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.
SYNOPSIS: These studies propose a detailed analysis of epigenetic modifications in the 20kb region of chromosome 19q which contains the AAVS1 locus. This locus is of significance because at least under some circumstances this is where AAV virus can integrate. The experiments will analyze both histone and non-histone based chromatin modifications in this region, and compare the results with transcriptome data. INNOVATION AND SIGNIFICANCE: The significance of mapping the AAVS1 locus is highly questionable for the stated goals of future genetic modification of hES cells, since the underlying assumption of the investigator that this is a useful locus for targeting transfer of genes into hESC by AAV vectors is flawed. There is some basic significance to the studies, because little information is available that charcterizes epigenetic modifications of any genomic region in hES cells in great depth. Therefore, some useful information may be obtained, particularly if the PI will integrate with proposed studies on transcriptional activity and with larger scale datasets obtained from other studies (e.g., Young and Jaenisch laboratories). Overall the level of innovation is low, and the significance is at least questionable. STRENGTHS: The studies to define the chromatin modifications in the 20kb region of the 19q locus are fairly strightforward, and well within the expertise of the PI's laboratory. Detailed analyses of a single chromosomal region in comparison with transcriptional activity will provide some useful insights. It would have been more relevant if the PI had chosen to apply the detailed technologies to a locus more clearly relevant to hES cells (for example, the Nanog locus). The justification of analyzing the AAVS1 locus is questionable. Of merit are the experiments that will analyze the chromatin in the presence of Rep protein. The PI will transiently espress Rep in hESC. Thus, while the ultimate application of these studies to gene therapy approaches are questionable, documenting chromatin alterations as a function of Rep expression may be more useful. WEAKNESSES: The major problem with this proposal is the choice of the AAVS1 locus on chromosomal 19 for detailed physical and epigenetic analyses and its questionable relevance to hESC biology. There is no reason to suspect that the AAVS1 locus plays an important role in hES cell biology and the relevance of AAV vector integration is questionable. AAV-based vectors do not in general integrate because they do not carry the viral gene required for integration, rep. Mostly they are maintained in an episomal state that is not very well characterized. After strong selection, integration can be observed but there is little, if any, site selectivity. The investigators clearly do not know enough about gene therapy and the fact that AAV vectors behave very differently from the AAV wildtype virus. Nevertheless, AAV vectors have been widely suggested for gene therapy applications, and thus further definition of their integration requirements is warranted. However, at this time these types of studies do not need to be performed in a system as complex as hESC. The authors also have no experience working with ES cells, and it is not clear that they have lined up collaborators to work with them. DISCUSSION: There was no discussion following the reviewers' comments.