A pig-a knock-out model of pnh: rescue of hematopoiesis follows transfer of gpi-anchored proteins

Acquired mutations in the X-linked gene PIG-A lead to loss of expression of glycosyl phosphatidylinositol-anchored proteins [GPI-AP], and are responsible for the hemolytic diathesis in patients with the disease paroxysmal nocturnal hemoglobinuria (PNH1. The causal relationship of these mutations to bone marrow failure-the other cardinal feature of PNH-remains, however, uncertain. In order to create an in vitro model system for this disease, we inserted a neomycin resistance cassette into the second exon of the mouse PIG-A gene and created, by homologous recombination, a PIG-A knock-out embryonic stem cell JESCJ. termed C25. As would be predicted, surface expression of CD24, a GPI-AP present on normal ESC. was lost. Embryoid bodies [EB] derived from C25 ESC exhibited arrested development in that they did not grow beyond an Intermediate size and did not develop cystic structures characteristic of wild type EB, Secondary hematopoletic colonies tailed to develop from disaggregated C25 EB. Chimenc EB comprised of normal D(UB C2S ESC. however aoneared normal and PIG-A ESC-denved hematopoiesis (i.e. G418-resistant colony formation) was reconstituted to levels equivalent to that seen with control chtmeric EB (Table I). Thus absence of a functional PIG-A gene does nof result in an intrinsic defect in hematopoiesis. Hence the autoimmune bone marrow suppression that often accompanies PNH may in fact be a predisposing condition that selects for expansion of Rematopoietic stem cells harboring PIG-A mutations. Transfer in situ of GPI-AP from normal to knock-out cells was demonstrated by two-color fluorescent analysis, suggesting a possible mechanism for the functional reconstitution of hematopoiesis observed in the chimeric EB. Hematopoietic cells with mutated PIG-A genes in patients with PNH may be subject to comparable pathophysiologic processes ana amenable to similar therapeutic protein transfers. Table. 1 Hematopoietic progenitor wnumeration neo ₊ C25, and chimeric EB Cotonies Experiment ESC Unmixed 2 Chinenc 2 line 2 EB EB 1 neo 21/63 18/20 C25 0/0 12/14 2 neo R 30/35 7/16 C25 0/0 15/27 3 neo 33/63 12/13 C25 0/0 11/14 s Per 2× Viable pIMtd flnrt nunohf mpiWMi erythroid oobnes. secorw number nwwMwKts gwiutocyMnonocyM cotoo. 1 rwûdMWlMi contai ESC MlrantfKlid with iwoniycinrMittinncaiHine > -UmT <j n< MEBd n dfroim100WMrC25orrw)RESC Chnnnc ttnXH EB dnvml from 80% nom2 ESC plus 20% aithtr C2S or neoRESC.