Lipid nanoparticle-mediated messenger RNA delivery for ex vivo engineering of natural killer cells

Natural killer (NK) cells participate in the immune system by eliminating cancer and virally infected cells through germline-encoded surface receptors. Their independence from prior activation as well as their significantly lower toxicity have placed them in the spotlight as an alternative to T cells for adoptive cell therapy (ACT). Engineering NK cells with mRNA has shown great potential in ACT by enhancing their tumor targeting and cytotoxicity. However, mRNA transfection of NK cells is challenging, as the most common delivery methods, such as electroporation, show limitations. Therefore, an alternative non-viral delivery system that enables high mRNA transfection efficiency with preservation of the cell viability would be beneficial for the development of NK cell therapies. In this study, we investigated both polymeric and lipid nanoparticle (LNP) formulations for eGFP-mRNA delivery to NK cells, based on a dimethylethanolamine and diethylethanolamine polymeric library and on different ionizable lipids, respectively. The mRNA nanoparticles based on cationic polymers showed limited internalization by NK cells and low transfection efficiency. On the other hand, mRNA-LNP formulations were optimized by tailoring the lipid composition and the microfluidic parameters, resulting in a high transfection efficiency (∼100%) and high protein expression in NK cells. In conclusion, compared to polyplexes and electroporation, the optimized LNPs show a greater transfection efficiency and higher overall eGFP expression, when tested in NK (KHYG-1) and T (Jurkat) cell lines, and cord blood-derived NK cells. Thus, LNP-based mRNA delivery represents a promising strategy to further develop novel NK cell therapies

The protofilament architecture of a de novo designed coiled coil-based amyloidogenic peptide

International audienceAmyloid fibrils are polymers formed by proteins under specific conditions and in many cases they are related to pathogenesis, such as Parkinson's and Alzheimer's diseases. Their hallmark is the presence of a β-sheet structure. High resolution structural data on these systems as well as information gathered from multiple complementary analytical techniques is needed, from both a fundamental and a pharmaceutical perspective. Here, a previously reported de novo designed, pH-switchable coiled coil-based peptide that undergoes structural transitions resulting in fibril formation under physiological conditions has been exhaustively characterized by transmission electron microscopy (TEM), cryo-TEM, atomic force microscopy (AFM), wide-angle X-ray scattering (WAXS) and solid-state NMR (ssNMR). Overall, a unique 2-dimensional carpet-like assembly composed of large coexisiting ribbon-like, tubular and funnel-like structures with a clearly resolved protofilament substructure is observed. Whereas electron microscopy and scattering data point somewhat more to a hairpin model of β-fibrils, ssNMR data obtained from samples with selectively labelled peptides are in agreement with both, hairpin structures and linear arrangements

Coulomb dissociation of O-16 into He-4 and C-12

We measured the Coulomb dissociation of O-16 into He-4 and C-12 within the FAIR Phase-0 program at GSI Helmholtzzentrum fur Schwerionenforschung Darmstadt, Germany. From this we will extract the photon dissociation cross section O-16(alpha,gamma)C-12, which is the time reversed reaction to C-12(alpha,gamma)O-16. With this indirect method, we aim to improve on the accuracy of the experimental data at lower energies than measured so far. The expected low cross section for the Coulomb dissociation reaction and close magnetic rigidity of beam and fragments demand a high precision measurement. Hence, new detector systems were built and radical changes to the (RB)-B-3 setup were necessary to cope with the high-intensity O-16 beam. All tracking detectors were designed to let the unreacted O-16 ions pass, while detecting the C-12 and He-4

Inhibition of amyloid Formation

1 EINLEITUNG ... 3 2 STRUKTUR AMYLOIDER FIBRILLEN ... 7 2.1 MAKROSKOPISCHE STRUKTUR DER AMYLOIDFIBRILLEN ... 7 2.2 INTERNE STRUKTUR AMYLOIDER PROTOFILAMENTE... 8 2.3 MOLEKULARE GRUNDLAGEN UND INTERNE STRUKTUR AMYLOIDER FIBRILLEN ... 10 2.3.1 Amyloidogene Kernsequenzen... 11 2.3.2 Allgemeines Sequenz-Muster amyloidogener Kernsequenzen... 13 2.3.3 Modell des sterischen Reißverschlusses („steric zipper“)... 14 2.3.4 Die Rolle aromatischer Seitenketten ... 15 3 MECHANISMEN DER BILDUNG AMYLOIDER FIBRILLEN... 17 3.1 MODELLE DER GESTEIGERTEN INTERAKTION („GAIN-OF-INTERACTION“) ... 18 4 KINETIK DER BILDUNG AMYLOIDER FIBRILLEN ... 21 5 AMYLOIDOGENE OLIGOMERE UND IHRE PATHOLOGISCHE BEDEUTUNG... 24 5.1 DIE BEDEUTUNG α−HELIKALER INTERMEDIATE ... 25 6 EINGRIFF IN DEN PROZESS DER BILDUNG AMYLOIDER AGGREGATE ... 28 6.1 INHIBITION DER BILDUNG AMYLOIDOGENER PROTEINE UND PEPTIDE... 30 6.2 FÖRDERUNG DER DEGRADATION AMYLOIDOGENER PROTEINE UND PEPTIDE ... 32 6.3 INHIBITION DER AGGREGATION AMYLOIDOGENER PROTEINE UND PEPTIDE ... 32 6.3.1 Strategie der Stabilisierung der α-helikalen Konformation ... 34 6.3.2 Nicht-peptidische Inhibitoren ... 36 6.3.2.1 Unspezifische Modulatoren der Aggregation ... 37 6.3.2.2 Natürliche Polyphenole ... 39 6.3.2.3 Kinetische Stabilisierung des nativen Zustands... 41 6.3.2.4 Rationales Design und Wirkung von Metall- Chelatoren ... 42 6.3.2.5 Stabilisierung der α-helikalen Konformation... 44 6.3.2.6 Mimetika der β-Faltblattstruktur... 46 6.3.2.7 Inhibition durch Nanopartikel... 47 6.3.2.8 Nicht-peptidische Verbindungen in klinischen Studien ... 47 6.3.3 Peptidbasierte Inhibitoren ... 50 6.3.3.1 Screening von inhibitorisch wirksamen Peptiden... 51 6.3.3.2 Rationales Design von peptidbasierten Inhibitoren... 53 6.3.3.2.1 Austausch von Aminosäuren ... 54 6.3.3.2.2 Modifikation der Termini... 57 6.3.3.2.3 Modifikation des Peptidrückgrats ... 59 6.3.3.2.4 Zyklisierung von Peptiden... 70 6.3.3.2.5 Multivalente Inhibitoren ... 73 6.3.3.2.6 Strukturbasiertes Design... 74 6.3.3.2.6 Stabilisierung der α-helikalen Konformation ... 75 6.3.3.2.7 Stabilisierung von β-Faltblatt-Strukturen im nicht amyloiden Zustand... 79 6.4 KONTROLLE DER PROTEINFALTUNG ... 82 6.4.1 Proteine und Moleküle mit Chaperone- Funktion... 83 6.4.2 Die BRICHOS-Domäne ... 84 6.5 IMMUNOTHERAPIE ... 84 6.5.1 Formen der Immunisierung... 85 6.5.2 Wirkungsmechanismen immunotherapeutischer Ansätze... 85 6.5.3 Generierung spezifischer anti-amyloidogener Antikörper... 86 6.5.4 Ergebnisse und Studien... 87 6.6 ZUSAMMENFASSUNG... 88 7 AMYLOIDE MODELLPEPTIDE... 91 7.1 FRAGMENTE AMYLOIDOGENER PEPTIDE UND PROTEINE ... 92 7.2 DE NOVO-DESIGN AMYLOIDOGENER MODELLPEPTIDE ... 92 7.2.1 Design struktureller Ambivalenz ... 93 7.2.2 Coiled Coil-basierte Switch-Peptide ... 95 7.3 AMYLOIDOGENE KERNSEQUENZEN IN KOMBINATION MIT DEM SWITCH-PEPTID-ANSATZ ... 97 8 ZIELE DER ARBEIT ... 99 9 DESIGN DER VERWENDETEN MODELLPEPTIDE... 100 9.1 MODELLPEPTIDE BASIEREND AUF DEM COILED COIL-FALTUNGS-MOTIV ... 100 9.2 MODELLPEPTIDE AUF BASIS EINER AMPHIPATHISCHEN HELIX ... 101 10 CHARAKTERISIERUNG DER MODELLPEPTIDE.... 103 10.1 COILED COIL-BASIERTES MODELLPEPTID VW01 ... 103 10.2 MODELLPEPTID VW01-RAN... 107 10.3 COILED COIL- BASIERTES MODELLPEPTID VW18... 109 10.4 COILED COIL-BASIERTES MODELLPEPTID VW19... 114 10.5 COILED COIL-BASIERTES MODELLPEPTID RR01... 117 10.5.1 Aufklärung der supramolekularen Struktur des Modellpeptids RR01... 120 10.6 MODELLPEPTIDE BASIEREND AUF EINER AMPHIPATHISCHEN HELIX... 131 10.7 NIAD-4 FLUORESZENZ BEI HELIKALEN FASERN UND AMYLOIDEN FIBRILLEN ... 135 11 INHIBITION DER BILDUNG AMYLOIDER AGGREGATE ... 143 11.1 MISCHUNGSSYSTEM VW18 UND VW01 ... 143 11.2 MISCHUNGSSYSTEM VW19 UND VW01 ... 150 11.3 MISCHUNGSSYSTEM RR01 UND VW01... 158 11.4 MISCHUNGSSYSTEM RR01 UND FF03... 161 11.5 MISCHUNGSSYSTEM α-AH UND β-AH-V ... 164 11.6 MISCHUNGSSYSTEM PSEUDOPEPTID PP1 MIT VW18 UND RR01... 166 11.7 EINFÜHRUNG DES AMYLOIDOGENEN MODELLPEPTIDS VW18 IN EINE ZELLULÄRE UMGEBUNG ... 170 12 DISKUSSION ... 176 12.1 DISKUSSION DER INHIBITION DURCH STABILISIERUNG DER α-HELIKALEN KONFORMATION ... 176 12.2 DISKUSSION DER INHIBITION DURCH PSEUDOPEPTID PP1 ... 187 13 ZUSAMMENFASSUNG UND AUSBLICK... 189 14 EXPERIMENTELLE BESCHREIBUNG... 193 14.1 PEPTID SYNTHESE, REINIGUNG UND CHARAKTERISIERUNG ... 193 14.1.1 Festphasenpeptidsynthese... 193 14.1.2 Reinigung und Charakterisierung... 195 14.1.3 Synthetisierte Peptide... 196 14.2 FALTUNGSSTUDIEN ... 197 14.2.1 Allgemeine Bedingungen... 197 14.2.2 Konzentrationsbestimmung ... 198 14.2.3 Bestimmung der Nettoladung eines Peptids ... 199 14.2.4 Probenpräparation ... 199 14.2.5 CD-Spektroskopie... 200 14.2.6 Fluoreszenz-Assays ... 202 14.2.6.1 Thioflavin T ... 202 14.2.6.2 NIAD-4... 204 14.2.7 Inhibitionsstudien mit Hilfe der CD-Spektroskopie und Fluoreszenz-Assays... 205 14.2.8 Größenausschlusschromatographie... 205 14.2.9 Analytische Ultrazentrifugation... 206 14.2.10 Limitierte Proteolyse... 208 14.2.11 Computer-Algorithmen zur Vorhersage des Faltungsverhaltens ... 209 14.2.11.1 AGADIR... 209 14.2.11.2 TANGO... 209 14.3 STRUKTURELLE CHARAKTERISIERUNG ... 210 14.3.1 Elektronenmikroskopie ... 210 14.3.2 Röntgenbeugung... 211 14.3.3 Festphasen-Kernresonanzspektroskopie (ssNMR)... 213 15 ABKÜRZUNGSVERZEICHNIS... 215 16 LITERATUR ... 217Die Aggregation einer ganzen Reihe von verschiedenen Proteinen steht in einem engen Zusammenhang mit verschiedenen Krankheiten, wie z.B. Alzheimer. Obwohl die dafür verantwortlichen Proteine sich in ihrer Ausgangsstruktur und ihrer Sequenz unterscheiden, zeigen sie alle die strukturelle Veränderung zu β -Faltblatt-reichen fibrillären Aggregaten mit der charakteristischen cross-β-Struktur. Der zugrunde liegende Mechanismus verläuft über mehrere Stufen, indem sich instabile präfibrilläre Oligomere über fibrilläre Vorstufen schließlich zu amyloiden Fibrillen zusammenlagern. Die mechanistischen Details sind nur wenig verstanden und viele verschiedene Strategien zur Entwicklung von Inhibitoren gegen die amyloide Aggregation sind bisher verfolgt worden. In den meisten Fällen binden die chemisch diversen Verbindungen an bereits aggregierte Formen des amyloidogenen Proteins in der β-Faltblattkonformation. Das Screening von Substanzbibliotheken und das rationale Design führt in den meisten Fällen zu Inhibitoren die an die aggregierte Spezies binden und die weiter Polymerisation durch die Blockierung der Addition weiterer Monomere unterbinden. Zahlreiche Studien in der Entwicklung peptidbasierter Inhibitoren gehen von amyloiden Kernsequenzen der jeweiligen Proteine aus, da sie dafür bekannt sind, an sich selbst zu binden. Peptidbasierte Inhibitoren binden meist an das amyloidogene Peptidrückgrat in β-Faltblattkonformation. Einmal gebunden, blockieren diese Inhibitoren das Fibrillenwachstum durch sterische Effekte, elektrostatische Abstoßung oder durch das Unterbinden der Ausbildung von Wasserstoffbrückenbindungen. Doch diese Strategie kann möglicherweise die Menge an präfibrillären Oligomeren erhöhen, welche als wesentliche toxische Spezies des Amyloidbildungsprozesses diskutiert werden. Daher sind alternative Ansätze notwendig. Der Übergang vom löslichen Monomer in β-Faltblatt-reiche Spezies ist hierbei der entscheidende Schritt. Einige natürliche amyloidogene Peptidsequenzen bergen ebenfalls Elemente einer α-Helix oder bilden während des Aggregationsprozesses helikale Intermediate. Die Stabilisierung der helikalen Konformation zur Vermeidung des entscheidenden Schritts ist daher ein viel versprechender Ansatz in der Inhibition. Im Rahmen dieser Arbeit werden Studien beschrieben, die das Potential dieser Strategie anhand von Modellpeptiden demonstrieren. Der Ansatz beruht auf helikalen peptidischen Inhibitoren, die designt worden sind, die amyloidogenen Modellpeptide in einem helikalen Komplex zu stabilisieren und auf diese Weise den Übergang zur β-Faltblattkonformation und daraus folgend die Bildung von amyloiden Fibrillen zu inhibieren. Darüber hinaus konnte gezeigt werden, dass helikale peptidische Inhibitoren in der Lage sind, bereits gebildet amyloide Fibrillen wieder aufzulösen.The formation of amyloid aggregates is responsible for a wide range of diseases, for example Alzheimer’s disease. Although the amyloid forming proteins have natively different structures and different sequences, all undergo a structural change to form amyloid aggregates that have a characteristic cross-β-structure. The mechanism is a multistep process in which unstable prefibrillar oligomers assemble via fibrillar intermediates into amyloid fibrils. The mechanistic details of this process are poorly understood and different strategies to develop inhibitors of amyloid formation are followed to date. In most cases, chemically diverse compounds bind to an elongated form of the protein in a β-strand conformation and thereby exert their therapeutic effect. The screening of libraries of small molecules and the rational design leads in most cases to inhibitors that bind to aggregated species and prevent further polymerization by blocking the addition of peptide monomers. Numerous studies in the development of peptide based amyloid aggregation inhibitors have taken the core sequence of the target peptide as a lead as it is already known to bind to itself. The peptide based inhibitors were designed to bind to the peptide backbone in β-sheet conformation. They block further elongation by steric effects, electrostatic repulsion, or inhibition of the formation of hydrogen- bonds. However, blocking fibril formation with this strategy could increase the amount of prefibrillar oligomeric forms, which are thought to be the toxic species in the amyloid formation process. Therefore, alternative approaches are necessary. The conversion from the soluble monomeric form into β-sheet rich aggregated morphologies appears to be the key event. Several naturally occurring amyloidogenic peptides that form β- sheet rich fibrils harbor also an α-helix in the primary structure or appear to form helical intermediates during amyloid formation under certain conditions. Therefore, one promising approach is the stabilization of the helical conformation to prevent the key-event, the conversion to β-sheet and the formation amyloids. This thesis presents the investigation of the potential of this approach using model peptides. The approach consists of a helical inhibitor peptides which are designed to engage amyloid forming model peptides in a stable helical arrangement, thus to prevent rearrangement into a β-sheet conformation and the subsequent formation of amyloid-like fibrils. Moreover, the helix forming peptide is able to disassemble mature amyloid-like fibrils

Vibron band structure in chlorinated benzene crystals; lattice dynamics calculations and Raman spectra of 1,4-dichlorobenzene

Commercially available Off The Shelf (COTS) multicores have been assessed as the baseline computing platform even in the most conservative real-time domains. Multicore contention arising on shared hardware resources, with its circular dependence with scheduling, is among the most challenging issues that require urgent attention before multicores can be fully embraced for real-time computing. In the context of static scheduling, still the most used scheduling approach in real-time industries, we propose an ILP formulation for computing the worst-case contention delay suffered by a task due to interference on a shared bus. Our model provides accurate contention delay bounds that avoid unnecessary over-accounting of conflicts between bus requests, by considering contention effects at system-level (i.e., across tasks) rather than at task-level only. This allows precisely capturing the interdependence between timing interference of conflicting requests, issued in parallel by other cores (tasks), and the identification of the particular set of tasks co-running on those cores. We assess our technique both analytically and empirically on a real COTS multicore platform. We show, via extensive evaluation, that jointly accounting for worst-case task overlapping and request distribution scenarios always provides tighter contention bounds when compared to state-of-the-art solutions

Formation of α‑Helical Nanofibers by Mixing β‑Structured and α‑Helical Coiled Coil Peptides

The helical coiled coil is a well-studied folding motif that can be used for the design of nanometer-sized bioinspired fibrous structures with potential applications as functional materials. A two-component system of coiled coil based model peptides is investigated, which forms, under acidic conditions, uniform, hundreds of nanometers long, and ∼2.6 nm thick trimeric α-helical fibers. In the absence of the other component and under the same solvent conditions, one model peptide forms β-sheet-rich amyloid fibrils and the other forms stable trimeric α-helical coiled coils, respectively. These observations reveal that the complementary interactions driving helical folding are much stronger here than those promoting the intermolecular β-sheet formation. The results of this study are important in the context of amyloid inhibition but also open up new avenues for the design of novel fibrous peptidic materials

A Self-Assembling Peptide Scaffold for the Multivalent Presentation of Antigens

Self-assembling peptides can be used to create tunable higher-order structures for the multivalent presentation of a variety of ligands. We describe a novel, fiber-forming coiled-coil-based peptide that assembles to display, simultaneously, carbohydrate and peptide ligands recognized by biomacromolecules. Preassembly decoration of the scaffold with a diphtheria toxin peptide epitope or a mannose motif did not interfere with self-assembly of the nanostructure. The resulting multivalent display led to tighter binding by antidiphtheria toxin antibodies and mannose-specific carbohydrate binding proteins, respectively. The potential of this self-assembling peptide to display ligands in bioanalytical assays is illustrated by its decoration with a disaccharide glycotope from the Leishmania parasite. Carbohydrate-specific antibodies produced in response to a Leishmania infection are detected more sensitively in human and canine sera due to the multivalent presentation on the self-assembled scaffold. Thus, nanofibers based on coiled-coil peptides are a powerful tool for the development of bioassays and diagnostics