Gene Therapy (1997) 4, 63–75 1997 Stockton Press All rights reserved 0969-7128/97 $12.00 Uptake of a-(L)-iduronidase produced by retrovirally transduced fibroblasts into neuronal and glial cells in vitro K Stewart1, OA Brown1, AE Morelli1, LJ Fairbairn2, LS Lashford2,3, A Cooper4, CE Hatton4, TM Dexter2, MG Castro1 and PR Lowenstein1 1Molecular Medicine Unit, Department of Medicine, University of Manchester School of Medicine; 2CRC Department of Experimental Haematology, Paterson Institute for Cancer Research, Christie Hospital NHS Trust, Manchester; 3Academic Unit of Pediatric Oncology, Christie Hospital NHS Trust, Manchester; and 4Willink Biochemical Genetics Unit, Royal Manchester Children’s Hospital, Manchester, UK The uptake of recombinant a-(L)-iduronidase into glial and higher in actively dividing or immature brain cells. Conse- neuronal cells, produced by retrovirally transduced NIH3T3 quently, (1) neuronal metabolism ought to be capable of fibroblasts, was studied. We demonstrate that: (1) neuronal cross correction by enzyme provided by genetically engine- and glial cells take up a-(L)-iduronidase released into the ered and transplanted cells provided by bone marrow medium by retrovirally transduced fibroblasts expressing transplantation (BMT); (2) that BMT could have a more high levels of a-(L)-iduronidase; (2) both glial and neuronal beneficial effect on neurological function if performed as cells express the cation independent mannose-6-phos- early as possible; and (3) given that the uptake mechanism phate receptor responsible for lysosomal enzyme uptake; of glial cells has a higher capacity, it might be easier to and (3) uptake of the lysosomal enzyme can be blocked target diseases like the leukodystrophies in which lysoso- by excess free mannose-6-phosphate, but not glucose-6- mal enzymes are needed in glial cells, compared to dis- phosphate. Thus, various brain cells take up a-(L)-iduronid- eases where lysosomal enzymes ought to be delivered ase, possibly through a cation independent mannose-6- into neurons. phosphate receptor mediated pathway, and this uptake is Keywords: mucopolysaccharidoses; brain; macrophages; gene therapy Introduction the neurons of individuals suffering from Sheie syn- drome.2 Mucopolysaccharidosis type 1 (MPS 1), the most fre- Although a complete genotype–phenotype correlation quently occurring MPS, is an autosomal recessive disease for mutations of the gene encoding a-(l)-iduronidase has a l due to mutations in the gene encoding -( )-iduronidase. not yet been established, some nonsense mutations which The gene defect causes a specific lysosomal enzyme correlate with the most severe Hurler’s disease pheno- deficiency that results in the intracellular accumulation type have been identified (mutations W402X and Q70X).3 and storage of unprocessed glycosaminoglycans (GAGS), Establishing a complete genotype–phenotype correlation dermatan sulfate and heparan sulfate. Patients suffering should greatly aid in the accurate implementation of ther- from MPS 1 show variable clinical manifestations, the apies like bone marrow transplantation (BMT), since the most severe of which is Hurler’s syndrome, with pro- genotype will help in the decision on whether a parti- gressive neurological dysfunction, skeletal and soft tissue cular patient ought to be treated by complex treatments deformities, and leading to death within the first decade. like BMT or gene therapy. In less severe forms of this deficiency, such as the inter- BMT is used clinically to treat children suffering from mediate Hurler–Sheie syndrome, or the mild Scheie syn- various inherited lysosomal enzyme disorders, such as drome, no mental retardation and only milder symptoms the mucopolysaccharidoses and the leukodystrophies. 1 a l occur. Importantly, the levels of -( )-iduronidase While BMT during the very early stages of the disease enzyme remaining are inversely correlated with lysoso- has been shown to reverse the peripheral (eg liver and mal distension of neurons and CNS functional deterio- spleen) pathology (ie the accumulation of unmetabolized ration; ie severe distension in the neurons of patients intermediary metabolites), the effectiveness of this treat- suffering from the Hurler syndrome, and little if any in ment for preventing the progression of established neuro- logical pathology and dysfunction remains controversial. The reversal of the peripheral lesions, however, appears Correspondence: Professor PR Lowenstein, Molecular Medicine Unit, Room 1.302 Stopford Building, Department of Medicine, University of to be achieved by a combination of the repopulation of Manchester School of Medicine, Oxford Road, Manchester M13 9PT, UK target tissues by donor-derived macrophages, and by Received 6 September 1996; accepted 16 October 1996 receptor-mediated uptake of secreted lysosomal enzyme.4 Transfer of a-(L)-iduronidase into brain cells K Stewart et al 64 Similar mechanisms of action have also been postulated 50 to be effective in delivering enzyme activity to the central nervous system (CNS).5 However, it is unclear whether and to what extent donor macrophages invade the host brain in human patients, and if neurons and glial cells :2 have the capacity to take up lysosomal enzyme released :§" 40 Q) into the extracellular medium. 0... Currently, BMT is the only therapeutic intervention for C. 6 MPS-1 to have shown clinical effectiveness. BMT in C) :::,. patients or in experimental animals can lead to correction 0 of the enzyme defect, a decrease in abnormal lysosomes E in soft tisssues, and in some cases stabilisation of the -=so w neurological status.6–25 However, while there is general en <C agreement on the effectiveness of BMT for the treatment Cl of liver, spleen or soft tissue disease, there is no consen- z 0 sus on whether it has a beneficial effect on the disease a: induced progressive deterioration of brain function. The 20 outcome of the neurological manifestations appears to ....I' depend on the initial mental status, the age of the patient, <C' :I: the engraftment of the grafted bone marrow and the a.. development of graft-versus-host diseases (GVHD).6 So ....I <C far, BMT has also been ineffective for correcting all of the 10 skeletal abnormalities.26 Lysosomal enzymes are post-translationally processed to contain highly phosphorylated mannose residues. These bind to mannose-6-phosphate receptors (M6P-R) which target lysosomal enzymes to lysosomes. M6P-R are also present on the cell membrane and are able to bind 0 circulating or extracellular lysosomal enzymes and deliver them to the lysosomes;27,28 these constitute the E20 PND1 PND4 PND6 PND14 ADULT cation independent mannose 6 phosphate receptors (CI- M6P-R). Thus, lysosomal enzyme replacement was sug- DEVELOPMENTAL TIME gested for the treatment of lysosomal storage diseases, but so far has only been implemented for the treatment of Figure 1 Levels of a-(l)-iduronidase were measured in different brain Gaucher’s disease.29 The use of a-(l)-iduronidase enzyme regions of the rat throughout development. Embryonic day 1 (1) was taken a l replacement for the treatment of Hurler’s disease remains to be the day plugs first appeared in the mothers. Statistics and -( )- at the experimental stage and has shown, in dogs lacking iduronidase levels were measured as described in Materials and methods. There was no statistically significant difference for the values obtained for all enzyme production, to lead to important immune cortex and striatum throughout the period studied. In the hippocampus, responses. a-(l)-iduronidase levels at days PND1 and 4 were statistically different It has previously been shown that recombinant retro- from levels at E20 and adult, demonstrating the existence of a postnatal virus expressing the full-length human a-(l)-iduronidase peak at days 1–4 after birth, which then decreases towards adulthood. In a l cDNA can correct the enzymatic defect in MPS-1 skin the cerebellum, -( )-iduronidase levels at PND4 were statistically differ- 30 ent from PND1 and adult, thus also demonstrating the existence of a fibroblast cultures, and also in long-term cultures of a l 31 postnatal peak in the levels of cerebellar -( )-iduronidase. human bone marrow from Hurler’s patients. Our PND = postnatal day; `, cortex; a, striatum; M, hippocampus; l, cere- experiments were designed to assess whether neuronal bellum. and glial cells could take up a-(l)-iduronidase from the extracellular medium and retain it intracellularly, when a-(l)-iduronidase was being produced by fibroblasts Immunocytochemical distribution of the cation transduced with a-(l)-iduronidase cDNA. independent mannose-6-phosphate receptor: in primary cultures of neurons, glial cells, the neuronal cell line Results Neuro2A and the glioma cell line C6 Glial and neuronal cells were incubated with a rabbit Developmental study polyclonal antibody raised against the CI-M6P-R, and all Levels of a-(l)-iduronidase were examined in four brain samples tested displayed specific immunoreactivity. Pri- areas (cortex, striatum, hippocampus and cerebellum) mary cultures of neurons and glial cells were double between embryonic day 20 and adulthood (Figure 1). immunolabelled in order to identify the cell type: neu- Results were analysed statistically utilising the analysis rons were identified using antibodies raised against the of variance (ANOVA) and Tukey–Kramer multiple com- neuron-specific protein microtubule associated protein 2 parison’s test (for a description of the methods see (MAP2), while glial cells were identified by their immu-