Promoter Optimisation of Lentiviral Vectors for E fficient Insulin Gene Expression in Canine Mesenchymal Stromal Cells: Potential Surrogate Beta Cells
Authors
Gautam, Pratigya
Foale, Robert D
Recino, Asha
Zhao, Jing
Gan, Shu Uin
Wallberg, Maja
Calne, Roy
Lever, Andrew
Publication Date
2016-08-30Journal Title
The Journal of Gene Medicine
ISSN
1099-498X
Publisher
Wiley
Volume
18
Pages
312-321
Language
English
Type
Article
This Version
AM
Metadata
Show full item recordCitation
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
Abstract
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.
Keywords
insulin, gene therapy, mesenchymal stromal cells, MSCs, diabetes, lentivirus
Sponsorship
The study was funded by the Lollipop Trust. Work in the laboratory is supported by the Biomedical Research Centre.
Funder references
MRC (MR/N022939/1)
MRC (G0800142)
British Heart Foundation (PG/14/16/30699)
NC3Rs (NC/M001083/1)
Identifiers
External DOI: https://doi.org/10.1002/jgm.2900
This record's URL: https://www.repository.cam.ac.uk/handle/1810/260839
Rights
Attribution-NonCommercial-NoDerivatives 4.0 International, Attribution-NonCommercial-NoDerivatives 4.0 International, Attribution-NonCommercial-NoDerivatives 4.0 International, Attribution-NonCommercial-NoDerivatives 4.0 International