Levering af eksogen kunstigt syntetiseret miRNA-efterligning til nyrerne ved hjælp af polyethylenaminnanopartikler i flere nyresygdomsmusemodeller
Summary
Her leverer vi eksogene kunstigt syntetiserede miRNA-efterligninger til nyrerne via haleveneinjektion af en ikke-viral vektor og polyethylenaminnanopartikler i flere nyresygdomsmusemodeller. Dette førte til signifikant overekspression af mål-miRNA i nyrerne, hvilket resulterede i hæmmet progression af nyresygdom i flere musemodeller.
Abstract
microRNA’er (miRNA’er), små ikke-kodende RNA’er (21-25 baser), der ikke oversættes til proteiner, hæmmer masser af target messenger RNA’er (mRNA’er) ved at destabilisere og hæmme deres oversættelse i forskellige nyresygdomme. Derfor er veksling af miRNA-ekspression ved eksogene kunstigt syntetiserede miRNA-efterligninger en potentielt nyttig behandlingsmulighed til at hæmme udviklingen af mange nyresygdomme. Men fordi serum-RNAase straks nedbryder systematisk administrerede eksogene miRNA-efterligninger in vivo, er levering af miRNA til nyrerne fortsat en udfordring. Derfor er vektorer, der kan beskytte eksogene miRNA-efterligninger mod nedbrydning af RNAase og levere dem signifikant til nyrerne, nødvendige. Mange undersøgelser har brugt virale vektorer til at levere eksogene miRNA-efterligninger eller hæmmere til nyrerne. Imidlertid kan virale vektorer forårsage interferonrespons og / eller genetisk ustabilitet. Derfor er udviklingen af virale vektorer også en hindring for den kliniske anvendelse af eksogene miRNA-efterligninger eller hæmmere. For at overvinde disse bekymringer vedrørende virale vektorer udviklede vi en ikke-viral vektormetode til at levere miRNA-efterligninger til nyrerne ved hjælp af haleveneinjektion af polyethylenaminnanopartikler (PEI-NP’er), hvilket førte til signifikant overekspression af mål-miRNA’er i flere musemodeller af nyresygdom.
Introduction
miRNA’er, små ikke-kodende RNA’er (21-25 baser), der ikke oversættes til proteiner, hæmmer masser af målbudbringer-RNA’er (mRNA’er) ved at destabilisere dem og hæmme deres oversættelse i forskellige nyresygdomme 1,2. Derfor er genterapi ved hjælp af eksogene kunstigt syntetiserede miRNA-efterligninger eller -hæmmere en potentiel ny mulighed for at hæmme udviklingen af mange nyresygdomme 3,4,5.
På trods af løftet om miRNA-efterligninger eller hæmmere til genterapi er levering til målorganer fortsat en stor hindring for in vivo-eksperimenter for at udvikle deres kliniske potentiale. Fordi kunstigt syntetiserede miRNA-efterligninger eller -hæmmere udsættes for øjeblikkelig nedbrydning af serum-RNase, forkortes deres halveringstid ved systemisk administration in vivo6. Derudover er effektiviteten af miRNA-efterligninger eller hæmmere til at krydse plasmamembranen og transfektcytoplasma generelt meget lavere uden passende vektorer 7,8. Disse beviser tyder på, at udviklingen af miRNA-efterligninger eller inhibitorer leveringssystem til nyrerne er påkrævet for at muliggøre deres anvendelse i kliniske omgivelser og gøre dem til en ny behandlingsmulighed for patienter med forskellige nyresygdomme.
Virale vektorer er blevet brugt som bærere til at levere eksogene miRNA-efterligninger eller hæmmere til nyrerne 9,10. Selvom de er udviklet til biosikkerhed og transfektionseffektivitet, kan virale vektorer stadig forårsage interferonrespons og / eller genetisk ustabilitet11,12. For at overvinde disse bekymringer udviklede vi et miRNA-efterligningsleveringssystem til nyrerne ved hjælp af polyethylenaminnanopartikler (PEI-NP’er), en ikke-viral vektor, i flere musemodeller af nyresygdom13,14,15.
PEI-NP’er er lineære polymerbaserede NP’er, der effektivt kan levere oligonukleotider, herunder miRNA-efterligninger, til nyrerne og anses for at foretrække til fremstilling af ikke-virale vektorer på grund af deres langsigtede sikkerhed og biokompatibilitet13,16,17.
Denne undersøgelse demonstrerer virkningerne af systematisk eksogen miRNA-efterligning med PEI-NP’er via haleveneinjektion i nyrefibrosemodelmus produceret ved ensidig ureterobstruktion (UUO). Derudover demonstrerer vi virkningerne af systematisk eksogen miRNA-efterligning med PEI-NP’er via haleveneinjektion i diabetiske nyresygdomsmodelmus (db / db-mus: C57BLKS / J Iar – + Lepr db / + Leprdb) og akut nyreskade modelmus produceret af renal iskæmi-reperfusionsskade (IRI).
Protocol
Representative Results
Discussion
Ved hjælp af protokollen præsenteret i dette manuskript kan PEI-NP’er levere miRNA-efterligninger til nyrerne for at inducere overekspression af mål-miRNA’er, hvilket resulterer i behandlingseffekter i in vivo-musemodeller af flere nyresygdomme, herunder nyrefibrose, diabetisk nyresygdom og AKI.
Metoden til at forberede komplekset af PEI-NP’er og miRNA-efterligning er meget enkel. Den positivt ladede overflade af PEI-NP’er fanger miRNA-efterligningen, når de bare blandes 13,14,15,…
Disclosures
The authors have nothing to disclose.
Acknowledgements
Dette arbejde blev delvist støttet af JSPS KAKENHI (tilskud nr. 21K08233). Vi takker Edanz (https://jp.edanz.com/ac) for at redigere udkast til dette manuskript.
Materials
| 4’,6-diamidino-2-phenylindole for staining to nucleus | Thermo Fisher Scientific | D-1306 | |
| Buffer RPE | Qiagen | 79216 | Wash buffer 2 |
| Buffer RWT | Qiagen | 1067933 | Wash buffer 1 |
| Control-miRNA-mimic (artificially synthesized miRNA) | Thermo Fisher Scientific | Not assigned | 5’-UUCUCCGAACGUGUCACGUTT- 3’ (sense) 5’-ACGUGACACGUUCGGAGAATT-3′ (antisense) |
| Cy3-labeled double-strand oligonucleotides | Takara Bio Inc. | MIR7900 | |
| Fluorescein-labeled Lotus tetragonolobus lectin | Vector Laboratories Inc | FL-1321 | |
| In vivo-jetPEI | Polyplus | 101000021 | |
| MicroAmp Optical 96-well reaction plate for qRT-PCR | Thermo Fisher Scientific | 4316813 | 96-well reaction plate |
| MicroAmp Optical Adhesive Film | Thermo Fisher Scientific | 4311971 | Adhesive film for 96-well reaction plate |
| miRNA-146a-5p mimic (artificially synthesized miRNA) | Thermo Fisher Scientific | Not assigned | 5’-UGAGAACUGAAUUCCAUGGGU UT-3′ (sense) 5’-CCCAUGGAAUUCAGUUCUCAUU -3′ (antisense) |
| miRNA-146a-5p primer | Qiagen | MS00001638 | Not available because Qiagen has changed qRT-PCR kits (from miScript miRNA PCR system to miRCURY LNA miRNA PCR System from May 2021) |
| miRNA-181b-5p mimic (artificially synthesized miRNA) | Gene design | Not assigned | 5’-AACAUUCAUUGCUGUCGGUGG GUU-3’ |
| miRNA-181b-5p primer | Qiagen | MS00006083 | Not available because Qiagen has changed qRT-PCR kits (from miScript miRNA PCR system to miRCURY LNA miRNA PCR System from May 2021) |
| miRNA-5100-mimic (artificially synthesized miRNA) | Gene design | Not assigned | 5’-UCGAAUCCCAGCGGUGCCUCU -3′ |
| miRNA-5100-primer | Qiagen | MS00042952 | Not available because Qiagen has changed qRT-PCR kits (from miScript miRNA PCR system to miRCURY LNA miRNA PCR System from May 2021) |
| miRNeasy Mini kit | Qiagen | 217004 | Membrane anchored spin column in a 2.0-mL collection tube |
| miScript II RT kit | Qiagen | 218161 | Not available because Qiagen has changed qRT-PCR kits (from miScript miRNA PCR system to miRCURY LNA miRNA PCR System from May 2021) |
| miScript SYBR Green PCR kit | Qiagen | 218073 | Not available because Qiagen has changed qRT-PCR kits (from miScript miRNA PCR system to miRCURY LNA miRNA PCR System from May 2021) |
| QIA shredder | Qiagen | 79654 | Biopolymer spin columns in a 2.0-mL collection tube |
| QIAzol Lysis Reagent | Qiagen | 79306 | Phenol/guanidine-based lysis reagent |
| QuantStudio 12K Flex Flex Real-Time PCR system | Thermo Fisher Scientific | 4472380 | Real-time PCR instrument |
| QuantStudio 12K Flex Software version 1.2.1. | Thermo Fisher Scientific | 4472380 | Real-time PCR instrument software |
| RNase-free water | Qiagen | 129112 | |
| RNU6-2 primer | Qiagen | MS00033740 | Not available because Qiagen has changed qRT-PCR kits (from miScript miRNA PCR system to miRCURY LNA miRNA PCR System from May 2021) |
| Tissue-Tek OCT (Optimal Cutting Temperature Compound) | Sakura Finetek Japan Co.,Ltd. | Not assigned |
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