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 the 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 potency of mesenchymal stem cells (MSC) to develop more effective treatments. Genetic modification of MSC has been demonstrated to significantly improve 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) DNA vector containing human angiopoietin 1 (MC-ANGPT-1) can provide more effective platform for gene-enhanced MSC therapy of ALI/ARDS. Results: At 24 hours after transfection, nuclear-targeted electroporation using a 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±567pg/mL vs. 552.1±33.5pg/mL). In the lipopolysaccharide (LPS)-induced ALI model, administration of pANGPT1 transfected MSC significantly reduced bronchoalveolar lavage (BAL) neutrophil counts by 57%, while MC-ANGPT1 transfected MSC reduced it by 71% (p<0.001) by Holm-Sidak’s multiple comparison test. Moreover, compared to pANGPT1, the MC-ANGPT1 transfected MSC significantly reduced pulmonary inflammation, as observed in decreased levels of proinflammatory cytokines tumor necrosis factor alpha (TNF-α), interferron gamma (IFN-γ), interleukin-6 (IL-6), monocyte chemoattractant protein-1 (MCP-1) and macrophage inflammatory protein-2 (MIP-2). pANGPT1 transfected MSC significantly reduced BAL albumin levels by 71% , while MC-ANGPT1 transfected MSC reduced it by 85%. Coclusions: Overall, using a minicircle vector, we demonstrated an efficient and sustained expression of the ANGPT1 transgene in MSC and enhanced therapeutic effect on ALI model compared to plasmid. These results support the potential benefits of the MC-ANGPT1 gene enhancement of MSC therapy to treat ARDS.

The Expression of Angiopoietin-1 and −2 in the Osteogenesis of Mesenchymal Stem Cells

“…When it is significantly improved, the signal pathways of Ang-1 and Tie-2 can produce endogenous new neurons and other effects. Xu et al [Florian et al, 2021] showed that the bone marrow mesenchymal stem cells with high expression of the target gene Ang-1 constructed by genetic technology can detect the transformation and repair of MSCs in the lung after transplantation, which is a good gene carrier for the treatment of lung disease.…”