Gene Therapy For Type Research Articles

Nanobiotechnology-Modified Cellular and Molecular Therapy as a Novel Approach for Autoimmune Diabetes Management.

Several cellular and molecular therapies such as stem cell therapy, cell replacement therapy, gene modification therapy, and tolerance induction therapy have been researched to procure a permanent cure for Type 1 Diabetes. However, due to the induction of undesirable side effects, their clinical utility is questionable. These anti-diabetic therapies can be modified with nanotechnological tools for reducing adverse effects by selectively targeting genes and/or receptors involved directly or indirectly in diabetes pathogenesis, such as the glucagon-like peptide 1 receptor, epidermal growth factor receptor, human leukocyte antigen (HLA) gene, miRNA gene and hepatocyte growth factor (HGF) gene. This paper will review the utilities of nanotechnology in stem cell therapy, cell replacement therapy, beta-cell proliferation strategies, immune tolerance induction strategies, and gene therapy for type 1 diabetes management.

Immune Cell Infiltration and Identifying Genes of Prognostic Value in the Papillary Renal Cell Carcinoma Microenvironment by Bioinformatics Analysis.

Papillary renal cell carcinoma (PRCC) is one of the most common histological subtypes of renal cell carcinoma. Type 1 and type 2 PRCC are reported to be clinically and biologically distinct. However, little is known about immune infiltration and the expression patterns of immune-related genes in these two histologic subtypes, thereby limiting the development of immunotherapy for PRCC. Thus, we analyzed the expression of 22 immune cells in type 1 and type 2 PRCC tissues by combining The Cancer Genome Atlas (TCGA) database with the ESTIMATE and CIBERSORT algorithms. Subsequently, we extracted a list of differentially expressed genes associated with the immune microenvironment. Multichip mRNA microarray data sets for PRCC were downloaded from the Gene Expression Omnibus (GEO) to further validate our findings. We found that the immune scores and stromal scores were associated with overall survival in patients with type 2 PRCC rather than type 1 PRCC. Tumor-infiltrating M1 and M2 macrophages could predict the clinical outcome by reflecting the host's immune capacity against type 2 PRCC. Furthermore, CCL19/CCR7, CXCL12/CXCR4, and CCL20/CCR6 were shown to be potential new targets for tumor gene therapy in type 2 PRCC. Our findings provide valuable resources for improving immunotherapy for PRCC.

Chaperone mediated gene therapy for type I sialidosis

Chaperone mediated gene therapy for type I sialidosis

Effect of decoy receptor-3 and glutamic acid decarboxylase-65 recombinant adenovirus on type 1 diabetes mice

The decoy receptor-3 ( DcR3 ) and glutamic acid decarboxylase-65 ( GAD65 ) recombinant adenovirus was construced and transduced into denlritic cells (DC). After the transduced DC were utilized to immunize NOD mice,the CD+8 T cells and blood glucose were analyzed. The results showed that recombinant adenovirus inhibited the proliferation and cytokine release of GAD65 specific T cells,and delayed the incidence of diabetes.Both interferon-γ[ (50.5±7.2)vs(95.4±6.9) and(91.2±6.5) pg/ml] and interleukin-2 [ (46.3±5.1 )vs ( 86.1 ±5.2 ) and ( 80.3 ± 7.3 ) pg/ml ] were decreased compared to those in negative and blank controls ( all P＜0.05 ).The results suggest that DcR3 and GAD65 recombinant adenovirus might provide a promising way for gene therapy of type 1 diabetes. Key words: Decoy receptor-3; Glutamic acid decarboxylase-65; Diabetes mellitus, experimental; Gene therapy

Effects of adenovirus-mediated interleukin-10 gene transfer on apoptosis and insulin secretion function of beta cell

To evaluate the efficacy of adenovirus vector-mediated murine interleukin-10 (mIL-10) gene transfer to rat beta cell-RINm5F cells in vitro and to explore the potential value of gene therapy in type 1 diabetes mellitus. The recombinant adenovirus vector Ad-mIL-10 was constructed and transfected into RINm5F cells (Ad-mIL-10 group). Untransfected RINm5F cells and Ad-eGFP-transfected cells were used as controls. The expression of mIL-10 was examined by RT-PCR and Western blot. The levels of IL-10 in supernatant were measured by enzyme-linked immunosorbent assay (ELISA). For a determination of insulin release, the cells were cultured with high glucose (16.7 mmol/L) for a 1-hour co-incubation. Then radioimmunoassay was used to detect the level of insulin in supernatant. After induction of IL-1beta, the levels of nitric oxide (NO) and nitric oxide synthase (NOS) were measured, the apoptosis of transfected cells was detected by Hoechst 33258 staining and Fas expression by flow cytometry. Both mRNA and protein of Ad-mIL-10 were successfully expressed in RINm5F cells. Expression of mIL-10 gene resulted in significant increases in insulin secretion in response to high glucose. Compared with uninfected control and Ad-eGFP infected group, Ad-mIL-10 infected group had decreased levels of NO and NOS induced by IL-1beta [NO level (nmol/10(6) cells): 52.9 +/- 3.2 vs. 227.3 +/- 26.4, 235.1 +/- 28.6, both P < 0.05; NOS level (U/10(6) cells): 9.3 +/- 1.2 vs. 29.8 +/- 2.5, 30.5 +/- 2.8, both P < 0.05]. Furthermore, Ad-mIL-10 gene transfer led to a profound reduction of Fas-expressing islet cells under the induction of IL-1beta. Fas-expressing islet cells in Ad-mIL-10 group were significantly lower than those in uninfected group and Ad-eGFP-transfected group (24.6% +/- 1.0% vs. 33.3% +/- 5.1%, 32.6% +/- 1.1%) (P < 0.05). The apoptotic rates of Ad-mIL-10 group were lower in comparison with the other two groups (9.4% +/- 1.1% vs. 19.2% +/- 2.2%, 20.6% +/- 2.3%, P < 0.05). IL-10 gene transfer to islet beta cells may be beneficial in maintaining beta cells function, protecting islet cells from IL-1beta-mediated apoptosis and promoting islet cells survival. It is a potential therapy for type 1 diabetes mellitus.

BSGT 2010 Invited Speaker Presentations

Gaucher disease is an autosomal recessive lysosomal storage disorder caused by mutations in the glucosidase, beta, acid gene that encodes the lysosomal enzyme glucosylceramidase (GCase). GCase deficiency leads to characteristic visceral pathologic conditions and, in a minority of patients, to lethal neurologic manifestations. Previous mouse models with GCase deficiency have either been lethal in the perinatal period or viable without displaying clinical features of Gaucher disease. We have generated viable mice with characteristic clinical symptoms of type 1 Gaucher disease (eg, hepatosplenomegaly, anemia) by conditionally deleting GCase exons 9 through 11 on induction postnatally.! Both transplantation of wild-type bone marrow and gene therapy through retroviral transduction of bone marrow from Gaucher disease mice prevented development of disease, as well as corrected an already established Gaucher disease phenotype. The gene therapy approach produced considerably higher GCase activity than transplantation of wild-type bone marrow.! Both therapeutic modalities normalized glucosylceramide levels, and practically no infiltration of Gaucher cells was seen in the bone marrow, spleen, or liver, demonstrating correction 5 to 6 months after treatment. The findings suggest, for the first time, the feasibility of gene therapy for type 1 Gaucher disease in vivo. Using similar approaches, we have generated mouse models for severe neuronopathic Gaucher disease.? To circumvent the lethal skin phenotype observed in several of the previous GCase-deficient animals.l-I we genetically engineered a mouse model with a strong reduction in GCase activity in all tissues except the skin. These mice exhibited rapid motor dysfunction associated with severe neurodegeneration and apoptotic cell death within the brain, which is reminiscent of neuronopathic Gaucher disease. In addition, we created a distinct mouse model in which GCase deficiency was restricted to neural and glial cell progenitors and progeny. These mice developed pathologic conditions similar to the first mouse model but with a delayed onset and slower disease progression, indicating that GCase deficiency within microglial cells, which are of hematopoietic origin, is not the primary determinant of the central nervous system pathology. These findings strongly suggest that normal microglial cells cannot rescue this serious neurodegenerative disease. These mouse models may be used to investigate pathologic mechanisms and develop novel therapies for all types of Gaucher disease. Recently, we used nonmyeloablative conditioning of type 1 Gaucher mice and transplanted them with normal bone marrow cells. Based on the results, ::;10% engraftment is enough to correct the pathology of the disease. Collectively, these findings indicate that gene therapy in type 1 Gaucher disease is likely to be successful and may lead to a cure of the disease.

981. Gene Therapy for Type 1 Diabetes by Expressing Insulin and Glucokinase (GK) in Skeletal Muscle: Pre-Clinical Studies in Diabetic Dogs

981. Gene Therapy for Type 1 Diabetes by Expressing Insulin and Glucokinase (GK) in Skeletal Muscle: Pre-Clinical Studies in Diabetic Dogs

641. Gene Therapy for Type II Diabetes by Adeno-Assoicated Viral-Mediated Gene Transfer of AntiGFAT1

641. Gene Therapy for Type II Diabetes by Adeno-Assoicated Viral-Mediated Gene Transfer of AntiGFAT1

Gene therapy for type 1 diabetes mellitus in rats by gastrointestinal administration of chitosan nanoparticles containing human insulin gene

To study the expression of human insulin gene in gastrointestinal tracts of diabetic rats. pCMV.Ins, an expression plasmid of the human insulin gene, wrapped with chitosan nanoparticles, was transfected to the diabetic rats through lavage and coloclysis, respectively. Fasting blood glucose and plasma insulin levels were measured for 7 d. Reverse transcription polymerase chain reaction (RT-PCR) analysis and Western blot analysis were performed to confirm the expression of human insulin gene. Compared with the control group, the fasting blood glucose levels in the lavage and coloclysis groups were decreased significantly in 4 d (5.63 +/- 0.48 mmol/L and 5.07 +/- 0.37 mmol/L vs 22.12 +/- 1.31 mmol/L, respectively, P < 0.01), while the plasma insulin levels were much higher (32.26 +/- 1.81 microIU/mL and 32.79 +/- 1.84 microIU/mL vs 14.23 +/- 1.38 microIU/mL, respectively, P < 0.01). The human insulin gene mRNA and human insulin were only detected in the lavage and coloclysis groups. Human insulin gene wrapped with chitosan nanoparticles can be successfully transfected to rats through gastrointestinal tract, indicating that chitosan is a promising non-viral vector.

Treatment of type 1 diabetes by transplantation of bone-derived mesenchymal stem cells expressing human insulin gene: experiment with mice

To evaluate the effect of treatment of type 1 diabetes by transplantation of bone-derived stem cells expressing human insulin gene. Murine bone marrow-derived stem cells expressing green fluorescent protein (GFP-mMSCs) were isolated from 4/6-week-old GFP mice and transfected with a recombinant retrovirus-murine stem cell virus (MSCV) encoding human insulin gene, thus constructing the GFP-mMSCs-MCV-insulin. 16 C57BL/6J mice were injected with streptozotocin so as to establish models of type 1 diabetes and then randomly divided into 4 equal groups: Group A, undergoing injection into the liver with GFP-mMSC-MCV-insulin 1 week after the establishment of the model, Group B, undergoing intrahepatic transplantation of the GFP-mMSCs transfected with blank vector, Group C, undergoing intrahepatic transplantation of untransfected GFP-mMSCs, and Group D, undergoing intrahepatic transplantation of phosphate-buffered saline (PBS). Another 4 normal mice were used as controls and underwent intrahepatic transplantation of PBS too. After the transplantation the blood glucose, serum insulin, and body weight were detected everyday. 6 weeks later immunohistochemistry was used to detect the expression of human insulin in the mice liver tissues. The body weight of Group A increased by 6% within 6 weeks after treatment, and the average blood glucose level 7 d and 42 d after transplantation were (10.4 +/- 2.8) mmol/L and (6.5 +/- 0.9) mmol/L respectively, both significantly lower than those of Group D [(26.8 +/- 2.5) mmol/L and (25.4 +/- 4.1) mmol/L respectively, both P < 0.05]. Immunohistochemistry showed secretion of human insulin in serum and liver. The clinical manifestations of diabetes can be relieved effectively by intrahepatic transplantation of mMSCs expressing human insulin gene. This study implies a novel approach of gene therapy for type 1 diabetes.

Phenotypic correction of von Willebrand disease type 3 blood-derived endothelial cells with lentiviral vectors expressing von Willebrand factor

AbstractVon Willebrand disease (VWD) is an inherited bleeding disorder, caused by quantitative (type 1 and 3) or qualitative (type 2) defects in von Willebrand factor (VWF). Gene therapy is an appealing strategy for treatment of VWD because it is caused by a single gene defect and because VWF is secreted into the circulation, obviating the need for targeting specific organs or tissues. However, development of gene therapy for VWD has been hampered by the considerable length of the VWF cDNA (8.4 kb [kilobase]) and the inherent complexity of the VWF protein that requires extensive posttranslational processing. In this study, a gene-based approach for VWD was developed using lentiviral transduction of blood-outgrowth endothelial cells (BOECs) to express functional VWF. A lentiviral vector encoding complete human VWF was used to transduce BOECs isolated from type 3 VWD dogs resulting in high-transduction efficiencies (95.6% ± 2.2%). Transduced VWD BOECs efficiently expressed functional vector-encoded VWF (4.6 ± 0.4 U/24 hour per 106 cells), with normal binding to GPIbα and collagen and synthesis of a broad range of multimers resulting in phenotypic correction of these cells. These results indicate for the first time that gene therapy of type 3 VWD is feasible and that BOECs are attractive target cells for this purpose.

Gene Therapy for Type 1 Diabetes: Is it Ready for the Clinic?

This review, in addition to updating the growing list of type 1 diabetes- relevant gene therapies, offers an outline of short-term objectives that can readily be met to move, at least, adenoviral and adeno-associated viral-based protocols into the clinic, first as a means of facilitating islet allografts as well as platforms with which to introduce immunoregulatory transgenes. A wide array of genes have been tested to restore insulin production, to drive the differentiation of insulin-producing progenitors, and to confer immunosuppression in an antigen- and tissue-restricted manner.

Suppression of Hyperglycemia in NOD Mice After Inoculation With Recombinant Vaccinia Viruses

In autoimmune (type 1) diabetes, autoreactive lymphocytes destroy pancreatic beta-cells responsible for insulin synthesis. To assess the feasibility of gene therapy for type 1 diabetes, recombinant vaccinia virus (rVV) vectors were constructed expressing pancreatic islet autoantigens proinsulin (INS) and a 55-kDa immunogenic peptide from glutamic acid decarboxylase (GAD), and the immunomodulatory cytokine interleukin (IL)-10. To augment the beneficial effects of recombinant virus therapy, the INS and GAD genes were fused to the C terminus of the cholera toxin B subunit (CTB). Five-week-old non-obese diabetic (NOD) mice were injected once with rVV. Humoral antibody immune responses and hyperglycemia in the infected mice were analyzed. Only 20% of the mice inoculated with rVV expressing the CTB::INS fusion protein developed hyperglycemia, in comparison to 70% of the mice in the uninoculated animal group. Islets from pancreatic tissues isolated from euglycemic mice from this animal group showed no sign of inflammatory lymphocyte invasion. Inoculation with rVV producing CTB::GAD or IL-10 was somewhat less effective in reducing diabetes. Humoral antibody isotypes of hyperglycemic and euglycemic mice from all treated groups possessed similar IgG1/IgG2c antibody titer ratios from 19 to 32 wk after virus inoculation. In comparison with uninoculated mice, 11-wk-old NOD mice injected with virus expressing CTB::INS were delayed in diabetes onset by more than 4 wk. The experimental results demonstrate the feasibility of using rVV expressing CTB::INS fusion protein to generate significant protection and therapy against type 1 diabetes onset and progression.

Baculovirus p35 increases pancreatic β-cell resistance to apoptosis

β-cells die by apoptosis in type 1 diabetes as a result of autoimmune attack mediated by cytokines, and in type 2 diabetes by various perpetrators including human islet amyloid polypeptide (hIAPP). The cascade of apoptotic events induced by cytokines and hIAPP is mediated through caspases and reactive oxygen species. The baculovirus p35 protein is a potent anti-apoptotic agent shown to be effective in a variety of species and able to inhibit a number of apoptotic pathways. Here, we aimed at determining the protective potential of p35 in β-cells exposed to cytokines and hIAPP, as well as the effects of p35 on β-cell function. The p35 gene was introduced into βTC-tet cells, a differentiated murine β-cell line capable of undergoing inducible growth-arrest. Both proliferating and growth-arrested cells expressing p35 manifested increased resistance to cytokines and hIAPP, compared with control cells, as judged by cell viability, DNA fragmentation, and caspase-3 activity assays. p35 was significantly more protective in growth-arrested, compared with proliferating, cells. No significant differences were observed in proliferation and insulin content between cells expressing p35 and control cells. In contrast, p35 manifested a perturbing effect on glucose-induced insulin secretion. These findings suggest that p35 could be incorporated as part of a multi-pronged approach of immunoprotective strategies to provide protection from recurring autoimmunity for transplanted β-cells, as well as in preventive gene therapy in type 1 diabetes. p35 may also be protective from β-cell damage caused by hIAPP in type 2 diabetes.

Gene therapy for type 1 diabetes: a novel approach for targeted treatment of autoimmunity

Gene therapy for type 1 diabetes: a novel approach for targeted treatment of autoimmunity

155. Non-Viral Gene Therapy for Type 1 Diabetes by Electrotransfer of Insulin and Glucokinase Genes to Skeletal Muscle

Alpha1-antitrypsin (α1-AT) deficiency is caused by mutations in the α1-AT gene, a protease inhibitor, and can result in emphysema and/or liver disease. The lung disease is caused by a decrease in secreted α1-AT enzyme, a loss-of-function which results in tissue damage from unchecked leukocyte proteases. In contrast, the liver disease occurs by a gain-of-function, whereby mutant protein polymers are retained in the endoplasmic reticulum of hepatocytes. In persuit of an alternative to the only current treatment for the liver disease, to transplant the organ, we are investigating a genetic therapy that would combine the technologies of RNA interference (RNAi) with the Sleeping Beauty transposon system. The RNAi pathway consists of endogenous enzymes that destroy mRNAs selectively in response to small RNAs that are complimentary in sequence. Small inhibitory RNAs (siRNAs), which can be transcribed from plasmid constructs introduced into cells, have been shown to mediate RNAi in vitro and in vivo. Moreover, siRNAs can mediate long-term repression of a target gene if they are constitutively expressed from the genome. The Sleeping Beauty (SB) transposon system can integrate DNA transgenes into the genome for long-term expression in vitro and in vivo, and is being developed as a novel, non-viral gene therapy tool. A SB transposon vector was constructed to express siRNAs that target human α1-AT mRNA. The results demonstrate that siRNA transposons can be used to reduce wildtype and mutant α1-AT expression in cultured cells, whereas the house-keeping gene GAPDH was not affected. Next, we plan to test these siRNA transposons in vivo, in the transgenic mouse model of α1-AT liver disease. This gene therapy model combines two technologies, RNAi and the SB transposon, for a potential new therapeutic for the liver disease associated with mutant α1-AT.

1079. Evaluation of Glucagon-Like Peptide 1 (GLP-1) as a Therapeutic Transgene for Gutless Adenoviral Vector-Mediated Gene Therapy of Type 2 Diabetes

1079. Evaluation of Glucagon-Like Peptide 1 (GLP-1) as a Therapeutic Transgene for Gutless Adenoviral Vector-Mediated Gene Therapy of Type 2 Diabetes

Prospects for gene therapy of insulin-dependent diabetes mellitus.

The application of gene therapy to Type I (insulin-dependent) diabetes mellitus awaits improvements in gene transfer technologies and the development of better tools for accurate diagnosis of pre-diabetic people. Identification of the most promising candidate genes for gene transfer requires further elucidation of the molecular events involved in beta-cell autoimmune destruction, islet ontogeny and differentiation, and beta-cell function. This review outlines a number of possible targets for gene therapy in Type I diabetes, which could help prevent the autoimmune damage to islets, induce islet regeneration, and restore insulin production through engineering of self non-beta cells or beta-cell transplantation. It also evaluates their potential merits and drawbacks.

Glucose-stimulated genes and prospects of gene therapy for type I diabetes.

I. Introduction II. Transcriptional Regulation of Glucose-Responsive Genes A. The insulin gene 1. Glucose-signaling pathway in pancreas β-cells 2. E elements of the insulin gene promoter 3. A elements of the insulin gene promoter 4. Role of the E and A boxes of the insulin gene promoters in glucose-dependent transcriptional regulation 5. Role of the cAMP pathway in glucose responsiveness of the insulin gene promoters B. Transcriptional regulation of glucose-responsive genes in the liver 1. The general outlook for glucose metabolism in the liver 2. Transcriptional glucose signaling mechanisms in the liver 3. Glucose/carbohydrate response elements III. Cell and Gene Therapies for IDDM A. Islet and cell transplantations 1. Transplantation of islets 2. Transplantation of established lines B. Bioartificial pancreas C. Transplantation of genetically engineered cells 1. The “ideal” engineered cell for glucose-dependent insulin secretion 2. Engineering established neuroendocrine cells 3. Engineering endogenous he...