528 Modulation of the Human Rhodopsin Expression by Artificial Zinc Finger Transcription Factors To Treat Dominant Retinal Degenerations disease Todate, in vivoexperimentalmodelsofHlVencephalopa thy h[.]
disease To date, in vivoexperimentalmodelsofHlVencephalopathy have relied on acute injection of recombinant HIV proteins or, less predictably, on primate infection with SIV Developmentofa system of chronic HlV-induced neurotoxicity is necessary before effective therapies can bc devised and tested, Wc devised such a model, in which SV(gpI20), a recombinant SV40-derived vector expressing HIV-INL4-3 envelope glycoprotein, gp120, is administered stereotaxically into the rat caudate-putamen Continued expression of gp120 leads to ongoing neuron apoptosis for weeks to months Wehave employed this system to test the elTectiveness of antioxidant gene delivery as a potential treatment of chronic gpl20-induccd neuron ccll death For this purpose, we uscd two Tag-deleted SV40-derived vectors (rSV40s) that carry Cu/Zn superoxide dismutasc (SV(SODI» and glutathione peroxidase (SV(GPxl), each driven by the Rous sarcoma virus long terminal repeat (RSV-LTR) as a promoter SODI converts the free radical oxygenspeciessuperoxideto peroxideand GPx1convertsperoxide to water SV(SODI) and SV(GPxl) were administered to rats via direct stereotaxic injection into the caudate-putamen (CP, where SV(gpI20) would be administeredafterwards) To characterizethe SV(gpI20)-induccd damage, analysis was performed at varying time points after SV(gpI20) administration To study the putative elTect of antioxidant molecules, SV(gpI20) was administered into the CPone monthafter injection ofSV(SOD I) or SV(GPxI) Brains were harvested one week after the challenge Neuron apoptosis was measured by TUNEL assay, and neuronal lipid peroxidation (a marker of HIV-induced brain damage) was visualized by assaying for 4-hydroxy-noncnal (HNE) and quantitated by calorimetric malonaldehydc (MDA) assay In rats not injected with SV(SODI) or SV(GPxI), SV(gp120)induced considerable neuronapoptosis, as well as ongoingneuron injuryby lipidperoxidation Prior administration ofSV(SOD I) or SV(GPxI) significantly protectedthe brain fromthis ongoingdamageelicitedby HIV-I envelopeglycoprotein Fewerapoptoticcells wereobservedwhenSV(SODI) or SV(GPxI) (reduction by 85 and 70% comparedto untreatedrats respectively) wcrc administered one monthbefore SV(gpI20) was injectedin thc CP As well, prior rSV40 delivery of these antioxidant enzymes reduced lipid peroxidation Thus, in a new model of chronic neurotoxicity due to long-term HIV Env expression, antioxidant gene delivery using SV40-derived vectors protects neurons and reduces oxidant-related damage as measured by lipid peroxidation The possibility that such gene delivery may be useful in other chronic neurodcgenerative diseases is under study 527 Macrophage Delivery of GDNF Ameliorates Neurodegeneration in Parkinson's Disease Qing Zhou, Sycd Z Imam, Weijing He, Zhiqiang Liu, Robert A Clark, James L Roberts, Senlin Li 'Medicine and Pharmacology, UTHSCSA and ST Veterans Health Care System, San Antonio, TX Parkinson's disease (PO) is among the most prevalent neurological disorders The key pathologic feature of PO is progressive degeneration ofnigrostriatal dopaminergic neurons Glial-cell-linederived neurotrophic factor (GDNF) can support the survival of these neurons,and therefore is an attactive therapeutictarget,since existingtreatments provideonlysymptomatic reliefand havesignificant side elTects GDNF has shown neuroprotectiveand restorative effectsinanimalmodelsof POand demonstrated promising resultsin two clinicaltrials, althougha latestudy failedto show clear clinical benefits Neurosurgerywas requiredas GDNF is unableto penetrate the blood-brainbarrier(BBB).On the other hand, numerousstudies have shown that blood macrophages can pass the BBB and then migrate to and accumulate in the diseased sites in various neurodeMolecular Therapy Volume15,Supplement I May 2007 (it"11C Therapy Copyright © Th e American Society o f generative disorders, ineludingPD Macrophagesare derived from hematopoieticstem cells (HSC), which are readily accessible and can repopulatethe blood, includingmonocytes/macrophages, after geneticallyengineeredwith a therapeuticgene.These HSC-derived macrophagesmay thus be an ideal vehicle for delivering GDNF to the lesion sites of PD In HSC-based macrophage gene therapies, a powerful macrophage promoter is critical to drive therapeutic levels of gene expression We previously developed a series of super macrophage promoters (SMP), which are much more active than the currentlyavailablemacrophagepromoters Wehypothesize that highly effective CNS delivery ofGDNF can be achieved with the usc of our SMP and this will amelioratethe pathologicchanges and neurological defects of PD Here we present our preliminary data of the study Bone marrow stem cells collected from C57B/6 EGFP transgenicmice were transplantedinto lethally irradiated6week old mice Five weeks post transplantation groups of randomly selected recipientmice receiveda dose of 4x13 mg/kg body weight of MPTP-HCI or saline injected subcutaneously at hr intervals One week after MPTPadministration,the mice were intracardially perfused and the brains collected for immunohistochemistry with Ibal (microglia marker) antibody Following MPTP lesion, the infiltratingGFP-positivccells were seen predominantly in the substantia nigra, the specific location of the MPTP injury About 70% ofGFP-positive cells were identified as macrophage/microglia In similar experiments, when donor HSC were infected with either lentivectorLenti-SP-GDNF (expressingGDNFin macrophages) or Lenti-SP-GFP (expressingGFP in macrophages), the recipientsthat receivedLenti-SP-GDNF-transduced HSCdemonstrated protection of tyrosine hydroxylase-positive dopamine neurons in substantia nigra of MPTP-treated mice, compared with the recipients that received Lenti-SP-GFP-transduced HSC.In addition, HPLCanalysis of striatal dopamineandmetabolite (DA,DOPAC, and I,IVA) showed the formergroupof recipients had higherlevelsthanthe la1.1.er group, consistent with GDNF protection of the dopaminergicneurons In summary, this study suggestsa noveltreatmentfor PD patientsthat would halt or slow down the degenerationof diseased neurons 528 Modulation of the Human Rhodopsin Expression by Artificial Zinc Finger Transcription Factors To Treat Dominant Retinal Degenerations Claudio Mussolino,' Daniela Sanges.P ValeriaMarigo,' Germana Moroni,' Enrico M Surace.' 'Telethon Institute ofGenetics and Medicine, TlGEM Napoli, Italy; 2Department ofBiomedical Sciences, University ofModena and Reggio Emilia, Modena, Italy Retinitis Pigmentosa (RP) represents a group of retinal degenerations with dilTerent patterns of inheritance, characterized by a widegenetic and clinicalheterogeneity Rhodopsin is the gene most commonlyassociated withautosomal dominantform ofRP (ADRP; 25-50% of cases) with over 150 mutations identified While gene replacement-based therapies forautosomal recessiveRP(ARRP)are close to be tested in humans, the design of effectivetherapeuticapproachestor dominantformsstillrepresents a greatchallenge In this study we used a novel strategy to treatADRp, based on engineered zinc-fingers transcription factors technologyto selectively silence, at the transcriptional level, the rhodopsin gene This approach has the advantageto be mutationindependent, thus potentiallyallowing the treatmentof all ADRPdue to rhodopsinmutations Wedesigned 10 different zinc-fingers based DNA binding domains targeting the human rhodopsin promoter and we fused them to the generic activator VP64 For the first round of selection we used an in vitro luciferase-transactivation assay of the human rhodopsin promoter Three artificial zinc-fingertranscriptionfactors, ZF2, ZF6 and ZF7 strongly transactivated (15 to 20 folds) the luciferase expression The DNA binding domains ofZF2, ZF6 and ZF7 were then fused S203 to the KRAB repressor domain and tested in Y79 retinoblastoma cells, which express human rhodopsin Real time PCR and western blot analysis showed a robust repression of the transcript and the protein respectively, in particular with ZF2 and ZF6 constructs To verify their therapeutic potential we cxplanted and cultured retinal stem cells from adult P347S AORP mouse model Once differentiated into photoreceptors, these retinal stem cells die because of the expression ofa P347S mutated copy ofthe human rhodopsin gene The transduction ofP347S retinal stem cells with retroviral vectors carrying ZF2, ZF6 or ZF7 resulted into a significant reduction of photoreceptors degeneration 45% of the untransduced cells were found TUNEL staining positive while 10%, 4,5% and 25% were TUNEL positive after transduction with ZF2 , ZF6 or ZF7 respectively Wc arc currently testing whether Adeno-associated viral vectors- (AAY) mediated delivery ofZF2 or ZF6to photo receptors of the P347S mouse silence the human rhodopsin expression in vivo, thus inhibiting photoreceptor degeneration 529 rAAV Mediated Parkin Over-Expression Partially Ameliorates the Behavioral Effects of the 6-Hydroxy Dopamine Lesion Fredric P Manfredsson.P Alfred S Lewin.t-' Corinna Burgen ' Nicholas Muzyczka,2.3 Ronald J Mandel.'? 'Neuroscience, University ofFlorida College ofMedicine, Gainesville FL; 2Molecular Genetics and Microbiology, University ofFlorida College ofMedicine, Gainesville , FL; JPowell Gene Therapy Center, University 0/Florida College ofMedicine, Gainesville , FL Parkinson's disease (PO) is a common neurodegcnerative disease affecting a large portion of the elderly population Although the cause of idiopathic PO is largely unknown, insight into the molecular events has been achieved through the identification of certain familial forms of the disease One such form, autosomal-recessive juvenile PO (AR-lP) has been linked to various mutations in the gene encoding the E3 ligase parkin Here we investigated ifparkin overexpression during the course ofa 6-0HOA lesion would alleviate the pathology that arises due to the acute oxidative insult of the toxin rAAY encoding the human form of parkin was injected unilaterally in the substantia nigra ofadult rats weeks after the rAAY injection the animals were subjected to a unilateral 4-site striatal 6-0HOA lesion and subjected to a battery of behavioral tests The parkin treated animals showed a significant improvement in amphetamine induced rotations as well as in the cylinder test as compared to the injection control Surprisingly, upon histological examination we observed no significant improvement in cell survival in the parkin treated animals However, biochemical evalu ation of the animals revealed increased levels of dopamine and tyrosine hydroxylase (Tll) in the striata ofthe parkin treated animals An additional group of animals was treated with parkin similarly to that of the initial group , but received no lesion This group of animals displayed increased contraversive amphetamine induced rotational behavior, and biochemical analysis showed significantly higher levels ofTl-i as well as increased dopamine levels in the parkin-treated striata Our results indicate that parkin over-expression does not improve cell survival, but instead, the observed improvement in behavior is due to increased levels ofTH and subsequent elevated dopamine levels in surviving cells of the substantia nigra S204 530 Protecting from Abnormal Blood Brain Barrier Permeability by Antioxidant Gene Delivery to the eNS Using SV40-Derived Vectors lean-Pierre Louboutin,' Lokesh Agrawal, ' Celestine Wanjalla, I Beverly A S Reyes,' Elisabeth J van Bockstaele.' David S Strayer.' I Pathology; Thomas Jefferson University Philadelphia PA; /Neurosurgery and Farber Institute for Neurosciences, Thomas Jefferson University, Philadelphia PA Blood-brain barrier (BBB) abnormalities may be prominent in many systemic and CNS disease processes We studied BBB dysfunction using exposure to HIY-I proteins as a model, and tested the protective effectiveness of antioxidant gene delivery We injected 500 ng/microl ofHIY-1 envelope glycoprotein, gp120 , stereotaxically into the caudate-putamen (CP) of the rat brain, or saline as a control rSY40-derived vectors were designed to carry Cu/Zn superoxide dismutase (SY(SOD I» or glutathione peroxidase (SY(OPxl » SY(BUOT) was used as an unrelated rSY40 control Hemorrhagic lesions were seen after injection ofgp I20 in the Cp' suggesting BBB damage To sec if the BBB had in fact been disrupted by gpI20, wc first administered the vital dye Evans Blue (EB) intravenously (i.v.), before injecting gpl20 into the CPo Within 15 minutes after the latter injection, EB was seen in the CP, suggesting that gpl20 partly destroyed the BBB and that there was an extravasation ofEB through the damaged vessels At 1hand 24h after gp 120 inoculation, EB was detected by fluorescence microscopy in the injected CPo No extravascular EY was seen on the contralateral side, or when gpI20 was replaced by saline EB concentration was measured by spectrophotometry: EB was higher in the brains injected with gp 120 than in those given saline To study the impact of gp 120 on brain microvessels, we studied expression ofiCAM-l and YCAM-I, as well as c1audin-5, respectively markers of rnicrovessels and tight junctions Within hour of gp 120 injection into the CP, numbers of ICAM-I-, YCAM-l- and c1audin-5-positive structures were decreased in EB-positive areas No such reduction was seen when gp 120 was replaced by saline Gp 120 was immunolocalizcd in the walls of microvessels, and coimmunostained with CD31, a marker of endothelial cells, and ICAM-I Matrix metalloproteinases linked to BBB abnormalities, MMP-2 and MMP-9, were overexpressed in the gp 120-injected CPs, compared to controls Pathologic BBB opening has occasionally been linked to the R-I subunit of the NMDA receptor To test whether NMOAR-l was involved in the opening of the BBB caused by gp120, we administered memantine i.p (30 mglkg) an inhibitorofNMOAR-l , before injecting EB i.v, and thcn gp 120 into the CPo Animals injected with memantine exhibited less EB extravasation into the CP than did control animals given saline i.p before EB and gp120 We finally administered SY(SODI) or SY(OPxl) into the Cp' to 24 weeks before injecting EB i.v, and gp 120 into the CPo Both antioxidant vectors significantly reduced EB extravasation These results suggest that : I) gpl20 induces disturbances of the BBB after injection in the CP; 2) gpI20-mediated abnormalities of the BBB can be related to Icsions of brain rnicrovcsscls, degradation of vascular basement membrane by MMPs and involvement of NMDAR-I; and 3) antioxidant gene delivery using rSY40 vectors may protect against abnormal BBI3 opening due to HIY-I gp120 Molecular Therapy Volume 15.Sup plement I ~b y 2007 Copyright © The American Society o f Gene Therapy ... whether Adeno-associated viral vectors- (AAY) mediated delivery ofZF2 or ZF 6to photo receptors of the P347S mouse silence the human rhodopsin expression in vivo, thus inhibiting photoreceptor... die because of the expression ofa P347S mutated copy ofthe human rhodopsin gene The transduction ofP347S retinal stem cells with retroviral vectors carrying ZF2, ZF6 or ZF7 resulted into a significant... oxidative insult of the toxin rAAY encoding the human form of parkin was injected unilaterally in the substantia nigra ofadult rats weeks after the rAAY injection the animals were subjected to a unilateral