Trimeric Small Interfering RNAs and Their Cholesterol-Containing Conjugates Exhibit Improved Accumulation in Tumors, but Dramatically Reduced Silencing Activity

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.

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

“…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 ]. Human lung carcinoma A549 cells were chosen as possible target cells of tumor origin.…”

“…KB-8-5 cells were selected for further investigations because as it was mentioned above a number of antisense oligonucleotides and siRNAs were targeted MDR1 mRNA overexpressed by these cells [ 56 , 57 , 58 , 59 , 60 ]. Thus these cells represent a well-studied model to determine the silencing activity of antisense oligonucleotides delivered by tONs.…”

“…The sequence within MDR1 mRNA to which the antisense oligonucleotides were directed was successfully used by us previously [ 56 ]. Drug resistant KB-8-5 cells that overexpress the MDR1 gene and can grow in the presence of vinblastine were used to evaluate the silencing activity of duplexes tON/asONs since these cells are a convenient model to assess the biological activity of therapeutic nucleic acids [ 57 , 58 , 59 , 60 ].…”

“…Since oligonucleotides containing phosphorothioate modifications could interact with intracellular proteins and phosphorothioate oligonucleotides are toxic to cells at concentrations over 5 × 10 −6 M [32], only non-modified ON-PO and phosphoryl guanidine-modified ON-PX were selected for further experiments. KB-8-5 cells were selected for further investigations because as it was mentioned above a number of antisense oligonucleotides and siRNAs were targeted MDR1 mRNA overexpressed by these cells [56][57][58][59][60]. Thus these cells represent a well-studied model to determine the silencing activity of antisense oligonucleotides delivered by tONs.…”

Delivery of Oligonucleotides: Efficiency with Lipid Conjugation and Clinical Outcome

“…Chernolovskaya group delved into silencing MDR1 (multidrug resistance protein), which overexpressed in oncogenic cells to efflux impending drug and amplify resistance [65,66]. 21-mer MDR1 targeting siRNA both as monomer and trimer (63-mer) were compared in vivo demonstrating monomeric siRNA obtained superior silencing efficacy while trimeric derivative accumulated highly in tumors [67,68]. Additional examples of other applicable disease would be including Huntington's disease [37,69,70], diabetes nephropathy [71], and herpes simplex virus-2 protection [72].…”