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

Abstract

Intraocular inflammation has been recognized as a major factor leading to blindness. Because tumor necrosis factor-a (TNF-a) 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-a 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-a in a rat experimental model of endotoxininduced 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-a soluble receptors induces a local immunomodulation enabling the control of intraocular inflammation.

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

“…42 Its efficacy was shown over the shortterm 41,43,44 and mid-term 44 in two rat models of intraocular inflammation, by using secreted tumor necrosis factor-a soluble Posterior segment containing the sclera (sc), the choroid (ch) and the retina (ret) was discarded and the lens (l) was carefully removed from the rest of anterior segment. (a3) Anterior segment explants made of the cornea (co), the iris (ir) and the ciliary body (cb), the latter containing the transfected ciliary muscle fibers, were incubated for 15 additional minutes at 37 1C in conditioning medium before being cultured ex vivo.…”

“…42 This technique was shown to be efficient on the shortterm 41,43,44 and mid-term 44 to deliver anti-tumor necrosis factor molecules in two rat models of intraocular inflammation.…”

The ciliary smooth muscle electrotransfer: basic principles and potential for sustained intraocular production of therapeutic proteins

“…A semi‐annular platinum/iridium sheet (return anode electrode, Figure 1F, black arrow) was placed on the scleral surface facing the active cathode (Figures 1B and 1D). Electrotransfer was performed with eight square‐wave electrical pulses (15 V voltage, 20 ms in duration, 5 Hz frequency) generated by the 830 BTX electropulsator (Genetronics, San Diego, CA, USA) (Figure 1E), as described previously 30–32. These conditions were safe for ocular tissues 30.…”

“…Freshly enucleated eyes were incised at the limbus and fixed for 1 h at 4 °C in PBS containing 2% paraformaldehyde (PFA) and 0.2% glutaraldehyde. They were rinsed three times in PBS before being incubated overnight at room temperature with 1 mg/ml X‐gal (5‐bromo‐4‐chloro‐3‐indolyl galactopyranoside; Sigma‐Aldrich, Saint‐Quentin Fallavier, France) in PBS containing 5 m M of K 3 Fe(CN) 6 , 5 m M of K 4 Fe(CN) 6 , 2 m M of MgCl 2 and 0.02% NP40, as detailed previously 32. After washing with PBS, direct imaging from the outside of the eyes was realized using a numerized camera (Coolpix, Nikon, Fnac, Paris, France).…”

“…In our initial studies, we have demonstrated that this tissue was a good candidate for pDNA gene transfer by means of ET and could effectively be used to secrete encoded proteins in ocular cavities. Proofs of principle of its usefulness as a drug delivery system over the short‐ and mid‐term were given in two rat models of intraocular inflammation, uveitis, by secreting anti‐tumor necrosis factor (TNF) molecules 30–32.…”