scholarly journals Transgene codon usage drives viral fitness and therapeutic efficacy in oncolytic adenoviruses

NAR Cancer ◽  
2021 ◽  
Vol 3 (2) ◽  
Author(s):  
Estela Núñez-Manchón ◽  
Martí Farrera-Sal ◽  
Marc Otero-Mateo ◽  
Giancarlo Castellano ◽  
Rafael Moreno ◽  
...  
Keyword(s):  
Viral Replication ◽  
Codon Usage ◽  
Transgene Expression ◽  
Rational Design ◽  
Viral Fitness ◽  
Fine Tuning ◽  
Oncolytic Adenoviruses ◽  
Oncolytic Activity

Abstract Arming oncolytic adenoviruses with therapeutic transgenes is a well-established strategy for multimodal tumour attack. However, this strategy sometimes leads to unexpected attenuated viral replication and a loss of oncolytic effects, preventing these viruses from reaching the clinic. Previous work has shown that altering codon usage in viral genes can hamper viral fitness. Here, we have analysed how transgene codon usage impacts viral replication and oncolytic activity. We observe that, although transgenes with optimized codons show high expression levels at the first round of infection, they impair viral fitness and are therefore not expressed in a sustained manner. Conversely, transgenes encoded by suboptimal codons do not compromise viral replication and are thus stably expressed over time, allowing a greater oncolytic activity both in vitro and in vivo. Altogether, our work shows that fine-tuning codon usage leads to a concerted optimization of transgene expression and viral replication paving the way for the rational design of more efficacious oncolytic therapies.

scholarly journals Transgene codon usage drives viral fitness and therapeutic efficacy in oncolytic adenoviruses

2020 ◽  
Author(s):  
Estela Núñez-Manchón ◽  
Martí Farrera-Sal ◽  
Giancarlo Castellano ◽  
David Medel ◽  
Ramon Alemany ◽  
...  
Keyword(s):  
Viral Replication ◽  
Codon Usage ◽  
Transgene Expression ◽  
Rational Design ◽  
Viral Fitness ◽  
Fine Tuning ◽  
Oncolytic Adenoviruses ◽  
Oncolytic Activity

AbstractArming oncolytic adenoviruses with therapeutic transgenes is a well-established strategy for multimodal tumour attack. However, this strategy sometimes leads to unexpected attenuated viral replication and a loss of oncolytic effects, preventing these viruses from reaching the clinic. Previous work has shown that altering codon usage in viral genes can hamper viral fitness. Here, we have analysed how transgene codon usage impacts viral replication and oncolytic activity. We observe that, although transgenes with optimised codons show high expression levels at a first round of infection, they impair viral fitness and are therefore not expressed in a sustained manner. Conversely, transgenes encoded by suboptimal codons do not compromise viral replication and are thus stably expressed over time allowing a greater oncolytic activity both in vitro and in vivo. Altogether, our work shows that fine-tuning codon usage leads to a concerted optimisation of transgene expression and viral replication paving the way for the rational design of more efficacious oncolytic therapies.

scholarly journals 99mTc Labelling Strategies for the Development of Potential Nitroimidazolic Hypoxia Imaging Agents

Inorganics ◽  
2019 ◽  
Vol 7 (11) ◽  
pp. 128 ◽  
Author(s):  
Giglio ◽  
Rey
Keyword(s):  
Rational Design ◽  
Biological Properties ◽  
Fine Tuning ◽  
Oxidation States ◽  
Hypoxia Imaging ◽  
Biological Target ◽  
99Mtc Radiopharmaceuticals ◽  
99Mtc Labelling

Technetium-99m has a rich coordination chemistry that offers many possibilities in terms of oxidation states and donor atom sets. Modifications in the structure of the technetium complexes could be very useful for fine tuning the physicochemical and biological properties of potential 99mTc radiopharmaceuticals. However, systematic study of the influence of the labelling strategy on the “in vitro” and “in vivo” behaviour is necessary for a rational design of radiopharmaceuticals. Herein we present a review of the influence of the Tc complexes’ molecular structure on the biodistribution and the interaction with the biological target of potential nitroimidazolic hypoxia imaging radiopharmaceuticals presented in the literature from 2010 to the present. Comparison with the gold standard [18F]Fluoromisonidazole (FMISO) is also presented.

scholarly journals Regulated hAAT Expression from a Novel rAAV Vector and Its Application in the Prevention of Type 1 Diabetes

2019 ◽  
Vol 8 (9) ◽  
pp. 1321 ◽  
Author(s):  
Hongxia Ma ◽  
Yuanqing Lu ◽  
Keith Lowe ◽  
Lonneke van der Meijden-Erkelens ◽  
Clive Wasserfall ◽  
...  
Keyword(s):  
Type 1 Diabetes ◽  
Transgene Expression ◽  
Viral Vectors ◽  
Fine Tuning ◽  
Vector System ◽  
Regulation System ◽  
Raav Vector

We, and others, have previously achieved high and sustained levels of transgene expression from viral vectors, such as recombinant adeno-associated virus (rAAV). However, regulatable transgene expression may be preferred in gene therapy for diseases, such as type 1 diabetes (T1D) and rheumatoid arthritis (RA), in which the timing and dosing of the therapeutic gene product play critical roles. In the present study, we generated a positive feedback regulation system for human alpha 1 antitrypsin (hAAT) expression in the rAAV vector. We performed quantitative kinetics studies in vitro and in vivo demonstrating that this vector system can mediate high levels of inducible transgene expression. Transgene induction could be tailored to occur rapidly or gradually, depending on the dose of the inducing drug, doxycycline (Dox). Conversely, after withdrawal of Dox, the silencing of transgene expression occurred slowly over the course of several weeks. Importantly, rAAV delivery of inducible hAAT significantly prevented T1D development in non-obese diabetic (NOD) mice. These results indicate that this Dox-inducible vector system may facilitate the fine-tuning of transgene expression, particularly for hAAT treatment of human autoimmune diseases, including T1D.

scholarly journals TMOD-16. A NOVEL ADENOVIRAL-PERMISSIVE, IMMUNOCOMPETENT HAMSTER GLIOMA MODEL TO EVALUATE ONCOLYTIC ADENOVIRAL THERAPY

2020 ◽  
Vol 22 (Supplement_2) ◽  
pp. ii231-ii231
Author(s):  
Lynette Phillips ◽  
Joy Gumin ◽  
Shoudong Li ◽  
Marc Daou ◽  
Daniel Ledbetter ◽  
...  
Keyword(s):  
Immune Response ◽  
Viral Replication ◽  
Tumor Infiltrating Lymphocytes ◽  
Peripheral Blood Lymphocytes ◽  
T Antigen ◽  
Sv40 Large T Antigen ◽  
Oncolytic Adenoviruses ◽  
Glioma Model

Abstract Oncolytic adenoviruses, including Delta-24-RGD, target tumors by direct tumor cell oncolysis and by activation of an anti-tumor immune response. Due to the species selectivity of oncolytic adenoviruses, there is currently no single preclinical animal model of glioma that supports viral replication, tumor oncolysis, and virus-mediated immune responses. To address this gap, we took advantage of the Syrian hamster to develop the first glioma model that is both adenovirus replication-permissive and immunocompetent. Hamster glioma stem-like cells (GSCs), transformed by forced expression of hTERT, SV40 large T antigen, and h-RasV12, reproducibly form intracranial tumors in hamsters. In vitro, electron microscopy and cytopathic effect assays demonstrated that hamster GSCs supported viral replication and were susceptible to Delta-24-RGD-mediated cell death. In vivo, hamster GSCs consistently developed into highly proliferative tumors resembling high-grade gliomas. Following intratumoral delivery of Delta-24-RGD, immunohistochemistry for viral proteins demonstrated viral infectivity and replication in hamster gliomas. Flow cytometry revealed increased T cell infiltration in Delta-24-RGD-infected tumors. Delta-24-RGD treatment of tumor-bearing hamsters led to significantly increased survival compared with hamsters treated with PBS. Using this model, we evaluated the effects of corticosteroid-mediated immunosuppression on Delta-24-RGD efficacy. Dexamethasone treatment significantly decreased peripheral blood lymphocytes, decreased tumor-infiltrating lymphocytes, and suppressed the levels of serum anti-adenovirus antibodies. Dexamethasone reduced the number of long-term survivors and decreased the median survival (50 days for Delta-24-RGD + dexamethasone vs undetermined for Delta-24-RGD alone). In summary, we have developed the first adenovirus-permissive, immunocompetent hamster glioma model, addressing a critical need for a model in which to study the role of direct oncolysis in driving immune mediated viral clearance versus driving an antiglioma immune response. Understanding these mechanisms is critical to optimizing the success of oncolytic adenoviral therapy in the clinic.

Novel group C oncolytic adenoviruses carrying a microRNA inhibitor demonstrate enhanced oncolytic activity in vitro and in vivo

2022 ◽  
pp. molcanther.MCT-21-0240-A.2021
Author(s):  
Johannes Doerner ◽  
Erwan Sallard ◽  
Wenli Zhang ◽  
Manish Solanki ◽  
Jing Liu ◽  
...  
Keyword(s):  
Oncolytic Adenoviruses ◽  
Oncolytic Activity

scholarly journals Uncovering the functional residues ofArabidopsisisoprenoid biosynthesis enzyme HDS

2019 ◽  
Vol 117 (1) ◽  
pp. 355-361 ◽  
Author(s):  
Jin-Zheng Wang ◽  
Yongxing Lei ◽  
Yanmei Xiao ◽  
Xiang He ◽  
Jiubo Liang ◽  
...  
Keyword(s):  
Rational Design ◽  
Binding Pocket ◽  
Mep Pathway ◽  
Fine Tuning ◽  
Ligand Docking ◽  
Stunted Growth ◽  
Mutant Phenotypes ◽  
Retrograde Signal

The methylerythritol phosphate (MEP) pathway is responsible for producing isoprenoids, metabolites with essential functions in the bacterial kingdom and plastid-bearing organisms including plants and Apicomplexa. Additionally, the MEP-pathway intermediate methylerythritol cyclodiphosphate (MEcPP) serves as a plastid-to-nucleus retrograde signal. A suppressor screen of the high MEcPP accumulating mutant plant (ceh1) led to the isolation of 3 revertants (designatedRceh1–3) resulting from independent intragenic substitutions of conserved amino acids in the penultimate MEP-pathway enzyme, hydroxymethylbutenyl diphosphate synthase (HDS). The revertants accumulate varying MEcPP levels, lower than that ofceh1, and exhibit partial or full recovery of MEcPP-mediated phenotypes, including stunted growth and induced expression of stress response genes and the corresponding metabolites. Structural modeling of HDS and ligand docking spatially position the substituted residues at the MEcPP binding pocket and cofactor binding domain of the enzyme. Complementation assays confirm the role of these residues in suppressing theceh1mutant phenotypes, albeit to different degrees. In vitro enzyme assays of wild type and HDS variants exhibit differential activities and reveal an unanticipated mismatch between enzyme kinetics and the in vivo MEcPP levels in the correspondingRcehlines. Additional analyses attribute the mismatch, in part, to the abundance of the first and rate-limiting MEP-pathway enzyme, DXS, and further suggest MEcPP as a rheostat for abundance of the upstream enzyme instrumental in fine-tuning of the pathway flux. Collectively, this study identifies critical residues of a key MEP-pathway enzyme, HDS, valuable for synthetic engineering of isoprenoids, and as potential targets for rational design of antiinfective drugs.

scholarly journals 742 Multi-armed myxoma virus induces potent anti-tumor responses in vitro and in vivo

2021 ◽  
Vol 9 (Suppl 3) ◽  
pp. A773-A773
Author(s):  
Wazir Abdullahi ◽  
Lina Franco ◽  
Christopher Fraser ◽  
Heather Hrach ◽  
Nicole Grigaitis ◽  
...  
Keyword(s):  
Tumor Growth ◽  
Transgene Expression ◽  
Caspase 3 ◽  
Cellular Responses ◽  
Myxoma Virus ◽  
Mouse Tumor ◽  
Tgf Beta ◽  
Oncolytic Activity

BackgroundMyxoma virus (MYXV) has been shown to selectively infect cancer cells in humans in vitro and inhibit tumor growth in mice. The genome of MYXV is large and amenable to engineering for expression of multiple transgenes. We armed MYXV with mouse or human IL-12 and human decorin. IL-12 is an immune modulator. Cellular responses to decorin include tumor cell intrinsic signaling effects, tumor matrix remodeling, and inhibition of the TGF-beta pathway. We hypothesized that MYXV armed with decorin and IL-12 would be an effective anti-tumor therapy. The current work describes the oncolytic activity and transgene expression, following exposure to armed MYXV in human cancer cell lines in vitro and efficacy in in vivo in murine models, as single agents and in combination with immune checkpoint inhibition.MethodsCytotoxicity was measured by a cell viability assay. ELISAs were used to detect transgene expression, Caspase-3 activation, and TGF-beta induced SMAD phosphorylation. Mouse tumor models were treated with vehicle control or the indicated virus.ResultsMYXV carrying payloads of decorin and mouse IL-12 (vMYX-mIL-12/Dec) or human IL-12 (vMYX-hIL-12/Dec) were tested. Human tumor cell lines infected with vMYX-hIL-12/Dec in vitro showed independent effects when levels of transgene expression and cytotoxicity were compared, suggesting that oncolytic activity and transgene expression differentially contribute to MYXV activity. Virus-free supernatants derived from infected cells suggested a decorin specific response in caspase-3 activation, and inhibition of TGF-beta signaling. Human IL-12 is not active on mouse immune cells giving the opportunity to evaluate the role of decorin in tumor regression. B16-F10 murine melanoma mice treated with vMYX-mIL-12/Dec showed a robust response while vMYX-hIL-12/Dec showed an intermediate anti-tumor response suggesting decorin has cancer inhibitory activity and synergized with IL-12. We tested anti-PD-1 and vMYX-mIL-12/Dec in the colon adenocarcinoma model MC38. We observed that the combination for multi-armed MYXV with an immune checkpoint inhibitor showed dramatically reduced tumor growth and improved survival.ConclusionsOur data demonstrates that MYXV with IL-12 and decorin payloads have cytotoxic activity in vitro and inhibit tumor growth in vivo. Cellular responses to decorin in vitro included inhibition of processes intrinsic to tumor progression. In mouse tumor models decorin played a role in inhibiting tumor progression and synergized with IL-12 implying the combination has immune-modulatory activity. Interestingly, MYXV with IL-12 and decorin payloads significantly synergized with anti-PD-1 in preventing tumor growth, suggesting a potentially new approach towards anti-cancer therapy.Ethics ApprovalAll studies and procedures involving animals were carried out under the institutional guidelines of Translational Drug Development Institutional Animal Care and Use Committee

scholarly journals miR-155 induction is a marker of murine norovirus infection but does not contribute to control of replication in vivo

2018 ◽  
Vol 3 ◽  
pp. 42 ◽  
Author(s):  
Lucy Thorne ◽  
Jia Lu ◽  
Yasmin Chaudhry ◽  
Ian Goodfellow
Keyword(s):  
Bone Marrow ◽  
Viral Replication ◽  
Cell Line ◽  
Cell Lines ◽  
Cellular Protein ◽  
Fine Tuning ◽  
Murine Norovirus ◽  
Serum Igg

Background: Due to their role in fine-tuning cellular protein expression, microRNAs both promote viral replication and contribute to antiviral responses, for a range of viruses. The interactions between norovirus and the microRNA machinery have not yet been studied. Here, we investigated the changes that occur in microRNA expression during murine norovirus (MNV) infection. Methods: Using RT-qPCR-based arrays, we analysed changes in miRNA expression during infection with the acute strain MNV-1 in two permissive cell lines, a murine macrophage cell line, RAW264.7, and a murine microglial cell line, BV-2. By RT-qPCR, we further confirmed and analysed the changes in miR-155 expression in the infected cell lines, bone-marrow derived macrophage, and tissues harvested from mice infected with the persistent strain MNV-3. Using miR-155 knockout (KO) mice, we investigated whether loss of miR-155 affected viral replication and pathogenesis during persistent MNV-3 infection in vivo and monitored development of a serum IgG response by ELISA. Results: We identified cell-specific panels of miRNAs whose expression were increased or decreased during infection. Only two miRNAs, miR-687 and miR-155, were induced in both cell lines. miR-155, implicated in innate immunity, was also upregulated in bone-marrow derived macrophage and infected tissues. MNV-3 established a persistent infection in miR-155 knockout (KO) mice, with comparable levels of secreted virus and tissue replication observed as for wildtype mice. However, serum anti-MNV IgG levels were significantly reduced in miR-155 KO mice compared to wildtype mice. Conclusions: We have identified a panel of miRNAs whose expression changes with MNV infection. miR-155 induction is a marker of MNV infection in vitro and in vivo, however it does not contribute to the control of persistent infections in vivo. This finding suggests that the immune defects associated with miR-155 deletion, such as lower serum IgG levels, are also not important for control of persistent MNV-3 infection.

scholarly journals Arming Oncolytic Adenoviruses: Effect of Insertion Site and Splice Acceptor on Transgene Expression and Viral Fitness

2020 ◽  
Vol 21 (14) ◽  
pp. 5158
Author(s):  
Martí Farrera-Sal ◽  
Jana de Sostoa ◽  
Estela Nuñez-Manchón ◽  
Rafael Moreno ◽  
Cristina Fillat ◽  
...  
Keyword(s):  
Transgene Expression ◽  
Therapeutic Potential ◽  
Insertion Site ◽  
Viral Fitness ◽  
Late Promoter ◽  
Oncolytic Adenoviruses ◽  
Major Late Promoter ◽  
Insertion Sites ◽  
Main Strategy

Oncolytic adenoviruses (OAds) present limited efficacy in clinics. The insertion of therapeutic transgenes into OAds genomes, known as “arming OAds”, has been the main strategy to improve their therapeutic potential. Different approaches were published in the decade of the 2000s, but with few comparisons. Most armed OAds have complete or partial E3 deletions, leading to a shorter half-life in vivo. We generated E3+ OAds using two insertion sites, After-fiber and After-E4, and two different splice acceptors linked to the major late promoter, either the Ad5 protein IIIa acceptor (IIIaSA) or the Ad40 long fiber acceptor (40SA). The highest transgene levels were obtained with the After-fiber location and 40SA. However, the set of codons of the transgene affected viral fitness, highlighting the relevance of transgene codon usage when arming OAds using the major late promoter.

scholarly journals High-throughput screening and rational design of biofunctionalized surfaces with optimized biocompatibility and antimicrobial activity

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Zhou Fang ◽  
Junjian Chen ◽  
Ye Zhu ◽  
Guansong Hu ◽  
Haoqian Xin ◽  
...  
Keyword(s):  
Antimicrobial Activity ◽  
High Throughput ◽  
High Throughput Screening ◽  
Rational Design ◽  
Click Reaction ◽  
Reaction Times ◽  
Great Promise ◽  
Biomaterial Surfaces

AbstractPeptides are widely used for surface modification to develop improved implants, such as cell adhesion RGD peptide and antimicrobial peptide (AMP). However, it is a daunting challenge to identify an optimized condition with the two peptides showing their intended activities and the parameters for reaching such a condition. Herein, we develop a high-throughput strategy, preparing titanium (Ti) surfaces with a gradient in peptide density by click reaction as a platform, to screen the positions with desired functions. Such positions are corresponding to optimized molecular parameters (peptide densities/ratios) and associated preparation parameters (reaction times/reactant concentrations). These parameters are then extracted to prepare nongradient mono- and dual-peptide functionalized Ti surfaces with desired biocompatibility or/and antimicrobial activity in vitro and in vivo. We also demonstrate this strategy could be extended to other materials. Here, we show that the high-throughput versatile strategy holds great promise for rational design and preparation of functional biomaterial surfaces.

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