(655) Respiratory cell-type affinity and absolute CFTR expression in the primate airway upon nebulization of KB407

Background: Recent drug development efforts in cystic fibrosis (CF) has largely focused on modulation of specific cystic fibrosis transmembrane conductance regulator (CFTR) dysfunctions, thereby treating only a subset of the population. Regrettably, no mutation-agnostic corrective therapy is approved. To this end, we engineered KB407, a replication-defective herpes simplex virus type 1 (HSV-1)-based vector encoding full-length human CFTR, for the treatment of CF. Previously, KB407 was shown to transduce primary CF patient airway epithelial cells in 2D culture, molecularly correct multiple CFTR defects in clinically relevant CF patient-derived organoids, and direct localized expression of human CFTR in the airways of immunocompetent mice and non-human primates (NHPs). Here, we investigated the cell type(s) of the primate airway epithelium transduced by KB407 after nebulization, as well as performed an absolute quantification of exogenous human CFTR and endogenous cyno CFTR transcripts in multiple NHP lung biopsies.

Methods: Biopsies harvested from the conducting airways of cynomolgus macaques exposed to nebulized KB407 or vehicle control were formalin-fixed, paraffin embedded, and sectioned for immunofluorescent analysis. Slides were co-stained for an HSV-1-specific viral marker and SCGB1A1 (club cells), MUC5AC (goblet cells), CK5 (basal cells), or AC-tubulin (ciliated cells), and images were captured using identical exposure settings. Right cranial and caudal lung lobe biopsies were harvested from an NHP 48-hours post-KB407 exposure and processed for qRT-PCR analysis. Absolute quantification of exogenous human and endogenous cyno CFTR expression was performed using custom, gene-specific primer/probe pairs and standard curves constructed from a dilution series of double-stranded gBlock DNA containing a known copy number of the target CFTR sequences.

Results: Staining of the viral marker in the airway epithelium was limited to KB407-exposed NHPs with no fluorescence in vehicle treated animals. In KB407 treated primates, positive colocalization of the viral marker and the cell type-specific stains for ciliated and secretory cells (both club and goblet cells) was observed, suggesting that each of these cell types is susceptible to vector transduction through their apical membranes. A majority of KB407-positive respiratory epithelium was identified as ciliated cells, which is consistent with the observation that ciliated cells are the predominant cell type found in the conducting airways¹. No significant colocalization of the viral and basal cell markers was detected, indicating that transduction was limited to air-exposed cells. Absolute quantification of exogenous human CFTR transcripts in multiple lung tissue biopsies were found to be 106%-440% of the endogenous levels observed in otherwise healthy primate airways.

Conclusions: Taken together, these data indicate that KB407 capably transduces multiple cell types that contribute to native CFTR function in the primate airways and expresses the transgene at physiologically relevant levels, providing further support for the potential of KB407 as an inhalable gene therapy for the treatment of CF.

Acknowledgements: These studies were funded by Krystal Biotech, Inc. We thank Lovelace Biomedical for their contributions to the work presented here.

References: [1] Okuda, K. et al. 2021. Am J Respir Crit Care Med 203(10): 1275-1289.