This Article 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 O’Brien, T. Search for Related Content PubMed PubMed Citation Articles by O’Brien, T.

(Arteriosclerosis, Thrombosis, and Vascular Biology. 2000;20:1414.)
© 2000 American Heart Association, Inc.

Editorials

Adenoviral Vectors and Gene Transfer to the Blood Vessel Wall

Timothy O’Brien

From the Mayo Clinic and Foundation, Rochester, Minn.

Correspondence to Timothy O’Brien, 200 First St SW, Rochester, MN 55905. E-mail obrien.timothy{at}mayo.edu

Key Words: adenovirus • gene therapy • gene transfer

Gene therapy may be defined as the treatment of human disease via the delivery of genes to human tissues. The transferred gene may replace a defective gene or may introduce a new function to a cell. Alternatively, the transgene may enhance an existing cellular function or manifest this function at a time it would not normally be exhibited. Local delivery of genes to the vascular wall is a promising approach to a number of vascular disorders. Overexpression of therapeutic genes within the vessel wall may avoid side effects associated with systemic delivery of the product and result in higher concentrations locally at the site of disease. Inhibition of restenosis after vascular injury^{1 2 3 4 5} and enhancement of new vessel formation after gene transfer has been demonstrated in numerous animal models.^{6 7 8 9} Phase 1 clinical trials of vascular gene therapy have also recently been reported.^{10 11} Two major obstacles to the clinical application of this technology include limitations of the currently available vectors and difficulties delivering genes to the vessel wall in vivo by percutaneous methods. Although direct gene delivery to a surgically isolated segment of the blood vessel has been demonstrated, efficient percutaneous catheter-mediated delivery to the coronary vasculature is more challenging.¹²

A number of vector systems have been used to deliver genes to the blood vessel wall. The simplest involves the use of plasmid alone or plasmid complexed with liposomes. These methods result in relatively inefficient gene transfer. However, if the therapeutic product is a potent secreted protein, biological effects may be observed after plasmid-based gene delivery. Viral vectors have been used extensively in vascular gene transfer, adenovirus vectors being the most commonly used system. Other vector systems with promise include adeno-associated virus (AAV) and lentivirus vectors, but few reports exist concerning their use in the vasculature.

Advantages of adenovirus vectors include the relative ease with which the viral genome can be manipulated, the efficiency of gene transfer, and the ability to transduce quiescent cells.¹³ The latter point is particularly important in the case of vascular gene delivery in which target cells are often nondividing. However, this vector system also has a number of significant drawbacks. Two major limitations are an associated inflammatory response to the vector and the transient nature of transgene expression. It has been presumed that these factors are linked and that the transient nature of transgene expression is due to an antigen-specific immune response to the virally-transduced cells.¹⁴ Furthermore, it may be hypothesized that ablation of the inflammatory response may lead to more persistent transgene expression. The article in this issue by Wen et al casts doubt on this assumption.¹⁵

Inflammation has been reported after adenoviral mediated gene transfer^{16 17 18 19} due to an immune response to virally transduced cells that express adenoviral proteins. Thus, deletion of these cells may limit the duration of transgene expression. It has been hoped that modifications of the adenoviral genome resulting in less viral protein expression may decrease the immune response and thus prolong the duration of transgene expression. Indeed this has been shown to be the case in other systems.^{20 21} Furthermore, immunosuppression and delivery of vector to immunodeficient mice is associated with prolonged transgene expression.^{22 23 24 25 26 27} Initial adenoviral vectors (first generation) had deletions in the E1a and/or E3 regions of the adenoviral genome. However, the majority of the adenoviral genome is still present and low-level protein expression occurs.

A number of further modifications of the adenoviral genome, resulting in 2nd generation adenoviral vectors, have been described. The use of these vectors in gene transfer to the blood vessel wall is reported in this issue of the journal.¹⁵ The vectors studied include one with a temperature-sensitive mutation in the adenovirus E2A DNA-binding protein, one that expresses the immunomodulatory adenoviral 19 kDa glycoprotein (gp 19k), and another with a deletion of E2A. Disappointingly, use of these vectors did not significantly prolong transgene expression or decrease vascular wall inflammation. Furthermore, the administration of cyclophosphamide abolished the inflammatory response to the vector but did not prolong the duration of transgene expression to any significant extent. These results suggest that vascular wall inflammation is not the cause of transient transgene expression after adenoviral-mediated gene transfer to the blood vessel wall and that other factors are responsible.

Thus, attempts at engineering the vector to reduce the inflammatory response may not be sufficient to prolong transgene expression in the blood vessel wall. A puzzling aspect of these results is the fact that immunosuppressive approaches after adenoviral-mediated gene transfer in other organ systems has been associated with decreased inflammation and prolongation of transgene expression. The discrepancy between results in other systems and the vessel wall suggests that different mechanisms may be responsible for the brevity of expression in the vessel wall. Further work will be needed to clarify the mechanism of DNA loss after adenoviral-mediated gene transfer to the blood vessel wall.

Another important observation in the current study is that vector-induced inflammation is dose-dependent. Thus, when first- and second-generation adenoviral vectors are used, it will be critical to determine the lowest vector dose associated with a beneficial biological effect for the specific transgene being studied. For instance, nitric oxide synthase (NOS) gene transfer has been suggested to be a useful strategy for many vascular diseases.²⁸ The amount of nitric oxide generated after NOS gene transfer will depend on the specific isoform used. Thus, an isoform associated with greater generation of nitric oxide may allow lower doses of adenoviral vector to be used. Indeed, an adenoviral vector with inducible nitric oxide synthase (AdiNOS) has been shown to prevent intimal hyperplasia when used at a very low dose.^{29 30} Carefully performed studies to find the lowest possible dose for any given transgene will therefore be important. First-generation vectors may still be useful if a dose not associated with inflammation but resulting in a therapeutic effect can be determined.

In the meantime, what are the implications of these results for the use of adenoviral vectors in vascular gene delivery? First-generation vectors have been used to examine the effect of gene overexpression in the vessel wall. With attention to vector dose and the time after transduction, we have not observed vessel wall inflammation or abnormal endothelium-dependent vasorelaxation after adenoviral mediated gene transfer.^{31 32} Thus, experiments assessing the effect of short-term gene expression are feasible using adenoviral vectors, although the effects of vector-induced inflammation should be considered in the design and interpretation of these experiments. The results of the current study suggest that second-generation adenoviral vectors are also associated with vessel wall inflammation and limited duration of transgene expression. Results of vascular gene delivery using third-generation adenoviral vectors are eagerly awaited. In other systems, these vectors have been associated with less inflammation and prolonged transgene expression.^{21 33 34 35 36} The results of the study by Wen et al suggest that one should not extrapolate from the experience with other organ systems to the blood vessel wall, and therefore, experiments with these vectors are necessary. If these vectors are not associated with inflammation and the duration of transgene expression remains brief, the case for noninflammatory causes limiting the duration of transgene expression will be strengthened.

Thus, gene transfer to the blood vessel wall using first- and second-generation adenoviral vectors results in transient transgene expression and inflammation. The latter may be prevented by immunosuppression or the use of low doses of vector, but the duration of transgene expression is not prolonged. When a longer duration of transgene expression is needed, other vectors will be required. AAV have been associated with prolonged transgene expression in the absence of inflammation in neurons, skeletal muscle, and liver.³⁷ However, there is limited experience with this vector system in the vessel wall.^{38 39} Lentiviral vectors are also capable of stable transgene expression without tissue inflammation.⁴⁰

This report by Wen et al provides important information on the use of adenoviral vectors for gene transfer to the vessel wall. First- and second-generation adenoviral vectors are associated with dose-dependent inflammation and limited duration of transgene expression. Although one may avoid inflammation with lower doses of these vectors or with immunosuppression, the duration of transgene expression remains limited. Although immunosuppression to reduce inflammation will not be practical outside of transplantation, use of a low dose of vector (if this is still associated with a biological response) is a useful strategy when short-term transgene expression is sought. In addition, gene transfer experiments to the vessel wall with other vectors including 3rd-generation adenovirus, AAV, and lentivirus are awaited.

References

1. Ohno T, Gordon D, San H, Pompili VJ, Imperiale MJ, Nabel GJ, Nabel EG. Gene therapy for vascular smooth muscle cell proliferation after arterial injury. Science. 1994;265:781–784.[Abstract/Free Full Text]
2. Chang MW, Barr E, Seltzer J, Jiang Y, Nabel GJ, Nabel EG, Parmacek MS, Leiden JM. Cystostatic gene therapy for vascular proliferative disorders with a constitutively active form of the retinoblastoma gene product. Science.. 1995;267:518–522.[Abstract/Free Full Text]
3. Varenne O, Pislaru S, Gillijns H, van Pelt N, Gerard RD, Zoldhelyi P, Van de Werf F, Collen D, Janssens S. Local adenovirus-mediated transfer of human endothelial nitric oxide synthase reduces luminal narrowing after coronary angioplasty in pigs. Circulation. 1998;98:919–926.[Abstract/Free Full Text]
4. Janssens S, Flaherty D, Nong Z, Varenne O, van Pelt N, Haustermans C, Zoldhelyi P, Gerard RD, Collen D. Human endothelial nitric oxide synthase gene transfer inhibits vascular smooth muscle cell proliferation and neointimal formation after balloon injury in rats. Circulation. 1998;97:1274–1281.[Abstract/Free Full Text]
5. Von der Leyen H, Gibbons G, Morishita R, Nakajima M, Kaneda Y, Cooke J, Dzau VJ. Gene therapy inhibiting neointimal vascular lesions: in vivo transfer of endothelial cell nitric oxide synthase gene. Proc Natl Acad Sci U S A. 1995;92:1137–1141.[Abstract/Free Full Text]
6. Garcia-Martinez C, Opolon P, Trochon V, Chianale C, Musset K, Lu H, Abitbol M, Perricaudet M, Ragot T. Angiogenesis induced in muscle by a recombinant adenovirus expressing functional isoforms of basic fibroblast growth factor. Gene Ther. 1999;6:1210–1221.[Medline] [Order article via Infotrieve]
7. Shyu K, Manor O, Magner M, Yancopoulos GD, Isner JM. Direct intramuscular injection of plasmid DNA encoding angiopoietin-1 but not angiopoietin-2 augments revascularization in the rabbit ischemic hindlimb. Circulation. 1998;98:2081–2087.[Abstract/Free Full Text]
8. Tsurumi Y, Takeshita S, Chen D, Kearney M, Rossow ST, Passeri J, Horowitz JR, Symes JF, Isner JM. Direct intramuscular gene transfer of naked DNA encoding vascular endothelial growth factor augments collateral development and tissue perfusion. Circulation. 1996;94:3281–3290.[Abstract/Free Full Text]
9. Takeshita S, Weir L, Chen D, Zheng LP, Riessen R, Bauters C, Symes JF, Ferrara N, Isner JM. Therapeutic angiogenesis following arterial gene transfer of vascular endothelial growth factor in a rabbit model of hindlimb ischemia. Biochem Biophys Res Commun. 1996;227:628–635.[Medline] [Order article via Infotrieve]
10. Losordo DW, Vale PR, Symes JF, Dunnington CH, Esakof DD, Maysky M, Ashare AB, Lathi K, Isner JM. Gene therapy for myocardial angiogenesis: initial clinical results with direct myocardial injection of phVEGF165 as sole therapy for myocardial ischemia. Circulation. 1998;98:2800–2804.[Abstract/Free Full Text]
11. Rosengart TK, Lee LY, Patel SR, Sanborn TA, Parikh M, Bergman GW, Hachamovitch R, Szulc M, Kligfield PD, Okin PM, Hahn RT, Devereux RB, Post MR, Hackett NR, Foster T, Grasso TM, Lesser ML, Isom OW, Crystal RG. Angiogenesis gene therapy: Phase I assessment of direct intramyocardial administration of an adenovirus vector expressing VEGF121 cDNA to individuals with clinically significant severe coronary artery disease. Circulation. 1999;100:468–474.[Abstract/Free Full Text]
12. Abaci A, Oguzhan A, Kahraman S, Eryol NK, Ünal S, Arinc H, Ergin A. Effect of diabetes mellitus on formation of coronary collateral vessels. Circulation. 1999;99:2239–2242.[Abstract/Free Full Text]
13. Benihoud K, Yeh P, Perricaudet M. Adenovirus vectors for gene delivery. Curr Opin Biotechnol.. 1999;10:440–447.[Medline] [Order article via Infotrieve]
14. Yang Y, Li Q, Ertl HCJ, Wilson JM. Cellular and humoral immune responses to viral antigens create barriers to lung-directed gene therapy with recombinant adenoviruses. J Virol. 1995;69:2004–2015.[Abstract]
15. Wen S, Schneider DB, Driscoll RM, Vassalli G, Sassani AB, Dichek DA. Second-generation adenoviral vectors do not prevent rapid loss of transgene expression and vector DNA from the arterial wall. Arterioscler Thromb Vasc Biol. 2000;101:1452–1458.
16. Yang Y, Nunes FA, Berencsi K, Furth EE, Gönczöl E, Wilson JM. Cellular immunity to viral antigens limits E1-deleted adenoviruses for gene therapy. Proc Natl Acad Sci U S A. 1994;91:4407–4411.[Abstract/Free Full Text]
17. Zabner J, Petersen DM, Puga AP, Graham SM, Couture LA, Keyes LD, Lukason MJ, St George JA, Gregory RJ, Smith AE. Safety and efficacy of repetitive adenovirus-mediated transfer of CFTR cDNA to airway epithelia of primates and cotton rats. Nat Genet. 1994;6:75–83.[Medline] [Order article via Infotrieve]
18. Engelhardt JF, Simon RH, Yang Y, Zepeda M, Weber-Pendleton S, Doranz B, Grossman M, Wilson JM. Adenovirus-mediated transfer of the CFTR gene to lung of nonhuman primates: biological efficacy study. Hum Gen Ther. 1993;4:759–769.[Medline] [Order article via Infotrieve]
19. Newman KD, Dunn PF, Owens JW, Schulick AH, Virmani R, Sukhova G, Libby P, Dichek DA. Adenovirus-mediated gene transfer into normal rabbit arteries results in prolonged vascular cell activation, inflammation, and neointimal hyperplasia. Journal of Clinical Investigation. 1995;96:2955–2965.
20. 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 U S A. 1994;91:6196–6200.[Abstract/Free Full Text]
21. Morsy MA, Gu M, Motzel S, Zhao J, Lin J, Su Q, Allen H, Franlin L, Parks RJ, Graham FL, Kochanek S, Bett AJ, Caskey CT. An adenoviral vector deleted for all viral coding sequences results in enhanced safety and extended expression of a leptin transgene. Proc Natl Acad Sci U S A. 1998;95:7866–7871.[Abstract/Free Full Text]
22. Zsengeller ZK, Wert SE, Hull WM, Hu X, Yei S, Trapnell BC, Whitsett JA. Persistence of replication-deficient adenovirus-mediated gene transfer in lungs of immune-deficient (nu/nu) mice. Hum Gen Ther.. 1995;6:457–467.[Medline] [Order article via Infotrieve]
23. Cichon G, Strauss M. Transient immunosuppression with 15-deoxyspergualin prolongs reporter gene expression and reduces humoral immune response after adenoviral gene transfer. Gene Ther. 1998;5:85–90.[Medline] [Order article via Infotrieve]
24. Jooss K, Yang Y, Wilson JM. Cyclophosphamide diminishes inflammation and prolongs transgene expression following delivery of adenoviral vectors to mouse liver and lung. Hum Gene Ther. 1996;7:1555–1566.[Medline] [Order article via Infotrieve]
25. Fang B, Eisensmith RC, Wang H, Kay MA, Cross RE, Landen CN, Gordon G, Bellinger DA, Read MS, Hu PC, Brinkhous KM, Woo SLC. Gene therapy for hemophilia B: Host immunosuppression prolongs the therapeutic effect of adenovirus-mediated Factor IX expression. Hum Gene Ther. 1995;6:1039–1044.[Medline] [Order article via Infotrieve]
26. Quiñones MJ, Leor J, Kloner RA, Masamichi I, Patterson M, Witke WF, Kedes L. Avoidance of immune response prolongs expression of genes delivered to the adult rat myocardium by replication-defective adenovirus. Circulation. 1996;94:1394–1401.[Abstract/Free Full Text]
27. Yap J, O’Brien T, Tazelaar HD, McGregor CGA. Immunosuppression prolongs adenoviral mediated transgene expression in cardiac allograft transplantation. Cardiovasc Res. 1997;35:529–535.[Abstract/Free Full Text]
28. Chen AFY, O’Brien T, Katusic ZS. Transfer and expression of recombinant nitric oxide synthase genes in the cardiovascular system. Trends Pharmacol Sci. 1998;19:276–286.[Medline] [Order article via Infotrieve]
29. Shears L, Kibbe M, Murdock A, Billiar T, Lizonova A, Kovesdi I, Watkins S, Tzeng E. Efficient inhibition of intimal hyperplasia by adenovirus-mediated inducible nitric oxide synthase gene transfer to rats and pigs in vivo. J Am Coll Surg. 1998;187:295–306.[Medline] [Order article via Infotrieve]
30. Shears L, Kawaharada N, Tzeng E, Billiar TR, Watkins SC, Koveski I, Lizonova A, Pham SM. Inducible nitric oxide synthase suppresses the development of allograft arteriosclerosis. J Clin Invest. 1997;100:2035–2042.[Medline] [Order article via Infotrieve]
31. Kullo IJ, Mozes G, Schwartz RS, Gloviczki P, Tsutsui M, Crotty TB, Barber DA, Katusic ZS, O’Brien T. Adventitial gene transfer of recombinant endothelial nitric oxide synthase to rabbit carotid arteries alters vascular reactivity. Circulation. 1997;96:2254–2261.[Abstract/Free Full Text]
32. Kullo IJ, Mozes G, Schwartz RS, Gloviczki P, Tsutsui M, Katusic ZS, O’Brien T. Enhanced endothelium-dependent relaxations after gene transfer of recombinant endothelial nitric oxide synthase to rabbit carotid arteries. Hypertension. 1997;30:314–320.[Abstract/Free Full Text]
33. Chen HH, Mack LM, Kelly R, Ontell M, Kochanek S, Clemens PR. Persistence in muscle of an adenoviral vector that lacks all viral genes. Proc Natl Acad Sci U S A. 1997;94:1645–1650.[Abstract/Free Full Text]
34. Schiedner G, Morral N, Parks RJ, Wu Y, Koopmans SC, Langston C, Graham FL, Beaudet AL, Kochanek S. Genomic DNA transfer with a high-capacity adenovirus vector results in improved in vivo gene expression and decreased toxicity. Nat Genet. 1998;18:180–183.[Medline] [Order article via Infotrieve]
35. Morral N, Parks RJ, Zhou H, Langston C, Schiedner G, Quinones J, Graham FL, Kochanek S, Beaudet AL. High doses of a helper-dependent adenoviral vector yield supraphysiological levels of alpha1-antitrypsin with negligible toxicity. Hum Gene Ther. 1998;9:2709–2716.[Medline] [Order article via Infotrieve]
36. Morral N, O’Neal W, Rice K, Leland M, Kaplan J, Piedra PA, Zhou H, Parks RJ, Velji R, Aguilar-Cordova E, Wadsworth S, Graham FL, Kochanek S, Carey KD, Beaudet AL. Administration of helper-dependent adenoviral vectors and sequential delivery of different vector serotype for long-term liver-directed gene transfer in baboons. Proc Natl Acad Sci U S A. 1999;96:12816–12821.[Abstract/Free Full Text]
37. Bueler H. Adeno-associated viral vectors for gene transfer and gene therapy. Biol Chem. 1999;380:613–622.[Medline] [Order article via Infotrieve]
38. Lynch CM, Hara PS, Leonard JC, Williams JK, Dean RH, Geary RL. Adeno-associated virus vectors for vascular gene delivery. Circ Res. 1997;80:497–505.
39. Arnold TE, Gnatenko D, Bahou WF. In vivo gene transfer into rat arterial walls with novel adeno-associated virus vectors. J Vasc Surg. 1997;25:347–355.[Medline] [Order article via Infotrieve]
40. Federico M. Lentiviruses as gene delivery vectors. Curr Opin Biotechnol. 1999;10:448–453.[Medline] [Order article via Infotrieve]

This article has been cited by other articles:

				F. E. Rey and P. J. Pagano The Reactive Adventitia: Fibroblast Oxidase in Vascular Function Arterioscler. Thromb. Vasc. Biol., December 1, 2002; 22(12): 1962 - 1971. [Abstract] [Full Text] [PDF]

				X. Yin, C. Yutani, Y. Ikeda, K. Enjyoji, H. Ishibashi-Ueda, S. Yasuda, Y. Tsukamoto, H. Nonogi, Y. Kaneda, and H. Kato Tissue factor pathway inhibitor gene delivery using HVJ-AVE liposomes markedly reduces restenosis in atherosclerotic arteries Cardiovasc Res, December 1, 2002; 56(3): 454 - 463. [Abstract] [Full Text] [PDF]

				M O'Sullivan and M R Bennett Gene therapy for coronary restenosis: is the enthusiasm justified? Heart, November 1, 2001; 86(5): 491 - 493. [Full Text] [PDF]

				N. van Royen, J. J. Piek, I. Buschmann, I. Hoefer, M. Voskuil, and W. Schaper Stimulation of arteriogenesis; a new concept for the treatment of arterial occlusive disease Cardiovasc Res, February 16, 2001; 49(3): 543 - 553. [Abstract] [Full Text] [PDF]

				Y. Taniyama, K. Tachibana, K. Hiraoka, T. Namba, K. Yamasaki, N. Hashiya, M. Aoki, T. Ogihara, K. Yasufumi, and R. Morishita Local Delivery of Plasmid DNA Into Rat Carotid Artery Using Ultrasound Circulation, March 12, 2002; 105(10): 1233 - 1239. [Abstract] [Full Text] [PDF]

This Article 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 O’Brien, T. Search for Related Content PubMed PubMed Citation Articles by O’Brien, T.