112. Novel Nonviral Plasmid Vectors With Minimalized Bacterial Backbones Dramatically Increase Transgene Expression Level In Vivo

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, 6 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 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 1 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 0 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 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 6 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 3 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