mRNA lipid nanoparticle formulation, characterization and evaluation
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Anionic lipids direct efficient microfluidic encapsulation of stable and functionally active proteins in lipid nanoparticles
Because proteins do not efficiently pass through the plasma membrane, protein therapeutics are limited to target ligands located at the cell surface or in serum. Lipid nanoparticles can facilitate delivery of polar molecules across a membrane. We hypothesized that because most proteins are amphoteric ionizable polycations, proteins would associate with anionic lipids, enabling microfluidic chip assembly of stable EP-LNPs (Encapsulated Proteins in Lipid NanoParticles). Here, by employing anionic lipids we were able to efficiently load proteins into EP-LNPs at protein:lipid w:w ratios of 1:20. Several proteins with diverse molecular weights and isoelectric points were encapsulated at efficiencies of 70 75%–90% and remained packaged for several months. Proteins packaged in EP-LNPs efficiently entered mammalian cells and fungal cells with cell walls. The proteins delivered intracellularly were functional. EP-LNPs technology should improve cellular delivery of medicinal antibodies, enzymes, peptide antimetabolites, and dominant negative proteins, opening new fields of protein therapeutics
Nature-inspired platform nanotechnology for RNA delivery to myeloid cells and their bone marrow progenitors
Nucleic acid therapeutics are used for silencing, expressing or editing genes in vivo. However, their systemic stability and targeted delivery to bone marrow resident cells remains a challenge. In this study we present a nanotechnology platform based on natural lipoproteins, designed for delivering small interfering RNA (siRNA), antisense oligonucleotides and messenger RNA to myeloid cells and haematopoietic stem and progenitor cells in the bone marrow. We developed a prototype apolipoprotein nanoparticle (aNP) that stably incorporates siRNA into its core. We then created a comprehensive library of aNP formulations and extensively characterized their physicochemical properties and in vitro performance. From this library, we selected eight representative aNP-siRNA formulations and evaluated their ability to silence lysosomal-associated membrane protein 1 (Lamp1) expression in immune cell subsets in mice after intravenous administration. Using the most effective aNP identified from the screening process, we tested the platform’s potential for therapeutic gene silencing in a syngeneic murine tumour model. We also demonstrated the aNP platform’s suitability for splice-switching with antisense oligonucleotides and for protein production with messenger RNA by myeloid progenitor cells in the bone marrow. Our data indicate that the aNP platform holds translational potential for delivering various types of nucleic acid therapeutics to myeloid cells and their progenitors.
Cholesterol homeostasis and lipid raft dynamics at the basis of tumor-induced immune dysfunction in chronic lymphocytic leukemia
Autologous T-cell therapies show limited efficacy in chronic lymphocytic leukemia (CLL), where acquired immune dysfunction prevails. In CLL, disturbed mitochondrial metabolism has been linked to defective T-cell activation and proliferation. Recent research suggests that lipid metabolism regulates mitochondrial function and differentiation in T cells, yet its role in CLL remains unexplored. This comprehensive study compares T-cell lipid metabolism in CLL patients and healthy donors, revealing critical dependence on exogenous cholesterol for human T-cell expansion following TCR-mediated activation. Using multi-omics and functional assays, we found that T cells present in viably frozen samples of patients with CLL (CLL T cells) showed impaired adaptation to cholesterol deprivation and inadequate upregulation of key lipid metabolism transcription factors. CLL T cells exhibited altered lipid storage, with increased triacylglycerols and decreased cholesterol, and inefficient fatty acid oxidation (FAO). Functional consequences of reduced FAO in T cells were studied using samples from patients with inherent FAO disorders. Reduced FAO was associated with lower T-cell activation but did not affect proliferation. This implicates low cholesterol levels as a primary factor limiting T-cell proliferation in CLL. CLL T cells displayed fewer and less clustered lipid rafts, potentially explaining the impaired immune synapse formation observed in these patients. Our findings highlight significant disruptions in lipid metabolism as drivers of functional deficiencies in CLL T cells, underscoring the pivotal role of cholesterol in T-cell proliferation. This study suggests that modulating cholesterol metabolism could enhance T-cell function in CLL, presenting novel immunotherapeutic approaches to improve outcome in this challenging disease.
Iron homeostasis and ferroptosis in muscle diseases and disorders: mechanisms and therapeutic prospects
The muscular system plays a critical role in the human body by governing skeletal movement, cardiovascular function, and the activities of digestive organs. Additionally, muscle tissues serve an endocrine function by secreting myogenic cytokines, thereby regulating metabolism throughout the entire body. Maintaining muscle function requires iron homeostasis. Recent studies suggest that disruptions in iron metabolism and ferroptosis, a form of iron-dependent cell death, are essential contributors to the progression of a wide range of muscle diseases and disorders, including sarcopenia, cardiomyopathy, and amyotrophic lateral sclerosis. Thus, a comprehensive overview of the mechanisms regulating iron metabolism and ferroptosis in these conditions is crucial for identifying potential therapeutic targets and developing new strategies for disease treatment and/or prevention. This review aims to summarize recent advances in understanding the molecular mechanisms underlying ferroptosis in the context of muscle injury, as well as associated muscle diseases and disorders. Moreover, we discuss potential targets within the ferroptosis pathway and possible strategies for managing muscle disorders. Finally, we shed new light on current limitations and future prospects for therapeutic interventions targeting ferroptosis.
A capless hairpin-protected mRNA vaccine encoding the full-length Influenza A hemagglutinin protects mice against a lethal Influenza A infection
The success of mRNA vaccines in controlling the COVID 19 pandemic has confirmed the efficacy of synthetically synthesized mRNA in humans and has also provided a blueprint on how to design them in terms of molecular structure and cost. We describe a mRNA vector that, unlike linear mRNAs used in current vaccines/therapeutics, does not require a 5′ cap to function. The described mRNA vector initiates translation from an internal ribosomal entry site (IRES) and contains specially designed self-folding secondary structures (hairpins) to protect the 5′ end against degradation, dramatically improving its stability. The produced mRNA did not require any additional modifications for functionality. The 5′ hairpins completely inhibited cap-dependent translation, and all vectors containing them required an IRES to express protein. When this capless mRNA vector was constructed to express the full-length Influenza A membrane protein hemagglutinin (HA), complexed with pre-formed lipid-based nanoparticles, and then injected into mice as a vaccine, it generated high titers of anti-HA antibodies and protected mice against a lethal dose of Influenza A.
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