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Summary
Abstract
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Protocol
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Bioengenharia

Generation af kationiske Nanoliposomes til effektiv levering af In Vitro transskriberet Messenger RNA

Published: February 01, 2019
doi:
10.3791/58444
, , , , , , ,
1Department of Thoracic and Cardiovascular Surgery, Clinical Research Laboratory,University Medical Center, 2Atherothrombosis and Vascular Biology,Baker Heart & Diabetes Institute, 3Department of Medicine,Monash University

Summary

Her beskriver vi en protokol for generation af kationiske nanoliposomes, som er baseret på metoden tør-film og kan bruges til sikker og effektiv levering af in vitro- transskriberet messenger RNA.

Abstract

Udviklingen af messenger RNA (mRNA)-baseret terapi til behandling af forskellige sygdomme bliver mere og mere vigtig på grund af positive egenskaber af in vitro transskriberet (IVT) mRNA. Ved hjælp af IVT mRNA, kan være fremkaldt de novo -syntesen af en ønskede protein uden ændring af målcellen fysiologiske tilstand. Derudover kan proteinsyntese styres netop på grund af forbigående effekten af IVT mRNA.

For den effektive Transfektion af celler, kan nanoliposomes (NLps) repræsenterer en sikker og effektiv levering køretøj for terapeutisk mRNA. Denne undersøgelse beskriver en protokol for at generere sikre og effektive kationiske NLps bestående af DC-kolesterol og 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) som en levering vektor for IVT mRNA. NLps har en defineret størrelse, en homogen fordeling og en høj compleksering kapacitet og kan produceres ved hjælp af metoden tør-film. Desuden præsenterer vi forskellige testsystemer for at analysere deres kompleks og Transfektion efficacies ved hjælp af syntetiske forbedret grøn fluorescerende proteiner (eGFP) mRNA, samt deres effekt på cellernes levedygtighed. Samlet set giver præsenteres protokollen en effektiv og sikker tilgang til mRNA kompleks, som kan fremme og forbedre forvaltningen af terapeutiske mRNA.

Introduction

Brug af modificerede mRNA for terapeutisk anvendelse har vist stort potentiale i de sidste par år. I hjerte-kar-, provokerende og monogenetic sygdomme, samt i udviklingen af vacciner, er mRNA en lovende terapeutisk agent1.

Protein substitutionsterapi med mRNA tilbyder flere fordele i forhold til den klassiske genterapi, som er baseret på DNA Transfektion i target celler2. Funktionen mRNA indleder direkte i cytosol. Selvom plasmid DNA (pDNA), en konstruktion af dobbelt-strenget, cirkulært kan DNA indeholdende en promotor-regionen og en gensekvens, kodning den terapeutiske protein3, også handlinger i cytosol, det kun indgå i celler, som går gennem mitosen på tidspunktet for Transfektion. Dette reducerer antallet af transfekteret celler i væv1,4. Specifikt er Transfektion af væv med svag mitose aktivitet, såsom hjerte celler, vanskeligt5. I modsætning til pDNA forekomme Transfektion og oversættelse af mRNA i mitotiske og ikke-mitotiske celler i væv1,6. Viral integration af DNA i host-genom kan komme med mutagene virkninger eller immun reaktioner7,8, men efter Transfektion af celler med en protein-kodning mRNA, de novo -syntesen af det ønskede protein starter autonomt9,10. Derudover kan proteinsyntese justeres præcist til patientens behov gennem individuelle doser, uden at gribe ind i genomet og risikere mutagene virkninger11. Den immun aktivering potentiale af syntetisk genererede mRNA kunne sænkes drastisk ved hjælp af pseudo-uridine og 5′-methylcytidine i stedet for uridine og cytidine12. Pseudo-uridine modificerede mRNA har også vist sig at have en øget biologiske stabilitet og en betydeligt højere translationel kapacitet13.

For at kunne drage fordel af de lovende egenskaber af mRNA-baseret terapi i kliniske anvendelser, er det vigtigt at skabe et passende køretøj til transport af mRNA ind i cellen. Dette køretøj skal være forsynet med ikke-toksiske egenskaber i in vitro og i vivo, beskytte mRNA mod nukleasen-nedbrydning og give tilstrækkelig cellulære optagelse for en langvarig ledighed og oversættelse af mRNA14.

Blandt alle mulige carrier typer for i vivo drug delivery, såsom kulstof-nanorør, quantum dots og Liposomer, sidstnævnte har været studeret mest15,16. Liposomer er vesikler bestående af en lipid tolagede10. De er amphiphilic med en hydrofob og en hydrofil sektion, og gennem selvstændig ordningen af disse molekyler, en sfærisk dobbelt lag er dannet17. Inde i Liposomer, kan terapeutiske agenter eller narkotika være indkapslet og dermed beskyttet mod Enzymatisk nedbrydning18. Liposomer som indeholder N-[1-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium chlorid (DOTMA)19, [1,2-bis (oleoyloxy) -3-(trimethylammonio) propan] (DOTAP)20, og dioctadecylamidoglycylspermine (hunde)21, eller DC-kolesterol22, er godt præget og ofte benyttes til cellulære Transfektion med DNA eller RNA.

Kationiske Liposomer består af et positivt ladede lipid og et tomt phospholipid23. Transfektion via kationiske Liposomer er en af de mest almindelige metoder til transport af nukleinsyrer i celler24,25. Kationiske lipid partikler danner komplekser med negativt ladede fosfat-grupper i rygraden i nukleinsyre molekyler26. Disse såkaldte lipoplexes tillægger overfladen af cellemembranen og komme ind i cellen via endocytose eller endocytose-lignende-mekanismer27.

I 1989, Malone mfl. korrekt beskrevet kationiske lipid-medieret mRNA Transfektion28. Men ved hjælp af en blanding af DOTMA og 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), gruppen fandt at DOTMA manifesteret cytotoksiske virkninger28. Derudover viste Zohra et al. , at DOTAP (1,2-dioleoyloxy-3-trimethylammoniumformiat-propan chlorid) kan bruges som et mRNA Transfektion reagens29. For den effektive Transfektion af celler, bør DOTAP anvendes i kombination med andre reagenser, såsom fibronektin29 eller DOPE30. Hidtil har var DOTMA den første kationiske lipid på markedet anvendes til gen levering31. Andre lipider er brugt som terapeutisk luftfartsselskaber eller testes i forskellige stadier af kliniske forsøg, (f.eks. EndoTAG-I, som indeholder DOTAP som lipid transportør), undersøges i øjeblikket i et fase II kliniske forsøg32.

Dette arbejde beskriver en protokol for generation af NLps indeholdende DC-kolesterol og DOPE. Denne metode er nem at udføre og giver mulighed for generation af NLps i forskellige størrelser. Det generelle mål med NLp generation ved hjælp af metoden tør-film er at skabe Liposomer til mRNA kompleks, således at effektiv og biokompatible celle Transfektion in vitro-14,33.

Protocol

1. generation af kationiske Nanoliposomes (figur 1) Opløse lipider DC-kolesterol (3β-[N-(N′,N′-dimethylaminoethane)-carbamoyl] kolesterol hydrochlorid) og DOPE (dioleoyl phosphatidylethanolamin), leveres som et pulver i chloroform at opnå en endelig koncentration på 25 mg/mL.Bemærk: Gemme de opløste lipider ved-20 ° C. Arbejde med 25 mg/mL stamopløsning af både lipider. Mix 40 µL af den opløste DC-kolesterol og 80 µL af den opløste DOPE i et glas kolb…

Representative Results

Ved hjælp af protokollen som beskrevet, blev NLps bestående af lipider DC-kolesterol og DOPE udarbejdet ved hjælp af metoden tør-film (figur 1). Under forberedelsen viser nanoliposome løsning forskellige stadier i turbiditet (figur 2). Indkapsling effekten af NLps kan derefter analyseres efter indkapsling af 1 µg for eGFP-kodning mRNA ved at analysere den gratis m…

Discussion

Den præsenterede protokol beskriver generation af NLps med høj indkapsling effekten for syntetisk modificerede mRNA, samt den pålidelige Transfektion af celler i vitro. Desuden, NLps sikre frigivelsen af mRNA, som til gengæld er oversat til en funktionel proteiner inde i cellerne. Derudover transfections ved hjælp af NLps kan udføres i regelmæssig celle medium, resulterer i høj celle viabilities under Transfektion, og vare op til tre dage efter Transfektion.

For at bruge mRNA …

Declarações

The authors have nothing to disclose.

Acknowledgements

Ingen

Materials

(3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) AppliChem, Darmstadt, Germany A2231
(3β-[N-(N′,N′-dimethylaminoethane)-carbamoyl]cholesterol hydrochloride (DC-Cholesterol) Avanti, Alabama, USA 700001
4 ′,6-diamidino-2-phenylindole (DAPI) Thermo Fisher Scientific, Darmstadt, Germany D1306
BD FACScan system BD Biosciences, Heidelberg, Germany
Cell Fix (10x) BD Biosciences, Heidelberg, Germany 340181
Chloroform Merck, Darmstadt, Germany 102445
Dimethyl sulfoxid (DMSO) Serva Electrophoresis GmbH, Heidelberg, Germany 20385.02
Dioleoyl phosphatidylethanolamine (DOPE) Avanti, Alabama, USA 850725
Fluorescence microscope Zeiss Axio, Oberkochen, Germany
Lipofectamine 2000 Thermo Fisher Scientific, Darmstadt, Germany 11668019
Mini extruder Avanti, Alabama, USA
Nuclease-free water Qiagen, Hilden, Germany 129114
Opti-Mem Thermo Fisher Scientific, Darmstadt, Germany 11058021
PBS buffer (w/o Ca2+/Mg2+) Thermo Fisher Scientific, Darmstadt, Germany 70011044
Quant-iT Ribo Green RNA reagent kit Thermo Fisher Scientific, Darmstadt, Germany Q33140
RPMI (w/o phenol red) Thermo Fisher Scientific, Darmstadt, Germany 11835030
Silica gel Carl Roth, Karlsruhe, Germany P077
Trypsin/EDTA (0.05%) Thermo Fisher Scientific, Darmstadt, Germany 25300054
HotStar HiFidelity Polymerase Kit Qiagen, Hilden, Germany 202602
QIAquick PCR Purification Kit Qiagen, Hilden, Germany 28104

Pseudouridine-5'-Triphosphate (Ψ-UTP)		 TriLink Biotechnologies, San Diego, USA N-1019
5-Methylcytidine-5'-Triphosphate (Methyl-CTP) TriLink Biotechnologies, San Diego, USA N-1014
Cyanine 3-CTP PerkinElmer, Baesweiler, Germany NEL580001EA
RNeasy Mini Kit Qiagen, Hilden, Germany 74104
MEGAscript T7 Transcription Kit Thermo Fisher Scientific, Darmstadt, Germany AM1333
3´-O-Me-m7G(5')ppp(5')G RNA Cap Structure Analog New England Biolabs, Ipswich, USA S1411L
Antarctic Phosphatase New England Biolabs, Ipswich, USA M0289S
Agarose Thermo Fisher Scientific, Darmstadt, Germany 16500-500
GelRed Biotium, Fremont, USA 41003
peqGOLD DNA ladder mix VWR, Pennsylvania, USA 25-2040
Invitrogen 0.5-10kb RNA ladder Fisher Scientific, Göteborg,
Sweden		 11528766
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Tags

Cationic NanoliposomesMRNA DeliveryTransfectionNanoparticleLipid FilmExtrusionNanolipoplexA549 Cells
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Michel, T., Link, A., Abraham, M., Schlensak, C., Peter, K., Wendel, H., Wang, X., Krajewski, S. Generation of Cationic Nanoliposomes for the Efficient Delivery of In Vitro Transcribed Messenger RNA. J. Vis. Exp. (144), e58444, doi:10.3791/58444 (2019).
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