Polarized AAVR expression determines infectivity by AAV gene therapy vectors

Area of Science:

• Gene Therapy
• Virology
• Pulmonology

Background:

• Adeno-associated virus (AAV) vectors are explored for cystic fibrosis (CF) gene therapy, aiming to deliver the cystic fibrosis transmembrane conductance regulator (CFTR) gene to airways.
• Inhaled AAV2-CFTR is safe but inefficient for CF patients, with poor transduction of the apical surface of airway epithelia.
• AAV2 efficiently transduces airway epithelia from the basolateral side, suggesting receptor localization is key.

Purpose of the Study:

• To investigate the localization of the AAV receptor (AAVR) in human airway epithelia.
• To determine the transduction mechanisms of AAV2 and a novel capsid, AAV2.5T, on airway epithelia.
• To identify why AAV2.5T achieves efficient apical transduction, unlike AAV2.

Main Methods:

• Western blot and RNA-Seq to detect AAVR in airway epithelia.
• Immunocytochemistry to visualize AAVR localization, with and without overexpression.
• Functional assays using anti-AAVR antibodies and CRISPR/Cas9 knockout of AAVR to assess transduction efficiency of AAV2 and AAV2.5T.

Main Results:

• AAVR was detected in human airway epithelia, localizing to the basolateral membrane upon overexpression and enhancing transduction.
• Anti-AAVR antibodies blocked basolateral AAV2 transduction but not apical AAV2.5T transduction.
• CRISPR knockout of AAVR blocked AAV2 infection but not AAV2.5T infection in cell lines.

Conclusions:

• The absence of apical AAVR limits AAV2 transduction efficiency in airway epithelia.
• AAV2.5T achieves efficient apical transduction via an AAVR-independent pathway, suggesting a distinct apical receptor.
• These findings are crucial for developing improved AAV-based gene therapies for cystic fibrosis and other applications.