Antisense oligonucleotides ameliorate kidney dysfunction in podocyte-specific APOL1 risk variant mice.

Coding variants (named G1 and G2) in Apolipoprotein L1 (APOL1) can explain most excess risk of kidney disease observed in African American individuals. It has been proposed that risk variant APOL1 dose, such as increased risk variant APOL1 level serves as a trigger (second hit) for disease development. The goal of this study was to determine whether lowering risk variant APOL1 levels protects from disease development in a podocyte-specific transgenic mouse disease model. We administered antisense oligonucleotides (ASO) targeting APOL1 to podocyte-specific G2APOL1 mice and observed efficient reduction of APOL1 levels. APOL1 ASO1, which more efficiently lowered APOL1 transcript levels, protected mice from albuminuria, glomerulosclerosis, tubulointerstitial fibrosis, and renal failure. Administration of APOL1 ASO1 was effective even for established disease in the NEFTA-rtTA/TRE-G2APOL1 (NEFTA/G2APOL1) mice. We observed a strong correlation between APOL1 transcript level and disease severity. We concluded that APOL1 ASO1 may be an effective therapeutic approach for APOL1-associated glomerular disease.

The nuclear-retained noncoding RNA MALAT1 regulates alternative splicing by modulating SR splicing factor phosphorylation.

Alternative splicing (AS) of pre-mRNA is utilized by higher eukaryotes to achieve increased transcriptome and proteomic complexity. The serine/arginine (SR) splicing factors regulate tissue- or cell-type-specific AS in a concentration- and phosphorylation-dependent manner. However, the mechanisms that modulate the cellular levels of active SR proteins remain to be elucidated. In the present study, we provide evidence for a role for the long nuclear-retained regulatory RNA (nrRNA), MALAT1 in AS regulation. MALAT1 interacts with SR proteins and influences the distribution of these and other splicing factors in nuclear speckle domains. Depletion of MALAT1 or overexpression of an SR protein changes the AS of a similar set of endogenous pre-mRNAs. Furthermore, MALAT1 regulates cellular levels of phosphorylated forms of SR proteins. Taken together, our results suggest that MALAT1 regulates AS by modulating the levels of active SR proteins. Our results further highlight the role for an nrRNA in the regulation of gene expression.

Targeted degradation of sense and antisense C9orf72 RNA foci as therapy for ALS and frontotemporal degeneration.

Expanded hexanucleotide repeats in the chromosome 9 open reading frame 72 (C9orf72) gene are the most common genetic cause of ALS and frontotemporal degeneration (FTD). Here, we identify nuclear RNA foci containing the hexanucleotide expansion (GGGGCC) in patient cells, including white blood cells, fibroblasts, glia, and multiple neuronal cell types (spinal motor, cortical, hippocampal, and cerebellar neurons). RNA foci are not present in sporadic ALS, familial ALS/FTD caused by other mutations (SOD1, TDP-43, or tau), Parkinson disease, or nonneurological controls. Antisense oligonucleotides (ASOs) are identified that reduce GGGGCC-containing nuclear foci without altering overall C9orf72 RNA levels. By contrast, siRNAs fail to reduce nuclear RNA foci despite marked reduction in overall C9orf72 RNAs. Sustained ASO-mediated lowering of C9orf72 RNAs throughout the CNS of mice is demonstrated to be well tolerated, producing no behavioral or pathological features characteristic of ALS/FTD and only limited RNA expression alterations. Genome-wide RNA profiling identifies an RNA signature in fibroblasts from patients with C9orf72 expansion. ASOs targeting sense strand repeat-containing RNAs do not correct this signature, a failure that may be explained, at least in part, by discovery of abundant RNA foci with C9orf72 repeats transcribed in the antisense (GGCCCC) direction, which are not affected by sense strand-targeting ASOs. Taken together, these findings support a therapeutic approach by ASO administration to reduce hexanucleotide repeat-containing RNAs and raise the potential importance of targeting expanded RNAs transcribed in both directions.

In vivo evaluation of candidate allele-specific mutant huntingtin gene silencing antisense oligonucleotides.

Huntington disease (HD) is a dominant, genetic neurodegenerative disease characterized by progressive loss of voluntary motor control, psychiatric disturbance, and cognitive decline, for which there is currently no disease-modifying therapy. HD is caused by the expansion of a CAG tract in the huntingtin (HTT) gene. The mutant HTT protein (muHTT) acquires toxic functions, and there is significant evidence that muHTT lowering would be therapeutically efficacious. However, the wild-type HTT protein (wtHTT) serves vital functions, making allele-specific muHTT lowering strategies potentially safer than nonselective strategies. CAG tract expansion is associated with single nucleotide polymorphisms (SNPs) that can be targeted by gene silencing reagents such as antisense oligonucleotides (ASOs) to accomplish allele-specific muHTT lowering. Here we evaluate ASOs targeted to HD-associated SNPs in acute in vivo studies including screening, distribution, duration of action and dosing, using a humanized mouse model of HD, Hu97/18, that is heterozygous for the targeted SNPs. We have identified four well-tolerated lead ASOs that potently and selectively silence muHTT at a broad range of doses throughout the central nervous system for 16 weeks or more after a single intracerebroventricular (ICV) injection. With further validation, these ASOs could provide a therapeutic option for individuals afflicted with HD.

Rational design of antisense oligonucleotides targeting single nucleotide polymorphisms for potent and allele selective suppression of mutant Huntingtin in the CNS.

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.

An exocyclic methylene group acts as a bioisostere of the 2'-oxygen atom in LNA.

We show for the first time that it is possible to obtain LNA-like (Locked Nucleic Acid 1) binding affinity and biological activity with carbocyclic LNA (cLNA) analogs by replacing the 2'-oxygen atom in LNA with an exocyclic methylene group. Synthesis of the methylene-cLNA nucleoside was accomplished by an intramolecular cyclization reaction between a radical at the 2'-position and a propynyl group at the C-4' position. Only methylene-cLNA modified oligonucleotides showed similar thermal stability and mismatch discrimination properties for complementary nucleic acids as LNA. In contrast, the close structurally related methyl-cLNA analogs showed diminished hybridization properties. Analysis of crystal structures of cLNA modified self-complementary DNA decamer duplexes revealed that the methylene group participates in a tight interaction with a 2'-deoxyribose residue of the 5'-terminal G of a neighboring duplex, resulting in the formation of a CH...O type hydrogen bond. This indicates that the methylene group retains a negative polarization at the edge of the minor groove in the absence of a hydrophilic 2'-substituent and provides a rationale for the superior thermal stability of this modification. In animal experiments, methylene-cLNA antisense oligonucleotides (ASOs) showed similar in vivo activity but reduced toxicity as compared to LNA ASOs. Our work highlights the interchangeable role of oxygen and unsaturated moieties in nucleic acid structure and emphasizes greater use of this bioisostere to improve the properties of nucleic acids for therapeutic and diagnostic applications.

Likelihood of Nonspecific Activity of Gapmer Antisense Oligonucleotides Is Associated with Relative Hybridization Free Energy.

Reduction of matched and nearly complementary unintended transcripts was evaluated for 96 antisense oligonucleotides (ASOs) and 832 nearly matched unintended transcripts. The ASOs were 16-20 nucleotide "gapmers" with a gap of 8-10 DNA residues and 2'-O-methoxy-ethyl or constrained-ethyl substitutions in the wings. Most unintended transcripts were not reduced or were reduced with a potency more than 10-fold weaker than the intended transcript. For the unintended transcripts that were reduced, a strong correlation between relative potency of the intended versus the unintended transcript with predicted free energy of hybridization was observed. These results suggest ASO selectivity should be evaluated by testing for reduction of the unintended transcripts predicted to bind most stably to the ASO.

Antisense oligonucleotide treatment ameliorates IFN-γ-induced proteinuria in APOL1-transgenic mice.

African Americans develop end-stage renal disease at a higher rate compared with European Americans due to 2 polymorphisms (G1 and G2 risk variants) in the apolipoprotein L1 (APOL1) gene common in people of African ancestry. Although this compelling genetic evidence provides an exciting opportunity for personalized medicine in chronic kidney disease, drug discovery efforts have been greatly hindered by the fact that APOL1 expression is lacking in rodents. Here, we describe a potentially novel physiologically relevant genomic mouse model of APOL1-associated renal disease that expresses human APOL1 from the endogenous human promoter, resulting in expression in similar tissues and at similar relative levels as humans. While naive APOL1-transgenic mice did not exhibit a renal disease phenotype, administration of IFN-γ was sufficient to robustly induce proteinuria only in APOL1 G1 mice, despite inducing kidney APOL1 expression in both G0 and G1 mice, serving as a clinically relevant "second hit." Treatment of APOL1 G1 mice with IONIS-APOL1Rx, an antisense oligonucleotide (ASO) targeting APOL1 mRNA, prior to IFN-γ challenge robustly and dose-dependently inhibited kidney and liver APOL1 expression and protected against IFN-γ-induced proteinuria, indicating that the disease-relevant cell types are sensitive to ASO treatment. Therefore, IONIS-APOL1Rx may be an effective therapeutic for APOL1 nephropathies and warrants further development.

Analysis of the androgen receptor-regulated lncRNA landscape identifies a role for ARLNC1 in prostate cancer progression.

The androgen receptor (AR) plays a critical role in the development of the normal prostate as well as prostate cancer. Using an integrative transcriptomic analysis of prostate cancer cell lines and tissues, we identified ARLNC1 (AR-regulated long noncoding RNA 1) as an important long noncoding RNA that is strongly associated with AR signaling in prostate cancer progression. Not only was ARLNC1 induced by the AR protein, but ARLNC1 stabilized the AR transcript via RNA-RNA interaction. ARLNC1 knockdown suppressed AR expression, global AR signaling and prostate cancer growth in vitro and in vivo. Taken together, these data support a role for ARLNC1 in maintaining a positive feedback loop that potentiates AR signaling during prostate cancer progression and identify ARLNC1 as a novel therapeutic target.

Analgesic profile of intrathecal P2X(3) antisense oligonucleotide treatment in chronic inflammatory and neuropathic pain states in rats.

Extracellular adenosine triphosphate (ATP), acting at P2X ionotropic receptors, is implicated in numerous sensory processes. Exogenous ATP has been shown to be algogenic in both animals and humans. Research focus has been directed towards the P2X(3) receptor, as it is preferentially expressed on nociceptive C-fibers and its implication in pain processing is supported by an altered nociceptive phenotype in P2X(3) knock-out mice. In order to further characterize the role of P2X(3) receptor activation in nociception, we evaluated the effects of continuous intrathecal administration of P2X(3) antisense oligonucleotides for 7 days in the rat. P2X(3) receptor antisense oligonucleotide treatment significantly decreased nociceptive behaviors observed after injection of complete Freund's adjuvant (CFA), formalin or alphabeta-methylene ATP into the rat's hind paw. The anti-hyperalgesic effects of the antisense treatment in the CFA model of inflammatory pain were dose related. Similar effects were observed with two distinct P2X(3) antisense oligonucleotides. These behavioral effects were significantly correlated with a decrease in P2X(3) receptor protein expression in the dorsal root ganglia (DRG). In contrast, a decrease in P2X(3) receptor protein expression in the DRG did not affect nociceptive behavior in the carrageenan model of acute thermal hyperalgesia. P2X(3) receptor antisense oligonucleotide treatment also significantly reduced mechanical allodynia observed after spinal nerve ligation. Overall, the present data demonstrate that activation of P2X(3) receptors contribute to the expression of chronic inflammatory and neuropathic pain states and that relief form these forms of chronic pain might be achieved by selective blockade of P2X(3 )receptor expression or activation.

In vitro and in vivo effects of an alpha3 neuronal nicotinic acetylcholine receptor antisense oligonucleotide.

In the mammalian central nervous system (CNS), a family of alpha and beta subunits (alpha2-7, beta2-4) assemble to form both hetero- and homopentameric neuronal nicotinic acetylcholine receptors (nAChRs). In contrast to alpha4beta2 and alpha7, the predominant brain subtypes, far less is known regarding the functional expression and significance of alpha3-containing nAChRs in the CNS. In trying to better understand the role alpha3 in the CNS, an antisense knockdown strategy was utilized in the present studies. Specifically, Isis 106567 was identified out of 80 antisense oligonucleotides (aONs) designed and screened for their ability to reduce alpha3 mRNA expression in PC-12 cells. In addition to reducing alpha3 mRNA by greater than 75%, Isis 106567 attenuated nicotine-induced calcium influx in alpha3-expressing F11 cells. In vivo studies revealed significant reduction of alpha3 mRNA levels in both thalamus and medial habenula, regions known to express alpha3, following continuous (7 days) intracerebroventricular (i.c.v.) infusion of Isis 106567 in rats. Consistent with functional alpha3 knockdown, epibatidine-induced c-Fos expression in the medial habenula was attenuated in aON-treated rats. Known physiological responses elicited by epibatidine, such as hypothermia and micturition, were not affected by alpha3 aON treatment. However, the incidence of epibatidine-induced seizures was reduced in alpha3-antisense aON-treated rats, suggesting that alpha3 may be involved in mediating seizures produced by the nAChR agonist. Results of our studies suggest that Isis 106567 may be a useful in vivo tool for characterizing the functional significance of alpha3 expression in the CNS.

Allele-specific suppression of mutant huntingtin using antisense oligonucleotides: providing a therapeutic option for all Huntington disease patients.

Huntington disease (HD) is an inherited, fatal neurodegenerative disorder caused by a CAG repeat expansion in the huntingtin gene. The mutant protein causes neuronal dysfunction and degeneration resulting in motor dysfunction, cognitive decline, and psychiatric disturbances. Currently, there is no disease altering treatment, and symptomatic therapy has limited benefit. The pathogenesis of HD is complicated and multiple pathways are compromised. Addressing the problem at its genetic root by suppressing mutant huntingtin expression is a promising therapeutic strategy for HD. We have developed and evaluated antisense oligonucleotides (ASOs) targeting single nucleotide polymorphisms that are significantly enriched on HD alleles (HD-SNPs). We describe our structure-activity relationship studies for ASO design and find that adjusting the SNP position within the gap, chemical modifications of the wings, and shortening the unmodified gap are critical for potent, specific, and well tolerated silencing of mutant huntingtin. Finally, we show that using two distinct ASO drugs targeting the two allelic variants of an HD-SNP could provide a therapeutic option for all persons with HD; allele-specifically for roughly half, and non-specifically for the remainder.

Control of RNA processing by a large non-coding RNA over-expressed in carcinomas.

RNA processing is vital for the high fidelity and diversity of eukaryotic transcriptomes and the encoded proteomes. However, control of RNA processing is not fully established. Σ RNA is a class of conserved large non-coding RNAs (murine Hepcarcin; human MALAT-1) up-regulated in carcinomas. Using antisense technology, we identified that RNA post-transcriptional modification is the most significant global function of Σ RNA. Specifically, processing of the pre-mRNAs of genes including Tissue Factor and Endoglin was altered by hydrolysis of Σ RNA/MALAT-1. These results support the hypothesis that Σ RNA/MALAT-1 is a regulatory molecule exerting roles in RNA post-transcriptional modification.

Antisense oligonucleotide inhibition of Bcl-xL and Bid expression in liver regulates responses in a mouse model of Fas-induced fulminant hepatitis.

Activation of the cell-surface receptor Fas can lead to apoptosis in parenchymal cells in the liver, and if severe enough, result in fulminant hepatic failure and animal death. In the present study, we have examined the roles played by the Bcl-2 family members Bcl-xL and Bid in regulating this response. To do this, we have developed chemically modified 2'-O-(2-methoxy) ethyl antisense inhibitors of both Bid and Bcl-xL expression. In Balb/c mice, dosing with these antisense oligonucleotides reduced expression of the targeted mRNA by greater than 80% in the liver. This reduction was highly dependent upon oligonucleotide sequence and oligonucleotide dose. Reduction of Bcl-xL expression resulted in a potentiation of Fas-mediated apoptosis in liver and significant increase of the lethality of Fas-mediated fulminant hepatitis (p < 0.0001). In contrast, reduction of Bid expression protected the animals against Fas-mediated fulminant hepatitis and death (p < 0.0001). Simultaneous dosing of mice with Bcl-xL and Bid-targeting antisense oligonucleotides resulted in an inhibition of expression of both targeted proteins and protection of the animals from Fas-mediated apoptosis. These results demonstrate, for the first time, the role of Bcl-xL in regulating responses to proapoptotic Fas signaling in mouse liver. In addition, this is the first reported example demonstrating the ability of antisense inhibitors to reduce expression of multiple proteins in animals by simultaneous dosing.