Perinuclear positioning of endosomes can affect PS-ASO activities

Abstract

Phosphorothioate (PS) modified antisense oligonucleotide (ASO) drugs that act on cellular RNAs must enter cells and be released from endocytic organelles to elicit antisense activity. It has been shown that PS-ASOs are mainly released by late endosomes. However, it is unclear how endosome movement in cells contributes to PS-ASO activity. Here, we show that PS-ASOs in early endosomes display Brownian type motion and migrate only short distances, whereas PS-ASOs in late endosomes (LEs) move linearly along microtubules with substantial distances. In cells with normal microtubules and LE movement, PS-ASO-loaded LEs tend to congregate perinuclearly. Disruption of perinuclear positioning of LEs by reduction of dynein 1 decreased PS-ASO activity, without affecting PS-ASO cellular uptake. Similarly, disruption of perinuclear positioning of PS-ASO-LE foci by reduction of ER tethering proteins RNF26, SQSTM1 and UBE2J1, or by overexpression of P50 all decreased PS-ASO activity. However, enhancing perinuclear positioning through reduction of USP15 or over-expression of RNF26 modestly increased PS-ASO activity, indicating that LE perinuclear positioning is required for ensuring efficient PS-ASO release. Together, these observations suggest that LE movement along microtubules and perinuclear positioning affect PS-ASO productive release.

Peptide Conjugates of a 2′-O-Methoxyethyl Phosphorothioate Splice-Switching Oligonucleotide Show Increased Entrapment in Endosomes

“…We reconcile these observations by speculating that our nuclear fractions are contaminated by the perinucleus, a membraneless organelle that extends a few micrometers from the nucleus . The perinucleus contains 15 to 18% of the total proteins of the mammalian cell and is a site for the efficient maturation of endosomes. , Previous reports have shown that ASOs and ASO-loaded endosomes localize to the perinuclear region. Future studies may elaborate on our findings by employing a higher-resolution subcellular fractionation protocol to dissect the cell into nucleus, perinucleus, and cytosol …”

“… 69 The perinucleus contains 15 to 18% of the total proteins of the mammalian cell and is a site for the efficient maturation of endosomes. 69 , 70 Previous reports have shown that ASOs 32 and ASO-loaded endosomes 71 localize to the perinuclear region. Future studies may elaborate on our findings by employing a higher-resolution subcellular fractionation protocol to dissect the cell into nucleus, perinucleus, and cytosol.…”

Nonviral delivery systems for antisense oligonucleotide therapeutics

“…Due to hindered cell uptake [15,16], off-target effects [17,18], undesirable on-target effects [19], short halflife, immune clearance, and other disadvantages that free ASOs cannot avoid in vivo, researchers have proposed a variety of modifications to improve the stability and extend the half-life of ASOs [20,21]. Phosphorothioate allows the nonbridging oxygen of the phosphate group in ASOs to be replaced by a sulfur group, resulting in the formation of a phosphorothioate bond, which is resistant to nuclease-based degradation [22]. In addition, the phosphorodiamidate morpholino modification increases the water solubility of ASOs [23], a peptide nucleic acid is an artificial mimic capable of self-assembly to form a backbone structure [24], and a locked nucleic acid is more commonly used today and can greatly increase the stability of ASOs [25], and 2′-O-methoxyethyl-(2′-O-MOE) and 2′-O-[2-(methylamino)-2-oxoethyl] improve the binding affinity of ASOs and provide resistance to enzymatic degradation [26].…”