Higher Luciferase Activity Research Articles

The Biodistribution of Replication-Defective Simian Adenovirus 1 Vector in a Mouse Model.

The administration route affects the biodistribution of a gene transfer vector and the expression of a transgene. A simian adenovirus 1 vector carrying firefly luciferase and GFP reporter genes (SAdV1-GFluc) were constructed, and its biodistribution was investigated in a mouse model by bioluminescence imaging and virus DNA tracking with real-time PCR. Luciferase activity and virus DNA were mainly found in the liver and spleen after the intravenous administration of SAdV1-GFluc. The results of flow cytometry illustrated that macrophages in the liver and spleen as well as hepatocytes were the target cells. Repeated inoculation was noneffective because of the stimulated serum neutralizing antibodies (NAbs) against SAdV-1. A transient, local expression of low-level luciferase was detected after intragastric administration, and the administration could be repeated without compromising the expression of the reporter gene. Intranasal administration led to a moderate, constant expression of a transgene in the whole respiratory tract and could be repeated one more time without a significant increase in the NAb titer. An immunohistochemistry assay showed that respiratory epithelial cells and macrophages in the lungs were transduced. High luciferase activity was restricted at the injection site and sustained for a week after intramuscular administration. A compromised transgene expression was observed after a repeated injection. When these mice were intramuscularly injected for a third time with the human adenovirus 5 (HAdV-5) vector carrying a luciferase gene, the luciferase activity recovered and reached the initial level, suggesting that the sequential use of SAdV-1 and HAdV-5 vectors was practicable. In short, the intranasal inoculation or intramuscular injection may be the preferred administration routes for the novel SAdV-1 vector in vaccine development.

Abstract 4327: Development of bioluminescent biosensors to monitor the interaction betweenGSDMD and caspase-1 in living cells

Abstract Gasdermin D (GSDMD) is the key executioner of pyroptosis, an inflammatory form of programmed cell death implicated in various inflammation-driven diseases, including cancer, heart failure and strokes. The interaction between GSDMD and caspase-1 (CASP1) orchestrates the critical pathway of GSDMD-mediated pyroptosis. During pyroptosis, pyroptosis stimuli activate CASP1, which subsequently binds to and cleave GSDMD between GSDMD’s N- and C-terminal domains. The free N-terminal domains oligomerize and translocate to the cell membrane, forming pores and releasing proinflammatory cytokines and causing cellular lysis. Key interaction sites have been identified for GSDMD recognition and engagement by CASP1; therefore, blocking functionally significant residues presents a new therapeutic strategy for inhibiting CASP1-induced GSDMD cleavage and pyroptosis. Currently, there is no small molecule capable of specifically disrupting the GSDMD and CASP1 interaction. In response, the ongoing pursuit of identifying potential inhibitors is a driving force for developing a biosensor capable of monitoring this interaction and their roles in downstream pyroptotic effects. In this study, we utilized the NanoLuc Binary Technology (NanoBiT) to construct several bioluminescent biosensors comprising two NanoLuc luciferase enzyme fragments (LgBiT and SmBiT) fused to GSDMD and CASP1, respectively, in various combinations. Further validation of these biosensors by luciferase assays in living cells identified a GSDMD-CASP1 biosensor with highest luciferase activity. This newly developed biosensor paves the way for high-throughput screens to identify novel small molecule inhibitors disrupting the GSDMD-CASP1 interplay. These small molecule inhibitors will effectively impede GSDMD-mediated pyroptosis, holding promise for novel therapies of various human diseases. Citation Format: Tynan Kelly. Development of bioluminescent biosensors to monitor the interaction betweenGSDMD and caspase-1 in living cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4327.

Abstract 2065: Development of a genetically validated, cell-based reporter assay for ADAR1 editing activity

Abstract Adenosine Deaminase Acting on RNA (ADAR) enzymes perform adenosine to inosine base editing in RNA, particularly targeting adenosines located within a specific double-stranded stem-loop motif. The ADAR1 gene encodes both a universally expressed isoform (p110) and an interferon-inducible isoform (p150), which plays a role in innate immunity by mediating interferon signaling. In the context of healthy, uninfected cells, ADAR1 performs A-to-I editing on endogenous double-stranded RNA to prevent it from activating downstream dsRNA sensors RIG-I and MDA5, which in-turn activate a pro-inflammatory response. Loss of function mutations in ADAR1 result in aberrant activation of the dsRNA sensors and are involved in autoimmune disorders. ADAR1 dysfunction also impacts cancer cell growth, proliferation, and response to immunotherapy. ADAR1 expression is increased in several types of cancer and ADAR1 knock-out has been demonstrated to improve the response to certain immunotherapies such PD-1/PD-L1 blockade and to circumvent tumor immunotherapy resistance mechanisms, making ADAR1 an attractive target for therapeutic development. We have developed and validated a high-throughput, cell-based assay for measuring ADAR1 editing activity. Several ADAR editing reporter constructs were designed and evaluated for their relative response to ADAR1 over-expression. They all feature an ADAR1 hairpin target with a stop codon (UAG) which is susceptible to ADAR1-mediated editing to a tryptophan (UUG), located upstream of a firefly luciferase gene. We show that increasing transient expression of ADAR1 led to proportional increased activity of the downstream firefly luciferase, indicating dose-dependent transcript editing by ADAR1. Selected reporters were used in conjunction with ADAR2 overexpression to identify an ADAR1-biased reporter. Thus, a reporter with low luciferase activity in response to ADAR2 expression was chosen to establish stable HEK293 cell lines (which express low levels of ADAR1) with and without constitutively expressed ADAR1 protein. ADAR1-overexpressing HEK293 cells displayed high luciferase activity, in accordance with high ADAR1-mediated editing of the reporter transcript, which was decreased in response to siRNA-mediated knockdown of ADAR1 expression or pharmacological inhibition of ADAR1 activity. Sequencing of the reporter RNA confirmed that changes in reporter activity were correlated with editing of the RNA transcript. Finally, these validated ADAR1 editing reporter cell lines were further engineered to constitutively express Renilla luciferase under the control of a CMV promoter, serving as an internal control for the toxicity of ADAR1 inhibitors. In conclusion, our robust cell-based assay provides a well-validated resource to identify modulators of ADAR1 editing activity in a cellular context. Citation Format: Valerie Sapp, Jim Bilakovics, Ava Burr, Fernando Martins, Pavel Shashkin, Veronique Baron, Henry Zhu, Junguk Park. Development of a genetically validated, cell-based reporter assay for ADAR1 editing activity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2065.

Abstract 4051: Optimizing breast cancer therapy by modulating oxygen consumption

Abstract Immune checkpoint blockade (ICB) has achieved great clinical responses in multiple cancers. However, only a subset of patients with triple negative breast cancer (TNBC), a highly hypoxic tumor, respond to ICB. In TNBC, the immune checkpoints PD-1/PD-L1 and adenosine receptor (A2aR) are often activated to escape anti-tumor responses. These two immune checkpoints are sensitive to hypoxia. We propose that blocking tumor oxygen consumption using deferiprone (DFP), phenformin (Phen) and metformin (Met) will improve the efficacy of PD-1/PD-L1 and A2aR blockade. We generated two mouse breast cancer cell lines (4T1 and E0771) expressing green fluorescent protein (GFP) fused to Firefly luciferase under the control of the hypoxia reporter element (HRE). This construct, which we called HRE/TGL, serves as a hypoxia reporter, with GFP expression and luciferase activity increasing in hypoxic conditions. To induce hypoxia in vitro, we used two approaches: 1) we incubated cells in a hypoxia chamber (3% oxygen), and 2) treated cells with cobalt chloride, which chemically mimics hypoxia by inducing the expression of the hypoxia master regulator HIF1α. Both 4T1-HRE/TGL and E0771-HRE/TGL cells exhibited increased GFP expression and luciferase activity within hypoxia conditions. We orthotopically implanted these cells into mice. We measured luciferase activity in vivo using the IVIS spectrum imaging system overtime after tumor implantation. We observed high luciferase activity in 4T1-HRE/TGL tumors. Next, we examined the effects of DFP, Phen and Met on cell hypoxic responses by performing immunoblotting against HIF1α and phosphorylated AMPK (p-AMPK) on 4T1 and E0771 cells. We observed that, unlike Phen or Met, DFP increased HIF1α and p-AMPK expression. Binding of HIF1 proteins to HREs is known to activate the expression of the HRE promoter. To confirm our results, we treated 4T1-HRE/TGL and E0771-HRE/TGL cells with low (10μM) or high (100μM) doses of the drugs in vitro. We observed that, while DFP increased GFP expression and luciferase activity, Phen had no consistent measurable effect. We observed similar results in vivo. Finally, we examined the effect of DFP, Phen and Met either alone or in combination with anti-PD-1 on tumor progression and survival in mice bearing 4T1 or E0771 tumors. Contrary to what expected, we did not observe better tumor control and survival after treatment with these drugs. In addition, DFP, Phen and Met failed to improve the anti-tumor effect of anti-PD-1 treatment in both TNBC models. Future experiments will focus on charactering the adenosine pathway in vivo in 4T1 and E0771 tumors and examine how drugs targeting A2aR and oxygen consumption (DFP, Phen and Met) influence the activation of immune cells within the tumor microenvironment and potentially combinate these two strategies to improve their anti-tumor efficacy. Citation Format: Anais Assouvie, Sadna Budhu, Mayuresh Mane, Mamadou Bah, Juan Zurita, Inna Serganova, Soe Min, Jason Koutcher, Vladimir Ponomarev, Jedd Wolchok, Taha Merghoub. Optimizing breast cancer therapy by modulating oxygen consumption [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4051.

Development of a Gene and Nucleic Acid Delivery System for Skeletal Muscle Administration via Limb Perfusion Using Nanobubbles and Ultrasound.

Strategies for gene and nucleic acid delivery to skeletal muscles have been extensively explored to treat Duchenne muscular dystrophy (DMD) and other neuromuscular diseases. Of these, effective intravascular delivery of naked plasmid DNA (pDNA) and nucleic acids into muscles is an attractive approach, given the high capillary density in close contact with myofibers. We developed lipid-based nanobubbles (NBs) using polyethylene-glycol-modified liposomes and an echo-contrast gas and found that these NBs could improve tissue permeability by ultrasound (US)-induced cavitation. Herein, we delivered naked pDNA or antisense phosphorodiamidate morpholino oligomers (PMOs) into the regional hindlimb muscle via limb perfusion using NBs and US exposure. pDNA encoding the luciferase gene was injected with NBs via limb perfusion into normal mice with application of US. High luciferase activity was achieved in a wide area of the limb muscle. DMD model mice were administered PMOs, designed to skip the mutated exon 23 of the dystrophin gene, with NBs via intravenous limb perfusion, followed by US exposure. The number of dystrophin-positive fibers increased in the muscles of mdx mice. Combining NBs and US exposure, which can be widely delivered to the hind limb muscles via the limb vein, could be an effective therapeutic approach for DMD and other neuromuscular disorders.

Optimization of transfection methods for human lymphoblast TK6 cell line

Transfection has recently gained attention in the field of biomedical research due to its ability to manipulate gene expression. Every mammalian cell type has a characteristic set of requirements for optimal transfection. Some cells can be difficult to transfect and require optimization for successful transfection. Human lymphoblast TK6 cell line, an important cell line for genotoxic studies, is known to be extremely hard to transfect. Thus, optimizing transfection methods for human lymphoblast TK6 are increasingly important. To accomplish this, TK6 human lymphoblasts were transfected with plasmid constructs that expressed green fluorescent protein (GFP) and NanoLuc® activity. We compared the transfection efficiency of three commercially available transfection reagents, including Amaxa 96-well Nucleofection procedure using various solutions (SF, SE, and SG), Lipofectamine LTX, and Metafectene Pro®. The transfection efficiency and toxicity of various reagents were tested by fluorescence microscopy, luciferase activity, and cell viability assays. Amaxa 96-well Nucleofection Solution SF was identified as the best transfection reagent due to its relatively high luciferase activity, acceptable cell viability (80%), and GFP transfection efficiency (80%). Optimal conditions for transfection utilized with this reagent included 0.4 μg of plasmid DNA, 1.8 × 105 cells, and using the DS 137 Nucleofector program.

861P Association of genetic variants in JAK/STAT signaling pathway with cutaneous melanoma susceptibility

Janus kinase signal transducer and activator of transcription (JAK/STAT) signaling pathway is involved in cell proliferation, angiogenesis, apoptosis inhibition, metastasis, and immune suppression. Due to these multiple functions, this pathway regulates cutaneous melanoma (CM) development and progression. The JAK1, JAK2 and STAT3 proteins are encoded by polymorphic genes. This study aimed to verify whether single-nucleotide variants (SNVs) in JAK1 (c.1648+1272G>A, c.991-27C>T), JAK2 (c.-1132G>T, c.-139G>A) and STAT3 (c.*1671T>C, c.-1937C>G) altered risk, clinicopathological aspects, survival of CM patients, as well as protein activity. CM patients (N = 248) and controls (N = 274) were included in this study. Genotyping was performed by real-time polymerase chain reaction (PCR) and JAK1, JAK2 and STAT3 expression was assessed by quantitative PCR (qPCR). STAT3 c.-1937C>G SNV was investigated by luciferase, qPCR, western blot, apoptosis, and cell cycle assays in SKMEL-28 cells with CC or GG genotype. In addition, fourteen melanoma cell lines with different genotypes were used to analyze STAT3 expression with non-intervention. Individuals with STAT3 c.*1671TT and c.-1937CC genotypes, and TC haplotype of both SNVs were under 2.0-fold increased risk of CM. Specific JAK1, JAK2 and STAT3 combined genotypes were associated with up to 4.0-fold increased risk of CM. Higher luciferase activity (4,013.34 vs. 2,463.32 arbitrary unit (AU); p = 0.009), STAT3 expression by qPCR (649.20 vs. 0.04 AU; p = 0.003) and western blot (1.69 vs. 1.16 AU; p = 0.01), and percentage of cells in S phase of cell cycle (57.54 vs. 30.73 %; p = 0.04) were more frequent in SKMEL-28 with STAT3 c.-1937CC than with GG genotype. CM cell lines with CC genotype presented higher STAT3 protein levels than those with GG genotype (1.93 versus 1.27 AU, p = 0.0027). Our data present for the first-time preliminary evidence that JAK1, JAK2 and STAT3 SNVs alter risk and clinical aspects of CM patients. If validated in a further epidemiological study, our data can be used to select individuals at high-risk of CM, who should receive special attention in tumor prevention and early detection.

Association of JAK/STAT genetic variants with cutaneous melanoma.

BackgroundThe Janus-activated kinase (JAK)-signal transducer and activator of transcription (STAT) signaling pathway regulates cutaneous melanoma (CM) development and progression. The JAK1, JAK2, and STAT3 proteins are encoded by polymorphic genes. This study aimed to verify whether single-nucleotide variants (SNVs) in JAK1 (c.1648+1272G>A, c.991-27C>T), JAK2 (c.-1132G>T, c.-139G>A), and STAT3 (c.*1671T>C, c.-1937C>G) altered the risk, clinicopathological aspects, and survival of CM patients as well as protein activity.MethodsCM patients (N = 248) and controls (N = 274) were enrolled in this study. Genotyping was performed by real-time polymerase chain reaction (PCR), and JAK1, JAK2, and STAT3 expression was assessed by quantitative PCR (qPCR). STAT3 c.-1937C>G SNV was investigated by luciferase, qPCR, western blot, apoptosis, and cell cycle assays in SKMEL-28 cells with CC or GG genotype.ResultsIndividuals with STAT3 c.*1671TT and c.-1937CC genotypes and TC haplotype of both SNVs were under about 2.0-fold increased risk of CM. Specific JAK1, JAK2, and STAT3 combined genotypes were associated with up to 4.0-fold increased risk of CM. Higher luciferase activity [4,013.34 vs. 2,463.32 arbitrary units (AU); p = 0.004], STAT3 expression by qPCR (649.20 vs. 0.03 AU; p = 0.003) and western blot (1.69 vs. 1.16 AU; p = 0.01), and percentage of cells in the S phase of the cell cycle (57.54 vs. 30.73%; p = 0.04) were more frequent in SKMEL-28 with STAT3 c.-1937CC than with GG genotype. CM cell line with CC genotype presented higher STAT3 protein levels than the one with GG genotype (1.93 versus 1.27 AU, p = 0.0027).ConclusionOur data present preliminary evidence that inherited abnormalities in the JAK/STAT pathway can be used to identify individuals at a high risk of CM, who deserve additional attention for tumor prevention and early detection.

Abstract 960: HAPLN1 increases peritoneal carcinomatosis by inducing tumor cell hyperplasticity

Abstract Pancreatic Ductal Adenocarcinoma (PDAC) frequently metastasizes into the peritoneum forming peritoneal carcinomatosis, which are so far not treatable effectively. Metastasis-initiating cells need to acquire beneficial traits including cell plasticity, immune evasion, dormancy state control and organ colonization. These characteristics can be summarized in broad terms into two main processes, epithelial-to-mesenchymal transition (EMT) and stemness. Hyaluronic acid (HA), an extracellular matrix component, is a crucial factor in regulating these processes in PDAC, but it is so far not successfully targetable. Analyzing publicly available databases by gene set enrichment analysis (GSEA), a signature related to HA binding was enriched in tumor samples compared to normal tissue. Hyaluronan And Proteoglycan Link Protein 1 (HAPLN1) was the top contributor to the enrichment score, being the 8th most enriched gene overall. We found that higher HAPLN1 expression correlated with shorter overall survival and that HAPLN1high patients had both, basal subtype and EMT signatures enriched. Moreover, these patients had a signature for peritoneal metastasis significantly enriched, suggesting a higher risk for peritoneal carcinomatosis. To study the role of HAPLN1 on PDAC in vitro, we stably overexpressed HAPLN1 in the murine PDAC cell line KPC. KPC-HAPLN1 cells expressed more EMT markers, more stem-related genes and changed the proteoglycan production from Aggrecan to Versican, which is known to be pro-metastatic. We found that spheroid formation, a feature of stemness, was improved in KPC-HAPLN1 vs KPC. Additionally, embedding these spheroids into matrigel led to an increased invasion of KPC-HAPLN1 cells. KPC-HAPLN1 cells improved KPC cell invasion capacities when co-cultured, indicating a paracrine effect. In vivo, intraperitoneal injection of luciferase expressing KPC cells resulted in higher luciferase activity when tumor cells expressed HAPLN1. Analyzing the peritoneal lavage (PL) from these mice, we obtained significantly more tumor cells in KPC-HAPLN1 injected mice. RNAseq data of tumor cells isolated from tumor nodules and PL showed that KPC-HAPLN1 cells acquired an increased metastatic potential and a strong immunomodulatory phenotype. Thus, we evaluated the immune cell composition of the PL by flow cytometry. Neutrophil and monocyte percentages were drastically reduced in KPC-HAPLN1 bearing mice. On the contrary, these mice had a significant increase in macrophages, which showed a reduction in pro-inflammatory gene expression. We conclude that HAPLN1 expression in tumor cells promotes a hyperplastic phenotype that facilitates invasion and colonization of the peritoneum, among others by creation of a pro-tumoral immune microenvironment. Citation Format: Lena Wiedmann, Francesca De Angelis Rigotti, Nuria Vaquero-Siguero, Elisa Donato, Elisa Espinet, Andreas Trumpp, Andreas Fischer, Juan Rodriguez-Vita. HAPLN1 increases peritoneal carcinomatosis by inducing tumor cell hyperplasticity [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 960.

Reduction of Cell Fusion by Deletion in the Hypervariable Region of the Spike Protein of Mouse Hepatitis Virus.

Deletions in the spike gene of mouse hepatitis virus (MHV) produce several variants with diverse biological characteristics, highlighting the significance of the spike gene in viral pathogenesis. In this study, we characterized the JHM-X strain, which has a deletion in the hypervariable region (HVR) of the spike gene, compared with the cl-2 strain, which has a full spike gene. Cytopathic effects (CPEs) induced by the two strains revealed that the size of the CPE produced by cl-2 is much greater than that produced by JHM-X in delayed brain tumor (DBT) cells. Thus, this finding explains the greater fusion activity of cl-2 than JHM-X in cultured cells, and we speculate that the deletion region of the spike protein is involved in the fusion activity differences. In contrast with the fusion activity, a comparison of the virus growth kinetics revealed that the titer of JHM-X was approximately 100 times higher than that of cl-2. We found that the deletion region of the spike protein was involved in fusion activity differences, whereas cl-2 produced significantly higher luciferase activity than JHM-X upon similar expression levels of the spike protein. However, the reason behind the growth difference is still unknown. Overall, we discovered that deletion in the HVR of the spike gene could be involved in the fusion activity differences between the two strains.

Functional genetic variants in complement component 7 confer susceptibility to gastric cancer.

BackgroundComplement system plays an important role in innate immunity which involved in the changes tumor immune microenvironment by mediating the inflammatory response. This study aims to explore the relationship between complement component 7 (C7) polymorphisms and the risk of gastric cancer (GC).Materials and MethodsAll selected SNPs of C7 were genotyped in 471 patients and 471 controls using the polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP). Odds ratios (ORs) and 95% confidence intervals (CIs) were calculated by unconditional Logistic regression to analyze the relationship between each genotype and the genetic susceptibility to gastric cancer. The level of C7 expression in GC was analyzed by Gene Expression Profiling Interactive Analysis (GEPIA) and detected by Enzyme Linked Immunosorbent Assay. Kaplan–Meier plotter were used to reveal C7 of prognostic value in GC. We examined SNPs associated with the expression of C7 using the GTEx database. The effect of C7 polymorphisms on the regulatory activity of C7 was detected by luciferase reporter assay.ResultsUnconditional logistic regression showed that individuals with C7 rs1376178 AA or CA genotype had a higher risk of GC with OR (95% CI) of 2.09 (1.43–3.03) and 1.88 (1.35–2.63), respectively. For C7 rs1061429 C > A polymorphism, AA genotype was associated with the elevated risk for developing gastric cancer (OR = 2.16, 95% CI [1.37–3.38]). In stratified analysis, C7 rs1376178 AA genotype increased the risk of GC among males (OR = 2.88, 95% CI [1.81–4.58]), but not among females (OR = 1.06, 95% CI [0.55–2.06]). Individuals carrying rs1061429 AA significantly increased the risk of gastric cancer among youngers (OR = 2.84, 95% CI [1.39–5.80]) and non-smokers (OR = 2.79, 95% CI [1.63–4.77]). C7 was overexpressed in gastric cancer tissues and serum of cancer patients and was significantly associated with the prognosis. C7 rs1061429 C > A variant contributed to reduced protein level of C7 (P = 0.029), but rs1376178 didn’t. Luciferase reporter assay showed that rs1376178C-containing plasmid exhibited 2.86-fold higher luciferase activity than rs1376178 A-containing plasmid (P < 0.001). We also found that rs1061429A allele contributed 1.34-fold increased luciferase activity than rs1061429C allele when co-transfected with miR-591 (P = 0.0012).ConclusionsThese findings highlight the role of C7 in the development of gastric cancer.

Development and in vitro evaluation of recombinant chicken promoters to efficiently drive transgene expression in chicken oviduct cells

Virus injection into EGK-X embryos is a well-defined approach in avian transgenesis. This system uses a chicken ovalbumin gene promoter to induce transgene expression in the chicken oviduct. Although a reconstructed chicken ovalbumin promoter that links an ovalbumin promoter and estrogen-responsive enhancer element (ERE) is useful, a large viral vector containing the ovalbumin promoter and a target gene restricts viral packaging capacity and produces low-titer virus particles. We newly developed recombinant chicken promoters by linking regulatory regions of ovalbumin and other oviduct-specific genes. Putative enhancer fragments of the genes, such as ovotransferrin (TF), ovomucin alpha subunit (OVOA), and ovalbumin-related protein X (OVALX), were placed at the 5`-flanking region of the 2.8-kb ovalbumin promoter. Basal promoter fragments of the genes, namely, pTF, lysozyme (pLYZ), and ovomucoid (pOVM), were placed at the 3`-flanking region of the 1.6-kb ovalbumin ERE. The recombinant promoters cloned into each reporter vector were evaluated using a dual luciferase assay in human and chicken somatic cells, and LMH/2A cells treated with 0-1,000 nM estrogen, and cultured primary chicken oviduct cells. The recombinant promoters with linking ovalbumin and TF, OVOA, pOVM, and pLYZ regulatory regions had 2.1- to 19.5-fold (P < 0.05) higher luciferase activity than the reconstructed ovalbumin promoter in chicken oviduct cells. Therefore, recombinant promoters may be used to efficiently drive transgene expression in transgenic chickens.

Loss of function of the carbon catabolite repressor CreA leads to low but inducer\u2010independent expression from the feruloyl esterase B promoter in Aspergillus niger

ObjectiveWith the aim to decipher the mechanisms involved in the transcriptional regulation of feruloyl esterase encoded by faeB, a genetic screen was performed to isolate A. niger mutants displaying inducer-independent expression from the faeB promoter.ResultPfaeB-amdS and PfaeB-lux dual reporter strains were constructed and used to isolate trans-acting mutants in which the expression of both reporters was increased, based on the ability to grow on acetamide plates and higher luciferase activity, respectively. The genetic screen on the non-inducing carbon source D-fructose yielded in total 111 trans-acting mutants. The genome of one of the mutants was sequenced and revealed several SNPs, including a point mutation in the creA gene encoding a transcription factor known to be involved in carbon catabolite repression. Subsequently, all mutants were analyzed for defects in carbon catabolite repression by determining sensitivity towards allyl alcohol. All except four of the 111 mutants were sensitive to allyl alcohol, indicating that the vast majority of the mutants are defective in carbon catabolite repression. The creA gene of 32 allyl alcohol sensitive mutants was sequenced and 27 of them indeed contained a mutation in the creA gene. Targeted deletion of creA in the reporter strain confirmed that the loss of CreA results in constitutive expression from the faeB promoter.ConclusionLoss of function of CreA leads to low but inducer-independent expression from the faeB promoter in A. niger.

TLR4 promoter rs1927914 variant contributes to the susceptibility of esophageal squamous cell carcinoma in the Chinese population.

BackgroundToll-like receptor 4 (TLR4), as a key regulator of both innate and acquired immunity, has been linked with the development of various cancers, including esophageal cancer. This study aims to analyze the association of potential functional genetic polymorphisms in TLR4 with the risk of esophageal cancer.MethodsThis case-control study involved in 480 ESCC patients and 480 health controls. Polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) was used to genotype TLR4 rs1927914 polymorphism. Taqman probe method was used to determine the genotypes of TLR4 rs11536891 and rs7873784 variants. The relationship between TLR4 genetic variation and ESCC risk was analyzed by Logistic regression model by calculating the odds ratio (OR) and 95% confidence interval (95% CI).ResultsCompared with TLR4 rs1927914 AA genotype carriers, GG carriers had a lower ESCC risk (OR = 0.59, 95% CI [0.38–0.93], P = 0.023). Stratification analysis by age showed that TLR4 rs1927914 GG could affect the risk of ESCC in elderly people (OR = 0.59, 95% CI [0.36–0.97]). Smoking stratification analysis indicated that rs1927914 GG carriers were related to ESCC susceptibility among non-smokers (OR = 0.36, 95% CI [0.18–0.73]). Dual luciferase reporter assay suggested that rs1927914 G-containing TLR4 promoter displayed a 1.76-fold higher luciferase activity than rs1927914 A-containing counterpart in KYSE30 cells. Electrophoretic mobility shift assay (EMSA) showed the KYSE30 cell nuclear extract was able to bind the probe with rs1927914 G allele and this DNA-protein interaction could be eliminated by competition assays with unlabeled rs1927914 G probe, which indicating that the binding is sequence-specific. Our results also showed that TLR4 rs7873784 (G>C) and rs11536891 (T>C) conformed to complete genetic linkage. The genotype distributions of TLR4 rs11536891 variant among ESCC patients and normal controls have no statistical significance.ConclusionThe TLR4 rs1927914 variant contributes to the ESCC risk by effecting the promoter activity.

Suprachoroidally Delivered DNA Nanoparticles Transfect Retina and Retinal Pigment Epithelium/Choroid in Rabbits.

PurposeThis study evaluated ocular tolerability and transfectability of nonviral DNA nanoparticles (DNPs) after microneedle-based suprachoroidal (SC) administration, in comparison to subretinal (SR) administration.MethodsThe DNPs consisted of a single copy of plasmid DNA with a polyubiquitin C/luciferase transcriptional cassette compacted with 10 kDa PEG-substituted lysine 30-mer peptides (CK30PEG10k). New Zealand White rabbits (n = 4 per group) received a unilateral SC injection (0.1 mL via a microneedle technique) of ellipsoid-shaped DNPs, rod-shaped DNPs, or saline (negative control). A cohort of rabbits (n = 4) also received a single unilateral SR injection (0.05 mL via a transvitreal approach) of rod-shaped DNPs. At day 7, luciferase activity was measured in the retina and retinal pigment epithelium (RPE)–choroid via bioluminescence assay. A cohort of rabbits received a SC injection of analogous DNPs to assess spread of DNP injectate in the suprachoroidal space (SCS) via optical coherent tomography and histology.ResultsSuprachoroidal injection of DNPs resulted in reversible opening of the SCS circumferentially and posteriorly and was generally well tolerated, with no significant ocular examination score changes, intraocular pressure abnormalities, or changes in electroretinography amplitudes on day 7 compared to the baseline. High luciferase activity was observed in the retina and RPE-choroid of eyes that received SC DNPs (rod and ellipsoid shape) and SR DNPs (rod shape) compared to controls. The mean luciferase activity in RPE-choroid and retina was comparable between SC and SR administrations. Transfection in the RPE-choroid was approximately 10-fold higher than in the retina after either SC or SR administration of DNPs.ConclusionsSuprachoroidal and SR administration of DNPs resulted in comparable transfection of retina and RPE-choroid.Translational RelevanceSuprachoroidal delivery of DNPs offers the potential to precisely target chorioretinal tissues while avoiding surgical risks associated with SR injection, and it may offer an office-based nonsurgical gene therapy option for the treatment of retinal diseases.

SIRT1 Gene SNP rs932658 Is Associated With Medication-Related Osteonecrosis of the Jaw

Medication-related osteonecrosis of the jaw (MRONJ) is a rare but serious adverse drug reaction. Our previous whole-exome sequencing study found SIRT1 intronic region single-nucleotide polymorphism (SNP) rs7896005 to be associated with MRONJ in cancer patients treated with intravenous (iv) bisphosphonates (BPs). This study aimed to identify causal variants for this association. In silico analyses identified three SNPs (rs3758391, rs932658, and rs2394443) in the SIRT1 promoter region that are in high linkage disequilibrium (r2 > 0.8) with rs7896005. To validate the association between these SNPs and MRONJ, we genotyped these three SNPs on the germline DNA from 104 cancer patients of European ancestry treated with iv BPs (46 cases and 58 controls). Multivariable logistic regression analysis showed the minor alleles of these three SNPs were associated with lower odds for MRONJ. The odds ratios (95% confidence interval) and p values were 0.351 (0.164-0.751; p = 0.007) for rs3758391, 0.351 (0.164-0.751; p = 0.007) for rs932658, and 0.331 (0.157-0.697; p = 0.0036) for rs2394443, respectively. In the reporter gene assays, constructs containing rs932658 with variant allele A had higher luciferase activity than the reference allele, whereas constructs containing SNP rs3758391 and/or rs2394443 did not significantly affect activity. These results indicate that the promoter SNP rs932658 regulates the expression of SIRT1 and presumably lowers the risk of MRONJ by increasing SIRT1 expression. © 2020 American Society for Bone and Mineral Research (ASBMR).

Intra-tracheal delivery of AAV6 vectors results in sustained transduction in murine lungs without genomic integration.

Despite the progress made in AAV-based gene therapy targeting different organ systems, lung-targeted gene therapy using AAV vectors has not been effective, mostly due to the poor transduction and un-sustained gene expression in airway epithelium. Furthermore, concerns over possible harmful insertional mutagenesis seen in other cell types, particularly hepatocytes, raised a question about AAV safety. In this study, we evaluate the long-term persistence of this vector in mouse lungs and any possible harmful integration of these vectors into the host genome. AAV6 vectors expressing reporter gene (firefly luciferase) were delivered to the lungs of C57BL/6 mice through intra-tracheal intubation. Despite the large variation among individual animals, most animals had high and sustained luciferase activity with a peak from 2 to 3 weeks post-transduction before a significant decline between 15 and 19 weeks post-transduction. More importantly, even after its decline, most animals maintained detectable luciferase expression for 150 days or more, which was confirmed by post-necropsy qPCR analysis of luciferase gene expression. At the termination point of experiments, an average of one copy of AAV expression cassette per mouse genome was detected. We also found that partial overlaps between the AAV6 expression cassette and the mouse genome were distributed broadly with no apparent systematic preference in any mouse chromosomal map location. In summary, our data suggest that AAV6 mediated long-term gene expression in the lungs with no evidence of genomic integration, and thus, any insertional mutagenesis.

Novel lncRNA lncFAM200B: Molecular Characteristics and Effects of Genetic Variants on Promoter Activity and Cattle Body Measurement Traits.

Skeletal muscle is one of the three major muscle types in an organism and has key roles in the motor system, metabolism, and homeostasis. RNA-Seq analysis showed that novel lncRNA, lncFAM200B, was differentially expressed in embryonic, neonatal, and adult cattle skeletal muscles. The main aim of this study was to investigate the molecular and expression characteristics of lncFAM200B along with its crucial genetic variations. Our results showed that bovine lncFAM200B was a 472 nucleotide (nt) non-coding RNA containing two exons. The transcription factor binding site prediction analysis found that lncFAM200B promoter region was enriched with SP1 transcription factor, which promotes the binding of myogenic regulatory factor MyoD and DNA sequence. The mRNA expression analysis showed that lncFAM200B was differentially expressed in embryonic, neonatal, adult bovine muscle tissues, and the lncFAM200B expression trend positively correlated with that of MyoG and Myf5 in myoblast proliferation and differential stages. To identify the promoter active region of lncFAM200B, we constructed promoter luciferase reporter gene vector pGL3-Basic plasmids containing lncFAM200B promoter sequences and transfected them into 293T, C2C12, and 3T3-L1 cells. Our results suggested that lncFAM200B promoter active region was from −403 to −139 (264 nt) of its transcription start site, covering 6 SP1 potential binding sites. Furthermore, we found a novel C-T variation, named as SNP2 (ERZ990081 in European Variation Archive) in the promoter active region, which was linked to the nearby SNP1 (rs456951291 in Ensembl database). The genotypes of SNP1 and combined genotypes of SNP1 and SNP2 were significantly associated with Jinnan cattle hip height. The luciferase activity analysis found that the SNP1-SNP2 haplotype CC had the highest luciferase activity, which was consistent with the association analysis result that the combined genotype CC-CC carriers had the highest hip height in Jinnan cattle. In conclusion, our data showed that lncFAM200B is a positive regulator of muscle development and that SNP1 and SNP2 could be used as genetic markers for marker-assisted selection (MAS) breeding of beef cattle.

Ultrasound and Microbubble Gene Delivery for Targeting Altered Placental MicroRNAs in Preeclampsia

Ultrasound (US) and microbubble (MB) gene delivery has attracted growing interest as a clinically applicable gene therapy (GT). Though preclinical studies have investigated the system in various tissues, there is limited research in targeting the placenta. This is a potential therapeutic strategy for preeclampsia (PE), which has an underlying genetic basis and ineffective management strategies. Differentially expressed placental microRNAs (miRNAs) in PE may represent suitable targets for GT. Microbubbles (SonoVue) and plasmid (pGL3 or pGL4.13) were administered systemically to CD1 mice, followed by exposure of the heart to US (H14, 1.8 M.I., 1cm focal depth, 2 minutes), using Siemens Acuson Sequoia-512 system and 15L8 probe. Luciferase assays were performed to evaluate gene transfection. Significantly differentially expressed placental miRNAs in PE patients were identified as candidates based on detection by three or more screening studies. Expression of candidate miRNAs was measured by qRT-PCR in PE rat model placentas. In trial 1, low levels of luciferase activity were detected in the heart of treatment mouse 1, 2 and 3. In trial 2, luciferase activity was evident in the atria of treatment mouse 2. In trial 3, higher luciferase activity was detected in the ventricles of the treatment mouse and activity was also detected in the atria. The literature review identified eight candidate miRNAs. MiR-223 (1.46-fold increase) and miR-181a (0.81-fold decrease) were significantly differentially expressed in PE rat model placentas. MiR-223 and -181a may represent targets for US and MB gene delivery. Future studies will apply the US and MB gene delivery protocol for translation to targeting the placenta in our PE rodent model.

Abstract 3553: Targeting miR-361-3p suppresses the growth of human oral squamous cell carcinoma cells in vitro &amp; in vivo

Abstract MicroRNAs (miRNAs) are small non-coding RNAs with size of 20-25 nucleotides, and inhibit protein translation by binding the 3’-untranslated region (3’-UTR) of target mRNA. Each miRNA can regulate multiple mRNAs and each mRNA can be targeted by a number of miRNAs. In cancer, miRNAs can act as not only tumor suppressor genes but also oncogenes. Addiction to oncogenic miRNAs (OncomiRs) may provide therapeutic opportunities in human cancers such as oncogene addiction. In this study, we have attempted to identify OncomiRs in human oral squamous cell carcinoma (OSCC) cells and considered whether targeting miRNAs can be possible for cancer therapy. Functional screening for OncomiRs in human OSCC cells was performed with the use of miRCURY LNATM microRNA Knockdown Library-Human v12.0 (EXIQON). We transfected 918 locked nucleic acid (LNA)/DNA antisense oligonucleotides for specific human mature miRNAs into human OSCC cells (GFP-SAS). After 80 hours, each cell growth was evaluated by WST-8 assay. We identified LNA/DNA antisense oligonucleotide against miR-361-3p (LNA-miR-361-3p) which showed remarkable growth inhibition in GFP-SAS cells. Subsequently, we confirmed the target specificity of LNA-miR-361-3p by quantitative RT-PCR (qRT-PCR). LNA-miR-361-3p significantly reduced the expression of miR-361-3p. Co-transfection of synthetic mature miR-361-3p abrogated the growth inhibitory effect of LNA-miR-361-3p in GFP-SAS cells. Furthermore, transfection of synthetic mature miR-361-3p resulted in 20% increase of cell growth in GFP-SAS cells. Next, we identified odd-skipped related 2 (OSR2) as a target gene of miR-361-3p by the use of microarray, GeneSpring GX, and Ingenuity Pathway Analysis (IPA). The expression of OSR2 after transfection with LNA-miR-361-3p was significantly induced, and co-transfection of the OSR2 3’-UTR luciferase reporter plasmid and LNA-miR-361-3p into GFP-SAS cells produced higher luciferase activity than cells in co-transfected with LNA-non target (LNA-NT). Finally, we assessed the effect of LNA-miR-361-3p on the in vivo growth of GFP-SAS cells. We administered LNA-miR-361-3p into the xenograft tumors every 3 days. We found that LNA-miR-361-3p significantly reduced the size of subcutaneously xenografted GFP-SAS tumors, compared to the control group treated with LNA-NT. The expression of miR-361-3p in excised tumors was examined by qRT-PCR. LNA-miR-361-3p suppressed the expression levels of miR-361-3p compared with the control tumors. These results suggest that miR-361-3p support the growth of human OSCC cells and that targeting miR-361-3p may be a useful therapeutic approach for patients with OSCC. Citation Format: Norihiko Tokuzen, Koh-ichi Nakashiro, Himiko Ogawa, Hiroyuki Goda. Targeting miR-361-3p suppresses the growth of human oral squamous cell carcinoma cells in vitro &amp; in vivo [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3553.