Endocytic Profiling of Cancer Cell Models Reveals Critical Factors Influencing LNP-Mediated mRNA Delivery and Protein Expression.

Lipid nanoparticles have great potential for delivering nucleic-acid-based therapeutics, but low efficiency limits their broad clinical translation. Differences in transfection capacity between in vitro models used for nanoparticle pre-clinical testing are poorly understood. To address this, using a clinically relevant lipid nanoparticle (LNP) delivering mRNA, we highlight specific endosomal characteristics in in vitro tumor models that impact protein expression. A 30-cell line LNP-mRNA transfection screen identified three cell lines having low, medium, and high transfection that correlated with protein expression when they were analyzed in tumor models. Endocytic profiling of these cell lines identified major differences in endolysosomal morphology, localization, endocytic uptake, trafficking, recycling, and endolysosomal pH, identified using a novel pH probe. High-transfecting cells showed rapid LNP uptake and trafficking through an organized endocytic pathway to lysosomes or rapid exocytosis. Low-transfecting cells demonstrated slower endosomal LNP trafficking to lysosomes and defective endocytic organization and acidification. Our data establish that efficient LNP-mRNA transfection relies on an early and narrow endosomal escape window prior to lysosomal sequestration and/or exocytosis. Endocytic profiling should form an important pre-clinical evaluation step for nucleic acid delivery systems to inform model selection and guide delivery-system design for improved clinical translation.

Extracellular vesicles induce minimal hepatotoxicity and immunogenicity.

Extracellular vesicles (EVs) mediate cellular communication through the transfer of active biomolecules, raising interest in using them as biological delivery vehicles for therapeutic drugs. For drug delivery applications, it is important to understand the intrinsic safety and toxicity liabilities of EVs. Nanoparticles, including EVs, typically demonstrate significant accumulation in the liver after systemic administration in vivo. We confirmed uptake of EVs derived from Expi293F cells into HepG2 cells and did not detect any signs of hepatotoxicity measured by cell viability, functional secretion of albumin, plasma membrane integrity, and mitochondrial and lysosomal activity even at high exposures of up to 5 × 1010 EVs per mL. Whole genome transcriptome analysis was used to measure potential effects on the gene expression in the recipient HepG2 cells at 24 h following exposure to EVs. Only 0.6% of all genes were found to be differentially expressed displaying less than 2-fold expression change, with genes related to inflammation or toxicity being unaffected. EVs did not trigger any proinflammatory cytokine response in HepG2 cells. However, minor changes were noted in human blood for interleukin (IL)-8, IL-6, and monocyte chemotactic protein 1 (MCP-1). Administration of 5 × 1010 Expi293F-derived EVs to BALB/c mice did not result in any histopathological changes or increases of liver transaminases or cytokine levels, apart from a modest increase in keratinocyte chemoattractant (KC). The absence of any significant toxicity associated with EVs in vitro and in vivo supports the prospective use of EVs for therapeutic applications and for drug delivery.

Deconvoluting Kinase Inhibitor Induced Cardiotoxicity.

Many drugs designed to inhibit kinases have their clinical utility limited by cardiotoxicity-related label warnings or prescribing restrictions. While this liability is widely recognized, designing safer kinase inhibitors (KI) requires knowledge of the causative kinase(s). Efforts to unravel the kinases have encountered pharmacology with nearly prohibitive complexity. At therapeutically relevant concentrations, KIs show promiscuity distributed across the kinome. Here, to overcome this complexity, 65 KIs with known kinome-scale polypharmacology profiles were assessed for effects on cardiomyocyte (CM) beating. Changes in human iPSC-CM beat rate and amplitude were measured using label-free cellular impedance. Correlations between beat effects and kinase inhibition profiles were mined by computation analysis (Matthews Correlation Coefficient) to identify associated kinases. Thirty kinases met criteria of having (1) pharmacological inhibition correlated with CM beat changes, (2) expression in both human-induced pluripotent stem cell-derived cardiomyocytes and adult heart tissue, and (3) effects on CM beating following single gene knockdown. A subset of these 30 kinases were selected for mechanistic follow up. Examples of kinases regulating processes spanning the excitation-contraction cascade were identified, including calcium flux (RPS6KA3, IKBKE) and action potential duration (MAP4K2). Finally, a simple model was created to predict functional cardiotoxicity whereby inactivity at three sentinel kinases (RPS6KB1, FAK, STK35) showed exceptional accuracy in vitro and translated to clinical KI safety data. For drug discovery, identifying causative kinases and introducing a predictive model should transform the ability to design safer KI medicines. For cardiovascular biology, discovering kinases previously unrecognized as influencing cardiovascular biology should stimulate investigation of underappreciated signaling pathways.

Structure Based Design of Non-Natural Peptidic Macrocyclic Mcl-1 Inhibitors.

Mcl-1 is a pro-apoptotic BH3 protein family member similar to Bcl-2 and Bcl-xL. Overexpression of Mcl-1 is often seen in various tumors and allows cancer cells to evade apoptosis. Here we report the discovery and optimization of a series of non-natural peptide Mcl-1 inhibitors. Screening of DNA-encoded libraries resulted in hit compound 1, a 1.5 µM Mcl-1 inhibitor. A subsequent crystal structure demonstrated that compound 1 bound to Mcl-1 in a ß-turn conformation, such that the two ends of the peptide were close together. This proximity allowed for the linking of the two ends of the peptide to form a macrocycle. Macrocyclization resulted in an approximately 10-fold improvement in binding potency. Further exploration of a key hydrophobic interaction with Mcl-1 protein and also with the moiety that engages Arg256 led to additional potency improvements. The use of protein-ligand crystal structures and binding kinetics contributed to the design and understanding of the potency gains. Optimized compound 26 is a <3 nM Mcl-1 inhibitor, while inhibiting Bcl-2 at only 5 µM and Bcl-xL at >99 µM, and induces cleaved caspase-3 in MV4-11 cells with an IC50 of 3 µM after 6 h.