ANTISENSE OLIGONUCLEIC ACID
FIG3 is a graph showing the results of hepatotoxicity of antisense oligonucleic acid and GR gene expression-suppressing activity evaluated in the Example described later
With regard to each of the obtained antisense oligonucleic acids, the ALT concentration in mouse serum was measured in the same manner as in the above-mentioned Example 1 (2). The results are shown in FIG3 (1). In addition, the GR gene expression-suppressing activity of each antisense oligonucleic acid was measured. To be specific, after blood was collected in the above-mentioned hepatotoxicity test, the mouse was euthanized by freezing, and the liver was removed and the weight was measured. About 100 mg was collected as a sample from a site where no abnormality was found macroscopically in the excised liver. The weight of the collected sample was measured, the sample was frozen in liquid nitrogen and stored in an ultra low temperature freezer. The cryopreserved liver sample was homogenized as much as possible using a homogenizer (Shake Master auto, Bio Medical Science Inc.) and TRIzol reagent (Thermo Fisher Scientific) under ice-cooling and total RNA was extracted. The UV absorption spectrum of the extracted total RNA was measured using Nano Vue or Nano Vue Plus (GE Healthcare), and the purity was calculated from the RNA concentration and O.D.260/O.D.280 ratio. Quantitative PCR was performed on total RNA using One Step SYBR PrimeScript RT-PCR Kit (Takara) and Applied Biosystems 7500 (Life Technologies Japan Ltd),the expression ratio of the target gene in the antisense oligonucleic acid administration group relative to the saline administration group was calculated, and the activity was evaluated. The results are shown in FIG3 (2). As shown in the results of FIG3 (1), the hepatotoxicity by Posi12 was reduced to 40% by only changing cytosine, which is in one of the 2',4'-non-bridged nucleic acid residues in the middle part, to 5-hydroxycytosine. On the other hand, as shown in the results of FIG3 (2), GR gene expression-suppressing activity did not change even when cytosine, which is in one of the 2',4'-non-bridged nucleic acid residues in the middle part of Posi12, was changed to 5-hydroxycytosine. Thus, according to the present invention, hepatotoxicity can be reduced without reducing the activity of antisense oligonucleic acid.
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["1. An antisense oligonucleic acid having a base length of not less than 7nt and not more than 30nt, wherein nucleic acid residues of not less than 1nt and not more than 5nt respectively from the both terminals are 2',4'-bridged nucleic acids, 2',4'-non-bridged nucleic acid residue(s) is(are) present between the both terminals, and one or more bases in the nucleic acid residue(s) of the 2',4'-non-bridged nucleic acid residue(s) is/are modified."]