U.S. patent application number 11/329295 was filed with the patent office on 2006-08-17 for vector for expressing alpha-n-acetyl-galactosaminidase and method of treating mps i by stereotactic injection into the brain of a mammal.
This patent application is currently assigned to Institut Pasteur. Invention is credited to Nathalie Desmaris, Jean Michel Heard.
Application Number | 20060182717 11/329295 |
Document ID | / |
Family ID | 31186406 |
Filed Date | 2006-08-17 |
United States Patent
Application |
20060182717 |
Kind Code |
A1 |
Desmaris; Nathalie ; et
al. |
August 17, 2006 |
Vector for expressing alpha-N-acetyl-galactosaminidase and method
of treating MPS I by stereotactic injection into the brain of a
mammal
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.
Inventors: |
Desmaris; Nathalie;
(Roissy-en-Brie, FR) ; Heard; Jean Michel; (Paris,
FR) |
Correspondence
Address: |
FINNEGAN, HENDERSON, FARABOW, GARRETT & DUNNER;LLP
901 NEW YORK AVENUE, NW
WASHINGTON
DC
20001-4413
US
|
Assignee: |
Institut Pasteur
Paris
FR
|
Family ID: |
31186406 |
Appl. No.: |
11/329295 |
Filed: |
January 11, 2006 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
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10176066 |
Jun 21, 2002 |
|
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11329295 |
Jan 11, 2006 |
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Current U.S.
Class: |
424/93.2 ;
424/93.21; 435/252.3; 435/366; 435/456 |
Current CPC
Class: |
C12N 9/16 20130101; A61K
48/00 20130101; C12N 9/2462 20130101; C12Y 302/01076 20130101; C07H
21/04 20130101; C12N 9/2402 20130101 |
Class at
Publication: |
424/093.2 ;
424/093.21; 435/366; 435/456; 435/252.3 |
International
Class: |
A61K 48/00 20060101
A61K048/00; C12N 15/867 20060101 C12N015/867; C12N 1/21 20060101
C12N001/21; C12N 5/08 20060101 C12N005/08 |
Claims
1-22. (canceled)
23. An AAV vector for the expression of
.alpha.-N-acetyl-glucosaminidase, wherein the vector comprises (a)
the nucleic acid coding sequence for
.alpha.-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 .alpha.-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 .alpha.-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 .alpha.-L-iduronidase-encoding
sequence operably linked to (a) and (b), wherein brain cells local
to the injection site express .alpha.-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 .alpha.-N-acetyl-glucosaminidase-encoding sequence
operably linked to (a) and (b), wherein brain cells local to the
injection site express .alpha.-N-acetyl-glucosaminidase from the
vector sequences.
28. A method of providing .alpha.-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 .alpha.-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 .alpha.-N-acetyl-glucosaminidase is delivered to areas of the
brain of the mammal distal to the injection site.
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, 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 .alpha.-L-iduronidase (IDUA) and
.alpha.-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 (>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 exanple, 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] FIG. 1. Structure of the AAV-PGK-IDUA and the AAV-PGK-NaGLU
vectors.
[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 .mu.m 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] 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 .mu.m)
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] 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 .beta.-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 .beta.-glucuronidase, or
received a functional .beta.-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 .beta.-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 .beta.-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.
[0035] Material and Methods
[0036] Gene Transfer Vectors
[0037] Investigations in MPS I and MPS IIIb mice were performed
with the AAV-PGK-IDUA and the AAV-PGK-NaGLU vectors,
respectively.
[0038] 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: [0039] 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). [0040] 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).
[0041] 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. [0042] 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 Geneve) has been isolated
from a plasmid, kindly provided to us by Dr. Naldini (Universita di
Torino). [0043] 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).
[0044] Vector Preparation
[0045] Vectors stocks were prepared in the Laboratoire de Therapie
Genique, CHU Hotel-Dieu, Nantes, by triple transfection into 293-T
cells, as described in Salvetti et al. (Salvetti et al., 1998).
[0046] Vector Administration
[0047] Vectors were administrated by stereotactic injection in the
brain tissue. In the mouse, a single injection of 5 .mu.L
containing 2.times.10.sup.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 .mu.L injection was
performed.
[0048] Investigations in MPS Mouse Models
[0049] MPS I and MPS IIIb mice have been obtained by a selective
disruption of the genes coding for .alpha.-L-iduronidase
(IDUA)(Clarke et al., 1997) and .alpha.-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.
[0050] Investigations in MPS I Dogs
[0051] 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).
[0052] 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.
[0053] MPS I dogs are genotyped and homozygous animals are
transferred to the Centre de Boisbonne of the Ecole Nationale
Veterinaire de Nantes at weaning. Surgery is performed at the
Centre de Boisbonne.
[0054] Enzyme Activity, Diffusion and Correction of Storage Lesions
in MPS I Mouse Brains.
[0055] 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.
[0056] 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-US-00001 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
[0057] 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
.mu.m 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.
[0058] Enzyme Activity and Diffusion in the Brain of a MPS I
Dog.
[0059] A 40 .mu.L 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.
[0060] 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 .beta.-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.
[0061] 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.
[0062] 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.
[0063] 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.
[0064] 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.
[0065] 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:
TABLE-US-00002 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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Sequence CWU 1
1
2 1 7648 DNA Artificial Sequence Description of Artificial Sequence
vector AAV-PGK-IDUA 1 cagcagctgc gcgctcgctc gctcactgag gccgcccggg
caaagcccgg gcgtcgggcg 60 acctttggtc gcccggcctc agtgagcgag
cgagcgcgca gagagggagt ggccaactcc 120 atcactaggg gttccttgta
gttaatgatt aacccgccat gctacttatc tactcgagaa 180 ttctaccggg
taggggaggc gcttttccca aggcagtctg gagcatgcgc tttagcagcc 240
ccgctggcac ttggcgctac acaagtggcc tctggcctcg cacacattcc acatccaccg
300 gtagcgccaa ccggctccgt tctttggtgg ccccttcgcg ccaccttcta
ctcctcccct 360 agtcaggaag ttccccccgc cccgcagctc gcgtcgtgca
ggacgtgaca aatggaagta 420 gcacgtctca ctagtctcgt gcagatggac
agcaccgctg agcaatggaa gcgggtaggc 480 ctttggggca gcggccaata
gcagctttgc tccttcgctt tctgggctca gaggctggga 540 aggggtgggt
ccgggggcgg gctcaggggc gggctcaggg gcggggcggg cgcgaaggtc 600
ctccggagcc cggcattctg cacgcttcaa aagcgcacgt ctgccgcgct gttctcctct
660 tcctcatctc cgggcctttc gaccggatca gatcgaattc cccgaagccc
cgcagtcccc 720 gagcacgcgt ggccatgcgt cccctgcgcc cccgcgccgc
gctgctggcg ctcctggcct 780 cgctcctggc cgcgcccccg gtggccccgg
ccgaggcccc gcacctggtg catgtggacg 840 cggcccgcgc gctgtggccc
ctgcggcgct tctggaggag cacaggcttc tgccccccgc 900 tgccacacag
ccaggctgac cagtacgtcc tcagctggga ccagcagctc aacctcgcct 960
atgtgggcgc cgtccctcac cgcggcatca agcaggtccg gacccactgg ctgctggagc
1020 ttgtcaccac cagggggtcc actggacggg gcctgagcta caacttcacc
cacctggacg 1080 ggtacttgga ccttctcagg gagaaccagc tcctcccagg
gtttgagctg atgggcagcg 1140 cctcgggcca cttcactgac tttgaggaca
agcaggtgtt tgagtggaag gacttggtct 1200 ccagcctggc caggagatac
atcggtaggt acggactggc gcatgtttcc aagtggaact 1260 tcgagacgtg
gaatgagcca gaccaccacg actttgacaa cgtctccatg accatgcaag 1320
gcttcctgaa ctactacgat gcctgctcgg agggtctgcg cgccgccagc cccgccctgc
1380 ggctgggagg ccccggcgac tccttccaca ccccaccgcg atccccgctg
agctggggcc 1440 tcctgcgcca ctgccacgac ggtaccaact tcttcactgg
ggaggcgggc gtgcggctgg 1500 actacatctc cctccacagg aagggtgcgc
gcagctccat ctccatcctg gagcaggaga 1560 aggtcgtcgc gcagcagatc
cggcagctct tccccaagtt cgcggacacc cccatttaca 1620 acgacgaggc
ggacccgctg gtgggctggt ccctgccaca gccgtggagg gcggacgtga 1680
cctacgcggc catggtggtg aaggtcatcg cgcagcatca gaacctgcta ctggccaaca
1740 ccacctccgc cttcccctac gcgctcctga gcaacgacaa tgccttcctg
agctaccacc 1800 cgcacccctt cgcgcagcgc acgctcaccg cgcgcttcca
ggtcaacaac acccgcccgc 1860 cgcacgtgca gctgttgcgc aagccggtgc
tcacggccat ggggctgctg gcgctgctgg 1920 atgaggagca gctctgggcc
gaagtgtcgc aggccgggac cgtcctggac agcaaccaca 1980 cggtgggcgt
cctggccagc gcccaccgcc cccagggccc ggccgacgcc tggcgcgccg 2040
cggtgctgat ctacgcgagc gacgacaccc gcgcccaccc caaccgcagc gtcgcggtga
2100 ccctgcggct gcgcggggtg ccccccggcc cgggcctggt ctacgtcacg
cgctacctgg 2160 acaacgggct ctgcagcccc gacggcgagt ggcggcgcct
gggccggccc gtcttcccca 2220 cggcagagca gttccggcgc atgcgcgcgg
ctgaggaccc ggtggccgcg gcgccccgcc 2280 ccttacccgc cggcggccgc
ctgaccctgc gccccgcgct gcggctgccg tcgcttttgc 2340 tggtgcacgt
gtgtgcgcgc cccgagaagc cgcccgggca ggtcacgcgg ctccgcgccc 2400
tgcccctgac ccaagggcag ctggttctgg tctggtcgga tgaacacgtg ggctccaagt
2460 gcctgtggac atacgagatc cagttctctc aggacggtaa ggcgtacacc
ccggtcagca 2520 ggaagccatc gaccttcaac ctctttgtgt tcagcccaga
cacaggtgct gtctctggct 2580 cctaccgagt tcgagccctg gactactggg
cccgaccagg ccccttctcg gaccctgtgc 2640 cgtacctgga ggtccctgtg
ccaagagggc ccccatcccc gggcaatcca tgagcctgtg 2700 ctgagcccca
gtgggttggc gattagtcca atttgttaaa gacaggatat cagtggtcca 2760
ggctctagtt ttgactcaac aatatcacca gctgaagcct atagagtacg agccatagat
2820 aaaataaaag attttattta gtctccagaa aaagggggga atgaaagacc
ccacctgtag 2880 gtttggcaag ctagtctagt aacggccgcc agtgtgctgg
aattctgcag atatccatca 2940 cactggcggc cgctcgagca tgcatctaga
gcgataatca acctctggat tacaaaattt 3000 gtgaaagatt gactggtatt
cttaactatg ttgctccttt tacgctatgt ggatacgctg 3060 ctttaatgcc
tttgtatcat gctattgctt cccgtatggc tttcattttc tcctccttgt 3120
ataaatcctg gttgctgtct ctttatgagg agttgtggcc cgttgtcagg caacgtggcg
3180 tggtgtgcac tgtgtttgct gacgcaaccc ccactggttg gggcattgcc
accacctgtc 3240 agctcctttc cgggactttc gctttccccc tccctattgc
cacggcggaa ctcatcgccg 3300 cctgccttgc ccgctgctgg acaggggctc
ggctgttggg cactgacaat tccgtggtgt 3360 tgtcggggaa gctgacgtcc
tttccatggc tgctcgcctg tgttgccacc tggattctgc 3420 gcgggacgtc
cttctgctac gtcccttcgg ccctcaatcc agcggacctt ccttcccgcg 3480
gcctgctgcc ggctctgcgg cctcttccgc gtcttcgcct tcgccctgag acgagtcgga
3540 tctccctttg ggccgcctcc ccgcatcgct attctatagt gtcacctaaa
tgctagagct 3600 cgctgatcag cctcgactgt gccttctagt tgccagccat
ctgttgtttg cccctccccc 3660 gtgccttcct tgaccctgga aggtgccact
cccactgtcc tttcctaata aaatgaggaa 3720 attgcatcgc attgtctgag
taggtgtcat tctattctgg ggggtggggt ggggcaggac 3780 agcaaggggg
aggattggga agacaatagc aggcatgctg gggatgcggt gggctctatg 3840
gcttctgagg cggaaagaac caggtagata agtagcatgg cgggttaatc attaactaca
3900 aggaacccct agtgatggag ttggccactc cctctctgcg cgctcgctcg
ctcactgagg 3960 ccgggcgacc aaaggtcgcc cgacgcccgg gctttgcccg
ggcggcctca gtgagcgagc 4020 gagcgcgcag ctggcgtaat agcgaagagg
cccgcaccga tcgcccttcc caacagttgc 4080 gcagcctgaa tggcgaatgg
cgattccgtt gcaatggctg gcggtaatat tgttctggat 4140 attaccagca
aggccgatag tttgagttct tctactcagg caagtgatgt tattactaat 4200
caaagaagta ttgcgacaac ggttaatttg cgtgatggac agactctttt actcggtggc
4260 ctcactgatt ataaaaacac ttctcaggat tctggcgtac cgttcctgtc
taaaatccct 4320 ttaatcggcc tcctgtttag ctcccgctct gattctaacg
aggaaagcac gttatacgtg 4380 ctcgtcaaag caaccatagt acgcgccctg
tagcggcgca ttaagcgcgg cgggtgtggt 4440 ggttacgcgc agcgtgaccg
ctacacttgc cagcgcccta gcgcccgctc ctttcgcttt 4500 cttcccttcc
tttctcgcca cgttcgccgg ctttccccgt caagctctaa atcgggggct 4560
ccctttaggg ttccgattta gtgctttacg gcacctcgac cccaaaaaac ttgattaggg
4620 tgatggttca cgtagtgggc catcgccctg atagacggtt tttcgccctt
tgacgttgga 4680 gtccacgttc tttaatagtg gactcttgtt ccaaactgga
acaacactca accctatctc 4740 ggtctattct tttgatttat aagggatttt
gccgatttcg gcctattggt taaaaaatga 4800 gctgatttaa caaaaattta
acgcgaattt taacaaaata ttaacgctta caatttaaat 4860 atttgcttat
acaatcttcc tgtttttggg gcttttctga ttatcaaccg gggtacatat 4920
gattgacatg ctagttttac gattaccgtt catcgattct cttgtttgct ccagactctc
4980 aggcaatgac ctgatagcct ttgtagagac ctctcaaaaa tagctaccct
ctccggcatg 5040 aatttatcag ctagaacggt tgaatatcat attgatggtg
atttgactgt ctccggcctt 5100 tctcacccgt ttgaatcttt acctacacat
tactcaggca ttgcatttaa aatatatgag 5160 ggttctaaaa atttttatcc
ttgcgttgaa ataaaggctt ctcccgcaaa agtattacag 5220 ggtcataatg
tttttggtac aaccgattta gctttatgct ctgaggcttt attgcttaat 5280
tttgctaatt ctttgccttg cctgtatgat ttattggatg ttggaatcgc ctgatgcggt
5340 attttctcct tacgcatctg tgcggtattt cacaccgcat atggtgcact
ctcagtacaa 5400 tctgctctga tgccgcatag ttaagccagc cccgacaccc
gccaacaccc gctgacgcgc 5460 cctgacgggc ttgtctgctc ccggcatccg
cttacagaca agctgtgacc gtctccggga 5520 gctgcatgtg tcagaggttt
tcaccgtcat caccgaaacg cgcgagacga aagggcctcg 5580 tgatacgcct
atttttatag gttaatgtca tgataataat ggtttcttag acgtcaggtg 5640
gcacttttcg gggaaatgtg cgcggaaccc ctatttgttt atttttctaa atacattcaa
5700 atatgtatcc gctcatgaga caataaccct gataaatgct tcaataatat
tgaaaaagga 5760 agagtatgag tattcaacat ttccgtgtcg cccttattcc
cttttttgcg gcattttgcc 5820 ttcctgtttt tgctcaccca gaaacgctgg
tgaaagtaaa agatgctgaa gatcagttgg 5880 gtgcacgagt gggttacatc
gaactggatc tcaacagcgg taagatcctt gagagttttc 5940 gccccgaaga
acgttttcca atgatgagca cttttaaagt tctgctatgt ggcgcggtat 6000
tatcccgtat tgacgccggg caagagcaac tcggtcgccg catacactat tctcagaatg
6060 acttggttga gtactcacca gtcacagaaa agcatcttac ggatggcatg
acagtaagag 6120 aattatgcag tgctgccata accatgagtg ataacactgc
ggccaactta cttctgacaa 6180 cgatcggagg accgaaggag ctaaccgctt
ttttgcacaa catgggggat catgtaactc 6240 gccttgatcg ttgggaaccg
gagctgaatg aagccatacc aaacgacgag cgtgacacca 6300 cgatgcctgt
agcaatggca acaacgttgc gcaaactatt aactggcgaa ctacttactc 6360
tagcttcccg gcaacaatta atagactgga tggaggcgga taaagttgca ggaccacttc
6420 tgcgctcggc ccttccggct ggctggttta ttgctgataa atctggagcc
ggtgagcgtg 6480 ggtctcgcgg tatcattgca gcactggggc cagatggtaa
gccctcccgt atcgtagtta 6540 tctacacgac ggggagtcag gcaactatgg
atgaacgaaa tagacagatc gctgagatag 6600 gtgcctcact gattaagcat
tggtaactgt cagaccaagt ttactcatat atactttaga 6660 ttgatttaaa
acttcatttt taatttaaaa ggatctaggt gaagatcctt tttgataatc 6720
tcatgaccaa aatcccttaa cgtgagtttt cgttccactg agcgtcagac cccgtagaaa
6780 agatcaaagg atcttcttga gatccttttt ttctgcgcgt aatctgctgc
ttgcaaacaa 6840 aaaaaccacc gctaccagcg gtggtttgtt tgccggatca
agagctacca actctttttc 6900 cgaaggtaac tggcttcagc agagcgcaga
taccaaatac tgttcttcta gtgtagccgt 6960 agttaggcca ccacttcaag
aactctgtag caccgcctac atacctcgct ctgctaatcc 7020 tgttaccagt
ggctgctgcc agtggcgata agtcgtgtct taccgggttg gactcaagac 7080
gatagttacc ggataaggcg cagcggtcgg gctgaacggg gggttcgtgc acacagccca
7140 gcttggagcg aacgacctac accgaactga gatacctaca gcgtgagcta
tgagaaagcg 7200 ccacgcttcc cgaagggaga aaggcggaca ggtatccggt
aagcggcagg gtcggaacag 7260 gagagcgcac gagggagctt ccagggggga
acgcctggta tctttatagt cctgtcgggt 7320 ttcgccacct ctgacttgag
cgtcgatttt tgtgatgctc gtcagggggg cggagcctat 7380 ggaaaaacgc
cagcaacgcg gcctttttac ggttcctggc cttttgctgg ccttttgctc 7440
acatgttctt tcctgcgtta tcccctgatt ctgtggataa ccgtattacc gcctttgagt
7500 gagctgatac cgctcgccgc agccgaacga ccgagcgcag cgagtcagtg
agcgaggaag 7560 cggaagagcg cccaatacgc aaaccgcctc tccccgcgcg
ttggccgatt cattaatgca 7620 gctgcgcgct cgctcgctca ctgaggcc 7648 2
8092 DNA Artificial Sequence Description of Artificial Sequence
vector AAV-PGK-NaGLU 2 cagcagctgc gcgctcgctc gctcactgag gccgcccggg
caaagcccgg gcgtcgggcg 60 acctttggtc gcccggcctc agtgagcgag
cgagcgcgca gagagggagt ggccaactcc 120 atcactaggg gttccttgta
gttaatgatt aacccgccat gctacttatc tactcgagaa 180 ttctaccggg
taggggaggc gcttttccca aggcagtctg gagcatgcgc tttagcagcc 240
ccgctggcac ttggcgctac acaagtggcc tctggcctcg cacacattcc acatccaccg
300 gtagcgccaa ccggctccgt tctttggtgg ccccttcgcg ccaccttcta
ctcctcccct 360 agtcaggaag ttccccccgc cccgcagctc gcgtcgtgca
ggacgtgaca aatggaagta 420 gcacgtctca ctagtctcgt gcagatggac
agcaccgctg agcaatggaa gcgggtaggc 480 ctttggggca gcggccaata
gcagctttgc tccttcgctt tctgggctca gaggctggga 540 aggggtgggt
ccgggggcgg gctcaggggc gggctcaggg gcggggcggg cgcgaaggtc 600
ctccggagcc cggcattctg cacgcttcaa aagcgcacgt ctgccgcgct gttctcctct
660 tcctcatctc cgggcctttc gaccggatcc cccgggctgc aggaattccg
agaccatgga 720 ggcggtggcg gtggccgcgg cggtgggggt ccttctcctg
gccggggccg ggggcgcggc 780 aggcgacgag gcccgggagg cggcggccgt
gcgggcgctc gtggcccggc tgctggggcc 840 aggccccgcg gccgacttct
ccgtgtcggt ggagcgcgct ctggctgcca agccgggctt 900 ggacacctac
agcctgggcg gcggcggcgc ggcgcgcgtg cgggtgcgcg gctccacggg 960
cgtggcggcc gccgcggggc tgcaccgcta cctgcgcgac ttctgtggct gccacgtggc
1020 ctggtccggc tctcagctgc gcctgccgcg gccactgcca gccgtgccgg
gggagctgac 1080 cgaggccacg cccaacaggt accgctatta ccagaatgtg
tgcacgcaaa gctactcctt 1140 cgtgtggtgg gactgggccc gctgggagcg
agagatagac tggatggcgc tgaatggcat 1200 caacctggca ctggcctgga
gcggccagga ggccatctgg cagcgggtgt acctggcctt 1260 gggcctgacc
caggcagaga tcaatgagtt ctttactggt cctgccttcc tggcctgggg 1320
gcgaatgggc aacctgcaca cctgggatgg ccccctgccc ccctcctggc acatcaagca
1380 gctttacctg cagcaccggg tcctggacca gatgcgctcc ttcggcatga
ccccagtgct 1440 gcctgcattc gcggggcatg ttcccgaggc tgtcaccagg
gtgttccctc aggtcaatgt 1500 cacgaagatg ggcagttggg gccactttaa
ctgttcctac tcctgctcct tccttctggc 1560 tccggaagac cccatattcc
ccatcatcgg gagcctcttc ctgcgagagc tgatcaaaga 1620 gtttggcaca
gaccacatct atggggccga cactttcaat gagatgcagc caccttcctc 1680
agagccctcc taccttgccg cagccaccac tgccgtctat gaggccatga ctgcagtgga
1740 tactgaggct gtgtggctgc tccaaggctg gctcttccag caccagccgc
agttctgggg 1800 gcccgcccag atcagggctg tgctgggagc tgtgccccgt
ggccgcctcc tggttctgga 1860 cctgtttgct gagagccagc ctgtgtatac
ccgcactgcc tccttccagg gccagccctt 1920 catctggtgc atgctgcaca
actttggggg aaaccatggt ctttttggag ccctagaggc 1980 tgtgaacgga
ggcccagaag ctgcccgcct cttccccaac tccaccatgg taggcacggg 2040
catggccccc gagggcatca gccagaacga agtggtctat tccctcatgg ctgagctggg
2100 ctggcgaaag gaccagctgc cagatttggc agcctgggtg accagctttg
ccgcccggcg 2160 gtatggggtc tcccacccgg acgcaggggc agcgtggagg
ctactgctcc ggagtgtgta 2220 caactgctcc ggggaggcct gcaggggcca
caatcgtagc ccgctggtca ggcggccgtc 2280 cctacagatg aataccagca
tctggtacaa ccgatctgat gtgtttgagg cctggcggct 2340 gctgctcaca
tctgctccct ccctggccac cagccccgcc ttccgctacg acctgctgga 2400
cctcactcgg caggcagtgc aggagctggt cagcttgtac tatgaggagg caagaagcgc
2460 ctacctgagc aaggagctgg cctccctgtt gagggctgga ggcgtcctgg
cctatgagct 2520 gctgccggca ctggacgagg tgctggctag tgacagccgc
ttcttgctgg gcagctggct 2580 agagcaggcc cgagcagcgg cagtcagtga
ggccgaggcc gatttctacg agcagaacag 2640 ccgctaccag ctgaccttgt
gggggccaga aggcaacatc ctggactatg ccaacaagca 2700 gctggcgggg
ttggtggcca actactacac ccctcgctgg cggcttttcc tggaggcgct 2760
ggttgacagt gtggcccagg gcatcccttt ccaacagcac cagtttgaca aaaatgtctt
2820 ccaactggag caggccttcg ttctcagcaa gcagaggtac cccagccagc
cgcgaggaga 2880 cactgtggac ctggccaaga agatcttcct caaatattac
cccggctggg tggccggctc 2940 ttggtgatag attcgccacc actgggcctt
gttttccgct aattccaggg cagattccag 3000 ggcccagagc tggacagaca
tcacaggata acccaggcct gggaggaggc cccacggcct 3060 gctggtgggg
tctgacctgg ggggattgga gggaaatgac ctgccctcca ccaccaccca 3120
aagtgtggga ttaaagtagc ttggtaccga gctcggatcc ggcgattagt ccaatttgtt
3180 aaagacagga tatcagtggt ccaggctcta gttttgactc aacaatatca
ccagctgaag 3240 cctatagagt acgagccata gataaaataa aagattttat
ttagtctcca gaaaaagggg 3300 ggaatgaaag accccacctg taggtttggc
aagctagcgc tagtaacggc cgccagtgtg 3360 ctggaattct gcagatatcc
atcacactgg cggccgctcg agcatgcatc tagagcgata 3420 atcaacctct
ggattacaaa atttgtgaaa gattgactgg tattcttaac tatgttgctc 3480
cttttacgct atgtggatac gctgctttaa tgcctttgta tcatgctatt gcttcccgta
3540 tggctttcat tttctcctcc ttgtataaat cctggttgct gtctctttat
gaggagttgt 3600 ggcccgttgt caggcaacgt ggcgtggtgt gcactgtgtt
tgctgacgca acccccactg 3660 gttggggcat tgccaccacc tgtcagctcc
tttccgggac tttcgctttc cccctcccta 3720 ttgccacggc ggaactcatc
gccgcctgcc ttgcccgctg ctggacaggg gctcggctgt 3780 tgggcactga
caattccgtg gtgttgtcgg ggaagctgac gtcctttcca tggctgctcg 3840
cctgtgttgc cacctggatt ctgcgcggga cgtccttctg ctacgtccct tcggccctca
3900 atccagcgga ccttccttcc cgcggcctgc tgccggctct gcggcctctt
ccgcgtcttc 3960 gccttcgccc tgagacgagt cggatctccc tttgggccgc
ctccccgcat cgctattcta 4020 tagtgtcacc taaatgctag agctcgctga
tcagcctcga ctgtgccttc tagttgccag 4080 ccatctgttg tttgcccctc
ccccgtgcct tccttgaccc tggaaggtgc cactcccact 4140 gtcctttcct
aataaaatga ggaaattgca tcgcattgtc tgagtaggtg tcattctatt 4200
ctggggggtg gggtggggca ggacagcaag ggggaggatt gggaagacaa tagcaggcat
4260 gctggggatg cggtgggctc tatggcttct gaggcggaaa gaaccaggta
gataagtagc 4320 atggcgggtt aatcattaac tacaaggaac ccctagtgat
ggagttggcc actccctctc 4380 tgcgcgctcg ctcgctcact gaggccgggc
gaccaaaggt cgcccgacgc ccgggctttg 4440 cccgggcggc ctcagtgagc
gagcgagcgc gcagctggcg taatagcgaa gaggcccgca 4500 ccgatcgccc
ttcccaacag ttgcgcagcc tgaatggcga atggcgattc cgttgcaatg 4560
gctggcggta atattgttct ggatattacc agcaaggccg atagtttgag ttcttctact
4620 caggcaagtg atgttattac taatcaaaga agtattgcga caacggttaa
tttgcgtgat 4680 ggacagactc ttttactcgg tggcctcact gattataaaa
acacttctca ggattctggc 4740 gtaccgttcc tgtctaaaat ccctttaatc
ggcctcctgt ttagctcccg ctctgattct 4800 aacgaggaaa gcacgttata
cgtgctcgtc aaagcaacca tagtacgcgc cctgtagcgg 4860 cgcattaagc
gcggcgggtg tggtggttac gcgcagcgtg accgctacac ttgccagcgc 4920
cctagcgccc gctcctttcg ctttcttccc ttcctttctc gccacgttcg ccggctttcc
4980 ccgtcaagct ctaaatcggg ggctcccttt agggttccga tttagtgctt
tacggcacct 5040 cgaccccaaa aaacttgatt agggtgatgg ttcacgtagt
gggccatcgc cctgatagac 5100 ggtttttcgc cctttgacgt tggagtccac
gttctttaat agtggactct tgttccaaac 5160 tggaacaaca ctcaacccta
tctcggtcta ttcttttgat ttataaggga ttttgccgat 5220 ttcggcctat
tggttaaaaa atgagctgat ttaacaaaaa tttaacgcga attttaacaa 5280
aatattaacg cttacaattt aaatatttgc ttatacaatc ttcctgtttt tggggctttt
5340 ctgattatca accggggtac atatgattga catgctagtt ttacgattac
cgttcatcga 5400 ttctcttgtt tgctccagac tctcaggcaa tgacctgata
gcctttgtag agacctctca 5460 aaaatagcta ccctctccgg catgaattta
tcagctagaa cggttgaata tcatattgat 5520 ggtgatttga ctgtctccgg
cctttctcac ccgtttgaat ctttacctac acattactca 5580 ggcattgcat
ttaaaatata tgagggttct aaaaattttt atccttgcgt tgaaataaag 5640
gcttctcccg caaaagtatt acagggtcat aatgtttttg gtacaaccga tttagcttta
5700 tgctctgagg ctttattgct taattttgct aattctttgc cttgcctgta
tgatttattg 5760 gatgttggaa tcgcctgatg cggtattttc tccttacgca
tctgtgcggt atttcacacc 5820 gcatatggtg cactctcagt acaatctgct
ctgatgccgc atagttaagc cagccccgac 5880 acccgccaac acccgctgac
gcgccctgac gggcttgtct gctcccggca tccgcttaca 5940 gacaagctgt
gaccgtctcc gggagctgca tgtgtcagag gttttcaccg tcatcaccga 6000
aacgcgcgag acgaaagggc ctcgtgatac gcctattttt ataggttaat gtcatgataa
6060 taatggtttc ttagacgtca ggtggcactt ttcggggaaa tgtgcgcgga
acccctattt 6120 gtttattttt ctaaatacat tcaaatatgt atccgctcat
gagacaataa ccctgataaa 6180 tgcttcaata atattgaaaa aggaagagta
tgagtattca acatttccgt gtcgccctta 6240 ttcccttttt tgcggcattt
tgccttcctg tttttgctca cccagaaacg ctggtgaaag 6300 taaaagatgc
tgaagatcag ttgggtgcac gagtgggtta catcgaactg gatctcaaca 6360
gcggtaagat ccttgagagt tttcgccccg aagaacgttt tccaatgatg agcactttta
6420 aagttctgct atgtggcgcg gtattatccc gtattgacgc cgggcaagag
caactcggtc 6480 gccgcataca ctattctcag aatgacttgg ttgagtactc
accagtcaca gaaaagcatc 6540 ttacggatgg catgacagta agagaattat
gcagtgctgc cataaccatg agtgataaca 6600 ctgcggccaa cttacttctg
acaacgatcg gaggaccgaa ggagctaacc gcttttttgc 6660 acaacatggg
ggatcatgta actcgccttg atcgttggga accggagctg aatgaagcca 6720
taccaaacga cgagcgtgac accacgatgc ctgtagcaat ggcaacaacg ttgcgcaaac
6780 tattaactgg cgaactactt actctagctt cccggcaaca attaatagac
tggatggagg 6840 cggataaagt tgcaggacca cttctgcgct cggcccttcc
ggctggctgg tttattgctg 6900 ataaatctgg agccggtgag cgtgggtctc
gcggtatcat tgcagcactg gggccagatg 6960 gtaagccctc ccgtatcgta
gttatctaca cgacggggag tcaggcaact atggatgaac 7020 gaaatagaca
gatcgctgag ataggtgcct cactgattaa gcattggtaa ctgtcagacc 7080
aagtttactc atatatactt tagattgatt taaaacttca tttttaattt aaaaggatct
7140 aggtgaagat cctttttgat aatctcatga ccaaaatccc ttaacgtgag
ttttcgttcc 7200 actgagcgtc agaccccgta
gaaaagatca aaggatcttc ttgagatcct ttttttctgc 7260 gcgtaatctg
ctgcttgcaa acaaaaaaac caccgctacc agcggtggtt tgtttgccgg 7320
atcaagagct accaactctt tttccgaagg taactggctt cagcagagcg cagataccaa
7380 atactgttct tctagtgtag ccgtagttag gccaccactt caagaactct
gtagcaccgc 7440 ctacatacct cgctctgcta atcctgttac cagtggctgc
tgccagtggc gataagtcgt 7500 gtcttaccgg gttggactca agacgatagt
taccggataa ggcgcagcgg tcgggctgaa 7560 cggggggttc gtgcacacag
cccagcttgg agcgaacgac ctacaccgaa ctgagatacc 7620 tacagcgtga
gctatgagaa agcgccacgc ttcccgaagg gagaaaggcg gacaggtatc 7680
cggtaagcgg cagggtcgga acaggagagc gcacgaggga gcttccaggg ggaaacgcct
7740 ggtatcttta tagtcctgtc gggtttcgcc acctctgact tgagcgtcga
tttttgtgat 7800 gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa
cgcggccttt ttacggttcc 7860 tggccttttg ctggcctttt gctcacatgt
tctttcctgc gttatcccct gattctgtgg 7920 ataaccgtat taccgccttt
gagtgagctg ataccgctcg ccgcagccga acgaccgagc 7980 gcagcgagtc
agtgagcgag gaagcggaag agcgcccaat acgcaaaccg cctctccccg 8040
cgcgttggcc gattcattaa tgcagctgcg cgctcgctcg ctcactgagg cc 8092
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