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Next Article 
Blood, Vol. 95 No. 12 (June 15), 2000:
pp. 3645-3651
PLENARY PAPER
Megakaryocyte-targeted synthesis of the integrin
 3-subunit results in the phenotypic correction of
Glanzmann thrombasthenia
David A. Wilcox,
John C. Olsen,
Lori Ishizawa,
Paul F. Bray,
Deborah L. French,
Douglas A. Steeber,
William R. Bell,
Michael Griffith, and
Gilbert C. White II
From the Center for Thrombosis and Hemostasis, Departments of
Medicine and Pharmacology, Cystic Fibrosis/Pulmonary Research and
Treatment Center, University of North Carolina, Chapel Hill, NC; Nexell
Therapeutics Inc., Irvine, CA; the Department of Medicine, Johns
Hopkins University, Baltimore, MD; the Department of Medicine, Mount
Sinai School of Medicine, New York, NY; and the Department of
Immunology, Duke University Medical Center, Durham, NC.
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Abstract |
Glanzmann thrombasthenia is an inherited bleeding disorder
characterized by qualitative or quantitative defects of the
platelet-specific integrin,  IIb 3. As a result,
IIb3 cannot be activated and cannot bind to
fibrinogen, leading to a loss of platelet aggregation. Thrombasthenia
is clinically characterized by mucocutaneous hemorrhage with episodes
of intracranial and gastrointestinal bleeding. To develop methods for
gene therapy of Glanzmann thrombasthenia, a murine leukemia virus
(MuLV)-derived vector,  889PlA23, was
transduced into peripheral blood CD34+ cells from 2 patients with thrombasthenia with defects in the 3 gene. The human IIb promoter
was used in this vector to drive megakaryocyte-targeted expression of
the wild-type 3 subunit. Proviral DNA and
IIb3 biosynthesis were detected after in vitro
differentiation of transduced thrombasthenic CD34+ cells
with megakaryocyte growth and development factor. Flow cytometric
analysis of transduced patient samples indicated that 19% of
megakaryocyte progeny expressed IIb3 on the surface
at 34% of normal receptor levels. Treatment of transduced
megakaryocytes with a combination of agonists including epinephrine and
the thrombin receptor-activating peptide induced the
IIb3 complex to form an activated conformation
capable of binding fibrinogen as measured by PAC-1 antibody binding.
Transduced cells retracted a fibrin clot in vitro similar to
megakaryocytes derived from a normal nonthrombasthenic individual.
These results demonstrate ex vivo phenotypic correction of Glanzmann
thrombasthenia and support the potential use of hematopoietic
CD34+ cells as targets for IIb promoter-driven MuLV
vectors for gene therapy of platelet disorders.
(Blood. 2000;95:3645-3651)
© 2000 by The American Society of Hematology.
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Introduction |
Glanzmann thrombasthenia is a rare, autosomal recessive
bleeding disorder characterized by an absence or dysfunction of the platelet receptor for fibrinogen, integrin  IIb3
(glycoproteins [GP] IIb-IIIa). To date, about 50 distinct mutations
associated with Glanzmann thrombasthenia have been localized with
relatively equal occurrence on either the IIb or
3 gene.1 As in many
genetic disorders, the molecular abnormalities range from major
deletions and inversions to single point mutations.1 Although the percentage of abnormal IIb3 expressed on
the platelet surface may vary with the type of defect, all
thrombasthenic platelets are functionally indistinguishable as
characterized by the failure of defective IIb3 to
bind fibrinogen resulting in the absence of platelet aggregation after
stimulation by physiologic agonists.
Clinically, Glanzmann thrombasthenia is characterized by irregular
bleeding from mucous membranes with easy bruising, epistaxis, gingival
bleeding, and menorrhagia, which usually appears at an early age and
recurs throughout the individual's life.2 These individuals occasionally experience severe intracranial or
gastrointestinal bleeding that may result in death. Platelet
transfusions are used to treat severe cases of bleeding associated with
Glanzmann thrombasthenia, although many patients become refractory to transfusions.
Glanzmann thrombasthenia occurs at a low rate internationally, but
certain geographically restricted groups have a high carrier rate for
thrombasthenia including Iraqi Jews, distinct Arab populations of the
Middle East, French gypsies, and individuals from southern India.2 Approximately 2.3% of the 270 000 Iraqi Jews
living in Israel are carriers for thrombasthenia.3
Individuals of Arabic decent also have a prevalence for this disorder,
with more than 13 patients identified from 5 kindreds. The discovery of the molecular genetic defects in relatively large, "high-risk" populations has influenced the development of rapid DNA-based diagnostic assays,4 which provides opportunities for
genetic counseling and family planning for suspected carriers of the
abnormal IIb or
3 gene, and could also help to
identify prospective candidates for gene therapy.
The CD34+ hematopoietic cells are potential targets for
gene therapy strategies because these cells can be safely collected from the body, genetically modified, and reinfused into the
patient.5,6 This cell population has the capacity to
generate an engrafting cell mass capable of establishing hematopoiesis
with progeny cells of multilineages expressing the transferred gene for
the life span of the graft recipient. The use of CD34+
cells in vitro to develop a strategy for gene therapy of platelet disorders was facilitated by the discovery of techniques7
that induce pluripotent CD34+ progenitor cells to
proliferate and differentiate into megakaryocytes in vitro and in vivo
using c-Mpl-ligand or megakaryocyte growth and development factor
(MGDF),8 flk2/flt3 ligand, interleukin (IL)-3, IL-6, IL-11,
and stem cell factor (SCF). Among other effects, MGDF plays a direct
role in increasing the transcriptional activity of the integrin
IIb gene in megakaryocytes9; therefore, an IIb promoter-directed expression system may be activated by MGDF within transduced CD34+ cells. Thus, CD34+
cells can be transduced with genes driven by the IIb promoter and
induced to form megakaryocytes that can be examined for
targeted proviral gene expression.
This study examined the use of peripheral blood CD34+ cells
from patients with thrombasthenia as a model system for gene therapy of
disorders affecting platelets. This investigation used the murine
leukemia virus (MuLV)-derived vector,
889PlA23, in which the transcription
of 3 is controlled by an 889-nucleotide fragment of the
human IIb promoter. We previously used this vector to direct
megakaryocyte-specific expression of the platelet alloantigen 2 (PlA2) form of 3 in human cell lines and
expression of the proviral IIb3 complex in
megakaryocyte progeny of transduced CD34+ cells derived
from a normal (nonthrombasthenic) individual.10 Alloantigens were used in that study to allow us to distinguish the
biosynthesis of proviral (PlA2)
3 from endogenous (PlA1) 3 in
the normal human cells. The present study extends the use of the
889PlA23 vector to examine expression
of the integrin IIb3 complex on the surface of
megakaryocytes following transduction and MGDF-induced differentiation
of thrombasthenic CD34+ cells. Functional studies
demonstrated that transduced thrombasthenic cells were capable of
agonist-induced IIb3 activation and
retraction of a fibrin clot indicating ex vivo phenotypic correction of
Glanzmann thrombasthenia. These results indicate that ex vivo gene
transfer of a megakaryocyte-targeted gene expression system into
peripheral blood CD34+ cells, followed by reinfusion of
transduced cells, may be appropriate for gene therapy for disorders of platelets.
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Patients, materials, and methods |
Patients
Two patients with type I Glanzmann thrombasthenia,
RS 93,11 and EAY115C3, who
is a member of a previously reported kindred,12 volunteered
for the study. Both have lifelong bleeding episodes characterized by
prominent mucous membrane bleeding and bleeding secondary to surgery or
trauma. In each case, the diagnosis of thrombasthenia was established
by a prolonged bleeding time, the failure of platelets to aggregate
with physiologic agonists (adenosine diphosphate [ADP], epinephrine,
collagen, and thrombin), and the failure of platelets to retract a
fibrin clot. Platelets from each subject contained less than 5%
detectable IIb3, which is consistent with type I
Glanzmann thrombasthenia. Distinct defects in
3 have been defined at the molecular level:
patient E.A. has a novel nucleotide substitution in
3 resulting in a single amino acid
substitution of tyrosine to cysteine at residue 115 (Y115C), and
patient R.S. has a previously reported single nucleotide substitution in 3 that affects the splice-donor site of exon 9 resulting in the deletion of 45 amino acids (9). All of
these studies have been conducted with patient consent and approval by
the human rights committees of Johns Hopkins University and the
University of North Carolina and conducted according to the principles
expressed in the Declaration of Helsinki.
Antibodies and reagents
Polyclonal antibodies specific for IIb and 3 and a
monoclonal antibody that recognizes an epitope on 3,
AP3,13 were gifts from Dr Peter J. Newman (Blood Research
Institute, Milwaukee, WI). The fluorescein isothiocyante
(FITC)-conjugated monoclonal antibody, PAC-1, which recognizes an
epitope on the activated IIb3 complex was purchased
from Becton Dickinson (San Jose, CA). The monoclonal antibody,
AP2,14 which recognizes an epitope on the
IIb3 complex, was provided by Dr Robert R. Montgomery (Blood Research Institute, Milwaukee, WI). The monoclonal antibody, 6D1,15 which recognizes an epitope on glycoprotein (GP)Ib
was provided by Dr Barry Coller (Mt Sinai School of Medicine, New York,
NY). Phycoerythrin (PE)-conjugated anti-GPIb antibody (mouse antihuman CD42b) and isotype standards (PE-IgG, FITC-IgM) were purchased from PharMingen (San Diego, CA). ADP was purchased from Fisher (Pittsburgh, PA), and epinephrine was from Bio/Data Corp (Horsham, PA). Thrombin receptor-activating peptide (TRAP) and an
Arg-Gly-Asp-containing peptide (GRGDW) were synthesized at the Blood
Research Institute Core Facility (Milwaukee, WI).
Retroviral construct p-889PlA23
As previously described,10 a fragment of the IIb
promoter beginning at nucleotide 889 was used to drive
transcription of complementary DNA (cDNA) encoding the platelet
alloantigen 2 (PlA2) form of 3 (provided by
Dr Peter J. Newman, Blood Research Institute, Milwaukee,
WI).16 The 889PlA23 DNA
cassette was positioned within the MuLV-derived retroviral vector,
pHIT-SIN, which encodes a 3' long terminal repeat (LTR) sequence
(provided by Dr Estuardo Aguilar-Cordova, Baylor College of Medicine,
Houston, TX)17 lacking the viral enhancer/promoter so that
the IIb promoter could be used to promote megakaryocyte-targeted gene transcription.
Retrovirus production
Human 293 cells were transiently transfected on 10-cm plates with 15 µg each of pCI-GPZ, pCI-VSV-G helper plasmids, and
p-889PlA23 using the Calcium Transfection
System (Life Technologies, Gaithersburg, MD).10 Media
containing retrovirus was concentrated 500-fold and resuspended in
Iscove's modified Dulbecco's Eagle medium (IMDM). Viral preps were
stored at 80°C until needed. Replication competent virus was
not detected in 889PlA23 viral
preparations using extended marker rescue assays as previously described.18
Selection of CD34+ cells from peripheral blood
Peripheral blood collection was performed after obtaining written
informed consent from adult Glanzmann thrombasthenic volunteers enrolled in a protocol approved by the University of North Carolina and
Johns Hopkins University Committees on the Protection of the Rights of
Human Subjects. Subjects were given granulocyte colony-stimulating factor (Amgen, Thousand Oaks, CA) at 10 µg/kg/d subcutaneously for 4 days and peripheral blood cell collection was performed on day 5 using
a COBE Spectra Blood Cell Separator. CD34+ cells were
immunoselected from the apheresis product on an Isolex 300i Magnetic
Cell Separator (Nexell Therapeutics, Irvine, CA, distribution through
Baxter Healthcare) as previously described.19 Cell yields
from both patients were approximately 600 million total nucleated
cells, with a final recovery of 150 million CD34+ cells
(85% CD34+ purity). Selected cells were suspended in
X-VIVO 10 (Biowhittaker, Walkersville, MD) containing 1% (w/v)
human serum albumin, frozen in 10% (v/v) DMSO at
5 × 106 cells/mL, and stored in liquid nitrogen.
Transduction of CD34+ cells
Human CD34+ cells were transduced as previously
described.10 Briefly, cells were prestimulated in IMDM
containing 20% fetal bovine serum (FBS), 10 U/mL recombinant human
(rh) IL-3, 100 U/mL rhIL-6, 30 U/mL recombinant murine SCF (Genetics
Institute, Cambridge, MA) and 10 ng/mL flk2/flt3 ligand (R&D Systems,
Minneapolis, MN) for 48 hours at 37°C in 5% CO2. Cells
were transduced at 5 × 105 per well of a sterile,
24-well nontissue culture-treated plate (Falcon-Becton Dickinson,
Franklin Lakes, NJ) coated with 20 µg/cm2
RetroNectin20,21 (Takara Shuzo, Otsu, Shiga, Japan) with an estimated multiplicity of infection of 10 retrovirus
(889PlA23) per cell in IMDM plus 20%
FCS and rhIL-3, rhIL-6, SCF, and flk2/flt3 ligand. Fresh viral
supernatant was added after 2 hours. This procedure was repeated 1 time
24 hours later. Twenty-four hours after the final transduction,
megakaryocyte formation was induced similar to a previously described
method.7 Cells were resuspended at
5 × 105/mL in IMDM containing 10% platelet poor
plasma and rhIL-3, rhIL-6, SCF, flk2/flt3 ligand plus 100 ng/mL rhIL-11
(Genetics Institute) and 100 ng/mL rhMGDF (Amgen) for up to 17 days.
Cells were collected, washed twice in phosphate-buffered saline (PBS),
and solubilized in 1 mL of lysis buffer and stored at 80°C.
Detection of proviral DNA in transduced cells by polymerase chain
reaction
Cells (1.2 × 105) were split into 3 aliquots and
DNA was amplified in 3 separate polymerase chain reaction (PCR)
reactions: (1) amplification of IIb genomic DNA from nucleotides
13 703 to 14 064 (361 base product)22 was performed as a
positive control to detect genomic DNA from untransduced and transduced
samples; (2) PCR of plasmid vector backbone DNA
(P889PlA23) was performed using sense
primer 5'-TGACTGGTGAGTACTCAACC-3' from nucleotide 1861 to
1880 and antisense primer 5'-TTCACACCGCATACAGGTGGC-3', which consisted of nucleotides 2323 to 2303 (462 base product) outside
of the region packaged into retroviral capsids as a negative control
that demonstrated retroviral plasmid DNA was not transfected into the
cells during transduction; and (3) plasmid vector
(P889PlA23) sequence packaged into
retroviral capsids was amplified by PCR using sense primer
"OL-psi-1" 5'-TTGAACCTCCTCGTTCGAC-3' beginning 111 nucleotides upstream of 889PlA23 DNA cassette and
antisense primer 5'-ACTCCTCCTCCGTCTTGAGCC-3', which
consisted of nucleotides 572 to 592 of the IIb
promoter (428 base product) to detect proviral DNA in transduced
samples. The PCR products were separated by electrophoresis
on a 2.0% agarose gel and visualized by ethidium bromide staining.
Immunoprecipitation analysis
Immunoprecipitaion analysis was performed as previously
described.10 Precleared lysates were immunoprecipitated for
1 hour at 25°C with AP2 coupled to Affi-gel Hz (Bio-Rad, Hercules,
CA). Immunoprecipitates were electrophoresed on a 7% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gel under nonreducing conditions and proteins were transferred to Immobilon-P (Millipore, Bedford, MA) at 200 mA for 3 hours and blocked for 12 hours
at 4°C in 10% FBS in TBS-Tween. Protein was detected using rabbit
polyclonal antibodies specific for IIb (5 µg/mL) and
3 (4 µg/mL) and a peroxidase-conjugated
F(ab')2 fragment donkey antirabbit IgG (H+L) (Jackson
ImmunoResearch, West Grove, PA) at a 1:20 000 dilution followed by
chemiluminescence detection and exposure to autoradiography film from 1 second to 10 minutes.
Indirect immunofluorescence
Transduced and untransduced cells (5 × 105) were
blocked for 15 minutes in 2% bovine serum albumin (BSA) and 0.1 mmol/L
Ca/Mg in PBS and incubated with 5 µg AP2 for 20 minutes at 25°C,
then treated with PE-conjugated F(ab')2 donkey
antimouse secondary antibody (Jackson ImmunoResearch) for 20 minutes on
ice. Cells were resuspended in 200 µL of 2% formaldehyde, 0.2%
glutaraldehyde in PBS and fixed to single wells of a 24-well tissue
culture-treated plate for 15 minutes at 25°C while centrifuging at
230g. Positively staining cells were detected and photographed
using a Zeiss Axiovert 10 fluorescence microscope at × 320 magnification.
Flow cytometric analysis
Transduced and untransduced cells (1.5 × 105)
were blocked for 15 minutes in 2% BSA and 0.1 mmol/L Ca/Mg in PBS;
incubated with 2.5 µg of AP2, AP3, 6D1, mouse IgG for 20 minutes at
25°C; and treated with PE-conjugated F(ab')2
donkey antimouse secondary antibody (Jackson ImmunoResearch) for 20 minutes on ice. Cells were resuspended in 200 µL of 1%
paraformaldehyde in PBS and analyzed on a FACScan flow cytometer
(Becton Dickinson) using CellQuest software. A minimum of
2 × 103 cells exhibiting light scattering
properties of megakaryocytes were used for the analysis. Identification
of megakaryocytes in this cell population was further confirmed with
antibodies directed against glycoprotein GPIb. Megakaryocytes
expressing IIb3 were determined using samples stained
with AP2 and the secondary antibody described above. Cells expressing
3 were determined using samples stained with AP3 and
secondary antibody. Negative populations of cells were determined using
unreactive isotype-specific antibodies as a control for background
staining. Fluorescence contours are shown as 50% log density plots.
The efficiency of 889PlA23 to
transduce CD34+ cells was calculated by comparing the
percent of the cell population that expressed the
IIb3 complex following transduction with untransduced and normal nonthrombasthenic megakaryocytes under identical culture conditions. The mean fluorescence intensity of AP2
and AP3 staining was measured at saturating antibody
levels13,14 to determine receptor expression level and
estimate receptor density on the cell samples.
Agonist-induced IIb3 activation
Culture cells were harvested for physiologic studies of
IIb3 function 8 to 13 days after transduction. Cells
(1.5 × 106/mL) were incubated with PE-GPIb and
FITC-PAC1 (5 µg/mL) antibodies in modified Tyrode buffer containing 1 mmol/L CaCl2, 1 mmol/L MgCl2, 50 µmol/L each
of TRAP, ADP, and epinephrine for 15 minutes at 25°C. FITC-PAC1
binding was monitored in the FL1 channel of the flow cytometer on the
gated subset of cells that expressed GPIb (FL2). The specificity of
FITC-PAC1 binding was determined by co-incubation of samples with a
blocking peptide containing Arg-Gly-Asp (GRGDW, 2.5 mmol/L). Samples
were diluted 10-fold with buffer and examined using flow cytometry and
2-color analysis performed as described above.
Clot retraction
Clot retraction assays were performed using a slightly modified
version of a previously described method.23 Ten to 14 days after transduction, cultured cells (1.5 × 106/mL)
were resuspended in serum-free IMDM containing 60 µg/mL human fibrinogen in a standard aggregometry tube. Clot formation was initiated by the addition of 2.5 U/mL thrombin. Tubes were incubated at
37°C for up to 12 hours and photographed.
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Results |
Formation of the IIb3 complex following
transduction of thrombasthenic CD34+ cells
Patients R.S.11 and E.A.12 have type I
Glanzmann thrombasthenia due to defects in 3 associated
with undetectable surface expression of the IIb3
complex, failure of platelets to bind fibrinogen, absence of
platelet-platelet aggregates, and inability to retract a fibrin clot.
Patient R.S. has a previously reported single nucleotide substitution
that affects the splice-donor site of exon 9 of 3
resulting in the deletion of 45 amino acids (RS93), and patient E.A. has a novel nucleotide substitution of
3 resulting in a single amino acid substitution of
tyrosine to cysteine at residue 115 (EAY115C3). To
assess the feasibility of human gene therapy of Glanzmann
thrombasthenia, granulocyte colony-stimulating factor mobilized,
peripheral blood CD34+ cells were collected from each
patient and transduced with MuLV-derived vector
889PlA23. The transduced
CD34+ cells were subjected to in vitro expansion and
differentiation with IL-3, IL-6, IL-11, flk2/flt3 ligand, SCF, and
MGDF, and examined for the presence of proviral DNA by PCR analysis.
After 10 days of cytokine treatment, successful transduction of
CD34+ cells from RS93 and
EAY115C3 was indicated by the detection of proviral DNA
by PCR in 889PlA23-transduced cells
but not in untransduced cells (not shown, see "Materials and
methods").
Immunoblot analysis was performed to determine if the
IIb3 complex was synthesized in megakaryocyte progeny
of 889PlA23-transduced
CD34+ cells from RS93 and
EAY115C3. On days 10 and 15 after differentiation, respectively, the IIb3 receptor was immunoprecipitated from cellular lysates with an IIb3 complex-specific
monoclonal antibody (AP2) and detected using a mixture of
well-characterized polyclonal antibodies specific for the IIb and
3 integrin subunits. Both IIb
(Mr = 145 000) and 3
(Mr = 95 000) were detected in the 3-transduced cells (Figure
1) indicating that there was
proviral-derived, IIb promoter-directed synthesis of
PlA23 resulting in formation of the
IIb3 complex. The IIb3 complex was
not detected in untransduced thrombasthenic CD34+ cells
(Figure 1) nor in cells transduced with a similar MuLV-derived vector
(889nLacz), which encoded the -galactosidase gene in place of PlA23 (not shown).
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| Fig 1.
Immunoprecipitation analysis of
889PlA23 transduced CD34+
cells from patient RS 93 and
EAY115C3.
Cells (5 × 105) were collected 10 and 15 days,
respectively, after transduction and detergent lysates were
immunoprecipitated with 10 µg of an
IIb3 complex-specific antibody (AP2). The
complexed proteins were separated on a 7% SDS-PAGE gel under
nonreducing conditions. Immunoanalysis using polyclonal antibodies to
IIb and 3 followed by chemiluminescence
detection showed that transduction with the
889PlA23 vector resulted
in the synthesis of detectable levels of 3
and IIb (arrows on right), whereas untransduced samples did not have
detectable protein. Molecular mass markers are in kilodaltons (left).
Additional bands appearing equally in each lane are nonspecific
background resulting from chemiluminescence detection using a murine
monoclonal antibody for immunoprecipitation and rabbit polyclonal
antibodies and a horseradish peroxidase-conjugated donkey antirabbit
antibody for analysis.
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Surface expression of IIb3 on transduced
thrombasthenic megakaryocytes
Indirect immunofluorescence was performed to examine expression of
IIb3 on the surface of megakaryocytes following
889PlA23 transduction of
CD34+ cells from RS93 and
EAY115C3. Five to 10 days after ex vivo expansion and
differentiation of CD34+ cells, megakaryocytes were
identified in cell cultures by detection of the megakaryocyte-specific
glycoprotein (GP)Ib using the monoclonal antibody 6D1. GPIb was
detected on the surface of megakaryocytes in untransduced
thrombasthenic, transduced thrombasthenic, and normal
(nonthrombasthenic) samples (Figure 2, top
row), and cells transduced with 889nLacz (not shown). Only
megakaryocytes derived from
889PlA23- transduced
CD34+ cells expressed IIb3 receptors on
the cell surface. This expression was qualitatively similar to
megakaryocyte progeny of CD34+ cells from a normal
nonthrombasthenic individual as demonstrated by detectable AP2 staining
(Figure 2, bottom row).
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| Fig 2.
Indirect immunofluorescence analysis of thrombasthenic
CD34+ cells transduced with
889PlA23.
The RS93 and EAY115C3
CD34+ cells were transduced with
889PlA23, induced to form
megakaryocytes ex vivo, and then examined by indirect
immunofluorescence analysis for IIb3
surface expression. Cells were blocked in 2% BSA, and incubated with 5 µg monoclonal antibody 6D1 that recognizes megakaryocyte-specific
glycoprotein (GP)Ib (top panels) or 5 µg AP2, which recognizes
IIb3 (bottom panels), and detected with a
PE-conjugated F(ab')2 donkey antimouse secondary
antibody. Five to 10 days after transduction, megakaryocytes were
present in untransduced and 3-transduced cell cultures
as demonstrated with the anti-GPIb antibody; however, only
3-transduced cells demonstrated detectable
IIb3 complex on the surface of derived
megakaryocytes similar to cultured megakaryocytes from a normal
individual (control). There are at least 3 cells in each field of
untransduced thrombasthenic cells stained with AP2 for
IIb3.
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To quantitate the efficiency of transduction and estimate
IIb3 receptor density on megakaryocytes, flow
cytometric analysis was performed following transduction of patient
CD34+ cells with
889PlA23. After day 9 of ex vivo
cellular expansion and differentiation, typically 38% of the large
cells from each culture sample differentiated to megakaryocytes
expressing GPIb (not shown). In Figure 3,
megakaryocytes that expressed IIb3 on the surface
were identified on contour plots as the cells that emitted a high
fluorescence intensity with AP2 at saturating antibody
levels.14 An unreactive isotype-specific antibody was used
as a control for background staining of normal and patient cells.
Normal nonthrombasthenic CD34+ cells were induced to form
megakaryocytes with detectable IIb3 in 41% of the
cell population (Figure 3), whereas untransduced CD34+
cells from EAY115C3 and RS93 showed no
detectable IIb3 expression above background levels
(Figure 3). Megakaryocytes expressing IIb3 were
detected in 10% and 7% of the transduced cell population from
EAY115C3 and RS93, respectively
(Figure 3). This indicates that approximately 19% of patient
megakaryocytes were transduced with
889PlA23, when the percent of
megakaryocytes expressing IIb3 in the transduced cell
populations were compared with untransduced and normal
nonthrombasthenic cultures (Figure 3). Transduced patient megakaryocytes had a mean fluorescence intensity of AP2 staining that
was 34% of IIb3 receptor expression level on normal
(nonthrombasthenic)-derived megakaryocytes indicating a reduced
receptor density on transduced patient cells (Figure 3). Flow
cytometric analysis using a monoclonal antibody (AP3) specific for the
3 subunit demonstrated 3 expression on
approximately 5% of transduced patient cells at 28% of normal receptor density (Figure 3). Because the percent of the hematopoietic population expressing 3 (which normally pairs with V
on monocytes and lymphocytes as well as megakaryocytes) is nearly
identical with data obtained for IIb3 expression on
transduced patient cells, these results suggest that the IIb
promoter directed megakaryocyte-specific expression of the
3 subunit.
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| Fig 3.
Flow cytometric analysis following
889PlA23 transduction of thrombasthenic
CD34+ cells.
Untransduced and transduced cells were induced to form megakaryocytes
for 9 days ex vivo, and then examined by flow cytometric analysis for
surface expression of the 3 subunit. Shown in the first
set of panels are megakaryocytes expressing IIb3 as
detected with complex-specific antibody AP2 and a PE-conjugated
F(ab')2 donkey antimouse secondary antibody. The
second set of panels are cells expressing the 3 subunit
as detected with monoclonal antibody, AP3, and secondary antibody.
Fluorescence contour plots are presented for normal nonthrombasthenic
cells (upper right), untransduced and transduced cells from
EAY115C3 (middle panels), and untransduced and
transduced cells from RS93 (lower panels). The x-axis
depicts cell size as measured with forward scatter on a linear scale,
and the y-axis is relative fluorescence intensity of PE-AP2 or PE-AP3.
Megakaryocytes that expressed IIb3 on the
cell surface were detected in the upper right quadrant as were cells
that expressed 3. Normal and patient cells incubated
with an isotype nonspecific antibody and secondary antibody were
presented as controls for background staining (upper left panels).
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IIb3 signaling in 3-transduced
thrombasthenic megakaryocytes
Inside-out signaling in 3-transduced cells was
measured by performing agonist-dependent binding assays with the
fibrinogen mimetic antibody, PAC1, to determine if
IIb3 could be induced to form an activated
conformation capable of binding fibrinogen. Activation of
IIb3 was measured 9 days after ex vivo cellular expansion and differentiation using megakaryocytes selected with a
PE-GPIb antibody following stimulation of cells with TRAP, ADP, and
epinephrine. Results of these studies are shown in Figure 4 and demonstrate that in the presence of
agonist, 3-transduced megakaryocytes bound FITC-PAC1 at
a peak value that was on the average 10-fold above levels from
untransduced patient cells. Similar to Figure 3,
3-transduced megakaryocytes bound antibody at a reduced
fluorescence intensity that was approximately 9% of the peak value
level on normal control megakaryocytes (Figure 4). FITC-PAC1 was
inhibited from binding 3-transduced and normal control
megakaryocytes in the presence of an Arg-Gly-Asp-containing peptide
that is known to block the specific-binding of IIb3 to PAC1 and fibrinogen (Figure 4). In contrast, untransduced
thrombasthenic megakaryocytes did not bind FITC-PAC1 in the presence or
absence of the inhibitor peptide (Figure 4). These results indicate
that IIb3 expressed on 3-transduced
megakaryocytes functions similar to the integrin complex on normal
nonthrombasthenic megakaryocytes because excitatory agonists stimulated
PAC1 binding and an inhibitory peptide blocked antibody
recognition.
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| Fig 4.
Analysis of agonist induced activation of
IIb3 on 3-transduced thrombasthenic
megakaryocytes.
Cultured cells were harvested for physiologic studies of
IIb3 function at 9 days after transduction with
889PlA23. Untransduced and
3-transduced cells (1.5 × 106/mL)
were incubated with PE-GPIb and FITC-PAC1 antibodies in modified
Tyrode buffer containing TRAP, ADP, and epinephrine agonists. Binding
of the IIb3 activation-sensitive antibody, FITC-PAC1,
was monitored in the FL1 channel of the flow cytometer on the gated
subset of megakaryocytes that expressed GPIb (FL2). In each panel,
FITC-PAC1 binding was measured in the absence (shaded histograms) and
presence (unshaded histograms) of an Arg-Gly-Asp-containing peptide
(GRGDW) that blocks FITC-PAC1 binding specifically to activated
IIb3. The 3-transduced megakaryocytes
from patients R.S. and E.A. bound FITC-PAC1 at a fluorescence intensity
peak value of 13 and 7, respectively, which is on average 10-fold
higher than the FITC-PAC1 peak value of 1 for untransduced
megakaryocytes from R.S. and E.A. (shaded). The
3-transduced samples from R.S. and E.A. bound FITC-PAC1
at a fluorescence intensity peak value that was approximately 9% of
the peak value of 110 for normal nonthrombasthenic megakaryocytes
(shaded top). In the presence of the GRGDW peptide, FITC-PAC1 did not
bind to megakaryocytes from 3-transduced, untransduced,
or normal samples as demonstrated with fluorescence intensity peak
value of 1 for each sample (unshaded) and 3-transduced
megakaryocytes from R.S. that had a peak value of 2.
|
|
Transduced thrombasthenic cells retract a fibrin clot
To further identify the function of the expressed
IIb3, CD34+ cells from
RS93 and EAY115C3 were transduced with
889PlA23, treated with cytokines for
10 days to form megakaryocytes, and examined for the ability to retract
a fibrin clot (Figure 5). The
889PlA23 transduced cells mediated
clot retraction (middle panels) that was similar to cultured
CD34+ cells from a nonthrombasthenic individual (normal
control, right panels), whereas untransduced thrombasthenic cells were
unable to retract a fibrin clot (left panels). The data suggest that the expressed IIb3 receptors are
functional in mediating clot retraction, implying an ex vivo correction
of the Glanzmann thrombasthenia phenotype.
View larger version (84K):
[in this window]
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| Fig 5.
Fibrin clot retraction assay following
889PlA23 transduction of thrombasthenic
CD34+ cells.
The RS93 and EAY115C3
CD34+ cells were transduced with
889PlA23, induced for 10 to 14 days
to form megakaryocytes in vitro, and then examined for the ability to
retract a fibrin clot. Cells (1.5 × 106/mL) were
resuspended in IMDM containing 60 µg/mL human fibrinogen in a
standard aggregometry tube. Clot formation was initiated by the
addition of 2.5 U/mL thrombin. Tubes were incubated at 37°C for up
to 12 hours and photographed. The 3-transduced cells
were able to mediate clot retraction in vitro similar to the
nonthrombasthenic cells (normal control), whereas untransduced patient
samples were not able to retract a fibrin clot. A normal control was
included for the time each patient sample was assayed.
|
|
| |
Discussion |
The results of this investigation demonstrate successful
transduction of peripheral blood CD34+ cells from 2 patients with Glanzmann thrombasthenia using the MuLV-derived vector,
889PlA23. Integrin 3
subunit synthesis, IIb3 complex formation, and surface expression were demonstrated on transduced megakaryocytes derived from thrombasthenic patients R.S. and E.A. Although FACS analysis indicated a subnormal IIb3 receptor density
on the surface of megakaryocytes, these
889PlA23 transduced cells
demonstrated function by mediating retraction of a fibrin clot and
binding the IIb3 activation-dependent PAC1 antibody
on cellular stimulation with agonists, which is consistent with the ex
vivo correction of the Glanzmann thrombasthenic phenotype. Proplatelet
formation was observed in transduced cell culture, but the quantity of
platelets were too few to test aggregation. Our in vitro results show
that despite suboptimal transduction efficiency of CD34+
cells and reduced IIb3 receptor density on patient
megakaryocytes, there is correction of Glanzmann thrombasthenia.
This is consistent with the fact that genetic carriers for Glanzmann
thrombasthenia (about 50% normal IIb3 receptor
density levels) are disease free with normal platelet aggregation and
clot retraction. We have previously observed measurable platelet
aggregation and retraction of a fibrin clot using a mixture of 10%
normal platelets with 90% thrombasthenic platelets in vitro
(unpublished data). Thus, we might expect to see in vivo correction of
thrombasthenia with expression of IIb3 on less than
20% of transduced megakaryocytes as demonstrated in vitro; however, if
transduced progenitor cells were delivered to patients in the absence
of total marrow ablation, the actual percentage of corrected
megakaryocytes could be greatly reduced in the total cell population.
Nevertheless, our data suggest that compared to uncorrected platelets,
a circulating population of platelets derived from
3-transduced megakaryocytes have an increased potential
to aggregate at the site of vascular injury due to the expression of
IIb3 that can be induced by agonist to become
activated and bind fibrinogen.
One key aspect of this work is the use of a fragment of the IIb
promoter to target gene expression to human megakaryocytes. Regulatory
elements of the IIb promoter necessary for high level, megakaryocyte-specific gene transcription have been localized to the
first 800 nucleotides of the human IIb promoter using cell
lines,24-27 transfected rat primary cells,28
and transgenic mice.29,30 Megakaryocyte progenitor cells
express GATA and Ets factors that bind within this region to induce a
high level of transcription of the IIb
gene,31 while as yet undefined factors that play a role in
restricting transcription to developing megakaryocytes have been
localized between nucleotides 80 and 130 of the
IIb gene.28,32,33 An
MGDF-responsive element has also been recently identified that
increases the transcriptional activity of the integrin IIb
gene in megakaryocytes.9 Investigations with transgenic
mouse models have demonstrated megakaryocyte-targeted transcription in
vivo when the transcriptional activation of an 800-nucleotide fragment
of the human IIb promoter directed expression of the thymidine
kinase gene in multipotent hematopoietic cells leading to sustained
expression in megakaryocyte progeny and down-regulated expression
during erythroid and myeloid lineage differentiation.29,30 We have recently observed supporting results when the MuLV-derived expression vector, 889PlA23,
selectively targeted expression of 3 to transduced
promegakaryocyte cell lines, and a similarly controlled construct,
889nLacZ, preferentially directed -galactosidase activity to
megakaryocytes following transduction and MGDF-induced differentiation
of human CD34+ cells.10 The current
investigation demonstrates the use of an 889 nucleotide fragment of the
human IIb promoter for targeted gene expression to correct diseased
human megakaryocytes following transduction and MGDF induced
differentiation of thrombasthenic hematopoietic CD34+
cells. We detected levels of integrin 3 expression
nearly equal to IIb3 by flow cytometric analysis
suggesting megakaryocyte-specific expression of the transgene in
transduced patient cells. The cumulative data indicate that the
MuLV-derived IIb promoter expression system used in this report has
the capacity to direct expression of the 3 gene with
selective, MGDF-inducible expression in megakaryocyte progeny
suggesting this system may be ideal for gene therapy for platelet
disorders such as Glanzmann thrombasthenia.
Two potential challenges arise with our proposal to use the MuLV
construct, 889PlA23, which expresses
the rare PlA2 alloantigen form of 3 for
genetic correction of thrombasthenia. First, individuals have become
refractory to infused donor platelets due to the production of
antibodies against mismatched alloantigenic determinants of
3 resulting in clearance of the transfused
platelets34; therefore, epitopes of 3 may
have to be identically matched to result in acceptance of corrected
platelets as an additional requirement for gene therapy of
thrombasthenia. We speculate that further experimentation with
different platelet alloantigens of 3 for gene therapy of
Glanzmann thrombasthenia may help us overcome some principal problems
of transfusion medicine concerning antigen tolerance and correction of
platelet refractoriness. This could ultimately lead to a definitive
strategy that will pose as a viable alternative to the current
treatment of platelet transfusions for bleeding episodes.35
Second, individuals suffering from acute coronary thrombosis have been
noted with an increased prevalence for the presence of the
PlA2 allelic isoform of 3 on their
platelets36,37; alternatively, the PlA1 form
of 3 may be used for gene therapy of
Glanzmann thrombasthenia. These results indicate that expression of
different PlA1 or PlA2 forms of
3 in transduced thrombasthenic cells may lead to changes in the ability of platelets to activate and aggregate.
The results of this investigation suggest that transduction of altered
forms of integrin subunits into CD34+ cells of
thrombasthenics may allow elucidation of events important for integrin
expression and intracellular signaling in primary human hematopoietic
cells. Cultured cell lines transfected with recombinant
IIb3 subunits have been traditionally utilized to identify key steps that take place during integrin
biosynthesis,38-41 intracellular
signaling,23,42 and receptor activation.43 Although these studies have advanced our understanding concerning fundamental concepts of integrin biology, data suggest that
1, 2, and 3 integrins may
behave differently in transfected cells compared to natural host cells
due to the influence of cell type-specific cytosolic proteins in
governing signal transduction.44-46 Information gained from
our study may inspire new strategies for investigating integrin-mediated events via megakaryocyte-targeted gene expression during megakaryocytopoiesis of primary human hematopoietic cells.
The IIb promoter controlled expression system,
889PlA23, was developed to treat
thrombasthenics with 3 defects; however, this system may
be used for gene therapy of other molecular genetic defects of
platelets. The system could potentially be used for gene therapy of
thrombasthenics with IIb defects or individuals with defects
characterized in (GP)Ib-V-IX complex with Bernard Soulier syndrome,
platelet-type von Willebrand disease, and pseudo von Willebrand
disease.47 Other diseases may also benefit from the
expression of novel proteins in megakaryocytes, because this study
suggests that a platelet could potentially deliver other therapeutic
agents to the site of a vascular injury during a primary hemostasis response.
| |
Acknowledgments |
We would like to thank Suzanne Lyman for her technical assistance with
clot retraction assays in this study and Dr Susan Gidwitz for her
technical advice. We thank Dr Julie Oliver (Duke University, Durham,
NC) for technical assistance with flow cytometry and Dr Thomas F. Tedder for use of the flow cytometry facilities at Duke University
Medical Center. We thank Drs Masamichi Shiraga and Sanford Shattil
(Scripps Research Institute, La Jolla, CA) for technical advice for
megakaryocyte activation assays.
| |
Footnotes |
Submitted April 14, 1999; accepted February 7, 2000.
Supported by grants HL-45100 and HL-58931 (G.C.W.) from the National
Institutes of Health and by an American Heart Association (North
Carolina Affiliate) Postdoctoral Fellowship Award NC-95-FW-63 (D.A.W.).
Reprints: Gilbert C. White II, Division of
Hematology-Oncology, University of North Carolina, 932 Mary Ellen
Jones, 231H/CB#7035, Chapel Hill, NC 27599; e-mail:
gcwhite{at}med.unc.edu.
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.
| |
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Abstract
Introduction