Public Abstract:
The goal of our work is to understand the mechanisms and pathways that turn undifferentiated human stem or iPS cells into differentiated neurons that can be used in the brain as replacement for neurons lost in neurodegeneration. We will focus on Huntington's disease or chorea (HD), a incurable neurodegenerative disorder that manifests itself first in the middle age. HD is caused by a mutation in the gene, huntingtin, in which an expansion in the CAG coding for glutamine is expanded to above 36 repeats. Huntington’s disease is a dominantly inherited neurodegenerative disorder with massive loss of cells in the striatum- the cell type lost is medium spiny neurons. We will evaluate methods to induce cellular differentiation of ES or iPS into medium spiny neurons. We will also take iPS cells generated from HD skin fibroblasts and develop methods to correct the mutation in the gene huntingtin.
Statement of Benefit to California:
Huntington’s disease is a dominantly inherited neurodegenerative disorder with massive loss of cells in the striatum and replacement of these cells offers a possible therapy. We will use human embryonic stem cells and induced pluripotent stem (iPS) cell technologies with established methods to differentiate stem cells into pure medium spiny neurons and we will develop methods to genetically correct Huntington’s disease iPS cells for use therapeutically in patients.
The studies we perform should benefit the state of California in several ways:
1. We hope to increase the ability to generate medium spiny neurons that can be used in cell-replacement therapies for neurodegenerative diseases such as Huntington’s disease in which loss of these cells correlates with disease. This will directly benefit patients in California and elsewhere.
2. The human embryonic stem cell research we perform may bring new biotechnology jobs to California, thus increasing the state's visibility as a leader in stem cell technology.
3. New biochemical methods for genetically correcting disease iPS cells will have implications for all genetic diseases. Our discoveries could bring revenues to California due to the ability of the state to obtain licensing fees on technology generated using CIRM funds.
Review Summary:
EXECUTIVE SUMMARY
This study will focus on Huntington's disease (HD), a devastating and presently incurable neurodegenerative disorder. A hallmark of HD is the death and degeneration of medium spiny neurons (MSNs) in the striatum. The overall objective of this proposal is to develop methods to produce MSNs from human pluripotent stem cells (hPSC) in high yield and purity. The applicant proposes three aims to achieve this. In Aim 1, the applicant will optimize protocols for differentiating human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs) into MSNs. The goal of Aim 2 is to establish lines of hESCs and hiPSCs that stably express a drug-selectable marker under the control of a striatal specific promoter to enable selection of pure striatal cell populations. Finally, in Aim 3, the applicant proposes to use a gene targeting strategy to correct the expanded region of the diseased HD locus in fibroblasts or derived iPSCs from HD patients. This will generate gene corrected patient-specific striatal neurons/neural precursor cells for potential use as cell replacement therapy.
Reviewers agreed that the proposed research has significance and could have a major impact on the treatment of neurodegenerative diseases such as HD. Despite this, some reviewers found this to be a poorly focused proposal that fails to address key mechanisms related to stem cell biology or the mechanisms that underlie cellular demise in the HD brain. Although some reviewers found the concept of using hiPSCs to generate MSNs to be novel, others found the project lacked innovation, since very similar approaches focused on hESC/hiPSC differentiation into neurons, are being used by a large number of other labs worldwide.
Several reviewers questioned the overall feasibility of the proposal. One of the major criticisms was the fact that the preliminary data underpinning this study, although supportive, is not extensive and is not compelling. In particular, there is no evidence showing the generation of MSNs. Reviewers felt that considerably more work will be required to make a convincing argument that the hPSCs have differentiated into fully functional MSNs and they expressed concern as to whether the investigators will actually be able to generate the yield of MSNs that they anticipate. Whereas reviewers found that the methods proposed for Specific Aims 1 and 2 are standard and the experiments feasible, they found Specific Aim 3, which involves homologous recombination in hESCs, to be technically very challenging as very few groups have successfully applied homologous recombination to hESCs.
Reviewers found the applicant to have an excellent track record in the study of the pathogenesis of HD. However, reviewers found the lack of team experience in hESC/hiPSC biology to be a major source of concern, which ultimately weakened their confidence in the proposal. Reviewers would have liked to see an experienced hESC/hiPSC biologist on board as co-investigator or collaborator.
Overall, reviewers found this proposal to be of potentially high importance, but did not find the preliminary data to be compelling and they questioned the feasibility of this study.
