Résumé
mRNA is a new class of drugs that has the potential to revolutionize the treatment of brain tumors. Thanks to the COVID-19 mRNA vaccines and numerous therapy-based clinical trials, it is now clear that lipid nanoparticles (LNPs) are a clinically viable means to deliver RNA therapeutics. However, LNP-mediated mRNA delivery to brain tumors remains elusive. Over the past decade, numerous studies have shown that tumor cells communicate with each other via small extracellular vesicles, which are around 100 nm in diameter and consist of lipid bilayer membrane similar to synthetic lipidbased nanocarriers. We hypothesized that rationally designed LNPs based on extracellular vesicle mimicry would enable efficient delivery of RNA therapeutics to brain tumors without undue toxicity. We synthesized LNPs using four components similar to the formulation used in the mRNA COVID19 vaccines (Moderna and Pfizer): ionizable lipid, cholesterol, helper lipid and polyethylene glycol (PEG)-lipid. For the in vitro screen, we tested ten classes of helper lipids based on their abundance in extracellular vesicle membranes, commercial availability, and large-scale production feasibility while keeping rest of the LNP components unchanged. The transfection kinetics of GFP mRNA encapsulated in LNPs and doped with 16 mol% of helper lipids was tested using GL261, U87 and SIM-A9 cell lines. Several LNP formations resulted in stable transfection (upto 5 days) of GFP mRNA in all the cell lines tested in vitro. The successful LNP candidates (enabling >80% transfection efficacy) were then tested in vivo to deliver luciferase mRNA to brain tumors via intrathecal administration in a syngeneic glioblastoma (GBM) mouse model, which confirmed luciferase expression in brain tumors in the cortex. LNPs were then tested to deliver Cre recombinase mRNA in syngeneic GBM mouse model genetically modified to express tdTomato under LoxP marker cassette that enabled identification of LNP targeted cells. mRNA was successfully delivered to tumor cells (70-80% transfected) and a range of different cells in the tumor microenvironment, including tumor-associated macrophages (80-90% transfected), neurons (31- 40% transfected), neural stem cells (39-62% transfected), oligodendrocytes (70-80% transfected) and astrocytes (44-76% transfected). Then, LNP formulations were assessed for delivering Cas9 mRNA and CD81 sgRNA (model protein) in murine syngeneic GBM model to enable gene editing in brain tumor cells. Sanger sequencing showed that CRISPR-Cas9 editing was successful in ~94% of brain tumor cells in vivo. In conclusion, we have developed a library of safe LNPs that can transfect GBM cells in vivo with high efficacy. This technology can potentially be used to develop novel mRNA therapies for GBM by delivering single or multiple mRNAs and holds great potential as a tool to study brain tumor biology.
Résumé
Long-term care facilities for older adults present a high risk of outbreaks since they concentrate often more frail and vulnerable individuals. OBJECTIVE: To describe the epidemiological characteristics of influenza-like illness outbreaks and cases among older people in long-term care facilities in the state of S..o Paulo, Brazil. METODS: The analysis was performed through an exploratory and descriptive approach, with records from the outbreak module of the National System of Notifiable Diseases between January 2020 and June 2021. RESULTS: Outbreaks of influenza-like illness in this department represented 24.93% of all notifications. The highest concentration was seen in the state capital and metropolitan area. A total of 1 018 confirmed outbreaks were observed, involving 6 110 cases and 1 240 deaths among older people. Of these cases, 71.67% were confirmed for coronavirus disease 19 (COVID-19), 12.77% for the influenza virus, and 15.56% for other respiratory viruses. The percentages regarding death outcomes were similar, with a 20.29% lethality of influenza-like illness. Within the studied group, the older adults were the most affected. A statistical difference was observed between cases and deaths. CONCLUSIONS: Owing to the current scenario and the known vulnerabilities of these facilities, there is an urgent need for joint and articulated action by various administrative levels in order to minimize the devastating effects of influenza-like illness outbreaks (especially of COVID- 19) in older adults at long-term care homes. The strengthening of information systems and their interoperability are considered of utmost importance in order to improve the quality of information on outbreaks, which is essential during a pandemic.