Microparticle-mediated transfer of the viral receptors CAR and CD46, and the CFTR channel in a CHO cell model confers new functions to target cells

Abstract

Cell microparticles (MPs) released in the extracellular milieu can embark plasma membrane and intracellular components which are specific of their cellular origin, and transfer them to target cells. The MP-mediated, cell-to-cell transfer of three human membrane glycoproteins of different degrees of complexity was investigated in the present study, using a CHO cell model system. We first tested the delivery of CAR and CD46, two monospanins which act as adenovirus receptors, to target CHO cells. CHO cells lack CAR and CD46, high affinity receptors for human adenovirus serotype 5 (HAdV5), and serotype 35 (HAdV35), respectively. We found that MPs derived from CHO cells (MP-donor cells) constitutively expressing CAR (MP-CAR) or CD46 (MP-CD46) were able to transfer CAR and CD46 to target CHO cells, and conferred selective permissiveness to HAdV5 and HAdV35. In addition, target CHO cells incubated with MP-CD46 acquired the CD46-associated function in complement regulation. We also explored the MP-mediated delivery of a dodecaspanin membrane glycoprotein, the CFTR to target CHO cells. CFTR functions as a chloride channel in human cells and is implicated in the genetic disease cystic fibrosis. Target CHO cells incubated with MPs produced by CHO cells constitutively expressing GFP-tagged CFTR (MP-GFP-CFTR) were found to gain a new cellular function, the chloride channel activity associated to CFTR. Time-course analysis of the appearance of GFP-CFTR in target cells suggested that MPs could achieve the delivery of CFTR to target cells via two mechanisms: the transfer of mature, membrane-inserted CFTR glycoprotein, and the transfer of CFTR-encoding mRNA. These results confirmed that cell-derived MPs represent a new class of promising therapeutic vehicles for the delivery of bioactive macromolecules, proteins or mRNAs, the latter exerting the desired therapeutic effect in target cells via de novo synthesis of their encoded proteins.

Figure 1. Electron microscopy (EM) of MPs

isolated from CHO-CD46 cells. (A), Negative staining and immuno-EM of MP₃₀CD46. (B), Negative staining (a), and immuno-EM (b) of MP₁₀₀CD46. In (A) and (B, b), immunogold labeling was performed using anti-C46 antibody and 10 nm-gold tagged complementary antibody. MP-associated gold grains are indicated by arrows. (C), Ultrathin sections of pelletable complexes of MP₃₀CD46-HAdV5F35, shown at low (a) and high (b) magnifications. Particles of HAdV5F35 vector (70–80 nm in diameter) in complex with MP₃₀CD46 are indicated by the letter V.

Figure 2. EM of target cells incubated with control MP₃₀CHO or MP₃₀CD46 in complex with HAdV5F35.

(A), Ultrathin sections of target CHO cells incubated with MP₃₀CD46-HAdV5F35 complexes. V, particles of HAdV5F35 vector. (B), Ultrathin sections of target CHO cells incubated with control MP₃₀CHO from nontransduced CHO cells, mixed with HAdV5F35. Sections are shown at low (a) and high (b) magnifications. Note the absence of vector particles associated with control MP₃₀CHO.

Figure 3. MP₃₀-mediated transfer and functionality of (a) CAR, or (b) CD46 as adenoviral receptors in target cells.

Aliquots of CHO cells (target cells) were incubated with MP₃₀CAR (a) or MP₃₀CD46 (b) at different MP doses per cell, as indicated in the x-axis. At 72 h after MP₃₀-cell interaction, cells were infected with HAdV5-GFP or HAdV5F35-GFP vector, at the same MOI (500 vp/cell). The degree of CHO permissiveness to HAdV5-GFP or HAdV5F35-GFP vector was evaluated by flow cytometry analysis of the intracellular GFP signal. In (a), HAdV5F35-GFP, which does not recognize CAR as cellular receptor, was used as the negative control. In (b), HAdV5-GFP, which does not recognize CD46 as cellular receptor, was used as the negative control. MP₃₀ from nontransduced CHO cells (Control MP) served as the negative controls in both panels.

Figure 4. Functionality of exogenous CD46 in MP₃₀CD46-transduced CHO cells. (A), CD46 as complement C3 regulator.

CHO cells were harvested at 48 h posttransfer, and the CD46-induced protection against complement C3-mediated cell apoptosis was assayed by the percentage of Annexin V-positive cells determined by flow cytometry at increasing doses of complement C3. (B), Kinetics of the gain of adenoviral receptor function by MP₃₀CD46-transduced CHO cells. CHO cells were harvested at different times after MP₃₀CD46-transfer, and cell permissiveness to the HAdV5F35-GFP vector was assessed by infection with HAdV5F35-GFP at MOI 500. Cells were analyzed for GFP signal at 48 h postinfection. The degree of permissiveness to the vector was expressed as the percentage of GFP-positive cells (left y-axis), and the relative transduction efficiency (RTE; right y-axis). The RTE, in arbitrary units (AU), was given using the formula = (percentage of GFP-positive cells) x (MFI; mean fluorescence intensity).

Figure 5. Expression, immunoreactivity and functionality of GFP-CFTR protein expressed from an episomal plasmid in CHO cells (MP-producer cells).

(A), CHO cells harboring the GFP-CFTR-encoding pCEP4 episomal plasmid, were examined by phase-contrast (i) and epifluorescence microscopy (ii) of GFP-tagged CFTR protein. Positive controls, consisting of CHO cells transduced by the adenoviral vector HAdV5-GFP-CFTR were similarly examined by phase-contrast (iii) and epifluorescence microscopy (iv). Scale bars, 20 µm. (B), Surface expression and correct orientation of the GFP-tagged CFTR protein in pCEP4-GFP-CFTR harboring CHO cells, evaluated by flow cytometry using monoclonal antibody against the first extracellular loop of the CFTR ectodomain. (C), Chloride channel activity of CFTR in negative control CHO cells (dotted bars), positive control HAdV5F35-GFP-CFTR-transduced CHO-CD46 cells (hatched bars), and pCEP4-GFP-CFTR harboring CHO cells (black bars), evaluated using the fluorescent probe DiSBAC₂(3). The time-course analysis of DiSBAC₂(3) fluorescence changes in the presence of a cocktail of CFTR activators was monitored in regions of the cell monolayers corresponding to 20–30 cells. Bars represent the mean fluorescence value for each field of 20–30 cells ± SEM. Symbols: *, p<0.05; **, p<0.01; ns, not significant.

Figure 6. Time-course expression of GFP-CFTR in MP-transduced CHO cells.

(A), (B), GFP-positive cells (expressed as %) were determined by flow cytometry analysis of MP₃₀-transduced (A) and MP₁₀₀-transduced cells (B), harvested at different times post-transfer (pt). Bars represent mean values ± SEM (n = 3).

Figure 7. Time-course expression of GFP-CFTR glycoprotein at the surface of MP-transduced CHO cells.

Cells harvested at different times after MP-cell transfer were analyzed by flow cytometry for the immunoreactivity of the first N-terminal loop of the CFTR ectodomain with anti-CFTR monoclonal antibody. (A), MP₃₀-transduced CHO cells. (B), MP₁₀₀-transduced CHO cells. Bars represent mean values ± SEM (n = 3).

Figure 8. Occurrence of GFP-CFTR-encoding mRNA in MPs and MP-transduced CHO cells.

Bar graph representation of qRT-PCR assays of MP₃₀ (A), MP₁₀₀ (B), MP₃₀-transduced CHO cells (C), and MP₁₀₀-transduced CHO cells (D). Data shown in the graphs are mean values (m) ± SEM (n = 3). Symbols: *, p<0.05; **, p<0.01; ***, p<0.001; ns, not significant.

Figure 9. CFTR channel activity

in MP-transduced CHO recipient cells. (A), Evolution of the DiSBAC₂(3) fluorescence signal. The time-course analysis of DiSBAC₂(3) fluorescence changes was monitored in regions of the cell monolayer corresponding to 20–30 cells, taken at 72 h post-transfer, a time point corresponding to the maximal immunoreactivity of CFTR glycoprotein at the cell surface. Changes in the fluorescent signal were expressed as F_t/F₀ ratio values, in which F_t and F₀ were the fluorescence values at the times t and t ₀, respectively, and t ₀ the time when the cAMP-containing, CFTR-activating cocktail was added. The cocktail of CFTR activators was maintained throughout the experiment. The CFTR inhibitor GlyH-101 was added for 5 min (phase II, marked by two vertical arrows). Phase III shows reversibility of the CFTR block and recovery of the fluorescent signal. (B), Fluorescence microscopy. Photographs of cell monolayers were taken at different time-points corresponding to the four successive phases, as indicated on the curve by the letters (a), (b), (c) and (d).