Systemic Treatment for Postnatal, Juvenile, and Runted Adult Mice by Retrobulbar Sinus Injection

Our research involves the development of gene therapies for rare genetic disorders in particular with a focus on a group of metabolic disorders known as the organic acidemias. The goal of our research is to determine which approaches might be best suited to advance to human clinical trials. Multiple gene therapies are approved to treat a variety of genetic diseases which was not the case a decade ago.

In fact, several gene therapy clinical trials for organic acidemia are completed, ongoing or planned. Systemic delivery to mirroring models of severe genetic diseases can be challenging as the mice may not reach adult size. This retrobulbar injection protocol will allow for the testing of gene therapies in mirroring models with severe phenotypes and hopefully enable the development of new therapies for patients.

To begin, dilute the AAV to the desired injection volume and concentration with sterile PBS in a sterile 1.5 milliliter micro centrifuge tube for each injection. Prior to filling the single use sterile syringe move the plunger up and down several times to ensure the plunger can be depressed smoothly. Then fill the syringe to the desired volume ensuring there are no air bubbles.

If right handed, position the anesthetized mouse on its left side with the snout pointing towards the right hand to inject the right eye. Then, apply one or two drops of ophthalmic anesthetic to the eyeball to be injected. Using a sterile absorbent gauze pad remove any excess ophthalmic anesthetic solution.

Apply gentle pressure with fingertips to the skin, dorsal and ventral to the eye to partially protrude the mouse's eyeball from the socket. Now, insert the needle bevel down at a 30 degree angle into the medial canthus. Slowly and smoothly apply pressure to the syringe plunger to deliver the injectate.

Finally, remove the face mask from the mouse to allow from recovery from anesthesia. The expression of methylmalonyl coenzyme CoA mutase RNA was observed in hepatocytes indicating successful retrobulbar injection.