Alzheimer's Disease

Coding Dimension ID: 
304
Coding Dimension path name: 
Neurological Disorders / Alzheimer's Disease

Identifying Drugs for Alzheimer's Disease with Human Neurons Made From Human IPS cells

Funding Type: 
Early Translational III
Grant Number: 
TR3-05577
ICOC Funds Committed: 
$1 857 600
Disease Focus: 
Alzheimer's Disease
Neurological Disorders
Stem Cell Use: 
iPS Cell
Cell Line Generation: 
iPS Cell
oldStatus: 
Active
Public Abstract: 
We propose to discover new drug candidates for Alzheimer’s Disease (AD), which is common, fatal, and for which no effective disease-modifying drugs are available. Because no effective AD treatment is available or imminent, we propose to discover novel candidates by screening purified human brain cells made from human reprogrammed stem cells (human IPS cells or hIPSC) from patients that have rare and aggressive hereditary forms of AD. We have already discovered that such human brain cells exhibit an unique biochemical behavior that indicates early development of AD in a dish. Thus, we hope to find new drugs by using the new tools of human stem cells that were previously unavailable. We think that human brain cells in a dish will succeed where animal models and other types of cells have thus far failed.
Statement of Benefit to California: 
Alzheimer’s Disease (AD) is a fatal neurodegenerative disease that afflicts millions of Californians. The emotional and financial impact on families and on the state healthcare budget is enormous. This project seeks to find new drugs to treat this terrible disease. If we are successful our work in the long-term may help diminish the social and familial cost of AD, and lead to establishment of new businesses in California using our approaches to drug discovery for AD.
Progress Report: 
  • We have made steady and significant progress in developing a way to use human reprogrammed stem cells to develop drugs for Alzheimer's disease. In the more recent project term we have further refined our key assay, and generated sufficient cells to enable screening of 50,000 different chemical candidates that might reveal potential drugs for this terrible disease. With a little bit of additional refinement, we will be able to begin our search in earnest in collaboration with the Sanford-Burnham Prebys Screening Center.

A CIRM Disease Team to Develop Allopregnanolone for Prevention and Treatment of Alzheimer's Disease

Funding Type: 
Disease Team Therapy Planning I
Grant Number: 
DR2-05410
ICOC Funds Committed: 
$107 989
Disease Focus: 
Alzheimer's Disease
Neurological Disorders
oldStatus: 
Closed
Public Abstract: 
Alzheimer’s disease (AD) is now a nation-wide epidemic and California is at the epicenter of the epidemic. One-tenth of all people in the United States diagnosed with AD live in California. In the US, 5.4 million people have AD and another American develops AD every 69 seconds. No therapeutic strategies exist to prevent or treat AD. And the situation is worse than expected. Results of a recent two year clinical study show that the currently available medications for managing AD symptoms are ineffective in patients with mild cognitive impairment or mild AD. We seek to develop a small molecule therapeutic, allopregnanolone (APα) to prevent and treat AD. APα promotes the ability of brain to regenerate itself by increasing the number and survival of newly generated neurons. The APα-induced increase in newly generated neurons was associated with a reversal of cognitive deficits and restored learning and memory function to normal in a preclinical mouse model of AD. Further, APα reduced the amount of AD pathology in the brain. Importantly, when given peripherally either by injection under the skin or applied topically to the skin, APα was able to enter the brain to increase the generation of new neurons. The unique mechanism of APα action reduces the risk that APα would cause proliferation of other cells in the body. Because APα was efficacious in both pre-pathology and post-pathology stages of AD progression, APα has the potential to be effective for both the prevention of and early stage treatment of Alzheimer’s disease. Further, APα induced neurogenesis and restoration of cognitive function in normal aged mice suggesting that APα could be efficacious to sustain cognitive function and prevent development of AD in a normal aged population. In other clinical studies, APα has been proven safe in animals and humans and in both men and women. Together, these findings provide a strong foundation on which to plan a clinical trial of APα in persons with prodromal and diagnosed Alzheimer’s disease. To plan for a Phase I-IIa clinical trial to determine safety, dosing and clinical efficacy, we have assembled an interdisciplinary team of clinicians, scientists, therapeutic development, regulatory, data management and statistical analysis experts. The objectives of this proposal are to: a) develop allopregnanolone as a therapeutic for Alzheimer’s disease; to plan an early clinical development program for its use as a neurogenesis agent; b) file a complete and well-supported IND with the Food and Drug Administration (FDA); c) complete phase I/IIa clinical studies to evaluate safety, biological activity, and early efficacy in humans; and (d) complete a phase II clinical trial that will evaluate efficacy and lead to larger multisite clinical studies of efficacy.
Statement of Benefit to California: 
California is at the epicenter of the epidemic of Alzheimer’s disease (AD). Nationwide there are 5.4 million persons living with AD. Ten percent or over half a million Californians have AD. Among California’s baby boomers aged 55 and over, one in eight will develop AD. It is estimated that one in six Californians will develop a form of dementia. By 2030 the number of Californians living with AD will double to over 1.1 million. While all races and ethnic groups and regions of the state will be affected, not all regions within California will be equally affected. Los Angeles County has the greatest population in the state and thus will be the true epicenter of the Alzheimer’s epidemic in California. Alzheimer’s is a disease that affects an entire family, community and health care system. Nation-wide there are nearly 15 million Alzheimer and dementia care givers providing 17 billion hours of unpaid care per year. Total costs for caring for people with AD, totals $183 billion per year. California shouldered $18.3 billion of those costs and most of those costs were born by persons and health care services in Los Angeles County. Because of the psychological and physical toll of caring for people with Alzheimer’s, caregivers had $7.9 billion in additional health care costs. Proportionally that translates into $790 million of health care costs for Californians. In total, California spent over $19 billion per year for costs associated with Alzheimer’s disease. Multiple analyses indicate that a delay of just 5 years can reduce the number of persons diagnosed with Alzheimer’s by 50% and dramatically reduce the associated costs. We seek to develop a small molecule therapeutic, allopregnanolone (APα) to prevent and treat AD. APα promotes the innate regenerative capacity of the brain to increase the pool of neural progenitor cells. The APα-induced increase in neurogenesis was associated with a reversal of cognitive deficits and restored learning and memory function to normal in a preclinical mouse model of AD. Further, APα reduced the development of AD pathology. APα crosses the blood brain barrier and acts through a mechanism unique to neural progenitor cells and thus is unlikely to exert proliferative effects in other organs. Because APα was efficacious in both pre-pathology and post-pathology stages of AD progression, APα has the potential to be effective for both the prevention of and early stage treatment .
Progress Report: 
  • As a result of the planning grant award, the Allopregnanolone (APα) team accomplished the following that enabled submission of the CIRM Disease Team Therapy Development Research Awards Proposal:
  • 1) Created a team of experts in regeneration, neurology and Alzheimer's disease drug development to generate strategy and overall development plan. Through the team’s efforts we developed preclinical and clinical studies, determined correct dosing parameters for clinical studies, identified an optimal route of administration, developed chemistry, manufacturing and controls, and submitted our Pre-IND documents to the FDA.
  • 2) Filed a Pre-IND document with the FDA and held a Pre-IND meeting with the FDA and obtained feedback from the FDA on our program. FDA provided guidance on requirements for the preclinical plan along with input on the design of our two Phase 2 clinical studies. We also obtained agreement that we may cross-reference the existing IND of our academic partner, Michael Rogawski at UC Davis and utilize product manufactured at UC Davis.
  • 3) We developed an integrated CMC plan to manufacture allopregnanolone (clinical API) and established compliant processes to ensure material requirements are met for the preclinical and clinical studies. Manufacture of clinical API will be conducted at the UC Davis CIRM GMP facility.
  • 4) FDA required preclinical IND-enabling research strategy was developed. Teams at USC and a California-based CRO, were identified to conduct three studies: 1) Bridging Study: subcutaneous to IV dosing and administration to bridge from previous subcutaneous preclinical analyses to clinical studies using IV APα administration to determine a) optimal IV dose to promote neurogenesis and b) optimal infusion rate to achieve required peak of APα and area under the curve. 2) Cerebral Microhemorrhage: The FDA advised a safety test for the occurrence of cerebral microhemorrhages localized to the cerebral vasculature in areas of cerebral amyloid angiopathy with various anti-Aβ immunotherapies. 3) Chronic GLP Toxicity Analyses: Based on FDA guidance, safety studies will be required for chronic exposure of Alzheimer’s patients to APα. To initiate the chronic exposure Phase 2a Proof of Concept trial, chronic preclinical toxicology is required. We have designed 6-month and 9-month IV dose GLP toxicity studies in rat and dog, respectively. The studies include systemic toxicology and toxicokinetic evaluation.
  • 5) In support of developing ideal dosing parameters for the Phase 2 clinical studies, the California CRO, Simulations Plus was utilized. ADMET Predictor™ was used to estimate the biopharmaceutical properties of APα. Predictive modeling of optimal dosing regimen and expected human exposure in Alzheimer’s patients was performed.
  • 6) Designed two Phase 2 clinical trials, a Multiple Ascending Dose (MAD) and a Proof of Concept. A California-based CRO, Worldwide Clinical Trials and Alzheimer's clinical trials expert were identified to partner with USC to design and conduct our clinical trials. Phase 2 MAD study primary objectives are to evaluate safety, tolerability and pharmacokinetics. MAD exploratory objectives are to evaluate effect of allopregnanolone on MRI biomarker outcomes and cognition. Proposed MRI biomarkers include hippocampal volume, white matter integrity, and functional connectivity. Phase 2 Proof of Concept trial primary objectives are to evaluate safety and tolerability with long-term exposure. Therapeutic efficacy of allopregnanolone will be determined by outcomes on cognition and biomarkers of regeneration in brain.
  • 7) A Steering Committee and Advisory Board were established. Both advisory groups are composed of internationally recognized researchers, translational scientists, regulatory experts and therapeutic development experts. The charge of the Steering Committee is to provide oversight that CIRM allopregnanolone team progress is on track to meet milestones, ensure that processes and strategies are aligned. The Advisory Board is comprised of internationally recognized experts in Alzheimer’s disease and experts in stem cell biology. Advisory Board members will provide an objective evaluation of CIRM allopregnanolone project progress. The functions of Advisory Board are: 1) Advise CIRM allopregnanolone project leadership on identifying key milestones; 2) Review progress on meeting milestones and hitting development targets; 3) Provide strategic and tactical counsel to the Leadership team and Steering Committee.
  • 8) Generated viable commercial potential through partnership with SAGE Therapeutics. Ensured patent progression and prosecution through USC. Engaged key opinion leaders in the field and educated these experts regarding therapeutic potential of allopregnanolone as a first in class drug for neuroregeneration in Alzheimer's disease.

Neuroprotection to treat Alzheimer's: a new paradigm using human central nervous system cells

Funding Type: 
Disease Team Therapy Planning I
Grant Number: 
DR2-05416
ICOC Funds Committed: 
$98 050
Disease Focus: 
Alzheimer's Disease
Neurological Disorders
oldStatus: 
Closed
Public Abstract: 
Alzheimer’s disease (AD) is an incurable disorder that affects memory, social interaction and the ability to perform everyday activities. In the USA alone, the number of AD patients aged 65 and older has surpassed 5 million and that number may triple by 2050. Annual health care costs have been estimated to exceed 172 billion dollars, but do not reflect loss of income and stress caused to caregivers. Therefore, there is great hope for new therapies that will both improve symptoms and alleviate suffering. There are few FDA-approved medications to treat AD and none is capable of preventing, delaying onset or curing AD. Current medications mostly tend to temporarily slow the worsening of AD-associated symptoms such as sleep disturbances, depression and memory loss/disorientation. Pharmaceutical companies continue to develop new types of drugs or combination therapies that can better treat the symptoms or improve the quality of life of AD patients. There is also an ongoing effort to discover novel drugs that may prevent, reverse, or even cure AD. Unfortunately, the number of clinical studies addressing the possible benefit of such drugs is low, and agents that have shown initial promise have failed at later stage clinical testing, despite convincing preclinical data. There are ongoing studies in AD patients using vaccines and other biological compounds but it is unclear when data from these new trials will be available and more importantly, whether they will be successful. The need for divergent and innovative approaches to AD is clearly suggested by the failure of experimental drugs. Our proposal is to use brain stem cells to treat AD. This is a completely different approach to the more standard therapies described above such as drugs, vaccines, etc., and one that we hope will be beneficial for AD patients as a one-time intervention. AD is characterized by a dysfunction and eventual loss of neurons, the specialized cells that convey information in the brain. Death or dysfunction of neurons results in the characteristic memory loss, confusion and inability to solve new problems that AD patients experience. It is our hope that stem cells transplanted into the patient’s brain may provide factors that will protect neurons and preserve their function. Even a small improvement in memory and cognitive function could significantly alter quality of life in a patient with AD.
Statement of Benefit to California: 
Of the 5.4 million Americans affected with AD, 440,000 are California residents and, according to the Alzheimer’s Association, this number is projected to increase between 49.1 - 81.0% (second highest only to Northwestern states) between 2000 and 2025. Given that California is the most populous state, AD’s impact on state finances is proportionally high and will only increase as the population ages and AD incidence increases. The dementia resulting from this devastating disease disconnects patients from their community and loved ones by eroding memory and cognitive function. Patients gradually lose their ability to drive, work, cook and even carry out simple everyday tasks, and become totally dependent on others. The quality of life of AD patients is hugely affected and the burden on their families and caregivers is very costly to the state of California. There is no cure for AD and no way to prevent it. Most approved therapies only address symptomatic aspects of AD and disease modifying drugs are currently not available. By enacting Proposition 71, California voters acknowledged and supported the need to investigate the use of novel stem cell based therapies to treat currently incurable diseases such as AD. Our goal is to leverage our proven expertise in developing neural stem cell based therapies for human neurodegenerative disorders and apply it to AD. We propose that neural stem cell transplantation into select regions of the brain will have a beneficial impact on the patient. If successful, a single intervention may be sufficient to delay or stop progression of neuronal degeneration and preserve functional levels of cognition and memory. In a disease such as AD, any therapy that can exert even a modest impact on the patient’s ability to carry out some daily activities will have an exponential positive effect not only on patients but also on families, caregivers and the health care system. The potential economic impact of such type of therapeutic intervention for California could be tremendous, not only by reducing the high costs of care but also by becoming a vital world center for stem cell interventions in AD.
Progress Report: 
  • Alzheimer's disease (AD) is an incurable disorder that affects memory, social interaction, and the ability to perform everyday activities. The number of AD patients older than 65 has surpassed 5 million in the US and 600,000 in California, numbers that may triple by 2050. Annual health care costs related to AD have been estimated to exceed $172 billion in the US, even without reflecting either the loss of income or the physical and emotional stress experienced by caregivers. Efforts to discover novel and effective treatments for AD are ongoing, but unfortunately, the number of active clinical studies is low and many traditional approaches have failed in clinical testing. There is a great need for new therapies that will both improve symptoms and alleviate suffering.
  • AD is characterized by the dysfunction and eventual loss of neurons, the specialized cells that convey information in the brain. Death or dysfunction of neurons results in the characteristic memory loss, confusion, and inability to solve new problems that AD patients experience.
  • StemCells Inc. is embarking on an initiative to evaluate the use of its proprietary human neural stem cells to treat AD. We believe that neural stem cells transplanted into a patient’s brain may protect neurons and preserve their function. This represents an entirely new approach to standard therapeutic drug development for AD, which has so far resulted in drugs that only temporarily alleviate symptoms in some patients but that do not slow or change the course of the disease. We envision using neural stem cells as a one-time intervention that will improve memory and cognitive function in AD patients. Even a modest improvement in these symptoms could significantly alter the quality of life of a patient with AD.
  • StemCells Inc. received a Disease Team Planning (DTP) award from CIRM to establish a Disease Team for AD, and to begin organizing the activities required to submit a Disease Team Therapy Development (DTTD) award. We are reporting now on the successful completion of this DTP award. The main deliverables were (i) submission of a DTTD award application and (ii) development of a four year research plan that contemplates an Investigational New Drug (IND) submission to the FDA for the clinical study of neural stem cells in patients with AD, within four years.
  • To begin evaluating its proprietary human neural stem cells as a potential therapy for AD, StemCells Inc. and its collaborators from UC Irvine needed to first design IND-enabling safety and efficacy studies to test these stem cells in animal models relevant for AD. The DTP funding from CIRM helped support a series of telephone, email and face-to-face meetings over the last 6 months, between investigators at UCI and StemCells Inc., to present and evaluate existing data on neural stem cells and to share information about AD in order to design pilot and definitive efficacy and safety studies. During this time, the team also discussed the logistical details required to conduct these studies.
  • After a draft research plan had been outlined, StemCells Inc. and its principal collaborator at UCI, Dr. Frank LaFerla, enlisted the help of various experts in the field of AD, including both clinicians and academic scientists, to evaluate this plan. These experts attended a meeting at UCI and provided input into the experimental design of efficacy and safety studies. Many of these experts were also recruited by StemCells Inc. to participate in preclinical and clinical working groups hosted by the Company. These working groups will ultimately evaluate the preclinical experimental results and help design the protocol for the proposed clinical trial.
  • The DTP award also allowed StemCells Inc. to establish a “Project Team” consisting of highly trained and skilled personnel at UCI, StemCells Inc., and an established Contract Research Organization. This Project Team will be responsible for the production and supply of the human neural stem cells, the execution of all efficacy and safety studies, and the preparation and submission of IND documents to the FDA within the next 4 years.
  • Finally, the DTP award allowed StemCells Inc. to timely develop and submit its DTTD application to CIRM, in which the Company requested funding in the amount of up to $20 million to facilitate execution of IND-enabling safety and efficacy studies for its proposed breakthrough neural stem cell treatment for AD.

Developing a method for rapid identification of high-quality disease specific hIPSC lines

Funding Type: 
Tools and Technologies II
Grant Number: 
RT2-01927
ICOC Funds Committed: 
$1 816 157
Disease Focus: 
Alzheimer's Disease
Neurological Disorders
Stem Cell Use: 
iPS Cell
Cell Line Generation: 
iPS Cell
oldStatus: 
Active
Public Abstract: 
Elucidating how genetic variation contributes to disease susceptibility and drug response requires human Induced Pluripotent Stem Cell (hIPSC) lines from many human patients. Yet, current methods of hIPSC generation are labor-intensive and expensive. Thus, a cost-effective, non-labor intensive set of methods for hIPSC generation and characterization is essential to bring the translational potential of hIPSC to disease modeling, drug discovery, genomic analysis, etc. Our project combines technology development and scaling methods to increase throughput and reduce cost of hiPSC generation at least 10-fold, enabling the demonstration, and criterion for success, that we can generate 300 useful hiPSC lines (6 independent lines each for 50 individuals) by the end of this project. Thus, we propose to develop an efficient, cost effective, and minimally labor-intensive pipeline of methods for hIPSC identification and characterization that will enable routine generation of tens to hundreds of independent hIPSC lines from human patients. We will achieve this goal by adapting two simple and high throughput methods to enable analysis of many candidate hIPSC lines in large pools. These methods are already working in our labs and are called "fluorescence cell barcoding" (FCB) and expression cell barcoding (ECB). To reach a goal of generating 6 high quality hIPSC lines from one patient, as many as 60 candidate hIPSC colonies must be expanded and evaluated individually using labor and cost intensive methods. By improving culturing protocols, and implementing suitable pooled analysis strategies, we propose to increase throughput at least 10-fold with a substantial drop in cost. In outline, the pipeline we propose to develop will begin with the generation of 60 candidate hIPSC lines per patient directly in 96 well plates. All 60 will be analyzed for diagnostic hIPSC markers by FCB in 1 pooled sample. The 10 best candidates per patient will then be picked for expression and multilineage differentiation analyses with the goal of finding the best 6 from each patient for digital karyotype analyses. Success at 10-fold scaleup as proposed here may be the first step towards further scaleup once these methods are fully developed. Aim 1: To develop a cost-effective and minimally labor-intensive set of methods/pipeline for the generation and characterization high quality hIPSC lines from large numbers of human patients. We will test suitability/develop a set of methods that allow inexpensive and rapid characterization of 60 candidate hIPSC lines per patient at a time. Aim 2: To demonstrate/test/evaluate the success and cost-effectiveness of our pipeline by generating 6 high quality hIPSC lines from each of 50 human patients from [REDACTED]. We will obtain skin biopsies and expand fibroblasts from 50 patients, and generate and analyze a total of 6 independent hIPSC lines from each using the methods developed in Aim 1.
Statement of Benefit to California: 
Many Californians suffer from diseases whose origin is poorly understood, and which are not treatable in an effective or economically advantageous manner. Part of solving this problem relies upon elucidating how genetic variation contributes to disease susceptibility and drug response and better understanding disease mechanism. Achieving these goals can be accelerated through the use of human Induced Pluripotent Stem Cell (hIPSC) lines from many human patients. Yet, current methods of hIPSC generation are labor-intensive and expensive. Thus, a cost-effective, non-labor intensive set of methods for hIPSC generation and characterization is essential to bring the translational potential of hIPSC to disease modeling, drug discovery, genomic analysis, etc. If successful, our project will lead to breakthroughs in understanding of disease, development of better therapies, and economic development in California as businesses that use our methods are launched. In addition, new therapies will bring cost-savings in healthcare to Californians, stimulate employment since Californians will be employed in businesses that develop and sell these therapies, and relieve much suffering from the burdens of chronic disease.
Progress Report: 
  • An important problem in stem cell and regenerative medicine research has been the ability to quickly and cheaply generate and characterize reprogrammed stem cells from defined human patients. The primary goal of our project is to address this need by developing new technologies that allow stem cell lines to be characterized in large mixed pools as opposed to one by one. Our new methods use flow cytometry and highly sensitive methods for detecting the activity of genes in the cell lines. We made excellent progress in the first year and reduced flow cytometry methods to practice taking advantage of a method called fluorescence cell barcoding. Methods for analyzing activity of genes and chromosome number are in progress and being tested. Our ultimate goal is to reduce cost tenfold and increase speed by about tenfold and our methods development is on track to accomplish this aim.
  • A key bottleneck in reprogramming technology to make induced pluripotent stem (IPS) cell lines is the ability to make large numbers of lines from large numbers of patients in a way that is cost effective and minimizes labor. Our project has focused primarily on dropping the cost of characterization of candidate lines. We have made a number of discoveries about the behavior of candidate reprogrammed lines that allow us to drop cost and labor needed for candidate reprogrammed line characterization. We measured the frequency of candidate lines that were well-behaved in a large retroviral reprogramming experiment, which allows us to rigorously estimate how many candidate lines must be picked and analyzed if 4-6 high-quality lines are to be generated for every patient fibroblast sample subjected to typical retroviral reprogramming technology. We then continued our work on developing a combination of different array and microfluidic chip technologies to measure the chromosome number in each candidate line and the ability of each line to be pluripotent, i.e., to be able to generate many different type of cells similar to embryonic stem cells. We are optimistic that our work will simplify and drop the cost of the characterization process so that it costs far less than before our work was initiated.

Development of human ES cell lines as a model system for Alzheimer disease drug discovery

Funding Type: 
SEED Grant
Grant Number: 
RS1-00247
ICOC Funds Committed: 
$492 750
Disease Focus: 
Alzheimer's Disease
Neurological Disorders
Stem Cell Use: 
Embryonic Stem Cell
Cell Line Generation: 
Embryonic Stem Cell
oldStatus: 
Closed
Public Abstract: 
Alzheimer disease (AD) is a progressive neurodegenerative disorder that currently affects over 4.5 million Americans. By the middle of the century, the prevalence of AD in the USA is projected to almost quadruple. As current therapies do not abate the underlying disease process, it is very likely that AD will continue to be a clinical, social, and economic burden. Progress has been made in our understanding of AD pathogenesis by studying transgenic mouse models of the disease and by utilizing primary neuronal cell cultures derived from rodents. However, key proteins that are critical to the pathogenesis of this disease exhibit many species-specific differences at both a biophysical and functional level. Additional species differences in other as yet unidentified AD-related proteins are likely to also exist. Thus, there is an urgent need to develop novel models of AD that recapitulate the complex array of human proteins involved in this disease. Cell culture-based models that allow for rapid high-throughput screening and the identification of novel compounds and drug targets are also critically needed. To that end we propose to model both sporadic and familial forms of AD by generating two novel human embryonic stem cell lines (hES cells). Differentiation of these lines along a neuronal lineage will provide researchers with an easily accessible and reproducible neuronal cell culture model of AD. These cells will also allow high-throughput screening and experimentation in neuronal cells with a species-relevant complement of human proteins. In Aim 1 we will develop and characterize hES cell lines designed to model both sporadic and familial forms of AD. To model sporadic AD we will stably transfect HUES7 hES cells (developed by Douglas Melton) with lentiviral constructs coding for human wild type amyloid precursor protein (APP-695) under control of the human APP promoter. APP is well expressed within hES cells and upregulated upon neuronal differentiation. To model familial AD and generate cells that exhibit a more aggressive formation of oligomeric A species we will also develop a second hES cell line stably transfected with human APP that includes the Arctic (E693G) mutation.In Aim 2 we will utilize our wild-type APP hES cells to perform a high-throughput siRNA screen. We will utilize AMAXA reverse-nucleofection in conjunction with a human druggable genome siRNA array (Dharmacon) that targets 7309 genes considered to be potential therapeutic targets. Following transfection conditioned media will be examined by a sensitive ELISA to identify novel targets that modulate A levels. In addition a Thioflavin S assay will determine any effects on A aggregation. Follow-up experiments will confirm promising candidates identified in the high-throughput screen. Taken together these studies aim to establish novel AD-specific hES cell lines and identify promising new therapeutic targets for this devastating disease.
Statement of Benefit to California: 
Alzheimer disease (AD) is a progressive neurodegenerative disorder that currently affects over 500 thousand Californians. As the baby-boomer generation ages the prevalence of AD in California is projected to almost quadruple such that 1 in every 45 individuals will be afflicted. As current therapies do not abate the underlying disease process, it is very likely that AD will continue to be a major clinical, social, and economic burden. Some estimates have even suggested that AD alone may bankrupt the current Californian health care system. Progress has been made in our understanding of AD by studying rodent-based models of the disease. However, key proteins that are critical to the disease exhibit many species-specific differences at both a biophysical and functional level. Thus, there is an urgent need to develop novel models of AD that exhibit the complex array of human proteins involved in this disease. Cell culture-based models that also allow for rapid high-throughput screening and the identification of novel compounds and drug targets are also in critical need. The proposed studies aim to utilize human embryonic stem (hES) cells to establish a novel cell culture based model of Alzheimer’s disease. Once developed these cells will provide Californian researchers with a unique tool to investigate genes and proteins that influence the progress of AD. In this proposal we will also utilize these hES cells to perform a high-throughput screen of over 7300 genes to identify multiple novel drug targets that may critically regulate the development of this disease. Taken together these studies aim to establish novel AD-specific hES cell lines that can be utilized by multiple Californian researchers to identify promising new therapeutic targets for this devastating disease.
Progress Report: 
  • Alzheimer’s disease (AD) is the most common age-related neurodegenerative disorder. It is characterized by an irreversible loss of neurons accompanied by the accumulation of extracellular amyloid plaques and intraneuronal neurofibrillary tangles. Currently, 5.3 million Americans are afflicted with this insidious disorder, including over 588,000 in the State of California alone. Mouse models of AD have contributed significantly to our understanding of the proteins and factors involved in the pathology of AD. However, there are critical differences between mouse and human cell physiology that likely dramatically influence the development of AD-related pathologies. Hence, there is an urgent need to develop novel human neuronal cell-based models of AD.
  • To achieve this goal, we have generated stable human embryonic stem cell (hES) lines over-expressing the gene for human amyloid precursor protein (APP). We succeeded in creating several lines of hES cells that stably express either wild-type (unaltered) APP or APP that includes rare familial mutations known to cause early-onset cases of AD. In each line, transgene expression is driven under control of the human APP proximal promoter. Mutant versions of APP utilized include the “Swedish” mutation which increases production of Aß and the “Arctic” mutation which increases the assembly and accumulation of synaptotoxic Aß oligomers and protofibrils. The generation of lines that harbor familial mutations in APP both provides an aggressive model of AD, to facilitate the identification of targets that modulate not only Aß production but also the assembly of toxic oligomeric species.
  • In addition to generating stable HUES7 and H9 cell lines over-expressing mutant and wild type forms of APP, we also succeeded in establishing a neuronal differentiation protocol which results in 80% of cells adopting a mature neuronal fate. Importantly, we have also verified by biochemical measures that APP-overexpressing cells produce significantly elevated levels of Aß. As a result we are now preparing to utilize these novel cell lines to identify and examine genes that regulate Aß production and hence the development of AD.
  • Alzheimer’s disease (AD) is the most common age-related neurodegenerative disorder. Currently, 5.3 million individuals are afflicted with this insidious disorder, including over 588,000 in the State of California alone. Unfortunately, existing therapies provide only palliative relief. Although transgenic mouse models and cell culture experiments have contributed significantly to our understanding of the proteins and factors involved in the pathology of AD, these approaches are beset by certain critical limitations. Most notably, mouse models by definition are not based on human cells and cell culture models have been limited to non-human or non-neuronal cells. Hence, there is an urgent need to develop a human neuronal cell-based model of AD. To address this need, we have engineered human embryonic stem cell lines to overexpress mutant human genes that cause early-onset familial AD. These novel stem cell lines will provide a valuable system to test therapies and enhance our understanding of the mechanisms that mediate this devastating disease. Interestingly, we have found that overexpression of these AD-related genes can trigger the rapid differentiation of human embryonic stem cells into neuronal cells. We have examined the mechanisms involved and anticipate that our findings may provide a novel and rapid method to generate neurons from embryonic stem cells.

Generation of forebrain neurons from human embryonic stem cells

Funding Type: 
SEED Grant
Grant Number: 
RS1-00205
ICOC Funds Committed: 
$612 075
Disease Focus: 
Aging
Alzheimer's Disease
Neurological Disorders
Stem Cell Use: 
Embryonic Stem Cell
oldStatus: 
Closed
Public Abstract: 
The goal of this proposal is to generate forebrain neurons from human embryonic stem cells. Our general strategy is to sequentially expose ES cells to signals that lead to differentiation along a neuronal lineage, and to select for cells that display characteristics of forebrain neurons. These cells would then be used in transplantation experiments to determine if they are able to make synaptic connections with host neurons. If successful these experiments would provide a therapeutic strategy for the treatment of Alzheimer’s disease and other disorders that are characterized by loss of forebrain neurons. Currently there is no effective treatments for Alzheimer’s disease, and with an aging baby-boomer population, the incidence of this disease is likely to increase sharply. One of the few promising avenues to treat Alzheimer’s is the possibility of cell replacement therapy in which the neurons lost could be replaced by transplanted neurons. Embryonic stem cells, which have the ability to differentiate into various cells of the body, could be a key component of such a therapy if we can successfully differentiate them into forebrain neurons.
Statement of Benefit to California: 
Alzheimer’s disease is a devastating sporadic neurological disorder that places all of us at risk. As the California population ages, there will be a significant increase in the incidence of Alzheimer’s disease, and the medical and financial cost on the state will be severe. There are currently no effective treatments for this disorder, and one of the few promises is the possibility of transplantation therapy to replace the neurons that are lost in the disease. Being able to generate forebrain neurons from human embryonic stem cells would provide a key tool in the fight against this disease. Needless to say, the development of an effective cell replacement therapy would not only be of immense medical significance as we care for our senior population, it will also greatly relieve the financial burden associated with the care of Alzheimer’s patients, which is often borne by the state.
Progress Report: 
  • The goal of this proposal was to generate forebrain neurons from human embryonic stem cells. Our general strategy was to sequentially expose ES cells to signals that would lead the cells to acquire characteristics typical of differentiated brain cells that are lost in disorders such as Alzheimer's Disease. The most important advance of the research was our ability to achieve this goal. We now have a well-developed protocol that can be used to generate forebrain cells in culture. We have found that these cells not only express genes typical of these cells, they extend axons and dendrites and can make synaptic connections. These cells could be very useful for transplantation studies, as well as for developing cell culture models of Alzheimer's disease. Finally, we have discovered that the same protocol is effective in generating forebrain neurons from iPS cells, attesting to the general usefulness of this strategy.

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