This Article Abstract Full Text (PDF) All Versions of this Article: 23/12/2266    most recent 01.ATV.0000100403.78731.9Fv1 Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Herder, C. Articles by Seifried, E. Search for Related Content PubMed PubMed Citation Articles by Herder, C. Articles by Seifried, E. Related Collections Coagulation Gene therapy Gene expression

(Arteriosclerosis, Thrombosis, and Vascular Biology. 2003;23:2266.)
© 2003 American Heart Association, Inc.

Thrombosis

Sustained Expansion and Transgene Expression of Coagulation Factor VIII–Transduced Cord Blood–Derived Endothelial Progenitor Cells

Christian Herder; Torsten Tonn; Robert Oostendorp; Sven Becker; Ulrich Keller; Christian Peschel; Manuel Grez; Erhard Seifried

From the Institute for Transfusion Medicine and Immunohematology (C.H., T.T., S.B., E.S.), Red Cross Blood Donor Service Baden-Württemberg–Hessen, Frankfurt am Main; Georg-Speyer-Haus (C.H., M.G.), Institute for Biomedical Research, Frankfurt am Main; and III Medizinische Klinik (R.O., U.K., C.P.), Klinikum Rechts der Isar, Technical University Munich, Germany.

Correspondence to Dr Torsten Tonn, Institute for Transfusion Medicine and Immunohematology, Red Cross Blood Donor Service Baden-Württemberg–Hessen, Sandhofstr. 1, 60528 Frankfurt am Main, Germany. E-mail ttonn{at}bsdhessen.de

	Abstract

Top Abstract Introduction Methods Results Discussion References

 
Objective— Although hemophilia A seems particularly suitable for gene therapy because even low amounts of plasma coagulation factor VIII (FVIII) provide a significant clinical benefit to the patients, the ideal target cell for recombinant FVIII expression and gene therapy approaches remains to be identified. In this study, we tested the capacity of cord blood–derived endothelial progenitor cells (CBECs) for FVIII expression on stable lentiviral transduction.

Methods and Results— CD34⁺ endothelial progenitor cells (EPCs) from cord blood were differentiated into CBECs. Endothelial phenotype was characterized, and lentiviral transduction of early-passage CBECs with a vector encoding FVIII and EGFP did not alter their functional properties and proliferative potential. CBEC could be expanded by 5 to 9 orders of magnitude, thus allowing the expansion of up to 10¹⁵ FVIII-secreting CBECs, starting from as little as 10⁶ CD34⁺ cells. CBECs proved to be highly suitable for FVIII secretion, with 0.35 to 0.39 IU FVIII:C/5x10⁴ cells per 48 hours (7.0 to 7.8 IU FVIII:C/10⁶ cells per 48 hours), which remained stable over the expansion period.

Conclusions— Our data indicate that CBECs are attractive target cells for inherited coagulation disorders such as hemophilia A, which on lentiviral transduction can be readily expanded to large numbers of transplantable gene-modified cells in vitro.

Key Words: hemophilia • factor VIII • cord blood • endothelial progenitor cells • lentiviral gene therapy

	Introduction

Top Abstract Introduction Methods Results Discussion References

 
Hemophilia A is caused by insufficient levels or even the complete absence of functional coagulation factor VIII (FVIII) in the circulation. It is an X chromosome–linked bleeding disorder that affects 1 in 5000 to 10 000 males. The main symptom is the spontaneous occurrence of bleeding into joints, muscles, and internal organs, which can be life threatening.¹ Patients are presently treated by intravenous infusions of plasma-derived or recombinant FVIII.² This substitution therapy has become relatively efficacious during the last decade. However, because of several drawbacks, such as the potential transmission of infectious diseases by FVIII concentrates,³ alternate sources of recombinant FVIII or even a somatic gene therapy would be highly desirable.

See page 2117

Under physiological conditions, FVIII synthesis mainly occurs in hepatocytes and liver sinusoidal endothelial cells.^4–7 Because no FVIII-expressing cell lines derived from these cell types exist, recombinant FVIII protein for clinical use is presently produced at large scale in Chinese hamster ovary cells and baby hamster kidney cells.² Most studies for the gene therapy of hemophilia A focused on the in vivo transduction of liver cells with viral vectors. Several in vivo studies yielded promising results.^8,9 Long-lasting therapeutic FVIII plasma levels in hemophilia A mice could be achieved by systemic application of adenoviral, adeno-associated virus, retroviral, and lentiviral vectors. However, there remain concerns over the safety of these approaches. Potential side effects include adverse immunological reactions, vector-mediated cytotoxicity, and germ-line transmission.^10,11 Extrahepatic cells such as bone marrow stromal cells, fibroblasts, and keratinocytes (see the study by VandenDriessche et al⁸ and the references therein) may also be useful in gene therapeutic approaches. Indeed, the implantation of ex vivo modified fibroblasts that secreted FVIII was shown to be well tolerated and led to detectable FVIII plasma levels in patients with severe hemophilia A.¹²

Although endothelial progenitor cells (EPCs) have been shown to be of interest for use in vascular regeneration, they also seem interesting as alternate target cells for ectopic FVIII expression. They are easily accessible from bone marrow, peripheral blood, or cord blood (CB),^13–19 and they can be differentiated into long-lived progeny. However, although most of the studies have been conducted with bone marrow or peripheral blood–derived endothelial cell populations, only little is known about the capacity of CB-derived EPCs. Nonetheless, for inherited coagulation disorders such as hemophilia, CB may be a suitable source that is easily obtainable at the time of birth. The amount of EPCs in CB seems to be limited because of the restricted blood volume. Therefore, therapeutic applications, especially those using gene-modified EPCs, may require additional expansion and enrichment of gene-modified EPCs. In addition, whereas peripheral blood outgrowth endothelial cells transfected with a FVIII expression plasmid have been shown to efficiently cure hemophilia in a murine knockout model,²⁰ the impact of lentiviral transduction on the phenotype and proliferative potential of CB-derived EPCs (CBECs) has thus far not been addressed.

We therefore aimed to establish a lentiviral transduction protocol that allows highly efficient and stable transduction of CBECs using the enhanced green fluorescence protein (EGFP) gene as a marker and human B-domain–deleted FVIII, respectively. The deletion of the B domain increases the expression level of recombinant FVIII and does neither impair the procoagulant activity nor result in enhanced immunogenicity of the FVIII protein (see the study by Saenko et al¹⁸ and the references therein). We show that lentiviral transduction yields CBEC populations that have retained phenotypic characteristics of endothelial cells and that are readily expandable to large numbers of transplantable EPCs secreting high amounts of the FVIII transgene protein.

	Methods

Top Abstract Introduction Methods Results Discussion References

 
Isolation of CD34⁺ Cells From Cord Blood
CB mononuclear cells were obtained from healthy newborn donors by a protocol approved by the ethical committee of the Medical Faculty of the Technical University Munich. After Ficoll density separation, CD34⁺ cells were enriched to purities of 80% to 95% by magnetic-activated cell sorting using the Direct CD34 Progenitor Cell Isolation Kit (Miltenyi Biotec).

Differentiation of Endothelial Cells From EPCs
CD34⁺ cells were cultured in gelatin-coated plates in endothelial differentiation medium consisting of 80% basal Iscove medium (Biochrom), 10% horse serum (PAN Biotech), 10% heat-inactivated FCS (Biochrom), 2 mmol/L L-alanyl-L-glutamine, 100 U/mL penicillin, and 100 µg/mL streptomycin (all from BioWhittaker). The medium contained the recombinant human cytokines vascular endothelial growth factor (VEGF) 50 ng/mL, fibroblast growth factor-2 (FGF-2) 20 ng/mL, stem cell factor (SCF) 50 ng/mL (all from R&D Systems), and stem cell growth factor-ß (SCGF-ß) 20 ng/mL (PeproTech). Before reaching confluency (usually after 2 to 3 weeks), CBECs were transferred to gelatin-coated tissue culture flasks in endothelial cell basal medium-2 (EGM-2) supplemented with 2% FCS, VEGF, epidermal growth factor, FGF-2, R³-IGF-1 (a pharmacologically more available isoform of insulin-like growth factor), hydrocortisone, ascorbic acid, heparin, gentamicin, and amphotericin B (BioWhittaker). CBECs could be expanded most efficiently when grown at low density (1x10³/cm²).

Immunofluorescence
The following monoclonal antibodies were used: anti-CD14-PE, anti-CD34-PE, anti-CD45-FITC, anti-HLA-DR-FITC (all from BD Biosciences), anti-CD133-PE (Miltenyi Biotec), anti-CD146, anti-CD146-FITC, anti-E-cadherin (all from Chemicon), anti-34-PC5 (Immunotech), anti-CD31-FITC, and anti-KDR (both from Sigma). Unconjugated primary monoclonal antibodies were detected by RPE-conjugated F(ab')₂ fragment of rabbit anti-mouse immunoglobulin (Dako). Samples of cells were also stained with isotype-matched control antibodies (purchased from BD Biosciences and Immunotech). FACS analyses were performed using a FACScan flow cytometer (BD Biosciences) and Cell Quest software (BD Biosciences).

DiI-Ac-LDL Labeling
Fluorescent labeling of endothelial cells by uptake of acetylated (Ac)-LDL was performed by incubating CBECs with 2 µg/mL DiI-Ac-LDL (Harbor Bio-Products) in EGM-2 for 60 minutes at 37°C.

Lentiviral Constructs
We used 2 HIV-1–derived self-inactivating lentiviral gene transfer constructs. The EGFP-encoding vector pHR'SIN.cPPT-SEW (kindly provided by Adrian Thrasher, London, UK) has been described elsewhere.²¹ The vector cPPT-C(FVIIIB)IGWS is a derivative of the bicistronic vector C(FVIIIB)IGWS²² coding for human B-domain deleted FVIII (BDD FVIII) and EGFP. cPPT-C(FVIIIB)IGWS additionally contains the central polypurine tract and central termination sequence (cPPT/CTS)²³ from HIV-1 that was inserted 5' of the internal CMV promoter.

Production of Lentiviral Supernatants
Vector DNA was transiently introduced into 293T cells by triple cotransfection (calcium phosphate coprecipitation) with the packaging construct pCMVR8.91²⁴ and the pseudotyping construct pMD2.VSVG (kindly provided by Luigi Naldini, Turino, Italy)²⁵ coding for the vesicular stomatitis virus glycoprotein. Viral supernatants were concentrated by ultracentrifugation. Virus titers were determined as 293T-transducing units (transducing units per milliliter).

Transduction of CBEC
CBECs were transduced in the presence of 4 µg/mL protamine sulfate (Sigma) and 50 µmol/L dNTPs (New England Biolabs). After spinoculation (1250g, 90 minutes, 32°C), the cells were incubated for an additional 16 hours at 37°C with the virus. EGFP expression was analyzed by FACS analysis and fluorescence microscopy at various time points.

FVIII Quantification
FVIII antigen (FVIII:Ag) and FVIII activity (FVIII:C) were determined with the Immunozym FVIII:Ag ELISA and the Immunochrom FVIII:C chromogenic assay, respectively (Immuno). FVIII levels are given as international units (IU) per milliliter, with 150 ng/mL corresponding to 1.0 IU/mL. FVIII standards in both assays were calibrated against WHO plasma standards by the manufacturer.

FVIII Western Blot
Confluent layers of CBECs were cultured with EGM-2 without FCS for 15 to 24 hours, because the serum content would not have allowed efficient FVIII concentration. Supernatants were filtered (0.22 µm) and concentrated up to 400-fold by ultrafiltration in Vivaspin 20 concentrators (Sartorius) with a molecular weight cutoff of 30 kDa. After SDS-PAGE and blotting, FVIII protein was visualized by incubation with polyclonal sheep anti-human factor VIII:C antibody (Enzyme Research Laboratories) and peroxidase-conjugated donkey anti-sheep IgG secondary antibody (Sigma) followed by enhanced chemiluminescence (Pierce). As positive controls, we used recombinant B-domain–deleted (BDD) human FVIII ReFacto (Pharmacia and Upjohn) and plasma-derived human FVIII (Octanate; kindly provided by Lothar Biesert, Octapharma, Frankfurt, Germany).

In Vitro Matrigel Assay
Prechilled 24-well plates were coated with 500 µL Matrigel basement membrane matrix (BD Biosciences) per well and incubated for 1 hour at 37°C. CBECs were seeded on top of the gelled Matrigel at 6x10⁴ to 1x10⁵ cells in 400 µL. Cultures were incubated at 37°C. After 8 to 10 hours, the cultures were checked for tube formation by phase contrast and fluorescence microscopy (Nikon Eclipse TE300).

Statistical Analysis
Data are presented as mean±SD. The means of transduction efficiencies and FVIII secretion levels of cells transduced at different multiplicities of infection (MOIs) were compared with the use of a 2-tailed paired Student’s t test. Statistical analysis was performed using the GraphPad Prism 3.0 software.

	Results

Top Abstract Introduction Methods Results Discussion References

 
Differentiation and Culture of CBECs
To investigate whether EPCs could be derived from CB, CB CD34⁺ cells were cultured in endothelial differentiation medium containing VEGF, FGF-2, SCF, and SCGF-ß. Proliferating adherent cells with endothelial morphology were passaged before reaching confluency. With an input of 10⁵ to 10⁷ CD34⁺ cells, adherent cells could be expanded up to 10⁹-fold during a total culture time of 8 weeks. Figure 1 shows the growth curve of a representative experiment, with CBEC proliferating with a doubling time of 30 to 35 hours (denoted as WT).

View larger version (25K): [in this window] [in a new window]   Figure 1. Cumulative growth curve of CBECs. In the representative experiment shown, 106 CD34+ cells from cord blood obtained from a pool of donors were differentiated into CBECs and cultured until senescence. The graph defined by the black squares (WT; ) gives the number of CBECs plotted against the time after the enrichment of CD34+ cells. On day 37, 104 cells were plated for transduction 48 hours later with the constructs cPPT-C(FVIIIB)IGWS [C(F8)IGWS; ] and pHR'SIN.cPPT-SEW (SEW; ).

Cultures with CB mononuclear cells before CD34 isolation and with the CD34-depleted fraction either did not yield any detectable CBEC differentiation under otherwise identical conditions or yielded a considerably lower cell number (<1%). This strongly indicates that the isolation of CD34-expressing cells was important to enrich for CBECs or that CD34-negative cells may have inhibited endothelial cell differentiation. Although we did not attempt to prove this on a single-cell level, our data suggest that the CBECs were derived from CD34⁺ progenitor cells.

Phenotypic Characterization of CBECs
The adherent cells grew as monolayers of spindle-shaped flat cells (Figure 2). The cells were able to incorporate DiI-Ac-LDL (Figure 2) and showed formation of tubules in the Matrigel assay (see below). The cells were found to be uniformly positive for E-cadherin (CD144), CD146, and CD31, which are typical of endothelial cells. The cells were uniformly negative for the hematopoietic markers CD45, CD14, CD133, and HLA-DR (Figure 2). FACS analysis of 5 batches of endothelial cells showed that the cells obtained with our protocol were heterogeneous concerning the expression of CD34 and KDR/VEGF receptor (Figure 2). CD34 expression was detected on 5% to 45% of cells, whereas KDR was weakly expressed by less than 5% of CBECs, as estimated by dot blot analysis (data not shown). The expression of the aforementioned cell-surface markers seemed to be unchanged throughout cell expansion (data not shown), suggesting that a population of endothelial progenitor cells was maintained throughout the expansion, which gave rise to the CBECs. Additionally, the expression of the endothelial marker von Willebrand factor and binding of Ulex europaeus agglutinin could be demonstrated by immunohistochemical analysis (data not shown).

View larger version (50K): [in this window] [in a new window]   Figure 2. Morphology and phenotypic characterization of CBEC. CBECs were analyzed for uptake of DiI-Ac-LDL and expression of cell-surface markers by flow cytometry at various time points during culture. The x-axis gives the fluorescence intensity of the analyzed cells. Cells that were labeled with DiI-Ac-LDL or antibodies are represented by the thick lines, whereas the thin lines represent the appropriate negative controls.

Transduction Efficiency
CBECs from 4 different pools of donors were transduced with the constructs pHR'SIN.cPPT-SEW encoding EGFP and cPPT-C(FVIIIB)IGWS encoding human BDD FVIII and EGFP. MOIs of 10 yielded transduction efficiencies of 88.2%±7.6% and 76.9%±5.0% with pHR'SIN.cPPT-SEW and cPPT-C(FVIIIB)IGWS, respectively. When the MOIs were raised to 100, there was no statistically significant increase in transduction efficiencies (90.4%±9.5% and 90.6%±9.7%, respectively; P>0.05 for both vectors). The percentage of EGFP-expressing cells as well as the EGFP expression level remained relatively unchanged throughout the expansion, suggesting that endothelial progenitor cells have been successfully transduced. Figure 3 shows the EGFP expression of transduced versus nontransduced cells 30 days after transduction with MOIs of 10 in a representative experiment.

View larger version (34K): [in this window] [in a new window]   Figure 3. Lentiviral transduction of CBECs. In 4 independent experiments, CBECs at passages 3 through 8 were transduced with constructs pHR'SIN.cPPT-SEW (SEW) and cPPT-C(FVIIIB)IGWS [C(F8)IGWS] at MOIs of 10 and 100, and transduction efficiencies were determined by analysis of EGFP expression with untransduced cells as negative controls. The histogram illustrates the level of EGFP expression in a representative experiment as detected by FACS 30 days after transduction with C(F8)IGWS (MOI 10; black) and SEW (MOI 10; gray). Untransduced cells served as negative controls (white).

Influence of Lentiviral Transduction on CBEC Phenotype and Proliferation
We observed some cell death during and after transduction (below 10% with the lower MOI and 20% to 50% with the higher MOI) and a lag phase before the cells recovered to take up again the proliferation rate of untransduced control cells, as illustrated in Figure 1. To investigate whether delayed proliferation may be accompanied by alterations of the cell phenotype, we analyzed the expression of several surface markers of transduced cells several weeks after transduction. The expression of CD146, CD34, and KDR was unchanged in comparison to untransduced cells (data not shown), as was the uptake of DiI-Ac-LDL (Figures 4A and 4B). In addition, transduced CBECs retained their ability to form tubes in the Matrigel assay (Figures 4C and 4D), which is characteristic of functional endothelial cells.

View larger version (70K): [in this window] [in a new window]   Figure 4. Phenotype of transduced CBECs. Incorporation of DiI-Ac-LDL and in vitro tube formation. A and B, Five weeks after transduction with pHR'SIN.cPPT-SEW (MOI 100), cells were incubated with DiI-Ac-LDL and examined for EGFP expression (A) and DiI-Ac-LDL uptake (B) by fluorescence microscopy. C and D, For the tube formation assay, transduced CBECs were plated on Matrigel and examined for the formation of angiogenic tubes (C, phase contrast; D, EGFP expression).

Quantification of Secreted FVIII Protein and Coagulation Activity
To investigate whether the transduction of CBECs with the BDD FVIII cDNA has also included endothelial progenitor cells, which would be necessary to allow expansion of these cells, the capacity of CBECs to express BDD FVIII was investigated at 5 time points throughout the expansion period. Supernatants of cells transduced with construct cPPT-C(FVIIIB)IGWS were analyzed for FVIII coagulation activity (FVIII:C) using a chromogenic assay. It seemed that the expression of FVIII was relatively constant over the expansion period, with high amounts of FVIII secreted. During the first 4 weeks after transduction of cells from 3 pools of donors, mean FVIII:C levels were 0.35 to 0.39 IU/5x10⁴ cells per 48 hours at an MOI of 10, corresponding to 7.0 to 7.8 IU/10⁶ cells per 48 hours (Figure 5A). FVIII:C secretion then decreased in all cultures, which was accompanied by reduced proliferation and finally senescence of CBECs.

View larger version (43K): [in this window] [in a new window]   Figure 5. Detection of FVIII in CBEC supernatants. A, CBECs from 3 pools of donors were transduced with the BDD FVIII-encoding construct cPPT-C(FVIIIB)IGWS at an MOI of 10. FVIII:C levels were determined using a chromogenic assay. Every assay included supernatants from cells transduced with pHR'SIN.cPPT-SEW and from untransduced cells. No FVIII:C could be detected in these controls. B, Detection of FVIII protein in concentrated CBEC supernatants by immunoblotting. To detect signals of various intensities, the autoradiography film was exposed for 1 minute (1a through 4a) and 20 minutes (1b through 4b). Lane 1, Octanate (Oct) (plasma-derived full-length FVIII); lane 2, ReFacto (ReF) (recombinant BDD FVIII); lane 3, cPPT-C(FVIIIB)IGWS-transduced CBECs (BDD FVIII); lane 4, untransduced CBECs.

In a second set of experiments, it was analyzed whether higher MOIs additionally increase the amount of FVIII protein secreted. However, raising the MOI from 10 to 100 led only to a slight increase in FVIII:C secretion (P>0.05, n=4). Cells that were transduced with the control construct pHR'SIN.cPPT-SEW and untransduced cells did not secrete detectable amounts of FVIII:C (all <0.01 IU/5x10⁴ cells per 48 hours; n=7).

To compare the amount of coagulation-active FVIII protein (FVIII:C) to the total amount of FVIII antigen secreted (FVIII:Ag), the supernatants were additionally analyzed using an FVIII:Ag-specific ELISA. In fact, the amount of FVIII protein secreted exceeded that of the functionally active FVIII. FVIII:Ag levels in the same CBEC supernatants were 0.45 to 0.66 IU/5x10⁴ cells per 48 hours at an MOI of 10 compared with FVIII:C levels of 0.35 to 0.39 IU/5x10⁴ cells per 48 hours (n=3). This resulted in mean ratios of FVIII:C/FVIII:Ag of 0.54 to 0.83 (n=3).

Characterization of Secreted FVIII by Western Blot Analysis
To additionally characterize the recombinant coagulation FVIII released by CBECs, Western blot analysis of the FVIII protein was performed. FVIII was enriched from CBEC supernatants by ultrafiltration and analyzed by SDS-PAGE and immunoblotting. As reference, we used plasma-derived FVIII (Octanate) and recombinant BDD FVIII (ReFacto). Octanate consists of a doublet of light chains of 80 kDa and heavy chains of various sizes attributable to multiple proteolytic steps involving the B domain in vivo. ReFacto is composed of an 80-kDa doublet of light chains and a 90-kDa heavy chain. The deletion of the B domain used to express ReFacto is almost identical to the deletion in construct cPPT-C(FVIIIB)IGWS. cPPT-C(FVIIIB)IGWS-transduced CBECs secreted FVIII protein correctly cleaved into heavy chain (90 kDa) and light chain (80 kDa) with only a small fraction of uncleaved FVIII protein (170 kDa) (Figure 5B), indicating that the intracellular cleavage of FVIII into heavy chain and light chain might not have been complete. No FVIII protein could be detected in concentrated supernatants of untransduced cells.

	Discussion

Top Abstract Introduction Methods Results Discussion References

 
We examined whether CBECs could be expanded significantly in vitro and possess the capacity for high-level transgene expression. After enrichment of CD34⁺ cells, our cultures containing VEGF, FGF-2, SCF, and SCGF-ß yielded adherent cells that had an endothelial phenotype and a very high proliferative potential. To test whether CBECs would be suitable vectors for the expression of transgenes, we established a transduction protocol using integrating lentiviral vectors encoding the marker gene EGFP and the therapeutic gene FVIII. A moderate MOI of 10 proved to be sufficient to achieve transduction rates of 75% to 95% without impairing the in vitro proliferation and expansion properties. The transductions resulted in very high levels of FVIII secretion that were determined by a chromogenic assay and by ELISA. FVIII:C levels corresponded to 7.0 to 7.8 IU/10⁶ cells per 48 hours and were among the highest levels reported for FVIII so far in other recombinant systems.^12,26 We could also achieve comparable levels of FVIII secretion in HUVECs transduced with the same vector but 10- to 20-fold lower levels in hematopoietic cell lines that were transduced with the same transgene cassette.²² In addition, the level of FVIII secretion in CBECs exceeded by more than 10-fold that observed by Chuah et al,²⁷ who used a retroviral vector for FVIII expression in HUVECs.

The amount of FVIII secreted was even higher when the total FVIII protein was analyzed by ELISA, which showed that only 54% to 83% of the secreted FVIII protein was active. This reduction in specific activity, which was not observed in HUVECs, could be caused by incomplete intracellular proteolysis, because Western blotting of concentrated cell supernatant demonstrated that a small part of the FVIII protein was secreted as a 170-kDa precursor whereas the major part of the FVIII protein was already cleaved into the active heavy and light chains of 90 and 80 kDa, respectively. It can be assumed that the FVIII protein secreted as a precursor protein will be additionally cleaved in the plasma of hemophilia patients when clinically applied. The high levels of FVIII secretion by CBECs might reflect the fact that at least 1 subtype of endothelial cells, the liver sinusoidal endothelial cells, is one of the physiological sites of FVIII expression in vivo.^6,7 The data presented here (together with the study by Lin et al²⁰) suggest that endothelial cells from extrahepatic origin possess the complex set of proteins necessary for the posttranslational processing of FVIII, which includes folding, glycosylation, sulfation, and proteolytic cleavage. Hence, this study indicates that CBECs seem to be particularly suited for the recombinant expression of FVIII and that the lentiviral vector allows for considerably higher and more efficient recombinant FVIII expression than previously used viral or nonviral expression vectors.

In addition, we propose CBECs as alternate cell source for therapeutic applications in inherited coagulation disorders, such as hemophilia A gene therapy. The establishment of a successful ex vivo gene therapeutic approach to treat hemophilia A patients depends on the identification of target cells that meet most or all of the following demands. Apart from the crucial requirement to secrete procoagulant FVIII protein, the target cells have to be easily accessible and easy to transfect or transduce. They also need to be expandable in vitro or in vivo under controlled conditions, and they or their progeny should have direct access to the circulation. The use of CBECs for patients implies the use of large, constantly replenished storage banks because of the finite proliferative potential of cells. However, a finite proliferative potential may be a prerequisite for vector-mediated gene transfer as issues of insertional mutagenesis resurface.²⁸ Although CBECs seem to be suitable for autologous applications, they may also be valuable in allogeneic settings using immunoisolation devices or alginate encapsulation.²⁹ An important question in the context of gene therapy regards the immunogenicity of the transduced cells. The use of a self-inactivating lentiviral gene transfer vector with a deletion in the 3'-long terminal repeat entails that the vector cannot replicate within the transduced cells and that no viral proteins are expressed from vectors that are comparable to the ones used in this study. Therefore, the only immunologically relevant proteins are the FVIII protein and the EGFP protein, the latter of which would not be required for therapeutic applications. Whether FVIII-transduced CBECs can trigger FVIII-specific immune responses in vivo has not been investigated yet.

In extension of a previous study using blood outgrowth endothelial cells,²⁰ we have identified CB-derived EPCs and their progeny as potential novel target cells for gene therapy of hemophilia. The properties of EPCs isolated from different origins, such as peripheral blood, CB, or bone marrow, have not yet been thoroughly characterized and compared. Circulating CD34⁺ cells of all of these sources contribute to the formation of endothelium in vivo. However, the stimuli that induce mobilization of these cells and those that determine their migration into specific tissues are poorly understood. These stimuli seem to be released under pathological conditions, such as ischemia in myocardium or retina, after exposition to ionizing radiation or during tumor angiogenesis.^30–32 Although the development of therapeutic strategies using EPCs is very appealing, engraftment will depend on factors that favor the persistence of ex vivo–modified EPCs. The factors allowing optimal local engraftment of EPCs are presently unknown, as are the advantages or disadvantages of EPCs from different origins, such as CB and peripheral blood.

In recent studies, gene therapy using gene-modified endothelial cells was usually discussed in the context of cardiovascular diseases and the support of neovascularization in ischemic tissues³³ or the context of cancer therapy to inhibit angiogenesis in solid tumors.³⁴ This study presents a novel cell type that can readily be transduced with lentiviral vectors and that might therefore prove valuable for vascular regeneration and cancer gene therapy using gene-modified EPCs. However, the study emphasizes a wider field of application for gene-modified endothelial cells. In particular, we propose the use of CBECs for gene therapy of hemophilia A and other congenital disorders that are characterized by the absence of plasma proteins such as factor IX, von Willebrand factor, or ₁-antitrypsin.

	Acknowledgments

 
Acknowledgments

This work was supported by a grant of the Stiftung Hämotherapie-Forschung to T.T. and a grant from the Biotest AG (Dreieich, Germany). The authors thank the doctors and midwives of the Kreiskrankenhaus Neuperlach (Munich, Germany) for their support of the research and cord blood.

	Footnotes

 
C.H. and T.T. contributed equally to this study.

Received August 15, 2003; accepted September 23, 2003.

	References

Top Abstract Introduction Methods Results Discussion References

 

1. Mannucci PM, Tuddenham EGD. The hemophilias: from royal genes to gene therapy. N Engl J Med. 2001; 344: 1773–1779.[Free Full Text]
2. Mannucci PM, Giangrande PLF. Choice of replacement therapy for hemophilia: recombinant products only? Hematol J. 2001; 1: 72–76.[CrossRef]
3. Ragni MV. Safe passage: a plea for safety in hemophilia gene therapy. Mol Ther. 2002; 6: 436–440.[CrossRef][Medline] [Order article via Infotrieve]
4. Wion KL, Kelly D, Summerfield JA, Tuddenham EG, Lawn RM. Distribution of FVIII mRNA and antigen in human liver and other tissues. Nature. 1985; 317: 726–729.[CrossRef][Medline] [Order article via Infotrieve]
5. Zelechowska MG, van Mourik JA, Brodniewicz-Proba T. Ultrastructural localization of factor VIII procoagulant antigen in human liver hepatocytes. Nature. 1985; 317: 729–730.[CrossRef][Medline] [Order article via Infotrieve]
6. Do H, Healey JF, Waller EK, Lollar P. Expression of factor VIII by murine liver sinusoidal endothelial cells. J Biol Chem. 1999; 274: 19587–19592.[Abstract/Free Full Text]
7. Hollestelle MJ, Thinnes T, Crain K, Stiko A, Kruijt JK, van Berkel TJC, Loskutoff DJ, van Mourik JA. Tissue distribution of FVIII gene expression in vivo: a closer look. Thromb Haemost. 2001; 86: 855–861.[Medline] [Order article via Infotrieve]
8. VandenDriessche T, Collen D, Chuah MKL. Viral vector-mediated gene therapy for hemophilia. Curr Gene Ther. 2001; 1: 301–315.[CrossRef][Medline] [Order article via Infotrieve]
9. Kelley K, Verma I, Pierce GF. Gene therapy: reality or myth for the global bleeding disorders community? Haemophilia. 2002; 8: 261–267.[CrossRef][Medline] [Order article via Infotrieve]
10. Yang Y, Jooss KU, Su Q, Ertl HC, Wilson JM. Immune responses to viral antigens versus transgene product in the elimination of recombinant adenovirus-infected hepatocytes in vivo. Gene Ther. 1996; 3: 137–144.[Medline] [Order article via Infotrieve]
11. Lozier JN, Metzger ME, Donahue RE, Morgan RA. Adenovirus-mediated expression of human coagulation factor IX in the rhesus macaque is associated with dose-limiting toxicity. Blood. 1999; 94: 3968–3975.[Abstract/Free Full Text]
12. Roth DA, Tawa NE Jr, O’Brien JM, Treco DA, Selden RF. Nonviral transfer of the gene encoding coagulation factor VIII in patients with severe hemophilia. N Engl J Med. 2001; 344: 1735–1742.[Abstract/Free Full Text]
13. Murohara T, Ikeda H, Duan J, Shintani S, Sasaki K, Eguchi H, Onitsuka I, Matsui K, Imaizumi T. Transplanted cord blood-derived endothelial precursor cells augment postnatal neovascularization. J Clin Invest. 2000; 105: 1527–1536.[Medline] [Order article via Infotrieve]
14. Salven P, Mustjoki S, Alitalo R, Alitalo K, Rafii S. VEGFR-3 and CD133 identify a population of CD34⁺ lymphatic/vascular endothelial precursor cells. Blood. 2003; 101: 168–172.[Abstract/Free Full Text]
15. Burger PE, Coetzee S, McKeehan WL, Kan M, Cook P, Fan Y, Suda T, Hebbel RP, Novitzky N, Muller WA, Wilson EL. Fibroblast growth factor receptor-1 is expressed by endothelial progenitor cells. Blood. 2002; 100: 3527–3535.[Abstract/Free Full Text]
16. Yoo ES, Lee KE, Seo JW, Yoo EH, Lee MA, Im SA, Mun YC, Lee SN, Huh JW, Kim MJ, Jo DY, Ahn JY, Lee SM, Chung WS, Kim JH, Seong CM. Adherent cells generated during long-term culture of human umbilical cord blood CD34⁺ cells have characteristics of endothelial cells and beneficial effect on cord blood ex vivo expansion. Stem Cells. 2003; 21: 228–235.[Abstract/Free Full Text]
17. Eggermann J, Kliche S, Jarmy G, Hoffmann K, Mayr-Beyrle U, Debatin KM, Waltenberger J, Beltinger C. Endothelial progenitor cell culture and differentiation in vitro: a methodological comparison using human umbilical cord blood. Cardiovasc Res. 2003; 58: 478–486.[Abstract/Free Full Text]
18. Saenko EL, Ananyeva NM, Moayeri M, Ramezani A, Hawley RG. Development of improved factor VIII molecules and new gene transfer approaches for hemophilia A. Curr Gene Ther. 2003; 3: 27–41.[CrossRef][Medline] [Order article via Infotrieve]
19. Rafii S, Lyden D. Therapeutic stem and progenitor cell transplantation for organ vascularization and regeneration. Nat Med. 2003; 9: 702–712.[CrossRef][Medline] [Order article via Infotrieve]
20. Lin Y, Chang L, Solovey A, Healey JF, Lollar P, Hebbel RP. Use of blood outgrowth endothelial cells for gene therapy for hemophilia A. Blood. 2002; 99: 457–462.[Abstract/Free Full Text]
21. Demaison C, Parsley K, Brouns G, Scherr M, Battmer K, Kinnon C, Grez M, Thrasher AJ. High-level transduction and gene expression in hematopoietic repopulating cells using a human immunodeficiency virus type 1-based lentiviral vector containing an internal spleen focus forming virus promoter. Hum Gene Ther. 2002; 13: 803–813.[CrossRef][Medline] [Order article via Infotrieve]
22. Tonn T, Herder C, Becker S, Seifried E, Grez M. Generation and characterization of human hematopoietic cell lines expressing factor VIII. J Hematother Stem Cell Res. 2002; 11: 695–704.[CrossRef][Medline] [Order article via Infotrieve]
23. Zennou V, Petit C, Guetard D, Nehrbass U, Montagnier L, Charneau P. HIV-1 genome nuclear import is mediated by a central DNA flap. Cell. 2000; 101: 173–185.[CrossRef][Medline] [Order article via Infotrieve]
24. Zufferey R, Nagy D, Mandel RJ, Naldini L, Trono D. Multiply attenuated lentiviral vector achieves efficient gene delivery in vivo. Nature Biotechnol. 1997; 15: 871–875.[CrossRef][Medline] [Order article via Infotrieve]
25. Follenzi A, Naldini L. Generation of HIV-1 derived lentiviral vectors. Methods Enzymol. 2002; 346: 454–465.[Medline] [Order article via Infotrieve]
26. Andrews JL, Weaver L, Kaleko M, Connelly S. Efficient adenoviral vector transduction and expression of functional human factor VIII in cultured primary human hepatocytes. Haemophilia. 1999; 5: 160–168.[CrossRef][Medline] [Order article via Infotrieve]
27. Chuah MKL, VandenDriessche T, Morgan RA. Development and analysis of retroviral vectors expressing human factor VIII as a potential gene therapy for hemophilia A. Hum Gene Ther. 1995; 6: 1363–1377.[Medline] [Order article via Infotrieve]
28. Schröder ARW, Shinn P, Chen H, Berry C, Ecker JR, Bushman F. HIV-1 integration in the human genome favors active genes and local hotspots. Cell. 2002; 110: 521–529.[CrossRef][Medline] [Order article via Infotrieve]
29. Garcia-Martin C, Chuah MKL, van Damme A, Robinson KE, Vanzieleghem B, Saint-Remy JM, Gallardo D, Ofosu FA, VandenDriessche T, Hortelano G. Therapeutic levels of human factor VIII in mice implanted with encapsulated cells: potential for gene therapy of hemophilia A. J Gene Med. 2002; 4: 215–223.[CrossRef][Medline] [Order article via Infotrieve]
30. Asahara T, Murohara T, Sullivan A, Silver M, van der Zee R, Li T, Witzenbichler B, Schatteman G, Isner JM. Isolation of putative progenitor endothelial cells for angiogenesis. Science. 1997; 275: 964–967.[Abstract/Free Full Text]
31. Jackson KA, Majka SM, Wang H, Pocius J, Hartley CJ, Majewsky MW, Entman ML, Michael LH, Hirschi KK, Goodell MA. Regeneration of ischemic cardiac muscle and vascular endothelium by adult stem cells. J Clin Invest. 2001; 107: 1395–1402.[CrossRef][Medline] [Order article via Infotrieve]
32. Grant MB, May WS, Caballero S, Brown GAJ, Guthrie SM, Mames RN, Byrne BJ, Vaught T, Spoerri PE, Peck AB, Scott EW. Adult hematopoietic stem cells provide functional hemangioblast activity during retinal neovascularization. Nat Med. 2002; 6: 607–612.
33. Iwaguro H, Yamaguchi J, Kalka C, Murasawa S, Masuda H, Hayashi S, Silver M, Li T, Isner JM, Asahara T. Endothelial progenitor cell VEGF gene transfer for vascular regeneration. Circulation. 2002; 105: 732–738.[Abstract/Free Full Text]
34. Ferrari N, Glod J, Lee J, Kobiler D, Fine HA. Bone marrow-derived, endothelial progenitor-like cells as angiogenesis-selective gene-targeting vectors. Gene Ther. 2003; 10: 647–656.[CrossRef][Medline] [Order article via Infotrieve]

This article has been cited by other articles:

				S. F. De Meyer, K. Vanhoorelbeke, M. K. Chuah, I. Pareyn, V. Gillijns, R. P. Hebbel, D. Collen, H. Deckmyn, and T. VandenDriessche Phenotypic correction of von Willebrand disease type 3 blood-derived endothelial cells with lentiviral vectors expressing von Willebrand factor Blood, June 15, 2006; 107(12): 4728 - 4736. [Abstract] [Full Text] [PDF]

				J. W. Liu, G. Pernod, S. Dunoyer-Geindre, R. J. Fish, H. Yang, H. Bounameaux, and E. K. O. Kruithof Promoter Dependence of Transgene Expression by Lentivirus-Transduced Human Blood-Derived Endothelial Progenitor Cells Stem Cells, January 1, 2006; 24(1): 199 - 208. [Abstract] [Full Text] [PDF]

				L. G. Melo, M. Gnecchi, A. S. Pachori, D. Kong, K. Wang, X. Liu, R. E. Pratt, and V. J. Dzau Endothelium-Targeted Gene and Cell-Based Therapies for Cardiovascular Disease Arterioscler. Thromb. Vasc. Biol., October 1, 2004; 24(10): 1761 - 1774. [Abstract] [Full Text] [PDF]

				J. D. Pearson Using Endothelial Progenitor Cells for Gene Therapy Arterioscler. Thromb. Vasc. Biol., December 1, 2003; 23(12): 2117 - 2118. [Full Text] [PDF]

This Article Abstract Full Text (PDF) All Versions of this Article: 23/12/2266    most recent 01.ATV.0000100403.78731.9Fv1 Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Herder, C. Articles by Seifried, E. Search for Related Content PubMed PubMed Citation Articles by Herder, C. Articles by Seifried, E. Related Collections Coagulation Gene therapy Gene expression