Intramuscular Doxycycline-Regulated Insulin Gene Therapy For Diabetes Mellitus

Background: Insulin gene therapy is still an experimental approach to replace subcutaneous insulin therapy for diabetes mellitus, in which the artificial insulin gene is transferred into somatic cells and kept in situ to produce insulin. Some critical techniques pertaining to insulin gene therapy need to be solved before it can be used to treat human diabetes mellitus. Firstly, insulin gene should be delivered safely and kept stable in target tissue cells. Secondly the expression and production of insulin should be under tight control in a regulated way. Skeletal muscle is currently regarded as a favorable host tissue for foreign gene transfer and expression in vivo. It has following advantages: (1)Skeletal muscle is easy for foreign gene to be delivered to because of its huge mass and superficial location; (2)The synthetized therapeutic biological molecule is easy to be secreted into circulation for its abundant vascular supply; (3)The foreign gene is easy to be kept stable in number and duration of expression in muscle in that the muscle fibers are terminally differentiated cells with post-mitotic nuclei, and individual fibers could persist for much of the lifetime of the individual, and most nuclei are kept intact even short segments of muscle fibers are damaged; (4)Foreign gene can be easily dispersed to neighboring nucleic within the fiber for the syncytial nature of the myocyte; (5) Plasmid can be used as a foreign gene delivery and expression vector, and is practical in application because plasmid is easy and cheap to be constructed and produced in large scale for gene therapy. Further more, in vivo delivery of plasmid is safe, and injections can be repeated for its slight host immune reaction. However, shortcoming exists for skeletal muscle as host tissue of insulin gene concerning the special relation between insulin and blood glucose control. Because the muscle cells lack glucose-induced responsive element to drive the transcription of insulin gene, and sustained production and secretion of insulin might leads to severe and fatal hypoglycemia. Recently, recombinant tetracycline regulated system derived from prokaryocyte tetracycline resistant gene by Bujard et al. from Germany was found could be successfully used in mammal cell to guide the foreign gene expression under the control of tetracycline or its analogues. The expression of foreign gene can be switched on and off through a very low toxic and small antibiotics which has been used for nearly a century. The system had been applied to promote the expression of erythropoietin production regulated by doxycycline in mouse skeletal muscle in vivo. Therefore, the tetracycline regulated gene expression system might be feasible to be applied for the regulated production of insulin gene in skeletal muscle.Perspectives: To construct a single plasmid vector that can mediates doxycycline-regulated expression of furin-cut proinsulin for intramuscular insulin gene therapy. To testify firstly its expression and regulation of insulin production in myotube cell in vitro. Secondly to study its hypoglycemic efficacy in streptozotocin (STZ)-induced diabetic mice, and finally to study if the transfer rate can be enhanced by electroporation.Methods: 1. Construction of the doxycycline-regulated insulin gene expression plasmid and its expression in cultured myotube cells in vitro. (1)Construction of plasmid ptet-mINS. The insulin cDNA cut from pINS-ABC with BamHI and ScaI was inserted downstream the tetracycline activator binding motif (ptet4) of plasmid pUHG102-8 digested with the same restriction enzymes (RE). After the two fragments ligated with T4 in E Coli, the resultant ptet-mINS plasmid was checked if it was inserted correctly in direction with BamHI and ClaI.(2)Construction of plasmid prTA-tet4-mINS. PhCMV-rtTA2-1/poly(A) fragment from XhoI and HindIII digestion of plasmid prtTA2-1 was cloned to the XhoI digested plasmid ptet-mINS. The resultant plasmid was designed as prTA-ptet4-mINS.(3)Construction of the final plasmid prTA-tet4-rhINS. A 5'-LTR-...