Patent Application
20140377246
The invention relates to methods of treating mucopolysaccharidoses using enzyme replacement therapy and chemically modified lysosomal enzymes.
The mucopolysaccharidoses (MPS) are a group of lysosomal storage disorders (LSDs) that result from a deficiency of lysosomal enzymes necessary for the degradation of glycosaminoglycans (GAGs). In mucopolysaccharidosis type VII (MPS VII; Sly syndrome) the GAGs, dermatan sulfate, heparan sulfate, chondroitin 4-sulfate, and chondroitin 6-sulfate, accumulate in lysosomes in the absence of the catabolic enzyme β-glucuronidase (GUS)(1). Around 50 different mutations in the GUS gene have been identified producing a wide range of clinical severity (2). MPS VII is characterized by short stature, dysmorphic features, corneal clouding, hepatomegaly, skeletal abnormalities collectively referred to as dysostosis multiplex, and developmental delay. These clinical manifestations become progressively worse over time if left untreated. MPS VII patients with the most severe phenotype have hydrops fetalis prenatally and often are stillborn or survive only a few months. At the other extreme, patients with attenuated manifestations of MPS VII have survived into the fifth decade of life.
Murine models of MPS VII have characteristics similar to the human disease (3,4). MPS VII mice show GAG storage in lysosomes of visceral organs, skeleton, and brain. They have facial dysmorphism, growth retardation, deafness, behavioral deficits, and a shortened lifespan. Radiographic analysis showed significant bone dysplasia including shortened and thick long bones, sclerosis of the calvarium, and a narrow thorax. Microscopically, the epiphyseal growth plate is hypercellular and irregular and osteoblasts in the bone marrow contain vacuoles. In addition, synovial proliferation, vacuolated synovial cells, and articular-synovial synechiae have been described.
Several LSDs have been treated with enzyme replacement therapies (ERTs), which rely on mannose 6-phosphate receptor (M6PR) or mannose receptor-mediated uptake of enzymes into target cells (5-8). This receptor-mediated ERT strategy has been used with substantial success to treat storage in visceral organs in murine MPS VII. However, GAG storage in the central nervous system (CNS) has been resistant to clearance by ERT using conventional doses of enzyme unless begun during the newborn period (9, 10). In several disease models partial correction in some areas of the brain followed repeated injections of large doses of enzyme (11-14). Grubb et al. reported that a chemically modified form of GUS referred to herein as PerT modified β-glucuronidase or PerT-GUS, which was more resistant to clearance from the blood by mannose and mannose 6-phosphate receptors, and showed prolonged circulation (half-life over 18 hours) and was more effective than native enzyme at clearing storage from cortical and hippocampal neurons. Higher levels of enzyme in other tissues suggested improved delivery to other organs as well (15). The mechanism, by which PerT-GUS enzyme escapes uptake by the mannose and mannose 6-phosphate receptors, relies on chemical inactivation of its terminal sugars by treatment of sodium metaperiodate followed by borohydride reduction. How long-circulating PerT-GUS gains entry to some cell types remains unknown. This chemically modified form of GUS (PerT-GUS), may escaped clearance by mannose 6-phosphate and mannose receptors, and reduce CNS storage more effectively than native GUS. However clearance of GAG storage material in bone is limited by the avascularity of the growth plate. In this disclosure, the Inventors compared the skeletal response of MPS VII mice to treatment with 12 weeks of either PerT-GUS or native GUS ERT where treatment began 5 weeks after birth. The Inventors also assessed the skeletal effects of long-term treatment of MPS VII mouse models with PerT-GUS ERT. Micro-CT, radiographs, and quantitative histopathology were used in parallel to define the bone pathology in MPS VII mice and their response to treatments. In addition, some subjects after receiving repeated intravenous injections or infusions suffer from collapsed veins and have difficulty with continued intravenous treatment. The Inventors have addressed this problem by disclosing a method of ERT which uses intraperitoneal administration.
A method of treating mucopolysaccharidoses by using enzyme replacement therapy with chemically modified lysosomal enzymes by intraperitoneal injection. A method of treating type VII mucopolysaccharidoses by administering a chemically modified β-glucuronidase by intraperitoneal injection. A method of treating type VII mucopolysaccharidoses using enzyme replacement therapy by administering a chemically modified β-glucuronidase, beginning at 5 weeks after birth. A method of treating a type VII mucopolysaccharidoses related bone lesion using enzyme replacement therapy beginning about 5 weeks after birth wherein bone mineral density improves.
Mucopolysaccharidosis (MPS) type VII is a lysosomal storage disease caused by deficiency of the lysosomal enzyme β-glucuronidase (GUS), which is involved in the degradation of glycosaminoglycans (GAGs). In order to increase the time of exposure to high levels of β-glucuronidase (GUS) in the blood and tissues, β-glucuronidase was chemically modified by sodium metaperiodate followed by sodium borohydride reduction (PerT GUS) to prevent uptake by the M6P or mannose receptor pathway. While not wishing to be bound by theory, it is thought that this chemical modification results in removal or modification of mannose and mannose-6-phosphate exposed sugars on GUS. The result is that PerT GUS is not bound and removed from the blood by cell surface mannose and mannose-6-phosphate receptors located on the luminal surface of the blood vessels. Unmodified native GUS was cleared with a t1/2of 11.7 min by the MR and the M6PR clearance systems while the clearance of modified β-glucuronidase (PerT-GUS) was dramatically prolonged to a t1/2of 18.5 h. PerT-GUS ERT therapy was also able to clear GAG storage material from the CNS in a mouse model of MPS VII. (15) However, because clearance of GAG storage material from bone is thought to be limited by the avascularity of the growth plate, the effectiveness of PerT-GUS on the treatment of bone lesions remains unknown. In addition, it is sometimes difficult for clinicians to find veins suitable for intravenous injection in subjects receiving repeated intravenous injections or infusions. Particularly when the subject is of small size. The term “subject” as used herein is meant to refer to mammalian subjects including experiential animals, and human subjects, including newborn human subjects. Intraperitoneal administration of enzyme replacement therapy was not considered feasible because absorption of enzymes from the intraperitoneal cavity was either unknown or thought to be unlikely. This was confirmed by the Inventors (unpublished results) The Inventors disclose a method of ERT administration that utilizes a PerT modified enzyme and intraperitoneal injection. In the particular example disclose below, a PerT modified GUS is administered intraperitoneally in a mouse model of MPS VII.
Treatment of Bone Lesions: Short Term Protocol
A limitation of ERT for LSDs has been the inability to correct bone pathology because of the avascularity of the growth plate. It was also thought that unless ERT treatment began in the neonatal period it will not be effective (9, 16, 24). In previous studies, the response to treatment of MPS VII from birth with intravenous native GUS enzyme was shown to improve growth, fertility, longevity, and histology of visceral organs. However, the response of bone (chondrocytes) to ERT was limited even if treatment began at birth (9,11,16). Given the greatly prolonged blood clearance of PerT-GUS, the inventors reasoned that PerT-GUS may be effective when administered outside of this neonatal window. To this end the Inventors designed a short term treatment protocol whereby a MPS VII mouse model was treated with intravenous injections of either native GUS or Pert-GUS weekly, at 2 mg per kilogram of the subject to be treated, for 12 weeks, starting 5 weeks after birth. In this short term protocol, several quantitative measurements of histopathology showed significant improvement in mice treated with PerT-GUS compared with native GUS treatment. These results indicate that when using Pert-GUS, treatment need not start at birth to provide beneficial therapeutic effects.
The direct comparison of native GUS and PerT-GUS confirmed that PerT-GUS treated mice have significantly reduced storage material at the growth plate and while not statistically significant, storage material was also reduced in the articular cartilage, as indicated by cell area measurements (
Micro-CT studies showed greater reduction in bone mineral density (BMD) with PerT-GUS treatment. These findings were supported by the X-ray findings of lower radiodensity in PerT-GUS treated mouse legs as well as reduced femur thickness compared to those of GUS treated mice. Histopathological analysis also showed reduced storage material and a more organized growth plate in PerT-GUS treated mice compared with GUS treated mice.
Intraperitoneal Treatment
The Inventors made the surprising discovery that PerT modification of GUS enabled the enzyme to be suitable for intraperitoneal administration. The Inventors observed that the subjects' response to multiple intraperitoneal injections of PerT-GUS were equivalent to those receiving multiple intravenous injections. Despite the fact that PerT-GUS was taken up poorly by peritoneal lining cells, intraperitoneally infused enzyme reached the same concentrations in the blood as intravenously infused PerT-GUS, after a 30-60 minute delay (data not shown). This contrasts with earlier observations that the native enzyme was much less effective if administered IP because much of the delivered dose was taken up by peritoneal lining cells and never reached the circulation. While not wishing to be bound by theory, it is thought that because PerT-GUS is not taken up by cells of the peritoneal lining it may enter the subject's circulation by way of the lymphatic system. The Inventors believe that PerT modification is responsible for providing these new properties to GUS, and that PerT modification of other lysosomal enzymes would provide the same or similar properties to those enzymes, including prolonged half-life in circulation and enabled delivered by intraperitoneal administration. It is envisioned that subjects suffering from type VII mucopolysaccharidoses may receive intraperitoneal administration of PerT-GUS during a course of enzyme replacement therapy. Similarly, it is envisioned that subjects suffering from other LSDs may receive intraperitoneal administration of a PerT modified lysosomal enzyme during a course of enzyme replacement therapy. Non-limiting examples of LSDs and their respective deficient lysosomal enzyme which are expected to be provided with prolonged half-life in circulation and enabled delivered by intraperitoneal administration after PerT modification include: Morquio syndrome, deficient in N-acetylgalactosamine-6-sulfatase; Hurler syndrome, deficient in Iduronidase; Hunter syndrome, deficient in Iduronate-2-sulfatase; Sanfilippo syndrome, deficient in Alpha-N-acetylglucosaminidase; Gaucher's disease, deficient in beta-glucosidase; Fabry disease, deficient in alpha-galactosidase; Hurler syndrome, deficient in α-L-iduronidase; Maroteaux-Lamy syndrome, deficient in N-acetylgalactosamine 4-sulfatase; and Pompe disease deficient in acid alpha-glucosidase. A preferred example is type VII mucopolysaccharidoses, deficient in β-glucuronidase, as exemplified in this disclosure.
The use of intraperitoneal administration to deliver ERT will be a benefit to subjects who receive repeated administrations of enzymes, in particular those which are of a small size. It is anticipated that most subjects will begin ERT therapy early in life and these subjects will include neonatal subjects. It is also envisioned that IP administration may be accomplished with the aid of various pumps and/or peritoneal infusion or injections ports, well known in other methods of treatment, by way of example the delivery of chemotherapy to cancer patients. Intraperitoneal delivery of ERT by way of a peritoneal port would allow repeated access to the intraperitoneal cavity by the clinician with minimum trauma to the subject.
Treatment of Bone Lesions: Long Term Protocol
It was unknown whether long term PerT-GUS treatment would be effective enough to reduce bone lesions due to MPS VII, to a state comparable to a non-disease subject. To that end the Inventors designed a treatment protocol whereby a MPS VII mouse model was treated with PerT-GUS at 2 mg per kilogram of the subject to be treated, weekly from birth until 6 weeks, then with PerT-GUS at 2 mg per kilogram of the subject to be treated, administered every other week until 57 weeks of age. Based on the observation described above, all treatments were administered by intraperitoneal injection. Effectiveness was evaluated using micro-CT, X-rays, and histopathology. After 57 weeks, The quantitative histological analysis showed that long-term IP injected PerT-GUS ERT improves epiphyseal growth plate organization and GAG storage and reduces growth plate thickness, cell size of chondrocytes, perimeter/length ratio of growth plate, and abnormal proliferation of articular and meniscal cartilage and connective tissue in knee joints. This study showed significant reduction in size of chondrocytes (both articular and epiphyseal chondrocytes, which were half of the size of untreated chondrocytes:
long term PerT-GUS ERT therapy showed the correction of skeletal pathology in a mouse model of MPS VII. In MPS VII mice treated with PerT-GUS ERT. Micro-CT and X-rays demonstrated marked radiographical improvements in bone lesions of legs, ribs, and spine. Histopathology also showed substantial improvements in skeletal GAG storage and morphology. In the long term protocol, micro-CT and radiographs analysis demonstrated that MPS VII mice treated with IP injected PerT-GUS from birth had substantial correction of bone pathology. The Inventors show that PerT-GUS treatment from birth to more than 6 months of age reduced cortical bone thickening and reduced the amount of shortening seen in long bones of the leg. In addition, PerT-GUS reduced exophytic bone formation, diminished spinal stenosis, and normalized radiodensity of the cervical spine and ribs in the MPS VII mice. The BMD of PerT-GUS treated MPS VII mice was reduced to the level of wild-type mice. Thus, IP injected PerT-GUS treatment addresses major components of the dysostosis multiplex associated with MPS VII. The Inventors have shown that long-term PerT-GUS treatment prevents skeletal pathology to an extent that would impact the quality of life of a human subject Similar results in humans may reduce the need for corrective surgeries and improve the quality of life in MPS VII patients.
PerT-GUS
PerT-GUS was prepared as previously described in U.S. application Ser. No. 12/042,601, published as U.S. Published Application No. US 2009/0041741 A1, and incorporated herein by reference in its entirety. In summary, isolated GUS was treated with periodate and borohydride without significantly reducing the enzymatic activity or stability. It is expected that these same methodology may be applied to other lysosomal enzymes to produce other PerT modified lysosomal enzymes.
Generation of Stable Cell Lines Secreting GUS
Using DNA cloning techniques, the cDNA sequence encoding the full length cDNA for human -glucuronidase (GUS) (Genbank Accession # NM 000181)(SEQ ID NO:1) was sub cloned into the mammalian expression vector pCXN (32) The plasmid was introduced into the Chinese hamster ovary cell line, CHO-KI (33) by electroporation (34). After selection in growth medium, high level expressing clones were identified by measuring GUS activity secreted into the conditioned medium. The highest-producing clone was scaled up and secreted enzyme was collected in protein-free collection medium PF-CHO. Conditioned medium collected in this way was pooled, centrifuged at 5000×g for 20 min and the supernatant was collected and frozen at 20° F. GUS was then isolated using conventional column chromatography or antibody affinity techniques.
Treatment of Purified GUS with Periodate and Borohydride: PerT Modification
In order to inactivate the mannose and mannose 6-phosphate recognition sites on GUS, the enzyme was treated by a well-established procedure utilizing reaction with sodium meta-periodate followed by sodium borohydride (35, 36). Approximately 10 mg of purified GUS was treated with a final concentration of 20 mM sodium meta-periodate in 20 mM sodium phosphate, 100 mM NaCl pH 6.0 for 6.5 h on ice in the dark. The reaction was quenched by the addition of 200 mM final concentration ethylene glycol and incubated for an additional 15 min on ice in the dark. Afterwards, this mixture was dialyzed against 2 changes of 20 mM sodium phosphate, 100 mM NaCl pH 6.0 at 4° C. The periodate treated, dialyzed enzyme was then treated with the addition of 100 mM final concentration sodium borohydride overnight on ice in the dark to reduce reactive aldehyde groups. After this treatment, the enzyme was dialyzed against two changes of 20 mM sodium phosphate, 100 mM NaCl, pH 7.5 at 4° C. The final dialyzed enzyme was stored in this buffer at 4° C. where it was stable indefinitely.
Treatment Amounts and Treatment Periods
The above treatment regimens and dosages of PerT GUS administered by intravenous or intraperitoneal infusion are non-limiting. A skilled artisan may determine the treatment dosages based on a particular subject and the severalty of the GUS deficiency or the severity of bone lesions being treated. It is anticipated that treatment regimens of PerT-GUS will vary. Treatment amounts may be from about 0.1 mg/kg to about 1 mg/kg, about 1 mg/kg to about 2 mg/kg, about 2 mg/kg to about 3 mg/kg, about 3 mg/kg to about 4 mg/kg, about 4 mg/kg to about 6 mg/kg about 6 mg/kg to about 12 mg/kg of the subject being treated. A preferable amount of PerT GUS administered is about 2 mg/kg of the subject being treated and a preferred route of administration is by one or more intraperitoneal injections.
The treatment periods described herein are non-limiting. A skilled artisan may determine the start and duration of the treatment period based on a particular subject and the severalty of the bone lesions being treated. It is anticipated that treatment periods would be between about 12 and about 57 weeks and may start at or after birth. By way of example, a treatment period my start 5 weeks after birth.
Preferred embodiments of the invention are described in the following examples. Other embodiments within the scope of the claims herein will be apparent to one skilled in the art from consideration of the specification or practice of the invention as disclosed herein. It is intended that the specification, together with the examples, be considered exemplary only, with the scope and spirit of the invention being indicated by the claims, which follow the examples.
MPS VII Tolerant Mouse
A tolerant mouse model for MPS VII (4) was developed from the original Birkenmeir GUS deficient mouse (gusmps/mps) (17) and has been used for evaluation of the effectiveness of a variety of experimental treatments (11,15, 18-20). This mouse has characteristics similar to humans with MPS VII including a shortened face, facial dysmorphism, growth retardation, deafness, shortened lifespan, and behavioral deficits. In addition, it is immunotolerant to administered human GUS.
Purification of GUS
GUS was purified by a multistep procedure with conventional column chromatography as described (15). Purified enzyme was frozen at −80° C. where it was stable indefinitely until thawed for treatment with periodate.
Treatment of GUS with Periodate and Borohydride
The M6P and mannose recognition sites on GUS are contained in the oligosaccharide side chains of the enzyme. To inactivate the exposed carbohydrates, the enzyme was treated with sodium metaperiodate followed by sodium borohydride (15). At the final step, the enzyme was dialyzed against two changes of 20 mM sodium phosphate, 100 mM NaCl (pH 7.5) at 4° C., and was stable stored in this buffer at 4° C. before use.
Short Term Protocol: Comparison of Response to Treatment of MPS VII Mice with Native GUS and PerT-GUS
MPS VII mice were treated intravenously (IV) with 2 mg/kg native GUS (n=4) or PerT-GUS (n=4) for 12 weeks beginning at 5 weeks of age. One week after the last treatment, mice were euthanized and tissues were treated in using the protocol described below for long-term treatment with PerT-GUS (Examples 1-3).
Long-Term Protocol: Treatment of MPS VII Mice with PerT-GUS Intraperitoneally
MPS VII mice were treated Intraperitoneally (IP) with a fixed dose of 30,000 units (7 micrograms) at birth (0-1 day) and at 7 days of age, and with 2 mg/kg weekly on days 14, 21, 28, 35 and 42 with IP infusions of PerT-GUS (2 mg/kg body weight). After 6 weeks of age, mice received 2 mg/kg of the enzyme IP every other week until 27-57 weeks of age. One week after the last infusion, tissues from untreated (n=4) or PerT-GUS treated MPS VII mice (n=5; ages 27, 38, 41, 57, and 57 weeks old) were perfused at necropsy with 25 mM Tris and 140 mM NaCl (pH 7.2), fixed in 2% paraformaldehyde and 4% glutaraldehyde, postfixed in osmium tetroxide, and embedded in Spurr resin. For evaluation of lysosomal storage, toluidine blue-stained 0.5-μm-thick sections of knee joints were assessed by light microscopy. The Inventors also euthanized untreated MPS VII mice at 1 day old and ages 2.5, 5, 10, 23, 29, 32, and 36 weeks old and age-matched wild-type mice to understand the progression of the disease (Examples 4-8).
Micro-CT Analysis and Radiography
Mice were euthanized using CO2. At dissection, leg bones, spines, and ribs were placed in 95% ethanol. A micro-CT scan was performed on each bone using a Scanco μCT40 system (Scanco Medical; Brüttisellen, Switzerland) according to manufacturer's instructions (21). Scans were focused on cervical vertebrae 1 and 2 and the knee joint. The bones were then fixed in formalin in preparation for the micro-CT imaging, which was performed on a micro-CT scanner at 16-μm isotropic voxel size, with 250 projections, integration time of 300 msec, photon energy of 50 keV, and current of 160 μA. A three dimensional reconstruction of each bone was made and the bone mineral density (BMD) of each knee joint was measured. Radiographs were also done for each leg, spine, and ribcage and compared. Leg measurements were recorded using plain radiographs (
Quantitative Analysis of Histopathology
Cartilage thickness: The thickness of the tibia growth plate or articular cartilage was measured at five different places and averaged. This average for each mouse was then used to calculate the mean cartilage thickness for wild-type, untreated MPS VII, GUS treated MPS VII, and PerT-GUS-treated MPS VII groups.
Cellularity: The number of cells in three predetermined areas of equal size in the tibia growth plate proliferative zone and articular cartilage were counted and averaged. The values reported are means and standard deviations of the average cellularity for the mice in each group.
Cell Area: Cells in the proliferative zone of tibia growth plate and articular cartilage were outlined as shown in
Cells/Column: The number of cells stacked in columns perpendicular to the long axis of the tibia growth plate was counted, and the mean value was reported.
Perimeter/Length Ratio: The length and perimeter of the tibia growth plate region were measured as shown in
Short Term Protocol: GUS and PerT-GUS ERT Treatment of MPS VII Mice
Examples 1-3 were preformed to examine the effects of the short term treatment protocol with PerT-GUS.
Growth Plate and Articular Cartilage Histology
Growth Plate: The resting, proliferative, and hypertrophic zones of the growth plates in GUS and PerT-GUS treated mice contained enlarged and vacuolated cells (
Articular Cartilage: Cells of the articular cartilage and meniscus were enlarged and vacuolated in both GUS and PerT-GUS treated mice. The articular cartilage chondrocytes were moderately smaller in PerT-GUS treated mice than in GUS treated mice. Cells in the meniscus of PerT-GUS treated mice contained noticeably less storage material compared with meniscal chondrocytes in GUS treated mice.
Quantitative Histopathological Analysis
To assess the morphology of the growth plate and articular cartilage in GUS and PerT-GUS treated MPS VII mice, the Inventors measured the thickness of the cartilage layer in the growth plate and articular cartilage, the cellularity in the articular cartilage and proliferative zone of the growth plate, a cross-sectional area of chondrocytes in the articular cartilage and proliferative zone of the growth plate as an estimate of cell volume, the mean number of cells aligned in columns perpendicular to the growth plate, and the ratio of the perimeter of the growth plate to its length as an indication of the amount of irregularity in the morphology of the growth plate (
These measurements supported our histological observations. The thickness of the articular cartilage in GUS and PerT-GUS treated mice was similar, however the growth plates in GUS treated MPS VII mice showed a trend towards increased thickness compared with PerT-GUS treated mice (p=0.51;
Micro-CT and Radiographic Findings
Micro-CT analysis of the bones of the knee joint (
Long Term Protocol: Effects of Long-Term Treatment with PerT-GUS
Examples 4-8 were preformed to examine the effects of the long term treatment protocol with PerT-GUS.
Micro-CT Findings
Micro-CT analysis of the bones of the knee joint (
Knee Joints:
At 5 weeks of age, bones in the knee joints of untreated MPS VII mice had modest changes from those of wild-type: 1) less ossified bone, and 2) reduced amounts of trabecular bone (
Treatment effects: PerT-GUS treated MPS VII mice showed marked improvements of the knee joint when compared with those of untreated MPS VII mice. Thicknesses of cortical bone of the femur and tibia were normalized and there were fewer periosteal bone formations, although the knee joints were still distinguishable from wild-type mouse knee joints (
Cervical Spine:
At 5 weeks of age, the vertebrae of untreated MPS VII mice appeared to have less ossified bone than those of the wild-type mice (
Treatment effects: A micro-CT scan (n=1) of the spine of a 38-week-old treated MPS VII mouse showed less abnormal thickening of the bone than in untreated MPS VII mice (n=4), resulting in less spinal canal narrowing. In addition, the vertebral bodies were not abnormally wide like those in untreated MPS VII mice (
Only one cervical spine from a PerT-GUS long-term therapy mouse was available for micro-CT study due to dissection-related damage to CV1-2 on the other specimens.
Radiographic Analysis
Radiographs comparing the lower extremities of wild-type, untreated MPS VII, and PerT-GUS-treated MPS VII mice are presented in
Treatment effects: The tibia length of treated MPS VII mice (1.73±0.03 cm) was significantly increased compared with untreated MPS VII mice (1.54±0.09 cm, p<0.05). In addition, the ribs of treated mice were longer and had significantly reduced radiodensity compared with those of untreated mice. The cervical vertebrae of treated MPS VII mice had significantly reduced radiodensity compared with those in untreated mice (
Histopathologic Analysis of Knee Joints
Untreated MPS VII Mice
Articular cartilage: The knee joints of affected mice showed noticeable lysosomal storage within the articular cartilage even in the newborn mouse (day 1 or 2). Most articular chondrocytes had vacuoles, although the structure was organized (
Growth plate: The growth plate region in 1- or 2-day-old MPS VII mice had ballooned vacuolated chondrocytes in resting and proliferative zones. By 2.5 weeks of age, the growth plate was thickened but showed normal resting and proliferative zonal organization (
At 10 weeks of age, the growth plates were thicker and their boundaries became irregular. The column structure through all layers of the growth plate was disorganized. The chondrocytes were ballooned with vacuoles. The osteoblasts surrounding diaphyseal bone trabeculae and the cells lining bone marrow sinusoids contained a large amount of clear cytoplasmic vacuoles (data not shown).
At 32 weeks of age, the column structure through all layers of the growth plate was markedly disorganized and all chondrocytes were prominently ballooned with vacuoles (
PerT-GUS Treated MPS VII Mice:
PerT-GUS treatment from birth to older than 6 months provided substantial improvement in bone pathology. The articular cartilage region showed reduced cellularity and improvement in irregular articular surfaces, although reduction of storage materials in chondrocytes was limited at all cartilage layers. Marked improvement was observed in the abnormal proliferation of articular and meniscal cartilage, leading to reduced articular-meniscal-synovial fusion (
The growth plate region in treated mice showed the following: 1) improvement of architecture by reduction of thickened cartilage layer and irregular surface, and 2) reduced cell area in the proliferative zone, although vacuolated chondrocytes with lysosomal distension remained obvious (
Quantitative Analysis of Histopatholoqy
Quantitative analysis of the histopathology of wild-type and untreated and treated MPS VII mouse knees was carried out using the same methods described for the comparison of GUS and PerT-GUS treated mice. These measurements supported our histopathological observations. Untreated MPS VII mice (n=4) had thicker growth plates (p<0.005) and articular cartilage (p<0.02) compared with those in wild-type mice (n=5). PerT-GUS treatment (n=5) reduced the thickness of both the growth plate (p=0.14) and articular cartilage (p=0.24) compared with those in the untreated MPS VII mice (
The articular cartilage cellularity in untreated MPS VII mice was increased (p<0.001) compared with that in wild-type mice and was significantly reduced by PerT-GUS treatment (p<0.03;
All publications and patents cited in this specification are hereby incorporated by reference in their entirety. The discussion of the references herein is intended merely to summarize the assertions made by the authors and no admission is made that any reference constitutes prior art. Applicants reserve the right to challenge the accuracy and pertinence of the cited references.
This application claims priority to provisional application 61/837,141, filed Jun. 19, 2013, which is hereby incorporated by reference in its entirety.
This work was supported by National Institutes of Health grant no. GM34182. The government of the United States may have certain rights in this invention.
| Number | Date | Country | |
|---|---|---|---|
| 61837141 | Jun 2013 | US |
