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Adeno-associated virus vector-mediated anti-angiogenic gene therapy for collagen-induced arthritis in mice

H. Takahashi^1,3*, K. Kato^1,3, K. Miyake^1,2, Y. Hirai^1,2, S. Yoshino³, T. Shimada^1,2

¹Department of Biochemistry and Molecular Biology; ²Division of Gene Therapy Research, Center for Advanced Medical Technology; and ³Department of Joint Disease and Rheumatism, Nippon Medical School, Tokyo, Japan.

ABSTRACT
Objective
The goal of this study was to determine the utility of adeno-associated virus (AAV) vectors for anti-angiogenic gene therapy in a mouse model of collagen-induced arthritis (CIA).

Methods
CIA mice were generated by immunization with bovine type-II collagen and Freund’s complete adjuvant. AAV vectors containing angiostatin and enhanced green fluorescent protein (eGFP) expression units (AAV-Ang/GFP) or the GFP and neomycin phosphotransferase (NeoR) expression units (AAV-GFP/NeoR) were injected into mouse knee joints before development of arthritis. The expression of transgenes was confirmed by reverse transcriptase polymerase chain reaction (RT-PCR) and immunostaining, and the incidence and severity of arthritis was determined histologically via assessment of synovial hyperplasia, cartilage erosion and bone erosion. Vascularity in the knee joint was evaluated by immunohistochemical staining with anti-von Willebrand factor antibody.

Results
AAV vectors were capable of efficient gene transfer into chondrocytes and synovial cells, and the extent of synovial hyperplasia and other parameters of arthritis were significantly reduced in the knee joints injected with AAV-Ang/GFP compared with the joints treated with either AAV-GFP/NeoR or phosphate-buffered solution (PBS). Reduction in the number of vessels was confirmed in AAV-Ang/GFP-treated joints.

Conclusion
AAV-vector-mediated local expression of angiostatin efficiently inhibited the development of collagen-induced arthritis in the treated joint. Anti-angiogenic gene therapy using AAV vector may provide a new approach for the effective treatment of rheumatoid arthritis.

Key words
Angiostatin, adeno-associated virus vector, gene therapy, rheumatoid arthritis, collagen-induced arthritis, synovial neovascularization.

Hiroshi Takahashi, MD; Ko Kato, MD, PhD; Koichi Miyake, MD, PhD, Assistant Professor; Yukihiko Hirai, PhD, Assistant Professor; Shinichi Yoshino, MD, PhD, Professor; and Takashi Shimada, MD, PhD, Professor.
This work was supported in part by grants from the Ministry of Health and Welfare of Japan and the Ministry of Education, Science, and Culture of Japan.
Please address for reprint requests and correspondence to: Takashi Shimada, MD, PhD, Professor, Department of Biochemistry and Molecular Biology, Nippon Medical School, 1-1-5 Sendagi, Bunkyo-ku, Tokyo 113-8602, Japan.
E-mail: tshimada@nms.ac.jp