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536 Long Lasting Vision Following One Time Gene Therapy Partnership of Brain and Retina Revealed By fMRI Molecular Therapy Volume 22, Supplement 1, May 2014 Copyright © The American Society of Gene &[.]
SENSORY (OPHTHALMIC AND AUDITORY) DISEASES 534 Evaluating Competition of Delivered Small Hairpin RNAs with Endogenous microRNAs for Safe Effective Gene Knockdown Paul N Valdmanis,1 Shuo Gu,2 Leszek Lisowski,1 Lan Jin,2 Kirk Chu,1 Feijie Zhang,1 Yong Huang,1 Mark A Kay.1 Pedatrics and Genetics, Stanford University, Stanford, CA; Center for Cancer Research, National Cancer Institute, Frederick, MD RNA interference (RNAi)-based therapeutics have the potential to revolutionize the way medicine, in particular personalized medicine, is provided and delivered Knockdown of a disease gene by small hairpin RNA (shRNA) delivery involves a delicate ratio between effective concentrations of the shRNA within the cell and enabling sustained normal activities within the cellular milieu Too much expressed shRNAs can lead to toxicity and lethality in mice; thus an evaluation the toxicity of existing delivery mechanisms is relevant in this context such that this toxicity can be recognized and avoided We have harnessed the potential of high throughput sequencing to identify an endogenous signature of shRNA toxicity in the liver following adeno-associated viral vector delivery and are working to establish the downstream effects that result from this toxicity Mice were monitored for elevated serum enzymes indicative of toxicity and the livers of these mice were subjected to high throughput sequencing of small RNAs including microRNAs As the level of small RNAs associated with our delivered vectors increased, we noted a concomitant decrease in the liver-specific microRNA, miR-122, while all other microRNAs were unaffected in terms of relative expression in the liver Upon evaluation of length differences of mature miR-122-5p we identified a specific loss of the 22 nucleotide isoform of miR-122 in toxic liver samples RNA sequencing of the same livers indicates that predicted and validated targets of miR-122 are de-repressed in affected livers leading to displaced metabolic pathways, in particular related to glucose and cholesterol metabolism We have validated some of these miR-122 targets and are evaluating how the perturbation of these genes relates to the cell death and toxicity we observe By understanding the endogenous mechanisms that are compromised in shRNA delivery, we can ensure that they are monitored and sustain their expression to ensure safe and effective RNAi gene therapy We systematically determined the relative normal/mutant allele discriminating knockdown activity of a series of constructs by sequential evaluation of guide strand position effect The single nucleotide mutation at positions 2-5 of the guide strand to be the most effective Similar constructs were made and tested for G12D, G12V, G12R (comprising 91% of PDAC KRAS mutations) and G12C mutations (2.9%) G12D and G12C selective bi-shRNA expression vectors did not inhibit HEK-293 (KRASwt) growth in contrast to cell growth reduction of PANC-1 (KRASwt/G12D) and MiaPaCa2 (KRASwt/ G12C ) By adopting the miR-17-92 polycistronic miRNA backbone, multiplex knockdown targeting G12D, G12C and G12R were determined to be effective for multiple expressive KRAS mutant alleles with a single vector construct A second multiplex construct targeting G12C, G12D and G12V achieved similar results Mice are a biorelevant species for the triple bi-shRNA KRAS lipoplex Preliminary safety testing revealed no toxic effect in vivo following a single intravenous dose of liposome encased bi-shRNA KRAS triplexes In vivo efficacy studies with PANC-1 or MIAPaCa-2 pancreatic cancer models will be reported Application of this therapeutic formulation with combination to EGFR targeting agents will also be examined Sensory (Ophthalmic and Auditory) Diseases 536 Long-Lasting Vision Following One-Time Gene Therapy: Partnership of Brain and Retina Revealed By fMRI 535 In Vivo Study of a Bi-Functional ShortHairpin RNA (bi-shRNA) Targeting Multiple SingleNucleotide KRAS Mutations in Pancreatic Cancer Manzar Ashtari,1 Amanda Viands,1 Kathleen Marshall,1 Kenneth Shindler,2 Albert Maguire,2 Jean Bennett.2 Children’s Hospital of Philadelphia, Philadelphia; 2University of Pennsylvania, Philadelphia KRAS is a member of guanine nucleotide-binding protein family and acts as a key molecular switch regulating multiple intracellular signaling pathways including epidermal growth factor receptor (EGFR) KRAS mutations essentially result in constitutively active KRAS and unregulated downstream signaling The majority of pancreatic ductal adenocarcinoma (PDAC) patients carry a singlenucleotide KRAS mutation, >90% of which are single amino acid substitutions of Glycine at codon 12 in exon The five-year survival rate of PDAC is less than 5% KRAS mutation-specific targeting remains elusive and recent small molecule therapeutics demonstrates weak affinity or rapid degradation We have recently developed a novel bi-functional shRNA (bi-shRNA) RNA interference (RNAi) technology that achieves in vitro selective knockdown of mutant KRAS expression (Figure 1) Background:Human LCA2 patients treated with subretinal gene therapy (GT) have shown dramatically improved and stable vision for a long time These improvements have been mostly attributed to the rescued retinal cells, but vision develops from combined work between the eye and brain and the role that the brain plays in the success of GT is not well studied Utilizing fMRI, we previously showed that GT enhances visual cortex responsiveness to visual inputs In this report, we examine the long-term effects of GT on plasticity of the visual pathway and the role it may play in augmenting vision Methods: Follow-up neuroimaging studies were performed in six LCA2 patients who underwent a second GT in their untreated eye Subjects received fMRI before the second eye GT (baseline), 1,3, and months as well as 1,2, and years after re-administration Visual stimuli consisted of high contrast checkerboard, and to account for variability in disease fMRI data was analyzed individually Results: None of the subjects showed significant activation within the primary visual cortex (V1) on their baseline fMRI, except one young participant One month after GT, results showed dramatic increases in V1 activation that continuously improved over time Consistent with their cortical activation patterns, subjects showed no Donald D Rao,1,3 Chris M Jay,1,3 Zhaohui Wang,1,3 Padmasini Kumar,1,3 Neil Senzer,1,2,3 John Nemunaitis.1,2,3 Gradalis, Inc., Dallas, TX; 2Mary Crowley Cancer Research Centers, Dallas, TX; 3Strike Bio, Inc., Dallas, TX S208 Molecular Therapy Volume 22, Supplement 1, May 2014 Copyright © The American Society of Gene & Cell Therapy SENSORY (OPHTHALMIC AND AUDITORY) DISEASES LGN activation at baseline At one month follow up, LGN activation unexpectedly initially appeared only on the side of the previously treated eye, and by the end of two years fMRI follow up results showed the expected bilateral LGN activation, as normally observed in sighted controls fMRI results for all LCA2 patients showed a similar pattern of activations However, younger subjects showed even greater improvement over time than older participants Conclusions: The eye and brain work in concert to enable vision Using fMRI, we demonstrate the vital role that the brain plays in securing long-lasting vision after one-time GT Visual improvement was detected one month after GT and continually enhanced 2-3 years later LGN responses suggest that brain at first uses previously strengthened pathways promoted by the GT of the first eye Over time, brain plasticity was detected by observing increased activations for ipsilateral LGN and bilateral V1 In summary, fMRI results demonstrate that while GT initiates the sight in LCA2 patients, their continual visual experiences promote long-term neural plasticity, which in turn further enhances their vision Results suggest one-time subretinal injection promotes continuous brain plasticity, leading to progressive enhancement of visual pathway function and long-term visual improvement Longer follow up studies are needed to further support this hypothesis 537 Gene Therapy for X-Linked Retinitis Pigmentosa: A Long-Term Efficacy Study in a Mouse Model of RPGR Deficiency Zhijian Wu,1 Suja Hiriyanna,1 Haohua Qian,1 Suddhasil Mookherjee,1 Kayleigh Kaneshiro,1 Maria Campos,1 Chun Gao,1 Robert Fariss,1 Tiansen Li,1 Peter Colosi,1 Anand Swaroop.1 National Eye Institute, National Institutes of Health, Bethesda, MD Retinitis pigmentosa (RP) refers to a diverse group of hereditary retinal dystrophies characterized by a progressive loss of photoreceptor cells, resulting in vision loss or blindness The prevalence of the disease is approximately 1/3500 worldwide X-linked RP (XLRP) accounts for 15% of all RP cases and is one of the more severe forms of retinal degeneration Over 70% of XLRP cases are caused by mutations on the Retinitis Pigmentosa GTPase Regulator (RPGR) gene, one of the most common causes of RP among the over sixty RP-causing genes identified to date To develop a gene replacement therapy for this disease, we designed and generated AAV vectors carrying mouse or human RPGRorf15 full-length cDNA, and conducted a long-term efficacy study in an Rpgr knock-out (KO) mouse model This model recapitulates the major features of the human disease over a 20 month frame AAV8 or AAV9 vectors were injected subretinally into to week- or 12 month-old Rpgr-KO mice at doses ranging between 1e8 and 1e10 vector genomes (vg) per eye The mice were monitored for retinal function and structural changes by electroretinography (ERG) and optical coherence tomography (OCT) and were sacrificed at 20-24 months for histological and immunohistochemical analyses Our results showed that preservation of retinal function and structure in Rpgr-KO mice was achieved with either mouse or human RPGRorf15 vector administration Among all tested doses, 3e8 vg or 1e9 vg per eye appeared to be optimal for the mouse or human RPGRorf15 vector injection, respectively The mice receiving these vector doses displayed significantly larger scotopic and photopic ERG amplitudes in their vector-injected eyes compared to fellow control eyes, at 18 months post-injection OCT analyses revealed the preservation of outer nuclear layer (ONL) in the vector-injected retina, which was later confirmed by histology Cone opsin mislocalization was partially corrected as well Interestingly, the mice injected at year of age also displayed larger ERG amplitude and a greater ONL thickness The mice receiving other doses of the vectors generally exhibited suboptimal therapeutic effects, however very high doses, such as 1e10 vg of the mouse or human RPGRorf15 Molecular Therapy Volume 22, Supplement 1, May 2014 Copyright © The American Society of Gene & Cell Therapy vector, produced pronounced retinal toxicity We conclude that both mouse and human RPGRorf15 AAV vectors are able to prevent photoreceptor degeneration and preserve rod and cone function in the Rpgr-KO mouse retina Our studies also suggest that patients with RPGR mutations could benefit even when treated at an advanced age 538 Gene Therapy With the Mitochondrial Heat Shock Protein 70 Suppresses Axonal Degeneration and Vision Loss in Experimental Optic Neuritis Venu Talla,1 Vittorio Porciatti,1 Vince Chiodo,2 Sanford L Boye,2 William W Hauswirth,2 John Guy.1 Ophthalmology, Bascom Palmer Eye Institute, Miami, FL; Department of Ophthalmology, University of Florida, Gainsville, FL PURPOSE: To rescue visual loss and optic neuropathy in the experimental autoimmune encephalomyelitis (EAE) mouse model using gene therapy with the mtHSP70 chaperone responsible for import of proteins into the mitochondria METHODS: EAE was induced in DBA/1J (n=20) mice by subdermal injection of 0.1 ml homologous spinal cord emulsion in complete Freunds adjuvant Ten mice were rescued by intravitreal injection of ssAAV-CBA-mtHSP70 with a Flag tag, 10 were injected with the scAAV-Cox8-mCherry as injection controls Unsensitized DBA1J mice (n=10) were injected with scAAV-Cox8-mCherry as a control Visual function was assessed by pattern electroretinograms (PERG) Spectral domain OCT evaluated the thickness of the inner plexiform layer + nerve fiber layers at 1, and months post injection (MPI) All mice were euthanized at 6MPI Retinas and optic nerves (ONs) were dissected for histological and ultrastructural evaluation Mitochondrial complexes I+III, III and I activities were analyzed in the retinas obtained from MPI control, EAE-mCherry and EAEmtHSP70 rescued mice Expression of mtHSP70Flag in the retina and ONs was evaluated at MPI by immunofluorescence (IF), RT-PCR and western blotting (WB) Mitochondrial import of Cox8-mCherry in retina of EAE mice with and without mtHSP70 rescue was analyzed RESULTS: IF revealed a typical punctate and perinuclear expression of Flag-HSP70 which colocalized with porin and thy1.2 RT-PCR, WB confirmed HSP70 expression in the retina and ONs PERG analysis at and 6MPI showed a 42% and 45% reduction in amplitude in EAE-mCherry compared to control mCherry mice (p