Targeting of cellular transduction and regulation of gene expression

The results of experiments in previous chapters demonstrate that AAV-mediated gene transfer of the VEGF inhibitor sFlt-1 reduces neovascularisation in short-lived animal models and offers a powerful approach to the local, sustained delivery of angiostatic proteins for the treatment of ocular neovascular disorders. The possibility that

uncontrolled sustained expression of sFlt-1 may cause adverse effects in the long term might be minimised by the development of strategies for the targeting of cellular transduction and for the appropriate regulation of gene expression. Specific tissues may be targeted by an appropriate choice of vector and by the accurate delivery of vector suspension. Retinal pigment epithelial cells, for example, may be specifically targeted by lenti viral vectors delivered to the subretinal space.^^' Proliferating cells in any tissue may be targeted by retroviral vectors since these specifically transduce cells during cell division.^"^^'^^ The different time-courses of expression mediated by different vectors may be exploited to optimise the timing of therapeutic protein delivery. Adenovirus-mediated expression, for example, is rapid in onset but typically of limited duration as a result of the induction of an immune response.^AA V -m ediated expression, on the other hand, is delayed in onset^^° and subsequently sustained for months or years. Although vectors that

Chapter 5 HRE-driven gene expression in experimental retinal and choroidal neovascularisation mediate only short-term expression, such as adenoviral vectors, may be less likely to cause long-term adverse effects, their limited duration of expression also risks an increased likelihood of disease recurrence.

The efficiency of gene expression by a transduced cell is modulated by promoter sequences and regulatory elements. The design and incorporation of these elements in therapeutic constructs offers a valuable opportunity to achieve appropriate control of transgene expression. Cell-specific promoters can be incorporated to restrict expression to a given cell type. The use of an opsin promoter, for example, results in efficient expression only in photoreceptor cells while minimising inappropriate expression from transduced retinal pigment epithelial cells. The timing and level of gene expression can be regulated through the incorporation of inducible promoters that are responsive to the administration of exogenous pharmacological agents. In such systems transgene

expression can be induced, for example, by the systemic administration of a drug and the level of gene expression regulated by adjusting its dose according to therapeutic or adverse effects. Tetracycline-inducible systems, for example, can facilitate regulation of gene expression in the retina using adenoviral^^' or vectors. Intraocular

expression following AAV-mediated transduction may similarly be regulated by rapamycin-inducible systems.^^ These pharmacological approaches are dependent, however, on regular clinical observation to determine appropriate dosing and may be limited by inefficient drug penetration across the blood-retinal barrier.

An alternative strategy to regulate gene expression is to couple transgene expression to changes in the local tissue environment. Since retinal neovascularisation is typically the result of local tissue hypoxia, the possibility of hypoxia-regulated expression of

angiostatic molecules by the incorporation of a hypoxia response element (HRE) presents an attractive scenario. A synthetic HRE developed by Oxford Biomedica combines low basal expression in normoxic conditions with high-level activated expression when the oxygen concentration is low.^^° In this study the HRE was used to target AAV-mediated reporter gene expression to sites of experimental retinal and choroidal neovascularisation.

Chapter 5 HRE-driven gene expression in experimental retinal and choroidal neovascularisation

5.4.2 HRE-driven expression in expérimentai retinai and

choroidai neovascularisation

In i schaemi a-i nduced retinal neovascularisation CMV-driven expression was observed across a wide area of the retina and was sustained over time beyond the period of active angiogenesis. In contrast, HRE-driven expression was evident only at the posterior pole in a pattern that is consistent with the temporal and spatial features of retinal capillary non-perfusion. ^ Furthermore, HRE-driven expression was not sustained beyond the period of hypoxia-driven, VEGF-induced angiogenesis that is well characterised in this m o d e l.T h u s AAV-mediated HRE-driven reporter gene expression is targeted both spatially and temporally to the areas of retinal ischaemia in this model.

Following laser rupture of Bruch’s membrane, CMV-driven GFP expression was evident constitutively in photoreceptor cells and retinal pigment epithelial cells regardless of the timing or site of laser rupture. HRE-driven expression however was restricted to

photoreceptor cells overlying the developing choroidal neovascular complex; expression was not significant prior to laser rupture and was not sustained after 4 weeks even at the site of laser rupture. Though laser rupture resulted in a limited amount of autofluorescent material in the subretinal space regardless of vector delivery, HRE-driven GFP

expression was also observed in RPE cells specifically at sites of laser injury. While HRE-driven expression in this model is consistent with a possible role for hypoxia in the evolution of choroidal neovascularisation following laser rupture of Bruch’s membrane, alternative mechanisms may be involved. HREs may be activated by transcription factors other than HIE-F^"* and it is possible that HRE-driven expression may occur as part of a stress or wound-healing response in the context of laser injury. VEGF expression is well- described in laser-induced choroidal neovascularisation,^^^'^^^^^^ and peaks at up to 7 days after wounding.^^^ Retinal VEGF expression may also be upregulated, however, in conditions other than ischaemia^^^ in an mechanism that is independent of hypoxia- induced activation of HIF-1. Further experiments to investigate the possible role of

Chapter 5 HRE-driven gene expression in experimental retinal and choroidal neovascularisation hypoxia in this model using the bioreductive marker nitro imidazole theophylline (NITP) are planned. Whatever is the mechanism in this experimental model, a role for ischaemia has been suggested in the pathogenesis of choroidal neovascularisation in human

patients.'^’*® Retinal hypoxia may result from thickening of Bruch’s membrane or abnormalities of choroidal perfusion,*'^'^ leading to an upregulation of VEGF expression retinal pigment epithelial cells that has been demonstrated in vitro^^^ and there is reason to believe that HRE-driven expression might facilitate the effective targeting of

angiogenesis in the clinical context.