Stem cells, like all transplants not derived from an identical twin, are subject to scrutiny by the immune system and, without medical interventions that suppress the immune system, are usually killed after transplantation. However, rare exceptions to this rule exist because a small fraction of transplant patients has been able to maintain their transplant in the absence of immunosuppressive drug therapy and developed "operational tolerance" towards the foreign graft. Our team has extensively studied these patients and identified a number of genes that are characteristically overexpressed or silenced in these patients. Other instances of tolerance towards foreign cells also occur naturally, e.g. during pregnancy. While numerous genes that correlate with operational tolerance are known, it is less clear whether they actively contribute to tolerance and how they compare in their effectiveness. We will therefore transfer this collection of genes, one-by-one or as combinations, into mouse embryonic stem cells using gene therapy methods and identify those genes that can best protect the cells from rejection by the immune system.
To accurately monitor the survival of transplanted stem cells in mice over time we will use in vivo bioluminescence imaging (BLI). For this, we label the stem cells with luciferase, a protein from the firefly that emits light. These bioluminescent stem cells are transplanted into recipient mice (whose background luminescence is negligible) where the cells can be repeatedly and non-invasively visualized with a highly sensitive camera system. Thus, the cellular survival, growth and migration can be assessed over time and under various conditions, and we can determine whether the introduced genes affect the survival of stem cell transplants positively or negatively, and how they compare and, hopefully, cooperate.
Our preliminary data show that we can detect differences in survival of such engineered cells. This indicates that the proposed studies will succeed in prolonging the survival of mouse stem cell transplants, and that these studies are greatly accelerated by the use of BLI and of gene transfer methods developed over the past several years in our and other laboratories. The potential impact of this proposal is substantial, in that successful completion of the specific aims will both be an important step towards tissue replacement and regeneration using stem cells, and the first demonstration of a multiplexed gene screen in mice. If the genes found to modulate the immune system locally and to protect stem cell transplants in mice can be translated to the bedside, e.g. developed into small molecule drugs that are safe to administer, there is promise that we can reduce the untoward effects of systemically delivered drugs, and extend the lifespan of stem cell and organ transplants without the need for chronic immunosuppression. This would have a substantial impact of the management of a variety of medical conditions.
The California Institute of Regenerative Medicine is seeking to discover new therapeutic approaches that use stem cells for a wide range of diseases and to critically evaluate these for the citizens of California. Unfortunately, however, transplanted stem cells derived from genetically unrelated donors are recognized as foreign by the recipient's immune system and usually destroyed within a month. Currently, the only treatment option for stem cell and organ transplants consists of immunosuppressive drug therapy that is costly and due to its systemic and nonspecific effects carries substantial risks of infection and cancer.
Tolerance to foreign tissues, nevertheless, can develop naturally, for example in women during pregnancy where the partially mismatched fetus is tolerated, and in rare patients who are fortunate enough to maintain their mismatched grafts in the absence of immunosuppressive drug therapy. It is from these instances, which our group has studied extensively, that we will take our clues to develop a comprehensive understanding of transplant tolerance and its genetic basis. Because without this and without an effective means of transferring this tolerance to stem cells, the tremendous potential of the new stem cell therapies may not be realized. Even autologous stem cell therapies, in which patients receive induced pluripotent stem cells (iPSCs), which are derived from their own cells and therefore not mismatched, if they were to succeed otherwise, will not benefit patients with autoimmune disorders, like type 1 diabetes, who will still react to and destroy any transplanted tissue. Therefore, alternatives that modulate the immune response at the site of the engrafted cells or tissues are clearly needed.
Our plan is to select out of the mouse genome the quintessential set of genes whose up- or down-regulation is necessary and sufficient for the long-term protection of stem cell transplants in genetically mismatched mice. In this innovative and comprehensive genetic approach, we will modify the expression of individual genes or groups of genes in mouse stem cells, and observe the cells’ fate after transplantation with powerful imaging technologies that are non-invasive and highly sensitive. Thus, the effect of each gene on the survival of a stem cell transplant can be easily measured and comparatively evaluated, as well as each gene be examined for cooperativity with other genes in the induction of tolerance.
Acquiring this genetic knowledge and translating it into effective stem cell therapies for human patients will be the critical steps in a continuum of research that will clearly benefit citizens in California and elsewhere. Because making stem cell transplantation more efficient and better tolerated will not only advance the fields of stem cell biology and medicine but also that of organ transplantation in general so that less suffering and costs will be incurred in the future in terms of lost lives and funds.
The goal of this proposal is to define a set of immunoregulatory genes that, when their function is modulated in stem cells, can prevent allogeneic rejection. Investigators will perform a series of in vivo functional genomic screens utilizing graft survival as the readout. Genes will be expressed in embryonic stem cells (ESC) using a transposon system and will include a label that enables real time assessment of graft survival in vivo. Cells will be transplanted in allogeneic hosts and assessed for prolonged graft survival. Genetic screens will increase in complexity as the work proceeds from single and combinatorial screens of a targeted series of tolerance-associated genes to single and combinatorial screens of cDNA and shRNA libraries. The group will then assess the effects of their hits and hit combinations upon the behavior and composition of both circulating and graft-infiltrating host immune cells.
Overall, reviewers were enthusiastic about the proposed program They found the proposal’s use of both combinatorial genetics and functional in vivo screening innovative, comprehensive and powerful. If successful, the program could form the foundation of future tolerance inducing strategies. The work should enable identification of genes, and potentially synergistic combinations of genes, that can prolong graft survival. In addition, the work will provide mechanistic insight into how these genes subvert immune rejection. Reviewers did note that embryonic stem cells themselves are unlikely cell therapy candidates and questioned the translatability of these findings to other cell types. However, they also highlighted that achieving tolerance to such a mixed population could identify common mechanisms that would apply to a range of cell types.
Reviewers noted that the scientific literature provides solid support for the proposal’s rationale; both genes expressed in naturally acquired and operational tolerance have been associated with improved graft survival in vivo. Preliminary data suggest that the team possesses both the tools and the expertise required to perform the proposed studies. Furthermore, reviewers appreciated the presentation of potential pitfalls and alternative plans as well as its logical aims. Reviewers suggested that the applicants should give more consideration to the potential effects of insertional mutagenesis and gene expression changes on the behavior of therapeutic cells. The panel also noted that teratoma formation limits the amount of time one can follow acceptance of engrafted ESC clones and should be considered. Reviewers valued the validated discovery approach and absence of bias offered by library screens. However, reviewers agreed this proposal would benefit from a narrower focus, and felt it unlikely all the variables presented could reasonably be screened in vivo given the three-year time frame of the award. Still, they noted that even if only part of the work were performed, the team would likely achieve their goal of identifying good gene candidates to promote graft survival.
The reviewers uniformly praised the PI’s leadership ability and outstanding experience in developing and implementing in vivo imaging technology. The PI will commit 10% effort to the project. The co-investigator has expertise in transplant immunology, operational tolerance and gene expression analysis. Together, the team possesses stem cell biology, immunology, molecular biology and imaging experience that uniquely positions them to successfully execute the proposed studies.
In summary, this program will identify strategies to promote graft survival in allogeneic hosts that could ultimately inform future drug discovery efforts. While the group will likely need to narrow the focus of the work, the project has a high likelihood of success.