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Blood, Vol. 95 No. 12 (June 15), 2000: pp. 3645-3651

PLENARY PAPER

Megakaryocyte-targeted synthesis of the integrin beta 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.


    Abstract
Top
Abstract
Introduction
Patients, materials, and...
Results
Discussion
References

Glanzmann thrombasthenia is an inherited bleeding disorder characterized by qualitative or quantitative defects of the platelet-specific integrin, alpha IIbbeta 3. As a result, alpha IIbbeta 3 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, -889PlA2beta 3, was transduced into peripheral blood CD34+ cells from 2 patients with thrombasthenia with defects in the beta 3 gene. The human alpha IIb promoter was used in this vector to drive megakaryocyte-targeted expression of the wild-type beta 3 subunit. Proviral DNA and alpha IIbbeta 3 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 alpha IIbbeta 3 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 alpha IIbbeta 3 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 alpha IIb promoter-driven MuLV vectors for gene therapy of platelet disorders. (Blood. 2000;95:3645-3651)

© 2000 by The American Society of Hematology.


    Introduction
Top
Abstract
Introduction
Patients, materials, and...
Results
Discussion
References

Glanzmann thrombasthenia is a rare, autosomal recessive bleeding disorder characterized by an absence or dysfunction of the platelet receptor for fibrinogen, integrin alpha IIbbeta 3 (glycoproteins [GP] IIb-IIIa). To date, about 50 distinct mutations associated with Glanzmann thrombasthenia have been localized with relatively equal occurrence on either the alpha IIb or beta 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 alpha IIbbeta 3 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 alpha IIbbeta 3 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 alpha IIb or beta 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 alpha IIb gene in megakaryocytes9; therefore, an alpha 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 alpha 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, -889PlA2beta 3, in which the transcription of beta 3 is controlled by an 889-nucleotide fragment of the human alpha IIb promoter. We previously used this vector to direct megakaryocyte-specific expression of the platelet alloantigen 2 (PlA2) form of beta 3 in human cell lines and expression of the proviral alpha IIbbeta 3 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) beta 3 from endogenous (PlA1) beta 3 in the normal human cells. The present study extends the use of the -889PlA2beta 3 vector to examine expression of the integrin alpha IIbbeta 3 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 alpha IIbbeta 3 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.


    Patients, materials, and methods
Top
Abstract
Introduction
Patients, materials, and...
Results
Discussion
References

Patients

Two patients with type I Glanzmann thrombasthenia, RSDelta 9beta 3,11 and EAY115Cbeta 3, 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 alpha IIbbeta 3, which is consistent with type I Glanzmann thrombasthenia. Distinct defects in beta 3 have been defined at the molecular level: patient E.A. has a novel nucleotide substitution in beta 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 beta 3 that affects the splice-donor site of exon 9 resulting in the deletion of 45 amino acids (Delta 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 alpha IIb and beta 3 and a monoclonal antibody that recognizes an epitope on beta 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 alpha IIbbeta 3 complex was purchased from Becton Dickinson (San Jose, CA). The monoclonal antibody, AP2,14 which recognizes an epitope on the alpha IIbbeta 3 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-GPIbalpha 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-889PlA2beta 3

As previously described,10 a fragment of the alpha IIb promoter beginning at nucleotide -889 was used to drive transcription of complementary DNA (cDNA) encoding the platelet alloantigen 2 (PlA2) form of beta 3 (provided by Dr Peter J. Newman, Blood Research Institute, Milwaukee, WI).16 The -889PlA2beta 3 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 alpha 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-889PlA2beta 3 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 -889PlA2beta 3 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 (-889PlA2beta 3) 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 alpha 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 (P-889PlA2beta 3) 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 (P-889PlA2beta 3) sequence packaged into retroviral capsids was amplified by PCR using sense primer "OL-psi-1" 5'-TTGAACCTCCTCGTTCGAC-3' beginning 111 nucleotides upstream of -889PlA2beta 3 DNA cassette and antisense primer 5'-ACTCCTCCTCCGTCTTGAGCC-3', which consisted of nucleotides -572 to -592 of the alpha 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 alpha IIb (5 µg/mL) and beta 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 GPIbalpha . Megakaryocytes expressing alpha IIbbeta 3 were determined using samples stained with AP2 and the secondary antibody described above. Cells expressing beta 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 -889PlA2beta 3 to transduce CD34+ cells was calculated by comparing the percent of the cell population that expressed the alpha IIbbeta 3 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 alpha IIbbeta 3 activation

Culture cells were harvested for physiologic studies of alpha IIbbeta 3 function 8 to 13 days after transduction. Cells (1.5 × 106/mL) were incubated with PE-GPIbalpha 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 GPIbalpha (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.


    Results
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Abstract
Introduction
Patients, materials, and...
Results
Discussion
References

Formation of the alpha IIbbeta 3 complex following transduction of thrombasthenic CD34+ cells

Patients R.S.11 and E.A.12 have type I Glanzmann thrombasthenia due to defects in beta 3 associated with undetectable surface expression of the alpha IIbbeta 3 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 beta 3 resulting in the deletion of 45 amino acids (RSDelta 9beta 3), and patient E.A. has a novel nucleotide substitution of beta 3 resulting in a single amino acid substitution of tyrosine to cysteine at residue 115 (EAY115Cbeta 3). 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 -889PlA2beta 3. 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 RSDelta 9beta 3 and EAY115Cbeta 3 was indicated by the detection of proviral DNA by PCR in -889PlA2beta 3-transduced cells but not in untransduced cells (not shown, see "Materials and methods").

Immunoblot analysis was performed to determine if the alpha IIbbeta 3 complex was synthesized in megakaryocyte progeny of -889PlA2beta 3-transduced CD34+ cells from RSDelta 9beta 3 and EAY115Cbeta 3. On days 10 and 15 after differentiation, respectively, the alpha IIbbeta 3 receptor was immunoprecipitated from cellular lysates with an alpha IIbbeta 3 complex-specific monoclonal antibody (AP2) and detected using a mixture of well-characterized polyclonal antibodies specific for the alpha IIb and beta 3 integrin subunits. Both alpha IIb (Mr = 145 000) and beta 3 (Mr = 95 000) were detected in the beta 3-transduced cells (Figure 1) indicating that there was proviral-derived, alpha IIb promoter-directed synthesis of PlA2beta 3 resulting in formation of the alpha IIbbeta 3 complex. The alpha IIbbeta 3 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 beta -galactosidase gene in place of PlA2beta 3 (not shown).


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Fig 1. Immunoprecipitation analysis of -889PlA2beta 3 transduced CD34+ cells from patient RStriangle 9beta 3 and EAY115Cbeta 3. Cells (5 × 105) were collected 10 and 15 days, respectively, after transduction and detergent lysates were immunoprecipitated with 10 µg of an alpha IIbbeta 3 complex-specific antibody (AP2). The complexed proteins were separated on a 7% SDS-PAGE gel under nonreducing conditions. Immunoanalysis using polyclonal antibodies to alpha IIb and beta 3 followed by chemiluminescence detection showed that transduction with the -889PlA2beta 3 vector resulted in the synthesis of detectable levels of beta 3 and alpha 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.

Surface expression of alpha IIbbeta 3 on transduced thrombasthenic megakaryocytes

Indirect immunofluorescence was performed to examine expression of alpha IIbbeta 3 on the surface of megakaryocytes following -889PlA2beta 3 transduction of CD34+ cells from RSDelta 9beta 3 and EAY115Cbeta 3. 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 -889PlA2beta 3- transduced CD34+ cells expressed alpha IIbbeta 3 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 -889PlA2beta 3. The RSDelta 9beta 3 and EAY115Cbeta 3 CD34+ cells were transduced with -889PlA2beta 3, induced to form megakaryocytes ex vivo, and then examined by indirect immunofluorescence analysis for alpha IIbbeta 3 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 alpha IIbbeta 3 (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 beta 3-transduced cell cultures as demonstrated with the anti-GPIb antibody; however, only beta 3-transduced cells demonstrated detectable alpha IIbbeta 3 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 alpha IIbbeta 3.

To quantitate the efficiency of transduction and estimate alpha IIbbeta 3 receptor density on megakaryocytes, flow cytometric analysis was performed following transduction of patient CD34+ cells with -889PlA2beta 3. 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 alpha IIbbeta 3 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 alpha IIbbeta 3 in 41% of the cell population (Figure 3), whereas untransduced CD34+ cells from EAY115Cbeta 3 and RSDelta 9beta 3 showed no detectable alpha IIbbeta 3 expression above background levels (Figure 3). Megakaryocytes expressing alpha IIbbeta 3 were detected in 10% and 7% of the transduced cell population from EAY115Cbeta 3 and RSDelta 9beta 3, respectively (Figure 3). This indicates that approximately 19% of patient megakaryocytes were transduced with -889PlA2beta 3, when the percent of megakaryocytes expressing alpha IIbbeta 3 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 alpha IIbbeta 3 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 beta 3 subunit demonstrated beta 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 beta 3 (which normally pairs with alpha V on monocytes and lymphocytes as well as megakaryocytes) is nearly identical with data obtained for alpha IIbbeta 3 expression on transduced patient cells, these results suggest that the alpha IIb promoter directed megakaryocyte-specific expression of the beta 3 subunit.


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Fig 3. Flow cytometric analysis following -889PlA2beta 3 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 beta 3 subunit. Shown in the first set of panels are megakaryocytes expressing alpha IIbbeta 3 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 beta 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 EAY115Cbeta 3 (middle panels), and untransduced and transduced cells from RSDelta 9beta 3 (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 alpha IIbbeta 3 on the cell surface were detected in the upper right quadrant as were cells that expressed beta 3. Normal and patient cells incubated with an isotype nonspecific antibody and secondary antibody were presented as controls for background staining (upper left panels).

alpha IIbbeta 3 signaling in beta 3-transduced thrombasthenic megakaryocytes

Inside-out signaling in beta 3-transduced cells was measured by performing agonist-dependent binding assays with the fibrinogen mimetic antibody, PAC1, to determine if alpha IIbbeta 3 could be induced to form an activated conformation capable of binding fibrinogen. Activation of alpha IIbbeta 3 was measured 9 days after ex vivo cellular expansion and differentiation using megakaryocytes selected with a PE-GPIbalpha 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, beta 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, beta 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 beta 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 alpha IIbbeta 3 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 alpha IIbbeta 3 expressed on beta 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 alpha IIbbeta 3 on beta 3-transduced thrombasthenic megakaryocytes. Cultured cells were harvested for physiologic studies of alpha IIbbeta 3 function at 9 days after transduction with -889PlA2beta 3. Untransduced and beta 3-transduced cells (1.5 × 106/mL) were incubated with PE-GPIbalpha and FITC-PAC1 antibodies in modified Tyrode buffer containing TRAP, ADP, and epinephrine agonists. Binding of the alpha IIbbeta 3 activation-sensitive antibody, FITC-PAC1, was monitored in the FL1 channel of the flow cytometer on the gated subset of megakaryocytes that expressed GPIbalpha (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 alpha IIbbeta 3. The beta 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 beta 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 beta 3-transduced, untransduced, or normal samples as demonstrated with fluorescence intensity peak value of 1 for each sample (unshaded) and beta 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 alpha IIbbeta 3, CD34+ cells from RSDelta 9beta 3 and EAY115Cbeta 3 were transduced with -889PlA2beta 3, treated with cytokines for 10 days to form megakaryocytes, and examined for the ability to retract a fibrin clot (Figure 5). The -889PlA2beta 3 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 alpha IIbbeta 3 receptors are functional in mediating clot retraction, implying an ex vivo correction of the Glanzmann thrombasthenia phenotype.


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Fig 5. Fibrin clot retraction assay following -889PlA2beta 3 transduction of thrombasthenic CD34+ cells. The RSDelta 9beta 3 and EAY115Cbeta 3 CD34+ cells were transduced with -889PlA2beta 3, 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 beta 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
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Abstract
Introduction
Patients, materials, and...
Results
Discussion
References

The results of this investigation demonstrate successful transduction of peripheral blood CD34+ cells from 2 patients with Glanzmann thrombasthenia using the MuLV-derived vector, -889PlA2beta 3. Integrin beta 3 subunit synthesis, alpha IIbbeta 3 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 alpha IIbbeta 3 receptor density on the surface of megakaryocytes, these -889PlA2beta 3 transduced cells demonstrated function by mediating retraction of a fibrin clot and binding the alpha IIbbeta 3 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 alpha IIbbeta 3 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 alpha IIbbeta 3 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 alpha IIbbeta 3 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 beta 3-transduced megakaryocytes have an increased potential to aggregate at the site of vascular injury due to the expression of alpha IIbbeta 3 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 alpha IIb promoter to target gene expression to human megakaryocytes. Regulatory elements of the alpha IIb promoter necessary for high level, megakaryocyte-specific gene transcription have been localized to the first 800 nucleotides of the human alpha 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 alpha 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 alpha IIb gene.28,32,33 An MGDF-responsive element has also been recently identified that increases the transcriptional activity of the integrin alpha 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 alpha 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, -889PlA2beta 3, selectively targeted expression of beta 3 to transduced promegakaryocyte c