lysosomal disorders in general , and mps i in particular have long been considered amenable to treatment by exogenous enzyme that would enter the deficient cells by endocytosis ( fratantoni et al ., 1968 ; kaplan et al ., 1977 ; sando and neufeld , 1977 ). exogenous enzyme eliminates the abnormal accumulation of gags in cultures mps i fibroblasts . high efficient enzyme uptake relies on the presence of specific sugars , which are recognized by their cognate receptor . these include the mannose - 6 - phosphate receptor ( m6pr ) which is ubiquitously expressed , the galactose receptor of hepatocytes and the mannose receptor of macrophages . the latter is used with success for treating gaucher type i patients with a modified glucocerebrosidase enzyme preparation targeting the macrophages ( barton et al ., 1991 ; grabowski et al ., 1998 ). trials have recently been performed with enzyme targeting the m6pr in patients with diseases that do not affect the brain , as fabry disease ( eng et al ., 2000 ; schiffmann et al ., 2000 ), pompe disease ( van der hout et al ., 2000 ) and mild forms of mps i ( scheie disease ). as the infused enzyme does not cross the blood brain barrier , no benefit can be expected on brain damages . thus , etiological treatment is currently proposed only for patients in whom a neurological disease is not anticipated . in the most frequent situation of a disease known to affect the brain , no treatment can be proposed at the present time . gene therapy appears as the only option that could lead to a therapeutic strategy targeted to the brain . various approaches have been considered with the aim to obtain in situ enzyme delivery in the brain parenchyme . cells genetically - modified ex vivo in order to over - express and secrete the missing lysosomal enzyme were implanted in the brain . direct intracranial injections of gene transfer vectors by stereotactic methods were performed with the aim to inducing enzyme over - expression and secretion from resident neurons and glial cells . these experiments were performed in a mouse model of lysosomal storage diseases . the β - glucuronidase deficient mouse ( mps vii ) resumes the clinical features of human mucopolysaccharidosis , including abnormal skeletal development , corneal clouding and deafness ( birkenmeier et al ., 1989 ). considerable lysosomal storage occurs in every tissue , and especially in the brain . animals die around 6 months of age , apparently from both progressive neurological degradation and locomotor disability . animals were either engrafted with cell genetically - modified to over - express β - glucuronidase , or received a functional β - glucuronidase cdna by the mean of a gene transfer vector which could be adenovirus vectors , aav vectors or lentivirus vectors . consistent results provided evidence that enzyme expression was not restricted to the area where the cells or the vector had been injected ( ghodsi et al ., 1998 ; snyder et al ., 1995 ; taylor and wolfe , 1997 ). activity could be demonstrated in far remote locations , including in the contralateral hemisphere when injection was unilateral . these data indicated that brain cells were able to take up enzyme from the extracellular environment and more importantly , suggest that β - glucuronidase could be transported over long distances in the brain by retrograde axonal transport . these studies also demonstrated that gene therapy could prevent the development of lesions and reverse pre - existing damages . the feasibility of preventing the development lesions was demonstrated in newborn mps vii mice . this was shown either in animals engrafted in situ with immortalized enzyme - secreting cells ( snyder et al ., 1995 ); or injected intravenously at birth with purified enzyme ( sands et al ., 1994 ; sands et al ., 1997 ; vogler et al ., 1993 ; vogler et al ., 1996 ) or with a recombinant adeno - associated vector encoding β - glucuronidase ( daly et al ., 1999a ; daly et al ., 1999b ). the reversion of pre - existing lesions in adult animals has also been demonstrated . transient correction was reported after the engraftment of enzyme - secreting primary cells ( taylor and wolfe , 1997 ) or the in situ injection of an adenovirus vector ( ghodsi et al ., 1998 ; stein et al ., 1999 ). others and ourselves have shown a sustained correction after the in situ injection of an adeno - associated virus ( aav ) vector ( bosch et al ., 2000a ; skorupa et al ., 1999 ). using lentivirus - based vector we have documented enzyme delivery and reversal of pathology in the entire brain of mps vii mice ( bosch et al ., 2000b ). the efficacy of direct gene transfer into the brain has recently been documented another mouse model of lysosomal storage disease . the mld mouse has been created by the selective destruction of the asa gene . mice develop a mild pathology reminiscent of that associated with human mld after 8 to 10 months , with typical storage lesions in the white matter ( hess et al ., 1996 ). this pathology can locally be prevented and reversed by the delivery of lentivirus - derived gene transfer vector encoding asa in the fimbria ( consiglio et al ., 2001 ). a controversy remains about whether this treatment actually improves mouse behavior and with regards to the relevance of correcting fimbria neurons in a disease that is mostly a demyelinating process . achievements in the brain of mps vii mice stereotactically injected with aav or lentivirus vector reached the requisites for an effective treatment . the current issue consists in passing through the various stages from mouse experimentation to clinical application . as gene therapy targeted to the brain is very innovative , these stages must be cautiously designed . as mps i affects both the central nervous system and the peripheral organs , gene therapy trial targeted to the brain in this disease will have to be combined with enzyme replacement therapy in the periphery . the choice of mps iiib and mld as diseases in which a clinical trial will be considered first , is based on the predominance of neurological symptoms , the relative high frequency of the disorders among lysosomal storage diseases and the absence of efficacy of bone marrow transplantation . on the other hand , it is important to consider that whereas excellent mouse and dog models are available for mps i and mps iiib , there is no convenient animal model for mld . indeed , the mld mouse develops late and mild pathology , which delays and hampers accurate assessment of disease correction . our strategy therefore is to perform most of the preclinical investigations proposed in this program in the available mps i and mps iiib animal models . it is well documented in the literature that mps i and mps iii share common pathophysiology with mld . thus feasibility studies performed in the mps i and mps iiib models will provide relevant information for application in mld patients . the final objective of the pre - clinical studies is the design of a phase i / ii protocols for the assessment of tolerance and therapeutic potential of intracranial injections of gene transfer vectors in children with mps i and mps iiib . pre - clinical studies in animal models are mandatory to designing a clinical trial protocol . investigations in mps i and mps iiib mice were performed with the aav - pgk - idua and the aav - pgk - naglu vectors , respectively . these vectors were derived from aav serotype 2 ( aav - 2 ). vector genomes are similarly organized for both vectors , the only difference resides in the cdna sequence that is expressed . structure is shown in fig1 : itr are repeated aav sequences present at both extremities that are important for packaging , and genome replication . in the aav - pgk - idua and the aav - pgk - naglu vectors , these sequences consist in 181 bp from plasmid psub 201 isolated by dr . r . samulski ( samulski et al ., 1987 ). the promoter of the mouse phosphoglycerate kinase gene ( adra et al ., 1987 ) is inserted downstream of the 5 ′ itr . this is a 500 bp xbai / mlui fragment from plasmid m48 ( salvetti et al ., 1995 ). this promoter is highly active in brain cells ( kardower et al ., 2000 ). a human cdna is inserted downstream of the mouse pgk promoter . in aav - pgk - idua , this cdna encodes human idua . it has been inserted as a 2165 bp mlui / nhei fragment from plasmid m48 . this cdna was isolated by us , using the published sequence ( scott et al ., 1991 ). in the aav - pgk - naglu vector the cdna encodes human naglu . this cdna was isolated by pr . e . neufeld ( ucla ) ( zhao et al ., 1996 ) who kindly provided it to us . a woodchuck enhancer ( wpre ) sequence is inserted downstream of the human cdna ( zufferey et al ., 1999 ). this 639 bp sequence , originally described in the laboratory of dr . d . trono ( cmu genève ) has been isolated from a plasmid , kindly provided to us by dr . naldini ( università di torino ). a polyadenylation site from the bovine growth hormone gene is inserted downstream of wpre . this is a 382 bp sequence orignally described by goodwin et al . ( goodwin and rottman , 1992 ). vectors stocks were prepared in the laboratoire de thérapie génique , chu hôtel - dieu , nantes , by triple transfection into 293 - t cells , as described in salvetti et al . ( salvetti et al ., 1998 ). vectors were administrated by stereotactic injection in the brain tissue . in the mouse , a single injection of 5 μl containing 2 × 10 9 physical particles of aav vector was performed in the putamen . animals were treated at 6 - 8 weeks of age . in dogs , a single intrastriatal 40 μl injection was performed . mps i and mps iiib mice have been obtained by a selective disruption of the genes coding for α - l - iduronidase ( idua )( clarke et al ., 1997 ) and α - n - acetyl - galactosaminidase ( naglu )( li et al ., 1999 ), respectively . we obtained these animals from pr . e . neufeld ( ucla ). homozygous mutants exhibit a total absence of catalytic activity of the targeted enzymes . they develop typical lysosomal storage pathology over the first 6 months of life , including lysosomal storage lesions in brain cells . a colony of dogs deficient for idua has been raised and maintained at the university of tennessee ( shull et al ., 1982 ; spellacy et al ., 1983 ). we obtained 10 breeders from dr . e . kakis ( ucla ). dogs have been installed in france with the support of the afm . these animals have a point mutation in the first exon / intron border of the idua gene ( menon et al ., 1992 ). dogs homozygous for the mutation exhibit a total enzyme deficiency . they develop a characteristic hurler / scheie disease during the course of their first year of life , associating severe abnormalities of the skeleton and intense lysosomal storage lesions in various tissues , including in the brain ( constantopoulos et al ., 1985 ; walkley et al ., 1988 ). mps i dogs have been extensively studied in the past . clinical benefit has been demonstrated after allogeneic bone marrow transplantation ( shull et al ., 1987 ). enzyme infusion in the periphery improves lysosomal storage significantly ( shull et al ., 1994 ). however , all animals develop an immune response against the infused human enzyme ( kakkis et al ., 1996 ; lutzko et al ., 1999 ). in the absence of any detectable idua activity in these animals , it is expected that immunization will occur with the canine enzyme as well . to our knowledge , no attempt has been made so far with the aim to treat the brain pathology in these dogs . mps i dogs are genotyped and homozygous animals are transferred to the centre de boisbonne of the ecole nationale vétérinaire de nantes at weaning . surgery is performed at the centre de boisbonne . enzyme activity , diffusion and correction of storage lesions in mps i mouse brains . forty young adult idua - deficient mps i mice received a single intrastriatal injection of the aav - pgk - idua vector . animals were sacrificed 2 , 6 , 16 , 20 or 26 weeks after injection . in a first group of treated mice , we measured enzyme activity in tissue extracts from the injected hemisphere , the contralateral hemisphere and the caudal part of the encephalon including the cerebellum and the brain stem . results are shown in table 1 . these experiments revealed high enzyme activity in the injected hemisphere ( 3 to 4 folds more than in normal mice ), and significant levels in more remote locations ( 10 to 30 % of normal mouse levels ). activities were stable over the 7 - month follow up . in a second series of mice , serial coronal brain sections ( 100 μm or 1 mm ) were performed and activity was measured in extracts . this experiment allowed drawing of a precise map of the location of enzyme activity throughout the brain over time . it showed that enzyme progressively spreads , from week 2 to 16 , from the injection site to remote locations ( fig2 ). at 16 weeks after injection , in most mice , enzyme activity could be detected all over brain , except in the most rostral and caudal regions of the contralateral hemisphere . a third series of mice was used to examine enzyme activity and disease correction in adjacent coronal sections . it revealed a complete correction of storage lesions in areas where enzyme was detectable , but also in region where the enzyme assay was negative . corrected areas progressively increased in size with time ( fig3 ). at 26 weeks , only very limited areas of the contralateral olfactive bulb and the cerebellum still showed minimal storage lesions . these results clearly demonstrate that idua is produced from cells genetically modified with the aav - idua vector and delivered to far distant locations from the vector injection site . spreading over the brain increases with time . enzyme delivery allows a correction the histological lesions associated with the disease . such an efficient delivery of a lysosomal enzyme in the brain parenchyme has not been reported previously . enzyme activity and diffusion in the brain of a mps i dog . a 40 μl injection of the aav - pgk - idua vector was performed in the striatum of one mps i dog . the animal received cyclosporine for 3 days before treatment and until sacrifice 12 weeks after the injection . for analysis of enzyme spreading in the brain , the entire encephalon was cut in 16 slices and each slice separated in four sections . tissue extracts were prepared from every second sections and idua activity was measured . results are shown in fig4 . they indicate high enzyme activity at the injection site and in adjacent areas . enzyme spreading could be demonstrated over 7 slices , which represent a maximal extension of 2 . 8 cm . histological analysis is currently performed to assess the extend of disease correction . with respect to the short term follow up of the animal , the limited amount of injected vector and our knowledge that correction extents further than detected enzyme activity , it may be anticipated that four stereotactic injections ( two in each hemisphere ) might be sufficient for disease correction in the entire dog brain . this hypothesis will be investigated in the next available mps i dogs . results from these experiments will help designing a therapeutic protocol in affected children . in summary , lysosomal storage disease can be corrected through the delivery of the missing enzyme . for those diseases affecting the central nervous system , which are the more frequent ones , intracerebral delivery supposes in situ enzyme secretion . this can be obtained by gene therapy methods . stereotactic injection of aav - based vectors encoding the missing enzyme in the brain leads to inducing enzyme secretion in a small number of genetically - modified cells that provide an intra - cerebral source of enzyme . enzyme can be transported to remote locations leading to the definitive correction of storage lesions in the entire brain . we obtained these results in the mouse model of mps vii , which is deficient for β - glucuronidase , and now in the mps i mouse , which is deficient for alpha - l - iduronidase ( idua ) and which provides a model for hurler &# 39 ; s disease , a disorder relatively frequent in children . correction in mps i mice was obtained by using an aav - 2 derived vector ( aav - pkg - idua ). expression levels with this vector , and spreading of the activity through out the brain was much more efficient than with previously described aav vectors . efficiency seems related in the use of a murine phosphoglycerate promoter ( pgk ) and the addition of sequences called wpre for woodchuck hepatitis virus posttranscriptional regulatory element , which are known to increase mrna stability and traductability . though the concept that the stereotactic injection of aav vector can cure lysosomal storage lesions in the brain of mice with mucopolysaccharidosis has been largely publicized , these results with aav - pgk - idua provide the first demonstration that this is effective in mpss i , which is one of the most attractive target for clinical application . enzyme activity levels attained in the brain of mps i mice with the aav - pgk - idua vector were much higher than previously reported with aav vectors in different models . the volume of brain tissue in which activity was detected , and the volume in which a correction of lesions was observed were much broader than previously reported in different models of affected mice . expression levels were achieved allowing a therapeutic effect in the entire brain with a single vector injection , which is clinically relevant result , whereas similar achievement required multiple injections in previous reports . the aav - pgk - idua vector has also recently been used in a canine model of mps i . we could confirm in dogs the efficient spreading of enzyme activity in the brain following a single intrstriatal vector injection . recombinant bacteria containing nucleic acid molecules of the invention have been deposited at the collection nationale de cultures de microorganismes (“ c . n . c . m .”) institute pasteur , 28 , rue du docteur roux , 75724 paris cedex 15 , france , as follows : plasmid accession no . deposit date aav2 - mpgk - hnaglu - wpre - pa i - 2891 jun . 20 , 2002 aav2 - mpgk - idua - wpre - pa i - 2892 jun . 20 , 2002 the following references are cited herein . the entire disclosure of each reference is relied upon and incorporated by reference herein . adra , c . n ., boer , p . h ., and mcburney , m . 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