This protocol offers a method of gene therapy delivery to multiple ocular tissues in a safe, easy, and effective manner. It is also adaptable for intrastromal and subretinal injections in small animals. Use of the subconjunctival route of injection is minimal invasive.
It poses fewer risks and holds great promise for AV delivery to treat multiple ocular diseases. With over 1 billion people worldwide suffering from visual impairment, using a safe and easy method for drug delivery offers broad applicability for the treatment of many different causes of blindness. The gene delivery methods described in this protocol are of particular interest to those who are developing potential intervention strategies and practicing ophthalmologists, who will eventually deliver these drugs to patients.
The biggest challenge for someone who is performing this protocol for the first time is likely the insertion of the needle into the concept conjunctival space. To begin, assemble the injection system by placing a stereo microscope and a syringe pump in a bio-safety cabinet. Next, cut the polyethylene tubing to a length of approximately 50 centimeters.
Insert the end of a 36-gauge needle into one end of the tubing. Then fill a disposable 3-milliliter syringe with sterile water and insert it into the side of the tube opposite the needle. Flush the tubing three times, alternating between sterile water and 70%alcohol to disinfect the tubing and ensure that there are no leaks, clogs, or damage throughout the tubing.
Using the disposable 3-milliliter syringe, fill the tubing with sterile water and leave the tubing attached to the disposable syringe. Then place a piece of parafoam on the surface of the bio-safety cabinet, and add a pool of sterile water to it. Submerge the portion of the tubing connected to the needle into the pool of sterile water.
Next, pull the disposable syringe out from the tube opening to prevent any air from entering the tubing system upon removal of the syringe. Then fill a 10-microliter Hamilton syringe with sterile water. Next, connect the Hamilton syringe needle to the open end of the tubing by submerging the needle tip of the Hamilton syringe into the pool of sterile water on the para film.
Press and hold the fast reverse button on the pump screen to move the pusher blocks to the approximate length of the syringe. Next, unscrew the bracket clamping knobs to loosen the the retaining brackets on the pusher and the syringe holder blocks. Subsequently, load the Hamilton syringe onto the syringe holder block and secure the syringe following the manufacturer's instructions.
To adjust the parameters in the pump setting screen, press the Force"button and set the force level at 30%Then accept the changes to go back to the setting screen. Press the Quick Start"button, select Method, and then select the Infuse Only"option. For the syringe option, select Hamilton 1700, then click on Glass"and select 10 microliters.
Later, select the infusion rate and the injection volume. Eject the water from the Hamilton syringe while leaving the tubing and injection needle full of water by pressing the Infuse"button. Next, withdraw the virus by placing the injection needle into an aliquot of the virus stock.
Then slightly pull back on the Hamilton syringe by pressing the reverse button to introduce a small air bubble in the tubing. After anesthetizing the animal, grab the conjunctiva with forceps. Then insert the needle into the conjunctiva until the bevel is entirely covered by the conjunctival membrane, and lay the needle against the globe.
Simultaneously, start the injection by pressing the Start"button using the foot switch. To examine the distribution of the AAV vector, the ocular and surrounding tissues were stained with hematoxylin and eosin. The representative sagittal sections demonstrated that the dispersion of India ink occurred mainly adjacent to the extraocular muscles in the outer surface of the sclera in the periocular loose connective tissues.
To confirm transduction of self-complimentary AAV 8 at eight weeks post-injection, green fluorescence protein abundance in the cross-sections was examined by immunofluorescence staining. The results revealed that AAV 8 vectors efficiently transduced the periocular muscles posterior to the eye and the cornea. To analyze the vector bio-distribution, vector genome copy numbers and distinct eye compartments in other organs, including liver and heart, were examined by quantitative polymerase chain reaction using primers and probes specific to the transgene.
Expression of the transgene was confirmed by quantitative reverse transcription polymerase chain reaction, which suggested that the subconjunctival injection of AAV 8 results in transgene expression in the eyelid, conjunctiva, cornea, and optic nerve. The air bubble will serve as a barrier between the water in the tube and the therapeutic drug. This technique allows other researchers to explore the use of the subconjunctival route of administration for both ophthalmic solutions and ocular gene therapies for multiple diseases.
