US20060182717A1

US20060182717A1 - Vector for expressing alpha-N-acetyl-galactosaminidase and method of treating MPS I by stereotactic injection into the brain of a mammal

Info

Publication number: US20060182717A1
Application number: US11/329,295
Authority: US
Inventors: Nathalie Desmaris; Jean Heard
Original assignee: Institut Pasteur
Current assignee: Institut Pasteur
Priority date: 2002-06-21
Filing date: 2006-01-11
Publication date: 2006-08-17
Also published as: US20040023218A1; US7090836B2

Abstract

A purified nucleic acid molecule which is capable of expressing a lysosomal enzyme wherein said nucleic acid molecule comprises at least a sequence coding for said lysosomal enzyme and a promoter highly active in the brain inserted upstream from said sequence.

Description

BACKGROUND OF THE INVENTION

This invention relates to a purified nucleic acid molecule, which is capable of expressing a lysosomal enzyme, wherein the nucleic acid molecule comprises at least a sequence coding for the lysosomal enzyme and a promoter highly active in the brain inserted upstream from the sequence.
Lysosomal storage diseases form a group of more than 30 metabolic disorders in which the function of one or several lysosomal hydrolases is deficient. Although the prevalence of each disease is low, prevalence of lysosomal storage diseases as a whole may be equivalent to that of cystic fibrosis in the general population (1:2500). In France, the most frequent lysosomal storage diseases are Gaucher type I disease, Hurler disease (MPS I), Hunter disease (MPS II), Sanfilippo disease (MPS III) and metachromatic leucodystrophy (MLD). They represent 10 to 50 births every year. With the exception of Gaucher type I disease, Pompe disease, Fabry disease and mild forms of MPS I, there is no etiological treatment available for lysosomal storage diseases so far. Bone marrow transplantation, which may be an option in some MPS I patients, is not effective in MPS III and MLD.
Lysosomal enzyme deficiencies induce the accumulation of intermediate catabolites in lysosomes, which progressively alters cell function and survival. Although deficiencies affect every tissue, clinical expression varies depending on the missing enzyme. Neurological symptoms are often predominant. They include severe motor impairments and mental retardation. Histopathology reveals characteristic vacuolizations in both neurons, glia and perivascular cells, without known predominance in specific locations. Other frequent symptoms include hepatomegaly, skeletal abnormalities, comeal clouding and respiratory, cardiac or renal dysfunctions leading to premature death. There is a need in the art for a treatment of the central nervous system pathology in lysosomal storage diseases in which neurological symptoms are either predominant, as in MPS III and MLD, or highly determinant for the clinical prognosis, as in MPS I. MPS I and MPS IIIb are autosomal recessive lysosomal storage diseases classified among mucopolysaccharidosis. These diseases are caused by a defect in the degradation pathway of glycosaminoglycans (GAGs). In MPS I and MPS IIIb, the degradation of heparan sulfates is interrupted by the deficiency of α-L-iduronidase (IDUA) and α-N-acetyl-glucosaminidase (NaGlu), respectively. Complete IDUA deficiency is associated with mutations W402X, Q70X and is responsible for severe forms of MPS I, in which skeletal abnormalities can be recognized at birth and neurological symptoms may occur before the age of 2-3 years. Milder forms exist in which the neurological disease is delayed and less severe (mild forms of MPS I or Hurler-Scheie disease) or even absent (Scheie disease). Except a frequent hepatomegaly, peripheral abnormalities are absent in MPS IIIb. Symptomatology appears in children between the age 2 and 6 as behavioral troubles, which progressively lead to a severe mental and motor degradation.
MLD is an autosomal recessive lysosomal storage disorder classified among the lipidoss. It is caused by a deficiency of arylsulphatase A (ASA) that leads to demyelination in the central and peripheral nervous system. Deficiency of ASA causes intralysosomal storage of the sphingolipid cerebroside sulphate. This lipid is abundant in myelin and its accumulation leads to the death of oligodendrocytes. ASA catalyses the first step in the degradation of the sphingolipid cerebrosisde 3-sulphate by removing the sulphate from the polar head of this lipid, which is a galactose 3-sulphate moiety. If this step does not occur, owing to a deficiency of ASA, this lipid cannot be degraded and accumulates into lysosomes. MLD may appear at any age. The three main clinical forms that correlate with the genotype can be distinguished: infantile, juvenile and adult forms. Allogenic BMT has no effect in the most frequent infantile form of MLD (>60% of the MLD cases) and limited effect in juvenile MLD.

SUMMARY OF THE INVENTION

This invention provides a purified nucleic acid molecule, which is capable of expressing a lysosomal enzyme, wherein the nucleic acid molecule comprises at least a sequence coding for the lysosomal enzyme and a promoter highly active in the brain inserted upstream from the sequence. The nucleic acid molecule can further comprise a posttranscriptional regulatory element inserted downstream from the sequence. In one embodiment, the promoter highly active in the brain is the promoter of the phosphoglycerate kinase gene. In another embodiment, the posttranscriptional regulatory element is a hepatitis virus posttranscriptional regulatory element. The sequence can code, for example, for an iduronidase (IDUA) or an arylsulphatase (ASA).
In a further embodiment of the invention, the nucleic acid molecule further comprises at least one repeated AAV sequence involved in packaging and genome replication placed upstream from the promoter and/or downstream from the sequence coding for the lysosomal enzyme.
In another embodiment, the nucleic acid molecule further comprises at least one repeated AAV sequence involved in packaging and genome replication placed upstream from the promoter and/or downstream from the sequence coding for the posttranscriptional regulatory element.
This invention also provides a recombinant bacteria containing the nucleic acid molecule of the invention, wherein the recombinant bacteria has been deposited at CNCM on Jun. 20, 2002 under the reference I-2891.
This invention also provides a recombinant bacteria containing the nucleic acid molecule of the invention, wherein the recombinant bacteria has been deposited at CNCM on Jun. 20, 2002 under the reference I-2892.
In addition, this invention provides a vector for the expression of a lysosomal enzyme, wherein the vector comprises the nucleic acid molecule of the invention.
The vector is, for exanple, an adenovirus vector (AAV), or a lentivirus vector.
Still further, this invention provides a cell transformed with the nucleic acid molecule of the invention. The cell can be a mammalian cell, and the cell can be transformed ex vivo.
This invention provides a method for preventing or treating a lysosomal storage disease in a mammal, wherein the method comprises administering the nucleic acid molecule of the invention to a mammalian host. In one embodiment, the mammal is a human. The disease can be, for example, MPS I or MPS IIIb.
This invention also provides a method for preventing or treating a lysosomal storage disease in a mammal, wherein the method comprises administering a vector of the invention to a mammalian host. The vector can be administered by stereotactic method.
This invention also provides a method for preventing or treating a lysosomal storage disease in a mammal, wherein the method comprises the transfer of a cell of the invention into said mammalian host.

BRIEF DESCRIPTION OF THE DRAWINGS

This invention will be described with reference to the drawings in which:
FIG. 1. Structure of the AAV-PGK-IDUA and the AAV-PGK-NaGLU vectors.
FIG. 2. IDUA spreading in the brain of treated MPS I mice.
Treated mice were sacrificed, 2, 6, 16, 20 or 26 weeks after vector injection. Coronal 100 μm or 1 mm brain sections were prepared and IDUA activity was measured in tissue extract from these sections. Data are shown as a schematic representation of the brain and of the analyzed sections. Activity levels are shown according to the indicated color code. The vector injection site is indicated as a red dot in the right hemisphere. Results demonstrate IDUA spreading in brain tissues from the injection site to the ipsi and contralateral hemispheres.
FIG. 3. Disease correction in treated MPS I mouse brain.
Samples were taken from mouse brain section, fixed with glutaradehyde and embedded in Epoxy. Semi-thin sections (1 μm) were prepared and stained with toluidine blue. The intensity of lysosomal storage lesions in the various analyzed part of the brains is indicated as: −, lesions were not observed; +, moderate lesions; or ++, severe lesions. PV: perivascular area, PR: parenchymal area. Controls are untreated MPS I mice. Lesions were detected in these animals as early as one month of age and progressively aggravated with time. Treated animals analyzed after 6 weeks were 3 month old, after 16 weeks, 6 month old and after 26 weeks, 8 month old. Data show a progressive regression of the lesions with time in treated mice.
FIG. 4. Enzyme spreading in MPS I dog brain.
Brain was cut into 16 slices. Every second slice was used for IDUA detection. The alternate slice was used for histology. Each slice was divided into four samples for each hemisphere, from which tissue extracts were prepared for IDUA assay. A total of 64 samples were measured. The site of vector injection is indicated by a red dot. Data are enzyme activity levels for the injected (IL) and contralateral (CL) hemispheres.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS

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.
Material and Methods
Gene Transfer Vectors
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 FIG. 1:

- 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).

Vector Preparation
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).
Vector Administration
Vectors were administrated by stereotactic injection in the brain tissue. In the mouse, a single injection of 5 μL containing 2×10⁹physical 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.
Investigations in MPS Mouse Models
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.
Investigations in MPS I Dogs
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.

TABLE 1


IDUA activity in brain extracts of normal mice (+/+), heterozygote
mice (+/−) and untreated (−/−) or treated IDUA-deficient
mutant MPS I mice. Treated mice were sacrificed at 2, 6-16, 20 or
26 weeks after a single vector injection in the striatum.

IDUA

		BRAIN	CR + BRAINSTEM

+/+	2.38 ± 0.12	(n = 6)	2.33 ± 0.47	(n = 3)
+/−	1.23 ± 0.22	(n = 24)	1.16 ± 0.27	(n = 13)
−/−	0	(n = 14)	0	(n = 7)

	IL	CL

2 WKS	2-1	6	ND	ND
	2-2	7.97	ND	ND
	2-3	7.93	0.38	0.55
	2-4	8.22	0.55	0.20
	2-5	9.07	1.11	0.19
	2-6	7.8	0.1	0
	2-7	8.9	0.4	0.8
6 WKS	6-1	10	0.40	0.10
	6-2	8.6	0.90	0.30
	6-3	9.41	0.44	0.44
	6-4	9.78	0.44	0.15
	6-5	9.93	1.56	0.44
	6-6	0.85	0	0
	6-7	0.7	0.01	0
	6-8	3.34	0	0.6
	6-9	6.68	0.2	0
16 WKS	16-1	10.5	1.5	0.30
	16-2	2.9	0.41	0.34
	16-3	6.3	ND	ND
	16-4	21.7	0.53	0.35
	16-5	9	9.7*	0.18
	16-6	0.6	0	0
	16-7	3.59	1.18	0.12
	16-8	6.46	0.38	ND
	16-9	11.94	4.96	1.8
	16-10	8.55	1.36	12.15
	16-11	0.01	0	0
20 WKS	20-1	1.21	0	0
	20-2	1.74	0	0
	20-3	1.73	0.18	0
	20-4	0	0	0
	26-5	0.53	0	0
26 WKS	26-1	23.6	0.20	0.53
	26-2	3.5	0.35	ND
	26-3	17.5	0.6	0
	26-4	0.42	0	0
	26-5	0.6	0	0
	26-6	5.7	0.82	0
	26-7	0	0	0
	26-8	0.46	0	0
	26-9	2.73	0	0

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 (FIG. 2). 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 (FIG. 3). 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 FIG. 4. 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'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

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The following references are cited herein. The entire disclosure of each reference is relied upon and incorporated by reference herein.

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Claims

1-22. (canceled)

23. An AAV vector for the expression of α-N-acetyl-glucosaminidase, wherein the vector comprises (a) the nucleic acid coding sequence for α-N-acetyl-glucosaminidase, as found in C.N.C.M I-2891; (b) a phosphoglycerate kinase gene promoter; and (c) a woodchuck hepatitis virus posttranscriptional regulatory element, and wherein the coding sequence is operably linked to (b) and (c).

24. A method of transforming a brain cell of a mammal in vivo comprising: (a) providing a vector comprising: (i) a nucleic acid sequence encoding α-N-acetyl-glucosaminidase, as found in C.N.C.M. I-2891; (ii) a phosphoglycerate kinase gene promoter; and (iii) a woodchuck hepatitis virus posttranscriptional regulatory element; and (b) delivering the vector to the brain of the mammal by stereotactic injection; wherein the nucleic acid, promoter, and regulatory element are in operable linkage and the cell is local to the stereotactic injection site.

25. A cell transformed in vitro with the vector of claim 37, wherein the cell expresses α-N-acetyl-glucosaminidase from the vector sequence.

26. A method of treating or preventing the accumulation of plaques associated with MPS I, wherein the method comprises administering by stereotactic injection, into the brain of the mammal, an AAV vector comprising (a) a phosphoglycerate kinase gene promoter sequence; (b) a woodchuck hepatitis virus posttranscriptional regulatory element; and (c) an α-L-iduronidase-encoding sequence operably linked to (a) and (b), wherein brain cells local to the injection site express α-N-acetyl-glucosaminidase from the vector sequences.

27. A method of treating or preventing MPS III disease in a mammal, wherein the method comprises administering by stereotactic injection into the brain of the mammal, an AAV vector comprising (a) a phosphoglycerate kinase gene promoter sequence; (b) a woodchuck hepatitis virus posttranscriptional regulatory element; and (c) an α-N-acetyl-glucosaminidase-encoding sequence operably linked to (a) and (b), wherein brain cells local to the injection site express α-N-acetyl-glucosaminidase from the vector sequences.

28. A method of providing α-N-acetyl-glucosaminidase to the brain of a mammal comprising administering, by stereotactic injection into the brain of the mammal, an AAV vector comprising (a) a sequence encoding α-N-acetyl-glucosaminidase; (b) an operably linked phosphoglycerate kinase gene promoter sequence located upstream from (a); (c) an operably linked woodchuck hepatitis virus posttranscriptional regulatory element; and (d) two AAV terminal repeat sequences flanking (a), (b), and (c), wherein the α-N-acetyl-glucosaminidase is delivered to areas of the brain of the mammal distal to the injection site.