Concordant activity of transgene expression cassettes inserted into E1, E3 and E4 cloning sites in the adenovirus genome

Abstract

Background Expression cassettes can be inserted at several positions into recombinant adenoviral genomes but the implications of this choice for transgene expression level have not been determined. Knowledge of the relative expression levels of transgenes inserted at different sites in the adenoviral genome is of particular significance for transgene expression monitoring approaches that rely on the concordant expression of a marker transgene inserted elsewhere in the viral genome. Methods Three expression cassettes, each comprising a cytomegalovirus promoter driving one of three marker peptides [serum carcinoembryonic antigen (sCEA), beta subunit of human chorionic gonadotropin (βhCG) or human sodium iodide symporter (hNIS)], were inserted into E1, E3 or E4 cloning sites in a recombinant adenoviral vector backbone. High titer stocks of bicistronic adenoviral vectors coding for combinations of marker peptides were prepared. A panel of human cells of various lineages was infected with the vectors and expression ratios of the transgene-encoded proteins were analysed. Serum levels of the soluble proteins and hepatic uptake of radioactive iodine were also compared in vivo in nude rats after intravenous vector infusion. Results High concordance of expression between the inserted transgenes was observed in all of the bicistronic vectors irrespective of whether the expression cassettes were placed in the E1, E3 or E4 regions. Concordance was maintained across multiple cell lineages. In vivo, in athymic rats, blood and urine levels of βhCG were highly concordant with serum levels of sCEA at all timepoints after intravenous infusion of the bicistronic vectors encoding both of these soluble markers. Hepatic radioiodine uptake was concordant with serum CEA concentration in mice infused with a bicistronic vector expressing CEA and NIS. Conclusions The expression level of a given transgene in an adenoviral vector genome can be accurately and quantitatively inferred from the expression of a marker protein encoded by a second transgene inserted elsewhere in the vector genome.

Secreted blood reporters: Insights and applications

“…Edmonston vaccine strain of measles virus (MV-Edm) has been effectively engineered to express different soluble marker peptides, including the ones derived from the human carcinoembryonic antigen (hCEA) and β subunit of human chorionic gonadotropin (βhCG) (Peng et al, 2002b; Pham et al, 2009). The replication-competent virus was engineered to express the soluble extracellular N-terminal domain of hCEA or βhCG as an additional transcription unit before the viral N gene.…”

“…Secreted blood reporter proteins are valuable tools for rapid and sensitive detection, quantification, and non-invasive monitoring of biological phenomena in pre-clinical animal experiments (Hughes et al, 2009; Pham et al, 2009; Wurdinger et al, 2008). Traditional enzyme-based reporter systems using cytosolic markers are sensitive, but require either a tissue lysis step which prevents their use for long-term monitoring, or frequent systemic anesthesia and substrate injection for detection of photon absorption.…”

“…More recently, virus-encoded soluble marker peptides have been exploited to monitor viral therapy. Edmonston vaccine strain of measles virus (MV-Edm) was engineered to express a soluble peptide derived from the human carcinoembryonic antigen (hCEA) or human chorionic gonadotropin (hCG) (Liu et al, 2007; Peng et al, 2002b; Pham et al, 2009). Infection of this oncolytic virus was easily detected by measuring the concentration of these polypeptides in tissue culture supernatant or in body fluids (Msaouel et al, 2009; Peng et al, 2002a).…”

Homologous Recombination in E3 Genes of Human Adenovirus Species D

“…Finally, recognition of promiscuous recombination among CR1 genes in HAdV-D E3 should be of interest to those investigators using HAdV-D for development of viral gene vectors (76)(77)(78)(79), particularly when the transgene is placed within the E3 cassette (77,(80)(81)(82)(83)(84). HAdV-D-based gene vectors with a transgene within the E3 region might be susceptible to recombination into wild-type HAdV-D during a coincidental coinfection, resulting in an infectious, replication-competent virus with potential expression of the transgene.…”

Adeno-Associated Virus (AAV)-2 Genome in Arthrobacter sp. LS16?

“…J00152.1) was also identified between a multiple cloning site. It must be noted that GLYCA is used as a quantitative serum expression marker for in vivo studies [4]. To further understand if the presence of foreign DNA sequence in Arthrobacter is mediated by integration elements we screened for the presence of repeat elements.…”