Testing av in vitro og in vivo effektivitet av mRNA-lipid nanopartikler formulert ved mikrofluidisk blanding
Summary
Her presenteres en protokoll for formulering av lipid nanopartikler (LNP) som innkapsler mRNA som koder for firefly luciferase. Disse LNP-ene ble testet for deres styrke in vitro i HepG2-celler og in vivo i C57BL/6-mus.
Abstract
Lipid nanopartikler (LNP) har tiltrukket seg stor oppmerksomhet nylig med den vellykkede utviklingen av COVID-19 mRNA-vaksinene av Moderna og Pfizer / BioNTech. Disse vaksinene har demonstrert effekten av mRNA-LNP-terapeutika og åpnet døren for fremtidige kliniske applikasjoner. I mRNA-LNP-systemer fungerer LNP-ene som leveringsplattformer som beskytter mRNA-lasten mot nedbrytning av nukleaser og medierer deres intracellulære levering. LNPene består vanligvis av fire komponenter: et ioniserbart lipid, et fosfolipid, kolesterol og et lipidforankret polyetylenglykol (PEG) konjugat (lipid-PEG). Her formuleres LNP-er som innkapsler mRNA-kodende for firefly luciferase ved mikrofluidisk blanding av den organiske fasen som inneholder LNP-lipidkomponenter og den vandige fasen som inneholder mRNA. Disse mRNA-LNP-ene testes deretter in vitro for å evaluere deres transfeksjonseffektivitet i HepG2-celler ved hjelp av en bioluminescerende platebasert analyse. I tillegg evalueres mRNA-LNP in vivo hos C57BL/6-mus etter en intravenøs injeksjon via den laterale halevenen. Helkroppsbioluminescensavbildning utføres ved bruk av et in vivo bildesystem. Representative resultater er vist for mRNA-LNP-egenskapene, deres transfeksjonseffektivitet i HepG2-celler og den totale luminescerende fluksen i C57BL/6-mus.
Introduction
Lipid nanopartikler (LNP) har vist stort løfte de siste årene innen ikke-viral genterapi. I 2018 godkjente USAs Food and Drug Administration (FDA) den aller første RNA-interferens (RNAi) terapeutisk, Onpattro by Alnylam, for behandling av arvelig transtyretinamyloidose 1,2,3,4. Dette var et viktig skritt fremover for lipid nanopartikler og RNA-baserte terapier. Nylig mottok Moderna og Pfizer/BioNTech FDA-godkjenninger for sine mRNA-LNP-vaksiner mot SARS-CoV-2 4,5. I hver av disse LNP-baserte nukleinsyreterapiene tjener LNP til å beskytte lasten mot nedbrytning av nukleaser og legge til rette for potent intracellulær levering 6,7. Mens LNP har sett suksess i RNAi-terapier og vaksineapplikasjoner, har mRNA-LNP også blitt utforsket for bruk i proteinutskiftningsterapier8, samt for samlevering av Cas9 mRNA og veiledende RNA for levering av CRISPR-Cas9-systemet for genredigering9. Det finnes imidlertid ingen spesifikk formulering som er velegnet for alle anvendelser, og subtile endringer i LNP-formuleringsparametrene kan i stor grad påvirke potens og biodistribusjon in vivo 8,10,11. Dermed må individuelle mRNA-LNP utvikles og evalueres for å bestemme den optimale formuleringen for hver LNP-basert terapi.
LNP er vanligvis formulert med fire lipidkomponenter: et ioniserbart lipid, et fosfolipid, kolesterol og et lipidforankret polyetylenglykol (PEG) konjugat (lipid-PEG)11,12,13. Den potente intracellulære leveransen tilrettelagt av LNP er delvis avhengig av den ioniserbare lipidkomponenten12. Denne komponenten er nøytral ved fysiologisk pH, men blir positivt ladet i det sure miljøet til endosomet11. Denne endringen i ionisk ladning antas å være en viktig bidragsyter til endosomal flukt12,14,15. I tillegg til det ioniserbare lipidet forbedrer fosfolipidkomponenten (hjelperlipid) innkapslingen av lasten og hjelpemidler i endosomal flukt, kolesterolet gir stabilitet og forbedrer membranfusjon, og lipid-PEG minimerer LNP-aggregering og opsonisering i omløp10,11,14,16. For å formulere LNP kombineres disse lipidkomponentene i en organisk fase, typisk etanol, og blandes med en vandig fase som inneholder nukleinsyrelasten. LNP-formuleringsprosessen er svært allsidig ved at den gjør det mulig for forskjellige komponenter å enkelt substitueres og kombineres ved forskjellige molforhold for å formulere mange LNP-formuleringer med en rekke fysisk-kjemiske egenskaper10,17. Men når du utforsker dette store utvalget av LNP-er, er det avgjørende at hver formulering evalueres ved hjelp av en standardisert prosedyre for nøyaktig å måle forskjellene i karakterisering og ytelse.
Her er den komplette arbeidsflyten for formulering av mRNA-LNP og vurdering av deres ytelse i celler og dyr skissert.
Protocol
Representative Results
Discussion
Med denne arbeidsflyten kan en rekke mRNA-LNP-er formuleres og testes for in vitro og in vivo-effektivitet. Ioniserbare lipider og hjelpestoffer kan byttes ut og kombineres ved forskjellige molare forhold og forskjellige ioniserbare lipid til mRNA-vektforhold for å produsere mRNA-LNP med forskjellige fysisk-kjemiske egenskaper22. Her formulerte vi C12-200 mRNA-LNP med et molforhold på 35/16/46.5/2.5 (ioniserbart lipid: hjelper lipid: kolesterol: lipid-PEG) ved et 10: 1 ioniserb…
Disclosures
The authors have nothing to disclose.
Acknowledgements
MJM anerkjenner støtte fra en US National Institutes of Health (NIH) direktørens 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 tilleggsfinansiering 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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