Neurological Disorders

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

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.
  • In this third year of progress, we have made final design refinements to the Radially Branched Deployment (RBD) cell transplantation device, which is fully compatible with currently employed interventional MRI stereotactic (iMRI) neurosurgical targeting systems. These design changes increase the "usability" of the device and enhance patient safety. The iMRI-guided RBD technology advances our ability to properly “tailor” the distribution of cell delivery to larger brain target volumes that vary in size and shape due to individual patient anatomy and different disease states. Furthermore, iMRI-guided RBD may increase patient safety by enabling intraoperative MRI monitoring. Importantly, this platform technology is easy-to-use and has a low barrier to implementation, as it can be performed “inside” essentially any typical diagnostic 1.5T MRI scanner found in most hospitals. We believe that this ease of access to the technology will facilitate the conduct of multi-site clinical trials and the future adoption of successful cellular therapies for patient care worldwide. In summary, by improving intracerebral cell delivery to the human brain, iMRI-guided RBD may have a transformative impact on the safety and efficacy of cellular therapeutics for a wide range of neurological disorders, helping ensure that basic science results are not lost in clinical translation.
  • Working with a California-based medical device manufacturer, we have developed manufacturing and testing procedures that are now being compiled into a design history file, which is a document required for eventual commercial use of the device. We are also working with an FDA regulatory consultant to prepare a 510K application to seek marketing clearance from the FDA.

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.

Banking transplant ready dopaminergic neurons using a scalable process

Funding Type: 
Early Translational II
Grant Number: 
TR2-01856
ICOC Funds Committed: 
$6 016 624
Disease Focus: 
Parkinson's Disease
Neurological Disorders
Collaborative Funder: 
Maryland
Stem Cell Use: 
Embryonic Stem Cell
oldStatus: 
Active
Public Abstract: 
Parkinson's disease (PD) is a devastating movement disorder caused by the death of dopaminergic neurons (a type of nerve cells in the central nervous system) present in the midbrain. These neurons secrete dopamine (a signaling molecule) and are a critical component of the motor circuit that ensures movements are smooth and coordinated. All current treatments attempt to overcome the loss of these neurons by either replacing the lost dopamine, or modulating other parts of the circuit to balance this loss or attempting to halt or delay the loss of dopaminergic neurons. Cell replacement therapy (that is, transplantation of dopaminergic neurons into the brain to replace lost cells and restore function) as proposed in this application attempts to use cells as small pumps of dopamine that will be secreted locally and in a regulated way, and will therefore avoid the complications of other modes of treatment. Indeed, cell therapy using fetal tissue-derived cells have been shown to be successful in multiple transplant studies. Work in the field has been limited however, partially due to the limited availability of cells for transplantation (e.g., 6-10 fetuses of 6-10 weeks post-conception are required for a single patient). We believe that human embryonic stem cells (hESCs) may offer a potentially unlimited source of the right kind of cell required for cell replacement therapy. Work in our laboratories and in others has allowed us to develop a process of directing hESC differentiation into dopaminergic neurons. To move forward stem cell-based therapy development it is important to develop scale-up GMP-compatible process of generating therapeutically relevant cells (dopaminergic neurons in this case). The overall goal of this proposal is to develop a hESC-based therapeutic candidate (dopaminergic neurons) by developing enabling reagents/tools/processes that will allow us to translate our efforts into clinical use. We have used PD as a model but throughout the application have focused on generalized enabling tools. The tools, reagents and processes we will develop in this project will allow us to move towards translational therapy and establish processes that could be applied to future IND-enabling projects. In addition, the processes we will develop would be of benefit to the CIRM community.
Statement of Benefit to California: 
Parkinson’s disease affects more than a million patients United States with a large fraction being present in California. California, which is the home of the Parkinson’s Institute and several Parkinson’s related foundations and patient advocacy groups, has been at the forefront of this research and a large number of California based scientists supported by these foundations and CIRM have contributed to significant breakthroughs in this field. In this application we and our collaborators in California aim propose to develop a hESC-based therapeutic candidate (dopaminergic neurons) that will allow us to move towards translational therapy and establish processes that could be applied to future IND-enabling projects for this currently non-curable disorder. We believe that this proposal includes the basic elements that are required for the translation of basic research to clinical research. We believe these experiments not only provide a blueprint for moving Parkinson’s disease towards the clinic for people suffering with the disorder but also a generalized blueprint for the development of stem cell therapy for multiple neurological disorders including motor neuron diseases and spinal cord injury. The tools and reagents that we develop will be made widely available to Californian researchers. We expect that the money expended on this research will benefit the Californian research community and the tools and reagents we develop will help accelerate the research of our colleagues in both California and worldwide.
Progress Report: 
  • Parkinson's disease (PD) is a devastating movement disorder caused by the death of dopaminergic neurons (a type of nerve cells in the central nervous system) present in the midbrain. These neurons secrete dopamine (a signaling molecule) and are a critical component of the motor circuit that ensures movements are smooth and coordinated.
  • All current treatments attempt to overcome the loss of these neurons by either replacing the lost dopamine, or modulating other parts of the circuit to balance this loss or attempting to halt or delay the loss of dopaminergic neurons. Cell replacement therapy (that is, transplantation of dopaminergic neurons into the brain to replace lost cells and restore function) as proposed in this application attempts to use cells as small pumps of dopamine that will be secreted locally and in a regulated way, and will therefore avoid the complications of other modes of treatment. Indeed, cell therapy using fetal tissue-derived cells have been shown to be successful in multiple transplant studies. Work in the field has been limited however, partially due to the limited availability of cells for transplantation (e.g., 6-10 fetuses of 6-10 weeks post-conception are required for a single patient).
  • We believe that human embryonic stem cells (hESCs) may offer a potentially unlimited source of the right kind of cell required for cell replacement therapy. Work in our laboratories and in others has allowed us to develop a process of directing hESC differentiation into dopaminergic neurons. To move forward stem cell-based therapy development it is important to develop scale-up GMP-compatible process of generating therapeutically relevant cells (dopaminergic neurons in this case).
  • The overall goal of this proposal is to develop a hESC-based therapeutic candidate (dopaminergic neurons) by developing enabling reagents/tools/processes that will allow us to translate our efforts into clinical use. We have used PD as a model but throughout the application have focused on generalized enabling tools. The tools, reagents and processes we will develop in this project will allow us to move towards translational therapy and establish processes that could be applied to future IND-enabling projects. In addition, the processes we will develop would be of benefit to the CIRM community.
  • Parkinson's disease (PD) is a devastating movement disorder caused by the death of dopaminergic neurons (a type of nerve cells in the central nervous system) present in the midbrain. These neurons secrete dopamine (a signaling molecule) and are a critical component of the motor circuit that ensures movements are smooth and coordinated.
  • All current treatments attempt to overcome the loss of these neurons by either replacing the lost dopamine, or modulating other parts of the circuit to balance this loss or attempting to halt or delay the loss of dopaminergic neurons. Cell replacement therapy (that is, transplantation of dopaminergic neurons into the brain to replace lost cells and restore function) as proposed in this application attempts to use cells as small pumps of dopamine that will be secreted locally and in a regulated way, and will therefore avoid the complications of other modes of treatment. Indeed, cell therapy using fetal tissue-derived cells have been shown to be successful in multiple transplant studies. Work in the field has been limited however, partially due to the limited availability of cells for transplantation (e.g., 6-10 fetuses of 6-10 weeks post-conception are required for a single patient).
  • We believe that human pluripotent stem cells (PSC) may offer a potentially unlimited source of the right kind of cell required for cell replacement therapy. Work in our laboratories and in others has allowed us to develop a process of directing PSC differentiation into dopaminergic neurons. To move forward stem cell-based therapy development it is important to develop scale-up GMP-compatible process of generating therapeutically relevant cells (dopaminergic neurons in this case).
  • During this grant, we have optimized a step-wise scalable process for generating authentic dopaminergic neurons in defined media from human PSC, and have determined the time point at which dopaminergic neurons can be frozen, shipped, thawed and transplanted without compromising their ability to mature and provide therapeutic benefit in animal models. Our process has been successfully transferred to a GMP facility and we have manufactured multiple lots of GMP-equivalent cells using this process. Importantly, we have shown functional equivalency of the manufactured cells in appropriate models. The tools, reagents and processes we have developed in this project allow us to move towards translational therapy and establish processes that could be applied to future IND-enabling projects. In addition, the processes we have developed would be of benefit to the CIRM community.

New Drug Discovery for SMA using Patient-derived Induced Pluripotent Stem Cells

Funding Type: 
Early Translational II
Grant Number: 
TR2-01844
Investigator: 
ICOC Funds Committed: 
$5 665 887
Disease Focus: 
Spinal Muscular Atrophy
Neurological Disorders
Pediatrics
Stem Cell Use: 
iPS Cell
oldStatus: 
Closed
Public Abstract: 
Spinal muscular atrophy (SMA) is the leading genetic cause of infant death in the U.S. This devastating disease affects 1 child in every 6,000-10,000 live births, with a North American prevalence of approximately 14,000 individuals. The disease is characterized by the death of spinal cord cells called motor neurons that connect the brain to muscle. Death of these cells causes muscle weakness and atrophy, which progresses to paralysis, respiratory failure and frequently death. The three different types of SMA differ in severity and prognosis, with Type I being the most severe. SMA is caused by a genetic defect that leads to reduced levels of a single protein called SMN. There are currently no approved therapies for the disease. The existing treatments for SMA consist of supportive care for the respiratory and nutritional deficits, for example ventilation and feeding tubes. Previous attempts to develop drugs using conventional technologies, such as cultured cancer cells or cells derived from animals have been unsuccessful. These failures are likely due the fact that previous attempts used cell types that don’t reflect the disease or aren’t affected by low levels of the SMN protein. Our approach uses patient-derived motor neurons, the specific cell type that dies. We will conduct drug discovery experiments using these motor neurons to find potential therapeutics that increase the levels of the SMN protein in these diseased cells. Induced pluripotent stem cell (iPSC) technology allows us to take skin cells from patients with SMA, grow them in a dish, and turn them into motor neurons. We are conducting high-throughput screens of potential drugs with these cells to identify drug candidates that increase SMN protein levels in motor neurons derived from SMA patients. An added advantage to our approach is that we can test our drug candidates in motor neurons from many different patients, with different disease subtypes and from different ethnic backgrounds. We have generated iPSCs from many patients with SMA and we will test compounds for effectiveness against this cohort. These studies will give us an indication of the effectiveness of our compounds across patients before moving into costly and lengthy clinical trials. If our drug candidate is successful, it could be the first effective therapeutic available for SMA. It will increase the amount of SMN protein and prevent motor neuron death. Halting the death of spinal cord motor neurons prevents the progressive weakness and muscle atrophy. We anticipate that this would prevent disability in Type III patients. For Type I and II patients, we believe such a therapy would mitigate respiratory and feeding challenges and allow lifespan increase. The sponsoring institution has integrated iPSC-based drug discovery capabilities, ranging from stem cell line production, high throughput drug screening and medicinal chemistry. Accordingly, this institution is uniquely positioned to achieve the aims of this grant.
Statement of Benefit to California: 
Spinal muscular atrophy (SMA) is the second-most common autosomal-recessive disorder and leading genetic cause of death of infants in the U.S. We estimate that there are up to 1,500 SMA patients currently living in California, with 100 new cases diagnosed in California every year. The CIRM Early Translational II Awards is intended to fund studies that will propel drug discovery forward for many devastating diseases. In keeping with this mission, we propose to leverage iPSC technology to generate disease-relevant cell types from patients themselves for a high throughput drug screen. A successful therapy for SMA would lead to significant cost savings to California’s health care system, and would provide relief to families of patients with this devastating disorder. Given that there are not many successful drugs in the making for neurological diseases such as ALS, SMA, Parkinson’s disease or Alzheimer’s disease, our project should significantly impact drug discovery in this area by introducing iPSC technologies as a valid drug discovery and development platform. The application of iPSC-based disease modeling and drug discovery to SMA is highly innovative and represents the opportunity to establish worldwide leadership for California in this emerging field. Furthermore, the sponsoring institution will fund over 70% of the direct costs during the timeframe of this award. Accordingly, the 3:1 leverage provides great opportunity to magnify the effect of a CIRM award. Our research program will also create new, high-paying jobs in California, and will stimulate California’s economy by creating new research and clinical tools. These activities will continue to strengthen California’s leadership position at the forefront of the stem cell and regenerative medical revolution of the 21st century.
Progress Report: 
  • Spinal muscular atrophy (SMA) is the leading genetic cause of infant death in the U.S. This devastating disease affects 1 child in every 6,000-10,000 live births, with a North American prevalence of approximately 14,000 individuals. The disease is characterized by the death of spinal cord cells called motor neurons that connect the brain to muscle. Death of these cells causes muscle weakness and atrophy, which progresses to paralysis, respiratory failure and frequently death. The three different types of SMA differ in severity and prognosis, with Type I being the most severe. SMA is caused by a genetic defect that leads to reduced levels of a single protein called SMN. There are currently no approved therapies for the disease.
  • Existing treatments for SMA consist of supportive care for the respiratory and nutritional deficits, for example ventilation and feeding tubes. Previous attempts to develop drugs using conventional technologies, such as cultured cancer cells or cells derived from animals have been unsuccessful. These failures are likely due to the fact that previous attempts used cell types that do not reflect the disease or are not affected by low levels of the SMN protein. Our approach uses patient-derived motor neurons, the specific cell type that dies in SMA.
  • An added advantage to our approach is that we can test our drug candidates in motor neurons from many different patients and different disease subtypes. We have generated iPSCs from many patients with SMA and we will test compounds for effectiveness against this cohort. These studies will give us an indication of the effectiveness of our compounds across patients before moving into costly and lengthy clinical trials. It will increase the amount of SMN protein and prevent motor neuron death. Halting the death of spinal cord motor neurons prevents the progressive weakness and muscle atrophy. We anticipate that this would prevent disability in Type III patients. For Type I and II patients, we believe such a therapy would mitigate respiratory and feeding challenges and allow an increase in lifespan.
  • In the past year, we conducted drug discovery experiments using these motor neurons to find potential therapeutics that increase the levels of the SMN protein in these diseased cells. Induced pluripotent stem cell (iPSC) technology allows us to take skin cells from patients with SMA, grow them in a dish, and turn them into SMA motor neurons. We conducted high-throughput screens of potential drugs with these cells to identify drug candidates that increase SMN protein levels in motor neurons derived from SMA patients. Despite the high quality of these screens, no suitable drug candidate was identified. We have modified our strategy and developed a method to identify, in parallel, all targets in the “druggable” genome that regulate SMN protein levels. An exhaustive screen currently is being performed to identify such a target and will be completed by end April 2012. Once a target is identified, it will be developed into a lead and validated in animals.

Repair of Conus Medullaris/Cauda Equina Injury using Human ES Cell-Derived Motor Neurons

Funding Type: 
Early Translational II
Grant Number: 
TR2-01785
ICOC Funds Committed: 
$1 614 441
Disease Focus: 
Spinal Cord Injury
Neurological Disorders
Stem Cell Use: 
Embryonic Stem Cell
oldStatus: 
Active
Public Abstract: 
Injuries to the spinal cord commonly result from motor vehicle accidents, traumatic falls, diving, surfing, skiing, and snowboarding accidents, other forms of sports injuries, as well as from gunshot injuries in victims of violent crimes. Injuries to the anatomically lowest part of the spinal cord, the lumbosacral portion and its associated nerve roots commonly cause paralysis, loss of sensation, severe pain, as well as loss of bladder, bowel, and sexual function. Lumbosacral injuries represent approximately one-fifth of all traumatic lesions to the human spinal cord. As a result of the direct injury to the lumbosacral portion of the spinal cord, there is degeneration and death of spinal cord nerve cells, which control muscles in the legs as well as bladder, bowel, and sexual function. No treatments are presently available in clinical practice to reverse the effects of these devastating injuries. In order to reverse the loss of function after lumbosacral spinal cord injury, replacement of the lost nerve cells is required. Recent research studies have identified some properties that are shared by spinal cord neurons responsible for muscle and bladder control. Human embryonic stem cells can now be prepared in research laboratories to develop properties that are shared between nerve cells controlling muscle and bladder function. Such nerve cells are particularly at risk of degeneration and death as a result of injuries to the lumbosacral spinal cord. Human embryonic stem cells, which have undergone treatment to obtain properties of muscle and bladder controlling nerve cells, are now very attractive development candidates for new cell replacement therapies after lumbosacral spinal cord injuries. The proposed feasibility studies will study the properties of such cells in a clinically relevant rat model for lumbosacral spinal cord injuries. In Specific Aim 1, we will determine whether ACUTE transplantation of human embryonic stem cells, which have been treated to develop properties of specific lumbosacral spinal cord neurons, may replace lost nerve cells and result in a return of bladder function in a rat model of lumbosacral spinal cord injury and repair. In Specific Aim 2, we will determine whether DELAYED transplantation of human embryonic stem cells, which have been treated to develop properties of specific lumbosacral spinal cord neurons, may replace lost nerve cells and result in a return of bladder function in a rat model of lumbosacral spinal cord injury and repair. A variety of functional studies will determine the effect of the cell transplantation on bladder function, walking, and pain. We will also use detailed anatomical studies to determine in microscopes whether the transplanted cells have grown processes to connect with pelvic target tissues, including the lower urinary tract. If successful, the proposed experiments may lead to a new treatment strategy for patients with lumbosacral spinal cord injuries.
Statement of Benefit to California: 
There are presently about 250,000 patients living with neurological impairments from spinal cord injuries (SCIs) in the United States, and approximately 11,000 new cases present every year. SCIs typically result in paralysis, loss of sensation, pain as well as bladder, bowel, and sexual dysfunction. No successful treatments are available to reverse the neurological deficits that result from SCI. Common causes for SCIs include car and motorcycle accidents, skiing, diving, surfing, and snow boarding injuries, traumatic falls, sports injuries, and acts of violence. California medical centers encounter a large proportion of the overall cases in the U.S. because of our large population, extensive network of freeways, and an active life style with recreational activities taking place both along the Californian coastline and in the mountains. The proposed development candidate feasibility project will capitalize on recent progress in human stem cell science and surgical repair of conus medullaris/cauda equina (CM/CE) forms of SCI. Human embryonic stem cell-derived neurons and neuronal progenitors, which express the transcription factor Hb9, will be transplanted into the conus medullaris in attempts to replace lost motor and autonomic neurons after a lumbosacral ventral root avulsion injury in rats. Surgical replantation of avulsed lumbosacral ventral roots into the spinal cord will also be performed in this clinically relevant model for CM/CE injury and repair. If successful, our development candidate may reinnervate muscles and pelvic organs, including the lower urinary tract after CM/CE forms of SCI. Return of functional bladder control represents one of the absolute top priorities among the spinal cord injured population (Anderson, J Neurotrauma, 2004; 21, 1371-83). Successful recovery of bladder function after SCI is expected to have very significant impact on the quality of life of spinal cord injured subjects and markedly reduce health care costs. Recovery of bladder function in spinal cord injured subjects would markedly reduce or eliminate the need for intermittent bladder catheterizations and indwelling bladder catheters. The number of visits in physicians’ offices and already over-crowded California emergency rooms for bladder infections and other complications would be markedly reduced, thereby significantly reducing health care costs for both patients and our state. Improved neurological function among the SCI population is also expected to reduce care giver needs, thereby further reducing health care costs. The increased independence that will result from improved bladder control and concomitant possible recovery of other neurological functions, for instance in transfers and locomotion, will promote return to and participation in the work force for many individuals with SCI. These effects are also expected to bring a very positive effect to the California economy and increased quality of life for those living with an SCI.
Progress Report: 
  • Injuries to the lowest portion of the spine and the spinal cord commonly results in paralysis and impairment of bladder , bowel, and sexual functions. These injuries are usually referred to as conus medullaris and cauda equina forms of spinal cord injuries. Presently, no treatments are available to reverse the neurological deficits that result from these injuries.
  • In this project, we aim to reverse neurological deficits, including bladder function, in a rat model of spinal cord injury, which affects the lowermost portion of the spinal cord. This part of the spinal cord and the associated nerve roots are called the conus medullaris and cauda equina. In our experimental model, nerve roots carrying fibers that control muscle function and pelvic organs, such as the bladder and bowel, are injured at the surface of the spinal cord. This injury mimics many of the neurological deficits encountered in human cases.
  • For treatment purposes, we transplant human derived embryonic stem cells, which have been prepared to acquire properties of motor neurons, into the lowermost portion of the rat spinal cord after injury and surgical repair of nerve roots carrying motor fibers. The studies will evaluate both acute and delayed transplantation of human embryonoic
  • During the first year of the studies, we have developed improved protocols to increase our ability to produce large number of motor neurons from human embryonic stem cells. We have also developed improved methods to detect motor neurons during the neuron production process by using fluorescent reporters inside of the cells. The latter development is of great help when sorting and preparing cells with desired properties for transplantation studies. In addition, we have refined our surgical methods to make it less invasive, using a one-sided injury model instead of lesions on both sides of the spinal cord in rats. Specifically, bladder dysfunction can be assess after a one sided injury of nerve roots and be evaluated using a combination of bladder pressure recorings and electrical recordings referred to as electromyography (EMG) from muscles along the urethra. The revised procedure is well tolerated by the rats and is a suitable approach for studies of chronic injury and cell-based long-term treatments. A research manuscript describing this improved experimental method and refinement has been submitted to a scientific journal and reviewed, and the manuscript is currently undergoing our revisions before being considered for publication. The experimental refinement will greatly assist with our long-term studies on the effects of transplanted motor neurons derived from human embryonic stem cells. We have also begun experiments using our refined model and cells, which now can be produced in high numbers and be identifiable during both the cell culture steps and during the animal studies. Initial tissues have been harvested and are being processed for morphological analyses.
  • Injuries to the lowest portion of the spine and the spinal cord commonly results in paralysis and impairment of bladder , bowel, and sexual functions. These injuries are usually referred to as conus medullaris and cauda equina forms of spinal cord injuries. Presently, no treatments are available to reverse the neurological deficits that result from these injuries.
  • In this project, we aim to reverse neurological deficits, including bladder function, in a rat model of spinal cord injury, which affects the lowermost portion of the spinal cord. This part of the spinal cord and the associated nerve roots are called the conus medullaris and cauda equina. In our experimental model, nerve roots carrying fibers that control muscle function and pelvic organs, such as the bladder and bowel, are injured at the surface of the spinal cord. This injury mimics many of the neurological deficits encountered in human cases.
  • For treatment purposes, we transplant human derived embryonic stem cells, which have been prepared to acquire properties of motor neurons, into the lowermost portion of the rat spinal cord after injury and surgical repair of nerve roots carrying motor fibers. The studies will evaluate both acute and delayed transplantation of human embryonic stem cells, which have acquired properties of motor neurons.
  • During the second year of the studies, we have developed improved protocols to increase our ability to produce large number of motor neurons from human embryonic stem cells. We have also developed improved methods to detect motor neurons during the neuron production process by using fluorescent reporters inside of the cells. The latter development is of great help when sorting and preparing cells with desired properties for transplantation studies. In addition, we have refined our surgical methods to make it less invasive, using a one-sided injury model instead of lesions on both sides of the spinal cord in rats. Specifically, bladder dysfunction can be assessed after a one sided injury of nerve roots and be evaluated using a combination of bladder pressure recordings and electrical recordings referred to as electromyography (EMG) from muscles along the urethra. The revised procedure is well tolerated by the rats and is a suitable approach for studies of chronic injury and cell-based long-term treatments. A research manuscript describing this improved experimental method and refinement has been published. The experimental refinement will greatly assist with our long-term studies on the effects of transplanted motor neurons derived from human embryonic stem cells. We have also performed transplantations of embryonic human stem cell derived motor neurons into the rat spinal cord and demonstrated surgical feasibility as well as survival of large numbers of neurons in the rat spinal cord. Some of the transplanted cells also demonstrate anatomical markers for motor neurons after transplantation.

Crosstalk: Inflammation in Parkinson’s disease (PD) in a humanized in vitro model

Funding Type: 
Early Translational II
Grant Number: 
TR2-01778
ICOC Funds Committed: 
$2 472 839
Disease Focus: 
Parkinson's Disease
Neurological Disorders
Collaborative Funder: 
Germany
Stem Cell Use: 
iPS Cell
Cell Line Generation: 
iPS Cell
oldStatus: 
Active
Public Abstract: 
Parkinson’s Disease (PD) is the most common neurodegenerative movement disorder. It is characterized by motor impairment such as slowness of movements, shaking and gait disturbances. Age is the most consistent risk factor for PD, and as we have an aging population, it is of upmost importance that we find therapies to limit the social, economic and emotional burden of this disease. Most of the studies to find better drugs for PD have been done in rodents. However, many of these drugs failed when tested in PD patients. One problem is that we can only investigate the diseased neurons of the brain after the PD patients have died. We propose to use skin cells from PD patients and reprogram these into neurons and other surrounding cells in the brain called glia. This is a model to study the disease while the patient is still alive. We will investigate how the glial surrounding cells affect the survival of neurons. We will also test drugs that are protective for glial cells and neurons. Overall, this approach is advantageous because it allows for the study of pathological development of PD in a human system. The goal of this project is to identify key molecular events involved at early stages in PD and exploit these as potential points of therapeutic intervention.
Statement of Benefit to California: 
The goal of this proposal is to create human cell-based models for neurodegenerative disease using transgenic human embryonic stem cells and induced pluripotent stem cells reprogrammed from skin samples of highly clinically characterized Parkinson’s Disease (PD) patients and age-matched controls. Given that age is the most consistent risk factor for PD, and we have an aging population, it is of utmost importance that we unravel the cellular, molecular, and genetic causes of the highly specific cell death characteristic of PD. New drugs can be developed out of these studies that will also benefit the citizens of the State of California. In addition, if our strategy can go into preclinical development, this approach would most likely be performed in a pharmaceutical company based in California.
Progress Report: 
  • In the first year of our CIRM Early Translational II Award we have largely accomplished the first two aims put forth in our proposal “Crosstalk: Inflammation in Parkinson’s disease (PD) in a humanized in vitro model.” Dr. Juergen Winkler, in Erlangen, Germany, has enrolled 10 patients and 6 controls in this project, most of which have had a biopsy of their skin cells sent to The Salk Institute in La Jolla. In Dr. Gage’s lab at The Salk Institute these patient fibroblasts are being reprogrammed into induced pluripotent stem cells (iPSCs), and initial attempts at differentiation into dopaminergic neurons are underway. Additionally, patient blood cells have been sent from Dr. Winkler’s clinic to the lab of Dr. Glass at UC San Diego, where their gene expression profile is being determined. In this initial reporting period we are successfully building the cellular tools necessary to investigate the role of nuclear receptors and inflammation in Parkinson’s Disease.
  • In the second year of our CIRM Early Translational II Award we are making substantial progress towards completing all three aims put forth in our proposal. Dr. Juergen Winkler, our German collaborator, has completed the patient recruitment phase of this project, and skin cells from all 16 subjects (10 with PD and six controls) have been reprogrammed into induced pluripotent stem cells (iPSCs) at the Salk Institute in La Jolla. The patient-specific iPSCs have been differentiated into well-characterized neural stem cells, which the Gage lab is further differentiating into both dopaminergic neurons and astrocytes. In addition to collecting patient skin cells, Dr. Winkler’s group has collected blood cells which are currently being analyzed for gene expression differences by Dr. Glass’ lab at UCSD using state-of-the-art RNA sequencing technology. We have identified a compound that is anti-inflammatory in human cells that we will test on the patient-specific cells once we finish building the cellular tools required to investigate the role of nuclear receptors and inflammation in Parkinson’s Disease.
  • In the final year of our CIRM Early Translational II Award we made considerable progress towards completing all three aims put forth in our proposal. Dr. Juergen Winkler, our German collaborator, has completed the patient recruitment phase of this project, and skin cells from all 16 subjects (10 with PD and six controls) have been reprogrammed into induced pluripotent stem cells (iPSCs) at the Salk Institute in La Jolla. The patient-specific iPSCs have been differentiated into well-characterized neural stem cells, which the Gage lab is further differentiating into both dopaminergic neurons and astrocytes. In addition to collecting patient skin cells, Dr. Winkler’s group has collected blood cells which are currently being analyzed for gene expression differences by Dr. Glass’ lab at UCSD using state-of-the-art RNA sequencing technology. We have identified a compound that is anti-inflammatory in human cells that can reduce inflammation in patient-specific cells, and we are beginning to look at its effects on neuronal survival. This award has allowed us to build the cellular tools required to investigate the role of nuclear receptors and inflammation in Parkinson’s disease, which is a model with endless potential.

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.

ES-Derived Cells for the Treatment of Alzheimer's Disease

Funding Type: 
New Faculty I
Grant Number: 
RN1-00538-A
ICOC Funds Committed: 
$2 120 833
Disease Focus: 
Aging
Alzheimer's Disease
Neurological Disorders
Stem Cell Use: 
Embryonic Stem Cell
Public Abstract: 
Alzheimer’s disease is the most common cause of dementia in the elderly, affecting over 5 million people in the US alone. Boosting immune responses to beta-Amyloid (Aβ) has proven beneficial in mouse models and Alzheimer’s disease (AD) patients. Vaccinating Alzheimer’s mice with Aβ improves cognitive performance and lessens pathological features within the brain, such as Aβ plaque loads. However, human trials with direct Aβ vaccination had to be halted to brain inflammation in some patients. We have demonstrated that T cell immunotherapy also provides cognitive benefits in a mouse model for Alzheimer’s disease, and without any detectable brain inflammation. Translating this approach into a clinical setting requires that we first develop a method to stimulate the proliferation of Aβ-specific T cells without triggering generalized inflammatory response, as happens with vaccinations. Adaptive immune responses are provided by T cells and B cells, which are regulated by the innate immune system through antigen presenting cells, such as mature dendritic cells. We propose to leverage the power of embryonic stem (ES) cells by engineering dendritic cells that express a recombinant transgene that will specifically activate Aβ-specific T cells. We will test the effectiveness of this targeted stimulation strategy using real human T cells. If successful, this approach could provide a direct method to activate beneficial immune responses that may improve cognitive decline in Alzheimer’s disease.
Statement of Benefit to California: 
Alzheimer’s disease is the most common cause of dementia in the elderly, affecting more than 5 million people in the US. In addition to being home to more than 1 in 8 Americans, California is a retirement destination so a proportionately higher percentage of our residents are afflicted with Alzheimer’s disease. It has been estimated that the number of Alzheimer’s patients in the US will grow to 13 million by 2050, so Alzheimer’s disease is a pending health care crisis. Greater still is the emotional toll that Alzheimer’s disease takes on it’s patients, their families and loved one. Currently, there is no effective treatment or cure for Alzheimer’s disease. The research proposed here builds on more than 7 years of work showing that the body’s own immune responses keep Alzheimer’s in check in young and unaffected individuals, but deficiencies in T cell responses to beta-amyloid peptide facilitate disease progression. We have shown that boosting a very specific T cell immune response can provide cognitive and other benefits in mouse models for Alzheimer’s disease. Here we propose to use stem cell research to propel these findings into the clinical domain. This research may provide an effective therapeutic approach to treating and/or preventing Alzheimer’s disease, which will alleviate some of the financial burden caused by this disease and free those health care dollars to be spent for the well-being of all Californians.
Progress Report: 
  • We have developed new proteins that will stimulate immune responses to a major factor in Alzheimer's disease. Previous studies from our lab and others indicate that those responses can be improve memory deficits and brain pathology that occurs in Alzheimer's patients, and in Alzheimer's mice. To stimulate these immune responses the new proteins must be expressed by specific immune cells called, dendritic cells. Viruses have been made that carry the codes for these new proteins and we have confirmed that those viruses can deliver them into dendritic cells. To optimize these procedures we have made dendritic cells from human embryonic stem cells, and we developed methods to accomplish that step in our laboratory. At the end of year 2 we are nearing the completion of our preclinical studies and are poised to begin introducing the new proteins into immune cells that are derived from human blood, within the next year. The over-arching goal of this project is to develop method to trigger Alzheimer's-specific immune responses in a safe and reliable manner that could provide beneficial effects with minimal side-effects. This CIRM-funded project is on track to be completed within the 5 year time-frame.

ES-Derived Cells for the Treatment of Alzheimer's Disease

Funding Type: 
New Faculty I
Grant Number: 
RN1-00538-B
ICOC Funds Committed: 
$2 120 833
Disease Focus: 
Aging
Alzheimer's Disease
Neurological Disorders
Stem Cell Use: 
Embryonic Stem Cell
oldStatus: 
Closed
Public Abstract: 
Alzheimer’s disease is the most common cause of dementia in the elderly, affecting over 5 million people in the US alone. Boosting immune responses to beta-Amyloid (Aβ) has proven beneficial in mouse models and Alzheimer’s disease (AD) patients. Vaccinating Alzheimer’s mice with Aβ improves cognitive performance and lessens pathological features within the brain, such as Aβ plaque loads. However, human trials with direct Aβ vaccination had to be halted to brain inflammation in some patients. We have demonstrated that T cell immunotherapy also provides cognitive benefits in a mouse model for Alzheimer’s disease, and without any detectable brain inflammation. Translating this approach into a clinical setting requires that we first develop a method to stimulate the proliferation of Aβ-specific T cells without triggering generalized inflammatory response, as happens with vaccinations. Adaptive immune responses are provided by T cells and B cells, which are regulated by the innate immune system through antigen presenting cells, such as mature dendritic cells. We propose to leverage the power of embryonic stem (ES) cells by engineering dendritic cells that express a recombinant transgene that will specifically activate Aβ-specific T cells. We will test the effectiveness of this targeted stimulation strategy using real human T cells. If successful, this approach could provide a direct method to activate beneficial immune responses that may improve cognitive decline in Alzheimer’s disease.
Statement of Benefit to California: 
Alzheimer’s disease is the most common cause of dementia in the elderly, affecting more than 5 million people in the US. In addition to being home to more than 1 in 8 Americans, California is a retirement destination so a proportionately higher percentage of our residents are afflicted with Alzheimer’s disease. It has been estimated that the number of Alzheimer’s patients in the US will grow to 13 million by 2050, so Alzheimer’s disease is a pending health care crisis. Greater still is the emotional toll that Alzheimer’s disease takes on it’s patients, their families and loved one. Currently, there is no effective treatment or cure for Alzheimer’s disease. The research proposed here builds on more than 7 years of work showing that the body’s own immune responses keep Alzheimer’s in check in young and unaffected individuals, but deficiencies in T cell responses to beta-amyloid peptide facilitate disease progression. We have shown that boosting a very specific T cell immune response can provide cognitive and other benefits in mouse models for Alzheimer’s disease. Here we propose to use stem cell research to propel these findings into the clinical domain. This research may provide an effective therapeutic approach to treating and/or preventing Alzheimer’s disease, which will alleviate some of the financial burden caused by this disease and free those health care dollars to be spent for the well-being of all Californians.
Progress Report: 
  • Alzheimer’s disease remains the most common cause of dementia in California and the US with more than 5 million cases nationwide, a number that is expected to exceed 13 million by 2050 if treatments are not developed. We, and others, showed that T cells responses to beta-amyloid can provide beneficial effects in mouse models of this disease. However, a clinical trial of Abeta vaccination was halted due to immune cell infiltration of the meninges and consequent brain swelling. Most of the other patients seemed to benefit from the vaccination, but the uncontrolled robustness of the immune response to vaccination makes those trials unfeasible. This project aims to refine and control Abeta-specific T cell responses using antigen presenting cells derived from human embryonic stem cells (hESC). If we are successful, then we would be able to deliver only the beneficial cells responsible for the beneficial effects, and do so in a controlled manner so as to avoid encephalitogenic complications.
  • During the first 4 years of this CIRM grant, my lab developed novel methods to assess adaptive immune responses to the Alzheimer’s-linked peptide, amyloid-beta/Abeta, in human blood samples. This technique relies on the use of pluripotent stem cells to produce specific immune-modulating cells in a complicated differentiation process that takes ~50 days. Over the past year we have found that this technology can employ both human embryonic stem cells and induced-pluripotent stem cells (iPSC), the latter of which were developed in my lab through other funding sources. We have now confirmed that this method provides consistent and robust readouts. Over the past year we have moved into the clinical phase of this project and assessed these responses in over 60 human subjects. Control subjects (not affected by Alzheimer’s disease) were recruited from the university community. Initially, we looked for age-dependent changes in these responses with a cohort of >50 research subjects who ranged in age from 20-88 years. Interesting patterns emerged from that study, which are currently being prepared for publication, and will remain confidential until publication; further details are not provided in this report as it will become public record. Several Alzheimer’s patients have also been assessed. We recently entered into an agreement with a local Alzheimer’s assessment center that will allow us to expand our study by including subjects with a presumptive diagnosis of Alzheimer’s disease, as well as individuals with mild cognitive impairment (MCI) and other causes of dementia such as Fronto-temporal Dementia, Dementia with Lewy bodies and Vascular Dementia. It will be interesting to determine if the assay we have developed will be able to distinguish subjects with developing Alzheimer's pathology from those with other causes of dementia, using a small blood sample. Overall, our progress is on-track for this project to be completed at the end of year 5, with many more subject samples analyzed than were originally proposed. In the approved grant it was proposed that spleen samples from 6-8 organ donors would be assessed, but as we developed this technology it became clear that we can detect these responses using 20 mL whole blood samples from living human subjects. At present, we have used our assay to assess more than 60 human subjects – 10 times what was proposed - and by this time next year we estimate that number will double. Information gained from this research is providing exciting new insights into immune changes associated with Alzheimer’s disease. The Western University of Health Sciences is engaged in patent processes to secure intellectual property and commercialize this technology.
  • Alzheimer’s disease affects more than 5.5 million people in the USA. Problems with memory correspond with the appearance of insoluble plaques in certain brain regions, and these plaques large consist of a peptide called, amyloid-beta. For more than a decade it has known that certain immune responses to amyloid-beta improve memory in mouse models of Alzheimer’s disease, yet in humans little is known about how those responses normally occur or if they may a beneficial therapeutic strategy. In this grant we have used stem cell technology to pioneer a new method to isolate and characterize those cells using only 20 cc of whole blood from a variety of human subjects. We have found that these immune responses increase dramatically in when high-risk people are in their late 40’s and early 50’s. Those responses may provide protection against Alzheimer’s disease progression as they diminish as memory problems begin to develop. This technology will be developed as an early diagnostic test for Alzheimer's disease with private equity partners. A patent application covering this technology was submitted by the Western University of Health Sciences.
  • This CIRM grant allowed my group to translate findings from our Alzheimer’s research from mouse to man. Over several years my group, an others, showed that boosting T cell responses to a peptide found in the plaques of Alzheimer’s patients could reduce disease pathology and memory problems in mouse models of this disease. Interestingly, at least some people carry T cells in their immune system, but it was unknown who has them or if they are lost over the course of Alzheimer’s disease. In this CIRM-funded project we used stem cells to develop a new technology, called CD4see, to identify and quantify those T cells using a small sample of human blood, roughly the same amount taken for a standard blood panel. After years of development and testing of CD4see, we used it to look for and quantify those plaque-specific T cells in over 70 human subjects. We found an age-dependent decline of Aβ-specific CD4+ T cells that occurred earlier in women than in men. Men showed a 50% decline around the age of 70, but women reached the same level before the age of 60. Notably, women who carried the AD risk marker apolipoproteinE-ε4 (ApoE4) showed the earliest decline, with a precipitous drop that coincided with an age when menopause usually begins. This assay requires a sample of whole blood that is similar to standard blood panels, making it suitable as a routine test to evaluate adaptive immunity to Aβ in at-risk individuals as an early diagnostic test for Alzheimer’s disease. In future applications CD4see can be used to isolate those cells in the lab, expand them to millions of cells, and then return them back to the same person--our earlier mouse studies showed those T cells counter Alzheimer’s pathology and memory impairment, so this technology may lead to a new therapeutic approach. I am grateful to CIRM and California taxpayers for supporting young scientists and funding innovative research.

Sustained siRNA production from human MSC to treat Huntingtons Disease and other neurodegenerative disorders

Funding Type: 
Early Translational I
Grant Number: 
TR1-01257
ICOC Funds Committed: 
$2 753 559
Disease Focus: 
Huntington's Disease
Neurological Disorders
Stem Cell Use: 
Adult Stem Cell
Embryonic Stem Cell
Cell Line Generation: 
Adult Stem Cell
oldStatus: 
Closed
Public Abstract: 
One in every ten thousand people in the USA have Huntington's Disease, and it impacts many more. Multiple generations within a family can inherit the disease, resulting in escalating health care costs and draining family resources. This highly devastating and fatal disease touches all races and socioeconomic levels, and there are currently no cures. Screening for the mutant HD gene is available, but the at-risk children of an affected parent often do not wish to be tested since there are currently no early prevention strategies or effective treatments. HD is a challenging disease to treat. Not only do the affected, dying neurons need to be salvaged or replaced, but also the levels of the toxic mutant protein must be diminished to prevent further neural damage and to halt progression of the movement disorders and physical and mental decline that is associated with HD. Our application is focused on developing a safe and effective therapeutic strategy to reduce levels of the harmful mutant protein in damaged or at-risk neurons. We are using an RNA interference strategy – “small interfering RNA (siRNA)” to prevent the mutant protein from being produced in the cell. This strategy has been shown to be highly effective in animal models of HD. However, the inability to deliver the therapeutic molecules into the human brain in a robust and durable manner has thwarted scale-up of this potentially curative therapy into human trials. We are using mesenchymal stem cells, the “paramedics of the body”, to deliver the therapeutic siRNA directly into damaged cells. We have discovered that these stem cells are remarkably effective delivery vehicles, moving robustly through the tissue and infusing therapeutic molecules into each damaged cell that they contact. Thus we are utilizing nature's own paramedic system, but we are arming them with a new tool to also reduce mutant protein levels. Our novel system will allow the therapy to be carefully tested in preparation for future human cellular therapy trials for HD. The significance of our studies is very high because there are currently no treatments to diminish the amount of toxic mutant htt protein in the neurons of patients affected by Huntington’s Disease. There are no cures or successful clinical trials for HD. Our therapeutic strategy is initially examining models to treat HD, since the need is so acute. But this biological delivery system could also be used, in the future, for other neurodegenerative disorders such as amyotrophic lateral sclerosis (ALS), spinocerebellar ataxia (SCA1), Alzheimer's Disease, and some forms of Parkinson's Disease, where reduction of the levels of a mutant or disease-activating protein could be curative. Development of this novel stem cell therapeutic and effective siRNA delivery system is extremely important for the community of HD and neurodegenerative disease researchers, patients, and families.
Statement of Benefit to California: 
It is estimated that one in 10,000 CA residents have Huntington’s Disease (HD). While the financial burden of Huntington’s Disease is estimated to be in the billions, the emotional burden on the friends and families of HD patients is immeasurable. Health care costs are extremely high for HD patients due to the decline in both body and mind. The lost ability of HD patients to remain in the CA workforce and to support their families causes additional financial strain on the state’s economy. HD is inherited as an autosomal dominant trait, which means that 50% of the children of an HD patient will inherit the disease and will in turn pass it on to 50% of their children. Individuals diagnosed through genetic testing are at risk of losing insurance coverage. Since there are currently no cures or successful clinical trials for HD, many are reluctant to be tested. The proposed project is designed in an effort to reach out to these individuals who, given that HD is given an orphan disease designation, may feel that they are completely forgotten and thus have little or no hope for their future or that of their families. To combat this devastating disease, we are using an RNA interference strategy, “small interfering RNA (siRNA),” to prevent the mutant htt protein from being produced in the cell. This strategy has been shown to be highly effective in animal models of HD. However the siRNA needs to be delivered to the brain or central nervous system in a continual manner, to destroy the toxic gene products as they are produced. There are currently no methods to infuse or produce siRNA in the brain, in a safe and sustained manner. Therefore the practical clinical use of this dramatically effective potential therapeutic application is currently thwarted. Here we propose a solution, using adult mesenchymal stem cells (MSC) modified to infuse siRNA directly into diseased or at-risk neurons in the striata of HD patients, to decrease the levels of the toxic mutant htt protein. MSC are known as the “paramedics of the body" and have been demonstrated through clinical trials to be safe and to have curative effects on damaged tissue. Even without the modification to reduce the mutant protein levels, the infused MSC will help repair the damaged brain tissue by promoting endogenous neuronal growth through secreted growth factors, secreting anti-apoptotic factors, and regulating inflammation. Our therapeutic strategy will initially examine models to treat HD, since the need is so acute. But our biological delivery system could also be applied to other neurodegenerative disorders such as ALS, some forms of Parkinson’s Disease, and Alzheimer’s Disease, by using siRNA to interfere with key pathways in development of the pathology. This would be the first cellular therapy for HD patients and would have a major impact on those affected in California. In addition, the methods that we are developing will have far-reaching effects for other neurodegenerative disorders.
Progress Report: 
  • During the first year of funding we have made significant progress toward the goals of the funded CIRM grant TR1-01257: Sustained siRNA production from human MSC to treat Huntington’s disease and other neurodegenerative disorders.
  • The overall goal of the grant is to use human mesenchymal stem cells (MSC) as safe delivery vehicles to knock down levels of the mutant Huntingtin (htt) RNA and protein in the brain. There is mounting evidence in trinucleotide repeat disorders that the RNA, as well as the protein, is toxic and thus we will need to significantly reduce levels of both in order to have a durable impact on this devastating disease.
  • This year we have shown that human MSC engineered to produce anti-htt siRNA can directly transfer enough RNA interfering molecules into neurons in vitro to achieve significant reduction in levels of the htt protein. This is a significant achievement and a primary goal of our proposed studies, and demonstrates that the hypothesis for our proposed studies is valid. The transfer occurs through direct cell-to-cell transfer of siRNA, and we have filed an international patent for this process, working closely with our Innovation Access Program at UC Davis. A manuscript documenting the results of these studies is in preparation.
  • We continue to explore the precise methods by which the cell-to-cell transfer of small RNA molecules occurs, working in close collaboration with the national Center for Biophotonics Science and Technology at UC Davis. This Center is located across the street from our CIRM-funded Institute for Regenerative Cures (IRC) where our laboratory is located, and has equipment that allows visualization of protein-protein interactions in high clarity and detail. The proximity of our HD team researchers in the IRC to the Center for Biophotonics has been an important asset to our project and a collaborative manuscript is in preparation.
  • During year two of the proposed studies we will continue to document levels of reduction of the toxic htt protein in different types of neurons, including medium spiny neurons (MSN) derived from HD patient induced pluripotent stem cells (iPSC). We have made significant advances in developing the tools for these studies, including HD iPSC line generation and MSN maturation from human pluripotent cells in culture. A manuscript on improved techniques for generating MSN from pluripotent cells is in preparation. We have also worked closely with our colleagues at the UC Davis MIND Institute to achieve improved maturation and electrical activity in neurons derived from human pluripotent stem cells in vitro, and we are examining the impact of human MSC on enhancing survival of damaged human neurons.
  • In the second year of funding we will test efficacy of the siRNA-mediated knockdown of the mutant human htt RNA and protein in the brains of our newly developed strain of immune deficient Huntington's disease mice. This strain was developed by our teams at UC Davis to allow testing of human cells in the mice, since the current strains of HD mice will reject human stem cells. A manuscript describing generation of this novel HD mouse strain is in preparation, in collaboration with our nationally prominent Center for Mouse Biology.
  • Behavioral studies will be conducted in this strain with and without the MSC/siRNA-mediated knockdown of the mutant protein, through years 2-3, in collaboration with our well established mouse neurobehavioral core at the UC Davis Center for Neurosciences. We have documented the safety of intrastriatal injection of human MSC in immune deficient mice and will next test the efficacy of human MSC engineered to continually produce the siRNA to knock down the mutant htt protein in vivo.
  • As added leverage for this grant program, and supported entirely by philanthropic donations from the community committed to curing HD, we have performed IND-enabling studies in support of an initial planned clinical trial that will use normal donor MSC (non-engineered) to validate their significant neurotrophic effects in the brain. These trophic effects have been documented in animal models. The planned study will be a phase 1 safety trial. We have completed the clinical protocol design and have received feedback from the Food and Drug Administration. We will be conducting additional studies in response to their queries, over the next 6-10 months, through a pilot grant obtained from our Clinical Translational Science Center (CTSC), which is located in the same building as our Institute. Upon completion of these additional studies we will submit the updated IND application to the FDA. MSCs for this project have been expanded and banked using standard operating procedures in place in the Good Manufacturing Practice Facility in the CIRM/UC Davis Institute for Regenerative Cures.
  • From the funded studies 4 manuscripts are now in preparation, a chapter is in press and a review paper on MSC to treat neurodegenerative diseases is in press.
  • During the second year of funding we have made significant progress toward the goals of the funded CIRM grant TR1-01257: Sustained siRNA production from human MSC to treat Huntington’s disease and other neurodegenerative disorders.
  • The overall goal of the grant is to use human mesenchymal stem cells (MSC) as safe delivery vehicles to knock down levels of the mutant Huntingtin (htt) RNA and protein in the brain. During the second year we have more fully characterized our development candidate; MSC/anti-htt. We have documented that normal human donor MSC engineered to produce anti-htt siRNA can directly transfer enough RNA interfering molecules into neurons in vitro to achieve significant reduction in levels of the htt protein. We reported this work at the Annual meeting of the American Academy of Neurology (G Mitchell, S Olson, K Pollock, A Kambal, W Cary, K Pepper, S Kalomoiris, and J Nolta. Mesenchymal Stem Cells as a Delivery Vehicle for Intercellular Delivery of RNAi to Treat Huntington's disease. AAN IN10-1.010, 2011) and have recently completed and submitted a manuscript describing these results (S Olson, A Kambal, K Pollock, G Mitchell, H Stewart, S Kalomoiris, W Cary, C Nacey, K Pepper, J Nolta. Mesenchymal stem cell-mediated RNAi transfer to Huntington's disease affected neuronal cells for reduction of huntingtin. Submitted, In Review, July 2011).
  • We have explored the molecular methods by which the cell-to-cell transfer of small RNA molecules occurs, working in close collaboration with the national Center for Biophotonics Science and Technology at UC Davis. This Center is located across the street from our CIRM-funded Institute for Regenerative Cures (IRC) where our laboratory is located, and has equipment that allows visualization of protein-siRNA interactions in high clarity and detail. The proximity of our HD team researchers in the IRC to the Center for Biophotonics has been an important asset to our project. This work was also presented at AAN 2011, and a collaborative manuscript is in preparation for submission (S Olson, G McNerny, K Pollock, F Chuang, T Huser and J Nolta, Visualization of siRNA Complexed to RISC Machinery: Demonstrating Intercellular siRNA Transfer by Imaging Activity. MS in preparation, Presented at AAN 2011: IN4-1.014).
  • In the second year of funding we developed the models for in vivo efficacy testing of the siRNA-mediated knockdown of the mutant human htt RNA and protein in the brains of established and new strains of Huntington's disease mice. Behavioral studies were conducted in two strains, the R6/2 immune competent mice and our new immune deficient strain, the NSG/HD, in comparison to normal littermate controls that are not affected by HD. We established the batteries of behavioral tests that are now needed to test efficacy of our development candidate in the brain, in year three. Established tests include rotarod, treadscan, pawgrip, spontaneous activity, nesting, locomotor activity, and the characteristic HD mouse hindlimb clasping phenotype. In addition we monitor the status of weight and tremor, grooming, eyes, hair, body position, and tail position, which all change over time in HD mice. These tests are conducted at 48 hour intervals by two highly trained technicians who are blinded to the treatment that the mouse had received. These behavioral and phenotypic tests have been established at the level of Good laboratory practices in our new Institute for Regenerative Cures shower-in barrier facility vivarium. We have documented the biosafety of intrastriatal injection of human MSC in immune deficient mice and are now examining the in vivo efficacy of the development candidate: human MSC engineered to continually produce the siRNA to knock down the mutant htt protein in vivo, which will be completed in year three.
  • As added leverage for this funded grant program, and supported entirely by philanthropic donations from the community committed to curing HD, we have performed IND-enabling studies in support of an initial planned clinical trial that will use normal donor MSC (non-engineered) to validate their significant neurotrophic effects in the brain. These trophic effects have been documented in animal models. The planned study will be a phase 1 safety trial. We have completed the clinical protocol design and have received feedback from the Food and Drug Administration. We will be conducting additional studies in response to their queries, over the next 6-10 months, through a pilot grant obtained from our Clinical Translational Science Center (CTSC), which is located in the same building as our Institute. Upon completion of these additional studies we will submit the updated IND application to the FDA. MSCs for this project have been expanded and banked using standard operating procedures in place in the Good Manufacturing Practice Facility in the CIRM/UC Davis Institute for Regenerative Cures.
  • During the three years of funding we made significant progress toward the goals of the funded CIRM grant TR1-01257: Sustained siRNA production from human MSC to treat Huntington’s disease and other neurodegenerative disorders.
  • The overall goal of the grant is to use human mesenchymal stem cells (MSC) as safe delivery vehicles to knock down levels of the mutant Huntingtin (htt) RNA and protein in the brain. There is mounting evidence in trinucleotide repeat disorders that the RNA, as well as the protein, is toxic and thus we will need to significantly reduce levels of both in order to have a durable impact on this devastating disease.
  • We initially demonstrated that human MSC engineered to produce anti-htt siRNA can directly transfer enough RNA interfering molecules into neuronal cells in vitro to achieve significant reduction in levels of the htt protein. This is a significant achievement and a primary goal of our proposed studies, and demonstrates that the hypothesis for our proposed studies is valid. The transfer occurs either through direct cell-to-cell transfer of siRNA or through exosome transfer, and we filed an international patent for this process, working closely with our Innovation Access Program at UC Davis. This patent has IP sharing with CIRM.
  • An NIH transformative grant was awarded to Dr. Nolta to further explore these exciting findings. This provides funding for five years to further define and optimize the siRNA transfer mechanism.
  • A manuscript documenting the results of these studies was published:
  • S Olson, A Kambal, K Pollock, G Mitchell, H Stewart, S Kalomoiris, W Cary, C Nacey, K Pepper, J Nolta. Examination of mesenchymal stem cell-mediated RNAi transfer to Huntington's disease affected neuronal cells for reduction of huntingtin. Molecular and Cellular Neuroscience; 49(3):271-81, 2012.
  • Also a review was published with our collaborator Dr. Gary Dunbar:
  • S Olson, K Pollock, A Kambal, W Cary, G Mitchell, J Tempkin, H Stewart, J McGee, G Bauer, T Tempkin, V Wheelock, G Annett, G Dunbar and J Nolta, Genetically Engineered Mesenchymal Stem Cells as a Proposed Therapeutic for Huntington’s disease. Molecular Neurobiology; 45(1):87-98, 2012.
  • We examined the potential efficacy of injecting relatively small numbers of MSCs engineered to produce ant-htt siRNA into the striata of the HD mouse strain R6/2, in three series of experiments. Results of these experiments did not reach significance for the test agent as compared to controls. The slope of the decline in rotarod performance was less with the test agent, and development of clasping behavior was slightly delayed after injection of MSC/aHtt, but this caught up to the controls and was not significant after day 60.
  • Our conclusions are that the R6/2 strain is too rapidly progressing to see efficacy with the test agent, and also that improved methods of siRNA transfer from cell to cell are needed. We are currently working on this problem through the NIH transformative award, and will use the YAC 128 strain, which has a more slowly progressing phenotype, for all future studies. These mice are now bred and in use in our vivarium, for the MSC/BDNF studies funded through our disease team grant.
  • Through this translational grant funding we have also developed in vitro potency assays, using human embryonic stem cell-derived neurons and medium spiny neurons, as we have described in prior reports. The differentiation techniques (funded through other grants to our group) have now been published:1-3
  • 1. Liu J, Githinji J, McLaughlin B, Wilczek K, Nolta J. Role of miRNAs in Neuronal Differentiation from Human Embryonic Stem Cell-Derived Neural Stem Cells. Stem Cell Rev;8(4):1129-37, 2012.
  • 2. Jun-feng Feng, Jing Liu, Xiu-zhen Zhang, Lei Zhang, Ji-yao Jiang, Nolta J, Min Zhao. Guided Migration of Neural Stem Cells Derived from Human Embryonic Stem Cells by an Electric Field. Stem Cells. Feb; 30(2):349-55, 2012.
  • 3. Liu J, Koscielska KA, Cao Z, Hulsizer S, Grace N, Mitchell G, Nacey C, Githinji J, McGee J, Garcia-Arocena D, Hagerman RJ, Nolta J, Pessah I, Hagerman PJ. Signaling defects in iPSC-derived fragile X premutation neurons. Hum Mol Genet. 21(17):3795-805. 2012.

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