SYNOPSIS: This proposal aims to test human Embryonic Stem Cells (hESCs) for their efficacy in treating Recessive Dystrophic Epidermolysis Bullosa (RDEB), an incurable blistering disease that results from a lack of collagen type VII (C7) in the skin. hESC-derived progenitor cells will be obtained from Advanced Cell Technologies (ACT) and transduced with lentivectors to drive C7 expression. Cells will be studied in vitro for their ability to incorporate into human skin cultures and in vivo, using a C7 deficient mouse model, or a xenogeneic model in which human skin (normal or RDEB) is grafted onto immunodeficient mice. IMPACT & SIGNIFICANCE: RDEB is an incurable and fatal disease resulting from a known genetic lesion. This is a rare skin disease that has not received the attention of pharmaceutical companies because the market is small. Literature indicates prevalence rates of ~ 10 to 20 patients per million and the prevalence of epidermolyis bullosa total cases in Japan in one report was only 393 patients. These data give a rough estimate of the number of patients that could benefit from such therapy in California. RDEB is well characterized clinically and pathophysiologically and is due to a defect in type VII collagen and results in blistering wounds in the skin and esophagus which heal by scarring. There is also potential for squamous cell cancer developing at a young age. Success of this project would obviously be significant for RDEB patients, and also could provide a novel approach to treating other chronic skin disorders. The therapies that are being investigated in this project may also be applicable to chronic non-healing skin wounds which are more prevalent and related to venous insufficiency, diabetes, and other causes. There are currently other therapies that have been developed over the years which are used clinically for chronic non-healing skin wounds, including engineered composite tissue grafts. It is important to note that the pathophysiology is different for chronic wounds and RDEB. While this proposal has the specific goal of treating a rare genetic skin with hESC-derived dermal progenitors that have been genetically modified to over-express the missing collagen, a possible extension may be a similar application for chronic wounding for the elderly or diabetics. A more general goal is to test the use of hESC-derived cells as a carrier for gene therapy. Both the specific and general goals would have significant impact. The use of the hESC-derived progenitor cells from ACT is somewhat innovative, but these cells are not adequately described to enable evaluation of their likely relevance to this problem. QUALITY OF THE RESEARCH PLAN: This is a collaborative research plan that aims at developing therapies for a currently incurable skin disease. RDEB patients lack type VII collagen (C7) and as a result develop chronic skin blistering that eventually leads to aggressive, fatal carcinoma. The applicant's previous work in a C7 deficient mouse model of RDEB, and in mouse chimeras harboring human RDEB skin grafts, indicates that provision of C7 via direct intradermal injection or systemic means corrects the blistering phenotype because the C7 can "spontaneously" incorporate in the skin basement membrane. In previous studies, C7 has been delivered by gene-corrected keratinocytes or fibroblasts, which "home" to wounded skin. This proposal will test whether hESCs or their derivatives may be more effective in restoring C7 to RDEB skin, although the rationale for the use of hESCs and the choice of hESC-derived cells is not clearly explained. First, the applicant will generate C7-expressing lentiviral vectors and use these to transduce hESC-derived cells. The hESC-derived cells will be generated by Dr. West at Advanced Cell Technologies. While these are referred to as "dermal progenitor cells", little information is provided about these cells - how were they generated? What is their lifespan? Why were these chosen in particular? etc. In addition, it is unclear what impact C7 over-expression will have on the function and survival of these cells. To assay the therapeutic potential of these cells, the applicant will test their incorporation into in vitro human skin cultures provided by Xgene Corporation, and will assay their ability to hone to human skin after intravenous injection into immunodeficient mice harboring normal or RDEB skin grafts. For the in vivo studies, the cells will be tracked by GFP labeling (via viral transduction). Several different skin graft models will be tested, and tissue will be harvested at short and long timepoints for overall histological evaluation and analysis of GFP+ cell identity. Finally, C7 expressing progenitor cells will be injected into C7-deficient mice to analyze their ability to correct the blistering phenotype and prolong survival. No mention is made of potential problems with immune rejection of the human cells - will the mice be crossed to nu/nu or treated with immunosuppressants? A reviewer considers the major concerns with this study are that the cells the applicant proposes to test in skin repair models are very poorly characterized, and the experiments proposed are not described clearly enough. The application would be greatly improved by the provision of additional, relevant preliminary data about these cells and by reducing the scope of the study to focus on those that demonstrate promise in preliminary studies. A reviewer points out that this proposal builds on the longstanding expertise of the PI in studying experimental therapies for this skin disease. S/he has put together a synergistic group of collaborators which provides a unique opportunity to test the main hypothesis in this proposal, i.e. that hESC-derived skin progenitors can be administered intravenously to correct the gene defect in RDEB. This is a well-written and well-integrated proposal that has important strengths but also several weaknesses. Overall the research plan is carefully laid out and includes preliminary data on the systems used. The research team is a collaboration between two academic scientists with excellent track records in the field, a company with which they have collaborated on human skin equivalents, and a third stem cell company (ACT) that has numerous well-defined subclones of hESC-derived progenitors. However, concern is caused by several important underlying questions: Do the hESC-derived dermal progenitors maintain their stemness/proliferative capacity once in skin tissue, becoming (as implied) slowly dividing stem cells? Or, do they differentiate further and become a limited life span transient amplifying population? Are these hESC-derived dermal progenitors really immunologically stealth as implied? None of these questions are even posed. STRENGTHS: Reviewers highlighted several strengths of this proposal. The project focuses on a currently incurable human disease which is also the primary area of interest of the applicant. In addition, the PI has a track record of collaboration with this research team. The team of collaborators are all experts with unique and complementary technologies and expertise. Synergistic expertise in several different technologies that are established in several different laboratories are brought together. Collaborations with Dr. Michael West of Advanced Cell Technology, Dr. Warren Hoeffler of X-Gene Corporation, and Dr. Woodley of USC’s department of dermatology, are organized in a synergistic and unique way providing a beneficial opportunity to study the potential of hESCs for treatment of RDEB. In general, the research plan is well organized and described. Several groups of cell populations including controls are described, and pitfalls and alternative plans are considered. Time course studies are included and multiple independent models are also proposed to be studied. Multiple technologies, models and cell lines are in place to do the proposed studies. Human skin equivalents (HSE), particularly the RDEB HSE model developed by Dr. Warren Hoeffler, are an important strength of the proposal. In addition, the PI has established experience with RDEB animal models and lentiviral vector production. The collaboration with ACT will bring hESC expertise and potentially dermal progenitor cell lines to the proposed studies. In general, the models used (RDEB) mice and their fibroblasts, the human skin equivalents, the lentiviral overexpression of collagen 7) are all well-developed. Another advantage is that the administration of collagen 7 by various means corrects the anchoring of the epidermis to the dermis. The use of GFP marked cells is an important aspect to understanding the mechanisms of the observed effects and for tracking cells. WEAKNESSES: Several weaknesses with the proposal are evident. The rationale for use of hESC-derived cells is unclear. If fibroblasts are so effective (extending lifespan of RDEB model mice from 2 days to 2 months), why not use these? The applicant argues that the fibroblasts are not preferred because they are not maintained, and hESC-derived cells will be longer-lived, but no data is given to support this assertion. They also assert that hESC-derived progenitor cells are less immunogenic than fibroblasts, but again preliminary data is lacking to support this. Proof-of-concept preliminary data is needed to show that hESCs will be longer lived and less immunogenic. In fact, hESCs and their derivatives (dermal progenitor cells) may be more tumorigenic than dermal fibroblasts, a point which is not considered at all in the proposal. Again, proof-of-concept data would provide insurance for a potentially successful outcome. In the absence of these data, the proposal is deemed to be moderately high risk. There is insufficient preliminary data describing the hESC-derived progenitor cell lines identified and isolated by ACT that will be used. The hESC-derived dermal progenitor cell lines are not well characterized. Firstly, it is not proven that these are homogeneous and pure cell populations. Secondly, they have not been characterized for proliferative capacity or developmental potential which are the two hallmarks of progenitor cells. Except for collagen VII expression, and the gene expression profiling at the population level, the cells have not been characterized for gene or protein expression patterns that would be typical of a variety of epidermal and dermal cell types. In addition, the C7 staining and expression pattern is not convincing. Based on the poorly characterized dermal progenitors, the status of each of these six lines that are proposed to be tested is in question and a significant effort should be made to first characterize these lines. There is ambiguity in the research plan. It is not clear which cells will be transduced or at what time, if cells will be pre-selected before assaying, what the impact of C7 over-expression will be on these cells, etc. The applicant provides ample technical detail, but generally does not adequately describe the experimental design and data analysis methods to be used. In the preliminary data, claims are made of improved C7 expression and pathology in RDEB skin, after intravenous injection of C7-modified dermal fibroblasts; however, tracking studies were not performed to demonstrate migration of intravenously administered cells to the skin BMZ layer. Important tracking and mechanistic studies for the effect of intravenously injected cells have not yet been performed in a rigorous way. It is remarkable that there is homogeneous C7 expression in this treatment model. The question arises about if is this dose-dependent, time-dependent, homogeneous expression? Dose dependency studies are not described in the preliminary data nor are they proposed. The data in support of studies proposed on the healing of chronic non-healing skin wounds is weak. There is a modest effect, if one at all, in the human skin equivalent wounding athymic nude mouse model. This minimal effect as shown would probably not be clinically significant. It is not clear why over-expression of C7 would promote healing in this model. In addition, this model may not be predictive of the pathophysiology of chronic non-healing skin wounds as the model uses normal human skin. Important immunological aspects of the study such as the transplantation of human cells into an athymic mouse model where animals retain B cell activity and the possibility of xenograft rejection exists are not considered. In addition, the RDEB C7 knockout mouse model is an immunocompetent model and therefore it is likely that transplantation of human cells will result in immunologic destruction, again, a point that is not considered. The question of whether the hESC-derived progenitors will remain stealth to the immune system is key to the long-term goal but is not addressed. There is comment that in early gestation wound healing is scarless and that these progenitor cells are less immunogenic that post-gestational cells, thus providing the possibility of universal donor cells. If they remain stealth, is there a potential problem with time of tumor formation? Examination of the RDEB mice longer than 18 days after injection may be informative. The question of whether the hESC derived dermal progenitors will maintain their stemness is not examined. If they integrate so well in the skin, one reviewer would expect they would differentiate further rather than “behave like authentic slow cycling skin stem cells to continually generate renewed genetically corrected skin tissue” as described in this proposal. DISCUSSION: As with other applications, there was a question about what the lines from ACT are. The characterization of these lines represents one of the weakest parts of the proposal because while there is transcriptional data, there are no functional data to define the cells. If there are skin progenitors that could be identified from hESC, that would be great for the field. Significant effort should be undertaken at some point to characterize these cell lines; immunocytochemistry, proliferation, makeup, and determination if they give rise to other mesoderm derivations. Preliminary results on hC7 transgenic fibroblasts indicate there is little real benefit from transplanting to diabetic skin lesions (this is considered an extremely weak aspect of study; on the other hand, human skin equivalents are brought together nicely). Also, the GFP-marked cells used for honing is an important aspect of the proposal that was not emphasized. The investigators seem to under-appreciate that they’ll have immune activity against these cells. Reviewers were impressed with the team of collaborators and the longstanding experience of PI in this system; the expertise of the PI in the developed RDEB models makes this group uniquely qualified to do this work. The investigators are working with a nice, well-developed system to test issues, albeit only a small population of people has this disease. Finally, it is not clear that results with mice will translate to humans.