(G-266) Mucin–Targeted Gene Therapy for Asthma

Introduction:: Asthma is a globally burdensome respiratory disease, affecting millions worldwide. It is characterized by chronic airway inflammation, airway remodeling, and impaired mucociliary clearance (MCC)¹. Mucus comprises gel-forming mucin proteins mucin 5B (MUC5B) and mucin 5AC (MUC5AC). Healthy airway mucus is predominantly composed of MUC5B, with reduced MUC5AC. However, in individuals with asthma, biochemical analysis of their mucus revealed a shift from MUC5B to MUC5AC as the predominantly secreted mucin. These changes to mucus composition have been shown to impair MCC and obstruct airways due to increased mucus plugs in asthma patients². Given the overproduction of MUC5AC in the asthmatic airway, we hypothesized delivery of MUC5AC siRNA to the airway epithelium, could improve MCC, reduce airway hyperreactivity, and improve asthma symptoms in vitro and in vivo.

Materials and Methods:: Immortalized basal HAE cells (BCi-NS1.1) were differentiated into secretory and ciliated cells when cultured at the air-liquid interface (ALI). These cultures secrete mucus and possess ciliated cells that enable in vitro analyses of MCC function. Adeno-associated virus (AAV) serotype 6 (AAV6), which shows tissue tropism for lungs and has been seen to transduce airway epithelium in vitro and in vivo^3,4, was used in these experiments. Multiple particle tracking (MPT) analysis was used to compare AAV6 diffusion in mucus from standard ALI cultures and IL-13 exposed ALI cultures to establish an asthma-like phenotype. To characterize AAV6 transduction in HAE cells, AAV6 expressing enhanced green fluorescent protein (eGFP) was used and transduction efficacy was assessed. The efficacy of AAV6 carrying MUC5AC-siRNA to silence MUC5AC was evaluated using immunofluorescence and quantitative real-time polymerase chain reaction (qRT-PCR) using SYBR green chemistry and normalized to GAPDH levels using the ΔCt method.

Results, Conclusions, and Discussions:: MPT analysis of AAV6 in HAE mucus with and without IL-13 stimulation revealed that the mean squared displacement (MSD) of AAV6 was increased in IL-13 mucus showing that AAV6 can diffuse through asthma–like mucus barrier enriched with MUC5AC (Fig 1A). The AAV6 multiplicity of infection (MOI) of 10³, 10⁴, and 10⁵ were examined in undifferentiated, differentiated, and IL-13-stimulated differentiated HAE cells. AAV6-mediated GFP expression could be detected in undifferentiated, differentiated, and IL-13-stimulated HAE cells at MOIs 10⁴ and 10⁵ (Fig 1B). AAV6-MUC5AC-siRNA was administered in IL-13-stimulated HAE cells at MOI 10⁴ and MUC5AC expression was evaluated via immunofluorescence and qRT-PCR. Initial studies showed promise with expected trends of elevated MUC5AC mRNA expression in IL-13-exposed and an almost 2-fold reduction is seen in the AAV6-MUC5AC siRNA treatment group (Fig 1C). From these results, we can see that AAV6 is capable of transducing HAE ALI cultures with an asthma-like phenotype and delivery of MUC5AC-siRNA, resulting in reduced MUC5AC expression. Future studies include repeating qRT-PCR experiments and evaluating MCC in MUC5AC-treated groups. If successful, this gene delivery approach could open avenues for mucin-targeted therapies for asthma and other related chronic lung diseases.


Acknowledgements (Optional): :

References (Optional): : 1. Holgate, S. T. et al. Nat Rev Dis Primers 1, 15025 (2015).

2. Lachowicz-Scroggins, M. E. et al. Am J Respir Crit Care Med 194, 1296–1299 (2016).

3. Duncan et al. Mol Ther Methods Clin Dev 9, 296–304 (2018).

4. Halbert, C. L., Allen, J. M. & Miller, A. D. Journal of Virology 75, 6615–6624 (2001).