430 CEP290 minigene model of common splice site mutation in leber congenital amaurosis

430 CEP290 Minigene Model of Common Splice Site Mutation in Leber Congenital Amaurosis Molecular Therapy Volume 20, Supplement 1, May 2012 Copyright © The American Society of Gene & Cell Therapy S167[.]

NEUROLOGIC & OPHTHALMIC GENE & CELL THERAPY II Recent studies have shown that mutations in the CEP290 gene are the most common cause of LCA making it a major target gene in the development of new therapies The most frequent alteration is the intronic mutation c.2991+1655 A>G that creates a strong splice donor site and therefore creates the appearance of a cryptic exon in the messenger RNA of CEP290 This in turn modifies the reading frame and generates a premature stop codon In the present study, we investigated the feasibility of a therapeutic approach using exon skipping for the mutation c.2991+1655 A>G 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 NEUROLOGIC & OPHTHALMIC GENE & CELL THERAPY II myc and 3’ 3xFLAG epitope tags and conservative mutations near the end of exon 26 We have created cell lines with both wild-type and mutant minigenes as well as mutant transgenic mice The mutated and non-mutated versions of the minigene were introduced into 293T HEK cells using a retroviral vector Protein products derived from transcription and translation of exons 25, 26 and X (mutant) or exons 25, 26, and 27 (wild-type) would be 17.1 kb or 24.3 kb respectively Interestingly, only the 24.3 kb wild-type protein was detected, and it was found in both cells with the mutated and non-mutated versions of the minigene Mutant mRNA was detected only in cells with the mutant minigene, and wild-type transcripts were found in both cells with the wild-type and with the mutant minigene Using quantitative RT-PCR (TaqMan) to analyze cells expressing the mutant minigene, we found that wild-type transcripts outnumber mutant transcripts in both transiently transfected cells as well as cells stably expressing the mutant CEP290 minigene Transgenic mice were created by injection of 129S murine oocytes with a targeting vector comprising the CEP290 minigene under control of the murine rhodopsin promoter and followed by an internal ribosomal entry site (IRES) and eGFP cDNA Five transgenic founder animals (1 female and males) were created with transgenic copy numbers ranging from 10 to 186 All founders were bred to wild-type SV129 mice and the resultant litters were analyzed All but one founder produced pups that were positive for expression of mutant mRNA transcripts Most of the pups were also positive for eGFP expression as detected by fundoscopy and UV microscopy of retinal tissue sections All pups that were positive for mutant transcripts were also positive for expression of wild-type, but not mutant protein Finally, we performed quantitative RT-PCR (TaqMan) to quantify wild-type and mutant mRNA Unexpectedly, we found that the ratios of wild-type to mutant transcripts were skewed greatly in favor of wild-type transcripts These results are suggestive of nonsense-mediated decay and will be further investigated This work was performed as a part of the Project CEP290 Consortium 431 CCR5-Transduced Neural Stem Cells Confer Accelerated and Enhanced Therapeutic Effect on Experimental Autoimmune Encephalomyelitis Jingxian Yang,1 Yaping Yan,1 Ke Li,1 Bruno Gran,2 Hui Xu,1 Andro Zangaladze,1 Mark Curtis,3 Abdolmohamad Rostami,1 Guang-Xian Zhang.1 Neurology, Thomas Jefferson University, Philadelphia, PA; Neurology, University of Nottingham, United Kingdom; Pathology, Thomas Jefferson University, Philadelphia, PA Objective: To test the hypothesis that transducing a chemokine receptor on neural stem cells (NSCs) will enhance/accelerate their migration into CNS inflamed foci, thus producing a more rapid and enhanced therapeutic effect on experimental autoimmune encephalomyelitis (EAE) Methods: NSCs derived from bone marrow (BM) were transduced with CCR5, a receptor for CCL3, CCL4, and CCL5, all of which are abundantly produced in the CNS inflamed parenchyma of MS/EAE Their clinical and pathological effect after i.v injection to EAE mice, migration and distribution in the CNS, immunomodulation and in vivo neural cell differentiation were analyzed Results: The functional activity of CCR5-transduced NSCs was first confirmed by enhanced migration in vitro After i.v injection, these cells rapidly reached EAE foci, more effectively suppressed CNS inflammatory infiltration and myelin damage than GFP-NSCs, and suppressed clinical EAE Further, augmented remyelination and neuron/ oligodendrocyte repopulation were obtained in EAE mice treated with CCR5-NSCs than with PBS or GFP-NSCs Interpretation: The mechanisms underlying accelerated and enhanced effect of CCR5-transduced NSCs on EAE include: 1) rapid migration into the inflamed foci, thus more effectively exerting an immunomodulatory role, blocking further myelin/neuron S168 damage at an earlier stage, and creating a less hostile environment for remyelinating cells; 2) Increased exogenous NSCs in demyelinated foci may also participate in the remyelination/neural re-population process These effects, combined with the easy availability and autologous property of BM-NSCs (i.e., obtained from the patient’s own BM), may lay the groundwork for an innovative approach to rapid and highly effective MS therapy 432 Intraocular Gene Therapy with Erythropoietin: Identification of Therapeutic Doses Cody Richardson,1 Jessica Hines-Beard,1 Siddharth Desai,1 Scott Parker,1 Tonia S Rex.1 Ophthalmology, University of Tennessee Health Science Center, Memphis, TN Retinal degeneration is a long-term, progressive disease that can be caused by mutations in over 180 genes making it a good candidate for neuroprotective gene therapy Systemic gene delivery of erythropoietin (Epo) protects photoreceptor cells in the retinal degeneration slow mouse, but intraocular gene delivery was ineffective Our previous studies also show that the lack of efficacy was likely due to production of Epo at levels above the therapeutic dose range Our goal in this study was to identify therapeutic doses of Epo in the eye We used recombinant adeno-associated viral vector (rAAV) mediated delivery of a mutated form of Epo (Epo-mut) that is as neuroprotective as Epo, but has attenuated erythropoietic activity, under the control of the tetracycline inducible promoter Subretinal injections of the vector were performed in postnatal day 5-7 retinal degeneration slow mice that express the tetracycline transactivators (rtTA) from the retinal pigment epithelium specific vitelliform macular dystrophy promoter Immediately upon weaning, mice were treated with escalating doses of doxycycline via their drinking water At postnatal day 60, Epo-mut levels in the eye were quantified and photoreceptor protection was assessed As levels of doxycycline were increased, the amount of Epo-mut in the eye increased Further, there was an increase in the level of photoreceptor protection with increasing doses of doxycycline We have identified the low end of the intraocular dose range for Epo-mut in the eye 433 A Highly Effective Equine Infectious Anaemia Virus-Based Lentiviral Gene Therapy Platform for the Treatment of Ocular Diseases Kyriacos Mitrophanous,1 James Miskin,1 Katie Binley,1 Jackie de Belin,1 Julie Loader,1 Georgina Ferrige,1 Diana Angell-Manning,1 Marie Carlucci,1 Michelle Kelleher,1 Cherry Lucas,1 Scott Ellis.1 Oxford BioMedica (UK) Ltd, Oxford, United Kingdom Oxford BioMedica has developed ocular gene therapies based on the recombinant Equine Infectious Anaemia Virus (EIAV) Currently we have four ocular therapies: RetinoStat®, StarGen™, UshStat® and EncorStat®, for the treatment of age-related macular degeneration, Stargardt macula dystrophy, Usher Syndrome 1B, and the prevention of corneal transplant rejection respectively RetinoStat®, StarGen™ and UshStat® have received regulatory approval in the US and France (IND/CTA) and are currently in clinical evaluation The development of lentiviral vector-based gene therapies to treat eye diseases is an attractive option due to the vector’s innate ability to express therapeutic genes for extended periods, either to correct inherited disease, or to interfere with disease aetiology The accessibility of the eye both for administration and evaluating therapeutic benefit are significant advantages The lentiviral vector system has been extensively characterized in GLP safety and biodistribution studies following subretinal administration, as part of the RetinoStat®, StarGen™ and UshStat® programmes These studies were conducted in rabbits and NHP for up to months duration Subretinal administration of all three products caused only mild-to-moderate ocular inflammation Molecular Therapy Volume 20, Supplement 1, May 2012 Copyright © The American Society of Gene & Cell Therapy ... levels of doxycycline were increased, the amount of Epo-mut in the eye increased Further, there was an increase in the level of photoreceptor protection with increasing doses of doxycycline We... transcripts in both transiently transfected cells as well as cells stably expressing the mutant CEP290 minigene Transgenic mice were created by injection of 129S murine oocytes with a targeting vector... oocytes with a targeting vector comprising the CEP290 minigene under control of the murine rhodopsin promoter and followed by an internal ribosomal entry site (IRES) and eGFP cDNA Five transgenic