Funding opportunities

Creating stem cells for treatment of neurological disease: a novel microRNA-based method for safe, efficient induction of pluripotence in patient-specific cells

Funding Type: 
New Cell Lines
Grant Number: 
Funds requested: 
$1 706 171
Funding Recommendations: 
Not recommended
Grant approved: 
Public Abstract: 
Replacement of degenerated tissues by stem cells is a dream shared by victims of incurable disease, their families, and the scientists who devote their careers to advancing medical research. One of the greatest challenges for developing stem cell replacement therapies is to develop stem cell lines that won't be rejected by the patient's own immune system. The best solution for this problem would be to turn the patient's own cells- skin cells or bone marrow cells, for example- into a more useful cell type that can be used to repair other tissues such as brain, heart, or pancreas. There have been recent advances in several laboratories around the world that indicate that this will be possible. In several laboratories, skin cells have been "reprogrammed" so that they become more like the stem cells that can be turned into many different kinds of tissues. The successful strategy to convert skin cells to stem cells is based on a scientific technique called "phenocopying," in which the molecular composition (proteins, DNA, RNA) of one cell type is changed in specific ways that make it more similar to another cell type. The application of this approach so far has been through genetically engineering the skin cells so that they make a different set of "transcription factors", which are proteins that turn on the production of many other proteins. The transcription factors selected by the researchers come from a list of such proteins that are abundant in human embryonic stem cells. This list was generated in large part by work of our laboratory and our collaborators, and we have far more information about pluripotent cells than is currently available in the scientific literature. Analysis of our database of more than 500 samples of human embryonic stem cells, adult stem cells, and non-stem cells (like skin cells) has given us insight into the inner workings of pluripotent cells. We have discovered that pluripotent cells are unique not only in their transcription factors, but also in other types of molecules that are termed "regulatory molecules" because they control the production of many other molecules. In our search for the ideal way to phenocopy pluripotent cells, we have decided that using a higher level regulatory molecule would be more powerful than using transcription factors. The molecules we decided to use are microRNAs, which are recently discovered small molecules within cells that are powerful regulators of other molecules, including transcription factors. Based on our discovery of a unique set of microRNAs that are characteristic of human ES cells, we propose to reprogram skin cells (and other non-pluripotent cells) by phenocopying the microRNA profile of pluripotent cells. Since these molecules are small, we do not necessarily need to use viruses to introduce them, and we will not require any cancer-causing genes. Thus our approach may make this method safer for patients receiving transplants.
Statement of Benefit to California: 
California has a large and diverse population that poses challenges for the future of medical care. A major goal for California's supporters of stem cell research is development of stem cell-based products that have broad medical use. Stem cells have the potential to improve the health of all Californians, but they are especially important for research and development of treatments for diseases that have no cure. Neurological diseases are especially difficult to treat with classical medicines, and these diseases are increasing in prevalence. This proposed project is designed to convert an individual's skin or bone marrow cells into stem cells. It focuses on three neurological diseases that affect a wide range of people of all ages and ethnicities, and are increasing in the California population: autism, amyotrophic lateral sclerosis (ALS), and Alzheimer's disease (AD). Autism is a severe developmental disorder that is becoming more prevalent in California, affecting 10-12 people in 10,000. There is very little known about the causes of autism, although there is evidence for genetic predisposition, and there is no effective treatment. Stem cells will help researchers understand the causes of autism, and guide development of treatments. ALS, commonly called Lou Gehrig's disease is a progressive degenerative disease of nerve cells that control muscles. For most of the cases there is no known cause, but the some cases appear to be inherited. There is no treatment available for ALS, and life expectancy after onset can be as little as 2- 5 years. Stem cells can be used to screen promising drugs, and for cell therapy to reverse the degeneration. AD is another neurological disease that is increasing exponentially. AD is the most common form of dementia, currently affecting more than half a million Californians. The number of AD cases is expected to triple by 2050, and it is no longer just a disease of the aged. There is a growing number of cases of early-onset AD (occurring before the age of 65). AD, like autism and ALS, is untreatable with conventional drugs. Stem cells can be used to test potential AD drugs, and to deliver supportive molecules to the brain. Development of treatments for these incurable diseases will increase the quality of life for all Californians.
Review Summary: 
Executive Summary This is a proposal to generate induced pluripotent stem (iPS) cell lines using a novel derivation technique. The goal is to alter gene expression profiles and epigenetic states of human fibroblasts by manipulating the expression of human embryonic stem cell (hESC)-specific microRNAs. The proposal has three specific aims. The first is to identify pluripotency-associated microRNAs. The second aim is to test microRNA-mediated induction of pluripotency. The third is to analyze the reprogrammed cells for characteristics of hESCs. It is hypothesized that manipulation of the microRNAs will result in changes at the transcriptional level in differentiated cells, possibly reprogramming the cells toward pluripotency. The research proposed in this application is as exciting and innovative as it is speculative. Understanding the role of microRNAs during reprogramming is important, and generating iPS cells from diseased differentiated cells using microRNAs could be significant. Since microRNAs are small, it might be possible to deliver them to cells without the use of viral vectors, which would be significant. However, the proposed microRNA approach has not been successfully accomplished even in normal fibroblasts. Important proof of concept studies are missing from the proposal, so reviewers questioned the feasibility of the work. Specific concerns raised by the reviewers included the fact that it is not known how many microRNAs (of the hundreds identified as upregulated in hESC) would be needed to achieve reprogramming, nor at what concentrations they are needed. The applicant claims to have identified a microRNA seed sequence that “dominates in the hESC-upregulated group” of microRNAs, but does not provide enough information for the reviewers to determine whether this will aid in accomplishing the goals of this proposal or narrow down the microRNAs that will be investigated. In addition, the applicant proposes to use lentiviruses to introduce the microRNAs, with all the known problems of a viral delivery system. Although s/he does discuss the possibility of using episomal, non-integrating delivery, there is no evidence that this would lead to sufficiently high expression levels. Also, microRNAs are not very specific and can affect multiple targets, so this approach could have complex effects. These issues of feasibility were not addressed by the preliminary data. Finally, the reprogramming of diseased cells is not even presented on the three year timeline, so it is unclear that disease-specific cell lines would be produced under this grant. The personnel and the principal investigator are highly qualified, and have significant experience with microRNA research. Although overall reviewers found this to be an exciting and innovative proposal, the issues of feasibility and thus potential failure to produce any new cell lines made the work unfundable under this RFA. Reviewer Synopsis This proposal is the fourth grant in my pile targeting induced pluripotent stem (iPS) cells. This application targets the molecular basis of reprogramming somatic cells into ES-like pluripotent cells. The authors’ goal is to alter gene expression and epigenetic state by overexpression of “hESC-specific” microRNAs along with repression of “fibroblast-specific” microRNAs in human fibroblast cells, and thereby generating an iPS cell line with a novel derivation technique. It is hypothesized that such manipulation will result in changes at the transcriptional level in differentiated cells, possibly reprogramming the cell toward pluripotency. Reviewer One Comments Significance: The use of miRNAs to create iPSC from diseased differentiated cells would be very useful, however, this approach has not been successfully accomplished even in normal fibroblasts. While it would be useful to understand the role of miRNAs in hESCs, as a technique to create new cells lines there are currently no clear advantages over using the current transcription factor-based approach. Feasibility: The proposal to alter miRNA expression to reprogram cells has some advantages over the current transcription factor-based approach. For example, altering a single miRNA could potentially alter the expression of 100-1000s of genes. There are also a number of disadvantages to this approach. 1. Unlike the current approach that uses 4, or fewer, transcription factors to reprogram cells it is unknown how many miRNAs would be needed to obtain reprogramming. The PI states that they have identified 250 miRNAs that are “tightly regulated” in hESCs. There is no discussion on how they will choose which of these they will work with. It is currently not possible to alter expression of all 250 in a single cell (nor would you want to). 2. The applicant claims to have identified a miRNA seed sequence that “dominates in the hESC upregulated group” of miRNAs, but does not describe the sequence or how it was identified. There is also no information regarding whether this seed sequence belongs to a single miRNA or a large family. Lacking this information it is impossible to determine if the identification of a putative seed sequence will aid in accomplishing the goals of this proposal or narrow down the miRNAs that will be investigated. 3. The delivery systems that will be used are very similar to the ones currently used for creating iPSC using transcription factors. It is clear that there are huge disadvantages to using viral/integration-based approaches. The PI has also proposed to use transient overexpression but as pointed out in the proposal there are some major drawbacks using this approach. This proposal suffers from a lack of focus. The general idea, to alter expression of miRNAs to aid in the reprogramming of cells, is potentially interesting from the standpoint of miRNA biology but not as a mechanism to create new cells lines. Using miRNAs to reprogram cells has all the drawbacks of the transcription factor approach with additional disadvantages (see above). For the development of new iPS and hES cell lines it is currently not clear why miRNAs should be pursued. Responsiveness to RFA: The successful completion of the aims of this project will yield iPS lines from fibroblast and foreskin. These cell lines are not really “new”, only the way that they will be derived is novel. The PI also would like to create iPS cell lines from patients with autism, ALS and Alzheimer’s disease. However, it is very unclear if this will be feasible since the approach that is proposed has not been successfully demonstrated in normal fibroblasts (the reprogramming of diseased cells is not even on their three year outline). Reviewer Two Comments Significance: Significance and Innovation: Research conducted by this group and others has identified a unique set of micro RNA sequences that are selectively expressed in pluripotent stem cells, and additional micro RNA sequences that are specifically expressed in differentiated cells. The author suggests that they can use the micro RNAs to reprogram somatic cells in a manner similar to that described for iPS lines. The advantage of the micro RNA approach is that the micro RNAs might regulate the expression of a large number of genes including the transcription factors that are normally over-expressed to induce iPS lines. Another advantage could be that since they are small (19-25 nt) it might be possible to deliver them to cells without the use of viral vectors. Additionally if they could be expressed for sufficiently long periods, they might be able to reprogram cells without integrating genetic sequences. The authors propose to identify those micro RNAs enriched in pluripotent cell lines, reintroduce them into somatic cells using a variety of techniques, some integrating for testing purposes and others designed to be more transient, and then score for iPS generation by morphology, gene expression profiling and biological assays. The project is highly innovative. Feasibility: Design and Feasibility of the Research Plan: The PI has significant experience with micro RNA research. The team has apparently identified approximately 250 micro RNA (out of 800 total) that seem to be over-expressed in pluripotent cell lines. Because of the small size they should be relatively easy to introduce into somatic cells. Important proof of concept studies are still missing. The authors need to demonstrate that they can reprogram cells by this technology even if it is with integrating vectors. Even data with one iPS line would encourage considerable support for the application. The authors plan for screening for pluripotency is presented and is probably adequate. It is mentioned, but there is little in the application concerning the generation of specific cell lines from diseased individuals or how these would be used and nothing about moving the technology to a cGMP level. All of these decrease enthusiasm for the project. PI and Personnel: The Pi is Jeanne Loring, Professor and director of the Center for Regenerative Medicine, Scripps Inst. She got her Ph.D. in 1979 from the Univ Oregon, Eugene in developmental biology. She was in different High level scientific positions with biotechnology companies including Gen Pharm, Molecular Dynamics, Incyte. She was Chief Scientific officer and founder for ARG genomics, now Novovell. She was Co-director of the stem cell center Burnham Inst, La Jolla, CA prior to her present position. She is highly qualified. Additional personnel are also highly qualified. Overall Evaluation: The research proposed in this application is as exciting and innovative as it is speculative. Positive results on iPS generation by the techniques they propose would significantly improve the application. Responsiveness to RFA: Adequate Reviewer Three Comments Significance: This proposal is highly innovative, but raises some significant concerns, as detailed below. Feasibility: This proposal has three specific aims. The first is to identify pluripotence-associated microRNAs. The second aim is to test microRNA mediated induction of pluripotence. The third is to analyze the reprogrammed cells for characteristics of human ES cells. Dr. Loring has a good track record of productivity. It is not altogether clear that these aims are feasible, particularly given that it has not been accomplished in normal fibroblasts, much less diseased fibroblasts. This approach also lacks the simplicity of the transcription factor approach, requiring the manipulation of as yet unknown numbers of microRNAs. Dr. Loring is an expert in microRNA research, and her track record of publications is good. The facilities at Scripps Research Institute are an excellent setting for the studies proposed in this grant application. This proposal includes a collaboration, with Dr. Philip Schwartz (Children’s Hospital of Orange Country), who will provide a neural precursor cell line derived from the postmortem brain of a severely autistic child as well as fibroblasts and/or mesenchymal stem cells from other autism patients. Responsiveness to RFA: Given that the proposed iPS lines have not been characterized, it is impossible to ascertain whether they will have the ability to differentiate into all three germ layers. Plans for sharing cell lines and training the recipients is adequate.

© 2013 California Institute for Regenerative Medicine