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.

Public Health Relevance

Recombinant adeno-associated virus 2.5T (rAAV2.5T) vector has been developed to deliver the cystic fibrosis conductance regulator (CFTR) gene in human airway epithelia. The proposed study will identify the cellular receptor and the entry and trafficking pathways of the rAAV2.5T vector, which could be targeted for enhancement of rAAV2.5T gene delivery in human airway epithelia.

Agency
National Institute of Health (NIH)
Institute
National Institute of Allergy and Infectious Diseases (NIAID)
Type
Exploratory/Developmental Grants (R21)
Project #
5R21AI151542-02
Application #
10106576
Study Section
Gene and Drug Delivery Systems Study Section (GDD)
Program Officer
Dyall, Julie
Project Start
2020-02-13
Project End
2022-01-31
Budget Start
2021-02-01
Budget End
2022-01-31
Support Year
2
Fiscal Year
2021
Total Cost
Indirect Cost
Name
University of Kansas
Department
Microbiology/Immun/Virology
Type
Schools of Medicine
DUNS #
016060860
City
Kansas City
State
KS
Country
United States
Zip Code
66160