Oncogenic Role of THOR, a Conserved Cancer/Testis Long Non-coding RNA.

Large-scale transcriptome sequencing efforts have vastly expanded the catalog of long non-coding RNAs (lncRNAs) with varying evolutionary conservation, lineage expression, and cancer specificity. Here, we functionally characterize a novel ultraconserved lncRNA, THOR (ENSG00000226856), which exhibits expression exclusively in testis and a broad range of human cancers. THOR knockdown and overexpression in multiple cell lines and animal models alters cell or tumor growth supporting an oncogenic role. We discovered a conserved interaction of THOR with IGF2BP1 and show that THOR contributes to the mRNA stabilization activities of IGF2BP1. Notably, transgenic THOR knockout produced fertilization defects in zebrafish and also conferred a resistance to melanoma onset. Likewise, ectopic expression of human THOR in zebrafish accelerated the onset of melanoma. THOR represents a novel class of functionally important cancer/testis lncRNAs whose structure and function have undergone positive evolutionary selection.

A functional interplay between the two BMP-SMAD pathway inhibitors TMPRSS6 and FKBP12 regulates hepcidin expression in vivo.

The Activin A Receptor type I (ALK2) is a critical component of BMP-SMAD signaling that, in the presence of ligands, phosphorylates cytosolic SMAD1/5/8 and modulates important biological processes, including bone formation and iron metabolism. In hepatocytes, the BMP-SMAD pathway controls the expression of hepcidin, the liver peptide hormone that regulates body iron homeostasis via the BMP receptors ALK2 and ALK3, and the hemochromatosis proteins. The main negative regulator of the pathway in the liver is transmembrane serine protease 6 (TMPRSS6), which downregulates hepcidin by cleaving the BMP coreceptor hemojuvelin. ALK2 function is inhibited also by the immunophilin FKBP12, which maintains the receptor in an inactive conformation. FKBP12 sequestration by tacrolimus or its silencing upregulates hepcidin in primary hepatocytes and in vivo in acute but not chronic settings. Interestingly, gain-of-function mutations in ALK2 that impair FKBP12 binding to the receptor and activate the pathway cause a bone phenotype in patients affected by Fibrodysplasia Ossificans Progressiva but not hepcidin and iron metabolism dysfunction. This observation suggests that additional mechanisms are active in the liver to compensate for the increased BMP-SMAD signaling. Here we demonstrate that Fkbp12 downregulation in hepatocytes by antisense oligonucleotide treatment upregulates the expression of the main hepcidin inhibitor Tmprss6, thus counteracting the ALK2-mediated activation of the pathway. Combined downregulation of both Fkbp12 and Tmprss6 blocks this compensatory mechanism. Our findings reveal a previously unrecognized functional cross talk between FKBP12 and TMPRSS6, the main BMP-SMAD pathway inhibitors, in the control of hepcidin transcription.NEW & NOTEWORTHY This study uncovers a previously unrecognized mechanism of hepcidin and BMP-SMAD pathway regulation in hepatocytes mediated by the immunophilin FKBP12 and the transmembrane serine protease TMPRSS6.

Combination of a TGF-ß ligand trap (RAP-GRL) and TMPRSS6-ASO is superior for correcting ß-thalassemia.

A recently approved drug that induces erythroid cell maturation (luspatercept) has been shown to improve anemia and reduce the need for blood transfusion in non-transfusion-dependent as well as transfusion-dependent ß-thalassemia (BT) patients. Although these results were predominantly positive, not all the patients showed the expected increase in hemoglobin (Hb) levels or transfusion burden reduction. Additional studies indicated that administration of luspatercept in transfusion-dependent BT was associated with increased erythropoietic markers, decreased hepcidin levels, and increased liver iron content. Altogether, these studies suggest that luspatercept may necessitate additional drugs for improved erythroid and iron management. As luspatercept does not appear to directly affect iron metabolism, we hypothesized that TMPRSS6-ASO could improve iron parameters and iron overload when co-administered with luspatercept. We used an agent analogous to murine luspatercept (RAP-GRL) and another novel therapeutic, IONIS TMPRSS6-LRx (TMPRSS6-ASO), a hepcidin inducer, to treat non-transfusion-dependent BT-intermedia mice. Our study shows that RAP-GRL alone improved red blood cell (RBC) production, with no or limited effect on splenomegaly and iron parameters. In contrast, TMPRSS6-ASO improved RBC measurements, ameliorated splenomegaly, and improved iron overload most effectively. Our results provide pre-clinical support for combining TMPRSS6-ASO and luspatercept in treating BT, as these drugs together show potential for simultaneously improving both erythroid and iron parameters in BT patients.

A single approach to targeting transferrin receptor 2 corrects iron and erythropoietic defects in murine models of anemia of inflammation and chronic kidney disease.

Anemia is a common complication of systemic inflammation. Proinflammatory cytokines both decrease erythroblast sensitivity to erythropoietin (EPO) and increase the levels of the hepatic hormone hepcidin, sequestering iron in stores and causing functional iron deficiency. Anemia of chronic kidney disease (CKD) is a peculiar form of anemia of inflammation, characterized by impaired EPO production paralleling progressive kidney damage. Traditional therapy based on increased EPO (often in combination with iron) may have off-target effects due to EPO interaction with its non-erythroid receptors. Transferrin Receptor 2 (Tfr2) is a mediator of the iron-erythropoiesis crosstalk. Its deletion in the liver hampers hepcidin production, increasing iron absorption, whereas its deletion in the hematopoietic compartment increases erythroid EPO sensitivity and red blood cell production. Here, we show that selective hematopoietic Tfr2 deletion ameliorates anemia in mice with sterile inflammation in the presence of normal kidney function, promoting EPO responsiveness and erythropoiesis without increasing serum EPO levels. In mice with CKD, characterized by absolute rather than functional iron deficiency, Tfr2 hematopoietic deletion had a similar effect on erythropoiesis but anemia improvement was transient because of limited iron availability. Also, increasing iron levels by downregulating only hepatic Tfr2 had a minor effect on anemia. However, simultaneous deletion of hematopoietic and hepatic Tfr2, stimulating erythropoiesis and increased iron supply, was sufficient to ameliorate anemia for the entire protocol. Thus, our results suggest that combined targeting of hematopoietic and hepatic Tfr2 may be a therapeutic option to balance erythropoiesis stimulation and iron increase, without affecting EPO levels.

Mediator1 involved in functional integration of Smad3 and Notch1 promoting enamel mineralization.

Enamel hypoplasia is a tooth development defection due to the disruption of enamel matrix mineralization, manifesting as chalky white phenotype. Multiple genes may be involved in this tooth agenesis. It has been proved that ablation of coactivator Mediator1 (Med1) switches the cell fate of dental epithelia, resulting in abnormal tooth development via Notch1 signaling. Smad3 (-/-) mice displays the similar chalky white incisors. However, the expression of Smad3 in Med1 ablation mice and the impact of Med1 on functional integration between Smad3 and Notch1 remains unclear. Cre-loxP-based C57/BL6 mice with epithelial-specific Med1 knockout (Med1 KO) backgrounds were generated. Mandibles and dental epithelial stem cells (DE-SCs) from incisors cervical loop (CL) were isolated from wild-type (CON) mice and Med1 KO mice. Transcriptome sequencing was used to analyze the differences of CL tissue between KO and CON mice. The results revealed the enrichment of TGF-ß signaling pathway. qRT-PCR and western blot were performed to show the gene and protein expression of Smad3, pSmad3, Notch1 and NICD, the key regulators of TGF-ß and Notch1 signaling pathway. Expression of Notch1 and Smad3 was confirmed to be down-regulated in Med1 KO cells. Using activators of Smad3 and Notch1 on Med1 KO cells, both pSmad3 and NICD were rescued. Moreover, adding inhibitors and activators of Smad3 and Notch1 to cells of CON groups respectively, the protein expressions of Smad3, pSmad3, Notch1 and NICD were synergistically affected. In summary, Med1 participates in the functional integration of Smad3 and Notch1, thus promoting enamel mineralization.

HOTAIR lncRNA promotes epithelial-mesenchymal transition by redistributing LSD1 at regulatory chromatin regions.

Epithelial-to-mesenchymal transition (EMT) describes the loss of epithelial traits and gain of mesenchymal traits by normal cells during development and by neoplastic cells during cancer metastasis. The long noncoding RNA HOTAIR triggers EMT, in part by serving as a scaffold for PRC2 and thus promoting repressive histone H3K27 methylation. In addition to PRC2, HOTAIR interacts with the LSD1 lysine demethylase, an epigenetic regulator of cell fate during development and differentiation, but little is known about the role of LSD1 in HOTAIR function during EMT. Here, we show that HOTAIR requires its LSD1-interacting domain, but not its PRC2-interacting domain, to promote the migration of epithelial cells. This activity is suppressed by LSD1 overexpression. LSD1-HOTAIR interactions induce partial reprogramming of the epithelial transcriptome altering LSD1 distribution at promoter and enhancer regions. Thus, we uncover an unexpected role of HOTAIR in EMT as an LSD1 decommissioning factor, counteracting its activity in the control of epithelial identity.

Heat Shock Proteins in Tooth Development and Injury Repair.

Heat shock proteins (HSPs) are a class of molecular chaperones with expression increased in response to heat or other stresses. HSPs regulate cell homeostasis by modulating the folding and maturation of intracellular proteins. Tooth development is a complex process that involves many cell activities. During tooth preparation or trauma, teeth can be damaged. The damaged teeth start their repair process by remineralizing and regenerating tissue. During tooth development and injury repair, different HSPs have different expression patterns and play a special role in odontoblast differentiation and ameloblast secretion by mediating signaling pathways or participating in protein transport. This review explores the expression patterns and potential mechanisms of HSPs, particularly HSP25, HSP60 and HSP70, in tooth development and injury repair.

[Dietary micronutrient intake of children aged 2-5 years in China in 2019-2021].

OBJECTIVE: To describe dietary intake of calcium, iron, zinc, selenium, vitamin A, vitamin B_1, vitamin B_2 and vitamin C and compare the intake between urban and rural areas among preschool children aged 2-5 years based on the data from the National Nutrition and Health Systematic Survey for Children 0-17 Years of Age in China. METHODS: Children from 14 provinces were selected by multi-stage stratified random cluster sampling, and the dietary data of preschool children aged 2-5 years were recorded using the 3 day 24-hour weighted food records method. SAS 9.4 was used to calculate dietary intake of these micronutrients based on the Chinese Food Composition Table and to compare the intake between urban and rural areas. The risk of insufficient or excessive intake of micronutrients among Chinese children aged 2-5 years was assessed according to the Chinese Dietary Reference Intakes(DRIs) 2013 edition. RESULTS: A total of 820 children aged 2 to 5 years were selected. The median daily dietary calcium intake of children aged 2-5 years in China was 433.7, 338.9, 356.4 and 347.4 mg, respectively. The median daily dietary intake of vitamin B_1 of children aged 2-5 years was 0.5 mg for all age groups. The median daily dietary vitamin B_2 intake of children aged 2-5 years was 0.7, 0.6, 0.6 and 0.6 mg, respectively. The median daily dietary intake of vitamin C of children aged 2-5 years was 37.9, 37.4, 44.0 and 40.0 mg, respectively. The median daily dietary selenium intake of children aged 2-5 years was 17.1, 20.5, 22.7 and 22.3 µg, respectively. Dietary calcium intake for aged 2-5 years, dietary vitamin B_2 intake for aged 2-4 years, dietary iron, selenium, zinc and vitamin B_1 intake for aged 2-3 years was significantly greater in urban children than rural children. Among all nutrients, the proportion of dietary calcium intakes below the estimated average requirement(EAR) was the highest in aged 2-5 years(61.4%, 76.4%, 91.4% and 91.5%, respectively). The proportions of dietary vitamin B_1, vitamin C and selenium intake lower than EAR of children aged 2-5 years in China were 52.4%-63.2%, 42.8%-50.2% and 46.6%-58.7%, respectively. COUCLUSION: The dietary calcium intake of children aged 2-5 years in China remains insufficient for these children, especially for rural children. Dietary vitamin B_1, vitamin C and selenium intake should be improved.

Correcting ß-thalassemia by combined therapies that restrict iron and modulate erythropoietin activity.

ß-Thalassemia intermedia is a disorder characterized by ineffective erythropoiesis (IE), anemia, splenomegaly, and systemic iron overload. Novel approaches are being explored based on the modulation of pathways that reduce iron absorption (ie, using hepcidin activators like Tmprss6-antisense oligonucleotides [ASOs]) or increase erythropoiesis (by erythropoietin [EPO] administration or modulating the ability of transferrin receptor 2 [Tfr2] to control red blood cell [RBC] synthesis). Targeting Tmprss6 messenger RNA by Tmprss6-ASO was proven to be effective in improving IE and splenomegaly by inducing iron restriction. However, we postulated that combinatorial strategies might be superior to single therapies. Here, we combined Tmprss6-ASO with EPO administration or removal of a single Tfr2 allele in the bone marrow of animals affected by ß-thalassemia intermedia (Hbbth3/+). EPO administration alone or removal of a single Tfr2 allele increased hemoglobin levels and RBCs. However, EPO or Tfr2 single-allele deletion alone, respectively, exacerbated or did not improve splenomegaly in ß-thalassemic mice. To overcome this issue, we postulated that some level of iron restriction (by targeting Tmprss6) would improve splenomegaly while preserving the beneficial effects on RBC production mediated by EPO or Tfr2 deletion. While administration of Tmprss6-ASO alone improved the anemia, the combination of Tmprss6-ASO + EPO or Tmprss6-ASO + Tfr2 single-allele deletion produced significantly higher hemoglobin levels and reduced splenomegaly. In conclusion, our results clearly indicate that these combinatorial approaches are superior to single treatments in ameliorating IE and anemia in ß-thalassemia and could provide guidance to translate some of these approaches into viable therapies.

Population pharmacokinetic/pharmacodynamic modelling of eplontersen, an antisense oligonucleotide in development for transthyretin amyloidosis.

AIMS: Transthyretin-mediated amyloidosis is a progressive and fatal disease caused by the build-up of misfolded transthyretin (TTR) protein. Eplontersen is a triantennary N-acetyl galactosamine (GalNAc3)-conjugated antisense oligonucleotide targeting TTR messenger ribonucleic acid (mRNA) to inhibit production of both variant and wild-type TTR. We aimed to develop a population pharmacokinetic/pharmacodynamic (PK/PD) model for eplontersen and to evaluate the impact of covariates on exposure and response. METHODS: Plasma eplontersen and serum TTR concentration data were obtained from two phase 1 studies in healthy volunteers (ClinicalTrials.gov: NCT03728634, NCT04302064). Model development was conducted using a nonlinear mixed-effects approach. RESULTS: Eplontersen PK was well described by a two-compartment model. Evaluation of demographics identified significant covariates of lean body mass on clearance and body weight on intercompartmental clearance and volumes of distribution. Population PK modelling showed the absorption rate was 29.6% greater with injection into the abdomen versus the arm. The typical population terminal elimination half-life was 25.5 days. Serum TTR was well described by an indirect response model with inhibition of TTR production by eplontersen. Maximum fractional inhibition (Imax ) was 0.970 (0.549%RSE) and the half maximal inhibitory concentration (IC50 ) was 0.0283 ng/ml (13.3%RSE). Simulations showed subjects with lower weight had higher exposure (AUC, Cmax ), while higher Cmax was observed when comparing site of administration (ratio abdomen/arm = 1.18), but differences in exposure did not significantly impact response at evaluated doses. CONCLUSION: The exposure-response relationship of eplontersen was well characterised by the PKPD model. Weight and injection site were found to affect systemic exposure, but this effect does not seem to result in clinically relevant variation in response.

MED1 Ablation Promotes Oral Mucosal Wound Healing via JNK Signaling Pathway.

Mediator complex subunit 1 (MED1) is a coactivator of multiple transcription factors and plays a key role in regulating epidermal homeostasis as well as skin wound healing. It is unknown, however, whether it plays a role in healing oral mucosal wounds. In this study, we investigate MED1's functional effects on oral mucosal wound healing and its underlying mechanism. The epithelial-specific MED1 null (Med1epi-/-) mice were established using the Cre-loxP system with C57/BL6 background. A 3 mm diameter wound was made in the cheek mucosa of the 8-week-old mice. In vivo experiments were conducted using HE staining and immunostaining with Ki67 and uPAR antibodies. The in vitro study used lentiviral transduction, scratch assays, qRT-PCR, and Western blotting to reveal the underlying mechanisms. The results showed that ablation of MED1 accelerated oral mucosal wound healing in 8-week-old mice. As a result of ablation of MED1, Activin A/Follistatin expression was altered, resulting in an activation of the JNK/c-Jun pathway. Similarly, knockdown of MED1 enhanced the proliferation and migration of keratinocytes in vitro, promoting re-epithelialization, which accelerates the healing of oral mucosal wounds. Our study reveals a novel role for MED1 in oral keratinocytes, providing a new molecular therapeutic target for accelerated wound healing.

[Relationship between sugary food intake and myopia in 11-14 years old Chinese children in 2019-2021].

OBJECTIVE: To investigate the relationship between the intake of sugary foods and the occurrence and development of myopia in children aged 11-14 in China. METHODS: In the 28 urban and rural survey sites in 14 provinces that implemented the "China Children's Nutrition and Health System Survey and Application for 0-18 Years Old" project, a multi-stage stratified random cluster sampling method was adopted, and finally 12 397 adolescent children aged 11 to 14 were selected in the analysis. Demographic characteristics, myopia information and the intake of sugary food(cakes, preserved fruits, candies, chocolates and ice cream) were collected through questionnaires. Multifactor Logistic regression was used to analyze the relationship between sugary food intake and myopia in children. RESULTS: The median daily intake of sugary foods(cakes, preserved fruits, candies, chocolates and ice cream)of 11-14 year old boys and girls in China was 11.4 g and 11.2 g respectively, 33.9% of boys and 34.2% of girls consumed ≥22 g of sugary food every day. The myopia rates of boys with intakes of <2 g, 2-21 g and ≥22 g were 36.0%, 39.2% and 41.6%, and girls' myopia rates were 45.9%, 51.8% and 55.1%. The result of Logistic regression analysis showed that after controlling for confounding factors such as age, gender, region, weekly high-intensity physical activity time, daily screen time, daily sleep time and daily intake of sugary beverages, compared with boys whose daily intake of sugary food was less than 2 g, the risk of myopia for boys whose daily intake of sugary food reached 2-21 g and ≥22 g was 1.18 and 1.23 times, for girls whose daily intake was less than 2 g, the risk of myopia was 1.27 times and 1.38 times for girls whose intake reached 2-21 g and ≥22 g(P<0.05). No matter whether confounding factors were controlled or not, there was no correlation between the intake of sugary foods and the degree of myopia(P>0.05). CONCLUSION: The consumption of sugary food such as cakes, preserved fruits, candies, chocolates and ice cream among children aged 11 to 14 in China is common, and there is a certain degree of positive correlation with the prevalence of myopia, but no correlation is observed with the degree of myopia.

Safety, Pharmacokinetic, and Pharmacodynamic Evaluation of a 2'-(2-Methoxyethyl)-D-ribose Antisense Oligonucleotide-Triantenarry N-Acetyl-galactosamine Conjugate that Targets the Human Transmembrane Protease Serine 6.

Cellular uptake of antisense oligonucleotides (ASOs) is one of the main determinants of in vivo activity and potency. A significant advancement in improving uptake into cells has come through the conjugation of ASOs to triantenarry N-acetyl-galactosamine (GalNAc3), a ligand for the asialoglycoprotein receptor on hepatocytes. The impact for antisense oligonucleotides, which are already taken up into hepatocytes, is a 10-fold improvement in potency in mice and up to a 30-fold potency improvement in humans, resulting in overall lower effective dose and exposure levels. 2'-Methoxyethyl-modified antisense oligonucleotide conjugated to GalNAc3 (ISIS 702843) is specific for human transmembrane protease serine 6 and is currently in clinical trials for the treatment of ß-thalassemia. This report summarizes a chronic toxicity study of ISIS 702843 in nonhuman primates (NHPs), including pharmacokinetic and pharmacology assessments. Suprapharmacologic doses of ISIS 702843 were well tolerated in NHPs after chronic dosing, and the data indicate that the overall safety profile is very similar to that of the unconjugated 2'-(2-methoxyethyl)-D-ribose (2'-MOE) ASOs. Notably, the GalNAc3 moiety did not cause any new toxicities nor exacerbate the known nonspecific class effects of the 2'-MOE ASOs. This observation was confirmed with multiple GalNAc3-MOE conjugates by querying a data base of monkey studies containing both GalNAc3-conjugated and unconjugated 2'-MOE ASOs. SIGNIFICANCE STATEMENT: This report documents the potency, pharmacology, and overall tolerability profile of a triantenarry N-acetyl-galactosamine (GalNAc3)-conjugated 2'-(2-methoxyethyl)-D-ribose (2'-MOE) antisense oligonucleotide (ASO) specific to transmembrane protease serine 6 after chronic treatment in the cynomolgus monkey. Collective analysis of 15 independent GalNAc3-conjugated and unconjugated 2'-MOE ASOs shows the consistency in the dose response and character of hepatic and platelet tolerability across sequences that will result in much larger safety margins for the GalNAc3-conjugated 2'-MOE ASOs when compared with the unconjugated 2'-MOE ASOs given the increased potency.

SERPINA1 modulates expression of amyloidogenic transthyretin.

Hereditary transthyretin amyloidosis (ATTR) is caused by amyloid deposition of misfolded transthyretin (TTR) in various tissues. Recently, reduction of circulating serum TTR, achieved via silencing oligonucleotides, was introduced as therapy of ATTR amyloidosis. We explored the impact of Serpin Family A Member 1 (SERPINA1) on TTR mRNA and protein expression. Oncostatin M (OSM) induced SERPINA1 in hepatoma cells and mice, while concomitantly TTR expression was significantly reduced. SERPINA1 knockdown resulted in specific elevated TTR expression in hepatoma cells; however other genes belonging to the group of acute phase proteins were unaffected. In mice, serum TTR was elevated after mSERPINA1 knockdown throughout antisense treatment. Following SERPINA1 knockdown, TTR deposition in several tissues, including dorsal root ganglia and intestine, was found to be increased, however numbers did not exceed significance levels. The data suggest that SERPINA1 is a co-factor of TTR expression. Our findings provide novel insight in the regulation of TTR and reveal a role of SERPINA1 in the pathogenesis of ATTR amyloidosis.

A novel and translational role for autophagy in antisense oligonucleotide trafficking and activity.

Endocytosis is a mechanism by which cells sense their environment and internalize various nutrients, growth factors and signaling molecules. This process initiates at the plasma membrane, converges with autophagy, and terminates at the lysosome. It is well-established that cellular uptake of antisense oligonucleotides (ASOs) proceeds through the endocytic pathway; however, only a small fraction escapes endosomal trafficking while the majority are rendered inactive in the lysosome. Since these pathways converge and share common molecular machinery, it is unclear if autophagy-related trafficking participates in ASO uptake or whether modulation of autophagy affects ASO activity and localization. To address these questions, we investigated the effects of autophagy modulation on ASO activity in cells and mice. We found that enhancing autophagy through small-molecule mTOR inhibition, serum-starvation/fasting, and ketogenic diet, increased ASO-mediated target reduction in vitro and in vivo. Additionally, autophagy activation enhanced the localization of ASOs into autophagosomes without altering intracellular concentrations or trafficking to other compartments. These results support a novel role for autophagy and the autophagosome as a previously unidentified compartment that participates in and contributes to enhanced ASO activity. Further, we demonstrate non-chemical methods to enhance autophagy and subsequent ASO activity using translatable approaches such as fasting or ketogenic diet.

Reducing dynamin 2 (DNM2) rescues DNM2-related dominant centronuclear myopathy.

Centronuclear myopathies (CNM) are a group of severe muscle diseases for which no effective therapy is currently available. We have previously shown that reduction of the large GTPase DNM2 in a mouse model of the X-linked form, due to loss of myotubularin phosphatase MTM1, prevents the development of the skeletal muscle pathophysiology. As DNM2 is mutated in autosomal dominant forms, here we tested whether DNM2 reduction can rescue DNM2-related CNM in a knock-in mouse harboring the p.R465W mutation (Dnm2RW/+) and displaying a mild CNM phenotype similar to patients with the same mutation. A single intramuscular injection of adeno-associated virus-shRNA targeting Dnm2 resulted in reduction in protein levels 5 wk post injection, with a corresponding improvement in muscle mass and fiber size distribution, as well as an improvement in histopathological CNM features. To establish a systemic treatment, weekly i.p. injections of antisense oligonucleotides targeting Dnm2 were administered to Dnm2RW/+mice for 5 wk. While muscle mass, histopathology, and muscle ultrastructure were perturbed in Dnm2RW/+mice compared with wild-type mice, these features were indistinguishable from wild-type mice after reducing DNM2. Therefore, DNM2 knockdown via two different strategies can efficiently correct the myopathy due to DNM2 mutations, and it provides a common therapeutic strategy for several forms of centronuclear myopathy. Furthermore, we provide an example of treating a dominant disease by targeting both alleles, suggesting that this strategy may be applied to other dominant diseases.

In Vitro and In Vivo Effects of SerpinA1 on the Modulation of Transthyretin Proteolysis.

Transthyretin (TTR) proteolysis has been recognized as a complementary mechanism contributing to transthyretin-related amyloidosis (ATTR amyloidosis). Accordingly, amyloid deposits can be composed mainly of full-length TTR or contain a mixture of both cleaved and full-length TTR, particularly in the heart. The fragmentation pattern at Lys48 suggests the involvement of a serine protease, such as plasmin. The most common TTR variant, TTR V30M, is susceptible to plasmin-mediated proteolysis, and the presence of TTR fragments facilitates TTR amyloidogenesis. Recent studies revealed that the serine protease inhibitor, SerpinA1, was differentially expressed in hepatocyte-like cells (HLCs) from ATTR patients. In this work, we evaluated the effects of SerpinA1 on in vitro and in vivo modulation of TTR V30M proteolysis, aggregation, and deposition. We found that plasmin-mediated TTR proteolysis and aggregation are partially inhibited by SerpinA1. Furthermore, in vivo downregulation of SerpinA1 increased TTR levels in mice plasma and deposition in the cardiac tissue of older animals. The presence of TTR fragments was observed in the heart of young and old mice but not in other tissues following SerpinA1 knockdown. Increased proteolytic activity, particularly plasmin activity, was detected in mice plasmas. Overall, our results indicate that SerpinA1 modulates TTR proteolysis and aggregation in vitro and in vivo.

Steric Inhibition of 5' UTR Regulatory Elements Results in Upregulation of Human CFTR.

Cystic fibrosis (CF) is an autosomal recessive monogenic disease caused by mutations in the CFTR gene. Therapeutic approaches that are focused on correcting CFTR protein face the challenge of the heterogeneity in CFTR mutations and resulting defects. Thus, while several small molecules directed at CFTR show benefit in the clinic for subsets of CF patients, these drugs cannot treat all CF patients. Additionally, the clinical benefit from treatment with these modulators could be enhanced with novel therapies. To address this unmet need, we utilized an approach to increase CFTR protein levels through antisense oligonucleotide (ASO)-mediated steric inhibition of 5' UTR regulatory elements. We identified ASOs to upregulate CFTR protein expression and confirmed the regulatory role of the sites amenable to ASO-mediated upregulation. Two ASOs were investigated further, and both increased CFTR protein expression and function in cell lines and primary human bronchial epithelial cells with distinct CF genotypes. ASO treatment further increased CFTR function in almost all CF genotypes tested on top of treatment with the FDA approved drug Symdeko (ivacaftor and tezacaftor). Thus, we present a novel approach to CFTR therapeutic intervention, through ASO-mediated modulation of translation.

MXD3 antisense oligonucleotide with superparamagnetic iron oxide nanoparticles: A new targeted approach for neuroblastoma.

Neuroblastoma (NB) is the most common extracranial solid tumor in children. The outcomes for aggressive forms of NB remain poor. The aim of this study was to develop a new molecular-targeted therapy for NB using an antisense oligonucleotide (ASO) and superparamagnetic iron oxide (SPIO) nanoparticles (NPs), as a delivery vehicle, targeting the transcription regulator MAX dimerization protein 3 (MXD3). We previously discovered that MXD3 was highly expressed in high-risk NB, acting as an anti-apoptotic factor; therefore, it can be a good therapeutic target. In this study, we developed two ASO-NP complexes using electrostatic conjugation to polyethylenimine-coated SPIO NPs and chemical conjugation to amphiphilic polymers on amine-functionalized SPIO NPs. Both ASO-NP complexes demonstrated MXD3 knockdown, which resulted in apoptosis in NB cells. ASO chemically-conjugated NP complexes have the potential to be used in the clinic as they showed great efficacy with minimum NP-associated cytotoxicity.