Abstract
Cholesterol derivatives of nuclease-resistant, anti-MDR1 small-interfering RNAs were designed to contain a 2’-OMe-modified 21-bp siRNA and a 63-bp TsiRNA in order to investigate their accumulation and silencing activity in vitro and in vivo. The results showed that increasing the length of the RNA duplex in such a conjugate increases its biological activity when delivered using a transfection agent. However, the efficiency of accumulation in human drug-resistant KB-8-5 cells during delivery in vitro in a carrier-free mode was reduced as well as efficiency of target gene silencing. TsiRNAs demonstrated a similar biodistribution in KB-8-5 xenograft tumor-bearing SCID mice with more efficient accumulation in organs and tumors than cholesterol-conjugated canonical siRNAs; however, this accumulation did not provide a silencing effect. The lack of correlation between the accumulation in the organ and the silencing activity of cholesterol conjugates of siRNAs of different lengths can be attributed to the fact that trimeric Ch-TsiRNA lags mainly in the intercellular space and does not penetrate sufficiently into the cytoplasm of the cell. Increased accumulation in the organs and in the tumor, by itself, shows that using siRNA with increased molecular weight is an effective approach to control biodistribution and delivery to the target organ.
Highlights
Small interfering RNAs are considered promising drugs that can effectively and selectively suppress the expression of genes associated with diseases
Anti-MDR1 monomeric and trimeric Small interfering RNAs (siRNA) and their conjugates with cholesterol connected though C6 linker (Tables 1 and 2) were synthesized as described previously [9,10] The C6 linker was selected because the monomeric siRNA conjugate with this linker showed the highest biological activity compared to the conjugates with other linkers [10]. 2’-O-Methyl modifications were introduced into nuclease-sensitive sites according to the previously developed algorithm [11] in order to prevent degradation of carrier-free siRNA in the presence of serum and in the bloodstream
Two different trimeric siRNAs were used in this study: trimeric small-interfering RNA (TsiRNA)-1 containing the sequence of the monomeric siRNA repeated three times, which, as we showed earlier, possessed higher silencing activity than monomer when it was transfected into cells using Lipofectamine 2000
Summary
Small interfering RNAs (siRNA) are considered promising drugs that can effectively and selectively suppress the expression of genes associated with diseases. Various approaches to increase their permanence in the bloodstream and improve accumulation in target organs are being actively developed on a worldwide basis Such approaches include the formation of complexes with lipids and polymers [1,2], the attachment of ligands to siRNAs that interact with lipoproteins and other blood proteins [3], the use of specific ligands that facilitate the interaction of siRNAs with cells [4,5], as well as the development of formulations for their local administration and controlled release [6]. We designed a multimeric nuclease-resistant 63-bp trimeric small-interfering RNA (TsiRNA) comprising in one duplex the sequences of siRNAs targeting the mRNAs of the MDR1, LMP2, and LMP7 genes [9]. In in vivo experiments with healthy and tumor-bearing mice, cholesterol-containing trimeric TsiRNAs demonstrated more efficient accumulation in organs and tumors than the same canonical siRNA derivatives; this accumulation did not provide an appreciable silencing effect
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