Modulation of collagen-induced arthritis by adenovirus-mediated intra-articular expression of modified collagen type II

Tang et al. Arthritis Research & Therapy RMeseoardchuarltiacletion of collagen-induced arthritis by adenovirus-mediated intra-articular expression of modified collagen type II Bo Tang 3 David L Cullins 3 Jing Zhou 3 Janice A Zawaski 2 Hyelee Park 1 3 David D Brand 3 4 Karen A Hasty 1 M Waleed Gaber 2 John M Stuart 3 4 Andrew H Kang 3 4 Linda K Myers 0 0 Department of Pediatrics, University of Tennessee Health Science Center , 50 North Dunlap, Room 401, Memphis TN 38163 USA 1 Department of Orthopedics, University of Tennessee Health Science Center , 1211 Union Avenue, Suite 520, Memphis, Tennessee 38104 USA 2 Department of Biomedical Engineering, University of Tennessee Health Science Center , 920 Madison, Suite 407, Memphis, Tennessee 38163 USA 3 Department of Medicine, University of Tennessee Health Science Center , 956 Court Avenue, Memphis, Tennessee 38163 , USA 4 Research Service, Veterans Affairs Medical Center , 1030 Jefferson Avenue, Memphis TN 38104 USA Introduction: Rheumatoid arthritis (RA) is a systemic disease manifested by chronic inflammation in multiple articular joints, including the knees and small joints of the hands and feet. We have developed a unique modification to a clinically accepted method for delivering therapies directly to the synovium. Our therapy is based on our previous discovery of an analog peptide (A9) with amino acid substitutions made at positions 260 (I to A), 261 (A to B), and 263 (F to N) that could profoundly suppress immunity to type II collagen (CII) and arthritis in the collagen-induced arthritis model (CIA). Methods: We engineered an adenoviral vector to contain the CB11 portion of recombinant type II collagen and used PCR to introduce point mutations at three sites within (CII124-402, 260A, 261B, 263D), (rCB11-A9) so that the resulting molecule contained the A9 sequence at the exact site of the wild-type sequence. Results: We used this construct to target intra-articular tissues of mice and utilized the collagen-induced arthritis model to show that this treatment strategy provided a sustained, local therapy for individual arthritic joints, effective whether given to prevent arthritis or as a treatment. We also developed a novel system for in vivo bioimaging, using the firefly luciferase reporter gene to allow serial bioluminescence imaging to show that luciferase can be detected as late as 18 days post injection into the joint. Conclusions: Our therapy is unique in that we target synovial cells to ultimately shut down T cell-mediated inflammation. Its effectiveness is based on its ability to transform potential inflammatory T cells and/or bystander T cells into therapeutic (regulatory-like) T cells which secrete interleukin (IL)-4. We believe this approach has potential to effectively suppress RA with minimal side effects. - Introduction Rheumatoid arthritis (RA) is a systemic disease with polyarticular manifestation of chronic inflammation in multiple articular joints, including the knees and small joints of the hands and feet. The current systemic anti-TNF- therapies ameliorate disease in 60% to 70% of RA patients [1]. However, biologics must be given systemically in relatively high dosages to achieve constant therapeutic levels in the joints, and significant side effects have been reported [2]. Gene therapy may provide an effective alternative to drug delivery for the treatment of arthritis [3]. Although various strategies have been tested, those that target gene delivery to the synovial lining of joints have made the most experimental progress [3,4]. This strategy has shown efficacy in several experimental models of RA [57]. For this reason, we have developed a unique modification to a clinically acceptable method of gene delivery to allow delivery of the gene product directly to the synovium. Our therapy is based on our previous discovery of an analog peptide (A9) of type II collagen (CII) with amino acid substitutions made at positions 260 (I to A), 261 (A to B), and 263 (F to N) that could profoundly suppress immunity to CII and arthritis in the collageninduced arthritis (CIA) model [8]. Such collagen peptides containing specially designed substitutions and expressed as a gene products may provide an ideal choice to be delivered to the joints. We engineered an adenoviral gene-based therapy and showed that this treatment strategy provided a sustained, local therapy for individual arthritic joints. Our therapy is unique in that we target synovial cells to ultimately shut down T cell-mediated inflammation. Its effectiveness is based on its ability to transform potential inflammatory T cells and/or bystander T cells into therapeutic (regulatory-like) T cells [8]. They are potentially safer than current therapies because they contain a modification of an endogenous naturally occurring protein, used to interrupt the autoimmune T cell attack and allow for tissue repair. We believe this approach has the potential to become applicable for treatment of RA. Materials and methods Preparation of tissue-derived type II collagen Native CII was solubilized from fetal calf articular cartilage by limited pepsin-digestion and purified as described earlier [9]. The purified collagen was dissolved in cold 0.01 M acetic acid at 4 mg/ml and stored frozen at -70C until used. DBA/1 mice were obtained from the Jackson Laboratories and raised in our animal facility. They were fed standard rodent chow (Ralston Purina Co., St. Louis, MO, USA) and water ad libitum. The environment was specific pathogen-free and sentinel mice were tested routinely for mouse hepatitis and Sendai viruses. All animals were kept until the age of 7 to 10 weeks before being used for experiments, which were conducted in accordance with approved Institutional Animal Care and Use Committee (IACUC) protocols. CII was solubilized in 0.01 M acetic acid at a concentration of 4 mg/ml and emulsified with an equal volume of complete Freund's adjuvant (CFA) containing 4 mg/ml of Mycobacterium tuberculosis strain H37Ra (Difco Microbiology Products, Becton Dickinson, NJ, USA) [10]. Each mouse received 100 g of CII emulsified in CFA intradermally at the base of the tail. Generation of replication-defective, recombinant adenoviral vector expressing modified CB11 Recombinant adenovirus carrying cDNA for rCB11-A9 was generated using a BD Adeno-X Expression System (BD Biosciences Clontech (San Jose, California, USA)), which incorporates the rCB11-A9 expression cassette into a replication-incompetent (E1/E3) human adenoviral type 5 (Ad5) genome. All work was conducted in accordance with approved Institutional Biosafety Committee (IBC) protocols. In brief, an 834 bp of full-length murine CB11 gene was PCR-amplified from murine spleen cDNA and cloned into the PCR2 vector (Invitrogen, Carlsbad, California, USA). We introduced three point mutations (I260A, A261B, and F263N) within the immunodominant T cell determinant of CB11 (CII124-402) to generate an rCB11-A9 construct. The rCB11 (...truncated)