Gene engineered mesenchymal stem cells: greater transgene expression and efficacy with minicircle vs. plasmid DNA vectors in a mouse model of acute lung injury

Abstract

Background: Acute lung injury (ALI) and in its severe form, acute respiratory distress syndrome (ARDS), results in increased pulmonary vascular inflammation and permeability and is a major cause of mortality in many critically ill patients. Although cell-based therapies have shown promise in experimental ALI, strategies are needed to enhance the potency of mesenchymal stem cells (MSCs) to develop more effective treatments. Genetic modification of MSCs has been demonstrated to significantly improve the therapeutic benefits of these cells; however, the optimal vector for gene transfer is not clear. Given the acute nature of ARDS, transient transfection is desirable to avoid off-target effects of long-term transgene expression, as well as the potential adverse consequences of genomic integration.

Methods: Here, we explored whether a minicircle DNA (MC) vector containing human angiopoietin 1 (MC-ANGPT1) can provide a more effective platform for gene-enhanced MSC therapy of ALI/ARDS.

Results: At 24 h after transfection, nuclear-targeted electroporation using an MC-ANGPT1 vector resulted in a 3.7-fold greater increase in human ANGPT1 protein in MSC conditioned media compared to the use of a plasmid ANGPT1 (pANGPT1) vector (2048 ± 567 pg/mL vs. 552.1 ± 33.5 pg/mL). In the lipopolysaccharide (LPS)-induced ALI model, administration of pANGPT1 transfected MSCs significantly reduced bronchoalveolar lavage (BAL) neutrophil counts by 57%, while MC-ANGPT1 transfected MSCs reduced it by 71% (p < 0.001) by Holm-Sidak's multiple comparison test. Moreover, compared to pANGPT1, the MC-ANGPT1 transfected MSCs significantly reduced pulmonary inflammation, as observed in decreased levels of proinflammatory cytokines, such as tumor necrosis factor-alpha (TNF-α), interferon-gamma (IFN-γ), interleukin-6 (IL-6), monocyte chemoattractant protein-1 (MCP-1), and macrophage inflammatory protein-2 (MIP-2). pANGPT1-transfected MSCs significantly reduced BAL albumin levels by 71%, while MC-ANGPT1-transfected MSCs reduced it by 85%.

Conclusions: Overall, using a minicircle vector, we demonstrated an efficient and sustained expression of the ANGPT1 transgene in MSCs and enhanced the therapeutic effect on the ALI model compared to plasmid. These results support the potential benefits of MC-ANGPT1 gene enhancement of MSC therapy to treat ARDS.

Keywords: Acute lung injury; Acute respiratory distress syndrome; Mesenchymal stem cell; Minicircle.

Fig. 1

Comparison of plasmid or MC-based transfection in mouse MSCs. a Schematic illustration of method in generating minicircles (adapted from www.systembio.com). b Nuclear-targeted electroporation (Amaxa) of MC-ANGPT1 resulted in a 3.7-fold increase in human ANGPT1 protein in conditioned media using ELISA (R&D Systems) at 24 h after transfection, compared to the same amount of plasmid ANGPT1 (pANGPT1). n = 4 experiments. c MSCs transfected with nucleofection using reagent only (mock), empty plasmid (pVAX), human ANGPT1 plasmid (pANGPT1), empty minicircle DNA (MC), or human ANGPT1 minicircle DNA (MC-ANGPT1). MC-ANGPT1 peak values at D1 and D2 compared to pANGPT1 and mock transfected. n = 2 experiments. d MSC viability evaluated by trypan blue exclusion (bar graph) and cell morphology (photomicrographs) at 24 h after transfection. n = 5 experiments. Group comparisons were analyzed by one-way ANOVA with Holm-Sidak’s post hoc test. *p < 0.05, mock-transfection vs. MC-ANGPT1-transfected MSCs. #p < 0.05, pANGPT1-transfection versus MC-ANGPT1-transfected MSCs

Fig. 2

Therapeutic efficacy of MSCs alone or transfected with ANGPT1 in reducing LPS-induced lung inflammation in mice. a Experimental design for in vivo study. C57Bl/6J mice were first challenged by intratracheal instillation of LPS, followed by respective treatment after 30 min. Mice were then sacrificed 3 days after LPS to evaluate therapeutic efficacy. b Number of neutrophils in bronchoalveolar lavage (BAL) fluid were obtained from differential counts via cytospin to evaluate lung airspace inflammation. Group comparisons were analyzed by one-way ANOVA with Bonferroni post hoc test. ^####p < 0.0001, sham/vehicle versus LPS/vehicle group. **p < 0.01 and ****p < 0.0001, LPS/vehicle vs. each treated group

Fig. 3

Effect of MSCs alone or transfected with ANGPT1 (plasmid vs. MC) on LPS-induced pulmonary inflammation. Pulmonary inflammation was assessed by measurement of multiple cytokines in BAL fluids using multiplex luminex immunoassays kit (Bio-Rad). Group comparisons were analyzed by one-way ANOVA with Bonferroni post hoc test. ^####p < 0.0001, sham/vehicle vs. LPS/vehicle group. ****p < 0.0001, LPS/vehicle vs. each treated group

Fig. 4

Effect of MSCs alone or ANGPT1-transfected (plasmid vs. MC) on LPS-induced pulmonary vascular permeability. Pulmonary vascular permeability was assessed by measurement of a albumin and b IgM in BAL fluids using ELISAs. Group comparisons were analyzed by one-way ANOVA with Bonferroni post hoc test. ^####p < 0.0001, sham/vehicle vs. LPS/vehicle group. ***p < 0.001 and ****p < 0.0001, LPS/vehicle vs. each treated group