ABO gene editing for the conversion of blood type A to universal type O in Rh_null donor‐derived human‐induced pluripotent stem cells

Abstract

The limited availability of red cells with extremely rare blood group phenotypes is one of the global challenges in transfusion medicine that has prompted the search for alternative self‐renewable pluripotent cell sources for the in vitro generation of red cells with rare blood group types. One such phenotype is the Rh null , which lacks all the Rh antigens on the red cell membrane and represents one of the rarest blood types in the world with only a few active blood donors available worldwide. Rh null red cells are critical for the transfusion of immunized patients carrying the same phenotype, besides its utility in the diagnosis of Rh alloimmunization when a high‐prevalence Rh specificity is suspected in a patient or a pregnant woman. In both scenarios, the potential use of human‐induced pluripotent stem cell (hiPSC)‐derived Rh null red cells is also dependent on ABO compatibility. Here, we present a CRISPR/Cas9‐mediated ABO gene edition strategy for the conversion of blood type A to universal type O, which we have applied to an Rh null donor‐derived hiPSC line, originally carrying blood group A. This work provides a paradigmatic example of an approach potentially applicable to other hiPSC lines derived from rare blood donors not carrying blood type O.

Generation of Rh D‐negative blood using CRISPR/Cas9

“…Later on, new strategies came out as multiple knockouts of rare blood type antigens from BEL‐A or hiPSCs, and RHAG knockout was taken instead of RHD knockout to derive a Rh null phenotype 29,30 . Besides, a Rh null hiPSC line was chosen for the creation of universal RBCs 31 . However, O‐type Rh‐D‐negative RBCs have not yet been successfully identified.…”

“…The CRISPR/Cas9 based RHD knockout could be further improved by deleting exogenous selection markers when the gene editing procedure has been completed 42 . To deplete RHD expression, some recent studies chose RHAG , which is the processor of RHD and RHCE , as target gene, or directly took RHD and RHCE double mutant hiPSCs (Rh null ) as seed cells 29–31 . However, the deletion of RHAG or Rh null phenotype would result in membrane deformability and gas transport function defection, subsequently haematological abnormal 26,43,44 .…”

“…29,30 Besides, a Rh null hiPSC line was chosen for the creation of universal RBCs. 31 The selection of efficient seed cells is one of the key steps in producing RBCs. Although haematopoietic stem cells (HSCs) from cord blood, bone marrow or peripheral blood, pluripotent stem cells (PSCs), including embryonic stem cells and iPSCs, and immortalized erythroid progenitors have all shown the potential to be induced into RBCs in vitro, there are still shortcomings that remain to be overcome before clinical translation.…”

“…(Rh null ) as seed cells. [29][30][31] However, the deletion of RHAG or Rh null phenotype would result in membrane deformability and gas transport function defection, subsequently haematological abnormal. 26,43,44 Considering Rh null phenotype occurs in only 1 from 600,000 peoples, 26 RHD specific knockout might be more applicable in clinical transfusion, meanwhile perform better in functional RBC derivation in vitro.…”

Generation of red blood cells from induced pluripotent stem cells

“…iPSC-derived type O Rh null RBCs and those with other rare antigen phenotypes can be an alternative source of blood for alloimmunized patients. As proof of principle, iPSCs derived from a rare Rh null individual with blood type A were converted to the universal donor type O using a targeted gene editing strategy that reproduced the c.261delG polymorphism present in the inactive ABOÃO.01 allele [34]. This approach can be applied to other iPSCs reprogrammed from somatic cells of individuals with rare red cell antigen phenotypes who are not blood type O.…”