Suppression Of Target Genes Research Articles

Novel Methods for Inducing Cardiac Tissue Regeneration Following Ischemic Injury

Cardiac disease continues to be among the most prevalent causes of death worldwide. Presently, surgeries such as angioplasties, stents, and bypasses pose many risks. To improve outcomes in treating ischemia, researchers have been pursuing minimally invasive, biocompatible treatments such as gene therapies. Gene therapies are treatments that enhance or suppress target genes to alleviate illness. There are various applications for gene therapies, including, but not limited to, the treatment of cancers, diabetes, and heart disease. Gene and stem cell therapies can regenerate cardiac tissue that is damaged due to ischemia. Furthermore, gene therapies intended to evade the immune system may decrease infection risks due to the new tissue being better accepted by the body as it is created from patients’ own cells. While DNA treatments show poor results in treating cardiac illness, stem cells, such as mesenchymal stem cells and induced pluripotent cells, can differentiate into cardiomyocytes, and mRNA can be modified to express angiogenesis growth factors around the affected tissue. Although further research is needed to adapt these techniques for safe clinical use, they show potential for inducing cardiac tissue regeneration in ischemic injury. This paper conducts a review of the emerging techniques and evaluates gene therapy as a potential treatment for ischemic injury.

HOTAIR interacts with PRC2 complex regulating the regional preadipocyte transcriptome and human fat distribution.

Mechanisms governing regional human adipose tissue (AT) development remain undefined. Here, we show that the long non-coding RNA HOTAIR (HOX transcript antisense RNA) is exclusively expressed in gluteofemoral AT, where it is essential for adipocyte development. We find that HOTAIR interacts with polycomb repressive complex 2 (PRC2) and we identify core HOTAIR-PRC2 target genes involved in adipocyte lineage determination. Repression of target genes coincides with PRC2 promoter occupancy and H3K27 trimethylation. HOTAIR is also involved in modifying the gluteal adipocyte transcriptome through alternative splicing. Gluteal-specific expression of HOTAIR is maintained by defined regions of open chromatin across the HOTAIR promoter. HOTAIR expression levels can be modified by hormonal (estrogen, glucocorticoids) and genetic variation (rs1443512 is a HOTAIR eQTL associated with reduced gynoid fat mass). These data identify HOTAIR as a dynamic regulator of the gluteal adipocyte transcriptome and epigenome with functional importance for human regional AT development.

Abstract 1488: The role of epithelial miR-149 in immune modulation and progression of bronchial premalignant lesions

Abstract The molecular events involved in the development of bronchial premalignant lesions (PMLs), and their progression to lung squamous cell carcinoma, are not well understood. Prior work characterized lung PML molecular subtypes by identifying co-expressed gene modules associated with histologic severity and progression/persistence. The proliferative subtype was enriched for PMLs with dysplasia. Genes related to interferon signaling and antigen processing/presentation (Module 9) were decreased in progressive/persistent lesions within this subtype, suggesting early immune suppression is related to PML progression. However, the mechanisms that drive these alterations are unclear. We investigated the role of microRNAs (miRNAs) in regulating gene expression associated with PML outcomes. mRNA and miRNA were extracted and sequenced from longitudinally collected endobronchial biopsies from patients with PMLs (148 samples, 30 patients). miRNAs targeting each gene co-expression module were identified based on target gene enrichment and the degree of negative correlation between the miRNA expression and its targets. Expression association with outcome within the proliferative subtype was tested with a mixed effects model adjusting for batch and patient as random effect. Cell type specificity of miRNA expression was tested based on cell type specific sequencing data from FANTOM5 and cell type marker correlation analysis. Genes regulated by NLRC5 were identified with ChIPseq data from Ludigs et al. Target gene suppression was confirmed by transfecting SW900 cells with miR-149-5p. miR-149-5p level in PML biopsies was examined by miRNA in situ hybridization (miR-ISH). miR-149-5p is identified as potential regulator of Module 9 gene expression and is significantly up-regulated in progressive/persistent PMLs. Its expression is highly enriched in epithelial cells in FANTOM5 and positively correlates with basal cell markers within PMLs. Predicted targets of miR-149-5p are down-regulated in the progressive PMLs in both our and data from Merrick et al. MHC-I and related gene expressions are down-regulated in progressing/persistent PMLs. These genes are regulated by the transcriptional coactivator NLRC5 which is a predicted target gene of miR-149-5p. We find that overexpressing miR-149-5p in SW900 cells decreases the expression levels of both NLRC5 and NLRC5 regulated genes. Additionally, miR-ISH targeting miR-149-5p in proliferative biopsy samples confirm its expression in epithelium compartment and association with outcome. Our data suggest epithelial miR-149-5p might be a key regulator of gene expression contributing to PML progression. We hypothesize that by suppressing NLRC5, miR-149-5p inhibits MHC-I gene expression of epithelial cells, promoting early immune depletion and lesion progression. miR-149-5p might therefore be a therapeutic target for preventing PML progression. Citation Format: Boting Ning, Roxana M. Pfefferkorn, Gang Liu, Sherry Zhang, Hanqiao Liu, Christopher Stevenson, Sarah A. Mazzilli, Avrum E. Spira, Marc E. Lenburg, Jennifer E. Beane. The role of epithelial miR-149 in immune modulation and progression of bronchial premalignant lesions [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1488.

Abstract 1493: Development of 5-FU-modified tumor suppressor microRNAs as a platform for novel microRNA-based cancer therapeutics

Abstract MicroRNA (miRNAs) are small non-coding RNAs that regulate gene expression and are frequently dysregulated, affecting many key oncogenic pathways. Drug resistance is one of the key barriers in the treatment of cancer, contributing to the poor prognoses in patients. To overcome this hurdle, we sought to exploit the inherent multi-targeted nature of miRNAs for the development of cancer therapeutics. Despite its potential, it remains a challenge to deliver nucleic acid-based therapies, including miRNAs, to cancer cells. We recently developed a novel approach to modify miRNAs by replacing its uracil bases with 5-fluorouracil (5-FU) in the guide strand of tumor suppressor miRNAs. This modification combines the therapeutic effects of a traditional chemotherapeutic with the effect of a tumor suppressor miRNA to create a potent, multi-targeted therapeutic molecule. We have previously demonstrated that 5-FU-modified miR-15a (where the prefix 5-FU- refers to the modified miRNA mimetic, i.e., 5-FU-miR-15a) and 5-FU-miR-129 have several key features such as the retention of target gene specificity, vehicle-free delivery, enhanced potency, and stability in colon and pancreatic cancer. To demonstrate the general applicability of this approach for other tumor suppressor miRNAs we selected 12 well-studied tumor suppressor miRNAs and modified them with 5-FU. The efficacy of these 12 different miRNA mimetics were screened in several cancer types including gastric, lung, breast, pancreatic, and blood cancer. Our results show that 5-FU-modified miRNAs are more effective at inhibiting cancer cell proliferation than its unmodified counterparts. In gastric cancer, there is an 8.4-fold increase in efficacy after modifying let-7a (let-7a IC50 = 59.5 nM vs. 5-FU-let-7a IC50 = 7.1 nM) and a 4.3-fold increase for miR-145 (miR-145 IC50 = 20.9 nM vs. 5-FU-miR-145 IC50 = 4.9 nM). Similarly in lung cancer, there is a 6.3-fold increase in efficacy after modifying let-7a (let-7a IC50 = 143.5 nM vs. 5-FU-let-7a IC50 = 22.8 nM) and a 13.8-fold increase (miR-145 IC50 = 51.7 nM vs. 5-FU-miR-145 IC50 = 11.0 nM). We also show that these miRNA mimetics can be delivered into cancer cells without the aid of a delivery vehicle. Additionally, this modification preserves miRNA target gene suppression activity and has 5-FU activity from its breakdown products. Lastly, in a CT26 syngeneic mouse colon cancer model, we demonstrate that 5-FU-miR-15a inhibits tumor growth by 58.2% without vehicle and is enhanced to 97.2% with a low concentration of vehicle. Notably, there was no observable toxicity in the animal studies. This work demonstrates the potential of fluoropyrimidine-modification that can be applicable to a broad spectrum of tumor suppressor miRNAs in different cancer types and represents an advancement in the development of therapeutic miRNAs for cancer treatment. Citation Format: John G. Yuen, Andrew Fesler, Ga-Ram Hwang, Thomas Dahl, Lan-Bo Chen, Henry Chen, Jingfang Ju. Development of 5-FU-modified tumor suppressor microRNAs as a platform for novel microRNA-based cancer therapeutics [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1493.

Abstract 6304: Targeting HIF2a with siRNA: From preclinical models to the clinic

Abstract Hypoxia-inducible factor 2 alpha (HIF2a) is arguably the most important driver of kidney cancer. HIF2a is constitutively activated following von Hippel-Lindau (VHL) gene inactivation, the signature event of the most common type of kidney cancer, clear cell renal cell carcinoma (ccRCC). HIF2a functions as a heterodimeric transcription factor and regulates a program of gene expression that promotes cell proliferation, stemness, and angiogenesis. Using a highly specific inhibitor designed to target a structural vulnerability in HIF2a (PT2399), we previously showed that approximately 50% of ccRCCs are dependent on HIF2a. However, prolonged drug exposure results in resistance and the acquisition of gatekeeper mutations, which we reported first in patient-derived xenografts (PDXs) and subsequently in humans. Using the same PDX platform that previously validated PT2399, we show that HIF2a can be effectively inhibited using a tumor-directed siRNA (siHIF2). Referring herein to both first- and second-generation (ARO-HIF2) siRNA drugs, siHIF2 is specifically taken up by human ccRCC tumors transplanted in mice, where it depletes HIF2a inhibiting target gene expression and tumor growth. Through orthogonal RNA-seq studies integrating both PT2399 and siHIF2 in PDXs, we provide unprecedented detail on the HIF2a effector transcriptome, which we further dissect by incorporating ChIP-seq. A PDX line was generated from a ccRCC patient who had paraneoplastic polycythemia (a HIF2a dependent syndrome due to erythropoietin [Epo] secretion by the tumor) and participated in the phase I trial of ARO-HIF2 (NCT04169711). We show that siHIF2 effectively depleted HIF2a in both the PDX as well as in the patient, that it normalized Epo (and hemoglobin), and that it inhibited tumor growth. siHIF2 has activity against both wild-type and drug-resistant mutant HIF2a and is expected to be active in patients progressing on PT2977 (belzutifan), a PT2399-related drug recently approved by the FDA. To our knowledge, this is the first example of functional inactivation of an oncoprotein with a tumor-directed siRNA in humans. In summary, these studies provide unique insight into HIF2a (the only known core dependency in ccRCC), illustrate how it can be effectively inhibited by an siRNA drug, and establish a paradigm for the development of tumor directed siRNA-based therapeutics. Citation Format: Yuanqing Ma, Christina Stevens, Olivia Brandenburg, Vanina Toffessi Tcheuyap, Quyen N. Do, Faeze Saatchi, Tanner Hardy, Oluwatomilade Fatunde, Alyssa Macchiaroli, Jeffrey Miyata, Deyssy Carrillo, Thomas Schluep, So Wong, Alana Christie, Payal Kapur, Ivan Pedrosa, James Hamilton, James Brugarolas. Targeting HIF2a with siRNA: From preclinical models to the clinic [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 6304.

A study of relacorilant in combination with nab-paclitaxel in patients with metastatic pancreatic ductal adenocarcinoma.

4140 Background: Pancreatic cancer remains the third-leading cause of cancer-related death in the US. Average overall survival is only one year, and no standard therapies exist beyond second line. Chemotherapy resistance is one reason for the poor outcomes in pancreatic cancer, which can be caused by, among other factors, excess tumor expression of the glucocorticoid receptor (GR). Nonclinical and clinical data indicate that GR antagonism may enhance or restore chemotherapy sensitivity. Here, we report the interim analysis of RELIANT, a trial evaluating the efficacy and safety of relacorilant, a selective GR modulator, with nab-paclitaxel in patients with metastatic pancreatic ductal adenocarcinoma (mPDAC). Methods: RELIANT (NCT04329949) was a single-arm, open-label, multicenter study of relacorilant (100 mg QD) + nab-paclitaxel (80 mg/m2 on days 1, 8, and 15 of each 28-day cycle) in patients with histologically confirmed mPDAC. Based on tolerability, relacorilant doses were escalated up to 150 mg. Patients with 2+ prior lines of therapy, including prior gemcitabine- and fluoropyrimidine-based therapy, were enrolled. Planned enrollment was 80 patients. The study included a planned interim analysis after approximately 40 patients had completed 12 weeks of treatment or discontinued study treatment due to disease progression or toxicity. Objective response rate (ORR) by blinded independent central review was the primary endpoint. At the interim analysis, ORR was assessed by the investigator. Results: At the interim analysis, 43 heavily pretreated patients with a median age of 64 years (range: 43–78; 56% male) had been enrolled. 27/43 (63%) patients had received ≥2 prior lines of therapy (range: 2–5), and all but 3 patients had received prior treatment with nab-paclitaxel. Twelve patients (28%) did not have a post-baseline radiographic tumor assessment and were hence not efficacy evaluable. Most common reasons for discontinuation from relacorilant were disease progression (n = 16), adverse event (AE, n = 8), and patient decision (n = 9). Relacorilant + nab-paclitaxel demonstrated antitumor activity with 15/43 (35%) patients showing decreases in target lesion size, 10/43 (23%) achieving disease control for at least 12 weeks, and 17/43 (40%) having decreases in CA 19-9. Of note, one patient has been on study treatment for > 15 months. No confirmed responses by RECIST (CR or PR) were observed, and enrollment was thus stopped after the interim analysis. No new safety signals were identified. The most common AEs were fatigue, nausea, and decreased appetite. Suppression of GR target genes was also observed. Conclusions: Modest antitumor activity of relacorilant + nab-paclitaxel was observed in this heavily pretreated patient population, with a safety profile similar to that observed for relacorilant in other oncology studies. Clinical trial information: NCT04329949.

Role of the Two-Component System CiaRH in the Regulation of Efflux Pump SatAB and Its Correlation with Fluoroquinolone Susceptibility.

ABSTRACTStreptococcus suis is an important pathogen in both pigs and humans. Although the diseases associated with S. suis can typically be treated with antibiotics, such use has resulted in a sustained increase in drug resistance. Bacteria can sense and respond to antibiotics via two-component systems (TCSs). In this study, the TCS CiaRH was identified as playing an important role in the susceptibility of S. suis to fluoroquinolones (FQs). We found that a ΔciaRH mutant possessed lower susceptibility to FQs than the wild-type strain, with no observed growth defects at the tested concentrations and lower levels of intracellular drugs and dye. Proteomic data revealed that the levels of SatA and SatB expression were upregulated in the ΔciaRH mutant compared with their levels in the wild-type strain. The satA and satB genes encode a narrow-spectrum FQ efflux pump. The phenomena associated with combined ciaRH-and-satAB deletion mutations almost returned the ΔciaRH ΔsatAB mutant to the phenotype of the wild-type strain compared to the phenotype of the ΔciaRH mutant, suggesting that the resistance of the ΔciaRH strain to FQs could be attributed to satAB overexpression. Moreover, SatAB expression was regulated by CiaR (a response regulator of CiaRH) and SatR (a regulator of the MarR family). The ciaRH genes were consistently downregulated in response to antibiotic stress. The results of electrophoretic mobility shift assays (EMSAs) and affinity assays revealed that both regulator proteins directly controlled the ABC transporter proteins SatAB. Together, the results show that cascade-mediated regulation of antibiotic export by CiaRH is crucial for the ability of S. suis to adapt to conditions of antibiotic pressure. Our study may provide a new target for future antibiotic research and development.IMPORTANCE Streptococcus suis is a zoonotic pathogen with high incidence and mortality rates in both swine and humans. Following antibiotic treatment, the organism has evolved many resistance mechanisms, among which efflux pump overexpression can promote drug extrusion from the cell. This study clarified the role of CiaRH in fluoroquinolone resistance. A mutant with the ciaRH genes deleted showed decreased susceptibility to the antibiotics tested, an invariant growth rate, and reduced intracellular efflux pump substrates. This research also demonstrated that overexpression of the efflux pump SatAB was the main cause of ΔciaRH resistance. In addition, CiaR could combine with the promoter region of satAB to further directly suppress target gene transcription. Simultaneously, satAB was also directly regulated by SatR. Our findings may provide novel insights for the development of drug targets and help to exploit corresponding inhibitors to combat bacterial multidrug resistance.

GM-CSF-miRNA-Jak2/Stat3 Signaling Mediates Chemotherapy-Induced Cancer Cell Stemness in Gastric Cancer.

Chemotherapy serves as the first choice in clinic to treat advanced gastric cancer. However, emerging evidence indicated the induction of drug resistance and cancer stem cells occasionally by chemotherapy, which seriously limit the therapeutic effects, but the regulatory mechanism remains unclear. Here we treated two human gastric cancer cell lines SGC7901 and BGC823 with 5-Fluorouracil (5-Fu) or Cisplatin (DDP) in vitro. The survived cells showed significant increase of drug resistance, cell stemness and cytokine GM-CSF expression and secretion. As such, GM-CSF was applied to stimulate gastric cancer cells, followed by the subpopulation of CD133 + CSC analysis, sphere formation assay and stemness genes expression analysis. As a result, CSCs showed induction by GM-CSF treatment. A gastric cancer animal model further indicated that the gastric cancer cells significantly promoted tumor growth after GM-CSF treatment in vivo. High-throughput miRNA and mRNA sequencing analyses identified a subset of miRNAs and mRNAs under regulation of both 5-Fu and GM-CSF in gastric cancer cells, including upregulation of miR-877-3p and downregulation of SOCS2. Targeted overexpression or knockdown of miR-877-3p in gastric cancer cells revealed the oncogenic function of miR-877-3p in regulating gastric cancer by suppressing target gene SOCS2. Jak2/Stat3 signaling pathway, as a downstream target of SOCS2, showed activation in vitro and in vivo after treatment with miR-877-3p or GM-CSF. Our findings not only revealed a novel mechanism through which chemotherapy induced CSCs in gastric cancer via GM-CSF-miRNA-Jak2/Stat3 signaling, but also provided an experimental evidence for appropriate dose reduction of adjuvant chemotherapy in treatment of cancer patients.

Aerosol delivery of star polymer-siRNA nanoparticles as a therapeutic strategy to inhibit lung tumor growth

Lung cancer is a major contributor to cancer-related death worldwide. siRNA nanomedicines are powerful tools for cancer therapeutics. However, there are challenges to overcome to increase siRNA delivery to solid tumors, including penetration of nanoparticles into a complex microenvironment following systemic delivery while avoiding rapid clearance by the reticuloendothelial system, and limited siRNA release from endosomes once inside the cell. Here we characterized cell uptake, intracellular trafficking, and gene silencing activity of miktoarm star polymer (PDMAEMA-POEGMA) nanoparticles (star nanoparticles) complexed to siRNA in lung cancer cells. We investigated the potential of nebulized star-siRNA nanoparticles to accumulate into orthotopic mouse lung tumors to inhibit expression of two genes [βIII-tubulin, Polo-Like Kinase 1 (PLK1)] which: 1) are upregulated in lung cancer cells; 2) promote tumor growth; and 3) are difficult to inhibit using chemical drugs. Star-siRNA nanoparticles internalized into lung cancer cells and escaped the endo-lysosomal pathway to inhibit target gene expression in lung cancer cells in vitro. Nebulized star-siRNA nanoparticles accumulated into lungs and silenced the expression of βIII-tubulin and PLK1 in mouse lung tumors, delaying aggressive tumor growth. These results demonstrate a proof-of-concept for aerosol delivery of star-siRNA nanoparticles as a novel therapeutic strategy to inhibit lung tumor growth.

Identification and Validation of Chromobox Family Members as Potential Prognostic Biomarkers and Therapeutic Targets for Human Esophageal Cancer.

Background: Chromobox family proteins (CBXs) are vital components of epigenetic regulation complexes and transcriptionally inhibit target genes by modifying the chromatin. Accumulating evidence indicates that CBXs are involved in the initiation and progression of multiple malignancies. However, the expression, function, and clinical relevance such as the prognostic and diagnostic values of different CBXs in esophageal carcinoma (ESCA) are still unclear. Methods: We applied Oncomine, TCGA, GEO, GEPIA, UALCAN, Kaplan–Meier plotter, cBioPortal, Metascape, and TIMER to investigate the roles of CBX family members in ESCA. Additionally, quantitative real-time PCR (RT-PCR), western blot, and immunofluorescence were used to verify the expression of CBX family members in ESCA clinical samples. Results: Compared with normal tissues, the mRNA expression levels of CBX1/3/8 were significantly increased in ESCA, whereas CBX7 mRNA expression was reduced in both the TCGA cohort and GEO cohort. In the TCGA cohort, ROC curves suggested that CBX1/2/3/4/8 had great diagnostic value in ESCA, and the AUCs were above 0.9. Furthermore, upregulation of CBX1/3/8 and downregulation of CBX7 were closely related to the clinicopathological parameters in ESCA patients, such as tumor grades, tumor nodal metastasis status, and TP53 mutation status. The survival analysis indicated that higher CBX1/3/8 mRNA expressions and lower CBX7 expression suggested an unfavorable prognosis in ESCA. High genetic change rate (52%) of CBXs was found in ESCA patients. Functions and pathways of mutations in CBXs and their 50 frequently altered neighbor genes in ESCA patients were investigated; the results showed that DNA repair and DNA replication were correlated to CBX alterations. Moreover, we found a significant correlation between the expression level of CBX family members and the infiltration of immune cells in ESCA. Finally, we verified the expression of CBX family members in clinical samples and found the results were consistent with the databases. Conclusion: Our study implied that CBX1/3/7/8 are potential targets of precision therapy for ESCA patients and new biomarkers for the prognosis.

MiR-99a-5p inhibits target gene FZD5 expression and steroid hormone secretion from goat ovarian granulosa cells

MicroRNA (miRNA) has vital regulatory effects on the proliferation, differentiation and secretion of ovarian granulosa cells, but the role of miR-99a-5p in goat ovarian granulosa cells (GCs) is unclear. Both miR-99a-5p and Frizzled-5 (FZD5) were found to be expressed in GCs in goat ovaries via fluorescence in situ hybridization and immunohistochemistry, respectively, and FZD5 was verified (P<0.001) as a target gene of miR-99a-5p by double luciferase reporter gene experiments. Furthermore, FZD5 mRNA and protein expression were both found to be regulated (P<0.05) by miR-99a-5p in GCs. Moreover, the overexpression of miR-99a-5p or knockdown of FZD5 suppressed (P<0.05) estradiol and progesterone secretion from the GCs, as determined by ELISA. In summary, miR-99a-5p inhibits target gene FZD5 expression and estradiol and progesterone synthesis in GCs. Our study thus provides seminal data and new insights into the regulatory mechanisms of follicular development in the goat and other animals.

ANP32A represses Wnt signaling across tissues thereby protecting against osteoarthritis and heart disease

To investigate how ANP32A, previously linked to the antioxidant response, regulates Wnt signaling as unraveled by transcriptome analysis of Anp32a-deficient mouse articular cartilage, and its implications for osteoarthritis (OA) and diseases beyond the joint. Anp32a knockdown chondrogenic ATDC5 cells were cultured in micromasses. Wnt target genes, differentiation markers and matrix deposition were quantified. Wnt target genes were determined in articular cartilage from Anp32a-deficient mice and primary human articular chondrocytes upon ANP32A silencing, using qPCR, luciferase assays and immunohistochemistry. Co-immunoprecipitation, immunofluorescence and chromatin-immunoprecipitation quantitative PCR probed the molecular mechanism via which ANP32A regulates Wnt signaling. Anp32a-deficient mice were subjected to the destabilization of the medial meniscus (DMM) OA model and treated with a Wnt inhibitor and an antioxidant. Severity of OA was assessed by cartilage damage and osteophyte formation. Human Protein Atlas data analysis identified additional organs where ANP32A may regulate Wnt signaling. Wnt target genes were determined in heart and hippocampus from Anp32a-deficient mice, and cardiac hypertrophy and fibrosis quantified. Anp32a loss triggered Wnt signaling hyper-activation in articular cartilage. Mechanistically, ANP32A inhibited target gene expression via histone acetylation masking. Wnt antagonist treatment reduced OA severity in Anp32a-deficient mice by preventing osteophyte formation but not cartilage degradation, contrasting with antioxidant treatment. Dual therapy ameliorated more OA features than individual treatments. Anp32a-deficient mice also showed Wnt hyper-activation in the heart, potentially explaining the cardiac hypertrophy phenotype found. ANP32A is a novel translationally relevant repressor of Wnt signaling impacting osteoarthritis and cardiac disease.

Perspective: It is time to consider new ways to attack unpesticidable (undruggable) target sites by designing peptide pesticides.

Evolved resistance and regulatory deregistration have severely limited farmers' pesticide options. Many potential new pesticide target sites have been elucidated using targeted gene suppression and mutational tools, but few small molecules could be found that inhibit the target enzymes; the targets were considered 'undruggable'. Some organisms from all biological kingdoms use toxic peptides to ward off or kill enemies, and the agrochemical industry has used a few peptide analogs (glufosinate and bialophos) for field application. Conversely, pharmaceutical scientists have been using three-dimensional target protein structure to discover and synthesize short peptides that bind tightly to the surfaces of, and inhibit previously undruggable targets. New computational tools to quickly elucidate 3-D protein structure from amino acid sequence have just emerged. They replace crystallizing target proteins and performing X-ray crystallography to elucidate 3-D structure. These new tools allow prediction of peptides that will bind to the target proteins. They have further modified such peptides to enhance penetration, translocation and temperature stability. There is reason to assume that the same pioneering techniques can be used to develop peptide pesticides as well as pesticide synergists that act against undruggable targets and have excellent environmental and toxicological profiles. © 2022 Society of Chemical Industry.

Antisense Oligonucleotide Technologies to Combat Obesity and Fatty Liver Disease.

Synthetic oligonucleotide technologies are DNA or RNA based molecular compounds that are utilized to disrupt gene transcription or translation in target tissues or cells. Optimally, oligonucleotides are 10–30 base pairs in length, and mediate target gene suppression through directed sequence homology with messenger RNA (mRNA), leading to mRNA degradation. Examples of specific oligonucleotide technologies include antisense oligonucleotides (ASO), short hairpin RNAs (shRNA), and small interfering RNAs (siRNA). In vitro and in vivo studies that model obesity related disorders have demonstrated that oligonucleotide technologies can be implemented to improve the metabolism of cells and tissues, exemplified by improvements in fat utilization and hepatic insulin signaling, respectively. Oligonucleotide therapy has also been associated with reductions in lipid accumulation in both the liver and adipose tissue in models of diet-induced obesity. Recent advances in oligonucleotide technologies include the addition of chemical modifications such as N-acetylgalactosamine (GalNAc) conjugates that have been successful at achieving affinity for the liver, in turn improving specificity, and thus reducing off target effects. However, some challenges are still yet to be overcome relating to hepatic injury and off-target effects that have been reported with some compounds, including ASOs. In summary, oligonucleotide-based therapies are an effective tool to elucidate mechanistic insights into metabolic pathways and provide an attractive avenue for translational research into the clinic.

MiRNA-126a-3p participates in hippocampal memory via alzheimer’s disease-related proteins

Memory formation and consolidation necessitate gene expression and new protein synthesis. MicroRNAs (miRNAs), a family of small noncoding RNAs that inhibit target gene mRNA expression, are involved in new memory formation. In this study, elevated miR-126a-3p (miR-126) levels were found to contribute to the consolidation of contextual fear memory. Using different commonly mined algorithms and luciferase reporter assay, we found two Alzheimer's disease (AD)-related proteins, namely EFHD2 and BACE1, but not ADAM9, were the targets downregulated by miR-126 after CFC training. Moreover, we indicated that upregulated miR-126 could promote the formation of contextual fear memory by modulating its target EFHD2. Finally, we demonstrated that miR-126 overexpression in dentate gyrus of hippocampus could reduce Aβ plaque area and neuroinflammation, as well as rescue the hippocampal memory deficits in APP/PS1 mice. This study adds to the growing body of evidence for the role of miRNAs in memory formation and demonstrates the implication of EFHD2 protein regulated by miR-126 in the adult brain.

Targeted gene suppression through double-stranded RNA application using easy-to-use methods in Arabidopsis thaliana

RNA interference (RNAi) is an RNA-dependent gene silencing process that is regulated by the interaction between the RNA-induced silencing complex (RISC) and double-stranded RNA (dsRNA). Exogenous dsRNAs are imported directly into the cytoplasm, where they are cleaved by Dicer into short dsRNA fragments of 20–25 base pairs. These short dsRNA fragments, called small interfering RNAs (siRNAs) have sequence-specific interaction with target genes. The guide strand, onto which siRNAs are incorporated in the RISC interacts with the target mRNA sequence, thereby inducing cleavage and degradation of target messenger RNAs (mRNAs) by ribonucleases. Recent studies have shown that plant dsRNA treatments can induce RNAi. However, the dsRNA application methods and delivery systems involved have not been well examined. In this study, dsRNA was introduced to Arabidopsis thaliana by two methods: dipping and spray. We synthesized two dsRNAs designed to target mRNAs encoding enhanced green fluorescent protein (EGFP). After applying dsRNAs that target EGFP, we found an obvious reduction in GFP expression. This was determined using fluorescence microscopy and quantitative reverse transcription PCR to assess the mRNA levels of the auxin-sensitive reporter DR5-EGFP Arabidopsis thaliana. Our data revealed that applying target gene-specific exogenous dsRNAs can induce suppression of target genes of interest whether the dipping or spray method is used. This study therefore provides a foundation for understanding how to apply and deliver dsRNAs in plants.

A Method for Developing RNAi-Derived Resistance in Cowpea Against Geminiviruses.

In plants, RNA interference (RNAi) is triggered by double-stranded RNA (dsRNA). Accordingly, various RNA silencing technologies involving hpRNA, artificial microRNA (miRNA), and virus-induced gene silencing (VIGS) are used for controlling the expression of genes. Such manipulations help understanding gene functions and crop improvement biotechnology. A typical hpRNA construct is comprised of an intron splicable perfect inverted repeat of the target gene sequences under the control of a strong promoter. Geminiviruses, especially Mungbean Yellow Mosaic India Virus (MYMIV) cause devastating diseases in legume plants including cowpea, incurring severe crop loss. RNAi, involving hpRNA construct as transgene, is used to control these diseases at the early stages of geminivirus infection in the host, preventing symptom development and viral DNA accumulation. In this chapter, we describe a detailed protocol for the identification of geminivirus isolates from the filed grown cowpea plants, characterization of virus isolates under the laboratory conditions, design and construct RNAi vectors for effective suppression of viral target genes, and consequent development of transgenic cowpea using Agrobacterium-mediated transformation protocol. These transgenics are subsequently evaluated for resistance to MYMIV.

MicroRNA-155-5p Targets SKP2, Activates IKKβ, Increases Aβ Aggregation, and Aggravates a Mouse Alzheimer Disease Model.

The nuclear factor kappa B (NF-κB) pathway and inhibitor of NF-κB kinase β (IKKβ) are involved in Alzheimer disease (AD) pathogenesis. This study explored the mechanisms underlying IKKβ-mediated Aβ aggregation and neuron regeneration in APP.PS1 mice. Adenoviral transduction particles were injected into the hippocampal CA1 region of the mice to knock down or inhibit target genes. Morris water maze was performed to evaluate the cognitive function of the mice. Aβ deposition was determined by histological examination. sh-IKKβ plasmids and microRNA (miR)-155-5p inhibitor were transfected into Aβ1-42-induced N2a cells. The expressions of AD-related proteins were detected by Western blot. The interaction between S-phase kinase-associated protein 2 (SKP2) and IKKβ was assessed by co-immunoprecipitation. IKKβ knockdown (KD) and miR-155-5p inhibition ameliorated cognitive impairment, improved neuron regeneration, and attenuated Aβ deposition in APP/PS1 mice. SKP2 KD aggravated cognitive impairment, inhibited neuron regeneration, and promoted Aβ deposition in the mice. SKP2 regulated the stability of IKKβ protein via ubiquitination. MiR-155-5p regulates Aβ deposition and the expression of Aβ generation-related proteins in N2a cells via targeting SKP2. These results indicate that the miR-155-5p/SKP2/IKKβ axis was critical for pathogenesis in this AD model and suggest the potential of miR-155-5p as a target for AD treatment.

Identification of Pax protein inhibitors that suppress target gene expression and cancer cell proliferation

The Pax family of developmental control genes are frequently deregulated in human disease. In the kidney, Pax2 is expressed in developing nephrons but not in adult proximal and distal tubules, whereas polycystic kidney epithelia or renal cell carcinoma continues to express high levels. Pax2 reduction in mice or cell culture can slow proliferation of cystic epithelial cells or renal cancer cells. Thus, inhibition of Pax activity may be a viable, cell-type-specific therapy. We designed an unbiased, cell-based, high-throughput screen that identified triazolo pyrimidine derivatives that attenuate Pax transactivation ability. We show that BG-1 inhibits Pax2-positive cancer cell growth and target gene expression but has little effect on Pax2-negative cells. Chromatin immunoprecipitation suggests that these inhibitors prevent Pax protein interactions with the histone H3K4 methylation complex at Pax target genes in renal cells. Thus, these compounds may provide structural scaffolds for kidney-specific inhibitors with therapeutic potential.

BAd-CRISPR: Inducible gene knockout in interscapular brown adipose tissue of adult mice

CRISPR/Cas9 has enabled inducible gene knockout in numerous tissues; however, its use has not been reported in brown adipose tissue (BAT). Here, we developed the brown adipocyte CRISPR (BAd-CRISPR) methodology to rapidly interrogate the function of one or multiple genes. With BAd-CRISPR, an adeno-associated virus (AAV8) expressing a single guide RNA (sgRNA) is administered directly to BAT of mice expressing Cas9 in brown adipocytes. We show that the local administration of AAV8-sgRNA to interscapular BAT of adult mice robustly transduced brown adipocytes and ablated expression of adiponectin, adipose triglyceride lipase, fatty acid synthase, perilipin 1, or stearoyl-CoA desaturase 1 by >90%. Administration of multiple AAV8 sgRNAs led to simultaneous knockout of up to three genes. BAd-CRISPR induced frameshift mutations and suppressed target gene mRNA expression but did not lead to substantial accumulation of off-target mutations in BAT. We used BAd-CRISPR to create an inducible uncoupling protein 1 (Ucp1) knockout mouse to assess the effects of UCP1 loss on adaptive thermogenesis in adult mice. Inducible Ucp1 knockout did not alter core body temperature; however, BAd-CRISPR Ucp1 mice had elevated circulating concentrations of fibroblast growth factor 21 and changes in BAT gene expression consistent with heat production through increased peroxisomal lipid oxidation. Other molecular adaptations predict additional cellular inefficiencies with an increase in both protein synthesis and turnover, and mitochondria with reduced reliance on mitochondrial-encodedgene expression and increased expression of nuclear-encoded mitochondrial genes. These data suggest that BAd-CRISPR is an efficient tool to speed discoveries in adipose tissue biology.