QR-1011 restores defective ABCA4 splicing caused by multiple severe ABCA4 variants underlying Stargardt disease.

Stargardt disease type 1 (STGD1), the most common form of hereditary macular dystrophy, can be caused by biallelic combinations of over 2200 variants in the ABCA4 gene. This leads to reduced or absent ABCA4 protein activity, resulting in toxic metabolite accumulation in the retina and damage of the retinal pigment epithelium and photoreceptors. Approximately 21% of all ABCA4 variants that contribute to disease influence ABCA4 pre-mRNA splicing. This emphasizes the need for therapies to restore disrupted ABCA4 splicing and halt STGD1 progression. Previously, QR-1011, an antisense oligonucleotide (AON), successfully corrected splicing abnormalities and restored normal ABCA4 protein translation in human retinal organoids carrying the prevalent disease-causing variant c.5461-10T>C in ABCA4. Here, we investigated whether QR-1011 could also correct splicing in four less common non-canonical splice site (NCSS) variants flanking ABCA4 exon 39: c.5461-8T>G, c.5461-6T>C, c.5584+5G>A and c.5584+6T>C. We administered QR-1011 and three other AONs to midigene-transfected cells and demonstrate that QR-1011 had the most pronounced effect on splicing compared to the others. Moreover, QR-1011 significantly increased full-length ABCA4 transcript levels for c.5461-8T>G and c.5584+6T>C. Splicing restoration could not be achieved in the other two variants, suggesting their more severe effect on splicing. Overall, QR-1011, initially developed for a single ABCA4 variant, exhibited potent splice correction capabilities for two additional severe NCSS variants nearby. This suggests the possibility of a broader therapeutic impact of QR-1011 extending beyond its original target and highlights the potential for treating a larger population of STGD1 patients affected by multiple severe ABCA4 variants with a single AON.

Targeting transforming growth factor-ß2 by antisense oligodeoxynucleotide accelerates T cell-mediated tumor rejection in a humanized mouse model of triple-negative breast cancer.

Transforming growth factor-beta (TGF-ß) pathway mediates suppression of antitumor immunity and is associated with poor prognosis in triple-negative breast cancer (TNBC). In this study, we generated a humanized animal model by transplanting human peripheral blood mononuclear cells into immunodeficient mice followed by inoculation of MDA-MB-231 cells and subsequently analyzed the role of TGF-ß2 in the interaction between human T cells and human tumor cells. Following reconstitution of the human immune system, inhibition of TGF-ß signaling by TGF-ß2 antisense oligodeoxynucleotide (TASO) resulted in accelerated tumor growth inhibition. TGF-ß2 inhibition also resulted in downregulation of peripheral Foxp3 + regulatory T cells (Treg), whereas no effect was seen in the expression of CD8 + cytotoxic T cells. Analysis of the TASO-treated mice serum revealed elevated levels of human IFN-γ and reduced levels of human IL-10 and TGF-ß2. Moreover, TGF-ß2 inhibition resulted in increased CD8 + T cell infiltration, whereas the reduced infiltration of Tregs into the tumor partly resulted from decreased expression of CCL22. Decreased intratumoral Tregs facilitated the activation of cytotoxic T cells, associated with increased granzyme B expression. These results indicate that TASO potentiated T cell-mediated antitumor immunity, and it is proposed that TGF-ß2 may be a promising target in the immunotherapeutic strategy of TNBC.

Antisense oligonucleotide activity in tumour cells is influenced by intracellular LBPA distribution and extracellular vesicle recycling.

Next generation modified antisense oligonucleotides (ASOs) are commercially approved new therapeutic modalities, yet poor productive uptake and endosomal entrapment in tumour cells limit their broad application. Here we compare intracellular traffic of anti KRAS antisense oligonucleotide (AZD4785) in tumour cell lines PC9 and LK2, with good and poor productive uptake, respectively. We find that the majority of AZD4785 is rapidly delivered to CD63+late endosomes (LE) in both cell lines. Importantly, lysobisphosphatidic acid (LBPA) that triggers ASO LE escape is presented in CD63+LE in PC9 but not in LK2 cells. Moreover, both cell lines recycle AZD4785 in extracellular vesicles (EVs); however, AZD4785 quantification by advanced mass spectrometry and proteomic analysis reveals that LK2 recycles more AZD4785 and RNA-binding proteins. Finally, stimulating LBPA intracellular production or blocking EV recycling enhances AZD4785 activity in LK2 but not in PC9 cells thus offering a possible strategy to enhance ASO potency in tumour cells with poor productive uptake of ASOs.

A Novel Peptide-Equipped Exosomes Platform for Delivery of Antisense Oligonucleotides.

Exosomes are natural delivery vehicles because of their original feature such as low immunogenicity, excellent biocompatibility, and migration capability. Engineering exosomes with appropriate ligands are effective approaches to improve the low cellular uptake efficiency of exosomes. However, current strategies face considerable challenges due to the tedious and labor-intensive operational process. Here, we designed a novel peptides-equipped exosomes platform which can be assembled under convenient and mild reaction condition. Cell-penetrating peptides (CPPs) was conjugated on HepG2 cells-derived exosomes surface which can not only enhance the penetrating capacity of exosomes but also assist exosomes in loading antisense oligonucleotides (ASOs). The cellular uptake mechanism was investigated and we compared the difference between natural exosomes and modified exosomes. The resulting nanosystem demonstrated a preferential tropism for cells that are parented to their source tumor cells and could remarkably increase the cellular delivery of G3139 with efficient downregulation of antiapoptotic Bcl-2. This work developed a rapid strategy for intracellular delivery of nucleic acids, thus providing more possibilities toward personalized cancer medicine.

Defective heart chamber growth and myofibrillogenesis after knockout of adprhl1 gene function by targeted disruption of the ancestral catalytic active site.

ADP-ribosylhydrolase-like 1 (Adprhl1) is a pseudoenzyme expressed in the developing heart myocardium of all vertebrates. In the amphibian Xenopus laevis, knockdown of the two cardiac Adprhl1 protein species (40 and 23 kDa) causes failure of chamber outgrowth but this has only been demonstrated using antisense morpholinos that interfere with RNA-splicing. Transgenic production of 40 kDa Adprhl1 provides only part rescue of these defects. CRISPR/Cas9 technology now enables targeted mutation of the adprhl1 gene in G0-generation embryos with routine cleavage of all alleles. Testing multiple gRNAs distributed across the locus reveals exonic locations that encode critical amino acids for Adprhl1 function. The gRNA recording the highest frequency of a specific ventricle outgrowth phenotype directs Cas9 cleavage of an exon 6 sequence, where microhomology mediated end-joining biases subsequent DNA repairs towards three small in-frame deletions. Mutant alleles encode discrete loss of 1, 3 or 4 amino acids from a di-arginine (Arg271-Arg272) containing peptide loop at the centre of the ancestral ADP-ribosylhydrolase site. Thus despite lacking catalytic activity, it is the modified (adenosine-ribose) substrate binding cleft of Adprhl1 that fulfils an essential role during heart formation. Mutation results in striking loss of myofibril assembly in ventricle cardiomyocytes. The defects suggest Adprhl1 participation from the earliest stage of cardiac myofibrillogenesis and are consistent with previous MO results and Adprhl1 protein localization to actin filament Z-disc boundaries. A single nucleotide change to the gRNA sequence renders it inactive. Mice lacking Adprhl1 exons 3-4 are normal but production of the smaller ADPRHL1 species is unaffected, providing further evidence that cardiac activity is concentrated at the C-terminal protein portion.

Rational design of antisense oligonucleotides modulating the activity of TLR7/8 agonists.

Oligonucleotide-based therapeutics have become a reality, and are set to transform management of many diseases. Nevertheless, the modulatory activities of these molecules on immune responses remain incompletely defined. Here, we show that gene targeting 2'-O-methyl (2'OMe) gapmer antisense oligonucleotides (ASOs) can have opposing activities on Toll-Like Receptors 7 and 8 (TLR7/8), leading to divergent suppression of TLR7 and activation of TLR8, in a sequence-dependent manner. Surprisingly, TLR8 potentiation by the gapmer ASOs was blunted by locked nucleic acid (LNA) and 2'-methoxyethyl (2'MOE) modifications. Through a screen of 192 2'OMe ASOs and sequence mutants, we characterized the structural and sequence determinants of these activities. Importantly, we identified core motifs preventing the immunosuppressive activities of 2'OMe ASOs on TLR7. Based on these observations, we designed oligonucleotides strongly potentiating TLR8 sensing of Resiquimod, which preserve TLR7 function, and promote strong activation of phagocytes and immune cells. We also provide proof-of-principle data that gene-targeting ASOs can be selected to synergize with TLR8 agonists currently under investigation as immunotherapies, and show that rational ASO selection can be used to prevent unintended immune suppression of TLR7. Taken together, our work characterizes the immumodulatory effects of ASOs to advance their therapeutic development.

MicroRNA-153 impairs hippocampal synaptic vesicle trafficking via downregulation of synapsin I in rats following chronic cerebral hypoperfusion.

Chronic cerebral hypoperfusion (CCH) promotes the development of Alzheimer's pathology. However, whether and how CCH impairs the synaptic vesicle trafficking is still unclear. In the present study, we found that the hippocampal glutamatergic vesicle trafficking was impaired as indicated by a significant shortened delayed response enhancement (DRE) phase in CA3-CA1 circuit and decreased synapsin I in CCH rats suffering from bilateral common carotid artery occlusion (2VO). Further study showed an upregulated miR-153 in the hippocampus of 2VO rats. In vitro, overexpression of miR-153 downregulated synapsin I by binding the 3'UTRs of SYN1 mRNAs, which was prevented by its antisense AMO-153 and miRNA-masking antisense oligodeoxynucleotides (SYN1-ODN). In vivo, the upregulation of miR-153 elicited similar reduced DRE phase and synapsin I deficiency as CCH. Furthermore, miR-153 knockdown rescued the downregulated synapsin I and shortened DRE phase in 2VO rats. Our results demonstrate that CCH impairs hippocampal glutamatergic vesicle trafficking by upregulating miR-153, which suppresses the expression of synapsin I at the post-transcriptional level. These results will provide important references for drug research and treatment of vascular dementia.

Antisense oligonucleotide-mediated correction of CFTR splicing improves chloride secretion in cystic fibrosis patient-derived bronchial epithelial cells.

Cystic fibrosis (CF) is an autosomal recessive disorder caused by mutations in the CF transmembrane conductance regulator (CFTR) gene, encoding an anion channel that conducts chloride and bicarbonate across epithelial membranes. Mutations that disrupt pre-mRNA splicing occur in >15% of CF cases. One common CFTR splicing mutation is CFTR c.3718-2477C>T (3849+10 kb C>T), which creates a new 5' splice site, resulting in splicing to a cryptic exon with a premature termination codon. Splice-switching antisense oligonucleotides (ASOs) have emerged as an effective therapeutic strategy to block aberrant splicing. We test an ASO targeting the CFTR c.3718-2477C>T mutation and show that it effectively blocks aberrant splicing in primary bronchial epithelial (hBE) cells from CF patients with the mutation. ASO treatment results in long-term improvement in CFTR activity in hBE cells, as demonstrated by a recovery of chloride secretion and apical membrane conductance. We also show that the ASO is more effective at recovering chloride secretion in our assay than ivacaftor, the potentiator treatment currently available to these patients. Our findings demonstrate the utility of ASOs in correcting CFTR expression and channel activity in a manner expected to be therapeutic in patients.

Evaluation of cell-penetrating peptide-peptide nucleic acid effect in the inhibition of cagA in Helicobacter pylori.

Helicobacter pylori is the most common cause of chronic infection in human and is associated with gastritis, peptic ulcer disease, and adenocarcinoma of mucosa-associated lymphoid tissue cells. Peptide nucleic acid (PNA) is a synthetic compound, which can inhibit the production of a particular gene. This study aimed to investigate the effect of PNA on inhibiting the expression of cagA. After confirmation of the desired gene by polymerase chain reaction (PCR), the antisense sequence was designed against cagA gene. The minimum inhibitory concentrations of conjugated PNA against H. pylori was determined. The effect of the compound on the expression level of the cagA was investigated in HT29 cell culture using real-time PCR. The results showed 2 and 3 log reduction in bacterial count after 8- and 24-h treatment with 4 and 8 µM of the compound, respectively. The lowest expression level of the cagA gene was observed at a concentration of 8 µM after 6 h. The results of this study showed that cell-penetrating peptide antisense can be employed as effective tools for inhibiting the target gene mRNA for various purposes. Moreover, further research is necessary to assess the potency, safety, and pharmacokinetics of CPP-PNAs for clinical prevention and treatment of infections due to H. pylori.

Therapeutic IDOL Reduction Ameliorates Amyloidosis and Improves Cognitive Function in APP/PS1 Mice.

Brain lipoprotein receptors have been shown to regulate the metabolism of ApoE and ß-amyloid (Aß) and are potential therapeutic targets for Alzheimer's disease (AD). Previously, we identified E3 ubiquitin ligase IDOL as a negative regulator of brain lipoprotein receptors. Genetic ablation of Idol increases low-density lipoprotein receptor protein levels, which facilitates Aß uptake and clearance by microglia. In this study, we utilized an antisense oligonucleotide (ASO) to reduce IDOL expression therapeutically in the brains of APP/PS1 male mice. ASO treatment led to decreased Aß pathology and improved spatial learning and memory. Single-cell transcriptomic analysis of hippocampus revealed that IDOL inhibition upregulated lysosomal/phagocytic genes in microglia. Furthermore, clustering of microglia revealed that IDOL-ASO treatment shifted the composition of the microglia population by increasing the prevalence of disease-associated microglia. Our results suggest that reducing IDOL expression in the adult brain promotes the phagocytic clearance of Aß and ameliorates Aß-dependent pathology. Pharmacological inhibition of IDOL activity in the brain may represent a therapeutic strategy for the treatment of AD.

Inotersen preserves or improves quality of life in hereditary transthyretin amyloidosis.

OBJECTIVE: To examine the impact on quality of life (QOL) of patients with hATTR amyloidosis with polyneuropathy treated with inotersen (Tegsedi™) versus placebo. METHODS: Data were from the NEURO-TTR trial (ClinicalTrials.gov Identifier: NCT01737398), a phase 3, multinational, randomized, double-blind, placebo-controlled study of inotersen in patients with hATTR amyloidosis with polyneuropathy. At baseline and week 66, QOL measures-the Norfolk-QOL-Diabetic Neuropathy (DN) questionnaire and SF-36v2® Health Survey (SF-36v2)-were assessed. Treatment differences in mean changes in QOL from baseline to week 66 were tested using mixed-effect models with repeated measures. Responder analyses compared the percentages of patients whose QOL meaningfully improved or worsened from baseline to week 66 in inotersen and placebo arms. Descriptive analysis of item responses examined treatment differences in specific activities and functions at week 66. RESULTS: Statistically significant mean differences between treatment arms were observed for three of five Norfolk-QOL-DN domains and five of eight SF-36v2 domains, with better outcomes for inotersen than placebo in physical functioning, activities of daily living, neuropathic symptoms, pain, role limitations due to health problems, and social functioning. A larger percentage of patients in the inotersen arm than the placebo arm showed preservation or improvement in Norfolk-QOL-DN and SF-36v2 scores from baseline to week 66. Responses at week 66 showed more substantial problems with daily activities and functioning for patients in the placebo arm than in the inotersen arm. CONCLUSION: Patients with hATTR amyloidosis with polyneuropathy treated with inotersen showed preserved or improved QOL at 66 weeks compared to those who received placebo.

Inhibition of MicroRNA-9 Improves Fracture Healing by Modulating the Bone Morphogenetic Protein-7 Pathway.

We evaluated the effect of microRNA (miR)-9 inhibition on fracture healing in a rat model of femoral fracture. The rats were divided into sham, negative control and miR-9 inhibitor groups. The miR-9 inhibitor group received 30 pmol/mL inhibitor intrathecally for 8 consecutive weeks following surgery-induced femoral fracture. The effect of miR-9 inhibition on fracture healing was estimated by determining the bone mineral density (BMD) and by performing X-ray analysis of the fractured bone. The serum levels of markers of bone formation were estimated by enzyme-linked immunosorbent assay and reverse transcription polymerase chain reaction, and western blotting and immunohistochemical analysis were performed to assess the effect of miR-9 inhibition on fracture healing. The BMD at the fracture site was significantly higher in the miR-9 inhibitor group than in the negative control group. Inhibition of miR-9 blocked the fracture gap and resulted in new callus formation at the fracture site. The serum levels of osteocalcin and bone GLA protein were increased and that of alkaline phosphatase was decreased by inhibition of miR-9 compared to levels in the negative control. However, inhibition of miR-9 significantly increased the mRNA levels of runt-related transcription factor 2 (Runx2) and bone morphogenetic protein 7 (BMP-7) in the bone tissue at the fracture site compared to the negative control group; this result was confirmed by western blotting. In conclusion, -miR-9 inhibition enhanced fracture healing by modulating the BMP-7/Runx2 signalling pathway in a rat model of femoral fracture.

Peptide-conjugated oligonucleotides evoke long-lasting myotonic dystrophy correction in patient-derived cells and mice.

Antisense oligonucleotides (ASOs) targeting pathologic RNAs have shown promising therapeutic corrections for many genetic diseases including myotonic dystrophy (DM1). Thus, ASO strategies for DM1 can abolish the toxic RNA gain-of-function mechanism caused by nucleus-retained mutant DMPK (DM1 protein kinase) transcripts containing CUG expansions (CUGexps). However, systemic use of ASOs for this muscular disease remains challenging due to poor drug distribution to skeletal muscle. To overcome this limitation, we test an arginine-rich Pip6a cell-penetrating peptide and show that Pip6a-conjugated morpholino phosphorodiamidate oligomer (PMO) dramatically enhanced ASO delivery into striated muscles of DM1 mice following systemic administration in comparison with unconjugated PMO and other ASO strategies. Thus, low-dose treatment with Pip6a-PMO-CAG targeting pathologic expansions is sufficient to reverse both splicing defects and myotonia in DM1 mice and normalizes the overall disease transcriptome. Moreover, treated DM1 patient-derived muscle cells showed that Pip6a-PMO-CAG specifically targets mutant CUGexp-DMPK transcripts to abrogate the detrimental sequestration of MBNL1 splicing factor by nuclear RNA foci and consequently MBNL1 functional loss, responsible for splicing defects and muscle dysfunction. Our results demonstrate that Pip6a-PMO-CAG induces long-lasting correction with high efficacy of DM1-associated phenotypes at both molecular and functional levels, and strongly support the use of advanced peptide conjugates for systemic corrective therapy in DM1.

Inotersen for the treatment of adults with polyneuropathy caused by hereditary transthyretin-mediated amyloidosis.

Introduction: Hereditary transthyretin-mediated amyloidosis (ATTRv; v for variant) is an underdiagnosed, progressive, and fatal multisystemic disease with a heterogenous clinical phenotype that is caused by TTR gene mutations that destabilize the TTR protein, resulting in its misfolding, aggregation, and deposition in tissues throughout the body. Areas covered: Inotersen, an antisense oligonucleotide inhibitor, was recently approved in the United States and Europe for the treatment of the polyneuropathy of ATTRv based on the positive results obtained in the pivotal phase 3 trial, NEURO-TTR. This review will discuss the mechanism of action of inotersen and its pharmacology, clinical efficacy, and safety and tolerability. A PubMed search using the terms 'inotersen,' 'AG10,' 'antisense oligonucleotide,' 'hereditary transthyretin amyloidosis,' 'familial amyloid polyneuropathy,' and 'familial amyloid cardiomyopathy' was performed, and the results were screened for the most relevant English language publications. The bibliographies of all retrieved articles were manually searched to identify additional studies of relevance. Expert opinion: Inotersen targets the disease-forming protein, TTR, and has been shown to improve quality of life and neuropathy progression in patients with stage 1 or 2 ATTRv with polyneuropathy. Inotersen is well tolerated, with a manageable safety profile through regular monitoring for the development of glomerulonephritis or thrombocytopenia.

Specific Delivery of Oligonucleotides to the Cell Nucleus via Gentle Compression and Attachment of Polythymidine.

Nonviral delivery of nucleic acids to the cell nucleus typically requires chemical methods that do not guarantee specific delivery (e.g., transfection agent) or physical methods that may require extensive fabrication (e.g., microfluidics) or an elevated pressure (e.g., 105 Pa for microneedles). We report a method of delivering oligonucleotides to the nucleus with high specificity (relative to the cytosol) by synergistically combining chemical and physical approaches. Particularly, we demonstrate that DNA oligonucleotides appended with a polythymidine [poly(T)] segment (chemical) profusely accumulate inside the nucleus when the cells are under gentle compression imposed by the weight of a single glass coverslip (physical; ∼2.2 Pa). Our "compression-cum-poly(T)" delivery method is simple, can be generalizable to three "hard-to-transfect" cell types, and does not induce significant levels of cytotoxicity or long-term oxidative stress to the treated cells when provided the use of suitable compression times and oligonucleotide concentrations. In bEnd.3 endothelial cells, compression-aided intranuclear delivery of poly(T) is primarily mediated by importin ß and nucleoporin 62. Our method significantly enhances the intranuclear delivery of antisense oligonucleotides to bEnd.3 endothelioma cells and the inhibition of two target genes, including a reporter gene encoding the enhanced green fluorescent protein and an intranuclear lncRNA oncogene (metastasis-associated lung adenocarcinoma transcript 1), when compared with delivery without gentle compression or poly(T) attachment. Our data underscore the critical roles of pressure and nucleotide sequence on the intranuclear delivery of nucleic acids.

Increased apolipoprotein C3 drives cardiovascular risk in type 1 diabetes.

Type 1 diabetes mellitus (T1DM) increases the risk of atherosclerotic cardiovascular disease (CVD) in humans by poorly understood mechanisms. Using mouse models of T1DM-accelerated atherosclerosis, we found that relative insulin deficiency rather than hyperglycemia elevated levels of apolipoprotein C3 (APOC3), an apolipoprotein that prevents clearance of triglyceride-rich lipoproteins (TRLs) and their remnants. We then showed that serum APOC3 levels predict incident CVD events in subjects with T1DM in the Coronary Artery Calcification in Type 1 Diabetes (CACTI) study. To explore underlying mechanisms, we investigated the impact of Apoc3 antisense oligonucleotides (ASOs) on lipoprotein metabolism and atherosclerosis in a mouse model of T1DM. Apoc3 ASO treatment abolished the increased hepatic Apoc3 expression in diabetic mice - resulting in lower levels of TRLs - without improving glycemic control. APOC3 suppression also prevented arterial accumulation of APOC3-containing lipoprotein particles, macrophage foam cell formation, and the accelerated atherosclerosis in diabetic mice. Our observations demonstrate that relative insulin deficiency increases APOC3 and that this results in elevated levels of TRLs and accelerated atherosclerosis in a mouse model of T1DM. Because serum levels of APOC3 predicted incident CVD events in the CACTI study, inhibiting APOC3 might reduce CVD risk in T1DM patients.

Antisense Oligonucleotides Targeting Lipoprotein(a).

PURPOSE OF REVIEW: High lipoprotein(a) levels are observationally and causally, from human genetics, associated with increased risk of cardiovascular disease including myocardial infarction and aortic valve stenosis. The European Atherosclerosis Society recommends screening for elevated lipoprotein(a) levels in high-risk patients. Different therapies have been suggested and some are used to treat elevated lipoprotein(a) levels such as niacin, PCSK9 inhibitors, and CETP inhibitors; however, to date, no randomized controlled trial has demonstrated that lowering of lipoprotein(a) leads to lower risk of cardiovascular disease. RECENT FINDINGS: Synthetic oligonucleotides can be used to inactivate genes involved in disease processes. To lower lipoprotein(a), two antisense oligonucleotides have been developed, one targeting apolipoprotein B and one targeting apolipoprotein(a). Mipomersen is an antisense oligonucleotide targeting apolipoprotein B and thereby reducing levels of all apolipoprotein B containing lipoproteins in the circulation. Mipomersen has been shown to lower lipoprotein(a) by 20-50% in phase 3 studies. AKCEA-APO(a)-LRx is the most recent antisense oligonucleotide targeting apolipoprotein(a) and thereby uniquely targeting lipoprotein(a). It has been tested in a phase 2 study and has shown to lower lipoprotein(a) levels by 50-80%. The treatment of elevated lipoprotein(a) levels with the newest antisense oligonucleotides seems promising; however, no improvement in cardiovascular disease risk has yet been shown. However, a phase 3 study of AKCEA-APO(a)-LRx is being planned with cardiovascular disease as outcome, and results are awaited with great anticipation.

DNA insecticide developed from the Lymantria dispar 5.8S ribosomal RNA gene provides a novel biotechnology for plant protection.

Having observed how botanicals and other natural compounds are used by nature to control pests in the environment, we began investigating natural polymers, DNA and RNA, as promising tools for insect pest management. Over the last decade, unmodified short antisense DNA oligonucleotides have shown a clear potential for use as insecticides. Our research has concentrated mainly on Lymantria dispar larvae using an antisense oligoRING sequence from its inhibitor-of-apoptosis gene. In this article, we propose a novel biotechnology to protect plants from insect pests using DNA insecticide with improved insecticidal activity based on a new antisense oligoRIBO-11 sequence from the 5.8S ribosomal RNA gene. This investigational oligoRIBO-11 insecticide causes higher mortality among both L. dispar larvae grown in the lab and those collected from the forest; in addition, it is more affordable and faster acting, which makes it a prospective candidate for use in the development of a ready-to-use preparation.

In Vitro and In Vivo Activity of a Novel Antisense Peptide Nucleic Acid Compound Against Multidrug-Resistant Acinetobacter baumannii.

Multidrug-resistant (MDR) Acinetobacter baumannii is a difficult pathogen due to its propensity to develop resistance to antibiotics. Antisense nucleic acid analogs have been proposed as a potential alternative anti-infective approach. We developed a peptide nucleic acid (PNA) compound that targets the essential Acinetobacter gene carA. The PNA oligomer was conjugated to the cell-penetrating peptide (RXR)4XB. In vitro testing of the PNA conjugate against four clinical strains of MDR-A. baumannii in minimal medium demonstrated that all four strains were inhibited at a concentration of 1.25 µM. In vivo testing of the PNA conjugate was done using a Galleria mellonella model of sepsis caused by one of the clinical strains. Preliminary testing of a variety of inocula demonstrated that an inoculum of 1 × 106 cfu was lethal to the majority of caterpillars by day 3, but not within 24 hours. The PNA compound was administered 30 minutes after an inoculum of 1 × 106 cfu at doses estimated to produce concentrations of ∼5 and 20 µM. The PNA compound had no effect at the lower dose. However, the higher dose reduced mortality from 5/28 (18%) to 0/28 (0%) at day 1 (p = 0.051) and from 19/28 (68%) to 9/28 (32%) at day 6 (p = 0.015). Antisense therapy is a novel approach to dealing with difficult MDR pathogens that could circumvent the problem of progressive resistance to available antibiotics. Further studies need to be done with additional strains and more complex in vivo model systems.

Tankyrase1 antisense oligodeoxynucleotides suppress the proliferation, migration and invasion through Hippo/YAP pathway in human osteosarcoma cells.

Osteosarcoma is the most common malignant tumor of bone with a high potential for metastasis and poor prognosis. This study intends to explore the effect of tankyrase1 (TANK1) in the development of osteosarcoma cells and the underlying mechanism. The osteosarcoma cell line MG-63 cells were cultured and transfected with tankyrase1 antisense oligodeoxynucleotides (TANK1-ASODN). Cell proliferation was detected with CCK-8 and immunofluorescence. Cell migration and invasion were examined by wound healing assay and Transwell assay, respectively. Reverse transcription-quantitative polymerase chain reaction was performed to detect the mRNA level of TANK1 and western blot was conducted to detect relative protein expression during the research. As a result, we demonstrated that TANK1 was upregulated in osteosarcoma. The TANK1-ASODN inhibited MG-63 cell proliferation, migration and invasion. The progress of epithelial-mesenchymal transition (EMT) was also suppressed in TANK1-ASODN transfected MG-63 cells compared to control group. Besides, the TANK1-ASODN activated and modulated the Hippo/YAP signaling which might be the pathway that TANK1 depended on. Overall, our finding supported that TANK1-ASODN slowed down the progress of osteosarcoma by suppressing cell proliferation, migration, invasion and EMT through Hippo/YAP pathway.