of genetically modifying mouse embryonic stem cells that is more efficient than traditional methods. During the second year of this project, we took advantage of new methods using the CRISPR/Cas9 system to develop novel approaches to modifying human embryonic stem cells. We have also created reversible gene trap mutations in a diversity of human embryonic stem cell genes that can be used to better harness human embryonic stem cells for modeling, or even treating, human diseases.

Triplet Repeat Instability in Human iPSCs

Funding Type: 
Basic Biology III
Grant Number: 
RB3-05022
ICOC Funds Committed: 
$1 755 861
Disease Focus: 
Huntington's Disease
Neurological Disorders
Stem Cell Use: 
iPS Cell
Cell Line Generation: 
iPS Cell
oldStatus: 
Active
Public Abstract: 
Over twenty human genetic diseases are caused by expansion of simple DNA sequences composed of repeats of three nucleotides (such as CAG, CTG, CGG and GAA) within essential genes. These repeats can occur within the region of a gene that encodes the protein, generally resulting in proteins with large stretches of repeats of just one amino acid, such as runs of glutamine. These proteins are toxic, cause the death of specific types of brain cells and result in diseases such as Huntington’s disease (HD) and many of the spinocerebellar ataxias (a type of movement disorder). Other repeats can be in regions of genes that do not code for the protein itself, but are copied into messenger RNA, which is a copy of the gene that serves to generate the protein. These RNAs with expanded repeats are also toxic to cells, and sometimes these RNAs sequester essential cellular proteins. One example of this type of disease is Myotonic Dystrophy type 1, a form of muscular dystrophy. Lastly, there are two examples of repeat disorders where the repeats silence the genes harboring these mutations: these are Friedreich’s ataxia (FRDA) and Fragile X syndrome (FXS). One limitation in the development of drugs to treat these diseases is the lack of appropriate cell models that represent the types of cells that are affected in these human diseases. With the advent of the technology to produce induced pluripotent stem cells from patient skin cells, and our ability to turn iPSCs into any cell type, such as neurons (brain cells) that are affected in these triplet repeat diseases, such cellular models are now becoming available. Our laboratories have generated iPSCs from fibroblasts obtained from patients with HD, FXS and FRDA. By comparing cells before and after reprogramming, we found that triplet repeats were expanded in the FRDA iPSCs, but not in HD iPSCs. This application is aimed at the understanding the molecular basis underlying triplet repeat expansion/instability that we have observed during the establishment and propagation of iPSCs from disease-specific fibroblasts. While artificial systems with reporter gene constructs have reproduced triplet repeat expansion in bacteria, yeast and mammalian cells, no cellular models have previously been reported that recapitulate repeat expansions at the endogenous cellular genes involved in these diseases. Therefore, our observations that repeat expansion is found in FRDA iPSCs provides the first opportunity to dissect the mechanisms involved in expansion at the molecular level for the authentic cellular genes in their natural chromatin environment. Repeat expansion is the central basis for these diseases, no matter what the outcome of the expansion (toxic protein or RNA or gene silencing), and a fuller understanding of how repeats expand may lead to new drugs to treat these diseases.
Statement of Benefit to California: 
A major obstacle in the development of new drugs for human diseases is our lack of cell models that represent the tissues or organs that are affected in these diseases. Examples of such diseases are the triplet-repeat neurodegenerative diseases, such as Huntington’s disease, the spinocerebellar ataxias, forms of muscular dystrophy, Fragile X syndrome and Friederich’s ataxia. These diseases, although relatively rare compared to cancer or heart disease, affect thousands of individuals in California. Recent advances in stem cell biology now make it possible to generate cells that reflect the cell types at risk in these diseases (such as brain, heart and muscle cells), starting from patient skin cells. Skin cells can be turned into stem cell-like cells (induced pluripotent stem cells or iPSCs), which can then give rise to just about any cell type in the human body. During the course of our studies, we found that iPSCs derived from Friedreich’s ataxia patient skin cells mimic the behavior of the genetic mutation in this disease. A simple repeat of the DNA sequence GAA is found in the gene encoding an essential protein called frataxin, and this repeat increases in length between generations in human families carrying this mutation. Over a certain threshold, the repeats silence this gene. It is also known that the repeats expand in brain cells in individuals with this disease. With the advent of patient derived iPSCs and neurons, we now have human model systems in which to study the mechanisms responsible for repeat expansion. We have already identified one set of proteins involved in repeat expansion and we now wish to delve more deeply into how the repeats expand. In this way, we may be able to identify new targets for drug development. We will extend our studies to Huntington’s disease and Fragile X syndrome. We have identified two possible therapeutic approaches for Friedreich’s ataxia, and identified molecules that either reactivate the silent gene or block repeat expansion. Our studies in related diseases may provide possible therapeutic strategies for these other disorders as well, which will be of benefit to patients suffering from these diseases, both in California and world-wide.
Progress Report: 
  • Over twenty human genetic diseases are caused by expansion of simple trinucleotide repeat sequences within essential genes, resulting in toxic proteins (as in the polyglutamine expansion diseases, such as Huntington’s disease (HD)), toxic RNAs (as in Myotonic Dystrophy type 1), or gene repression (as in Friedreich’s ataxia (FRDA) and Fragile X syndrome (FXS)). Our laboratories have generated induced pluripotent stem cells (iPSCs) from fibroblasts obtained from patients with Huntington’s disease (HD), Fragile X syndrome (FXS), Myotonic dystrophy type 1 (DM1) and Friedreich’s ataxia (FRDA). By comparing cells before and after reprogramming, we found that triplet repeats were expanded in the FRDA and DM1 iPSCs, but not in HD iPSCs. During growth of the iPSCs in culture, the repeats continue to expand, suggesting that expansion might be linked to DNA replication in these cells. The expansion we observe in iPSCs does not occur in the fibroblast (skin cells) from which the iPSCs were derived. Similarly, on differentiation of the FRDA iPSCs into neurons (brain cells), repeat expansion stops. This observation suggests that some cellular factors necessary for expansion may be selectively expressed in iPSCs, but not in fibroblasts or neurons.
  • Over the past year, our studies have been aimed at the understanding the molecular basis underlying triplet repeat expansion/instability that we have observed during the establishment and propagation of iPSCs from disease-specific fibroblasts. Previous studies have implicated the mismatch repair (MMR) enzymes in repeat expansion in mouse models for HD and DM1. We find that silencing of the MSH2 gene, encoding one of the subunits of the MMR enzymes, impedes repeat expansion in human FRDA iPSCs. We find that components of the human mismatch repair (MMR) system are associated with the disease alleles in the FRDA and DM1 iPSCs, and that silencing of these genes at the level of their messenger RNAs is sufficient to suppress repeat expansion. Moreover, we have monitored the levels of the MMR enzymes in fibroblasts, iPSCs and neurons, and as expected these enzymes are present at higher amounts in the iPSCs, suggesting that it is the availability of these enzymes in iPSCs that may be responsible for repeat expansion.
  • We wish to determine whether it is the DNA structure of triplet-repeats or protein recognition of the repeats that recruits the MMR enzymes to triplet repeats in iPSCs. To this end, we used a series of small molecule probes that can be designed to target particular DNA sequences in the human genome, and we find that a molecule that targets the GAA-TTC repeats in the FRDA frataxin gene displaces MMR enzymes and prevents repeat expansion. We are currently exploring the mechanism whereby this molecule displaces the MMR enzymes. A deeper understanding of the molecular events that lead to repeat expansion at the endogenous cellular genes responsible for these diseases will likely lead to discoveries of new therapeutic strategies for these currently untreatable disorders.
  • Over the past year, our research efforts have focused on the generality of the results we found in human induced pluripotent stem cells derived from patients with the neurodegenerative disease Friedreich's ataxia (FRDA). FRDA is one of the trinucleotide repeat (TNR) diseases, and our major previous finding was that the GAA•TCC trinucleotide repeats that cause FRDA expand during isolation and propagation of FRDA hiPSCs. This expansion was shown to be dependent on enzymes that are involved in the repair of mismatches in the human genome. To extend these studies, we have now focused on hiPSCs from the related TNR diseases myotonic dystrophy, Huntington's disease and Fragile X syndrome. Myotonic dystrophy type 1 (DM1) is an inherited dominant muscular dystrophy caused by expanded CTG•CAG triplet repeats in the 3’ UTR of the DMPK1 gene, which produces a toxic gain-of-function CUG RNA. It has been shown that the severity of disease symptoms, age of onset and progression are related to the length of the triplet repeats. However, the mechanism(s) of CTG•CAG triplet-repeat instability is not fully understood. Human induced pluripotent stem cells (iPSCs) were generated from DM1 and Huntington’s disease (HD) patient fibroblasts. We isolated 41 iPSC clones from DM1 fibroblasts, all showing different CTG•CAG repeat lengths, thus demonstrating somatic instability within the initial fibroblast population. During propagation of the iPSCs, the repeats expanded in a manner analogous to the intergenerational expansion observed in DM1 patient families. The correlation between repeat length and expansion rate identified the interval between 57 and 126 repeats as being an important length threshold where expansion rates dramatically increased. Moreover, longer repeats showed faster triplet-repeat expansion. The relatively short repeats in the gene responsible for Huntington's disease are below this threshold and hence do not expand in the iPSCs. The overall tendency of triplet repeats to expand ceased on differentiation into differentiated embryoid body or neurospheres. The mismatch repair components MSH2, MSH3 and MSH6 were highly expressed in iPSCs compared to fibroblasts, and only occupied the DMPK1 gene harboring longer CTG•CAG triplet repeats. In addition, shRNA silencing of MSH2 impeded CTG•CAG triplet-repeat expansion. We have also generated hiPSC lines from seven male subjects clinically diagnosed with fragile X syndrome. These hiPSCs have been thoroughly characterized with respect to pluripotency, DNA methylation status at the FMR1 gene, CGG repeat length, FMR1 expression and neuronal differentiation. The information gained from these studies provides new insight into a general mechanism of triplet repeat expansion in iPSCs.
  • Over the past year, our research efforts have focused on the generality of the results we found in human induced pluripotent stem cells derived from patients with the neurodegenerative disease Friedreich's ataxia (FRDA). FRDA is one of the trinucleotide repeat (TNR) diseases, and our major previous finding was that the GAA•TCC trinucleotide repeats that cause FRDA expand during isolation and propagation of FRDA hiPSCs. This expansion was shown to be dependent on enzymes that are involved in the repair of mismatches in the human genome. To extend these studies, we have focused on hiPSCs from the related TNR diseases myotonic dystrophy type 1 (DM1), Huntington's disease (HD), Fragile X syndrome (FXS), and Fuchs endothelial corneal dystrophy (FECD). DM1 is an inherited dominant muscular dystrophy caused by expanded CTG•CAG triplet repeats in the DMPK gene, which produces a toxic gain-of-function CUG RNA. It has been shown that the severity of disease symptoms, age of onset and progression are related to the length of the triplet repeats. However, the mechanism(s) of CTG•CAG triplet-repeat instability is not fully understood. hiPSCs were generated from DM1 and HD patient fibroblasts. Similar to our results in FRDA, DM1 hiPSCs show repeat instability, and repeat expansion is again dependent on the DNA mismatch repair system. We defined a threshold of repeat lengths where repeat expansion occurs. The relatively short repeats in the gene responsible for Huntington's disease are below this threshold and hence do not expand in the iPSCs. We have also generated hiPSC lines from seven male subjects clinically diagnosed with fragile X syndrome. These hiPSCs have been thoroughly characterized with respect to pluripotency, DNA methylation status at the FMR1 gene, CGG repeat length, FMR1 expression and neuronal differentiation. In recent studies, we have turned our attention to the common eye disease FECD, where ~75% or so of Caucassian patients have a CTG•CAG triplet-repeat in an intron of the gene encoding the essential transcription factor TCF4. We find repeat instability in fibroblasts from FECD patient fibroblasts, and repeat expansion in the corresponding hiPSCs. Importantly, similar to DM1 with the same repeat sequence as in FECD, the pathological mechanism in both diseases appear to be similar, namely RNA toxicity caused by sequestering essential messenger RNA processing factors. We have also identified a potential small molecule therapeutic that binds CTG•CAG triplet-repeats and are currently testing this molecule in the relevant patient iPSC-derived cell types. The information gained from these studies provides new insight into a general mechanism of triplet repeat expansion in iPSCs and has revealed a new therapeutic approach for these diseases.

Optimization of guidance response in human embryonic stem cell derived midbrain dopaminergic neurons in development and disease

Funding Type: 
SEED Grant
Grant Number: 
RS1-00271
ICOC Funds Committed: 
$633 170
Disease Focus: 
Parkinson's Disease
Neurological Disorders
Stem Cell Use: 
Embryonic Stem Cell
oldStatus: 
Closed
Public Abstract: 
A promising approach to alleviating the symptoms of Parkinson’s disease is to transplant healthy dopaminergic neurons into the brains of these patients. Due to the large number of transplant neurons required for each patient and the difficulty in obtaining these neurons from human tissue, the most viable transplantation strategy will utilize not fetal dopaminergic neurons but dopaminergic neurons derived from human stem cell lines. While transplantation has been promising, it has had limited success, in part due to the ability of the new neurons to find their correct targets in the brain. This incorrect targeting may be due to the lack of appropriate growth and guidance cues as well as to inflammation in the brain that occurs in response to transplantation, or to a combination of the two. Cytokines released upon inflammation can affect the ability of the new neurons to connect, and thus ultimately will affect their biological function. In out laboratory we have had ongoing efforts to determine the which guidance molecules are required for proper targeting of dopaminergic neurons during normal development and we have identified necessary cues. We now plan to extend these studies to determine how these critical guidance cues affect human stem cell derived dopaminergic neurons, the cells that will be used in transplantation. In addition, we will examine how these guidance cues affect both normal and stem cell derived dopaminergic neurons under conditions that are similar to the diseased and transplanted brain, specifically when the brain is inflamed. Ultimately, an understanding of how the environment of the transplanted brain influences the ability of the healthy new neurons to connect to their correct targets will lead to genetic, and/or drug-based strategies for optimizing transplantation therapy.
Statement of Benefit to California: 
The goal of our work is to further optimize our ability to turn undifferentiated human stem cells into differentiated neurons that the brain can use as replacement for neurons damaged by disease. We focus onParkinson’s disease, a neurodegenerative disease that afflicts 4-6 million people worldwide in all geographical locations, but which is more common in rural farm communities compared to urban areas (Van Den Eeden et al., 2003), a criteria important for California’s large farming population. In Parkinson’s patients, a small, well-defined subset of neurons, the midbrain dopaminergic neurons have died, and one therapeutic strategy is to transplant healthy replacement neurons to the patient. Our work will further our understanding of the biology of these neurons in normal animals. This will allow us to refine the process of turning human ES cells onto biologically active dopaminergic neurons that can be used in transplantation therapy. Our work will be of benefit to all Parkinson’s patients including afflicted Californians. In addition to the direct benefit in improving PD therapies, discoveries from this work are also likely to generate substantial intellectual property and further boost clinical and biotechnical development efforts in California.
Progress Report: 
  • A promising approach to alleviating the symptoms of Parkinson's disease is to transplant healthy dopaminergic neurons into the brains of these patients. Due to the large number of transplant neurons required for each patient and the difficulty in obtaining these neurons from human tissue, the most viable transplantation strategy will utilize not fetal dopaminergic neurons but dopaminergic neurons derived from human stem cell lines. While transplantation has been promising, it has had limited success, in part due to the ability of the new neurons to find their correct targets in the brain. This incorrect targeting may be due to the lack of appropriate growth and guidance cues as well as to inflammation in the brain that occurs in response to transplantation, or to a combination of the two. Cytokines released upon inflammation can affect the ability of the new neurons to connect, and thus ultimately will affect their biological function. In out laboratory we have been examining which guidance molecules are required for proper targeting of dopaminergic neurons during normal development and have identified necessary cues. We have now extended these studies to determine that two of the molecules have dramitc effects on dopaminergic neurons made from human embryonic stem cellls and that at least in vitro, cytokines do not mask these effects. Ultimately, an understanding of how the environment of the transplanted brain influences the ability of the healthy new neurons to connect to their correct targets will lead to genetic, and/or drug-based strategies for optimizing transplantation therapy.

Neural and general splicing factors control self-renewal, neural survival and differentiation

Funding Type: 
Basic Biology III
Grant Number: 
RB3-05009
ICOC Funds Committed: 
$1 372 660
Disease Focus: 
Amyotrophic Lateral Sclerosis
Neurological Disorders
Dementia
Stem Cell Use: 
Embryonic Stem Cell
Cell Line Generation: 
iPS Cell
oldStatus: 
Active
Public Abstract: 
Human embryonic and patient-specific induced pluripotent stem cells have the remarkable capacity to differentiate into many cell-types, including neurons, thus enabling the modeling of human neurological diseases in vitro, and permit the screening of molecules to correct diseases. Maintaining the pluripotent state of the stem cell, directing the stem cell towards a neuronal lineage, keeping the neuronal progenitor and stem cells alive - these are all maintained by thousands of different proteins in the cell at these different "stages". Thus the levels and types of proteins are highly controlled by gene regulatory mechanisms. Genes produce pre-messenger RNA (mRNA) transcripts in the nucleus, which undergo a process of refinement called splicing, whereby long (1,000-100,000 bases) stretches of nucleotides are excised, and much shorter pieces (150 bases) are ligated together to form mature messenger RNA to eventually make proteins in the cytoplasm. Strikingly, some pieces of RNA are used in a particular cell-type, but not another, in a process called "alternative splicing". This is the most prevalent form of generating transcriptome diversity in the human genome, and is important for pushing cells from one state to another i.e. stem cells to neurons, maintaining a cell state i.e. keeping a stem cell pluripotent, or a neuron alive and functioning. Alternative splicing is highly controlled by the recognition of even smaller stretches (6-10 bases) of RNA binding sites) by proteins that bind directly to RNA called splicing factors. The goal of the proposed research is to produce a regulatory map of where these splicing factors bind within pre-mRNAs across the entire human genome with unprecedented resolution using a high-throughput biochemical strategy. Furthermore, using advanced genomic technologies, we will deduce what happens to splicing when these factors do not bind to their binding sites. Finally, using molecular and imaging methods, we will analyze what happens to survival of stem and neuronal cells when these factors are depleted or over-expressed, and if stem cells are induced to make neurons if the levels of these factors are altered. Completion of the proposed research is expected to transform our understanding of the regulatory mechanisms underlying transcriptome complexity important for neurological disease modeling, especially human neurodegeneration, and stem cell biology. In turn, this will facilitate more accurate comparisons of diseased states of neurons from stem-cell models of Amyotrophic Lateral Sclerosis (ALS), Myotonic Dystropy, Spinal Muscular Atrophy (SMA), Parkinson’s and Alzheimer’s to identify mis-spliced genes and the splicing factors responsible for therapeutic intervention.
Statement of Benefit to California: 
Our research provides the foundation for decoding the mechanisms that control the transcriptome complexity of stem cells and neurons derived from stem cells. Our work has direct application in the design of novel strategies to understand the impact of splicing factor misregulation, or mutations within the binding sites for these splicing factors in neurological diseases that heavily impact Californians, such as Amyotrophic Lateral Sclerosis (ALS), Myotonic Dystropy, Spinal Muscular Atrophy (SMA), Parkinson’s and Alzheimer’s. Our research has and will continue to serve as a basis for understanding deviations from "normal" stem and neuronal cells, enabling us to make inroards to understanding neurological disease modeling using neurons differentiated from reprogammed patient-specific lines. Such disease modeling will have great potential for California health care patients, pharmaceutical and biotechnology industries in terms of improved human models for drug discovery and toxicology testing. Our improved knowledge base will support our efforts as well as other Californian researchers to study stem cell models of neurological disease and regenerative medicine, and for the design of new diagnostics and treatments, thereby maintaining California's position as a leader in clinical and biomedical research.
Progress Report: 
  • The overwhelming majority of human genes undergo extensive alternative splicing, but save for several dozens of these regulated splicing events, it is not known which proteins are responsible for controlling these key splicing decisions. Furthermore, mutations in several of these proteins, known as splicing factors, have recently been shown to be causative of neurodegeneration. In this proposal we aim to understand the importance of splicing factor regulation of alternative splicing in controlling pluripotency, fate decision towards the neural lineage and neuronal survival. In our recent publication in Cell Reports, Huelga et al demonstrated that the ubiquitously expressed heterogeneous nuclear ribonucleoproteins (hnRNPs) commonly cooperate and antagonize one another to regulate alternative splicing in a somatic human cell-line. In year one of this grant, we have interrogated several key members of these hnRNP proteins in human neural progenitor and differentiated neurons from embryonic stem cells and induced pluripotent stem cells.
  • The overwhelming majority of human genes undergo extensive alternative splicing, but save for several dozens of these regulated splicing events, it is not known which proteins are responsible for controlling these key splicing decisions. Furthermore, mutations in several of these proteins, known as splicing factors, have recently been shown to be causative of neurodegeneration. In this proposal we aim to understand the importance of splicing factor regulation of alternative splicing in controlling pluripotency, fate decision towards the neural lineage and neuronal survival. In years one and two, we have made significant progress in analyzing the functions of three hnRNP proteins, namely TAF15, EWSR1 and hnRNP A2/B1. All three have been associated with neurological diseases, in particular ALS and FTD. We have also made progress in generating and successfully validating reagents to deplete the larger class of RNA binding proteins in human neural progenitors. Finally, we are making slower but steady progress in depleting RBFOX proteins in human neurons.
  • The overwhelming majority of human genes undergo extensive alternative splicing, but save for several dozens of these regulated splicing events, it is not known which proteins are responsible for controlling these key splicing decisions. Furthermore, mutations in several of these proteins, known as splicing factors, have recently been shown to be causative of neurodegeneration. In this proposal we aim to understand the importance of splicing factor regulation of alternative splicing in controlling pluripotency, fate decision towards the neural lineage and neuronal survival. In year 3 of the proposal, we have completed a deeper analysis of hnRNP A2/B1 which we are preparing for manuscript submission. HnRNP A2/B1 is implicated in neurological diseases such as ALS and FTD.

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