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427 Antisense Oligonucleotide Mediated Exon Skipping Restores Primary Cilia Assembly in Fibroblasts Harbouring the Common Leber Congenital Amaurosis CEP290 Mutation Molecular Therapy Volume 20, Supple[.]
NEUROLOGIC & OPHTHALMIC GENE & CELL THERAPY II showed a significantly higher number of regenerated axons compared with other conduits This corresponded to the increased neurotrophin-3 level seen in neural tissue implanted with the conduits Anti-IL-10 antibody staining showed Il-10 expression in the regenerated neural tissue implanted with conduits delivering polyplexed plasmids encoding IL-10 BBB scores increased significantly at weeks, but no significant change from weeks to weeks post-surgery This study suggests that multichannel collagen conduits provide structural guidance for neural tissue regeneration and a reservoir for sustained gene delivery Collagen neural conduits functionalized with a nonviral therapeutic gene can effectively improve spinal cord axonal regeneration 425 The Aurora A Kinase Inhibitor MLN8237 Significantly Enhances the Antitumor Activity of Oncolytic Measles Virus Derivatives in the Treatment of Glioblastoma Cory Allen,1 Ianko Iankov,1 Mateusz Opyrchal,1 Lucas A G Keith,1 Mark A Schroeder,1 Jann N Sarkaria,1 Jeffrey Ecsedy,2 Evanthia Galanis.1 Mayo Clinic College Of Medicine, Rochester, MN; 2Millenium Pharmaceuticals, Cambridge, MA Glioblastoma (GBM), the most common primary brain tumor in adults, has a dismal prognosis despite aggressive use of multi-modality treatment approaches Oncolytic derivatives of measles virus (a negative strand RNA virus) have significant antitumor activity against GBM lines and xenografts and are currently being tested in clinical trials Aurora kinases play a key role in regulating mitotic division and are frequently overexpressed in tumors including gliomas We therefore hypothesized that the combination of oncolytic measles virus derivatives with the Aurora A kinase inhibitor MLN8237 can further increase the antitumor activity of measles strains in glioma treatment Methods/Results: Three primary glioma xenograft lines derived from GBM patients (GBM6, 10 and 12) were treated with concentrations of MLN8237 (MLN) ranging from 0.004-1.0 uM in order to determine the IC50 per line and this concentration was used in in vitro combination experiments with the measles virus derivative MV-Lambda-NAP (MOIs 0.1 and 0.05) employing MTT assays Significant increase in cytotoxicity was observed in combination treated cells in all lines tested; this effect was independent of the virus / MLN sequence An orthotopic GBM6 model was next used to examine the in vivo activity of this approach Animals (n=8-10 mice per group) were treated with single agent MLN (30 mg/kg/day x 21days) versus single agent MV (3.6e5/dose x doses) versus the combination Control mice were treated with UV inactivated virus + MLN vehicle Survival was statistically increased in all treatment groups as compared to the control group (P=0.001, p=0.0002 and pG Our results indicate that after transfection of antisense oligonucleotides in patient cells, the amount of wild type CEP290 mRNA increases while that of the mutant transcript decreases Exon skipping also resulted in an increased amount of wild type protein and the restoration of ciliation in fibroblasts of affected patients Taken together, our results indicate that exon skipping represents a therapeutic strategy for bypassing the c.2991+1655 A>G mutation 428 The Development of Non-Viral Vectors for Choroideremia Richard P Harbotte,1 Elham Ostad-Saffari,1 Mariya Moosajee,1 Suet Ping Wong,1 Dhani Tracey-White,1 Tanya Tolmachova,1 Miguel Seabra.1 Molecular Medicine, Imperial College London, United Kingdom Purpose: Non-viral vectors are attractive alternatives to viral gene delivery systems due to their low toxicity, relatively easy production, and great versatility Recently, we developed a novel plasmid vector employing a scaffold/matrix attachment region (S/MAR) to provide functional episomal persistence within cells and clinically relevant levels of transgene expression, in vivo, without vector toxicity We will provide evidence for the utility of S/MAR vectors in the eye Methods: We have employed our S/MAR vector technology for the development of novel non-viral persistently expressing vectors for Choroideremia (CHM) gene therapy We have created a series of constructs with either the reporter genes EGFP and Luciferase encoded along with REP1, with various promoters, with and without an S/MAR element Results: We performed initial experiments to evaluate the expression of the transgenes in vitro in a range of cell lines and demonstrated that the S/MAR plasmids provide long-term transgene expression in vitro We subsequently optimised a procedure for the delivery and expression of these non-viral constructs in vivo via subretinal injection of formulated DNA We will present data showing the longitudinal transgene expression of constructs measured using a bioluminescence bioimager for over 12 months Additionally, long term expression of transgenes were also observed by rtPCR and Western blot analysis of protein levels We will also show that evidence for the persistent episomal maintenance of these vectors in the eye We show that the S/MAR motif in these vectors is essential for providing long term expression and maintenance in the eye We also demonstrate the lack of toxicity within the eye and show that fundus examinations as well as detailed histological examinations of retinal sections not elicit an inflammatory response to our plasmids once subretinally injected in the eye Conclusions: In conclusion, an ideal non-viral vector for gene therapy should be non-toxic, have mitotic stability, allow non-integrative establishment and provide persistent therapeutic expression levels The S/MAR-containing vector that we have developed and applied fulfils these requirements and provides proof of principle for a new class of vector systems for ocular gene therapy Molecular Therapy Volume 20, Supplement 1, May 2012 Copyright © The American Society of Gene & Cell Therapy 429 Adenoviral and Lentiviral Vectors for Efficient Gene Transfer to Mouse Retina Agostina Puppo,1 Giulia Cesi,1 Donna J Palmer,3 Pasquale Piccolo,1 Robin J Parks,4 Philip Ng,3 Nicola Brunetti-Pierri,1 Alberto Auricchio.1,2 TIGEM-Telethon Institute of Genetics and Medicine, Naples, Italy; 2Department of Pediatrics, Medical Genetics, Federico II University, Naples, Italy; 3Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, TX; 4Ottawa Hospital Research Institute, Ottawa, ON, Canada Photoreceptors (PR) are important targets of gene therapy as these are affected cells in inherited retinal degenerations (IRD) Several forms of IRD are due to defects in large genes which cannot be accommodated into AAV vectors, which so far have demonstrated the greatest potential in preclinical animal models and human clinical trials PR are refractory to transduction by the most studied Adenoviral (Ad) and Lentiviral (Lv) vectors, namely Ad5 and Lv-VSVG which have the unique ability to transfer large DNA sequences The use of Ad and Lv vectors would allow to either transfer therapeutic genes with large coding sequences or to deliver whole genomic loci including their endogenous regulatory regions Here, we aimed at the identification of Lv and Ad vectors with high PR tropism and transduction efficiency in preparation for further testing in animal models of severe inherited PR diseases In order to this, we collected 16 different Ad serotypes which were either isolated as naturally infectants of humans and chimpanzees or genetically capsid modified Ad5 We have also collected seven different Lv pseudotypes with heterologous envelope proteins The vector containing either a CMV-eGFP or lacZ expression cassette were injected in adult C57/Bl6 or Cd1 mice and harvested at either four or 14 days post-injection Transgene expression was evaluated by indirect ophthalmoscopy or by analysis of retinal sections to visualize eGFP or lacZ expression We identified five vectors either based on Ad serotypes or on Lv pseudotypes that appear promising for efficient murine PR transduction, showing a PR transduction efficiency higher than Ad5 and Lv-VSVG respectively Since the promoter used is ubiquitous, RPE, PR, and cells from the inner nuclear layer were transduced Some vectors achieved substantial levels and extension of PR transduction of up to 40-50% of the retinal sections To better define the potential of these vectors for retinal gene therapy, we are currently performing experiments using an expression cassette including the PR-specific rhodopsin promoter In addition, we will test these promising vectors in the pig retina, a large model retina which is highly cone-enriched 430 CEP290 Minigene Model of Common Splice Site Mutation in Leber Congenital Amaurosis Jeannette L Bennicelli,1 Vidyullatha Vassireddy,1 Jean Bennett.1 F.M Kirby Center for Molecular Ophthalmology, Scheie Eye Institute, University of Pennsylvania, Philadelphia, PA Mutations in the human CEP290 gene are a frequent cause of Leber Congenital Amaurosis, with the mutation c.2991+1655A>G often identified The mutation occurs bp downstream of a cryptic exon X, located in intron 26, and creates a strong splice donor site The result is the insertion of exon X and introduction of a premature stop codon immediately downstream of exon 26 To test various gene correction strategies that lead to correct mRNA splicing and expression of CEP290 protein, we created both cell lines and transgenic mice that contain a synthetic, mutant CEP290 minigene comprising the contiguous cDNA sequences of exons 25 and 26, followed by the entire intron 26 and exon 27 Both the CEP290 minigene and expressed CEP290 mini-protein contain engineered features that facilitate analysis of both mRNA splicing and protein expression and allow engineered wild-type and mutant products to be distinguished from potential endogenous products These include 5’ S167 ... resulted in an increased amount of wild type protein and the restoration of ciliation in fibroblasts of affected patients Taken together, our results indicate that exon skipping represents a therapeutic... rhodopsin promoter In addition, we will test these promising vectors in the pig retina, a large model retina which is highly cone-enriched 430 CEP290 Minigene Model of Common Splice Site Mutation in. .. followed by the entire intron 26 and exon 27 Both the CEP290 minigene and expressed CEP290 mini-protein contain engineered features that facilitate analysis of both mRNA splicing and protein expression