Promoter Optimisation of Lentiviral Vectors for E fficient Insulin Gene Expression in Canine Mesenchymal Stromal Cells: Potential Surrogate Beta Cells
Foale, Robert D
Gan, Shu Uin
The Journal of Gene Medicine
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Gautam, P., Foale, R. D., Recino, A., Zhao, J., Gan, S. U., Wallberg, M., Calne, R., & et al. (2016). Promoter Optimisation of Lentiviral Vectors for E fficient Insulin Gene Expression in Canine Mesenchymal Stromal Cells: Potential Surrogate Beta Cells. The Journal of Gene Medicine, 18 312-321. https://doi.org/10.1002/jgm.2900
Background: The lack of an ideal cell type that can be easily acquired, modified to produce insulin, and re-implanted has been a limitation for ex vivo insulin gene therapy. Canine diabetes is currently treated with human insulin and is a good model for human diabetes. Mesenchymal stromal cells (MSCs) are a promising candidate cell type for gene therapy. Here we optimised insulin production using lentiviral transduced canine MSCs aiming to evaluate their ability for use as surrogate beta cells. Method: Canine MSCs were derived from bone marrow and validated by measuring expression of MSC lineage specific markers. Lentivirus vectors encoding the proinsulin gene (with or without a Kozak sequence) under the control of SFFV, CMV, EF1α and SV40 promotors were generated and used to transduce primary cMSCs and a hepatocyte cell line. The insulin producing capacity of transduced primary canine MSCs was assessed by measuring the concentration of C-peptide produced. Result: Primary canine MSC could be readily expanded in culture and efficiently transduced using lentiviral vectors encoding proinsulin. Increasing the multiplicity of infection from 3 to 20, led to an increase in C-peptide secretion (1700 pmol/l to 4000 pmol/l). The SFFV promoter conferred the strongest transcriptional ability. Conclusion: Our results suggest that optimised lentiviral transduction of the insulin gene into primary canine MSCs renders these cells capable of secreting insulin both short- and long-term, in sufficient quantities in vitro to support their potential use in insulin gene therapy.
insulin, gene therapy, mesenchymal stromal cells, MSCs, diabetes, lentivirus
The study was funded by the Lollipop Trust. Work in the laboratory is supported by the Biomedical Research Centre.
British Heart Foundation (PG/14/16/30699)
External DOI: https://doi.org/10.1002/jgm.2900
This record's URL: https://www.repository.cam.ac.uk/handle/1810/260839
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