Plasmid electrotransfer of eye ciliary muscle: principles and therapeutic efficacy using hTNF‐α soluble receptor in uveitis

Abstract

Due to its small size and particular isolating barriers, the eye is an ideal target for local therapy. Recombinant protein ocular delivery requires invasive and painful repeated injections. Alternatively, a transfected tissue might be used as a local producer of transgene-encoded therapeutic protein. We have developed a nondamaging electrically mediated plasmid delivery technique (electrotransfer) targeted to the ciliary muscle, which is used as a reservoir tissue for the long-lasting expression and secretion of therapeutic proteins. High and long-lasting reporter gene expression was observed, which was restricted to the ciliary muscle. Chimeric TNF-alpha soluble receptor (hTNFR-Is) electrotransfer led to elevated protein secretion in aqueous humor and to drastic inhibition of clinical and histological inflammation scores in rats with endotoxin-induced uveitis. No hTNFR-Is was detected in the serum, demonstrating the local delivery of proteins using this method. Plasmid electrotransfer to the ciliary muscle, as performed in this study, did not induce any ocular pathology or structural damage. Local and sustained therapeutic protein production through ciliary muscle electrotransfer is a promising alternative to repeated intraocular protein administration for a large number of inflammatory, degenerative, or angiogenic diseases.

Soluble TNF-α receptor secretion from healthy or dystrophic mice after AAV6-mediated muscle gene transfer

“…23 After this control experiment, we performed assays using a recombinant AAV6 vector encoding the human TNF-a-soluble receptor I linked to the Fc (fragment crystallizable) fragment of the mouse immunoglobulin G1, which has previously shown its efficacy in both a rheumatoid arthritis mouse model 13 and a rat uveitis model. 14 In agreement with muSeAP experiments, results showed a reduced muscle transduction efficiency and secretion of hTNFR-Is/mIgG1 in mdx as compared with B10 mice. Another difference between mdx and B10 was the transient nature of the expression in mdx.…”

“…Notably, the human chimeric protein has already proven its efficacy in a rheumatoid arthritis mouse model, 13 as well as in a rat uveitis model. 14 Increasing amounts of pseudotyped 2/6 AAV particles encoding for chimeric proteins were injected into the skeletal muscle of mdx and C57BL/10 mice (the wild-type genetic background of mdx mice, hereafter named B10). By following the expression levels of TNFR-Is levels, we found that injection of the same dose of AAV6 did not lead to the same expression levels in healthy and diseased muscle.…”

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

“…20 Extending this preliminary proof-of-principle findings, we undertook the present study. Transfecting the ocular ciliary Nonviral anti-TNF gene therapy for eye disease E Touchard et al muscle with plasmids encoding for different variants of hTNFR-Is (p55), we observed a high and long-term production of these proteins in the intraocular fluids.…”

“…Furthermore, we have shown that high levels of this protein were found in the ocular media of rat eyes after ciliary muscle electrotransfer (ET) transfection. 20 In the present study, we are reporting the results demonstrating that ciliary muscle ET of a plasmid encoding for different variants of TNF-a soluble p55 receptor markedly inhibited the development of EIU in the treated rat eyes, through mechanisms similar to those observed after systemic administration of anti-TNF-a, without any circulating TNF soluble receptors.…”

“…Electrotransfer was performed as previously described, 20 with a slight modification of the route of injection. Briefly, the plasmid (3-30 mg in 10 ml saline) was injected in the ciliary muscle using a 30-gauge disposable needle (BD Micro-Fine syringe, NM Médical, Asnière, France) transsclerally posterior to the limbus (Figure 1a).…”