Antisense Nucleotides Research Articles

Effective lowering of α-synuclein expression by targeting G-quadruplex structures within the SNCA gene

Alpha-synuclein, encoded by the SNCA gene, is a pivotal protein implicated in the pathogenesis of synucleinopathies, including Parkinson's disease. Current approaches for modulating alpha-synuclein levels involve antisense nucleotides, siRNAs, and small molecules targeting SNCA's 5′-UTR mRNA. Here, we propose a groundbreaking strategy targeting G-quadruplex structures to effectively modulate SNCA gene expression and lowering alpha-synuclein amount. Novel G-quadruplex sequences, identified on the SNCA gene's transcription starting site and 5′-UTR of SNCA mRNAs, were experimentally confirmed for their stability through biophysical assays and in vitro experiments on human genomic DNA. Biological validation in differentiated SH-SY5Y cells revealed that well-known G-quadruplex ligands remarkably stabilized these structures, inducing the modulation of SNCA mRNAs expression, and the effective decrease in alpha-synuclein amount. Besides, a novel peptide nucleic acid conjugate, designed to selectively disrupt of G-quadruplex within the SNCA gene promoter, caused a promising lowering of both SNCA mRNA and alpha-synuclein protein. Altogether our findings highlight G-quadruplexes' key role as intriguing biological targets in achieving a notable and successful reduction in alpha-synuclein expression, pointing to a novel approach against synucleinopathies.

RNA-Based Antipsoriatic Gene Therapy: An Updated Review Focusing on Evidence from Animal Models.

Psoriasis presents as a complex genetic skin disorder, characterized by the interaction between infiltrated immune cells and keratinocytes. Substantial progress has been made in understanding the molecular mechanisms of both coding and non-coding genes, which has positively impacted clinical treatment approaches. Despite extensive research into the genetic aspects of psoriasis pathogenesis, fully grasping its epigenetic component remains a challenging endeavor. In response to the pressing demand for innovative treatments to alleviate inflammatory skin disorders, various novel strategies are under consideration. These include gene therapy employing antisense nucleotides, silencing RNA complexes, stem cell therapy, and antibody-based therapy. There is a pressing requirement for a psoriasis-like animal model that replicates human psoriasis to facilitate early preclinical evaluations of these novel treatments. The authors conduct a comprehensive review of various gene therapy in different psoriasis-like animal models utilized in psoriasis research. The animals included in the list underwent skin treatments such as imiquimod application, as well as genetic and biologic injections, and the results of these interventions are detailed. Animal models play a crucial role in translating drug discoveries from the laboratory to clinical practice, and these models aid in improving the reproducibility and clinical applicability of preclinical data. Numerous animal models with characteristics similar to those of human psoriasis have proven to be useful in understanding the development of psoriasis. In this review, the article focuses on RNA-based gene therapy exploration in different types of psoriasis-like animal models to improve the treatment of psoriasis.

Modern gene therapy drugs

Gene therapy is a modern and effective approach for treating diseases previously considered incurable by traditional methods. Its main strategy is to transfer the genetic material into the patient’s somatic cells to inhibit or promote the expression of a target gene/protein associated with disease development. This type of therapy is used in patients with few or no alternative treatment options.
 Over the past two decades, gene therapy has produced promising clinical results, with many products approved for treating severe oncologic, hematologic, infectious, orphan, and/or inherited diseases, including monogenic and metabolic diseases. The scope of clinical indications and tissue targets for gene therapy is expanding every year.
 This review examines new genetically engineered drugs recently introduced in global and domestic markets. These drugs are based on molecules of small interfering RNA (siRNA), mRNA, antisense nucleotides, and viral and plasmid vectors. Each group of drugs is unique and has its mechanism of action, depending on the purpose of the therapy.

Lipoprotein dysfunction in patients with chronic kidney disease (CKD). Pathogenesis and treatment of CKD dyslipidemia (literature review)

Dyslipidemia develops in the initial stages of chronic kidney disease (CKD) and worsens as nephropathy progresses. The main manifestation of dyslipidemia is hypercholesterolemia, especially in nephrotic syndrome. However, with CKD of stages 4-5, it is replaced by hypertriglyceridemia in combination with an increase in blood levels of lipoproteins low and very low density. Such changes are closely related to the development of cardiovascular pathology with high mortality. The content of high-density lipoproteins (HDL) in the blood is gradually decreasing, as well as the reversible transport of cholesterol. Thus, their anti-atherogenic, antioxidant and anti-inflammatory functions are lost. The main components of HDL – apolipoproteins ApoA-I and ApoA-II, which provide functionality, are replaced by acute-phase proteins, and HDL lose their cardioprotective potential and acquire a proinflammatory and proatherogenic phenotype. According to modern concepts, HDL dysfunction, along with metabolic shifts, is largely due to epigenetic disorders affecting gene expression and partially eliminated by prescribing drugs containing microRNAs (mRNAs) or antisense nucleotides. Drugs with interfering RNAs created in recent years have been successfully used not only for the treatment of dyslipidemia in nephrological patients, but also in patients with neoplastic processes, inflammatory arthritis, degenerative diseases of the central nervous system, porphyria, hemophilia and many other diseases. The proposed review is devoted to the mechanisms of disorders of the structure and functions of HDL in patients with CKD and the correction of these disorders.

Exosomal miR-223 promotes ARDS by targeting insulin-like growth factor 1 receptor: A cell communication study

Background Acute respiratory distress syndrome (ARDS) is a respiratory failure syndrome characterized by hypoxemia and changes in the respiratory system. ARDS is the most common cause of death in COVID-19 deaths was ARDS. In this study, we explored the role of miR-223 in exosomes in ARDS. Methods Exosomes were purified from the supernatants of macrophages. qPCR was used to detect relative mRNA levels. A luciferase reporter assay was performed to verify the miRNA target genes. Western blotting was used to detect the activation of inflammatory pathways. Flow cytometry was performed to assess apoptosis. An LPS-induced ARDS mouse model was used to assess the function of miR-223 in ARDS. Results Exosomes secreted by macrophages promoted apoptosis in A549 cells. Macrophages and exosomes contain high levels of miR-223. Exogenous miR-223 can decrease the expression of insulin-like growth factor 1 receptor (IGF-1R) in A549 and promote the apoptosis of A549.Transfection of anti-miR223 antisense nucleotides effectively reduced the level of miR-223 in macrophages and exosomes and eliminated the pro-apoptotic effect of A549. In vivo, LPS stimulation increased inflammatory cell infiltration in the lungs of mice, whereas knockdown of miR-223 in mice resulted in significantly reduced eosinophil infiltration. Conclusions Macrophages can secrete exosomes containing miR-223 and promote apoptosis by targeting the IGF-1R/Akt/mTOR signaling pathway in A549 cells and mouse models, suggesting that miR-223 is a potential target for treating COVID-19 induced ARDS.

LncRNA Malat1 suppresses pyroptosis and T cell-mediated killing of incipient metastatic cells.

The contribution of antitumor immunity to metastatic dormancy is poorly understood. Here we show that the long noncoding RNA Malat1 is required for tumor initiation and metastatic reactivation in mouse models of breast cancer and other tumor types. Malat1 localizes to nuclear speckles to couple transcription, splicing and mRNA maturation. In metastatic cells, Malat1 induces WNT ligands, autocrine loops to promote self-renewal and the expression of Serpin protease inhibitors. Through inhibition of caspase-1 and cathepsin G, SERPINB6B prevents gasdermin D-mediated induction of pyroptosis. In this way, SERPINB6B suppresses immunogenic cell death and confers evasion of T cell-mediated tumor lysis of incipient metastatic cells. On-target inhibition of Malat1 using therapeutic antisense nucleotides suppresses metastasis in a SERPINB6B-dependent manner. These results suggest that Malat1-induced expression of SERPINB6B can titrate pyroptosis and immune recognition at metastatic sites. Thus, Malat1 is at the nexus of tumor initiation, reactivation and immune evasion and represents a tractable and clinically relevant drug target.

Inhibition of Angiopoietin-Like Protein 3 or 3/8 Complex and ApoC-III in Severe Hypertriglyceridemia.

The role of the inhibition of ANGPTL3 in severe or refractory hypercholesterolemia is well documented, less in severe hyperTG. This review focuses on the preclinical and clinical development of ApoC-III inhibitors and ANGPTL3, 4, and 3/8 complex inhibitors for the treatment of severe or refractory forms of hypertriglyceridemia to prevent cardiovascular disease or other morbidities. APOC3 and ANGPTL3 became targets for drug development following the identification of naturally occurring loss of function variants in families with a favorable lipid profile and low cardiovascular risk. The inhibition of ANGPTL3 covers a broad spectrum of lipid disorders from severe hypercholesterolemia to severe hypertriglyceridemia, while the inhibition of ApoC-III can treat hypertriglyceridemia regardless of the severity. Preclinical and clinical data suggest that ApoC-III inhibitors, ANGPTL3 inhibitors, and inhibitors of the ANGPTL3/8 complex that is formed postprandially are highly effective for the treatment of severe or refractory hypertriglyceridemia. Inhibition of ANGPTL3 or the ANGPTL3/8 complex upregulates LPL and facilitates the hydrolysis and clearance of triglyceride-rich lipoproteins (TRL) (LPL-dependent mechanisms), whereas ApoC-III inhibitors contribute to the management and clearance of TRL through both LPL-dependent and LPL-independent mechanisms making it possible to successfully lower TG in subjects completely lacking LPL (familial chylomicronemia syndrome). Most of these agents are biologicals including monoclonal antibodies (mAb), antisense nucleotides (ASO), small interfering RNA (siRNA), or CRISPR-cas gene editing strategies.

Blocking the interaction between circTNRC18 and LIN28A promotes trophoblast epithelial–mesenchymal transformation and alleviates preeclampsia

Defects in migration and invasion caused by dysregulation of trophoblastic epithelial–mesenchymal transformation (EMT) play a vital role in preeclampsia (PE). We have previously shown that circTNRC18 inhibits the migration and EMT of trophoblasts; however, its role in PE remains unknown. Herein, we demonstrate that circTNRC18 interacts with an RNA-binding protein, lin-28 homolog A (LIN28A), and this interaction is enhanced in PE placental tissue. LIN28A overexpression suppresses circTNRC18-mediated inhibition of trophoblast migration, invasion, and EMT, whereas LIN28A knockdown promotes them. The intracellular distribution of LIN28A is regulated by circTNRC18, where it promotes the expression of insulin-like growth factor II by stabilizing its mRNA. circTNRC18 also promotes complex formation between GATA-binding factor 1 (GATA1) and sine oculis homeobox 1 (SIX1) by inhibiting LIN28A–GATA1 interaction. GATA1–SIX1 promotes transcription of grainyhead-like protein 2 homolog and circTNRC18-mediated regulation of cell migration and invasion. Moreover, blocking circTNRC18–LIN28A interaction with antisense nucleotides alleviates PE in a mouse model of reduced uterine perfusion pressure. Thus, targeting the circTNRC18–LIN28A regulatory axis may be a novel PE treatment method.

RNA in situ conformation sequencing reveals novel long-range RNA structures with impact on splicing.

Over recent years, long-range RNA structure has emerged as a factor that is fundamental to alternative splicing regulation. An increasing number of human disorders are now being associated with splicing defects; hence it is essential to develop methods that assess long-range RNA structure experimentally. RNA in situ conformation sequencing (RIC-seq) is a method that recapitulates RNA structure within physiological RNA-protein complexes. In this work, we juxtapose pairs of conserved complementary regions (PCCRs) that were predicted in silico with the results of RIC-seq experiments conducted in seven human cell lines. We show statistically that RIC-seq support of PCCRs correlates with their properties, such as equilibrium free energy, presence of compensatory substitutions, and occurrence of A-to-I RNA editing sites and forked eCLIP peaks. Exons enclosed in PCCRs that are supported by RIC-seq tend to have weaker splice sites and lower inclusion rates, which is indicative of post-transcriptional splicing regulation mediated by RNA structure. Based on these findings, we prioritize PCCRs according to their RIC-seq support and show, using antisense nucleotides and minigene mutagenesis, that PCCRs in two disease-associated human genes, PHF20L1 and CASK, and also PCCRs in their murine orthologs, impact alternative splicing. In sum, we demonstrate how RIC-seq experiments can be used to discover functional long-range RNA structures, and particularly those that regulate alternative splicing.

Overexpression of Pericentromeric HSAT2 DNA Increases Expression of EMT Markers in Human Epithelial Cancer Cell Lines.

Pericentromeric tandemly repeated DNA of human satellites 1, 2, and 3 (HS1, HS2, and HS3) is actively transcribed in some cells. However, the functionality of the transcription remains obscure. Studies in this area have been hampered by the absence of a gapless genome assembly. The aim of our study was to map a transcript that we have previously described as HS2/HS3 on chromosomes using a newly published gapless genome assembly T2T-CHM13, and create a plasmid overexpressing the transcript to assess the influence of HS2/HS3 transcription on cancer cells. We report here that the sequence of the transcript is tandemly repeated on nine chromosomes (1, 2, 7, 9, 10, 16, 17, 22, and Y). A detailed analysis of its genomic localization and annotation in the T2T-CHM13 assembly revealed that the sequence belonged to HSAT2 (HS2) but not to the HS3 family of tandemly repeated DNA. The transcript was found on both strands of HSAT2 arrays. The overexpression of the HSAT2 transcript increased the transcription of the genes encoding the proteins involved in the epithelial-to-mesenchymal transition, EMT (SNAI1, ZEB1, and SNAI2), and the genes that mark cancer-associated fibroblasts (VIM, COL1A1, COL11A1, and ACTA2) in cancer cell lines A549 and HeLa. Co-transfection of the overexpression plasmid and antisense nucleotides eliminated the transcription of EMT genes observed after HSAT2 overexpression. Antisense oligonucleotides also decreased transcription of the EMT genes induced by tumor growth factor beta 1 (TGFβ1). Thus, our study suggests HSAT2 lncRNA transcribed from the pericentromeric tandemly repeated DNA is involved in EMT regulation in cancer cells.

Epilepsy in Dravet Syndrome—Current and Future Therapeutic Opportunities

Dravet Syndrome (DS) is a developmental epileptic encephalopathy characterized by drug-resistant seizures and other clinical features, including intellectual disability and behavioral, sleep, and gait problems. The pathogenesis is strongly connected to voltage-gated sodium channel dysfunction. The current consensus of seizure management in DS consists of a combination of conventional and recently approved drugs such as stiripentol, cannabidiol, and fenfluramine. Despite promising results in randomized clinical trials and extension studies, the prognosis of the developmental outcomes of patients with DS remains unfavorable. The article summarizes recent changes in the therapeutic approach to DS and discusses ongoing clinical research directions. Serotonergic agents under investigation show promising results and may replace less DS-specific medicines. The use of antisense nucleotides and gene therapy is focused not only on symptom relief but primarily addresses the underlying cause of the syndrome. Novel compounds, after expected safe and successful implementation in clinical practice, will open a new era for patients with DS. The main goal of causative treatment is to modify the natural course of the disease and provide the best neurodevelopmental outcome with minimum neurological deficit.

An overview of structural approaches to study therapeutic RNAs.

RNAs provide considerable opportunities as therapeutic agent to expand the plethora of classical therapeutic targets, from extracellular and surface proteins to intracellular nucleic acids and its regulators, in a wide range of diseases. RNA versatility can be exploited to recognize cell types, perform cell therapy, and develop new vaccine classes. Therapeutic RNAs (aptamers, antisense nucleotides, siRNA, miRNA, mRNA and CRISPR-Cas9) can modulate or induce protein expression, inhibit molecular interactions, achieve genome editing as well as exon-skipping. A common RNA thread, which makes it very promising for therapeutic applications, is its structure, flexibility, and binding specificity. Moreover, RNA displays peculiar structural plasticity compared to proteins as well as to DNA. Here we summarize the recent advances and applications of therapeutic RNAs, and the experimental and computational methods to analyze their structure, by biophysical techniques (liquid-state NMR, scattering, reactivity, and computational simulations), with a focus on dynamic and flexibility aspects and to binding analysis. This will provide insights on the currently available RNA therapeutic applications and on the best techniques to evaluate its dynamics and reactivity.

O012 The role of the anti-miR-135/Spock1 axis on metabolism and drug response in intestinal/colon tumour organoids

Abstract Introduction Little is known about the role of microRNAs (miRNAs) in rewiring the metabolism within tumours and adjacent non-tumour bearing normal tissue and their potential in cancer therapy. This study aimed to investigate the relationship between deregulated miRNAs and metabolic components in murine duodenal polyps and non-polyp-derived organoids (mPOs and mNPOs) from a double mutant Apc/Fbxw7 mouse model of intestinal/colorectal cancer (CRC). Methods We measured glucose consumption and lactate production, and analysed the expression of 373 miRNAs and 12 metabolic genes using qRT-PCR and Western blotting in mPOs and mNPOs. The overexpressed miRNAs systematically target metabolic genes explored by the TargetScan web server. Organoid viability was assessed using MTT and Sulforhodamine-B (SRB) colorimetric assays. Results The tumour and adjacent non-tumour organoids showed differential metabolic activity. mPOs showed elevated lactate and consumption of glucose in the medium. Murine Apc/Fbxw7 duodenal polyp-derived organoids mimicked colorectal cancer patient-derived organoids. Our findings revealed miR-135b might target Spock1. Upregulation of SPOCK1 correlated with advanced stages of CRCs. Knockdown of miR-135b decreased the expression level of SPOCK1, glucose consumption and lactic secretion in CRC patient-derived tumours organoids (CRC tPDOs). Increased SPOCK1 induced by miR-135b overexpression promoted the Warburg effect and consequently antitumour effect of 5-fluorouracil. Conclusion Combination with miR-135b antisense nucleotides may represent a novel strategy to sensitise CRC to the chemo-reagent based treatment. Take-home message Our data highlight the therapeutic potential of targeting miR-135/SPOCK1 to enhance the treatment of colorectal cancer.

Genetically modified animal models of hereditary diseases for testing of gene-directed therapy

Disease-causing genes have been identified for many severe muscular and neurological genetic disorders. Advances in the gene therapy field offer promising solutions for drug development to treat these life-threatening conditions. Depend­ing on how the mutation affects the function of the gene product, different gene therapy approaches may be beneficial. Gene replacement therapy is appropriate for diseases caused by mutations that result in the deficiency of the functional protein. Gene suppression strategy is suggested for disorders caused by the toxic product of the mutant gene. Splicing modulators, genome editing, and base editing techniques can be applied to disorders with different types of underlying mutations. Testing potential drugs in animal models of human diseases is an indispensable step of development. Given the specific gene therapy approach, appropriate animal models can be generated using a variety of technologies ranging from transgenesis to precise genome editing. In this review, we discuss technologies used to generate small and large animal models of the most common muscular and neurological genetic disorders. We specifically focus on animal mod­els that were used to test gene therapies based on adeno-associated vectors and antisense nucleotides.

Targeted Molecular Strategies for Genetic Neurodevelopmental Disorders: Emerging Lessons from Dravet Syndrome.

Dravet syndrome is a severe developmental and epileptic encephalopathy mostly caused by heterozygous mutation of the SCN1A gene encoding the voltage-gated sodium channel α subunit Nav1.1. Multiple seizure types, cognitive deterioration, behavioral disturbances, ataxia, and sudden unexpected death associated with epilepsy are a hallmark of the disease. Recently approved antiseizure medications such as fenfluramine and cannabidiol have been shown to reduce seizure burden. However, patients with Dravet syndrome are still medically refractory in the majority of cases, and there is a high demand for new therapies aiming to improve behavioral and cognitive outcome. Drug-repurposing approaches for SCN1A-related Dravet syndrome are currently under investigation (i.e., lorcaserin, clemizole, and ataluren). New therapeutic concepts also arise from the field of precision medicine by upregulating functional SCN1A or by activating Nav1.1. These include antisense nucleotides directed against the nonproductive transcript of SCN1A with the poison exon 20N and against an inhibitory noncoding antisense RNA of SCN1A. Gene therapy approaches such as adeno-associated virus-based upregulation of SCN1A using a transcriptional activator (ETX101) or CRISPR/dCas technologies show promising results in preclinical studies. Although these new treatment concepts still need further clinical research, they offer great potential for precise and disease modifying treatment of Dravet syndrome.

RNA Therapeutics: the Next Generation of Drugs for Cardiovascular Diseases.

Purpose of ReviewRNA therapeutics are a new and rapidly expanding class of drugs to prevent or treat a wide spectrum of diseases. We discuss the defining characteristics of the diverse family of molecules under the RNA therapeutics umbrella.Recent FindingsRNA therapeutics are designed to regulate gene expression in a transient manner. For example, depending upon the strategy employed, RNA therapies offer the versatility to replace, supplement, correct, suppress, or eliminate the expression of a targeted gene. RNA therapies include antisense nucleotides, microRNAs and small interfering RNAs, RNA aptamers, and messenger RNAs. Further, we discuss the mechanism(s) by which different RNA therapies either reduce or increase the expression of their targets.SummaryWe review the RNA therapeutics approved (and those in trials) to treat cardiovascular indications. RNA-based therapeutics are a new, rapidly growing class of drugs that will offer new alternatives for an increasing array of cardiovascular conditions.

Anti-miR-135/SPOCK1 axis antagonizes the influence of metabolism on drug response in intestinal/colon tumour organoids

Little is known about the role of microRNAs (miRNAs) in rewiring the metabolism within tumours and adjacent non-tumour bearing normal tissue and their potential in cancer therapy. This study aimed to investigate the relationship between deregulated miRNAs and metabolic components in murine duodenal polyps and non-polyp-derived organoids (mPOs and mNPOs) from a double-mutant ApcMinFbxw7∆G mouse model of intestinal/colorectal cancer (CRC). We analysed the expression of 373 miRNAs and 12 deregulated metabolic genes in mPOs and mNPOs. Our findings revealed miR-135b might target Spock1. Upregulation of SPOCK1 correlated with advanced stages of CRCs. Knockdown of miR-135b decreased the expression level of SPOCK1, glucose consumption and lactic secretion in CRC patient-derived tumours organoids (CRC tPDOs). Increased SPOCK1 induced by miR-135b overexpression promoted the Warburg effect and consequently antitumour effect of 5-fluorouracil. Thus, combination with miR-135b antisense nucleotides may represent a novel strategy to sensitise CRC to the chemo-reagent based treatment.

Potential Genes Associated with COVID-19 and Comorbidity

Hypertension, diabetes mellitus, and coronary artery disease are common comorbidities and dangerous factors for infection and serious COVID-19. Polymorphisms in genes associated with comorbidities may help observe susceptibility and disease severity variation. However, specific genetic factors and the extent to which they can explain variation in susceptibility of severity are unclear. Therefore, we evaluated candidate genes associated with COVID-19 and hypertension, diabetes mellitus, and coronary artery disease. In particular, we performed searches against OMIM, NCBI, and other databases, protein-protein interaction network construction, and GO and KEGG pathway enrichment analyses. Results showed that the associated overlapping genes were TLR4, NLRP3, MBL2, IL6, IL1RN, IL1B, CX3CR1, CCR5, AGT, ACE, and F2. GO and KEGG analyses yielded 302 GO terms (q < 0.05) and 29 signaling pathways (q < 0.05), respectively, mainly including coronavirus disease-COVID-19 and cytokine-cytokine receptor interaction. IL6 and AGT were central in the PPI, with 8 and 5 connections, respectively. In this study, we identified 11 genes associated with both COVID-19 and three comorbidities that may contribute to infection and disease severity. The key genes IL6 and AGT are involved in regulating immune response, cytokine activity, and viral infection. Therefore, RAAS inhibitors, AGT antisense nucleotides, cytokine inhibitors, vitamin D, fenofibrate, and vaccines regulating non-immune and immune factors could be potential strategies to prevent and cure COVID-19. The study provides a basis for further investigation of genes and pathways with predictive value for the risk of infection and prognosis and could help guide drug and vaccine development to improve treatment efficacy and the development of personalised treatments, especially for COVID-19 individuals with common comorbidities.

Selective Inhibition of STAT6 With Antisense Nucleotides Enhances Systemic Antitumor Effect of Hypofractionated Radiotherapy and Anti-PD1 in Metastatic Non-Small Cell Lung Cancer

Despite advances in radiotherapy (RT) and immunotherapy, their antitumor activity remains suboptimal in metastatic non-small cell lung cancer (NSCLC). The tumor microenvironment after hypofractionated RT (hRT) is largely immunosuppressive and replete with M2 tumor-associated macrophages. STAT6 is a highly activated transcription factor that contributes to M2 macrophage polarization but has been difficult to target therapeutically. We selectively targeted STAT6 with antisense nucleotides (ASOs), hypothesizing that using ASOs to inhibit STAT6 in combination with hRT and anti-PD1 would inhibit M2 polarization and improve systemic antitumor effects in NSCLC.We tested three bilaterally established lung cancer murine models: Lewis lung carcinoma in B6.Foxp3EGFP mice, and 344SQ-parental or 344SQ anti-PD1-resistant adenocarcinomas in 129Sv/Ev mice. The primary tumor was treated with hRT (3 fractions of 12 Gy each) and secondary tumors were observed for evidence of abscopal effects. STAT6 knockdown was determined by Western blotting and qRT-PCR. Tumor-infiltrating leukocytes were characterized by immunohistochemical tissue microarray-based immunofluorescence, flow cytometry, and 770 gene-Immune NanoString analyses. Also, serum TGF-β was measured by ELISA.The combination of hRT and STAT6 ASOs reduced the volumes of both primary and secondary tumors and extended survival (vs. either treatment alone). The addition of anti-PD1 enhanced the systemic antitumor effect (vs. STAT6 ASOs + hRT or hRT+anti-PD1) and led to decreased STAT6, increased M1 genes (IL1R, iNOS, CD80, CD86), and lowered M2 genes (Arg-1, Fizz-1, CD163, CCR2) in tumor-infiltrating leukocytes by qRT-PCR. Treatment with hRT+STAT6 ASOs ± anti-PD1 also enhanced tumor infiltration by macrophages and Th1 cells, improved T-cell and macrophage function, and slowed cancer progression. Flow cytometry and tissue microarray confirmed the hRT (vs. control) led to higher STAT6 and lower M1/M2 ratio, but hRT+ STAT6 ASOs ± anti-PD1 led to decreased STAT6, reduced Tregs, increased M1/M2 ratio, and lowered TGF-β levels. Depletion of macrophages by anti-F4/80 abrogated the observed antitumor effect.The combination of STAT6 ASOs and hRT reduces immunosuppressive M2 macrophage infiltration into tumors and enhances the systemic antitumor effect of hRT; this radio-immunotherapy combination may provide an alternative therapeutic approach for NSCLC patients with PD1-resistance.

Selected Molecular Targets for Antiepileptogenesis.

The term epileptogenesis defines the usually durable process of converting normal brain into an epileptic one. The resistance of a significant proportion of patients with epilepsy to the available pharmacotherapy prompted the concept of a causative treatment option consisting in stopping or modifying the progress of epileptogenesis. Most antiepileptic drugs possess only a weak or no antiepileptogenic potential at all, but a few of them appear promising in this regard; these include, for example, eslicarbazepine (a sodium and T-type channel blocker), lamotrigine (a sodium channel blocker and glutamate antagonist) or levetiracetam (a ligand of synaptic vehicle protein SV2A). Among the approved non-antiepileptic drugs, antiepileptogenic potential seems to reside in losartan (a blocker of angiotensin II type 1 receptors), biperiden (an antiparkinsonian drug), nonsteroidal anti-inflammatory drugs, antioxidative drugs and minocycline (a second-generation tetracycline with anti-inflammatory and antioxidant properties). Among other possible antiepileptogenic compounds, antisense nucleotides have been considered, among these an antagomir targeting microRNA-134. The drugs and agents mentioned above have been evaluated in post-status epilepticus models of epileptogenesis, so their preventive efficacy must be verified. Limited clinical data indicate that biperiden in patients with brain injuries is well-tolerated and seems to reduce the incidence of post-traumatic epilepsy. Exceptionally, in this regard, our own original data presented here point to c-Fos as an early seizure duration, but not seizure intensity-related, marker of early epileptogenesis. Further research of reliable markers of early epileptogenesis is definitely needed to improve the process of designing adequate antiepileptogenic therapies.