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

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1 original article Neonatal Gene Therapy With a Gamma Retroviral Vector in Mucopolysaccharidosis VI Cats Katherine P Ponder,2, Thomas M O Malley 3,4, Ping Wang 3,4, Patricia A O Donnell 3,4, Anne M Traas 3,4, Van W Knox 3,4, Gustavo A Aguirre 3,4, N Matthew Ellinwood 3,4, Jason A Metcalf,2, Bin Wang,2, Emma J Parkinson-Lawrence 5, Meg M Sleeper 3,4, Doug A Brooks 5, John J Hopwood 5 and Mark E Haskins 3,4 Department of Internal Medicine, Washington University School of Medicine, St Louis, Missouri, USA; 2 Department of Biochemistry and Molecular Biophysics, Washington University School of Medicine, St Louis, Missouri, USA; 3 Department of Pathobiology, School of Veterinary Medicine, University of Pennsylvania, Philadelphia, Pennsylvania, USA; 4 Department of Clinical Studies, School of Veterinary Medicine, University of Pennsylvania, Philadelphia, Pennsylvania, USA; 5 Lysosomal Diseases Research Unit, SA Pathology at Women s and Children s Hospital, North Adelaide, South Australia 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 I in mice and dogs. However, sulfatases require post-translational modification by sulfatase-modifying factors. cats were injected intravenously (i.v.) with a gamma RV-expressing feline 4S, resulting in 5 3 copies of RV per 00 cells in liver. Liver and serum 4S activity were,450,720 U/mg (26-fold normal) and U/ml (3-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 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 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. Received 29 October 20; accepted 3 January 202; advance online publication 6 March 202. doi:0.038/mt INTRODUCTION The mucopolysaccharidoses (MPS) are lysosomal storage diseases that result from deficient activity of enzymes that degrade glycosaminoglycans (GAGs). (Maroteaux Lamy syndrome; Online Mendelian Inheritance in Man #253200) is due to a deficiency in the activity of N-acetylgalactosamine 4-sulfatase (4S), also known as arylsulfatase B. Although dermatan sulfate is the major GAG seen in the urine of patients, 4S also contributes to the degradation of chondroitin sulfate A, which contains N-acetylgalactosamine that is sulfated at the 4 position. MPS VI has an incidence of :248,000 2 and can result in bone abnormalities collectively referred to as dysostosis multiplex that affect growth, gait, and appearance. In addition, patients can develop hepatosplenomegaly, corneal clouding, and cardiac valve abnormalities, although neurological disease is generally believed to be absent. 3 9 cats are homozygous for a single nucleotide change that replaces a leucine with a proline at amino acid 476 of the 533 amino acid feline 4S precursor protein. 0 Features in MPS VI cats resemble those in humans, except hepatosplenomegaly is less severe 4 and some neurons develop lysosomal storage. 5 The pathogenesis of and other types of MPS might involve activation of the innate immune system via the Toll-like receptor and/or complement 9 signaling pathways by GAGs, resulting in upregulation of inflammatory cytokines and destructive proteases. Some clinical aspects of such as hepatosplenomegaly, ear infections, and poor mobility can be reduced in humans by hematopoietic stem cell transplantation as enzyme with mannose 6-phosphate (M6P) secreted locally by blood cells can be internalized via the M6P receptor present on the surface of most cells. 23 However, heart disease, bone disease, and corneal clouding are not resolved with this therapy. Hematopoietic stem cell transplantation has also demonstrated some efficacy in rats with. 24 Alternatively, can be treated by intravenous (i.v.) injection of recombinant enzyme, a procedure referred to as enzyme replacement therapy (ERT) that was approved for use in 2005 in the United States. ERT has improved mobility and pulmonary function in humans with, although ERT does not reverse established skeletal, cardiac, or ocular disease, and its long-term efficacy is still being investigated Initiation of ERT very shortly after birth is more effective than initiation at older ages in humans 30 and in cats. 3,32 Systemic gene therapy enables transduced cells to continuously secrete 4S with M6P targeting signals into the bloodstream and is being tested in animal models of lysosomal storage disease with a variety of vectors. 33 Achieving >5% of normal activity in cells is believed to be a reasonable target for prevention of disease. 34 I.v. injection of adeno-associated viral (AAV) vectors expressing 4S has reduced some clinical manifestations of the disease in Correspondence: Katherine P Ponder, Department of Internal Medicine, Washington University School of Medicine, 660 South Euclid Avenue, St Louis, Missouri 630, USA. kponder@dom.wustl.edu vol. 20 no. 5, may 202

2 mice and cats. 35,36 We have previously demonstrated that neonatal gene therapy with a gamma retroviral vector (RV) could achieve stable and high-level expression of canine -L-iduronidase (IDUA) and -glucuronidase from the liver in mice, cats, and dogs with MPS I or I, and that lower expression was achieved in spleen and blood cells The level of expression that we have achieved with a gamma RV exceeded that reported in MPS I or I mice with AAV or lentiviral vectors, 33 although multiple factors other than the vector per se could contribute to this difference. However, it was unclear if gene therapy would be as effective for a gene that encodes a sulfatase protein, as all sulfatases are post-translationally modified in the endoplasmic reticulum by sulfatase-modifying factor (SUMF) or SUMF2, which change a cysteine to the active site c- -formylglycine required for catalytic activity. 40,4 Indeed, overexpression of 4S has resulted in multiple sulfatase deficiency in cultured cells due to saturation of this modification pathway 42 while coexpression of SUMF enhanced enzyme activity of sulfatases in vivo in muscle, 43 although there appeared to be sufficient SUMF activity to achieve near-normal 4S enzyme activity in liver. Herein, we report high-enzyme activity and clinical improvements in cats that were treated with a feline 4S-expressing RV as neonates and followed for 6 months to 8 years. RESULTS The RV designated haat-f4s-wpre is diagramed in Figure. Six cats from group were treated with transducing units (TU)/kg of this amphotropic RV within 4 days after birth, as detailed in the Materials and Methods section. Serum 4S activity was tested to determine the level of expression over time. Group RV-treated cats had U/ml of serum 4S activity (N = 6), as shown with the open circles in Figure 2, which was 3-fold the value in homozygous normal cats and 60-fold that in untreated cats; background activity in untreated cats was likely due to the activity of other sulfatases against the nonspecific 4-methylumbelliferone-sulfate substrate. Average serum activity in individual cats varied substantially from 39 to 88 U/ml. Several years later, group 2 RV-treated cats were injected as newborns with transducing units/kg of a different batch of RV, which was approximately threefold the dose given to group, resulting in an average serum 4S activity of U/ml (N = 6), as shown with the closed circles in Figure 2. The treatment was welltolerated with no elevation in serum transaminases or changes in blood cell counts at 24 hours after the first dose of RV (data not shown). The amount of circulating 4S with the M6P moiety was 26 3% of the total 4S activity for 4 RV-treated cats with high haat-f4s-wpre LTR haat Feline 4-sulfatase WPRE LTR Figure Retroviral vector (RV) diagram. haat-f4s-wpre contains intact long-terminal repeats (LTRs), an extended packaging signal ( + ), the human -antitrypsin (haat) promoter, the feline N-acetylgalactosamine 4-sulfatase (f4s) cdna, and the woodchuck hepatitis virus post-transcriptional regulatory element (WPRE). The arrows indicate that transcription can initiate from the LTR or the haat promoter. serum 4S activity. The limited sensitivity of the 4-methylumbelliferone-sulfate assay and the background due to other sulfatases precluded accurate determination of the percent of phosphorylated 4S in normal or untreated cats. 4S activity, -glucuronidase activity, and GAGs in organs and urine Liver 4S activity was determined for all six RV-treated cats from group using samples collected at 4 6 months, as shown in Figure 3a. Liver 4S activity in crude extracts was high at,450,720 U/mg (26-fold normal) and values in individual RV-treated cats correlated with each animal s serum activity. Activity in other organs was determined for samples from the RV-treated cats 6600 and 663, which were sacrificed at 6 months after birth. These cats had serum 4S activities of 49 and 9 U/ml, respectively, which were only 65% of the average serum 4S activity for group but were still eightfold the normal value of 8 3 U/ml. 4S activity in crude extracts of spleens from RV-treated cats was fourfold normal. 4S activities in kidney, lung, heart, aorta, skeletal muscle, pancreas, and white blood cells (WBC) from RV-treated cats were 9 85% of the values in normal cats, demonstrating that supranormal serum 4S does not necessarily translate into normal levels of 4S in tissues, as enzyme needs to diffuse to cells and be taken-up via the M6P receptor. 4S activity in these organs in RV-treated cats was two- to ninefold that in untreated cats (Figure 3a), although only some of the values in RV-treated cats were significantly higher than for untreated cats, as the background activity in untreated cats of 7 3% of normal complicated measurements. No increase in 4S activity was detected in crude extracts from the brain of RV-treated cats above the background in untreated cats. MPS animals exhibit elevations in other lysosomal enzymes and in GAG levels; normalization of these biochemical parameters is an excellent measure of disease correction. Activity of the lysosomal enzyme -glucuronidase and GAG levels were reduced to normal levels in several tissues that were analyzed U/ml, Serum 4-sulfatase activity Years after birth Group Group Figure 2 Serum4-sulfatase (4S) activity. Mucopolysaccharidosis (MPS) VI cats shown in the left column of the key were injected intravenously (i.v.) with transducing units (TU)/kg of haat-f4s-wpre at 2 4 days after birth, and are designated group in the text and are depicted with open symbols. cats shown in the right column were injected i.v. with TU/kg of a different batch of vector, and are designated group 2 in the text, and are depicted with closed symbols. Serum was tested for 4S activity using the substrate 4-methylumbelliferonesulfate (4MU-S) at the indicated years after birth. Each line shows activity for an individual cat. Mean values ± SD in adult homozygous normal cats were 8.2 ± 2.6 U/ml and those in untreated cats were 2.6 ±.0 U/ml as indicated with gray and yellow boxes, respectively, in the online color version, and as indicated by the dark and light grey boxes in the print version. Individual cats are identified with different colors in the print version. Molecular Therapy vol. 20 no. 5 may

3 a 4S U/mg b GUSB U/mg, NS,000 4-Sulfatase activity (no IP) -Glucuronidase activity RV d ug GAG/ mg Cr, Urine GAG RV c g GAG/ mg protein f RV 4S Endog 4S C/EBP Liver Spleen Glycosaminoglycans MPS VI 6600 Kidney-C Kidney-M Lung Heart Aorta Muscle Brain Pancreas WBC Liver RV e 4S U/mg 00,000 0,000, Sulfatase activity after IP Kidney-C Kidney-M g % protein on immunblot Muscle Brain Pancreas WBC Liver 4S activity versus Liver 4S protein 20 0 R 2 = 0.93 P = 0 0 2,000 4,000 Liver sulfatase activity (U/mg) Figure 3 Biochemistry of liver, other organs and urine. Samples from liver of all six of the group retroviral vector (RV)-treated cats were obtained at biopsy or postmortem at 4 7 months. Samples from other tissues of two RV-treated cats from group, 6600 and 663, were collected at 7 and 6 months, respectively. Urine samples were collected at years after birth. (a c) Analysis of crude extracts of organs. The mean ± SD of 4-sulfatase (4S) activity without immunocapture (no IP), -glucuronidase (GUSB) activity, and glycosaminoglycan (GAG) levels per mg of protein was determined as detailed in the Materials and Methods section. A large X instead of data for a particular organ indicates that an assay was not performed for that organ. Samples from homozygous normal (N = 3) and homozygous mucopolysaccharidosis (MPS) VI (N = 4) cats were age-matched. (d) Urine GAG. Urine GAGs were normalized to the amount of creatinine in the sample for four normal, four, and six RV-treated cats. (e) Organ 4S activity after immunocapture. Samples were immunocaptured with a feline 4S-specific antibody, and the feline 4S activity per mg wet weight of the original sample was determined with a 4-methylumbelliferone-sulfate (4MU-S) substrate. Samples from three normal, four, and two RV-treated cats were evaluated. For all biochemical assays in (a e), P of and P < 0.0 for comparison of the two groups linked with a bracket using ANOVA with Tukey post-hoc analysis. (f) Liver 4S protein levels. Immunoblot was performed for feline 4S protein levels using liver samples from normal, untreated, or RV-treated cats. After probing for feline 4S, the blot was stripped and reprobed for the liver protein C/EBP for normalization. Samples from RV-treated cats are organized from left to right according to lowest to highest liver 4S activity. The position of the endogenous 65 kda feline 4S band found in normal and cats (Endog 4S) and the 68 kda band found in RV-treated cats (RV-4S) are indicated. (g) Liver 4S activity versus 4S protein. For individual RV-treated cats, liver 4S activity was plotted versus liver protein levels. The latter was determined from scanning the immunoblot for feline 4S protein with normalization to the signal for C/EBP. The identification number for each cat is indicated at the left. A linear regression line for the data is shown with the R 2 value. (Figure 3b and c). The reduced GAG levels in brain suggest that 4S enzyme diffuses into brain from blood, 4S is expressed from blood cells that migrate into the brain, or that there is some transduction of cells in the brain after neonatal i.v. injection, as has been discussed previously after a similar gene therapy approach for MPS VII in dogs. 39 GAG levels in urine are elevated in untreated MPS VI patients and normalization is a commonly used biochemical marker of disease correction. Indeed, GAG levels in urine samples from untreated cats were 5.4-fold normal (P = 0.0) and were normalized in RV-treated cats (P = versus untreated ; P not significant versus normal), as shown in Figure 3d. The 4S background activity in untreated cats averaged 3% of normal activity in Figure 3a, which is likely due to cleavage of the fluorogenic substrate 4-methylumbelliferone-sulfate by other sulfatases in the crude samples. To determine enzyme activity more accurately, 4S from the homogenates was captured with an anti-4s antibody and then tested for activity (Figure 3e). With this assay, 4S activity in untreated cats was % of that in normal cats, and activity in RV-treated cats in kidney, skeletal muscle, and pancreas was 4% of normal. Although 4S activity in brain of RV-treated cats was low at only 24 5 U/mg wet weight (0.2% of normal), the activity was 3-fold the value found in untreated cats, which was not statistically significant, probably due to the small number of animals evaluated. Approximately tenfold higher activity in the immune-capture assay compared with the activity in crude extracts may reflect different homogenization or assay conditions. Livers were also tested for the amount of 4S protein by immunoblot (Figure 3f). Samples from both normal and cats had a band at ~65 kda on nonreducing polyacrylamide gel electrophoresis, which corresponded to the expected size of feline 4S. 0,44 RV-treated cats had a band that migrated at ~68 kda, slightly slower than the 4S protein present in normal and cats, vol. 20 no. 5 may 202

4 for reasons that were unclear. The amount of feline 4S protein on immunoblot was 20-fold normal and was directly proportional to the liver 4S activity, as shown in Figure 3g (R 2 value of 0.93). These data suggest that most of the 4S produced by the liver was functional and, thus, had converted the cysteine at the active site to C- -formyl glycine, which implies that there was no saturation of the SUMF activity needed for this modification at the levels of expression that were achieved. Nucleic acids in liver, spleen, and WBC RV DNA and RNA levels in liver, spleen, and WBCs were determined by real-time PCR for the Woodchuck hepatitis virus post-transcriptional regulatory element (WPRE) of the RV with a Copies per 00 cells 8 DNA Liver Spleen WBC Percent -actin RNA Liver Spleen WBC Figure 4 Retroviral vector (RV) nucleic acid levels in RV-treated cats. (a) RV DNA levels. RV DNA levels were determined by real-time PCR for the WPRE of the RV at 4 6 months after transduction for samples from group RV-treated mucopolysaccharidosis (MPS) VI cats, and normalized to the copies of -actin to determine the copies per cell. Five samples from group were evaluated from liver (values for cat 6437 are not shown as the DNA was of poor quality), 2 samples were evaluated from spleen (6600 and 663), and 6 samples from group were evaluated from white blood cells (WBC). (b) RV RNA levels. RNA was evaluated for the same samples as in a, except liver samples from cats 6437 and 6438 were not included due to poor RNA quality. RV DNA and RNA sequences were not detected for samples from normal and untreated cats (data not shown). b normalization to -actin, as shown in Figure 4. The RV DNA levels in RV-treated cats in liver, spleen, and WBCs were 5 3, 2, and 4 2 copies/00 cells, respectively. cats and untreated cats had no detectable RV copies, as expected (data not shown). The RV RNA level in liver in RV-treated cats was 0 % the level of -actin in liver, which was fivefold the level in spleen, and,900-fold the level in WBCs. Although WBCs produced only small amounts of 4S RNA, this expression was >00-fold the background activity in WBCs for cats that did not receive RV, and expression in blood cells could contribute to the observed clinical effect. Clinical evaluation The clinical course of group RV-treated cats is summarized in Table. Cats from group 2 are not shown in this table, as their time of evaluation was only year. cats from the same colony generally live more than 0 years. Some untreated MPS VI cats live for up to 7 years, although some develop spinal cord compression due to bony proliferation in the thoracolumbar spine at years of age and require euthanasia. 2 Untreated MPS VI cats can also develop debilitating arthritis at 2 years or later that can require euthanasia, which manifests as an inability to get in and out of the litterbox as well as general difficulty in moving. RV-treated cats 6600 and 663 were sacrificed at years old while in good health to obtain organs for biochemical analyses. Cat 6438 contracted feline infectious peritonitis, an infection seen in colony cats, and was euthanized at.4 years. Cat 6598 was euthanized at 3.5 years due to sudden onset of paraplegia, probably due to spinal cord compression from thoracolumbar spine instability, 2 although radiographs failed to identify a cause. The remaining two RV-treated cats from group remain alive and well at 8 years of age, albeit with some reduction in mobility. For group 2, RV-treated cats, one animal died at 3 weeks of age from a patent ductus arteriosus, which has no known relationship to, Table Summary of results in group RV-treated cats Liver 4S activity at 4 to 6 months in U/mg (fold normal) 4S Activity Dose of RV in Cat 0 9 TU/kg a Clinical evaluation 6437 Female 6.3 Alive at 8.3 years with slight reduction in mobility 4,709 (86-fold) 64 (20-fold),936 (35-fold) 88 (23-fold) 6438 Female 6.3 Euthanized at.4 years for feline infectious peritonitis, good mobility prior to death Average serum 4S activity in U/ml (fold normal) 6597 Male 4.6 Alive at 7.8 years with moderate reduction in mobility 425 (8-fold) 39 (5-fold) 6598 Male 4.6 Euthanized at 3.5 years with paraplegia and loss of 382 (7-fold) 3 (4-fold) sensation in hind-limbs due to spinal cord compression between T3 and L Male 4.2 Sacrificed at 0.7 years for tissue when alive and well 87 (3-fold) 49 (6-fold) 663 Male 3.4 Sacrificed at 0.6 years for tissue when alive and well,064 (9-fold) 9 (-fold) None Alive and well (-fold normal; N = 8) (-fold normal; N = 36) None Of 0 cats, was euthanized at 0.5 years with spinal cord compression, was euthanized at 4 years for congestive heart failure, 7 were euthanized at 2-5 years for arthritis, and cat is alive at 7 years with poor mobility 7 2 (0.-fold normal; N = 4) (0.2-fold normal; N = 30) Abbreviations: 4S, 4-sulfatase; haat, human -antitrypsin; MPS, mucopolysaccharidosis; RV, retroviral vector; TU, transducing units; WPRE, Woodchuck hepatitis virus post-transcriptional regulatory element; a Group cats received the indicated dose in billion transducing units of haat-f4s-wpre per kg at 2 4 days after birth. Molecular Therapy vol. 20 no. 5 may

5 and the other cats are doing well at year of age. The RV-treated males 6597 and 6598 fathered 85 and 28 kittens, respectively, while the RV-treated female 6437 mothered 32 kittens from 0 litters. None of 93 samples from progeny of these RV-treated cats had RV sequences detected with real-time PCR of WBC DNA for the WPRE of the vector, demonstrating an absence of germline transmission in animals evaluated to date. Effect of gene therapy on facial dysmorphia, growth, skeletal radiographs, and gait Cats with have small ears and a broad and flat face, which is similar to the facial dysmorphia and low-set ears observed in children with. Figure 5a d and Supplementary Figure S illustrate that the faces of two RV-treated cats were indistinguishable from normal, and were clearly different from an untreated littermate. The body weights of RV-treated males and RV-treated females were statistically greater than for untreated cats of the same gender (P < 0.04), and did not differ from values in normal cats (Figure 5e). Bone abnormalities are an important feature of. Figure 5f shows measurements of lengths of the femur and C3 vertebral body; Figure 5g shows scoring of various parameters of abnormalities in the cervical spine; and Figure 5h o show representative radiographs of the cervical spine. Femur lengths were normalized to those of gender-matched cats in the same colony, and the average ratio to normal was determined. Untreated MPS VI cats had femur lengths that were 88 3% of normal (P < 0.00 versus normal), and RV-treated cats had normalization of femur lengths to 03 2% of normal, which was statistically greater than the value in untreated cats (P < 0.00), but was not significantly different from the value in normal cats. MPS VI cats had C3 vertebral body heights that were only 44 8% normal (P < 0.00). RV-treated cats had C3 heights that remained markedly abnormal at 58 8% normal (P < 0.00 versus normal; not significant versus ). The images of radiographs in Figure 5h o illustrate the reduced heights of the C3 vertebral bodies in untreated as well as RV-treated cats. Radiographs also demonstrate that untreated cats have widening of the articular processes (Figure 5i), a bony bridge connecting the dorsal aspects of adjacent vertebrae (Figure 5m) and beaking of the caudoventral aspect of the vertebral body (Figure 5m), which represents bony proliferation that resembles a bird s beak. These and other parameters of the cervical spine radiographs were scored subjectively from 0 (normal) to +2 (severely abnormal) for cats at 2 3 years of age, when radiographs from 3 RV-treated cats were available for evaluation. Beaking and articular process widening were significantly improved in the RV-treated cats compared with the untreated cats, but remained abnormal. Tipping (see legend for definition), articular facet fusion and vertebral body shortening were not improved in RV-treated compared with untreated cats. Nevertheless, the quality of the vertebral bone was improved in RV-treated cats, as there was less bone lucency and bony proliferation, and the end-plates were better defined than for untreated cats. Figure 5p shows scoring of various parameters in the carpus (wrist). Articular cartilage erosion and epiphyseal dysplasia/lucency were improved in RV-treated cats compared with untreated cats but, other parameters were not improved. Magnetic resonance imaging (MRI; Figure 6) was performed at 7 8 years of age to evaluate further the cervical spine. For the normal cat, the nucleus pulposus of the intervertebral discs appeared hyperintense and thus, bright white, indicating hydration. In contrast, the intervertebral discs in both the untreated and the RV-treated cats were hypointense or dark, and thus were dehydrated and degenerated, and there was slight impingement of the spinal cord at some levels. The untreated cat had compression of the spinal cord due to dorsal angulation of the dens (the most cranial portion of C2) and regional ligamentous hypertrophy. The transverse images demonstrate remarkable thickening of the transverse and spinous processes of the vertebral body and the laminae that connect these processes in an untreated cat, which was substantially reduced in the RV-treated cats. These MRI data confirm that RV-treated cats have reduced vertebral body abnormalities compared with untreated cats, but still have severe intervertebral disc disease. As shown in Supplementary Video S, the RV-treated MPS VI cat 6438 was agile while an untreated cat was not at year of age. By 7 years of age, an untreated cat (6723) had very limited movement, as shown in Supplementary Videos S2, S5, and S6. One RV-treated cat (6597) showed moderate limitations in movement at 7.5 years, while a different RV-treated cat (6437) showed mild limitations in movement at 8 years, as shown in Supplementary Videos S3 S6. Cardiac manifestations Cardiac disease is a major manifestation of in patients 6 and in cats. 3 Representative examples of echocardiograms performed on normal, untreated and RV-treated cats at 6 years of age are shown in Figure 7a i, while average values for several cats of each group are shown in Figure 7j and k. An untreated cat had aortic regurgitation during diastole (Figure 7b), which was absent in a normal cat (Figure 7a), and was reduced in an RV-treated cat (Figure 7c). Aortic dilatation at the sinus of Valsalva was seen in an cat (Figure 7e), where marked thickening of the aortic valve leaflets can also be appreciated. Both abnormalities were reduced in an RV-treated cat (Figure 7f). Finally, dilatation of the aorta at the sinotubular junction can be seen in an untreated cat (Figure 7h), which was reduced in an RV-treated cat (Figure 7i). Figure 7j and k quantify the echocardiogram results for cats at 3 years of age, when there were sufficient numbers of untreated and RV-treated cats still alive to perform statistical analyses. The diameter of the aorta at the sinus of Valsalva was cm in untreated cats, which was.5-fold the value of cm in normal (P = 0.03), and.4-fold the value of cm in RV-treated (P = 0.05) cats. Similarly, the diameter of the aorta at the sinotubular junction was increased in cats compared with normal (P = 0.02) and RV-treated (P = 0.05) cats. Thickening of and regurgitation at the aortic and mitral valves were given a score of 0 (normal) to +4 (severely abnormal). cats had substantial thickening of the aortic valve leaflets with a score of , which was vol. 20 no. 5 may 202

6 significantly less in both normal and RV-treated cats (P < 0.00). The aortic valve thickening was associated with an aortic regurgitation score of.2 in cats, which was not significantly different from the values in normal or RV-treated cats. The mitral valve was thickened in MPS VI cats, with a score of This was substantially greater than in the normal cats with a score of 0 0 (P = 0.004), but was not significantly different from the values of RV-treated cats of Mitral valve regurgitation was minimal in all groups. We conclude that dilatation of the aorta and aortic valve thickening are consistent findings in untreated cats that were significantly reduced with neonatal gene therapy. DISCUSSION This study demonstrates that neonatal i.v. injection of an RV-expressing feline 4S can transduce 5% of liver cells and achieve mean serum 4S activity of 07 U/ml (3-fold normal), which was stable during the time of evaluation, up to 8 years in some animals. The liver expressed at least 23-fold as much RNA as did the spleen based upon the experimental finding that liver, which weighs at least fivefold as much as spleen in cats (M.E. Haskins, unpublished results), had fivefold as much RV RNA relative to -actin than did spleen. Thus, the major effect of this gene therapy approach probably involves secretion of M6P-modified enzyme from liver into blood, after which 4S can be taken-up by other organs in a manner similar to that of ERT, although expression in spleen may contribute to serum 4S activity. Expression from the long-terminal repeat (LTR) of the RV in blood cells that migrate into tissues could also contribute to a therapeutic effect, as RV RNA was detected in WBCs, although this level was very low at 0.% that of the liver. One concern for gene therapy involving a sulfatase deficiency was that SUMF and/or SUMF2 might be limiting. Indeed, overexpression of a sulfatase reduced expression of other sulfatases in cultured cells, 4,42 while coexpression of SUMF enhanced 4S activity after transduction of muscle. 43 This does not appear to be e Kg 3 2 Body weight at 5 months NI VI RV NI VI RV RV U/ml RV U/ml f Ratio to normal Males Females Bone lengths >0.7 years NI VI RV NI VI RV g Femur C3 height Cervical spine at 3 years Severity score p Severity of score Beaking 4 3 Laxity Tipping RV Spinous process widening Facet fusion Vertebral body shortening Carpus at 3 years Swelling/ effusion Articular cartilage erosion Abnormal carpal shape Bone lucency Epiphyseal dysplasia/ lucency Long bone shortening/ curving Molecular Therapy vol. 20 no. 5 may

7 a problem, as we demonstrated that enzyme activity in liver was directly proportional to the amount of protein observed with an immunoblot, suggesting that all protein was active. This is consistent with a previous study with AAV vectors in mice and cats demonstrating that functional enzyme was produced by liver, 36 although the level of expression here was higher than in the previous study. These data suggest that expression of SUMF and/or SUMF2 are higher in liver than in muscle and cultured fibroblasts. cats did not appear to generate an immune response to transduced cells or to the normal feline 4S protein, as expression was stable without immunosuppression. This is in contrast to our previous study in MPS I cats that received an RV expressing the canine IDUA cdna, where a CTL response developed to the canine IDUA protein, which was associated with a loss of expression without immunosuppression. 45 The stable expression observed here may relate to the use of a feline cdna that encoded a protein with a single amino acid difference from the mutant protein. Similarly, use of the feline gene may have reduced the chance of an antibody response to feline 4S in these cats compared with that seen after ERT with human 4S. 44 Efficacy of RV-mediated gene therapy RV-treated cats had reduced GAG accumulation in several tissues, improvements in some aspects of bone disease such as femur length, articular cartilage erosion, cervical vertebral articular process widening, and mobility, and reductions in aortic dilatation and aortic valve thickening compared with untreated cats. However, RV-treated cats still had short cervical vertebrae, cervical vertebral fusion, and intervertebral disc degeneration. The failure to prevent axial bone disease likely stems from poor diffusion of enzyme into relatively avascular tissues such as cartilage and intervertebral discs and represents an important limitation to the efficacy of gene therapy and ERT. However, the appendicular skeleton, which can progress to severe osteoarthritis, was dramatically improved in RV-treated cats compared with untreated cats, a finding that recapitulates what occurs in RV-treated I dogs. 46 An RV-treated cat with very high expression appeared to have somewhat better results long-term in the cervical spine than an RV-treated cat with lower expression, although the small number of animals evaluated long-term to date limits our ability to draw a firm conclusion. Evaluation of additional cats from group 2, which have currently been followed for a year, should help to define the relationship between serum 4S activity and a therapeutic effect on bone. Future studies will also try to determine the role of inflammation in bone disease and if inflammation can be reduced with gene and protein therapies. The level of serum expression observed in this study with RV-mediated gene therapy in cats was approximately fivefold the level observed at late times after administration of vector genomes/kg of an AAV8 vector with the liverspecific thyroglobulin promoter to newborns, and was approximately fivefold the level achieved with a dose of vector genomes/kg to 50 day-old cats. 36 The higher expression achieved in this study was associated with a relatively low RV DNA copy number of 5 copies per 00 cells, which was less than the values of 30 and 20 copies per 00 cells in the neonatal AAV and the juvenile AAV cats, respectively. It is unclear if the higher expression observed here is related to the enhancer activity of the LTR of the RV, the use of the human -antitrypsin (haat) instead of the thyroglobulin promoter, or to other vector differences. Implications for gene therapy Neonatal RV-mediated gene therapy was effective, simple, and did not appear to result in germline transduction or cause insertional Figure 5 Facial appearance, body weights, and skeletal radiographs. (a d) Facial appearance. The normal cat (a) was photographed at 6 years of age. An untreated mucopolysaccharidosis (MPS) VI cat (b) has short ears and a broad face at year. Two retroviral vector (RV)-treated cats from the same litter as the untreated cat (c d) look essentially normal at year. (e) Body weight at 5 months. Body weights for the indicated number of cats in each group were determined separately for male and female animals at 5 months of age. Values in normal and RV-treated cats were compared with those in untreated cats of the same gender using ANOVA with Tukey post-hoc analysis; P = , and P < 0.0. (f) Femur and C3 bone lengths. The length of the femur and height of the third cervical vertebrae (C3) were measured as detailed in the Methods section from radiographs obtained at years after birth, and the ratio to normal determined for the indicated number of cats of each group and the mean ± SD determined. For the RV-treated cats, the average serum 4-sulfatase (4S) activity in the cats that were evaluated here was 26 ± 66 U/ml (5-fold normal). (g) Cervical spine radiograph scores. Radiographs like those shown in panels h to o obtained at 2 3 years after birth were scored for abnormalities in the cervical spine, where 0 represents normal and +2 represents severely abnormal. This age was chosen as more untreated (N = 4) and RV-treated (N = 3) cats were available for analysis at this age than when older. Values that are statistically different between untreated and RV-treated cats are indicated. Values for normal cats are generally 0, but are not shown here. (h o) Representative images of the cervical spine. Radiographs were obtained at 6 years after birth (as indicated with 6Y in the lower right corner), to illustrate better the ability of gene therapy to result in long-term bone improvements. The top panels show ventrodorsal images, while the bottom panels show lateral images. The position of the second cervical vertebrae (C2) is shown, and the cranial aspect is at the top for all images. Beaking refers to a bony proliferation of the caudoventral aspect of the vertebral body and is best seen in the lateral radiograph in panel n, where it is identified with a white arrow. Beaking is present but is more difficult to identify in panels m and o due to a reduced bone quality in these animals. Tipping refers to a rotation of one vertebral body relative to the other, so that the cranial and caudal aspects do not align; tipping is difficult to identify in these particular radiographs. Articular facet widening refers to an increased diameter of the articular facets, which are identified with the black brackets for C4 on the ventrodorsal images; widening is best appreciated in panel i. Facet fusion refers to a fusion of the bone in one articular facet with that in the adjacent one. The black arrows identify bony bridges between C2 and C3 in panels m and o. In addition, the dorsal to ventral dimensions of the laminae are markedly increased in the untreated cat, and increased to a lesser extent in the RV-treated cats. Vertebral body shortening can be seen as the loss of vertebral body length, a feature best appreciated on the lateral image of untreated and RV-treated cats. Widening of the vertebral bodies can also be appreciated on the ventrodorsal images of both untreated and RV-treated cats. (p) Radiograph scores of the carpal region. Radiographs of the carpal (wrist) region were scored at 2 3 years of age for abnormalities in four untreated and 3 RV-treated cats, where 0 represents normal and +2 represents markedly abnormal. The carpus was scored for joint laxity, soft tissue swelling or joint effusions, articular cartilage erosion,abnormal carpal shape, bone lucency, epiphyseal dysplasia or lucency, and long bone shortening or curving. Values in normal cats are generally vol. 20 no. 5 may 202

8 8 years 7 years Sagittal Transverse diagnosed at birth, although implementation of newborn screening 49 should identify patients at a younger age. MATERIALS AND METHODS Reagents. Reagents were obtained from Sigma-Aldrich (St Louis, MO) unless otherwise specified. The RV designated haat-wpre contains an intact Molony murine leukemia virus LTR. The,939 base pairs feline 4S cdna 50 with 3 nucleotides of 5 untranslated and 328 nucleotides of 3 untranslated sequences was removed from farsb3 after restriction with HindIII and EcoRI, and blunt-end ligated into the Not I site downstream of the 420 base pairs haat promoter of haat-wpre to generate the vector designated haat-f4s-wpre. The 59 base pairs WPRE was located downstream of the cdna. haat-f4s-wpre was packaged in the amphotropic GP+AM2 cells as previously described U/ml 8 years U/ml 8 years Figure 6 Magnetic resonance imaging (MRI) of the cervical spine. MRIs were obtained at the indicated age. For the retroviral vector (RV)- treated mucopolysaccharidosis (MPS) VI cats 6437 and 6598, the average serum 4-sulfatase (4S) activity is indicated. A sagittal view is shown in the left panels, with the dorsum at the top and ventrum at the bottom; the cerebellum (C), the intervertebral discs (IVD) at C2-C3 and C3 C4, and the spinal cord (SC) are indicated. For the untreated cat, the thin white arrow identifies compression of the spinal cord due to dorsal dens angulation and ligamentous hypertrophy. The right panels show transverse images. The spinal cord (SC) and the spinous (S) and transverse (T) processes of the vertebrae are indicated. mutagenesis. However, in a previous study in I dogs, one 8-year-old RV-treated animal developed a hemangioma, which is a benign splenic tumor (T.M. O Malley, K.P. Ponder, M.E. Haskins, unpublished results). Since the hemangioma expressed RV sequences, this raises concerns regarding the long-term risks of this vector. The major issue with gamma RVs appears to relate to the enhancer activity of the LTR, which is present in our vector. 47 We are currently attempting to create a self-inactivating RV, but to date the level of gene transfer and expression have been inferior to that of the LTR-intact RV. 48 The absence of malignant tumors in any of >20 dogs or >5 cats that received neonatal i.v. injection of an RV and have been followed for years suggests that the risk of malignant transformation with this gene therapy approach is low. Future studies will need to weigh the risks and benefits of this or other vectors before proceeding with a clinical trial in humans. Currently, most patients with MPS are not Analysis of animals. Cats maintained at the School of Veterinary Medicine, University of Pennsylvania followed NIH and USDA guidelines for the care and use of animals in research. All experiments were approved by the authors Institutional Animal Care and Use Committee. Most RV-treated cats received three i.v. administrations of ~2 ml over ~ minute via an external jugular vein catheter separated by 4 hours over a single day, while a few cats received three injections each day for 2 days. Liver biopsies were performed as previously described. 45 Radiographs and MRIs were taken after intramusular injection of 0.02 mg/kg of atropine (Phoenix Pharmaceutical, St Joseph, MO) and an i.v. injection of 2 mg/ kg of propofol (Abbott Laboratories, Chicago, IL); inhaled isoflurane (IsoVet; Scherin-Plough, Omaha, NE) was also given for MRIs. Scoring of radiographs and MRIs was performed by a board-certified veterinary radiologist (V.W.K.). Femur lengths were obtained by measuring the side of the animal that was against the table from lateral radiographs that were obtained at 2 years of age, and the distance from the proximal femoral head to the condyle was determined. Values in normal males and females of a similar lineage were determined, and the ratio of the length in other animals to that in normal animals of the same gender was determined to calculate the percent normal. Heights of the C3 cervical vertebrae were similarly determined from lateral radiographs. For MRIs, fast-spin echo T2-weighted images were obtained without the injection of contrast using a General Electic.5 tesla magnet. Echocardiograms were performed as described previously 3 by a board-certified cardiologist (M.M.S.). Euthanasia was performed using 80 mg/kg of sodium pentobarbital (Veterinary Laboratories, Lenexa, KA) in accordance with the American Veterinary Medical Association guidelines, and tissue samples were frozen on dry ice within 30 minutes of death. Analysis of serum and organs. Serum 4S activity was determined by incubating 5 μl of serum in a final volume of 200 μl in a solution containing 5 mmol/l of 4-methylumbelliferone-sulfate in 50 mmol/l sodium acetate, ph 5.6, with 0.3 mmol/l silver nitrate as detailed in the Supplementary Materials and Methods section. One unit of enzyme produces nmol of product in hour. The percentage of 4S that contained M6P was determined by capture on a column containing the cationic-independent M6P receptor coupled to Sepharose and elution with M6P. For organ analysis, samples were homogenized and enzyme activity or GAG levels were normalized to the protein concentration as determined with the Bradford assay (BioRad Laboratories, Hercules, CA). For the immune-capture assay, samples were homogenized in a different fashion and 4S was captured with an antibody that recognized the feline 4S and enzyme activity was determined. Immunoblot was performed as detailed in the Supplementary Materials and Methods. Analysis of DNA and RNA. DNA and RNA were isolated from organs as described. 39 RNA was reverse transcribed with oligo-dt. Samples were analyzed with real-time PCR using Taqman reagents as described previously with feline -actin primers and probe 45 and WPRE primers and probe. 48 Molecular Therapy vol. 20 no. 5 may

9 U/ml a b c j.5 Aorta diameter Aortic regurg Diameter (cm) N = 4 N = 5 N = d e f SV STJ Aortic valve g h i k Score 3 2 Cardiac scores at 3 years RV 0 Aorta Thick Regurg Thick Regurg Aortic valve Mitral valve Figure 7 Echocardiograms. (a i) Representative images of echocardiograms. Echocardiograms were obtained from normal, untreated mucopolysaccharidosis (MPS) VI, or retroviral vector (RV)-treated cats as indicated at 6 years of age, as noted by the term 6Y in the lower right corner, to allow abnormalities a maximal time to develop. (a c) Color Flow Doppler of the aortic valve during diastole. These panels show color Flow Doppler analysis of the aortic valve during diastole. The left ventricle (LV), aorta (Ao) and a size marker for cm are indicated. A regurgitant jet across the aortic valve appears multicolored and is best visualized in the untreated cat in panel b. (d f) Short axis 2D images of the aortic valve during diastole. The aorta is seen at the level of the sinus of Valsalva. The yellow arrows identify the coaptation of the three leaflets in some panels. For the untreated cat in panel e, marked thickening of the leaflets is seen and the diameter of the aorta is markedly increased. (g i) Long axis 2D images of the aorta during diastole. The inner diameter of the aorta at the sinotubular junction is indicated by the double-pointed arrow. The aorta at this position is dilated for the untreated cat in panel h. (j) Aortic diameters. The aortic diameter was obtained at the sinus of Valsalva (SV) or the sinotubular junction (STJ) at 3 years of age, when more RV-treated cats were available for evaluation (four normal, five untreated, and three RV-treated cats). Values were compared in other groups with those in untreated cats using ANOVA with Tukey post-hoc analysis. (k) Echocardiogram scores. Echocardiograms were obtained at 3 years of age, and were scored subjectively for aortic valve thickening, aortic valve regurgitation, mitral valve thickening and mitral valve regurgitation from 0 (normal) to +4 (severely abnormal); the mean ± SD are shown. Values in other groups were compared with those in untreated cats using ANOVA on ranks; P value and P value of <0.0. Statistical analyses. Averages the SD were calculated for all values. Sigma Stat 3. software (Systat Software, Point Richmond CA) was used for statistical analyses. ANOVA with Tukey post-hoc analysis or the Student s t-test was used to compare values in three or two groups, respectively, when samples had continuous values, while ANOVA on ranks or Mann Whitney rank sum test were used when only integer numbers were used to score parameters. SUPPLEMENTARY MATERIAL Figure S. Side view of facial appearance. Video S. This shows a female RV-treated cat called Tina Turner (cat 6438), and her untreated female littermate Dar Williams (cat 6439) at year of age. Video S2. At 7 years of age, Adrian (cat 6723) is gray and white, with a little bit of orange in the face. Video S3. At 7.5 years of age, Jean-Luc (cat 6597) is orange with some white. Video S4. At 8 years of age, Janet Jackson (cat 6437) has long fur that is white with a little bit of gray Video S5. This shows RV-treated cat 6597 at 7.5 years of age (orange and white) and untreated cat 6723 at 7 years of age (gray and white) together in a cage with a toy on a string. Video S6. RV-treated cat 6597 at 7.5 years of age (orange and white) and untreated cat 6723 at 7 years of age (gray and white) were joined by normal cat Kuri (cat 7393; calico in color), who was 5 years old when the video was obtained. Supplementary Materials and Methods. ACKNOWLEDGMENTS From the University of Pennsylvania, we thank James Hayden for facial illustrations, Meg Weil and Tracey Sikora for the videos, the memory of Karyn Cullen for assistance with animal care, and veterinary students and the University Laboratory Animal Resources staff for assistance with animal care. This work was supported by grants from the National MPS Society awarded to M.E.H. and K.P.P., grants from the National Institutes of Health (HD06879 awarded to K.P.P., DK25759 and RR0252 awarded to M.E.H., RR07063 awarded to N.M.E.), and grants from the National Health and Medical Research Council of Australia awarded to J.J.H. REFERENCES. Neufeld, EF and Muenzer, J. (200). The Mucopolysaccharidoses. In: C.R. Scriver, A.L. Beaudet, W.S. Sly and D. Valle (eds). Metabolic and Molecular Basis of Inherited Disease. New York, McGraw Hill, pp Meikle, PJ, Hopwood, JJ, Clague, AE and Carey, WF (999). Prevalence of lysosomal storage disorders. 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