Sunday, May 25, 2025
5/25/2025
Alloimmune-compatible stem cells for ocular diseases - SUMMARY: Induced pluripotent stem cells (iPSCs) possess remarkable ability to transform into various cell types found in the eye, making them an exceptional cellular resource for regenerating ocular tissues. However, while the use of autologous iPSCs ensures immune compatibility, it still has no clinical applicability to date, given the time and costs associated with generating patient-specific cells. Hence, there is a critical unmet need to develop universally immune-compatible iPSCs that will eliminate the risk of immune rejection and provide an off- the-shelf source of compatible cells readily available for regenerative applications in various eye conditions. The success of allogeneic cell transplantation is hindered by the expression of the human leukocyte antigen class 1 (HLA-I) molecule present on the surface of all nucleated cells. When cells are transplanted to a host with a mismatched HLA-I, they present antigenic peptides via HLA-I to host CD8+T cells, triggering cytotoxic activity. Conversely, the absence of HLA-I triggers natural killer (NK) cell-mediated cell lysis via the missing-self response. Since cells expressing HLA-I without antigenic peptide or loaded with low-affinity peptide cannot be recognized by cytotoxic CD8+T cells but are still protected from the NK cell response due to the presence of HLA-1, in this project, we aim to develop iPSCs expressing HLA-I molecules which are “peptide-free” or loaded with “low-affinity” peptides. Specifically, we have used the CRISPR/Cas9 system to delete: (i) ERp57 and CALR, which keep the HLA-I molecule in a peptide-receptive state, and (ii) ERAP1 and ERAP2, which trim peptides to fit into the HLA-I groove with high affinity. Our robust preliminary studies demonstrate that these gene-edited iPSCs preserve their pluripotency, differentiate into corneal limbal epithelial stem cells (LESCs) and are protected in vitro from allogeneic immune cell cytotoxicity. We thus hypothesize that iPSCs expressing peptide- free HLA-I or HLA-I with low-affinity peptides are protected from both CD8+T and NK cell-mediated cytotoxicity. We propose 3 specific aims to validate our hypothesis. In Aim 1, we will determine if ERp57-/- and CALR-/- iPSCs express peptide-free HLA-I molecules and escape allogeneic CD8+T and NK cell-mediated cytotoxicity while preserving their pluripotency. In Aim 2, we will determine if ERAP1-/- and ERAP2-/- iPSCs lose their ability to present high-affinity peptides and are protected from allogeneic CD8+T and NK cell-mediated death. In Aim 3, we will evaluate in-vivo the ability of select gene knockout iPSCs and their differentiated LESCs to escape alloimmune rejection in humanized immunocompetent mice. While performing pilot studies, we have generated and standardized the key tools required to successfully complete the proposed study. The proposed project is highly novel and represents a substantial departure from the status quo by shifting the focus from deleting HLA- I or overexpressing non-specific inhibitory factors to a system that fine-tunes HLA-I–peptide complex function to regulate the cytotoxic immunity. This project would pave the way for generating universally immunocompatible, transgene-free iPSCs, enabling widely accessible allogeneic stem cell therapies for various eye conditions.
HHS’ Tracking Accountability in Government Grants System (TAGGS) website provides access to detailed descriptions of grants, loans, aggregated direct payments and other types of financial assistance awarded by the HHS Operating Divisions (OPDIVs) and the Office of the Secretary Staff Divisions (STAFFDIVs).