A sensitive AAV transduction inhibition assay assists evaluation of critical factors for detection and concordance of pre-existing antibodies.

Autor:	Pan Y; Sangamo Therapeutics, Inc, 7000 Marina Boulevard, Brisbane, CA 94005, USA., Rohde M; Sangamo Therapeutics, Inc, 7000 Marina Boulevard, Brisbane, CA 94005, USA., Zeitler J; Sangamo Therapeutics, Inc, 7000 Marina Boulevard, Brisbane, CA 94005, USA., Namburi SVS; Sangamo Therapeutics, Inc, 7000 Marina Boulevard, Brisbane, CA 94005, USA., Cao L; Sangamo Therapeutics, Inc, 7000 Marina Boulevard, Brisbane, CA 94005, USA., Hu J; Sangamo Therapeutics, Inc, 7000 Marina Boulevard, Brisbane, CA 94005, USA., Meyer K; Sangamo Therapeutics, Inc, 7000 Marina Boulevard, Brisbane, CA 94005, USA., Lu Y; Sangamo Therapeutics, Inc, 7000 Marina Boulevard, Brisbane, CA 94005, USA.
Jazyk:	angličtina
Zdroj:	Molecular therapy. Methods & clinical development [Mol Ther Methods Clin Dev] 2023 Oct 10; Vol. 31, pp. 101126. Date of Electronic Publication: 2023 Oct 10 (Print Publication: 2023).
DOI:	10.1016/j.omtm.2023.101126
Abstrakt:	Pre-existing antibodies to viral capsids may have a negative impact on the efficacy and safety of adeno-associated virus (AAV)-based gene therapies. Total antibody (TAb) and/or cell-based transduction inhibition (TI) assays have been used to exclude seropositive individuals in clinical studies. Published AAV seroprevalence and patient enrollment criteria regarding antibody status lack comparability between assay formats, hindering a direct cross-study comparison. To identify critical factors impacting TI assay detection of AAV neutralizing antibodies (NAbs), we created a reporter construct expressing NanoLuc® luciferase (Nluc) that enabled a more sensitive and robust detection of AAV6 NAbs than using firefly luciferase. Assessment of additional factors including multiplicity of infection, cell lines, viral production, and capsid purity revealed the reporter is the major determinant of assay sensitivity impacting NAb detection. The Nluc reporter was further used to assess seroprevalence to AAV5, 8, and 9. Last, we compared AAV6 Nluc TI with two TAb assay formats. A higher correlation of Nluc TI was observed with direct binding (90%) than with the more sensitive bridging TAb assay (65%), suggesting both assay sensitivity and TAb formats contribute to AAV seropositivity concordance. Our results support a need to standardize assay formats to ensure proper assessment of pre-existing AAV immunity. Competing Interests: The authors declare the following competing financial interests: Y.P, M.R., J.Z., S.N., L.C., J.H., K.M., and Y.L. are full-time current or former employees and own stocks of Sangamo Therapeutics, Inc. (© 2023 The Author(s).)
Databáze:	MEDLINE
Externí odkaz:	Zobrazit plný text záznamu