Antisense oligonucleotides to therapeutically target SARS-CoV-2 infection.

Although the COVID-19 pandemic began over three years ago, the virus responsible for the disease, SARS-CoV-2, continues to infect people across the globe. As such, there remains a critical need for development of novel therapeutics against SARS-CoV-2. One technology that has remained relatively unexplored in COVID-19 is the use of antisense oligonucleotides (ASOs)-short single-stranded nucleic acids that bind to target RNA transcripts to modulate their expression. In this study, ASOs targeted against the SARS-CoV-2 genome and host entry factors, ACE2 and TMPRSS2, were designed and tested for their ability to inhibit cellular infection by SARS-CoV-2. Using our previously developed SARS-CoV-2 bioassay platform, we screened 180 total ASOs targeting various regions of the SARS-CoV-2 genome and validated several ASOs that potently blocked SARS-CoV-2 infection in vitro. Notably, select ASOs retained activity against both the WA1 and B.1.1.7 (commonly known as alpha) variants. Screening of ACE2 and TMPRSS2 ASOs showed that targeting of ACE2 also potently prevented infection by the WA1 and B.1.1.7 SARS-CoV-2 viruses in the tested cell lines. Combined with the demonstrated success of ASOs in other disease indications, these results support further research into the development of ASOs targeting SARS-CoV-2 and host entry factors as potential COVID-19 therapeutics.

Associations between schizotypal traits and antisocial behaviours in a sub-Saharan sample.

Schizophrenic symptoms have often been associated with antisocial behaviours (ASBs). Most studies have focused on violence. The association between schizophrenia and violence is often confounded by socio demographic characteristics of patients. Overemphasis on diagnosis, rather than traits, and the traditional focus on violence may have obscured associations between psychotic features and antisocial behaviour. In this study, an analogue sample (n = 604, females = 54.4%, mean age = 21.46, SD = 5.89) completed self-report measures of schizotypy and antisocial behaviours. The aim was to identify the strength of associations between different schizotypal traits/symptoms (unusual experiences, cognitive disorganisation, impulsivity & introvertive anhedonia) and different forms of antisocial behaviour (rule breaking, physical aggression & social aggression). Differential patterns of association between genders were also considered. Traits akin to positive symptoms were more strongly associated with antisocial behaviours. Impulsive non-conformity was associated with rule breaking, physical aggression and social aggression for both males and females whilst unusual experiences traits were associated with physical and social aggression in both males and females but had only a weak (gender-nonspecific) association with rule breaking. Finally, cognitive disorganisation was not associated with any class of ASB for males but was associated with physical and social aggression in females. These specific associations could inform the development of more targeted treatment approaches for specific types of ASBs in males and females.

2'-O-methoxyethyl splice-switching oligos correct splicing from IVS2-745 ß-thalassemia patient cells restoring HbA production and chain rebalance.

ß-thalassemia is a disorder caused by altered hemoglobin protein synthesis and affects individuals worldwide. Severe forms of the disease, left untreated, can result in death before the age of 3 years (1). The standard of care consists of chronic and costly palliative treatment by blood transfusion combined with iron chelation. This dual approach suppresses anemia and reduces iron-related toxicities in patients. Allogeneic bone marrow transplant is an option, but limited by the availability of a highly compatible HSC donor. While gene therapy is been explored in several trials, its use is highly limited to developed regions with centers of excellence and well-established healthcare systems (2). Hence, there remains a tremendous unmet medical need to develop alternative treatment strategies for ß-thalassemia (3). Occurrence of aberrant splicing is one of the processes that affects ß-globin synthesis in ß-thalassemia. The (C>G) IVS-2-745 is a splicing mutation within intron 2 of the ß-globin gene. It leads to an aberrantly spliced mRNA that incorporates an intron fragment. This results in an in-frame premature termination codon that inhibits ß-globin production. Here, we propose the use of uniform 2'-O-methoxyethyl (2'-MOE) splice switching oligos (SSOs) to reverse this aberrant splicing in the pre-mRNA. With these lead SSOs we show aberrant to wild type splice switching. This switching leads to an increase of adult hemoglobin (HbA) up to 80% in erythroid cells from patients with the IVS-2-745 mutation. Furthermore, we demonstrate a restoration of the balance between ß-like- and α-globin chains, and up to an 87% reduction in toxic α-heme aggregates. While examining the potential benefit of 2'-MOE-SSOs in a mixed sickle-thalassemic phenotypic setting, we found reduced HbS synthesis and sickle cell formation due to HbA induction. In summary, 2'-MOE-SSOs are a promising therapy for forms of ß-thalassemia caused by mutations leading to aberrant splicing.

Suppressing transthyretin production in mice, monkeys and humans using 2nd-Generation antisense oligonucleotides.

Transthyretin amyloidosis (ATTR amyloidosis) is a rare disease that results from the deposition of misfolded transthyretin (TTR) protein from the plasma into tissues as amyloid fibrils, leading to polyneuropathy and cardiomyopathy. IONIS-TTRRx (ISIS 420915) is a 2nd-Generation 2'-O-(2-methoxyethyl) modified "2'-MOE" antisense oligonucleotide (ASO) that targets the TTR RNA transcript and reduces the levels of the TTR transcript through an RNaseH1 mechanism of action, leading to reductions in both mutant and wild-type TTR protein. The activity of IONIS-TTRRx to decrease TTR protein levels was studied in transgenic mice bearing the Ile84Ser human TTR mutant, in cynomolgus monkeys and in healthy human volunteers. Robust (>80%) reductions of plasma TTR protein were obtained in all three species treated with IONIS-TTRRx, which in mice and monkeys was associated with substantial reductions in hepatic TTR RNA levels. These effects were dose-dependent and lasted for weeks post-dosing. In a Phase 1 healthy volunteer study, treatment with IONIS-TTRRx for four weeks was well tolerated without any remarkable safety issues. TTR protein reductions up to 96% in plasma were observed. These nonclinical and clinical results support the ongoing Phase 3 development of IONIS-TTRRx in patients with ATTR amyloidosis.

Gain of Toxicity from ALS/FTD-Linked Repeat Expansions in C9ORF72 Is Alleviated by Antisense Oligonucleotides Targeting GGGGCC-Containing RNAs.

Hexanucleotide expansions in C9ORF72 are the most frequent genetic cause of amyotrophic lateral sclerosis and frontotemporal dementia. Disease mechanisms were evaluated in mice expressing C9ORF72 RNAs with up to 450 GGGGCC repeats or with one or both C9orf72 alleles inactivated. Chronic 50% reduction of C9ORF72 did not provoke disease, while its absence produced splenomegaly, enlarged lymph nodes, and mild social interaction deficits, but not motor dysfunction. Hexanucleotide expansions caused age-, repeat-length-, and expression-level-dependent accumulation of RNA foci and dipeptide-repeat proteins synthesized by AUG-independent translation, accompanied by loss of hippocampal neurons, increased anxiety, and impaired cognitive function. Single-dose injection of antisense oligonucleotides (ASOs) that target repeat-containing RNAs but preserve levels of mRNAs encoding C9ORF72 produced sustained reductions in RNA foci and dipeptide-repeat proteins, and ameliorated behavioral deficits. These efforts identify gain of toxicity as a central disease mechanism caused by repeat-expanded C9ORF72 and establish the feasibility of ASO-mediated therapy.

Hepatotoxicity of high affinity gapmer antisense oligonucleotides is mediated by RNase H1 dependent promiscuous reduction of very long pre-mRNA transcripts.

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.

Allele-Specific Inhibition of Rhodopsin With an Antisense Oligonucleotide Slows Photoreceptor Cell Degeneration.

PURPOSE: To preserve photoreceptor cell structure and function in a rodent model of retinitis pigmentosa with P23H rhodopsin by selective inhibition of the mutant rhodopsin allele using a second generation antisense oligonucleotide (ASO). METHODS: Wild-type mice and rats were treated with ASO by intravitreal (IVT) injection and rhodopsin mRNA and protein expression were measured. Transgenic rats expressing the murine P23H rhodopsin gene (P23H transgenic rat Line 1) were administered either a mouse-specific P23H ASO or a control ASO. The contralateral eye was injected with PBS and used as a comparator control. Electroretinography (ERG) measurements and analyses of the retinal outer nuclear layer were conducted and correlated with rhodopsin mRNA levels. RESULTS: Rhodopsin mRNA and protein expression was reduced after a single ASO injection in wild-type mice with a rhodopsin-specific ASO. Transgenic rat eyes that express a murine P23H rhodopsin gene injected with a murine P23H ASO had a 181 ± 39% better maximum amplitude response (scotopic a-wave) as compared with contralateral PBS-injected eyes; the response in control ASO eyes was not significantly different from comparator contralateral eyes. Morphometric analysis of the outer nuclear layer showed a significantly thicker nuclear layer in eyes injected with murine P23H ASO (18%) versus contralateral PBS-injected eyes. CONCLUSIONS: Allele-specific ASO-mediated knockdown of mutant P23H rhodopsin expression slowed the rate of photoreceptor degeneration and preserved the function of photoreceptor cells in eyes of the P23H rhodopsin transgenic rat. Our data indicate that ASO treatment is a potentially effective therapy for the treatment of retinitis pigmentosa.

Partial Hepatectomy Induced Long Noncoding RNA Inhibits Hepatocyte Proliferation during Liver Regeneration.

Liver regeneration after partial hepatectomy (PHx) is a complex and well-orchestrated biological process in which synchronized cell proliferation is induced in response to the loss of liver mass. To define long noncoding RNAs (lncRNAs) that participate in the regulation of liver regeneration, we performed microarray analysis and identified more than 400 lncRNAs exhibiting significantly altered expression. Of these, one lncRNA, LncPHx2 (Long noncoding RNA induced by PHx 2), was highly upregulated during liver regeneration. Depletion of LncPHx2 during liver regeneration using antisense oligonucleotides led to a transient increase in hepatocyte proliferation and more rapid liver regeneration. Gene expression analysis showed that LncPHx2 depletion resulted in upregulation of mRNAs encoding proteins known to promote cell proliferation, including MCM components, DNA polymerases, histone proteins, and transcription factors. LncPHx2 interacts with the mRNAs of MCM components, making it a candidate to regulate the expression of MCMs and other genes post-transcriptionally. Collectively, our data demonstrate that LncPHx2 is a key lncRNA that participates in a negative feedback loop modulating hepatocyte proliferation through RNA-RNA interactions.

Rev-Erbs repress macrophage gene expression by inhibiting enhancer-directed transcription.

Rev-Erb-α and Rev-Erb-ß are nuclear receptors that regulate the expression of genes involved in the control of circadian rhythm, metabolism and inflammatory responses. Rev-Erbs function as transcriptional repressors by recruiting nuclear receptor co-repressor (NCoR)-HDAC3 complexes to Rev-Erb response elements in enhancers and promoters of target genes, but the molecular basis for cell-specific programs of repression is not known. Here we present evidence that in mouse macrophages Rev-Erbs regulate target gene expression by inhibiting the functions of distal enhancers that are selected by macrophage-lineage-determining factors, thereby establishing a macrophage-specific program of repression. Remarkably, the repressive functions of Rev-Erbs are associated with their ability to inhibit the transcription of enhancer-derived RNAs (eRNAs). Furthermore, targeted degradation of eRNAs at two enhancers subject to negative regulation by Rev-Erbs resulted in reduced expression of nearby messenger RNAs, suggesting a direct role of these eRNAs in enhancer function. By precisely defining eRNA start sites using a modified form of global run-on sequencing that quantifies nascent 5' ends, we show that transfer of full enhancer activity to a target promoter requires both the sequences mediating transcription-factor binding and the specific sequences encoding the eRNA transcript. These studies provide evidence for a direct role of eRNAs in contributing to enhancer functions and suggest that Rev-Erbs act to suppress gene expression at a distance by repressing eRNA transcription.

Reducing TMPRSS6 ameliorates hemochromatosis and ß-thalassemia in mice.

ß-Thalassemia and HFE-related hemochromatosis are 2 of the most frequently inherited disorders worldwide. Both disorders are characterized by low levels of hepcidin (HAMP), the hormone that regulates iron absorption. As a consequence, patients affected by these disorders exhibit iron overload, which is the main cause of morbidity and mortality. HAMP expression is controlled by activation of the SMAD1,5,8/SMAD4 complex. TMPRSS6 is a serine protease that reduces SMAD activation and blocks HAMP expression. We identified second generation antisense oligonucleotides (ASOs) targeting mouse Tmprss6. ASO treatment in mice affected by hemochromatosis (Hfe(-/-)) significantly decreased serum iron, transferrin saturation and liver iron accumulation. Furthermore, ASO treatment of mice affected by ß-thalassemia (HBB(th3/+) mice, referred to hereafter as th3/+ mice) decreased the formation of insoluble membrane-bound globins, ROS, and apoptosis, and improved anemia. These animals also exhibited lower erythropoietin levels, a significant amelioration of ineffective erythropoiesis (IE) and splenomegaly, and an increase in total hemoglobin levels. These data suggest that ASOs targeting Tmprss6 could be beneficial in individuals with hemochromatosis, ß-thalassemia, and related disorders.

Divergent roles of ALS-linked proteins FUS/TLS and TDP-43 intersect in processing long pre-mRNAs.

FUS/TLS (fused in sarcoma/translocated in liposarcoma) and TDP-43 are integrally involved in amyotrophic lateral sclerosis (ALS) and frontotemporal dementia. We found that FUS/TLS binds to RNAs from >5,500 genes in mouse and human brain, primarily through a GUGGU-binding motif. We identified a sawtooth-like binding pattern, consistent with co-transcriptional deposition of FUS/TLS. Depletion of FUS/TLS from the adult nervous system altered the levels or splicing of >950 mRNAs, most of which are distinct from RNAs dependent on TDP-43. Abundance of only 45 RNAs was reduced after depletion of either TDP-43 or FUS/TLS from mouse brain, but among these were mRNAs that were transcribed from genes with exceptionally long introns and that encode proteins that are essential for neuronal integrity. Expression levels of a subset of these were lowered after TDP-43 or FUS/TLS depletion in stem cell-derived human neurons and in TDP-43 aggregate-containing motor neurons in sporadic ALS, supporting a common loss-of-function pathway as one component underlying motor neuron death from misregulation of TDP-43 or FUS/TLS.