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Nucleic acid therapeutics (NATs) have proven useful in promoting the degradation of specific transcripts, modifying gene expression, and regulating mRNA splicing. In each situation, efficient delivery of nucleic acids to cells, tissues and intracellular compartments is crucial—both for optimizing efficacy and reducing side effects. Despite successes in NATs, our understanding of their cellular uptake and distribution in tissues is limited. Current methods have yielded insights into distribution of NATs within cells and tissues, but the sensitivity and resolution of these approaches are limited. Here, we show that nanoscale secondary ion mass spectrometry (NanoSIMS) imaging can be used to define the distribution of 5-bromo-2′-deoxythymidine (5-BrdT) modified antisense oligonucleotides (ASO) in cells and tissues with high sensitivity and spatial resolution. This approach makes it possible to define ASO uptake and distribution in different subcellular compartments and to quantify the impact of targeting ligands designed to promote ASO uptake by cells. Our studies showed that phosphorothioate ASOs are associated with filopodia and the inner nuclear membrane in cultured cells, and also revealed substantial cellular and subcellular heterogeneity of ASO uptake in mouse tissues. NanoSIMS imaging represents a significant advance in visualizing uptake and distribution of NATs; this approach will be useful in optimizing efficacy and delivery of NATs for treating human disease.

We determined the effect of attaching palmitate, tocopherol or cholesterol to PS ASOs and their effects on plasma protein binding and on enhancing ASO potency in the muscle of rodents and monkeys. We found that cholesterol ASO conjugates showed 5-fold potency enhancement in the muscle of rodents relative to unconjugated ASOs. However, they were toxic in mice and as a result were not evaluated in the monkey. In contrast, palmitate and tocopherol-conjugated ASOs showed enhanced potency in the skeletal muscle of rodents and modest enhancements in potency in the monkey. Analysis of the plasma-protein binding profiles of the ASO-conjugates by size-exclusion chromatography revealed distinct and species-specific differences in their association with plasma proteins which likely rationalizes their behavior in animals. Overall, our data suggest that modulating binding to plasma proteins can influence ASO activity and distribution to extra-hepatic tissues in a species-dependent manner and sets the stage to identify other strategies to enhance ASO potency in muscle tissues.

Spinal muscular atrophy (SMA) is a motor neuron disease. Nusinersen, a splice-switching antisense oligonucleotide (ASO), was the first approved drug to treat SMA. Based on prior preclinical studies, both 2′-O-methoxyethyl (MOE) with a phosphorothioate backbone and morpholino with a phosphorodiamidate backbone—with the same or extended target sequence as nusinersen—displayed efficient rescue of SMA mouse models. Here, we compared the therapeutic efficacy of these two modification chemistries in rescue of a severe mouse model using ASO10-29—a 2-nt longer version of nusinersen—via subcutaneous injection. Although both chemistries efficiently corrected SMN2 splicing in various tissues, restored motor function and improved the integrity of neuromuscular junctions, MOE-modified ASO10-29 (MOE10-29) was more efficacious than morpholino-modified ASO10-29 (PMO10-29) at the same molar dose, as seen by longer survival, greater body-weight gain and better preservation of motor neurons. Time-course analysis revealed that MOE10-29 had more persistent effects than PMO10-29. On the other hand, PMO10-29 appears to more readily cross an immature blood-brain barrier following systemic administration, showing more robust initial effects on SMN2 exon 7 inclusion, but less persistence in the central nervous system. We conclude that both modifications can be effective as splice-switching ASOs in the context of SMA and potentially other diseases, and discuss the advantages and disadvantages of each.

Splice-switching antisense oligonucleotides (ASOs), which bind specific RNA-target sequences and modulate pre-mRNA splicing by sterically blocking the binding of splicing factors to the pre-mRNA, are a promising therapeutic modality to treat a range of genetic diseases. ASOs are typically 15–25 nt long and considered to be highly specific towards their intended target sequence, typically elements that control exon definition and/or splice-site recognition. However, whether or not splice-modulating ASOs also induce hybridization-dependent mis-splicing of unintended targets has not been systematically studied. Here, we tested the in vitro effects of splice-modulating ASOs on 108 potential off-targets predicted on the basis of sequence complementarity, and identified 17 mis-splicing events for one of the ASOs tested. Based on analysis of data from two overlapping ASO sequences, we conclude that off-target effects are difficult to predict, and the choice of ASO chemistry influences the extent of off-target activity. The off-target events caused by the uniformly modified ASOs tested in this study were significantly reduced with mixed-chemistry ASOs of the same sequence. Furthermore, using shorter ASOs, combining two ASOs, and delivering ASOs by free uptake also reduced off-target activity. Finally, ASOs with strategically placed mismatches can be used to reduce unwanted off-target splicing events.

Antisense oligonucleotides (ASOs) are synthetic oligonucleotides that alter expression of disease-associated transcripts via Watson–Crick hybridization. ASOs that function through RNase H or the RNA-induced silencing complex (RISC) result in enzymatic degradation of target RNA. ASOs designed to sterically block access of proteins to the RNA modulate mRNA metabolism but do not typically cause degradation. Here, we rationally design steric blocking ASOs to promote mRNA reduction and characterize the terminating mechanism. Transfection of ASOs complementary to constitutive exons in STAT3 and Sod1 results in greater than 70% reduction of mRNA and protein. The ASOs promote aberrant exon skipping and generation of premature termination codon (PTC)-containing mRNAs. We inhibit the nonsense-mediated mRNA decay (NMD) pathway and show that the PTC-containing mRNAs are recognized by the UPF1 ATPase, cleaved by the SMG6 endonuclease and degraded by the XRN1 cytoplasmic exonuclease. NMD surveillance, however, does not entirely explain the mechanism of decreased STAT3 expression. In addition to exon skipping, ASO treatment causes intron retention and reduction of chromatin-associated STAT3 mRNA. The application of steric blocking ASOs to promote RNA degradation allows one to explore more nucleotide modifications than tolerated by RNase H or RISC-dependent ASOs, with the goal of improving ASO drug properties.

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.

Chemically modified antisense oligonucleotides (ASOs) are widely used as a tool to functionalize microRNAs (miRNAs). Reduction of miRNA level after ASO inhibition is commonly reported to show efficacy. Whether this is the most relevant endpoint for measuring miRNA inhibition has not been adequately addressed in the field although it has important implications for evaluating miRNA targeting studies. Using a novel approach to quantitate miRNA levels in the presence of excess ASO, we have discovered that the outcome of miRNA inhibition can vary depending on the chemical modification of the ASO. Although some miRNA inhibitors cause a decrease in mature miRNA levels, we have identified a novel 2′-fluoro/2′-methoxyethyl modified ASO motif with dramatically improved in vivo potency which does not. These studies show there are multiple mechanisms of miRNA inhibition by ASOs and that evaluation of secondary endpoints is crucial for interpreting miRNA inhibition studies.

High affinity antisense oligonucleotides (ASOs) containing bicylic modifications (BNA) such as locked nucleic acid (LNA) designed to induce target RNA cleavage have been shown to have enhanced potency along with a higher propensity to cause hepatotoxicity. In order to understand the mechanism of this hepatotoxicity, transcriptional profiles were collected from the livers of mice treated with a panel of highly efficacious hepatotoxic or non-hepatotoxic LNA ASOs. We observed highly selective transcript knockdown in mice treated with non-hepatotoxic LNA ASOs, while the levels of many unintended transcripts were reduced in mice treated with hepatotoxic LNA ASOs. This transcriptional signature was concurrent with on-target RNA reduction and preceded transaminitis. Remarkably, the mRNA transcripts commonly reduced by toxic LNA ASOs were generally not strongly associated with any particular biological process, cellular component or functional group. However, they tended to have much longer pre-mRNA transcripts. We also demonstrate that the off-target RNA knockdown and hepatotoxicity is attenuated by RNase H1 knockdown, and that this effect can be generalized to high affinity modifications beyond LNA. This suggests that for a certain set of ASOs containing high affinity modifications such as LNA, hepatotoxicity can occur as a result of unintended off-target RNase H1 dependent RNA degradation.

A series of antisense oligonucleotides (ASOs) containing either 2′-O-methoxyethylribose (MOE) or locked nucleic acid (LNA) modifications were designed to investigate whether LNA antisense oligonucleotides (ASOs) have the potential to improve upon MOE based ASO therapeutics. Some, but not all, LNA containing oligonucleotides increased potency for reducing target mRNA in mouse liver up to 5-fold relative to the corresponding MOE containing ASOs. However, they also showed profound hepatotoxicity as measured by serum transaminases, organ weights and body weights. This toxicity was evident for multiple sequences targeting three different biological targets, as well as in mismatch control sequences having no known mRNA targets. Histopathological evaluation of tissues from LNA treated animals confirmed the hepatocellular involvement. Toxicity was observed as early as 4 days after a single administration. In contrast, the corresponding MOE ASOs showed no evidence for toxicity while maintaining the ability to reduce target mRNA. These studies suggest that while LNA ASOs have the potential to improve potency, they impose a significant risk of hepatotoxicity.

In this study, we have examined the potential of second-generation antisense chimeric 2 0 -O(2-methoxy)ethyl/DNA phosphorothioate oligonucleotides (ONs) to affect cell growth through non-antisense mechanisms. Evaluation of a series of ONs demonstrated that only a small number were cytotoxic at concentrations close to those required for antisense activity. Toxicity of the ONs appeared to be sequence dependent and could be affected by base and backbone modifications. Caspase-3 activation occurs with some ONs and it is most likely secondary to necrosis rather than apoptosis, since cells treated with toxic ONs did not show chromatin condensation, but did exhibit high-extracellular lactate dehydrogenase activity. Caspase-3 activation does not correlate with and appears not to be required for the inhibition of cell proliferation. Toxicity was only observed when ONs were delivered intracellularly. The mechanism by which one of the most cytotoxic ON produces cytotoxicity was investigated in more detail. Treatment with the cytotoxic ON caused disruption of lysosomes and Pepstatin A, a specific inhibitor of aspartic proteases, reduced the cytotoxicity of the ON. Reduction of lysosomal aspartic protease cathepsin D by prior treatment with cathepsin D-specific antisense ON did not attenuate the cytotoxicity, suggesting that other aspartic proteases play a crucial role in the cellular proliferation inhibition by ONs.

Cognate recognition between the CD40 receptor and its ligand, CD154, is thought to play a central role in the initiation and propagation of immune responses. We describe the specific down regulation of cell surface associated CD40 protein expression by use of a peptide nucleic acid (PNA) antisense inhibitor, ISIS 208529, that is designed to bind to the 3' end of the exon 6 splice junction within the primary CD40 transcript. Binding of ISIS 208529 was found to alter constitutive splicing, leading to the accumulation of a transcript lacking exon 6. The resulting protein product lacks the transmembrane domain. ISIS 208529-mediated CD40 protein depletion was found to be sequence specific and dose dependent, and was dependent on the length of the PNA oligomer. CD40-dependent induction of IL-12 in primary murine macrophages was attenuated in cells treated with ISIS 208529. Oligolysine conjugation to the PNA inhibitor produced an inhibitor, ISIS 278647, which maintained its specificity and displayed efficacy in BCL1 cells and in primary murine macrophages in the absence of delivery agents. These results demonstrate that PNA oligomers can be effective inhibitors of CD40 expression and hence may be useful as novel immuno-modulatory agents.

Autosomal dominant diseases such as Huntington's disease (HD) are caused by a gain of function mutant protein and/or RNA. An ideal treatment for these diseases is to selectively suppress expression of the mutant allele while preserving expression of the wild-type variant. RNase H active antisense oligonucleotides (ASOs) or small interfering RNAs can achieve allele selective suppression of gene expression by targeting single nucleotide polymorphisms (SNPs) associated with the repeat expansion. ASOs have been previously shown to discriminate single nucleotide changes in targeted RNAs with ∼5-fold selectivity. Based on RNase H enzymology, we enhanced single nucleotide discrimination by positional incorporation of chemical modifications within the oligonucleotide to limit RNase H cleavage of the non-targeted transcript. The resulting oligonucleotides demonstrate >100-fold discrimination for a single nucleotide change at an SNP site in the disease causing huntingtin mRNA, in patient cells and in a completely humanized mouse model of HD. The modified ASOs were also well tolerated after injection into the central nervous system of wild-type animals, suggesting that their tolerability profile is suitable for advancement as potential allele-selective HD therapeutics. Our findings lay the foundation for efficient allele-selective downregulation of gene expression using ASOs-an outcome with broad application to HD and other dominant genetic disorders.

Phosphorothioate oligonucleotides were identified which directly inhibited human type II phospholipase A2 (PLA2) enzyme activity in a sequence specific manner. The minimum pharmacophore common to all oligonucleotides which inhibited PLA2 enzyme activity consisted of two sets of three or more consecutive guanosine residues in a row. These oligonucleotides appear to form G quartets resulting in the formation of oligonucleotide aggregates. Additionally, a phosphorothioate backbone was required to be effective inhibitors of type II PLA2. The activity of one oligodeoxynucleotide, IP 3196 (5'-GGGTGGGTATAGAAGGGCTCC-3') has been characterized in more detail. IP 3196 inhibited PLA2 enzyme activity when the substrate was presented in the form of a phospholipid bilayer but not when presented in the form of a mixed micelle with anionic detergents. Human type II PLA2 was 50-fold more sensitive to inhibition by IP 3196 than venom and pancreatic type I enzymes. These data demonstrate that phosphorothioate oligonucleotides can specifically inhibit human type II PLA2 enzyme activity in a sequence specific manner.

Phosphorothioate oligodeoxynucleotides (P=S ODNs) are frequently used as antisense agents to specifically interfere with the expression of cellular target genes. However, the cell biological properties of P=S ODNs are poorly understood. Here we show that P=S ODNs were able to continuously shuttle between the nucleus and the cytoplasm and that shuttling P=S ODNs retained their ability to act as antisense agents. The shuttling process shares characteristics with active transport since it was inhibited by chilling and ATP depletion in vivo. Transport was carrier-mediated as it was saturable, and nuclear pore complex-mediated as it was sensitive to treatment with wheatgerm agglutinin. Oligonucleotides without a P=S backbone chemistry were only weakly restricted in their migration by chilling, ATP depletion and wheatgerm agglutinin and thus moved by diffusion. P=S ODN shuttling was only moderately affected by disruption of the Ran/RCC1 system. We propose that P=S ODNs shuttle through their binding to yet unidentified cellular molecules that undergo nucleocytoplasmic transport via a pathway that is not as strongly dependent on the Ran/RCC1 system as nuclear export signal-mediated protein export, U-snRNA, tRNA and mRNA export. The shuttling property of P=S ODNs must be taken into account when considering the mode and site of action of these antisense agents.

Antisense oligonucleotides complementary to specific mRNA sequences are widely used inhibitors of gene expression in vitro and in vivo . In vitro cationic lipids have been demonstrated to increase the pharmacological activity of antisense oligonucleotides by increasing cellular uptake and facilitating nuclear accumulation. We have investigated the intracellular fate of oligonucleotide/cationic lipid complexes using fluorescently labeled lipids and oligonucleotides targeted to protein kinase C-alpha. After addition to cells the lipids initially co-localized with the oligonucleotide on the cell surface and in fine punctate structures within the cytoplasm. At later times the oligonucleotide began to accumulate in the nucleus as well as the cytoplasm. In contrast, the cationic lipid remained localized to the cell surface and the cytoplasm and was never found in the nucleus. Expression of protein kinase C-alpha mRNA did not begin to decline until after oligonucleotide was seen in the nucleus. This was also coincident with the transient appearance of a smaller mRNA transcript believed to result from RNase H cleavage of protein kinase C-alpha mRNA. These data suggest that although cationic lipids facilitate uptake of oligonucleotides, the complex must disassociate before the oligonucleotide can gain access to the nucleus and induce degradation of targeted mRNA.

Systemically administered phosphorothioate antisense oligodeoxynucleotides can specifically affect the expression of their target genes, which affords an exciting new strategy for therapeutic intervention. Earlier studies point to a major role of the liver in the disposition of these oligonucleotides. The aim of the present study was to identify the cell type(s) responsible for the liver uptake of phosphorothioate oligodeoxynucleotides and to examine the mechanisms involved. In our study we used ISIS-3082, a phosphorothioate antisense oligodeoxynucleotide specific for murine ICAM-1. Intravenously injected [3H]ISIS-3082 (dose: 1 mg/kg) was cleared from the circulation of rats with a half-life of 23.3+/-3.8 min. At 90 min after injection (>90% of [3H]ISIS-3082 cleared), the liver contained the most radioactivity, whereas the second-highest amount was recovered in the kidneys (40.5+/-1.4% and 17.9+/-1.3% of the dose, respectively). Of the remaining tissues, only spleen and bone marrow actively accumulated [3H]ISIS-3082. By injecting different doses of [3H]ISIS-3082, it was found that uptake by liver, spleen, bone marrow, and kidneys is saturable, which points to a receptor-mediated process. Subcellular fractionation of the liver indicates that ISIS-3082 is internalized and delivered to the lysosomes. Liver uptake occurs mainly (for 56.1+/-3.0%) by endothelial cells, whereas parenchymal and Kupffer cells account for 39.6+/-4.5 and 4.3+/-1.7% of the total liver uptake, respectively. Preinjection of polyinosinic acid substantially reduced uptake by liver and bone marrow, whereas polyadenylic acid was ineffective, which indicates that in these tissues scavenger receptors are involved in uptake. Polyadenylic acid, but not polyinosinic acid, reduced uptake by kidneys, which suggests renal uptake by scavenger receptors different from those in the liver. We conclude that scavenger receptors on rat liver endothelial cells play a predominant role in the plasma clearance of ISIS-3082. As scavenger receptors are also expressed on human endothelial liver cells, our findings are probably highly relevant for the therapeutic application of phosphorothioate oligodeoxynucleotides in humans. If the target gene is not localized in endothelial liver cells, the therapeutic effectiveness might be improved by developing delivery strategies that redirect the oligonucleotides to the actual target cells.