Bioengineered Mesenchymal-Stromal-Cell-Derived Extracellular Vesicles as an Improved Drug Delivery System: Methods and Applications.

Extracellular vesicles (EVs) are cell-derived nano-sized lipid membranous structures that modulate cell-cell communication by transporting a variety of biologically active cellular components. The potential of EVs in delivering functional cargos to targeted cells, their capacity to cross biological barriers, as well as their high modification flexibility, make them promising drug delivery vehicles for cell-free therapies. Mesenchymal stromal cells (MSCs) are known for their great paracrine trophic activity, which is largely sustained by the secretion of EVs. MSC-derived EVs (MSC-EVs) retain important features of the parental cells and can be bioengineered to improve their therapeutic payload and target specificity, demonstrating increased therapeutic potential in numerous pre-clinical animal models, including in the treatment of cancer and several degenerative diseases. Here, we review the fundamentals of EV biology and the bioengineering strategies currently available to maximize the therapeutic value of EVs, focusing on their cargo and surface manipulation. Then, a comprehensive overview of the methods and applications of bioengineered MSC-EVs is presented, while discussing the technical hurdles yet to be addressed before their clinical translation as therapeutic agents.

Mesenchymal Stromal Cells (MSCs): A Promising Tool for Cell-Based Angiogenic Therapy.

The Mesenchymal stromal cells (MSCs) are a diverse subset of adult multipotent precursors, known for their potential therapeutic properties in regenerative medicine mainly sustained by paracrine effects through secretion of a variety of biologically active molecules. MSC secretome includes a wide range of soluble protein factors, composed of growth factors and cytokines, and vesicular components, which transfer proteins and genetic material modulating the host microenvironment. In particular, MSC-derived secretome mediates the different steps of the angiogenic process, inducing endothelial cell functions in vitro and promoting angiogenesis in vivo. As a result, MSCs have been widely explored as a promising cell-based therapy in diseases caused by insufficient angiogenesis. Numerous studies of myocardial infarction, ischemic stroke, and critical limb ischemia in animals have shown that human MSCs can enhance angiogenesis and accelerate tissue regeneration. This extensive preclinical work encouraged the study of these remarkable cells for the treatment of these disorders in human clinical settings. The present review provides a comprehensive overview of the pro-angiogenic potential of MSCs and paracrine effectors of their secretome. In addition, bioengineering strategies, including ex vivo preconditioning and genetic modification approaches, to enhance MSC innate angiogenic properties, and thereby therapeutic potency, will be presented. Finally, an update on completed preclinical and clinical studies with MSCs for the treatment of ischemia-related diseases will be discussed.

Minicircle-based expression of vascular endothelial growth factor in mesenchymal stromal cells from diverse human tissues.

BACKGROUND: Mesenchymal stromal cells (MSC) have been exploited for the treatment of ischemic diseases given their angiogenic potential. Despite bone marrow (BM) being the most studied tissue source, cells with similar intrinsic properties can be isolated from adipose tissue (AT) and umbilical cord matrix (UCM). The present study aims to compare the angiogenic potential of MSC obtained from BM, AT and UCM that were genetically modified with vascular endothelial growth factor (VEGF)-encoding minicircle (MC) vectors. The overexpression of VEGF combined with the intrinsic properties of MSC could represent a promising strategy towards angiogenic therapies. METHODS: We established a microporation-based protocol to transfect human MSC using VEGF-encoding MC (MC-VEGF). VEGF production levels were measured by an enzyme-linked immunosorbent assay and a quantitative polymerase chain reaction. The in vitro angiogenic potential of transfected cells was quantified using cell tube formation and migration functional studies. RESULTS: MSC isolated from BM, AT or UCM showed similar levels of VEGF secretion after transfection with MC-VEGF. Those values were significantly higher when compared to non-transfected cells, indicating an effective enhancement of VEGF production. Transfected cells displayed higher in vitro angiogenic potential than non-transfected controls, as demonstrated by functional in vitro assays. No significant differences were observed among cells from different sources. CONCLUSIONS: Minicircles can be successfully used to transiently overexpress VEGF in human MSC, regardless of the cell tissue source, representing an important advantage in a clinical context (i.e., angiogenic therapy) because a standard protocol might be applied to MSC of different tissue sources, which can be differentially selected according to the application (e.g., autologous versus allogeneic settings).

Engineering of Human Mesenchymal Stem/Stromal Cells with Vascular Endothelial Growth Factor-Encoding Minicircles for Angiogenic Ex Vivo Gene Therapy.

Peripheral artery disease (PAD) is a debilitating and prevalent condition characterized by blockage of the arteries, leading to limb amputation in more severe cases. Mesenchymal stem/stromal cells (MSC) are known to have intrinsic regenerative properties that can be potentiated by the introduction of pro-angiogenic genes such as the vascular endothelial growth factor (VEGF). Herein, the use of human bone marrow MSC transiently transfected with minicircles encoding for VEGF is proposed as an ex vivo gene therapy strategy to enhance angiogenesis in PAD patients. The VEGF gene was cloned in minicircle and conventional plasmid vectors and used to transfect bone marrow-derived MSC ex vivo. VEGF expression was evaluated both by quantitative polymerase chain reaction and enzyme-linked immunosorbent assay. The number of VEGF transcripts following MSC transfection with minicircles increased 130-fold relative to the expression in non-transfected MSC, whereas for the plasmid (pVAX1)-based transfection, the increase was 50-fold. Compared to the VEGF basal levels secreted by MSC (11.1 ± 3.4 pg/1,000 cells/day), significantly higher values were detected by enzyme-linked immunosorbent assay after both minicircle and pVAX1 transfection (644.8 ± 82.5 and 508.3 ± 164.0 pg/1,000 cells/day, respectively). The VEGF overexpression improved the angiogenic potential of MSC in vitro, as confirmed by endothelial cell tube formation and cell migration assays, without affecting the expansion potential ex vivo, as well as multilineage differentiation capacity or immunophenotype of MSC. Although preclinical in vivo studies are required, these results suggest that minicircle-mediated VEGF gene delivery, combined with the unique properties of human MSC, could represent a promising ex vivo gene therapy approach for an improved angiogenesis in the context of PAD.

Plasmid DNA production with Escherichia coli GALG20, a pgi-gene knockout strain: fermentation strategies and impact on downstream processing.

The market development of plasmid biopharmaceuticals for gene therapy and DNA vaccination applications is critically dependent on the availability of cost-effective manufacturing processes capable of delivering large amounts of high-quality plasmid DNA (pDNA) for clinical trials and commercialization. The producer host strain used in these processes must be designed to meet the upstream and downstream processing challenges characteristic of large scale pDNA production. The goal of the present study was to investigate the effect of different glucose feeding strategies (batch and fed-batch) on the pDNA productivity of GALG20, a pgi Escherichia coli strain potentially useful in industrial fermentations, which uses the pentose phosphate pathway (PPP) as the main route for glucose metabolism. The parental strain, MG1655ΔendAΔrecA, and the common laboratory strain, DH5α, were used for comparison purposes and pVAX1GFP, a ColE1-type plasmid, was tested as a model. GALG20 produced 3-fold more pDNA (∼141 mg/L) than MG1655ΔendAΔrecA (∼48 mg/L) and DH5α (∼40 mg/L) in glucose-based fed-batch fermentations. The amount of pDNA in lysates obtained from these cells was also larger for GALG20 (41%) when compared with MG1655ΔendAΔrecA (31%) and DH5α (26%). However, the final quality of pDNA preparations obtained with a process that explores precipitation, hydrophobic interaction chromatography and size exclusion was not significantly affected by strain genotype. Finally, high cell density fed-batch cultures were performed with GALG20, this time using another ColE1-type plasmid, NTC7482-41H-HA, in pre-industrial facilities using glucose and glycerol. These experiments demonstrated the ability of GALG20 to produce high pDNA yields of the order of 2100-2200 mg/L.

Comparative analysis of antigen-targeting sequences used in DNA vaccines.

Plasmid vectors can be optimized by including specific signals that promote antigen targeting to the major antigen presentation and processing pathways, increasing the immunogenicity and potency of DNA vaccines. A pVAX1-based backbone was used to encode the Green Fluorescence Protein (GFP) reporter gene fused either to ISG (Invariant Surface Glycoprotein) or to TSA (trans-sialidase) Trypanosoma brucei genes. The plasmids were further engineered to carry antigen-targeting sequences, which promote protein transport to the extracellular space (secretion signal), lysosomes (LAMP-1) and to the endoplasmic reticulum (adenovirus e1a). Transfection efficiency was not affected by differences in the size between each construct as no differences in the plasmid copy number per cell were found. This finding also suggests that the addition of both ISG gene and targeting sequences did not add sensitive regions prone to nuclease attack to the plasmid. Cells transfected with pVAX1GFP had a significant higher number of transcripts. This could be a result of lower mRNA stability and/or a lower transcription rate associated with the bigger transcripts. On the other hand, no differences were found between transcript levels of each ISG-GFP plasmids. Therefore, the addition of these targeting sequences does not affect the maturation/stability of the transcripts. Microscopy analysis showed differences in protein localization and fluorescent levels of cells transfected with pVAX1GFP and ISG constructs. Moreover, cells transfected with the lamp and secretory sequences presented a distinct distribution pattern when compared with ISG protein. Protein expression was quantified by flow cytometry. Higher cell fluorescence was observed in cells expressing the cytoplasmic fusion protein (ISG-GFP or TSA-GFP) compared with cells where the protein was transported to the lysosomal pathway. Protein transport to the endoplasmic reticulum does not lead to a decrease in the mean fluorescence values. The secretion signal was only effective when used in conjunction with TSA gene. Therefore, the characteristics of each protein (e.g., presence of transmembrane domains) might influence the efficacy of its cellular transport. This analysis constitutes a useful tool for the optimization of the design of DNA vaccines.

Development of a candidate DNA vaccine against Maedi-Visna virus.

DNA vaccine candidates against Maedi-Visna virus (MVV) infection in ovines were developed as an alternative to conventional vaccines. Candidates were constructed by cloning genes encoding the MVV gag polyprotein and gag proteins p16 and p25 fused to a beta-galactosidase reporter in a plasmid backbone. Transfection of different ovine cells showed a higher protein expression with plasmid lacZp16, which was hence further optimised by (i) removing a putative inhibitory sequence via reduction of the AU-content in the p16 gene or by (ii) introducing a secretory signal (Sc) to promote antigen secretion and increase its presentation to APCs. Unexpectedly, plasmids constructed on the basis of the first strategy by mutagenesis of lacZp16 (lacZp16mut(24)), led to a reduction in the expression of the antigen/reporter fusion in cultured ovine cells. This indicates that the high AU content in MVV does not inhibit protein expression. However, mice primed with lacZp16mut(24) and boosted with MVV protein displayed higher humoral response when compared with control lacZp16. The addition of the Sc signal (Sc-p16) led to lower amounts of intracellular antigen/reporter fusion in transfected ovine cells, thus confirming secretion. These findings correlate with in vivo experiments, which showed that mice primed with Sc-p16 and boosted with MVV exhibited stronger antibody responses when compared with control mice primed with lacZp16 and boosted with MVV. Stronger humoral responses were recorded by immunising mice with (i) Sc-p16 and lacZp16mut(24) plasmids together or with (ii) one plasmid containing both the mutations and the Sc signal.