Human Leukocyte Antigens (HLA) are proteins that are expressed on the surface of almost all cells in the body. Because HLA sequences are highly variable and each person generally has a different set of HLA gene sequences, these cell surface markers serve as the identifiers of “self” vs. “non-self”. If immune cells in the body encounter foreign cells transplanted from a different individual, in most cases these foreign cells are recognized due to their display of a different “non-self” HLA on their cell surfaces, and attacked by the immune system. However, because it is difficult to obtain donors with precise matches, many patients succumb to their disease while on a waiting list for matched bone marrow or organs. Even one mismatch in HLA can result in immune responses against the transplant graft, making it necessary to administer immunosuppressive drugs for the lifetime of the patient.Initially it was thought that human embryonic stem cell (hESC)-derived cells and tissues might not be attacked by the immune system because these cells do not have much HLA on their surfaces in their primitive state. However, it is now known that once hESC start to develop into mature adult-type cells, they also start to increase their display of HLA, marking them as foreign “non-self” transplants. Thus, for hESC-derived cell and tissue transplants face the same problem of immune rejection as adult organ transplants.Gene therapy is a promising new treatment approach that involves the delivery of genetic material such as DNA or RNA directly into cells, thus altering their genetic configuration and “re-programming” them to change the pattern of cellular protein expression. Long-term genetic re-programming can be efficiently achieved with the use of certain types of virus, chiefly retroviruses, which insert themselves directly into the chromosomes of the infected cell, becoming a permanent part of that cell’s genome. Lentiviruses are a type of retrovirus which includes pathogens such as HIV, but as gene delivery vehicles (“vectors”), they have been completely disabled by removal of the viral genes, and replacing them with the therapeutic sequences we want them to deliver, thus turning viral foes into friends. We propose to use this approach to deliver a newly discovered class of “small interfering RNA” (siRNA) that can be designed to target and down-regulate specific sequences in the cell, thereby silencing expression of specific genes such as HLA, without affecting other cellular proteins. Since the genetically re-programmed hESC-derived transplants will no longer display their own HLA due to siRNA-mediated silencing, this gene therapy approach may make it possible to create “universal” donor cells by erasing the HLA identifiers completely, or at least may expand the usefulness of existing hESC-derived donor cells and tissues by nullifying certain subsets of HLA sequences and thus making it easier to find matches with the remaining HLA sequences.
Statement of Benefit to California:
This project proposes to develop novel strategies and technologies that will reduce the likelihood that human embryonic stem cell (hESC)-derived cell and tissue transplants might be rejected by the patient’s immune system, by eliminating the cell surface identifiers (Human Leukocyte Antigens (HLA)) that allow the immune system to discriminate between “self” vs. “non-self”. By allowing the hESC-derived transplants to survive without rejection for longer periods of time, these strategies will improve the effectiveness of regenerative medicine. In fact, immune rejection due to recognition of “non-self” HLA is the same problem that is encountered in the field of adult tissue and organ transplantation of bone marrow, kidneys, etc., and so if the proposed strategies prove successful, there is potential to benefit not only patients who need hESC-derived cells and tissues, but patients waiting to be HLA-matched for conventional adult organ transplants as well. The need suitable HLA-matches between the donor and recipient greatly limits the availability of transplantable organs and tissues, therefore our proposed strategy has the potential to greatly increase the accessibility of transplantation-based treatments, thereby improving healthcare for the Californian population.Since even one mismatch in HLA can result in immune responses against the transplant graft, making it necessary to administer immunosuppressive drugs for the lifetime of the patient, the development of strategies to nullify HLA altogether would greatly reduce morbidity and mortality due to the side effects of post-transplant immunosuppression. These side effects include increased susceptibility to infections and malignancies, therefore reducing the incidence of these adverse events would also improve the health and productivity of Californians, and reduce health care costs. Furthermore, for certain transplant patients on the waiting list, currently it is often difficult to obtain donors with precise HLA matches due to ethnic differences in HLA types. Therefore, development of these novel methods for nullifying HLA identifiers on transplanted cells would eliminate otherwise unavoidable racial or ethnic differences in availability of life-saving treatments, and thus has the potential to improve the health of underserved populations in California.
SYNOPSIS: The applicant proposes to genetically modify hESC to down regulate Class I HLA to achieve immunological evasion without the need for severe immunosuppression treatments. Aim 1 proposes to test lentiviral-mediated transduction of hESC with siRNA targeting all or specific Class I HLA alleles. Aim 2 proposes to characterize the level of HLA inhibition upon differentiation of siRNA transduced hESC into hematopoietic lineages. Aim 3 proposes to establish a fully reconstituted SCID-hu mouse model for in vivo testing of strategies to prevent allograft rejection of hESC-derived cell transplants. INNOVATION AND SIGNIFICANCE: This is a very innovative and potentially highly significant study to use siRNA methods to inhibit Class 1 HLA expression in human ESC-derived cells and to test the immune response to these cells in vitro and in vivo. In general, these studies are well conceived and have a good chance at yielding important and significant results. STRENGTHS: This proposal demonstrates that the PI is well versed on multiple fields, including siRNA knockdown methodologies, human ESC, and immunology-based studies. In general, this is a coherent set of experiments that does a good job of fitting the model for these SEED grants. The aims to first test lentivirus-mediated knockdown of Class 1 HLA is appropriate and the scope of the siRNAs to be tested using either antigen-specific or pan-specific siRNAs provides a good chance of success. The second aim to use hematopoietic differentiation should be achievable and the in vitro immune testing using pre-screened T-cell lines and NK cells is reasonable. The third aim to test in vivo response is more exploratory but a logical follow-up to the other studies. There is a good discussion of potential problems and proposed solutions. There is strong preliminary data, including design of the lentiviral vectors and the effective use of this siRNA method in 293 cells. WEAKNESSES: More information about the known Class 1 HLA expression on the H1 cell line could be provided; it is unclear whether these cells are HLA-A2 positive. The PI states that they will get the H1 cells from Dr. Zach, their collaborator. As the PI is an independent investigator, she should make arrangements to obtain cells independently. The studies proposed in aim 3 using the in vivo model, in collaboration with Dr. Zach, may have a hard time demonstrating effective immune response against these cell lines as T-cell or other immune cell engraftment of these mice is limited, though important other information can be obtained. DISCUSSION: The PI has extensive experience with siRNA work and presents a sound and well-ordered experimental strategy. The work is novel because although several groups are using siRNAs in ESC work, but not many are doing immune suppression studies.