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.

Keywords: CFTR; exosome, cystic fibrosis, MSC, Zinc Finger Protein.

FIGURE 1

CFTR gene activation using Zinc Finger activator protein. (A) A schematic of the zinc finger binding domain targeted by CFZF in the CFTR promoter. The target sequence is shown along with its genomic location as determined from the UCSC genome browser. (B) Schematic of vector expressing the FLAG‐CFZF‐VPR. A CMV Pol II promoter expresses a FLAG‐tagged CFZF targeted to the CFTR promoter with a VPR activation domain. (C) ChIP assays were performed and enrichment relative to control (Neg) is shown as a fraction of input. (D) CFTR mRNA levels as determined by qRT‐PCR in HuBECs from healthy donor (HuBEC CFTR‐WT), and (E) HuBECs from CF patient with the F508del mutation (HuBEC CFTR‐F508del). HuBEC non‐transfected were used as negative control (Neg control). Experiments show the standard error of the mean and p values are represented (paired two‐sided Student's T‐test, *P = 0.01, **P < 0.02)

FIGURE 2

MSC Exosome mediated delivery of Zinc Finger Protein Activator increases the expression of CFTR. (A) TEM micrograph of exosomes isolated from the culture medium of MSC‐CFZF‐VPR transfected cells. Exosomes were measured by using Nanosight NS 300 system in the supernatant from cultures cells. The histogram represents particle size distribution. (B) Western blot analysis for exosome markers in CFZF‐VPR transfected MSC (CFZF) and non‐transfected MSCs (Neg) derived exosomes. (C) Western blot of FLAG‐tagged CFZF‐VPR protein enriched in exosomes from left: MSC‐CFZF‐VPR or right: non‐transfected MSC‐exosomes (M‐Ex), HEK293‐exosomes (H‐Ex) and corresponding MSC cell lysate (M‐CL) samples, respectively. The Image J values from the Flag‐tag (Flag) relative to Beta Actin (B‐Act) are shown below the blot. (D) Evaluation by qRT‐PCR of mRNA expression of exosomes from CFZF‐VPR/Cx43‐transfected MSCs. The results from triplicate exosomes collected from three different CFZF‐VPR/Cx43 transfected MSCs are shown. (E) Light microscopy immunofluorescence images of HuBECs uptake of MSCs‐CFZF‐VPR exosomes labelled with BODIPY TR ceramide (red), Nuclei (Blue), Actin (Green). Scale bar, 10 μm. (F) CFTR expression was determined by qRT‐PCR following treatment with MSC exosomes directed to the CFTR promoter (MSC‐CFZF‐VPR) or Control (MSC‐Neg) in HuBECs. For E and F experiments were performed in triplicate with 10e+03 HuBECs treated with 5e+10 exosomes. Experiment shown the standard error of the means and p values from a paired two‐sided T‐test, *P = 0.01

FIGURE 3

Exosome mediated delivery of CFZF‐VPR increases CFTR protein expression and enhanced Chloride transport. CFTR ELISA analysis of CFTR protein expression in (A) HuBECs from healthy donor HuBEC–WT and (B) in patient cells with F508del mutation following transfection with CFZF‐VPR plasmid or treated with MSC‐derived exosomes carrying CFZF‐VPR. (C) A schematic is shown depicting the halide assay used to assess chloride transport. Fluorescence decrease was evaluated at 0, 2, and 60 s in response to exchange of 25 mM of sodium iodide in cells treated with 0.3 μM of forskolin and combination of VX‐809 and VX‐770, current drugs used for treatment of CF patients. (D) HuBEC CFTR‐WT and (E) HuBEC F508del cells were transfected with CFZF‐VPR plasmid or treated with MSC‐derived exosomes carrying CFZF‐VPR and assessed 48 h post‐treatment using the halide assay. Experiments were performed in triplicate with 10e+03 HuBEC treated with 5e+10 exosomes. ELISA and halide experiments were performed in triplicate in cells shown with the standard error of the means and p values from a paired two‐sided T‐test, *P < 0.05

FIGURE 4

Underlying mechanism of MSC‐CFZF‐VRP exosome mediated activation CFTR. MSC‐exosome are generated to contain a CFZF‐VPR targeted to the CFTR promoter and are internalized into human bronchial epithelial cells whereby the CFZF‐VPR protein and CFZF‐VPR mRNA are released into the intracellular environment by the action of Cx43 and presumably interactions with other endogenous exosome pathway proteins. The result of this Cx43 mediated delivery of CFZF‐VPR is increased expression and functional activation of CFTR