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.
Highlights
Cystic fibrosis (CF) is an autosomal recessive genetic disease caused by mutations in the CFTR gene [Riordan et al, 1989)
To test whether CFZF-VPR binds directly to the CFTR promoter, a FLAG-tag zinc finger proteins (ZFP)-VPR vector was constructed with a CMV promoter used to express the transgene (Figure 1B), and used in a FIGURE CFTR gene activation using Zinc Finger activator protein. (A) A schematic of the zinc finger binding domain targeted by CFZF in the CFTR promoter
A CMV Pol II promoter expresses a FLAG-tagged CFZF targeted to the CFTR promoter with a VPR activation domain. (C) Chromatin Immunoprecipitation assay (ChIP) assays were performed and enrichment relative to control (Neg) is shown as a fraction of input. (D) CFTR mRNA levels as determined by Quantitative realtime polymerase chain reaction (qRT-PCR) in Human Bronchial Epithelial cells (HuBEC) from healthy donor (HuBEC CFTR-WT), and (E) HuBECs from CF patient with the F508del mutation (HuBEC CFTR-F508del)
Summary
Cystic fibrosis (CF) is an autosomal recessive genetic disease caused by mutations in the CFTR gene [Riordan et al, 1989). Mutations in the CFTR gene result in a frameshift mutation in the mRNA coding sequence that impinges protein processing, channel gating, conductance function and results in the expression of a relatively non-functional CFTR protein. The most prevalent CFTR mutation is caused by the deletion of a phenylalanine residue at position 508 (F508del) in the CFTR protein, which affects ∼70% of patients (Bobadilla et al, 2002). A small proportion of this misfolded F508del protein is released from the Golgi and presented on the plasma membrane with a reduction in channel gating activity (Du et al, 2005, Kim & Skach, 2012, Thomas et al, 1992). There are a plethora of other CFTR mutations (Romey, 2006), which result in diminished CFTR protein function, and increasing its expression could improve the CF phenotype. A means to target and activate CFTR, irrespective of the variation in CFTR mutations, could prove therapeutically relevant in CF patients
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