Gene therapy is a fundamental strategy that delivers normal genes to human cells to treat diseases caused by gene defects and abnormalities. Undergoing 30-year development, gene therapy has become a key means to treat various human genetic diseases, among which adeno-associated viruses mediated gene therapy (AAV -mediated gene therapy) is worth mentioning.
AAV vector is one of the most advanced, promising, and commonly used viral vectors in somatic gene therapy because it is considered to be safe that can infect a wide range of tissues with low immunogenicity and integration ability. According to the World Vision Report released by the World Health Organization, at least 2.2 billion people are afflicted with visual impairment or blindness globally, among which 1 billion people can prevent it or accept treatment.
The eye has become a very popular organ in AAV-mediated gene therapy due to the following reasons:
- Eyes are in small sizes, so a small dose of AAV vector can achieve robust transduction of target gene.
- The existence of the blood-retinal barrier makes the eye immune-privileged, relatively independent from other tissues, ensuring the safety of local administration.
- Most fundus diseases are single-gene genetic diseases.
Currently, ophthalmology AAV-mediated gene therapy applies to treating Inherited Retinal Diseases (IRDs) related to single-gene mutation. That means patients with these diseases, such as retinitis pigmentosa, choroideremia, Leber hereditary optic neuropathy (LHON), Leber congenital amaurosis (LCA), Stargardt disease, Achromatopsia (ACHM), X-linked retinoschisis (XLRS) and age-related macular degeneration (AMD), can accept treatment.
Great medical needs exist because patients afflicted with these diseases struggle to live independently, let alone ensure daily activities. Therefore, research on eye AAV-mediated gene therapy has been at the forefront of gene therapy research, which accounts for one-quarter of AAV-mediated gene therapy clinical trials.
Vector Serotypes and Injection Methods Selection
Currently, there are various targeted cell types in ophthalmology AAV-mediated gene therapy, such as retinal pigment epithelium (RPE), ganglion cell layer, and photoreceptor cell layer. The majority of the vectors used in clinical trials are AAV2, 5, and 8 serotypes. However, AAV2-7M8, AAV2-TYF, and scAAV commonly used in recent years are applied to clinical studies.
Based on different therapeutic purposes, eye gene transduction with AAV vector mainly includes intravitreal injection, subretinal injection, anterior chamber injection, and subconjunctival injection, among which intravitreal injection and subretinal injection are the most common ones.
In various animal models, it has been shown that vitreous injection with the AAV-mediated gene therapy primarily infects ganglion cells, resulting in humoral immune responses to AAV capsid proteins and intraocular inflammation. Subretinal injection mainly infects retinal pigment epithelial cells and photoreceptor cells. Although it does not trigger a humoral immune response, attention should be paid to avoiding injection-induced bleeding damage and the potential risk of retinal detachment.
Examples of Injection Methods
This article will give a detailed introduction to common injection methods for eye AAV-mediated therapy, which can serve as a reference when people choose and operate the injection methods with AAV-mediated gene therapy.
1. Anterior Chamber Injection
The anterior chamber is the space between the cornea and the iris, filled with aqueous humor. Due to the giant lens that bulges into the anterior chamber, the anterior chamber of mice is relatively much shallower than a human's. Inject liquid or gas into the anterior chamber of mice can increase intraocular pressure, which is one of the methods for making glaucoma models. The specific operation steps are as follows:
- Preparation before the Experiment
Anesthetized mice: After being weighed, mice will be intraperitoneally injected with an appropriate amount of 1% pentobarbital sodium solution and placed in the feeding cage, waiting 5 to 10 minutes for anesthetization. Next, the pupil dilator should be dripped onto the surface of the eyeball to dilate the pupil.
- Anterior Chamber Injection
Absorb a moderate amount of virus by using a microinjector, then put the anesthetized mice on the operation table. Then, carry out local anesthetization of the eye with lidocaine hydrochloride. Use the left hand to hold the head and make the eye outstanding while using the right hand to hold the microinjector to insert the anterior chamber away from the 1-2 mm of iris. The needlepoint must be parallel to enter, then quickly push the virus and make bubbles on the needle to prevent leakage. When the injection is finished, the needle should be slowly removed.
- Mice Resuscitation
Put the mice back into the cage and wait until they wake up. Be sure to keep them warm.
2. Intravitreal Injection
- Preparation before the Experiment
Anesthetized mice: After being weighed, mice mice will be intraperitoneally injected with an appropriate amount of 1% pentobarbital sodium solution and placed in the feeding cage, waiting 5 to 10 minutes for anesthetization. Next, the pupil dilator should be dripped onto the surface of the eyeball to dilate the pupil.
- Vitreous Cavity Injection
Absorb a moderate amount of virus by using a microinjector, then put the anesthetized mice on the operation table. Then, carry out local anesthetization of the eye with lidocaine hydrochloride. These steps can carry out the anesthetization process:
- Use the left hand to hold the head and make the eye outstanding while using the right hand to hold the microinjector to insert the vitreous cavity from 1-2 mm away from the back of the iris.
- The needlepoint must be vertically into the vitreous cavity first, then tilt to push the virus slowly.
- When the virus is injected, the needle should be slowly removed.
. Mice Resuscitation
Put the mice back into the cage and wait until they wake up. Be sure to keep them warm.
3. Subretinal Injection
The posterior eye wall of mice is divided into retina, choroid, and sclera from inside to outside. The structure of the mouse's retina is similar to that of the human. The specific procedures for injection are as follows:
- Preparation before the Experiment
Anesthetized mice: After being weighed, mice mice will be intraperitoneally injected with an appropriate amount of 1% pentobarbital sodium solution and placed in the feeding cage, waiting 5 to 10 minutes for anesthetization. Next, the pupil dilator should be dripped onto the surface of the eyeball to dilate the pupil.
- Virus Injection
Absorb a moderate amount of virus by using a microinjector, then put the anesthetized mice on the operation table. Then, carry out local anesthetization of the eye with lidocaine hydrochloride. The anesthetization process can be carried out by these steps:
- Use the left hand to hold the head and make the eye outstanding while using the right hand to hold the microinjector to insert the vitreous cavity from 1 mm away from the back of the iris.
- Ensure the needlepoint is vertically first, then tilt until it reaches the contralateral retina. (Through vitreous cavity injection)
- When there is resistance, stop inserting the needle but push the virus slowly. Alternatively, a syringe needle with an angle of 15° to the eye axis should puncture the sclera but not penetrate the RPE, in which the sclera of the exposed animal should be 1-2 mm away from the corneal margin.
- The needle injection (through the sclera injection) can be stopped if the oblique surface of the needle is totally inserted, and the virus should be injected slowly.
- When the virus is injected, slowly remove the needle.
- Mice Resuscitation
Put the mice back into the cage and wait until they wake up. Be sure to keep them warm.
4. Subconjunctival Injection
The subconjunctival injection is a common method for topical administration for the eyes. The detailed procedures are as follows:
- Preparation before the Experiment
After anesthetizing the mice, wait for another 5 to 10 minutes, then drip the pupil dilator.
- Virus Injection
Absorb a moderate amount of virus by using a microinjector, then put the anesthetized mice on the operation table. Then, carry out local anesthetization of the eye with lidocaine hydrochloride. These steps can carry out the anesthetization process:
- Use a tweezer to pick up the conjunctiva and pierce the inside of the conjunctival for 3 to 4 mm. In this way, people can observe how's the needle going through the conjunctival. Then slowly inject viruses.
- When the virus is injected, stay for a few seconds before pulling out the needle; after that, the bulge in the ball subconjunctival is visible with proptosis.
- Mice Resuscitation
Put the mice back into the cage and wait until they wake up. Be sure to keep them warm.
References:
[1]She, K., Su, J., Wang, Q., Liu, Y., Zhong, X., Jin, X., ... & Wei, Y. (2022). Delivery of nVEGFi using AAV8 for the treatment of neovascular age-related macular degeneration. Molecular Therapy-Methods & Clinical Development, 24, 210-221.
[2]Buck, T. M., & Wijnholds, J. (2020). Recombinant adeno-associated viral vectors (rAAV)-vector elements in ocular gene therapy clinical trials and transgene expression and bioactivity assays. International journal of molecular sciences, 21(12), 4197.
[3]Telias, M., Denlinger, B., Helft, Z., Thornton, C., Beckwith-Cohen, B., & Kramer, R. H. (2019). Retinoic acid induces hyperactivity, and blocking its receptor unmasks light responses and augments vision in retinal degeneration. Neuron, 102(3), 574-586.
[4]Lee, S. H., Kim, Y. S., Nah, S. K., Kim, H. J., Park, H. Y., Yang, J. Y., ... & Park, T. K. (2018). Transduction patterns of adeno-associated viral vectors in a laser-induced choroidal neovascularization mouse model. Molecular Therapy-Methods & Clinical Development, 9, 90-98.
