Efficient retrovirus-mediated PIG-A gene transfer and stable restoration of GPI-anchored protein expression in cells with the PNH phenotype

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 phosphatidylinosi...

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Bibliographic Details
Published in:Blood 2001-05, Vol.97 (10), p.3004-3010
Main Authors: Nishimura, Jun-ichi, Phillips, Ken L., Ware, Russell E., Hall, Sharon, Wilson, Lee, Gentry, Tracy L., Howard, Thad A., Murakami, Yoshiko, Shibano, Masaru, Machii, Takashi, Gilboa, Eli, Kanakura, Yuzuru, Takeda, Junji, Kinoshita, Taroh, Rosse, Wendell F., Smith, Clay A.
Format: Article
Language:English
Subjects:
3T3 Cells
Animals
B-Lymphocytes - metabolism
Biological and medical sciences
Biotechnology
Bone Marrow Cells - metabolism
Cell Line
Cell Line, Transformed
Fundamental and applied biological sciences. Psychology
Gene Expression
Gene therapy
Genetic Vectors
Glycosylphosphatidylinositols - genetics
Health. Pharmaceutical industry
Hematopoietic Stem Cells - metabolism
Hemoglobinuria, Paroxysmal - blood
Hemoglobinuria, Paroxysmal - genetics
Hemoglobinuria, Paroxysmal - metabolism
Hemolysis
Herpesvirus 4, Human
Industrial applications and implications. Economical aspects
Membrane Proteins - deficiency
Membrane Proteins - genetics
Membrane Proteins - physiology
Mice
Mutation
Nerve Growth Factor - analysis
Nerve Growth Factor - genetics
Phenotype
Retroviridae - genetics
Transfection
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Summary: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.
ISSN:0006-4971
1528-0020
DOI:10.1182/blood.V97.10.3004