One of the exciting frontiers of science is the potential to use human embryonic stem (hES) cells to provide relief to patients with terrible diseases like Parkinson’s, spinal chord injury, diabetes, and a host of other congenital, developmental, and degenerative disorders. ES cells have two remarkable traits: 1) the ability to develop into mature cells that can function as neurons, muscles, bones, blood, heart tissue etc., and 2) a limitless self-renewal capacity, thus offering an inexhaustible source of precursor cells. In addition to being clinically important and relevant for cures of diseases, hES cells offer a wonderful opportunity to study molecular mechanisms of differentiation and self-renewal. To correct a genetic defect, one needs to introduce a functional gene into regenerated tissue of the patient. For example, therapeutic cloning from a diabetes patient lacking the insulin gene to regenerate the pancreas will require a functional gene for making insulin to be inserted in the patient. But how will one introduce a gene into a stem cell that divides to produce a large progeny? If the transduced gene is not integrated in the genome, the gene will be lost in the subsequent progeny. Thus, a delivery vehicle that will become part and parcel of the chromosome and transcribe the transgene in the progeny cells as well as in the self-renewal cells is essential. During the last decade, my laboratory has developed HIV based lentiviral vectors (LV) that have the ability to introduce genes in both dividing and nondividing cells. Furthermore, LV can integrate in the chromosome to provide sustained production of the transgene product. In preliminary experiments we can show that genes can be transferred to some hES cells. To be sure that this property is not unique to a few experimental hES cell lines, we need to test them in many different hES cell lines. Therefore it is essential that we have access to additional hES cell lines, most of which are not NIH approved, hence the experiments can be performed only in a special stem cell facility with funds provided by CIRM.
Recently a new technology referred to as RNA interference, for which the 2006 Nobel Prize in Physiology and Medicine was awarded, has emerged which offers new ways to study the mechanisms involved in hES cell differentiation. It appears that small noncoding RNAs (micro RNA) play an important role in differentiation of a variety of cell types, including ES cells. We are planning to use a novel combination of strategies to identify such as micro RNAs, as they will play an important role in understanding the differentiation potential of hES cells.
Finally, in this proposal we will generate a number of tools which will be freely available to scientists working in the field of human stem cell biology.
Statement of Benefit to California:
Regenerative medicine is the next frontier of medicine. It not only has the potential to cure diseases like diabetes and Parkinson’s, but can also have a significant impact on patients suffering from spinal cord injuries, Alzheimer’s, and other neurological disorders. The potential of reducing the burden of incurable and hopeless physical afflictions is rewarding in itself. However, fruits of human stem cell research can create new biotech and instrumentation companies that will have a very positive effect on the economy of the state of California. The work we are proposing will provide some of the seminal tools to achieve success in regenerative medicine.
Two specific aims are proposed: 1) development of regulated Lentiviral vectors and 2) Role of miRNAs in hES cell maintenance and differentiation. The investigator proposes to build on the extensive experience of his lab with lentiviral vectors to determine whether they can be used to achieve regulatable expression of miRNAs directed toward artificial mRNA with the appropriate target sequence. This approach will allow the testing of the vector system in a variety of hESC lines to determine the consistencey of expression. In the second aim, viral proteins which protect against miRNA supression will be expressed in hESC via lentiviral vector mediated gene transfer. Inducible expression will be utilized to turn on and off the miRNA in attempt to correlate expression with specific phenotypic features related to differentiation into specific lineages. Follow-up studies will utilize microarray analysis in an effort to identify specific proteins whose expression is influenced by the miRNA.
SIGNIFICANCE AND INNOVATION: The proposal involves the use of state of the art techniques already being developed in the investigator's laboratory. As such it can be considered an extension of on-going work with a new focus on hESC. Although miRNA is likely to have important roles in hESC proliferation and differentiation, the work proposed is largely exploratory so that the potential significance cannot be judged. If successful, specific hypotheses will emerge that could lead to specific insights in critical asppects of hESC biology.
The proposed experiments will lead to the development of new vectors that will be important for genetic modification of human ES cells, and the experiments will further our understanding of the role that a very important biological pathway, miRNA-mediated gene suppression, plays in ES cell differentiation.
STRENGTHS: The applicants are world leaders in gene therapy and in the development of vectors necessary to modify gene expression.
WEAKNESSES: There are two major weaknesses to this proposal. First Aim 1 is already funded, and experiments are already underway, in NIH-approved human ES cell lines. Since the goal of this aim is vector development I see little justification for repeating these experiments in non-approved ES cell lines. In Aim 2 the applicant will use a viral protein to suppress the RNAi pathway in human ES cells, and examine the effect this has on differentiation. The applicant has no method for determining the specific genes that are affected and thus will be unable to pursue this beyond a purely descriptive level. Overall, there is a lack of experimental detail and sharp focus in the proposed experiments.
DISCUSSION: There was no discussion beyond the reviewers' comments.