Mesenchymal Stem Cell exosome delivered Zinc Finger Protein activation of cystic fibrosis transmembrane conductance regulator

Abstract

Cystic fibrosis is a genetic disorder that results in a multi-organ disease with progressive respiratory decline which leads to premature death. Mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene disrupts the capacity of the protein to function as a channel, transporting chloride ions and bicarbonate across epithelial cell membranes. Small molecule treatments targeted at potentiating or correcting CFTR have shown clinical benefits, but are only effective for a small percentage of individuals with specific CFTR mutations. To overcome this limitation, we engineered stromal-derived mesenchymal stem cells (MSC) and HEK293 cells to produce exosomes containing a novel CFTR Zinc Finger Protein fusion with transcriptional activation domains VP64, P65 and Rta to target the CFTR promoter (CFZF-VPR) and activate transcription. Treatment with CFZF-VPR results in robust activation of CFTR transcription in patient derived Human Bronchial Epithelial cells (HuBEC). We also find that CFZF-VPR can be packaged into MSC and HEK293 cell exosomes and delivered to HuBEC cells to potently activate CFTR expression. Connexin 43 appeared to be required for functional release of CFZF-VPR from exosomes. The observations presented here demonstrate that MSC derived exosomes can be used to deliver a packaged zinc finger activator to target cells and activate CFTR. The novel approach presented here offers a next-generation genetic therapy that may one day prove effective in treating patients afflicted with Cystic fibrosis. K E Y WO R D S CFTR, exosome, cystic fibrosis, MSC, Zinc Finger Protein  SIGNIFICANCE STATEMENT Exosomes are important mediators that facilitate intercellular communication and transfer of genetic material to recipient cells. Here, we take advantage of this inherent property and engineer MSCs to produce exosomes packaged with a Zinc Finger Activator targeted to the CFTR gene promoter. We find that treatment of CF patient derived HuBEC with the CFZF-VPR containing exosomes results in significant activation of CFTR. The novel approach presented here represents an effective way to deliver therapeutic proteins to patients afflicted with CF. This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited.

Cellular Therapy for the Treatment of Paediatric Respiratory Disease

“…They replaced the chloride ions in the cells with iodide ions and incubated them with forskolin to stimulator CTFR prior to further culture in the presence of the CTFR potentiator (VX770) and corrector (VX809). In both wild type HuBECs and ∆F508 CTFR HuBECs that had been treated with CFZF-VPR exosomes, there was a significant decrease in fluorescence compared to untreated controls [71]. This confirmed the ability for MSC CFZF-VPR exosomes to be used to provide a functional repair of the CTFR mutation in HuBEC cells.…”

“…A recent study by Villamizar and colleagues was carried out to determine the potential of MS-derived exosomes for delivery of zinc finger activators targeted to the CFTR gene promoter (CFZF-VPR) [71]. Human BMMSCs (6 × 10 6 ) were co-transfected with CFZP-VPR and Connexin 43 (Cx43), and after 48 h, exosomes were harvested from the medium.…”

Mesenchymal stem cells-derived exosomes for drug delivery

“…Therefore, further exploring the mechanism of RNA and protein in exosomes is necessary. Other studies also confirmed that MSC-EXOs are feasible as a protein transport carrier [122,[125][126][127][128] (Table 4).…”

Treatment of Cystic Fibrosis: From Gene- to Cell-Based Therapies

“…Furthermore, recent studies suggest that MSCs function transiently to reduce inflammation via the secretion of extra-cellular vesicles such as exosomes (Zhu et al, 2014;Zulueta et al, 2018) that can be used for delivery of drugs or gene editing material (reviewed in (Almeida-Porada et al, 2020)). Exosomes derived from MSCs genetically engineered to carry a transcription activator protein have demonstrated success in targeting and activating CFTR transcription in primary human bronchial epithelial cells from patients with CF (Villamizar et al, 2021). Further in vitro and in vivo studies are needed before this approach is considered viable.…”