109 efficiency of electroporation delivery of peptide nucleic acid (PNA) depends on PNA charge and electrotransfer protocol

109 Efficiency of Electroporation Delivery of Peptide Nucleic Acid (PNA) Depends on PNA Charge and Electrotransfer Protocol Molecular Therapy Volume 18, Supplement 1, May 2010 Copyright © The American[.]

PHYSICAL METHODS OF DELIVERY 106 Ca2+ Electrotransfer; a Quick Stop Procedure Against Transgenic Expression Pernille Hojman,1,2 Iben Spanggaard,1 Julie Gehl,1 Hanne Gissel.3 Department of Oncology, Copenhagen University Hospital Herlev, Herlev, Denmark; 2Center of Inammation and Metabolism, Copenhagen University Hospital, Copenhagen, Denmark; Institute of Physiology and Biophysics, Unversity of Aarhus, Aarhus, Denmark Introduction DNA electrotransfer to muscle tissue yields high-level, long-term expression of the transferred products This is advantageous in many treatment regiments, but also poses a concern of how to control accelerated transgenic expression During electrotransfer, the cell membrane becomes permeable, resulting of inux of ions, i.e Na+ and Ca2+, down their electrochemical gradient Excessive inux of, in particular, Ca2+ results in activation of various cellular processes, which eventually leads to induction of cell death and cell degradation Thus, we aimed to test if Ca2+ electrotransfer could be used to turn off transgenic expression in muscles expressing a transgenic product Methods Muscles of C57black/C mice respectively Wistar rats were transfected with the uorescent marker, Katuskha, or erythropoietin (EPO) Ten days after transfection, transgenic expression was evaluated by in vivo imaging or blood sampling to ensure efcient transgenic expression Next, the muscles were electrotransferred with 20 µl Calcium-Sandoz diluted to 168 mM, which is isoosmotic with physiological NaCl Calcium uptake was measured using Ca-45 Gene expression was followed by in vivo imaging, and blood sampling for measurement of hemoglobin and serum EPO levels Results Maximal Ca-45 uptake in muscles was obtained using electric parameters of HV pulses of 1000 V/cm, and injection before pulsing Using these parameters, EPO expression in muscles was eliminated Hemoglobin levels were signicantly elevated two weeks after EPO gene electrotransfer (EPO: 9.6 ± 1.0, control: 7.6 ± 0.4, P < 0.01), and returned to basal level within a week after Ca2+ electrotransfer (EPO: 7.7 ± 0.5, control: 7.8 ± 0.2, P > 0.05) Same pattern was observed for serum EPO In vivo imaging of the uorescent marker Katuskha showed that transgenic expression was signicantly decreased hours after Ca2+ electrotransfer, and completely eliminated within days Conclusion Electrotransfer of isotonic CaCl2 is an efcient method to rapidly terminate expression of transgenic proteins 107 In Vivo Gene Delivery to the Skin by Electroporation Using a Multiple Electrode Array Siqi Guo, Amy Donate, Gaurav Basu, Kathryn Lundberg, Richard Heller Frank Reidy Research Center for Bioelectrics, Old Dominion University, Norfolk, VA The efciency and advantages of in vivo electroporation (EP) for gene delivery have been demonstrated by our and other groups Because of its large surface area and easy access for both delivery and monitoring, the skin is an attractive target for gene therapy for cutaneous diseases, vaccine and several metabolic disorders Suitable electrodes or the corresponding optimal delivery parameters for skin EP gene delivery have not yet been completely dened Previously, we demonstrated the effectiveness of a new multielectrode array (MEA) In this study, we have further characterized and evaluated the MEA Work was performed in guinea pig skin Several aspects of the electrode design were evaluated including type of material and shape of the electrode tip Evaluation of the MEA has also included the correlation between the expression level and amount of area treated We found that the gene expression levels could be further enhanced by increasing the size of the treated area Interestingly, the gene-expressing cells were exclusively within the epidermal layer demonstrated by histology after skin EP with GFP-plasmid Importantly, we observed by gross observation and histology that Molecular Therapy Volume 18, Supplement 1, May 2010 Copyright © The American Society of Gene & Cell Therapy the skin damage caused by the EP was limited and completely recoverable under the appropriate treatment parameters Currently, we are performing experiments to observe whether we can achieve the long-term in vivo gene expression by multiple DNA EP treatments with the new MEA applicator 108 Hydrodynamic Gene Delivery Efciency Is Reduced in Fibrotic Liver in Rats Tian Zhou, Dexi Liu Department of Pharmaceutical Sciences, University of Pittsburgh, Pittsburgh, PA Liver brosis is commonly seen during the progression of many chronic liver diseases, and may result in liver failure if not controlled properly While anti-brotic gene therapy has been proposed for the reversal of liver brosis, the appropriate gene transfer remains a critical issue for therapeutic success The current study aimed at assessing the efciency of hydrodynamic gene delivery (HDG) into brotic liver in Sprague-Dawley rats established by twice-weekly, intra-peritoneal injection of CCl4 for 4-8 weeks Efficiency of hydrodynamic gene delivery to brotic liver by tail vein injection (injection volume, 7% body weight; injection time, 10 sec) was examined using reporter construct (pCMV-Luc) and compared to that of control animals Compared to control animals that showed the level of luciferase activity at 108 to 109 RLU/mg of liver proteins hr after hydrodynamic delivery, the level of luciferase activity obtained in animals with brotic liver was 105 to 107 RLU/mg, up to approximately 2,000 fold lower than that of normal animal depending on the severity of brosis The negative impact of liver brosis on HGD efciency was correlated with formation of brous septa surrounding liver lobules in CCl4-treated rats, reduced liver fenestrae, and signicantly higher intra-hepatic pressure upon hydrodynamic injection Transmission and scanning electron microscopy showed that the sinusoidal endothelium in the brotic liver were less stretched and fenestrae less enlarged by hydrodynamic pressure, suggesting that reduced HGD efciency is likely caused by decreased enlargement of fenestrae and reduced permeabilization of hepatocytes These results suggest that efciency of hydrodynamic gene delivery is dependent on tissue structure of the target organ and application of hydrodynamic gene delivery to treatment of liver brosis requires careful evaluation on the severity of brosis 109 Efciency of Electroporation Delivery of Peptide Nucleic Acid (PNA) Depends on PNA Charge and Electrotransfer Protocol Mette Joergensen,1 Birgit Agerholm-Larsen,2 Peter E Nielsen,3 Julie Gehl.1 Copenhagen University Hospital Herlev, Herlev, Denmark; Copenhagen University Hospital Glostrup, Glostrup, Denmark; Health Science Faculty, University of Copenhagen, Copenhagen, Denmark Background Peptide nucleic acids are synthetic DNA mimics, having a peptide backbone instead of a sugar phosphate backbone They are highly potent antisense and antigene molecules, down regulating protein expression at the level of translation and transcription respectively The special structure of the PNA gives the molecule several advantages compared to other antisense and antigene oligonucleotide types, most importantly is the very high biological (and chemical) stability as well as easy access to a wide range of chemical modications in a medicinal chemistry context However, despite these apparent advantages, biological applications of PNA molecules is limited by their inherently poor cellular uptake, being a relatively hydrophilic molecule which does not readily cross cell membranes Electroporation is potentially a very powerful technique for both in vitro cellular and in vivo drug delivery, in S41 PHYSICAL METHODS OF DELIVERY particular relating to oligonucleotides and their analogues for genetic therapy, and it is currently used clinically for drug and gene delivery in cancer treatment Methods A special designed system based on a recombinant luciferase gene, interrupted by a mutated human beta-globin intron 2, is used to detect the uptake of PNA in HeLa pLuc705 cells The mutation in the intron causes aberrant splicing of luciferase pre-mRNA, preventing translation of luciferase Cells which have taken up PNA targeted to the aberrant splice site induce correct splicing, thereby restoring luciferase activity We used two different electroporation systems, a cuvette system (Cliniporator) and a microtiter plate electrotransfer system, Cellaxess (Cellectricon), and PNA of negative -, positive – and neutral charge was included Results Electrotransfer efciently enabled PNA entry, veried by luciferase activity and splice correction quantied in RT-PCR Interestingly, for an adherent cell electrotransfer system (Cellaxess) charge neutral PNA were favourably transferred (factor 31, p