657 Knockdown of GTP Cyclohydrolase I Expression by siRNA Based Gene Silencing Modality Molecular Therapy Volume 17, Supplement 1, May 2009 Copyright © The American Society of Gene Therapy S250 GENE R[.]
GENE REGULATION: ORGANOTROPIC APPROACHES - HEART, MUSCLE, LIVER, NEURO, BLOOD patients but cannot fully compensate for the loss of SMN1 due to a C to T nucleotide transition in the first exon splicing enhancer element of exon This translationally silent transition disrupts binding of the ASF/SF2 splicing factor, resulting in transcripts thal lack exon and only small amounts that contain it Varying SMN2 copy number in patients and animal models creates a titration of SMN protein and ultimately a gradient of disease severity Many clinical trials for SMA aim to upregulate SMN expression yet we know very little about when and where SMN expression is critical for normal development and maintenance of motor neurons To uncover the SMN expression requirements in vivo we developed animal models that can be used to regulate SMN expression spatially and temporally We created two inducible alleles which mimic SMN2 splicing in the mouse by engineering discrete mutations within the endogenous Smn locus The SmnCT-Neo mutant allele mimics the C-T transition within human SMN2 while the Smn2B-Neo allele mutates a splice enhancer within exon The presence of a LoxP flanked selectable marker within intron causes nearly all Smn transcripts from the mutant alleles to lack exon Mutant embryos are embryonic lethal, indicating that more than 20% FLSmn transcript is required for embryonic development Expression of CRE up-regulates distinct levels of full length transcripts through excision of the floxed selectable marker Utilizing a tamoxifen inducible Cre allele we were able to increase SMN expression above 50%; bringing the transcript level to that of a phenotypically normal mouse, and thus rescuing embryonic lethality These novel Smn mutant mice can be used to determine the timing, location, and amount of Smn protein required for normal development, maintenance and survival of motor neurons In vivo regulation of SMN allows us to identify the timepoints and expression levels which should be aimed for in clinical trials 655 Enhancement of the Expression of Genes Electrotransferred in Cells in Culture and in Skeletal Muscle by Nanosecond Electric Pulses Vanessa Joubert,1,2 Julien Villemejane,1,2,3 Lluis M Mir.1,2 UMR 8121 CNRS - Institut Gustave Roussy, Villejuif, France; UMR 8121- University Paris Sud, Villejuif, France; 3UMR 8029, CNRS-SATIE-ENS de Cachan, Cachan, France The objective of this study was to explore whether nanosecond electric pulses (nsPEF) affect the expression of electrotransferred genes, not only in vitro but also in vivo, and to explore the parameters affecting such effect to start the analysis of the underlying mechanisms The experiments were performed in vitro (on DC-3F and LPB cells) and in vivo (on skeletal muscle) In vitro: DNA coding for the luciferase was electrotransferred using classical procedures with CliniporatorTM (IGEA, Italy) Then nsPEF (10 ns duration) were delivered or not with a nsPEF generator (FID Technology, Russia) In vivo: DNA coding for the luciferase was electrotransferred into skeletal muscle (tibialis cranialis) of mice using combinations of microseconds (HV) and milliseconds (LV) pulses Then tissues were exposed or not to nsPEF with a new exposure technique using insulated electrodes Muscles were processed 48 or 72 hours after Measurement of the luciferase expression was performed using a luminometer and results were expressed as pg luciferase/µg total protein (in vitro) or pg luciferase/mg muscle (in vivo) In vitro, an increase of three-fold of the luciferase expression in the extracts can be achieved with the application of 20 nsPEF (10 ns, Hz, 60 kV/ cm) 60 minutes after plasmid electrotransfer, in an electroporation cuvette with mm of gap between the electrodes The effects are also dependent of the nsPEF repetition frequency: 100 Hz are more effective than Hz Contrary to these parameters, the time between DNA electrotransfer and nsPEF delivery, or the amount of DNA per cuvette not seem crucial No loss of viability is associated to the exposure under these conditions In vivo: When 30000 nsPEF were delivered immediately after or immediately before DNA S250 electrotransfer (combination of HV and LV), a statistically significant increase of about seven- to eight-fold in the luciferase activity was observed in the muscles treated by DNA electrotransfer + nsPEF compared to DNA electrotransfer only In contrast, when nsPEF were delivered 60 after DNA electrotransfer, we observed a non significant increase of about two-fold in muscles treated by nsPEF compared to DNA electrotransfer only In muscles exposed to 30000 nsPEF without HV and LV, no difference in the luciferase expression was observed with respect to the DNA injection alone Studies on nsPEF mechanisms are still ongoing, but our preliminary results seem to show that nsPEF does not have an effect on nuclear pore Moreover, nsPEF does not seem to destabilize enough the plasma membrane to allow plasmid DNA to enter inside the cells In this study we demonstrate that cell manipulation by means of electric nsPEF delivery may increase the overall efficiency of gene electrotransfer An increase of three-fold in the production of the reporter gene was achieved in vitro and of seven- to eight-fold in vivo 656 The Role of miR-21 during Liver Regeneration in Mice Rebecca T Marquez,1 Sarmistha Bhattacharya,1 Kathy Keck,1 Erik Wendlandt,1 Scott Ebert,1 Anton P McCaffrey.1 Internal Medicine, University of Iowa, Iowa City, IA The liver has an enormous capacity to regenerate upon injury Healthy hepatocytes rarely divide 2,3 However, upon injury, hepatocytes proliferate to maintain liver function The partial hepatectomy is a common small animal liver regeneration model where ∼60% of the liver is removed Remarkably, all the hepatocytes in the liver divide within seven days The liver mass, including the complex architecture of the regenerated liver, is completely restored Relatively little is known about what triggers the gene expression programs directing liver regeneration Using a candidate-based approach, we determined that the proliferative miRNA, miR-21, was upregulated during liver regeneration We have conducted in situ hybridization to localize miR-21 within the liver miR-21 is predicted to target a number of components of the transforming growth factor-β (TGF-β) signaling pathway TGF-β is produced early after partial hepatectomy Paradoxically, TGF-β inhibits hepatocyte division For poorly understood reasons, during liver regeneration, hepatocytes become resistant to TGF-β Later, it appears that TGF-β signaling contributes to the termination of hepatocyte proliferation as well as remodeling of the liver Our hypothesis is that miRNAs regulate components of the TGF-β pathway during liver regeneration Using luciferase reporter assays, we have shown that miR-21 directly targets a protein known as TGF-β-Induced (TGFβI) Furthermore, we demonstrated that this interaction was direct by relieving miR-21 repression of TGFβI using an antisense target protector oligonucleotide The validity of other predicted miR-21 targets in the TGF-β pathway will be discussed 657 Knockdown of GTP Cyclohydrolase I Expression by siRNA-Based Gene Silencing Modality Sung Jin Kim,1 Won Il Lee,1 Yoon Sun Lee,2 DongHou Kim,3 Seong Who Kim,2 Heuiran Lee.1 Microbiology, Research Institute for Biomacromolecules, University of Ulsan College of Medicine, Seoul, Korea, Republic of; 2Biochemistry & Molecular Biology, Research Institute for Biomacromolecules, University of Ulsan College of Medicine, Seoul, Korea, Republic of; 3& Cell Biology, Research Institute for Biomacromolecules, University of Ulsan College of Medicine, Seoul, Korea, Republic of Background & Aim : GTP cyclohydrolase (GCH1) is a rate-limiting enzyme in the biosynthesis of tetrahydrobiopterin Molecular Therapy Volume 17, Supplement 1, May 2009 Copyright © The American Society of Gene Therapy GENE REGULATION: ORGANOTROPIC APPROACHES - HEART, MUSCLE, LIVER, NEURO, BLOOD (BH4) BH4 has been known as a cofactor for aromatic amino acid hydroxylase and nitric oxide synthase, which synthesize certain neurotransmitters and nitric oxide (NO), respectively Indeed, there have been many reports that either GCH1 genetic defect or malfunction is deeply involved in several human diseases, such as hypertension, neuropathic pain, liver cirrhosis, and even diabetes However, the detailed mechanisms of GCH1 involvement to these human diseases are needed to be investigated To achieve this aim, we attempted to explore the GCH1 activity in transcriptional level Methods & Results : We designed siRNA targeting various exons of GCH1 mRNA and validated their GCH1 down-regulation effects WB analysis showed that all the siRNAs effectively down-regulated GCH1 expression To generate their corresponding short hairpin RNAs (shRNAs), siRNA sequence was cloned into pSP72 vector under H1 promoter Additionally, we simultaneously expressed GFP in the identical vector, ensuring shRNA expression (pSP72-shGCH1/ GFP) As expected, pSP72-shGCH1/GFP not only expressed GFP, but also sharply decreased GCH1 protein level Furthermore, we generated a recombinant adeno-associated virus encoding shGCH1/ GFP expression cassette, which efficiently reduced GCH1 expression Conclusion : These data suggest that siRNA-based gene silencing strategy is applicable to effectively downregulate GCH1 expression in various exon regions Moreover, a newly-developed bidirectional rAAV-shRNA/GFP gene delivery system can be valuable tool to explore the activity of persistent expression of a certain shRNA both in vivo and in vitro Key words: GTP cyclohydrolase 1, exon, siGCH1, shRNA, bidirectional expression, rAAV a These authors contributed equally to this work; * Corresponding authors contributed equally to this work 658 Neuropathic Pain Can Be Effectively Attenuated by Down-Regulating GCH1 Expression Using rAAV Harboring shGCH1 Sung Jin Kim,1 Won Il Lee,1 Yoon Sun Lee,2 Jin Woo Chang,5 DongHou Kim,3 Seong Who Kim,2 Heuiran Lee.1 Microbiology, Research Institute for Biomacromolecules, University of Ulsan College of Medicine, Seoul, Korea, Republic of; 2Biochemistry & Molecular Biology, Research Institute for Biomacromolecules, University of Ulsan College of Medicine, Seoul, Korea, Republic of; 3Anatomy & Cell Biology, Research Institute for Biomacromolecules, University of Ulsan College of Medicine, Seoul, Korea, Republic of; 4Neurosurgery, Yonsei University College of Medicine, Seoul, Korea, Republic of Background: Transcriptional activation of GTP cyclohydrolase I (GCH1) after nerve injury has been reported to associate with the development of neuropathic pain, as well as its persistency Therefore, we hypothesized that inhibition of GCH1 expression can be a potential strategy to attenuate neuropathic pain Aim: To test this hypothesis, we constructed a recombinant adeno-associated virus encoding both GFP and a small hairpin RNA against GCH1 (rAAV-shGCH1) to suppress GCH1 induction Then, we validated its therapeutic potential in animal model Methods & Results: rAAV-shGCH1 was introduced into the sciatic nerve of rats before injury (pre-injection) and after injury (post-injection) GFP expression in dorsal root ganglia (DRG) under fluorescent microscope indicated that rAAV administration through the sciatic nerve could effectively deliver transgene to DRG and in turn significantly attenuate neuropathic pain Immunoblotting and immunohistochemical analysis demonstrated that GCH1 levels were significantly down-regulated in rAAV-shGCH1 injected groups In addition to GCH1 down-regulation, suppression of GCH1 using shRNA inhibited the activation of macrophages/microglia and p38 in DRG as well as spinal cord Taken together, the data suggest that inhibition of GCH1 expression in DRG sharply relieves neuropathic pain Similarly, post-injection of rAAV-shGCH1 significantly alleviated pain symptom by reducing GCH1 level and interfering the Molecular Therapy Volume 17, Supplement 1, May 2009 Copyright © The American Society of Gene Therapy activation of microglia and p38 Conclusion: These data demonstrate that the blockage of GCH1 induction by rAAV encoding shGCH1 could not only prevent the development of neuropathic pain, but also attenuate the persistent pain The results further suggest that pain symptom relief is closely associated with the decreased level of inflammation In conclusion, these findings provide strong evidence that down-regulation of GCH1 level would be potentially useful strategy to treat neuropathic pain a These authors contributed equally to this work; * Corresponding authors contributed equally to this work 659 Long-Term Growth Factor-Independent Control of Canine Hematopoiesis Using Chemical Inducers of Dimerization Kiyoshi Okazuka,1 Brian Beard,2 Kerstin Schwarzwaelder,3 Christopf von Kalle,3 Hans-Peter Kiem,2 C Anthony Blau.1 Institute for Stem Cell and Regenerative Medicine, University of Washington, Seattle, WA; 2Transplantation Biology, Fred Hutchinson Cancer Research Cancer, Seattle, WA; 3Nationales Centrum für Tumorerkrankungen (NCT) Heidelberg und Abteilung für Translationale Onkologie, Deutsches Krebsforschungszentrum (DKFZ) Heidelberg, Heidelberg, Germany Methods for specifically controlling transplanted cells have many theoretical applications in gene and cell therapy In 2001 and 2002 we transplanted two dogs with autologous CD34+ cells modified to express a derivative of the thrombopoietin receptor, F36VMpl, that is resistant to activation by endogenous ligands but can instead be activated by small molecule drugs called chemical inducers of dimerization (CIDs) Two 30 day courses of a CID (AP20187 – Ariad Pharmaceuticals) were administered to both dogs within the first year post transplant, and the resulting hematopoietic responses have been reported previously (Blood 100: 2026-31, 2002) In brief, both dogs exhibited reversible rises in GFP+ red cells, white blood cells (mostly B cells) and platelets Both dogs subsequently received a single subcutaneous course of AP20187, administered daily (5 mg/ kg) for consecutive days While both dogs again responded, the subcutaneous route of AP20187 administration was accompanied by significant soft tissue inflammation Our gene transfer protocol had used an oncoretroviral vector, and we therefore monitored both dogs for longer than years without CID Both animals remained hematologically normal, and neither exhibited spontaneous rises in GFP+ cells over time In both animals, GFP+ red cells, platelets and granulocytes fell to very low levels (