Janina Buck

Systemic RNA delivery to dendritic cells exploits antiviral defence for cancer immunotherapy

Lymphoid organs, in which antigen presenting cells (APCs) are in close proximity to T cells, are the ideal microenvironment for efficient priming and amplification of T-cell responses. However, the systemic delivery of vaccine antigens into dendritic cells (DCs) is hampered by various technical challenges. Here we show that DCs can be targeted precisely and effectively in vivo using intravenously administered RNA-lipoplexes (RNA-LPX) based on well-known lipid carriers by optimally adjusting net charge, without the need for functionalization of particles with molecular ligands. The LPX protects RNA from extracellular ribonucleases and mediates its efficient uptake and expression of the encoded antigen by DC populations and macrophages in various lymphoid compartments. RNA-LPX triggers interferon-α (IFNα) release by plasmacytoid DCs and macrophages. Consequently, DC maturation in situ and inflammatory immune mechanisms reminiscent of those in the early systemic phase of viral infection are activated. We show that RNA-LPX encoding viral or mutant neo-antigens or endogenous self-antigens induce strong effector and memory T-cell responses, and mediate potent IFNα-dependent rejection of progressive tumours. A phase I dose-escalation trial testing RNA-LPX that encode shared tumour antigens is ongoing. In the first three melanoma patients treated at a low-dose level, IFNα and strong antigen-specific T-cell responses were induced, supporting the identified mode of action and potency. As any polypeptide-based antigen can be encoded as RNA, RNA-LPX represent a universally applicable vaccine class for systemic DC targeting and synchronized induction of both highly potent adaptive as well as type-I-IFN-mediated innate immune mechanisms for cancer immunotherapy.

Personalized RNA mutanome vaccines mobilize poly-specific therapeutic immunity against cancer

T cells directed against mutant neo-epitopes drive cancer immunity. However, spontaneous immune recognition of mutations is inefficient. We recently introduced the concept of individualized mutanome vaccines and implemented an RNA-based poly-neo-epitope approach to mobilize immunity against a spectrum of cancer mutations. Here we report the first-in-human application of this concept in melanoma. We set up a process comprising comprehensive identification of individual mutations, computational prediction of neo-epitopes, and design and manufacturing of a vaccine unique for each patient. All patients developed T cell responses against multiple vaccine neo-epitopes at up to high single-digit percentages. Vaccine-induced T cell infiltration and neo-epitope-specific killing of autologous tumour cells were shown in post-vaccination resected metastases from two patients. The cumulative rate of metastatic events was highly significantly reduced after the start of vaccination, resulting in a sustained progression-free survival. Two of the five patients with metastatic disease experienced vaccine-related objective responses. One of these patients had a late relapse owing to outgrowth of β2-microglobulin-deficient melanoma cells as an acquired resistance mechanism. A third patient developed a complete response to vaccination in combination with PD-1 blockade therapy. Our study demonstrates that individual mutations can be exploited, thereby opening a path to personalized immunotherapy for patients with cancer.

Interplay of ‘induced fit’ and preorganization in the ligand induced folding of the aptamer domain of the guanine binding riboswitch

Riboswitches are highly structured elements in the 5′-untranslated regions (5′-UTRs) of messenger RNA that control gene expression by specifically binding to small metabolite molecules. They consist of an aptamer domain responsible for ligand binding and an expression platform. Ligand binding in the aptamer domain leads to conformational changes in the expression platform that result in transcription termination or abolish ribosome binding. The guanine riboswitch binds with high-specificity to guanine and hypoxanthine and is among the smallest riboswitches described so far. The X-ray-structure of its aptamer domain in complex with guanine/hypoxanthine reveals an intricate RNA-fold consisting of a three-helix junction stabilized by long-range base pairing interactions. We analyzed the conformational transitions of the aptamer domain induced by binding of hypoxanthine using high-resolution NMR-spectroscopy in solution. We found that the long-range base pairing interactions are already present in the free RNA and preorganize its global fold. The ligand binding core region is lacking hydrogen bonding interactions and therefore likely to be unstructured in the absence of ligand. Mg2+-ions are not essential for ligand binding and do not change the structure of the RNA-ligand complex but stabilize the structure at elevated temperatures. We identified a mutant RNA where the long-range base pairing interactions are disrupted in the free form of the RNA but form upon ligand binding in an Mg2+-dependent fashion. The tertiary interaction motif is stable outside the riboswitch context.

Time-resolved NMR methods resolving ligand-induced RNA folding at atomic resolution

Structural transitions of RNA between alternate conformations with similar stabilities are associated with important aspects of cellular function. Few techniques presently exist that are capable of monitoring such transitions and thereby provide insight into RNA dynamics and function at atomic resolution. Riboswitches are found in the 5′-UTR of mRNA and control gene expression through structural transitions after ligand recognition. A time-resolved NMR strategy was established in conjunction with laser-triggered release of the ligand from a photocaged derivative in situ to monitor the hypoxanthine-induced folding of the guanine-sensing riboswitch aptamer domain of the Bacillus subtilis xpt-pbuX operon at atomic resolution. Combining selective isotope labeling of the RNA with NMR filter techniques resulted in significant spectral resolution and allowed kinetic analysis of the buildup rates for individual nucleotides in real time. Three distinct kinetic steps associated with the ligand-induced folding were delineated. After initial complex encounter the ligand-binding pocket is formed and results in subsequent stabilization of a remote long-range loop–loop interaction. Incorporation of NMR data into experimentally restrained molecular dynamics simulations provided insight into the RNA structural ensembles involved during the conformational transition.

Evaluation of Parameters Critical for Observing Nucleic Acids Inside Living Xenopus laevis Oocytes by In-Cell NMR Spectroscopy

In-cell NMR spectroscopy of proteins in different cellular environments is a well-established technique that, however, has not been applied to nucleic acids so far. Here, we show that isotopically labeled DNA and RNA can be observed inside the eukaryotic environment of Xenopus laevis oocytes by in-cell NMR spectroscopy. One limiting factor for the observation of nucleic acids in Xenopus oocytes is their reduced stability. We demonstrate that chemical modification of DNA and RNA can protect them from degradation and can significantly enhance their lifetime. Finally, we show that the imino region of the NMR spectrum is devoid of any oocyte background signals enabling the detection even of isotopically nonlabeled molecules.

Mapping the Landscape of RNA Dynamics with NMR Spectroscopy

Among the three major classes of biomacromolecules (DNA, RNA, and proteins) RNAs pronounced dynamics are the most explicitly linked to its wide variety of functions, which include catalysis and the regulation of transcription, translation, and splicing. These functions are mediated by a range of RNA biomachinery, including such varied examples as macromolecular noncoding RNAs, microRNAs, small interfering RNAs, riboswitch RNAs, and RNA thermometers. In each case, the functional dynamics of an interconversion is characterized by an associated rate constant. In this Account, we provide an introduction to NMR spectroscopic characterization of the landscape of RNA dynamics. We introduce strategies for measuring NMR parameters at various time scales as well as the underlying models for describing the corresponding rate constants. RNA exhibits significant dynamic motion, which can be modulated by (i) intermolecular interactions, including specific and nonspecific binding of ions (such as Mg(2+) and tertiary amines), (ii) metabolites in riboswitches or RNA aptamers, and (iii) macromolecular interactions within ribonucleic protein particles, including the ribosome and the spliceosome. Our understanding of the nature of these dynamic changes in RNA targets is now being incorporated into RNA-specific approaches in the design of RNA inhibitors. Interactions of RNA with proteins, other RNAs, or small molecules often occur through binding mechanisms that follow an induced fit mechanism or a conformational selection mechanism, in which one of several populated RNA conformations is selected through ligand binding. The extent of functional dynamics, including the kinetic formation of a specific RNA tertiary fold, is dependent on the messenger RNA (mRNA) chain length. Thus, during de novo synthesis of mRNA, both in prokaryotes and eukaryotes, nascent mRNA of various lengths will adopt different secondary and tertiary structures. The speed of transcription has a critical influence on the functional dynamics of the RNA being synthesized. In addition to modulating the local dynamics of a conformational RNA ensemble, a given RNA sequence may adopt more than one global, three-dimensional structure. RNA modification is one way to select among these alternative structures, which are often characterized by nearly equal stability, but with high energy barriers for conformational interconversion. The refolding of different secondary and tertiary structures has been found to be a major regulatory mechanism for transcription and translation. These conformational transitions can be characterized with NMR spectroscopy, for any given RNA sequence, in response to external stimuli.

Dissecting the influence of Mg2+ on 3D architecture and ligand-binding of the guanine-sensing riboswitch aptamer domain

Long-range tertiary interactions determine the three-dimensional structure of a number of metabolite-binding riboswitch RNA elements and were found to be important for their regulatory function. For the guanine-sensing riboswitch of the Bacillus subtilis xpt-pbuX operon, our previous NMR-spectroscopic studies indicated pre-formation of long-range tertiary contacts in the ligand-free state of its aptamer domain. Loss of the structural pre-organization in a mutant of this RNA (G37A/C61U) resulted in the requirement of Mg2+ for ligand binding. Here, we investigate structural and stability aspects of the wild-type aptamer domain (Gsw) and the G37A/C61U-mutant (Gswloop) of the guanine-sensing riboswitch and their Mg2+-induced folding characteristics to dissect the role of long-range tertiary interactions, the link between pre-formation of structural elements and ligand-binding properties and the functional stability. Destabilization of the long-range interactions as a result of the introduced mutations for Gswloop or the increase in temperature for both Gsw and Gswloop involves pronounced alterations of the conformational ensemble characteristics of the ligand-free state of the riboswitch. The increased flexibility of the conformational ensemble can, however, be compensated by Mg2+. We propose that reduction of conformational dynamics in remote regions of the riboswitch aptamer domain is the minimal pre-requisite to pre-organize the core region for specific ligand binding.

Synthesis, properties, and biological activity of boranophosphate analogs of the mRNA cap: versatile tools for manipulation of therapeutically relevant cap-dependent processes

Modified mRNA cap analogs aid in the study of mRNA-related processes and may enable creation of novel therapeutic interventions. We report the synthesis and properties of 11 dinucleotide cap analogs bearing a single boranophosphate modification at either the α-, β- or γ-position of the 5′,5′-triphosphate chain. The compounds can potentially serve either as inhibitors of translation in cancer cells or reagents for increasing expression of therapeutic proteins in vivo from exogenous mRNAs. The BH3-analogs were tested as substrates and binding partners for two major cytoplasmic cap-binding proteins, DcpS, a decapping pyrophosphatase, and eIF4E, a translation initiation factor. The susceptibility to DcpS was different between BH3-analogs and the corresponding analogs containing S instead of BH3 (S-analogs). Depending on its placement, the boranophosphate group weakened the interaction with DcpS but stabilized the interaction with eIF4E. The first of the properties makes the BH3-analogs more stable and the second, more potent as inhibitors of protein biosynthesis. Protein expression in dendritic cells was 2.2- and 1.7-fold higher for mRNAs capped with m27,2′-OGppBH3pG D1 and m27,2′-OGppBH3pG D2, respectively, than for in vitro transcribed mRNA capped with m27,3′-OGpppG. Higher expression of cancer antigens would make mRNAs containing m27,2′-OGppBH3pG D1 and m27,2′-OGppBH3pG D2 favorable for anticancer immunization.

Structure and dynamics of the deoxyguanosine-sensing riboswitch studied by NMR-spectroscopy

The mfl-riboswitch regulates expression of ribonucleotide reductase subunit in Mesoplasma florum by binding to 2′-deoxyguanosine and thereby promoting transcription termination. We characterized the structure of the ligand-bound aptamer domain by NMR spectroscopy and compared the mfl-aptamer to the aptamer domain of the closely related purine-sensing riboswitches. We show that the mfl-aptamer accommodates the extra 2′-deoxyribose unit of the ligand by forming a more relaxed binding pocket than these found in the purine-sensing riboswitches. Tertiary structures of the xpt-aptamer bound to guanine and of the mfl-aptamer bound to 2′-deoxyguanosine exhibit very similar features, although the sequence of the mfl-aptamer contains several alterations compared to the purine-aptamer consensus sequence. These alterations include the truncation of a hairpin loop which is crucial for complex formation in all purine-sensing riboswitches characterized to date. We further defined structural features and ligand binding requirements of the free mfl-aptamer and found that the presence of Mg2+ is not essential for complex formation, but facilitates ligand binding by promoting pre-organization of key structural motifs in the free aptamer.

13C-direct detected NMR experiments for the sequential J-based resonance assignment of RNA oligonucleotides

We present here a set of 13C-direct detected NMR experiments to facilitate the resonance assignment of RNA oligonucleotides. Three experiments have been developed: (1) the (H)CC-TOCSY-experiment utilizing a virtual decoupling scheme to assign the intraresidual ribose 13C-spins, (2) the (H)CPC-experiment that correlates each phosphorus with the C4′ nuclei of adjacent nucleotides via J(C,P) couplings and (3) the (H)CPC-CCH-TOCSY-experiment that correlates the phosphorus nuclei with the respective C1′,H1′ ribose signals. The experiments were applied to two RNA hairpin structures. The current set of 13C-direct detected experiments allows direct and unambiguous assignment of the majority of the hetero nuclei and the identification of the individual ribose moieties following their sequential assignment. Thus, 13C-direct detected NMR methods constitute useful complements to the conventional 1H-detected approach for the resonance assignment of oligonucleotides that is often hindered by the limited chemical shift dispersion. The developed methods can also be applied to large deuterated RNAs.