scholarly journals Durable multitransgene expression in vivo using systemic, nonviral DNA delivery

2019 ◽  
Vol 5 (11) ◽  
pp. eaax0217 ◽  
Author(s):  
Chakkrapong Handumrongkul ◽  
Alice L. Ye ◽  
Stephen A. Chmura ◽  
Liliana Soroceanu ◽  
Marissa Mack ◽  
...  
Keyword(s):  
Immune Responses ◽  
Therapeutic Potential ◽  
Expression System ◽  
System A ◽  
Human Proteins ◽  
Delivery Platform ◽  
Immunocompetent Mice ◽  
High Level ◽  
Deficiency Diseases

Recombinant adeno-associated virus (AAV) vectors are transforming therapies for rare human monogenic deficiency diseases. However, adaptive immune responses to AAV and its limited DNA insert capacity, restrict their therapeutic potential. HEDGES (high-level extended duration gene expression system), a nonviral DNA- and liposome-based gene delivery platform, overcomes these limitations in immunocompetent mice. Specifically, one systemic HEDGES injection durably produces therapeutic levels of transgene-encoded human proteins, including FDA–approved cytokines and monoclonal antibodies, without detectable integration into genomic DNA. HEDGES also controls protein production duration from <3 weeks to >1.5 years, does not induce anti-vector immune responses, is reexpressed for prolonged periods following reinjection, and produces only transient minimal toxicity. HEDGES can produce extended therapeutic levels of multiple transgene-encoded therapeutic human proteins from DNA inserts >1.5-fold larger than AAV-based therapeutics, thus creating combinatorial interventions to effectively treat common polygenic diseases driven by multigenic abnormalities.

Nanoparticle delivery of microRNA-146a regulates mechanotransduction in lung macrophages and mitigates lung injury during mechanical ventilation

2019 ◽  
Author(s):  
Christopher Bobba ◽  
Qinqin Fei ◽  
Vasudha Shukla ◽  
Hyunwook Lee ◽  
Pragi Patel ◽  
...  
Keyword(s):  
Mechanical Ventilation ◽  
Lung Injury ◽  
Therapeutic Potential ◽  
Compensatory Response ◽  
Nanoparticle Delivery ◽  
Biophysical Forces ◽  
Delivery Platform ◽  
In Vitro Systems

ABSTRACTDuring mechanical ventilation, injurious biophysical forces exacerbate lung injury. These forces disrupt alveolar capillary barrier integrity, trigger proinflammatory mediator release, and differentially regulate genes and non-coding oligonucleotides such as microRNAs. In this study, we identify miR-146a as a mechanosensitive microRNA in alveolar macrophages that has therapeutic potential to mitigate lung injury during mechanical ventilation. We used humanized in-vitro systems, mouse models, and biospecimens from mechanically ventilated patients to elucidate the expression dynamics of miR-146a that might be required to decrease lung injury during mechanical ventilation. We found that the endogenous increase in miR-146a following injurious was relatively modest and not sufficient to prevent lung injury. However, when miR-146a was highly overexpressed using a nanoparticle-based delivery platform in vivo, it was sufficient to prevent lung injury. These data indicate that the endogenous increase in microRNA-146a during MV is a compensatory response that only partially limits VILI and that nanoparticle delivery approaches that significantly over-express microRNA-146a in AMs is an effective strategy for mitigating VILI.

Novel monoclonal antibody therapeutics for metastatic castration resistant prostate cancer.

2019 ◽  
Vol 37 (15_suppl) ◽  
pp. e14222-e14222
Author(s):  
Maloy Ghosh ◽  
Kavitha Iyer Rodrigues ◽  
Sunit Maity ◽  
Sanghamitra Bhattacharjee ◽  
Yogendra Manjunath ◽  
...  
Keyword(s):  
Prostate Cancer ◽  
Immune Responses ◽  
Nk Cells ◽  
Nk Cell ◽  
Therapeutic Potential ◽  
Target Cells ◽  
Castration Resistant Prostate Cancer ◽  
Growth Reduction ◽  
Castration Resistant

e14222 Background: Therapeutic potential of innate immunity comprising Natural killer cell based targets are beginning to unravel the complexity of immune responses. NK cells recognize and induce cytotoxicity of wide range of target cells, such as, tumor cells without prior antigen sensitization. In this study, we have studied Lectin-like transcript 1 (LLT1), a member of the C-type lectin super family, is expressed on target cells and various immune cells. LLT1 isoform 1, is known to interact with CD161, a critical receptor on NK cells. CD161 is expressed on most of human NK cells, NK-T cells, γδ T cells and so on. Tumors exploit the CD161- LLT1 interaction to evade host defense mechanism (“DO NOT KILL” signal); indicating LLT1 as an attractive immunotherapeutic strategy. Methods: Prostate cancer cell lines and other tumor cell lines were used to evaluate novel anti LLT1 antibodies for therapeutic potential - IFNγ production assays and tumor cell death assays were carried out. In vivo efficacy of these antibodies were established using PC3 xenograft in humanized mouse model (HuNOG-EXL). Results: Human androgen independent prostate cancer cell line, PC3 was studied for LLT1 expression and interactions with immune cells, to understand role of LLT1 in metastatic castration resistant prostate cancer (mCRPC). Overexpression of LLT1 on tumor cells was influenced by cytokines and various TLRs. Inhibition of CD161-LLT1 interaction with novel anti LLT1 antibodies leads to IFNγ production and consequent NK cell mediated cytotoxicity – hall mark of anti-tumor responses. Disruption of LLT1 - CD161 innate immunity axis with anti LLT1 antibody releases the break on NK cell cytotoxicity and hence, established a new therapeutic option. PC3 xenograft on HuNOG mouse revealed in vivo efficacy of LLT1 antibody. Significant tumor growth reduction was observed with specific anti LLT1 antibodies alone and in combination with check point antibodies. Thus, synergistic tumor growth reduction was established by combinatorial application of anti LLT1 antibody and PD1/PDL1 axis inhibitors. Conclusions: PC3 xenograft study and other results point to therapeutic opportunities in metastatic castration resistant prostate cancer, a disease condition which needs improved patient outcomes. The ligation of CD161/LLT1 will serve as a new immuno-oncology pair regulating innate and adaptive immune responses; novel human antibodies against LLT1 described here will bring therapeutic benefit to patients in need.

scholarly journals Frutapin, a lectin from Artocarpus incisa (breadfruit): cloning, expression and molecular insights

2017 ◽  
Vol 37 (4) ◽  
Author(s):  
Felipe Domingos de Sousa ◽  
Bruno Bezerra da Silva ◽  
Gilvan Pessoa Furtado ◽  
Igor de Sa Carneiro ◽  
Marina Duarte Pinto Lobo ◽  
...  
Keyword(s):  
Biomarker Discovery ◽  
Therapeutic Potential ◽  
Expression System ◽  
Mouse Fibroblast ◽  
Biologically Active ◽  
Carbohydrate Binding ◽  
X Rays ◽  
Minimal Concentration ◽  
High Level ◽  
Artocarpus Incisa

Artocarpus incisa (breadfruit) seeds contain three different lectins (Frutalin, Frutapin (FTP) and Frutackin) with distinct carbohydrate specificities. The most abundant lectin is Frutalin, an α-D-galactose-specific carbohydrate-binding glycoprotein with antitumour properties and potential for tumour biomarker discovery as already reported. FTP is the second most abundant, but proved difficult to purify with very low yields and contamination with Frutalin frustrating its characterization. Here, we report for the first time high-level production and isolation of biologically active recombinant FTP in Escherichia coli BL21, optimizing conditions with the best set yielding >40 mg/l culture of soluble active FTP. The minimal concentration for agglutination of red blood cells was 62.5 µg/ml of FTP, a process effectively inhibited by mannose. Apo-FTP, FTP–mannose and FTP–glucose crystals were obtained, and they diffracted X-rays to a resolution of 1.58 (P212121), 1.70 (P3121) and 1.60 (P3121) Å respectively. The best solution showed four monomers per asymmetric unit. Molecular dynamics (MD) simulation suggested that FTP displays higher affinity for mannose than glucose. Cell studies revealed that FTP was non-cytotoxic to cultured mouse fibroblast 3T3 cells below 0.5 mg/ml and was also capable of stimulating cell migration at 50 µg/ml. In conclusion, our optimized expression system allowed high amounts of correctly folded soluble FTP to be isolated. This recombinant bioactive lectin will now be tested in future studies for therapeutic potential; for example in wound healing and tissue regeneration.

scholarly journals Mutations in the GNAS Gene Prevent the Cell Invasion by Activating the MEG3/Wnt/β-catenin Axia in Growth Hormone-secreting Pituitary Adenoma

2021 ◽  
Author(s):  
Chao Tang ◽  
Chunyu Zhong ◽  
Junhao Zhu ◽  
Feng Yuan ◽  
Jin Yang ◽  
...  
Keyword(s):  
Growth Hormone ◽  
Pituitary Adenoma ◽  
Signaling Pathway ◽  
Cell Invasion ◽  
Expression System ◽  
Gene Mutations ◽  
Gh3 Cells ◽  
Α Subunit ◽  
High Level

Abstract Approximately 30–40% of growth hormone-secreting pituitary adenoma (GHPA) harbor somatic mutations in the GNAS (α subunit of the stimulatory G protein) gene. However, the latent functional role of the mutations and relative molecular mechanism in GHPA remain unknown. The GNAS gene mutations were detected in GHPAs using a standard PCR-sequencing procedure. The mutation-associated MEG3 expression was measured by RT-qPCR. MEG3 was manipulated in GH3 cells using a lentiviral expression system. Alterations in mRNA profiles in the MEG3-overexpressed cells were analyzed by RNA-seq. The cell invasion ability was measured using a Transwell assay, and the EMT-associated proteins were quantified by immunofluorescence and western blot. Finally, a tumor cell xenograft mouse model was applied to verify the effect of MEG3 on tumor growth and invasiveness. The percentage of invasive tumors was significantly declined in GNAS-mutated GHPA tumors with the GNAS mutations compared to those tumors with the wild-type of GNAS. Consistently, the GH3 cell invasion capacity was decreased by expressing the mutant GNAS. MEG3 is uniquely expressed at high levels in GHPA harboring the mutated GNAS gene. Accordingly, the upregulation of MEG3 resulted in inhibiting cell invasion; and vice versa, the downregulation of MEG3 led to enhancing cell invasion. Mechanistically, the high level of MEG3 in mutated GNAS cells prevented the cell invasion via inactivation of the Wnt/β-catenin signaling pathway, which was further validated in vivo. The GNAS mutations inhibit the invasiveness of GHPA cells via inactivation of the MEG3/Wnt/β-catenin signaling pathway.

scholarly journals Complete Genome Sequence of Escherichia coli BL21-AI

2020 ◽  
Vol 9 (10) ◽  
Author(s):  
Niketa Bhawsinghka ◽  
Katie F. Glenn ◽  
Roel M. Schaaper
Keyword(s):  
Escherichia Coli ◽  
Genome Sequence ◽  
Complete Genome Sequence ◽  
Complete Genome ◽  
Expression System ◽  
High Yield ◽  
Content Type ◽  
System A ◽  
Phage T7 ◽  
High Level

Escherichia coli BL21-AI is a commercially available strain possessing a phage T7-based protein-expression system. A combination of tight regulation and high yield makes it widely used for high-level expression of toxic recombinant proteins. Here, we present the complete genome sequence of BL21-AI and provide insights on its genome.

scholarly journals Interleukin 35 induced Th2 and Tregs bias under normal conditions in mice

PeerJ ◽  
2018 ◽  
Vol 6 ◽  
pp. e5638 ◽  
Author(s):  
Xiaoning Zhang ◽  
Zhiqiang Zhang ◽  
Zhiqiang He ◽  
Mingyan Ju ◽  
Jiaci Li ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Therapeutic Potential ◽  
T Cell Subsets ◽  
Effector Molecules ◽  
Dna Injection ◽  
High Level ◽  
Cell Inhibition

Objective The benefits of IL-35 treatment have been verified in multiple animal models of diseases, while its influence on T cells immunity under normal condition still needs to be elucidated. The present study was designed to investigate the effects modulating IL-35 levels in vivo and in vitro on T cells, response and also the effects on T cells subsets in normal mice. Methods A plasmid pMSCV-IL-35-GFP carrying mouse linear IL-35 fragment with two subunits joint together was constructed and the heterodimer expression was confirmed. Normal mice were randomly divided into three groups and received an intravenous injection of PBS, pMSCV-GFP and pMSCV-IL-35-GFP respectively. After 72 h, spleen tissues and peripheral blood were harvested for following analysis. Meanwhile, splenic T cells were isolated and incubated with 10, 30, or 50 ng/mL recombinant IL-35 factor for 24 h with the addition of anti-CD3/CD28 in vitro. T-cell subsets were assessed by Fluorescence activated cell sorting (FACS) and related cytokines together with effector molecules were determined by real time PCR. Results Western blotting confirmed a 52 kDa band in the cell lysate of HEK 293T transducted with pMSCV-IL-35-GFP plasmid, indicating a successful expression of IL-35. Ebi3 and IL-12A, two subunits of IL-35, could be identified 72 h post DNA injection. IL-35 upregulation in vivo effectively inhibit CD4+ and CD8+ T cell proliferation and Th1 cytokine secretion. Effector molecules of CD8+ T cells were also remarkably suppressed. On the contrary, high level of IL-35 significantly induced CD4+ CD25+ Tregs and Th2 enhancement. The in vitro study provided similar results. Conclusion The results indicated Th1 and CD8+ T cell inhibition and Th2 and Tregs bias in the presence of IL-35 under a normal state which partly contributed to its therapeutic potential.

scholarly journals First report of a tetracycline-inducible gene expression system for mollicutes

Microbiology ◽  
2010 ◽  
Vol 156 (1) ◽  
pp. 198-205 ◽  
Author(s):  
Marc Breton ◽  
Evelyne Sagné ◽  
Sybille Duret ◽  
Laure Béven ◽  
Christine Citti ◽  
...  
Keyword(s):  
Gene Expression ◽  
Expression System ◽  
Inducible Promoter ◽  
High Level Expression ◽  
Spiroplasma Citri ◽  
High Level ◽  
Tetr Repressor ◽  
Promoter System ◽  
Inducible Gene

Inducible promoter systems are powerful tools for studying gene function in prokaryotes but have never been shown to function in mollicutes. In this study we evaluated the efficacy of the tetracycline-inducible promoter Pxyl/tetO2 from Bacillus subtilis in controlling gene expression in two mollicutes, the plant pathogen Spiroplasma citri and the animal pathogen Mycoplasma agalactiae. An S. citri plasmid carrying the spiralin gene under the control of the xyl/tetO2 tetracycline-inducible promoter and the TetR repressor gene under the control of a constitutive spiroplasmal promoter was introduced into the spiralin-less S. citri mutant GII3-9a3. In the absence of tetracycline, expression of TetR almost completely abolished expression of spiralin from the xyl/tetO2 promoter. Adding tetracycline (>50 ng ml−1) to the medium induced high-level expression of spiralin. Interestingly, inducible expression of spiralin was also detected in vivo: in S. citri-infected leafhoppers fed on tetracycline-containing medium and in S. citri-infected plants watered with tetracycline. A similar construct was introduced into the M. agalactiae chromosome through transposition. Tetracycline-induced expression of spiralin proved the TetR-Pxyl/tetO2 system to be functional in the ruminant pathogen, suggesting that this tetracycline-inducible promoter system might be of general use in mollicutes.

scholarly journals The Selenoprotein MsrB1 Instructs Dendritic Cells to Induce T-Helper 1 Immune Responses

Antioxidants ◽  
2020 ◽  
Vol 9 (10) ◽  
pp. 1021
Author(s):  
Ho-Jae Lee ◽  
Joon Seok Park ◽  
Hyun Jung Yoo ◽  
Hae Min Lee ◽  
Byung Cheon Lee ◽  
...  
Keyword(s):  
Dendritic Cells ◽  
T Cell ◽  
Immune Responses ◽  
Therapeutic Potential ◽  
Methionine Sulfoxide Reductase ◽  
Interleukin 12 ◽  
T Helper ◽  
T Helper 1

Immune activation associates with the intracellular generation of reactive oxygen species (ROS). To elicit effective immune responses, ROS levels must be balanced. Emerging evidence shows that ROS-mediated signal transduction can be regulated by selenoproteins such as methionine sulfoxide reductase B1 (MsrB1). However, how the selenoprotein shapes immunity remains poorly understood. Here, we demonstrated that MsrB1 plays a crucial role in the ability of dendritic cells (DCs) to provide the antigen presentation and costimulation that are needed for cluster of differentiation antigen four (CD4) T-cell priming in mice. We found that MsrB1 regulated signal transducer and activator of transcription-6 (STAT6) phosphorylation in DCs. Moreover, both in vitro and in vivo, MsrB1 potentiated the lipopolysaccharide (LPS)-induced Interleukin-12 (IL-12) production by DCs and drove T-helper 1 (Th1) differentiation after immunization. We propose that MsrB1 activates the STAT6 pathway in DCs, thereby inducing the DC maturation and IL-12 production that promotes Th1 differentiation. Additionally, we showed that MsrB1 promoted follicular helper T-cell (Tfh) differentiation when mice were immunized with sheep red blood cells. This study unveils as yet unappreciated roles of the MsrB1 selenoprotein in the innate control of adaptive immunity. Targeting MsrB1 may have therapeutic potential in terms of controlling immune reactions.

scholarly journals Interferon α/β and Interleukin 12 Responses to Viral Infections

2002 ◽  
Vol 195 (4) ◽  
pp. 517-528 ◽  
Author(s):  
Marc Dalod ◽  
Thais P. Salazar-Mather ◽  
Lene Malmgaard ◽  
Casey Lewis ◽  
Carine Asselin-Paturel ◽  
...  
Keyword(s):  
Immune Responses ◽  
Viral Infections ◽  
Lymphocytic Choriomeningitis ◽  
Murine Cytomegalovirus ◽  
Interleukin 12 ◽  
Interferon Α ◽  
Type I ◽  
Type I Ifns ◽  
Immunocompetent Mice

Interferon (IFN)-α/β and interleukin (IL)-12 are cytokines critical in defense against viruses, but their cellular sources and mechanisms of regulation for in vivo expression remain poorly characterized. The studies presented here identified a novel subset of dendritic cells (DCs) as major producers of the cytokines during murine cytomegalovirus (MCMV) but not lymphocytic choriomeningitis virus (LCMV) infections. These DCs differed from those activated by Toxoplasma antigen but were related to plasmacytoid cells, as assessed by their CD8α+Ly6G/C+CD11b− phenotype. Another DC subset (CD8α2Ly6G/C−CD11b+) also contributed to IL-12 production in MCMV-infected immunocompetent mice, modestly. However, it dramatically increased IL-12 expression in the absence of IFN-α/β functions. Conversely, IFN-α/β production was greatly reduced under these conditions. Thus, a cross-regulation of DC subset cytokine responses was defined, whereby secretion of type I IFNs by CD8α+ DCs resulted in responses limiting IL-12 expression by CD11b+ DCs but enhancing overall IFN-α/β production. Taken together, these data indicate that CD8α+Ly6G/C+CD11b− DCs play important roles in limiting viral replication and regulating immune responses, through cytokine production, in some but not all viral infections. They also illustrate the plasticity of cellular sources for innate cytokines in vivo and provide new insights into the roles of IFNs in shaping immune responses to viruses.

scholarly journals Tyrosine Phosphorylation of the Proto-Oncoprotein Raf-1 Is Regulated by Raf-1 Itself and the Phosphatase Cdc25A

1999 ◽  
Vol 19 (7) ◽  
pp. 4819-4824 ◽  
Author(s):  
Kai Xia ◽  
Robert S. Lee ◽  
Radha P. Narsimhan ◽  
Nishit K. Mukhopadhyay ◽  
Benjamin G. Neel ◽  
...  
Keyword(s):  
Tyrosine Phosphorylation ◽  
Expression System ◽  
Baculovirus Expression System ◽  
Wild Type ◽  
Phosphatase Inhibitors ◽  
System A ◽  
New Findings ◽  
Nih 3T3 ◽  
Tyrosine Phosphate

ABSTRACT There is a growing body of evidence demonstrating that Raf-1 is phosphorylated on tyrosines upon stimulation of a variety of receptors. Although detection of Raf-1 tyrosine phosphorylation has remained elusive, genetic analyses have demonstrated it to be important for Raf-1 activation. Here we report new findings which indicate that Raf-1 tyrosine phosphorylation is regulated in vivo. In both a mammalian and baculovirus expression system, a kinase-inactive allele of Raf-1 was found to be tyrosine phosphorylated at levels much greater than that of wild-type Raf-1. The level of tyrosine phosphate on Raf-1 was markedly increased upon treatment with phosphatase inhibitors either before or after cell lysis. Cdc25A was found to dephosphorylate Raf-1 on tyrosines that resulted in a significant decrease in Raf-1 kinase activity. In NIH 3T3 cells, coexpression of wild-type Raf-1 and phosphatase-inactive Cdc25A led to a marked increase in Raf-1 tyrosine phosphorylation in response to platelet-derived growth factor. These data suggest that the tyrosine phosphorylation of Raf-1 is regulated not only by itself but also by Cdc25A.

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