Test af in vitro - og in vivo-effektiviteten af mRNA-lipidnanopartikler formuleret ved mikrofluidisk blanding
Summary
Her præsenteres en protokol til formulering af lipidnanopartikler (LNP’er), der indkapsler mRNA-kodning af ildflueluciferase. Disse LNP’er blev testet for deres styrke in vitro i HepG2-celler og in vivo i C57BL/6-mus.
Abstract
Lipidnanopartikler (LNP’er) har tiltrukket sig stor opmærksomhed for nylig med den vellykkede udvikling af COVID-19 mRNA-vaccinerne af Moderna og Pfizer/BioNTech. Disse vacciner har vist effektiviteten af mRNA-LNP-terapi og åbnet døren for fremtidige kliniske anvendelser. I mRNA-LNP-systemer fungerer LNP’erne som leveringsplatforme, der beskytter mRNA-lasten mod nedbrydning af nukleaser og formidler deres intracellulære levering. LNP’erne består typisk af fire komponenter: et ioniserbart lipid, et phospholipid, kolesterol og et lipidforankret polyethylenglycol (PEG) konjugat (lipid-PEG). Her formuleres LNP’er, der indkapsler mRNA-kodende firefly luciferase ved mikrofluidisk blanding af den organiske fase indeholdende LNP-lipidkomponenter og den vandige fase indeholdende mRNA. Disse mRNA-LNP’er testes derefter in vitro for at evaluere deres transfektionseffektivitet i HepG2-celler ved hjælp af et bioluminescerende pladebaseret assay. Derudover evalueres mRNA-LNP’er in vivo i C57BL/6-mus efter en intravenøs injektion via den laterale halevene. Helkropsbioluminescensbilleddannelse udføres ved hjælp af et in vivo-billeddannelsessystem. Repræsentative resultater vises for mRNA-LNP-egenskaberne, deres transfektionseffektivitet i HepG2-celler og den totale selvlysende flux i C57BL/6-mus.
Introduction
Lipidnanopartikler (LNP’er) har vist sig meget lovende i de senere år inden for ikke-viral genterapi. I 2018 godkendte United States Food and Drug Administration (FDA) den første RNA-interferens (RNAi) terapeutiske, Onpattro af Alnylam, til behandling af arvelig transthyretin amyloidose 1,2,3,4. Dette var et vigtigt skridt fremad for lipidnanopartikler og RNA-baserede terapier. For nylig modtog Moderna og Pfizer/BioNTech FDA-godkendelser for deres mRNA-LNP-vacciner mod SARS-CoV-2 4,5. I hver af disse LNP-baserede nukleinsyreterapier tjener LNP til at beskytte sin last mod nedbrydning af nukleaser og lette potent intracellulær levering 6,7. Mens LNP’er har set succes i RNAi-terapier og vaccineapplikationer, er mRNA-LNP’er også blevet undersøgt til brug i proteinerstatningsterapier8 samt til co-levering af Cas9 mRNA og guide-RNA til levering af CRISPR-Cas9-systemet til genredigering9. Der er imidlertid ingen specifik formulering, der er velegnet til alle applikationer, og subtile ændringer i LNP-formuleringsparametrene kan i høj grad påvirke styrken og biodistributionen in vivo 8,10,11. Således skal individuelle mRNA-LNP’er udvikles og evalueres for at bestemme den optimale formulering for hver LNP-baseret terapi.
LNP’er formuleres almindeligvis med fire lipidkomponenter: et ioniserbart lipid, et phospholipid, kolesterol og et lipidforankret polyethylenglycol (PEG) konjugat (lipid-PEG)11,12,13. Den potente intracellulære levering lettet af LNP’er er delvis afhængig af den ioniserbare lipidkomponent12. Denne komponent er neutral ved fysiologisk pH, men bliver positivt ladet i det sure miljø i endosomet11. Denne ændring i ionisk ladning menes at være en vigtig bidragyder til endosomal flugt12,14,15. Ud over det ioniserbare lipid forbedrer phospholipidkomponenten (hjælperlipid) indkapslingen af lasten og hjælper med endosomal flugt, kolesterolet giver stabilitet og forbedrer membranfusion, og lipid-PEG minimerer LNP-aggregering og opsonisering i omløb10,11,14,16. For at formulere LNP kombineres disse lipidkomponenter i en organisk fase, typisk ethanol, og blandes med en vandig fase indeholdende nukleinsyrelasten. LNP-formuleringsprocessen er meget alsidig, idet den gør det muligt let at erstatte og kombinere forskellige komponenter i forskellige molære forhold for at formulere mange LNP-formuleringer med en lang række fysisk-kemiske egenskaber10,17. Men når man udforsker dette store udvalg af LNP’er, er det afgørende, at hver formulering evalueres ved hjælp af en standardiseret procedure for nøjagtigt at måle forskellene i karakterisering og ydeevne.
Her skitseres den komplette arbejdsgang til formulering af mRNA-LNP’er og vurderingen af deres ydeevne i celler og dyr.
Protocol
Representative Results
Discussion
Med denne arbejdsgang kan en række mRNA-LNP’er formuleres og testes for deres in vitro– og in vivo-effektivitet. Ioniserbare lipider og hjælpestoffer kan udskiftes og kombineres ved forskellige molære forhold og forskellige ioniserbare lipid til mRNA-vægtforhold for at producere mRNA-LNP’er med forskellige fysisk-kemiske egenskaber22. Her formulerede vi C12-200 mRNA-LNP’er med et molært forhold på 35/16/46,5/2,5 (ioniserbart lipid:hjælperlipid:kolesterol:lipid-PEG) ved et …
Disclosures
The authors have nothing to disclose.
Acknowledgements
M.J.M. anerkender støtte fra en US National Institutes of Health (NIH) Director’s New Innovator Award (DP2 TR002776), en Burroughs Wellcome Fund Career Award ved Scientific Interface (CASI), en US National Science Foundation CAREER-pris (CBET-2145491) og yderligere finansiering fra National Institutes of Health (NCI R01 CA241661, NCI R37 CA244911 og NIDDK R01 DK123049).
Materials
| 0.1 M Hydrochloric Acid | Sigma | 7647-01-0 | |
| 0.22 μm Syringe Filters | Genesee | 25-243 | |
| 1 mL BD Slip Tip Syringe | BD | 309659 | |
| 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000] (ammonium salt) (C14-PEG2000) | Avanti Polar Lipids | 880150P | |
| 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) | Avanti Polar Lipids | 850725P | |
| 1.5 mL Eppendorf Tubes | Fisher Scientific | 05-408-129 | |
| 15 mL Conical Tubes | Fisher Scientific | 14-959-70C | |
| 200 proof Ethanol | Decon Labs | 2716 | |
| 23G Needles | Fisher Scientific | 14-826-6C | |
| 3 mL BD Disposable Syringes with Luer-Lok tips | Fisher Scientific | 14-823-435 | |
| 3 mL Dialysis Cassettes | Thermo Scientific | A52976 | |
| 96 Well Black Wall Black Bottom Plate | Fisher Scientific | 07-000-135 | |
| 96 Well White/Clear Bottom Plate, TC Surface | Thermo Scientific | 165306 | |
| Ammonium Acetate, 1 Kilogram | Research Products International | 631-61-8 | |
| Ammonium Citrate dibasic | SIgma | 3012-65-5 | |
| BD Luer-Lok Syringe sterile, single use, 5 mL | BD | 309646 | |
| C12-200 Ionizable Lipid | Cayman Chemical | 36699 | |
| C57BL/6 Mice | Jackson Laboratory | 000664 | |
| Cholesterol | Sigma | 57-88-5 | |
| CleanCap FLuc mRNA (5moU) | TriLink Biotechnologies | L-7202 | |
| Disposable cuvettes | Fisher Scientific | 14955129 | |
| D-Luciferin, Potassium Salt | Thermo Scientific | L2916 | |
| DMEM, high glucose | Thermofisher Scientific | 11965-084 | |
| Exel Insulin Syringes – 0.5 mL | Fisher Scientific | 1484132 | |
| Fetal Bovine Serum | Corning | 35-010-CV | |
| Hep G2 [HEPG2] | ATCC | HB-8065 | |
| HyPure Molecular Biology Grade Water | Cytiva | SH30538.03 | |
| Infinite 200 PRO Plate Reader | Tecan | N/A | |
| IVIS Spectrum In Vivo Imaging System | Perkin Elmer | N/A | |
| Large Kimwipes | Fisher Scientific | 06-666-11D | |
| Luciferase Assay Kit | Promega | E4550 | |
| NanoAssemblr Ignite Cartridges – Classic – 100 Pack | Precision Nanosystems | NIN0065 | |
| NanoAssemblr Ignite Instrument | Precision Nanosystems | NIN0001 | |
| PBS – Phosphate-Buffered Saline (10x) pH 7.4, RNase-free | Thermo Scientific | AM9624 | |
| Penicillin-Streptomycin | Thermofisher Scientific | 15140122 | |
| QB Citrate Buffer, (Citrate 100 mM) pH 3.0 | Teknova | Q2442 | |
| Quant-it RiboGreen RNA Assay Kit | Thermo Scientific | R11490 | |
| Reporter Lysis 5x Buffer | Promega | E3971 | |
| RNase Away Surface Decontaminant | Thermofisher Scientific | 7000TS1 | |
| Sodium Chloride | Sigma | 7647-14-5 | |
| Sodium Hydroxide | Sigma | 1310-73-2 | |
| Sodium Phosphate | Sigma | 7601-54-9 | |
| TNS reagent (6-(p-Toluidino)-2-naphthalenesulfonic acid sodium salt) | Sigma | T9792 | |
| Triton X-100 | Sigma | 9036-19-5 | |
| Zetasizer | Malvern Panalytical | NanoZS |
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