111 Exons 45 55 Skipping of Human Dystrophin Transcripts Using Cocktail Antisense Oligonucleotides Molecular Therapy Volume 22, Supplement 1, May 2014 Copyright © The American Society of Gene & Cell T[.]
GENE REGULATION AND TARGETS (PEI) with different degree of lipid substitutions The linoleic acid and caprylic acid substituted PEIs have given the most successful silencing of proteins To explore the potential cell cycle proteins as therapeutic targets, we screened an siRNA library in MDA-MB-231 and MDA-MB-435 cells using linoleic acid-substituted kDa PEI to deliver siRNA Out of 169 cell cycle protein targets, the siRNA against cell division cycle protein 20 (CDC20), RAD51, and serinethreonine protein kinase CHK1 diminished the cell growth most significantly in MDA-MB-435 cells These identified targets along with another well-studied cell cycle protein, kinesin spindle protein (KSP), were then evaluated in MDA-MB-435 and MCF7 cells using independently prepared siRNAs The synergistic effect was not seen in the combinational siRNA delivery of cell cycle proteins Surprisingly, the treatment of siRNA against cell cycle proteins stopped the cell proliferation in MDA-MB-435, but not in MCF7 cells However, quantitative-PCR and digital-PCR results indicated the down-regulation of mRNA transcript of cell cycle proteins in both cell-lines Moreover, flow cytometry results indicated similar uptake of siRNA-polymer complexes in both cells On the contrary, the uptake study by confocal microscopy suggested that the chosen polymer has delivered more complexes in MDA-MB-435 compared to MCF7 cells We speculate that MCF7 cells may have used different survival pathway and escaped the siRNA treatment, and conclude that linoleic acid-substituted PEI is more effective polymer for siRNA delivery to breast cancer cells 109 Designing miRNA Sequences for Huntington Disease Therapeutics Alejandro Mas Monteys,1 Ryan M Spengler,1 Matt S Wilson,1 Clayton K Oakley,1 Matt J Sowada,1 Beverly L Davidson.1 Internal Medicine, Neurology and Molecular Phisiology & Biophisics, University of Iowa, Iowa City, IA Huntington’s disease (HD) is a fatal autosomal dominant neurodegenerative disease caused by the expression of a polyglutamine-expanded form of the huntingtin (Htt) protein There is no cure for HD, but reducing the expression of the mutant Htt with RNA interference (RNAi) offers the possibility of a new treatment by directly targeting the disease-causing gene We previously developed a strategy to design miHDS1, an artificial miRNA sequence against Htt with high on-target silencing efficacy, and minimized off-target potential over unintended genes Previous studies in non-human primates demonstrated that miHDS1 expression is tolerable when expressed in caudate and putamen As a requirement for moving into humans, testing in normal mice is required, even though the sequence is designed to be safest in human brain To fulfil this requirement, we injected wild type mice with AAV vectors to direct miHDS1 expression in mouse striatum, and performed motor coordination studies, immunohistochemical studies and biochemical analyses to assess miHDS1 tolerability We found that, in contrast to monkeys, neurological deficits developed months after AAV injection, and were attributed to off-target silencing We subsequently developed several strategies to resolve miHDS1 toxicity to address the conundrum of preclinical testing of nucleic acid-based therapies designed to be safe in the context of a human genome, in a different species, while maintaining miHDS1-silencing efficacy In summary, our study highlights how a miRNA safety and tolerability profile is species specific, and emphasizes the careful interpretation of initial studies using mouse models of disease, and show putative strategies to resolve off-target toxicity associated with miRNA sequences Gene Regulation and Targets 110 RT-Aptamer Interactions Govern Antiviral Specificity and Viral Encapsidation of Anti-HIV RT Aptamers Margaret J Lange,1 Phuong D M Nguyen,2 Donald H Burke.1,2 Molecular Microbiology & Immunology, University of Missouri, Columbia, MO; 2Biochemistry, University of Missouri, Columbia, MO Our laboratory uses RNA aptamers that target HIV reverse transcriptase (RT) to understand HIV biology and inhibit the replication of the virus RNA aptamers bind at low nanomolar concentrations and significantly inhibit RT in biochemical assays and in cell culture models of HIV infection where the aptamer is packaged within the viral particle These qualities make aptamers attractive therapeutic candidates and useful tools for dissecting viral pathogenesis However, the interactions between aptamer and virus that govern antiviral specificity and aptamer encapsidation into the viral particle have not been defined We hypothesized that the aptamer interacts specifically with the RT (p66-p66 homodimer) in the cytoplasm of the producer cell, and that this interaction allows the aptamer to encapsidate while bound to the RT and then inhibit RT activity in the newly infected target cell To test this hypothesis, we exploited our observation that aptamers inhibit only specific subtypes of HIV RT We engineered recombinant reporter virus plasmids to encode phylogenetically diverse HIV RT sequences (subtypes A and B, Group O, and HIV-2) The rest of the viral backbone is held constant to rule out contributions from other viral components Each of the proviral constructs exhibited normal DNA replication and protein production kinetics and infection efficiencies We tested aptamers from several different secondary structural families against each of the new proviral constructs We found that aptamer-mediated inhibition of the constructs correlated with our observed biochemical results For example, aptamer 70.05 was found to significantly inhibit RT from viral strain HXB2 (Group M, subtype B), but not RT from strain MVP5180 (Group O) When aptamer 70.05 was tested against the corresponding proviral constructs, 70.05 inhibited the provirus expressing HXB2, but not MVP5180 RT In addition, we found that a single point mutation of R277K in the provirus expressing HXB2 RT was enough to completely abolish inhibition for some of our aptamers, matching our previous biochemical results These results correlated positively with aptamer encapsidation levels within the virus Thus, this work demonstrates that the aptamer-RT interaction governs antiviral specificity and viral encapsidation of anti-HIV RT aptamers Notably, these experiments not only demonstrate that the aptamer-RT interaction is required for inhibition, but they are the very first demonstration of aptamer-specific viral resistance in cell culture by a viable virus 111 Exons 45-55 Skipping of Human Dystrophin Transcripts Using Cocktail Antisense Oligonucleotides Yusuke Echigoya,1 William Duddy,2 Joshua Lee,1 Vincent Mouly,2 Toshifumi Yokota.1,3 Department of Medical Genetics, University of Alberta Faculty of Medicine and Dentistry, Edmonton, AB, Canada; 2Therapies for Striated Muscle Disease, Institute of Myology, Paris 6, Paris, France; 3Muscular Dystrophy Canada Research Chair, Edmonton, AB, Canada Duchenne muscular dystrophy (DMD), one of the most common fatal genetic disorders, is caused by mutations in the dystrophin (DMD) gene Currently, antisense oligonucleotide (AO)-mediated exon skipping, which restores the reading frame by eliminating specific exons, is a promising therapeutic approach for DMD Molecular Therapy Volume 22, Supplement 1, May 2014 Copyright © The American Society of Gene & Cell Therapy S41 GENE REGULATION AND TARGETS Remaining challenges of the current single exon skipping are the restricted applicability to patients and unclear stability/function of resulting in-frame short dystrophin proteins A potential solution is multiple exon skipping targeting exons 45-55 at the mutation hot spot region It is particularly noted that skipping of exons 45-55 could treat approximately 63% of DMD patients with deletion mutations in theory In addition, the entire exons 45-55 deletion is reported to be mostly associated with milder symptoms or an asymptomatic course, indicating that the shortened dystrophin lacking this specific region is more stable/functional Recently, we have demonstrated a proof-of-concept of exons 45-55 skipping with cocktail AOs in a dystrophic mouse model However, sequence-specific AOs have to be optimized for the human DMD gene In the present study, we designed AOs for the human dystrophin transcript with a new predictive tool Immortalized human DMD skeletal muscle cells harboring deletion mutations within exons 45-55 (deletion of exons 45-52, exons 48-50, and exon 52) were transfected with cocktails containing 3, or 10 phosphorodiamidate morpholino oligomers (PMOs) using EndoPorter transfection reagent Skipped dystrophin transcripts with the junction of exons 44 and 56 were observed in all the DMD cell lines we tested, accompanied by dystrophin protein expression We demonstrate for the first time that skipping of the entire exons 45-55 region is feasible by using the optimal AO cocktails in the human DMD gene Our observation will facilitate clinical development of exons 45-55 skipping therapy for the treatment of DMD 112 Novel Nonviral Plasmid Vectors With Minimalized Bacterial Backbones Dramatically Increase Transgene Expression Level In Vivo Jeremy Luke,1 Aaron Carnes,1 Frank Lay,2 Donald Rees,2 Lixin Liu,2 Guy Marti,2 John Harmon,2 James Williams.1 R&D, Nature Technology Corporation, Lincoln, NE; 2Department of Surgery and Hendrix Burn/Wound Laboratory, Johns Hopkins University, Baltimore, MD Extensive research to improve plasmid delivery has resulted in advanced methods such as electroporation and needle free jet injectors that dramatically improve gene transfer compared with naked DNA delivery However further increases in transgene expression level are needed to meet efficacy requirements for various non viral gene therapy and DNA vaccination applications We report herein that reduction of the spacer region linking the 5’ and 3’ ends of the transgene expression cassette to less than 500 bp remarkably increases plasmid-mediated transgene expression Expression improvement is not promoter, tissue or transgene specific These minimalized new generation vectors, NanoplasmidsTM, utilize antibiotic free RNA-OUT antisense RNA selection (150 bp antisense transcription unit) and further replace the traditional large self replicating 1000 bp pUC replication origin with novel temperature inducible specialized ColE2 (100 bp) or R6K (300 bp) derived miniorigins Novel heat inducible high copy ColE2 or R6K replication protein expressing host strains have been developed for selection and propagation of ColE2 or R6K miniorigin NanoplasmidsTM This is an additional Nanoplasmid™ safety factor since miniorigin vectors can only replicate within the engineered Rep protein expressing E coli host strain NanoplasmidTM production is also high yielding: plasmid titers of up to g/L have been obtained in the HyperGROTM fermentation process RNA-OUT-R6K (466 bp spacer region) and RNA-OUT-ColE2 (281bp spacer region) NanoplasmidsTM have dramatically improved in vivo transgene expression after intramuscular and intradermal delivery (Fig 1) compared to conventional plasmid vectors Improved expression after intradermal delivery is also observed with NanoplasmidsTM with bp spacer regions (RNA-OUT-R6K or RNA-OUT-ColE2 cloned within an intron of the transgene expression cassette; 3’ untranslated region encoded R6K miniorigin, S42 intron encoded RNA-OUT) or a 300 bp spacer region (spacer region encoded R6K miniorigin, intron encoded RNA-OUT) These results suggest improved expression level is a general property of short spacer region non viral vectors While the mechanism to explain transgene expression enhancement is unknown, it may be the result of reduced formation of inhibitory chromatin on nontranscribed spacer region sequences These novel vectors have exciting application to improve non viral gene therapy and DNA vaccine performance and safety 113 Lentivirus Mediated Knockdown of SENP1 Inhibits Tumorigenic Properties and Enhances Chemosensitivity of Chronic Myeloid Leukemia Stem/Progenitor cells Huiyan Sun,1 Jun Xu,1 Fengjun Xiao,1 Yue Yin,1 Hua Wang,1 Lisheng Wang.1 Department of Experimental Hematology, Beijing Institute of Radiation Medicine, Beijing, China Small ubiquitin-like modifier (SUMO) modification plays important roles in protein function regulation SENP1 (SUMO-specific protease 1) is a key enzyme in the protein sumoylation regulation, which can affect the cell cycle, proliferation and differentiation However the function of SNEP1 in regulating leukemic stem/progenitor cell growth is not well understood Using microarray-based mRNA profiling we discovered that significantly up regulation of SENP1 in CML compared to normal CD34+ cells QPCR confirmed expression of SENP1 highest in purified stem cell population isolated from CML Retrovirus mediated bcr-abl transduction increase the SENP1 expression in cord blood CD34+ cells Transduction of lentivirus expressing shRNA against SENP1 displayed significant inhibition of its expression in both normal and CML blood CD34+ cells CML CD34+ cells transduced SENP1-shRNA generated obviously decreased numbers of cells (11 fold) in culture with SCF, IL-3, GM-CSF, G-CSF and EPO (GEMM culture) for days Whereas normal CD34+ cells transduced SENP1-shRNA generated modestly decreased numbers of cells (1.7 fold) Meanwhile, SENP1 inhibition reduces the erythroid differentiation of CML CD34+ cells cultured in GEMM for days (GPA+ cells: 47.9% to Ctrl vs 6.9% to SENP1 shRNA) SENP1-shRNA transduction promoted the apoptosis of CML CD34+ cells significantly SENP1 knockdown also enhances chemosensitivity of TF-1 cells harbored BCR-ABL T315I mutant to Imatinib (Imatinib induced apoptosis: 7.1% to ctrl-shRNA vs 58.1% to SENP1-shRNA) Similar results were obtained in CML CD34+ cells, K562 cells and KCL22 cells transduced with SENP1-shRNA We also investigated that SENP1 inhibition induces sumoylation of Stat5 in CML cells Our results indicated that SENP1-shRNA transduction Molecular Therapy Volume 22, Supplement 1, May 2014 Copyright © The American Society of Gene & Cell Therapy ... solution is multiple exon skipping targeting exons 45- 55 at the mutation hot spot region It is particularly noted that skipping of exons 45- 55 could treat approximately 63% of DMD patients with deletion... for the human dystrophin transcript with a new predictive tool Immortalized human DMD skeletal muscle cells harboring deletion mutations within exons 45- 55 (deletion of exons 45- 52, exons 48-50,... have demonstrated a proof -of- concept of exons 45- 55 skipping with cocktail AOs in a dystrophic mouse model However, sequence-specific AOs have to be optimized for the human DMD gene In the present