title: Expression of recombinant transmembrane CD59 in paroxysmal nocturnal hemoglobinuria B cells confers resistance to human complement.
abstract: Paroxysmal nocturnal hemoglobinuria (PNH) is an acquired hematopoietic disorder characterized by complement-mediated hemolytic anemia, pancytopenia, and venous thrombosis. These clinical manifestations arise from an underlying molecular defect of bone marrow stem cells. Specifically, somatic mutations in the phosphatidylinositol glycan class A gene result in the ability of blood cells to anchor complement-regulatory proteins (CD59 and DAF) to the cell surface via glycosyl phosphatidylinositol (GPI). In an attempt to circumvent the functional defect in PNH cells, a recombinant transmembrane form of CD59 (CD59-TM) was analyzed for the ability to regulate complement activity. Balb/3T3 stable transfectants expressing similar levels of either CD59-TM or native CD59 (CD59-GPI) were equally protected against human complement-mediated membrane damage. Treatment of these cells with phosphatidylinositol-specific phospholipase C failed to release CD59-TM from the cell surface. Retroviral transduction of GPI-anchoring deficient mouse L cells with CD59-TM resulted in surface expression of the protein and rendered these cells resistant to human complement-mediated membrane damage. Conversely, L cells transduced with CD59-GPI failed to express this protein on the cell surface. A GPI-anchoring deficient complement-sensitive B-cell line derived from a PNH patient was successfully transduced with CD59-TM, resulting in surface expression of the protein. The PNH B cells expressing CD59-TM were protected against classical complement-mediated membrane damage by human serum. Taken together, these data establish that a functional recombinant transmembrane form of CD59 can be expressed on the surface of GPI-anchoring deficient PNH cells and suggest that retroviral gene therapy with this molecule could provide a treatment for PNH patients.
authors.affiliation: Alexion Pharmaceuticals Inc, New Haven, CT.
authors.forename: R P
authors.initials: RP
authors.lastname: Rother
authors.forename: S A
authors.initials: SA
authors.lastname: Rollins
authors.forename: J
authors.initials: J
authors.lastname: Mennone
authors.forename: A
authors.initials: A
authors.lastname: Chodera
authors.forename: S A
authors.initials: SA
authors.lastname: Fidel
authors.forename: M
authors.initials: M
authors.lastname: Bessler
authors.forename: P
authors.initials: P
authors.lastname: Hillmen
authors.forename: S P
authors.initials: SP
authors.lastname: Squinto
mesh_terms: D016475:3T3 Cells; D000818:Animals; D015703:Antigens, CD / Q000235:genetics*; D001402:B-Lymphocytes / Q000276:immunology*; D001483:Base Sequence; D018957:CD59 Antigens; D002460:Cell Line; D002461:Cell Line, Transformed; D003165:Complement System Proteins / Q000276:immunology*; D015870:Gene Expression; D018014:Gene Transfer Techniques; D017261:Glycosylphosphatidylinositols / Q000378:metabolism; D006457:Hemoglobinuria, Paroxysmal / Q000276:immunology*; D004854:Herpesvirus 4, Human; D006801:Humans; D007739:L Cells / Q000378:metabolism; D008562:Membrane Glycoproteins / Q000235:genetics*; D051379:Mice; D008807:Mice, Inbred BALB C; D008969:Molecular Sequence Data; D011994:Recombinant Proteins
chemical_list: D015703:Antigens, CD; D018957:CD59 Antigens; D017261:Glycosylphosphatidylinositols; D008562:Membrane Glycoproteins; D011994:Recombinant Proteins; D003165:Complement System Proteins