On the Horizon: Genetic Therapies for AMD
DANIEL F. KIERNAN, MD · THEODORE K. LIN, MD · RAMA D. JAGER, MD, FACS
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.
GENETICS AND AMD
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 firstname.lastname@example.org.|
DRY AMD THERAPIES
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 (Clinicaltrials.gov number NCT00447954). POT-4 is derived from compstatin, which inhibits complement-initiated inflammation.14 A phase 1 study is also under way (Clinicaltrials.gov 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 (Clinicaltrials.gov 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 (Clinicaltrials.gov 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.
PIGMENT EPITHELIUM–DERIVED FACTOR
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.
ZINC FINGER PROTEINS
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
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
- Jager RD, Mieler WF, Miller JW. Age-related macular degeneration. N Engl J Med. 2008;358:2606-2617.
- Congdon N, O'Colmain B, Klaver CC, et al. Causes and prevalence of visual impairment among adults in the United States. Arch Ophthalmol. 2004;122:477-485.
- Seddon JM, Ajani UA, Mitchell BD. Familial aggregation of age-related maculopathy. Am J Ophthalmol. 1997;123:199-206.
- Klaver CC, Wolfs RC, Assink JJ, et al. Genetic risk of age-related maculopathy: population-based familial aggregation study. Arch Ophthalmol. 1998;116:1646-1651.
- Seddon JM, Cote J, Page WF, et al. The US twin study of age-related macular degeneration. Arch Ophthalmol. 2005;123:321-327.
- Klein RJ, Zeiss C, Chew EY, et al. Complement factor H polymorphism in age-related macular degeneration. Science. 2005;308:385-389.
- Edwards AO, Ritter R III, Abel KJ, et al. Complement factor H polymorphism and age-related macular degeneration. Science. 2005;308:421-424.
- Haines JL, Hauser MA, Schmidt S, et al. Complement factor H variant increases the risk of age-related macular degeneration. Science. 2005;308:419-421.
- Hageman GS, Anderson DH, Johnson LV, et al. A common haplotype in the complement regulatory gene factor H (HF1/CFH) predisposes individuals to age-related macular degeneration. Proc Natl Acad Sci USA. 2005;102:7227-7232.
- Gold B, Merriam JE, Zernant J, et al. Variation in factor B (BF) and complement component 2 (C2) genes is associated with age-related macular degeneration. Nat Genet. 2006;38:458-462.
- Kanda A, Chen W, Othman M, et al. A variant of mitochondrial protein LOC387715/ARMS2, not HTRA1, is strongly associated with age-related macular degeneration. Proc Natl Acad Sci USA. 2007;104:16227-16232.
- Tao W, Wen R, Goddard MB, et al. Encapsulated cell-based delivery of CNTF reduces photoreceptor degeneration in animal models of retinitis pigmentosa. Invest Ophthalmol Vis Sci. 2002;43:3292-3298.
- Sieving PA, Caruso RC, Tao W, et al. Ciliary neurotrophic factor (CNTF) for human retinal degeneration: phase I trial of CNTF delivered by encapsulated cell intraocular implants. Proc Natl Acad Sci USA. 2006;103:3896-3890.
- Janssen BJ, Halff EF, Lambris JD, Gros P. Structure of compstatin in complex with complement component C3c reveals a new mechanism of complement inhibition. J Biol Chem. 2007;282:29241-29247.
- Tanito M, Li F, Elliott MH, Dittmar M, Anderson RE. Protective effect of TEMPOL derivatives against light-induced retinal damage in rats. Invest Ophthalmol Vis Sci. 2007;48:1900-1905.
- Brantley MA Jr, Fang AM, King JM, et al. Association of complement factor H and LOC387715 genotypes with response of exudative age-related macular degeneration to intravitreal bevacizumab. Ophthalmology. 2007;114:2168-2173.
- Tomanin R, Scarpa, M. Why do we need new gene therapy viral vectors? Characteristics, limitations and future perspectives of viral vector transduction. Curr Gene Ther. 2004;4:357-372.
- Hacein-Bey-Abina S, Von Kalle C, Schmidt M. LMO2-associated clonal proliferation in two patients after gene therapy for SCID-X1. Science. 2003;302:415-419.
- He Y, Smith SK, Day KA, et al. Alternative splicing of vascular endothelial growth factor (VEGF)-R1 (FLT-1) pre-mRNA is important for the regulation of VEGF activity. Mol Endocrinol. 1999;13:537-545.
- Kendall RL, Wang GL, Thomas KA. Identification of a natural soluble form of the vascular endothelial growth factor receptor, FLT-1, and its heterodimerization with KDR. Biochem Biophys Res Comm. 1996;226:324-328.
- Honda M, Sakamoto T, Ishibashi T, et al. Experimental subretinal neovascularization is inhibited by adenovirusmediated soluble VEGF.flt-1 receptor gene transfection: a role of VEGF and possible treatment for SRN in age-related macular degeneration. Gene Ther. 2000;7:978-985.
- Rota R, Riccioni T, Zaccarini M, et al. Marked inhibition of retinal neovascularization in rats following soluble-flt-1 gene transfer. J Gene Med. 2004;6:992-1002.
- Lai CM, Shen WY, Brankov M, et al. Long-term evaluation of AAV-mediated sFlt-1 gene therapy for ocular neovascularization in mice and monkeys. Mol Ther. 2005;12:659-668.
- Lai YK, Shen WY, Brankov M, et al. Potential long-term inhibition of ocular neovascularization by recombinant adeno-associated virus-mediated secretion gene therapy. Gene Ther. 2002;9:804-813.
- Gehlbach P, Demetriades AM, Yamamoto S, et al. Periocular injection of an adenoviral vector encoding pigment epithelium-derived factor inhibits choroidal neovascularization. Gene Ther. 2003;10:637-646.
- Alon T, Hemo I, Itin A, et al. Vascular endothelial growth factor acts as a survival factor for newly formed retinal vessels and has implications for retinopathy of prematurity. Nat Med. 1995;1:1024-1028.
- Baffert F, Le T, Sennino B, Thruston et al. Cellular changes in normal blood capillaries undergoing regression after inhibition of VEGF signaling. Am J Physiol Heart Circ Physiol. 2006;290:547-559.
- Baffert F, Thurston G, Rochon-Duck M, et al. Age-related changes in vascular endothelial growth factor dependency and angiopoietin-1-induced plasticity of adult blood vessels. Circ Res. 2004;94:984-992.
- Kamba T, Tam BY, Hashizume H, et al. VEGF-dependent plasticity of fenestrated capillaries in the normal adult microvasculature. Am J Physiol Heart Circ Physiol. 2006;290:560-576.
- Oosthuyse B, Moons L, Storkebaum E, et al. Deletion of the hypoxia-response element in the vascular endothelial growth factor promoter causes motor neuron degeneration. Nat Genet. 2001;28:131-138.
- Timmons L, Tabara H, Mello CC, et al. Inducible systemic RNA silencing in Caenorhabditis elegans. Mol Biol Cell. 2003;14:2972-2983.
- Reich SJ, Fosnot J, Kuroki A, et al. Small interfering RNA (siRNA) targeting VEGF effectively inhibits ocular neovascularization in a mouse model. Mol Vis. 2003;9:210-216.
- Shen J, Samul R, Lima e Silva R, et al. Suppression of ocular neovascularization with siRNA targeting VEGF receptor 1. Gene Ther. 2005;13:225-234.
- Cashman SM, Bowman L, Christofferson J, et al. Inhibition of choroidal neovascularization by adenovirus-mediated delivery of short hairpin RNAs targeting VEGF as a potential therapy for AMD. Invest Ophthalmol Vis Sci. 2006;47:3496-3504.
- Araki T, Taniwaki T, Becerra SP, et al. Pigment epithelium-derived factor (PEDF) differentially protects immature but not mature cerebellar granule cells against apoptotic cell death. J Neurosci Res. 1998;53:7-15.
- Bilak MM, Corse AM, Bilak SR, et al. Pigment epithelium-derived factor (PEDF) protects motor neurons from chronic glutamate-mediated neurodegeneration. J Neuropathol Exp Neurol. 1999;58:719-728.
- Cao W, Tombran-Tink J, Elias R, et al. In vivo protection of photoreceptors from light damage by pigment epithelium-derived factor. Invest Opthalmol Vis Sci. 2001;42:1642-1652.
- Steele FR, Chader GJ, Johnson LV, et al. Pigment epithelium-derived factor: neurotrophic activity and identification as a member or the serine protease inhibitor gene family. Proc Natl Acad Sci USA. 1993;90:1526-1530.
- Mori K, Duh E, Gehlbach P, et al. Pigment epithelium-derived factor inhibits retinal and choroidal neovascularization. J Cell Physiol. 2001;188:253-263.
- Mori K, Gehlbach P, Ando A, et al. Regression of ocular neovascularization by increased expression of pigment epithelium-derived factor. Invest Ophthalmol Vis Sci. 2001:43: 2428-2434.
- Saishin Y, Lima Silva R, Saishin Y, et al. Periocular injection of microspheres containing PKC412 inhibits choroidal neovascularization in a porcine model. Invest Ophthalmol Vis Sci. 2003;44:4989-4993.
- Rasmussen H, Chu KW, Campochiaro P, et al. An open-label, phase I, single administration, dose-escalation study of ADGVPEDF.11D (ADPEDF) in neovascular age-related macular degeneration (AMD). Hum Gene Ther. 2001;12:2029-2032.
- Tan S, Guschin D, Davalos A, et al. Zinc-finger protein-targeted gene regulation: genomewide single-gene specificity. Proc Natl Acad Sci USA. 2003;100:11997-12002.
- Zhang HS, Kachi S, Kachi M, et al. Engineered zinc finger protein transcription factors as a potential therapy for ocular neovascularization. Paper presented at: Annual Meeting of the Association of Research in Vision and Ophthalmology, Ft. Lauderdale, FL, April 30–May 4, 2006; abstract 1786.
- Joussen AM, Poulaki V, Tsujikawa A, et al. Suppression of diabetic retinopathy with angiopoietin-1. Am J Pathol. 2002;160:1683-1693.
- Tait CR, Jones PF. Angiopoietins in tumours: the angiogenic switch. J Pathol. 2004;204:1-10.
- Davis S, Aldrich TH, Jones P, et al. Isolation of angiopoietin-1, a ligand for the TIE2 receptor, by secretion trap expression cloning. Cell. 1996;87:1161-1169.
- Maisonpierre PC, Suri C, Jones PF, et al. Angiopoietin-2, a natural antagonist for Tie2 that disrupts in vivo angiogenesis. Science. 1997;277:55-60.
- Oshima Y, Oshima S, Nambu H, et al. Different effects of angiopoietin 2 in different vascular beds in the eye: new vessels are most sensitive. FASEB J. 2005;19:963-965.
- Nambu H, Nambu R, Oshima Y, et al. Angiopoietin 1 inhibits ocular neovascularization and breakdown of the blood-retinal barrier. Gene Ther. 2004;11:865-873.
- Nambu H, Umeda N, Kachi S, et al. Angiopoietin 1 prevents retinal detachment in an aggressive model of proliferative retinopathy, but has no effect on established neovascularization. J Cell Physiol. 2005;204:227-235.
- O'Reilly MS, Holmgren L, Shing Y, et al. Angiostatin: a novel angiogenesis inhibitor that mediates the suppression of metastases by a Lewis lung carcinoma. Cell. 1994;79:315-328.
- Tonini T, Rossi F, Claudio PP. Molecular basis of angiogenesis and cancer. Oncogene. 2003;22:6549-6556.
- Sima J, Zhang SX, Shao C, et al. The effect of angiostatin on vascular leakage and VEGF expression in rat retina. FEBS Lett. 2004;564:19-23.
- Lai CC, Wu WC, Chen SL, et al. Suppression of choroidal neovascularization by adeno-associated virus vector expressing angiostatin. Invest Ophthalmol Vis Sci. 2001;42:2401-2407.
- Igarashi T, Miyake K, Kato K, et al. Lentivirus-mediated expression of angiostatin efficiently inhibits neovascularization in a murine proliferative retinopathy model. Gene Ther. 2003;10:219-226.
- Raisler BJ, Berns KI, Grant MB, et al. Adeno-associated virus type 2 expression of pigmented epithelium-derived factor or Kringles 1-3 of angiostatin reduce retinal neovascularization. Proc Natl Acad Sci USA. 2002;99:8909-8914.
- Morgunova E, Tuuttila A, BergmannU, et al. Structure of human pro-matrix metalloproteinase-2: activation mechanism revealed. Science. 1999;284:1667-1670.
- Ciardella AP, Donsoff IM, Guyer DR, et al. Antiangiogenesis agents. Ophthalmol Clin North Am. 2002;15:453-458.
- De La Paz MA, Itoh Y, Toth CA, et al. Matrix metalloproteinases and their inhibitors in human vitreous. Invest Ophthalmol Vis Sci. 1998;39:1256-1260.
- Lambert V, Munaut C, Jost M, et al. Matrix metalloproteinase-9 contributes to choroidal neovascularization. Am J Pathol. 2002;161:1247-1253.
- Lambert V, Wielockx B, Munaut C, et al. MMP-2 and MMP-9 synergize in promoting choroidal neovascularization. FASEB J. 2003;17:2290-2292.
- Takahashi T, Nakamura T, Hayashi A, et al. Inhibition of experimental choroidal neovascularization by overexpression of tissue inhibitor of metalloproteinases-3 in retinal pigment epithelium. Am J Ophthalmol. 2000;130:774-781.
- Cheng L, Rivero ME, Garcia CR, et al. Evaluation of intraocular pharmacokinetics and toxicity of prinomastat (AG3340) in the rabbit. J Ocul Pharmacol Ther. 2001;17:295-304.
- Griffioen AW. AG-3340 (Agouron Pharmaceuticals Inc). IDrugs. 2000;3:336-345.
- Blodi BA, Group AS. The angiogenesis inhibitor, prinomastat (AG3340), in the treatment of age-related macular degeneration: study design and baseline characteristics. Invest Ophthalmol Vis Sci. 2001;42:S311.
- Weber BHF, Vogt G, Pruett RC, et al. Mutations in the tissue inhibitor of metalloproteinases-3 (TIMP3) in patients with Sorsby's fundus dystrophy. Nat Genet. 1994;8:352-356.
- Fariss RN, Apte SS, Suthert PJ, et al. Accumulation of tissue inhibitor of metalloproteinases-3 in human eyes with Sorsby's fundus dystrophy or retinitis pigmentosa. Br J Ophthalmol. 1998;82:1329-1334.
- Soboleva G, Geis B, Schrewe H, et al. Sorsby fundus dystrophy mutation Timp3s156C affects the morphological and biochemical phenotype but not metalloproteinase homeostasis. J Cell Physiol 2003;197:149-156.
- Apte SS, Olsen BR, Murphy G. The gene structure of tissue inhibitor of metalloproteinases (TIMP)-3 and its inhibitory activities define the distinct TIMP gene family. J Biol Chem. 1995;270:14313-14318.
- Green WR, Wilson, D. J. Choroidal neovascularization. Ophthalmology. 1986;93:1169-1176.
- Carmeliet P, Jain RK. Angiogenesis in cancer and other diseases. Nature. 2000;407:249-257.
- Grimm C, Wenzel A, Groszer M, et al. HIF-1-induced erythropoietin in the hypoxic retina protects against light-induced retinal degeneration. Nat Med. 2002;8:718-724.
- Forsythe JA, Jiang BH, Iyer NV, et al. Activation of vascular endothelial growth factor gene transcription by hypoxia-inducible factor 1. Mol Cell Biol. 1996;16:4604-4613.
- Wang B, Li H, Yan H, et al. Genistein inhibited hypoxia- inducible factor-1alpha expression induced by hypoxia and cobalt chloride in human retinal pigment epithelium cells. Methods Find Exp Clin Pharmacol. 2005;27:179-184.
- Ando A, Yang A, Nambu H, et al. Blockade of nitric-oxide synthase reduces choroidal neovascularization. Mol Pharmacol. 2002;62:539-544.
- Ando A, Yang A, Mori K, et al. Nitric oxide is proangiogenic in the retina and choroid. J Cell Physiol. 2002;191:116-124.
- Hynes RO. A reevaluation of integrins as regulators of angiogenesis. Nat Med. 2002;8:918-921.