Systemic Treatment for Postnatal, Juvenile, and Runted Adult Mice by Retrobulbar Sinus Injection
May 17th, 2024
This article provides a protocol and an accompanying video for the retrobulbar sinus injection of up to a total volume of 150 µL for postnatal, juvenile, and runted adult mice. This procedure is particularly well suited for the injection of small mice (15 g) when tail vein injection is not feasible.
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.
Overview
This article presents a protocol for retrobulbar sinus injection in mice, facilitating gene therapy research for metabolic disorders. The method is particularly useful for small or juvenile mice when other injection techniques are impractical.
Key Study Components
Area of Science
- Gene therapy
- Metabolic disorders
- Animal models
Background
- Gene therapies are increasingly approved for genetic diseases.
- Organic acidemias represent a group of metabolic disorders targeted by these therapies.
- Systemic delivery in small animal models poses challenges.
- Retrobulbar injection offers a solution for effective delivery in small mice.
Purpose of Study
- To develop a reliable injection protocol for gene therapy testing.
- To enable research on severe phenotypes in animal models.
- To advance potential therapies for human clinical trials.
Methods Used
- Dilution of AAV with sterile PBS for injection.
- Preparation of a sterile syringe for injection.
- Application of ophthalmic anesthetic to the injection site.
- Insertion of the needle at a specific angle for retrobulbar injection.
Main Results
- Successful delivery of gene therapy agents to the target site.
- Observation of gene expression in hepatocytes post-injection.
- Demonstration of the protocol's effectiveness for small mice.
- Potential for advancing gene therapy research in metabolic disorders.
Conclusions
- The retrobulbar injection method is effective for small mice.
- This technique can facilitate the testing of gene therapies.
- It holds promise for developing new treatments for genetic disorders.
