441 AAV Mediated RNAi and Antisense Knockdown of Intranuclear DMPK Transcripts in DM1 Cells Molecular Therapy Volume 20, Supplement 1, May 2012 Copyright © The American Society of Gene & Cell Therapy[.]
MUSCULO-SKELETAL GENE & CELL THERAPY II plasmid, as a transfection control, were transfected into non-attached equine articular chondrocytes with FuGeneHD The chondrocytes were plated in 6-well ultra-low attachment plates and allowed to grow for 48 hours LPS challenge and RNA purification were then done Expression of IL1b, TNFa, aggrecan and collagen type IIb were determined by qRT PCR Results: Sequencing yielded four identical LHp plasmids for testing in chondrocytes Overall, LHp plasmids had 62% lower TNFa expression than positive control Additionally, LHp plasmids had 40% lower IL1b expression than positive control and all LPS treated cells had higher IL1b expression than untreated controls At 36 hours, long hairpin treated chondrocytes had no significant TNFa knockdown when in 3D culture, whereas IL-1beta expression was 32% lower, compared with controls (Fig 1) Aggrecan expression was 2.3-fold and 2.4-fold higher in LPS and non-LPS treated chondrocytes with pLHp than control, respectively (Fig 2) Collagen type IIb expression was not significantly different between LPS treated cells but was 2.7-fold higher in non-LPS pLHp cells than control Discussion: A long hairpin clone was used to deliver RNAi motifs to control IL-1 and TNFa expression in chondrocyte cultures Four clonally isolated long hairpin plasmids were confirmed and tested in LPS stimulated chondrocytes grown in monolayer All four LHp plasmids knocked down IL1b expression 40% and TNFa expression 62% Long hairpin plasmids were then tested in lps stimulated chondrocytes grown in non-attached, 3D conditions Long hairpin plasmid treated cells had 2.3 to 2.4-fold higher aggrecan expression regardless of LPS treatment Collagen type IIb expression was 2.7 fold higher without LPS treatment 440 Pathogenesis of Lumbar Spine Disease in Mucopolysaccaridosis and Response to Neonatal Gene Therapy Lachlan J Smith,1 John T Martin,1 Guilherme Baldo,2 Susan Wu,2 Yuli Liu,2 Mark E Haskins,3 Dawn M Elliott,4 Katherine P Ponder.2 Orthopaedic Surgery, University of Pennsylvania, Philadelphia, PA; 2Internal Medicine, Washington University School of Medicine, St Louis, MO; 3Pathobiology, School of Veterinary Medicine, University of Pennsylvania, Philadelphia, PA; 4Biomedical Engineering, University of Delaware, Newark, DE Mucopolysaccharidoses (MPS) are lysosomal storage diseases that are due to deficiency of enzymes that degrade glycosaminoglycans, with an incidence of 1:27,000 MPS is associated with bone and joint abnormalities of the spine, although the severity can vary with the specific type of MPS Approximately 10% of patients require spine surgery for spinal cord compression, which can be due to bone instability or thickening, disc degeneration, or thickening of ligaments or other tissues The goals of this project were: 1) determine the pathogenesis of spine disease in MPS; and 2) determine if neonatal systemic gene therapy with a gamma retroviral vector could prevent it These studies used the dog model of MPS VII, which has a missense mutation in the β-glucuronidase (GUSB) gene that results in the accumulation of heparin, dermatan, and chondroitin sulfate Evaluation of radiographs demonstrated that calcification within the epiphysis was markedly reduced in the lumbar spine at one month of age in untreated MPS VII dogs, which was associated with large, cartilaginous lesions within the vertebral epiphyses Radiographs remained abnormal at older ages In addition, spine segments from MPS VII dogs had significantly reduced stiffness and increased range of motion at months, consistent with structural instability Finally, spine tissues from MPS VII dogs had increased enzyme activity and mRNA for cathepsins, which are enzymes that can degrade extracellular matrix proteins We propose that spine instability is due to abnormal conversion of cartilage to bone during development, and to overexpression of enzymes that degrade extracellular matrix components and weaken the bone and intervertebral discs Some MPS VII dogs received neonatal intravenous injection of a gamma retroviral vector expressing the canine GUSB gene This resulted in transduction of 2% of hepatocytes, and serum GUSB activity that was above normal for the duration of evaluation, up to a decade in some animals However, abnormal calcification of the epiphysis and structural stability was at most marginally improved at months in RV-treated MPS VII dogs Evaluation of an 8-year-old RV-treated MPS VII dog demonstrated complete desiccation of the nucleus pulposus of the intervertebral disc and continued reduced calcification in regions of the epiphysis of vertebral bodies This study suggests that neonatal gene therapy does not prevent spine disease in MPS VII, which likely reflects poor diffusion of the large molecular weight GUSB (240 kD) into the relatively avascular spine The upregulation of destructive proteases in the spine in MPS VII dogs may be due to induction of signal transduction by GAGs via the TLR4 or the complement pathways, as proposed by others previously Inhibition of these enzymes or signal transduction pathways might be targets for treatment of spine disease in the future 441 AAV Mediated RNAi and Antisense Knockdown of Intranuclear DMPK Transcripts in DM1 Cells Syed-Rehan A Hussain,1 K Reed Clark.1 Center for Gene Therapy, The Research Institute at Nationwide Children’s Hospital, Columbus, OH Introduction: Myotonic dystrophy type (DM1) is the most common form of adult-onset muscular dystrophy and is caused by a Molecular Therapy Volume 20, Supplement 1, May 2012 Copyright © The American Society of Gene & Cell Therapy S171 MUSCULO-SKELETAL GENE & CELL THERAPY II CUG triplet repeat expansion (CUGexp) in the 3’-untranslated region of the DMPK gene Disease manifestations in DM1 are due to RNA splicing abnormalities caused by trans-splicing factor dysregulation As a consequence, DM1 patients demonstrate an increased presence of fetal protein isoforms for a host of alternatively spliced genes, which is thought to be responsible for the clinical disease Currently there is no effective treatment for DM1, therefore we are developing rAAV vector based therapeutic approaches to target the mutant CUGexp in human DM1 cells as an initial step to evaluate its therapeutic potential Our objective in this study was to obtain proof-of-concept (POC) in support of antisense or RNAi to target the expanded DMPK mutant transcript within the nucleus Methods: Self-complimentary rAAV vectors (either AAV2 or AAV8) with a U6 pol III promoter was used to drive expression of a [CAG]8 antisense RNA or short hairpin RNA constructs targeting the 3’ UTR of human DMPK (sc2577 and sc2683) The constructs co-express the eGFP reporter to monitor transduction efficiency DM1 fibroblast cells were converted to myotubes by forced MyoD over expression using either Ad5-MyoD transduction (moi=3,000)(Coriell Institute, NJ, USA) or doxycycline induction (AS308-DM1-hT-MyoD, a gift from Dr D Furling, Institut De Myologie, France) Differentiated cells were transduced with rAAV2.U6.shRNA vectors (moi = 104 vg) or rAAV8.U6.[CAG]8 (moi = 5x104 vg) Fluorescent in situ hybridization (FISH) using Cy3-[CAG]7-Ome probe was performed to quantify nuclear foci using ImageJ software qRT-PCR and northern blot analysis was performed on RNA isolated from nuclear and cytoplasmic fractions 5-10 days post-transduction Results: We consistently observed approximately 40-60% knockdown of DMPK expression in both nuclear and cytosolic fractions using rAAV2-shRNA vectors by RT qPCR Consistent with the qPCR data, northern blot analysis showed clear CUGexp transcript knockdown along with a 35% reduction in nuclear foci following rAAV2-shRNA vector transduction Moreover, rAAV8.U6.[CAG]8 vector transduction of immortalized DM1 cells resulted in 40-50% reduction of the DMPK transcript in total RNA by qPCR with concurrent reduction in CUG nuclear foci Conclusions: POC data was obtained demonstrating the ability of AAV vectors to target the intranuclear mutant DMPK transcript in DM1 patient cells We will now test the ability of these AAV vectors to reduce mutant DMPK RNA levels following local or systemic vector delivery in mouse models of DM1 442 An Approach for Systemic Delivery of Embryonic Stem Cells into Mouse Skeletal Muscle Shigemi Kimura,1 Kowashi Yoshioka,1 Shiro Ozasa.1 Child Development, Kumamoto University Graduate School, Kumamoto, Japan We established genetically engineered ES (ZHTc6-MyoD) cells that harbor a tetracycline-regulated expression vector encoding myogenic transcriptional factor MyoD, for the therapy of muscle diseases, especially Duchenne muscular dystrophy (DMD) Almost all the ZHTc6-MyoD cells were induced into muscle lineage after removal of tetracycline The undifferentiated ZHTc6-MyoD cells are Sca-1+ and c-kit+, but CD34-, all well-known markers for mouse hematopoietic stem cells In addition, they are able to maintain themselves in the undifferentiated state, even after one month of culture Therefore, it is possible to obtain a large quantity of ZHTc6-MyoD cells in the undifferentiated state that maintain the potential to differentiate only into muscle lineage Additionally, the cells were infected with lentiviral vector carrying GFP gene as maker We injected the infected cells into vein of mdx mice, a model mouse of DMD, for systemic delivery A few GFP positive myofibers were detected in the muscle of the mice In addition, GFP positive cells were isolated from skeletal muscle of the injected mice, and differentiated to muscle lineage Finally the myotubes beat Therefore, our ES cells have considerable therapeutic potential for treating muscle diseases S172 443 Autologous Muscle-Derived Stem Cell Treatment for Female Stress Urinary Incontinence: A 1-Year Follow-Up of Polish Investigation Klaudia Stangel-Wojcikiewicz,1 Monika Piwowar,3 Antoni Basta,1 Danuta Jarocha,2 Marcin Majka.2 Gynecology and Oncology, Jagiellonian University School of Medicine, Krakow, Poland; 2Transplantation, Jagiellonian University School of Medicine, Krakow, Poland; 3Bioinformatics and Telemedicine, Jagiellonian University School of Medicine, Krakow, Poland Purpose: We evaluated the effects of muscle-derived stem cells therapy among women with stress urinary incontinence (SUI) Material and methods: Muscle-derived stem cells were isolated from upper arm muscle biopsy from eighteen women with SUI Cells were expanded and after weeks injected trans urethral into the urethral rhabdosphincter of each women Endoscopic guidance procedure is a single-dose injection of a cell count in range of 0.9 up to 15 x 106 MDSC circumferential at 9, 12 and o’clock under local anesthesia Results: Initial results of SUI treatment with adult muscle-derived stem cells suggest that perspectives of this method are encouraging Conclusions: Stem cell therapy is promising to become minimally invasive method for reconstruction of the urethral rhabdosphincter muscle 444 Efficient Myoblast Expansion for Urinary Incontinence Treatment Danuta J Jarocha,1 Klaudia Stangel-Wojcikiewicz,2 Antoni Basta,2 Marcin M Majka.1 Department of Transplantation, Jagiellonian University School of Medicine, Krakow, Poland; 2Department of Gynecology and Oncology, Jagiellonian University School of Medicine, Krakow, Poland Cellular therapy using expanded autologous myoblasts of skeletal muscle biopsies is an evolving investigational treatment modality in regenerative medicine for urinary incontinence patients First report of using autologous myoblasts to treat urinary incontinence shows dependency of the therapeutic effect on the number of cells In our study we compared usefulness of commercially available medium with medium, designed by our group to obtain sufficient number of skeletal myoblasts Muscle biopsies were taken by needle biopsy under local anesthesia from women with Stress Urinary Incontinence Institutional review board approval and informed consent were obtained Satellite cells were isolated by enzymatic digestion Obtained cells were divided into two groups and expanded in commercial SKBM-2 medium or in medium composed by our group: DMEM/F-12 with dexamethasone, insulin, 18% FBS and growth factors HGF, EGF and FGF (DMEM/F12) and cultured in 5% CO2 and 95% humidity conditions with medium exchange every 2-3 days to the first passage and every 3-4 days with further passages Number of myoblasts colonies and cell number were assessed to evaluate proliferation potential Morphology of the cells was evaluated using inverted microscope Differentiation was induced by withdrawal of growth factors and low serum content to check if the expanded cells have ability to fuse Expression of myogenin, MYF-5 and myostatin genes was performed by Real-Time method Medium designed by us DMEM/F12 resulted in obtaining about 50% more myoblast colonies than when commercially available medium SKBM-2 was used Cultures led in DMEM/F12 medium resulted in times more cells than cultures led in SKBM-2 medium at first passage After second passage the difference in number of obtained cells was even higher reaching about times more cells in DMEM/F12 medium Finally after third passage the difference was the highest reaching times more cells cultured in DMEM/F12 medium Expression of myogenin, MYF-5 and myostatin genes did not differ significantly between Molecular Therapy Volume 20, Supplement 1, May 2012 Copyright © The American Society of Gene & Cell Therapy ... objective in this study was to obtain proof -of- concept (POC) in support of antisense or RNAi to target the expanded DMPK mutant transcript within the nucleus Methods: Self-complimentary rAAV vectors... injected the infected cells into vein of mdx mice, a model mouse of DMD, for systemic delivery A few GFP positive myofibers were detected in the muscle of the mice In addition, GFP positive cells. .. Cells were expanded and after weeks injected trans urethral into the urethral rhabdosphincter of each women Endoscopic guidance procedure is a single-dose injection of a cell count in range of