Generation and comprehensive characterization of induced pluripotent stem cells from somatic adult cells, without the use of viral vectors.
Early Translational I
$2 499 977
In 2007, there was great excitement in both the general and scientific communities when it was announced that it had been discovered how to "reprogram" adult cells into a state almost indistinguishable from true embryonic stem cells. These cells were named induced pluripotent stem cells, or iPS. The public was appropriately excited about the possibility of retaining all the potentials benefits of embryonic stem cells, without the need for destruction of embryos. The scientific world immediately saw the possibilities of iPS including the generation of patient-specific and disease-specific pluripotent cell lines, and the possibility of deriving non-immunogenic ("self") replacement cells for diseased and degenerative post-natal organs. The original iPS were produced by using a virus to introduce the necessary four genes into cells to reprogram them. However, cells changed in this way could never be re-introduced into a human because it is not safe to do so. Another method for introducing the effect of these genes must be found as a pre-requisite to their clinical use. The current proposal is an idea which may be an effective solution to this bottleneck in the path of iPS from the science laboratory to a potential human treatment. Instead of using virus vectors to introduce the genes, the necessary factors are engineered so that part of the protein is recognized by the cells and carried to the nucleus. In this way, the factors can exert their effect, without causing permanent changes to the DNA of the cell (as the viral method does). If this proposed technique is successful, there implications are very large. The ability to safely reprogram adult cells into a "stem cell" state has the potential to transform transplantation medicine, by supplying a readily available source of "self" cells that may be potentially used as replacement cells for diseased or degenerative organs.
Statement of Benefit to California:
Pluripotent stem cells have the potential to transform medicine, because of the possbilit to regenerate diseased or degenerative organs. This is because pluripotent cells retain the capability to become any type of specialized adult cell (for example nerve cell, muscle cell, skin cell etc), through a process known as differentiation. Until recently, the only source of such cells was a human embryo, but very recently it was discovered how to induce normal adult cells to return to this state - so called "induced pluripotent stem cells". The current proposal describes a plausible variation on the method to produce induced pluripotent stem cells that makes them much safer for eventual therapeutic use. (In brief, the new method dispenses with the need for laboratory viruses to deliver the genes into the cell necessary to generate the induced pluripotent stem cells). Potentially such pluripotent cells might be used to derive cells for the treatment of progressive and degenerative diseases once thought incurable. For example diseases such as Parkinson's disease and Alzheimer's disease might be alleviated by producing new neurons, type I diabetes by producing new insulin-producing pancreatic cells, or heart failure by producing new cardiac muscle cells, and delivering them into the respective diseased organs. Another enormous potential benefit to the citizens of California, is the possibility of producing disease-specific stem cells. These cells would be a great research tool for investigating causes and treatments of many diseases in the laboratory. In addition to the potential benefits to humankind, any treatments discovered would likely lead to development of new Californian companies and Californian jobs.
This proposal addresses a bottleneck facing the therapeutic potential of induced pluripotent stem cells (iPSC) generated by lentiviral or retroviral transduction of transcription factors. Such cells are unsafe for therapeutic use in humans because of the integration of the vector into the genome. The applicant proposes that adult somatic cells can be transformed into iPSCs by fusing transcription factors to a cell penetration peptide that translocates rapidly and efficiently to the mammalian cell nucleus. In this proposal, the peptide is used to deliver pluripotency inducing proteins to the nucleus. If this approach is successful, the applicant proposes further experiments to determine the minimum number, concentration, and time of exposure to the proteins necessary to induce pluripotency. The applicant then proposes to optimize cardiac differentiation from iPSCs generated by the fusion protein transduction, and the resultant cells will be functionally tested in mouse and large animal models of myocardial infarction. In general, reviewers agreed this proposal is an innovative approach to an important problem; however, for technical reasons and because of extremely rapid developments in this field, this recombinant fusion molecule approach was not considered a promising approach by reviewers. They noted that there has been recent progress in the use of transient, non-integrating transfections as well as the use of small molecules to reprogram somatic cells. On the other hand, although the use of fusion proteins has been widely considered in the literature over the last 10 years, there has been little demonstrated success in translating this idea into widespread, useful protocols. Finally, although many of the factors required for reprogramming are known to be transcription factors, Lin28 at least has not been shown to have transcription factor activity. Lin28 is primarily a cytoplasmic protein that enhances translation of certain mRNAs and can inhibit specific miRNAs, and driving it into the nucleus would at first glance appear to be counter-productive. A combination of fusion proteins and other (such as small molecule) approaches might have resulted in a more convincing proposal. In addition to concerns regarding the potential impact of the technology, reviewers expressed serious concerns regarding the execution of the research and the research team’s experience in the field. The preliminary data supporting the research plan was not strong and suggested that the project was at too early a stage to be considered translational. Significantly, the applicant has not made or tested any of the fusion constructs proposed. The composition of the research team also raised questions regarding the project’s feasibility. The principal applicant is an accomplished cardiologist, but reviewers noted a lack of pluripotent stem cell expertise on the team. The one member with iPSC experience is listed at 1% effort, which is far too little for the scope of this application. One reviewer noted that the applicant did not seem to be well-acquainted with the biology of iPSCs, as the rationale behind coupling Lin28 to the peptide domain for nuclear transport, for instance, was not well developed. There has also been progress recently in determining the number of genes necessary for reprogramming, which was not discussed in the application. Finally, reviewers were not comforted by the high degree of interdependency of the specific aims, as a failure early in this study could have detrimental effects downstream and there was neither consideration of potential pitfalls nor discussion of alternative methods. Both the primary applicant’s and the collaborators’ institution have excellent facilities and reviewers were confident that the team would have access to the necessary equipment. In summary, the goal of this project is to overcome a bottleneck for iPSC-based therapies by utilizing fusion proteins, thereby bypassing viral vectors. While the environment and resources for carrying out the animal model work are outstanding, reviewers expressed serious concerns regarding the project’s feasibility and the team’s stem cell experience, particularly in the face of the lack of robust preliminary data.