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However, rAds containing the RGD-4C peptide in the HI loop of the fiber knob domain tend to aggregate during 2\u2009\u00d7\u2009CsCl 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\u2009\u00d7\u2009CsCl 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\u2009\u00d7\u2009CsCl 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\u2009\u00d7\u2009CsCl 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\u2009\u00d7\u2009CsCl 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.<\/jats:p>","DOI":"10.1038\/s41598-021-94573-y","type":"journal-article","created":{"date-parts":[[2021,8,9]],"date-time":"2021-08-09T10:10:36Z","timestamp":1628503836000},"update-policy":"https:\/\/doi.org\/10.1007\/springer_crossmark_policy","source":"Crossref","is-referenced-by-count":24,"title":["A new insight into aggregation of oncolytic adenovirus Ad5-delta-24-RGD during CsCl gradient ultracentrifugation"],"prefix":"10.1038","volume":"11","author":[{"given":"Aleksei A.","family":"Stepanenko","sequence":"first","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Anastasiia O.","family":"Sosnovtseva","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Marat P.","family":"Valikhov","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Vladimir P.","family":"Chekhonin","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"297","published-online":{"date-parts":[[2021,8,9]]},"reference":[{"key":"94573_CR1","doi-asserted-by":"publisher","first-page":"652","DOI":"10.1093\/jnci\/95.9.652","volume":"95","author":"J Fueyo","year":"2003","unstructured":"Fueyo, J. et al. 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