Familial aggregation studies, segregation analyses, and twin studies have provided strong evidence for a significant genetic role in the etiology of age-related macular degeneration (AMD) (Surv Ophthalmol. 2006;51:313-363). It is unlikely that researchers will pinpoint a single, causative “defective” gene that is responsible for the pathophysiology that leads to choroidal neovascularization (CNV) and/or geographic atrophy in AMD patients. Rather, the disease is likely caused by a complex interaction between a variety of environmental factors and several genes or gene products. The success of monoclonal antibodies and aptamers against vascular endothelial growth factor (VEGF) gene products have supported the concept that there may be a few proteins key to disease progression,3,4 and this has contributed to the emergence of 2 new approaches toward treating AMD. The first involves the modulation of disease-causing proteins, while the second involves promoting therapeutic gene expression.
Modulation of VEGF via RNA Interference
In AMD, the presence of VEGF does not automatically mean that a gene is “defective.” Elevated VEGF mRNA levels are due to the pathologic upregulation of VEGF, which, in turn, may be a result of free radical damage, inflammation, and hypoxia in the retina and choroid (Endocr Rev. 2004;25:581-611). The discovery of the process called ribonucleic acid interference (RNAi) by the Nobel Prize-winning team of Fine and Mello, however, means that it is possible to interrupt this process. This is because RNAi allows for the silencing of a specific disease-associated gene without interfering with other genes. RNAi is the process whereby small RNA molecules suppress or silence gene activity at a post-transcriptional, pre-translational level by inactivating messenger RNA. Interfering RNAs exist in nature and may have evolved for several reasons including as a means of gene control during development, to secure genome stability, and as a defense against infectious viral genomes (Nature. 2004;431:338-342).
Unpublished data from Acuity Pharmaceuticals suggest that Bevasiranib sodium (formerly Cand5), a siRNA directed against VEGF mRNA, was safe in a phase 2 clinical trial. The company also reported decreases in CNV following treatment lasting at least 12 weeks. Meanwhile, a different siRNA, Sirna-027, has been designed to cleave the mRNA of a specific VEGF receptor, VEGFR1. In a mouse model, both intravitreal and periocular injections of Sirna-027 reduced retinal and choroidal neovascularization, suggesting the potential silencing effect of Sirna-027 as well as the benefit of targeting the VEGF1 receptor (Gene Ther. 2006;13:225-234). The use of Sirna-027 is currently undergoing a phase I clinical trial.
Modulation of Disease Genes via Ribozyme Gene Therapy
With the sequence of the human genome being continuously refined, extensive work is being done to identify genes involved in the pathogenesis of AMD. Ribozymes (“ribonucleic enzymes”) are catalytic RNA molecules capable of cleaving specific mRNA sequences. Ribozyme gene therapy often employs a vector to introduce sequences that, when transcribed, are directed to cleave specific disease-causing mRNAs. Ribozyme gene therapy may be utilized to neutralize the production of harmful gene products (Curr Mol Med. 2004;4:489-506). Autosomal dominant diseases often involve a single allele, which generates a gene product responsible for disease. For those autosomal dominant diseases where the second allele is unaffected, cells may continue to produce the naturally occurring normal gene product, although usually at reduced levels. In one model of autosomal dominant retinitis pigmentosa, introduction of ribozyme against a known P23H mutation in rhodopsin resulted in slowed cellular apoptosis (Nat Med. 1998;4:967-971). While AMD rarely behaves like a classic, single-gene, autosomally transmitted disease, it is possible that disease-influencing genes may be dominantly transmitted. Thus, this technique may have a role for AMD therapy in the future.
Therapeutic Gene Expression via AdPEDF
The adenoviral delivery of the pigment epithelial derived growth factor (PEDF) gene is an example of therapeutic gene transfer. PEDF is a neurotrophic and neuroprotective factor shown to protect the retinal pigment epithelial cells as well as demonstrating potent anti-angiogenic properties.10,11 These features make it a potential, multi-faceted therapeutic agent for patients with AMD. Adenovectors, which are designed with 3 key viral genes deleted to prevent viral replication, are effective in transducing ocular cells following intravitreal injection. These modified viruses infiltrate human cells and introduce the beneficial PEDF gene. Intravitreal and subretinal injection of adenoviral vectors carrying cDNA encoding human PEDF (AdPEDF.11) has demonstrated ocular neovascular regression in animal models (Carrión et al, ARVO, May 5, 2005).
A recently completed phase I clinical trial with 28 patients receiving intravitreal injections of AdPEDF.11 demonstrated no evidence of dose limiting toxicity and suggested the possibility of anti-angiogenic activity lasting several months after a single injection (Hum Gene Ther. 2006;17:177-179). Unfortunately, adenovector derived transgene expression of PEDF decreases with time. However, there appear to be no associated systemic immune responses, so repeated treatments may be possible (Wei et al, ARVO, April 29, 2004).
Therapeutic Gene Expression via Encapsulated Cell Technology
An alternative approach to the delivery of therapeutic products to the eye involves in situ production via encapsulated cell technology (ECT). ECT employs an intraocular implant housing human cells genetically engineered to secrete diffusible therapeutic factors (Proc Natl Acad Sci USA. 2006;1033:896-901). A 6-mm long, semi-permeable membrane is anchored to the sclera and allows oxygen and nutrients into the tube to nurture the cells while preventing larger immune system components from interfering. ECT delivery of ciliary neurotrophic factor (CNTF) slowed the loss of photoreceptor cells during retinal degeneration in animal models (Invest Ophthalmol Vis Sci. 2002;43:3292-3298). Clinical trials to evaluate ECT-mediated release of CNTF on retinitis pigmentosa and non-neovascular atrophic macular degeneration are underway.
Stem Cell Therapy
Stem cell therapy has remarkable potential and may permit both therapeutic gene expression and the replacement of lost cells and tissue. Ocular stem cells may come from bone marrow, embryos, umbilical cord, and other such adult tissues as iris pigment epithelium. These immature cells have the potential to become other cells such as photoreceptors or retinal pigment epithelial cells and may be capable of replacing diseased tissue (Ophthalmol Clin North Am. 2003;16:575-582). Such replacement may protect patients from further vision loss or even potentially restore lost vision entirely. There is much that is still unknown about this technology; significant challenges include identifying pluripotent cells, controlling their differentiation, and understanding the relationships between modified cells and the immune system.
Future Implications
The use of gene therapy to treat ocular diseases like AMD has certain advantages over the application of these techniques to other parts of the body due to the relatively immune privileged condition of the eye and the eye’s compartmentalized structure, which is amenable to low dosages delivered in a local fashion. Therapeutic prescriptions for the eye are also non-invasively measurable through a variety of subjective and objective tests (Ophthalmol Clin North Am. 2003;16:575-582). In the case of AMD, the condition appears to be especially amenable to gene therapy, because the etiology of AMD is multifactorial, complicated, and associated with such risk factors as increasing age, cigarette smoking, and heredity. Further understanding of the pathophysiology of both neovascular and non-neovascular AMD at the molecular level will lead to the development of new and different treatments for AMD, as new genes involved in the pathogenesis of the disease are identified, and screening programs aimed at identifying at-risk patients early enough for timely intervention are established. One can envision a future where therapy is specifically tailored to individuals based on their genotype and proteomic profile.
References
1. |
Francis PJ, Stout JT. Gene therapy and control of angiogenesis. Ophthalmol Clin North Am. 2003;16:575-582. |
2. |
Haddad S, Chen CA, Santangelo SL, Seddon JM. The genetics of age-related macular degeneration: a review of progress to date. Surv Ophthalmol. 2006;51:313-363. |
3. |
Gragoudas ES, Adamis AP, Cunningham ET Jr, Feinsod M, Guyer DR; VEGF Inhibition Study in Ocular Neovascularization Clinical Trial Group. Pegaptanib for neovascular age-related macular degeneration. N Engl J Med. 2004;351:2805-2816. |
4. |
Rosenfeld PJ, Villate N, Feuer WJ, Puliafito CA, McCluskey ER. RhuFav V2 (Anti-VEGF antibody fragment) in neovascular AMD: safety, tolerability, and efficacy of multiple, escalating dose intravitreal injections. Invest Ophthalmol Vis Sci. 2003; 44:970. |
5. |
Ferrara N. Vascular endothelial growth factor: basic science and clinical progress. Endocr Rev. 2004;25:581-611. |
6. |
Mello CC, Conte D Jr. Revealing the world of RNA interference. Nature. 2004;431:338-342. |
7. |
Shen J, Samul R, Silva RL, et al. Suppression of ocular neovascularization with siRNA targeting VEGF receptor 1. Gene Ther. 2006;13:225-234. |
8. |
Baqheri S, Kashani-Sabet M. Ribozymes in the age of molecular therapeutics. Curr Mol Med. 2004;4:489-506. |
9. |
Lewin AS, Drenser KA, Hauswirth WW, et al. Ribozyme rescue of photoreceptor cells in a transgenic rat model of autosomal dominant retinitis pigmentosa. Nat Med. 1998;4:967-971. |
10. |
Steele FR, Chader GJ, Johnson LV, Tombran-Tink J. Pigment epithelium-derived factor: neurotrophic activity and identification as a member of the serine protease inhibitor gene family. Proc Natl Acad Sci USA. 1993;90:1526-1530. |
11. |
Mori K, Gehlbach P, Ando A, McVey D, Wei L, Campochiaro PA. Regression of ocular neovascularization in response to increased expression of pigment epithelium-derived factor. Invest Ophthalmol Vis Sci. 2002;43:2428-2434. |
12. |
Carrión ME, Hamilton MM, Harris B, King R, Mori K, Wei LL. Ocular pharmacokinetics of pigment epithelium-derived factor (PEDF) following adenovector-based gene delivery indicate that low doses of PEDF are therapeutic. Poster presented at: annual meeting of the Association of Research for Vision in Ophthalmology (ARVO); May 5, 2005. |
13. |
Campochiaro PA, Nguyen QD, Shah SM, et al. Adenoviral vector-delivered pigment epithelium derived factor for neovascular age-related macular degeneration: results of a phase I clinical trial. Hum Gene Ther. 2006;17:177-179. |
14. |
Wei LL, Hamilton M, McVey D, King CR. Repeat dosing of adenovector in the eye. Poster presented at: annual meeting of the Association of Research for Vision in Ophthalmology (ARVO); April 29, 2004. |
15. |
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;1033:896-901. |
16. |
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. |
Author Disclosure
The authors state that they have no financial relationship with the manufacturer or provider of any product or service discussed in this article or with the manufacturer or provider of any competing product or service.