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 Phillips, M. I. Search for Related Content PubMed PubMed Citation Articles by Phillips, M. I. Pubmed/NCBI databases Compound via MeSH Substance via MeSH Medline Plus Health Information Genes and Gene Therapy High Blood Pressure

(Hypertension. 1997;29:177.)
© 1997 American Heart Association, Inc.

State-of-the-Art Lecture

Antisense Inhibition and Adeno-Associated Viral Vector Delivery for Reducing Hypertension

M. Ian Phillips

From the Department of Physiology, College of Medicine, University of Florida, Gainesville.

Correspondence to M. Ian Phillips, Department of Physiology, College of Medicine, Box 100274, University of Florida, Gainesville, FL 32610-0274. E-mail MIP{at}phys.med.ufl.edu

	Abstract

Top Abstract Introduction Designing Antisense Molecules Antisense and Hypertension Delivery of Antisense Summary References

 
Antisense oligodeoxynucleotides have been designed to inhibit the production of specific proteins. In models of hypertension, we have targeted the renin-angiotensin system at the level of synthesis (angiotensinogen) and the receptor (AT₁ receptor). The design of antisense oligonucleotides requires choosing a site to inhibit mRNA processing or translation. The strategy we use is to make three oligonucleotides of antisense sequences, upstream and downstream from the AUG site and over the AUG site. The oligonucleotides are tested in a screening test. Antisense oligonucleotides to AT₁-receptor mRNA and to angiotensinogen mRNA reduce blood pressure in spontaneously hypertensive rats when injected into the brain. They significantly reduce the concentration of the appropriate protein. The oligonucleotides are also effective when administered systemically. The decrease in blood pressure with antisense oligonucleotides delivered in blood or brain lasts 3 to 7 days. To prolong the action, direct injection of naked DNA and injection of DNA in liposome carriers have been tested. Viral vectors have been developed to deliver antisense DNA. The viral vectors available include retroviruses and adenovirus, but the adeno-associated virus (AAV) vector is the vector of choice for ultimate use in gene therapy. It offers safety because it is nonpathogenic, has longevity because it integrates into the genome, and has sufficient carrying capacity to carry up to 4.5 kb antisense or gene in a recombinant AAV. Using rAAV-antisense to AT₁ mRNA, there is efficient transfection into cells and an inhibition of AT₁ receptor number. In in vivo tests, rAAV-AS AT₁-receptor when injected into the brains of SHR reduces blood pressure for more than 2 months. In young rats (3 weeks old), rAAV-AS AT₁-receptor decreases blood pressure and slows the development of hypertension. While further experiments need to be done on dose-response relationships and on the cellular mechanisms of these effects, the results show the feasibility of AAV as a vector for antisense inhibition, which may ultimately be used in gene therapy for hypertension.

Key Words: antisense oligonucleotide • adeno-associated viral vector • AT₁ receptor • angiotensin

Abbreviations: AAV = adeno-associated virus • AAV-AS = adeno-associated virus-antisense • Ang = angiotensin • AS-ODN = antisense oligodeoxynucleotide • AT = angiotensin type II • AVP = arginine vasopressin • CMV = cytomegalovirus • ITR = inverted terminal repeat • LTR = long-terminal repeat • ODN = oligodeoxynucleotide • paAo = plasmid for angiotensinogen antisense • paAT₁ = plasmid for AT₁-receptor antisense • PCR = polymerase chain reaction • rAAV = recombinant adeno-associated virus • rAAV-AS = recombinant adeno-associated virus-antisense • RAS = renin-angiotensin system • RT-PCR = reverse transcription-polymerase chain reaction • SHR = spontaneously hypertensive rats • TGF = transforming growth factor • TRH = thyrotropin-releasing hormone • VSMC = vascular smooth muscle cell • WKY = Wistar Kyoto rats • wtAAV = wild-type adeno-associated virus

	Introduction

Top Abstract Introduction Designing Antisense Molecules Antisense and Hypertension Delivery of Antisense Summary References

 
Hypertension is one of the most important risk factors for myocardial infarction, stroke, congestive heart failure, end-stage renal disease, and peripheral vascular disease^1,2 Twenty-four percent (an estimated 43 million) of the adult, civilian, noninstitutionalized population of the United States has hypertension.³ The incidence of hypertension is slightly more prevalent among men than women. Many drugs are available for hypertension control, including new drugs that inhibit the Ang receptor or inhibit calcium channels. All present drug regimens suffer from one obvious disadvantage. Since the disease of hypertension is lifelong, treatments have to be repeated daily. In the case of drug treatment, compliance with a daily ration of pills by the patients is difficult to maintain. Our goal in developing a gene therapy approach to hypertension is to find effective ways of modifying genes chronically in vivo. We have used anti-sense sequences and delivery systems that allow a single dose of a specific gene antimessage to have prolonged effects over several days, weeks, or months.

Although hypertension is a polygenic disease, single genes may play significant roles that can be modified by gene regulation. Lifton⁴ has contributed greatly to identifying rare examples of single-gene causes of hypertension and isolated various defects in the fundamental cellular processes, such as the Na⁺-Cl^- transporter gene (Gitelman’s syndrome), epithelial Na⁺ channel subunits (Liddle’s syndrome and pseudohypoaldosteronism-1), and Na⁺-K⁺-2Cl^- cotransporter, which is mutated in Bartter’s syndrome. Hypertension is a polygenic disease with genes overexpressing or underexpressing steroids, peptides, and lipids. In the expression of genes encoding components of the RAS, at least two mutations have been linked to hypertension. These include the angiotensin-converting enzyme (ACE) genes, D (deletion) mutation, and the angiotensinogen T235 variant.⁵ Inserting genes such as the renin gene to produce transgenic animals results in the animals developing hypertension. The opposite to gene insertion is gene knockout. Knockout genes for angiotensinogen production result in hypotension in mice.^6,7 Antisense inhibition offers a different approach to knockout because it can be used in adult animals, whereas gene knockout must be done in embryos, and therefore the adult knockout animal is one that has survived development without a specific gene. A new idea with potential has been the production of transgenic rats with an antisense gene to angiotensinogen.⁸

In SHR, Ang II is increased in the brain but normal in the periphery.⁹ In addition, AT₁ receptors are overabundantly expressed in the brain of the hypertensive SHR compared with that of the normotensive rat.¹⁰

The apparent increased activity of Ang in brain tissue of SHR gave us a starting point for testing the effectiveness of antisense inhibition in lowering blood pressure in hypertensive animals. Earlier experiments¹¹ had shown that a single injection of the nonspecific Ang antagonist Sar¹Ile⁸ Ang II injected into the brain produced a brief but significant decrease in blood pressure at a dose that was ineffective when given peripherally. Normotensive rats did not respond to central injections of Ang II antagonists. Therefore, it was hypothesized that in the SHR, the hypertension is associated with an overactive brain tissue system. We designed antisense oligonucleotides to specifically inhibit Ang II receptors or angiotensinogen to produce longer-lasting effects than a single injection of the pharmacological antagonist. The brain was convenient tissue to work on because of the relatively small volumes of oligonucleotides required.

Antisense inhibition has been developed, particularly in cell culture applications, to the point where it is being tested in clinical trials for HIV and cancer.^12,13 The pros and cons of its use have been reviewed elsewhere.¹⁴ Before 1992, however, antisense had not been applied in vivo with any success. There was much concern about the efficiency of cellular uptake of oligonucleotides. As so often happens in science, three or four or more labs simultaneously and independently made and tested anti-sense by injections into the brain.^15–18 In the brain, cellular uptake was not a limiting factor. The first demonstration of antisense inhibition for hypertension was antisense designed to inhibit angiotensinogen mRNA and Ang to AT₁-receptor mRNA in the brain.¹⁹

	Designing Antisense Molecules

Top Abstract Introduction Designing Antisense Molecules Antisense and Hypertension Delivery of Antisense Summary References

 
Table 1 lists the characteristics of AS-ODNs that need to be incorporated into their design and use. The concept of antisense inhibition assumes that a short DNA sequence in the antisense direction binds to the specific mRNA of the target protein in the cytoplasm and prevents either ribosomal assembly or read-through of the message.²⁰ Most AS-ODNs are therefore targeted to the gene initiation codon (AUG) or part of the coding region downstream from it. Antisense oligonucleotides are 15 to 20 bases long, but longer or full-length DNA in the antisense direction is used in viral vectors. When designing antisense molecules, one has to consider two antagonistic factors: the affinity of oligonucleotide to its target sequence, which is dependent on the number and composition of complementary bases, and the availability of the target sequence, which is dependent on the folding of the mRNA molecule.²¹

View this table: [in this window] [in a new window]   TABLE 1. Conditions for Antisense Oligonucleotide Inhibition

Several reports suggested that AS-ODNs targeted to different regions of the RNA have unequal efficiencies.^22,23 These differences may be related to the predicted secondary structure of the target mRNA.^24–26 The folding of the mRNA influences target-sequence availability. The RNA double helices that are responsible for the secondary structure of the mRNA incorporate a weaker G-U base pairing next to A-U and G-C and are generally short and rarely perfect. Therefore, the design should avoid G repeats. Burgess and Farrell²⁷ showed that when repeated G sequences appear in the oligonucleotide, the effects produced can be due to nonantisense mechanisms. Proper testing of antisense requires a sense ODN and a mismatch ODN control for every AS-ODN. An ODN that has strong (Watson-Crick) base pairing with 100% complementariness will form the more thermodynamically favorable structure with its target RNA.²⁸

AS-ODNs have several potential sites of action. AS-ODNs inhibit translation by hybridizing to the specific mRNA for which they are designed, and the hybridization prevents either ribosomal assembly or ribosomal sliding along the mRNA. This kind of action assumes that AS-ODNs are acting in the cytosol and do not affect measurable mRNA levels. Indeed, there are several articles reporting antisense effects without detectable change in target mRNA levels.¹⁷

Alternatively, the mechanism can be by reduction of mRNA. Decreased mRNA levels can occur by RNase H digestion of the RNA portion of the mRNA-antisense DNA hybrid. RNase H is found in the cytoplasm as well as in the nucleus, and it is normally involved in DNA duplication. The role of RNase H is to cleave RNA that has bound to DNA. Activation of RNase H is advantageous because the enzyme leaves the AS-ODN intact so it is free to hybridize with another mRNA, making the reaction catalytic rather than stoichiometric. Besides inhibition of translation, other possible antisense mechanisms of action have been proposed. Based on studies of cellular uptake of labeled oligonucleotides, the picture emerged that most AS-ODNs quickly migrate to the cell nucleus, suggesting an intranuclear site of action.²⁹

Antisense DNA can hybridize to its target mRNA or pre-mRNA in the nucleus, forming a partially double-stranded structure that would inhibit its transport out of the nucleus into the cytoplasm, thus preventing translation. AS-ODNs targeted to intron-exon junction sites prevent the splicing process and consequently the maturation of the transcript. Therefore, antisense molecules might inhibit pre-RNA splicing or the transport of mRNA from the nucleus to the cytoplasm. An alternate anti-gene strategy is to target the DNA with triplex-forming oligonucleotides to block DNA transcription. Effective AS-ODNs have been designed targeting exon-intron splicing sites³⁰ or the major groove of the DNA,³¹ but triplex formation is corrected by DNA repair mechanisms.

Currently, the three regions that are considered to be the best targets for designing effective AS-ODNs are the 5' cap region, the AUG translation initiation codon, and the 3' untranslated region of the mRNA.^32–34 Since most mRNAs have an AUG initiator codon site, targeting 12 to 15 of the neighboring bases should produce inhibitory ODNs. Routinely, all designed oligonucleotides must be checked with the GenBank for existing sequences to avoid any homology with other mRNAs. Essentially, antisense design boils down to trial-and-error testing in a model first. A general rule suggested by our own experience is that for a 15-mer oligonucleotide, three different sites should be tested with the expectation that at least one will work. Obviously, it is desirable to have a rapid screening test in vitro or in vivo for the specific protein that the AS-ODN has been designed to inhibit. Controls are sense ODN and mismatch (one or more nucleotides different from AS) or scrambled, where the entire sequence is random.

Stability of Oligonucleotides
Oligonucleotides in their natural form as phosphodiesters are subject to rapid degradation in the blood, intracellular fluid, or cerebrospinal fluid by exonucleases and endonucleases. The half-life of phosphodiester oligonucleotides is in the range of minutes in blood and tissue culture media. The half-life of oligonucleotides is somewhat longer in cerebrospinal fluid, and intact oligonucleotides can be detected 24 hours after injection into the cerebral ventricles.³⁴ Several chemical modifications have been proposed to prolong the half-life of oligonucleotides in biological fluids and enhance their uptake while retaining their activity and specificity.

The most widely used modified oligonucleotides are phosphorothioates, in which one of the oxygen atoms in the phosphodiester bond between nucleotides is replaced with a sulfur atom. These phosphorothioate oligonucleotides have greater stability in biological fluids than normal oligonucleotides. The half-life of a 15-mer phosphorothioate oligonucleotide is 9 hours in human serum, 4 days in tissue culture media,³⁵ and 19 hours in cerebrospinal fluid.³⁶ Phosphorothioate oligonucleotides can be synthesized with automated DNA synthesizers, but the product may contain impurities unless purified on an affinity gel. Oligonucleotides should be checked to ensure that they are pure.

One or more of the oxygen atoms in the phosphodiester bond can be replaced with a variety of other compounds, such as methyl groups (methylophosphoriate), alkyl phosphotriester, phosphoramidate, or boranophosphate, all of which expand the half-life of oligonucleotides in in vivo experiments. Some clever new designs are being tried in which the ODN has a dumbbell shape produced by a hairpin extension at the 3' end.³⁷ It is hoped that these third- or fourthgeneration ODNs will provide longer-lasting stability, enhanced uptake kinetics, and affinity for the target.³⁸

Cellular Uptake of Oligonucleotides
To hybridize with the target mRNA, AS-ODNs have to cross the cell membrane. Saturable uptake of oligonucleotides reaches a plateau within 50 hours, occurs rapidly, and depending on the cells, the uptake can be efficient.³⁹ Uptake is faster for shorter oligonucleotides than for longer ones.³⁹ Decreasing the temperature prevents oligonucleotide uptake, indicating that there is an active uptake mechanism. An 80-kD oligonucleotide-binding protein has been proposed to be the receptor molecular for oligonucleotide uptake.³⁹ An efflux mechanism has also been described indicating temperature-dependent secretion of the oligonucleotides from the cells to the extracellular space.³⁵

Pharmacology of AS-ODNs
Antisense inhibition can be considered pharmacologically a drug-receptor interaction in which the oligonucleotide is the drug and the target sequence is the receptor. For binding to occur between the two, a minimum level of affinity is required, which is provided by hydrogen bonding between the Watson-Crick base pairs and base stacking in the double helix that is formed. To achieve pharmacological activity, a minimum number of 12 to 15 bases can provide the minimum level of affinity.⁴⁰ Longer sequences are more specific, but above 20 bases, problems of cell uptake begin to reduce the effectiveness of ODNs.

One of the main advantages of antisense inhibition is the specificity of the AS-ODN target-sequence interaction provided by the Watson-Crick base pairing. An oligonucleotide 12 to 15 nucleotides long is specific enough statistically to be complementary to a single sequence.¹³ Increasing the length of the antisense should result in a higher level of specificity, but it decreases its uptake into cells.³⁹ With viral vectors, however, the uptake problem is overcome because the virus freely enters cells by binding to viral receptors on cell membranes. Therefore, in a viral vector a full-length DNA-antisense sequence can be used. The mechanism of action of antisense DNA is different from that of the AS-ODN. The antisense DNA produces an antisense mRNA that competes negatively with mRNA in the cytoplasm.

Toxicity
AS-ODNs can inhibit protein synthesis in cultured cells in nanomolar doses. The therapeutic window for AS-ODNs is rather narrow⁴⁰: when testing for the optimal dose, small increments in the high nanomolar range should be tested.³⁴ High concentrations may produce nonspecific binding to cytosolic proteins and give misleading results.

Phosphodiester oligonucleotides are degraded to their naturally occurring nucleotide building blocks relatively quickly; therefore, no toxic reaction is expected from even high doses of phosphodiester. Studies on phosphorothioated oligonucleotides in rats show that after intravenous injection, phosphorothioated oligonucleotides are taken up from the plasma mainly by the liver, fat, and muscle tissues. Phosphorothioate oligonucleotides are excreted through the urine in 3 days, mainly in their original form. An apparent mild increase in plasma lactate dehydrogenase, and to a lesser extent indicators of a possible transient liver-toxicity, has been noted with very high doses of phosphorothioated oligonucleotides.⁴¹ Whole new classes of oligonucleotide backbone modifications of phosphorothioate oligonucleotides are being developed to avoid the possible liver toxicity in humans.^38,40

	Antisense and Hypertension

Top Abstract Introduction Designing Antisense Molecules Antisense and Hypertension Delivery of Antisense Summary References

 
The role of candidate genes in the pathogenesis of hypertension can be studied by inhibition of the specific, overexpressed target gene with AS-ODNs. This approach is being developed in rat models of hypertension. Table 2 lists all the current in vivo studies of antisense inhibition in which blood pressure has been measured.

View this table: [in this window] [in a new window]   TABLE 2. Use of Antisense Oligodeoxynucleotides to Reduce Hypertension In Vivo

The first study on antisense and hypertension was by Gyurko et al,¹⁹ who used synthetic AS-ODN, delivered intracerebroventricularly, in SHR with established hypertension. Based on data indicating an overactive brain RAS is involved in the hypertension of this rat model, we applied antisense inhibition to the RAS (Fig 1). We designed an AS-ODN constructed to bases -5 to +13 of angiotensinogen mRNA (18 mer) and a 15-mer oligonucleotide to bases +63 to +77 of AT₁-receptor mRNA.⁴² The AT₁-receptor AS-ODN was phosphorothioated, and the angiotensinogen mRNA AS-ODN was a phosphodiester. In both cases, hypertension was significantly reduced by the application of a single dose of 50 µg AS-ODN. The mean arterial blood pressure recorded chronically was reduced by up to 35 mm Hg, in most cases lowering the high blood pressure to normotensive levels. These effects were measured 8 and 24 hours after treatment. The effects began at 8 hours and were significant at 24 hours. The single dose of AS-ODN to the AT₁-receptor mRNA produced a long-lasting, 7-day decrease in blood pressure. AT₁ receptors were measured to test that the targeted protein had been decreased after this treatment. AT₁-receptors were significantly reduced in the paraventricular nucleus and in the anterior third ventricle area. After administration of anti-sense to angiotensinogen mRNA, brain Ang II levels were significantly reduced (P<.05), indicating inhibition of the brain renin-Ang II synthesis system. Blood pressure in these animals also decreased significantly for several days. The results indicated that by inhibiting the brain RAS by AS-ODNs to the major substrate or the major receptor, high blood pressure was effectively lowered. Follow-up studies investigated how much decrease there was in the receptor and angiotensinogen levels with these oligonucleotides and also investigated the other effects of Ang II. Ambühl et al⁴⁷ quantified the reduction in Ang receptor after AS-ODN against mRNA for the AT₁ receptor. We used autoradiography, quantitative analysis, and membrane binding. Control injections contained sense or scrambled oligonucleotides or saline vehicle. Three daily injections of AS-ODN into the third ventricle of Sprague-Dawley rats decreased AT₁-receptor numbers significantly by 25% in hypothalamic tissue block. AT₂ receptors were not affected by the AT₁ antisense. Autoradiography showed a decrease in AT₁-receptor number in the hypothalamic nuclei and the anterior third ventricle area after the antisense treatment. Since AT₂ receptors were not reduced, the AT₁ AS-ODN was shown to be specific. In a second series of experiments, single injections of AS-ODN into the lateral ventricles of SHR rats were tested. AS-ODN produced a significant decrease in receptor number in the hypothalamic area. Sense and scrambled oligonucleotides did not decrease the receptor numbers significantly. The extent of the decrease after injection of AS-ODN was between 15% and 30%. These relatively modest changes apparently accounted for the physiological effects on hypertension seen by Gyurko et al.¹⁹ Wielbo et al⁴⁸ followed up the initial finding with a phosphodiester AS-ODN to angiotensinogen mRNA by developing a phosphorothioated ODN. This was administered intracerebroventricularly in unanesthetized SHR, and blood pressure was recorded. The antisense lowered hypertensive blood pressure significantly, while the control sense ODN had no effect. The phosphorothioated antisense produced a decrease in blood pressure lasting several days, in contrast to the short 1-day effect of phosphorodiesters. This undoubtedly reflects the longer-lasting action of the phosphorothioated ODN due to its resistance to nuclease. Direct measurements of angiotensinogen concentration showed that there was a significant decrease in the hypothalamus and also a decrease in the brain stem. Again, this supported the concept of centrally mediated regulation of hypertension by an overactive brain Ang system. The study also included an experiment using FITC-labeled oligonucleotide to locate where the cellular uptake of the oligonucleotide occurred. Much of it was close to the site of injection and the surrounding brain nuclei. This reflects the rapid uptake of ODN by brain cells. No decreases in mRNA were found, and it was concluded that the angiotensinogen antisense inhibited mRNA translation and not transcription.

View larger version (27K): [in this window] [in a new window]   FIG 1. The principal of antisense inhibition applied to the RAS to decrease blood pressure in hypertension. Top, Ang is synthesized in cells in various tissues and secreted or released into blood. Antisense oligonucleotide targeted to angiotensinogen mRNA will block the synthesis of angiotensinogen, which is the substrate for renin to produce Ang I. Reduction of Ang II levels lowers blood pressure via the effector cells. Bottom, The effector cells that lead to the vasoconstrictor actions of Ang II. These include contractile cells, such as VSMCs, but also noncontractile cells, such as brain and glial cells, which trigger physiological circuits leading to blood pressure increase. Application of anti-sense directed to the AT1 mRNA specifically inhibits the synthesis of AT1 receptors. Reduction of the AT1 receptors reduces the responsiveness of these types of cells to Ang II.

Injections in the brain, however, are obviously not practical for hypertension therapy. A more practical route is by oral intake or injection into the peripheral blood. Tomita et al⁴⁴ demonstrated a decrease in high blood pressure by AS-ODN against rat angiotensinogen mRNA by delivering three antisense ODNs into the portal vein. The ODNs were encapsulated in liposomes that contained viral agglutinins. The viral carrier, in this case, was a Sendai virus that had been shown to be efficient in promoting fusion with target cells by Kaneda et al.⁴⁹ The effect of the ODNs was to produce a transient decrease in plasma angiotensinogen levels in SHR. This decrease in protein was correlated to a decrease in blood pressure and a reduction in hepatic angiotensinogen mRNA. Plasma Ang II concentration was also decreased in these rats. The reduction in blood pressure, however, was shorter lasting (1 to 4 days) than the reductions in hormone (1 to 7 days). This discrepancy has not been explained. Recently Wielbo et al,⁴³ in a follow-up study of injecting antisense centrally, succeeded in showing a reduction in blood pressure by a single intravenous injection of angiotensinogen ODN encapsulated in a liposome (cholesterol plus phospholipids). Using an FITC label, they showed that the intravenous route delivered the ODN mostly to the liver. Liver angiotensinogen was reduced after antisense treatment. This study opens the possibility of effective direct delivery through intravenous injection with antisense prepared in a liposome. Preliminary work in a different model of hypertension indicates that naked DNA is also effective. Galli et al⁵⁰ injected AS-ODN to angiotensinogen mRNA in 2-kidney, 1 clip rats. These rats have high circulating Ang II, which causes the hypertension. The AS-ODN in a single dose (100 µg IV) lowered blood pressure significantly for several (7) days.

Antisense TRH receptor has also been shown to reduce arterial blood pressure in SHR.⁴⁵ Here, an intrathecal approach was used to deliver AS-ODN complementary to TRH receptor mRNA. The rats were WKY and SHR. The antisense was an 18-base phosphodiester, and the rats received 100 µg per day for 3 days. Twenty-four hours after the last injection, the magnitude of the response to an intrathecal TRH injection (10 µg) was significantly reduced in the antisense-treated group. In separate experiments, mean resting arterial blood pressure was significantly reduced by the antisense treatment in SHR but was not affected in WKY. The blood pressure in these rats was relatively low (156±4.8 mm Hg) but was reduced to 119±8.8 mm Hg (P<.01). No efforts were made to measure a change in the TRH receptors after the treatment in this study. Without showing that TRH was reduced, one cannot be sure that the antisense has reduced blood pressure by the mechanism of inhibiting receptor protein synthesis.

In another study by the same group, Suzuki et al⁴⁶ injected antisense to c-fos mRNA in the rostral ventral medullar of SHR rats, and mean arterial pressure in anesthetized rats was significantly reduced at 6 hours compared with sense-treated controls. In this experiment, the protein (c-fos) was shown to be reduced in the brains of the anti-sense-treated rats.

In all the studies on SHR in which WKY normotensive controls have been used, including our own, no effects of antisense have been found on the WKY. This indicates that antisense is useful for reducing overactive hormonal systems, such as the RAS, but does not interfere with the normal physiological functions of the hormonal system.

Several studies report applying AS-ODNs to cell cultures relevant to hypertensive mechanisms. Smooth muscle cell proliferation is a critical feature of many forms of hypertension. The oncogene c-myb and the protein myosin are believed to be involved in this process. Simons et al⁵¹ used antisense to nonmuscle myosin heavy chain or c-myb phosphorothioated ODNs to inhibit proliferation of smooth muscle cells in vitro. The growth suppression correlated with reductions in the concentrations of nonmuscle myosin heavy chain and c-myb RNA and their corresponding proteins. Sense controls were without effect. The effects were long lasting, up to 72 hours. TGF-ß is also implicated in the faster growth observed in VSMCs from SHR compared with normotensive controls. AS-ODN complimentary to TGF-ß-1 mRNA significantly suppressed VSMC number in SHR but not in WKY. Plasmin, an activator of TGF-ß, did not stimulate DNA synthesis in the presence of the AS-ODN. This cellular study suggests that endogenous TGF-ß plays a role in the hypertension of SHR through the increased growth of VSMCs.

Important pathophysiological events, such as arterial restenosis after balloon catheterization, have been effectively influenced with AS-ODNs. Inhibition of two cell-cycle regulatory enzymes, cyclin-dependent kinase 2 kinase and cell division cycle 2 kinase, with AS-ODNs resulted in a nearly complete abolition of neointima formation, which is thought to be responsible for arterial restenosis.⁵² Application of c-myc AS-ODNs in a pluronic gel to the outside of balloon-catheter-injured blood vessel resulted in a 75% decrease in c-myc expression and significantly reduced neointima formation.⁵³ This result also suggests the remarkable penetrating ability of the oligonucleotides by which they reached the neointima of the rat carotid from the outside of the vessel.

The outcome of cerebral infarction can be influenced with AS-ODNs. Synthesis of NMDA receptors, which are thought to be responsible for neuronal death after cerebral vascular occlusion, can be inhibited by antisense treatment. Such an inhibition reduces the volume of the infarction produced experimentally by the occlusion of the middle cerebral artery.¹⁷

	Delivery of Antisense

Top Abstract Introduction Designing Antisense Molecules Antisense and Hypertension Delivery of Antisense Summary References

 
Table 3 shows the currently used methods of delivering antisense for hypertension and some of their advantages and disadvantages.

View this table: [in this window] [in a new window]   TABLE 3. Vectors for Gene Transfer in Hypertension

Naked DNA
Direct injection of the antisense DNA has been used in the experiments described above.⁵⁴ For injections into the brain, naked DNA appears to be very successful. In a number of studies using different AS-ODNs, there has been efficient uptake and effective reduction in protein and inhibition of the physiological parameters studied. Uptake is so efficient that one difficulty with intracerebroventricular injections is that the DNA tends to be taken up close to the site of injection and not to spread to other parts of the brain. While this has little impact for hypertension therapy, it is an important consideration in antisense strategies for the treatment of brain diseases, such as Parkinson’s, thalamic pain, Alzheimer’s disease, and gliomas.

Lin and colleagues⁵⁵ have reported outstanding success with direct gene delivery of human tissue kallikrein in reducing blood pressure of SHR. They used naked DNA constructs, one with a metallothionine metal response element, the other with a Rous sarcoma virus 3' LTR promoter. They injected the DNA intravenously. Expression of human kallikrein was identified in several tissues. Blood pressure was significantly reduced for 6 weeks and reached a maximum of -46 mm Hg reduction.⁵⁴ They have also shown that intravenous injection of ANP gene as naked ANP reduces hypertension in young SHR (but not in adult SHR).⁵⁵ Preliminary work with a single intravenous injection of naked AS-ODN directed to angiotensinogen mRNA in the two-kidney, one clip rat reduced renovascular hypertension for several days.⁵⁰ There was uptake of fluorescent antisense oligonucleotide in blood vessels, kidney, and liver. However, with this route of administration, it is difficult to locate where in the body the antisense is having its major effect and whether the effect is direct or indirect.

Liposomes
Liposomes, which are self-assembling particles of bilipid layers, have been used for encapsulating AS-ODN for delivery in blood. Antisense directed to angiotensinogen mRNA in liposomes has had successful results. Tomita et al⁴⁴ used liposome encapsulation of angiotensinogen antisense and a Sendai virus injected in the portal vein. Blood pressure decreased for several days. However, they did not compare their results to naked DNA effects. Wielbo et al⁴³ compared liposome-encapsulated antisense and naked DNA given intra-arterially. They found that only liposome encapsulation was effective, whereas naked DNA was not, under the same conditions. Twenty-four hours after 50 µg liposome-encapsulated AS-ODN, blood pressure decreased 25 mm Hg. Empty liposomes showed no effect, and liposome-encapsulated scrambled ODN had no significant action. Unencapsulated AS-ODN also had no significant effect on blood pressure. Confocal micrographs of rat liver tissue 1 hour after intra-arterial injection of 50 µg unencapsulated FITC antisense or liposome-encapsulated FITC-conjugated antisense showed intense fluorescence in liver tissue sinusoids with the liposome-encapsulated ODN. Levels of protein (angiotensinogen and Ang II in the plasma) were significantly reduced in the liposome-encapsulated ODN group. Antisense alone, lipids alone, and scrambled ODN in liposomes were without effect on protein levels.

Liposome development with cationic lipids allows high transfection efficiency of plasmid DNA. The short, single-stranded AS-ODNs are not actually encapsulated but are complexed with microlamellar vesicles by electrostatic interactions. This simplifies the production of antisense delivery system and allows for a variety of routes of delivery, including aerosol nasal sprays and parenteral injections.

Viral Vectors
There are several viruses that have been tested for gene delivery, and each has its advantages but does not fit perfectly to the description of the "ideal viral vector." To be the perfect vector, a virus should fulfill all of the following criteria:

1. The vector should be safe. This means that it cannot be a virus known to cause disease, or it has to be reengineered to be harmless. The viral vector should not elicit an immune or inflammatory response. It should not integrate into the genome, randomly carrying the risk of disrupting other cellular genes and mutagenesis. The virus also has to be replication-deficient for the prevention of the spread to other tissues or infection of other individuals. An ideal vector would deliver a defined gene copy number into each infected cell.

2. In addition, the vector must be efficiently taken up in target tissue. The virus has to infect the target cells with high frequency to achieve biological effect.

3. To be practical, the vector should be easy to manipulate and produce in pure form. The virus should to be able to accommodate the gene of interest, along with its regulatory sequences, and the recombinant DNA has to be packaged with high efficiency into the viral capsid proteins.

Retroviruses
These have been used primarily because of their high efficiency in delivering genes to dividing cells.⁶¹ Retroviruses permit insertion and stable integration of single-copy genes. Although effective in cell culture systems, they randomly integrate into the genome, which raises concerns about their safety for practical use in vivo. Retroviruses can only act in dividing cells, which makes them ideal for tumor therapy but less desirable where other cells are dividing that need to be protected. In hypertension research, they are being investigated in developing SHR. Researchers at the University of Florida delivered a retrovirus vector (LNSV) containing an antisense DNA to AT_1b-receptor mRNA.⁵⁷ Injections in the heart of 6-week-old SHR resulted in effective long-term inhibition of AT₁-receptor mRNA and significant inhibition of the development of hypertension. Several measures indicated that the AT₁ receptors in vessels were reduced in responsiveness to the treatment.⁵⁷

Adenoviruses
Adenovirus vectors have been tested successfully in their natural host cells, the respiratory endothelia, as well as other tissues such as vascular smooth and striated muscle and brain.^62–64 Adenovirus is a double-stranded DNA with 2700 distinct adenoviral gene products. The virus infects most mammalian cell types because most cells have membrane receptors. Viruses enter the cell by a receptor-induced endocytosis and translocate to the nucleus. Most adenovirus vectors in their current form are episomal, that is, they do not integrate into the host DNA. They provide high levels of expression, but the episomal DNA will invariably turn inactive. In some species, eg, mice, this may be a long time compared with their life span, but in humans it is a limitation of the virus as a vector. Repeated infections result in inflammatory response with consequent tissue damage. This is because the adenovirus expresses genes that lead to immune cell attacks. This further limitation makes current recombinant adenovirus vectors unsuitable for long-term treatment, and several gene therapy trials using adenovirus have failed to produce acceptable results. Preliminary studies with adenovirus vectors for delivery of AT₁-receptors in mRNA antisense have been tested in rats and reduce developing hypertension in SHR.⁵⁶ The adenovirus is easy to produce and therefore useful for animal studies of mechanisms. However, the adenovirus as a vector has too many limitations at present to be successful in human gene therapy. Further engineering of the adenovirus may eventually avoid these limitations.

Adeno-Associated Virus
The AAV has been gaining attention because of its safety and efficiency.⁶⁵ It has been successfully used for delivering antisense RNA against -globin⁶⁶ and HIV LTR,⁶⁷ and it is our vector of choice for delivering anti-sense targeted to the AT-1 receptor in hypertensive rat models.

AAV is a parvovirus, discovered as a contamination of adenoviral stocks. It is a ubiquitous virus (antibodies are present in 85% of the US human population) that has not been linked to any disease. It is also classified as a dependovirus, because its replication is dependent on the presence of a helper virus, such as adenovirus. Five serotypes have been isolated, of which AAV-2 is the best characterized. AAV has a single-stranded linear DNA that is encapsidated into capsid proteins VP1, VP2, and VP3 to form an icosahedral virion of 20 to 24 nm in diameter.⁶⁵

The AAV DNA is approximately 4.7 kilobases long. It contains two open reading frames and is flanked by two ITRs (Fig 2). There are two major genes in the AAV genome: rep and cap. rep codes for proteins responsible for viral replication, whereas cap codes for capsid protein VP1-3. Each ITR forms a T-shaped hairpin structure. These terminal repeats are the only essential cis components of the AAV for chromosomal integration. Therefore, the AAV can be used as a vector with all viral coding sequences removed and replaced by the cassette of genes for delivery. Three viral promoters have been identified and named p5, p19, and p40, according to their map position. Transcription from p5 and p19 results in production of rep proteins, and transcription from p40 produces the capsid proteins.⁵⁶ For more powerful expression, we have inserted a CMV promoter. Other promoters are being tested that are specific to certain cells, including AVP promoter for cells synthesizing AVP, neuron-specific enolase, and glial fibrillary acid protein.

View larger version (22K): [in this window] [in a new window]   FIG 2. Schematic of wtAAV and rAAV vectors. In rAAV-gfp vector, almost all of the parental wtAAV genome has been deleted except the terminal repeats and replaced with gfp, driven by a CMV promoter (p). Also, neomycin resistance (neor) gene has been inserted with a thymidine kinase promoter (TKp). The neor serves for selection in vitro and as a reporter gene in vitro or in vivo when PCR and RT-PCR are used to detect it. In pAAV-AS vector, the gfp gene is replaced by a 750-bp fragment of the AT1A receptor gene with cDNA in antisense orientation. CMVp indicates human CMV early promoter; gfp, A. Victoria GFP gene, and neor, neomycin phosphotransferase gene from Tn5. Other promoters have been substituted for CMV including AVP, neuron specific enolase, and glial fibrillary acid protein promoters.

Upon infection of a human cell, the wtAAV integrates to the q arm of chromosome 19.^68,69 Although chromosomal integration requires the terminal repeats, the viral components responsible for site-specific integration have been recently targeted to the rep proteins.⁷⁰ With no helper virus present, AAV infection remains latent indefinitely. Upon superinfection of the cell with helper virus, the AAV genome is excised, replicated, packaged into virions, and released to the extracellular fluid. This fact is the basis of rAAV production for research.

There are several factors that prompted researchers to study the possibility of using rAAV as an expression vector. One is that the requirements for delivering a gene to integrate into the host chromosome are surprisingly few. It is necessary to have the 145-bp ITRs, which are only 6% of the AAV genome. This leaves room in the vector to assemble a 4.5-kb DNA insertion. While this carrying capacity may prevent the AAV from delivering large genes, it is amply suited to delivering small genes and antisense cDNA. Capacity is sufficient to ensure a specific gene response, a potent promoter, and a marker and/or selector gene, such as a neomycin resistance gene.

The second characteristics that makes AAV a good vector candidate is its safety. There is a relatively complicated rescue mechanism. Not only adenovirus (wild type) but also AAV genes are required to mobilize the rAAV. The spread of rAAV vectors to nontarget areas can be limited to certain tissues. AAV is not pathogenic and is not associated with disease. The removal of viral coding sequences in producing a +AAV minimizes immune reactions to viral gene expression, and therefore rAAV does not evoke an inflammatory response (in contrast to the recombinant adenovirus).

AAV is also a good candidate for gene therapy because it has a very broad host range. AAV infects all mammalian tissues tested, with the only exception apparently being vascular endothelial cells.⁷¹ The AAV remains intact for long periods of time.

The limitation of AAV is its production. Although it can be purified and concentrated, which are advantages, it also has to be rendered free of adenovirus, and therefore production is more complicated than for other vectors.

The advantages, particularly its safety, make AAV appear to be one of the best candidates currently for delivery of genes for long-term therapy. Recently, Flotte and colleagues⁷² have established gene therapy phase I trails for cystic fibrosis using AAV gene delivery in patients.

Expression of Antisense Sequences With Vectors
The general concept for antisense gene delivery in the AAV vector and the steps involved are shown in Fig 3. To illustrate these steps, a brief description is given that is applicable to hypertension. Further details are presented in Reference 72.

View larger version (29K): [in this window] [in a new window]   FIG 3. Gene delivery of antisense with AAV vector. 1, Gene packaging: the plasmid containing the LTRs characteristic of AAV are recombined with a 1300-bp DNA in the antisense direction. The final cassette also contains promoters such as CMV promoter. The AAV is grown in cells in the presence of adenovirus as a helper virus, and the cassette is packaged in the adenovirus. 2, Endocytosis: the viral vector fuses with the cell membrane by binding to adhesion molecules and becomes an endosome within the bilipid layer. 3, Cytoplasmic entry: the vesicle opens in the cytoplasm, releasing the vector, which is transported to and enters the nucleus. 4, Gene integration: AAV integrates with chromosome 19 in culture. It is not known if the addition of foreign DNA interferes with this integration. 5, Gene expression competition: genomic message in chromosome 19 produces an antisense RNA. This competes with the natural mRNA and prevents it from producing its product. 6, Inhibition of protein: a binding of antisense RNA to the mRNA prevents translation through the ribosomal assembly to produce protein. The result of this gene delivery system should be reduction of the protein specifically targeted by the antisense DNA. AS indicates antisense.

We constructed plasmids for both AT₁-receptor antisense (paAT₁) and angiotensinogen antisense (paAo) into the AAV-derived expression vector. Initially we used a plasmid-containing AAV genome and 750 bp DNA inserted into the AAV in the antisense direction downstream from the promoter. The plasmid was transfected with lipofectamine into NG108-15 cells (for the paAT₁) or hepatoma H4 cells for the paAo.^59,73 In both cases, there were significant reductions in the appropriate proteins, namely AT₁ receptor and angiotensinogen. To test that the cells expressed AAV, we used the rep gene product as a marker. Immunocytochemical staining with a rep protein antibody showed that the majority of cells in culture fully expressed the vector. A further development of the AAV was the insertion of more powerful and specific promoters than the p40 promoter. AAV with CMV promoter and neomycin resistance (neo^r) gene as a selectable marker is now being used in our current experiments on antisense AT₁-receptor mRNA. The AAV cassette contains either full-length DNA in the antisense direction or markers, green fluorescent protein (gfp) gene or lacZ gene.⁶⁰ The NG108-15 cells with AAV plasmid containing the neo^r were selected by antibiotic, G418 (600 µg/mL), and the selected clones viewed for gfp expression. Two weeks after transfection, all of the cells were expressing gfp (Fig 4). There is a latency for AAV integration of 1 to 2 weeks, then expression increases. Transfection efficiency of this AAV/gfp in different cell lines including ATt20 (mouse pituitary cells), L929 (mouse fibroblasts), HEK239 (human embryonic kidney cells), and NG108-15 (neoblastoma cells) is over 50%.⁷⁴ Expression in vivo was tested by direct injection into the brain. An AAV with an AVP to drive a lacZ gene was constructed. The vector expressed ß-galactosidase in neurons of the paraventricular nucleus and supraoptic nucleus. The expression was in magnocellular cells, which normally express AVP⁵⁹ (Fig 5). The expression was observed at 1 day, 1 week, and 1 month with no diminution of signal. This is an example of how AAV can be developed for specific tissue and/or cell gene expression and shows that AAV vectors can deliver foreign genes into adult brain for long periods of time.

View larger version (142K): [in this window] [in a new window]   FIG 4. Reporter genes for a single injection of rAAV-lacZ. The blue color indicates expression of ß-galactosidase in magnocellular cells of the supraoptic nucleus of the hypothalamus 1 month after injection. The result shows that rAAV injected into the brain is taken up by cells and expressed for several weeks.

View larger version (17K): [in this window] [in a new window]   FIG 5. Comparison of the effectiveness of intracerebroventricular injection of AT1 AS-ODN and an intracerebroventricular injection of rAAV-ASAT1 on the systolic blood pressure of separate groups of adult SHR rats. Both injections lower significantly the blood pressure after 3 to 7 days of administration. The AS-ODN group decreased mean arterial blood pressure (measured by implanted catheters) below control levels for up to 7 days. The rAAV-ASAT1 group had systolic blood pressure (measured by tailcuff plethysmography) significantly lower than pretreatment level for up to 9 weeks.

Functional Tests
To prepare the antisense vector, a large fragment of the Ang receptor cDNA (750 bp) was amplified using PCR and ligated to an AAV-derived vector in the antisense orientation, in place of gfp. The resulting vector (rAAV-AS) contained AAV ITRs, a CMV promoter (CMVp), the DNA encoding AT₁ receptor mRNA in the antisense direction, and a neomycin resistance gene (neo^r). The rAAV-AS vector was used to transform Escherichia coli JC8111, and the plasmid DNA was purified on CsCl gradient.

To prepare rAAV stocks, recombinant viruses were obtained using HEK293 cells infected with adenovirus type 5 and transfected with our antisense vector together with helper plasmid delivering in trans rep and cap genes, necessary for AAV replication and encapsidation.^60,69 Because AAV was to be injected into animals, it required the CsCl purification procedure, separation from adenovirus, and heat inactivation to eliminate any residual adenovirus.

The vector was tested for AT₁-receptor inhibition in vitro. Selection of the vector-transfected cells was based on the presence of the neomycin resistance gene. The cells had a significant (P<.01) decrease in Ang II AT₁ receptor number compared with the control, untransfected cells. No effect was seen on the AT₂ receptors.

To test for effectiveness in vivo, rAAV-AS plasmid vector was microinfused into the lateral ventricles of adult male SHR. Control rats received AAV with gfp reporter gene but without the AS gene ("mock" vector) in vehicle, which was artificial cerebrospinal fluid. Blood pressure was measured by the tail-cuff method. There was a significant decrease in systolic blood pressure in the group of rats that received the rAAV-AS vector. No effect was observed in the controls. Systolic blood pressure decreased by 23±2 mm Hg in the first week after administration. This drop in blood pressure was prolonged in 4 rats for 9 weeks, whereas controls had no reduction in blood pressure. This was considerably longer than the longest effect observed with antisense oligonucleotide (Fig 5). This result demonstrates that rAAV-AS in a single application is effective in chronically reducing hypertension. Furthermore, intracardiac injection of rAAV-AS in young SHR (3 weeks old) reduced blood pressure and slowed the development of hypertension.⁷⁴ The results show the feasibility of the rAAV-AS in reducing hypertension. They encourage further research on gene regulation in hypertension and exploration of the most effective routes of delivery applicable to humans.

	Summary

Top Abstract Introduction Designing Antisense Molecules Antisense and Hypertension Delivery of Antisense Summary References

 
Long-term suppression of synthesis of individual proteins is desirable in conditions in which overexpression of endogenous genes or expression of foreign genes is thought to be the cause of the disease. Expression of antisense vectors produces antisense RNA, which can hybridize with the target mRNA and prevent its translation by either promoting its degradation, preventing its transport from the nucleus to the cytoplasm, or interfering with ribosomal function.

There are many problems to overcome in the use of antisense technology as a treatment for hypertension that are related to uptake efficiency in various tissues and its stability and activity once inside the cell. However, AS-ODNs to either AT₁-receptor mRNA or angiotensinogen mRNA are effective in lowering hypertension in the SHR to normotensive levels. A single injection in the brain or systemically in lipid-encapsulated form is effective for up to 7 days. The antisense approach appears to be working through the expected mechanisms of inhibiting mRNA translation as the appropriate proteins are reduced in correlation to the physiological effect. More models of hypertension need to be tested, and the effectiveness of repeated doses to maintain reductions in blood pressure needs study. The long-term goal of prolonged blood pressure control by antisense may lie in the delivery of anti-sense DNA with viral vectors. The case for using rAAV as the vector of choice is based on safety, simplicity, and effectiveness. The carrying capacity is large enough for antisense cDNA. Results with AAV vectors in vitro demonstrate uptake in specific cells (NG108-15) and expression as shown by PCR and RT-PCR. Preliminary results with rAAV-AS produce long-lasting decreases in blood pressure. The blood pressure of adult SHR was reduced significantly for more than 9 weeks by a single intraventricular injection of rAAV-AS. In young rats, the development of hypertension was slowed by rAAV-AS. We hope these early studies with the AAV vector and hypertension will provide a viable basis for a gene therapy approach to hypertension.

	Acknowledgments

 
This work has been supported by NIH (MERIT) grant HL-23774 and grants from the American Heart Association, Florida Affiliate. I am grateful to colleagues who have been so helpful, including Drs Nicholas Muzyczka, Robert Gyurko, Ping Wu, Dagamara Mohuczy-Dominiak, Tibor Zelles, Ed Meyer, Sara M. Galli, Jeff Hughes, and Alyson Peele; technical help from Birgitta Kimura and Leping Shen; students Mark Coffey, Jon Bui, Adrian Varela, Bing Li, and Hongbin Meng; and Gayle Butters for patience in word processing.

	References

Top Abstract Introduction Designing Antisense Molecules Antisense and Hypertension Delivery of Antisense Summary References

 
1. Whelton PK. Epidemiology of hypertension. Lancet. 1994; 334 : 101 –106.

2. Stamler J, Stamler R, Neaton JD. Blood pressure, systolic and diastolic and cardiovascular risks: US population data. Arch Intern Med. 1993; 153 : 598 –615.[Abstract/Free Full Text]

3. Burt VL, Whelton PK, Roccella EJ, Brown C, Cutler JA, Higgins M, Horan MJ, Labarth D. Prevalence of hypertension in the US adult population: results from the 3rd National Health and Nutrition Examination Survey, 1988–1991. Hypertension. 1995; 25 : 305 –313.[Abstract/Free Full Text]

4. Lifton RP. Molecular genetics of human blood pressure variation. Science. 1996; 272 : 676 –680.[Abstract]

5. Junemaitre X, Soubrier F, Kotelvtsev YV, Lifton RP, Williams CS, Charru A, Hunt SC, Hopkins PN, William RR, Lalouel JM, Corvol P. Molecular basis of human hypertension: role of angiotensinogen. Cell. 1992; 71 : 169 –180.[Medline] [Order article via Infotrieve]

6. Tanimoto K, Sugiyama F, Goto Y, Ishida J, Takimoto E, Yagami K, Fukamizu A, Murakami K. Angiotensinogen-deficient mice with hypotension. J Biol Chem. 1994; 269 : 31334 –31337.[Abstract/Free Full Text]

7. Kim HS, Krege JH, Kluckman KD, Hagaman JR, Hodgin JB, Best CF, Jennette JC, Coffman TM, Maeda N, Smithies O. Genetic control of blood pressure and the angiotensinogen locus. Proc Natl Acad Sci U S A. 1995; 92 : 2735 –2739.[Abstract/Free Full Text]

8. Schinke M, Bohm M, Bricca G, Ganten D, Bader M. Permanent inhibition of angiotensinogen synthesis of antisense RNA expression. Hypertension. 1996; 27 : 508 –513.[Abstract/Free Full Text]

9. Phillips MI, Kimura B. Brain angiotensin in the developing spontaneously hypertensive rat. J Hypertens. 1988; 6 : 607 –612.[Medline] [Order article via Infotrieve]

10. Nazarali AJ, Gutkind JS, Correa FM, Saavedra JM. Decreased angiotensin II receptors in subfornical organ of spontaneously hypertensive rats after chronic antihypertensive treatment with enalapril. Am J Hypertens. 1990; 3 : 59 –61.[Medline] [Order article via Infotrieve]

11. Phillips MI, Mann JFE, Haebara H, Hoffman WE, Dietz R, Schelling P, Ganten D. Lowering of hypertension by central saralasin in the absence of plasma renin. Nature. 1977; 270 : 445 –447.[Medline] [Order article via Infotrieve]

12. Wagner RW. Gene inhibition using antisense oligodeoxynucleotides. Nature. 1994; 372 : 333 –335.[Medline] [Order article via Infotrieve]

13. Crooke ST. Therapeutic applications of oligonucleotides. Annu Rev Pharmacol Toxicol. 1992; 32 : 329 –376.[Medline] [Order article via Infotrieve]

14. Stein CA, Cheng Y-C. Antisense oligonucleotides: is the bullet really magical? Science. 1993; 261 : 1004 –1012.[Abstract/Free Full Text]

15. Chiasson BJ, Hooper ML, Murphy PR, Robertson HA. Antisense oligonucleotide eliminates in vivo expression of c-fos in mammalian brain. Eur J Pharmacol. 1992; 277 : 451 –453.

16. Wahlestedt C, Pich EM, Koob GF, Yee F, Heilig M. Modulation of anxiety and neuropeptide Y-Y1 receptors by antisense oligodeoxynucleotides. Science. 1993; 259 : 528 –531.[Abstract/Free Full Text]

17. Wahlestedt C, Golanov E, Yamamoto S, Yee F, Ericson H, Yoo H, Inturrisi CE, Reis DJ. Antisense oligonucleotides to NMDA-R1 receptor channel protect cortical neurons from excitotoxicity and reduce focal ischemic infarctions. Nature. 1993; 363 : 260 –263.[Medline] [Order article via Infotrieve]

18. McCarthy MM, Masters DB, Rimvall K, Schwartz-Giblin S, Pfaff DW. Intracerebral administration of antisense oligodeoxynucleotides to GAD65 and GAD67 mRNAs modulates reproductive behavior in the female rat. Brain Res. 1994; 636 : 209 –220.[Medline] [Order article via Infotrieve]

19. Gyurko R, Wielbo D, Phillips MI. Antisense inhibition of AT₁ receptor mRNA and angiotensinogen mRNA in the brain of spontaneously hypertensive rats reduces hypertension of neurogenic origin. Regul Pep. 1993; 49 : 167 –174.[Medline] [Order article via Infotrieve]

20. Simons RW. Naturally occurring antisense RNA control: a brief review. Gene. 1988; 72 : 35 –44.[Medline] [Order article via Infotrieve]

21. Stull RA, Taylor LA, Szoka FC. Predicting antisense oligonucleotide inhibitory efficacy: a computational approach using histograms and thermodynamic indices. Nucl Acids Res. 1992; 20 : 3501 –3508.[Abstract/Free Full Text]

22. Cowsert LM, Fox MC, Zon G, Mirabelli CK. In vitro evaluation of phosphorothioate oligonucleotides targeted to the E2 mRNA of Papillomavirus: potential treatment for genital warts. Antimicrob Agents Chemother. 1993; 37 : 171 –177.[Abstract/Free Full Text]

23. Wakita T, Wands JR. Specific inhibition of hepatitis C virus expression by antisense oligonucleotides: in vitro model for selection of target sequence. J Biol Chem. 1994; 269 : 14205 –14210.[Abstract/Free Full Text]

24. Lima WF, Monia BP, Ecker DJ, Freier SM. Implication of RNA structure on antisense oligonucleotide hybridization kinetics. Biochemistry. 1992; 31 : 12055 –12061.[Medline] [Order article via Infotrieve]

25. Rittner K, Sczakiel G. Identification and analysis of antisense RNA target regions of the human immunodeficiency virus type 1. Nucl Acid Res. 1991; 19 : 1421 –1426.[Abstract/Free Full Text]

26. Jaroszevski JW, Syi JL, Ghosh M, Ghosh K, Cohen JS. Targeting of antisense DNA: comparison of activity of anti-rabbit beta-globin oligodeoxyribonucleoside phosphorothioates with computer predictions of mRNA folding. Antisense Res Dev. 1993; 3 : 339 –348.[Medline] [Order article via Infotrieve]

27. Burgess T, Farrell C. The antiproliferative activity of c-myb and ac-myb antisense oligonucleotides in smooth muscle cells is caused by a non-antisense mechanism. Proc Natl Acad Sci U S A. 1995; 92 : 4051 –4055.[Abstract/Free Full Text]

28. Singer M, Berg P. In: Genes and Genomes. Mill Valley, Calif: University Science Books; 1992: 54 –59.

29. Iversen PL, Zhu S, Meyer A, Zon G. Cellular uptake and subcellular distribution of phosphorothioate oligonucleotides into cultured cells. Antisense Res Dev. 1992; 2 : 211 –222.[Medline] [Order article via Infotrieve]

30. Colige A, Sokolov BP, Nugent P, Baserge R, Prockop DJ. Use of an antisense oligonucleotide to inhibit expression of a mutated human procollagen gene (COL1A1) in transfected mouse 3T3 cells. Biochemistry. 1993; 32 : 7 –11.[Medline] [Order article via Infotrieve]

31. Helene C. The anti-gene strategy: control of gene expression by triplex-forming-oligonucleotides. Anticancer Drug Res. 1991; 6 : 569 –584.[Medline] [Order article via Infotrieve]

32. Bennett CF, Condon TP, Grimm S, Chan H, Chiang MY. Inhibition of endothelial cell adhesion molecule expression with antisense oligonucleotides. J Immunol. 1994; 152 : 3530 –3540.[Abstract]

33. Bacon TA, Wickstrom E. Walking along human c-myc mRNA with antisense oligonucleotides: maximum efficacy at the 5' cap region. Oncogene Res. 1991; 6 : 13 –19.[Medline] [Order article via Infotrieve]

34. Wahlestedt C. Antisense oligonucleotide strategies in neuropharmacology. Trends Pharmacol Sci. 1994; 15 : 42 –46.[Medline] [Order article via Infotrieve]

35. Li B, Hughes JA, Phillips MI. Uptake and efflux of intact antisense phosphorothioate deoxyoligonucleotide directed against angiotensin receptors in bovine adrenal cells. Neurochem Int. In press.

36. Campbell JM, Bacon TA, Wickstrom E. Oligodeoxynucleotide phosphorothioate stability in subcellular extracts, culture media, ser and cerebrospinal fluid. J Biochem Biophys Methods. 1990; 20 : 259 –267.[Medline] [Order article via Infotrieve]

37. Takaku H. Properties and anti-HIV activity of nicked dumbbell oligonucleotides. Neurotide. 1996; 15 : 519 –529.

38. Stein CA. Exploiting the potential of antisense: beyond phosphorothioate oligodeoxynucleotides. Chem Biol. 1996; 3 : 319 –323.[Medline] [Order article via Infotrieve]

39. Loke SL, Stein CA, Zhang XH, Mori K, Nakanishi M, Subasinghe C, Cohen JS, Neckers LM. Characterization of oligonucleotide transport into living cells. Proc Natl Acad Sci U S A. 1989; 86 : 3474 –3478.[Abstract/Free Full Text]

40. Wagner RW, Mattenci MD, Lewis JG, Gutierrez AJ, Moulds C, Froehler BC. Antisense gene inhibition by oligonucleotides containing C-5 mopyne primidines. Science. 1993; 260 : 1510 –1513.[Abstract/Free Full Text]

41. Iversen PL, Mata J, Tracewell WG, Zon G. Pharmacokinetics of an antisense phosphorothioate oligodeoxynucleotide against rev from human immunodeficiency virus type 1 in the adult male rat following single injections and continuous infusion. Antisense Res Dev. 1994; 4 : 43 –52.[Medline] [Order article via Infotrieve]

42. Phillips MI, Wielbo D, Gyurko R. Antisense inhibition of hypertension: a new strategy for renin-angiotensin candidate genes. Kidney Int. 1994; 46 : 1554 –1556.[Medline] [Order article via Infotrieve]

43. Wielbo D, Simon A, Phillips MI, Toffolo S. Inhibition of hypertension by peripheral administration of antisense oligodeoxynucleotides. Hypertension. 1996; 28 : 147 –151.[Abstract/Free Full Text]

44. Tomita N, Morishita R, Higaki J, Kaneda Y, Mikami H, Ogihara T. In vivo transfer of antisense oligonucleotide against rat angiotensinogen with HJV-liposome delivery resulted in reduction of blood pressure in SHR. Hypertension. 1994; 24 : 397 .

45. Suzuki S, Pilowsky P, Minson J, Arnolda L, Llewellyn-Smith I, Chalmers J. c-fos antisense in rostral ventral medulla reduces arterial blood pressure. Am J Physiol. 1994; 35 : R1418 –R1422.

46. Suzuki S, Pilowsky P, Minson J, Arnolda L, Llewellyn-Smith I, Chalmers J. Antisense to thyrotropin releasing hormone receptor reduces arterial blood pressure in spontaneously hypertensive rats. Clin Res. 1995; 77 : 679 –683.

47. Ambühl P, Gyurko R, Phillips MI. A decrease of angiotensin receptor number in rat brain nuclei by antisense oligonucleotides against the angiotensin AT_1A receptor. Regul Pep. 1995; 59 : 171 –182.[Medline] [Order article via Infotrieve]

48. Wielbo D, Sernia C, Gyurko R, Phillips MI. Antisense inhibition of hypertension in the spontaneously hypertensive rat. Hypertension. 1995; 25 : 314 –319.[Abstract/Free Full Text]

49. Kaneda Y, Iwai K, Uchida T. Increased expression of DNA co-introduced with nuclear protein in adult rat liver. Science. 1989; 243 : 375 –378.[Abstract/Free Full Text]

50. Galli SM, Varela A, Phillips MI. Lowering of blood pressure and catecholamines in chronic but not in acute 2 kidney-1 clip rats by antisense oligonucleotide to angiotensin mRNA. Hypertension. 1995; 26 : 557 . Abstract.

51. Simons M, Edelman ER, DeKeyser J-L, Langer R, Rosenberg RD. Antisense c-myb oligonucleotides inhibit intimal arterial smooth muscle cell accumulation in vivo. Nature. 1992; 359 : 67 –70.[Medline] [Order article via Infotrieve]

52. Morishita R, Gibbons GH, Kaneda Y, Ogihara T, Dzau VJ. Pharmacokinetics of antisense oligodeoxyribonucleotides (cyclin B1 and CDC 2 kinase) in the vessel wall in vivo: enhanced therapeutic utility for restenosis by HVJ-liposome delivery. Gene. 1994; 149 : 13 –19.[Medline] [Order article via Infotrieve]

53. Bennett MR, Anglin S, McEwan JR, Jagoe R, Newby AC, Evan GI. Inhibition of vascular smooth muscle cell proliferation in vitro and in vivo by c-myc antisense oligodeoxynucleotides. J Clin Invest. 1994; 93 : 820 –828.[Medline] [Order article via Infotrieve]

54. Wang C, Chao L, Chao J. Direct gene delivery of human tissue kallikrein reduces blood pressure in spontaneously hypertensive rats. J Clin Invest. 1995; 95 : 1710 –1716.[Medline] [Order article via Infotrieve]

55. Lin K-F, Chao J, Chao L. Human atrial natriuretic peptide gene delivery reduces blood pressure in hypertensive rats. Hypertension. 1995; 26 : 847 –853.[Abstract/Free Full Text]

56. Lu D, Yu K, Raizada MK. Retrovirus-mediated transfer of an angiotensin type 1 receptor (AT₁-R) antisense sequence decreases AT₁ receptors and angiotensin II action in astroglial and neuronal cells in primary cultures from the brain. Proc Natl Acad Sci U S A. 1995; 92 : 1162 –1166.[Abstract/Free Full Text]

57. Katovich MJ, Lu D, Lyer S, Raizada MK. Chronic attenuation of high blood pressure in spontaneously hypertensive rat by retrovirally mediated delivery of antisense cDNA sequence of AT₁ receptor. Exp Biol. 1996; 9 : 1558 . Abstract.

58. Willard JE, Jesse ME, Gerard RD, Meidell RS. Recombinant adenovirus is an efficient vector for in vivo gene transfer and can be preferentially directed at vascular endothelium or smooth muscle cells. Circulation. 1992; 86 : 473 .

59. Wu P, Du B, Phillips MI, Terwilliger EF. Efficient and long-term gene delivery into rat brain using adeno-associated viral vectors. Soc Neuroscience. 1996; 22 : 133.2 . Abstract.

60. Zolothukhin S, Potter M, Hauswirth WW, Guy J, Muzyczka N. A ‘humanized’ green fluorescent protein cDNA adapted for high-level expression in mammalian cells. J Virol. 1996; 70 : 4646 –4654.[Abstract]

61. Mulligan RC. The basic science of gene therapy. Science. 1993; 260 : 926 –932.[Abstract/Free Full Text]

62. Brody SL, Jaffe HA, Eissa NT, Daniel C. Administration of an adenovirus containing the human CFTR cDNA to the respiratory tract of individuals with cystic fibrosis. Nat Genet. 1994; 8 : 42 –51.[Medline] [Order article via Infotrieve]

63. Quantin B, Perricaudet LD, Tajbakhsh S, Mandel J-L. Adenovirus as an expression vector in muscle cells in vivo. Proc Natl Acad Sci U S A. 1992; 89 : 2581 –2584.[Abstract/Free Full Text]

64. Le Gal La Salle G, Robert JJ, Berrard S, Ridoux V, Stratford-Perricaudet LD, Perricaudet M, Mallet J. An adenovirus vector for gene transfer into the neurons and glia in the brain. Science. 1993; 259 : 988 –990.[Abstract]

65. Muzyczka N, McLaughlin S. Use of adeno-associated virus as a mammalian transduction vector. In: Gluzman Y, Hughes SH, eds. Current Communications in Molecular Biology: Viral Vectors. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press; 1988: 39 –44.

66. Ponnazhagan S, Nallari ML, Srivastava A. Suppression of human aglobin gene expression mediated by the recombinant adeno-associated virus 2-based antisense vectors. J Exp Med. 1994; 179 : 733 –738.[Abstract/Free Full Text]

67. Chatterjee S, Johnson PR, Wong KK. Dual-target inhibition of HIV-1 in vitro by means of an adeno-associated virus antisense vector. Science. 1992; 258 : 1485 –1488.[Abstract/Free Full Text]

68. Muzyczka N. Use of adeno-associated virus as a general transduction vector for mammalian cells. In: Current Topics in Microbiology and Immunology. Berlin, Germany: Springer-Verlag; 1992; 158 : 97 –129.

69. Samulski RJ, Zhu X, Xiao X, Brook JD, Housman DE, Epstein N, Hunter LA. Targeted integration of adeno-associated virus (AAV) into human chromosome 19. EMBO J. 1991; 10 : 3941 –3950.[Medline] [Order article via Infotrieve]

70. Linden RM, Winocour E, Berns KI. The recombination signals for adeno-associated virus site-specific integration. Proc Natl Acad Sci U S A. 1996; 93 : 7966 –7972.[Abstract/Free Full Text]

71. Lebkowski JS, McNally MM, Okarma TB, Lerch B. Adeno-associated virus: a vector system for efficient introduction and integration of DNA into a variety of mammalian cell types. Mol Cell Biol. 1988; 8 : 3988 –3996.[Abstract/Free Full Text]

72. Flotte TR, Carter B, Conrad C, Guggino W, Reynolds T, Rosenstein B, Taylor G, Walden S, Wetzel R. A phase I study of an adeno-associated virus-CTFR gene vector in adult CF patients with mild lung disease. Hum Gene Ther. 1996; 7 : 1145 –1159.[Medline] [Order article via Infotrieve]

73. Gyurko R, Phillips MI. Antisense expression vector decreases angiotensin receptor binding in NG108-15 cells. Exp Biol. 1995; 9 : 1915 . Abstract.

74. Phillips MI, Mohuczy-Dominiak D, Coffey M, Galli SM, Kimura B, Wu P, Zelles T. Prolonged reduction of high blood pressure with an in vivo, nonpathogenic, adeno-associated viral vector delivery of AT₁-R mRNA antisense. Hypertension. 1997; 28 (part 2): 374 –380.

This article has been cited by other articles:

				Z. Sun, M. Bello-Roufai, and X. Wang RNAi inhibition of mineralocorticoid receptors prevents the development of cold-induced hypertension Am J Physiol Heart Circ Physiol, April 1, 2008; 294(4): H1880 - H1887. [Abstract] [Full Text] [PDF]

				Y. Feng, X. Yue, H. Xia, S. M. Bindom, P. J. Hickman, C. M. Filipeanu, G. Wu, and E. Lazartigues Angiotensin-Converting Enzyme 2 Overexpression in the Subfornical Organ Prevents the Angiotensin II-Mediated Pressor and Drinking Responses and Is Associated With Angiotensin II Type 1 Receptor Downregulation Circ. Res., March 28, 2008; 102(6): 729 - 736. [Abstract] [Full Text] [PDF]

				K. Sakai, Y. Hirooka, H. Shigematsu, T. Kishi, K. Ito, H. Shimokawa, A. Takeshita, and K. Sunagawa Overexpression of eNOS in brain stem reduces enhanced sympathetic drive in mice with myocardial infarction Am J Physiol Heart Circ Physiol, November 1, 2005; 289(5): H2159 - H2166. [Abstract] [Full Text] [PDF]

				M. Kikuchi, D. L. Harris, Y. Obara, T. Senoo, and N. C. Joyce p27kip1 Antisense-Induced Proliferative Activity of Rat Corneal Endothelial Cells Invest. Ophthalmol. Vis. Sci., June 1, 2004; 45(6): 1763 - 1770. [Abstract] [Full Text] [PDF]

				Q. Xie, W. Bu, S. Bhatia, J. Hare, T. Somasundaram, A. Azzi, and M. S. Chapman The atomic structure of adeno-associated virus (AAV-2), a vector for human gene therapy PNAS, August 6, 2002; 99(16): 10405 - 10410. [Abstract] [Full Text] [PDF]

				M. I. Phillips, Y. Tang, K. Schmidt-Ott, K. Qian, and S. Kagiyama Vigilant Vector: Heart-Specific Promoter in an Adeno-Associated Virus Vector for Cardioprotection Hypertension, February 1, 2002; 39(2): 651 - 655. [Abstract] [Full Text] [PDF]

				M. I. Phillips Gene Therapy for Hypertension: The Preclinical Data Hypertension, September 1, 2001; 38(3): 543 - 548. [Abstract] [Full Text] [PDF]

				S. M. Galli and M. I. Phillips Angiotensin II AT1A Receptor Antisense Lowers Blood Pressure in Acute 2-Kidney, 1-Clip Hypertension Hypertension, September 1, 2001; 38(3): 674 - 678. [Abstract] [Full Text] [PDF]

				J. Culman, J. Baulmann, A. Blume, and T. Unger Review: The renin-angiotensin system in the brain: an update Journal of Renin-Angiotensin-Aldosterone System, June 1, 2001; 2(2): 96 - 102. [PDF]

				S. Kagiyama, A. Varela, M. I. Phillips, and S. M. Galli Antisense Inhibition of Brain Renin-Angiotensin System Decreased Blood Pressure in Chronic 2-Kidney, 1 Clip Hypertensive Rats Hypertension, February 1, 2001; 37(2): 371 - 375. [Abstract] [Full Text] [PDF]

				B. Kimura, D. Mohuczy, X. Tang, and M. I. Phillips Attenuation of Hypertension and Heart Hypertrophy by Adeno-Associated Virus Delivering Angiotensinogen Antisense Hypertension, February 1, 2001; 37(2): 376 - 380. [Abstract] [Full Text] [PDF]

				H. Chen, Y. C. Zhang, D. Li, M. I. Phillips, P. Mehta, M. Shi, and J. L. Mehta Protection against Myocardial Dysfunction Induced by Global Ischemia-Reperfusion by Antisense-Oligodeoxynucleotides Directed at beta 1-Adrenoceptor mRNA J. Pharmacol. Exp. Ther., August 1, 2000; 294(2): 722 - 727. [Abstract] [Full Text]

				C. Lanneau, M. T. Bluet-Pajot, P. Zizzari, Z. Csaba, P. Dournaud, L. Helboe, D. Hoyer, E. Pellegrini, G. S. Tannenbaum, J. Epelbaum, et al. Involvement of the Sst1 Somatostatin Receptor Subtype in the Intrahypothalamic Neuronal Network Regulating Growth Hormone Secretion: An in Vitro and in Vivo Antisense Study Endocrinology, March 1, 2000; 141(3): 967 - 979. [Abstract] [Full Text] [PDF]

				Y. C. Zhang, J. D. Bui, L. Shen, and M. I. Phillips Antisense Inhibition of {beta}1-Adrenergic Receptor mRNA in a Single Dose Produces a Profound and Prolonged Reduction in High Blood Pressure in Spontaneously Hypertensive Rats Circulation, February 15, 2000; 101(6): 682 - 688. [Abstract] [Full Text] [PDF]

				Y. Clare Zhang, B. Kimura, L. Shen, and M. I. Phillips New {beta}-Blocker : Prolonged Reduction in High Blood Pressure With {beta}1 Antisense Oligodeoxynucleotides Hypertension, January 1, 2000; 35(1): 219 - 224. [Abstract] [Full Text] [PDF]

				X. Tang, D. Mohuczy, Y. C. Zhang, B. Kimura, S. M. Galli, and M. I. Phillips Intravenous angiotensinogen antisense in AAV-based vector decreases hypertension Am J Physiol Heart Circ Physiol, December 1, 1999; 277(6): H2392 - H2399. [Abstract] [Full Text] [PDF]

				J. Zicha and J. Kunes Ontogenetic Aspects of Hypertension Development: Analysis in the Rat Physiol Rev, October 1, 1999; 79(4): 1227 - 1282. [Abstract] [Full Text] [PDF]

				S. Nakamura, A. Moriguchi, R. Morishita, K. Yamada, T. Nishii, N. Tomita, M. Ohishi, Y. Kaneda, J. Higaki, and T. Ogihara Activation of the Brain Angiotensin System by In Vivo Human Angiotensin-Converting Enzyme Gene Transfer in Rats Hypertension, August 1, 1999; 34(2): 302 - 308. [Abstract] [Full Text] [PDF]

				M. I. Phillips Is Gene Therapy for Hypertension Possible? Hypertension, January 1, 1999; 33(1): 8 - 13. [Full Text] [PDF]

				D. Mohuczy, C. H. Gelband, and M. I. Phillips Antisense Inhibition of AT1 Receptor in Vascular Smooth Muscle Cells Using Adeno-Associated Virus-Based Vector Hypertension, January 1, 1999; 33(1): 354 - 359. [Abstract] [Full Text] [PDF]

				Z.-Q. Wang, R. A. Felder, and R. M. Carey Selective Inhibition of the Renal Dopamine Subtype D1A Receptor Induces Antinatriuresis in Conscious Rats Hypertension, January 1, 1999; 33(1): 504 - 510. [Abstract] [Full Text] [PDF]

				B. C. Yang, M. I. Phillips, Y. C. Zhang, B. Kimura, L. P. Shen, P. Mehta, and J. L. Mehta Critical Role of AT1 Receptor Expression After Ischemia/Reperfusion in Isolated Rat Hearts : Beneficial Effect of Antisense Oligodeoxynucleotides Directed at AT1 Receptor mRNA Circ. Res., September 7, 1998; 83(5): 552 - 559. [Abstract] [Full Text] [PDF]

				J.-F. Peng, B. Kimura, M. J. Fregly, and M. I. Phillips Reduction of Cold-Induced Hypertension by Antisense Oligodeoxynucleotides to Angiotensinogen mRNA and AT1-Receptor mRNA in Brain and Blood Hypertension, June 1, 1998; 31(6): 1317 - 1323. [Abstract] [Full Text] [PDF]

				P. F. Dillon, R. S. Root-Bernstein, and D. D. Holsworth Augmentation of Aortic Ring Contractions by Angiotensin II Antisense Peptide Hypertension, March 1, 1998; 31(3): 854 - 860. [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 Phillips, M. I. Search for Related Content PubMed PubMed Citation Articles by Phillips, M. I. Pubmed/NCBI databases Compound via MeSH Substance via MeSH Medline Plus Health Information Genes and Gene Therapy High Blood Pressure