Gene Therapy and Molecular Biology Vol 3, page 249
Gene Ther Mol Biol Vol 3, 249-256. August 1999.
Antisense gene therapy in the long-term control of hypertension
Craig H. Gelband1, Michael J. Katovich 2, Mohan K. Raizada1*
1Departments of Physiology and 2Pharmacodynamics , Colleges of Medicine and Pharmacy, University of Florida, Gainesville, FL 32610
*Correspondence : Mohan K. Raizada, Ph.D., Professor of Physiology, University of Florida, College of Medicine, P.O. Box
100274, Gainesville, FL 32610, USA. Tel: 352-392-9299; Fax: 352-846-0270; E-mail: firstname.lastname@example.org
Key words : AT1 receptor antisense, hypertension, viral vectors, cardiac and renal pathophysiology, long-term prevention
Abbreviations: RAS , renin-angiotensin system; ACE, angiotensin converting enzyme; AT1 R, angiotensin II type 1 receptor;
AT1 R-AS angiotensin II type 1 receptor antisense; Ang , angiotensin; SHR, spontaneously hypertensive rat
Received: 7 December 1998; accepted 10 December 1998
Studies from the last two decades have established that both circulating and tissue renin-angiotensin system (RAS) are important. Their coordinated interaction is essential in the regulation of blood pressure and play a key role in the development, establishment and maintenance of hypertension. Interruption of the RAS pathway, either by preventing the formation of Ang II (i.e. ACE inhibitor) or by blocking its actions at the level of the receptor (i.e. AT 1 receptor antagonists), has been shown to reduce BP and protect against target-organ injury. Since there are problems associated with pharmacological control of high blood pressure, we developed a viral gene delivery approach to target hypertension. It was our intention to try and interrupt the RAS at the genetic level in order to achieve long term control of hypertension and reversal of pathophysiology associated with the disease. In general, delivery of antisense to the AT 1R was able to prevent (for up to 18 months) or reverse the elevated blood pressure, and the alterations in vascular calcium homeostasis, alterations in ion channel activity, and cardiac vascular ultrastructure. These results demonstrate that antisense gene delivery is useful in the long-term treatment of hypertension.
I. Current pharmacological treatment for hypertension
A stepped care regimen, starting with drugs of lowest toxicity and adding drugs from other groups, is often used to manage hypertension. First line therapy is the use of diuretics including the thiazides. If response to the thiazides is inadequate to control the hypertension, a beta-adrenoceptor ( -blocker) would then be added to the regimen. If the antagonist response to the diuretic and the -blocker is inadequate at tolerated doses then a direct vasodilator (calcium channel blocker) is generally added. Finally, if this combination does not work or is not
tolerated an ACE inhibitor is then substituted. In actuality, the ACE inhibitors are widely prescribed drugs of choice and have been beneficial in a wide groups of patients with primary hypertension. The reason for such a success using ACE inhibitors is that they not only attenuate vasoconstriction but have some important vasoprotective effects. These vasoprotective effects include: an antiatherogenic effect, an antiproliferative and antimigratory effect, improves/restores endothelial function, antiplatelet effect, enhances fribrinolysis, and improves arterial compliance (Lonn et al., 1994). Thus it is not surprising that emphasis has been placed in developing a strategy aimed at the RAS.
Gene Therapy and Molecular Biology Vol 3, page 250
Table 1 . Pharmacological therapy for the treatment of angiotensin II-dependent hypertension
II. Renin-angiotensin system and its role in hypertension
Primary human hypertension is characterized by normal cardiac output and an increase in total peripheral resistance (Khalil et al., 1990). Hypertension is one of the most important risk factors for stroke, congestive heart failure, myocardial infarction, end-stage renal diseases and peripheral vascular disease (Stamler et al., 1993; Kang et al., 1994; Whelton 1994). Studies from the last two decades have established that both circulating and tissue renin-angiotensin system (RAS) are important, that their coordinated interaction is essential in the regulation of blood pressure and they play a key role in the development, establishment and maintenance of hypertension (Brunner et al.,1993; Whelton 1994; Hsueh et al.,1995).
The relevance of the RAS to blood pressure control is further supported by reports that various genes that encode renin, angiotensinogen, angiotensin converting enzyme (ACE), and the angiotensin II type 1 receptor (AT1R) have been associated with hypertension in both human and animal models (Kurtz et al.,1990; Jeunemaitre et al.,1992; Bonnardeaux et al.,
1994). Interruption of the RAS pathway, either by preventing the formation of Ang II (i.e. ACE inhibitor) or by blocking its actions at the level of the peptide receptor (i.e. AT1 receptor antagonists), has been shown to reduce BP and protect against target-organ injury (Vogt et al.,1993; Kang et al., 1994; Kaneko et al., 1996 and Table 1 ). In fact, blockade of the RAS has become a well-accepted treatment for Ang-dependent hypertension and congestive heart failure (Vogt et al.,1993). Since ACE inhibition and AT1R blockade are standard means to treat hypertension and that AT1R encoding gene polymorphism is coupled with hypertension in both humans and in animal models of hypertension (Kurtz et al.,1990;
Jeunemaitre et al.,1992; Brunner et al., 1993; Bonnardeaux et al., 1994), it would only appear logical that AT1R is an important target in the intervention of high blood pressure and hypertension. Although major strides have been made in developing drugs which interfere with either Ang II formation or its action toward the management of Ang-dependent hypertension, there is neither long-term prevention nor a cure for this disease.
There are a number of limitations in the current pharmacological therapy to treat Ang-dependent forms of hypertension as summarized in Table 2 . ACE inhibitors and AT1R antagonists must be administered chronically to achieve long term antihypertensive benefits. Required daily dosing and undesirable side effects such as sexual dysfunction, coughing, and lethargy, increased serum Ang II levels (with AT1R antagonists), and diminish patient compliance. Finally the attenuation or delay of non-hemodynamic pathophysiological impairments with these agents does not entirely reduce the risk to hypertensive patients (de Divitiis et al.,1993; Vogt et al.,1993). In other words current pharmacological therapies do not cure hypertension; only control the disease.
Table 2 : Why administer gene therapy for the treatment of hypertension?
Gene Therapy and Molecular Biology Vol 3, page 251
Table 3 . Gene therapy and hypertension sense approach.
Table 4 . Gene therapy and hypertension antisense approach.
Therefore to circumvent the above problems associated with pharmacological control of high blood pressure, a number of research groups have used a gene delivery approach to target hypertension (Tables 3 and 4 ). Two approaches have been used to target hypertension using gene therapy; namely a sense and an antisense approach. Using the sense approach, Dr. Chao and her colleagues have been successful in over expressing genes relevant to vasodilatory effects. The genes have been delivered in hypertensive rats either in the form of naked DNA or by a viral mediated transduction system (Table 3 ). For example, genes encoding kallikrein, ANP, eNOS and adrenomedullin have been successfully delivered and have had short-term reduction in high blood pressure and other beneficial effects on pathophysiological parameters associated with hypertension (Lin et al., 1995; 1997; Xiong et al., 1995; Chao et al., 1997; Yayama et al., 1998). The laboratories of Phillips
and Tomita independently have utilized an antisense oligodeoxynucloetide or naked DNA delivery approach to interrupting the RAS in order to target hypertension (Table 4 ; Gyurko et al., 1993; Wielbo et al., 1995; Tomita et al.,1995). However, the effects were short lived and did not present a major advance over the traditional pharmacological therapy. Later it was shown that viral delivery systems could extend the duration of antihypertensive action (Table 4 , Phillips 1997). Although these studies did not produce desired long-term effects and thus were limited in scope, they were highly relevant in setting the stage indicating that a gene therapy strategy hold great potential in the treatment and cure of hypertension.
Our objective has been to extend these concepts and investigate the feasibility of the antisense gene therapy approach in order to achieve long term control of hypertension and reversal of pathophysiology associated with the disease.
Gene Therapy and Molecular Biology Vol 3, page 252
Table 5 . Protocol for AT1 -R-AS gene delivery.
III. Retroviral (LNSV)-mediated gene delivery system as a model to study AT1R- AS therapy
We chose the LNSV retrovirus because of its high infectivity, ability to effectively integrate into cells particularly, in slowly and rapidly diving cells, and its potential for long-term expression of an introduced gene (Lu and Raizada,
1995, Lu et al., 1995). In addition, the vector has been shown to influence non-dividing cells to a limited degree (Murata et al., 1998; personal communication N. Muzyczka, Univ. Florida). These properties, coupled with the fact that significant remodeling occurs during the development and establishment of hypertension, we argued that retroviruses may be an excellent vector for such purposes. With this in hand, our first attempts were to show that retroviral mediated gene delivery of AT1R- AS in vitro would be successful. Astroglial cells in primary cultures were chosen first to demonstrate the efficiency of gene transduction mediated by this vector. The viral particles were able to infect >96% of the cells as evidenced by the detection of AT1R-AS transcript using RT- in situ PCR and Northern analysis. This was associated with a significant decrease in the number of AT1 receptors and AT1 receptor-mediated actions in these cells (Lu et al., 1995b). Next, primary neuronal cultures from hypothalamus and brainstem were used since neurons in culture have limited capacity to multiply and since neurons form the SHR show an increased expression of the AT1R gene, an increased Ang II-dependent norepinephrine (NE) uptake, increased stimulation of mRNA for c-fos and the NE transporter when compared to the normotensive control (Lu et al. 1995a, 1995b). Infection of neuronal cultures with the LNSV containing AT1R-AS resulted in decrease in AT1R number, an inhibition of AT1R-mediated stimulation of both
c-fos and NE transporter mRNA, as well as NE uptake in the SHR neurons (Lu et al., 1995a, 1995b). These data not only showed the retrovirally mediated delivery of AT1R-AS could be used to selectively control the actions of Ang II but laid the framework for the in vivo studies.
IV. Prevention of the development of high blood pressure and associated pathophysiology using in vivo AT1R-AS gene delivery
The first in vivo approach that we used was based on the hypothesis that interruption in the activity of the RAS at a âcriticalâ stage in the development would prevent the onset of high blood pressure and other pathophysiology alterations associated with hypertension on a permanent basis. We used the spontaneously hypertensive rat (SHR) which is the most widely used animal model for studying human primary hypertension. As stated previously pharmacological intervention has been relatively successful in normalizing the elevation in blood pressure associated with hypertension in this model. However, the assumption that reduction of blood pressure will totally reverse hypertension-induced pathophysiological changes remains unclear. The protocol used for AT1R-AS gene delivery was to give a single intracardiac injection of the antisense into the ventricle of a 5 day old rat (Table 5 ). This route of administration insured that the antisense was delivered through out the periphery .
Indeed, using this route of administration, the AT1R-AS is expressed in a number of physiologically relevant tissue, including adrenals, heart, mesenteric arteries, kidney, and liver (Iyer et al., 1996). With the knowledge that the AT1R-AS is expressed in a number of different tissue types we next
Gene Therapy and Molecular Biology Vol 3, page 253
investigated whether the AT1R-AS had any effect on blood pressure and other cardiovascular pathophysiological alterations associated with the SHR (Figure 1 and Table 6 ). We first reported that AT1R-AS can prevent the onset of high blood pressure for up to 90 days after a single injection (Figure 1 , Iyer et al., 1996). We have extended those studies and now show an extension of up to 120 days (Figure 1, Martens et al.,
1998), 210 days (Figure 1 , Gelband et al., 1999) and 18 months (Reaves et al., 1999). This prevention of an increase in blood pressure is associated with a decrease in the specific binding of Ang II to the AT1R (Iyer et al.,1996). Similarly the AT1R-AS gene delivery prevented the Ang II dependent stimulation of blood pressure and the Ang II-stimulated increase in drinking in the SHR (Iyer et al.,1996). A number of cardiovascular pathophysiological alterations are exhibited in hypertension. These include altered renal resistance and arteriolar contractile sensitivity to circulating agents (i.e. Ang II and norepinephrine) as well as voltage dependent stimuli (KCl), endothelial dysfunction, increased smooth muscle cell Ca2+ current, increased Ca2+ release from the sarcoplasmic reticulum of smooth muscle cells, decreased smooth muscle cell voltage-dependent potassium channel (Kv) activity, increased left ventricular to body weight ratios and increased cardiac fibrosis. AT1R-AS gene delivery prevented all cardiovascular pathophysiological alterations associated with the disease mentioned above but caused no visible inflammatory response (Martens et al., 1998; Gelband et al.,
V. Reversal of the development of high blood pressure and associated pathophysiology using in vivo AT1R-AS gene delivery
Although we have used this gene delivery approach to prevent the development of high blood pressure and cardiovascular pathophysiology in the developing SHR, the ultimate strategy would be the reversal of these actions in the adult SHR. Therefore we performed in vivo gene delivery studies in the adult SHR to determine if we could reverse the pathophysiology associated with hypertension. A similar protocol was used for gene delivery except the AT1R-AS was injected into the adult SHR six days in a row instead of a single injection (Gelband et al., 1998). This protocol resulted in a significant lowering of blood pressure for up to 45 days. At day 45 the blood pressure of the SHR treated with AT1R-AS was similar to the control SHR. In renal resistance arterioles the enhanced contractile response to KCl, norepinephrine, and angiotensin II as well as decreased endothelium-dependent relaxation was reversed in the SHR treated with AT1R-AS. Finally, the left ventricular weight to body weight ratio, an index of hypertension, was reversed in the adult SHR treated with AT1R-AS. These results demonstrated the potential use of a similar gene transfer approach for long term reversal of
VI. Future directions
Is antisense gene therapy targeting the RAS a therapeutic step forward? In short, the answer is yes. It results in the prevention and reversal of the increase in mean blood pressure and the associated pathophysiological impairments in hypertension. It also offers an alternative to the compliance problem and complications of vascular and target-organ injury. Finally, the AT1R-AS therapy does not produce a significant increase in plasma Ang II levels compared with losartan, the AT1R antagonist (Lu et al., 1997). Therefore, AT1R-AS gene delivery and therapy does have prolonged antihypertensive effects without the possible adverse side effects produced by traditional pharmacological therapies.
Yet, there is a still question regarding the method of delivery. Conventional wisdom states that the LNSV retrovirus should only be successful in a population of cells undergoing cell division. Yet we find that there is an effect in the adult SHR. This leads to our first future direction and that is the development of a better viral gene delivery tool. The ideal viral vector should have the following characteristics for its successful use in a long-term reversal of hypertension: (i ) high titer should be achieved reproducibly and conveniently; (ii ) chromosome specific integration; (iii ) long-term expression; (iv ) cell specificity and (v ) no immune response. To date the ideal viral vector does not exist, but with genetic engineering it is only a matter of time before it is developed. At the present time the virus of choice may be a lenti or adeno-associated virus (AAV)-based vectors. A lentiviral based vector, for example, has the potential to be highly infective, can integrate into the host genome, has long term expression and little immune response. However, they are poorly defined at the present time. In contrast, AAV vectors are not highly infective but elicit a small immune response.
In order for this approach to be successful for consideration in humans, it needs to demonstrate its effectiveness in many other forms of hypertension. Thus, our alternative direction would be to examine the feasibility of this approach in both non-genetic models of hypertension (such as the two kidney, one-clip Goldblatt model and the DOCA salt model of hypertension) as well as a monogenetic model of hypertension (such as the renin-transgenic rat). Other components of the RAS, such as antisense to ACE and angiotensinogen should also be tested in the prevention/reversal of hypertension. Antisense to ACE is of particular importance since ACE inhibitors have been shown to be beneficial not only as antihypertensive agents but also to play an important role in protecting against myocardial infarction, kidney failure, and the restenosis/remodeling that occurs after balloon injury in angioplasty. The latter would
Gene Therapy and Molecular Biology Vol 3, page 254
Figure 1 . Time course of the change in blood pressure after antisense gene delivery. There is no change in the blood pressure in the control or antisense treated WKY rats. However there is a signifncat decrease in blood pressure in the SHRs that were treated with antisense. P<0.05, n>
be clinically beneficial to those who are not only hypertensive but undergo coronary balloon angioplasty every year. Taken together gene therapy holds promise for a single dose, long- term treatment of hypertension and other potentially lethal cardiovascular disorders.
Bonnardeaux, A., Davies, E., Jeunemaitre, X., Fery, I., Charru, A., Clauser, E., Tiret, L., Cambien, F., Corvol, P. and Soubrier, F. (1994 ) Angiotensin II type 1 receptor gene polymorphism in human essential hypertension. Hypertension 24, 63-69.
Brunner, H.R., Nussberger, J. and Waeber, B. (1993 ) Angiotensin
II blockade compared with other methods of inhibiting the renin-angiotensin system. J. Hyperten ., 11, 553-558.
Chao J, Jin L, Lin KF, Chao L ( 1997 ) Adrenomedullin gene delivery reduces blood pressure in spontaneously hypertensive rats. Hypertens. Res . 20, 269-277.
Christopherson KS, Bredt DS (1997 ) Nitric oxide in excitable tissues, physiological roles and disease. J Clin Invest 100,
de Divitiis O, Celentano A, De Simone G, Di Somma S, Galderisi M, Liguori V, de Divitiis M, Petitto M (1993 ) Management of the patient with left ventricular hypertrophy. Eur Heart J Suppl D, 22-32.
Gene Therapy and Molecular Biology Vol 3, page 255
Table 6 . Effect of AT1 -RAS in developing rats
Gelband, C.H. Reaves, P.Y. Evans, J. Wang, H, Katovich, M.J. and Raizada M.K. Angiotensin II Type 1 Receptor Antisense Gene Therapy Prevents Altered Renal Vascular Calcium Homeostasis in Hypertension. Hypertension (In Press.)
Gelband, C.H., Reaves, P.Y., Dang, H., Wang, H., Raizada, M.K., and Katovich, M.J. (1998 ) Reversal of hypertension by retroviral-mediated (LNSV) delivery of angiotensin II type 1 receptor antisense (AT1 R-AS) in the adult spontaneously hypertensive rat (SHR). Circulation 98, I-320.
Gyurko R, Wielbo D, Phillips MI (1993 ) Antisense inhibition of AT1 receptor mRNA and angiotensinogen mRNA in the brain of spontaneously hypertensive rats reduces hypertension of neurogenic origin. Reg. Pep . 49, 167-174.
Hsueh, W.A., Do, Y-S., Anderson, P.W., and Law, R.E. (1995 ) Angiotensin II in cell growth and matrix production. IN, Tissue renin-angiotensin system. (Mukhopadhyay, A. and Raizada, M.K., eds) Plenum Press, New York, pp.217-223.
Iyer, S.N., Lu, D., Katovich, M.J., Raizada, M.K. (199 6) Chronic control of high blood pressure in the spontaneouslyhypertensive rat by delivery of angiotensin type 1 receptor antisense. Proc. Natl. Acad. Sci. U.S.A.
Jeunemaitre, X., Soubrier, F., Kotelevtsev, Y.V., Liffon, R.P., Williams, C.S., Charru, A., Hunt, S.C., Hopkins, P.N., Williams, R.R., Label, J.M. and Corvol, P. (199 2) Molecular basis of human hypertension, Role of angiotensinogen. Cell
Kaneko, K., Susic, D., Nunez, E. and Frohlich, E.D. (1996 ) Losartan reduces cardiac mass and improves coronary flow reserves in the spontaneously hypertensive rat. J. Hypertens . 14, 645-653.
Kang, P.M., Landau, A.J., Eberhardt, R.T. and Fishman, W.H. (1994 ) Angiotensin II receptor antagonists, A new approach to blockade of renin angiotensin system. Am. Heart J. 127,
Khalil, R. A., Lodge, N. J., Gelband, C. H., and van Breemen, C. (1990 ) in Hypertension, Pathophysiology, Diagnosis, and Management, eds. Laragh, J. H. & Brenner, B. M., (Raven Press), pp. 547-567.
Kurtz, T.W., Simonet, L., Kabra, P.N., Wolfe, S., Chen, L. and HjeIIe, B.L. (1990 ) Consegregation of the renin allele of the spontaneously hypertensive rat with an increase in blood
Gelband et al: Antisense gene therapy for hypertension
pressure. J. Clin. Invest . 85, 1328-1332.
Lin KF, Chao J, Chao L (1995 ) Human atrial natriuretic peptide gene delivery reduces blood pressure in hypertensive rats. Hypertension 26, 847-853.
Lin KF, Chao L, Chao J (199 7) Prolonged reduction of high blood pressure with human nitric oxide synthase gene delivery. Hypertension 30, 307-313.
Lonn EM, Yusuf S, Jha P, Montague TJ, Teo KK, Benedict CR, Pitt B (1994 ) Emerging role of angiotensin-converting enzyme inhibitors in cardiac and vascular protection. Circulation
Lu, D., and Raizada, M.K. (1995 ) Delivery of angiotensin type 1 receptor antisense inhibits angiotensin action in neurons from hypertensive rat brain. Proc. Natl. Acad. Sci. U.S.A .,
Lu, D., Raizada, M.K., Iyer, S., Reaves, P., Yang, H., Katovich, M.J. (1997 ) Losartan vesus gene therapy, chronic control of high blood pressure in spontaneously hypertensive rats. Hypertension 30, 363-370.
Lu, D., Yu, K. and Raizada, M.K. (1995 ) Retrovirus meidated transfer of an angiotensin type 1 receptor antisense sequence decreases AT1 -Rs and angiotensin II action in astroglial and neuronal cells in primary culture from the brain. Proc. Natl. Acad. Sci. U.S.A ., 92, 1162-1166.
Martens, J.R., Reaves, P.Y., Lu,D., Berecek, K.H. Bishop, S.P.
Katovich, M.J., Raizada, M.K., and Gelband, C.H. (1998 ) Prevention of cardiac and renovascular pathophysiological changes in hypertension by AT1 receptor antisense gene therapy. Proc. Natl. Acad. Sci. USA 95, 2664-2669.
Murata, T., Hoffmann, S. Ishibashi, T., Spee, C., Gordon, E.M., Anderson, W.F., Hinton, D.R., and Ryan, S.J. (1998 ) Retrovirus-mediated gene transfer targeted to retinal photocoagulation sites. Diabetologia 41, 500-506.
Phillips MI. (1997 ) Antisense inhibition and adeno-associated viral vector delivery for reducing hypertension. Hypertension 29, 177-187.
Reaves, P.Y. H. Wang, D. Lu, H. Yang, M. J. Katovich, M.K.
Raizada and C.H. Gelband. (1999 ) Permanent reversal of hypertension and altered renal vascular Ca2+ homeostasis by angiotensin II type 1 receptor antisense (AT1 R-AS) gene therapy. Biophys. J . (In Press).
Stamler, J., Stamler, R., Neaton, J.D. (1993 ) Blood pressure, systolic and diastolic and cardiovascular risks, US population data. Arch. Intern. Med ., 153, 598-615.
Tomita N, Morishita R, Higaki J, Aoki M, Nakamura Y, Mikami H, Fukamizu A, Murakami K, Kaneda Y, Ogihara T (1995 ) Transient decrease in high blood pressure by in vivo transfer of antisense oligodeoxynucleotides against rat angiotensinogen. Hypertension 26, 131-136.
Vogt, M., Motz, W.H., Schwartzkopf, B., and Strauer, B. E. (1993 ) Pathophysiology and clinical aspects of hypertensive hypertrophy. Eur. Heart. J. 14, 2-7. 12.
Whelton, P.K. (1994 ) Epidemiology of hypertension. Lancet
Wielbo D, Sernia C, Gyurko R, Phillips MI (1995 ) Antisense inhibition of hypertension in the spontaneously hypertensive rat. Hypertension 25, 314-319.
Xiong W, Chao J, Chao L ( 1995 ) Muscle delivery of human kallikrein gene reduces blood pressure in hypertensive rats. Hypertension 25, 715-719.
Yayama K, Wang C, Chao L, Chao J ( 1998 ) Kallikrein gene delivery attenuates hypertension and cardiac hypertrophy and enhances renal function in Goldblatt hypertensive rats. Hypertension 31, 1104-1110.