Human Proinsulin Gene Research Articles

Unpredictive Expression of recombinant proteins under the control of different promoters in different expression systems

Synthetic human proinsulin gene with E.coli preferred codon and extra methionine was expressed as 30% of total cellular protein. Fusing seven aminoacids of well expressed protein to the N’-terminus did not alter the expression of recombinant proinsulin. Doubling the copies of proinsulin gene will not doubles the expression. The protein was expressed as inclusion bodies. Changing the distances between ribosome binding site and start codon effect the expression in an unexplained way. Ten nucleotide distance gave expression of 2-4% and with 12 nucleotides, expression decreasaes up to 1-2%.. Incorporating 10 different nucleotides at the start of proinsulin gene decreases the mRNA secondary structure but did not alter the expression. Metabolic instability, rapid degradation of mRNA or accumulation of protein may down regulate expression of mRNA. High expression of azurin gene in pET22b vector was obtained when fused with 19 aminoacid signal peptide in BL21-Codon Plus (DE3) cells. PE38KDEL can be expressed in huge quantities in Escherichia coli by using the recombinant vector PE38KDEL/pET under control of T7 promoter and E. coli host strain BL21 (DE3) CodonPlus. The protein expressed as soluble and insoluble inclusion bodies the expression in soluble form was more pronounced. When human interferone alpha2|B gene and E.coli amino peptidase genes were expressed bicistronically in expression plasmid pET-21a (+). There was negligible expression of human interferone alpha 2B. PelB fused DNA polymerase I was expressed as soluble fraction with maximum expression with 0.5mM IPTG and 20mM lactose. Recombinant protein expression in expression host can be improved by mRNA stability, codon bias, and controlling inclusion bodies formation and changing genetic tools in different combinations. There are other controlling factors of protein expression i.e., metabolic instability, rapid degradation of mRNA or accumulation of protein may downregulate expression of mRNA.

Production of genetically modified pigs expressing human insulin and C-peptide as a source of islets for xenotransplantation.

Islet xenotransplantation is a promising treatment for type I diabetes. Numerous studies of islet xenotransplantation have used pig-to-nonhuman primate transplantation models. Some studies reported long-term survival and successful function of porcine islets in diabetic monkeys. Genetic engineering techniques may improve the survival and function of porcine islets. A recent study reported the generation of transgenic pigs expressing human insulin rather than porcine insulin by changing one amino acid at the end of the β-chain in insulin. However, C-peptide from pigs still existed. In this study, we generated transgenic pigs expressing human proinsulin to express human insulin and C-peptide using fibroblasts from proinsulin knockout pigs as donor cells for somatic cell nuclear transfer. Eleven live piglets were delivered from three surrogates and characterized to confirm the genotype and phenotype of the generated piglets. Genotype analysis of the generated piglets showed that five of the eleven piglets contained the human proinsulin gene. Insulin expression was confirmed in the serum and pancreas in two of the five piglets. C-peptide derived from human proinsulin was also confirmed by liquid chromatography tandem mass spectrometry. Non-fasting blood glucose level was measured to verify the function of the insulin derived from the human proinsulin. Two piglets expressing insulin showed normal glucose levels similar to that in the wild-type control. In conclusion, human insulin- and C-peptide-expressing pigs without porcine insulin and C-peptide were successfully established. These pigs can be used as a source of islets for islet xenotransplantation.

Thioflavin T Monitoring of Guanine Quadruplex Formation in the rs689-Dependent INS Intron 1

The human proinsulin gene (INS) contains a thymine-to-adenine variant (rs689) located in the 3′ splice site (3′ ss) recognition motif of the first intron. The adenine at rs689 is strongly associated with type 1 diabetes. By weakening the polypyrimidine tract, the adenine allele reduces the efficiency of intron 1 splicing, which can be ameliorated by antisense oligonucleotides blocking a splicing silencer located upstream of the 3′ ss. The silencer is surrounded by guanine-rich tracts that may form guanine quadruplexes (G4s) and modulate the accessibility of the silencer. Here, we employed thioflavin T (ThT) to monitor G4 formation in synthetic DNAs and RNAs derived from INS intron 1. We show that the antisense target is surrounded by ThT-positive segments in each direction, with oligoribonucleotides exhibiting consistently higher fluorescence than their DNA counterparts. The signal was reduced for ThT-positive oligonucleotides that were extended into the silencer, indicating that flanking G4s have a potential to mask target accessibility. Real-time monitoring of ThT fluorescence during INS transcription in vitro revealed a negative correlation with ex vivo splicing activities of corresponding INS constructs. Together, these results provide a better characterization of antisense targets in INS primary transcripts for restorative strategies designed to improve the INS splicing defect associated with type 1 diabetes.

Proinsulin protects against age-related cognitive loss through anti-inflammatory convergent pathways

Brain inflammaging is increasingly considered as contributing to age-related cognitive loss and neurodegeneration. Despite intensive research in multiple models, no clinically effective pharmacological treatment has been found yet. Here, in the mouse model of brain senescence SAMP8, we tested the effects of proinsulin, a promising neuroprotective agent that was previously proven to be effective in mouse models of retinal neurodegeneration. Proinsulin is the precursor of the hormone insulin but also upholds developmental physiological effects, particularly as a survival factor for neural cells. Adeno-associated viral vectors of serotype 1 bearing the human proinsulin gene were administered intramuscularly to obtain a sustained release of proinsulin into the blood stream, which was able to reach the target area of the hippocampus. SAMP8 mice and the control strain SAMR1 were treated at 1 month of age. At 6 months, behavioral testing exhibited cognitive loss in SAMP8 mice treated with the null vector. Remarkably, the cognitive performance achieved in spatial and recognition tasks by SAMP8 mice treated with proinsulin was similar to that of SAMR1 mice. In the hippocampus, proinsulin induced the activation of neuroprotective pathways and the downstream signaling cascade, leading to the decrease of neuroinflammatory markers. Furthermore, the decrease of astrocyte reactivity was a central effect, as demonstrated in the connectome network of changes induced by proinsulin. Therefore, the neuroprotective effects of human proinsulin unveil a new pharmacological potential therapy in the fight against cognitive loss in the elderly.

Transfer of human proinsulin gene into Cucumber (Cucumis sativus L.) via agrobacterium method

Nowadays, approximately 5.8% in adult population around the world are suffering by diabetes. It can be caused by an increase in risk factors such as being overweight. Also it has been estimated that the number of patients will be doubled in near future and the demands for insulin hormone will be growing up by 3 to 4 % annually. Therefore, it?s necessary to develop new methods for hormone production with high rate of capacity in future. By advanced technology of transgenic DNA, the transgenic plants are introduced as an attractive system for expression and production of many kinds of pharmaceutical proteins. In this study, we investigated transfer of Human Proinsulin Gene into the Cucumber (Cucumissativus L.). Transgenic cucumber could be a great prospect for future source of eatable insulin pharmaceutical drugs to be taken by patients.Agrobacterium tumefaciensstrain LBA4404 carrying proinsulin genes with CaMV 35S promoter was used for the transformation purpose. The transgenic plants were analyzed by PCR, RT-PCR, SDS-PAGE, Dot blot and Electrochemiluminescence techniques. Production of proinsulin in cucumber could be a great prospect in molecular farming of human proinsulin.

Correction of Murine Diabetic Hyperglycaemia With A Single Systemic Administration of An AAV2/8 Vector Containing A Novel Codon Optimized Human Insulin Gene.

We report the correction of hyperglycemia of STZ induced diabetic mice using one intravenous systemic administration of a single stranded serotype 8 pseudotyped adeno-associated virus (ssAAV2/8) vector encoding the human proinsulin gene under a constitutive liver specific promoter. In vivo dose titration experiments were carried out and we identified an optimal range that achieved maintenance of euglycaemia or a mild diabetic condition for at least 9 months and ongoing to beyond 1 year for some animals, accompanied by human C-peptide secretion and weight gain. Further DNA codon optimization of the insulin gene construct resulted in approximately 3-10 times more human C-peptide secreted in the blood of codon optimized treated animals thereby reducing the number of vector particles required to achieve the same extent of reduction in blood glucose levels as the non-codon optimized vector. The constitutive secretion of insulin achieved with a single administration of the vector could be of therapeutic value for some diabetic patients.

A double built-in containment strategy for production of recombinant proteins in transgenic rice.

Using transgenic rice as a bioreactor for mass production of pharmaceutical proteins could potentially reduce the cost of production significantly. However, a major concern over the bioreactor transgenic rice is the risk of its unintended spreading into environment and into food or feed supplies. Here we report a mitigating method to prevent unwanted transgenic rice spreading by a double built-in containment strategy, which sets a selectively termination method and a visual tag technology in the T-DNA for transformation. We created transgenic rice with an inserted T-DNA that harbors a human proinsulin gene fused with the far-red fluorescent protein gene mKate_S158A, an RNAi cassette suppressing the expression of the rice bentazon detoxification enzyme CYP81A6, and an EPSPS gene as the selection marker for transformation. Herbicide spray tests indicated that such transgenic rice plants can be killed selectively by a spray of bentazon at regular field application dosage for rice weed control. Moreover, the transgenic rice seeds were bright red in color due to the fused far-red fluorescent protein, and could be easily visualized under daylight by naked eyes. Thus, the transgenic rice plants reported in this study could be selectively killed by a commonly used herbicide during their growth stage, and their seeds may be detected visually during processing and consumption after harvest. This double built-in containment strategy may greatly enhance the confinement of the transgenic rice.

Root and shoot parts of strawberry: factories for production of functional human pro-insulin.

Diabetes, a disease caused by excessive blood sugar, is caused by the lack of insulin. For commercial production, insulin is made in bacteria or yeast by protein recombinant technology. The focus of this research is evaluating another resource and producing of recombinant insulin protein in as strawberry as this plant has high potential in production of pharmaceutical proteins. Strawberry is a suitable bioreactor for production of recombinant proteins especially edible vaccines. In this research, human pro-insulin gene was cloned in pCAMBIA1304 vector under CaMV35S promoter and NOS terminator. Agrobacterium tumefaciens LBA4404, AGL1, EHA105, EHA101, C58, C58 (pGV2260) and C58 (pGV3101) strains were used for transformation of pro-insulin gene into strawberry cv. Camarosa, Selva, Sarian Hybrid, Pajaro, Paros, Gaviota, Alpine. Additionally, Agrobacterium rhizogenes K599, R1000, A4 and MSU440 strains were utilized for gene transformation into hairy roots. PCR analysis indicated the presence of transformed human pro-insulin gene in the strawberry and hairy roots. Also, its transcription was confirmed using RT-PCR. Furthermore, the analysis of plants, fruits and hairy roots at the level of proteins using dot blot, ELISA, SDS-PAGE and ECL tests re-confirmed the expression of this protein in the transgenic plants as well as hairy roots. Protein purification of human pro-insulin from transgenic tissues was performed using affinity chromatography. Finally, the bioassay of recombinant pro-insulin was performed. The analysis of second generations of transgenic plants (T1) at DNA and protein levels was also performed as a complementary experiment. This study opens a new avenue in molecular farming of human pro-insulin through its mass production in roots and shoots of strawberry.

Failure to Achieve Normal Metabolic Response in Non-Obese Diabetic Mice and Streptozotocin-Induced Diabetic Mice After Transplantation of Primary Murine Hepatocytes Electroporated With the Human Proinsulin Gene (p3MTChins)

BackgroundA recent study by Chen et al described a therapy for diabetes that involved electroporation of primary hepatocytes with human proinsulin cDNA, p3MTChins. Intrahepatic transplantation of treated hepatocytes into streptozotocin (STZ) murine and porcine models led to euglycemia, weight maintenance, and normal insulin production. We tested the repeatability of their basic experiments and transplantation technique and expanded the study to include an autoimmune model. MethodsHepatocytes were isolated from B6 mice, electroporated with p3MTChins, and glucose-challenged or were injected into hepatic or spleen parenchyma of STZ-diabetic B6 and non-obese diabetic mice. Outcomes included survival, serum glucose levels, insulin, and c-peptide release. Untransfected primary hepatocytes and mice transplanted with these cells served as controls. Resultsp3MTChins-hepatocytes secreted insulin during glucose challenge, but glucose levels did not change with increasing glucose concentrations. Direct hepatic injection led to high mortality rates. Mice that underwent intrasplenic injection survived for >50 days (control = 4 days) and had a mild but stable improvement in hyperglycemia. C-peptide in both mouse models was detectable but eventually declined to baseline in the non-obese diabetic mice. ConclusionsHepatocytes can be transfected with p3MTChins to produce human insulin but may lack the proper glucose-sensing or complex storage and secretion capabilities that allow for a finely tuned dynamic insulin response. Treatment is subtherapeutic, and p3MTChins-hepatocyte function may not endure in an autoimmune model. Without successful preliminary findings, cell therapy involving electroporation of p3MTChins does not appear to be practical as a therapy for diabetes and may not be a strategy to pursue at this time.

Dicistronic expression of human proinsulin–protein A fusion in tobacco chloroplast

Different expression systems such as bacteria and mammalian cells have been used to produce pharmaceutical proteins. In recent years, the use of plants as bioreactors offers efficient and economical systems in recombinant protein production. Furthermore, because of the large number of plastid copies in plants, chloroplast engineering functions as an effective method to increase recombinant protein expression. Because the commercially available insulin for treatment does not contain C-peptide, which is of great importance for type 1 diabetic patients, the current study introduces the human proinsulin gene fused with protein A into the tobacco chloroplast genome using the biolistic method. To achieve homoplasmy, three rounds of selection and regeneration of transforming cells were performed on the medium that contained spectinomycin antibiotic and hormones. The PCR analysis indicated the presence of the proinsulin gene in transplastomic plants. The reverse-transcription PCR analysis confirmed the expression of the proinsulin-proteinA fusion at the transcription level. Immunoblot assays of leaf-derived protein extracts confirmed that the target gene expression is up to 0.2% of the total soluble protein. Our study showed that protein A fusion is not as efficient as other reported fusions. The transplastomic plants were also confirmed for homoplasmy using Southern blot analysis.

Distinct Effects of Metformin on Pdx-1 Before and After Birth

In a recent article (Int J Endocrinol Metab 8:211-214, 2010), Hashemitabar et al. (1) have shown that intraperitoneal injection of metformin in pregnant mice induces an increase in the levels of Pdx-1 and insulin mRNA in neonatal mice but not in 19.5 embryonic fetuses. On the basis of these results, the authors concluded that metformin affects the regulatory region of the insulin gene only after birth. The transcriptional factor Pdx-1 is expressed in both endocrine and exocrine tissues and is essential for pancreatic development in the embryonic stage (2). Mice with knocked out Pdx-1 gene do not develop a pancreas; therefore, this deletion is lethal (3). However, after pancreatic development, Pdx-1 is expressed only in insulin-producing β cells and somatostatin-producing δ cells (4). Pdx-1 transactivates the insulin gene in β cells obtained from mature animals (5). However, the expression of the insulin gene is not regulated by Pdx-1 in the embryonic stage. Insulinexpressing cells are observed even in Pdx-1 homozygous mutant mice (6). Metformin has 2 distinct effects on the levels of Pdx-1 protein. First, although metformin does not regulate Pdx-1 gene expression, it increases Pdx-1 protein levels after 24 h in isolated rat islets (7), suggesting that metformin has a post-transcriptional effect on Pdx-1 production. Second, metformin induces the translocation of Pdx-1 from the cytoplasm to the nucleus of MIN6 pancreatic cells and increases binding of Pdx-1 to the reporter gene construct that includes the promoter for the human proinsulin gene (7). Therefore, the ability of metformin to increase the expression levels of the insulin gene in mature pancreatic β cells could be partly explained by the synergistic effect of the 2 above-mentioned mechanisms involved in the metformin-mediated regulation of Pdx-1 protein levels. This hypothesis is consistent with the findings of Hashemitabar et al., who have clearly shown that the main effect of metformin on Pdx-1 takes place after birth and that metformin does not interfere with the embryonic development of the pancreas.

Characterization of Human Umbilical Cord Lining-Derived Epithelial Cells and Transplantation Potential

In this study we describe the derivation and immunological characterization of a primary epithelial cell type from the human umbilical cord membrane. These cord lining epithelial cells (CLECs) expressed and/or secreted isoforms of the nonclassical human leukocyte antigen class I (HLA-1b) glycoproteins, HLA-G and E. Conditioned media from CLECs inhibited mitogen-stimulated T-lymphocyte responses, and in a mixed leukocyte reaction (MLR) assay, cocultured CLECs inhibited allogeneic responses with a concomitant reduction in proinflammatory cytokines. Using a transwell coculture system, it was demonstrated that these immunoregulatory effects were mediated by soluble factors secreted by CLECs, in a dose-dependent manner. Functional studies using HLA-G blocking antibody showed that the effects of CLEC-secreted products could be inhibited, thus demonstrating a significant and important role for soluble HLA-G. In vivo, we show that transplanted CLECs could be maintained for extended periods in immunocompetent mice where xenorejection rapidly destroyed primary keratinocytes, a control human epithelial cell type. Additionally, CLECs delayed the rejection of keratinocytes and extended their survival when cotransplanted, indicating an ability to protect adjacent human cell types that would otherwise be rejected if transplanted alone. We also show that CLECs transduced with a modified human proinsulin gene were transplanted intraperitoneally into streptozotocin (STZ)-induced diabetic mice, resulting in significantly lower levels of serum glucose compared to control mice. This study has characterized the immunological properties of CLECs and tested a potential therapeutic application in the treatment of a type 1 diabetes mouse model.

Paternally Inherited Proinsulin Mutations May Result in Earlier Onset of Monogenic Diabetes Mutation Identity Effect in Monogenic Diabetes

Mutations within the human proinsulin gene ( INS ) have been reported to cause neonatal, maturity onset diabetes of the young (MODY), and antibody-negative idiopathic type 1 diabetes (1–3). However, because the expression of maternally or paternally transmitted INS-IGF2 alleles is different as a result of selective methylation (imprinting) (4), we surmised that the effect of INS mutations may depend on the origin of the mutated allele. To verify this hypothesis, pairs comprising an affected child and parent carrying the same heterozygous INS mutation were studied. A literature search was performed and yielded eight relevant articles reporting heterozygous INS mutations in neonatal, MODY, or antibody-negative type 1 diabetes (supplementary Table 1, available in an online appendix at http://care.diabetesjournals.org/cgi/content/full/dc10-1142/DC1). We also included an unpublished case …

Short communication Correct targeting of proinsulin in protein storage vacuoles of transgenic soybean seeds

Soybean plants are promising bioreactors for the expression of biochemically complex proteins that cannot be produced in a safe and/or economically viable way in microorganisms, eukaryotic culture cells or secreted by transgenic animal glands. Soybeans present many desirable agronomic characteristics for high scale protein production, such as high productivity, short reproductive cycle, photoperiod sensitivity, and natural organs destined for protein accumulation in the seeds. The significant similarities between plant and human cells in terms of protein synthesis processes, folding, assembly, and post-translational processing are important for efficient accumulation of recombinant proteins. We obtained two transgenic lines using biolystics, incorporating the human proinsulin gene under control of the monocot tissue-specific promoter from sorghum gamma-kafirin seed storage protein gene and the alpha-coixin cotyledonary vacuolar signal peptide from Coix lacryma-jobi (Poaceae). Transgenic plants expressed the proinsulin gene and accumulated the polypeptide in mature seeds. Protein targeting to cotyledonary protein storage vacuoles was successfully achieved and confirmed with immunocytochemistry assays. The combination of different regulatory sequences was apparently responsible for high stability in protein accumulation, since human proinsulin was detected after seven years under room temperature storage conditions.

Fusion Expression of Antimicrobial Peptide GK1 in Escherichia coli

The antimicrobial peptide GK1, a derivative of cecropin A, shows high antimicrobial activities against Gram-positive and Gram-negative bacteria. To avoid the lethal effects on Escherichia coli host cells during its expression, the GK1 gene was combined with a modified human proinsulin ( mhPI) gene, inserted into the vector pET28a to construct the recombinant expression plasmid (pET28a-mhPI-GK1) and then transformed into BL21 (DE3). A fusion protein was expressed and resulted in insoluble inclusion bodies counting 20% ( W/W) of total cellular proteins. Recombinant GK1 was cleaved from the mhPI-GK1 fusion protein by cyanogen bromide (CNBr) and purified by cation exchange chromatography and reverse-phase high-performance liquid chromatography. The final concentration of recombinant GK1 was 5.7 mg/L E. coli culture, and the purity was above 97%. Its molecular weight measured using electrospray ionization mass spectrometry (ESI-MS) was 2794 D, similar to that of the synthetic GK1. In addition, they have similar antibacterial activity. The results show that mhPI is capable of mediating high-level GK1 gene expression.

Human Krüppel-like factor 11 inhibits human proinsulin promoter activity in pancreatic beta cells

The Krüppel-like factor 11 (KLF11; TIEG2), a pancreas-enriched Sp1-like transcription factor, is a known negative regulator of pancreatic exocrine cell growth. A recent study indicated KLF11-induced activation of the human proinsulin promoter (hInsP). We investigated the functional role of KLF11 in pancreatic beta cells. Endogenous KLF11 mRNA expression was found in whole rat pancreas, human pancreatic islets and INS-1E beta cells and was profoundly reduced by high glucose in INS-1E. Cotransfections of INS-1E and beta-TC3 beta cells with a human (h)KLF11 expression plasmid and an hInsP-driven reporter plasmid resulted in a substantial dose-dependent and glucose-independent inhibition of proinsulin promoter activity. 5'-deletion of hInsP demonstrated that hKLF11 acts via DNA sequences upstream of -173 and requires the beta cell-specific transcription machinery, since hKLF11-mediated inhibition of promoter activity was abolished in HEK293 cells. Besides a previously described GC box, we further identified a CACCC box within the hInsP, both putative KLF11-binding motifs. Electrophoretic mobility shift analysis (EMSA) verified binding of in vitro translated hKLF11 to the GC box, but neither hKLF11-induced inhibition nor basal hInsP activity was altered by mutation or 5'-deletion of the GC box. In contrast, CACCC box mutation substantially reduced basal promoter activity and partially diminished hKLF11 inhibition, although binding of in vitro translated hKLF11 to the CACCC box could not be verified by EMSA. In rodent beta cell lines, we demonstrate hKLF11overexpression-mediated inhibition [corrected] of human proinsulin gene expression and characterise a prominent role for the CACCC box in maintaining basal proinsulin promoter activity.

Krueppel-like transcription factor 11 (KLF11) inhibits activity of the human proinsulin promoter in pancreatic beta-cells

KLF11 (TIEG2) is a member of the Sp1/KLF transcription factor family which plays an important role in mammalian gene regulation. KLF11 is a pancreas-enriched transcription factor that has elicited significant attention because of its role as negative regulator of exocrine cell growth in vitro and in vivo. However, its functional role in the endocrine pancreas remains to be established. Here we investigated endogenous expression of KLF11 in pancreatic beta-cells and analyzed the effects of KLF11 overexpression on human proinsulin promoter activity. RT-PCR revealed endogenous KLF11 mRNA expression in whole rat pancreas, human pancreatic islets and rat INS-1 beta-cells. Glucose stimulation does not seem to modulate KLF11 expression in INS-1 beta-cells. Transient co-transfection of human proinsulin promoter driven secreted alkaline phosphatase (SEAP) reporter gene vectors with KLF11 expression plasmids in INS-1 and betaTC3 beta-cells demonstrates dose-dependent, but glucose-independent KLF11-mediated repression of insulin promoter activity. 5((prime))-deletion analysis of the human proinsulin promoter sequence and gel-shift assays (EMSA) of protein/DNA binding indicate specific GC and CACCC boxes within the human proinsulin promoter as potential KLF11 binding sites. Further, KLF11 may interfere with SP1 binding sites at these response elements. In summary, we have identified Krueppel-like transcription factor 11 (KLF11) as a novel negative transcriptional regulator of human proinsulin gene expression in pancreatic beta-cells. Further we have characterized specific KLF11 DNA-response elements within the human proinsulin promoter. Impairment of KLF11-dependent transcriptional regulation of proinsulin expression may contribute to beta-cell dysfunction seen in the pathogenesis of diabetes mellitus.

Gene therapy for diabetes mellitus in rats by intramuscular injection of lentivirus containing insulin gene

We assessed therapeutic potential of intramuscular insulin gene delivery in a diabetic murine model. The human proinsulin gene cDNA engineered with concensus furin cleavage sequences was inserted into an advanced lentiviral vector that contained CMV early promoter. After injection of concentrated lentiviral vector (3.5 μg p24 Gag antigen) carrying the insulin gene into the thigh muscle, treated rats demonstrated an increase in body weight, increased survivability, attenuated the hyperglycemic response as well as prevented the formation of ketoacidosis. For these reasons, the intraparenchymal injection of lentiviral vectors into the skeletal muscle to ectopically produce insulin may be an easy and therapeutic treatment modality for type 1 diabetes mellitus.

932. Gene Therapy for Diabetes Mellitus in Rats by Intramuscular Injection of Lentivirus Containing Insulin Gene

Top of pageAbstract Type 1 diabetes mellitus (DM) is an auto-immune disease in which activated T-lymphocytes infiltrate the islets of Langerhans and completely destroy the insulin-producing pancreatic |[beta]|-cells. We assessed therapeutic potential of intramuscular insulin gene delivery in a diabetic murine model. The human proinsulin gene cDNA engineered with concensus furin cleavage sequences was inserted into an advanced lentiviral vector that contained CMV early promoter. After injection of concentrated lentiviral vector (3.5 microgram p24 Gag antigen) carrying the insulin gene into the thigh muscle, treated rats demonstrated an increase in body weight, increased survivability, attenuated the hyperglycemic response as well as prevented the formation of ketoacidosis. For these reasons, the intraparenchymal injection of lentiviral vectors into the skeletal muscle to ectopically produce insulin may be an easy and therapeutic treatment modality for type 1 diabetes mellitus.

LONG–TERM REVERSAL OF TYPE 1 DIABETES IN RATS AFTER IN VIVO DELIVERY OF THE HUMAN INSULIN GENE

O197 Aims: Attempts to replace the β-cells lost in type 1 diabetes include pancreas and islet transplantation, derivation of β-cells from stem cells, and gene therapy. Despite these varied approaches, permanent reversal of type 1 diabetes is yet to be achieved. The aim of this study was to reverse streptozotocin (STZ)-induced diabetes in Wistar rats. Methods: The human proinsulin gene was cloned into a viral vector. Transduction efficiency was assessed by flow cytometry. We introduced the viral vector which expressed the insulin gene into male Wistar rats (250-300 g in body weight) via the portal circulation. Prior to vector delivery, diabetes was induced by the intraperitoneal injection of STZ (75 mg/ kg body weight). Following delivery of the insulin gene or empty vector, rats were monitored for changes in body weight and blood glucose levels. An intravenous glucose tolerance test was performed after a 14 h fast in untreated (n = 3), empty vector-transduced (n = 4) and insulin vector-trransduced (n=6) rats. At various times (0, 2, 5, 10, 15, 30, 60, 120 min) after the infusion of glucose (0.5 g /kg body weight), blood samples were collected from the tail vein and assayed for glucose and insulin. Two months after vector infusion, liver, pancreas, kidney, spleen and lung were harvested. Expression of insulin was determined by RT-PCR and immunohistochemistry. Results: The ideal amount of vector for successful delivery was 4 X 106 transduction units. Rats treated with the insulin-expressing vector, exhibited normalization of blood glucose levels within 48 h and normoglycaemia was maintained for over 11 months. Insulin was expressed only in the liver of insulin-transduced rats. Glucose tolerance tests revealed that insulin-transduced rats showed similar responses to normal rats. In contrast, diabetic rats that received the empty vector remained hyperglycaemic (blood glucose > 24mmol/L) and failed to increase body weight. Conclusions: This study shows that hepatic insulin gene therapy can induce the long-term reversal of diabetes. Importantly, hepatocytes transfected with transgenic insulin are sufficiently responsive to cure STZ–induced diabetes without inducing hypoglycemia.