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Blood, 15 May 2001, Vol. 97, No. 10, pp. 3004-3010
GENE THERAPY
Efficient retrovirus-mediated PIG-A gene transfer and
stable restoration of GPI-anchored protein expression in cells with the
PNH phenotype
Jun-ichi Nishimura,
Ken L. Phillips,
Russell E. Ware,
Sharon Hall,
Lee Wilson,
Tracy L. Gentry,
Thad A. Howard,
Yoshiko Murakami,
Masaru Shibano,
Takashi Machii,
Eli Gilboa,
Yuzuru Kanakura,
Junji Takeda,
Taroh Kinoshita,
Wendell F. Rosse, and
Clay A. Smith
From the Division of Hematology and Medical Oncology,
Department of Medicine; Division of Experimental Surgery, Department of
Surgery; and the Division of Hematology-Oncology, Department of
Pediatrics, Duke University Medical Center, Durham, NC; the Department
of Immunoregulation, Research Institute for Microbial Diseases, Osaka
University; and the Department of Hematology and Oncology and the
Department of Environmental Medicine, Osaka University Graduate School
of Medicine, Japan.
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.
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