HLA-A, B and C are polymorphic class I proteins expressed by most nucleated cells that must be matched for histocompatibility. The Beta-2 Microglobulin (B2M) gene encodes a common subunit essential for cell surface expression of all HLA class I heterodimers (the other subunits are the heavy chains for HLA-A, B, C, E, F, or G), so B2M-/- cells are class I-deficient. We edit both B2M genes to create human pluripotent cells that lack polymorphic class I proteins. These editing steps also introduce a Thymidine Kinase (TK) suicide gene to allow for in vivo elimination of transplanted cells.
For some cell types, a lack of class I expression leads to lysis by Natural Killer (NK) cells. To overcome this “missing self” response, we simultaneously knock in the heavy chain of the non-polymorphic HLA-E gene using HLA single chain technology (see below). This provides a class I-positive signal to inhibit NK cells. These class I-engineered stem cells can serve as universal donor cells in applications where the differentiated cell product does not express HLA class II.