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(Hypertension. 2001;38:65.)
© 2001 American Heart Association, Inc.

Scientific Contributions

Analysis of Cell-Specific Promoters for Viral Gene Therapy Targeted at the Vascular Endothelium

Stuart A. Nicklin; Paul N. Reynolds; M. Julia Brosnan; Steve J. White; David T. Curiel; Anna F. Dominiczak; Andrew H. Baker

From the Department of Medicine and Therapeutics, University of Glasgow, Western Infirmary (S.A.N., M.J.B., A.F.D., A.H.B.), Glasgow, United Kingdom; The Gene Therapy Center at the University of Alabama at Birmingham (P.N.R., D.T.C.); and Bristol Heart Institute, University of Bristol, Bristol Royal Infirmary (S.J.W.), Bristol, United Kingdom.

Correspondence to Dr A.H. Baker, Department of Medicine and Therapeutics, University of Glasgow, Glasgow G11 6NT, United Kingdom. E-mail A.H.Baker{at}clinmed.gla.ac.uk

	Abstract

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Abstract— The use of viral vectors for vascular gene therapy targeted at the endothelium is limited by the promiscuous tropism of vectors and nonspecificity of viral promoters, resulting in high-level transgene expression in multiple tissues. To evaluate suitable endothelial cell (EC)–specific promoters for vascular gene therapy, we directly compared the ability of the fms-like tyrosine kinase-1 (FLT-1), intercellular adhesion molecule-2 (ICAM-2), and von Willebrand factor (vWF) promoters to drive EC-restricted transcription after cloning into adenoviral vectors upstream of lacZ. Vastly different expression profiles were observed. Whereas both FLT-1 and ICAM-2 promoters generated transgene expression levels similar to cytomegalovirus in ECs in vitro, vWF expression levels were extremely low. Analysis of non-EC types revealed that ICAM-2 but not FLT-1 evoked leaky transgene expression, thus identifying FLT-1 as the most selective promoter. With an ex vivo human gene therapy model, the FLT-1 promoter demonstrated EC-specific transgene expression in intact human vein but no detectable expression from infected exposed smooth muscle cells in EC-denuded vein. Furthermore, when adenoviruses were systemically administered to mice, the FLT-1 promoter demonstrated extremely low-level gene expression in the liver, the major target organ for adenoviral transduction in vivo. This study highlights the potential of using the FLT-1 promoter for local and systemic human gene therapy in hypertension and its complications.

Key Words: adenovirus • promoter • gene therapy • endothelium • cell adhesion molecules

	Introduction

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The vascular endothelium is an attractive target for clinical gene therapy. Its proximity to circulating blood and the vessel wall throughout the body underlies the need to generate gene therapeutic vehicles aimed specifically at the endothelium. The endothelium plays a fundamental role in many vascular pathologies, including endothelial dysfunction associated with hypertension and early atherogenesis, plaque rupture, postangioplasty restenosis, and late vein graft failure. However, current gene-delivery vectors, both viral and nonviral, demonstrate poor selectivity to endothelium after local or systemic delivery. There is therefore an urgent need to develop endothelial cell (EC)–specific vascular gene-transfer vectors, which would be applicable to prevention or attenuation of vascular complications of hypertension

We recently demonstrated¹ the ability to retarget adenoviral tropism to ECs, resulting in high-level and selective targeting at the level of virus-cell interaction. Although this results in EC-specific infection with adenoviral vectors, the inclusion of cell-specific promoters would further enhance selectivity and hence safety. Transgene production by the use of viral promoters such as the cytomegalovirus (CMV) immediate early promoter or the Rous sarcoma virus (RSV) evokes high-level gene expression in all cell types transduced, which clearly may be deleterious in clinical situations. The promoters for a number of genes with transcriptions restricted to endothelium have been sequenced and partially characterized; these include vascular cell adhesion molecule-1 (VCAM-1),² endothelial nitric oxide synthase (eNOS),³ von Willebrand factor (vWF),⁴ fms-like tyrosine kinase-1 (FLT-1),⁵ tyrosine kinase with immunoglobulin and epidermal growth factor homology domains (TIE),⁶ kinase-like domain receptor (KDR),⁷ and intercellular adhesion molecule-2 (ICAM-2).⁸ To date, no studies have directly compared candidate EC-specific promoters within the same viral gene-delivery system. Here, we document the gene expression profiles of adenoviral vectors using the candidate vWF, FLT-1, and ICAM-2 promoters in vitro, ex vivo, and in vivo.

	Methods

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Cell Culture
For this study, 293 cells, human primary foreskin fibroblasts (used between passages 3 and 6; a generous gift from Dr Mark Bond, Bristol Heart Institute, University of Bristol, UK), HeLa, and HepG2 cell lines were maintained in minimal essential media (MEM) supplemented with 100 IU/mL penicillin, 100 µg/mL streptomycin, 2 mmol/L L-glutamine, and 10% (vol/vol) fetal calf serum (FCS). Human umbilical vein endothelial cells (HUVECs) and human saphenous vein endothelial cells (HSVECs) were isolated by a modified version described by Jaffe⁹ and used below passage 5. ECs were identified by immunofluorescence for vWF. Vascular smooth muscle cells (VSMCs) were from medial explants of human saphenous vein obtained from patients undergoing bypass surgery.¹⁰ VSMCs were cultured until first passage in SmGM BulletKit media (Clonetics). At passage 1, they were confirmed as VSMCs by immunofluorescence for smooth muscle cell -actin. For subsequent passages, VSMCs were cultured in Dulbecco’s modified Eagle’s medium (4500 mg/L glucose, glutamax-1) supplemented with 100 IU/mL penicillin, 100 µg/mL streptomycin, and 20% (vol/vol) FCS. The IP-IB murine simian virus-40 (SV-40) transformed EC line (American Type Tissue Culture collection) was maintained as for VSMC except with 10% FCS. All cells were maintained at 37°C under a mixture of 95% air and 5% CO₂.

Adenoviral Constructs
The adenoviruses RAdCMV¹¹ and RAdLUC (obtained from Robert Gerard) express the bacterial LacZ and firefly luciferase genes, respectively, under the control of the CMV promoter. Polymerase chain reaction was used to clone EC-specific promoters for FLT-1 (-748 to 284),⁵ ICAM-2 (-367 to -34),⁸ and vWF (-487 to 247)⁴ into adenoviral vectors. Oligonucleotide primers were designed spanning the above sequences (FLT-1 sense 5'-CCC GCA TGC CTT CTA GGA AGC AGA AGA CTG AGG A-3', antisense 5'-CCC TCT AGA GTG AGC GCG ACG CGG CCT GCT CGC C-3'; ICAM-2 sense 5'-CCA TGG GAT TTG GGG TTC CC-3', anti-sense 5'-CCA AGG GCT GCC TGG AGG GA-3'; and vWF sense 5'-CCC GCA TGC ATC TTT AGC CGA TCC ATT CAA CCC T-3', antisense 5'-CCC TCT AGA CCC CTG CAA ATG AGG GCT GCG GCT A-3'). An SphI site and clamp (underlined) was synthesized at the 5' end of the FLT-1 and vWF sense primers and an XbaI site and clamp (underlined) at the 5' end of each FLT-1 and vWF antisense primer to create unique SphI and XbaI cloning sites. For ICAM-2, an NcoI site was engineered at the 5' end of the sense primer. For amplification of vWF, 1 ng of HUVEC DNA template was amplified with Vent DNA polymerase (New England Biolabs) for 35 cycles at 94°C for 1 minute, 54°C for 1 minute, and 72°C for 1 minute. FLT-1 was amplified from 1 ng of plasmid DNA template (a gift from L. Williams, University of California at San Francisco) under the same conditions as for vWF except that annealing was at 56°C. ICAM-2 was amplified from human genomic DNA with annealing at 61°C. FLT-1 and vWF promoters were cloned into the SphI/XbaI site of pMV10¹¹ upstream of LacZ and the CMV polyadenylation signal. Sequencing confirmed that no polymerase chain reaction–induced mutations were present. Expression cassettes were excised and cloned into the HindIII site of the adenovirus shuttle pMV60. ICAM-2 was cloned into pGEM-T-Easy (Promega) and sequenced. The fragment was then subcloned as an HinDIII-SmaI fragment and transferred into pE1sp1B (Microbix Biosystems), with the lacZ gene excised from pCA17 (Microbi- Biosystems) and confirmed by sequencing. Recombinant adenoviruses were generated by homologous recombination with pJM17 in low-passage 293 cells.¹² Recombinant adenoviruses designated RAdFLT-1, RAdvWF, and RAdICAM-2 were plaque purified, propagated on 293 cells, cesium chloride banded, and titered by standard techniques.¹³ Recombinant adenoviruses were assessed for lack of replication-competent adenovirus by plaque titration on nonpermissive HeLa cells and immunofluorescence for E1a after infection of HeLa.

Infection Protocols
Cells were trypsinized and plated into 24-well plates at 5x10⁴ cells/well (2.5x10⁴ for IP-IB cells). Immediately before infection, an accurate cell count was determined. We used a dose range to assess ß-galactosidase production in each cell type tested. Because of differing levels of both adenovirus cell entry receptors, _vß_3/5 integrins and the Coxsackie/adenovirus receptor (CAR), on different cell types, initial experiments were performed to allow direct comparison of each promoter. The quantity of virus required to achieve 100% infection into each cell type was first determined with the constitutive viral promoter in RAdCMV. Once this optimal level was determined, transgene levels were further characterized at 50% and 10% infection for each cell type. HUVECs, HSVECs, and IP-IB were infected with 100, 500, and 1000 pfu/cell of each recombinant adenovirus in triplicate cultures, VSMCs with 60, 300, or 600 pfu/cell, fibroblasts with 30, 150, or 300 pfu/cell, and HepG2s and HeLa with 10, 50, or 100 pfu/cell. A media change was performed 16 hours after infection. Cells were incubated for an additional 48 hours in complete media before either being fixed and stained with X-gal stain (5-bromo-4-chloro-3-indolyl-ß-D-galactopyranoside; 100 mmol/L sodium phosphate, pH 7.3 [77 mmol/L Na₂HPO₄, 23 mmol/L NaH₂PO₄], 1.3 mmol/L MgCl₂, 3 mmol/L K₃Fe(CN)₆, 3 mmol/L K₄Fe(CN)₆, and 20 mg/mL X-gal), as described previously,¹⁴ or being harvested for quantification (see below). All photomicrographs were taken randomly.

Quantitative ß-Galactosidase Assay
ß-Galactosidase production was quantified with a chemiluminescent reporter gene assay (Galacto-Light Plus, Tropix). Forty-eight hours after infection, cells were lysed for 10 minutes at 4°C in 50 µL of lysis buffer. Two to 20 µL of each resulting cell lysate was analyzed for ß-galactosidase levels according to the manufacturer’s recommendations. Each sample was quantified within the linear range of a standard curve.

In Situ Infection of Human Saphenous Vein
Freshly isolated (EC-intact) and surgically prepared (EC-denuded) vein segments were obtained from patients undergoing bypass surgery and prepared as described previously.^15,16 Surgically prepared vein segments that had undergone manual distension were obtained after storage in patients’ heparinized blood for 60 to 120 minutes. Freshly isolated vein was obtained after removal from the patient with minimal handling and before distension. Vein segments were cannulated and infected with 120 µL of adenovirus (1.2x10¹⁰ pfu/mL) as described previously.¹⁷ This protocol results in 39+7% transduction of exposed lumenal surface cells.¹⁷ Veins were pinned with the lumenal surface uppermost and cultured for 7 days in complete culture media (RPMI 1640, 100 IU/mL penicillin, 100 µg/mL streptomycin, 8 µg/mL gentamicin, 2 mmol/L L-glutamine, and 30% FCS).^15,16 ß-Galactosidase production was assessed at day 7 by X-gal staining and frozen serial sections.

In Vivo Analysis of Promoter Activity
Athymic nude mice (female, 8 weeks old; Frederick Cancer Research, Ft Detrick US Army Base, Md) were injected with 5x10¹⁰ particles of RAdCMV, RAdFLT-1, or RAdLUC (negative control) in 200 µL of PBS. Three days after infection, livers were harvested, frozen, and ground to a fine powder with a pestle and mortar. Lysed extracts were assayed for ß-galactosidase activity with Galacto-light Plus according to the manufacturer’s recommendations.

Statistical Analysis
All data were analyzed by an unpaired Student’s t test and are shown as mean±SEM.

	Results

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Analysis of ß-Galactosidase Production In Vitro
To evaluate the ability of RAdFLT-1, RAdICAM-2, or RAdvWF to drive cell-specific gene expression, histological analysis of ß-galactosidase production from each adenovirus was performed. As expected, primary HUVECs, HSVECs, VSMCs, and fibroblast cells required higher titers than HepG2 and HeLa cells (Figure 1). Infection of cells with RAdFLT-1, RAdICAM-2, or RAdvWF, however, produced vastly different ß-galactosidase staining compared with that from RAdCMV. In HUVECs and HSVECs, RAdFLT-1 and RAdICAM-2 but not RAdvWF produced high levels of ß-galactosidase, comparable to that evoked by RAdCMV (Figure 1). Evaluation of primary VSMCs and human fibroblasts demonstrated that RAdFLT-1 and RAdvWF produced lower levels of ß-galactosidase–positive cells than RAdCMV (Figure 1). However, RAdICAM-2, unlike RAdFLT-1 and RAdvWF, appeared to evoke high-level transgene expression in VSMCs, HeLa, fibroblasts, and HepG2 cells (Figure 1).

View larger version (105K): [in this window] [in a new window]   Figure 1. ß-Galactosidase staining in isolated endothelial and nonendothelial cells. Phase contrast pictures of cells stained for ß-galactosidase expression after infection with RAdCMV, RAdFLT-1, RAdvWF, or RAdICAM-2. Primary human VSMCs (300 pfu/cell), primary human fibroblasts (100 pfu/cell), HepG2 hepatocytes (100 pfu/cell), HeLa (100 pfu/cell), primary HUVECs (500 pfu/cell), and primary HSVECs (1000 pfu/cell) are shown. Representative of 3 independent experiments, each performed in triplicate.

We next quantified ß-galactosidase levels in cell extracts from both ECs (Figure 2) and non-ECs (Figure 3). In ECs, RAdFLT-1 and RAdICAM-2 produced high levels of ß-galactosidase. Interestingly, RAdICAM-2 evoked levels significantly higher than those from CMV (Figure 2). In accordance with histological analysis, vWF induced very low levels of ß-galactosidase in both HSVECs and HUVECs (Figure 2), as expected. Analysis of non-ECs revealed divergent results from each promoter (Figure 3). RAdICAM-2 was extremely active in VSMCs, HeLa, fibroblasts, and HepG2 cells, but levels from RAdFLT-1 were significantly lower than CMV (Figure 3). Similar to ECs, vWF demonstrated low-level activity in non-EC types, although some ß-galactosidase was observed in VSMCs (Figure 3). These data demonstrate that RAdFLT-1 induces high-level and selective EC-specific gene expression in vitro, and it was therefore selected for ex vivo and in vivo experiments.

View larger version (15K): [in this window] [in a new window]   Figure 2. Promoter activity in primary human ECs. ß-Galactosidase was quantified in cell lysates from primary ECs (HUVEC and HSVEC) infected with RAdCMV, RAdFLT-1, RAdvWF, or RAdICAM-2 at the MOI shown. *Statistical significance vs CMV at the same MOI (P<0.05). Data are representative of 3 independent experiments.

View larger version (16K): [in this window] [in a new window]   Figure 3. Promoter activity in non-ECs. ß-Galactosidase was quantified in cell lysates from primary non-ECs, including VSMCs, dermal fibroblasts, and nonendothelial cell lines HepG2 and HeLa infected with RAdCMV, RAdFLT-1, RAdvWF, or RAdICAM-2. *Statistical significance vs CMV at the same MOI (P<0.05). Representative of 3 independent experiments.

Promoter Activity in Human Saphenous Vein Ex Vivo
We further sought to define whether RAdFLT-1 could evoke similarly high-level EC-specific transcription in a clinically appropriate human model in which reporter gene expression can be evaluated in both endothelial (undamaged, freshly isolated vein) and nonendothelial (endothelium-denuded, surgically prepared vein) cell types. As expected, localized lumenal-specific delivery of RAdCMV to human saphenous vein demonstrated widespread lumenal surface staining of cells for ß-galactosidase in both freshly isolated and surgically prepared human saphenous vein (Figure 4). Histological cross sections revealed transgene expression at the lumenal surface of both vein types (Figure 4). Conversely, RAdFLT-1 demonstrated widespread staining in freshly isolated saphenous vein but minimal lumenal surface staining in surgically prepared vein (Figure 4). Analysis of histological sections demonstrated this selective expression profile with substantial staining in freshly isolated vein but no staining in surgically prepared vein (Figure 4). This demonstrates the ability of FLT-1 to evoke high-level EC-specific gene expression after local delivery into human vein.

View larger version (108K): [in this window] [in a new window]   Figure 4. CMV and FLT-1 promoter activity in human saphenous vein ex vivo. A, B, E, and F, En face ß-galactosidase staining of isolated segments of freshly isolated (A, E) or surgically prepared (B, F) human saphenous vein 7 days after lumenal specific infection. C, D, G, and H, Representative frozen cross sections of freshly isolated (C, G) or surgically prepared (D, H) vein. Scale bar in C represents 25 µm and is applicable to panels C, D, G, and H. Examples of ß-galactosidase–positive cells are indicated with arrows. Representative of 6 independent experiments/treatment.

Analysis of Promoter Activity In Vivo
For many vascular gene-delivery protocols based on both local and systemic delivery approaches, a potential deleterious effect of the transgene on nontarget tissue may be apparent if the vector has access to its primary site(s) of infection, either through leakage (in the case of local delivery) or through the bloodstream (systemic delivery). Therefore, we evaluated the activity of the FLT-1 and CMV promoters in liver in vivo, because this is the primary site for adenoviral infection. We first defined that the human FLT-1 promoter was functional in murine ECs. Although significantly lower than RAdCMV, RAdFLT-1 activity in IP-1B cells was 40% of that produced by RAdCMV, which demonstrates functional FLT-1 activity in murine ECs (Figure 5A). We next evaluated ß-galactosidase levels in the livers of mice injected with RAdCMV, RAdFLT-1, or RAdLUC (negative control). As expected, levels of ß-galactosidase were very high in livers of mice injected with RAdCMV; livers from RAdFLT-1–injected mice, however, demonstrated extremely low levels of ß-galactosidase production (Figure 5B).

View larger version (15K): [in this window] [in a new window]   Figure 5. FLT-1 promoter activity in murine ECs in vitro and liver in vivo. A, FLT-1 and CMV activity in the mouse EC-line IP-1B. Representative of 3 experiments. B, ß-Galactosidase production in murine liver tissue extracted 3 days after infection (n=5 mice/promoter). RLU indicates relative light units. *P<0.05 vs CMV.

	Discussion

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Effective use of gene therapy vectors within clinical settings requires the development of suitable targeting strategies. One of the major challenges is to develop vectors that incorporate targeting both at the level of vector-cell interaction and at the level of transcription. In the present study, we used an adenoviral system to directly compare transcription from 3 candidate endothelium-specific promoters in isolated cells in a relevant human vein model and in vivo. We demonstrated high-level reporter gene expression in human ECs from the FLT-1 and ICAM-2 promoter but not the vWF promoter. Whereas expression from the FLT-1 promoter was restricted to ECs, the ICAM-2 promoter was extremely leaky. The FLT-1 promoter also drove high-level, EC-restricted expression in human vein after local delivery and did not demonstrate any activity in hepatocytes in vivo after systemic delivery. We were surprised that the activity of the vWF promoter in endothelial cells was extremely low. Original characterization of this promoter with plasmid-based vectors demonstrated that expression levels in bovine ECs from the -487/247-bp vWF fragment were 90% of the level achieved with the core fragment (-90 to 155 bp).⁴ In non-EC types, the core promoter fragment achieved levels in bovine SMC and HeLa cells similar to that observed in ECs, but the levels from the -487/247-bp fragment were negligible.⁴ We demonstrated in the present study, however, that expression levels from the -487/247-bp vWF promoter were significantly lower than other candidate promoters when engineered into adenoviral vectors, presumably owing to the distinct influences the promoter is exposed to within adenoviral vectors, as has been observed for the human ventricular/slow myosin light chain 1 promoter.¹⁸ The 334-bp ICAM-2 promoter used in the present study contains 2 GATA motifs, 3 ETS, 1 SP1, and 1 CACCC,⁸ which compares favorably with the structure of the mouse ICAM-2 promoter (1 GATA motif and 2 ETS motifs oriented as the human promoter and with a CACCC motif).¹⁹ We observed that this promoter drove high-level gene expression in ECs and in non-EC types. These results differ from those published previously.^8,20 In transgenic animals, the same ICAM-2 promoter demonstrated high-level consistent transgene expression in ECs of all blood vessels in the heart, kidney, lung, liver, and pancreas, with negligible expression in other cell types, except neutrophils and monocytes.⁸ Another study using the same fragment in plasmid vectors showed high-level activity in bovine ECs but not in COS cells, although no human ECs were tested.²⁰ Therefore, when reengineered into viral vectors, ICAM-2 promoter activity and selectivity are markedly altered, similar to vWF. Although our data have demonstrated the limitations of the vWF and ICAM-2 promoters for endothelium-specific gene transcription in adenoviral systems, it is clear that the findings from isolated cell cultures cannot be directly compared with previous in vivo findings that used other gene-transfer and transgenic systems.

Transgene expression in vitro from the FLT-1 promoter was very high and selective to ECs, in agreement with the study by Morishita et al,⁵ who analyzed expression in isolated cells in vitro. Direct comparison with the vWF and ICAM-2 promoters confirmed the potential for this promoter. We extended these findings to include relevant models and demonstrated that the FLT-1 promoter may be useful clinically for vascular gene therapy. In evaluating the EC-specific activity of the FLT-1 promoter in human saphenous vein, we demonstrated potential clinical utility for FLT-1 when delivered locally into human vessels for delivery of therapeutic genes, such as metalloproteinase inhibitors.^17,21 Furthermore, it is clear that systemic dissemination of virus may have deleterious consequences, particularly for prodeath or proangiogenic genes.^22–26 We found that FLT-1 activity in hepatocytes in vitro and in vivo was extremely low, which indicates that if vector dissemination occurred during local delivery to the vessel wall, the use of FLT-1 would avoid undesirable transgene expression in the liver.

Because our in vitro and ex vivo experiments were performed in the presence of serum, it is clear that cells will be at different stages in the cell cycle, and this will vary considerably between different cell types. It will therefore be important to define promoter activity in cells where activity is observed based on transgene expression and cell-cycle characteristics. Furthermore, in the context of human saphenous vein, it will be important to document FLT-1 promoter activity in quiescent and damaged endothelium when considering endothelium-restricted gene expression in coronary artery bypass grafts. In summary, we have demonstrated the ability of the FLT-1 promoter to drive EC-restricted expression in vitro and in human vein ex vivo. Furthermore, FLT-1 was shown to be inactive in hepatocytes after systemic delivery into mice in vivo. These data clearly identify FLT-1 as a candidate EC-selective promoter for gene therapy protocols that target the endothelium using both local and systemic delivery approaches. The ability to target the endothelium provides the first step toward refined local and systemic gene transfer in hypertension and its complications.

	Acknowledgments

 
This work was supported by the British Heart Foundation (PG97/018 and PG99/097), the Biotechnology and Biological Sciences Research Council (17GTH12582), and grants from the United States Department of Defense (PC 970193, PC 991018), the CapCure Foundation, and an American Heart Association Scientist Development Grant to P.N.R.

Received September 26, 2000; first decision November 6, 2000; accepted January 17, 2001.

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				P. I. Teng, M. R. Dichiara, L. G. Komuves, K. Abe, T. Quertermous, and J. N. Topper Inducible and selective transgene expression in murine vascular endothelium Physiol Genomics, October 29, 2002; 11(2): 99 - 107. [Abstract] [Full Text] [PDF]

				B. D MacNeill, I. Pomerantseva, H. C Lowe, S. N Oesterle, and J. P Vacanti Toward a new blood vessel Vascular Medicine, August 1, 2002; 7(3): 241 - 246. [Abstract] [PDF]

				G. J. Bauerschmitz, D. M. Nettelbeck, A. Kanerva, A. H. Baker, A. Hemminki, P. N. Reynolds, and D. T. Curiel The flt-1 Promoter for Transcriptional Targeting of Teratocarcinoma Cancer Res., March 1, 2002; 62(5): 1271 - 1274. [Abstract] [Full Text] [PDF]

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