The interleukin-1 receptor antagonist (IL-1Ra) is an endogenous protein that can prevent the binding of IL-1 to its cellsurface receptors. Among a number of techniques for gene transfer in vivo, the direct injection of naked DNA into muscle is simple, inexpensive and safe. In this study, we evaluated the potential of intramuscular gene therapy with plasmid DNA containing the cDNA for IL-1Ra in the prevention of murine collagen-induced arthritis (CIA). DBA/1 mice were immunized with bovine type II collagen. At 4 weeks after the initial immunization, expression plasmid for IL-1Ra was injected into four selected sites in the thigh and calf muscles of DBA/1 mice. Control mice received the same plasmid, but lacking the IL-1Ra coding sequence. Macroscopic analysis of paws for redness, swelling and deformities showed that the onset of moderate to severe CIA in the paws of mice injected with IL-1Ra DNA was significantly prevented (Po0.05). In addition, both the synovitis and the cartilage erosion in knee joints were dramatically reduced in mice treated with IL-1Ra DNA (Po0.05). The expression of IL-1b was significantly decreased in the ankle joints of mice treated with IL-1Ra (Po0.01). Interestingly, the levels of IL-1Ra in sera and joints after intramuscular injection of IL-1Ra DNA were significantly lower than when protein had been used in previous reports, suggesting that the therapeutic effect may be achieved by an alternative mechanism(s) rather than by systemic elevation of IL-1Ra. These observations provide the first evidence that direct intramuscular injection of expression plasmid for IL-1Ra may effectively suppress the inflammatory pathology in arthritis.

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We made gene therapeutics for X-chronic granulomatous disease (CGD) by transducing murine bone marrow-derived stem cells with MT-gp91 retrovirus and evaluated possible toxicity in mice as a prerequisite for human clinical trials. Male C57BL/6 mice were injected intravenously with gene therapeutics for X-CGD twice at an interval of two weeks at 5 × 107 cells/kg and sacrificed 2 weeks after the last administration. Significant changes noted in gene therapeutics for X-CGD-treated animals were an increase in white blood cell counts and a slight decrease in albumin/globulin ratio. The red pulp hyperplasia in the spleen accompanied with an increase in organ weight was considered to result from the accumulation of gene therapeutics for X-CGD, bone marrow-derived stem cells, in the spleen. No anti-gp91 antibody was detected in the sera collected from the animals treated with gene therapeutics for X-CGD. No integration of gp91 DNA from retroviral vector was detected in chromosomal DNA of gonads in animals dosed with the test substance, indicating no potential of genomic integration. In conclusion, the repeated dose of gene therapeutics for X-CGD exerted no toxicity. The splenic red pulp hyperplasia and the increase observed in white blood cell counts and in spleen weights were considered as pharmacological changes induced by the treatment.

Chronic active hepatitis was selectively induced in mice by the feeding of a diet containing 3-hydroxy-4-pyrone (0.5% by weight) for periods of 6 weeks and longer. This model should be of particular value in elucidating the pathogenesis of drug-induced forms of chronic active hepatitis. Maltol (3-hydroxy-2-methyl-4-pyrone) did not produce any liver lesion.