The necessity of gene transfer into target cells is remarkably increasing in the fields of research. Each type of transduction has its own property to infect and express certain transgenes in target cells, but its efficiency depends on types of vectors and cells to be transduced. It is therefore important to evaluate the ability of the designed vectors to transduce various types of human cells. Viral vectors are attractive tools for gene delivery in terms of their high efficiency and stable transgene expression. The most commonly used viral vectors are recombinant adenoviral vectors derived from human adenoviruses and retroviral vectors from oncoretroviruses. Adenoviral vectors have high efficiency of transduction and strong expression of transgenes; however, lack of the integration into the host genome and the potent immune response directed against adenoviral structural proteins render the expression of transgene temporary. 1 Retroviral vectors derived from moloney murine leukemia virus (MoMLV) integrate stably into the genome and do not express any viral proteins. On the other hand, disappearance of nuclear membrane during cell division is required for such integration. 2 Recently developed lentiviral vectors derived from human immunodeficiency virus type 1 (HIV-1) have been considered to be novel. Lentiviral preintegration complexes possess karyophilic properties that allow their active transport through the nucleopore, resulting in transduction of nondividing cells. 3 Corresponding to such property, lentiviral vectors can mediate the efficient gene delivery, integration, and long term expression of transgenes in nondividing cells in vitro or in vivo. 4,5 Thus the present authors examined the potential of a lentiviral vector expressing reporter genes to transduce human liver endothelial cells (HLECs).
Materials and Methods
Three Plasmid System
The vesicular stomatitis virus G protein (VSV-G) pseudotyped lentiviral vector encoding green fluorescent protein (GFP), LtV-GFP, was produced with three plasmids, including packaging plasmid HPV 274, envelope plasmid YN15, and vector plasmids HPV 306 (PGK-GFP) or HPV335 (EF1α-GFP), as shown in Figure 1A. All experiments performed in the present study were approved by the Ethical Committee of Okayama University Graduate School of Medicine and Dentistry and were performed under its guidelines.
Production of LtV-GFP
LtV-GFP was produced by both FuGENE 6 method and calcium phosphate (CP) method. Calcium Phosphate Transfection System (Gibco BRL, Rockville, MD) was used for the generation of lentiviral vectors as previously described. 6 FuGENE 6 Transfection Reagent (Roche Diagnostics, Germany) was applied to generate LtV-GFP according to the manufacturer’s protocol with minor modification, as shown in Figure 1B. Briefly, 24 μl of FuGENE 6 Transfection Reagent was diluted with 160 μl of media, and a total of 8 μg of plasmid-DNAs was added to the diluted FuGENE 6 Transfection Reagent containing 4.0 μg of HPV274, 0.4 μg of YN15, and 3.6 μg of HPV306 or 3.6 μg of HPV335. The culture medium was changed 2 hours before transfection with Dulbecco’s Modified Eagle Medium (DMEM) with 10% fetal bovine serum (FBS) and no antibiotics, and then the FuGENE 6 DNA mixture was introduced directly into subconfluent 293T cells. Twelve hours after the transfection, the medium was replaced with the DMEM media with 10% FBS supplemented with 100 units/ml of penicillin-streptomycin. The culture supernatant was collected at 48 hours after the transfection and then filtered through a 0.45 μm filter.
Concentration of LtV-GFP
Polyethylene glycol 8000 (PEG 8000) precipitation method was applied to concentrate viral titers. PEG 8000 (ICN Biomedicals, Inc., Aurora, OH) and NaCl solution was added to the filtered supernatant containing LtV-GFP at the final concentration of 5% and 0.15 M, respectively. The mixtures were incubated at 4°C overnight and centrifuged at 5,000 rpm for 10 minutes in a bench top centrifuge. The resulting viral pellet was resuspended in a low volume of DMEM and stored at −80°C in small aliquots before use.
Titration of LtV-GFP
The viral stocks were quantified by flow cytometric analysis (FCM). Briefly, 293T cells were transduced with various concentration of LtV-GFP obtained from viral stocks and incubated for 72 hours. LtV-GFP infected cells were trypsinized, fixed in 4% paraformaldehyde, washed twice in PBS, and subjected to FCM for GFP. The viral titer was calculated according to GFP positive cell percentage and dilution ratio of LtV-GFP used. At least three experiments were conducted for the titration assay.
Transduction of HLECs with LtV-GFP
HLECs were purchased from Cell Systems Co. (Seattle, WA) and maintained with CS-C medium (Cell Systems Co.). HLECs were plated on the wells of six well plates and transduced with LtV-GFP at a multiplicity of infection (MOI) of 1 to 10 in the presence of 12 mg/ml polybrene (Sigma, St. Louis, MO) in 1 ml of medium for 3 hours. Two milliliters of medium was added into the wells 12 hours after the transduction, and cells were incubated for another 36 hours without medium replacement. Seventy-two hours after LtV-GFP infection, the cells were subjected to FCM and fluorescent microscopic examination (FM). Transduction efficiency of LtV-GFP was compared among several transducing systems, including CP method, lipofection method, adenoviral and retroviral GFP transduction. We used Calcium Phosphate Transfection System (Gibco BRL) for CP method, and Lipofectamin 2000 (Gibco BRL) for lipofection method under the manufacturer’s protocol. A recombinant adenovirus vector encoding GFP cDNA under the CMV (human cytomegalovirus) promoter, AdV-GFP (provided by Dr. J. A. Roth, University of Texas MD Anderson Cancer Center, Houston, TX), was previously constructed and characterized in detail. 7 The viral stocks were quantified by a plaque forming assay with 293 cells and then stored at −80°C. An MoMLV based retroviral vector expressing GFP cDNA, RtV-GFP (provided by Dr. P. Leboulch, Harvard-Massachusetts Institute of Technology, Cambridge, MA), was also used for GFP transduction experiment in HLECs. Normal HLECs at passage 2 were transduced with AdV-GFP for 1 hour at MOIs of 1 to 10, or with RtV-GFP for 72 hours at MOIs of 1 to 10 in the presence of 12 mg/ml polybrene, as control experiments. The GFP expression of transduced HLECs was analyzed 72 hours after the transduction, and the cells were carefully observed for at least 1 month for GFP expression and endothelial phenotype study.
Flow Cytometric Analysis and Fluorescent Microscopy
FCM and FM were used to evaluate transduction efficacy of LtV-GFP in HLECs. HLECs treated with LtV-GFP were trypsinized and washed in PBS. The cells were fixed in 4% paraformaldehyde, washed twice in PBS, and resuspended into single cells to a concentration of 106 cells/ml. FCM for GFP expression was performed with FACSCalibur (Becton Dickinson, Mountain View, CA) using CellQuest program (Becton Dickinson). LtV-GFP treated HLECs were also fixed in 4% paraformaldehyde in the culture flask, washed twice with PBS, and subjected to FM. The cells were observed and photographed using an inverted fluorescent microscope. Unmodified HLECs were used as a control in the GFP expression assay. GFP expressing HLECs were recovered with FACSCalibur and used for the following experiments, including gene expression and Matrigel angiogenic assay.
Gene Expression of Endothelial Cell Markers
LtV-GFP transduced HLECs were analyzed by reverse transcription-polymerase chain reaction (RT-PCR) for CD 34, factor VIII, flt-1, KDR/flk-1, and HGF. Total RNA was isolated from 5 × 106 of unmodified and LtV-GFP transduced HLECs with RNAzol (Cinna/Bio Tecx, Friendswood, TX) and used as templates. Reverse transcription was performed at 22°C for 10 minutes and then at 42°C for 20 minutes using 2.0 μg of RNA per reaction to ensure that the amount of amplified DNA was proportional to that of specific mRNA in the original sample. PCR was performed with specific primers in volumes of 50 μl containing 2.0 μl RT products according to the manufacturer’s protocol (Perkin-Elmer, Foster City, CA). The amplification reaction involved denaturation at 95°C for 30 seconds, annealing at 60°C for 30 seconds, and at 72°C for 30 seconds with a thermal cycler (Perkin-Elmer). The PCR products were resolved on 1% agarose gels and visualized by ethidium bromide staining. The human β-actin gene served as an internal control for the efficiency of mRNA isolation and cDNA synthesis. Primers used were as follows: CD34 (499 base pair, bp), sense, 5′-TTCCCAAAAGACCCTGATTG-3′, anti-sense, 5′-GGAGCCGA- ATGTGTAAAGGA-3′; factor VIII (500 bp), sense, 5′-CTGACCAGGGTCCTATTCCA-3′, anti-sense, 5′-TGGTTATCCCAAGCAAGAGG-3′; flt-1 (496 bp), sense, 5′-TCTCAACAAGGATGCAGCAC-3′, anti-sense, 5′-GGAGAAGATTTCCCACAGCA-3′; KDR/flk-1 (501 bp), sense, 5′-CCCACCCCCAGAAATAAAAT-3′, anti-sense, 5′-ACATTTGCCGCTTGGATAAC-3′; HGF (493 bp), sense, 5′-CAAATGTCAGCCCTGGAGTT-3′, anti-sense, 5′-TTC- TCCTTGACCTTGGATGC-3′; human β-actin (610 bp), sense, 5′-TGACGGGGTCACCCACACTGTGCCCATCTA-3′, anti-sense, 5′-CTAGAAGCATTTGCGGTGGACGATGGAGGG-3′.
To examine in vitro angiogenic potential, LtV-GFP transduced HLECs were subjected to a Matrigel assay. Matrigel (Collaborative Biomedical Product, Waltham, MA) was layered on prechilled six well plates and then placed in an incubator at 37°C for 30 minutes until solidified. The 3 × 105 untransduced and LtV-GFP infected cells were seeded on Matrigel, allowed to incubate at 37°C in a 5% CO2 environment, and observed for 72 hours for the capillary web formation. Cells in Matrigel culture were then fixed in 4% paraformaldehyde, washed twice with PBS, and subjected to FM for GFP expression analysis.
Replication Competent Virus Assay
Gag p24 ELISA and mobilization assay were applied to detect replication competent virus (RCV) (Figure 2). 293T cells were transduced with a lentiviral vector expressing the genes of LacZ and neomycin resistance flanked by a pair of complete long terminal repeat (LTR). Transduced 293T cells were cultured in DMEM containing 400 μg/ml G418, neomycin analogue (Gibco BRL), and resultant G418 resistant cells (293T-LacZ/Neo) were used for the subsequent RCV assay. The 293T-LacZ/Neo cells were infected with LtV-GFP and cultured for 25 passages; the conditioned medium obtained after such culture was used for Gag p24 assay and serial infection to unmodified 293T cells. Measurement of p24 in the conditioned medium was performed by RETRO-TEK HIV-1 p24 Antigen ELISA (Zeptometrix Co., Buffalo, NY) under manufacturer’s protocol. After the serial infection of naive 293T cells with the conditioned medium, GFP expression of the cells was analyzed by FCM and FM.
Production, Concentration, and Titration of LtV-GFP
The resultant titers of LtV-GFP generated by means of FuGENE 6 and CP method were summarized in Table 1. Although there was no significant difference in LtV-GFP titers between FuGENE 6 and CP method, the FuGENE 6 method offered easy handling and showed less cytotoxicity to 293T cells compared to CP transduction system. Comparing an EF1α-GFP vector plasmid HPV335 with a PGK-GFP vector plasmid HPV 306, the titer of LtV-GFP generated with HPV335 was nearly 20-fold higher than that obtained with HPV306. LtV-GFP titers were concentrated up to approximately 100 times when PEG 8000 precipitation method was applied.
Transduction of HLECs with LtV-GFP
To examine the transduction efficiency of LtV-GFP, transduced HLECs were subjected to FCM. LtV-GFP was efficiently able to transfer the GFP gene into HLECs on an MOI dependent manner (Figure 3A–D). In contrast, transduction efficiency of nonviral methods, including CP and lipofection method, was nearly 0% (Table 2). Adenovirus mediated GFP gene delivery was more effective in HLECs compared with lentiviral GFP transduction, but GFP expression of HLECs dramatically decreased 1 month after adenoviral transduction. Notably, LtV-GFP infected HLECs stably expressed GFP over 2 months after transduction (Fig. 3E).
Expression of Endothelial Cell Markers
We examined the gene expression of endothelial markers in LtV-GFP transduced HLECs recovered with FACSCalibur. There was no significant difference in gene expression profile, including CD 34, factor VIII, flt-1, KDR/flk-1, and HGF between unmodified parental HLECs and LtV-GFP transduced cells (Figure 4A,B).
Angiogenesis Matrigel Assay
To evaluate the angiogenic potential of GFP expressing HLECs sorted with FACSCalibur after LtV-GFP transduction, the present authors cultured the cells on Matrigel-layered plates. LtV-GFP transduced HLECs were capable of morphologically differentiating into capillary tubes on the Matrigel, which was paralleled to web formation of primarily cultured cells (Figure 4C,E). LtV-GFP transduced HLECs rapidly adhered on Matrigel, formed abundant networks of branching and anastomosing cords of cells with showing uniform green fluorescence within 12 hours (Figure 4F). These findings showed that LtV-GFP infected HLECs properly maintained their ability to differentiate into angiogenic tubes.
Gag p24 Assay and Mobilization Assay
An assay for Gag p24, one of lentiviral structural proteins, and a mobilization study were conducted to detect whether RCV was generated. In these analyses, emergence of RCV has not occurred whole through the experiments for up to 25 passages, ensuring the safety of the lentiviral production system used in the current study.
The essential role performed by liver HLECs under pathophysiologic conditions is becoming more apparent in various types of liver diseases. In addition, the involvement of HLECs has been recognized in inflammatory responses, graft rejection, and ischemia reperfusion injury in liver transplantation. 8,9 Therefore, much attention has been paid to HLECs as a potential target of gene therapy or regenerative medicine in the liver. Thus establishment of efficient transduction system in HLECs is highly desirable for research and clinical applications. The currently developed HIV-1 derived lentiviral vectors can transport viral genome through nucleopore and integrate into the host genomes without altering immunogenicity even in nondividing cells. 10–12 These reports prompted the present authors to examine an efficacy of lentivirus mediated GFP gene delivery into HLECs. After LtV-GFP infection, almost all of the transduced HLECs were positive for GFP expression even 2 months later. Our previous observations demonstrated that a lentiviral vector expressing an Escherichia coli LacZ gene tagged with nuclear localization signal (NLS), LtV-NLS/LacZ, was able to transduce senescent human umbilical vein endothelial cells as well as human umbilical vein endothelial cells at early stage. 6 These findings suggest that therapeutic genes such as growth factors and antiapoptotic molecules could be transferred into HLECs at any PDLs using lentiviral transduction system for clinical cell and gene therapies.
When considered in clinical applications, it is of extreme importance to stably generate a certain amount of high titer lentiviral particles. In the present study, the authors adopted FuGENE 6 Transfection Reagent, which is described by the manufacturer as a nonliposomal blend of lipids, for stable generation of recombinant lentiviruses. FuGENE 6 method was found to be easy handling and noncytotoxic to 293T cells, seemed to be suitable for recombinant lentivirus production. The authors also used an EF1α promoter for achieving high titers. The titer of LtV-GFP generated with EF1α containing GFP vector plasmid HPV335 was nearly 20-fold higher than that obtained with PGK promoter driving GFP vector plasmid HPV306. Ultra centrifugation system is currently used to concentrate recombinant viruses, but it requires a large device. Instead of use of ultracentrifugation, PEG 8000 precipitation, which is a common method to precipitate DNA, was used in this study. The use of PEG 8000 precipitation method requires only a bench top centrifuge and is found to be a convenient means to concentrate lentiviruses. Development of easy and stable generation system for lentiviral vectors can facilitate the advances in the field of lentiviral gene transfer.
Issues regarding biosafety, including emergence of wild type HIV-1 and RCV, should be considered when lentiviral vector systems are developed. In the authors’ lentiviral vector system, the accessory genes vif, vpu, and nef are deleted, and application of VSV-G instead of env gene reduces the possibility to generate wild type HIV-1. 13–15 Division of lentiviral construct into three parts renders the generation of RCV extremely unlikely because RCV emergence requires multiple recombination events among different plasmids. The self inactivating vectors that carry an almost complete deletion in the U3 region of 3′LTR without affecting vector function are recently documented. The system can diminish the concerns for unexpected activation of protooncogenes of host genomes by a promoter activity existing in proviral LTRs. 16,17 One of the other important aspects in handling of lentiviral vectors is to establish a detection scheme of RCV. 14,18 We have screened RCV emergence with Gag p24 and mobilization assays. Detection of p24 in Gag p24 assay or GFP positive 293T cells in mobilization assay indicates RCV generation. Fortunately, RCV has never been detected in the whole experiments conducted in our institute. It is of great importance to monitor RCV at regular intervals for in vivo animal study. Accumulation of such data and further safety considerations would make lentiviral vectors be more valuable in animal experiments and further clinical applications.
In the present work, the authors have demonstrated that HIV-1 based lentiviral vectors can efficiently transduce HLECs without affecting cell functions. These findings provide evidence that lentiviral vectors are a promising tool for gene delivery and expression in HLECs that could be target cells in gene an cell therapies and regenerative medicine in the liver. Therefore, further investigation is required to determine if genetically modified HLECs could be a general source for reendothelialization in sites actively undergoing vascularization or will be home only to the liver under specific circumstances. Establishment of such a system that allows for LtV-GFP-transduced HLEC engraftment into an area actively undergoing angiogenesis, like during liver regeneration or coincident with liver transplantation after long periods of cold ischemic time, is an attractive avenue of experimentation.
This work was supported in part by a Grant-in-Aid for Scientific Research from the Ministry of Education, Science and Culture of Japan. The authors thank Drs. Philippe Leboulch and Karen A. Westerman, Harvard and Massachusetts Institute of Technology, for providing the plasmids, and Dr. Hiroyuki Miyoshi, RIKEN Tsukuba Institute, for advising the details of RCV assay.
1. Yang Y, Nunes FA, Berencsi K, Furth EE, Gonczol E, Wilson JM: Cellular immunity to viral antigens limits E1-deleted adenoviruses for gene therapy. Proc Natl Acad Sci U S A 91: 4407–4411, 1994.
2. Gordon EM, Anderson WF: Gene therapy using retroviral vectors. Curr Opin Biotechnol 5: 611–616, 1994.
3. Naldini L, Blomer U, Gallay P, et al: In vivo gene delivery and stable transduction of nondividing cells by a lentiviral vector. Science 272: 263–267, 1996.
4. Blomer U, Naldini L, Kafri T, Trono D, Verma IM, Gage FH: Highly efficient and sustained gene transfer in adult neurons with a lentivirus vector. J Virol 71: 6641–6649, 1997.
5. Kafri T, Blomer U, Peterson DA, Gage FH, Verma IM: Sustained expression of genes delivered directly into liver and muscle by lentiviral vectors. Nat Genet 17: 314–317, 1997.
6. Totsugawa T, Kobayashi N, Okitsu T, et al: Lentiviral transfer of the LacZ gene into human endothelial cells and human bone marrow mesenchymal stem cells. Cell Transplant 11: 481–488, 2002.
7. Jian G, Kagawa S, Takakura M, et al: Tumor-specific transgene expression from the human telomerase reverse transcriptase promoter enables targeting of the therapeutic effects of the Bax gene to cancers. Cancer Res 60: 5359–5364, 2000.
8. Kojima N, Sato M, Suzuki A, et al: Enhanced expression of B7–1, B7–2, and intercellular adhesion molecule 1 in sinusoidal endothelial cells by warm ischemia/ reperfusion injury in rat liver. Hepatology 34: 751–757, 2001.
9. Yang H, Majno P, Morel P, et al: Prostaglandin E1
protects human liver sinusoidal endothelial cell from apoptosis induced by hypoxia reoxygenation. Microvasc Res 64: 94–103, 2002.
10. Deglon N, Tseng JL, Bensadoun JC, et al: Self-inactivating lentiviral vectors with enhanced transgene expression as potential gene transfer system in Parkinson’s Disease. Hum Gene Ther 11: 179–190, 2000.
11. Giannoukakis N, Mi Z, Gambotto A, et al: Infection of intact human islets by a lentiviral vector. Gene Ther 6: 1545–1551, 1999.
12. Leibowitz G, Beattie GM, Kafri T, et al: Gene transfer to human pancreatic endocrine cells using viral vectors. Diabetes 48: 745–753, 1999.
13. Burns JC, Friedmann T, Driever W, Burrascano M, Yee JK: Vesicular stomatitis virus G glycoprotein pseudotyped retroviral vectors: concentration to a very high titer and efficient gene transfer into mammalian and nonmammalian cell. Proc Natl Acad Sci U S A 90: 8033–8037, 1993.
14. Chang LJ, Urlacher V, Iwakuma T, Cui Y, Zucali J: Efficient and safety analyses of a recombinant human immunodeficiency virus type I derived vector system. Gene Ther 6: 715–728, 1999.
15. Zufferey R, Nagy D, Mandel RJ, Naldini L, Trono D: Multiply attenuated lentiviral vector achieves efficient gene delivery in vivo. Nat Biotechnol 15: 871–875, 1997.
16. Iwakuma T, Cui Y, Chang LJ: Self-inactivating lentiviral vectors with U3 and U5 modifications. Virology 261: 120–132, 1999.
17. Zufferey R, Dull T, Mandel RJ, et al: Self-inactivating lentivirus vector for safe and efficient in vivo gene delivery. J Virol 72: 9873–9880, 1998.
18. Kafri T, van Praag H, Ouyang L, Gage FH, Verma IM: A packaging cell line for lentivirus vectors. J Virol 73: 576–584, 1999.