Stable expression of shRNA for the control of recombinant adenovirus replication

Author(s)

Lana, M. V. G. ; Antunes, F. ; Tessarollo, N. G. ; Strauss, B. E.

• Document type: Journal article
• Source: Brazilian Journal of Medical and Biological Research; v. 56, p. 8-pg., 2023-01-01.

Abstract

Preventing the replication of adenovirus could have practical uses, such as controlling infection with wild-type virus or in applications involving recombinant vectors. Mainly transient methods have been used to inhibit adenovirus replication, including siRNA or drugs. Here, we tested whether stable expression of shRNA designed to target hexon, Iva2, or pol can inhibit the replication of a recombinant adenoviral vector, Ad-LacZ (serotype 5, E1/E3 deleted), in 293T cells. Significant knockdown correlating with reduced Ad-LacZ replication was achieved only when hexon was targeted. Cell sorting and isolation of cellular clones further accentuated knockdown of the hexon transcript, reduced protein levels by more than 90%, and diminished adenovirus production. As visualized by transmission electron microscopy, the cellular clone expressing the hexon-specific shRNA yielded 89.2% fewer particles compared to the parental 293T cells. Full scale production followed by purification revealed a 90.2% reduction in Ad-LacZ biological titer. These results support the notion that stable expression of shRNA can be used as a means to control adenovirus replication. (AU)

• FAPESP's process: 18/04800-5 - The role of p73 isoforms in the response of B16F10 cells to transduction with adenoviral vectors carrying p19Arf and IFNbeta cDNAs.
• Grantee: Fernanda Antunes
• Support Opportunities: Scholarships in Brazil - Post-Doctoral
• FAPESP's process: 17/25290-2 - A study of the role of neoantigens in the modulation of the anti-tumor response promoted by the treatment of B16 cells with p19Arf and IFN-beta.
• Grantee: Nayara Gusmão Tessarollo
• Support Opportunities: Scholarships in Brazil - Post-Doctoral
• FAPESP's process: 12/05066-7 - Use of shRNA anti-hexon e anti-IVa2 during the production of adeno-associated virus as a strategy for eliminating helper adenovirus: Proof of principle
• Grantee: Marlous Vinícius Gomes Lana
• Support Opportunities: Scholarships in Brazil - Master
• FAPESP's process: 15/26580-9 - Cancer gene therapy: strategic positioning for translational studies
• Grantee: Bryan Eric Strauss
• Support Opportunities: Research Projects - Thematic Grants