Extracellular barriers in respiratory gene therapy

Abstract

Respiratory gene therapy has been considered for the treatment of a broad range of pulmonary disorders. However, respiratory secretions form an important barrier towards the pulmonary delivery of therapeutic nucleic acids. In this review we will start with a brief description of the biophysical properties of respiratory mucus and alveolar fluid. This must allow the reader to gain insights into the mechanisms by which respiratory secretions may impede the gene transfer efficiency of nucleic acid containing nanoparticles (NANs). Subsequently, we will summarize the efforts that have been done to understand the barrier properties of respiratory mucus and alveolar fluid towards the respiratory delivery of therapeutic nucleic acids. Finally, new and current strategies that can overcome the inhibitory effects of respiratory secretions are discussed.

Fig. 1

Schematic representation of human respiratory mucins and their carbohydrate side chains. Mucins consist out of up to 5 subunits attached to each other by disulfide bonds. These subunits are highly glycosylated proteins with non-glycosylated ends. The depicted carbohydrate chain is only illustrative. Ala, Gly, Pro, Thr, Ga, GaN, Gn, and NA correspond respectively to alanine, glycine, proline, threonine, galactose, N-acetylgalactosamine, N-acetylglucosamine, and N-acetylneuraminic acid (adapted from Ref. [120]).

Fig. 2

Confocal image of a network of polymerized actin filaments (0.01 mg/ml) in the presence of MgCl₂ (80 mM) (A) and atomic force microscopy image of a DNA network (100 µg/ml) (B) (reproduced from Refs. and [35]).

Fig. 3

Photo (A) and schematic drawing (B) of the vertical diffusion chambers. A thin CF sputum layer was placed between the donor and acceptor sides using modified snapwells. Snapwells containing a polycarbonate membrane (pore size 3 µm) were modified by gluing a 220 µm thick ring on their borders. This ring was filled with CF sputum and sealed with a second polycarbonate membrane. Finally, the modified snapwell was placed between the donor and acceptor side. The diffusion experiments were started by filling the donor side with nanoparticles or NANs and the acceptor side with buffer. Subsequently the transport of nanoparticles or NANs through the sputum layer was followed by measuring, at the desired time points, their concentration at the acceptor side.

Fig. 4

Percentages of nanospheres and DOTAP/DOPE lipoplexes that crossed a 220 µm thick layer of CF sputum after 150 min as a function of the average size of the particles (n = 4) (reproduced from Ref. [53]).

Fig. 5

Scanning electron microscopic image of CF sputum showing the meshes in the biopolymer network. The CF sputum samples were processed as previously described (reproduced from Ref. [49]). Bar = 0.5 µm.

Fig. 6

Percentages of nanospheres (per cm²) that crossed a 220 µm thick layer of CF or COPD (gray circle) sputum after 150 min as a function of the elastic moduli of the sputum samples (n = 4) (reproduced from Ref. [49]).

Fig. 7

Principle of multiple particle tracking. In a MPT experiment a high-speed film is recorded of particles. If the particle size is below the microscope's resolution limit, they are seen as dots of light with a Gaussian intensity distribution. However, with suitable image processing software, one can determine the centre of the particles with very high accuracy, typically in the order of tens of nanometers. By determining the position of the particles in all frames of the MPT film, trajectories can be calculated for each individual particle as shown in A (colored lines). The mean square displacements (MSD) of the trajectories can be used for further quantitative analysis in terms of the mode of motion (free diffusion, directed transport, etc.) and the corresponding quantitative parameters (diffusion coefficient, velocity). In B the trajectories of free-diffusing (red) and confined (green) particles are shown. The graph on the right depicts the corresponding MSD of a particle as a function of lag time. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Fig. 8

Effect of CF sputum (upper panels) and normal respiratory mucus (lower panels) on the in vitro gene expression of cationic GL67/DOPE based lipoplexes (A and C) and adenoviral vectors (B and D) in sheep tracheae. Error bars indicate standard error of the mean. (n = 6, ⁎p < 0.05, ⁎⁎ p < 0.01) (reproduced from Ref. [65]).