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153. Elucidating the Expression Kinetics and Infiltration Resulting from Gene Delivery Enhanced by Dermal Electroporation
PHYSICAL METHODS OF DELIVERY 151. Non-Viral Enteral Hormone Gastrin Gene Therapy Improves the Function and Stimulates the Proliferation of Pancreatic beta Cells
Jeonghyun Park,1,2 Hyesook Jung,2 Na Han,1,2 Taekyun Kim,1,2 Minjung Kwon,1,2 Soonhee Lee,1 Byungdoo Rhee,1 Mikyung Kim.1,2 1 Department of Internal Medicine, College of Medicine, Inje University, Busan, Republic of Korea; 2Paik Institute for Clinical Research, Inje University, Busan, Republic of Korea.
AIM: Type 2 diabetes mellitus is caused by increased insulin resistance and decreased endogenous insulin secretion. By increasing the amount of functioning beta cell mass in the islets of pancreas, we can expect the “remission” of type 2 diabetes mellitus. To increase the amount of beta cell mass, various kinds of growth factors and signaling molecules were employed in the past. Recently, the functional connections between gastrointestinal tract and endocrine pancreas via various hormones including GLP-1, GIP, gastrin and cholecystokinin and neural network have been revealed. We cloned gastrointestinal hormone gastrin cDNA containing novel plasmid pCMV-gastrin, and tested its capacity not only to augment the beta cell function but also stimulates the proliferation of beta cells. METHOD: We cloned rat gastrin cDNA containing signal peptide sequence from the gastrointestinal tract of SD rat by RT-PCR. Cloned gastrin cDNA was inserted into pCI (Promega) to make pCMV-gastrin. The production of gastrin by pCMV-gastrin was verified by ELISA after transfection into HEK293 cells. Gene transfections were performed using BPEI (25kDa) and sonoporation into cultured INS-1 cells and isolated primary islets from SD rats. GSIS (glucose stimulated insulin secretion), Ki-67, insulin and glucagon stainings were perfomed to verify the improved function and the proliferation of beta cells. RESULTS: pCMV-gastrin produced sufficient gastrin from the transfected HEK293 cells. Non-viral gene transfection of pCMVgastrin into cultured INS-1 cells and primary isolated rat pancreatic islets clearly improved GSIS, and stimulates the proliferation of beta cells. Gastrin gene therapy could be used to treat diabetes mellitus via increasing the amount of functioning beta cell mass.
152. Initial Toxicology of High Dose GNE Plasmid in Rodent and Canine Animal Species
Yadira Valles-Ayoub,1,2 Rosangela Carbajo,1,3 Lucia Sandoval,1,3 Zeshan Khokher,1,2,3 Jorge Garcia-Figueroa,1,2,3 Sarah Stein,1 Daniel No,1,2,3 Babak Darvish,2 Daniel Darvish.1,2 1 HIBM Research Group, Reseda, CA; 2VA Greater Los Angeles (VA-GLA/UCLA), Los Angeles, CA; 3California State University Northridge, Northridge, CA. Naked DNA is an attractive non-viral vector because of its inherent simplicity of manufacturing, prior safety record in human trials, and ability to repeat administration. It shows little to no vector gene expression at distant sites following delivery and can be re-administered multiple times without inducing neutralizing antibody host response. Thus, it is an attractive option for GNE Myopathy, or Hereditary Inclusion Body Myopathy (HIBM), which is caused by hypomorphic UDP-N-acetylglucosamine 2-epimerase/ N-acetylmannosamine kinase (GNE) enzyme, decreased sialic biosynthesis, and hyposialylation of muscle glycoproteins. For assessing safety, high dose high pressure regional limb delivery of GNE plasmid (0.7mg/mL) was tested in Beagle dogs, and very high dose (2.5mg/mL, o.5mL BID) slow tail vein infusion was tested in mice. No toxicity or death was observed, and no observable adverse effect level (NOAEL) dose is higher than the tested doses. Real-time RT-qPCR analysis demonstrated recombinant human GNE mRNA expression in canine muscles 30 days after treatment. In mice, even slow tail vein infusion of high dose plasmid resulted in expression in quadriceps muscles at one day after treatment, but declined rapidly Molecular Therapy Volume 21, Supplement 1, May 2013 Copyright © The American Society of Gene & Cell Therapy
showing no vector gene expression in muscle at 30 days. High pressure regional delivery of high dose GNE plasmid is feasible and expected to be reasonably safe for human clinical trials.
153. Elucidating the Expression Kinetics and Infiltration Resulting from Gene Delivery Enhanced by Dermal Electroporation
Janess M. Mendoza,1 Gleb Kichaev,1 Dinah Amante,1 Christine Knott,1 Trevor R. F. Smith,1 Niranjan Y. Sardesai,1 Kate E. Broderick.1 1 Research and Development, Inovio Pharmaceuticals Inc., San Diego, CA.
The skin is an attractive tissue for vaccination in a clinical setting due to the accessibility of the target, the ease of monitoring and most importantly the immunocompetent nature of the dermis. While dermal electroporation offers an exciting and novel future methodology for the delivery of DNA vaccines in the clinic, little is known about the mechanism of the approach and the elucidation of the resulting immune responses. To further understand the mechanism of the platform we wanted to investigate the expression kinetics and localization of a reporter plasmid delivered via a dermal electroporation (EP) device, as well as the effect treatment had on the tissue resident immune cells. Initially a time course (day 0 to 21) was produced to observe the localization of GFP expression that occurred through enhanced gene delivery with EP and the kinetics of its appearance and clearance. Gross imaging revealed that GFP expression was first detected on the surface of the skin 8 hours after treatment. However, histological analysis by fluorescent microscopy revealed GFP positive cells as early as 1 hour after plasmid delivery and electroporation. Peak GFP expression was observed at 48 hours and expression was maintained in skin for up to 7 days. HE staining of treated skin sections demonstrated an influx of monocytes and granulocytes at the EP site starting at 24 hours and persisting up to day 21 post treatment. In mice, immunological staining revealed a significant migration of Langerhans cells to the EP site. In conclusion, this study provided insights into expression kinetics following EPenhanced DNA delivery targeting the dermal space. These findings may have implications in the future when designing efficient DNA vaccination strategies for the clinic.
154. The Optimization of Electroporation Parameters To Enhance the Efficiency of Plasmid Gene Delivery in Muscle
Ningqing Wang,1,2 Yong Diao.1,2 School of Biomedical Science, Huaqiao University, Quanzhou, China; 2Institutes of Molecular Medicine, Huaqiao University, Quanzhou, China. 1
Plasmid DNA-based gene delivery has inherent advantages such as improved safety, reduced immunogenicity, reduced potential for integration into the genome, however, the lack of efficient delivery across cell membranes and low level of expression in many tissues limits its application. Electroporation of plasmid has been accepted as an effective physical method for transferring therapeutic genes in vivo, and has been applied to many tissues including skin, muscle, liver, tumors. The effectiveness of electroporation and consequently of gene electrotransfer depends on pulse parameters, such as amplitude, duration, number, pulse repetition frequency and geometric properties of electrode and tissue configuration. In order to improve gene transfer efficiency, the parameters of electric pulses have to be optimized depending on the type of electrodes used and specifically adjusted for each target tissue. In our present study, we used the simplest twin needle electrodes to determine the best electrical parameters for efficient gene electrotransfer into muscle tissue, which is regarded by many as the “tissue of choice” for gene electrotransfer based gene S61
Jeonghyun Park,1,2 Hyesook Jung,2 Na Han,1,2 Taekyun Kim,1,2 Minjung Kwon,1,2 Soonhee Lee,1 Byungdoo Rhee,1 Mikyung Kim.1,2 1 Department of Internal Medicine, College of Medicine, Inje University, Busan, Republic of Korea; 2Paik Institute for Clinical Research, Inje University, Busan, Republic of Korea.
AIM: Type 2 diabetes mellitus is caused by increased insulin resistance and decreased endogenous insulin secretion. By increasing the amount of functioning beta cell mass in the islets of pancreas, we can expect the “remission” of type 2 diabetes mellitus. To increase the amount of beta cell mass, various kinds of growth factors and signaling molecules were employed in the past. Recently, the functional connections between gastrointestinal tract and endocrine pancreas via various hormones including GLP-1, GIP, gastrin and cholecystokinin and neural network have been revealed. We cloned gastrointestinal hormone gastrin cDNA containing novel plasmid pCMV-gastrin, and tested its capacity not only to augment the beta cell function but also stimulates the proliferation of beta cells. METHOD: We cloned rat gastrin cDNA containing signal peptide sequence from the gastrointestinal tract of SD rat by RT-PCR. Cloned gastrin cDNA was inserted into pCI (Promega) to make pCMV-gastrin. The production of gastrin by pCMV-gastrin was verified by ELISA after transfection into HEK293 cells. Gene transfections were performed using BPEI (25kDa) and sonoporation into cultured INS-1 cells and isolated primary islets from SD rats. GSIS (glucose stimulated insulin secretion), Ki-67, insulin and glucagon stainings were perfomed to verify the improved function and the proliferation of beta cells. RESULTS: pCMV-gastrin produced sufficient gastrin from the transfected HEK293 cells. Non-viral gene transfection of pCMVgastrin into cultured INS-1 cells and primary isolated rat pancreatic islets clearly improved GSIS, and stimulates the proliferation of beta cells. Gastrin gene therapy could be used to treat diabetes mellitus via increasing the amount of functioning beta cell mass.
152. Initial Toxicology of High Dose GNE Plasmid in Rodent and Canine Animal Species
Yadira Valles-Ayoub,1,2 Rosangela Carbajo,1,3 Lucia Sandoval,1,3 Zeshan Khokher,1,2,3 Jorge Garcia-Figueroa,1,2,3 Sarah Stein,1 Daniel No,1,2,3 Babak Darvish,2 Daniel Darvish.1,2 1 HIBM Research Group, Reseda, CA; 2VA Greater Los Angeles (VA-GLA/UCLA), Los Angeles, CA; 3California State University Northridge, Northridge, CA. Naked DNA is an attractive non-viral vector because of its inherent simplicity of manufacturing, prior safety record in human trials, and ability to repeat administration. It shows little to no vector gene expression at distant sites following delivery and can be re-administered multiple times without inducing neutralizing antibody host response. Thus, it is an attractive option for GNE Myopathy, or Hereditary Inclusion Body Myopathy (HIBM), which is caused by hypomorphic UDP-N-acetylglucosamine 2-epimerase/ N-acetylmannosamine kinase (GNE) enzyme, decreased sialic biosynthesis, and hyposialylation of muscle glycoproteins. For assessing safety, high dose high pressure regional limb delivery of GNE plasmid (0.7mg/mL) was tested in Beagle dogs, and very high dose (2.5mg/mL, o.5mL BID) slow tail vein infusion was tested in mice. No toxicity or death was observed, and no observable adverse effect level (NOAEL) dose is higher than the tested doses. Real-time RT-qPCR analysis demonstrated recombinant human GNE mRNA expression in canine muscles 30 days after treatment. In mice, even slow tail vein infusion of high dose plasmid resulted in expression in quadriceps muscles at one day after treatment, but declined rapidly Molecular Therapy Volume 21, Supplement 1, May 2013 Copyright © The American Society of Gene & Cell Therapy
showing no vector gene expression in muscle at 30 days. High pressure regional delivery of high dose GNE plasmid is feasible and expected to be reasonably safe for human clinical trials.
153. Elucidating the Expression Kinetics and Infiltration Resulting from Gene Delivery Enhanced by Dermal Electroporation
Janess M. Mendoza,1 Gleb Kichaev,1 Dinah Amante,1 Christine Knott,1 Trevor R. F. Smith,1 Niranjan Y. Sardesai,1 Kate E. Broderick.1 1 Research and Development, Inovio Pharmaceuticals Inc., San Diego, CA.
The skin is an attractive tissue for vaccination in a clinical setting due to the accessibility of the target, the ease of monitoring and most importantly the immunocompetent nature of the dermis. While dermal electroporation offers an exciting and novel future methodology for the delivery of DNA vaccines in the clinic, little is known about the mechanism of the approach and the elucidation of the resulting immune responses. To further understand the mechanism of the platform we wanted to investigate the expression kinetics and localization of a reporter plasmid delivered via a dermal electroporation (EP) device, as well as the effect treatment had on the tissue resident immune cells. Initially a time course (day 0 to 21) was produced to observe the localization of GFP expression that occurred through enhanced gene delivery with EP and the kinetics of its appearance and clearance. Gross imaging revealed that GFP expression was first detected on the surface of the skin 8 hours after treatment. However, histological analysis by fluorescent microscopy revealed GFP positive cells as early as 1 hour after plasmid delivery and electroporation. Peak GFP expression was observed at 48 hours and expression was maintained in skin for up to 7 days. HE staining of treated skin sections demonstrated an influx of monocytes and granulocytes at the EP site starting at 24 hours and persisting up to day 21 post treatment. In mice, immunological staining revealed a significant migration of Langerhans cells to the EP site. In conclusion, this study provided insights into expression kinetics following EPenhanced DNA delivery targeting the dermal space. These findings may have implications in the future when designing efficient DNA vaccination strategies for the clinic.
154. The Optimization of Electroporation Parameters To Enhance the Efficiency of Plasmid Gene Delivery in Muscle
Ningqing Wang,1,2 Yong Diao.1,2 School of Biomedical Science, Huaqiao University, Quanzhou, China; 2Institutes of Molecular Medicine, Huaqiao University, Quanzhou, China. 1
Plasmid DNA-based gene delivery has inherent advantages such as improved safety, reduced immunogenicity, reduced potential for integration into the genome, however, the lack of efficient delivery across cell membranes and low level of expression in many tissues limits its application. Electroporation of plasmid has been accepted as an effective physical method for transferring therapeutic genes in vivo, and has been applied to many tissues including skin, muscle, liver, tumors. The effectiveness of electroporation and consequently of gene electrotransfer depends on pulse parameters, such as amplitude, duration, number, pulse repetition frequency and geometric properties of electrode and tissue configuration. In order to improve gene transfer efficiency, the parameters of electric pulses have to be optimized depending on the type of electrodes used and specifically adjusted for each target tissue. In our present study, we used the simplest twin needle electrodes to determine the best electrical parameters for efficient gene electrotransfer into muscle tissue, which is regarded by many as the “tissue of choice” for gene electrotransfer based gene S61