The authors acknowledge Ann Anu Kurian&#160;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

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.
<ol>
	<li>Order the plasmid templates (Table of Materials) and generate a clean PCR product to use as the mRNA template.</li>
	<li>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) ppp(50)G, 75 mM guanosine triphosphate, 75 mM adenosine triphosphate, 75 mM cytidine triphosphate, 100 mM pseudouridine-5-triphosphate, and T7 DNase enzyme.</li>
	<li>Purify the modRNA made in step 1.2 with a transcription cleanup kit (Table of Materials) and treat with antarctic phosphatase. Next, repurify the modRNA with the transcription cleanup kit and quantify it using a spectrophotometer.</li>
	<li>Precipitate the modRNA with ethanol and ammonium acetate and resuspend in 10 mM Tris-HCl and 1 mM EDTA.</li>
	<li>Concentrate the modRNA for in vivo delivery by centrifugal filters and measure the quality using an automated electrophoresis platform (Table of Materials).</li>
</ol>
2. Preparation of modRNA injection for in vivo delivery
<ol>
	<li>Calculate the volume needed for 100 &#181;g of luciferase (Luc)/Cre modRNA injection based on the modRNA concentration.</li>
	<li>Mix the calculated volume of modRNA with equal parts of sucrose solution prepared in nuclease free water (0.3 g/mL) and 0.1 M citrate solution (pH = 7) (20 &#181;L each recommended).</li>
	<li>Bring the final volume of the injection to 60 &#181;L by adding saline solution.</li>
</ol>
3. Myocardial infarction surgery
<ol>
	<li>Anesthesia and preparation of the animal 
	NOTE: This study uses male and female CFW mice 8&#8722;12 weeks old.
	<ol>
		<li>Anesthetize the mouse with 2% isoflurane using an induction chamber and place the animal on its back in a dorsal position with a facemask over its nose and mouth to keep up the anesthesia. Maintain the anesthesia by mechanical ventilation using a respirator equipped with a 7&#8722;8 mm airway tube and a filter.</li>
		<li>To immobilize the mouse on the surgery platform, secure its limbs with surgical tape. shave the neck area and the left side of the ribcage and disinfect using 70% ethanol and povidone iodine. Repeat the disinfecting step twice. Check its reflexes, pinching the tail and hind feet to assess the depth of anesthesia.</li>
		<li>Continuously monitor and maintain the core body temperature at normothermia (37.5&#8722;38 &#176;C).</li>
		<li>Maintain an open airway by performing intratracheal intubation. To do this, place the mouse in a ventral position with its mouth facing towards the experimenter and gently pull out the tongue. Adjust the angle of the neck and straighten the intubation path and push in the intubation cannula.</li>
		<li>In case the animal is unable to breathe through it, tracheostomy can be performed. Perform a cervical incision along the midline isolating the skin, muscle, and tissue outlining the trachea using a microscope. When the trachea is clearly visible, insert the endotracheal tube into the tissue between two cartridge rings below the glottis by making a small hole and holding the cranial part of the trachea using microsurgical forceps.</li>
		<li>Observe the thoracic movement of the mouse to verify that both lungs are receiving oxygen. The respiratory rate (RR) should be approximately 120 breaths per minute, with an inspiratory pressure of 17&#8722;18 cm H2O.</li>
	</ol>
	</li>
	<li>Left anterior descending (LAD) artery ligation
	<ol>
		<li>To perform the LAD ligation, turn the mouse carefully so it is lying on its right side. Lift the skin and perform a left sided thoracotomy between the 3rd and the 4th rib and dissect the tissue and muscle carefully. Use a cautery to prevent bleeding.</li>
		<li>Open the thorax carefully and once it is open, find the heart, without touching the lung with any sharp object. Place the retractors into the incision to keep the thoracic cavity open and have a clear view of the heart. Now move the lungs to the edge of the incision and remove the part of the pericardial sac that is covering the heart to access the anterior surface of the heart.</li>
		<li>Carefully identify the LAD, which can be seen as a deep light red vessel located between the pulmonary artery and the left auricle. Ligate the LAD proximal with one single suture of a 7-0 silk suture. Place a chest tube (28 G, venal catheter), between the 4th and the 5th rib.</li>
		<li>Close the thoracic incision in layers. Use the 6-0 silk running sutures to adapt the ribs and use 5-0 silk running sutures to close the skin. Make sure to leave a proper window for future echocardiographic measurement.</li>
		<li>Carefully drain the thorax with warm isotonic solution (9 g of sodium chloride in 1 L of water) using a 2 mL syringe. Place the mouse on its back. If performing tracheostomy, take the endotracheal tube out and using 7-0 silk sutures adapt the tracheal cartridge rings with one single stitch. Place the face mask on the mouse and close the skin using 5-0 silk running sutures.</li>
		<li>For the recovery phase, disconnect the intubation cannula from the ventilator and allow spontaneous breathing. Place the animal under a heat lamp until it attains consciousness. Do not leave the animal unattended after surgery until it is fully awake.</li>
		<li>Manage pain therapy with buprenophine at 0.1 mg/kg body weight, injected subcutaneously for the next 3 days at 12 h intervals.</li>
	</ol>
	</li>
</ol>
4. Cardiac delivery of modRNA
<ol>
	<li>Deliver a total of 60 &#181;L of the modRNA prepared in step 2.3 intramuscularly (IM) using an insulin syringe (31 G) following an MI.
	<ol>
		<li>Inject 20 &#181;L of the modRNA at three different sites of the heart muscle surrounding the infarct area (two on either side of ligation and one in the apex). Perform the injections immediately after the LAD, before closing the chest. 
		NOTE: Representative images of modRNA injection sites can be seen in the Figure 1.</li>
	</ol>
	</li>
</ol>
5. Protein expression validation in heart post MI
<ol>
	<li>Luciferase modRNA expression using a bioluminescence imaging system 
	NOTE: A total of 100 &#181;g of Luc mRNA prepared in 60 &#181;L of the sucrose citrate buffer is directly injected into the heart of CFW mice after the MI.
	<ol>
		<li>At 24 h post MI and modRNA injection, anesthetize the mice with 2% isoflurane using an induction chamber and intraperitoneally inject luciferin (150 mg/g body weight) to validate the Luc signal in vivo.</li>
		<li>Image the mice using a bioluminescence imaging system every 2 min until the Luc signal reaches saturation.</li>
		<li>Quantify the collected imaging data with imaging analysis software. Use the mice injected with saline only as a baseline reading for Luc expression and subtract the background signal collected from the saline-injected mice. 
		NOTE: Luc signal is expressed in p/s/cm2/sr x 106.</li>
	</ol>
	</li>
	<li>Immunostaining for Cre transfection validation
	<ol>
		<li>To validate the transfection with Cre, sacrifice the animals 24 h postinjection using an intraperitoneal injection of 100 mg/kg ketamine and 10 mg/kg xylazine followed by cervical dislocation. Before making an incision, disinfect the chest and abdomen using a 70% alcohol swab.</li>
		<li>Open the thoracic cavity by making a transverse incision ~1 cm lower than the sternum and move the scissors towards the head, cutting through the rib cage.</li>
		<li>Open the chest and inject 1 mL of sterile PBS in the right ventricular chamber to remove excess blood. Excise the heart immediately and place it in the sterile PBS to wash away the remaining blood.</li>
		<li>Fix the heart in 4% paraformaldehyde (PFA) for 24 h, followed by overnight incubation in 30% sucrose solution at 4 &#176;C. The following day embed the hearts in optimal cutting temperature medium and section them at a thickness of 10 &#181;m using a cryostat.</li>
		<li>Stain the sections with primary antibody against cardiac troponin I (cTNI) and label them with a fluorescent secondary antibody. To identify the nucleus, stain with 4&#39;,6-diamidino-2-phenylindole (DAPI) for 5 min. Image the slides using a fluorescent microscope.</li>
	</ol>
	</li>
</ol>
6. Statistical analysis
<ol>
	<li>Plot a bar graph based on the Luc expression in saline and Luc-injected mice and report the values as mean &#177; SEM. Compare the two groups using an unpaired t-test (****p &#60; 0.0001).</li>
</ol>

<ol>
	<li>Muruve, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+innate+immune+response+to+adenovirus+vectors.">The innate immune response to adenovirus vectors.</a> Human Gene Therapy. 15 (12), 1157-1166 (2004).</li><li>Donsante, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Observed+incidence+of+tumorigenesis+in+long-term+rodent+studies+of+rAAV+vectors.">Observed incidence of tumorigenesis in long-term rodent studies of rAAV vectors.</a> Gene Therapy. 8 (17), 1343-1346 (2001).</li><li>Calcedo, R., Wilson, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Humoral+Immune+Response+to+AAV.">Humoral Immune Response to AAV.</a> Frontiers in Immunology. 4, 341 (2013).</li><li>Diebold, S. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nucleic+acid+agonists+for+Toll-like+receptor+7+are+defined+by+the+presence+of+uridine+ribonucleotides.">Nucleic acid agonists for Toll-like receptor 7 are defined by the presence of uridine ribonucleotides.</a> European Journal of Immunology. 36 (12), 3256-3267 (2006).</li><li>Magadum, A., Kaur, K., Zangi, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=mRNA-Based+Protein+Replacement+Therapy+for+the+Heart.">mRNA-Based Protein Replacement Therapy for the Heart.</a> Molecular Therapy. 27 (4), 785-793 (2019).</li><li>Kariko, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Incorporation+of+pseudouridine+into+mRNA+yields+superior+nonimmunogenic+vector+with+increased+translational+capacity+and+biological+stability.">Incorporation of pseudouridine into mRNA yields superior nonimmunogenic vector with increased translational capacity and biological stability.</a> Molecular Therapy. 16 (11), 1833-1840 (2008).</li><li>Hadas, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optimizing+Modified+mRNA+In+Vitro+Synthesis+Protocol+for+Heart+Gene+Therapy.">Optimizing Modified mRNA In Vitro Synthesis Protocol for Heart Gene Therapy.</a> Molecular Therapy- Methods and Clinical Development. 14, 300-305 (2019).</li><li>Sultana, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optimizing+Cardiac+Delivery+of+Modified+mRNA.">Optimizing Cardiac Delivery of Modified mRNA.</a> Molecular Therapy. 25 (6), 1306-1315 (2017).</li><li>Zangi, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modified+mRNA+directs+the+fate+of+heart+progenitor+cells+and+induces+vascular+regeneration+after+myocardial+infarction.">Modified mRNA directs the fate of heart progenitor cells and induces vascular regeneration after myocardial infarction.</a> Nature Biotechnology. 31 (10), 898-907 (2013).</li><li>Carlsson, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Purified+VEGF-A+mRNA+Improves+Cardiac+Function+after+Intracardiac+Injection+1+Week+Post-myocardial+Infarction+in+Swine.">Purified VEGF-A mRNA Improves Cardiac Function after Intracardiac Injection 1 Week Post-myocardial Infarction in Swine.</a> Molecular Therapy Methods Clinical Development. 9, 330-346 (2018).</li><li>Huang, C. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synthetic+chemically+modified+mRNA-based+delivery+of+cytoprotective+factor+promotes+early+cardiomyocyte+survival+post-acute+myocardial+infarction.">Synthetic chemically modified mRNA-based delivery of cytoprotective factor promotes early cardiomyocyte survival post-acute myocardial infarction.</a> Molecular Pharmaceutics. 12 (3), 991-996 (2015).</li><li>Magadum, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ablation+of+a+Single+N-Glycosylation+Site+in+Human+FSTL+1+Induces+Cardiomyocyte+Proliferation+and+Cardiac+Regeneration.">Ablation of a Single N-Glycosylation Site in Human FSTL 1 Induces Cardiomyocyte Proliferation and Cardiac Regeneration.</a> Molecular Therapy - Nucleic Acids. 13, 133-143 (2018).</li><li>Kondrat, J., Sultana, N., Zangi, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synthesis+of+Modified+mRNA+for+Myocardial+Delivery.">Synthesis of Modified mRNA for Myocardial Delivery.</a> Methods in Molecular Biology. 1521, 127-138 (2017).</li><li>Gan, L. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intradermal+delivery+of+modified+mRNA+encoding+VEGF-A+in+patients+with+type+2+diabetes.">Intradermal delivery of modified mRNA encoding VEGF-A in patients with type 2 diabetes.</a> Nature Communication. 10 (1), 871 (2019).</li></ol>

The authors have nothing to disclose.

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&#8217; 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.

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.

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

delivery of modified mrna in a myocardial infarction mouse model

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.

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 &#181;g 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&#8722;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 &#956;g 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).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/60832/60832fig01.jpg" /> 
Figure 1: LAD ligation and cardiac delivery of modRNA.&#160;(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 &#181;g of modRNA dissolved in 60 &#181;L 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. <a href="https://www.jove.com/files/ftp_upload/60832/60832fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/60832/60832fig02.jpg" /> 
Figure 2: Luc expression analysis post modRNA injection.&#160;Sucrose citrate buffer containing 100 &#181;g 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 &#60; 0.0001. <a href="https://www.jove.com/files/ftp_upload/60832/60832fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/60832/60832fig03.jpg" /> 
Figure 3: Validation of Cre expression in vivo.&#160;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. <a href="https://www.jove.com/files/ftp_upload/60832/60832fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Delivery of Modified mRNA in a Myocardial Infarction Mouse Model at JoVE.com

Delivery of Modified mRNA in a Myocardial Infarction Mouse Model

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 ...

delivery-of-modified-mrna-in-a-myocardial-infarction-mouse-model

Nanyang Technological University

Research

JoVE Journal

Genetics

9.0K Views.  Icahn School of Medicine at Mount Sinai. Gemodificeerd RNA is een nieuwe methode voor genoverdracht naar het hart en andere organen. Dit protocol zal nauwkeurige aanpak van het leveren van gewenste genen tijdelijk naar het hart, post myocardiaal infarct. Deze methode is niet beperkt tot een hartinfarct, maar kan ook worden toegepast op ischemie reperfusie, en transversale aorta vernauwing modellen.Gemodificeerd RNA wordt gebruikt in verschillende menselijke klinische studies. Er is echter geen gestandaardiseerd protocol voor ...

Video: Delivery of Modified mRNA in a Myocardial Infarction Mouse Model

Coronary Artery Ligation and Intramyocardial Injection in a Murine Model of Infarction

Mouse models are a valuable tool for studying acute injury and chronic remodeling of the myocardium in vivo. With the advent of genetic modifications to the whole organism or the myocardium and an array of biological and/or synthetic materials, there is great potential for any combination of these to assuage the extent of acute ischemic injury and impede the onset of heart failure pursuant to myocardial remodeling. 
Here we present the methods and materials used to reliably perform this microsurgery and the modifications involved for temporary (with reperfusion) or permanent coronary artery occlusion studies as well as intramyocardial injections. The effects on the heart that can be seen during the procedure and at the termination of the experiment in addition to histological evaluation will verify efficacy. 
Briefly, surgical preparation involves anesthetizing the mice, removing the fur on the chest, and then disinfecting the surgical area. Intratracheal intubation is achieved by transesophageal illumination using a fiber optic light. The tubing is then connected to a ventilator. An incision made on the chest exposes the pectoral muscles which will be cut to view the ribs. For ischemia/reperfusion studies, a 1 cm piece of PE tubing placed over the heart is used to tie the ligature to so that occlusion/reperfusion can be customized. For intramyocardial injections, a Hamilton syringe with sterile 30gauge beveled needle is used. When the myocardial manipulations are complete, the rib cage, the pectoral muscles, and the skin are closed sequentially. Line block analgesia is effected by 0.25% marcaine in sterile saline which is applied to muscle layer prior to closure of the skin. The mice are given a subcutaneous injection of saline and placed in a warming chamber until they are sternally recumbent. They are then returned to the vivarium and housed under standard conditions until the time of tissue collection. At the time of sacrifice, the mice are anesthetized, the heart is arrested in diastole with KCl or BDM, rinsed with saline, and immersed in fixative. Subsequently, routine procedures for processing, embedding, sectioning, and histological staining are performed. 
Nonsurgical intubation of a mouse and the microsurgical manipulations described make this a technically challenging model to learn and achieve reproducibility. These procedures, combined with the difficulty in performing consistent manipulations of the ligature for timed occlusion(s) and reperfusion or intramyocardial injections, can also affect the survival rate so optimization and consistency are critical.

Numerous genetic manipulations and/or intramyocardial injections of genes, proteins, cells, and/or biomaterials are superimposed upon the dimension of time in studies of acute ischemia/ reperfusion injury and chronic remodeling in mice. This video illustrates the microsurgical procedures for ischemia/reperfusion, permanent coronary artery ligation, and intramyocardial injection studies.

Mouse models are a valuable tool for studying acute injury and chronic remodeling of the myocardium in vivo.  With the advent of genetic modifications to ...

Medicine

Gene Transfer for Ischemic Heart Failure in a Preclinical Model

Various emerging technologies are being developed for patients with heart failure. Well-established preclinical evaluations are necessary to determine their efficacy and safety.
Gene therapy using viral vectors is one of the most promising approaches for treating cardiac diseases. Viral delivery of various different genes by changing the carrier gene has immeasurable therapeutic potential.
In this video, the full process of an animal model of heart failure creation followed by gene transfer is presented using a swine model. First, myocardial infarction is created by occluding the proximal left anterior descending coronary artery. Heart remodeling results in chronic heart failure. Unique to our model is a fairly large scar which truly reflects patients with severe heart failure who require aggressive therapy for positive outcomes. After myocardial infarct creation and development of scar tissue, an intracoronary injection of virus is demonstrated with simultaneous nitroglycerine infusion. Our injection method provides simple and efficient gene transfer with enhanced gene expression. This combination of a myocardial infarct swine model with intracoronary virus delivery has proven to be a consistent and reproducible methodology, which helps not only to test the effect of individual gene, but also compare the efficacy of many genes as therapeutic candidates.

A method of gene transfer for the treatment of ischemic heart failure is described using a swine model of myocardial infarction. Our simple and reproducible method enables us to readily evaluate the efficacy of various gene transfers with a very simple and reproducible way.

Various emerging technologies are being developed for patients with heart failure. Well-established preclinical evaluations are necessary to determine ...

A Murine Model of Myocardial Ischemia-reperfusion Injury through Ligation of the Left Anterior Descending Artery

Acute or chronic myocardial infarction (MI) are cardiovascular events resulting in high morbidity and mortality. Establishing the pathological mechanisms at work during MI and developing effective therapeutic approaches requires methodology to reproducibly simulate the clinical incidence and reflect the pathophysiological changes associated with MI. Here, we describe a surgical method to induce MI in mouse models that can be used for short-term ischemia-reperfusion (I/R) injury as well as permanent ligation. The major advantage of this method is to facilitate location of the left anterior descending artery (LAD) to allow for accurate ligation of this artery to induce ischemia in the left ventricle of the mouse heart. Accurate positioning of the ligature on the LAD increases reproducibility of infarct size and thus produces more reliable results. Greater precision in placement of the ligature will improve the standard surgical approaches to simulate MI in mice, thus reducing the number of experimental animals necessary for statistically relevant studies and improving our understanding of the mechanisms producing cardiac dysfunction following MI. This mouse model of MI is also useful for the preclinical testing of treatments targeting myocardial damage following MI.

We introduce a surgical method to induce experimental ischemia/reperfusion (I/R) injury to simulate myocardial infarction (MI) in mouse models that allows for more clarity in positioning of the ligation on the left anterior descending artery (LAD) to increase the reproducibility of MI experiments in mice.

Acute or chronic myocardial infarction (MI) are cardiovascular events resulting in high morbidity and mortality. Establishing the pathological mechanisms ...

Ultrasound-guided Transthoracic Intramyocardial Injection in Mice

Murine models of cardiovascular disease are important for investigating pathophysiological mechanisms and exploring potential regenerative therapies. Experiments involving myocardial injection are currently performed by direct surgical access through a thoracotomy. While convenient when performed at the time of another experimental manipulation such as coronary artery ligation, the need for an invasive procedure for intramyocardial delivery limits potential experimental designs. With ever improving ultrasound resolution and advanced noninvasive imaging modalities, it is now feasible to routinely perform ultrasound-guided, percutaneous intramyocardial injection. This modality efficiently and reliably delivers agents to a targeted region of myocardium. Advantages of this technique include the avoidance of surgical morbidity, the facility to target regions of myocardium selectively under ultrasound guidance, and the opportunity to deliver injectate to the myocardium at multiple, predetermined time intervals. With practiced technique, complications from intramyocardial injection are rare, and mice quickly return to normal activity on recovery from anesthetic. Following the steps outlined in this protocol, the operator with basic echocardiography experience can quickly become competent in this versatile, minimally invasive technique.

Echocardiography-guided percutaneous intramyocardial injection represents an efficient, reliable, and targetable modality for the delivery of gene transfer agents or cells into the murine heart. Following the steps outlined in this protocol, the operator can quickly become competent in this versatile, minimally invasive technique.

Murine models of cardiovascular disease are important for investigating pathophysiological mechanisms and exploring potential regenerative therapies. ...

Transplantation of a 3D Bioprinted Patch in a Murine Model of Myocardial Infarction

Testing regenerative properties of 3D bioprinted cardiac patches in vivo using murine models of heart failure via permanent left anterior descending (LAD) ligation is a challenging procedure and has a high mortality rate due to its nature. We developed a method to consistently transplant bioprinted patches of cells and hydrogels onto the epicardium of an infarcted mouse heart to test their regenerative properties in a robust and feasible way. First, a deeply anesthetized mouse is carefully intubated and ventilated. Following left lateral thoracotomy (surgical opening of the chest), the exposed LAD is permanently ligated and the bioprinted patch transplanted onto the epicardium. The mouse quickly recovers from the procedure after chest closure. The advantages of this robust and quick approach include a predicted 28-day mortality rate of up to 30% (lower than the 44% reported by other studies using a similar model of permanent LAD ligation in mice). Moreover, the approach described in this protocol is versatile and could be adapted to test bioprinted patches using different cell types or hydrogels where high numbers of animals are needed to optimally power studies. Overall, we present this as an advantageous approach which may change preclinical testing in future studies for the field of cardiac regeneration and tissue engineering.

This protocol aims to transplant a 3D bioprinted patch onto the epicardium of infarcted mice modeling heart failure. It includes details regarding anesthesia, the surgical chest opening, permanent ligation of the left anterior descending (LAD) coronary artery and application of a bioprinted patch onto the infarcted area of the heart.

Testing regenerative properties of 3D bioprinted cardiac patches in vivo using murine models of heart failure via permanent left anterior descending (LAD) ...

Bioengineering

Murine Myocardial Infarction Model using Permanent Ligation of Left Anterior Descending Coronary Artery

Myocardial infarction (MI) and acute coronary diseases are among the most prominent causes of death in population with western lifestyle. The murine models of MI with permanent ligation of left-anterior descending (LAD) coronary artery closely mimics MI in humans. Murine models benefit from the extensive genetic engineering available nowadays. Here we propose a reproducible murine surgical model of myocardial infarction by permanent LAD coronary ligation. Our technique comprises anesthesia with ketamine/xylazine that can be rapidly reversed by administration of an antagonist, intubation without tracheotomy for mechanical-assisted ventilation, ventilation with application of extrinsic positive end-expiratory pressure (PEEP) to avoid alveolar collapse, a thoracotomy method limiting to the minimum surgical lesions made to skeletal muscles, and lung inflation without thoracentesis. This method is sparsely invasive, reproducible and reduces post-surgery mortality and complications.

Herein we describe a surgical procedure showing how to achieve permanent ligation of the left-anterior descending coronary artery in mice. This model is of high relevance to investigate the pathophysiology of myocardial infarction and the concomitant biological processes.

Myocardial infarction (MI) and acute coronary diseases are among the most prominent causes of death in population with western lifestyle. The murine ...

A Modified Simple Method for Induction of Myocardial Infarction in Mice

Myocardial infarction (MI) represents one of the leading causes of death. MI models are widely used for investigating the pathomechanisms of post-MI remodeling and evaluation of novel therapeutics. Different methods (e.g., isoproterenol treatment, cryoinjury, coronary artery ligation, etc.) have been used to induce MI. Compared with isoproterenol treatment and cryoinjury, coronary artery ligation may better reflect the ischemic response and chronic remodeling after MI. However, traditional methods for coronary ligation in mice are technically challenging. The current study describes a simple and efficient process for induction of MI in mice with readily available materials. The mouse chest skin was cut open under stable anesthesia. The heart was immediately externalized through the intercostal space after blunt separation of the pectoralis major and pectoralis minor. The left anterior descending branch (LAD) was ligated with a 6-0 suture 3 mm from its origin. Following LAD ligation, staining with 2,3,5-Triphenyltetrazolium chloride (TTC) indicated successful induction of MI and temporal changes of post-MI scar size. Meanwhile, survival analysis results showed overt mortality within 7 days after MI, mainly due to cardiac rupture. Moreover, post-MI echocardiographic assessment demonstrated successful induction of contractile dysfunction and ventricular remodeling. Once mastered, an MI model can be established in mice within 2-3 min with readily available materials.

Under adequate anesthesia, the mouse heart was externalized through the intercostal space, and myocardial infarction was successfully induced by ligating the left anterior descending artery (LAD) using materials readily available in most laboratories.

Myocardial infarction (MI) represents one of the leading causes of death. MI models are widely used for investigating the pathomechanisms of post-MI ...

Induction of Myocardial Infarction and Myocardial Ischemia-Reperfusion Injury in Mice

Acute myocardial infarction is a common cardiovascular disease with high mortality. Myocardial reperfusion injury can counteract the beneficial effects of heart reflow and induce secondary myocardial injury. A simple and reproducible model of myocardial infarction and myocardial ischemia-reperfusion injury is a good tool for researchers. Here, a customizable method to create a myocardial infarction (MI) model and MIRI by precision ligation of the left anterior descending coronary artery (LAD) through micromanipulation is described. Accurate and reproducible ligature positioning of the LAD helps obtain consistent results for heart injury. ST-segment changes can help to identify model accuracy. The serum level of cardiac troponin T (cTnT) is used to assess the myocardial injury, cardiac ultrasound is employed to evaluate the myocardial systolic function, and Evans-Blue/triphenyl tetrazolium chloride staining is used to measure infarct size. In general, this protocol reduces procedure duration, ensures controllable infarct size, and improves mouse survival.

Here we describe a simple and reproducible method that can induce myocardial infarction or myocardial ischemia-reperfusion injury in mice by precision ligation of the left anterior descending coronary artery through micromanipulation.

Acute myocardial infarction is a common cardiovascular disease with high mortality. Myocardial reperfusion injury can counteract the beneficial effects of ...

Ultrasound-Guided Induced Pluripotent Stem Cell-Derived Cardiomyocyte Implantation in Myocardial Infarcted Mice

The key objective of cell therapy after myocardial infarction (MI) is to effectively enhance the cell grafted rate, and human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) are a promising cell source for cardiac repair after ischemic damage. However, a low grafted rate is a significant obstacle for effective cardiac tissue regeneration after transplantation. This protocol shows that multiple hiPSC-CM ultrasound-guided percutaneous injections into an MI area effectively increase cell transplantation rates. The study also describes the entire hiPSC-CM culture process, pretreatment, and ultrasound-guided percutaneous delivery methods. In addition, the use of human mitochondrial DNA help detect the absence of hiPSC-CMs in other mouse organs. Lastly, this paper describes the changes in cardiac function, angiogenesis, cell size, and apoptosis at the infarcted border zone in mice 4 weeks after cell delivery. It can be concluded that echocardiography-guided percutaneous injection of the left ventricular myocardium is a feasible, relatively invasive, satisfactory, repeatable, and effective cellular therapy.

Ultrasound-guided cell delivery around the site of myocardial infarction in mice is a safe, effective, and convenient way of cell transplantation.

The key objective of cell therapy after myocardial infarction (MI) is to effectively enhance the cell grafted rate, and human induced pluripotent stem ...

A Simple and Efficient Method for In Vivo Cardiac-specific Gene Manipulation by Intramyocardial Injection in Mice

Gene manipulation specifically in the heart significantly potentiate the investigation of cardiac disease pathomechanisms and their therapeutic potential. In vivo cardiac-specific gene delivery is commonly achieved by either systemic or local delivery. Systemic injection via tail vein is easy and efficient in manipulating cardiac gene expression by using recombinant adeno-associated virus 9 (AAV9). However, this method requires a relatively high amount of vector for efficient transduction, and may result in nontarget organ gene transduction. Here, we describe a simple, efficient, and time-saving method of intramyocardial injection for in vivo cardiac-specific gene manipulation in mice. Under anesthesia (without ventilation), the pectoral major and minor muscles were bluntly dissected, and the mouse heart was quickly exposed by manual externalization through a small incision at the fourth intercostal space. Subsequently, adenovirus encoding luciferase (Luc) and vitamin D receptor (VDR), or short hairpin RNA (shRNA) targeting VDR, was injected with a Hamilton syringe into the myocardium. Subsequent in vivo imaging demonstrated that luciferase was successfully overexpressed specifically in the heart. Moreover, Western blot analysis confirmed the successful overexpression or silencing of VDR in the mouse heart. Once mastered, this technique can be used for gene manipulation, as well as injection of cells or other materials such as nanogels in the mouse heart.

A Simple and Efficient Method for In Vivo Cardiac-specific Gene Manipulation by Intramyocardial Injection in Mice

Here we present a protocol for cardiac-specific gene manipulation in mice. Under anesthesia, the mouse hearts were externalized through the fourth intercostal space. Subsequently, adenoviruses encoding specific genes were injected with a syringe into the myocardium, followed by protein expression measurement via in vivo imaging and Western blot analysis.

Gene manipulation specifically in the heart significantly potentiate the investigation of cardiac disease pathomechanisms and their therapeutic potential. ...

Delivery of Modified mRNA in a Myocardial Infarction Mouse Model

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