Rarely comes along a new technology that has the potential to make a major impact on human health. The idea that embryonic stem cells (ES cells) have the ability to differentiate into a variety of cell types, tissues, and organs, opens the possibility of tissue engineering, replacement, and even organ transplantation to cure diseases ranging from Parkinson’s, Alzheimer’s, diabetes, thalassemias, and a host of other debilitating disorders. Recently researchers have discovered methods to reprogram adult skin (or other) cells back into a cell that appears indistinguishable from an ES cell. This is accomplished without the need for embryo destruction and offers great potential to alleviate the problems of immune rejection in cell or tissue transplantation by allowing a patient’s own cells to be reprogrammed, expanded then used in therapeutic applications.
A recent research study using a mouse model of the human sickle cell-blood disorder has successfully demonstrated the ability to reprogram an adult skin cell, correct the defective gene, then expand and differentiate these cells back into blood type cells and use them for transplantation to cure the mouse. While this work represents a power proof of concept, it also highlights a barrier to use in human therapies in that nearly a third of the mice treated developed tumors. These tumors likely developed as a result of shortcomings in the nascent technology of reprogramming. In this proposal we aim to make substantial improvements in the core technology for creating reprogrammed cells.
To fully realize the therapeutic potential of reprogrammed cells we must also develop improvements in the technology of targeted gene therapy. The basic concept is disarmingly simple—introduce the gene, and its product should cure a disease, alleviate a defect, or slow down the progression of a disease. Encompassed within this definition are a number of different goals, including the treatment of both inherited and acquired disease. Treatment requires a technology capable of gene transfer in a wide variety of cells, tissues, and whole organs. The therapeutic response may range from a total correction of symptoms to an improvement in the quality of life of a patient. Central to this notion is the ability to transfer genes, but the delivery vehicles needed to ferry genetic material into a cell still represent the “Achilles’ heel” of gene therapy. In this proposal we also aim to make technology developments specifically targeted to performing gene therapy in human reprogrammed cells.
Statement of Benefit to California:
The goal of this proposal is to make technology advancements in human stem cell research that aim to improve the capacity and functionality of these cells in clinical or pre-clinical research settings. The benefit to the people of California will be realized as our technology developments are moved into clinical research where they will provide safer and more efficacious means for treating a wide range of diseases and disorders that affect the citizens of our state. We will be developing technology improvements for reprogramming adult cells back to a cell type that appear to have the full therapeutic potential of embryonic stem cells without having to involve embryo destruction. There are many California researchers who are or will be employing this powerful new technology, and our developments and ready willingness to share them will benefit the strong research community in our state.
The applicants propose to develop a series of technological advances to provide improved human pluripotent stem cells for preclinical and clinical research. First, the applicants address deficiencies in reprogramming technology by developing vectors that achieve both the expression of pluripotency factors and the subsequent removal of those genes. In addition to these vectors, the applicants will investigate the use of mRNA as a means of generating induced pluripotent stem (iPS) cells. Next, a recombinant “kill switch” will be engineered in order to provide a means for eliminating undifferentiated pluripotent stem cells from a differentiated cell population. Finally, a series of integration-deficient lentiviral vectors will be created for more efficient homologous recombination in human fibroblasts and iPS cells.
iPS cells generated by current methods are unsafe as therapeutic agents, largely due to the risks of tumor formation that are specific to these procedures. Moreover, the retention of undifferentiated pluripotent cells within differentiated cell populations poses an additional threat of tumorigenesis. The reviewers agreed that the proposed methodologies would address these roadblocks directly and could therefore move human iPS and embryonic stem cell (ESC) technologies toward therapeutic applications.
In terms of feasibility, the reviewers expressed confidence in the experience and capabilities of the investigators. The proposed experiments were perceived as highly ambitious but achievable. However, reviewers argued that the success of this effort rests upon a number of uncertain assumptions. For example, it is unknown whether the gene copy number or the proposed stoichiometry of reprogramming factors would be sufficient to induce pluripotency. In addition, the utility of the mRNA approach was unclear, as the applicants did not discuss how the appropriate doses would be maintained as cells divide over the course of the experiment. The proposed “kill switch” concept is not novel since others have published it, and the reviewers were uncertain how this proposal would make a new contribution in this regard. Finally, although preliminary data demonstrate the ability of the team to generate mouse iPS cells, evidence of human iPS experience was lacking.
The principal investigator (PI) has world-renowned expertise in retroviral biology and genetic manipulation of mammalian cells. However, the reviewers raised concerns about the applicant’s lack of experience with pluripotent cells, and especially with generation of human iPS cells. In addition, some reviewers questioned whether the PI would be able to commit sufficient time to this effort.
In summary, while the proposed technology addresses key roadblocks in stem cell biology, the reviewers were doubtful of its overall feasibility.