Thomas O'Malley

Therapeutic neonatal hepatic gene therapy in mucopolysaccharidosis VII dogs.

Dogs with mucopolysaccharidosis VII (MPS VII) were injected intravenously at 2–3 days of age with a retroviral vector (RV) expressing canine β-glucuronidase (cGUSB). Five animals received RV alone, and two dogs received hepatocyte growth factor (HGF) before RV in an attempt to increase transduction efficiency. Transduced hepatocytes expanded clonally during normal liver growth and secreted enzyme with mannose 6-phosphate. Serum GUSB activity was stable for up to 14 months at normal levels for the RV-treated dogs, and for 17 months at 67-fold normal for the HGF/RV-treated dog. GUSB activity in other organs was 1.5–60% of normal at 6 months for two RV-treated dogs, which was likely because of uptake of enzyme from blood by the mannose 6-phosphate receptor. The body weights of untreated MPS VII dogs are 50% of normal at 6 months. MPS VII dogs cannot walk or stand after 6 months, and progressively develop eye and heart disease. RV- and HGF/RV-treated MPS VII dogs achieved 87% and 84% of normal body weight, respectively. Treated animals could run at all times of evaluation for 6–17 months because of improvements in bone and joint abnormalities, and had little or no corneal clouding and no mitral valve thickening. Despite higher GUSB expression, the clinical improvements in the HGF/RV-treated dog were similar to those in the RV-treated animals. This is the first successful application of gene therapy in preventing the clinical manifestations of a lysosomal storage disease in a large animal.

Long-term amelioration of feline Mucopolysaccharidosis VI after AAV-mediated liver gene transfer.

Mucopolysaccharidosis VI (MPS VI) is caused by deficient arylsulfatase B (ARSB) activity resulting in lysosomal storage of glycosaminoglycans (GAGs). MPS VI is characterized by dysostosis multiplex, organomegaly, corneal clouding, and heart valve thickening. Gene transfer to a factory organ like liver may provide a lifetime source of secreted ARSB. We show that intravascular administration of adeno-associated viral vectors (AAV) 2/8-TBG-felineARSB in MPS VI cats resulted in ARSB expression up to 1 year, the last time point of the study. In newborn cats, normal circulating ARSB activity was achieved following delivery of high vector doses (6 × 10(13) genome copies (gc)/kg) whereas delivery of AAV2/8 vector doses as low as 2 × 10(12) gc/kg resulted in higher than normal serum ARSB levels in juvenile MPS VI cats. In MPS VI cats showing high serum ARSB levels, independent of the age at treatment, we observed: (i) clearance of GAG storage, (ii) improvement of long bone length, (iii) reduction of heart valve thickness, and (iv) improvement in spontaneous mobility. Thus, AAV2/ 8-mediated liver gene transfer represents a promising therapeutic strategy for MPS VI patients.

Gene therapy for mucopolysaccharidosis type VI is effective in cats without pre-existing immunity to AAV8.

Liver gene transfer with adeno-associated viral (AAV) 2/8 vectors is being considered for therapy of systemic diseases like mucopolysaccharidosis type VI (MPS VI), a lysosomal storage disease due to deficiency of arylsulfatase B (ARSB). We have previously reported that liver gene transfer with AAV2/8 results in sustained yet variable expression of ARSB. We hypothesized that the variability we observed could be due to pre-existing immunity to wild-type AAV8. To test this, we compared the levels of AAV2/8-mediated transduction in MPS VI cats with and without pre-existing immunity to AAV8. In addition, since levels of lysosomal enzymes as low as 5% of normal are expected to be therapeutic, we evaluated the impact of pre-existing immunity on MPS VI phenotypic rescue. AAV2/8 administration to MPS VI cats without pre-existing neutralizing antibodies to AAV8 resulted in consistent and dose-dependent expression of ARSB, urinary glycosaminoglycan (GAG) reduction, and femur length amelioration. Conversely, animals with pre-existing immunity to AAV8 showed low levels of ARSB expression and limited phenotypic improvement. Our data support the use of AAV2/8-mediated gene transfer for MPS VI and other systemic diseases, and highlight that pre-existing immunity to AAV8 should be considered in determining subject eligibility for therapy.

Neonatal Gene Therapy With a Gamma Retroviral Vector in Mucopolysaccharidosis VI Cats

Mucopolysaccharidosis (MPS) VI is due to a deficiency in the activity of N-acetylgalactosamine 4-sulfatase (4S), also known as arylsulfatase B. Previously, retroviral vector (RV)-mediated neonatal gene therapy reduced the clinical manifestations of MPS I and MPS VII in mice and dogs. However, sulfatases require post-translational modification by sulfatase-modifying factors. MPS VI cats were injected intravenously (i.v.) with a gamma RV-expressing feline 4S, resulting in 5 ± 3 copies of RV per 100 cells in liver. Liver and serum 4S activity were 1,450 ± 1,720 U/mg (26-fold normal) and 107 ± 60 U/ml (13-fold normal), respectively, and were directly proportional to the liver 4S protein levels for individual cats. This study suggests that sulfatase-modifying factor (SUMF) activity in liver was sufficient to result in active enzyme despite overexpression of 4S. RV-treated MPS VI cats achieved higher body weights and longer appendicular skeleton lengths, had reduced articular cartilage erosion, and reduced aortic valve thickening and aortic dilatation compared with untreated MPS VI cats, although cervical vertebral bone lengths were not improved. This demonstrates that therapeutic expression of a functional sulfatase protein can be achieved with neonatal gene therapy using a gamma RV, but some aspects of bone disease remain difficult to treat.

247. Gene Therapy with a Retroviral Vector Expressing Canine |[beta]|-Glucuronidase to Juvenile (7 Week Old ) Dogs Improves the Biochemical Manifestations of MPS VII

Top of pageAbstract Mucopolysaccharidosis VII (MPS VII) is a lysosomal storage disease due to deficiency of |[beta]|-glucuronidase (GUSB). Manifestations include bone and joint disease, heart disease, and neurological dysfunction. The MPS VII dog has a missense mutation in GUSB (R166H) that results in clinical manifestations that resemble those in humans. We have previously demonstrated that neonatal gene therapy with 3|[times]|109 TU/kg of a retroviral vector (RV) expressing canine GUSB from the human |[alpha]|1-antitrypsin promoter (hAAT-cGUSB-WPRE) resulted in transduction of hepatocytes and 195+/-36 U/ml of GUSB in serum. This resulted in a marked improvement in cardiac, bone and joint, and other manifestations. However, most patients with MPS VII are not identified at birth, and it will be necessary to determine the effect of transfer into older animals. Seven week-old MPS VII dogs were injected IV with 1|[times]|1010 transducing units (TU)/kg of hAAT-cGUSB-WPRE. Some received a cumulative dose of hepatocyte growth factor (HGF) over 24 hours of 2.5 mg/kg, then were injected with RV at 24, 48, 72, and 96 hours after the first dose of HGF (HGF/RV). Others received RV once a day for 4 days without preceding HGF (RV alone). Dogs that received HGF/RV or RV alone at 7 weeks had transduced hepatocytes and achieved stable expression of GUSB in serum at 185+/-54 U/ml or 51+/-5 U/ml for up to 1 year, respectively, although differences were not statistically significant. The serum GUSB achieved per TU/kg given was 28% and 8%, respectively, of that achieved after neonatal gene transfer, which likely reflects lower levels of hepatocyte replication in juveniles. Some animals were sacrificed at 6 months after gene transfer, and organs analyzed for biochemical correction of disease. The results in both juvenile transfer groups were similar and were pooled for statistical analyses. MPS VII results in elevation of the secondary lysosomal enzyme |[beta]|-hexosaminodase (|[beta]|-hex) and glycosaminoglycan (GAG) levels, and normalization of these occurs with successful therapies. In liver, spleen, jejunum, and lung, GUSB activity was 1% to 15% of normal levels and there was a marked reduction in |[beta]|-hex and GAG levels. In thymus, pancreas, kidney, and muscle, GUSB activity was 0.25% to 1% of normal and there was a marked or partial reduction in |[beta]|-hex and GAG levels. However, brain GUSB activity was only 0.2% of normal and there was little reduction in |[beta]|-hex (GAG was not evaluable as untreated MPS VII dogs do not have elevated brain GAG levels). Despite these biochemical improvements, the mobility of the dogs was only slightly improved and all were unable to walk by 12 months or earlier. We conclude that gene therapy into juvenile dogs results in expression similar to that observed after neonatal gene transfer, and marked biochemical improvements in somatic organs. This is the first demonstration of using juvenile gene therapy to improve disease in a large animal model for LSD. However, there was little biochemical improvement in brain, and the bone disease remained severe.

421. Liver Contains Sufficient Sulfatase Modifying Factor for 4-Sulfatase and Correction of MPS VI with Neonatal Retroviral Gene Therapy in Cats

Sulfatases are enzymes that remove a sulfate from a substrate. 4-sulfatase (4S; also known as arylsulfatase B) is a lysosomal enzyme that removes a sulfate from the 4 position of N-acetylgalactosamine, a component of the glycosomaminoglycan (GAG) dermatan sulfate. Deficient activity of 4S results in mucopolysaccharidosis VI (MPS VI), a disorder that includes bone and joint abnormalities, corneal clouding, and cardiac disease, but does not usually involve the brain. The lack of neurological involvement makes this an excellent candidate for somatic therapies. All sulfatases are modified post-translationally by enzymes designated as sulfatase modifying factors (SUMF) that convert a cysteine in the active site to a C-alpha-formylglycine, which is absolutely essential for enzyme activity. SUMF is readily saturated after over expression of a sulfatase in fibroblasts in vitro, resulting in limitations in the amount of enzyme that can be produced and a reduction in activity of other sulfatases that require the same modification. It was, therefore, unclear if hepatocytes would contain sufficient SUMF activity to modify the 4S produced by hepatocytes after liver-directed gene therapy for MPS VI. A retroviral vector (RV) designated hAAT-f4S-WPRE expressing feline 4S was generated. 4S-deficient feline fibroblasts were transduced in vitro with increasing multiplicities of infection. The 4S enzyme activity (4MU-sulfate assay) and 4S protein levels (immunoblot) were directly proportional to the DNA copy number, which leveled off at about 2 copies per cell despite using higher multiplicities of infection. This likely reflects an inhibition of transduction when large amounts of vector are used, which has been described previously, suggesting that SUMF is not limiting when relatively low copy numbers are achieved. hAAT-f4S-WPRE (4.2-3.4X10E9 TU/kg) was injected IV into 6 newborn cats with MPS VI. They achieved stable expression at 109+/-49 U/ml in serum, which is 10-fold normal and has been maintained for up to 1.5 years in the oldest animals. Analysis of liver biopsies demonstrated that the 4S enzyme activity was directly proportional to the amount of 4S protein, suggesting that all of the enzyme was functional, and that modification by SUMF was not limiting. The cats have marked improvement in the clinical skeletal manifestations. The cats also have biochemical evidence of improvement, which includes high levels of 4S and a reduction in GAG and secondary lysosomal enzyme activity in other organs. We conclude that liver contains sufficient SUMF to modify 4S, and that liver-directed gene therapy can markedly improve the clinical manifestations of this disorder.

857. Intravenous Retroviral Gene Therapy in Neonatal Mucopolysaccharidosis VI Cats|[ast]|

Mucopolysaccharidosis (MPS) VI is a lysosomal storage disease caused by deficient activity of N-acetylgalactosamine 4-sulfatase (4S) resulting in an inability to degrade the glycosaminoglycan (GAG) dermatan sulfate, which accumulates in the lysosomes. Clinical features include skeletal abnormalities, growth retardation, facial dysmorphia, and corneal clouding. Two MPS VI cats were injected intravenously at 4 days of age with a Moloney murine leukemia virus-based retroviral vector containing the human alpha-1-antitrypsin promoter, the feline 4S (f4S) cDNA (kindly provided by John J. Hopwood), and the woodchuck hepatitis virus posttranscriptional regulatory element. Both animals received 7.2X10E9 transducing units/kg of body weight. Stable f4S activity has been detected in serum throughout the study and the most recent values from samples taken at 6 months were 23 and 9 times normal. The f4S activities in liver biopsies taken at 5 months were 82 and 38 times normal and the concentration of GAG in the biopsies was reduced to normal levels, 1.2 and 1.8 ug/mg protein, respectively. In comparison, the GAG concentration in liver biopsies from the untreated MPS VI littermates was 34.9 and 20.6 ug/mg. The body weights of the treated cats are 130% and 122% that of their two untreated MPS VI sex-matched control littermates (average of 1.89 kg) and are not different from normal unrelated age- and sex-matched controls. There are profound differences in the appearance of the treated and untreated cats. The MPS VI controls have short ears and tails and their faces are broad and flat. They have joint stiffness and major locomotor difficulties. The treated cats have only mild facial dysmorphia, a much freer range of pelvic motion, near normal gaits, and are more physically active than their MPS VI littermates. The cat with the higher f4S activity is nearly indistinguishable from a phenotypically normal cat except for the eyes, as there was no significant improvement in corneal clouding. MPS VI is particularly suited to liver-directed therapy because unlike other MPS disorders, CNS involvement is minimal. Because MPS VI is a progressive disease, the full extent of the clinical benefit will only become evident over time. We will present a 10-month update, including radiographs comparing the skeletons of normal, treated, and untreated MPS VI cats.

162. Neonatal gene therapy induces tolerance in dogs but not cats

Immune responses after gene therapy could include an antibody response that blocks the activity of a blood protein, or a cytotoxic T lymphocyte (CTL) response that destroys transduced cells. Either could reduce the efficacy of gene therapy. We have previously shown that high dose neonatal gene therapy resulted in tolerance to canine Factor IX (cFIX), human FIX (hFIX), and canine β-glucuronidase in mice. However, since large animals may have a more mature immune system at birth, we evaluated immune responses after neonatal gene therapy in dogs and cats. Neonatal normal dogs that were transduced with a medium (3 × 109 transducing units (TU)/kg) or a low (8 × 107 TU/kg) dose of an RV expressing hFIX achieved stable expression of hFIX for over 6 months at 494 +/− 132 ng/ml (10% of normal) and 26 +/− 12 ng/ml (0.5% of normal), respectively. None of the neonatal RV-treated dogs developed anti-hFIX IgG. Further, the low dose group did not develop antibodies after infusion of 10 doses of hFIX (30 IU/kg/dose, given once per week starting at 2 months after birth), and thus were truly tolerant. Similar results were obtained in one hemophilia B dog that was transduced with 3 × 109 TU/kg of RV at birth and achieved 220 ng/ml of hFIX, as he did not develop anti-hFIX antibodies either before or after stimulation. In contrast, normal dogs that did not receive neonatal gene therapy developed high levels of anti-hFIX antibodies in response to an identical regimen of hFIX protein infusion. We conclude that neonatal gene therapy results in tolerance to hFIX in dogs. A similar gene therapy approach was tested in cats with mucopolysaccharidosis I (MPS I). Six MPS I cats received 1 × 109 TU/kg of an RV expressing canine α-L-iduronidase (IDUA) shortly after birth. All achieved detectable IDUA activity in blood within 2 weeks after gene transfer, with average serum levels of 25.2 units (U)/ml. However, serum activity declined to undetectable levels (<0.2 U/ml) by 2 months. This decline was not associated with anti-cIDUA antibodies. None of 5 cats analyzed at 2 months had detectable IDUA activity or RV DNA sequences in the liver, although these were high at 10 days in 1 cat (DNA copy number 0.25 copies/cell). Cats are capable of expressing this RV long-term, as MPS VI cats that received neonatal injection of an otherwise-identical vector expressing the feline N-acetylgalactosamine 4-sulfatase have maintained expression for 4 months. We infer that the cats most likely developed a CTL response to cIDUA after neonatal gene therapy. Interestingly, 3 of 18 MPS I mice that received 1 × 108 TU/kg of the cIDUA-expressing RV at birth probably developed a CTL response, although 0 of 25 MPS I mice that received 1 × 109 TU/kg did so. We conclude that cats develop a potent immune response to cIDUA but dogs do not develop an immune response to hFIX after neonatal gene therapy. It is possible that the intracellular cIDUA is more potent at inducing a CTL response than is the secreted hFIX. Alternatively, cats may have more mature immune systems at birth than do dogs. Further experiments will determine if cats produce immune responses to hFIX after neonatal gene therapy, and will further evaluate the CTL response to cIDUA in cats.