ICOC Funds Committed:
Development and differentiation is regulated by spatial and temporal regulation of genes. Genes in the nucleus are found associated with proteins and this is called chromatin, which regulates genes. Genes in stem cells are also regulated by chromatin and the structure of chromatin undergoes changes during differentiation. Understanding the sequence of events that occur in specific chromatin domains during stem cell self-renewal and differentiation becomes vital before we can begin to use these in regenerative medicine. Genetically modifying stem cells may be necessary prior to their use in therapy. The non-pathogenic virus AAV is employed as a vector in numerous gene therapy trials and holds promise for use in modifying stem cells. This virus establishes a latent infection by integrating into a specific region of the human genome called AAVS1. This is in contrast to other viruses used in gene therapy that randomly insert into the genome and thus can be mutagenic. We propose to investigate the chromatin structure at AAVS1 so that AAV based vectors can be used optimally in regenerative medicine. This proposal will improve our toolkit for modifying stem cells using gene therapy. One way to reverse the effects of dysfunctional genes is to deliver a corrected copy to the affected individual. By virtue of their ability to propagate indefinitely, stem cells offer an unlimited supply of healthy genes but undifferentiated stem cells transplanted into patients give rise to problems. These problems can potentially be circumvented by genetically manipulating stem cells in vitro to direct their differentiation into the lineage of choice prior to transplantation but will necessitate integrating transgenes into these cells. The proposed experiments will allow us to better genetically modify stem cells. The experiments outlined in this proposal will characterize the chromatin domains around the AAVS1 region in depth. We will determine how the AAVS1 genomic locus changes with respect to its chromatin structure as stem cells undergo differentiation into specific lineages. Furthermore, we will establish the chromatin determinants that (i) promote the stable integration of AAV into a specific region of the genome and (ii) allow stable expression of transgenes in stem cells. As our long-term goal we will study the changes that occur in the chromatin structure of the AAVS1 region in stem cells expressing an AAV-mediated transgene that induces these cells to differentiate along a specific lineage. These studies will enable the development of vectors for the expression of specific transgenes in stem cells that will direct their differentiation into specific cell types. Such a system could then be exploited to generate large cell banks with diverse histocompatibilities for use in patients with hereditary disorders.
Statement of Benefit to California:
This proposal seeks to combine the potential of two of the most promising approaches in modern medicine: stem cell and gene therapy. Over 1800 genes have been determined to cause hereditary disorders and the most obvious way to reverse the effects of such dysfunctional genes is to deliver a corrected copy to the affected individual. By virtue of their ability to propagate indefinitely, stem cells offer an unlimited supply of healthy genes. However, when undifferentiated embryonic stem cells are transplanted into the patient they have the potential to form teratomas while adult stem cells can potentially give rise to tissues that are not desirable at the site of transplantation. These problems can potentially be circumvented by genetically manipulating stem cells in vitro to direct and control their differentiation into the lineage of choice prior to transplantation. In the future one can envision CA-based large therapeutic cell bank repositories of different lineages and immune characteristics that would enable physicians to find immunologically compatible cells for corrective cell therapy. Results from experiments in this proposal will allow the stable expression of proteins and growth factors that can direct stem cell differentiation without being subjected to position effects resulting from random integrations and can therefore be utilized for generating cell banks. A second application for the proposed research is in gene transfer therapy where stem cells derived from the patient are corrected for the defective gene, expanded, characterized and allowed to differentiate prior to re-transplantation into that patient thus avoiding immune rejection. Although this approach requires heavy logistics and might be limited to small numbers of patients, therapies such as these could be developed from the proposed research and will have the advantage that the integrated genes will not be subject to variations in expression by gene silencing and additionally will avoid the problems of histocompatibility mismatches and immune rejection. Knowledge from this research will also spur growth in new biotechnology firms to develop gene delivery vectors in stem cells thus offering a direct advantage to the state in terms of revenue and employment opportunities. This research will also put the state of California at the forefront of stem cell technology along with other nations.