doi:10.1006/mthe.2002.0594, available online at http://www.idealibrary.com on IDEAL

Enhanced Secretion and Uptake of �-GlucuronidaseImproves Adeno-associated Viral-Mediated Gene Therapy

of Mucopolysaccharidosis Type VII MiceSusan S. Elliger,* Carl A. Elliger, Chen Lang, and Gordon L. Watson

Children’s Hospital Oakland Research Institute, Oakland, California 94609, USA

*To whom correspondence and reprint requests should be addressed. Fax: (510) 450-7910. E-mail: selliger@chori.org.

Previous treatment of mucopolysaccharidosis type VII mice (Sly syndrome) with AAV vectors hasresulted in increased levels of �-glucuronidase (GUS) enzyme in some tissues with reduction ofglycosaminoglycan storage granules and improved health. By adding coding sequences for secre-tion (Ig�) and uptake (HIV-1 TAT) signals to the GUS gene delivered by AAV, and treating miceboth intrathecally and intravenously as newborns, we have increased the GUS enzyme levels inmore tissues and have improved the health of the mice so much that they are able to breed. Thelevels of GUS in the serum were above normal in some mice, which caused reduction of storagein the spleen, a nontransduced tissue. The heart and aorta showed therapeutic levels of GUSenzyme. AAV GUS DNA was found in brain and liver, which showed no storage. Phenotypicallythe treated mice were more active and showed less stunted skeletal growth. The pups born tothese mice were not affected by the gene therapy, as shown by mutant levels of GUS enzyme intheir tissues and the absence of AAV GUS DNA. However, they were resistant to intravenoustreatment with AAV GUS due to the mother’s antibodies, but not to intrathecal treatment.

Key Words: lysosomal storage disease, mucopolysaccharidosis, MPS VII, �-glucuronidase,adeno-associated virus, AAV, protein transduction domain, secretion signal, intrathecal

ARTICLE

INTRODUCTION

Replacement of a defective gene to restore health to ananimal provides a good demonstration of the potentialusefulness of gene therapy. Here, the gene of interestencodes �-glucuronidase (GUS), whose absence or func-tional defectiveness causes a buildup of glycosaminogly-can (GAG) storage granules within the lysosomes of mostcell types, leading to the symptoms of mucopolysaccha-ridosis (MPS) type VII, Sly syndrome [1]. In humans thisdisease is characterized by mental retardation, abnormalbone development, distorted features, and organ mal-functions leading to organ failure and early death [2]. Anaturally occurring mutation of GUS in mice has pro-vided an animal model for this disease [3,4]. MPS VII inthe mouse model mimics the human disease and themost visible symptoms are stunted growth, lethargy, poorgrooming, and death before 1 year of age. Neither malenor female mice that are homozygous for the mutationbreed, consequently the mutation is maintained by heterozygous breeding. Several investigators [5–12] haveused adeno-associated virus (AAV) to deliver GUS cDNAto these mice. In general their health was improved, espe-cially as evidenced by the reduction in accumulated

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storage granules in specific tissues. In addition, Daly etal. [13] reported that male mice treated with an AAV vec-tor as newborns developed the ability to breed success-fully with normal female mice. Also, using restoration ofbreeding ability as an indicator of health, success hasbeen demonstrated after treatment of neonatal mice byenzyme replacement [14] or by syngeneic bone marrowtransplant [15]. Both of these latter treatments, however,have immunological limitations.

A problem with treating MPS VII, as well as manyother lysosomal storage diseases, is that the therapy tar-get is virtually all cells in an organism. Clearly, it is notfeasible to transduce all cells with any known vector.Fortunately, however, this is not necessary when cross-correction can occur. It has been demonstrated that sev-eral lysosomal enzymes, including GUS, can be secretedby producing cells and taken up by nonproducing cells[16]. Enzyme that enters the circulatory system, eitherfrom overproducing cells or by direct enzyme therapy,can be taken up by most cell types via the mannose-6-phosphate receptor [5,17,18]. Another favorable factor isthat only a small fraction of the normal lysosomalenzyme level is usually sufficient to prevent or eliminatestorage. For MPS VII in mice, we estimate that only about

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TABLE 1: Effect of enzyme modification on GUS secretion

Specific activityAverage specific activitiesa ratiob

No. SerumVector name Construct of mice Serum Heart Liver Spleen Kidney Aorta Liver

AAVmGus mGus 3 0.11 0.011 0.25 0.013 0.010 0.006 0.43 ± 0.05

AAVmGus�6 mGus �6 2 2.5 0.180 4.2 0.11 0.024 0.008 0.62 ± 0.07

AAVIg�mGus�6 Ig� mGus �6 2 1.9 0.065 2.3 0.071 0.019 0.014 0.87 ± 0.11

AAVmGus SU2 Ig� mGus �6TAT 4 14 0.15 2.5 0.50 0.069 0.022 7.1 ± 2.3

AAVmGus U mGus �6TAT 3 4.4 0.068 1.1 0.18 0.034 0.023 5.1 ± 1.9

AAV U mGus TAT mGus �6 3 0.53 0.034 1.0 0.059 0.015 0.006 0.52 ± 0.20

AAV U mGus U TAT mGus �6TAT 3 6.1 0.044 1.2 0.24 0.036 0.013 5.4 ± 1.1

Two-month-old mps/mps mice were treated IV with the various AAV vectors. The tissues were assayed after 7 weeks treatment. All enzyme assays were done in triplicate.aTissue-specific activities are U/g tissue (wet weight), whereas serum activity is shown as mU/mL.bSerum/liver ratios were calculated for each animal and then averaged for each construct. Shown are means ± SD.

1% of the normal level of GUS is sufficient [8], whileoverproduction of GUS has no apparent deleterious effectas judged by transgenic mice expressing GUS at 20-foldthe normal level [19]. In principle, then, a limited num-ber of overproducing cells should be able to provide suf-ficient enzyme to the rest of the organism. There are twoproblems: first, GUS is not efficiently secreted; and sec-ond, circulating enzyme does not cross the blood/brainbarrier. Previously we have circumvented the blood/brainbarrier by administering AAV vector via intrathecal injec-tion into the cerebrospinal fluid [7]. To further broadenthe distribution of GUS within an organism, we havenow altered the GUS protein. We modified the GUS genedelivered by AAV by adding coding sequences for secre-tion and/or uptake signals. Several vector constructs weretested and one was chosen for more extensive study. Thisconstruct contains truncated mouse GUS cDNA with anIgk secretion signal peptide at the amino terminus andthe protein transduction domain (PTD) from HIV-1 TATat the carboxy terminus. The resulting modified GUS pro-tein was secreted into the serum at high levels and takenup by tissues that are usually resistant to AAV treatment,such as the aorta, kidney, and spleen. Uptake of the mod-ified GUS from serum was at least as fast as the uptake ofnormal GUS. We amplified GUS mRNA in tissues show-ing decreased lysosomal storage and increased GUSenzyme activity to determine if the enzyme was comingfrom the serum or being made in the tissue.

There were several indicators that this modified GUSwas improving the health of the MPS VII mice, but per-haps the clearest was that treated male and femalemutants were now able to breed and rear their youngsuccessfully. When offspring were tested for the presenceof AAV vector, it was uniformly absent.

Xia et al. [20] have shown recently that human GUSwith the PTD attached at the C terminus also has

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extended distribution in the MPS VII mouse. In that case,an adenovirus vector was used to deliver the modifiedgene and modification was limited to the C terminus.

RESULTS

Modifying the GUS VectorSeveral vectors were made in which the sequences codingfor the N-terminal and/or C-terminal peptides of GUSwere altered (Table 1). For most constructs the codingregion for the C-terminal six amino acids was deleted.This eliminated the esterase 22 (also known as egasyn)binding site that anchors mouse GUS to endoplasmicreticulum and delays the entry of GUS into lysosomes[21]. In the lysosomal form of GUS these terminal aminoacids are proteolytically removed without change in theenzyme activity of GUS. By deleting the coding sequencefor these terminal amino acids, we eliminated microsomalbinding of GUS and a potential hindrance to GUS secre-tion. For some constructs a PTD sequence was added tothe 3� end of the truncated GUS coding sequence. Thisdomain, from the HIV-1 TAT protein, has been shown tofacilitate the passage of proteins across cell membranes[22,23]. At the N terminus of GUS we have added thesecretion signal peptide from Ig�. The Ig� peptide wasused because it has been well established as an effectivesecretion signal for heterologous proteins. Alternatively,the PTD from TAT protein was added to the N terminusof GUS. All of the constructs (Table 1) produced reason-able physical titers of the rAAV using the triple transfec-tion protocol and all produced enzymatically active GUSprotein.

To test the different rAAV vectors, adult MPS VII micewere injected intravenously (IV). After 7 weeks the ani-mals were killed and GUS activity was measured in sev-eral tissues including serum. For all of the constructs the

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FIG. 1. Histopathology of brain,liver, and spleen from mutantstreated IT and IV with AAVmGUSSU2 as newborns compared withuntreated mutant and normal mice.All mice are 4 to 6 months old. Allphotomicrographs were originallytaken at �200. Note that AAV treat-ment reduces storage to normal lev-els in brain and liver and to minimallevels in spleen.

liver was the major site of GUS activity, whereas the brainhad virtually no GUS. Other tissues varied between thesetwo extremes. For a given vector construct there was oftenconsiderable animal-to-animal variation in the extent ofGUS expression. However, as also noted by Daly et al.[24], there was a strong correlation between the amountof enzyme in the liver and circulating enzyme in theserum. This implied that the liver was the primary sourceof serum enzyme. To assess the effects on secretion causedby the different modifications to GUS, the ratios of serumactivity to liver activity were compared (Table 1). As thespleen also showed significant differences in GUS expres-sion among the different vectors, we looked at the num-ber of storage granules present after treatment with threeof the vectors. Spleens from mice treated with AAV mGUS(unmodified) or AAV mGUS�6 (truncated) both had asmuch storage as an untreated mutant (Fig. 1). However,treatment with AAV mGUS SU2 reduced the storage tominimal levels, as it did when the mice were treated asnewborns (Fig. 1). Regardless of N-terminal modifications,the most dramatic increases in secretion were seen in thethree constructs having the TAT sequence added to the Cterminus. The vector engineered to provide GUS with Ig�at the N-terminal end and TAT at the C-terminal end,AAVmGUS SU2, was chosen for more extensive studiesbecause its use resulted in the highest levels of GUSenzyme in most of the tissues and in the serum.

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Characterization of mGUS SU2Thermostability and electrophoretic mobility can be sen-sitive indicators of changes in GUS protein structure [25].GUS from genetically normal mice and mGUS SU2 fromtreated MPS VII mice were both stable at elevated tem-peratures and had inactivation half times of 5.5 and 7.5

FIG. 2. Western blot comparing secreted GUS generated by AAV transduced293 cells (lanes 1 and 2) with native GUS isolated from mouse kidney (lane3). The lanes were loaded with equal amounts of GUS enzyme based on activ-ity. The primary antibody used for detection was specific for mouse GUS. Notethat the GUS activity to protein ratios are similar for all three enzymes.

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minutes, respectively, at 73�C. In native gel elec-trophoresis [26], mGUS SU2 migrated slightly faster thannormal GUS. These small differences suggested that thetwo mature, processed proteins were similar, but notidentical.

The difference in proteins, which might include dif-ferences in glycosylation, raised the question as to whetheror not mGUS and mGUS SU2 were equally capable ofbeing taken up from serum. To generate material for adirect test of uptake, protein was partially purified from themedium of 293 cells transduced with either AAVmGUS orAAVmGUS SU2. The amount of modified GUS secretedinto the medium by the cells was four times greater thanthe amount of unmodified enzyme. These two secretedforms of GUS were compared with native GUS from mousekidney using SDS-PAGE followed by western blot (Fig. 2).The two secreted GUS proteins were indistinguishable insize and had similar ratios of enzyme activity to antibodyrecognizable protein. The GUS activity to protein ratio wasalso similar for the native enzyme, but the latter was pre-dominantly in the slightly smaller, processed, lysosomalform [reviewed in 27].

When the partially purified mGUS or mGUS SU2secreted from transduced cells was injected IV into MPSVII mice, each was rapidly cleared from the serum witha half time of 8 to 11 minutes (Fig. 3). After 2 hours,when very little of the injected enzyme remained in theserum, the mice were killed and GUS activity in varioustissues was measured (Table 2). Nearly half of the admin-istered enzyme was in the liver, and the spleen also hadsubstantially elevated enzyme activity. Most other tis-sues had marginally elevated enzyme, and the brain wasnot significantly above untreated MPS VII animals.Neither mGUS nor mGUS SU2 was taken up consistentlymore efficiently than the other.

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Treatment of Newborn MiceTreated MPS mice reported up to this point were all treatedas adults. To maximize the benefits of gene therapy, MPSVII mice (three males and three females) were treated withAAVmGUS SU2 shortly after birth (2 to 3 days), which isbefore major developmental damage due to lysosomalstorage has occurred. IV injection of vector has limitedeffectiveness in the brain [7], therefore, the mice were alsogiven intrathecal (IT) injections. IT administration ofAAVmGUS to provide vector to the brain was previouslyshown to be effective in both adults and newborns [7].

When the treated mice were killed at 3–6 months ofage, their tissues and serum were assayed for GUS enzymeactivity. The results for five mice (one died of unknowncauses 2 weeks after giving birth) are shown in Table 3. Ofthe 10 tissues assayed, only the lung, kidney, spleen, andgonads showed less than a therapeutic level of GUSenzyme, that is, below 1% of normal [8]. (The lung andgonads showed mutant levels.) The brain, spinal cord,heart, liver, spleen, and serum were all substantially higherin GUS activity than untreated mutants [8].

Tissues of the brain, heart, liver, and spleen were fixed,sectioned, and stained to examine the magnitude of gly-cosaminoglycan storage in the lysosomes. In untreatedmutants the heart (muscle) did not show a buildup of stor-age granules (data not shown), but excessive storage wasevident in the brain, liver, and spleen (Fig. 1). Treatmentwith AAVmGUS SU2 caused the brain and liver of mutantsto appear identical to normal tissues, and GAG storage inthe spleen was much decreased (Fig. 1).

TABLE 2: Distribution of GUS activity following IV adminis-tration of enzyme to MPS VII mice

Tissue mGUS mGUS SU2

Brain 0.015 ± 0.012 0.010 ± 0.002

Heart 0.070 ± 0.048 0.029 ± 0.003

Aorta 0.017 ± 0.005 0.016 ± 0.007

Lung 0.066 ± 0.027 0.038 ± 0.009

Liver 1.4 ± 0.53 2.0 ± 0.89

Kidney 0.051 ± 0.017 0.046 ± 0.011

Spleen 0.40 ± 0.19 0.43 ± 0.17

Ovaries 0.048 0.083

Testes 0.045 ± 0.017 0.025 ± 0.007

Muscle 0.028 ± 0.015 0.011 ± 0.005

MPS VII mice injected with 4.0 units of mGUS or mGUS SU2 were killed after 2 hours, andenzyme-specific activity was measured for the tissues listed. For most tissues the number ofmice, n, was 3; for aorta and testes, n = 2; for ovaries, n = 1. Means ± SD are in U/g tissue.

FIG. 3. Serum levels of GUS following IV injection of enzyme. GUS enzymewas partially purified from the medium of 293 cells transduced with AAVmGUSor AAVmGUS SU2. Note that both enzymes were rapidly taken up by the tis-sues (removed from the serum) with the half times indicated.

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A further improvement of the mutantphenotype could be seen in the bones.Because the mice were treated as new-borns, skeletal growth was affected andthe mice grew to greater body lengththan mutants and had longer noses(Table 4). However, the treated mice didnot show completely normal bonedevelopment, but rather were interme-diate between untreated mutants andnormal mice.

The improved health of theAAVmGUS SU2 treated mice was alsoevident in their behavior. The mice wereactive and had sleek, groomed coats.They formed three breeding pairs pro-ducing litters averaging four pups each.One pair was maintained for 5 months,during which time the mice produced21 offspring in five litters. None of thepups had AAV DNA in their cells asshown by PCR (Fig. 4).

Pups from these treated mutants weretested for GUS activity 1, 24, and 48hours after birth and compared withmutants born to untreated mps/+ par-

ents to determine if the increased level of GUS enzymein the mother’s serum would give rise to higher amountsof GUS in tissues of her offspring. The mother of thepups had 75% of normal GUS levels in her serum whenthey were born; mps/+ parents have 50% of normal. Wefound that GUS enzyme levels in the pups’ tissues werethe same as that of mutants born to mps/+ parents (datanot shown). When progeny from treated mice wereallowed to mature (2–6 months old), they displayed themutant phenotype and also showed only mutant levelsof GUS enzyme in 10 tissues plus serum (data notshown).

One major difference between the two sets of mutantsjust described became evident when some of the pups ofAAVmGUS SU2 treated parents were in turn treated withAAVmGUS SU2 as newborns, IT and IV. Levels of GUSenzyme above mutant levels were only seen in brain,spinal cord, and serum (Table 5).

RNA was extracted from brain, liver, and spleen ofthe treated breeders. These tissues had displayed bothdecreased lysosomal storage and increased GUS enzymeactivity. The RNA was DNase treated, and RT-PCR wasperformed using primers to amplify segments of the GUSmRNA generated by the AAV vector. Segments of themutant GUS mRNA were also amplified and provided abackground control. The samples were also run withoutreverse transcriptase to control for DNA contamination.The Southern analysis of these PCR products is shown inFig. 5. mGUS SU2 RNA derived from AAV-delivered DNAwas present in brain and liver, but not in spleen.

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TABLE 3: GUS enzyme activity in tissues of breeders

GUS activity: U/g tissue or mL serum

Pair I Pair II Pair III % ofnormal activity

Tissue (mean, n = 5)

Brain 0.52 0.30 0.40 0.22 0.30 15.5

Spinal Cord 1.7 1.2 0.65 0.22 13 370

Heart 0.035 0.13 0.044 0.096 0.26 7.5

Aorta 0.017 0.02 0.010 0.012 0.029 1.5

Lung 0.022 0.036 0.029 0.027 0.061 0.3

Liver 0.079 1.5 0.47 0.82 1.9 2.6

Kidney 0.013 0.07 0.042 0.027 0.066 0.3

Spleen 0.022 0.21 0.12 0.13 0.33 0.5

Ovaries 0.018 0.038 0.4

Testes 0.012 0.006 0.010 0.1

Muscle 0.009 0.018 0.005 0.006 0.022 1.2

Serum 0.0001 0.0071 0.0044 0.0033 0.014 98

Mps/mps mice were treated with AAVmGUS SU2 as newborns, IT and IV. The mice were allowed to breed and thenassayed at 3–6 months of age. All enzyme assays were done in triplicate. (The female of pair III died of unknowncauses.)

O+ O O+ O O

Parts

DISCUSSION

Modification of GUS can alter its secretion from trans-duced cells in vivo and can increase the redistribution ofthe enzyme. The results shown here confirm and extendthe observations of Xia et al. [20], who showed that sys-temic administration of an adenoviral vector making aTAT-modified GUS resulted in increased enzyme in non-transduced tissues. Furthermore, they showed that afterintrastriatal or intraventricular administration of vector,the diffusion of modified GUS from transduced cells toother parts of the brain was substantially increased.

The first 22 amino acids of intact GUS normally func-tion as a leader sequence that aids in directing GUS throughmicrosomal membranes and into lysosomes. The leadersequence is cleaved off during the process. Like many lyso-somal enzymes (including the mannose-6-phosphate moi-eties, which help target the protein to lysosomes via man-nose-6-phosphate receptors), GUS is glycosylated. Normally,only a small fraction of GUS is secreted, allowing it to beredistributed to other cells which can take up GUS via man-nose-6-phosphate receptors on the cell surface. To increasesecretion we added a known secretion signal, Igk, to the Nterminus of GUS leaving the normal cleavage site intact.This N-terminal modified version of GUS demonstratedonly modest increases in secretion as evidenced by the ratioof circulating serum enzyme to liver enzyme.

We also modified the C terminus of GUS in ourattempts to increase the redistribution of enzyme. Innative mouse GUS, the C terminus is recognized by

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TABLE 4: Measurements of mouse skeletons

Normal Treated mutant Mutant

Skull bones

A Nasal length 0.76 ± 0.04 0.632 ± 0.05a 0.612 ± 0.03

B Rostrum width 0.399 ± 0.01 0.413 ± 0.01b 0.457 ± 0.03

C Zygomatic arch depth 0.125 ± 0.01 0.161 ± 0.01 0.178 ± 0.01

D Skull width 1.23 ± 0.02 1.29 ± 0.03 1.35 ± 0.04

Limb bones

1 Femur 1.57 ± 0.04 1.37 ± 0.06 1.15 ± 0.11

2 Tibia and fibula 1.80 ± 0.06 1.61 ± 0.07 1.50 ± 0.06

3 Humerus 1.20 ± 0.05 1.12 ± 0.09b 1.0 ± 0.07

4 Radius and ulna 1.40 ± 0.04 1.31 ± 0.04 1.22 ± 0.02

Three representative sets of bones from a normal (I), AAVmGUS SU2 treated mutant breeder (II), and anuntreated mutant (III) are shown in the photo. Bone measurements are shown in the table for five animals of eachset in centimeters. The bones are from mixed males and females, 3–6 months old. The diagram shows where theskull measurements were taken, while the limbs were compared by length. All differences between groups in pair-wise comparisons were statistically significant (t-test, P < 0.05) except as noted. The treated mutants were inter-mediate between normal and untreated mutant mice.aNot significantly different from normal by t-test (P > 0.05).bNot significantly different from mutant by t-test (P > 0.05).

esterase 22, which tends to anchor GUS to the endoplas-mic reticulum. Thus, at any given time a significant frac-tion of intracellular GUS is sequestered outside the lyso-somes [27]. In the processing of GUS to the mature,lysosomal form, the C-terminal amino acids are cleaved.Zhen et al. [21] showed that deletion of the sequenceencoding the last six amino acids resulted in GUS proteinthat was no longer sequestered in the endoplasmic retic-ulum. They also showed that the C-terminal amino acidsof human GUS, which differ from mouse GUS, are notrecognized by esterase 22. We find here that deletion ofthe six terminal amino acids from mouse GUS by itself hadlittle effect on secretion of the enzyme, as evidenced by

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the serum-to-liver ratio. To this truncatedGUS sequence we then added the PTD fromthe HIV-1 TAT protein [22,23]. This pep-tide, attached to a variety of proteins, hasbeen shown to aid the cellular uptake ofthose proteins. Here, C-terminal TAT, eitheralone or in combination with N-terminalmodifications, increased GUS secretion intoserum roughly tenfold. However, TATadded to the N terminus was ineffectual.Although the TAT peptide is thought of asan uptake signal, it clearly aids secretionwhen attached to the C-terminal end ofGUS. Presumably, it can aid the transportof protein across the cell membrane ineither direction. With the concentration ofGUS within a transduced cell being rela-tively high, the net flow of enzyme is out,whereas the net flow for a nontransducedcell should be in.

The modified protein with N-terminalIg� and C-terminal TAT, mGUS SU2, wascharacterized in some detail. Comparedwith endogenous GUS from normal mice,it was slightly more thermostable andslightly more acidic in electrophoreticmobility at neutral pH. This suggested pos-sible differences in posttranslational pro-cessing, which includes glycosylation andphosphorylation in addition to peptidecleavage. This in turn suggested that cellu-lar uptake might be altered. Xia et al. [20]showed that the PTD, when added tohuman GUS, did not interfere with uptakevia the mannose-6-phosphate receptor, butdid provide a second, independent mecha-nism for transporting the enzyme acrosscell membranes. To address the question ofuptake directly, mGUS and mGUS SU2were partially purified from the medium oftransduced cells in culture. Western blotanalysis of these secreted proteins com-pared with partially purified kidney GUS

indicated that in each case the enzyme activity per pro-tein molecule was similar. However, the two secreted pro-teins derived from the AAV vectors were in the larger,prelysosomal form, whereas the native kidney enzyme waspredominantly the smaller, lysosomal form with only aminor band at the larger size. When infused into MPS VIImice, both mGUS and mGUS SU2 were rapidly taken upby tissues, especially liver. Although mGUS SU2 appearedto be cleared from serum slightly faster than mGUS, thedifference was within the standard error of the data. Thus,the modifications to GUS clearly do not inhibit its cellu-lar uptake. If the TAT peptide provides an additional routefor uptake, as indicated by Xia et al. [20], the kinetics of

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this TAT-mediated uptake must be relatively slow com-pared with uptake via the mannose-6-phosphate receptor.

From the present data, we can estimate how efficientlythe modified GUS is secreted. In the serum, mGUS SU2had a half-life (t1/2) of about 10 minutes (Fig. 3), which isvery short compared with the half-life of GUS in tissues(1–5 days). To maintain the steady-state serum enzymelevel shown in Table 1, mGUS SU2 must be secreted intothe serum at a rate of 2.8 units per day (rate of secretioninto serum = ln 2 / t1/2 � total serum enzyme). Assuminga conservative half-life in liver of 1 day, this implies thata substantial fraction, about 60%, of the GUS SU2 madein the liver was secreted into the serum. A longer half-lifein liver would mean an even higher fraction

of enzyme was secreted. Making similar calcu-lations, about 10% and 15% of liver enzymewas secreted into the serum of mice treatedwith AAVmGUS and AAVmGUS�6, respec-tively, whereas in untreated wild-type miceGUS secretion into the serum seems to beabout 5% of what is made in the liver.

To assess the physiological and potentiallyclinical value of increased secretion, we chosethe vector construct having Ig� at the N ter-minus and TAT at the truncated C terminus.This vector generated both high expressionand high secretion of GUS. To maximize thebenefit to treated MPS VII mice, vector wasadministered to newborns both IV and IT.

As with the results treating adult mice, weobserved that the fusion protein was secretedmore effectively into the serum. Furthermore,organs like the aorta, which were previouslyunaffected by vectors carrying native GUS,now showed therapeutic levels of GUSenzyme. The kidney and spleen also showedhigher levels of GUS enzyme but still averaged

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less than 1% of normal, although the GUS activity in thespleen of one mouse was 1.1%. Analysis of GUS mRNAindicated that the spleen accumulated GUS enzyme froman extracellular source, whereas the brain and liver wereable to make GUS intracellularly. The levels of GUS in theserum varied widely from mouse to mouse, but the higherthe level in the serum, the higher the level in the spleen.The brain of each treated animal showed 9–22% of nor-mal levels of GUS. This was more than in any other tissueexcept the spinal cord, which varied widely, perhaps dueto variability in the intrathecal injection.

The improvement in the health of the treated mutantmice was shown on several levels. GAG storage granulesin the brain and liver were reduced below the level ofmicroscopic detection, and the spleen showed very littlestorage. Bone growth was improved and the mice weremore active. Treated mice, both males and females, weresuccessful breeders. The mothers were able to bear andnurse an average of four pups per litter. All pups werehomozygous for the mps mutation. It is rare to find fourmps/mps pups in a litter from an mps/+ cross; one to twois usual. The amount of time between mating and the firstlitters and the time between litters was the same as formps/+ crosses, further showing good health in the treatedmutants.

FIG. 4. PCR assay for AAV DNA in pups born to AAV-treated parents. Theprimers used in lanes 1–8 identify the GUS genotype of the mouse. The primersused in lanes 9–14 test for the presence of the CMV promoter from AAV.mGUS SU2 plasmid was used as a positive control in lanes 7 and 13; water asa negative control in lanes 8 and 14. Four pups (one litter), numbers 1–4, weretested. Note the absence of the AAV DNA (mGUS SU2) in the pups with bothsets of primers.

TABLE 5: GUS enzyme activity in treated offspring of breeders

GUS activity: U/g tissue or mL serum% of

normal activityTissue 1 2 3 4 5 (mean, n = 5)

Brain 0.12 0.005 0.038 0.096 0.044 2.6

Spinal Cord 5.7 0.023 0.059 2.6 0.19 190

Heart 0.007 0.011 0.011 0.011 0.009 0.7

Aorta 0.006 0.005 0.006 0.009 0.006 0.5

Lung 0.024 0.025 0.027 0.019 0.015 0.2

Liver 0.047 0.052 0.059 0.069 0.058 0.2

Kidney 0.009 0.011 0.014 0.012 0.010 0.08

Spleen 0.014 0.010 0.011 0.022 0.009 0.04

Ovaries 0.016 0.015 0.2

Testes 0.005 0.003 0.006 0.03

Muscle 0.001 0.003 0.005 0.014 0.004 0.5

Serum 0.00027 0.00018 0.00035 0.00051 0.00021 5.2

Pups born to AAVmGUS SU2 treated mps/mps parents were in turn treated with AAVmGUS SU2 as newborns,IT and IV. Data shown are for five mice 3–8 months of age. All enzyme assays were done in triplicate.

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FIG. 5. Southern analysis of RT-PCR products. RNA was extracted from the brain, liver, and spleen of the AAVmGUS SU2 treated breeders (O 1, O+1, O 2) andfrom untreated normal (+/+) and mutant (mps/mps) mice. RT-PCR was performed on the DNased RNA with (+) and without (–) reverse transcriptase to controlfor any DNA contamination. Water was included as a negative control. The Gusa delivered by AAVmGUS SU2 is also present in the normal strain used here andgives a 324-bp product. Gusb is present in mps/mps mice and serves as the background control, giving a 274-bp product. RNA derived from AAVmGUS SU2 isseen in the brain and liver of the three breeders but not in the untreated mutants (as expected) or in the spleen.

The benefits of the AAVmGUS SU2 therapy extendedonly to the treated animals and not to their progeny. Wefound that gonadal tissue in treated animals did not haveenzyme levels above untreated mutant levels, so it seemsunlikely that germ cells were transduced. Neither vectornor elevated GUS was detected in 21 progeny of treatedanimals; however, we cannot exclude the possibility thata rare germline transmission event might occur if a largernumber of progeny were tested. The increased levels ofGUS circulating in the mother’s serum also did not elevatethe levels in the newborn pups’ tissues above levels seenin mutant mice born to heterozygous parents. The onlyeffect the pups received was a resistance to AAV treatment,which presumably resulted from the mother’s vector-spe-cific IgGs crossing the placenta and/or IgAs in her milk.This antibody-conferred resistance did not extend to thebrain and spinal cord.

Although there was considerable variability in tissuelevels of GUS among treated mice, the resulting phenotypeshowed much improvement. Our goal for treating lysoso-mal storage disease by gene therapy is to effectively treatthe whole organism without affecting the germ line andthe next generation. Combined IV and IT administrationof vector, plus the use of a modified sequence yielding asecretion-enhanced protein, have brought us closer to thisgoal.

MATERIALS AND METHODS

Recombinant AAV preparation. Vector construction was similar to thatpreviously reported [8] using either mouse GUS cDNA or mouse GUS cDNAaltered by the deletion of the final six codons. The latter construct was sub-jected to several modifications by addition of coding sequences for a 5�secretion signal and/or 5� and 3� protein transduction domain(s). TheseGUS sequences were situated within the multiple cloning site of the pre-viously used plasmid, pV4.1c, which contains a CMV promoter and AAVITRs. Thus, truncated mouse GUS cDNA from plasmid hpcDNA�6, a gift

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of Richard T. Swank [21], was excised by digestion with XbaI and clonedinto the AAV plasmid to give pV4.1c mGUS�6. AAV plasmid bearing theV-J2-C signal peptide sequence of mouse Ig� (bases 905–967 of pSecTag2,Invitrogen Inc., Carlsbad, CA), attached in-frame at the start codon of thetruncated mouse GUS cDNA, was prepared by generating a PCR fragmentbearing an SfiI restriction site immediately upstream from the start codonof mGUS�6 and an EcoRI site downstream from the termination codon.This fragment was inserted into pSecTag2 at the relevant sites, and theresulting plasmid was then cut with NheI and XhoI to give a 2050-bp frag-ment. This was then inserted into pV4.1c between XbaI and XhoI yieldingpV4.1c Ig� mGus�6. The protein transduction domain (PTD) sequence ofHIV-1 TAT protein [22,23] was appended in-frame to this construct by PCRand is located immediately upstream of the original stop codon. The result-ing plasmid is termed pV4.1c mGUS SU2.

For preparation of the construct having only a 3� PTD, pV4.1c mGUSSU2 was digested sequentially with BglII and BsiWI to give a 503-bp frag-ment containing TAT sequence, which was then inserted into pV4.1cmGus�6 at the same sites to give pV4.1c mGUS U. The analogous con-struct having a 5� PTD was prepared by generating a 1979-bp PCR frag-ment containing an XbaI site at the 5� end followed by the Kozak con-sensus sequence, CCACCATGGGC, which includes a glycine codon,sequence for the 11 amino acids of TAT region 3, and an additionalglycine. The downstream primer contained the 3� sequence of mGUS�6and an XhoI site. This fragment was inserted into pV4.1c at XbaI and XhoIto give pV4.1c U mGUS. For preparation of the analog having two TATregions, the 658-bp fragment XbaI-HindIII containing the 5� sequence ofU mGUS was inserted into pV4.1c mGUS U at XbaI and HindIII sites togive pV4.1c U mGUS U.

Adeno-associated virus was packaged and purified as follows: 293 cellsat 75% confluence were cotransfected with a plasmid carrying the GUSDNA construct, a helper plasmid containing essential adenoviral functions,and a helper plasmid containing AAV rep and cap coding sequences [28]by the calcium phosphate method [29]. After 6 hours the medium wasreplaced with DMEM without fetal bovine serum. The cells were harvested48 hours later, pelleted, resuspended in 20 mM Tris, pH 8, 150 mM NaCl,1 mM MgCl2, and frozen in dry ice/ethanol. After thawing at 37�C, sodiumdeoxycholate (to 0.5%) and Benzonase (Sigma; to 50 u/mL) were added andthe solution was incubated at 37�C for 30 minutes [30]. After centrifuga-tion at 3600g to clarify the supernatant, 1 M NaCl was added, followed byadditional centrifugation to clarify it further. The supernatant was thenpurified on an iodixanol density step gradient (without 1 M NaCl) fol-lowed by a Heparin (Poros 20 HE, PerSeptive Biosystems) column [31]. TheAAV was dialyzed into a final solution of 20 mM Tris, pH 7.4, 150 mMNaCl, 1 mM MgCl2.

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Recombinant AAV titer was determined by a standard DNase diges-tion/dot blot assay [32]. A relative functional titer was determined by trans-ducing 293 cells at 50–80% confluence and measuring GUS enzyme activ-ity 24 hours later [33] using a 30-minute incubation time.

Purification and analysis of secreted enzyme. mGUS and mGUS SU2enzymes were isolated from the medium of transduced 293 cells. The cellswere grown to 75% confluence in flasks. Then the medium was replacedwith DMEM without fetal bovine serum and containing 4 � 109 particlesof AAVmGUS or AAVmGUS SU2 per mL. The medium was harvested 75hours later and stored at –70�C. To crudely purify the enzymes, the mediumwas thawed and heated at 56�C for 20 minutes (�-glucuronidase does notlose activity at this temperature, but many other contaminating proteinsare denatured). Centrifugation at 12,000g was carried out to remove anydebris. The supernatant was concentrated approximately 40-fold by evap-oration in a Pierce Slide-A-Lyzer, then dialyzed against PBS, pH 7.4. Specificactivities, expressed in U/mL, were determined by GUS enzyme assay. Theenzymes were characterized by separation on a 7.5% SDS polyacrylamidegel followed by western blot and detection using an antibody to mouseGUS.

To measure uptake, mps/mps mice were injected with 4 U/mouse mGUSor mGUS SU2 enzyme, 250 �L in the tail vein under anesthesia. Within 1minute blood was drawn using a retro-orbital sinus puncture and serumwas collected. Blood was drawn again the same way 10, 20, 60, and 120minutes later. Then the mice were killed and nine tissues were collected.A GUS enzyme assay was performed on the tissues and serum with incu-bation times of 2 hours for the tissues and 30 minutes for the serum.

Mice. MPS VII mice used were homozygous for the mutant allele, mps, ofthe �-glucuronidase gene, Gus, and were obtained from heterozygouscrosses. (As previously described [8], these heterozygous mice carried awild-type Gusa allele and a mutant (mps) Gusb allele. This aided genotyp-ing, as Gusa has a 199-bp insert in intron 4 that is missing from Gusb.) Forcomparison, normal levels of GUS enzyme were measured in B6.A congenicmice in which Gusa/Gusa alleles are in a C57BL6/J genetic background [25].

To compare vector constructs, adult MPS VII mice were injected with2 to 5 � 1011 particles AAV vector via the tail vein using 200 �L from stockshaving relative titers of 1 to 2.5 � 1012 particles/mL. Their tissues were ana-lyzed 7 weeks later.

For treatment of newborns, pups from heterozygous crosses were toe-clipped and screened by PCR (see next section) on the day after birth (day2). The mutants were anesthetized by isoflurane inhalation and treated byintrathecal injection into the cerebrospinal fluid [7] with 3 � 1010 parti-cles AAVmGUS SU2 on day 3. On day 4, 1 � 1011 particles were injectedintravenously into the superficial temporal vein [34]. Males and femaleswere mated at 4 weeks of age and the first litter was born 5 weeks later.

All animal protocols were approved by the Institutional Animal Careand Use Committee and conformed to the NIH Guidelines.

Treatment of tissues. GUS enzyme activity was measured in tissuehomogenates using a fluorometric assay as described [8]. It should be notedthat this assay, using 4-methylumbelliferyl-�-glucuronide as substrate, isextremely sensitive and can detect as little as 1 � unit of GUS activity [35].As reported previously, MPS VII mice have very low, but readily measura-ble, residual levels of GUS activity that are sensitive to highly specific anti-body against mouse GUS [8]. Mutant homogenates were incubated for 2hours at 37�C, whereas enzyme levels in normal animals were determinedafter 30 minutes. One unit (U) of enzyme activity hydrolyzes 1 �mole ofsubstrate per hour. For accuracy and continuity, specific activities arereported as U (or mU) per gram (wet weight) of tissue or per mL serum.

To measure levels of GUS enzyme in the serum, the mice were anes-thetized with Avertin and blood was drawn using a retro-orbital sinus punc-ture [36]. The red blood cells were spun out and the serum was assayed forGUS activity using a 24-hour incubation time.

For histopathology, tissues were fixed in 2.3% glutaraldehyde, 0.05 Mcacodylate, pH 7.4, embedded in Epoxy resin, and 1 m sections were stainedwith 1% toluidine blue, 1% sodium borate.

Total RNA was isolated from tissues quick-frozen in liquid N2 using the“Perfect RNA Isolation Kit” (Eppendorf). The RNA was incubated with 0.5u/�g RNase-free DNase (Promega) for 30 minutes at 37�C, phenol-chloro-form extracted, and ethanol precipitated.

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RT-PCR was performed on 800 ng RNA using the Gene AMP Gold RNAPCR Kit (Applied Biosystems) with and without Multiscribe ReverseTranscriptase. To distinguish between mRNA transcribed from vector DNA(which codes for GUS A) and any transcript from the endogenous gene(GUS B with the mps mutation), we took advantage of slight differencesbetween the GUS A and GUS B sequences [37]. The two forward primersdiffered from each other by the two bases at their 3� ends, which corre-spond to a polymorphism at bases 806 and 807 in the GUS mRNA tran-script. The two reverse primers were designed to give different length PCRproducts and each incorporated a polymorphism in the 3� terminal baseof the primer (base numbers 1095 for GUS A and 1047 for GUS B). Allprimers were upstream from the mps mutation. Positioning the polymor-phic bases at the 3� ends of the primers allows the PCR to distinguishbetween GUS A (from AAVmGUS SU2) and GUS B (the mutant back-ground). The primers were as follows: GUS A, forward, 5�-CAACTTTTG-GATGAGGAC-3�, reverse, 5�-GTTGAAATCCTTTACCAGT-3�, 324 bp PCRproduct; GUS B, forward, 5�-CAACTTTTGGATGAGGGT-3�, reverse, 5’-CTGAATCCTCGTGCTTA-3�, 274-bp PCR product. The PCR products wereexamined by Southern analysis using the cDNA of mouse GUS as a probe.

Mouse pups were screened by PCR. A toe was clipped on day 2 andDNA was isolated using a GNT-K buffer [38]. The following set of primersin GUS exon 4 and exon 5 spans intron 4, which identifies the genotypeof the mouse and confirms the presence or absence of AAVmGUS vector:forward, 5�-TGTCCAGGACACAAGCTTTG-3�; reverse, 5�-ATGCTCATG-CATCAGGTAAGG-3�. PCR with these primers gives bands of 899 bp forGusa wild type, 700 bp for Gusb carrying the mps mutation, and 289 bp ifAAVGUS (no intron) is present. Another set of primers covers the CMV pro-moter, which is not present in the normal mouse genome, and gives a 412-bp product only if AAV vector DNA is present: forward, 5�-TCCATAGAA-GACACCGGGAC-3�; reverse, 5�-AATCCAGCCTTATCCCAACC-3�.

The mouse carcasses were prepared for skeletonization by gutting andremoval of most tissue, soaked in water for 2 days at 4�C, and then cleanedby a colony of dermestid beetles, Dermestes longicaudus, overnight.

ACKNOWLEDGMENTSWe thank Amy Jess (Children’s Hospital Oakland Research Institute) for precisesurgical skills and knowledge of anatomical measurements; Eve Clausnitzer(Children’s Hospital Oakland Research Institute) for preparing the tissue sec-tions; and Lise Thomsen of the California Academy of Sciences for cleaning themouse skeletons. This work was supported by NIH grant DK54258.

RECEIVED FOR PUBLICATION AUGUST 13, 2001; ACCEPTED FEBRUARY 22, 2002.

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