Soluble complement receptor 1 (CD35) delivered by retrovirally infected syngeneic cells or by naked DNA injection prevents the progression of collagen-induced arthritis

Abstract

Objective. The complement system is important in the development of autoimmune inflammation, including rheumatoid arthritis (RA) and collagen-induced arthritis (CIA). Complement receptor 1 (CR1) is involved in regulation of complement activity. Studies on models of autoimmunity have demonstrated that soluble CR1 (sCR1) is a potent therapeutic agent. The present study was thus undertaken to investigate the feasibility of antiinflammatory gene therapy to prevent CIA by delivery of genes encoding truncated sCR1 (tsCR1) and dimeric tsCR1-Ig.Methods. Syngeneic fibroblasts or arthritogenic splenocytes, engineered to express tsCR1 using retrovirus-mediated gene transfer, were injected into DBA/1 recipients that had been immunized with bovine type II collagen (CII). In separate experiments, naked DNA containing tsCR1 and tsCR1-Ig genes was injected intramuscularly into the immunized animals. The clinical development of arthritis was monitored, anti-CII levels measured, and antigenic T cell response studied. Affinity-purified tsCR1-Ig was assayed for its inhibitory effect on the alternative complement pathway in mouse serum.Results. Treatment of CII-immunized mice with the tsCR1-expressing cells inhibited development of CIA, reduced anti-CII antibody levels, and inhibited T cell response to CII in vitro. Intramuscular injections of DNA encoding the CR1 genes prevented the progression of disease. Furthermore, compared with full-length sCR1, purified tsCR1-Ig was more active in inhibiting the murine alternative complement pathway.Conclusion. Our findings demonstrated that tsCR1 and tsCR1-Ig, when delivered via gene therapy, had a beneficial effect on autoimmune inflammation. These results indicate that targeting the complement system in RA patients may be of clinical importance.

Protection against collagen-induced arthritis by intramuscular gene therapy with an expression plasmid for the interleukin-1 receptor antagonist

“…receptor could effectively inhibit the development of arthritis, 34,35 the present study suggested that gene therapy for RA using the intramuscular gene delivery with a plasmid vector might be a therapeutically plausible form of RA treatment. This direct DNA transfer method has several advantages over viral vectors.…”

Complement activation by both classical and alternative pathways is critical for the effector phase of arthritis

“…Difference between the C3 -/-group and the FB -/-group, p X 0.005. complement activation [23,24]. We have previously shown that both of these pathways are deleterious in CIA in mice [25].…”

A novel doxycycline inducible autoregulatory plasmid which displays ‘on’/‘off’ regulation suited to gene therapy applications

“…In addition we have been able to extend these observations to a therapeutic gene, sCR1 which is a potent inhibitor of the complement cascade 21 with proven effect by gene therapy in a mouse arthritis model. 22 Regulated expression of sCR1 from per-manent transfectants with the construct GTRTC displayed maximal regulation in excess of 70-fold. The improved regulation observed with permanent transfectants probably occurs via favourable interactions with DNA sequences adjacent to the site of integration which reduce basal expression and enable good induction of gene expression.…”

“…22 The sCR1 gene was removed from this vector by the restriction NcoI-blunted-NotI, and cloned into the pGT vector cut with EcoRV-NotI, forming the inducible vector pGTC1. The autoregulatory vector expressing sCR1 required transfer of the tetP-sCR1 cassette (restricted NheI-BspMI) from pGTC1 and cloned into pGTRTL cut with the same enzymes, producing the autoregulatory vector pGTRTC (Figure 1).…”

“…22 ELISA plates were coated overnight with anti-CR1 mAb J3.D3 (Serotec, Oxford, UK) diluted 1:2000 with PBS. The following day plates were blocked with a 1% bovine serum albumin (Sigma, St Louis, MO, USA) solution in PBS for 2 h. Standards of full length sCR1 (gift from Professor D Fearon, Cambridge University) in the range 5 g/ml to 160 pg/ml, (lowest level of detection is 320 pg/ml) and samples were then loaded on washed plates and incubated for 3 h. Plates were again washed and sCR1 detected with a polyclonal rabbit antisera (gift from Dr R Smith, AdProtec, Royston, UK) diluted 1:2000 with PBS for 1 h. Second layer detection was performed with antirabbit Ig, horseradish-peroxidase linked F(ab) 2 fragment from donkey (Amersham, Buckingham, UK) which was diluted 1:2000 with PBS and incubated for 1 h. Signal was detected using the TMB microwell substrate system (Kirkegaard and Perry Laboratories, Gaithersburg, MD, USA) and the reaction stopped by addition of 1 m phosphoric acid.…”