RNA interference is a powerful tool for target-specific knockdown of gene expression. However, efficient and safe in vivo delivery of short interfering RNA (siRNA) to the target organ, which is essential for therapeutic applications, has not been established. In this study we used alpha-tocopherol (vitamin E), which has its own physiological transport pathway to most of the organs, as a carrier molecule of siRNA in vivo. The alpha-tocopherol was covalently bound to the antisense strand of 27/29-mer siRNA at the 5'-end (Toc-siRNA). The 27/29-mer Toc-siRNA was designed to be cleaved by Dicer, producing a mature form of 21/21-mer siRNA after releasing alpha-tocopherol. The C6 hydroxyl group of alpha-tocopherol, associated with antioxidant activity, was abolished. Using this new vector, intravenous injection of 2 mg/kg of Toc-siRNA, targeting apolipoprotein B (apoB), achieved efficient reduction of endogenous apoB messenger RNA (mRNA) in the liver. The downregulation of apoB mRNA was confirmed by the accumulation of lipid droplets in the liver as a phenotype. Neither induction of interferons (IFNs) nor other overt side effects were revealed by biochemical and pathological analyses. These findings indicate that Toc-siRNA is effective and safe for RNA interference-mediated gene silencing in vivo.

We originally reported the use of vitamin E (a-tocopherol) as an in vivo vector of short-interfering RNA (siRNA) to the liver. Here, we apply our strategy to the brain. By combining high-density lipoprotein (HDL) as a second carrier with a-tocopherol-conjugated siRNA (Toc-siRNA) in the brain, we achieved dramatic improvement of siRNA delivery to neurons. After direct intracerebroventricular (ICV) infusion of Toc-siRNA/HDL for 7 days, extensive and specific knock-down of a target gene, b-site amyloid precursor protein cleaving enzyme 1 (BACE1), was observed in both mRNA and protein levels, especially in the cerebral cortex and hippocampus. This new delivery method achieved a much more prominent down-regulation effect than conventional silencing methods of the brain gene, i.e., ICV infusion of nonconjugated siRNA or oligonucleotides. With only 3 nmol Toc-siRNA with HDL, BACE1 mRNA in the parietal cortex could be reduced by *70%. We suppose that this dramatic improvement of siRNA delivery to the brain is due to the use of lipoprotein receptor-mediated endocytosis because the silencing efficiency was significantly increased by binding of Toc-siRNA to the lipoprotein, and in contrast, was clearly decreased in lipoprotein-receptor knockout mice. These results suggest exogenous siRNA could be used clinically for otherwise incurable neurological diseases.

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SynopsisPoly(styrene-co-methyl methacrylate) copolymers of narrow chemical composition distributions were separated according to composition by linear gradient elution at column temperatures between 10 and 80°C using silica gel as an adsorbent. The initial mobile phase was a mixture of chloroform and ethanol (99.0 : 1.0, v/v) and the final one (95.5 : 4.5). The relationship of retention volume and the composition of the copolymers was obtained. Higher column temperature (and/or less ethanol content in the mobile phase) is preferable for the separation of copolymers having less MMA and vice versa. Molecular weight dependence on retention volume was not observed. The chromatographed copolymers were still chemical heterogeneous: the compositional difference of the front and rear halves of an adsorption chromatogram of a copolymer was 2.1%. The front half had a higher styrene content and lower molecular weight averages than those for the rear half of the peak. This separation method was reproducible and the relative standard deviation was about 0.6%. Recovery of fractions was 98.2% by isocratic elution and 96.4% by gradient elution.

applied pressures on the reagent side without reaching the plateau level.In practice, both the membrane reactors and the screen-tee mixer gave reproducible peak areas and good peak shapes and operated reliably over eluent flow rates of 0.5-2.0 mlsrnin-l. LITERATURE CITED(1) Spackman, D. H.; Stein, W. H.; Moore, S. Anal. Chem. 1958, 30, (2) Reaction Defectors in Liquid Chromatography; Krull, I. S., Ed.; Academic: New York, in press. (3) Knight, C. H.; Cassidy, R. M.; Recoskie, 8. M.; Green, L. W Anal. Chem. 1984, 56, 474-478. (4) Cassidy, e. M.; Elchuk, S.; Elliot, N. L.; Green, L. W.; Knight, C. H.; Recoskii, 0. M. Anal.The copolymers of a large range of cmposnlon were separated wlth a mlxture of chloroform (or 1,P-dkhloroethane (DCE)) and ethanol on a sillca gel column by linear gradient elutlon. Chloroform (and DCE) wlthout ethanol retatned the copolymers in the column. By the addltlon of ethanol to chloroform, copolymers having less methyl methacrylate (MMA) started to elute, and wlth lncreaslng ethanol content In chlorofann, those having more MMA could be eluted. The copolymers tend to adsorb on the column at hlgher column temperature, and thaw having more MMA require a lower column temperature for elutlon. Ethanol content or column temperature dld not affect peak retentlon volume for the copolymers. The effects of both ethanol concentratlon and column temperature were attrlbuted to the change of populatlon of free sUanol groups on the surface of sillca gel, because the hydrogen bonding of carbonyl groups in the copolymers to the sllanol groups was the main mechanlsm of thls separation.The accurate determination of the chemical composition distribution (CCD) for copolymers is very important for the characterization of copolymers. Among several techniques to measure CCD, high-performance liquid chromatography (HPLC) holds great promise because of its high efficiency. There are a number of published papers in this area, e.g., separations of styrene-methyl acrylate copolymers on a silica gel column ( I ) , styrene-acrylonitrile copolymers by precipitation liquid chromatography (2), styrene-butadiene copolymers on a polyacrylonitrile gel column (3), styrenemethyl methacrylate copolymers on a silica gel column ( 4 ) , styrenemethyl methacrylate block copolymers by column adsorption chromatography using a 50-mm-i.d. cylindrical column ( 5 ) , and styrene-n-butyl methacrylate copolymers by orthogonal chromatography (6).In a previous paper (7), separation of styrene-methyl methacrylate random copolymers (P(S-MMA)) according to chemical composition by liquid adsorption chromatography (LAC) was reported. Silica gel was used as an adsorbent. The copolymers were separated by stepwise gradient elution using chloroform and 1,2-dichloroethane (DCE) as mobile phases. When DCE was used as the mobile phase, the copolymers adsorbed on the surface of silica gel, though polystyrene (PS) eluted from the column. When chloroform was used as the mobile phase instead of DCE, the copolymers having a methyl methacrylate (MMA) compone...