Use Of Antisense Oligonucleotides Research Articles

Medicinal Chemistry of Antisense Oligonucleotides for Therapeutic Use in SARS-CoV-2: Design Strategies and Challenges for Targeted Delivery

Background: The evolution of novel Severe Acute Respiratory Syndrome-related Coronavirus 2 (SARS-CoV-2) strains with greater degrees of infectivity, resistance to vaccine-induced acquired immunity, and more severe morbidity have contributed to the recent spread of COVID-19. In light of this, novel therapeutic alternatives with improved effectiveness and fewer side effects have become a necessity. Despite many new or repurposed antiviral agents recommended for Coronavirus disease (COVID-19) therapy, this objective remains unfulfilled. Under these circumstances, the scientific community holds the significant responsibility to develop classes of novel therapeutic modalities to combat SARS-CoV-2 with the least harmful side effects. Objective: Antisense Oligonucleotides (ASOs) are short single-stranded oligonucleotides that allow the specific targeting of RNA, leading to its degradation. They may also prevent cellular factors or machinery from binding to the target RNA. It is possible to improve the pharmacokinetics and pharmacodynamics of ASOs by chemical modification or bioconjugation, which may provide conditions for customization of a particular clinical target. This study aimed to outline the potential use of ASOs in the treatment of COVID-19 disease, along with the use of antisense stabilization and transfer methods, as well as future challenges and limitations. Methods: We have reviewed the structure and properties of ASOs containing nucleobase, sugar, or backbone modifications, and provided an overview of the therapeutic potential, delivery challenges, and strategies of ASOs in the treatment of COVID-19. Results: The first-line therapy for COVID-19-infected individuals, as well as the development of oligonucleotide-based drugs, warrants further investigation. Chemical changes in the oligonucleotide structure can affect the biological processes. These chemical alterations may lead to enhanced potency, while changing the pharmacokinetics and pharmacodynamics. Conclusion: ASOs can be designed to target both coding and non-coding regions of the viral genome to disrupt or completely degrade the genomic RNA and thereby eliminate SARS-CoV-2. They may be very effective in areas, where vaccine distribution is challenging, and they may be helpful for future coronavirus pandemics.

METTL3-Mediated N6-Methyladenosine mRNA Modification and cGAS-STING Pathway Activity in Kidney Fibrosis

Key Points METTL3 was upregulated in CKD, leading to increased m6A modification levels.M6A modifications were enriched in genes related to the cyclic guanosine monophosphate–AMP synthase-stimulator of IFN genes pathway in CKD.Normalizing heightened METTL3 and m6A modification levels showed promise as a strategy to combat kidney disease. Background Chemical modifications on RNA profoundly affect RNA function and regulation. m6A, the most abundant RNA modification in eukaryotes, plays a pivotal role in diverse cellular processes and disease mechanisms. However, its importance is understudied in human CKD samples regarding its influence on pathological mechanisms. Methods Liquid chromatography–tandem mass spectrometry and methylated RNA immunoprecipitation sequencing were used to examine alterations in m6A levels and patterns in CKD samples. Overexpression of the m6A writer METTL3 in cultured kidney tubular cells was performed to confirm the effect of m6A in tubular cells and explore the biological functions of m6A modification on target genes. In addition, tubule-specific deletion of Mettl3 (Ksp-Cre Mettl3f/f) mice and antisense oligonucleotides inhibiting Mettl3 expression were used to reduce m6A modification in an animal kidney disease model. Results By examining 127 human CKD samples, we observed a significant increase in m6A modification and METTL3 expression in diseased kidneys. Epitranscriptomic analysis unveiled an enrichment of m6A modifications in transcripts associated with the activation of inflammatory signaling pathways, particularly the cyclic guanosine monophosphate–AMP synthase (cGAS)-stimulator of IFN genes (STING) pathway. m6A hypermethylation increased mRNA stability in cGAS and STING1 as well as elevated the expression of key proteins within the cGAS-STING pathway. Both the tubule-specific deletion of Mettl3 and the use of antisense oligonucleotides to inhibit Mettl3 expression protected mice from inflammation, reduced cytokine expression, decreased immune cell recruitment, and attenuated kidney fibrosis. Conclusions Our research revealed heightened METTL3-mediated m6A modification in fibrotic kidneys, particularly enriching the cGAS-STING pathway. This hypermethylation increased mRNA stability for cGAS and STING1, leading to sterile inflammation and fibrosis.

Review of the Use of Antisense Oligonucleotides as Therapy for Huntington's Disease

Introduction: Huntington's disease is a neurodegenerative disorder characterized by the progressive degeneration of nerve cells in the brain, and whose current treatment focuses on the control of symptoms, which is why in recent years several therapies have been evaluated, including Antisense oligonucleotides a promising strategy, thanks to their ability to modulate the expression of the mutated huntingtin protein.Methods:A bibliographic search was carried out using the PRISMA methodology in databases such as PubMed, limited to documents in English published during the last 5 years, using keywords such as "Huntington's Disease", "Antisense Oligonucleotides", "Treatment" “Tominersen”, selecting only documents such as original articles, bibliographic reviews and clinical trial reports related to the use of Antisense Oligonucleotides as therapy for Huntington's Disease.Results:150 articles were collected and analyzed, of which 30 documents were excluded due to their age and 40 due to lack of access and quality of information, leaving a total of 80 articles to which inclusion criteria were applied, selecting 25 articles for completion. of this literature review on the use of antisense oligonucleotides in Huntington's diseaseConclusion: Antisense oligonucleotides show great therapeutic potential for Huntington's disease by directly attacking the underlying cause of the disease, mutated huntingtin. However, significant challenges still remain, which is why better research is needed to ensure efficacy. and safety of this therapy in the long term.

Allele-specific antisense oligonucleotides for the treatment of BEST1-related dominantly inherited retinal diseases: An in vitro model

Retinal dystrophies are a common health problem worldwide that are currently incurable due to the inability of retinal cells to regenerate. Inherited retinal diseases (IRDs) are a diverse group of disorders characterized by progressive vision loss caused by photoreceptor cell dysfunction. The eye has always been an attractive organ for the development of novel therapies due to its independent access to the systemic pathway. Moreover, anti-sense oligonucleotides (ASOs), which facilitate manipulation of unwanted mRNAs via degradation or splicing, are undergoing rapid development and have been clinically deployed for the treatment of several diseases. The primary aim of this study was to establish a reliable in vitro model utilizing induced photoreceptor-like cells (PRCs) for assessing the efficacy and safety of ASOs targeting the BEST1 gene. Despite advances in gene therapy, effective treatments for a broad range of IRDs remain limited. An additional aim was to develop an in vitro model for evaluating RNA-based therapeutics, specifically ASOs, for the treatment in IRDs.Firstly, a cell culture model was established by induction of PRCs from dermal fibroblasts via direct programming. The induced PRCs were characterized at both the transcriptomic and protein level. Then, a common single nucleotide polymorphism (SNP) was identified in the BEST1 gene (rs1800007) for targeting with ASOs. ASOs were designed using the GapmeR strategy to target multiple alleles of this SNP, which is potentially suitable for a large proportion of the population. The efficacy and possible off-target effects of these ASOs were also analyzed in the induced PRC model.The findings show that the selected ASOs achieved allele-specific mRNA degradation with virtually no off-target effects on the global transcriptome profile, indicating their potential as safe and effective therapeutic agents. The presented in vitro model is a valuable platform for testing personalized IRD treatments and should inspire further research on RNA-based therapeutics. To the best of our knowledge this study is the first to test RNA-based therapeutics involving the use of ASOs in an induced PRC model. Based on the present findings, it will be possible to establish an ex vivo disease model using dermal fibroblast samples from affected individuals. In other words, the disease model and the ASOs that were successfully designed in this study can serve as a useful platform for the testing of personalized treatments for IRDs.

Outfitting COVID-19: An Effective Therapeutic Approach

Use of antisense oligonucleotides of the type 3′-(N)x-AAAUUUG-(N)x-5′ against slippery sequence and polynucleotides against pseudoknots forming sequences of SARS-CoV-2 RNA would block the first translation of ORF1a and ORF1b and hence dwindle the virus replication. It is easy to synthesize and deliver the antisense oligonucleotides to the target by directly injecting the nano formulation into the blood.

Evaluating the efficacy of purchased antisense oligonucleotides to reduce mouse and human tau in vivo.

Many preclinical and clinical studies support the use of antisense oligonucleotides (ASOs) as effective therapeutic strategies. However, acquiring ASOs for research purposes may be limited by partnerships with the pharmaceutical companies. Our lab previously developed an effective ASO strategy to lower human tau and reverse pathology in aged tauopathy model mice. Testing the efficacy of purchased tau lowering ASOs would provide support for these reagents as broad research tools. Purchased mouse and human tau lowering ASOs were infused or injected intracerebroventricularly into wildtype and tau transgenic mice. Following treatment, brain tissue evaluated for ASO distribution and levels of tau mRNA, protein, and phosphorylated tau. We show that purchased ASOs enter cell types of the brain and effectively decrease mouse or human tau mRNA and protein levels. Human tau lowering ASO treatment in PS19 mice decreased phosphorylated tau and gliosis relative to saline-treated PS19 mice, consistent with our previous study using a non-commercial tau lowering ASO. The results of this study demonstrate the efficacy of purchased tau targeting ASOs in vivo to support their broad use by researchers.

Roadmap for C9ORF72 in Frontotemporal Dementia and Amyotrophic Lateral Sclerosis: Report on the C9ORF72 FTD/ALS Summit.

A summit held March 2023 in Scottsdale, Arizona (USA) focused on the intronic hexanucleotide expansion in the C9ORF72 gene and its relevance in frontotemporal dementia (FTD) and amyotrophic lateral sclerosis (ALS; C9ORF72-FTD/ALS). The goal of this summit was to connect basic scientists, clinical researchers, drug developers, and individuals affected by C9ORF72-FTD/ALS to evaluate how collaborative efforts across the FTD-ALS disease spectrum might break down existing disease silos. Presentations and discussions covered recent discoveries in C9ORF72-FTD/ALS disease mechanisms, availability of disease biomarkers and recent advances in therapeutic development, and clinical trial design for prevention and treatment for individuals affected by C9ORF72-FTD/ALS and asymptomatic pathological expansion carriers. The C9ORF72-associated hexanucleotide repeat expansion is an important locus for both ALS and FTD. C9ORF72-FTD/ALS may be characterized by loss of function of the C9ORF72 protein and toxic gain of functions caused by both dipeptide repeat (DPR) proteins and hexanucleotide repeat RNA. C9ORF72-FTD/ALS therapeutic strategies discussed at the summit included the use of antisense oligonucleotides, adeno-associated virus (AAV)-mediated gene silencing and gene delivery, and engineered small molecules targeting RNA structures associated with the C9ORF72 expansion. Neurofilament light chain, DPR proteins, and transactive response (TAR) DNA-binding protein 43 (TDP-43)-associated molecular changes were presented as biomarker candidates. Similarly, brain imaging modalities (i.e., magnetic resonance imaging [MRI] and positron emission tomography [PET]) measuring structural, functional, and metabolic changes were discussed as important tools to monitor individuals affected with C9ORF72-FTD/ALS, at both pre-symptomatic and symptomatic disease stages. Finally, summit attendees evaluated current clinical trial designs available for FTD or ALS patients and concluded that therapeutics relevant to FTD/ALS patients, such as those specifically targeting C9ORF72, may need to be tested with composite endpoints covering clinical symptoms of both FTD and ALS. The latter will require novel clinical trial designs to be inclusive of all patient subgroups spanning the FTD/ALS spectrum.

Antisense Therapy for Infectious Diseases.

Infectious diseases, particularly Tuberculosis (TB) caused by Mycobacterium tuberculosis, pose a significant global health challenge, with 1.6 million reported deaths in 2021, making it the most fatal disease caused by a single infectious agent. The rise of drug-resistant infectious diseases adds to the urgency of finding effective and safe intervention therapies. Antisense therapy uses antisense oligonucleotides (ASOs) that are short, chemically modified, single-stranded deoxyribonucleotide molecules complementary to their mRNA target. Due to their designed target specificity and inhibition of a disease-causing gene at the mRNA level, antisense therapy has gained interest as a potential therapeutic approach. This type of therapy is currently utilized in numerous diseases, such as cancer and genetic disorders. Currently, there are limited but steadily increasing studies available that report on the use of ASOs as treatment for infectious diseases. This review explores the sustainability of FDA-approved and preclinically tested ASOs as a treatment for infectious diseases and the adaptability of ASOs for chemical modifications resulting in reduced side effects with improved drug delivery; thus, highlighting the potential therapeutic uses of ASOs for treating infectious diseases.

The Biotechnological “Provence” of the Future Provided by Antisense Oligoilators and Olinscides for Horticulturae

Successful management of the synthesis of secondary metabolites of essential oil plants is the basis for the economic growth of the essential oil industry. Against the backdrop of a growing global population and a decrease in land available for cultivation, simple and effective ways to increase the content of certain components in essential oils are becoming increasingly important. Selection is no longer keeping pace with market needs, which stimulates the search for faster methods to control the biosynthesis of secondary metabolites. In this article, using the genera Lavandula and Mentha as examples, we consider the prospects for use of antisense oligonucleotides (ASO), oligoilators, to rapidly increase the concentration of valuable components in essential oil and its yield. This article discusses the use of unmodified ASOs as regulators of a plant’s secondary metabolism to increase the synthesis of individual valuable components, presenting a completely new way to increase the yield of valuable substances based on unique nucleotide sequences. The proposed approach is effective, affordable, safe, and significantly reduces the time needed to obtain plants that synthesize the required concentrations of target substances. Oligoilators can be used with oligonucleotide insecticides (olinscides) in complex formulations used for green agriculture. Further investigation is needed to determine maximum economic efficiency for this approach.

Gene therapy for selected neuromuscular and trinucleotide repeat disorders - An insight to subsume South Asia for multicenter clinical trials.

In this article, the authors discuss how they utilized the genetic mutation data in Sri Lankan Duchenne muscular dystrophy (DMD), Spinal muscular atrophy (SMA), Spinocerebellar ataxia (SCA) and Huntington's disease (HD) patients and compare the available literature from South Asian countries to identifying potential candidates for available gene therapy for DMD, SMA, SCA and HD patients. Rare disease patients (n=623) with the characteristic clinical findings suspected of HD, SCA, SMA and Muscular Dystrophy were genetically confirmed using Multiplex Ligation Dependent Probe Amplification (MLPA), and single plex PCR. A survey was conducted in the "Wiley database on Gene Therapy Trials Worldwide" to identify DMD, SMA, SCA, and HD gene therapy clinical trials performed worldwide up to April 2021. In order to identify candidates for gene therapy in other neighboring countries we compared our findings with available literature from India and Pakistan which has utilized the same molecular diagnostic protocol to our study. From the overall cohort of 623 rare disease patients with the characteristic clinical findings suspected of HD, SCA, SMA and Muscular Dystrophy, n=343 (55%) [Muscular Dystrophy- 65%; (DMD-139, Becker Muscular Dystrophy -BMD-11), SCA type 1-3-53% (SCA1-61,SCA2- 23, SCA3- 39), HD- 52% (45) and SMA- 34% (22)] patients were positive for molecular diagnostics by MLPA and single plex PCR. A total of 147 patients in Sri Lanka amenable to available gene therapy; [DMD-83, SMA-15 and HD-49] were identified. A comparison of Sri Lankan finding with available literature from India and Pakistan identified a total of 1257 patients [DMD-1076, SMA- 57, and HD-124] from these three South Asian Countries as amenable for existing gene therapy trials. DMD, SMA, and HD gene therapy clinical trials (113 studies) performed worldwide up to April 2021 were concentrated mostly (99%) in High Income Countries (HIC) and Upper Middle-Income Countries (UMIC). However, studies on the potential use of anti-sense oligonucleotides (ASO) for treatment of SCAs have yet to reach clinical trials. Most genetic therapies for neurodegenerative and neuromuscular disorders have been evaluated for efficacy primarily in Western populations. No multicenter gene therapy clinical trial sites for DMD, SMA and HD in the South Asian region, leading to lack of knowledge on the safety and efficacy of such personalized therapies in other populations, including South Asians. By fostering collaboration between researchers, clinicians, patient advocacy groups, government and industry in gene therapy initiatives for the inherited-diseases community in the developing world would link the Global North and Global South and breathe life into the motto "Together we can make a difference".

Novel and future lipid-modulating therapies for the prevention of cardiovascular disease.

Lowering the levels of LDL cholesterol in the plasma has been shown to reduce the risk of atherosclerotic cardiovascular disease (ASCVD). Several other lipoproteins, such as triglyceride-rich lipoproteins, HDL and lipoprotein(a) are associated with atherosclerosis and ASCVD, with strong evidence supporting causality for some. In this Review, we discuss novel and upcoming therapeutic strategies targeting different pathways in lipid metabolism to potentially attenuate the risk of cardiovascular events. Key proteins involved in lipoprotein metabolism, such as PCSK9, angiopoietin-related protein 3, cholesteryl ester transfer protein and apolipoprotein(a), have been identified as viable targets for therapeutic intervention through observational and genetic studies. These proteins can be targeted using a variety of approaches, such as protein inhibition or interference, inhibition of translation at the mRNA level (with the use of antisense oligonucleotides or small interfering RNA), and the introduction of loss-of-function mutations through base editing. These novel and upcoming strategies are complementary to and could work synergistically with existing therapies, or in some cases could potentially replace therapies, offering unprecedented opportunities to prevent ASCVD. Moreover, a major challenge in the prevention and treatment of non-communicable diseases is how to achieve safe, long-lasting reductions in causal exposures. This challenge might be overcome with approaches such as small interfering RNAs or genome editing, which shows how far the field has advanced from when the burden of achieving this goal was placed upon patients through rigorous adherence to daily small-molecule drug regimens.

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.

The contribution of amyloid deposition in the aortic valve to calcification and aortic stenosis.

Calcific aortic valve disease (CAVD) and stenosis have a complex pathogenesis, and no therapies are available that can halt or slow their progression. Several studies have shown the presence of apolipoprotein-related amyloid deposits in close proximity to calcified areas in diseased aortic valves. In this Perspective, we explore a possible relationship between amyloid deposits, calcification and the development of aortic valve stenosis. These amyloid deposits might contribute to the amplification of the inflammatory cycle in the aortic valve, including extracellular matrix remodelling and myofibroblast and osteoblast-like cell proliferation. Further investigation in this area is needed to characterize the amyloid deposits associated with CAVD, which could allow the use of antisense oligonucleotides and/or isotype gene therapies for the prevention and/or treatment of CAVD.

Molecular Basis of the Schuurs-Hoeijmakers Syndrome: What We Know about the Gene and the PACS-1 Protein and Novel Therapeutic Approaches.

The Schuurs–Hoeijmakers syndrome (SHMS) or PACS1 Neurodevelopment Disorder (PACS1-NDD) is a rare autosomal dominant disease caused by mutations in the PACS1 gene. To date, only 87 patients have been reported and, surprisingly, most of them carry the same variant (c.607C>T; p.R203W). The most relevant clinical features of the syndrome include neurodevelopment delay, seizures or a recognizable facial phenotype. Moreover, some of these characteristics overlap with other syndromes, such as the PACS2 or Wdr37 syndromes. The encoded protein phosphofurin acid cluster sorting 1 (PACS-1) is able to bind to different client proteins and direct them to their subcellular final locations. Therefore, although its main function is protein trafficking, it could perform other roles related to its client proteins. In patients with PACS1-NDD, a gain-of-function or a dominant negative mechanism for the mutated protein has been suggested. This, together with the fact that most of the patients carry the same genetic variant, makes it a good candidate for novel therapeutic approaches directed to decreasing the toxic effect of the mutated protein. Some of these strategies include the use of antisense oligonucleotides (ASOs) or targeting of its client proteins.

Transcript-Targeted Therapy Based on RNA Interference and Antisense Oligonucleotides: Current Applications and Novel Molecular Targets

The development of novel target therapies based on the use of RNA interference (RNAi) and antisense oligonucleotides (ASOs) is growing in an exponential way, challenging the chance for the treatment of the genetic diseases and cancer by hitting selectively targeted RNA in a sequence-dependent manner. Multiple opportunities are taking shape, able to remove defective protein by silencing RNA (e.g., Inclisiran targets mRNA of protein PCSK9, permitting a longer half-life of LDL receptors in heterozygous familial hypercholesteremia), by arresting mRNA translation (i.e., Fomivirsen that binds to UL123-RNA and blocks the translation into IE2 protein in CMV-retinitis), or by reactivating modified functional protein (e.g., Eteplirsen able to restore a functional shorter dystrophin by skipping the exon 51 in Duchenne muscular dystrophy) or a not very functional protein. In this last case, the use of ASOs permits modifying the expression of specific proteins by modulating splicing of specific pre-RNAs (e.g., Nusinersen acts on the splicing of exon 7 in SMN2 mRNA normally not expressed; it is used for spinal muscular atrophy) or by downregulation of transcript levels (e.g., Inotersen acts on the transthryretin mRNA to reduce its expression; it is prescribed for the treatment of hereditary transthyretin amyloidosis) in order to restore the biochemical/physiological condition and ameliorate quality of life. In the era of precision medicine, recently, an experimental splice-modulating antisense oligonucleotide, Milasen, was designed and used to treat an 8-year-old girl affected by a rare, fatal, progressive form of neurodegenerative disease leading to death during adolescence. In this review, we summarize the main transcriptional therapeutic drugs approved to date for the treatment of genetic diseases by principal regulatory government agencies and recent clinical trials aimed at the treatment of cancer. Their mechanism of action, chemical structure, administration, and biomedical performance are predominantly discussed.

1342-P: Reduction of Glucokinase and Glucokinase Regulatory Protein Expression by siRNA Is Associated with the Reduction of Lactate and Pyruvate Export from Canine Hepatocytes

Glucokinase (GCK) mediates hepatic glucose uptake during hyperglycemia and is crucial for the regulation of glucose-responsive glycolysis genes. Mutations in GCK are responsible for maturity-onset diabetes of the young, and glucokinase regulatory protein (GKRP) , the regulator of hepatic GCK, is also a diabetes susceptibility locus. Recently in in vivo experiments, we used hepatic lactate export as a surrogate for GCK activity, which was a measure of “glucose effectiveness.” We hypothesized that in vitro GCK and GKRP suppression in hepatocytes promotes a reduction in lactate and pyruvate export from the liver. We used cryopreserved canine hepatocytes and knocked down GCK and GKRP via lipid delivery of siRNA. A control group did not receive siRNA. We measured lactate dehydrogenase D (LDHD) and pyruvate kinase (PK) gene expression and lactate and pyruvate release into the medium 24 and 48 h after siRNA delivery. Selective blocking of GCK and GKRP did not change cell proliferation but reduced the expression of these genes by 90% and 85%, respectively (P<0.0vs. controls; n=4) . GCK and GKRP knockdown reduced LDHD by 71% and 74%, respectively (P<0.001) ; similarly, PK was reduced by 84% and 82%, respectively (P<0.001) . Lactate exported into the medium was reduced by 36% (P<0.001) and 26% (P<0.05) by GCK and GKRP siRNA, respectively, after 48 h of hepatocyte culture. However, pyruvate was reduced by 17% only after 48 h culture of GCK siRNA (P<0.05) . Thus, we showed that direct suppression of GCK and GKRP genes by siRNA in vitro affects glucose-responsive glycolysis genes, consequently reducing lactate export which can be used as a surrogate for reduced glucokinase activity in hepatocytes. The use of antisense oligonucleotides or CRISPR gene-editing methods in animals will be useful methods to study the glucose-lactate relationship in vivo and will evaluate the role of glucose effectiveness in the pathogenesis of type 2 diabetes. Disclosure M.Kabir: None. M.Ader: None. R.N.Bergman: Consultant; Fractyl Health, Inc., Novo Nordisk, Research Support; AstraZeneca, Janssen Research & Development, LLC. Funding National Institutes of Health R01- DK027619-28

Dysregulated miRNAs as Biomarkers and Therapeutical Targets in Neurodegenerative Diseases.

Alzheimer’s disease (AD), Parkinson’s disease (PD), and Amyotrophic Lateral Sclerosis (ALS) are representative neurodegenerative diseases (NDs) characterized by degeneration of selective neurons, as well as the lack of effective biomarkers and therapeutic treatments. In the last decade, microRNAs (miRNAs) have gained considerable interest in diagnostics and therapy of NDs, owing to their aberrant expression and their ability to target multiple molecules and pathways. Here, we provide an overview of dysregulated miRNAs in fluids (blood or cerebrospinal fluid) and nervous tissue of AD, PD, and ALS patients. By emphasizing those that are commonly dysregulated in these NDs, we highlight their potential role as biomarkers or therapeutical targets and describe the use of antisense oligonucleotides as miRNA therapies.

Long Noncoding RNAs in Lung Cancer: From Disease Markers to Treatment Roles.

There is an urgent need to identify reliable biomarkers that can be used in early diagnosis, prognostication prediction and as possible therapeutic targets for lung cancer due to its current poor prognosis. Long noncoding RNAs (lncRNAs) have recently attracted additional attention due to their potential role in carcinogenesis, invasion and metastasis. Issues involved in the biofunctions and regulatory mechanisms of oncogenic and tumor-suppressive lncRNAs in lung cancer are discussed. Some lncRNAs have shown good diagnostic value, especially in combination with conventional serum protein markers. The use of antisense oligonucleotides, small molecules and RNA interference techniques have shown promise as direct therapeutic tools for targeting lncRNAs in preclinical studies. The biomarker function of lncRNAs may also indirectly involved in tumor therapy as a reference to conventional therapy. Overall, the concept of using lncRNAs as biomarkers for prognostication and intervention in lung cancer is still in its infancy, and only with more in-depth studies could they have a significant impact.

Polyglutamine diseases.

Polyglutamine diseases are a collection of nine CAG trinucleotide expansion disorders, presenting with a spectrum of neurological and clinical phenotypes. Recent human, mouse and cell studies of Huntington's diseasehave highlighted the role of DNA repair genes in somatic expansion of the CAG repeat region, modifying disease pathogenesis. Incomplete splicing of the HTT gene has also been shown to occur in humans, with the resulting exon 1 fragment most probably contributing to the Huntington's disease phenotype. In the spinocerebellar ataxias, studies have converged on transcriptional dysregulation of ion channels as a key disease modifier. In addition, advances have been made in understanding how increased levels of toxic, polyglutamine-expanded proteins can arise in the spinocerebellar ataxias through post-transcriptional and -translational modifications and autophagic mechanisms. Recent studies in spinal and bulbar muscular atrophyimplicate similar pathogenic pathways to the more common polyglutamine diseases, highlighting autophagy stimulation as a potential therapeutic target. Finally, the therapeutic use of antisense oligonucleotides in several polyglutamine diseases has shown preclinical benefits and serves as potential future therapies in humans.

Open reading frame correction using splice-switching antisense oligonucleotides for the treatment of cystic fibrosis

CFTR gene mutations that result in the introduction of premature termination codons (PTCs) are common in cystic fibrosis (CF). This mutation type causes a severe form of the disease, likely because of low CFTR messenger RNA (mRNA) expression as a result of nonsense-mediated mRNA decay, as well as the production of a nonfunctional, truncated CFTR protein. Current therapeutics for CF, which target residual protein function, are less effective in patients with these types of mutations due in part to low CFTR protein levels. Splice-switching antisense oligonucleotides (ASOs), designed to induce skipping of exons in order to restore the mRNA open reading frame, have shown therapeutic promise preclinically and clinically for a number of diseases. We hypothesized that ASO-mediated skipping of CFTR exon 23 would recover CFTR activity associated with terminating mutations in the exon, including CFTR p.W1282X, the fifth most common mutation in CF. Here, we show that CFTR lacking the amino acids encoding exon 23 is partially functional and responsive to corrector and modulator drugs currently in clinical use. ASO-induced exon 23 skipping rescued CFTR expression and chloride current in primary human bronchial epithelial cells isolated from a homozygote CFTR-W1282X patient. These results support the use of ASOs in treating CF patients with CFTR class I mutations in exon 23 that result in unstable CFTR mRNA and truncations of the CFTR protein.