In Silico Development of a Targeted Small Interfering RNA (siRNA) Sequence to Silence Goblet Cell Mucin-5AC Overexpression in Cystic Fibrosis

Cystic fibrosis (CF) is characterized by persistent airway obstruction, frequent infection, and worsening lung function. Although CFTR channel-modulating therapy has improved outcomes, mucus hypersecretion morbidity remains present in most patients. Mucin 5AC (MUC5AC) is the main actor, a gel-forming mucin whose expression is highly upregulated in airway inflammation. Mucolytic and anti-inflammatory drug therapy reduces symptoms but does not prevent overproduction of mucin. We present here the in silico design and computational evaluation of a chemically synthesized small interfering RNA (siRNA) against MUC5AC mRNA. The duplex candidate has four modifications, 5' phosphorylation, site-specific 2'-O-methylation, amino-terminal conjugation, and hexadecyl lipid conjugation, aimed at promoting Argonaute 2 (Ago2) loading, increasing nuclease resistance, suppressing innate immune recognition, and enhancing membrane interaction. The computational approach was validated using a GL2 positive control and an inverted negative control sequence, which showed the expected stable and unstable binding behaviors, respectively. In 20-ns molecular dynamics simulations on OpenMM, the experimental siRNA-Ago2 complex showed stable binding (root-mean-square deviation (RMSD) 1.44±1.35 Å) comparable to the GL2 positive control (0.26±0.77 Å). The negative control complex, on the other hand, showed structural instability (11.94±2.29 Å). In addition, the experimental complex achieved energetically favorable binding with a converged binding energy of -11.2±0.21 kcal/mol at approximately 12.5 ns, demonstrating stable complex formation comparable to the positive control complex (-41.9±0.27 kcal/mol at about 12 ns). In contrast, the negative control failed to converge, oscillating around 0.2±0.9 kcal/mol without achieving definitively favorable binding. In summation, this paper attempts to provide a computational basis for the development of MUC5AC-targeted siRNA as a therapeutic treatment for CF.