NEW THERAPIES IN DIABETIC RETINOPATHY AND AMD

A gene therapy approach using an oxygen sensitive “on/off†switch may produce new therapies in diabetic retinopathy and agerelated macular degeneration (AMD), report US researchers. Several research groups worldwide have focused significant efforts into fine-tuning the mechanics of how and when to turn genes on and off for maximal therapeutic benefit. New studies from a US research team, based at the Centre for Complex Systems and Brain Sciences at Florida Atlantic University, report the construction of a novel gene promoter regulated by oxygen concentration (IOVS; DOI:10.1167/ iovs.10-6835). The promoter, which may be incorporated upstream of a therapeutic gene, could ultimately be used in the treatment of hypoxia-related retinal disorders, including diabetic retinopathy and AMD.

Gene therapy has become an attractive therapeutic approach to dealing with retinal disorders, not least of all due to the retina’s relatively immune privilege status, its surgical access and the presence of the blood-brain barrier, minimising any “leakage†of delivered genes and their vectors. Recent human and animal clinical successes have boosted the field over the last three years and in recent months the first gene therapy approval was announced by the European Medicines Agency following the development of a product aimed at delivering a functional lipoprotein lipase gene to treat lipoprotein lipase deficiency (LPLD).

A key challenge arising in many of the human and animal trials to date is to design a system that controls the level and timing of gene expression. One ideal way to achieve such control is to use perturbations arising from the pathology itself to trigger the “cureâ€. In a number of ocular disorders oxygen sensitivity is critical to pathogenesis and a reduction in O2 levels may serve as a trigger of pathology, for example in diabetic retinopathy and AMD. The Florida Atlantic University based team, led by Prof Janet Blanks, used such knowledge to harness the environmental trigger to drive the expression of an AAV (adeno-associated virus) delivered transgene in retinal Müller cells. The genebased technology uses a regulatory domain that incorporates multiple hypoxia (oxygensensing) responsive elements (HREs) known to bind the transcription factor HIF-1, a key component of the oxygen-dependent sensing system. Under hypoxic conditions HIF-1 dimerizes with HIF-1 beta and translocates to the nucleus activating gene transcription of target genes. Combining the HREs with a Müller cell specific promoter – the human glial fibrillary acidic protein (GFAP) sequence – facilitates the expression of the transgene under hypoxic conditions within a specific cell type.

In experimental models the US research team delivered AAV vectors intra-vitreally containing the HRE-GFAP sequence upstream of a green fluorescent protein (GFP) marker. For cell-based work the team used primary cultures of Müller cells that were transfected to show a lack of gene (GFP) expression under normoxic conditions but high levels of expression under hypoxic conditions. However, in testing the system under in vivo conditions the oxygen-induced retinopathy (OIR) model was employed where post-natal experimental models are exposed to high levels of oxygen between day seven and 12 and then returned to normal air for five days.

During the five-day regression phase retinal cells produce several HIF-1 mediated pro-angiogenic factors leading to neovascularisation by day 17. Expression of HIF-1 in the day-17 hypoxic retina is 31 times greater than the normoxic retina. The engineered promoter remained silent under aerobic conditions however, induction of hypoxia induced a 12-fold (in primary Müller cells) and a 16-fold increase (in human Müller cell lines) in gene promoter activity indicating the effect of oxygen depletion on gene expression.

Intravitreal injection of the engineered promoter at post-natal day-seven produced high levels of GFP expression only in retinal Müller cells at day-17 but was absent in retinas exposed to room air only. In essence the in vivo studies confirmed for the researchers that gene expression was silenced in normoxic conditions, induced under hypoxic conditions and only expressed in the specific cells receiving the regulated promoter. By tethering the expression of the therapeutic to the cell type in which oxygen levels become depleted, “delivery†of the treatment may be triggered hopefully long before the macroscopic symptoms become apparent to either patient or clinician. Several research groups are active in using oxygen sensitive regulators upstream of a range of beneficial “productsâ€, such as growth factors, including bFGF and VEGF, antioxidant components, anti-angiogenic factors including angiostatin and proapoptotic transcripts such as Bax (Bcl- 2 associated X protein). All such systems have been designed to exploit the biology of the HREs that bind the transcription factor HIF-1.

In conclusion to the study (published in IOVS; DOI:10.1167/iovs.10-6835) the authors commented that, “our hypoxiaregulated, retinal glial cell-specific vector is likely to be applicable to a range of diseasesâ€, including AMD where the technology may be used to induce a range of neurotrophic factors once hypoxic conditions are detected. The authors additionally proposed that, “activation of the promoter by the oxidative/ inflammatory environment contributing to geographic atrophy in dry AMD would provide an efficient means to deliver neurotrophic therapy only to the pathologic regions of the retinaâ€. The technology could be applied to a number of retinal gene therapy approaches using a variety of gene products such as pro-survival kinases, antioxidant enzymes and secreted factors to block angiogenesis or promote neuroprotection. Establishing proofof- principle for inherited photoreceptor degenerations, diabetic retinopathy and glaucoma are expected to represent key applications for this platform approach.