Third-generation lentivirus vectors efficiently transduce and phenotypically modify vascular cells: implications for gene therapy

Journal of Molecular and Cellular Cardiology 35 (2003) 739–748 www.elsevier.com/locate/yjmcc

Original Article

Third-generation lentivirus vectors efficiently transduce and phenotypically modify vascular cells: implications for gene therapy Kate L. Dishart a, Laura Denby a, Sarah J. George b, Stuart A. Nicklin a, Satya Yendluri c, Melanie J. Tuerk c, Michael P. Kelley c, Brian A. Donahue c, Andrew C. Newby b, Thomas Harding c, Andrew H. Baker a,* a

Division of Cardiovascular and Medical Sciences, University of Glasgow, Church Street, Glasgow G11 6NT, UK b Bristol Heart Institute, Bristol, UK c Cell Genesys Inc, Foster City, CA, USA Received 21 January 2003; received in revised form 5 March 2003; accepted 26 March 2003

Abstract Grafting of saphenous vein (SV) conduits into the arterial circulation triggers a number of adaptive pathological changes characterized by progressive medial thickening, neointima formation and accelerated atheroma. Previous studies have shown that modification of vein graft biology is possible by adenovirus (Ad)-mediated gene transfer, although gene expression is transient. Advancement of vascular gene therapy to the clinic is compromised by the lack of safe and efficient vector systems that provide sustained therapeutic gene delivery to the vasculature. Due to inadequacies of both Ad and adeno-associated virus (AAV) serotype-2 (AAV-2) systems, we have evaluated gene delivery to endothelial cells (ECs) and smooth muscle cells (SMCs) using alternate AAV serotypes and a third-generation vesicular stomatis virus glycoprotein-pseudotyped lentiviral system. Transduction of both primary human SV EC and SMC was lower using all alternate AAV serotypes compared to AAV-2. However, transduction of both cell types by lentivirus was efficient even at clinically relevant exposure times (15 min), was without toxicity and was promoter sensitive. Transduction levels at lower doses were further enhanced with the addition of the surfactant Poloxamer-407 (P-407). Direct comparison with Ad and AAV-2 confirmed the unique potential for this system. Moreover, we constructed and overexpressed the therapeutic gene tissue inhibitor of metalloproteinase-3 (TIMP-3) using lentivirus and demonstrated transgene production comparable to Ad with concomitant blockade of SMC migration and induction of cell death. We have demonstrated for the first time the potential for third-generation lentiviral vectors, but not alternate AAV serotypes, as efficient vascular gene delivery vectors. © 2003 Elsevier Science Ltd. All rights reserved. Keywords: Vascular gene therapy; Adeno-associated virus; Adenovirus; Lentivirus; Endothelial cells; Smooth muscle cells; Tissue inhibitor of metalloproteinases; Vein grafts

1. Introduction Coronary artery bypass graft (CABG) surgery alleviates angina and prolongs patient survival but patency rates remain low with a 50% failure rate within 10 years [1]. Insertion of vein grafts into the arterial circulation triggers adaptive physiological changes and these characteristically include acute thrombotic occlusions in early failure. Contractile medial vascular smooth muscle cell (SMC) migration to the graft lumen with resultant proliferation and secretion of ex* Corresponding author. Tel.: +44-141-211-2100/2116; fax: +44-141-211-1763. E-mail address: [email protected] (A.H. Baker). © 2003 Elsevier Science Ltd. All rights reserved. DOI: 10.1016/S0022-2828(03)00136-6

tracellular matrix proteins to form a neointima and subsequent accelerated atheroma are both symptomatic of late vein graft failure [2]. Since SMC migration and proliferation are key factors in the development of vein graft failure, the majority of gene therapy strategies have been focused on these targets. Modification of vein graft biology has been shown in both in vivo animal and ex vivo human models using adenovirus (Ad)-mediated gene transfer, although gene expression is transient [2]. There is currently no effective pharmacological treatment for vein graft failure and the surgical procedure provides a unique window for ex vivo gene therapy. Gene therapy for cardiovascular disease would benefit from the availability of vector systems that allow efficient and long-term transgene expression in vascular

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SMC and/or vascular endothelial cell (EC), since low gene transfer efficiency to blood vessels using standard viral and non-viral systems is still a significant problem. Viral systems, particularly Ad vectors, are commonly used but drive transient gene expression and demonstrate poor vascular tropism, particularly vascular SMC, which lack significant levels of the coxsackie and adenovirus receptor (CAR) [3]. However, Ad-mediated gene transfer produces a marked inflammatory and immunogenic response limiting gene expression, which remains transient [4]. Hence, in the context of the cardiovascular system, Ad transduction levels are relatively poor, especially when compared to the hepatic system [5]. Adeno-associated virus (AAV) vectors are a minimally immunogenic alternative vector system. The potential of AAV-2 is principally extrapolated from excellent progress in gene therapy for diseases, such as hemophilia [6], and combined with the ability to transduce non-dividing and dividing cells, provide a broad spectrum of target disease applications. Although there has been success in the myocardium using AAV-2 [7], low-level gene transfer to EC using AAV-2 vectors has limited progress in the vasculature [8]. The identification of an alternative gene delivery system is of paramount importance for transition to the clinic. Possibilities include other AAV serotypes, which demonstrate enhanced infectivity compared to AAV-2 in certain target tissues [9,10]. Likewise, lentivirus, derived from human immunodeficiency virus-1 (HIV-1) (pseudotyped with the vesicular stomatis virus glycoprotein (VSV-G) to broaden its tropism), has shown therapeutic promise for a number of target diseases including retinal disorders, treatment of hemophilia and Fanconi anemia, as well as the management of neurodegenerative disorders, such as Parkinson’s disease [11–14]. Importantly, lentiviral-mediated gene transfer of anti-angiogenic factors to T24 human bladder cancer cells demonstrated efficacy for the treatment of cancer, where adenoviral vectors had previously failed [15]. The stable and persistent nature of lentiviruses for in vivo administration may facilitate the transduction of cells, otherwise refractory to commonly used vector systems. Third-generation lentiviruses are additionally deleted of the majority of their native genome, thus improving their safety and applicability due to the reduction in generation of replication-competent lentiviruses (RCLs). Here, we have demonstrated the potential for third-generation lentiviral vectors, but not alternate AAV serotypes, as efficient vascular gene delivery vectors. 2. Methods All materials were from Sigma (Poole, UK) unless otherwise stated. Recombinant-enhanced green fluorescent protein (eGFP) was from Clontech (Basingstoke, UK), culture reagents from Invitrogen (Paisley, UK) or JRH Biosciences (Lanexa, Kansas) except fetal calf serum (PAA, Dorset, UK). HeLa and 293-T cells were from ATCC (Rockville, Maryland), human umbilical vein endothelial cells (HUVECs) from TCS Cellworks (Botolph Clayden, UK) and human

SV. EC and SMC were prepared as described [8] and used below passage 6. 2.1. Viruses 2.1.1. AAV AAV serotypes-2–6 expressing human factor IX (hFIX) as a sensitive reporter gene from the MFG or the cytomegalovirus (CMV) IE enhancer/chicken a-actin/rabbit b-globin (CAG) promoters were utilized for AAV serotype analysis and were produced and titrated as described [16]. 2.1.2. Ad RAd-eGFP and RAd-T-3 express eGFP and tissue inhibitor of metalloproteinase-3 (TIMP-3) from the CMV promoter, respectively. RAd60 contains no transgene. Production of Ad is described elsewhere [3]. 2.1.3. Lentivirus The third-generation, self-inactivating plasmids pRRLsin.hPGK.EGFP.WPRE and pRRLsinpptCMV.EGFP.WPRE have been described previously [17,18]. Plasmid pRRLsinpptCAG.EGPF.WPRE was constructed by replacing CMV with CAG from pBacMam-2 (Novogen, WI). Lentiviral vector expressing human TIMP-3 (Lenti-T-3) plasmid was generated by removal of eGFP from pRRLsinpptCMV.EGFP.WPRE by AgeI/SalI digestion and ligation of human TIMP-3 cDNA amplified with Pfu polymerase (Promega, UK) using primers with AgeI and SalI sites. Lentiviral vectors were produced by co-transfection of 293-T cells and the virus harvested by ultracentrifugation of the supernatant [17,19]. Viral p24 concentration was determined by immunocapture (Alliance, Perkin Elmer Life Sciences Inc., Boston, MA) and transducing activity quantified. 2.2. Virus infections Cells were plated 24 h prior to infection, incubated with fresh media containing the required multiplicity of infection (MOI)/cell of virus (as indicated), left for the required time, washed and maintained until harvesting. All lentivirus infections were performed in the presence of 8 µg/ml polybrene (Sigma, Poole, UK). eGFP expression was visualized by fluorescence microscopy before cells were lysed in 0.2% (v/v) Triton-X-100 PBS at 7-d post-infection and eGFP expression quantified by plate assay using a Wallac Victor 2 luminometer and recombinant eGFP as a standard [8]. A BCA assay (Perbio, Tattenhall, UK) on the lysates was performed to determine protein concentration, and the results were expressed as RFU/mg protein. Factor IX production was measured in conditioned media using ELISA [20]. 2.3. Western blotting and immunofluorescence TIMP-3 production was evaluated by western blot analysis and immunofluorescence on HeLa cells using an antihuman TIMP-3 antibody (Chemicon, Harrow, UK). Cells

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Fig. 1. Transduction of human EC and SMC with AAV serotypes. Human SV EC, HUVEC and SMC were exposed to 20,000 genomic particles/cell of each AAV serotypes (2–6) expressing hFIX from the MFG promoter. Cells were washed after 24 h and conditioned media collected for 5 d. Factor IX production was quantified by ELISA [20] and expressed as a percent of the levels achieved by AAV-2.

were harvested in 2× sample reducing buffer (100 mM Tris pH 6.8, 4% (w/v) SDS, 20% (v/v) glycerol, 0.2% (w/v) bromophenol blue, 5% (v/v) b-mercaptoethanol) at 4-d postinfection. Cell lysates were electrophoresed on 12% (v/v) polyacrylamide gels and transferred to nylon membranes (Hybond P+, Amersham, Little Chalfont, UK). Membranes were incubated in blocking solution (1× TBS, 5% (w/v) milk powder, 0.1% (v/v) Tween 20) overnight and incubated with 1:2500 TIMP-3 antibody followed by repeated washing in 1 × TBS (0.1% Tween-20) before incubation in 1:2000 pig anti-rabbit HRP (Dako, Ely, UK) secondary antibody and detection by ECL (Amersham, Little Chalfont, UK). For indirect immunofluorescence, cells were fixed for 30 min in 4% (w/v) paraformaldehyde, washed in PBS and permeabilized for 30 min in 0.1% Triton-X-100 PBS. Cells were incubated for 30 min in 1:500 of TIMP-3 antibody containing 20% porcine serum or 1:100 rabbit IgG containing 20% porcine serum before repeated washing in PBS. Detection was by incubation in 1:200 pig anti-rabbit FITC (Dako, Ely, UK) containing 20% porcine serum before repeated washing in PBS. Cells were mounted on propidiumiodide containing Vectashield (Vector Laboratories, California) before visualization with a fluorescence microscope. 2.4. Migration and cell viability assays Cells were trypsinized 48-h post-infection and placed in Boyden chambers pre-coated with Matrigel™, as described previously [2]; 24 h later, cells that had invaded the matrix towards the chemoattractant (10% fetal calf serum) were quantified.

Cell viability was quantified using the CellTiter 96® nonradioactive viable cell number assay (Promega, Poole, UK) as per the manufacturer’s instructions. 2.5. Statistical methods Experiments were performed in triplicate on three independent occasions. Error bars shown are the standard error of the mean. All data were subject to statistical analysis using Student’s t-test and considered significant at P < 0.05.

3. Results We first tested whether alternative AAV serotypes have increased infectivity for human EC and SMC compared to AAV-2. To assess this, we used AAV vectors expressing hFIX as a sensitive reporter gene from the MFG promoter and quantified transgene expresssion in cell supernatants by ELISA. Disappointingly, all alternate AAV serotypes (AAV3–6) evoked lower levels of hFIX secretion than AAV-2 (Fig. 1), a finding observed at all MOIs tested and using both MFG (Fig. 1) and CAG promoters (data not shown). We next tested third-generation HIV-1-derived VSV-Gpseudotyped lentiviruses for transduction of both SMC and EC and compared this to permissive HeLa cells. In direct contrast to AAV, lentivirus expressing eGFP (Lenti-eGFP) from the CMV promoter efficiently transduced both EC and SMC, as quantified by fluorimetry of cell lysates (Fig. 2A–C) and visualized by fluorescence microscopy (Fig. 2D). Although 18-h exposure times to the virus (data not shown) evoked the highest level of reporter gene expression for each

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cell type, short exposure times (as low as 15 min) still evoked appreciable levels of transgene production (Fig. 2A–C). Interestingly, in contrast to HeLa cells (Fig. 2C), levels of transgene expression were not strictly dose- and timedependent, when short exposure times were evaluated for EC and SMC (Fig. 2A,B) indicating possible saturation of transduction pathways. Since VSV-G binding to target cells involves interaction with membrane phospholipids [21], we assessed whether surfactant-based agents could further enhance transduction. Expression levels could be significantly enhanced in EC by 5% P-407 (Fig. 3) at both short (30-min) and prolonged (18-h) exposure times. Interestingly, human SMC transduction was only enhanced at prolonged exposure times by P-407 (Fig. 3). To define the potential for alternative promoters, we quantified lentivirus-mediated transduction of vascular cells using CAG and PGK promoters in comparison to CMV (Fig. 4A,B). Following 30-min exposure to lentivirus vectors, the CMV promoter was significantly more active, particularly in EC, than both CAG and PGK at all MOIs evaluated (1–100/cell) (Fig. 4A,B). The CAG promoter, but not PGK,

was also active in both EC and SMC albeit at lower levels than CMV (Fig. 4A,B). Based on the efficiency of transduction of vascular cells by lentiviral vectors, we directly compared lentivirus with Ad and AAV-2 gene delivery systems using equal MOIs of each virus, all of which expressed eGFP from the CMV promoter (Fig. 5A,B). In human EC, startling differences were observed between the three viral systems. Lentiviral vectors displayed efficiencies significantly lower than Ad vectors in EC but higher than AAV-2 (Fig. 5A), evaluated using both 30-min and 18-h exposure times. Transduction of SMC with lentivirus was significantly higher than both Ad and AAV-2 (Fig. 5B). This resulted in the transduction of 96 ± 2.7% for lentivirus against 9 ± 2.5% for Ad infection. Based on these observations and the requirement for highlevel TIMP-3 production to induce the required anti-migratory and pro-apoptotic effects on vascular cells [2], we generated a novel Lenti-T-3. The full-length human DNA for TIMP-3 was cloned downstream of the CMV promoter (Fig. 6A) and lentivirus produced. Western blot analysis (Fig. 6B) and indirect immunofluorescence (Fig. 6C) on Lenti-T-3transduced HeLa cells at 4-d post-infection demonstrated

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Fig. 2. Third-generation lentivirus efficiently transduce human vascular cells (A–C). Eighty-percent confluent (A) human EC, (B) human SMC and (C) HeLa cells in triplicate wells of a 48-well plate were transduced with increasing MOI (50, 100 or 200 transducing units (tu)/cell) of Lenti-eGFP for 15, 30 or 45 min. eGFP expression was quantified by fluorimetry and normalized for protein as described in Section 2. Striped bars represent uninfected, solid bars an MOI of 50, hatched bars an MOI of 100 and open bars an MOI of 200 tu/cell; * indicates P < 0.05 for higher transduction of 50 MOI vs. 100 and ** indicates P < 0.05 for higher transduction of 100 MOI vs. 200. (D) Representative micrographs of Lenti-eGFP-transduced cells (EC or SMC) taken at 7-d post-infection at ×40 magnification. The scale bar represents 100 µm.

high-level TIMP-3 production, at levels comparable to Admediated gene delivery with production of the expected glycosylated and non-glycosylated forms (Fig. 6B) [22]. We next evaluated the phenotypic effect of Lenti-T-3 overexpression on migration and cell death. Using established

Matrigel™-coated Boyden chamber assays [2] Lenti-T-3 was equipotent to Ad-mediated overexpression of TIMP-3 and evoked a significant reduction in human SMC migration (Fig. 7A). Importantly, Lenti-T-3, like Ad-mediated TIMP-3 gene delivery, also significantly induced cell death (Fig. 7B).

Fig. 3. Enhanced levels of transduction in the presence of P-407. Human EC and SMC were transduced in triplicate wells of a 24-well plate with an MOI of 30 tu/cell in the absence and presence of 5% (w/v) P-407 for 30 min or 18 h. eGFP expression was quantified by fluorimetry and normalized for protein as described in Section 2. The presence of P-407 is shown by a +/– on the lower axis; * indicates P < 0.05 vs. transduction in the absence of P-407.

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Fig. 4. Influence of promoters and direct comparison of lentiviral vectors with other viral gene delivery systems. (A) Human EC and (B) human SMC were transduced in triplicate wells of a 48-well plate with increasing MOI (between 1 and 100 tu/cell) of lentiviral vectors expressing eGFP from CMV, CAG or PGK promoters for 30 min, washed and eGFP levels quantified by fluorimetry and normalized for protein at 7-d post-infection; * indicates P < 0.05 for higher transduction of CMV vs. both PGK and CAG and ** indicates P < 0.05 for higher transduction of CAG vs. PGK.

4. Discussion Identification and testing of efficient vascular gene delivery vectors is a prerequisite for the future application of gene therapy in the clinical setting. We have evaluated gene deliv-

ery to human EC and SMC using alternative AAV serotype vectors and third-generation lentiviruses. Disappointingly, we show that alternate AAV serotypes show no benefit for EC or SMC. However, we demonstrate that third-generation lentiviruses are highly efficient for both cell types. Importantly,

Fig. 5. Direct comparison of viral vector systems. Comparison of transduction of (A) human EC and (B) human SMC by eGFP expressing lentiviral, AAV-2 and Ad vectors for 30 min (MOI = 50 tu/cell for lentivirus and AAV-2 and 50 pfu/cell for Ad) with expression quantified by fluorimetry and normalized for protein at 7-d post-infection. Solid bars represent lentivirus, open bars AAV-2 and hatched bars Ad; * indicates P < 0.05 for higher lentiviral transduction vs. AAV and ** indicates P < 0.05 for higher lentiviral transduction vs. Ad.

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Fig. 6. Functional expression of a therapeutic gene from lentivirus. (A) Plasmid map of pRRLsinpptCMV.T-3, the construct used to produce Lenti-T-3. (B) HeLa cells were transduced in 48-well plates with either an MOI of 3 or 30 tu/cell of Lenti-eGFP/T-3 or pfu/cell RAd60/T-3 for 18 h. Cells were harvested at 4-d post-infection and a western blot performed as described in Section 2. The large arrow in (B) represents unglycosylated TIMP-3 and the small arrow is glycosylated TIMP-3. (C) Indirect immunofluorescence of RAd-T-3, Lenti-b-Gal and Lenti-T-3 infected HeLa cells was examined at 4-d post-infection as described in Section 2 after identical infection procedures as for the western blot analysis with the exception that cells were grown on coverslips. The magnification is ×400 and the scale bar represents 20 µm.

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Fig. 7. Effect of TIMP-3 on migration and proliferation. (A) Human SMC were transduced for 18 h with the null virus RAd60 or RAd-T-3 at an MOI of 100 pfu/cell or Lenti-eGFP/T-3 at an MOI of 30 or 100 tu/cell and maintained for 48 h to allow transgene expression to occur. Migration assays were then performed as described in Section 2. (B) HeLa cells in 48 well plates were transduced with either 100 tu/cell Lenti-eGFP/T-3 or 100 pfu/cell RAd60/RAd-T-3 for 18 h and cultured for 7 d. Cell death indicated by a significant reduction in viable cell number (as represented by a lower absorbance reading at 570 nm) was quantified following transduction using the CellTiter 96 ® non-radioactive viable cell number assay. * P < 0.05 vs. respective control.

transduction of SMC by lentiviruses was significantly higher than Ad-mediated delivery. We also demonstrate efficient production of TIMP-3 with associated phenotypic effects. The finding that AAV-2 evokes higher levels of transgene expression than alternate AAV serotypes is in contrast to other tissues, such as lung epithelia, where AAV-5 shows increased infectivity to apical surfaces compared to AAV-2 [23]. These findings may undermine the potential of the AAV vector system for gene delivery to EC and/or SMC. The lower hFIX levels observed with serotypes other than AAV-2 may also reflect the lack of primary and/or co-receptor expression required for individual AAV serotype transduction on human primary EC and SMC. Although previous studies have demonstrated reporter gene expression from AAV-2 vectors in the vessel wall using high titers [24–26], to date there is a clear lack of functional studies using AAV vectors. This contrasts the efficiency of AAV-2 for phenotypic modu-

lation in the myocardial system [27], but underscores the need to find more efficient vascular gene delivery vectors. Lentiviral vectors, derived from HIV (pseudotyped with the VSV-G to broaden their tropism), have shown promise for gene delivery to organs including brain and liver [28,29]. Third-generation lentiviruses are also deleted of the majority of their native genome, thus improving their safety and applicability [17]. In direct contrast to other vectors, thirdgeneration lentiviruses were efficient at gene delivery to both HSVEC and SMC. This is consistent with previous findings in HUVECs, whereby 70% of infected cells were positive for lentivirus-mediated LacZ expression [30]. Appreciable levels of transgene expression even at short exposure times may have important clinical implications since many cardiovascular gene therapy protocols, such as vein graft failure, require the use of vectors that can efficiently transduce target cells in a minimal surgical therapeutic window. In addition,

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the broad tropism of VSV-G-pseudotyped lentiviruses may, therefore, be advantageous for local delivery to vascular cells, for example during CABG or via stents, and this likely reflects the fusogenic nature of the VSV-G–cell interaction. Exposure of cells to lentivirus in the presence of P-407, an agent previously been shown to enhance Ad-mediated gene delivery to vascular cells in vitro and in vivo, increased levels of transgene expression [31,32]. Long-term gene expression in the vasculature may require the use of alternate promoters to CMV, since promoter activity can be strongly influenced by different vector systems in host cell types. We show high activity of the CMV promoter and lower levels of the hybrid CAG and PGK promoters in EC and SMC, providing scope for alternate promoter usage. As with other systems, further regulation using cell-selective promoters will be possible and may provide added efficiency and safety. However, low levels of transgene expression observed with CAG- and PGK-derived lentiviral vectors may limit their use for vascular gene therapy regimens, since levels of transgene expression are critically important and differ between individual applications. For example, vascular endothelial growth factor is highly potent at low concentrations [33], whereas TIMP-3 only induces its pro-apoptotic effect at mid-nM concentrations, thus requiring high-level transgene overexpression [34,35]. The high levels of Ad-mediated transgene expression in EC most likely reflect the expression of the CAR and avb3 or avb5 integrins on human EC [3,36]. In contrast, human SMC, which have insufficient CAR levels to support CARdependent gene transfer [3], have a different transduction profile (Fig. 5B). Lentiviral vectors were significantly more efficient than both Ad and AAV systems. The high levels of reporter gene expression observed in vitro correlated well with the phenotypic effect from Lenti-TIMP-3-transduced cells and confirmed that lentiviruses may well be of use in the clinical setting. From these results it will be important to evaluate the longevity of gene expression in vivo within transduced blood vessels. In other systems, the integrative mechanism of lentiviruses evokes stable, long-term gene expression [37,38]. Together, this indicates the broad potential of thirdgeneration lentivirus vectors for vascular gene therapy.

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Acknowledgements We wish to thank Margaret Cunningham for technical support and the Medical Research Council (UK) for funding.

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