Tissue-Specific Transgene Expression in Induced Pluripotent Stem (iPS) Cell-Derived Megakaryocytes: Correction of Glanzmann Thrombasthenia (GT)

Abstract 387

Megakaryocyte-specific transgene expression in patient-derived iPS cells offers a new approach to study and potentially treat disorders that affect megakaryocytes and platelets. The feasibility of this method was demonstrated in GT, a disorder resulting from an absence of functional platelet integrin alphaIIb/beta3, leading to impaired platelet aggregation and clinically presenting with severe bleeding. While rare, GT can serve as a model of an inherited platelet disorder that would benefit from corrective cell therapy. For this study, iPS cells were generated from the peripheral blood of healthy controls and two patients with GT. Patient 1 is homozygous for a novel alphaIIb exon/intron 4 splice site mutation, leading to an alternatively spliced transcript and premature stop codon. Patient 2 is homozygous for a previously reported alphaIIb missense mutation in exon 8 (p.G273D), leading to impaired intracellular transport and surface expression of the heterodimer. Both patients express <5% alphaIIb/beta3 on the surface of their platelets, which was confirmed in IPS cell-derived megakaryocytes. To achieve megakaryocyte-specific expression, transgene constructs were created using the murine proximal GPIbalpha promoter driving either an eGFP reporter or wild type alphaIIb cDNA. These constructs were inserted into the AAVS1 safe harbor locus using zinc finger nuclease-mediated homologous recombination. When targeted to a single allele of the AAVS1 locus in human ES cells, the GPIbalpha promoter driving eGFP showed a high-level of expression in ES cell-derived megakaryocytes. Inserting the GPIbalpha promoter driving alphaIIb into a single allele of the AAVS1 locus in GT iPS cells restored ∼60% surface expression of integrin alphaIIb/beta3 when compared to control iPS cell-derived megakaryocytes. Transgenic alphaIIb mRNA levels were similar to endogenous alphaIIb mRNA levels in differentiated megakaryocytes. When stimulated with thrombin, corrected GT iPS cell-derived megakaryocytes demonstrated both PAC-1 antibody binding and fibrinogen binding. This study shows the GPIbalpha promoter construct targeted to the AAVS1 locus drives a high-level of expression in iPS cell-derived megakaryocytes, which offers a novel tool to study megakaryocyte and platelet biology in samples obtained from affected individuals. For GT, this study demonstrates a higher level of correction than seen with a lentiviral approach. To our knowledge, this is the first report of the generation and correction of iPS cell lines from patients with a disease affecting platelet function.