Unassisted Cellular Uptake of Oligonucleotides

Cellular internalization of free ONs has frequently been observed. Binding of ASOs to a cell membrane receptor can trigger their endocytotic or pinocytotic uptake,30,31,32 and a number of research groups have investigated uptake of ligand-ASO conjugates. , , In addition, Li and co-workers reported the transport of unassisted ONs through anion channels in cultured bovine adrenal cells.36 Cellular uptake is, however, only the first barrier to be overcome. Following uptake, ONs are initially trapped in the endosomal-lysosomal compartment and only a fraction are released into the cytoplasm intact. Therefore, unassisted ON-mediated gene silencing is ineffective, even at high ON concentrations. , , Nevertheless, a great number of reports support the pharmacological activity of chemically stabilized, but otherwise unassisted, ASOs in vivo.

Following systemic injection, thiolated ASOs distribute mainly to kidney cortex, liver, spleen, and lymph nodes.40 In addition, accumulation and antisense activity have been observed in adipose tissue.41 At the sub-tissue level, a dose-dependent compartmentalized uptake was observed in rat liver. ASO dosages equal to or lower than 10mgkg_1 resulted in a selective uptake in Kupffer cells and endothelial cells, and dosages of 25mgkg_1 or more were needed to penetrate into the liver parenchyma and achieve localization in hepatocytes.42 Pharmacological responses in rodents match these results, and the ASO-mediated knockdown of hepatocellular targets, such as ApoB-100 and DGAT2, have been achieved with dosages of 10-50 mgkg-1.43,44

Second-generation ASOs, such as 2'-MOE gapmer ASOs, exhibit a remarkable longevity in target tissues. Depending on the dosage and the organ, tissue elimination half-lives of 11-19 days have been reported.45,46 In clinical settings, this feature enables convenient, intermittent dosing regimens while keeping maintenance dosages at low levels.

siRNA molecules cannot passively penetrate cell membranes because, in comparison to ASOs, siRNAs carry approximately twice the charge and are approximately twice the size. Although some reports claim entry of siRNA molecules into cells of the target tissue following i.v. injection, the vast majority of reports confirm that naked siRNA is not active in vivo.41 Moreover, naked siRNA is used by many researchers as a negative control, which fails to produce silencing effects after i.v. injection. Hydrodynamic injections have enabled experimental work in vivo using siRNA. This harsh and painful administration procedure lacks, however, any clinical relevance.48,49

Important progress for the in vivo application of siRNAs was made in 2004 by Soutschek and co-workers, who attached a modified cholesterol molecule at the 3'-end of the sense strand of a partially phosphorothioated and 2'-OMe-modified siRNA.28 Following i.v. administration of this hybrid molecule, ApoB-100 levels were reduced in the liver and jejunum on both the mRNA and protein levels. Most convincingly, ApoB-100 mRNA was cleaved within the sequence targeted by the siRNA. In addition, the reduction of ApoB-100 levels was paralleled by a reduction in cholesterol, high-density lipoprotein, low-density lipoprotein, and chylomicron plasma levels. Despite these impressive results, the dosage of chemically modified siRNAs was relatively high, at 50mgkg_1.

The extended persistence of 2'-MOE-modified ASOs in tissues and the improved potency of these molecules in contrast to first-generation ASOs have lowered the dosages needed for a therapeutic effect substantially, and even made convenient subcutaneous (s.c.) injections a reality for selected indications. However, the need for improvement has been widely recognized. In clinical studies, ASOs accessed sites of inflammation but clinical endpoints have so far not been met.50 In addition, recruitment and uptake of ASOs in cancer is poor and requires high dosages.51,52,53 While ASOs are therapeutically active at sufficiently high doses, the hurdles for the delivery of siRNA are much higher and are not outweighed by the higher potency of siRNA molecules.

Therefore, assisted delivery of siRNA molecules into the cytosol is so far a necessary condition for systemic application.

To date, significant progress has been made in the construction of delivery systems that enable cytosolic delivery or nuclear uptake of ONs without affecting cellular integrity. Section 10.4 reviews some of the most advanced delivery vectors for ONs and highlights common principles.

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