(Hypertension. 1997;29:374.)
© 1997 American Heart Association, Inc.
State-of-the-Art-Lecture |
Prolonged Reduction of High Blood Pressure With an In Vivo, Nonpathogenic, Adeno-Associated Viral Vector Delivery of AT1-R mRNA Antisense
From the Department of Physiology, College of Medicine, University of Florida, Gainesville, Fla, and Harvard Medical School (P.W.), Boston, Mass.
Correspondence to Dr M.I. Phillips, Department of Physiology, College of Medicine, University of Florida, Gainesville, FL 32610. E-mail MIP{at}phys.med.ufl.edu
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Abstract |
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To produce a prolonged decrease in blood pressure, we have developed a nonpathogenic adeno-associated viral vector (AAV) with the antisense DNA for AT1-R. AAV has many advantages over other viral vectors. AAV does not stimulate inflammation or immune reaction. AAV enters nondividing cells and does not replicate. Therefore, it is an appropriate choice for gene therapy. Recombinant AAV was prepared with a cassette containing a cytomegalovirus promoter and the cDNA for the AT1 receptor inserted in the antisense direction. The cassette was packaged in the virion. Stable transfection of NG108-15 cells with the pAAV-AS (plasmid AAV) antisense to AT1-R produced a significant reduction in AT1 receptors. A single injection of the rAAV-AS (viral vector) was made in adult spontaneously hypertensive rats, either directly in the hypothalamus (1 µL) or in the lateral ventricles (5 µL). The result shows that there is a significant decrease of blood pressure (
23±2 mm Hg) for up to 9 weeks after injection. Control injections of mock vector produced no change in blood pressure during the same time period in age-matched controls. In young spontaneously hypertensive rats (3 weeks), a single intracardiac injection of recombinant rAAV-AS reduced blood pressure and slowed the development of hypertension compared with controls (P<.01). The results suggest that a prolonged reduction in high blood pressure can be achieved with AAV vectors delivering antisense to inhibit AT1 receptors with a single administration.
Key Words: recombinant adeno-associated virus (AAV) vector • AT1 receptor • antisense • hypertension • NG108-15 cells • angiotensin
Abbreviations: AAV = adeno-associated virus • Ang = angiotensin • AT = angiotensin II type • AT1-R = angiotensin II type 1 receptor • CMV = cytomegalovirus • GFP = green fluorescent protein • ICV = intracerebral ventricular • ITR = inverted terminal repeat • ODN = oligodeoxynucleotide • pAAV-AS = plasmid adeno-associated virus antisense • PCR = polymerase chain reaction • rAAV = recombinant adeno-associated virus • rAAV-AS = recombinant adeno-associated virus antisense • RT-PCR = reverse transcription-polymerase chain reaction • SBP = systolic blood pressure • SHR = spontaneously hypertensive rats
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Introduction |
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We have previously demonstrated that antisense oligonucleotides, directed to either AT1-R mRNA or to angiotensinogen mRNA, significantly reduce blood pressure in hypertensive animals with a single injection into the brain.1–3 Although the administration of antisense in the brain proved that anti-sense can reduce high blood pressure of neurogenic origin, it obviously is not an acceptable route for treatment of human hypertension. To demonstrate that antisense acts via a systemic route of delivery, we have shown that antisense delivered intravenously4 or intra-arterially5 can also reduce blood pressure in hypertensive rats. Antisense AT1 mRNA significantly decreased the blood pressure in 2-kidney, 1 clip rats, in which circulating renin-Ang levels are high.4 Angiotensinogen mRNA-directed antisense oligonucleotide, in a liposome carrier injected intravenously in SHR, also decreased hypertension.5 The uptake of antisense was predominantly in the liver, as shown by fluorescent-tagged antisense. A similar approach was taken by Tomita et al,6 who prepared three angiotensinogen mRNA-directed antisense oligonucleotides and delivered them in liposomes and Sendai virus by direct injection into the hepatic portal vein. They also noted a decrease in blood pressure in SHR. While these results have been encouraging for the use of antisense as a potential treatment of hypertension, the maximum effectiveness of a single injection lasts for 7 days. Although this is impressively longer than the response to a single dose of any antihypertensive drug currently available, it is our hope that we can extend the effectiveness of the antisense approach by delivering antisense in a viral vector that will produce a prolonged reduction in blood pressure for weeks or months.
There are several viral vectors to choose from, including retroviruses, adenovirus, herpes virus, polio virus, and AAV. All have disadvantages and some advantages, but the AAV offers the most attractive advantages and the fewest disadvantages. AAV is safe to use. It does not induce any pathogenic response and does not replicate inside cells. The AAV is a defective parvovirus and cannot replicate in cells without the presence of wild-type adenovirus.7,8 The AAV is effective as a vector because it either integrates into the genome or remains in the nucleus as a stable episome.9 rAAV does not produce viral proteins that stimulate inflammatory reaction, and the vector contains sufficient carrying capacity for the insertion of an antisense cDNA for AT1-R. In this study, we report the results of using AAV vectors that we constructed to deliver AT1-R mRNA antisense (pAAV-AS and rAAV-AS). The objectives of the present study were: (1) to demonstrate in vitro the effectiveness of the pAAV-AS stably transfected into NG108-15 cells that express AT1-Rs; (2) to study the effect of pAAV-AS on Ang receptor (AT1 and AT2) binding and to test for specificity and efficacy; (3) to demonstrate the expression of the viral vector in brain tissue using the reporter gene lacZ; and (4) to study in vivo the effect on blood pressure of a single injection of the rAAV-AS into the brain of adult SHR and into the heart of young SHR (3 weeks) and to compare its effect on the development of hypertension at adulthood and at early stage of development. The results are encouraging, showing significant long-term decreases in blood pressure in hypertensive rats.
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Methods |
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Construction of Plasmid AAV
The AAV-derived vector can be adapted for several genes and promoters between the ITRs at each end (Fig 1). The AAV genome is removed, leaving only the ITRs. The vector has a 4.7-kb carrying capacity. Foreign genes, such as antisense DNA and marker genes, can be inserted together with foreign promoters. The plasmid AAV with the (gfp) gene was designed by Zolotukhin et al10 (provided by N. Muzyczka) and designated pAAV-gfp. The plasmid has a CMV promoter-driven gfp gene and also contains a neomycin (aminoglycoside phosphotransferase) gene, neor, driven by a thymidine kinase promoter, for selection by geneticin (G418) in transduced cells.
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For the antisense vector, a 749-bp fragment (-183 to 566) of rat vascular AT1-R cDNA (provided by T.J. Murphy in the pKSCa18b plasmid)11 was amplified with PCR and ligated to pAAV vector in the antisense orientation in place of gfp and designated pAAV-AS. Recombinant viruses (Fig 2) were produced using pAAV-AS and pAAV-gfp.
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Method to Prepare Recombinant AAV
To prepare recombinant AAV, Human Embryonic Kidney (HEK293) cells were transfected with plasmid vector containing Ang receptor antisense and AAV terminal repeats (pAAV-AS), together with helper plasmid-delivering rep and cap genes (necessary for AAV replication) in trans using the calcium phosphate method. Eight hours after transfection, adenovirus was added at a multiplicity of infection of 5. When cells developed cytopathic effects (usually after 2 to 3 days), they were harvested in media and centrifuged at 900g at 4°C for 15 minutes. Pellet was resuspended in 50 mmol/L Tris/Cl (pH 8.4) and 150 mmol/L NaCl and frozen-thawed three times. Cell debris was removed by centrifugation at 2000g at 4°C for 10 minutes, and supernatant was subjected to heating at 56°C for 30 minutes to inactivate adenovirus. After cooling, precipitate was separated at 3700g at 4°C for 10 minutes. CaCl2 (25 mmol/L) was added to supernatant and incubated at 4°C for 1 hour. After spinning at 15 300g at 4°C for 20 minutes, the supernatant was removed and equal volume of ammonium sulfate (pH 7.0) saturated at 4°C, was added dropwise and incubated on ice for 30 minutes. The solution was centrifuged at 20 000 rpm in an SW28 rotor at 4°C for 30 minutes. The floating pellet was dissolved in HEPES-buffered saline and purified through 1.39 g/cm3 CsCl gradient with 1.5 g/cm3 CsCl cushion. The gradient was fractionated, and aliquots were hybridized with [32P]dCTP-labeled random primed AAV probe. Positive fractions were pooled and concentrated using Centricon-30.
Virus Titer Assay
Titer of the virus was assayed by dot-blot analysis. Two samples of 2 µL of virus each were diluted in 200 µL of DMEM. One sample was digested with 10 U of DNase I at 37°C for 30 minutes; the second sample served as a control. The above samples were diluted 1:1 with 20 mmol/L Tris/Cl (pH 8.0), 20 mmol/L EDTA (pH 8.0), 1% SDS, and digested with 200 µg of proteinase K at 37°C for 1 hour. Samples were phenol/chloroform extracted and ethanol precipitated with addition of 40 µg glycogen as the carrier. Precipitates were dissolved in 0.4 mol/L NaOH, 10 mmol/L EDTA (pH 8.0), and loaded on the Zeta-probe membrane together with AAV DNA standards. Membrane was prehybridized in 7% SDS, 0.25 mol/L sodium phosphate buffer (pH 7.1), 1 mmol/L EDTA (pH 8.0) at 65°C for 10 minutes and then hybridized with [32P]dCTP-labeled random primed AAV probe at 65°C overnight. Membrane was washed in 1% SDS, 40 mmol/L sodium phosphate buffer (pH 7.1), and 1 mmol/L EDTA and autoradiographed. The intensity of the DNase-digested samples was compared with standards, and virus titer was calculated based on the equation 1 ng DNA=1x1012 DNA bases=2x108 copies of 5-kb ssDNA and on the fact that infectious viruses are 1% of the total virus particles.
Cell Culture
Mouse neuroblastomaxrat glioma hybrid cells (NG108-15) were grown in DMEM with L-glutamine and 4.5 g/L glucose) supplemented with 10% fetal bovine serum, 100 µmol/L hypo-xanthine, 0.4 µmol/L aminopterin, 16 µmol/L thymidine, 100 U/mL penicillin, and 100 µg/mL streptomycin in an incubator (Quene Systems, Inc) with humidified atmosphere of 5% CO2 and 95% air at 37°C. Cells were used between passages 35 and 50. The medium was replaced every day except the day immediately following subcultivation, when it was not changed.
Transfection of NG108-15 Cells by the pAAV-AS
NG108-15 cells were plated (day 1) in six-well plates and transfected the following day (day 2) at a confluence of 75%. Plasmid (2 µg) and 10 µL lipofectamine (2 mg/mL) were added to 100 µL Opti-MEM each. The two solutions were gently mixed and kept for 30 minutes at room temperature. The mixture was then filled up to 1 mL with Opti-MEM, gently mixed, and added to cells previously washed with Opti-MEM. Cells were placed into the incubator. After 5 hours, the normal medium (heat-inactivated fetal bovine serum) was given to the cells. On day 3, the cells were subcultivated and pAAV-AS-transfected cells were selected for G418 (600 µg/mL) resistance. This concentration of G418 is required to kill 100% of nontransfected NG108-15 in 4 to 7 days. Three weeks after selection, binding experiments were started on pAAV-AS-transfected and non-transfected control cells.
GFP Detection
NG108-15 cells were grown on a cover slide, embedded in a tissue culture dish. Pictures were taken using a laser-scanning confocal system, BioRad MRC-600 mounted on an Olympus IMT-2 inverted microscope.
Detection of Neomycin Resistance Gene in Transfected NG108-15 Cells by PCR and RT-PCR
To confirm that the transfected cells expressed pAAV-AS, RNA and DNA from NG108-15 cells transfected with pAAV-AS after 2 weeks on G418 selection were isolated by the method of Chomczynski.12 Control consisted of cells treated with lipofectamine only during transfection. One hundred nanograms of DNA was analyzed by PCR. Presence of neomycin resistance gene was detected using a set of primers yielding a 757-bp fragment; the 5' primer was 5'-GGATTGCACGCAGGTTCTCCG-3' and a 3' primer was 5' -CGATAGAAGGCGATGCGCTGC-3'.13 Amplification was performed for 40 cycles with annealing at 65°C. As a positive control, 1 pg of the plasmid-containing neomycin resistance gene was amplified.
RNA (2.5 µg) was subjected to RT-PCR using SuperScript II Reverse Transcriptase (Gibco BRL). Two microliters from the RT reaction was added to 100 µL final volume of the PCR reaction, and amplification was performed as described above.
Amplification products were analyzed on 1% agarose gel stained with ethidium bromide.
125I-[Sar1, Ile8]Ang II Binding to NG108-15 Membranes
Membrane Preparation
Confluent NG108-15 cells were washed twice with ice-cold phosphate-buffered saline, then dislodged and homogenized (Potter-Elvehjem homogenizer) in ice-cold Tris-HCI buffer (50 mmol/L Tris, 5 mmol/L EDTA, 150 mmol/L NaCl) and centrifuged at 51500g for 30 minutes at 4°C. The pellet was resuspended in the same buffer and homogenized again. The protein content was diluted to 1 mg/mL, and the membranes were used immediately for experiments.
Binding Assay
One hundred micrograms of membrane proteins were incubated with 0.2 nmol/L of the radiolabeled Ang II antagonist 125I-[Sar1,IIe8]Ang II in the absence and presence of 1 µmol/L cold Ang II, PD123319, or losartan to determine the total, non-specific AT1 and AT2 receptor binding, respectively. The membrane proteins were incubated in triplicate with the receptor ligands in 500 µL Tris-HCl buffer containing 0.1% bacitracin, 10 µmol/L o-phenanthroline, and 0.1% BSA for 90 minutes at room temperature. At the end of the incubation procedure, membrane-bound ligand was collected on Whatman GF/B filter paper using a Brandel cell harvester (Biomedical Research and Development Laboratories, Inc) and the radioactivity was determined with a Beckman 
-counter. An AT1-R saturation assay using a range of 0.01 to 10 nmol/L 125I-[Sar1,Ile8] Ang II performed on control, nontransfected cells revealed Kd=1.0 nmol/L and Bmax=21.8 fmol/mg protein (based on two experiments done in triplicate). For the binding experiment, 0.2 nmol/L iodinated ligand was used, based on the saturation curve.
Protein Determination
Protein content was measured by the method of Bradford using BSA as a standard.14
Animals
Adult (weight, 250 to 275 g) and young male (weight, 55 g; 3-week-old) SHR were purchased from Harlan (Indianapolis, Ind). The rats were kept in cages in a 12 hour light-dark cycle. They were fed standard laboratory rat chow and tap water ad libitum.
Blood Pressure Measurement
SBP was measured with an electrosphygmomanometer (Narco Bio-Systems, division of International Biomedical, Inc) using the indirect tail-cuff method. Unanesthetized rats were placed in a plastic holder that was mounted on a heating strip that was thermostatically controlled to maintain 37°C during measurement. Ten readings per rat were taken and the SBPs calculated. The adult rats were recorded for 1 month (n=7 experimental animals and 7 controls). After 1 month, SBP measurements were continued with four experimental rats.
Delivery of Antisense in Rats
ICV Injection
The adult rats were anesthetized with pentobarbital (65 mg/kg IP) and placed in a stereotactic instrument. After removal of scalp skin and cleaning of the exposed dorsal cranium, a hole was refined on the coordinates of -1 mm posterior to the bregma and -1.2 mm lateral to the bregma. A Hamilton syringe with a fine needle (33 gauge) was lowered 5 mm into the brain, and in the experimental group (n=7), 5 µL of rAAV-AS viral vector (2x109 infectious particles/mL) was injected into the lateral ventricles. In the control group (n=7), mock rAAV (5 µL) was injected in the same way. "Mock" virus was AAV containing gfp in place of antisense DNA. In all other respects, the construct was the same as the rAAV-AS. The needle was removed and the hole sealed by bone wax. Skin flaps were sutured, and the animals were returned to their cages for recovery. Blood pressure was recorded 1 week before injection and then at weekly intervals after the injection of rAAV-AS.
Systemic Injection
For the intracardiac injections in the young rats, blood pressures were measured from 3 weeks of age. The young SHR were divided into two groups. For the rAAV-AS cardiac injection (n=6), each animal was anesthetized with methoxyflurane (metafane), and the injection was made by passing a needle below the sternum at a slight angle dorsally and laterally to the left side of the animal. A pulse felt through the needle indicated that the injection site was in the heart. rAAV-AS 25 µL (2x109 infectious particles/mL) was injected into the heart. In one rat, the injection failed to enter the heart. This rat was monitored by SBP recordings with the other groups but was not included in the final analysis. Control rats (n=5) received intracardiac injection of vehicle (0.9% saline).
Losartan injections were made in a separate group of 4-week-old SHR (n=5). Each animal received a dorsal subcutaneous injection of losartan 10 mg/kg. The injections were given every 2 days for 2 weeks.
Expression of rAAV-lacZ Vector In Vivo
To test the duration of the vector in the brain tissue, rAAV-lacZ15 was injected into the lateral ventricles of Sprague-Dawley rats. Animals were killed 3 days, 1 week, or 1 month after injection. The hearts were perfused with saline and the brains removed and frozen. Sections were cut on a Microtome Cryostat (International Equipment Company) (30 µm) and incubated with 5-bromo-4-chloro-3-indolyl-ß-galactoside (x-gal) solution. The presence of ß-galactosidase indicated by blue staining was observed directly by light microscopy.
Statistical Analysis
Data were analyzed with the use of standard statistical methods. Repeated blood pressure measurements were taken after gene delivery, and animals were compared with controls with their baseline measurements. ANOVA and Neumann-Keuls tests were applied. Group data are expressed as mean±SEM. Significance was set at the P<.05 value. All experimental protocols were approved by the Institutional Animal Care and Use Committee of the University of Florida.
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Results |
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Expression of GFP in Cultured Cells
The first test was to assess transfection efficiency of pAAV-gfp in different cell lines. These included ATt20 (mouse pituitary cells) and L929 (mouse fibroblasts). Expression of GFP in the cells was measured 24 hours after transfection by comparing green fluorescence of the transfected cells to cells treated only with lipofectamine. The results showed that there was a 50% transfection efficiency in the cells.
Transfection in NG108-15 (rat glioma-mouse neuro-blastoma hybrid cells) was also efficient as measured by GFP expression. Fig 2 illustrates examples of transfected NG108-15 cells expressing GFP.
Detection of the Neomycin Resistance Gene in Transfected NG108-15 Cells by PCR and RT-PCR
Presence of the neomycin resistance gene in host DNA was confirmed by PCR of DNA extracted from NG108-15 cells transfected with pAAV-AS, 2 weeks after the experiment (Fig 3). A 757-bp fragment was also detected in RNA extract of pAAV-AS-transfected cells after RT-PCR was performed (Fig 3). No visible band was present in control cells.
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Effect of pAAV-AS Transfection on Ang II Receptor Binding in NG108-15 Cells
The pAAV-AS plasmid-transfected NG108-15 cells showed 41% reduction in AT1-R number assayed at 0.2 nmol/L 125I-[Sar1,Ile8] Ang II compared with the nontransfected cells (1.9±0.3 fmol/mg versus 3.2±0.6 fmol/mg; n=8; P<.001). The AT2-R number was not changed. Fig 4 shows the significant reduction in AT1-R number determined at 3 to 5 weeks after transfection (four consecutive passages).
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Expression of rAAV-lacZ Vector In Vivo After Gene Delivery
Fig 5 shows the presence of ß-galactosidase in the para-ventricular nucleus and supraoptic nucleus 1 month after injection of rAAV-lacZ in adult SHR. Many labeled cells were found spread over other tissues including cortex, amygdala, piriform cortex, and periventricular sites.
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Hypotensive Effect of ICV rAAV-AS Gene Delivery in Adult SHR
The effect of rAAV-AS delivered by a single injection (5 µL) in the lateral cerebral ventricles of adult SHR was monitored weekly for 9 weeks. The results show that compared with the baseline levels (181 mm Hg), the treated rats had a significant reduction in blood pressure (23±2 mm Hg; P<.05) 1 week later (Fig 6). A persistent reduction in blood pressure continued to be maintained for up to 9 weeks. The controls were tested weekly for 4 weeks. Their blood pressures showed no reduction from baseline, and over the 4-week period, the hypertension was well established.
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) is compared with the a control group of age-matched SHR injected with rAAV mock (
) vector (containing GFP and no antisense DNA). In this control group, the hypertension is well established over a period of 4 weeks, while in the rAAV-AS group, blood pressure significantly decreased from 181 mm Hg baseline levels and was lower than controls for 4 weeks. A group of 4 vector-treated rats were tested for a further 5 weeks and continued to have reduced blood pressure.Compared with the mock vector control rats, there were significant differences at all times for all 4 weeks tested (P<.05). The original protocol was to test the animals for 1 month, and the control animals were killed after 1 month, but because 66.6% of the experimental group had maintained significantly lower blood pressure than their base-line or control levels, they were recorded for an additional 5 weeks. At 9 weeks, the blood pressure still had not reached the original baseline of the treated group. No effect on heart rate due to the vector was observed. The animals did not appear unhealthy and had no reduction in body weight.
Hypotensive Effect of Intracardiac rAAV-AS Gene Delivery in Young SHR
Intracardiac injection of rAAV-AS at the dose used (25 µL; 2x109 infectious particles/mL) slowed the development of hypertension in young SHR (Fig 7). We compared the effects of the rAAV vector-carrying antisense with a nontreated control. The SBP of the rAAV-AS animals was significantly below the level of the control animals for 5 weeks after the rAAV-AS injection (P<.01). Losartan given to a separate group of SHR by subcutaneous injections every 2 days for 2 weeks also lowered blood pressure compared with untreated controls (P<.05) (Fig 8). However, the effect of rAAV-AS lasted longer with a single injection than the losartan-treated group with multiple injections.
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Discussion |
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The results show that gene delivery with a recombinant AAV vector of an antisense DNA to the AT1-R has potential for chronically reducing hypertension in adult animals and slowing the development of hypertension in young animals. Decreased blood pressure was sustained and persisted for over 2 months in the adult SHR, and hypertension development was slowed in young SHR. These findings, while preliminary, suggest the feasibility of rAAV-AS for gene therapy of hypertension. Obviously, the intracranial route of injection would be unacceptable for treatment of human hypertension, but the experiment in young rats with intracardiac injections suggests that the rAAV-AS will also be effective through systemic delivery. We are exploring nasal sprays and intramuscular injections as alternate routes.
Further testing requires not only systemic routes of delivery, but also higher titers and vector modification with tissue-specific promoters. The CMV promoter has been shown to stimulate gene expression in different parts of the brain for up to 3 months.16 In the present study, AAV with a CMV promoter was effective for at least 1 month. At that time, brains were expressing ß-galactosidase in several cells, including hypothalamic cells. In non-brain tissue, such as blood vessels, more specific promoters need to be tested.
The experiments are a development from our previous work using antisense oligonucleotides. In the first report of the use of antisense oligonucleotides to reduce hypertension,1 we demonstrated that AT1-R mRNA-directed antisense and angiotensinogen mRNA-targeted antisense when injected into SHR were both effective in reducing blood pressure significantly. The reduction in blood pressure was simultaneous with a reduction in receptor binding17 in key regions of the brain that have been associated with cardiovascular regulation. In the original report, the effects on blood pressure were only measured for 24 hours after injection. In a later study, we demonstrated that the effects lasted for up to 7 days with a single administration of AS-ODN.2 While the brain offers several advantages from a research point of view for studying the feasibility of antisense inhibition, the ultimate goal is to provide a safe, efficient method of delivery for humans. Therefore, in subsequent studies we injected antisense systemically5 and demonstrated that angiotensinogen antisense ODN injected intra-arterially in liposome encapsulation was taken up by the liver and reduced the levels of angiotensinogen with simultaneous reduction in blood pressure in SHR. A similar but more complex study was carried out by Tomita et al,6 who delivered three antisense oligonucleotides to angiotensinogen via the hepatic portal vein and also demonstrated a transient decrease in blood pressure.
These positive results with antisense oligonucleotides stimulated us to test viral vector delivery to produce long-term hypotension with antisense inhibition. We chose to use the AAV because of its many advantages compared with other vectors such as retroviruses or adenovirus. The properties of AAV vectors include safety, because AAV is nonpathogenic and does not replicate. It produces no viral genes that stimulate inflammatory responses. It has site-specific integration in the genome of chromosome 19.8,9 It can be concentrated and is stable in tissue. The vector can easily be modified to carry foreign genes and tissue-specific promoters. Therefore, we constructed an rAAV with antisense to AT1-R. The in vitro studies indicated efficient transfection of foreign genes (gfp and lacZ) into cultured cells. The PCR and RT-PCR detection of neomycin resistance gene confirmed that the pAAV-AS was being expressed in the transfected cells. There was a decrease in AT1-R number in the NG108-15 cells in the pAAV-AS-transfected cells. It is not known at this stage whether rAAV integrates into the genome and is expressing AS mRNA or whether the expression is episomal. Further studies with low-molecular-weight DNA18 will resolve this. The rAAV-AS induced a prolonged decrease in blood pressure for as long as we recorded it in this experiment (2 months). More experiments are needed to confirm the result, but taking the data from adult and young SHR together, the rAAV-AS does appear to be effective in chronically lowering blood pressure. ß-Gal gene expression from the rAAV-lacZ lasted at least 1 month, suggesting that the rAAV-AS was also expressed for this time. Kaplitt et al16 and McCowan et al19 have reported that the rAAV vector with CMV promoter expresses ß-galactosidase in the brain for up to 3 months. Therefore, it seems likely that the rAAV-AS was expressing the antisense DNA during the 2-month period. In the young rats given rAAV-AS by intracardiac injection, it is not known in which tissue the rAAV was sequestered and expressed. Further analysis of RT-PCR in multiple tissue samples is necessary to determine the fate of the AAV. At present, the data suggest that the reduction in blood pressure compared with controls and the retardation of hypertension development over 5 weeks was due to AT1-R decrease. The data obtained with the AT1-R antagonist losartan indicate that it is the blockade of AT1-Rs that underlies the reduction in blood pressure. The advantage of the rAAV-AS for possible therapy is that it can be effective when given once rather than repeated for several weeks. The experiment is being repeated with younger SHR to test if there is a critical period in the development of Ang-mediated peripheral resistance that, when inhibited, would permanently prevent the development of hypertension. Several studies indicate this is possible. We showed that levels of Ang II are elevated in 4-week-old SHR compared with Wistar-Kyoto rats.20 Harrap et al21 found that ACE inhibition in 6- to 10-week-old SHR prevents hypertension and proposed a critical period spanning those weeks. Our data show that the rise in blood pressure in the control rats is very rapid in weeks 3, 4, and 5 of life. By 6 to 10 weeks, hypertension is established, so it would seem that the early weeks of age would be more vulnerable to Ang II-receptor changes.
Several recent studies have applied gene therapy to hypertension since the antisense studies began in 1993.1–3 Lin et al22 showed that human ANP, delivered as a naked DNA construct, fused to the Rous sarcoma virus 3' long-terminal repeat and injected intravenously into SHR through the tail vein, produced decreased blood pressure in 4-week-old SHR that lasted for 7 weeks. The maximal reduction in pressure was 21 mm Hg. These results are strikingly similar to the present results in young SHR using quite different gene-modification procedures: one to introduce a gene associated with vasodilation and diuresis (ANP) and the other an antisense to inhibition of a vasoconstrictor (Ang II). Both approaches raise the feasibility of gene therapy for treatment in human hypertension with safe delivery systems and open new possibilities for research.
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Acknowledgments |
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This work was supported by NIH (MERIT) award HL-23774. We thank Nick Muzycka and Serge Zolotukhin for supplying adeno-associated virus.
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References |
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