Long-term efficacy of ciliary muscle gene transfer of three sFlt-1 variants in a rat model of laser-induced choroidal neovascularization.

Inhibition of vascular endothelial growth factor (VEGF) has become the standard of care for patients presenting with wet age-related macular degeneration. However, monthly intravitreal injections are required for optimal efficacy. We have previously shown that electroporation enabled ciliary muscle gene transfer results in sustained protein secretion into the vitreous for up to 9 months. Here, we evaluated the long-term efficacy of ciliary muscle gene transfer of three soluble VEGF receptor-1 (sFlt-1) variants in a rat model of laser-induced choroidal neovascularization (CNV). All three sFlt-1 variants significantly diminished vascular leakage and neovascularization as measured by fluorescein angiography (FA) and flatmount choroid at 3 weeks. FA and infracyanine angiography demonstrated that inhibition of CNV was maintained for up to 6 months after gene transfer of the two shortest sFlt-1 variants. Throughout, clinical efficacy was correlated with sustained VEGF neutralization in the ocular media. Interestingly, treatment with sFlt-1 induced a 50% downregulation of VEGF messenger RNA levels in the retinal pigment epithelium and the choroid. We demonstrate for the first time that non-viral gene transfer can achieve a long-term reduction of VEGF levels and efficacy in the treatment of CNV.

Non-viral gene therapy for GDNF production in RCS rat: the crucial role of the plasmid dose.

Glial cell line-derived neurotrophic factor (GDNF) is one of the candidate molecules among neurotrophic factors proposed for a potential treatment of retinitis pigmentosa (RP). It must be administered repeatedly or through sustained releasing systems to exert prolonged neuroprotective effects. In the dystrophic Royal College of Surgeon's (RCS) rat model of RP, we found that endogenous GDNF levels dropped during retinal degeneration time course, opening a therapeutic window for GDNF supplementation. We showed that after a single electrotransfer of 30 µg of GDNF-encoding plasmid in the rat ciliary muscle, GDNF was produced for at least 7 months. Morphometric, electroretinographic and optokinetic analyses highlighted that this continuous release of GDNF delayed photoreceptors (PRs) as well as retinal functions loss until at least 70 days of age in RCS rats. Unexpectedly, increasing the GDNF secretion level accelerated PR degeneration and the loss of electrophysiological responses. This is the first report: (i) demonstrating the efficacy of GDNF delivery through non-viral gene therapy in RP; (ii) establishing the efficacy of intravitreal administration of GDNF in RP associated with a mutation in the retinal pigment epithelium; and (iii) warning against potential toxic effects of GDNF within the eye/retina.

Protein delivery for retinal diseases: from basic considerations to clinical applications.

Because the eye is protected by ocular barriers but is also easily accessible, direct intravitreous injections of therapeutic proteins allow for specific and targeted treatment of retinal diseases. Low doses of proteins are required in this confined environment and a long time of residency in the vitreous is expected, making the eye the ideal organ for local proteic therapies. Monthly intravitreous injection of Ranibizumab, an anti-VEGF Fab has become the standard of care for patients presenting wet AMD. It has brought the proof of concept that administering proteins into the physiologically low proteic concentration vitreous can be performed safely. Other antibodies, Fab, peptides and growth factors have been shown to exert beneficial effects on animal models when administered within the therapeutic and safe window. To extend the use of such biomolecules in the ophthalmology practice, optimization of treatment regimens and efficacy is required. Basic knowledge remains to be increased on how different proteins/peptides penetrate into the eye and the ocular tissues, distribute in the vitreous, penetrate into the retinal layers and/or cells, are eliminated from the eye or metabolized. This should serve as a basis for designing novel drug delivery systems. The later should be non-or minimally invasive and should allow for a controlled, scalable and sustained release of the therapeutic proteins in the ocular media. This paper reviews the actual knowledge regarding protein delivery for eye diseases and describes novel non-viral gene therapy technologies particularly adapted for this purpose.

Effects of ciliary muscle plasmid electrotransfer of TNF-alpha soluble receptor variants in experimental uveitis.

Intraocular inflammation has been recognized as a major factor leading to blindness. Because tumor necrosis factor-alpha (TNF-alpha) enhances intraocular cytotoxic events, systemic anti-TNF therapies have been introduced in the treatment of severe intraocular inflammation, but frequent re-injections are needed and are associated with severe side effects. We have devised a local intraocular nonviral gene therapy to deliver effective and sustained anti-TNF therapy in inflamed eyes. In this study, we show that transfection of the ciliary muscle by plasmids encoding for three different variants of the p55 TNF-alpha soluble receptor, using electrotransfer, resulted in sustained intraocular secretion of the encoded proteins, without any detection in the serum. In the eye, even the shorter monomeric variant resulted in efficient neutralization of TNF-alpha in a rat experimental model of endotoxin-induced uveitis, as long as 3 months after transfection. A subsequent downregulation of interleukin (IL)-6 and iNOS and upregulation of IL-10 expression was observed together with a decreased rolling of inflammatory cells in anterior segment vessels and reduced infiltration within the ocular tissues. Our results indicate that using a nonviral gene therapy strategy, the local self-production of monomeric TNF-alpha soluble receptors induces a local immunomodulation enabling the control of intraocular inflammation.

[Drug delivery systems for intraocular applications]. / Dispositifs de délivrance de principes actifs pour des applications ophtalmologiques.

Numerous drug delivery systems (DDSs) can be used as intraocular tools to provide a sustained and calibrated release for a specific drug. Great progress has been made on the design, biocompatibility, bioavailability, and efficacy of DDSs. Although several of them are undergoing clinical trials, a few are already on the market and could be of a routine use in clinical practice. Moreover, miniaturization of the implants makes them less and less traumatic for the eye tissues and some DDSs are now able to target certain cells or tissues specifically. An overview of ocular implants with therapeutic application potentials is provided.

Non-viral ocular gene therapy: potential ocular therapeutic avenues.

Non-viral vectors for potential gene replacement and therapy have been developed in order to overcome the drawbacks of viral vectors. The diversity of non-viral vectors allows for a wide range of various products, flexibility of application, ease of use, low-cost of production and enhanced "genomic" safety. Using non-viral strategies, oligonucleotides (ODNs) can be delivered naked (less efficient) or entrapped in cationic lipids, polymers or peptides forming slow release delivery systems, which can be adapted according to the organ targeted and the therapy purposes. Tissue and cell internalization can be further enhanced by changing by physical or chemical means. Moreover, a specific vector can be selected according to disease course and intensity of manifestations fulfilling specific requirements such as the duration of drug release and its level along with cells and tissues specific targeting. From accumulating knowledge and experience, it appears that combination of several non-viral techniques may increase the efficacy and ensure the safety of these evolving and interesting gene therapy strategies.