Modified Concatemeric Oligonucleotide Complexes: New System for Efficient Oligonucleotide Transfer into Mammalian Cells

Abstract

Antisense oligonucleotides and double-stranded small interfering RNAs have become an important instrument for the manipulation of gene expression in molecular biology experiments and a promising tool for the development of gene-targeted therapeutics. One of the main impediments in the use of oligonucleotide-based therapeutics is their poor uptake by target cells. The formation of supramolecular concatemeric complexes by oligonucleotides was shown to promote their binding to various mammalian cells [Simonova, O.N.,Vladimirova, A.V., Zenkova, M.A., and Vlassov, V.V. (2006). Biochim. Biophys. Acta 1758, 413-418]. We attempted to improve the efficiency of oligonucleotide concatemer delivery into cells by the attachment of lipophilic cholesterol molecules to the components of concatemeric complexes. Uptake, cellular distribution, and biological activity of the supramolecular complexes formed by delivered antisense oligonucleotides and cholesterol-modified "carrier" oligonucleotides were studied. Our results demonstrate that incorporation of an antisense oligonucleotide into the self-assembling concatemeric system promotes its delivery into cells without the addition of any supplementary transfection agents and allows achieving specific inhibition of the target gene expression.

Transport Oligonucleotides—A Novel System for Intracellular Delivery of Antisense Therapeutics

“…To assess the ability of tONs to enter the cells in the absence of any delivery vehicles, the intracellular accumulation of FAM-labeled tONs in A549, KB-8-5 and HEK293 cells was studied by flow cytometry. Human embryonic kidney HEK293 cells were used because these cells have been widely used in experiments with various transfectants [ 43 , 50 , 51 , 52 , 53 , 54 , 55 ]. Human epidermoid carcinoma cells KB-8-5 exhibiting multi-drug resistance phenotype were used because several types of antisense oligonucleotides and siRNAs were targeted to MDR1 mRNA overexpressed by these cells [ 56 , 57 , 58 , 59 , 60 ].…”

“…Despite the perspective of this approach, the redundant modifications of asONs with such molecules, especially in combination with modifications of the oligonucleotide backbone to improve their nuclease resistance, can significantly affect their biological activity, create problems in adaptation of length and composition of linker groups and, finally, complicate the scheme of asON synthesis and isolation as well as increase their cost. On the other hand, it was demonstrated that formation by asONs various supramolecular structures, such as concatemeric complexes with partially complementary lipophilic oligonucleotides [ 43 , 44 ] or aggregates [ 45 ], significantly enhances their intracellular accumulation and bio-performance.…”

Amphiphilic “Like-A-Brush” Oligonucleotide Conjugates with Three Dodecyl Chains: Self-Assembly Features of Novel Scaffold Compounds for Nucleic Acids Delivery

“…Cargo systems can be based as well on the attachment of lipophilic groups directly to a specific antisense oligonucleotide [ 10 , 24 , 25 ], to one of the siRNA strands [ 11 , 21 , 26 , 27 ] or more complex delivery formulations [ 13 , 23 , 28 , 29 ]. Furthermore, LOCs suit for the construction of oligonucleotide delivery system based on the liposomal-type spherical nucleic acids (SNA) [ 30 , 31 , 32 ].…”