Analytical and Functional Characterization of Plasmid DNA Topological Forms and Multimers.

Plasmid DNA (pDNA) is an essential tool in genetic engineering that has gained prevalence in cell and gene therapies. Plasmids exist as supercoiled (SC), open circular (OC), and linear forms. Plasmid multimerization can also occur during the manufacturing process. Even though the SC forms are thought to provide optimal knock-in (KI) efficiency, there is no strong consensus on the effect of the topological forms and multimers on the functional activity. In addition, the results obtained for conventional pDNAs (>5 kbp) do not necessarily translate to smaller pDNAs (∼3 kbp). In this study, a workflow was developed for the analytical and functional characterization of pDNA topological forms and multimers. An anion exchange chromatography (AEC) method was first developed to quantify the topological forms and multimers. Four AEC columns were initially compared, one of which was found to provide superior chromatographic performance. The effect of mobile phase pH, various salts, column temperature, and acetonitrile content on the separation performance was systematically studied. The method performance, including precision and accuracy, was evaluated. The final AEC method was compared to capillary gel electrophoresis (CGE) by analyzing several pDNA sequences and lots. A forced degradation study revealed unexpectedly high degradation of the SC forms. Finally, the KI efficiency was compared for the SC and OC forms, and the multimers.

Comprehensive Impurity Profiling of mRNA: Evaluating Current Technologies and Advanced Analytical Techniques.

In vitro transcription (IVT) of mRNA is a versatile platform for a broad range of biotechnological applications. Its rapid, scalable, and cost-effective production makes it a compelling choice for the development of mRNA-based cancer therapies and vaccines against infectious diseases. The impurities generated during mRNA production can potentially impact the safety and efficacy of mRNA therapeutics, but their structural complexity has not been investigated in detail yet. This study pioneers a comprehensive profiling of IVT mRNA impurities, integrating current technologies with innovative analytical tools. We have developed highly reproducible, efficient, and stability-indicating ion-pair reversed-phase liquid chromatography and capillary gel electrophoresis methods to determine the purity of mRNA from different suppliers. Furthermore, we introduced the applicability of microcapillary electrophoresis for high-throughput (<1.5 min analysis time per sample) mRNA impurity profiling. Our findings revealed that impurities are mainly attributed to mRNA variants with different poly(A) tail lengths due to aborted additions or partial hydrolysis and the presence of double-stranded mRNA (dsRNA) byproducts, particularly the dsRNA 3'-loop back form. We also implemented mass photometry and native mass spectrometry for the characterization of mRNA and its related product impurities. Mass photometry enabled the determination of the number of nucleotides of different mRNAs with high accuracy as well as the detection of their size variants [i.e., aggregates and partial and/or total absence of the poly(A) tail], thus providing valuable information on mRNA identity and integrity. In addition, native mass spectrometry provided insights into mRNA intact mass, heterogeneity, and important sequence features such as poly(A) tail length and distribution. This study highlights the existing bottlenecks and opportunities for improvement in the analytical characterization of IVT mRNA, thus contributing to the refinement and streamlining of mRNA production, paving the way for continued advancements in biotechnological applications.

Use of artificial intelligence-guided echocardiography to detect cardiac dysfunction and heart valve disease in rural and remote areas: Rationale and design of the AGILE-echo trial.

BACKGROUND: Transthoracic echocardiography (TTE) is essential in the diagnosis of cardiovascular diseases (CVD), including but not limited to heart failure (HF) and heart valve disease (HVD). However, its dependence on expert acquisition means that its accessibility in rural areas may be limited, leading to delayed management decisions and potential missed diagnoses. Artificial intelligence-guided (AI)-TTE offers a solution by permitting non-expert image acquisition. The impact of AI-TTE on the timing of diagnosis and early initiation of cardioprotection is undefined. METHODS: AGILE-Echo (use of Artificial intelligence-Guided echocardiography to assIst cardiovascuLar patient managEment) is a randomized-controlled trial conducted in 5 rural and remote areas around Australia. Adults with CV risk factors and exercise intolerance, or concerns regarding HVD are randomized into AI-TTE or usual care (UC). AI-TTE participants may have a cardiovascular problem excluded, identified (leading to AI-guided interventions) or unresolved (leading to conventional TTE). UC participants undergo usual management, including referral for standard TTE. The primary endpoint is a composite of HVD or HF diagnosis at 12-months. Subgroup analysis, stratified based on age range and sex, will be conducted. All statistical analyses will be conducted using R. RESULTS: Of the first 157 participants, 78 have been randomized into AI-TTE (median age 68 [IQR 17]) and 79 to UC (median age 65 [IQR 17], P = .034). HVD was the primary concern in 37 participants (23.6%) while 84.7% (n = 133) experienced exercise intolerance. The overall 10-year HF incidence risk was 13.4% and 20.0% (P = .089) for UC and AI-TTE arm respectively. Atrial remodeling, left ventricular remodeling and valvular regurgitation were the most common findings. Thirty-three patients (42.3%) showed no abnormalities. CONCLUSIONS: This randomized-controlled trial of AI-TTE will provide proof-of-concept for the role of AI-TTE in identifying pre-symptomatic HF or HVD when access to TTE is limited. Additionally, this could promote the usage of AI-TTE in rural or remote areas, ultimately improving health and quality of life of community dwelling adults with risks, signs or symptoms of cardiac dysfunction.

Restoring microvascular circulation with diagnostic ultrasound and contrast agent: rationale and design of the REDUCE trial.

OBJECTIVES: This study aims to evaluate the efficacy and cost-effectiveness of sonothrombolysis delivered pre and post primary percutaneous coronary intervention (pPCI) on infarct size assessed by cardiac MRI, in patients presenting with STEMI, when compared against sham procedure. BACKGROUND: More than a half of patients with successful pPCI have significant microvascular obstruction and residual infarction. Sonothrombolysis is a therapeutic use of ultrasound with contrast enhancement that may improve microcirculation and infarct size. The benefits and real time physiological effects of sonothrombolysis in a multicentre setting are unclear. METHODS: The REDUCE (Restoring microvascular circulation with diagnostic ultrasound and contrast agent) trial is a prospective, multicentre, patient and outcome blinded, sham-controlled trial. Patients presenting with STEMI will be randomized to one of 2 treatment arms, to receive either sonothrombolysis treatment or sham echocardiography before and after pPCI. This tailored design is based on preliminary pilot data from our centre, showing that sonothrombolysis can be safely delivered, without prolonging door to balloon time. Our primary endpoint will be infarct size assessed on day 4±2 on Cardiac Magnetic Resonance (CMR). Patients will be followed up for 6 months post pPCI to assess secondary endpoints. Sample size calculations indicate we will need 150 patients recruited in total. CONCLUSIONS: This multicentre trial will test whether sonothrombolysis delivered pre and post primary PCI can improve patient outcomes and is cost-effective, when compared with sham ultrasound delivered with primary PCI. The results from this trial may provide evidence for the utilization of sonothrombolysis as an adjunct therapy to pPCI to improve cardiovascular outcomes in STEMI. ANZ Clinical Trial Registration number: ACTRN 12620000807954.

Prophylaxis to Prevent Cardiotoxicity in Patients Receiving Anthracycline for Breast Cancer: A Combined Bayesian and Frequentist Network Meta-Analysis of Randomised Controlled Trials.

BACKGROUND: The benefits in survivorship gained with anthracycline (ANT)-based chemotherapies for breast cancer are unfortunately mitigated for some patients by irreversible cardiotoxicity. Randomised controlled trials (RCTs) have explored multiple cardioprotection options, however, it remains unclear which drug is most effective in preserving left ventricular ejection fraction (LVEF). This study aimed to perform a systematic review and network meta-analysis, using Bayesian and frequentist approaches, of RCTs evaluating cardioprotective agents. METHODS: Two authors searched four databases (CENTRAL, Cochrane Reviews, MEDLINE, SCOPUS), to find RCTs evaluating cardioprotective agents. Trial populations were limited to patients with breast cancer without prior ANT exposure. The primary outcome was mean LVEF change pre and post ANT dosing. Our primary analysis utilised a Bayesian approach, while our sensitivity analysis used frequentist methodology (Prospero registration number CRD42020199580). RESULTS: From 4,007 search results, we identified 12 RCTs, with their various trial arms considered separately-nine beta-blocker (BB), two angiotensin-converting enzyme inhibitor /angiotensin receptor blockers [(AA)+BB=AABB], one AA, one spironolactone, one statin-evaluating 1,126 patients (age 50.5 years). Bayesian network meta-analysis showed no difference in LVEF preservation between AA (1.3%, 95% credible interval [-0.20, 2.9]), BB (0.77, [-0.21, 1.8]), AABB (0.84 [-1.1, 2.8]), spironolactone (0.72, [-2.3, 3.7]) or statin (0.60, [-2.4, 3.6]) when compared against placebo. However, the frequentist analysis showed benefits from using AA (mean difference, 1.32% [0.32, 2.33]) and BB (mean difference, 0.76% [0.12, 1.4]). CONCLUSIONS: There is insufficient evidence to support prophylactic cardioprotection to prevent EF reduction. However, frequentist analysis suggested that AA or BBs provide cardioprotection. Thus, for those already on other anti-hypertensives, switching to AA or BBs could be considered.

Coronary Flow Velocity Reserve Declines After Anthracycline Therapy in Breast Cancer Patients.

Anthracycline therapy (ANT) is associated with cancer therapy-related cardiac dysfunction. Coronary flow velocity reserve (CFVR) has shown prognostic utility in non-cancer cohorts, but no data have been obtained in a cardio-oncology setting. We investigated the acute effect of ANT on CFVR in breast cancer patients. A total of 12 female breast cancer patients undergoing ANT had pre- and post-ANT CFVR assessment. A significant decline in CFVR occurred (baseline: 2.66 ± 0.41 vs post-ANT: 2.47 ± 0.37, P = 0.016). This prospective study is the first to identify ANT-related coronary physiology changes in humans. Further studies are required to determine their clinical significance.

Le traitement par l'anthracycline est associé à une dysfonction cardiaque liée au traitement anticancéreux. La réserve de débit coronaire a démontré son utilité pronostique dans les cohortes sans cancer, mais aucune donnée n'a été obtenue dans un contexte de cardio-oncologie. Nous avons étudié l'effet aigu de l'anthracycline sur la réserve de débit coronaire chez des patientes atteintes d'un cancer du sein. La réserve de débit coronaire a été évaluée avant et après le traitement par l'anthracycline chez un total de 12 femmes atteintes d'un cancer du sein. Un déclin important de la réserve de débit coronaire est survenu (valeur initiale de 2,66 ± 0,41 par rapport à 2,47 ± 0,37 après le traitement par l'anthracycline, p = 0,016). Cette étude prospective est la première à déceler des changements dans la physiologie coronarienne liés à l'anthracycline chez les humains. D'autres études sont nécessaires pour en déterminer la portée clinique.

Sonothrombolysis Before and After Percutaneous Coronary Intervention Provides the Largest Myocardial Salvage in ST Segment Elevation Myocardial Infarction.

BACKGROUND: Sonothrombolysis is a therapeutic application of ultrasound with ultrasound contrast for patients with ST elevation myocardial infarction (STEMI). Recent trials demonstrated that sonothrombolysis, delivered before and after primary percutaneous coronary intervention (pPCI), increases infarct vessel patency, improves microvascular flow, reduces infarct size, and improves ejection fraction. However, it is unclear whether pre-pPCI sonothrombolysis is essential for therapeutic benefit. We designed a parallel 3-arm sham-controlled randomized controlled trial to address this. METHODS: Patients presenting with first STEMI undergoing pPCI within 6 hours of symptom onset were randomized 1:1:1 into 3 arms: sonothrombolysis pre-/post-pPCI (group 1), sham pre- sonothrombolysis post-pPCI (group 2), and sham pre-/post-pPCI (group 3). Our primary end point was infarct size (percentage of left ventricular mass) assessed by cardiac magnetic resonance imaging at day 4 ± 2. Secondary end points included myocardial salvage index (MSI) and echocardiographic parameters at day 4 ± 2 and 6 months. RESULTS: Our trial was ceased early due to the COVID pandemic. From 122 patients screened between September 2020 and June 2021, 51 patients (age 60, male 82%) were included postrandomization. Median sonothrombolysis took 5 minutes pre-pPCI and 15 minutes post-, without significant door-to-balloon delay. There was a trend toward reduction in median infarct size between group 1 (8% [interquartile range, 4,11]), group 2 (11% [7, 19]), or group 3 (15% [9, 22]). Similarly there was a trend toward improved MSI in group 1 (79% [64, 85]) compared to groups 2 (51% [45, 70]) and 3 (48% [37, 73]) No major adverse cardiac events occurred during hospitalization. CONCLUSIONS: Pre-pPCI sonothrombolysis may be key to improving MSI in STEMI. Multicenter trials and health economic analyses are required before clinical translation.