The role of biological sex in pre-clinical (mouse) mRNA vaccine studies

In this study, we consider the influence of biological sex-specific immune responses on the assessment of mRNA vaccines in pre-clinical murine studies. Recognising the established disparities in immune function attributed to genetic and hormonal differences between individuals of different biological sexes, we compared the mRNA expression and immune responses in mice of both biological sexes after intramuscular injection with mRNA incorporated within lipid nanoparticles. Regarding mRNA expression, no significant difference in protein (luciferase) expression at the injection site was observed between female and male mice following intramuscular administration; however, we found that female BALB/c mice exhibit significantly greater total IgG responses across the concentration range of mRNA lipid nanoparticles (LNPs) in comparison to their male counterparts. This study not only contributes to the scientific understanding of mRNA vaccine evaluation but also emphasizes the importance of considering biological sex in vaccine study designs during pre-clinical evaluation in murine studies

Synthetic cells synthesize therapeutic proteins inside tumors

The existing dogma is that protein medicines need to be produced in large factories, and then injected to the patient. We propose that miniature artificial inert factories can be injected to the patient, to produce a protein of interest directly in the diseased tissue. We engineered artificial cell-like particles with an autonomous capacity to synthesize protein drugs after receiving an external signal. The protein is tuned to the patient\u27s needs based on a predetermined DNA code we incorporate inside the particles. This approach increases treatment efficiency and reduces adverse effects to healthy tissues. We developed a new T7-S30 based cell-free protein synthesis system, which contains all the transcription and translation machines and molecules required for protein production (Krinsky et al., PloS one 2016). This system was used to prepare liposomes that act as artificial cells, capable of producing proteins autonomously in response to a physical trigger. Functional enzymes (luciferase and tyrosinase) and fluorescent proteins (GFP) were successfully produced using the new cell-free protein synthesis system and inside the particles both in vitro and in vivo. In addition, we demonstrated the therapeutic capabilities of the protein producing particles by producing Pseudomonas exotoxin A, an extremely potent protein, for treating cancer. Applying the particles on 4T1 cells (a triple-negative breast cancer cell-line) in vitro or injecting them into a 4T1-induced tumor in vivo, resulted in high cytotoxicity due to the effective production of the therapeutic protein inside the vesicles (Krinsky et al. Advanced Healthcare Materials, 2017). Synthetic cells serve as autonomous, trigger-able, artificial particles that produces a variety of proteins. This platform has promise to address a wide range of fundamental questions associated with protein synthesis in nature, as well as applicative protein delivery needs. Please click Additional Files below to see the full abstract

Remotely Activated Protein-Producing Nanoparticles

The development of responsive nanomaterials, nanoscale systems that actively respond to stimuli, is one general goal of nanotechnology. Here we develop nanoparticles that can be controllably triggered to synthesize proteins. The nanoparticles consist of lipid vesicles filled with the cellular machinery responsible for transcription and translation, including amino acids, ribosomes, and DNA caged with a photolabile protecting group. These particles served as nanofactories capable of producing proteins including green fluorescent protein (GFP) and enzymatically active luciferase. In vitro and in vivo, protein synthesis was spatially and temporally controllable, and could be initiated by irradiating micrometer-scale regions on the time scale of milliseconds. The ability to control protein synthesis inside nanomaterials may enable new strategies to facilitate the study of orthogonal proteins in a confined environment and for remotely activated drug delivery.National Cancer Institute (U.S.) (MIT-Harvard Center for Cancer Nanotechnology Excellence Grant U54 CA151884)Marie D. and Pierre Casimir-Lambert FundNational Cancer Institute (U.S.) (Cancer Center Support (Core) Grant P30-CA14051)National Institutes of Health (U.S.) (Grant EB000244

Esterase-Responsive Polyglycerol-Based Nanogels for Intracellular Drug Delivery in Rare Gastrointestinal Stromal Tumors

Rare gastrointestinal stromal tumors (GISTs) are caused by mutations in the KIT and PDGFRA genes. Avapritinib (BLU-285) is a targeted selective inhibitor for mutated KIT and PDGFRA receptors that can be used to treat these tumors. However, there are subtypes of GISTs that exhibit resistance against BLU-285 and thus require other treatment strategies. This can be addressed by employing a drug delivery system that transports a combination of drugs with distinct cell targets. In this work, we present the synthesis of esterase-responsive polyglycerol-based nanogels (NGs) to overcome drug resistance in rare GISTs. Using inverse nanoprecipitation mediated with inverse electron-demand Diels–Alder cyclizations (iEDDA) between dPG-methyl tetrazine and dPG-norbornene, multi-drug-loaded NGs were formed based on a surfactant-free encapsulation protocol. The obtained NGs displayed great stability in the presence of fetal bovine serum (FBS) and did not trigger hemolysis in red blood cells over a period of 24 h. Exposing the NGs to Candida Antarctica Lipase B (CALB) led to the degradation of the NG network, indicating the capability of targeted drug release. The bioactivity of the loaded NGs was tested in vitro on various cell lines of the GIST-T1 family, which exhibit different drug resistances. Cell internalization with comparable uptake kinetics of the NGs could be confirmed by confocal laser scanning microscopy (CLSM) and flow cytometry for all cell lines. Cell viability and live cell imaging studies revealed that the loaded NGs are capable of intracellular drug release by showing similar IC50 values to those of the free drugs. Furthermore, multi-drug-loaded NGs were capable of overcoming BLU-285 resistance in T1-α-D842V + G680R cells, demonstrating the utility of this carrier system

Nanodelivery of nucleic acids

Funding: This work was supported by the European Research Council (ERC) Starting Grant (ERC-StG-2019-848325 to J. Conde) and the Fundação para a Ciência e a Tecnologia FCT Grant (PTDC/BTM-MAT/4738/2020 to J. Conde). J.S. acknowledges US National Institute of Health (NIH) grants (R01CA200900, R01HL156362 and R01HL159012), the US DoD PRCRP Idea Award with Special Focus (W81XWH1910482), the Lung Cancer Discovery Award from the American Lung Association and the Innovation Discovery Grants award from the Mass General Brigham. H.L., D.Y. and X.Z. were supported by the National Key R&D Program of China (no. 2020YFA0710700), the National Natural Science Foundation of China (nos 21991132, 52003264, 52021002 and 52033010) and the Fundamental Research Funds for the Central Universities (no. WK2060000027).There is growing need for a safe, efficient, specific and non-pathogenic means for delivery of gene therapy materials. Nanomaterials for nucleic acid delivery offer an unprecedented opportunity to overcome these drawbacks; owing to their tunability with diverse physico-chemical properties, they can readily be functionalized with any type of biomolecules/moieties for selective targeting. Nucleic acid therapeutics such as antisense DNA, mRNA, small interfering RNA (siRNA) or microRNA (miRNA) have been widely explored to modulate DNA or RNA expression Strikingly, gene therapies combined with nanoscale delivery systems have broadened the therapeutic and biomedical applications of these molecules, such as bioanalysis, gene silencing, protein replacement and vaccines. Here, we overview how to design smart nucleic acid delivery methods, which provide functionality and efficacy in the layout of molecular diagnostics and therapeutic systems. It is crucial to outline some of the general design considerations of nucleic acid delivery nanoparticles, their extraordinary properties and the structure–function relationships of these nanomaterials with biological systems and diseased cells and tissues.publishersversionpublishe

Targeting FGFR4 Inhibits Hepatocellular Carcinoma in Preclinical Mouse Models

The fibroblast growth factor (FGF)-FGF receptor (FGFR) signaling system plays critical roles in a variety of normal developmental and physiological processes. It is also well documented that dysregulation of FGF-FGFR signaling may have important roles in tumor development and progression. The FGFR4–FGF19 signaling axis has been implicated in the development of hepatocellular carcinomas (HCCs) in mice, and potentially in humans. In this study, we demonstrate that FGFR4 is required for hepatocarcinogenesis; the progeny of FGF19 transgenic mice, which have previously been shown to develop HCCs, bred with FGFR4 knockout mice fail to develop liver tumors. To further test the importance of FGFR4 in HCC, we developed a blocking anti-FGFR4 monoclonal antibody (LD1). LD1 inhibited: 1) FGF1 and FGF19 binding to FGFR4, 2) FGFR4–mediated signaling, colony formation, and proliferation in vitro, and 3) tumor growth in a preclinical model of liver cancer in vivo. Finally, we show that FGFR4 expression is elevated in several types of cancer, including liver cancer, as compared to normal tissues. These findings suggest a modulatory role for FGFR4 in the development and progression of hepatocellular carcinoma and that FGFR4 may be an important and novel therapeutic target in treating this disease

Efficiency of siRNA delivery by lipid nanoparticles is limited by endocytic recycling

Despite substantial efforts to understand the interactions between nanoparticles and cells, the cellular processes that determine the efficiency of intracellular drug delivery remain largely unclear. Here we examined cellular uptake of siRNA delivered in lipid nanoparticles (LNPs) using cellular trafficking probes in combination with automated high-throughput confocal microscopy as well as defined perturbations of cellular pathways paired with systems biology approaches to uncover protein-protein and protein-small molecule interactions. We show that multiple cell signaling effectors are required for initial cellular entry of LNPs through macropinocytosis, including proton pumps, mTOR, and cathepsins. SiRNA delivery is substantially reduced as ≅70% of the internalized siRNA undergoes exocytosis through egress of LNPs from late endosomes/lysosomes. Niemann Pick type C1 (NPC1) is shown to be an important regulator of the major recycling pathways of LNP-delivered siRNAs. NPC1-deficient cells show enhanced cellular retention of LNPs inside late endosomes/lysosomes and increased gene silencing of the target gene. Our data suggests that siRNA delivery efficiency might be improved by designing delivery vehicles that can escape the recycling pathways

Resolving identity ambiguity through transcending fandom

Identity construction involves accumulating cultural, social, and symbolic capital, with initial endowments being accrued through socialization into one’s habitus. This research explores the experiences of individuals that feel a lack of capital, which leads to ambiguity regarding their identities and places in the world. Through in-depth interviews, this interpretive research shows that such individuals may turn to fandom for gaining status and belonging. Fandoms are consumption fields with clear, limited forms of cultural capital. Through serial fandom and engagement with fandom in different ways, individuals were able to learn the skill of identifying and accruing relevant cultural capital. The skill became decontextualized and recontextualized, allowing individuals to transcend fandom and accrue general forms of cultural capital. Learning the skill aids individuals in dealing with the simultaneously debilitating and empowering freedom of contemporary consumer culture. Moreover, gaining cultural capital could be altogether developing into the form of the process we describe

Barbarians at the British Museum: Anglo-Saxon Art, Race and Religion

A critical historiographical overview of art historical approaches to early medieval material culture, with a focus on the British Museum collections and their connections to religion