This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Svensson, E. C. Articles by Leiden, J. M. Search for Related Content PubMed PubMed Citation Articles by Svensson, E. C. Articles by Leiden, J. M. Pubmed/NCBI databases Substance via MeSH Related Collections Gene therapy Cell signalling/signal transduction

(Circulation. 1999;99:201-205.)
© 1999 American Heart Association, Inc.

Brief Rapid Communications

Efficient and Stable Transduction of Cardiomyocytes After Intramyocardial Injection or Intracoronary Perfusion With Recombinant Adeno-Associated Virus Vectors

Eric C. Svensson, MD, PhD; Deborah J. Marshall, PhD; Karen Woodard, BS; Hua Lin, MD; Fang Jiang, MD; Lein Chu, MS; Jeffrey M. Leiden, MD, PhD

From the Departments of Medicine (E.C.S., D.J.M., K.W., H.L., F.J., L.C.) and Pathology (J.M.L.), University of Chicago, Chicago, Ill.

Correspondence to Jeffrey M. Leiden, MD, PhD, University of Chicago, Room B608 MC 6080, 5841 S Maryland Ave, Chicago, IL 60637. E-mail jleiden{at}medicine.bsd.uchicago.edu

	Abstract

Top Abstract Introduction Methods Results Discussion References

 
Background—The delivery of recombinant genes to cardiomyocytes holds promise for the treatment of a variety of cardiovascular diseases. Previous gene transfer approaches that used direct injection of plasmid DNA or replication-defective adenovirus vectors have been limited by low transduction frequencies and transient transgene expression due to immune responses, respectively. In this report, we have tested the feasibility of using intramyocardial injection or intracoronary infusions of recombinant adeno-associated virus (rAAV) vectors to program transgene expression in murine cardiomyocytes in vivo.

Methods and Results—We constructed an rAAV containing the LacZ gene under the transcriptional control of the cytomegalovirus (CMV) promoter (AAV_CMV-LacZ). We then injected 1x10⁸ infectious units (IU) of this virus into the left ventricular myocardium of adult CD-1 mice. Control hearts were injected with the Ad_CMV-LacZ adenovirus vector. Hearts harvested 2, 4, and 8 weeks after AAV_CMV-LacZ injection demonstrated stable ß-galactosidase (ß-gal) expression in large numbers of cardiomyocytes without evidence of myocardial inflammation or myocyte necrosis. In contrast, the Ad_CMV-LacZ-injected hearts displayed transient ß-gal expression, which was undetectable by 4 weeks after injection. Explanted C57BL/6 mouse hearts were also perfused via the coronary arteries with 1.5x10⁹ IU of AAV_CMV-LacZ and assayed 2, 4, and 8 weeks later for ß-gal expression. ß-Gal expression was detected in <1% of cardiomyocytes at 2 weeks after perfusion but was detected in up to 50% of cardiomyocytes 4 to 8 weeks after perfusion.

Conclusions—Direct intramyocardial injection or coronary artery perfusion with rAAV vectors can be used to program stable transgene expression in cardiomyocytes in vivo. rAAV appears to represent a useful vector for the delivery of therapeutic genes to the myocardium.

Key Words: myocardium • genes • molecular biology

	Introduction

Top Abstract Introduction Methods Results Discussion References

 
Myocardial gene therapy holds promise for the treatment of a number of cardiovascular diseases, including ischemic cardiomyopathies, congestive heart failure, and malignant arrhythmias.¹ The ideal vector for myocardial gene delivery would allow the efficient and stable transduction of cardiomyocytes with a variety of transgenes after either direct intramyocardial injection or infusion into the coronary arteries. Plasmid DNA vectors injected directly into the left ventricular myocardium are expressed for 6 months by cardiomyocytes adjacent to the area of injection.^{2 3 4} To date, however, the therapeutic usefulness of this approach has been limited by the low efficiency of cardiomyocyte transduction (0.1% to 1.0% of cardiomyocytes in the area of injection). Both intramyocardial injection and intracoronary infusion of replication-defective adenovirus (RDAd) vectors can be used to efficiently transduce cardiomyocytes in rodents, rabbits, and pigs in vivo.^{4 5 6} However, the feasibility of adenovirus-mediated gene transfer has been limited by immune responses to viral and foreign transgene proteins, which cause significant myocardial inflammation, eliminate virus-transduced cells within 30 days of infection, and thereby result in transient recombinant gene expression in immunocompetent hosts.⁶

Recently, recombinant adeno-associated virus (rAAV) vectors have been shown to program efficient and stable recombinant gene expression in skeletal muscle and liver in both rodents and primates.^{7 8 9} Unlike RDAd, rAAV vectors do not encode viral proteins and have not been associated with immune responses to foreign transgene proteins. A previous report has shown that rAAV can transduce cardiomyocytes in vivo.¹⁰ However, in that study, the efficiency of rAAV-mediated transgene expression in the heart was low (0.2%). In the present report, we have assessed the efficiency and stability of rAAV-mediated gene transfer in the heart after both intramyocardial injection and intracoronary infusion. Our results suggest that rAAV vectors may be useful vectors for myocardial gene delivery.

	Methods

Top Abstract Introduction Methods Results Discussion References

 
Plasmids and Viruses
The structure of pAAV_CMV-LacZ is shown in Figure 1A. Ad_CMV-LacZ and the E3-deleted adenovirus, Ad_dl309, were propagated and purified as described previously.⁶

View larger version (63K): [in this window] [in a new window]   Figure 1. rAAV-mediated gene transfer into primary cardiomyocytes in vitro. A, Schematic of AAVCMV-LacZ. ITR indicates inverted terminal repeat; BGH pA, bovine growth hormone polyadenylation signal; CMV Pr, CMV immediate-early promoter; LacZ, bacterial LacZ gene. B and C, LacZ expression in primary rat neonatal cardiomyocytes 48 hours after infection in vitro with 100 IU/cell of AAVCMV-LacZ (B) or 24 hours after infection in vitro with 100 IU/cell of AAVCMV-LacZ plus 2 pfu/cell Addl309 (C).

Propagation and Purification of rAAV
rAAV was prepared as described.¹¹ We determined viral titer by using a dot blot hybridization assay to determine the number of viral genomes per milliliter and by infecting HeLa cells with the virus and staining with X-gal 24 hours after infection. All viral preparations had titers of 1 to 2x10¹¹ genomes/mL and 2 to 3x10⁹ infectious units (IU)/mL.

In Vitro Infection of Neonatal Rat Cardiomyocytes
Cultures of primary neonatal rat cardiomyocytes¹² (4x10⁶ cells) were infected with 4x10⁸ IU of rAAV with or without 3x10⁷ plaque-forming units (pfu) of Ad_dl309 (n=3 for each group). After 24 to 48 hours, cells were fixed and stained with X-gal.

Intramyocardial Injection of rAAV or RDAd
Six- to 8-week-old CD-1 mice were anesthetized, intubated, and mechanically ventilated. AAV_CMV-LacZ (1x10⁸ IU) or Ad_CMV-LacZ (2x10⁹ pfu) in a volume of 50 µL was injected into the apex of the left ventricle with a 30-gauge needle (n=3 for each time point).

Intracoronary Perfusion With rAAV
Adult C57BL/6 mouse hearts were perfused via the left carotid artery with cardioplegia solution (110 mmol/L NaCl, 25 mmol/L KCl, 22 mmol/L NaHCO₃, 16 mmol/L MgCl₂, 0.8 mmol/L CaCl₂, 40 mmol/L glucose) at 4°C until they stopped beating. They were then perfused ex vivo for 15 minutes with 1.5x10⁹ IU of AAV_CMV-LacZ in 0.5 mL of PBS at a rate of 33 µL/min at 4°C. After perfusion, the hearts were transplanted into the neck of a syngeneic host with anastomosis of the donor aorta to the right common carotid artery of the host and anastomosis of the donor pulmonary artery to the right external jugular vein¹³ (n=3 for each time point).

X-Gal Staining
Freshly isolated hearts were fixed in PBS plus 1.25% glutaraldehyde for 10 minutes at room temperature, stained overnight with X-gal,² and counterstained with eosin.

ß-Galactosidase Activity
Cardiac homogenates were assayed for ß-galactosidase (ß-gal) activity and protein concentration.² ß-Gal activities were normalized for total protein and for the number of infectious rAAV or RDAd particles injected.

	Results

Top Abstract Introduction Methods Results Discussion References

 
Efficient In Vitro Transduction of Cardiomyocytes by rAAV Vectors
To test the capacity of rAAV vectors to transduce cardiomyocytes in vitro, we constructed an rAAV (AAV_CMV-LacZ) containing the LacZ gene under the transcriptional control of the cytomegalovirus (CMV) immediate-early promoter (Figure 1A). This virus was used to infect cultures of primary neonatal rat cardiomyocytes at a multiplicity of infection (MOI) of 100 IU/cell. Duplicate cultures were infected with AAV_CMV-LacZ (MOI=100 IU/cell) plus 2 pfu/cell Ad_dl309. In other cell types, coinfection with adenovirus has been reported to facilitate the conversion of rAAV genomes to double-stranded DNA and to thereby increase the efficiency of rAAV-mediated transgene expression.¹⁴ Twenty-four to 48 hours after infection, ß-gal expression was detected by staining with X-gal. In the absence of added adenovirus, 10% of cardiomyocytes expressed ß-gal (Figure 1B). The addition of adenovirus increased transduction frequencies to 50% (Figure 1C). These results demonstrate that rAAV vectors can be used to transduce primary cardiomyocytes in vitro.

Stable Transduction of Cardiomyocytes In Vivo After Direct Intramyocardial Injection of rAAV Vectors
To determine if rAAV could be used to stably transduce cardiomyocytes in vivo, 1x10⁸ IU of AAV_CMV-LacZ was injected directly into the left ventricular apical myocardium of 8-week-old immunocompetent CD-1 mice. Mice were killed 2, 4, and 8 weeks after injection, and ß-gal expression in the myocardium was assayed by X-gal staining. As shown in Figure 2A, ß-gal expression was detected in a small number of cells surrounding the site of injection at 2 weeks after injection and in a larger number of cells at 4 and 8 weeks after injection. The majority of the ß-gal–positive cells were cardiomyocytes, as evidenced by their readily identifiable myofibers (Figure 2A). Furthermore, the rAAV-injected hearts did not display detectable myocardial inflammation (assessed by hematoxylin and eosin staining) or myocyte necrosis (Figure 2A and data not shown), as has been seen after myocardial injection of RDAd.^{3 4 5 6} To directly compare the efficiency and stability of rAAV-mediated gene transfer with those of RDAd, adult CD-1 mouse hearts were injected with either AAV_CMV-LacZ or Ad_CMV-LacZ and quantitatively assayed for ß-gal activity at different times after injection (Figure 2B). Consistent with previous reports, direct intramyocardial injection of RDAd resulted in transient transgene expression, with peak levels of ß-gal activity seen 1 week after injection.^{3 4 5 6} By 4 weeks after injection, transgene expression was undetectable in the Ad_CMV-LacZ-injected hearts. In contrast, ß-gal activity in the AAV_CMV-LacZ-injected hearts exceeded that seen in the Ad_CMV-LacZ-injected hearts at both 2 and 4 weeks after injection. Peak levels of ß-gal activity in the AAV_CMV-LacZ-injected hearts were 25% of those seen with Ad_CMV-LacZ. Thus, rAAV vectors can be used to stably transduce cardiomyocytes in vivo without significant myocardial inflammation and with an efficiency of at least 25% relative to adenovirus vectors.

View larger version (47K): [in this window] [in a new window]   Figure 2. Gene transfer into cardiomyocytes in vivo after intramyocardial injection or coronary artery perfusion with AAVCMV-LacZ. A, Gross sections (left) and photomicrographs (right) of mouse hearts after intramyocardial injection with 1x108 IU of AAVCMV-LacZ and staining with X-gal. B, ß-Gal activity in heart lysates at different times after intramyocardial injection of AAVCMV-LacZ (n=3) or AdCMV-LacZ (n=3). Data are presented as mean±SEM. C, Gross sections (left) and photomicrographs (right) of mouse hearts after coronary artery perfusion with 1.5x109 IU of AAVCMV-LacZ and staining with X-gal. Bar=25 µm.

Stable and Efficient Transduction of Cardiomyocytes After Intracoronary Perfusion With rAAV
Many clinical applications of myocardial gene therapy will require the stable and efficient transduction of cardiomyocytes distributed throughout large areas of myocardium. Coronary artery infusions of RDAd have been shown to result in the efficient transduction of cardiomyocytes throughout the region of perfused myocardium.⁶ To test whether rAAV is similarly capable of transducing cardiomyocytes after coronary artery perfusion, we explanted hearts from C57BL/6 mice and perfused them with 1.5x10⁹ IU of AAV_CMV-LacZ for 15 minutes at 4°C via a catheter placed in the left common carotid artery. These perfused hearts were then transplanted into syngeneic hosts, and the arterial circulation was reestablished by anastomosis of the transplanted aorta to the recipient carotid artery. Such transplanted and revascularized hearts resumed beating and continued to do so until the recipient mice were killed 2, 4, or 8 weeks after perfusion. Two weeks after perfusion, small numbers (<1%) of ß-gal–positive cardiomyocytes were detected throughout the myocardium of the rAAV-perfused hearts (Figure 2C). By 4 weeks after perfusion, 40% of the cardiomyocytes were ß-gal positive. This high level of transduction was stable at 8 weeks after perfusion, with >50% of the cardiomyocytes continuing to express ß-gal. Similar increases in recombinant gene expression over the first several weeks after rAAV infection have been observed in skeletal muscle.^{7 8} It has been postulated that such increases may reflect the gradual process of conversion of the single-stranded AAV genome into a double-stranded DNA molecule that is competent for transcription of the transgene.¹⁴ Thus, rAAV delivered by coronary artery perfusion can be used to stably transduce cardiomyocytes throughout the myocardium.

	Discussion

Top Abstract Introduction Methods Results Discussion References

 
In this report, we have demonstrated efficient and stable transduction of cardiac myocytes in vivo after intramyocardial injection or intracoronary infusions of rAAV vectors. Our results suggest that rAAV displays significant advantages for myocardial gene transfer compared with either plasmid DNA or adenovirus vectors. Plasmid DNA vectors produce low-efficiency (albeit stable) cardiomyocyte transduction and can only be administered by direct intramyocardial injection.^{2 3 4 5} In contrast, rAAV allows efficient transduction of cardiomyocytes and can be administered by either intramyocardial injection or intracoronary infusion. Adenovirus vectors allow highly efficient cardiomyocyte gene transfer but produce intense intramyocardial inflammation and myocyte necrosis, thereby resulting in transient recombinant gene expression in immunocompetent hosts in vivo.^{3 4 5 6} In contrast, rAAV vectors program stable expression of foreign transgenes in immunocompetent hosts. The stability of transgene expression observed with rAAV even after expression of a foreign transgene protein likely reflects the fact that rAAV vectors, unlike their adenovirus counterparts, do not express any viral gene products and are therefore significantly less immunogenic. This lack of immunogenicity represents a major advantage of rAAV for myocardial gene transfer.

Despite these advantages, there are several issues that might limit the utility of rAAV for cardiovascular gene therapy. First, this vector can only accept transgenes less than 4.5 kb in length. Second, current techniques do not allow the convenient production of large amounts of rAAV. Finally, although our studies have demonstrated efficient transduction of cardiomyocytes after 15 minutes of coronary artery perfusion with rAAV, it remains unclear if similar high-efficiency transduction can be obtained by catheter-mediated intracoronary infusions of this vector. Despite these caveats, our results suggest that rAAV vectors will significantly enhance our ability to stably express recombinant genes in cardiomyocytes in vivo and, as such, may represent an important vector system for myocardial gene therapy.

	Acknowledgments

 
This work was supported in part by grants from the National Institutes of Health (DK-48987, AR-42885, and HL-54592) to Dr Leiden.

Received September 24, 1998; revision received November 9, 1998; accepted November 12, 1998.

	References

Top Abstract Introduction Methods Results Discussion References

 

1. Nabel EG. Gene therapy for cardiovascular disease. Circulation. 1995;91:541–548.[Free Full Text]
   
2. Lin H, Parmacek MS, Morle G, Bolling S, Leiden JM. Expression of recombinant genes in myocardium in vivo after direct injection of DNA. Circulation. 1990;82:2217–2221.[Abstract/Free Full Text]
   
3. Kass EA, Falck PE, Alvira M, Rivera J, Buttrick PM, Wittenberg BA, Cipriani L, Leinwand LA. Quantitative determination of adenovirus-mediated gene delivery to rat cardiac myocytes in vitro and in vivo. Proc Natl Acad Sci U S A. 1993;90:11498–11502.[Abstract/Free Full Text]
   
4. 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]
   
5. French BA, Mazur W, Geske RS, Bolli R. Direct in vivo gene transfer into porcine myocardium using replication-deficient adenoviral vectors. Circulation. 1994;90:2414–2424.[Abstract/Free Full Text]
   
6. Barr E, Carroll J, Kalynych AM, Tripathy SK, Kozarsky K, Wilson JM, Leiden JM. Efficient catheter-mediated gene transfer into the heart using replication-defective adenovirus. Gene Ther. 1994;1:51–58.[Medline] [Order article via Infotrieve]
   
7. Fisher KJ, Jooss K, Alston J, Yang Y, Haecker SE, High K, Pathak R, Raper SE, Wilson JM. Recombinant adeno-associated virus for muscle directed gene therapy. Nat Med. 1997;3:306–312.[Medline] [Order article via Infotrieve]
   
8. Kessler PD, Podsakoff GM, Chen X, McQuiston SA, Colosi PC, Matelis LA, Kurtzman GJ, Byrne BJ. Gene delivery to skeletal muscle results in sustained expression and systemic delivery of a therapeutic protein. Proc Natl Acad Sci U S A. 1996;93:14082–14087.[Abstract/Free Full Text]
   
9. Snyder RO, Miao CH, Patijn GA, Spratt SK, Danos O, Nagy D, Gown AM, Winther B, Meuse L, Cohen LK, Thompson AR, Kay MA. Persistent and therapeutic concentrations of human factor IX in mice after hepatic gene transfer of recombinant AAV vectors. Nat Genet. 1997;16:270–276.[Medline] [Order article via Infotrieve]
   
10. Kaplitt MG, Xiao X, Samulski RJ, Li J, Ojamaa K, Klein IL, Makimura H, Kaplitt MJ, Strumpf RK, Diethrich EB. Long-term gene transfer in porcine myocardium after coronary infusion of an adeno-associated virus vector. Ann Thorac Surg. 1996;62:1669–1676.[Abstract/Free Full Text]
    
11. Rolling F, Samulski RJ. AAV as a viral vector for human gene therapy: generation of recombinant virus. Mol Biotechnol. 1995;3:9–15.[Medline] [Order article via Infotrieve]
    
12. Parmacek MS, Vora AJ, Shen T, Barr E, Jung F, Leiden JM. Identification and characterization of a cardiac-specific transcriptional regulatory element in the slow/cardiac troponin C gene. Mol Cell Biol. 1992;12:1967–1976.[Abstract/Free Full Text]
    
13. Lin H, Iammattoni MD, Modblum JR, Bolling SF. Experimental heterotopic heart transplantation without ischemia or reperfusion. J Heart Transplant. 1990;9:720–723.[Medline] [Order article via Infotrieve]
    
14. Ferrari FK, Samulski T, Shenk T, Samulski RJ. Second-strand synthesis is a rate-limiting step for efficient transduction by recombinant adeno-associated virus vectors. J Virol. 1996;70:3227–3234.We tested the feasibility of using recombinant adeno-associated virus (rAAV) vectors to transduce murine cardiomyocytes in vivo. Intramyocardial injection or coronary artery perfusion of mouse hearts with a rAAV encoding the LacZ gene produced efficient transduction of cardiomyocytes that was stable for 8 weeks. rAAV infection was not associated with detectable myocardial inflammation or myocyte necrosis. Thus, rAAV may be a useful vector for the stable expression of therapeutic genes in the myocardium.[Abstract]
    

This article has been cited by other articles:

				L. E. Vinge, P. W. Raake, and W. J. Koch Gene Therapy in Heart Failure Circ. Res., June 20, 2008; 102(12): 1458 - 1470. [Abstract] [Full Text] [PDF]

				M. S. Penn and A. A. Mangi Genetic Enhancement of Stem Cell Engraftment, Survival, and Efficacy Circ. Res., June 20, 2008; 102(12): 1471 - 1482. [Abstract] [Full Text] [PDF]

				A R Lyon, M Sato, R J Hajjar, R J Samulski, and S E Harding Gene therapy: targeting the myocardium Heart, January 1, 2008; 94(1): 89 - 99. [Abstract] [Full Text] [PDF]

				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]

				A. I. Nykanen, K. Pajusola, R. Krebs, M. A.I. Keranen, O. Raisky, P. K. Koskinen, K. Alitalo, and K. B. Lemstrom Common Protective and Diverse Smooth Muscle Cell Effects of AAV-Mediated Angiopoietin-1 and -2 Expression in Rat Cardiac Allograft Vasculopathy Circ. Res., June 9, 2006; 98(11): 1373 - 1380. [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]

				A. Chandiwal, V. Balasubramanian, Z. K. Baldwin, M. S. Conte, and L. B. Schwartz Gene Therapy for the Extension of Vein Graft Patency: A Review Vascular and Endovascular Surgery, January 1, 2005; 39(1): 1 - 14. [Abstract] [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]

				L. G. Melo, A. S. Pachori, D. Kong, M. Gnecchi, K. Wang, R. E. Pratt, and V. J. Dzau Molecular and Cell-Based Therapies for Protection, Rescue, and Repair of Ischemic Myocardium: Reasons for Cautious Optimism Circulation, May 25, 2004; 109(20): 2386 - 2393. [Full Text] [PDF]

				Y. Yue, Z. Li, S. Q. Harper, R. L. Davisson, J. S. Chamberlain, and D. Duan Microdystrophin Gene Therapy of Cardiomyopathy Restores Dystrophin-Glycoprotein Complex and Improves Sarcolemma Integrity in the Mdx Mouse Heart Circulation, September 30, 2003; 108(13): 1626 - 1632. [Abstract] [Full Text] [PDF]

				D. Chu, C. C. Sullivan, M. D. Weitzman, L. Du, P. L. Wolf, S. W. Jamieson, and P. A. Thistlethwaite Direct comparison of efficiency and stability of gene transfer into the mammalian heart using adeno-associated virus versus adenovirus vectors J. Thorac. Cardiovasc. Surg., September 1, 2003; 126(3): 671 - 679. [Abstract] [Full Text] [PDF]

				C. R. Bridges, J. M. Burkman, R. Malekan, S. M. Konig, H. Chen, C. B. Yarnall, T. J. Gardner, A. S. Stewart, M. M. Stecker, T. Patterson, et al. Global cardiac-specific transgene expression using cardiopulmonary bypass with cardiac isolation Ann. Thorac. Surg., June 1, 2002; 73(6): 1939 - 1946. [Abstract] [Full Text] [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. Shimpo, U. Ikeda, Y. Maeda, M. Takahashi, H. Miyashita, H. Mizukami, M. Urabe, A. Kume, T. Takizawa, M. Shibuya, et al. AAV-mediated VEGF gene transfer into skeletal muscle stimulates angiogenesis and improves blood flow in a rat hindlimb ischemia model Cardiovasc Res, March 1, 2002; 53(4): 993 - 1001. [Abstract] [Full Text] [PDF]

				T. Kawada, M. Nakazawa, S. Nakauchi, K. Yamazaki, R. Shimamoto, M. Urabe, J. Nakata, C. Hemmi, F. Masui, T. Nakajima, et al. Rescue of hereditary form of dilated cardiomyopathy by rAAV-mediated somatic gene therapy: Amelioration of morphological findings, sarcolemmal permeability, cardiac performances, and the prognosis of TO-2 hamsters PNAS, January 22, 2002; 99(2): 901 - 906. [Abstract] [Full Text] [PDF]

				V. J. Dzau, M. J. Mann, A. Ehsan, and D. P. Griese Gene therapy and genomic strategies for cardiovascular surgery: The emerging field of surgiomics J. Thorac. Cardiovasc. Surg., February 1, 2001; 121(2): 0206 - 216. [Full Text] [PDF]

				H. Su, R. Lu, and Y. W. Kan Adeno-associated viral vector-mediated vascular endothelial growth factor gene transfer induces neovascular formation in ischemic heart PNAS, November 22, 2000; (2000) 250488097. [Abstract] [Full Text]

				S. L. Meyerson, C. L. Skelly, M. A. Curi, and L. B. Schwartz Gene Therapy for Cardiovascular Disease Seminars in Cardiothoracic and Vascular Anesthesia, November 1, 2000; 4(4): 289 - 300. [Abstract] [PDF]

				A. Ehsan and M. J Mann Antisense and gene therapy to prevent restenosis Vascular Medicine, May 1, 2000; 5(2): 103 - 114. [Abstract] [PDF]

				M. RICHTER, A. IWATA, J. NYHUIS, Y. NITTA, A. D. MILLER, C. L. HALBERT, and M. D. ALLEN Adeno-associated virus vector transduction of vascular smooth muscle cells in vivo Physiol Genomics, April 27, 2000; 2(3): 117 - 127. [Abstract] [Full Text] [PDF]

				R. J. Hajjar, F. del Monte, T. Matsui, and A. Rosenzweig Prospects for Gene Therapy for Heart Failure Circ. Res., March 31, 2000; 86(6): 616 - 621. [Abstract] [Full Text] [PDF]

				A. S. Shah, R. E. Lilly, A. P. Kypson, O. Tai, J. A. Hata, A. Pippen, S. C. Silvestry, R. J. Lefkowitz, D. D. Glower, and W. J. Koch Intracoronary Adenovirus-Mediated Delivery and Overexpression of the {beta}2-Adrenergic Receptor in the Heart : Prospects for Molecular Ventricular Assistance Circulation, February 1, 2000; 101(4): 408 - 414. [Abstract] [Full Text] [PDF]

				R. Kornowski, S. Fuchs, M. B. Leon, and S. E. Epstein Delivery Strategies to Achieve Therapeutic Myocardial Angiogenesis Circulation, February 1, 2000; 101(4): 454 - 458. [Abstract] [Full Text] [PDF]

				M. I. Miyamoto, F. del Monte, U. Schmidt, T. S. DiSalvo, Z. B. Kang, T. Matsui, J. L. Guerrero, J. K. Gwathmey, A. Rosenzweig, and R. J. Hajjar Adenoviral gene transfer of SERCA2a improves left-ventricular function in aortic-banded rats in transition to heart failure PNAS, January 18, 2000; 97(2): 793 - 798. [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]

				H. Su, R. Lu, and Y. W. Kan Adeno-associated viral vector-mediated vascular endothelial growth factor gene transfer induces neovascular formation in ischemic heart PNAS, December 5, 2000; 97(25): 13801 - 13806. [Abstract] [Full Text] [PDF]

				S. C. FRANCIS, M. K. RAIZADA, A. A. MANGI, L. G. MELO, V. J. DZAU, P. R. VALE, J. M. ISNER, D. W. LOSORDO, J. CHAO, M. J. KATOVICH, et al. Genetic targeting for cardiovascular therapeutics: are we near the summit or just beginning the climb? Physiol Genomics, December 21, 2001; 7(2): 79 - 94. [Abstract] [Full Text] [PDF]

				L. G. Melo, R. Agrawal, L. Zhang, M. Rezvani, A. A. Mangi, A. Ehsan, D. P. Griese, G. Dell'Acqua, M. J. Mann, J. Oyama, et al. Gene Therapy Strategy for Long-Term Myocardial Protection Using Adeno-Associated Virus-Mediated Delivery of Heme Oxygenase Gene Circulation, February 5, 2002; 105(5): 602 - 607. [Abstract] [Full Text] [PDF]

This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Svensson, E. C. Articles by Leiden, J. M. Search for Related Content PubMed PubMed Citation Articles by Svensson, E. C. Articles by Leiden, J. M. Pubmed/NCBI databases Substance via MeSH Related Collections Gene therapy Cell signalling/signal transduction