U.S. patent application number 11/614970 was filed with the patent office on 2007-09-06 for enhancing the effect of therapeutic proteins on the central nervous system.
This patent application is currently assigned to Saint Louis University, a non-profit organization. Invention is credited to Jeffrey H. Grubb, Monica A. Gutierrez, Adriana Montano, Tatsuo Nishioka, Amelia Ortigoza Rodriguez, William S. Sly, Shunji Tomatsu.
Application Number | 20070207139 11/614970 |
Document ID | / |
Family ID | 46326906 |
Filed Date | 2007-09-06 |
United States Patent
Application |
20070207139 |
Kind Code |
A1 |
Tomatsu; Shunji ; et
al. |
September 6, 2007 |
ENHANCING THE EFFECT OF THERAPEUTIC PROTEINS ON THE CENTRAL NERVOUS
SYSTEM
Abstract
The present invention provides a polypeptide therapeutic agent,
useful in enzyme replacement therapy, with increased therapeutic
benefits for the central nervous system. The invention provides a
method of enhancing the effect of a polypeptide or protein on the
central nervous system by the attachment of a short acidic amino
acid sequence. Specifically the inventors disclose the attachment
of a 4-15 acidic amino acid sequence to human .beta.-glucuronidase
by construction of a fusion protein. This molecule is useful in the
treatment of type VII mucopolysaccharidosis when administered to a
patient.
Inventors: |
Tomatsu; Shunji; (St. Louis,
MO) ; Montano; Adriana; (Hyogo, JP) ;
Nishioka; Tatsuo; (Ishikawa, JP) ; Grubb; Jeffrey
H.; (St. Louis, MO) ; Sly; William S.; (St.
Louis, MO) ; Gutierrez; Monica A.; (US) ;
Rodriguez; Amelia Ortigoza; (Cucuta Norte de Santander,
CO) |
Correspondence
Address: |
SAINT LOUIS UNIVERSITY;OFFICE OF INNOVATION AND INTELLECTUAL PROPERTY
3556 CAROLINE MALL
SUITE C208
ST. LOUIS
MO
63104
US
|
Assignee: |
Saint Louis University, a
non-profit organization
St. Louis
MO
|
Family ID: |
46326906 |
Appl. No.: |
11/614970 |
Filed: |
December 21, 2006 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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11245424 |
Oct 7, 2005 |
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11614970 |
Dec 21, 2006 |
|
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10864758 |
Jun 10, 2004 |
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11245424 |
Oct 7, 2005 |
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Current U.S.
Class: |
424/94.61 ;
435/209; 514/17.7 |
Current CPC
Class: |
C12N 15/625 20130101;
C07K 2319/02 20130101; C12N 9/96 20130101; C12Y 302/01031 20130101;
A61K 38/00 20130101; A61P 25/00 20180101; C07K 2319/31 20130101;
C12N 9/2402 20130101 |
Class at
Publication: |
424/094.61 ;
514/015; 514/016; 514/017; 514/018; 435/209 |
International
Class: |
A61K 38/47 20060101
A61K038/47; C12N 9/42 20060101 C12N009/42 |
Goverment Interests
GOVERNMENT SUPPORT CLAUSE
[0004] This work was supported by the National Institutes of Health
grant number GM34182, and International Morquio Organization. U.S.
Government has certain rights in this invention.
Claims
1. A polypeptide therapeutic agent with increased central nervous
system therapeutic activity comprised of a) a physiologically
active protein with therapeutic benefits for the central nervous
system, and b) a short peptide which consisting of 4-15 acidic
amino acids attached to the physiology active protein via the
N-terminus thereof.
2. A polypeptide therapeutic agent as in claim 1 wherein said
physiologically active protein is an enzyme.
3. A polypeptide therapeutic agent as in claim 1 wherein said
physiologically active protein is an enzyme known to be therapeutic
in treatment of lysosomal storage disease.
4. A polypeptide therapeutic agent as in claim 1 wherein said
physiologically active protein is human .beta.-glucuronidase.
5. A polypeptide therapeutic agent as in claim 1 wherein attaching
said acid amino acid sequence of 4-15 amino acids to the N terminus
of said polypeptide increases clearance time in the blood.
6. A polypeptide therapeutic agent as in claim 1 whereby attached
comprises produced a fusion protein through genetic
engineering.
7. A polypeptide therapeutic agent as in claim 1 whereby attached
comprises chemically linking at least two molecules.
8. A polypeptide therapeutic agent as in claim 1 whereby attached
is via a linker peptide.
9. A method of increasing therapeutic benefits of a physiologically
active protein on the central nervous system wherein the method
comprises, a) a physiologically active protein with therapeutic
benefits for the central nervous system, and b) a short peptide
consisting of 4-15 acidic amino acids, which is c) attached to said
physiology active protein via the N-terminus thereof.
10. A method of increasing therapeutic benefits as in claim 9
whereby said physiologically active protein is an enzyme.
11. A method of increasing therapeutic effects as in claim 9
whereby said physiologically active protein is an enzyme known to
be therapeutic in treatment of lysosomal storage disease.
12. A method of increasing therapeutic effects as in claim 9
whereby said physiologically active protein is human
.beta.-glucuronidase.
13. A method of increasing therapeutic effects as in claim 9
whereby attaching said acid amino acid sequence of 4-15 amino acids
to the N terminus of said polypeptide increases clearance time in
the blood.
14. A method of increasing therapeutic effects as in claim 9
whereby attaching comprises a fusion protein produced through
genetic engineering.
15. A method of increasing therapeutic effects as in claim 9
whereby attaching comprises chemically linking at least two
molecules.
16. A method of increasing therapeutic effects as in claim 9
whereby attaching is via a linker peptide.
17. A method of treating a patient with a central nervous system
disease by administering an effective amount of a physiologically
active protein with therapeutic benefits for the central nervous
system, and b) a short peptide which consists of 4-15 acidic amino
acids, which is c) attached to the physiology active protein on the
N-terminus thereof.
18. A method as in claim 17 whereby said physiologically active
protein is human .beta.-glucuronidase.
19. A method as in claim 17 whereby said central nervous system
disease is a lysosomal storage disease.
20. A method as in claim 17 whereby said central nervous system
disease is type VII mucopolysaccharidosis.
21. A method as in claim 17 whereby said physiologically active
protein with said short peptide attached further comprises
increased clearance time in the blood.
Description
PARENT CASE TEXT
[0001] This application claims benefit of priority to a
continuation in part of U.S. application Ser. No. 11/245,424, filed
Oct. 7, 2005, and also a continuation in part of U.S. application
Ser. No. 10/864,758, filed Jun. 10, 2004.
[0002] Sequence Listing
[0003] A paper copy of the sequence listing and a computer readable
form of the same sequence listing are disclosed in U.S. application
Ser. No. 11/245,424, filed Oct. 7, 2005 to which this application
claims benefit of priority as a continuation in part, and is herein
incorporated by reference.
BACKGROUND OF THE INVENTION
[0005] 1. Field of the Invention
[0006] The present invention relates to endowing therapeutic
protein agents with increased in vivo stability and effectiveness
on the central nervous system (CNS). More specifically, the present
invention relates to endowing .beta.-glucuronidase protein (GUS)
with improved stability in the blood and enhanced ability to affect
the CNS, in a therapeutic capacity by attaching a short peptide of
acidic amino acids to the N-terminus of the protein.
[0007] 2. Description of the Related Art
[0008] Lysosomal storage diseases (LSDs) are a class of forty rare
genetic disorders, each of which is caused by a deficiency in a
specific lysosomal enzyme. As a consequence of the progressive
accumulation of unmetabolized macromolecules in the lysosomes of
cells in various tissues, the disease manifestations worsen over
time..sup.1 Individuals afflicted with an LSD can suffer from mild
to severe physical and/or neurological abnormalities or can die at
an early age. A therapeutic paradigm for the treatment of LSDs was
established with the success of enzyme-replacement therapy (ERT)
for the treatment of Gaucher disease..sup.2,3 In the case of
Gaucher disease, delivery of the enzyme to the affected cells was
achieved by modifying the N-linked carbohydrate on the enzyme. This
exposed core mannose residues,.sup.4,5 enabling the enzyme to bind
to the MR, which is highly abundant on cells of the
reticuloendothelial system..sup.6,7 These findings led to clinical
management of Gaucher disease by ERT..sup.8 Over 3,500 patients
have been treated with dramatic clinical results..sup.9
[0009] Meanwhile there is a problem that pharmaceutical
preparations of physiologically active proteins like enzymes and
peptide hormones are generally made unstable when they are
administered to the body, and thus undergo relatively rapid
inactivation by, e.g., enzymatic degradation. For pharmaceutical
preparations of a physiologically active protein, a method for
increasing the stability of the physiologically active protein in
the body is known which is based on coupling the proteins to
polyethylene glycol..sup.10
[0010] Sly's syndrome is an autosomal recessive, genetic lysosomal
storage disease caused by an anomaly in the gene for a lysosomal
enzyme, .beta.-glucuronidase (hereinafter referred to as GUS)
.sup.11(6), and is classified as type VII mucopolysaccharidosis
(hereinafter referred to as MPS VII). In lysosomes, GUS acts as an
exoglycosidase to remove glucuronic acid residues from the
non-reducing termini of GAGs (glycosaminoglycans), such as dermatan
sulfate (DS), heparan sulfate (HS), and chondroitin sulfate (CS).
In the absence of GUS, GAGs are only partially degraded and
accumulate in lysosomes of a variety of tissues. Progressive
accumulation of undegraded GAGs in lysosomes affects the spleen,
liver, kidney, cornea, brain, heart valves, and the skeletal
system, leading to facial dysmorphism, growth retardation, systemic
bone dysplasia, deafness, mental retardation, and shortened
lifespan.
[0011] No effective remedy is currently available for MPS VII.
Enzyme replacement therapy (ERT) has been considered to be the
potential remedy for MPS VII. Considering its rapid inactivation in
the body, however, native GUS is not expected to give any
satisfactory effect.
[0012] The challenge is to improve joint and brain-related
pathology since most of the enzyme-based drugs are delivered to
major visceral organs like liver and spleen and only a small amount
of enzyme is delivered to bone and brain. Many lysosomal enzymes
have a short half-life when injected into the bloodstream because
of rapid clearance in the liver by carbohydrate-recognizing
receptors, particularly the mannose receptor that is highly
abundant on Kupffer cells..sup.12 Although a part of the enzyme
reaches the bone marrow, there is no way to guarantee that the
enzyme will reach the brain since the blood brain barrier presents
a formidable obstacle. As a result, current ERT doses not work
efficiently on the bone and brain lesions.
[0013] The inventors have sought to address the problem of
stability of therapeutic proteins in vivo and the inability of some
proteins to effectively cross the blood brain barrier. The
inventors have previously disclosed the use of short peptides of
acidic amino acids to target proteins to bone tissue for use in
Enzyme Replacement Therapy (ERT)..sup.13 The inventors have also
disclosed the use of short peptides of acidic amino acids to
improve stability of physiological active proteins in the
blood.
[0014] The addition of 4-15 acidic amino acids to GUS results in an
increase in molecular weight which generally, would not be expected
to increase functional activity of proteins to the CNS. In fact,
higher molecular weigh molecules are more effectively excluded from
the brain by an ineffectual crossing the blood brain barrier.
Similarly, an increase in the hydrophilic nature of a molecule is
also thought to exclude molecules at the blood brain barrier. The
inventors have made the surprising discovery that despite causing
an apparent increase in molecular weight and increase in
hydrophilic nature, the addition of an acid amino acid leader to
GUS has allowed enhanced therapeutic benefits on the brain.
SUMMARY OF THE INVENTION
[0015] An object of the present invention is a method to increase
in vivo stability of a physiologically active peptide or protein by
the addition of a short acidic amino acid leader, and thereby
increase its therapeutic effects on the CNS for treatment of CNS
related disease.
[0016] Previously, the inventors made the unexpected discovery that
N-acetylgalactosamine-6-sulfate sulfatase (GALNS),
tissue-nonspecific alkaline phosphatase (TNSALP) and GUS with a
short amino acidic peptide (AAA) attached to the N-terminus
increased targeting and deposition of these enzymes to bone. They
further discovered that GALNS and GUS, with this short acidic amino
acidic peptide attached possessed improved in vivo stability in the
blood. The inventors have now further discovered that AAA-GUS
possessed improved functional activity to tissues of the CNS, when
administered to a patient with MPS VII.
[0017] The addition of a short amino acidic peptide attached to the
N-terminus of GUS or other physiology active proteins possessing
CNS therapeutic activity will endow these molecules with enhanced
therapeutic benefits for the treatment for patients with CNS
disorders. Compared with native physiologically active GUS, the
present invention described above provides a physiologically active
fusion protein with increased stability in the blood and increased
therapeutic effects on the brain when administered to a patient
with MPS VII.
[0018] Therefore, an object of this invention is 1) a polypeptide
therapeutic agent with increased benefits for the CNS, 2) a method
of increasing beneficial effects on the CNS, of a protein or
polypeptide possessing CNS therapeutic activity, by attaching a
4-15 acid amino acid leader through chemical modification or
genetic engineering of a fusion protein and 3) a method of
treatment for patients suffering from CNS related diseases with the
afore mentioned preparation.
BRIEF DESCRIPTION OF THE DRAWINGS
[0019] FIG. 1: is a schematic diagram illustrating pCXN vector and
the cloning site in the vector for the cDNA encoding native GUS or
the GUS fusion protein
[0020] FIG. 2: illustrates the steps for the construction of an
expression vector for the production of the GUS and GUS fusion
protein.
[0021] FIG. 3: is a graph showing the time profiles of the blood
activity levels of native GUS and GUS fusion protein after they are
intravascularly administered in an equivalent amount.
[0022] FIG. 4: shows light microscopy of neocortex from native GUS
and D.sub.6-GUS treated mice.
[0023] The cortical neuron, hippocampus, and glia cell sections
show a reduction of storage (S) in D.sub.6-GUS treated compared to
GUS treated MPS VII mice.
DETAILED DESCRIPTION OF THE INVENTION
[0024] Mucopolysaccharidoses (MPS) are a group of lysosomal storage
disorders (LSDs) caused by deficiency of the lysosomal enzymes
needed to degrade glycosaminoglycans (GAGs)..sup.14 In MPS, the
undegraded GAGs are stored in lysosomes and/or secreted into the
blood stream.sup.15,16, and excreted in urine. MPS involve the
deficiency of one of 11 enzymes needed for the stepwise degradation
of DS, HS, KS, and/or CS.
[0025] ERT is an established means of treating MPS. However,
improving bone and brain pathology is still an unmet challenge
because only a small fraction of enzyme is delivered to bone and
brain. Most of the enzyme-based drugs are delivered to major
visceral organs like liver and spleen. Although some of the enzyme
reaches bone marrow, only small amounts of the enzyme go to bone or
brain. The blood brain barrier provides a formidable obstacle for
enzymes to reach brain. Therefore, the improvement of bone and
brain lesions is quite limited, even after long-term treatment with
ERT. We have tested an acidic oligopeptide-based targeting system
for delivery of enzymes to tissues in murine MPS IVA and VII
models. This strategy is based on tagging a short peptide
consisting of acidic amino acids (AAA) to the mature enzyme. The
AAA-tagged enzyme had five to ten times prolonged blood clearance
compared with the untagged enzymes. The tagged enzyme was delivered
effectively to bone, bone marrow, and brain in MPS VII mice and was
more effective in reversing the storage pathology than the untagged
enzyme.
[0026] Others have shown therapeutic responses in brain of mouse
models MPS VII, aspartylglycosaminuria and .beta.-mannosidosis when
higher doses and longer treatment with enzyme is made
possible..sup.17,18,19 These results indicate that when therapeutic
enzyme is administered over a sufficient period, at doses higher
than those used in conventional ERT trials such a therapeutic dose
has a beneficial effect in an adult mouse. The present invention
allows such beneficial effects to be achieved with the
administration of less therapeutic enzyme.
[0027] Therefore, the present invention discloses 1) an enzyme with
therapeutic benefits for the CNS whereby said benefits are enhanced
by the attachment of an AAA sequence, 2) a method of attaching an
acidic amino acid sequence to a therapeutic enzyme with benefits
for the CNS so as to allow said benefits to be delivered to the CNS
under conditions which would otherwise be ineffectual, 3) a method
of treating a patient with an CNS related disease using the
aforementioned AAA-therapeutic enzyme.
[0028] The inventors have previous disclosed AAA-GALNS which is
hereby incorporated by reference..sup.20 This reference discloses a
fusion protein for the treatment of disease, and a method of
increasing the stability of a therapeutic protein in blood and
transfer of said protein to bone. More specifically the therapeutic
protein is GALNS and the disease is Morquio disease.
[0029] The inventors have also previously disclosed an AAA-GUS,
described in detail bellow, and herein incorporated by
reference..sup.21 This reference discloses a fusion protein AAA-GUS
for the treatment of disease with improved in vivo stability and a
method for treating a patient with MPS VII.
[0030] The term "polypeptide therapeutic agent" refereed to in the
present invention means any polypeptide, oligopeptide or protein
which will benefit a patient suffering from disease when
administered to the patient.
[0031] The term "acidic amino acid" or "AAA" referred to the
present invention means glutamic acid or aspartic acid. As the
employment of these acidic amino acids in the present invention is
for the purpose of constructing an acidic short peptide, they may
be used in any arbitrary combination including a simple use of one
or the other of them alone for construction of such a short
peptide. The number of the acidic amino acids forming a short
peptide is preferably 4-15, more preferably 4-12, and still more
preferably 4-8.
[0032] A short peptide consisting of acidic amino acids may be
directly attached to the N-terminus of physiologically active human
GUS via a peptide bond or like, or, instead, it may be attached via
a linker peptide.
[0033] In the present invention "a linker peptide" is not an
indispensable component, for it is usable only for convenience in
attaching a short peptide consisting of acidic amino acids to
N-terminus of physiologically active GUS. Where it is used, a
linker peptide may be a short peptide consisting e.g., preferably
of 15 or less, more preferably of 10 or less, and still more
preferably of 6 or less amino acids. Such a linker that consists of
a single amino acid molecule and linking between the acidic short
peptide and physiologically active GUS via peptide bonds is also
included in the definition of a linker peptide. A linker peptide
may be made of any one or more amino acids desired.
[0034] In the present invention, though there is no specific
limitation as to the method for attaching an acidic short peptide
to physiologically active GUS, it is of advantage, e.g., to form
and use a transformant cell expressing the fusion protein
consisting of the short peptide and physiologically active GUS.
[0035] In the present invention "attachment" in reference to acidic
amino acids or AAA and therapeutic proteins or peptides or enzymes
refers to creation of a covalent bond either through the creation
of a fusion protein or through the use of chemical agents or
manipulation to achieve the same result.
[0036] A fusion protein of the present invention may include a
non-acidic amino acid or a sequence of several (e.g., 3) non-acidic
amino acids at N-terminus of the short peptide consisting of acidic
amino acids.
[0037] A fusion protein of the present invention may be formulated
into a pharmaceutical composition containing the fusion protein
dissolved or dispersed in a pharmaceutically acceptable carrier
well known to those skilled in the art, for parenteral
administration by e.g., intravenous, subcutaneous, or intramuscular
injection or by intravenous drip infusion.
[0038] For pharmaceutical compositions for parenteral
administration, any conventional additives may be used such as
excipients, binders, disintegrants, dispersing agent, lubricants,
diluents, absorption enhancers, buffering agents, surfactants,
solubilizing agents, preservatives, emulsifiers, isotonizers,
stabilizers, solubilizers for injection, pH adjusting agents,
etc.
[0039] A fusion protein of the present invention may be used
advantageously in place of the conventional native enzyme protein
in a substitution therapy for the treatment of MPS VII. In the
treatment, the fusion protein may be administered intravenously,
subcutaneously, or intramuscularly. Doses and frequencies of
administration are to be determined by the physician in charge in
accordance with the condition of his or her patient.
[0040] Preferred embodiments of the invention are described in the
following examples. Other embodiments within the scope of the
claims herein will be apparent to one skilled in the art from
consideration of the specification or practice of the invention as
disclosed herein. It is intended that the specification, together
with the examples, be considered exemplary only, with the scope and
spirit of the invention being indicated by the claims, which follow
the examples.
EXAMPLE 1
Method for Construction of Expression Vectors
[0041] Vector pCXN had been constructed in accordance with a
literature (7) and was offered to us by Prof. Miyazaki at Osaka
University. An expression vector for native human GUS, pCXN-GUS,
was constructed by using human GUS cDNA that had been reported by
Oshima et al. (8)(Accession No. of GenBank for the Amino acid and
cDNA sequence of Human GUS is BC014142.). An expression vector for
human GUS to the N-terminus of which is attached (via a linker
peptide) a short peptide (N-terminal bone tag: NBT) consisting of
acidic amino acids (NBT-GUS), was constructed starting with
pCXN-GUS in the following manner. FIGS. 1 and 2 schematically
illustrate the process for construction.
[0042] Using pCXN-GUS as a template, PCR was carried out using
LA-Taq (Takara) to amplify .DELTA.sig GUS cDNA (the sequence, nt
67-1956, left behind after removal of the sequence of nt 1-66
corresponding to a secretion signal, from the ORF region of the
sequence set forth as SEQ ID NO:1) (for human GUS without signal
sequence, see SEQ ID NO:2), to the 5'-terminus of which is attached
an AgeI cleavage sequence. The PCR was carried out according to the
instruction for use of LA-Taq, i.e., through the cycles consisting
of 30 seconds at 94.degree. C., (30 seconds at 94.degree. C., 30
seconds at 60.degree. C., and 2 minutes at 72.degree. C.).times.25,
and then 3 minutes at 72.degree. C., with primer 1 (SEQ ID NO:3),
and primer 2 (SEQ ID NO:4). The cDNA thus amplified was inserted
into pT7Blue vector (Novagen) to construct pT7-.DELTA.sig GUS.
[0043] The N-terminal bone tag (NBT) cDNA to be attached to the
5'-terminus then was constructed by PCR using LA-Taq (Takara).
Briefly, primer 3 (SEQ ID NO:5) and primer 4 (SEQ ID NO:6) were
used for the construction of NBT-E6 cDNA, primer 5 (SEQ ID NO:7)
and primer 4 (SEQ ID NO:6) for the construction of NBT-E8 cDNA,
primer 6 (SEQ ID NO:8) and primer 4 (SEQ ID NO:6) for the
construction of NBT-D6 cDNA, and primer 7 (SEQ ID NO:9) and primer
4 (SEQ ID NO:6) for the construction of NBT-D8 cDNA. In the names
of the NBT cDNAs, "E6" or "E8" indicate that the NBT is made up of
6 or 8 serially connected glutamic acid residues, respectively.
Likewise, "D6" or "D8" indicates that the NBT is made up of 6 or 8
connected aspartic acid residues, respectively.
[0044] Employing each pair of the above primers, which contained a
portion complementary to each other, PCR was carried out through
the cycles consisting of 30 seconds at 94.degree. C., (30 seconds
at 94.degree. C., 30 seconds at 60.degree. C., 30 seconds at
72.degree. C.).times.20 minutes, and then one minute at 72.degree.
C. The thus amplified DNA fragments were inserted into pT7Blue
vector (Novagen) to construct pT7-NBTs.
[0045] A human GUS cDNA recovered as a fragment of pT7
pT7-.DELTA.sig GUS cleaved with AgeI and XbaI was inserted into the
AgeI-XbaI site of pT7-NBTs to construct pT7-NBT-GUSs.
[0046] Then each of pT7-NBT-GUSs was cleaved with BclI, blunt-ended
with T4 DNA polymerase, and cleaved with XbaI to recover NBT-GUS
cDNAs.
[0047] pST-RAP-GUSB (a vector comprising the p97 signal sequence,
provided by Tomatsu at Saint Louis University) was cleaved with
BamHI and XbaI, into which then was inserted the NBT-GUS cDNAs
recovered above to construct pST-p97-NBT-GUSs.
[0048] pST-p97-NBT-GUSs were cleaved with EcoRI to recover
respective p97-NBT-GUS cDNAs, each of which then was inserted into
the EcoRI site of pCXN to construct a NBT-GUS expression vector,
pCXN-p97-NBT-GUS. The DNA sequence of the expression vectors'
region corresponding to the p97-NBT-D6-GUS, p97-NBT-D8-GUS,
p97-NBT-E6-GUS and p97-NBT-E8-GUS cDNAs are shown in the Sequence
Listing (SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16,)
along with their corresponding amino acid sequences (SEQ ID NO:11,
SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17), respectively.
[0049] SEQ ID NO:10 shows part of the sequence containing the
NBT-E6-GUS cDNA of pCXN-p97-NBT-E6-GUS. Its nt 1-57 encode the p97
signal sequence, nt 61-78 a poly Glu, nt 79-96 a linker sequence,
and nt 97-1983 GUS without the signal sequence.
[0050] SEQ ID NO:11 shows the NBT-E6-GUS amino acid sequence with
the p97 signal sequence. Aa 1-19: p97 signal sequence, aa 21-26:
poly Glu, aa 27-32: linker sequence, aa 33-661: GUS without signal
sequence.
[0051] SEQ ID NO:12 shows part of the sequence containing the
NBT-E8-GUS cDNA of pCXN-p97-NBT-E8-GUS. Its nt 1-57 encode the p97
signal sequence, nt 61-84 a poly Glu, nt 85-102 a linker sequence,
and nt 103-1989 GUS without the signal sequence.
[0052] SEQ ID NO:13 shows the NBT-E8-GUS amino acid sequence with
attached p97 signal sequence. Aa 1-19: p97 signal sequence, aa
21-28: poly Glu, aa 29-34: linker sequence, aa 35-663: GUS without
signal sequence.
[0053] SEQ ID NO:14 shows part of the sequence containing the
NBT-D6-GUS cDNA of pCXN-p97-NBT-D6-GUS. Its nt 1-57 encode the p97
signal sequence, nt 61-78 a poly Asp, nt 79-96 a linker sequence,
and nt 97-1983 GUS without the signal sequence.
[0054] SEQ ID NO:15 shows the NBT-D6-GUS amino acid sequence with
attached p97 signal sequence. Aa 1-19: p97 signal sequence, aa
21-26: poly Asp, aa 27-32: linker sequence, aa 33-661: GUS without
signal sequence.
[0055] SEQ ID NO:16 shows part of the sequence containing the
NBT-D8-GUS cDNA of pCXN-p97-NBT-D8-GUS. Its nt 1-57 encode the p97
signal sequence, nt 61-84 a poly Asp, nt 85-102 a linker sequence,
and nt 103-1989 GUS without the signal sequence.
[0056] SEQ ID NO:17 shows the NBT-D8-GUS amino acid sequence with
attached p97 signal sequence. Aa 1-19: p97 signal sequence, aa
21-28: poly Asp, aa 29-34: linker sequence, aa 35-663: GUS without
signal sequence.
[0057] The proteins set forth as SEQ ID NO:11, SEQ ID NO:13, SEQ ID
NO:15 and SEQ ID NO:17 contain the p97 secretion signal sequence.
The signal sequence is removed during secretion process from the
cell and the fusion proteins are thus recovered as NBT-GUS in the
medium.
[0058] p97 is a cell-surface glycoprotein occurring in most human
melanomas and its signal sequence consists of 19 amino acids(9).
The aforementioned pCXN-p97-NBT-GUSs containing the cDNA encoding
this signal sequence may also be constructed by the following
method. Briefly, a cDNA containing the p97 signal sequence is
synthesized through the process of primers annealing and PCR
amplification. LA-Taq is used as an enzyme for PCR. As primers
having mutually complementary portions, primer 8 (SEQ ID NO:18) and
primer 9 (SEQ ID NO:19) are used. PCR is performed through the
cycles of 30 seconds at 94.degree. C., (30 seconds at 94.degree.
C., 30 seconds at 60.degree. C., 30 seconds at 72.degree.
C.).times.20, and one minute at 72.degree. C. The amplified cDNA
containing the p97 signal sequence is cleaved with BamHI and EcoRI.
Into the pCXN vector, after cleaved with EcoRI, are simultaneously
incorporated the aforementioned NBT-GUSs cDNA recovered after the
enzyme treatment and cDNA for the p97 signal sequence, giving
pCXN-p97-NBT-GUSs.
[0059] SEQ ID No:18 is a forward primer, in which nt 1-5 comprise a
random synthetic sequence, and nt 6-52 comprise part of the
sequence encoding the p97 signal.
[0060] SEQ ID No:19 is a reverse primer, in which nt 1-6 comprise a
random synthetic sequence, and nt 7-52 comprise part of the
sequence encoding the p97 signal.
[0061] Establishment of Expression Cells
[0062] Nunclon delta-MultiDish 6 Well was inoculated with CHO-K1
cells. After an overnight culture in DMEM/F12/FBS medium [DMEM/F12
medium(Gibco) supplemented with 10% fetal bovine serum (Thermo
Trace)], each of the expression vector constructed above was
introduced into the cells using Lipofectamine 2000 reagent. For
experimental procedures, the instruction manual attached to the
Lipofectamine 2000 reagent was followed. After a two-day incubation
at 37.degree. C. in 5% CO.sub.2, the cells were added to
75-cm.sup.2 tissue culture flasks (Iwaki) and incubated until
colonies of resistant cells were formed with Genetcin (Gibco) added
to the DMEM/F12/FBS medium at the final concentration of 1 mg/mL.
After formation of colonies was confirmed under a microscope, cells
which exhibited stable expression were cloned by the limiting
dilution-culture method. Screening for expression cells were
performed by GUS-specific enzyme activity assay of the culture
supernatants. Cell lines thus established were subcultured in
DMEM/F12/FBS medium supplemented with 0.2 mg/mL Geneticin.
[0063] Method for Measurement of GUS-Specific Enzyme Activity
[0064] After intravenous administration of native-or NBT-GUS to
mice, GUS activity in the blood was determined as follows. Briefly,
12.5 uL of plasma sample from the mice was added to 50 uL of a
solution of 10 mM 4-methylumbelliferyl-.beta.-D-glucuronide (Sigma
Chemical Co., St. Louis, Mo., cat #M9130) which had been prepared
using determination buffer (0.1M sodium acetate buffer pH 4.8), and
reaction was allowed for 1 hr at 37.degree. C. Then, 950 uL of stop
buffer (1 M Glycine-HCl, pH 10.5) was added and mixed to stop the
enzyme reaction. Samples of the reaction mixture were transferred
to a fluorometer for measurement with excitation 366 nm/emission
450 nm.
[0065] Expression and Purification of Native GUS and GUS Fusion
Protein
[0066] Native GUS and GUS fusion proteins were produced in
overexpressing CHO cells, which were grown to confluency and fed
with low-serum medium (Waymouth's MB 752/1 medium, supplemented
with 2% FBS/1.2 mM glutamine/1 mM pyruvate) (Gibco) for
purification every 24 hr. The media of the culture were pooled,
centrifuged at 5,000.times.g for 20 min at 4.degree. C., and frozen
at -20.degree. C. Purification was performed using affinity
chromatography (10). Briefly, the conditioned medium from cells
overexpressing the Native GUS or a GUS fusion protein was filtered,
and NaCl was added to the medium at the final concentration of 0.5
M. The medium was applied to a 5 ml column of Affi-Gel 10 (BioRad)
which carried an anti-human GUS monoclonal antibody and had been
pre-equilibrated with wash buffer. The column was washed at 36
mL/hour with 20-column volumes of wash buffer. The column was
eluted at 36 mL/hour with 50 ml of 10 mM sodium phosphate (pH 5.0)
containing 3.5 M MgCl.sub.2. Fractions were collected and subjected
to GUS activity assay. Fractions containing the enzyme activity
were pooled for each of the Native or fusion proteins, diluted with
an equal volume of P6 buffer (25 mM Tris, pH 7.5/1 mM
.beta.-glycerol phosphate/0.15 mM NaCl/0.025% sodium azide), and
desalted over a BioGel P6 column (BioRad) pre-equilibrated with P6
buffer. Fractions containing GUS activity were pooled, and the
finally purified active protein was stored at -80.degree. C.
EXAMPLE 2
Stability in the Blood
[0067] Per 1 g of body weight, 1,000 U of native GUS or one of the
NBT-GUSs, both purified, were administered to male, 4-month old
C57BL mice (3 animals/group) in the tail vein. Samples of venous
blood were collected at 2 min, 5 min, 10 min, 20 min, 30 min, 1 hr,
2 hr, 6 hr, 24 hr after the administration, and GUS activity in the
serum was measured. The results are shown in FIG. 3. Comparison
between the NBT-GUSs-administered groups and the native
GUS-administered group reveales that, at 2 min after the
administration, the enzyme activity in the blood was 2-fold higher
in the NBT-GUSs-administered groups as compared with the native
GUS-administered group. While the enzyme activity in the blood at
30 min after the administration was almost disappeared in the
native GUS-administered group, the NBT-GUSs-administered groups
retained activity levels, which were even higher than the activity
level found at 2 min in the native GUS-administered group.
Afterwards, the NBT-GUSs-administered groups continued to show
remarkably slower reduction in the enzyme activity levels in the
blood as compared with those found in the native GUS-administered
group. Even 24 hr (1440 min) after the administration, the residual
enzyme activity was detectable in the NBT-GUSs-administered group.
A half-life time of the enzyme activity in blood in the native
GUS-administered group was 4.9 min, while a half-life time in blood
in the NBT-GUS-administered group was prolonged 5-6 times. The
results demonstrate that the stability of GUS in the body is
remarkably increased by attaching a short peptide of acidic amino
acids to the N-terminus of native GUS.
EXAMPLE 3
Effects of GUS on Brain Tissue
[0068] To compare the effectiveness of AAA-tagged and untagged GUS
at clearing storage from affected tissues in the MPS VII mouse, the
inventors used a protocol in which enzyme was given in 12 weekly
treatments with 1 mg/kg enzyme. There were notable differences in
which the D.sub.6-GUS appeared to be more effective in clearing the
storage material. The parietal neocortical neurons and glia had
less storage in the D.sub.6-GUS-treated MPS VII mice. In brain, the
AAA-tagged enzyme showed improved clearance of storage from
parietal neocortical neurons and glial cells, where as storage
showed minimal or no clearance response to untagged enzyme at the
same dose. FIG. 4 shows light microscopy of tissues from native GUS
and D.sub.6-GUS treated MPS VII mice. The cortical neuron,
hippocampus, and glia cell sections show a reduction of storage (S)
in D.sub.6-GUS treated compared to GUS treated mice.
[0069] In these studies, MPS VII/E540A.sup.tg mice were
used..sup.22 These mice carry a GUS transgene that encodes an
inactive enzyme, which confers immunotolerance to the human
protein. To define the clearance from the blood circulation, 1,000
units per g of body weight of D6-GUS, D8-GUS or untagged GUS were
administered to 4-month-old MPS VII mice (3 animals/group) via the
tail vein. Samples of venous blood were collected at 2 min, 5 min,
10 min, 20 min, 30 min, 1 h, 2 h, 6 h, and 24 h after
administration, and GUS activity in the serum was measured.
[0070] To determine the effectiveness of D6-GUS, D8-GUS, and
untagged enzyme at reversing storage pathology, three adult animals
in each group received twelve weekly doses (5,000 units/g) of
D6-GUS, D8-GUS, untagged enzyme or PBS by injection in the lateral
tail vein.
[0071] Animals were killed 1 week after the 12th injection, and the
organs were removed for histopathology analysis with light or
electron microscopy.
[0072] For morphological evaluation, liver, spleen, kidney, brain,
heart, femur, and bone marrow from 4-5 month old MPS VII mice
treated with D6-GUS (n=2), D8-GUS (n=3), and untagged enzyme (n=3),
or buffer (n=2) were collected at necropsy, immersion-fixed in 4%
paraformaldehyde/2% glutaraldehyde in PBS, postfixed in osmium
tetroxide, and embedded in Spurr's resin. For evaluation of
lysosomal storage by light microscopy, toluidine blue-stained
0.5-.mu.m-thick sections were examined. One mouse treated by D6-GUS
died immediately after the 12th weekly infusion and was not
evaluated morphologically. Tissues from the treated and untreated
mice were evaluated for reduction in storage without knowledge of
their treatment. Two pathologists (CV, BL) independently evaluated
the brain for lysosomal storage.
[0073] Some individual elements of the inventors' methodology are
generally known or described in detail in numerous laboratory
protocols, one of which is Molecular Cloning 2nd edition, (1989)
Sambrook, J., Fritsch, E. F. and Maniatis, J., Cold Spring Harbor.
As such detailed discussion of their composition and methodology is
superfluous.
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