Hypertension is a common disease with a prevalence of 20-30 % adults. Although antihypertensive drugs have been relatively successful in attenuating elevated blood pressure (BP) and in reducing adverse outcomes, control of BP depends on continuation of therapy. Drugs may have undesirable side effects which diminish compliance and BP may be resistant to treatment. Gene transfer approaches may potentially provide a tool to control BP. It was hypothesised that a lentivirus derived vector (either a naked lentiviral plasmid or a pseudotyped lentiviral vector) incorporating rat atrial natriuretic peptide (rANP) gene could be developed to produce long-term BP control. The spontaneous hypertensive rat (SHR) was used as a model for human hypertension.
Aims: To achieve this goal (i) an accurate radiotelemetry BP recording method was developed for use in the SHR, (ii) the expression of lentiviral rANP plasmid in vitro and in vivo was determined, (iii) a molecular method for titration of pseudotyped lentiviral rANP vector stock was established, and (iv) the in vivo effect of systemic delivery of lentiviral rANP plasmid on BP was measured.
Methods: Continuous 24 h arterial BP was recorded by radiotelemetry using Maclab hardware and a transducer fixed in the abdominal aorta connected to a transmitter in the abdominal cavity. Data was analysed using software specifically written for the project. Reverse transcriptase polymerase chain reaction (RT-PCR) was used to detect rANP transcripts in cells transfected with lentiviral rANP plasmid and in tissues following in vivo rANP gene delivery. Radioimmunoassay was employed to determine rANP levels in supernatant derived from post transfected cells and in plasma following in vivo gene delivery. Pseudotyped lentiviral rANP vector stock was titrated using viral supernatant via real-time quantitative RT-PCR (RQ-RTPCR). BP was monitored weekly for 9 weeks following 1.5 mg lentiviral rANP plasmid delivery into 9-month-old SHR by tailvein injection. SHR injected with lentiviral enhanced green fluorescent protein (eGFP) plasmid or saline served as controls.
Results: Weekly 24 h BP was successfully recorded for up to 16 weeks. Following transfection with lentiviral rANP plasmid in vitro, expression of rANP in transfected cells was determined by RT-PCR and by radioimmunoassay in the supernatant (at 48 h 776.2±5.4 pg/mL, n=2). Furthermore, RQ-RTPCR enabled direct titration of lentiviral rANP vector stock (eg in one sample of stock, the titre of lentiviral rANP vector = 9.9±0.4 X 109 genomes/mL). Following systemic delivery of lentiviral rANP plasmid into middle-aged SHR there was significant mean arterial BP reduction over the first 6 weeks compared with control groups [130±5 (rANP group) vs 144±3 (eGFP group) and 146±2 (saline group) mmHg, rANP group significantly different (p<0.05) from either eGFP or saline control groups, values at 6 weeks after injection, n=7 in each group]. BP returned towards pre-injection values during the final 3 weeks of monitoring. There was a significant increase in plasma immunoreactive rANP levels following injection of rANP plasmid [832±38 (rANP group) vs 662±37 (eGFP group) pg/mL, values at 6 weeks after injection, n=5 in each group, p<0.05] and an inverse correlation between plasma rANP levels and BP (r = -0.03, n=40, p<0.05). Exogenous rANP gene was expressed in the liver and kidney.
Conclusions: Radiotelemetry BP recording provided a reliable measure of long-term BP. Transfection of cells with lentiviral rANP plasmid resulted in detection of rANP transcription in transfected cells and expression of rANP secreted into cellular supernatant. RQ-RTPCR for direct titration of lentiviral vector stock independent of the transgene was developed. The titration method did not need transduction procedures nor require gene expression, or DNA or RNA isolation. It was convenient and quick to perform. Significant BP reduction was induced over 6 weeks in a group of middle-aged SHR following systemic delivery of lentiviral plasmid incorporating the rANP gene. This correlated with significant increase of plasma immunoreactive rANP levels. The presence of exogenous rANP transcripts in the liver further supported the role of the gene therapy approach in producing a reduction in BP.
In summary, the thesis lays the foundation for lentiviral vector mediated rANP gene delivery as a therapeutic strategy for hypertension. Future work should consider the possible benefits of lentiviral rANP plasmid containing a regulated tissue-selective promoter and explore approaches which might extend the time during which the hypotensive effect is present.