Promoter optimisation of lentiviral vectors for efficient insulin gene expression in canine mesenchymal stromal cells: potential surrogate beta cells

作者：Gautam Pratigya; Recino Asha; Foale Robert D; Zhao Jing; Gan Shu Uin; Wallberg Maja; Calne Roy; Lever Andrew M L^*

来源：JOURNAL OF GENE MEDICINE, 2016, 18(10): 312-321.

DOI：10.1002/jgm.2900

• diabetes
• gene therapy
• insulin
• lentivirus
• mesenchymal stromal cells

BackgroundThe 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. In the present study, we optimised insulin production using lentiviral transduced canine MSCs (cMSCs), aiming to evaluate their ability for use as surrogate beta cells.
MethodsCanine MSCs were derived from bone marrow and validated by measuring the expression of MSC lineage specific markers. Lentivirus vectors encoding the proinsulin gene (with or without a Kozak sequence) under the control of spleen focus forming virus, cytomegalovirus, elongation factor 1 and simian virus 40 promotors were generated and used to transduce primary cMSCs and a hepatocyte cell line. The insulin-producing capacity of transduced primary cMSCs was assessed by measuring the concentration of C-peptide produced.
ResultsPrimary cMSC 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 (from 1700 to 4000pmol/l). The spleen focus forming virus promoter conferred the strongest transcriptional ability.
ConclusionsThe results of the present study suggest that optimised lentiviral transduction of the insulin gene into primary cMSCs renders these cells capable of secreting insulin over both the short- and long-term, in sufficient quantities in vitro to support their potential use in insulin gene therapy.

• 出版日期2016-10