scholarly journals Regulation of the Target Protein (Transgene) Expression in the Adenovirus Vector Using Agonists of Toll-Like Receptors

Acta Naturae ◽  
2014 ◽  
Vol 6 (4) ◽  
pp. 27-39 ◽  
Author(s):  
A. V. Bagaev ◽  
A. V. Pichugin ◽  
E. S. Lebedeva ◽  
A. A. Lysenko ◽  
M. M. Shmarov ◽  
...  
Keyword(s):  
Gene Therapy ◽  
Transgene Expression ◽  
Target Genes ◽  
Dominant Role ◽  
Adenoviral Vectors ◽  
Target Protein ◽  
Toll Like Receptors ◽  
Tlr Agonists ◽  
Gene Vaccination ◽  
In Cells

Replication-defective adenoviral vectors are effective molecular tools for both gene therapy and gene vaccination. Using such vectors one can deliver and express target genes in different epithelial, liver, hematopoietic and immune system cells of animal and human origin. The success of gene therapy and gene vaccination depends on the production intensity of the target protein encoded by the transgene. In this work, we studied influence of Toll-like receptors (TLR) agonists on transduction and expression efficacy of adenoviral vectors in animal and human antigen-presenting cells. We found that agonists of TLR2, 4, 5, 7, 8 and 9 significantly enhance a production of the target protein in cells transduced with adenoviral vector having the target gene insert. The enhancement was observed in dendritic cells and macrophages expressing cytoplasmic (GFP), membrane (HA) or secretory (SEAP) proteins encoded by the respective rAd-vectors. Experiments in mice showed that enhancement of the transgene expression can be achieved in the organism of animals using a pharmaceutical-grade TLR4-agonist. In contrast to other TLR-agonists, the agonist of TLR3 substantially suppressed the expression of transgene in cells transduced with adenoviral vectors having insert of GFP or SEAP target genes. We propose that the enhancement of transgene expression is linked to the activation of MyD88 NF-kB, while the inhibition of transgene expression depends on TRIF IRF signaling pathways. Both of these pathways jointly exploited by TLR4-agonists lead to the enhancement of transgene expression due to the dominant role of the MyD88 NF-kB signaling.

scholarly journals Gene expression induced by Toll-like receptors in macrophages requires the transcription factor NFAT5

2012 ◽  
Vol 209 (2) ◽  
pp. 379-393 ◽  
Author(s):  
Maria Buxadé ◽  
Giulia Lunazzi ◽  
Jordi Minguillón ◽  
Salvador Iborra ◽  
Rosa Berga-Bolaños ◽  
...  
Keyword(s):  
Histone Deacetylases ◽  
Target Genes ◽  
Leishmania Major ◽  
De Novo ◽  
Specific Gene ◽  
Toll Like Receptors ◽  
Tlr Agonists ◽  
High Doses ◽  
Pathogen Burden

Toll-like receptors (TLRs) engage networks of transcriptional regulators to induce genes essential for antimicrobial immunity. We report that NFAT5, previously characterized as an osmostress responsive factor, regulates the expression of multiple TLR-induced genes in macrophages independently of osmotic stress. NFAT5 was essential for the induction of the key antimicrobial gene Nos2 (inducible nitric oxide synthase [iNOS]) in response to low and high doses of TLR agonists but is required for Tnf and Il6 mainly under mild stimulatory conditions, indicating that NFAT5 could regulate specific gene patterns depending on pathogen burden intensity. NFAT5 exhibited two modes of association with target genes, as it was constitutively bound to Tnf and other genes regardless of TLR stimulation, whereas its recruitment to Nos2 or Il6 required TLR activation. Further analysis revealed that TLR-induced recruitment of NFAT5 to Nos2 was dependent on inhibitor of κB kinase (IKK) β activity and de novo protein synthesis, and was sensitive to histone deacetylases. In vivo, NFAT5 was necessary for effective immunity against Leishmania major, a parasite whose clearance requires TLRs and iNOS expression in macrophages. These findings identify NFAT5 as a novel regulator of mammalian anti-pathogen responses.

scholarly journals Helper-dependent adenoviral vectors in experimental gene therapy.

2005 ◽  
Vol 52 (3) ◽  
pp. 589-599 ◽  
Author(s):  
Alicja Józkowicz ◽  
Józef Dulak
Keyword(s):  
Gene Therapy ◽  
Transgene Expression ◽  
Inflammatory Responses ◽  
Adenoviral Vectors ◽  
Strong Immune Response ◽  
Basic Features ◽  
High Level ◽  
Effective Transfer

In the majority of potential applications gene therapy will require an effective transfer of a transgene in vivo resulting in high-level and long-term transgene expression, all in the absence of significant toxicity or inflammatory responses. The most efficient vehicles for delivery of foreign genes to the target tissues are modified adenoviruses. Adenoviral vectors of the first generation, despite the high infection efficacy, have an essential drawback: they induce strong immune response, which leads to short term expression of the transgene, and limits their usefulness in clinical trials. In contrast, helper-dependent adenoviral vectors (HdAd) lacking all viral coding sequences display only minimal immunogenicity and negligible side-effects, allowing for long-term transgene expression. Thus, HdAd vehicles have become the carrier of choice for adenoviral vector-mediated experimental gene therapy, effectively used in animal models for delivery of transgenes into the liver, skeletal muscle, myocardium or brain. Strong and long-lasting expression of therapeutic genes has allowed for successful treatment of dyslipidemias, muscular dystrophy, obesity, hemophilia, and diabetes. Additionally, the large cloning capacity of HdAd, up to 37 kb, facilitates the use of physiologically regulated, endogenous promoters, instead of artificial viral promoter sequences. This enables also generation of the single vectors expressing multiple genes, which can be potentially useful for treatment of polygenic diseases. In this review we characterize the basic features of HdAd vectors and describe some of their experimental and potential clinical applications.

O‐GlcNAc Engineering on a Target Protein in Cells with Nanobody‐OGT and Nanobody‐splitOGA

2021 ◽  
Vol 1 (5) ◽  
Author(s):  
Daniel H. Ramirez ◽  
Yun Ge ◽  
Christina M. Woo
Keyword(s):  
Target Protein ◽  
In Cells

scholarly journals The efficiency of murine MLL-ENL–driven leukemia initiation changes with age and peaks during neonatal development

2019 ◽  
Vol 3 (15) ◽  
pp. 2388-2399 ◽  
Author(s):  
Theresa Okeyo-Owuor ◽  
Yanan Li ◽  
Riddhi M. Patel ◽  
Wei Yang ◽  
Emily B. Casey ◽  
...  
Keyword(s):  
Myeloid Leukemia ◽  
Transgene Expression ◽  
Target Genes ◽  
Lymphoblastic Leukemia ◽  
Human Infant ◽  
Driver Mutations ◽  
Hematopoietic Progenitors ◽  
Adult Mice ◽  
Almost All ◽  
Infant Leukemia

Abstract MLL rearrangements are translocation mutations that cause both acute lymphoblastic leukemia and acute myeloid leukemia (AML). These translocations can occur as sole clonal driver mutations in infant leukemias, suggesting that fetal or neonatal hematopoietic progenitors may be exquisitely sensitive to transformation by MLL fusion proteins. To test this possibility, we used transgenic mice to induce one translocation product, MLL-ENL, during fetal, neonatal, juvenile and adult stages of life. When MLL-ENL was induced in fetal or neonatal mice, almost all died of AML. In contrast, when MLL-ENL was induced in adult mice, most survived for >1 year despite sustained transgene expression. AML initiation was most efficient when MLL-ENL was induced in neonates, and even transient suppression of MLL-ENL in neonates could prevent AML in most mice. MLL-ENL target genes were induced more efficiently in neonatal progenitors than in adult progenitors, consistent with the distinct AML initiation efficiencies. Interestingly, transplantation stress mitigated the developmental barrier to leukemogenesis. Since fetal/neonatal progenitors were highly competent to initiate MLL-ENL–driven AML, we tested whether Lin28b, a fetal master regulator, could accelerate leukemogenesis. Surprisingly, Lin28b suppressed AML initiation rather than accelerating it. This may explain why MLL rearrangements often occur before birth in human infant leukemia patients, but transformation usually does not occur until after birth, when Lin28b levels decline. Our findings show that the efficiency of MLL-ENL–driven AML initiation changes through the course of pre- and postnatal development, and developmental programs can be manipulated to impede transformation.

scholarly journals Efficient and Selective Gene Transfer into Primary Human Brain Tumors by Using Single-Chain Antibody-Targeted Adenoviral Vectors with Native Tropism Abolished

2002 ◽  
Vol 76 (6) ◽  
pp. 2753-2762 ◽  
Author(s):  
Victor W. van Beusechem ◽  
Jacques Grill ◽  
D. C. Jeroen Mastenbroek ◽  
Thomas J. Wickham ◽  
Peter W. Roelvink ◽  
...  
Keyword(s):  
Gene Therapy ◽  
Gene Transfer ◽  
Brain Tumors ◽  
Human Brain ◽  
Cancer Gene Therapy ◽  
Adenoviral Vectors ◽  
Receptor Expression ◽  
Normal Brain ◽  
Cancer Gene ◽  
Single Chain

ABSTRACT The application of adenoviral vectors in cancer gene therapy is hampered by low receptor expression on tumor cells and high receptor expression on normal epithelial cells. Targeting adenoviral vectors toward tumor cells may improve cancer gene therapy procedures by providing augmented tumor transduction and decreased toxicity to normal tissues. Targeting requires both the complete abolition of native tropism and the addition of a new specific binding ligand onto the viral capsid. Here we accomplished this by using doubly ablated adenoviral vectors, lacking coxsackievirus-adenovirus receptor and αv integrin binding capacities, together with bispecific single-chain antibodies targeted toward human epidermal growth factor receptor (EGFR) or the epithelial cell adhesion molecule. These vectors efficiently and selectively targeted both alternative receptors on the surface of human cancer cells. Targeted doubly ablated adenoviral vectors were also very efficient and specific with primary human tumor specimens. With primary glioma cell cultures, EGFR targeting augmented the median gene transfer efficiency of doubly ablated adenoviral vectors 123-fold. Moreover, EGFR-targeted doubly ablated vectors were selective for human brain tumors versus the surrounding normal brain tissue. They transduced organotypic glioma and meningioma spheroids with efficiencies similar to those of native adenoviral vectors, while exhibiting greater-than-10-fold-reduced background levels on normal brain explants from the same patients. As a result, EGFR-targeted doubly ablated adenoviral vectors had a 5- to 38-fold-improved tumor-to-normal brain targeting index compared to native vectors. Hence, single-chain targeted doubly ablated adenoviral vectors are promising tools for cancer gene therapy. They should provide an improved therapeutic index with efficient tumor transduction and effective protection of normal tissue.

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