Delivery of Exogenous Artificially Synthesized miRNA Mimic to the Kidney Using Polyethylenimine Nanoparticles in Several Kidney Disease Mouse Models

Summary

Here, we deliver exogenous artificially synthesized miRNA mimics to the kidney via tail vein injection of a nonviral vector and polyethylenimine nanoparticles in several kidney disease mouse models. This led to significant overexpression of target miRNA in the kidney, resulting in inhibited progression of kidney disease in several mouse models.

Abstract

microRNAs (miRNAs), small noncoding RNAs (21-25 bases) that are not translated into proteins, inhibit lots of target messenger RNAs (mRNAs) by destabilizing and inhibiting their translation in various kidney diseases. Therefore, alternation of miRNA expression by exogenous artificially synthesized miRNA mimics is a potentially useful treatment option for inhibiting the development of many kidney diseases. However, because serum RNAase immediately degrades systematically administered exogenous miRNA mimics in vivo, delivery of miRNA to the kidney remains a challenge. Therefore, vectors that can protect exogenous miRNA mimics from degradation by RNAase and significantly deliver them to the kidney are necessary. Many studies have used viral vectors to deliver exogenous miRNA mimics or inhibitors to the kidney. However, viral vectors may cause an interferon response and/or genetic instability. Therefore, the development of viral vectors is also a hurdle for the clinical use of exogenous miRNA mimics or inhibitors. To overcome these concerns regarding viral vectors, we developed a nonviral vector method to deliver miRNA mimics to the kidney using tail vein injection of polyethylenimine nanoparticles (PEI-NPs), which led to significant overexpression of target miRNAs in several mouse models of kidney disease.

Introduction

miRNAs, small noncoding RNAs (21-25 bases) that are not translated into proteins, inhibit lots of target messenger RNAs (mRNAs) by destabilizing them and inhibiting their translation in various kidney diseases^1,2. Therefore, gene therapy employing exogenous artificially synthesized miRNA mimics or inhibitors is a potential new option for inhibiting the development of many kidney diseases^3,4,5.

Despite the promise of miRNA mimics or inhibitors for gene therapy, delivery to target organs rema....

Protocol

All animal experimental protocols were approved by the animal ethics committee of Jichi Medical University and performed in accordance with Use and Care of Experimental Animals guidelines from the Jichi Medical University Guide for Laboratory Animals. Here, we demonstrated miRNA mimic delivery to the kidney resulting in its overexpression using UUO mice. This study was approved by the Ethics Committee of Jichi Medical University [Approval Nos. 19-12 for renal fibrosis, 17-024 for acute kidney infection (AKI), and 19-11 f.......

Discussion

Using the protocol presented in this manuscript, PEI-NPs can deliver miRNA mimics to the kidney to induce overexpression of target miRNAs, resulting in treatment effects in in vivo mouse models of several renal diseases, including renal fibrosis, diabetic kidney disease, and AKI.

The method to prepare the complex of PEI-NPs and miRNA mimic is very simple. The positively charged surface of PEI-NPs entraps the miRNA mimic when they are just mixed^13,

Materials

Name	Company	Catalog Number	Comments
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