'Advanced' generation lentiviruses as efficient vectors for ... - Nature

Gene Therapy (2003) 10, 630–636 & 2003 Nature Publishing Group All rights reserved 0969-7128/03 $25.00 www.nature.com/gt

RESEARCH ARTICLE

‘Advanced’ generation lentiviruses as efficient vectors for cardiomyocyte gene transduction in vitro and in vivo D Bonci1, A Cittadini2, MVG Latronico1,3, U Borello4,5, JK Aycock1, A Drusco3, A Innocenzi4, A Follenzi6, M Lavitrano7, MG Monti2, J RossJr8, L Naldini6, C Peschle1, G Cossu4,5 and G Condorelli3,9 Laboratory of Hematology-Oncology, Istituto Superiore di Sanita`, Rome, Italy; 2Department of Clinical Medicine and Cardiovascular Science, University ‘Federico II’, Naples, Italy; 3Kimmel Cancer Center, Thomas Jefferson University, Philadelphia, PA, USA; 4Stem Cell Research Institute, H. S. Raffaele, Milan, Italy; 5Department of Histology and Medical Embryology, University La Sapienza, Rome, Italy; 6 Gene Transfer and Therapy and Tumor Immunology, IRCCS, Institute for Cancer Research and Treatment, University of Turin Medical School, Candiolo (TO), Italy; 7Department of Environmental and Experimental Medicine and Medical Biotechnologies, University of Milano-Bicocca, Monza (MI), Italy; 8Department of Medicine, UCSD School of Medicine La Jolla, CA, USA; and 9II Medical School, IRCCS Neuromed, University ‘La Sapienza’, 00161 Rome, Italy 1

Efficient gene transduction in cardiomyocytes is a task that can be accomplished only by viral vectors. Up to now, the most commonly used vectors for this purpose have been adenoviral-derived ones. Recently, it has been demonstrated that lentiviral vectors can transduce growth-arrested cells, such as hematopoietic stem cells. Moreover, a modified form of lentiviral vector (the ‘advanced’ generation), containing an mRNA-stabilizer sequence and a nuclear import sequence, has been shown to significantly improve gene transduction in growth-arrested cells as compared to the third-generation vector. Therefore, we tested whether the ‘advanced’ generation lentivirus is capable of infecting and transducing cardiomyocytes both in vitro and in vivo, comparing efficacy

in vitro against the third-generation of the same vector. Here we report that ‘advanced’ generation lentiviral vectors infected most (480%) cardiomyocytes in culture, as demonstrated by immunofluorescence and FACS analyses: in contrast the percentage of cardiomyocytes infected by third-generation lentivirus was three- to four-fold lower. Moreover, ‘advanced’ generation lentivirus was also capable of infecting and inducing stable gene expression in adult myocardium in vivo. Thus, ‘advanced’ generation lentiviral vectors can be used for both in vitro and in vivo gene expression studies in the cardiomyocyte. Gene Therapy (2003) 10, 630–636. doi:10.1038/sj.gt.3301936

Keywords: lentivirus; gene therapy; cardiomyocytes; cardiovascular diseases

Introduction Gene transduction in the cardiomyocyte has always represented a major technical difficulty that has hampered detailed study of specific biochemical pathways in this cell type. In fact, conventional methods such as calcium phosphate transfections and liposome or lipofectamine-mediated gene transduction work poorly in cardiomyocytes. While these methods are useful for gene reporter studies, they are of little value when the aim is to introduce a gene into a substantial percentage of cardiomyocytes. For this purpose, adenovirus (Ad) type 5 is the preferred vector.1 However, drawbacks of Ad vectors are their generation process, which is difficult and lengthy, and their immunogenicity, which prevents their use for long-term in vivo experiments.1 Another vector, the adeno-associated virus (AAV), is suitable for in vivo myocardial gene transduction, because of its low Correspondence: Dr G Condorelli, Kimmel Cancer Center, Thomas Jefferson University, 233 S. 10th Street, room 1006, Philadelphia, PA 19107, USA Received 11 June 2002; accepted 18 September 2002

or absent immunogenic potential,2 but the use of AAV for in vitro studies is limited by the low gene expression (less then 10% of cardiomyocytes in culture) achieved during the first weeks of infection.2 Thus, although adenoviral vectors have contributed to the advancement of studies in cardiovascular pathophysiology, they are still not optimal for both in vitro and in vivo studies. Retroviridae are RNA viruses whose RNA has to be reverse transcribed into DNA in order to be integrated into the nucleus.3 g-Retroviruses, represented by Moloney virus, and lentiviruses, represented by HIV, are genuses of the Retroviridae family. Moloney virus requires nuclear breakdown, and therefore M phase, in order to be integrated into host genomic DNA, while lentivirus can enter the nucleus even without mitosis.4,5 The ability of third-generation lentiviral vectors to infect growth-arrested cells (such as cardiomyocytes) has been shown in principle,6,7 but their transducing efficiency has never been analyzed in detail. Moreover, there are no reports demonstrating whether these vectors can be used for myocardial gene expression in vivo. Recently, a new variant of third-generation lentivirus (the ‘advanced’ generation) has been described5 in which sequences of

Lentiviral gene transduction in cardiomyocytes D Bonci et al

the pol gene of HIV-1 (cPPT) and of the post-transcriptional regulatory element of woodchuck hepatitis virus (WPRE) have been inserted. cPPT is a cis element, which has been associated with more efficient gene expression in growth-arrested human hematopoietic progenitor cells.5 Here, we describe the use of ‘advanced’ generation lentiviral vectors as an extremely efficient and simple method of inducing gene expression both in vitro, in neonatal rat cardiac myocytes and in vivo in the adult rat heart.

Results ‘Advanced’ generation lentivirus efficiently transduces neonatal rat cardiomyocytes Neonatal rat cardiomyocytes (2.5 104/well) were exposed to 1 ml supernatant containing the ‘advanced’ generation lentiviral vector (100 ng p24) carrying a GFP reporter gene. Cells were exposed to lentivirus for either 2 h or ON and GFP expression assessed by FACS analysis 12 or 48 h later (Figure 1a and b). In cells exposed for 2 h, the percentage of GFP-positive cells rose from 63% at 12 h to 72 % at 48 h. In cells exposed to the virus ON, the percentage of infection increased from 64 to 84% at 12 and 48 h, respectively. To determine the infection efficiency of this viral vector, scalar cell concentrations were exposed to the same amount of virus (100 ng p24/ml). The percentage

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of infection was evaluated by FACS analysis after exposing the cells for 2 h and detecting GFP after 48 h (Figure 1c). Results show that with this concentration of virus, infection was highest (470%) when 2.5 104 cells were used. Viral stocks concentrated more than 100 ng of p24/ml produced toxicity. In some experiments, expression of the muscle-specific marker, myosin heavy chain (MHC), was detected by indirect immunofluorescence using a monoclonal antibody, MF20, and visualized by a commercial TRITClabeled goat-anti-mouse-IgG secondary antibody (Sigma). Double-labeled cells represented infected cardiomyocytes (Figure 1d). Thus, these experiments show that the lentiviral vector, at a concentration of 100 ng p24, can efficiently infect neonatal rat cardiomyocytes in vitro.

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Comparison of gene transduction efficiencies of ‘advanced’ and third-generation lentiviral vectors in cardiomyocytes In order to compare the infection capacity of the two generations, cardiomyocytes were seeded at 5 104/ well, a number at which GFP expression reaches approximately 50% with 100 ng p24 of ‘advanced’ viral supernatant. Cells were infected for 2 h with p24 concentrations of 100, 10, 1 or 0.1 ng/ml (106,105,104 and 103 TU, respectively) of either ‘advanced’ or thirdgeneration lentivirus (Figure 2c) and GFP expression determined by FACS analysis after 48 h. The efficiency of infection was markedly different in the two types of

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Figure 1 Time-dependent and dose–response effects of ‘advanced’ generation lentiviral (pRRLcPPT.hPGK.EGFP.WPRE) gene transduction in cardiomyocytes in vitro. (a) Quantification of GFP expression by FACS analysis after viral exposure of 2 h or ON and detection after 12 or 48 h (2.5 104 cells infected with 100 ng p24/ml). (b) FACS profile: b.1: mock-infected cells; b.2: 2 h viral exposure and detection 12 h later; b.3: ON exposure and detection 48 h later. (c) FACS analysis of the transduction efficiency of 100 ng p24/ml with different cell concentrations. (d) Confocal microscopy of neonatal rat cardiomyocytes infected with lentivirus. Green: direct GFP fluorescence (d.1); Red: indirect fluorescence with an anti-HMC antibody (d.2); Yellow: a merged image of the two fluorescences, showing coexpression of GFP and myosin in the same cell (d.3). Gene Therapy


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Figure 2 Comparison of the gene transduction efficiencies of third-generation and ‘advanced’ generation lentiviruses in cardiomyocytes. Cardiomyocytes were infected at a concentration of 5 104 cells/ml. (a) The viral supernatants of third (black line) or ‘advanced’ (gray line) lentiviruses were used at the specified concentrations and FACS analysis performed after 48 h. Cells were incubated for 2 h with viral particles. (b) FACS profile of cells infected with third-generation (pRRL.CMV.GFP, b.2–5) or advanced generation (pRRLcppt.CMV.GFP.WPRE, b.6–9) lentivirus. b.1: mock-infected cells; b.2/6: cells infected with 0.1 ng p24/ml supernatant; b.3/7: cells infected with 1 ng p24/ml; b.4/8: cells infected with 10 ng p24/ml; b.5/9: cells infected with 100 ng p24/ ml. (c) Schematic drawing of the vectors used here. c.1: Third generation lentivirus: the vectors carry an internal cassette for the enhanced green fluorescent protein (EGFP) driven by the CMV promoter. The LTR regions with a deletion of 400 bp including the enhancer and promoter from U3 are indicated by AU3, R,U5; major splice donor site (sd); encapsidation signal (f) including the 50 portion of the gag gene (GA); Rev-response element (RRE); splice acceptor sites (sa); c.2: ‘Advanced’ generation lentivirus: the inserted cPPT and WPRE sequences are shown with arrows.

lentivirus. In fact, the ‘advanced’ generation lentivirus showed a four-fold increased efficiency at 100 ng p24/ml as compared to the third-generation lentivirus, and remained at least three-fold higher at lower dilutions (Figure 2a and b).

Role of promoters in mediating the transduction efficiency of lentiviral vectors We tested the effect of two different promoters in determining gene transduction in cardiomyocytes. Lentiviral vectors containing either the human PGK or the CMV promoter in the expression cassette were produced (Figure 3a). Cells were exposed to virus for either 2 h or ON and FACS analysis of GFP expression performed 48 h later. Results demonstrate that the viruses with these two different promoters had comparable efficiencies of infection. In fact, the percentage of infection reached more than 75% using viruses containing either one of the two types of promoters in cells exposed ON and Gene Therapy

approximately 70% in cells exposed for 2 h (Figure 3b and c). The efficiencies of infection were similar also when using diluted supernatants. Mean fluorescence intensity (MFI), a parametrer which corresponds to the strength of the promoter, was also measured. MFI was higher using the lentiviral vector constructed with the CMV promoter with respect to that with the hPGK one (Figure 3d).

In vivo efficiency of lentiviral vectors Once the efficiency of lentiviral vectors was established in vitro, experiments were performed in order to address whether they are able to induce efficient and long-lasting expression in myocardial cells in the intact animal. For this purpose, the hPGK promoter variant of the ‘advanced’ generation lentivirus was used. A previous report on third generation lentiviral vectors in CNS infections demonstrated that lentiviral DNA had to be integrated into the host’s genome for gene expression to


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Figure 3 Comparison of the efficiency of cardiomyocyte gene transduction in vitro of ‘advanced’ lentiviruses with PGK or CMV promoters. Cells (2.5 104/ml) were exposed to different dilutions of either virus. (a) Schematic drawing of the vectors used in these experiments. The vectors differ only in the promoter region. EGFP: enhanced green fluorescent protein. PGK: promoter of the human phosphoglycerate kinase gene. CMV: Cytomegalovirus promoter. cPPT: nuclear import sequence. WPRE: regulatory element of woodchuck hepatitis virus. AU3, R, U5 are the LTR regions, with a deletion of 400 bp, including the enhancer and promoter from U3; SD: major splice donor site. f: encapsidation signal including the 50 portion of the gag gene (GA). RRE: Rev-response element. SA: splice acceptor sites. (b) Percentage of GPF positive cells treated for 2 h or ON and analyzed after 48 h to either PGKcontaining (darker line) or CMV-containing (lighter line) lentiviruses. (c) FACS profile of: c.1: Mock-infected cells; c.2: PGK-promoter or; c.3: CMVpromoter lentivirus. (d) Mean fluorescence intensity (MFI) expressed in relative arbitrary units analyzing cells at 48 h after transduction. We compared MFI of the two promoters using different concentrations of p24.

take place in long-term in vivo experiments.8 Thus, we used the same vector without integrase (int), an enzyme critical for integration of lentiviral DNA with that of the host, as a control. The ability of integrase + (int+) and – (int) lentiviral vectors to infect and induce persistent GFP expression was first tested on the TF1 leukemic cell line. These cells were selected since they are suitable for long-term (more than 2 weeks) experiments in vitro. FACS analysis after 48 h showed that more than 80% of TF1 cells infected with int+ ‘advanced’ lentivirus scored positive for GFP, while only around 40% of cells were infected with the int form. After 15 days, virtually no cells infected with the int vector scored positive for GFP at FACS analysis, while the percentage of cells infected with the int+ lentivirus remained similar to that of 48 h (Figure 4b). These two types of lentiviruses were then used for in vivo experiments. A volume of 200 ml of a 250 concentrated viral solution was injected into the left ventricle of rats while the pulmonary artery and aorta were being clamped during immersion hypothermia, as described in the Methods section. This procedure allows diffusion of the virus throughout the myocardium via the coronary arteries. GFP-positivity was analyzed on sections of myocardial tissue 5 weeks after treatment. Results showed a sustained, diffuse transmural expression of GFP after 5 weeks in the myocardium of

the group of rats (n¼3) treated with the int+ vector. In fact, most cardiomyocytes of int+ virus-infected rats, staining positive to MHC, expressed GFP, while GFP was absent in int- lentivirus-infected rats (n¼3) (Figure 4a).

Discussion Here, we report on the efficacy of a modified lentiviral vector as an easy and efficient way of inducing genes into the cardiomyocyte, both in vitro and in vivo. Our comparison of third-generation and ‘advanced’ lentiviral vectors demonstrates that the latter has an improved effect in transducing cardiomyocytes in vitro. Our data also describe the conditions of infection needed for optimal gene transduction in vitro, showing that ‘advanced’ lentiviral vectors induce a high percentage of GFP positivity in cardiomyocytes after only 2 h of exposure. Previous reports have shown that lentiviruses can infect neonatal rat cardiomyocytes in vitro.6,7 In one of these studies, a high percentage of infection was achieved, but the exact amount of virus needed for extensive infection was not specified.7 In both cases, precise dosage and quantification of infection was not determined, nor was it assessed whether lentivirus induces extensive infection in vivo. In our hands, the Gene Therapy


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Figure 4 Myocardial infection with active integrase (int+) or an integrase-defective (int) virus. (a) Sections of myocardium (20 a.1/5, a.2/6; 40 a.3/7, a.4/8) from rats injected with int+ (a.1/5, a.3/7) or int (a.2/6, a.4/8) lentiviral particles. a.1–4: direct GFP fluorescence; a.5–8: indirect fluorescence for MHC. (b) Percentage of GFP-positive TF1 cells after 48 h or 15 days of int (darker bar) or int+ (lighter bar) lentiviral treatments.

third-generation lentivirus did not achieve satisfactory results, since the percentage of GFP-expressing cells was generally three-fold lower than that achieved with the ‘advanced’ generation vector, thus rendering impractical the use of this vector for studies requiring transduction of a high percentage of cardiomyocytes. On the other hand, ‘advanced’ generation lentiviral vectors were very efficient, transducing more than 80% of cells in vitro after 48 h with only one round of infection. The differences observed between ‘advanced’ and third-generation lentiviruses lie in the 118 bp preceding the promoter region in the shuttle vector, as well as in the WPRE sequence at the 30 end of the expression cassette. Thus, even in this study these two elements have been shown to be critical in enhancing transduction efficiency. We also used two different promoters, CMV and hPGK. A similar percentage of gene transduction was achieved with both, showing that either is suitable for studies in cardiomyocytes. It seems though, by analysis of MFI, that the expression driven by CMV is stronger than that of PGK. Furthermore, the lack of expression seen in vivo with the int– variant of ‘advanced’ generation lentivirus strongly suggests that lentiviral DNA must be integrated with cardiomyocyte DNA in order to achieve a sustained and lasting expression in this cell type, as has been demonstrated also for other growth-arrested cells by Follenzi et al.5 Gene Therapy

In conclusion, the data reported in this work demonstrate that the ‘advanced’ generation lentiviral vector is an excellent cDNA carrier for cardiomyocytes, driving long-term gene expression both in vitro and in vivo. Since lentiviruses are not immunogenic, it is possible to foresee their use in myocardial gene therapy studies in the future.

Methods Cell cultures Neonatal rat cardiomyocytes were obtained utilizing a modification of an original protocol.9–11 Cells were cultured in DMEM-Medium199 (4:1) supplemented with 5% FBS, 5% HS, 1% L-glutamine and 1% penicillin/ streptomycin. Cells were treated with Mitomycin C (Sigma) to prevent fibroblast cell growth. Final cell populations contained more than 95% growth-arrested cardiomyocytes, as assessed by immunofluorescence analysis. 293 T cells (originally called 293tsA1609ne36) were grown in Iscove’s modified Dulbecco’s medium (IMDM, Gibco) supplemented with 10% FBS (Hyclone) and Lglutamine (50 U/ml), penicillin and streptomycin (50 U/ ml). They derive from 293 cells, a continuous human embryonic kidney cell line, transformed with sheared Type 5 Adenovirus DNA, and by transfection with the


tsA 1609 mutant gene of SV40 Large T Antigen and the Neor gene of E. coli. TF1, human erythroleukemia cells, were cultured in RPMI with 10 ng/ml GM-CSF and 10% FBS.

Plasmids The three-plasmid expression system used to generate lentiviral vectors by transient transfection was used as previously described.5,12 The three plasmids were: the packaging plasmid, pCMVDR8.74 designed to provide the HIV proteins needed to produce the virus particle; the envelope-coding plasmid, pMD.G, for pseudotyping the virion with VSV-G, and; the self-inactivating (SIN) transfer vector plasmid (pRRLcPPT.hPGK.EGFP.WPRE or pRRLcPPT.CMV.EGFP.WPRE). The transfer vector plasmid contains the enhanced GFP marker gene driven by either the human phosphoglycerate kinase promoter (hPGK) or the Cytomegalovirus promoter (CMV) and has been described before for assembling ‘advanced’ third-generation lentivirus.5 It has an additional DNA sequence of 118 bp (cPPT) situated before the PGK/ CMV-EGFP cassette, taken from the pol gene, which has been shown to be required in cis. The third-generation transfer vector used for some experiments was pRRL.CMV.EGFP. This vector is identical to that of the ‘advanced’ generation apart from the absence of the cPPT and WPRE sequences. Virus production We produced vector stocks by calcium phosphate transient transfection, cotransfecting the three plasmids in 293 T human embryonic kidney cells, since these cells are good DNA recipients. The calcium phosphate–DNA precipitate was allowed to stay on the cells for 14–16 h, after which the medium was replaced, collected 48 h later, centrifuged at 1000 rpm for 5 min at room temperature and filtered through 0.22 mm pore nitrocellulose filters. Determination of viral titer and transfection efficiency In order to determine the viral particle concentration of the supernatants from 293 T cells, p24 antigen was analyzed by HIV-1 p24 Core profile ELISA (Abbott Diagnostics or NENTM Life Science Products) following the manufacturer’s instructions. Moreover, the supernatants were used to infect TF1 cells to determine biological efficiency before experimentation on cardiomyocytes. Transduction experiments were performed by adding serial dilutions of viral supernatant to 5 104 TF1cells/well in 24-well plates in the presence of Polybrene (4 mg/ml). Transduced cells were analyzed by FACS (FACS Calibur, Becton Dickinson Immunocytometry Systems) and CellQuest (Becton Dickinson) or WindMDI (Microsoft) software used. Typical supernatants contained approximately 104 transfection units (TU) per ng p24 making the titer usually in the range of 106–107 TU/ml. Viral stocks were usually prepared at 106 TU/ml, corresponding to 100 ng p24/ml. Usually, cells were infected at a concentration of 5 104/ml, for non-saturating conditions or 2.5 104/ml, for saturating conditions, with a supernatant titer of 100 ng p24/ml. Relative mean fluorescence intensity (MFI) was calculated using FACS analysis parameters.

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Concentration of viral supernatants For in vivo experiments, the supernatant was concentrated 250-fold. To obtain high-titer vector stocks, medium collected from infected 293 T cells was ultracentrifuged at 50 000 g for 90 min at 41C. Pellets were resuspended with PBS containing 0.5% BSA, pooled and stored under liquid N2. Transduction of cardiomyocytes Cardiomyocytes were plated 2 days before exposure to the virus. Cells (2.5 104) were plated in 24-well plates. On the day of infection, the medium was removed and replaced with viral supernatant to which 4 mg/ml of Polybrene had been added. Cells were then centrifuged in their plate for 45 min in a Beckman GS-6KR centrifuge, at 1800 rpm and 321C. After centrifugation, cells were kept for either 1 h 15 min or ON in a 5% CO2 incubator at 32 or 371C, respectively. After exposure, cells were washed twice with cold PBS and fresh medium added. At either 12 or 48 h after the infection, cells were washed with PBS, harvested with trypsin/EDTA and analyzed by FACS. Infection and detection of GFP in myocardial cells in vivo For in vivo experiments, a preparation of the virus 250fold more concentrated than the regular viral supernatant was used. A method based on that reported by Hajjar et al13 and subsequently modified by Ikeda et al14 was used to deliver the virus. The validity of the method was first determined by gene transduction using adenoviral b-galactosidase (not shown). Briefly, preliminary experiments were performed on male Sprague– Dawley rats (Charles River, Italy) anesthetized with a mixture of ketamine hydrochloride (Sigma, 50 mg/kg BW) and Xylazine (Sigma, 10 mg/kg BW), and then orally intubated and ventilated. The animals were subsequently cooled with ice-bags and their temperature monitored with a thermistor catheter. When the animal’s temperature reached 301C, an anterior thoracotomy was performed, the heart was exteriorized and a 7.0 suture placed on the apex of the left ventricle. A 22 G catheter containing 200 ml of viral solution was then gently introduced into the left ventricle. The aortic root and pulmonary artery were identified and the catheter advanced through the left ventricle into the aortic root. The aorta and pulmonary artery were clamped gently, distal to the site of the catheter and the viral solution injected. The period of total occlusion (time for viral injection and the postinjection period) lasted 20 s, allowing the solution to circulate down the coronary arteries. Then the aortic and pulmonary clamps and the catheter were removed, the pneumothorax evacuated and the chest closed. Animals were transferred back to their cages where they were allowed to recover. The same procedure was followed for sham-operated rats, but the catheter was filled with saline. The extension of b-galactosidase adenoviral infection was determined by 6Br-2-naphtyl-b-D-galactopyranoside/Fast Blue tissue staining after 15 days and compared to control. Most cells in the left ventricle showed blue granular condensations in the b-galactosidase infected hearts, which were absent in shamGene Therapy


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operated animals and in control sections (not shown). Similar experiments were also performed with a vital dye, demonstrating a uniform distribution of the dye throughout the myocardium (not shown).

Acknowledgements The following sources of support are acknowledged: American Heart Association (GLC), Italian Association for Cancer Research (GLC), Fondi 1% Ministero della Sanita’-Italy (GC, LN and GLC), Telethon Association (GC and LN), European Community (GC) and Progetto Terapia Tumori Italy-USA (GLC)

References 1 Hajjar RJ, del Monte F, Matsui T, Rosenzweig A. Prospects for gene therapy for heart failure. Circ Res 2000; 86: 616–621. 2 Svensson EC et al. Efficient and stable transduction of cardiomyocytes after intramyocardial injection or intracoronary perfusion with recombinant adeno-associated virus vectors. Circulation 1999; 99: 201–205. 3 Kay MA, Glorioso JC, Naldini L. Viral vectors for gene therapy: the art of turning infectious agents into vehicles of therapeutics. Nat Med 2001; 7: 33–40. 4 Zennou V et al. HIV-1 genome nuclear import is mediated by a central DNA flap. Cell 2000; 101: 173–185. 5 Follenzi A et al. Gene transfer by lentiviral vectors is limited by nuclear translocation and rescued by HIV-1 pol sequences. Nat Genet 2000; 25: 217–222.

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6 Mochizuki H et al. High-titer human immunodeficiency virus type 1-based vector systems for gene delivery into nondividing cells. J Virol 1998; 72: 8873–8883. 7 Sakoda T, Kasahara N, Hamamori Y, Kedes L. A high-titer lentiviral production system mediates efficient transduction of differentiated cells including beating cardiac myocytes. J Mol Cell Cardiol 1999; 31: 2037–2047. 8 Naldini L et al. Efficient transfer, integration, and sustained long-term expression of the transgene in adult rat brains injected with a lentiviral vector. Proc Natl Acad Sci USA 1996; 93: 11382–11388. 9 Sen A et al. Terminally differentiated neonatal rat myocardial cells proliferate and maintain specific differentiated functions following expression of SV40 large T antigen. J Biol Chem 1988; 263: 19132–19136. 10 De Luca A et al. Characterization of caveolae from rat heart: localization of postreceptor signal transduction molecules and their rearrangement after norepinephrine stimulation. J Cell Biochem 2000; 77: 529–539. 11 Condorelli G et al. Cardiomyocytes induce endothelial cells to trans-differentiate into cardiac muscle: implications for myocardium regeneration. Proc Natl Acad Sci USA 2001; 98: 10733– 10738. 12 Dull T et al. A third generation lentivirus vector with a conditional packaging system. J Virol 1998; 71: 8463–8471. 13 Hajjar RJ et al. Modulation of ventricular function through gene transfer in vivo. Proc Natl Acad Sci USA 1998; 95: 5251–5256. 14 Ikeda Y et al. Restoration of deficient membrane proteins in the cardiomyopathic hamster by in vivo cardiac gene transfer. Circulation 2002; 105: 502–508.