Neurological Disorders

Coding Dimension ID: 
303
Coding Dimension path name: 
Neurological Disorders
Funding Type: 
Early Translational IV
Grant Number: 
TR4-06788-A
Investigator: 
ICOC Funds Committed: 
$2 124 000
Disease Focus: 
Stroke
Neurological Disorders
Stem Cell Use: 
Embryonic Stem Cell
oldStatus: 
Active
Public Abstract: 

The goal of this project is to produce a stem cell-based therapy for stroke (also known as an ischemic cerebral infarct). Stroke is the third leading cause of death in the USA, and a leading cause of disability among adults. Currently, there are no effective treatments once a stroke has occurred (termed completed stroke). In this proposal, we aim to develop human stem cells for therapeutic transplantation to treat stroke. Potential benefits will outweigh risks because only patients with severe strokes that have compromised activities of daily living to an extreme degree will initially be treated. Using a novel approach, we will generate stem cells that do not form tumors, but instead only make new nerve cells. We will give drugs to avoid rejection of the transplanted cells. Thus, the treatment should be safe. We will first test the cells in stroke models in rodents (mice and rats) in preparation for a human clinical trial. We will collect comprehensive data on the mice and rats to determine if the stem cells indeed become new nerve cells to replace the damaged tissue and to assess if the behavior of the mice and rats has improved. If successfully developed and commercialized, this approach has the potential for revolutionizing stroke therapy.

Statement of Benefit to California: 

The goal of this project is to produce a stem cell-based therapy for stroke (also known as an ischemic cerebral infarct). Stroke is the third leading cause of death in the State of California, and a leading cause of disability among adults. Currently, there are no effective treatments once a stroke has occurred (termed completed stroke), and the quality of life is severely compromised in those that survive the malady. In this proposal, we aim to develop human stem cells for therapeutic transplantation to treat stroke. Using a novel approach, we will generate stem cells that do not form tumors, but instead only make new nerve cells. If successfully developed and commercialized, this approach could provide a therapeutic candidate for the unmet medical need, which would have a tremendous impact on the quality of life for the patient, his or her family, and for the economic and emotional burden on the State of California and its citizens.

Progress Report: 
  • Stroke is the third leading cause of death in the USA and remains a great medical problem in California. Currently, there is no effective treatment for patients with a stroke who are seen several hours after the event. The goal of this project is to establish the feasibility of using a stem cell line for cell-replacement therapy to target stroke (cerebral ischemia). Using a novel, genetic pre-programming approach, we will generate human neural stem/progenitor cells (hNSC/NPCs) that are resistant to apoptotic cell death and destined to become nerve cells (or neurons). Our approach also avoids tumor formation, which can occur if stem cells that are not programmed to become neurons are injected into the brain. We will achieve our goals by introducing a constitutively active form of the transcription factor MEF2C (MEF2CA) into human embryonic stem cell (hESC)-derived hNPCs. For this purpose it is critical to identify a viral vector system that is the safest and most effective in producing MEF2CA-programmed hNPCs. We decided to use an adenoviral-associated virus (AAV) vector system because unlike other viral delivery methods (e.g., lentiviral, retroviral), an AAV system allows us to achieve MEF2CA expression in an integration-free and transient manner, as required for proper neuronal differentiation from NPCs. After in vitro characterization of these cells, including their neurogenic capacity, scalability etc., we will transplant them into rodent models of focal stroke. We are analyzing transplanted rats with immunohistochemical, electrophysiological, and behavioral methods to determine whether MEF2CA-programmed hNPCs can successfully differentiate into functional, integrated neurons into the host brain and ameliorate stroke-induced behavioral deficits. To assess the robustness of the AAV approach, we will also compare the results obtained from this system to those obtained using a hESC line that is stably programmed (resulting in permanent insertion of the MEF2C transgene into the genome of the cell, as opposed to transient MEF2C expression achieved with the AAV system). These studies will allow us to determine the effectiveness of the integration-free AAV system vs. stable integration of MEF2C on hNPCs developed for cell replacement therapy. During the current reporting period (Year 01), we have efficiently produced an AAV vector that transduces the MEF2CA transgene into hESC-derived NPCs. FACS analysis revealed that we have robustly infected the hNPCs with this AAV-based construct (~95% of cells infected). In vitro evaluation for protein and mRNA (through immunocytochemistry, and qRT-PCR assays) from the cells infected with AAV-MEF2CA revealed their neural progenitor cell identity and that the MEF2CA transgene is active in these cells. We have begun to transplant these cells into the brain of the spontaneously hypertensive (SHR) rat model of focal stroke. We have begun to compare the effects in stroke of the AAV-MEF2CA and stable-MEF2CA cell lines. In vitro characterization of the stable MEF2CA stem cell line demonstrated that we can differentiate these hNPCs into neurons. For example, protein, mRNA, and morphological analyses revealed robust differentiation of these hNPCs into mature cerebrocortical neurons. Electrophysiological analysis further confirmed the expression of functional neuronal channels and synaptic currents. Behavioral evaluation performed 12-weeks after transplant into the stroked brain with the stable-MEF2CA hNPC line vs. control revealed promising behavioral improvements in the MEF2CA-NPC transplanted group compared to control without apparent side effects.
Funding Type: 
Early Translational III
Grant Number: 
TR3-05676
Investigator: 
Name: 
Type: 
PI
ICOC Funds Committed: 
$1 654 830
Disease Focus: 
Amyotrophic Lateral Sclerosis
Neurological Disorders
Stem Cell Use: 
iPS Cell
Cell Line Generation: 
iPS Cell
oldStatus: 
Active
Public Abstract: 

Approximately 5,600 people in the U.S. are diagnosed with ALS each year. The incidence of ALS is two per 100,000 people, and it is estimated that as many as 30,000 Americans may have the disease at any given time. There are no effective therapies of ALS to-date. Recent genetic discoveries have pinpointed mutations that lead to the aberrant function of two proteins that bind to RNA transcripts in neurons. Misregulation of these RNA binding proteins is responsible for the aberrant levels and processing of hundreds of RNA representing genes that are important for neuronal survival and function. In this proposal, we will use neurons generated from patient cells that harbor the mutations in these RNA binding proteins to (1) prioritize a RNA “signature” unique to neurons suffering from the toxic function of these proteins and (2) as an abundant source of raw material to enable high-throughput screens of drug-like compounds that will bypass the mutations in the proteins and “correct” the RNA signature to resemble that of a healthy neuron. If successful, our unconventional approach that uses hundreds of parallel measurements of specific RNA events, will identify drugs that will treat ALS patients.

Statement of Benefit to California: 

Our research aims to develop drug-like compounds that are aimed to treat Amyotrophic Lateral Sclerosis (ALS), which may be applicable to other neurological diseases that heavily impact Californians, such as Frontotemporal Lobar Degeneration, Parkinson’s and Alzheimer’s. The cellular resources and genomic assays that we are developing in this research will have great potential for future research and can be applied to other disease areas. The cells, in particular will be beneficial to 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 design new diagnostics and treatments, thereby maintaining California's position as a leader in clinical research.

Progress Report: 
  • Our research aims to develop drug-like compounds that are aimed to treat Amyotrophic Lateral Sclerosis (ALS), which may be applicable to other neurological diseases that heavily impact Californians, such as Frontotemporal Lobar Degeneration, Parkinson’s and Alzheimer’s. In the first year, we have succeeded in improving the efficiency of motor neuron differentiation to generate high-quality motor neurons from induced pluripotent stem cells. We have generated RNA signatures from motor neurons differentiated from induced pluripotent stem cells from normal, healthy individuals whereby key proteins implicated in ALS are depleted using RNAi technology. We have also generated motor neurons from induced pluripotent stem cells that contained mutations in these key proteins and are in the process of applying genomic technologies to compare these cells to ones where we have depleted the proteins themselves. In parallel, we have started to optimize conditions for a small molecule screen to identify previously FDA-approved compounds that may alter aberrant and ALS-associated phenotypes in human cell lines.
  • In this reporting period, we have successfully generated lines that we have used to identify small molecules that alter the formation of aggregates in human neural progenitors and non-neuronal cell lines. These molecules will be tested for the reversal of aberrant RNA signatures in motor neurons from patients with ALS-associated mutations.
Funding Type: 
Early Translational IV
Grant Number: 
TR4-06847
Investigator: 
Institution: 
Type: 
PI
ICOC Funds Committed: 
$1 333 795
Disease Focus: 
Huntington's Disease
Neurological Disorders
Stem Cell Use: 
iPS Cell
oldStatus: 
Active
Public Abstract: 

The long-term objective of this project is to develop a drug to treat Huntington’s disease (HD), the most common inherited neurodegenerative disorder. Characterized by involuntary movements, personality changes and dementia, HD is a devastatingly progressive disease that results in death 10–20 years after disease onset and diagnosis. No therapy presently exists for HD; therefore, this project is highly innovative and ultimately aims to deliver something transformative for the HD patient population. The specific goal of the proposed research will be to achieve preclinical proof-of-concept with a novel small molecule that binds to and ameliorates the neurotoxicity of the mutant huntingtin (mHtt) protein that causes HD. Rationale for development of such compounds comes from previous research that found that mHtt assumes a shape that is selectively toxic to neurons, and that small molecules that disrupt this shape can reduce mHtt’s toxicity in primary neurons. Critical to the proposed studies will be assays that employ human striatal neurons derived from adult and juvenile HD patients and generated with induced pluripotent stem cell (iPSC) technology. These HD i-neurons display many characteristics that are also observed in striatal neurons of HD patients, including reduced survival times. They provide the most genetically precise preclinical system available to test for both drug efficacy and safety.

Statement of Benefit to California: 

The long-term objective of this project is to develop a first-in-class, disease-modifying drug to treat Huntington’s disease (HD), a devastatingly progressive genetic disorder that results in death 10–20 years after disease onset and diagnosis. No therapy presently exists for HD; therefore, this highly innovative project aims to deliver a medical breakthrough that will provide significant benefit for California’s estimated > 2000 HD patients and the family members, friends and medical system that care for them. The proposed research will be performed at a biotechnology startup, a leading academic research center and two contract research organizations, all of which are California-based. The work will over time involve more than 10 California scientists, thereby helping to employ tax-paying citizens and maintain the State’s advanced technical base. Finally, an effective, proprietary drug for the treatment of HD is expected to be highly valuable and to attract favorable financial terms upon out-licensing for development and commercialization. These revenues would flow to the California companies and institutions (including CIRM) that would have a stake in the proceeds.

Progress Report: 
  • The long-term objective for this project was to develop a first-in-class, disease-modifying drug to treat Huntington's disease (HD). This drug would comprise a small molecule that binds to and ameliorates the neurotoxicity of the mutant huntingtin protein (mHtt) that causes HD.
  • The goal of the research conducted under the CIRM Award was to demonstrate development candidate feasibility in vitro with a novel small molecule mHtt detoxifier early lead compound that is potent and efficacious in neurons from HD patients generated using stem cell technology (HD i-neurons) as well as suitable for use in mice as experimental models for HD.
  • The original project strategy was to 1) acquire or synthesize new samples of compounds identified as potential mHtt detoxifiers in the screening campaign conducted 7 years ago; 2) establish or re-establish the cell-free and cultured neuron biological assays needed to characterize potential small molecule mHtt detoxifiers (this work was carried out in the laboratory of our collaborator, Dr. Steven Finkbeiner of the J. David Gladstone Institutes); 3) acquire or synthesize new/novel analogs of the initial hits; 4) test new/novel compounds for activity in a cell-free assay for potential mHtt detoxifier activity; 5) test hits for efficacy in HD and non-HD i-neurons; and 6) profile the in vitro and in vivo pharmacokinetics and absorption, distribution, metabolism and elimination (PK/ADME) profiles of compounds that displayed selective neuroprotection toward HD i-neurons.
  • Specific achievements of the first year of the Project include:
  • • Acquiring 205 previously identified hits or analogs thereof from commercial sources;
  • • Synthesizing an additional 84 novel, designed analogs;
  • • Generating the reagents, re-establishing and implementing the screening assay;
  • • Testing all compounds acquired or synthesized in the screening assay;
  • • Establishing a counterscreen for false positives in the screening assay;
  • • Preliminary screening 48 previously reported hits in the counterscreen;
  • • Testing 14 previously or newly identified hits side-by-side in full concentration-response assays in both the screening and counterscreening assays;
  • • Profiling 11 diverse hits in in vitro PK/ADME assays;
  • • Testing 17 compounds for their ability to ameliorate neurotoxicity in a rodent primary neuron model; and
  • • Preliminary testing 2 previously identified hits in human HD i-neurons.
  • Unfortunately and surprisingly, we observed that all compounds displayed essentially identical profiles in full concentration-response studies in both the screening and counterscreening assays. We interpret this result to indicate that these compounds and structurally related compounds that we considered to be most promising and tested do not in fact bind to mHtt, i.e., they are all false positives. Since no valid starting points exist for continued work, the Project will be terminated after the first award period.
Funding Type: 
Tools and Technologies III
Grant Number: 
RT3-07948
Investigator: 
Institution: 
Type: 
PI
Institution: 
Type: 
Co-PI
ICOC Funds Committed: 
$1 452 708
Disease Focus: 
Spinal Cord Injury
Neurological Disorders
Stem Cell Use: 
iPS Cell
Public Abstract: 

One critical bottleneck in the translation of regenerative medicine into the clinic is the efficient delivery and engraftment of transplanted cells. While direct injection is the least invasive method for cell delivery, it commonly results in the survival of only 5-20% of cells. Studies suggest that delivery within a carrier gel may enhance cell viability, but most of the gels used previously were naturally derived materials that have complex and unknown compositions. In our previous CIRM-funded work, we discovered that pre-encapsulating cells in very weak hydrogels offers the best protection during injection; however, those gels may be too compliant to support long-term cell survival. To address these limitations, we propose the design of a fully defined, customizable, and injectable material that initially forms a weak gel that then stiffens post-injection. We focus our studies on the delivery of human induced pluripotent stem cell-derived neural progenitors for the treatment of spinal cord injury (SCI). There are ~12,000 new SCI patients in the US each year, primarily young adults. SCI commonly results in paralysis, and the estimated lifetime cost for a patient can rise above $4 million dollars. In preclinical models of SCI, stem cell therapies have resulted in partial regeneration; however, reproducible delivery and engraftment of sufficient cells remain difficult and unmet challenges. This award potentially develops transformational regenerative therapies for SCI.

Statement of Benefit to California: 

The annual incidence of spinal cord injuries (SCI) in the United States is estimated at 12,000 new cases per year, with motor vehicle crashes accounting for up to a third of these cases. SCI has devastating impacts not only on the quality of life for the victims and their families, but also on their economic security – the estimated lifetime cost of an SCI patient can rise to over $4 million dollars depending on the severity and age at which the injury was sustained, not including the loss of wages and productivity. Although the most prevalent types of SCIs are those sustained at either the cervical or thoracic vertebrae, there are currently no definitive therapies approved for the chronic management of these SCI. Stem cell-based therapies have recently been shown to be mildly successful in several clinical and pre-clinical trials in various injuries and diseases, and a number of trials are ongoing for applications in SCI. In our proposal, we seek to advance the stem cell-based approach to the treatments of SCI. The potential benefit of this proposal to the state of California and its citizens include 1) the provision of a better medical prognosis for patients with spinal cord injuries, 2) the improved quality of life for SCI patients and their families, 3) the reduction of the burden of health care costs, 4) the creation and maintenance of jobs in the stem cell technology field, and 5) preserving California’s prominence in the field of stem cell research.

Funding Type: 
Preclinical Development Awards
Grant Number: 
PC1-08086
Investigator: 
ICOC Funds Committed: 
$1 737 271
Disease Focus: 
Alzheimer's Disease
Neurological Disorders
Stem Cell Use: 
Embryonic Stem Cell
iPS Cell
Cell Line Generation: 
Embryonic Stem Cell
iPS Cell
Public Abstract: 

Over 6 million people in the US suffer from Alzheimer’s disease (AD). There are no drugs that prevent the death of nerve cells in AD, nor has any drug been identified that can stimulate nerve cell replacement in aged human brain. Importantly, even if nerve cells could be replaced, the toxic environment of the AD brain which caused the disease in the first place will likely kill any cells that are born into that environment unless they are resistant to those conditions or can be protected by a drug. Therefore, drugs that stimulate the generation of new neurons (neurogenesis) alone will not be effective. A drug with both neurogenic and neuroprotective properties is required. With the ability to use cells derived from human neural precursor cells (hNPCs) derived from human embryonic stem cells (hESCs) as a screen for neurogenic compounds, we have shown that it is possible to identify and tailor drugs for therapeutic use in AD. With the support of CIRM, we have recently made a very potent AD drug candidate that is exceptionally effective in promoting the making of new nerve cells from human embryonic stem cells. It is both neurogenic and has therapeutic efficacy in a rodent model of AD. However, this molecule needs more preclinical development work before it can start the formal FDA pre clinical toxicity screening protocols. This work will optimize the chances for its true therapeutic potential in AD, and presents a unique opportunity to expand the use of hESCs for the development of a therapeutic for a disease for which there is no cure.

Statement of Benefit to California: 

Over 6 million people in the US suffer from AD, and unless a viable therapeutic is identified it is estimated that this number will increase to at least 16 million by 2050, with a cost of well over $1 trillion per year, likely overwhelming both the California and national health care systems. There is no treatment to prevent, cure or slow down this condition. In this application we have used the new human stem cell technologies to develop an AD drug candidate that stimulates the multiplication of nerve precursor cells derived from human embryonic stem cells. This approach presents a unique opportunity to expand the use of human embryonic stems cells for the development of a therapeutic for a disease for which there is no cure, and could lead to a paradigm shift in the treatment of neurodegenerative disease. Since our AD drug discovery approach is fundamentally different from the unsuccessful approaches used by the pharmaceutical industry. It could also stimulate new biotech. The work in this proposal addresses one of the most important medical problems of California as well as the rest of the world, and if successful would benefit all.

Funding Type: 
Preclinical Development Awards
Grant Number: 
PC1-08117
Investigator: 
Type: 
PI
ICOC Funds Committed: 
$4 951 623
Disease Focus: 
Huntington's Disease
Neurological Disorders
Stem Cell Use: 
Embryonic Stem Cell
Public Abstract: 

Huntington’s disease (HD) is a devastating degenerative brain disease with at least 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 care-giving. 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) and their derivatives 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 tissue in HD patients suggests that replacing neurons that are lost may also be effective. The ability to differentiate hESCs into neural populations offers a powerful and sustainable alternative to provide neuroprotection to the brain with the possibility of cell replacement. We have assembled a multidisciplinary team of investigators and consultants with expertise in basic, translational and clinical development and have identified a lead developmental candidate, ESI-017 neural stem cells, that have disease modifying activity in HD mice with sufficient promise to perform systematic efficacy and safety studies in HD mice with cells generated for this project. We will utilize the collaborative research team, additional preclinical and clinical investigators, stem cell experts and FDA consultants to finalize work that will lead to a productive pre-IND meeting with the FDA and a path forward for clinical trials with the neural stem cell development candidate.

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, we have brought together a team of investigators highly experienced in HD basic science and preclinical development, stem cell research, HD clinical trials and FDA regulatory activities to evaluate a human stem cell derived neural stem cell line, ESI-107 NSC in HD mouse models. This selection of this development candidate is based on efficacy in behavioral and electrophysiology measurements in a rapidly progressing mouse model of HD. 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 NSCs 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 proposed studies to move ESI-017 NSCs to the point of a productive pre-IND meeting with the FDA to ultimately move this clinical product into Investigative New Drug-enabling (IND) activities with the goal of performing clinical trials in HD subjects. 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 substantial.

Funding Type: 
Tools and Technologies III
Grant Number: 
RT3-07914
Investigator: 
Name: 
Type: 
PI
ICOC Funds Committed: 
$1 818 751
Disease Focus: 
Intestinal Disease
Pediatrics
Neurological Disorders
Stem Cell Use: 
Adult Stem Cell
Public Abstract: 

The intestine performs the essential function of absorbing food and water into the body. Without a functional intestine, children and adults cannot eat normal meals, and these patients depend on intravenous nutrition to sustain life. Many of these patients do not have a neural system that coordinates the function of the intestine. These patients have a poor quality of life, and the cost of medical care is over $200,000 per year for each patient. Stem cell therapies offer potential cures for these patients while avoiding the risks of invasive procedures and hazardous treatments. A novel approach to treat these patients is to use stem cells derived from the patient’s own skin to generate the neural system. This has been shown to be feasible in small animals, and the next step hinges on the demonstration of these results in a large animal model of intestinal dysfunction. We will develop a model in large animals that can be used to test the ability of skin-derived stem cells to form the neural system. Skin-derived stem cells will be isolated from large animal models and human skin to demonstrate their potential to generate a functional neural system. These cells will be transplanted into the animal model to determine the best way for these cells to make the intestine function properly. This research will gather critical information needed to begin a clinical trial using skin-derived cells to treat intestinal dysfunction.

Statement of Benefit to California: 

Gastrointestinal dysfunction destroys the lives of thousands of Californians. These Californians have frequent and prolonged hospitalizations and lost wages due to their chronic illness. It is estimated that the health care cost of Californians with gastrointestinal neuromuscular dysfunction is over 400 million dollars annually. Currently, most of these patients are covered by the state’s insurance agency. Stem cell therapies offer potential cures for these patients and reduce this economic burden. The proposed research will obtain critical information needed to begin a clinical trial using skin-derived cells to treat patients with intestinal dysfunction. The California economy will significantly benefit from this research through the reduced costs for health care and increased quality of life of the affected Californians. Additionally, this work will add to the state’s growing stem cell industry and will increase employment opportunities and revenue by the state of California. The educational benefit to Californians involved in this research project will also maintain California’s position in leading the stem cell effort in the future.

Funding Type: 
Tools and Technologies II
Grant Number: 
RT2-01965
Investigator: 
Institution: 
Type: 
PI
ICOC Funds Committed: 
$1 327 983
Disease Focus: 
Parkinson's Disease
Neurological Disorders
Stem Cell Use: 
iPS Cell
Cell Line Generation: 
iPS Cell
oldStatus: 
Active
Public Abstract: 

The goal of this proposal is to establish a novel research tool to explore the molecular basis of Parkinson’s disease (PD) - a critical step toward the development of new therapy. To date, a small handful of specific genes and associated mutations have been causally linked to the development of PD. However, how these mutations provoke the degeneration of specific neurons in the brain remains poorly understood. Moreover, conducting such genotype-phenotype studies has been hampered by two significant experimental problems. First, we have historically lacked the ability to model the relevant human cell types carrying the appropriate gene mutation. Second, the genetic variation between individuals means that the comparison of a cell from a disease-carrier to a cell derived from a normal subject is confounded by the many thousands of genetic changes that normally differentiate two individuals from one another. Here we propose to combine two powerful techniques – one genetic and one cellular – to overcome these barriers and drive a detailed understanding of the molecular basis of PD. Specifically, we propose to use zinc finger nucleases (ZFNs) in patient-derived induced pluripotent stem cells (iPSC) to accelerate the generation of a panel of genetically identical cell lines differing only in the presence or absence of a single disease-linked gene mutation. iPSCs have the potential to differentiate into many cell types – including dopaminergic neurons that become defective in PD. Merging these two technologies will thus allow us to study activity of either the wild-type or the mutant gene product in cells derived from the same individual, which is critical for elucidating the function of these disease-related genes and mutations. We anticipate that the generation of these isogenic cells will accelerate our understanding of the molecular causes of PD, and that such cellular models could become important tools for developing novel therapies.

Statement of Benefit to California: 

Approx. 36,000-60,000 people in the State of California are affected with Parkinson’s disease (PD) – a number that is estimated to double by the year 2030. This debilitating neurodegenerative disease causes a high degree of disability and financial burden for our health care system.

Importantly, recent work has identified specific gene mutations that are directly linked to the development of PD. Here we propose to exploit the plasticity of human induced pluripotent stem cells (iPSC) to establish models of diseased and normal tissues relevant to PD. Specifically, we propose to take advantage of recent developments allowing the derivation of stem cells from PD patients carrying specific mutations. Our goal is to establish advanced stem cell models of the disease by literally “correcting” the mutated form of the gene in patient cells, therefore allowing for direct comparison of the mutant cells with its genetically “repaired” yet otherwise identical counterpart. These stem cells will be differentiated into dopaminergic neurons, the cells that degenerate in the brain of PD patients, permitting us to study the effect of correcting the genetic defect in the disease relevant cell type as well as provide a basis for the establishment of curative stem cells therapies.

This collaborative project provides substantial benefit to the state of California and its citizens by pioneering a new stem cell based approach for understanding the role of disease causing mutations via “gene repair” technology, which could ultimately lead to advanced stem cell therapies for Parkinson’s disease – an unmet medical need without cure or adequate long-term therapy.

Progress Report: 
  • The goal of this proposal was to establish a novel research tool to explore the molecular basis of Parkinson’s disease (PD) - a critical step toward the development of new therapy. To date, a small handful of specific genes and associated mutations have been causally linked to the development of PD. However, how these mutations provoke the degeneration of specific neurons in the brain remains poorly understood.
  • In the first year of the grant, we have successfully modified the LRRK2 G2019S mutation in patient-derived induced pluripotent stem cells (iPSC) using zinc-finger technology. We created several clonal lines with the gene correction and also with a knockdown of the LRRK2 gene.
  • We characterized these lines for pluripotency, karyotype, and differentiation potential and currently, we are testing the lines for functional differences in the next reporting period and will generate iPSCs with specific LRRK2 mutations introduced using zinc-finger technology.
  • Despite the growing number of diseases linked to single gene mutations, determining the molecular mechanisms by which such errors result in disease pathology has proven surprisingly difficult. The ability to correlate disease phenotypes with a specific mutation can be confounded by background of genetic and epigenomic differences between patient and control cells. To address this problem, we employed zinc finger nucleases-based genome editing in combination with a newly developed high-efficiency editing protocol to generate isogenic patient-derived induced pluripotent stem cells (iPSC) differing only at the most common mutation for Parkinson's disease (PD), LRRK2 p.G2019S. We show that correction of the LRRK2 p.G2019S mutation rescues a panel of neuronal cell phenotypes including reduced dopaminergic cell number, impaired neurite outgrowth and mitochondrial dysfunction. These data reveal that PD-relevant cellular pathophysiology can be reversed by genetic repair, thus confirming the causative role of this prevalent mutation – a result with potential translational implications.
  • The goal of this proposal has been to establish a novel research tool to explore the molecular basis of Parkinson’s disease (PD) - a critical step toward the development of new therapies. To date, a small handful of specific genes and associated mutations have been causally linked to the development of PD. However, how these mutations provoke the degeneration of specific neurons in the brain remains poorly understood.
  • Moreover, conducting such genotype-phenotype studies has been hampered by two significant experimental problems. First, we have historically lacked the ability to model the relevant human cell types carrying the appropriate gene mutation. Second, the genetic variation between individuals means that the comparison of a cell from a disease-carrier to a cell derived from a normal subject is confounded by the many thousands of genetic changes that normally differentiate two individuals from one another.
  • We proposed to use zinc finger nucleases (ZFNs) in patient-derived induced pluripotent stem cells (iPSC) to accelerate the generation of a panel of genetically identical cell lines differing only in the presence or absence of a single disease-linked gene mutation.
  • To this end, we have successfully generated a panel of LRRK2 isogenic cell lines that differ only in "one building block" in the genomic DNA of a cell which can cause PD, therefore we genetically 'cured' the cells in the culture dish. These lines are invaluable because they are a set of tools that allow to study the effect of this mutation in the context of neurodegeneration and cell death. We received interest from many outside academic laboratories and industry to distribute these novel tools and these cell lines will hopefully lead to the discovery of new drugs that can halt or even reverse PD.
  • Being afflicted with a chronic, progressive disease means that it never stops, it is there in the morning when you wake up and it is the last thing at night that you feel when you are falling asleep. Parkinson’s disease (PD) makes you slowly lose body functions that you once took for granted. Eating tasks become more challenging as well as chewing and swallowing, simple motor movements such as turning in bed or getting out of the car or a deep chair takes a lot of extra effort. You might also show signs of depression, anxiety, even hallucinations or just feeling indifferent towards hobbies/activities or being with loved ones. Autonomic functions are affected with lightheadedness, constipation, or urine control. You might lose your sense of smell, have changes in heart rate, and sleep problems. All these changes can occur at once or become apparent over time. Not everyone with PD is experiencing all of these symptoms. Every disease is different and the symptoms can be diverse. PD is a “designer disease” and needs a targeted approach clinically and scientifically.
  • In this CIRM project, we focused on the clinical and genetic variability and used gene editing technology to modify the genome at precise positions (“correct genetic mutations”) known to cause clinically and neuropathologically PD. The newly created patient-derived pluripotent stem cell lines only differ at the known positions and “off-target” modifications were excluded and we were able to experimentally show that the change in the genetic sequence is “rescuing” the cellular changes relevant for PD.
  • The advantage of these patient-specific cell lines are that specific genetic changes can be directly investigated without the experimental noise in control cell lines. This approach has been adopted by many laboratories in the field of disease modeling and will probably become the gold standard for stem cell modeling and drug discovery.
Funding Type: 
Tools and Technologies II
Grant Number: 
RT2-01881
Investigator: 
ICOC Funds Committed: 
$1 825 613
Disease Focus: 
Stroke
Neurological Disorders
Stem Cell Use: 
iPS Cell
oldStatus: 
Active
Public Abstract: 

Stroke is the leading cause of adult disability. Most patients survive their initial stroke, but do not recover fully. Because of incomplete recovery, up to 1/3 of stroke patients are taken from independence to a nursing home or assisted living environment, and most are left with some disability in strength or control of the arms or legs. There is no treatment that promotes brain repair and recovery in this disease. Recent studies have shown that stem cell transplantation into the brain can promote repair and recovery in animal models of stroke. However, a stem cell therapy for stroke has not reached the clinic. There are at least three limitations to the development of a human stroke stem cell therapy: most of the transplanted cells die, most of the cells that survive do not interact with the surrounding brain, and the process of injecting stem cells into the brain may damage the normal brain tissue that is near the stroke site. The studies in this grant develop a novel investigative team and research approach to achieve a solution to these limits. Using the combined expertise of engineering, stem cell biology and stroke scientists the studies in this grant will develop tissue bioengineering systems for a stem cell therapy in stroke. The studies will develop a biopolymer hydrogel that provides a pro-growth and pro-survival environment for stem cells when injected with them into the brain. This approach has three unique aspects. First, the hydrogel system utilizes biological components that mimic the normal brain environment and releases specific growth factors that enhance transplanted stem cell survival. Second, these growth factors will also likely stimulate the normal brain to undergo repair and recovery, providing a dual mechanism for neural repair after stroke. Third, this approach allows targeting of the stroke cavity for a stem cell transplant, and not normal brain. The stroke cavity is an ideal target for a stroke stem cell therapy, as it is a cavity and can receive a stem cell transplant without displacing normal brain, and it lies adjacent to the site in the brain of most recovery in this disease—placing the stem cell transplant near the target brain region for repair in stroke.
The progress from stroke stem cell research has identified stem cell transplantation as a promising treatment for stroke. The research in this grant develops a next generation in stem cell therapies for the brain by combining new bioengineering techniques to develop an integrated hydrogel/stem cell system for transplantation, survival and neural repair in this disease.

Statement of Benefit to California: 

Advances in the early treatment of stroke have led to a decline in the death rate from this disease. At the same time, the overall incidence of stroke is projected to substantially increase because of the aging population. These two facts mean that stroke will not be lethal, but instead produce a greater number of disabled survivors. A 2006 estimate placed over half of the annual cost in stroke as committed to disabled stroke survivors, and exceeding $30 billion per year in the United States. The studies in this grant develop a novel stem cell therapy in stroke by focusing on one major bottleneck in this disease: the inability of most stem cell therapies to survive and repair the injured brain. With its large population California accounts for roughly 24% of all stroke hospital discharges in the Unites States. The development of a new stem cell therapy approach for this disease will lead to a direct benefit to the State of California.

Progress Report: 
  • This grant develops a tissue bioengineering approach to stem cell transplantation as a treatment for brain repair and recovery in stroke. Stem cell transplantation has shown promise as a therapy that promotes recovery in stroke. Stem cell transplantation in stroke has been limited by poor survival of the transplanted cells. The studies in this grant utilize a multidisciplinary team of bioengineers, neuroscientists/neurologists and stem cell biologists to develop an approach in which stem or progenitor cells can be transplanted into the site of the stroke within a biopolymer hydrogel that provides an environment which supports cell survival and treatment of the injured brain. These hydrogels need to contain naturally occurring brain molecules, so that they do not release foreign or toxic components when they degrade. Further, the hydrogels have to remain liquid so that the injection approach can be minimally invasive, and then gel within the brain. In the past year the fundamental properties of the hydrogels have been determined and the optimal physical characteristics, such as elasticity, identified. Hydrogels have been modified to contain molecules which stem or progenitor cells will recognize and support survival, and to contain growth factors that will both immediately release and, using a novel nanoparticle approach, more slowly release. These have been tested in culture systems and advanced to testing in rodent stroke models. This grant also tests the concept that the stem/progenitor cell that is more closely related to the area within the brain that receives the transplant will provide a greater degree of neural repair and recovery. Progress has been made in the past year in differentiating induced pluripotent stem cells along a lineage that more closely resembles the part of the brain injured in this stroke model, the cerebral cortex.
  • This grant determines the effect of a tissue bioengineering approach to stem cell survival and engraftment after stroke, as means of improving functional recovery in this disease. Stem cell transplantation in stroke has been limited by the poor survival of transplanted cells and their lack of differentiation in the brain. These studies use a biopolymer hydrogel, made of naturally occurring molecules, to provide a pro-survival matrix to the transplanted cells. The studies in the past year developed the chemical characteristics of the hydrogel that promote survival of the cells. These characteristics include the modification of the hydrogel so that it contains specific amounts of protein signals which resemble those seen in the normal stem cell environment. Systematic variation of the levels of these protein signals determined an optimal concentration to promote stem cell survival in vitro. Next, the studies identified the chemistry and release characteristics from the hydrogel of stem cell growth factors that normally promotes survival and differentiation of stem cells. Two growth factors have been tested, with the release characteristics more completely defined with one specific growth factor. The studies then progressed to determine which hydrogels supported stem cell survival in vivo in a mouse model of stroke. Tests of several hydrogels determined that some provide poor cell survival, but one that combines the protein signals, or “motifs”, that were studied in vitro provided improved survival in vivo. These hydrogels did not provoke any additional scarring or inflammation in surrounding tissue after stroke. Studies in the coming year will now determine if these stem cell/hydrogel matrices promote recovery of function after stroke, testing both the protein motif hydrogels and those that contain these motifs plus specific growth factors.
  • This grant determines the effect of a tissue bioengineering approach to stem cell survival and engraftment after stroke, as means of improving functional recovery in this disease. Stem cell transplantation in stroke has been limited by the poor survival of transplanted cells and their lack of differentiation in the brain. These studies use a biopolymer hydrogel, made of naturally occurring molecules, to provide a pro-survival matrix to the transplanted cells. The studies in past years developed the two chemical characteristics of hydrogels that contain recognition or signal elements for stem cells: “protein motifs” that resemble molecules in the normal stem cell environment and growth factors that normally communicate to stem cells in the brain. The hydrogels were engineered so that they contain these familiar stem cell protein motifs and growth factors and release the growth factors over a slow and sustained time course. In the past year on this grant, we tested the effects of hydrogels that had the combined characteristics of these protein motifs and growth factors, at varying concentrations, for their effect on induced pluripotent neural precursor cells (iPS-NPCs) in culture. We identified an optimum concentration for cell survival and for differentiation into immature neurons. We then initiated studies of the effects of this optimized hydrogel in vivo in a mouse model of stroke. These studies are ongoing. They will determine the cell biological effect of this hydrogel on adjacent tissue and on the transplanted cells—determining how the hydrogel enhances engraftment of the transplant. The behavioral studies, also under way, will determine if this optimized hydrogel/iPS-NPC transplant enhances recovery of movement, or motor, function after stroke.
Funding Type: 
Early Translational III
Grant Number: 
TR3-05628
Investigator: 
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.

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