CRISPR/Cas9-mediated conversion of human platelet alloantigen allotypes. Blood 2016 Feb 11;127(6):675-80
Date
12/05/2015Pubmed ID
26634302Pubmed Central ID
PMC4751021DOI
10.1182/blood-2015-10-675751Scopus ID
2-s2.0-84959330331 (requires institutional sign-in at Scopus site) 43 CitationsAbstract
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.
Author List
Zhang N, Zhi H, Curtis BR, Rao S, Jobaliya C, Poncz M, French DL, Newman PJAuthors
Brian Curtis PhD Director in the Platelet & Neutrophil Immunology Laboratory department at BloodCenter of WisconsinPeter J. Newman PhD Professor in the Pharmacology and Toxicology department at Medical College of Wisconsin
Sridhar Rao MD, PhD Associate Professor in the Pediatrics department at Medical College of Wisconsin
MESH terms used to index this publication - Major topics in bold
Antigens, Human PlateletBase Sequence
CRISPR-Associated Proteins
Cells, Cultured
Clustered Regularly Interspaced Short Palindromic Repeats
Epitopes
Humans
Integrin beta3
Isoantibodies
Isoantigens
Platelet Glycoprotein GPIIb-IIIa Complex
Platelet Membrane Glycoproteins
Polymorphism, Single Nucleotide