GENETIC AND METABOLIC DISEASES: PART ONE 196. Neonatal Retroviral Vector-Mediated Gene Therapy for MPS I in Mice and Cats Yuli Liu,1 Lingfei Xu,1 N. Matthew Ellinwood,2 Thomas O’Malley,2 Mark E. Haskins,2 Katherine P. Ponder.1 1 Internal Medicine, Washington University School of Medicine, St. Louis, MO, United States; 2Pathobiology, University of Pennsylvania School of Veterinary Medicine, Philadelphia, PA, United States. Mucopolysaccharidosis I (MPS I) is a lysosomal storage disease due to deficient α-L-iduronidase (IDUA) activity. MPS I results in growth retardation, cardiac valve abnormalities, upper respiratory disease, bone and joint abnormalities, and corneal clouding. Severely affected human patients are mentally retarded and usually die before age 10 if not treated with bone marrow transplantation. There are mouse, cat, and dog models of MPS I that are homologs of the human disease. The goal of this project is to determine if neonatal liver-directed retroviral vector (RV)-mediated gene therapy can prevent the clinical manifestations of MPS I. An RV designated hAAT-cIDUA-WPRE was generated that expressed the canine IDUA cDNA from the human α1-antitrypsin promoter. A large scale preparation of amphotropic hAAT-cIDUA-WPRE had a titer of 2x107 transducing units (TU)/ml, which is 10-fold lower than what was observed with an otherwise-identical canine βglucuronidase-expressing vector. Mice with MPS I were injected IV with 2.8x109 TU/kg at 2-3 days after birth. Two RV-treated MPS I mice have expressed stable IDUA serum levels of 1169+/-127 U/ml for 4-6 months after injection, a level that is 687-fold higher than seen in homozygous normal mice. IDUA activity remained undetectable in untreated MPS I mice. To assess the uptake of IDUA by other organs, normal mice were either untreated or were injected with RV at birth, and were sacrificed at 6 weeks of age after perfusion with saline to remove enzyme activity in blood. In the RV-treated normal mice, liver IDUA activity was 158 U/mg, which was 88-fold that in untreated normal mice. IDUA activity in RVtreated mice relative to untreated normal contols was increased 125fold in spleen, 78-fold in kidney, 49-fold in thymus, 34-fold in lung, 31-fold in heart, 3.6-fold in muscle, and 2.9-fold in brain. We conclude that the enzyme present in blood can be taken up by other organs, likely via the mannose 6-phosphate receptor. Both RV-treated MPS I mice have normal-appearing faces and paws, while age-matched untreated MPS I mice have broadened faces and thickened digits. Additional data on the clinical effect of the RV treatment on MPS I is underway and will be presented. With the high enzyme activity achieved in other organs in normal mice, a beneficial effect on most of the major clinical manifestations will likely be achieved. We have also initiated similar studies in a large animal model of MPS I. Four MPS I cats were injected at 2-3 days after birth with 0.8 to 2.5x109 TU/kg. This is 28% to 89% of the dose that was given to mice. At 2 to 12 days after transduction, serum IDUA activity was 21.4 +/12.7 (SD) U/ml, which is 60-fold higher than in the untreated MPS I cats (0.25 +/- 0.2 U/ml), and 1.7-fold that of homozygous normal controls (10.25+/-7.3 U/ml). We conclude that neonatal injection of an RV expressing cIDUA results in high levels of enzyme in serum, and will likely confer a clinical benefit in mice and cats. This work was supported by the Ryan Foundation, the National MPS Society, and the NIH (DK25759 and RR07063).
Molecular Therapy Vol. 7, No. 5, May 2003, Part 2 of 2 Parts Copyright © The American Society of Gene Therapy
197. Neonatal Retroviral Vector-Mediated Gene Therapy Causes Pathological Improvements and Prolonged Clinical Benefits without Germline Transmission in MPS VII Dogs Katherine P. Ponder,1 Margaret M. Sleeper,2 Lingfei Xu,1 N. Matthew Ellinwood,2 Charles H. Vite,2 Thomas M. O’Malley,2 Mark E. Haskins.2 1 Internal Medicine, Washington University School of Medicine, St. Louis, MO, United States; 2Pathobiology and Clinical Studies, University of Pennsylvania School of Veterinary Medicine, Philadelphia, PA, United States. Mucopolysaccharidosis VII (MPS VII) is a lysosomal storage disease due to deficient β-glucuronidase (GUSB) activity. We previously reported that IV injection of 3x109 transducing units (TU)/kg of a retroviral vector (RV) expressing GUSB into 5 newborn MPS VII dogs without any stimulus for hepatocyte replication (RV-treated) results in transduction of hepatocytes, which secrete GUSB into blood. One MPS VII animal that was treated with hepatocyte growth factor (HGF) prior to neonatal IV injection of 12x109 TU/kg of the same RV (HGF/RV-treated) had higher serum levels, but it was unclear if this was due to the HGF or the higher dose of RV. Enzyme could be taken up by other organs, which resulted in clinical benefits at 14 months. We report herein: 1) followup at 2 years after transduction for 3 RV-treated dogs and 1 HGF/ RV treated dog; 2) pathological analyses for 2 RV-treated dogs at 6 months; and 3) an absence of germline transmission in transduced males. At 2 years after transduction, serum GUSB levels remain stable at 210 +/- 128 Units/ml (78% of normal) for the RV-treated dogs, and 14,253 U/ml for the HGF/RV-treated dog, demonstrating that transduced hepatocytes are long-lived. All animals have good range of motion and no effusions in their joints, and can run. Echocardiography at 23 months shows normal valvular function and no valve thickening. The dogs appear normal neurologically, although it is unclear if untreated MPS VII dogs develop neurological disease due to their limited lifespan. We conclude that the clinical benefit of this neonatal gene therapy approach is maintained at 2 years. Two RV-treated dogs were sacrificed at 6 months after transduction. Histochemical staining demonstrated that GUSB activity was high in liver and spleen, and was detectable in the glomeruli but not tubules of the kidney, the laminia propria and submucosal region of the intestines, the bone marrow, and the cortex of the thymus. In the brain, there were only very rare cells with apparent enzyme activity. The RV-treated dogs had no detectable lysosomal storage material in the liver, spleen, or kidney glomeruli, and reduced amounts of lysosomal storage material in the heart valves, aorta, kidney tubules, intestines, thymus, and meninges, as compared with samples from age-matched untreated MPS VII dogs. Storage material was reduced in the neurons of one RV-treated dog, but was at most marginally affected in the second RV-treated dog. The RV-treated and HGF/RV-treated males have fathered 72 and 21 offspring, respectively, none of which contain RV sequences in their blood cells by PCR. Sperm from these dogs does not contain RV sequences. This demonstrates that germline transmission has not occurred. We conclude that neonatal gene therapy with an RV expressing GUSB results in pathological improvements at 6 months and a remarkable clinical effect for at least 2 years, and does not cause germline transmission. If further analyses confirm the clinical efficacy and absence of adverse effects, this approach might be used to treat patients with MPS VII or other lysosomal storage diseases.