Properties of Nucleosyl Amino Acid-Modified Oligonucleotides

Antisense technology has the potential to evolve into a powerful biomedical tool. By utilizing the unique hybridization behavior of antisense oligonucleotides (ON) the antisense approach facilitates a specific intervention in cellular processes that would be barely feasible by using conventional drugs. However, the tremendous potential that comes with the antisense strategy is paired with the insufficient pharmacokinetic profile of native DNA ONs. Thus, an overall low stability in vivo as well as limited cellular uptake due to the extremely polar DNA structure, represent two of the main obstacles. Consequently, alterations of the native DNA structure are required to obtain applicable, drug-like substances. The reported work focuses on the synthesis and evaluation of modified ONs carrying the nucleosyl amino acid (NAA)-modification, a peptide-like internucleoside structure featuring a free amino group, within their backbone. Part A of this thesis concentrates on the biological stability of partially zwitterionic NAA-modified ONs in the presence of 3'→5'- and 5'→3'-exonucleases as well as human plasma and whole cell lysate. In part B, the hybridization properties of cationic, fully NAA-modified ONs in presence of matched and mismatched counterstrands were analyzed and validated. Finally, part C focuses on the synthesis and evaluation of a chimeric NAA/LNA-gapmer structure with the aim to obtain a selective, high-affinity precursor of an antisense ON.Die Antisense-Technologie hat das Potenzial, sich zu einem leistungsfähigen biomedizinischen Werkzeug zu entwickeln. Durch die Nutzung des einzigartigen Hybridisierungsverhaltens von Antisense-Oligonucleotiden (ON) ermöglicht sie einen gezielten Eingriff in zelluläre Prozesse, der mit herkömmlichen Methoden kaum durchführbar wäre. Das enorme Potenzial, das mit dem Antisense-Ansatz einhergeht, ist jedoch an das unzureichende pharmakokinetische Profil nativer DNA-ON geknüpft. Hierbei stellen eine insgesamt geringe in-vivo-Stabilität sowie eine erschwerte zelluläre Aufnahme aufgrund der polaren DNA-Struktur zwei der Hauptprobleme dar. Folglich sind Veränderungen der nativen DNA-Struktur erforderlich, um applizierbare Substanzen zu erhalten. Daher konzentriert sich die folgende Arbeit auf die Synthese und Evaluierung modifizierter ON, die in ihrem Rückgrat die Nucleosylaminosäure-(NAA)-Modifikation, eine peptidähnliche Internucleosid-Struktur mit einer freien Aminogruppe, tragen. Teil A dieser Arbeit befasst sich mit der biologischen Stabilität von teilweise zwitterionischen NAA-modifizierten ON in Gegenwart von 3'→5'- und 5'→3'-Exonukleasen sowie humanem Plasma und Zelllysat. In Teil B wurden die Hybridisierungseigenschaften von kationischen, vollständig NAA-modifizierten ON analysiert und validiert. Zuletzt befasst sich Teil C mit der Synthese und Untersuchung einer chimären NAA/LNA-Gapmer-Struktur mit dem Ziel, eine selektive, hochaffine Vorstufe eines Antisense-ON zu erhalten

Towards Zwitterionic Oligonucleotides with Improved Properties: the NAA/LNA-Gapmer Approach

Oligonucleotides (ON) are promising therapeutic candidates, for instance by blocking endogenous mRNA (antisense mechanism). However, ON usually require structural modifications of the native nucleic acid backbone to ensure satisfying pharmacokinetic properties. One such strategy to design novel antisense oligonucleotides is to replace native phosphate diester units by positively charged artificial linkages, thus leading to (partially) zwitterionic backbone structures. Herein, we report a “gapmer” architecture comprised of one zwitterionic central segment (“gap”) containing nucleosyl amino acid (NAA) modifications and two outer segments of locked nucleic acid (LNA). This NAA/LNA‐gapmer approach furnished a partially zwitterionic ON with optimised properties: i) the formation of stable ON‐RNA duplexes with base‐pairing fidelity and superior target selectivity at 37 °C; and ii) excellent stability in complex biological media. Overall, the NAA/LNA‐gapmer approach is thus established as a strategy to design partially zwitterionic ON for the future development of novel antisense agents

Global burden of 369 diseases and injuries in 204 countries and territories, 1990–2019: a systematic analysis for the Global Burden of Disease Study 2019

Background: In an era of shifting global agendas and expanded emphasis on non-communicable diseases and injuries along with communicable diseases, sound evidence on trends by cause at the national level is essential. The Global Burden of Diseases, Injuries, and Risk Factors Study (GBD) provides a systematic scientific assessment of published, publicly available, and contributed data on incidence, prevalence, and mortality for a mutually exclusive and collectively exhaustive list of diseases and injuries. Methods: GBD estimates incidence, prevalence, mortality, years of life lost (YLLs), years lived with disability (YLDs), and disability-adjusted life-years (DALYs) due to 369 diseases and injuries, for two sexes, and for 204 countries and territories. Input data were extracted from censuses, household surveys, civil registration and vital statistics, disease registries, health service use, air pollution monitors, satellite imaging, disease notifications, and other sources. Cause-specific death rates and cause fractions were calculated using the Cause of Death Ensemble model and spatiotemporal Gaussian process regression. Cause-specific deaths were adjusted to match the total all-cause deaths calculated as part of the GBD population, fertility, and mortality estimates. Deaths were multiplied by standard life expectancy at each age to calculate YLLs. A Bayesian meta-regression modelling tool, DisMod-MR 2.1, was used to ensure consistency between incidence, prevalence, remission, excess mortality, and cause-specific mortality for most causes. Prevalence estimates were multiplied by disability weights for mutually exclusive sequelae of diseases and injuries to calculate YLDs. We considered results in the context of the Socio-demographic Index (SDI), a composite indicator of income per capita, years of schooling, and fertility rate in females younger than 25 years. Uncertainty intervals (UIs) were generated for every metric using the 25th and 975th ordered 1000 draw values of the posterior distribution. Findings: Global health has steadily improved over the past 30 years as measured by age-standardised DALY rates. After taking into account population growth and ageing, the absolute number of DALYs has remained stable. Since 2010, the pace of decline in global age-standardised DALY rates has accelerated in age groups younger than 50 years compared with the 1990–2010 time period, with the greatest annualised rate of decline occurring in the 0–9-year age group. Six infectious diseases were among the top ten causes of DALYs in children younger than 10 years in 2019: lower respiratory infections (ranked second), diarrhoeal diseases (third), malaria (fifth), meningitis (sixth), whooping cough (ninth), and sexually transmitted infections (which, in this age group, is fully accounted for by congenital syphilis; ranked tenth). In adolescents aged 10–24 years, three injury causes were among the top causes of DALYs: road injuries (ranked first), self-harm (third), and interpersonal violence (fifth). Five of the causes that were in the top ten for ages 10–24 years were also in the top ten in the 25–49-year age group: road injuries (ranked first), HIV/AIDS (second), low back pain (fourth), headache disorders (fifth), and depressive disorders (sixth). In 2019, ischaemic heart disease and stroke were the top-ranked causes of DALYs in both the 50–74-year and 75-years-and-older age groups. Since 1990, there has been a marked shift towards a greater proportion of burden due to YLDs from non-communicable diseases and injuries. In 2019, there were 11 countries where non-communicable disease and injury YLDs constituted more than half of all disease burden. Decreases in age-standardised DALY rates have accelerated over the past decade in countries at the lower end of the SDI range, while improvements have started to stagnate or even reverse in countries with higher SDI. Interpretation: As disability becomes an increasingly large component of disease burden and a larger component of health expenditure, greater research and developm nt investment is needed to identify new, more effective intervention strategies. With a rapidly ageing global population, the demands on health services to deal with disabling outcomes, which increase with age, will require policy makers to anticipate these changes. The mix of universal and more geographically specific influences on health reinforces the need for regular reporting on population health in detail and by underlying cause to help decision makers to identify success stories of disease control to emulate, as well as opportunities to improve. Funding: Bill & Melinda Gates Foundation. © 2020 The Author(s). Published by Elsevier Ltd. This is an Open Access article under the CC BY 4.0 licens

Towards Zwitterionic Oligonucleotides with Improved Properties: the NAA/LNA‐Gapmer Approach

Oligonucleotides (ON) are promising therapeutic candidates, for instance by blocking endogenous mRNA (antisense mechanism). However, ON usually require structural modifications of the native nucleic acid backbone to ensure satisfying pharmacokinetic properties. One such strategy to design novel antisense oligonucleotides is to replace native phosphate diester units by positively charged artificial linkages, thus leading to (partially) zwitterionic backbone structures. Herein, we report a “gapmer” architecture comprised of one zwitterionic central segment (“gap”) containing nucleosyl amino acid (NAA) modifications and two outer segments of locked nucleic acid (LNA). This NAA/LNA‐gapmer approach furnished a partially zwitterionic ON with optimised properties: i) the formation of stable ON‐RNA duplexes with base‐pairing fidelity and superior target selectivity at 37 °C; and ii) excellent stability in complex biological media. Overall, the NAA/LNA‐gapmer approach is thus established as a strategy to design partially zwitterionic ON for the future development of novel antisense agents

Clinical Data Combined With Modeling and Simulation Indicate Unchanged Drug-Drug Interaction Magnitudes in the Elderly

Age-related comorbidities and consequently polypharmacy are highly prevalent in the elderly, resulting in an increased risk for drug-drug interactions (DDIs). The effect of aging on DDI magnitudes is mostly uncertain, leading to missing guidance regarding the clinical DDI management in the elderly. Clinical data obtained in aging people living with HIV ≥ 55 years, who participated in the Swiss HIV Cohort Study, demonstrated unchanged DDI magnitudes with advanced aging for four studied DDI scenarios. These data plus published data for midazolam in the presence of clarithromycin and rifampicin in elderly individuals assessed the predictive potential of the used physiologically-based pharmacokinetic (PBPK) model to simulate DDIs in the elderly. All clinically observed data were generally predicted within the 95% confidence interval of the PBPK simulations. The verified model predicted subsequently the magnitude of 50 DDIs across adulthood (20-99 years) with 42 scenarios being only verified in adults aged 20-50 years in the absence of clinically observed data in the elderly. DDI magnitudes were not impacted by aging regardless of the involved drugs, DDI mechanism, mediators of DDIs, or the sex of the investigated individuals. The prediction of unchanged DDI magnitudes with advanced aging were proofed by 17 published, independent DDIs that were investigated in young and elderly subjects. In conclusion, this study demonstrated by combining clinically observed data with modeling and simulation that aging does not impact DDI magnitudes and thus, clinical management of DDIs can a priori be similar in aging men and women in the absence of severe comorbidities