Parkinson's Disease

Coding Dimension ID: 
313
Coding Dimension path name: 
Neurological Disorders / Parkinson's Disease

Stem Cell Pathologies in Parkinson’s disease as a key to Regenerative Strategies

Funding Type: 
Research Leadership 10
Grant Number: 
LA1_C10-06535
ICOC Funds Committed: 
$6 718 471
Disease Focus: 
Parkinson's Disease
Neurological Disorders
oldStatus: 
Closed
Public Abstract: 
Protection and cell repair strategies for neurodegenerative diseases such as Parkinson’s Disease (“PD”) depend on well-characterized candidate human stem cells that are robust and show promise for generating the neurons of interest following stimulation of inherent brain stem cells or after cell transplantation. These stem cells must also be expandable in the culture dish without unwanted growth and differentiation into cancer cells, they must survive the transplantation process or, if endogenous brain stem cells are stimulated, they should insinuate themselves in established brain networks and hopefully ameliorate the disease course. The studies proposed for the CIRM Research Leadership Award have three major components that will help better understand the importance and uses of stem cells for the treatment of PD, and at the same time get a better insight into their role in disease repair and causation. First, we will characterize adult human neural stem cells from control and PD brain specimens to distinguish their genetic signatures and physiological properties of these cells. This will allow us to determine if there are stem cells that are pathological and fail in their supportive role in repairing the nervous system. Next, we will investigate a completely novel disease initiation and propagation mechanism, based on the concept that secreted vesicles from cells (also known as “exosomes”) containing a PD-associated protein, alpha-synuclein, propagate from cell-to cell. Our hypothesis is that these exosomes carry toxic forms of alpha-synuclein from cell to cell in the brain, thereby accounting disease spread. They may do the same with cells transplanted in patients with PD, thereby causing these newly transplanted cells designed to cure the disease, to be affected by the same process that causes the disease itself. This is a bottleneck that needs to be overcome for neurotransplantation to take its place as a standard treatment for PD. Our studies will address disease-associated toxicity of exosomal transmission of aggregated proteins in human neural precursor stem cells. Importantly, exosomes in spinal fluid or other peripheral tissues such as blood might represent a potentially early and reliable disease biomarker as well as a new target for molecular therapies aimed at blocking transcellular transmission of PD-associated molecules. Finally, we have chosen pre-clinical models with α-synucleinopathies to test human neural precursor stem cells as cell replacement donors for PD as well as interrogate, for the first time, their potential susceptibility to PD and contribution to disease transmission. These studies will provide a new standard of analysis of human neural precursor cells at risk for and contributing to pathology (so-called “stem cell pathologies”) in PD and other neurodegenerative diseases via transmission of altered or toxic proteins from one cell to another.
Statement of Benefit to California: 
According to the National Institute of Health, Parkinson’s disease (PD) is the second most common neurodegenerative disease in California and the United States (one in 100 people over 60 is affected) second only to Alzheimer’s Disease. Millions of Americans are challenged by PD, and according to the Parkinson’s Action Network, every 9 minutes a new case of PD is diagnosed. The cause of the majority of idiopathic PD is unknown. Identified genetic factors are responsible for less than 5% of cases and environmental factors such as pesticides and industrial toxins have been repeatedly linked to the disease. However, the vast majority of PD is thought to be etiologically multi-factorial, resulting from both genetic and environmental risk factors. Important events leading to PD probably occur in early or mid adult life. According to the Michael J. Fox Foundation, “…there is no objective test, or reliable biomarker for PD, so rate of misdiagnosis is high, and there is a seriously pressing need to develop better early detection approaches to be able to attempt disease-halting protocols at a non-symptomatic, so-called prodromal stage.” The proposed innovative and transformative research program will have a major direct impact for patients who live in California and suffer from PD and other related neurodegenerative diseases. If these high-risk high-pay-off studies are deemed successful, this new program will have tackled major culprits in the PD field. They could lead to a better understanding of the role of stem cells in health and disease. Furthermore they could greatly advance our knowledge of how the disease spreads throughout the brain which in turn could lead to entire new strategies to halt disease progression. In a similar manner these studies could lead to ways to prevent the disease from spreading to cells that have been transplanted to the brain of Parkinson’s patients in an attempt to cure their disease. This is critical for neurotransplantation to thrive as a therapeutic approach to treating PD. In addition, if we extend the cell-to-cell transmissible disease hypothesis to other neurodegenerative diseases, and cancer, the studies proposed here represent a new diagnostic approach and therapeutic targets for many diseases affecting Californians and humankind in general. This CIRM Research Leadership Award will not only have an enormous impact on understanding the cause of PD and developing new therapeutic strategies using stem cells and its technologies, this award will also be the foundation of creating a new Center for Translational Stem Cell Research within California. This could lead to further growth at the academic level and for the biotechnology industry, particularly in the area regenerative medicine.

Neural Stem Cell-Based Therapy For Parkinson’s Disease

Funding Type: 
Disease Team Therapy Planning I
Grant Number: 
DR2-05431
ICOC Funds Committed: 
$99 976
Disease Focus: 
Parkinson's Disease
Neurological Disorders
oldStatus: 
Closed
Public Abstract: 
Ongoing degeneration of dopaminergic (DA) neurons in the midbrain is the hallmark of Parkinson’s disease (PD), a movement disorder that manifests with tremor, bradykinesia and rigidity. One million Americans live with PD and 60,000 are diagnosed with this disease each year. Although the cost is $25 billion per year in the United States alone, existing therapies for PD are only palliative and treat the symptoms but do not address the underlying cause. Levodopa, the gold standard pharmacological treatment to restore dopamine, is compromised over time by decreased efficacy and particularly increased side effects over time. Neural transplantation is a promising strategy for improving dopaminergic dysfunction in PD. The rationale behind neural transplantation is that grafting cells that produce DA into the denervated striatum will reestablish regulated neurotransmission and restore function. Indeed, over 20 years of research using fetal mesencephalic tissue as a source of DA neurons has demonstrated the therapeutic potential of cell transplantation therapy in animal model of PD and in human patients. However, there are limitations associated with primary human fetal tissue transplantation, including high tissue variability, lack of scalability, ethical concerns and inability to obtain an epidemiologically meaningful quantity of tissue. Thus, the control of the identity, purity and potency of these cells becomes exceedingly difficult and jeopardizes both the safety of the patient and the efficacy of the therapy. Thus the search of self-renewable sources of cells is a very worthwhile goal with societal importance and commercial application. Human neural stem cells are currently the only potential reliable and continuous source of homogenous and qualified populations of DA neurons for cell therapy for PD. Such cell source is ideal for developing a consistently safe and efficacious cellular product for treating large number of PD patients in California and throughout the world We have developed a human neural stem cell line with midbrain dopaminergic properties and the technology to make 75% of the neuronal population express dopamine. We have also shown that these cells are efficacious in the most authentic animal model of PD. We now propose to conduct the manufacturing of these cells in conjunction with the safety and efficacy testing to bring this much needed cellular product to PD patients and treat this devastating disease.
Statement of Benefit to California: 
In this grant application we propose to develop a unique technology to manufacture neurons that will be used to treat patients suffering from Parkinson’s disease. One million Americans live with PD and 60,000 are diagnosed with this disease each year. Although the cost is $25 billion per year in the United States alone, existing therapies for PD are only palliative and treat the symptoms but do not address the underlying cause. Levodopa, the gold standard pharmacological treatment to restore dopamine, is compromised over time by decreased efficacy and increased side effects. Human stem cells are currently the only potential reliable and continuous source of homogenous and qualified populations of DA neurons for cell therapy for PD. Such cell source is ideal for developing a consistently safe and efficacious cellular product for treating large number of PD patients in California and throughout the world We have developed a human neural stem cell line with midbrain dopaminergic properties and the technology to make 75% of the neuronal population express dopamine. We have also shown that these cells are efficacious in the most authentic animal model of PD. We now propose to conduct the manufacturing of these cells and safety and efficacy testing to bring this cell product to PD patients and treat this devastating disease. The CIRM grant will help us create further intellectual property pertaining to the optimization of the process of manufacturing of the cellular product we developed to treat PD. The grant will also create jobs at Californian institutions and contract companies we will work with to develop this product. Importantly, the intellectual property will be made available for licensing to biotechnology companies here in California to develop this product to treat the over 10 million people afflicted with PD world wide. Revenues from such a product will be beneficial to the California economy.
Progress Report: 
  • The planning award allowed the PI and members of the disease team to identify gaps in studies performed to date and strategically plan manufacturing and preclinical IND enabling studies to lead into a phase I clinical trial
  • The PI, Marcel Daadi, PhD assembled a team comprised of neurosurgeons, neurologists and scientists with expertise in Parkinson’s disease, a contract manufacturing organization (CMO) for cell production, a contract research organization (CRO) for the pharmacology and toxicology studies, and accomplished regulatory and project management consultants to work together on developing a cellular product for treating Parkinson’s disease.
  • Together with the members of the disease team, the PI established a detailed strategy to meet the overall goal of the project, to develop a human neural stem cell (NSC) line for transplantation into patients. The team put together a plan to manufacture the cells that included seven stages:
  • STAGE 1: Product manufacturing and process development in the PI laboratory, with CMO’s participation, in preparation for technology transfer including material sourcing, gap analysis of the current manufacturing and analytical process, development of product characterization profile, refinement of manufacturing and analytical procedures and development of requisite documentation.
  • STAGE 2: Technology transfer to CMO, comprised of training and establishing the necessary resources, perform the manufacturing process in house, demonstrate tech transfer and perform runs to manufacture GMP-like cell product suitable for non-GLP animal studies at the CRO facility.
  • STAGE 3: Manufacturing of GLP materials for use in the pre-clinical studies.
  • STAGE 4: Early pre-clinical non-GLP studies using materials that meet product release criteria. The preclinical studies will address critical issues such as delivery devise and approach, immuno-suppression regiment, dose-range finding study, imaging MRI/PET, micro-dialysis, immune response, behavioral outcome, dyskinesias, immunohistopathology and biochemical analysis.
  • STAGE 5: Formal GLP pre-clinical studies using the GMP materials manufactured at CMO with primary efficacy endpoint that is a significant change in the PD score without appearance of dyskinesias.
  • STAGE 6: Regulatory support activities, including pre-pre IND and pre-IND meetings, and compilation and filing of the IND.
  • STAGE 7: Full Process Qualification at the CMO, and manufacture of the GMP cell bank.
  • Among preclinical development studies proposed are a definitive single-dose toxicity and toxicokinetic study in rats with functional observation battery, a one year recovery period (GLP), tumorigenicity in NOD-SCID mice and study to determine dose-range for efficacy and safety in non-human primates.

hESC-derived NPCs Programmed with MEF2C for Cell Transplantation in Parkinson’s Disease

Funding Type: 
Disease Team Therapy Planning I
Grant Number: 
DR2-05272
ICOC Funds Committed: 
$96 448
Disease Focus: 
Parkinson's Disease
Neurological Disorders
oldStatus: 
Closed
Public Abstract: 
We proposes to use human embryonic stem cells (hESCs) differentiated into neural progenitor/stem cells (NPCs), but modified by transiently programming the cells with the transcription factor MEF2C to drive them more specifically towards dopaminergic (DA) neurons, representing the cells lost in Parkinson’s disease. We will select Parkinson’s patients that no longer respond to L-DOPA and related therapy for our study, because no alternative treatment is currently available. The transplantation of cells that become DA neurons in the brain will create a population of cells that secrete dopamine, which may stop or slow the progression of the disease. In this way, moderate to severely affected Parkinson’s patients will benefit. The impact of development of a successful cell-based therapy for late-stage Parkinson’s patients would be very significant. There are approximately one million people in the United States with Parkinson’s disease (PD) and about ten million worldwide. Though L-DOPA therapy controls symptoms in many patients for a period of time, most reach a point where they fail to respond to this treatment. This is a very devastating time for sufferers and their families as the symptoms then become much worse. A cell-based therapy that restores production of dopamine and/or the ability to effectively use L-DOPA would greatly improve the lives of these patients. Because of our extensive preclinical experience and the clinical acumen of our Disease Team, we will be able to quickly adapt our procedures to human patients and be able to seek an IND from the FDA within four years.
Statement of Benefit to California: 
It is estimated that the cost per year for a Parkinson’s patient averages over $10,000 in direct costs and over $21,000 in total cost to society (in 2007 dollars). With nearly 40 million people in California and with one in 500 estimated to have Parkinson’s (1.5-2% of the population over 60 years of age), there are approximately 80,000 people in California with Parkinson’s disease. Thus, Parkinson’s disease is a significant burden to California, not to mention the devastating effect on those who have the disease and their families. A therapy that could halt the progression or reverse Parkinson’s disease would be of great benefit to the state and its residents. It would be particularly advantageous if the disease could be halted or reversed to an early stage, since the most severe symptoms and highest costs of care are associated with the late stages of the disease. Cell-based therapies offer the hope of achieving this goal.
Progress Report: 
  • A distinguished group of scientists was assembled by Dr. Stuart Lipton to plan a strategy to develop a human embryonic stem cell line expressing a constitutively active form of the transcription factor MEF2 (MEF2CA) into a therapeutic for treatment of Parkinson’s disease (PD), as funded by this planning grant. Preliminary data presented showed directed differentiation of the stem cells into mature dopaminergic cells and a positive outcome, histologically, electrophysiologically and behaviorally, when transplanted into a rat model. The salient features of the preliminary data show that the cells showed a strong propensity to differentiate into dopaminergic neurons, remaining endogenous dopaminergic neurons were saved from death or recruited to synthesize more dopamine through trophic interactions, and the behavioral readout showed that the rats’ neuromotor deficits were improved. An additional feature of the transplanted cells produced by the presented strategy was that none of the MEF2CA-expressing cells were hyperproliferative, indicating that tumor formation will not be a problem with their use. A strategy to further develop the cells under GMP conditions, test in rat and monkey models of PD and begin regulatory compliance for FDA approval was developed. Importantly, insertion of the Mef2CA gene in the stable stem cell line was verified by sequencing to occur at non-essential site of integration.

Understanding the role of LRRK2 in iPSC cell models of Parkinson's Disease

Funding Type: 
Basic Biology III
Grant Number: 
RB3-02221
ICOC Funds Committed: 
$1 482 822
Disease Focus: 
Parkinson's Disease
Neurological Disorders
Stem Cell Use: 
iPS Cell
oldStatus: 
Active
Public Abstract: 
The goal of this research is to utilize novel research tools to investigate the molecular mechanisms that cause Parkinson’s disease (PD). The proposed work builds on previous funding from CIRM that directed the developed patient derived models of PD. The majority of PD patients suffer from sporadic disease with no clear etiology. However some PD patients harbor specific inherited mutations have been shown to cause PD. The most frequently observed form of genetic parkinsonism is caused by the LRRK2 G2019S mutation it the most common. This mutation accounts for approximately 1.5-2% of patients with apparently sporadic PD, increasing to 4-6% of patients with a family history of PD, and even higher in isolated populations. Importantly, LRRK2 induced PD is clinically and pathologically largely indistinguishable from sporadic PD. This proposal focuses on studying the most frequent cause of familial PD and induces disease that is clinically and pathologically identical to sporadic PD cases. It is likely that LRRK2 regulates a pathway(s) that is important in the more common sporadic form of PD as well. Therefore by employing relevant models of PD, we hope to drive the biological understanding of LRRK2 in a direction that facilitates the development of disease therapeutics in the future. We ascertained patients harboring mutations in LRRK2 [heterozygous (+/G2019S) and homozygous (G2019S/G2019S)] as well as sporadic cases and age matched controls. We have successfully derived iPSCs from each genotype and differentiated these to DA neurons. We will use these as a model system to investigate these LRRK2 based models of PD. We will adapt current biochemical assays of LRRK2, which are source material intensive, to the small culture volumes required for the differentiation of iPSCs to DA neurons. This is a crucial necessity for development for utilizing iPSC derived DA neurons as tractable models of LRRK2 based PD. We will then probe the roles of LRRK2 in neuronal cell differentiation and survival. We will also ask whether the mutant LRRK2 induces changes in autophagy, as this has been postulated as a mechanism of LRRK2 induced pathogenesis. By studying wild-type and disease mutant LRRK2, in DA models of PD we hope to provide crucial understanding of the role mutant LRRK2 has in disease.
Statement of Benefit to California: 
It is estimated that by the year 2030, 75,000-120,000 Californians will be affected by Parkinson’s disease. Currently, there is no cure, early detection mechanism, preventative treatment, or effective way to slow disease progression. The increasing disability caused by the progression of disease burdens the patients, their caregivers as well as society in terms of healthcare costs. The majority of PD patients suffer from sporadic disease with no clear etiology, and a in a handful of these patients specific inherited mutations have been shown to cause PD. The most frequently mutated gene is called Leucine Rich Repeat Kinase 2 (LRRK2). Our goal is to study the mutated gene product in patient based models of Parkinson’s disease. In previous CIRM funding, we have developed patient derived induced pluripotent stem cells (iPSCs) from patients harboring mutations in LRRK2. We have been successful in differentiating populations these iPSCs into the neurons that are depleted in PD. The next step is to utilize these cells as models of mutation induced PD ‘in a dish’. We will employ these pertinent disease models to answer basic biology questions that remain about the function of LRRK2. This project brings together scientists previously funded by CIRM with scientists well versed in the study of LRRK2. This multidisciplinary approach to studying the causes of PD is a natural benefit to the State of California and its citizens. By bringing a better understanding of the role of LRRK2 in the cells that are lost in the progression of PD, we will bring more concrete knowledge of PD as a whole, bringing more hope for the development of a therapeutic for disease.
Progress Report: 
  • The overarching goal of this work is to utilize models of Parkinson's disease (PD) that originate from cells of PD affected patients harboring mutations within the LRRK2 gene so that we may discern the role of mutated LRRK2 in disease. Mutations in LRRK2 are the most common cause of familial PD. The disease presentation of patients with LRRK2 mutation is typically clinically indistinguishable from sporadic PD cases, making the onset of disease due to LRRK2 dysfunction clinically relevant. We have employed stem cells derived from these patients to generate neuronal cells in which we can determine the roles of LRRK2 in the PD mutated and the unmutated state. We have focused on a cellular process called autophagy that regulates the cell response to nutrient deprivation and plays a role in the selective degradation of proteins within the cell.
  • In the first year of funding we have analyzed the expression of the protein LRRK2 in induced pluripotent stem cells, neuronal precursor cells and have begun to differentiate the neuronal precursors to dopaminergic cells of the type lost in PD (a difficult task in itself). We have applied a novel method for detection of LRRK2 in situ by marrying the protein detection of antibodies and the sensitivity of nucleic acid amplification. We will continue to develop this methodology for maximum sensitivity to LRRK2. We have established assays to assess the effects of the LRRK2 mutant on autophagy that are relevant to PD and neurological diseases in general. We have met or made great progress on most of our anticipated milestones and are eager to proceed to the next phase of the project.
  • The overarching goal of this work is to utilize stem cell based models of Parkinson's disease (PD) derived from cells of PD affected patients that harbor mutations in the LRRK2 gene so that we may elucidate the deleterious role of mutated LRRK2 in disease. Mutations in LRRK2 are the most common cause of familial PD. The disease presentation for these patients with LRRK2 mutation is typically clinically similar to those with sporadic disease, making the onset of disease due to LRRK2 dysfunction clinically relevant. We have utilized stem cells harboring a mutation in LRRK2 and also daughter cells of that line in which genomic editing techniques have been applied to correct the PD mutation or disrupt the LRRK2 gene. We have generated the same kind of cells in culture that are lost during PD and hope that next, we can determine how these mutations that eventually cause disease disrupt normal neuronal function. We have made great progress in the understanding the expression of LRRK2 in early differentiation of stem cells to neurons and his will inform our future studies on mutation caused dysfunctions.

Engineering Defined and Scaleable Systems for Dopaminergic Neuron Differentiation of hPSCs

Funding Type: 
Tools and Technologies II
Grant Number: 
RT2-02022
ICOC Funds Committed: 
$1 493 928
Disease Focus: 
Parkinson's Disease
Neurological Disorders
Stem Cell Use: 
Embryonic Stem Cell
iPS Cell
oldStatus: 
Active
Public Abstract: 
Human pluripotent stem cells (hPSC) have the capacity to differentiate into every cell in the adult body, and they are thus a highly promising source of differentiated cells for the investigation and treatment of numerous human diseases. For example, neurodegenerative disorders are an increasing healthcare problem that affect the lives of millions of Americans, and Parkinson's Disease (PD) in particular exacts enormous personal and economic tolls. Expanding hPSCs and directing their differentiation into dopaminergic neurons, the cell type predominantly lost in PD, promises to yield cells that can be used in cell replacement therapies. However, developing technologies to create the enormous numbers of safe and healthy dopaminergic neurons required for clinical development and implementation represents a bottleneck in the field, because the current systems for expanding and differentiating hPSCs face numerous challenges including difficulty in scaling up cell production, concerns with the safety of some materials used in the current cell culture systems, and limited reproducibility of such systems. An emerging principle in stem cell engineering is that basic advances in stem cell biology can be translated towards the creation of “synthetic stem cell niches” that emulate the properties of natural microenvironments and tissues. We have made considerable progress in engineering bioactive materials to support hESC expansion and dopaminergic differentiation. For example, basic knowledge of how hESCs interact with the matrix that surrounds them has led to progress in synthetic, biomimetic hydrogels that have biochemical and mechanical properties to support hESC expansion. Furthermore, biology often presents biochemical signals that are patterned or structured at the nanometer scale, and our application of materials chemistry has yielded synthetic materials that imitate the nanostructured properties of endogenous ligands and thereby promise to enhance the potency of growth factors and morphogens for cell differentiation. We propose to build upon this progress to create general platforms for hPSC expansion and differentiation through two specific aims: 1) To determine whether a fully defined, three dimensional (3D) synthetic matrix for expanding immature hPSCs can rapidly and scaleably generate large cell numbers for subsequent differentiation into potentially any cell , and 2) To investigate whether a 3D, synthetic matrix can support differentiation into healthy, implantable human DA neurons in high quantities and yields. This blend of stem cell biology, neurobiology, materials science, and bioengineering to create “synthetic stem cell niche” technologies with broad applicability therefore addresses critical challenges in regenerative medicine.
Statement of Benefit to California: 
This proposal will develop novel tools and capabilities that will strongly enhance the scientific, technological, and economic development of stem cell therapeutics in California. The most important net benefit will be for the treatment of human diseases. Efficiently expanding immature hPSCs in a scaleable, safe, and economical manner is a greatly enabling capability that would impact many downstream medical applications. The development of platforms for scaleable and safe cell differentiation will benefit therapeutic efforts for Parkinson’s Disease. Furthermore, the technologies developed in this proposal are designed to be tunable, such that they can be readily adapted to numerous downstream applications. The resulting technologies have strong potential to benefit human health. Furthermore, this proposal directly addresses several research targets of this RFA – the development and validation of stem cell scale-up technologies including novel cell expansion methods and bioreactors for both human pluripotent cells and differentiated cell types – indicating that CIRM believes that the proposed capabilities are a priority for California’s stem cell effort. While the potential applications of the proposed technology are broad, we will apply it to a specific and urgent biomedical problem: developing systems for generating clinically relevant quantities of dopaminergic neurons from hPSCs, part of a critical path towards developing therapies for Parkinson’s disease. This proposal would therefore work towards developing capabilities that are critical for hPSC-based regenerative medicine applications in the nervous system to clinically succeed. The principal investigator and co-investigator have a strong record of translating basic science and engineering into practice through interactions with industry, particularly within California. Finally, this collaborative project will focus diverse research groups with many students on an important interdisciplinary project at the interface of science and engineering, thereby training future employees and contributing to the technological and economic development of California.
Progress Report: 
  • Human pluripotent stem cells (hPSC) have the capacity to differentiate into every cell in the adult body, and they are thus a highly promising source of differentiated cells for the investigation and treatment of numerous human diseases. For example, neurodegenerative disorders are an increasing healthcare problem that affect the lives of millions of Americans, and Parkinson's Disease (PD) in particular exacts enormous personal and economic tolls. Expanding hPSCs and directing their differentiation into dopaminergic neurons, the cell type predominantly lost in PD, promises to yield cells that can be used in cell replacement therapies. However, developing technologies to create the enormous numbers of safe and healthy dopaminergic neurons required for clinical development and implementation represents a bottleneck in the field, because the current systems for expanding and differentiating hPSCs face numerous challenges including difficulty in scaling up cell production, concerns with the safety of some materials used in the current cell culture systems, and limited reproducibility of such systems.
  • This project has two central aims: 1) To determine whether a fully defined, three dimensional (3D) synthetic matrix for expanding immature hPSCs can rapidly and scaleably generate large cell numbers for subsequent differentiation into potentially any cell , and 2) To investigate whether a 3D, synthetic matrix can support differentiation into healthy, implantable human DA neurons in high quantities and yields. In the first year of this project, we have made progress in both aims. Specifically, we are conducting high throughput studies to optimize matrix properties in aim 1, and we have developed a material formulation in aim 2 that supports a level of DA differentiation that we are now beginning to optimize with a high throughput approach.
  • This blend of stem cell biology, neurobiology, materials science, and bioengineering to create “synthetic stem cell niche” technologies with broad applicability therefore addresses critical challenges in regenerative medicine.
  • Human pluripotent stem cells (hPSC) have the capacity to differentiate into every cell in the adult body, and they are thus a highly promising source of differentiated cells for the investigation and treatment of numerous human diseases. For example, neurodegenerative disorders are an increasing healthcare problem that affect the lives of millions of Americans, and Parkinson's Disease (PD) in particular exacts enormous personal and economic tolls. Expanding hPSCs and directing their differentiation into dopaminergic neurons, the cell type predominantly lost in PD, promises to yield cells that can be used in cell replacement therapies. However, developing technologies to create the enormous numbers of safe and healthy dopaminergic neurons required for clinical development and implementation represents a bottleneck in the field, because the current systems for expanding and differentiating hPSCs face numerous challenges including difficulty in scaling up cell production, concerns with the safety of some materials used in the current cell culture systems, and limited reproducibility of such systems.
  • This project has two central aims: 1) To determine whether a fully defined, three dimensional (3D) synthetic matrix for expanding immature hPSCs can rapidly and scaleably generate large cell numbers for subsequent differentiation into potentially any cell , and 2) To investigate whether a 3D, synthetic matrix can support differentiation into healthy, implantable human DA neurons in high quantities and yields. In the first year of this project, we have made progress in both aims. Specifically, we are conducting high throughput studies to optimize matrix properties in aim 1, and we have developed a material formulation in aim 2 that supports a level of DA differentiation that we are now beginning to optimize with a high throughput approach.
  • This blend of stem cell biology, neurobiology, materials science, and bioengineering to create “synthetic stem cell niche” technologies with broad applicability therefore addresses critical challenges in regenerative medicine.

Development and preclinical testing of new devices for cell transplantation to the brain.

Funding Type: 
Tools and Technologies II
Grant Number: 
RT2-01975
ICOC Funds Committed: 
$1 831 723
Disease Focus: 
Neurological Disorders
Parkinson's Disease
oldStatus: 
Active
Public Abstract: 
The surgical tools currently available to transplant cells to the human brain are crude and underdeveloped. In current clinical trials, a syringe and needle device has been used to inject living cells into the brain. Because cells do not spread through the brain tissue after implantation, multiple brain penetrations (more than ten separate needle insertions in some patients) have been required to distribute cells in the diseased brain region. Every separate brain penetration carries a significant risk of bleeding and brain injury. Furthermore, this approach does not result in effective distribution of cells. Thus, our lack of appropriate surgical tools and techniques for clinical cell transplantation represents a significant roadblock to the treatment of brain diseases with stem cell based therapies. A more ideal device would be one that can distribute cells to large brain areas through a single initial brain penetration. In rodents, cell transplantation has successfully treated a great number of different brain disorders such as Parkinson’s disease, epilepsy, traumatic brain injury, multiple sclerosis, and stroke. However, the human brain is about 500 times larger than the mouse brain. While the syringe and needle transplantation technique works well in mice and rats, using this approach may not succeed in the much larger human brain, and this may result in failure of clinical trials for technical reasons. We believe that the poor design of current surgical tools used for cell delivery is from inadequate interactions between basic stem cell scientists, medical device engineers, and neurosurgeons. Using a multidisciplinary approach, we will first use standard engineering principles to design, fabricate, refine, and validate an innovative cell delivery device that can transplant cells to a large region of the human brain through a single brain penetration. We will then test this new prototype in a large animal brain to ensure that the device is safe and effective. Furthermore, we will create a document containing engineering drawings, manufacturing instructions, surgical details, and preclinical data to ensure that this device is readily available for inclusion in future clinical trials. By improving the safety and efficacy of cell delivery to the brain, the development of a superior device for cell transplantation may be a crucial step on the road to stem cell therapies for a wide range of brain diseases. In addition, devices and surgical techniques developed here may also be advantageous for use in other diseased organs.
Statement of Benefit to California: 
The citizens of California have invested generously into stem cell research for the treatment of human diseases. While significant progress has been made in our ability to produce appropriate cell types in clinically relevant numbers for transplantation to the brain, these efforts to cure disease may fail because of our inability to effectively deliver the cells. Our proposed development of a superior device for cell transplantation may thus be a crucial step on the road to stem cell therapies for a wide range of brain disorders, such as Parkinson’s disease, stroke, brain tumors, epilepsy, multiple sclerosis, and traumatic brain injury. Furthermore, devices and surgical techniques developed in our work may also be advantageous for use in other diseased organs. Thus, with successful completion of our proposal, the broad community of stem cell researchers and physician-scientists will gain access to superior surgical tools with which to better leverage our investment into stem cell therapy.
Progress Report: 
  • The surgical tools currently available to transplant cells to the human brain are crude and underdeveloped. In current clinical trials, a syringe and needle device has been used to inject living cells into the brain. Because cells do not spread through the brain tissue after implantation, multiple brain penetrations (more than ten separate needle insertions in some patients) have been required to distribute cells in the diseased brain region. Every separate brain penetration carries a significant risk of bleeding and brain injury. Furthermore, this approach does not result in effective distribution of cells. Thus, our lack of appropriate surgical tools and techniques for clinical cell transplantation represents a significant roadblock to the treatment of brain diseases with stem cell based therapies. A more ideal device would be one that can distribute cells to large brain areas through a single initial brain penetration.
  • In this first year of progress, we have designed, prototyped, and tested a stereotactic neurosurgical device capable of delivering cells to a volumetrically large target region through a single cortical brain penetration. We compared the performance of our device to a currently used cell transplantation implement – a 20G cannula with dual side ports. Through a single initial penetration, our device could transplant materials to a region greater than 4 cubic centimeters. Modeling with neurosurgical planning software indicated that our device could distribute cells within the entire human putamen – a target used in Parkinson’s disease trials – via a single transcortical penetration. While reflux of material along the penetration tract was problematic with the 20G cannula, resulting in nearly 80% loss of cell delivery, our device was resistant to reflux. We also innovated an additional system that facilitates small and precise volumes of injection. Both dilute and highly concentrated neural precursor cell populations tolerated transit through the device with high viability and unaffected developmental potential. Our device design is compatible with currently employed frame-based, frameless, and intraoperative MRI stereotactic neurosurgical targeting systems.
  • The surgical tools currently available to transplant cells to the human brain are crude and underdeveloped. In current clinical trials, a syringe and needle device has been used to inject living cells into the brain. Because cells do not spread through the brain tissue after implantation, multiple brain penetrations (more than ten separate needle insertions in some patients) have been required to distribute cells in the diseased brain region. Every separate brain penetration carries a significant risk of bleeding and brain injury. Furthermore, this approach does not result in effective distribution of cells. Thus, our lack of appropriate surgical tools and techniques for clinical cell transplantation represents a significant roadblock to the treatment of brain diseases with stem cell based therapies. A more ideal device would be one that can distribute cells to large and anatomically complex brain areas through a single initial brain penetration.
  • In the first year of progress, we designed, prototyped, and tested a stereotactic neurosurgical device capable of delivering cells to a volumetrically large target region through a single cortical brain penetration. We compared the performance of our device to a currently used cell transplantation implement – a 20G cannula with dual side ports. Through a single initial penetration, our device could transplant materials to a region greater than 4 cubic centimeters. Modeling with neurosurgical planning software indicated that our device could distribute cells within the entire human putamen – a target used in Parkinson’s disease trials – via a single transcortical penetration. While reflux of material along the penetration tract was problematic with the 20G cannula, resulting in nearly 80% loss of cell delivery, our device was resistant to reflux. We also innovated an additional system that facilitates small and precise volumes of injection. Both dilute and highly concentrated neural precursor cell populations tolerated transit through the device with high viability and unaffected developmental potential. Our device design is compatible with currently employed frame-based, frameless, and intraoperative MRI stereotactic (iMRI) neurosurgical targeting systems.
  • In this second year of progress, we have produced and tested the iMRI compatible version of our cell delivery device. The device components are fabricated from materials that are FDA-approved for use in medical devices, and we have assembled the device under Good Manufacturing Practice (GMP) conditions. Our device functions seamlessly with an FDA-approved stereotactic iMRI neurosurgical platform and computer-aided targeting system, and we have demonstrated that this iMRI-compatible system can deliver to the volume and shape of the human putamen through a single initial brain penetration. Thus, by using modern materials and manufacturing techniques, we have produced a neurosurgical device and technique that enables clinicians to “tailor” cell delivery to individual patient anatomical characteristics and specific disease states. This modern and “easy to use” platform technology furthermore allows “real-time” monitoring of cell delivery and unprecedented complication avoidance, increasing patient safety.

Editing of Parkinson’s disease mutation in patient-derived iPSCs by zinc-finger nucleases

Funding Type: 
Tools and Technologies II
Grant Number: 
RT2-01965
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.

Site-specific integration of Lmx1a, FoxA2, & Otx2 to optimize dopaminergic differentiation

Funding Type: 
Tools and Technologies II
Grant Number: 
RT2-01880
ICOC Funds Committed: 
$1 619 627
Disease Focus: 
Parkinson's Disease
Neurological Disorders
Stem Cell Use: 
iPS Cell
Cell Line Generation: 
iPS Cell
Embryonic Stem Cell
oldStatus: 
Active
Public Abstract: 
The objective of this study is to develop a new, optimized technology to obtain a homogenous population of midbrain dopaminergic (mDA) neurons in a culture dish through neuronal differentiation. Dopaminergic neurons of the midbrain are the main source of dopamine in the mammalian central nervous system. Their loss is associated with one of the most prominent human neurological disorders, Parkinson's disease (PD). There is no cure for PD, or good long-term therapeutics without deleterious side effects. Therefore, there is a great need for novel drugs and therapies to halt or reverse the disease. Recent groundbreaking discoveries allow us to use adult human skin cells, transduce them with specific genes, and generate cells that exhibit virtually all characteristics of embryonic stem cells, termed induced pluripotent stem cells (iPSCs). These cell lines, when derived from PD patient skin cells, can be used as an experimental pre-clinical model to study disease mechanisms unique to PD. These cells will not only serve as an ‘authentic’ model for PD when further differentiated into the specific dopaminergic neurons, but that these cells are actually pathologically affected with PD. All of the current protocols for directed neuronal differentiation from iPSCs are lengthy and suboptimal in terms of efficiency and reproducibility of defined cell populations. This hinders the ability to establish a robust model in-a-dish for the disease of interest, in our case PD-related neurodegeneration. We will use a new, efficient gene integration technology to induce expression of midbrain specific transcription factors in iPSC lines derived from a patient with PD and a sibling control. Forced expression of these midbrain transcription factors will direct iPSCs to differentiate into DA neurons in cell culture. We aim at achieving higher efficiency and reproducibility in generating a homogenous population of midbrain DA neurons, which will lay the foundation for successfully modeling PD and improving hit rates of future drug screening approaches. Our study could also set a milestone towards the establishment of efficient, stable, and reproducible neuronal differentiation using a technology that has proven to be safe and is therefore suitable for cell replacement therapies in human. The absence of cellular models of Parkinson’s disease represents a major bottleneck in the scientific field of Parkinson’s disease, which, if solved, would be instantly translated into a wide range of clinical applications, including drug discovery. This is an essential avenue if we want to offer our patients a new therapeutic approach that can give them a near normal life after being diagnosed with this progressively disabling disease.
Statement of Benefit to California: 
The proposed research could lead to a robust model in-a-dish for Parkinson’s disease (PD)-related neurodegeneration. This outcome would deliver a variety of benefits to the state of California. First, there would be a profound personal impact on patients and their families if the current inevitable decline of PD patients could be halted or reversed. This would bring great happiness and satisfaction to the tens of thousands of Californians affected directly or indirectly by PD. Progress toward a cure for PD is also likely to accelerate the development of treatments for other degenerative disorders. The technology for PD modeling in-a-dish could be applied to other cell types such as cardiomyocytes (for heart diseases) and beta-cells (for diabetes). The impact would likely stimulate medical progress on a variety of conditions in which stem cell based drug screening and therapy could be beneficial. An effective drug and therapy for PD would also bring economic benefits to the state. Currently, there is a huge burden of costs associated with the care of patients with long-term degenerative disorders like PD, which afflict tens of thousands of patients statewide. If the clinical condition of these patients could be improved, the cost of maintenance would be reduced, saving billions in medical costs. Many of these patients would be more able to contribute to the workforce and pay taxes. Another benefit is the effect of novel, cutting-edge technologies developed in California on the business economy of the state. Such technologies can have a profound effect on the competitiveness of California through the formation of new manufacturing and health care delivery facilities that would employ California citizens and bring new sources of revenue to the state. Therefore, this project has the potential to bring health and economic benefits to California that is highly desirable for the state.
Progress Report: 
  • Dopaminergic (DA) neurons of the midbrain are the main source of dopamine in the mammalian central nervous system. Their loss is associated with a prominent human neurological disorder, Parkinson's disease (PD). There is no cure for PD, nor are there any good long-term therapeutics without deleterious side effects. Therefore, there is a great need for novel therapies to halt or reverse the disease. The objective of this study is to develop a new technology to obtain a purer, more abundant population of midbrain DA neurons in a culture dish. Such cells would be useful for disease modeling, drug screening, and development of cell therapies.
  • Recent discoveries allow us to use adult human skin cells, introduce specific genes into them, and generate cells, termed induced pluripotent stem cells (iPSC), that exhibit the characteristics of embryonic stem cells. These iPSC, when derived from PD patient skin cells, can be used as an experimental model to study disease mechanisms that are unique to PD. When differentiated into DA neurons, and these cells are actually pathologically affected with PD.
  • The current methods for directed DA neuronal differentiation from iPSC are inadequate in terms of efficiency and reproducibility. This situation hinders the ability to establish a robust model for PD-related neurodegeneration. In this study, we use a new, efficient gene integration technology to induce expression of midbrain-specific genes in iPSC lines derived from a patient with PD and a normal sibling. Forced expression of these midbrain transcription factor genes directs iPSC to differentiate into DA neurons in cell culture. A purer population of midbrain DA neurons may lay the foundation for successfully modeling PD and improving hit rates in drug screening approaches.
  • The milestones for the first year of the project were to establish PD-specific iPSC lines that contain genomic “docking” sites, termed “attP” sites. In year 2, these iPSC/attP cell lines will be used to insert midbrain-specific transcription factors with high efficiency, mediated by enzymes called integrases. We previously established an improved, high-efficiency, site-specific DNA integration technology in mice. This technology combines the integrase system with newly identified, actively expressed locations in the genome and ensures efficient, uniform gene expression.
  • The PD patient-specific iPSC lines we used were PI-1754, which contains a severe mutation in the SNCA (synuclein alpha) gene, and an unaffected sibling line, PI-1761. The SNCA mutation causes dramatic clinical symptoms of PD, with early-onset progressive disease. We use a homologous recombination-based procedure to place the “docking” site, attP, at well-expressed locations in the SNCA and control iPSC lines (Aim 1.1). We also included a human embryonic stem cell line, H9, to monitor our experimental procedures. The genomic locations we chose for placement of the attP sites included a site on chromosome 22 (Chr22) and a second, backup site on chromosome 19 (Chr19). These two sites were chosen based on mouse studies, in which mouse equivalents of both locations conferred strong gene expression. In order to perform recombination, we constructed targeting vectors, each containing an attP cassette flanked by 5’ and 3’ homologous fragments corresponding to the human genomic location we want to target. For the Chr22 locus, we were able to obtain all 3 targeting constructs for the PI-1754, PI-1761 and H9 cell lines. For technical reasons, we were not able to obtain constructs for the Chr19 location Thus, we decided to focus on the Chr22 locus and move to the next step.
  • We introduced the targeting vectors into the cells and selected for positive clones by both drug selection and green fluorescent protein expression. For the H9 cells, we obtained 110 double positive clones and analyzed 98 of them. We found 8 clones that had targeted the attP site precisely to the Chr22 locus. For the PI-1761 sibling control line, we obtained 44 clones, and 1 of them had the attP site inserted at the Chr22 locus. The PI-1754 SNCA mutant line, on the other hand, grows slowly in cell culture. We are in the process of obtaining enough cells to perform the recombination experiment in that cell line.
  • In summary, we demonstrated that the experimental strategy proposed in the grant indeed worked. We were successful in obtaining iPSC lines with a “docking” site placed in a pre-selected human genomic location. These cell lines are the necessary materials that set the stage for us to fulfill the milestones of year 2.
  • Parkinson's disease (PD) is caused by the loss of dopaminergic (DA) neurons in the midbrain. These DA neurons are the main source of dopamine, an important chemical in the central nervous system. PD is a common neurological disorder, affecting 1% of those at 60 years old and 4% of those over 80. Unfortunately, there is no cure for PD, nor are there any long-term therapeutics without harmful side effects. Therefore, there is a need for new therapies to halt or reverse the disease. The goal of this study is to develop a new technology that helps us obtain a purer, more abundant population of DA neurons in a culture dish and to characterize the resulting cells. These cells will be useful for studying the disease, screening potential drugs, and developing cell therapies.
  • Due to recent discoveries, we can introduce specific genes into adult human skin cells and generate cells similar to embryonic stem cells, termed induced pluripotent stem cells (iPSC). These iPSC, when derived from PD patients, can be used as an experimental model to study disease mechanisms that are unique to PD, because when differentiated into DA neurons, these cells are actually pathologically affected with PD. We are using a PD iPSC line called PI-1754 derived from a patient with a severe mutation in the SNCA gene, which encodes alpha-synuclein. The SNCA mutation causes dramatic clinical symptoms of PD, with early-onset progressive disease. For comparison we are using a normal, unaffected sibling iPSC line PI-1761. We are also using a normal human embryonic stem cell (ESC) line H9 as the gold standard for differentiation.
  • The current methods for differentiating iPSC into DA neurons are not adequate in terms of efficiency and reliability. Our hypothesis is that forced expression of certain midbrain-specific genes called transcription factors will direct iPSC to differentiate more effectively into DA neurons in cell culture. We use transcription factors called Lmx1a, Otx2, and FoxA2, abbreviated L, O, and F. In this project, we have developed a new, efficient gene integration technology that allows us rapidly to introduce and express these transcription factor genes in various combinations, in order to test whether they stimulate the differentiation of iPSC into DA neurons.
  • In the first year of the project, we began establishing iPSC and ESC lines that contained a genomic “landing pad” site for insertion of the transcription factor genes. We carefully chose a location for placement of the genes based on previous work in mouse that suggested that a site on human chromosome 22 would provide strong and constant gene expression. We initially used ordinary homologous recombination to place the landing pad into this site. By the end of year 1 of the project, this method was successful in the normal iPSC and in the ESC, but not in the more difficult-to-grow PD iPSC. To solve this problem, in year 2 we introduced a new and more powerful recombination technology, called TALENs, and were successful in placing the landing pad in the correct position in all three of the lines, including the PD iPSC.
  • We were now in a position to insert the midbrain-specific transcription factor genes with high efficiency. For this step, we developed a new genome engineering methodology called DICE, for dual integrase cassette exchange. In this technology, we use two site-specific integrase enzymes, called phiC31 and Bxb1, to catalyze precise placement of the transcription factor genes into the desired place in the genome.
  • We constructed gene cassettes carrying all pair-wise combinations of the L, O, and F transcription factors, LO, LF, and OF, and the triple combination, LOF. We successfully demonstrated the power of this technology by rapidly generating a large set of iPSC and ESC that contained all the above combinations of transcription factors, as well as lines that contained no transcription factors, as negative controls for comparison. Two examples of each type of line for the 1754 and 1761 iPSC and the H9 ESC were chosen for differentiation and functional characterization studies. Initial results from these studies have demonstrated correct differentiation of neural stem cells and expression of the introduced transcription factor genes.
  • In summary, we were successful in obtaining ESC and iPSC lines from normal and PD patient cells that carry a landing pad in a pre-selected genomic location chosen and validated for strong gene expression. These lines are valuable reagents. We then modified these lines to add DA-associated transcription factors in four combinations. All these lines are currently undergoing differentiation studies in accordance with the year two and three timelines. During year three of the project, the correlation between expression of various transcription factors and the level of DA differentiation will be established. Furthermore, functional studies with the PD versus normal lines will be carried out.

Directed Evolution of Novel AAV Variants for Enhanced Gene Targeting in Pluripotent Human Stem Cells and Investigation of Dopaminergic Neuron Differentiation

Funding Type: 
Tools and Technologies I
Grant Number: 
RT1-01021
ICOC Funds Committed: 
$918 000
Disease Focus: 
Parkinson's Disease
Neurological Disorders
Stem Cell Use: 
Embryonic Stem Cell
Cell Line Generation: 
iPS Cell
oldStatus: 
Closed
Public Abstract: 
Human embryonic stem cells (hESCs) and induced pluripotent stem (iPS) cells have considerable potential as sources of differentiated cells for numerous biomedical applications. The ability to introduce targeted changes into the DNA of these cells – a process known as gene targeting – would have very broad implications. For example, mutations could readily be introduced into genes to study their roles in stem cell propagation and differentiation, to analyze mechanisms of human disease, and to develop disease models to aid in creating new therapies. Unfortunately, gene targeting efficiency in hESCs is very low. To meet this urgent need, we propose to develop new molecular tools and novel technologies for high efficiency gene targeting in hES and iPS cells. Importantly, this approach will be coupled with genome-wide identification and functional analysis of genes involved in the process in dopaminergic neuron development, work with fundamental implications for Parkinson's disease. Barriers to targeted genetic modification include the effective delivery of gene targeting constructs into cells and the introduction of defined changes into the genome. We have developed a high throughput approach to engineer novel properties into a highly promising, safe, and clinically relevant gene delivery vehicle. For example, we have engineered variants of this vehicle with highly efficient gene delivery to neural stem cells (NSCs), and the resulting vehicles can mediate efficient gene targeting. We now propose to engineer novel gene delivery and targeting vehicles optimized for use in hESCs and iPS cells. One application of such an improved vector system will be to study the mechanism of ESC differentiation into dopaminergic neurons aided by the key transcription factor Lmx1a. We propose to identify target genes that are regulated by Lmx1a during dopaminergic neuron differentiation using the newly developed technique of ChIP-seq, in combination with RNA expression and bioinformatics analysis. This work will identify essential control genes that drive dopaminergic neuron differentiation. Furthermore, our improved gene delivery and targeting system will be used for overexpressing candidate genes, knocking them down via RNA interference, and knocking in reporter genes to analyze gene expression networks during neuronal differentiation. The generation of efficient targeting technologies, in combination with genome wide analysis of gene regulation networks, will provide a general method for identifying and testing key regulatory genes for stem cell self-renewal and differentiation, as well as generating stem cell-based models of human disease. This blend of bioengineering and cell biology therefore has strong potential to create an important new capability for basic and applied stem cell research.
Statement of Benefit to California: 
This proposal will develop novel molecular tools and methodologies that will strongly enhance the scientific, technological, and economic development of stem cell therapeutics in California. The most important net benefit will be for the treatment of human diseases. Efficiently introducing specific genetic modifications into a stem cell genome is a greatly enabling technology that would impact many downstream medical applications. This capability will further enable investigations of self-renewal and differentiation, two defining properties of human stem cells. New tools to introduce targeted alterations of ES and iPS cells will also yield key model systems to elucidate mechanisms of human disease, and most importantly enable the generation of mutant cell lines to serve as models of human disease and systems for high throughput screening to develop novel therapies. Finally, the reverse process, the repair of genetic lesions responsible for disease, can in the long run enable the generation of patent-specific stem cell lines for therapeutic application. Each of these applications will directly benefit biomedical knowledge and human health. Furthermore, this proposal directly addresses several research targets of this RFA – the development and utilization of efficient homologous recombination techniques for gene targeting in human stem cells, the development of safer and more effective viral vectors for gene transduction in human stem cells, and the development and analysis of human embryonic stem cell lines with reporter genes inserted into key loci – indicating that CIRM believes that the proposed capabilities are a priority for California’s stem cell effort. While the potential applications of the proposed technology are broad, we will apply it to a specific and urgent biomedical problem: elucidating mechanisms of ES cell differentiation into dopaminergic neurons, part of a critical path towards developing therapies for Parkinson’s disease. While hESCs clearly have this capacity, the underlying mechanisms are incompletely understood, and the efficiency of this process must be improved. We will elucidate transcriptional networks that underlie this process, and utilize our novel gene targeting system to identify and analyze key components of these networks. This work will lead to a better fundamental understanding of mechanisms regulating stem cell differentiation, as well as enhance our ability to control this complex process for biomedical application. The co-investigators have a strong record of translating basic science and engineering into practice through interactions with industry, including the founding of biotech companies in California. Finally, this collaborative project will focus diverse research groups with many students on an important interdisciplinary project at the interface of science and engineering, thereby training future employees and contributing to the technological and economic development of California.
Progress Report: 
  • The central goal of this is to develop enhanced vehicles for gene delivery to human embryonic stem cells, both to modulate gene expression and to edit the cellular genome via homologous recombination. We have been using a novel directed evolution technology to improve the properties of a promising viral vehicle, and we are in the progress of progressively increasing gene delivery efficiency. In particular, we have isolated several viral vector variants with enhanced gene delivery to human embryonic stem cells.
  • In parallel, we have a strong interest in understanding and elucidating mechanisms of human pluripotent stem cell differentiation into dopaminergic neurons, with implications for Parkinson's Disease. In particular, the transcription factor Lmx1a plays a role in this fate specification, but the underlying mechanisms are largely unknown. We are conducting chromatin immunoprecipitation coupled with next generation DNA sequencing to identify the genes in the cellular genome that this factor regulates. We have generated an antibody to isolate this protein from cells and are in the process of pulling down DNA bound to this factor within cells undergoing dopaminergic specification. Once we have identified relevant target genes, we will use the new gene delivery technology to study their functional role in dopaminergic specification of human embryonic stem cells.
  • The central goal of this is to develop enhanced vehicles for gene delivery to human embryonic stem cells, both to modulate gene expression and to edit the cellular genome via homologous recombination. We have been using a novel directed evolution technology to improve the properties of a promising viral vehicle, and we are in the progress of progressively increasing gene delivery efficiency. In particular, we have isolated several viral vector variants with enhanced gene delivery to human embryonic stem cells.
  • In parallel, we have a strong interest in understanding and elucidating mechanisms of human pluripotent stem cell differentiation into dopaminergic neurons, with implications for Parkinson's Disease. In particular, the transcription factor Lmx1a plays a role in this fate specification, but the underlying mechanisms are largely unknown. We are conducting chromatin immunoprecipitation coupled with next generation DNA sequencing to identify the genes in the cellular genome that this factor regulates. We have generated an antibody to isolate this protein from cells and are in the process of pulling down DNA bound to this factor within cells undergoing dopaminergic specification. Once we have identified relevant target genes, we will use the new gene delivery technology to study their functional role in dopaminergic specification of human embryonic stem cells.
  • The central goal of this project is to develop enhanced vehicles for gene delivery to human embryonic stem cells, both to modulate gene expression and to edit the cellular genome via homologous recombination. Harnessing a novel directed evolution technology we have developed to improve the properties of a promising viral vehicle, we have significantly increased its gene delivery efficiency to human embryonic and human induced pluripotent stem cells. Furthermore, this advance resulted in considerable improvements in the efficiency of gene targeting (i.e. editing) in the genomes of these cells.
  • In parallel, we have a strong interest in understanding and elucidating mechanisms of luripotent stem cell differentiation into neurons, with for example implications for Parkinson's Disease. In particular, the transcription factor Lmx1a plays a role in this fate specification, but the underlying mechanisms are largely unknown. We attempted chromatin immunoprecipitation coupled with next generation DNA sequencing to identify the genes in the cellular genome that this factor regulates. Progress in this objective was ultimately hampered by the lack of a suitable antibody against Lmx1a. However, in parallel we have used an analogous approach to investigate mechanisms by which RNA transcription is regulated during the differentiation of embryonic stem cells into neurons, including motor neurons. These basic results can now be applied to enhance the efficiency of neuronal differentiation.

Derivation of Parkinson's Disease Coded-Stem Cells (PD-SCs)

Funding Type: 
New Cell Lines
Grant Number: 
RL1-00682
ICOC Funds Committed: 
$1 589 760
Disease Focus: 
Parkinson's Disease
Neurological Disorders
Stem Cell Use: 
Embryonic Stem Cell
iPS Cell
Cell Line Generation: 
iPS Cell
oldStatus: 
Closed
Public Abstract: 
Parkinson's disease (PD) is currently the most common neurodegenerative movement disorder, severely debilitating approximately 1-2% of the US population. The disease is caused by a selective loss of dopamine-producing neurons located in a specific region of the brain. This loss leads to significant motor function impairment and age-dependent tremors. Unfortunately there is currently no cure for PD, however a synthetic dopamine treatment (L-DOPA), temporarily alleviates symptoms. The mechanisms of PD progression are currently unknown. However, genetic studies have identified that mutations (changes) in seven genes, including ?-synuclein, LRRK2, uchL1, parkin, PINK1, DJ-1 and ATP13A2 cause familial PD. Although the familial form of PD only affects a small portion of PD cases, uncovering the function of these genes may provide insight into the mechanisms that lead to the majority of PD cases. One of the best strategies to study PD mechanisms is to generate experimental models that mimic the initiation and progression of PD. A number of cellular and animal models have been developed for PD research. However, a model, which closely resembles the human degeneration process of PD, is currently not available because human neurons are unable to continuously propagate (grow) in culture. Human stem cells provide an opportunity to fulfill this task because these cells can grow and be programmed to generate dopamine nerve cells (the neurons under assault in PD patients). In this study, we propose to create stem cell lines that possess PD-associated mutations in two causative genes, PINK1 and parkin, using either rejected early stage embryos or cultured patient fibroblasts. These cell lines will in effect, represent a model of human PD degeneration of dopaminergic neurons. Our working hypothesis is that PD-associated abnormal parkin or PINK1 genes cause degeneration of stem cell-derived dopaminergic neurons, and dopaminergic neurons in vivo via the same mechanism. We will fulfill three tasks in this study; 1/ To generate the PD-stem cell (PD-SCs) line which harbor abnormal or mutant parkin or PINK1 genes; 2/ To determine the whether the PD-SCs cell lines can form into midbrain dopaminergic nerve cells; 3/ To determine whether mutations in parkin and PINK1 effect the survival of dopaminergic neurons which are derived from the PD-SCs cells. Successful completion of this study will yield novel cellular models for studying the mechanisms involved in PD initiation and progression, and further screening remedies for PD treatment.
Statement of Benefit to California: 
Parkinson's disease (PD) is the second leading neurodegenerative disease with no current cure available. Compared to other states, California is the highest in the incidence of this particular disease. First, California growers use approximately 250 million pounds of pesticides annually, about a quarter of all pesticides used in the US (Cal Pesticide use reporting system). A commonly used herbicide, paraquat, has been shown to induce parkinsonism in both animals and human. Other pesticides are also proposed as potential causative agents for PD. Studies have shown increased PD-caused mortality in agricultural pesticide-use counties in comparison to those non-use counties in California. Second, California has the largest Hispanic population. Studies suggest that incidence of PD is the highest among Hispanics (Van Den Eeden et al, American Journal of Epidemiology, Vol 157, pages 1015-1022, 2003). Thus, finding effective treatments of PD will significantly benefit citizens in California.
Progress Report: 
  • Parkinson’s disease (PD) is currently the most common neurodegenerative movement disorder, severely debilitating approximately 1-2% of the US population. The disease is caused by a selective loss of dopamine-producing neurons located in a specific region of the brain. This loss leads to significant motor function impairment and age-dependent tremors. Unfortunately there is currently no cure for PD, however a synthetic dopamine treatment (L-DOPA), temporarily alleviates symptoms.
  • The mechanism of PD progression are currently unknown. However, genetic studies have identified that mutations (changes) in multiple genes, including α-synuclein, LRRK2, uchL1, parkin, PINK1, DJ-1 and ATP13A2 cause familial PD. Although the familial form of PD only affects a small portion of PD cases, uncovering the function of these genes may provide insight into the mechanisms that lead to the majority of PD cases.
  • One of the best strategies to study PD mechanisms is to generate experimental models that mimic the initiation and progression of PD. A number of cellular and animal models have been developed for PD research. However, a model, which closely resembles the human degeneration process of PD, is currently not available because human neurons are unable to continuously propagate (grow) in culture. Human stem cells provide an opportunity to fulfill this task because these cells can grow and be programmed to generate dopamine nerve cells (the neurons under assault in PD patients).
  • In this study, we propose to create stem cell lines that possess PD-associated mutations in two causative genes, PINK1 and parkin, using either rejected early stage embryos or cultured patient fibroblasts. These cell lines will in effect, represent a model of human PD degeneration of dopaminergic neurons. Our working hypothesis is that PD-associated abnormal parkin or PINK1 genes cause degeneration of stem cell-derived dopaminergic neurons, and dopaminergic neurons in vivo via the same mechanism. We will fulfill three tasks in this study; 1/ To generate the PD-stem cell (PD-SCs) line which harbor abnormal or mutant parkin or PINK1 genes; 2/ To determine the whether the PD-SCs cell lines can form into midbrain dopaminergic nerve cells; 3/ To determine whether mutations in parkin and PINK1 effect the survival of dopaminergic neurons which are derived from the PD-SCs cells. Successful completion of this study will yield novel cellular models for studying the mechanisms involved in PD initiation and progression, and further screening remedies for PD treatment.
  • During last year, we have successfully generated primary skin fibroblast cultures from PD patients harboring mutations of parkin, PINK1, and DJ-1 genes, as well as sporadic PD patients and normal individuals. By using these cells, we have already generated four induced stem cell lines expressing multiple pluripotent markers (two from PD patients and two from normal individuals. These lines can also form teratomas with cells from three germ layers using mouse as host. These findings suggest that the induced pluripotent cell lines generated in the lab are likely PD patient specific stem cells.
  • During the next report year, we will continue to generate more PD patient-specific induced pluripotent stem cells. We will carefully characterize all lines generated in the lab as proposed. Furthermore, we will adapt protocols to differentiate the new lines into dopaminergic neurons.
  • Public Summary of Scientific Progress
  • Parkinson’s disease (PD) is currently the most common neurodegenerative movement disorder affecting approximately 1-2% of the US population. The disease is caused by a selective loss of dopamine-producing neurons located in a specific region of the brain. This loss leads to significant motor function impairment and age-dependent tremors. Unfortunately, there is currently no cure for PD, however a synthetic dopamine treatment (L-DOPA), temporarily alleviates symptoms.
  • Genetic studies have identified that mutations (changes) in multiple genes cause familial PD. Although the familial form of PD only affects a small portion of PD cases, uncovering the function of these genes in PD-affected dopamine-secretion neurons may provide insight into the mechanisms that lead to the majority of PD cases.
  • One of the best strategies to study PD mechanisms is to generate experimental models that mimic the initiation and progression of PD. A number of cellular and animal models have been developed for PD research. However, a model, which closely resembles the human degeneration process of PD, is currently not available because human neurons are unable to continuously propagate (grow) in culture. Human stem cells provide an opportunity to fulfill this task because these cells can grow and be programmed to generate dopamine nerve cells (the neurons under assault in PD patients).
  • In this study, we propose to create stem cell lines that possess PD-associated mutations in two causative genes, PINK1 and parkin, using either rejected early stage embryos or cultured patient fibroblasts. These cell lines will in effect, represent a model of human PD degeneration of dopaminergic neurons. Our working hypothesis is that PD-associated abnormal parkin or PINK1 genes cause degeneration of stem cell-derived dopaminergic neurons, and dopaminergic neurons in vivo via the same mechanism. We will fulfill three tasks in this study; 1/ To generate the PD-stem cell (PD-SCs) line which harbor abnormal or mutant parkin or PINK1 genes; 2/ To determine the whether the PD-SCs cell lines can form into midbrain dopaminergic nerve cells; 3/ To determine whether mutations in parkin and PINK1 effect the survival of dopaminergic neurons which are derived from the PD-SCs cells. Successful completion of this study will yield novel cellular models for studying the mechanisms involved in PD initiation and progression, and further screening remedies for PD treatment.
  • During last year, we have successfully obtained more primary skin fibroblast cultures from PD patients harboring mutations of parkin, PINK1, DJ-1 and PLA2G6 genes, as well as sporadic PD patients and normal control individuals. By using these cells, we have already generated 9 induced stem cell lines expressing multiple pluripotent markers (7 from PD patients and 2 from normal individuals). These lines can also form teratomas with cells from three germ layers using mouse as host. These findings suggest that the induced pluripotent cell lines generated in the lab are likely PD patient specific stem cells.
  • During the next report year, we will continue to generate more PD patient-specific induced pluripotent stem cells. We will carefully characterize all lines generated in the lab as proposed. Furthermore, we will adapt protocols to differentiate the new lines into dopaminergic neurons.
  • Parkinson’s disease (PD) is currently the most common neurodegenerative movement disorder, severely debilitating approximately 1-2% of the US population. The disease is caused by a selective loss of dopamine-producing neurons located in a specific region of the brain. This loss leads to significant motor function impairment and age-dependent tremors. Unfortunately there is currently no cure for PD, however a synthetic dopamine treatment (L-DOPA), temporarily alleviates symptoms.
  • The mechanism of PD progression is currently unknown. However, genetic studies have identified that mutations (changes) in multiple genes, including α-synuclein, LRRK2, uchL1, parkin, PINK1, DJ-1 and ATP13A2 cause familial PD. Although the familial form of PD only affects a small portion of PD cases, uncovering the function of these genes may provide insight into the mechanisms that lead to the majority of PD cases.
  • One of the best strategies to study PD mechanisms is to generate experimental models that mimic the initiation and progression of PD. A number of cellular and animal models have been developed for PD research. However, a model, which closely resembles the human degeneration process of PD, is currently not available because human neurons are unable to continuously propagate (grow) in culture. Human stem cells provide an opportunity to fulfill this task because these cells can grow and be programmed to generate dopamine nerve cells (the neurons under assault in PD patients).
  • In this study, we propose to create stem cell lines that either have the genetic background of sporadic PD patients or possess PD-associated mutations using cultured patient fibroblasts. These cell lines will in effect, represent a model of human PD degeneration of dopaminergic neurons. Our working hypothesis is that the degeneration of stem cell-derived dopaminergic neurons and dopaminergic neurons in vivo via the same mechanism. We will fulfill three tasks in this study; 1/ To generate the PD-stem cell (PD-SCs) line which either have the genetic background of sporadic PD patients or harbor PD specific gene mutantions; 2/ To determine the whether the PD-SCs cell lines can form into midbrain dopaminergic nerve cells; 3/ To determine whether mutations in parkin and PINK1 effect the survival of dopaminergic neurons which are derived from the PD-SCs cells. Successful completion of this study will yield novel cellular models for studying the mechanisms involved in PD initiation and progression, and further screening remedies for PD treatment.
  • During last year, we have finished to develop 15 lines of iPSCs. These include 5 lines from normal control individuals, 5 lines from sporadic Parkinson disease patients, and 5 lines from Parkinson disease patients harboring disease related mutations of PINK1, DJ-1 and PLA2G6 genes. These lines provide an unique opportunity to systematically study comparative pathophysiology of Parkinson disease using sporadic and genetic cases. Moreover, we indeed spent more than a year in optimizing the condition for differentiation of these lines. It is recognized that iPSCs are more difficult to differentiate than the hESCs. We are now able to finalize the protocols to have all lines be differentiated in vitro. Therefore, we will be able to compare differences among the controls, sporadic PD and genetic PD at the level of cell biology and molecular biology.
  • During the next report year, we will differentiate all lines into DA neurons and carefully the functional changes of these cells. We hope that the results will reveal some molecular basis of PD pathogenesis from these human neurons.
  • Parkinson’s disease (PD) is currently the most common neurodegenerative movement disorder, severely debilitating approximately 1-2% of the US population. The disease is caused by a selective loss of dopamine-producing neurons located in a specific region of the brain. This loss leads to significant motor function impairment and age-dependent tremors. Unfortunately there is currently no cure for PD, however a synthetic dopamine treatment (L-DOPA), temporarily alleviates symptoms.
  • The mechanism of PD progression is currently unknown. However, genetic studies have identified that mutations (changes) in multiple genes, including α-synuclein, LRRK2, uchL1, parkin, PINK1, DJ-1 and ATP13A2 cause familial PD. Although the familial form of PD only affects a small portion of PD cases, uncovering the function of these genes may provide insight into the mechanisms that lead to the majority of PD cases.
  • One of the best strategies to study PD mechanisms is to generate experimental models that mimic the initiation and progression of PD. A number of cellular and animal models have been developed for PD research. However, a model, which closely resembles the human degeneration process of PD, is currently not available because human neurons are unable to continuously propagate (grow) in culture. Human stem cells provide an opportunity to fulfill this task because these cells can grow and be programmed to generate dopamine nerve cells (the neurons under assault in PD patients).
  • In this study, we propose to create stem cell lines that either have the genetic background of sporadic PD patients or possess PD-associated mutations using cultured patient fibroblasts. These cell lines will in effect, represent a model of human PD degeneration of dopaminergic neurons. Our working hypothesis is that the degeneration of stem cell-derived dopaminergic neurons and dopaminergic neurons in vivo via the same mechanism. We will fulfill three tasks in this study; 1/ To generate the PD-stem cell (PD-SCs) line which either have the genetic background of sporadic PD patients or harbor PD specific gene mutantions; 2/ To determine the whether the PD-SCs cell lines can form into midbrain dopaminergic nerve cells; 3/ To determine whether mutations in parkin and PINK1 effect the survival of dopaminergic neurons which are derived from the PD-SCs cells. Successful completion of this study will yield novel cellular models for studying the mechanisms involved in PD initiation and progression, and further screening remedies for PD treatment.
  • During last four years, we have finished to develop 15 lines of iPSCs. These include 5 lines from normal control individuals, 5 lines from sporadic Parkinson disease patients, and 5 lines from Parkinson disease patients harboring disease related mutations of PINK1, DJ-1 and PLA2G6 genes. These iPS lines are shown to have biochemical and genomic characteristics of human ES cells. These lines provide an unique opportunity to systematically study comparative pathophysiology of Parkinson disease using sporadic and genetic cases. Using these lines, we have identified a group of genes differentially expressed and differentially methylated between iPS cells derived from PD patients and iPS cells derived from normal control individuals. However, we recognize that iPSCs are more difficult to differentiate than the hESCs. We are yet to finalize the protocols to have all lines be differentiated in vitro. Our goal is to compare differences among the controls, sporadic PD and genetic PD at the level of cell biology and molecular biology.

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