Neurological Disorders

Coding Dimension ID: 
303
Coding Dimension path name: 
Neurological Disorders
Funding Type: 
Early Translational II
Grant Number: 
TR2-01778
Investigator: 
Name: 
Type: 
PI
Type: 
Partner-PI
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.
Funding Type: 
Early Translational II
Grant Number: 
TR2-01749
Investigator: 
ICOC Funds Committed: 
$1 752 058
Disease Focus: 
Neurological Disorders
Epilepsy
Pediatrics
Stem Cell Use: 
Embryonic Stem Cell
oldStatus: 
Active
Public Abstract: 

Many neurological disorders are characterized by an imbalance between excitation and inhibition. Our ultimate goal: to develop a cell-based therapy to modulate aberrant brain activity in the treatment of these disorders. Our initial focus is on epilepsy. In 20-30% of these patients, seizures are unresponsive to drugs, requiring invasive surgical resection of brain regions with aberrant activity. The candidate cells we propose to develop can inhibit hyperactive neural circuits after implantation into the damaged brain. As such, these cells could provide an effective treatment not just for epilepsy, but also for a variety of other neurological conditions like Parkinson's, traumatic brain injury, and spasticity after spinal cord injury. We propose to bring a development candidate, a neuronal cell therapy, to the point of preclinical development. The neurons that normally inhibit brain circuits originate from a region of the developing brain called the medial ganglionic eminence (MGE). When MGE cells are grafted into the postnatal or adult brain, they disperse seamlessly and form inhibitory neurons that modulate local circuits. This property of MGE cells has not been shown for any other type of neural precursor. Our recent studies demonstrate that MGE cells grafted into an animal model of epilepsy can significantly decrease the number and severity of seizures. Other "proof-of-principle" studies suggest that these progenitor cells can be effective treatments in Parkinson's. In a separate effort, we are developing methods to differentiate large numbers of human MGE (H-MGE) cells from embryonic stem (ES) cells. To translate this therapy to humans, we need to determine how many MGE cells are required to increase inhibition after grafting and establish that this transplantation does not have unwanted side-effects. In addition, we need simple assays and reagents to test preparations of H-MGE cells to determine that they have the desired migratory properties and differentiate into nerve cells with the expected inhibitory properties. At present, these issues hinder development of this cell-based therapy in California and worldwide. We propose: (1) to perform "dose-response" experiments using different graft sizes of MGE cells and determine the minimal amount needed to increase inhibition; (2) to test whether MGE transplantation affects the survival of host neurons or has unexpected side-effects on the behavior of the grafted animals; (3) to develop simple in vitro assays (and identify reagents) to test H-MGE cells before transplantation. Our application takes advantage of an established multi-lab collaboration between basic scientists and clinicians. We also have the advice of neurosurgeons, epilepsy neurologists and a laboratory with expertise in animal behavior. If a safe cell-based therapy to replace lost inhibitory interneurons can be developed and validated, then clinical trials in patients destined for invasive neurosurgical resections could proceed.

Statement of Benefit to California: 

This proposal is designed to accelerate progress toward development of a novel cell-based therapy with potentially broad benefit for the treatment of multiple neurological diseases. The potential to translate our basic science findings into a treatment that could benefit patients is our primary focus and our initial target disease is epilepsy. This work will provide benefits to the State of California in the following areas:

* California epilepsy patients and patients with other neurological diseases will benefit from improved therapies. The number of patients refractory to available medications is significant: a recent report from the Center for Disease Control and Prevention [www.cdc.gov/epilepsy/] estimates that 1 out of 100 adults have epilepsy and up to one-third of these patients are not receiving adequate treatment. In California, it is one of the most common disabling neurological conditions. In most states, including California, epileptic patients whose seizures aren't well-controlled cannot obtain a driver's license or work certain jobs -- truck driving, air traffic control, firefighting, law enforcement, and piloting. The annual cost estimates to treat epilepsy range from $12 to $16 billion in the U.S. Current therapies curb seizures through pharmacological management but are not designed to modify brain circuits that are damaged or dysfunctional. The goals of our research program is to develop a novel cell-based therapy with the potential to eliminate seizures and improve the quality of life for this patient population, as well as decrease the financial burden to the patients' families, private insurers, and state agencies. Since MGE cells can mediate inhibition in other neurological and psychiatric diseases, the neural based therapy we are proposing is likely to have a therapeutic and financial impact that is much broader.

* Technology transfer in California. Historically, California institutions have developed and implemented a steady flow of technology transfer. Based on these precedents and the translational potential of our research goals, both to provide bioassays and potentially useful markers to follow the differentiation of MGE cells, this program is likely to result in licensing of further technology to the corporate sector. This will have an impact on the overall competitiveness of our state's technology sector and the resulting potential for creation of new jobs.

* Stem cell scientists training and recruitment in California. As part of this proposal we will train a student, technicians, and associated postdocs in MGE progenitor derivation, transplantation, and cell-based therapy for brain repair. Moreover, the translational nature of the disease-oriented proposal will result in new technology which we expect to be transferable to industry partners for facilitate development into new clinical alternatives.

Progress Report: 
  • Advances in stem cell research and regenerative medicine have led to the potential use of stem cell therapies for neurodegenerative, developmental and acquired brain disease. The Alvarez-Buylla lab at UCSF is part of a collaboration that is pioneering the investigation of therapeutic interneuron replacement for the correction of neurological disorders arising from defects in neural excitation/inhibition. Our preliminary data suggests that grafting interneuron precursors into the postnatal rodent brain allows for up to a 35% increase in the number of cortical interneurons. Interneuron replacement has been used in animal models to modify plasticity, prevent spontaneous epileptic seizures, ameliorate hemiparkinsonian motor symptoms, and prevent PCP-induced cognitive deficits. Transplantation of interneuron precursors therefore holds therapeutic potential for treatment of human neurological diseases involving an imbalance in circuit inhibition/excitation.
  • The goal of the research in progress here is to ultimately prepare human interneuron precursors for clinical trials. Towards the therapeutic development of inhibitory neuron precursor transplantation for human neurological disorders, we have made significant progress in the differentiation of these cells from human ESCs and will complete optimization of this protocol. We will continue our investigation of rodent-derived interneuron transplantation to obtain relevant preclinical data for dose response, safety and efficacy in animal models. These dosing and safety data will then serve as the baseline for comparison with human interneuron precursors and inform design of preclinical studies of these cells in immunosuppressed mice. Together, these data will provide essential information for developing a plan for clinical trials using human interneuron precursors.
  • During this first year, we have made considerable headway in the optimization of the human interneuron precursor differentiation protocol, verified functional engraftment of these cells in mice, and begun to collect dose, safety and efficacy data for rodent-derived interneuron transplantation. Importantly, we have achieved the development of a protocol that robustly generates interneuron-like progenitor cells from human ES cells and demonstrated that these progenitors mature in vitro and in vivo into GABAergic inhibitory interneurons with functional potential. We have also compared the behavior of primary fetal cells to these human interneuron precursor-like cells both in vivo and in vitro. As we continue to optimize our ES cell differentiation protocol, these primary interneuron precursors will enable initial human cell dose response and behavior experiments and, along with rodent-derived cells, will provide important baseline measures.
  • In sum, this work will provide essential knowledge for the therapeutic development of inhibitory neuron transplantation. The experiments underway will yield insights that will be critical to the development of a clinical trial using human interneuron precursors.
  • During the reporting period, we have developed methods to enable the optimization of inhibitory nerve precursor cell, or MGE cell, derivation from human pluripotent stem cells (hPSCs). Optimization encompassed increasing MGE cell motility, enhancing MGE cell maturation into inhibitory nerve subtypes, and elimination of tumors post-transplantation into the rodent brain. Furthermore, we demonstrated that the injected hPSC-MGE cells functionally matured into inhibitory nerves with advanced physiological properties that integrated into the rodent brain. In addition, we determined an optimum dose of injected mouse MGE cells in rodent. Moreover, following injection of either the optimum dose or a 10-fold higher dose of mouse MGE cells, we found no detectable behavioral side effects from MGE cell transplantation.
  • In this reporting period, we continued to improve the acquisition of migratory medial ganglionic eminence (MGE)-type interneurons from human embryonic stem cells (hESC). We compared alternative procedures by testing MGE marker expression, and we developed additional tests to measure cell division and migration of MGE cell made from hESCs. We also extended our methods to clinical-grade hESC lines. With these optimized procedures, both research and clinical grade lines were transplanted into the rodent brain. In addition, we completed an evaluation of human fetal MGE transplants after-injection into the rodent brain. Finally, we report safety, survival, and neuronal differentiation of both hESC-MGE and human Fetal-MGE grafts at three months post-injection into the brain of the non-human primate rhesus macaque. A manuscript is in preparation concerning this work. Our work has continued to show the viability of using a cell therapy technique in the potential treatment of brain-related disease.
Funding Type: 
New Faculty I
Grant Number: 
RN1-00577
Investigator: 
Name: 
Type: 
PI
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.
Funding Type: 
New Faculty I
Grant Number: 
RN1-00564
Investigator: 
ICOC Funds Committed: 
$2 229 427
Disease Focus: 
Rett's Syndrome
Neurological Disorders
Stem Cell Use: 
iPS Cell
Cell Line Generation: 
iPS Cell
oldStatus: 
Active
Public Abstract: 

Embryonic stem (ES) cells are remarkable cells in that they can replicate themselves indefinitely and have the potential to turn into all possible cell type of the body under appropriate environmental conditions. These characteristics make ES cells a unique tool to study development in the culture dish and put them at center stage for regenerative medicine. Two techniques, one called somatic cell nuclear transfer (SCNT) and the other in vitro reprogramming, have shown that adult cells from the mouse can be reverted to an ES like state. In SCNT, adult cell nuclei are transferred into oocytes and allowed to develop as early embryos from which ES cells can be derived, while in the in vitro method four genes are ectopically activated in the adult cell nucleus to induce an embryonic state in the culture dish. Key requirement for both processes is to erase the memory of the adult cell that specifies it as an adult cell and set up the ES cell program. How this happens remains unclear, and if it can be reproduced with human adult cells is an open question. Therefore, we will attempt to use the in vitro reprogramming method to generate human ES cells from adult cells and begin to understand the mechanism of the reprogramming process in both human and mouse cells. In addition to being integral to improving our understanding of how ES cells develop, if successful, this work will provide an important milestone for regenerative medicine. Many debilitating diseases and conditions are caused by damage to cells and tissue. In vitro reprogramming could provide a way to generate patient-specific stem cells that, in culture, could be turned into the type of cell or tissue needed to cure the patient’s disease or injury and transplanted back into the patient’s body. For example, Parkinson’s disease is caused by the loss or destruction of nerve cells. If reprogramming becomes possible, we could take a skin biopsy from a patient with Parkinson’s disease, induce the embryonic state in those skin cells to then be able to turn them into nerve cells and transplant them back into the same donor patient. Reprogramming could also be used to repair spinal cord injuries, allowing people who are paralyzed by accidents to walk again, or be helpful for patients with juvenile diabetes. One important advantage of patient-specific self-transplants is that they obviate the need for immunosuppression, which is often problematic for the patient. In addition, human cell reprogramming could be a new way to study how diseases progress at the cellular level as reprogramming could generate ES cells from patients with complex diseases that can be studied in detail for what makes them go awry during development. This knowledge could speed the search for new treatments and possibly cures for some of the most complex diseases that affect societies. We hope that the knowledge gained from our studies on reprogramming can, someday, support research that will help to put these idea to clinical use.

Statement of Benefit to California: 

Donated organs and tissues are often used to replace those that are diseased or destroyed, but unfortunately, the number of people needing a transplant exceeds the number of organs available for transplantation. Embryonic stem (ES) cells can be propagated in the laboratory for an unlimited period of time and can turn into all the specialized cell types that make us a human being. Therefore, ES cells offer the possibility of a renewable source of replacement cells and tissues to treat diseases, conditions, and disabilities such as Parkinson’s and Alzheimer’s, spinal cord injury, stroke, burns, heart disease, diabetes, osteoarthritis and rheumatoid arthritis. Our research is aimed to generate ES cells from adult cells through a method called in vitro reprogramming and to understand the mechanism by which the ES cell program can be reinstated in the adult cells. This work will not only provide the foundation for a better understanding of how human ES cells develop, but, if successful, be an important milestone for regenerative medicine. The advantage of using ES cells derived from adult cells by in vitro reprogramming would be that the patient’s own cells could be reprogrammed to an ES cell state and therefore, when transplanted back into the patient, not be attacked and destroyed by the body’s immune system. This would be beneficial to the people of California as tens of millions of Americans suffer from diseases and injuries that could benefit from research of in vitro reprogramming. Such advances would benefit the health as well as the economy of the state of California.

Progress Report: 
  • The discovery of induced pluripotent stem (iPS) cells by Shinya Yamanaka in 2006 marks a major landmark in the fields of stem cell biology and regenerative medicine. iPS cells can be obtained by co‐expression of four transcription factors in differentiated cells. The reprogramming process takes 2‐3 weeks and is very inefficient with about 1 in a 1000 somatic cells giving rise to an iPS cell. In previous work, we and others had demonstrated that mouse iPS cells are highly similar to ES cells in their molecular and functional characteristics as they for example can support adult chimerism with germline
  • contribution. The goal of the New Faculty Award proposal is to understand the molecular mechanisms underlying transcription factor‐ induced reprogramming of differentiated cells and to define the iPS cell state.
  • During this funding period, our efforts have focused on all three Aims. Within Aim 1, we have addressed a range of technical strategies to improve the reprogramming process. In Aim 2, we have analyzed human and mouse iPS cells in comparison to ES cells and attempted a better definition of the iPS cell state. In Aims 3, we are currently attempting to define barriers of the reprogramming process and begin to understand the transcriptional network that leads to reprogrammed cells.
  • The discovery of induced pluripotent stem (iPS) cells, which are derived from differentiated cells by simply overexpression a few transcription factors, by Shinya Yamanaka in 2006 marks a major landmark in the fields of stem cell biology and regenerative medicine. To unfold the full potential of reprogramming for disease studies and regenerative medicine, we believe that it is important to understand the molecular mechanisms underlying transcription factor‐ induced reprogramming and to carefully characterize the iPS cell state. To this end, during the third year of funding, we have devised a novel screen to identify factors important for the reprogramming process and allow replacement of the original reprogramming factors. We also studied the role of candidate transcriptional and chromatin regulators in the reprogramming process, which led us to identify novel barriers of the reprogramming process and to a better understanding of how chromatin interferes with the reprogramming process. We have also made progress in understanding the function of the reprogramming factors. Regarding human iPS cell lines, we have derived iPS cells from patients carrying X-linked diseases, and are beginning to characterize them molecularly. Together, we hope that our work will contribute to a better understanding of the reprogramming process.
  • Cellular reprogramming and the generation of induced pluripotent stem cells (iPSCs) from differentiated cells has enabled the creation of patient-specific stem cells for use in disease modeling. Reprogramming to the induced pluripotent state can be achieved through the ectopic expression of Oct4, Sox2, Klf4 and cMyc. Insight into the role that the reprogramming factors, various signaling pathways and epigenetic mechanisms play during the different stages of reprogramming remains limited, partly due to the low efficiency with which somatic cells convert to pluripotency. During the past year we have made great progress in (i) defining the molecular requirement for the reprogramming factors; (ii) gaining a better understanding of how repressive chromatin states control the reprogramming process; (iii) determining the differential regulation of chromatin states during reprogramming; (iv) identifying novel reprogramming stages; (v) assessing the three-dimensional organization of the genome during reprogramming; and (vi) determining the influence of a specific signaling pathway and its downstream effectors on different stages of the reprogramming process. Together, our findings provide novel mechanistic insights into the reprogramming process, which will form the basis of approaches to approve the efficiency of the process.
  • When this grant was awarded in 2008, reprogramming to the induced pluripotent state was just achieved by Shinya Yamanaka through the ectopic expression of Oct4, Sox2, Klf4 and cMyc in mouse fibroblasts. The overall goal of this proposal was to understand the molecular mechanisms underlying in vitro reprogramming of somatic cells of the mouse to iPSCs and to apply this knowledge to the reprogramming of human somatic cells. During the last funding period, our work particularly aimed at mechanistic questions: (i) determining the molecular origin of the spatio-temporal demarcation of the DNA binding sites of the reprogramming factors, and how the reprogramming factors induce chromatin changes, employing systematic and comprehensive mapping approaches; (ii) defining how the reprogramming factors induce a specific transcriptional output on target genes; (iii) identifying the steps of the reprogramming process to mouse iPSCs, which revealed an unprecedented detail of the reprogramming process and established that transition through a multitude of hierarchical stages is a fundamental feature of the reprogramming process; (iv) determining the dynamics of DNA methylation in reprogramming; (v) gaining a better understanding of how repressive Polycomb proteins control the reprogramming process; (vi) assessing the three-dimensional organization of the genome during reprogramming; and (vii) using the human iPSC approach for disease studies. Together, our findings provide novel mechanistic insights into the reprogramming process.
Funding Type: 
New Faculty I
Grant Number: 
RN1-00538-A
Investigator: 
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.
Funding Type: 
New Faculty I
Grant Number: 
RN1-00538-B
Investigator: 
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.
Funding Type: 
New Faculty I
Grant Number: 
RN1-00530
Investigator: 
Name: 
Type: 
PI
ICOC Funds Committed: 
$2 200 715
Disease Focus: 
Amyotrophic Lateral Sclerosis
Neurological Disorders
Stem Cell Use: 
Adult Stem Cell
Cell Line Generation: 
Adult Stem Cell
oldStatus: 
Active
Public Abstract: 

One of the most exciting possibilities in stem cell biology is the potential to replace damaged or diseased neural tissues affected by neurodegenerative disorders. Stem-cell-derived neurons provide a potentially limitless supply of replacement cells to repair damaged or diseased neurons. Typically, only one or a very few types of neurons are affected in most neurodegenerative diseases, and simply transplanting stem cells directly into a degenerating or damaged brain will not guarantee that the stem cells will differentiate into the specific neurons types needed. In fact, they may instead cause tumor formation. Thus, we must learn how to guide stem cells, cultured in a laboratory, toward a specific differentiation pathway that will produce neurons of the specified type. These cells would then provide a safe, effective way to treat neurodegenerative diseases and central nervous system injuries.

Since there are hundreds or thousands of types of neurons in the cerebral cortex, functionally repairing damaged neurons in the cortex will require a detailed understanding of the mechanisms controlling differentiation, survival, and connectivity of specific neuronal subtypes. In this proposal, I propose to investigate the molecular mechanisms that guide the neural stem cells in developing embryonic brains to generate two specific types of neurons – corticospinal motor neurons (CSMNs) and corticothalamic projection neurons (CTNs).

Our first goal is to understand what regulates the development of CSMNs. CSMNs are clinically important neurons that degenerate in Amyotrophic Lateral Sclerosis (ALS), and are damaged in spinal cord injuries. With our current technology, replacing damaged CSMNs has been impossible, due largely to a lack of understanding of what signals regulate their development. Our second goal is to identify genes that direct the neural stem cells to generate the CTNs. Despite their essential importance in sensory processing and involvement in epilepsy, mechanisms governing the development of CTNs have not yet been revealed. CSMNs and CTNs express many identical genes, and are generated from common neural stem cells in the embryonic brains. Yet it is unclear how they are specified from common stem cells. Our third goal is to identify transcription factor codes that neural stem cells employ to specifically generate either CSMNs or CTNs.

Currently, there is no cure for neurodegenerative diseases. Understanding how CSMNs and CTNs are generated during development provides the opportunity to design procedures to direct the stem cells cultured in a laboratory to specifically produce CSMNs or CTNs, which can then be used to replaced damaged or diseased neurons, such as those affected by ALS, or spinal cord injuries.

Statement of Benefit to California: 

Neurodegenerative diseases, including Amyotrophic Lateral Sclerosis (ALS), affect tens of thousands of Californians. There are no cures for these devastating diseases, nor effective treatments that consistently slow or stop them. The research proposed in this application may provide the basis for a novel, cost-effective, cell replacement therapy for ALS, thereby benefiting the State of California and its citizens.

Stem cells offer a potential renewable source of a wide range of cell types that could be used to replace damaged cells involved in neurodegenerative diseases or in spinal cord injuries. At present, transplanting stem cells directly into patients is problematic, because this approach may instead cause tumor growth. To support safe and effective cell transplants, it is important to differentiate stem cells prior to the therapy into the specific cell types affected by the diseases. Understanding how different types of neurons are generated during development provides an opportunity to develop new methods to guide the differentiation of stem cells into the proper neuron types.

In this application, we propose to uncover the mechanisms that regulate the neural stem cells in developing mouse brains to generate different neuronal types in the cerebral cortex, including the corticospinal motor neurons (CSMNs) and the corticothalamic neurons (CTNs). CSMNs are the neurons that degenerate in ALS and are affected in spinal cord injuries. Dysfunction of CTNs has been implicated in epilepsy. Understanding the mechanisms regulating neural stem cells to generate CSMNs and CTNs in vivo will help scientists and physicians to direct stems cells to produce CSMNs or CTNs to replace damaged neurons in patients with neurodegenerative conditions.

Progress Report: 
  • In this reporting period, we have been continuing our work to identify genes that regulate neural stem cells to produce different types of neurons in the brain.
  • In the past grant period, we have identified Tbr1 as the major cell fate-determing gene for the corticothalamic neurons.
  • In year 4 of the grant period, we continue to explore the molecular mechanisms that regulate neural stem cells to generate various types of cortical projection neurons, in particular the corticospinal motor neurons and the corticothalamic neurons. We have identified a novel transcription factor that regulates neural stem cell differentiation.
  • During the last grant period, we continue to explore the molecular mechanisms that regulate neural stem cells to generate different types of neurons in the mammalian brains. We have identified a transcription factor that is essential for neural stem cell differentiation, neuronal migration and axon projection.
  • We have continued our study to identify the molecular mechanisms that regulate cortical neuron fate specification. We have discovered/confirmed that (1) Early cortical progenitors are multipotent, and they give rise to different types of cortical project neurons and glia based on birthdates. There is no evidence of intrinsically lineage-restricted early neural stem cells; (2) expression of Fezf2, a major cell fate determining gene for cortical neurons, is regulated by multiple enhancers and promoters. These enhancers and promotor region have distinct and sometimes overlapping activity; (3) transcription factor Nfib is essential for the differentiation of neural stem cells and required for the cortical neurons to extend corticofugal axons; and (4) splicing factor Tra2b is essential for the survival and differentiation of cortical neural progenitor cells. These results provide novel insights into the development of cortical neurons.
Funding Type: 
New Faculty I
Grant Number: 
RN1-00527
Investigator: 
Institution: 
Type: 
PI
ICOC Funds Committed: 
$2 348 520
Disease Focus: 
Aging
Neurological Disorders
Stem Cell Use: 
Adult Stem Cell
Embryonic Stem Cell
Cell Line Generation: 
Adult Stem Cell
oldStatus: 
Active
Public Abstract: 

The adult brain contains a pool of stem cells, termed adult neural stem cells, that could be used for regenerative purposes in diseases that affect the nervous system. The goal of this proposal is to understand the mechanisms that promote the maintenance of adult neural stem cells as an organism ages. Understanding the factors that maintain the pool of adult neural stem cells should open new avenues to prevent age-dependent decline in brain functions and to use these cells for therapeutic purposes in neurological and neurodegenerative diseases, such as Alzheimer’s or Parkinson’s diseases.

Our general strategy is to use genes that play a central role in organismal aging as we have recently discovered that two of these genes, Foxo and Sirt1, have profound effects on the maintenance and self-renewal of adult neural stem cells. We propose to use these genes as a molecular handle to understand the mechanisms of maintenance of neural stem cells. Harnessing the regenerative power of stem cells by acting on genes that govern aging will provide a novel angle to identify stem cell therapeutics for neurological and neurodegenerative diseases, most of which are age-dependent.

Statement of Benefit to California: 

As the population of the State of California ages, neurodegenerative diseases such as Alzheimer’s and Parkinson’s disease affect increasing numbers of patients. There are no efficient treatments of cures for these diseases. In addition to the devastating effects of neurodegenerative diseases on the patients and their relatives, the cost of caring for California’s Alzheimer patients—about $22.4 billion in 2000—has been estimated to triple by 2040 due to the aging of the baby-boomer’s generation.

Stem cells from the brain, or neural stem cells, hold the promise of treatments and cures for these neurodegenerative diseases. One therapeutic strategy will be to replace degenerating cells in patients with stem cells. Another approach would be to identify strategy to better maintain the pool of neural stem cell with age. Both approaches will only be possible when the mechanisms controlling the maintenance of these stem cells and their capacity to produce their functional progeny are better understood in young and old individuals.

We propose to study the mode of action in neural stem cells of two genes, Foxo and Sirt, that are known to play major roles to extend lifespan in a variety of species. These genes are major targets for the development of stem cell therapeutic strategies that will benefit a wide range of patients suffering from age-dependent neurodegenerative disorders.

The development of effective replacement therapies in neurodegenerative diseases will be a benefit for the rapidly aging population of California; it will also alleviate the financial burden that these age-related disorders create for the State of California.

Progress Report: 
  • Aging is accompanied by a decline in the number and the function of adult stem cells in several tissues. In the brain, the depletion of adult neural stem cells (NSC) may underlie impaired cognitive performance associated with aging. Discovering the factors that govern the maintenance of adult NSC during aging should allow us to harness their regenerative potential for therapeutic purposes during normal aging and age-related neurodegenerative disorders. We have recently found that two 'longevity genes', Foxo3 and Sirt1, are critical for adult NSC function. In the past year, we have published a manuscript showing that Foxo3 is necessary for the maintenance of NSC in the adult brain. We have also started to explore the critical mechanisms by which Foxo3 maintains adult neural stem cells in the brain. We have used ultra-high throughput sequencing approach to reveal that Foxo3 is recruited to the regulatory regions of 3,000 genes in the adult neural stem cells, thereby triggering a gene expression network that regulates both the ability of neural stem cells to divide and their ability to give rise to progeny. Finally, we have obtained new results in the past year, showing that Sirt1, another 'longevity gene' is critical for the proper function of neural stem cells in the adult brain, and their ability to give rise to differentiated cells. Together, our results will help understand the regulation of neural stem cell maintenance in aging individuals and will provide new avenues to preserve the pool of these cells in the brain. Modulating longevity genes to harness the regenerative power of stem cells will provide new avenues for stem cell therapeutics for neurological and neurodegenerative diseases, most of which are age-dependent.
  • The adult brain contains pools of stem cells called neural stem cells that are critical for
  • the formation of new neurons in the adult brain. During aging, the number of neural stem
  • cells and their ability to give rise to new neurons strikingly decline. This decline could
  • underlie at least in part memory deterioration that occurs during aging and age-related
  • neurodegenerative disease such as Alzheimer’s disease. We have been interested over
  • the years in the importance of genes that regulate overall longevity in the control of the
  • pool of neural stem cells. We made the important discovery that Foxo3, a gene that has
  • been implicated in human exceptional longevity, is necessary for preserving the neural
  • stem cell pool. In the past year, we have made extensive progress in characterizing the
  • ensemble of genes regulated by Foxo3 in adult neural stem cells, a key step in
  • unraveling the mechanisms by which neural stem cells are maintained intact. In the past
  • year, we have observed that in the absence of another gene important for longevity
  • Sirt1, there is an unexpected increase in oligodendrocyte progenitors, which are cells
  • that are important for myelination of neurons, which is important for the proper
  • propagation of the neuronal information. Defects in myelination, which happen for
  • example in multiple sclerosis, have devastating consequences on the neurological
  • function. In the past year, we have made progress to understand the cellular and
  • molecular mechanism of action that enhances the production of oligodendrocytes in the
  • absence of Sirt1. Finally, we have made progress in initiating a project in human stem
  • cells that can be reprogrammed from adult cells, to extend our findings from mice to
  • humans, in particular as it relates to human diseases that have an age-dependent
  • component.
  • The number and function of adult stem cells decrease with age in a number of tissues. In the nervous system, the depletion of functional adult neural stem cells (NSC) may be responsible for impaired cognitive performance associated with normal or pathological aging. Understanding the factors that govern the maintenance of adult NSC should provide insights into their regenerative potential and open new avenues to use these cells for therapeutic purposes during normal aging and age-related neurodegenerative disorders.
  • Clues to key regulators of stem cell functions may come from studies of the genetics of aging, as genes that regulate longevity may do so by maintaining stem cells. To date, the most compelling examples for genes that control aging in a variety of organisms include the insulin-Akt-Foxo transcription factor pathway and the Sirt deacetylases. We have recently found that Foxo3 regulates a network of genes in adult NSC and interact with another transcription factor, called Ascl1, to preserve the integrity of the NSC pool and prevent the premature exhaustion of this important pool of cells. In the past year, we have also made the surprising discovery that inactivating Sirt1 in adult neural stem cells leads to the increased production of oligodendrocyte progenitors, which are cells that are crucial for myelination and could help demyelinating diseases, such as multiple sclerosis, or demyeliating injuries such as spinal cord injuries. Importantly, the enzymatic activity of Sirt1 can be targeted by small molecules, underscoring the potential for Sirt1 as a therapeutic target in stem cell and oligodendrocyte production. In the last year, we have also made significant progress in using cellular reprogramming to investigate the role of longevity genes in human cells. Our work examines the mechanisms by which ‘longevity genes’ regulate stem cell function and maintenance. Harnessing the regenerative power of stem cells by acting on longevity genes will provide a novel angle to identify stem cell therapeutics for regenerative medicine.
  • The adult brain contains reservoirs of neural stem cells that are critical for the formation of new neurons, oligodendrocytes, and astrocytes in the adult brain. During aging, the number of neural stem cells and their ability to give rise to new neurons strikingly decline. This decline could underlie at least in part the decline in memory that occurs during aging. We are interested in the importance of genes that regulate organismal longevity in the control of the reservoir of neural stem cells. We discovered that Foxo3, a transcription factor that has been implicated in human exceptional longevity, is important for regulating the neural stem cell pool pool. In the past year, we have made extensive progress in characterizing the interaction between Foxo3 and specific chromatin states at target genes in adult neural stem cells, which provides us with a mechanistic view onto how longevity genes can affect specific networks of target genes in neural stem cells in adult organisms. In the past year, we have made significant progress in testing the role of a gene involved in healthspan and longevity in a number of organisms, the deacetylase Sirt1, in adult neural stem cell function. We have observed that Sirt1 inactivation, whether genetic or pharmacological, leads to an increase in oligodendrocyte progenitors, which are cells that are important for myelination of axons. We have found that Sirt1 inactivation is beneficial for models of demyelinating injuries and diseases, which has important consequences for multiple sclerosis. Finally, we are making progress in reprogramming adult human fibroblasts into induced pluripotent stem cells and induced NSCs, with the aim to test the importance of longevity genes in this process.
Funding Type: 
Early Translational I
Grant Number: 
TR1-01267
Investigator: 
Type: 
Partner-PI
ICOC Funds Committed: 
$5 416 003
Disease Focus: 
Parkinson's Disease
Neurological Disorders
Collaborative Funder: 
Victoria, Australia
Stem Cell Use: 
Adult Stem Cell
Embryonic Stem Cell
iPS Cell
oldStatus: 
Active
Public Abstract: 

Parkinson's Disease (PD) is a devastating disorder, stealing vitality from vibrant, productive adults & draining our health care dollars. It is also an excellent model for studying other neurodegenerative conditions. We have discovered that human neural stem cells (hNSCs) may exert a significant beneficial impact in the most authentic, representative, & predictive animal model of actual human PD. Interestingly, we have learned that, while some of the hNSCs differentiate into replacement dopamine (DA) neurons, much of the therapeutic benefit derived from a stem cell action we discovered a called the “Chaperone Effect” – even hNSC-derived cells that do not become DA neurons contributed to the reversal of severe Parkinsonian symptoms by protecting endangered host DA neurons & their connections, restoring equipoise to the host nigrostriatal system, and reducing pathological hallmark of PD. While the ultimate goal may someday be to replace dead DA neurons, the Chaperone Effect represents a more tractable near-term method of using cells to address this serious condition. However, many questions remain in the process of developing these cellular therapeutic candidates. A major question is what is the best (safest, most efficacious) way to generate hNSCs? Directly from the fetal brain? From human embryonic stem cells? From skin cells reprogrammed to act like stem cells? Also, would benefits be even greater if, in addition to harnessing the Chaperone Effect, the number of stem cell-derived DA neurons was also increased? And could choosing the right stem cell type &/or providing the right supportive molecules help achieve this? This study seeks to answer these questions. Importantly, we will do so using the most representative model of human PD, a model that not only mimics all of the human symptomatology but also all the side-effects of treatment; inattention to this latter aspect plagued earlier clinical trials in PD. A successful therapy for PD would not only be of great benefit for the many patients who now suffer from the disease, or who are likely to develop it as they age, but the results will help with other potential disease applications due to greater understanding of stem cell biology (particularly the Chaperone Effect, which represents “low hanging fruit”) as well as their potential complications and side effects.

Statement of Benefit to California: 

Not only is Parkinson's Disease (PD) a devastating disease in its own right-- impairing typically vibrant productive adults & draining our health care dollars -- but it is also an excellent model for studying other neurodegenerative diseases. We have discovered that stem cells may actually exert a beneficial impact independent of dopamine neuron replacement. As a result of a multiyear study performed by our team, implanting human neural stem cells (hNSCs) into the most authentic, representative, and predictive animal model of actual human PD, we learned that the cells could reverse severe Parkinsonian symptoms by protecting endangered host dopaminergic (DA) neurons, restoring equipoise to the cytoarchitecture, preserving the host nigrostriatal pathway, and reducing alpha-synuclein aggregations (a pathological hallmark of PD). This action, called the "Chaperone Effect" represents a more tractible near-term method of using cells to address an unmet medical need. However, many questions remain in the process of developing these cellular therapeutic candidates. A major question is what is the best (safest & most efficacious way) to generate hNSCs? Directly from the fetal brain? From human embryonic stem cells? From human induced pluripotent cells? Also, would benefits be even greater if, in addition to harnessing the Chaperone Effect, the number of donor-derived DA neurons was also increased? And could choosing the right stem cell type &/or providing the right supportive molecules help achieve this? This study seeks to answer these questions. Importantly, we will continue to use the most representative model of human PD to do so, a model that not only mimics all of the human symptomatology but also all the side-effects of treatment; inattention to this latter aspect plagued earlier clinical trials in PD. Because of the unique team enlisted, these studies can be done at a fraction of the normal cost, allowing for parsimony in the use of research dollars, clearly a benefit to California taxpayers. Not only might California patients benefit in terms of their well-being, and the economy benefit from productive adults re-entering the work force & aging adults remaining in the work force, but it is likely that new intellectual property will emerge that will provide additional financial benefit to California stakeholders, both citizens & companies.

Progress Report: 
  • Parkinson's Disease (PD) is a devastating disorder, stealing vitality from vibrant, productive adults & draining our health care dollars. It is also an excellent model for studying other neurodegenerative conditions. We have discovered that human neural stem cells (hNSCs) may exert a significant beneficial impact in the most authentic, representative, & predictive animal model of actual human PD (the adult African/St. Kitts Green Monkeys exposed systemically to the neurotoxin MPTP). Interestingly, we have learned that, while some of the hNSCs differentiate into replacement dopamine (DA) neurons, much of the therapeutic benefit derived from a stem cell action we discovered called the “Chaperone Effect” – even hNSC-derived cells that do not become DA neurons contributed to the reversal of severe Parkinsonian symptoms by protecting endangered host DA neurons & their connections, restoring equipoise to the host nigrostriatal system, and reducing pathological hallmark of PD. While the ultimate goal may someday be to replace dead DA neurons, the Chaperone Effect represents a more tractable near-term method of using cells to address this serious condition. However, many questions remain in the process of developing these cellular therapeutic candidates. A major question is what is the best (safest, most efficacious) way to generate hNSCs? Directly from the fetal brain? From human embryonic stem cells? From skin cells reprogrammed to act like stem cells? Also, would benefits be even greater if, in addition to harnessing the Chaperone Effect, the number of stem cell-derived DA neurons was also increased? And could choosing the right stem cell type &/or providing the right supportive molecules help achieve this? This international study – which involves scientists from California, Madrid, Melbourne -- has been seeking to answer these questions. Importantly, we have been doing so using the most representative model of human PD, a model that not only mimics all of the human symptomatology but also all the side-effects of treatment; inattention to this latter aspect plagued earlier clinical trials in PD. A successful therapy for PD would not only be of great benefit for the many patients who now suffer from the disease, or who are likely to develop it as they age, but the results will help with other potential disease applications due to greater understanding of stem cell biology (particularly the Chaperone Effect, which represents “low hanging fruit”) as well as their potential complications and side effects.
  • To date, we have transplanted nearly 40 Parkinsonian non-human primates (NHPs) with a range of the different stem cell types described above. We have been able to generate neurons from some of these stem cells that appear to have the characteristics of the desired A9-type midbrain dopaminergic neuron lost in PD. Following transplantation, some of these stem cell derivatives appear to survive, integrate, & behave like dopaminergic neurons. Preliminary behavioral analysis of some engrafted NHPs offers encouraging results, suggesting an improvement in the Parkinsonism score in some of the animals. These NHPs will need to be followed for 1 year to insure that improvement continues & that no adverse events intervene. Over the next year, more stem cell candidates will be tested as we further optimize their preparation & differentiation.
  • We have made substantial progress in what will amount to the largest and most comprehensive head-to-head behavioral analysis of stem cell transplanted MPTP-NHPs to date and have identified cell types that show dramatic improvement in this model. Compared to the improvement observed with undifferentiated fetal CNS-derived hNSCs (the stem cell type in used Redmond et al, PNAS, 2007), 3 human stem cell candidates have shown a larger improvement in PS.
  • Summary of Achievements for this reporting period
  • • Comprehensive Behavioral data collection of 84 monkeys comprising over 10,000 observation data points
  • • Statistical analysis of Behavioral data collected to date identifies striking and statistically significant improvements in PS for several stem cell types. (Accordingly, NO-GO (or near NO-GO) cell types have been identified via comparison of levels of improvement or no improvement) [Figure 1]
  • • DNA samples collected in order to pursue the first ever complete genome sequencing of the Vervet in collaboration with the Washington University Genome Center
  • • Biochemistry sample processing and data collection of a 2nd large batch of samples completed.
  • The identification and development of an ideal cell-based therapy for a complex neurodegenerative disease requires the rigorous evaluation of both efficacy and safety of different sources and subtypes of hNSCs. The objective of this project has been to fully evaluate and identify the optimal stem cell type for a cell based therapy for refractory Parkinson’s Disease (PD) using the systemically MPTP-lesioned Old World non-human primate (NHP) (the St. Kitts Green Monkey) the most authentic animal model of the actual human disease. Among a list of plausible potentially therapeutic stem cell sources, 7 candidates have been evaluated head-to-head. The intent has been that the stem cell type (and its derivatives) safely producing the largest improvement in behavioral scores (based on a well-established NHP PD score – the Parkinson’s Factor Score [PFS] or ParkScore (which closely parallels the Hoehn–Yahr scale used in human patients, and is an accurate functional read-out of nigrostriatal dopamine [DA] activity) -- as well as a Healthy Behaviors Score [HBS] (similar to the activities-of-daily-living [ADL] on the major Parkinson’s rating scale and allows quantification of adverse events) -- will be advanced towards IND-enabling studies, to an actual IND filing, and ultimately a clinical trial.
  • Candidate cells have been transplanted into specific sub-regions of the nigrostriatal pathway of MPTP-lesioned NHPs. Animals undergo behavioral scoring for analysis of severity of Parkinsonian behavior at multiple time points pre- and post-cell transplantation. At sacrifice, biochemical measurements of DA content are made. Tissue is also analyzed to determine the fate of donor cells; the status of the host nigrostriatal pathway; the number of alpha-synuclein aggregates; degree of inflammation; any evidence of adverse events (e.g., tumor formation, cell overgrowth, emergence of cells inappropriate to the CNS).
  • We have made substantial progress in what will amount to the largest and most comprehensive head-to-head analysis of stem cell transplanted into any disease model to date, let alone behavioral analysis into a primate model of PD. Behavioral data have been collected on ~100 monkeys comprising >10,000 observation data points. We have identified a single Developmental Candidate (DC) that shows consistent and dramatic improvement in severely Parkinsonian NHPs (i.e., a significant decrease in Parkinsonian symptoms over the entire evaluation period), reflecting a restitution of DA function – human embryonic stem cell (hESC-derived) ventral mesencephalic (VM) precursors. We also suggest adding a mechanism to these cells for insuring unambiguous safety and invariant lineage commitment (a construct already generated and inserted into this DC, and recently engrafted into some initial monkeys).
  • We believe are ready for IND-enabling studies, including additional long-term pre-clinical behavioral studies of hESC-derived hVM cells that bear the above-mentioned “safety construct” – combined with additional biochemical assays of DA metabolism, histological assessments, serial profiling to insure genomic stability. Scale-up conditions for this DC are defined and reproducible and a working cell bank has been established.
  • Parkinson's Disease (PD) is a devastating disorder that is caused by the loss of a particular type of neuron in the brain. PD patients show movement abnormalities which worsen over time and significantly reduce the quality of life. Current treatments reduce the severity of these problems but very often the efficacy of these treatments gradually weakens over time leaving patients with few therapeutic options, some of which carry significant unwanted side effects. Since the development of growing undifferentiated human stem cells in the late 1990’s, much has been learned in regards to how to make these cells develop into neuronal cells, in particular the same type of neuron that is lost in a PD patient. Therefore, a cellular therapy has been envisioned for the treatment of PD, however, the complex nature of this disease requires higher level models in which potential therapies can be accurately evaluated before moving a therapy to clinical trials.
  • Previous work using human fetal tissue showed improvement of PD symptoms in an animal model and human clinical trials, however, distinctive movement abnormalities arose from the use of this treatment and combined with the ethical issues, it is not a viable therapeutic strategy. Recent work suggests that the use of embryonic stem cells for the treatment of PD may be possible but a direct comparison of the different types of cells derived from these was lacking. Additionally, tumors caused by these cells have been reported.
  • Our research efforts funded by this CIRM award allowed us to complete the largest stem cell therapy comparison for PD using the most accurate disease model available. Over the last 3 years we have evaluated the efficacy of 8 potential therapeutic cell types and 2 control cell types (in addition to various other control groups to rule out any possibility that the observations may have resulted from something other than cells). From these efforts we have confidently identified a strategy for producing cells that show a dramatic reduction in the PD symptoms in this model and these cells will be developed for clinical trials. Furthermore, we have incorporated a critical step for ensuring the safety of this cell therapy by including a purification technique that removes cells that may give rise to tumors or produce unknown or unwanted effects.
Funding Type: 
Early Translational I
Grant Number: 
TR1-01257
Investigator: 
Name: 
Type: 
PI
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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