Transgenic Insulin Research Articles

Direct Analysis of Insulin-Specific T Cells Provides New Insights.

Type 1 diabetes (T1D) is an autoimmune disease in which destruction of insulin-producing β-cells leads to dependence on exogenous insulin (1). T1D progression involves progressive loss of tolerance to β-cell antigens, but this has been challenging to observe in human disease. Loss of self-tolerance is most evident through development of islet cell antibodies, among which insulin antibodies typically appear first (2). Given strong genetic association of T1D with HLA-DQ8, DQ8-restricted CD4+ T cells activated by self-peptides are thought to play a crucial role in disease. Key evidence from the nonobese diabetic (NOD) mouse model supports a primary role for recognition of an epitope within the insulin B chain amino acids 9–23 (InsB9–23) peptide in disease initiation. The majority of CD4+ T cells isolated from pancreatic islets of NOD mice were shown to respond to this peptide (3). Furthermore, expression of an insulin transgene with a substitution within this peptide abrogated diabetes development (4). Efforts to directly visualize and study insulin-specific T cells in human subjects have been hampered by the biochemical instability of the HLA-DQ8 protein (5). However, recent reports highlight the development of HLA-DQ8 tetramer reagents and their application to study T1D, affirming that InsB11–23 peptide is analogously presented by IAg7 and HLA-DQ8 (6,7). In addition, two recent reports documented the presence …

Long-term glycemic control with hepatic insulin gene therapy in streptozotocin-diabetic mice.

Insulin self-administration is burdensome and can produce dangerous hypoglycemia. Insulin gene therapy may improve and simplify the treatment of diabetes mellitus. In rats, metabolically responsive hepatic insulin gene therapy (HIGT) delivered by adenovirus normalizes random blood sugars but with a limited duration. To prolong glycemic control, we delivered a metabolically regulated insulin transgene by adeno-associated virus (AAV). We administered increasing doses of self-complementary (SC), pseudotyped AAV8 expressing the (GlRE)3 BP1-2xfur insulin transgene to streptozotocin-diabetic CD-1 mice, and monitored blood sugar and body weight. We also compared responses to intraperitoneal glucose and chow withdrawal, assessed for viral genomes in liver by Southern blotting, and measured hepatic glycogen. Glucose lowering required the combination of SC genomes and AAV capsid pseudotyping. HIGT controlled glycemia in diabetic mice (DM) for > 1 year. However, glycemic responses were variable. Approximately 30% of mice succumbed to hypoglycemia, and approximately 30% of mice again became hyperglycemic. During an intraperitoneal glucose tolerance test, blood sugars declined to normal within 180 min in HIGT-treated DM compared to 90 min in control mice. Hypoglycemia was common among HIGT-treated mice during a 24-h fast. However, HIGT mice lost less weight than either diabetic or nondiabetic controls as a result of increased water intake. HIGT treatment reduced the hepatic glycogen content of fed mice. Our studies demonstrate the possibility for long-term glycemic correction following AAV-mediated HIGT in mice. However, the dose-response relationship is irregular, and metabolic responsiveness may be less than that observed in rats.

Hepatic insulin gene therapy prevents diabetic enteropathy in STZ-treated CD-1 mice

Depending on the population examined, from 6 to 83% of people with diabetes mellitus exhibit symptoms of altered gut motility, manifesting as dysphagia, reflux, early satiety, nausea, abdominal pain, diarrhea, or constipation. Hyperglycemia-induced cell loss within the enteric nervous system has been demonstrated in both diabetic rodents and patients with diabetes. Glycemic control is recommended to prevent diabetic gastroenteropathy but is often difficult to achieve with current treatment modalities. We asked if hepatic insulin gene therapy (HIGT) could inhibit the development of diabetic gastroenteropathy in mice. Bowel length, bowel transit, colonic muscle relaxation, and the numbers of both stimulatory and inhibitory neurons in the colonic myenteric plexus were compared in groups of diabetic mice (DM), control nondiabetic mice (Con), and diabetic mice treated with HIGT (HIGT). Delivery of a metabolically responsive insulin transgene to the liver of STZ-diabetic mice with an adeno-associated virus, sero-type 8 (AAV8) produced near-normal blood sugars for over 1 month and prevented anatomic, functional, and neurohistologic changes observed in diabetic mice. We conclude that in addition to normalizing oxidative metabolism in diabetic rodents, HIGT is sufficient to prevent the development of diabetic gastroenteropathy.

Regulation of glucose homeostasis using radiogenetics and magnetogenetics in mice.

Endogenous expression of tailored nanoparticles in cells followed by application of low-frequency radio waves or a magnetic field can be used to noninvasively modulate gene expression. This approach successfully induces insulin transgene expression in diabetic mice.

Remote regulation of glucose homeostasis in mice using genetically encoded nanoparticles.

Means for temporally regulating gene expression and cellular activity are invaluable for elucidating underlying physiological processes and would have therapeutic implications. Here we report the development of a genetically encoded system for remote regulation of gene expression by low-frequency radio waves (RFs) or a magnetic field. Iron oxide nanoparticles are synthesized intracellularly as a GFP-tagged ferritin heavy and light chain fusion. The ferritin nanoparticles associate with a camelid anti-GFP-transient receptor potential vanilloid 1 fusion protein, αGFP-TRPV1, and can transduce noninvasive RF or magnetic fields into channel activation, also showing that TRPV1 can transduce a mechanical stimulus. This, in turn, initiates calcium-dependent transgene expression. In mice with stem cell or viral expression of these genetically encoded components, remote stimulation of insulin transgene expression with RF or a magnet lowers blood glucose. This robust, repeatable method for remote regulation in vivo may ultimately have applications in basic science, technology and therapeutics.

Combinatorial insulin secretion dynamics of recombinant hepatic and enteroendocrine cells

One of the most promising cell-based therapies for combating insulin-dependent diabetes entails the use of genetically engineered non-β cells that secrete insulin in response to physiologic stimuli. A normal pancreatic β cell secretes insulin in a biphasic manner in response to glucose. The first phase is characterized by a transient stimulation of insulin to rapidly lower the blood glucose levels, which is followed by a second phase of insulin secretion to sustain the lowered blood glucose levels over a longer period of time. Previous studies have demonstrated hepatic and enteroendocrine cells to be appropriate hosts for recombinant insulin expression. Due to different insulin secretion kinetics from these cells, we hypothesized that a combination of the two cell types would mimic the biphasic insulin secretion of normal β cells with higher fidelity than either cell type alone. In this study, insulin secretion experiments were conducted with two hepatic cell lines (HepG2 and H4IIE) transduced with 1 of 3 adenoviruses expressing the insulin transgene and with a stably transfected recombinant intestinal cell line (GLUTag-INS). Insulin secretion was stimulated by exposing the cells to glucose only (hepatic cells), meat hydrolysate only (GLUTag-INS), or to a cocktail of the two secretagogues. It was found experimentally that the recombinant hepatic cells secreted insulin in a more sustained manner, whereas the recombinant intestinal cell line exhibited rapid insulin secretion kinetics upon stimulation. The insulin secretion profiles were computationally combined at different cell ratios to arrive at the combinatorial kinetics. Results indicate that combinations of these two cell types allow for tuning the first and second phase of insulin secretion better than either cell type alone. This work provides the basic framework in understanding the secretion kinetics of the combined system and advances it towards preclinical studies.

Insulin expressed from endogenously active glucose-responsive EGR1 promoter in bone marrow mesenchymal stromal cells as diabetes therapy

Advances in islet transplantation have encouraged efforts to create alternative insulin-secreting cells that overcome limitations associated with current therapies. We have recently demonstrated durable correction of murine and porcine diabetes by syngeneic and autologous implantation, respectively, of primary hepatocytes non-virally modified with a glucose-responsive promoter-regulated insulin transgene. As surgical procurement of hepatocytes may be clinically unappealing, we here describe primary bone marrow-derived mesenchymal stromal cells (BMMSC) as alternative insulin-secreting bioimplants. BMMSC are abundant and less invasively procured for clinical autologous transplantation. Electroporation achieved high transgene transfection efficiencies in human BMMSC (HBMMSC) and porcine BMMSC (PBMMSC). We transcriptomically identified an HBMMSC glucose-responsive promoter, EGR1. This endogenously active promoter drove rapid glucose-induced transgene secretions in BMMSC with near-physiological characteristics during static and kinetic induction assays simulating normal human islets. Preparatory to preclinical transplantation, PBMMSC transfected with the circular insulin transgene vector or stably integrated with the linearized vector were evaluated by intrahepatic or intraperitoneal xenotransplantation in streptozotocin-diabetic and non-diabetic NOD-SCID mice. Hyperglycemia, glucose tolerance and body weight were corrected in a dose-responsive manner. Hypoglycemia was not observed even in identically implanted non-diabetic mice. These results establish human EGR1 promoter-insulin construct-modified BMMSC as safe and efficient insulin-secreting bioimplants for diabetes treatment.

Directed overexpression of insulin in Leydig cells causes a progressive loss of germ cells

The primary goal of this study was to determine the 5'region of the Insl3 gene that specifically targets the expression of human insulin to Leydig cells, and to explore whether the testicular proinsulin is efficiently processed to insulin that is able to rescue the diabetes in different mouse models of diabetes. We show here that the sequence between nucleotides -690 and +4 of mouse Insl3 promoter is sufficient to direct the Leydig cell-specific expression of the human insulin transgene (Insl3-hIns). We also found that the 3'untranslated region (3'UTR) of Insl3 was effective in enhancing transgene expression of the insulin in vivo. Expression analysis revealed that the temporal expression pattern of the hIns transgene in Leydig cells of transgenic testes is roughly the same as that of the endogenous Insl3. Despite the Leydig cells translate human proinsulin and secrete a significant level of free C-peptide into the serum, the Leydig cell-derived insulin is not able to overcome the diabetes in different mouse models of diabetes, suggesting a lack of glucose sensing mechanisms in the Leydig cells. A consequence of overexpression of the human proinsulin in Leydig cells was the decrease of fertility of transgenic males at older ages. Germ cells in transgenic males were able to initiate and complete spermatogenesis. However, there was a progressive and age-dependent degeneration of the germ cells that lead to male infertility with increasing age.

Hepatic Insulin Gene Therapy Normalizes Diurnal Fluctuation of Oxidative Metabolism in Diabetic BB/Wor Rats

Previous studies of hepatic insulin gene therapy (HIGT) focused on glycemic effects of insulin produced from hepatocytes. In this study, we extend the observations of glycemic control with metabolically regulated HIGT to include systemic responses and whole-body metabolism. An insulin transgene was administered with an adenoviral vector [Ad/(GlRE)(3)BP1-2xfur] to livers of BB/Wor rats made diabetic with polyinosinic polycytidilic acid (poly-I:C) (HIGT group), and results compared with nondiabetic controls (non-DM), and diabetic rats receiving different doses of continuous-release insulin implants (DM-low BG and DM-high BG). Blood glucose and growth normalized in HIGT, with lower systemic insulin levels, elevated glucagon, and increased heat production compared with non-DM. Minimal regulation of systemic insulin levels were observed with HIGT, yet the animals maintained normal switching from carbohydrate to lipid metabolism determined by respiratory quotients (RQs), and tolerated 24-hour fasts without severe hypoglycemia. HIGT did not restore serum lipids as we observed increased triglycerides (TGs) and increased free fatty acids, but reduced weight of visceral fat pads despite normal total body fat content and retroperitoneal fat depots. HIGT favorably affects blood glucose, normalizes metabolic switching in diabetic rats, and reduces intra-abdominal fat deposition.

Targeted Ablation of Glucose-dependent Insulinotropic Polypeptide-producing Cells in Transgenic Mice Reduces Obesity and Insulin Resistance Induced by a High Fat Diet

The K cell is a specific sub-type of enteroendocrine cell located in the proximal small intestine that produces glucose-dependent insulinotropic polypeptide (GIP), xenin, and potentially other unknown hormones. Because GIP promotes weight gain and insulin resistance, reducing hormone release from K cells could lead to weight loss and increased insulin sensitivity. However, the consequences of coordinately reducing circulating levels of all K cell-derived hormones are unknown. To reduce the number of functioning K cells, regulatory elements from the rat GIP promoter/gene were used to express an attenuated diphtheria toxin A chain in transgenic mice. K cell number, GIP transcripts, and plasma GIP levels were profoundly reduced in the GIP/DT transgenic mice. Other enteroendocrine cell types were not ablated. Food intake, body weight, and blood glucose levels in response to insulin or intraperitoneal glucose were similar in control and GIP/DT mice fed standard chow. In contrast to single or double incretin receptor knock-out mice, the incretin response was absent in GIP/DT animals suggesting K cells produce GIP plus an additional incretin hormone. Following high fat feeding for 21-35 weeks, the incretin response was partially restored in GIP/DT mice. Transgenic versus wild-type mice demonstrated significantly reduced body weight (25%), plasma leptin levels (77%), and daily food intake (16%) plus enhanced energy expenditure (10%) and insulin sensitivity. Regardless of diet, long term glucose homeostasis was not grossly perturbed in the transgenic animals. In conclusion, studies using GIP/DT mice demonstrate an important role for K cells in the regulation of body weight and insulin sensitivity.

Immunohistochemical staining for tilapia and human insulin demonstrates that a tilapia transgenic for humanized insulin is a mosaic

In a previous paper we described the production of transgenic tilapia grown from fertilized eggs microinjected with a tilapia insulin transgene ‘‘humanized’’ by site directed mutagenesis; founder male NT 56 was able to pass this to his F1 offspring, and we were able to measure high levels of circulating humanized insulin in many of these offspring (Pohajdak et al. 2004) and subsequently F2 offspring (unpublished observation). In NT 56’s offspring, the size, shape, and distributions of cells staining for tilapia insulin and human insulin appeared to be identical, suggesting that they are the same cells expressing both peptides—as would be expected. Curiously, we were never able to demonstrate humanized insulin in the serum of NT56, suggesting that production of the transgene was silenced or highly inhibited, possibly due to mosaic integration in the genome. After siring many offspring, NT56 died and was necropsied. As shown in Fig. 1, immunoperoxidase staining of NT 56’s islet tissue for (a) tilapia insulin, using a monoclonal antibody specific for tilapia insulin (Snowdon 2003) and for (b) human insulin (Pohajdak et al. 2004) showed that, unlike his offspring, there were two distinct populations of insulin-positive b-cells. The b-cells that stained for tilapia insulin possessed abundant cytoplasm and were arranged in repetitive units of small tight clusters of multiple b-cells, surrounded by concentric layers of nonb-cells; this pattern is typical for wild-type tilapia (Yang et al. 1999) and for teleost fish in general. In stark contrast, the ‘‘b-cells’’ staining for human insulin were smaller, much less numerous, tended to occur as single cells rather than discrete clusters, and appeared excessively densely granulated (i.e., ‘‘constipated’’ with insulin). Studies with mammalian islets have shown that disruption of normal b-cell interrelationships (i.e., islet architecture), inhibits insulin secretion, so the abnormal architecture of the cells staining for human insulin may account for absence of measurable humanized insulin in NT56’s serum. J. R. Wright Jr (&) Department of Pathology & Laboratory Medicine (Calgary Laboratory Services) and the Julia McFarlane Diabetes Research Centre, Faculty of Medicine, University of Calgary, Room C410, Diagnostic & Scientific Centre, 9, 3535 Research Road NW, Calgary, AB, Canada T2L 2K8 e-mail: jim.wright@cls.ab.ca

Adeno-associated viral delivery of a metabolically regulated insulin transgene to hepatocytes

Transduction with a liver specific, metabolically responsive insulin transgene produces near-normal blood sugars in STZ-diabetic rats. To overcome the limited duration of hepatic transgene expression induced by E1A-deleted adenoviral vectors, we evaluated recombinant adeno-associated virus (rAAV2) for cell type specificity and glucose responsiveness in vitro. Co-infection of AAV2 containing the glucose responsive, liver-specific (GlRE) 3BP-1 promoter with an empty adenovirus enhanced transduction efficiency, and shortened the duration of transgene expression in HepG2 hepatoma cells, but not primary hepatocytes. However, in the context of rAAV2, (GlRE) 3BP-1 promoter activity remained confined to cells of hepatocyte lineage, and retained glucose responsiveness. While isolated infection with an insulin expressing rAAV2 failed to attenuate blood sugars in diabetic mice, adenoviral co-administration with the same rAAV2 induced transient, near-normal random blood sugars in a diabetic animal. We conclude that rAAV2 can induce metabolically responsive insulin secretion from hepatocytes in vitro and in vivo. However, alternative AAV serotypes will likely be required to efficiently deliver therapeutic genes to the liver for the treatment of diabetes mellitus.

Long‐Term Prevention of Diabetes and Marked Suppression of Insulin Autoantibodies and Insulitis in Mice Lacking Native Insulin B9–23 Sequence

We analyzed double native insulin gene knockout NOD mice with a mutated (B16:alanine) proinsulin transgene at multiple ages for the development of insulin autoantibodies, insulitis, and diabetes. In contrast to mice with at least one copy of a native insulin gene that expressed insulin antibodies, only 2 out of 21 (10%) double native insulin gene knockout mice with a mutated insulin transgene developed insulin autoantibodies. Of 21 double insulin knockout mice sacrificed between 10 to 48 weeks of age, only 5 showed minimal insulitis versus 100% of wild-type NOD and more than 90% of insulin 1 knockout mice. Consistent with robust suppression of insulin autoantibodies and insulitis, no double insulin knockout mice developed diabetes. In that the B9-23 peptide with B16A is an altered peptide ligand inducing Th2 responses, we analyzed transfer of splenocytes into NOD.SCID mice. There was no evidence for regulatory T cells able to inhibit transfer of diabetes by diabetogenic NOD splenocytes. Insulin peptide B9-23 is likely a crucial target for initiation of islet autoimmunity and further mutation of the sequence will be tested to attempt to eliminate all anti-islet autoimmunity.

Su.35. The Destruction of Islets Lacking Native Insulin Genes with Mutated B16:a Insulin Transgene By Diabetic Nod Mice

Su.35. The Destruction of Islets Lacking Native Insulin Genes with Mutated B16:a Insulin Transgene By Diabetic Nod Mice

Host Cells Reduce Glucose Uptake and Glycogen Deposition in Response to Hepatic Insulin Gene Therapy

BackgroundHepatic insulin gene therapy (HIGT) restores weight gain and near-normal glycemia in rodent models of insulin- deficient diabetes mellitus. However, the effect of transgenic insulin on endogenous genes and recipient...

Plasmid-electroporated primary hepatocytes acquire quasi-physiological secretion of human insulin and restore euglycemia in diabetic mice

We describe the durable correction of streptozotocin-induced murine diabetes by in vivo implantation of primary mouse hepatocytes electroporated ex vivo with a human proinsulin cDNA plasmid construct controlled by glucose and zinc regulatory elements. Transfected hepatocytes increased insulin transgene transcription and secretion within 10-20 min of exposure to 25 mM glucose or 60 microM zinc. Insulin release did not occur from secretory granules. Electroporated Rosa26 hepatocytes ( approximately 8 x 10(5) viable cells) were implanted in C57BL/6J diabetic mice in one of three sites: unresected liver, regenerating liver or mesentery. Control diabetic mice were implanted with untransfected hepatocytes. At 30 days after implantation, 8/15 control mice were alive, while 19/19 treated mice were alive. The ratio of body weight on day 30/nadir body weight was significantly higher for all treated groups compared with controls. All eight surviving control mice were hyperglycemic 30 days post-implantation, while 16/19 treated diabetic mice remained normoglycemic. Treated mice had lower mean glucose values (P< or =0.001) without fasting hypoglycemia and better glucose tolerance (P< or =0.0003) than untreated controls. All (6/6) diabetic mice implanted in regenerating liver and 71% (5/7) implanted in unresected liver were alive 77 days after implantation. Engrafted hepatocytes were identified, mainly around central veins, by staining for beta-galactosidase activity and with anti-human insulin antibody.

Enhanced insulin secretion from engineered 3T3-L1 preadipocytes by induction of cellular differentiation

We have established insulin-secreting cell line, L1-INS/fur cells, by engineering 3T3-L1 murine preadipocytes with human preproinsulin cDNA. Analysis with HPLC, mass spectrometry and immunological assay identified human insulin in the culture medium. Notably, secretion of insulin from L1-INS/fur cell was increased 8.2 times higher after induction of cellular differentiation. The increment of insulin secretion during differentiation was further enhanced by additive treatment with thiazolidinedione, a promoting agent of adipocyte differentiation. This observation strongly suggests that the enhancement of insulin secretion is tightly associated with cellular differentiation process itself. Expression rate of the insulin transgene was not changed after the additional treatment with thiazolidinedione. On the other hand, furin gene expression by Northern analysis showed an increase, and Western analysis revealed even more reduction in cellular content of proinsulin. These results indicate that mechanism of the observed enhancement in insulin secretion during the differentiation is mainly due to increased capacity of the proinsulin processing by induction of furin. Results of our present study will provide important information on cell-based therapy using undifferentiated progenitors and tissue stem cells.

Establishment of Native Insulin‐Negative NOD Mice and the Methodology to Distinguish Specific Insulin Knockout Genotypes and a B:16 Alanine Preproinsulin Transgene

We hypothesize that NOD mice without native insulin, but with an altered insulin B:9-23 sequence, will be completely protected from diabetes/insulitis if insulin B:9-23 is an essential T cell epitope. To investigate this hypothesis, we have established initial insulin 1- and 2-negative NOD mice with a transgene directing production of preproinsulin with alanine at position B:16 rather than the native tyrosine of both insulin 1 and insulin 2. Sets of primers for PCR-based assays have been created and validated. They are able to distinguish the presence or absence of the insulin gene knockouts and of both native insulin 1 and insulin 2 (and thus distinguish heterozygous versus homozygous knockouts), as well as the presence of the altered insulin transgene, B:16 alanine preproinsulin. Four B:16 alanine transgenic founders were produced directly in NOD mice and, by intercrossing, initial live native insulin-negative B:16 alanine transgenic mice have been generated.

376. A Metabolically Responsive Insulin Transgene Delivered by AAV2/8 Pseudotyped, Self-Complementary AAV (SC-AAV2/8) Controls Glycemia in STZ-Diabetic Mice

Metabolically responsive insulin gene therapy promises advances in the treatment of type 1 diabetes mellitus. We have shown that hepatic expression of a transcriptionally regulated human insulin transgene successfully treats diabetes in rodents. However, consistent with first generation adenoviral delivery transgenic insulin production is transient in vivo. In contrast, transgenes delivered by recombinant adeno-associated virus (rAAV) express in murine liver for more than a year. Despite their advantages application of AAV has been limited. Transgene expression is delayed, a factor that can complicate experiments in vivo. Moreover, producing large quantities of AAV at high titers remains costly and technically demanding. Finally, common vectors infect some tissues poorly, or express weakly due to limited second-strand synthesis. Two recent developments in rAAV technology address these issues. A novel serotype, rAAV8 was shown to infect hepatocytes 10-100 fold more efficiently than rAAV2. Separately, self-complimentary (SC)-AAV particles produced by eliminating the terminal resolution sequence (TRS) from one inverted terminal repeat (ITR) were shown to accelerate and enhance transgene expression in vivo. Here we describe the combination of SC and pseudotyping strategies to obtain long-term glycemic control in diabetic mice. We created pSC-AAV2(GlRE)3BP-1 2fur by inserting a previously described glucose and insulin responsive insulin transgene into an AAV2 transfer plasmid from which the TRS had been removed from the left-ITR. Compared to vector derived from a longer, AAV2(GlRE)3BP-1 2furGFP, or similarly short sequence, AAV2(GlRE)3BP-1 2fur, infection with SC-AAV2(GlRE)3BP-1 2fur accelerated, and more than tripled secretion of human insulin from primary hepatocytes. Co-transfection of 293 cells with pSC-AAV2(GlRE)3BP-1 2fur, and a REP2/CAP8 expressing plasmid produced pseudotyped SC-AAV2/8 vector. Twenty-three male, STZ-diabetic CD-1 (BG>200 mg/dl) mice received a portal vein injection of either SC-AAV2(GlRE)3BP-1 2fur, or increasing doses of SC-AAV2/8(GlRE)3BP-1 2fur. Despite enhanced transgene expression in vitro, SC-AAV2(GlRE)3BP-1 2fur administration (3.4-4.8×1010vg, n=4) failed to reduce hyperglycemic by 18 days. In contrast, SC-AAV2/8(GlRE)3BP-1 2fur administration lowered blood glucose in 18 of 19 diabetic mice by 7 days, beginning with doses as low as 1.3×1010vg. Six of seven mice receiving the three highest doses (5.2×1010vg n=3, 7.8×1010vg n=1, 1.3×1011vg n=3) succumbed to hypoglycemia within 4 days. Two animals receiving 2.6×1010vg (n=6) died of hypoglycemia at day 34. However, 9 of the remaining 10 mice receiving 1.3-2.6×1010vg continue to grow normally and maintain near euglycemia (105±6 mg/dl). In conclusion, we have combined SC-AAV, AAV2/8 pseudotyping techniques, and metabolically responsive hepatic insulin gene therapy to treat STZ-diabetic mice in vivo. Effective viral doses may be 10-fold less than standard, non-pseudotyped vector delivering the same transgene. On-going investigations will determine the long-stability of glycemic control in this model.

1080. Adenoviral Transduction with a Glucose Responsive Insulin Transgene Reduces Expression of Glucokinase and GLUT2 in Primary Cultured Hepatocytes

1080. Adenoviral Transduction with a Glucose Responsive Insulin Transgene Reduces Expression of Glucokinase and GLUT2 in Primary Cultured Hepatocytes