Funding opportunities

Establishment of Human Embryonic Stem Cells Modeling Human Disease by Somatic Cell Nuclear Transfer

Funding Type: 
New Cell Lines
Grant Number: 
RL1-00659
Funds requested: 
$1 532 277
Funding Recommendations: 
Not recommended
Grant approved: 
No
Public Abstract: 
To date, most basic and applied research on human disease is performed with animal models, the majority being mice. Unfortunately, most of what is learned from mouse models is not associated with human disease. Moreover, even with current advanced genetic technology, it is nearly impossible to generate models of rare diseases such as Amyotrophic Lateral Sclerosis (ALS), also known as Lou Gehrig’s disease. “Therapeutic Cloning” will provide a novel opportunity to study patient specific stem cells allowing one to understand the genetic causes of disease. It is a process known as somatic cell nuclear transfer (SCNT) in which the nucleus of a donor cell is transferred into an egg that has been emptied of its chromosomes. That egg now contains an exact duplicate of the donor’s genome, and if all goes well when it is cultured in incubator for five to seven days, an SCNT-embryo will develop into a stage in the incubator for derivation of embryonic stem cells (ESC) which can differentiate into any cell type a patient may need for possible therapy. However, although the potential of combining SCNT technology with ESC research for the future therapies in animals has been demonstrated, it has not yet been accomplished in humans, although members of our team are closing in on this accomplishment. While we understand and respect those who oppose SCNT-ESC research, we are equally sincere in our belief that the life-and-death medical needs of suffering children and adults might be ameliorated by insights derived from SCNT-ESC. This proposal marks the beginning of the effort to use human embryonic stem cells (hESC) in a series of experiments whose principle has already been proven in animals and even in early stages in human by members of our team. The focus of our research is to gain a better understanding of ALS. By developing ALS hESC lines, particularly recreating the neuronal niche thought to be awry, researchers will have a novel tool for studying the mechanism of disease. Furthermore, this presumed new model for ALS may also be used to test potential therapies; high throughput screening allows the rapid examination of millions of compounds and may be used to examine gene manipulations and it’s impact on disease. Members of our team have recently reported the first successful derivation of SCNT-embryos from cultured adult cells (skin) in the human. Other members have derived new hESC cell lines under very defined conditions and feel poised to accomplish the next step; the derivation of SCNT derived hESC lines. Upon completion of this project, we may be one step closer to understanding and perhaps stopping the progression of this horrific disease.
Statement of Benefit to California: 
The proposed research involves three cutting edge technologies: human embryonic stem cell research, somatic cell nuclear transfer, and patient specific cell lines. These technologies which are complementary to each other, have yet to be developed and optimized in humans, and when combined, will offer great potential in medicine. The ability to stay close to emerging technologies is paramount to keeping California business at the leading technological edge. In addition to the likely economic benefits to the State of such a commercial program, it is critical to point to the paramount medical benefits to the public of such collaboration in terms of the development of milestone medical therapies for future cell therapy. Upon completion of this project, we will be able to prove the concept of SCNT-ESC research as a tool for developing therapies. Stem cells derived from SCNT-embryos will likely be a conduit to drug development, eventually leading to new pharmaceuticals. Furthermore, producing such stem cell lines would establish a novel resource to study the role of individual genes in disease development. With the improvement of cloning methodologies as described in our proposal, SCNT-ES cells could be used as a cellular transplantation resource. Cell therapy technologies overlap with those of gene therapy, cancer vaccines, drug delivery, tissue engineering and regenerative medicine. Collectively, the value of the cell-based markets is estimated to be $26.6 billion in 2005, $56.2 billion in 2010, and $96.3 billion in 2015. The largest expansion will be in diseases of the nervous system and cancer. Skin and soft tissue repair as well as diabetes mellitus will be other major markets. The number of companies involved in cell therapy has increased remarkably during the past few years. In 2006, more than 500 companies have been identified to be involved in cell therapy. Of these, 104 are involved in stem cell therapy. If the proposed research is successful as anticipated in contributing to the development of cell therapy products, there are likely to be substantial benefits to two critical sectors of the California economy, health care and pharmaceuticals. Additionally, our proposed research will benefit through further refinement and expansion of its SCNT-ESC technology and through collaboration in other areas of biotechnology, including molecular biology and immunobiology. Furthermore, the advancement of ESC research together with SCNT technique will enhance partnership-developing programs for start-up as well as established biotechnology companies. Support for this project will help maintain our leadership in embryonic stem cell and cloning technologies and will help a California business to be better equipped to compete for a multi-billion dollar market.
Review Summary: 
Executive Summary This applicant proposes to develop methods to generate human embryonic stem cell (hESC) lines using somatic cell nuclear transfer (SCNT), in order to allow the derivation of disease-specific cell lines. The applicant states that nuclear transfer is the “gold standard” method of reprogramming and will remain that way until proven otherwise. Optimization of SCNT technology to humans is described in Aims 1 and 2. Once SCNT methods are reliable in humans, the applicant proposes to derive hESCs from individuals with known genetic versions of motor neuron disease (amyotrophic lateral sclerosis, ALS), as well as patients with idiopathic motor neuron disease. This is an interesting and highly focused application with two goals 1) to develop SCNT in humans and 2) to study motor neuron disease. The recent publication of the derivation of non-human primate ES cells by SCNT certainly advanced knowledge on ES cell derivation. To be able to accomplish this with human cells would be an important breakthrough. Even if transcription factor-mediated somatic cell reprogramming becomes the technique of choice, a comparison between that method and SCNT-mediated reprogramming may yield useful data for making reprogramming more efficient. In addition, if successful, this application could lead to the generation of hESC encoding important genetic information regarding the pathogenesis of motor neuron disease, which could provide new therapies specific to this disease. In spite of the potential significance of the work, and therefore its potential fundability, reviewers were not supportive of this application due to significant weaknesses in the project and the lack of experience of the principal investigator on the project. Reviewers were very critical of the fact that, if Aim 1 fails to produce blastocysts from SCNT embryos, Aim2 and 3 cannot be performed. Overall, the preliminary data presented was weak and the project was poorly described. Aim 1 is an assembly of different protocols for oocyte activation and delivery of the nucleus into the oocyte, with no sound rationale other than a handful of manuscripts gathered from experience in farm animals. The applicant describes the use of chromatin transfer but there is no explanation of how this is done or reference of work done by the applicant or some of the collaborators. In Aim 2, the applicant is proposing to use growth factors to enhance the development of the SCNT embryos. The applicant proposes to use factors that favor the development of fertilized embryos, but does not take into account the work done by others that demonstrated that SCNT embryos need culture systems that resemble more those from somatic cells rather than embryo culture. The availability and source of oocytes was also of concern. Reviewers were unclear about how many oocytes were needed, how many oocytes would be used in each aim, and how many of these oocytes would be used fresh or be supplied frozen. This information impacts the overall budget of the proposal and the statistical power of the results. Furthermore, one reviewer commented that the applicant did not provide evidence that s/he has access to a supply of oocytes, as the applicant acknowledges this is a limiting factor. Reviewers were also concerned about whether the project was feasible as described. As the applicant points out, the group has considerable experience in the technology to be used and much of the proposed work seems standard. However, although the project lists good and appropriate collaborators, the role of the collaborators is not clear. In addition, the applicant appears to have no experience managing a major project. The applicant also failed to cite key publications on motor-neuron disease, perhaps reflecting lack of understanding/knowledge of the field. Finally, one reviewer noted that although the gene expression and proteomic analyses they plan on neurons derived from ALS patients with familial and sporadic disease might identify significant differences, it could equally yield a long list of gene expression differences that will be hard to decipher. Reviewer One Comments Feasibility: Strengths: - PI has some experience with embryo manipulations and oocyte activation - One of the collaborators recently published the first human embryo produced by SCNT Weaknesses: - Aim one is an assembly of different protocols for oocyte activation and delivery of the nucleus into the oocyte with not sound rationale other than a handful of manuscripts gathered from experience in farm animals. - Aim 1.2 describes the use of chromatin transfer but there is no explanation of how this is done or reference of work done by the PI or some of the collaborators - How many oocytes will be used in each aim? How many will be used fresh vs. frozen? How many are needed? This will impact the overall budget of the proposal and the statistical power of the results. At least the PI should have included the desired number and worked from there. - Aim 2.2. The PI is proposing to use growth factors to enhance the development of the SCNT embryos. It is proposed to use factors that favor fertilized embryos to develop. PI does not take into account the work done by Latham’s group that demonstrated that SCNT embryos need culture systems that resemble more those from somatic cells rather than embryo culture. - If Aim 1 fails to produce blastocysts from SCNT embryos, Aim2 and 3 cannot be performed. Responsiveness to RFA: yes Reviewer Two Comments Significance: The goal behind this proposal is to establish hESC lines from blastocysts using SCNT. The applicant proposes that this method is the “gold standard” and will remain that way until proven otherwise. If reprogramming becomes the technique of choice, a comparison between the two methods may provide useful data in making reprogramming more efficient. The publication of the derivation of non-human primate ES cells by SCNT by Byrne et al (2007) certainly advanced knowledge on ES cell derivation. To be able to accomplish this with human cells would be an important breakthrough. The question however will be whether iPS cell technology really takes over the field, making SCNT redundant. However this remains to be proven and our current understanding of hESCs and their differentiation potential means that iPS cells have to catch up. As the applicant points out, the group here has considerable experience in the technology to be used and much of the proposed work seems standard and not necessarily innovative. Feasibility: Much of this proposal is technically rich from a group of investigators who know what they are doing in cloning and SCNT in rodents and in large animals. They have three Specific Aims which seem well organized and appropriate. It might have been helpful to have a letter of collaboration from an IVF clinic ensuring them of a supply of oocytes as they acknowledge this as a limiting factor. Throughout the Research Design they provide alternative approaches if the initial plan does not work. I could not find any note however of their using any of the techniques used by Byrne et al in their paper – could they not learn something from this study? They have chosen ALS as the disease that they would like to model with their hESCs. The Co-PI, Dr. Snyder has considerable experience with in vitro neural stem cell differentiation. Unless I have missed this, they did not cite the two papers from Nature Neuroscience on mutant astrocytes in the SOD-1 transgenic mice that were derived from ES cells. The co-cultures they note here are similar to the studies in Nature and should have been cited. It might be that the gene expression and proteomic analyses they plan on neurons derived from ALS patients with familial and sporadic disease might identify significant differences or simply a long list of gene expression differences that will be hard to decipher. The preliminary data that they present is dense and somewhat disorganized getting down to the size of figures where over-strength reading glasses are called for! It would have been much better to organize this data according to the Specific Aims. The team is strong and they are technologically accomplished. The PI is a junior Research Assistant Professor. He has some quite significant publications in the field but no funding at present. While they name a number of senior scientists in San Diego and elsewhere who are collaborators, letters of support are missing. I wonder what exactly I. Wilmut will actually do here, apart from providing his name. It also might be noted that I. Wilmut has publicly given up on SCNT as a means of deriving disease-specific hES cells in favor of the iPS approach. Responsiveness to RFA: Yes, they will demonstrate pluripotency of their cells appropriately and share them. Reviewer Three Comments Significance: The work in this application is focused on the generation of motor neuron disease specific cell lines in patients with both genetic and non genetic forms of ALS. If successful, this application could provide human embryonic stem cells encoding important genetic information regarding the pathogenesis of motor neuron disease which could provide new therapies specific to this disease. Feasibility: This is a highly focused proposal specifically targeted on the generation of motor neuron disease specific human embryonic stem cell lines using somatic cell nuclear transfer. The applicants have provided a relatively precise and detailed methodology for the generation and characterization of the resultant cell lines. Moreover, they have also provided detailed methodology and preliminary data regarding differentiation of these cells toward neural lineages. To date no group has successfully generated cloned human embryonic stem cells through SCNT although two of the lead applicants are authors on a recent publication demonstrating successful nuclear transfer and propagation of cloned embryos to the blastocyst stage. Overall the applicants would appear to have the necessary research expertise in the various required aspects of the project including cloning technology, neurobiology, and human embryology. However the principal applicant is a very junior research scientist, currently holding a research assistant professor at the Burnham with no demonstrated ability to manage a project of this size. It is very unclear why he is leading on this project. Another criticism is that the role of Professor Yang and Professor Wilmut in this application are ill-defined. Responsiveness to RFA: Assuming that the applicants can successfully generate expandable populations of cells derived from patients with motor neuron disease, the characterization and differentiation of these cells as outlined in the application is appropriate. Moreover the applicants have considerable expertise in neural differentiation of human embryonic stem cells, as well as other non-neural populations. Unfortunately in this application there does not appear to be any consideration of how these cell lines will be provided for use by other research groups and/or commercialized, with the exception of a single line that says “All reagents and products will be made available to research groups cost free.”
Conflicts: 

© 2013 California Institute for Regenerative Medicine