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Novel diabetes mellitus treatment: mature canine insulin production by canine striated muscle through gene therapy

Lookup NU author(s): Dr Susan Campbell, Dr Ali Aldibbiat, Christopher Huggins, Dr Audrey Brown, Professor James Shaw

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Abstract

Muscle-targeted gene therapy using insulin genes has the potential to provide an inexpensive, low maintenance alternative or adjunctive treatment method for canine diabetes mellitus. A canine skeletal muscle cell line was established through primary culture, as well as through transdifferentiation of canine fibroblasts after infection with a myo-differentiation gene containing adenovirus vector. A novel mutant furin-cleavable canine preproinsulin gene insert (cppI4) was designed and created through de novo gene synthesis. Various cell lines, including the generated canine muscle cell line, were transfected with nonviral plasmids containing cppI4. Insulin and desmin immunostaining were used to prove insulin production by muscle cells and specific canine insulin ELISA to prove mature insulin secretion into the medium. The canine myoblast cultures proved positive on desmin immunostaining. All cells tolerated transfection with cppI4-containing plasmid, and double immunostaining for insulin and desmin proved present in the canine cells. Canine insulin ELISA assessment of medium of cppI4-transfected murine myoblasts and canine myoblast and fibroblast mixture proved presence of mature fully processed canine insulin, 24 and 48 h after transfection. The present study provides proof of principle that canine muscle cells can be induced to produce and secrete canine insulin on transfection with nonviral plasmid DNA containing a novel mutant canine preproinsulin gene that produces furin-cleavable canine preproinsulin. This technology could be developed to provide an alternative canine diabetes mellitus treatment option or to provide a constant source for background insulin, as well as C-peptide, alongside current treatment options. (c) 2012 Elsevier Inc. All rights reserved.


Publication metadata

Author(s): Niessen SJM, Fernandez-Fuente M, Mahmoud A, Campbell SC, Aldibbiat A, Huggins C, Brown AE, Holder A, Piercy RJ, Catchpole B, Shaw JAM, Church DB

Publication type: Article

Publication status: Published

Journal: Domestic Animal Endocrinology

Year: 2012

Volume: 43

Issue: 1

Pages: 16-25

Print publication date: 21/02/2012

ISSN (print): 0739-7240

ISSN (electronic): 1879-0054

Publisher: Elsevier Inc.

URL: http://dx.doi.org/10.1016/j.domaniend.2012.01.006

DOI: 10.1016/j.domaniend.2012.01.006


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