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Bioengineering a non-genotoxic vector for genetic modification of mesenchymal stem cells.

ABSTRACT: Vectors used for stem cell transfection must be non-genotoxic, in addition to possessing high efficiency, because they could potentially transform normal stem cells into cancer-initiating cells. The objective of this research was to bioengineer an efficient vector that can be used for genetic modification of stem cells without any negative somatic or genetic impact. Two types of multifunctional vectors, namely targeted and non-targeted were genetically engineered and purified from E. coli. The targeted vectors were designed to enter stem cells via overexpressed receptors. The non-targeted vectors were equipped with MPG and Pep1 cell penetrating peptides. A series of commercial synthetic non-viral vectors and an adenoviral vector were used as controls. All vectors were evaluated for their efficiency and impact on metabolic activity, cell membrane integrity, chromosomal aberrations (micronuclei formation), gene dysregulation, and differentiation ability of stem cells. The results of this study showed that the bioengineered vector utilizing VEGFR-1 receptors for cellular entry could transfect mesenchymal stem cells with high efficiency without inducing genotoxicity, negative impact on gene function, or ability to differentiate. Overall, the vectors that utilized receptors as ports for cellular entry (viral and non-viral) showed considerably better somato- and genosafety profiles in comparison to those that entered through electrostatic interaction with cellular membrane. The genetically engineered vector in this study demonstrated that it can be safely and efficiently used to genetically modify stem cells with potential applications in tissue engineering and cancer therapy.

SUBMITTER: Chen X

PROVIDER: S-EPMC5671363 | biostudies-literature | 2018 Jan

REPOSITORIES: biostudies-literature

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Bioengineering a non-genotoxic vector for genetic modification of mesenchymal stem cells.

Chen Xuguang X Nomani Alireza A Patel Niket N Nouri Faranak S FS Hatefi Arash A

Biomaterials 20171020

Vectors used for stem cell transfection must be non-genotoxic, in addition to possessing high efficiency, because they could potentially transform normal stem cells into cancer-initiating cells. The objective of this research was to bioengineer an efficient vector that can be used for genetic modification of stem cells without any negative somatic or genetic impact. Two types of multifunctional vectors, namely targeted and non-targeted were genetically engineered and purified from E. coli. The t ...[more]

PMID: 29078136

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Project description:IntroductionA combination of gene and cell therapies has the potential to significantly enhance the therapeutic value of mesenchymal stem cells (MSCs). The development of efficient gene delivery methods is essential if MSCs are to be of benefit using such an approach. Achieving high levels of transgene expression for the required period of time, without adversely affecting cell viability and differentiation capacity, is crucial. In the present study, we investigate lentiviral vector-mediated genetic modification of rat bone-marrow derived MSCs and examine any functional effect of such genetic modification in an in vitro model of ischaemia.MethodsTransduction efficiency and transgene persistence of second and third generation rHIV-1 based lentiviral vectors were tested using reporter gene constructs. Use of the rHIV-pWPT-EF1-?-GFP-W vector was optimised in terms of dose, toxicity, cell species, and storage. The in vivo condition of ischaemia was modelled in vitro by separation into its associated constituent parts i.e. hypoxia, serum and glucose deprivation, in which the effect of therapeutic gene over-expression on MSC survival was investigated.ResultsThe second generation lentiviral vector rHIV-pWPT-EF1-?-GFP-W, was the most efficient and provided the most durable transgene expression of the vectors tested. Transduction with this vector did not adversely affect MSC morphology, viability or differentiation potential, and transgene expression levels were unaffected by cryopreservation of transduced cells. Over-expression of HSP70 resulted in enhanced MSC survival and increased resistance to apoptosis in conditions of hypoxia and ischaemia. MSC differentiation capacity was significantly reduced after oxygen deprivation, but was preserved with HSP70 over-expression.ConclusionsCollectively, these data validate the use of lentiviral vectors for efficient in vitro gene delivery to MSCs and suggest that lentiviral vector transduction can facilitate sustained therapeutic gene expression, providing an efficient tool for ex vivo MSC modification. Furthermore, lentiviral mediated over-expression of therapeutic genes in MSCs may provide protection in an ischaemic environment and enable MSCs to function in a regenerative manner, in part through maintaining the ability to differentiate. This finding may have considerable significance in improving the efficacy of MSC-based therapies.

Project description:Background:The therapeutic capacity of mesenchymal stem cells (also known as mesenchymal stromal cells/MSCs) depends on their ability to respond to the need of the damaged tissue by secreting beneficial paracrine factors. MSCs can be genetically engineered to express certain beneficial factors. The aim of this systematic review is to compile and analyze published scientific literatures that report the use of engineered MSCs for the treatment of various diseases/conditions, to discuss the mechanisms of action, and to assess the efficacy of engineered MSC treatment. Methods:We retrieved all published studies in PubMed/MEDLINE and Cochrane Library on July 27, 2019, without time restriction using the following keywords: "engineered MSC" and "therapy" or "manipulated MSC" and "therapy." In addition, relevant articles that were found during full text search were added. We identified 85 articles that were reviewed in this paper. Results:Of the 85 articles reviewed, 51 studies reported the use of engineered MSCs to treat tumor/cancer/malignancy/metastasis, whereas the other 34 studies tested engineered MSCs in treating non-tumor conditions. Most of the studies reported the use of MSCs in animal models, with only one study reporting a trial in human subjects. Thirty nine studies showed that the expression of beneficial paracrine factors would significantly enhance the therapeutic effects of the MSCs, whereas thirty three studies showed moderate effects, and one study in humans reported no effect. The mechanisms of action for MSC-based cancer treatment include the expression of "suicide genes," induction of tumor cell apoptosis, and delivery of cytokines to induce an immune response against cancer cells. In the context of the treatment of non-cancerous diseases, the mechanism described in the reviewed papers included the expression of angiogenic, osteogenic, and growth factors. Conclusion:The therapeutic capacity of MSCs can be enhanced by inducing the expression of certain paracrine factors by genetic modification. Genetically engineered MSCs have been used successfully in various animal models of diseases. However, the results should be interpreted cautiously because animal models might not perfectly represent real human diseases. Therefore, further studies are needed to explore the translational potential of genetically engineered MSCs.

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Bioengineering a non-genotoxic vector for genetic modification of mesenchymal stem cells.

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Bioengineering a non-genotoxic vector for genetic modification of mesenchymal stem cells.

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