Generation and Use of an Alloantigen-Specific Preclinical Humanized Mouse Model of Fetal and Neonatal Alloimmune...

Fetal/neonatal alloimmune thrombocytopenia (FNAIT) is a life-threatening bleeding disorder caused by maternal antibodies targeting a paternally inherited alloantigen present on the surface of fetal platelets. The human platelet alloantigen HPA-1a/HPA-1b, also known as the Pl^A1/Pl^A2 alloantigen, is the most frequently implicated HPA for causing FNAIT in Caucasians. A single Leu33Pro amino acid polymorphism residing within the plexin/semaphorin/integrin (PSI) domain of the integrin β3 subunit (platelet membrane glycoprotein (GP), GPIIIa) is responsible for generating the HPA-1a and HPA-1b epitopes. In mice, the GPIIIa gene encodes a glutamine at position 33, making it challenging to model the alloimmune response, thereby hindering studies on the underlying mechanisms and therapeutic interventions of this alloimmune disorder. This chapter outlines the development and use of a humanized, alloantigen-specific preclinical mouse model of FNAIT, aiming to advance basic and translational research in this field. Basic protocols are provided for (1) generating an HPA-1a transgenic mouse by introducing humanized residues A₃₀P₃₂L₃₃D₃₉ within the PSI domain and Q₄₇₀ in the EGF1 domain of mouse integrin β3 using CRISPR/Cas9 gene editing technology, (2) developing a preclinical alloantigen-specific humanized mouse model of FNAIT using the HPA-1a transgenic APLDQ mice, and (3) examining the efficacy of a prophylactic treatment regimen in preventing alloimmunization and FNAIT development using this alloantigen-specific mouse model. This humanized mouse model faithfully recapitulates many clinically important aspects of human FNAIT, serving as a valuable tool for studying the pathophysiological mechanisms underlying the alloimmune response and assessing prophylactic and therapeutic interventions in translational research.