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

• Published in: Scientific reports Vol. 11; no. 1; pp. 16088 - 15
• Main Authors: Stepanenko, Aleksei A., Sosnovtseva, Anastasiia O., Valikhov, Marat P., Chekhonin, Vladimir P.
• Format: Journal Article
• Language: English
• Published: London Nature Publishing Group UK 09.08.2021; Nature Publishing Group; Nature Portfolio
• Subjects: 631/1647; 631/208; 631/337; 631/45; 631/61; 631/67; 692/4028; A549 Cells; Adenoviridae - isolation & purification; Adenoviridae Infections - therapy; Adenoviruses; Animals; Antioxidants; Antioxidants - chemistry; Antitumor activity; C-Terminus; Cell Line; Cell Line, Tumor; Cesium; Cesium - chemistry; Chlorides - chemistry; Coxsackie and Adenovirus Receptor-Like Membrane Protein - genetics; Cysteine; Disulfide bonds; Expression vectors; Gene therapy; Genetic Vectors - genetics; Glioma; Glioma - therapy; Glioma - virology; HEK293 Cells; Humanities and Social Sciences; Humans; Immune response; Mice; multidisciplinary; Oligopeptides - genetics; Oncolysis; Oncolytic Virotherapy - methods; Peptides; Purification; Rats; Science; Science (multidisciplinary); Triiodobenzoic Acids - chemistry; Tumor cell lines; Ultracentrifugation; Ultracentrifugation - methods
• ISSN: 2045-2322; 2045-2322
• DOI: 10.1038/s41598-021-94573-y

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.