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Intracerebroventricular infusion of acid sphingomyelinase corrects CNS manifestations in a mouse model of Niemann–Pick A disease
Experimental Neurology 215 (2009) 349–357
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Experimental Neurology j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / yex n r
Intracerebroventricular infusion of acid sphingomyelinase corrects CNS manifestations in a mouse model of Niemann–Pick A disease James C. Dodge a,⁎, Jennifer Clarke a, Christopher M. Treleaven a, Tatyana V. Taksir a, Denise A. Griffiths a, Wendy Yang a, Jonathan A. Fidler a, Marco A. Passini a, Kenneth P. Karey a, Edward H. Schuchman b, Seng H. Cheng a, Lamya S. Shihabuddin a a b
Genzyme Corporation, 49 New York Avenue, Framingham, MA 01701, USA Mount Sinai School of Medicine, New York, NY 10029, USA
a r t i c l e
i n f o
Article history: Received 15 September 2008 Revised 29 October 2008 Accepted 30 October 2008 Available online 14 November 2008 Keywords: Intracerebroventricular delivery Acid sphingomyelinase Sphingomyelin Lysosomal storage disease Acid sphingomyelinase knockout mouse
a b s t r a c t Niemann–Pick A (NPA) disease is a lysosomal storage disorder (LSD) caused by a deficiency in acid sphingomyelinase (ASM) activity. Previously, we showed that the storage pathology in the ASM knockout (ASMKO) mouse brain could be corrected by intracerebral injections of cell, gene and protein based therapies. However, except for instances where distal areas were targeted with viral vectors, correction of lysosomal storage pathology was typically limited to a region within a few millimeters from the injection site. As NPA is a global neurometabolic disease, the development of delivery strategies that maximize the distribution of the enzyme throughout the CNS is likely necessary to arrest or delay progression of the disease. To address this challenge, we evaluated the effectiveness of intracerebroventricular (ICV) delivery of recombinant human ASM into ASMKO mice. Our findings showed that ICV delivery of the enzyme led to widespread distribution of the hydrolase throughout the CNS. Moreover, a significant reduction in lysosomal accumulation of sphingomyelin was observed throughout the brain and also within the spinal cord and viscera. Importantly, we demonstrated that repeated ICV infusions of ASM were effective at improving the disease phenotype in the ASMKO mouse as indicated by a partial alleviation of the motor abnormalities. These findings support the continued exploration of ICV delivery of recombinant lysosomal enzymes as a therapeutic modality for LSDs such as NPA that manifests substrate accumulation within the CNS. © 2008 Elsevier Inc. All rights reserved.
Introduction Niemann–Pick A (NPA) disease is a rare lysosomal storage disease (LSD) caused by a deficiency in acid sphingomyelinase (ASM) activity. Loss of ASM activity results in progressive accumulation of sphingomyelin (SPM) and secondary metabolites such as cholesterol in the lysosomes. These aberrations lead to cellular dysfunction in various organ systems including the central nervous system (CNS) which in turn lead to death by early childhood (Brady et al., 1966; Leventhal et al., 2001; Schuchman, 2001). Enzyme replacement therapy (ERT) by intravenous infusion of the normal, active enzyme has been successfully implemented to treat the visceral disease of a number of different LSDs (Amalfitano et al., 2001; Barton et al., 1991; Barton et al., 1990; Harmatz et al., 2004; Kakkis et al., 2001; Schiffmann et al., 2001). However, LSDs that exhibit storage accumulation within the CNS such as NPA are generally unresponsive to this mode of therapy due to the
⁎ Corresponding author. Fax: +1 508 271 4776. E-mail address: [email protected] (J.C. Dodge). 0014-4886/$ – see front matter © 2008 Elsevier Inc. All rights reserved. doi:10.1016/j.expneurol.2008.10.021
inability of the enzyme to traverse the blood brain barrier (Miranda et al., 2000; Sly and Vogler, 2002). Indeed, studies of intravenous administrations of human ASM (hASM) into a mouse model of NPA disease (ASMKO mice) showed therapeutic correction of the storage pathology in the visceral organs but not the brain (Miranda et al., 2000). Recently, it has been reported that intracerebral injections of gene, cell and protein based therapies to the ASMKO mouse can reduce the burden of SPM accumulation in the CNS (Dodge et al., 2005; Jin et al., 2002; Jin and Schuchman, 2003; Passini et al., 2007; Passini et al., 2005; Shihabuddin et al., 2004; Yang et al., 2007). However, except for instances where distal regions were targeted with viral vectors, correction of lysosomal storage pathology was typically restricted to an area within a few millimeters from the site of injection. As NPA is a global neurometabolic disease, the development of treatment strategies that result in widespread correction of the disease pathology in the CNS are likely optimal to address this disease. One potential approach to enhance enzyme distribution within the CNS is to exploit the natural flow of cerebrospinal fluid (CSF) throughout the CNS following either intrathecal or ICV delivery. Both injection strategies reportedly are effective in facilitating distribution of lysosomal enzymes within the CNS of animal models
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of LSDs to reduce storage pathology (Chang et al., 2008; Hemsley et al., 2007; Kakkis et al., 2004; Lee et al., 2007). Successful penetration of active enzyme into surrounding brain parenchyma of LSD patients, however, will likely be dependent upon the infusion parameters used during enzyme delivery. For example, an attempt to alter the storage disease in the CNS of Gaucher patients by administering a bolus ICV injection of alglucerase was unsuccessful (Bembi et al., 1995). Whether or not a continuous ICV infusion approach could be used to achieve widespread distribution of a lysosomal enzyme within the CNS for the treatment of LSDs such as NPA remains unknown and warrants further investigation. In our current experiments, therefore, we examined the distribution of hASM in the CNS of ASMKO mouse following continuous ICV infusion of the enzyme over a 6 h period. In addition to evaluating the impact of ICV enzyme infusion on storage accumulation within the ASMKO mouse brain, we also assessed for the first time in an animal model of LSD whether ICV infusion of enzyme could be applied to treating the disease in the spinal cord and visceral organs. Furthermore, we also determined the rate of substrate re-accumulation following treatment and assessed the effect of recurring ICV infusions of hASM on disease progression in ASMKO mice. Our results showed that ICV administration of hASM into ASMKO mice led to widespread enzyme distribution throughout the CNS and to a significant reduction in SPM Levels in both the CNS and visceral organs. Our findings also showed for the first time that repeated ICV infusions of enzyme were able to partially improve the disease phenotype in an animal model of LSD. Methods Animals The ASMKO mouse model was established by gene targeting as described previously (Horinouchi et al., 1995). Animals were maintained under a 12 h/12 h light/dark cycle and given water and food ad libitum. All procedures were accomplished using a protocol approved by the Institutional Animal Care and Use Committee. Stereotaxic surgery Mice were anesthetized with 3% isofluorane and placed in a stereotaxic frame for placement of an indwelling guide cannula (Plastics One, Roanoke, VA) to a position that was 1 mm dorsal to the right lateral ventricle (A–P: −.4 from bregma, M–L: −1.0 from bregma, D–V: − 1.05 from dura, incisor bar: 0.0). Guide cannulas were permanently affixed to the skull using anchor screws (CMA Microdialysis Inc., North Chelmsford, MA) and dental acrylic (CMA Microdialysis Inc., North Chelmsford, MA). A dummy cannula (Plastics One, Roanoke, VA) was inserted into the guide cannula to maintain cannula patency prior to insertion of the infusion probe. One hour before and twenty-four hours after surgery mice were given ketoprofen (5 mg/kg; SC) for analgesia. Intracerebroventricular infusion On the day of enzyme infusion, the dummy cannula was removed and replaced with an infusion probe (Plastics One, Roanoke, VA) that extended 1 mm ventral to the tip of the guide cannula. Infusion probes were connected to a swivel (Instech Laboratories Inc., Plymouth Meeting, PA) with a tubing that permitted 360°of movement during the infusion procedure. Swivels were connected with tubing to a Hamilton gastight syringe mounted on a Harvard Apparatus infusion pump (Harvard Apparatus, Holliston, Massachusetts) set to deliver enzyme (at variable concentrations depending on the experiment) at a rate of 5 μl/h for up to 6 h. Upon completion of the infusion procedure, mice were returned to their cages, the
infusion probes were then removed and replaced with dummy cannulas. Animal perfusion and tissue collection Animals were killed according to a humane protocol approved by the Institutional Animal Care and Use Committee. Animals destined for biochemical analysis were perfused through the heart with phosphate-buffered saline (PBS) to remove all blood. Brain, spinal cord, liver and lung tissue were harvested and snap frozen in liquid nitrogen as reported (Dodge et al., 2005; Passini et al., 2007). Prior to being snap frozen, brain tissue was cut transversely into five sections (S1, S2, S3, S4 and S5) using a mouse brain matrix (ASI Instruments, Inc., MI). Sections 1 to 4 were approximately 2 mm apart from each other with S1 being the most rostral and S4 the most caudal. Section 5 (S5) contained the cerebellum and brainstem. Animals destined for histological analyses were perfused with 4% paraformaldehyde, and the tissues sectioned on a vibratome as reported previously (Shihabuddin et al., 2004). Histological and biochemical assays Brain sections were stained for hASM with a biotinylated antihASM monoclonal antibody (Genzyme, Framingham, MA) at a dilution of 1:200 and visualized as reported (Shihabuddin et al., 2004). Cholesterol was detected in brain sections with a filipin-staining protocol (Shihabuddin et al., 2004). Quantification of SPM levels in tissue samples was performed using the Amplex Red sphingomyelinase kit (Molecular Probes, Eugene, OR), as described previously (Shihabuddin et al., 2004). Briefly, SPM was hydrolyzed by the bacterial enzyme to yield ceramide and phosphorylcholine. The latter was further hydrolyzed to choline, which in turn was oxidized to betaine and hydrogen peroxide. The released hydrogen peroxide was quantitated by reacting with Amplex Red to generate a highly fluorescent resorufin that could be detected by fluorescence emission at 590 nm. Purified SPM C18 (Matreya, Pleasant Gap, PA) was used as a standard. An ELISA specific for hASM (Genzyme, Framingham, MA) was used to quantify the level of the enzyme in serum samples as reported (Dodge et al., 2005). Foot fault, gait analysis and grip strength tests Foot placement coordination was assessed using a foot misplacement apparatus (Columbus Instruments, Columbus, OH) that is comprised of a stainless steel horizontal ladder with rungs spaced to accommodate traversing by mice. At one end of the ladder, a dark chamber is placed to entice mice to walk toward an area of perceived “safety” offered by the cover of darkness. After the animal is trained to walk toward the dark chamber, an actual test is performed by placing the animal on one end of the ladder, and counting the number of missteps it makes as it moves toward the dark compartment at the other end of the ladder. Each mouse is given 2 test trials and the average number of missteps between the two trials is reported. Gait analysis was conducted by collecting digital images at 80 frames per second with a high-speed video camera located beneath a transparent treadmill moving at a speed of 10 cm/s (Digi-Gait Imaging Systems, Boston, MA) as reported (Cabrera-Salazar et al., 2007). Hindlimb grip strength was measured using a grip strength meter (Columbus Instruments) as reported (Dodge et al., 2008). Statistics Latency to fall on the rotarod, hASM protein levels, and SPM levels were analyzed with separate one way ANOVAs. Follow-up analyses were conducted with Bonferroni post-hoc test where appropriate. All values were considered significant if p b 0.05.
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Results ICV infusion of hASM into ASMKO mice resulted in widespread biodistribution of the enzyme in the brain Fourteen week-old ASMKO mice (n = 24) received an ICV infusion of either hASM (0.025 mg/mouse) or artificial cerebrospinal fluid (aCSF) and then sacrificed at the following time intervals after infusion (n = 3 mice/treatment/time point): immediately, 24 h, 1 week, 2 weeks and 3 weeks. Fourteen week-old mice were used because previous histological analysis had shown that ASMKO mice display significant storage pathology within the CNS by this age (Passini et al., 2005). Positive staining for hASM was observed throughout the brains of ASMKO mice administered hASM but not aCSF at 24 h post-treatment (Fig. 1A; S1 = most rostral section and S5 = most caudal section). Areas within S2 (site of cannula placement) and in S1 and S3 (which were adjacent to the site of infusion) displayed the strongest hASM staining. Regions that were more caudal, such as S5, showed less intense staining for hASM. The signals observed in mice that were killed immediately after infusion appeared more diffused (Fig. 1B) compared to those that were processed at later time points. The staining pattern in the brains of mice killed 24 h post-infusion appeared intracellular, presumably in the lysosomal compartment. Positive intracellular hASM staining in the cortex and hippocampal regions were still evident at 3 weeks post infusion (Fig. 1B).
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ICV infusion of hASM reduced sphingomyelin levels in the brain and visceral organs of ASMKO mice To evaluate the effect of ICV infusion of hASM on SPM levels, 14 week-old ASMKO mice (n = 7 animals/dose) were administered the following doses of hASM/mouse: 0.250 mg, 0.025 mg and 0.0075 mg. As controls, a cohort of ASMKO (n = 7) and another of wild type mice (n = 7) were infused with aCSF. Brain tissues were collected from the mice at 7 days post infusion, divided into 5 sections along the rostral caudal axis and analyzed for SPM levels. As CSF circulates back into venous circulation (Fig. 2D), we also analyzed SPM levels in the visceral tissues (i.e., liver and lung). Amounts of hASM, levels of which were dose-dependent, could be detected in the serum of the hASMtreated mice using an ELISA (Fig. 2D). Our results showed that ICV infusion of the highest dose of hASM (0.250 mg/mouse) led to a significant (p b 0.05) reduction in SPM levels throughout the entire brain (Fig. 2A). The SPM levels in the ASMKO mice treated at this high dose were reduced to those observed in otherwise normal and healthy wild type (WT) animals. ASMKO mice treated with the middle (0.025 mg/mouse) and lowest (0.0075 mg/mouse) doses also showed a significant (p b 0.05) reduction in SPM levels throughout the brain. However, at the lowest dose tested, SPM levels in the most caudal section (S5) were not significantly reduced (Fig. 2A). This finding is in agreement with the observation noted above showing more limited hASM distribution in brain sections that were most caudal to the
Fig. 1. (A) Distribution of hASM staining (1× magnification) in the ASMKO mouse brain following a 6 h intraventricular infusion of hASM or aCSF (S1 = most rostral section and S5 = most caudal section; cannula placement was in S2). Positive hASM staining was observed throughout the brain following treatment with hASM. (B) Time course analysis of hASM staining (20× magnification) in the ASMKO mouse brain. Shown are sections from the motor cortex and CA3 region of the hippocampus. Immediately after infusion hASM staining appeared to be diffuse; however, 24 h post infusion, intracellular accumulation of hASM became evident. Positive intracellular hASM staining was still present 3 weeks post infusion.
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Fig. 2. CNS and visceral sphingomyelin (SPM) levels in ASMKO mice following a 6 h intraventricular infusion of hASM at different doses. Shown are SPM levels for (A) brain (S1 = most rostral brain section and S5 = most caudal brain section), (B) liver and (C) lung tissue. ⁎Significantly different from untreated ASMKO (KO) tissue (p b 0.05). SPM levels were reduced to that of wild type (WT) control levels throughout the brain (S1–S5) in mice infused with 0.25 mg or 0.025 mg of hASM. Brain SPM levels in mice treated with 0.0075 mg of hASM showed significant reductions in S1–S4, but not in S5. Brain SPM levels in mice treated with 0.0075 mg hASM were significantly reduced, but not to WT levels. In the liver and lung, SPM levels were reduced to that of WT control tissue levels at the 0.25 mg dose. In mice treated with 0.025 mg of hASM, significant reductions in liver and lung SPM levels were also observed; however, SPM levels were significantly higher than WT tissue levels. (D) Serum levels of hASM following intracerebroventricular infusion of hASM.
Fig. 3. Time course analysis of filipin staining (20× magnification) in the CNS following a 6 h intracerebroventricular infusion of hASM (0.025 mg/mouse). Shown are sections stained with filipin from forebrain, hippocampus and lumbar spinal cord.
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Fig. 4. Rate of sphingomyelin (SPM) re-accumulation in the CNS and viscera of ASMKO mice following a 6 h intracerebroventricular infusion of hASM (0.025 mg/mouse). Shown are rates of SPM re-accumulation for (A) brain (S1 = most rostral brain section and S5 = most caudal brain section), (B) spinal cord, (C) liver and (D) lung. ⁎Significantly different from untreated ASMKO (KO) tissue (p b 0.05).
infusion site (Fig. 1A). As hASM levels were detected in the serum of enzyme-treated ASMKO mice (Fig. 2D), there were also measurable dose-dependent reductions in SPM levels (p b 0.05) in both the liver and lung (Figs. 2B and C). At the highest dose tested, SPM levels in the liver and lung of ASMKO mice were reduced to those observed in wild type mice. ASMKO mice treated with the mid dose of hASM also showed significant (p b 0.05) reductions in SPM in both organs; however, in ASMKO mice treated at the lowest dose, only the liver showed a significant (p b 0.05) reduction in SPM. ICV infusion of hASM reduced the aberrant lysosomal accumulation of cholesterol in ASMKO mice Fourteen week-old ASMKO mice (n = 12) were infused with either hASM (0.025 mg/mouse) or aCSF (n = 12) and then killed at the following time intervals post-infusion (n = 3 mice/treatment/time
point): immediately, 24 h, 1 week, 2 weeks and 3 weeks. To assess cholesterol levels, ASMKO mouse brains were stained with filipin, an autofluorescent molecule isolated from Streptomyces filipinensis (Bergy and Eble, 1968). CNS tissue from aCSF-treated ASMKO mice showed the highest level of filipin-stained cholesterol deposits (Fig. 3). By contrast, a reduction in the number and intensity of filipin-stained cells were observed within 24 h post-treatment with hASM in both brain and spinal cord tissue. Analysis of hASM-treated mice after 3 weeks showed indications of re-accumulation of cholesterol to levels noted in untreated ASMKO mice (data not shown). Rate of re-accumulation of SPM in the CNS of ASMKO mice following a single ICV infusion of hASM Prior to testing the therapeutic efficacy of repeated ICV infusions of hASM in ASMKO mice, we sought to determine the rate of SPM re-
Fig. 5. Rate of sphingomyelin accumulation in the CNS and viscera of ASMKO mice as function of age. Shown are SPM levels for brain, spinal cord, liver and lung. Regardless of tissue analyzed, significant SPM accumulation was apparent by 6 weeks of age and continued to accumulate until 30 weeks of age. ⁎Significantly different from wild type control tissue (p b 0.05).
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accumulation following a single administration to determine the optimal interval for readministration. Fourteen week-old ASMKO mice (n = 14 mice/time point) were administered 0.025 mg hASM/mouse by ICV injection and then killed 1, 2 and 3 weeks later for assessment of SPM levels. ASMKO and age-matched WT mice (n = 14 mice/group) were also infused with aCSF and sacrificed at 1 week and 3 weeks post infusion. Fig. 4A shows that ICV infusion of hASM led to a significant reduction in SPM levels (p b 0.05) throughout the brain when they were analyzed at 1 and 2 weeks post-treatment. After 3 weeks, levels of SPM were still reduced in most regions of the brain except for the most caudal region (S5), which showed re-accumulation to levels noted in untreated ASMKO mice (Fig. 4A). In the spinal cord, a similar pattern of reaccumulation of the substrate was noted. Significant reductions in SPM levels (p b 0.05) were realized for up to 2 weeks post-treatment but animals analyzed after 3 weeks showed indications of re-accumulation to levels observed in untreated ASMKO mice (Fig. 4B). In the liver and lung, SPM levels were still significantly (p b 0.05) reduced at 3 weeks post infusion (Figs. 4C and D).
Accumulation of sphingomyelin in the CNS and viscera of ASMKO mouse was progressive with age Prior to evaluating the efficacy of repeated ICV infusions of hASM in ASMKO mice we also determined the kinetics of SPM accumulation in the CNS and viscera of ASMKO mice as a function of age. It was hoped that this information would allow for a more informed decision regarding the timing for initiating therapy. To mimic the situation in humans, the intent was to start treatment only after the animals had exhibited significant accumulation of the substrate. Tissues were collected from ASMKO mice (n = 7 animals/time point) at 6, 18, 24 and 30 weeks of age. As controls, tissues were also collected from 6 and 30 week-old wild type mice. We observed significantly higher accumulation of SPM in the CNS and viscera of ASMKO mice than in wild type animals (p b 0.05) as early as 6 weeks of age (Figs. 5A and B). Consequently, we elected six week-old mice as the earliest time point for therapeutic intervention. It should also be noted that for practical reasons the earliest age we were able to perform stereotaxic guide
Fig. 6. CNS sphingomyelin levels and behavioral outcome in ASMKO mice following recurring intracerebroventricular administration of hASM (0.025 mg hASM/mouse administered every 2 weeks starting at 6 weeks of age). Shown are SPM levels in the (A) brain and (B) spinal cord of ASMKO mice that received either recurring intracerebroventricular (ICV) infusions or tail vein injection (IV) of hASM. ⁎Significantly different from untreated ASMKO (KO) tissue (p b 0.05). Regardless of tissue analyzed, significant reductions in SPM were observed in mice that received repeated intracerebroventricular infusions of hASM, but not repeated tail vein injections of hASM. Repeated intracerebroventricular infusions of hASM in ASMKO mice also led to improved function on the (C) foot fault and (D, E) gait analysis tests. ⁎Significantly different from untreated ASMKO (KO) performance (p b 0.05).
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cannula implantation surgery on a mouse was when they were 4 weeks of age. After a 2 week recovery period from surgery, the mice were then subjected to ICV infusion of enzyme (i.e., at 6 weeks of age). Thirty weeks was chosen as the final time point because ASMKO mice were typically deemed moribund at this age and because SPM accumulation was close to maximal (Fig. 5) at this time point (Passini et al., 2007). The CNS showed regional differences in the extent of SPM accumulation with the brain showing greater accumulation than the spinal cord. Differences in visceral SPM accumulation were also apparent with liver tissue showing greater accumulation than lung tissue. Relative to the CNS (depending on the region analyzed) SPM accumulation was 2–5 fold higher in the visceral tissues. Repeated ICV infusions of hASM slowed disease progression in ASMKO mice As SPM and cholesterol levels in the hindbrain and spinal cord reaccumulated back to untreated levels by 3 weeks post-treatment, we elected to evaluate the efficacy of biweekly infusions of hASM at slowing disease progression in ASMKO mice. Specifically, we measured the effect of repeated treatments on tissue SPM levels and on the performance of the ASMKO mice on various behavioral tests (i.e., foot fault, gait analysis and grip strength) designed to assess motor function. ASMKO mice (n = 14) received ICV infusions of hASM (0.025 mg/mouse) starting at 6 weeks of age and every two weeks thereafter for 8 weeks. As controls, a cohort of ASMKO mice (n = 14) was administered aCSF by ICV infusion and another with hASM (0.025 mg/mouse) administered via tail vein injections (n = 14). The latter was included as an experimental group because we wanted to be sure that potential improvements in motor function could be ascribed to the actions of hASM in the CNS and not the visceral organs. Foot fault, gait and hindlimb grip strength testing began when the mice were 7 weeks of age and these tests were repeated every 2 weeks thereafter. All mice were sacrificed at 14 weeks of age for concern that an immune response to the human enzyme may circumvent the ability to measure biochemical efficacy. Analysis of the animals at the end of the study period showed significant reductions in SPM levels in the brain and spinal cord (p b 0.05) in hASM- but not aCSF-treated ASMKO mice (Figs. 6A and B). As expected (due to presence of the BBB) ASMKO mice administered hASM via tail vein injections did not significantly lower the accumulation of SPM in the CNS. Correlated with this observation, ASMKO mice treated with ICV infusions of hASM made significantly (p b 0.05) fewer missteps on the foot fault test at 13 weeks of age when compared to ASMKO mice that received aCSF infusions (Fig. 6C). No significant improvement on the foot fault test was observed in ASMKO mice treated by intravenous administrations of hASM. Gait analysis showed similar significant (p b 0.05) improvements in forelimb and hindlimb stance width in ASMKO mice treated by ICV infusions of hASM but not in those treated by aCSF or those treated by tail vein injections of hASM (Figs. 6D and E). On the grip strength test, however, ASMKO mice treated with hASM regardless of the route of delivery (ICV or tail vein injections) did not show significant improvements in muscle strength (Fig. 6F). Discussion Intravenous infusion of normal, active recombinant enzymes has been successfully implemented to treat a number of different LSDs including Gaucher, Fabry, MPS I, MPS II, MPS VI and Pompe disease (Amalfitano et al., 2001; Barton et al., 1991; Barton et al.,1990; Harmatz et al., 2004; Kakkis et al., 2001; Schiffmann et al., 2001). Although ERT has proven to be effective in reducing substrate accumulation in the visceral organs, the presence of the blood brain barrier has prevented this therapeutic approach from being used to treat LSDs with CNS manifestations such as NPA disease. Recently, intracerebral injection has been used to deliver gene, cell and protein based therapies to the
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CNS of mouse models of LSDs including ASMKO mice in a manner that led to significant reductions in lysosomal accumulation of the substrate (Dodge et al., 2005; Jin et al., 2002; Jin and Schuchman, 2003; Passini et al., 2007; Passini et al., 2005; Shihabuddin et al., 2004; Yang et al., 2007). However, depending on the therapeutic platform used, correction of lysosomal storage accumulation was typically circumscribed to an area of a few millimeters from the sites of injection. Although this limitation could possibly be circumvented in part by using injection strategies that enhance diffusion of the therapeutic agent (Bankiewicz et al., 2000; Bobo et al., 1994; Lonser et al., 2005) or by targeting highly connected regions of the brain to enable vector axonal transport to distal regions (Dodge et al., 2005; Passini et al., 2005), multiple intracerebral injections would likely still be needed to achieve global delivery of the enzyme throughout the CNS (Cabrera-Salazar et al., 2007; Passini et al., 2007). Therefore, we initiated an effort to explore alternative enzyme delivery strategies to address the lysosomal storage pathology within the CNS. Delivery of drugs to the CSF using semi-permanent indwelling catheters has been shown to be a viable approach to address CNS diseases in humans (Anderson and Burchiel, 1999). Moreover, delivery of lysosomal enzymes into the CSF either through intrathecal or ICV injection has been shown to be effective in distributing the enzyme broadly within the brain of animal models of LSDs (Chang et al., 2008; Horinouchi et al., 1995; Kakkis et al., 2001; Lonser et al., 2005). Successful application of this approach to LSD patients, however, will likely be dependent upon the infusion parameters used during enzyme delivery. For example, this may explain why a previous attempt to treat the CNS storage disease in Gaucher patients using a bolus ICV injection of the aglucerase (the enzyme that is deficient in Gaucher disease) was unsuccessful (Bembi et al.,1995). Therefore, in our current experiments we elected to evaluate the efficacy of continuous ICV infusion of hASM over a 6 h period in a mouse model of NPA disease. Following infusion of hASM into the lateral ventricle of ASMKO mice, immunostaining for hASM in brain sections showed bilateral enzyme distribution to widespread regions of the brain including the forebrain, midbrain and hindbrain. Similar patterns of enzyme distribution have been reported following a 2 week continuous ICV infusion of recombinant tripeptidyl peptidase I in a mouse model of late-infantile Batten disease (Chang et al., 2008) and with a bolus ICV injection of galactocerebrosidase into 20 day old twitcher mice (Lee et al., 2007). Our experiments, however, were the first to examine the kinetics of enzyme spread and intracellular uptake within the brain following an ICV infusion. Immediately after infusion, we observed a diffuse pattern of hASM staining throughout the brain with cellular uptake of hASM occurring within 24 h. Interestingly, hASM was still apparent 3 weeks post-infusion suggesting that hASM has a relatively long half-life in cells of the brain parenchyma. Similar to what was reported in a mouse model of Krabbe disease (Lee et al., 2007), ICV infusion of hASM in ASMKO mice also led to a significant reduction in the offending substrate throughout the brain. Our experiment, however, was the first to examine the effectiveness of this therapeutic approach as a function of dose. Treatment with the highest dose reduced substrate levels throughout the brain to those observed in wild type animals. As expected, the lowest dose was least effective in reducing substrate particularly in the regions that were most distal (e.g., hindbrain) to the site of infusion. We also demonstrated for the first time using both biochemical and histological measures that ICV infusion of a lysosomal enzyme was equally effective in reducing accumulated substrate not only in the brain but also within the spinal cord. More specifically, we observed a significant reduction in SPM tissue levels and filipin staining (a stain used to detect cholesterol accumulation, a secondary metabolite that accumulates in NPA disease) throughout the spinal cord. Given that CSF circulates back into venous circulation we also analyzed visceral tissues (i.e., liver and lung) for potential reductions in SPM levels following ICV infusion of hASM. Our results showed for the first time that ICV enzyme infusion
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could also be applied to treating storage pathology observed in the visceral organs of LSDs with CNS pathological manifestations, as we observed a significant reduction in SPM in both liver and lung tissue in a dose dependent manner. This feature of ICV enzyme delivery will likely provide added benefit to NPA patients, as maximal improvements in both function and survival are achieved in ASMKO mice following treatments that address both the visceral and CNS disease (Passini et al., 2007). Prior to evaluating the efficacy of repeated ICV administrations of hASM in ASMKO mice, we sought to characterize the rate of SPM accumulation as a function of age in ASMKO mice and determined the kinetics of SPM re-accumulation following a single ICV hASM infusion. Assessment of SPM accumulation in ASMKO mice as a function of age showed that 6 week-old ASMKO mice displayed significant SPM accumulation in both CNS and visceral tissue. Interestingly, the rate of SPM accumulation was tissue specific with visceral tissues showing significantly greater SPM accumulation than CNS tissue. Within the CNS, SPM accumulation was significantly greater in brain than in spinal cord tissue. Whether or not this phenomenon also occurs in humans is unknown and warrants further investigation. However, since SPM levels in normal mice were similar in all the tissues analyzed this suggests (at least in ASMKO mice) that a tissue's metabolic environment may play an important role in modulating SPM accumulation. More importantly, understanding parameters that govern tissue specific differences in SPM accumulation may lead to the development of adjunct therapies to ERT for the treatment of NPA disease. Nevertheless, our results demonstrated that SPM accumulation was rapid, attaining significant levels by 6 weeks of age in ASMKO mice. Our efforts to examine the rate of SPM re-accumulation following a single ICV infusion of hASM showed that SPM levels returned to those observed in untreated ASMKO mice within 2– 3 weeks post-treatment, particularly in regions that were most distal to the infusion site (e.g., hindbrain and spinal cord). Interestingly, SPM levels in the visceral tissues were still relatively low after 3 weeks post-treatment suggesting that visceral organs were more sensitive to exogenous hASM than the CNS or that the rate of re-accumulation of SPM was slower in the visceral tissues. As SPM in the CNS re-accumulated back to untreated levels by 3 weeks post-treatment, we elected to test the ability of recurring hASM treatments given every 2 weeks starting at 6 weeks of age to slow disease progression in ASMKO mice. Our findings showed that repeated ICV infusions of hASM were effective in reducing SPM accumulation within the CNS and partially improving motor function in ASMKO mice. Specifically, we observed a significant reduction in SPM levels in the brain and spinal cord of ASMKO mice that received recurring ICV infusions of hASM. Animals treated in this manner also showed improved performance on tests designed to assess motor coordination and balance (i.e., foot fault and gait analysis). However, it should be noted that improved muscle strength was not seen using this therapeutic regimen (as measured by the grip strength test) suggesting either inadequate targeting of the enzyme to the diseased muscle or motor neurons. Whether or not this is a limitation of ICV infusion or to the dose used in our current experiment remains to be determined. It was also difficult to compare these results with our previous studies examining the efficacy of gene delivery of hASM. Use of certain combinations of viral vectors and transcriptional cassettes immunotolerized mice to the human lysosomal enzymes and allowed for improved substrate clearance (Barbon et al., 2005; Passini et al., 2007; Ziegler et al., 2007). In addition, as gene therapy facilitated continuous enzyme exposure, this limited substrate re-accumulation and therefore, any potential pathophysiology initiated by cyclic changes (i.e., repeated clearance followed by re-accumulation) in cellular SPM levels. Future experiments examining the merits of ICV infusion of enzyme in mouse models of LSD should be performed under conditions that permit similar immunotolerization to the enzyme and greater exposure of the enzyme (e.g. by continuous delivery of the
enzyme) to assess the relative benefits of these two different technologies. Nevertheless, our results are encouraging as we observed an alleviation of disease phenotype in ASMKO mice even with a limited number of ICV infusions of hASM. In conclusion, the results of our studies illustrate that continuous ICV infusion of hASM is an effective approach to address the pathological manifestations observed in the CNS of a mouse model of NPA disease. Our findings support the continued exploration of ICV delivery of enzymes as a therapeutic modality for LSDs such as NPA that exhibit lysosomal accumulation of substrate within the CNS. Acknowledgments We would like to thank Leah Curtin and the staff at DCM for their technical support.
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Contents lists available at ScienceDirect
Experimental Neurology j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / yex n r
Intracerebroventricular infusion of acid sphingomyelinase corrects CNS manifestations in a mouse model of Niemann–Pick A disease James C. Dodge a,⁎, Jennifer Clarke a, Christopher M. Treleaven a, Tatyana V. Taksir a, Denise A. Griffiths a, Wendy Yang a, Jonathan A. Fidler a, Marco A. Passini a, Kenneth P. Karey a, Edward H. Schuchman b, Seng H. Cheng a, Lamya S. Shihabuddin a a b
Genzyme Corporation, 49 New York Avenue, Framingham, MA 01701, USA Mount Sinai School of Medicine, New York, NY 10029, USA
a r t i c l e
i n f o
Article history: Received 15 September 2008 Revised 29 October 2008 Accepted 30 October 2008 Available online 14 November 2008 Keywords: Intracerebroventricular delivery Acid sphingomyelinase Sphingomyelin Lysosomal storage disease Acid sphingomyelinase knockout mouse
a b s t r a c t Niemann–Pick A (NPA) disease is a lysosomal storage disorder (LSD) caused by a deficiency in acid sphingomyelinase (ASM) activity. Previously, we showed that the storage pathology in the ASM knockout (ASMKO) mouse brain could be corrected by intracerebral injections of cell, gene and protein based therapies. However, except for instances where distal areas were targeted with viral vectors, correction of lysosomal storage pathology was typically limited to a region within a few millimeters from the injection site. As NPA is a global neurometabolic disease, the development of delivery strategies that maximize the distribution of the enzyme throughout the CNS is likely necessary to arrest or delay progression of the disease. To address this challenge, we evaluated the effectiveness of intracerebroventricular (ICV) delivery of recombinant human ASM into ASMKO mice. Our findings showed that ICV delivery of the enzyme led to widespread distribution of the hydrolase throughout the CNS. Moreover, a significant reduction in lysosomal accumulation of sphingomyelin was observed throughout the brain and also within the spinal cord and viscera. Importantly, we demonstrated that repeated ICV infusions of ASM were effective at improving the disease phenotype in the ASMKO mouse as indicated by a partial alleviation of the motor abnormalities. These findings support the continued exploration of ICV delivery of recombinant lysosomal enzymes as a therapeutic modality for LSDs such as NPA that manifests substrate accumulation within the CNS. © 2008 Elsevier Inc. All rights reserved.
Introduction Niemann–Pick A (NPA) disease is a rare lysosomal storage disease (LSD) caused by a deficiency in acid sphingomyelinase (ASM) activity. Loss of ASM activity results in progressive accumulation of sphingomyelin (SPM) and secondary metabolites such as cholesterol in the lysosomes. These aberrations lead to cellular dysfunction in various organ systems including the central nervous system (CNS) which in turn lead to death by early childhood (Brady et al., 1966; Leventhal et al., 2001; Schuchman, 2001). Enzyme replacement therapy (ERT) by intravenous infusion of the normal, active enzyme has been successfully implemented to treat the visceral disease of a number of different LSDs (Amalfitano et al., 2001; Barton et al., 1991; Barton et al., 1990; Harmatz et al., 2004; Kakkis et al., 2001; Schiffmann et al., 2001). However, LSDs that exhibit storage accumulation within the CNS such as NPA are generally unresponsive to this mode of therapy due to the
⁎ Corresponding author. Fax: +1 508 271 4776. E-mail address: [email protected] (J.C. Dodge). 0014-4886/$ – see front matter © 2008 Elsevier Inc. All rights reserved. doi:10.1016/j.expneurol.2008.10.021
inability of the enzyme to traverse the blood brain barrier (Miranda et al., 2000; Sly and Vogler, 2002). Indeed, studies of intravenous administrations of human ASM (hASM) into a mouse model of NPA disease (ASMKO mice) showed therapeutic correction of the storage pathology in the visceral organs but not the brain (Miranda et al., 2000). Recently, it has been reported that intracerebral injections of gene, cell and protein based therapies to the ASMKO mouse can reduce the burden of SPM accumulation in the CNS (Dodge et al., 2005; Jin et al., 2002; Jin and Schuchman, 2003; Passini et al., 2007; Passini et al., 2005; Shihabuddin et al., 2004; Yang et al., 2007). However, except for instances where distal regions were targeted with viral vectors, correction of lysosomal storage pathology was typically restricted to an area within a few millimeters from the site of injection. As NPA is a global neurometabolic disease, the development of treatment strategies that result in widespread correction of the disease pathology in the CNS are likely optimal to address this disease. One potential approach to enhance enzyme distribution within the CNS is to exploit the natural flow of cerebrospinal fluid (CSF) throughout the CNS following either intrathecal or ICV delivery. Both injection strategies reportedly are effective in facilitating distribution of lysosomal enzymes within the CNS of animal models
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of LSDs to reduce storage pathology (Chang et al., 2008; Hemsley et al., 2007; Kakkis et al., 2004; Lee et al., 2007). Successful penetration of active enzyme into surrounding brain parenchyma of LSD patients, however, will likely be dependent upon the infusion parameters used during enzyme delivery. For example, an attempt to alter the storage disease in the CNS of Gaucher patients by administering a bolus ICV injection of alglucerase was unsuccessful (Bembi et al., 1995). Whether or not a continuous ICV infusion approach could be used to achieve widespread distribution of a lysosomal enzyme within the CNS for the treatment of LSDs such as NPA remains unknown and warrants further investigation. In our current experiments, therefore, we examined the distribution of hASM in the CNS of ASMKO mouse following continuous ICV infusion of the enzyme over a 6 h period. In addition to evaluating the impact of ICV enzyme infusion on storage accumulation within the ASMKO mouse brain, we also assessed for the first time in an animal model of LSD whether ICV infusion of enzyme could be applied to treating the disease in the spinal cord and visceral organs. Furthermore, we also determined the rate of substrate re-accumulation following treatment and assessed the effect of recurring ICV infusions of hASM on disease progression in ASMKO mice. Our results showed that ICV administration of hASM into ASMKO mice led to widespread enzyme distribution throughout the CNS and to a significant reduction in SPM Levels in both the CNS and visceral organs. Our findings also showed for the first time that repeated ICV infusions of enzyme were able to partially improve the disease phenotype in an animal model of LSD. Methods Animals The ASMKO mouse model was established by gene targeting as described previously (Horinouchi et al., 1995). Animals were maintained under a 12 h/12 h light/dark cycle and given water and food ad libitum. All procedures were accomplished using a protocol approved by the Institutional Animal Care and Use Committee. Stereotaxic surgery Mice were anesthetized with 3% isofluorane and placed in a stereotaxic frame for placement of an indwelling guide cannula (Plastics One, Roanoke, VA) to a position that was 1 mm dorsal to the right lateral ventricle (A–P: −.4 from bregma, M–L: −1.0 from bregma, D–V: − 1.05 from dura, incisor bar: 0.0). Guide cannulas were permanently affixed to the skull using anchor screws (CMA Microdialysis Inc., North Chelmsford, MA) and dental acrylic (CMA Microdialysis Inc., North Chelmsford, MA). A dummy cannula (Plastics One, Roanoke, VA) was inserted into the guide cannula to maintain cannula patency prior to insertion of the infusion probe. One hour before and twenty-four hours after surgery mice were given ketoprofen (5 mg/kg; SC) for analgesia. Intracerebroventricular infusion On the day of enzyme infusion, the dummy cannula was removed and replaced with an infusion probe (Plastics One, Roanoke, VA) that extended 1 mm ventral to the tip of the guide cannula. Infusion probes were connected to a swivel (Instech Laboratories Inc., Plymouth Meeting, PA) with a tubing that permitted 360°of movement during the infusion procedure. Swivels were connected with tubing to a Hamilton gastight syringe mounted on a Harvard Apparatus infusion pump (Harvard Apparatus, Holliston, Massachusetts) set to deliver enzyme (at variable concentrations depending on the experiment) at a rate of 5 μl/h for up to 6 h. Upon completion of the infusion procedure, mice were returned to their cages, the
infusion probes were then removed and replaced with dummy cannulas. Animal perfusion and tissue collection Animals were killed according to a humane protocol approved by the Institutional Animal Care and Use Committee. Animals destined for biochemical analysis were perfused through the heart with phosphate-buffered saline (PBS) to remove all blood. Brain, spinal cord, liver and lung tissue were harvested and snap frozen in liquid nitrogen as reported (Dodge et al., 2005; Passini et al., 2007). Prior to being snap frozen, brain tissue was cut transversely into five sections (S1, S2, S3, S4 and S5) using a mouse brain matrix (ASI Instruments, Inc., MI). Sections 1 to 4 were approximately 2 mm apart from each other with S1 being the most rostral and S4 the most caudal. Section 5 (S5) contained the cerebellum and brainstem. Animals destined for histological analyses were perfused with 4% paraformaldehyde, and the tissues sectioned on a vibratome as reported previously (Shihabuddin et al., 2004). Histological and biochemical assays Brain sections were stained for hASM with a biotinylated antihASM monoclonal antibody (Genzyme, Framingham, MA) at a dilution of 1:200 and visualized as reported (Shihabuddin et al., 2004). Cholesterol was detected in brain sections with a filipin-staining protocol (Shihabuddin et al., 2004). Quantification of SPM levels in tissue samples was performed using the Amplex Red sphingomyelinase kit (Molecular Probes, Eugene, OR), as described previously (Shihabuddin et al., 2004). Briefly, SPM was hydrolyzed by the bacterial enzyme to yield ceramide and phosphorylcholine. The latter was further hydrolyzed to choline, which in turn was oxidized to betaine and hydrogen peroxide. The released hydrogen peroxide was quantitated by reacting with Amplex Red to generate a highly fluorescent resorufin that could be detected by fluorescence emission at 590 nm. Purified SPM C18 (Matreya, Pleasant Gap, PA) was used as a standard. An ELISA specific for hASM (Genzyme, Framingham, MA) was used to quantify the level of the enzyme in serum samples as reported (Dodge et al., 2005). Foot fault, gait analysis and grip strength tests Foot placement coordination was assessed using a foot misplacement apparatus (Columbus Instruments, Columbus, OH) that is comprised of a stainless steel horizontal ladder with rungs spaced to accommodate traversing by mice. At one end of the ladder, a dark chamber is placed to entice mice to walk toward an area of perceived “safety” offered by the cover of darkness. After the animal is trained to walk toward the dark chamber, an actual test is performed by placing the animal on one end of the ladder, and counting the number of missteps it makes as it moves toward the dark compartment at the other end of the ladder. Each mouse is given 2 test trials and the average number of missteps between the two trials is reported. Gait analysis was conducted by collecting digital images at 80 frames per second with a high-speed video camera located beneath a transparent treadmill moving at a speed of 10 cm/s (Digi-Gait Imaging Systems, Boston, MA) as reported (Cabrera-Salazar et al., 2007). Hindlimb grip strength was measured using a grip strength meter (Columbus Instruments) as reported (Dodge et al., 2008). Statistics Latency to fall on the rotarod, hASM protein levels, and SPM levels were analyzed with separate one way ANOVAs. Follow-up analyses were conducted with Bonferroni post-hoc test where appropriate. All values were considered significant if p b 0.05.
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Results ICV infusion of hASM into ASMKO mice resulted in widespread biodistribution of the enzyme in the brain Fourteen week-old ASMKO mice (n = 24) received an ICV infusion of either hASM (0.025 mg/mouse) or artificial cerebrospinal fluid (aCSF) and then sacrificed at the following time intervals after infusion (n = 3 mice/treatment/time point): immediately, 24 h, 1 week, 2 weeks and 3 weeks. Fourteen week-old mice were used because previous histological analysis had shown that ASMKO mice display significant storage pathology within the CNS by this age (Passini et al., 2005). Positive staining for hASM was observed throughout the brains of ASMKO mice administered hASM but not aCSF at 24 h post-treatment (Fig. 1A; S1 = most rostral section and S5 = most caudal section). Areas within S2 (site of cannula placement) and in S1 and S3 (which were adjacent to the site of infusion) displayed the strongest hASM staining. Regions that were more caudal, such as S5, showed less intense staining for hASM. The signals observed in mice that were killed immediately after infusion appeared more diffused (Fig. 1B) compared to those that were processed at later time points. The staining pattern in the brains of mice killed 24 h post-infusion appeared intracellular, presumably in the lysosomal compartment. Positive intracellular hASM staining in the cortex and hippocampal regions were still evident at 3 weeks post infusion (Fig. 1B).
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ICV infusion of hASM reduced sphingomyelin levels in the brain and visceral organs of ASMKO mice To evaluate the effect of ICV infusion of hASM on SPM levels, 14 week-old ASMKO mice (n = 7 animals/dose) were administered the following doses of hASM/mouse: 0.250 mg, 0.025 mg and 0.0075 mg. As controls, a cohort of ASMKO (n = 7) and another of wild type mice (n = 7) were infused with aCSF. Brain tissues were collected from the mice at 7 days post infusion, divided into 5 sections along the rostral caudal axis and analyzed for SPM levels. As CSF circulates back into venous circulation (Fig. 2D), we also analyzed SPM levels in the visceral tissues (i.e., liver and lung). Amounts of hASM, levels of which were dose-dependent, could be detected in the serum of the hASMtreated mice using an ELISA (Fig. 2D). Our results showed that ICV infusion of the highest dose of hASM (0.250 mg/mouse) led to a significant (p b 0.05) reduction in SPM levels throughout the entire brain (Fig. 2A). The SPM levels in the ASMKO mice treated at this high dose were reduced to those observed in otherwise normal and healthy wild type (WT) animals. ASMKO mice treated with the middle (0.025 mg/mouse) and lowest (0.0075 mg/mouse) doses also showed a significant (p b 0.05) reduction in SPM levels throughout the brain. However, at the lowest dose tested, SPM levels in the most caudal section (S5) were not significantly reduced (Fig. 2A). This finding is in agreement with the observation noted above showing more limited hASM distribution in brain sections that were most caudal to the
Fig. 1. (A) Distribution of hASM staining (1× magnification) in the ASMKO mouse brain following a 6 h intraventricular infusion of hASM or aCSF (S1 = most rostral section and S5 = most caudal section; cannula placement was in S2). Positive hASM staining was observed throughout the brain following treatment with hASM. (B) Time course analysis of hASM staining (20× magnification) in the ASMKO mouse brain. Shown are sections from the motor cortex and CA3 region of the hippocampus. Immediately after infusion hASM staining appeared to be diffuse; however, 24 h post infusion, intracellular accumulation of hASM became evident. Positive intracellular hASM staining was still present 3 weeks post infusion.
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Fig. 2. CNS and visceral sphingomyelin (SPM) levels in ASMKO mice following a 6 h intraventricular infusion of hASM at different doses. Shown are SPM levels for (A) brain (S1 = most rostral brain section and S5 = most caudal brain section), (B) liver and (C) lung tissue. ⁎Significantly different from untreated ASMKO (KO) tissue (p b 0.05). SPM levels were reduced to that of wild type (WT) control levels throughout the brain (S1–S5) in mice infused with 0.25 mg or 0.025 mg of hASM. Brain SPM levels in mice treated with 0.0075 mg of hASM showed significant reductions in S1–S4, but not in S5. Brain SPM levels in mice treated with 0.0075 mg hASM were significantly reduced, but not to WT levels. In the liver and lung, SPM levels were reduced to that of WT control tissue levels at the 0.25 mg dose. In mice treated with 0.025 mg of hASM, significant reductions in liver and lung SPM levels were also observed; however, SPM levels were significantly higher than WT tissue levels. (D) Serum levels of hASM following intracerebroventricular infusion of hASM.
Fig. 3. Time course analysis of filipin staining (20× magnification) in the CNS following a 6 h intracerebroventricular infusion of hASM (0.025 mg/mouse). Shown are sections stained with filipin from forebrain, hippocampus and lumbar spinal cord.
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Fig. 4. Rate of sphingomyelin (SPM) re-accumulation in the CNS and viscera of ASMKO mice following a 6 h intracerebroventricular infusion of hASM (0.025 mg/mouse). Shown are rates of SPM re-accumulation for (A) brain (S1 = most rostral brain section and S5 = most caudal brain section), (B) spinal cord, (C) liver and (D) lung. ⁎Significantly different from untreated ASMKO (KO) tissue (p b 0.05).
infusion site (Fig. 1A). As hASM levels were detected in the serum of enzyme-treated ASMKO mice (Fig. 2D), there were also measurable dose-dependent reductions in SPM levels (p b 0.05) in both the liver and lung (Figs. 2B and C). At the highest dose tested, SPM levels in the liver and lung of ASMKO mice were reduced to those observed in wild type mice. ASMKO mice treated with the mid dose of hASM also showed significant (p b 0.05) reductions in SPM in both organs; however, in ASMKO mice treated at the lowest dose, only the liver showed a significant (p b 0.05) reduction in SPM. ICV infusion of hASM reduced the aberrant lysosomal accumulation of cholesterol in ASMKO mice Fourteen week-old ASMKO mice (n = 12) were infused with either hASM (0.025 mg/mouse) or aCSF (n = 12) and then killed at the following time intervals post-infusion (n = 3 mice/treatment/time
point): immediately, 24 h, 1 week, 2 weeks and 3 weeks. To assess cholesterol levels, ASMKO mouse brains were stained with filipin, an autofluorescent molecule isolated from Streptomyces filipinensis (Bergy and Eble, 1968). CNS tissue from aCSF-treated ASMKO mice showed the highest level of filipin-stained cholesterol deposits (Fig. 3). By contrast, a reduction in the number and intensity of filipin-stained cells were observed within 24 h post-treatment with hASM in both brain and spinal cord tissue. Analysis of hASM-treated mice after 3 weeks showed indications of re-accumulation of cholesterol to levels noted in untreated ASMKO mice (data not shown). Rate of re-accumulation of SPM in the CNS of ASMKO mice following a single ICV infusion of hASM Prior to testing the therapeutic efficacy of repeated ICV infusions of hASM in ASMKO mice, we sought to determine the rate of SPM re-
Fig. 5. Rate of sphingomyelin accumulation in the CNS and viscera of ASMKO mice as function of age. Shown are SPM levels for brain, spinal cord, liver and lung. Regardless of tissue analyzed, significant SPM accumulation was apparent by 6 weeks of age and continued to accumulate until 30 weeks of age. ⁎Significantly different from wild type control tissue (p b 0.05).
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accumulation following a single administration to determine the optimal interval for readministration. Fourteen week-old ASMKO mice (n = 14 mice/time point) were administered 0.025 mg hASM/mouse by ICV injection and then killed 1, 2 and 3 weeks later for assessment of SPM levels. ASMKO and age-matched WT mice (n = 14 mice/group) were also infused with aCSF and sacrificed at 1 week and 3 weeks post infusion. Fig. 4A shows that ICV infusion of hASM led to a significant reduction in SPM levels (p b 0.05) throughout the brain when they were analyzed at 1 and 2 weeks post-treatment. After 3 weeks, levels of SPM were still reduced in most regions of the brain except for the most caudal region (S5), which showed re-accumulation to levels noted in untreated ASMKO mice (Fig. 4A). In the spinal cord, a similar pattern of reaccumulation of the substrate was noted. Significant reductions in SPM levels (p b 0.05) were realized for up to 2 weeks post-treatment but animals analyzed after 3 weeks showed indications of re-accumulation to levels observed in untreated ASMKO mice (Fig. 4B). In the liver and lung, SPM levels were still significantly (p b 0.05) reduced at 3 weeks post infusion (Figs. 4C and D).
Accumulation of sphingomyelin in the CNS and viscera of ASMKO mouse was progressive with age Prior to evaluating the efficacy of repeated ICV infusions of hASM in ASMKO mice we also determined the kinetics of SPM accumulation in the CNS and viscera of ASMKO mice as a function of age. It was hoped that this information would allow for a more informed decision regarding the timing for initiating therapy. To mimic the situation in humans, the intent was to start treatment only after the animals had exhibited significant accumulation of the substrate. Tissues were collected from ASMKO mice (n = 7 animals/time point) at 6, 18, 24 and 30 weeks of age. As controls, tissues were also collected from 6 and 30 week-old wild type mice. We observed significantly higher accumulation of SPM in the CNS and viscera of ASMKO mice than in wild type animals (p b 0.05) as early as 6 weeks of age (Figs. 5A and B). Consequently, we elected six week-old mice as the earliest time point for therapeutic intervention. It should also be noted that for practical reasons the earliest age we were able to perform stereotaxic guide
Fig. 6. CNS sphingomyelin levels and behavioral outcome in ASMKO mice following recurring intracerebroventricular administration of hASM (0.025 mg hASM/mouse administered every 2 weeks starting at 6 weeks of age). Shown are SPM levels in the (A) brain and (B) spinal cord of ASMKO mice that received either recurring intracerebroventricular (ICV) infusions or tail vein injection (IV) of hASM. ⁎Significantly different from untreated ASMKO (KO) tissue (p b 0.05). Regardless of tissue analyzed, significant reductions in SPM were observed in mice that received repeated intracerebroventricular infusions of hASM, but not repeated tail vein injections of hASM. Repeated intracerebroventricular infusions of hASM in ASMKO mice also led to improved function on the (C) foot fault and (D, E) gait analysis tests. ⁎Significantly different from untreated ASMKO (KO) performance (p b 0.05).
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cannula implantation surgery on a mouse was when they were 4 weeks of age. After a 2 week recovery period from surgery, the mice were then subjected to ICV infusion of enzyme (i.e., at 6 weeks of age). Thirty weeks was chosen as the final time point because ASMKO mice were typically deemed moribund at this age and because SPM accumulation was close to maximal (Fig. 5) at this time point (Passini et al., 2007). The CNS showed regional differences in the extent of SPM accumulation with the brain showing greater accumulation than the spinal cord. Differences in visceral SPM accumulation were also apparent with liver tissue showing greater accumulation than lung tissue. Relative to the CNS (depending on the region analyzed) SPM accumulation was 2–5 fold higher in the visceral tissues. Repeated ICV infusions of hASM slowed disease progression in ASMKO mice As SPM and cholesterol levels in the hindbrain and spinal cord reaccumulated back to untreated levels by 3 weeks post-treatment, we elected to evaluate the efficacy of biweekly infusions of hASM at slowing disease progression in ASMKO mice. Specifically, we measured the effect of repeated treatments on tissue SPM levels and on the performance of the ASMKO mice on various behavioral tests (i.e., foot fault, gait analysis and grip strength) designed to assess motor function. ASMKO mice (n = 14) received ICV infusions of hASM (0.025 mg/mouse) starting at 6 weeks of age and every two weeks thereafter for 8 weeks. As controls, a cohort of ASMKO mice (n = 14) was administered aCSF by ICV infusion and another with hASM (0.025 mg/mouse) administered via tail vein injections (n = 14). The latter was included as an experimental group because we wanted to be sure that potential improvements in motor function could be ascribed to the actions of hASM in the CNS and not the visceral organs. Foot fault, gait and hindlimb grip strength testing began when the mice were 7 weeks of age and these tests were repeated every 2 weeks thereafter. All mice were sacrificed at 14 weeks of age for concern that an immune response to the human enzyme may circumvent the ability to measure biochemical efficacy. Analysis of the animals at the end of the study period showed significant reductions in SPM levels in the brain and spinal cord (p b 0.05) in hASM- but not aCSF-treated ASMKO mice (Figs. 6A and B). As expected (due to presence of the BBB) ASMKO mice administered hASM via tail vein injections did not significantly lower the accumulation of SPM in the CNS. Correlated with this observation, ASMKO mice treated with ICV infusions of hASM made significantly (p b 0.05) fewer missteps on the foot fault test at 13 weeks of age when compared to ASMKO mice that received aCSF infusions (Fig. 6C). No significant improvement on the foot fault test was observed in ASMKO mice treated by intravenous administrations of hASM. Gait analysis showed similar significant (p b 0.05) improvements in forelimb and hindlimb stance width in ASMKO mice treated by ICV infusions of hASM but not in those treated by aCSF or those treated by tail vein injections of hASM (Figs. 6D and E). On the grip strength test, however, ASMKO mice treated with hASM regardless of the route of delivery (ICV or tail vein injections) did not show significant improvements in muscle strength (Fig. 6F). Discussion Intravenous infusion of normal, active recombinant enzymes has been successfully implemented to treat a number of different LSDs including Gaucher, Fabry, MPS I, MPS II, MPS VI and Pompe disease (Amalfitano et al., 2001; Barton et al., 1991; Barton et al.,1990; Harmatz et al., 2004; Kakkis et al., 2001; Schiffmann et al., 2001). Although ERT has proven to be effective in reducing substrate accumulation in the visceral organs, the presence of the blood brain barrier has prevented this therapeutic approach from being used to treat LSDs with CNS manifestations such as NPA disease. Recently, intracerebral injection has been used to deliver gene, cell and protein based therapies to the
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CNS of mouse models of LSDs including ASMKO mice in a manner that led to significant reductions in lysosomal accumulation of the substrate (Dodge et al., 2005; Jin et al., 2002; Jin and Schuchman, 2003; Passini et al., 2007; Passini et al., 2005; Shihabuddin et al., 2004; Yang et al., 2007). However, depending on the therapeutic platform used, correction of lysosomal storage accumulation was typically circumscribed to an area of a few millimeters from the sites of injection. Although this limitation could possibly be circumvented in part by using injection strategies that enhance diffusion of the therapeutic agent (Bankiewicz et al., 2000; Bobo et al., 1994; Lonser et al., 2005) or by targeting highly connected regions of the brain to enable vector axonal transport to distal regions (Dodge et al., 2005; Passini et al., 2005), multiple intracerebral injections would likely still be needed to achieve global delivery of the enzyme throughout the CNS (Cabrera-Salazar et al., 2007; Passini et al., 2007). Therefore, we initiated an effort to explore alternative enzyme delivery strategies to address the lysosomal storage pathology within the CNS. Delivery of drugs to the CSF using semi-permanent indwelling catheters has been shown to be a viable approach to address CNS diseases in humans (Anderson and Burchiel, 1999). Moreover, delivery of lysosomal enzymes into the CSF either through intrathecal or ICV injection has been shown to be effective in distributing the enzyme broadly within the brain of animal models of LSDs (Chang et al., 2008; Horinouchi et al., 1995; Kakkis et al., 2001; Lonser et al., 2005). Successful application of this approach to LSD patients, however, will likely be dependent upon the infusion parameters used during enzyme delivery. For example, this may explain why a previous attempt to treat the CNS storage disease in Gaucher patients using a bolus ICV injection of the aglucerase (the enzyme that is deficient in Gaucher disease) was unsuccessful (Bembi et al.,1995). Therefore, in our current experiments we elected to evaluate the efficacy of continuous ICV infusion of hASM over a 6 h period in a mouse model of NPA disease. Following infusion of hASM into the lateral ventricle of ASMKO mice, immunostaining for hASM in brain sections showed bilateral enzyme distribution to widespread regions of the brain including the forebrain, midbrain and hindbrain. Similar patterns of enzyme distribution have been reported following a 2 week continuous ICV infusion of recombinant tripeptidyl peptidase I in a mouse model of late-infantile Batten disease (Chang et al., 2008) and with a bolus ICV injection of galactocerebrosidase into 20 day old twitcher mice (Lee et al., 2007). Our experiments, however, were the first to examine the kinetics of enzyme spread and intracellular uptake within the brain following an ICV infusion. Immediately after infusion, we observed a diffuse pattern of hASM staining throughout the brain with cellular uptake of hASM occurring within 24 h. Interestingly, hASM was still apparent 3 weeks post-infusion suggesting that hASM has a relatively long half-life in cells of the brain parenchyma. Similar to what was reported in a mouse model of Krabbe disease (Lee et al., 2007), ICV infusion of hASM in ASMKO mice also led to a significant reduction in the offending substrate throughout the brain. Our experiment, however, was the first to examine the effectiveness of this therapeutic approach as a function of dose. Treatment with the highest dose reduced substrate levels throughout the brain to those observed in wild type animals. As expected, the lowest dose was least effective in reducing substrate particularly in the regions that were most distal (e.g., hindbrain) to the site of infusion. We also demonstrated for the first time using both biochemical and histological measures that ICV infusion of a lysosomal enzyme was equally effective in reducing accumulated substrate not only in the brain but also within the spinal cord. More specifically, we observed a significant reduction in SPM tissue levels and filipin staining (a stain used to detect cholesterol accumulation, a secondary metabolite that accumulates in NPA disease) throughout the spinal cord. Given that CSF circulates back into venous circulation we also analyzed visceral tissues (i.e., liver and lung) for potential reductions in SPM levels following ICV infusion of hASM. Our results showed for the first time that ICV enzyme infusion
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could also be applied to treating storage pathology observed in the visceral organs of LSDs with CNS pathological manifestations, as we observed a significant reduction in SPM in both liver and lung tissue in a dose dependent manner. This feature of ICV enzyme delivery will likely provide added benefit to NPA patients, as maximal improvements in both function and survival are achieved in ASMKO mice following treatments that address both the visceral and CNS disease (Passini et al., 2007). Prior to evaluating the efficacy of repeated ICV administrations of hASM in ASMKO mice, we sought to characterize the rate of SPM accumulation as a function of age in ASMKO mice and determined the kinetics of SPM re-accumulation following a single ICV hASM infusion. Assessment of SPM accumulation in ASMKO mice as a function of age showed that 6 week-old ASMKO mice displayed significant SPM accumulation in both CNS and visceral tissue. Interestingly, the rate of SPM accumulation was tissue specific with visceral tissues showing significantly greater SPM accumulation than CNS tissue. Within the CNS, SPM accumulation was significantly greater in brain than in spinal cord tissue. Whether or not this phenomenon also occurs in humans is unknown and warrants further investigation. However, since SPM levels in normal mice were similar in all the tissues analyzed this suggests (at least in ASMKO mice) that a tissue's metabolic environment may play an important role in modulating SPM accumulation. More importantly, understanding parameters that govern tissue specific differences in SPM accumulation may lead to the development of adjunct therapies to ERT for the treatment of NPA disease. Nevertheless, our results demonstrated that SPM accumulation was rapid, attaining significant levels by 6 weeks of age in ASMKO mice. Our efforts to examine the rate of SPM re-accumulation following a single ICV infusion of hASM showed that SPM levels returned to those observed in untreated ASMKO mice within 2– 3 weeks post-treatment, particularly in regions that were most distal to the infusion site (e.g., hindbrain and spinal cord). Interestingly, SPM levels in the visceral tissues were still relatively low after 3 weeks post-treatment suggesting that visceral organs were more sensitive to exogenous hASM than the CNS or that the rate of re-accumulation of SPM was slower in the visceral tissues. As SPM in the CNS re-accumulated back to untreated levels by 3 weeks post-treatment, we elected to test the ability of recurring hASM treatments given every 2 weeks starting at 6 weeks of age to slow disease progression in ASMKO mice. Our findings showed that repeated ICV infusions of hASM were effective in reducing SPM accumulation within the CNS and partially improving motor function in ASMKO mice. Specifically, we observed a significant reduction in SPM levels in the brain and spinal cord of ASMKO mice that received recurring ICV infusions of hASM. Animals treated in this manner also showed improved performance on tests designed to assess motor coordination and balance (i.e., foot fault and gait analysis). However, it should be noted that improved muscle strength was not seen using this therapeutic regimen (as measured by the grip strength test) suggesting either inadequate targeting of the enzyme to the diseased muscle or motor neurons. Whether or not this is a limitation of ICV infusion or to the dose used in our current experiment remains to be determined. It was also difficult to compare these results with our previous studies examining the efficacy of gene delivery of hASM. Use of certain combinations of viral vectors and transcriptional cassettes immunotolerized mice to the human lysosomal enzymes and allowed for improved substrate clearance (Barbon et al., 2005; Passini et al., 2007; Ziegler et al., 2007). In addition, as gene therapy facilitated continuous enzyme exposure, this limited substrate re-accumulation and therefore, any potential pathophysiology initiated by cyclic changes (i.e., repeated clearance followed by re-accumulation) in cellular SPM levels. Future experiments examining the merits of ICV infusion of enzyme in mouse models of LSD should be performed under conditions that permit similar immunotolerization to the enzyme and greater exposure of the enzyme (e.g. by continuous delivery of the
enzyme) to assess the relative benefits of these two different technologies. Nevertheless, our results are encouraging as we observed an alleviation of disease phenotype in ASMKO mice even with a limited number of ICV infusions of hASM. In conclusion, the results of our studies illustrate that continuous ICV infusion of hASM is an effective approach to address the pathological manifestations observed in the CNS of a mouse model of NPA disease. Our findings support the continued exploration of ICV delivery of enzymes as a therapeutic modality for LSDs such as NPA that exhibit lysosomal accumulation of substrate within the CNS. Acknowledgments We would like to thank Leah Curtin and the staff at DCM for their technical support.
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