U.S. patent application number 11/234876 was filed with the patent office on 2006-06-08 for methods and compositions for targeting proteins across the blood brain barrier.
This patent application is currently assigned to ZyStor Therapeutics, Inc.. Invention is credited to Jonathan LeBowitz.
Application Number | 20060121018 11/234876 |
Document ID | / |
Family ID | 26834495 |
Filed Date | 2006-06-08 |
United States Patent
Application |
20060121018 |
Kind Code |
A1 |
LeBowitz; Jonathan |
June 8, 2006 |
Methods and compositions for targeting proteins across the blood
brain barrier
Abstract
Disclosed are methods and compositions for targeting therapeutic
proteins to the brain. Methods and compositions of the invention
involve associating an IGF moiety with a therapeutic protein in
order to target the therapeutic protein to the brain. Soluble
fusion proteins that include an IGF targeting moiety are
transported to neural tissue in the brain from blood. Methods and
compositions of the invention include therapeutic applications for
treating lysosomal storage diseases. The invention also provides
nucleic acids and cells for expressing IGF fusion proteins.
Inventors: |
LeBowitz; Jonathan;
(Whitefish Bay, WI) |
Correspondence
Address: |
KIRKPATRICK & LOCKHART NICHOLSON GRAHAM LLP
STATE STREET FINANCIAL CENTER
ONE LINCOLN STREET
BOSTON
MA
02111-2950
US
|
Assignee: |
ZyStor Therapeutics, Inc.
Milwaukee
WI
|
Family ID: |
26834495 |
Appl. No.: |
11/234876 |
Filed: |
September 23, 2005 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10136639 |
Apr 30, 2002 |
|
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11234876 |
Sep 23, 2005 |
|
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60329650 |
Oct 16, 2001 |
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Current U.S.
Class: |
424/94.61 ;
514/1.2; 514/17.7; 514/8.5; 514/8.6 |
Current CPC
Class: |
C07K 2319/02 20130101;
A61K 38/30 20130101; C07K 2319/74 20130101; C07K 2319/50 20130101;
A61K 2300/00 20130101; A61K 38/30 20130101; C07K 2319/00
20130101 |
Class at
Publication: |
424/094.61 ;
514/012 |
International
Class: |
A61K 38/47 20060101
A61K038/47; A61K 38/30 20060101 A61K038/30 |
Claims
1-6. (canceled)
7. A method for targeting a therapeutic to lysosomes of cells in
the central nervous system, the method comprising the step of
administering a targeted therapeutic comprising: (i) a therapeutic
agent that is therapeutically active in a lysosome and (ii) means
for traversing the blood-brain barrier.
8. The method of claim 7, wherein the means for traversing the
blood-brain barrier comprises an insulin-like growth factor (IGF)
moiety.
9. The method of claim 8, wherein the IGF moiety is an intact IGF-I
protein.
10. The method of claim 8, wherein the IGF moiety comprises at
least one of the A, B, C, or D domains, or the C-terminal region or
a portion thereof, of either IGF-I or IGF-II.
11. The method of claim 7, wherein the means for traversing the
blood-brain barrier comprises a polypeptide sufficiently
duplicative of IGF-II such that it binds an extracellular domain of
human cation-independent mannose-6-phosphate/IGF-II receptor.
12. The method of claim 7, wherein the therapeutic agent is a
lysosomal enzyme.
13. A method for targeting a therapeutic to lysosomes of cells of
the central nervous system, the method comprising the step of
administering a targeted therapeutic to the patient, wherein the
targeted therapeutic comprises a therapeutic agent that is
therapeutically active in lysosome and a targeting moiety
comprising a polypeptide comprising a sequence sufficiently
duplicative of at least one of the A, B, C, or D domains, or the
C-terminal region or a portion thereof, of human IGF-I to be
targeted to the central nervous system.
14. The method of claim 13, wherein the targeting moiety comprises
a mutein of the A domain of human IGF-I in which amino acids 55 and
56 are changed.
15. The method of claim 14, wherein amino acids 55 and 56 of human
IGF-I are changed to hydrophobic amino acids.
16. The method of claim 15, wherein amino acids 55 and 56 are
changed to Ala and Leu, respectively.
17. The method of claim 13, wherein the targeting moiety comprises
a polypeptide sufficiently duplicative of IGF-II such that it binds
an extracellular domain of human cation-independent
mannose-6-phosphate IGF-II receptor.
18. The method of claim 13, wherein the therapeutic agent is a
lysosomal enzyme.
19. A method for targeting a therapeutic to lysosomes of cells of
the central nervous system, the method comprising the step of
administering a targeted therapeutic to a patient, wherein the
targeted therapeutic comprises a therapeutic agent that is
therapeutically active in a human lysosome and a targeting moiety
comprising a polypeptide comprising a sequence of human IGF-I or a
mutein of the sequence of human IGF-I, wherein the sequence is
selected from a group consisting of: (i) amino acids 1 to 25 of
human IGF-I; (ii) amino acids 25 to 40 of human IGF-I; (iii) amino
acids 40 to 65 of human IGF-I; and (iv) amino acids 65 to 70 of
human IGF-I.
20. The method of claim 19, wherein targeting moiety comprises a
mutein of the sequence of amino acids 1 to 25 of human IGF-I, in
which amino acid 24 is changed to Leu.
21. The method of claim 19, wherein targeting moiety comprises a
mutein of the sequence of amino acids 1 to 25 of human IGF-I, in
which amino acids 1-3 are deleted.
22. The method of claim 19, wherein targeting moiety comprises a
mutein of the sequence of amino acids 40 to 65 of human IGF-I, in
which amino acid 60 is changed to Leu.
23. The method of claim 19, wherein targeting moiety comprises a
mutein of the sequence of amino acids 40 to 65 of human IGF-I, in
which amino acids 55 and 56 are changed to hydrophobic amino
acids.
24. The method of claim 19, wherein targeting moiety comprises a
mutein of the sequence of amino acids 40 to 65 of human IGF-I, in
which amino acids 55 and 56 are changed to Ala and Leu,
respectively.
25. The method of claim 19, wherein the targeting moiety comprises
a polypeptide sufficiently duplicative of IGF-II such that it binds
an extracellular domain of human cation-independent
mannose-6-phosphate/IGF-II receptor.
26. The method of claim 19, wherein the therapeutic agent is a
lysosomal enzyme.
Description
RELATED APPLICATIONS
[0001] This application claims the benefit of U.S. Ser. No.
60/329,650, filed Oct. 16, 2001, the entire disclosure of which is
incorporated herein by reference.
FIELD OF THE INVENTION
[0002] This invention provides a means for specifically delivering
proteins to the brain. The ability to target proteins to the brain
is of great utility in the treatment of neurological diseases.
Methods and compositions of the invention are useful to target
proteins to cells across the blood brain barrier, and in
particular, to target proteins to the lysosomes of cells in the
CNS, including neuronal cells, macrophage cells, and other cell
types. Accordingly, the invention provides methods and compositions
to deliver therapeutically useful proteins to treat lysosomal
storage diseases that affect the CNS.
BACKGROUND
[0003] The blood-brain barrier maintains a homeostatic environment
in the central nervous system (CNS). The capillaries that supply
the blood to the brain have tight junctions which block passage of
most molecules through the capillary endothelial membranes. While
the membranes do allow passage of lipid soluble materials, water
soluble materials such as glucose, proteins and amino acids do not
pass through the blood brain barrier. Mediated transport mechanisms
exist to transport glucose and essential amino acids across the
blood brain barrier. Active transport mechanisms remove molecules
which become in excess, such as potassium, from the brain. However,
the blood brain barrier impedes the delivery of drugs to the
CNS.
[0004] Many neurological diseases result from cellular defects in
the CNS. In particular, many lysosomal storage diseases affect
cells of the CNS and result in mild to serious neurological
symptoms. Accordingly, the ability to deliver therapeutic
compositions to the CNS is an important aspect of an effective
treatment for many diseases, including many lysosomal storage
diseases.
[0005] Methods have been designed to deliver needed drugs to the
CNS such as direct delivery within the CNS by intrathecal delivery.
However, methods are not available in the art to efficiently
deliver drugs, and particularly protein-based drugs, from the blood
stream to the CNS through the blood brain barrier.
[0006] Therefore, there is a need in the art for methods to deliver
proteins to the brain parenchyma on the CNS side of the blood brain
barrier, and in particular to deliver proteins to the lysosomes of
cells in the CNS.
SUMMARY OF THE INVENTION
[0007] The present invention provides general methods and
compositions for targeting compositions from the blood stream to
the brain or CNS. According to the invention, an IGF moiety is used
to target a molecule from the blood stream to the brain parenchyma
on the other side of the blood brain barrier. Preferred molecules
are therapeutic polypeptides.
[0008] Accordingly, the invention relates in one aspect to a
protein including a therapeutic agent attached to an insulin-like
growth factor (IGF) moiety or tag. In one embodiment, the protein
is expressed as a fusion protein along with the IGF tag. In a
preferred embodiment, the fusion protein also includes a lysosomal
targeting portion sufficiently duplicative of IGF-II such that the
targeting portion binds the cation independent
mannose-6-phosphate/IGF-II receptor to mediate uptake by a
lysosome. In another embodiment, the fusion protein also comprises
mannose-6-phosphate in order to target the protein to the
lysosomes.
[0009] Preferred IGF moieties or tags are IGF-I or IGF-II tags.
Most preferred IGF tags are IGF-I tags. In one aspect, the IGF tag
is an intact IGF-I or IGF-II protein. Alternatively, an IGF tag is
a portion of an IGF-I or IGF-II protein that is sufficient for
targeting through the blood brain barrier. Preferred portions
comprise at least one of the A, B, C, or D domains, or the
C-terminal region or a portion thereof, of either IGF-I or IGF-II.
In one embodiment, an IGF tag includes both an A and a B domain.
According to the invention, the A and B domains provide core
structural features of a preferred IGF moiety. The A and B domains
may be linked by a linker peptide. Alternatively, the A and B
domains may be provided as separate peptides that dimerize to form
an IGF tag. Preferably, A and B domains from the same IGF protein
are used. However, an A domain from IGF-I can be associated with a
B domain from IGF-II. Similarly, an A domain from IGF-II can be
associated with a B domain from IGF-I. Accordingly, composition of
the invention include chimeric IGF-I/IGF-II molecules. For example,
an A domain from one IGF protein can be joined to the C and B
domains of another IGF protein. Alternative combinations of A, B,
and C domains are also useful. In further embodiments, the A domain
of one IGF protein can be joined directly to the domain of another
IGF protein, for example by using an amino acid bridge such as a
two amino acid bridge.
[0010] A most preferred IGF moiety comprises an IGF-I portion
selected from the group consisting of IGF-I fragments from about
residue 1 to about residue 25, IGF-I fragments from about residue
25 to about residue 40, IGF-I fragments from about residue 40 to
about residue 65, and IGF-I fragments from about residue 65 to
about residue 70 of the IGF-I sequence shown in FIG. 1. Alternative
preferred regions of IGF-I and IGF-II comprise regions of homology
between IGF-I and IGF-II such as those shown in FIG. 1 for human
IGF-I and IGF-II. The sequences shown in FIG. 1 relate to mature
IGF-I and IGF-II proteins. Specific IGF variants described herein
refer to the mature amino acid sequence numbering shown in FIG. 1.
In a further embodiment, an IGF tag comprises the C-terminal
fragment of an IGF protein, for example the region C-terminal to
the D domain shown in FIG. 2. A preferred IGF tag includes an IGF-I
C-terminal fragment. In addition, according to the invention, IGF
tags include peptide tags with a sequence that is sufficiently
duplicative of the IGF tags described herein to effectively target
compositions of the invention to the brain parenchyma across the
blood brain barrier. In some embodiments, an IGF tag includes at
least one peptide sequence from an IGF-I protein and one from an
IGF-II protein.
[0011] Most preferred IGF tags are based on human IGF proteins.
However, IGF tags based on IGF proteins from other mammals, such as
mouse, rabbit, monkey, and pig IGF proteins, are also useful
according to the invention. Preferred IGF tags such as the IGF
fragments, peptides, or domains described herein are between 1 and
100 amino acids long, more preferably between 10 and 50 amino acids
long, and even more preferably about 25 amino acids long, and are
sufficient for targeting associated peptides to the brain.
Preferred IGF fragments, peptides, or domains are based on the
mature IGF-I and IGF-II sequences.
[0012] IGF tags of the invention can be fused to a therapeutic
peptide at its N-terminus, C-terminus, within the body of the
therapeutic peptide, or a combination of the above. When an IGF
moiety is fused to the N-terminus of a therapeutic protein, an IGF
signal peptide is preferably included in the expression construct.
However, an IGF signal peptide can also be included at the
N-terminus when the IGF targeting moiety is located at the
C-terminus or within the body of the therapeutic protein. In a
preferred embodiment, the IGF tag is fused to the C-terminal end of
a peptide. In one embodiment, a first domain of an IGF tag is fused
to a therapeutic peptide, and a second domain of the IGF tag is
provided in a form that dimerizes with the first domain resulting
in a protein that is targeted to the brain. For example, the
therapeutic peptide can be fused to the A domain of an IGF protein,
and dimerized with a B domain that is provided separately.
Alternatively, the therapeutic peptide can be fused to the B domain
of an IGF protein, and dimerized with an A domain that is provided
separately.
[0013] The invention also relates to methods for identifying
IGF-based peptide fragments that can reach neuronal tissue from
blood and are useful to target an associated protein to the brain
or CNS. According to the invention, the effectiveness of IGF-based
tags can be assayed using methods described herein, such as
localization assays based on radioactive labels or histochemical
staining.
[0014] The invention also relates to a nucleic acid encoding an IGF
tag or a protein fused to an IGF tag, and to a cell (e.g., a cell
cultured in vitro including a mammalian cell culture such as a CHO
cell culture, and/or a unicellular organism such as E. coli or
Leishmania) containing such a nucleic acid.
[0015] In another aspect, the invention relates to a method of
producing a therapeutic agent for targeting across the blood brain
barrier, and in particular to the lysosomes of cells in the CNS.
The agent is produced by culturing a cell expressing a nucleic acid
encoding a protein containing both a therapeutic agent and an IGF
tag effective to target the protein across the blood brain barrier.
The protein is then harvested (e.g. from the milieu about the cell,
or by lysing the cell). The invention also relates to protein
compositions described herein.
[0016] The invention also relates to methods of treating a patient
(e.g. a patient with a disorder in the CNS, and preferably a CNS
disorder resulting from a lysosomal storage disorder) by
administering, for example, a protein including a therapeutic agent
effective in the mammalian CNS and an IGF tag to target the protein
to the CNS. Preferably, the protein also comprises a lysosomal
targeting portion such as those described in attorney docket number
SYM-007 entitled "Methods and Compositions for Lysosomal Targeting"
filed on Apr. 30, 2002, or mannose-6-phosphate to target the
protein to the lysosomes of deficient cells in the CNS. Similarly,
the invention relates to methods of treating a patient by
administering a nucleic acid encoding such a protein and/or by
administering a cell (e.g. a human cell, or an organism such as
Leishmania) containing a nucleic acid encoding such a protein.
BRIEF DESCRIPTION OF THE DRAWINGS
[0017] FIG. 1 shows a sequence alignment of mature human IGF-I and
IGF-II, indicating regions of homology and the A, B, C, and D
domains.
[0018] FIG. 2 is a two-dimensional representation of an IGF protein
showing the signal sequence, the A, B, C, and D domains, and the C
terminal sequence.
[0019] FIG. 3 is a depiction the 3 dimensional structure of an IGF
protein.
[0020] FIG. 4 shows protein (FIG. 4A) and nucleic acid (FIG. 4B)
sequences for human IGF-I mRNA.
DETAILED DESCRIPTION OF THE INVENTION
CNS Targeting Portion
[0021] According to the invention, an IGF moiety is useful for
targeting a composition, preferably a protein composition, to the
CNS, across the blood brain barrier. Preferably, an IGF tag is used
to target a composition to the brain parenchyma. According to the
invention, a composition may enter the CNS or brain parenchyma
either directly across the blood-brain barrier (the BBB) or
indirectly across the blood-cerebrospinal fluid barrier (the BCB).
The BBB is formed by capillary endothelial cells and the BCB is
formed by epithelial cells of the choroid plexus. Transport across
either barrier typically involves transcytosis. According to the
invention, a composition that is targeted across the BCB to the CSF
can subsequently reach the brain parenchyma. The CSF and brain
parenchyma are separated by the ependyma, and diffusion or bulk
flow can transport substances between these two compartments.
[0022] The invention exploits, in part, the recognition that [125I]
IGF-I and IGF-II can be detected in the brain when infused into the
carotid artery, and that IGF-I and analogs administered
subcutaneously can be found in the cerebrospinal fluid. According
to the invention, this suggests that both can traverse the BBB or
BCB. According to the invention, the observed saturation of the
transport process suggests that the process is carrier
mediated.
[0023] However, experimental analysis using a series of IGF-I
analogs suggests that the IGF-I receptor, the IGF-II receptor, and
IGF binding proteins-1, -3, -4, or 5 do not play a role in the
blood brain barrier transport.
[0024] According to one aspect of the invention, preferred
therapeutic compositions include a therapeutic peptide fused to an
IGF tag. Preferred therapeutic composition include IGF-I tags that
will direct LSD (lysosomal storage disease) proteins to which they
are fused across the blood brain barrier. In this instance, the tag
will not necessarily direct the protein to the lysosome of multiple
cell types. However, by expressing such fusion proteins in
mammalian cell culture systems, the invention exploits the
endogenous M6P signal for lysosomal localization and uses the IGF-I
tag to traverse the blood brain barrier. In preferred embodiments
of the invention, a human IGF-I tag is used. In alternative
embodiments, methods and compositions of the invention involve
using allelic, species or other sequence variants of an IGF-I tag.
Preferred sequence variants include mutations that lessen binding
of the IGF tag to the IGF-I receptor and/or IGF binding proteins
such as Leu.sup.60-IGF-I, or Leu.sup.24 IGF-I which have diminished
binding to the IGF-I receptor or .DELTA.1-3 IGF-I which has
diminished binding to IGF-binding proteins. Additional useful
sequence variants include IGF-I variants with amino acid
replacements of Arg.sup.55 and Arg.sup.56. Other mutant IGF protein
tags with similar properties are also useful.
[0025] IGF-II based tags are also useful to target proteins to the
brain. IGF-II has been reported to be transported across the blood
brain barrier via transcytosis (Bickel et al. (2001) Adv. Drug
Deliv. Rev. 46(1-3):247-79). According to the invention, preferred
IGF-II-based tags target proteins to the brain and also target
proteins to the lysosome via receptor binding in order to treat
neurological symptoms associated with lysosomal storage diseases.
Preferred variants of IGF-II have an amino acid replacement at
Leu.sup.24.
[0026] In another aspect of the invention, chimeric tags are used
that include fragments of IGF-I and IGF-II, conferring preferred
functional properties of each protein. In one embodiment, the
retained portion of IGF-II includes regions of IGF-II known to be
critical for binding to the IGF-II M6P receptor while the remainder
of IGF-II would be substituted for the corresponding regions of
IGF-I. This embodiment, is particularly useful where IGF-I is more
active as a tag for traversing the blood brain barrier. In this
embodiment, the tag has optimized activity for lysosomal targeting
in addition to brain targeting. A recombinant form of this
embodiment could be made in any expression system.
[0027] In a further aspect of the invention, a useful recombinant
LSD protein includes any one of the different IGF-based lysosomal
targeting tags described in attorney docket number SYM-007 entitled
"Methods and Compositions for Lysosomal Targeting" filed on Apr.
30, 2002.
[0028] In preferred embodiments, recombinant proteins of the
invention including IGF-II tags are expressed in a mammalian
expression system such as a CHO cell expression system. According
to the invention, the endogenous M6P signal added in the mammalian
cell culture enhances the lysosomal targeting that may be provided
by an IGF-II tag.
[0029] According to the invention, useful minimal IGF tags and
variant IGF tags can be identified based on known IGF-I and IGF-II
sequences by testing minimal or variant IGF fragments in a CNS
localization assay such as one described herein.
[0030] A preferred IGF tag is sufficiently duplicative of IGF-I to
be targeted to the brain, but has reduced binding affinity for the
IGF-I receptor thereby removing the mitogenic properties of IGF-I.
However, a preferred IGF tag does bind to the IGF-II receptor in
order to be targeted to lysosomes. Accordingly, in one embodiment,
an IGF tag is based on the IGF-I sequence but includes two
hydrophobic IGF-II residues at positions 54 and 55 instead of the
IGF-I Arg residues at these positions.
Structure of IGF-II
[0031] NMR structures of IGF-II have been solved by two groups
(see, e.g., Protein Data Bank record 1IGL). The general features of
the IGF-II structure are similar to IGF-I and insulin. The A and B
domains of IGF-II correspond to the A and B chains of insulin.
Secondary structural features include an alpha helix from residues
11-21 of the B region connected by a reverse turn in residues 22-25
to a short beta strand in residues 26-28. Residues 25-27 appear to
form a small antiparallel beta sheet; residues 59-61 and residues
26-28 may also participate in intermolecular beta-sheet formation.
In the A domain of IGF-II, alpha helices spanning residues 42-49
and 53-59 are arranged in an antiparallel configuration
perpendicular to the B-domain helix. Hydrophobic clusters formed by
two of the three disulfide bridges and conserved hydrophobic
residues stabilize these secondary structure features. The N and C
termini remain poorly defined as is the region between residues
31-40.
[0032] IGF-II binds to the IGF-II/M6P and IGF-I receptors with
relatively high affinity and binds with lower affinity to the
insulin receptor. IGF-II also interacts with a number if serum
IGFBPs.
Binding to the IGF-II/M6P Receptor
[0033] Substitution of IGF-II residues 48-50 (Phe Arg Ser) with the
corresponding residues from insulin, (Thr Ser Ile), or substitution
of residues 54-55 (Ala Leu) with the corresponding residues from
IGF-I (Arg Arg) result in loss of binding to the IGF-II/M6P
receptor but retention of binding to the IGF-I and insulin
receptors.
[0034] IGF-I and IGF-II share identical sequences and structures in
the region of residues 48-50 yet have a 1000-fold difference in
affinity for the IGF-II receptor. The NMR structure reveals a
structural difference between IGF-I and IGF-II in the region of
IGF-II residues 53-58 (IGF-I residues 54-59): the alpha-helix is
better defined in IGF-II than in IGF-I and, unlike IGF-I, there is
no bend in the backbone around residues 53 and 54. This structural
difference correlates with the substitution of Ala 54 and Leu 55 in
IGF-II with Arg 55 and Arg 56 in IGF-I. It is possible either that
binding to the IGF-II receptor is disrupted directly by the
presence of charged residues in this region or that changes in the
structure engendered by the charged residues yield the changes in
binding for the IGF-II receptor. In any case, substitution of
uncharged residues for the two Arg residues in IGF-I resulted in
higher affinities for the IGF-II receptor. Thus the presence of
positively charged residues in these positions correlates with loss
of binding to the IGF-II receptor.
[0035] IGF-II binds to repeat 11 of the cation-independent M6P
receptor. Indeed, a minireceptor in which only repeat 11 is fused
to the transmembrane and cytoplasmic domains of the
cation-independent M6P receptor is capable of binding IGF-II (with
an affinity approximately one tenth the affinity of the full length
receptor) and mediating internalization of IGF-II and its delivery
to lysosomes (Grimme et al. (2000) J. Biol. Chem.
275(43):33697-33703). The structure of domain 11 of the M6P
receptor is known (Protein Data Base entries 1GP0 and 1GP3; Brown
et al. (2002) EMBO J. 21(5):1054-1062). The putative IGF-II binding
site is a hydrophobic pocket believed to interact with hydrophobic
amino acids of IGF-II; candidate amino acids of IGF-II include
leucine 8, phenylalanine 48, alanine 54, and leucine 55. Although
repeat 11 is sufficient for IGF-II binding, constructs including
larger portions of the cation-independent M6P receptor (e.g.
repeats 10-13, or 1-15) generally bind IGF-II with greater affinity
and with increased pH dependence (see, for example, Linnell et al.
(2001) J. Biol. Chem. 276(26):23986-23991).
Binding to the IGF-I Receptor
[0036] Substitution of IGF-II residues Tyr 27 with Leu, Leu 43 with
Val or Ser 26 with Phe diminishes the affinity of IGF-II for the
IGF-I receptor by 94-, 56-, and 4-fold respectively. Deletion of
residues 1-7 of human IGF-II resulted in a 30-fold decrease in
affinity for the human IGF-I receptor and a concomitant 12 fold
increase in affinity for the rat IGF-II receptor. The NMR structure
of IGF-II shows that Thr 7 is located near residues 48 Phe and 50
Ser as well as near the 9 Cys-47 Cys disulfide bridge. It is
thought that interaction of Thr 7 with these residues can stabilize
the flexible N-terminal hexapeptide required for IGF-I receptor
binding. At the same time this interaction can modulate binding to
the IGF-II receptor.
[0037] Truncation of the C-terminus of IGF-II (residues 62-67) also
appear to lower the affinity of IGF-II for the IGF-I receptor by 5
fold.
Deletion Mutants of IGF-II
[0038] The binding surfaces for the IGF-I and cation-independent
M6P receptors are on separate faces of IGF-II. Based on structural
and mutational data, functional cation-independent M6P binding
domains can be constructed that are substantially smaller than
human IGF-II. For example, the amino terminal amino acids 1-7
and/or the carboxy terminal residues 62-67 can be deleted or
replaced. Additionally, amino acids 29-40 can likely be eliminated
or replaced without altering the folding of the remainder of the
polypeptide or binding to the cation-independent M6P receptor.
Thus, a targeting moiety including amino acids 8-28 and 41-61 can
be constructed. These stretches of amino acids could perhaps be
joined directly or separated by a linker. Alternatively, amino
acids 8-28 and 41-61 can be provided on separate polypeptide
chains. Comparable domains of insulin, which is homologous to
IGF-II and has a tertiary structure closely related to the
structure of IGF-II, have sufficient structural information to
permit proper refolding into the appropriate tertiary structure,
even when present in separate polypeptide chains (Wang et al.
(1991) Trends Biochem. Sci. 279-281). Thus, for example, amino
acids 8-28, or a conservative substitution variant thereof, could
be fused to a therapeutic agent; the resulting fusion protein could
be admixed with amino acids 41-61, or a conservative substitution
variant thereof, and administered to a patient.
Binding to IGF Binding Proteins
[0039] IGF-II and related constructs can be modified to diminish
their affinity for IGFBPs, thereby increasing the bioavailability
of the tagged proteins.
[0040] Substitution of IGF-II residue phenylalanine 26 with serine
reduces binding to IGFBPs 1-5 by 5-75 fold. Replacement of IGF-II
residues 48-50 with threonine-serine-isoleucine reduces binding by
more than 100 fold to most of the IGFBPs; these residues are,
however, also important for binding to the cation-independent
mannose-6-phosphate receptor. The Y27L substitution that disrupts
binding to the IGF-I receptor interferes with formation of the
ternary complex with IGFBP3 and acid labile subunit; this ternary
complex accounts for most of the IGF-II in the circulation.
Deletion of the first six residues of IGF-II also interferes with
IGFBP binding.
[0041] Studies on IGF-I interaction with IGFBPs revealed
additionally that substitution of serine for phenylalanine 16 did
not effect secondary structure but decreased IGFBP binding by
between 40 and 300 fold. Changing glutamate 9 to lysine also
resulted in a significant decrease in IGFBP binding. Furthermore,
the double mutant lysine 9/serine 16 exhibited the lowest affinity
for IGFBPs. Although these mutations have not previously been
tested in IGF-II, the conservation of sequence between this region
of IGF-I and IGF-II suggests that a similar effect will be observed
when the analogous mutations are made in IGF-II (glutamate 12
lysine/phenylalanine 19 serine).
IGF Homologs
[0042] The amino acid sequence of human IGF-I, IGF-II, or a portion
thereof affecting transport into the brain, may be used as a
reference sequence to determine whether a candidate sequence
possesses sufficient amino acid similarity to have a reasonable
expectation of success in the methods of the present invention.
Preferably, variant sequences are at least 70% similar or 60%
identical, more preferably at least 75% similar or 65% identical,
and most preferably 80% similar or 70% identical to human IGF-I or
IGF-II.
[0043] To determine whether a candidate peptide region has the
requisite percentage similarity or identity to human IGF-I or
IGF-II, the candidate amino acid sequence and human IGF-I or IGF-II
are first aligned using the dynamic programming algorithm described
in Smith and Waterman (1981) J. Mol. Biol. 147:195-197, in
combination with the BLOSUM62 substitution matrix described in FIG.
2 of Henikoff and Henikoff(1992) PNAS 89:10915-10919. For the
present invention, an appropriate value for the gap insertion
penalty is -12, and an appropriate value for the gap extension
penalty is -4. Computer programs performing alignments using the
algorithm of Smith-Waternan and the BLOSUM62 matrix, such as the
GCG program suite (Oxford Molecular Group, Oxford, England), are
commercially available and widely used by those skilled in the
art.
[0044] Once the alignment between the candidate and reference
sequence is made, a percent similarity score may be calculated. The
individual amino-acids of each sequence are compared sequentially
according to their similarity to each other. If the value in the
BLOSUM62 matrix corresponding to the two aligned amino acids is
zero or a negative number, the pairwise similarity score is zero;
otherwise the pairwise similarity score is 1.0. The raw similarity
score is the sum of the pairwise similarity scores of the aligned
amino acids. The raw score is then normalized by dividing it by the
number of amino acids in the smaller of the candidate or reference
sequences. The normalized raw score is the percent similarity.
Alternatively, to calculate a percent identity, the aligned amino
acids of each sequence are again compared sequentially. If the
amino acids are non-identical, the pairwise identity score is zero;
otherwise the pairwise identity score is 1.0. The raw identity
score is the sum of the identical aligned amino acids. The raw
score is then normalized by dividing it by the number of amino
acids in the smaller of the candidate or reference sequences. The
normalized raw score is the percent identity. Insertions and
deletions are ignored for the purposes of calculating percent
similarity and identity. Accordingly, gap penalties are not used in
this calculation, although they are used in the initial
alignment.
IGF Structural Analogs
[0045] The known structures of human IGF proteins permit the design
of IGF analogs using computer-assisted design principles such as
those discussed in U.S. Pat. Nos. 6,226,603 and 6,273,598. For
example, the known atomic coordinates of IGF-II can be provided to
a computer equipped with a conventional computer modeling program,
such as INSIGHTII, DISCOVER, or DELPHI, commercially available from
Biosym, Technologies Inc., or QUANTA, or CHARMM, commercially
available from Molecular Simulations, Inc. These and other software
programs allow analysis of molecular structures and simulations
that predict the effect of molecular changes on structure and on
intermolecular interactions. For example, the software can be used
to identify modified analogs with the ability to form additional
intermolecular hydrogen or ionic bonds, improving the affinity of
the analog for the target receptor.
[0046] The software also permits the design of peptides and organic
molecules with structural and chemical features that mimic the same
features displayed on at least part of an IGF surface that is
sufficient for targeting to the CNS. A preferred embodiment of the
present invention relates to designing and producing a synthetic
organic molecule having a framework that carries chemically
interactive moieties in a spatial relationship that mimics the
spatial relationship of the chemical moieties disposed on the amino
acid sidechains which are identified as associated with CNS
targeting as described herein.
[0047] For example, upon identification of relevant chemical
groups, the skilled artisan using a conventional computer program
can design a small molecule having appropriate chemical moieties
disposed upon a suitable carrier framework. Useful computer
programs are described in, for example, Dixon (1992) Tibtech 10:
357-363; Tschinke et al. (1993) J. Med. Chem 36: 3863-3870; and
Eisen el al. (1994) Proteins: Structure, Function, and Genetics
19:199-221, the disclosures of which are incorporated herein by
reference.
[0048] One particular computer program entitled "CAVEAT" searches a
database, for example, the Cambridge Structural Database, for
structures which have desired spatial orientations of chemical
moieties (Bartlett et al. (1989) in "Molecular Recognition:
Chemical and Biological Problems" (Roberts, S. M., ed) pp 182-196).
The CAVEAT program has been used to design analogs of tendamistat,
a 74 residue inhibitor of .alpha.-amylase, based on the orientation
of selected amino acid side chains in the three-dimensional
structure of tendamistat (Bartlett et al. (1989) supra).
[0049] Alternatively, upon identification of a series of analogs
which target transport to the CNS, the skilled artisan may use a
variety of computer programs which assist the skilled artisan to
develop quantitative structure activity relationships (QSAR) and
further to assist in the de novo design of additional analogs.
Other useful computer programs are described in, for example,
Connolly-Martin (1991) Methods in Enzymology 203:587-613; Dixon
(1992) supra; and Waszkowycz et al. (1994) J. Med. Chenm. 37:
3994-4002.
Therapeutic Agent
[0050] While methods and compositions of the invention are useful
for producing and delivering any therapeutic agent to the CNS, the
invention is particularly useful for gene products that overcome
enzymatic defects associated with lysosomal storage diseases.
[0051] Preferred LSD genes are shown in Table 1. In a preferred
embodiment, a wild-type LSD gene product is delivered to a patient
suffering from a defect in the same LSD gene. In alternative
embodiments, a functional sequence or species variant of the LSD
gene is used. In further embodiments, a gene coding for a different
enzyme that can rescue an LSD gene defect is used according to
methods of the invention. TABLE-US-00001 TABLE 1 Lysospmal Storage
Diseases and associated enzyme defects Substance Disease Name
Enzyme Defect Stored A. Glycogenosis Disorders Pompe Disease
Acid-a1, 4- Glycogen .alpha. 1-4 linked Glucosidase
Oligosaccharides B. Glycolipidosis Disorders GM1 Gangliodsidosis
.beta.-Galactosidase GM.sub.1 Ganliosides Tay-Sachs Disease
.beta.-Hexosaminidase A GM.sub.2 Ganglioside GM2 Gangliosidosis:
GM.sub.2 Activator GM.sub.2 Ganglioside AB Variant Protein Sandhoff
Disease .beta.-Hexosaminidase GM.sub.2 Ganglioside A&B Fabry
Disease .alpha.-Galactosidase A Globosides Gaucher Disease
Glucocerebrosidase Glucosylceramide Metachromatic Arylsulfatase A
Sulphatides Leukodystrophy Krabbe Disease Galactosylceramidase
Galactocerebroside Niemann-Pick, Types Acid Sphingomyelin A and B
Sphingomyelinase Niemann-Pick, Type Cholesterol Sphingomyelin C
Esterification Defect Nieman-Pick, Type D Unknown Sphingomyelin
Farber Disease Acid Ceramidase Ceramide Wolman Disease Acid Lipase
Cholesteryl Esters C. Mucopolysaccharide Disorders Hurler Syndrome
.alpha.-L-Iduronidase Heparan & Dermatan (MPS IH) Sulfates
Scheie Syndrome .alpha.-L-Iduronidase Heparan & Dermatan, (MPS
IS) Sulfates Hurler-Scheie .alpha.-L-Iduronidase Heparan &
Dermatan (MPS IH/S) Sulfates Hunter Syndrome Iduronate Sulfatase
Heparan & Dermatan (MPS II) Sulfates Sanfilippo A Heparan
N-Sulfatase Heparan Sulfate (MPS IIIA) Sanfilippo B .alpha.-N-
Heparan Sulfate (MPS IIIB) Acetylglucosaminidase Sanfilippo C
Acetyl-CoA- Heparan Sulfate (MPS IIIC) Glucosaminide
Acetyltransferase Sanfilippo D N-Acetylglucosamine- Heparan Sulfate
(MPS IIID) 6-Sulfatase Morquio A Galactosamine-6- Keratan Sulfate
(MPS IVA) Sulfatase Morquio B .beta.-Galactosidase Keratan Sulfate
(MPS IVB) Maroteaux-Lamy Arylsulfatase B Dermatan Sulfate (MPS VI)
Sly Syndrome .beta.-Glucuronidase (MPS VII) D.
Oligosaccharide/Glycoprotein Disorders .alpha.-Mannosidosis
.alpha.-Mannosidase Mannose/Oligosaccha- rides .beta.-Mannosidosis
.beta.-Mannosidase Mannose/Oligosaccha- rides Fucosidosis
.alpha.-L-Fucosidase Fucosyl Oligosaccharides Asparylglucosaminuria
N-Aspartyl-.beta.- Asparylglucosamine Glucosaminidase Asparagines
Sialidosis .alpha.-Neuraminidase Sialyloligosaccharides
(Mucolipidosis I) Galactosialidosis Lysosomal Protective
Sialyloligosaccharides (Goldberg Syndrome) Protein Deficiency
Schindler Disease .alpha.-N-Acetyl- Galactosaminidase E. Lysosomal
Enzyme Transport Disorders Mucolipidosis II (I-
N-Acetylglucosamine- Heparan Sulfate Cell Disease)
1-Phosphotransferase Mucolipidosis III Same as ML II (Pseudo-Hurler
Polydystrophy) F. Lysosomal Membrane Transport Disorders Cystinosis
Cystine Transport Free Cystine Protein Salla Disease Sialic Acid
Transport Free Sialic Acid and Protein Glucuronic Acid Infantile
Sialic Acid Sialic Acid Transport Free Sialic Acid and Storage
Disease Protein Glucuronic Acid G. Other Batten Disease Unknown
Lipofuscins (Juvenile Neuronal Ceroid Lipofuscinosis) Infantile
Neuronal Palmitoyl-Protein Lipofuscins Ceroid Lipofuscinosis
Thioesterase Mucolipidosis IV Unknown Gangliosides & Hyaluronic
Acid Prosaposin Saposins A, B, C or D
[0052] In one embodiment, the therapeutic agent is
glucocerebrosidase, currently manufactured by Genzyme as an
effective enzyme replacement therapy for Gaucher Disease.
Currently, the enzyme is prepared with exposed mannose residues,
which targets the protein specifically to cells of the macrophage
lineage. Although the primary pathology in type 1 Gaucher patients
are due to macrophage accumulating glucocerebroside, there may be
therapeutic advantage to delivering glucocerebrosidase to other
cell types. Targeting glucocerebrosidase to lysosomes using the
present invention would target the agent to multiple cell types and
may have a therapeutic advantage compared to other
preparations.
Association Between Targeting Portion and Therapeutic Portion
[0053] The therapeutic portion and the targeting portion of
compositions of the invention are necessarily associated, directly
or indirectly. In one embodiment, the therapeutic portion and the
targeting portion are non-covalently associated. For example, the
targeting portion could be biotinylated and bind an avidin moiety
associated with the therapeutic portion. Alternatively, the
targeting portion and the therapeutic portion could each be
associated (e.g. as fusion proteins) with different subunits of a
multimeric protein. In another embodiment, the targeting portion
and the therapeutic portion are crosslinked to each other (e.g.
using a chemical crosslinking agent).
[0054] In a preferred embodiment, the therapeutic portion is fused
to the targeting portion as a fusion protein. The targeting portion
may be at the amino-terminus of the fusion protein, the
carboxy-terminus, or may be inserted within the sequence of the
therapeutic portion at a position where the presence of the
targeting portion does not unduly interfere with the therapeutic
activity of the therapeutic portion.
[0055] Where the therapeutically active moiety is a heteromeric
protein, one or more of the subunits may be associated with a
targeting portion. Hexosaminidase A, for example, a lysosomal
protein affected in Tay-Sachs disease, includes an alpha subunit
and a beta subunit. Either the alpha subunit, or the beta subunit,
or both may be associated with a targeting portion in accordance
with the present invention. If, for example, the alpha subunit is
associated with a targeting portion and is coexpressed with the
beta subunit, an active complex is formed and targeted to the
lysosome.
Methods
[0056] Methods and compositions of the invention are useful in the
context of many different expression systems. For example, a
protein of the invention can be targeted to the CNS, and preferably
taken up by lysosomes, whether it is expressed and isolated from
Leishmania, baculovirus, yeast or bacteria. Thus, the invention
permits great flexibility in protein production. For example, if a
protein to be produced includes one or more disulfide bonds, an
appropriate expression system can be selected and modified, if
appropriate, to further improve yield of properly folded protein.
For example, one useful IGF targeting portion has three
intramolecular disulfide bonds. Fusion proteins of the invention
expressed in E. coli may be constructed to direct the protein to
the periplasmic space. IGF tags, when fused to the C-terminus of
another protein, can be secreted in an active form in the periplasm
of E. coli (Wadensten, Ekebacke et al. 1991). To facilitate optimal
folding of the IGF moiety, appropriate concentrations of reduced
and oxidized glutathione are preferably added to the cellular
milieu to promote disulfide bond formation. In the event that a
fusion protein with disulfide bonds is incompletely soluble, any
insoluble material is preferably treated with a chaotropic agent
such as urea to solubilize denatured protein and refolded in a
buffer having appropriate concentrations of reduced and oxidized
glutathione, or other oxidizing and reducing agents, to facilitate
formation of appropriate disulfide bonds (Smith, Cook et al. 1989).
For example, IGF-I has been refolded using 6M guanidine-HCl and 0.1
M tris(2-carboxyethyl)phosphine reducing agent for denaturation and
reduction of IGF-II (Yang, Wu et al. 1999). Refolding of proteins
was accomplished in 0.1M Tris-HCl buffer (pH8.7) containing 1 mM
oxidized glutathione, 10 mM reduced glutathione, 0.2M KCl and 1 mM
EDTA.
[0057] Methods of the invention are also useful to target a protein
directly to the CNS of a mammal without requiring a purification
step. In one embodiment, an IGF fusion protein is expressed in a
symbiotic or parasitic organism that is administered to a host. The
expressed IGF fusion protein is secreted by the organism into the
blood stream and delivered across the blood brain barrier.
[0058] In some embodiments of the invention, CNS targeted proteins
are delivered in situ via live Leishmania secreting the proteins
into the lysosomes of infected macrophage. From this organelle, it
leaves the cell and may be delivered across the blood brain
barrier. Thus, the IGF tag and the therapeutic agent necessarily
remain intact while the protein resides in the macrophage lysosome.
Accordingly, when proteins designed for delivery to lysosomes in
the CNS are expressed in situ, they are preferably modified to
ensure compatibility with the lysosomal environment. In alternative
embodiments, therapeutic proteins of the invention can be delivered
by expression in T. brucei which can penetrate the BCB.
EXAMPLES
Example 1
Fusion Protein Expressing Constructs
[0059] Nucleic acid constructs for expressing therapeutic protein
fusions of the invention can be made recombinantly according to
methods known in the art. For example, oligonucleotides
complementary to genes encoding the different components described
herein can be used to make synthetic genes or to amplify the
natural genes and construct gene fusions. In preferred embodiments,
proteins of the invention are expressed from a recombinant gene
comprising a signal sequence. Examples of useful nucleic acids
include nucleic acids that encode IGF targeting moieties of the
invention. Such nucleic acids can be based on the sequences of
IGF-1 shown in FIG. 4.
Example 2
Expression and Purification Methods
[0060] Expression product can also be isolated from serum free
media using other protozoa, including other Leishmania species. In
general, the expression strain is grown in medium with serum,
diluted into serum free medium, and allowed to grow for several
generations, preferably 2-5 generations, before the expression
product is isolated. For example, production of secreted
recombinant LSD proteins can be isolated from Leishmania mexicana
promastigotes that are cultured initially in 50 mL 1.times.M199
medium in a 75 cm2 flask at 27.degree. C. When the cell density
reaches 1-3.times.10.sup.7/mL, the culture is used to inoculate 1.2
L of M199 media. When the density of this culture reaches about
5.times.10.sup.6/mL, the cells were harvested by centrifugation,
resuspended in 180 mL of the supernatant and used to inoculate 12 L
of "Zima" medium in a 16 L spinner flask. The initial cell density
of this culture is typically about 5.times.10.sup.5/mL. This
culture is expanded to a cell density of about
1.0-1.7.times.10e.sup.7 cells/mL. When this cell density is
reached, the cells are separated from the culture medium by
centrifugation and the supernatant is filtered at 4.degree. C.
through a 0.2 .mu. filter to remove residual promastigotes. The
filtered media was concentrated from 12.0 L to 500 mL using a
tangential flow filtration device (MILLIPORE Prep/Scale-TFF
cartridge).
[0061] Preferred growth media for this method are M199 and "Zima"
growth media. However, other serum containing and serum free media
are also useful. M199 growth media is as follows: (1 L batch)=200
mL 5.times.M199 (with phenol pH indicator) mixed at 5.times.+637 mL
H.sub.2O, 50.0 mL FBS, 50.0 mL EF, 20.0 mL of 50 g/mL SAT, 2.0 mL
of 0.25% hemin in 50% triethanolamine, 10 mL of 10 mM adenine in 50
mM Hepes pH 7.5, 40.0 mL of 1M Hepes pH 7.5, 1 mL of 0.1% biotin in
95% ethanol, 10.0 mL of penicillin/streptomycin. All serums used
are inactivated by heat. The final volume=1 L and is filter
sterilized. "Zima" modified M199 media is as follows: (20.0 L
batch)=217.8 g M199 powder (-)phenol red+7.0 g sodium bicarbonate,
200.0 mL of 10 mM adenine in 50 mM Hepes pH 7.5, 800.0 mL 0 f Hepes
free acid pH 7.5, 20.0 mL 0.1% biotin in 95% ethanol, 200.0 mL
penicillin/streptomycin, 2780.0 mL H20 Final volume=20.0 L and is
filter sterilized.
[0062] According to one aspect of the invention, LSD proteins
secreted from Leishmania and containing carbohydrate with terminal
mannose residues can be purified as follows. For example,
recombinant .beta.-glucuronidase from Leishmania mexicana
containing plasmsid pXSAP0-GUS was grown in M199 culture medium
with a small amount of serum proteins. When the culture reached a
density of >1.0.times.10.sup.7 promastigotes/mL the L. mexicana
were removed by centrifugation, 10 min at 500.times.g. The
harvested culture medium was passed through a 0.2 .mu.m filter to
remove particulates before being loaded directly onto a
Concanavalin A (ConA)-agarose column (4% cross-linked beaded
agarose, Sigma). The ConA-agarose column was pretreated with 1 M
NaCl, 20 mM Tris pH 7.4, 5 mM each of CaCl.sub.2, MgCl.sub.2 and
MnCl.sub.2 and then equilibrated with 5 volumes of column buffer
(20 mM Tris pH 7.4, 1 mM CaCl.sub.2, and 1 mM MnCl.sub.2). A total
of 179,800 units (nmol/hr) of GUS activity (in 2 L) in culture
medium was loaded onto a 22 mL ConA agarose column. No activity was
detectable in the flow through or wash. The GUS activity was eluted
with column buffer containing 200 mM methyl mannopyranoside. Eluted
fractions containing the activity peak were pooled and
concentrated: 143900 units of GUS activity were recovered from the
column (80% recovery of activity loaded onto the column). This
demonstrates that the recombinant .beta.-GUS secreted from L.
mexicana possesses carbohydrate with terminal mannose residues and
further points out the potential for using the interaction of
mannose with ConA as the basis for an affinity purification step.
Accordingly, the presence of high mannose carbohydrate can serve as
the basis of an affinity step in the purification of recombinant
LSD proteins using lectin affinity chromatography.
Example 3
Assays for Crossing the Blood Brain Barrier
[0063] According to the invention, a useful model system to
determine whether a protein, particularly an LSD protein, tagged
with an IGF tag crosses the blood-brain barrier, is the MPSVII
mouse model (Wolfe and Sands (1996) Protocols for Gene Transfer in
Neuroscience: Towards Gene Therapy of Neurological Disorders
Chapter 20: 263-273). For example, recombinant human
.beta.-glucuronidase fused to an IGF tag can be produced in any
convenient expression system such as Leishmania, yeast, mammalian,
bacculovirus and other expression systems. L. mexicana expressing
and secreting .beta.-GUS is grown at 26.degree. C. in 100 ml
Standard Promastigote medium (M199 with 40 mM HEPES, pH 7.5, 0.1 mM
adenine, 0.0005% hemin, 0.0001% biotin, 5% fetal bovine serum, 5%
embryonic fluid, 50 units/ml penicillin, 50 .mu.g/ml streptomycin
and 50.mu.g/ml nourseothricin). After reaching a density of
approximately 5.times.10.sup.6 promastigotes/ml, the promastigotes
is collected by centrifugation for 10 min. at 1000 .times.g at room
temperature; these promastigotes were used to inoculate 1 liter of
low protein medium (M199 supplemented with 0.1 mM adenine, 0.0001%
biotin, 50 units/ml penicillin and 50 .mu.g/ml streptomycin) at
room temperature. The 1 liter cultures are contained in 2 liter
capped flasks with a sterile stir bar so that the cultures could be
incubated at 26.degree. C. with gentle stirring. The 1 liter
cultures are aerated twice a day by moving them into a laminar flow
hood, removing the caps and swirling vigorously before replacing
the caps. When the cultures reach a density of 2-3.times.10.sup.7
promastigotes/ml, the cultures are centrifuged as previously
described except the promastigote pellet is discarded and the media
decanted into sterile flasks. The addition of 434 g
(NH.sub.4).sub.2SO.sub.4 per liter precipitates active GUS protein
from the medium; the salted out medium is stored at 4.degree. C.
overnight. Precipitated proteins are harvested either by
centrifugation at 10,500 .times.g for 30 min. or filtration through
Gelman Supor-800 membrane; the proteins are resuspended in 10 mM
Tris pH 8, 1 mM CaCl.sub.2 and stored at -80.degree. C. until
dialysis. The crude preparations from several liters of medium are
thawed, pooled, placed in dialysis tubing (Spectra/Por -7, MWCO
25,000), and dialyzed overnight against two 1 liter volumes of DMEM
with bicarbonate (Dulbecco's Modified Eagle's Medium). The ammonium
sulfate fraction is further purified on a ConA column.
[0064] GUS minus mice generated by heterozygous matings of
B6.C-H-2.sup.bml/ByBIR-gus.sup.mps/+ mice are used to assess the
effectiveness of GUS-IGF fusion proteins or derivatives in enzyme
replacement therapy. Two formats are used. In one format, 3-4
animals are given a single injection of 20,000 U of enzyme in 100
.mu.l enzyme dilution buffer (150 mM NaCl, 10 mM Tris, pH7.5). Mice
are killed 72-96 hours later to assess the efficacy of the therapy.
In a second format, mice are given weekly injections of 20,000
units over 3-4 weeks and are killed 1 week after the final
injection. Histochemical and histopathologic analysis of liver,
spleen and brain are carried out by published methods. In the
absence of therapy, cells (e.g. macrophages and Kupffer cells) of
GUS minus mice develop large intracellular storage compartments
resulting from the buildup of waste products in the lysosomes. It
is anticipated that in cells in mice treated with GUS fusion
constructs of the invention, the size of these compartments will be
visibly reduced or the compartments will shrink until they are no
longer visible with a light microscope.
[0065] According to the invention, newborn mice do not possess a
complete blood brain barrier. However, by day 15 the blood brain
barrier is formed to the point that .beta.-glucuronidase no longer
can be detected in the brain. Accordingly, the above experiments
are preferably performed on mice that are at day 15 or greater.
[0066] According to one embodiment of the invention, experiments
first assess the ability of complete IGF-I and IGF-II tags to
direct proteins across the blood brain barrier. Next, specific
mutant versions of the proteins that disrupt receptor or IGF
binding protein binding are assayed. For domain swaps, the B domain
of IGF-II (residues 1-28 of the mature protein) contains only two
differences from IGF-I that could conceivably alter transport
across the blood brain barrier G11 and T16. Altering these residues
in IGF-II would is essentially a domain B swap. Another swap of
regions between residues 28 and 41 of IGF-II and the corresponding
region of IGF-I can also be tested. This essentially swaps the C
domains of the two proteins which contains the most divergent
regions of the two proteins. An alternative swap switches the
C-terminal 15 residues with the corresponding region of IGF-I.
These three chimeras provide an essentially complete picture of how
any differences in uptake across the blood brain barrier between
IGF-I and IGF-II correlate with sequence/structural differences
between the two proteins.
Example 4
Assays for Protein Accumulation in the Brain or CNS
[0067] Radioactive assays can be used to monitor the accumulation
of protein product in the brain. For example, the uptake and
accumulation of a radioactively labeled protein in the brain
parenchyma can be assayed as disclosed in Reinhardt and Bondy
(1994) Endocrinology 135:1753-1761.
[0068] Enzyme assays can also be used to monitor the accumulation
of protein product in the brain. Enzyme assays are particularly
useful when the therapeutic protein moiety is an enzyme for which
there is an assay that is applicable for histochemical staining.
Useful enzyme assays for lysosomal storage disease proteins include
assays disclosed in Sly at al. (2001) P.N.A.S. 98(5): 2205-2210,
and in Wolfe and Sands (1996) Protocols for Gene Transfer in
Neuroscience: Towards Gene Therapy of Neurological Disorders
Chapter 20: 263-273.
Example 5
In Vivo Therapy
[0069] GUS minus mice generated by heterozygous matings of
B6.C-H-2.sup.bml/ByBIR-gus.sup.mps/+ mice (Birkenmeier, Davisson et
al. 1989) are used to assess the effectiveness of compositions of
the invention in enzyme replacement therapy. Two formats are used.
In one format, 3-4 animals are given a single injection of 20,000 U
of enzyme in 100 .mu.l enzyme dilution buffer (150 mM NaCl, 10 mM
Tris, pH7.5). Mice are killed 72-96 hours later to assess the
efficacy of the therapy. In a second format, mice are given weekly
injections of 20,000 units over 3-4 weeks and are killed 1 week
after the final injection. Histochemical and histopathologic
analysis of liver, spleen and brain are carried out by published
methods (Birkenmeier, Barker et al. 1991; Sands, Vogler et al.
1994; Daly, Vogler et al. 1999). In the absence of therapy, cells
(e.g. macrophages and Kupffer cells) of GUS minus mice develop
large intracellular storage compartments resulting from the buildup
of waste products in the lysosomes. It is anticipated that in cells
in mice treated with compositions of the invention, the size of
these compartments will be visibly reduced or the compartments will
shrink until they are no longer visible with a light
microscope.
[0070] Similarly, humans with lysosomal storage diseases will be
treated using constructs targeting an appropriate therapeutic
portion to their CNS and in particular to lysosomes within the CNS.
In some instances, treatment will take the form of regular (e.g.
weekly) injections of a fusion protein of the invention. In other
instances, treatment will be achieved through administration of a
nucleic acid to permit persistent in vivo expression of the fusion
protein, or through administration of a cell (e.g. a human cell, or
a unicellular organism) expressing the fusion protein in the
patient. For example, a protein the invention may be expressed in
situ using a Leishmania vector as described in U.S. Pat. No.
6,020,144, issued Feb. 1, 2000; and PCT Serial No. PCT/US01/44935,
filed Nov. 30, 2001.
Equivalents
[0071] The invention may be embodied in other specific forms
without departing from the spirit or essential characteristics
thereof. The foregoing embodiments are therefore to be considered
in all respects illustrative rather than limiting on the invention
described herein. Scope of the invention is thus indicated by the
appended claims rather than by the foregoing description, and all
changes which come within the meaning and range of equivalency of
the claims are intended to be embraced therein.
INCORPORATION BY REFERENCE
[0072] The disclosure of each of the patent documents and
scientific publications disclosed herein, and U.S. Ser. No.
60/250,446 filed Nov. 30, 2000; U.S. Ser. No. 60/250,444 filed Nov.
30, 2000; U.S. Ser. No. 60/290,281 filed May 11, 2001; U.S. Ser.
No. 60/287,531, filed Apr. 30, 2001; U.S. Ser. No. 60/304,609,
filed Jul. 10, 2001; U.S. Ser. No. 60/329,461, filed Oct. 15, 2001,
a U.S. Ser. No. 60/351,276, filed Jan. 23, 2002; and attorney
docket number SYM-007 entitled "Methods and Compositions for
Lysosomal Targeting" filed on Apr. 30, 2002; PCT Serial No.
PCT/US01/44935, filed Nov. 30, 2001; are incorporated by reference
into this application in their entirety.
Sequence CWU 1
1
4 1 70 PRT Homo sapiens 1 Gly Pro Glu Thr Leu Cys Gly Ala Glu Leu
Val Asp Ala Leu Gln Phe 1 5 10 15 Val Cys Gly Asp Arg Gly Phe Tyr
Phe Asn Lys Pro Thr Gly Tyr Gly 20 25 30 Ser Ser Ser Arg Arg Ala
Pro Gln Thr Gly Ile Val Asp Glu Cys Cys 35 40 45 Phe Arg Ser Cys
Asp Leu Arg Arg Leu Glu Met Tyr Cys Ala Pro Leu 50 55 60 Lys Pro
Ala Lys Ser Ala 65 70 2 67 PRT Homo sapiens 2 Ala Tyr Arg Pro Ser
Glu Thr Leu Cys Gly Gly Glu Leu Val Asp Thr 1 5 10 15 Leu Gln Phe
Val Cys Gly Asp Arg Gly Phe Tyr Phe Ser Arg Pro Ala 20 25 30 Ser
Arg Val Ser Arg Arg Ser Arg Gly Ile Val Glu Glu Cys Cys Phe 35 40
45 Arg Ser Cys Asp Leu Ala Leu Leu Glu Thr Tyr Cys Ala Thr Pro Ala
50 55 60 Lys Ser Glu 65 3 153 PRT Homo sapiens 3 Met Gly Lys Ile
Ser Ser Leu Pro Thr Gln Leu Phe Lys Cys Cys Phe 1 5 10 15 Cys Asp
Phe Leu Lys Val Lys Met His Thr Met Ser Ser Ser His Leu 20 25 30
Phe Tyr Leu Ala Leu Cys Leu Leu Thr Phe Thr Ser Ser Ala Thr Ala 35
40 45 Gly Pro Glu Thr Leu Cys Gly Ala Glu Leu Val Asp Ala Leu Gln
Phe 50 55 60 Val Cys Gly Asp Arg Gly Phe Tyr Phe Asn Lys Pro Thr
Gly Tyr Gly 65 70 75 80 Ser Ser Ser Arg Arg Ala Pro Gln Thr Gly Ile
Val Asp Glu Cys Cys 85 90 95 Phe Arg Ser Cys Asp Leu Arg Arg Leu
Glu Met Tyr Cys Ala Pro Leu 100 105 110 Lys Pro Ala Lys Ser Ala Arg
Ser Val Arg Ala Gln Arg His Thr Asp 115 120 125 Met Pro Lys Thr Gln
Lys Glu Val His Leu Lys Asn Ala Ser Arg Gly 130 135 140 Ser Ala Gly
Asn Lys Asn Tyr Arg Met 145 150 4 7260 DNA Homo sapiens 4
tcactgtcac tgctaaattc agagcagatt agagcctgcg caatggaata aagtcctcaa
60 aattgaaatg tgacattgct ctcaacatct cccatctctc tggatttcct
tttgcttcat 120 tattcctgct aaccaattca ttttcagact ttgtacttca
gaagcaatgg gaaaaatcag 180 cagtcttcca acccaattat ttaagtgctg
cttttgtgat ttcttgaagg tgaagatgca 240 caccatgtcc tcctcgcatc
tcttctacct ggcgctgtgc ctgctcacct tcaccagctc 300 tgccacggct
ggaccggaga cgctctgcgg ggctgagctg gtggatgctc ttcagttcgt 360
gtgtggagac aggggctttt atttcaacaa gcccacaggg tatggctcca gcagtcggag
420 ggcgcctcag acaggcatcg tggatgagtg ctgcttccgg agctgtgatc
taaggaggct 480 ggagatgtat tgcgcacccc tcaagcctgc caagtcagct
cgctctgtcc gtgcccagcg 540 ccacaccgac atgcccaaga cccagaagga
agtacatttg aagaacgcaa gtagagggag 600 tgcaggaaac aagaactaca
ggatgtagga agaccctcct gaggagtgaa gagtgacatg 660 ccaccgcagg
atcctttgct ctgcacgagt tacctgttaa actttggaac acctaccaaa 720
aaataagttt gataacattt aaaagatggg cgtttccccc aatgaaatac acaagtaaac
780 attccaacat tgtctttagg agtgatttgc accttgcaaa aatggtcctg
gagttggtag 840 attgctgttg atcttttatc aataatgttc tatagaaaag
aaaaaaaaat atatatatat 900 atatatctta gtccctgcct ctcaagagcc
acaaatgcat gggtgttgta tagatccagt 960 tgcactaaat tcctctctga
atcttggctg ctggagccat tcattcagca accttgtcta 1020 agtggtttat
gaattgtttc cttatttgca cttctttcta cacaactcgg gctgtttgtt 1080
ttacagtgtc tgataatctt gttagtctat acccaccacc tcccttcata acctttatat
1140 ttgccgaatt tggcctcctc aaaagcagca gcaagtcgtc aagaagcaca
ccaattctaa 1200 cccacaagat tccatctgtg gcatttgtac caaatataag
ttggatgcat tttattttag 1260 acacaaagct ttatttttcc acatcatgct
tacaaaaaag aataatgcaa atagttgcaa 1320 ctttgaggcc aatcattttt
aggcatatgt tttaaacata gaaagtttct tcaactcaaa 1380 agagttcctt
caaatgatga gttaatgtgc aacctaatta gtaactttcc tctttttatt 1440
ttttccatat agagcactat gtaaatttag catatcaatt atacaggata tatcaaacag
1500 tatgtaaaac tctgtttttt agtataatgg tgctattttg tagtttgtta
tatgaaagag 1560 tctggccaaa acggtaatac gtgaaagcaa aacaataggg
gaagcctgga gccaaagatg 1620 acacaagggg aagggtactg aaaacaccat
ccatttggga aagaaggcaa agtcccccca 1680 gttatgcctt ccaagaggaa
cttcagacac aaaagtccac tgatgcaaat tggactggcg 1740 agtccagaga
ggaaactgtg gaatggaaaa agcagaaggc taggaatttt agcagtcctg 1800
gtttcttttt ctcatggaag aaatgaacat ctgccagctg tgtcatggac tcaccactgt
1860 gtgaccttgg gcaagtcact tcacctctct gtgcctcagt ttcctcatct
gcaaaatggg 1920 ggcaatatgt catctaccta cctcaaaggg gtggtataag
gtttaaaaag ataaagattc 1980 agattttttt accctgggtt gctgtaaggg
tgcaacatca gggcgcttga gttgctgaga 2040 tgcaaggaat tctataaata
acccattcat agcatagcta gagattggtg aattgaatgc 2100 tcctgacatc
tcagttcttg tcagtgaagc tatccaaata actggccaac tagttgttaa 2160
aagctaacag ctcaatctct taaaacactt ttcaaaatat gtgggaagca tttgattttc
2220 aatttgattt tgaattctgc atttggtttt atgaatacaa agataagtga
aaagagagaa 2280 aggaaaagaa aaaggagaaa aacaaagaga tttctaccag
tgaaagggga attaattact 2340 ctttgttagc actcactgac tcttctatgc
agttactaca tatctagtaa aaccttgttt 2400 aatactataa ataatattct
attcattttg aaaaacacaa tgattccttc ttttctaggc 2460 aatataagga
aagtgatcca aaatttgaaa tattaaaata atatctaata aaaagtcaca 2520
aagttatctt ctttaacaaa ctttactctt attcttagct gtatatacat ttttttaaaa
2580 agtttgttaa aatatgcttg actagagttt cagttgaaag gcaaaaactt
ccatcacaac 2640 aagaaatttc ccatgcctgc tcagaagggt agcccctagc
tctctgtgaa tgtgttttat 2700 ccattcaact gaaaattggt atcaagaaag
tccactggtt agtgtactag tccatcatag 2760 cctagaaaat gatccctatc
tgcagatcaa gattttctca ttagaacaat gaattatcca 2820 gcattcagat
ctttctagtc accttagaac tttttggtta aaagtaccca ggcttgatta 2880
tttcatgcaa attctatatt ttacattctt ggaaagtcta tatgaaaaac aaaaataaca
2940 tcttcagttt ttctcccact gggtcacctc aaggatcaga ggccaggaaa
aaaaaaaaag 3000 actccctgga tctctgaata tatgcaaaaa gaaggcccca
tttagtggag ccagcaatcc 3060 tgttcagtca acaagtattt taactctcag
tccaacatta tttgaattga gcacctcaag 3120 catgcttagc aatgttctaa
tcactatgga cagatgtaaa agaaactata catcattttt 3180 gccctctgcc
tgttttccag acatacaggt tctgtggaat aagatactgg actcctcttc 3240
ccaagatggc acttcttttt atttcttgtc cccagtgtgt accttttaaa attattccct
3300 ctcaacaaaa ctttataggc agtcttctgc agacttaaca tgttttctgt
catagttaga 3360 tgtgataatt ctaagagtgt ctatgactta tttccttcac
ttaattctat ccacagtcaa 3420 aaatccccca aggaggaaag ctgaaagatg
caactgccaa tattatcttt cttaactttt 3480 tccaacacat aatcctctcc
aactggatta taaataaatt gaaaataact cattatacca 3540 attcactatt
ttatttttta atgaattaaa actagaaaac aaattgatgc aaaccctgga 3600
agtcagttga ttactatata ctacagcaga atgactcaga tttcatagaa aggagcaacc
3660 aaaatgtcac aaccaaaact ttacaagctt tgcttcagaa ttagattgct
ttataattct 3720 tgaatgaggc aatttcaaga tatttgtaaa agaacagtaa
acattggtaa gaatgagctt 3780 tcaactcata ggcttatttc caatttaatt
gaccatactg gatacttagg tcaaatttct 3840 gttctctctt gcccaaataa
tattaaagta ttatttgaac tttttaagat gaggcagttc 3900 ccctgaaaaa
gttaatgcag ctctccatca gaatccactc ttctagggat atgaaaatct 3960
cttaacaccc accctacata cacagacaca cacacacaca cacacacaca cacacacaca
4020 cacacattca ccctaaggat ccaatggaat actgaaaaga aatcacttcc
ttgaaaattt 4080 tattaaaaaa caaacaaaca aacaaaaagc ctgtccaccc
ttgagaatcc ttcctctcct 4140 tggaacgtca atgtttgtgt agatgaaacc
atctcatgct ctgtggctcc agggtttctg 4200 ttactatttt atgcacttgg
gagaaggctt agaataaaag atgtagcaca ttttgctttc 4260 ccatttattg
tttggccagc tatgccaatg tggtgctatt gtttctttaa gaaagtactt 4320
gactaaaaaa aaaagaaaaa aagaaaaaaa agaaagcata gacatatttt tttaaagtat
4380 aaaaacaaca attctataga tagatggctt aataaaatag cattaggtct
atctagccac 4440 caccaccttt caacttttta tcactcacaa gtagtgtact
gttcaccaaa ttgtgaattt 4500 gggggtgcag gggcaggagt tggaaatttt
ttaaagttag aaggctccat tgttttgttg 4560 gctctcaaac ttagcaaaat
tagcaatata ttatccaatc ttctgaactt gatcaagagc 4620 atggagaata
aacgcgggaa aaaagatctt ataggcaaat agaagaattt aaaagataag 4680
taagttcctt attgattttt gtgcactctg ctctaaaaca gatattcagc aagtggagaa
4740 aataagaaca aagagaaaaa atacatagat ttacctgcaa aaaatagctt
ctgccaaatc 4800 ccccttgggt attctttggc atttactggt ttatagaaga
cattctccct tcacccagac 4860 atctcaaaga gcagtagctc tcatgaaaag
caatcactga tctcatttgg gaaatgttgg 4920 aaagtatttc cttatgagat
gggggttatc tactgataaa gaaagaattt atgagaaatt 4980 gttgaaagag
atggctaaca atctgtgaag attttttgtt tcttggtttt gttttttttt 5040
ttttttttac tttatacagt ctttatgaat ttcttaatgt tcaaaatgac ttggttcttt
5100 tcttcttttt tttatatcag aatgaggaat aataagttaa acccacatag
actctttaaa 5160 actataggct agatagaaat gtatgtttga cttgttgaag
ctataatcag actatttaaa 5220 atgttttgct atttttaatc ttaaaagatt
gtgctaattt attagagcag aacctgtttg 5280 gctctcctca gaagaaagaa
tctttccatt caaatcacat ggctttccac caatattttc 5340 aaaagataaa
tctgatttat gcaatggcat catttatttt aaaacagaag aattgtgaaa 5400
gtttatgccc ctcccttgca aagaccataa agtccagatc tggtaggggg gcaacaacaa
5460 aaggaaaatg ttgttgattc ttggttttgg attttgtttt gttttcaatg
ctagtgttta 5520 atcctgtagt acatatttgc ttattgctat tttaatattt
tataagacct tcctgttagg 5580 tattagaaag tgatacatag atatcttttt
tgtgtaattt ctatttaaaa aagagagaag 5640 actgtcagaa gctttaagtg
catatggtac aggataaaga tatcaattta aataaccaat 5700 tcctatctgg
aacaatgctt ttgtttttta aagaaacctc tcacagataa gacagaggcc 5760
caggggattt ttgaagctgt ctttattctg cccccatccc aacccagccc ttattatttt
5820 agtatctgcc tcagaatttt atagagggct gaccaagctg aaactctaga
attaaaggaa 5880 cctcactgaa aacatatatt tcacgtgttc cctctctttt
ttttcctttt tgtgagatgg 5940 ggtctcgcac tgtcccccag gctggagtgc
agtggcatga tctcggctca ctgcaacctc 6000 cacctcctgg gtttaagcga
ttctcctgcc tcagcctcct gagtagctgg gattacaggc 6060 acccaccact
atgcccggct aattttttgg atttttaata gagacggggt tttaccatgt 6120
tggccaggtt ggactcaaac tcctgacctt gtgatttgcc cgcctcagcc tcccaaattg
6180 ctgggattac aggcatgagc caccacaccc tgcccatgtg ttccctctta
atgtatgatt 6240 acatggatct taaacatgat ccttctctcc tcattcttca
actatctttg atggggtctt 6300 tcaaggggaa aaaaatccaa gcttttttaa
agtaaaaaaa aaaaaagaga ggacacaaaa 6360 ccaaatgtta ctgctcaact
gaaatatgag ttaagatgga gacagagttt ctcctaataa 6420 ccggagctga
attacctttc actttcaaaa acatgacctt ccacaatcct tagaatctgc 6480
ctttttttat attactgagg cctaaaagta aacattactc attttatttt gcccaaaatg
6540 cactgatgta aagtaggaaa aataaaaaca gagctctaaa atccctttca
agccacccat 6600 tgaccccact caccaactca tagcaaagtc acttctgtta
atcccttaat ctgattttgt 6660 ttggatattt atcttgtacc cgctgctaaa
cacactgcag gagggactct gaaacctcaa 6720 gctgtctact tacatctttt
atctgtgtct gtgtatcatg aaaatgtcta ttcaaaatat 6780 caaaaccttt
caaatatcac gcagcttata ttcagtttac ataaaggccc caaataccat 6840
gtcagatctt tttggtaaaa gagttaatga actatgagaa ttgggattac atcatgtatt
6900 ttgcctcatg tatttttatc acacttatag gccaagtgtg ataaataaac
ttacagacac 6960 tgaattaatt tcccctgcta ctttgaaacc agaaaataat
gactggccat tcgttacatc 7020 tgtcttagtt gaaaagcata ttttttatta
aattaattct gattgtattt gaaattatta 7080 ttcaattcac ttatggcaga
ggaatatcaa tcctaatgac ttctaaaaat gtaactaatt 7140 gaatcattat
cttacattta ctgtttaata agcatatttt gaaaatgtat ggctagagtg 7200
tcataataaa atggtatatc tttctttagt aattacaaaa aaaaaaaaaa aaaaaaaaaa
7260
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