Gene therapy of streptozotocin-induced diabetes by intramuscular delivery of modified preproinsulin genes.
BACKGROUND
Despite improvements in insulin preparation and delivery, physiological normoglycemia is not easily achieved in diabetics. Therefore, there has been considerable interest in developing gene therapy approaches to supply insulin. We studied a nonviral muscle-based method of gene therapy and demonstrated that it could prevent hyperglycemia in murine streptozotocin (STZ)-induced diabetes.


METHODS
A plasmid encoding mouse furin-cleavable preproinsulin II cDNA (FI), or its B10-analogue (B10FI), and a plasmid encoding furin were coinjected into muscle of CD-1 mice, who were treated a day later with STZ to induce diabetes. Electroporation was applied to increase gene transfer. Blood glucose was measured in fed and fasting mice, and fasting plasma insulin was measured by radioimmunoassay. The form of insulin produced and the presence of C-peptide were analyzed by gel filtration chromatography.


RESULTS
A B10FI plasmid codelivered with a furin plasmid reduced fed and fasting blood glucose levels in STZ-treated diabetic mice. The (pro)insulin levels in plasma were increased by up to 70-fold versus blank plasmid-treated diabetic mice. The administration of FI with furin was less effective. (Pro)insulin levels were greatly increased by using two plasmids carrying different promoter elements (CMV and SV40). Insulin was identified in muscle cells by immunohistochemistry. In plasma, 40-70% of the (pro)insulin was processed to the mature form and free C-peptide was identified. Insulin gene-treated mice had improved growth rates and appeared healthier. A single injection of B10FI with SV40Furin DNA increased plasma (pro)insulin for at least 8 weeks and reduced fed blood glucose levels for 5 weeks and fasting levels for 8 weeks.


CONCLUSIONS
This is the first report that electroporation-enhanced intramuscular gene therapy with B10FI can prevent hyperglycemia in murine STZ-induced diabetes. Gene therapy using various routes and methods of furin-cleavable insulin gene delivery has been previously explored but, in muscle, results comparable to ours have not been reported.
Croze
France
F
Department of Pathology, McGill University, 3775 University St., Room B13, Montreal, Quebec, Canada H3A 2B4.
Prud'homme
Gérald J
GJ
D000818:Animals; D001786:Blood Glucose / Q000378:metabolism; D001835:Body Weight; D002096:C-Peptide / Q000097:blood; D002850:Chromatography, Gel; D018076:DNA, Complementary / Q000378:metabolism; D003921:Diabetes Mellitus, Experimental / Q000628:therapy*; D018274:Electroporation; D045683:Furin / Q000235:genetics; D018014:Gene Transfer Techniques; D015316:Genetic Therapy / Q000379:methods*; D005822:Genetic Vectors / Q000235:genetics; D006943:Hyperglycemia / Q000517:prevention & control; D007150:Immunohistochemistry; D007273:Injections, Intramuscular; D007328:Insulin / Q000235:genetics / Q000378:metabolism; D051379:Mice; D009154:Mutation; D010179:Pancreas / Q000378:metabolism; D010957:Plasmids / Q000378:metabolism; D011384:Proinsulin / Q000235:genetics*; D011498:Protein Precursors / Q000235:genetics*; D011863:Radioimmunoassay; D013311:Streptozocin / Q000494:pharmacology; D013997:Time Factors
D001786:Blood Glucose; D002096:C-Peptide; D018076:DNA, Complementary; D007328:Insulin; D011498:Protein Precursors; D013311:Streptozocin; C013737:preproinsulin; D011384:Proinsulin; D045683:Furin