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

• Published in: Molecular therapy. Methods & clinical development Vol. 31; p. 101126
• Main Authors: Pan, Yonghua, Rohde, Michelle, Zeitler, Jennifer, Namburi, Sai Valli Srujana, Cao, Liching, Hu, Jing, Meyer, Kathleen, Lu, Yanmei
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 14.12.2023; American Society of Gene & Cell Therapy; Elsevier
• Subjects: adeno-associated virus; clinical enrollment assay; comparative analysis; neutralizing antibodies; Original; pre-existing humoral immunity; seropositivity concordance; total antibodies; total antibody assay; transduction inhibition assay; comparative analysis; pre-existing humoral immunity; total antibody assay; clinical enrollment assay; neutralizing antibodies; adeno-associated virus; transduction inhibition assay; total antibodies; seropositivity concordance
• ISSN: 2329-0501; 2329-0501
• DOI: 10.1016/j.omtm.2023.101126

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. [Display omitted] Pan and colleagues developed a sensitive cell-based AAV6 TI assay to assist identification of critical TI assay factors and compared it to two different AAV6 TAb assay formats. The results revealed impacts of assay sensitivity and assay formats on detection of pre-existing anti-AAV6 antibodies in human sera.