Parkinson's Disease

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

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.
  • The objective of this project is to develop approaches and technologies that will improve neuronal differentiation of stem cells into midbrain dopaminergic (DA) neurons. DA neurons are of central importance in the project, because they are that cells that are impaired in patients with Parkinson’s disease (PD). Current differentiation methods typically produce low yields of DA neurons. The methods also give variable results, and cell populations contain many types of cells. These impediments have hampered the study of disease mechanisms for PD, as well as other uses for the cells, such as drug screening and cell replacement therapy. Our strategy is to develop a novel method to introduce genes into the genome at a specific place, so we can rapidly add genes that might help in the differentiation of DA neurons. The genes we would like to add are called transcription factors, which are proteins involved differentiation of stem cells into DA neurons. We have placed the genes for three transcription factors into a safe, active position on human chromosome 22 in the cell lines we are studying. These cells, called pluripotent stem cells, have the potential to differentiate into almost any type of cell. We are using embryonic stem cells in our study, as well as induced pluripotent stem cells (iPSC), which are similar, but are derived from adult cells, rather than an embryo. We are using iPSC derived from a PD patient, as well as iPSC from a normal person, for comparison. By forced expression of these neuronal transcription factors, we may achieve more efficient and reproducible generation of DA neurons. The effects of expressing different combinations of the three transcription factors called Lmx1a, FoxA2, and Otx2 on DA neuronal differentiation will be evaluated in the context of embryonic stem cells (ESC) as the gold standard, as well as in iPSC derived from a PD patient with a severe mutation in alpha-synuclein and iPSC derived from a normal control. Comparative functional assays of the resulting DA neurons will complete the analysis.
  • To date, this project has created a novel technology for modifying the genome. The strategy developed out of the one that we originally proposed, but contains several innovations that make it more powerful and useful. The new methodology, called DICE for Dual Integrase Cassette Exchange, allowed us to generate “master” or recipient cell lines for ESC, normal iPSC, and PD iPSC. These recipient cell lines contain a “landing pad” placed into a newly-identified actively-expressed location on human chromosome 22 called H11 that permits robust expression of genes placed into it. We then generated a series of cell lines by "cassette exchange" at the H11 locus. In cassette exchange, the new genes we want to add take the place of the landing pad we originally put into the cells. Cassette exchange is a good way to introduce various genes into the same place in the chromosomes. We created cell lines expressing three neuronal transcription factors suspected to be involved in DA neuronal differentiation, in all pair-wise combinations, including lines with expression of all three factors, and negative control lines with no transcription factors added. This collection of modified human pluripotent stem cell lines is now being used to study neural differentiation. The modified ESC have undergone differentiation into DA neurons and are being evaluated for the effects of the different transcription factor combinations on DA neuronal differentiation. During the final year of the project, this differentiation analysis will be completed, and we will also analyze functional properties of the differentiated DA neurons, with special emphasis on disease-related features of the cells derived from PD iPSC.

Genetic Encoding Novel Amino Acids in Embryonic Stem Cells for Molecular Understanding of Differentiation to Dopamine Neurons

Funding Type: 
New Faculty I
Grant Number: 
RN1-00577
ICOC Funds Committed: 
$2 626 937
Disease Focus: 
Parkinson's Disease
Neurological Disorders
oldStatus: 
Closed
Public Abstract: 
Embryonic stem cells have the capacity to self-renew and differentiate into other cell types. Understanding how this is regulated on the molecular level would enable us to manipulate the process and guide stem cells to generate specific types of cells for safe transplantation. However, complex networks of intracellular cofactors and external signals from the environment all affect the fate of stem cells. Dissecting these molecular interactions in stem cells is a very challenging task and calls for innovative new strategies. We propose to genetically incorporate novel amino acids into proteins directly in stem cells. Through these amino acids we will be able to introduce new chemical or physical properties selectively into target proteins for precise biological study in stem cells. Nurr1 is a nuclear hormone receptor that has been associated with Parkinson’s disease (PD), which occurs when dopamine (DA) neurons begin to malfunction and die. Overexpression of Nurr1 and other proteins can induce the differentiation of neural stem cells and embryonic stem cells to dopamine (DA) neurons. However, these DA neurons did not survive well in a PD mouse model after transplantation. In addition, it is unclear how Nurr1 regulates the differentiation process and what other cofactors are involved. We propose to genetically introduce a novel amino acid that carries a photocrosslinking group into Nurr1 in stem cells. Upon illumination, molecules interacting with Nurr1 will be permanently linked for identification by mass spectrometry. Using this approach, we aim to identify unknown cofactors that regulate Nurr1 function or are controlled by Nurr1, and to map sites on Nurr1 that can bind agonists. The function of identified cofactors in DA neuron specification and maturation will be tested in mouse and human embryonic stem cells. These cofactors will be varied in combination to search for more efficient ways to induce embryonic stem cells to generate a pure population of DA neurons. The generated DA neurons will be evaluated in a mouse model of PD. Additionally, the identification of the agonist binding site on Nurr1 will facilitate future design and optimization of potent drugs.
Statement of Benefit to California: 
Parkinson’s disease (PD) is the second most common human neurodegenerative disorder, and primarily results from the selective and progressive degeneration of ventral midbrain dopamine (DA) neurons. Cell transplantation of DA neurons differentiated from neural stem cells or embryonic stem cells raised great hope for an improved treatment for PD patients. However, DA neurons derived using current protocols do not survive well in mouse PD models, and the details of DA neuron development from stem cells are unclear. Our proposed research will identify unknown cofactors that regulate the differentiation of embryonic stem cells to DA neurons, and determine how agonists activate Nurr1, an essential nuclear hormone receptor for DA neuron specification and maturation. This study may yield new drug targets and inspire novel preventive or therapeutic strategies for PD. These discoveries may be exploited by California’s biotech industry and benefit Californians economically. In addition, we will search for more efficient methods to differentiate human embryonic stem cells into DA neurons, and evaluate their therapeutic effects in PD mouse models. Therefore, the proposed research will also directly benefit California residents suffering from PD.
Progress Report: 
  • Patients with Parkinson’s disease have malfunctioning or dying dopaminergic (DA) neurons. Human embryonic stem cells can be differentiated into DA neurons for transplantation with the potential to cure this disease, yet the differentiation mechanism is not very clear. A nuclear hormone receptor named Nurr1 is found to regulate the differentiation process. To study the regulation mechanism, we proposed to genetically incorporate nonnatural amino acids into Nurr1 in stem cells, and use the novel properties of these amino acids to identify the interacting protein partners of Nurr1. Once these partners are discovered, effective protocols can be developed to generate high purity DA neurons for therapeutic purposes. In the past year, we made significant progress in genetically inserting nonnatural amino acids in stem cells. We are in the process of making stem cell lines that have this capacity. We also set up functional assays for studying Nurr1 and its mutants containing nonnatural amino acids. These results paved the way for our future identification of Nurr1 interacting networks in stem cells.
  • Patients with Parkinson’s disease have malfunctioning or dying dopaminergic (DA) neurons. Human embryonic stem cells can be differentiated into DA neurons for transplantation with the potential to cure this disease, yet the differentiation mechanism is not very clear. A nuclear hormone receptor named Nurr1 is found to regulate the differentiation process. To study the regulation mechanism, we proposed to genetically incorporate nonnatural amino acids into Nurr1 in stem cells, and use the novel properties of these amino acids to identify the interacting protein partners of Nurr1. Once these partners are discovered, effective protocols can be developed to generate high purity DA neurons for therapeutic purposes. In the past year, we figured out several mechanisms that prevent the efficient incorporation of nonnatural amino acids into proteins in stem cells. We now have developed new strategies to overcome these difficulties. In the meantime, we developed another complementary approach in order to detect unknown proteins that interact with Nurr1 during the differentiation process of stem cells. We are employing these new methods to identify Nurr1 interacting networks in stem cells.
  • Patients with Parkinson’s disease have malfunctioning or dying dopaminergic (DA) neurons. Human embryonic stem cells can be differentiated into DA neurons for transplantation with the potential to cure this disease, yet the differentiation mechanism is not very clear. The differentiation of embryonic stem cells to DA neurons has been found to be regulated by a nuclear hormone receptor Nurr1, but how Nurr1 involves in this complicated process remains unclear - no ligands or protein partners have been uncovered for Nurr1. To understand the regulation mechanism in molecular details, we proposed to incorporate non-natural amino acids into Nurr1 directly in stem cells, and use the novel properties of these amino acids to identify the protein partners of Nurr1. Once these partners are discovered, effective protocols can be developed to generate high purity DA neurons for therapeutic purposes. In the past year, we figured out a right solution for generating stem cell lines capable of incorporating non-natural amino acids. We also created a novel bacterial strain for efficient producing Nurr1 proteins with the non-natural amino acids inserted. With these progresses we are now probing proteins that interact with Nurr1 during the differentiation of stem cells, which should eventually enable us to come up with new strategies for making DA neurons from embryonic stem cells.
  • Patients with Parkinson’s disease have malfunctioning or dying dopaminergic (DA) neurons. Human embryonic stem cells can be differentiated into DA neurons for transplantation with the potential to cure this disease, yet the differentiation mechanism is not very clear. The differentiation of embryonic stem cells to DA neurons has been found to be regulated by a nuclear hormone receptor Nurr1, but how Nurr1 is involved in this complicated process remains unclear - no ligands or protein partners have been uncovered for Nurr1. To understand the regulation mechanism in molecular details, we proposed to incorporate non-natural amino acids into Nurr1 directly in stem cells, and use the novel properties of these amino acids to identify the protein partners of Nurr1. Once these partners are discovered, effective protocols can be developed to generate high purity DA neurons for therapeutic purposes. In the past year, after testing numerous conditions in various cell lines, we discovered that photo-crosslinking is inefficient in capturing proteins interacting with Nurr1, possibly because the affinity between the unknown target protein and Nurr1 is too low. To overcome this challenge, we developed a new strategy of capture interacting proteins based on a novel class of non-natural amino acids, which do not require additional reagents nor external stimuli to function. We demonstrated the ability of these amino acids to crosslink proteins in the process of interacting with other proteins in live cells. We have also generated stable cell lines that are able to incorporate such non-natural amino acids. Using these new methods, we have been probing proteins that interact with Nurr1 during the differentiation of stem cells, which should eventually enable us to come up with new strategies for making DA neurons from embryonic stem cells.

Misregulated Mitophagy in Parkinsonian Neurodegeneration

Funding Type: 
Basic Biology V
Grant Number: 
RB5-06935
ICOC Funds Committed: 
$1 174 943
Disease Focus: 
Parkinson's Disease
Neurological Disorders
Stem Cell Use: 
iPS Cell
oldStatus: 
Closed
Public Abstract: 
Parkinson’s disease (PD), is one of the leading causes of disabilities and death and afflicting millions of people worldwide. Effective treatments are desperately needed but the underlying molecular and cellular mechanisms of Parkinson’s destructive path are poorly understood. Mitochondria are cell’s power plants that provide almost all the energy a cell needs. When these cellular power plants are damaged by stressful factors present in aging neurons, they release toxins (reactive oxygen species) to the rest of the neuron that can cause neuronal cell death (neurodegeneration). Healthy cells have an elegant mitochondrial quality control system to clear dysfunctional mitochondria and prevent their resultant devastation. Based on my work that Parkinson’s associated proteins PINK1 and Parkin control mitochondrial transport that might be essential for damaged mitochondrial clearance, I hypothesize that in Parkinson’s mutant neurons mitochondrial quality control is impaired thereby leading to neurodegeneration. I will test this hypothesis in iPSC (inducible pluripotent stem cells) from Parkinson’s patients. This work will be a major step forward in understanding the cellular dysfunctions underlying Parkinson’s etiology, and promise hopes to battle against this overwhelming health danger to our aging population.
Statement of Benefit to California: 
Parkinson's disease (PD), one of the most common neurodegenerative diseases, afflicts millions of people worldwide with tremendous global economic and societal burdens. About 500,000 people are currently living with PD in the U.S, and approximate 1/10 of them live in California. The number continues to soar as our population continues to age. An effective treatment is desperately needed but the underlying molecular and cellular mechanisms of PD’s destructive path remain poorly understood. This proposal aims to explore an innovative and critical cellular mechanism that controls mitochondrial transport and clearance via mitophagy in PD pathogenesis with elegant employment of bold and creative approaches to live image mitochondria in iPSC (inducible pluripotent stem cells)-derived dopaminergic neurons from Parkinson’s patients. This study is closely relevant to public health of the state of California and will greatly benefit its citizens, as it will illuminate the pathological causes of PD and provide novel targets for therapuetic intervention.

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.

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.

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