C70-34 Local Airway Mechanical Preconditioning (LAMP) Safely Removes the Airway Epithelium for Transplantation of Gene-Edited Stem Cells, Enabling Durable Engraftment and Restoration of Epithelial Function

Rationale

Cystic fibrosis (CF) is caused by mutations in CF Transmembrane Conductance Regulator (CFTR), leading to airway epithelial dysfunction. Our group developed an efficient method to genetically restore CFTR function in airway basal stem cells (ABC) for autologous cell transplantation. However, translating this approach into the clinic requires a preconditioning method to safely remove the host epithelium and support successful engraftment of donor cells. Current preconditioning methods using detergent/chemical-induced injury allow cell engraftment but cause off-target damage in the alveoli, limiting their clinical application. Here, we developed a bronchoscopy-adaptable local airway mechanical preconditioning (LAMP) method to safely remove the airway epithelium with a nylon brush, avoiding off-target effects and supporting durable ABC engraftment.

Methods

Safety of LAMP was first assessed in immunocompetent (C57BL/6; n = 5), immunocompromised (NOD scid; n = 7-9), and CF-like (ΔF508; n = 5) mice. Efficiency of de-epithelization and signs of fibrosis/stenosis were evaluated using Masson’s trichrome staining. Engraftment was optimized by comparing different encapsulation matrices (PBS, fibrin, and collagen; n = 5/group) with mouse GFP⁺ABC. Furthermore, ABC from people with CF were either gene-edited to express luciferase or were corrected to insert full-length CFTR cDNA with a truncated CD19 marker (77%± 4.7%) and transplanted to assess translational relevance (n = 3 patients, n = 5 mice/group). Restoration of epithelial function was determined by maintenance of the basal cell pool (KRT5⁺), and differentiation into ciliated (ACT⁺) and secretory (CCSP⁺) lineages.

Results

LAMP was well tolerated in immunocompetent, immunocompromised, and CF-like mice, achieving localized airway epithelial denudation without injury to adjacent tissue or development of fibrosis. At 1-month post-transplantation, GFP⁺ABC comprised up to 80% of the recipient epithelium, and fibrin notably increased their engraftment efficiency compared to collagen or PBS (2-fold and 4-fold, respectively). At 6 months, engrafted GFP⁺ABC showed maintenance of basal progenitor pools and reconstitution of a differentiated epithelium, indicating significant epithelial restoration. Likewise, radiance measurement demonstrated successful engraftment of gene-edited luciferase+ human ABC encapsulated in fibrin (8.27-fold compared to PBS) and retention for at least 6 months. After efficient CFTR correction, human ABC were transplanted and colocalization with specific cell markers indicated that the engrafted cells differentiated into ciliated and secretory cells.

Conclusions

These results demonstrate that LAMP is an efficient and clinically translatable preconditioning approach, which facilitates durable engraftment of transplanted gene-corrected ABC and preserved their epithelial regeneration capacity. This strategy can be implemented with bronchoscopy assistance in humans, supporting the development of cell-based therapies for airway disorders such as CF.

This abstract is funded by: NIH