© 1997 by European Society of Cardiology
Copyright © 1997, European Society of Cardiology
Analysis of tissue-specific gene delivery by recombinant adenoviruses containing cardiac-specific promoters
aMedizinische Klinik II, Medizinische Universität zu Lübeck, Ratzeburger, Allee 160, 23538 Lübeck, Germany
bAngewandte Tumorvirologie, Deutsches Krebsforschungszentrum, Heidelberg, Germany
* Corresponding author. Tel.: +49 451 5002734; Fax: +49 451 5006437; E-mail: franz@medinf.mu-luebeck.de
Received 10 February 1997; accepted 29 May 1997
 |
Abstract |
|---|
 Top Abstract 1 Introduction2 Methods3 Results4 DiscussionReferences |
|---|
Objective: To approach heart muscle diseases by gene transfer, an adenoviral vector system was intended to be established suitable for gene expression in ventricular and/or atrial myocardium. Methods: Two adenoviral vectors (Ad-mhcLuc, Ad-mlcLuc) were constructed, in which the luciferase reporter gene is under control of either the ventricle-specific myosin light chain-2 (mlc-2v) or the atrial- and ventricular-specific
-myosin heavy chain (-mhc) promoter. For controls, a recombinant adenovirus without promoter (Ad-Luc) and one with the Rous sarcoma virus (rsv) promoter (Ad-rsvLuc) were generated. A volume of 20 µl containing 2x109 plaque forming units (pfu) of the recombinant adenoviruses Ad-mhcLuc, Ad-mlcLuc, Ad-rsvLuc or Ad-Luc was injected into the cardiac cavity or the quadriceps femoris muscle of neonatal rats. After five days animals were sacrificed and nine different tissues were analyzed for reporter gene expression by detection of light activity relative to mg of tissue. Results: Injections of recombinant adenoviruses into the cardiac cavity of neonatal rats resulted in heart-specific gene expression of Ad-mlcLuc (20 fold of Ad-Luc; 11% of Ad-rsvLuc), whereas Ad-mhcLuc gave mainly luciferase activity in the heart (6.5 fold of Ad-Luc; 3% of Ad-rsvLuc) with additional activity in lung and liver (2–4 fold of Ad-Luc). In the ventricular tissue Ad-mlcLuc revealed a 35-fold higher luciferase activity, whereas Ad-mhcLuc, Ad-rsvLuc and Ad-Luc showed only 2-fold higher luciferase activities compared to the atrium. Viral DNA in atrial and ventricular tissue was detected by PCR at approximately the same abundance independent of the injected type of adenovirus. Direct injection of Ad-mhcLuc and Ad-mlcLuc into the thigh muscle revealed only background luciferase activities. Conclusions: In the adenoviral system only the mlc-2v promoter may fulfil the safety requirements for a myocardial specific gene expression with a high selectivity for the ventricular myocardium, thus providing a promising tool for future gene therapy of cardiomyopathies.
KEYWORDS Adenovirus; Gene therapy; Myocardium; Heart; Myosin light chain; Myosin heavy chain; Promoter; Rat
|
1 Introduction |
|---|
|
TopAbstract1 Introduction2 Methods3 Results4 DiscussionReferences |
|---|
Somatic gene therapy represents a promising approach for the treatment of inherited heart diseases mainly by substitution of an affected gene using a safe and efficient delivery system for the therapeutic gene. This approach was taken recently to modify excitability of myocardial cells or to improve blood flow in the heart of experimental animals by adenoviral gene transfer [1, 2]. The results of these experimental studies may be applicable in the treatment of patients with long QT syndrome or ischemic heart diseases. So far, recombinant adenoviruses have been shown to be the most potent vector system for the transfer of foreign genes to the myocardium in the experimental setting [3–5]. In contrast to retroviruses, adenoviruses can infect differentiated cardiomyocytes with a 50–100 fold higher transduction efficiency of the rat heart compared to the injection of naked plasmid DNA [3]. However, in most adenoviral vector systems gene expression is transient due to an immune response and the gene expression is not restricted to myocardial tissue [5–7]. Second and third generation adenoviral vectors may reduce the immune response and prolong gene expression and thus facilitate the use of this transfer system. However, these newly developed adenoviruses have not yet been characterized in respect to their persistence in the myocardium [8–12]. So far, first generation adenoviral vectors have been tested by different methods of injection for their efficiency to infect heart muscle cells in vivo. After infusion of adenoviruses into the tail vein of mice only 0.2% of cardiomyocytes were transfected and the virus had infected different tissues throughout the body [13]. Direct injection of viral particles into the myocardium, the cardiac cavity, or the local injection by percutaneous transluminal coronary gene transfer (PTGT) have been proven to be more efficient for gene delivery to the myocardium [3–5, 7, 14]. In previous gene transfer approaches non-cardiomyocytes were also transduced. Direct injection of adenoviruses into the heart lead to infection and transgene expression in many other tissues including thymus, lung and liver [3, 4, 7, 14]. Percutaneous transluminal gene therapy (PTGT) administration of viral particles results in transduction of the coronary vasculature and non-myocyte connective tissue cells throughout the region of the myocardium supplied by the injected coronary artery [5]. Clearly the expression of a putative therapeutic gene in non-cardiomyocytes may have undesired side effects. With respect to the risk-to-benefit ratio of gene therapy, which is higher for patients with cardiomyopathy, where alternative treatments such as heart transplantation or cardiomyoplasty exist, than for patients suffering from cancer or AIDS, the safety of the vector system is a major concern. An improved adenoviral vector system should be immunologically tolerated and gene expression should be restricted to cardiomyocytes and if possible to a defined region such as atrium or ventricle. A cardiac specific gene expression may be achieved by transgene expression under the control of a specific promoter element.
The best characterized promoters for a tissue-specific expression in embryonic and adult ventricular myocardium derive from the myosin light chain-2 (mlc-2v) and the alpha-myosin heavy chain (-mhc) genes. In a transgenic animal model, we previously demonstrated the ventricular specific gene expression under the control of the 2.1 kb myosin light chain-2 (mlc-2v) promoter [15, 16]. Based on these results, we were recently able to target gene expression exclusively to the myocardium using the 0.8 kb mlc-2v promoter as indicated by the luciferase reporter gene in an adenoviral vector construct (Ad-mlcLuc) [17]. In another transgenic animal model the -mhc promoter was shown to be active in ventricular and atrial tissues [18, 19]. In order to target genes and the corresponding proteins to defined regions within the heart such as atrium and/or ventricle, we compared the rat cardiac -mhc promoter with the mlc-2v promoter with regard to their cardiac selectivity and their differences in atrial and ventricular gene expression.
|
2 Methods |
|---|
|
TopAbstract1 Introduction2 Methods3 Results4 DiscussionReferences |
|---|
2.1 Construction of recombinant plasmids pAd-rsvLuc, pAd-mlcLuc, pAd-mhcLuc and pAd-Luc
Plasmids pAd-rsvLuc, pAd-mlcLuc, pAd-mhcLuc and pAd-Luc are derivates of the plasmid pAd.RSV-βgal, in which the BamHI/KpnI RSV-βgal cassette is exchanged for luciferase cDNA containing the endogenous polyadenylation signal [13]. The construction of the plasmids pAd-Luc, pAd-rsvLuc, pAd-mlcLuc and pBluescript-Luc was reported previously [17]. For the construction of plasmid pAd-mhcLuc the EcoRI/HindIII
-mhc promoter fragment (–612 bp to +420 bp) plus 32 bp of the 5' multiple cloning side of plasmid pGCATCalphaMHC were excised by BamHI and HindIII. The 1064 bp fragment was subcloned into the BamHI/HindIII sites of pBluescript-Luc, which resulted in the generation of plasmid pBluescript-mhcLuc [20]. The BamHI/KpnI luciferase fragment of subclone pBluescript-Luc was then cloned into the BamHI/KpnI site of plasmid pAd.RSV-βgal resulting in plasmid pAd-mhcLuc.
2.2 Construction of recombinant adenoviruses
Recombinant viruses Ad-rsvLuc, Ad-mlcLuc and Ad-Luc were constructed as previously described [17]. Virus Ad-mhcLuc was generated after homologous recombination between plasmid pAd-mhcLuc and plasmid pBHG10 in 911 cells [21, 22]. Briefly, 1x106 911 cells were cotransfected with 5 µg AatII linearized pAd-mhcLuc and 5 µg of pBHG10. After overlaying with agar for 8–10 days and incubation at 37°C, plaques containing recombinant viruses were picked and screened for the correct integration of the transgene by restriction enzyme analysis. Recombinant adenoviruses were again plaque purified before being propagated in 911 cells and purified by two rounds of cesium chloride density centrifugation [13]. Finally, viruses were dialyzed against TD-buffer (137 mM NaCl, 5 mM KCl, 0.7 mM Na2HPO4, 0.5 mM CaCl2, 1mM MgCl2, 10% (v/v) glycerol, 25 mM Tris–HCl pH 7.4) and stored at –70°C. The titer of the frozen viral stocks was determined by plaque assay using 911 cells [13]. All recombinant adenoviruses had a titer of approximately 1011 pfu/ml DNA of the viral stocks. They were isolated and analyzed by restriction enzyme analysis and PCR for the correct integration of the insert. In addition, viral stocks were tested by PCR for wildtype Ad-5, showing no contamination in 50 ng of recombinant viral DNA [23].
2.3 In vivo injections into the cardiac cavity and thigh muscle
For the injection experiments, two days old Sprague–Dawley rats were used. The investigation conforms with the Guide for the Care and Use of Laboratory Animals published by the US National Institute of Health (NIH Publication No. 8523, revised 1985). Prior to injections, rats were anaesthetized by a 3–5 min inhalation of methoxyfluran (Metofane, Janssen GmbH). 2x109 pfu of the recombinant adenoviruses Ad-Luc, Ad-rsvLuc, Ad-mhcLuc or Ad-mlcLuc were injected in a 20 µl volume using a 271/2 gauge tuberculin syringe as previously described [17]. Injections were carried out by transthoracal puncture of the cardiac cavity. The flashback of pulsatile blood into the needle indicated the intracavitary position of the needle tip. A slow rate of injection (20 µl/min) was achieved using a self-constructed device. Injection into the quadriceps femoris was performed accordingly.
2.4 Luciferase assay
For in vivo studies, animals were sacrificed five days post injection by decapitation. In a first set of experiments (data represented in Table 1) several tissues were dissected (total heart, intercostal muscle, thymus, lung, diaphragm, abdominal wall muscle, liver, stomach, spleen, quadriceps femoris) and in a second set of experiments (data represented in Fig. 2) only ventricular and atrial tissues were excised using a stereo microscope. After dissection tissues were immediately frozen in liquid nitrogen. Samples were weighed, homogenized in lysis buffer (1% (v/v) Triton X-100, 1 mM DTT, 100 mM potassium phosphate pH 7.8), centrifuged and the supernatant was used to perform luciferase assays as described [24]. The luciferase activity is given in relative light units (RLU) per mg wet tissue after correction for background luciferase activity measured in tissues of uninfected control animals.
|
|
2.5 PCR assay
Genomic DNA was extracted from all tissues investigated as well as the same ventricular and atrial specimen used for the luciferase assay applying the QIAamp tissue kit (QIAGEN, Germany). Eight representative animals, two for each of the four different adenoviruses, were analyzed for tissue distribution of the viruses using polymerase chain reaction (PCR) as previously described [17]. Gel electrophoresis of the PCR product revealed a specific 860 bp band.
|
3 Results |
|---|
|
TopAbstract1 Introduction2 Methods3 Results4 DiscussionReferences |
|---|
3.1 Construction of recombinant adenoviruses
To assess the specificity of gene expression driven by the
-mhc and mlc-2v promoter in the adenoviral vector system, four types of recombinant adenoviruses were constructed as shown in Fig. 1. Each of the replication defective adenoviruses contains the luciferase reporter gene under the control of a different upstream regulatory region, such as the 1.0 kb -mhc, 0.8 kb mlc-2v and the 0.6 kb rsv promoter, substituting the E1 region 3' of the encapsidation signal 
[20, 25–28]. The resulting recombinant adenoviruses Ad-mhcLuc, Ad-mlcLuc and Ad-rsvLuc were named according to their respective promoter. The adenoviral construct Ad-Luc (Fig. 1D) containing only the luciferase reporter gene served as a negative control virus.
|
3.2 Transgene expression in neonatal rats after injection of recombinant adenoviruses into the cardiac cavity and into the thigh muscle
In order to test whether the recombinant adenoviruses Ad-mhcLuc and Ad-mlcLuc are able to direct luciferase expression specifically into the heart muscle, a volume of 20 µl containing 2x109 pfu was injected into the cardiac cavity of newborn rats. The positive control virus Ad-rsvLuc and the negative control virus Ad-Luc were administered via the same route of injection. Luciferase activities determined five days after injection for nine different tissues are summarized in Table 1. Injection of Ad-rsvLuc led to the highest luciferase activity in heart and intercostal muscle and showed a substantial level of reporter gene expression in thymus, diaphragm, lung and a lower abundance in the abdominal wall muscle, liver and spleen. This is consistent with the previously demonstrated pattern of adenoviral DNA distribution after intracavitary injection of recombinant adenovirus [17]. The negative control virus Ad-Luc showed a low level luciferase activity in all tissues with a slightly higher value in diaphragm, heart, intercostal muscle and thymus due to the transthoracal gene transfer method. Adenovirus Ad-mhcLuc and Ad-mlcLuc harboring the heart muscle specific promoters revealed highest reporter gene expression in the heart. Compared to the promoterless control virus Ad-Luc, Ad-mhcLuc infected animals showed additional luciferase activity in lung and liver tissue. By contrast, adenoviral Ad-mlcLuc transfer resulted in heart specific gene expression with only background activities below the negative control virus Ad-Luc. Light activity levels of Ad-mlcLuc in the heart were 20 fold higher in comparison to Ad-Luc and reached 11% of the activity obtained by Ad-rsvLuc. Ad-mhcLuc gave only a 6.5 fold higher luciferase activity in the heart compared to Ad-Luc. This level corresponds to 3% of the Ad-rsvLuc activity. These data demonstrate, that in contrast to the 0.8 kb mlc-2v promoter the 1.0 kb
-mhc promoter does not provide a cardiac-specific gene expression.
To test whether the adenoviral vector Ad-mhcLuc reveals reporter gene activity in skeletal muscle after a direct needle injection, a volume of 20 µl containing 2x10 [9]pfu of recombinant adenovirus was injected into the right quadriceps femoris muscle. A luciferase activity of 3,4±0,8 RLU per 10–3 mg tissue was measured five days after injection (n = 4). This result was comparable to the negative control virus Ad-Luc and Ad-mlcLuc (data not shown) as previously published [17]. Adenoviruses containing the -mhc or the mlc-2v promoter showed only background activities comparable to Ad-Luc. These data suggest, that in the adenoviral context the -mhc promoter, like the mlc-2v promoter, has no skeletal muscle activity.
3.3 Ventricle versus atrium specific gene expression after injection of recombinant adenoviruses into the cardiac cavity
To study the regional differences of reporter gene expression in atrial and ventricular myocardium the four recombinant adenoviruses were injected into the cardiac cavity accordingly. Five days after injection, luciferase activity was determined in heart ventricle and atrium (see Fig. 2A and 2B). While Ad-mlcLuc showed high luciferase activity in the ventricle (Fig. 2A), the luciferase expression level in the atrium was comparable to the negative control Ad-Luc (Fig. 2B). Fig. 2C shows the ratio of activity of the four recombinant adenoviruses in ventricle to atrium. While Ad-rsvLuc, Ad-mhcLuc and Ad-Luc injection led to an approximately 2-fold higher luciferase activity in the ventricle, Ad-mlcLuc injection gave a 35-fold higher luciferase activity in the ventricular compared to the atrial tissue (Fig. 2C). These data demonstrate that Ad-mlcLuc is specifically active in the ventricle of neonatal rats.
3.4 Tissue infection in neonatal rats after intracardiac injection of recombinant adenoviruses
To evaluate the efficiency of atrial and ventricular gene transfer following intracardiac injection of the four recombinant viruses, genomic DNA from atrial and ventricular heart tissue tested in the luciferase assay was analyzed for the presence of adenoviral DNA sequences by PCR. Genomic DNA from eight representative animals (two for each recombinant type of virus) was assayed for the presence of adenoviral sequences by PCR. The sensitivity of the semiquantitative PCR-assay was assessed previously [17]. In adenovirus infected animals, viral DNA was detected to similar amounts both in ventricular and atrial tissue. This could be repeated independently of the injected recombinant adenovirus (Fig. 3) indicating a similar intracardiac viral distribution after intracavitary application. The extent of gene transfer to noncardiac tissue following injection into the cardiac cavity revealed a pattern of viral distribution as described [17]. The bulk of adenoviral DNA was detected in lung, thymus, intercostal muscle, diaphragm and liver (data not shown). Thus, gene expression of Ad-mhcLuc and Ad-mlcLuc may result from the regulatory promoter sequence of -mhc and mlc-2v and may not be due to a difference in tissue infection or tissue specific differences in local viral concentration.
|
|
4 Discussion |
|---|
|
TopAbstract1 Introduction2 Methods3 Results4 DiscussionReferences |
|---|
We could previously show that the heart-muscle specific myosin-light-chain 2 (mlc-2v) promoter retains its in vivo specificity of gene expression in the myocardium [15, 16]even after incorporation into an adenoviral vector, Ad-mlcLuc [17]. In this report we describe, that the reporter gene of Ad-mlcLuc is exclusively expressed in the ventricles of neonatal rats in vivo. By contrast using the adenoviral shuttle vector Ad-mhcLuc, where the
-myosin heavy chain promoter was used to drive the transgene, the reporter gene was active in ventricular and atrial myocardium, and revealed an ectopic expression in lung as well as liver tissue.
It has been shown by several groups that adenoviruses are very efficient vectors to transduce skeletal muscle and cardiac muscle cells in vitro and in vivo [3–5, 29]. However, the control of a tissue specific gene expression has remained a major difficulty. So far, only two skeletal muscle-specific promoters have been tested in adenoviral vectors [30, 31]. These two are the mouse skeletal -actin promoter enhanced by 300 bp of the mouse myosin light chain 1/3 locus and the chicken acetylcholin receptor (AChR) 1 subunit promoter. Both have been shown to be active in skeletal muscle in vivo. Tissue specificity of gene expression, however, was not assessed in these studies.
In concordance with the results reported earlier [17], we could show that injection of the recombinant viruses into the cardiac cavity of neonatal rats results in a heart muscle-specific gene expression in vivo, when the virus contains a 800 bp fragment of the mlc-2 promoter substituting the E1 region. After intracavitary application of Ad-mlcLuc myocardial light activity was 11% in relation to Ad-rsvLuc infection and 20 fold above the luciferase activity of the negative control virus Ad-Luc (Table 1). In the other 9 tissues tested, the mlc-2v promoter was inactive and light activity did not exceed the expression level of the promoterless construct Ad-Luc. Interestingly, Admlc-Luc showed only background activity in atrial myocardium, but was restricted in its activity to the ventricular myocardium of neonatal rats. The negative control construct Ad-Luc and the positive control construct Ad-rsvLuc revealed an approximately two times higher activity in ventricular as compared to atrial myocardium. However, Ad-mlcLuc was 35 times more active in the ventricular compartment (Fig. 2C). The detection of adenoviral DNA by a specific PCR in atrial and ventricular tissue did not dependent on the injected type of adenovirus indicating a ventricle-specific regulation of Ad-mlcLuc by the mlc-2v promoter (Fig. 3). Therefore we conclude that in adenoviral vectors the mlc-2v promoter retains its ventricle specificity first demonstrated in the transgenic system [15, 16].
The two fold higher luciferase activity of the control constructs in the ventricular as compared to the atrial myocardium can be explained by the mode of intracavitary application. After injection of a recombinant adenovirus expressing the β-galactosidase (Ad.RSVβgal) reporter gene, we reported recently that half of the β-galactosidase activity in the myocardium resulted from viral accumulation along the transventricular needle track [17], whereas in the remaining myocardium only 1–2% of the cardiomyocytes stained blue. Despite of limitations in efficient gene delivery, the neonatal rat model was used successfully by Huard to study adenoviral gene transfer [7]. Furthermore, we demonstrated this model to be useful for the in vivo evaluation of promoter specificity within the adenoviral context [17]. For an efficient gene therapy approach, however, a catheter guided delivery system such as percutaneous transluminal gene transfer (PTGT) should be applied [5], which is not feasible in neonatal rats.
For gene therapy of cardiovascular diseases it is useful to target recombinant gene expression to the myocardium. Previous attempts of adenoviral gene transfer have not allowed a precise gene expression in cardiac cells [3, 7, 14]. The finding that administration of recombinant adenovirus resulted in infection and expression of the transgene in many non-cardiac tissues raises important safety concerns. For example, a strong expression of the Drosophila Shaker potassium channel was reported in liver after intracardiac injection of the recombinant adenovirus AdShK [1]. As noted by the authors, this may have phenotypic consequences since the hepatocyte membrane potential determines the rate of bile acid uptake. Such undesired effects could be avoided by using the here described adenoviral vector Ad-mlcLuc, which allows a ventricular muscle-specific gene expression. It may be necessary to induce gene expression both in ventricular and atrial tissue. Based on transgenic experiments showing a ventricular and atrial activity of the -mhc promoter [19], we constructed the adenoviral vector Ad-mhcLuc where the rat -mhc promoter is driving the luciferase reporter gene [20]. After intracavitary injection of the recombinant adenovirus Ad-mhcLuc the highest luciferase activity was found in the myocardium, which was 3–4 fold less compared to Ad-mlcLuc. In contrast to Ad-mlcLuc, luciferase activity under control of the -mhc promoter was also detected at significant levels in atrium (Fig. 2B), lung and liver (Table 1). It has been shown before, that the -mhc promoter drives gene expression in the lung of transgenic animals [18]. An expression in the liver, however, has not yet been reported. The non-specific activity of Ad-mhcLuc in the liver might be an unique sequela of the adenoviral vector system. In skeletal muscle tissue, however, Ad-mhcLuc was inactive and showed only background luciferase activity (0.05% of Ad-rsvLuc) even when injected directly into the thigh muscle. We, therefore, believe that only the mlc-2v promoter but not the -mhc promoter may be potentially useful within adenoviral vectors to target gene expression to heart and ventricular muscle tissue respectively. To characterize the usefullness of this novel adenoviral gene shuttle system with potential ventricular specificity further investigations in adult rat myocardium and larger animals such as rabbit or pig are necessary.
The low level of luciferase expression driven by the -mhc promoter in comparison to the mlc-2v promoter may be due to the chosen neonatal rat model because of two promoter specific properties. First, it is known that neonatal rat hearts express primarily the β-mhc rather than the -mhc gene [32]. Secondly, mlc-2v promoter activity has been demonstrated to be higher in neonatal compared to adult mice [15]. Both effects may account for the 3 to 4 fold lower expression level of the Ad-mhcLuc viruses in the neonatal rat model.
In this study a first generation adenoviral vector was used [13]. Within the time frame of five days no cell necroses, mononuclear cell infiltrations or other signs of inflammation were observed as described previously [17]. The problem of a transient transgene expression may be overcome by second and third generation adenoviral vector systems [8–12, 14]. When long lasting gene expression becomes an established reality, tissue specificity will be of critical importance to circumvent side effects caused by a long lasting gene expression. Combination of the tissue specificity of the mlc-2v promoter with improved adenoviral vectors may facilitate the use of recombinant adenoviruses to treat heart muscle diseases such as dilated or hypertrophic cardiomyopathies.
Time for primary review 21 days.
|
Acknowledgements |
|---|
We thank Professor Dr. Alex van der Eb (University of Leiden) for providing us the cell line 911, Professor Dr. Martin Paul (Free University of Berlin) for the gift of the 1.0 kb
-MHC promoter and Yvonne Müller for excellent technical assistance. The generous support of Professor Dr. Harald zur Hausen is highly appreciated. This work was supported by the Deutsche Forschungsgemeinschaft (SFB 320/B6). |
Notes |
|---|
1 Both authors contributed equally.
|
References |
|---|
|
TopAbstract1 Introduction2 Methods3 Results4 DiscussionReferences |
|---|
- Johns DC, Nuss HB, Chiamvimonvat N, Ramza BM, Marban E, Lawrence JH. Adenovirus-mediated expression of a voltage-gated potassium channel in vitro (rat cardiac myocytes) and in vivo (rat liver). J Clin Invest (1995) 96:1152–1158.[Web of Science][Medline]
- Giordano FJ, Ping P, McKirnan MD, et al. Intracoronary gene transfer of fibroblast growth factor-5 increases blood flow and contractile function in an ischemic region of the heart. Nat Med (1996) 2:534–539.[CrossRef][Web of Science][Medline]
- Kass Eisler A, Falck Pedersen E, Alvira M, et al. Quantitative determination of adenovirus-mediated gene delivery to rat cardiac myocytes in vitro and in vivo. Proc Natl Acad Sci USA (1993) 90:11498–11502.
[Abstract/Free Full Text] - Guzman RJ, Lemarchand P, Crystal RG, Epstein SE, Finkel T. Efficient gene transfer into myocardium by direct injection of adenovirus vectors. Circ Res (1993) 73:1202–1207.
[Abstract/Free Full Text] - Barr E, Carroll J, Kalynych AM, et al. Efficient catheter-mediated gene transfer into the heart using replication defective adenovirus. Gene Ther (1994) 1:51–58.[Web of Science][Medline]
- Yang Y, Nunes FA, Berencsi K, Furth EE, Gonczol E, Wilson JM. Cellular immunity to viral antigens limits E1-deleted adenovirus for gene therapy. Proc Natl Acad Sci USA (1994) 91:4407–4411.
[Abstract/Free Full Text] - Huard J, Lochmüller H, Acsadi G, Jani A, Massie B, Karpati G. The route of administration is a major determinant of the transduction efficiency of rat tissues by adenoviral recombinants. Gene Ther (1995) 2:107–115.[Web of Science][Medline]
- Krougliak V, Graham FL. Development of cell lines capable of complementing E1, E4 and protein IX defective adenovirus type 5 mutants. Hum Gene Ther (1995) 6:1575–1586.[Web of Science][Medline]
- Wang Q, Jia XC, Finer MH. A packaging cell line for propagation of recombinant adenovirus containing two lethal gene-region deletions. Gene Ther (1995) 2:775–783.[Web of Science][Medline]
- Yeh P, Dedieu JF, Orsini C, Vigne E, Denefle P, Perricaudet M. Efficient dual transcomplementation of adenovirus E1 and E4 regions from a 293-derived cell line expressing a minimal E4 functional unit. J Virol (1996) 70:559–565.[Abstract]
- Engelhardt JF, Ye X, Doranz B, Wilson JM. Ablation of E2a in recombinant adenoviruses improves transgene persistence and decreases inflammatory response in mouse liver. Proc Natl Acad Sci USA (1994) 91:6196–6200.
[Abstract/Free Full Text] - Yang Y, Nunes FA, Berencsi K, Gonczol E, Engelhardt JF, Wilson JM. Inactivation of E2a in recombinant adenoviruses improves the prospect for gene therapy in cystic fibrosis. Nat Gen (1994) 7:362–369.[CrossRef][Web of Science][Medline]
- Stradford-Perricaudet LD, Makeh I, Perricaudet M, Briand P. Widespread long-term gene transfer to mouse skeletal muscles and heart. J Clin Invest (1992) 90:626–630.[Web of Science][Medline]
- Kass Eisler A, Falck Pedersen E, Elfenbein DH, Alvira M, Buttrick PM, Leinwand LA. The impact of developmental stage, route of administration and the immune system on adenovirus mediated gene transfer. Gene Ther (1994) 1:395–402.[Web of Science][Medline]
- Franz WM, Breves D, Klingel K, Brem G, Hofschneider PH, Kandolf R. Heart-specific targeting of firefly luciferase by the myosin light chain-2 promoter and developmental regulation in transgenic mice. Circ Res (1993) 73:629–638.
[Abstract/Free Full Text] - Franz WM, Brem G, Katus HA, Klingel K, Hofschneider PH, Kandolf R. Characterization of a cardiac-selective and developmentally upregulated promoter in transgenic mice. Cardioscience (1994) 5:235–243.[Web of Science][Medline]
- Rothmann T, Katus HA, Hartong R, Perricaudet M, Franz WM. Heart muscle-specific gene expression using replication defective recombinant adenoviurs. Gene Ther (1996) 3:919–926.[Web of Science][Medline]
- Subramaniam A, Jones WK, Gulick J, Wert S, Neumann J, Robbins J. Tissue specific regulation of the alpha-myosin heavy chain gene promoter in transgenic mice. J Biol Chem (1991) 266:24613–24620.
[Abstract/Free Full Text] - Subramaniam A, Gulick J, Neumann J, Knotts S, Robbins J. Transgenic analysis of the thryoid elements in the alpha-cardiac myosin heavy chain gene promoter. J Biol Chem (1993) 268:4331–4336.
[Abstract/Free Full Text] - Mahdavi V, Cahmber AP, Nadal-Ginard B. Cardiac alpha- and beta-myosin heavy chain genes are organized in tandem. Proc Natl Acad Sci USA (1984) 81:2626–2630.
[Abstract/Free Full Text] - Fallaux FJ, Kranenburg O, Cramer SJ, et al. Characterization of 911: A new helper cell line for the titration and propagation of early region 1-deleted adenoviral vectors. Hum Gene Ther (1996) 7:215–222.[Web of Science][Medline]
- Bett AJ, Haddara W, Prevec L, Graham FL. An efficient and flexible system for construction of adenovirus vectors with insertions or deletions in early regions 1 and 3. Proc Natl Acad Sci USA (1994) 91:8802–8806.
[Abstract/Free Full Text] - Zhang WW, Koch PE, Roth JA. Detection of wild-type contamination in a recombinant adenoviral preparation by PCR. BioTechniques (1995) 18:444–447.[Web of Science][Medline]
- Ausbel JM, Brent R, Kingston RR, et al. Current Protocols in Molecular Biology. New York: Greene and Wiley, 1989.
- Hearing P, Shenk T. The adenovirus type 5 E1A transcriptional control region contains a duplicated enhancer element. Cell (1983) 33:695–703.[CrossRef][Web of Science][Medline]
- Hearing P, Samulski RJ, Wishart WL, Shenk T. Identification of a repeated sequence element required for efficient encapsidation of the adenovirus type 5 chromosome. J Virol (1987) 61:2555–2558.
[Abstract/Free Full Text] - de Wet JR, Wood KV, DeLuca M, Helinski DR, Subramani S. Firefly luciferase gene: Structure and expression in mammalian cells. Mol Cell Biol (1987) 7:725–737.
[Abstract/Free Full Text] - Henderson SA, Spencer M, Sen A, Kumar C, Siddiqui MA, Chien KR. Structure, organization, and expression of the rat cardiac myosin light chain-2 gene. J Biol Chem (1989) 264:1842–1848.
- Acsadi G, Jani A, Massie B, et al. A differential efficiency of adenovirus-mediated in vivo gene transfer into skeletal muscle cells of different maturity. Hum Mol Gen (1994) 3:579–584.
[Abstract/Free Full Text] - Quantin B, Perricaudet LD, Tajabkhsh S, Mandel JL. Adenovirus as an expression vector in muscle cells in vivo. Proc Natl Acad Sci USA (1992) 89:2581–2584.
[Abstract/Free Full Text] - Bessereau JL, Stratford-Perricaudet LD, Piette J, Le Poupon C, Changeux JP. In vivo and in vitro analysis of electrical activity-dependent expression of muscle acetylcholione receptor genes using adenovirus. Proc Natl Acad Sci USA (1994) 91:1304–1308.
[Abstract/Free Full Text] - Lompre AM, Nadal-Ginard B, Mahdavi V. Expression of the cardiac ventricular - and β-myosin heavy chain genes is developmentally and hormonally regulated. J Biol Chem (1984) 259:6437–6446.
[Abstract/Free Full Text]
CiteULike 
Connotea 
Del.icio.us What's this?
This article has been cited by other articles:
|
|
 |
|
  O. J. Muller, H. A. Katus, and R. Bekeredjian Targeting the heart with gene therapy-optimized gene delivery methods Cardiovasc Res, February 1, 2007; 73(3): 453 - 462. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 R. Zhou, P. D. Acton, and V. A. Ferrari Imaging Stem Cells Implanted in Infarcted Myocardium J. Am. Coll. Cardiol., November 21, 2006; 48(10): 2094 - 2106. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
O. J. Muller, B. Leuchs, S. T. Pleger, D. Grimm, W.-M. Franz, H. A. Katus, and J. A. Kleinschmidt Improved cardiac gene transfer by transcriptional and transductional targeting of adeno-associated viral vectors Cardiovasc Res, April 1, 2006; 70(1): 70 - 78. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. Miyagawa, M. Beyer, B. Wagner, M. Anton, C. Spitzweg, B. Gansbacher, M. Schwaiger, and F. M. Bengel Cardiac reporter gene imaging using the human sodium/iodide symporter gene Cardiovasc Res, January 1, 2005; 65(1): 195 - 202. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. M. Jones, J. A. Petrofski, K. H. Wilson, C. Steenbergen, W. J. Koch, and C. A. Milano {beta}2 Adrenoceptor gene therapy ameliorates left ventricular dysfunction following cardiac surgery Eur. J. Cardiothorac. Surg., December 1, 2004; 26(6): 1161 - 1168. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 H. Su, S. Joho, Y. Huang, A. Barcena, J. Arakawa-Hoyt, W. Grossman, and Y. W. Kan Adeno-associated viral vector delivers cardiac-specific and hypoxia-inducible VEGF expression in ischemic mouse hearts PNAS, November 16, 2004; 101(46): 16280 - 16285. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. Miyagawa, M. Anton, R. Haubner, M. V. Simoes, C. Stadele, W. Erhardt, S. Reder, T. Lehner, B. Wagner, S. Noll, et al. PET of Cardiac Transgene Expression: Comparison of 2 Approaches Based on Herpesviral Thymidine Kinase Reporter Gene J. Nucl. Med., November 1, 2004; 45(11): 1917 - 1923. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
G. Vassalli, S. Fleury, J. Li, J.-J. Goy, L. Kappenberger, and L. K. von Segesser Gene transfer of cytoprotective and immunomodulatory molecules for prevention of cardiac allograft rejection Eur. J. Cardiothorac. Surg., November 1, 2003; 24(5): 794 - 806. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. G. Nir, R. David, M. Zaruba, W.-M. Franz, and J. Itskovitz-Eldor Human embryonic stem cells for cardiovascular repair Cardiovasc Res, May 1, 2003; 58(2): 313 - 323. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 W. Poller, H. Fechner, M. Noutsias, C. Tschoepe, M. Pauschinger, and H.-P. Schultheiss The molecular basis of cardiotropic viral infections Eur. Heart J. Suppl., December 1, 2002; 4(suppl_I): I18 - I30. [Abstract] [PDF]
|
|
|
|
 |
|
 R. Aikawa, G. S. Huggins, and R. O. Snyder Cardiomyocyte-specific Gene Expression Following Recombinant Adeno-associated Viral Vector Transduction J. Biol. Chem., May 17, 2002; 277(21): 18979 - 18985. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. I. Phillips, Y. Tang, K. Schmidt-Ott, K. Qian, and S. Kagiyama Vigilant Vector: Heart-Specific Promoter in an Adeno-Associated Virus Vector for Cardioprotection Hypertension, February 1, 2002; 39(2): 651 - 655. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
R. Khurana, J. F. Martin, and I. Zachary Gene Therapy for Cardiovascular Disease: A Case for Cautious Optimism Hypertension, November 1, 2001; 38(5): 1210 - 1216. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. E. Lehnart, P. M. L. Janssen, W. M. Franz, J. K. Donahue, J. H. Lawrence, E. Marban, J. Prestle, and G. Hasenfuss Preservation of myocardial function after adenoviral gene transfer in isolated myocardium Am J Physiol Heart Circ Physiol, September 1, 2000; 279(3): H986 - H991. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 K. Eizema, H. Fechner, K. Bezstarosti, S. Schneider-Rasp, A. van der Laarse, H. Wang, H.-P. Schultheiss, W. C. Poller, and J. M. J. Lamers Adenovirus-Based Phospholamban Antisense Expression as a Novel Approach to Improve Cardiac Contractile Dysfunction : Comparison of a Constitutive Viral Versus an Endothelin-1-Responsive Cardiac Promoter Circulation, May 9, 2000; 101(18): 2193 - 2199. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
P. Sinnaeve, O. Varenne, D. Collen, and S. Janssens Gene therapy in the cardiovascular system: an update Cardiovasc Res, December 1, 1999; 44(3): 498 - 506. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
W. M Franz, O. J Mueller, M. Fleischmann, P. Babij, N. Frey, M. Mueller, U. Besenfelder, A. F.M Moorman, G. Brem, and H. A Katus The 2.3 kb smooth muscle myosin heavy chain promoter directs gene expression into the vascular system of transgenic mice and rabbits Cardiovasc Res, September 1, 1999; 43(4): 1040 - 1048. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. C. Wu, M. Inubushi, G. Sundaresan, H. R. Schelbert, and S. S. Gambhir Optical Imaging of Cardiac Reporter Gene Expression in Living Rats Circulation, April 9, 2002; 105(14): 1631 - 1634. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||