203 insulin gene therapy of diabetes in mice by glucose regulated helper dependent adenoviral vectors

203 Insulin Gene Therapy of Diabetes in Mice by Glucose Regulated Helper Dependent Adenoviral Vectors Molecular Therapy �������� ��� ���� ���������������� �������� ���� ������© ����������� �!����� ���[.]

GENETIC AND METABOLIC DISEASES: PART ONE levels decreased to the normal range within weeks after treatment and normal levels were maintained for at least months, whereas untreated diabetic ob/ob mice remained hyperglycemic We examined the presence of the LPK-MI genome in various tissues by PCR at one month after treatment with rAAV-LPK-MI We detected the LPK-MI genome in all tested tissues including the liver, kidney, spleen, lung, and heart, with the highest amounts in the liver and spleen When we examined the expression of MI mRNA in various tissues by reverse transcriptase-PCR, we found that MI mRNA was expressed mainly in the liver, suggesting that the expression of MI mRNA is dependent on the liver-specific L-type pyruvate kinase promoter activity To determine whether rAAV-LPK-MI-treated ob/ob mice clear glucose from the blood, we performed glucose tolerance tests at weeks after rAAV-LPK-MI administration We found that the glucose level peaked at 30 after glucose injection and returned to normal within 180 min, whereas the blood glucose levels of untreated diabetic ob/ob mice were not normalized In addition, we performed insulin tolerance tests to determine the effect of rAAV-LPK-MI treatment on insulin resistance Although insulin resistance in rAAV-LPK-MI-treated ob/ob mice was significantly alleviated compared to untreated diabetic ob/ob mice, some insulin resistance was still present as compared to heterozygous ob (+/-) mice These results suggest that this insulin gene therapy may have potential therapeutic value for the treatment of obese type diabetes 201 Ectopic Expression of Glucagon-Like Peptide for Gene Therapy of Type Diabetes Donna Armentano,1 Geoffrey Parsons,1 David Souza,1 Dan Yu,1 Samuel Wadsworth,1 Richard Gregory.1 Genzyme Corporation, Framingham, MA, United States Glucagon-like peptide (GLP-1) is an incretin hormone produced in the gut in response to nutrient intake and acts in several ways to reduce post-prandial blood glucose excursions It is a potent stimulator of glucose-induced insulin secretion, delays gastric emptying, suppresses glucagon secretion, improves insulin sensitivity in peripheral tissues and more recently has been shown to stimulate β-cell neogenesis The glucose-dependent insulinoptropic actions of GLP-1 have made it a promising candidate for the treatment of type diabetes However, the short in vivo half-life of GLP-1, due to inactivation by dipeptidylpeptidase IV (DPPIV), has made protein-based treatments challenging requiring repeat subcutaneous injections or continuous infusion of peptide A gene therapy approach using vectors to express a DPPIV resistant analog is one way to circumvent the rapid turnover of GLP-1 and need for repeat administration We have developed GLP-1 chimeric expression vectors encoding a DDPIV-resistant 31-amino acid peptide linked to leader sequences required for secretion of GLP-1 Plasmid or adenoviral expression vectors were administered via tail vein to diabetic db/db mice in both the C57BL/KsJ (more severe diabetes) and C57BL/6 (less severe diabetes) genetic backgrounds Plasma GLP-1, blood glucose, insulin, body weight and %HbA1c were monitored over time Constitutive secretion of GLP-1 in vivo resulted in plasma levels of GLP-1 >1nM which were sustained for periods longer than month Expression of GLP-1 led to lowering either fasting or random fed hyperglycemia, and consequently, %HbA1c in these mice These results demonstrate proof-of-concept for GLP-1 gene therapy of type diabetes using GLP-1 expression vectors and provide a means for circumventing the need for continuous infusion of GLP-1 or subcutaneous injection 202 Constitutive Expression of Insulin in the Liver by Systemic Administration of a Recombinant Adenovirus Expressing a FurinCleavable Mouse Preproinsulin Can Remit Diabetes in Diabetic NOD Mice and Evade Autoimmune Attack Seungjin Shin,1 Jaeseok Han,1 Hee-Sook Jun,1 Ji-Won Yoon.1 Julia McFarlane Diabetes Research Centre, The University of Calgary, Calgary, AB, Canada Type diabetes results from the destruction of pancreatic beta cells caused by autoimmune-mediated immune responses Restoration of beta cells by islet transplantation or regeneration of pancreatic beta cells from ductal stem cells are attractive methods for the cure of diabetes; however, it is difficult to overcome the hostile beta cell-specific autoimmune responses that might eventually destroy the transplanted or regenerated beta cells Since the liver is an attractive target for insulin gene therapy, this investigation was initiated to determine whether transduced, insulin-producing hepatocytes can evade autoimmune attack and remit hyperglycemia in the nonobese diabetic (NOD) mouse, which spontaneously develops autoimmune diabetes We constructed a recombinant adenovirus expressing a furin-cleavable mouse preproinsulin under the cytomegalovirus promoter and the beta-globin/IgG chimeric intron, which will constitutively express insulin (rAd-CMV-mPPI) In vitro studies showed that an immortalized hepatocyte cell line transduced with rAd-CMV-mPPI produced biologically active insulin To test this gene construct in vivo, we administered rAd-CMVmPPI (5 × 109 particles) intravenously through the tail vein of spontaneously diabetic NOD mice (blood glucose > 400 mg/dl) and found that blood glucose was reduced to normal levels within days after administration and maintained at normoglycemic levels for one month, after which time the experiment was terminated We examined the expression of insulin in various tissues, including the liver, kidney, spleen, stomach, heart, pancreas, and lung, and found that insulin was mainly expressed in the liver, as compared to other organs, suggesting that systemic administration of rAd-CMV-mPPI resulted in delivery mainly to liver tissue To determine whether these insulinproducing hepatocytes are attacked by existing effector T cells in the recipients, we examined the liver at 7, 14, 21, and 30 days after rAd-CMV-mPPI administration We found that the liver was not invaded by effector cells such as T cells and macrophages When we adoptively transferred splenic lymphocytes from the rAd-CMVmPPI-treated NOD mice into NOD.scid mice, over 90% of the recipients developed diabetes within weeks, indicating that effector cells in the rAd-CMV-mPPI-treated NOD mice were active Based on these observations, we conclude that constitutive expression of insulin in the liver by systemic administration of rAd-CMV-mPPI can remit diabetes and evade autoimmune attack in diabetic NOD mice 203 Insulin-Gene Therapy of Diabetes in Mice by Glucose-Regulated Helper-Dependent Adenoviral Vectors Paola Corbella,1 Laura Perani,1 Francesca Mingozzi,1 Alessandra Recchia,1 Fulvio Mavilio,3 Maria-Grazia Roncarolo,1 Luca Falqui.1,2 San Raffaele-Telethon Institute for Gene Therapy, San Raffaele Hospital Scientific Institute, Milan, Italy; 2Medicine, Endocrinology and Diabetes Unit, San Raffaele Hospital Scientific Institute, Milan, Italy; 3Department of Biomedical Science, University of Modena School of Medicine, Modena, Italy Type diabetes is an autoimmune disease characterized by destruction of insulin-producing pancreatic beta cells, resulting in Molecular Therapy Vol 7, No 5, May 2003, Part of Parts Copyright © The American Society of Gene Therapy S79 GENETIC AND METABOLIC DISEASES: PART ONE deficient insulin secretion and hyperglycemia Despite intensive insulin therapy, properly controlled glucose values are hardly achieved, therefore degenerative chronic complications may arise In search for alternative therapeutic options, this project aims at engineering non-beta cells for the production of insulin in a glucoseregulated manner The liver is the candidate target organ for this purpose, as hepatocytes are able to ‘sense’ extracellular glucose variations and modulate consequentially gene expression To obtain efficient insulin expression in the liver, we are exploring HelperDependent Adenoviral Vectors (HD-AdV), which should also provide a better safety profile We generated a vector coding for Furine cleavable-Human Proinsulin (FurHPI) under the control of the glucose-responsive liver-specific Pyruvate Kinase gene promoter (L-PKp) coupled to the SV40 enhancer The vector was injected into STZ-induced diabetic immunodeficient’nude’ mice (n=4/group) at two different doses: 1.4x10e11 (low dose) or 2,8x10e11 (high dose) vp/mouse Human insulin was detectable as early as the first week after virus injection in both groups, reaching a maximum between the 2nd and 3rd week of 1.1-3.6 μU/ml in the low-dose group and of 129-192 μU/ml in the high-dose group, and remaining at similar levels thereafter Mice injected with the low dose remained hyperglycemic whereas with the high dose blood glucose returned to normal value, starting from the 2nd week At weeks, the high dose produced exceedingly high insulin levels (normal insulin values in fed nude mice =49.3+8.1 μU/ml) Consequently, blood glucose level was reduced to moderate-severe hypoglycemic value (53±8 mg/dl) Glucose loads and fasting tests demonstrated that L-PKp displays a slow and weak (3.6-fold over h) induction and 6-fold repression of insulin synthesis upon glucose increase/decrease We then generated a second vector containing a Hybrid(L-PK/Spot14) promoter, which displayed a stronger response to glucose than the L-PKp once transfected in rat hepatocytes We linked this promoter to the nuclear LacZ reporter gene in order to quantify liver expression and tissue specificity This vector was injected into ‘nude’ mice (n=2) at the dose of 2,8x10e11 particles/mouse Animals were sacrificed and tissues collected weeks post-gene transfer and analyzed for histochemistry and β-Galactosidase activity Liver sections showed ∼15% transduced hepatocytes β-Galactosidase activity was detected in liver extracts only, indicating that this promoter maintains liver-restricted expression Hepatocytes toxicity was determined by liver enzyme function test AST and ALT peaked (x5) at day after injection, then returned to normal level at day The reporter gene will be replaced with FurHPI to obtain faster glucose-responsive induction of insulin synthesis in liver cells These results indicate that HD-AdVv are suitable vectors to pursue a finetuned glucose-responsive insulin expression in the liver for gene therapy of type diabetes 204 Systemic Gene Therapy with Interleukin10 (IL-10) for Prevention of Type Diabetes in NOD Mice: Comparison of Mutant (I87A Substitution) to Recombinant Murine IL-10 Matthias H Kapturczak,1 Clive H Wasserfall,2 Scott Loiler,3 Martha Campbell-Thompson,2 Marda Scott-Jorgensen,2 Jeff Cross,2 James M Crawford,2 Tamir M Ellis,2 Terence Flotte,3 Mark A Atkinson.2 Medicine, University of Florida, Gainesville, FL; 2Pathology, Immunology and Laboratory Medicine, University of Florida, Gainesville, FL; 3Pediatrics, University of Florida, Gainesville, FL Type diabetes results from the autoimmne destruction of the insulin producing β cells While the immunological mechanisms underlying this process are unclear, immunoregulatory defects appear to be associated with genetic susceptibility and disease progression Previous studies have demonstrated that a single intramuscular (I.M) S80 injection of recombinant adeno-associated virus (rAAV) vector (serotype 1) containing the murine IL-10 gene, in a dose and timedependent fashion, consistently and dependably prevents type diabetes and insulitis development in female non-obese diabetic (NOD) mice However, histological evaluation of the muscle injection sites receiving rAAV-IL-10 revealed long term evidence of myositis and atrophic muscular changes Additional studies have suggested that a substitution of isoleucine at position 87 of IL-10 with alanine inhibits some of the immunostimulatory functions of IL-10, rendering the mutated molecule a potentially more safe agent for studies aimed at disease prevention To compare IL-10 with I87A-IL10 in terms of diabetes prevention and side effect profiles, groups of week-old female NOD mice (n=13 animals per group) were I.M injected with 109 infectious units (IU) of rAAV-IL-10, 109 IU of rAAV-I87A-IL10, 107 IU of rAAV-I87A-IL-10, 109 IU of rAAV-delta-IL-10 (encoding the first 33 amino acids of IL-10 and not biologically active) and saline as controls Urine glucose levels were monitored weekly for disease development, with disease rates assessed by life-table (Kaplan-Meier) analysis At 14 weeks post injection, animals from each group were sacrificed for histological and immunological assesments At 38 weeks post injection, diabetes frequency was as follows: IL-10 0% p=0.0016 vs saline, 0.0671 vs delta-IL-10), 109 IU I87A 20% NS vs controls), 107 IU I87A 60%, delta-IL-10 30% and saline 50% In terms of the prevention of insulitis, the degree of infiltration essentially mirrored that of diabetes development with high-dose IL-10 proving most effective.Unlike the saline and delta-IL-10 groups, the IL-10 group and both I87AIL-10 groups showed development of significant infiltrate of the muscle at the injection site Phenotyic analysis (CD3, CD4, CD8, macrophage, B-lymphocyte) revealed this inflitrate to be predominantly B-lymphocyte in origin Significant muscle fiber atrophy, however, was seen only in the IL-10 group Our study demonstrates that I87A-IL-10, was less efficient than IL-10 in terms of diabetes prevention Furthermore, such substitution did not abrogate the chemotactic properties of IL-10 Studies of other mutations in the IL-10 structure will be necessary to select for only the desirable parts of the IL-10 function repertoire; allowing for effective disease prevention and devoid of sequence allowing for potentially deleterious inflammation 205 Intrathecal Administration of AAV Vector for the Treatment of Lysosomal Storage Disease in the Brains of MPS I Mice Gordon Watson,1 Jacob Bastacky,1 Steve Jungles,2 Michael Vellard,2 Pavel Belichenko,3 Emil Kakkis.2 Children’s Hospital Oakland Research Institute, Oakland, CA; BioMarin Pharmaceutical, Novato, CA; 3Stanford University, Stanford, CA Mucopolysaccharidosis Type I (MPS I) is caused by an inherited deficiency of α-l-iduronidase (IDU) The result is a progressive, lysosomal storage disease that includes CNS as well as systemic involvement Enzyme replacement therapy by periodic intravenous infusion of purified IDU has been shown to be effective in treating the non-CNS manifestations of the disease; however, the blood brain barrier excludes therapeutic levels of IDU in the brain Thus, our goal was to develop gene therapy for MPS I specifically targeting the brain Although the blood brain barrier also excludes viral vectors that are administered intravenously, initial studies with a similar storage disease, MPS VII, indicated that intrathecal administration of an AAV vector could circumvent this problem For MPS VII experiments, which eliminated storage vacuoles throughout the brain, a relatively high dose of x 1011 vector particles per adult mouse was used To treat MPS I mice, AAV-IDU vectors with and without the woodchuck hepatitis virus posttranslational regulatory element (WPRE) were constructed These vectors contained human IDU Molecular Therapy Vol 7, No 5, May 2003, Part of Parts Copyright © The American Society of Gene Therapy ... finetuned glucose- responsive insulin expression in the liver for gene therapy of type diabetes 204 Systemic Gene Therapy with Interleukin10 (IL-10) for Prevention of Type Diabetes in NOD Mice: Comparison... reporter gene will be replaced with FurHPI to obtain faster glucose- responsive induction of insulin synthesis in liver cells These results indicate that HD-AdVv are suitable vectors to pursue a finetuned... ‘sense’ extracellular glucose variations and modulate consequentially gene expression To obtain efficient insulin expression in the liver, we are exploring HelperDependent Adenoviral Vectors (HD-AdV),