Promoter Optimisation of Lentiviral Vectors for E fficient Insulin Gene Expression in Canine Mesenchymal Stromal Cells: Potential Surrogate Beta Cells

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.