A Study of Musical and Extra-Musical Imagery in Rachmaninoff\u27s Etudes-Tableaux , Opus 39.

The nine Etudes-Tableaux, op. 39 (1916-17) for piano solo are the last important works written by Sergei Rachmaninoff (1873-1943) before his exile from Russia in 1917. The use of the word Tableaux in the title suggests an association with pictures, paintings, or scenes. Although the composer often wrote under the external influence of extra-musical sources, he rarely revealed them. This study examines the musical and extra-musical imagery which influenced Rachmaninoff\u27s compositional style as observed in op. 39, the most important elements being the Dies irae from the Roman Catholic Mass for the Dead, Russian chant, bell sonorities, and paintings by the Swiss artist Arnold Bocklin (1827-1901). In addition, Rachmaninoff\u27s love of nature and of his homeland is reflected throughout the study, and an explanation is given for the dark, somber sentiment that permeates many of the etudes

mRNA-LNP vaccines tuned for systemic immunization induce strong antitumor immunity by engaging splenic immune cells

mRNA vaccines have recently proved to be highly effective against SARS-CoV-2. Key to their success is the lipid-based nanoparticle (LNP), which enables efficient mRNA expression and endows the vaccine with adjuvant properties that drive potent antibody responses. Effective cancer vaccines require long-lived, qualitative CD8 T cell responses instead of antibody responses. Systemic vaccination appears to be the most effective route, but necessitates adaptation of LNP composition to deliver mRNA to antigen-presenting cells. Using a design-of-experiments methodology, we tailored mRNA-LNP compositions to achieve high-magnitude tumor-specific CD8 T cell responses within a single round of optimization. Optimized LNP compositions resulted in enhanced mRNA uptake by multiple splenic immune cell populations. Type I interferon and phagocytes were found to be essential for the T cell response. Surprisingly, we also discovered a yet unidentified role of B cells in stimulating the vaccine-elicited CD8 T cell response. Optimized LNPs displayed a similar, spleen-centered biodistribution profile in non-human primates and did not trigger histopathological changes in liver and spleen, warranting their further assessment in clinical studies. Taken together, our study clarifies the relationship between nanoparticle composition and their T cell stimulatory capacity and provides novel insights into the underlying mechanisms of effective mRNA-LNP-based antitumor immunotherapy

Recombinant phosphatidylserine-binding nanobodies for targeting of extracellular vesicles to tumor cells : a plug-and-play approach

Extracellular vesicles (EVs) are increasingly being recognized as candidate drug delivery systems due to their ability to functionally transfer biological cargo between cells. However, manipulation of targeting properties of EVs through engineering of the producer cells can be challenging and time-consuming. As a novel approach to confer tumor targeting properties to isolated EVs, we generated recombinant fusion proteins of nanobodies against the epidermal growth factor receptor (EGFR) fused to phosphatidylserine (PS)-binding domains of lactadherin (C1C2). C1C2-nanobody fusion proteins were expressed in HEK293 cells and isolated from culture medium with near-complete purity as determined by SDS-PAGE. Fusion proteins specifically bound PS and showed no affinity for other common EV membrane lipids. Furthermore, C1C2 fused to anti-EGFR nanobodies (EGa1-C1C2) bound EGFR with high affinity and competed with binding of its natural ligand EGF, as opposed to C1C2 fused to non-targeting control nanobodies (R2-C1C2). Both proteins readily self-associated onto membranes of EVs derived from erythrocytes and Neuro2A cells without affecting EV size and integrity. EV-bound R2-C1C2 did not influence EV-cell interactions, whereas EV-bound EGa1-C1C2 dose-dependently enhanced specific binding and uptake of EVs by EGFR-overexpressing tumor cells. In conclusion, we developed a novel strategy to efficiently and universally confer tumor targeting properties to PS-exposing EVs after their isolation, without affecting EV characteristics, circumventing the need to modify EV-secreting cells. This strategy may also be employed to decorate EVs with other moieties, including imaging probes or therapeutic proteins

Tuning LNPs to target antigen presenting cells in spleen induces CD8 T-cell responses and tumor regression in mice

International audienceIntravenous (i‧v.) delivery of vaccines is emerging as an appealing approach to evoke high-quality T-cell responses needed to combat tumors. Messenger RNA (mRNA) holds huge potential to use in vaccination because of its ease of production, intrinsic adjuvant activity and the high versatility in antigen design, but also requires a potent delivery vehicle to enable antigen expression and immune activation. Lipid based nanoparticles (LNP) are currently the clinically most advanced tool to protect mRNA from degradation and efficiently deliver it inside cells. Yet, i‧v administered LNPs generally tend to distribute to the liver. We hypothesized LNPs can be altered to target antigen presenting cells in the spleen and hereby orchestrate specific and durable immune responses against cancer antigens. By using a design of experiment methodology we were able to screen LNPs in a cost and time effective manner. LNP compositions that were evaluated solely differed in the molar ratios of ionizable lipid, phospholipid, cholesterol and PEG-lipid and the choice of the PEG-lipid. Bayesian Regression modeling enabled us to identify LNP compositions conferring maximum immunogenicity upon i‧v. administration. To link LNP compositions to T cell responses and biodistribution, we prepared the same LNPs packaging either the viral oncoprotein E7 as an antigen or Cy-5 labelled luciferase mRNA. The optimized LNP compositions showed increased localization in the spleen (of mice and non-human primates) and uptake by antigen presenting cells. Furthermore, they induced high magnitude CD8 T cell responses that conferred strong antitumor immunity and prolonged survival of TC-1 tumor bearing mice. Mechanistically, type I interferons and phagocytes were found to be essential for eliciting strong T-cell responses. Unexpectedly, we identified B cells as major mediators of the vaccine- elicited T-cell response, revealing a previously undocumented role of these cells in the immune response to mRNA LNPs. Our data highlight the potential of optimizing LNP compositions by tailoring of the molar ratio of the lipids compromising the LNPs and provide insight in the cell types involved in the mRNA LNP-induced immune response. Collectively, our results illustrate the great promise of intravenous mRNA LNP vaccination for the treatment of cancer

Rationally designed mRNA-loaded lipid nanoparticles provoke strong antitumor T cell immunity which critically depends on specific immune cell subsets

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A CRISPR-Cas9-based reporter system for single-cell detection of extracellular vesicle-mediated functional transfer of RNA

Extracellular vesicles (EVs) form an endogenous transport system for intercellular transfer of biological cargo, including RNA, that plays a pivotal role in physiological and pathological processes. Unfortunately, whereas biological effects of EV-mediated RNA transfer are abundantly studied, regulatory pathways and mechanisms remain poorly defined due to a lack of suitable readout systems. Here, we describe a highly-sensitive CRISPR-Cas9-based reporter system that allows direct functional study of EV-mediated transfer of small non-coding RNA molecules at single-cell resolution. Using this CRISPR operated stoplight system for functional intercellular RNA exchange (CROSS-FIRE) we uncover various genes involved in EV subtype biogenesis that play a regulatory role in RNA transfer. Moreover we identify multiple genes involved in endocytosis and intracellular membrane trafficking that strongly regulate EV-mediated functional RNA delivery. Altogether, this approach allows the elucidation of regulatory mechanisms in EV-mediated RNA transfer at the level of EV biogenesis, endocytosis, intracellular trafficking, and RNA delivery