528 modulation of the human rhodopsin expression by artificial zinc finger transcription factors to treat dominant retinal degenerations

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 Todate, in

vivoexperimentalmodelsofHlVencephalopa-thy have relied on acute injection of recombinantHIV 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

admin-istered 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 Roussarcoma 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 monthafterinjection ofSV(SODI) 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

assay-ing 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 ongoingneuroninjuryby lipidperoxidation

Prioradminis-tration ofSV(SOD I) or SV(GPxI) significantly protectedthe brain

fromthis ongoingdamageelicitedby HIV-I envelopeglycoprotein

Fewerapoptoticcells wereobservedwhenSV(SODI) or SV(GPxI)

(reductionby 85 and 70% comparedto untreatedrats respectively)

wcrc administered one monthbeforeSV(gpI20) was injectedin thc

CP As well, prior rSV40 delivery of these antioxidant enzymes

reduced lipid peroxidation Thus, in a new model of chronic

neu-rotoxicity due to long-term HIV Env expression, antioxidant gene

delivery using SV40-derived vectors protects neuronsand reduces

oxidant-related damage as measured by lipid peroxidation The

possibilitythat 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

neuro-logical disorders.The key pathologic feature of PO is progressive

degeneration ofnigrostriatal dopaminergic neurons

Glial-cell-line-derived neurotrophic factor (GDNF) can support the survival of

these neurons,and therefore is an attactive therapeutictarget,since

existingtreatments provideonlysymptomatic reliefand

havesignifi-cant side elTects GDNF has shown neuroprotectiveand restorative

effectsinanimalmodelsof POanddemonstrated promising resultsin

two clinicaltrials, althougha latestudy failedto show clear clinical

benefits Neurosurgerywas requiredas GDNF is unableto penetrate

the blood-brainbarrier(BBB).On the otherhand,numerousstudies

have shown that blood macrophages can pass the BBB and then

migrate to and accumulate in the diseased sites in various

neurode-Molecular Therapy Volume15,Suppl ement I Ma y 2007

C opyright © Th e Americ an So ciety o f (it"11C Therapy

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 thanthe 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 irradiated6-week old mice Fiveirradiated6-weekspost transplantation groups of randomly selected recipientmice receiveda dose of 4x13 mg/kg body weight

of MPTP-HCI or saline injected subcutaneously at 2 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 sub-stantia 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 (expressingGFPin 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 recipientsthat re-ceived Lenti-SP-GFP-transduced HSC.In addition,HPLCanalysis

of striatal dopamineandmetabolite (DA,DOPAC,andI,IVA)showed the formergroupof recipients hadhigherlevelsthanthe 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 ofGenetic s and Medicine , TlGEM Napoli, Italy; 2Department ofBiomedical Sciences, University ofModena and Reggio Emilia, Modena, Italy

Retinitis Pigmentosa (RP) represents a group of retinal degen-erations with dilTerent patterns of inheritance, characterized by a widegeneticand clinicalheterogeneity Rhodopsin is the gene most commonlyassociatedwithautosomal 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 effectivetherapeuticap-proachestor dominantformsstillrepresents a greatchallenge Inthis study we used a novel strategy to treatADRp, basedon engineered zinc-fingers transcription factors technologyto selectivelysilence,

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-transactivationassay of the human rhodopsin promoter Three artificial zinc-fingertranscriptionfactors,ZF2,ZF6 and ZF7

stronglytransactivated (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

differenti-ated 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

respec-tively Wcarc currently testing whether Adeno-associated viral

vec-tors- (AAY) mediated delivery ofZF2 or ZF6to photo receptors of

the P347S mouse silence the human rhodopsin expression in vivo,

thus inhibiting photoreceptor degeneration

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 RonaldJ.Mandel.'?

'Neuroscience, University ofFlorida College ofMedicine,

Gainesville FL; 2Molecular Genetics and Microbiology,

Uni-versity ofFlorida College ofMedicine, Gainesville , FL; JPowell

Gene Therapy Center, University0/Florida College ofMedicine,

Gainesville , FL.

Parkinson'sdisease (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

molecu-lar 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

over-expression 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 ofparkin was injected unilaterally

in the substantia nigra ofadult rats 6 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 inthe parkin

treated animals.However, biochemical evaluationof 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

Barrier Permeability by Antioxidant Gene Delivery

to the eNS Using SV40-Derived Vectors

lean-Pierre Louboutin,' Lokesh Agrawal,'Celestine Wanjalla,I BeverlyA.S.Reyes,'Elisabeth J vanBockstaele.'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 dys-function 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, stereotaxi-cally into the caudate-putamen (CP) of the rat brain,or saline as a control rSY40-derived vectors were designed to carryCu/Zn super-oxide 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 inocula-tion, 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 1 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 open-ing 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' 4 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-medi-ated abnormalities of the BBB can be relgpI20-medi-ated 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 S upplement I ~b y 2007