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RL1-00649-1: Development of Induced Pluripotent Stem Cells for Modeling Human Disease

Recommendation: Not recommended for funding. Recommended to be available for funding only if the ICOC makes a programmatic determination to fund this application
Scientific Score: 69

First Year Funds Requested: $579,240
Total Funds Requested: $1,737,720

Public Abstract (provided by applicant)

Human embryonic stem cells (hESC) hold great promise in regenerative medicine and cell replacement therapies because of their unique ability to self-renew and their developmental potential to form all cell lineages in the body. Traditional techniques for generating hESC rely on surplus IVF embryos and are incompatible with the generation of genetically diverse, patient or disease specific stem cells. Recently, it was reported that adult human skin cells could be induced to revert back to earlier stages of development and exhibit properties of authentic hES cells. The exact method for “reprogramming” has not been optimized but currently involves putting multiple genes into skin cells and then exposing the cells to specific chemical environments tailored to hES cell growth. While these cells appear to have similar developmental potential as hES cells, they are not derived from human embryos. To distinguish these reprogrammed cells from the embryonic sourced hES cells, they are termed induced pluripotent stem (iPS) cells. Validating and optimizing the reprogramming method would prove very useful for the generation of individual cell lines from many different patients to study the nature and complexity of disease. In addition, the problems of immune rejection for future therapeutic applications of this work will be greatly relieved by being able to generate reprogrammed cells from individual patients. We have initiated a series of studies to reprogram human and mouse fibroblasts to iPS cells using the genes that have already been suggested. While induction of these genes in various combinations have been reported to reprogram human cells, we plan to optimize conditions for generating iPS cells using methods that can control the level of the “reprogramming” genes, and also can be used to excise the inducing genes once reprogramming is complete; thus avoiding unanticipated effects on the iPS cells. Once we have optimized the methods of inducing human iPS cells from human fibroblasts, we will make iPS cells from patients with 2 different neurological diseases. We will then coax these iPS cells into specific types of neurons using methods pioneered and established in our lab to explore the biological processes that lead to these neurological diseases. Once we generate these cell based models of neural diseases, we can use these cells to screen for drugs that block the progress, or reverse the detrimental effects of neural degeneration. Additionally, we will use the reprogramming technique to study models of human blood and liver disease. In these cases, genetically healthy skin cells will be reprogrammed to iPS cells, followed by introduction of the deficient gene and then coaxed to differentiate into therapeutic cell types to be used in transplantation studies in animal models of these diseases. The ability of the reprogrammed cell types to rescue the disease state will serve as a proof of principle for therapeutic grafting in treating human disease.

Statement of Benefit to California (provided by applicant)

It has been close to a decade since the culture of human embryonic stem (hES) cells was first established. To this day there are still a fairly limited number of stem cell lines that are available for study due in part to historic federal funding restrictions and the challenges associated with deriving hES cell lines from human female egg cells or discarded embryos. In this proposal we aim to advance the revolutionary new reprogramming technique for generating new stem cell lines from adult cells, thus avoiding the technical and ethical challenges associated with the use of human eggs or embryos, and creating the tools and environment to generate the much needed next generation of human stem cell lines. Stem cells offer a great potential to treat a vast array of diseases that affect the citizens of our state. The establishment of these reprogramming techniques will enable the development of cellular models of human disease via the creation of new cell lines with genetic predisposition for specific diseases. Our proposal aims to establish cellular models of two specific neurological diseases, as well as developing methods for studying blood and liver disorders that can be alleviated by stem cell therapies. California has thrived as a state with a diverse population, but the stem cell lines currently available represent a very limited genetic diversity. In order to understand the variation in response to therapeutics, we need to generate cell lines that match the rich genetic diversity of our state. The generation of disease-specific and genetically diverse stem cell lines will represent great potential not only for CA health care patients but also for our state’s pharmaceutical and biotechnology industries in terms of improved models for drug discovery and toxicological testing. California is a strong leader in clinical research developments. To maintain this position we need to be able to create stem cell lines that are specific to individual patients to overcome the challenges of immune rejection and create safe and effective transplantation therapies. Our proposal advances the very technology needed to address these issues. As a further benefit to California stem cell researchers, we will be making available the new stem cell lines created by our work.

Review

This is a proposal to improve somatic cell reprogramming in order to generate induced pluripotent stem cells (iPS cells) for use in cell-based disease models. In Aim 1, the applicant proposes to test both inducible vectors and a system that will allow excision of genes after they have induced reprogramming. Successful methods will be used in Aim 2 to generate disease-specific iPS cells in order to study the biological basis of Rett syndrome and amyotrophic lateral sclerosis (ALS). Finally, the applicant proposes to induce iPS cells into hematopoietic or hepatic lineages in Aim 3, in order to explore therapy of blood and liver diseases.

This is an application from a highly accomplished investigator who proposes to collaborate with a second outstanding scientist to improve somatic cell reprogramming. The inducible method described is novel and, according to the preliminary data described, has already been accomplished. The application provides good preliminary data, and the in vitro model of Rett syndrome would be a great asset to understanding this complex disorder. However, reviewers commented that the application was overly ambitious and unfocused. They also commented that the proposal itself was not well-crafted and Aim 3 was only superficially thought-out.

A major strength of this proposal is that the two groups involved in the experiments are recognized as leaders in the field and have the experience and the technical know-how to carry out the work described. The proposed method for generating iPS cells is novel and would be a significant advance in iPS cell technology. The cell lines to be generated through reprogramming will be appropriately tested for their pluripotency. Each part of Specific Aim 1, if accomplished, would advance the field.

In Specific Aim 2, the major advance would be in the development of an in vitro system of the complex disorder Rett syndrome. This would be significant and novel, and the applicant is an expert in many aspects of the investigation as proposed. It will be important to determine whether the Rett syndrome results obtained in mice are repeatable in human iPS cells. The contribution of the ALS work is less clear, and although the applicant does not cite them, there are recent publications of similar results in a mouse model of ALS. Reviewers were unclear that anything new would be gained from the proposed ALS experiments.

In Aim 3, the applicant moves away from neuroscience and proposes to investigate blood disorders and hemophilia, moving into mouse models for cell therapy. The applicant does not have much expertise in this area. Reviewers commented, for instance, that Factor VIII is not produced by hepatocytes as stated by the applicant but mostly in large vessel endothelial cells in the liver. This aim was a distraction and suggested a lack of focus.

During programmatic review, reviewers discussed this application’s principal investigator, the potential importance of the new techniques for generating iPS cells, and the fact that the proposal addresses an important disorder (Rett syndrome). Overall, however, reviewers remained concerned at the lack of focus of the proposal and therefore did not make a motion to move it into the funding category.

The following Working Group members had a conflict of interest with this application and were therefore recused from participating in review of, discussion of, and voting on the application:

• Cowan, Chad
• Minger, Stephen