Sialic Acid Deposition Impairs the Utility of AAV9, but Not Peptide-modified AAVs for Brain Gene Therapy in a Mouse Model of Lysosomal Storage Disease

original article

© The American Society of Gene & Cell Therapy

Sialic Acid Deposition Impairs the Utility of AAV9, but Not Peptide-modified AAVs for Brain Gene Therapy in a Mouse Model of Lysosomal Storage Disease Yong Hong Chen1, Kristin Claflin1, James C Geoghegan1 and Beverly L Davidson1–3 1 Department of Internal Medicine, University of Iowa, Iowa City, Iowa, USA; 2Department of Molecular Physiology and Biophysics, University of Iowa, Iowa City, Iowa, USA; 3Department of Neurology, University of Iowa, Iowa City, Iowa, USA

Recombinant vector systems have been recently identified that when delivered systemically can transduce neurons, glia, and endothelia in the central nervous system (CNS), providing an opportunity to develop therapies for diseases affecting the brain without performing direct intracranial injections. Vector systems based on adenoassociated virus (AAV) include AAV serotype 9 (AAV9) and AAVs that have been re-engineered at the capsid level for CNS tropism. Here, we performed a head-tohead comparison of AAV9 and a capsid modified AAV for their abilities to rescue CNS and peripheral disease in an animal model of lysosomal storage disease (LSD), the mucopolysacharidoses (MPS) VII mouse. While the ­peptide-modified AAV reversed cognitive deficits, improved storage burden in the brain, and substantially prolonged survival, we were surprised to find that AAV9 provided no CNS benefit. Additional experiments demonstrated that sialic acid, a known inhibitor of AAV9, is elevated in the CNS of MPS VII mice. These studies highlight how disease manifestations can dramatically impact the known tropism of recombinant vectors, and raise awareness to assuming similar transduction profiles between normal and disease models. Received 5 January 2012; accepted 18 April 2012; advance online publication 15 May 2012. doi:10.1038/mt.2012.100

Introduction The incidence of lysosomal storage diseases (LSDs) is estimated to be greater than 1 in 10,000 live births, with >60% inducing central nervous system (CNS) involvement.1 Therapeutic approaches that have been developed and tested in animal models for the LSDs include bone marrow transplantation,2,3 enzyme replacement therapy,4–7 and gene therapy.8 Success relies on the mechanism of cross-correction9 whereby nonmembrane-bound lysosomal enzymes can be endocytosed by cells via mannose- or mannose-6-phosphate receptors. In the setting of gene-corrected cells expressing recombinant protein, a proportion of the expressed product is secreted into the external milieu for uptake by the same or other cells.8

Although bone marrow transplantation, enzyme replacement therapy, and gene therapy are effective for targeting ­systemic disease, the blood–brain barrier raises practical issues for all approaches. Bone marrow transplantations effectiveness may require extensive influx of bone marrow derived cells during the disease course,3 and enzyme replacement therapy for brain correction would need repeated delivery of enzyme to the ­cerebrospinal fluid for distribution.10 Gene transfer with vectors conferring stable, long-term correction could provide sustained therapy if a sufficient level of enzyme was secreted from an appropriate cellular reservoir within the brain.11 Recently, studies demonstrated that adeno-associated virus serotype 9 (AAV9) could cross the blood– brain barrier and transduce neurons and astrocytes after systemic delivery to adult mice.12,13 AAV9 administration also transduces visceral tissues including liver, heart and muscle. Thus, AAV9 may be optimal for LSDs affecting both CNS and peripheral tissues, as was recently demonstrated in a mouse model of the LSD, mucopolysacharidoses (MPS) IIIB.14 AAV tropism can also be affected by inserting targeting peptides into the capsid region critical for receptor binding. Such peptide insertions can abrogate the natural tropism of AAV capsids and result in the vector targeting new tissues, including the CNS.15–18 The LSD MPS VII is caused by a genetic deficiency of β-glucuronidase,19 with resultant accumulation of glycosaminoglycans (GAGs) in the brain and visceral tissues including the liver, kidney, spleen, and bone.20 GAG accumulation induces progressive neurological disease, skeletal deformities, and a shortened lifespan. In previous work, we used in vivo biopanning to identify epitopes specific to the brain vascular endothelia of a mouse model of MPS VII.15 When the epitope was cloned into an AAV2 capsid, the tropism of the AAV was altered from predominantly liver targeting to predominately brain vascular targeting. The recombinant enzyme was expressed in endothelia, and secreted basolaterally into the underlying brain parenchyma. However, the utility of this modified AAV (AAV-PFG) for treating disease manifestations of β-glucuronidase deficiency were not addressed. In the present study, we compared the efficacy of systemically delivered AAV-PFG and AAV9 expressing β-glucuronidase in affected adult MPS VII mice. While AAV-PFG provided

Correspondence: Beverly L Davidson, Department of Internal Medicine, University of Iowa, Room 200, Eckstein Medical Research Building, Iowa City, Iowa 52242, USA. E-mail: [email protected] Molecular Therapy vol. 20 no. 7, 1393–1399 july 2012 

1393

© The American Society of Gene & Cell Therapy

Brain Phenotypes Impact AAV Gene Transfer

profound improvements in CNS deficits and prolonged survival, AAV9 did not. Interestingly, we found that deposition of molecules shown previously to block AAV9 transduction were elevated in MPS VII brain, likely impairing the general utility of this vector system in mice with β-glucuronidase deficiency. These studies demonstrate important differences between wild type and disease models and have relevance to the broad utility of vectors whose tropism is determined in nondiseased animals.

a Freezing time (second)

25 20 15 10 5

1394

0 mice virus

Het −

MPS VII AAV-PFG AAV9

−

Difference of freezing between context 1 and 2 (second)

b

c

* *

35

*

30 25 20 15 10 5 0

mice virus

Percent survival

Impact of systemic injection of AAV-PFG.βgluc or AAV9.βgluc on animal behavior and survival The progressive impairment in the CNS and peripheral tissues of MPS VII mice causes measurable behavioral changes.11 We used fear-conditioning assays as a measure to assess hippocampal-dependent function, and activity chamber assay to evaluate overall mobility (Figure 1 and Supplementary Table S1). For the fear-conditioning assays, mice were placed to the chamber on day 1 with a negative stimulus, the foot shock.11 On day 2, they were placed into the same chamber and freezing time recorded. Freezing time was quantified for untreated normal, heterozygous mice from our MPS VII mouse model breeding colony (gus+/mps) and disease MPS VII mice (gusmps/mps), or MPS VII mice given AAV-PFG.βgluc or AAV9.βgluc. AAV-PFG.βgluctreated MPS VII mice were indistinguishable from heterozygous mice (P = 0.2098). In contrast, AAV9.βgluc treated and untreated MPS VII mice had significantly decreased freezing time relative to both normal (P < 0.001; Dunnett’s post hoc) or AAV-PFG.βgluctreated mice (P < 0.001; Dunnett’s post hoc; Figure 1a). We next assessed context discrimination by transferring the mice into a chamber with modified olfactory, tactile, and visual cues. As show in Figure 1b, the heterozygous and AAV-PFG.βgluc-treated MPS VII mice froze less in context 2 relative to context 1 compared with untreated and AAV9.βgluc-treated disease mice (P < 0.001; Dunnett’s post hoc). The decrease in freezing time indicates that heterozygous and AAV-PFG.βgluc-treated MPS VII mice can distinguish between the two contexts. Untreated and AAV9.βgluctreated MPS VII mice were unable to discriminate contexts and froze for a similar duration in context 1 and 2. General locomotor activity and exploratory behavior in a novel environment were assessed using the activity chamber. As compared to heterozygous mice, untreated MPS VII mice had deficiencies in exploratory movements, featured by less jumps, reduced ambulatory activity, reduced stereotypic activity and increased resting time. Systemic injection of either AAV-PFG. βgluc or AAV9.βgluc partially alleviated these defects, but they remained impaired relative to nondiseased heterozygous mice (Supplementary Table S1).

*

*

30

Results To compare the therapeutic efficacy of systemic AAV-PFG and AAV9 gene delivery, 6-week old of MPS VII mice were treated with a single intravenous injection of AAV-PFG.βgluc or AAV9.β-Gluc (1.0 × 1012 gp/mouse). After 6 weeks, the effects of the treatment on behavior were evaluated using context fear-conditioning and activity chamber assay, and effects on neuropathology determined 8 weeks after viral injection.

*

35

Het −

−

MPS VII AAV-PFG AAV9 MPS VII MPSVII+AAV-PFG MPSVII+AAV9

150

100

50

0

0

100

200 Days

300

400

Figure 1 The effects of peripheral delivery of AAV-PFG.βGluc and AAV9.βGluc on context fear-conditioning assays and survival in mucopolysacharidoses (MPS) VII mice. (a) Freezing time from baseline after placement in context 1 on day 2 was measured. MPS VII mice treated with AAV-PFG.βgluc were significantly increased compared to untreated MPS VII mice. (*P < 0.001; one-way ANOVA, Dunnett’s post hoc), unlike AAV9.βGluc-treated mice. (b) The difference in freezing time between context 1 and 2 was significantly improved in MPS VII mice treated with AAV-PFG.βGluc compared to untreated mice (*P < 0.001; one-way ANOVA, Dunnett’s post hoc), or AAV9.βGluc-treated mice. n = 8–11 per group. (c) Extended survival in MPS VII mice after systemic AAVPFG.βGluc treatment (n = 9 per group). AAV, adeno-associated virus.

MPS VII mice have a shorter lifespan relative to normal mice. In our laboratory, the median survival of untreated MPS VII mice is 154 days. AAV-PFG.βgluc treated disease mice showed significantly longer survival relative to untreated and AAV9.βgluctreated MPS VII mice. (P < 0.001; log-rank (Mantel–Cox) test). The median age of survival was 160.5 and 302 days for AAV9. βgluc and AAV-PFG.βgluc-treated MPS VII mice, respectively (Figure 1c). www.moleculartherapy.org vol. 20 no. 7 july 2012

© The American Society of Gene & Cell Therapy

Brain Phenotypes Impact AAV Gene Transfer

Enzyme activity and viral genome distribution after systemic delivery of AAV-PFG.βgluc or AAV9.βgluc We used in situ enzyme activity assays11 to determine the distribution of β-glucuronidase activity in the brain (Figure  2a). Heterozygous mice show limited endogenous activity, as noted before for this insensitive assay, which requires overexpressed enzyme levels for detection using these short-term conditions.11 Heterozygous mice treated with AAV-PFG do not display detectable enzyme activity (Figure  2a) as the brain tropism of this vector is specific to the MPSVII mouse.15 However, and as seen previously in AAV-PFG treated MPS VII mice,15 β-glucuronidase activity was evident in large and small diameter vessels throughout the brain (Figure  2a).12 AAV9.βgluc injected heterozygous mice also showed scattered β-glucuronidase activity, consistent with the described ability of AAV9 vectors to transduce endothelia and astrocytes in the brain after peripheral injection (Figure 2a). The number of β-glucuronidase positive cells in the brain was reduced relative to reporters expressed from self-complimentary genomes in our (unpublished observations) and prior work;12 the

a

AAV-PFG

AAV9

Het

MPSVII

250

Het MPSVII MPSVII+PMAAV MPSVII+AAV9

200 150

Correction of lysosomal storage pathology Progressive lysosomal storage and distension is a hallmark of the pathological changes in MPS VII patients and β-glucuronidasedeficient mice. We injected AAV-PFG.βgluc and AAV9.βgluc via tail vein into MPS VII mice at 6 weeks of age, as disease phenotypes, including lysosomal storage deposits, are apparent at this time. Eight weeks later, we evaluated lysosomal storage and cellular distension in the brain and several peripheral organs including liver, spleen, and kidney. For the brain, AAV-PFG.βgluc, but not AAV9.βgluc treated MPS VII mice exhibited reduced levels of lysosomal storage in different regions, including the cerebral cortex, hippocampus and striatum (Figure3a). Quantitative analysis of lysosomal storage showed that AAV-PFG.βgluc significantly

100 50 0

c

108

AAV copies/µg

β-Glucurondase activity (% of het)

b

β-glucuronidase expression cassette is too large for a self-complementary genome. Contrary to AAV9.βgluc-treated heterozygous mice, we observed negligible β-glucuronidase positive cells in the brains of AAV9.βgluc-treated MPS VII mice. Quantitation of viral genomes in the brains of treated animals supported this finding. Heterozygous mice treated with AAV9.βgluc had significantly more viral genomes in the brain relative to MPS VII mice treated with the same preparation (3.41 × 104 ± 1.6 × 104 and 1.2 × 102 ± 7.6 × 101, respectively; P < 0.05 by Student’s t-test). Subsequently, we quantified β-glucuronidase activity by fluorometric assay11 in brain and peripheral tissues. Enzyme levels were high in the brains of AAV-PFG.βgluc-treated MPS VII mice, whereas in AAV9. βgluc-treated MPS VII mice, enzyme levels were low in brain, but near heterozygous levels in heart and liver. The level of enzyme activity was similar in serum, spleen, lung and kidney between AAV-PFG.βgluc and AAV9.βgluc-treated groups (Figure 2b). We further analyzed the biodistribution of viral genome by quantitative real-time PCR. After peripheral injection into MPS VII mice, AAV-PFG.βgluc viral genomes were predominantly localized to MPS VII mice brain while AAV9.βgluc viral genomes were enriched in liver, followed by heart, spleen, and kidney, and very low in brain after systemic administration (Figure 2c). These data suggest that AAV-PFG but not AAV9 can transduce and restore β-glucuronidase levels in the brains of deficient mice, while AAV9, at least in this disease model, provides better systemic delivery.

106

Serum

Brain

Heart

Liver Spleen

Lung Kidney PMAAV AAV9

104

102 100 Brain

Heart

Liver

Molecular Therapy vol. 20 no. 7 july 2012

Spleen

Lung

Kidney

Figure 2  Biodistribution of β-glucuronidase enzyme and vector. (a) Representative sections stained for β-glucuronidase activity in situ (red precipitate), show activity in brain cerebral cortex after systemic injection of AAV-PFG.βgluc (right images) or AAV9.βgluc (left images) into heterozygous (top images) or mucopolysacharidoses (MPS) VII (bottom images) mice. n = 4 mice per group. Bar = 100 µm. (b) β-Glucuronidase activity in brain and peripheral organs 8 weeks after AAV-PFG.βgluc or AAV9.βgluc injection into mice. Enzyme activity in brains of AAV-PFG. βgluc-injected mice was significantly higher than for mice injected with AAV9.βgluc mice (P < 0.001; Bonferroni’s post hoc). The liver and heart of AAV9.βgluc-treated MPS VII mice had significantly higher enzyme activity compared to AAV-PFG.βgluc-treated mice. (P < 0.001; Bonferroni’s post hoc). Enzyme activity levels were similar in serum, spleen, lung, and kidney between AAV-PFG.βgluc- and AAV9.βgluc-treated mice. (c) Quantification of vector copy number in brain and peripheral organs 8 weeks after tail vein injection of AAV-PFG.βgluc or AAV9.βgluc. Vector copy number was significantly higher in brain of MPS VII mice injected with AAV-PFG.βgluc compared to those injected with AAV9.βgluc. Vector copy number for AAV9.βgluc was significantly higher than for AAV-PFG. βgluc in heart, liver, spleen, and kidney. n = 3 mice per group. Data are presented as mean ± s.e.m. AAV, adeno-associated virus.

1395

© The American Society of Gene & Cell Therapy

Brain Phenotypes Impact AAV Gene Transfer

AAV-PFG

AAV9

120

No virus

100

AAV-PFG AAV9

90 60 40 20 0

*

** Cortex

**

Hippocampus

Striatum

d

Striatum

Liver No virus AAV-PFG AAV9 No virus

AAV-PFG

3+ 1 1

Spleen

Kidney

Red White Cortex Medulla 3 1 1

2 1 1

2+ 1 1

3 1 1

AAV9

Kidney

Spleen

Liver

c

b % Cells with Iysosomal storage

Hippocampus Cerebral cortex

a

Figure 3 Central nervous system (CNS) pathology after systemic injection of AAV-PFG.βgluc or AAV9.βgluc to mucopolysacharidoses (MPS) VII mice. (a) Representative images from cerebral cortex, hippocampus and striatum of MPS VII mice, collected 8 weeks after tail vein injection with either AAV-PFG.βgluc (left) or AAV9.βgluc (right). Yellow arrows denote regions magnified in insets (lower right, all images) for better visualization of storage vacuoles. Bar 20 µm. Inset magnification ×2. n = 4 mice per group. (b) Quantification of vacuolar storage in various brain regions. Tail vein injection of AAV-PFG.βgluc but not AAV9.βgluc significantly reduced lysosomal storage vacuoles in cortex, hippocampus and striatum (*P < 0.01, **P < 0.001, Bonferroni’s post hoc) n = 4 mice per group. (c) Representative images from liver, spleen and kidney of untreated MPS VII mice (left), or AAV-PFG.βgluc- (middle), and AAV9.βgluc- (right) treated mice. (d) Semiquantification of vacuolar storage in various peripheral organs. Rankings are 1, small cytoplasmic vacuoles in scattered cells; 2, cytoplasmic vacuoles in most cells; 3, large, abundant vacuoles filling the cell cytoplasm. Tail vein injection of AAV-PFG.βgluc or AAV9.βgluc to MPS VII mice improved the pathology of peripheral organs (n = 4 mice per group). AAV, adenoassociated virus.

lowered the number of cells laden with storage vacuoles relative to untreated and AAV9.βgluc-treated MPS VII mice (Figure 3b). In somatic tissues, we observed reduction of lysosomal storage in liver, spleen, and kidney in both AAV-PFG.βgluc- and AAV9. βgluc-treated MPS VII mice (Figure 3c,d).

Assessing inhibition of AAV9 transduction in disease mice brain Our data are curious in light of the noted ability of AAV9 to transduce CNS cells after peripheral delivery,12 and to improve disease in MPS IIIB mice.14 We found limited transduction of the brain and no improvements in CNS phenotypes with AAV9.βgluc. Recent reports show that AAV9 uses terminal galactose on cell-surface 1396

carbohydrates as the primary receptor, and showed that sialic acid can block AAV9 transduction.21,22 We hypothesized that disease progression in MPS VII mice might modify galactose or sialic acid distribution and impact AAV9’s ability to transduce brain. To test this rhodamin-labeled RCA I staining was done. Qualitative assessment of galactose did not reveal any differences between sections from heterozygous and disease mouse brains (data not shown). Brain sections were next incubated with fluorescein­labeled Sambucus nigra lectin (SNA) which preferentially detects α2,6-sialic acid. We found enhanced fluorescein-SNA staining in MPS VII brain sections relative to those obtained from heterozygous mice (Figure 4a). The staining appeared vascular in nature, and colabeling with von Willibrand factor and NeuN showed that www.moleculartherapy.org vol. 20 no. 7 july 2012

© The American Society of Gene & Cell Therapy

Brain Phenotypes Impact AAV Gene Transfer

the bulk of elevated sialic acid localized to endothelia (Figure 4b). SNA-staining reflected no differences between MPS VII mice and heterozygous mice in visceral organs including liver, lung and heart (data not shown). We next quantified sialic acid levels in brain vasculature and whole brain. As shown in Figure 4c, MPS VII brain vasculature has significantly higher levels of sialic acid relative to that in heterozygous brain. Taken together, the enrichment of sialic acid in the brain vasculature in MPS VII mice may underlie the inefficiency of AAV9 transduction in this model.

Discussion Our initial goal was to compare the general utility of two braintropic vectors, AAV9 and an AAV that had been modified to present

a

b

Salic acid (µg/mg protein)

c

250 * 200

Het MPS VII

150 100 50 0

Brain vasculature

Whole brain

Figure 4 Sialic acid deposition in mucopolysacharidoses (MPS) VII mice brain vasculature. (a) Representative sections, stained with fluorescein labeled SNA, which recognizes predominantly α-2,6 sialic acid, shows elevated sialic acid staining in MPS VII mice brain sections (bottom) relative to sections from heterozygous mice (top). Bar 50 µm. (b)  Representative confocal photomicrograph from a cortical section from MPS VII mice stained with fluorescein-labeled SNA (green), antibody to the vasculature marker vWF (red) and the neuronal marker NeuN (blue). Colocalized vascular and sialic acid staining is indicated (arrows). Bar 20 µm. (c) Quantification of total sialic acid in whole brain and brain vasculature. (*P < 0.001; Bonferroni’s post hoc). Sialic acid levels in the vasculature from MPS VII mice brain were significantly greater than those in heterozygous mice (n = 4 per group). Data are presented as means ± s.e.m.

Molecular Therapy vol. 20 no. 7 july 2012

a brain-targeting peptide on the viral capsid, AAV-PFG.15 We were surprised to find that only the AAV-PFG provided CNS benefits. Peripheral delivery of AAV-PFG conferred elevated enzyme levels in brain, reduction of lysosomal storage, reversal of cognitive deficits, and improved survival. Notably, this is the first study in which the lifespan of adult MPS VII mice was prolonged by peripheral delivery of an AAV encoding β-glucuronidase. Previous experiments that demonstrated increased lifespan required AAV delivery at a fetal or neonatal stage.23,24 In contrast to AAV-PFG, AAV9 expressing β-glucuronidase only impacted peripheral disease pathology. We found that sialic acid deposition, which has been shown previously to block AAV9 transduction, is likely responsible for the reduced transduction efficiency of AAV9 in this model. Interestingly, we also found that compared to controls, sialic acid deposition was elevated in brain tissue sections from dogs deficient in the lysosomal protease tripeptidyl peptidase 1, a model of late infantile onset Batten disease (Y.H. Chen and B.L. Davidson, unpublished results). Sialic acid deposition is not a general phenomenon of the LSD brain, however, as mice with CLN3 deficiency, a model of juvenile onset Batten disease, do not display this phenotype (Y.H. Chen and B.L. Davidson, unpublished results). Phage display panning in vivo typically selects for peptides that interact with proteins on the cell-surface.25,26 However, phage display libraries have been used to identify peptides that bind to other biomolecules including small molecules, nucleic acids, and specific glycan structures associated with proteoglycans.27–29 In our previous work we found that AAV-PFG binding to brain endothelial tissue is mediated in part by interaction with the GAG, chondroitin sulfate.15 This observation is consistent with the hypothesis that the increased deposition of GAGs caused by β-glucuronidase deficiency creates a disease-specific cell-surface epitope with which displayed peptides or AAV-PFG can interact. Similarly, AAV9 was recently shown to use terminal galactose residues on carbohydrate chains as a cellular receptor.21,22 Resialyation of sialic acid-deficient cells inhibited AAV9 transduction. Further, the Wilson lab demonstrated that coadministration of AAV9 with neuraminidase to mouse lung in vivo improved transduction by revealing galactose residues.22 Collectively, these studies demonstrated that AAV9 binding is blocked when galactose is in the penultimate position and sialic acid is the terminal residue, a common configuration occurring in glycan chains.30 Therefore, in contrast to our AAV-PFG, which utilizes the upregulation of brain endothelial glycan structures to facilitate transduction, AAV9 appears to be hindered by this disease phenotype. Although AAV9 transduction of MPS VII brain may be inhibited by sialic acid, we found that AAV9 was more efficient than AAV-PFG with respect to systemic delivery to peripheral tissues. Given this, it would be interesting to evaluate whether introduction of the brain-targeting PFG peptide into the capsid of AA9 would yield a superior vector that mediates robust gene transfer to both CNS and non-CNS tissues. Recent work has shown that AAV9, similar to AAV2, can accommodate peptide epitopes.31,32 Such a vector could prove to be useful for gene delivery in diseases such as MPS VII and in similar scenarios in which terminal galactose residues are masked or unavailable to act as an AAV9 receptor. An alternative means to achieving efficient delivery to both CNS and peripheral tissues could be to utilize the GMN peptide-modified AAV that we previously developed for gene delivery to the brain 1397

Brain Phenotypes Impact AAV Gene Transfer

in a mouse model of tripeptidyl peptidase 1 deficiency.15 In contrast to the AAV-PFG in this study, the GMN-modified AAV was an effective vector for delivery to both peripheral organs and to the brain. Indeed, in our original panning in the MPS VII mouse, we did identify a phage which, when cloned onto AAV capsids, showed equivalent distribution to brain and peripheral tissues.15 Before our study there have been no reports of increased sialic acid levels in MPS VII mouse models. However, the accumulation of molecules that are not substrates for the deficient enzymes in LSDs has been noted, including the build-up of gangliosides in MPS VII brain.33 Sialic acid accumulation is a hallmark of Salla disease and infantile sialic acid storage disease but in these disorders sialic acid accumulates in the lysosome due to transporter dysfunction.34,35 In addition to understanding why extracellular sialic acid is increased, an outstanding question is why the abundance of sialic acid is increased in the MPS VII brain but not in peripheral tissues. In a mouse model of MPS IIIB, a related disorder, it was found that there was a differential distribution of heparin sulfate GAGs in the CNS and that increased expression levels of the enzymes responsible for the synthesis and modification of the GAGs were upregulated.36 It would be interesting to compare the expression of sialidases, sialytransferases, and other glycan modifying enzymes between MPS VII CNS tissue and peripheral organs. A possible mechanism by which sialic acid levels are increased is that GAG accumulation in lysosomes and subsequent lysosomal dysfunction alters the activity of resident sialidase enzymes. In support of this, it was found that in galactosialidosis, abnormal intralysosomal processing of sialidases leads to formation of catalytically inactive isoforms that are rapidly degraded.37 Future studies are required to test whether lysosomal dysfunction in MPS VII leads to decreased sialidase expression or function and the eventual accumulation of sialic acid. Studies have also shown that vascular beds in different organs display unique repertoires of cell-surface proteins, supporting the idea that the biology of endothelia differs between tissues.38–40 The endothelia of the brain, which compose the blood–brain barrier, are particularly unique in that they form an exceptionally regulated barrier via tight junctions and membrane transporter systems.41 Neurovascular dysfunction has been established as an important pathological component in several neurodegenerative disorders including Alzheimer’s disease.41,42 An intriguing notion is that GAG and sialic acid deposition in brain endothelium are indicative of blood–brain barrier dysregulation that may be contributing to the mechanism underlying MPS VII neural dysfunction. In conclusion, we show that systemic injection of either AAVPFG or AAV9 could improve motor function and decrease the pathology in viscera organs of MPS VII mice. However, only AAV-PFG improved CNS function and prolonged survival. The inability of AAV9 to efficiently transduce brain correlated with sialic acid accumulation in the brain vasculature of disease mice. This observation raises the question as to the general utility of vectors whose tropism is determined in wild-type animals. In normal, nonhuman primates, AAV9 can cross the blood–brain barrier, though glial transduction is favored over neuronal.43,44 These data are encouraging but in light of the findings presented here, it will be important to assess vector performance in large animal models of the disease to which the therapy is intended. 1398

© The American Society of Gene & Cell Therapy

Materials and Methods Experimental animals. MPS VII (b6.C-H-2bm1/byBir-gusmps/+) mice

and heterozygous controls were obtained from the Jackson Laboratory (Bar Harbor, ME) and from our own breeding colonies. Animal maintenance conditions and experimental protocols were approved by the University of Iowa Animal Care and Use Committee.

Viral vectors. Both AAV-PFG.βGluc and AAV9.βGluc were produced by tri-

ple-plasmid cotransfection of human HEK 293 cells as described in Chen et al.15 Both viruses were purified by purified by Mustang Q membrane cassettes after iodixanol gradient centrifugation. For western blot analysis, 1010 viral particles of AAV-PFG.βGluc and AAV9.βGluc were subjected to SDS-PAGE and transferred to PVDF membrane. The membrane was probed with anti-AAV VP1, VP2, VP3 mouse monoclonal antibody clone B1 (American Research Products, Waltham, MA) diluted 1:5,000, followed by HRP conjugated antimouse IgG secondary antibody. ECL Plus western reagent (GE Healthcare Waukesha, WI) was used to visualize bound antibody. Supplementary Figure S1 demonstrates that the preparations are equivalent. Context fear conditioning. The experiments were performed in a fear-con-

ditioning chamber as described previously.11 Fear response was measured by freezing, which was defined as no movement other than respiratory activity. Freezing in the first 3 minutes after placement into the chamber was recorded during the training and again 24 hours later in two contexts. Context 1 was the one used for training. Context 2 was a modified chamber with new olfactory, tactile, and visual cues. Animals stayed in their home cages during the 2-hour interval between testing in the two contexts. Activity chamber assay. Activity assay took place in a square arena (43.2

× 43.2 cm) that comprised three planes of infrared detectors within a specially designed sound attenuating chamber (66 × 55.9 × 55.9 cm) under dim light (Med Associates, St Albans, VT). The animal was placed in the center of the testing arena and allowed to move freely while being tracked by an automated tracking system.

In vivo biodistribution of virus. Mice were sacrificed and perfused with

20-ml cold phosphate-buffered saline (Invitrogen, Grand Island, NY). The tissues were harvested and snap frozen. Genomic DNA from representative organs was extracted using a Qiagen DNA extraction kit (Qiagen, Valencia, CA). AAV copies/organ were determined by quantitative-PCR for each sample, in duplicate.

Analysis of lysosomal storage. After perfusion with cold phosphate-­buffered saline and 4% paraformaldehyde, organs were dissected and fixed in 4% paraformaldehyde at 4 °C overnight and blocked. After washing, dehydration, and infiltration, samples were embedded in JB4. Sections (2 µm) were stained with toluidine blue solution and analyzed for lysosomal storage. Histological sections were evaluated morphologically by light microscopy. Sections were examined further for semiquantitative evaluation of lysosomal storage. For the hippocampus, the cerebral cortex, and the striatum, ~200 cells were examined per region per brain. For liver, spleen, and kidney, Storage was graded as follows: (i) small cytoplasmic vacuoles in scattered cells; (ii) cytoplasmic vacuoles in most cells; or (iii) large, abundant vacuoles filling the cell cytoplasm. Preparation of brain vasculature. Brain vasculature tissue was prepared as previously described.45 Briefly, saline-perfused mouse brains were cut with a razor blade to ~1 mm3 pieces and then homogenized in Hanks Buffer (Invitrogen) using a Dounce homogenizer. The homogenate was spun at 1,000g for 5 minutes at 4 °C and the supernatant discarded. The pellet was resuspended in Hanks Buffer containing 18% Dextran and then centrifuged at 10,000g for 10 minutes to separate vessels from brain parenchyma. The supernatant was removed and the pellet containing vascular tissue was washed with Hanks Buffer. Immunohistology and lectin staining. Mice were perfused with 4% para-

formaldehyde. Tissues were harvested and embedded in OCT. Sections www.moleculartherapy.org vol. 20 no. 7 july 2012

© The American Society of Gene & Cell Therapy

were blocked with 0.5% polyvinyl alcohol (Sigma, St Louis, MO) for 1 hour and incubated with 15 μg/ml rhodamine-labeled RCA I and 7.5 μg/ ml fluorescein-labeled SNA (Vector Laboratories, Burlingame, CA) for 30 minutes at room temperature. Slides were then washed twice in phosphate-buffered saline. For co-staining with von Willebrand Factor (vWF; Dako Cytomation, Carpinteria, CA) and neuronal nuclei marker (NeuN; Millipore, Billerica, MA), after lectin staining, slides were incubated with rabbit anti vWF (1:200) and mouse anti NeuN (1:500) primary antibodies at 4 °C overnight followed by fluorescent-labeled anti-rabbit and antimouse secondary antibody (1:500; Vector Laboratories). Sialic acid assay (thiobarbituric acid method). Assays for sialic acid were

performed as described.46 Briefly, glycoprotein samples (~200 µg) and N-acetyl-neuraminic acid (10 µg) as a standard were hydrolyzed in 450 µl of 50 mmol/l sulfuric acid at 80 °C for 60 minutes to release sialic acid, followed by neutralization with 1 mol/l sodium hydroxide. Samples were treated with 250 µl of periodic acid solution (25 mmol/l in 62.5 mmol/l sulfuric acid) at 37 °C for 30 minutes and reaction terminated by the addition of 100 µl of 50 mmol/l sodium thiosulfate solution. After 3 minutes, 200 µl was mixed with 800 µl thiobarbituric acid (0.1 mol/l, pH 9.0), and the resultant solution heated in a boiling water bath for 7.5 minutes. The solution was then cooled in ice water, centrifuged at 15,000 rpm for 10 minutes, and 100 µl of the supernatant transferred to 96 plates and read at 550 nm.

SUPPLEMENTARY MATERIAL Figure  S1.  Western blot analysis of 1010 viral particles of AAV9 (lane 1), and AAV-PFG (lane 2) capsid proteins VP1, VP2, and VP3. Table  S1.  Activity chamber data were collected 6 weeks after ­treatment (n = 9 per group).

ACKNOWLEDGMENTS The authors like to acknowledge the members of the Davidson Lab for discussion and technical advice. We thank Gene Transfer Vector Core for vectors and the Central Microscopy Research Facilities at the University of Iowa for advice and assistance. This work was supported by NIH grants DK 54759, HD33531 and NS34568, and the Roy J Carver Trust.

REFERENCES

1. Fuller, M, Meikle, PJ. and Hopwood, JJ. In: Fabry Disease: Perspectives from 5 Years of FOS (eds. A. Mehta, M. Beck, and G. Sunder-Plassmann) (2006). 2. Biffi, A, De Palma, M, Quattrini, A, Del Carro, U, Amadio, S, Visigalli, I et al. (2004). Correction of metachromatic leukodystrophy in the mouse model by transplantation of genetically modified hematopoietic stem cells. J Clin Invest 113: 1118–1129. 3. Cartier, N, Hacein-Bey-Abina, S, Bartholomae, CC, Veres, G, Schmidt, M, Kutschera, I et al. (2009). Hematopoietic stem cell gene therapy with a lentiviral vector in X-linked adrenoleukodystrophy. Science 326: 818–823. 4. Xu, S, Wang, L, El-Banna, M, Sohar, I, Sleat, DE and Lobel, P (2011). Large-volume intrathecal enzyme delivery increases survival of a mouse model of late infantile neuronal ceroid lipofuscinosis. Mol Ther 19: 1842–1848. 5. Kakkis, ED (2002). Enzyme replacement therapy for the mucopolysaccharide storage disorders. Expert Opin Investig Drugs 11: 675–685. 6. O’Connor, LH, Erway, LC, Vogler, CA, Sly, WS, Nicholes, A, Grubb, J et al. (1998). Enzyme replacement therapy for murine mucopolysaccharidosis type VII leads to improvements in behavior and auditory function. J Clin Invest 101: 1394–1400. 7. Chang, M, Cooper, JD, Sleat, DE, Cheng, SH, Dodge, JC, Passini, MA et al. (2008). Intraventricular enzyme replacement improves disease phenotypes in a mouse model of late infantile neuronal ceroid lipofuscinosis. Mol Ther 16: 649–656. 8. Sands, MS and Davidson, BL (2006). Gene therapy for lysosomal storage diseases. Mol Ther 13: 839–849. 9. Fratantoni, JC, Hall, CW and Neufeld, EF (1968). Hurler and Hunter syndromes: mutual correction of the defect in cultured fibroblasts. Science 162: 570–572. 10. Macauley, SL and Sands, MS (2009). Promising CNS-directed enzyme replacement therapy for lysosomal storage diseases. Exp Neurol 218: 5–8. 11. Liu, G, Martins, I, Wemmie, JA, Chiorini, JA and Davidson, BL (2005). Functional correction of CNS phenotypes in a lysosomal storage disease model using adeno-associated virus type 4 vectors. J Neurosci 25: 9321–9327. 12. Foust, KD, Nurre, E, Montgomery, CL, Hernandez, A, Chan, CM and Kaspar, BK (2009). Intravascular AAV9 preferentially targets neonatal neurons and adult astrocytes. Nat Biotechnol 27: 59–65. 13. Cearley, CN, Vandenberghe, LH, Parente, MK, Carnish, ER, Wilson, JM and Wolfe, JH (2008). Expanded repertoire of AAV vector serotypes mediate unique patterns of transduction in mouse brain. Mol Ther 16: 1710–1718. 14. Fu, H, Dirosario, J, Killedar, S, Zaraspe, K and McCarty, DM (2011). Correction of neurological disease of mucopolysaccharidosis IIIB in adult mice by rAAV9 trans-bloodbrain barrier gene delivery. Mol Ther 19: 1025–1033.

Molecular Therapy vol. 20 no. 7 july 2012

Brain Phenotypes Impact AAV Gene Transfer

15. Chen, YH, Chang, M and Davidson, BL (2009). Molecular signatures of disease brain endothelia provide new sites for CNS-directed enzyme therapy. Nat Med 15: 1215–1218. 16. Büning, H, Perabo, L, Coutelle, O, Quadt-Humme, S and Hallek, M (2008). Recent developments in adeno-associated virus vector technology. J Gene Med 10: 717–733. 17. Grifman, M, Trepel, M, Speece, P, Gilbert, LB, Arap, W, Pasqualini, R et al. (2001). Incorporation of tumor-targeting peptides into recombinant adeno-associated virus capsids. Mol Ther 3: 964–975. 18. Work, LM, Büning, H, Hunt, E, Nicklin, SA, Denby, L, Britton, N et al. (2006). Vascular bed-targeted in vivo gene delivery using tropism-modified adeno-associated viruses. Mol Ther 13: 683–693. 19. Sly, WS, Quinton, BA, McAlister, WH and Rimoin, DL (1973). Beta glucuronidase deficiency: report of clinical, radiologic, and biochemical features of a new mucopolysaccharidosis. J Pediatr 82: 249–257. 20. Neufeld, EF and Muenzer, J. In: Part 16: Lysosomal Storage Disoders (eds Beaudet Valle, Vogelshtein, Kinnzler, Antonarakis, Balabio) (Schriver, Online, 2011). 21. Shen, S, Bryant, KD, Brown, SM, Randell, SH and Asokan, A (2011) Terminal N-linked galactose is the primary receptor for adeno-associated virus 9. J Biol Chem 286: 13532–13540. 22. Bell, CL, Vandenberghe, LH, Bell, P, Limberis, MP, Gao, GP, Van Vliet, K et al. (2011). The AAV9 receptor and its modification to improve in vivo lung gene transfer in mice. J Clin Invest 121: 2427–2435. 23. Karolewski, BA and Wolfe, JH (2006). Genetic correction of the fetal brain increases the lifespan of mice with the severe multisystemic disease mucopolysaccharidosis type VII. Mol Ther 14: 14–24. 24. Daly, TM, Ohlemiller, KK, Roberts, MS, Vogler, CA and Sands, MS (2001). Prevention of systemic clinical disease in MPS VII mice following AAV-mediated neonatal gene transfer. Gene Ther 8: 1291–1298. 25. Balestrieri, ML and Napoli, C (2007). Novel challenges in exploring peptide ligands and corresponding tissue-specific endothelial receptors. Eur J Cancer 43: 1242–1250. 26. Hajitou, A, Pasqualini, R and Arap, W (2006). Vascular targeting: recent advances and therapeutic perspectives. Trends Cardiovasc Med 16: 80–88. 27. Burg, MA, Pasqualini, R, Arap, W, Ruoslahti, E and Stallcup, WB (1999). NG2 proteoglycan-binding peptides target tumor neovasculature. Cancer Res 59: 2869–2874. 28. Yabe, T, Hosoda-Yabe, R, Kanamaru, Y and Kiso, M (2011). A peptide found by phage display discriminates a specific structure of a trisaccharide in heparin. J Biol Chem 286: 12397–12406. 29. Wölcke, J and Weinhold, E (2001). A DNA-binding peptide from a phage display library. Nucleosides Nucleotides Nucleic Acids 20: 1239–1241. 30. Traving, C and Schauer, R (1998). Structure, function and metabolism of sialic acids. Cell Mol Life Sci 54: 1330–1349. 31. Michelfelder, S, Varadi, K, Raupp, C, Hunger, A, Körbelin, J, Pahrmann, C et al. (2011). Peptide ligands incorporated into the threefold spike capsid domain to re-direct gene transduction of AAV8 and AAV9 in vivo. PLoS ONE 6: e23101. 32. Varadi, K, Michelfelder, S, Korff, T, Hecker, M, Trepel, M, Katus, HA et al. (2011). Novel random peptide libraries displayed on AAV serotype 9 for selection of endothelial cell-directed gene transfer vectors. Gene Ther. (doi:10.1038/gt. 2011.143). 33. Frisella, WA, O’Connor, LH, Vogler, CA, Roberts, M, Walkley, S, Levy, B et al. (2001). Intracranial injection of recombinant adeno-associated virus improves cognitive function in a murine model of mucopolysaccharidosis type VII. Mol Ther 3: 351–358. 34. Renlund, M, Tietze, F and Gahl, WA (1986). Defective sialic acid egress from isolated fibroblast lysosomes of patients with Salla disease. Science 232: 759–762. 35. Tietze, F, Seppala, R, Renlund, M, Hopwood, JJ, Harper, GS, Thomas, GH et al. (1989). Defective lysosomal egress of free sialic acid (N-acetylneuraminic acid) in fibroblasts of patients with infantile free sialic acid storage disease. J Biol Chem 264: 15316–15322. 36. McCarty, DM, DiRosario, J, Gulaid, K, Killedar, S, Oosterhof, A, van Kuppevelt, TH et al. (2011). Differential distribution of heparan sulfate glycoforms and elevated expression of heparan sulfate biosynthetic enzyme genes in the brain of mucopolysaccharidosis IIIB mice. Metab Brain Dis 26: 9–19. 37. Vinogradova, MV, Michaud, L, Mezentsev, AV, Lukong, KE, El-Alfy, M, Morales, CR et al. (1998). Molecular mechanism of lysosomal sialidase deficiency in galactosialidosis involves its rapid degradation. Biochem J 330 (Pt 2): 641–650. 38. Rajotte, D, Arap, W, Hagedorn, M, Koivunen, E, Pasqualini, R and Ruoslahti, E (1998). Molecular heterogeneity of the vascular endothelium revealed by in vivo phage display. J Clin Invest 102: 430–437. 39. Kolonin, M, Pasqualini, R and Arap, W (2001). Molecular addresses in blood vessels as targets for therapy. Curr Opin Chem Biol 5: 308–313. 40. Arap, W, Kolonin, MG, Trepel, M, Lahdenranta, J, Cardó-Vila, M, Giordano, RJ et al. (2002). Steps toward mapping the human vasculature by phage display. Nat Med 8: 121–127. 41. Neuwelt, EA, Bauer, B, Fahlke, C, Fricker, G, Iadecola, C, Janigro, D et al. (2011). Engaging neuroscience to advance translational research in brain barrier biology. Nat Rev Neurosci 12: 169–182. 42. Zlokovic, BV (2011). Neurovascular pathways to neurodegeneration in Alzheimer’s disease and other disorders. Nat Rev Neurosci 12: 723–738. 43. Bevan, AK, Duque, S, Foust, KD, Morales, PR, Braun, L, Schmelzer, L et al. (2011). Systemic gene delivery in large species for targeting spinal cord, brain, and peripheral tissues for pediatric disorders. Mol Ther 19: 1971–1980. 44. Gray, SJ, Matagne, V, Bachaboina, L, Yadav, S, Ojeda, SR and Samulski, RJ (2011). Preclinical differences of intravascular AAV9 delivery to neurons and glia: a comparative study of adult mice and nonhuman primates. Mol Ther 19: 1058–1069. 45. Song, L and Pachter, JS (2003). Culture of murine brain microvascular endothelial cells that maintain expression and cytoskeletal association of tight junction-associated proteins. In Vitro Cell Dev Biol Anim 39: 313–320. 46. Warren, L (1959). The thiobarbituric acid assay of sialic acids. J Biol Chem 234: 1971–1975.

1399