Enzyme replacement therapy in α-mannosidosis guinea-pigs

Molecular Genetics and Metabolism 89 (2006) 48–57 www.elsevier.com/locate/ymgme

Enzyme replacement therapy in -mannosidosis guinea-pigs Allison C. Crawley ¤, Barbara King, Thomas Berg 1, Peter J. Meikle, John J. Hopwood Lysosomal Diseases Research Unit, Department of Genetic Medicine, Children, Youth and Women’s Health Service, 72 King William Rd., North Adelaide, South Australia 5006, Australia Received 20 March 2006; received in revised form 13 May 2006; accepted 13 May 2006 Available online 27 June 2006

Abstract -Mannosidosis is a lysosomal storage disorder caused by deWcient activity of lysosomal -mannosidase and is characterised by massive accumulation of mannose-containing oligosaccharides in aVected individuals. Patients develop behaviour and learning diYculties, skeletal abnormalities, immune deWciency and hearing impairment. Disease in -mannosidosis guinea-pigs resembles the clinical, histopathological, biochemical and molecular features of the human disease. We have used the guinea-pig model to investigate eYcacy of enzyme replacement therapy as a treatment for -mannosidosis. Intravenous recombinant human lysosomal -mannosidase, administered at a dose of 1 mg/kg, was cleared from circulation with a half-life of 53 h, with signiWcant enzyme activity (1.4£ normal levels) detected in circulation one week post-injection. -Mannosidase administered to -mannosidosis guinea-pigs at 1 mg/kg (onset at birth or »30 days) and 10 mg/kg (at birth) was distributed widely amongst tissues, including to capillary depleted brain. By monitoring with tandem mass spectrometry, enzyme replacement therapy was found to be eVective in reducing stored substrates in peripheral tissues at both dose rates, and in brain by up to 39% at the 10 mg/kg dose, compared with untreated -mannosidosis controls. Reductions of up to 60% of urinary mannose containing oligosaccharides were also observed. No histological improvements were seen in the brain at either dose, however marked decreases in lysosomal vacuolation in liver, kidney, spleen and endocrine pancreas, as well as a signiWcant reduction in trigeminal ganglion neurons were observed. Multiple injections of 1 mg/kg recombinant enzyme in -mannosidosis guinea-pigs induced a very rapid humoral immune response precluding long-term intravenous treatment. © 2006 Elsevier Inc. All rights reserved. Keywords: Lysosomal storage disease; -Mannosidase; Animal model; Guinea-pig; Therapy; Enzyme replacement; Genetic disease

Introduction -Mannosidosis is a lysosomal storage disorder (LSD) caused by the deWcient activity of lysosomal -mannosidase (EC 3.2.1.24), an essential enzyme in the degradation of glycoproteins. Due to impaired ability to hydrolyse -linked mannoside residues in oligosaccharides attached to glycoproteins, -mannosidosis patients accumulate mannosecontaining oligosaccharides in tissues and body Xuids. Accumulation of these oligosaccharides leads to the many symptoms associated with -mannosidosis, typically *

Corresponding author. Fax: +61 08 81617100. E-mail address: [email protected] (A.C. Crawley). 1 Present address: Department of Pathology, University Hospital of Northern Norway 9038, Tromsø, Norway. 1096-7192/$ - see front matter © 2006 Elsevier Inc. All rights reserved. doi:10.1016/j.ymgme.2006.05.005

including behavioural and learning diYculties, hepatomegaly, immune deWciency and hearing impairment [1]. A guinea-pig model of -mannosidosis was found to mirror clinical and histopathological features of the human disease [2]. Mild neurological changes could be detected in aVected guinea-pigs as early as 1 month of age. Neurological disease increased in severity with age, and end-stage disease was usually between 10 to 14 months of age, invariably due to inability to prehend, masticate and swallow food eYciently (A. Crawley, unpublished observations). The -mannosidosis guinea-pig also resembles the human disease at the molecular level, as the oligosaccharide storage products are identical to those found in human patients [2], and the mutation causing -mannosidosis in the guinea-pig (R227W) is identical to a missense mutation found in human patients [3].

A.C. Crawley et al. / Molecular Genetics and Metabolism 89 (2006) 48–57

Except for general symptomatic management, there are limited therapeutic options for LSD with central nervous system (CNS) involvement, including -mannosidosis. Hematopoietic stem cell transplantation (HSCT) in a cat model of -mannosidosis was shown to ameliorate the disease in the CNS and visceral organs [4]. Clinical improvement and an apparent stabilization of neurocognitive function were also found following HSCT in human mannosidosis patients [5,6]. This indicates that HSCT may be an adequate therapeutic option for some patients, however, HSCT carries a signiWcant risk of morbidity and mortality, hence the need for new therapeutic options to be developed. EVective gene therapy has recently been demonstrated in a feline -mannosidosis model using intracerebral injections of an AAV1 vector into multiple sites, however further development is necessary before its use could be applied to humans [7]. Enzyme replacement therapy (ERT) is emerging as an alternative therapy for some LSD. The success of ERT for non-neuronopathic Gaucher disease [8,9], together with successful results obtained in animal studies with other LSD, have led to human ERT clinical trials for several LSD, including mucopolysaccharidoses (MPS type I, type II and type VI), Pompe disease, and Fabry disease [10–17], with FDA approval now granted for MPS I, MPS VI, and Fabry. Generally, these studies have shown that ERT reduces storage in visceral tissues and produces considerable gains in quality of life. SigniWcantly, the patients successfully treated in the aforementioned trials have not suVered from CNS involvement, which is a feature in approximately two-thirds of LSD. Overcoming the blood–brain barrier and targeting the appropriate lysosomal enzyme to the CNS is required for ERT to be eVective for LSD with CNS involvement. The potential to treat -mannosidosis by ERT was demonstrated in cell culture experiments showing mannose-6phosphate receptor mediated uptake of recombinant human -mannosidase (rh-man) by -mannosidase deWcient Wbroblasts [3]. This rh-man uptake normalised -mannosidase activity and removed storage of mannosideoligosaccharides. However, due to the structure of the blood–brain barrier, except those substances utilising speciWc transport processes, most large molecules such as naturally occurring plasma proteins or intravenously administered lysosomal proteins are believed to be excluded from passage into the adult CNS. Several ERT studies in newborn mouse LSD models have directly or indirectly demonstrated enzyme passage across the blood– brain barrier in the newborn period (MPS VII [18,19] and MPS IIIA [20]). This has recently been suggested to be due to the presence of mannose-6-phosphate/insulin-like growth factor II receptors in the murine blood–brain barrier at this age, which signiWcantly enhances transport of mannose-6-phosphorylated lysosomal enzymes from circulation into the brain [21]. A number of recent ERT studies in several diVerent adult murine LSD models have challenged the adult

49

blood–brain barrier concept, demonstrating biochemical evidence of reduction of stored substrates in the CNS following intravenous ERT at either high enzyme doses or short dose intervals [22,23]. In some cases, these Wndings were supported by histological reductions in pathology [24,25]. One of these studies in a knock-out -mannosidosis mouse model used various sources of -mannosidase to evaluate the eYcacy of ERT [23]. Intravenous injection of two doses of rh-man (approximately 25 mg/kg) into adult mice cleared the storage of neutral mannosides in liver, kidney and heart. Surprisingly they also reported these neutral mannosides were reduced in whole brain homogenates to less than 30% of that in untreated mannosidosis controls. The mechanisms by which the administered lysosomal enzymes gained entry to the adult CNS to reduce stored substrates in brain are currently unclear. To determine the in vivo potential of ERT for -mannosidosis in our model system, this study investigated the uptake and tissue distribution of rh-man in normal guinea-pigs and evaluated the therapeutic eYcacy of rhman in -mannosidosis guinea-pigs. The work reported here provides baseline data for future experiments designed to correct CNS pathology by supplying the brain with rhman. Methods Animals All guinea-pigs used in these studies were bred in a non-barrier colony established from heterozygotes related to the two -mannosidosis aVected guinea-pigs as described previously [2]. Diagnosis of aVected and heterozygous animals was based on the presence of the 679C > T mutation (R227W) [3]. Four-week-old (250-400 g) homozygous unaVected (+/+) or heterozygous (+/¡) unaVected guinea-pigs were used in the plasma and tissue distribution studies. ERT of homozygous -mannosidosis aVected (¡/¡) animals was started at 1 day (n D 2, 1 mg/kg; n D 4, 10 mg/kg) or 30 days (n D 2, 1 mg/kg) after birth. Plasma distribution studies were also performed in Sprague–Dawley rats (200 g). All experimental procedures involving animals were approved by the Children, Youth and Women’s Health Service, Animal Ethics Committee.

Preparation of enzyme Rh-man was produced in Chinese hamster ovary cells and immunoaYnity puriWed as described previously [26]. The speciWc activity of the preparations ranged between 38 and 42 U/mg. -Mannosidase activity was measured at pH 4.5 using p-nitrophenyl -D-mannopyranoside (Sigma, Castle Hill, Australia). One unit of activity corresponded to the amount of enzyme which releases 1 mol of p-nitrophenol per min. PuriWed enzyme was Wlter sterilised and stored at ¡70 °C in phosphate-buVered saline (PBS) at a concentration of 0.5–1 mg/ml.

Enzyme administration and sample collection Guinea-pigs were given subcutaneous prophylactic antihistamine (chlorpheniramine maleate, Niramine® Jurox Pty. Ltd., Silverwater, Australia, 1 mg/kg) 45 min before rh-man infusion. For plasma distribution, normal control guinea-pigs (+/+,+/¡) and rats were anaesthetised and maintained under isoXurane inhalational anaesthesia for 2 and 1 h, respectively. Rh-man was injected over 30 s into the cephalic vein.

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Serial venous blood samples were collected from a jugular catheter (guinea-pigs) or from the tail vein (rats). Blood samples taken more than 1–2 h after rh-man infusion were collected from the ear capillaries (guinea-pigs) or the tail vein (rats) without anaesthesia. For tissue distribution studies, 12 control guinea-pigs (+/+) were administered rh-man intravenously (1 mg/kg) over 30 s into the cephalic vein under isoXurane anaesthesia. At speciWed time points animals were anaesthetised, then perfused intracardially with cold saline and tissues weighed and collected onto ice. Tissues were then homogenised in buVer (0.02 M Tris– HCl containing 0.5 M NaCl, pH 7–7.5), freeze-thawed six times and kept at ¡20 °C until analysed for rh-man activity, and protein concentration. Capillary depletion of brain tissues was according to Triguero et al. [27]. Plasma and tissue half-lives were calculated using standard mathematical calculations [28]. One to two day old and 30–31 day old, aVected guinea-pigs (¡/¡) were injected intravenously under anaesthesia with either a single dose of rhman at 10 mg/kg (n D 4) and were sacriWced 7 days later, as above; or were injected weekly for 5 weeks with rh-man at 1 mg/kg (n D 4) and sacriWced 2 days after the last injection at 31 or »60 days of age. One animal in each dose group was perfused with fresh 4% paraformaldehyde in 0.1 M Cacodylate buVer, pH 7.2, and tissues collected for histological analysis. The remaining animals were perfused with saline as above, and tissues collected for determination of rh-man activity and oligosaccharide storage in tissues using tandem mass spectrometry. Two additional untreated aVected (¡/¡) guinea-pigs (7 and 31 days old) were perfused with Wxative as above as age–matched histological controls. A further four untreated aVected (¡/¡) guinea-pigs (61 days old, n D 1; 8 days old, n D 3) were perfused with saline as above, as age-matched controls for oligosaccharide storage using tandem mass spectrometry.

Histological analysis Tissues for light microscopy in ERT treated -mannosidosis guineapigs (¡/¡) and age-matched untreated controls (+/+ and ¡/¡) were Wxed, processed and embedded into Spurrs resin as described previously [2]. The degree of vacuolation due to lysosomal storage was examined by light microscopy at 400–1000£ magniWcation on 1 m thick, resin embedded sections stained with toluidine blue, and was compared and graded accordingly with age-matched untreated -mannosidosis controls.

Immunoassay of anti-rh-man antibodies An ELISA assay was used to determine the presence of anti-rh-man antibodies in plasma from aVected (¡/¡) guinea-pigs undergoing weekly injections of 1 mg/kg rh-man. Polyvinylchloride plates were coated with 0.5 g rh-man/well and blocked with 1% (w/v) ovalbumin. Plasma samples were pre-absorbed overnight with ovalbumin/BSA coupled AY-Gel 10 (10 mg of each protein was coupled to 1 ml of AY-Gel 10, using the manufacturers instructions), then serially diluted in 0.02 M Tris–HCl, pH 7.2, containing 0.25 M NaCl and 1% (w/v) ovalbumin and applied to the plates. A peroxidase-labelled rabbit anti-guinea-pig IgG (Silenus/Chemicon, Boronia, Australia) secondary antibody was then added to the plates followed by colour development with a peroxidase substrate solution (ABTS substrate kit, Bio-Rad, Regent’s Park, Australia). Colour development was quantiWed by measuring absorbancy at 450 nm on an automated ELISA reader (Ceres 900 Hdi, Bio-Tek Instruments, Winooski, USA).

Results

Determination of tissue rh-man activity

Plasma clearance and tissue distribution of rh-man

Due to the high level of endogenous (non-lysosomal) -mannosidase activities in guinea-pig tissues, species speciWc rh-man activity was detected using an immune capture assay, based on a monoclonal antibody to rh-man. BrieXy, microtitre wells were coated with sheep antimouse polyclonal antibody (10 g/ml) in 0.1 M NaHCO3 (100 l/well). A second layer of an anti-rh-man monoclonal antibody (4C5) in culture supernatant, was then added [26]. Homogenised tissue samples, diluted in 1% skim milk in 0.02 M Tris–HCl, 0.25 M NaCl, pH 7.5, were added to the wells and incubated overnight at 4 °C. The plate was washed (3£) with 0.02 M Tris–HCl, 0.25 M NaCl, pH 7.5, and 4 mM p-nitrophenyl-D-mannoside, 0.1 M CH 3COONa/CH3COOH, pH 4.5 (100 L), was added to each well. After incubation (16 h, 37 °C), the absorbance was determined at  D 410. A calibration curve prepared using rh-man produced in CHO cells was used to calculate the level of rh-man in each sample. The activity values were normalised to protein concentration, determined with a micro BCA Protein kit (Sigma, Castle Hill, Australia) and expressed as nmol/min/mg protein. Limits of detection for the assay were 0.1 nmol/min/mg.

High levels of rh-man were detected in circulation after intravenous injection of rh-man into control guinea-pigs (Fig. 1 and Table 1). The plasma half-life was approximately »53 h. SigniWcant levels of activity could be measured in circulation one week after the injection at both low and high doses of rh-man, with immunocaptured rh-man levels of 7 and 41 nmol/min/ml, respectively. Prior to enzyme infusion, total plasma -mannosidase activity (pH 4.5) in normal guinea-pigs was 4.93 § 0.97 nmol/min/ml (n D 3). Similar plasma half-life results were obtained from studies in rats (t1/2 of »37 h, data not shown). Analysis of guinea-pig tissues revealed rh-man activity in all tissues examined, 2 h following a single injection of a low

Detection of storage substrates Oligosaccharides in tissues extracts, urine and plasma were derivatised with 1-phenyl-3-methyl-5 pyrazolone (PMP, Tokyo Kasei Co. Ltd. Tokyo, Japan) and analysed by electrospray ionisation-tandem mass spectrometry [26]. The signal intensities of relevant oligosaccharides were calculated relative to the signal intensity of an internal standard, 1 nmol of methyllactose (4-0-(2-0-methyl--D-galactopyranosyl)-D-glucopyranose, Sigma– Aldrich, Castle Hill, Australia), which was added to each sample at a concentration of 1 M. This value was designated the relative intensity (ri). A calibration curve was calculated for the trisaccharide mannose-mannoseN-acetylglucosamine (Man-Man-GlcNAc), which was available in a puriWed form (a gift from J.M. Michalski, Lille, France). This calibration curve was used to calculate the concentration of Hex-Hex-HexNAc in each sample, which by deduction, would be Man-Man-GlcNAc. A student t-test was used to test statistical diVerences between dose groups and agematched untreated -mannosidosis controls.

Fig. 1. Plasma clearance of rh-man in normal guinea-pigs up to 7 days following intravenous administration of 1 mg/kg rh-man. N D 2 to 5 per timepoint except n D 1 at 120 and 144 h. Error bars D 1SD.

A.C. Crawley et al. / Molecular Genetics and Metabolism 89 (2006) 48–57

51

Table 1 Mean tissue rh-man activity in normal control guinea-pigs at various time-intervals up to 7 days following a single 1 mg/kg dose IV injection of rh-man, compared with -mannosidosis guinea-pigs given a single 10 mg/kg dose or 5 £ weekly 1 mg/kg doses of rh-man (nmol/min/mg protein; n D 3 all groups unless indicated)

Dose:

Control

-Mannosidosis

1 £ 1 mg/kg

1 £ 10 mg/kg

5 £ 1 mg/kg (weekly)

Interval:

2h

2 days

4 days

7 days

7 days

2 days

Liver Kidney Lung Spleen Heart Lymph node Pancreas Cerebral hemisphere/cd Cerebellum/cd Brain stem/cd Bone marrow Plasma⵩ Urine⵩

4654 § 657 241 § 98 146 § 29 589 § 112 60 § 14 95 § 71 30 § 19 1.5,2.6* 4.0** 3.1** 1546 § 230 314 § 99 0.2**

370 § 86 37 § 8 20 § 4 80 § 26 29 § 11 32 § 11 14 § 4 13.6,1.3,0 0,1.7* 0,0* 131 § 128 30 § 12 0

164 § 74 13 § 4 13 § 5 30 § 11 11 § 3 17 § 6 5§2 0 0 0 26 § 7 21 § 8 0

24 § 14 10 § 9 4§2 19 § 6 3§2 11 § 2 2§1 0 0 0 6§2 7§2 0

227 § 135 113 § 49 42 § 32 64 § 40 24 § 15 44 § 30 26 § 5 0 0 0 47* 41 § 16 0.2 § 0

44 § 14 2§1 1§2 25* 0 1* 2§2 0,0.2,1.3 0,0,3 0,0,7 8 § 14

§1SD; ¤n D 2; ¤¤n D 1; cd, capillary depleted; ⵩nmol/min/ml.

dose (1 mg/kg) of rh-man (Table 1). Highest activity was found in the liver, followed by bone marrow, spleen, kidney and lung, with much lower but signiWcant activity detected in capillary depleted brain. Two days following a single IV injection, tissues contained approximately 25% (6–48%) of the activity levels found 2 h after injection (Table 1) with low to trace amounts still detectable in two out of three brain samples. The rate at which enzyme activity decreased was similar in all non-CNS tissues analysed (Fig. 2), with an estimated tissue half-life of 1.2 days for liver and 1.5 days for spleen. Activity was present in all of these tissues except brain, 7 days after a single dose of 1 mg/kg rh-man. -Mannosidosis guinea-pigs analysed 7 days after a single high dose of enzyme (10 mg/kg) at birth, displayed approximately 10-fold higher levels of rh-man activity in non-neurological tissues when compared to single low dose guinea-pig tissues at 7 days (Table 1). As seen for the single low dose at 7 days, no rh-man activity was detected in the

Fig. 2. Tissue pharmacokinetics of rh-man in normal guinea-pigs, 2 h to 7 days following a single intravenous dose of 1 mg/kg rh-man (see Table 1 also). N D 3 per timepoint. Error bars D 1SD.

brain. In contrast, signiWcantly lower levels of rh-man activity were detected in most tissues at 2 days post-injection, from -mannosidosis animals receiving 5 weekly injections of 1 mg/kg compared with tissues from 2 day single low dose controls (Table 1). This reduction is most likely due to development of antibody titres against rh-man in multiple dose animals which interfered with the ELISA based assay (see below). EVects of rh-man ERT on -mannoside-oligosaccharide accumulation in tissues and excretion in urine Mass spectrometry of PMP derived oligosaccharides revealed that compared to untreated aVected guinea-pigs, animals receiving the 5 weekly injections (1 mg/kg) had reduction of the trisaccharide Hex-Hex-HexNAc in liver, kidney, spleen, lung, heart, mesenteric lymph node, and pancreas (Fig. 3A). Reductions in trisaccharide levels for these tissues of between 33–80% (mean D 58%) of those found in the untreated -mannosidosis control were observed. Overall greater reduction of Hex-Hex-HexNAc in the same tissues were observed following a single high dose (10 mg/kg) of rhman (mean reduction 81%, range 62–92%; p < 0.05 most tissues) (Fig. 3A). Non-capillary depleted cerebrum from the 5 £ 1 mg/kg dose animals revealed no signiWcant diVerences in Hex-Hex-HexNAc levels compared with untreated mannosidosis animals, with a mean reduction of only 6% observed (Fig. 3B). However, statistically signiWcant reductions were detected in capillary depleted cerebrum (35%; p D 0.05) and brainstem (39%; p < 0.05) from the 10 mg/kg dose animals where ERT was given at birth, but not in cerebellum (3%)(Fig. 3B), compared with untreated -mannosidosis controls. Negligible levels of Hex-Hex-HexNAc were found in normal control brain. The trisaccharide Hex-Hex-HexNAc is elevated in urine from untreated -mannosidosis guinea-pigs (n D 9) to

52

A.C. Crawley et al. / Molecular Genetics and Metabolism 89 (2006) 48–57

Fig. 3. Levels of storage of the trisaccharide Hex-Hex-HexNAc (deduced to be Man-Man-GlcNAc) using tandem mass spectrometry in tissues from mannosidosis guinea-pigs following either Wve weekly IV injections of 1 mg/kg rh-man from »30 days old (and euthanasia at »60 days old), or one IV injection of 10 mg/kg rh-man at birth, compared with age-matched normal and untreated -mannosidosis guinea-pigs. (A) Peripheral tissues; (B) Central nervous system. Sample sizes: normal n D 1; 60-day old untreated -mannosidosis guinea-pigs n D 1; 8-day old untreated -mannosidosis guinea-pigs n D 3; 5 £ 1 mg/kg n D 3; 1 £ 10 mg/kg n D 3; *,p < 0.05; **cerebrum from the 5 £ 1 mg/kg dose and age-matched untreated control was not capillary depleted, and comparably prepared brainstem and cerebellum were not available for analysis. Error bars D 1 SD.

approximately 20 times that of normal (n D 4). Analysis of urine samples, collected one week after each injection of rh-man, revealed reduced levels of trisaccharide. An initial decrease in urine levels of Hex-Hex-HexNAc was evident in animals receiving multiple injections (Fig. 4), with levels appearing to subsequently stabilise at 25% below those in untreated aVected guinea-pigs. A single high dose injection (10 mg/kg) resulted in a »60% reduction of trisaccharide levels found in untreated aVected guinea-pigs, which is still 7.5 times above normal. Tissue pathology One m, resin-embedded tissue sections were evaluated by light microscopy after single (10 mg/kg) or multiple

(5 £ 1 mg/kg) injections of rh-man. Compared to agematched untreated aVected animals, there was a marked reduction in lysosomal vacuolation in liver, kidney, spleen and endocrine pancreas in an animal receiving a single dose of 10 mg/kg of rh-man (Table 2). In addition, signiWcant reduction of lysosomal vacuolation was observed in trigeminal ganglion neurons and adjacent associated cell types (endothelial cells, oligodendrocytes, interstitial macrophages; Fig. 5A–D). The multiple low dose injections (5 £ 1 mg/kg) only reversed pathology in reticuloendothelial cells (liver and spleen) and had very limited eVect in kidney and pancreas (Table 2). No obvious reductions in storage pathology were observed in frontal cortex, cerebellum, thalamus and cervical spinal cord at either dose rate (Table 2).

A.C. Crawley et al. / Molecular Genetics and Metabolism 89 (2006) 48–57

53

Fig. 4. Levels of the trisaccharide Hex-Hex-HexNAc (deduced to be Man-Man-GlcNAc) in urine of -mannosidosis guinea-pigs, following treatment with either weekly injections of 1 mg/kg rh-man (n D 3 to 4 per group), or one injection of 10 mg/kg rh-man (n D 2) compared with normal controls (n D 4), using tandem mass spectrometry. Samples were collected 1 week after the previous injection of recombinant protein for both the 5 £ 1 mg/kg and 10 mg/kg groups, with the Wnal sample for the 5 £ 1 mg/kg group taken 2 days after the Wnal injection. Error bars D 1 SD.

Antibody response An ELISA based method was used to determine the antibody titre in -mannosidosis animals receiving multiple injections of rh-man. All animals (n D 4) developed an antibody response after two or three injections of rh-man, despite onset of ERT at birth in half of the animals compared with onset at 30 days of age. The highest antibody titre reached 1 in 1.26 £ 106 (Fig. 6). No adverse hypersensitivity reactions were observed during infusions under anaesthesia. Antibody titres before treatment and in untreated normal (+/+, +/¡) and aVected (¡/¡) animals were very low (<1/640), (data not shown). Discussion Therapies to target CNS pathology in LSD are being actively pursued. As a precursor to further studies to deliver recombinant enzyme to the CNS, we investigated the distribution and fate of rh-man in normal guinea-pigs and eYcacy of ERT in -mannosidosis guinea-pigs. The plasma half-life of »53 h for rh-man in guinea-pigs is exceptionally long, with plasma half-lives generally less than 60 min observed for other lysosomal enzymes in other LSD animal models (MPS IIIB, 18 min [29]; MPS IIID, 5– 31 min [30]; MPS VI, 11 min [31]; MPS I, 19 min [32]; Aspartylglycosaminuria, 39 min [22]; metachromatic leukodystrophy, 40 min [24]). Half-lives for puriWed bovine kidney -mannosidase, column puriWed CHO expressed recombinant human -mannosidase, and mouse embryonic Wbroblast expressed recombinant mouse -mannosidase injected intravenously into -mannosidosis mice were 4, 8 and 47 min respectively [23]. In our studies, the signiWcantly

longer half-life of monoclonal antibody column aYnity puriWed rh-man also occurred in rats (data not shown), suggesting that this was not a species-speciWc phenomenon. It is unknown why such a signiWcantly longer half-life was observed for rh-man in our study compared with previous observations for other -mannosidase preparations and other lysosomal enzymes. A slow clearance phase of rh-man may reXect speciWc or special characteristics of this enzyme, or a portion of this enzyme, thereby allowing it to remain in circulation, such as carbohydrates containing terminal sialic acid, or low aYnity for the mannose-6-phosphate/insulin-like growth factor II receptor or other receptors. However, this observation is surprising, as the deduced amino acid sequence indicates 11 potential N-linked glycosylation sites [33] compared with other lysosomal enzymes which more commonly have 4–8 potential sites. Earlier work in our laboratory indicated that rh-man from the same expression system and puriWcation protocol had a high degree of N-glycosylation and used mannose-6-phosphate mediated uptake into patient Wbroblasts [26]. Further studies are needed to conWrm the degree of mannose-6phosphorylation of the enzyme. Other carbohydrate speciWc receptors such as mannose and asialoglycoprotein receptors can eYciently mediate lysosomal enzyme uptake as demonstrated in Mr 46 000 mannose-6-phosphate receptor deWcient mice [34]. Therefore, saturation of all available mannose-6-phosphate receptors alone, is an unlikely explanation for the prolonged plasma half-life. A long plasma half-life increases the likelihood of greater enzyme access to non-hepatic tissues including the CNS. To get a more accurate assessment of whether ERT had delivered recombinant enzyme (and hence enzyme activity) across the blood–brain barrier, saline perfusion

54

A.C. Crawley et al. / Molecular Genetics and Metabolism 89 (2006) 48–57

Table 2 Evaluation of lysosomal storage in 1 m toluidine blue stained sections in -mannosidosis guinea-pigs given a single 10 mg/kg dose or 5 £ weekly 1 mg/kg doses of rh-man from birth compared with age-matched untreated control -mannosidosis guinea-pigs Tissue Cell type Liver KupVer cells Kidney Podocytes Interstitial cells Tubules Spleen Sinusoid cells Pancreas Exocrine Endocrine Trigeminal ganglion Neurons Cerebellum Purkinje cells Granule cells Golgi type II Thalamus Large neurons Frontal cortex Neurons Neuroglia Cervical spinal cord Ventral horn cells

-Mannosidosis 1 £ 10 mg/kg

5 £ 1 mg/kg

###

###

### ### ##

## # NC

###

###

# ###

NC #

##¤

NC

NC NC NC

NC NC NC

NC

NC

NC NC

NC NC

NC

NC

###

Marked decrease in lysosomal vacuolation compared with agematched untreated -mannosidosis guinea-pigs, indistinguishable from normal. ## Moderate decrease in lysosomal vacuolation compared with agematched untreated -mannosidosis guinea-pigs. # Slight decrease in lysosomal vacuolation compared with age-matched untreated -mannosidosis guinea-pigs. NC, no change, similar to age-matched untreated -mannosidosis guineapigs. ¤ Most neurons with only occasional or no vacuoles; isolated neurons with moderate numbers of vacuoles (see Fig. 5C).

and capillary depletion of brain was employed to remove residual enzyme activity in plasma, and also enzyme bound to or within capillary endothelial cells. Low but signiWcant enzyme activity was present in brain up to 2 days following 1 mg/kg rh-man, with signiWcant reduction in stored oligosaccharides (35–39%) in two major CNS regions at the 10 mg/kg dose rate, indicating cellular uptake and metabolic function of rh-man. The most likely explanation for enzyme passage across the blood–brain barrier at the higher dose rate was of increased mannose-6-phosphate/ insulin-like growth factor II receptor mediated transport as seen in newborn mice [21], as the 10 mg/kg dose guinea-pigs were injected intravenously at birth. The increased presence of these receptors in guinea-pigs is somewhat surprising due to the much more precocious nature of the newborn guinea-pig compared to a mouse pup, and therefore the more advanced brain development at birth. Further studies are needed in the newborn guinea-pig to conWrm these observations.

In a recent ERT study in adult MPS VII mice immunotolerant to recombinant human -glucuronidase (rhGUS), low but signiWcant enzyme activity was also detected in the CNS 7 days after the last IV injection of 13 doses of enzyme, at relatively low dose rates (1-4 mg/kg, weekly) [25]. Compared to our studies, it is interesting that plasma half-life for rhGUS was much shorter (enzyme activity completely cleared from circulation by less than 24 hours), however tissue half-lives were much longer (»100 h). A major Wnding in the MPS VII study was that longer duration of therapy was imperative to see histological correction of lysosomal storage in the CNS. No CNS changes were observed at three doses of 20 mg/kg rhGUS over 1 week, however four weekly doses (at 20 mg/kg) resulted in moderate reductions in lysosomal storage in neurons in the cortex, in meninges and perivascular cells, and slight decreases in glia. Thirteen weekly doses at 4 mg/kg resulted in almost identical observations [25]. In our study, as biochemical evidence of reduction in stored substrates at the 10 mg/kg dose was observed, a longer therapy protocol may therefore have also revealed histological changes in the CNS. Passage of large molecules such as proteins into the CNS is considered negligible in sites like the circumventricular organs and choroid plexus where the blood–brain barrier characteristics are absent or modiWed [35]. The mechanism(s) by which recombinant enzymes gained access to the CNS in some recent ERT studies in adult murine LSD animal models was not elucidated and clearly requires speciWc and detailed evaluation in future studies. Using very high dose rates (10–25 mg/kg; [22–25]), or high cumulative dose rates [25], these studies clearly demonstrated biochemical evidence of reduction of stored substrates in the CNS, following intravenous administration of recombinant enzyme. In our studies, blood–nerve barrier permeability in ganglion [36] enabled passage of rh-man into the trigeminal ganglion, with reduction in lysosomal vacuolation observed histologically in the single high-dose (10 mg/kg) treated mannosidosis guinea-pig. This observation suggests that a mechanism such as retrograde axonal transport [37,38] along the trigeminal nerve may be able to deliver low levels of recombinant enzyme to limited regions of the CNS, together with intracellular transfer to adjacent cells via mannose-6-phosphate mediated transport mechanisms to eVect a reduction in stored substrates. This hypothesis requires further investigation in future ERT studies. Our studies demonstrate the sensitivity of tandem mass spectrometry in detecting reduction of stored substrates in tissues compared with histological evaluation of resin embedded toluidine blue stained sections. In other ERT studies in a feline MPS VI model, dose-related reductions in urinary oligosaccharides were detected [39], as seen in the -mannosidosis guinea-pig model. Use of this sensitive technology to monitor eYcacy of therapies using easily obtained patient samples such as urine or plasma is currently under development for a number of diVerent LSD [40–43]. Sequence analysis of the guinea-pig lysosomal -mannosidase gene revealed 82–85% identity with the same

A.C. Crawley et al. / Molecular Genetics and Metabolism 89 (2006) 48–57

55

Fig. 5. Trigeminal ganglion neurons in resin embedded sections from: (A) an 8-day old untreated -mannosidosis guinea-pig where multiple Wne cytoplasmic vacuoles are evident in neurons; (B) a 31-day old -mannosidosis guinea-pig treated for 5 weekly doses with 1 mg/kg rh-man enzyme from birth. Numerous Wne clear vacuoles are present in the cytoplasm, indistinguishable from the age-matched untreated -mannosidosis control. (C) an 8-day old mannosidosis guinea-pig treated at birth with a single dose of 10 mg/kg rh-man, where a signiWcant reduction in lysosomal vacuolation was observed in neurons and adjacent cell types. (D) No vacuoles were observed in normal control guinea-pigs. Bar D 20 m.

Fig. 6. Development of antibody titres in -mannosidosis guinea-pigs treated for 5 weekly doses with 1 mg/kg rh-man enzyme intravenously from 1 day (animals A&B) or 30 days of age (animals C&D). Samples 1–5 were collected 7 days after the previous rh-man injection, directly prior to the next treatment, except for sample 6, which was collected 2 days after the Wnal enzyme injection.

genes in humans, mice, cats and cattle. In addition, the guinea-pig mutation R227W is an amino acid substitution resulting in a full-length inactive protein [3]. Therefore, although -mannosidosis guinea-pigs have a fulllength lysosomal -mannosidase protein, the degree of diVerence in guinea-pig sequence homology compared with rh-man induced an immune response, as seen with ERT in other LSD animal models [32,44–46]. In other studies, onset of ERT at birth in a feline MPS VI model

using recombinant human protein resulted in lack of immune response compared with onset of therapy at a later age [47]. However, starting therapy at birth in the guinea-pig -mannosidosis model did not abrogate this immune response. Further studies using recombinant guinea-pig -mannosidase would likely overcome immunological issues, and would enable long-term eVects of ERT on CNS pathology and clinical function to be evaluated.

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Acknowledgments This work was supported by grants from the Norwegian Research Council to Thomas Berg (Grant 125842/410), the Women’s and Children’s Hospital’s Research Foundation and the National Health and Medical Research Council of Australia. We are very grateful to Dr. Dyane Auclair for technical assistance and management of the guinea-pig colony, and to Dr. Kim Hemsley for helpful discussions. We thank J.M. Michalski for mannoside standard and staV of the Children, Youth and Women’s Health Service animal house facility for their care of the guinea-pigs and rats, and Dr. Lynda Bonning for the original donation of heterozygous animals used to establish the -mannosidosis guineapig colony.

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