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Biomedical subjects

M E Haskins

Publications and source records attributed to M E Haskins.

At least 19 recordsLinked to original sources
PubMed2001

Enzyme replacement therapy in feline mucopolysaccharidosis I.

Enzyme replacement therapy (ERT) has long been considered an approach to treating lysosomal storage disorders caused by deficiency of lysosomal enzymes. ERT is currently used to treat Gaucher disease and is being developed for several lysosomal storage disorders now that recombinant sources of the enzymes have become available. We have continued development of ERT for mucopolysaccharidosis I (MPS I) using the feline model. Recombinant alpha-L-iduronidase was administered intravenously at low dose (approximately 0.1 mg/kg or 25,000 units/kg) to four cats and high dose (0.5 mg/kg or 125,000 units/kg) to two cats on a weekly basis for 3- or 6-month terms. Clinical examinations showed distinct clearing of corneal clouding in one cat although clinical effects in the others were not evident. Biochemical studies of the cats showed that the enzyme was distributed to a variety of tissues although the liver and spleen contained the highest enzyme activities. Glycosaminoglycan storage was decreased in liver and spleen, and the histologic appearance improved in liver, spleen, and renal cortex. Enzyme was not consistently detected in cerebral cortex, brainstem, or cerebellum and the histologic appearance and ganglioside profiles did not improve. A variety of other tissues showed low variable uptake of enzyme and no distinct improvement. IgG antibodies to alpha-L-iduronidase were observed in five cats with higher titers noted when higher doses were administered. Mild complement activation occurred in three cats. Enzyme replacement therapy was effective in reversing storage in some tissues at the biochemical and histologic level in MPS I cats but an improved tissue distribution and prevention of a significant immune response could make the therapy more effective.

Animals↗
PubMed2001

Articular chondrocytes from animals with a dermatan sulfate storage disease undergo a high rate of apoptosis and release nitric oxide and inflammatory cytokines: a possible mechanism underlying degenerative joint disease in the mucopolysaccharidoses.

Mucopolysaccharidosis (MPS) Type VI (Maroteaux-Lamy Disease) is the lysosomal storage disease characterized by deficient arylsulfatase B activity and the resultant accumulation of dermatan sulfate-containing glycosaminoglycans (GAGs). A major feature of this and other MPS disorders is abnormal cartilage and bone development leading to short stature, dysostosis multiplex, and degenerative joint disease. To investigate the underlying cause(s) of degenerative joint disease in the MPS disorders, articular cartilage and cultured articular chondrocytes were examined from rats and cats with MPS VI. An age-progressive increase in the number of apoptotic chondrocytes was identified in the MPS animals by terminal transferase nick-end translation (TUNEL) staining and by immunohistochemical staining with anti-poly (ADP-ribose) polymerase (PARP) antibodies. Articular chondrocytes grown from these animals also released more nitric oxide (NO) and tumor necrosis factor alpha (TNF-alpha) into the culture media than did control chondrocytes. Notably, dermatan sulfate, the GAG that accumulates in MPS VI cells, induced NO release from normal chondrocytes, suggesting that GAG accumulation was responsible, in part, for the enhanced cell death in the MPS cells. Coculture of normal chondrocytes with MPS VI cells reduced the amount of NO release, presumably because of the release of arylsulfatase B by the normal cells and reuptake by the mutant cells. As a result of the enhanced chondrocyte death, marked proteoglycan and collagen depletion was observed in the MPS articular cartilage matrix. These results demonstrate that MPS VI articular chondrocytes undergo cell death at a higher rate than normal cells, because of either increased levels of dermatan sulfate and/or the presence of inflammatory cytokines in the MPS joints. In turn, this leads to abnormal cartilage matrix homeostasis in the MPS individuals, which further exacerbates the joint deformities characteristic of these disorders.

Animals↗
PubMed2001

Adenoviral vector-mediated beta-glucuronidase cDNA transfer to treat MPS VII RPE in vitro.

PURPOSE: To develop an effective therapy for treating glycosaminoglycan (GAG) storage in mucopolysaccharidosis VII (MPS VII) retinal pigment epithelium (RPE) in vitro using adenoviral vector mediated human beta-glucuronidase cDNA (Ad-GUSB) transfer. METHODS: Ad-GUSB was used to infect RPE at confluency. The transduction condition was optimized varying time of infection and number of infectious particles. The beta-glucuronidase (GUSB) activity was measured in transduced cells and media using a fluorogenic substrate. The GAG profiles were examined by metabolically labeling RPE with (35)Na(2)SO(4). RESULTS: Transduced RPE, irrespective of species or disease status, expressed a high level of beta-glucuronidase. The expressed enzyme restored normal levels of GAGs in the RPE cells of homozygous affected MPS VII dogs by metabolizing stored GAGs. The over-expressed enzyme (>10 000 nmoles/hr/mg) failed to restore normal level of GAGs. A high level of GUSB expression was maintained in vitro at least nine weeks. CONCLUSIONS: Adenoviral vector could mediate transfer of GUSB in MPS VII affected RPE and RPE of various species, and the expression was observed to be stable in vitro. However, controlled expression of GUSB was essential for the metabolism of stored GAGs to achieve normal levels.

Adenoviridae↗
PubMed2001

Histopathology, electrodiagnostic testing, and magnetic resonance imaging show significant peripheral and central nervous system myelin abnormalities in the cat model of alpha-mannosidosis.

Alpha-mannosidosis is a disease caused by the deficient activity of alpha-mannosidase, a lysosomal hydrolase involved in the degradation of glycoproteins. The disease is characterized by the accumulation of mannose-rich oligosaccharides within lysosomes. The purpose of this study was to characterize the peripheral nervous system (PNS) and central nervous system (CNS) myelin abnormalities in cats from a breeding colony with a uniform mutation in the gene encoding alpha-mannosidase. Three affected cats and 3 normal cats from 2 litters were examined weekly from 4 to 18 wk of age. Progressively worsening neurological signs developed in affected cats that included tremors, loss of balance, and nystagmus. In the PNS, affected cats showed slow motor nerve conduction velocity and increased F-wave latency. Single nerve fiber teasing revealed significant demyelination/remyelination in affected cats. Mean G-ratios of nerves showed a significant increase in affected cats compared to normal cats. Magnetic resonance imaging of the CNS revealed diffuse white matter signal abnormalities throughout the brain of affected cats. Quantitative magnetization transfer imaging showed a 8%-16% decrease in the magnetization transfer ratio in brain white matter of affected cats compared to normal cats, consistent with myelin abnormalities. Histology confirmed myelin loss throughout the cerebrum and cerebellum. Thus, histology, electrodiagnostic testing, and magnetic resonance imaging identified significant myelination abnormalities in both the PNS and CNS that have not been described previously in alpha-mannosidosis.

Animals↗
PubMed2000

Delivery of a retroviral vector expressing human beta-glucuronidase to the liver and spleen decreases lysosomal storage in mucopolysaccharidosis VII mice.

Mucopolysaccharidosis VII (MPS VII) is caused by beta-glucuronidase (beta-gluc) deficiency and results in lysosomal storage due to the inability to degrade glycosaminoglycans. Transfer of a beta-gluc gene into the liver reduces hepatic pathology as well as storage in other organs via uptake of secreted protein. A Moloney murine leukemia-based retroviral vector expressing the human beta-gluc cDNA was injected intravascularly into MPS VII mice during hepatocyte replication, which was induced with im injection of an adenoviral vector that transiently expressed hepatocyte growth factor (Ad.CMV. HGF). This procedure resulted in transduction of approximately 1% of hepatocytes, 1% of normal liver enzyme activity, and a reduction in lysosomal storage in the liver at 3.5 months. Surprisingly, controls that received retroviral vector without HGF had transduction of nonparenchymal cells in the liver, significant levels of enzyme and RNA in the liver at 2 but not 3.5 months, and reduced lysosomal storage at 3.5 months. Transduction was also achieved in the replicating cells of the spleen, where lysosomal storage was reduced. An approach using a retroviral vector without a growth factor might temporarily reduce lysosomal storage in the liver and spleen in humans. Addition of HGF might be used to augment and prolong gene transfer.

Animals↗
PubMed2000

Gene transfer of low levels of beta-glucuronidase corrects hepatic lysosomal storage in a large animal model of mucopolysaccharidosis VII.

Gene therapy has been at least partially effective in several mouse disease models, but treatment of large mammals has been more difficult to achieve. One major limitation is that only low levels of expression of the corrective gene are often maintained in vivo. In a mouse model of the lysosomal storage disease mucopolysaccharidosis (MPS) type VII (Sly disease) with a null mutation in beta-glucuronidase, gene transfer experiments have shown that only 1-2% of normal beta-glucuronidase can correct the storage in some major organs. In contrast, MPS VII dogs, cats, and humans that have residual beta-glucuronidase activity levels in this range are affected. Thus, higher levels of transferred gene expression may be needed to achieve a therapeutic effect in large animals and humans. We tested this by examining liver pathology in MPS VII dogs after intraperitoneal transplantation of neo-organs containing retrovirus vector-corrected autologous fibroblasts that expressed low levels of beta-glucuronidase. The enzyme secreted from the neo-organs was taken up by the liver and significantly reduced the substrate content compared with untreated dogs. This suggests that small amounts of normal enzyme, when delivered to target tissues, may be therapeutically effective in human MPS VII patients.

Animals↗
PubMed1999

Molecular basis of feline beta-glucuronidase deficiency: an animal model of mucopolysaccharidosis VII.

A family of domestic cats was found that exhibited clinical and biochemical abnormalities consistent with mucopolysaccharidosis VII, an autosomal recessive lysosomal storage disorder caused by beta-glucuronidase deficiency. beta-Glucuronidase activity was undetectable in affected cat fibroblasts and restored by retroviral gene transfer of rat beta-glucuronidase cDNA. beta-Glucuronidase mRNA was normal in affected cat testis by Northern blot analysis. Normal feline beta-glucuronidase cDNA was cloned and characterized, and amplified from affected cat fibroblasts by reverse transcription coupled polymerase chain reaction. There was a G-to-A transition in the affected cat cDNA that predicted an E351K substitution, destroyed a BssSI site, and eliminated GUSB enzymatic activity in expression studies. Multiple species comparison and the crystal structure of human beta-glucuronidase indicated that E351 is a highly conserved residue most likely essential in maintenance of the enzyme's conformation. BssSI digestion of polymerase chain reaction products amplified from genomic DNA indicated that affected cats were homozygous and cats with half-normal beta-glucuronidase activity were heterozygous for the missense mutation. Carriers identified in this manner produced affected kittens in prospective breedings, and a feline MPS VII breeding colony has been established.

Amino Acid Sequence↗
PubMed1999

Retrovirus vector-mediated correction and cross-correction of lysosomal alpha-mannosidase deficiency in human and feline fibroblasts.

Lysosomal alpha-mannosidase (EC 3.2.1.24) is an exoglycosidase in the glycoprotein degradation pathway. A deficiency of this enzyme causes the lysosomal storage disease alpha-mannosidosis. Retrovirus vector transfer of a new human alpha-mannosidase cDNA resulted in high-level expression of alpha-mannosidase enzymatic activity in deficient human and feline fibroblasts. The expressed alpha-mannosidase had the same biochemical properties (thermal stability, pH profile, inhibitor/activator sensitivity) as the native enzyme expressed in normal cells. The transferred enzyme colocalized with a control lysosomal hydrolase in cell fractionation experiments. The vector-encoded enzyme also was released at high levels from the corrected cells, and was taken up by untreated mutant cells via the mannose 6-phosphate receptor-mediated endocytic pathway (cross-correction). It is envisioned that genetic correction of a subset of cells (e.g., hematopoietic stem cells) in patients will provide a source of corrective enzyme for other affected tissues in this multisystem disease. Development of a vector expressing high levels of alpha-mannosidase that cross-corrects mutant cells will enable somatic gene transfer experiments in the cat model of human alpha-mannosidosis.

Animals↗
PubMed1999

Neonatal gene transfer leads to widespread correction of pathology in a murine model of lysosomal storage disease.

For many inborn errors of metabolism, early treatment is critical to prevent long-term developmental sequelae. We have used a gene-therapy approach to demonstrate this concept in a murine model of mucopolysaccharidosis type VII (MPS VII). Newborn MPS VII mice received a single intravenous injection with 5.4 x 10(6) infectious units of recombinant adeno-associated virus encoding the human beta-glucuronidase (GUSB) cDNA. Therapeutic levels of GUSB expression were achieved by 1 week of age in liver, heart, lung, spleen, kidney, brain, and retina. GUSB expression persisted in most organs for the 16-week duration of the study at levels sufficient to either reduce or prevent completely lysosomal storage. Of particular significance, neurons, microglia, and meninges of the central nervous system were virtually cleared of disease. In addition, neonatal treatment of MPS VII mice provided access to the central nervous system via an intravenous route, avoiding a more invasive procedure later in life. These data suggest that gene transfer mediated by adeno-associated virus can achieve therapeutically relevant levels of enzyme very early in life and that the rapid growth and differentiation of tissues does not limit long-term expression.

Animals↗
PubMed1999

Neonatal intramuscular injection with recombinant adeno-associated virus results in prolonged beta-glucuronidase expression in situ and correction of liver pathology in mucopolysaccharidosis type VII mice.

For many metabolic diseases, early correction of the inherited deficiency is required to prevent long-term sequelae. We examined the ability of adeno-associated virus (AAV) to mediate efficient gene transfer during the neonatal period in mice with the lysosomal storage disease mucopolysaccharidosis type VII (MPS VII). Quadriceps of newborn MPS VII mice were injected with an AAV vector containing human beta-glucuronidase (GUSB) cDNA. High-level intramuscular GUSB expression was seen as early as 2 weeks of age, and persisted for at least 16 weeks with no reduction in activity. In addition, GUSB activity was detected in both liver and spleen at later time points. The level of GUSB activity resulted in a significant reduction in lysosomal storage in the liver and a minimal reduction in the spleen at 16 weeks. However, the temporal and spatial pattern of hepatic GUSB activity, coupled with the presence of GUSB cDNA in liver sections, suggests that hematogenous dissemination of virus at the time of injection led to gene transfer to hepatic cells. These results demonstrate that AAV vectors can successfully infect neonatal muscle and persist through the rapid growth phase following birth. However, GUSB secretion from an intramuscular source is inefficient, limiting the therapeutic efficacy of this approach.

Age Factors↗
PubMed1999

Identification and characterization of the molecular lesion causing mucopolysaccharidosis type I in cats.

Mucopolysaccharidosis Type I (MPS I) is the lysosomal storage disease caused by the deficient activity of alpha-L-iduronidase (IDUA). In man, MPS I can occur in severe, mild, or intermediate forms known as the Hurler, Scheie, or Hurler/Scheie syndromes, respectively. MPS I also has been described in cats, dogs, and mice. This manuscript reports the identification and characterization of the mutation causing MPS I in cats. To obtain wild-type feline IDUA cDNAs, two PCR-based strategies were used. PCR primers were constructed from a conserved region of the published human and dog sequences and used to amplify a 224-bp IDUA fragment from normal cat genomic DNA. This fragment was then used to screen a feline uterus cDNA library. PCR also was used to directly amplify IDUA fragments from the same cDNA library. Two overlapping feline IDUA cDNAs encoding 466 amino acid residues of the feline IDUA polypeptide ( approximately 85% of the mature protein based on comparison to the human, dog, and mouse sequences) were obtained by these strategies. To identify the mutation causing MPS I in cats, DNA sequencing was carried out on the corresponding IDUA region from several affected animals. A 3-bp deletion was found on both IDUA alleles in each of the MPS I animals, predicting the deletion of a single aspartate residue from the feline IDUA polypeptide. To confirm the authenticity of this mutation, heteroduplex, SSCP, and transient expression studies were carried out. Over 100 animals from the MPS I colony were screened for the presence of the mutation by heteroduplex and SSCP analyses-in all cases the presence of the 3-bp deletion was 100% concordant with the disease phenotype. For transient expression studies, the two partial, overlapping feline cDNAs were combined and joined in-frame to the 5' end of the canine IDUA cDNA. This wild-type, hybrid cDNA expressed IDUA activity up to sixfold over endogenous levels after transfection into COS-1 cells. A modified full-length IDUA cDNA containing the 3-bp deletion did not express IDUA activity in a transient expression system, providing proof that this lesion was the cause of feline MPS I.

Animals↗
PubMed1999

Autologous transplantation of retrovirally transduced bone marrow or neonatal blood cells into cats can lead to long-term engraftment in the absence of myeloablation.

Autologous transplantation of retrovirally transduced bone marrow (BM) or neonatal blood cells was carried out on eight cats (ranging in age from 2 weeks to 12 months) with mucopolysaccharidosis type VI (MPS VI). The transducing vector contained the full-length cDNA encoding human arylsulfatase B (hASB), the enzymatic activity deficient in this lysosomal storage disorder. Following transplantation, the persistence of transduced cells and enzymatic expression were monitored for more than 2 years. Five of the cats received no myeloablative preconditioning, two cats received 370-390 cGy of total body irradiation (TBI), and one cat received 190 cGy TBI. Evidence of transduced cells, as judged by enzymatic activity and PCR detection of the provirus, was demonstrated in granulocytes, lymphocytes, or BM cells of the treated animals up to 31 months after transplantation. Radiation preconditioning was not required to achieve these results, nor were they dependent on the recipient's age. However, despite the long-term persistence of transduced cells, the levels of ASB activity in the transplanted animals was low, and no clinical improvements were detected. These data provide evidence for the long-term persistence of retrovirally transduced feline hematopoietic cells, and further documentation that engraftment of transduced cells can be achieved in the absence of myeloablation. Consistent with previous bone marrow transplantation studies, these results also suggest that to achieve clinical improvement of MPS VI, particularly in the skeletal system, high-level expression of ASB must be achieved in the treated animals and improved techniques for targeting the expressed enzyme to specific sites of pathology (e.g. chondrocytes) must be developed.

Animals↗
PubMed1998

Retroviral marking of canine bone marrow: long-term, high-level expression of human interleukin-2 receptor common gamma chain in canine lymphocytes.

Optimization of retroviral gene transfer into hematopoietic cells of the dog will facilitate gene therapy of canine X-linked severe combined immunodeficiency (XSCID) and in turn advance similar efforts to treat human XSCID. Both canine and human XSCID are caused by defects in the common gamma chain, gammac, of receptors for interleukin-2 and other cytokines. In this study, normal dogs were given retrovirally transduced bone marrow cells with and without preharvest mobilization by the canine growth factors granulocyte colony-stimulating factor (G-CSF) and stem cell factor (SCF). Harvey sarcoma virus and Moloney murine leukemia virus constructs were used, both containing cDNA encoding human gammac. The Harvey-based vector transduced into cytokine-primed marrow yielded persistent detectable provirus in bone marrow and blood and expression of human gammac on peripheral lymphocytes. In three dogs, human gammac expression disappeared after 19 to 34 weeks but reappeared and was sustained, in one dog beyond 16 months posttransplantation, upon immunosuppression with cyclosporin A and prednisone, with up to 25% of lymphocytes expressing human gammac. The long-term expression of human gammac in a high proportion of normal canine lymphocytes predicts that retrovirus-mediated gene correction of hematopoietic cells may prove to be of clinical benefit in humans affected with this XSCID. This is a US government work. There are no restrictions on its use.

Animals↗
PubMed1998

Biochemical, pathological, and clinical response to transplantation of normal bone marrow cells into acid sphingomyelinase-deficient mice.

BACKGROUND: Acid sphingomyelinase knock-out (ASMKO) mice are a model of types A and B Niemann-Pick disease. In the present study, we evaluated whether bone marrow transplantation (BMT) carried out on newborn ASMKO mice could prevent or alter the Niemann-Pick disease phenotype. METHODS: Previous work from our laboratory had shown that ASMKO mice were highly susceptible to irradiation-induced death. Therefore, we preconditioned 1-day-old ASMKO (n=35) mice with a "sublethal" dose of 200 cGy of total body irradiation before BMT. The transplantation effects were then analyzed by biochemical, pathological, and clinical approaches. RESULTS: Engraftment ranging from 7% to 100% was achieved in 97% of the transplanted animals. Growth of the engrafted animals was improved, and their survival was increased (from a mean of 5 months to 9 months). The onset of ataxia also was delayed in most of the engrafted animals. In accordance with these observations, biochemical and pathological analysis revealed significant changes in the transplanted group as compared with nontransplanted animals. Lipid storage was reduced in several organs, and there was evidence of histologic improvement seen throughout the reticuloendothelial system, even in animals that were engrafted as low as 14%. In the central nervous system, lipid storage also was reduced, and the Purkinje cells, which are almost absent in ASMKO mice, were present in certain areas of the transplanted animals cerebella. CONCLUSIONS: These results demonstrated that BMT could alter the pathologic phenotype in ASMKO mice, but that this procedure alone was not sufficient to elicit a complete therapeutic effect.

Animals↗
PubMed1998

Cloning of the canine beta-glucuronidase cDNA, mutation identification in canine MPS VII, and retroviral vector-mediated correction of MPS VII cells.

Mucopolysaccharidosis type VII (MPS VII) is an inherited disease resulting from deficient activity of the lysosomal acid hydrolase beta-glucuronidase (GUSB) and has been reported in humans, mice, cats, and dogs. To characterize canine MPS VII, we have isolated and sequenced the canine GUSB cDNA from normal and affected animals. A single nucleotide substitution was detected in the GUSB cDNA derived from MPS VII dogs. This guanosine to adenine base change at nucleotide position 559 in the canine cDNA sequence causes an arginine to histidine substitution at amino acid position 166. Introduction of the G to A substitution at position 559 in a mammalian expression vector containing the normal canine GUSB cDNA nearly eliminated the GUSB enzymatic activity, demonstrating that this mutation is the cause of canine MPS VII. A retroviral vector expressing the full-length canine beta-glucuronidase cDNA corrected the deficiency in MPS VII cells.

Amino Acid Sequence↗
PubMed1998

MRI and electrophysiological abnormalities in a case of canine globoid cell leucodystrophy.

A six-month-old West Highland white terrier with progressive, multifocal neurological disease was diagnosed with canine globoid cell leucodystrophy (GCL). Magnetic resonance imaging (MRI) of the brain was performed, as well as electrophysiological testing (including brainstem auditory evoked response, peripheral nerve conduction velocity, repetitive stimulation, F wave analysis and electromyography). MRI findings were consistent with diffuse, symmetrical white matter disease. Electrodiagnostic testing revealed evidence of peripheral neuropathy and an abnormal brainstem auditory evoked response. These observations were consistent with the pathological changes in central and peripheral white matter described for canine GCL, and resembled what has been described in human patients. It is believed that the tests may raise the suspicion of GCL in dogs and may aid in monitoring disease progression.

Animals↗
PubMed1998

Retroviral cDNA transfer to the RPE: stable expression and modification of metabolism.

PURPOSE: To determine the versatility of retroviral vector-mediated rat beta-glucuronidase cDNA expression in the normal retinal pigment epithelium (RPE) of eyes of various species and in RPE of eyes with three types of mucopolysaccharidosis (MPS types I, VI, and VII) and to evaluate the effect of multiple transductions and long-term stable expression in the RPE. METHODS: A retroviral construct containing a rat beta-glucuronidase cDNA (NTK-BGEO) was used to infect RPE cells at subconfluence. The transduced cells were selected in G418, an antibiotic toxic to normal mammalian cells. Beta-glucuronidase activity was measured in transduced cells and media, using a fluorogenic substrate. Glycosaminoglycan profiles were examined by metabolically labeling RPE with Na2(35)SO4. RESULTS: Transduced RPE cells, regardless of species or disease status, expressed rat beta-glucuronidase. The expressed enzyme restored normal levels of glycosaminoglycans in the RPE cells of homozygous MPS VII-affected dogs by metabolizing stored glycosaminoglycans. The expressed enzyme failed to metabolize stored glycosaminoglycans of MPS I and MPS VI, indicating that overexpression could not bypass the exoglycosidase restriction. Multiple transductions increased beta-glucuronidase activity several times in the cell layer and in the media. The expression was stable in vitro for at least 12 weeks. CONCLUSIONS: A retroviral vector can mediate transfer of beta-glucuronidase in various species of normal and MPS-affected RPE. The expression is stable in vitro. The metabolism of stored glycosaminoglycans in MPS needs replacement of only the deficient enzyme to reverse the storage.

Animals↗