title: Efficient retrovirus-mediated PIG-A gene transfer and stable restoration of GPI-anchored protein expression in cells with the PNH phenotype.
abstract: Paroxysmal nocturnal hemoglobinuria (PNH) is a clonal hematopoietic stem cell disorder characterized by complement-mediated hemolysis due to deficiencies of glycosylphosphatidylinositol-anchored proteins (GPI-APs) in subpopulations of blood cells. Acquired mutations in the X-linked phosphatidylinositol glycan-class A (PIG-A) gene appear to be the characteristic and pathogenetic cause of PNH. To develop a gene therapy approach for PNH, a retroviral vector construct, termed MPIN, was made containing the PIG-A complementary DNA along with an internal ribosome entry site and the nerve growth factor receptor (NGFR) as a selectable marker. MPIN transduction led to efficient and stable PIG-A and NGFR gene expression in a PIG-A-deficient B-cell line (JY5), a PIG-A-deficient K562 cell line, an Epstein-Barr virus-transformed B-cell line (TK-14(-)) established from a patient with PNH, as well as peripheral blood (PB) mononuclear cells from a patient with PNH. PIG-A expression in these cell lines stably restored GPI-AP expression. MPIN was transduced into bone marrow mononuclear cells from a patient with PNH, and myeloid/erythroid colonies and erythroid cells were derived. These transduced erythroid cells restored surface expression of GPI-APs and resistance to hemolysis. These results indicate that MPIN is capable of efficient and stable functional restoration of GPI-APs in a variety of PIG-A-deficient hematopoietic cell types. Furthermore, MPIN also transduced into PB CD34(+) cells from a normal donor, indicating that MPIN can transduce primitive human progenitors. These findings set the stage for determining whether MPIN can restore PIG-A function in multipotential stem cells, thereby providing a potential new therapeutic option in PNH.
authors.affiliation: Division of Hematology and Medical Oncology, Department of Medicine, Duke University Medical Center, Durham, NC, USA. junnishi@acpub.duke.edu
authors.lastname: Nishimura Ji
authors.forename: K L
authors.initials: KL
authors.lastname: Phillips
authors.forename: R E
authors.initials: RE
authors.lastname: Ware
authors.forename: S
authors.initials: S
authors.lastname: Hall
authors.forename: L
authors.initials: L
authors.lastname: Wilson
authors.forename: T L
authors.initials: TL
authors.lastname: Gentry
authors.forename: T A
authors.initials: TA
authors.lastname: Howard
authors.forename: Y
authors.initials: Y
authors.lastname: Murakami
authors.forename: M
authors.initials: M
authors.lastname: Shibano
authors.forename: T
authors.initials: T
authors.lastname: Machii
authors.forename: E
authors.initials: E
authors.lastname: Gilboa
authors.forename: Y
authors.initials: Y
authors.lastname: Kanakura
authors.forename: J
authors.initials: J
authors.lastname: Takeda
authors.forename: T
authors.initials: T
authors.lastname: Kinoshita
authors.forename: W F
authors.initials: WF
authors.lastname: Rosse
authors.forename: C A
authors.initials: CA
authors.lastname: Smith
mesh_terms: D016475:3T3 Cells; D000818:Animals; D001402:B-Lymphocytes / Q000378:metabolism; D001854:Bone Marrow Cells / Q000378:metabolism; D002460:Cell Line; D002461:Cell Line, Transformed; D015870:Gene Expression; D005822:Genetic Vectors; D017261:Glycosylphosphatidylinositols / Q000235:genetics*; D006412:Hematopoietic Stem Cells / Q000378:metabolism; D006457:Hemoglobinuria, Paroxysmal / Q000097:blood / Q000235:genetics* / Q000378:metabolism; D006461:Hemolysis; D004854:Herpesvirus 4, Human; D008565:Membrane Proteins / Q000172:deficiency / Q000235:genetics* / Q000502:physiology; D051379:Mice; D009154:Mutation; D020932:Nerve Growth Factor / Q000032:analysis / Q000235:genetics; D010641:Phenotype; D012190:Retroviridae / Q000235:genetics*; D014162:Transfection
chemical_list: D017261:Glycosylphosphatidylinositols; D008565:Membrane Proteins; C080086:phosphatidylinositol glycan-class A protein; D020932:Nerve Growth Factor