Delivery of Modified mRNA in a Myocardial Infarction Mouse Model

Summary

This protocol presents a simple and coherent way to transiently upregulate a gene of interest using modRNA after myocardial infarction in mice.

Abstract

Myocardial infarction (MI) is a leading cause of morbidity and mortality in the Western world. In the past decade, gene therapy has become a promising treatment option for heart disease, owing to its efficiency and exceptional therapeutic effects. In an effort to repair the damaged tissue post-MI, various studies have employed DNA-based or viral gene therapy but have faced considerable hurdles due to the poor and uncontrolled expression of the delivered genes, edema, arrhythmia, and cardiac hypertrophy. Synthetic modified mRNA (modRNA) presents a novel gene therapy approach that offers high, transient, safe, nonimmunogenic, and controlled mRNA delivery to the heart tissue without any risk of genomic integration. Due to these remarkable characteristics combined with its bell-shaped pharmacokinetics in the heart, modRNA has become an attractive approach for the treatment of heart disease. However, to increase its effectiveness in vivo, a consistent and reliable delivery method needs to be followed. Hence, to maximize modRNA delivery efficiency and yield consistency in modRNA use for in vivo applications, an optimized method of preparation and delivery of modRNA intracardiac injection in a mouse MI model is presented. This protocol will make modRNA delivery more accessible for basic and translational research.

Introduction

Gene therapy is a powerful tool involving delivery of nucleic acids for the treatment, cure, or prevention of human diseases. Despite the progress in the diagnostic and therapeutic approaches for heart disease, there has been limited success in the delivery of genes in myocardial infarction (MI) and heart failure (HF). As straightforward as the process of gene therapy seems, it is a markedly complex approach considering the many factors that need to be optimized before employing a particular delivery vehicle. The correct delivery vector should be non-immunogenic, efficient, and stable inside the human body. Efforts in this field have generated two types of delivery sy....

Protocol

All animal procedures outlined here have been approved by Icahn School of Medicine at Mount Sinai Institutional Care and Use Committee.

1. Synthesis of modRNA

NOTE: The details of modRNA synthesis can be found in Kondrat et al.¹³.

1. Order the plasmid templates (Table of Materials) and generate a clean PCR product to use as the mRNA template.
2. Prepare the modRNAs by transcription in vitro with a customized ribonucleoside blend of the following (Table of Materials): T7 transcription kit, 6 mM anti-reverse cap analog (ARCA) 30-O-Me-m7G(50) ....

Results

Eight to ten-week-old mice were anesthetized with isoflurane and intubated. After the animal was under anesthesia, the left thoracic region was shaved and sterilized with ethanol, and the heart was exposed for LAD ligation. The left coronary artery was occluded by firmly knotting the suture under the artery (diagram representation Figure 1A). After a successful infarction (indicated by the paling of the left ventricular free wall), a direct injection of 100 µg of Luc or Cre modRNA dissolved .......

Discussion

Gene therapy has shown tremendous potential to significantly advance the treatment of cardiac disease. However, traditional tools employed in the initial clinical trials for treatment of HF have shown limited success and are associated with severe side effects. Modified RNA presents a nonviral gene delivery that is continuously gaining popularity as a gene transfer tool in the heart. ModRNA requires no nuclear localization of genes for translation, and thus offers an efficient and fast expression of the protein. Further,.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Adenosine triphosphate	Invitrogen	AMB13345	Included in Megascript kit
Antarctic Phosphatase	New England Biolabs	M0289L
Anti-reverse cap analog, 30-O-Mem7G(50) ppp(50)G	TriLink Biotechnologies	N-7003
Bioluminescense imaging system	Perkin Elmer	124262	IVIS100 charge-coupled device imaging system
Blunt retractors	FST	18200-09
Cardiac tropnin I	Abcam	47003
Cytidine triphosphate	Invitrogen	AMB13345	Included in Megascript kit
Dual Anesthesia System	Harvard Apparatus	75-2001
Forceps- Adson	FST	91106-12
Forceps- Dumont #7	FST	91197-00
Guanosine triphosphate	Invitrogen	AMB13345	Included in Megascript kit
In vitro transcription kit	Invitrogen	AMB13345	5X MEGAscript T7 Kit
Intubation cannula	Harvard Apparatus
Megaclear kit	Life Technologies
Mouse ventilator	Harvard Apparatus	73-4279
N1-methylpseudouridine-5-triphosphate	TriLink Biotechnologies	N-1081
NanoDrop Spectrometer	Thermo Scientific
Olsen hegar needle holder with suture scissors	FST	12002-12
Plasmid templates	GeneArt, Thermo Fisher Scientific
Sharp-Pointed Dissecting Scissors	FST	14200-12
Stereomicroscope	Zeiss
Sutures	Ethicon	Y433H	5.00
Sutures	Ethicon	Y432H	6.00
Sutures	Ethicon	7733G	7.00
T7 DNase enzyme	Invitrogen	AMB13345	Included in Megascript kit
Tape station	Aligent	4200
Transcription clean up kit	Invitrogen	AM1908	Megaclear
Ultra-4 centrifugal filters 10k	Amicon	UFC801096