title: Promoter optimisation of lentiviral vectors for efficient 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. 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.
  
  
  METHODS
  Canine 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 1α 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.
  
  
  RESULTS
  Primary 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 4000 pmol/l). The spleen focus forming virus promoter conferred the strongest transcriptional ability.
  
  
  CONCLUSIONS
  The 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.
authors: Department of Medicine, University of Cambridge, Cambridge, UK.
authors: Pratigya
authors: P
authors: Gautam
authors: Department of Pathology, University of Cambridge, Cambridge, UK.
authors: Asha
authors: A
authors: Recino
authors: Dick White Referrals, Station Farm, Six Mile Bottom, Suffolk, UK.
authors: Robert D
authors: RD
authors: Foale
authors: Department of Medicine, University of Cambridge, Cambridge, UK.
authors: Jing
authors: J
authors: Zhao
authors: Department of Surgery, National Institute of Singapore, Singapore.
authors: Shu Uin
authors: SU
authors: Gan
authors: Dick White Referrals, Station Farm, Six Mile Bottom, Suffolk, UK.
authors: Maja
authors: M
authors: Wallberg
authors: Department of Surgery, University of Cambridge, Cambridge, UK.
authors: Roy
authors: R
authors: Calne
authors: Department of Medicine, University of Cambridge, Cambridge, UK. amll1@medschl.cam.ac.uk.
authors: Andrew M L
authors: AM
authors: Lever
mesh_terms: D000818:Animals; D001854:Bone Marrow Cells / Q000378:metabolism; D045744:Cell Line, Tumor; D002478:Cells, Cultured; D004285:Dogs; D015870:Gene Expression; D018014:Gene Transfer Techniques; D015316:Genetic Therapy / Q000379:methods; D005822:Genetic Vectors / Q000235:genetics; D057809:HEK293 Cells; D022781:Hepatocytes / Q000378:metabolism; D006801:Humans; D007328:Insulin / Q000235:genetics* / Q000378:metabolism; D050417:Insulin-Secreting Cells / Q000378:metabolism; D016086:Lentivirus / Q000235:genetics*; D059630:Mesenchymal Stem Cells / Q000378:metabolism*; D011384:Proinsulin / Q000235:genetics / Q000378:metabolism; D011401:Promoter Regions, Genetic / Q000235:genetics*
chemical_list: D007328:Insulin; D011384:Proinsulin
keywords: diabetes; gene therapy; insulin; lentivirus; mesenchymal stromal cells