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Lentiviral expression of wildtype LAMA3A restores cell adhesion in airway basal cells from children with epidermolysis bullosa

ABSTRACT: The hallmark of epidermolysis bullosa (EB) is the fragile attachment of epithelia to underlying tissues due to genetic variants in genes involved in cell adhesion. A variety of epithelial surfaces can be affected, and EB patients who develop airway involvement suffer from a high degree of morbidity and mortality from laryngotracheal stenosis. Here, we describe 15 EB patients that were treated in the Ear, Nose and Throat department of a tertiary pediatric hospital linked to the United Kingdom’s National EB unit between 1992 and 2023. Investigation of the underlying genotypic status revealed that variants in LAMA3 were common in EB patients managed with airway complications (9/14 patients where genotype was available). Since LAMA3 encodes a subunit of the laminin-332 heterotrimeric extracellular matrix protein complex and is expressed by airway basal epithelial stem cells, we investigated the possibility of correcting LAMA3 in EB patient basal cell cultures. Airway basal cell cultures initiated from EB patients with variants affecting LAMA3 demonstrated fragile adherence to culture flasks but were otherwise expanded similarly to non-EB patient cell cultures. For genetic modification, we generated a lentiviral vector containing LAMA3A under the control of a constitutively active promoter sequence. In vitro transduction of primary EB patient-derived basal cell cultures restored cell attachment to levels comparable to a healthy control donor culture. Transduced cells could be expanded extensively in cell culture, suggesting their potential as an airway cell therapy for EB. We anticipate that our proof-of-concept data will be a starting point for translational combined cell and gene therapy for this rare patient group.

ORGANISM(S): Homo sapiens

PROVIDER: GSE249910 | GEO | 2024/02/01

REPOSITORIES: GEO

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Project description:Background. The human airway epithelium consists of 4 major cell types: ciliated, secretory, columnar and basal cells. During natural turnover and in response to injury, the airway basal cells function as stem / progenitor cells for the other airway cell types. The objective of this study is to better understand basal cell biology by defining the subset of expressed genes that characterize the signature of human airway epithelial basal cells. Methodology / Principal Findings. Microarrays were used to assess the transcriptome of basal cells purified from the airway epithelium of healthy nonsmokers obtained by bronchial brushings in comparison to the transcriptome of the complete differentiated airway epithelium. This analysis identified the “human airway basal cell signature” as 1,161 unique genes with >5-fold higher expression level in basal cells compared to the differentiated epithelium. The basal cell signature was suppressed when the basal cells differentiated into a ciliated airway epithelium in vitro. The human airway basal cell signature displayed extensive overlap with genes expressed in basal cells from other human tissues and murine airway basal cells. Consistent with self-modulation as well as signaling to other airway cell types, the airway basal cell signature was characterized by genes encoding extracellular matrix components, and growth factors and growth factor receptors, including genes related to EGFR and VEGFR signaling. However, while human airway basal cells share similarity with basal-like cells of other organs, the human airway basal cell signature has features not previously associated with this cell type, including a unique pattern of genes encoding extracellular matrix components, integrins, G protein-coupled receptors, neuroactive ligands and receptors, and ion channels. Conclusion / Significance. The human airway epithelial basal cells signature identified in the present study provides novel insights into the ontogeny, molecular phenotype and biology of the stem / progenitor cells of the human airway epithelium. This study was designed to distinguish the transcriptome of the airway epithelium basal cell from that of differentiated airway epithelium. A basal cell signature was derived and analyzed for functional significance. The signature was also evaluated as basal cells differentiated into ciliated epithelium in vitro.

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Lentiviral expression of wildtype LAMA3A restores cell adhesion in airway basal cells from children with epidermolysis bullosa

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