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 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 Characterization of New Virulence Factors Involved in the Intracellular Growth and Survival of Burkholderia pseudomallei

2015 ◽  
Vol 84 (3) ◽  
pp. 701-710 ◽  
Author(s):  
Madeleine G. Moule ◽  
Natasha Spink ◽  
Sam Willcocks ◽  
Jiali Lim ◽  
José Afonso Guerra-Assunção ◽  
...  
Keyword(s):  
Burkholderia Pseudomallei ◽  
Insertion Site ◽  
Wild Type ◽  
Content Type ◽  
Growth And Survival ◽  
New Genes ◽  
Insertion Sites

Burkholderia pseudomallei, the causative agent of melioidosis, has complex and poorly understood extracellular and intracellular lifestyles. We used transposon-directed insertion site sequencing (TraDIS) to retrospectively analyze a transposon library that had previously been screened through a BALB/c mouse model to identify genes important for growth and survivalin vivo. This allowed us to identify the insertion sites and phenotypes of negatively selected mutants that were previously overlooked due to technical constraints. All 23 unique genes identified in the original screen were confirmed by TraDIS, and an additional 105 mutants with various degrees of attenuationin vivowere identified. Five of the newly identified genes were chosen for further characterization, and clean, unmarkedbpsl2248,tex,rpiR,bpsl1728, andbpss1528deletion mutants were constructed from the wild-type strain K96243. Each of these mutants was testedin vitroandin vivoto confirm their attenuated phenotypes and investigate the nature of the attenuation. Our results confirm that we have identified new genes important toin vivovirulence with roles in different stages ofB. pseudomalleipathogenesis, including extracellular and intracellular survival. Of particular interest, deletion of the transcription accessory protein Tex was shown to be highly attenuating, and thetexmutant was capable of providing protective immunity against challenge with wild-typeB. pseudomallei, suggesting that the genes identified in our TraDIS screen have the potential to be investigated as live vaccine candidates.

scholarly journals Functional Analysis of the CAAT Box in the Major Late Promoter of the Subgroup C Human Adenoviruses

1998 ◽  
Vol 72 (4) ◽  
pp. 3213-3220 ◽  
Author(s):  
Byeongwoon Song ◽  
C. S. H. Young
Keyword(s):  
Binding Site ◽  
Transcription Initiation ◽  
Mutational Analysis ◽  
Wild Type ◽  
Late Promoter ◽  
Phenotypic Effect ◽  
Partial Restoration ◽  
Wild Type Phenotype ◽  
Major Late Promoter

ABSTRACT Comparisons among sequences predicted to encode the major late promoter (MLP) of adenoviruses from a wide variety of host species show that an inverted CAAT box is among the most highly conserved transcription elements found in the putative MLPs. The high degree of conservation suggests that the CAAT box plays an important role in the function of the MLP in vivo, an idea supported by a previous mutational analysis of the core CCAAT sequence. To address the importance of the CAAT box, in terms both of quantitative levels of transcription and of specificity, a further set of mutations was created and examined in the context of the viral genome. One mutation, CAAT5, contains individual changes at five positions, four of which correspond to invariant residues in a CAAT box consensus derived either by computer analysis or empirically. The CAAT5 mutation had no discernible phenotype by itself but when coupled with the previously described USF0 mutation, which disrupts binding of the upstream stimulating factor (USF) but is otherwise phenotypically silent, gave rise to virus with a severe replication deficiency. Nuclear run-on assays showed that transcription initiation at the mutant MLP was significantly reduced compared with that of the wild type or the virus containing CAAT5 alone. Replication of the double mutant was lower than that of the previously described USF0::CCCAT virus, suggesting that the additional mutations in the CAAT box had further lowered the binding of transcription factor CP1 (also called CBF, NF-Y). Replacement of the CAAT box by an ATF binding site or an OCT1 binding site had no phenotypic effect in an otherwise wild-type background, but replacement in a USF0::CCCAT background led to only partial restoration of the wild-type phenotype. The failure to restore the functional redundancy normally exhibited by the CAAT box and the proximal upstream activating element is consistent with the idea that in the adenovirus MLP the CAAT box is preferred over others as the distal transcriptional element.

scholarly journals Lentiviral vectors containing an enhancer-less ubiquitously acting chromatin opening element (UCOE) provide highly reproducible and stable transgene expression in hematopoietic cells

Blood ◽  
2007 ◽  
Vol 110 (5) ◽  
pp. 1448-1457 ◽  
Author(s):  
Fang Zhang ◽  
Susannah I. Thornhill ◽  
Steven J. Howe ◽  
Meera Ulaganathan ◽  
Axel Schambach ◽  
...  
Keyword(s):  
Transgene Expression ◽  
Lentiviral Vector ◽  
Severe Combined Immunodeficiency ◽  
Insertion Site ◽  
Housekeeping Genes ◽  
Enhancer Activity ◽  
Position Effects ◽  
Chromatin Opening

AbstractUbiquitously acting chromatin opening elements (UCOEs) consist of methylation-free CpG islands encompassing dual divergently transcribed promoters of housekeeping genes that have been shown to confer resistance to transcriptional silencing and to produce consistent and stable transgene expression in tissue culture systems. To develop improved strategies for hematopoietic cell gene therapy, we have assessed the potential of the novel human HNRPA2B1-CBX3 UCOE (A2UCOE) within the context of a self-inactivating (SIN) lentiviral vector. Unlike viral promoters, the enhancer-less A2UCOE gave rise to populations of cells that expressed a reporter transgene at a highly reproducible level. The efficiency of expression per vector genome was also markedly increased in vivo compared with vectors incorporating either spleen focus-forming virus (SFFV) or cytomegalovirus (CMV) promoters, suggesting a relative resistance to silencing. Furthermore, an A2UCOE-IL2RG vector fully restored the IL-2 signaling pathway within IL2RG-deficient human cells in vitro and successfully rescued the X-linked severe combined immunodeficiency (SCID-X1) phenotype in a mouse model of this disease. These data indicate that the A2UCOE displays highly reliable transcriptional activity within a lentiviral vector, largely overcoming insertion-site position effects and giving rise to therapeutically relevant levels of gene expression. These properties are achieved in the absence of classic enhancer activity and therefore may confer a high safety profile.

scholarly journals 653. The Controlled Transgene Expression in Oncolytic Adenoviral Vectors with Major Late Promoter for Therapy of Cancer

2006 ◽  
Vol 13 ◽  
pp. S251
Author(s):  
Wei Jiang ◽  
Rong Cai ◽  
Guanghua Yang ◽  
Kai Chen ◽  
Yu Chen ◽  
...  
Keyword(s):  
Transgene Expression ◽  
Adenoviral Vectors ◽  
Late Promoter ◽  
Major Late Promoter
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