U.S. patent application number 15/429309 was filed with the patent office on 2017-08-03 for recombinant human naglu protein and uses thereof.
The applicant listed for this patent is Alexion Pharmaceuticals, Inc.. Invention is credited to Markley C. Leavitt, ANTHONY QUINN, Zhinan Xia.
Application Number | 20170216412 15/429309 |
Document ID | / |
Family ID | 48082738 |
Filed Date | 2017-08-03 |
United States Patent
Application |
20170216412 |
Kind Code |
A1 |
QUINN; ANTHONY ; et
al. |
August 3, 2017 |
RECOMBINANT HUMAN NAGLU PROTEIN AND USES THEREOF
Abstract
The present invention provides compositions comprising an
isolated mixture of recombinant human NaGlu proteins in which a
substantial amount of the NaGlu proteins in the mixture has
increased levels of phosphorylated mannose that confer the proteins
to be efficiently internalized into human cells. The present
invention also provides methods of producing such mixture of NaGlu
proteins, vectors used in transgenesis and expression, host cells
harboring such vectors, and methods of isolating and purifying the
mixture of NaGlu proteins. The invention further provides methods
of treating NaGlu associated diseases.
Inventors: |
QUINN; ANTHONY; (Chestnut
Hill, MA) ; Leavitt; Markley C.; (Watkinsville,
GA) ; Xia; Zhinan; (Wellesley, MA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Alexion Pharmaceuticals, Inc. |
New Haven |
CT |
US |
|
|
Family ID: |
48082738 |
Appl. No.: |
15/429309 |
Filed: |
February 10, 2017 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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13649595 |
Oct 11, 2012 |
9579366 |
|
|
15429309 |
|
|
|
|
61546248 |
Oct 12, 2011 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61P 1/00 20180101; A61K
9/0019 20130101; A61P 1/12 20180101; A61P 7/02 20180101; A61K
31/7024 20130101; A61P 25/00 20180101; A61P 17/00 20180101; A61K
45/06 20130101; A61P 1/02 20180101; C07K 7/08 20130101; A61P 37/06
20180101; A61P 25/20 20180101; A61P 3/00 20180101; C12Y 302/0105
20130101; A61P 1/16 20180101; A61K 31/7024 20130101; A61P 19/02
20180101; A61P 43/00 20180101; C12N 9/2402 20130101; C12N 9/2474
20130101; C07K 7/06 20130101; A61K 38/47 20130101; A61K 38/47
20130101; A61K 2300/00 20130101; A61K 2300/00 20130101 |
International
Class: |
A61K 38/47 20060101
A61K038/47; C12N 9/24 20060101 C12N009/24; A61K 9/00 20060101
A61K009/00 |
Claims
1. A composition comprising an isolated mixture of recombinant
human N-acetyl-5 alpha-D-glucosaminidase (rhNaGlu) comprising the
amino acid sequence 24-743 of SEQ ID NO: 1, wherein at least 10% of
said rhNaGlu in said mixture comprises at least one glycan
structure having mannose-6-phosphate (M6P).
2.-16. (canceled)
17. A method of producing a recombinant human NaGlu (rhNaGlu)
comprising: a) producing a transgenic avian comprising a trans gene
having a promoter component operably linked to a nucleic acid
sequence encoding the rhNaGlu set forth in 24-743 of SEQ ID NO:1,
wherein said transgene is contained in the genome of the transgenic
avian and expressed in an oviduct cell, such that the rhNaGlu is
glycosylated in the oviduct cell of the transgenic avian, secreted
into lumen of oviduct and deposited in egg white of an egg laid of
the transgenic avian; and b) isolating said rhNaGlu from said egg
white.
18.-29. (canceled)
30. A method of delivering recombinant human NaGlu protein to the
brain of a subject suffering from NaGlu deficiency, the method
comprising intravenously administering recombinant human NaGlu
protein to the subject.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation of U.S. patent
application Ser. No. 13/649,595, filed Oct. 11, 2012, which claims
the benefit of U.S. Provisional Application No. 61/546,248, filed
Oct. 12, 2011, the entire contents of which are expressly
incorporated herein by this reference.
SEQUENCE LISTING
[0002] The instant application contains a sequence listing which
has been submitted electronically in ASCII format and is hereby
incorporated by reference in its entirety. Said ASCII copy, created
on Feb. 10, 2017, is named 081245-0227_SL.txt and is 25,284 bytes
in size.
BACKGROUND OF THE INVENTION
[0003] Sanfilippo Syndrome B is an autosomal recessive lysosomal
storage disease (LSD) caused by a deficiency in a lysosomal enzyme
known as N-acetyl-alpha-D-glucosaminidase (NaGlu). NaGlu is
required for the degradation of heparan sulfate as part of the
stepwise breakdown of glycosaminoglycans (GAG) in the lysosome. The
deficiency or absence of NaGlu leads to accumulation and urinary
excretion of heparan sulfate. With over 70 different mutations
identified to date, Sanfilippo Syndrome B exhibits extensive
molecular and genetic heterogeneity.
[0004] Approximately 1 out of 200,000 births is affected by
Sanfilippo Syndrome B and the deficiency mainly manifests in young
children. After initial symptom-free interval, patients suffering
from Sanfilippo Syndrome B usually present with a slowing of mental
development and behavioral problems, followed by progressive
intellectual decline resulting in severe mental retardation,
dementia and motor disease. Acquisition of speech is slow and
incomplete. Profoundly affected patients may present delayed
psychomotor and speech development as early as 2 years of age. The
disease usually progresses to increasing behavioral disturbance and
sleep disturbance. Although the clinical features are mainly
neurological, patients often develop diarrhea, carious teeth, an
enlarged liver and spleen, stiff joints, hirsteness and/or coarse
hair and may exhibit blood-clotting problems. In the final stage of
the illness, patients become immobile and unresponsive and develop
swallowing difficulties and seizure. The life-span of an affected
child typically does not extend beyond late teens to early
twenties.
[0005] Different approaches have been attempted to provide the
missing enzyme in patients. To produce NaGlu for enzyme replacement
therapy (ERT), human NaGlu has been expressed in various mammalian
cell culture systems. However, in contrast to the naturally
occurring NaGlu which trafficks to the lysosome intracellularly,
recombinant NaGlu proteins produced and secreted from mammalian
cells were found to contain no or only a trace amount of mannose
6-phosphate (M6P). The absence or scarcity of M6P moieties in the
secreted NaGlu has been known to prevent its efficient
internalization into target cells (e.g., human skin fibroblasts),
which have M6P receptors on the surface on its plasma membrane
(see, Zhao et al., Protein Expression and Purification, 19:202-211
(2000); and Weber et al., Protein Expression and Purification,
21:251-259 (2001)). The low degree of phosphorylation was seen in
secreted mouse NaGlu expressed in CHO cells, secreted human NaGlu
expressed in HeLa cells, secreted human NaGlu expressed in human
fibroblasts, and secreted human NaGlu expressed in human embryonic
kidney (HEK) cell line 293 (see, Zhao et al., Protein Expression
and Purification, 19:202-211 (2000); Yogalingam et al., Biochim
Biophys. Acta 1502: 415-425; and Weber et al., Protein Expression
and Purification, 21:251-259 (2001)). No or weak phosphorylation of
N-glycans in the NaGlu proteins secreted from the mammalian cells
has posed a major obstacle for the development of a recombinant
human NaGlu protein suitable for enzyme replacement therapy as all
the aforementioned attempts has failed to produce an enzyme which
is efficiently taken up by target cells as the concentration of the
internalized proteins, if detectable at all, was nearly a thousand
times less than wild-type levels (see, Zhao et al., Protein
Expression and Purification, 19:202-211 (2000)). To date, no
approved product is available for the treatment of Sanfilippo
Syndrome B.
[0006] Direct administration of mammalian cell-produced recombinant
human NaGlu protein (rhNaGlu) having the native amino acid sequence
into the central nervous system (CNS) (e.g., intrathecal
administration into the cerebrospinal fluid (CSF)) of NaGlu
deficient mice has been attempted, but failed to demonstrate
successful biodistribution of the enzyme to the brain due to
excessive accumulation of the protein on the ependymal ling of the
ventricles as well as lack of requisite M6P residues for efficient
cellular uptake. Similarly, systemic administration (i.e.,
intravenous (IV) injection) of mammalian cell-produced rhNaGlu
having the native amino acid sequence also failed to demonstrate
successful localization of the protein to the brain. In addition to
known risks associated with highly invasive intrathecal
administration, these obstacles in targeting rhNaGlu to the brain
have been too great a challenge to achieve effective therapy for
the treatment of Sanfilippo Syndrome B.
[0007] Therefore, there is a need to provide a stable NaGlu protein
which is enzymatically active and has physical properties that
allow for the protein to cross the blood brain barrier (BBB) and
for effective internalization of the protein into the lysosomes of
target cells. There is also a need for a high expressing and robust
protein production platform which can provide a recombinant human
NaGlu that effectively crosses the blood brain barrier and is
efficiently internalized into human target cells.
SUMMARY OF THE INVENTION
[0008] The present invention is drawn to compositions comprising
recombinant human NaGlu protein (rhNaGlu) useful for therapy, for
example, in the treatment of Sanfilippo Syndrome B. The present
invention is based on the surprising and unexpected discovery that
the rhNaGlu described herein has one or more glycosylation patterns
that allow the rhNaGlu to efficiently cross the blood brain barrier
(BBB), and be taken up into cells within the central nervous system
(CNS) of animals deficient in the enzyme, resulting in a dramatic
increase in .alpha.-N-acetylglucosaminidase activity in the brain,
as well as a reduction of substrate levels. Moreover, the rhNaGlu
described herein is efficiently taken up into a mammalian cell
(e.g., human cell), resulting in an increased enzymatic activity as
compared to NaGlu proteins produced and secreted from unmodified
mammalian cells that are not designed to produce specific
glycosylation. The increased cellular uptake of the NaGlu protein
also provides benefits for the use in enzyme replacement therapy
for a human patient suffering from Sanfilippo Syndrome B by
minimizing the need for an increased amount and frequency of dose,
and thereby greatly reducing the potential risk of
immunogenicity.
[0009] The rhNaGlu protein described herein contains sufficient
amount of oligosaccharides (e.g., mannose and phosphorylated
mannose (i.e., M6P)) to allow efficient cellular uptake via mannose
and/or M6P receptor-mediated endocytosis and be correctly targeted
into human cells. In one embodiment, the rhNaGlu contains at least
one mole of protein, for example, 1, 2, 3, 4, 5 or 6 moles of M6P
per mole of protein. In one embodiment, rhNaGlu can be internalized
into a NaGlu deficient human cell such that the internalized
protein fully (100% or more) restores normal levels (i.e.,
wild-type levels) of NaGlu activity in the NaGlu deficient
cell.
[0010] Also disclosed herein are methods for producing a transgenic
avian that expresses rhNaGlu which benefits from phosphorylation of
mannose. In particular, a transgenic avian that expresses rhNaGlu
protein in oviduct cells, secretes into the lumen of the oviduct
and deposits the protein into egg white. Avian eggs that contain
such rhNaGlu are also included in the present invention.
[0011] The present invention also contemplates vectors and host
cells that contain a transgene encoding rhNaGlu as well as
pharmaceutical compositions comprising rhNaGlu to be used in the
application of such rhNaGlu for the treatment of Sanfilippo
Syndrome B.
[0012] In one aspect, the invention provides a composition
comprising an isolated mixture of recombinant human
N-acetyl-alpha-D-glucosaminidase (rhNaGlu) comprising the amino
acid sequence 24-743 of SEQ ID NO:1, wherein at least 10% of the
rhNaGlu in the mixture comprises at least one glycan structure
having mannose-6-phosphate (M6P). In one embodiment, the rhNaGlu
having M6P is capable of being taken up into a mammalian cell
deficient in NaGlu such that internalized rhNaGlu restores at least
50%, 60%, 70%, 80%, 90% or 100% of normal NaGlu activity observed
in a wild-type mammalian cell of the same type. In another
embodiment, the glycan structure is an N-linked glycan.
[0013] In one embodiment, the rhNaGlu contains at least 1 mole of
M6P per mole of protein. In another embodiment, the rhNaGlu
contains between about 1 and about 6 moles of M6P per mole of
protein. In another embodiment, the rhNaGlu contains about 2 moles
of M6P per mole of protein. In yet another embodiment, the rhNaGlu
contains about 3 moles of M6P per mole of protein. In another
embodiment, the rhNaGlu contains about 4 moles of M6P per mole of
protein. In another embodiment, the rhNaGlu contains about 5 moles
of M6P per mole of protein. In yet another embodiment, the rhNaGlu
contains about 6 moles of M6P per mole of protein.
[0014] In one embodiment, the mammalian cell deficient in NaGlu is
a human cell. In another embodiment, the human cell deficient in
NaGlu is a skin fibroblast, a hepatocyte or a macrophage. In one
embodiment, the human cell deficient in NaGlu is a neuronal
cell.
[0015] In one embodiment, the rhNaGlu is effectively delivered to
the brain of a mammal having NaGlu deficiency when systemically
administered. In one particular embodiment, the rhNaGlu is
effectively delivered to the brain of a mammal having NaGlu
deficiency when intravenously administered. In one embodiment, the
rhNaGlu is effectively delivered to the brain of a mammal having
NaGlu deficiency when administered intrathecally.
[0016] In one embodiment, the rhNaGlu having M6P is internalized by
a NaGlu deficient cell and restores at least 100% of normal NaGlu
activity in vivo. In one embodiment, the rhNaGlu having M6P
contains at least 25 moles of mannose per mole of protein.
[0017] In one embodiment, at least 20%, 30%, 40%, 50%, 60%, 70%,
80%, 90% or 95% of the rhNaGlu in the mixture contains M6P. In
another embodiment, at least 20% of the rhNaGlu in the mixture
contains at least one M6P. In another embodiment, at least 30% of
the rhNaGlu in the mixture contains at least one M6P. In another
embodiment, at least 40% of the rhNaGlu in the mixture contains at
least one M6P. In another embodiment, at least 50% of the rhNaGlu
in the mixture contains at least one M6P. In another embodiment, at
least 60% of the rhNaGlu in the mixture contains at least one
M6P.
[0018] In another aspect, the invention provides a composition
comprising an isolated mixture of recombinant human
N-acetyl-alpha-D-glucosaminidase (rhNaGlu) comprising the amino
acid sequence 24-743 of SEQ ID NO: 1, wherein the mixture comprises
a sufficient amount of rhNaGlu containing one or more glycan
structures comprising mannose-6-phosphate (M6P) such that the
rhNaGlu containing M6P is internalized into a mammalian cell having
NaGlu deficiency via M6P receptor-mediated endocytosis and restores
at least 50% of NaGlu activity observed in a wild-type cell of the
same type expressing endogenous NaGlu. In one embodiment, the
rhNaGlu is N-linked glycosylated. In another embodiment, the
rhNaGlu is O-linked glycosylated.
[0019] In one embodiment, the rhNaGlu comprises at least 1 moles of
M6P per mole of rhNaGlu. In another embodiment, the rhNaGlu
comprises about 1, 2, 3, 4, 5 or 6 moles of M6P per mole of
rhNaGlu. In another embodiment, the rhNaGlu comprises about 3 moles
of M6P per mole of rhNaGlu. In another embodiment, the rhNaGlu
comprises about 4 moles of M6P per mole of rhNaGlu.
[0020] In one embodiment, the rhNaGlu comprises mannose. In another
embodiment, the rhNaGlu comprises N-acetylglucosamine (GlcNAc). In
another embodiment, the rhNaGlu comprises galactose. In another
embodiment, the rhNaGlu comprises N-acetylgalactosamine (GalNAc).
In another embodiment, the rhNaGlu contains no fucose. In another
embodiment, the rhNaGlu contains no glucose. In one embodiment, the
rhNaGlu restores at least 60, 70, 80, 90, 95 or 100% of normal
NaGlu enzymatic activity.
[0021] In another embodiment, the rhNaGlu is effectively delivered
to the brain of a mammal having NaGlu deficiency when administered
systemically. In one embodiment, the rhNaGlu is effectively
delivered to the brain of a mammal having NaGlu deficiency when
administered intravenously. In another embodiment, the rhNaGlu is
effectively delivered to the brain of a mammal having NaGlu
deficiency when administered intrathecally.
[0022] In one embodiment, the mammalian cell deficient in NaGlu is
a human cell. In another embodiment, the human cell is a skin
fibroblast, a hepatocyte or a macrophage. In one embodiment, the
human cell deficient in NaGlu is a neuronal cell.
[0023] In one embodiment, the rhNaGlu is a fusion protein
comprising a second moiety. In one embodiment, the second moiety is
a polypeptide. In another embodiment, the polypeptide is selected
from the group consisting of transferrin receptor ligand (TfRL),
insulin-like growth factor receptor (IGF2R) ligand, low density
lipoprotein (LDL) receptor ligand and acidic amino acid (AAA)
residues.
[0024] In one embodiment, the rhNaGlu is produced from a transgenic
avian. In one embodiment, the transgenic avian is a chicken, a
turkey, a duck or a quail. In one embodiment, the transgenic avian
is a chicken. In one embodiment, the rhNaGlu is produced from an
oviduct cell.
[0025] In another aspect, the invention provides a composition
comprising an isolated recombinant human
N-acetyl-alpha-D-glucosaminidase (rhNaGlu) comprising one or more
glycan structures having sufficient amount of mannose-6-phosphate
(M6P) that allows for internalization of the rhNaGlu into a
mammalian cell having NaGlu deficiency via M6P receptor-mediated
endocytosis, such that when internalized in vivo, the rhNaGlu
restores at least 50% of NaGlu activity observed in a wild-type
cell of the same type expressing endogenous NaGlu.
[0026] In one embodiment, the rhNaGlu protein is N-linked
glycosylated. In another embodiment, the rhNaGlu protein is
O-linked glycosylated. In one embodiment, the rhNaGlu comprises
about 2, 3, 4, 5 or 6 moles of M6P per mole of rhNaGlu.
[0027] In one embodiment, the rhNaGlu is effectively delivered to
the brain of a mammal having NaGlu deficiency when administered
systemically. In another embodiment, the rhNaGlu is effectively
delivered to the brain of a mammal having NaGlu deficiency when
administered intravenously. In another embodiment, the rhNaGlu is
effectively delivered to the brain of a mammal having NaGlu
deficiency when administered intrathecally.
[0028] In another aspect, the invention provides a transgenic avian
comprising a transgene containing a promoter operably linked to a
nucleic acid sequence encoding a recombinant human NaGlu (rhNaGlu),
wherein the transgene is contained in the genome of the transgenic
avian and expressed in an oviduct cell such that the rhNaGlu is
glycosylated in the oviduct cell of the transgenic avian, secreted
into lumen of oviduct and deposited in egg white of an egg of the
transgenic avian.
[0029] In one embodiment, the rhNaGlu comprises about 2, 3, 4 or 6
moles of M6P per mole of rhNaGlu. In another embodiment, the
promoter component is an oviduct-specific promoter. In another
embodiment, the oviduct-specific promoter is an ovalbumin promoter.
In yet another embodiment, the transgenic avian is selected from
the group consisting of a chicken, a turkey, a duck and a
quail.
[0030] In another aspect, the invention provides an egg produced by
the transgenic avian of the invention.
[0031] In yet another aspect, the invention provides a method of
producing a recombinant human NaGlu (rhNaGlu) comprising: a)
producing a transgenic avian comprising a transgene having a
promoter component operably linked to a nucleic acid sequence
encoding the rhNaGlu set forth in 24-743 of SEQ ID NO: 1, wherein
the transgene is contained in the genome of the transgenic avian
and expressed in an oviduct cell, such that the rhNaGlu is
glycosylated in the oviduct cell of the transgenic avian, secreted
into lumen of oviduct and deposited in egg white of an egg laid by
the transgenic avian; and b) isolating the rhNaGlu from the egg
white.
[0032] In one embodiment, the promoter component is an
oviduct-specific promoter. In another embodiment, the
oviduct-specific promoter is an ovalbumin promoter. In one
embodiment, the avian is selected from the group consisting of a
chicken, a turkey, a duck and a quail. In one embodiment, the avian
is chicken.
[0033] In another aspect, the invention provides a vector
comprising a nucleotide sequence encoding a human NaGlu operably
linked to an ovalbumin promoter. In another aspect, the invention
provides a host cell comprising the vector of the invention. In
another aspect, the invention provides an isolated nucleic acid
comprising the nucleic acid sequence of 5232-10248 of SEQ ID
NO:4.
[0034] In one aspect, the invention provides a pharmaceutical
formulation comprising a composition of the invention in
combination with a pharmaceutically acceptable carrier, diluent or
excipient.
[0035] In another aspect, the invention provides a composition
comprising recombinant human NaGlu protein that crosses the blood
brain barrier of a mammal having NaGlu deficiency when administered
intravenously.
[0036] In yet another aspect, the invention provides a method of
treating a subject suffering from NaGlu deficiency, the method
comprising administering to the subject a therapeutically effective
amount of the composition of the invention.
[0037] In yet another aspect, the invention provides a method of
delivering recombinant human NaGlu protein to the brain of a
subject suffering from NaGlu deficiency, the method comprising
intravenously administering recombinant human NaGlu protein to the
subject.
[0038] In another aspect, the invention provides a method of
transporting a recombinant human NaGlu protein from the circulation
across the blood brain barrier in a therapeutically effective
amount, the method comprising intravenously administering a
recombinant human NaGlu protein to a subject having NaGlu
deficiency.
[0039] In one embodiment, the NaGlu deficiency is Sanfilippo
Syndrome B. In another embodiment, the subject is a human.
[0040] In another embodiment, the recombinant human NaGlu protein
is administered intravenously to the subject at a dosage of about
0.5 to about 50 mg/kg body weight. In another embodiment, the
recombinant human NaGlu protein is administered intravenously to
the subject at a dosage of about 1 to about 30 mg/kg body weight.
In another embodiment, the recombinant human NaGlu protein is
administered intravenously to the subject at a dosage of about 6 to
about 27 mg/kg body weight.
[0041] In yet another embodiment, the recombinant human NaGlu
protein is intrathecally administered to the subject. In one
embodiment, the recombinant human NaGlu protein is intrathecally
administered at a dosage of at least about 0.3, 0.4, 0.5, 0.6, 0.7,
0.8, or 0.9 mg/kg body weight. In another embodiment, the
recombinant human NaGlu protein is intrathecally administered at a
dosage of about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 mg/kg body weight.
In another embodiment, the recombinant human NaGlu protein is
administered intrathecally at a dosage of about 10 to about 30
mg/kg body weight.
[0042] In another embodiment, the therapeutically effective amount
is an amount effective to reduce heparan sulfate levels in the
brain, the kidney, or the liver of the subject. In another
embodiment, the therapeutically effective amount is an amount
effective to increase NaGlu activity in the brain or the liver of
the subject.
[0043] In another embodiment, the method further comprises
administering a second therapeutic agent. In one embodiment, the
second therapeutic is an immunosuppressant.
BRIEF DESCRIPTION OF THE FIGURES
[0044] FIG. 1 depicts the amino acid sequence of human recombinant
NaGlu (amino acid residues 1-23, signal peptide).
[0045] FIG. 2 depicts the nucleic acid sequence (cDNA) of human
recombinant NaGlu, including the nucleic acid sequence encoding the
signal peptide.
[0046] FIG. 3 depicts the nucleic acid sequence of 1.1 kb ovalbumin
promoter.
[0047] FIGS. 4A-D depict the nucleic acid sequence of
pSIN-OV-1.1-I-rhNaGlu vector used in transgenesis of an avian.
[0048] FIG. 5 is a schematic representation of
pSIN-OV-1.1-I-rhNaGlu vector.
[0049] FIG. 6 depicts Western analysis of rhNaGlu isolated and
purified from egg white of a transgenic Gallus.
[0050] FIG. 7 depicts the average concentration of rhNaGlu
deposited in egg white of transgenic Gallus.
[0051] FIG. 8 depicts an oligosaccharide profile of rhNaGlu
produced from a transgenic Gallus using HPAEC-PAD.
[0052] FIG. 9 depicts uptake analysis of rhNaGlu by human skin
fibroblasts (MPS IIIB, NaGlu deficient; Normal, wild-type human
skin fibroblast; 1 U of enzymatic activity=nmol of protein/hr).
[0053] FIG. 10 depicts uptake inhibition analysis of rhNaGlu
(Gallus) using various concentrations of M6P monosaccharide (1U of
enzymatic activity=1 .mu.mol of protein/min).
[0054] FIG. 11 depicts a schematic representation of pTT22 vector
containing a recombinant human NaGlu fusion construct (AAA-NaGlu:
acidic amino acid residues fused to N-terminus of the full length
NaGlu).
[0055] FIG. 12 depicts a schematic representation of pTT22 vector
containing a recombinant human NaGlu fusion construct (NaGlu-TfRL:
transferrin receptor ligand fused to C-terminus of the full length
NaGlu).
[0056] FIG. 13 depicts enzymatic activity of AAA-NaGlu produced
from HEK293 as compared to rhNaGlu produced from Gallus.
[0057] FIG. 14 depicts enzymatic activity of NaGlu-TfRL produced
from HEK293 as compared to AAA-NaGlu produced from HEK293.
[0058] FIG. 15 depicts uptake levels of rhNaGlu (Gallus) into a
macrophage cell line (NR8383) over time (48 hours). Cellular NaGlu
activity was measured in units/mg of protein.
[0059] FIG. 16 depicts heparan sulfate substrate levels (ag/mg
tissue) in the kidney of naglu (.sup.-/-) mice following
intravenous administration of vehicle (KO); rhNaGlu gallus at a
dosage concentration of 6.25 mg/kg; or rhNaGlu gallus at a dosage
concentration of 27 mg/kg. Wild type (WT) mice were untreated.
[0060] FIG. 17 depicts heparan sulfate substrate levels (ag/mg
tissue) in the brain of naglu (.sup.-/-) mice following intravenous
administration of vehicle (KO); rhNaGlu gallus at a dosage
concentration 6.25 mg/kg; or rhNaGlu gallus at a dosage
concentration of 27 mg/kg. Wild type (WT) mice were untreated.
[0061] FIG. 18 depicts heparan sulfate substrate levels (ag/mg
tissue) in the liver of naglu (.sup.-/-) mice following intravenous
administration of vehicle (KO); rhNaGlu gallus at a dosage
concentration of 6.25 mg/kg; or rhNaGlu gallus at a dosage
concentration of 27 mg/kg. Wild type (WT) mice were untreated.
[0062] FIG. 19 depicts heparan sulfate substrate levels (ag/mg
tissue) in the brain of naglu (.sup.-/-) mice following intrathecal
administration of vehicle (KO) or rhNaGlu gallus at a dosage
concentration of 0.31 mg/kg. Wild type (WT) mice were
untreated.
DETAILED DESCRIPTION OF THE INVENTION
[0063] The present invention provides compositions comprising
recombinant human NaGlu protein (rhNaGlu) useful for therapy, for
example, in the treatment of NaGlu associated diseases, e.g.,
Sanfilippo Syndrome B. The present invention is based on a
discovery that the rhNaGlu protein described herein contains
sufficient amount of oligosaccharides (e.g., mannose and
phosphorylated mannose (i.e., M6P)) to allow efficient cellular
uptake via mannose and/or M6P receptor-mediated endocytosis and be
correctly targeted into human cells. Since the rhNaGlu of the
invention is more efficiently taken up into a human cell, the
rhNaGlu of the invention exhibits increased enzymatic activity as
compared to NaGlu proteins produced and secreted from unmodified
mammalian cells that are not designed to produce specific
glycosylation. Additionally, the rhNaGlu described herein has one
or more glycosylation patterns that allow the rhNaGlu to
efficiently cross the blood brain barrier (BBB) when administered
intravenously. The increased cellular uptake of the rhNaGlu protein
of the invention minimizes the need for large and frequent dosing,
thereby greatly reducing the potential risk of immunogenicity.
[0064] Some of the definitions and abbreviations used herein
include the following: aa, amino acid(s); bp, base pair(s); CDS,
coding sequence cDNA, DNA complementary to an RNA; GalNac,
N-acetylgalactosamine; Gal, galactose; GlcNac, N-acetylglucosamine;
nt, nucleotide(s); kb, 1,000 base pairs; .mu.g, microgram; mL,
milliliter; ng, nanogram; and nt, nucleotide.
[0065] Certain definitions are set forth herein to illustrate and
define the meaning and scope of the various terms used to describe
the invention herein.
[0066] The term "avian" as used herein refers to any species,
subspecies or strain of organism of the taxonomic class ava, such
as, but not limited to, chicken, turkey, duck, goose, quail,
pheasants, parrots, finches, hawks, crows and ratites including
ostrich, emu and cassowary. The term includes the various known
strains of Gallus gallus, or chickens, (for example, White Leghorn,
Brown Leghorn, Barred-Rock, Sussex, N.H., Rhode Island,
Ausstralorp, Minorca, Amrox, California Gray, Italian
Partridge-colored), as well as strains of turkeys, pheasants,
quails, duck, ostriches and other poultry commonly bred in
commercial quantities.
[0067] The phrases "based on" and "derived from" typically mean
obtained from, in whole or in part. For example, a retroviral
vector being based on or derived from a particular retrovirus or
based on a nucleotide sequence of a particular retrovirus mean that
the genome of the retroviral vector contains a substantial portion
of the nucleotide sequence of the genome of the particular
retrovirus. The substantial portion can be a particular gene or
nucleotide sequence such as the nucleotide sequence encoding the
gag, pol and/or env proteins or other structural or functional
nucleotide sequence of the virus genome such as sequences encoding
the long terminal repeats (LTRs) or can be substantially the
complete retrovirus genome, for example, most (e.g., more than 60%
or more than 70% or more than 80% or more than 90%) or all of the
retrovirus genome, as will be apparent from the context in the
specification as the knowledge of one skilled in the art. Examples
of retroviral vectors that are based on or derived from a
retrovirus are the NL retroviral vectors (e.g., NLB) which are
derived from the avian leukosis retrovirus ("ALV") as disclosed in
Cosset et al., Journal of Virology (1991) vol. 65, p 3388-3394.
[0068] The term "coding sequence" and "coding region" as used
herein refer to nucleotide sequences and nucleic acid sequences,
including both RNA and DNA, that encode genetic information for the
synthesis of an RNA, a protein, or any portion of an RNA or
protein.
[0069] Nucleotide sequences that are not naturally part of a
particular organism's genome or are introduced at a non-native site
in the organism's genome are referred to as "foreign" nucleotide
sequences, "heterologous" nucleotide sequences, "recombinant"
nucleotide sequences or "exogenous" nucleotide sequences. In
addition, a nucleotide sequence that has been isolated and then
reintroduced into the same type (e.g., same species) of organism is
not considered to be a naturally occurring part of a particular
organism's genome and is therefore considered exogenous or
heterologous. "Heterologous proteins" or "exogenous proteins" can
be proteins encoded by foreign, heterologous or exogenous
nucleotide sequences and therefore are often not naturally
expressed in a cell of the host organism.
[0070] As used herein, the terms "exogenous," "heterologous" and
"foreign" with reference to nucleic acids, such as DNA and RNA, are
used interchangeably and refer to nucleic acid that does not occur
naturally as part of a chromosome, a genome or cell in which it is
present or which is found in a location(s) and/or in amounts that
differ from the location(s) and/or amounts in which it occurs in
nature. It can be nucleic acid that is not endogenous to the
genome, chromosome or cell and has been exogenously introduced into
the genome, chromosome or cell. Examples of heterologous DNA
include, but are not limited to, DNA that encodes a gene product or
gene product(s) of interest, for example, for production of an
encoded protein. Examples of heterologous DNA include, but are not
limited to, DNA that encodes traceable marker proteins, DNA that
encodes therapeutic proteins. The terms "heterologous" and
"exogenous" can refer to a biomolecule such as a nucleic acid or a
protein which is not normally found in a certain cell, tissue or
substance produced by an organism or is not normally found in a
certain cell, tissue or substance produced by an organism in an
amount or location the same as that found to occur naturally. For
example, a protein that is heterologous or exogenous to an egg is a
protein that is not normally found in the egg.
[0071] The term "construct" as used herein refers to a linear or
circular nucleotide sequence such as DNA that has been assembled
from more than one segments of nucleotide sequence which have been
isolated from a natural source or have been chemically synthesized,
or combinations thereof.
[0072] The term "complementary" as used herein refers to two
nucleic acid molecules that can form specific interactions with one
another. In the specific interactions, an adenine base within one
strand of a nucleic acid can form two hydrogen bonds with thymine
within a second nucleic acid strand when the two nucleic acid
strands are in opposing polarities. Also in the specific
interactions, a guanine base within one strand of a nucleic acid
can form three hydrogen bonds with cytosine within a second nucleic
acid strand when the two nucleic acid strands are in opposing
polarities. Complementary nucleic acids as referred to herein, can
further comprise modified bases wherein a modified adenine may form
hydrogen bonds with a thymine or modified thymine, and a modified
cytosine may form hydrogen bonds with a guanine or a modified
guanine.
[0073] The term "expressed" or "expression" as used herein refers
to the transcription of a coding sequence to yield an RNA molecule
at least complementary in part to a region of one of the two
nucleic acid strands of the coding sequence. The term "expressed"
or "expression" as used herein can also refer to the translation of
an mRNA to produce a protein or peptide.
[0074] The term "expression vector" as used herein refers to a
nucleic acid vector that comprises a gene expression controlling
region, such as a promoter or promoter component, operably linked
to a nucleotide sequence encoding at least one polypeptide.
[0075] The term "fragment" as used herein can refer to, for
example, an at least about 10, 20, 50, 75, 100, 150, 200, 250, 300,
500, 1000, 2000, 5000, 6,000, 8,000, 10,000, 20,000, 30,000,
40,000, 50,000 or 60,000 nucleotide long portion of a nucleic acid
that has been constructed artificially (e.g., by chemical
synthesis) or by cleaving a natural product into multiple pieces,
using restriction endonucleases or mechanical shearing, or
enzymatically, for example, by PCR or any other polymerizing
technique known in the art, or expressed in a host cell by
recombinant nucleic acid technology known to one of skill in the
art. The term "fragment" as used herein can also refer to, for
example, an at least about 5, 10, 15, 20, 25, 30, 40, or 50 amino
acid residues less than a full length amino acid sequence for NaGlu
(i.e., amino acid sequence 24-743 of SEQ ID NO: 1), which portion
is cleaved from a naturally occurring amino acid sequence by
proteolytic cleavage by at least one protease, or is a portion of
the naturally occurring amino acid sequence synthesized by chemical
methods or using recombinant DNA technology (e.g., expressed from a
portion of the nucleotide sequence encoding the naturally occurring
amino acid sequence) known to one of skill in the art. "Fragment"
may also refer to a portion, for example, of about 50%, about 60%,
about 70%, about 80%, about 90%, about 95% or about 99% of a
particular nucleotide sequence or amino acid sequence.
[0076] "Functional portion" and "functional fragment" can be used
interchangeably and as used herein mean a portion or fragment of a
whole capable of performing, in whole or in part, a function of the
whole. For example, a biologically functional portion of a molecule
means a portion of the molecule that performs a biological function
of the whole or intact molecule. Functional portions may be of any
useful size. For example, a functional fragment may range in size
from about 20 bases in length to a length equal to the entire
length of the specified sequence minus one nucleotide. In another
example, a functional fragment may range in size from about 50
bases in length to a length equal to the entire length of the
specified sequence minus one nucleotide. In another example, a
functional fragment may range in size from about 50 bases in length
to about 20 kb in length. In another example, a functional fragment
may range in size from about 500 bases in length to about 20 kb in
length. In another example, a functional fragment may range in size
from about 1 kb in length to about 20 kb in length. In another
example, a functional fragment may range in size from about 0.1 kb
in length to about 10 kb in length. In another example, a
functional fragment may range in size from about 20 bases kb in
length to about 10 kb in length.
[0077] The term "fully transgenic" or "germline transgenic" refers
to an animal such as an avian that contains at least one copy of a
transgene in essentially all of its cells.
[0078] The term "gene expression controlling region" as used herein
refers to nucleotide sequences that are associated with a coding
sequence and which regulate, in whole or in part, expression of the
coding sequence, for example, regulate, in whole or in part, the
transcription of the coding sequence. Gene expression controlling
regions may be isolated from a naturally occurring source or may be
chemically synthesized and can be incorporated into a nucleic acid
vector to enable regulated transcription in appropriate cells. The
"gene expression controlling regions" may precede, but is not
limited to preceding, the region of a nucleic acid sequence that is
in the region 5' of the end of a coding sequence that may be
transcribed into mRNA.
[0079] As used herein, "host cells" refers to cells that harbor
vectors constructed using recombinant DNA techniques and encoding
at least one heterologous gene.
[0080] The term "isolated nucleic acid" as used herein covers, for
example, (a) a DNA which has the sequence of part of a naturally
occurring genomic molecule but is not flanked by at least one of
the sequences that flank that part of the molecule in the genome of
the species in which it naturally occurs; (b) a nucleic acid which
has been incorporated into a vector or into the genomic DNA of a
prokaryote or eukaryote in a manner such that the resulting vector
or genomic DNA is not identical to naturally occurring DNA from
which the nucleic acid was obtained; (c) a separate molecule such
as a cDNA, a genomic fragment, a fragment produced by polymerase
chain reaction (PCR), ligase chain reaction (LCR) or chemical
synthesis, or a restriction fragment; (d) a recombinant nucleotide
sequence that is part of a hybrid gene, i.e., a gene encoding a
fusion protein, and (e) a recombinant nucleotide sequence that is
part of a hybrid sequence that is not naturally occurring. Isolated
nucleic acid molecules of the present invention can include, for
example, natural allelic variants as well as nucleic acid molecules
modified by nucleotide deletions, insertions, inversions, or
substitutions.
[0081] The term "nucleic acid" as used herein refers to any linear
or sequential array of nucleotides and nucleosides, for example
cDNA, genomic DNA, mRNA, tRNA, oligonucleotides, oligonucleosides
and derivatives thereof. For ease of discussion, non-naturally
occurring nucleic acids may be referred to herein as constructs.
Nucleic acids can include bacterial plasmid vectors including
expression, cloning, cosmid and transformation vectors such as,
animal viral vectors such as, but not limited to, modified
adenovirus, influenza virus, polio virus, pox virus, retroviruses
such as avian leukosis virus (ALV) retroviral vector, a murine
leukemia virus (MLV) retroviral vector, and a lentivirus vector,
and the like and fragments thereof. In addition, the nucleic acid
can be an LTR of an avian leukosis virus (ALV) retroviral vector, a
murine leukemia virus (MLV) retroviral vector, or a lentivirus
vector and fragments thereof. Nucleic acids can also include NL
vectors such as NLB, NLD and NLA and fragments thereof and
synthetic oligonucleotides such as chemically synthesized DNA or
RNA. Nucleic acids can include modified or derivatized nucleotides
and nucleosides such as, but not limited to, halogenated
nucleotides such as, but not only, 5-bromouracil, and derivatized
nucleotides such as biotin-labeled nucleotides.
[0082] As used herein, the terms "glycan," "glycan structure,"
"glycan moiety," "oligosaccharide," "oligosaccharide structure,"
"glycosylation pattern," "glycosylation profile," and
"glycosylation structure" have essentially the same meaning and
each refers to one or more structures which are formed from sugar
residues and are attached to glycosylated protein such as human
NaGlu. For example, "N-glycan" or "N-linked glycan" refers to a
glycan structure attached to a nitrogen of asparagine or arginine
side-chain of the glycosylated protein. "O-glycan" or "O-linked
glycan" refers to a glycan structure attached to the hydroxyl
oxygen of serine, threonine, tyrosine, hydroxylysine, or
hydroxyproline side chain of the glycosylate protein.
[0083] The term "vector" and "nucleic acid vector" as used herein
refers to a natural or synthetic single or double stranded plasmid
or viral nucleic acid molecule that can be transfected or
transformed into cells and replicate independently of, or within,
the host cell genome. A circular double stranded vector can be
linearized by treatment with an appropriate restriction enzyme
based on the nucleotide sequence of the vector. A nucleic acid can
be inserted into a vector by cutting the vector with restriction
enzymes and ligating the desired pieces together, as is understood
in the art. A typical vector can be comprised of the following
elements operatively linked at appropriate distances for allowing
functional gene expression: replication origin, promoter, enhancer,
5' mRNA leader sequence, ribosomal binding site, nucleic acid
cassette, termination and polyadenylation sites, and selectable
marker sequences. One or more of these elements can be omitted in
specific applications. The nucleic acid cassette can include a
restriction site for insertion of the nucleic acid sequence to be
expressed. In a functional vector the nucleic acid cassette
contains the nucleic acid sequence to be expressed including
translation initiation and termination sites. An intron optionally
can be included in the construct, for example, 5' to the coding
sequence. A vector is constructed so that the particular coding
sequence is located in the vector with the appropriate regulatory
sequences, the positioning and orientation of the coding sequence
with respect to the control sequences being such that the coding
sequence is transcribed under the "control" of the control or
regulatory sequences. Modification of the sequences encoding the
particular protein of interest can be desirable to achieve this
end. For example, in some cases it can be necessary to modify the
sequence so that it can be attached to the control sequences with
the appropriate orientation, or to maintain the reading frame. The
control sequences and other regulatory sequences can be ligated to
the coding sequence prior to insertion into a vector.
Alternatively, the coding sequence can be cloned directly into an
expression vector which already contains the control sequences and
an appropriate restriction site which is in reading frame with and
under regulatory control of the control sequences.
[0084] The term "operably linked" refers to an arrangement of
elements wherein the components so described are configured so as
to perform their usual function. Gene expression controlling
regions or promoter(s) (e.g., promoter components) operably linked
to a coding sequence are capable of effecting the expression of the
coding sequence. The controlling sequence(s) or promoter need not
be contiguous with the coding sequence, so long as they function to
direct the expression thereof. Thus, for example, intervening
untranslated yet transcribed sequences can be present between a
promoter sequence and the coding sequence and the promoter sequence
can still be considered "operably linked" to the coding
sequence.
[0085] "Overexpression", as used herein, refers to the production
of a gene product in transgenic organisms that exceeds levels of
production in normal or non-transformed organisms.
[0086] The term "oviduct" or "oviduct tissue" refers to a tissue of
an avian oviduct, such as the magnum, e.g., tubular gland cells,
where proteins are produced with N-linked oligosaccharides that
contain increased amounts of mannose and mammose-6-phosphate (M6P)
and substantially reduced amounts of galactose and/or sialic acid
relative to that of proteins produced in other tissue of the avian
such as liver or kidney tissue.
[0087] The term "oviduct-specific promoter" as used herein refers
to promoters and promoter components which are functional, i.e.,
provide for transcription of a coding sequence, to a large extent,
for example, primarily (i.e., more than 50% of the transcription
product produced in the animal by a particular promoter type being
produced in oviduct cells) or exclusively in oviduct cells of a
bird. Examples of oviduct specific promoters include, but are not
limited to, ovalbumin promoter, ovomucoid promoter, ovoinhibitor
promoter, lysozyme promoter and ovotransferrin promoter and
functional portions of these promoters, e.g., promoter components.
By limiting the expression of NaGlu protein to the magnum using
oviduct specific promoters, deleterious physiological effects to
the bird as result of expression of these enzymes in other tissues
of the bird can be minimized.
[0088] The terms "percent sequence identity," "percent identity,"
"% identity," "percent sequence homology," "percent homology," "%
homology" and "percent sequence similarity" can each refer to the
degree of sequence matching between two nucleic acid sequences or
two amino acid sequences. Such sequence matching can be determined
using the algorithm of Karlin & Altschul (1990) Proc. Natl.
Acad. Sci. 87: 2264-2268, modified as in Karlin & Altschul
(1993) Proc. Natl. Acad. Sci. 90: 5873-5877. Such an algorithm is
incorporated into the NBLAST and XBLAST programs of Altschul et al.
(1990) T. Mol. Biol. Q15: 403-410. BLAST nucleotide searches are
performed with the NBLAST program, score=100, wordlength=12, to
obtain nucleotide sequences homologous to a nucleic acid molecule
of the invention. BLAST protein searches are performed with the
XBLAST program, score=50, wordlength=3, to obtain amino acid
sequences homologous to a reference amino acid sequence. To obtain
gapped alignments for comparison purposes, Gapped BLAST is utilized
as described in Altschul et al. (1997) Nucl. Acids Res. 25:
3389-3402. When utilizing BLAST and Gapped BLAST programs, the
default parameters of the respective programs (e.g., XBLAST and
NBLAST) are used. Other algorithms, programs and default settings
may also be suitable such as, but not only, the GCG-Sequence
Analysis Package of the U.K. Human Genome Mapping Project Resource
Centre that includes programs for nucleotide or amino acid sequence
comparisons. A sequence may be at least 50%, 60%, 70%, 75%, 80%,
85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more
identical to another sequence, e.g., the NaGlu protein sequence
identified herein.
[0089] The term "avian derived" refers to a composition or
substance produced by or obtained from a bird, poultry or avian.
"Avian" refers to birds that can be kept as livestock, including
but not limited to, chickens, duck, turkey, quail and ratites. For
example, "avian derived" can refer to chicken derived, turkey
derived and/or quail derived.
[0090] The terms "polynucleotide," "oligonucleotide", "nucleotide
sequence" and "nucleic acid sequence" can be used interchangeably
herein and include, but are not limited to, coding sequences, i.e.,
polynucleotide(s) or nucleic acid sequence(s) which are transcribed
and translated into polypeptide in vitro or in vivo when placed
under the control of appropriate regulatory or control sequences;
controlling sequences, e.g., translational start and stop codons,
promoter sequences, ribosome binding sites, polyadenylation
signals, transcription factor binding sites, transcription
termination sequences, upstream and downstream regulatory domains,
enhancers, silencers, DNA sequences to which a transcription
factor(s) binds and alters the activity of a gene's promoter either
positively (induction) or negatively (repression) and the like. No
limitations as to length or to synthetic origin are suggested by
the terms described herein.
[0091] As used herein the terms "polypeptide" and "protein" refer
to a polymer of amino acids, for example, three or more amino
acids, in a serial array, linked through peptide bonds. The term
"polypeptide" includes proteins, protein fragments, protein
analogues, oligopeptides and the like. The term "polypeptides"
includes polypeptides as defined above that are encoded by nucleic
acids, produced through recombinant technology (e.g., isolated from
a transgenic bird), or synthesized. The term "polypeptides` further
contemplates polypeptides as defined above that include chemically
modified amino acids or amino acids covalently or noncovalently
linked to labeling ligands.
[0092] The term "promoter" as used herein refers to a DNA sequence
useful to initiate transcription by an RNA polymerase in an avian
cell. A "promoter component" is a DNA sequence that can, by itself
or in combination with other DNA sequences, effect or facilitate
transcription. Promoter components can be functional fragments of
promoters.
[0093] The terms "recombinant nucleic acid" and "recombinant DNA"
as used herein refer to combinations of at least two nucleic acid
sequences that are not naturally found in a eukaryotic or
prokaryotic cell. The nucleic acid sequences may include, but are
not limited to, nucleic acid vectors, gene expression regulatory
elements, origins of replication, suitable gene sequences that when
expressed confer antibiotic resistance, protein-encoding sequences
and the like. The term "recombinant polypeptide" is meant to
include a polypeptide produced by recombinant DNA techniques such
that it is distinct from a naturally occurring polypeptide either
in its location, purity or structure. Generally, such a recombinant
polypeptide will be present in a cell in an amount different from
that normally observed in nature.
[0094] As used herein, the term "regulatory" sequences or elements
include promoters, enhancers, terminators, stop codons, and other
elements that can control gene expression.
[0095] A "retrovirus", "retroviral particle," "transducing
particle," or "transduction particle" refers to a
replication-defective or replication-competent virus capable of
transducing non-viral DNA or RNA into a cell.
[0096] A "SIN vector" refers to a self-inactivating vector. In
particular, a SIN vector is a retroviral vector having an altered
genome such that upon integration into genomic DNA of the target
cell (e.g., avian embryo cells), the 5' LTR of the integrated
retroviral vector will not function as a promoter. For example, a
portion or all of the nucleotide sequence of the retroviral vector
that results in the U3 region of the 5' LTR of the retroviral
vector once integrated can be deleted or altered in order to reduce
or eliminate promoter activity of the 5' LTR. In certain examples,
deletion of the CAAT box and/or the TAATA box from U3 of the 5' LTR
can result in a SIN vector, as is understood in the art.
[0097] The term "sense strand" as used herein refers to a single
stranded DNA molecule from a genomic DNA that can be transcribed
into RNA and translated into the natural polypeptide product of the
gene. The term "antisense strand" as used herein refers to the
single strand DNA molecule of a genomic DNA that is complementary
with the sense strand of the gene.
[0098] A "therapeutic protein" or "pharmaceutical protein" is a
substance that, in whole or in part, makes up a drug. In
particular, "therapeutic proteins" and "pharmaceutical proteins"
include an amino acid sequence which in whole or in part makes up a
drug.
[0099] The terms "promoter," "transcription regulatory sequence"
and "promoter component" as used herein refer to nucleotide which
regulates the transcriptional expression of a coding sequence.
Exemplary transcription regulatory sequences include enhancer
elements, hormone response elements, steroid response elements,
negative regulatory elements, and the like. The "transcription
regulatory sequence" can be isolated and incorporated into a vector
to enable regulated transcription in appropriate cells of portions
of the vector DNA. The "transcription regulatory sequence" can
precede, but is not limited to, the region of a nucleic acid
sequence that is in the region 5' of the end of a protein coding
sequence that is transcribed into mRNA. Transcriptional regulatory
sequence can also be located within a protein coding region, for
example, in regions of a gene that are identified as "intron"
regions.
[0100] The terms "transformation" and "transfection" as used herein
refer to the process of inserting a nucleic acid into a host. Many
techniques are well known to those skilled in the art to facilitate
transformation or transfection of a nucleic acid into a prokaryotic
or eukaryotic organism. These methods involve a variety of
techniques, such as treating the cells with certain concentrations
of salt, for example, but without limitation, a calcium or
magnesium salt, or exposing the cells to an electric field,
detergent, or liposome material, to render the host cell competent
for the uptake of the nucleic acid molecules.
[0101] As used herein, a "transgenic animal" is any non-human
animal, such as an avian species, including the chicken, in which
one or more of the cells of the animal contain heterologous nucleic
acid introduced by way of human intervention, such as by transgenic
techniques known in the art (see, for example, U.S. patent
publication No. 2007/0243165, published Oct. 18, 2007, the
disclosure of which is incorporated in its entirety herein by
reference) including those disclosed herein. The nucleic acid is
introduced into an animal, directly or indirectly by introduction
into a cell (e.g., egg or embryo cell) by way of deliberate genetic
manipulation, such as by microinjection or by infection with a
recombinant virus. The term genetic manipulation does not include
classical cross-breeding, or in vitro fertilization, but rather is
directed to the introduction of a recombinant DNA molecule. This
molecule can be integrated within a chromosome, or it may be
extrachromosomally replicating DNA. In the typical transgenic
animal, the transgene can cause cells to express a recombinant form
of the target protein or polypeptide. The terms "chimeric animal"
or "mosaic animal" are used herein to refer to animals in which a
transgene is found, or in which the recombinant nucleotide sequence
is expressed, in some but not all cells of the animal. A germ-line
chimeric animal contains a transgene in its germ cells and can give
rise to an offspring transgenic animal in which most or all cells
of the offspring will contain the transgene.
[0102] As used herein, the term "transgene" means a nucleic acid
sequence (encoding, for example, a human NaGlu protein) that is
partly or entirely heterologous, i.e., foreign, to the animal or
cell into which it is introduced, or, is partly or entirely
homologous to an endogenous gene of the transgenic animal or cell
into which it is introduced, but which is designed to be inserted,
or is inserted, into the animal or cell genome in such a way as to
alter the genome of the organism into which it is inserted (e.g.,
it is inserted at a location which differs from that of the natural
gene or its insertion results in a knockout).
[0103] As used herein, the term "enzyme replacement therapy (ERT)"
refers to a therapeutic strategy for correcting an enzyme
deficiency in a subject by administering the missing enzyme to a
subject. For lysosomal enzyme replacement therapy to be effective,
the therapeutic enzyme must be delivered to lysosomes in the
appropriate cells in tissues where the storage defect is
manifested. In one embodiment, the enzyme may be administered to
the subject intravenously, intrathecally, intracerebrally,
intraventricularly, or intraparenchymaly. In one embodiment, the
enzyme is able to cross the blood brain barrier (BBB). Without
intending to be limited by mechanism, it is believed that as the
blood perfuses patient tissues, enzyme is taken up by cells and
transported to the lysosome, where the enzyme acts to eliminate
material that has accumulated in the lysosomes due to the enzyme
deficiency.
[0104] I. Composition of NaGlu
[0105] The present invention provides novel compositions of
recombinant human NaGlu (rhNaGlu or NaGlu) (amino acid sequence
24-743 set forth in SEQ ID NO: 1) having patterns of glycosylation
that confer an increased cellular uptake and an increased
subcellular activity which are particularly useful for therapy, for
example, in the treatment of Sanfilippo Syndrome B
(mucopolysaccharidosis (MPS) IIIB).
[0106] In some aspects, the composition can be an isolated mixture
of rhNaGlu comprising the amino acid sequence 24-743 of SEQ ID NO:
1. In one embodiment, the mixture contains a sufficient amount of
rhNaGlu having at least one glycan structure that contains
phosphorylated mannose (e.g., M6P) or mannose such that the rhNaGlu
containing M6P or mannose is internalized into a human cell
deficient in NaGlu and restores at least 50% of NaGlu activity
observed in a wild-type human cell of the same type that actively
expresses endogenous NaGlu. In one aspect, at least 10%, 20%, 30%,
40%, 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93% 94%, 95%, 96%, 97%, 98%
or 99% of rhNaGlu in the mixture contains at least one glycan
structure having phosphorylated mannose and/or mannose. In one
embodiment, at least 10% of rhNaGlu in the mixture contains at
least one glycan structure having phosphorylated mannose and/or
mannose. In one embodiment, at least 20% of rhNaGlu in the mixture
contains at least one glycan structure having phosphorylated
mannose and/or mannose. In one embodiment, at least 30% of rhNaGlu
in the mixture contains at least one glycan structure having
phosphorylated mannose and/or mannose. In one embodiment, at least
30% of rhNaGlu in the mixture contains at least one glycan
structure having phosphorylated mannose and/or mannose. In one
embodiment, at least 40% of rhNaGlu in the mixture contains at
least one glycan structure having phosphorylated mannose and/or
mannose. In one embodiment, at least 50% of rhNaGlu in the mixture
contains at least one glycan structure having phosphorylated
mannose and/or mannose. In one embodiment, at least 60% of rhNaGlu
in the mixture contains at least one glycan structure having
phosphorylated mannose and/or mannose.
[0107] In some aspects, the NaGlu contains one or more N-linked
glycan structure. The NaGlu contains at least one phosphorylated
mannose (e.g., M6P or bis-M6P) which allows the protein to be
recognized by the Mannose 6-phosphate receptor (M6P receptor), and
subsequently taken up into a human cell, including but not limited
to, a skin fibroblast, an endothelial, a neuronal cell, a
hepatocyte, a macrophage or any cell that expresses M6P receptor on
the cell surface via M6P receptor-mediated endocytosis. In one
embodiment, the NaGlu contains at least one mannose (Man). In
another embodiment, the NaGlu contains at least one
N-acetylglucosamine (GlcNAc).
[0108] In some aspects, the NaGlu contains a glycan structure
comprising a phosphorylated mannose (M6P). As used herein, M6P can
encompass any phosphorylated mannose residue and includes mono- and
bis-phosphorylated mannose. In one embodiment, the M6P is present
at a concentration that is about 1, about 2, about 3, about 4,
about 5 or about 6 mole(s) per mole of protein. In one embodiment,
the NaGlu contains M6P at a concentration that is about 2, about 3,
about 4, or about 5 moles per mole of protein. In one embodiment,
the NaGlu contains M6P at a concentration that is about 2 moles per
mole of protein. In one embodiment, the NaGlu contains M6P at a
concentration that is about 3 moles per mole of protein. In one
embodiment, the NaGlu contains M6P at a concentration that is about
4 moles per mole of protein. In one embodiment, the NaGlu contains
M6P at a concentration that is about 5 moles per mole of protein.
In one embodiment, the NaGlu contains M6P at a concentration that
is about 6 moles per mole of protein.
[0109] In some aspects, the rhNaGlu contains a sufficient amount of
M6P for cellular uptake into a human cell having a M6P receptor on
the cell surface via M6P receptor-mediated endocytosis. In one
embodiment, a sufficient amount of M6P for uptake into a human cell
is about 1, 2, 3, 4, 5 or 6 moles per mole of protein. The rhNaGlu
can be internalized into a human cell deficient in NaGlu such that
the internalized protein fully (100% or more) restores a normal
level of NaGlu activity in the human cell deficient in NaGlu. In
one embodiment, the internalized rhNaGlu protein fully restores a
normal level of NaGlu activity in the human cell at a concentration
that is at least 0.5, 0.6, 0.7, 0.8, 0.9 or 1.0 .mu.g/mL. In one
embodiment, the internalized protein fully restores a normal level
of NaGlu activity in the human cell deficient in NaGlu at a
concentration that is at least 2, 3, 4, 5, 6, 7, 8, 9 or 10
.mu.g/mL. In one embodiment, the internalized protein fully
restores a normal level of NaGlu activity in the human cell at a
concentration that is at least 20, 30, 40, 50, 60, 70, 80, 90 or
100 .mu.g/mL. As used herein, the normal level of NaGlu activity is
a level of NaGlu activity measured in a wild-type human cell of the
same type that actively expresses a normal NaGlu enzyme.
[0110] In some aspects, the rhNaGlu can be internalized into a
human cell deficient in NaGlu such that the protein restores at
least about 50%, about 60%, about 70%, about 80%, about 90% or
about 95% of NaGlu activity of a normal human cell of the same
type. In some embodiments, the rhNaGlu can be internalized into a
human cell deficient in NaGlu such that the internalized rhNaGlu
provides a higher enzymatic activity than that observed in a normal
human cell of the same type. In one embodiment, the rhNaGlu is
internalized into a human cell deficient in NaGlu such that the
internalized rhNaGlu provides about 2, about 3, about 4, about 5,
about 6, about 7, about 8, about 9 and about 10-fold higher
activity than that observed in a normal human cell of the same
type. In one embodiment, the rhNaGlu is internalized into a human
cell deficient in NaGlu such that the internalized rhNaGlu provides
about 15, about 20, about 25, about 30, about 40, 0, about 60,
about 70, about 80, about 90 or about 100-fold higher activity than
that observed in a normal human cell.
[0111] In one embodiment, the human cell deficient in NaGlu is any
human cell deficient in NaGlu that expresses one or more M6P
receptors on the cell surface. In one embodiment, the human cell
deficient in NaGlu is a human mucopolysaccharidosis (MPS) IIIB
fibroblast that accumulates heparan sulfate. In one embodiment, the
human cell deficient in NaGlu is a hepatocyte. In one embodiment,
the human cell deficient in NaGlu is a neuronal cell. In one
embodiment, the human cell deficient in NaGlu is an endothelial
cell. In one embodiment, the human cell deficient in NaGlu is a
macrophage.
[0112] In some aspects, uptake of rhNaGlu into a human cell is
inhibited by the presence of about 1, about 2, about 3, about 4,
about 5, about 6, about 7, about 8, about 9 or about 10 mM of
competing M6P monosaccharide. In some aspects, the cellular uptake
of rhNaGlu is inhibited by the presence of about 0.1, about 0.2,
about 0.3, about 0.4, about 0.5, about 0.6, about 0.7, about 0.8,
about 0.9 or about 1.0 mM of M6P monosaccharide. In one embodiment,
the cellular uptake of rhNaGlu is inhibited by the presence of
about 0.01, about 0.02, about 0.03, about 0.04, about 0.05, about
0.06, about 0.07, about 0.08, or about 0.09 mM of M6P
monosaccharide.
[0113] In some aspects, the rhNaGlu contains mannose in its glycan
structures at a concentration that is about 17, 18, 19, 20, 21, 22,
23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34 or 35 moles per mole
of protein. In one embodiment, the rhNaGlu has mannose at a
concentration that is about 20, 21, 22, 23, 24, 25, 26, 27, 28, 29
or 30 moles per mole of protein. The rhNaGlu contains mannose at a
concentration that is about 22, 23, 24, 25, 26, 27 or 28 moles per
mole of protein. The rhNaGlu contains mannose at a concentration
that is about 24 moles per mole of protein. The rhNaGlu protein
contains mannose at a concentration that is about 25 moles per mole
of protein. The rhNaGlu contains mannose at a concentration that is
about 26 moles per mole of protein. The rhNaGlu contains mannose at
a concentration that is about 27 moles per mole of protein. In one
embodiment, the rhNaGlu has mannose at a concentration that is
between about 20 and about 30 moles per mole of protein.
[0114] In some aspects, the rhNaGlu comprises N-acetylglucosamine
(GlcNAc). In one embodiment, the rhNaGlu contains GlcNAc at a
concentration that is between about 28 and about 42 moles per mole
of protein. In one embodiment, the NaGlu protein has GlcNAc at a
concentration that is between about 30 and about 40 moles per mole
of protein. In one embodiment, the NaGlu protein comprises GlcNAc
at a concentration that is between about 32 and about 38 moles per
mole of protein. In one embodiment, the NaGlu protein comprises
GlcNAc at a concentration that is between about 34 and about 36
moles per mole of protein. In one embodiment, the NaGlu protein has
GlcNAc at a concentration that is about 35 moles per mole of
protein. In one embodiment, the rhNaGlu protein contains GlcNAc at
a concentration that is about 30, 31, 32, 33, 34, 35, 36, 37, 38,
39 or 40 moles per mole of protein.
[0115] In some aspects, the rhNaGlu contains N-acetylgalactosamine
(GalNAc) and/or galactose (Gal). The presence of the GalNAc and Gal
typically indicates that the NaGlu may contain one or more O-linked
glycan structures which are added to the protein in the Golgi
compartment. Accordingly, the present invention optionally includes
a composition comprising a recombinant human NaGlu that contains
one or more O-linked glycan structure.
[0116] In one embodiment, the rhNaGlu contains galactose at a
concentration that is about 1, 2, 3, 4, 5, 6 or 7 moles per mole of
protein. In one embodiment, the rhNaGlu has galactose at a
concentration that is about 2, 3, 4, 5 or 6 moles per mole of
protein. In one embodiment, the rhNaGlu has galactose at a
concentration that is about 3 moles per mole of protein. In one
embodiment, the rhNaGlu has galactose at a concentration that is
about 4 moles per mole of protein.
[0117] In one embodiment, the NaGlu comprises at least one GalNAc
molecule per mole of protein. In one embodiment, the NaGlu
comprises GalNAc at a concentration that is about 1 or 2 moles per
mole of protein.
[0118] In one embodiment, the NaGlu contains no fucose. In yet
another embodiment, the NaGlu contains no glucose. In yet another
embodiment, rhNaGlu contains neither fucose nor glucose.
[0119] The present invention also contemplates compositions of
modified rhNaGlu proteins produced from modified nucleic sequences
of rhNaGlu. The modified nucleic acid sequences include deletions,
insertions, or substitutions of different nucleotides resulting in
a polynucleotide that encodes a functionally equivalent
polynucleotide or polypeptide. The encoded protein may also contain
deletions, insertions, or substitutions of amino acid residues that
produce a silent change and result in a functionally equivalent
protein or polypeptide. Deliberate amino acid substitutions can be
made on the basis of similarity in polarity, charge, solubility,
hydrophobicity, hydrophilicity, and/or the amphipathic nature of
the residues as long as the biological activity of the NaGlu is
retained. For example, negatively charged amino acids can include
aspartic acid and glutamic acid; positively charged amino acids can
include lysine and arginine; and amino acids with uncharged polar
head groups having similar hydrophilicity values can include
leucine, isoleucine, and valine; glycine and alanine; asparagine
and glutamine; serine and threonine; phenylalanine and
tyrosine.
[0120] In other aspects, the rhNaGlu can be modified such that it
contains an additional moiety or second peptide. Although
unmodified NaGlu protein may cross the blood brain barrier at a
high serum concentration, modifications of the protein can be
performed to increase the efficiency of central nervous system
(CNS) targeting. In one embodiment, transferrin receptor ligand
(TfRL) can be attached to human NaGlu at N- or C-terminus of NaGlu
protein. A non-limiting example of TrRL is THRPPMWSPVWP (SEQ ID
NO:5). In one embodiment, the transferrin receptor ligand can be
attached to human NaGlu C-terminus of the NaGlu protein. In another
embodiment, human NaGlu is fused to insulin-like growth factor
receptor (IGF2R) ligand at N- or C-terminus of the NaGlu protein.
In yet another embodiment, the NaGlu protein is fused to low
density lipoprotein (LDL) receptor ligand at N- or C-terminus of
the NaGlu protein. In one embodiment, the NaGlu protein is fused to
a stretch of five to ten consecutive acidic amino acid residues.
The acidic amino acid residues can include aspartic acid (D) or
glutamic acid (E).
[0121] In one embodiment, the rhNaGlu is produced in a transgenic
avian that contains a transgene encoding the NaGlu protein. In one
embodiment, the rhNaGlu is produced in an oviduct cell (e.g., a
tubular gland cell) of a transgenic avian (e.g., chicken (Gallus)).
In one embodiment, the rhNaGlu is glycosylated in the oviduct cell
(e.g., tubular gland cell) of the transgenic avian. In one
embodiment, the rhNaGlu has a glycosylation pattern resulting from
the rhNaGlu being produced in an oviduct cell of a transgenic
avian. In one embodiment, the rhNaGlu can be isolated and purified
from the content of the hard shell eggs laid by the transgenic
avian. In one embodiment, the rhNaGlu can be isolated and purified
from egg white of the transgenic avian.
[0122] The present invention also includes compositions of an
isolated mixture of NaGlu proteins, such as a mixture of one or
more fragments and full-length rhNaGlu (e.g., 24-743 set forth in
SEQ ID NO:1). In one embodiment, a substantial portion of the
mixture contains phosphorylated M6P. In one embodiment, at least
10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90% 95%, 97%, 98% or
99% of the rhNaGlu in the mixture contains M6P. In yet another
embodiment, at least 50% of the isolated rhNaGlu in the mixture
contains M6P. In yet another embodiment, at least 60% of the
isolated rhNaGlu in the mixture contains M6P. In yet another
embodiment, at least 70% of the isolated rhNaGlu in the mixture
contains M6P. In yet another embodiment, at least 80% of the
isolated rhNaGlu in the mixture contains M6P. In yet another
embodiment, at least 90% of the isolated rhNaGlu in the mixture
contains M6P. In yet another embodiment, at least 95% of the
isolated rhNaGlu in the mixture contains M6P. In yet another
embodiment, at least 96% of the isolated rhNaGlu in the mixture
contains M6P. In yet another embodiment, at least 97% of the
isolated rhNaGlu in the mixture contains M6P. In yet another
embodiment, at least 98% of the isolated rhNaGlu in the mixture
contains M6P. In yet another embodiment, at least 99% of the
isolated rhNaGlu in the mixture contains M6P.
[0123] Optionally, the rhNaGlu protein produced from an avian or
mammalian expression system (e.g., CHO, HEK293, or human skin
fibroblast cell-line) can be further modified to achieve a
favorable glycosylation pattern (i.e., an increased amount of M6P)
for cellular uptake while retaining the biological activity.
Additional terminal M6P can be introduced to the rhNaGlu by the
general methods applied to other hydrolases as described in U.S.
Pat. No. 6,679,165, U.S. Pat. No. 7,138,262, or U.S. Publication
No. 2009/0022702, the entire teachings of each of which are
incorporated herein by reference. For example, a highly
phosphorylated mannopyranosyl oligosaccharide compound can be
derivatized with a chemical compound containing a carbonyl-reactive
group, followed by oxidizing the rhNaGlu protein to generate
carbonyl (aldehyde) group on one glycan structure of the protein,
and reacting the oxidized NaGlu protein with the glycan with the
derivatized highly phosphorylated mannopyranosyl oligosaccharide
compound to form a new compound having hydrazine bond.
[0124] II. Vectors
[0125] Methods which are well-known to those skilled in the art can
be used to construct expression vectors containing sequences
encoding NaGlu and appropriate transcriptional and translational
control elements. These methods include in vitro recombinant DNA
techniques, synthetic techniques, and in vivo genetic
recombination. Such techniques are described, for example, in
Sambrook, J. et al. (1989) Molecular Cloning, A Laboratory Manual,
Cold Spring Harbor Press, Plainview, N.Y., and Ausubel, F. M. et
al. (1989) Current Protocols in Molecular Biology, John Wiley &
Sons, New York, N.Y., the entire teachings of which are
incorporated herein by reference.
[0126] A variety of expression vector/host systems can be utilized
to express nucleic acid sequences encoding rhNaGlu. These include,
but are not limited to, microorganisms such as bacteria transformed
with recombinant bacteriophage, plasmid, or cosmid DNA expression
vectors; yeast transformed with yeast expression vectors; insect
cell systems infected with virus expression vectors (e.g.,
baculovirus) or with bacterial expression vectors (e.g., Ti or
pBR322 plasmids); or mammalian cell culture systems (e.g., pTT22
vector). Non-limiting examples of the pTT22 vector containing human
NaGlu cDNA fused to a nucleic acid sequence encoding acidic amino
acid residue and TfRL are shown in FIGS. 11 and 12.
[0127] Polynucleotide and nucleic acid coding regions of the
present invention may be associated with additional coding regions
which encode secretory or signal peptides, which direct the
secretion of a polypeptide encoded by a polynucleotide of the
present invention. According to the signal hypothesis, proteins
secreted by vertebrate (e.g., avian or mammalian) cells have a
signal peptide or secretory leader sequence which is cleaved from
the mature protein once export of the growing protein chain across
the rough endoplasmic reticulum (ER) has been initiated. Those of
ordinary skill in the art are aware that polypeptides produced in
the ER by vertebrate cells generally have a signal peptide fused to
the N-terminus of the polypeptide, which is cleaved from the
complete or "full length" polypeptide to produce a secreted or
"mature" form of the polypeptide. In certain embodiments, the
native signal peptide, e.g., the MEAVAVAAAVGVLLLAGAGGAAG (1-23 of
SEQ ID NO:1) signal peptide of human NaGlu is used, or a functional
derivative of that sequence that retains the ability to direct the
secretion of the polypeptide that is operably associated with it.
Alternatively, a heterologous signal peptide (e.g., a heterologous
mammalian or avian signal peptide), or a functional derivative
thereof, may be used. For example, the wild-type leader sequence
may be substituted with the leader sequence of, for example, human
tissue plasminogen activator (tPA) or mouse
.beta.-glucuronidase.
[0128] The control elements or regulatory sequences can includes
those non-translated regions of the vector-enhancers, promoters, 5'
and 3' untranslated regions that interact with host cellular
proteins to carry out transcription and translation. Such elements
can vary in their strength and specificity. Depending on the vector
system and host cell utilized, any number of suitable transcription
and translation elements can be used. For example, when cloning in
bacterial systems, inducible promoters such as the hybrid lac-Z
promoter of the Bluescript.TM. phagemid (Stratagene, LaJolla,
Calif.) or pSport1.TM. plasmid (Gibco BRL) and the like can be
used. In mammalian cell systems, promoters from mammalian genes or
from mammalian viruses are preferred. If it is necessary to
generate a cell line that contains multiple copies of the sequence
encoding NaGlu, vectors based on SV40 or EBV can be also used with
an appropriate selectable marker such as puromycin and ampicillin
(see, e.g., FIGS. 11 and 12).
[0129] When the rhNaGlu is produced in a transgenic avian, the
present invention contemplates that the rhNaGlu sequence be placed
downstream of a promoter such that the sequence encoding the
rhNaGlu can be expressed in a tissue-specific manner in a
transgenic avian. For example, the promoter can be an
oviduct-specific promoter that is largely, but not entirely,
specific to the magnum, such as the oviduct-specific promoter,
including but not limited to, ovalbumin, lysozyme, conalbumin,
ovomucoid, ovomucoid, ovomucin and ovotransferrin promoters. In one
embodiment, the promoter is an ovalbumin promoter, a lysozyme
promoter, a conalbumin promoter, an ovomucoid promoter, an ovomucin
promoter and/or an ovotransferrin promoter or any functional
portion thereof.
[0130] Alternatively, a constitutive promoter can be used to
express the coding sequence of human NaGlu in an avian. In this
case, expression is not limited to the magnum; expression also
occurs in other tissues within the avian (e.g., blood). The use of
such a transgene, which includes a constitutive promoter and the
coding sequence of NaGlu, is also suitable for effecting or driving
the expression of a protein in the oviduct and the subsequent
secretion of the protein into the egg. In one embodiment, the
constitutive promoter can be, for example, a cytomegalovirus (CMV)
promoter, a rous-sarcoma virus (RSV) promoter, a murine leukemia
virus (MLV) promoter, and 3-actin promoter. In one embodiment, the
promoter is a CMV promoter, a MDOT promoter, a RSV promoter, a MLV
promoter, or a mouse mammary tumor virus (MMTV) promoter of any
functional portion thereof.
[0131] The invention also contemplates any useful fragment or
component of the promoters described herein. The promoter can be at
least one segment, fragment or component of a promoter region, such
as a segment of the ovalbumin, lysozyme, conalbumin, ovomucoid,
ovomucin, ovotransferrin, CMV, RSV or MLV promoter region. In a
preferred embodiment, the promoter is a segment of the
oviduct-specific promoter region which contains essential elements
to direct expression of the coding sequence in the tubular gland
cells. For example, included in the scope of the present invention
is a segment, portion or fragment of an oviduct-specific promoter
and/or condensing the critical regulatory elements of the
oviduct-specific promoter so that it retains sequences required for
expression in the tubular gland cells of the magnum of the oviduct.
In one embodiment, a segment of the ovalbumin promoter region is
used. This segment comprises the 5'-flanking region of the
ovalbumin gene.
[0132] A vector that contains a coding sequence for human NaGlu can
be used for transfecting blastodermal cells of an avian or
mammalian cell to generate stable integrations into the avian or
mammalian genome and to create a germline transgenic avian or
mammalian cell line. A non-limiting example of such vector is shown
in FIGS. 4A-D and 5. In the avian expression system, the human
NaGlu coding sequence is operably linked to a promoter in a
positional relationship to express the coding sequence in a
transgenic avian, particularly in the tubular gland cell of the
magnum of the avian oviduct, such that the recombinant human NaGlu
protein is expressed and deposited in egg white of a hard shell egg
laid by the transgenic avian. Additional suitable vectors and
methods to making vectors for expressing rhNaGlu in an avian system
are also disclosed in U.S. Pat. No. 6,730,822; U.S. Pat. No.
6,825,396; U.S. Pat. No. 6,875,588; U.S. Pat. No. 7,294,507; U.S.
Pat. No. 7,521,591; U.S. Pat. No. 7,534,929; U.S. Publication No.
2008/0064862A1; and U.S. Patent Publication No. 2006/0185024, the
entire teachings of which are incorporated herein by reference.
Non-limiting examples of other promoters which can be also useful
in the present invention include Pol III promoters (for example,
type 1, type 2 and type 3 Pol III promoters) such as H1 promoters,
U6 promoters, tRNA promoters, RNase MPR promoters and functional
portions of each of these promoters. Typically, functional
terminator sequences are selected for use in the present invention
in accordance with the promoter that is employed.
[0133] In one embodiment, the vector is a retroviral vector, in
which the coding sequence and the promoter are both positioned
between the 5' and 3' LTRs of the retroviral vector. In one useful
embodiment, the LTRs or retroviral vector is derived from an avian
leukosis virus (ALV), a murine leukemia virus (MLV) or a
lentivirus. One useful retrovirus for randomly introducing a
transgene into the avian genome is the replication-deficient ALV,
the replication-deficient MLV, or the replication-deficient
lentivirus.
[0134] The present invention also contemplates the use of
self-inactivating (SIN) vectors. SIN vectors can be useful for
increasing the quantity of human NaGlu produced in the oviduct of a
transgenic avian. This effect can be further enhanced when the SIN
vector does not contain any selectable marker cassette with a
functional promoter (SIN/SC negative vector). In one embodiment, a
SIN vector is a retroviral vector having altered genome so that the
5' LTR of the integrated retroviral vector does not function as a
promoter. In one particular embodiment, a portion or all of the
nucleotide sequence of the retroviral vector that results in the U3
region of the 5' LTR of the retroviral vector once integrated can
be deleted or altered in order to reduce or eliminate promoter
activity of the 5' LTR. A non-limiting example of SIN vector which
contains an ovalbumin promoter region fused to the coding sequence
of human rhNaGlu is shown in FIGS. 4A-D and 5. Functional
components of the vector are also tabulated in Table 1.
TABLE-US-00001 TABLE 1 Functional components in
pSIN-OV-1.1kb-I-rhNaGlu Functional components Nucleotide Sequence
in SEQ ID NO: 4 poly A site 634-639 Partial gag 692-945 LTR (RAV2)
1243-1588 Partial LTR (RAV2) 4691-4863 ALV CTE 4899-4986 1.1 kb
Ovalbumin promoter 5232-6363 DHS II 5334-5714 DHS I 6064-6364 Exon
L 6364-6410 Intron 1 6411-7999 NaGlu 8017-10248
[0135] Any of the vectors described herein can include a sequence
encoding a signal peptide that directs secretion of the protein
expressed by the vector's coding sequence from, for example, the
tubular gland cells of the oviduct of an avian. Where a recombinant
human NaGlu protein would not otherwise be secreted, the vector
containing the coding sequence is modified to comprise a DNA
sequence comprising about 60 bp encoding a signal peptide from, for
example, the lysozyme gene. The DNA sequence encoding the signal
peptide is inserted in the vector such that it is located at the
N-terminus of the rhNaGlu protein encoded by the DNA.
[0136] Further, the coding sequences of vectors used in any of the
methods of the present invention can be provided with a 3'
untranslated region (3' UTR) to confer stability to the RNA
produced. When a 3' UTR is added to a retroviral vector, the
orientation of the promoter, the coding sequence and the 3' UTR is
preferably reversed with respect to the direction of the 3' UTR, so
that the addition of the 3' UTR does not interfere with
transcription of the full-length genomic RNA. In one embodiment,
the 3' UTR may be that of the ovalbumin gene, lysozyme gene or any
3' UTR that is functional in a magnum cell, i.e., the SV40 late
region.
[0137] III. Transgenic Avians
[0138] Transgenes described herein can be introduced into avian
embryonic blastodermal cells to produce a transgenic chicken,
transgenic turkey, transgenic quail and other avian species that
carry the transgene encoding recombinant human NaGlu in the genome
of its germ-line tissue. In one aspect of the invention, a
transgenic avian that produces rhNaGlu is created by transduction
of embryonic blastodermal cells with replication-defective or
replication-competent retroviral particles carrying the transgene
between the 5' and 3' LTRs of the retroviral vector. For instance,
an avian leukosis virus (ALV) retroviral vector or a murine
leukemia virus (MLV) retroviral vector can be used. An RNA copy of
the modified retroviral vector packaged into viral particles can be
used to infect embryonic blastoderms which develop into transgenic
avians.
[0139] By the methods of the present invention, transgenes can be
introduced into embryonic blastodermal cells of various avian
species. For example, the methods can be applied to produce a
transgenic chicken, transgenic turkey, transgenic quail, transgenic
duct, and other avian species, that carry the transgene in the
genome of its germ-line tissue in order to produce proteins of the
invention. The blastodermal cells are typically stage VII-XII cells
as defined by Eyal-Giladi and Kochav (1976), or the equivalent
thereof. In a preferred embodiment, the blastoderm cells are at or
near stage X.
[0140] In one method of transfecting blastodermal cells, a packaged
retroviral-based vector can be used to deliver the vector into
embryonic blastodermal cells so that the vector is integrated into
the avian genome. Such viral particles (i.e., transduction
particles) are produced for the vector and titered to determine the
appropriate concentration that can be used to inject embryos. In
one embodiment, avian eggs are windowed according to the procedure
described in U.S. Pat. No. 5,897,998, the disclosure of which is
incorporated herein by reference in its entirety, and the eggs are
injected with transducing particles at or near stage X.
[0141] The transgenic avians of the invention which produce rhNaGlu
are developed from the blastodermal cells into which the vector has
been introduced. The resulting embryo is allowed to develop and the
chick allowed to mature. At this stage, the transgenic avian
produced from blastodermal cells is known as a founder and is
chimeric with respect to the cells carrying the transgene and is
referred to G0. G0 founder avians are typically chimeric for each
inserted transgene. That is, only some of the cells of the G0
transgenic bird contain the transgene. Some founders carry the
transgene in tubular gland cells in the magnum of their oviducts.
These avians express the rhNaGlu protein encoded by the transgene
in their oviducts. The NaGlu protein may also be expressed in other
tissues (e.g., blood) in addition to the oviduct. Some founders are
germ-line founders that carry the transgene in the genome of the
germ-line tissues, and may also carry the transgene in oviduct
magnum tubular gland cells that express the exogenous protein.
[0142] The transgenic avian can carry the transgene in its
germ-line providing transmission of the exogenous transgene to the
avian's offspring stably in a Mendelian fashion. The G0 generation
is typically hemizygous for the transgene encoding rhNaGlu. The G0
generation can be bred to non-transgenic animals to give rise to G1
transgenic offspring which are also hemizygous for the transgene
and contain the transgene in essentially all of the bird's cells.
The G1 hemizygous offspring can be bred to non-transgenic animals
giving rise to G2 hemizygous offspring or may be bred together to
give rise to G2 offspring homozygous for the transgene.
Substantially all of the cells of avians which are positive for the
transgene that are derived from G1 offspring contain the transgene.
In one embodiment, hemizygotic G2 offspring from the same line can
be bred to produce G3 offspring homozygous for the transgene. In
another embodiment, hemizygous G0 or G1 animals, for example, are
bred together to give rise to homozygous G1 offspring containing
two copies of the transgene(s) in each cell of the animal. These
are merely examples of certain useful breeding methods and the
present invention contemplates the employment of any useful
breeding method such as those known to individuals of ordinary
skill in the art.
[0143] IV. Production of rhNaGlu
[0144] The rhNaGlu can be produced using a transgenic avian that
contains in the genome a transgene encoding rhNaGlu. In one
embodiment, the transgenic avian is a germline transgenic chicken,
quail, duck or turkey. In one particularly useful embodiment, the
invention is drawn to the production of NaGlu which can be produced
in the oviduct of a chicken.
[0145] Production of rhNaGlu with or without modification in the
avian system (e.g., in the avian oviduct) is within the scope of
the invention. In one embodiment, the unmodified rhNaGlu comprises
the wild-type amino acid sequence (24-743 of SEQ ID NO: 1) with a
glycosylation structure (i.e., M6P) that enables efficient uptake
by human cells. In another embodiment, the modified protein can be
an rhNaGlu fusion protein having a glycosylation pattern (i.e.,
M6P) that enables efficient uptake by human cells.
[0146] A suitable avian vector that contains a nucleic acid
sequence encoding a NaGlu protein, operably linked to a
tissue-specific or constitutive promoter that drives expression of
the encoding sequence in the chicken oviduct are introduced into
chicken embryonic cells at or near stage X as described herein. The
transformed embryonic cells are incubated under conditions
conducive to hatching live chicks. Live chicks are nurtured into a
mature chimeric chicken which are mated with a non-transgenic
chicken naturally or via artificial insemination. A transgenic
chicken is identified by screening progeny for germline
incorporation of the protein encoding sequence. The transgenic
progeny can be mated with another transgenic or a non-transgenic
chicken to produce a fully germline transgenic hen that lays
eggs.
[0147] The rhNaGlu can be produced in a tissue-specific manner. For
example, rhNaGlu can be expressed in the oviduct, blood and/or
other cells or tissues of the transgenic avian. In one embodiment,
the NaGlu is expressed in the tubular gland cells of the magnum of
the oviduct of the transgenic avian, secreted into the lumen of the
oviduct, and deposited into egg white. In one embodiment, egg white
containing rhNaGlu is harvested and stored in bulk at a temperature
ranging from 4.degree. C. to -20.degree. C. The NaGlu is then
isolated and purified from the contents of the eggs using various
methods known in the art.
[0148] One aspect of the present invention relates to avian hard
shell eggs (e.g., chicken hard shell eggs) which contain the
rhNaGlu protein. The rhNaGlu produced and secreted by the
transgenic avian is glycosylated in a manner favorable to cellular
uptake by a human cell. The protein may be present in any useful
amount. In one embodiment, the protein is present in an amount in a
range between about 0.01 .mu.g per hard-shell egg and about 1 gram
per hard-shell egg. In another embodiment, the protein is present
in an amount in a range of between about 1 .mu.g per hard-shell egg
and about 1 gram per hard-shell egg. For example, the protein may
be present in an amount in a range of between about 10 .mu.g per
hard-shell egg and about 1 gram per hard-shell egg (e.g., a range
of between about 10 .mu.g per hard-shell egg and about 400
milligrams per hard-shell egg).
[0149] In one embodiment, the rhNaGlu is present in the egg white
of the egg. In one embodiment, the rhNaGlu is present in an amount
in a range of between about 1 ng per milliliter of egg white and
about 0.2 gram per milliliter of egg white. For example, the
rhNaGlu may be present in an amount in a range of between about 0.1
.mu.g per milliliter of egg white and about 0.2 gram per milliliter
of egg white (e.g., the rhNaGlu may be present in an amount in a
range of between about 1 .mu.g per milliliter of egg white and
about 100 milligrams per milliliter of egg white. In one
embodiment, the rhNaGlu is present in an amount in a range of
between about 1 .mu.g per milliliter of egg white and about 50
milligrams per milliliter of egg white. For example, the rhNaGlu
may be present in an amount in a range of about 1 .mu.g per
milliliter of egg white and about 10 milligrams per milliliter of
egg white (e.g., the rhNaGlu may be present in an amount in a range
of between about 1 .mu.g per milliliter of egg white and about 1
milligrams per milliliter of egg white). In one embodiment, the
rhNaGlu is present in an amount of more than 0.1 .mu.g per
milliliter of egg white. In one embodiment, the rhNaGlu is present
in an amount of more than 0.5 .mu.g per milliliter of egg white. In
one embodiment, the rhNaGlu is present in an amount of more than 1
.mu.g per milliliter of egg white. In one embodiment, the protein
is present in an amount of more than 1.5 .mu.g per milliliter of
egg white. In one embodiment, the rhNaGlu is present in an amount
of more than 0.5 .mu.g per milliliter of egg white. In one
embodiment, the protein is present in an amount of more than 0.1
.mu.g per milliliter of egg white.
[0150] In one embodiment, the rhNaGlu is present in an amount of 20
mg/L, 30 mg/L, 40 mg/L, 50 mg/L, 60 mg/L, 70 mg/L, 80 mg/L, 90
mg/L, 100 mg/L, 120 mg/L, 130 mg/L, 140 mg/L, 150 mg/L, 160 mg/L,
170 mg/L, 200 mg/L, 300 mg/L, 400 mg/L, 500 mg/L, 600 mg/L, 700
mg/L, 800 mg/L, 900 mg/L, or 1,000 mg/L egg white. In one
embodiment, the rhNaGlu is present in an amount of about 100 mg/L
of egg white. In one embodiment, the rhNaGlu is present in an
amount of about 200 mg/L of egg white.
[0151] V. Host Cells
[0152] The present invention also contemplates rhNaGlu produced in
any useful protein expression system including, without limitation,
cell culture (e.g., avian cells, CHO cells, HEK293 cells and COS
cells), yeast, bacteria, and plants.
[0153] A host cell strain can be chosen for its ability to modulate
the expression of the inserted sequences or to process the
expressed NaGlu in the desired fashion. Such modifications of the
polypeptide of NaGlu include, without limitation, glycosylation,
phosphorylation, or lipidation. Different host cells such as CHO,
COS, HeLa, MDCK, HEK293 and W138, which have specific cellular
machinery and characteristic mechanisms for such post-translational
activities, can be chosen to ensure the correct modification and
processing of the fusion protein of the present invention. An avian
tumor cell line is also contemplated as a host cell for expressing
the polypeptide of the present invention. Examples of a useful
avian cell line (e.g., an avian oviduct tumor cell line) are
described in U.S. Pat. Publication No. 2009/0253176, the entire
teachings of which are incorporated herein by reference.
[0154] VI. Pharmaceutical Compositions
[0155] The present invention also features pharmaceutical
compositions comprising isolated and substantially purified rhNaGlu
or a pharmaceutically acceptable salt thereof. The recombinant
human NaGlu proteins may be administered using one or more
carriers, e.g., as part of a pharmaceutical formulation, or without
a carrier. The carrier(s) must be "acceptable" in the sense of
being compatible with the other ingredients of the formulation and
not deleterious to the recipient thereof. Compositions comprising
such carriers, including composite molecules, are formulated by
well-known conventional methods (see, for example, Remington's
Pharmaceutical Sciences, 14th Ed., Mack Publishing Co., Easton,
Pa.), the entire teachings of which are incorporated herein by
reference. The carrier may comprise a diluent. In one embodiment,
the pharmaceutical carrier can be a liquid and the protein may be
in the form of a solution. The pharmaceutical carrier can be wax,
fat, or alcohol. In another embodiment, the pharmaceutically
acceptable carrier may be a solid in the form of a powder, a
lyophilized powder, or a tablet. In one embodiment, the carrier may
comprise a liposome or a microcapsule.
[0156] In some embodiments, a pharmaceutical composition comprising
recombinant human NaGlu protein further comprises a buffer.
Exemplary buffers include acetate, phosphate, citrate and glutamate
buffers. Exemplary buffers also include lithium citrate, sodium
citrate, potassium citrate, calcium citrate, lithium lactate,
sodium lactate, potassium lactate, calcium lactate, lithium
phosphate, sodium phosphate, potassium phosphate, calcium
phosphate, lithium maleate, sodium maleate, potassium maleate,
calcium maleate, lithium tartarate, sodium tartarate, potassium
tartarate, calcium tartarate, lithium succinate, sodium succinate,
potassium succinate, calcium succinate, lithium acetate, sodium
acetate, potassium acetate, calcium acetate, and mixtures thereof.
In some embodiments, the buffer is trisodium citrate dihydrate. In
some embodiments, the buffer is citric acid monohydrate. In some
embodiments, a pharmaceutical composition comprises trisodium
citrate dehydrate and citric acid monohydrate.
[0157] In some embodiments, a pharmaceutical composition comprising
recombinant human NaGlu protein further comprises a stabilizer.
Exemplary stabilizers include albumin, trehalose, sugars, amino
acids, polyols, cyclodextrins, salts such as sodium chloride,
magnesium chloride, and calcium chloride, lyoprotectants, and
mixtures thereof. In some embodiments, a pharmaceutical composition
comprises human serum albumin.
[0158] In some embodiments, it is desirable to add a surfactant to
the pharmaceutical composition. Exemplary surfactants include
nonionic surfactants such as Polysorbates (e.g., Polysorbates 20 or
80); poloxamers (e.g., poloxamer 188); Triton; sodium dodecyl
sulfate (SDS); sodium laurel sulfate; sodium octyl glycoside;
lauryl-, myristyl-, linoleyl-, or stearyl-sulfobetaine; lauryl-,
myristyl-, linoleyl- or stearyl-sarcosine; linoleyl-, myristyl-, or
cetyl-betaine; lauroamidopropyl-, cocamidopropyl-,
linoleamidopropyl-, myristamidopropyl-, palmidopropyl-, or
isostearamidopropyl-betaine (e.g., lauroamidopropyl);
myristamidopropyl-, palmidopropyl-, or
isostearamidopropyl-dimethylamine; sodium methyl cocoyl-, or
disodium methyl ofeyl-taurate; and the MONAQUAT.TM. series (Mona
Industries, Inc., Paterson, N.J.), polyethyl glycol, polypropyl
glycol, and copolymers of ethylene and propylene glycol (e.g.,
Pluronics, PF68, etc). Typically, the amount of surfactant added is
such that it reduces aggregation of the protein and minimizes the
formation of particulates or effervescences. For example, a
surfactant may be present in a formulation at a concentration from
about 0.001-0.5% (e.g., about 0.005-0.05%, or 0.005-0.01%). In
particular, a surfactant may be present in a formulation at a
concentration of approximately 0.005%, 0.01%, 0.02%, 0.1%, 0.2%,
0.3%, 0.4%, or 0.5%, etc. Ranges and values intermediate to the
above recited ranges and values are also contemplated to be part of
the invention.
[0159] In some embodiments, suitable pharmaceutical compositions of
the invention may further include one or more bulking agents, in
particular, for lyophilized formulations. A "bulking agent" is a
compound which adds mass to the lyophilized mixture and contributes
to the physical structure of the lyophilized cake. For example, a
bulking agent may improve the appearance of lyophilized cake (e.g.,
essentially uniform lyophilized cake). Suitable bulking agents
include, but are not limited to, sodium chloride, lactose,
mannitol, glycine, sucrose, trehalose, hydroxyethyl starch.
Exemplary concentrations of bulking agents are from about 1% to
about 10% (e.g., 1.0%, 1.5%, 2.0%, 2.5%, 3.0%, 3.5%, 4.0%, 4.5%,
5.0%, 5.5%, 6.0%, 6.5%, 7.0%, 7.5%, 8.0%, 8.5%, 9.0%, 9.5%, and
10.0%). Ranges and values intermediate to the above recited ranges
and values are also contemplated to be part of the invention. The
pharmaceutical compositions can be in the form of a sterile
lyophilized powder for injection upon reconstitution with a
diluent. The diluent can be water for injection, bacteriostatic
water for injection, or sterile saline. The lyophilized powder may
be produced by freeze drying a solution of the fusion protein to
produce the protein in dry form. As is known in the art, the
lyophilized protein generally has increased stability and a longer
shelf-life than a liquid solution of the protein.
[0160] Pharmaceutical formulations include those suitable for oral,
rectal, nasal, topical (including buccal and sub-lingual), vaginal
or parenteral administration. Preferably, the pharmaceutical
formulations of the invention include those suitable for
administration by injection including intrathecal,
intraparenchymal, intracerebral, intraventricular, intramuscular,
sub-cutaneous and intravenous administration. In one embodiment,
the formulations of the invention are suitable for intravenous
administration. In another embodiment, the formulations of the
invention are suitable for intrathecal administration. The
pharmaceutical formulations of the invention also include those
suitable for administration by inhalation or insufflation. The
formulations can, where appropriate, be conveniently presented in
discrete dosage units and can be prepared by any of the methods
well known in the art of pharmacy. The methods of producing the
pharmaceutical formulations typically include the step of bringing
the therapeutic proteins into association with liquid carriers or
finely divided solid carriers or both and then, if necessary,
shaping the product into the desired formulation.
[0161] Recombinant human NaGlu proteins of the invention can also
be formulated for parenteral administration (e.g., by injection,
for example bolus injection or continuous infusion) and can be
presented in unit dose form in ampoules, pre-filled syringes, small
volume infusion or in multi-dose containers with an added
preservative. The therapeutic proteins can be injected by, for
example, subcutaneous injections, intramuscular injections,
intrathecal injections, intracerebral injections, intraparenchymal
injections, intraventricular injections, and intravenous (IV)
infusions or injections.
[0162] In one embodiment, the recombinant human NaGlu protein is
administered intravenously by IV infusion by any useful method. In
one example, the recombinant human NaGlu protein can be
administered by intravenous infusion through a peripheral line. In
another example, the recombinant human NaGlu protein can be
administered by intravenous infusion through a peripherally
inserted central catheter. In another example, the recombinant
human NaGlu protein can be administered by intravenous infusion
facilitated by an ambulatory infusion machine attached to a venous
vascular access port. In one embodiment of intravenous infusion,
the medication is administered over a period of 1 to 8 hours
depending on the amount of medication to be infused and the
patient's previous infusion-related reaction history, as determined
by a physician skilled in the art. In another embodiment, the
recombinant human NaGlu protein is administered intravenously by IV
injection. In another embodiment, the recombinant human NaGlu
protein can be administered via intraperitoneal or intrathecal
injection.
[0163] In some embodiments, the therapeutic proteins are
administered by infusion, and the infusion can occur over an
extended time period, for example, 30 minutes to 10 hours. Thus,
the infusion can occur, for example, over a period of about 1 hour,
about 2 hours, about 3 hours, about 4 hours, or about 5 hours. The
infusion can also occur at various rates. Thus, for example, the
infusion rate can be about 1 mL per hour to about 20 mL per hour.
In some embodiments, the infusion rate is 5 mL to 10 mL per hour.
In one embodiment, the infusion rate is 1, 2, 3, 4, 5, 6, 7, 8, 9,
10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 mL per hour. In one
embodiment, the infusion rate is 0.1 to 5 mg/kg/hr. In one
embodiment, the infusion rate is about 0.1, about 0.2, about 0.3,
about 0.5, about 1.0, about 1.5, about 2.0, or about 3 mg/kg/hr.
Ranges and values intermediate to the above recited ranges and
values are also contemplated to be part of the invention.
[0164] The therapeutic proteins can take such forms as suspensions,
solutions, or emulsions in oily or aqueous vehicles, and can
contain formulatory agents such as suspending, stabilizing and/or
dispersing agents. The recombinant human NaGlu proteins can be in
powder form, obtained by aseptic isolation of sterile solid or by
lyophilization from solution, for constitution with a suitable
vehicle, e.g., sterile, pyrogen-free water, before use.
[0165] Formulations in accordance with the present invention can be
assessed based on product quality analysis, reconstitution time (if
lyophilized), quality of reconstitution (if lyophilized), high
molecular weight, moisture, and glass transition temperature.
Typically, protein quality and product analysis include product
degradation rate analysis using methods including, but not limited
to, size exclusion HPLC (SE-HPLC), cation exchange-HPLC (CEX-HPLC),
X-ray diffraction (XRD), modulated differential scanning
calorimetry (mDSC), reversed phase HPLC (RP-HPLC), multi-angle
light scattering (MALS), fluorescence, ultraviolet absorption,
nephelometry, capillary electrophoresis (CE), SDS-PAGE, and
combinations thereof. In some embodiments, evaluation of product in
accordance with the present invention may include a step of
evaluating appearance (either liquid or cake appearance).
[0166] Generally, formulations (lyophilized or aqueous) can be
stored for extended periods of time at room temperature. Storage
temperature may typically range from 0.degree. C. to 45.degree. C.
(e.g., 4.degree. C., 20.degree. C., 25.degree. C. or 45.degree.
C.). Formulations may be stored for a period of months to a period
of years. Storage time generally will be 24 months, 12 months, 6
months, 4.5 months, 3 months, 2 months or 1 month. Formulations can
be stored directly in the container used for administration,
eliminating transfer steps. Ranges and values intermediate to the
above recited ranges and values are also contemplated to be part of
the invention.
[0167] Formulations can be stored directly in the lyophilization
container (if lyophilized), which may also function as the
reconstitution vessel, eliminating transfer steps. Alternatively,
lyophilized product formulations may be measured into smaller
increments for storage. Storage should generally avoid
circumstances that lead to degradation of the proteins, including
but not limited to exposure to sunlight, UV radiation, other forms
of electromagnetic radiation, excessive heat or cold, rapid thermal
shock, and mechanical shock. The pharmaceutical compositions
according to the invention can also contain other active
ingredients such as immunosuppressive agents, antimicrobial agents,
or preservatives, discussed in more detail below.
[0168] VII. Methods of Treatment
[0169] The present invention also provides methods of treating
NaGlu-associated diseases, e.g., Sanfilippo Syndrome B. Recombinant
NaGlu employed in accordance with the invention includes
recombinant NaGlu which can be produced in any useful protein
expression system including, without limitation, cell culture
(e.g., CHO cells, COS cells), bacteria such as E. coli, transgenic
animals such as mammals and avians (e.g., chickens, duck, and
turkey) and in plant systems (e.g., duck weed and tobacco plants).
In one embodiment, the recombinant NaGlu is produced in a
transgenic animal, such as an avian.
[0170] In one embodiment, the method comprises administering to the
subject a recombinant human NaGlu protein (rhNaGlu), for instance a
recombinant human NaGlu protein containing a sufficient amount of
oligosaccharides (e.g., mannose and phosphorylated mannose (i.e.,
M6P)), in an amount sufficient to treat (e.g., reduce, ameliorate)
or prevent one or more symptoms of a NaGlu deficiency or NaGlu
associated disease. The recombinant human NaGlu protein can be
administered therapeutically or prophylactically, or both. The
recombinant human NaGlu protein (rhNaGlu) can be administered to
the subject, alone or in combination with other therapeutic
modalities as described herein.
[0171] The terms "treat," "treating," and "treatment" refer to
methods of alleviating, abating, or ameliorating a disease or
symptom, preventing an additional symptom, ameliorating or
preventing an underlying cause of a symptom, inhibiting a disease
or condition, arresting the development of a disease or condition,
relieving a disease or condition, causing regression of a disease
or condition, relieving a condition caused by the disease or
condition, or stopping a symptom of the disease or condition either
prophylactically and/or after the symptom has occurred.
[0172] "Therapeutically effective dose" as used herein refers to
the dose (e.g., amount and/or interval) of drug required to produce
an intended therapeutic response (e.g., reduction of heparan
sulfate levels and/or increase in NaGlu activity in a target
tissue). A therapeutically effective dose refers to a dose that, as
compared to a corresponding subject who has not received such a
dose, results in improved treatment, healing, prevention, or
amelioration of a disease, disorder, or side effect, or a decrease
in the rate of the occurrence or advancement of a disease or
disorder. The term also includes within its scope, doses effective
to enhance physiological functions.
[0173] As used herein, the term "subject" or "patient" is intended
to include human and non-human animals. Non-human animals include
all vertebrates, e.g., mammals and non-mammals, such as non-human
primates, sheep, dogs, cats, cows, horses, chickens, amphibians,
and reptiles. Preferred subjects include human subjects having a
NaGlu deficiency or NaGlu associated disease.
[0174] As used herein a "NaGlu associated disease" is a disease or
condition which is mediated by NaGlu activity or is associated with
aberrant NaGlu expression or activity. An example of an NaGlu
associated disease includes, but is not limited to, NaGlu
deficiency such as Sanflippo Syndrome B (also known as
mucopolysaccharidosis type IIIB).
[0175] The therapeutic methods of the present invention encompass
any route of administration which facilitates the uptake or
transport of the recombinant human NaGlu protein into the pertinent
organs and tissues. In one embodiment, the methods of the invention
include delivering the recombinant human NaGlu proteins of the
invention to the CNS (central nervous system), the kidney, or the
liver of a subject for the treatment of a NaGlu associated disease
(e.g., NaGlu deficiency). For example, the recombinant human NaGlu
protein may be administered to the patient intravenously (e.g., via
intravenous injection or intravenous infusion) and surprisingly
crosses the blood brain barrier (BBB) of the subject having NaGlu
deficiency. In another embodiment of the invention, the recombinant
human NaGlu protein is administered to the patient
intrathecally.
[0176] A. Device for Intrathecal Delivery
[0177] Various devices may be used for intrathecal delivery
according to the present invention. In some embodiments, a device
for intrathecal administration contains a fluid access port (e.g.,
injectable port); a hollow body (e.g., catheter) having a first
flow orifice in fluid communication with the fluid access port and
a second flow orifice configured for insertion into spinal cord;
and a securing mechanism for securing the insertion of the hollow
body in the spinal cord. As a non-limiting example, a suitable
securing mechanism contains one or more nobs mounted on the surface
of the hollow body and a sutured ring adjustable over the one or
more nobs to prevent the hollow body (e.g., catheter) from slipping
out of the spinal cord. In various embodiments, the fluid access
port comprises a reservoir. In some embodiments, the fluid access
port comprises a mechanical pump (e.g., an infusion pump). In some
embodiments, an implanted catheter is connected to either a
reservoir (e.g., for bolus delivery), or an infusion pump. The
fluid access port may be implanted or external.
[0178] In some embodiments, intrathecal administration may be
performed by either lumbar puncture (i.e., slow bolus) or via a
port-catheter delivery system (i.e., infusion or bolus). In some
embodiments, the catheter is inserted between the laminae of the
lumbar vertebrae and the tip is threaded up the thecal space to the
desired level (generally L3-L4).
[0179] Relative to intravenous administration, a single dose volume
suitable for intrathecal administration is typically small.
Typically, intrathecal delivery according to the present invention
maintains the balance of the composition of the CSF as well as the
intracranial pressure of the subject. In some embodiments,
intrathecal delivery is performed absent the corresponding removal
of CSF from a subject. In some embodiments, a suitable single dose
volume may be e.g., less than about 10 mL, 8 mL, 6 mL, 5 mL, 4 mL,
3 mL, 2 mL, 1.5 mL, 1 mL, or 0.5 mL. In some embodiments, a
suitable single dose volume may be about 0.5-5 mL, 0.5-4 mL, 0.5-3
mL, 0.5-2 mL, 0.5-1 mL, 1-3 mL, 1-5 mL, 1.5-3 mL, 1-4 mL, or
0.5-1.5 mL. In some embodiments, intrathecal delivery according to
the present invention involves a step of removing a desired amount
of CSF first. In some embodiments, less than about 10 mL (e.g.,
less than about 9 mL, 8 mL, 7 mL, 6 mL, 5 mL, 4 mL, 3 mL, 2 mL, 1
mL) of CSF is first removed before intrathecal administration. In
those cases, a suitable single dose volume may be e.g., more than
about 3 mL, 4 mL, 5 mL, 6 mL, 7 mL, 8 mL, 9 mL, 10 mL, 15 mL, or 20
mL. Ranges and values intermediate to the above recited ranges and
values are also contemplated to be part of the invention.
[0180] Various other devices may be used to effect intrathecal
administration of a therapeutic composition. For example,
formulations containing desired enzymes may be given using an
Ommaya reservoir which is in common use for intrathecally
administering drugs for meningeal carcinomatosis (Lancet 2: 983-84,
1963). More specifically, in this method, a ventricular tube is
inserted through a hole formed in the anterior horn and is
connected to an Ommaya reservoir installed under the scalp, and the
reservoir is subcutaneously punctured to intrathecally deliver the
particular enzyme being replaced, which is injected into the
reservoir. Other devices for intrathecal administration of
therapeutic compositions or formulations to an individual are
described in U.S. Pat. No. 6,217,552, the entire contents of which,
as they relate to these devices, are incorporated herein by
reference. Alternatively, the drug may be intrathecally given, for
example, by a single injection, or continuous infusion. It should
be understood that the dosage treatment may be in the form of a
single dose administration or multiple doses.
[0181] For injection, formulations of the invention can be
formulated in liquid solutions. In addition, the NaGlu enzyme may
be formulated in solid form and re-dissolved or suspended
immediately prior to use. Lyophilized forms are also included. The
injection can be, for example, in the form of a bolus injection or
continuous infusion (e.g., using infusion pumps) of the NaGlu
enzyme.
[0182] In one embodiment of the invention, the NaGlu enzyme is
administered by lateral cerebro ventricular injection into the
brain of a subject. The injection can be made, for example, through
a burr hole made in the subject's skull. In another embodiment, the
enzyme and/or other pharmaceutical formulation is administered
through a surgically inserted shunt into the cerebral ventricle of
a subject. For example, the injection can be made into the lateral
ventricles, which are larger. In some embodiments, injection into
the third and fourth smaller ventricles can also be made.
[0183] In yet another embodiment, the pharmaceutical compositions
used in the present invention are administered by injection into
the cisterna magna, or lumbar area of a subject.
[0184] In another embodiment of the method of the invention, the
pharmaceutically acceptable formulation provides sustained
delivery, e.g., "slow release" of the enzyme or other
pharmaceutical composition used in the present invention, to a
subject for at least one, two, three, four weeks or longer periods
of time after the pharmaceutically acceptable formulation is
administered to the subject.
[0185] As used herein, the term "sustained delivery" refers to
continual delivery of a pharmaceutical formulation of the invention
in vivo over a period of time following administration, preferably
at least several days, a week or several weeks. Sustained delivery
of the composition can be demonstrated by, for example, the
continued therapeutic effect of the enzyme over time (e.g.,
sustained delivery of the enzyme can be demonstrated by continued
reduced amount of storage granules in the subject). Alternatively,
sustained delivery of the enzyme may be demonstrated by detecting
the presence of the enzyme in vivo over time.
[0186] B. Intravenous Delivery
[0187] As discussed above, one of the surprising features of the
present invention is that the recombinant human NaGlu proteins of
the invention are able to effectively and extensively diffuse
across the blood brain barrier (BBB) and brain surface and
penetrate various layers or regions of the brain, including deep
brain regions, when administered intravenously. The methods of the
present invention effectively deliver the rhNaGlu proteins to
various tissues, neurons or cells of the central nervous system
(CNS), which are hard to target by existing CNS delivery methods.
Furthermore, the methods of the present invention deliver
sufficient amounts of the recombinant human NaGlu proteins to the
blood stream and various peripheral organs and tissues.
[0188] "Intravenous injection," often medically referred to as IV
push or bolus injection, refers to a route of administration in
which a syringe is connected to the IV access device and the
medication is injected directly, typically rapidly and occasionally
up to a period of 15 minutes if it might cause irritation of the
vein or a too-rapid effect. Once a medicine has been injected into
the fluid stream of the IV tubing, there must be some means of
ensuring that it gets from the tubing to the patient. Usually this
is accomplished by allowing the fluid stream to flow normally and
thereby carry the medicine into the bloodstream. However, in some
cases a second fluid injection, sometimes called a "flush," is used
following the first injection to facilitate the entering of the
medicine into the bloodstream.
[0189] "Intravenous infusion" refers to a route of administration
in which medication is delivered over an extended period of time.
For example, the medication can be delivered to a patient over a
period of time between 1 and 8 hours. The medication can also be
delivered to a patient over a period of about 1, about 2, about 3,
about 4, about 5, about 6, about 7, or about 8 hours. To accomplish
an intravenous infusion, an IV gravity drip or an IV pump can be
used. IV infusion is typically used when a patient requires
medications only at certain times and does not require additional
intravenous fluids (e.g., water solutions which can contain sodium,
chloride, glucose, or any combination thereof) such as those that
restore electrolytes, blood sugar, and water loss.
[0190] C. Target Tissues
[0191] In some embodiments, the rhNaGlu of the invention is
delivered to the central nervous system (CNS) of a subject. In some
embodiments, the rhNaGlu of the invention is delivered to one or
more of target tissues of brain, spinal cord, and/or peripheral
organs. As used herein, the term "target tissue" refers to any
tissue that is affected by the NaGlu associated disease to be
treated or any tissue in which the deficient NaGlu is normally
expressed. In some embodiments, target tissues include those
tissues in which there is a detectable or abnormally high amount of
enzyme substrate, for example stored in the cellular lysosomes of
the tissue, in patients suffering from or susceptible to the NaGlu
associated disease. In some embodiments, target tissues include
those tissues that display a disease-associated pathology, symptom,
or feature. In some embodiments, target tissues include those
tissues in which the deficient NaGlu is normally expressed at an
elevated level. As used herein, a target tissue may be a brain
target tissue, a spinal cord target tissue and/or a peripheral
target tissue. Exemplary target tissues are described in detail
below.
[0192] D. Brain Target Tissues
[0193] In general, the brain can be divided into different regions,
layers and tissues. For example, meningeal tissue is a system of
membranes which envelops the central nervous system, including the
brain. The meninges contain three layers, including dura matter,
arachnoid matter, and pia matter. In general, the primary function
of the meninges and of the cerebrospinal fluid is to protect the
central nervous system. In some embodiments, a therapeutic protein
in accordance with the present invention is delivered to one or
more layers of the meninges.
[0194] The brain has three primary subdivisions, including the
cerebrum, cerebellum, and brain stem. The cerebral hemispheres,
which are situated above most other brain structures and are
covered with a cortical layer. Underneath the cerebrum lies the
brainstem, which resembles a stalk on which the cerebrum is
attached. At the rear of the brain, beneath the cerebrum and behind
the brainstem, is the cerebellum.
[0195] The diencephalon, which is located near the midline of the
brain and above the mesencephalon, contains the thalamus,
metathalamus, hypothalamus, epithalamus, prethalamus, and
pretectum. The mesencephalon, also called the midbrain, contains
the tectum, tegumentum, ventricular mesocoelia, and cerebral
peduncels, the red nucleus, and the cranial nerve III nucleus. The
mesencephalon is associated with vision, hearing, motor control,
sleep/wake, alertness, and temperature regulation.
[0196] Regions of tissues of the central nervous system, including
the brain, can be characterized based on the depth of the tissues.
For example, CNS (e.g., brain) tissues can be characterized as
surface or shallow tissues, mid-depth tissues, and/or deep
tissues.
[0197] According to the present invention, the rhNaGlu of the
invention may be delivered to any appropriate brain target
tissue(s) associated with a particular disease to be treated in a
subject. In some embodiments, the rhNaGlu of the invention is
delivered to surface or shallow brain target tissue. In some
embodiments, the rhNaGlu of the invention is delivered to mid-depth
brain target tissue. In some embodiments, the rhNaGlu of the
invention is delivered to deep brain target tissue. In some
embodiments, the rhNaGlu of the invention is delivered to a
combination of surface or shallow brain target tissue, mid-depth
brain target tissue, and/or deep brain target tissue. In some
embodiments, the rhNaGlu of the invention is delivered to a deep
brain tissue at least 4 mm, 5 mm, 6 mm, 7 mm, 8 mm, 9 mm, 10 mm or
more below (or internal to) the external surface of the brain.
Ranges and values intermediate to the above recited ranges and
values are also contemplated to be part of the invention.
[0198] In some embodiments, the rhNaGlu of the invention is
delivered to one or more surface or shallow tissues of cerebrum. In
some embodiments, the targeted surface or shallow tissues of the
cerebrum are located within 4 mm from the surface of the cerebrum.
In some embodiments, the targeted surface or shallow tissues of the
cerebrum are selected from pia mater tissues, cerebral cortical
ribbon tissues, hippocampus, Virchow Robin space, blood vessels
within the VR space, the hippocampus, portions of the hypothalamus
on the inferior surface of the brain, the optic nerves and tracts,
the olfactory bulb and projections, and combinations thereof.
[0199] In some embodiments, the rhNaGlu of the invention is
delivered to one or more deep tissues of the cerebrum. In some
embodiments, the targeted surface or shallow tissues of the
cerebrum are located at least 4 mm (e.g., 5 mm, 6 mm, 7 mm, 8 mm, 9
mm, or 10 mm) below (or internal to) the surface of the cerebrum.
In some embodiments, targeted deep tissues of the cerebrum include
the cerebral cortical ribbon. In some embodiments, targeted deep
tissues of the cerebrum include one or more of the diencephalon
(e.g., the hypothalamus, thalamus, prethalamus or subthalamus),
metencephalon, lentiform nuclei, the basal ganglia, caudate,
putamen, amygdala, globus pallidus, and combinations thereof.
[0200] In some embodiments, the rhNaGlu of the invention is
delivered to one or more tissues of the cerebellum. In certain
embodiments, the targeted one or more tissues of the cerebellum are
selected from the group consisting of tissues of the molecular
layer, tissues of the Purkinje cell layer, tissues of the Granular
cell layer, cerebellar peduncles, and combination thereof. In some
embodiments, therapeutic agents (e.g., enzymes) are delivered to
one or more deep tissues of the cerebellum including, but not
limited to, tissues of the Purkinje cell layer, tissues of the
Granular cell layer, deep cerebellar white matter tissue (e.g.,
deep relative to the Granular cell layer), and deep cerebellar
nuclei tissue.
[0201] In some embodiments, the rhNaGlu of the invention is
delivered to one or more tissues of the brainstem. In some
embodiments, the targeted one or more tissues of the brainstem
include brain stem white matter tissue and/or brain stem nuclei
tissue.
[0202] In some embodiments, the rhNaGlu of the invention is
delivered to various brain tissues including, but not limited to,
gray matter, white matter, periventricular areas, pia-arachnoid,
meninges, neocortex, cerebellum, deep tissues in cerebral cortex,
molecular layer, caudate/putamen region, midbrain, deep regions of
the pons or medulla, and combinations thereof.
[0203] In some embodiments, the rhNaGlu of the invention is
delivered to various cells in the brain including, but not limited
to, neurons, glial cells, perivascular cells and/or meningeal
cells. In some embodiments, a therapeutic protein is delivered to
oligodendrocytes of deep white matter.
[0204] E. Spinal Cord Target Tissue
[0205] In general, regions or tissues of the spinal cord can be
characterized based on the depth of the tissues. For example,
spinal cord tissues can be characterized as surface or shallow
tissues, mid-depth tissues, and/or deep tissues.
[0206] In some embodiments, the rhNaGlu of the invention are
delivered to one or more surface or shallow tissues of the spinal
cord. In some embodiments, a targeted surface or shallow tissue of
the spinal cord is located within 4 mm from the surface of the
spinal cord. In some embodiments, a targeted surface or shallow
tissue of the spinal cord contains pia matter and/or the tracts of
white matter.
[0207] In some embodiments, the rhNaGlu of the invention are
delivered to one or more deep tissues of the spinal cord. In some
embodiments, a targeted deep tissue of the spinal cord is located
internal to 4 mm from the surface of the spinal cord. In some
embodiments, a targeted deep tissue of the spinal cord contains
spinal cord grey matter and/or ependymal cells.
[0208] In some embodiments, replacement enzymes (e.g., a NaGlu
fusion protein) are delivered to neurons of the spinal cord.
[0209] F. Peripheral Target Tissues
[0210] As used herein, peripheral organs or tissues refer to any
organs or tissues that are not part of the central nervous system
(CNS). Peripheral target tissues may include, but are not limited
to, blood system, liver, kidney, heart, endothelium, bone marrow
and bone marrow derived cells, spleen, lung, lymph node, bone,
cartilage, ovary and testis. In some embodiments, the rhNaGlu of
the invention is delivered to one or more of the peripheral target
tissues.
[0211] G. Biodistribution and Bioavailability
[0212] In various embodiments, once delivered to the target tissue,
the rhNaGlu of the invention is localized intracellularly. For
example, the rhNaGlu of the invention may be localized to exons,
axons, lysosomes, mitochondria or vacuoles of a target cell (e.g.,
neurons such as Purkinje cells). For example, in some embodiments
the rhNaGlu of the invention demonstrates translocation dynamics
such that the rhNaGlu moves within the perivascular space (e.g., by
pulsation-assisted convective mechanisms). In addition, active
axonal transport mechanisms relating to the association of the
administered protein or enzyme with neurofilaments may also
contribute to or otherwise facilitate the distribution of the
rhNaGlu proteins of the invention into the deeper tissues of the
central nervous system.
[0213] In some embodiments, the rhNaGlu of the invention delivered
according to the present invention may achieve therapeutically or
clinically effective levels or activities in various targets
tissues described herein. As used herein, a therapeutically or
clinically effective level or activity is a level or activity
sufficient to confer a therapeutic effect in a target tissue. The
therapeutic effect may be objective (i.e., measurable by some test
or marker) or subjective (i.e., subject gives an indication of or
feels an effect). For example, a therapeutically or clinically
effective level or activity may be an enzymatic level or activity
that is sufficient to ameliorate symptoms associated with the
disease in the target tissue (e.g., GAG storage).
[0214] In some embodiments, the rhNaGlu of the invention delivered
according to the present invention may achieve an enzymatic level
or activity that is at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%,
80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% of the
normal level or activity of the corresponding NaGlu enzyme in the
target tissue. In some embodiments, the rhNaGlu of the invention
delivered according to the present invention may achieve an
enzymatic level or activity that is increased by at least 1-fold,
2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold or
10-fold as compared to a control (e.g., endogenous levels or
activities without the treatment). In some embodiments, the rhNaGlu
delivered according to the present invention may achieve an
increased enzymatic level or activity at least approximately 10
nmol/hr/mg, 20 nmol/hr/mg, 40 nmol/hr/mg, 50 nmol/hr/mg, 60
nmol/hr/mg, 70 nmol/hr/mg, 80 nmol/hr/mg, 90 nmol/hr/mg, 100
nmol/hr/mg, 150 nmol/hr/mg, 200 nmol/hr/mg, 250 nmol/hr/mg, 300
nmol/hr/mg, 350 nmol/hr/mg, 400 nmol/hr/mg, 450 nmol/hr/mg, 500
nmol/hr/mg, 550 nmol/hr/mg or 600 nmol/hr/mg in a target tissue.
Ranges and values intermediate to the above recited ranges and
values are also contemplated to be part of the invention.
[0215] In some embodiments, inventive methods according to the
present invention are particularly useful for targeting the lumbar
region. In some embodiments, the rhNaGlu delivered according to the
present invention may achieve an increased enzymatic level or
activity in the lumbar region of at least approximately 500
nmol/hr/mg, 600 nmol/hr/mg, 700 nmol/hr/mg, 800 nmol/hr/mg, 900
nmol/hr/mg, 1000 nmol/hr/mg, 1500 nmol/hr/mg, 2000 nmol/hr/mg, 3000
nmol/hr/mg, 4000 nmol/hr/mg, 5000 nmol/hr/mg, 6000 nmol/hr/mg, 7000
nmol/hr/mg, 8000 nmol/hr/mg, 9000 nmol/hr/mg, or 10,000 nmol/hr/mg.
Ranges and values intermediate to the above recited ranges and
values are also contemplated to be part of the invention.
[0216] In general, therapeutic agents (e.g., the rhNaGlu) delivered
according to the present invention have sufficiently long half time
in CSF and target tissues of the brain, spinal cord, and peripheral
organs. In some embodiments, the rhNaGlu delivered according to the
present invention may have a half-life of at least approximately 30
minutes, 45 minutes, 60 minutes, 90 minutes, 2 hours, 3 hours, 4
hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 12
hours, 16 hours, 18 hours, 20 hours, 25 hours, 30 hours, 35 hours,
40 hours, up to 3 days, up to 7 days, up to 14 days, up to 21 days
or up to a month. In some embodiments, In some embodiments, the
rhNaGlu delivered according to the present invention may retain
detectable level or activity in CSF or bloodstream after 12 hours,
24 hours, 30 hours, 36 hours, 42 hours, 48 hours, 54 hours, 60
hours, 66 hours, 72 hours, 78 hours, 84 hours, 90 hours, 96 hours,
102 hours, or a week following administration. Detectable level or
activity may be determined using various methods known in the art.
Ranges and values intermediate to the above recited ranges and
values are also contemplated to be part of the invention. In
certain embodiments, the rhNaGlu delivered according to the present
invention achieves a concentration of at least 30 .mu.g/mL in the
CNS tissues and cells of the subject following administration
(e.g., one week, 3 days, 48 hours, 36 hours, 24 hours, 18 hours, 12
hours, 8 hours, 6 hours, 4 hours, 3 hours, 2 hours, 1 hour, 30
minutes, or less, following administration of the pharmaceutical
composition to the subject). In certain embodiments, the rhNaGlu
delivered according to the present invention achieves a
concentration of at least 2 .mu.g/mL, at least 15 .mu.g/mL, at
least 1 .mu.g/mL, at least 7 .mu.g/mL, at least 5 .mu.g/mL, at
least 2 .mu.g/mL, at least 1 .mu.g/mL or at least 0.5 .mu.g/mL in
the targeted tissues or cells of the subject (e.g., brain tissues
or neurons) following administration to such subject (e.g., one
week, 3 days, 48 hours, 36 hours, 24 hours, 18 hours, 12 hours, 8
hours, 6 hours, 4 hours, 3 hours, 2 hours, 1 hour, 30 minutes, or
less following administration of such pharmaceutical compositions
to the subject). Ranges and values intermediate to the above
recited ranges and values are also contemplated to be part of the
invention.
[0217] H. Treatment of Sanfilippo Syndrome
[0218] Sanfilippo syndrome, or mucopolysaccharidosis III (MPS III),
is a rare genetic disorder characterized by the deficiency of
enzymes involved in the degradation of glycosaminoglycans (GAG). In
the absence of enzyme, partially degraded GAG molecules cannot be
cleared from the body and accumulate in lysosomes of various
tissues, resulting in progressive widespread somatic dysfunction
(Neufeld and Muenzer, 2001).
[0219] Four distinct forms of MPS III, designated MPS IIIA, B, C,
and D, have been identified. Each represents a deficiency in one of
four enzymes involved in the degradation of the GAG heparan
sulfate. All forms include varying degrees of the same clinical
symptoms, including coarse facial features, hepatosplenomegaly,
corneal clouding and skeletal deformities. Most notably, however,
is the severe and progressive loss of cognitive ability, which is
tied not only to the accumulation of heparan sulfate in neurons,
but also the subsequent elevation of the gangliosides GM2, GM3 and
GD2 caused by primary GAG accumulation (Walkley 1998).
[0220] Mucopolysaccharidosis type IIIB (MPS IIIB; Sanfilippo
syndrome B) is an autosomal recessive disorder that is
characterized by a deficiency of the enzyme
alpha-N-acetyl-glucosaminidase (NaGlu). In the absence of this
enzyme, GAG heparan sulfate accumulates in lysosomes of neurons and
glial cells, with lesser accumulation outside the brain.
[0221] A defining clinical feature of this disorder is central
nervous system (CNS) degeneration, which results in loss of, or
failure to attain, major developmental milestones. The progressive
cognitive decline culminates in dementia and premature mortality.
The disease typically manifests itself in young children, and the
lifespan of an affected individual generally does not extend beyond
late teens to early twenties.
[0222] Compositions and methods of the present invention may be
used to effectively treat individuals suffering from or susceptible
to Sanfilippo syndrome B. The terms, "treat" or "treatment," as
used herein, refers to amelioration of one or more symptoms
associated with the disease, prevention or delay of the onset of
one or more symptoms of the disease, and/or lessening of the
severity or frequency of one or more symptoms of the disease.
[0223] In some embodiments, treatment refers to partial or complete
alleviation, amelioration, relief, inhibition, delaying onset,
reducing severity and/or incidence of neurological impairment in a
Sanfilippo syndrome B patient. As used herein, the term
"neurological impairment" includes various symptoms associated with
impairment of the central nervous system (e.g., the brain and
spinal cord). Symptoms of neurological impairment may include, for
example, developmental delay, progressive cognitive impairment,
hearing loss, impaired speech development, deficits in motor
skills, hyperactivity, aggressiveness and/or sleep disturbances,
among others.
[0224] Thus, in some embodiments, treatment refers to decreased
lysosomal storage (e.g., of GAG) in various tissues. In some
embodiments, treatment refers to decreased lysosomal storage in
brain target tissues, spinal cord neurons, and/or peripheral target
tissues. In certain embodiments, lysosomal storage is decreased by
about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%,
65%, 70%, 75%, 80%, 85%, 90%, 95%, 100% or more as compared to a
control. In some embodiments, lysosomal storage is decreased by at
least 1-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold,
8-fold, 9-fold or 10-fold as compared to a control. In some
embodiments, lysosomal storage is determined by LAMP-1 staining.
Ranges and values intermediate to the above recited ranges and
values are also contemplated to be part of the invention.
[0225] In some embodiments, treatment refers to reduced
vacuolization in neurons (e.g., neurons containing Purkinje cells).
In certain embodiments, vacuolization in neurons is decreased by
about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%,
65%, 70%, 75%, 80%, 85%, 90%, 95%, 100% or more as compared to a
control. In some embodiments, vacuolization is decreased by at
least 1-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold,
8-fold, 9-fold or 10-fold as compared to a control. Ranges and
values intermediate to the above recited ranges and values are also
contemplated to be part of the invention.
[0226] In some embodiments, treatment refers to increased NaGlu
enzyme activity in various tissues. In some embodiments, treatment
refers to increased NaGlu enzyme activity in brain target tissues,
spinal cord neurons and/or peripheral target tissues. In some
embodiments, NaGlu enzyme activity is increased by about 5%, 10%,
15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%,
80%, 85%, 90%, 95%, 100%, 200%, 300%, 400%, 500%, 600%, 700%, 800%,
900% 1000% or more as compared to a control. In some embodiments,
NaGlu enzyme activity is increased by at least 1-fold, 2-fold,
3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold or 10-fold
as compared to a control. In some embodiments, increased NaGlu
enzymatic activity is at least approximately 10 nmol/hr/mg, 20
nmol/hr/mg, 40 nmol/hr/mg, 50 nmol/hr/mg, 60 nmol/hr/mg, 70
nmol/hr/mg, 80 nmol/hr/mg, 90 nmol/hr/mg, 100 nmol/hr/mg, 150
nmol/hr/mg, 200 nmol/hr/mg, 250 nmol/hr/mg, 300 nmol/hr/mg, 350
nmol/hr/mg, 400 nmol/hr/mg, 450 nmol/hr/mg, 500 nmol/hr/mg, 550
nmol/hr/mg, 600 nmol/hr/mg or more. In some embodiments, NaGlu
enzymatic activity is increased in the lumbar region. In some
embodiments, increased NaGlu enzymatic activity in the lumbar
region is at least approximately 2000 nmol/hr/mg, 3000 nmol/hr/mg,
4000 nmol/hr/mg, 5000 nmol/hr/mg, 6000 nmol/hr/mg, 7000 nmol/hr/mg,
8000 nmol/hr/mg, 9000 nmol/hr/mg, 10,000 nmol/hr/mg, or more.
Ranges and values intermediate to the above recited ranges and
values are also contemplated to be part of the invention.
[0227] In certain embodiments, treatment according to the present
invention results in a reduction (e.g., about a 5%, 10%, 15%, 20%,
25%, 30%, 40%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 90%, 95%, 97.5%,
99% or more reduction) or a complete elimination of the presence,
or alternatively the accumulation, of one or more pathological or
biological markers which are associated with the NaGlu associated
disease. Such reduction or elimination may be particularly evident
in the cells and tissues of the CNS (e.g., neurons and
oligodendrocytes). For example, in some embodiments, upon
administration to a subject the pharmaceutical compositions of the
present invention demonstrate or achieve a reduction in the
accumulation of the biomarker lysosomal associated membrane protein
1 (LAMP1) in the CNS cells and tissues of the subject (e.g., in the
cerebral cortex, cerebellum, caudate nucleus and putamen, white
matter and/or thalamus). LAMP1 is a glycoprotein highly expressed
in lysosomal membranes and its presence is elevated many patients
with a lysosomal storage disorder (Meikle et al., Clin. Chem.
(1997) 43:1325-1335). The presence or absence of LAMP1 in patients
(e.g., as determined by LAMP staining) with a lysosomal storage
disease therefore may provide a useful indicator of lysosomal
activity and a marker for both the diagnosis and monitoring of
lysosomal storage diseases.
[0228] Accordingly, some embodiments of the present invention
relate to methods of reducing or otherwise eliminating the presence
or accumulation of one or more pathological or biological markers
associated with the NaGlu associated disease. Similarly, some
embodiments of the invention relate to methods of increasing the
degradation (or the rate of degradation) of one or more
pathological or biological markers (e.g., LAMP1) associated with
lysosomal storage diseases.
[0229] In some embodiments, treatment refers to decreased
progression of loss of cognitive ability. In certain embodiments,
progression of loss of cognitive ability is decreased by about 5%,
10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%,
75%, 80%, 85%, 90%, 95%, 100% or more as compared to a control. In
some embodiments, treatment refers to decreased developmental
delay. In certain embodiments, developmental delay is decreased by
about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%,
65%, 70%, 75%, 80%, 85%, 90%, 95%, 100% or more as compared to a
control. Ranges and values intermediate to the above recited ranges
and values are also contemplated to be part of the invention.
[0230] In some embodiments, treatment refers to increased survival
(e.g., survival time). For example, treatment can result in an
increased life expectancy of a patient. In some embodiments,
treatment according to the present invention results in an
increased life expectancy of a patient by more than about 5%, about
10%, about 15%, about 20%, about 25%, about 30%, about 35%, 0%, a
40%, about 45%, about 50%, about 55%, about 60%, about 65%, about
70%, about 75%, about 80%, about 85%, about 90%, about 95%, about
100%, about 105%, about 110%, about 115%, about 120%, about 125%,
about 130%, about 135%, about 140%, about 145%, about 150%, about
155%, about 160%, about 165%, about 170%, about 175%, about 180%,
about 185%, about 190%, about 195%, about 200% or more, as compared
to the average life expectancy of one or more control individuals
with similar disease without treatment. In some embodiments,
treatment according to the present invention results in an
increased life expectancy of a patient by more than about 6 month,
about 7 months, about 8 months, about 9 months, about 10 months,
about 11 months, about 12 months, about 2 years, about 3 years,
about 4 years, about 5 years, about 6 years, about 7 years, about 8
years, about 9 years, about 10 years or more, as compared to the
average life expectancy of one or more control individuals with
similar disease without treatment. In some embodiments, treatment
according to the present invention results in long term survival of
a patient. As used herein, the term "long term survival" refers to
a survival time or life expectancy longer than about 40 years, 45
years, 50 years, 55 years, 60 years, or longer. Ranges and values
intermediate to the above recited ranges and values are also
contemplated to be part of the invention.
[0231] The terms, "improve," "increase" or "reduce," as used
herein, indicate values that are relative to a control. In some
embodiments, a suitable control is a baseline measurement, such as
a measurement in the same individual prior to initiation of the
treatment described herein, or a measurement in a control
individual (or multiple control individuals) in the absence of the
treatment described herein. A "control individual" is an individual
afflicted with Sanfilippo syndrome B, who is about the same age
and/or gender as the individual being treated (to ensure that the
stages of the disease in the treated individual and the control
individual(s) are comparable).
[0232] The individual (also referred to as "patient" or "subject")
being treated is an individual (fetus, infant, child, adolescent,
or adult human) having Sanfilippo syndrome B or having the
potential to develop Sanfilippo syndrome B. The individual can have
residual endogenous NaGlu expression and/or activity, or no
measurable activity. For example, the individual having Sanfilippo
Syndrome B may have NaGlu expression levels that are less than
about 30-50%, less than about 25-30%, less than about 20-25%, less
than about 15-20%, less than about 10-15%, less than about 5-10%,
less than about 0.1-5% of normal NaGlu expression levels. Ranges
and values intermediate to the above recited ranges and values are
also contemplated to be part of the invention.
[0233] In some embodiments, the individual is an individual who has
been recently diagnosed with the disease. Typically, early
treatment (treatment commencing as soon as possible after
diagnosis) is important to minimize the effects of the disease and
to maximize the benefits of treatment.
[0234] I. Combination Therapies
[0235] Recombinant human NaGlu proteins, for instance a recombinant
human NaGlu protein containing a sufficient amount of
oligosaccharides (e.g., mannose and phosphorylated mannose (i.e.,
M6P)), can be used alone or in combination to treat NaGlu
associated diseases (e.g., Sanfilippo Syndrome B). It should be
understood that the recombinant human NaGlu proteins of the
invention can be used alone or in combination with an additional
procedure, e.g., surgical procedure, or agent, e.g., therapeutic
agent, the additional procedure or agent being selected by the
skilled artisan for its intended purpose. For instance, the
additional procedure or agent can be a therapeutic procedure or
agent art-recognized as being useful to treat the disease or
condition being treated by the recombinant human NaGlu protein of
the present invention. The additional procedure or agent also can
be an agent that imparts a beneficial attribute to the therapeutic
composition, e.g., an agent which affects the viscosity of the
composition.
[0236] It should also be understood that the combinations which are
included within this invention are those combinations useful for
their intended purpose. The agents and procedures set forth below
are for illustrative purposes and not intended to be limiting to
the present invention. The combinations, which are part of this
invention, can be the recombinant human NaGlu proteins of the
present invention and at least one additional agent or procedure
selected from the lists below. The combination can also include
more than one additional agent or procedure, e.g., two or three
additional agents if the combination is such that the formed
composition can perform its intended function.
[0237] The combination therapy can include surgical procedures,
gene therapy, or enzyme-replacement therapy. Additionally, the
recombinant human NaGlu protein can be coformulated with one or
more additional therapeutic agents, e.g., other recombinant
proteins or antibodies or drugs capable of preventing or reducing
the accumulation of undegraded substrates (e.g., substrate
reduction therapy).
[0238] In one or more embodiments, the combination therapy can
include co-administration with immunosuppresants, as discussed in
further detail below. Immunosuppresants such as, but not limited
to, antihistamines, corticosteroids, sirolimus, voclosporin,
ciclosporin, methotrexate, IL-2 receptor directed antibodies,
T-cell receptor directed antibodies, TNF-alpha directed antibodies
or fusion proteins (e.g., infliximab, etanercept, or adalimumab),
CTLA-4-Ig (e.g., abatacept), anti-OX-40 antibodies can also be
administered before, during, or after administration of a
recombinant human protein, such as a recombinant human NaGlu
protein, for example, if an anaphylactic reaction or adverse immune
response is expected or experienced by a patient.
[0239] J. Immunogenicity
[0240] The pharmaceutical compositions of the present invention are
characterized by their tolerability. As used herein, the terms
"tolerable" and "tolerability" refer to the ability of the
pharmaceutical compositions of the present invention to not elicit
an adverse reaction in the subject to whom such composition is
administered, or alternatively not to elicit a serious adverse
reaction in the subject to whom such composition is administered.
In some embodiments, the pharmaceutical compositions of the present
invention are well tolerated by the subject to whom such
compositions is administered.
[0241] Generally, administration of a rhNaGlu protein according to
the present invention does not result in severe adverse effects in
the subject. As used herein, severe adverse effects induce, but are
not limited to, substantial immune response, toxicity, or death. As
used herein, the term "substantial immune response" refers to
severe or serious immune responses, such as adaptive T-cell immune
responses.
[0242] Thus, in many embodiments, inventive methods according to
the present invention do not involve concurrent immunosuppressant
therapy (i.e., any immunosuppressant therapy used as
pre-treatment/pre-conditioning or in parallel to the method). In
some embodiments, inventive methods according to the present
invention do not involve an immune tolerance induction in the
subject being treated. In some embodiments, inventive methods
according to the present invention do not involve a pre-treatment
or preconditioning of the subject using T-cell immunosuppressive
agent.
[0243] However, in some embodiments, a subject mounts an immune
response after being administered the rhNaGlu of the invention.
Thus, in some embodiments, it may be useful to render the subject
receiving the rhNaGlu of the invention tolerant to the enzyme
replacement therapy. Immune tolerance may be induced using various
methods known in the art. For example, an initial 30-60 day regimen
of a T-cell immunosuppressive agent such as cyclosporin A (CsA) and
an antiproliferative agent, such as, azathioprine (Aza), combined
with weekly intrathecal infusions of low doses of a desired
replacement enzyme may be used.
[0244] Any immunosuppressant agent known to the skilled artisan may
be employed together with a combination therapy of the invention.
Such immunosuppressant agents include but are not limited to
cyclosporine, FK506, rapamycin, CTLA4-Ig, and anti-TNF agents such
as etanercept (see e.g., Moder, 2000, Ann. Allergy Asthma Immunol.
84, 280-284; Nevins, 2000, Curr. Opin. Pediatr. 12, 146-150;
Kurlberg et al., 2000, Scand. J. Immunol. 51, 224-230; Ideguchi et
al., 2000, Neuroscience 95, 217-226; Potter et al., 1999, Ann. N.Y.
Acad. Sci. 875, 159-174; Slavik et al., 1999, Immunol. Res. 19,
1-24; Gaziev et al., 1999, Bone Marrow Transplant. 25, 689-696;
Henry, 1999, Clin. Transplant. 13, 209-220; Gummert et al., 1999,
J. Am. Soc. Nephrol. 10, 1366-1380; Qi et al., 2000,
Transplantation 69, 1275-1283). The anti-IL2 receptor
(.alpha.-subunit) antibody daclizumab (e.g., Zenapax.TM.), which
has been demonstrated effective in transplant patients, can also be
used as an immunosuppressant agent (see e.g., Wiseman et al., 1999,
Drugs 58, 1029-1042; Beniaminovitz et al., 2000, N. Engl J. Med.
342, 613-619; Ponticelli et al., 1999, Drugs R. D. 1, 55-60; Berard
et al., 1999, Pharmacotherapy 19, 1 127-1 137; Eckhoff et al.,
2000, Transplantation 69, 1867-1872; Ekberg et al., 2000, Transpl.
Int. 13, 151-159). Additional immunosuppressant agents include but
are not limited to anti-CD2 (Branco et al., 1999, Transplantation
68, 1588-1596; Przepiorka et al., 1998, Blood 92, 4066-4071),
anti-CD4 (Marinova-Mutafchieva et al., 2000, Arthritis Rheum. 43,
638-644; Fishwild et al., 1999, Clin. Immunol. 92, 138-152), and
anti-CD40 ligand (Hong et al., 2000, Semin. Nephrol. 20, 108-125;
Chirmule et al., 2000, J. Virol. 74, 3345-3352; Ito et al., 2000,
J. Immunol. 164, 1230-1235).
[0245] In other embodiments, the invention includes methods
comprising co-administration of the NaGlu proteins of the present
invention with agents which decrease or suppress an immune response
to the NaGlu protein, e.g., immunosuppresants. Immunosuppresants
such as, but not limited to, antihistamines, corticosteroids,
sirolimus, voclosporin, ciclosporin, methotrexate, IL-2 receptor
directed antibodies, T-cell receptor directed antibodies, TNF-alpha
directed antibodies or fusion proteins (e.g., infliximab,
etanercept, or adalimumab), CTLA-4-Ig (e.g., abatacept), anti-OX-40
antibodies can also be administered before, during, or after
administration of a recombinant human protein, such as a
recombinant human NaGlu protein, for example, if an anaphylactic
reaction or adverse immune response is expected or experienced by a
patient.
[0246] In one embodiment, the invention provides for a pretreatment
procedure to minimize or prevent any potential anaphylactic
reactions that can be incurred by administration of the recombinant
protein in accordance with the invention. In one embodiment, to
prevent a potential anaphylactic reaction, an H-1 receptor
antagonist, also known as an antihistamine (e.g., diphenhydramine)
is administered to the patient. In one embodiment, the H-1 receptor
antagonist is administered in a dose of about 1 mg to about 10 mg
per kilogram of body weight. For example, an antihistamine can be
administered in a dose of about 5 mg per kilogram. In one
embodiment, the antihistamine is administered in a dose of between
about 0.1 mg and about 10 mg per kilogram of body weight. In one
embodiment, the antihistamine is administered in a dose between
about 1 mg and about 5 mg per kilogram of body weight. For example
the dose can be 1 mg, 2 mg, 3 mg, 4 mg, or 5 mg per kilogram of
body weight. The antihistamine can be administered by any useful
method. In one embodiment, the antihistamine is administered
intravenously. In another embodiment, the antihistamine is
administered in pharmaceutically acceptable capsules.
[0247] Administration of the antihistamine can be prior to the
administration of the recombinant NaGlu in accordance with the
invention. In one embodiment, the H-1 receptor antagonist is
administered about 10 to about 90 minutes, for example, about 30 to
about 60 minutes prior to the administration of recombinant NaGlu.
The H-1 receptor antagonist can be administered using an ambulatory
system connected to a vascular access port. In one embodiment, the
antihistamine is administered about 90 minutes prior to the
administration of recombinant NaGlu. In one embodiment, the
antihistamine is administered between about 10 and about 60 minutes
prior to the administration of recombinant NaGlu. In another
embodiment, the antihistamine is administered between about 20 and
about 40 minutes prior to administering recombinant NaGlu. For
example, the antihistamine can be administered 20, 25, 30, 35, or
40 minutes prior to the administration of recombinant NaGlu.
[0248] In one embodiment, the antihistamine administered is
diphenhydramine. Any useful antihistamine can be used. Such
antihistamines include, without limitation, clemastine, doxylamine,
loratidine, desloratidine, fexofenadine, pheniramine, cetirizine,
ebastine, promethazine, chlorpheniramine, levocetirizine,
olopatadine, quetiapine, meclizine, dimenhydrinate, embramine,
dimethidene, and dexchloropheniramine.
[0249] In another embodiment, with reference to intravenous
infusion, the potential for anaphylactic reactions can be reduced
by administering the infusions using a ramp-up protocol. In this
context, a ramp-up protocol refers to slowly increasing the rate of
the infusion over the course of the infusion in order to
desensitize the patient to the infusion of the medication.
[0250] K. Administration
[0251] The methods of the present invention contemplate single as
well as multiple administrations of a therapeutically effective
amount of the rhNaGlu of the invention described herein. The
rhNaGlu of the invention can be administered at regular intervals,
depending on the nature, severity and extent of the subject's
condition. In some embodiments, a therapeutically effective amount
of the rhNaGlu protein of the present invention may be administered
intravenously or intrathecally periodically at regular intervals
(e.g., once every year, once every six months, once every five
months, once every three months, bimonthly (once every two months),
monthly (once every month), biweekly (once every two weeks) or
weekly).
[0252] In some embodiments, intrathecal administration may be used
in conjunction with other routes of administration (e.g.,
intravenous, subcutaneously, intramuscularly, parenterally, trans
dermally, or transmucosally (e.g., orally or nasally)). In some
embodiments, those other routes of administration (e.g.,
intravenous administration) may be performed no more frequent than
biweekly, monthly, once every two months, once every three months,
once every four months, once every five months, once every six
months, annually administration.
[0253] As used herein, the term "therapeutically effective amount"
is largely determined based on the total amount of the therapeutic
agent contained in the pharmaceutical compositions of the present
invention. Generally, a therapeutically effective amount is
sufficient to achieve a meaningful benefit to the subject (e.g.,
treating, modulating, curing, preventing and/or ameliorating the
underlying disease or condition). For example, a therapeutically
effective amount may be an amount sufficient to achieve a desired
therapeutic and/or prophylactic effect, such as an amount
sufficient to modulate lysosomal enzyme receptors or their activity
to thereby treat such lysosomal storage disease or the symptoms
thereof (e.g., a reduction in or elimination of the presence or
incidence of "zebra bodies" or cellular vacuolization following the
administration of the compositions of the present invention to a
subject). Generally, the amount of a therapeutic agent (e.g., the
rhNaGlu of the invention) administered to a subject in need thereof
will depend upon the characteristics of the subject. Such
characteristics include the condition, disease severity, general
health, age, sex and body weight of the subject. One of ordinary
skill in the art will be readily able to determine appropriate
dosages depending on these and other related factors. In addition,
both objective and subjective assays may optionally be employed to
identify optimal dosage ranges.
[0254] A therapeutically effective amount is commonly administered
in a dosing regimen that may comprise multiple unit doses. For any
particular therapeutic protein, a therapeutically effective amount
(and/or an appropriate unit dose within an effective dosing
regimen) may vary, for example, depending on route of
administration, on combination with other pharmaceutical agents.
Also, the specific therapeutically effective amount (and/or unit
dose) for any particular patient may depend upon a variety of
factors including the disorder being treated and the severity of
the disorder; the activity of the specific pharmaceutical agent
employed; the specific composition employed; the age, body weight,
general health, sex and diet of the patient; the time of
administration, route of administration, and/or rate of excretion
or metabolism of the specific fusion protein employed; the duration
of the treatment; and like factors as is well known in the medical
arts.
[0255] In some embodiments, the therapeutically effective dose
ranges from about 0.005 mg/kg body weight to 500 mg/kg body weight,
e.g., from about 0.005 mg/kg body weight to 400 mg/kg body weight,
from about 0.005 mg/kg body weight to 300 mg/kg body weight, from
about 0.005 mg/kg body weight to 200 mg/kg body weight, from about
0.005 mg/kg body weight to 100 mg/kg body weight, from about 0.005
mg/kg body weight to 90 mg/kg body weight, from about 0.005 mg/kg
body weight to 80 mg/kg body weight, from about 0.005 mg/kg body
weight to 70 mg/kg body weight, from about 0.005 mg/kg body weight
to 60 mg/kg body weight, from about 0.005 mg/kg body weight to 50
mg/kg body weight, from about 0.005 mg/kg body weight to 40 mg/kg
body weight, from about 0.005 mg/kg body weight to 30 mg/kg body
weight, from about 0.005 mg/kg body weight to 25 mg/kg body weight,
from about 0.005 mg/kg body weight to 20 mg/kg body weight, from
about 0.005 mg/kg body weight to 15 mg/kg body weight, from about
0.005 mg/kg body weight to 10 mg/kg bra body in weight. Ranges and
values intermediate to the above recited ranges and values (e.g.,
10-50 mg/kg, 1-5 mg/kg, 2-8 mg/kg, 5-10 mg/kg, 0.1-10 mg/kg, 0.3-30
mg/kg, 0.3-50 mg/kg, 0.5-10 mg/kg, 5-30 mg/kg, or 6-27 mg/kg) are
also contemplated to be part of the invention.
[0256] In some embodiments, the therapeutically effective dose is
greater than or at least about 0.1 mg/kg body weight, greater than
or at least about 0.2 mg/kg body weight, greater than or at least
about 0.3 mg/kg body weight, greater than or at least about 0.4
mg/kg body weight, greater than or at least about 0.5 mg/kg body
weight, greater than or at least about 1.0 mg/kg body weight,
greater than or at least about 3 mg/kg body weight, greater than or
at least about 5 mg/kg body weight, greater than or at least about
6 mg/kg body weight, greater than or at least about 7 mg/kg body
weight greater than or at least about 10 mg/kg body weight, greater
than or at least about 15 mg/kg body weight, greater than or at
least about 20 mg/kg body weight, greater than or at least about 30
mg/kg body weight, greater than or at least about 40 mg/kg body
weight, greater than or at least about 50 mg/kg body weight,
greater than or at least about 60 mg/kg body weight, greater than
or at least about 70 mg/kg body weight, greater than about or at
least 80 mg/kg body weight, greater than or at least about 90 mg/kg
body weight, greater than or at least about 100 mg/kg body weight.
Ranges and values intermediate to the above recited ranges and
values are also contemplated to be part of the invention.
[0257] In some embodiments, the therapeutically effective dose may
also be defined by mg/kg brain weight. As one skilled in the art
would appreciate, the brain weights and body weights can be
correlated (see, e.g., Dekaban A S. "Changes in brain weights
during the span of human life: relation of brain weights to body
heights and body weights," Ann Neurol 1978; 4:345-56).
[0258] In some embodiments, the therapeutically effective dose may
also be defined by mg/15 cc of CSF. As one skilled in the art would
appreciate, therapeutically effective doses based on brain weights
and body weights can be converted to mg/15 cc of CSF. For example,
the volume of CSF in adult humans is approximately 150 mL (Johanson
C E, et al. "Multiplicity of cerebrospinal fluid functions: New
challenges in health and disease," Cerebrospinal Fluid Res. 2008
May 14; 5: 10). Therefore, single dose injections of 0.1 mg to 50
mg protein to adults would be approximately 0.01 mg/15 cc of CSF
(0.1 mg) to 5.0 mg/15 cc of CSF (50 mg) doses in adults.
[0259] It is to be further understood that for any particular
subject, specific dosage regimens should be adjusted over time
according to the individual need and the professional judgment of
the person administering or supervising the administration of the
enzyme replacement therapy and that dosage ranges set forth herein
are exemplary only and are not intended to limit the scope or
practice of the claimed invention.
[0260] VIII. Kits
[0261] The present invention further provides kits or other
articles of manufacture which contain the recombinant human NaGlu
of the present invention and provide instructions for its
reconstitution (if lyophilized) and/or use. Kits or other articles
of manufacture may include a container, a catheter and any other
articles, devices or equipment useful in intrathecal administration
and associated surgery. Suitable containers include, for example,
bottles, vials, syringes (e.g., pre-filled syringes), ampules,
cartridges, reservoirs, or lyo-jects. The container may be formed
from a variety of materials such as glass or plastic. In some
embodiments, a container is a pre-filled syringe. Suitable
pre-filled syringes include, but are not limited to, borosilicate
glass syringes with baked silicone coating, borosilicate glass
syringes with sprayed silicone, or plastic resin syringes without
silicone.
[0262] Typically, a label on, or associated with, the container may
indicate directions for use and/or reconstitution. For example, the
label may indicate that the formulation is reconstituted to protein
concentrations as described above. The label may further indicate
that the formulation is useful or intended for, for example,
intravenous or intrathecal administration. In some embodiments, a
container may contain a single dose of a stable formulation
containing a replacement enzyme (e.g., a recombinant NaGlu
protein). In various embodiments, a single dose of the stable
formulation is present in a volume of less than about 15 mL, 10 mL,
5.0 mL, 4.0 mL, 3.5 mL, 3.0 mL, 2.5 mL, 2.0 mL, 1.5 mL, 1.0 mL, or
0.5 mL. Alternatively, a container holding the formulation may be a
multi-use vial, which allows for repeat administrations (e.g., from
2-6 administrations) of the formulation. Kits or other articles of
manufacture may further include a second container comprising a
suitable diluent (e.g., BWFI, saline, buffered saline). Upon mixing
of the diluent and the formulation, the final protein concentration
in the reconstituted formulation will generally be at least 1 mg/mL
(e.g., at least 5 mg/mL, at least 10 mg/mL, at least 25 mg/mL, at
least 50 mg/mL, at least 75 mg/mL, at least 100 mg/mL).
[0263] Kits or other articles of manufacture may further include
other materials desirable from a commercial and user standpoint,
including other buffers, diluents, filters, needles, catheters,
syringes, and package inserts with instructions for use. Ranges and
values intermediate to the above recited ranges and values are also
contemplated to be part of the invention.
EXAMPLES
[0264] The following specific examples are intended to illustrate
the invention and should not be construed as limiting the scope of
the claims. The contents of all figures and all references, patents
and published patent applications cited throughout this
application, as well as the Figures, are expressly incorporated
herein by reference in their entirety.
Example 1
[0265] Purification of rhNaGlu
[0266] rhNaGlu protein was purified by using methods known in the
art. Egg white (EW) containing rhNaGlu was solubilized at pH 6
overnight and clarified through centrifugation and/or depth
filtration. The EW was adjusted with 1 M NaOAc buffer (pH 4) to pH
6. For the depth filtration process, T2600 filter (Pall.TM., 40 um)
was used as a 1.sup.st filtration and then PDF1 (Pall.TM., K200P,
15 um+EKS, 0.22 um) as a 2.sup.nd filtration step. The filters are
single-use membrane with an optimized capacity 60 L EW/m.sup.2 for
each filter. The hold volume of membrane is 2 L/m.sup.2 for T2600
and 4-5 L/m.sup.2 for PDF1. In the process, the hold volume was
discarded before the filtered EW collected. The buffer (20 mM
Phosphate/137 mM NaCl, pH 6) equivalent to the membrane hold volume
was used to chase EW left on the filters.
[0267] A phenyl-HIC (hydrophobic interaction chromatography) column
was applied as a capture step. Since most of egg white proteins are
hydrophilic, 99% of egg white proteins passed through the HIC
column into flow through. rhNaGlu has a higher hydrophobicity
binding to phenyl-HIC.
[0268] Egg white containing rhNaGlu was loaded onto the column with
a ratio of 30:1. After completion of loading, the column was washed
with the equilibration buffer, 5 mM phosphate buffer, pH 6, and 5
mM Tris buffer, pH 7.2. rhNaGlu was eluted with 30% propylene
glycol, pH 7.2. After the completion of loading, the column was
washed with equilibration buffer and 5 mM phosphate buffer (pH 6).
rhNaGlu was eluted with 30% propylene glycol with 5 mM Tris buffer
(pH 7.2). The column binding capacity is approximately 4.5 mg/mL.
The purity of rhNaGlu through the phenyl-HIC column can be reached
to >95% (950 time increase). The recovery is approximately 80%
with 30% of propylene glycol elution.
[0269] The eluted rhNaGlu fraction was adjusted to pH 5 with 1 M
acetic acid and then loaded onto a GigaCap S column (EW:column
size=10:1). The column was equilibrated with 50 mM NaOAc buffer (pH
5). After completion of loading, the column was washed with the
equilibration buffer. The rhNaGlu was eluted with 50 mM NaOAc/60 mM
NaCl (pH 5).
[0270] The protein characterization was performed using purified
rhNaGlu. The molecular weight of rhNaGlu (.about.90 kDa) purified
from egg white was analyzed on SDS-PAGE (FIG. 6). The average
expression level of rhNaGlu in egg white is shown in FIG. 7. The
characteristics of rhNaGlu produced from the transgenic avian are
summarized in Table 2.
TABLE-US-00002 TABLE 2 rhNaGlu (Gallus) Apparent Molecular Weight
~90 kDa pI 6.1-6.9 pH Stability pH 5-8 Stability in Egg White
>50 days
Example 2
[0271] Stability of rhNaGlu in Egg White
[0272] A single egg was cracked 7 days post-lay and analyzed for
activity. Contents were divided in half and each half was subject
to standard egg white clarification. Both untreated and clarified
egg whites were aliquoted and stored at 4.degree. C. and
-20.degree. C. for enzyme activity stability. rhNaGlu in egg white
showed stable enzyme activity at least up to 50 days.
[0273] Freeze/thaw cycle stability was assessed. The purified
rhNaGlu was frozen in liquid nitrogen for 10 seconds and thawed at
37.degree. C. for 2 min. The enzyme activity showed no change for
10 cycles.
[0274] The purified rhNaGlu was dialyzed into different pH buffers
to measure the stability of pure enzyme. The results showed that
pure rhNaGlu was stable between pH 5-8 for 12 days.
Example 3
Oligosaccharide Profiling
[0275] Mannose-6-phosphate (M6P) is a terminal monosaccharide of
N-linked oligosaccharides that is an important part of the tertiary
structure of glycoprotein and, when incorporated in the
glycoprotein's final oligosaccharide, is recognized by and bound to
the M6P receptors present on the cell surface, subsequently
allowing internalization into the lysosomes. Thus, M6P is an
effective epitope for the targeting of glycoproteins to the
lysosomes.
[0276] Analysis of protein glycosylation is an important part of
glycoprotein characterization. Oligosaccharides can be linked to a
protein through a serine or a threonine as O-lined glycans or
through an asparagine as N-linked glycans.
[0277] To analyze the structure of oligosaccharides, various
chromatographic and spectroscopic techniques were performed.
High-performance anion exchange chromatography with pulsed
amperometric detection (HPAEC-PAD) was employed. Using this
technique, oligosaccharides were quickly separated into general
groups based on charge (i.e., neutral, singly charged, or multiply
charged) and their structures were determined by comparison to pure
standards.
[0278] All methods were based on protocols described by Hardy and
Townsend (Hardy, M. R., and Townsend, R. R., "High-pH
anion-exchange chromatography of glycoprotein-derived
carbohydrates", 1994, Methods Enzymol. 230: 208-225). Purified
samples of transgenic avian derived rhNaGlu were dialyzed using a
Tube-O-Dialyzer against nanopure water at 4.degree. C. for about 24
hours to remove salts and other contaminants. Nanopure water was
replaced four times during the entire dialysis period. After
dialysis, each of the samples was divided into three aliquots. The
aliquot intended for neutral and amino sugars analysis was
hydrolyzed with 2 N trifluoroacetic acid (TFA) at 100.degree. C.
for 4 hours and the aliquot for mannose-6-phosphate analysis was
hydrolyzed with 6.75 N TFA at 100.degree. C. for 1.5 hours. The
hydrolysates were then dried under N.sub.2, re-dissolved with 50
.mu.L H.sub.2O, sonicated for 7 min in ice and transferred to an
injection vial.
[0279] A mix of standards for neutral and amino sugars, and for
mannose-6-phosphate with a known number of moles was hydrolyzed in
the same manner and at the same time as the sample. Four different
concentrations of the neutral and amino sugar standard mix and
mannose-6-phosphate were prepared to establish a calibration
equation. The number of moles of each sugar in the sample was
quantified by linear interpolation from the calibration
equation.
[0280] The oligossacharide profile and mannose-6-phosphate profile
were analyzed separately by HPAEC-PAD. Instrument control and data
acquisition were accomplished using Dionex chromeleon software.
HPAEC-PAD analysis of hydrolyzed rhNaGlu detected M6P. The mean
measured amount of M6P was 3.8 .mu.g (CV 3.7%) per 210 .mu.g of
hydrolyzed protein. Converting to moles resulted in 13.4 nmol of
M6P per 2.8 nmol of protein which was equivalent to a ratio of 3.2
moles of M6P per mole of protein.
[0281] The oligosaccharide profile was also obtained for rhNaGlu
(Gallus) using HPAEC-PAD (see FIG. 8). The profiles demonstrated
good repeatability of the PNGase F reaction on the single sample.
Peak clusters were observed in regions corresponding to neutral
oligosaccharides (.about.10 min to .about.20 min). A group of
significantly smaller peaks eluting between .about.25 and .about.35
min were also observed, which were possibly attributed to singly
charged species.
[0282] The monosaccharide composition analysis results obtained
from samples of rhNaGlu produced from a transgenic avian (Gallus)
are summarized in Table 3, which tabulates the average molar ratio
of each monosaccharide analyzed for rhNaGlu.
TABLE-US-00003 TABLE 3 Monosaccharide Molar Ratios in rhNaGlu
(Gallus) N-acetylgalactosamine (GalNAc) 1.1* N-acetylglucosamine
(GlcNAc) 35.6* Galactose (Gal) 4* Mannose (Man) 25.5*
Mannose-6-phosphate (M6P) 3.2* Fucose Not detected Glucose Not
detected *mole of monosaccharide per mole of protein
Example 4
[0283] Cellular Uptake into Fibroblasts
[0284] Wild-type human fibroblasts and mucopolysaccharidosis III B
(NaGlu deficient) human fibroblasts were placed in a 24-well plate
(2.5.times.10.sup.4 cells per well) and incubated for overnight at
37.degree. C. in 5% CO.sub.2. Conditioned media containing
fibroblast basal medium and fibroblast growth kit having low serum
were used. Various amounts of rhNaGlu (30, 10, 3.0, 1.0, 0.3 and 0
.mu.g/mL) were co-incubated for 24 hours at 37.degree. C. with 5%
CO.sub.2 to determine levels of cellular uptake by the human
fibroblasts (see, FIG. 9). The wells were washed three times with
PBS. 100 .mu.L lysis buffer was added per well and the plate was
incubated for 10 min at 37.degree. C. Cell lysate was transferred
into 1.5 mL centrifuge tube. One cycle of freezing and thawing was
performed. The cell lysate was centrifuged at 10,000 rpm for 10
min. 25 .mu.L of supernatants were used for the assay. The assay
time was 2 hours. The enzyme activity was measured using the
methods known in the art and according to the methods described in
Marsh et al., Clinical Genetics (1985) 27: 258-262, Chow et al.,
Carbohydrate Research (1981) 96:87-93; Weber et al., Protein
Expression and Purification, (2001)21:251-259).
[0285] As shown in FIG. 9, negative control (i.e., MPS IIIB) did
not exhibit any NaGlu activity while positive control (i.e.,
wild-type human fibroblast) showed NaGlu activity. MPS IIIB cells
treated with 0.3 .mu.g/mL of rhNaGlu exhibited approximately 50% of
the normal activity level observed in wild-type fibroblast cells.
MPS IIIB cells treated with 1 .mu.g/mL of rhNaGlu demonstrated
NaGlu activity that was approximately 4-fold higher than that
observed in wild-type cells. Surprisingly, MPS IIIB cells treated
with 30 .mu.g/mL of rhNaGlu showed NaGlu activity that was at least
40-fold higher than that observed in wild-type cells. This result
indicated that rhNaGlu produced from a transgenic avian (Gallus)
was efficiently internalized into human fibroblasts at a high
level.
[0286] To determine whether internalization of rhNaGlu is via M6P
receptor mediate endocytosis, M6P inhibition assays were performed.
For the M6P inhibition assays, various concentrations of free M6P
were added to human MPS IIIB fibroblasts treated with 30 .mu.g/mL
of rhNaGlu and enzymatic activity was measured as described above.
As shown in FIG. 10, human MPS IIIB fibroblasts did not exhibit any
NaGlu activity, suggesting effective inhibition of NaGlu uptake by
free M6P. In contrast, MPSIII fibroblasts treated with 30 .mu.g/mL
of rhNaGlu in the absence of free M6P exhibited a high level of
enzymatic activity, suggesting that the protein was efficiently
internalized into the NaGlu deficient fibroblasts and retained
activity. This enzymatic activity was inhibited by the presence of
M6P monosaccharide in the medium at the concentration 0.03 mM and
higher. The presence of 1 mM of M6P monosaccharide in conditioned
medium inhibited more than 90% of cellular uptake of the
protein.
[0287] These results indicated that the rhNaGlu produced from a
transgenic avian was efficiently internalized into the MPS IIIB
fibroblasts via M6P receptor-mediated endocytosis and the rhNaGlu
competed with M6P monosaccharides for the receptor recognition. The
results were consistent with the glycan analysis that revealed the
presence of the M6P structures on the rhNaGlu produced from the
transgenic avian.
Example 5
Generation of Active NaGlu Fusion Proteins
[0288] Two different rhNaGlu fusion constructs were designed to
validate the feasibility of expressing rhNaGlu fusion proteins in
the avian expression system.
[0289] In one construct, a nucleic acid sequence encoding 8
consecutive aspartic acid residues (DDDDDDDD (SEQ ID NO: 6)) was
fused to the nucleic sequence encoding NaGlu protein at the 5' end
of the full-length NaGlu cDNA sequence (SEQ ID NO:2) using
conventional PCR and DNA recombinant technology. In another
construct, a nucleic acid sequence encoding TfRL (i.e.,
THRPPMWSPVWP; SEQ ID NO:5) was fused to the nucleic sequence
encoding NaGlu at the 3' end of the full-length NaGlu cDNA
sequence. The each construct was inserted into the pTT22 expression
vector using EcoRI and HindIII restriction sites. The resulting
vectors were each transfected into human embryonic kidney (HEK) 293
cells and stable clones expressing high levels of the fusion NaGlu
proteins were obtained. An rhNaGlu protein fused to a stretch of 8
consecutive aspartic acid residues (SEQ ID NO: 6) at N-terminus
(AAA-NaGlu) and an rhNaGlu protein fused to transferrin receptor
ligand (TfRL) at C-terminus (NaGlu-TfRL) were isolated from
conditioned media.
[0290] The enzymatic activity of AAA-NaGlu and NaGlu-TfRL was
measured using the methods known in the art (see, e.g., Marsh et
al., Clinical Genetics (1985) 27:258-262; Chow et al., Carbohydrate
Research, (1981) 96:87-93; Weber et al., Protein Expression and
Purification (2001) 21:251-259; Neufeld et al., Protein Expression
and Purification (2000) 19:202-211; and Weber et al., Human
Molecular Genetics (1996) 5:771-777.
[0291] As shown in FIGS. 13 and 14, AAA-NaGlu and NaGlu-TfRL fusion
proteins produced from HEK293 cells showed high levels of enzymatic
activity. These results confirmed the possibility that these
constructs can be used to produce NaGlu fusion proteins that have
increased levels of phosphorylated mannose while retaining
enzymatic activity from a transgenic avian expression system.
Example 6
[0292] Cellular Uptake into Macrophages
[0293] Internalization of rhNaGlu produced from Gallus into human
macrophage cells was also measured. NR8383 macrophage cells were
incubated with 10 .mu.g/mL of rhNaGlu in F12 growth media for 0, 4,
8, 24, 32 and 48 hours at 37.degree. C. with 5% CO.sub.2. Samples
were recovered and washed with PBS prior to lysis.
2.5.times.10.sup.5 cells were lysed in 1 mL of lysis buffer (10 mM
of Na Phosphate pH6.0, 0.05% NP40), and lysates transferred into
1.5 mL centrifuge tubes and centrifuged at 10,000 rpm for 10 min.
Protein concentration was determined by the Bradford assay and
aliquots were frozen for NaGlu enzyme assays.
[0294] Enzyme activity was measured using standard methods. 25 mM
of substrate (4-methylumbelliferyl
2-Acetamido-2-deoxy-a-D-glucopyranoside) was diluted to 2 mM in
nanopure water to form a working substrate stock. Dilutions of
samples were prepared in assay buffer (1% bovine serum albumin). 25
.mu.L of 200 mM sodium acetate was distributed to wells of a
multi-well plate. 25 .mu.L of standard and 25 .mu.L of samples were
added to designated wells. 50 .mu.L of the working substrate stock
was added to each well and the plate was gently tapped to mix. The
plate was sealed with adhesive film and incubated at 37.degree. C.
for 30 minutes. The reaction was then terminated by addition of 50
.mu.L of stop solution (1M Glycine pH 12.5). The plate was placed
on a microplate reader using a fluorescence bottom and the
intensity was measured at an excitation 360 nm and an emission 460
nm. The level of liberated 4-methylumbelliferone (4-MU) was
measured by comparison with standards of 4-MU at 0.25 mM, 0.125 mM,
0.0625 mM, 0.0312 mM, 0.0156 mM, and 0.0078 mM.
[0295] As depicted in FIG. 15, levels of the NaGlu activity in
macrophages incubated with 10 .mu.g/mL of rhNaGlu increased almost
linearly over a 48 hour period: The rhNaGlu uptake by macrophages
was rather slow, but steady throughout the entire time period
measured. The relatively slow, extended uptake of NaGlu activity
(as compared to other lysosomal enzymes containing M6P and/or
mannose in their glycosylation structures) was unexpected and
surprising. Equally surprising and unexpected was that a large
amount of rhNaGlu proteins was taken up into the macrophages over
the extended time period, resulting in intracellular enzymatic
activity levels at least 10, 50, 100, 200, 300, 500, or even
1,000-fold higher than the basal levels observed in wild-type
macrophages not exposed to rhNaGlu. The results demonstrate that
rhNaGlu is extremely stable in extracellular as well as
intracellular environments. Further, these results suggest that
rhNaGlu may possess physicochemical characteristics that allow for
longer serum half-life (e.g., longer circulation) and high serum
concentrations in vivo, properties which are ideal for enhanced
uptake into the central nervous system (CNS).
TABLE-US-00004 TABLE 4 Summary of NaGlu Characteristics Avian
(gallus) produced Natural human CHO produced rhNaGlu NaGlu human
NaGlu Apparent Molecular ~85-~90 ~86 ~79-~89 Mass (kDa) Enzymatic
Activity >1,000 ~500 ~1,057 (nmol/min/mg) Mannose-6-phosphate
High High None or very Low
Example 7
[0296] Administration of rhNaGlu into NaGlu Deficient Mice
[0297] Homozygous null mice were generated from breeding pairs of
the strain B6.129S6-NaGlu.sup.tm1Efn/J. Control wild-type mice were
generated in the same manner. Genotyping was performed according to
a standard PCR protocol. It is described in the art that at birth,
homozygous naglu(.sup.-/-) null mice are viable, normal in size,
and do not display any gross physical or behavioral abnormalities,
though they exhibited no NaGlu in all tissues (see, Li et al.,
(1999) Proc., Natl. Acad. Sci. USA 96:14505-14510). At one month of
age, vacuolated macrophages are found in most tissues. Epithelial
cells in kidney and neurons in some parts of the brain are also
affected. The vacuolation becomes more prominent with age. At 4-5
months, the mice show abnormal behavior in an open field test.
Older animals may have urinary retention and difficulty walking.
Typical life span of the homozygous null naglu.sup.-/- mice is 8-12
months (see, Li et al., (1999) Proc., Natl. Acad. Sci. USA
96:14505-14510).
Intravenous (IV) Administration
[0298] The intravenous administration of test article and vehicle
by tail vein injection was accomplished as follows. Before
injection vasodilation was achieved by gently warming the animal
with an incandescent lamp or by soaking the tail in warm water,
approximately 43.degree. C. The animal was then placed in restraint
device. The surface of the tail was disinfected with 70%
isopropanol prior to injection. The lateral veins of the tail are
located just under the skin and are identified in the distal part
of the tail with the application of tension. A 27 G needle, bevel
up, was inserted into the vein for 3-4 mm. The test article or
vehicle was then administered as a slow bolus injection over a
period of ten seconds as evidenced by the observed clearing of the
vein as the administered liquid momentarily occupies the vascular
space. After removal of needle, gentle pressure was applied to the
puncture site to provide hemostasis. The animal was monitored
immediately following procedure to assure normal activity.
Intrathecal (IT) Administration
[0299] The intrathecal administration of test article and vehicle
by lumbar puncture injection was accomplished as follows. Before
injection, animals were anesthetized using isoflurane that was
maintained via nose cone throughout the procedure. The site of
injection was prepared by shaving the fur, as necessary, prior to
each injection. The animal was placed in a prone position on a
platform, ensuring the hind limbs were straddling the platform
forming a convex curve of the animals back. The surface of the back
was swabbed with 70% isopropanol and allowed to dry prior to
injection. Spinal column and hip bones were palpated to locate the
L4-L5 or L5-L6 margin. A 30 G needle, bevel facing cranially, was
inserted into the intervertebral space. Placement was confirmed by
the observation of a tail flick. The test article or vehicle was
then administered as bolus injection. The animal was allowed to
recover from anesthesia and monitored immediately following
procedure to assure normal activity and use of limbs.
Results
[0300] Twelve-week old naglu.sup.-/- mice
(B6.129S6-Naglu.sup.tm1Efn/J) were administered rhNaGlu (Gallus) at
dose levels of 6.75 or 27 mg/kg via tail vein injection (IV
administration), once every other day, for a total of 5 doses (at
rhNaGlu concentrations of 1.125, or 4.5 mg/mL, respectively).
Similarly, twelve-week old naglu.sup.-/- mice were administered
with rhNaGlu (Gallus) at a dose level of 0.31 mg/kg via lumbar
puncture injection (IT administration), once every other day, for a
total of 5 doses at NaGlu concentrations of 1.54 mg/mL. Vehicle (10
mM phosphate buffer, 150 mM NaCl and 0.02% Tween80, pH 5.5-5.8) was
administered to naglu.sup.-/- knock-out mice at the same dose
concentration for 5 doses every other day. Untreated wild-type and
naglu.sup.-/- knock-out mice were also maintained for the duration
of the study.
[0301] Animals were sacrificed 4 hours after the fifth and final
injection. All animals were necropsied and the liver, brain,
spleen, heart, lung and kidneys were excised. Each organ was
divided sagittally, providing samples for both frozen (-80.degree.
C.) and formalin-fixed storage.
[0302] Tissue samples were analyzed for: (1) heparan sulfate
concentration using an analytical method based on SAX-HPLC analysis
of heparan sulfate disaccharides; and (2)
.alpha.-N-acetylglucosaminidase enzyme activity using a cell-based
enzyme activity assay.
[0303] Histopathologic evaluation of brain, liver, kidney, spleen,
heart and lung tissue was conducted using formalin-fixed tissue
samples, embedded in paraffin, sectioned at 4 .mu.m, mounted on
glass slides and stained with hematoxylin and eosin (H&E).
[0304] Following the repeated intravenous administration (5 doses
over a 10 day period) of rhNaglu (Gallus) to naglu.sup.-/- mice at
dose levels of 6.25 and 27 mg/kg body weight, there was an apparent
dose-dependent decrease in the concentration of Heparan Sulfate in
the brain, liver and kidney of naglu.sup.-/- mice (Table 5; FIGS.
16-18). The relative .alpha.-N-acetylglucosaminidase activity was
increased in the brain and liver following intravenous
administration (Table 6). These results were unexpected and
surprising because the NaGlu enzymatic activities and resulting
substrate clearance were observed in the brain of the treated
naglu.sup.-/- mice with the IV administration, suggesting that
rhNaGlu (Gallus) administered systemically was distributed to the
brain of the naglu.sup.-/- mice and effective to elicit efficacy
even in the present of the blood brain barrier (BBB).
[0305] Following the intrathecal administration (5 doses over a 10
day period) of rhNaGlu (Gallus) to naglu.sup.-/- mice at a dose
level of 0.31 mg/kg, there was a decrease in the concentration of
Heparan Sulfate in the brain of naglu.sup.-/- mice (Table 5; FIG.
19), suggesting that rhNaGlu (Gallus) was targeted to the brain and
effective in reducing the accumulated substrate in the brain of
naglu.sup.-/- mice.
TABLE-US-00005 TABLE 5 Tissue Substrate Level (rhNaGlu Gallus)
Heparan Age at Sulfate Animal sacrifice Dose ug/mg Tissue Number
Genotype (wks) Treatment (mg/kg) Route tissue mean sd KIDNEY 253 WT
4 na -- -- 0.1 155 WT 12 na -- -- 0.045 0.0725 0.038891 178 KO 12
na -- -- 1.882 242 KO 4 na -- -- 1.687 145 KO 13 na -- -- 1.904 474
KO 13 vehicle 0 IV 1.501 479 KO 13 vehicle 0 IV 1.983 484 KO 13
vehicle 0 IV 1.839 1.799333 0.175908 487 KO 13 rhNaGlu 6.25 IV
0.928 492 KO 13 rhNaGlu 6.25 IV 0.737 0.8325 0.135057 481 KO 13
rhNaGlu 27 IV 0.591 485 KO 13 rhNaGlu 27 IV 0.311 490 KO 13 rhNaGlu
27 IV 0.585 0.495667 0.159954 86 KO 15 vehicle 0 IT 2.105 91 KO 14
vehicle 0 IT 1.704 1.9045 0.28355 94 KO 14 rhNaGlu 0.31 IT 1.324
101 KO 14 rhNaGlu 0.31 IT 2.233 1.7785 0.64276 LIVER 253 WT 4 na --
-- 0.045 155 WT 12 na -- -- 0.092 0.0685 0.033234 243 WT 4 na -- --
0.045 178 KO 12 na -- -- 1.85 242 KO 4 na -- -- 2.263 2.0565
0.292035 255 KO 4 na -- -- 1.85 474 KO 13 vehicle 0 IV 1.822 479 KO
13 vehicle 0 IV 1.981 484 KO 13 vehicle 0 IV 2.004 1.961667
0.165779 487 KO 13 rhNaGlu 6.25 IV 0.748 492 KO 13 rhNaGlu 6.25 IV
0.444 504 KO 13 rhNaGlu 6.25 IV 0.494 0.562 0.163009 481 KO 13
rhNaGlu 27 IV 0.491 485 KO 13 rhNaGlu 27 IV 0.172 0.3315 0.225567
BRAIN 253 WT 4 na -- -- 0.021 155 WT 12 na -- -- 0.013 243 WT 4 na
-- -- 0.014308 10 WT 36 na -- -- 0.012649 0.015239 0.003906 239 KO
4 na -- -- 0.095 178 KO 12 na -- -- 0.084 242 KO 4 na -- -- 0.099
255 KO 4 na -- -- 0.094538 165 KO 24 na -- -- 0.084015 474 KO 13
vehicle 0 IV 0.085447 479 KO 13 vehicle 0 IV 0.072 484 KO 13
vehicle 0 IV 0.073 0.085875 0.009972 487 KO 13 rhNaGlu 6.25 IV
0.045 492 KO 13 rhNaGlu 6.25 IV 0.044119 504 KO 13 rhNaGlu 6.25 IV
0.044 0.044373 0.000546 481 KO 13 rhNaGlu 27 IV 0.017796 485 KO 13
rhNaGlu 27 IV 0.016668 490 KO 13 rhNaGlu 27 IV 0.028 0.020821
0.006242 86 KO 15 vehicle 0 IT 0.094521 91 KO 14 vehicle 0 IT
0.072623 0.083572 0.015484 94 KO 14 rhNaGlu 0.31 IT 0.038866 101 KO
14 rhNaGlu 0.31 IT 0.028229 0.033548 0.007521 na: Not applicable
(mice were untreated).
TABLE-US-00006 TABLE 6 Tissue enzymatic activity (rhNaGlu Gallus;
U/ng protein) Enzymatic Age at Activity Animal sacrifice Dose (U/ug
Tissue Number Genotype (wks) Treatment (mg/kg) Route protein) BRAIN
253 WT 4 na -- -- 7.7 178 KO 12 na -- -- 0 474 KO 13 vehicle 0 IV 0
479 KO 13 vehicle 0 IV 0 484 KO 13 vehicle 0 IV 0.575 487 KO 13
rhNaGlu 6.25 IV 10.58 492 KO 13 rhNaGlu 6.25 IV 5.066666667 504 KO
13 rhNaGlu 6.25 IV 4.033333333 481 KO 13 rhNaglu 27 IV 87.91666667
485 KO 13 rhNaGlu 27 IV 90.15 490 KO 13 rhNaGlu 27 IV 17.35 LIVER
253 WT 4 na -- -- 36.69 178 KO 12 na -- -- 0 474 KO 13 vehicle 0 IV
0 479 KO 13 vehicle 0 IV 0 484 KO 13 vehicle 0 IV 0 487 KO 13
rhNaGlu 6.25 IV 512.92 492 KO 13 rhNaGlu 6.25 IV 378.805 504 KO 13
rhNaGlu 6.25 IV 607.9225 481 KO 13 rhNaGlu 27 IV 659.6825 485 KO 13
rhNaGlu 27 IV 654.2475 490 KO 13 rhNaGlu 27 IV 677.8725 na: not
applicable (mice were untreated).
[0306] Each example in the above specification is provided by way
of explanation of the invention, not limitation of the invention.
In fact, it will be apparent to those skilled in the art that
various modifications, combinations, additions, deletions and
variations can be made in the present invention without departing
from the scope or spirit of the invention. For instance, features
illustrated or described as part of one embodiment can be used in
another embodiment to yield a still further embodiment. It is
intended that the present invention cover such modifications,
combinations, additions, deletions, and variations.
[0307] All publications, patents, patent applications, internet
sites, and accession numbers/database sequences (including both
polynucleotide and polypeptide sequences) cited herein are hereby
incorporated by reference in their entirety for all purposes to the
same extent as if each individual publication, patent, patent
application, internet site, or accession number/database sequence
were specifically and individually indicated to be so incorporated
by reference.
Sequence CWU 1
1
61743PRTHomo sapiens 1Met Glu Ala Val Ala Val Ala Ala Ala Val Gly
Val Leu Leu Leu Ala 1 5 10 15 Gly Ala Gly Gly Ala Ala Gly Asp Glu
Ala Arg Glu Ala Ala Ala Val 20 25 30 Arg Ala Leu Val Ala Arg Leu
Leu Gly Pro Gly Pro Ala Ala Asp Phe 35 40 45 Ser Val Ser Val Glu
Arg Ala Leu Ala Ala Lys Pro Gly Leu Asp Thr 50 55 60 Tyr Ser Leu
Gly Gly Gly Gly Ala Ala Arg Val Arg Val Arg Gly Ser 65 70 75 80 Thr
Gly Val Ala Ala Ala Ala Gly Leu His Arg Tyr Leu Arg Asp Phe 85 90
95 Cys Gly Cys His Val Ala Trp Ser Gly Ser Gln Leu Arg Leu Pro Arg
100 105 110 Pro Leu Pro Ala Val Pro Gly Glu Leu Thr Glu Ala Thr Pro
Asn Arg 115 120 125 Tyr Arg Tyr Tyr Gln Asn Val Cys Thr Gln Ser Tyr
Ser Phe Val Trp 130 135 140 Trp Asp Trp Ala Arg Trp Glu Arg Glu Ile
Asp Trp Met Ala Leu Asn 145 150 155 160 Gly Ile Asn Leu Ala Leu Ala
Trp Ser Gly Gln Glu Ala Ile Trp Gln 165 170 175 Arg Val Tyr Leu Ala
Leu Gly Leu Thr Gln Ala Glu Ile Asn Glu Phe 180 185 190 Phe Thr Gly
Pro Ala Phe Leu Ala Trp Gly Arg Met Gly Asn Leu His 195 200 205 Thr
Trp Asp Gly Pro Leu Pro Pro Ser Trp His Ile Lys Gln Leu Tyr 210 215
220 Leu Gln His Arg Val Leu Asp Gln Met Arg Ser Phe Gly Met Thr Pro
225 230 235 240 Val Leu Pro Ala Phe Ala Gly His Val Pro Glu Ala Val
Thr Arg Val 245 250 255 Phe Pro Gln Val Asn Val Thr Lys Met Gly Ser
Trp Gly His Phe Asn 260 265 270 Cys Ser Tyr Ser Cys Ser Phe Leu Leu
Ala Pro Glu Asp Pro Ile Phe 275 280 285 Pro Ile Ile Gly Ser Leu Phe
Leu Arg Glu Leu Ile Lys Glu Phe Gly 290 295 300 Thr Asp His Ile Tyr
Gly Ala Asp Thr Phe Asn Glu Met Gln Pro Pro 305 310 315 320 Ser Ser
Glu Pro Ser Tyr Leu Ala Ala Ala Thr Thr Ala Val Tyr Glu 325 330 335
Ala Met Thr Ala Val Asp Thr Glu Ala Val Trp Leu Leu Gln Gly Trp 340
345 350 Leu Phe Gln His Gln Pro Gln Phe Trp Gly Pro Ala Gln Ile Arg
Ala 355 360 365 Val Leu Gly Ala Val Pro Arg Gly Arg Leu Leu Val Leu
Asp Leu Phe 370 375 380 Ala Glu Ser Gln Pro Val Tyr Thr Arg Thr Ala
Ser Phe Gln Gly Gln 385 390 395 400 Pro Phe Ile Trp Cys Met Leu His
Asn Phe Gly Gly Asn His Gly Leu 405 410 415 Phe Gly Ala Leu Glu Ala
Val Asn Gly Gly Pro Glu Ala Ala Arg Leu 420 425 430 Phe Pro Asn Ser
Thr Met Val Gly Thr Gly Met Ala Pro Glu Gly Ile 435 440 445 Ser Gln
Asn Glu Val Val Tyr Ser Leu Met Ala Glu Leu Gly Trp Arg 450 455 460
Lys Asp Pro Val Pro Asp Leu Ala Ala Trp Val Thr Ser Phe Ala Ala 465
470 475 480 Arg Arg Tyr Gly Val Ser His Pro Asp Ala Gly Ala Ala Trp
Arg Leu 485 490 495 Leu Leu Arg Ser Val Tyr Asn Cys Ser Gly Glu Ala
Cys Arg Gly His 500 505 510 Asn Arg Ser Pro Leu Val Arg Arg Pro Ser
Leu Gln Met Asn Thr Ser 515 520 525 Ile Trp Tyr Asn Arg Ser Asp Val
Phe Glu Ala Trp Arg Leu Leu Leu 530 535 540 Thr Ser Ala Pro Ser Leu
Ala Thr Ser Pro Ala Phe Arg Tyr Asp Leu 545 550 555 560 Leu Asp Leu
Thr Arg Gln Ala Val Gln Glu Leu Val Ser Leu Tyr Tyr 565 570 575 Glu
Glu Ala Arg Ser Ala Tyr Leu Ser Lys Glu Leu Ala Ser Leu Leu 580 585
590 Arg Ala Gly Gly Val Leu Ala Tyr Glu Leu Leu Pro Ala Leu Asp Glu
595 600 605 Val Leu Ala Ser Asp Ser Arg Phe Leu Leu Gly Ser Trp Leu
Glu Gln 610 615 620 Ala Arg Ala Ala Ala Val Ser Glu Ala Glu Ala Asp
Phe Tyr Glu Gln 625 630 635 640 Asn Ser Arg Tyr Gln Leu Thr Leu Trp
Gly Pro Glu Gly Asn Ile Leu 645 650 655 Asp Tyr Ala Asn Lys Gln Leu
Ala Gly Leu Val Ala Asn Tyr Tyr Thr 660 665 670 Pro Arg Trp Arg Leu
Phe Leu Glu Ala Leu Val Asp Ser Val Ala Gln 675 680 685 Gly Ile Pro
Phe Gln Gln His Gln Phe Asp Lys Asn Val Phe Gln Leu 690 695 700 Glu
Gln Ala Phe Val Leu Ser Lys Gln Arg Tyr Pro Ser Gln Pro Arg 705 710
715 720 Gly Asp Thr Val Asp Leu Ala Lys Lys Ile Phe Leu Lys Tyr Tyr
Pro 725 730 735 Arg Trp Val Ala Gly Ser Trp 740 22234DNAHomo
sapiens 2atggaggcgg tggcggtggc cgcggcggtg ggggtccttc tcctggccgg
ggccgggggc 60gcggcaggcg acgaggcccg ggaggcggcg gccgtgcggg cgctcgtggc
ccggctgctg 120gggccaggcc ccgcggccga cttctccgtg tcggtggagc
gcgctctggc tgccaagccg 180ggcttggaca cctacagcct gggcggcggc
ggcgcggcgc gcgtgcgggt gcgcggctcc 240acgggcgtgg cagccgccgc
ggggctgcac cgctacctgc gcgacttctg tggctgccac 300gtggcctggt
ccggctctca gctgcgcctg ccgcggccac tgccagccgt gccgggggag
360ctgaccgagg ccacgcccaa caggtaccgc tattaccaga atgtgtgcac
gcaaagctac 420tctttcgtgt ggtgggactg ggcccggtgg gagcgagaga
tagactggat ggcgctgaat 480ggcatcaacc tggcactggc atggagcggc
caggaggcca tctggcagcg ggtgtacctg 540gccttgggcc tgacccaggc
agagatcaat gagttcttta ctggtcctgc cttcttggca 600tgggggcgaa
tgggcaacct gcacacctgg gatggccccc tgcccccctc ctggcacatc
660aagcagcttt atctgcagca ccgggtcctg gaccagatgc gctccttcgg
catgacccca 720gtgctgcctg cattcgcggg gcatgttccc gaggctgtca
ccagggtgtt ccctcaggtc 780aatgtcacga agatgggcag ttggggccac
tttaactgtt cctactcctg ctccttcctt 840ctggctccgg aagaccccat
attccccatc atcgggagcc tcttcttgcg agagctgatc 900aaagagtttg
gcacagacca catctatggg gccgacactt tcaatgagat gcagccacct
960tcctcagagc cctcctatct tgccgcagcc accactgccg tctatgaggc
catgactgca 1020gtggatactg aggctgtgtg gctgctccaa ggctggctct
tccagcacca gccgcagttc 1080tgggggcccg cccagatcag ggctgtgctg
ggagctgtgc cccgtggccg cctcctggtt 1140ctggacctgt ttgctgagag
ccagcctgtg tatacccgca ctgcctcctt ccaaggccag 1200cccttcatct
ggtgcatgct gcacaacttt gggggaaatc atggtctttt tggagccttg
1260gaggccgtga acggaggccc agaagctgcc cgcctcttcc ccaactccac
aatggtaggc 1320acgggcatgg cccccgaggg catcagccag aacgaagtgg
tctattccct catggctgag 1380ctgggctggc gaaaggaccc agtgccagat
ttggcagcct gggtgaccag ctttgccgcc 1440cggcggtatg gggtctccca
cccggacgca ggggcagcgt ggaggctact gctccggagt 1500gtgtacaact
gctccgggga ggcatgcagg ggccacaatc gtagcccgct ggtcaggcgg
1560ccgtccctac agatgaatac cagcatctgg tacaaccgat ctgatgtgtt
tgaggcctgg 1620cggctgctgc tcacatctgc tccctccctg gccaccagcc
ccgccttccg ctacgacctg 1680ctggacctca ctcggcaggc agtgcaggag
ctggtcagct tgtattatga ggaggcaaga 1740agcgcctatc tgagcaagga
gctggcctcc ttgttgaggg ctggaggcgt cctggcctat 1800gagctgctgc
cggcactgga cgaggtgctg gctagtgaca gccgcttctt gctgggcagc
1860tggctagagc aggcccgagc agcggcagtc agtgaggccg aggccgattt
ctacgagcag 1920aacagccgct accagctgac cttgtggggg ccagaaggca
acatcctgga ctatgccaac 1980aagcagctgg cggggttggt ggccaactac
tacacccctc gctggcggct tttcctggag 2040gcgctggttg acagtgtggc
ccagggcatc cctttccaac agcaccagtt tgacaaaaat 2100gtcttccaac
tggagcaggc cttcgttctc agcaagcaga ggtaccccag ccagccgcga
2160ggagacactg tggacctggc caagaagatc ttcctcaaat attacccccg
ctgggtggcc 2220ggctcttggt gatt 223431132DNAGallus gallus
3gttaagtcct cagacttggc aaggagaatg tagatttcca cagtatatat gttttcacaa
60aaggaaggag agaaacaaaa gaaaatggca ctgactaaac ttcagctagt ggtataggaa
120agtaattctg cttaacagag attgcagtga tctctatgta tgtcctgaag
aattatgttg 180tacttttttc ccccattttt aaatcaaaca gtgctttaca
gaggtcagaa tggtttcttt 240actgtttgtc aattctatta tttcaataca
gaacaatagc ttctataact gaaatatatt 300tgctattgta tattatgatt
gtccctcgaa ccatgaacac tcctccagct gaatttcaca 360attcctctgt
catctgccag gccattaagt tattcatgga agatctttga ggaacactgc
420aagttcatat cataaacaca tttgaaattg agtattgttt tgcattgtat
ggagctatgt 480tttgctgtat cctcagaata aaagtttgtt ataaagcatt
cacacccata aaaagataga 540tttaaatatt ccaactatag gaaagaaagt
gtgtctgctc ttcactctag tctcagttgg 600ctccttcaca tgcacgcttc
tttatttctc ctattttgtc aagaaaataa taggtcaagt 660cttgttctca
tttatgtcct gtctagcgtg gctcagatgc acattgtaca tacaagaagg
720atcaaatgaa acagacttct ggtctgttac tacaaccata gtaataagca
cactaactaa 780taattgctaa ttatgttttc catctccaag gttcccacat
ttttctgttt tcttaaagat 840cccattatct ggttgtaact gaagctcaat
ggaacatgag caatatttcc cagtcttctc 900tcccatccaa cagtcctgat
ggattagcag aacaggcaga aaacacattg ttacccagaa 960ttaaaaacta
atatttgctc tccattcaat ccaaaatgga cctattgaaa ctaaaatcta
1020acccaatccc attaaatgat ttctatggtg tcaaaggtca aacttctgaa
gggaacctgt 1080gggtgggtca caattcagac tatatattcc ccagggctca
gccagtgtct gt 1132410256DNAArtificial SequenceDescription of
Artificial Sequence Synthetic pSIN-OV-1.1-I-rhNaGlu polynucleotide
4ggccgcaaga agaaagctga aaaactctgt cccttccaac aagacccaga gcactgtagt
60atcaggggta aaatgaaaag tatgttatct gctgcatcca gacttcataa aagctggagc
120ttaattcaga aaaaaaatca gaaagaaatt acactgtgag aacaggtgca
attcactttt 180cctttacaca gagtaatact ggtaactcat ggatgaaggc
ttaagggaat gaaattggac 240tcacagtact gagtcatcac actgaaaaat
gcaacctgat acatcagcag aaggtttatg 300ggggaaaaat gcagccttcc
aattaagcca gatatctgta tgaccaagct gctccagaat 360tagtcactca
aaatctctca gattaaatta tcaactgtca ccaaccattc ctatgctgac
420aaggcaattg cttgttctct gtgttcctga tactacaagg ctcttcctga
cttcctaaag 480atgcattata aaaatcttat aattcacatt tctccctaaa
ctttgactca atcatggtat 540gttggcaaat atggtatatt actattcaaa
ttgttttcct tgtacccata tgtaatgggt 600cttgtgaatg tgctcttttg
ttcctttaat cataataaaa acatgtttaa gcaaacactt 660ttcacttgta
gtatttgaag gtaccggatc tcgagccgcc ttcaatgccc ccaaaaccaa
720tccccaggtt tttaactctc ccgattttcc aagtaccata gcccgctgag
agagcgccgc 780ggtaatggga tcccaggacc ccggggaata taagtctgag
ggggacgtaa gcaacccttc 840cttttgtaac agggacaaca tagcccctat
ttccttctta gaaggagagg ttttcccgca 900ataggtctta cacgcggacg
aaatcacctt tatgacggct tccatgcttg atccaccggg 960cgaccggaat
cacgcagagc aaccggaatc acgcctgggg tggaccgctc agtcgtcggg
1020cttccttccc gtcttccaac gactctctga gttctcggta gggtatgttg
gccccctgca 1080gtagggctcc ctccgacgcc actcagcttc tgccctccta
agccgcagcc ccctctacta 1140gggtcatcgt ccgctccccg aataagcgag
acggatgagg acaggatcgc cacgccgcct 1200gtggccgacc actattccct
aacgatcacg tcggggtcac caaatgaagc cttctgcttc 1260atgcatgtgc
tcgtagtcgt cagggaatca acggtccggc catcaaccca ggtgcacacc
1320aatgtggtga atggtcaaat ggcgtttatt gtatcgagct aggcacttaa
atacaatatc 1380tctgcaatgc ggaattcagt ggttcgtcca atccgtgtta
gacccgtctg ttgccttcct 1440aacaaggcac gatcatacca cgatcatacc
accttactcc caccaatcgg catgcacggt 1500gctttttctc tccttataag
gcatgttgct aactcatcgt tacataagca tgttgcaaga 1560ctacaagagt
attgcataag actacatttc cccctcccta tgcaaaagcg aaactactat
1620atcctgaggg gactcctaac cgcgtacaac cgaagccccg cttttcgcct
aaacatgcta 1680ttgtcccctc agtcaagcct tgcccgttac aacccgattc
gcaagccttg ccctccccac 1740attatccgta gcattatttc ctagcagtca
tcagagctac agaagatact ctatgctgta 1800gccaagtcta caagtttact
attcagcgac ctcctatatt ccgcgtgcca gccgatcaat 1860taccaatgcg
cgcttggcgt aatcatggtc atagctgttt cctgtgtgaa attgttatcc
1920gctcacaatt ccacacaaca tacgagccgg aagcataaag tgtaaagcct
ggggtgccta 1980atgagtgagc taactcacat taattgcgtt gcgctcactg
cccgctttcc agtcgggaaa 2040cctgtcgtgc cagctgcatt aatgaatcgg
ccaacgcgcg gggagaggcg gtttgcgtat 2100tgggcgctct tccgcttcct
cgctcactga ctcgctgcgc tcggtcgttc ggctgcggcg 2160agcggtatca
gctcactcaa aggcggtaat acggttatcc acagaatcag gggataacgc
2220aggaaagaac atgtgagcaa aaggccagca aaaggccagg aaccgtaaaa
aggccgcgtt 2280gctggcgttt ttccataggc tccgcccccc tgacgagcat
cacaaaaatc gacgctcaag 2340tcagaggtgg cgaaacccga caggactata
aagataccag gcgtttcccc ctggaagctc 2400cctcgtgcgc tctcctgttc
cgaccctgcc gcttaccgga tacctgtccg cctttctccc 2460ttcgggaagc
gtggcgcttt ctcatagctc acgctgtagg tatctcagtt cggtgtaggt
2520cgttcgctcc aagctgggct gtgtgcacga accccccgtt cagcccgacc
gctgcgcctt 2580atccggtaac tatcgtcttg agtccaaccc ggtaagacac
gacttatcgc cactggcagc 2640agccactggt aacaggatta gcagagcgag
gtatgtaggc ggtgctacag agttcttgaa 2700gtggtggcct aactacggct
acactagaag gacagtattt ggtatctgcg ctctgctgaa 2760gccagttacc
ttcggaaaaa gagttggtag ctcttgatcc ggcaaacaaa ccaccgctgg
2820tagcggtggt ttttttgttt gcaagcagca gattacgcgc agaaaaaaag
gatctcaaga 2880agatcctttg atcttttcta cggggtctga cgctcagtgg
aacgaaaact cacgttaagg 2940gattttggtc atgagattat caaaaaggat
cttcacctag atccttttaa attaaaaatg 3000aagttttaaa tcaatctaaa
gtatatatga gtaaacttgg tctgacagtt accaatgctt 3060aatcagtgag
gcacctatct cagcgatctg tctatttcgt tcatccatag ttgcctgact
3120ccccgtcgtg tagataacta cgatacggga gggcttacca tctggcccca
gtgctgcaat 3180gataccgcga gacccacgct caccggctcc agatttatca
gcaataaacc agccagccgg 3240aagggccgag cgcagaagtg gtcctgcaac
tttatccgcc tccatccagt ctattaattg 3300ttgccgggaa gctagagtaa
gtagttcgcc agttaatagt ttgcgcaacg ttgttgccat 3360tgctacaggc
atcgtggtgt cacgctcgtc gtttggtatg gcttcattca gctccggttc
3420ccaacgatca aggcgagtta catgatcccc catgttgtgc aaaaaagcgg
ttagctcctt 3480cggtcctccg atcgttgtca gaagtaagtt ggccgcagtg
ttatcactca tggttatggc 3540agcactgcat aattctctta ctgtcatgcc
atccgtaaga tgcttttctg tgactggtga 3600gtactcaacc aagtcattct
gagaatagtg tatgcggcga ccgagttgct cttgcccggc 3660gtcaatacgg
gataataccg cgccacatag cagaacttta aaagtgctca tcattggaaa
3720acgttcttcg gggcgaaaac tctcaaggat cttaccgctg ttgagatcca
gttcgatgta 3780acccactcgt gcacccaact gatcttcagc atcttttact
ttcaccagcg tttctgggtg 3840agcaaaaaca ggaaggcaaa atgccgcaaa
aaagggaata agggcgacac ggaaatgttg 3900aatactcata ctcttccttt
ttcaatatta ttgaagcatt tatcagggtt attgtctcat 3960gagcggatac
atatttgaat gtatttagaa aaataaacaa ataggggttc cgcgcacatt
4020tccccgaaaa gtgccacctg acgcgccctg tagcggcgca ttaagcgcgg
cgggtgtggt 4080ggttacgcgc agcgtgaccg ctacacttgc cagcgcccta
gcgcccgctc ctttcgcttt 4140cttcccttcc tttctcgcca cgttcgccgg
ctttccccgt caagctctaa atcgggggct 4200ccctttaggg ttccgattta
gtgctttacg gcacctcgac cccaaaaaac ttgattaggg 4260tgatggttca
cgtagtgggc catcgccctg atagacggtt tttcgccctt tgacgttgga
4320gtccacgttc tttaatagtg gactcttgtt ccaaactgga acaacactca
accctatctc 4380ggtctattct tttgatttat aagggatttt gccgatttcg
gcctattggt taaaaaatga 4440gctgatttaa caaaaattta acgcgaattt
taacaaaata ttaacgctta caatttccat 4500tcgccattca ggctgcgcaa
ctgttgggaa gggcgatcgg tgcgggcctc ttcgctatta 4560cgccagctgg
cgaaaggggg atgtgctgca aggcgattaa gttgggtaac gccagggttt
4620tcccagtcac gacgttgtaa aacgacggcc agtgagcgcg tattccctaa
cgatcacgtc 4680ggggtcacca aatgaagcct tctgcttcat gcatgtgctc
gtagtcgtca gggaatcaac 4740ggtccggcca tcaacccagg tgcacaccaa
tgtggtgaat ggtcaaatgg cgtttattgt 4800atcgagctag gcacttaaat
acaatatctc tgcaatgcgg aattcagtgg ttcgtccaat 4860ccgtccccct
ccctatgcaa aagcgaaact actatatcct gaggggactc ctaaccgcgt
4920acaaccgaag ccccgctttt cgcctaaaca tgctattgtc ccctcagtca
agccttgccc 4980gttacaaccc gattcgcaag ccttgccctc cccacattat
ccgtagcatt atttcctagc 5040agtcatcaga gctacagaag atactctatg
ctgtagccaa gtctacaagt ttactattca 5100gcgacctcct atattccgcg
tgccagccga tcaattacca atccaaccag ctatcacacg 5160gaatacaaga
actcgcctac gctcttcttt cgggctgctt ataagcctcc tgtaattttt
5220ttatattcct cgttaagtcc tcagacttgg caaggagaat gtagatttcc
acagtatata 5280tgttttcaca aaaggaagga gagaaacaaa agaaaatggc
actgactaaa cttcagctag 5340tggtatagga aagtaattct gcttaacaga
gattgcagtg atctctatgt atgtcctgaa 5400gaattatgtt gtactttttt
cccccatttt taaatcaaac agtgctttac agaggtcaga 5460atggtttctt
tactgtttgt caattctatt atttcaatac agaacaatag cttctataac
5520tgaaatatat ttgctattgt atattatgat tgtccctcga accatgaaca
ctcctccagc 5580tgaatttcac aattcctctg tcatctgcca ggccattaag
ttattcatgg aagatctttg 5640aggaacactg caagttcata tcataaacac
atttgaaatt gagtattgtt ttgcattgta 5700tggagctatg ttttgctgta
tcctcagaat aaaagtttgt tataaagcat tcacacccat 5760aaaaagatag
atttaaatat tccaactata ggaaagaaag tgtgtctgct cttcactcta
5820gtctcagttg gctccttcac atgcacgctt ctttatttct cctattttgt
caagaaaata 5880ataggtcaag tcttgttctc atttatgtcc tgtctagcgt
ggctcagatg cacattgtac 5940atacaagaag gatcaaatga aacagacttc
tggtctgtta ctacaaccat agtaataagc 6000acactaacta ataattgcta
attatgtttt ccatctccaa ggttcccaca tttttctgtt 6060ttcttaaaga
tcccattatc tggttgtaac tgaagctcaa tggaacatga gcaatatttc
6120ccagtcttct ctcccatcca acagtcctga tggattagca gaacaggcag
aaaacacatt 6180gttacccaga attaaaaact aatatttgct ctccattcaa
tccaaaatgg acctattgaa 6240actaaaatct aacccaatcc cattaaatga
tttctatggt gtcaaaggtc aaacttctga 6300agggaacctg tgggtgggtc
acaattcaga ctatatattc cccagggctc agccagtgtc 6360tgtacataca
gctagaaagc tgtattgcct ttagcagtca agctcgaaag gtaagcaact
6420ctctggaatt accttctctc tatattagct cttacttgca cctaaacttt
aaaaaattaa 6480caattattgt gctatgtgtt gtatctttaa gggtgaagta
cctgcgtgat accccctata 6540aaaacttctc acctgtgtat gcattctgca
ctattttatt atgtgtaaaa gctttgtgtt 6600tgttttcagg aggcttattc
tttgtgctta
aaatatgttt ttaatttcag aacatcttat 6660cctgtcgttc actatctgat
atgctttgca gtttgcttga ttaacttcta gccctacaga 6720gtgcacagag
agcaaaatca tggtgttcag tgaattctgg ggagttattt taatgtgaaa
6780attctctaga agtttaattc ctgcaaagtg cagctgctga tcactacaca
agataaaaat 6840gtggggggtg cataaacgta tattcttaca ataatagata
catgtgaact tatatacaga 6900aaagaaaatg agaaaaatgt gtgtgtgtat
actcacacac gtggtcagta aaaacttttg 6960aggggtttaa tacagaaaat
ccaatcctga ggccccagca ctcagtacgc atataaaggg 7020ctgggctctg
aaggacttct gactttcaca gattatataa atctcaggaa agcaactaga
7080ttcatgctgg ctccaaaagc tgtgctttat ataagcacac tggctataca
atagttgtac 7140agttcagctc tttataatag aaacagacag aacaagtata
aatcttctat tggtctatgt 7200catgaacaag aattcattca gtggctctgt
tttatagtaa acattgctat tttatcatgt 7260ctgcatttct cttctgtctg
aatgtcacca ctaaaattta actccacaga aagtttatac 7320tacagtacac
atgcatatct ttgagcaaag caaaccatac ctgaaagtgc aatagagcag
7380aatatgaatt acatgcgtgt ctttctccta gactacatga ccccatataa
attacattcc 7440ttatctattc tgccatcacc aaaacaaagg taaaaatact
tttgaagatc tactcatagc 7500aagtagtgtg caacaaacag atatttctct
acatttattt ttagggaata aaaataagaa 7560ataaaatagt cagcaagcct
ctgctttctc atatatctgt ccaaacctaa agtttactga 7620aatttgctct
ttgaatttcc agttttgcaa gcctatcaga ttgtgtttta atcagaggta
7680ctgaaaagta tcaatgaatt ctagctttca ctgaacaaaa atatgtagag
gcaactggct 7740tctgggacag tttgctaccc aaaagacaac tgaatgcaaa
tacataaata gatttatgaa 7800tatggttttg aacatgcaca tgagaggtgg
atatagcaac agacacatta ccacagaatt 7860actttaaaac tacttgttaa
catttaattg cctaaaaact gctcgtaatt tactgttgta 7920gcctaccata
gagtaccctg catggtacta tgtacagcat tccatcctta cattttcact
7980gttctgctgt ttgctctaga caactcagag ttcaccatgg aggcggtggc
ggtggccgcg 8040gcggtggggg tccttctcct ggccggggcc gggggcgcgg
caggcgacga ggcccgggag 8100gcggcggccg tgcgggcgct cgtggcccgg
ctgctggggc caggccccgc ggccgacttc 8160tccgtgtcgg tggagcgcgc
tctggctgcc aagccgggct tggacaccta cagcctgggc 8220ggcggcggcg
cggcgcgcgt gcgggtgcgc ggctccacgg gcgtggcagc cgccgcgggg
8280ctgcaccgct acctgcgcga cttctgtggc tgccacgtgg cctggtccgg
ctctcagctg 8340cgcctgccgc ggccactgcc agccgtgccg ggggagctga
ccgaggccac gcccaacagg 8400taccgctatt accagaatgt gtgcacgcaa
agctactctt tcgtgtggtg ggactgggcc 8460cggtgggagc gagagataga
ctggatggcg ctgaatggca tcaacctggc actggcatgg 8520agcggccagg
aggccatctg gcagcgggtg tacctggcct tgggcctgac ccaggcagag
8580atcaatgagt tctttactgg tcctgccttc ttggcatggg ggcgaatggg
caacctgcac 8640acctgggatg gccccctgcc cccctcctgg cacatcaagc
agctttatct gcagcaccgg 8700gtcctggacc agatgcgctc cttcggcatg
accccagtgc tgcctgcatt cgcggggcat 8760gttcccgagg ctgtcaccag
ggtgttccct caggtcaatg tcacgaagat gggcagttgg 8820ggccacttta
actgttccta ctcctgctcc ttccttctgg ctccggaaga ccccatattc
8880cccatcatcg ggagcctctt cttgcgagag ctgatcaaag agtttggcac
agaccacatc 8940tatggggccg acactttcaa tgagatgcag ccaccttcct
cagagccctc ctatcttgcc 9000gcagccacca ctgccgtcta tgaggccatg
actgcagtgg atactgaggc tgtgtggctg 9060ctccaaggct ggctcttcca
gcaccagccg cagttctggg ggcccgccca gatcagggct 9120gtgctgggag
ctgtgccccg tggccgcctc ctggttctgg acctgtttgc tgagagccag
9180cctgtgtata cccgcactgc ctccttccaa ggccagccct tcatctggtg
catgctgcac 9240aactttgggg gaaatcatgg tctttttgga gccttggagg
ccgtgaacgg aggcccagaa 9300gctgcccgcc tcttccccaa ctccacaatg
gtaggcacgg gcatggcccc cgagggcatc 9360agccagaacg aagtggtcta
ttccctcatg gctgagctgg gctggcgaaa ggacccagtg 9420ccagatttgg
cagcctgggt gaccagcttt gccgcccggc ggtatggggt ctcccacccg
9480gacgcagggg cagcgtggag gctactgctc cggagtgtgt acaactgctc
cggggaggca 9540tgcaggggcc acaatcgtag cccgctggtc aggcggccgt
ccctacagat gaataccagc 9600atctggtaca accgatctga tgtgtttgag
gcctggcggc tgctgctcac atctgctccc 9660tccctggcca ccagccccgc
cttccgctac gacctgctgg acctcactcg gcaggcagtg 9720caggagctgg
tcagcttgta ttatgaggag gcaagaagcg cctatctgag caaggagctg
9780gcctccttgt tgagggctgg aggcgtcctg gcctatgagc tgctgccggc
actggacgag 9840gtgctggcta gtgacagccg cttcttgctg ggcagctggc
tagagcaggc ccgagcagcg 9900gcagtcagtg aggccgaggc cgatttctac
gagcagaaca gccgctacca gctgaccttg 9960tgggggccag aaggcaacat
cctggactat gccaacaagc agctggcggg gttggtggcc 10020aactactaca
cccctcgctg gcggcttttc ctggaggcgc tggttgacag tgtggcccag
10080ggcatccctt tccaacagca ccagtttgac aaaaatgtct tccaactgga
gcaggccttc 10140gttctcagca agcagaggta ccccagccag ccgcgaggag
acactgtgga cctggccaag 10200aagatcttcc tcaaatatta cccccgctgg
gtggccggct cttggtgatt cgaagc 10256512PRTArtificial
SequenceDescription of Artificial Sequence Synthetic Transferrin
receptor ligand (TfRL) peptide 5Thr His Arg Pro Pro Met Trp Ser Pro
Val Trp Pro 1 5 10 68PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 6Asp Asp Asp Asp Asp Asp Asp
Asp 1 5
* * * * *