Modified RNA is a novel method for gene transfer to the heart, and other organs. This protocol will provide accurate approach of delivering desired genes transiently to the heart, post myocardial infarction. This method is not limited to myocardial infarction, but can also be applied to ischemia reperfusion, and transverse aortic constriction models.
Modified RNA is being used in several human clinical trials. However, there is no standardized protocol for gene transferred into the heart using this method. Visual demonstration will enable researchers to correctly inject modified RNA at appropriate sites in the heart.
Demonstrating the procedule will be Ajit Magadum, a postdoctoral fellow, and Elena Chepurko an animal supervisor from our laboratory. Based on the concentration of the previously prepared at modRNA, calculate the volume of modRNA needed for a 100 microgram injection. Makes equal volumes of sucrose and citrate solutions.
Then add the calculated volume of modRNA, and enough saline solution to bring the injection volume to 60 microliters. After anesthetizing the animal, place it ventral side up, with its mouth facing toward the experimenter. Then to maintain an open airway by performing intratracheal intubation, gently pull out the tongue.
Adjust the angle of the neck to straighten the intubation path, and push in the intubation cannula. Observe the thoracic movement of the mouse to verify that both lungs are receiving oxygen. The respiratory rate should be approximately 120 breaths per minute.
To begin the LAD ligation, turn the mouse carefully onto its right side. lift the skin, and perform a left-sided thoracotomy between the third and fourth ribs. Open the thorax carefully, without touching the lungs with any sharp objects.
Place retractors into the incision to keep the thoracic cavity open, providing a clear view of the heart. Next, move the lungs to the edge of the incision, and remove the part of the pericardial sac that is covering the heart. Carefully identify the LAD, a deep light red vessel, located between the pulmonary artery and the left auricle.
Ligate the proximal LAD with a single silk suture. Using an insulin syringe, deliver a total of 60 microliters of the modRNA solution to the heart. Inject 20 microliters intramuscularly, at three different sites.
One site on each side of the ligation and one in the apex of the heart. The best way to inject the modified RNA in the correct sites, is to observe the discoloration of the tissue under microscope, after the occlusion of LAD. Also when injected, do not go too deep into the tissue to reach the heart chambers into the circulation.
Close the thoracic incision in layers. First use was 6-0 silk running sutures to adapt the ribs. Then use 5-0 silk running sutures to close the skin.
For the recovery phase, remove the cannula, and allow spontaneous breathing. Place the animal on a heating pad until it regains consciousness. Do not leave the animal unattended until it is fully awake.
To manage postsurgical pain, inject buprenorphine subcutaneously at 12 hour intervals for three days. At a dose of 0.1 milligrams per kilogram of body weight. 24 hours after LAD ligation and injection of luciferase modRNA in CFW mice, transfection was validated by checking for luciferase protein expression using a bioluminescence imaging system.
The luciferase signal can be clearly detected in images comparing control mice, to mice injected with luciferase modRNA. Quantification of the luciferase signal shows significantly higher protein expression in treated mice than in control mice. Cre modRNA expression was validated by checking its translation and bio distribution in transgenic ROSA26 mTmG mice.
24 hours after surgery and cre modRNA injection, arts were fixed and processed for immunostaining with cardiomyocyte marker, cardiac troponin I, and nuclei marker DAPI. Successful cre expression was evident due to the appearance of green colored cardiomyocytes. A merged image shows cre activated cells around the two visible injection sites.
Blue is the nuclear stain DAPI. Modified RNA platform can deliver a single gene or gene combinations in variety of organs. And thus can be used for therapeutic purposes, such as correcting genetic disorders, or activating beneficial mechanisms in the body.
The unique pharmacokinetics of modified RNA enables us to evaluate gene function immediately after its injection. This enables researcher to target signaling pathways, which are altered in the first few hours of infarction.
