population, before amblyopia and retinal degeneration are established. In the naturally occurring animal model of LCA2, the Briard dog (with an rpe65 null mutation), RPE65 gene therapy has been shown to result in long-term rescue (over 5 years) of photoreceptor cells following a single injection.32 The first successful fetal gene therapy used rpe65 mice; it may be that fetal therapy following prenatal testing is therefore the future for LCA2 treatment.

While there are encouraging results from viral-mediated gene therapy, it is known to have a number of physical limitations, including random integration into the host’s genome, immunogenicity of the vector, and limitations in the DNA insert size. These have prompted the development of nonviral methods such as liposomes, compacted DNA nanoparticles, or combinations of both. Nanoparticles have been shown to be nonimmunogenic, noninflammatory, and nontoxic in various tissues, with higher transfection rates than any viral vector. They have had some success in phase I/II clinical trials for cystic fibrosis: ocular-specific nanoparticles for use in retinal dystrophies are being developed.34

DELIVERY OF GENES TO TARGET PATHOGENIC PATHWAYS

The multiplicity of mechanisms in the pathogenesis of RP reflecting its genetic heterogeneity prompted a search for therapeutic agents that might be effective in slowing photoreceptor cell death regardless of the causative genetic mutation. One such generic intervention that showed initial promise was through the delivery of the human ciliary neurotrophic factor (CNTF) gene within an encapsulated cell intraocular implant. Although found to be effective in retarding retinal degeneration in a number of transgenic animal models representing different genotypes of RP, it did not prevent photoreceptor cell death in an X-linked canine model. In a recent phase I clinical trial several subjects were reported to show an improvement in visual acuity following treatment, although the trial was not designed or powered to measure the significance of such outcomes. Further trials are required to evaluate whether this could provide either an alternative or an adjunct to primary gene-specific therapy.35

Most biological processes represent a balance between endogenous protein agonists and antagonists. When overactivity of a process is found to play a key role in the pathogenesis of a disease, an endogenous antagonist can be used to suppress the process. Gene therapy can therefore be used to transduce DNA into the host cell which stimulates expression of the antagonist. Such an approach circumvents problems with balancing specificity and toxicity of the therapeutic agent, and allows sustained delivery to a targeted organ without systemic effects. This method may be useful in a multifactorial disease such as AMD where no single specific primary genetic lesion may be targeted.

A common model for the wet form of AMD is induction of CNV by disrupting rodent or pig Bruch’s membrane with laser. This model has been used to test the delivery of genes to stimulate the endogenous antagonist to vascular endothelial growth factor (VEGF), namely pigment epithelial-derived growth factor (PEDF) using adenovirus as a vector, i.e., AdPEDF. Exogenous anti-VEGF treatments are the current most successful treatment for CNV in our limited armoury, but as they seem to have a relatively transient effect they require frequent administration through intraocular injections and are therefore not without risk. Although AdPEDF also currently requires such injections it carries the advantages of specificity and low toxicity mentioned above and it is hoped it will provide longer-term downregulation of VEGF. Results from animal studies have shown considerable success in suppressing neovascularization. A phase I clinical trial, although not designed to address efficacy, reported promising signs of sustained antiangiogenic activity in treated eyes.36,37 The risks associated with intraocular injections may be avoided if the development of a new platform for drug delivery is successful. Seagull Technologies is coupling nanotechnology and ultrasound to produce a noninvasive method of retinal drug delivery.38 Developed primarily for anti-VEGFs, it could also prove to be useful in gene therapy.

GENE-INDEPENDENT THERAPY

The considerable leaps in our understanding of the molecular basis of retinal disease have also allowed new gene-independent therapies to be developed which target specific pathophysiological pathways without stimulating or suppressing genes directly.

Professor Hageman and the company Optherion have worked to create novel therapies for AMD. Optherion’s primary therapeutic aim is to replace dysfunctional CFH with normal CFH. This principle has been already proven in another disease, atypical hemolytic–uremic syndrome (HUS). Some types of HUS are caused by factor H deficiency caused by a mutation in the CFH gene. Plasma exchange in these people, which replaces the CFH, is effective in preventing disease episodes and normalizes kidney function. Optherion’s products are still in the preclinical stage of development.39

Dr. Holer and Dr. Emlen are the co-founders of Taligen, which is another company currently developing AMD treatments that target the AP. Taligen’s technologies focus on controlling the amplification of complement activation: a monoclonal antibody which targets factor B (TA106) is designed to inhibit the activation of the alternative pathway of complement, and the targeted inhibitor program (TT30) allows for delivering endogenous inhibitors of the alternative pathway specifically to sites where complement activation is occurring. This targeting technology is designed to provide local regulation of complement activation without triggering systemic complement inhibition, potentially allowing application to a broad range of autoimmune and inflammatory diseases. Taligen has proof-of-concept data using this technology in animal models of CNV, and data demonstrating that targeted delivery of the complement-inhibitory portion of factor H to sites of complement activation with TT30 (CR2~factor H) is equally efficacious in animal models of CNV when given by systemic or by intraocular injection.40

The association between C. pneumoniae antibody titers, CFH alleles, and AMD risk prompts the question: could prophylactic antibiotics play a role in slowing the development of AMD? Over the last 20 years, the role of C. pneumoniae in another complex disease, atherosclerosis, has been evaluated, but it has proven difficult to establish a definite causal role for a common agent in a highly prevalent disease. Many seroepidemiological studies indicate an association between infection and cardiovascular events, yet a recent meta-analysis of 393 papers concluded there is no evidence for the use of antibiotics as a form of secondary prevention for cardiovascular disease.41,42 Considerable progress towards developing a C. pneumoniae vaccine has been made in the past decade, but a vaccine that offers long-lasting immunity in humans is not yet available. For AMD, more research is required to explore the possible role of infection in AMD, including other possible trigger pathogens which may represent therapeutic targets.

Targeting pathways shared by genetically heterogeneous diseases leading to cell death has been proposed as a potential therapy. In RP the final common pathway has been found to be apoptosis of photo­ receptors, although it is not yet known whether the same molecular mechanisms are activated during the cell demise in RP caused by different genetic lesions. Inhibiting enzymes involved in apoptosis could potentially provide a generic therapy for RP; further studies in animal models should indicate whether this is viable.43

GENOTYPE AS PREDICTOR OF

RESPONSE TO TREATMENT

Genotype variation can alter the response to drugs; this information is already in use in several systemic diseases such as human immuno­ deficiency virus (HIV), Stevens–Johnson, and melanoma. It is probable that genotyping will increasingly be used to guide treatment and predict outcomes in eye disease also. Pharmacogenetic studies in AMD have suggested that CFH genotype may play a role in response to antioxidants and zinc,44 and also in photodynamic therapy. There are currently studies under way examining possible associations between genotype and failure to respond to current anti-VEGF treatments. It is now technically feasible to identify genetic factors affecting drug

Retina in Sciences Basic • 1 section