Oligodendrocyte-Specific Gene Expressionin Mouse Brain: Use of a Myelin-FormingCell Type–Specific Promoter in anAdeno-Associated VirusHong Chen,1,3 Douglas M. McCarty,2 Andrew T. Bruce,2 Kunihiko Suzuki, 1,4

and Kinuko Suzuki1,3*1Neuroscience Center, University of North Carolina, Chapel Hill2Gene Therapy Center, University of North Carolina, Chapel Hill3Department of Pathology and Laboratory Medicine, University of North Carolina, Chapel Hill4Department of Neurology and Psychiatry, University of North Carolina, Chapel Hill

To explore the feasibility of cell type–specific geneexpression in oligodendrocytes as a possible therapeu-tic approach for demyelinating diseases, the cell speci-ficity, tissue specificity, and duration of gene expres-sion were investigated using recombinant adeno-associated viral vectors (rAAV) carrying a greenfluorescence protein (GFP) gene. Recombinant AAVvectors carrying either the myelin basic protein (MBP)promoter (rAAV-MBP-GFP) or the cytomegalovirus(CMV) immediate early promoter (rAAV-CMV-GFP)were semistereotactically injected into the brain ofC57BL/6J mice. Injection of the rAAV-MBP-GFPvector into or near the corpus callosum resulted inhigh levels of GFP expression in white matter regions.Double immunostaining with cell- specific markersproved that these GFP-expressing cells were oligoden-drocytes. Injection of the rAAV- MBP-GFP vector intogray matter rarely produced GFP expression. Incontrast, injection of the rAAV-CMV-GFP vectorresulted in few GFP-expressing cells in the whitematter, with most of the GFP-expressing cells beingneurons located in the cerebral cortex along the needletrack. The expression of the GFP driven by the MBPpromoter persisted for at least 3 months. J. Neurosci.Res. 55:504–513, 1999.r 1999 Wiley-Liss, Inc.

Key words: gene therapy; central nervous system;oligodendrocytes; rAAV; MBP promoter

INTRODUCTIONThe cytomegalovirus (CMV) immediate early pro-

moter and other viral promoters are strong constitutivepromoters and have been used widely to direct geneexpression in various cell types (Goodman et al., 1994;Anson et al., 1992; Miyanohara et al., 1992). In thecentral nervous system (CNS), genes under the control of

the CMV promoter were expressed in both glial andneuronal cells (Davidson et al., 1993; Caillaud et al.,1993). However, the recombinant adeno-associated virus(rAAV) vector carrying the CMV promoter transducedpredominantly neurons (Kaplitt et al., 1994; McCown etal., 1996). Several reports have indicated that geneexpression in a specific cell type could be achievedpotentially by modulation of viral capsid ligands to targetspecific cellular receptors or alternatively, by using a celltype–specific promoter (Barinaga, 1994; Kasahara et al.,1994; Andersen et al., 1992). In our previous in vitrostudy, we demonstrated that by using the myelin basicprotein (MBP) promoter, which is myelin-forming cell-specific, the rAAV vector preferentially transduced oligo-dendrocytes (Chen et al., 1998). In this in vivo study, wereport the differences in the specific expression of greenfluorescent protein (GFP) (Zolotukhin et al., 1996) be-tween the rAAV vector carrying the CMV promoter andthat carrying the MBP promoter in the mouse brain. Theexpression from the latter was specific in oligodendro-cytes, primarily in the white matter.

MATERIALS AND METHODSVector Construction and rAAV Generation

The structure of the pTR/MBP/GFP plasmid hasbeen described previously (Chen et al., 1998). In this

Contract grant sponsor: USPHS; Contract grant numbers: NS24453,NS24289, HD03110, and HD07201; Contract grant sponsor: March ofDimes; Contract grant number: FY94–0891.

*Correspondence to: Kinuko Suzuki, M.D., Department of Pathologyand Laboratory Medicine, CB#7525, Brinkhous-Bullitt Building, Uni-versity of North Carolina, Chapel Hill, NC 27599–7525.E-mail: KIS@med.unc.edu

Received 5 October 1998; Revised 4 November 1998; Accepted 5November 1998

Journal of Neuroscience Research 55:504–513 (1999)

r 1999 Wiley-Liss, Inc.

construct, the 1.9-kb fragment of the MBP transcriptionalcontrol region (Corbin et al., 1996) replaced the CMVpromoter fragment in the pTR-UF2 plasmid (Zolotukhinet al., 1996) (provided by Dr. Sergei Zolotukhin) andcontrolled the GFP transcription.

rAAV vectors were produced as described previ-ously (Samulski et al., 1989; Ferrari et al., 1996). Briefly,the virus particles were purified by isopycnic centrifuga-tion in 4.5 g/cm3 CsCl at 288,000g for 36 hr in SW41rotor followed by a second round of centrifugation at400,000g in NVT 65 rotor for 4 hr. The gradients werefractionated and assayed by dot blot hybridization (Sam-ulski et al., 1989). High peak fractions were dialyzedagainst 20 mM Hepes containing 15% glycerol or phos-phate-buffered saline (PBS) containing 10% glycerol at4°C and subjected to heat treatment for 30 min at 56°C toinactivate residual adenovirus. Based on the dot blothybridization analysis, the virus stock used for this studycontained 63 1012 physical particles/ml of the rAAV-MBP-GFP vector and 23 1013 particles/ml for therAAV-CMV-GFP vector. No significant wild type AAVwas detected based on the infectious center assay (Snyderet al., 1996). Plaque assay showed no detectable infec-tious adenovirus (at a sensitivity of,103/ml) after heatinactivation of the vector stock for 30 min at 56°C.

Animals and MicroinjectionC57BL/6J mice, 8 days old to young adult and

homozygous twitcher (C57BL/6J-twi) mice, 15–20 daysold, were used for these studies. The majority of experi-ments were carried out with wild-type mice, 20 to 30 daysof age. Mice were bred within the animal facility at theUniversity of North Carolina at Chapel Hill or purchasedfrom Harlan Sprague-Dawley, Inc. (Indianapolis, IN). Allsurgical and care procedures were preapproved by theInstitutional Animal Care and Use Committee at theUniversity. The microinjection procedures were based onthe technique described by McCown et al. (1996). Inbrief, the mice were anesthetized with intraperitonealinjection of 2.5% avertin (0.013–0.015 ml/g of bodyweight). Ether inhalation was used to anesthetize pups.The injection sites were determined based on the coordi-nates of the sagittal suture and lamboid suture and/orbregma. A small incision was made on the scalp and asmall hole was drilled into the skull with either a31-gauge or a 26-gauge needle depending on the age ofthe mice. The depth of the injector was modified from 1.2to 2 mm depending on the site of the injection and the ageof the mice. The recombinant AAV virus in 1 µl wasinjected into the left cerebral hemisphere over 5 mincontrolled by a syringe pump. The injector was left inplace for 3 min postinjection. The incision was thensutured. The operated pups were returned to the motherafter regaining consciousness. Mice were returned to the

animal facility and monitored daily for any abnormalbehavior. Daily observation of these mice receivinginjection of the rAAV vectors revealed no noticeablebehavioral or weight changes, or neurological abnormali-ties. They were sacrificed for immunocytochemical analy-sis at various days ranging from 6 days to 10 weekspostinjection.

ImmunocytochemistryAnimals were anesthetized with ether and perfused

through the heart with 4% paraformadehyde for 10 min.Approximately 6 hr postfixation, the brains were re-moved and transferred to PBS solution containing 20%sucrose and stored at 4°C overnight. Serial coronalsections were cut at 40–50-µm thickness with a vibra-tome. Tissues from every second to third section weremounted on Probe-on slides (Fisher Scientific, Pittsburgh,PA) and subjected to immunocytochemical analysis. Theremaining sections were mounted on slides and stored at280°C for later immunocytochemical studies.

Immunocytochemical analysis was carried out asdescribed previously (Chen et al., 1998). The followingprimary antibodies were used: polyclonal antibody againstGFP (Clontech, Palo Alto, CA, 1:150), mouse monoclo-nal antibodies against glial fibrillary acidic protein (GFAP,1:450; Sigma, St. Louis MO), NeuN (1:150; ChemiconInc., Temecula, CA), neurofilament (NF, 1:150; Sigma),F4/80 (1:100; Serotec, Washington DC), and 28,38-cyclicnucleotide 38-phosphodiesterase (CNPase, 1:50; Sigma).Double immunostaining was carried out with anti-GFPantibody and with various other monoclonal antibodiesdescribed above. Fluorescein isothiocyanate (FITC)-conjugated goat anti-rabbit IgG (1:50; Sigma) and tetra-methylrhodamine-5-(and-6)-isothiocyanate-conjugated(TRITC) goat anti-mouse IgG (1:100; Cappel, OrganonTeknika Corp., Durham, NC) were used as the secondaryantibodies.

In some instances, tissue sections were immuno-stained by the ABC (avidin-biotinylated enzyme com-plex) method using 3,38-diaminobenzidine (DAB) as thechromogen (Vector Laboratories, Inc., Burlingame, CA),then briefly counterstained with hematoxylin.

MicroscopySlides were coverslipped with anti-photobleaching

mounting medium (0. 25% DABCO, 2% n-propyl gallatein polyvinyl alcohol/glycerol) and examined under theNikon Microphoto FXA microscope equipped with aFITC, a TRITC, and a UV-FITC-TRITC triple filter(Nikon, Garden City, NJ). Some sections were analyzedon a confocal laser-scanning microscope, model LSM210 or LSM 410, (Carl Zeiss, Thornwood, NY) to better

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visualize cell morphology and overlapping immunostain-ing.

RESULTSDetection of GFP Expression

Expression of GFP was examined in mouse brainsat various time points between 6 days to 3 monthspostinjection of rAAV vector (Table I). The GFP-expressing cells were detected in all mice that receivedrAAV-MBP-GFP vector in the white matter. No GFP-expressing cells were observed in the brains of controlmice either without injection or those receiving bufferinjection only. Autofluorescence was observed at theinjection site and along the needle tract (Fig. 2). However,it was yellow-brown, not green, fluorescence under theFITC filter, and fluorescence was detected even underTRITC filter. The GFP-expressing cells emitted greenfluoresence and did not fluoresce under the TRITC filter.

Expression Varies With Different Injection SitesThe GFP expression driven by the rAAV-MBP-GFP

vector was examined at different locations within thebrains. Typically, injection into gray matter regionsyielded no GFP expression, while injection in the whitematter resulted in the cell type–specific GFP expression.The number of the GFP-expressing cells detected byimmunostaining varied among animals, presumably result-

ing from the individual variation, conditions of microin-jection, and deviation of injection sites to a great extent.Brain samples with needle tips in or slightly above thecorpus callosum between bregma 1.1 mm to bregma22.80 mm, as described in the mouse brain atlas(Franklin and Paxinos, 1997), showed the highest numberof GFP-expressing cells. On average, these brains re-vealed approximately 1,000 GFP-expressing cells cover-ing 37 sections (40 µm per section; Table I). Up toapproximately 2,000 GFP-expressing cells were esti-mated in one brain after injection of 63 109 rAAVparticles into the corpus callosum region (Fig. 1). TheseGFP-expressing cells were located almost exclusively inwhite matter including the corpus callosum, callosalradiation, fornix, fimbria, and subcortical white matterregions (Fig. 2). The GFP-expressing cells were usuallydetected in the ipsilateral hemisphere within approxi-mately 2 mm in radius from the injection site along thenerve fiber tract. Occasionally, however, GFP-expressingcells could be detected even in the contralateral cerebralhemisphere up to 4 mm away from the site of injection.Manual injections or deviation of injection site from thecorpus callosum often yield lower numbers of the GFP-expressing cells. Nevertherless, studies in.30 mice atvarious time points suggested that the GFP-expressingcells under the control of the MBP promoter locatedalmost exclusively in the white matter. Rarely, there werea few additional GFP-expressing cells scattered in thecerebral cortex in close proximity to the needle track. TheGFP-expressing cells in the cerebral cortex were gener-ally small. Some had intricate processes, morphologically

TABLE I. Expression of GFP Post-rAAV-MBP-GFP InjectionInto the Corpus Callosum*

Postinjectiontime

Total numberof GFP1 cells

(per mouse brain)

Number ofpositive sections(40 µM section)

6 days 960 3215 days 360 4512 days 1,980 406 days 483 3415 days 1,185 457 days 582 2821 days 1,050 32Average 943 37

1 month 96 41 month 102 61 month 225 151.5 months 450 122.5 months 1,131 13a

3 months 564 103 months 336 103 months 153 7Average 382 9

*GFP, green fluorescent protein; rAAV, recombinant adeno-associatedviral vectors; MBP, myelin basic protein.aThe brain was sectioned at 10 µM and the number was adjustedaccordingly.

Fig. 1. Distribution of green fluorescent protein (GFP)-expressing cells in the corpus callosum of a 28-day-old mouse16 days postinjection of the recombinant adeno-associatedvirus (rAAV)-myelin basic protein (MBP)-GFP vector. Everythird successive coronal section was immunostained with theanti-GFP antibody. The total number of GFP-expressing cells inthe brain, thus, equals approximately three times the observednumber. 0: site of injection.

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Fig. 2. Distribution of GFP-expressing cells in mouse brainsfollowing injection of the rAAV-MBP-GFP vector.a: GFP-expressing cells (arrows) are observed in subcortical whitematter, corpus callosum, and fornix regions and spread alongthe nerve fiber tract with fluorescent microscope. Arrowheadrepresents the needle track. Magnification,355 b: An adjacentsection to a. The GFP-expressing cells are detected immunocy-tochemically by the avidin-biotinylated enzyme complex (ABC)method with 3,38-diaminobenzidine (DAB) chromogen. Thesecells were arranged in parallel along the white matter fiber tract.Magnification, 3 47. Inset: Higher magnification of twooverlapping cells indicated with an arrow.c: A schematic

diagram of the corresponding coronal section of a. Hatchedregions represent the location of GFP-expressing cells. Anarrowhead indicates the site of injection.d: GFP-expressingcells in the white matter of the midbrain with fluorescentmicroscopy. Cells spread linearly in regions corresponding tothe bsc and opt fiber tracts. Magnification,395.e:A schematicdiagram of the corresponding coronal section of d. An arrow-head indicates the site of injection. bsc, brachium sup collicu-lus; opt, optic tract; c: cortex; cc, corpus callosum; cg,cingulum; LV, lateral ventricle; df, dorsal fornix; hip, hippocam-pus; fi, fimbria; hip, hippocampus; wm, white matter.

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similar to type I oligodendrocytes, which were describedas having spherical or slightly polygonal cell bodies withabundant and tenuous processes (Szuchet, 1995).

Though gene expression driven by rAAV carryingthe CMV promoter has been demonstrated in neurons inthe gray matter (McCown et al., 1996), it is unclearwhether injection of rAAV vector in the white matter willincrease transduction rate in oligodendrocytes. Therefore,attempt was made to inject rAAV-CMV-GFP vector intothe corpus callosum of eight mice. Examination of brainsrevealed GFP-expressing cells (5–14 per section) in sixmice. Higher number of GFP-expressing cells (up to 45)with the same vector were noted when the vector wasinjected into the hippocampus. Almost all of the GFP-expressing cells in the six mice were located in thecerebral cortex along the needle track. Morphologically,

the majority of these cells were identified as neurons (Fig.3b,d). Astrocyte-like cells were also noted.

The GFP expression in the gray matter was evalu-ated by injection of the rAAV-MBP-GFP vector intostriatum, thalamus, and cerebral cortex. Only occasion-ally, the GFP was expressed in some (less than 5 cells perslide and no more than 10 sections) oligodendrocyte-likecells. However, in one mouse, when the vector wasaccidentally injected into the hippocampus, the GFP-expressing cells were observed in both the polymorphiclayer of hippocampus and the fimbria hippocampi (datanot shown). In another mouse, the needle track went intothe lateral midbrain region and approximately 500 GFP-expressing cells were identified in the midbrain. In thismouse, the GFP-positive cells were found within thebrachium superior colliculus and optic fiber tract (Fig.

Fig. 3. GFP expression in cells with different morphology withrAAV vectors carrying (a, c) MBP promoter and (b, d)cytomegalovirus (CMV) promoter.a: The GFP expression incells with intricate cell processes in the subcortical whitematter. Magnification,3400. b: GFP expression in cells withlarge cell bodies and few long processes in the neocortex.Magnification,3320. a and b: Immunocytochemical stainingwith ABC method, observed under a FXA Nikon light micro-

scope.c: GFP-expressing cells with small bodies and longitudi-nal processes parallel to the nerve fiber tract after injection ofthe rAAV vector carrying the MBP promoter.d: GFP expres-sion in cells with larger cell bodies and a few long processes,showing Golgi-like staining in neurons after injection of therAAV vector carrying the CMV promoter. c and d: Negativeimages captured under Zeiss confocal microscope model LSM210. Bars, 25 µm.

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2d,e), spreading laterally,2 mm away from the injectionsite and an,3 mm region in a rostral-caudal direction.Furthermore, double immunostaining with anti–neuron-specific nuclear protein (NeuN; Mullen et al., 1992) andanti-CNPase (Sprinkle, 1996) antibodies proved thatthese GFP-expressing cells stained with anti-CNPase butnot with NeuN and thus were oligodendrocytes.

Cell Type–Specific Expression

The identification of the GFP-expressing cells wasbased on morphology, anatomic location, and immuno-staining with cell type–specific makers. The GFP-expressing cells in mouse brains receiving the rAAV-MBP-GFP vector typically were located in the corpus callosumand callosal radiation. Cells in these regions displayedoval-shaped cell bodies with parallel processes (Figs. 3cand 4). The nuclei of these GFP-expressing cells wereround or oval and eccentrically located or surrounded bya thin rim of cytoplasm (Fig. 4a,b), resembling oligoden-drocytes located in the nerve fiber tract (Szuchet, 1995;Butt et al., 1994; Suzuki and Raisman, 1992). These cellswere arranged in rows along the nerve fibers (Fig. 2b).Double imunostaining revealed colocalization of GFPand CNPase in almost all GFP-expressing cells under theconfocal microscope (Fig. 4a,b). However, there was nocolocalization of GFP and NF or/and NeuN (Fig. 4c,d),GFP and GFAP, or GFP and F4/80 (data not shown). Asreported (Sprinkle, 1996), the anti-CNPase antibodylabeled the perinuclear cytoplasm and cell processes ofoligodendrocytes while the GFP antibody signal waslocated in the nucleus and cytoplasm as well as cellprocesses. Overlapping staining was found in areas of theperinuclear cytoplasm and cell processes (Fig. 4a,b).

The GFP-expressing cells located in the subcorticalwhite matter or cerebral cortex, having spherical cellbodies and intricate processes (Fig. 3a) also showedoverlapping immunostaining with anti-GFP and anti-CNPase antibodies. In contrast, cells transduced by therAAV-CMV-GFP vector were more diverse morphologi-cally. Most of these cells appeared to be neurons, withlarge cell bodies and a few long processes (Fig. 3b,d).

Double immunostaining studies with anti-NeuNand anti-CNPase suggested that the GFP-expressing cellsobserved in the midbrain, as mentioned above, wereoligodendrocytes. In the particular mouse in which GFP-expressing cells were found in both the polymorphiclayer of hippocampus and in the fimbria hippocampi, thedouble immunostaining analysis did not colocalize theseGFP-expressing cells with anti-GFAP antibodies. Noimmunostaining was observed with anti-NF antibody.Thus, these cells were unlikely to be either neurons orastrocytes.

Duration of GFP ExpressionThe expression of GFP was analyzed at different

time points from 6 days to 3 months postinjection. Therewas no dramatic change in the number of GFP-expressingcells during the first 3 weeks postinjection. The totalnumber of GFP-expressing cells decreased with morelocalized expression after 1–2 months; however, up to,500 GFP-expressing cells in total were still found inmice with injection track located in the corpus callosum(Table I).

GFP Expression in Twitcher MiceAs a preliminary study, expression of the reporter

GFP gene was also examined in homozygous twitchermice. Injection of the rAAV-MBP-GFP vector was car-ried out at 15–20 postnatal days. The GFP expression wasobserved in the corpus callosum region in mice sacrificedat 25 or 30 days of age. However, the number ofGFP-expressing cells was far less than that found innormal C57BL/6J mice.

DISCUSSIONLe Gal La Salle et al. (1993), Akli et al. (1993), and

Davidson et al. (1993) reported a successful gene transferinto brain tissue using adenovirus vectors. These threegroups proved that direct transfer of the LacZ gene drivenby either the Rous sarcoma virus long-terminal-repeat(RSV LTR) promoter or CMV promoter resulted inexpression ofb-galactosidase in neurons in the caudatenucleus and putamen of young mice and neuron and glialcells in the substantia nigra and hippocampus in rats.More recent studies by Kaplitt et al. (1994) and McCownet al. (1996) with the rAAV vector suggested that directinjection of the rAAV vector into the central nervoussystem could produce long-term gene expression andphenotypic correction. Given its safety (Blacklow et al.,1968), lower immunogenicity, and ability to transducequiescent cells, the rAAV vector may prove to be themore suitable vector for gene therapy of neurologicaldisorders (Kaplitt et al., 1994; Karpati et al., 1996; Xiaoet al., 1997). However, similar to the results withadenovirus, studies with the rAAV vector using the CMVpromoter produced preferential neuronal expression inregions of hippocampus, cortex, caudate nucleus, andinferior colliculus. We hypothesized that a cell-specificpromoter may be necessary to achieve a high level oftransduction in oligodendrocytes.

The MBP promoter directs gene expression specifi-cally in the myelin-forming cell, i.e., oligodendrocytesand Schwann cells (Wrabetz et al., 1993). Transgenicmouse studies demonstrated that the LacZ gene con-trolled by a 1.9-kb fragment of the MBP transcriptional

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control region was expressed specifically in the whitematter such as callosal radiations, anterior commisure,fornix, and internal capsule (Gow et al., 1992). Using therAAV carrying this MBP promoter, we have demon-strated previously that the transgene was expressedpreferentially in the oligodendrocytes and had no detect-

able activity in type I astrocytes in cultured cells (Chen etal., 1998).

Oligodendrocytes are found widely in the CNS, buthighly populated in the white matter (Miller, 1996). Ourstudy showed that injection of the rAAV-MBP-GFPvector into or near the corpus callosum resulted in the

Fig. 4. Characterization of the GFP-expressing cells resultedfrom injection of the rAAV-MBP-GFP vector. Coronal tissuesections are double-labeled with antibodies against GFP (green)and 28,38-cyclic nucleotide 38-phosphodiesterase (CNPase; red)or neuron-specific nuclear protein (NeuN; red) and examinedunder the confocal microscope (Zeiss LSM 410). Fluorescencein both red and green channels was collected simultaneously.a:The anti-CNPase antibody (red) localizes only in the peri-nuclear cytoplasm and processes, while anti-GFP antibodylabeled both cytoplasm and nuclei (green). Colocalization withthe anti-CNPase antibody was observed in almost all the

GFP-expressing cells. Diffuse red fluorescence in the back-ground indicates CNPase localization in myelin.b: A closerview of the boxed region in a. The GFP-expressing cells haveoval-shaped nuclei located either eccentrically or surroundedby a thin rim of cytoplasm. Arrows: overlapping staining withanti-GFP and anti-CNPase antibodies observed in the cellprocesses.c and d: Sections adjacent to the section in adouble-labeled with anti-GFP (green) and anti-NeuN (red)antibodies. There was no NeuN immunostaining in any of theGFP-expressing cells. c, cerebral cortex; cc, corpus callosum;hip, hippocampus. Bars: (a, c, d) 50 µm; (b) 10 µm.

510 Chen et al.

GFP expression exclusively in oligodendrocytes mainlyin the subcortical white matter and corpus callosum. AsrAAV viral particles probably were concentrated near theneedle injection sites, it is possible that injection of therAAV vector into the nerve fiber tract might have limitedthe access of neurons to the viral particles. However, sucha possibility is unlikely, since injection of the vectorcarrying the CMV promoter into the same region resultedin a different transduction pattern. In these mice, fewGFP-expressing cells were present in the corpus callosumregion, and most GFP-positive cells were located withinthe cerebral cortex. These cells proved to be neurons bythe characteristic morphology and anti-NeuN immuno-staining (Mullen et al., 1992). Although the transductionof neurons in the cortical region by the rAAV vectorcarrying the CMV promoter was reported to be inefficient(McCown et al., 1996), this could not explain thedramatic difference between the distribution in differentcell types by these two different vectors. We believe thatthe inefficient transduction of neurons in the cerebralcortex and other regions was attributable to the celltype–specific character of the MBP promoter.

The possibility that the lack of transduction inneurons is due to limited neuronal access was furtherruled out by injection of the rAAV-MBP-GFP vector intogray matter regions. Neuron-rich regions such as cerebralcortex, caudate, thalamus, and hippocampus have beenreported to exhibit high levels of transgene expressionusing viral vectors carrying the CMV promotor (Mc-Cown et al., 1996). However, our study showed thatinjection of the rAAV-MBP-GFP vector into these regionsscarcely produced any GFP-expressing cells. The lowtransduction rate of perineuronal oligodendrocytes wasconsistent with the activity of the MBP promoter whichfunctions primarily in oligodendrocytes associated withmyelination. The GFP was expressed in cells in thehippocampus in one adult mouse that received accidentalinjection in this region. Expression of the LacZ genedriven by the MBP promoter in the hippocampus hasbeen reported previously in a transgenic mouse (Gow etal., 1992). Using the Rip antibody, numerous oligodendro-cytes were demonstrated in the hippocampus includingthe hilus region (Berger and Frostscher, 1994) in the adultrat. Thus, it is possible that the GFP-expressing cellsobserved in the hilus region of the hippocampus in thatmouse might be oligodendrocytes. Further studies arenecessary to clarify the exact nature of the GFP- express-ing cells in the hippocampus region.

An accidental injection of the rAAV-MBP-GFPvector into the midbrain of one mouse resulted in theexclusive expression of the GFP in oligodendrocytes inthe small fiber tracts (Fig. 2d,e). This finding is consistentwith our hypothesis that the transduction by the rAAV

carrying the MBP promoter was oligodendrocyte-specific.

Results from Peel et al. (1997) and our previous invitro study (Chen et al., 1998) indicated that immunocyto-chemical analysis using the anti-GFP antibody gave astronger signal than the intrinsic GFP fluorescence.Therefore, the GFP-expressing cells in this study wereidentified immunocytochemically with polyclonal anti-body against the GFP. However, the GFP-expressing cellstransduced by the rAAV-MBP-GFP emitted strong fluores-cence that was clearly visible without immunocytochem-istry even in sections that had been stored at –80°C formonths, suggesting that the expression directed by theMBP promoter was relatively efficient. Variation in thenumber of GFP-expressing cells was noted among micewith different infusion sites, suggesting that, in additionto the high titer of vector, accurate injection into whitematter was necessary and critical in order to achieve hightransduction rates for oligodendrocytes. With good injec-tion, long-term expression at a significant level is possible.

The twitcher mouse is an authentic murine modelfor human demyelinating disease-globoid cell leukodys-trophy (Suzuki et al., 1995; Suzuki and Taniike, 1996).The demyelination in twitcher is due to degeneration ofoligodendrocytes. Demyelination commences at around20 postnatal days in the CNS. Therefore, a gene transferexperiment for therapeutic purpose needs to be carriedout before postnatal day 20. The MBP and its mRNAwere expressed around the postnatal day 10 in mice(Wrabetz et al., 1993). Taking these factors together, therAAV-MBP-GFP was also tested in 8–12-day-oldC57BL/6J mice. Similar to the older C57BL/6J mice, theGFP expression was observed only in the white matter,i.e., corpus callosum (data not shown).

In preliminary studies with 20–25-day-old twitchermice, similar GFP-expressing cells were detected in thewhite matter following injection of rAAV-MBP-GFPvector, although the number of GFP-positive cells de-tected was far less than those found in normal C57BL/6Jmice. Degeneration of oligodendrocytes and depressedexpression of MBP mRNA during natural disease processin twitcher mouse (Suzuki and Taniike, 1996; Taniike etal., 1998) might be the reason for the low transductionrate. Nevertheless, this preliminary study suggested afeasibility of transgene expression in oligodendrocytes inthe twitcher mice. Studies with younger twitcher mice areplanned in future.

In conclusion, our studies using the MBP promoterdemonstrated cell type– and tissue-specific gene expres-sion. The GFP expression driven by the MBP promoteroccurred in the white matter in cells with morphology andspecific immunostaining of oligodendrocytes. This pat-tern of expression is different from that seen in the rAAV

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carrying a CMV promoter. Although expression directedby the CMV promoter has occasionally been observed inoligodendrocytes, our study with CMV promoter sug-gests that the transduction in oligodendrocytes wasinefficient even when the vector was injected into whitematter. Therefore, it is likely that the MBP promoter is amore suitable promoter for oligodendrocyte-specific ex-pression. The observation that expression was limited tothe cells in the inoculated nerve fiber tract suggests thatother delivery strategies in conjugation with vectorinjection may be required for therapeutic treatment.Usefulness of the vector carrying cell-specific promoterin gene therapy should be tested in animal models fordemyelinating diseases. However, the vector might beuseful for neurobiological studies of oligodendrocytesand myelination.

ACKNOWLEDGMENTSWe thank Dr. Sergei Zolotukhin for the pTR-UF2

construct and Dr. Joshua Corbin for the MBP-IFN-gplasmid. We thank Dr. Thomas McCown for his technicalassistance in microinjection and his generous donation ofmicro-injectors. We thank Dr. Robert Bagnell, Ms. Victo-ria Madden, and Dr. Tony Perdue for their assistance inmicroscopy, Ms. Carolyn Lloyd for her editing work, Ms.Shin-ja Kim and Ms. Clarita Langaman for their technicalassistance. We also thank Dr. Diane Armao for her criticaldiscussion in the morphological study.

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