Mechanisms of single-stranded phosphorothioate modified antisense oligonucleotide accumulation in hepatocytes

Abstract

Single-stranded antisense oligonucleotides (SSOs) are used to modulate the expression of genes in animal models and are being investigated as potential therapeutics. To better understand why synthetic SSOs accumulate in the same intracellular location as the target RNA, we have isolated a novel mouse hepatocellular SV40 large T-antigen carcinoma cell line, MHT that maintains the ability to efficiently take up SSOs over several years in culture. Sequence-specific antisense effects are demonstrated at low nanomolar concentrations. SSO accumulation into cells is both time and concentration dependent. At least two distinct cellular pathways are responsible for SSO accumulation in cells: a non-productive pathway resulting in accumulation in lysosomes, and a functional uptake pathway in which the SSO gains access to the targeted RNA. We demonstrate that functional uptake, as defined by a sequence-specific reduction in target mRNA, is inhibited by brefeldin A and chloroquine. Functional uptake is blocked by siRNA inhibitors of the adaptor protein AP2M1, but not by clathrin or caveolin. Furthermore, we document that treatment of mice with an AP2M1 siRNA blocks functional uptake into liver tissue. Functional uptake of SSO appears to be mediated by a novel clathrin- and caveolin-independent endocytotic process.

Involvement of an intracellular vesicular transport process in naked-sgRNA-mediated TRUE gene silencing

“…These results indicate that functional uptake by cells in culture does not appear to be mediated by clathrin-, caveolae-or lipid raft-dependent pathways. Similarly to our studies, Koller et al (14) reported that functional uptake of a single-stranded phosphorothioate modified antisense oligonucleotide is not mediated by clathrin or caveolin.…”

“…The present study suggested that functional uptake of sgRNA is an intracellular vesicular uptake process, since it is blocked by choroquine and brefeldin A. Recently, it has been reported that inhibition of the adaptor protein AP2M1 with small interfering RNA attenuated the antisense effects in cultured hepatocytes (14,24). Additional studies are required to determine whether AP2M1 mediates the effects of naked sgRNAs and to fully identify methods of sgRNA uptake.…”

“…However, little is known regarding the mechanism of naked sgRNA uptake into cultured cells. The 'gymnotic' mechanism (11)(12)(13) and/or the adaptor protein AP2M1 (14) are reported to be involved in the cellular uptake of naked oligonucleotides by cultured cells. In the present study, the effects of various inhibitors of apoptotic induction were investigated using naked sgRNA treatment in order to elucidate uptake pathways and mechanisms for sgRNA in cultured cells.…”

Comparison of the pharmacological profiles of murine antisense oligonucleotides targeting apolipoprotein B and microsomal triglyceride transfer protein

“…A series of uniform chimeric 20-mer phosphorothioate oligonucleotides containing 2'-O -methoxyethyl groups at positions 1-5 and 15-20 targeted to murine apoB, MTP, and a control ASO were synthesized and purifi ed on an automated DNA synthesizer using phosphoramidite chemistry as previously described (13). The sequences evaluated were as follows: apoB ASO-ISIS 147764 (5 ′ -GTCCC TGAAGATGTC AATGC -3 ′ ), MTP ASO-ISIS 144477 (5 ′ -CCCAG CACCTGGTTT GCCGT -3 ′ ), and one of two control ASOs-ASO 1 (5 ′ -AGCAT AGTTAACGAG CTCCC -3 ′ ) or ASO 2 (5 ′ -AGCAT AGTTAACGAG CTCCC -3 ′ ), with underlining indicating 2'-O -methoxyethyl-modifi ed bases.…”

RNA Therapeutics in Oncology: Advances, Challenges, and Future Directions

“…29,36 The precise mechanisms and molecular events resulting in ASO endocytotic uptake are only partially understood and are an intense area of research. 36 Nonetheless, the nucleic acid-based therapeutics has been extensively characterized in numerous biological systems, and it is now understood that, on systemic delivery, unformulated ASOs rapidly distribute to most tissues, and once taken up by cells, these stabilized molecules have prolonged tissue halflives of up to 2 weeks in rodents and in man. 29 The improvement in potency and ADME properties for second-generation ASOs in vivo over the firstgeneration chemistry has been shown to be as much as 10-fold, and, fortuitously, MOE Gapmers also benefit from a significantly reduced proinflammatory capacity, resulting in improved safety properties and a significantly increased therapeutic window (see Figure 2).…”