Gene Therapy for Ocular Diseases

In my last post, I introduced gene therapy and discussed potential applications in Duchenne muscular dystrophy. In this post, I'll be talking about gene therapy in relation to eye diseases.

What is gene therapy? How does it work?

See earlier post: Introduction to Gene Therapy

The Adeno-Associated Virus (AAV)

The main gene vector that has been tested for use in ocular diseases is the adeno-associated virus, or AAV. AAVs are small single-stranded DNA viruses that require adenovirus to be able to replicate (hence the name). Benefits of AAVs are that they are non-pathogenic, and have low immunogenicity, stable long-term expression and cell and tissue specificity. However, there are disadvantages- AAVs can only package a small amount of DNA, and due to endemic pre-existing immunity in humans, most people have high levels of circulating antibodies against AAVs. This latter problem isn't really an issue in ocular therapies, however, as the eye is relatively isolated from the immune system.

To make an AAV vector, you need two main ingredients: the gene of interest (plus the promoter and any other important stuff), and a packaging construct that includes adenoviral helper genes. Remember, AAVs need adenoviral genes in order to replicate.

Inherited Retinal Diseases

There are a lot of retinal diseases that can be inherited. Among them are retinitis pigmentosa, which affects peripheral vision, age-related macular degeneration, which affects central vision, and Leber's congenital amaurosis, which affects the whole field of vision. For the purpose of this post, we will be discussing age-related macular degeneration (AMD), which is the most common cause of vision loss in the developed world. Unlike some other genetic diseases where we can pin down the cause to a single gene, AMD is a multifactorial disease that is likely to rely on genetics, as well as environmental factors, such as smoking.

Age-Related Macular Degeneration (AMD)

AMD can be divided into two types: wet AMD and dry AMD. Wet AMD is more aggressive, and is responsible for 90% of cases of severe vision loss. Abnormal blood vessels grow, causing oedema and blotchy patches on the retina. Wet AMD is associated with VEGF (vascular endothelial growth factor), which I'll get back to later. Dry AMD, on the other hand, is associated with a more gradual loss of vision. It is caused by accumulation of Drusen particles which disrupt the retinal pigment epithelium (RPE), causing degeneration of photoreceptors.

Wet AMD Treatment Strategies

Since wet AMD is associated with VEGF, current treatments involve the use of recombinant humanised anti-VEGF antibody fragments, such as ranibizumab (Lucentis) and bevacizumab (Avastin). There are also soluble receptor decoys, such as aflibercept (Eylea). These medications are generally administered intravitreally, which is an injection into the vitreous fluid of the eye. (Doesn't sound like much fun.) In intravitreal delivery, ganglion cells are strongly targeted, but deeper layers of the eye, such as the RPE, get lower doses (if any) of the drug. (Some eye medications can be given via subretinal injections, in which the medication is injected into a small detachment created behind the retina. This strongly targets retinal cells, but the procedure is much more technically difficult than intravitreous surgery.)

One of the problems with anti-VEGF antibodies and soluble receptors is that these need to be administered every 4-8 weeks. This can be especially problematic for those living in remote areas as getting to a clinic can be troublesome. This is where gene therapy comes in!

The gene therapy that has been proposed is the use of a recombinant adeno-associated virus (rAAV) containing the DNA for sFlt-1, which is a soluble VEGF receptor and potent inhibitor of VEGF. The vector was tested for protein production efficiency in a variety of cell lines. To make sure that the sFlt-1 protein produced worked as predicted, a cell proliferation assay using human umbilical vein endothelial cells was performed. Normally, these cells proliferate via VEGF-regulated pathways, but this was blocked by the sFlt-1 produced.

To test the viability of this gene therapy, several animal models were used. Unfortunately, animals don't get AMD, so other kinds of models were used, such as corneal neovascularisation (CNV) in rats and primates, as well as the Kimba mouse (a model of chronic, progressive retinal neovascularisation and degeneration). Results showed that rAAV.sFlt-1 was non-toxic, caused regression of abnormal blood vessels and prevented the development of new blood vessels. Furthermore, this treatment seemed to work over the long term.

Phase 1 and 2a trials have also been performed in humans, with promising results. rAAV.sFlt-1 is safe, well-tolerated and appear to be efficacious in treating wet AMD. However, it takes around 6-8 weeks for anti-VEGF expression to become optimal, so a couple of doses of a standard treatment (such as Lucentis) still needs to be given at the commencement of therapy.