On the Horizon: Genetic Therapies for AMD

On the Horizon: Genetic Therapies for AMD


Age-related macular degeneration (AMD) is the leading cause of vision loss in people 50 years of age or older in the United States.1,2 Advances in research have led to a better understanding of the genetics of AMD and to new therapies designed to prevent and treat it. A handful of promising genetic therapies are in human trials, but questions regarding their efficacy and safety must be answered before they can achieve clinical application.


The heritability of AMD has been well documented in familial aggregation and twin studies. Familial aggregation studies have shown an increased prevalence of AMD in first-degree family members compared with first-degree family members in controls. In the US twin study, heritability estimates ranged from 0.46 to 0.71.3-5

In 2005, 3 independent groups reported that a polymorphism, Tyr402His, in the complement factor H (CFH) gene located on chromosome 1 substantially increases the risk of AMD in Caucasians.6-8 Those individuals who are heterozygous for the Tyr204His polymorphism have their risk of AMD increased by 2.1- to 4.6-fold, while in homozygous individuals, risk increases by 3.3- to 7.4-fold.9 Genes for complement factors B and C2 have also been associated with an increased risk of AMD. The 3 known complement associated polymorphisms may account for as much as 75% of AMD cases.10

The Ala69Ser polymorphism on the AMD-susceptibility 2 gene (ARMS2, also known as LOC387715), located on chromosome 10, has been associated with an increased risk of developing AMD independent of CFH.11

A recent retrospective analysis of patients treated with bevacizumab (Avastin, Genentech) for exudative AMD and later found to be either genetically heterozygous or homozygous for either CFH or LOC387715, showed a worse treatment response and larger choroidal lesion sizes in those homozygous for CFH.16 Commercially available tests designed to check for these polymorphisms in an individual's genome have recently become available and will help personalize targeted therapies for individuals at risk.

Daniel F. Kiernan, MD, is a resident in the Section of Ophthalmology and Visual Science of the Department of Surgery at the University of Chicago Hospitals. Theodore K. Lin, MD, is a retina fellow in the Section of Ophthalmology at Chicago. Rama D. Jager, MD, FACS, is clinical assistant professor in the Section of Ophthalmology at Chicago and a partner in University Retina and Macula Associates in Oak Forest, IL. The authors report no financial interests in any products mentioned in this article. Dr. Jager can be reached via e-mail at


Several investigations are ongoing that target oxidative photoreceptor damage, accumulation of toxic metabolites, and complement-associated inflammatory cascade mediators, which may lead to vision loss in dry AMD patients. Potential dry AMD therapies include ciliary neurotrophic factor (CNTF), intravitreal complement inhibitor (POT-4; Potentia Pharmaceuticals, Louisville, KY) and antioxidant eye drops (OT-551; Othera, Exton, PA). CNTF (Neurotech, Lincoln, RI) is a naturally occurring substance that, in animal models, protects against photoreceptor degeneration.12 A phase 1 study has been completed showing no significant toxicity,13 and a phase 2 study is under way with visual acuity (VA) as the primary efficacy end-point ( number NCT00447954). POT-4 is derived from compstatin, which inhibits complement-initiated inflammation.14 A phase 1 study is also under way ( number NCT00473928). OT-551 may prevent complement-mediated oxidative damage to the retinal pigment endothelium (RPE) and photoreceptors.15 A phase 2 trial to determine whether a topical preparation is efficacious at preventing progression of geographic atrophy ( number NCT00306488) is ongoing. Results from these studies will determine the efficacy and safety profile of these dry AMD treatments.


Choroidal neovascularization (CNV) is the major cause of severe vision loss in wet AMD patients.1 Considerable progress has been made identifying molecular signals that promote CNV, and it appears that imbalances between stimulatory and inhibitory proteins contribute to its development. Re-establishing this balance by ocular-gene transfer to either block stimulators or increased expression of endogenous inhibitors of CNV is an appealing therapeutic approach because it provides a potential means to achieve intraocular effects with little impact on the rest of the body.


Adenoviral-associated vectors are most commonly used in ocular gene therapy. They are relatively easy to produce, and they can provide good expression of specific proteins within cells. The potential disadvantages of adenoviral vectors include the possibility of enhanced immune response leading to tissue destruction, insufficient amounts of the protein production, requiring more treatments, or overexpression of the targeted gene with potential neoplastic consequences.17 In 2003, 2 patients in a clinical trial to treat X-linked severe combined immunodeficiency syndrome developed T-cell leukemia, halting the study. Initially thought to be more commonly associated with retroviral-based vectors, this is a rare but devastating potential downside to adenoviral vectors.18 Although no more reports of such adverse events have arisen, more research is required to ascertain whether targeted ocular therapy can result in systemic malignancies.


Vascular endothelial growth factor (VEGF) antagonists are currently the gold standard for wet AMD therapy. VEGF inhibition using genetic targets is currently under investigation. The secreted extracellular domain of VEGF receptor 1, sFlt-1, is a naturally occurring protein antagonist of VEGF formed by alternative splicing of the premRNA for the full-length receptor.19,20 Intraocular injection of an attenuated adenovirus vector containing sFlt-1 (Ad.sFlt-1) achieved long-term suppression of CNV in mice and monkeys.21-24 Periocular injection of Ad.sFlt-1 resulted in transduction of the gene into episcleral cells, high expression levels in the choroid, and suppression of CNV.25 These results indicate that gene transfer of sFlt-1 may provide efficacy in patients with exudative AMD.

However, as blood-ocular barrier breakdown is common in ophthalmic neovascular diseases, systemic penetration of such VEGF antagonists may occur, especially with frequent treatment. It is not certain whether long-term inhibition of all VEGF isoforms is safe, since VEGF plays an important role in a variety of systems.26-30 This uncertainty must be addressed before transfer of sFlt-1 can be used clinically.

The 2006 Nobel Prize in Medicine was awarded for the discovery of RNA interference in Caenorhabditis elegans.31 RNA interference is a mechanism that inhibits protein expression by hindering the transcription of specific genes by way of targeted mRNA cleavage, histone modification, and DNA methylation. Intraocular injection of small interfering RNA (siRNA) fragments targeting VEGF mRNA or VEGF receptor mRNA suppresses CNV and both of these approaches are being tested in clinical trials.32,33 Treatments with siRNA must enter cells to be effective, resulting in less efficiency than agents that target both extra- and intracellular VEGF isoforms. Additionally, siRNA is detectable in the retina for only 1 week,33 indicating that frequent injections or improved drug-delivery may be necessary to maintain therapeutic levels.

Prolonged effects may be achieved by using viral vectors to express short hairpin RNAs that target the mRNA of VEGF or its receptors, though this approach would have the same potential advantages and disadvantages as gene transfer of sFlt-1.34 Sustained delivery of siRNA would provide an alternative means of obtaining prolonged silencing, and since siRNA penetrates the sclera, such a device could be mounted on the outside of the eye.33 Currently OPKO Health (Miami, FL) is carrying out the the COBALT study ( number NCT00499590), a phase 3 trial examining whether different doses of bevasiranib, a siRNA molecule that targets VEGF genes, used in conjunction with ranibizumab is as efficacious as ranibizumab (Lucentis, Genentech) alone.


One well-studied protein that inhibits ocular neovascularization when expressed by gene transfer is pigment epithelium–derived growth factor (PEDF). PEDF has been shown to promote the survival of cultured neurons and protect photoreceptors from excessive light exposure.35-38 PEDF has antiangiogenic activity and could address 2 problems seen in patients with AMD: photoreceptor degeneration and CNV. Intravitreal or subretinal injection of an attenuated adenoviral vector expressing human PEDF (AdPEDF.11) has been shown to suppress the development of CNV and cause regression of established neovascularization.25,39 In animal models, AdPEDF.11 induced apoptosis of endothelial cells in new blood vessels, but not in normal blood vessels. Injection of AdPEDF.11 beneath the conjunctiva resulted in transduction of episcleral cells that produced PEDF, causing regression of CNV.40 Since subconjunctival injections are less invasive and associated with fewer serious adverse events than intravitreal injections, this approach may offer a favorable alternative to intraocular injections.

These results with AdPEDF led to a phase 1 trial in patients with CNV due to AMD. GenVec (Gaithersburg, MD) has developed an adenoviral vector encoding PEDF (AdGVPEDF.11D) and has completed the dose-escalation portion of a phase 1, multicenter trial evaluating the effects of a single intravitreal injection of the vector in 28 patients. No serious adverse events or dose-limiting toxicities arose, and 50% to 94% of patients had either no change or an improvement in lesion size from baseline after 3 to 6 months, suggesting that antiangiogenesis may last for several months after a single intravitreal injection.41 GenVec has also recently completed enrollment of 22 patients with less severe disease than in the phase 1 study. As with the first cohort, no dose-limiting toxicities or drug-related severe adverse events were observed.42 These reports were not designed to test clinical efficacy, however, but further studies investigating the efficacy of AdPEDF.11 in patients with wet AMD are ongoing.


Another approach for obtaining more physiologic levels of PEDF is expression of an engineered zinc finger protein transcription factor (ZFP-TF) to increase protein production by stimulating its endogenous promoter.43 This approach may better achieve physiologic concentrations and avoid paradoxical effects. Intravitreous or subretinal injection of adenovirus-associated vector ZFP-TF PEDF activator in mice resulted in increased levels of PEDF mRNA in the eye and suppressed CNV at Bruch's membrane rupture sites.44 This indicates methods to increase endogenous levels of antiangiogenic proteins in the eyes of AMD patients, but only in a murine model. Further studies are required to demonstrate an effect in humans.


Angiopoietins are a family of extracellular ligands that recognize and bind to the extracellular domain of Tie-2, an endothelial cell–specific receptor tyrosine kinase with endothelial growth factor homology domains.45,46 Tie-2 is stimulated by binding angiopoietin-1 and is blocked by angiopoietin-2.47,48 Blockade of Tie-2 by overexpression of angiopoietin-2 causes stimulation or regression of CNV, depending on whether levels of VEGF are high or low, respectively, compared to those of angiopoietin-2.49 In angiogenesis, angiopoietin-1 is a maturation factor promoting the recruitment of smooth muscle cells and pericytes to developing vessels,50,51 whereas angiopoietin-2 is a destabilizing factor seen before vessel sprouting or regression.45,46 Thus the ratio of angiopoietins may serve as an angiogenic switch. Angiogenesis occurs with higher angiopoietin-2 levels because it acts as a destabilizing factor, allowing the vasculature to sprout under the influence of VEGF or regress when VEGF is absent.46 Animal studies suggest that ocular neovascularization but not mature vessels are sensitive to angiopoietin-249 and that regression or stimulation of new vessels depends on the ratio of angiopoietin-2 to VEGF and the overall state of ischemia.46 Since overexpression of angiopoietin-1 or blockade of angiopoietin-2 inhibits CNV, gene transfer is a promising therapeutic approach, but may require concurrent treatment with direct VEGF inhibitors.


Angiostatin is a plasminogen cleavage product of fibrinogen that inhibits tumor angiogenesis and was among the first endogenous angiogenesis inhibitors recognized.52 It inhibits extracellular matrix–enhanced and tissue plasminogen activator–catalyzed plasminogen activation.52,53 Intravitreal injection of an angiostatin expression construct packaged in adenovirus-associated vectors suppressed CNV in rats with oxygen-induced retinopathy but not in normal rats.54 Subretinal injection of recombinant adeno-associated virus expressing mouse angiostatin significantly reduced CNV lesion size compared with controls in rats with laser-induced rupture of Bruch's membrane. Furthermore, recombinant adeno-associated virus angiostatin led to sustained expression of the angiostatin gene in chorioretinal tissue for up to 150 days and was not associated with inflammation.55-57 These findings suggest that angiostatin may be beneficial in the treatment of wet AMD, though human studies are needed to determine clinical relevance.


Matrix metalloproteinases (MMPs) are enzymes that degrade extracellular matrix and contribute to tissue remodeling.58 MMPs are thought to be involved in AMD, proliferative diabetic retinopathy, and retinopathy of prematurity.59 MMPs and tissue inhibitors of metalloproteinases were found in vitreous specimens of some patients with AMD and 2 subtypes, MMP-2 and MMP-9, were found to localize to areas of new vessel formation in surgically excised subfoveal CNV in 5 patients with AMD.60 MMP-9 expression was shown in a laser-induced model of CNV to be upregulated concomitantly with the appearance of inflammatory cells in the CNV lesion. In MMP-2, MMP-9, and MMP-2–MMP-9 deficient mice, CNV occurred at a reduced level.61,62 Moreover, overexpression of tissue inhibitor of MMP-1, MMP-2, or MMP-3 significantly decreased CNV compared with controls.62,63

Prinomastat (AG3340, Pfizer, New York) is an oral nonpeptide inhibitor of gelatinase types A and B (MMP-2 and MMP-9), MMP-14, and collagenase 3 that was originally tested as a chemotherapeutic agent and later found to be safe for intravitreal and subtenon injection in animals.64,65 Recruitment of patients with CNV caused by AMD was completed in 1999 for a study of the safety and efficacy of Prinomastat;66 however, the results of this study were never published and Pfizer is no longer pursuing phase 3 clinical trials. Further studies are needed to determine whether Prinomastat or other MMP inhibitors may be safe and useful as adjunctive agents in treating ocular neovascularization.


Patients with Sorsby dystrophy have mutations in tissue inhibitor of metalloproteinase (TIMP)-3 that results in retinal degeneration and CNV.67 Mutations in TIMP-3 may disrupt protein folding and patients with Sorsby dystrophy have thickened Bruch's membranes containing aggregates of TIMP-3.68,69 Recently, it has been shown that TIMP-3 also has antiangiogenic properties,70 suggesting that TIMP-3 may contribute in multiple ways to the barrier posed by Bruch's membrane that prevents vascular invasion into the subretinal space. Increased levels of TIMP-3 in RPE cells of rats induced by subretinal injection of virus containing a TIMP-3 expression construct suppressed CNV at Bruch's membrane rupture sites.63 This suggests that TIMP-3 is a candidate for gene therapy for wet AMD, but expression of an insoluble protein may carry greater risk than expression of a soluble protein. Overexpression of TIMP-3 may compromise its turnover and cause it to accumulate and thicken Bruch's membrane, which is associated with CNV in patients with AMD,71 as well as those with Sorsby dystrophy. Therefore, overexpression of insoluble proteins in RPE cells may limit the usefulness of this potential therapy.


Nuclear translocation of hypoxia-inducible factor–1 activates target genes, including VEGF and other genes involved in angiogenesis and vasodilation.72-74 Genistein, a soybean isolate, is a protein tyrosine kinase inhibitor that inhibits hypoxia-inducible factor–1 expression in RPE cells.75 Clinical studies are necessary to determine whether hypoxia-inducible factor–1 inhibition may be a feasible strategy for the treatment of ocular neovascularization.


Nitric oxide synthetase (NOS) is a signaling molecule that acts as a mediator of vasodilation and permeability.76 Three isoforms of NOS have been shown to have different pro- or antiangiogenic effects in both the retina and choroid, depending on whether the model was laser-induced rupture of Bruch's membrane or oxygen-induced ischemic retinopathy.76,77 Deficiency of any of the 3 isoforms in transgenic mice with increased VEGF expression in the photoreceptors caused a decrease in CNV but no alteration of VEGF expression.77 NG-monomethyl-Larginine is a broad-spectrum NOS inhibitor that has been shown to inhibit CNV in animals with laser-induced rupture of Bruch's membrane and in transgenic mice with expression of VEGF in the photoreceptors.76 These findings suggest that broad-spectrum NOS inhibition may have therapeutic potential in the treatment of CNV, but more studies are required to elucidate the role of each NOS isoform.


Thrombospondin-1 and thrombospondin-2 suppress angiogenesis by activating transforming growth factor–β, as a negative regulator of MMP-9 activation and as an activator of apoptotic pathways.78 Transforming growth factor–β upregulates tissue inhibitors of metalloproteinases, and MMP-9 activates angiogenesis by releasing VEGF from sequestered stores.78 More studies are needed to obtain clinically useful data for this potential treatment target.


Ongoing studies show the great promise of gene therapy for the treatment of wet AMD. Treatments have focused on blockade of VEGF, but a variety of mediators likely contribute to CNV, and many can serve as potential targets. Gene transfer of siRNA, PEDF, and angiostatin has shown beneficial effects in animal models and a small number of human trials. Whether or not these therapies are more efficacious than the current therapy remains to be seen. In vitro and animal models offer the possibility of many treatments that may not translate directly into human clinical trials. The rising cost of care for treating these patients with wet AMD should also factor into the optimal treatment. Further studies are needed to identify targets to prevent vision loss in these patients prior to reaching an exudative disease stage. RP


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