883. Non Viral Gene Therapy by Electrotransfer of hTNF-α Soluble Receptor-I Variants and Its Application to the Treatment of Experimental Arthritis
Authors: Carole Bloquel, Natacha Bessis, Christophe Boissier, Daniel Scherman, Pascal Bigey
Journal: Molecular Therapy (2004)

Objectives: Gene therapy using electrotransfer (ET) is a simple, efficient and safe method for transferring therapeutic transgenes in vivo. We have used this method to administer transgenes encoding three human TNF-α soluble receptor-I (hTNFR-Is) variants. Electrotransfer parameters and therapeutic effect in collagen-induced arthritis (CIA) in mice were studied. Methods: The following three plasmids were used: pCOR hTNFR-Is, pCOR hTNFR-Is/mIgG1, pCOR (hTNFR-Is)2, encoding respectively a monomeric (hTNFR-Is), chimeric (hTNFR-Is/mIgG1) or dimeric ((hTNFR-Is)2) form of hTNFR-Is. hTNFR-Is concentrations were determined in sera and in muscle lysates using a specific ELISA. Biological activity (TNF-a neutralization) of the protein encoded by each of the three plasmids was determined through inhibition of TNF-a using L929 cells. Detection of plasmid genome was determined by PCR with specific primers for pCOR backbone on genomic DNA from various mouse organs. CIA was induced by immunization of DBA/1 mice with bovine type II collagen in complete Freund adjuvant. Results: ET of the 3 plasmids (1 to 15 mg) allowed hTNFR-Is production in sera and muscle after 10 days. This expression was dependent on the dose of plasmid. Local expression in the muscle lasted for at least 6 months (stabilised at values of about 1000 pg hTNFR-Is/mg protein for the monomeric and dimeric forms, and around 30 000 pg/mg protein for the chimera. Systemic expression in the serum was detectable right from 1 mg for the hTNFR-Is/mIgG1 form. It lasted for at least 6 months for the hTNFR-Is/mIgG1 form (3100 pg/mL 6 months after electrotransfer of 15 mg plasmid) while it was shorter for the 2 other forms (about 3 weeks). After intramuscular ET, muscle lysates were able to inhibit TNF-a cytotoxicity on L929 cells ex vivo, which demonstrated the bioactivity of the three proteins produced by mouse muscle. No plasmid DNA was found in the organs distant from the injected muscle (liver, spleen, kidney, gonads, heart, lung brain and distant muscle). ET of 50 mg pCOR hTNFR-Is/mIgG1 plasmid at the onset of clinical disease induced a clear-cut decrease of clinical signs of clinical (p=0,0001 vs. empty pCOR electrotransferred group) and histological signs of arthritis. The dimeric (hTNFR-Is)2 form was also efficient (p=0,0378) although the effect was weaker than with the fusion protein. The monomeric form had no effect on arthritides. Repeated recombinant protein (etanercept) injections highlighted the interest of gene therapy approach, which leads to sustained therapeutic effect after single treatment. Conclusion: Intramuscular ET of plasmids encoding the three forms of hTNFR-Is (monomeric, dimeric and the IgG chimera) leads to a production of biologically active protein and, most importantly, is followed by a long-term secretion of hTNFR-Is in the serum. CIA is efficiently inhibited when ET of plasmids encoding either the chimera or the dimeric form of the hTNFR-Is was performed at the onset of the clinical signs.