TITLE: Systemic AAV administration through retro-orbital injection
AUTHORS: Gerard Michael Coughlin

[DESCRIPTION]
Adeno-associated viruses (AAVs) are popular tools for gene delivery to animal models. Natural and engineered AAVs can even achieve widespread transduction of target organs through systemic delivery. Fast (<3 minute per mouse) and reproducible transduction through systemic AAV delivery can be achieved with retro-orbital injection. This protocol describes how to systemically deliver AAVs via the retro-orbital sinus.

[STEPS]
SECTION: Systemic AAV administration through retro-orbital injection
2. Before beginning, ensure that all AAVs are prepared and recently (< 1 month) titered. If injecting multiple AAVs (either into the same animals, or animals in separate conditions), we recommend that all AAVs are titered in the same qPCR or ddPCR assay. 

Make a plan for diluting AAVs in the injection buffer, considering number of animals per group and total dose per animal. Plan to make slightly more than necessary to ensure that each animal gets the full volume (e.g. 10% extra, but appropriate surplus will depend on number of animals to inject). To reduce variability in injected dose due to error when measuring volume using a syringe, we recommend larger volume injections (up to 120 µL )

 

SECTION: Systemic AAV administration through retro-orbital injection
3. Dilute AAVs in the injection buffer. We use sterile saline to dilute AAVs for injection. Briefly vortex AAV dilutions, then briefly centrifuge to collect at bottom of tube. 

 

SECTION: Systemic AAV administration through retro-orbital injection
4. Prepare work area. Set up isoflurane anaesthesia system inside the biosafety cabinet (BSC), with an induction box. Ensure that you have easily access to biohazard sharps disposal. Place a small stack of paper towels near the front of the BSC. 
SECTION: Systemic AAV administration through retro-orbital injection
5. Transfer mouse to induction chamber and anaesthetize with 5% isoflurane in 1L/min oxygen. 
SECTION: Systemic AAV administration through retro-orbital injection
7. Once the mouse's breathing rate is about 1 breath per second, turn off isoflurane and flush chamber. Transfer anaesthetized mouse to stack of paper towels, with nose pointing towards the experimenters dominant hand and ventral side facing the experimenter. The eye to inject will be facing upwards.

Using the index finger and thumb of the non-dominant hand, draw back skin around the eye. This will cause the eye to protrude slightly from the socket. Using the dominant hand, insert the needle bevel down at a 30°-45° angle into the medial canthus and through the conjunctival membrane. The needle should be positioned behind the eye, in the retro-orbital sinus. Slowly inject the virus into the sinus, then remove the needle by pulling in the opposite direction as it was inserted. 

 

SECTION: Systemic AAV administration through retro-orbital injection
6. While animal is being anaesthetized, load a 31g insulin syringe with the virus. Draw up virus into the syringe, then gently eject virus back into the tube to expel air bubbles. Repeat this until no bubbles remain in the barrel of the syringe. Draw up virus to desired volume and place the syringe in a safe, sterile, and accessible place until needed.  
SECTION: Systemic AAV administration through retro-orbital injection
8. Apply 1-2 drops of 0.5% ophthalmic proparacaine to the corneal surface for local analgesia. Transfer animal to a clean cage and monitor until the animal is recovered. 

 

SECTION: Training
1. Though quick and less technically challenging than other injection routes, retro-orbital injections can still take practice to become proficient. Practicing by injecting adeno-associated viruses, especially at large doses, can be costly and time consuming. 

Injection of dyes can provide cheaper and faster paradigm for practicing retro-orbital injections, with more immediate feedback than needing to wait for AAV expression.

Injection of 50 µL of 1 mg/mL fluorophore-conjugated tomato lectin dye  (e.g. https://vectorlabs.com/products/dylight-649-lycopersicon-esculentum-tomato) will label vasculature. 60 min following injection, euthanize, perfuse (30 mLof heparinized 1x PBS, followed by 30 mL  of 4% PFA, in 1x PBS), then collect and post-fix target organs. Process tissue with appropriate techniques (slicing, tissue clearing, etc.). Clear vascular labeling should be evident under fluorescence microscopy.