Identification of AAV serotypes for lung gene therapy in human embryonic stem cell-derived lung organoids

Helena Meyer-Berg,Lucia Zhou Yang,María Pilar de Lucas,Deborah R. Gill,Alberto Zambrano,Stephen C. Hyde

doi:10.1186/s13287-020-01950-x

Abstract

Gene therapy is being investigated for a range of serious lung diseases, such as cystic fibrosis and emphysema. Recombinant adeno-associated virus (rAAV) is a well-established, safe, viral vector for gene delivery with multiple naturally occurring and artificial serotypes available displaying alternate cell, tissue, and species-specific tropisms. Efficient AAV serotypes for the transduction of the conducting airways have been identified for several species; however, efficient serotypes for human lung parenchyma have not yet been identified. Here, we screened the ability of multiple AAV serotypes to transduce lung bud organoids (LBOs)—a model of human lung parenchyma generated from human embryonic stem cells. Microinjection of LBOs allowed us to model transduction from the luminal surface, similar to dosing via vector inhalation. We identified the naturally occurring rAAV2 and rAAV6 serotypes, along with synthetic rAAV6 variants, as having tropism for the human lung parenchyma. Positive staining of LBOs for surfactant proteins B and C confirmed distal lung identity and suggested the suitability of these vectors for the transduction of alveolar type II cells. Our findings establish LBOs as a new model for pulmonary gene therapy and stress the relevance of LBOs as a viral infection model of the lung parenchyma as relevant in SARS-CoV-2 research.

Highlights

Recombinant adeno-associated virus is a wellestablished vector for gene delivery, currently in use clinically for gene therapy, with multiple, naturally occurring serotypes and artificial variants facilitating species-specific cell and tissue tropisms [1]
Analysis of enhanced green fluorescent protein (EGFP) brightness intensity confirmed that expression levels were highest for rAAV2, rAAV6 and variants rAAV6.2 and rAAV6.2FF which was significantly different from mock injection while rAAV5 and rAAV9 resulted in the lowest levels of brightness (Fig. 2k)
The generation of Lung bud organoid (LBO) from human embryonic stem cells (hESC) provides a 3D model of the human lung parenchyma, which has been shown to model aspects of embryonic development, Respiratory syncytial virus (RSV) infection and genetic diseases such as Hermansky-Pudlak syndrome [6, 13]

Summary

Introduction

Recombinant adeno-associated virus (rAAV) is a wellestablished vector for gene delivery, currently in use clinically for gene therapy, with multiple, naturally occurring serotypes and artificial variants facilitating species-specific cell and tissue tropisms [1]. We aimed to identify AAV serotypes that permit efficient gene delivery to the human lung parenchyma. The LBOs were generated from human embryonic stem cells (hESC) in this study because their differentiation efficiency is more robust compared to iPSC lines [7] as shown for the generation of progenitor lung cells from iPSC [8]. LBOs exhibit a strong bias towards the generation of lung parenchyma cell types, especially alveolar type II (ATII) pneumocytes [6] and provide a reproducible, in vitro model in which to study human/ viral vector interactions that is substantially more similar to the native tissue environment than traditional, immortalised, submerged cell culture models. The polarised 3D structure of LBOs allows for vector transduction from the luminal surface, mimicking vector administration by inhalation, and provides an attractive translational model for diseases of the human parenchyma

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