Gene therapy: State of the art. Ramon Gomis MD, PhD, MAE. Hospital Clínic. IDIBAPS. University of Barcelona.
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1 Gene therapy: State of the art Ramon Gomis MD, PhD, MAE. Hospital Clínic. IDIBAPS. University of Barcelona.
2
3 Gene Therapy: First Steps
4 Type 1 diabetes: An islet disease Alpha cell Beta cell Pancreatic Islet
5 Beta cell destruction Insulitis Amyloid deposits
6 Insulin analogs. Short acting Long acting
7 Inhaled insulin
8
9 Stem cells making insulin CPP matrigel, exendin-4 Glut2 Ins Pdx-1 tbp merge Insulin C-peptide E.Mato, A.Novials et alt Biochemical Journal 2009
10
11 Gene Therapy in diabetes 1.- Design of genes construction. 2.- Tissue target 3.-Protein expression 4.- Functionality 5.- Effectiveness
12 The four barriers to successful gene therapy 1.- Uptake, transport and uncoating. 2.- Vector gemome persistance 3.- Transcriptional activity 3.- The immune response can limit the viability of the transduced cells
13 Kay MA, Nature Reviews Genetics, 2011
14 Kay MA, Nature Reviews Genetics, 2011
15
16 Plasmid-based gene therapy for diabetes 1.- Therapy with cytokine inhibitors in autoimmune disease. 2.- DNA vaccination against type 1 diabetes 3.- Insulin delivered by gene therapeutic approaches. 4.- Leptin gene therapy in models of obesity and diabetes
17 Pivotal role of leptin-hypothalamus signaling in the ethiology of diabetes uncovered by gene therapy: a new therapeutic intervention? Kalra SP Gene Therapy (2011) 18,
18 Reinstatement of central leptin sufficiency by single systemic injection of recombinant adenovirus vector encoding leptin gene supressed hyperglycemia in rodent models of type 1 diabetes. Stable restoration of leptin sufficiency, solely in the hypothalamus, with biologically active leptin imposed euglycemia by stimulating glucose disposal in the periphery in models of diabetes type 1
19 Kalra SP, Gene Therapy, 2011
20 Non-viral-mediated hepatic expression of IGF-1 supresses autoimmune diabetes in mice. XM Anguela Diabetes 62: , 2013
21 Anguela XM, Diabetes, 2013
22 Anguela XM, Diabetes, 2013
23 Gene Therapy in diabetes 1.- Design of genes construction. 2.- Tissue target 3.-Protein expression 4.- Functionality 5.- Effectiveness
24 Bosch F, Diabetes, 2013
25 Bosch F, Diabetes, 2013
26
27 RNA interference (RNAi) is a powerful approach for reducing the expression of endogenously expressed proteins. It is used to silence mrnas encoding pathogenic proteins for therapy. Recently, RNAi-based gene silencing approaches have been demonstrated in humans, and ongoing clinical trials hold promise for treating fatal disorders or providing alternatives to traditional small molecule therapies. However, many hurdles remain for using these technologies for therapy.
28 Time course of Type 2 Diabetes pathogenesis Plasma FFA ( µmol/l) Fasting Insulin (pmol/l) Fasting Glucose (mm/l) ß-Cell Mass (% change) Normal Adaptation Glucose Intolerant Type-2 Diabetes Circulating FFA Fasting Serum Insulin Fasting Plasma Glucose Pancreatic ß-Cell Mass INCREASING AGE AND/0R DEGREE OF OBESITY (increasing peripheral insulin resistance)
29 Islets (10, 30 days) Obese (CAF) rats Control (STD) rats 5 arrays 5 arrays Affymetrix GeneChip RAE Statistical analysis Genes expressed differentially between obese and control rats
30 Microarray analysis mrna relative expression 3 2 STD CAF 1 0 Per2 Bcl2l1 sfrp5 * n=6 *, p<0,01
31
32 Results of PCR Arrays in CAF islets : sfrp5 and Wnt pathway
33 Knock down of sfrp5 in INS1E cells Proliferation : The decrease of sfrp5 expression in INS1E leads to an increase in proliferation.
34 To confirm the implication of sfrp5 in beta cell plasticity Knockdown of sfrp5 in dispersed cells from pancreatic islets (Wistar rats). Islets Dispersed cells from islets IF : Dispersed cells (Insulin)
35 Knockdown of sfrp5 in dispersed cells from islets Proliferation : Apoptosis : The decrease in sfrp5 expression in dispersed cells from islets promotes the increase in proliferation without effect on apoptosis.
36 pmes ADIPOSE TISSUE GENES + + SECRETOME METABOLITES + PROTEINS PANCREATIC ISLET GENES Triacylglycerol Degradation GLUT4 Transport and Fusion Transcriptional Regulation Lipid Transport Proteolysis Tissue Remodeling, Fibrinolysis Digestion, Proteolysis Circadian Rhythm Cholesterol Metabolism Acute-Phase Response Complement System Signaling Eating Behavior, Control of Feeding Chemokine, Cytokine Signaling Immune Response Signaling G-Protein Coupled Receptor Signaling Best scored network
37 Rosa Gasa Anna Novials Rita Malpique Sandra Rebuffat
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