HUMAN GENE THERAPY 16:781–791 (July 2005)© Mary Ann Liebert, Inc.

Review

Recombinant Adeno-Associated Viral Vectors in the Nervous System

CORINNA BURGER,1–3 KEVIN NASH,1,2 and RONALD J. MANDEL2–4

ABSTRACT

Recombinant adeno-associated virus 2 (rAAV2) has been extensively used as a gene delivery vector for thenervous system. It targets primarily neurons in the nervous system and results in sustained long-term ex-pression of transgenes. New rAAV serotypes have been characterized and demonstrated to have improvedtransduction efficiencies in various regions of the brain and spinal cord. This review discusses some proper-ties of rAAV that have been studied in the nervous system such as cell tropism, duration of transgene ex-pression, and distribution of viral transduction, as well as immunity and regulation of transgene expressionissues, all of which are important for optimization of the use of rAAV in the nervous system.

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INTRODUCTION

RECOMBINANT ADENO-ASSOCIATED VIRUS (rAAV) has been anattractive gene therapy vehicle since the wild-type virus

was genetically engineered in the early 1980s (Carter, 2004).The features of this human parvovirus that make it an attrac-tive tool for gene therapy include the fact that the wild-typevirus is nonpathogenic, that all viral genes can be removed fromthe recombinant virus, and that replication is dependent on coin-fection with a lytic helper virus (Muzyczka and Berns, 2001)and therefore reversion to replication-competent virus is an un-likely event in the context of gene therapy. Furthermore, rela-tively new purification methods have resulted in almost com-pletely pure rAAV vector stocks (Kaludov et al., 2002;Zolotukhin et al., 2002; Smith et al., 2003; Blouin et al., 2004;Davidoff et al., 2004). All these features, together with the factthat rAAV can target neurons in the nervous system, have madethis vector a valuable system for the delivery of genes to thebrain for the treatment of neurological disorders and for thestudy of nervous system function.

VIRAL CELL TROPISM IN THE NERVOUSSYSTEM: EFFICIENCY OF EXPRESSION INDIVERSE NEUROANATOMICAL REGIONS

There are approximately 35 known serotypes of AAV. Todate, at least 10 have been engineered into recombinant viralvectors (Gao et al., 2003; Grimm and Kay, 2003). rAAV2 hasbeen the most widely used AAV serotype to deliver genes tothe CNS (Kaplitt et al., 1994; Ali et al., 1996; McCown et al.,1996; Flannery et al., 1997; Peel et al., 1997; Klein et al., 1998;Glatzel et al., 2000). rAAV2 transduces mostly neurons (Mc-Cown et al., 1996; Peel et al., 1997; Bartlett et al., 1998; Kleinet al., 1998; Mandel et al., 1998), although it can infect somenonneuronal cell types at a significantly lower efficiency(Kaplitt et al., 1994; McCown et al., 1996; Peel et al., 1997;Klein et al., 1998; Lo et al., 1999; Davidson et al., 2000; Cuc-chiarini et al., 2003). AAV2 has been shown to efficiently trans-duce almost exclusively neurons in a number of brain structuresincluding the substantia nigra (Kaplitt et al., 1994; Mandel etal., 1997; Klein et al., 1998; Bjorklund et al., 2000; Furler et

1Department of Molecular Genetics and Microbiology, College of Medicine, University of Florida, Gainesville, FL 32610.2Powell Gene Therapy Center, College of Medicine, University of Florida, Gainesville, FL 32610.3McKnight Brain Institute, College of Medicine, University of Florida, Gainesville, FL 32610.4Department of Neuroscience, College of Medicine, University of Florida, Gainesville, FL 32610.

al., 2001; Wang et al., 2002; Burger et al., 2004; Paterna et al.,2004), septal area (Mandel et al., 1999a; Klein et al., 2000;Kaspar et al., 2002b; Landgraf et al., 2003), and globus pal-lidus (Tenenbaum et al., 2000; Burger et al., 2004), and slightlyless efficiently the neostriatum (Fan et al., 1998; Burger et al.,2004; Hadaczek et al., 2004; Dass et al., 2004).

rAAV2 has also been shown to strongly transduce neuronsin the dorsal root ganglia (DRG) and peripheral axons (Flem-ing et al., 2001; Xu et al., 2003). rAAV2 transduction in thehippocampus has been preferentially observed in the dentategyrus, with only weak transduction in the CA1 and CA3 fields(McCown et al., 1996; Freese et al., 1997; Bartlett et al., 1998;Klein et al., 1998; Shimazaki et al., 2000; Ehrengruber et al.,2001; Kaspar et al., 2002a; Klein et al., 2002; Passini et al.,2002, 2003; Haberman et al., 2003; Lin et al., 2003; Scammellet al., 2003; Ahmed et al., 2004; Burger et al., 2004).

Because different AAV serotypes enter cells via distinct cellsurface receptors (Summerford and Samulski, 1998; Qing et al.,1999; Summerford et al., 1999; Walters et al., 2001; DiPasquale et al., 2003), it was hypothesized that differentserotypes would target diverse cell types in the nervous system.Therefore, the identification of other serotypes and their de-velopment into recombinant AAV vectors sparked interest intesting these serotypes in the CNS. To date, rAAV1, rAAV2,rAAV4, rAAV5, and rAAV6 have been tested in the nervoussystem (Alisky et al., 2000; Davidson et al., 2000; Yang et al.,2002; Passini et al., 2003; Vite et al., 2003; Burger et al., 2004)(Fig. 1).

rAAV1 and rAAV5 have demonstrated a higher distributionand number of neurons transduced than rAAV2 in the CNS(Alisky et al., 2000; Davidson et al., 2000; Auricchio et al.,2001; Yang et al., 2002; Passini et al., 2003; Wang et al., 2003;Weber et al., 2003; Burger et al., 2004) (see Fig. 1). rAAV4has been shown to target ependymal cells exclusively; rAAV1rAAV2, and rAAV5 transduce both neurons and ependymalcells (Davidson et al., 2000; Wang et al., 2003). In the retina,rAAV2 and rAAV5 transduce retinal pigment epithelium (RPE)and photoreceptor cells, with higher transduction in the pho-toreceptor cells, whereas rAAV4 transduces RPE cells only(Weber et al., 2003).

rAAV1 and rAAV5 have been shown to have higher trans-duction efficiency and higher levels of transgene expressionthan rAAV2 in striatum, hippocampus, globus pallidus, sub-stantia nigra, spinal cord, and cerebellum (Alisky et al., 2000;Davidson et al., 2000; Wang et al., 2003; Burger et al., 2004;Paterna et al., 2004) (see Fig. 1). rAAV2 transduction of thehippocampus is limited to the hilar region; however, rAAV1and rAAV5 transduce the entire hippocampus including thepyramidal neurons in the CA1–CA3 region (Di Pasquale et al.,2003; Burger et al., 2004; Richichi et al., 2004; Klugmann etal., 2005) (see Fig. 1). rAAV1 has also been shown to have bet-ter transduction than rAAV2 in the neonatal mouse brain(Passini et al., 2003), and in the cat brain (where rAAV5 didnot transduce neurons to any significant level) (Passini et al.,2003).

rAAV serotypes 6–8 have shown greater transduction thanrAAV2 in other tissue organs such as lung, liver, pancreas, andmuscle (Halbert et al., 2001; Gao et al., 2002; Louboutin et al.,2004; Wang et al., 2004, 2005) and transduction in the liver

has been shown to be stable for more than 2 years with rAAV8(Wang et al., 2005). Indeed, rAAV8 appears to transduce stria-tum at a higher efficiency than rAAV1, rAAV2, and rAAV5(R.J. Mandel, C. Burger, N. Muzyczka, W.W. Hauswirth, F.Manfredsson, and S. Reimsnider, unpublished observations).

LONG-TERM PERSISTENCE OF TRANSGENEEXPRESSION IN VIVO

Early studies of long-term rAAV transduction in theCNS

rAAV2, the first and most commonly used serotype until afew years ago, was found to support long-term transgene ex-pression in the CNS (Kaplitt et al., 1994; McCown et al., 1996;Mandel et al., 1997; Peel et al., 1997; Bartlett et al., 1998; Kleinet al., 1998), although initial experiments with rAAV2 ex-pressing �-galactosidase (�-Gal) in the rat brain yielded vari-able results. Stable expression was observed in some regionsof the brain, such as the inferior colliculus; however, after only3 months, significant reduction of transgene expression levelswas observed in other regions such as the hippocampus (Mc-Cown et al., 1996). Similarly, Lo et al. (1999) injected rAAV2with �-Gal into BALB/c mouse caudate and observed expres-sion for as long as 1 year, although the amount of positive cellsdecreased significantly compared with levels seen 2 monthspostinjection. The low levels of expression in all these studieswere believed to be due to silencing of the cytomegalovirus(CMV) promoter, most likely due to hypermethylation (Proschet al., 1996; Lo et al., 1999).

This led to the investigation of other promoters, includingthe neuron-specific enolase (NSE) promoter, the platelet-de-rived growth factor � (PDGF-�) promoter, the myelin basicprotein (MBP) promoter, and two synthetic promoters (theCMV plus human growth hormone first intron enhancer, andthe hybrid CMV-chicken �-actin [CBA] promoter) (Niwa et al.,1991; Peel et al., 1997; Klein et al., 1998; Chen et al., 1999;Paterna et al., 2000; Fu et al., 2002; Owen et al., 2002; Idenoet al., 2003). In several studies, the MD promoter (CMV witha human �-globin intron) was also shown to support expres-sion for at least 1 year (Mandel et al., 1998; Leff et al., 1999;Szczypka et al., 1999). These promoters were found to be sig-nificantly better at consistent long-term expression (at least6–12 months) in the brain and therefore better candidates thanthe CMV promoter. Paterna et al. (2000) also tested the wood-chuck hepatitis virus posttranscriptional regulatory element(WPRE) (Donello et al., 1998; Loeb et al., 1999) in combina-tion with the PDGF-� promoter. The authors showed that greenfluorescent protein (GFP) expression was more robust with theWPRE and was stable for as long as 41 weeks in dopaminer-gic neurons and their projections in the striatum of rats.

In addition to the brain, rAAV has been found to transduceneurons in the spinal cord (Peel et al., 1997; Burger et al., 2004)(see Fig. 1) and cells in the peripheral nervous system (PNS)(Martinov et al., 2002; Xu et al., 2003). Long-term expressionin dorsal root ganglia (DRG) has been observed after injectionof rAAV2 into the sciatic nerve or DRG, which resulted instrong GFP expression of a large number of neurons of the

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DRG, dorsal root, and peripheral axons. This expression waspersistent for at least 8 months (Xu et al., 2003).

Long-term transduction in animal models ofneurological disorders

The long-term expression of rAAV-delivered genes has beenexploited to study the correction of various nervous system-re-lated diseases in rodent models. rAAV2 has been shown to cor-rect central diabetes insipidus for at least 50 weeks postinjec-tion (Ideno et al., 2003). rAAV2 expressing brain-derivedneurotrophic factor (BDNF) has been demonstrated to give per-sistent transduction of highly atrophic neurons for up to 18months after injury and the BDNF resulted in counteraction ofatrophy in both acute and chronic stages after injury (Ruiten-berg et al., 2004). In parkinsonian rat models, rAAV2 vectorsexpressing tyrosine hydroxylase (TH), aromatic-L-amino aciddecarboxylase (AADC), and guanosine triphosphate cyclohy-

drolase (GCH) successfully increased dopamine levels in stria-tum and improved rotational behavior, which persisted for atleast 12 months after intrastriatal injection (Mandel et al.,1999b; Shen et al., 2000; Kirik et al., 2002). Moreover, rAAV2vectors delivering L-3,4-dihydroxyphenylalanine (L-dopa) di-rectly to the striatum have been shown to block established L-dopa-induced dyskinesias in parkinsonian rats (Carlsson et al.,2005).

rAAV-mediated glial cell line-derived neurotrophic factor(GDNF) delivery has also been shown to be effective in rodentmodels of Parkinson’s disease (PD) (Kirik et al., 1998; Bjork-lund et al., 2000; Eslamboli et al., 2003, 2005). In these stud-ies with rats (Kirik et al., 1998) and marmosets (Eslamboli etal., 2003, 2005), pretreatment with the rAAV vector substan-tially protected the nigrostriatal tract and motor function. In ro-dents, GDNF was expressed for at least 6 months (Kirik et al.,1998) and in marmosets rAAV-mediated GDNF was expressedfor at least 17 weeks (Eslamboli et al., 2005).

RECOMBINANT AAV VECTORS IN THE NERVOUS SYSTEM 783

FIG. 1. Comparative transduction efficiencies of pseudotypedrAAV1, rAAV2, and rAAV5 capsids. The number of cells wasestimated for each vector serotype and CNS region in Burgeret al. (2004). (A–C) Representative photomicrographs fromhippocampal transductions of rAAV1 (A), rAAV2 (B), andrAAV5 (C) (1 � 1013 to 8 � 1013 vector genome copies).Green cells were visualized as native GFP fluorescence,whereas red cells were stained with a marker for terminally dif-ferentiated neurons, Neu-N (neuron-specific nuclear protein).rAAV1 and rAAV5 show higher transduction efficiency thanrAAV2 in the hippocampus. The scale bar in (B) represents 200�m and applies to (A) and (C). (D–F) Representative pho-tomicrographs from striatal transductions of rAAV1 (D),rAAV2 (E), and rAAV5 (F). Green cells were visualized asGFP fluorescence, and red cells are stained with Neu-N. As inthe hippocampus, rAAV1 and rAAV5 transduction rates instriatum are higher than the rAAV2 transduction rate. The scalebar in (F) represents 1 mm and applies to (D) and (E). (G–I)Representative photomicrographs of rAAV1 transduction (G),rAAV2 transduction (H), and rAAV5 transduction (I) in thesubstantia nigra (SN). In these panels green cells represent na-tive GFP fluorescence and red cells show tyrosine hydroxylaseimmunoreactivity. rAAV2 and rAAV5 are relatively specificfor the dopamine neurons in the SN, but rAAV1 displays wide-spread midbrain transduction in addition to transducing dopa-mine neurons. The scale bar in (I) represents 500 �m and ap-plies to (G) and (H). (J–L) Representative photomicrographsfrom injections into globus pallidus (GP) of rAAV1 (J), rAAV2(K), and rAAV5 (L). Green cells indicate native GFP fluores-cence, and red cells show Neu-N immunostaining. rAAV2 isrelatively specific for GP whereas rAAV1 and rAAV5 are moreefficient in striatum and therefore less specific for GP. All threeserotypes transduce GP efficiently. The scale bar in (L) repre-sents 1 mm and applies to (J) and (K). (M–O) Representativephotomicrographs of rAAV1 (M), rAAV2 (N), and rAAV5transduction (O) in the spinal cord. Green cells show nativeGFP fluorescence, and red cells are Neu-N immunoreactive. AllrAAV vector serotypes were efficiently transduced in spinalcord, with rAAV1 and rAAV5 displaying slightly improvedtransduction efficiency. The scale bar in (O) represents 100 �mand applies to (M) and (N). Reprinted with permission fromMolecular Therapy.

Various rAAV serotypes (rAAV1, rAAV2, and rAAV5)have also been effective in long-term (4–12 months) correctionof mucopolysaccharidosis (MPS) type I, II, and VII lysosomalstorage disease (Bosch et al., 2000; Fu et al., 2002; Passini etal., 2003; Desmaris et al., 2004). In transgenic knockout micefor Canavan’s disease, rAAV2 carrying the gene for asparto-acylase remarkably reduced the spongiform degeneration ob-served in the thalamus up to 5 months postinjection (Matalonet al., 2003).

Large animals have also been examined as a better model ofhuman viral transduction. Stable GFP expression has been ob-served in both RPE and photoreceptor cells in the eyes of bothdogs and cynomolgus macaques for at least 6 months postinjec-tion of rAAV2, rAAV4, and rAAV5 (Weber et al., 2003). Sub-retinal injections of rAAV2 carrying Rpe65 into Rpe65–/– dogsresulted in correction of eyesight for at least 9 months postinjec-tion (Narfstrom et al., 2003). In the treatment of Parkinson’s dis-ease, persistent behavioral recovery for up to 10 months postin-jection of rAAV2 vectors (encoding TH, AADC, and GCH) hasbeen observed in 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine-treated monkeys (Muramatsu et al., 2002). In conclusion, the var-ious rAAVs appear to sustain transgene expression for greatlengths of time, sometimes the lifetime of the animal, with no ob-vious side effects.

METHODS TO ENHANCE DISTRIBUTION OF TRANSDUCTION

Many neurological disorders caused by single-gene muta-tions such as leukodystrophies or lysosomal storage disorderswill require either global brain transduction or at least wide-spread transduction for successful treatment. One of the currentchallenges of virus-mediated gene therapy is the ability to trans-duce sufficient cells in the CNS to successfully treat disordersrequiring widespread transduction and, for the most part, rAAVcannot meet this challenge. As mentioned above, capsidserotypes such as rAAV1 and rAAV5 display increased trans-duction efficiency in many brain regions (Davidson et al., 2000;Burger et al., 2004) (see Fig. 1). In addition, some ancillarymethods to further increase distribution of rAAV transductionin the CNS have been studied. Infusion of rAAV2 with hep-arin, presumably to block one of the coreceptors for AAV bind-ing, has been reported to increase the distribution of rAAVtransduction (Nguyen et al., 2001; Mastakov et al., 2002). Asimilar approach has been used with basic fibroblast growthfactor, the other AAV coreceptor (Hadaczek et al., 2004). Amethod of slow, long-term infusion of rAAV, termed convec-tion-enhanced delivery, which essentially induces a controllededema in the brain, has also been demonstrated to greatly en-hance the distribution of rAAV-mediated transduction in thebrain (Nguyen et al., 2001). Finally, mannitol, a complex sugarthat induces hyperosmolality in the brain, thereby shrinking theinterstitial space and reversing the flow of cerebrospinal fluid(CSF), has been reported to increase both the efficiency and thedistribution of transduction when mixed with the vector stock(Mastakov et al., 2001) or after systemic injection (Fu et al.,2003; Burger et al., 2005) (Fig. 2).

BRAIN IMMUNE RESPONSE TO AAV

Long-term transgene expression is apparently not accompa-nied by any significant toxicity in naive animals with low trans-duction rates and relatively weak promoters (Mandel et al.,1997, 1998). However, there is evidence of immune responseswhen the animals have been preexposed to rAAV2 (Peden etal., 2004; Sanftner et al., 2004). Thus, peripheral preimmu-nization with either rAAV2 (Sanftner et al., 2004) or wild-typeAAV2 (Peden et al., 2004) can completely block brain trans-duction. However, preimmunization with rAAV2 does notblock subsequent striatal transduction with rAAV5-pseudo-typed vectors (Peden et al., 2004).

Furthermore, intrastriatal rAAV2 injections are sufficient toprime an immune response because a second injection ofrAAV2 in the opposite striatum either 2 weeks (C.S. Peden andR.J. Mandel, unpublished observations) or 4 weeks after thefirst injection led to highly significant inflammatory responseand infiltration of CD8a� cells (Peden et al., 2004). If the trans-gene is not immunogenic, transgene expression appears to berelatively unaffected, even at the second injection site (Pedenet al., 2004). However, if the expressed transgene is immuno-genic, such as humanized jellyfish GFP, there is significantclearance of transduced cells in both injection sites (C.S. Pe-den and R.J. Mandel, unpublished observations).

The immune effects of readministration in the brain are un-expected because no viral proteins are expressed in rAAV2-transduced cells. Without viral gene expression and the ex-pression of a nonimmunogenic protein, no presentation ofimmunogenic peptides on MHC class I should occur. More-over, readministration studies with high-capacity adenoviralvectors does not result in clearance of transduced cells after aninitial reduction of transgene expression associated with theearly inflammatory events peculiar to adenoviral infection(Lowenstein and Castro, 2003). It has been shown that rAAV2uncoats slowly, leading to MHC presentation of capsid peptidesover a prolonged period (Hauck et al., 2004; Thomas et al.,2004). It has been hypothesized that it is this slow uncoatingthat accounts for the unexpected immune reaction to readmin-istration of rAAV2 in the brain (Lowenstein, 2004).

In addition, these results regarding the brain immune re-sponse point out that the brain is not as immune privileged aspreviously believed and requires more detailed study as hasbeen performed for adenoviral vectors (Lowenstein and Castro,2003). Therefore, clinical protocols aimed at neurological dis-orders should probably include determination of peripheral cir-culating anti-AAV antibodies before intracerebral injection ofrAAV.

REGULATION OF TRANSGENE EXPRESSIONIN VIVO

The subject of regulation systems and rAAV in the CNS hasbeen discussed in detail (Haberman and McCown, 2002), andtherefore we briefly discuss only a few issues. As described inearlier sections, AAV can produce therapeutic proteins at rela-tively high levels for extended periods of time in the brain. Theconstitutive high-level expression of therapeutic proteins can

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cause undesirable side effects, such as in the case of growthfactors (Lang et al., 2004), which need to be expressed at lowlevels to produce physiological effects. Drug-regulated gene ex-pression systems provide the ability to control the levels of theprotein product quantitatively and temporally. Artificially reg-ulated gene expression systems could potentially avoid side ef-fects due to constitutive expression of the transgene and allowpharmacological control of expression. A number of artificialregulation systems have been reported for the control of trans-

gene expression in vivo, including tetracycline and its deriva-tive doxycycline (Gossen and Bujard, 1992; Gossen et al.,1995), ecdysone (No et al., 1996), the progesterone antagonistmifepristone (Wang et al., 1994), and rapamycin (Rivera et al.,1996). These systems rely on chimeric transcription factors thatbind promoters directing the expression of the transgene of in-terest. The transcription factor in turn responds to a drug thatcan be administered orally and the transgene expression can bedosed in this manner. Regulation of rAAV expression in the

RECOMBINANT AAV VECTORS IN THE NERVOUS SYSTEM 785

FIG. 2. Systemic administration of mannitol significantly enhances the transduction rate of rAAV2 in striatum. (a) Stereolog-ical estimation of the number of GFP-positive cells transduced with either systemic mannitol or saline control. (b) Volume ofGFP expression estimated by stereological method. (c) Low-power micrographs of representative serial coronal sections show-ing the anterior-to-posterior extent of transduction in the striatum. Error bars represent �1 SEM. Sections show GFP im-munoreactivity and illustrate the enhanced transgene distribution in the mannitol-infused brain (left), versus the saline controlbrain (right). The anterior–posterior (AP) measurements that correspond most closely to coronal sections in the Paxinos and Wat-son atlas (Paxinos and Watson, 1997) are indicated next to each section. Scale bar, 1 mm. For systemic injection of mannitol, 3ml of sterile 25% mannitol in 0.9% saline per 100 g body weight was injected intraperitoneally 15 min before intracerebral vec-tor injections (n � 5), as described above. Control animals received identical preinjections of 3 ml of 0.9% saline per 100 g bodyweight instead of mannitol (n � 5). Unbiased stereological estimation of the total number of cells in striatum was obtained bythe optical fractionator method, as previously described in detail (Burger et al., 2004; Peden et al., 2004). Actual cell countingwas performed with a �100 oil objective. The total number of neurons was estimated according to the optical fractionator for-mula (West et al., 1991) A coefficient of error (CE) was calculated according to Gundersen and Jensen (1987) for each animaland the estimation for each animal was accepted only when the CE was �0.1. Parametric analysis of variance (ANOVA) wasused to assess significant differences between treatments. Significance was accepted at the 95% probability level. Reprinted withpermission from Molecular Therapy.

A

C

B

brain, for gene therapy has focused mainly on the tetracycline-regulated system.

The tetracycline-regulated transcription system as developedby Gossen and Bujard (1992) has been the most studied as aneffective system for tight regulation of transgene expression forgene therapy applications (Urlinger et al., 2000). Other systemsare becoming more popular for gene therapy applications in thenervous system. The rapamycin system (Rivera et al., 1996)has been engineered into AAV and successfully used in mus-cle (Rivera et al., 1999; Ye et al., 1999; Johnston, 2003) andliver (Auricchio et al., 2002a). In the eye, intravitreous or sub-retinal injections of erythropoietin (Epo) (Auricchio et al.,2002b), allowed the control of expression of the transgene in areversible manner, although basal levels of Epo took severalweeks to decline after withdrawal of the drug. Tetracycline (Tet)regulation is the only other artificial regulation system that hasbeen engineered into rAAV to date.

One of the biggest issues in the regulation of gene expres-sion, using these artificial systems, concerns “leakiness” (or in-complete shutdown of transgene expression in the off-state).This is important when basal levels of expression of the trans-gene are unacceptable because expression of the gene can betoxic to the system. In the context of rAAV it has been arguedthat expression of the transgene in the absence of a transcrip-tional activator is probably due to the fact that AAV2 terminalrepeats (TRs) have endogenous enhancer/promoter functionthat is directing expression in the absence of a Tet activator.This has been demonstrated (Flotte et al., 1993; Haberman etal., 2000) and also discussed in Haberman and McCown (2002).Various expression cassettes have been tested to resolve thisbasal expression issue (Bohl et al., 1998; Fitzsimons et al.,2001; Haberman et al., 2002; Samakoglu et al., 2002; Chtartoet al., 2003; Folliot et al., 2003; Chenuaud et al., 2004; Gafniet al., 2004; Jiang et al., 2004), but no leak-proof constructshave been convincingly demonstrated in vivo. Until an optimalcassette that results in good induction and negligible basal ex-pression is achieved, it will not be safe to deliver genes to thenervous system for the cure of human neurological disorders.Therefore, in order for a gene therapy approach to reach theclinic, tight regulation of transgene expression needs to beachieved.

CONCLUDING REMARKS

Given that the safety and efficacy profile of rAAV2 vectorshas been encouraging, especially in the CNS, it is not surpris-ing that this vector has entered human clinical trials. There arecurrently five rAAV2-based clinical trials either underway orlate in the approval process (Mandel and Burger, 2004). Todate, none of these trials incorporate a regulated rAAV vector,nor have any of the trials explicitly dealt with immune re-sponses. Further development of the rAAV system for enhancedtransduction efficiency and targeting of various cellular phe-notypes is still highly warranted to improve the clinical utilityof this vector system. The capsid structure of AAV2 has beenresolved by electron cryomicroscopy (Kronenberg et al., 2001)and by X-ray crystallography (Xie et al., 2002); in addition,mutagenesis studies of the capsid protein have revealed the pu-tative cell receptor-binding sites (Wu et al., 2000; Opie et al.,

2003). These studies have been key to potential retargeting ofthe recombinant viral vector (Warrington et al., 2004). Contin-ued advancements in this field of study may allow targeting ofrAAV to specific neuronal phenotypes, or to glial cells. Also,improved delivery techniques will allow increasing retrogradetransport for better treatment of motoneuron disorders, or mas-sively augmenting transduction efficiency in the nervous sys-tem. Therefore, future advances in the areas discussed in thisreview may hold exciting developments in the field of genetherapy in the CNS.

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Address reprint requests to:Dr. Corinna Burger

Department of Molecular Genetics and Microbiologyand Powell Gene Therapy Center

College of MedicineP.O. Box 100266

University of FloridaGainesville, FL 32610

E-mail: cburger@ufl.edu

Received for publication March 10, 2005; accepted March 10,2005.

Published online: May 11, 2005.

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