CELL BASED DIABETES TREATMENT IN A PRECLINICAL MODEL WONG JEN-SAN {MBChB(UK), MRCS(Edinburgh), MMed(Surgery)} A THESIS SUBMITTED FOR THE DEGREE OF MASTER OF SCIENCE DEPARTMENT OF BIOCHEMISTRY NATIONAL UNIVERSITY OF SINGAPORE 2007 ACKNOWLEDGEMENTS I would like to express my deepest gratitude to my supervisor, Dr Kon Oi Lian, without whom none of this would have been possible. She kindly agreed to take me on and embark on this journey into uncharted territories with me. She designed the project, planned and arranged the resources, coordinated the various personnel of a multidisciplinary team, gave very helpful advice, encouragement and guidance when needed, and shared her vast knowledge throughout the course of the study. My sincere thanks also goes out to Mr Nelson Chen and Ms Irene Kee, my fellows in monkey business, for all the hard work and for sharing each step with me throughout this journey. I am also grateful to the all the staff at the Department of Experimental Surgery, Singapore General Hospital for providing all the assistance, facilities and husbandry for the monkeys. Many thanks also to the Department of General Surgery, Singapore General Hospital for allowing me to carry out this research part time and relieve me of my clinical commitments when required. And finally, I am forever thankful to my wife, Wen Chin, for all her love, advice, support, and encouragement all these years, and for our lovely daughter Chloe. i TABLE OF CONTENTS ACKNOWLEDGEMENTS i TABLE OF CONTENTS ii SUMMARY vi LIST OF FIGURES viii LIST OF TABLES ix ABBREVIATIONS x CHAPTER 1: INTRODUCTION 1 1.1 Diabetes Mellitus 1 1.1.1 Definition and Classification of Diabetes Mellitus 1 1.1.2 Aetiology and Genetics of Diabetes Mellitus 2 1.2 1.3 Type 1 Diabetes Mellitus 2 Type 2 Diabetes Mellitus 4 1.1.3 Burden of the Disease 5 Current Treatment for Diabetes Mellitus 6 1.2.1 Allogeneic Islet Transplantation 8 Experimental Approaches to Treatment of Diabetes Mellitus 9 1.3.1 Cell-Based Therapy for Diabetes Mellitus 9 1.3.1.1 Stem Cells 9 Embryonic Stem Cells 10 Adult Progenitor Cells 11 ii 1.3.1.2 Regeneration of Primary Pancreatic Beta Cells 12 1.3.1.3 Problems with Current Cell-based Therapy Approaches 13 1.3.2 Gene-Based Therapy for Diabetes Mellitus 1.3.2.1 In vivo Gene Therapy 16 Non-insulin genes 16 Glucose-responsive insulin genes and their variants 16 1.3.2.2 Ex vivo Gene Therapy 1.4 15 17 Our Approach 19 1.4.1 Plasmid Construct 19 1.4.2 Autologous Primary Hepatocytes 21 1.4.3 Transplantation Site 22 1.4.4 Primate Model of Diabetes 23 CHAPTER 2: MATERIALS AND METHODS 25 2.1 Plasmid Construct 25 2.2 Hepatocyte Isolation 25 2.3 Gene Transfer 26 2.4 In vitro Studies 27 2.4.1 Induction of Insulin Expression 27 2.4.2 Transcriptional Induction of Transgene (RT-PCR) 28 2.5 Hormone Radioimmunoassay 29 2.6 Animal (in vivo) Studies 29 2.6.1 Sedation and Anaesthesia 29 iii 2.7 2.6.2 Blood Glucose Monitoring and Venepuncture 30 2.6.3 Intravenous Glucose Tolerance Test (IVGTT) 30 2.6.4 Induction of Diabetes 31 2.6.5 Liver Resection and Hepatocyte Transplantation 32 Statistical Analysis 32 CHAPTER 3: RESULTS 33 3.1 Electroporation of Primary Hepatocytes 33 3.2 In vitro Studies 35 3.2.1 Static Induction of Human Insulin by Glucose and Zinc 35 3.2.2 Kinetics of Glucose-induced Insulin Secretion 38 3.2.3 Transcriptional Response of Transgene 40 Animal Studies 42 3.3.1 Induction of Diabetes 42 3.3.2 Hepatocyte Transplantation 50 3.3 3.3.2.1 Blood Glucose Levels and Insulin Requirements 51 3.3.2.2 Intravenous Glucose Tolerance Tests (IVGTT) 54 3.3.3 Liver and Renal Function 61 CHAPTER 4: DISCUSSION 67 4.1 Overview 67 4.2 Isolation and Electroporation of Primary Primate Hepatocytes 69 4.3 Preclinical Model of Diabetes Mellitus 70 iv 4.4 Engraftment and Function of Transplanted Hepatocytes 71 4.5 Limitations of Current Study 71 4.6 Future Work 72 4.7 Conclusions 72 REFERENCES 73 v SUMMARY Diabetes mellitus is a group of diseases characterized by chronic hyperglycaemia and disturbances of carbohydrate, fat and protein metabolism resulting from defects in insulin secretion, insulin action, or both. Despite the availability of exogenous insulin, insulin analogues and other pharmacological agents, a lifetime of good glycaemic control remains an elusive goal for a substantial majority of diabetics. Cell-based approaches are emerging strategies for diabetes treatment. Differentiation of stem/progenitor cells in beta cells appears promising, while the efficacy of a simpler approach of modifying autologous adult somatic cells is uncertain. Our approach was to electroporate autologous primary hepatocytes with a plasmid construct encoding human proinsulin cDNA driven by a bifunctional promoter comprising the human metallothionein IIA promoter linked to a single copy of the carbohydrate response element (ChoRE). Engineered hepatocytes were then transplanted into streptozotocininduced diabetic cynomolgus macaques. Daily fasting blood glucose and insulin requirements were monitored along with biochemical tests of liver and renal function. Intravenous glucose tolerance tests (IVGTT) were performed at 3 distinct stages of the study. In vitro studies confirmed the transfected hepatocytes had regulated insulin secretion in response to glucose and zinc. Diabetes was reliably induced in monkeys with a dose of 875 mg/m2 with mild derangement of liver and renal vi function. Diabetic monkeys transplanted with transfected hepatocytes had lower fasting blood glucose levels compared to controls and did not require exogenous insulin therapy to achieve this. Treated animals had post-transplant IVGTT curves for glucose similar to their pre-diabetic state but control animals had post-transplant IVGTT curves similar to their diabetic state. The area under the curve (AUC) for insulin was higher in the post-transplant state compared to the diabetic state in the treated monkeys although the curves themselves did not return to the prediabetic state. In controls, insulin AUC was lower post-transplant compared to the diabetic state. We have shown that hepatocytes transfected with the said plasmid construct were able to achieve regulated insulin secretion. When transplanted into diabetic monkeys, these engineered hepatocytes were able to partially correct hyperglycaemia and reduce insulin therapy. vii LIST OF FIGURES 1 Plasmid construct used in this study 20 2 Electroporation of primary primate hepatocytes 34 3 Glucose-induced insulin secretion 36 4 Zinc-induced insulin secretion 37 5 Kinetics of glucose-induced insulin secretion 39 6 Kinetics of glucose-induced insulin mRNA expression 41 7 Induction of diabetes with streptozotocin 47 8 Absence of insulin-positive cells in pancreas of diabetic monkeys 49 9 Daily fasting blood glucose levels of 4 monkeys in study 52 10 Blood glucose IVGTT curves of all 4 monkeys in study 55 11 Area under the curve for blood glucose IVGTT curves 57 12 Insulin IVGTT curves of all 4 monkeys in study 58 13 Area under the curve for insulin IVGTT curves 60 14 Liver function tests of 4 study animals 62 15 Renal function tests of all 4 study monkeys 64 viii LIST OF TABLES 1 2 Summary of sex, streptozotocin dose administered and resultant outcome of all monkeys in the study 44 Characteristics of 4 diabetic monkeys that underwent liver resection and hepatocyte transplantation 50 ix ABBREVIATIONS AAALAC Association for Assessment and Accreditation of Laboratory Animal Care ALT Alanine aminotransferase AST Aspartate aminotransferase AUC Area under the curve BSA Body surface area cDNA Complementary DNA ChoRE Carbohydrate response element DCCT Diabetes Control and Complications Trial DMEM Dulbecco’s Modified Eagle’s Medium EGF Epidermal growth factor EGTA Ethylene glycol tetraacetic acid FCS Foetal calf serum FDA Food and Drug Administration Gck Glucokinase GIP Glucose-dependent insulinotropic polypeptide GLP-1 Glucagon-like peptide-1 HLA Human leukocyte antigen IDDM Insulin dependent diabetes mellitus IVGTT Intravenous glucose tolerance test MHC Major histocompatibility complex NHP Nonhuman primate x NOD Nonobese diabetic pEGFP Plasmid expressing green fluorescent protein PTG Protein targeting to glycogen RNA Ribonucleic acid RT-PCR Reverse transcription polymerase chain reaction SEM Standard error of mean SGH Singapore General Hospital STZ Streptozotocin USA United States of America xi CHAPTER 1: INTRODUCTION 1.1 Diabetes Mellitus 1.1.1 Definition and Classification of Diabetes Mellitus Diabetes mellitus is a disorder of carbohydrate, fat and protein metabolism characterized by chronic hyperglycaemia. It results from defects in insulin secretion, insulin action, or both. The World Health Organization recognizes three main forms of diabetes: type 1, type 2 and gestational diabetes (occurring during pregnancy) [1]. Type 1 diabetes, previously called insulin-dependent diabetes mellitus (IDDM) or juvenile-onset diabetes, is caused by autoimmune destruction of the insulin-producing beta cells in the pancreatic islets of Langerhans leading to an absolute deficiency of insulin. This type comprises up to 10% of total cases of diabetes in North America and Europe. Type 2 diabetes, the most common form, was previously called non-insulin-dependent diabetes mellitus (NIDDM) or adultonset diabetes. It results from a combination of insulin resistance, whereby muscle, liver and fat cells do not use insulin properly, and beta cell dysfunction, leading to relative insulin deficiency [2]. As the need for insulin rises, the pancreas gradually loses its ability to produce it and ‘burns out’. Gestational diabetes develops in some women during the late stages of pregnancy. It is similar to type 2 diabetes in that it involves insulin resistance - the hormones of 1 pregnancy cause insulin resistance in women genetically predisposed to developing this condition. Diabetes can cause many complications. Acute complications such as hypoglycaemia, ketoacidosis or nonketotic hyperosmolar coma may occur if the disease is not adequately controlled. Serious long term complications are caused by chronic hyperglycaemia which damages nerves and blood vessels. This leads to heart disease, stroke, chronic renal failure (diabetic nephropathy is the main cause of end-stage renal disease in developed world adults), peripheral vascular disease, retinal damage leading to blindness, neuropathy, and microvascular damage which may cause erectile dysfunction and poor healing. Peripheral vascular disease, compounded by poor healing of wounds and neuropathy (particularly of the feet), can lead to gangrene which more often than not requires amputation. In fact, diabetes is the leading cause of non-traumatic amputation in adults in the developed world. 1.1.2 Aetiology and Genetics of Diabetes Mellitus The exact aetiology of diabetes mellitus is incompletely understood but it is widely accepted that it is multifactorial and that both genetic and environmental factors play a role. Type 1 Diabetes Mellitus Although type 1 diabetes is not a genetically predestined disease, an increased susceptibility can be inherited. This is evident by the fact that identical 2 (monozygotic) twins have a higher concordance rate (13%-47%) for type 1 diabetes compared to non-identical (dizygotic) twins (0%-8%) [3-5]. Further evidence of genetic involvement is that 95% of type 1 diabetics carry HLA-DR3, HLA-DR4 or both [6]. The most important genes contributing to disease susceptibility are located in the HLA class II locus on the short arm of chromosome 6 [7]. Nevertheless, only a relatively small proportion (95% homology at the genome level [145]. The spontaneous development of type 1 diabetes mellitus has been reported in nonhuman primates [146, 147]. Diabetes has been induced, for controlled experiments, in nonhuman primates with the use of streptozotocin (STZ), alloxan, or total pancreatectomy [146]. STZ has been used the most extensively because it is the least invasive and perhaps the most efficient way to induce diabetes. On the other hand, total pancreatectomy is associated with high surgical morbidity and mortality, and the exocrine pancreatic deficiency that is induced with this method results in reduced absorption and inconsistent drug levels [148]. In nonhuman primates, the administration of a low dose of streptozotocin (30–55 mg/kg) is unreliable because it inconsistently induces C peptide–negative diabetes [149-152]. The use of higher streptozotocin doses (100–150 mg/kg) has been effective in inducing C peptide–negative diabetes but is associated with major systemic side effects and therefore has generally been limited to juvenile primates [153-157]. 24 CHAPTER 2: MATERIALS AND METHODS 2.1 Plasmid Construct Human proinsulin cDNA sequence was modified to encode the HisB10Asp variant and to introduce furin cleavage sites at the B chain / C-peptide and Cpeptide / A chain junctions by PCR-based mutagenesis. The human proinsulin expression was driven by a bifunctional promoter comprised of a 3 kb fragment of the human metallothionein IIA promoter linked to a single copy of the carbohydrate response element (ChoRE). The resulting modified construct, p3MTChins, was used in all experiments. 2.2 Hepatocyte Isolation After partial resection of a liver lobe (see below in section 2.6.4), hepatocyte isolation was performed as described by Bumgardner et al [158] with the following modifications. The resected liver was sequentially perfused in situ with 2.5mM EGTA in calcium-free Dulbecco’s phosphate buffer (prepared with deionized water) and with 0.3% collagenase IV-S (Sigma-Aldrich) dissolved in the same buffer solution (prepared with distilled water) via a catheter placed in the main portal vein branch at the excised liver surface. The perfusion times were 8-10 and 15-20 minutes respectively, at a flow rate of 8-10 ml/min. 25 The perfused liver was then minced and scraped to release the cells. The cells were filtered through a cotton bag into fresh, ice-cold DMEM-high glucose supplemented with 12% foetal calf serum (FCS). A cellular fraction enriched in hepatocytes was obtained as a pellet after centrifugation (150g, 15 min at 4oC) on a discontinuous Percoll (GE Healthcare) gradient (15-30-45-60%). The number of viable cells was determined by trypan blue exclusion. 2.3 Gene Transfer Primary porcine hepatocytes were electroporated with a sterile solution, developed in our own laboratory, having the following composition: 19.8 mM KH2PO4 / 80.2 mM K2HPO4 / 2 mM NaCl, pH 7.6. Freshly prepared 2 mM ATP and 5 mM reduced L-glutathione were added to the solution just before use. Electroporation was performed in the Amaxa NucleofectorTM electroporation device (Amaxa GmbH, Germany). Various program settings were tested. Eight µg of endotoxin-free plasmid DNA (p3MTChins or pEGFP) was added to 4 x 106 viable hepatocytes in 0.2 ml electroporation solution before electrical pulsing. Cells were transferred to DMEM supplemented with 15% FCS (for in vitro characterization) or DMEM without phenol red and FCS (for transplantation in vivo) immediately after electroporation. 26 2.4 In vitro Studies 2.4.1 Induction of Insulin Expression After initial plating of 2 x 106 hepatocytes on collagen I-coated 35 mm dishes, non-adherent and dead cells were removed by medium change 3-5 hours later. The cells were cultured in DMEM-25 mM glucose (supplemented with 10% FCS, penicillin 10,000 units/ml and streptomycin 10 mg/ml) in 5% CO2 at 37°C for at least 16 hours. For static induction, electroporated hepatocytes were cultured in DMEM with increasing concentrations of glucose alone (2.5, 10, 15 and 25 mM) or zinc (5, 10, 20, and 60 µM) combined with 25 mM glucose. Glucose concentrations of 15 and 25 mM represent the conditions found in severe diabetes mellitus. Conditioned media (24 hours) of triplicate plates were assayed for human insulin. For a kinetic study of glucose-responsive insulin production, the culture medium of overnight plated hepatocytes was changed from DMEM-25 mM glucose to DMEM-2.5 mM glucose 3 hours before commencing induction. Replicate plates were then exposed to DMEM-25 mM glucose for 5, 10, and 60 minutes. The baseline time point of 20 minutes before cells were exposed to 25 mM glucose was taken as the unstimulated value. A parallel series of plates, after exposure to DMEM-25 mM glucose for 60 minutes, was returned to DMEM2.5 mM glucose for a further 20 and 60 minutes during the de-induction phase. At each time point during both induction and de-induction phases, each plate (3 plates per time point) was processed for human insulin assay in the conditioned 27 medium and total cellular RNA isolation (RNeasy Fibrous Tissue kit, Qiagen, Germany) according to the manufacturer’s protocol. 2.4.2 Transcriptional Induction of Transgene (RT-PCR) cDNA was generated from the RNA isolated at various time points using SuperScript II Rnase H reverse transcriptase (Invitrogen, USA) and the supplier’s recommended reaction protocol. Each 20 µl reaction contained 10 µl RNA. In all, 1 µl from the reverse transcriptase reaction was used as template for semiquantitative PCR comprising 0.8 µM each of forward and reverse primers, 1.6 mM magnesium chloride, 6.25 µl Quantitect SYBR Green PCR master mix (Qiagen, Germany) and sterile water to a final volume of 12.5 µl. Primer sequences for human insulin mRNA were 5' TTT GTG AAC CAA CAC CTG TGC 3' (forward) and 5' GGT TCA AGG GCT TTA TTC CAT CT 3' (reverse). Intron-spanning primer sequences for primate hypoxanthine phosphoribosyltransferase I (HPRT I) mRNA were 5' GGA TTA CAT CAA AGC ACT GAA TAG 3' (forward) and 5' GGC TTA TAT CCA ACA CTT CGT G 3' (reverse). Thermal cycling conditions (Opticon, MJ Research, USA) were 95oC for 15 minutes, followed by 49 cycles of 95oC x 30 seconds / 68oC x 30 seconds / 72oC x 20 seconds / 75oC x 1 second. Melting curves (65-95oC) confirmed amplification of a single product in all reactions. Data were analyzed using the Opticon Monitor Analysis software, version 1.07 (MJ Research, USA). Differences in input RNA amounts were normalized using Ct values obtained in parallel reactions for primate hypoxanthine 28 phosphoribosyltransferase (HPRT1) mRNA and spurious amplifications were corrected by minus RT controls. Corrected data are expressed as fold change of gene-specific mRNAs over basal mRNA. Human and primate insulins, HPRT1, and the respective minus RT controls were measured in triplicate samples. Each sample was assayed in quadruplicate RT-PCR reactions. 2.5 Hormone Radioimmunoassay Human insulin concentrations were determined using radioimmunoassay and the supplier’s protocols (Linco Research, USA) 2.6 Animal (in vivo) Studies Cynomolgus macaques (Macaca fascicularis) supplied by the SGH Animal Husbandry and Hospital, Sembawang, Singapore were used in our study. All animal handling procedures and animal husbandry were conducted in an AAALAC-accredited facility. The experimental protocol was approved by the Institutional Animal Care and Use Committee of the Singapore General Hospital. We used male and female macaques weighing 2.5 – 5.0 kg. They were fed twice daily (10 pellets of chow at 0900 hours and 8 pellets of chow and 1 fruit at 1530 hours) and allowed water ad libitum. 2.6.1 Sedation and Anaesthesia Intramuscular Ketamine (25 mg/kg) injections were used to sedate the animals for blood withdrawals and streptozotocin administration. For longer 29 procedures (intravenous glucose tolerance test) and surgical procedures, anaesthesia was maintained with isoflurane (2-3%) inhalation. 2.6.2 Blood Glucose Monitoring and Venepucture Capillary blood glucose was obtained by lancing either fingers, toes or the abdominal wall and measured with a glucometer (Ascensia ELITE®, Bayer Healthcare, Germany) in awake animals. Blood was drawn periodically, via venepuncture of the femoral vein under sedation, for measurements of human insulin, serum electrolytes, lipids (triglycerides and total cholesterol), biochemical tests of liver function (total protein, albumin, total bilirubin, alkaline phosphatase, serum alanine transaminase (ALT) and γ-glutamyl transferase (γGT) activities), renal function (urea and creatinine) and full blood counts (total and differential leucocyte counts, haemoglobin concentration, total red blood cell count and erythrocyte indices, and platelet count). 2.6.3 Intravenous Glucose Tolerance Test (IVGTT) We performed IVGTT on animals at three time points during the study – prediabetic (before streptozotocin injection), diabetic (after streptozotocin injection), and post-hepatocyte transplant. Animals were fasted overnight and anaesthetized. Two intravenous catheters were inserted: one into the saphenous vein for saline and glucose administration and one into the contralateral femoral vein for blood sampling. 30 To establish the baseline, a bolus injection of saline (equal to the volume of glucose solution) was administered over 3 minutes and blood was drawn 5 and 10 minutes later for blood glucose levels and plasma human insulin assays. IVGTT was initiated by a bolus injection of glucose (0.5 g/kg as a 25% w/v solution) over 3 minutes followed by a second saline (2 ml) injection to flush the line. One ml of blood was drawn 1, 3, 5, 10, 20, 30, 45 and 60 minutes after the intravenous glucose bolus for the same assays. In order to avoid possible artefacts caused by hypovolemia, one ml heparinized saline was injected after each blood sampling. Area under the curves (AUC) of glucose and insulin were calculated using the Trapezoidal Rule. The K-value or elimination rate constant of the curves was calculated by the formula: 0.693/half life. 2.6.4 Induction of Diabetes After hydration with subcutaneous normal saline (1 ml/kg/hr) for 16 hours the day before, intravenous streptozotocin (65-120 mg/kg, Zanosar, Pfizer) was administered as a bolus injection over 5 minutes via a femoral vein catheter under general anaesthesia. Hydration was continued for 16 hours after the streptozotocin injection with subcutaneous normal saline (1 ml/kg/hr). Exogenous insulin injections (Lantus, Sanofi Aventis; Actrapid, Novo Nordisk) were administered to diabetic monkeys if severely hyperglycaemic to avoid ketoacidosis. 31 2.6.5 Liver Resection and Hepatocyte Transplantation Under general anaesthesia, the abdominal cavity was entered through a left subcostal incision and the left liver lobe was mobilized. A non-crushing clamp was applied across the lobe and 30-50% of the left lobe was resected en masse (less than 13% of the total liver volume). The main portal vein branch was immediately cannulated and hepatocyte isolation and electroporation was performed as described above. Haemostasis of the remnant liver was achieved with 5/0 Prolene (Ethicon, USA) sutures and diathermy. Surgicel (Johnson & Johnson) was applied to the resected surface. Transfected hepatocytes in suspension were injected under direct vision into four sites (2 sites each in the right central and left central lobes) using a 23G needle (1-1.5 ml/site) at a depth of 5 to 10mm. The number of hepatocytes transplanted into each animal is reflected in Table 2 (page 50). Transplantation sites were marked with a 5/0 Prolene suture. The abdominal incision was closed using 3/0 Ethilon and the skin with 5/0 Vicryl (both Ethicon, USA). Post-operatively, antibiotic cover with enrofloxacin (5 mg/kg IM) or coamoxiclav (11 mg/kg IM) was given for 5 days. Buprenorphine (0.01 mg/kg IM) was also given for 5 days as analgesia. 2.7 Statistical Analysis Data were expressed as mean ± s.e.m. Statistical significance was determined by Student’s unpaired t test for independent samples. The difference between two groups was considered significant when p < 0.05. 32 CHAPTER 3: RESULTS 3.1 Electroporation of Primary Hepatocytes Transfection of primary hepatocytes was achieved using the Amaxa Nucleofector TM system apparatus and a sterile solution developed in our laboratory. We initially used various program settings on the Amaxa apparatus to transfect the cells with a reporter plasmid expressing green fluorescent protein (pEGFP) to assess transfection efficiency and cell viability (Figure 2). Program A23 was associated with the highest viability but low transfection efficiency with the hepatocytes producing low levels of human insulin. On the other hand, program T20 had the highest efficiency but with moderate 70% viability. We settled on program H22 for all future electroporation in the study as it provided acceptable cell viability with reasonable transfection efficiency (Figure 2). 33 Electroporated Primary Hepatocytes 60 100 Human insulin (pM) Viability (%) 50 40 60 30 40 20 20 10 0 0 H22 (a) H22 (b) T01 T27 T16 T20 G11 A23 A27 A10 O17 Program Figure 2. Electroporation of primary primate hepatocytes Electroporation of isolated primary primate hepatocytes was performed using the Amaxa Nucleofector system. Various program settings were tested with a reporter plasmid expressing green fluorescent protein (pEGFP) to assess transfection efficiency and cell viability. Program H22 provided a combination of acceptable cell viability with reasonable transfection efficiency and was used in all further electroporations. 34 Viability (%) Human insulin (pM) 80 3.2 In vitro Studies 3.2.1 Static Induction of Human Insulin by Glucose and Zinc Hepatocytes electroporated with p3MTChins were tested for glucose and zinc inducibility of the bifunctional promoter. Levels of human insulin secretion were measured in hepatocytes exposed to increasing glucose concentrations (2.5, 10, 15 and 25 mM) (Figure 3). Increasing ambient glucose concentration from 2.5 mM to 25 mM glucose increased insulin secretion by 3-fold (53.1±1.6 pM vs. 166.9±7.5 pM, p=0.006). 35 200 180 160 Human insulin (pM) 140 120 100 80 60 40 20 0 0 5 10 15 20 25 30 Glucose (mM) Figure 3. Glucose-induced insulin secretion Human insulin concentrations in 24h conditioned media of p3MTChinselectroporated hepatocytes cultured in increasing glucose concentrations (2.5, 10, 15 and 25mM). Data are the mean and s.e.m. of triplicate experiments Similarly, hepatocytes were exposed to increasing levels of zinc (5, 10, 20 and 60 µM), in the presence of 25 mM glucose, to measure the levels of insulin secreted (Figure 4). Like glucose stimulation, increasing zinc concentrations increased insulin secretion from transfected hepatocytes with 60 µM zinc producing a 2.3-fold increase in insulin secretion compared to 5 µM zinc (409.2±13.5 pM vs. 174.2±5.2 pM, p=0.004). 36 450 400 Human insulin (pM) 350 300 with 25m M glucose 250 200 150 100 50 0 0 10 20 30 40 50 60 70 Zinc (uM) Figure 4. Zinc-induced insulin secretion Human insulin concentrations in 24h conditioned media of p3MTChinselectroporated hepatocytes cultured in increasing zinc concentrations (5, 10, 20 and 60uM). Data are the mean and s.e.m. of triplicate experiments 37 3.2.2 Kinetics of Glucose-induced Insulin Secretion We characterized the kinetics of inducible insulin secretion of primary hepatocytes transfected with p3MTChins by measuring the amount of insulin secreted at various time points (5, 10 and 60 minutes) after exposure to 25 mM glucose. De-induction was measured in a parallel series of plates that was returned to 2.5 mM glucose after 60 minutes exposure to 25 mM glucose. Insulin secretion was measured at 20 and 60 minute time points in the de-induction plates (Figure 5). On exposure to the 25 mM glucose stimulus, insulin secretion by the hepatocytes rose and by 60 minutes, the insulin concentration was 4-fold above baseline (defined as the mean insulin concentration 20 minutes before exposure to 25 mM glucose) (145.3±10.7 pM vs. 34.6±2.7 pM, p=0.003). When returned to 2.5 mM glucose, insulin secretion by the hepatocytes declined such that the insulin concentration in the medium 60 minutes after de-induction was just above baseline. 38 180 160 2.5mM glucose140 25mM glucose 2.5mM glucose Human insulin (pM) 120 100 80 60 40 20 0 -40 -20 0 20 40 60 80 100 120 140 Time (min) Figure 5. Kinetics of glucose-induced insulin secretion Induction of insulin secretion in hepatocytes electroporated with p3MTChins by 25 mM glucose for 60min followed by downregulation in 2.5 mM glucose for a further 60min. Values are mean and s.e.m. of quadruplicate experiments 39 3.2.3 Transcriptional Response of Transgene To determine the transcriptional response of human insulin in p3MTChins to glucose stimulation, transgenic mRNA was assayed by RT-PCR at the same time points in the kinetic study above. Figure 6 shows the increase in insulin mRNA levels to be concordant with the kinetic study of insulin secretion in Figure 5. By 10 minutes after exposure to 25 mM glucose, the amount of insulin transcripts had risen by 1.6-fold over baseline. At 60 minutes after returning to 2.5 mM glucose, insulin mRNA levels had again decreased to almost baseline. 40 Human insulin transcript (arbitrary units) 3 2.5 2 changed to 25mM glucose changed to 2.5mM glucose 1.5 1 2.5mM glucose 25mM glucose 2.5mM glucose 0.5 0 -40 -20 0 20 40 60 80 100 120 140 Time (min) Figure 6. Kinetics of glucose-induced insulin mRNA expression Induction of human insulin transcription in electroporated hepatocytes by 25 mM glucose followed by downregulation in 2.5 mM glucose. Insulin RNA was quantified by real time RT-PCR after subtracting a parallel minus-RT control and normalizing to HPRT1 mRNA. Data are mean and s.e.m. of quadruplicate experiments 41 3.3 Animal Studies 3.3.1 Induction of Diabetes During the planning stages of this pilot study, the literature on inducing diabetes with streptozotocin in non-human primates, in particular cynomolgus monkeys, was reviewed. It became evident that a wide range of doses of streptozotocin had been used with varied success in establishing diabetes. This had to be balanced against the development of associated serious adverse effects (nephrotoxicity and hepatotoxicity) that also occurred with considerable variability. Stegall et al used a streptozotocin dose of 50 mg/kg body weight and reported 3 deaths from acute renal failure [151], while Litwak et al reported no deaths when using a dose of 30 mg/kg [152]. It should be noted that some monkeys required a second dose, and occasionally even a third dose, in order to develop diabetes in both these studies. Then in 1999, Theriault et al employed a single dose of 150 mg/kg and achieved a 100% success rate in inducing diabetes [153]. There was transient biochemical evidence of liver and renal dysfunction (raised AST, ALT and urea) but no deaths among the monkeys. In an attempt to determine the optimal dose of streptozotocin that would cause diabetes with minimal toxicity in cynomolgus monkeys, Koulmanda et al compared 55 mg/kg (low dose) versus 100 mg/kg (high dose) of streptozotocin [155]. In both groups, all monkeys became diabetic, some up to one year. However, the high-dose animals developed elevated liver function tests, urea 42 and creatinine, with steatosis of the liver and tubular injury in the kidneys evident on histology. The low-dose animals did not develop any adverse effects and they concluded that a low dose of 55 mg/kg was optimal. The following year, Wijkstrom and co-workers demonstrated that an even higher dose of 150 mg/kg could be used to induce diabetes without causing any deaths among the monkeys, albeit biochemical derangement of liver and renal function was again evident [156]. What was more impressive was that the monkeys could go on to receive immunosuppression without adverse effects. The same group then recommended administering streptozotocin based on body surface area (BSA) instead of weight to remove the confounding factor of age [157]. The dose was 1250 mg/m2 where BSA was calculated by: body weight0.67x12/10,000. Hence, it was not possible to develop a standard protocol for streptozotocin-induced diabetes in cynomolgus monkeys owing to the widely variable results of the above studies. What we could conclude was: a) either sex of animal could be used, b) a body weight of less than 3.5 kg is preferable, c) younger monkeys were better at tolerating higher streptozotocin doses, and d) hydration pre- and post-streptozotocin administration was important. The variability in diabetogenic action of streptozotocin and narrow window before the development of toxicity in monkeys was probably due to the genetic and ecologic diversity of the animals [149]. Our experience with the use of streptozotocin to induce diabetes in the course of this study is summarized in the following table: 43 Table 1. Summary of sex, streptozotocin dose administered and resultant outcome of all monkeys in the study Animal ID DM1 Sex Streptozotocin dose Outcome M DM2 F DM3 M 85/mg/kg body weight DM4/7 F 80.5/mg/kg body weight 1250 mg/m2 = 118.3 mg/kg body weight 1250 mg/m2 = 115.1 mg/kg body weight Diabetes successfully induced Died after complications of anaesthesia Diabetes was induced. However, biochemical evidence of renal failure appeared 4 days after streptozotocin. Placed in intensive care and full supportive care given: treated with subcutaneous insulin injections and hydration, sodium bicarbonate infusion to correct acidosis, intravenous antibiotics, tube feeding and intravenous furosemide. Generalised seizure on 6th day after streptozotocin. Euthanasia was advised on the 7th day because of significant neurological deterioration. Autopsy revealed florid acute tubular necrosis. Developed diabetes. Received autologous hepatocyte transplantation resulting in partial improvement in hyperglycemia for 65 days. Thereafter, subcutaneous insulin injections were instituted in an attempt to achieve body weight gain that was ultimately unsuccessful. Euthanasia was advised on the 138th experimental day when body weight fell below 70% of its starting body weight (which was approximately 80% of its body weight after streptozotocin administration.) Autopsy showed emaciation with significant reduction in body fat and no other notable pathology in the organs examined. Did not develop diabetes. 44 DM5 M 85.6/mg/kg body weight DM6 F DM8 M 85 mg/kg body weight 73 mg/kg body weight DM9 M 68 mg/kg body weight DM10 M 70 mg/kg body weight DM11 M 70 mg/kg body weight Developed diabetes. Biochemical evidence of hepatorenal toxicity 8 days after streptozotocin prompted daily hydration with subcutaneous physiological saline (1 mg/kg body weight/hour) and daily subcutaneous insulin injections to control hyperglycemia. Found dead in its cage 12 days after streptozotocin administration. Autopsy findings were consistent with acute renal tubular necrosis, hepatic steatosis and interstitial pneumonia. There were no other diagnostic pathological findings. Did not develop diabetes. Developed diabetes. This animal is assigned to the untreated control group of the study Clinically well. Developed diabetes. This animal has been assigned to the treatment group of the study. Clinically well. Developed diabetes. This animal is assigned to the untreated control group of the study Clinically well. Developed mild diabetes. This animal has been assigned to the treatment group of the study. Clinically well. The first two monkeys, DM1 and DM2, received a streptozotocin dose based on their body surface area as described by Wijkstrom et al [157] (see above). This translated to 115–118 mg/kg. Unfortunately, DM2 developed acute renal failure secondary to acute tubular necrosis and deteriorated despite full supportive therapy necessitating euthanasia. In view of this, the streptozotocin dose given was reduced to 80-85% of the full dose that was calculated (based on the body surface area) for the next 4 monkeys. This translated to 80-85 mg/kg. DM3 successfully became diabetic and 45 went on to undergo autologous hepatocyte transplantation that partially corrected the severe hyperglycaemia. However, it became emaciated which ultimately required euthanasia. Autopsy did not reveal any specific pathology. DM5 also became diabetic but developed biochemical evidence of hepatorenal toxicity. It was found dead in its cage 12 days later probably due to acute renal failure. DM4/7 and DM6 did not develop diabetes despite receiving 2 doses of streptozotocin each. Interestingly, both animals were female. They did not undergo further procedures and were returned to the husbandry unit. Faced with the severe hyperglycaemia that developed in DM3 and the death of DM5 from renal failure, the streptozotocin dose was reduced further in the subsequent 4 monkeys and only males were used. DM8, DM9, DM10 and DM11 received a dose of 70% of that calculated by their body surface area. They all became diabetic, without renal failure, and all proceeded to autologous hepatocyte transplantation as the study proper. Based on this initial experience with streptozocin-induced diabetes in cynomolgus monkeys, we can conclude that individual monkeys have varying susceptibility to beta-cell ablation by streptozotocin. The dose to induce moderate hyperglycaemia is in the range of 65-80 mg/kg body weight. Female monkeys appear to be more resistant to the diabetogenic effect of streptozotocin than their male counterparts. Also, some degree of hepatotoxicity and nephrotoxicity is probably unavoidable. Nevertheless, we were able to induce diabetes in 8 out of 10 monkeys. The average values of the intravenous glucose tolerance tests of these 8 animals 46 before and after streptozotocin treatment are shown in Figure 7. Poststreptozotocin, the monkeys had higher glucose levels when fasted and throughout the glucose tolerance tests. The rate of glucose clearance in these diabetic monkeys was also lower with a K-value of 0.4 compared to 1.36 before streptozocin treatment. 800 700 600 BG (mg/dl) 500 pre-STZ 400 post-STZ 300 200 100 0 -20 -10 0 10 20 30 40 50 60 70 Minutes Figure 7. Induction of diabetes with streptozotocin Intravenous glucose tolerance tests were performed before and after STZ treatment. After an overnight fast, 0.5 g/kg glucose was administered and blood sampled at 1, 3, 5, 10, 20, 30, 45 and 60 minutes later for blood glucose levels. Data are mean and s.e.m. of 8 monkeys 47 Further evidence of successful islet ablation was provided by histological assessment of pancreata removed at autopsy. In the micrographs below, the pancreas of diabetic monkeys DM8 and DM10 showed absence of insulinpositive cells in the islets while in the pancreas of DM4, which did not become diabetic, insulin-positive cells are present in the normal islets (Figure 8). 48 DM4 Not diabetic DM8 Diabetic DM10 Diabetic Figure 8. Absence of insulin-positive cells in pancreas of diabetic monkeys Pancreata harvested at autopsy were stained for insulin. Non-diabetic monkey DM4 had insulin-positive cells (arrowed) in the islets while diabetic monkeys DM8 and DM10 did not. 49 3.3.2 Hepatocyte Transplantation In the study proper, two monkeys, DM8 and DM10, were transplanted with autologous hepatocytes that were mock electroporated to serve as controls, while another two monkeys, DM9 and DM11, were transplanted with autologous hepatocytes electroporated with p3MTChins as the treated group. The characteristics of these 4 subject monkeys are summarized in the table below: Table 2. Characteristics of 4 diabetic monkeys that underwent liver resection and hepatocyte transplantation Monkey Sex Weight STZ Liver (kg) dose resection (mg/kg) Number of cells Arm transplanted DM8 M 3.95 73 30% left lobe 20 million Control DM9 M 4.63 68 50% left lobe 29 million Treated DM10 M 4.77 70 30% left lobe 18 million Control DM11 M 5.00 70 40% left lobe 19 million Treated The outcome of hepatocyte transplantation was assessed by daily fasting blood glucose levels and insulin requirements, intravenous glucose tolerance tests, and glycated haemoglobin levels. 50 3.3.2.1 Blood Glucose Levels and Insulin Requirements Fasting blood glucose levels were measured in the morning before feeding the animals. Exogenous insulin was administered if the blood glucose levels were high (>300mg/dl) to avoid ketoacidosis developing. The figures below summarize the blood glucose readings of the individual animals. 51 Controls: DM8 Daily fasting blood glucose 800 Average post-transplant daily insulin dose = 7.3 units Transplant 700 600 mg/dl 500 400 300 200 100 STZ 0 0 20 40 60 80 120 100 Day Figure 9a DM10 Daily fasting blood glucose Transplant 700 600 mg/dl 500 400 300 STZ 200 Average post-transplant daily insulin dose = 6.9 units 100 0 0 10 20 30 40 50 60 70 80 Day Figure 9b 52 Treated: DM9 Daily fasting blood glucose 500 Average post-transplant daily insulin dose = 0 units 450 Transplant 400 350 mg/dl 300 250 200 150 100 50 0 0 STZ 50 100 150 200 Day Figure 9c DM11 Daily fasting blood glucose 300 250 Transplant mg/dl 200 150 100 Average post-transplant daily insulin dose = 0 units STZ 50 0 0 20 40 60 80 100 120 140 Day Figure 9d Figure 9a-d. Daily fasting blood glucose levels of 4 monkeys in study Capillary blood glucose was measured in the morning before feeding by lancing the digits or abdominal wall 53 Overall, no trend can be found in these measurements but it should be noted that in the treated animals, the fasting blood glucose levels never exceeded 500 mg/dl despite no insulin being administered during the posttransplant period. However, DM11 was only mildly diabetic before the transplant. This partial correction in hyperglycaemia in DM9 was present for 113 days until the study was terminated. However, there was a slow deterioration in the effect. In contrast, the control group, DM8 and DM10, had blood glucose levels occasionally exceeding 500 mg/dl, even though the average daily insulin requirements post-transplant were 7.3 and 6.9 units respectively. 3.3.2.2 Intravenous Glucose Tolerance Tests (IVGTT) In order to better assess the outcome of hepatocyte transplantation, IVGTTs were performed on the animals at 3 stages during the study, namely normal (pre-STZ), diabetic (post-STZ), and post-transplant. After an overnight fast, a glucose load of 0.5 g/kg was administered to the animals intravenously. Blood was then sampled at 1, 3, 5, 10, 20, 30, 45 and 60 minutes thereafter to measure glucose and insulin levels. The results are shown in the figures below. 54 Controls: IVGTT and blood glucose DM8 IVGTT 800 Blood glucose (mg/dl) 700 600 500 Normal 400 Diabetic (post-STZ) 300 Post-transplant 200 100 0 -20 0 20 40 60 80 M inute s Figure 10a DM10 IVGTT 900 Blood glucose (mg/dl) 800 700 600 Normal 500 Diabetic (post-STZ) 400 Post-transplant 300 200 100 0 -20 0 20 40 60 80 M inute s Figure 10b 55 Treated: IVGTT and blood glucose DM9 IVGTT 600 Blood glucose (mg/dl) 500 400 Normal 300 Diabetic (post-STZ) Post-transplant 200 100 0 -20 0 20 40 60 80 M inute s Figure 10c DM11 IVGTT 500 Blood glucose (mg/dl) 450 400 350 300 Normal 250 Diabetic (post-STZ) 200 Post-transplant 150 100 50 0 -20 0 20 40 60 80 M inute s Figure 10d Figure 10a-d. Blood glucose IVGTT curves of all 4 monkeys in study Intravenous glucose tolerance tests were performed pre-STZ, post-STZ and post-hepatocyte transplant. After an overnight fast, 0.5 g/kg glucose was administered and blood sampled at 1, 3, 5, 10, 20, 30, 45 and 60 minutes later for blood glucose levels. 56 Blood glucose AUC 10-60mins 40000 35000 mg/dl.50min 30000 25000 Normal 20000 Diabetic (post-STZ) Post-transplant 15000 10000 5000 0 DM8 DM10 Controls DM9 DM11 Tre ate d Figure 11. Area under the curve for blood glucose IVGTT curves IVGTT was performed at normal (pre-STZ), diabetic (post-STZ) and posttransplant stages during the study. AUC was calculated with the Trapezoidal Rule In both controls, the post-transplant IVGTT curves followed that of the diabetic (pre-transplant) state which confirmed the mock electroporated hepatocytes status and islet ablation by streptozotocin. In the treated group, DM9 showed a post-transplant IVGTT curve which returned to the normal (pre-diabetic state) suggesting the hepatocytes electroporated with p3MTChins were able to function as surrogate islets to a certain extent. However, this was not seen in DM11 as it was only rendered mildly diabetic and all 3 IVGTT curves were the same. Further evidence came from calculating the area under curve (AUC) for the IVGTT curves, as shown above, where the controls had higher glucose AUCs 57 post-transplant compared to the diabetic period, while the treated animals had lower post-transplant glucose AUCs. Controls: IVGTT and insulin DM8 IVGTT 1200 1000 Insulin (pM) 800 Normal Diabetic (post-STZ) 600 Post-transplant 400 200 0 -20 0 20 40 60 80 Minutes Figure 12a DM 10 IVGTT Insulin (pM) 1800 1600 1400 1200 1000 800 600 400 200 0 -20 Normal Diabetic (post-STZ) Post-transplant 0 20 40 60 80 Minutes Figure 12b 58 Treated: IVGTT and insulin DM9 IVGTT 700 600 Insulin (pM) 500 Normal 400 Diabetic (post-STZ) 300 Post-transplant 200 100 0 -20 0 20 40 60 80 Minutes Figure 12c DM11 IVGTT 800 700 Insulin (pM) 600 500 Normal 400 Diabetic (post-STZ) Post-transplant 300 200 100 0 -20 0 20 40 60 80 Minutes Figure 12d Figure 12a-d. Insulin IVGTT curves of all 4 monkeys in study Intravenous glucose tolerance tests were performed pre-STZ, post-STZ and post-hepatocyte transplant. After an overnight fast, 0.5 g/kg glucose was administered and blood sampled at 1, 3, 5, 10, 20, 30, 45 and 60 minutes later for insulin levels. 59 Insulin released AUC 10-60mins 60000 50000 pM.50mins 40000 Normal Diabetic (post-STZ) 30000 Post-transplant 20000 10000 0 DM8 DM10 Controls DM9 DM11 Treated Figure 13. Area under the curve for insulin IVGTT curves IVGTT was performed at normal (pre-STZ), diabetic (post-STZ) and posttransplant stages during the study. AUC was calculated with the Trapezoidal Rule In terms of insulin release during the IVGTT, again the controls had posttransplant curves similar to that of the diabetic state. This goes to show again that neither the mock electroporated hepatocytes nor the native islets were producing insulin. As for the 2 treated animals, both showed an increase in insulin release during the IVGTT post-transplant compared to the diabetic phase, although it was not as high as the normal non-diabetic phase. This provides more evidence that the treated hepatocytes were able to respond to a change in ambient glucose levels and secrete insulin. 60 The AUC for insulin release during the IVGTTs confirms that the treated animals had higher insulin levels, in response to the glucose load, post-transplant compared to the diabetic state. In contrast, this was not seen in the controls. 3.3.3 Liver and Renal Function In all 4 animals, blood was sampled at regular intervals to conduct biochemical tests of liver and renal function. The results are displayed below: 61 Controls: Liver function DM8 Liver Function 600 160 140 500 120 100 300 80 STZ ALT (u/L) Alk Phos (u/L) Transplant 400 Alk Phos ALT 60 200 40 100 20 0 0 1 51 101 Day Figure 14a DM10 Liver function 1200 350 300 1000 Transplant 200 600 150 400 STZ 200 ALT (u/L) Alk Phos (u/L) 250 800 Alk Phos ALT 100 50 0 0 1 51 Day Figure 14b 62 Treated: Liver function DM9 Liver Function 80 600 70 500 STZ 60 50 40 300 ALT (u/L) ALk Phos (u/L) Trans plant 400 Alk Phos ALT 30 200 20 100 10 0 0 1 51 101 151 Day Figure 14c DM11 Liver Function 300 90 80 STZ 250 Transplant 70 60 50 150 40 100 ALT (u/L) Alk Phos (u/L) 200 Alk Phos ALT 30 20 50 10 0 0 1 51 101 Day Figure 14d Figure 14a-d. Liver function tests of 4 study animals Blood was sampled at regular intervals for measurement of liver function. ALT – alanine aminotransferase, Alk Phos – alkaline phosphatase 63 Controls: Renal function DM8 Renal Function 250 18 16 Transplant 14 12 STZ 150 10 8 100 6 Urea (mmol/L) Creat (umol/L) 200 Creat Urea 4 50 2 0 0 1 51 101 Day Figure 15a DM10 Renal Function 140 25 Transplant 120 20 100 15 80 60 10 Urea (mmol/L) Creat (umol/L) STZ Creat Urea 40 5 20 0 0 1 51 Day Figure 15b 64 Treated: Renal function DM9 Renal Function 140 12 120 STZ 8 80 6 60 4 Urea (mmol/L) 100 Creat (umol/L) 10 Transplant Creat Urea 40 2 20 0 0 1 51 101 151 Day Figure 15c DM11 Renal Function 80 7 70 6 5 50 4 40 3 Transplant 30 20 Urea (mmol/L) Creat (umol/L) 60 Creat Urea 2 STZ 1 10 0 0 1 51 101 Day Figure 15d Figure 15a-d. Renal function tests of all 4 study monkeys Blood was sampled at regular intervals for assessment of renal function. Creat – creatinine. 65 In the controls, liver AST and alkaline phospatase rose post-STZ treatment, this was not seen in the treated group. Following liver resection and hepatocyte transplantation, the AST and alkaline phosphatase in both groups increased further which was to be expected. However, fulminant liver failure, with grossly elevated liver enzymes and associated jaundice, anasarca, bleeding diathesis and encephalopathy, never developed in any of the animals. Renal function, in terms of blood urea and creatinine levels, worsened after streptozotocin exposure in all animals reflecting its nephrotoxic effects. However, in 3 out of 4 animals, namely DM8, DM9 and DM11, their renal function continued to slowly deteriorate after hepatocyte transplant. This may have been caused by the nephropathy associated with the diabetic state. 66 CHAPTER 4: DISCUSSION 4.1 Overview The objectives of this project were: 1) to optimize a method of harvesting, isolating and transfecting primary primate hepatocytes with a plasmid construct encoding human proinsulin cDNA, 2) to develop a reliable protocol to induce diabetes in monkeys using streptozotocin, and 3) to compare engraftment and functional effects of transplanted engineered autologous hepatocytes in streptozotocin-diabetic monkeys. For the first objective, a technique that minimized warm ischaemia time was employed successfully to partially resect a liver lobe in anaesthetized monkeys for hepatocyte harvest. Subsequently, hepatocyte isolation was carried out using a modified Bumgardner protocol which produced good viability. The Amaxa Nucleofector system apparatus was used to transfect the isolated primary hepatocytes obtained. As there was no recommended program setting available for primate hepatocytes, various settings were assessed with a reporter plasmid and program H22 was found to produce reasonable transfection efficiency with acceptable cell viability (Figure 2) and was used for all future transfections. Our in vitro results demonstrate successful transfection of primary primate hepatocytes with the plasmid construct p3MTChins. In static studies, inducibility of the bifunctional promoter of the plasmid construct by increasing glucose and zinc concentrations was evidenced by a corresponding increase in insulin secretion. Subsequent kinetic studies showed that both insulin mRNA expression 67 and insulin secretion were increased with exposure to high glucose concentrations and then decreased when glucose concentrations were reduced. Hence, the engineered hepatocytes were able to produce insulin in a regulated manner. After a trial of high dose streptozotocin, we settled on a dose of 875 mg/m2 where BSA was calculated by: body weight0.67x12/10,000. This dose was found to reliably induce diabetes in male monkeys with only transient biochemical derangement of liver and renal function. Intravenous glucose tolerance tests before and after streptozotocin treatment confirmed the development of diabetes in the 8 animals successfully induced. They had higher blood glucose levels throughout the tests post-streptozotocin and a lower K-value of glucose clearance. Histology showed absence of insulin staining in pancreata of diabetic monkeys. As regards the third objective, our animal study proper on 4 monkeys showed that the treated animals had overall lower fasting blood glucose levels and did not require exogenous insulin therapy to achieve this compared to the controls. Intravenous glucose tolerance tests at various stages of the study showed the treated animals having post-transplant curves of blood glucose similar to that of the non-diabetic state and post-transplant curves of insulin higher than that of the diabetic state. AUC levels of glucose were reduced and insulin increased post-transplant in the treated animals but the reverse was seen in the controls. 68 4.2 Isolation and Electroporation of Primary Primate Hepatocytes Autologous hepatocytes present a renewable source for a cell-based approach to diabetes treatment. In our approach, less than 13% of the total volume of the liver was resected for hepatocyte isolation, and this is within the regenerative capacity of the liver [124]. There was only transient biochemical evidence of liver dysfunction and no progression to liver failure. This overcomes the problem of donor scarcity as seen in allogeneic islet transplantation. Furthermore, using autologous hepatocytes obviates the need for long term immunosuppression and they are also not targets for recurrent autoimmune destruction [125] or increased beta cell apoptosis [159] of types 1 and 2 diabetes, respectively. We chose to use electroporation to transfect the isolated hepatocytes with a nonviral vector of human proinsulin cDNA for several reasons: it is rapid, simple, and produced reasonable efficiency. Isolation and transfection of autologous hepatocytes was performed in a reasonably short turnaround time of 2-3 hours which allowed us to transplant the engineered hepatocytes in the same period of general anaesthesia as the liver resection. This would prove more clinically acceptable as it would mean a single procedure and reduced anaesthetic exposure to the patient. As we did not perform ex vivo viral transduction and culture of primary hepatocytes, we avoided the attendant risks of producing chromosomal aberrations [160, 161] or co-implanting free viral particles with adverse immunogenic or genotoxic effects resulting in oncogenicity. In addition, plasmid 69 vectors are cheaper to produce, have low toxicity and immunogenicity, and are non-pathogenic compared to viral vectors. Although, in our study, we did not quantitate average plasmid copy number in transfected hepatocytes, others have reported on the transfer of vectors in high copy number (up to 800) by electroporation [162]. Furthermore, durable retention of plasmid vectors by nonmitotic hepatocytes [163] would sustain the therapeutic effects of the engineered hepatocytes and, again, make this approach more clinically acceptable. 4.3 Preclinical Model of Diabetes Mellitus We elected to assess our approach in a primate diabetes model as nonhuman primates have evolutionary proximity to humans and exhibit >95% homology at the genome level [145]. Previous attempts at gene- and cell-based diabetes treatment that were effective in murine models have not been successfully adapted to large animals. We felt utilizing a primate model would move our findings a step closer to clinical application. Induction of diabetes in cynomolgus macaques with streptozotocin was not that straightforward. Success with high dose streptozotocin reported by others [156, 157] was not reproducible in our study. We had to reduce the dose of streptozotocin to 70% of that described and this reliably induced diabetes with only transient derangement in liver and renal function. A likely explanation for the variability in susceptibility to streptozotocin and the development of toxic effects is that nonhuman primates tend to be outbred and wild-caught unlike commonly used species of small laboratory animals. They 70 are thus genetically diverse and disparate in terms of body weight, diet and general health [149]. 4.4 Engraftment and Function of Transplanted Engineered Hepatocytes We chose to transplant engineered hepatocytes directly into the liver parenchyma instead of infusion via the portal vein. Portal vein-infused hepatocytes are prone to destruction by blood macrophages [138] and often also lead to intraportal thrombosis and pulmonary embolism with infarction [134]. In contrast, hepatocytes implanted directly into the liver parenchyma did not distribute to other organs and integrated normally into liver lobules [140]. Therapeutic efficacy of transplanted hepatocytes was demonstrated by the overall lower fasting blood glucose levels in treated monkeys and without the need for exogenous insulin therapy to maintain it. More evidence came from challenge tests in the form of intravenous glucose tolerance tests - the treated animals having lower blood glucose and higher secreted insulin levels. Our attempts to immunostain liver sections from transplanted sites of treated animals using an antibody against human insulin were unsatisfactory because of the known cross-reactivity between human and monkey insulin. We were unable to locate a source of anti-human C-peptide antibody that might have provided clear evidence of transgenic insulin secretion in implanted hepatocytes. However, haematoxylin and eosin-stained sections of the transplanted sites showed entirely normal liver lobular architecture without evidence of fibrosis or inflammation. 71 4.5 Limitations of Current Study Being a pilot study, the number of animals used was too small to demonstrate any statistical significance and the follow-up period was too short to assess long term engraftment and function. Nevertheless, this project was able to demonstrate proof of concept in using engineered autologous hepatocytes as cell-based treatment for diabetes mellitus in a preclinical model. Obtaining hepatocytes by laparotomy and partial liver resection may be deemed too invasive by some. Laparoscopic resection may be a way of overcoming this. Optimising hepatocyte isolation further will allow for a smaller wedge of the liver to be resected. 4.6 Future Work Having demonstrated proof of concept, the next step would be to repeat the study in a larger cohort of monkeys. Varying the number of cells transplanted and assessing their therapeutic responses should also be studied in order to establish a treatment protocol. A longer period of follow-up and measurement of target organ injury in terms of retinopathy, nephropathy and neuropathy would also be ideal in the larger study to extend the assessment of the therapeutic efficacy of the transplants beyond just metabolic correction. 72 4.7 Conclusions • A moderate streptozotocin dose reliably induces diabetes in cynomolgus macaques with transient biochemical evidence of liver and renal dysfunction. • Autologous hepatocytes transfected with a human proinsulin cDNA construct are able to behave as surrogate beta cells in the treatment of a preclinical model of diabetes. 73 REFERENCES 1. WHO, Definition, Diagnosis and Classification of Diabetes Mellitus and its Complications: Report of a WHO Consultation. Part 1: Diagnosis and Classification of Diabetes Mellitus. 1999, World Health Organization: Geneva. 2. 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Adv Genet, 2005. 54: p. 3-20. 94 [...]...ABBREVIATIONS AAALAC Association for Assessment and Accreditation of Laboratory Animal Care ALT Alanine aminotransferase AST Aspartate aminotransferase AUC Area under the curve BSA Body surface area cDNA Complementary DNA ChoRE Carbohydrate response element DCCT Diabetes Control and Complications Trial DMEM Dulbecco’s Modified Eagle’s Medium EGF Epidermal growth factor EGTA Ethylene glycol tetraacetic... producing replacement beta cells for use in transplantation Islet cell clusters have a biphasic response to an increase in glucose levels: a rapid release of high concentrations of insulin and a slower release of lower concentrations of insulin In contrast, isolated beta cells do not release insulin in this manner Instead it is an all-or-nothing response with no fine regulation for intermediate concentrations... main cause of end-stage renal disease in developed world adults), peripheral vascular disease, retinal damage leading to blindness, neuropathy, and microvascular damage which may cause erectile dysfunction and poor healing Peripheral vascular disease, compounded by poor healing of wounds and neuropathy (particularly of the feet), can lead to gangrene which more often than not requires amputation In fact,... direct administration of insulin as their bodies do not produce enough (or even any) endogenous insulin Traditionally, insulin could only be administered by injections, but recently an inhaled form has been developed Exubera, an inhaled product, was approved by the Food and Drug Administration (FDA) and was made available in USA in 2006 Insulin therapy is supplemented with other measures including nutrition... hyperosmolar coma and hypoglycaemia, have become less common with the introduction of insulin and its various formulations As diabetics have begun to live longer, however, the chronic complications of diabetes, in the form of heart disease (angina and heart attacks), stroke, renal failure, neuropathy, peripheral vascular disease, amputations and blindness, have taken over as the principal causes of... cells, including nestin-positive ones, to generate beta-like cells that demonstrate glucose-dependent insulin release [53-55] More recently, pancreatic exocrine (acinar) cells have also been transdifferentiated to insulin-producing cells in vitro [56, 57] As the liver has a common embryonic origin with the pancreas, it is believed that it harbours cells that are capable of transdifferentiating to pancreatic... avoided Adult primary hepatocytes are not a cell line and, being non-mitotic, are a safer option with no malignancy risk and low probability of hyperinsulinaemic hypoglycaemia The risk of recurrence of disease due to autoimmune destruction of transplanted cells, as seen in islet transplantation for type 1 diabetes, is also not an issue [125] Hepatocyte transplantation has been conducted in animal models... prospect In the regeneration of primary pancreatic beta cells, the main hurdle seems to the scaling up of these processes to generate clinically relevant quantities of cells for therapies Large surface areas will need to be available to expand the cells 14 1.3.2 Gene -based Therapy for Diabetes Mellitus Gene therapy includes any approach that involves the introduction of an exogenous gene into any cell. .. functional beta cells Glucagon-like peptide-1 (GLP-1) and its analog exendin-4 can stimulate beta cell insulin secretion, inhibit apoptosis, and increase cell mass in rodents [77-80] Recent clinical trials of exendin-4 on type 2 diabetic patients showed improvement of glycaemic control associated with no weight gain [81, 82] Other factors that have been shown to increase beta cell proliferation and expand... Protein targeting to glycogen RNA Ribonucleic acid RT-PCR Reverse transcription polymerase chain reaction SEM Standard error of mean SGH Singapore General Hospital STZ Streptozotocin USA United States of America xi CHAPTER 1: INTRODUCTION 1.1 Diabetes Mellitus 1.1.1 Definition and Classification of Diabetes Mellitus Diabetes mellitus is a disorder of carbohydrate, fat and protein metabolism characterized ... diabetic monkeys that underwent liver resection and hepatocyte transplantation 50 ix ABBREVIATIONS AAALAC Association for Assessment and Accreditation of Laboratory Animal Care ALT Alanine aminotransferase... bilirubin, alkaline phosphatase, serum alanine transaminase (ALT) and γ-glutamyl transferase (γGT) activities), renal function (urea and creatinine) and full blood counts (total and differential... administered by injections, but recently an inhaled form has been developed Exubera, an inhaled product, was approved by the Food and Drug Administration (FDA) and was made available in USA in