Glucose transporter-2 (GLUT2) promoter mediated transgenic insulin production reduces hyperglycemia in diabetic mice.

Insulin production afforded by hepatic gene therapy (HGT) retains promise as a potential treatment for type 1 diabetes, but successful approaches have been limited. We employed a novel and previously untested promoter for this purpose, glucose transporter-2 (GLUT2) to drive insulin production via delivery by recombinant adeno-associated virus (rAAV). In vitro, the GLUT2 promoter was capable of robust glucose-responsive expression in transduced HepG2 human hepatoma cells. Therefore, rAAV constructs were designed to express the furin-cleavable human preproinsulin B10 gene, under the control of the murine GLUT2 promoter and packaged for delivery with rAAV expressing the type 5 capsid. Streptozotocin-induced diabetic mice were subjected to hepatic portal vein injection immediately followed by implantation of a sustained-release insulin pellet to allow time for transgenic expression. All mice injected with the rAAV5-GLUT2-fHPIB10 virus remained euglycemic for up to 35 days post-injection, with 50% euglycemic after 77 days post-injection. In contrast, mock-injected mice became hyperglycemic within 15 days post-injection following dissolution of the insulin pellet. Serum levels of both human insulin and C-peptide further confirmed successful transgenic delivery by the rAAV5-GLUT2-fHPIB10 virus. These findings indicate that the GLUT2 promoter may be a potential candidate for regulating transgenic insulin production for hepatic insulin gene therapy in the treatment of type I diabetes.

The pre-B-cell receptor induces silencing of VpreB and lambda5 transcription.

The pre-B-cell receptor (pre-BCR), composed of Ig heavy and surrogate light chain (SLC), signals pre-BII-cell proliferative expansion. We have investigated whether the pre-BCR also signals downregulation of the SLC genes (VpreB and lambda5), thereby limiting this expansion. We demonstrate that, as BM cells progress from the pre-BI to large pre-BII-cell stage, there is a shift from bi- to mono-allelic lambda5 transcription, while the second allele is silenced in small pre-BII cells. A VpreB1-promoter-driven transgene shows the same pattern, therefore suggesting that VpreB1 is similarly regulated and thereby defines the promoter as a target for transcriptional silencing. Analyses of pre-BCR-deficient mice show a temporal delay in lambda5 downregulation, thereby demonstrating that the pre-BCR is essential for monoallelic silencing at the large pre-BII-cell stage. Our data also suggest that SLP-65 is one of the signaling components important for this process. Furthermore, the VpreB1/lambda5 alleles undergo dynamic changes with respect to nuclear positioning and heterochromatin association, thereby providing a possible mechanism for their transcriptional silencing.