The Combination of Mesyl-Phosphoramidate Inter-Nucleotide Linkages and 2'-O-Methyl in Selected Positions in the Antisense Oligonucleotide Enhances the Performance of RNaseH1 Active PS-ASOs.

Antisense oligonucleotides (ASOs) that mediate RNA target degradation by RNase H1 are used as drugs to treat various diseases. Previously we found that introduction of a single 2'-O-methyl (2'-OMe) modification in position 2 of the central deoxynucleotide region of a gapmer phosphorothioate (PS) ASO, in which several residues at the termini are 2'-methoxyethyl, 2' constrained ethyl, or locked nucleic acid, dramatically reduced cytotoxicity with only modest effects on potency. More recently, we demonstrated that replacement of the PS linkage at position 2 or 3 in the gap with a mesyl-phosphoramidate (MsPA) linkage also significantly reduced toxicity without meaningful loss of potency and increased the elimination half-life of the ASOs. In this study, we evaluated the effects of the combination of MsPA linkages and 2'-OMe nucleotides on PS ASO performance. We found that two MsPA modifications at the 5' end of the gap or in the 3'-wing of a Gap 2'-OMe PS ASO substantially increased the activity of ASOs with OMe at position 2 of the gap without altering the safety profile. Such effects were observed with multiple sequences in cells and animals. Thus, the MsPA modification improves the RNase H1 cleavage rate of PS ASOs with a 2'-OMe in the gap, significantly reduces binding of proteins involved in cytotoxicity, and prolongs elimination half-lives.

Perinuclear positioning of endosomes can affect PS-ASO activities.

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.

Golgi-58K can re-localize to late endosomes upon cellular uptake of PS-ASOs and facilitates endosomal release of ASOs.

Phosphorothioate (PS) modified antisense oligonucleotide (ASO) drugs can trigger RNase H1 cleavage of cellular target RNAs to modulate gene expression. Internalized PS-ASOs must be released from membraned endosomal organelles, a rate limiting step that is not well understood. Recently we found that M6PR transport between Golgi and late endosomes facilitates productive release of PS-ASOs, raising the possibility that Golgi-mediated transport may play important roles in PS-ASO activity. Here we further evaluated the involvement of Golgi in PS-ASO activity by examining additional Golgi proteins. Reduction of certain Golgi proteins, including Golgi-58K, GCC1 and TGN46, decreased PS-ASO activity, without substantial effects on Golgi integrity. Upon PS-ASO cellular uptake, Golgi-58K was recruited to late endosomes where it colocalized with PS-ASOs. Reduction of Golgi-58K caused slower PS-ASO release from late endosomes, decreased GCC2 late endosome relocalization, and led to slower retrograde transport of M6PR from late endosomes to trans-Golgi. Late endosome relocalization of Golgi-58K requires Hsc70, and is most likely mediated by PS-ASO-protein interactions. Together, these results suggest a novel function of Golgi-58K in mediating Golgi-endosome transport and indicate that the Golgi apparatus plays an important role in endosomal release of PS-ASO, ensuring antisense activity.

Hsc70 Facilitates Mannose-6-Phosphate Receptor-Mediated Intracellular Trafficking and Enhances Endosomal Release of Phosphorothioate-Modified Antisense Oligonucleotides.

Phosphorothioate-modified antisense oligonucleotide (PS-ASO) drugs are commonly used to modulate gene expression through RNase H1-mediated cleavage of target RNAs. Upon internalization through endocytic pathways into cells, PS-ASOs must be released from membraned endosomal organelles to act on target RNAs, a limiting step of PS-ASO activity. Here we report that Hsc70 protein mediates productive release of PS-ASOs from endosomes. Hsc70 protein was enriched in endosome fractions shortly after PS-ASO incubation with cells. Reduction of Hsc70 significantly decreased the activities of PS-ASOs in reducing target RNAs. PS-ASO uptake and transport from early endosomes to late endosomes (LEs) were not affected upon Hsc70 reduction; however, endosomal release of PS-ASOs was impaired. Reduction of Hsc70 led to more scattered mannose-6-phosphate receptor (M6PR) localization at LEs in the cytoplasm, in contrast to the perinuclear localization at trans-Golgi network (TGN) in control cells, suggesting that retrograde transport of M6PR from LEs to TGN was affected. Consistently, reduction of Hsc70 increased colocalization of M6PR and PS-ASOs at LEs, and also delayed M6PR antibody transport from LE to TGN. Together, these results suggest that Hsc70 protein is involved in M6PR vesicle escape from LEs and may thus enhance PS-ASO release from LEs.

Some ASOs that bind in the coding region of mRNAs and induce RNase H1 cleavage can cause increases in the pre-mRNAs that may blunt total activity.

Antisense oligonucleotide (ASO) drugs that trigger RNase H1 cleavage of target RNAs have been developed to treat various diseases. Basic pharmacological principles suggest that the development of tolerance is a common response to pharmacological interventions. In this manuscript, for the first time we report a molecular mechanism of tolerance that occurs with some ASOs. Two observations stimulated our interest: some RNA targets are difficult to reduce with RNase H1 activating ASOs and some ASOs display a shorter duration of activity than the prolonged target reduction typically observed. We found that certain ASOs targeting the coding region of some mRNAs that initially reduce target mRNAs can surprisingly increase the levels of the corresponding pre-mRNAs. The increase in pre-mRNA is delayed and due to enhanced transcription and likely also slower processing. This process requires that the ASOs bind in the coding region and reduce the target mRNA by RNase H1 while the mRNA resides in the ribosomes. The pre-mRNA increase is dependent on UPF3A and independent of the NMD pathway or the XRN1-CNOT pathway. The response is consistent in multiple cell lines and independent of the methods used to introduce ASOs into cells.

Golgi-endosome transport mediated by M6PR facilitates release of antisense oligonucleotides from endosomes.

Release of phosphorothioate antisense oligonucleotides (PS-ASOs) from late endosomes (LEs) is a rate-limiting step and a poorly defined process for productive intracellular ASO drug delivery. Here, we examined the role of Golgi-endosome transport, specifically M6PR shuttling mediated by GCC2, in PS-ASO trafficking and activity. We found that reduction in cellular levels of GCC2 or M6PR impaired PS-ASO release from endosomes and decreased PS-ASO activity in human cells. GCC2 relocated to LEs upon PS-ASO treatment, and M6PR also co-localized with PS-ASOs in LEs or on LE membranes. These proteins act through the same pathway to influence PS-ASO activity, with GCC2 action preceding that of M6PR. Our data indicate that M6PR binds PS-ASOs and facilitates their vesicular escape. The co-localization of M6PR and of GCC2 with ASOs is influenced by the PS modifications, which have been shown to enhance the affinity of ASOs for proteins, suggesting that localization of these proteins to LEs is mediated by ASO-protein interactions. Reduction of M6PR levels also decreased PS-ASO activity in mouse cells and in livers of mice treated subcutaneously with PS-ASO, indicating a conserved mechanism. Together, these results demonstrate that the transport machinery between LE and Golgi facilitates PS-ASO release.

mRNA levels can be reduced by antisense oligonucleotides via no-go decay pathway.

Antisense technology can reduce gene expression via the RNase H1 or RISC pathways and can increase gene expression through modulation of splicing or translation. Here, we demonstrate that antisense oligonucleotides (ASOs) can reduce mRNA levels by acting through the no-go decay pathway. Phosphorothioate ASOs fully modified with 2'-O-methoxyethyl decreased mRNA levels when targeted to coding regions of mRNAs in a translation-dependent, RNase H1-independent manner. The ASOs that activated this decay pathway hybridized near the 3' end of the coding regions. Although some ASOs induced nonsense-mediated decay, others reduced mRNA levels through the no-go decay pathway, since depletion of PELO/HBS1L, proteins required for no-go decay pathway activity, decreased the activities of these ASOs. ASO length and chemical modification influenced the efficacy of these reagents. This non-gapmer ASO-induced mRNA reduction was observed for different transcripts and in different cell lines. Thus, our study identifies a new mechanism by which mRNAs can be degraded using ASOs, adding a new antisense approach to modulation of gene expression. It also helps explain why some fully modified ASOs cause RNA target to be reduced despite being unable to serve as substrates for RNase H1.

Site-specific replacement of phosphorothioate with alkyl phosphonate linkages enhances the therapeutic profile of gapmer ASOs by modulating interactions with cellular proteins.

Phosphorothioate-modified antisense oligonucleotides (PS-ASOs) interact with a host of plasma, cell-surface and intracellular proteins which govern their therapeutic properties. Given the importance of PS backbone for interaction with proteins, we systematically replaced anionic PS-linkages in toxic ASOs with charge-neutral alkylphosphonate linkages. Site-specific incorporation of alkyl phosphonates altered the RNaseH1 cleavage patterns but overall rates of cleavage and activity versus the on-target gene in cells and in mice were only minimally affected. However, replacing even one PS-linkage at position 2 or 3 from the 5'-side of the DNA-gap with alkylphosphonates reduced or eliminated toxicity of several hepatotoxic gapmer ASOs. The reduction in toxicity was accompanied by the absence of nucleolar mislocalization of paraspeckle protein P54nrb, ablation of P21 mRNA elevation and caspase activation in cells, and hepatotoxicity in mice. The generality of these observations was further demonstrated for several ASOs versus multiple gene targets. Our results add to the types of structural modifications that can be used in the gap-region to enhance ASO safety and provide insights into understanding the biochemistry of PS ASO protein interactions.

COPII vesicles can affect the activity of antisense oligonucleotides by facilitating the release of oligonucleotides from endocytic pathways.

RNase H1-dependent, phosphorothioate-modified antisense oligonucleotides (PS-ASOs) can enter cells through endocytic pathways and need to be released from the membrane-enclosed organelles, a limiting step for antisense activity. Accumulating evidence has suggested that productive PS-ASO release mainly occurs from late endosomes (LEs). However, how PS-ASOs escape from LEs is not well understood. Here, we report that upon PS-ASO incubation, COPII vesicles, normally involved in ER-Golgi transport, can re-locate to PS-ASO-containing LEs. Reduction of COPII coat proteins significantly decreased PS-ASO activity, without affecting the levels of PS-ASO uptake and early-to-late endosome transport, but caused slower PS-ASO release from LEs. COPII co-localization with PS-ASOs at LEs does not require de novo assembly of COPII at ER. Interestingly, reduction of STX5 and P115, proteins involved in tethering and fusion of COPII vesicles with Golgi membranes, impaired COPII re-localization to LEs and decreased PS-ASO activity. STX5 can re-locate to LEs upon PS-ASO incubation, can bind PS-ASOs, and the binding appears to be required for this pathway. Our study reveals a novel release pathway in which PS-ASO incubation causes LE re-localization of STX5, which mediates the recruitment of COPII vesicles to LEs to facilitate endosomal PS-ASO release, and identifies another key PS-ASO binding protein.

Translation can affect the antisense activity of RNase H1-dependent oligonucleotides targeting mRNAs.

RNase H1-dependent antisense oligonucleotides (ASOs) can degrade complementary RNAs in both the nucleus and the cytoplasm. Since cytoplasmic mRNAs are actively engaged in translation, ASO activity may thus be affected by translating ribosomes that scan the mRNAs. Here we show that mRNAs associated with ribosomes can be cleaved using ASOs and that translation can alter ASO activity. Translation inhibition tends to increase ASO activity when targeting the coding regions of efficiently translated mRNAs, but not nuclear non-coding RNAs or less efficiently translated mRNAs. Increasing the level of RNase H1 protein eliminated the enhancing effects of translation inhibition on ASO activity, suggesting that RNase H1 recruitment to ASO/mRNA heteroduplexes is a rate limiting step and that translating ribosomes can inhibit RNase H1 recruitment. Consistently, ASO activity was not increased by translation inhibition when targeting the 3' UTRs, independent of the translation efficiency of the mRNAs. Contrarily, the activity of 3' UTR-targeting ASOs tended to be reduced upon translation inhibition, likely due to decreased accessibility. These results indicate that ASO activity can be affected by the translation process, and the findings also provide important information toward helping better ASO drug design.

RNase H1-Dependent Antisense Oligonucleotides Are Robustly Active in Directing RNA Cleavage in Both the Cytoplasm and the Nucleus.

RNase H1-dependent antisense oligonucleotides (ASOs) are active in reducing levels of both cytoplasmic mRNAs and nuclear retained RNAs. Although ASO activity in the nucleus has been well demonstrated, the cytoplasmic activity of ASOs is less clear. Using kinetic and subcellular fractionation studies, we evaluated ASO activity in the cytoplasm. Upon transfection, ASOs targeting exonic regions rapidly reduced cytoplasmically enriched mRNAs, whereas an intron-targeting ASO that only degrades the nuclear pre-mRNA reduced mRNA levels at a slower rate, similar to normal mRNA decay. Importantly, some exon-targeting ASOs can rapidly and vigorously reduce mRNA levels without decreasing pre-mRNA levels, suggesting that pre-existing cytoplasmic mRNAs can be cleaved by RNase H1-ASO treatment. In addition, we expressed a cytoplasm-localized mutant 7SL RNA that contains a partial U16 small nucleolar RNA (snoRNA) sequence. Treatment with an ASO simultaneously reduced both the nuclear U16 snoRNA and the cytoplasmic 7SL mutant RNA as early as 30 min after transfection in an RNase H1-dependent manner. Both the 5' and 3' cleavage products of the 7SL mutant RNA were accumulated in the cytoplasm. Together, these results demonstrate that RNase H1-dependent ASOs are robustly active in both the cytoplasm and nucleus.

Hsp90 protein interacts with phosphorothioate oligonucleotides containing hydrophobic 2'-modifications and enhances antisense activity.

RNase H1-dependent antisense oligonucleotides (ASOs) are chemically modified to enhance pharmacological properties. Major modifications include phosphorothioate (PS) backbone and different 2'-modifications in 2-5 nucleotides at each end (wing) of an ASO. Chemical modifications can affect protein binding and understanding ASO-protein interactions is important for better drug design. Recently we identified many intracellular ASO-binding proteins and found that protein binding could affect ASO potency. Here, we analyzed the structure-activity-relationships of ASO-protein interactions and found 2'-modifications significantly affected protein binding, including La, P54nrb and NPM. PS-ASOs containing more hydrophobic 2'-modifications exhibit higher affinity for proteins in general, although certain proteins, e.g. Ku70/Ku80 and TCP1, are less affected by 2'-modifications. We found that Hsp90 protein binds PS-ASOs containing locked-nucleic-acid (LNA) or constrained-ethyl-bicyclic-nucleic-acid ((S)-cEt) modifications much more avidly than 2'-O-methoxyethyl (MOE). ASOs bind the mid-domain of Hsp90 protein. Hsp90 interacts with more hydrophobic 2' modifications, e.g. (S)-cEt or LNA, in the 5'-wing of the ASO. Reduction of Hsp90 protein decreased activity of PS-ASOs with 5'-LNA or 5'-cEt wings, but not with 5'-MOE wing. Together, our results indicate Hsp90 protein enhances the activity of PS/LNA or PS/(S)-cEt ASOs, and imply that altering protein binding of ASOs using different chemical modifications can improve therapeutic performance of PS-ASOs.