title: CRISPR/Cas9-mediated conversion of human platelet alloantigen allotypes.
abstract: Human platelet alloantigens (HPAs) reside on functionally important platelet membrane glycoproteins and are caused by single nucleotide polymorphisms in the genes that encode them. Antibodies that form against HPAs are responsible for several clinically important alloimmune bleeding disorders, including fetal and neonatal alloimmune thrombocytopenia and posttransfusion purpura. The HPA-1a/HPA-1b alloantigen system, also known as the Pl(A1)/Pl(A2) polymorphism, is the most frequently implicated HPA among whites, and a single Leu33Pro amino acid polymorphism within the integrin β3 subunit is responsible for generating the HPA-1a/HPA-1b alloantigenic epitopes. HPA-1b/b platelets, like those bearing other low-frequency platelet-specific alloantigens, are relatively rare in the population and difficult to obtain for purposes of transfusion therapy and diagnostic testing. We used CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats/CRISPR associated protein 9) gene-editing technology to transform Leu33 (+) megakaryocytelike DAMI cells and induced pluripotent stem cells (iPSCs) to the Pro33 allotype. CD41(+) megakaryocyte progenitors derived from these cells expressed the HPA-1b (Pl(A2)) alloantigenic epitope, as reported by diagnostic NciI restriction enzyme digestion, DNA sequencing, and western blot analysis using HPA-1b-specific human maternal alloantisera. Application of CRISPR/Cas9 technology to genetically edit this and other clinically-important HPAs holds great potential for production of designer platelets for diagnostic, investigative, and, ultimately, therapeutic use.
authors.affiliation: Blood Research Institute, BloodCenter of Wisconsin, Milwaukee, WI;
authors.forename: Nanyan
authors.initials: N
authors.lastname: Zhang
authors.affiliation: Blood Research Institute, BloodCenter of Wisconsin, Milwaukee, WI;
authors.forename: Huiying
authors.initials: H
authors.lastname: Zhi
authors.affiliation: Blood Research Institute, BloodCenter of Wisconsin, Milwaukee, WI;
authors.forename: Brian R
authors.initials: BR
authors.lastname: Curtis
authors.affiliation: Blood Research Institute, BloodCenter of Wisconsin, Milwaukee, WI; Department of Pediatrics, Medical College of Wisconsin, Milwaukee, WI;
authors.forename: Sridhar
authors.initials: S
authors.lastname: Rao
authors.affiliation: Center for Cellular and Molecular Therapeutics.
authors.forename: Chintan
authors.initials: C
authors.lastname: Jobaliya
authors.affiliation: Department of Pediatrics, and.
authors.forename: Mortimer
authors.initials: M
authors.lastname: Poncz
authors.affiliation: Center for Cellular and Molecular Therapeutics, Department of Pathology and Laboratory Medicine, Children's Hospital of Philadelphia, Philadelphia, PA; and.
authors.forename: Deborah L
authors.initials: DL
authors.lastname: French
authors.affiliation: Blood Research Institute, BloodCenter of Wisconsin, Milwaukee, WI; Department of Pharmacology and Department of Cell Biology, Medical College of Wisconsin, Milwaukee, WI.
authors.forename: Peter J
authors.initials: PJ
authors.lastname: Newman
mesh_terms: D016824:Antigens, Human Platelet / Q000235:genetics* / Q000276:immunology; D001483:Base Sequence; D064130:CRISPR-Associated Proteins / Q000235:genetics* / Q000276:immunology*; D002478:Cells, Cultured; D064112:Clustered Regularly Interspaced Short Palindromic Repeats / Q000235:genetics / Q000276:immunology; D000939:Epitopes / Q000235:genetics / Q000276:immunology; D006801:Humans; D039661:Integrin beta3 / Q000235:genetics / Q000276:immunology; D007518:Isoantibodies / Q000235:genetics / Q000276:immunology; D007519:Isoantigens / Q000235:genetics* / Q000276:immunology; D019039:Platelet Glycoprotein GPIIb-IIIa Complex / Q000235:genetics / Q000378:metabolism; D010980:Platelet Membrane Glycoproteins / Q000378:metabolism; D020641:Polymorphism, Single Nucleotide
chemical_list: D016824:Antigens, Human Platelet; D064130:CRISPR-Associated Proteins; D000939:Epitopes; C061248:ITGB3 protein, human; D039661:Integrin beta3; D007518:Isoantibodies; D007519:Isoantigens; D019039:Platelet Glycoprotein GPIIb-IIIa Complex; D010980:Platelet Membrane Glycoproteins; C083451:human platelet antigen 1b