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Therapeutic Potential of Transplanted human Embryonic Stem Cells Overexpressing Soluble APP in Treating Alzheimer's Disease

Funding Type: 
SEED Grant
Grant Number: 
RS1-00254
Funds requested: 
$758 999
Funding Recommendations: 
Not recommended
Grant approved: 
No
Public Abstract: 
Alzheimer disease (AD) afflicts over 5 million elderly Americans and is characterized by deposits of insoluble protein aggregates (amyloid plaques) and neurofibillary tangles) as well as massive neuronal loss in selected regions affecting learning and memory. Stem cell therapy represents a promising strategy for treating the chronic central nervous system (CNS) diseases such as AD by replacing damaged and lost neurons and thus restoring defective cognitive behaviors. Human embryonic neuronal stem cells (hES) transplanted into aged rodent brains are found to differentiate into neuronal cells and significantly improve the cognitive functions of the animals. However, ethical and practical issues remain which compel us to seek alternative strategies. Using a well-characterized human ES line in transplantation is an option which can be greatly enhanced by some potent neurotrophic factors to nourish neurons. In this application, we propose to combine hES with a natural soluble factor, the N-terminal portion of the amyloid precursor protein (sAPP) to create a superior stem cell agent for treating AD. sAPP is present normally in the cerebral spinal fluid (CSF) and its level is found to dramatically decline in AD patients, suggesting that this protein plays a critical role in preventing AD. Indeed, this is the best-characterized natural molecule that displays potent neuroprotective and neurotrophic actions on cultured neurons as well as in CNS cells upon infusion. We thus propose to engineer two human ES lines to secrete sAPP via lentivirus infection and to characterize these established lines for the effects of sAPP on differentiation and migration features of the transduced hES. Subsequently, we will transplant these cells into mouse brains at various ages to optimize a transplantation procedure. Finally, the efficacy of the transplanted hES secreting sAPP will be tested in reducing AD pathology in a selected mouse model that displays massive neuronal death/loss and impaired synaptic function. We hope this study will provide proof-of-concept for an established human ES line with a superior ability to differentiate and to stimulate neighboring neurons to proliferate into new neurons which can be further validated and used in future therapeutics.
Statement of Benefit to California: 
California's population is aging, and as people live longer the incidence of diseases caused by aging increases. This has an enormous economic impact on California, since the caregivers for the elderly are usually their children, who are in the peak of their productive years. Alzheimer's disease (AD), the number one dementia among the elderly, is especially devastating because the disease develops and worsens over a long period of time with no available cure . Stem cell replacement by transplantation represents a promising therapeutic option for treating AD. However, both the ethical and practical issues compel neuroscientists to seek alternative approaches (e.g., using human embryonic stem cell lines, hES lines herein) in addition to using primary human stem cells. The proposed studies to fully characterize and establish well-behaved hES lines with superior ability to replace damaged/lost neurons in AD brains upon transplantation will provide proof-of-concept for future transplantation feasibility in patients. Nationwide, an estimated 5 million Americans have AD. The number of Americans with AD has more than doubled since 1980 and continues to grow at an accelerated rate. California, as a paradise to retirees, accommodates the largest aging population and is estimated to have nearly 1 million people with AD. Additionally California farmers use approximately 250 million pounds of pesticides which is about a quarter of all pesticides used in the entire country. Pesticides have been proven to be neural toxic and linked to higher incidences of Parkinson’s disease and AD. Not to mention curing the disease, finding a treatment that could delay the onset of AD by five years alone could reduce the number of individuals with AD by nearly 50% after 50 years and thus greatly reduce the government’s medicare costs (which are expected to increase 75% from $11 billion in 2005 to $19 billion in 2010 in California).
Review Summary: 
SYNOPSIS: This proposal attempts to co-opt human embryonic stem cells (hESCs) to express soluble APP (sAPP) to effect benefit in AD models. The ultimate goal of this research is to develop a transplantable, hESC line for treating Alzheimer’s disease (AD). The working hypothesis is that hESCs engineered to overexpress and secrete sAPP may differentiate into specific types of post-mitotic neuronal cells, migrate to and incorporate into the affected regions upon transplantation. hESCs may also serve as excellent delivery vehicles for sAPP to protect neurons due to the potent neurogenic as well as neuroprotective actions of sAPP. To test this hypothesis, the PI proposes three specific aims: Aim1. Generate stable hESC lines secreting functional sAPP using a lentivirus approach (hES-sAPPa-EGFP) and characterize them in culture systems for neuronal differentiation and migration. Aim 2. Optimize a transplantation procedure in mice where hESC-sAPP-EGFP cells will be transplanted into the third ventricle or repeatedly transfused into peripheral blood (i.v.) for comparison to achieve a feasible procedure for chronic administration. Aim 3. Evaluate the effectiveness of hES-sAPP-EGFP cells transplantation in decreasing neurodegeneration in selected AD mouse model. Established transgenic mice which develop synaptic dysfunction associated with impaired LTP will be used for testing this hypothesis. INNOVATION & SIGNIFICANCE: Although the use of cells engineered to secrete soluble APP alpha is somewhat innovative, the motivation for using hESCs vs. some other, simpler cell type is unclear. The PI hopes that this strategy will lead to both neuroprotection and neuronal replacement. This work would not fundamentally advance the field in its current state of proposed direction. STRENGTHS: Many technical aspects are solid and the APP work makes sense. However, one reviewer is not convinced that the use of hESCs as a delivery vehicle is needed. The p25 transgenics from the Tsai lab provide an interesting model for neurofibrillary degeneration. WEAKNESSES: One reviewer felt that this work would not fundamentally advance the field in its current state of proposed direction and suggested that the PI re-think the fundamental directions and motivations of using hESCs. Another reviewer had questions about feasibility. How clear is it that the ESCs will differentiate into neural cells after transplantation? How reproducible will this differentiation be? Is the in vitro “neuralization” of ESC lines 100% efficient? That is, might there be other embryonic cell lineages represented in the cell population? How would the PI look for such cells? Although the PI notes that inflammation may be a problem, there are no controls that would be appropriate for the effects of the inflammation that the transplants will produce, or notes as to how inflammation will be assayed. The PI will examine brains for neuronal death and synaptic function, the former assessed by “histological studies”, the latter by PPF and LTP. Examining tissues for the differentiation of ESCs into neurons should not pose a problem, but determining if ESCs or ESCs secreting sAPPalpha have an effect on endogenous (mouse) neurogenesis or neuronal death is much more difficult. DISCUSSION: There was no further discussion following the reviewers' comments.
Conflicts: 

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