A new insight into aggregation of oncolytic adenovirus Ad5-delta-24-RGD during CsCl gradient ultracentrifugation.

Abstract

Two-cycle cesium chloride (2 × CsCl) gradient ultracentrifugation is a conventional approach for purifying recombinant adenoviruses (rAds) for research purposes (gene therapy, vaccines, and oncolytic vectors). However, rAds containing the RGD-4C peptide in the HI loop of the fiber knob domain tend to aggregate during 2 × CsCl gradient ultracentrifugation resulting in a low infectious titer yield or even purification failure. An iodixanol-based purification method preventing aggregation of the RGD4C-modified rAds has been proposed. However, the reason explaining aggregation of the RGD4C-modified rAds during 2 × CsCl but not iodixanol gradient ultracentrifugation has not been revealed. In the present study, we showed that rAds with the RGD-4C peptide in the HI loop but not at the C-terminus of the fiber knob domain were prone to aggregate during 2 × CsCl but not iodixanol gradient ultracentrifugation. The cysteine residues with free thiol groups after the RGD motif within the inserted RGD-4C peptide were responsible for formation of the interparticle disulfide bonds under atmospheric oxygen and aggregation of Ad5-delta-24-RGD4C-based rAds during 2 × CsCl gradient ultracentrifugation, which could be prevented using iodixanol gradient ultracentrifugation, most likely due to antioxidant properties of iodixanol. A cysteine-to-glycine substitution of the cysteine residues with free thiol groups (RGD-2C2G) prevented aggregation during 2 × CsCl gradient purification but in coxsackie and adenovirus receptor (CAR)-low/negative cancer cell lines of human and rodent origin, this reduced cytolytic efficacy to the levels observed for a fiber non-modified control vector. However, both Ad5-delta-24-RGD4C and Ad5-delta-24-RGD2C2G were equally effective in the murine immunocompetent CT-2A glioma model due to a primary role of antitumor immune responses in the therapeutic efficacy of oncolytic virotherapy.

• Authors: Aleksei A Stepanenko, Anastasiia O Sosnovtseva, Marat P Valikhov, Vladimir P Chekhonin
• Journal: Scientific reports (Sci Rep) Vol. 11 Issue 1 Pg. 16088 (08 09 2021) ISSN: 2045-2322 [Electronic] England
• PMID: 34373477 (Publication Type: Journal Article, Research Support, Non-U.S. Gov't)
• Copyright: © 2021. The Author(s).

Chemical References

• Antioxidants
• Chlorides
• Coxsackie and Adenovirus Receptor-Like Membrane Protein
• Oligopeptides
• Triiodobenzoic Acids
• Cesium
• cesium chloride
• iodixanol

Topics

• A549 Cells
• Adenoviridae (isolation & purification)
• Adenoviridae Infections (therapy)
• Animals
• Antioxidants (chemistry)
• Cell Line
• Cell Line, Tumor
• Cesium (chemistry)
• Chlorides (chemistry)
• Coxsackie and Adenovirus Receptor-Like Membrane Protein (genetics)
• Genetic Vectors (genetics)
• Glioma (therapy, virology)
• HEK293 Cells
• Humans
• Mice
• Oligopeptides (genetics)
• Oncolytic Virotherapy (methods)
• Rats
• Triiodobenzoic Acids (chemistry)
• Ultracentrifugation (methods)