PEGylated enhanced cell penetrating peptide nanoparticles for lung gene therapy

Abstract

The lung remains an attractive target for the gene therapy of monogenetic diseases such as cystic fibrosis (CF). Despite over 27 clinical trials, there are still very few gene therapy vectors that have shown any improvement in lung function; highlighting the need to develop formulations with improved gene transfer potency and the desirable physiochemical characteristics for efficacious therapy. Herein, we introduce a novel cell penetrating peptide (CPP)-based non-viral vector that utilises glycosaminoglycan (GAG)-binding enhanced transduction (GET) for highly efficient gene transfer. GET peptides couple directly with DNA through electrostatic interactions to form nanoparticles (NPs). In order to adapt the GET peptide for efficient in vivo delivery, we engineered PEGylated versions of the peptide and employed a strategy to form DNA NPs with different densities of PEG coatings. We were able to identify candidate formulations (PEGylation rates ≥40%) that shielded the positively charged surface of particles, maintained colloidal stability in bronchoalveolar lavage fluid (BALF) and retained gene transfer activity in human bronchial epithelial cell lines and precision cut lung slices (PCLS) in vitro. Using multiple particle tracking (MPT) technology, we demonstrated that PEG-GET complexes were able to navigate the mucus mesh and diffuse rapidly through patient CF sputum samples ex vivo. When tested in mouse lung models in vivo, PEGylated particles demonstrated superior biodistribution, improved safety profiles and efficient gene transfer of a reporter luciferase plasmid compared to non-PEGylated complexes. Furthermore, gene expression was significantly enhanced in comparison to polyethylenimine (PEI), a non-viral gene carrier that has been widely tested in pre-clinical settings. This work describes an innovative approach that combines novel GET peptides for enhanced transfection with a tuneable PEG coating for efficacious lung gene therapy.

Engineered amphiphilic peptides enable delivery of proteins and CRISPR-associated nucleases to airway epithelia

“…There is great interest in protein and nucleic acid-based therapeutics for airway diseases, but delivery to respiratory epithelial cells remains exceedingly challenging. Several viral vectors 3,6,46 , non-viral liposomes 21,47,48 , and other nanoparticle formulations are in development to address delivery challenges 49,50 , but none have advanced to widespread clinical utility. While some viral vectors overcome delivery barriers 4,5,51 , they are complex biologics and genetic payloads may exceed the capacity of some vectors.…”

CFTR Modulators: The Changing Face of Cystic Fibrosis in the Era of Precision Medicine

“…It remains nevertheless unclear how many CFTR modulators would be needed to reach such threshold in patients. In addition to CFTR modulators, progress has been made in developing cell-based (Barical et al, 2019;Hayes et al, 2019) and gene-based therapies (Donnelley and Parsons, 2018;Duncan et al, 2018;Lopes-Pacheco et al, 2018;Osman et al, 2018) for CF lung disease.…”

Cell Penetrating Peptides, Novel Vectors for Gene Therapy

“…These NPs, loaded with siRNA, accumulated within tumor cells upon near-infrared light illumination, resulting in increased antitumor efficacy in vivo [109]. Another approach employed was PEGylation of glycosaminoglycan-binding peptides coupled with DNA through electrostatic charge interactions, which formed NPs that targeted bronchial epithelial cell lines with precision cut lung slices in vitro showing that PEGylation rates of >40% were the optimal formulation [110]. Additionally, PEGylated supra-magnetic iron oxide NPs [111,112], Nobel metal NPs [113] or gold particles [114] have also been modified with CPPs to show internalization into tumor or stem cells.…”