This protocol presents a simple and coherent way to transiently upregulate a gene of interest using modRNA after myocardial infarction in mice.
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.
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 systems: viral or non-viral. The widely used viral systems, including gene transfer by adenovirus, retrovirus, lentivirus, or adeno-associated virus, have shown exceptional transduction capacity. However, their use in clinics is limited due to the strong immune response induced1, risk of tumorigenesis2, or the presence of neutralizing antibodies3, all of which remain a major obstacle to broad and effective application of viral vectors in human gene therapy. On the other hand, despite their impressive expression pattern, the delivery of naked plasmid DNA displays a low transfection efficiency, while mRNA transfer presents high immunogenicity and susceptibility to degradation by RNase4.
With the extensive research in the field of mRNA, modRNA has become an attractive tool for delivery of genes to the heart and various other organs due to its numerous advantages over traditional vectors5. Complete replacement of uridine with naturally occurring pseudouridine results in more robust and transient protein expression, with minimal induction of innate immune response and risk of genomic integration6. Recently established protocols use an optimized amount of anti-reverse cap analog (ARCA) that further enhances the protein translation by increasing the stability and transability of the synthetic mRNA7.
Previous reports have shown the expression of various reporter or functional genes delivered by modRNA in the rodent myocardium after MI. With modRNA applications, significant areas of the myocardium, including both cardiomyocytes and noncardiomyocytes, have been successfully transfected post-cardiac injury8 to induce angiogenesis9,10, cardiac cell survival11, and cardiomyocyte proliferation12. A single administration of modRNA encoded for mutated human follistatin-like 1 induces the proliferation of mouse adult CMs and significantly increases cardiac function, decreases scar size, and increases capillary density 4 weeks post-MI12. A more recent study reported improved cardiac function after MI with application of VEGFA modRNA in a swine model10.
Thus, with the increased popularity of modRNA in the cardiac field, it is essential to develop and optimize a protocol for the delivery of modRNA to the heart post-MI. Herein is a protocol describing the preparation and delivery of purified and optimized modRNA in a biocompatible citrate-saline formulation that provides robust, stable protein expression without stimulating any immune response. The method shown in this protocol and video demonstrates the standard surgical procedure of a mouse MI by permanent ligation of the left anterior descending artery (LAD), followed by three site intracardiac injections of modRNA. The aim for this paper is to clearly define a highly accurate and reproducible method of modRNA delivery to the murine myocardium to make modRNA application widely accessible for cardiac gene therapy.
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.
2. Preparation of modRNA injection for in vivo delivery
3. Myocardial infarction surgery
4. Cardiac delivery of modRNA
5. Protein expression validation in heart post MI
6. Statistical analysis
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 in sucrose citrate buffer was delivered directly into the myocardium at three different sites (Figure 1B) surrounding the injury area using an insulin syringe. The MI procedure with modRNA injections lasted for 30−45 min per animal. The animals showed approximately 90% survival rate postprocedure. After the procedure, the chest and the skin were firmly sutured in layers and the animal was removed from ventilation as soon as it started breathing normally.
After conducting the LAD ligation and subsequent delivery of Luc modRNA injection, we validated the Luc modRNA transfection by checking for Luc protein expression 24 h postinjection using a bioluminescence imaging system (Figure 2A). We established in previous publications that although the protein expression can be seen until day 6 posttransfection, the highest transfection efficiency of modRNA is observed at 24 h8. Similarly, we successfully detected the Luc signal in the heart treated with Luc modRNA injection (1.76 x 108) compared to the mice injected with sucrose citrate buffer after MI (3.0 x 105) (Figure 2B).
Further, we sought to validate the modRNA expression by checking its translation and biodistribution in a transgenic Rosa26mTmG mouse. This mouse model system expresses the cell membrane-localized tdTomato (mT) fluorescence expression in all body cells/tissues and changes to cell membrane-localized EGFP (mG) fluorescence expression upon Cre recombination. Thus, to observe the expression of the Cre modRNA, 100 μg Cre modRNA was injected directly into the myocardium post-MI in Rosa26mTmG male and female mice, and the animals were sacrificed 24 h postsurgery. Hearts were fixed and processed for immunostaining with cardiomyocyte marker cTNI and nuclear marker DAPI (Figure 3A). Successful Cre expression was evident due to the appearance of green colored cells (Figure 3B), which were a result of recombination with the Cre modRNA delivered to the mouse, represented by the change of the tdTomato color to EGFP around the Cre injection site (Figure 3C).
Figure 1: LAD ligation and cardiac delivery of modRNA. (A) Schematic diagram showing the area of LAD ligation and three sites of modRNA injection. (B) Representative images of the whole mouse heart post a successful MI induced by permanent ligation (a) and sites of modRNA injection following the MI. The 100 µg of modRNA dissolved in 60 µL of sucrose citrate buffer was delivered in the border zone area surrounding the infarction, two on either side of the ligation (b,c) and one in the apex (d). Scale bar = 1 cm. Please click here to view a larger version of this figure.
Figure 2: Luc expression analysis post modRNA injection. Sucrose citrate buffer containing 100 µg of Luc modRNA was injected directly into myocardium of CFW mice in an open-chest surgery. The bioluminescence imaging system was used to calculate Luc protein expression at 24 hours after injection. (A) Comparative bioluminescent images of control mice (transfected with buffer only) vs. mice injected with Luc modRNA. (B) Quantification of Luc signal compared with the control mice measured after 24 hours using bioluminescence imager. Error bar represents SEM with p < 0.0001. Please click here to view a larger version of this figure.
Figure 3: Validation of Cre expression in vivo. Representative images of transfected heart cross sections (short-axis view) validating the expression of Cre modRNA in Rosa26mTmG mouse 24 hours postinjection. (A) The cardiomyocytes immunostained with cTNI (red). (B) Green colored cells represent the Cre transfected cells. (C) Merged image showing Cre activated cells around the two injections. Blue is the nuclear stain DAPI. Scale bar = 1 cm. Please click here to view a larger version of this figure.
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, as the mRNA does not integrate into the genome host, gene delivery by modRNA skips the risk of mutagenesis. Consequently, owing to its advantages over conventional gene delivery vehicles, modRNA has become one of the most attractive platforms for delivery of single gene or gene combinations to the heart. However, to date there is no standardized protocol for the preparation and transfer of the modRNA into the heart postinjury. Thus, the aim here was to establish a standard and optimized protocol for intracardiac delivery of modRNA in rodent heart to improve the use of modRNA as a gene therapy tool and to make it suitable for therapeutic purposes.
In this protocol, modRNA is prepared by replacing uridine with pseudouridine, followed by capping with ARCA at the 5’ end. These changes in the secondary structure of mRNA have been shown to render higher protein translation compared to various other nucleotide modifications8. Moreover, this altered mRNA structure escapes the immunogenicity after IM injections in mice by limiting its recognition by toll-like receptors and nucleases6. Here, we showed that naked mRNA delivery with sucrose citrate buffer produces strong protein translation in the heart using a mouse MI model. In our previous studies, we established the superiority of modRNA delivered with sucrose citrate buffer in the heart in comparison to encapsulation of modRNA in nanoparticles, such as in vivo fectamine or in vivo jetPEI, which may hinder modRNA translation into protein8. The high preservation of the RNA by citrate and extra energy provided by the sucrose for endocytosis in addition of rescuing the single-stranded modRNA clumping could be the reason for the marked increase in translation of modRNA delivered in sucrose citrate buffer.
Although the use of modRNA has escalated in preclinical and clinical studies10,14 in the past decade, certain areas need to be investigated for improved success of modRNA in the clinic. First, synthesizing modRNA in the high quantities required for therapeutic investigations can be cost-prohibitive. Thus, it is essential to develop clinical-grade and cost-effective modRNA to move the field toward a clinical RNA therapy phase in humans. Second, a clinically applicable delivery method for modRNA needs to be identified. Considering the damage caused by the cardiac IM injections, less invasive catheter-based delivery methods might be more plausible in transferring the large amount of modRNA necessary to transfect large hearts.
In conclusion, this work demonstrated the successful delivery of RNA containing pseudouridine modifications in the mouse heart post MI. The delivery of modRNA in sucrose citrate buffer yielded a strong protein expression 24 hours after the injection. This protocol enables the researchers to follow a standardized delivery and protein evaluation in the heart post injury and thus provide more accessibility in the preparation and delivery of modRNA for their research.
The authors have nothing to disclose.
The authors acknowledge Ann Anu Kurian for her help with this manuscript. This work was funded by a cardiology start-up grant awarded to the Zangi laboratory and also by NIH grant R01 HL142768-01
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 |