240 spacio temporal mutagenesis in muscle by using AAV cre vectors: the example of the mtm1 gene

240 Spacio Temporal Mutagenesis in Muscle by Using AAV Cre Vectors The Example of the Mtm1 Gene Molecular Therapy Volume 21, Supplement 1, May 2013 Copyright © The American Society of Gene & Cell Ther[.]

GENE REGULATION technology, and followed by their which was then transplanted into a subcutaneous space of mice Transplantation of hFIX-transduced ADSC sheets (1 to sheets) was found to be successfully engrafted in mice, and we observed a linear increase in plasma hFIX levels proportional to the number of transplanted sheets The recipient mice that received ADSC sheets demonstrated plasma hFIX level of 33.1 ± 6.5 ng/ml at day 1, This plasma hFIX level of mice that received subcutaneous injection of ADSCs in suspension at the same cell number to the ADSC sheets was 17.1±3.1 ng/ml, which was significantly lower than the that of mice with ADSC sheets The plasma hFIX levels were found to decrease as a function of time, which was correlated with histological findings that the hFIX-positive ADSCs tended to diminish for several weeks The reasons for this hFIX decline are currently under investigation The present study showed that our tissue engineering approach based on transplantation of cell sheet format of SIV-transduced ADSCs could provide plasma hFIX levels in living mice Further modifications toward achieving longevity of the ADSC sheets and their higher transgene expression levels would establish this approach to be therapeutically valuable 238 Targeted PLGA Nanoparticles Encapsulating Histone Deacetylase Inhibitor, Panobinostat Effectively Control T Cell Leukemia Boyoung Choi,1 Jangwook Lee,1 Jeong-A Shin,1 Dahye Lee,1 Kuen Yong Lee,1 Priti Kumar,2 Sang-Kyung Lee.1 Bioengineering, Hanyang University, Seoul, Republic of Korea; Internal Medicine, Yale University, New Haven High levels of expression of histone deacetylases (HDACs) and hypo-acetylation of histone proteins are related with uncontrolled cell proliferation, differentiation, and pathogenesis of cancer Thus, histone deacetylase inhibitors (HDACi) can potentially be used as anti-cancer drugs HDACi are known to increase apoptosis of cancer cells by obstructing activity of HDACs, and increasing the accessibility of transcription factors for re-activating repressed tumor suppressor genes An opportunity for improving HDACi activity is through the use of delivery systems that allow targeted access to cell and tissue types that are not normally a characteristic of the incorporated drug In this study, we tested the efficacy of the HDACi, Panobinostat (LBH589) against human T cell leukemia through encapsulation in the biodegradable polymer, PLGA using a single chain variable fragment (scFv) antibody directed to the human CD7 molecule that is exclusively expressed on human T cells to achieve specific delivery of the drug The T cell-specific scFvCD7-conjugated drug-encapsulated PLGA nanoparticles - CD7-PLGA/LBH589 sizing at 200nm and loaded at 70% capacity, Further, systemic delivery of the formulation to tumors generated with the T cell leukemia line in immunodeficient mice controlled tumor growth through apoptosis induction through p21 gene expression, blockade of HDAC phosphorylation and acetylation of histone proteins 239 Radiosensitivity of Human Induced Pluripotent Stem Cells (hiPSC) Detectable by Cell Cycle Analysis Frank Houghton,1 Michael W Epperly,1 Xichen Zhang,1 Vishwajit Nimgaonkar,2 Joel S Greenberger.1 Radiation Oncology, University of Pittsburgh Cancer Institute, Pittsburgh, PA; 2Psychiatry, University of Pittsburgh Medical Center, Pittsburgh, PA Human induced pluripotent stem (hiPSC) cells are derived from differentiated somatic cells, such as fibroblasts, by over-expression of a suite of genes (OCT4, SOX2, NONOG and LIN28) Morphologically, and genetically these induced cells are indistinguishable from embryonic stem cells hiPSCs may have clinical potential in the areas of treatment of diseases through transplantation or repair; S92 in basic research in elucidating the factors controlling normal and abnormal differentiation; and in drug screening to identify genotoxic compounds hiPSCs may be also valuable in identifying new small molecule radiation mitigators and protectors We used cell cycle analysis for the determination of ionizing irradiation induced apoptosis and alteration of cell cycle distribution hiPSC cells were grown using mTeSR1 medium (Stem Cells Technology) on matrigelcoated 6-well plates in a humidified, 37oC incubator supplemented with 5% CO2 The hiPSC colonies were manually selected using a 200ul pipetter tip every 6-8 days Once selected, these colonies were gently agitated to reduce their size and replated in a new matrigelcoated 6-well plate The hiPSC colonies and the parent fibroblast cell line were irradiated to 0, 0.5, 1.0, 1.5, 2.0 and 2.5 Gy using a Varian linear accelerator with a dose rate of 300 cGy/min The 6-well plates were returned to the incubator The colonies were harvested at 24 and 48 hours post-irradiation as single cell suspensions using 0.05% Trypsin-EDTA (Invitrogen) and fixed in cold 70% ethanol Later, the cells were stained with propidium iodide and sorted using an Accuri C-6 flow cytometer For hiPSC cells by twenty-four hours after 1.0 Gy irradiation, the percent apoptosis increased from control levels of 7.4 + 1.7% to 21.5 + 3.6% (p = 0.0069), but decreased by 48 hrs to 18.0 + 4.1% (p = 0.0297) attributable to cell death In contrast, 1.0 Gy irradiation of the parent fibroblast line induced no detectable change in the percent apoptotic cells compared to nonirradiated cells (2.6 + 1.4%) Irradiation of parent fibroblasts to 2.5 Gy also showed no change in percent apoptosis at 24 or 48 hr (1.9 + 0.5 or 7.1 + 3.9%, p = 0.6841 or 0.384, respectively) While there was decreased viability in irradiated hiPSC cells after doses as low as 1.5 Gy (24.4 + 6.2%) compared to Gy control cells (49.3 + 5.4, p = 0.0250) No significant changes in viability were detected with parent fibroblasts after doses up to 2.5 Gy There was clear alteration in cell cycle distribution in irradiated hiPSC cells: a G1 phase arrest was seen 48 hr after 2.0 and 2.5 Gy with an increase in G1 cells from 32.5 + 3.9% for Gy to 47.0 + 3.6% or 45.1 + 4.4 (p = 0.0253 or 0.0275, respectively) In contrast parent fibroblasts showed no significant cell cycle changes at 24 or 48 hr after either irradiation dose Human iPSCs present a valuable system in which to identify the molecular signaling pathways controlling radiosensitivity 240 Spacio-Temporal Mutagenesis in Muscle by Using AAV-Cre Vectors: The Example of the Mtm1 Gene Romain Joubert,1 Alban Vignaud,1 Mickaël Le,1 Christelle Moal,1 Nadia Messaddeq,2 Anna Buj-Bello.1 R&D, Généthon, Evry, France; 2IGBMC, Illkirch, France Manipulation of the mouse genome by site-specific mutagenesis using transgenic animals has been extensively used to study gene function and model human disorders Mouse models of myotubular myopathy (XLMTM), a severe congenital muscular disorder due to loss-of-function mutations in the MTM1 gene, have been generated by homologous recombination and shown that myotubularin is essential for skeletal muscle However, since the Mtm1 deletion occurred constitutively or shortly after birth in these mice, it is not known whether myotubularin is required during adulthood, an important issue not only in the context of muscle biology but also therapies To delete the Mtm1 gene in adult muscle fibers, we constructed a recombinant adeno-associated vector (AAV) that expresses the Cre recombinase under the muscle-specific desmin promoter We report that a single injection of this vector into muscles of month-old Mtm1 conditional mice leads to a myotubular myopathy phenotype with myofiber atrophy, disorganization of organelle positioning and severe muscle weakness We establish the proof-of-concept that myotubularin is required for the proper function of skeletal muscle during adulthood, and provide a valuable tissue model that is useful to study pathogenesis and evaluate therapeutic strategies Therefore, Molecular Therapy Volume 21, Supplement 1, May 2013 Copyright © The American Society of Gene & Cell Therapy GENE REGULATION we demonstrate the power of utilizing an AAV-delivered Cre to temporally and spatially control gene knock out in muscle, allowing flexible development of localized disease models 241 Synergistic and Tunable Gene Activation by Combinations of Synthetic Transcription Factors Pablo Perez-Pinera,1 David G Ousterout,1 Jonathan M Brunger,1,3 Alicia M Farin,5 Katherine A Glass,1,3 Farshid Guilak,1,3 Gregory E Crawford,2,6 Alexander J Hartemink,2,4 Charles A Gersbach.1,2 Department of Biomedical Engineering, Duke University, Durham, NC; 2Institute for Genome Sciences and Policy, Duke University, Durham, NC; 3Department of Orthopaedic Surgery, Duke University Medical Center, Durham, NC; 4Department of Computer Science, Duke University, Durham, NC; 5Department of Medicine, Duke University Medical Center, Durham, NC; Department of Pediatrics, Duke University Medical Center, Durham, NC Gene therapies that use engineered transcription factors to regulate expression from endogenous genetic loci offer several advantages over exogenous gene delivery, including the ability to up-regulate all splice variants of a gene of interest Zinc-finger transcription factors (ZF-TFs) for different applications have been tested in humans and to date have an excellent safety profile However, in some cases ZFTFs have failed to induce robust expression that is sufficient to drive a therapeutic effect Furthermore, the assembly of ZF arrays can be costly and laborious and target sites are not always available The recent emergence of technologies for cloning transcription activatorlike effectors (TALEs) targeted to almost any DNA sequence provides a unique opportunity for engineering artificial transcription factors In the current study, the combinatorial regulation of endogenous mammalian genes in their natural chromosomal context is achieved by engineering several TALE transcription factors (TALE-TFs) to bind nearby sites upstream of the transcriptional start site for a target gene The composition of these combinations of independent TALE-TFs can be manipulated to control gene networks Synergistic regulation of gene expression by multiple transcriptional activators is known to occur via simultaneous binding and stabilization of components of the pre-initiation complex Building on this model, we activated endogenous genes with combinations of engineered transcription factors and could tune gene expression levels by systematically varying the components of the combinations The results of these experiments postulate new guidelines to achieve activation of endogenous gene expression by artificial transcription factors, and provide a new experimental platform for gene therapy, functional genomics, and synthetic biology 242 A Novel Enhancer Increases the Level and Duration of Transgene Expression in Mice Chunbo Zhang,1 Leping Li,2 Dexi Liu.1 Pharmaceutical and Biomedical Sciences, University of Georgia College of Pharmacy, Athens, GA; 2Biostatistics Branch, The National Institute of Environmental Health Sciences, Research Triangle Park, NC Bioinformatical analysis on human genomic database was performed to search for DNA sequences capable of enhancing transgene expression in mice driven by an albumin promoter Eleven fragments were identified (chr10:7744996-7745538, chr10:77424717742955, chr15:68592682-68592981, chr2:179334272-179334625, chr2:228678070-228678739, chr10:92691195-92691594, chr12:110386904-110387283, chr2:227424228-227424661, chr5:95200854-95201312, chr7:75482100-75482483, and chrX:109073944-109074495) and cloned into pLive plasmids (Mirus Bio, MIR5420) containing mouse interleukin 10 gene (mIL10) as a Molecular Therapy Volume 21, Supplement 1, May 2013 Copyright © The American Society of Gene & Cell Therapy reporter The level and persistency of mIL10 gene expression in mice after hydrodynamic gene delivery were compared between the parent and new plasmid constructs We demonstrated that mIL-10 expression was 3x higher than the parent plasmid with a plasmid containing an enhance sequence from chrX:109073944-109074495 fragment The expression levels of mIL-10 from plasmid containing DNA fragments of chr10:7742471-7742955 and chr15:68592682-68592981 were similar to that of parent pLIVE-mIL10 vector, while expression level resulted from other plasmids was lower The duration of mIL-10 expression in 11 plasmids was similar to that of pLIVE Sequence analysis of chrX:109073944-109074495 fragment reveals presence of DNA binding elements for liver specific transcription factor HNF4 alpha, HNF4 gamma, C/EBP-alpha, FOXA1, and FOXA2 These results suggest that chrX:109073944-109074495 fragment is a novel enhancer for high level and long-term gene expression in mouse liver 243 Design and Screening of Custom Zinc Finger Transcription Factors To Modulate Expression of Genes Controlling Cell Fate Ashley L Fischer,1 Qingzhou Ji,1 Erik R Eastlund,1 Mark A Gerber,1 Carol A Kreader,1 Scott W Knight.1 Life Sciences Research and Development, Sigma-Aldrich, Saint Louis, MO Modulating the expression of specific genes has been and continues to be a significant area of focus in the effort to ascertain gene function Development of new methods that allow researchers to control the expression of specific genes will impact the future of personalized medicine and molecular-based therapies Here, we demonstrate the use of ZF-TFs to specifically activate both coding and non-coding genes that regulate cell fate and development We show that target binding site and effector domain selection can be key factors in the ability to alter a given gene’s level of activity Under optimal conditions, we have been able to increase specific gene expression by a range of to over 70-fold, and have achieved these results in multiple cell lines Native gene activation with ZF-TFs ensures endogenous processing of all spliceoforms and allows for activation of long transcripts that are less amenable to cDNA cloning ZF-TFs also allow researchers to finely tune gene expression, rather than rely on forced overexpression of a cDNA when it is not needed, and thus provide a novel tool to approach research, medicine and patient treatment 244 TaqMan® Assays for Assessment of Genome, Epigenome and Quality of Pluripotent Stem Cells Andrew Fontes,1 Rene H Quintanilla,1 Jeffrey Fergus,1 Uma Lakshmipathy.1 Primary and Stem Cell Systems, Life Technologies, Carlsbad, CA The ability to generate footprint-free iPSC using platforms such as the CytotuneTM iPS Sendai Reprogramming Kit has created patient specific models for pathway and disease study providing traction for the translation of disease research The bottleneck in this process has now shifted from efficient methods of generation, to a lack of throughput tools for characterization that easily incorporate into the iPSC workflow Traditional characterization methods of iPS and ES cells neccisistate combination of in vitro and in vivo cellular analysis to confirm pluripotency and demonstrate differentiation potential into cell types representative of the three germ layers while maintaining a normal karyotype These methods are laborious with subjective measures and not amenable to high throughput analysis Molecular analysis platforms offer an appealing alternative for rapid generation of quantitative data to confirm quality of the generated clones based on genome and epigenome expression patterns Here, we have utilized TaqMan OpenArray qPCR and Taqman Array Human S93 ... endogenous genes with combinations of engineered transcription factors and could tune gene expression levels by systematically varying the components of the combinations The results of these experiments... (TALE-TFs) to bind nearby sites upstream of the transcriptional start site for a target gene The composition of these combinations of independent TALE-TFs can be manipulated to control gene networks.. .GENE REGULATION we demonstrate the power of utilizing an AAV- delivered Cre to temporally and spatially control gene knock out in muscle, allowing flexible development of localized