Overcoming the Cystic Fibrosis Sputum Barrier to Leading Adeno-associated Virus Gene Therapy Vectors

Abstract

Gene therapy has not yet improved cystic fibrosis (CF) patient lung function in human trials, despite promising preclinical studies. In the human CF lung, inhaled gene vectors must penetrate the viscoelastic secretions coating the airways to reach target cells in the underlying epithelium. We investigated whether CF sputum acts as a barrier to leading adeno-associated virus (AAV) gene vectors, including AAV2, the only serotype tested in CF clinical trials, and AAV1, a leading candidate for future trials. Using multiple particle tracking, we found that sputum strongly impeded diffusion of AAV, regardless of serotype, by adhesive interactions and steric obstruction. Approximately 50% of AAV vectors diffused >1,000-fold more slowly in sputum than in water, with large patient-to-patient variation. We thus tested two strategies to improve AAV diffusion in sputum. We showed that an AAV2 mutant engineered to have reduced heparin binding diffused twice as fast as AAV2 on average, presumably because of reduced adhesion to sputum. We also discovered that the mucolytic N-acetylcysteine could markedly enhance AAV diffusion by altering the sputum microstructure. These studies underscore that sputum is a major barrier to CF gene delivery, and offer strategies for increasing AAV penetration through sputum to improve clinical outcomes.

The Mucus Barrier to Inhaled Gene Therapy

“…86,88 Schuster et al, recently demonstrated that AAV2 adhesion to CF mucus may be partially mediated by specific binding to heparan sulfate on airway mucins. 11 This effect was demonstrated using a mutated AAV2 wherein specific binding to heparan sulfate was abolished, leading to a twofold increase in transport in CF mucus compared with wild-type AAV2. 11 Others have shown that the influenza virus is immobilized in airway mucus through adhesive interactions of the virus with sialic acid on mucins found in the gel layer, as influenza requires the same specific binding to sialic acid on cell-surface glycans to mediate epithelial cell entry.…”

“…5,17,18 Airway mucus is a viscoelastic gel 19,20 with a near neutral pH, 21 and possesses a porous structure as shown by electron microscopy (Figure 1b). 9,11,22,23 Mucins are continuously secreted into the airways where they form a gel layer that is continuously removed from the lungs to the throat via MCC, or is occasionally coughed out. Mucus cleared from the lungs via MCC is swallowed and its contents are sterilized in the stomach.…”

“…Adenovirus serotype 5 and adenoassociated virus (AAV) serotype 1, 2, and 5 all strongly adhere to the CF mucus gel (Figure 3a). 10,11 Adenovirus5 binds to heparan sulfate, 84,85 which is highly prevalent on airway mucins in the gel layer. AAV of various serotypes bind to cell-surface associated glycans, such as heparan sulfate and/or α 2,3/2,6-linked sialic acids, to infect host cells.…”

“…[2][3][4][5] The mucus layer traps inhaled particulates and facilitates their removal from the airways via mucociliary clearance (MCC) and other clearance mechanisms. [5][6][7] Likewise, many viral and nonviral gene vectors used in past inhaled gene therapy trials have recently been shown to be adhesive to human airway mucus [8][9][10][11] and, thus, are likely incapable of efficiently penetrating the mucus gel layer to reach the targeted underlying epithelial cells prior to clearance.…”

Highly compacted biodegradable DNA nanoparticles capable of overcoming the mucus barrier for inhaled lung gene therapy

“…patients to be 140 ± 50 nm (range 60-300 nm) (22). As a consequence of the elevated adhesivity and tighter mesh size of CF mucus, adenovirus (23,24), adeno-associated virus (AAV) serotypes 1, 2, and 5 (23,25), and a clinically tested polymeric nonviral gene vector (CK 30 PEG 10k DNA-NP) (26,27) are unable to efficiently penetrate the mucus gel layer, thereby limiting their success in clinical trials (20,22,28). In addition, conventional gene vectors formulated with cationic polymers, such as polyethylenimine (PEI) and poly-L-lysine (PLL), are immobilized in airway mucus regardless of their size due to the positively charged particle surface that interacts with the negatively charged mucus constituents (18).…”