The field of gene therapy is rapidly evolving, with antisense oligonucleotide therapies emerging as powerful tools for modulating gene expression. Antisense oligonucleotide therapies targeting the Renin-Angiotensin-Aldosterone System (RAAS) provide new opportunities for durable, upstream modulation of a key system involved regulation of blood volume, electrolyte balance, and systemic vascular resistance.
The successful development and application of ASO therapies hinge on robust analytical methods that quantify drug exposure at site, target engagement and expression of pharmacology.
- Exposure at site: Confirming that a therapy is bioavailable at the target site at the right time and at sufficient concentration
- Target engagement: Proof of the therapy’s engagement with its intended (and/or unintended) biological target
- Expression of pharmacology: Downstream pathway and system changes that predict or reflect clinical benefit/safety
Exposure at Site
Oligonucleotide therapeutics have revolutionised the field of medicine over the last decade due to their potential for treating a broad range of indications, particularly previously undruggable targets causing rare diseases. These oligonucleotides include a wide variety of synthetically modified ribonucleic acid (RNA) or a mixture of RNA and deoxyribonucleic acid (DNA) hybrids, specifically designed to target RNA sequences to alter RNA expression and/or protein expression.
Reliable and robust bioanalytical methods are essential for supporting preclinical and clinical investigations for novel oligonucleotides. However, these compounds present significant analytical challenges, including:
- High polarity and a strong negative charge, with molecular weights typically between 5-7 kDa per strand. Method development challenges include non-specific binding and low sensitivity due to high molecular weight, multiple charged species and adduct formation.
- Strong protein-binding, requiring specialist techniques for efficient recovery. A common approach to extraction from a biological matrix is to use a liquid/liquid extraction with dichloromethane and a phenol/chloroform mix. This is often a complex and laborious multi-step process.
At Synexa’s GLP/GCP facility in Manchester, we operate two ACQUITY Premier UPLC systems with the Xevo TQ Absolute Mass Spectrometer, offering advanced capabilities for oligonucleotide analysis. This technology provides high sensitivity in electrospray negative mode, minimises adduct formation, and eliminates the need for system passivation with acid, ensuring streamlined operation. Our standardised protocols enable efficient method development at a relatively low cost, supporting a broad range of oligonucleotide therapies. Additionally, critical reagents can be sourced directly from the vendor to ensure consistent quality. The workflow is optimised for efficiency, featuring a rapid 60-minute digestion followed by SPE and direct injection into the LC-MS/MS system. This approach delivers high recovery with minimal matrix effects, enhancing data reliability and reproducibility.
Target Engagement
The quantification of angiotensin peptides in biological matrices remains a formidable analytical challenge. These peptides play a central role in the renin-angiotensin system (RAS), a key regulator of blood pressure, fluid balance, and vascular function. However, their measurement is complicated by three major factors:
- Sensitivity – Angiotensins circulate at extremely low concentrations, often in the picomolar range
- Selectivity – All angiotensins are derived from the same amino acid sequence, making them structurally similar and difficult to distinguish
- Stability – Post-collection enzymatic activity continues to modify angiotensin peptides, leading to significant distortions in measured levels
At Attoquant’s facility, we developed a robust UPLC-MS/MS assay on a highly sensitive mass spectrometer (XEVO-TQ-S) to address these issues. Mass spectrometry measurement is based on the mass-to-charge ratio (m/z) of the analyte, allowing for the differentiation of angiotensin peptides without cross-reactivity. By additionally incorporating isotope-labelled internal standards for each individual angiotensin, the final signal of each angiotensin may be corrected for the extraction loss of analytes (recovery) and signal suppression (matrix effects). The sensitivity of modern triple quadrupole mass spectrometers enables the reliable detection of endogenous angiotensin levels as low as 1 pmol/L.
Stability is often the most underestimated challenge in angiotensin analysis. The RAAS is a dynamic system: angiotensinogen (AGT) is cleaved by renin to form angiotensin I (Ang I), which is rapidly processed by enzymes such as ACE, neprilysin, and aminopeptidases into downstream angiotensin metabolites, which are again subject to an ongoing turnover. This enzymatic cascade continues even after blood collection, leading to time-dependent changes in peptide concentrations.
To generate reproducible results, the ongoing formation and degradation of all angiotensins is enforced by incubating serum or heparin plasma at physiological temperature (37 °C). As AGT is present in a vast molar excess and is steadily truncated by renin to generate Ang I without significantly affecting the level of AGT itself or the turnover velocity of renin over time, the system is allowed to reach a steady-state equilibrium. This approach results in reproducible equilibrium angiotensin levels over several hours. These equilibrium levels reflect the activity of all RAS-related enzymes (e.g. renin / ACE / ACE2) and proteins (e.g. Angiotensinogen) present in the sample at the time of collection.
This approach enables the use of standard clinical matrices such as serum and heparin plasma. The samples remain stable for years when stored at –80°C, making the method compatible with biobank materials.
Expression of Pharmacology
Hypertension affects over 1.28 billion adults worldwide and is a leading cause of cardiovascular morbidity and mortality through end-organ damage (HMOD) to the heart, kidneys, brain, vasculature, and retina.
Early detection via plasma biomarkers can guide targeted antihypertensive therapy and improve prognosis, as traditional blood pressure measurements alone may not capture subclinical damage.
Cardiac Damage
Hypertensive heart disease often manifests as left ventricular hypertrophy (LVH) and heart failure. Key biomarkers include natriuretic peptides (e.g., NT-proBNP and BNP), which correlate with LVH, pulse wave velocity, and reduced glomerular filtration rate.
Cardiotrophin-1 (CT-1) levels rise with LVH, indicating early changes.
High-sensitivity cardiac troponin T (hs-cTnT) aids in risk stratification for LVH, while matrix metalloproteinases (MMP-2, MMP-9) reflect cardiac remodelling.
Renal Damage
Kidney injury in hypertension is characterised by inflammation and fibrosis. Neutrophil gelatinase-associated lipocalin (NGAL) is a sensitive early marker, with levels >144.3 ng/mL predicting damage and LVH.
Cystatin C detects early renal failure, and complement components (C3a, C5a) are elevated in progressive kidney disease.
The urine albumin/creatinine ratio (UACR) monitors progression in hypertensive renal disease.
Brain and Vascular Damage
Cerebral damage often stems from vascular remodelling. C-reactive protein (CRP) and high-sensitivity CRP (hs-CRP) are associated with arterial stiffness and atherosclerosis, potentially impacting brain health.
Interleukin-17A (IL-17A) promotes vascular inflammation and stiffness.
Emerging biomarkers like microRNAs (e.g., miR-92a, miR-7-5p) and circular RNAs (e.g., hsa_circ_0124782) indicate carotid plaque and vascular-brain involvement.
Emerging Biomarkers
Historical markers, such as plasma renin activity and aldosterone, remain essential for subtype diagnosis, while emerging ones, including adrenomedullin and microRNAs, offer promise for prognosis and monitoring.
No single biomarker suffices; a multi-marker strategy combining natriuretic peptides, interleukins, and CRP is recommended for comprehensive HMOD detection.
Co-written by Justin Devine, Synexa Scientific Director and Oliver Domenig, CEO, Attoquant.
About Synexa Life Sciences
Synexa Life Sciences is a biomarker and bioanalytical lab CRO, specialising in the development, validation and delivery of a wide range of complex and custom-designed assays.
With a team of over 200 staff across three global laboratory locations; Manchester, Turku (Finland) and Cape Town, we provide innovative solutions to support our customers to achieve their clinical milestones.
Our main areas of expertise include biomarker identification and development, large and small molecule clinical bioanalysis, (soluble) biomarker analysis (utilising MSD, LC-MS/MS, ELISA, RIA, fluorescence and luminescence-based technologies), cell biology (including flow cytometry, ELISpot and Fluorospot) and genomic services to support clinical trials and translational studies.
We pride ourselves on our deep scientific expertise and ability to tackle complex problems, translating them into robust and reliable assays to support clinical trial sample analysis.
About Attoquant
Attoquant Diagnostics is a global company providing high performance analysis of biomarkers supporting research and clinical management of hypertension. We specialize in the analytic challenges of angiotensin quantification for routine applications in clinical settings. Angiotensin-based biomarkers provide deep insights into the dynamic regulation of the Renin-Angiotensin-Aldosterone-System (RAAS). Our vision is to globally improve hypertension control by providing reliable angiotensin data for clinical research to identify the underlying causes of uncontrolled hypertension, allowing for a personalized and more effective therapy.
