- Email: [email protected]
678. Cell Permeable, Anti-Inflammatory Peptides Targeting NF-kB and NFAT Pathways Prevent the Onset of Diabetes in NOD Mice
677. AAV-Mediated IGF-1 Delivery to the Cervical Spinal Cord of the SOD1 Rat Model of ALS Via Intraparenchymallnjection Jun Yang,' Erin Carlton; Thais Federici ; Kathie M. Bishop.' Carey Backus,' Sang Su Oh,3 Qingshan Teng,' Jonathon Riley; Mehdi Gasmi.' Raymond T. Bartus." Eva L. Feldman,' Nicholas
M. Boulis.'
I Departments 0/ Neuroscience and Neurosurgery, Cleveland
Clinic/Lerner Research Institute, Cleveland, OU; 2Ceregene, Ceregene, Inc, San Diego, CA; 'Depanment ofNeurology; University ofMichigan, Ann Arbor; MI. Background: Vector mediated IGF-I expression prolongs survival in the SOD I mouse model of ALS when delivered by intramuscular injection. However, direct intraparenehymal delivery to the spinal cord may represent a more efficient means of achieving therapeutic levels of gene delivery in the spinal cords of larger animals. The present study tests the safety and efficiency of AAV2- mediated cervical spinal cord IGF-l delivery in the rat via direct intraparenchymal injection. Methods: 40 wild-type rats were divided into 4 groups receiving single and double unilateral or bilateral injections of 5 micro liter AAV2-IGF-1 into cervical spinal cord. Neuromuscular function was assessed by BBB score, grip strength and rotarod assays for six weeks following injection. Spinal cords were evaluated histologically for IGF-I distribution and MN density. Based on the results, 5 micro liters ofAAV2-IGF-I, AAV2-GFP, and PBS were injected into unilateral cervical spinal cord (C3 and C5 segments) of presymtomatie (80 days old) and symptomatic (115 days old) SOD I rats for evaluation of effects of IGF-I expression on preserving motor neurons and motor function in SOD I rat model ofALS . Results: Our results showed that double unilateral cervical spinal cord injections ofAAV2-IGF-1 (10 micro liter) resulted in robust transgene expression in the cervical spina l cord without detectable behavioral dysfunction. Motor neuron counts showed no significant differences between AAV2-IGF-1 injected and uninjected spinal cord in wild-type rats. Preliminary data from presymptomatic SODI rats injected withAAV2-IGF-1 unilaterally indicated a delayed decline ofmotor function in ipsilateral forelimb compared with the contralateral side. Conclusion: Cervical spinal cord injection ofAAV2-IGF-1 resulted in robust transgene expression for at least 6 weeks post-injection with no adverse effects on motor neuron number and motor function. Initial observations suggest that direct intraparenchymal delivery of AAV2-IGF-1 delays decline in motor performance of SOD I rats. On-going studies will further characterize the effects ofAAV2-IGF-1 in the SODI rat model ofALS. Key Words: Gene therapy; IGF-I ; Intraparenchymal injection. Support Contributed By: ALS Association.
678. Cell Permeable, Anti-Inflammatory Peptides Targeting NF-KB and NFAT Pathways Prevent the Onset of Diabetes in NOD Mice Khaja K. Rehman , Paul D. Robbins. Department ofMolecular Genetics and Biochemistry, University 0/ Pittsburgh School 0/ Medicine, Pittsburgh. I'll. I
Type I diabetes (1' ID) is a disease that results from autoimmune destruction of pancreatic ~-cells of islets of Langerhans. Autoreactive T cells directed against islet ~-cells become activated, expand clonally and entrain a cascade ofimmune process in the islets, finally leading to ~-cell destruction. Calcineurin-NFAT (nuclear factor of activated T cells) and NF-KB mediated signaling pathways are involved in diverse cellular reactions for regulating gene expression either positively or negatively. TID is associated with enhanced NF-KB activity in dendritic cells (DCs) as well as macrophages resulting in increased secretion of proinflammatory cytokines, influencing in macrophage APC function and also stimulating T S260
cells. The calcineurin inhibitors Cyclosporine A and FK-506 have been used for treatment of transplant rejection and autoimmune disorders, but with unwarranted side effects. In the present study in search for safer drugs for the treatment of autoimmune diabetes, we have used the technology of peptide transduction for delivery of peptides inhibitors ofNFAT and NF-KB activation in non-obese diabetic (NOD) mice. Intraperitoneal injections of NFAT or NFKB inhibitory peptides, either once or twice a week respectively, provided protection against spontaneous 1'1 D in four-week-old NOD mice. Peptide-based therapeutic targeting the NF-KB and NFAT represent a new strategy for the treatment of TID and may eventually be useful clinically.
679. Sensitive and Precise Regulation of Haemoglobin after Gene Transfer of Erythropoietin to Muscle Tissue Using Electroporation
Pernille H. Moller,' ? Hanne Gisscl,' Julie Gchl.' Dept. ofOncology; University ofCopenhagen at Herlev Hospital, Copenhagen, Denmark; 2Dep t. 0/Physiology and Biophysics, University 0/Aarhus, Aarhus, Denmark; JCenter 0/lrflammation and Metabolism. Copenhagen University Hospital. Copenhagen, Denmark I
DNA electrotransfer is gaining increasing momentum, in particular for muscle tissue, where long-term high-level expression is obtainable. In this study we investigated methods for precise and sensitive regulation of'transgene expression. Induction ofexpression using the Tet-On system has previously been established; however attempts to reach a predefined target dose - a prescription, have not been reported. We set 3 target haemoglobin levels (10, 12 and 14 rnrnol/l., base level was 8.2 mmol/L) , which three prescribers had to reach through DNA e1ectrotransfer of the erythropoietin (EPO) gene to mouse tibialis cranialis (TC) muscle. The prescribers could vary I) the DNA amount (0.25 - 10 Ilg), 2) muscle mass transfected (one or both TC muscles), and 3) induction with the Tel-On system (doxycycline concentration: 0.01 - 0.3 mg/ml). Results showed that: A) using GFP, luciferase and EPO low DNA amounts were needed. In tact we found that increasing the DNA amount for c1eetro gene transfer did not ensure better transgene expression. We were able to reduce the amount of DNA encoding EPO needed to obtain significant elevations in haemoglobin to 0.5 ug, which extrapolates to 1.4 mg in humans. B) the three prescribers were able to hit the target Hgb levels with average Hgb 01'10.5,12.0, and 13.7 mmolfL , C) different approaches could be employed to reach the same target concentration, D) undershooting the target Hgb could be corrected by retransferring new plasmid, E) overshooting the target Hgb could be alleviated by redueing the dose of inducer drug (doxycycline). In conclusion, we found that it was possible to prescribe precise levels oftherapeutic proteins (e.g. EPO) through regulation of 1) the amount of DNA transferred, 2) the area transfected , 3) regulating the inducer drug concentration, and 4) re-transfection, This finding that precise levels ofa therapeutic protein can be obtained , maintained and also be brought to a stop, after DNA eleetrotransfer is ofoutmost importance as it eliminates one ofthe main concerns ofgene therapy and is of great importance for future clinical use.
Molecular Therapy Yofume 15. Supplement I, .\by 2007 Copyright © '111C American Societyo f Gene TIICr.lpr
M. Boulis.'
I Departments 0/ Neuroscience and Neurosurgery, Cleveland
Clinic/Lerner Research Institute, Cleveland, OU; 2Ceregene, Ceregene, Inc, San Diego, CA; 'Depanment ofNeurology; University ofMichigan, Ann Arbor; MI. Background: Vector mediated IGF-I expression prolongs survival in the SOD I mouse model of ALS when delivered by intramuscular injection. However, direct intraparenehymal delivery to the spinal cord may represent a more efficient means of achieving therapeutic levels of gene delivery in the spinal cords of larger animals. The present study tests the safety and efficiency of AAV2- mediated cervical spinal cord IGF-l delivery in the rat via direct intraparenchymal injection. Methods: 40 wild-type rats were divided into 4 groups receiving single and double unilateral or bilateral injections of 5 micro liter AAV2-IGF-1 into cervical spinal cord. Neuromuscular function was assessed by BBB score, grip strength and rotarod assays for six weeks following injection. Spinal cords were evaluated histologically for IGF-I distribution and MN density. Based on the results, 5 micro liters ofAAV2-IGF-I, AAV2-GFP, and PBS were injected into unilateral cervical spinal cord (C3 and C5 segments) of presymtomatie (80 days old) and symptomatic (115 days old) SOD I rats for evaluation of effects of IGF-I expression on preserving motor neurons and motor function in SOD I rat model ofALS . Results: Our results showed that double unilateral cervical spinal cord injections ofAAV2-IGF-1 (10 micro liter) resulted in robust transgene expression in the cervical spina l cord without detectable behavioral dysfunction. Motor neuron counts showed no significant differences between AAV2-IGF-1 injected and uninjected spinal cord in wild-type rats. Preliminary data from presymptomatic SODI rats injected withAAV2-IGF-1 unilaterally indicated a delayed decline ofmotor function in ipsilateral forelimb compared with the contralateral side. Conclusion: Cervical spinal cord injection ofAAV2-IGF-1 resulted in robust transgene expression for at least 6 weeks post-injection with no adverse effects on motor neuron number and motor function. Initial observations suggest that direct intraparenchymal delivery of AAV2-IGF-1 delays decline in motor performance of SOD I rats. On-going studies will further characterize the effects ofAAV2-IGF-1 in the SODI rat model ofALS. Key Words: Gene therapy; IGF-I ; Intraparenchymal injection. Support Contributed By: ALS Association.
678. Cell Permeable, Anti-Inflammatory Peptides Targeting NF-KB and NFAT Pathways Prevent the Onset of Diabetes in NOD Mice Khaja K. Rehman , Paul D. Robbins. Department ofMolecular Genetics and Biochemistry, University 0/ Pittsburgh School 0/ Medicine, Pittsburgh. I'll. I
Type I diabetes (1' ID) is a disease that results from autoimmune destruction of pancreatic ~-cells of islets of Langerhans. Autoreactive T cells directed against islet ~-cells become activated, expand clonally and entrain a cascade ofimmune process in the islets, finally leading to ~-cell destruction. Calcineurin-NFAT (nuclear factor of activated T cells) and NF-KB mediated signaling pathways are involved in diverse cellular reactions for regulating gene expression either positively or negatively. TID is associated with enhanced NF-KB activity in dendritic cells (DCs) as well as macrophages resulting in increased secretion of proinflammatory cytokines, influencing in macrophage APC function and also stimulating T S260
cells. The calcineurin inhibitors Cyclosporine A and FK-506 have been used for treatment of transplant rejection and autoimmune disorders, but with unwarranted side effects. In the present study in search for safer drugs for the treatment of autoimmune diabetes, we have used the technology of peptide transduction for delivery of peptides inhibitors ofNFAT and NF-KB activation in non-obese diabetic (NOD) mice. Intraperitoneal injections of NFAT or NFKB inhibitory peptides, either once or twice a week respectively, provided protection against spontaneous 1'1 D in four-week-old NOD mice. Peptide-based therapeutic targeting the NF-KB and NFAT represent a new strategy for the treatment of TID and may eventually be useful clinically.
679. Sensitive and Precise Regulation of Haemoglobin after Gene Transfer of Erythropoietin to Muscle Tissue Using Electroporation
Pernille H. Moller,' ? Hanne Gisscl,' Julie Gchl.' Dept. ofOncology; University ofCopenhagen at Herlev Hospital, Copenhagen, Denmark; 2Dep t. 0/Physiology and Biophysics, University 0/Aarhus, Aarhus, Denmark; JCenter 0/lrflammation and Metabolism. Copenhagen University Hospital. Copenhagen, Denmark I
DNA electrotransfer is gaining increasing momentum, in particular for muscle tissue, where long-term high-level expression is obtainable. In this study we investigated methods for precise and sensitive regulation of'transgene expression. Induction ofexpression using the Tet-On system has previously been established; however attempts to reach a predefined target dose - a prescription, have not been reported. We set 3 target haemoglobin levels (10, 12 and 14 rnrnol/l., base level was 8.2 mmol/L) , which three prescribers had to reach through DNA e1ectrotransfer of the erythropoietin (EPO) gene to mouse tibialis cranialis (TC) muscle. The prescribers could vary I) the DNA amount (0.25 - 10 Ilg), 2) muscle mass transfected (one or both TC muscles), and 3) induction with the Tel-On system (doxycycline concentration: 0.01 - 0.3 mg/ml). Results showed that: A) using GFP, luciferase and EPO low DNA amounts were needed. In tact we found that increasing the DNA amount for c1eetro gene transfer did not ensure better transgene expression. We were able to reduce the amount of DNA encoding EPO needed to obtain significant elevations in haemoglobin to 0.5 ug, which extrapolates to 1.4 mg in humans. B) the three prescribers were able to hit the target Hgb levels with average Hgb 01'10.5,12.0, and 13.7 mmolfL , C) different approaches could be employed to reach the same target concentration, D) undershooting the target Hgb could be corrected by retransferring new plasmid, E) overshooting the target Hgb could be alleviated by redueing the dose of inducer drug (doxycycline). In conclusion, we found that it was possible to prescribe precise levels oftherapeutic proteins (e.g. EPO) through regulation of 1) the amount of DNA transferred, 2) the area transfected , 3) regulating the inducer drug concentration, and 4) re-transfection, This finding that precise levels ofa therapeutic protein can be obtained , maintained and also be brought to a stop, after DNA eleetrotransfer is ofoutmost importance as it eliminates one ofthe main concerns ofgene therapy and is of great importance for future clinical use.
Molecular Therapy Yofume 15. Supplement I, .\by 2007 Copyright © '111C American Societyo f Gene TIICr.lpr