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 doma...

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Autores principales:	Aleksei A. Stepanenko, Anastasiia O. Sosnovtseva, Marat P. Valikhov, Vladimir P. Chekhonin
Formato:	article
Lenguaje:	EN
Publicado:	Nature Portfolio 2021
Materias:	Medicine R Science Q
Acceso en línea:	https://doaj.org/article/10edaa59e24f4ba993cb64ff48bcec17
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spelling	oai:doaj.org-article:10edaa59e24f4ba993cb64ff48bcec172021-12-02T19:06:40ZA new insight into aggregation of oncolytic adenovirus Ad5-delta-24-RGD during CsCl gradient ultracentrifugation10.1038/s41598-021-94573-y2045-2322https://doaj.org/article/10edaa59e24f4ba993cb64ff48bcec172021-08-01T00:00:00Zhttps://doi.org/10.1038/s41598-021-94573-yhttps://doaj.org/toc/2045-2322Abstract 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.Aleksei A. StepanenkoAnastasiia O. SosnovtsevaMarat P. ValikhovVladimir P. ChekhoninNature PortfolioarticleMedicineRScienceQENScientific Reports, Vol 11, Iss 1, Pp 1-15 (2021)
institution	DOAJ
collection	DOAJ
language	EN
topic	Medicine R Science Q
spellingShingle	Medicine R Science Q Aleksei A. Stepanenko Anastasiia O. Sosnovtseva Marat P. Valikhov Vladimir P. Chekhonin A new insight into aggregation of oncolytic adenovirus Ad5-delta-24-RGD during CsCl gradient ultracentrifugation
description	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.
format	article
author	Aleksei A. Stepanenko Anastasiia O. Sosnovtseva Marat P. Valikhov Vladimir P. Chekhonin
author_facet	Aleksei A. Stepanenko Anastasiia O. Sosnovtseva Marat P. Valikhov Vladimir P. Chekhonin
author_sort	Aleksei A. Stepanenko
title	A new insight into aggregation of oncolytic adenovirus Ad5-delta-24-RGD during CsCl gradient ultracentrifugation
title_short	A new insight into aggregation of oncolytic adenovirus Ad5-delta-24-RGD during CsCl gradient ultracentrifugation
title_full	A new insight into aggregation of oncolytic adenovirus Ad5-delta-24-RGD during CsCl gradient ultracentrifugation
title_fullStr	A new insight into aggregation of oncolytic adenovirus Ad5-delta-24-RGD during CsCl gradient ultracentrifugation
title_full_unstemmed	A new insight into aggregation of oncolytic adenovirus Ad5-delta-24-RGD during CsCl gradient ultracentrifugation
title_sort	new insight into aggregation of oncolytic adenovirus ad5-delta-24-rgd during cscl gradient ultracentrifugation
publisher	Nature Portfolio
publishDate	2021
url	https://doaj.org/article/10edaa59e24f4ba993cb64ff48bcec17
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A new insight into aggregation of oncolytic adenovirus Ad5-delta-24-RGD during CsCl gradient ultracentrifugation

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