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  
         [0001]    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.  
           [0002]    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.  
           [0003]    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, corneal 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.  
           [0004]    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 (&gt;60% of the MLD cases) and limited effect in juvenile MLD.  
         SUMMARY OF THE INVENTION  
         [0005]    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).  
           [0006]    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.  
           [0007]    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.  
           [0008]    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.  
           [0009]    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.  
           [0010]    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.  
           [0011]    The vector is, for example, an adenovirus vector (AAV), or a lentivirus vector.  
           [0012]    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.  
           [0013]    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.  
           [0014]    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.  
           [0015]    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  
       [0016]    This invention will be described with reference to the drawings in which:  
         [0017]    [0017]FIG. 1. Structure of the AAV-PGK-IDUA and the AAV-PGK-NaGLU vectors.  
         [0018]    [0018]FIG. 2. IDUA spreading in the brain of treated MPS I mice.  
         [0019]    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.  
         [0020]    [0020]FIG. 3. Disease correction in treated MPS I mouse brain.  
         [0021]    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.  
         [0022]    [0022]FIG. 4. Enzyme spreading in MPS I dog brain.  
         [0023]    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  
       [0024]    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).  
         [0025]    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.  
         [0026]    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.  
         [0027]    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.  
         [0028]    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).  
         [0029]    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).  
         [0030]    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.  
         [0031]    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.  
         [0032]    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.  
         [0033]    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.  
         [0034]    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  
       [0035]    Gene Transfer Vectors  
         [0036]    Investigations in MPS I and MPS IIIb mice were performed with the AAV-PGK-IDUA and the AAV-PGK-NaGLU vectors, respectively.  
         [0037]    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:  
         [0038]    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).  
         [0039]    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).  
         [0040]    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.  
         [0041]    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).  
         [0042]    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). 
         
 
         [0043]    Vector Preparation  
         [0044]    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).  
         [0045]    Vector Administration  
         [0046]    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.  
         [0047]    Investigations in MPS Mouse Models  
         [0048]    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.  
         [0049]    Investigations in MPS I Dogs  
         [0050]    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).  
         [0051]    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.  
         [0052]    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.  
         [0053]    Enzyme Activity, Diffusion and Correction of Storage Lesions in MPS I Mouse Brains.  
         [0054]    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.  
         [0055]    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)               +/+    1.23 ± 0.22 (n = 24)   2.33 ± 0,47 (n = 3)           +/−   0 (n = 14)   1.16 ± 0,27 (n = 13)                −/−   IL   CL   0 (n = 7)                        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                      
 
         [0056]    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.  
         [0057]    Enzyme Activity and Diffusion in the Brain of a MPS I Dog.  
         [0058]    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 measuered. 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.  
         [0059]    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.  
         [0060]    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.  
         [0061]    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.  
         [0062]    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.  
         [0063]    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.  
         [0064]    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   1-2891   Jun. 20, 2002       AAV2-mPGK-IDUA-WPRE-pA   1-2892   Jun. 20, 2002                  
 
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                                           |   10     |   20     |   30     |   40     |   50     |   60     |   70     |   80     |   90     |  100               1   CAGCAGCTGC GCGCTCGCTC GCTCACTGAG GCCGCCCGGG CAAAGCCCGG GCGTCGGGCG ACCTTTGGTC GCCCGGCCTC AGTGAGCGAG CGAGCGCGCA   100               101   GAGAGGGAGT GGCCAACTCC ATCACTAGGG GTTCCTTGTA GTTAATGATT AACCCGCCAT GCTACTTATC TACTCGAGAA TTCTACCGGG TAGGGGAGGC   200               201   GCTTTTCCCA AGGCAGTCTG GAGCATGCGC TTTAGCAGCC CCGCTGGCAC TTGGCGCTAC ACAAGTGGCC TCTGGCCTCG CACACATTCC ACATCCACCG   300               301   GTAGCGCCAA CCGGCTCCGT TCTTTGGTGG CCCCTTCGCG CCACCTTCTA CTCCTCCCCT AGTCAGGAAG TTCCCCCCGC CCCGCAGCTC GCGTCGTGCA   400               401   GGACGTGACA AATGGAAGTA GCACGTCTCA CTAGTCTCGT GCAGATGGAC AGCACCGCTG AGCAATGGAA GCGGGTAGGC CTTTGGGGCA GCGGCCAATA   500               501   GCAGCTTTGC TCCTTCGCTT TCTGGGCTCA GAGGCTGGGA AGGGGTGGGT CCGGGGGCGG GCTCAGGGGC GGGCTCAGGG GCGGGGCGGG CGCGAAGGTC   600               601   CTCCGGAGCC CGGCATTCTG CACGCTTCAA AAGCGCACGT CTGCCGCGCT GTTCTCCTCT TCCTCATCTC CGGGCCTTTC GACCGGATCA GATCGAATTC   700               701   CCCGAAGCCC CGCAGTCCCC GAGCACGCGT GGCCATGCGT CCCCTGCGCC CCCGCGCCGC GCTGCTGGCG CTCCTGGCCT CGCTCCTGGC CGCGCCCCCG   800               801   GTGGCCCCGG CCGAGGCCCC GCACCTGGTG CATGTGGACG CGGCCCGCGC GCTGTGGCCC CTGCGGCGCT TCTGGAGGAG CACAGGCTTC TGCCCCCCGC   900               901   TGCCACACAG CCAGGCTGAC CAGTACGTCC TCAGCTGGGA CCAGCAGCTC AACCTCGCCT ATGTGGGCGC CGTCCCTCAC CGCGGCATCA AGCAGGTCCG   1000               1001   GACCCACTGG CTGCTGGAGC TTGTCACCAC CAGGGGGTCC ACTGGACGGG GCCTGAGCTA CAACTTCACC CACCTGGACG GGTACTTGGA CCTTCTCAGG   1100               1101   GAGAACCAGC TCCTCCCAGG GTTTGAGCTG ATGGGCAGCG CCTCGGGCCA CTTCACTGAC TTTGAGGACA AGCAGGTGTT TGAGTGGAAG GACTTGGTCT   1200               1201   CCAGCCTGGC CAGGAGATAC ATCGGTAGGT ACGGACTGGC GCATGTTTCC AAGTGGAACT TCGAGACGTG GAATGAGCCA GACCACCACG ACTTTGACAA   1300               1301   CGTCTCCATG ACCATGCAAG GCTTCCTGAA CTACTACGAT GCCTGCTCGG AGGGTCTGCG CGCCGCCAGC CCCGCCCTGC GGCTGGGAGG CCCCGGCGAC   1400               1401   TCCTTCCACA CCCCACCGCG ATCCCCGCTG AGCTGGGGCC TCCTGCGCCA CTGCCACGAC GGTACCAACT TCTTCACTGG GGAGGCGGGC GTGCGGCTGG   1500               1501   ACTACATCTC CCTCCACAGG AAGGGTGCGC GCAGCTCCAT CTCCATCCTG GACCAGGAGA AGGTCGTCGC GCAGCAGATC CGGCAGCTCT TCCCCAAGTT   1600               1601   CGCGGACACC CCCATTTACA ACGACGAGGC GGACCCGCTG GTGGGCTGGT CCCTGCCACA GCCGTGGAGG GCGGACGTGA CCTACGCGGC CATGGTGGTG   1700               1701   AAGGTCATCG CGCAGCATCA GAACCTGCTA CTGGCCAACA CCACCTCCGC CTTCCCCTAC GCGCTCCTGA GCAACGACAA TGCCTTCCTG AGCTACCACC   1800               1801   CGCACCCCTT CGCGCAGCGC ACGCTCACCG CGCGCTTCCA GGTCAACAAC ACCCGCCCGC CGCACGTGCA GCTGTTGCGC AAGCCGGTGC TCACGGCCAT   1900               1901   GGGGCTGCTG GCGCTGCTGG ATGAGGAGCA GCTCTGGGCC GAAGTGTCGC AGGCCGGGAC CGTCCTGGAC AGCAACCACA CGGTGGGCGT CCTGGCCAGC   2000               2001   GCCCACCGCC CCCAGGGCCC GGCCGACGCC TGGCGCGCCG CGGTGCTGAT CTACGCGAGC GACGACACCC GCGCCCACCC CAACCGCAGC GTCGCGGTGA   2100               2101   CCCTGCGGCT GCGCGGGGTG CCCCCCGGCC CGGGCCTGGT CTACGTCACG CGCTACCTGG ACAACGGGCT CTGCAGCCCC GACGGCGAGT GGCGGCGCCT   2200               2201   GGGCCGGCCC GTCTTCCCCA CGGCAGAGCA GTTCCGGCGC ATGCGCGCGG CTGAGGACCC GGTGGCCGCG GCGCCCCGCC CCTTACCCGC CGGCGGCCGC   2300               2301   CTGACCCTGC GCCCCGCGCT GCGGCTGCCG TCGCTTTTGC TGGTGCACGT GTGTGCGCGC CCCGAGAAGC CGCCCGGGCA GGTCACGCGG CTCCGCGCCC   2400               2401   TGCCCCTGAC CCAAGGGCAG CTGGTTCTGG TCTGGTCGGA TGAACACGTG GGCTCCAAGT GCCTGTGGAC ATACGAGATC CAGTTCTCTC AGGACGGTAA   2500               2501   GGCGTACACC CCGGTCAGCA GGAAGCCATC GACCTTCAAC CTCTTTGTGT TCAGCCCAGA CACAGGTGCT GTCTCTGGCT CCTACCGAGT TCGAGCCCTG   2600               2601   GACTACTGGG CCCGACCAGG CCCCTTCTCG GACCCTGTGC CGTACCTGGA GGTCCCTGTG CCAAGAGGGC CCCCATCCCC GGGCAATCCA TGAGCCTGTG   2700               2701   CTGAGCCCCA GTGGGTTGGC GATTAGTCCA ATTTGTTAAA GACAGGATAT CAGTGGTCCA GGCTCTAGTT TTGACTCAAC AATATCACCA GCTGAAGCCT   2800               2801   ATAGAGTACG AGCCATAGAT AAAATAAAAG ATTTTATTTA GTCTCCAGAA AAAGGGGGGA ATGAAAGACC CCACCTGTAG GTTTGGCAAG CTAGtCTAGT   2900               2901   AACGGCCGCC AGTGTGCTGG AATTCTGCAG ATATCCATCA CACTGGCGGC CGCTCGAGCA TGCATCTAGA Gcgataatca acctctggat tacaaaattt   3000               3001   gtgaaagatt gactggtatt cttaactatg ttgctccttt tacgctatgt ggatacgctg ctttaatgcc tttgtatcat gctattgctt cccgtatggc   3100               3101   tttcattttc tcctccttgt ataaatcctg gttgctgtct ctttatgagg agttgtggcc cgttgtcagg caacgtggcg tggtgtgcac tgtgtttgct   3200               3201   gacgcaaccc ccactggttg gggcattgcc accacctgtc agctcctttc cgggactttc gccttccccc tccctattgc cacggcggaa ctcatcgccg   3300               3301   cctgccttgc ccgctgctgg acaggggctc ggctgttggg cactgacaat tccgtggtgt tgtcggggaa gctgacgtcc tttccatggc tgctcgcctg   3400               3401   tgttgccacc tggattctgc gcgggacgtc cttctgctac gtcccttcgg ccctcaatcc agcggacctt ccttcccgcg gcctgctgcc ggctctgcgg   3500               3501   cctcttccgc gtcttcgcct tcgccctgag acgagtcgga tctccctttg ggccgcctcc ccgcatcgCT ATTCTATAGT GTCACCTAAA TGCTAGAGCT   3600               3601   CGCTGATCAG CCTCGACTGT GCCTTCTAGT TGCCAGCCAT CTGTTGTTTG CCCCTCCCCC GTGCCTTCCT TGACCCTGGA AGGTGCCACT CCCACTGTCC   3700               3701   TTTCCTAATA AAATGAGGAA ATTGCATCGC ATTGTCTGAG TAGGTGTCAT TCTATTCTGG GGGGTGGGGT GGGGCAGGAC AGCAAGGGGG AGGATTGGGA   3800               3801   AGACAATAGC AGGCATGCTG GGGATGCGGT GGGCTCTATG GCTTCTGAGG CGGAAAGAAC CAGGTAGATA AGTAGCATGG CGGGTTAATC ATTAACTACA   3900               3901   AGGAACCCCT AGTGATGGAG TTGGCCACTC CCTCTCTGCG CGCTCGCTCG CTCACTGAGG CCGGGCGACC AAAGGTCGCC CGACGCCCGG GCTTTGCCCG   4000               4001   GGCGGCCTCA GTGAGCGAGC GAGCGCGCAG CTGGCGTAAT AGCGAAGAGG CCCGCACCGA TCGCCCTTCC CAACAGTTGC GCAGCCTGAA TGGCGAATGG   4100               4101   CGATTCCGTT GCAATGGCTG GCGGTAATAT TGTTCTGGAT ATTACCAGCA AGGCCGATAG TTTGAGTTCT TCTACTCAGG CAAGTGATGT TATTACTAAT   4200               4201   CAAAGAAGTA TTGCGACAAC GGTTAATTTG CGTGATGGAC AGACTCTTTT ACTCGGTGGC CTCACTGATT ATAAAAACAC TTCTCAGGAT TCTGGCGTAC   4300               4301   CGTTCCTGTC TAAAATCCCT TTAATCGGCC TCCTGTTTAG CTCCCGCTCT GATTCTAACG AGGAAAGCAC GTTATACGTG CTCGTCAAAG CAACCATAGT   4400               4401   ACGCGCCCTG TAGCGGCGCA TTAAGCGCGG CGGGTGTGGT GGTTACGCGC AGCGTGACCG CTACACTTGC CAGCGCCCTA GCGCCCGCTC CTTTCGCTTT   4500               4501   CTTCCCTTCC TTTCTCGCCA CGTTCGCCGG CTTTCCCCGT CAAGCTCTAA ATCGGGGGCT CCCTTTAGGG TTCCGATTTA GTGCTTTACG GCACCTCGAC   4600               4601   CCCAAAAAAC TTGATTAGGG TGATGGTTCA CGTAGTGGGC CATCGCCCTG ATAGACGGTT TTTCGCCCTT TGACGTTGGA GTCCACGTTC TTTAATAGTG   4700               4701   GACTCTTGTT CCAAACTGGA ACAACACTCA ACCCTATCTC GGTCTATTCT TTTGATTTAT AAGGGATTTT GCCGATTTCG GCCTATTGGT TAAAAAATGA   4800               4801   GCTGATTTAA CAAAAATTTA ACGCGAATTT TAACAAAATA TTAACGCTTA CAATTTAAAT ATTTGCTTAT ACAATCTTCC TGTTTTTGGG GCTTTTCTGA   4900               4901   TTATCAACCG GGGTACATAT GATTGACATG CTAGTTTTAC GATTACCGTT CATCGATTCT CTTGTTTGCT CCAGACTCTC AGGCAATGAC CTGATAGCCT   5000               5001   TTGTAGAGAC CTCTCAAAAA TAGCTACCCT CTCCGGCATG AATTTATCAG CTAGAACGGT TGAATATCAT ATTGATGGTG ATTTGACTGT CTCCGGCCTT   5100               5101   TCTCACCCGT TTGAATCTTT ACCTACACAT TACTCAGGCA TTGCATTTAA AATATATGAG GGTTCTAAAA ATTTTTATCC TTGCGTTGAA ATAAAGGCTT   5200               5201   CTCCCGCAAA AGTATTACAG GGTCATAATG TTTTTGGTAC AACCGATTTA GCTTTATGCT CTGAGGCTTT ATTGCTTAAT TTTGCTAATT CTTTGCCTTG   5300               5301   CCTGTATGAT TTATTGGATG TTGGAATCGC CTGATGCGGT ATTTTCTCCT TACGCATCTG TGCGGTATTT CACACCGCAT ATGGTGCACT CTCAGTACAA   5400               5401   TCTGCTCTGA TGCCGCATAG TTAAGCCAGC CCCGACACCC GCCAACACCC GCTGACGCGC CCTGACGGGC TTGTCTGCTC CCGGCATCCG CTTACAGACA   5500               5501   AGCTGTGACC GTCTCCGGGA GCTGCATGTG TCAGAGGTTT TCACCGTCAT CACCGAAACG CGCGAGACGA AAGGGCCTCG TGATACGCCT ATTTTTATAG   5600               5601   GTTAATGTCA TGATAATAAT GGTTTCTTAG ACGTCAGGTG GCACTTTTCG GGGAAATGTG CGCGGAACCC CTATTTGTTT ATTTTTCTAA ATACATTCAA   5700               5701   ATATGTATCC GCTCATGAGA CAATAACCCT GATAAATGCT TCAATAATAT TGAAAAAGGA AGAGTATGAG TATTCAACAT TTCCGTGTCG CCCTTATTCC   5800               5801   CTTTTTTGCG GCATTTTGCC TTCCTGTTTT TGCTCACCCA GAAACGCTGG TGAAAGTAAA AGATGCTGAA GATCAGTTGG GTGCACGAGT GGGTTACATC   5900               5901   GAACTGGATC TCAACAGCGG TAAGATCCTT GAGAGTTTTC GCCCCGAAGA ACGTTTTCCA ATGATGAGCA CTTTTAAAGT TCTGCTATGT GGCGCGGTAT   6000               6001   TATCCCGTAT TGACGCCGGG CAAGAGCAAC TCGGTCGCCG CATACACTAT TCTCAGAATG ACTTGGTTGA GTACTCACCA GTCACAGAAA AGCATCTTAC   6100               6101   GGATGGCATG ACAGTAAGAG AATTATGCAG TGCTGCCATA ACCATGAGTG ATAACACTGC GGCCAACTTA CTTCTGACAA CGATCGGAGG ACCGAAGGAG   6200               6201   CTAACCGCTT TTTTGCACAA CATGGGGGAT CATGTAACTC GCCTTGATCG TTGGGAACCG GAGCTGAATG AAGCCATACC AAACGACGAG CGTGACACCA   6300               6301   CGATGCCTGT AGCAATGGCA ACAACGTTGC GCAAACTATT AACTGGCGAA CTACTTACTC TAGCTTCCCG GCAACAATTA ATAGACTGGA TGGAGGCGGA   6400               6401   TAAAGTTGCA GGACCACTTC TGCGCTCGGC CCTTCCGGCT GGCTGGTTTA TTGCTGATAA ATCTGGAGCC GGTGAGCGTG GGTCTCGCGG TATCATTGCA   6500               6501   GCACTGGGGC CAGATGGTAA GCCCTCCCGT ATCGTAGTTA TCTACACGAC GGGGAGTCAG GCAACTATGG ATGAACGAAA TAGACAGATC GCTGAGATAG   6600               6601   GTGCCTCACT GATTAAGCAT TGGTAACTGT CAGACCAAGT TTACTCATAT ATACTTTAGA TTGATTTAAA ACTTCATTTT TAATTTAAAA GGATCTAGGT   6700               6701   GAAGATCCTT TTTGATAATC TCATGACCAA AATCCCTTAA CGTGAGTTTT CGTTCCACTG AGCGTCAGAC CCCGTAGAAA AGATCAAAGG ATCTTCTTGA   6800               6801   GATCCTTTTT TTCTGCGCGT AATCTGCTGC TTGCAAACAA AAAAACCACC GCTACCAGCG GTGGTTTGTT TGCCGGATCA AGAGCTACCA ACTCTTTTTC   6900               6901   CGAAGGTAAC TGGCTTCAGC AGAGCGCAGA TACCAAATAC TGTTCTTCTA GTGTAGCCGT AGTTAGGCCA CCACTTCAAG AACTCTGTAG CACCGCCTAC   7000               7001   ATACCTCGCT CTGCTAATCC TGTTACCAGT GGCTGCTGCC AGTGGCGATA AGTCGTGTCT TACCGGGTTG GACTCAAGAC GATAGTTACC GGATAAGGCG   7100               7101   CAGCGGTCGG GCTGAACGGG GGGTTCGTGC ACACAGCCCA GCTTGGAGCG AACGACCTAC ACCGAACTGA GATACCTACA GCGTGAGCTA TGAGAAAGCG   7200               7201   CCACGCTTCC CGAAGGGAGA AAGGCGGACA GGTATCCGGT AAGCGGCAGG GTCGGAACAG GAGAGCGCAC GAGGGAGCTT CCAGGGGGAA ACGCCTGGTA   7300               7301   TCTTTATAGT CCTGTCGGGT TTCGCCACCT CTGACTTGAG CGTCGATTTT TGTGATGCTC GTCAGGGGGG CGGAGCCTAT GGAAAAACGC CAGCAACGCG   7400               7401   GCCTTTTTAC GGTTCCTGGC CTTTTGCTGG CCTTTTGCTC ACATGTTCTT TCCTGCGTTA TCCCCTGATT CTGTGGATAA CCGTATTACC GCCTTTGAGT   7500               7501   GAGCTGATAC CGCTCGCCGC AGCCGAACGA CCGAGCGCAG CGAGTCAGTG AGCGAGGAAG CGGAAGAGCG CCCAATACGC AAACCGCCTC TCCCCGCGCG   7600               7601   TTGGCCGATT CATTAATGCA GCTGCGCGCT CGCTCGCTCA CTGAGGCC   7648               |   10     |   20     |   30     |   40     |   50     |   60     |   70     |   80     |   90     |  100                       |   10     |   20     |   30     |   40     |   50     |   60     |   70     |   80     |   90     |  100       1   CAGCAGCTGC GCGCTCGCTC GCTCACTGAG GCCGCCCGGG CAAAGCCCGG GCGTCGGGCG ACCTTTGGTC GCCCGGCCTC AGTGAGCGAG CGAGCGCGCA   100               101   GAGAGGGAGT GGCCAACTCC ATCACTAGGG GTTCCTTGTA GTTAATGATT AACCCGCCAT GCTACTTATC TACTCGAGAA TTCTACCGGG TAGGGGAGGC   200               201   GCTTTTCCCA AGGCAGTCTG GAGCATGCGC TTTAGCAGCC CCGCTGGCAC TTGGCGCTAC ACAAGTGGCC TCTGGCCTCG CACACATTCC ACATCCACCG   300               301   GTAGCGCCAA CCGGCTCCGT TCTTTGGTGG CCCCTTCGCG CCACCTTCTA CTCCTCCCCT AGTCAGGAAG TTCCCCCCGC CCCGCAGCTC GCGTCGTGCA   400               401   GGACGTGACA AATGGAAGTA GCACGTCTCA CTAGTCTCGT GCAGATGGAC AGCACCGCTG AGCAATGGAA GCGGGTAGGC CTTTGGGGCA GCGGCCAATA   500               501   GCAGCTTTGC TCCTTCGCTT TCTGGGCTCA GAGGCTGGGA AGGGGTGGGT CCGGGGGCGG GCTCAGGGGC GGGCTCAGGG GCGGGGCGGG CGCGAAGGTC   600               601   CTCCGGAGCC CGGCATTCTG CACGCTTCAA AAGCGCACGT CTGCCGCGCT GTTCTCCTCT TCCTCATCTC CGGGCCTTTC GACCGGATCC CCCGGGCTGC   700               701   AGGAATTCCG AGACCATGGA GGCGGTGGCG GTGGCCGCGG CGGTGGGGGT CCTTCTCCTG GCCGGGGCCG GGGGCGCGGC AGGCGACGAG GCCCGGGAGG   900               801   CGGCGGCCGT GCGGGCGCTC GTGGCCCGGC TGCTGGGGCC AGGCCCCGCG GCCGACTTCT CCGTGTCGGT GGAGCGCGCT CTGGCTGCCA AGCCGGGCTT   800               901   GGACACCTAC AGCCTGGGCG GCGGCGGCGC GGCGCGCGTG CGGGTGCGCG GCTCCACGGG CGTGGCGGCC GCCGCGGGGC TGCACCGCTA CCTGCGCGAC   1000               1001   TTCTGTGGCT GCCACGTGGC CTGGTCCGGC TCTCAGCTGC GCCTGCCGCG GCCACTGCCA GCCGTGCCGG GGGAGCTGAC CGAGGCCACG CCCAACAGGT   1100               1101   ACCGCTATTA CCAGAATGTG TGCACGCAAA GCTACTCCTT CGTGTGGTGG GACTGGGCCC GCTGGGAGCG AGAGATAGAC TGGATGGCGC TGAATGGCAT   1200               1201   CAACCTGGCA CTGGCCTGGA GCGGCCAGGA GGCCATCTGG CAGCGGGTGT ACCTGGCCTT GGGCCTGACC CAGGCAGAGA TCAATGAGTT CTTTACTGGT   1300               1301   CCTGCCTTCC TGGCCTGGGG GCGAATGGGC AACCTGCACA CCTGGGATGG CCCCCTGCCC CCCTCCTGGC ACATCAAGCA GCTTTACCTG CAGCACCGGG   1400               1401   TCCTGGACCA GATGCGCTCC TTCGGCATGA CCCCAGTGCT GCCTGCATTC GCGGGGCATG TTCCCGAGGC TGTCACCAGG GTGTTCCCTC AGGTCAATGT   1500               1501   CACGAAGATG GGCAGTTGGG GCCACTTTAA CTGTTCCTAC TCCTGCTCCT TCCTTCTGGC TCCGGAAGAC CCCATATTCC CCATCATCGG GAGCCTCTTC   1600               1601   CTGCGAGAGC TGATCAAAGA GTTTGGCACA GACCACATCT ATGGGGCCGA CACTTTCAAT GAGATGCAGC CACCTTCCTC AGAGCCCTCC TACCTTGCCG   1700               1701   CAGCCACCAC TGCCGTCTAT GAGGCCATGA CTGCAGTGGA TACTGAGGCT GTGTGGCTGC TCCAAGGCTG GCTCTTCCAG CACCAGCCGC AGTTCTGGGG   1800               1801   GCCCGCCCAG ATCAGGGCTG TGCTGGGAGC TGTGCCCCGT GGCCGCCTCC TGGTTCTGGA CCTGTTTGCT GAGAGCCAGC CTGTGTATAC CCGCACTGCC   1900               1901   TCCTTCCAGG GCCAGCCCTT CATCTGGTGC ATGCTGCACA ACTTTGGGGG AAACCATGGT CTTTTTGGAG CCCTAGAGGC TGTGAACGGA GGCCCAGAAG   2000               2001   CTGCCCGCCT CTTCCCCAAC TCCACCATGG TAGGCACGGG CATGGCCCCC GAGGGCATCA GCCAGAACGA AGTGGTCTAT TCCCTCATGG CTGAGCTGGG   2100               2101   CTGGCGAAAG GACCCAGTGC CAGATTTGGC AGCCTGGGTG ACCAGCTTTG CCGCCCGGCG GTATGGGGTC TCCCACCCGG ACGCAGGGGC AGCGTGGAGG   2200               2201   CTACTGCTCC GGAGTGTGTA CAACTGCTCC GGGGAGGCCT GCAGGGGCCA CAATCGTAGC CCGCTGGTCA GGCGGCCGTC CCTACAGATG AATACCAGCA   2300               2301   TCTGGTACAA CCGATCTGAT GTGTTTGAGG CCTGGCGGCT GCTGCTCACA TCTGCTCCCT CCCTGGCCAC CAGCCCCGCC TTCCGCTACG ACCTGCTGGA   2400               2401   CCTCACTCGG CAGGCAGTGC AGGAGCTGGT CAGCTTGTAC TATGAGGAGG CAAGAAGCGC CTACCTGAGC AAGGAGCTGG CCTCCCTGTT GAGGGCTGGA   2500               2501   GGCGTCCTGG CCTATGAGCT GCTGCCGGCA CTGGACGAGG TGCTGGCTAG TGACAGCCGC TTCTTGCTGG GCAGCTGGCT AGAGCAGGCC CGAGCAGCGG   2600               2601   CAGTCAGTGA GGCCGAGGCC GATTTCTACG AGCAGAACAG CCGCTACCAG CTGACCTTGT GGGGGCCAGA AGGCAACATC CTGGACTATG CCAACAAGCA   2700               2701   GCTGGCGGGG TTGGTGGCCA ACTACTACAC CCCTCGCTGG CGGCTTTTCC TGGAGGCGCT GGTTGACAGT GTGGCCCAGG GCATCCCTTT CCAACAGCAC   2800               2801   CAGTTTGACA AAAATGTCTT CCAACTGGAG CAGGCCTTCG TTCTCAGCAA GCAGAGGTAC CCCAGCCAGC CGCGAGGAGA CACTGTGGAC CTGGCCAAGA   2900               2901   AGATCTTCCT CAAATATTAC CCCGGCTGGG TGGCCGGCTC TTGGTGATAG ATTCGCCACC ACTGGGCCTT GTTTTCCGCT AATTCCAGGG CAGATTCCAG   3000               3001   GGCCCAGAGC TGGACAGACA TCACAGGATA ACCCAGGCCT GGGAGGAGGC CCCACGGCCT GCTGGTGGGG TCTGACCTGG GGGGATTGGA GGGAAATGAC   3100               3101   CTGCCCTCCA CCACCACCCA AAGTGTGGGA TTAAAGTAGC TTGGTACCGA GCTCGGATCC GGCGATTAGT CCAATTTGTT AAAGACAGGA TATCAGTGGT   3200               3201   CCAGGCTCTA GTTTTGACTC AACAATATCA CCAGCTGAAG CCTATAGAGT ACGAGCCATA GATAAAATAA AAGATTTTAT TTAGTCTCCA GAAAAAGGGG   3300               3301   GGAATGAAAG ACCCCACCTG TAGGTTTGGC AAGCTAGCgC TAGTAACGGC CGCCAGTGTG CTGGAATTCT GCAGATATCC ATCACACTGG CGGCCGCTCG   3400               3401   AGCATGCATC TAGAGcgata atcaacctct ggattacaaa atttgtgaaa gattgactgg tattcttaac tatgttgctc cttttacgct atgtggatac   3500               3501   gctgctttaa tgcctttgta tcatgctatt gcttcccgta tggctttcat tttctcctcc ttgtataaat cctggttgct gtctctttat gaggagttgt   3600               3601   ggcccgttgt caggcaacgt ggcgtggtgt gcactgtgtt tgctgacgca acccccactg gttggggcat tgccaccacc tgccagctcc tttccgggac   3700               3701   tttcgctttc cccctcccta ttgccacggc ggaactcatc gccgcctgcc ttgcccgctg ctggacaggg gctcggctgt tgggcactga caattccgtg   3800               3801   gtgttgtcgg ggaagctgac gtcctttcca tggctgctcg cctgtgttgc cacctggatt ctgcgcggga cgtccttctg ctacgtccct tcggccctca   3900               3901   atccagcgga ccttccttcc cgcggcctgc tgccggctct gcggcctctt ccgcgtcttc gccttcgccc tgagacgagt cggatctccc tttgggccgc   4000               4001   ctccccgcat cgCTATTCTA TAGTGTCACC TAAATGCTAG AGCTCGCTGA TCAGCCTCGA CTGTGCCTTC TAGTTGCCAG CCATCTGTTG TTTGCCCCTC   4100               4101   CCCCGTGCCT TCCTTGACCC TGGAAGGTGC CACTCCCACT GTCCTTTCCT AATAAAATGA GGAAATTGCA TCGCATTGTC TGAGTAGGTG TCATTCTATT   4200               4201   CTGGGGGGTG GGGTGGGGCA GGACAGCAAG GGGGAGGATT GGGAAGACAA TAGCAGGCAT GCTGGGGATG CGGTGGGCTC TATGGCTTCT GAGGCGGAAA   4300               4301   GAACCAGGTA GATAAGTAGC ATGGCGGGTT AATCATTAAC TACAAGGAAC CCCTAGTGAT GGAGTTGGCC ACTCCCTCTC TGCGCGCTCG CTCGCTCACT   4400               4401   GAGGCCGGGC GACCAAAGGT CGCCCGACGC CCGGGCTTTG CCCGGGCGGC CTCAGTGAGC GAGCGAGCGC GCAGCTGGCG TAATAGCGAA GAGGCCCGCA   4500               4501   CCGATCGCCC TTCCCAACAG TTGCGCAGCC TGAATGGCGA ATGGCGATTC CGTTGCAATG GCTGGCGGTA ATATTGTTCT GGATATTACC AGCAAGGCCG   4600               4601   ATAGTTTGAG TTCTTCTACT CAGGCAAGTG ATGTTATTAC TAATCAAAGA AGTATTGCGA CAACGGTTAA TTTGCGTGAT GGACAGACTC TTTTACTCGG   4700               4701   TGGCCTCACT GATTATAAAA ACACTTCTCA GGATTCTGGC GTACCGTTCC TGTCTAAAAT CCCTTTAATC GGCCTCCTGT TTAGCTCCCG CTCTGATTCT   4800               4801   AACGAGGAAA GCACGTTATA CGTGCTCGTC AAAGCAACCA TAGTACGCGC CCTGTAGCGG CGCATTAAGC GCGGCGGGTG TGGTGGTTAC GCGCAGCGTG   4900               4901   ACCGCTACAC TTGCCAGCGC CCTAGCGCCC GCTCCTTTCG CTTTCTTCCC TTCCTTTCTC GCCACGTTCG CCGGCTTTCC CCGTCAAGCT CTAAATCGGG   5000               5001   GGCTCCCTTT AGGGTTCCGA TTTAGTGCTT TACGGCACCT CGACCCCAAA AAACTTGATT AGGGTGATGG TTCACGTAGT GGGCCATCGC CCTGATAGAC   5100               5101   GGTTTTTCGC CCTTTGACGT TGGAGTCCAC GTTCTTTAAT AGTGGACTCT TGTTCCAAAC TGGAACAACA CTCAACCCTA TCTCGGTCTA TTCTTTTGAT   5200               5201   TTATAAGGGA TTTTGCCGAT TTCGGCCTAT TGGTTAAAAA ATGAGCTGAT TTAACAAAAA TTTAACGCGA ATTTTAACAA AATATTAACG CTTACAATTT   5300               5301   AAATATTTGC TTATACAATC TTCCTGTTTT TGGGGCTTTT CTGATTATCA ACCGGGGTAC ATATGATTGA CATGCTAGTT TTACGATTAC CGTTCATCGA   5400               5401   TTCTCTTGTT TGCTCCAGAC TCTCAGGCAA TGACCTGATA GCCTTTGTAG AGACCTCTCA AAAATAGCTA CCCTCTCCGG CATGAATTTA TCAGCTAGAA   5500               5501   CGGTTGAATA TCATATTGAT GGTGATTTGA CTGTCTCCGG CCTTTCTCAC CCGTTTGAAT CTTTACCTAC ACATTACTCA GGCATTGCAT TTAAAATATA   5600               5601   TGAGGGTTCT AAAAATTTTT ATCCTTGCGT TGAAATAAAG GCTTCTCCCG CAAAAGTATT ACAGGGTCAT AATGTTTTTG GTACAACCGA TTTAGCTTTA   5700               5701   TGCTCTGAGG CTTTATTGCT TAATTTTGCT AATTCTTTGC CTTGCCTGTA TGATTTATTG GATGTTGGAA TCGCCTGATG CGGTATTTTC TCCTTACGCA   5800               5801   TCTGTGCGGT ATTTCACACC GCATATGGTG CACTCTCAGT ACAATCTGCT CTGATGCCGC ATAGTTAAGC CAGCCCCGAC ACCCGCCAAC ACCCGCTGAC   5900               5901   GCGCCCTGAC GGGCTTGTCT GCTCCCGGCA TCCGCTTACA GACAAGCTGT GACCGTCTCC GGGAGCTGCA TGTGTCAGAG GTTTTCACCG TCATCACCGA   6000               6001   AACGCGCGAG ACGAAAGGGC CTCGTGATAC GCCTATTTTT ATAGGTTAAT GTCATGATAA TAATGGTTTC TTAGACGTCA GGTGGCACTT TTCGGGGAAA   6100               6101   TCTGCGCGGA ACCCCTATTT GTTTATTTTT CTAAATACAT TCAAATATGT ATCCGCTCAT GAGACAATAA CCCTGATAAA TGCTTCAATA ATATTGAAAA   6200               6201   AGGAAGAGTA TGAGTATTCA ACATTTCCGT GTCGCCCTTA TTCCCTTTTT TGCGGCATTT TGCCTTCCTG TTTTTGCTCA CCCAGAAACG CTGGTGAAAG   6300               6301   TAAAAGATGC TGAAGATCAG TTGGGTGCAC GAGTGGGTTA CATCGAACTG GATCTCAACA GCGGTAAGAT CCTTGAGAGT TTTCGCCCCG AAGAACGTTT   6400               6401   TCCAATGATG AGCACTTTTA AAGTTCTGCT ATGTGGCGCG GTATTATCCC GTATTGACGC CGGGCAAGAG CAACTCGGTC GCCGCATACA CTATTCTCAG   6500               6501   AATGACTTGG TTGAGTACTC ACCAGTCACA GAAAAGCATC TTACGGATGG CATGACAGTA AGAGAATTAT GCAGTGCTGC CATAACCATG AGTGATAACA   6600               6601   CTGCGGCCAA CTTACTTCTG ACAACGATCG GAGGACCGAA GGAGCTAACC GCTTTTTTGC ACAACATGGG GGATCATGTA ACTCGCCTTG ATCGTTGGGA   6700               6701   ACCGGAGCTG AATGAAGCCA TACCAAACGA CGAGCGTGAC ACCACGATGC CTGTAGCAAT GGCAACAACG TTGCGCAAAC TATTAACTGG CGAACTACTT   6800               6801   ACTCTAGCTT CCCGGCAACA ATTAATAGAC TGGATGGAGG CGGATAAAGT TGCAGGACCA CTTCTGCGCT CGGCCCTTCC GGCTGGCTGG TTTATTGCTG   6900               6901   ATAAATCTGG AGCCGGTGAG CGTGGGTCTC GCGGTATCAT TGCAGCACTG GGGCCAGATG GTAAGCCCTC CCGTATCGTA GTTATCTACA CGACGGGGAG   7000               7001   TCAGGCAACT ATGGATGAAC GAAATAGACA GATCGCTGAG ATAGGTGCCT CACTGATTAA GCATTGGTAA CTGTCAGACC AAGTTTACTC ATATATACTT   7100               7101   TAGATTGATT TAAAACTTCA TTTTTAATTT AAAAGGATCT AGGTGAAGAT CCTTTTTGAT AATCTCATGA CCAAAATCCC TTAACGTGAG TTTTCGTTCC   7200               7201   ACTGAGCGTC AGACCCCGTA GAAAAGATCA AAGGATCTTC TTGAGATCCT TTTTTTCTGC GCGTAATCTG CTGCTTGCAA ACAAAAAAAC CACCGCTACC   7300               7301   AGCGGTGGTT TGTTTGCCGG ATCAAGAGCT ACCAACTCTT TTTCCGAAGG TAACTGGCTT CAGCAGAGCG CAGATACCAA ATACTGTTCT TCTAGTGTAG   7400               7401   CCGTAGTTAG GCCACCACTT CAAGAACTCT GTAGCACCGC CTACATACCT CGCTCTGCTA ATCCTGTTAC CAGTGGCTGC TGCCAGTGGC GATAAGTCGT   7500               7501   GTCTTACCGG GTTGGACTCA AGACGATAGT TACCGGATAA GGCGCAGCGG TCGGGCTGAA CGGGGGGTTC GTGCACACAG CCCAGCTTGG AGCGAACGAC   7600               7601   CTACACCGAA CTGAGATACC TACAGCGTGA GCTATGAGAA AGCGCCACGC TTCCCGAAGG GAGAAAGGCG GACAGGTATC CGGTAAGCGG CAGGGTCGGA   7700               7701   ACAGGAGAGC GCACGAGGGA GCTTCCAGGG GGAAACGCCT GGTATCTTTA TAGTCCTGTC GGGTTTCGCC ACCTCTGACT TGAGCGTCGA TTTTTGTGAT   7800               7801   GCTCGTCAGG GGGGCGGAGC CTATGGAAAA ACGCCAGCAA CGCGGCCTTT TTACGGTTCC TGGCCTTTTG CTGGCCTTTT GCTCACATGT TCTTTCCTGC   7900               7901   GTTATCCCCT GATTCTGTGG ATAACCGTAT TACCGCCTTT GAGTGAGCTG ATACCGCTCG CCGCAGCCGA ACGACCGAGC GCAGCGAGTC AGTGAGCGAG   8000               8001   GAAGCGGAAG AGCGCCCAAT ACGCAAACCG CCTCTCCCCG CGCGTTGGCC GATTCATTAA TGCAGCTGCG CGCTCGCTCG CTCACTGAGG CC   8092               |   10     |   20     |   30     |   40     |   50     |   60     |   70     |   80     |   90     |  100