Angelo Vertti, 18 de setembro de 2022

In contrast, when the same cells were propagated in serum-containing medium, higher transfectabilities were found for late-passage cells, while up to 40 % lower transfectabilities were observed for early-passage cells. The internalized LNPs diffuse through the cellular medium at a rate that is inversely proportional to their size. The solution depends on initial conditions. Chem. https://doi.org/10.1021/acs.molpharmaceut.9b01182 (2020). 5d). The methodology typically requires cell lysis and extraction to detect total metabolite concentrations, providing snapshots of cellular physiology over a period of hours to days that is inherently biased due to the multistep processes required to prepare samples12,13. Cells for NMR experiments were grown on 15cm plates in 20mL of DMEM/10% FBS to 6080% confluency. The metabolite cross peak intensities increased as labeled metabolites entered the free pool following biosynthesis and decreased as they were transiently bound or incorporated into larger biomolecules, or cleared from the free pool when labeled glucose was removed from the system. The time of acquisition for each transient spectrum was 47s. For each experiment a 13C-isotope edited 1H spectrum was collected prior to the injection of the 13C labeled glucose to establish a baseline reading of the metabolites present in the sample. The time of dilution, 1/DR~1min, is much shorter than tP and tC, the characteristic times of labeled metabolite production and clearance. Cells were harvested as described above60, passed through a 40m filter to reduce clumping and aggregation, and counted. 293T (or HEK 293T) is a derivative human cell line that expresses a mutant version of the SV40 large T antigen. The site is secure. 1b, Supplementary Table 2). The decrease in plateau signal intensity for alanine was analyzed by fitting the alanine plateau profiles to a linear function (Supplementary Table 6; Fig. 2, 26922703. 23, 14161417. Scaled kinetic flux profiles of metabolites in HEK 293T cells. Incorporation of mRNA into LNPs was shown to be necessary for strong gene expression. (A) The mRNA expressions of report gene (eGFP) and internal reference gene (GAPDH) were measured by qRT-PCR. The red and black traces represent biological replicate samples. Alshamleh, I. et al. https://doi.org/10.1101/gad.5.11.2108 (1991). The loop contained KH salts supplemented with 5mM 13C-glucose, allowing for uninterrupted transition to labeled medium. Transient gene expression in HEK293 cells: peptone addition posttransfection improves recombinant protein synthesis Gene expression by large-scale transfection of mammalian cells is becoming an established technology for the fast production of milligram and even gram amounts of recombinant proteins (r-proteins). To add a 3 poly(A) tail the mRNA was again denatured at 65C for 10min and the A-Plus Poly(A) Polymerase Tailing Kit (Cellscript) was used according to manufacturers guidelines. Anal. Swanson, R. A. The persistently slower clearance time for lactate and alanine reflected an increase in the concentration of free labeled metabolites as they exchanged out of the bound fraction and were utilized for ongoing metabolism. Breindel, L., Burz, D. S. & Shekhtman, A. NMR-based metabolomics of mammalian cell and tissue cultures. 2, 13691372. Insets show metabolite cross peak splitting and glycogen cross peaks. Ten L of each cell suspension was diluted into 245L of PBS and the number of cells counted manually using a hemocytometer. Webb, A. Open Access Published: 24 December 2022 Messenger RNA in lipid nanoparticles rescues HEK 293 cells from lipid-induced mitochondrial dysfunction as studied by real time pulse chase NMR, RTPC-NMR,. A rapid bead-based assay for screening of SARS-CoV-2 neutralizing antibodies. The effect of size and charge of lipid nanoparticles prepared by microfluidic mixing on their lymph node transitivity and distribution. Leukemia 30, 6573. 31P spectra were collected prior to the introduction of labeled medium and at the end of the experiment to assess the energy charge of the cells. Unauthorized use of these marks is strictly prohibited. Furthermore, these nucleotide ratios proved to be different for transfected and untransfected cells, providing a high potential tool to monitor the status of transfection under various culture conditions. Commun. Yuan, J., Bennett, B. D. & Rabinowitz, J. D. Kinetic flux profiling for quantitation of cellular metabolic fluxes. (7), In general the solution of (9) is possible only numerically. Majumder, S. et al. 4a). To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. Numerical solutions for Eqs. To that end the experimental temperature was adjusted to 300K to slow cellular metabolism37. Each experiment took~6h to complete and was repeated at least twice using biological replicates. Despite the chemically modified base, protein production was limited and the Luciferase-dependent signal rapidly diminished (Fig. Importantly, the term K A* exp(t) approaches zero when time goes to infinity, reflecting the fact that no net concentration change due to binding will be observed when the system is equilibrated with labeled metabolites. Moon, R. B. Importantly, the change in the concentration of bound metabolite over time can also be described by differentiating (19) and (20) and presenting the result as. No. This corresponded to 10 % of the total protein concentration in the cell-free supernatant of the cultures in protein-free medium. The transfections were incubated in a growth chamber for 5h in 5% CO2 at 37C. The short spectral acquisition time provides a temporal resolution of 47s, fast enough to observe the onset of rapid processes such as glycolysis. Biochemistry 57, 540546 (2018). Chem.-Int Edition 59, 23042308. Physiol. For lactate, alanine, and glutamate the two-phase association model from GraphPad Prism was used to fit intensities on the leading edge of the pulse, where K=1/tP, the reciprocal time constant for metabolite production and 0x1 is a parameter that assigns the percentage of each exponential component to the overall fit. Milo, R. & Philips, R. (2015) Cell Biology by the Numbers, 1st edition, pp 188192. 16, 18331840. ROS production in HEK 293T cells in response to LNP exposure with and without mRNA. Can these cells outcompete the surrounding cells for scarce nutrient resources in living tissues? This may explain how empty LNPs elicit greater ROS production than those that are cargo-laden and, in combination with the requirement for proper mitochondrial export of glutamate in the primary biosynthesis of alanine, why only the alanine profiles differed in the absence and presence of mRNA. Product Lipofectamine 2000 Reagent is a proprietary formulation Descriptionfor transfecting nucleic acids into a wide range of eukaryotic cells. CRISPR-Cas9 genome editing using targeted lipid nanoparticles for cancer therapy. Cell Dev. The average value of the slope resolved for cells treated with LF, 0.005min1, was trending 2.5 times greater than control and, significantly, 5 times greater than in cells treated with LF-mRNA (Supplementary Table 6) indicating a very slow process (Fig. The remarkable success of pseudouridine-modified mRNA lipid nanoparticle, LNP, -based, vaccines against COVID-19, which harnesses innate translational machinery powered by cellular metabolism, propels this technology to the forefront of medicine1,2,3,4,5,6. Transfection of HEK293-EBNA1 Cells in Suspension with 293fectin for Production of Recombinant Proteins CSH Protoc. Garland Science, Goa. HEK 293T cells were previously utilized to monitor the uptake and expression of Luciferase mRNA21,22. Pardi, N., Hogan, M. J. https://doi.org/10.1038/mt.2008.200 (2008). You are using a browser version with limited support for CSS. Breindel, L., DeMott, C., Burz, D. S. & Shekhtman, A. Real-time in-cell nuclear magnetic resonance: Ribosome-targeted antibiotics modulate quinary protein interactions. The 31P spectrum was centered at 10ppm, corresponding to 242.935MHz, and the spectral width in the 31P dimension was 30ppm. https://doi.org/10.1016/j.cell.2017.12.006 (2018). Zhang, L. Q. et al. The growth medium was changed to Opti-MEM/5% FBS and 0.5g of luciferase mRNA was combined with either 1.5L of FuGENE 6, 1.5L of FuGENE HD, 2L of ViaFect or 1L of LF in Opti-MEM and incubated at room temperature according the manufacturers recommendations. 121, 317a317a (2022). Control Release 217, 345351. It can be using any technology like. For some experiments the medium was removed after 8 or 12h of transfection and replaced with DMEM for further incubation. https://doi.org/10.1038/Nnano.2011.166 (2011). Mitochondrial dysfunction was largely abated by inclusion of mRNA in the LNPs, the presence of which increased the size and uniformity of the LNPs. where yo1, AoR and A1 are constants determined to fit the kinetic flux profiling at the leading edge (Rise) of 13C-glucose pulse. Biotechnol Bioeng. Statistical evaluation of differences between KFP parameters (a) production and clearance times, tP and tC; (b) characteristic times, tch=2/(1/tP+1/tC), (c) binding parameters, K=(1/tP1/tC)/2, for each metabolite, and (d) the slopes of alanine KFPs after treatment with LF and LF-mRNA. https://doi.org/10.1038/Nchembio.2077 (2016). Gutirrez-Granados S, Cervera L, Kamen AA, Gdia F. Crit Rev Biotechnol. Note that the characteristic time of the clearance in the NMR observed metabolite signal, tC=1/(J/A-K), is larger than tP. 4) cultured under serum-containing conditions (DMEM+FCS). Each well was seeded with 5104 cells in 0.5mL of DMEM/10% FBS 24h before transfection and incubated in a growth chamber in 5% CO2 at 37C. The non-overlapping peaks corresponding to lactate, alanine, and glutamate were of sufficient strength to reduce the acquisition time to 47s, which was the temporal resolution of the experiments. Extended gene expression by medium exchange and repeated transient transfection for recombinant protein production enhancement. (B) The mRNA . The purified transcript included a~150-nucleotide 3 poly(A) tail and 5 Cap124 to further facilitate protein translation (Supplementary Figure1). Question. [A*R]t=0=N k1 A*tot/(k1 [A]ss+k2). An official website of the United States government. All NMR spectra were recorded at 300K using a 600MHz Avance III NMR spectrometer equipped with a QCI-P cryoprobe (Bruker). In the absence of mRNA the LNP size distribution was asymmetric with mean particle size of 2912nm (Fig. 2, N.S., J.Y., and G.T. Lupitha SS, Darvin P, Chandrasekharan A, Varadarajan SN, Divakaran SJ, Easwaran S, Nelson-Sathi S, Umasankar PK, Jones S, Joseph I, Pillai MR, Santhoshkumar TR. Epub 2022 Oct 25. Pharmacol. At the appropriate times cells were centrifuged at 200g for 5min, washed with PBS and lysed as described above for the luminescence assay. Live monitoring of cellular metabolism and mitochondrial respiration in 3D cell culture system using NMR spectroscopy. HEK 293T cells treated with LNPs containing mRNA were able to access nutrients and produce protein for more than 24h implying that the inevitable release of pseudouridine upon mRNA degradation did not inhibit transcription of the mRNA product. They observed that an N- terminal fusion led to a 4-5 fold reduction in expression levels which was not observed in this study. The results suggest that alanine may function as a sensitive probe for LNP-induced toxicity. History [ edit] Cell 172, 358-+. Incorporation of pseudouridine into mRNA yields superior nonimmunogenic vector with increased translational capacity and biological stability. Kariko, K., Whitehead, K. & van der Meel, R. What does the success of mRNA vaccines tell us about the future of biological therapeutics?. To introduce the Cap1 structure on the 5 end, mRNA was denatured at 65C for 10min and the ScriptCap Cap1 Capping System (Cellscript) was used according to the manufacturers guidelines. CAS Cell Metab. Metabolic profiling, metabolomic and metabonomic procedures for NMR spectroscopy of urine, plasma, serum and tissue extracts. Linear PEI (25kDa) was purchased from Polysciences, rotenone from MP Biomedicals and Antimycin A from Sigma-Aldrich. Based on two different bicistronic model plasmids expressing the human erythropoietin gene (rHuEPO) in the first position and green fluorescent protein as reporter gene in the second position and vice versa, a completely serum-free transient transfection process was established. Trypsin was neutralized by using a fivefold dilution with complete medium, cells were counted and pelleted by centrifugation at 200g for 10min. HEK 293T cells were packaged as previously described into small,~0.6mm diameter, alginate beads by using an atomizer20 and modified KrebsHenseleit, KH, buffer34, a classical chemically defined35 serum-free medium used to study cardiomyocytes. The extent of ROS was significantly abated, and control behavior was largely restored by including mRNA in the LNPs. Article The cells were re-suspended in 80L of modified PBS and 10L were aliquoted into each of four wells of a black 384w plate (# 784,086, Greiner Bio-One). https://doi.org/10.1016/j.cmet.2018.03.002 (2018). Cell Syst. These differences are in part a result of the variation in cell packing density associated with each physical sample. The 31P peak intensity at11.5ppm contains -ATP, -ADP, NAD+ and NAD(H), which are all vital metabolites that can be used to assess cell vitality20. A completely randomized 10-nucleotide sequence preceding an upstream open reading frame (uORF) and downstream GFP drives a broad range of translational outputs and mRNA stability in mammalian. The resulting flow through was collected and equilibrated for 20min at room temperature to maximize transfection and facilitate cell recovery. Hertig, D. et al. Messenger RNA-based therapeutics utilize a complex set of biological molecules that have to be optimized for each application. where K=1/tC, the reciprocal time constant for metabolite clearance. However, if the concentration of bound metabolites is much smaller than the total metabolite concentration, Eqs. Tsuji, M., Allred, E., Jensen, F. & Holtzman, D. Phosphocreatine and atp regulation in the hypoxic developing rat-brain. At the leading edge of the 13C-glucose pulse, changes in the concentration of free 13C-labeled metabolite A*, with flux, J and isotopic steady state concentration A, over time t, can be described by a standard kinetic flux profiling, KFP, equation65,66 by taking into account the non-ideality of the 13C glucose pulse and adding a phenomenological term f1(A*, t) that describes the possibility that the metabolite is covalently or non-covalently bound to a receptor that is too large to be observed by NMR: The equation describes the time evolution of intracellular metabolites such as alanine and glutamate. https://doi.org/10.1002/anie.201912919 (2020). Biophys. The cell suspension was passed through the VECT device using a New Era Pump Systems Model 300 syringe pump at a flow rate of 100L/min. 11 and 13, Supplementary Table 5, Supplementary Figure9). Cells were centrifuged at 300g for 5min and washed twice with modified PBS and re-centrifuged. Meredith Packer, Dipendra Gyawali, Phil White, Michael J. Munson, Gwen ODriscoll, Alan Sabirsh, Huanzhen Ni, Marine Z. C. Hatit, James E. Dahlman, Manuel J. Carrasco, Suman Alishetty, Michael D. Buschmann, Marco Maugeri, Muhammad Nawaz, Hadi Valadi, K. A. Yamamoto, K. Blackburn, M. R. Soares, Audrey Gallud, Katharina Klditz, Bengt Fadeel, Scientific Reports Chem. Appl. The data suggest that even modified mRNAs are highly unstable, and that utilizing transfection reagents to protect mRNA from degradation is critical for sustained protein expression. (b). 5d). Metabolic plasticity in CLL: Adaptation to the hypoxic niche. The control contained no mRNA and no transfection reagent to assess baseline luminescence. Results showed no reduction in ATP levels over the course of each experiment (Supplementary Figure3). Probing protein quinary interactions by in-cell nuclear magnetic resonance spectroscopy. https://doi.org/10.1038/s41578-021-00358-0 (2021). Dots represent data points obtained using biological replicate samples. Lipid nanoparticle size is stabilized by mRNA. https://doi.org/10.1016/j.coi.2020.01.008 (2020). (14) and (15) are linear and can be solved exactly68. Today 27, 17631773 (2022). Nat. Pharm. One dimensional 13C-isotope edited 1H spectra were acquired with 32 scans using 13C edited heteronuclear single quantum coherence, HSQC, experiment43. Peak intensities were calculated as I=(I/HEPES)(Iref/HEPESref), where I/HEPES represents the normalized peak intensity before the background, Iref/HEPESref, was subtractedand HEPES is the peak intensity of HEPES at 3.08ppm. Lagziel, S., Gottlieb, E. & Shlomi, T. Mind your media. The project described was supported by Award Numbers 1P01HL146367-01 and R01GM085006 from the U.S. National Institute of Health. J. Lipofectamine 2000). Maximum protein expression was observed at~15h for continuous and 8h transfections, whereas the 12h transfection exhibited peak expression at~20h. All subsequent transfections were incubated for 10h to maximize protein production and for experimental convenience. 2500ng DNA 5ul of P3000/Lipo and. The kinetic flux profiles of lactate and alanine in cells treated with LF or LF-mRNA were qualitatively similar to controls (Fig. Please enable it to take advantage of the complete set of features! Both FAAH and FAAH-OUT had markedly reduced expression when the FOP2- and FOP3-guide pairs were used to induce a deletion in the FAAH-OUT promoter compared to cells transfected with . Dev. Tubes were incubated in the growing chamber at an angled position with open lids. Scientific Reports (Sci Rep) Article Electron leakage from the electron transport system on the inner membrane leads to partial reduction of oxygen to form superoxide, an ROS, that can ultimately result in mitophagy. https://doi.org/10.1152/jappl.1999.86.6.2013 (1999). The medium was replaced with 2mL of pre-warmed OptiMEM/5% FBS and the cells allowed to recover for 24h in the growth chamber (28h rest). N1-methylpseudouridine-triphosphate was purchased from TriLink Biotechnologies. Barney S. Graham Show authors Nature 586 , 567-571 ( 2020) Cite this article 292k Accesses 731 Citations 1949 Altmetric Metrics Abstract A vaccine for severe acute respiratory syndrome. Real-time detection of 13C NMR labeling kinetics in perfused EMT6 mouse mammary tumor cells and betaHC9 mouse insulinomas. Real time NMR spectroscopy uses natural 13C abundance or tracer-based analyses to identify metabolites by collecting 13C-isotope edited 1H spectra from cells, allowing for rapid data acquisition, thereby increasing the temporal resolution of the experiments to 10min or less14,15. A two-channel polydimethylsiloxane, PDMS, Volume Exchange for Convective Transfer, VECT, device with rigid, 9.6m microchannels was prepared as previously described61,62. However, the expression for IF is simplified when the metabolite binding is non-specific and t<<1. Will the lipids that constitute LNPs damage plasma and internal cell membranes? Nat. 10, e1003885. Safety of the BNT162b2 mRNA Covid-19 vaccine in a nationwide setting. https://doi.org/10.1016/j.mib.2018.02.004 (2018). 6b)51. where K=N k1, and =k1 N+k2. HEK293 Cells Humans RNA Stability RNA, Messenger / chemistry RNA, Messenger / genetics RNA, Messenger / therapeutic use Transfection Translational Research, Biomedical beta-Globins / genetics* 3' Untranslated Regions J. Med. Prog. Following injection, 500 spectra were collected consecutively over a 5.5h period allowing the disposition of the 13C-glucose to be monitored as it progressed through different metabolic pathways. 188, 114416. https://doi.org/10.1016/j.addr.2022.114416 (2022). National Library of Medicine The plasmid pTK305 used for in vitro transcription of firefly Luciferase mRNA was purchased from Addgene (plasmid # 66,812; http://n2t.net/addgene:66812; RRID:Addgene_66812). These 'nucleotide ratios' changed in an age-dependent manner and could be used to distinguish early- from late-passage cells. Cells were harvested from the wells with trypsinEDTA as described above and transferred to 1.5mL Eppendorf tubes, washed twice with warm modified PBS, PBS containing 1mM MgCl2, 1mM CaCl2 and 5.5mM glucose. 21, S59S59 (2013). To establish an analysis window for metabolic studies, the extent of protein expression was examined by incubating HEK 293T cells with LF-mRNA LNPs for different times. Sci. Expression kinetics of nucleoside-modified mRNA delivered in lipid nanoparticles to mice by various routes. Note that LF reagent used in these experiments is composed of a mixture of polycationic and phospholipids. Nat. Today (Kidlington) 21, 703712. Microbiol. 6, eabc9450. 385, 10781090. Provided by the Springer Nature SharedIt content-sharing initiative. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. There was a significant increase in tch for alanine metabolism in the presence of LF-mRNA relative to control cells (Fig. Cell mechanical and physiological behavior in the regime of rapid mechanical compressions that lead to cell volume change.

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