996 Intracerebral Delivery of AAV 2/5 Vectors To Treat Canine MPS IIIB 994 Neural Expression of G Protein Coupled Receptors GPR3, GPR6, and GPR12 Upregulates Cyclic AMP Levels and Promotes Neurite Out[.]
994 Neural Expression of G Protein-Coupled Receptors GPR3, GPR6, and GPR12 Upregulates Cyclic AMP Levels and Promotes Neurite Outgrowth Shigeru Tanaka,' Ken Ishii,' Kazue Kasai,' Sung Ok Yoon.' Yoshinaga Saeki.' /Dardinger Laboratoryfor Neuro-oncology and Neurosciences Department ofNeurological Surgery, The Ohio State University, Columbus, Oli; 2Department ofOrthopaedic Surge/Yo Keio University School ofMedicine , Shinjuku, Tokyo, Japan; "Department ofMolecular and Cellular Biochemistry, and Center for Molecu lar Neurobiology The Ohio State University Columbus OH Neurons in the adult mammalian central nervous system, unlike those in the embryonic or peripheral nervous system , cannot regenerate their axons after injury The failure of regeneration is attributable to the non-permissive environment for axonal growth , such as the presence of myelin-associated inhibitory molecules, formation of glial scar, and a deficiency in growth-promoting substrates and factors, as well as the intrinsic inability ofadult neurons to survive and regrow their axons after injury Intracellular level of cAMP was identified as onc of such intrinsic factors that could influence axonal regenerat ion Indeed, recent reports demonstrate that activating cAMP signaling by treating neurons with dibutyrylcAMP (a cell-permeable non-hydrolysable analogue ofcAMP) or a variety ofneurotrophins, such as brain-derived neurotrophic factor (BDNF) and neurotrophin-3 (NT-3), overcomes inhibition caused by myelin-associated glycoprotein (MAG) and myelin in vitro The activity of adenylate cyclases, a class of enzymes that synthesize cAMP from AT?, is regulated through a subset ofG protein-coupled receptors (GPCRs) GPR3, GPR6 , and GPRI2 make up a family of constitutively active GPCRs that share greater than 50% identity and 65% similarity at the amino acid level They are highly expressed in the central nervous system, and their expression in various cell lines results in constitutive stimulation of cAMP production When the constitutively active GPCRs were ovcrcxprcssed in rat cerebellar granule neurons in culture, the transfected neurons exhibited significantly enhanced neurite outgrowth and overcame growth inhibition caused by MAG GPR 12-mediated neurite outgrowth was the most prominent and was shown to depend on Gs and cAMP-dependent protein kinase (PKA) Moreo ver, the GPR 12-mediated rescue from MAG inhibition was attributable to PKA-mediated inhibition ofthe small GTPase, RhoA Among the receptors, GPR3 was revealed to be enriched in the developing rat cerebellar granule neurons When the endogenous GPR3 was knocked down , significant reduction of neurite growth was observed , which was reversed by expression of either GPR3 or GPR 12 Taken together, our results indicate that expression of the constitutively active GPCRs upregulates cAMP production in neurons, stimulates neurite outgrowth, and counteracts myelin inhibit ion Based on the present findings, upregulating expression ofthe constitutively active GPCRs in damaged neurons may hold potential as a therapeutic strategy to treat various neurological disorders, including spinal cord injury and stroke 995 Enhanced Gene Transfer to Photo receptors by Adenovirus Vectors Deleted in RGD Penton - Application to the Rescue of Photoreceptor Degeneration in Two Models of Retinitis Pigmentosa Siobhan Cashman,' Daly Gerard,' William Brunken,' Rajendra Kumar-Singh.' 'Ophthalmology, Tufts University School ofMedicine, Boston, AlA Purpose: Recent Phase I clinical trials indicate that adenovirus (Ad) vectors persist in human ocular tissues without adverse events and hence are a promising method of gene transfer to the retina Ad vectors can accommodate up to 36 Kb of'foreign DNA, making them useful for the inclusion oflarge upstream gene regulatory elements that arc necessary for appropriate Icvcls oftransgcnc expression- a factor shown to be important in animal models ofretinal degeneration However, like lentivirus vectors , Ad vectors only transduce photorecepto rs early during development and in adult mice transduce primarily the retinal pigment epithelium (RPE) upon subretinal delivery Since the majority of mutant gene products that cause retinal degeneration are expressed in the photoreceptors, vectors with large capacity and tropism for fully developed photoreccptors arc needed, Whilc it has been shown previously that pscudotyping of Ad can allow for photoreceptor transduction in adult mice, the levels of transduction are low relative to other viral vectors such as adeno associated virus (AAV) Methods: To enhance photoreceptor transduction by Ad, we investigated the interaction between the RGD domain in Ad penton base and the RPE We deleted the integrin-binding RGD domain in Ad penton and injected such Ad vectors expressing GFP into the subretinal space of adult mice In addition, we constructed Ad vectors expressing cGMP beta phosphodiesterase or rhodopsin and injected these vectors into rd I0 and rho -1- mice respectively at p 18, a stage post photoreceptor development Results: We found that deletion ofthe RGD domain permitted transgene expression in neural mouse retina approximately 700-fold more efficiently than with standard Ad5 vectors expressing GFP from the CMV promoter By qualitative fluorescence microscopy, it appeared that virtually every photoreceptor expressed transgene product in the region of injection and high levels of photoreceptor transduction were achieved in approximately 65% of the retina Two months after injection in rd 10 and rho -1- mice, the latest time point examined thus far, ERG responses and histology indicated that these novel vectors rescue retinal function as well as morphology in these two mouse models of retinitis pigmentosa Conclusions: Ad vectors with deletion in the RGD domain of penton base arc efficient at delivering transgenes to photoreceptor cells These vectors may be relevant for developing gene therapy approaches for individuals with retinal degeneration 996 Intracerebral Delivery of AAV-2/5 Vectors To Treat Canine MPS 11I8 N Matthew Ellinwood; Karen L Kline,' Marie-Anne Colle,' Elizabeth M Snella,' Jackie K Jens,' Song Lui,' Yan Cherel ' Jean Michel Heard.' /Animal Science & Veterinary Clinical Science, Iowa State University Ames, IA; 2Unite d'Anatomie Pathologique, UMR 703 INRAIENVN, Ecole Nationale Veterinaire de Nantes, Nantes, France; 'Unite Retrovirus et Transfert Genetique, INSERM U622 Institut Pasteur; Paris, France Mucopolysaccharidosis type IIIB (MPS IIIB), a fatal neuropathic lysosomal storage disease with pediatric onset, is characterized by autosomal inherited loss of N-acetyl-a-D-glucosaminidase activity (Naglu) , and heparan sulfate accumulation As an intermediary between mouse and human trials, and to evaluate vector efficacy, S380 Molecular Therapy Volume IS, Supplement I, ~br 2007 Copyright © 'J11c American Soder),o f G ene Therapj- we have conducted an AAV2.5 vector gene transfer via intracerebral injection in the MPS IIIB dog Three affected dogs were injected with an AAV-2/5 vector with the human Naglu cDNA under control ofa murine PGK promoter, with or without a WPRE One dog received WPRE+ vector without immunosuppresion Two dogs were immunosuppressed (cyclosporine and mycophenolatc mofetil), and received the WPRE+ or WPRE- vector Dogs received 5-lOx 1011 vector genomes in 320 ~I (8 deposits x 40 ~I) Deposits targeted putamen or centrum semiovale via injection sites, per hemisphere, with deposits per injection Minor post-surgical neurological sequella seen in one dog were substantially resolved one week post surgery At 3.5 months post injection animals were sacrificed, prior to which neurological assessments showed very minor deficits in dogs, different from the dog with post-surgical sequella The dog with the post-surgical sequella had gross lesions at one superficial deposit, consistent with hemorrhage Subsequently von Willebrand disease was found to segregate in this line, and surgical hemorrhage may have resulted form this Biochemical studies of brain hemisagital section quadrants demonstrated no Naglu activity in the non-immunosuppressed dog, while activity in immunosuppressed dogs was: (WPRE+) up to 40 X normal with 60% of quadrants positive, and (WPRE-) up to 30 X normal with 30% of quadrants positive Q-PCR results and secondary substrate analyses arc pending Histopathology confirmed grossly visible liquefactive necrosis with lymphocytic perivascular infiltrates in the non-immunosuppressed dog, and very mild lesions associated with injection trauma in the immunosuppressed dogs, with the exception ofthe limited necrotic lesion with hemorrhage Assessment of corrected storage awaits analysis ofcontrol animals, but significantly lower numbers ofcells with storage were scored adjacent to injection sites in the immunosuppressed dogs relative to the non-immunosuppressed dog These preliminary results demonstrate successful Naglu gene transfer and expression in a large MPS IIIB model Results confirm the need for efficient immunosuppression in this model as previously shown for MPS I dogs, and likely in any CRIM- patients Future studies will improve targeting and delivery and methods involving tolerized dogs undergoing long term follow up to demonstrate clinical efficacy and safety [Funded by Association Franeaise eontre les Myopathies, Iowa St Univ, Institut Pasteur, INSERM, The Ryan Foundation, The National MPS Society, and NIH (RR002512 colony foundation)) was designed to carry Cu/Zn superoxide dismutase (SV(SOD I» , an enzyme that converts the free radical oxygen species, superoxide, to peroxide An unrelated rSV40 vector, SV(BUGT), was used as a rSV40 control Rats were given vectors stereotaxically, either intraparenchymally into the caudate-putamen (CP) or into the lateral ventricle (LV) To sec ifrSV40 gene delivery to the brain following LV injection could be enhanced by blood brain barrier (BBB) disruption , we administered 30% mannitol intraperitoneally (i.p.) before injecting the vector into the LV SOD I expression was studied at different times by immunostaining of serial brain sections, and by immunoblotting After intraparenchymal administration, numerous transgene-expressing cells were seen , many as far as mm from the injection site Transgene expression remained strong throughout the month study period No SOD l-positivc cells were seen when SV(BUGT) was injected in the CP Coimmunostaining for lineage markers showed that neurons and, more rarely, microglial cells were tranduced; transgene expression was not detected in astrocytes and oligodendrocytes, One and two months after injection into the LV, high levels of transgene expression were detected throughout the frontal cortex by Western analysis Western blotting demonstrated that systemic mannitol-induced hypcrosmolarity further augmented LV transgene delivery one and two months after injection of the vector into the LV No expression of SODI was detected when SV(BUGT) was injected into the LV with or without prior i.p administration of mannitol These data demonstrate that high levels of long-term transgene expression can be achieved throughout the brain by combining the intraventricular route of administration, with or without systemic mannitol pretreatment, and SV40-derived vectors Long term transgene expression may be achieved locally by intraparenehymal administration of these vectors 997 Disseminated Gene Transfer to the CNS by Intraventricular Injection with Mannitol Pretreatment Using SV40-Derived Vectors Delivers Generalized Transgene Expression throughout the Brain Neuroaphtie pain has a medical impact and current treatments are often unsuccessful RNA interference (RNAi) represents a promising new approach to tackling this problem I-Iere , we report that intrathecal injection of a self-inactivating lentiviral vector delivering shRNA against the transient receptor potential vanilloid receptor I (TRPVI) and pain-related cation-channel P2X3 to alleviate neuropathic behavior Vectors were constructed with single (TRPVI or P2X3), double (two regions ofTRPVI , two regions of P2X3, one region ofTRPVI and one region ofP2X3) triple (two regions of TRPV I and one region of P2X3, two regions of P2X3 and one region ofTRPV I) cassettes expressing short hairpin RNAs (shRNAs) The shRNAs against P2X3 orTRPVI were shown to be effective in inhibiting gene expression in HEK 293 cells expressing P2X3 or TRPVI in vitro Simultaneous silencing of P2X3 and TRPVI significantly potentiated the reduction of mechanical allodynia compared to silencing of individual gene in vivo Triple shRNA vectors were showed to significantly increase the silencing efficiency in vitro However, no further reduction of mechanical allodynia was noted compared to double shRNA (P2X3 and TRPV I) vector The study demonstrates lentiviral delivery ofmultiple shRNA simultaneously against various targets has potential in the treatment of neuropathic pain Jean-Pierre Louboutin,' Lokesh Agrawal,' Beverly A S Reyes,' Elisabeth J van Bockstaele.' David S Strayer I I Department ofPathology Thomas Jefferson University, Philadelphia PA; 2Department ofNeurosurgery and Farber Institute for Neurosciences, Thomas Jefferson University; Philadelphia PA Gene transfer to CNS has been studied using various vectors There are applications for which generalized gene delivery throughout the brain is desirable, yet the challenge is to deliver vectors throughout the brain and achieve transgene expression at levels sufficient to provide the desired therapeutic effect In these studies, we tested the effectiveness of antioxidant gene expression delivered by vector administration into the lateral ventricle, with and without prior systemic treatment with mannitol, compared to locali zed inoculation, in protecting from a locali zed challenge with an oxidant stress Recombinant Tag-deleted SV40-derived vectors (rSV40s) transduce neurons and microglia very effectively in vitro, so we tested rSV40 gene transfer to the CNS in vivo We characterized the distribution, duration and cell types transduced An rSV40 Molecular Therapy volume 15.Supp lem ent I ~I; )" 2007 Co pr righ t © The American Soc,.; ety o f G ene Therapy 998 Simultaneous Silencing of Vanilloid Receptor TRPV1 and Pain-Related CationChannel P2X3 with a Single Lentiviral Vector Reduces Neuropathic Pain In Vivo Chung-Ren Lin ,' Kuan-Hung Chen,' Yi-Shen Chen, I Chien-To Lee.' Chien-Cheug Liu ' 'Anesthesiology; Gang Gung Memorial Hospital Ksohsiung, Taiwan; 'Intemal Medicine, Gang Gung Memorial Hospital, Ksohsiung, Taiwan S381 ... conducted an AAV2 .5 vector gene transfer via intracerebral injection in the MPS IIIB dog Three affected dogs were injected with an AAV- 2/5 vector with the human Naglu cDNA under control ofa murine... mannitol pretreatment, and SV40-derived vectors Long term transgene expression may be achieved locally by intraparenehymal administration of these vectors 997 Disseminated Gene Transfer to the... transgene delivery one and two months after injection of the vector into the LV No expression of SODI was detected when SV(BUGT) was injected into the LV with or without prior i.p administration of