doi: 10.1161/CIRCULATIONAHA.106.685255
(Circulation. 2007;115:1180-1183.)
© 2007 American Heart Association, Inc.
Editorial |
Therapeutic Gene Regulation
Targeting Transcription
From the Section of Cardiovascular Medicine, Yale University School of Medicine, New Haven, Conn.
Correspondence to Frank J. Giordano, MD, Cardiovascular Medicine, Yale University School of Medicine, 333 Cedar St, 3FMP, New Haven, CT 06520. E-mail frank.giordano{at}yale.edu
Key Words: Editorials • angiogenesis • gene therapy • genes • hypoxia • molecular biology • vasculature
In this issue of Circulation, Rajagopolan et al1 report the first clinical results of a gene therapy approach predicated on transcriptional activation of a patient’s own genes. This represents a second-generation gene therapy methodology for cardiovascular disease and is important for a number of reasons. We have witnessed in the past decade the primary sequencing of the human genome.2,3 One of the initial reactions to this milestone accomplishment was surprise at the relatively small number of definitive genes that are encoded by human DNA. Although the exact number is still uncertain, estimates as low as 23 299 have been made. In comparison, the genome of the worm Caenorhabditis elegans encodes approximately 19 000 genes, and the genome of the common fruit fly encodes approximately 18 000 genes, raising the question of how such significant differences in biological complexity and diversity are engendered by so few genes. A complete set of answers to this question is not currently in our grasp, but some crucial aspects are understood and are relevant to the clinical trial discussed here.
Article p 1234
One manner in which the biological effect of a finite number of genes is amplified is by alternative splicing. This is the process whereby a single gene encodes a number of alternative proteins by simply including or excluding specific exons within the coding sequence of that gene during mRNA transcription and maturation. The number of alternative splice variants that exist as transcriptional products of the human genome is not known, but this process alone likely increases the number of unique proteins that can be encoded by the genome 2- to 3-fold, possibly more. Interestingly, there are known human cardiovascular diseases caused by inappropriate splicing, including a cardiomyopathy and sudden death syndrome in children caused by deficient production of wild-type very-long-chain acyl-CoA dehydrogenase, the result of exclusion of an exon attributable to abnormal splicing.4 Relevant to the study by Rajagopolan et al are the alternative splice variants of the vascular endothelial growth factor gene (Vegf-A). There are 4 major alternative Vegf-A proteins encoded by the same Vegf-A gene because of alternative splicing—Vegf121, 165, 189, and 205—and additional minor splice variants also have been described. At least 2 of the major Vegf-A splice variants, Vegf121 and Vegf165, have been tested clinically as single agents in gene therapy trials for human peripheral arterial disease and coronary artery disease. It is now clear that the alternative Vegf-A splice variants are not biologically equivalent or redundant.5,6 Furthermore, blood vessels induced in response to single Vegf splice variants seem to be more permeable, and perhaps less mature, than those induced by activating the native Vegf-A gene with consequent expression of multiple Vegf splice variants.7,8 The clinical relevance of these differences is not yet clear, but they do illustrate how the determination of biological complexity does not stop at the primary DNA sequence.
In the study reported by Rajapogolan et al1 in this issue, the authors injected an adenovirus that encodes a stable and active form of the hypoxia-inducible transcription factor HIF-1
into the skeletal muscles of patients with severe peripheral arterial disease, in an effort to stimulate the growth of new blood vessels. HIF-1 is a basic helix-loop-helix transcription factor that regulates the expression of a wide repertoire of genes in response to decreased oxygen tension. When oxygen levels within a tissue or cell decrease, HIF-1 levels increase. HIF-1, as a heterodimer with HIF-1ß (aryl hydrocarbon nuclear transferase), then binds to specific sequences within the regulatory regions of these genes and alters their transcription.9 One of the genes regulated by HIF-1 is the Vegf-A gene. Thus, an expected, desired effect of this HIF-1 gene therapy approach is activation of the endogenous Vegf-A gene and consequent expression of all the major Vegf-A splice variants. This represents one potential advantage of this transcriptional approach to therapeutic angiogenesis.
Another important potential advantage of using this transcriptional approach is that it takes advantage of a biological pathway that has evolved naturally as a mechanism whereby tissues can adapt to decreased oxygen availability, including the ability of those tissues to grow new blood vessels. HIF-1 can be thought of as a master switch that coordinates the expression of a wide repertoire of genes involved in adaptive responses to hypoxia, including a significant number of genes involved in regulating vascular growth and reactivity. It is likely that the full complement of angiogenesis-associated genes regulated by HIF is not yet known, and thus "flipping the HIF switch," as in the approach by Rajagopolan and colleagues, may induce the expression of factors that have not yet been identified as contributors to the processes of angiogenesis and vascular remodeling. Thus, using this type of approach may unleash the therapeutic power of genes and biological pathways we do not yet understand, without a requisite requirement for knowing how it all fits together and works. The potential disadvantage of this approach is that HIF-1 has also evolved to regulate the expression of genes involved in other biological processes associated with adaptation to hypoxia, including genes that control cellular metabolism, glucose uptake, erythropoiesis, apoptosis, the cell cycle, and other processes. This biological permissiveness may theoretically lead to undesired clinical effects. Thus, the fact that HIF-1 gene therapy seems safe at all dose ranges tested is quite comforting and represents one of the major contributions of the study conducted by Rajagopolan and colleagues.
In a broader, more general sense, the HIF-1 clinical study by Rajagopalan et al1 highlights the importance of gene regulation, which is likely as significant to human health as are the gene mutations that have been the focus of human genetics to data and that are now the subject of intense, high-throughput, genome-wide searches. Genes are essentially our cellular instruction set for how and when to build specific proteins. How those instructions are read is of crucial biological importance. Every human cell type has the same genomic DNA sequences, yet some become retinal rods, whereas others become contractile cardiac myocytes. Exactly what determines in each specific cell type which genes are read, how they are read, and what proteins result is not yet clearly understood, but it involves a variety of processes that cardiovascular clinicians and scientists will undoubtedly be hearing much more about in the future, including DNA methylation, histone acetylation, gene silencing, transcriptional control, and other related topics. Many of these come under the general designation of epigenetics, the manner in which the function of genes is determined by factors other than their base sequences.10 An intriguing example of how epigenetics may affect cardiovascular health is the hypothesis that environmental factors within the womb during pregnancy may lead to alterations in DNA methylation or chromatin structure in fetal DNA, leading to long-term changes in gene expression that translate into a higher lifetime risk of cardiovascular disease in the offspring of these pregnancies. These epigenetic determinants of disease risk would not be found by searching for gene mutations, and it is prudent to consider these types of epigenetic disease determinants as we continue our search for the causes and best therapies for cardiovascular disease.
The first clinical trials of therapeutic induction of blood vessel growth began over a decade ago, and to date we have still not managed to develop an approach that yields the same tremendously robust, angiographically discernible collateral vessel development that occurs spontaneously in many fortunate patients. Still, the field remains quite promising, and the potential clinical benefits are of sufficient magnitude to warrant continued research and development in this area. We must, however, keep in mind that the biological control of blood vessel growth is quite complex and that therapeutically recapitulating this complex biology is, understandably, a difficult challenge. The HIF-1 approach by Rajagopolan and colleagues is a significantly different approach than those applied clinically to date, and it represents an important new direction in the field. Whether HIF-1 therapy will prove superior to the specific single-gene/single-protein approaches that have been clinically tested to date is not answered by this phase I study, but it will become evident in future efficacy trials. Irrespective of whether HIF-1 gene therapy proves clinically efficacious, it represents advancement in our thinking about how best to mimic human biology. Other approaches applying gene regulation as a therapeutic strategy are also in development, including the use of de novo engineered transcription factors targeted to specific genes7; these, too, may significantly advance the field. Defining the human genome sequence was a major accomplishment. Appropriately, we are moving forward past this identification of the instruction set and toward understanding how the instruction set defines biology and how it might be used therapeutically.
 |
Acknowledgments |
|---|
Sources of Funding
Dr Giordano is supported by National Institutes of Health grants HL075616 and HL64001.
Disclosures
Dr Giordano is a consultant for Edwards Lifesciences.
|
Footnotes |
|---|
The opinions expressed in this article are not necessarily those of the editors or of the American Heart Association.
|
References |
|---|
 Top References |
|---|
- Rajagopalan S, Olin J, Deitcher S, Pieczek A, Laird J, Grossman PM, Goldman CK, McKellin K, Kelley R, Chronos N. Use of a constitutively active hypoxia-inducible factor-1 transgene as a therapeutic strategy in no-option critical limb ischemia patients: phase 1 dose-escalation experience. Circulation. 2007; 115: 1234–1243.
[Abstract/Free Full Text] - Venter JC, Adams MD, Myers EW, Li PW, Mural RJ, Sutton GG, Smith HO, Yandell M, Evans CA, Holt RA, Gocayne JD, Amanatides P, Ballew RM, Huson DH, Wortman JR, Zhang Q, Kodira CD, Zheng XH, Chen L, Skupski M, Subramanian G, Thomas PD, Zhang J, Gabor Miklos GL, Nelson C, Broder S, Clark AG, Nadeau J, McKusick VA, Zinder N, Levine AJ, Roberts RJ, Simon M, Slayman C, Hunkapiller M, Bolanos R, Delcher A, Dew I, Fasulo D, Flanigan M, Florea L, Halpern A, Hannenhalli S, Kravitz S, Levy S, Mobarry C, Reinert K, Remington K, Abu-Threideh J, Beasley E, Biddick K, Bonazzi V, Brandon R, Cargill M, Chandramouliswaran I, Charlab R, Chaturvedi K, Deng Z, Di Francesco V, Dunn P, Eilbeck K, Evangelista C, Gabrielian AE, Gan W, Ge W, Gong F, Gu Z, Guan P, Heiman TJ, Higgins ME, Ji RR, Ke Z, Ketchum KA, Lai Z, Lei Y, Li Z, Li J, Liang Y, Lin X, Lu F, Merkulov GV, Milshina N, Moore HM, Naik AK, Narayan VA, Neelam B, Nusskern D, Rusch DB, Salzberg S, Shao W, Shue B, Sun J, Wang Z, Wang A, Wang X, Wang J, Wei M, Wides R, Xiao C, Yan C, Yao A, Ye J, Zhan M, Zhang W, Zhang H, Zhao Q, Zheng L, Zhong F, Zhong W, Zhu S, Zhao S, Gilbert D, Baumhueter S, Spier G, Carter C, Cravchik A, Woodage T, Ali F, An H, Awe A, Baldwin D, Baden H, Barnstead M, Barrow I, Beeson K, Busam D, Carver A, Center A, Cheng ML, Curry L, Danaher S, Davenport L, Desilets R, Dietz S, Dodson K, Doup L, Ferriera S, Garg N, Gluecksmann A, Hart B, Haynes J, Haynes C, Heiner C, Hladun S, Hostin D, Houck J, Howland T, Ibegwam C, Johnson J, Kalush F, Kline L, Koduru S, Love A, Mann F, May D, McCawley S, McIntosh T, McMullen I, Moy M, Moy L, Murphy B, Nelson K, Pfannkoch C, Pratts E, Puri V, Qureshi H, Reardon M, Rodriguez R, Rogers YH, Romblad D, Ruhfel B, Scott R, Sitter C, Smallwood M, Stewart E, Strong R, Suh E, Thomas R, Tint NN, Tse S, Vech C, Wang G, Wetter J, Williams S, Williams M, Windsor S, Winn-Deen E, Wolfe K, Zaveri J, Zaveri K, Abril JF, Guigo R, Campbell MJ, Sjolander KV, Karlak B, Kejariwal A, Mi H, Lazareva B, Hatton T, Narechania A, Diemer K, Muruganujan A, Guo N, Sato S, Bafna V, Istrail S, Lippert R, Schwartz R, Walenz B, Yooseph S, Allen D, Basu A, Baxendale J, Blick L, Caminha M, Carnes-Stine J, Caulk P, Chiang YH, Coyne M, Dahlke C, Mays A, Dombroski M, Donnelly M, Ely D, Esparham S, Fosler C, Gire H, Glanowski S, Glasser K, Glodek A, Gorokhov M, Graham K, Gropman B, Harris M, Heil J, Henderson S, Hoover J, Jennings D, Jordan C, Jordan J, Kasha J, Kagan L, Kraft C, Levitsky A, Lewis M, Liu X, Lopez J, Ma D, Majoros W, McDaniel J, Murphy S, Newman M, Nguyen T, Nguyen N, Nodell M, Pan S, Peck J, Peterson M, Rowe W, Sanders R, Scott J, Simpson M, Smith T, Sprague A, Stockwell T, Turner R, Venter E, Wang M, Wen M, Wu D, Wu M, Xia A, Zandieh A, Zhu X. The sequence of the human genome. Science. 2001; 291: 1304–1351.
[Abstract/Free Full Text] - Lander ES, Linton LM, Birren B, Nusbaum C, Zody MC, Baldwin J, Devon K, Dewar K, Doyle M, FitzHugh W, Funke R, Gage D, Harris K, Heaford A, Howland J, Kann L, Lehoczky J, LeVine R, McEwan P, McKernan K, Meldrim J, Mesirov JP, Miranda C, Morris W, Naylor J, Raymond C, Rosetti M, Santos R, Sheridan A, Sougnez C, Stange-Thomann N, Stojanovic N, Subramanian A, Wyman D, Rogers J, Sulston J, Ainscough R, Beck S, Bentley D, Burton J, Clee C, Carter N, Coulson A, Deadman R, Deloukas P, Dunham A, Dunham I, Durbin R, French L, Grafham D, Gregory S, Hubbard T, Humphray S, Hunt A, Jones M, Lloyd C, McMurray A, Matthews L, Mercer S, Milne S, Mullikin JC, Mungall A, Plumb R, Ross M, Shownkeen R, Sims S, Waterston RH, Wilson RK, Hillier LW, McPherson JD, Marra MA, Mardis ER, Fulton LA, Chinwalla AT, Pepin KH, Gish WR, Chissoe SL, Wendl MC, Delehaunty KD, Miner TL, Delehaunty A, Kramer JB, Cook LL, Fulton RS, Johnson DL, Minx PJ, Clifton SW, Hawkins T, Branscomb E, Predki P, Richardson P, Wenning S, Slezak T, Doggett N, Cheng JF, Olsen A, Lucas S, Elkin C, Uberbacher E, Frazier M, Gibbs RA, Muzny DM, Scherer SE, Bouck JB, Sodergren EJ, Worley KC, Rives CM, Gorrell JH, Metzker ML, Naylor SL, Kucherlapati RS, Nelson DL, Weinstock GM, Sakaki Y, Fujiyama A, Hattori M, Yada T, Toyoda A, Itoh T, Kawagoe C, Watanabe H, Totoki Y, Taylor T, Weissenbach J, Heilig R, Saurin W, Artiguenave F, Brottier P, Bruls T, Pelletier E, Robert C, Wincker P, Smith DR, Doucette-Stamm L, Rubenfield M, Weinstock K, Lee HM, Dubois J, Rosenthal A, Platzer M, Nyakatura G, Taudien S, Rump A, Yang H, Yu J, Wang J, Huang G, Gu J, Hood L, Rowen L, Madan A, Qin S, Davis RW, Federspiel NA, Abola AP, Proctor MJ, Myers RM, Schmutz J, Dickson M, Grimwood J, Cox DR, Olson MV, Kaul R, Raymond C, Shimizu N, Kawasaki K, Minoshima S, Evans GA, Athanasiou M, Schultz R, Roe BA, Chen F, Pan H, Ramser J, Lehrach H, Reinhardt R, McCombie WR, de la Bastide M, Dedhia N, Blocker H, Hornischer K, Nordsiek G, Agarwala R, Aravind L, Bailey JA, Bateman A, Batzoglou S, Birney E, Bork P, Brown DG, Burge CB, Cerutti L, Chen HC, Church D, Clamp M, Copley RR, Doerks T, Eddy SR, Eichler EE, Furey TS, Galagan J, Gilbert JG, Harmon C, Hayashizaki Y, Haussler D, Hermjakob H, Hokamp K, Jang W, Johnson LS, Jones TA, Kasif S, Kaspryzk A, Kennedy S, Kent WJ, Kitts P, Koonin EV, Korf I, Kulp D, Lancet D, Lowe TM, McLysaght A, Mikkelsen T, Moran JV, Mulder N, Pollara VJ, Ponting CP, Schuler G, Schultz J, Slater G, Smit AF, Stupka E, Szustakowski J, Thierry-Mieg D, Thierry-Mieg J, Wagner L, Wallis J, Wheeler R, Williams A, Wolf YI, Wolfe KH, Yang SP, Yeh RF, Collins F, Guyer MS, Peterson J, Felsenfeld A, Wetterstrand KA, Patrinos A, Morgan MJ, de Jong P, Catanese JJ, Osoegawa K, Shizuya H, Choi S, Chen YJ; International Human Genome Sequencing Consortium. Initial sequencing and analysis of the human genome. Nature. 2001; 409: 860–921.[CrossRef][Medline]
[Order article via Infotrieve]
- Orii KO, Aoyama T, Souri M, Orii KE, Kondo N, Orii T, Hashimoto T. Genomic DNA organization of human mitochondrial very-long-chain acyl-CoA dehydrogenase and mutation analysis. Biochem Biophys Res Commun. 1995; 217: 987–992.[CrossRef][Medline]
[Order article via Infotrieve]
- Grunstein J, Masbad JJ, Hickey R, Giordano F, Johnson RS. Isoforms of vascular endothelial growth factor act in a coordinate fashion to recruit and expand tumor vasculature. Mol Cell Biol. 2000; 20: 7282–7291.
[Abstract/Free Full Text] - Maes C, Carmeliet P, Moermans K, Stockmans I, Smets N, Collen D, Bouillon R, Carmeliet G. Impaired angiogenesis and endochondral bone formation in mice lacking the vascular endothelial growth factor isoforms VEGF164 and VEGF188. Mech Dev. 2002; 111: 61–73.[CrossRef][Medline]
[Order article via Infotrieve]
- Rebar EJ, Huang Y, Hickey R, Nath AK, Meoli D, Nath S, Chen B, Xu L, Liang Y, Jamieson AC, Zhang L, Spratt SK, Case CC, Wolffe A, Giordano FJ. Induction of angiogenesis in a mouse model using engineered transcription factors. Nat Med. 2002; 8: 1427–1432.[CrossRef][Medline]
[Order article via Infotrieve]
- Elson DA, Thurston G, Huang LE, Ginzinger DG, McDonald DM, Johnson RS, Arbeit JM. Induction of hypervascularity without leakage or inflammation in transgenic mice overexpressing hypoxia-inducible factor-1alpha. Genes Dev. 2001; 15: 2520–2532.
[Abstract/Free Full Text] - Giordano FJ. Oxygen, oxidative stress, hypoxia, and heart failure. J Clin Invest. 2005; 115: 500–508.[CrossRef][Medline]
[Order article via Infotrieve]
- Cho KS, Elizondo LI, Boerkoel CF. Advances in chromatin remodeling and human disease. Curr Opin Genet Dev. 2004; 14: 308–315.[CrossRef][Medline]
[Order article via Infotrieve]
This article has been cited by other articles:
 |
|
 |
|
  L. Lei, S. Mason, D. Liu, Y. Huang, C. Marks, R. Hickey, I. S. Jovin, M. Pypaert, R. S. Johnson, and F. J. Giordano Hypoxia-Inducible Factor-Dependent Degeneration, Failure, and Malignant Transformation of the Heart in the Absence of the von Hippel-Lindau Protein Mol. Cell. Biol., June 1, 2008; 28(11): 3790 - 3803. [Abstract] [Full Text] [PDF]
|
|
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||