New Cell Lines
$1 737 720
Recommended if funds allow
Statement of Benefit to California:
Executive Summary 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. Reviewer One Comments Significance: Methods to remove integrating genes in iPS cells are clearly required, and new technologies to do this need to be developed. The proposed use of 1) self deleting viruses and 2) a single virus with all pluripotency genes is a good idea that would, if it worked, have a significant impact on the field. Equally, the use of iPS cell derived hepatocytes or HSCs represent an attractive way to treat hemophilia or diseases such as SCID respectively. Feasibility/Research Plan. The strengths of this application are the PIs, the novelty and the significance. There are however a number of weaknesses The plan is very poorly focused – to examine 4 diseases is unrealistic and is accompanied by a lack of depth in the analysis. This superficiality is exemplified by the section on Rett syndrome. The interesting cross-genotype transplant experiments described in the aims are not detailed in the research plan. None of the translational applications are described in enough detail to be confident that progress could be made There is no plan B for the self-deleting viruses should they fail, and no preliminary data to support their use. Preliminary data is provided for Tet-inducible systems, but these are not a major focus of the proposal. In the single virus experiments, how will any necessary changes in the balance of expression be achieved? Responsiveness to RFA: Good Reviewer Two Comments Feasibility: Strengths: 1- The approach. PI will develop two new viral vector systems, much improved than the ones described by Yamanaka and Thomson’s groups. In one system, the authors use tetracycline inducible vectors. Upon the addition of tetracycline MEF and human cells are induced to de-differentiate. This is novel and according with the preliminary data described, has already been accomplished. The second approach will rely on the use of cre-lox system to remove the vectors after they have been used to dedifferentiate cells 2- The principal investigator. Dr Gage has a long record of accomplishments and will certainly produce most of the results proposed. 3- The collaborator. Dr Verma’s laboratory has demonstrated the capacity to built lentiviral vectors of all kinds as well as other viral systems. Weaknesses 1- Aim 2 and 3 are dependent on the success of aim 1. Regardless, the authors have already partially shown that they can generate iPS cells in their own laboratory. 2- Rett syndrome results obtained in mice may not be repeatable in human iPS cells (Aim 2). Unfortunately the text in aim 2 has the wrong numbers on the references. Responsiveness to RFA: Yes Reviewer Three Comments Significance: This proposal has significance to stem cell research in that it will 1) develop new techniques of reprogramming human fibroblasts to iPS cells using conditional vectors that will allow the excision of transgenes involved in the dedifferentiation of the cells. 2) They will generate iPS cells from patients with Rett Syndrome and ALS and then differentiate them into the appropriate neurons and glia. These cells could then be used to model the target disease. If this were accomplished, this would be a significant advance. 3) They will also induce iPS cells into hematopoietic or hepatic lineages to explore therapy of blood and liver diseases. Feasibility: If the work detailed in Specific Aim 1 is successful, this would be a significant advance in the iPS technology. A major strength of this proposal is that the two groups involved are both recognized as leaders in the field and have the experience and the technical know-how to carry it off. The cell lines generated 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 very novel and the PI is an expert at many aspects of the investigation as proposed. In contrast the ALS work is not so novel as two other groups have already published here and identified the potential role of mutant astrocytes in motor neuron death. While iPS cells can potentially be differentiated toward any cell type, I feel that the work described in Specific Aim 3 detracts from the overall focus. In my view it would have been a stronger and more focused proposal if it had been limited to the first two Specific Aims, perhaps choosing another neurodegenerative disease. Also while they state that they have differentiated hES cells to endoderm in the preliminary results, no actual data is shown. I was slightly disappointed with the preparation of this proposal. There were places where data should have been referenced (e.g. p5 – “A mouse model of the disease…”), a number of grammatical and/or spelling mistakes and inadequate figure labeling (Fig. 3). This figure also should really be in the Preliminary Results. To reiterate however, a huge strength of this proposal is the investigators themselves and the institution where they are located. Responsiveness to RFA: Yes on both counts.