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GENE THERAPY
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 Paroxysmal nocturnal hemoglobinuria (PNH) is an
acquired clonal hematopoietic stem cell disorder characterized by
intravascular hemolytic anemia.1,2 Abnormal blood cells
are deficient in glycosylphosphatidylinositol-anchored proteins
(GPI-APs).3-6 In the affected hematopoietic cells from
patients with PNH, the first step in biosynthesis of the GPI-anchor is
defective.7,8 At least 5 genes are involved in this
reaction step,9 and one of them, an X-linked gene termed
phosphatidylinositol glycan-class A (PIG-A),10
is mutated in affected cells.11,12 The PIG-A gene is mutated in every patient with PNH reported to date, and deficiency of GPI in PNH has thus been considered to be due solely to
the PIG-A mutation(s).3-6
The clinical manifestation of PNH is complex, involving primarily 3 sets of symptoms: hemolysis with acute exacerbation, cytopenia of
varying severity, and a tendency for thrombosis. PNH derives its name
from the episodes of brownish urine (hemoglobinuria) that frequently
appear in the morning13 and that are due to intravascular
hemolysis because of deficiencies of decay accelerating factor (DAF)
and CD59 in erythroid cells.4,5 However, the mechanism of
thrombosis and its relation to defects in PNH are not entirely clear.
Many patients with PNH have evidence of deficient hematopoiesis, and
the degree of bone marrow (BM) failure varies from subclinical
cytopenia to the development of severe aplastic anemia (AA). Also,
patients with AA have an increased risk of developing
PNH.14-17 Episodes of infection occur frequently and may
be attributable in part to leukopenia or to functional defects in
leukocytes, which may be due to impaired migration of
neutrophils18,19 and/or defective T-cell activation in
PNH.20,21 GPI-APs may thus function not only as complement
regulatory proteins, but also as receptors, or adhesion molecules, and
they may be involved in signal transduction.
Although PNH generally lasts for many years,22,23
thrombotic disease and hematopoietic failure are the major risk factors affecting survival.23,24 For example, Socie et
al23 reported that patients with thrombosis at presentation
had only a 40% survival rate at 4 years. Currently, allogeneic bone
marrow transplantation (BMT) is the only available cure for PNH;
however, this is associated with high levels of morbidity and
mortality. Gene therapy involving the transduction of the
PIG-A gene into autologous pluripotent stem cells is a
potential alternative strategy for treating PNH. In order to develop a
gene therapy approach for PNH, we have made a retroviral vector
construct, termed MPIN, containing the PIG-A complementary DNA (cDNA) along with internal ribosome entry sites (IRES) and the nerve growth factor receptor (NGFR) as a selectable marker. In the current study, we evaluated whether MPIN could restore
PIG-A function in various hematopoietic cells expressing the PNH phenotype.
Vector construction
The plasmid pCITE-1 (Novagen, Madison, WI), containing the
encephalomyocarditis virus-IRES sequence,28 was digested
by BalI (5') and XbaI (3'), and then a BalI/EagI oligonucleotide and an EagI (5')/XbaI (3') fragment from p The MN and MPIN amphotropic vector packaging line was generated by
transfection of the ecotropic retroviral vector producer line E86,
followed by infection of the amphotropic producer AM12. LNGFR-expressing AM12 cells (termed AM12/MN or MPIN) were then isolated
by fluorescence-activated cell sorting (FACS), and supernatant was
collected by growing AM12/MN or MPIN packaging cells to 80% confluence
and replacing the media with fresh media. For both supernatants, the
medium was Dulbecco's modified Eagle's medium (DMEM) (Gibco,
Gaithersburg, MD) containing 10% fetal calf serum (FCS) (Gibco), and
flasks with fresh media were incubated at 37°C for 16 hours, and the
supernatant was collected and centrifuged to remove cell debris. All
supernatants were aliquoted and frozen at Retroviral vector gene transfer
To introduce the PIG-A gene into various hematopoietic cells
expressing the PNH phenotype, generally 2 × 106 cells
were transduced by MN or MPIN supernatant, with the use of polybrene at
8 µg/mL or fibronectin (Takara, Ohtsu, Japan) at 100 µg/mL, with or
without centrifugation. In some experiments, the transduction procedure
was repeated 1 to 3 additional times. After transduction, cells were
immunophenotyped with various monoclonal antibodies.
Isolation and culture conditions of cells expressing the
PNH phenotype
FACS analysis and flow cytometric sorting Monoclonal antibodies (mAbs) included anti-CD59-fluorescein isothiocyanate (FITC), anti-CD55-phycoerythrin (PE), and anti-CD48-FITC (Pharmingen, San Diego, CA) for the detection of GPI-APs, anti-NGFR-PE (Chromoprobe, Mountain View, CA), anti-CD3-FITC (Pharmingen), anti-CD34-FITC (Pharmingen), and anti-CD45-peridinin chlorophyll protein (Pharmingen). For the detection of erythroid cells, E6,35 an antibody to red cell surface protein band 3 (generously provided by Dr Marilyn Telen, Duke University Medical Center, Durham, NC) as a first antibody and a secondary antibody conjugated to FITC (Jackson ImmunoResearch Lab, West Grove, PA) were used for staining. Antihuman IgG2a-FITC, antihuman IgG2a-PE and antihuman IgG1-PE (Pharmingen) were used for negative isotype controls. After staining, cells were analyzed with a FacsCalibur (Becton Dickinson). To separate CD59+, CD59 ,
NGFR+, and/or NGFR cells, the sample
was sorted on a FACStar Plus (Becton Dickinson) or a FACS Vantage
(Becton Dickinson) after staining.
Molecular analysis by PCR for PIG-A integration DNA was isolated from transduced (MN or MPIN) or untransduced (mock) NIH3T3 cell lines (JY5, K562 mutant, and TK-14),
BMMCs (primary erythroid cells), and colonies/bursts (mixture of
CFU-GM, CFU-mix, and BFU-E). A 1330-bp region including vector plus 5'
PIG-A was amplified by PCR with the use of the primer set of DG1 (5'-TCTCTCCCCCTTGAACC-3') and OX2
(5'-GCTCCCAAAAGACGCAC-3') as shown in Figure 1. For positive control,
the MPIN vector plasmid was used as a template, and Marker 6 (Takara)
was loaded as a molecular marker, in each experiment.
Small-scaled Ham test Serum was separated from whole blood of a blood-group-matched healthy volunteer. Serum was acidified with a 10% volume of 0.2 N HCl. Washed samples suspended in saline (3 × 106/0.01 mL) were incubated with 0.1 mL of the serum for 1 hour at 37°C. For 100% lysis, water was used in place of serum, and for 0% lysis, heat-inactivated serum was used. After incubation, each sample was centrifuged, and the optical density of hemoglobin in the supernatant was measured at 412 nm.36Aerolysin assay Cells (1 × 106) were incubated for 90 minutes at 37°C with aerolysin (1.5 × 108 M), which is a toxin
secreted by Aeromonas hydrophila and is capable of killing
target cells by binding to GPI-anchored receptors, produced by trypsin
activation of pro-aerolysin (Protox Biotech, Victoria, British
Columbia, Canada).37,38 For 100% lysis, water was used in
place of aerolysin, and for 0% lysis, PBS was used. After incubation,
each sample was centrifuged, and the optical density of hemoglobin in
the supernatant was measured at 412 nm.36
Retroviral vector MPIN restores PIG-A function in hematopoietic cell lines bearing PNH phenotype The MPIN vector was initially transduced into NIH3T3 cells. As shown in Figure 2A, 97.0% and 68.9% of NIH3T3 cells expressed LNGFR following transduction of MN and MPIN, respectively, yielding a titer of 4.8 × 106 infectious U/mL for MN and 3.4 × 106 infectious U/mL for MPIN. High levels of NGFR were expressed from MPIN, indicating that this could be a useful marker gene for analyzing gene transfer into hematopoietic cells. PCR analysis detected the region including PIG-A only in MPIN-transduced NIH3T3 cells (Figure 2B).
To evaluate whether MPIN would restore PIG-A function in hematopoietic
cells, we transduced it into several PIG-A-deficient cell lines. MPIN
was transduced into the PIG-A-deficient B-lymphoblastoid cell line
JY5, and then on days 10 and 20, NGFR+CD59+
cells were sorted to be enriched. On day 39, transduced cells were
stained with mAbs for NGFR and GPI-APs. In the selected JY5 cells, MPIN
expressed NGFR at high levels and restored stable and efficient
expression (greater than 95%) of GPI-APs including CD55, CD59, and
CD48 (Figure 3A). MPIN was also
transduced into a PIG-A-deficient K562 mutant cell line, and an
EBV-derived B cell line (TK-14
Retroviral vector gene transfer by MPIN restores PIG-A function in PBMCs with PNH phenotype To test MPIN in primary peripheral blood cells, CD59
PBMCs were isolated from a patient with PNH and transduced with MPIN after stimulation with IL-2, anti-CD3, and anti-CD28 as previously described.29 On day 19 in culture, 93.0% were positive
for CD3, a T-cell marker, and negative for CD19, a B-cell marker, and
the gene-transfer efficiency was 16.9% (16.8 ± 0.7%; range, 15.0% to approximately 18.4%; n = 4) as measured as a percentage of NGFR+ cells (Figure 4). MPIN
restored expression of GPI-APs at 8.8% (9.7 ± 0.4%; range, 8.8%
to approximately 10.6%; n = 4) as measured by
NGFR+CD59+ cells (Figure 4). Even though we
confirmed that the sorted PBMCs were mostly of the CD59
phenotype, 11.0% of cells in mock control expressed CD59. The most
likely explanation for this paradox is that contaminated CD59dim PBMCs brightly expressed after the culture with
stimulation. The CD59NGFR+ cells may also
have been observed because initiation or completion of translation did
not occur in the PIG-A sequence but, for currently unknown
reasons, did occur in the downstream NGFR sequence. Expression of both
NGFR and GPI-APs was stable for the duration of the cell cultures
(about a month). There was no significant difference in growth rates
between mock, MN-infected, and MPIN-infected cells, suggesting that
there was no toxicity from the virus.
Retroviral vector MPIN restores PIG-A function and confers resistance to hemolysis in PNH-phenotype primary erythroid cells from a patient with PNH One of the primary clinical manifestations of PNH is increased sensitivity of red cells (RBCs) to hemolysis by complement. To test whether transduction with MPIN would restore resistance to hemolysis in PNH RBCs, CD59 BMMCs were isolated from a patient with
PNH and transduced with MPIN. Cells were then cultured with flt-3
ligand, Epo, and IL-3, as previously described, to generate erythroid
progeny.33 Multiparameter FACS analysis performed on day
12 of culture demonstrated that 16.1% (19.2% ± 2.0%; range,
16.1% to approximately 24.9%; n = 4) of transduced cells were
CD59+NGFR+; 90.9% were positive for E6, an
erythroid marker; and most of the cells were negative for CD45, a
leukocyte marker (Figure 5A). The
integrated region including PIG-A was detected only in
MPIN-infected erythroid progeny (Figure 5B).
After the separation of CD59+NGFR+ and
CD59 Retroviral vector gene transfer by MPIN into PNH-phenotype and normal BMMCs BMMCs were also isolated from a patient with PNH and transduced with MPIN. Cells were then cultured in methylcellulose with Epo, SCF, GM-CSF, IL-3, and G-CSF to generate colonies and bursts. The numbers of colonies/bursts were scored on day 15 in culture, and the ratio of BFU-E to CFU-GM to CFU-mix was about 1:1:1 (data not shown). FACS analysis performed on day 15 of culture demonstrated that 13.6% of transduced cells were CD59+NGFR+ (Figure 6A). The integrated region including PIG-A was also detected only in MPIN-infected colonies/bursts (Figure 6B).
PB CD34+ cells were enriched from a G-CSF primed donor
after mobilization by means of StemSep and were transduced with MPIN. Gene-transfer efficiency in CD34+ cells was about 6% as
measured by NGFR+ cells on day 5 in culture (Figure
7).
BMT is currently the only available cure for PNH. Syngeneic and allogeneic BMTs, including eradication of the PNH clone, have been successful in several patients with PNH.39-41 In most, the indication for BMT was BM failure, and guidelines similar to those for AA have been proposed for the use of BMT in these patients. BMT was performed on one patient who had portal and hepatic vein thrombosis, with resolution of much of the thrombosis.42 Socie et al23 reported that thrombosis and hematopoietic failure were the major risk factors affecting survival. For example, patients with thrombosis at presentation have only a 40% survival rate at 4 years.23 Therefore, these patients are also good candidates for early and aggressive therapies. Autologous BMT, with or without depletion of GPI An alternative to BMT would be therapy designed to restore the surface
expression of GPI-APs, such as infusion of GPI-APs. Kooyman et
al43 demonstrated that GPI-APs expressed on the surface of
transgenic mouse red cells were transferred in a functional form to
endothelial cells in vivo. In addition, Dunn et al44 created a murine knock-out embryonic stem cell via targeted disruption of the Pig-a gene and demonstrated transfer in situ of
GPI-APs from normal to knock-out cells. Subsequently, Sloand et
al45 demonstrated that enriched DAF and CD59 reincorporated
into the membranes of PNH erythrocytes by means of high-density
lipoprotein were functional. At this time, however, strategies to
transfer therapeutic proteins to the GPI The discovery of PIG-A allows a new approach to therapy for patients with PNH, and molecular therapy to introduce a normal PIG-A gene into PIG-A mutant hematopoietic stem cells appears attractive. Autologous BMT after ex vivo transduction of a normal PIG-A gene into mutant hematopoietic stem cells could avoid both graft failure and graft-versus-host disease seen in allogeneic BMT. However, there remains the risk of recurrent PNH, caused by the re-establishment of clonal dominance by any residual PIG-A mutant stem cells. The mechanisms of clonal dominance of mutant stem cells in PNH are yet to be clarified. A possible mechanism is selection for PIG-A mutant cells, such as an immunological selection. For example, if cytotoxic T cells are involved in the autoimmunity, stem cells defective in the surface expression of GPI-APs may be less sensitive to the cytotoxic cells because the effector-target interaction may be inefficient owing to a lack of certain GPI-anchored types of adhesion molecules. In any case, selective killing of normal stem cells would result in selective survival and expansion of the GPI-deficient stem cell clone. Indeed, syngeneic BMT without conditioning in patients with PNH has not been universally successful because of an apparent survival advantage of mutant stem cells.46,47 If pretransplantation BM conditioning is effective in eliminating such selective conditions, then residual mutant stem cells would be eliminated or might not become dominant. The other possibility is that expanded clonal cells have a genetic abnormality in addition to PIG-A mutation and that the 2 in combination or the putative second abnormality may impart an ability to expand autonomously even in a normal bone marrow environment. In this case, the PIG-A-restored PNH stem cell clone might still maintain the ability to expand autonomously. More complete understanding of the pathogenesis of PNH is required as a scientific foundation for the rational design of further molecular therapeutic approaches. At this time, autologous BMT after the transduction of the normal PIG-A gene into mutant hematopoietic stem cells, along with depletion of residual mutant hematopoietic cells, seems the best molecular therapeutic approach to PNH. As proof of the principle of gene therapy for PNH, a functional recombinant transmembrane form of CD59 was expressed in GPI-deficient PNH B cells by means of retroviral gene transfer.48 This restored surface expression of CD59 and resistance to complement. In this study, we demonstrated that MPIN, a retroviral vector containing the full coding sequence of PIG-A, was able to restore GPI-AP expression and was capable of long-term stable gene transfer into various cell lines expressing the PNH phenotype. Subsequently, we demonstrated that MPIN was capable of efficient and stable gene transfer into primary peripheral T cells and progenitor cells from a patient with PNH. In addition, MPIN was capable of efficient functional restoration of GPI-APs that confer resistance to hemolytic action, indicating that MPIN can transduce erythroid progenitors. Finally, we also demonstrated that MPIN can transduce CD34+ primitive human progenitors. Taken together, our observations provide convincing evidence that stable and functional PIG-A gene transfer into human hematopoietic cells is possible. These findings set the stage for determining whether MPIN can restore PIG-A function in multipotential stem cells from patients with PNH. An additional advantage of MPIN vector is that the NGFR marker gene also permits easy determination of gene transfer efficiencies, as well as rapid isolation of transduced cells by means of a flow cytometric technique. Since we recently developed a PNH mouse model in which only the hematopoietic system has Pig-a-deficient cells,49 we could initiate the study for determining the restoration of the PIG-A function in multipotential stem cells using this mice model system, thereby providing a potential new therapeutic option in PNH.
We thank Dr Yasuhiko Horiguchi for advice on use of aerolysin; and Toshiyuki Hirota, Yvonne Ellis, Yuki Murakami, Keiko Kinoshita, and Reiko Fukuyama for their excellent technical assistance.
Submitted October 10, 2000; accepted January 18, 2001.
Partly supported by a grant from the Japan Intractable Diseases Research Foundation and The Osaka Medical Research Foundation for Incurable Diseases.
The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked "advertisement" in accordance with 18 U.S.C. section 1734.
Reprints: Jun-ichi Nishimura, Dept of Immunoregulation, Research Institute for Microbial Diseases, Osaka University, 3-1 Yamada-oka, Suita, Osaka 565-0871, Japan; e-mail: junnishi{at}acpub.duke.edu.
1. Rotoli B, Luzzatto L. Paroxysmal nocturnal haemoglobinuria. Baillieres Clin Haematol. 1989;2:113-138[CrossRef][Medline] [Order article via Infotrieve].
2.
Rosse WF.
Dr. Ham's test revisited.
Blood.
1991;78:547-550
3.
Rosse WF, Ware RE.
The molecular basis of paroxysmal nocturnal hemoglobinuria.
Blood.
1995;86:3277-3286 4. Kinoshita T, Inoue N, Takeda J. Defective glycosyl phosphatidylinositol anchor synthesis and paroxysmal nocturnal hemoglobinuria. Adv Immunol. 1995;60:57-103[Medline] [Order article via Infotrieve]. 5. Nishimura J, Smith CA, Phillips KL, Ware RE, Rosse WF. Paroxysmal nocturnal hemoglobinuria: molecular pathogenesis and molecular therapeutic approaches. Hematopathol Mol Hematol. 1998;11:119-146[Medline] [Order article via Infotrieve]. 6. Nishimura J, Murakami Y, Kinoshita T. Paroxysmal nocturnal hemoglobinuria: an aquired genetic disease. Am J Hematol. 1999;62:175-182[CrossRef][Medline] [Order article via Infotrieve].
7.
Takahashi M, Takeda J, Hirose S, et al.
Deficient biosynthesis of N-acetylglucosaminyl phosphatidylinositol, the first intermediate of glycosyl phosphatidylinositol anchor biosynthesis, in cell lines established from patients with paroxysmal nocturnal hemoglobinuria.
J Exp Med.
1993;177:517-521
8.
Hillmen P, Bessler M, Mason PJ, Watkins WM, Luzzatto L.
Specific defect in N-acetylglucosamine incorporation in the biosynthesis of the glycosylphosphatidylinositol anchor in cloned cell lines from patients with paroxysmal nocturnal hemoglobinuria.
Proc Natl Acad Sci U S A.
1993;90:5272-5276 9. Watanabe R, Murakami Y, Marmor MD, et al. Initial enzyme for glycosylphosphatidylinositol biosynthesis requires PIG-P and is regulated by DPM2. EMBO J. 2000;19:4402-4411[CrossRef][Medline] [Order article via Infotrieve].
10.
Miyata T, Takeda J, Iida Y, et al.
Cloning of PIG-A, a component in the early step of GPI-anchor biosynthesis.
Science.
1993;259:1318-1320 11. Takeda J, Miyata T, Kawagoe K, et al. Deficiency of the GPI anchor caused by a somatic mutation of the PIG-A gene in paroxysmal nocturnal hemoglobinuria. Cell. 1993;73:703-711[CrossRef][Medline] [Order article via Infotrieve].
12.
Miyata T, Yamada N, Iida Y, et al.
Abnormalities of PIG-A transcripts in granulocytes from patients with paroxysmal nocturnal hemoglobinuria.
N Engl J Med.
1994;330:249-255 13. Strubing P. Paroxysmale haemoglobinurie. Deutsche Med Wochenschrift. 1882;8:1-16.
14.
Schubert J, Vogt HG, Zielinska Skowronek M, et al.
Development of the glycosylphosphatitylinositol-anchoring defect characteristic for paroxysmal nocturnal hemoglobinuria in patients with aplastic anemia.
Blood.
1994;83:2323-2328
15.
Griscelli-Bennaceur A, Gluckman E, Scrobohaci ML, et al.
Aplastic anemia and paroxysmal nocturnal hemoglobinuria: search for a pathogenetic link.
Blood.
1995;85:1354-1363 16. Schrezenmeier H, Hertenstein B, Wagner B, Raghavachar A, Heimpel H. A pathogenetic link between aplastic anemia and paroxysmal nocturnal hemoglobinuria is suggested by a high frequency of aplastic anemia patients with a deficiency of phosphatidylinositol glycan anchored proteins. Exp Hematol. 1995;23:81-87[Medline] [Order article via Infotrieve]. 17. Azenishi Y, Ueda E, Machii T, et al. CD59-deficient blood cells and PIG-A gene abnormalities in Japanese patients with aplastic anaemia. Br J Haematol. 1999;104:523-529[CrossRef][Medline] [Order article via Infotrieve]. 18. Pedersen TL, Yong K, Pedersen JO, Hansen NE, Danø K, Plesner T. Impaired migration in vitro of neutrophils from patients with paroxysmal nocturnal haemoglobinuria. Br J Haematol. 1996;95:45-51[CrossRef][Medline] [Order article via Infotrieve].
19.
Gyetko MR, Sud S, Kendal T, Fuller JA, Newstead MW, Standiford TJ.
Urokinase receptor-deficient mice have impaired neutrophil recruitment in response to pulmonary pseudomonas aeruginosa infection.
J Immunol.
2000;165:1513-1519 20. Schubert J, Uciechowsky P, Zielinska-Skowronek M, Tietjen C, Leo R, Schmidt RE. Differences in activation of normal and glycosylphosphatidylinositol-negative lymphocytes derived from patients with paroxysmal nocturnal haemoglobinuria. J Immunol. 1992;148:3814-3819[Abstract].
21.
Romagnoli P, Bron C.
Defective TCR signaling events in glycosylphosphatidylinositol-deficient T cells derived from paroxysmal nocturnal hemoglobinuria patients.
Int Immunol.
1999;11:1411-1422
22.
Hillmen P, Lewis SM, Bessler M, Luzzatto L, Dacie JV.
Natural history of paroxysmal nocturnal hemoglobinuria.
N Engl J Med.
1995;333:1253-1258 23. Socie G, Mary JY, de-Gramont A, et al. Paroxysmal nocturnal haemoglobinuria: long-term follow-up and prognostic factors. Lancet. 1996;31:573-577. 24. Ware RE, Hall SE, Rosse WF. Paroxysmal nocturnal hemoglobinuria with onset in childhood and adolescence. N Engl J Med. 1991;325:991-996[Abstract].
25.
Mavilio F, Ferrari G, Rossini S, et al.
Peripheral blood lymphocytes as target cells of retroviral vector-mediated gene transfer.
Blood.
1994;83:1988-1997 26. McCowage GB, Phillips KL, Gentry TL, et al. Multiparameter fluorescence activated cell sorting analysis of retroviral vector gene transfer into primitive umbilical cord blood cells. Exp Hematol. 1998;26:288-298[Medline] [Order article via Infotrieve].
27.
Bonini C, Ferrari G, Verzeletti S, et al.
HSV-TK gene transfer into donor lymphocytes for control of allogeneic graft-versus-leukemia.
Science.
1997;276:1719-1724
28.
Kim DG, Kang HM, Jang SK, Shin HS.
Construction of a bifunctional mRNA in the mouse by using the internal ribosomal entry site of the encephalomyocarditis virus.
Mol Cell Biol.
1992;12:3636-3643 29. Rudoll T, Phillips K, Lee SW, et al. High-efficiency retroviral vector mediated gene transfer into human peripheral blood CD4+ T-lymphocytes. Gene Ther. 1996;3:695-705[Medline] [Order article via Infotrieve].
30.
Hirose S, Mohney RP, Mutka SC, et al.
Derivation and characterization of glycoinositol-phospholipid anchor-defective human K562 cell clones.
J Biol Chem.
1992;267:5272-5278
31.
Ueda E, Nishimura J, Kitani T, et al.
Deficient surface expression of glycosylphosphatidylinositol-anchored proteins in B cell lines established from patients with paroxysmal nocturnal hemoglobinuria.
Int Immunol.
1992;4:1263-1271 32. Nishimura J, Inoue N, Azenishi Y, et al. Analysis of PIG-A gene in a patient who developed reciprocal translocation of chromosome 12 and paroxysmal nocturnal hemoglobinuria during follow-up of aplastic anemia. Am J Hematol. 1996;51:229-233[CrossRef][Medline] [Order article via Infotrieve]. 33. Howrey RP, El-Alfondi M, Phillips KL, et al. An in vitro system for efficiently evaluating gene therapy approaches to hemoglobinopathies. Gene Ther. 2000;7:215-223[CrossRef][Medline] [Order article via Infotrieve]. 34. McNiece IK, Langley KE, Zsebo KM. Recombinant human stem cell factor synergizes with GM-CSF, G-CSF, IL-3 and Epo to stimulate human progenitor cells of the myeloid and erythroid lineages. Exp Hematol. 1991;19:226-231[Medline] [Order article via Infotrieve]. 35. Telen MJ, Scearce RM, Haynes BF. Human erythrocyte antigens, III: characterization of a panel of murine monoclonal antibodies that react with human erythrocyte and erythroid precursor membranes. Vox Sang. 1987;52:236-243[Medline] [Order article via Infotrieve].
36.
Pangburn MK, Schreiber RD, Trombold JS, Muller-Eberhard HJ.
Paroxysmal nocturnal hemoglobinuria: deficiency in factor H-like functions of the abnormal erythrocytes.
J Exp Med.
1983;157:1971-1980
37.
Garland WJ, Buckley JT.
The cytolytic toxin aerolysin must aggregate to disrupt erythrocytes, and aggregation is stimulated by human glycophorin.
Infect Immun.
1988;56:1249-1253
38.
Asao T, Kozaki S, Kato K, et al.
Purification and characterization of an aeromonas hydrophila hemolysin.
J Clin Microbiol.
1986;24:228-232 39. Saso R, Marsh J, Cevreska L, et al. Bone marrow transplants for paroxysmal nocturnal haemoglobinuria. Br J Haematol. 1999;104:392-396[CrossRef][Medline] [Order article via Infotrieve]. 40. Bemba M, Guardiola P, Garderet L, et al. Bone marrow transplantation for paroxysmal nocturnal haemoglobinuria. Br J Haematol. 1999;105:366-368[CrossRef][Medline] [Order article via Infotrieve].
41.
Raiola AM, Van-Lint MT, Lamparelli T, et al.
Bone marrow transplantation for paroxysmal nocturnal hemoglobinuria.
Haematologica.
2000;85:59-62 42. Graham ML, Rosse WF, Halperin EC, Miller CR, Ware RE. Resolution of Budd-Chiari syndrome following bone marrow transplantation for paroxysmal nocturnal haemoglobinuria. Br J Haematol. 1996;92:707-710[CrossRef][Medline] [Order article via Infotrieve].
43.
Kooyman DL, Byrne GW, McClellan S, et al.
In vivo transfer of GPI-linked complement restriction factors from erythrocytes to the endothelium.
Science.
1995;269:89-92
44.
Dunn DE, Yu J, Nagarajan S, et al.
A knock-out model of paroxysmal nocturnal hemoglobinuria: Pig-a(
45.
Sloand EM, Maciejewski JP, Dunn D, et al.
Correction of the PNH defect by GPI-anchored protein transfer.
Blood.
1998;92:4439-4445 46. Kawahara K, Witherspoon RP, Storb R. Marrow transplantation for paroxysmal nocturnal hemoglobinuria. Am J Hematol. 1992;39:283-288[Medline] [Order article via Infotrieve].
47.
Endo M, Beatty PG, Vreek TM, Wittwer CT, Singh SP, Parker CJ.
Syngeneic bone marrow transplantation without conditioning in a patient with paroxysmal nocturnal hemoglobinuria: in vivo evidence that the mutant stem cells have a survival advantage.
Blood.
1996;88:742-750
48.
Rother RP, Rollins SA, Mennone J, et al.
Expression of recombinant transmembrane CD59 in paroxysmal nocturnal hemoglobinuria B cells confers resistance to human complement.
Blood.
1994;84:2604-2611
49.
Murakami Y, Kinoshita T, Nakano T, Kosaka H, Takeda J.
Different roles of glycosylphosphatidylinositol in various hematopoietic cells as revealed by model mice of paroxysmal nocturnal hemoglobinuria.
Blood.
1999;94:2963-2970
© 2001 by The American Society of Hematology.
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  W. L. W. Hazenbos, Y. Murakami, J.-i. Nishimura, J. Takeda, and T. Kinoshita Enhanced Responses of Glycosylphosphatidylinositol Anchor-Deficient T Lymphocytes J. Immunol., September 15, 2004; 173(6): 3810 - 3815. [Abstract] [Full Text] [PDF]
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 W. F. Rosse, P. Hillmen, and A. D. Schreiber Immune-Mediated Hemolytic Anemia Hematology, January 1, 2004; 2004(1): 48 - 62. [Abstract] [Full Text] [PDF]
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 H. Wang, T. Chuhjo, S. Yasue, M. Omine, and S. Nakao Clinical significance of a minor population of paroxysmal nocturnal hemoglobinuria-type cells in bone marrow failure syndrome Blood, December 1, 2002; 100(12): 3897 - 3902. [Abstract] [Full Text] [PDF]
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S. Nagakura, S. Ishihara, D. E. Dunn, J.-i. Nishimura, T. Kawaguchi, K. Horikawa, M. Hidaka, T. Kagimoto, N. Eto, H. Mitsuya, et al. Decreased susceptibility of leukemic cells with PIG-A mutation to natural killer cells in vitro Blood, July 18, 2002; 100(3): 1031 - 1037. [Abstract] [Full Text] [PDF]
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Abstract
Introduction
LNGFR
= vector packaging sequence;
ATG = start codon.
70°C.
