# Mechanisms Underlying the Transduction Polarity of AAV in Polarized Human Airway Epithelium

> **NIH NIH R21** · UNIVERSITY OF KANSAS MEDICAL CENTER · 2021 · $191,925

## Abstract

PROJECT SUMMARY
Recombinant adeno-associated viral (rAAV) vectors have emerged as one of the preferred delivery agents for
clinical gene therapy. However, the gene therapy for cystic fibrosis lung disease has been hindered by the
inefficiency of rAAV transduction from airway lumen. Using the in vitro model of polarized human airway
epithelium cultured at an air-liquid interface (HAE-ALI), we found the transduction polarity of rAAV2 and rAAV5
that prefers basolateral infection. In spite of the apical transduction is low, over a thousand-fold augmentation
can be achieved when the cellular proteasome activity is transiently inhibited. rAAV2.5T is a novel AAV vector
developed from the directed evolution of a capsid library of shuffled AAV2 and AAV5 cap genes. rAAV2.5T
demonstrates altered transduction polarity with an efficiency of apical transduction over 10-fold higher than
those of rAAV2 and rAAV5 in polarized HAE, but its responses to proteasome inhibition still retain, suggesting
that vector intracellular trafficking remains the major post-entry barrier in transduction. Currently, the
mechanisms underlying the differences in the polarity of AAV transduction and the inefficient post-entry
trafficking toward productive transduction in polarized HAE remain unclear. Recently, a type I transmembrane
protein KIAA0319L, denoted hereafter as AAV receptor (AAVR), was identified as a proteinaceous receptor for
certain AAV serotypes, including AAV2 and AAV5, but not all. Importantly, AAV2.5T transduces HeLa cells in
the manners of both AAVR-dependent and independent entries. In polarized HAE, AAVR expresses only on
basolateral membrane. While AAVR is responsible for the basolateral infections of rAAV2 and rAAV2.5T, a
non-AAVR proteinaceous receptor for apical entry has not yet identified. We hypothesize that transduction
polarity of AAV in polarized HAE is determined by the polarized expression of an alternative (non-AAVR)
receptor on apical membrane and the AAVR on basolateral membrane, which divert the internalized vectors to
different intracellular trafficking pathways depending on apical or basolateral endocytosis. In this project, we
aim to identify the proteinaceous receptor that mediates apical infection of AAV2.5T, and to reveal vector entry
and intracellular trafficking of this AAVR-independent transduction pathway, in comparison with the AAVR-
mediated transduction from the basolateral membrane. We will also investigate how transient inhibition of the
proteasome activity reroutes the internalized vectors toward a pathway prone to productive transduction. The
preclinical studies of rAAV2.5T for cystic fibrosis (CF) gene therapy in CF ferret models are ongoing, the
outcomes of this proposal will benefit the development of an effective vector delivery approach to use this
vector in the gene therapy of human airway diseases, including CF.

## Key facts

- **NIH application ID:** 10106576
- **Project number:** 5R21AI151542-02
- **Recipient organization:** UNIVERSITY OF KANSAS MEDICAL CENTER
- **Principal Investigator:** Jianming Qiu
- **Activity code:** R21 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2021
- **Award amount:** $191,925
- **Award type:** 5
- **Project period:** 2020-02-13 → 2023-01-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10106576

## Citation

> US National Institutes of Health, RePORTER application 10106576, Mechanisms Underlying the Transduction Polarity of AAV in Polarized Human Airway Epithelium (5R21AI151542-02). Retrieved via AI Analytics 2026-05-23 from https://api.ai-analytics.org/grant/nih/10106576. Licensed CC0.

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