Adjuvant composition
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example 1
on of Nucleic Acids
[0038](1) Preparation of cM362-140 (Chemically Synthesized Nucleic Acid)
[0039]The single-stranded nucleic acids (S1, S2, S3, AS1, AS2 and AS3) shown in Table 1 were synthesized by GeneDesign, Inc. The RNA fragments were chemically synthesized using tBDMS RNA amidites, the DNA fragment was chemically synthesized using standard DNA amidites, and phosphorothioate linkages (sulfur modification) were introduced using PADS. The synthesis was performed based on the phosphoramidite method (Scaringe, S. A. et al, J Am Chem 1998; 120: 11820-11821) using a solid-phase carrier with optimized parameters. After completion of the synthesis, the protecting groups on the bases and on the 2′ position were removed by the usual method. The products were purified by reverse-phase HPLC and desalted to give the single-stranded nucleic acids.
TABLE 1Fragment RNASense MV-RNA sequenceAntisense MV-RNA sequenceS1 (SEQ ID NO: 6)5′-t{circumflex over ( )}g{circumflex over ( )}c{circumflex over (...
example 2
of Nucleic Acids
(1) Stability in Serum-Containing PBS
[0049]cM362-140 and cM362-139 were separately dissolved at 20 μg / mL in serum-free PBS, PBS containing 10% heat-inactivated fetal bovine serum (FBS), PBS containing 10% mouse serum (MS) or PBS containing 10% human serum (HS), and incubated at 37° C. or 42° C. for 60 minutes. Aliquots containing 0.1 μg of the treated nucleic acids were taken before start of incubation (0 minute-incubation, only for serum-free PBS) and after 5-, 15-, 30- and 60-minute incubation, mixed with 10× loading dye (Takara Bio), and electrophoresed on 4% agarose gel (Nusieve 3:1 Agarose, Lonza) containing ethidium bromide.
[0050]The results are shown in FIGS. 3A and 3B. FIGS. 3A and 3B show the electrophoretic patterns of cM362-140 and cM362-139, respectively. Both nucleic acids were stable during the 30-minute incubation at 37° C., but cM362-139, which was prepared by in vitro transcription, was slightly degraded during the 30-minute incubation in PBS contain...
example 3
n of IFN-β Promoter
[0054]HEK293 cells (8×105 cells / well) were seeded in 6-well culture plates. The HEK293 cells were transfected with a human TLR3 expression vector (400 ng / well) or an empty vector (400 ng / well) together with a reporter plasmid p-125 (400 ng / well) and an internal control vector phRL-TK (20 ng / well, Promega) using Lipofectamine 2000 (Invitrogen). The reporter plasmid p-125 containing the human IFN-β promoter region (−125 to +19) was provided by Dr. Taniguchi (the University of Tokyo). Dulbecco's Modified Eagle's medium (DMEM, Invitrogen) supplemented with 10% heat-inactivated fetal bovine serum (FCS, Invitrogen) and antibiotics was used as medium.
[0055]Twenty-four hours after transfection, the cells were recovered, resuspended in medium and seeded in 96-well culture plates. The nucleic acids, i.e., cM362-140, cM362-139, poly(I:C) (Amersham) and the double-stranded RNA portion (dsRNA140) of cM362-140, were separately added to a concentration of 10 μg / mL in the followi...
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