U.S. patent application number 16/866397 was filed with the patent office on 2021-01-21 for detection of oligosaccharides.
This patent application is currently assigned to BIOMARIN PHARMACEUTICAL INC.. The applicant listed for this patent is BIOMARIN PHARMACEUTICAL INC.. Invention is credited to Jillian R. BROWN, Brett E. CRAWFORD, Charles A. GLASS.
Application Number | 20210017570 16/866397 |
Document ID | / |
Family ID | 1000005134548 |
Filed Date | 2021-01-21 |
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United States Patent
Application |
20210017570 |
Kind Code |
A1 |
CRAWFORD; Brett E. ; et
al. |
January 21, 2021 |
DETECTION OF OLIGOSACCHARIDES
Abstract
Provided herein are processes for detecting oligosaccharides in
a biological sample. In specific instances, the biological sample
is provided from an individual suffering from a disorder associated
with abnormal glycosaminoglycan accumulation.
Inventors: |
CRAWFORD; Brett E.; (Poway,
CA) ; BROWN; Jillian R.; (Poway, CA) ; GLASS;
Charles A.; (San Diego, CA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
BIOMARIN PHARMACEUTICAL INC. |
Novato |
CA |
US |
|
|
Assignee: |
BIOMARIN PHARMACEUTICAL
INC.
Novato
CA
|
Family ID: |
1000005134548 |
Appl. No.: |
16/866397 |
Filed: |
May 4, 2020 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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15705677 |
Sep 15, 2017 |
10683530 |
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16866397 |
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14706794 |
May 7, 2015 |
9796999 |
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15705677 |
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12649094 |
Dec 29, 2009 |
9029530 |
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14706794 |
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61164365 |
Mar 27, 2009 |
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61142291 |
Jan 2, 2009 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
G01N 2400/40 20130101;
A61B 5/4842 20130101; C07H 11/00 20130101; C07H 3/06 20130101; A61B
5/4866 20130101; A61K 31/702 20130101; G01N 33/66 20130101; C07H
5/06 20130101; G01N 2800/042 20130101; G01N 2333/988 20130101; C12Q
1/527 20130101; C12N 9/88 20130101; C07H 3/04 20130101; C07H 13/04
20130101; G01N 2800/7071 20130101; G01N 2800/52 20130101; C07H
7/033 20130101 |
International
Class: |
C12Q 1/527 20060101
C12Q001/527; C07H 3/06 20060101 C07H003/06; C07H 3/04 20060101
C07H003/04; C07H 13/04 20060101 C07H013/04; C07H 5/06 20060101
C07H005/06; A61K 31/702 20060101 A61K031/702; A61B 5/00 20060101
A61B005/00; C12N 9/88 20060101 C12N009/88; G01N 33/66 20060101
G01N033/66; C07H 7/033 20060101 C07H007/033; C07H 11/00 20060101
C07H011/00 |
Claims
1-30. (canceled)
31. A method of determining in an individual the presence,
identity, and/or severity of an MPS IIIA or MPS IIIB disorder, the
method comprising: (a) generating a biomarker comprising one or
more saturated non-reducing end oligosaccharides, wherein the
biomarker is generated by treating a population of heparan sulfate
oligosaccharides, in or isolated from a biological sample from the
individual, with at least one digesting glycosaminoglycan lyase,
wherein prior to lyase treatment, the biomarker is not present in
abundance in samples from individuals with the MPS IIIA or MPS IIIB
disorder relative to individuals without the MPS IIIA or MPS IIIB
disorder; and (b) using an analytical instrument to detect the
presence of and/or measure the amount of the biomarker produced and
displaying or recording the presence of or the measure of the
biomarker produced; wherein the presence of and/or measure of the
amounts of the biomarker are utilized to determine the presence,
identity, and/or severity of the MPS III disorder; and wherein the
biomarker is selected from a group consisting of Formula III:
[GlcNS-IdoA-GlcN(Ac)0-1](SO.sub.3R)0-3; Formula IV:
[GlcNS-GlcA-GlcN(Ac)0-1](SO.sub.3R)0-2; Formula V:
[GlcNAc-IdoA-GlcN(Ac)0-1](SO.sub.3R)0-3; Formula VI:
[GlcNAc-GlcA-GlcN(Ac)0-1](SO.sub.3R)0-2; Formula VIII:
[GlcN-GlcA-GlcN(Ac)0-1](SO.sub.3R)0-4; Formula IX:
[GlcNAc6S-IdoA-GlcN(Ac)0-1](SO.sub.3R)0-3; Formula X:
[GlcNAc6S-GlcA-GlcN(Ac)0-1](SO.sub.3R)0-2; GlcN-IdoA-GlcNAc;
GlcN-IdoA2S-GlcNAc; GlcN-IdoA-GlcNS; GlcN-IdoA-GlcNAc6S;
GlcN-IdoA2-GlcNAc6S; and GlcN-IdoA-GlcNS6S.
32. The method of claim 31, wherein the biomarker is of Formula V:
[GlcNAc-IdoA-GlcN(Ac).sub.0-1](SO.sub.3R).sub.0-3; or Formula VI:
[GlcNAc-GlcA-GlcN(Ac).sub.0-1](SO.sub.3R).sub.0-2.
33. The method of claim 32, wherein the biomarker of Formula V is
selected from a group consisting of GlcNAc-IdoA-GlcNAc,
GlcNAc-IdoA2S-GlcNAc, GlcNAc-IdoA-GlcNS, GlcNAc-IdoA2S-GlcNS,
GlcNAc-IdoA-GlcNAc6S, GlcNAc-IdoA2S-GlcNAc6S, GlcNAc-IdoA-GlcNS6S,
and GlcNAc-IdoA2S-GlcNS6S.
34. The method of claim 32, wherein the biomarker of Formula VI is
selected from a group consisting of GlcNAc-GlcA-GlcNAc,
GlcNAc-GlcA-GlcNS, GlcNAc-GlcA-GlcNAc6S, and
GlcNAc-GlcA-GlcNS6S.
35. The method of claim 31, wherein the biomarker is of Formula
III: [GlcNS-IdoA-GlcN(Ac).sub.0-1](SO.sub.3R).sub.0-3.
36. The method of claim 35, wherein the biomarker of Formula III is
selected from a group consisting of GlcNS-IdoA-GlcNAc,
GlcNS-IdoA2S-GlcNAc, GlcNS-IdoA-GlcNS, GlcNS-IdoA2S-GlcNS,
GlcNS-IdoA-GlcNAc65, GlcNS-IdoA2S-GlcNAc65, GlcNS-IdoA-GlcNS6S, and
GlcNS-IdoA2S-GlcNS6S.
37. The method of claim 31, wherein the biomarker is of Formula IV:
[GlcNS-GlcA-GlcN(Ac).sub.0-1](SO.sub.3R).sub.0-2.
38. The method of claim 37, wherein the biomarker of Formula IV is
selected from a group consisting of GlcNS-GlcA-GlcNAc,
GlcNS-GlcA-GlcNS, GlcNS-GlcA-GlcNAc6S, or GlcNS-GlcA-GlcNS6S.
39. The method of claim 31, wherein the biomarker is selected from
a group consisting of GlcN-IdoA-GlcNAc, GlcN-IdoA2S-GlcNAc,
GlcN-IdoA-GlcNS, GlcN-IdoA-GlcNAc6S, GlcN-IdoA2S-GlcNAc6S, and
GlcN-IdoA-GlcNS6S.
40. The method of claim 31, wherein the biomarker is of Formula
VIII: [GlcN-GlcA-GlcN(Ac).sub.0-1](SO.sub.3R).sub.0-4.
41. The method of claim 40, wherein the biomarker of Formula VIII
is selected from a group consisting of GlcN-GlcA-GlcNAc,
GlcN-GlcA-GlcNS, GlcN-GlcA-GlcNAc6S, and GlcN-GlcA-GlcNS6S.
42. The method of claim 31, wherein the biomarker is of Formula IX:
[GlcNAc6S-IdoA-GlcN(Ac).sub.0-1](SO.sub.3R).sub.0-3.
43. The method of claim 32, wherein the biomarker of Formula IX is
selected from a group consisting of GlcNAc6s-IdoA-GlcNAc,
GlcNAc6s-IdoA2S-GlcNAc, GlcNAc6s-IdoA-GlcNS, GlcNAc6s-IdoA2S-GlcNS,
GlcNAc6s-IdoA-GlcNAc6S, GlcNAc6s-IdoA2S-GlcNAc6S,
GlcNAc6s-IdoA-GlcNS6S, and GlcNAc6s-IdoA2S-GlcNS6S.
44. The method of claim 31, wherein the biomarker is of Formula X:
[GlcNAc6S-GlcA-GlcN(Ac).sub.0-1](SO.sub.3R).sub.0-2.
45. The method of claim 44, wherein the biomarker of Formula X is
selected from a group consisting of GlcNAc6S-GlcA-GlcNAc,
GlcNAc6S-GlcA-GlcNS, GlcNAc6S-GlcA-GlcNAc6S, and
GlcNAc6S-GlcA-GlcNS6S.
46. The method of claim 31, wherein the method further comprises
purifying the biomarker generated in step (a) to yield an isolated
population of biomarkers.
47. The method of claim 46, wherein the biomarker is purified using
chromatography or electrophoresis.
48. The method of claim 31, wherein the method further comprises
tagging the biomarkers with a detectable label.
49. The method of claim 48, wherein the detectable label is a mass
label, a radio label, a fluorescent label, a chromophore label, or
affinity label.
50. The method of claim 1, wherein the method is used to determine
whether the individual is a heterozygous carrier of the MPS IIIA or
MPS IIIB disorder.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation application of U.S.
application Ser. No. 15/705,677, filed Sep. 15, 2017, which is a
continuation of U.S. application Ser. No. 14/706,794, filed May 7,
2015 (now U.S. Pat. No. 9,796,999, issued Oct. 24, 2017), which is
a continuation of U.S. application Ser. No. 12/649,094, filed Dec.
29, 2009 (now U.S. Pat. No. 9,029,530, issued May 12, 2015), which
claims the benefit of U.S. Provisional Application No. 61/164,365,
filed Mar. 27, 2009 and U.S. Provisional Application No.
61/142,291, filed Jan. 2, 2009, the disclosures of each of which is
incorporated by reference herein in its entirety.
BACKGROUND OF THE INVENTION
[0002] Glycosaminoglycans comprise a reducing end and a
non-reducing end. Normal biological processes degrade
glycosaminoglycans (such as heparan sulfate which has a normal
component of about 50-80 kDa) into monosaccharides. Disorders
associated with abnormal glycosaminoglycan degradation,
biosynthesis, and/or accumulation can result in an accumulation of
glycosaminoglycans and fragments thereof.
SUMMARY OF THE INVENTION
[0003] Described herein are populations of glycosaminoglycans that
are transformed into populations of oligosaccharides using
glycosaminoglycan lyases. Further described herein are the use of
analytical instruments to characterize the population of
oligosaccharides in order to provide relevant information about the
population of oligosaccharides, the population of
glycosaminoglycans and the biological sample that provided the
population of glycosaminoglycans.
[0004] Provided in certain embodiments herein is a process for
diagnosing the identity and/or severity of abnormal
glycosaminoglycan accumulation in an individual, or a disorder
thereof, the process comprising the steps of: [0005] a. using an
analytical instrument to detect the presence of and/or measure the
amount of a population of one or more oligosaccharides present in a
transformed biological sample that has been prepared by: [0006]
treating a population of glycosaminoglycans, in or isolated from a
biological sample from the individual to transform the
glycosaminoglycans into the population of the one or more
oligosaccharide; [0007] b. displaying or recording the presence of
or a measure of a population of one or more oligosaccharide.
[0008] In certain embodiments, provided is a process for diagnosing
the presence, identity, and/or severity of abnormal
glycosaminoglycan accumulation in an individual, or a disorder
thereof, the process comprising the steps of: [0009] a. generating
a biomarker comprising of one or more non-reducing end
oligosaccharides, wherein the biomarker is a saturated
oligosaccharide and is generated by treating a population of
glycosaminoglycans, in or isolated from a biological sample from
the individual, with at least one digesting glycosaminoglycan
lyases, wherein prior to lyase treatment, such oligosaccharide
biomarker is not present in abundance in samples from individuals
with abnormal glycosaminoglycan accumulation relative to
individuals with normal glycosaminoglycan; [0010] b. using an
analytical instrument to detect the presence of and/or measure the
amount of the biomarker produced and displaying or recording the
presence of or a measure of a population of the biomarker.
[0011] In specific embodiments, the presence of and/or measure the
amount of the biomarker is utilized to diagnose of the presence,
identity, and/or severity of abnormal glycosaminoglycan
accumulation.
[0012] In certain embodiments, the oligosaccharide(s) detected or
measured is one or more C4-C5 non-reducing end saturated
oligosaccharide(s).
[0013] In some embodiments, treating a population of
glycosaminoglycans to transform the glycosaminoglycans into the
population of the one or more oligosaccharide comprises contacting
the glycosaminoglycans with at least one digesting
glycosaminoglycan lyase. In some embodiments, the at least one
digesting glycosaminoglycan lyase is one or more heparin lyase, one
or more chondroitinase, one or more keratanase, one or more
hyaluronidase, or a combination thereof. In specific embodiments,
the at least one digesting glycosaminoglycan lyase is one or more
heparin lyase.
[0014] In certain embodiments, the one or more oligosaccharides
detected and/or measured are free of carbon-carbon unsaturation. In
various embodiments, the abnormal glycosaminoglycan accumulation
comprises abnormal heparan sulfate accumulation, abnormal
chondroitin sulfate accumulation, abnormal keratan sulfate
accumulation, abnormal hyaluronan accumulation, or a combination
thereof. In specific embodiments, the abnormal glycosaminoglycan
accumulation is abnormal heparan sulfate accumulation.
[0015] In various embodiments, any process descried herein of
preparing a transformed biological sample comprises purifying a
population of oligosaccharides in the biological sample that has
been treated with the at least one heparin lyase, the transformed
biological sample comprising the isolated population of
oligosaccharides. In some embodiments, any process described herein
of preparing a transformed biological sample comprises purifying a
population of glycosaminoglycans in the biological sample prior to
treatment with the at least one heparin lyase.
[0016] In certain embodiments, any process described herein of
detecting the presence of or measuring the amount of a population
of one or more oligosaccharide present in a transformed biological
sample comprises: [0017] a. isolating a subpopulation of one or
more oligosaccharides in the transformed biological sample; and
[0018] b. detecting the presence of and/or measuring the amount of
one or more oligosaccharides present in the subpopulation.
[0019] In specific embodiments, a subpopulation of one or more
oligosaccharides is isolated using, by way of non-limiting example,
chromatography or electrophoresis. In specific embodiments, the
chromatography is high performance liquid chromatography (HPLC),
gas chromatography (GC), column chromatography, affinity
chromatography, or thin layer chromatography (TLC). In some
embodiments, any process of detecting oligosaccharides described
herein comprises detecting oligosaccharides using mass
spectrometry.
[0020] In some embodiments, any process described herein of
preparing a transformed biological sample comprises tagging the
reducing end of a representative portion of the one or more
oligosaccharides in the transformed biological sample with a
detectable label. In specific embodiments, the detectable label is
a mass label, a radio label, a fluorescent label, a chromophore
label, or affinity label. In some embodiments, the tagged portion
of the one or more oligosaccharides is detected or measured using
UV-Vis spectroscopy, IR spectroscopy, mass spectrometry, or a
combination thereof
[0021] In certain embodiments, a digesting glycosaminoglycan lyase
utilized in any process described herein comprises heparan sulfate,
chondroitin sulfate, keratan sulfate, hyaluronan, or a combination
thereof. In specific embodiments, a digesting glycosaminoglycan
lyase utilized in any process described herein comprises heparan
sulfate.
[0022] In some embodiments, a process described herein comprises
detecting or measuring a disaccharide having the formula:
[IdoA-GlcN(Ac).sub.m](SO.sub.3R).sub.n, wherein m is 0-1, n is 0-3,
and R is H or a negative charge. In some embodiments, the term R
used in any formula described herein is H or a negative charge.
[0023] In specific embodiments, any process described herein
comprises detecting or measuring a disaccharide with the
formula:
##STR00001##
[0024] In some embodiments, the disaccharide above is detected
and/or measured in a process of diagnosing a disorder associated
with abnormal glycosaminoglycan degradation that is MPS I.
[0025] In some embodiments, any process described herein comprises
detecting or measuring a disaccharide with the formula:
##STR00002##
[0026] In some embodiments, the disaccharide above is detected
and/or measured in a process of diagnosing a disorder associated
with abnormal glycosaminoglycan degradation that is MPS II.
[0027] In some embodiments, any process described herein comprises
detecting or measuring a trisaccharide with the formula
[GlcN(Ac).sub.m-(IdoA/GlcA)-GlcN(Ac).sub.n]SO.sub.3R).sub.p,
wherein IdoA/GlcA is either IdoA or GlcA, m is 0-1, n is 0-1, p is
0-5, and R is H or a negative charge.
[0028] In certain embodiments, any process described herein
comprises detecting or measuring a trisaccharide with the
formula:
##STR00003##
[0029] In some embodiments, the trisaccharide above is detected
and/or measured in a process of diagnosing a disorder associated
with abnormal glycosaminoglycan degradation that is MPS IIIA.
[0030] In certain embodiments, any process described herein
comprises detecting or measuring a trisaccharide with the
formula:
##STR00004##
[0031] In some embodiments, the trisaccharide above is detected
and/or measured in a process of diagnosing a disorder associated
with abnormal glycosaminoglycan degradation that is MPS IIIB.
[0032] In certain embodiments, any process described herein
comprises detecting or measuring a trisaccharide with the
formula:
##STR00005##
[0033] In some embodiments, the trisaccharide above is detected
and/or measured in a process of diagnosing a disorder associated
with abnormal glycosaminoglycan degradation that is MPS IIIC.
[0034] In certain embodiments, any process described herein
comprises detecting or measuring a trisaccharide with the
formula:
##STR00006##
[0035] In some embodiments, the trisaccharide above is detected
and/or measured in a process of diagnosing a disorder associated
with abnormal glycosaminoglycan degradation that is MPS IIID
[0036] In certain embodiments, any process described herein
comprises detecting or measuring a disaccharide with the formula
[GlcA-GlcN(Ac).sub.n](SO.sub.3R).sub.m, wherein n is 0-1, m is 0-2,
and R is H or a negative charge.
[0037] In certain embodiments, any process described herein
comprises detecting or measuring a disaccharide with the
formula:
##STR00007##
[0038] In some embodiments, the disaccharide above is detected
and/or measured in a process of diagnosing a disorder associated
with abnormal glycosaminoglycan degradation that is MPS VII.
[0039] In various embodiments of the processes described herein, a
population of glycosaminoglycans treated with at least one
digesting glycosaminoglycan lyase comprises dermatan sulfate,
chondroitin sulfate, or a combination thereof. In certain
embodiments, any process described herein comprises detecting or
measuring a disaccharide with the formula:
[Ido-GalNAc](SO.sub.3R).sub.n, wherein n=0-2, and each R is
independently H or a negative charge. In some embodiments, any
process described herein comprises detecting or measuring a
trisaccharide with the formula: [GalNAc4S-Ido-GlcNAc](SO.sub.3R)n,
wherein n=0-3; and/or one or more trisaccharide with the formula:
[GalNAc4S-GlcA-GlcNAc](SO.sub.3R).sub.m, wherein m=0-1, and wherein
each R is independently H or a negative charge.
[0040] In certain embodiments, any process described herein
comprises detecting or measuring a disaccharide with the formula:
[Gal6S-GalNAc](SO.sub.3R).sub.n, wherein n=0-1, and wherein each R
is independently H or a negative charge. In specific embodiments,
detection and/or measurement of [Gal6S-GalNAc](SO.sub.3R).sub.n is
used in a method of diagnosing MPS IVA or the severity thereof. In
some embodiments, any process described herein comprises detecting
or measuring a disaccharide with the formula:
[Gal-GalNAc](SO.sub.3R)n, wherein n=0-1, and wherein each R is
independently H or a negative charge. In specific embodiments,
detection and/or measurement of [Gal-GalNAc](SO.sub.3R).sub.n is
used in a method of diagnosing MPS IVB or the severity thereof.
[0041] In some embodiments, any process described herein comprises:
[0042] a. comparing an amount of a population of one or more
oligosaccharide present in a transformed biological sample to an
amount of a population of one or more oligosaccharide present in a
control biological sample that has been treated in a manner
substantially similar to the transformed biological sample.
[0043] In certain embodiments, a control biological sample utilized
in any process described herein was provided from an individual
that does not have mucopolysaccharidosis (e.g., a non-MPS cell
line). In some embodiments, any control biological sample utilized
in a process described herein was provided from an individual that
has mucopolysaccharidosis. In specific embodiments, a control
biological sample was provided from an individual that has MPS I,
MPS II, MPS IIIA, MPS IIIB, MPS IIIC, MPS IIID, MPS IVA, MPS IVB,
MPS VII, MPS IX, or a combination thereof. In specific embodiments,
a control biological sample was provided from an individual that
has MPS I, MPS II, MPS IIIA, MPS IIIB, MPS IIIC, MPS IIID, MPS VII,
or a combination thereof.
[0044] Provided in certain embodiments herein is an analytical
sample comprising any oligosaccharide described herein, including
an oligosaccharide described herein and further attached to a
detectable label (e.g., at the reducing end of the
oligosaccharide). In specific embodiments, provided herein is an
analytical sample comprising one or more of any of FIGS. 4-23.
[0045] In specific embodiments, an analytical sample provided for
herein is for use in high performance liquid chromatography. In
some embodiments, an analytical sample provided for herein is for
use in mass spectrometry. In certain embodiments, an analytical
sample provided for herein is for use in gas chromatography. In
some embodiments, any analytical sample provided herein comprises
at least one disaccharide or trisaccharide from a transformed
biological sample from an individual with a disorder associated
with abnormal glycosaminoglycan accumulation.
[0046] Provided in some embodiments herein is an analytical method
comprising treating a biological sample that comprises
glycosaminoglycans with at least one digesting glycosaminoglycan
lyase to transform a representative portion of the
glycosaminoglycans into one or more oligosaccharides. In certain
embodiments an analytical method provided for herein comprises
purifying one or more oligosaccharides from other components of the
biological sample. In some embodiments, the purifying step includes
use of chromatography. In various embodiments, an analytical method
provided for herein comprises detecting and/or measuring the
presence of at least one of the oligosaccharides (e.g., after
purification). In certain embodiments, oligosaccharides are
detected and/or measured according to any process or method (used
interchangeably herein) described herein using UV-Vis spectroscopy,
IR spectroscopy, mass spectrometry, or a combination thereof. In
some embodiments, any process described herein comprises tagging at
least one of the oligosaccharides with a detectable label. In
certain embodiments, the at least one digesting glycosaminoglycan
lyase utilized in any process or method described herein comprises
one or more heparin lyase, one or more chondroitinase, one or more
keratanase, one or more hyaluronidase, or a combination thereof
[0047] In specific embodiments, an analytical method described
herein is used in a method of detecting and/or measuring one or
more oligosaccharides that are free of carbon-carbon
unsaturation.
[0048] In certain embodiments, any process described herein
comprises detecting or measuring a disaccharide with the formula:
[IdoA-GlcN(Ac).sub.m](SO.sub.3R).sub.p, wherein m is 0-1, n is 0-3,
and R is H or a negative charge. In some embodiments, any process
described herein comprises detecting or measuring a trisaccharide
with the formula:
[GlcN(Ac)m-(IdoA/GlcA)-GlcN(Ac).sub.p](SO.sub.3R).sub.p, wherein
IdoA/GlcA is either IdoA or GlcA, m is 0-1, n is 0-1, and p is 0-5.
In certain embodiments, any process described herein comprises
detecting or measuring a disaccharide with the formula:
[GlcA-GlcN(Ac).sub.n](SO.sub.3R).sub.m, wherein n is 0-1, and m is
0-2. In some embodiments, any process described herein comprises
detecting or measuring a disaccharide with the formula:
[Ido-GalNAc](SO.sub.3R).sub.n, wherein n=0-2, and each R is
independently H or a negative charge. In certain embodiments, any
process described herein comprises detecting or measuring a
trisaccharide with the formula:
[GalNAc4S-Ido-GalNAc](SO.sub.3R).sub.n, wherein n=0-3; and/or one
or more trisaccharide with the formula:
[GalNAc.sub.4S-GlcA-GalNAc](SO.sub.3R).sub.m, wherein m=0-2, and
wherein each R is independently H or a negative charge. In certain
embodiments, any process described herein comprises detecting or
measuring a disaccharide with the formula: GlcA-GlcNAc. In certain
embodiments, any process described herein comprises detecting or
measuring a disaccharide with the formula:
[GlcA-GalNAc](SO.sub.3R).sub.m, wherein m is 0-2 (e.g., for
diagnosing CS accumulation in MPS VII, or the severity thereof). In
some embodiments, any process described herein comprises detecting
or measuring a trisaccharide with the formula:
[GlcNAc6S-Gal-GlcNAc](SO.sub.3R).sub.p, wherein n=0-2, and wherein
each R is independently H or a negative charge. In certain
embodiments, any process described herein comprises detecting or
measuring a disaccharide with the formula:
[Gal6S-GlcNAc](SO.sub.3R).sub.n, wherein n=0-1, and wherein each R
is independently H or a negative charge. In certain embodiments,
any process described herein comprises detecting or measuring a
disaccharide with the formula: [Gal-GlcNAc](SO.sub.3R).sub.n,
wherein n=0-1, and wherein each R is independently H or a negative
charge.
[0049] In certain embodiments, a process described herein includes
a method of monitoring the treatment of disorders associated with
the abnormal degradation, biosynthesis and/or accumulation of
glycosaminoglycans (GAGs), the methods comprising: [0050] a.
following administration of an agent for treating MPS to an
individual in need thereof, generating a biomarker comprising of
one or more non-reducing end oligosaccharides, wherein the
biomarker is a saturated oligosaccharide and is generated by
treating a population of glycosaminoglycans, in or isolated from a
biological sample from the individual, with at least one digesting
glycosaminoglycan lyases, wherein prior to lyase treatment, such
oligosaccharide biomarker is not present in abundance in samples
from individuals with abnormal glycosaminoglycan accumulation
relative to individuals with normal glycosaminoglycan; [0051] b.
using an analytical instrument to detect the presence of and/or
measure the amount of the biomarker produced and displaying or
recording the presence of or a measure of a population of the
biomarker.
[0052] In specific embodiments, increases or decreases in the
amount of the biomarker measured (e.g., as compared to a biological
sample previously analyzed in a similar or identical manner) is
utilized to monitor the treatment of disorders associated with the
abnormal degradation, biosynthesis and/or accumulation of
glycosaminoglycans.
BRIEF DESCRIPTION OF THE DRAWINGS
[0053] The novel features of the invention are set forth with
particularity in the appended claims. A better understanding of the
features and advantages of the present invention will be obtained
by reference to the following detailed description that sets forth
illustrative embodiments, in which the principles of the invention
are utilized, and the accompanying drawings of which:
[0054] FIG. 1 illustrates the cleavage of the glycosaminoglycan
(GAG) heparan sulfate with a glycosaminoglycan lyase (heparinase
II).
[0055] FIG. 2 illustrates lyase liberation of three classes of di-
and trisaccharides from normal and MPS GAGs. The non-reducing end
(NRE) fragments are used as biomarkers in certain embodiments
herein.
[0056] FIGS. 3A-3C illustrate various oligosaccharide residues of
GAGs. FIG. 3A illustrates the heparan sulfate fragments that
accumulate in MPS II patients considering only the 3 most common
modifications (N-, 2-O, and 6-O sulfation). Even for a relatively
small nonasaccharides (n=4), there are well over 1,000 potential
structures. FIGS. 3B and 3C illustrate predicted non-reducing end
heparan sulfate biomarkers for 7 MPS classes.
[0057] FIG. 4 illustrates oligosaccharides of Formula I.
[0058] FIG. 5 illustrates oligosaccharides of Formula III.
[0059] FIG. 6 illustrates oligosaccharides of Formula IV.
[0060] FIG. 7 illustrates oligosaccharides of Formula V.
[0061] FIG. 8 illustrates oligosaccharides of Formula VI.
[0062] FIG. 9 illustrates oligosaccharides of Formula VII.
[0063] FIG. 10 illustrates oligosaccharides of Formula VIII.
[0064] FIG. 11 illustrates oligosaccharides of Formula IX.
[0065] FIG. 12 illustrates oligosaccharides of Formula X.
[0066] FIG. 13 illustrates oligosaccharides of Formula XI.
[0067] FIG. 14 illustrates oligosaccharides of Formula XII.
[0068] FIG. 15 illustrates oligosaccharides of Formula XIII.
[0069] FIG. 16 illustrates oligosaccharides of Formula XIV.
[0070] FIG. 17 illustrates oligosaccharides of Formula XV.
[0071] FIG. 18 illustrates oligosaccharides of Formula XVIII.
[0072] FIG. 19 illustrates oligosaccharides of Formula XVIX.
[0073] FIG. 20 illustrates oligosaccharides of Formula XX.
[0074] FIG. 21 illustrates oligosaccharides of Formula XXX.
[0075] FIG. 22 illustrates oligosaccharides of Formula XXXI.
[0076] FIG. 23 illustrates oligosaccharides of Formula XXXII.
[0077] FIG. 24 illustrates disaccharides isolated from heparan
sulfate from a normal human sample.
[0078] FIG. 25 illustrates disaccharides isolated from an MPS I
(iduronidase deficient) sample.
[0079] FIG. 26 illustrates the detection of oligosaccharides
described herein in individuals having an MPS disease state as
compared to individuals lacking an MPS disease state.
[0080] FIG. 27 illustrates mass spectrometry results for
non-reducing end oligosaccharides in a non-MPS sample.
[0081] FIG. 28A illustrates mass spectrometry results for
non-reducing end oligosaccharides in a MPS I sample. FIG. 28B is an
enlarged representation of the `28B` region of FIG. 28A.
[0082] FIG. 29A illustrates mass spectrometry results for
non-reducing end oligosaccharides in a MPS II sample. FIG. 29B is
an enlarged representation of the `29B` region of FIG. 29A. FIG.
29C is an enlarged representation of the `29C` region of FIG.
29A.
[0083] FIG. 30A illustrates mass spectrometry results for
non-reducing end oligosaccharides in a MPS IIIA sample. FIG. 30B is
an enlarged representation of the `30B` region of FIG. 30A.
[0084] FIG. 31A illustrates mass spectrometry results for
non-reducing end oligosaccharides in a MPS IIIB sample. FIG. 31B is
an enlarged representation of the `31B` region of FIG. 31A. FIG.
31C is an enlarged representation of the `31C` region of FIG.
31A.
[0085] FIG. 32 illustrates mass spectrometry results showing the
accumulation of GAG non-reducing end trisaccharide residues in the
liver of an MPS IIIB mouse.
[0086] FIG. 33 illustrates mass spectrometry results showing the
accumulation of GAG non-reducing end trisaccharide residues in the
brain of an MPS IIIB mouse.
[0087] FIG. 34 illustrates mass spectrometry results showing the
accumulation of GAG non-reducing end trisaccharide residues in the
kidney of an MPS IIIB mouse.
[0088] FIG. 35 illustrates the detection of MPS disease in urine
samples.
[0089] FIG. 36 illustrates the detection of MPS disease in serum
samples.
[0090] FIG. 37 illustrates the detection of MPS disease in CSF
samples.
[0091] FIG. 38 illustrates the detection of MPS disease in tissue
samples.
[0092] FIG. 39 illustrates monitoring and detecting the response to
therapy in serum from MPS patients.
[0093] FIG. 40 illustrates monitoring and detecting MPS disease
severity.
[0094] FIG. 41 illustrates monitoring and detecting the
differential response to therapy in CSF from MPS patients.
[0095] FIG. 42 illustrates monitoring and detecting response to
therapy in tissue samples from MPS patients.
[0096] FIGS. 43A, 43B, and 43C illustrate monitoring and detecting
the response to therapy in serum from individuals suffering from
MPS. FIGS. 43A, 43B, and 43C illustrate the treatment of different
patients.
[0097] FIG. 44 illustrates detecting carriers of the genetic cause
of MPS.
DETAILED DESCRIPTION OF THE INVENTION
[0098] While preferred embodiments of the present invention have
been shown and described herein, it will be obvious to those
skilled in the art that such embodiments are provided by way of
example only. Numerous variations, changes, and substitutions will
now occur to those skilled in the art without departing from the
invention. It should be understood that various alternatives to the
embodiments of the invention described herein may be employed in
practicing the invention. It is intended that the following claims
define the scope of the invention and that methods and structures
within the scope of these claims and their equivalents be covered
thereby.
[0099] Provided in certain embodiments herein are analytical
methods for detecting and/or identifying glycosaminoglycans (GAGs)
or other glycans (e.g., glycolipids) in biological sample. In
certain embodiments, the glycans, e.g., glycosaminoglycans (GAGs),
are present in cells within a biological sample (e.g., within a
lysosome thereof), and/or are present in a biological sample free
of cells. In certain embodiments, provided herein is a method of
diagnosing any disorder characterized by the accumulation of
glycosaminoglycans, such as a lysosomal storage disease (LSD). In
some embodiments, the glycosaminoglycan accumulation is a primary
accumulative effect. In certain instances, primary accumulative
effects include accumulation that is a direct result of an abnormal
biosynthetic process, such as abnormal production enzymes involved
in the glycan biosynthetic pathway (e.g., under-production or
production of poorly functioning enzymes), including glycan
bio-synthesis or depolymerization. In other embodiments, the
glycosaminoglycan accumulation is a secondary accumulative effect.
In certain embodiments, a secondary accumulative effect results
from a cascading effect, e.g., accumulation of other components,
such as GAGs or other glycans, such as glycolipids, causes the GAG
biosynthetic pathway to be hindered or interrupted.
[0100] In certain embodiments, glycosaminoglycans include, by way
of non-limiting example, heparan sulfate, heparin, chondroitin
sulfate, dermatan sulfate, hyaluronan, keratan sulfate, or the
like, or a combination thereof. In certain embodiments, an
analytical method provided herein comprising treating a biological
sample that comprises glycosaminoglycans with at least one agent
suitable for cleaving bonds between saccharide residues of
glycosaminoglycans. In specific embodiments, treating a biological
sample that comprises glycosaminoglycans with at least one agent
suitable for cleaving bonds between saccharide residues of
glycosaminoglycans comprises treating the biological sample with
one or more digesting glycosaminoglycan (GAG) lyase. In some
embodiments, any glycosaminoglycan (GAG) lyase suitable for
cleaving the bonds (e.g., the bonds linking saccharide residues of
the GAG to one another) of a glycosaminoglycan (GAG) analyze is
utilized. In some embodiments, the lyase is utilized to transform a
representative portion of the glycosaminoglycans into one or more
oligosaccharides. In certain embodiments, such glycosaminoglycan
(GAG) lyases are suitable for preparing di- and/or tri-saccharides
from the glycosaminoglycan present. Glycosaminoglycan (GAG) lyases
suitable for use in various embodiments provided herein include, by
way of non-limiting example, one or more heparin lyase
(heparinase), one or more chondroitinase, one or more keratanase,
one or more hyaluronidase, or a combination thereof. Other glycans
that are optionally detected by a method described herein include,
e.g., glycolipids.
[0101] In some embodiments, lyases utilized herein include, by way
of non-limiting example, Hyaluronate lyase, Pectate lyase,
Poly(beta-D-mannuronate) lyase, Chondroitin ABC lyase, Chondroitin
AC lyase, Oligogalacturonide lyase, Heparin lyase, Heparin-sulfate
lyase, Pectate disaccharide-lyase, Pectin lyase,
Poly(alpha-L-guluronate) lyase, Xanthan lyase,
Exo-(1.fwdarw.4)-alpha-D-glucan lyase, Glucuronan lyase,
Anhydrosialidase, Levan fructotransferase, Inulin
fructotransferase, Inulin fructotransferase, Chondroitin B lyase.
In certain instances, Hyaluronate lyase (EC 4.2.2.1) is an enzyme
that catalyzes the cleavage or hyaluronate chains at a
beta-D-GalNAc-(1.fwdarw.4)-beta-D-GlcA bond, ultimately breaking
the polysaccharide down to
3-(4-deoxy-beta-D-gluc-4-enuronosyl)-N-acetyl-D-glucosamine. In
some instances, Pectate lyase (EC 4.2.2.2) is an enzyme that
catalyzes the eliminative cleavage of
(1.fwdarw.4)-alpha-D-galacturonan to give oligosaccharides with
4-deoxy-alpha-D-galact-4-enuronosyl groups at their non-reducing
ends. In certain instances, Poly(beta-D-mannuronate) lyase (EC
4.2.2.3) is an enzyme that catalyzes the eliminative cleavage of
polysaccharides containing beta-D-mannuronate residues to give
oligosaccharides with
4-deoxy-alpha-L-erythro-hex-4-enopyranuronosyl groups at their
ends. In some instances, Chondroitin ABC lyase (EC 4.2.2.4) is an
enzyme that catalyzes the eliminative degradation of
polysaccharides containing 1,4-beta-D-hexosaminyl and
1,3-beta-D-glucuronosyl linkages to disaccharides containing
4-deoxy-beta-D-gluc-4-enuronosyl groups. In some instances,
Chondroitin ABC lyase (EC 4.2.2.4) also catalyzes the eliminative
cleavage of dermatan sulfate containing 1,4-beta-D-hexosaminyl and
1,3-beta-D-glucurosonyl or 1,3-alpha-L-iduronosyl linkages to
disaccharides containing 4-deoxy-beta-D-gluc-4-enuronosyl groups to
yield a 4,5-unsaturated dermatan-sulfate disaccharide
(deltaUA-GalNAc-4S). In certain instances, Chondroitin AC lyase (EC
4.2.2.5) is an enzyme that catalyzes the eliminative degradation of
polysaccharides containing 1,4-beta-D-hexosaminyl and
1,3-beta-D-glucuronosyl linkages to disaccharides containing
4-deoxy-beta-D-gluc-4-enuronosyl groups. In some instances,
Oligogalacturonide lyase (EC 4.2.2.6) is an enzyme that catalyzes
the cleavage of
4-(4-deoxy-beta-D-gluc-4-enuronosyl)-D-galacturonate into 2
5-dehydro-4-deoxy-D-glucuronate. In certain instances, Heparin
lyase (EC 4.2.2.7) is an enzyme that catalyzes the eliminative
cleavage of polysaccharides containing 1,4-linked D-glucuronate or
L-iduronate residues and 1,4-alpha-linked
2-sulfoamino-2-deoxy-6-sulfo-D-glucose residues to give
oligosaccharides with terminal 4-deoxy-alpha-D-gluc-4-enuronosyl
groups at their non-reducing ends. In some instances, Heparin lyase
(EC 4.2.2.7) tolerates alternative sulfation of the substrate. In
some instances, Heparin-sulfate lyase (EC 4.2.2.8) is an enzyme
that catalyzes the eliminative cleavage of polysaccharides
containing 1,4-linked D-glucuronate or L-iduronate residues and
1,4-alpha-linked 2-sulfoamino-2-deoxy-6-sulfo-D-glucose residues to
give oligosaccharides with terminal
4-deoxy-alpha-D-gluc-4-enuronosyl groups at their non-reducing
ends. In some instances, Heparin-sulfate lyase (EC 4.2.2.8)
tolerates alternative sulfation of the substrate. In certain
instances, Pectate disaccharide-lyase (EC 4.2.2.9) is an enzyme
that catalyzes the eliminative cleavage of
4-(4-deoxy-alpha-D-galact-4-enuronosyl)-D-galacturonate from the
reducing end of pectate, i.e. de-esterified pectin. In some
instances, Pectin lyase (EC 4.2.2.10) is an enzyme that catalyzes
the eliminative cleavage of (1->4)-alpha-D-galacturonan methyl
ester to give oligosaccharides with
4-deoxy-6-O-methyl-alpha-D-galact-4-enuronosyl groups at their
non-reducing ends. In certain instances, Poly(alpha-L-guluronate)
lyase (EC 4.2.2.11) is an enzyme that catalyzes the eliminative
cleavage of polysaccharides containing a terminal
alpha-L-guluronate group, to give oligosaccharides with
4-deoxy-alpha-L-erythro-hex-4-enuronosyl groups at their
non-reducing ends. In some instances, Xanthan lyase (EC 4.2.2.12)
is an enzyme that catalyzes the cleavage of the
beta-D-mannosyl-beta-D-1,4-glucuronosyl bond on the polysaccharide
xanthan. In certain instances, Exo-(1.fwdarw.4)-alpha-D-glucan
lyase (E.C. 4.2.2.13) is an enzyme that catalyzes the sequential
degradation of (1.fwdarw.4)-alpha-D-glucans from the non-reducing
end with the release of 1,5-anhydro-D-fructose. In some intances,
Glucuronan lyase (EC 4.2.2.14) is an enzyme that catalyzes the
eliminative cleavage of (1.fwdarw.4)-beta-D-glucuronans. This
produces either oligosaccharides with
4-deoxy-beta-D-gluc-4-enuronosyl groups at their non-reducing ends,
or, if the substrate is completely degraded, glucuronans produce
tetrasaccharides. In certain instances, Anhydrosialidase (EC
4.2.2.15) is an enzyme that catalyzes the elimination of
alpha-sialyl groups in N-acetylneuraminic acid glycosides,
releasing 2,7-anhydro-alpha-N-acetylneuraminate. In some instances,
Levan fructotransferase (DFA-IV-forming) (EC 4.2.2.16) is an enzyme
that produces di-beta-D-fructofuranose 2,6':2',6-dianhydride (DFA
IV) by successively eliminating the diminishing
(2.fwdarw.6)-beta-D-fructan (levan) chain from the terminal
D-fructosyl-D-fructosyl disaccharide. In certain instances, Inulin
fructotransferase (DFA-I-forming) (EC 4.2.2.17) is an enzyme that
produces alpha-D-fructofuranose beta-D-fructofuranose
1,2':2,1'-dianhydride (DFA I) by successively eliminating the
diminishing (2.fwdarw.1)-beta-D-fructan (inulin) chain from the
terminal D-fructosyl-D-fructosyl disaccharide. In some instances,
Inulin fructotransferase (DFA-III-forming) (EC 4.2.2.18) is an
enzyme that produces alpha-D-fructofuranose beta-D-fructofuranose
1,2':2,3'-dianhydride (DFA III) by successively eliminating the
diminishing (2.fwdarw.1)-beta-D-fructan (inulin) chain from the
terminal D-fructosyl-D-fructosyl disaccharide. In certain
instances, Chondroitin B lyase (EC 4.2.2.19) is an enzyme that
catalyzes the eliminative cleavage of dermatan sulfate containing
1,4-beta-D-hexosaminyl and 1,3-beta-D-glucurosonyl or
1,3-alpha-L-iduronosyl linkages to disaccharides containing
4-deoxy-beta-D-gluc-4-enuronosyl groups to yield a 4,5-unsaturated
dermatan-sulfate disaccharide (deltaUA-GalNAc-4S). Any other
suitable enzyme is also optionally utilized. For example, any
keratanase may be used, e.g., as isolated from bacteria or
evolved/designed from a related lyase.
[0102] In some embodiments, the analytical process comprises
detecting and/or measuring the one or more oligosaccharide present
in the biological sample after it has been treated with one or more
glycosaminoglycan lyase. In some embodiments, the one or more
oligosaccharide detected and/or measured is one or more
disaccharide and/or one or more trisaccharide. In certain
embodiments, the one or more oligosaccharides detected and/or
measured (e.g., one or more disaccharide and/or one or more
trisaccharide) are saturated at 4 and 5 carbons of the non-reducing
end saccharide residue. In some embodiments, the non-reducing end
residue of the one or more oligosaccharides detected and/or
measured (e.g., one or more disaccharide and/or one or more
trisaccharide) are free of carbon-carbon unsaturation. In certain
embodiments, the one or more oligosaccharides detected and/or
measured (e.g., one or more disaccharide and/or one or more
trisaccharide) are free of carbon-carbon unsaturation. Biological
samples suitable for analysis according to the methods and
processes described herein include, by way of non-limiting example,
blood, serum, urine, hair, saliva, skin, tissue, plasma,
cerebrospinal fluid (C SF), amniotic fluid, nipple aspirate,
sputum, feces, synovial fluid, nails, or the like. In specific
embodiments, the biological samples suitable for analysis according
to the methods and processes described herein include, by way of
non-limiting example, urine, serum, plasma, or CSF. In certain
embodiments, processes for detecting glycosoaminoglycans in a
sample comprise providing, from the individual, a test biological
sample that comprises glycosaminoglycans. In some embodiments,
providing a test biological sample from an individual includes
obtaining the sample from the individual or obtaining the sample
from another source (e.g., from a technician or institution that
obtained the sample from the individual). In some embodiments, the
biological sample is obtained from any suitable source, e.g., any
tissue or cell (e.g., urine, serum, plasma, or CSF) of an
individual. In certain embodiments, the tissue and/or cell from
which the GAGs are recovered is obtained from liver tissue or
cells, brain tissue or cells, kidney tissue or cells, or the
like.
[0103] FIG. 1 illustrates the cleavage of the glycosaminoglycan
(GAG) heparan sulfate with a glycosaminoglycan lyase (heparinase
II). As illustrated, in certain instances, internal cleavage of
glycosaminoglycans with glycosaminoglycan lyases provides
oligosaccharides with carbon-carbon unsaturation between the C4 and
C5 carbons of the non-reducing end of the oligosaccharide produce
(i.e., the newly created oligosaccharide). In some embodiments, the
one or more oligosaccharide detected and/or measured according to a
method described herein is one or more disaccharide and/or one or
more trisaccharide, each oligosaccharide being is comprised of two
or three saccharide residues that formed the original two or three
saccharide residues of a glycosaminoglycan (GAG) prior to treatment
with the one or more glycosaminoglycan (GAG) lyase.
[0104] In certain embodiments, analytical methods provided herein
further comprise methods of purification. In certain embodiments,
purification methods are performed prior to treating a biological
sample with a lyase, as described herein. In some embodiments,
purification methods are performed after treating a biological
sample with a lyase, as described herein. In certain embodiments,
purification methods are utilized before and after treating a
biological sample with a lyase, as described herein. In some
embodiments, purification methods include purifying one or more
glycosaminoglycan and/or one or more oligosaccharide from other
components (e.g., cells, cell parts, other polysaccharides, or the
like) of the biological sample. In certain embodiments,
purification methods include purifying one or more
glycosaminoglycan from other polysaccharides (e.g., other glycans,
other glycosaminoglycans, other sugars, or the like).
[0105] In certain instances the GAGs provided in a biological
sample are present in lysosomes of cells. In some embodiments, any
process described herein includes lysing a biological sample to
free the GAGs from the cells therein.
Diagnostics
[0106] Provided in some embodiments herein is a process for
diagnosing the identity and/or severity of abnormal
glycosaminoglycan (or other glycan, e.g., glycolipid) accumulation
in an individual, or a disorder thereof, the process comprising the
step of: detecting the presence of and/or measuring the amount of a
population of one or more oligosaccharides present in a transformed
biological sample (e.g., urine, serum, plasma, or CSF). In certain
embodiments, the process for diagnosing the identity and/or
severity of abnormal glycosaminoglycan accumulation in an
individual is a process of diagnosing the individual as an
individual suffering from, homozygous for, or symptomatic for such
a disorder. In other embodiments, the process for diagnosing the
identity and/or severity of abnormal glycosaminoglycan accumulation
in an individual is a process of diagnosing the individual as an
individual suffering from such a disorder as a carrier for, or
heterozygous for, such a disorder. In some embodiments, individuals
that are carriers for, or heterozygous for, such a disorder has an
elevated level of glycosaminoglycan accumulation (e.g., when
compared to a normal individual), but the elevated level is less
than an individual diagnosed with having the disorder. In certain
embodiments, individuals that are carriers for, or heterozygous
for, such a disorder has an elevated level of glycosaminoglycan
accumulation (e.g., when compared to a normal individual), but are
asymptomatic (including substantially asymptomatic) for a GAG
accumulation disorder. Carriers and individuals having a GAG
accumulation disease are identified utilizing any appropriate
procedure. For example, in certain embodiments, carriers or carrier
specimens may be identified as accumulating, e.g., 2-100 times more
GAG than a non-carrier or wild type specimen. Similarly, in some
exemplary embodiments, individuals that are symptomatic or have a
GAG accumulation disease state accumulate more than 2 times more
(e.g., 2-100.times.) GAG than a carrier. In some embodiments,
diagnosis of one or more carrier parent is optionally utilized to
make a progeny risk assessment (e.g., likelihood of a child being a
carrier for or having a disease state).
[0107] In some embodiments, provided herein is a process for
diagnosing abnormal glycosaminoglycan accumulation in an
individual, or a disorder thereof, the process comprising the step
of: using an analytical instrument to detect the presence of and/or
measure the amount of a population of one or more oligosaccharides
present in a transformed biological sample that has been prepared
by treating a population of glycosaminoglycans, in or isolated from
a biological sample from the individual, with at least one
digesting glycosaminoglycan lyase to transform the
glycosaminoglycans into the population of the one or more
oligosaccharide. In specific embodiments, the oligosaccharide(s)
detected or measured is one or more C4-C5 non-reducing end
saturated oligosaccharide(s).
[0108] In some embodiments, provided herein is a process for
diagnosing the identity (or type, e.g., heparan sulfate,
chondroitin sulfate, or any other glycosaminoglycan) of abnormal
glycosaminoglycan accumulation in an individual, or a disorder
thereof, the process comprising the step of: using an analytical
instrument to detect the presence of and/or measure the amount of a
population of one or more oligosaccharides present in a transformed
biological sample that has been prepared by treating a population
of glycosaminoglycans, in or isolated from a biological sample from
the individual, with at least one digesting glycosaminoglycan lyase
to transform the glycosaminoglycans into the population of the one
or more oligosaccharide. In specific embodiments, the
oligosaccharide(s) detected or measured is one or more C4-C5
non-reducing end saturated oligosaccharide(s).
[0109] In some embodiments, provided herein is a process for
diagnosing the severity of abnormal glycosaminoglycan accumulation
in an individual, or a disorder thereof, the process comprising the
step of: using an analytical instrument to detect the presence of
and/or measure the amount of a population of one or more
oligosaccharides present in a transformed biological sample that
has been prepared by treating a population of glycosaminoglycans,
in or isolated from a biological sample from the individual, with
at least one digesting glycosaminoglycan lyase to transform the
glycosaminoglycans into the population of the one or more
oligosaccharide. In specific embodiments, the oligosaccharide(s)
detected or measured is one or more C4-C5 non-reducing end
saturated oligosaccharide(s).
[0110] In some embodiments, provided herein is a process for
diagnosing an individual as being a carrier of a gene that causes
abnormal glycosaminoglycan accumulation in an individual, or a
disorder thereof, the process comprising the step of: using an
analytical instrument to detect the presence of and/or measure the
amount of a population of one or more oligosaccharides present in a
transformed biological sample that has been prepared by treating a
population of glycosaminoglycans, in or isolated from a biological
sample from the individual, with at least one digesting
glycosaminoglycan lyase to transform the glycosaminoglycans into
the population of the one or more oligosaccharide. In certain
instances, such a process involves determining the severity of
abnormal glycosaminoglycan accumulation, wherein such accumulation
is below a certain threshold (e.g., a predetermined level, a level
whereby the individual becomes symptomatic, or the like). In
specific embodiments, the oligosaccharide(s) detected or measured
is one or more C4-C5 non-reducing end saturated
oligosaccharide(s).
[0111] In some embodiments, provided herein is a process for
diagnosing abnormal glycosaminoglycan accumulation in a human
infant (e.g., a newborn) or fetus, or a disorder thereof, the
process comprising the step of: using an analytical instrument to
detect the presence of and/or measure the amount of a population of
one or more oligosaccharides present in a transformed biological
sample that has been prepared by treating a population of
glycosaminoglycans, in or isolated from a biological sample from
the individual, with at least one digesting glycosaminoglycan lyase
to transform the glycosaminoglycans into the population of the one
or more oligosaccharide. In specific embodiments, the
oligosaccharide(s) detected or measured is one or more C4-C5
non-reducing end saturated oligosaccharide(s).
[0112] In further embodiments, any of the processes described
herein further comprise the step of displaying or recording the
presence of or a measure of a population of one or more
oligosaccharide. The display may be on a computer screen or a paper
print out. The recording may be on any computer readable disk
(e.g., a hard drive, CD, DVD, portable memory device, such as a CF
device or SD device, or the like), a sheet of paper, or the
like.
[0113] In some embodiments, the transformed biological sample is
prepared by treating a population of glycosaminoglycans or other
glycan (e.g., glycolipid), the glycosaminoglycans or other glycan
(e.g., glycolipid) being present in or isolated from a biological
sample (e.g., urine, serum, plasma, or CSF) from an individual.
Diagnostics, methods and compositions of matter described herein
when referring to a GAG in general or a specific GAG, e.g., heparan
sulfate, is understood to contain disclosure for any suitable
glycan (e.g., a glycolipid). In certain embodiments, the
glycosaminoglycans are treated with at least one agent suitable for
cleaving bonds between saccharide residues of glycosaminoglycans.
In some embodiments, a process described herein comprises
transforming a biological sample by treating a population of
glycosaminoglycans, the glycosaminoglycans being present in or
isolated from a biological sample from an individual. In certain
embodiments, the glycosaminoglycans are treated with at least one
agent suitable for cleaving bonds between saccharide residues of
glycosaminoglycans. In specific embodiments, treating a biological
sample that comprises glycosaminoglycans with at least one agent
suitable for cleaving bonds between saccharide residues of
glycosaminoglycans comprises treating the biological sample with
one or more digesting glycosaminoglycan (GAG) lyase. In some
embodiments, the one or more digesting glycosaminoglycan lyase is
one or more heparin lyase, one or more chondroitinase, one or more
keratanase, one or more hyaluronidase, or a combination thereof. In
certain embodiments, treatment of the glycosaminoglycan with the
lyase provides to transform the glycosaminoglycans into the
population of the one or more oligosaccharide. In specific
embodiments, the at least one digesting glycosaminoglycan lyase is
one or more heparin lyase.
[0114] In certain embodiments, the abnormal glycosaminoglycan
accumulation comprises abnormal heparan sulfate accumulation,
abnormal chondroitin sulfate accumulation, abnormal keratan sulfate
accumulation, abnormal hyaluronan accumulation, abnormal dermatan
sulfate accumulation, or a combination thereof. In some
embodiments, disorders associated with abnormal glycosaminoglycan
accumulation include lysosomal storage diseases, such as, by way of
non-limiting example, mucopolysaccharidosis (MPS) (e.g., MPS I, MPS
II, MPS IIIA, MPS IIIB, MPS IIIC, MPS IIID, MPS IVA, MPS IVB, MPS
VI, MPS VII, MPS IX, or the like). In some embodiments, the process
of diagnosing the identity of or the severity of a disorder
associated with the accumulation of glycosaminoglycans is a
disorder associated with abnormal heparan sulfate accumulation. In
specific embodiments, disorders associated with abnormal heparan
sulfate accumulation include, by way of non-limiting example, MPS
I, MPS II, MPS IIIA, MPS IIIB, MPS IIIC, MPS IIID, MPS VII, or the
like. In some embodiments, the process of diagnosing the identity
of or the severity of a disorder associated with the accumulation
of glycosaminoglycans is a disorder associated with abnormal
dermatan sulfate accumulation (e.g., in some instances, MPS I, MPS
II, MPS VI, or the like). In certain embodiments, the process of
diagnosing the identity of or the severity of a disorder associated
with the accumulation of glycosaminoglycans is a disorder
associated with abnormal chondroitin sulfate accumulation (e.g., in
some instances, MPS VI, MPS VII, or the like). In some embodiments,
the process of diagnosing the identity of or the severity of a
disorder associated with the accumulation of glycosaminoglycans is
a disorder associated with abnormal keratan sulfate accumulation
(e.g., in some instances, MPS IVA, MPS IVB, or the like). In
certain embodiments, the process of diagnosing the identity of or
the severity of a disorder associated with the accumulation of
glycosaminoglycans is a disorder associated with abnormal
hyaluronan accumulation (e.g., in some instances, MPS VII, MPS IX,
or the like). In some embodiments, oligosaccharides provided by
treating the glycosaminoglycan with a suitable glycosaminoglycan
lyase are utilized in processes described herein to diagnose the
identity of and/or measure the severity of a disorder associated
with the abnormal accumulation of the particular glycosaminoglycan.
Specific oligosaccharides provided by treating various
glycosaminoglycans with glycosaminoglycan lyases are provided
herein in the oligosaccharide section.
[0115] Moreover, in certain embodiments, the diagnostic methods
described herein (or other method described herein) are suitable
for diagnosing (or measuring the efficacy of a treatment of) a
disorder in an individual involved with glycan (e.g., GAG)
accumulation or any disorder involved with altered GAG synthesis
and degradation (e.g., any disorder that provides a unique GAG or
population of GAGs that can be detected by a process described
herein). In some embodiments, such a disease includes Alzheimer's
Disease, wherein GAGs are present in plaques, and a biological
sample is taken from the plaque and analyzed according to a process
described herein. In other embodiments, such a disease includes
cancer.
[0116] In some embodiments, specific oligosaccharides are detected
and/or measured according to methods and/or processes described
herein to diagnose the identity and/or severity of a specific
disorder associated with glycosaminoglycan accumulation. In some
embodiments, such oligosaccharides are described herein. In
specific embodiments, a process for diagnosing the identity or
severity of a disorder associated with the accumulation of
glycosaminoglycans provided herein comprises detecting and/or
measuring one or more oligosaccharide set forth in Formulas I-XX or
any other oligosaccharide described in the figures. In certain
embodiments, the one or more oligosaccharides detected and/or
measured are free of carbon-carbon unsaturation. In some
embodiments, the one or more oligosaccharides detected and/or
measured are free of C4 and C5 carbon unsaturation on the
saccharide residue at the non-reducing end of the oligosaccharide.
In some embodiments, the oligosaccharide of any of Formulas I-XX is
a disaccharide or trisaccharide comprised of two or three
saccharide residues that formed the original two or three
saccharide residues of a glycosaminoglycan (GAG) prior to treatment
with the one or more glycosaminoglycan (GAG) lyase. In certain
instances, the amount of disaccharide or trisaccharide of any of
Formulas I-XX free of non-reducing end carbon-carbon (e.g., C4/C5)
is representative of the amount of accumulated glycosaminoglycans
comprising the same disaccharide or trisaccharide as residue
thereof, at its non-reducing end.
[0117] In certain instances, a diagnostic method described herein
is useful for analyzing the various different classes of MPS. In
some instances, the GAG accumulation provides a unique population
of GAGs depending on the specific MPS class. In specific instance,
the unique population of GAGs can be identified as being correlated
with a specific MPS class by detecting and/or measuring
oligosaccharides in a sample taken from an individual diagnosed
with or suspected of having an MPS disorder, the oligosaccharides
being free of C4 and C5 carbon unsaturation on the saccharide
residue at the non-reducing end of the oligosaccharide. In certain
instances, the oligosaccharides are digested with a suitable
enzyme, such as a lyase (e.g., a bacterial lyase or heparin lyase)
prior to detection/measurement and the resulting oligosaccharide
(shorter in certain instances than the sample oligosaccharide, such
as di- or tri-saccharides) are detected/measured. In certain
instances, the degradation enzymes (e.g., heparin lyase) work by an
eliminase mechanism which introduces an unsaturated bond on the
newly generated non-reducing end; whereas preexisting non-reducing
ends retain their full mass (e.g., these non-reducing ends are free
of C4 and C5 carbon unsaturation). Thus in certain embodiments, the
digested oligosaccharides comprising non-reducing ends that are
free of C4 and C5 carbon unsaturation are representative of the
total number of oligosaccharides present in the original sample
composition. In certain instances, the mechanism of digesting
(e.g., with a heparin lyase) effectively tags the preexisting ends
to allow for their identification by their unique mass (e.g., being
18 Daltons larger than the other oligosaccharides provided by
internal oligosaccharide residues). In some instances,
identification of these preexisting non-reducing ends are excellent
biomarkers because, e.g., in certain instances (1) they are
homogenous within an MPS class (e.g., in certain instances for MPS
II, they all end in 2-O sulfated uronic acid); (2) there are many
more non-reducing ends in GAGs from individuals suffering from MPS
than in non-MPS individuals); and/or (3) in non-MPS individuals the
non-reducing end saccharide residues are heterogeneous (see, e.g.,
FIG. 2). FIG. 3A illustrates the large number of oligosaccharide
residues found within GAGs and FIGS. 3B and 3C summarize the
predicted non-reducing end heparan sulfate oligosaccharide residues
(e.g., the biomarkers) for 7 MPS classes. In some embodiments, any
one or more of these oligosaccharides are detected and/or measured
in a method of diagnosing an individual suffering from the specific
MPS class described. In some embodiments, additional biomarkers for
these MPS classes that originate from chondroitin and dermatan can
also be analyzed using the same method. The same approach (using
chondroitinase, keratanase, and hyaluronidases) is provided for in
certain embodiments for other MPS classes that accumulate CS, DS,
KS, and/or HA.
[0118] In certain embodiments, a process for diagnosing the
identity or severity of a disorder associated with the accumulation
of glycosaminoglycans provided herein comprises detecting and/or
measuring one or more oligosaccharide set forth in Formulas
XXI-XXIX or in any of the figures described herein. In certain
embodiments, the one or more oligosaccharides detected and/or
measured comprise at least one point of carbon-carbon unsaturation.
In some embodiments, the one or more oligosaccharides detected
and/or measured comprise C4 and C5 carbon unsaturation on the
saccharide residue at the non-reducing end of the
oligosaccharide.
[0119] In certain embodiments, processes described herein,
including diagnostic processes, include preparing a transformed
biological sample by purifying a population of oligosaccharides in
a biological sample that has been treated with the at least one
glycosaminoglycan lyase (e.g., one or more heparin lyase), the
transformed biological sample comprising the isolated population of
oligosaccharides. In some embodiments, glycosaminoglycans of the
biological sample from an individual are purified prior to
treatment with the one or more glycosaminoglycan lyase.
[0120] In some embodiments, a diagnositic (including identity or
severity diagnostic) process provided herein comprises comparing a
detection or measurement according to the process to a control
reading. In some embodiments, the comparison to a control comprises
comparing the amount of the population of one or more
oligosaccharide present in the transformed biological sample to an
amount of a population of the one or more oligosaccharide present
in a control biological sample that has been treated in a manner
substantially similar to the transformed biological sample. In
specific embodiments, the control biological sample was provided
from an individual that does not have a disorder associated with
abnormal glycosaminoglycan accumulation (e.g.,
mucopolysaccharidosis (MPS)). In specific embodiments, the control
biological sample was provided from an individual that has a
disorder associated with abnormal glycosaminoglycan accumulation
(e.g., mucopolysaccharidosis (MPS)). In more specific embodiments,
the control is from an individual with an abnormal
glycosaminoglycan accumulation selected from, by way of
non-limiting example, MPS I, MPS II, MPS IIIA, MPS IIIB, MPS IIIC,
MPS IIID, MPS IVA, MPS IVB, MPS VI, MPS VII, MPS IX, and a
combination thereof (e.g., MPS I, MPS II, MPS IIIA, MPS IIIB, MPS
IIIC, MPS IIID, MPS VII, and a combination thereof).
[0121] In some embodiments, detecting the presence of or measuring
the amount of a population of one or more oligosaccharide present
in a transformed biological sample according to a process described
herein comprises: [0122] a. isolating a subpopulation of one or
more oligosaccharides in the transformed biological sample (e.g., a
transformed urine, serum, plasma, or CSF sample); and [0123] b.
detecting the presence of and/or measuring the amount of one or
more oligosaccharides present in the subpopulation.
[0124] Isolation of the subpopulation of one or more
oligosaccharides in the transformed biological sample is achieved
in any suitable manner, e.g., using a purification process
described herein (e.g., chromatography, electrophoresis,
filtration, centrifugation, etc.). Similarly, according to any
process described herein, the detection of and/or measuring the
presence of one or more oligosaccharide is achieved utilizing any
suitable process, including those detection processes set forth
herein (e.g., spectrometry, UV-Visible spectrometry, IR
spectrometry, NMR spectrometry, mass spectrometry, or the like). In
specific instances, prior to detecting and/or measuring the
oligosaccharide present, any process described herein further
comprises tagging the reducing end of a representative portion of
the one or more oligosaccharides in the transformed biological
sample with any suitable detectable label (e.g., a mass label, a
radio label, a fluorescent label, a chromophore label, affinity
label, etc.).
[0125] In certain embodiments, the detection of the presence and/or
measure of the amount of oligosaccharide is performed utilizing an
analytical instrument. In specific embodiments, the analytical
device comprises a spectrometer that detects and/or measures the
amount of a detectable label. In certain embodiments, the detection
and/or measurement of amounts of a detectable label serves as a
proxy to the presence or amounts of GAGs present. In more specific
embodiments, the spectrometer includes, by way of non-limiting
example, one or more of a mass spectrometer, a nuclear magnetic
resonance spectrometer, a UV-Vis spectrometer, an IR spectrometer,
a fluorimeter, a phosphorimeter, a radiation spectrometer, or the
like. In certain embodiments, the analytical device comprises a
purification device coupled to a detector or a measuring device
(e.g., a HPLC system coupled to a UV-Vis spectrometer). In certain
embodiments, an analytical device is a liquid chromatography mass
spectrometer (LC-MS) that detects and/or measures the mass of an
oligosaccharide.
[0126] In some embodiments, the presence detected and/or the
measure of the population of the oligosaccharide is displayed or
recorded. In some embodiments, the process comprises displaying or
recording the results of the characterization. In certain
embodiments, the results are displayed on a display monitor (e.g.,
a computer monitor, television, PDA, or the like), or print out. In
some embodiments, the results are recorded on an electronic medium
(e.g., a hard disk drive, magnetic storage drive, optical storage
drive or the like; a disk such as a floppy disk, CD, DVD, BLU-ray
or the like; a flash memory drive; removable drive or the
like).
[0127] In certain embodiments, the individual is a mammal, e.g., a
human. In some embodiments, the human is a newborn. In certain
embodiments, the human is an embryo in utero. In some embodiments,
the human has been diagnosed with a lysosomal storage disease. In
some embodiments, the human is suspected of suffering from a
lysosomal storage disease.
Analytical Samples
[0128] Provided in certain embodiments herein are compositions
comprising any one or more oligosaccharides provided herein. In
some embodiments, the composition provided herein is an analytical
sample, suitable analysis in any analytical device, e.g., one
provided herein (such as, by way of non-limiting example, high
performance liquid chromatography, mass spectrometry, gas
chromatography, or the like).
[0129] In certain embodiments, a composition provided herein
comprises at least one disaccharide or trisaccharide from a
transformed biological sample from an individual with a disorder
associated with abnormal glycosaminoglycan accumulation. In
specific embodiments, the transformed biological sample was
prepared by treating a biological sample comprising
glycosaminoglycans with one or more digesting glycosaminoglycan
lyase.
[0130] In some embodiments, an analytical sample provided herein
comprises one or more oligosaccharide of any of Formulas I to XX,
any one of Formulas XXI to XXIX, or any of Formulas I to XXIX. In
certain embodiments, an analytical sample provided herein comprises
one or more oligosaccharide of any of Formulas I to XX, any one of
Formulas XXI to XXIX, or any of Formulas I to XXIX, wherein the one
or more oligosaccharides further comprise a detectable label
attached (e.g., covalently and/or non-covalently) to the reducing
end of the one or more oligosaccharide.
[0131] In some embodiments, provided herein is a composition
comprising isolated glycans, wherein the glycans were isolated from
a biological sample, and one or more glycan degradation enzyme. In
certain embodiments, the composition further comprises one or more
biomarker generated according to any method described herein (e.g.,
wherein the biomarker is a non-reducing saturated oligosaccharide).
In certain embodiments, provided herein is an oligosaccharide
described herein (e.g., a labeled or non-labeled non-reducing
saturated oligosaccharide) and an analytical instrument or
chromatographic resin.
Oligosaccharides
[0132] In certain embodiments, methods and processes described
herein are utilized to detect and/or measure one or more biomarker.
In specific embodiments, such biomarkers comprise one or more
oligosaccharides (e.g., disaccharide(s) and/or trisaccharide(s)).
In certain embodiments, the one or more oligosaccharides comprise
any one or more of the oligosaccharides described herein.
[0133] As used herein, IdoA and are iduronic acid (e.g.,
.alpha.-L-iduronic acid) saccharide residues. As used herein, GlcA
and are glucuronic acid (e.g., .beta.-L-glucuronic acid) saccharide
residues. As used herein, is either an iduronic acid (e.g.,
.alpha.-L-iduronic acid) saccharide residue or a glucuronic acid
(e.g., .beta.-L-glucuronic acid) saccharide residue. As used
herein, GlcN and are glucosamine (e.g.,
2-deoxy-2-amino-.beta.-D-glucopyranosyl) saccharide residues. As
used herein, GlcN(Ac).sub.1 and are a glucosamine (e.g.,
2-deoxy-2-amino-.beta.-D-glucopyranosyl) saccharide residue wherein
the 2-amino group is acetylated. As used herein, Gal and
.largecircle. is a galactose saccharide residue. As used herein
GalNAc and .quadrature. represents an N-acetylgalactosamine
residue. As used herein and both represent N-sulfated (i.e.,
N-substituted with SO.sub.3R as described herein) glucosamine
(e.g., 2-deoxy-2-amino-.beta.-D-glucopyranosyl) saccharide residue.
In various specific instances, iduronic acid, glucuronic acid,
glucosamine, and/or galactose saccharide residues are saturated at
4 and 5 carbons of the non-reducing end saccharide residue, or are
free of carbon-carbon unsaturation. In other instances, any one or
more of the saccharide residues is unsaturated, e.g., at the 4 and
5 carbon positions of the saccharide residue at the non-reducing
end of an oligosaccharide provided herein. The symbolic
nomenclature used herein follows the "Symbol and Text Nomenclature
for Representation of Glycan Structure" as promulgated by the
Nomenclature Committee for the Consortium for Functional Glycomics,
as amended on October 2007. Recitation of an NS (e.g., above of
below any of the aforementioned structures) indicates that the
amino group thereof is substituted with (SO.sub.3R). If the NS is
associated with GlcN(Ac)m or above or below .box-solid., the
residue is GlcN(SO.sub.3R), wherein the amino group bears the
(SO.sub.3R). Recitation of a 2S (e.g., above or below any of the
aforementioned structures) indicates that the hydroxyl group at the
two carbon position of the indicated saccharide residue is
substituted with (SO.sub.3R). Recitation of a 3S (e.g., above of
below any of the aforementioned structures) indicates that the
hydroxyl group at the three carbon position of the indicated
saccharide residue is substituted with (SO.sub.3R). Recitation of a
4S (e.g., above of below any of the aforementioned structures)
indicates that the hydroxyl group at the four carbon position of
the indicated saccharide residue is substituted with (SO.sub.3R).
Recitation of a 6S (e.g., above of below any of the aforementioned
structures) indicates that the hydroxyl group at the six carbon
position of the indicated saccharide residue is substituted with
(SO.sub.3R).
[0134] In specific embodiments, methods and processes described
herein are utilized to detect and/or measure an oligosaccharide
(disaccharide) having the formula:
[IdoA-GlcN(Ac).sub.m](SO.sub.3R).sub.n (I)
[0135] In certain embodiments, oligosaccharides described herein,
e.g., those detected and/or measured according to the methods
and/or processes described herein, are disaccharides of Formula I,
wherein m is 0-1, and n is 0-3. As used herein, each R is
independently H or a negative charge. The disaccharide is
optionally sulfated with n SO.sub.3R groups in any suitable
location. In more specific embodiments, a disaccharide of Formula I
has a structure of IdoA-GlcNAc, IdoA-GlcNS, IdoA-GlcNS6S,
IdoA2S-GlcNAc, IdoA2S-GlcNS, IdoA2S-GlcNAc6S, IdoA2S-GlcNS6S or as
set forth in any of Formulas I-A to I-G, as set forth in FIG. 4. In
certain instances, compounds of Formulas I-A to I-G are provided by
treating the glycosaminoglycan heparan sulfate with a suitable
glycosaminoglycan lyase.
[0136] In some embodiments, the detection and/or measurement of any
one or more disaccharide of Formulas I-A to I-C is utilized in a
process for diagnosing MPS I. In certain embodiments, the detection
and/or measurement of any one or more disaccharide of Formulas I-D
to I-G is utilized in a process for diagnosing MPS II.
[0137] In certain embodiments, methods and processes described
herein are utilized to detect and/or measure an oligosaccharide
(trisaccharide) having the formula:
[GlcN(Ac)m-(IdoA/GlcA)-GlcN(Ac).sub.n](SO.sub.3R).sub.p (II)
[0138] In certain embodiments, oligosaccharides described herein,
e.g., those detected and/or measured according to the methods
and/or processes described herein, are trisaccharides of Formula
II, wherein IdoA/GlcA is either IdoA or GlcA, m is 0-1, n is 0-1,
and p is 0-5. The trisaccharide is optionally sulfated with p
SO.sub.3R groups in any suitable location.
[0139] In some specific embodiments, a compound of Formula II has a
structure as set forth in Formula III:
[GlcNS-IdoA-GlcN(Ac).sub.n](SO.sub.3R).sub.p (III)
[0140] In certain embodiments, oligosaccharides described herein,
e.g., those detected and/or measured according to the methods
and/or processes described herein, are trisaccharides of Formula
III, wherein n is 0-1, and p is 0-3. The trisaccharide is
optionally sulfated with p SO.sub.3R groups in any suitable
location. In more specific embodiments, a trisaccharide of Formula
III has a structure of GlcNS-IdoA-GlcNAc, GlcNS-IdoA2S-GlcNAc,
GlcNS-IdoA-GlcNS, GlcNS-IdoA2S-GlcNS, GlcNS-IdoA-GlcNAc6S,
GlcNS-IdoA2S-GlcNAc6S, GlcNS-IdoA-GlcNS6S, GlcNS-IdoA2S-GlcNS6S, or
as set forth in any of the trisaccharides of FIG. 5. In certain
instances, compounds of Formulas III-A to III-H are provided by
treating the glycosaminoglycan heparan sulfate with a suitable
glycosaminoglycan lyase.
[0141] In certain specific embodiments, a compound of Formula II
has a structure as set forth in Formula IV:
[GlcNS-GlcA-GlcN(Ac).sub.n](SO.sub.3R).sub.p (IV)
[0142] In certain embodiments, oligosaccharides described herein,
e.g., those detected and/or measured according to the methods
and/or processes described herein, are trisaccharides of Formula
IV, wherein n is 0-1, and p is 0-2. The trisaccharide is optionally
sulfated with p SO.sub.3R groups in any suitable location. In more
specific embodiments, a trisaccharide of Formula IV has a structure
of GlcNS-GlcA-GlcNAc, GlcNS-GlcA-GlcNS, GlcNS-GlcA-GlcNAc6S,
GlcNS-GlcA-GlcNS6S or as set forth in any of the trisaccharides of
FIG. 6. In certain instances, compounds of Formulas IV-A to IV-D
are provided by treating the glycosaminoglycan heparan sulfate with
a suitable glycosaminoglycan lyase.
[0143] In some embodiments, the detection and/or measurement of any
one or more trisaccharide of Formulas III and/or IV is utilized in
a process for diagnosing MPS IIIA.
[0144] In some specific embodiments, a compound of Formula II has a
structure as set forth in Formula V:
[GlcNAc-IdoA-GlcN(Ac).sub.n](SO.sub.3R).sub.p (V)
[0145] In certain embodiments, oligosaccharides described herein,
e.g., those detected and/or measured according to the methods
and/or processes described herein, are trisaccharides of Formula V,
wherein n is 0-1, and p is 0-3. The trisaccharide is optionally
sulfated with p SO.sub.3R groups in any suitable location. In more
specific embodiments, a trisaccharide of Formula V has a structure
of GlcNAc-IdoA-GlcNAc, GlcNAc-IdoA2S-GlcNAc, GlcNAc-IdoA-GlcNS,
GlcNAc-IdoA2S-GlcNS, GlcNAc-IdoA-GlcNAc6S, GlcNAc-IdoA2S-GlcNAc6S,
GlcNAc-IdoA-GlcNS6S, GlcNAc-IdoA2S-GlcNS6, or as set forth in any
of the trisaccharides of FIG. 7. In certain instances, compounds of
Formulas V-A to V-H are provided by treating the glycosaminoglycan
heparan sulfate with a suitable glycosaminoglycan lyase.
[0146] In certain specific embodiments, a compound of Formula II
has a structure as set forth in Formula VI:
[GlcNAc-GlcA-GlcN(Ac).sub.n](SO.sub.3R).sub.p (VI)
[0147] In certain embodiments, oligosaccharides described herein,
e.g., those detected and/or measured according to the methods
and/or processes described herein, are trisaccharides of Formula
VI, wherein n is 0-1, and p is 0-2. The trisaccharide is optionally
sulfated with p SO.sub.3R groups in any suitable location. In more
specific embodiments, a trisaccharide of Formula VI has a structure
of GlcNAc-GlcA-GlcNAc, GlcNAc-GlcA-GlcNS, GlcNAc-GlcA-GlcNAc6S,
GlcNAc-GlcA-GlcNS6S or as set forth in any of the trisaccharides of
FIG. 8. In certain instances, compounds of Formulas VI-A to VI-D
are provided by treating the glycosaminoglycan heparan sulfate with
a suitable glycosaminoglycan lyase.
[0148] In some embodiments, the detection and/or measurement of any
one or more trisaccharide of Formulas V and/or VI is utilized in a
process for diagnosing MPS IIIB.
[0149] In some specific embodiments, a compound of Formula II has a
structure as set forth in Formula VII:
[GlcN-Ido-GlcN(Ac).sub.n](SO.sub.3R).sub.p (VII)
[0150] In certain embodiments, oligosaccharides described herein,
e.g., those detected and/or measured according to the methods
and/or processes described herein, are trisaccharides of Formula
VII, wherein n is 0-1, and p is 0-4. The trisaccharide is
optionally sulfated with p SO.sub.3R groups in any suitable
location. In more specific embodiments, a trisaccharide of Formula
VII has a structure of GlcN-IdoA-GlcNAc, GlcN-IdoA2S-GlcNAc,
GlcN-IdoA-GlcNS, GlcN-IdoA2S-GlcNS, GlcN-IdoA-GlcNAc6S,
GlcN-IdoA2S-GlcNAc6S, GlcN-IdoA-GlcNS6S, GlcN-IdoA2S-GlcNS6S, or as
set forth in any of the trisaccharides of FIG. 9. In certain
instances, compounds of Formulas VII-A to VII-H are provided by
treating the glycosaminoglycan heparan sulfate with a suitable
glycosaminoglycan lyase.
[0151] In certain specific embodiments, a compound of Formula II
has a structure as set forth in Formula VIII:
[GlcN-GlcA-GlcN(Ac).sub.n](SO.sub.3R).sub.p (VIII)
[0152] In certain embodiments, oligosaccharides described herein,
e.g., those detected and/or measured according to the methods
and/or processes described herein, are trisaccharides of Formula
VIII, wherein n is 0-1, and p is 0-4. The trisaccharide is
optionally sulfated with p SO.sub.3R groups in any suitable
location. In more specific embodiments, a trisaccharide of Formula
VIII has a structure of GlcN-GlcA-GlcNAc, GlcN-GlcA-GlcNS,
GlcN-GlcA-GlcNAc6S, GlcN-GlcA-GlcNS6S, or as set forth in any of
the trisaccharides of FIG. 10. In certain instances, compounds of
Formulas VIII-A to VIII-D are provided by treating the
glycosaminoglycan heparan sulfate with a suitable glycosaminoglycan
lyase.
[0153] In some embodiments, the detection and/or measurement of any
one or more trisaccharide of Formulas VII and/or VIII is utilized
in a process for diagnosing MPS IIIC.
[0154] In some specific embodiments, a compound of Formula II has a
structure as set forth in Formula IX:
[GlcNAc6S-Ido-GlcN(Ac).sub.n](SO.sub.3R).sub.p (IX)
[0155] In certain embodiments, oligosaccharides described herein,
e.g., those detected and/or measured according to the methods
and/or processes described herein, are trisaccharides of Formula
IX, wherein n is 0-1, and p is 0-3. The trisaccharide is optionally
sulfated with p SO.sub.3R groups in any suitable location. In more
specific embodiments, a trisaccharide of Formula VII has a
structure of GlcNAc6S-IdoA-GlcNAc, GlcNAc6S-IdoA2S-GlcNAc,
GlcNAc6S-IdoA-GlcNS, GlcNAc6S-IdoA2S-GlcNS, GlcNAc6S-IdoA-GlcNAc6S,
GlcNAc6S-IdoA2S-GlcNAc6S, GlcNAc6S-IdoA-GlcNS6S,
GlcNAc6S-IdoA2S-GlcNS6S, or as set forth in any of the
trisaccharides of FIG. 11. In certain instances, compounds of
Formulas IX-A to IX-H are provided by treating the
glycosaminoglycan heparan sulfate with a suitable glycosaminoglycan
lyase.
[0156] In certain specific embodiments, a compound of Formula II
has a structure as set forth in Formula X:
[GlcNAc6S-GlcA-GlcN(Ac).sub.n](SO.sub.3R).sub.p (X)
[0157] In certain embodiments, oligosaccharides described herein,
e.g., those detected and/or measured according to the methods
and/or processes described herein, are trisaccharides of Formula X,
wherein n is 0-1, and p is 0-2. The trisaccharide is optionally
sulfated with p SO.sub.3R groups in any suitable location. In more
specific embodiments, a trisaccharide of Formula X has a structure
of GlcNAc6S-GlcA-GlcNAc, GlcNAc6S-GlcA-GlcNS,
GlcNAc6S-GlcA-GlcNAc6S, GlcNAc6S-GlcA-GlcNS6S, or as set forth in
any of the trisaccharides of FIG. 12. In certain instances,
compounds of Formulas X-A to X-D are provided by treating the
glycosaminoglycan heparan sulfate with a suitable glycosaminoglycan
lyase.
[0158] In some embodiments, the detection and/or measurement of any
one or more trisaccharide of Formulas IX and/or X is utilized in a
process for diagnosing MPS IIID.
[0159] In certain embodiments, methods and processes described
herein are utilized to detect and/or measure an oligosaccharide
(disaccharide) having the formula:
[GlcA-GlcN(Ac).sub.m](SO.sub.3R).sub.n (XI)
[0160] In certain embodiments, oligosaccharides described herein,
e.g., those detected and/or measured according to the methods
and/or processes described herein, are disaccharides of Formula XI,
wherein m is 0-1, and n is 0-2. The disaccharide is optionally
sulfated with n SO.sub.3R groups in any suitable location. In more
specific embodiments, a disaccharide of Formula XI has a structure
of GlcA-GlcNAc, GlcA-GlcNAc 6S, GlcA-GlcNS, GlcA-GlcNS6S, or as set
forth in any of the disaccharides of FIG. 13. In certain instances,
compounds of Formulas XI-A to XI-D are provided by treating the
glycosaminoglycan heparan sulfate with a suitable glycosaminoglycan
lyase.
[0161] In some embodiments, the detection and/or measurement of any
one or more disaccharide of Formula XI is utilized in a process for
diagnosing MPS VII.
[0162] In certain embodiments, methods and processes described
herein are utilized to detect and/or measure an oligosaccharide
(disaccharide) having the formula:
[IdoA-GalNAc](SO.sub.3R).sub.n (XII)
[0163] In certain embodiments, oligosaccharides described herein,
e.g., those detected and/or measured according to the methods
and/or processes described herein, are disaccharides of Formula
XII, wherein n is 0-2. The disaccharide is optionally sulfated with
n SO.sub.3R groups in any suitable location. In more specific
embodiments, a disaccharide of Formula XII has a structure of
IdoA-GalNAc, IdoA-GalNAc6S, IdoA-GalNAc4S, IdoA-GalNAc4S6S, or as
set forth in any of the disaccharides of FIG. 14. In certain
instances, compounds of Formulas XII-A to XII-D are provided by
treating the glycosaminoglycan dermatan sulfate with a suitable
glycosaminoglycan lyase.
[0164] In some embodiments, the detection and/or measurement of any
one or more disaccharide of Formula XII is utilized in a process
for diagnosing MPS I. In certain embodiments, the detection and/or
measurement of any one or more disaccharide of Formula XII and one
or more disaccharide of Formulas I-A to I-C is utilized in a
process for diagnosing MPS I.
[0165] In certain embodiments, methods and processes described
herein are utilized to detect and/or measure an oligosaccharide
(disaccharide) having the formula:
[IdoA2S-GalNAc](SO.sub.3R).sub.n (XIII)
[0166] In certain embodiments, oligosaccharides described herein,
e.g., those detected and/or measured according to the methods
and/or processes described herein, are disaccharides of Formula
XIII, wherein n is 0-2. The disaccharide is optionally sulfated
with n SO.sub.3R groups in any suitable location. In more specific
embodiments, a disaccharide of Formula XIII has a structure of
IdoA2S-GalNAc, IdoA2S-GalNAc6S, IdoA2S-GalNAc4S, IdoA2S-GalNAc4S6S,
or as set forth in any of the disaccharides of FIG. 15. In certain
instances, compounds of Formulas XIII-A to XIII-D are provided by
treating the glycosaminoglycan dermatan sulfate with a suitable
glycosaminoglycan lyase.
[0167] In some embodiments, the detection and/or measurement of any
one or more disaccharide of Formula XIII is utilized in a process
for diagnosing MPS II. In certain embodiments, the detection and/or
measurement of any one or more disaccharide of Formula XIII and one
or more disaccharide of Formulas I-D to I-G is utilized in a
process for diagnosing MPS II.
[0168] In certain embodiments, methods and processes described
herein are utilized to detect and/or measure an oligosaccharide
(trisaccharide) having the formula:
[GalNAc4S-IdoA-GalNAc](SO.sub.3R).sub.n (XIV)
[0169] In certain embodiments, oligosaccharides described herein,
e.g., those detected and/or measured according to the methods
and/or processes described herein, are trisaccharides of Formula
XIV, wherein n is 0-4. The trisaccharide is optionally sulfated
with n 503R groups in any suitable location. In more specific
embodiments, a disaccharide of Formula XIV has a structure of
GalNAc4S(.+-.6S)-IdoA(.+-.2S)-GalNAc(.+-.4S)(.+-.6S), or as set
forth in any of the disaccharides of FIG. 16, wherein .+-.indicates
that the indicated sulfation is independently present or absent. In
certain instances, compounds of Formulas XIV-A to XIV-P are
provided by treating the glycosaminoglycan dermatan sulfate with a
suitable glycosaminoglycan lyase.
[0170] In some embodiments, the detection and/or measurement of any
one or more trisaccharide of Formula XIV is utilized in a process
for diagnosing MPS VI.
[0171] In certain embodiments, methods and processes described
herein are utilized to detect and/or measure an oligosaccharide
(trisaccharide) having the formula:
[GalNAc4S-GlcA-GalNAc](SO.sub.3R).sub.n (XV)
[0172] In certain embodiments, oligosaccharides described herein,
e.g., those detected and/or measured according to the methods
and/or processes described herein, are trisaccharides of Formula
XV, wherein n is 0-3. The trisaccharide is optionally sulfated with
n SO.sub.3R groups in any suitable location. In more specific
embodiments, a disaccharide of Formula XV has a structure of
GalNAc4S(.+-.6S)-GlcA(.+-.2S)-GlcNAc(.+-.4S)(.+-.6S), or as set
forth in any of the trisaccharides of FIG. 17, wherein
.+-.indicates that the indicated sulfation is independently present
or absent. In certain instances, compounds of Formulas XV-A to XV-P
are provided by treating the glycosaminoglycan chondroitin sulfate
with a suitable glycosaminoglycan lyase.
[0173] In some embodiments, the detection and/or measurement of any
one or more trisaccharide of Formula XV is utilized in a process
for diagnosing MPS VI. In certain embodiments, the detection and/or
measurement of any one or more trisaccharide of Formula XIV and one
or more trisaccharide of Formula XV is utilized in a process for
diagnosing MPS VI.
[0174] In certain embodiments, methods and processes described
herein are utilized to detect and/or measure an oligosaccharide
(trisaccharide) having the formula:
GlcA-GlcNAc-GlcA (XVI)
[0175] In certain embodiments, oligosaccharides described herein,
e.g., those detected and/or measured according to the methods
and/or processes described herein, are trisaccharides of Formula
XVI. In certain instances, compounds of Formulas XVI are provided
by treating the glycosaminoglycan hyaluronan with a suitable
glycosaminoglycan lyase. In some embodiments, the detection and/or
measurement of any one or more trisaccharide of Formula XVI is
utilized in a process for diagnosing MPS IIIB. In certain
embodiments, the detection and/or measurement of any one or more
trisaccharide of Formula XVI and one or more trisaccharide of
either of Formulas V and/or VI is utilized in a process for
diagnosing MPS BIB.
[0176] In certain embodiments, methods and processes described
herein are utilized to detect and/or measure an oligosaccharide
(disaccharide) having the formula:
GlcA-GlcNAc (XVII)
[0177] In certain embodiments, oligosaccharides described herein,
e.g., those detected and/or measured according to the methods
and/or processes described herein, are disaccharides of Formula
XVII. In certain instances, compounds of Formulas XVII are provided
by treating the glycosaminoglycan hyaluronan with a suitable
glycosaminoglycan lyase. In some embodiments, the detection and/or
measurement of any one or more disaccharide of Formula XVII is
utilized in a process for diagnosing MPS VII or MPS IX. In certain
embodiments, the detection and/or measurement of a disaccharide of
Formula XVII and one or more disaccharide of Formula XI is utilized
in a process for diagnosing MPS VII. In some embodiments, the
detection and/or measurement of a disaccharide of Formula XVII and
the absence of one or more disaccharide of Formula XI is utilized
in a process for diagnosing MPS IX.
[0178] In certain embodiments, methods and processes described
herein are utilized to detect and/or measure an oligosaccharide
(trisaccharide) having the formula:
[GlcNAc6S-Gal-GlcNAc](SO.sub.3R).sub.n (XVIII)
[0179] In certain embodiments, oligosaccharides described herein,
e.g., those detected and/or measured according to the methods
and/or processes described herein, are trisaccharides of Formula
XVIII, and wherein n is 0-2. The trisaccharide is optionally
sulfated with n SO.sub.3R groups in any suitable location. In more
specific embodiments, a disaccharide of Formula XVIII has a
structure of GlcNAc6S-Gal-GlcNAc, GlcNAc6S-Gal6S-GlcNAc,
GlcNAc6S-Gal-GlcNAc6S, GlcNAc6S-Gal6S-GlcNAc6S, or as set forth in
any of the trisaccharides of FIG. 18. In certain instances,
compounds of Formulas XVIII-A to XVIII-D are provided by treating
the glycosaminoglycan keratan sulfate with a suitable
glycosaminoglycan lyase.
[0180] In some embodiments, the detection and/or measurement of any
one or more trisaccharide of Formula XVIII is utilized in a process
for diagnosing MPS IIID. In certain embodiments, the detection
and/or measurement of any one or more trisaccharide of Formula
XVIII and one or more trisaccharide of Formulas IX and/or X is
utilized in a process for diagnosing MPS IIID.
[0181] In certain embodiments, methods and processes described
herein are utilized to detect and/or measure an oligosaccharide
(disaccharide) having the formula:
[Gal6S-GlcNAc-(Gal).sub.m](SO.sub.3R).sub.n (XIX)
[0182] In certain embodiments, oligosaccharides described herein,
e.g., those detected and/or measured according to the methods
and/or processes described herein, are disaccharides of Formula
XIX, wherein n is 0-2 and m is 0-1. The disaccharide is optionally
sulfated with n SO.sub.3R groups in any suitable location. In more
specific embodiments, a disaccharide of Formula XIX has a structure
of Gal6S-GlcNAc, Gal6S-GlcNAc6S, or as set forth in any of the
disaccharides of FIG. 19. In certain instances, compounds of
Formulas XIX-A to XIX-B are provided by treating the
glycosaminoglycan keratan sulfate with a suitable glycosaminoglycan
lyase.
[0183] In some embodiments, the detection and/or measurement of any
one or more disaccharide of Formula XIX is utilized in a process
for diagnosing MPS IVA.
[0184] In certain embodiments, methods and processes described
herein are utilized to detect and/or measure an oligosaccharide
(disaccharide) having the formula:
[Gal-GlcNAc](SO.sub.3R).sub.n (XX)
[0185] In certain embodiments, oligosaccharides described herein,
e.g., those detected and/or measured according to the methods
and/or processes described herein, are disaccharides of Formula XX,
wherein n is 0-1. The disaccharide is optionally sulfated with n
SO.sub.3R groups in any suitable location. In more specific
embodiments, a disaccharide of Formula XX has a structure of
Gal-GlcNAc, Gal-GlcNAc6S, or as set forth in any of the
disaccharides of FIG. 20. In certain instances, compounds of
Formulas XX-A to XX-B are provided by treating the
glycosaminoglycan keratan sulfate with a suitable glycosaminoglycan
lyase.
[0186] In some embodiments, the detection and/or measurement of any
one or more disaccharide of Formula XX is utilized in a process for
diagnosing MPS IVB.
[0187] In certain embodiments, any analytical method or diagnostic
process described herein comprises detecting and/or measuring any
one or more of the oligosaccharides of any of Formulas I-XX,
wherein the non-reducing end saccharide residue is saturated at 4
and 5 carbons of the non-reducing end saccharide residue, the
non-reducing end saccharide residue is free of carbon-carbon
unsaturation, or the oligosaccharide is free of carbon-carbon
unsaturation. In certain instances, the non-reducing end saccharide
residue is on the left end of the oligosaccharides disclosed
herein. FIG. 1 illustrates the cleavage of the glycosaminoglycan
(GAG) heparan sulfate with a glycosaminoglycan lyase (heparinase
II). As illustrated, in certain instances, internal cleavage of
glycosaminoglycans with glycosaminoglycan lyases provides
oligosaccharides with carbon-carbon unsaturation between the C4 and
C5 carbons of the non-reducing end of the oligosaccharide produce
(i.e., the newly created oligosaccharide). In some embodiments, the
oligosaccharide of any of Formulas I-XX is a disaccharide or
trisaccharide comprised of two or three saccharide residues that
formed the original two or three saccharide residues of a
glycosaminoglycan (GAG) prior to treatment with the one or more
glycosaminoglycan (GAG) lyase.
[0188] In certain embodiments, methods and processes described
herein are utilized to detect and/or measure an oligosaccharide
(trisaccharide) having the formula:
[IdoA-GlcN(Ac).sub.m-IdoA](SO.sub.3R).sub.n (XXI)
[0189] In certain embodiments, oligosaccharides described herein,
e.g., those detected and/or measured according to the methods
and/or processes described herein, are trisaccharides of Formula
XXI, wherein m is 0-1 and n is 0-4. The trisaccharide is optionally
sulfated with n SO.sub.3R groups in any suitable location. In
certain instances, compounds of Formulas XXI are provided by
treating the glycosaminoglycan heparan sulfate with a suitable
glycosaminoglycan lyase.
[0190] In certain embodiments, methods and processes described
herein are utilized to detect and/or measure an oligosaccharide
(trisaccharide) having the formula:
[IdoA-GlcN(Ac).sub.m-GlcA](SO.sub.3R).sub.n (XXII)
[0191] In certain embodiments, oligosaccharides described herein,
e.g., those detected and/or measured according to the methods
and/or processes described herein, are trisaccharides of Formula
XXII, wherein m is 0-1 and n is 0-3. The trisaccharide is
optionally sulfated with n SO.sub.3R groups in any suitable
location. In certain instances, compounds of Formulas XXII are
provided by treating the glycosaminoglycan heparan sulfate with a
suitable glycosaminoglycan lyase.
[0192] In certain embodiments, methods and processes described
herein are utilized to detect and/or measure an oligosaccharide
(trisaccharide) having the formula:
[GlcA-GlcN(Ac).sub.m-GlcA](SO.sub.3R).sub.n (XXIII)
[0193] In certain embodiments, oligosaccharides described herein,
e.g., those detected and/or measured according to the methods
and/or processes described herein, are trisaccharides of Formula
XXIII, wherein m is 0-1 and n is 0-3. The trisaccharide is
optionally sulfated with n SO.sub.3R groups in any suitable
location. In certain instances, compounds of Formulas XXIII are
provided by treating the glycosaminoglycan heparan sulfate with a
suitable glycosaminoglycan lyase.
[0194] In some embodiments, the detection and/or measurement of any
one or more trisaccharide of any of Formulas XXI to XXIII is
utilized in a process for diagnosing a disorder associated with the
accumulation of heparan sulfate (e.g., a heparan sulfate lysosomal
storage disease such as, by way of non-limiting example, MPS I, MPS
II, MPS IIIA, MPS IIIB, MPS IIIC, MPS IIID, MPS VII, or a
combination thereof). In certain embodiments, an oligosaccharide of
any of Formulas XXI to XXIII is saturated or unsaturated at the C4
and C5 positions of the saccharide residue at the non-reducing end
of the oligosaccharide. In some embodiments, a process for
diagnosing a disorder associated with the accumulation of heparan
sulfate (e.g., MPS I, MPS II, MPS IIIA, MPS IIIB, MPS IIIC, MPS
IIID, MPS VII, or a combination thereof) further comprises
detecting one or more oligosaccharide, as set forth above and as
appropriate (e.g., any of Formulas I-XX, as they pertain to the
particular disorder being diagnosed).
[0195] In certain embodiments, methods and processes described
herein are utilized to detect and/or measure an oligosaccharide
(trisaccharide) having the formula:
[IdoA-GalNAc-Ido](SO.sub.3R).sub.n (XXIV)
[0196] In certain embodiments, oligosaccharides described herein,
e.g., those detected and/or measured according to the methods
and/or processes described herein, are trisaccharides of Formula
XXIV, wherein n is 0-4. The trisaccharide is optionally sulfated
with n SO.sub.3R groups in any suitable location. In certain
instances, compounds of Formulas XXIV are provided by treating the
glycosaminoglycan chondroitin and/or dermatan sulfate with a
suitable glycosaminoglycan lyase.
[0197] In certain embodiments, methods and processes described
herein are utilized to detect and/or measure an oligosaccharide
(trisaccharide) having the formula:
[IdoA-GalNAc-GlcA](SO.sub.3R).sub.n (XXV)
[0198] In certain embodiments, oligosaccharides described herein,
e.g., those detected and/or measured according to the methods
and/or processes described herein, are trisaccharides of Formula
XXV, n is 0-3. The trisaccharide is optionally sulfated with n
SO.sub.3R groups in any suitable location. In certain instances,
compounds of Formulas XXV are provided by treating the
glycosaminoglycan chondroitin and/or dermatan sulfate with a
suitable glycosaminoglycan lyase.
[0199] In certain embodiments, methods and processes described
herein are utilized to detect and/or measure an oligosaccharide
(trisaccharide) having the formula:
[GlcA-GalNAc-Ido](SO.sub.3R).sub.n (XXVI)
[0200] In certain embodiments, oligosaccharides described herein,
e.g., those detected and/or measured according to the methods
and/or processes described herein, are trisaccharides of Formula
XXVI, wherein n is 0-3. The trisaccharide is optionally sulfated
with n SO.sub.3R groups in any suitable location. In certain
instances, compounds of Formulas XXVI are provided by treating the
glycosaminoglycan chondroitin and/or dermatan sulfate with a
suitable glycosaminoglycan lyase.
[0201] In certain embodiments, methods and processes described
herein are utilized to detect and/or measure an oligosaccharide
(trisaccharide) having the formula:
[GlcA-GalNAc-GlcA](SO.sub.3R).sub.n (XXVII)
[0202] In certain embodiments, oligosaccharides described herein,
e.g., those detected and/or measured according to the methods
and/or processes described herein, are trisaccharides of Formula
XXVII, wherein n is 0-2. The trisaccharide is optionally sulfated
with n SO.sub.3R groups in any suitable location. In certain
instances, compounds of Formulas XXVII are provided by treating the
glycosaminoglycan chondroitin and/or dermatan sulfate with a
suitable glycosaminoglycan lyase.
[0203] In some embodiments, the detection and/or measurement of any
one or more trisaccharide of any of Formulas XXIV to XXVII is
utilized in a process for diagnosing a disorder associated with the
accumulation of chondroitin and/or dermatan sulfate (e.g., a
chondroitin and/or dermatan sulfate lysosomal storage disease). In
certain embodiments, an oligosaccharide of any of Formulas XXIV to
XXVII is saturated or unsaturated at the C4 and C5 positions of the
saccharide residue at the non-reducing end of the oligosaccharide.
In some embodiments, a process for diagnosing a disorder associated
with the accumulation of chondroitin and/or dermatan sulfate
further comprises detecting one or more oligosaccharide, as set
forth above and as appropriate (e.g., any of Formulas I-XX, as they
pertain to the particular disorder being diagnosed).
[0204] In certain embodiments, methods and processes described
herein are utilized to detect and/or measure an oligosaccharide
(trisaccharide) having the formula:
GlcA-GlcNAc-GlcA (XXVIII)
[0205] In certain embodiments, oligosaccharides described herein,
e.g., those detected and/or measured according to the methods
and/or processes described herein, are trisaccharides of Formula
XXVIII. In certain instances, compounds of Formulas XXVIII are
provided by treating the glycosaminoglycan hyaluronan with a
suitable glycosaminoglycan lyase.
[0206] In some embodiments, the detection and/or measurement of a
trisaccharide of Formula XXVIII is utilized in a process for
diagnosing a disorder associated with the accumulation of
hyaluronan (e.g., a hyaluronan lysosomal storage disease). In
certain embodiments, an oligosaccharide of Formula XXVIII is
saturated or unsaturated at the C4 and C5 positions of the
saccharide residue at the non-reducing end of the oligosaccharide.
In some embodiments, a process for diagnosing a disorder associated
with the accumulation of hyaluronan further comprises detecting one
or more oligosaccharide, as set forth above and as appropriate
(e.g., any of Formulas I-XX, as they pertain to the particular
disorder being diagnosed).
[0207] In certain embodiments, methods and processes described
herein are utilized to detect and/or measure an oligosaccharide
(trisaccharide) having the formula:
[Gal-GlcNAc-Gal](SO.sub.3R).sub.n (XXIX)
[0208] In certain embodiments, oligosaccharides described herein,
e.g., those detected and/or measured according to the methods
and/or processes described herein, are trisaccharides of Formula
XXIX, wherein n is 0-3. The trisaccharide is optionally sulfated
with n SO.sub.3R groups in any suitable location. In certain
instances, compounds of Formulas XXIX are provided by treating the
glycosaminoglycan keratan sulfate with a suitable glycosaminoglycan
lyase.
[0209] In some embodiments, the detection and/or measurement of any
one or more trisaccharide of Formula XXIX is utilized in a process
for diagnosing a disorder associated with the accumulation of
keratan sulfate (e.g., a keratan sulfate lysosomal storage
disease). In certain embodiments, an oligosaccharide of Formula
XXIX is saturated or unsaturated at the C4 and C5 positions of the
saccharide residue at the non-reducing end of the oligosaccharide.
In specific embodiments, the oligosaccharide is saturated at the C4
and C5 positions of the saccharide residue at the non-reducing end
of the oligosaccharide. In some embodiments, a process for
diagnosing a disorder associated with the accumulation of keratan
sulfate further comprises detecting one or more oligosaccharide, as
set forth above and as appropriate (e.g., any of Formulas I-XX, as
they pertain to the particular disorder being diagnosed).
[0210] In certain embodiments, methods and processes described
herein are utilized to detect and/or measure an oligosaccharide
(trisaccharide) having the formula:
[GalNAc6S-GlcA-(GalNAc).sub.n](SO.sub.3R).sub.n (XXX)
[0211] In certain embodiments, oligosaccharides described herein,
e.g., those detected and/or measured according to the methods
and/or processes described herein, are di- and tri-saccharides of
Formula XXX, wherein n is 0-3. The di- or tri-saccharide is
optionally sulfated with n SO.sub.3R groups in any suitable
location. In certain instances, compounds of Formulas XXX are
provided by treating the glycosaminoglycan chondroitin sulfate with
a suitable glycosaminoglycan lyase. In some embodiments, the
detection and/or measurement of any one or more disaccharide or
trisaccharide of Formula XXX is utilized in a process for
diagnosing MPS IVA. In some embodiments, a di- or tri-saccharide of
Formula XXX has a structure o as set forth in any of the
disaccharides of FIG. 21.
[0212] In certain embodiments, methods and processes described
herein are utilized to detect and/or measure an oligosaccharide
(trisaccharide) having the formula:
[GalNAc6S-IdoA-(GalNAc).sub.n](SO.sub.3R).sub.n (XXXI)
[0213] In certain embodiments, oligosaccharides described herein,
e.g., those detected and/or measured according to the methods
and/or processes described herein, are di- or tri-saccharides of
Formula XXXI, wherein n is 0-4 and m is 0-1. The di- or
tri-saccharide is optionally sulfated with n SO.sub.3R groups in
any suitable location. In more specific embodiments, a disaccharide
of Formula XVIII has a structure of any of the di- or
tri-saccharides of FIG. 22. In certain instances, compounds of
Formulas XXXI-A to XXXI-T are provided by treating the
glycosaminoglycan dermatan sulfate with a suitable
glycosaminoglycan lyase.
[0214] In some embodiments, the detection and/or measurement of any
one or more di- or tri-saccharide of Formula XXXI is utilized in a
process for diagnosing MPS IVA.
[0215] In certain embodiments, methods and processes described
herein are utilized to detect and/or measure an oligosaccharide
(disaccharide) having the formula:
[GlcA-GalNAc](SO.sub.3R).sub.n (XXXII)
[0216] In certain embodiments, oligosaccharides described herein,
e.g., those detected and/or measured according to the methods
and/or processes described herein, are disaccharides of Formula
XXXII, wherein n is 0-2. The disaccharide is optionally sulfated
with n SO.sub.3R groups in any suitable location. In more specific
embodiments, a disaccharide of Formula XXXII has a structure as set
forth in any of the disaccharides of FIG. 23. In certain instances,
compounds of Formulas XXXII-A to XXXII-C are provided by treating
the glycosaminoglycan chondroitin sulfate with a suitable
glycosaminoglycan lyase.
[0217] In some embodiments, the detection and/or measurement of any
one or more disaccharide of Formula XXXII is utilized in a process
for diagnosing MPS VII.
[0218] In certain embodiments, an oligosaccharide of any of
Formulas XXX to XXXII is saturated or unsaturated at the C4 and C5
positions of the saccharide residue at the non-reducing end of the
oligosaccharide.
[0219] In certain embodiments, any of the processes or methods
provided herein comprise detecting and/or measuring the amount of
any oligosaccharide provided in any of Formulas I to XX, any of
Formulas XXI to XXIX, or any of Formulas I to XXXII. In further
embodiments, any process or method provided herein comprises
detecting and/or measuring the amount of any oligosaccharide
provided in any of Formulas I to XX, any of Formulas XXI to XXIX,
or any of Formulas I to XXXII, wherein the oligosaccharide is
attached to a detectable label at the reducing end of the
oligosaccharide.
[0220] In certain embodiments, provided herein is a compound of any
of Formulas I to XX, any one of Formulas XXI to XXIX, or any of
Formulas I to XXXII. In some embodiments, provided herein is a
compound of any of FIGS. 4-23. In certain embodiments, the compound
of any of Formulas I to XXXII or FIGS. 4-23 is an isolated and/or
purified compound. In some embodiments, the isolated and/or
purified compound of any of Formulas I to XXXII or FIGS. 4-23 is
substantially free of oligosaccharides comprising a saccharide
residue at the non-reducing end of the oligosaccharide that is
unsaturated at the C4 and C5 positions. As an illustrative example
of non-reducing end saccharide residues that are saturated and
unsaturated at the C4 and C5 positions, an L-iduronic acid (IdoA)
residue that is saturated at the C4 and C5 positions has a
structure as follows:
##STR00008##
whereas an L-iduronic acid (IdoA) residue at the non-reducing end
of the oligosaccharide that is unsaturated at the C4 and C5
positions may have a structure as follows:
##STR00009##
or the like. Oligosaccharides having non-reducing end saccharide
residues that are saturated at the C4 and C5 position are referred
to herein as "C4-C5 non-reducing end saturated
oligosaccharides".
[0221] In certain embodiments, unsaturated saccharide residues
include the unsaturated residue of IdoA, which is denoted by and
the unsaturated residue of GlcA which is denoted by
[0222] In some embodiments, the isolated and/or purified compound
described herein comprises at least at least 50%, at least 60%, at
least 70%, at least 80%, at least 90%, at least 95%, at least 97%,
at least 98%, or at least 99% oligosaccharide (by weight). In
further or alternative embodiments, the isolated and/or purified
compound comprises 20%, at least 30%, at least 40%, at least 50%,
at least 60%, at least 70%, at least 80%, at least 90%, at least
95%, at least 97%, at least 98%, or at least 99% NRE-saturated
oligosaccharide (by weight).
[0223] In some embodiments, provided herein is a composition
comprising one or more isolated oligosaccharide of any of FIGS.
4-23. In certain embodiments, the oligosaccharide present in the
composition is less than 90% by weight non-reducing end unsaturated
oligosaccharide. In certain embodiments, the oligosaccharide
present in the composition is less than 80% by weight non-reducing
end unsaturated oligosaccharide. In certain embodiments, the
oligosaccharide present in the composition is less than 70% by
weight non-reducing end unsaturated oligosaccharide. In certain
embodiments, the oligosaccharide present in the composition is less
than 60% by weight non-reducing end unsaturated oligosaccharide. In
certain embodiments, the oligosaccharide present in the composition
is less than 50% by weight non-reducing end unsaturated
oligosaccharide. In certain embodiments, the oligosaccharide
present in the composition is less than 40% by weight non-reducing
end unsaturated oligosaccharide. In certain embodiments, the
oligosaccharide present in the composition is less than 30% by
weight non-reducing end unsaturated oligosaccharide. In certain
embodiments, the oligosaccharide present in the composition is less
than 25% by weight non-reducing end unsaturated oligosaccharide. In
certain embodiments, the oligosaccharide present in the composition
is less than 20% by weight non-reducing end unsaturated
oligosaccharide. In certain embodiments, the oligosaccharide
present in the composition is less than 15% by weight non-reducing
end unsaturated oligosaccharide. In certain embodiments, the
oligosaccharide present in the composition is less than 10% by
weight non-reducing end unsaturated oligosaccharide. In certain
embodiments, the oligosaccharide present in the composition is less
than 5% by weight non-reducing end unsaturated oligosaccharide. In
some embodiments, the composition further comprises chromatographic
resin or electrophoretic resin. In some embodiments, the resin is a
high performance liquid chromatographic resin, or the like.
[0224] The compounds of any of Formulas I-XXXII or FIGS. 4-23 are
obtained in any suitable manner, e.g., by generation and
purification from natural sources, chemical synthesis, or any other
suitable method. These structures do not naturally exist in an
isolated and/or purified form.
[0225] The described di- and trisaccharides are optionally
generated and purified from natural sources. In certain
embodiments, starting with glycosaminoglycans from natural sources
(for example, chondroitin sulfate, heparan sulfate, heparin, or
synthetic heparin (or heparan) like carbohydrates), these di- and
trisaccharides are generated by treating the glycan with
glycosaminoglycan lyases (for example heparin lyases or
chondroitinases) and purifying the di- and trisaccharides liberated
from the pre-existing non-reducing ends. In specific instances, the
di- and trisaccharides liberated from the pre-existing non-reducing
end are unique because they do not contain a c4-5 double bond due
to the action of the lyase.
[0226] In certain embodiments, the yield of the desired di- and
trisaccharides are increased by using glycosaminoglycans that have
been treated with glycan degradative enzymes (for example,
heparanase or lysosomal exo-enzymes such as the 2-sulfatase,
N-sulfatase, etc) to generate smaller fragments or fragments with a
greater number of desirable non-reducing ends. In further or
alternative embodiments, the yield is increased by starting with
glycosaminoglycans that have been isolated from an organism or cell
that has a defect in glycosaminoglycan degradation such that the
desirable non-reducing ends are enriched (for example,
glycosaminoglycans from iduronidase deficient systems, would be
enriched in glycosaminoglycans that terminal on the non-reducing
end with iduronate residues). As shown in FIGS. 24 and 25, these
unique non-reducing end disaccharides can be isolated by HPLC. FIG.
24 shows disaccharides isolated from heparan sulfate from a normal
human sample, while FIG. 25 shows disaccharides isolated from an
MPS I (iduronidase deficient) sample. The saturated disaccharides
(indicated with red arrows) can be isolated by collecting the
appropriate fractions from this or a similar preparative
chromatography.
[0227] Additional or alternative methods of obtaining such
compounds include, e.g., chemical transformation of the unsaturated
saccharides into the saturated saccharides. The described di- and
trisaccharides can be synthesized by chemical or chemoenzymatic
methods. Chemical methods for the synthesis of these saturated di-
and trisaccharides are optionally converted from the methods
described by Prabhu, Venot, and Boons (Organic Letters 2003 Vol. 5,
No. 26 4975-4978), which is incorporated herein for such
synthesis.
[0228] In further embodiments, provided herein is a compound of any
of Formulas I-XXXII or FIGS. 4-23, wherein the compound is tagged
with any label as described herein.
[0229] In some embodiments, an isolated or otherwise generated
oligosaccharide described herein has a molecular weight of less
than 2000 g/mol, less than 1500 g/mol, less than 1000 g/mol, less
than 500 g/mol, less than 400 g/mol, less than 300 g/mol, less than
260 g/mol, less than 200 g/mol, less than 100 g/mol, or the like
(e.g., prior to tagging with any detectable label that may be
included in a process described herein).
Detection/Measurement Methods
[0230] Oligosaccharides (including e.g., oligosaccharides tagged
with detectable labels) described herein are detected and/or
measured in processes described herein in any suitable manner. In
certain embodiments, detectable labels are detected and/or
quantified according to any process described herein using any
technique, particularly any technique suitable for the detectable
label utilized. In some embodiments, suitable detection techniques
include, by way of non-limiting example, one or more of a mass
spectrometer, a nuclear magnetic resonance spectrometer, a UV-Vis
spectrometer, an IR spectrometer, a fluorimeter, a phosphorimeter,
a radiation spectrometer (e.g., a scintillation counter), a thin
layer chromatographic technique, or the like. In certain
embodiments, in any process described herein, oligosaccharides are
optionally directly detected using a suitable technique, such as
quantitative nuclear magnetic resonance. Quantitative nuclear
magnetic resonance is also optionally utilized to quantify and/or
detect the presence of a detectable label. In certain embodiments,
one or more oligosaccharides are optionally detected using a
suitable liquid chromatography mass spectrometer (LC-MS).
[0231] In some embodiments, oligosaccharides are tagged with an
antibody or probe, and are quantified using any suitable method
(e.g., dot blot techniques, immune detection techniques (e.g.,
ELISA), or the like).
[0232] Various analytical methods useful for the processes
described herein include, by way of non-limiting example, mass
spectrometry, chromatography, HPLC, UPLC, TLC, GC, HPAEC-PAD,
electrophoresis--capillary or gel, or the like. In certain
embodiments, wherein a chromatographic technique is utilized, any
suitable solvent system is optionally employed. In certain
embodiments, a column (e.g., Cosmogel DEAE, Tsk Gel DEAE, Cosmogel
QA, Cosmogel CM, Cosmogel SP, 130A BEH particle Phenyl (1.7, 2.5,
3.5, 5, or 10 uM particle size), 130A BEH particle C18 (1.7, 2.5,
3.5, 5, or 10 uM particle size), HSS particle C18 (1.8, 3.5, or 5
uM particle size), 300A BEH particle C18 (1.7, 3.5, 5, 10 uM
particle size), or the like with suitable length and internal
diameter) is optionally loaded with an equilibrating solvent (e.g.,
a buffer or salt solution, such as a potassium acetate solution,
sodium chloride solution, sodium acetate solution, ammonium acetate
solution, or the like), e.g., with a pH of about 6, 7, or 8. In
some embodiments, the buffer or salt solution has a concentration
of about 10 mM, 20 mM, 30 mM, 50 mM, 100 mM, 500 mM, 1 M, 2 M, or
the like. Any suitable flow rate is used, e.g., 0.5 mL/min, 1 mL,
min, 1.5 mL/min, 2 mL/min, or the like. Following equilibration, a
linear gradient is optionally utilized. In some embodiments, the
linear gradient is run over 1-20 min, 1-10 min, 10-20 min, 1-5 min,
5-10 min, or the like. In certain embodiments, the gradient is a
buffer or salt solution, e.g., as described above (e.g., from 0 M
to 0.5 M, from 0 M to 3 M, from 0.5 M to 2 M, from 0 M to 2 M, from
1 M to 2 M, from 0 M to 3 M, from 2 M to 0 M, from 3 M to 0 M, or
the like). Once the gradient has reached a final concentration, the
eluent is optionally held at the final concentration for a suitable
period of time (e.g., 1-20 min, 5-10 min, 10-15 min, 1-5 min, 1-10
min, 15-20 min, or the like). After the optional holding of the
final concentration, the eluent may be switched to a second solvent
or solvent system (e.g., an alcohol, such as methanol, ethanol, or
isopropanol, acetonitrile, water, or the like). The switch to the
second solvent system may be over a period of time, e.g., 15
seconds, 30 seconds, 45 seconds, 60 seconds, 2 min, 3 min, or the
like. The second solvent system is optionally held for a period of
time, such as 1 min, 2 min, 3 min, 4 min, 5 min, 6 min, or the
like. Following the second solvent system cycle, the column is
optionally restored to initial solvent conditions.
[0233] In certain embodiments, detection or measurement of an
analytical device provides for diagnosis of a disease, diagnosis of
severity of a disease, of efficacy of a therapy, or analysis based
on other processes described herein. For example, in some
embodiments, absence of a peak or signal (e.g., a peak or signal
indicative of the presence of a particular oligosaccharide)
indicates that an individual is in a non-diseased state, in
remission for a disease state, or undergoing effective therapy for
a disease, depending on the circumstances of the diagnosis. In
certain embodiments, the presence and/or area of a peak or signal
(including, e.g., the presence of a certain signal or peak pattern
or fingerprint) is utilized to determine the severity of a disease.
In some embodiments, the presence and/or area of a peak or signal
is utilized to determine disease, disease severity, disease carrier
status or the like, based on a certain threshold value for the
disease, disease severity, disease carrier status. Such thresholds
are optionally determined in any suitable manner, e.g., by
analyzing control samples, such control samples coming from
non-diseased individuals, diseased individuals, or the like.
[0234] In certain embodiments, a control biological sample utilized
in any process described herein was provided from an individual
that does not suffer from a disorder being diagnosed. In other
embodiments, a control biological sample is taken from an
individual suffering from a disorder being diagnosed. In certain
embodiments, the result obtained from the control biological sample
is stored in a database. In such cases a test sample is optionally
compared to a plurality of control data in a database. Moreover in
certain embodiments, any diagnostic process described herein is
optionally utilized alone or in combination with other diagnostic
techniques. Other diagnostic techniques include, by way of
non-limiting example, symptom analysis, biopsies, detection of
accumulation of other compounds in biological samples, or the like.
In some embodiments, control biological samples are optionally
taken from the same individual at substantially the same time,
simply from a different location (e.g., one inflamed/arthritic
synovial joint fluid vs the contralateral non-arthritic synovial
joint). In other embodiments, control biological samples are
optionally taken from the same individual at different points in
time (e.g., before therapy and after therapy if the method being
utilized is a method of monitoring a treatment therapy).
Detectable Labels
[0235] In the various embodiments of any process or method
described herein, any suitable detectable label is optionally
utilized. In some embodiments, detectable labels useful in the
processes or methods described herein include, by way of
non-limiting example, mass labels, antibodies, affinity labels,
radiolabels, chromophores, fluorescent labels, or the like.
[0236] Fluorescent labels suitable for use in various embodiments
herein include, by way of non-limiting example, 2-aminopyridine
(2-AP), 2-aminobenzoic acid (2-AA), 2-aminobenzamide (2-AB),
2-aminoacridone (AMAC), p-aminobenzoic acid ethyl ester (ABEE),
p-aminobenzonitrile (ABN), 2-amino-6-cyanoethylpyridine (ACP),
7-amino-4-methylcoumarine (AMC),
8-aminonaphthalene-1,3,6-trisulfate (ANTS),
7-aminonaphthalene-1,3-disulfide (ANDS), and
8-aminopyrene-1,3,6-trisulfate (APTS), or the like. The fluorescent
labels can be attached by reductive amination with the fluorescent
label and sodium cyanoborohydride or the like.
[0237] Mass labels suitable for use in various embodiments herein
include, by way of non-limiting example, D-2-anthranilic acid,
D-2-aminopyridine, D-methyl iodide, .sup.13C methyl iodide,
deuterated-pyridyl-amine, D-biotin or the like. The mass labels can
be attached by permethylation or reductive amination by any method
that is known to those of skill in the art.
[0238] Affinity labels suitable for use in various embodiments
herein include, by way of non-limiting example, biotin and
derivatives.
[0239] Radio labels suitable for use in various embodiments herein
include, by way of non-limiting example, sodium borotritide
(NaB.sup.3H.sub.4), or the like.
[0240] Chromophores suitable for use in various embodiments herein
include, by way of non-limiting example, 4-amino-1,1'-azobenzene,
4'-N,N-dimethylamino-4-aminoazobenzene, aminoazobenzene,
diaminoazobenzene, Direct Red 16, CI Acid Red 57, CI Acid Blue 45,
CI Acid Blue 22, CL Mordant Brown 13, CI Direct Orange 75, or the
like. The chromophores may be labeled by any method that is known
to those of skill in the art, such as reductive amination with the
chromophore and sodium cyanoborohydride.
[0241] In some embodiments, the detectable label is an antibody. In
specific embodiments, the antibody is attached to a detectable
compound, such as mass labels, radiolabels, chromophores,
fluorescent labels, or the like. In some embodiments, antibodies
are themselves detected and/or are detectable in various manners,
e.g., as a chromophore, a fluorophore, or the like; or with a probe
(e.g., using dot blot techniques, immune-detection techniques, or
the like).
Purification Methods
[0242] In some embodiments, the processes described herein
comprises further treatment steps of the test and/or control
samples. For example, in some embodiments, the samples are
homogenized and/or purified. In specific embodiments homogenization
is achieved in any suitable manner including, by way of
non-limiting example, with a basic solution (e.g., 0.1 N NaOH),
sonication, tissue grinding or other chemical agents).
[0243] In certain embodiments, samples, including test samples
and/or control samples, described herein are optionally purified
prior to glycosaminoglycan processing (e.g., lyase treatment)
and/or characterization. Test samples and/or control samples (i.e.,
one or more or all of the glycans found therein) are optionally
purified using any suitable purification technique. Test samples
and/or control samples are optionally purified at any suitable
point in a process described herein, including before or after
tagging of the glycans founds within the sample. In certain
embodiments, purification techniques include centrifugation,
electrophoresis, chromatography (e.g., silica gel or alumina column
chromatography), gas chromatography, high performance liquid
chromatography (HPLC) (e.g., reverse phase HPLC on chiral or
achiral columns), thin layer chromatography, ion exchange
chromatography, gel chromatography (e.g., gel filtration or
permeation or size exclusion chromatography, gel electrophoresis),
molecular sieve chromatography, affinity chromatography, size
exclusion, filtration (e.g. through a florisil or activated
charcoal plug), precipitation, osmosis, recrystallization, fluorous
phase purification, distillation, extraction, chromatofocusing,
supercritical fluid extraction, preparative flash chromatography
(e.g., flash chromatography using a UV-Vis detector and/or a mass
spectrometer (e.g., using the Biotage.RTM. suite of products) or
the like.
[0244] In some embodiments, glycosaminoglycans, such as heparan
sulfate, are naturally found attached to a core protein (together
forming a proteoglycan). In some embodiments, provided herein are
purification processes of separating glycosaminoglycan fragments
(e.g., heparan sulfate fragments) from proteoglycans prior to
processing the glycosaminoglycans for detection.
Therapeutic Methods
[0245] Provided in certain embodiments are methods of treating
disorders associated with the abnormal degradation, biosynthesis
and/or accumulation of glycosaminoglycans (GAGs), the methods
comprising: [0246] a. administering an agent for treating MPS
(e.g., an agent that modulates (e.g., promotes and/or inhibits)
glycosaminoglycan biosynthesis and/or degradation) to an individual
in need thereof; [0247] b. monitoring the accumulation of
glycosaminoglycans in the individual using any process described
herein for detecting or quantifying the amount of oligosaccharides
(e.g., di-and/or tri-saccharides, such as GAG oligosaccharides,
including heparan sulfate fragments) present in a lyase digested
biological sample (e.g., urine, serum, plasma, or CSF sample)
according to any process described herein.
[0248] Provided in further or alternative embodiments are methods
of monitoring the treatment of disorders associated with the
abnormal degradation, biosynthesis and/or accumulation of
glycosaminoglycans (GAGs), the methods comprising: [0249] a.
following administration of an agent for treating MPS (e.g., an
agent that modulates (e.g., promotes and/or inhibits)
glycosaminoglycan biosynthesis and/or degradation) to an individual
in need thereof, using an analytical instrument to detect the
presence of and/or measure the amount of a population of one or
more C4-C5 non-reducing end saturated oligosaccharides present in a
transformed biological sample that has been prepared by: treating a
population of glycosaminoglycans, in or isolated from a biological
sample taken from the individual, with at least one digesting
glycosaminoglycan lyase to transform the glycosaminoglycans into
the population of the one or more C4-C5 non-reducing end saturated
oligosaccharide; [0250] b. displaying or recording the presence of
or a measure of a population of one or more oligosaccharide.
[0251] In specific embodiments, the oligosaccharide(s) detected or
measured is one or more C4-C5 non-reducing end saturated
oligosaccharide(s). In some embodiments, the agent is administered
one or more times. In certain embodiments, the agent is
administered multiple times. In some embodiments, the agent is
administered in a loading dose one or more times (e.g., in a
loading dosing schedule) and subsequently administered in a
maintenance dose (e.g., in a maintenance dosing schedule, such as
three times a day, twice a day, once a day, once every two days,
once every three days, once every four days, once a week, or the
like). In some embodiments, when GAG oligosaccharide accumulation
begins to increase or accelerate, the dose is optionally adjusted
(e.g., the maintenance dose is increased, or an additional loading
dose or dosing schedule is utilized).
[0252] In some embodiments, monitoring the accumulation of
glycosaminoglycans comprises repeating the step of: using an
analytical instrument to detect the presence of and/or measure the
amount of a population of one or more oligosaccharides present in a
transformed biological sample that has been prepared by treating a
population of glycosaminoglycans, in or isolated from a biological
sample from the individual, with at least one digesting
glycosaminoglycan lyase to transform the glycosaminoglycans into
the population of the one or more oligosaccharide. In specific
embodiments, the step is repeated at periodic intervals (e.g.,
every day, every other day, every 2 days, every 3 days, every 4
days, every week, every month, every 3 months, quarterly, every 6
months, yearly, or the like), at regular times following a dose
(e.g., 4 hours after a administration of the agent, 6 hours after
administration of the agent, 8 hours after administration of the
agent, 12 hours after administration of the agent, or the like),
prior to administration of the dose (e.g., immediately prior to
administration of the agent, 2 hours prior to administration of the
agent, or the like), or any other monitoring schedule.
[0253] In some embodiments, the monitoring of the accumulation of
glycosaminoglycans is conducted over a period of time, e.g., over a
week, two weeks, a month, two months, three months, six months, a
year, or the like. In some embodiments, the method for quantifying
the amount of one or more oligosaccharides in a lyase digested
biological sample (e.g., urine, serum, plasma, or CSF) comprises
detecting and/or measuring (e.g., with an analytical device), one
or more oligosaccharides within the lyase digested biological
sample from the individual after the biological sample obtained
from the individual has been treated with one or more
glycosaminoglycan lyases. In certain embodiments, such
glycosaminoglycan lyases are suitable for preparing di- and/or
tri-saccharides from the glycosaminoglycans present in the
biological sample obtained from the individual. In certain
instances, the reducing end of a representative portion of the one
or more oligosaccharides in the transformed biological sample is
tagged with any suitable detectable label (e.g., a mass label, a
radio label, a fluorescent label, a chromophore label, affinity
label, an antibody). In some embodiments, the process comprises
displaying or recording such a characterization of the population
of oligosaccharides and/or tagged oligosaccharides.
[0254] In some embodiments, the agent that modulates
glycosaminoglycan biosynthesis includes glycosaminoglycan
accumulation inhibitors, agents that promote glycosaminoglycan
degradation, agents that activate enzymes that degrade
glycosaminoglycans, agents that inhibit biosynthesis of
glycosaminoglycans, or the like. In some embodiments, the agent
that modulates glycosaminoglycan biosynthesis is an agent that
selectively modulates heparan sulfate biosynthesis, an agent that
selectively modulates chondroitin sulfate biosynthesis, an agent
that selectively modulates dermatan sulfate biosynthesis, an agent
that selectively modulates keratan sulfate biosynthesis, an agent
that selectively modulates hyaluronan biosynthesis, or a
combination thereof
[0255] In some instances, the detection and/or the quantification
of the identity and/or amount of oligosaccharides present in a
biological sample is used to identify and/or diagnose a disorder
associated with abnormal degradation, biosynthesis and/or
accumulation of glycosaminoglycans in an individual suspected of
having such a disorder.
[0256] In some instances, the detection and/or the quantification
of the identity and/or amount of oligosaccharides present in the
biological sample is used to monitor severity and course of the
disease in an individual diagnosed with or suspected of having a
disorder associated with the abnormal degradation, biosynthesis
and/or accumulation of glycosaminoglycans. In some instances, the
detection and/or the quantification of the identity and/or amount
of oligosaccharides present in the biological sample is used to
calculate the administered dose of an agent that modulates (e.g.,
promotes and/or inhibits) glycosaminoglycan biosynthesis and/or
degradation.
[0257] In certain instances, wherein following administration of a
selected dose of a an agent that modulates (e.g., promotes and/or
inhibits) glycosaminoglycan biosynthesis and/or degradation, an
individual's condition does not improve, the detection and/or the
quantification of the identity and/or amount of oligosaccharides
present in a biological sample provides for a treatment regimen to
be modified depending on the severity and course of the disease,
disorder or condition, previous therapy, the individual's health
status and response to the drugs, and the judgment of the treating
physician.
[0258] In certain embodiments, monitoring the accumulation of
glycosaminoglycans in the individual comprises detecting or
quantifying the amount of an oligosaccharide (or one or more
oligosaccharides) in a sample obtained from the individual (e.g.,
according to any method described herein) to obtain a first
accumulation result (e.g., an initial reading before treatment has
begun, or at any other time) and a second accumulation result that
is subsequent to obtaining the first result. In some embodiments,
the second result is compared to the first result to determine if
the treatment is effectively reducing, maintaining, or reducing the
rate of increasing the oligosaccharide levels in a substantially
identically obtained sample from the individual being treated. In
certain embodiments, depending on the difference between the first
and second results, the treatment can be altered, e.g., to increase
or decrease the amount of agent administered; to substitute the
therapeutic agent with an alternative therapeutic agent; or the
like. In certain embodiments, the dose of the therapeutic agent is
decreased to a maintenance level (e.g., if the oligosaccharide
level has been reduced sufficiently); further monitoring of
oligosaccharide levels is optional in such situation, e.g., to
ensure that reduced or maintained levels of oligosaccharide (e.g.,
GAG oligosaccharide(s)) are achieved.
[0259] Alternatively, provided herein is a method of detecting
response to therapy in an individual or a method of predicting
response to therapy in an individual comprising: [0260] a.
administering an agent for treating MPS (e.g., an agent that
modulates (e.g., promotes and/or inhibits) glycosaminoglycan
biosynthesis and/or degradation) to a plurality of cells from an
individual in need thereof (e.g., a plurality of fibroblasts,
serum, plasma, or CSF cells from a human suffering from MPS);
[0261] b. monitoring the accumulation of glycosaminoglycans in the
plurality of cells using any process described herein for detecting
or quantifying the amount of oligosaccharides (e.g., di- and/or
tri-saccharides, such as GAG oligosaccharides, including heparan
sulfate fragments) present in a lyase digested biological sample
from the plurality of cells according to any process described
herein.
[0262] In specific embodiments, the oligosaccharide(s) detected or
measured is one or more C4-C5 non-reducing end saturated
oligosaccharide(s). It is to be understood that a plurality of
cells from an individual includes cells that are directly taken
from the individual, and/or cells that are taken from an individual
followed by culturing to expand the population thereof.
[0263] As discussed above, FIGS. 24 and 25 illustrate that C4-C5
non-reducing end saturated oligosaccharides can be isolated by,
e.g., HPLC.
[0264] In some embodiments, monitoring the accumulation of
glycosaminoglycans comprises repeating the step of: using an
analytical instrument to detect the presence of and/or measure the
amount of a population of one or more oligosaccharides present in a
transformed biological sample that has been prepared by treating a
population of glycosaminoglycans, in or isolated from a biological
sample from the individual, with at least one digesting
glycosaminoglycan lyase to transform the glycosaminoglycans into
the population of the one or more oligosaccharide. In specific
embodiments, the step is repeated at periodic intervals (e.g.,
every day, every other day, every 2 days, every 3 days, every 4
days, every week, or the like), or any other monitoring
schedule.
Disorders
[0265] Disorders associated with the abnormal degradation,
biosynthesis and/or accumulation of glycosaminoglycans useful in
the treatment and diagnostic methods and processes described herein
include any disorder wherein accumulation of glycosaminoglycans
and/or fragments thereof can be detected in a biological sample
taken from an individual suffering from such a disorder. As
discussed herein, disorders associated with abnormal
glycosaminoglycan degradation, biosynthesis, and/or accumulation
include e.g., lysosomal storage diseases. In specific embodiments,
a lysosomal storage disease is mucopolysaccharidosis (MPS). In some
embodiments, a mucopolysaccharidosis (MPS) is MPS I, MPS II, MPS
IIIA, MPS IIIB, MPS IIIC, MPS IIID, MPS IVA, MPS IVB, MPS VI, MPS
VII, MPS IX, or a combination thereof.
[0266] In some instances, a lysosomal storage disease is caused by
abnormal glycosaminoglycan degradation. In some instances, a
lysosomal storage disease causes an accumulation of heparan sulfate
and/or dermatan sulfate (e.g., MPS I) and is caused by an
iduronidase deficiency. In some instances, a lysosomal storage
disease causes an accumulation of heparan sulfate and/or dermatan
sulfate (e.g., MPS II) and is caused by a 2-sulfatase deficiency.
FIG. 26 illustrates accumulation of oligosaccharides in MPS II
human fibroblast versus the accumulation of oligosaccharides in
normal human fibroblasts. In some instances, a lysosomal storage
disease causes an accumulation of heparan sulfate and/or hyaluronan
(e.g., MPS IIIB) and is caused by an N-acetylglulcosaminidase
deficiency. In some instances, a lysosomal storage disease causes
an accumulation of heparan sulfate (e.g., MPS IIIC) and is caused
by a glucosamine acetyltransferase deficiency. In some instances, a
lysosomal storage disease causes an accumulation of heparan sulfate
and/or keratan sulfate (e.g., MPS IIID) and is caused by a
N-acetylglucosamine 6-sulfatase deficiency. In some instances, a
lysosomal storage disease causes an accumulation of keratan sulfate
(e.g., MPS IVA) and is caused by a galactose 6-sulfatase
deficiency. In some instances, a lysosomal storage disease causes
an accumulation of keratan sulfate (e.g., MPS IVB) and is caused by
an N-acetylglactosamine 4-sulfatase deficiency. In some instances,
a lysosomal storage disease causes an accumulation of heparan
sulfate and/or hyaluronan (e.g., MPS VII) and is caused by a
glucuronidase deficiency. In some instances, a lysosomal storage
disease causes an accumulation of hyaluronan (e.g., MPS IX) and is
caused by a hyaluronidase deficiency.
[0267] In some embodiments, a MPS is Hunter's disease. In certain
embodiments, Hunter's disease causes an accumulation of dermatan
sulfate and heparan sulfate glycosaminoglycans. In certain
instances, the accumulation of dermatan sulfate and heparan sulfate
glycosaminoglycans in Hunter's disease is associated with a
deficiency in a sulfatase. In some embodiments, the MPS is Hurler's
disease. In certain instances, Hurler's disease causes an
accumulation of dermatan sulfate and heparan sulfate
glycosaminoglycans. In some instances, the accumulation of dermatan
sulfate and heparan sulfate glycosaminoglycans in Hurler's disease
is associated with a deficiency in an iduronidase.
[0268] In some embodiments, a disorder associated with abnormal
glycosaminoglycan degradation, biosynthesis and/or accumulation is
undesired angiogenesis (e.g., angiogenesis associated with cancer,
diabetic blindness, age-related macular degeneration, rheumatoid
arthritis, or psoriasis), insufficient angiogenesis (e.g., coronary
artery disease, stroke, or delayed wound healing), amyloidosis, a
spinal cord injury, hypertriglyceridemia, inflammation, or a
wound.
[0269] In some instances, amyloidosis is present in various
diseases including, e.g., Alzheimer's disease, Parkinson's disease,
type-2 diabetes, Huntington's disease, spongiform encephalopathies
(Creutzfeld-Jakob, Kuru, Mad Cow), diabetic amyloidosis, rheumatoid
arthritis, juvenile chronic arthritis, Ankylosing spondylitis,
psoriasis, psoriatic arthritis, adult still disease, Becet
syndrome, famalial Mediterranean fever, Crohn's disease, leprosy,
osteomyelitis, tuberculosis, chronic bronciectasis, Castleman
disease, Hodgkin's disease, renal cell carcinoma, carcinoma of the
gut, lung or urogenital tract. In some instances, the Alzheimer's
disease is associated with changes in the content and structure of
one or more GAG (e.g., heparan sulfate, keratan sulfate, or the
like).
[0270] In some embodiments, disorders associated with abnormal
glycosaminoglycan accumulation include disorders associated with
abnormal biosynthesis (e.g., polymerization and/or sulfation) of
glycosaminoglycans. In certain instances, the abnormal biosynthesis
of glycosaminoglycans results in glycosaminoglycans that are not
readily degraded by normal glycosaminoglycan degrading enzymes. In
some instances, disorders associated with abnormal GAG biosynthesis
include osteoarthritis. In certain instances, osteoarthritis is
associated with changes in sulfation of chondroitin sulfate,
changes in length of chondroitin sulfate, changes in expression
levels of chondroitin sulfate, or any combination of thereof. In
some instances, osteoarthritis is associated abnormal chondroitin
sulfate sulfotransferase. In certain instances, the osteoarthritis
is associated with changes in sulfation of dermatan sulfate,
changes in length of dermatan sulfate, changes in expression levels
of dermatan sulfate, or any combination of thereof. In certain
instances, the osteoarthritis is associated with changes in
sulfation of keratan sulfate, changes in length of keratan sulfate,
changes in expression levels of keratan sulfate, or any combination
of thereof.
[0271] In some embodiments, a disorder associated with abnormal
glycosaminoglycan degradation, biosynthesis and/or accumulation is
macular corneal dystrophy. In some instances, macular corneal
dystrophy is associated with low amounts of keratan sulfate. In
more specific embodiments, the keratan sulfate levels are due to
failure to initiate keratan sulfate synthesis, polymerize the
keratan sulfate chain length, or any combination thereof
[0272] In some embodiments, a disorder associated with abnormal
glycosaminoglycan degradation, biosynthesis and/or accumulation is
an infectious or viral disease. In some embodiments, the infectious
or viral disease includes herpes, diphtheria, papilloma virus,
hepatitis, HIV, coronavirus, or adenovirus.
[0273] In some embodiments, a disorder associated with abnormal
glycosaminoglycan degradation, biosynthesis and/or accumulation is
a cancer. In certain embodiments, the cancer is breast cancer,
ovarian cancer, colorectal cancer, cervical cancer, pancreatic
cancer, gastric cancer, esophageal cancer, head and neck cancer,
hepatocellular cancer, prostate cancer, melanoma, osteosarcoma,
endometrial cancer, multiple myeloma, gastric cancer, lung cancer,
glioma, kidney cancer, bladder cancer, thyroid cancer,
neuroblastoma, or non-Hodgkin lymphoma.
[0274] In certain instances, cancer is associated with abnormal
heparan sulfate depolymerization and degradation that results in
unbound, accumulated heparan sulfate. In some instances, abnormal
heparan sulfate depolymerization and degradation is associated with
melanomas, gliomas, multiple myelomas, ovarian, breast, colon,
cervical, pancreatic, gastric, and esophageal cancers. In certain
instances, abnormal heparan sulfate depolymerization and
degradation contributes to angiogenesis, metastasis and growth
factor mobilization. In some instances, the abnormal heparan
sulfate depolymerization and degradation is from increased activity
of a heparanase.
[0275] In certain instances, cancer is associated with abnormal
heparan sulfate sulfation that results in accumulated heparan
sulfate. In some instances, abnormal heparan sulfate sulfation is
associated with colon carcinoma, myeloma, ovarian cancer,
hepatocellular carcinoma, head and neck squamous cell carcinoma and
prostate cancer. In some instances, heparan sulfate sulfation is
decreased in certain cancers, while in other embodiments; the
heparan sulfate sulfation is increased. In some instances, abnormal
heparan sulfate sulfation is caused by abnormal heparan sulfate
sulfotransferase function.
[0276] In certain instances, cancer is associated with abnormal
chondroitin sulfate sulfation. In more specific embodiments, the
abnormal chondroitin sulfate sulfation is associated with lung
cancer. In some instances, the chondroitin sulfate sulfation is
increased in certain cancers. In certain instances, the abnormal
chondroitin sulfate sulfation is caused by abnormal chondroitin
sulfate sulfotransferase function. In some instances, increased
production of chondroitin sulfate is associated in breast cancer,
melanoma, and transformed fibroblasts.
[0277] In certain instances, cancer is associated with dermatan
sulfate epimerase expression. In some instances, the dermatan
sulfate epimerase expression is increased in squamous cell
carcinoma, glioma, gynecological cancer, pancreatic cancer,
colorectal carcinoma, and prostate cancer. In certain instances,
the cancer is associated with accumulation of dermatan sulfate
levels. In some instances, the dermatan sulfate levels are
increased in pancreatic cancer.
[0278] In certain instances, cancer is associated with abnormal
keratan sulfate sulfation. In some instances, the abnormal keratan
sulfate sulfation is associated with glioblastomas. In certain
instances, abnormal keratan sulfate sulfation is caused by abnormal
keratan sulfate sulfotransferase function. In some instances,
keratan sulfate expression is increased in glioblastomas.
[0279] In certain instances, cancer is associated with abnormal
hyaluronan accumulation. In some instances, abnormal hyaluronan
accumulation is associated with breast cancer, prostate cancer,
aggressive subtypes of non-Hodgkin lymphoma, and colorectal cancer.
In certain instances, hyaluronan accumulation contributes to
metastasis of certain cancers. In some instances, the hyaluronan
accumulation results from the over expression of a hyaluronan
synthase.
Drug Screens
[0280] Provided in certain embodiments herein is a process for
identifying an agent that inhibits the accumulation of
glycosaminoglycans in a cell, the process comprising: [0281] a.
contacting a plurality of mammalian cells with a compound, the
plurality of mammalian cells being of a cell line that accumulates
an abnormal amount of glycosaminoglycans; [0282] b. incubating the
mammalian cells with the compound; [0283] c. optionally
transforming the mammalian cells e.g., by isolating a population of
glycosaminoglycans from the cells (e.g., using any suitable method
described herein); [0284] d. contacting the mammalian cells and/or
the isolated population of glycosaminoglycans from step (c) with a
GAG lyase (e.g., heparin lyase); [0285] e. purifying a
sub-population of oligosaccharides from step (d) (e.g., using any
suitable method described herein); [0286] f. detecting the presence
of and/or measuring the amount of one or more oligosaccharides
present in the sub-population (e.g., using LC-MS or GC-MS); and
[0287] g. displaying or recording a characterization of the
sub-population of one or more oligosaccharides.
[0288] In certain instances, the mammalian cells are optionally
transformed by e.g., tagging a population of glycosaminoglycans on
and/or in the cells with a detectable label. In some instances, the
mammalian cells are optionally transformed by e.g., isolating a
population of glycosaminoglycans on and/or in the cells using any
suitable purification technique described herein.
[0289] In certain embodiments, the cell is present in an individual
(e.g., a human or other mammal) and is incubated at body
temperature. In some embodiments, the cell line that accumulates an
abnormal amount of glycosaminoglycans being a mucopolysaccharidosis
(MPS) cell line (e.g., a human MPS cell line). In more specific
embodiments, the MPS cell line is a cell line for MPS I, MPS II,
MPS IIIA, MPS TIM, MPS IIIC, MPS IIID, MPS IVA, MPS IVB, MPS VI,
MPS VII, MPS IX, or a combination thereof. In some embodiments, the
human MPS cell line is present in a human or other mammal. In some
embodiments, inhibitors of the accumulation of glycosaminoglycans
are compounds that reduce the rate of accumulation of
glycosaminoglycans in the cell, and/or agents that reduce the total
amount of glycosaminoglycans accumulated in the cell (i.e.,
diminish the amount of glycosaminoglycan that has been accumulated
in the cell). Agents that are optionally tested for the screening
process described herein include any compound such as, by way of
non-limiting example, a polynucleotide (e.g., siRNA), a
polypeptide, or a small molecule compound (e.g., having a molecular
weight of less than 2,000 g/mol).
EXAMPLES
[0290] Example 1--Purification: The biological sample (cells,
tissue, blood, serum, or the like) is homogenized and solubilized
in 0.1 - 1.0 N NaOH (e.g., 0.1 N, 0.2 N, 0.3 N, 0.4 N, 0.5 N, 0.6
N, 0.7 N, 0.8 N, 0.9 N, or 1.0 N) or acetic acid and then
neutralized with acetic acid or NaOH. Next a small sample is taken
to measure protein content of the sample using standard methods.
0.01-0.5 mg/mL (0.01 mg/mL, 0.07 mg/mL, 0.12 mg/mL, 0.17 mg/mL,
0.22 mg/mL, 0.27 mg/mL, 0.32 mg/mL, 0.37 mg/mL, 0.42 mg/mL, or 0.5
mg/mL) protease (trypsin, chymotrypsin, pepsin, pronase, papain, or
elastase) is treated in 0.1-0.5 M (e.g., 0.1 M, 0.16 M, 0.23 M,
0.32 M, 0.39 M, 0.44 M, or 0.5 M) NaCl, 0.01-0.1 M (e.g., 0.01 M,
0.02 M, 0.04 M, 0.06 M, 0.08 M, 0.1 M) NaOAc, at pH 5.5-7.5 (e.g.,
5.5, 6.0, 6.5, 7.0, or 7.5) and 25-40 C (e.g., 25 C, 30 C, 35 C, or
40 C) for 1-24 hours (e.g., 1 h, 2 h, 4 h, 6 h, 8h, 12 h, 18 h, 24
h). The sample is diluted to reduce the ionic strength and loaded
onto an ion exchange column in 5-100 mM (e.g., 5 mM, 10 mM, 20 mM,
30 mM, 40 mM, 50 mM, 60 mM, 70 mM, 75 mM, 80 mM, 90 mM, 95 mM, 100
mM) NaOAc pH 5-7 with 0-300 mM NaCl. After washing, the bound
glycosaminoglycans are eluted with 5-100 mM NaOAc pH 5-7 (e.g., 5,
5.5, 6, 6.5, 7) with 0.8-3 M (e.g., 0.8 M, 1 M, 1.2 M, 1.4 M, 1.6
M, 1.8 M, 2 M, 2.5 M, or 3 M)NaCl. The eluted glycans are then
concentrated and desalted by ethanol precipitation, size exclusion,
or other methods. The purified glycans are dried for further
analysis.
[0291] Example 2--Digestion: Lyase digestion: The purified glycans
are resuspended in 10 -300 mM sodium acetate, tris, phosphate, or
other suitable buffer, 0.02-1 mM (e.g., 0.02, 0.04, 0.06, 0.08,
0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, or 1) calcium acetate,
pH 5-8 (e.g., 5, 5.5, 6, 6.5, 7, 7.5, or 8), were digested with
heparin lyases I, II, III, I and II, I and III, II and III, or I,
II, and III (0.0.15-1.5 milliunits of each in 100-ul reactions,
IBEX, Montreal, Canada) at 25 to 37 .degree. C. for 1 to 24
hours.
[0292] Example 3--Tagging: Dried glycan sample is re-suspended in
2-100 .mu.L 0.003-0.1 M (e.g., 0.003 M, 0.003 M, 0.03 M, 0.06 M,
0.1 M) AB, AA, AMAC, or Bodipy dye and incubated at room
temperature for 1-120 minutes (e.g., 1-10 min, 10-15 min, 15-20
min, 20-25 min, 25-30 min, 30-40 min, 40-50 min, 50-60 min, 60-90
min, 90-120 min). Next, the reaction is initiated with 2-100 (2
.mu.L, 5 .mu.L, 10 .mu.L, 15 .mu.L, 20 .mu.L, 25 .mu.L, 30 .mu.L,
40 .mu.L, 50 .mu.L, 60 .mu.L, 70 .mu.L, 80 .mu.L, 90 .mu.L, or 100
.mu.L) 1 M NaCNBH.sub.4 and the reaction is allowed to proceed at
25-100 C. (e.g., 25 C, 30 C, 35 C, 40 C, 50 C, 60 C, 70 C, 80 C, 90
C, 100 C).
[0293] Example 4--Detecting: HPLC separation of tagged saccharides
was performed utilizing the following conditions: [0294] Column
types: 130A BEH particle Phenyl (1.7, 2.5, 3.5, 5, or 10 uM
particle size), 130A BEH particle C18 (1.7, 2.5, 3.5, 5, or 10 uM
particle size), HSS particle C18 (1.8, 3.5, or 5 uM particle size),
or 300A BEH particle C18 (1.7, 3.5, 5, 10 uM particle size) with
suitable length and internal diameter. [0295] Buffer Conditions:
[0296] A=10 mM Ammonium Acetate with 0-20% methanol [0297] B=100%
Methanol [0298] Initial Conditions: 70-95% A, 0-30% B [0299] Flow
Rate is constant at 0.05-1 ml/min [0300] Runs a gradient down to
70-90% A, 10-30% B over 5-65 min. [0301] At 8.1 min runs a gradient
to 0-20% A, 80-100% B over 5-20 min. [0302] 5-65 min returns to
initial conditions
[0303] Fluorescently tagged oligosaccharides (disaccharides) were
detected at various elution times depending on the specific marker
produced of interest. Elution order of unsaturated disaccharide
standards was: [0304] 2SNS6S [0305] NS6S [0306] 2SNS [0307] NS
[0308] NA6S [0309] NA
[0310] Example 5: Tested was the ability of a non-reducing end
detection method to identify and classify MPS samples by blindly
analyzing normal and MPS GAGs from cultured fibroblasts. Human
primary fibroblast cultures from MPS patients with MPS I, II, IIA,
and IIIB (GM01256, GM00298, GM00879, and GM01426, respectively)
were obtained from the NIGMS Human Genetic Cell Repository. Cells
were seeded at high density and allowed to accumulate GAGs for 7-14
days. At harvest, GAGs were isolated using the procedure described
in the specifications. Total GAGs were digested with heparin lyases
to completion. A portion of the digested GAG fragments were
analyzed by liquid chromatography tandem atmospheric pressure
electrospray mass spectrometry of aniline derivatized
oligosaccharides to determine if the diagnostic biomarkers were
present. FIGS. 27, 28A, 29A, 30A and 31A--Panel A shows
representative structures of predicted oligosaccharides for each
class of MPS. FIGS. 27, 28A, 29A, 30A and 31A--Panels B, C, D, and
E show the extracted ion currents for representative biomarker(s)
for MPS I (m/z=590.5-591.5+719.5-720.5), MPS II
(m/z=719.5-720.5+799.5-800.5), MPS IIIA
(m/z=831.5-832.5+960.5-961.5+1088.5-1089.5), and MPS IIIB
(m/z=793.5-794.5+873.5-874.5+922.5-923.5+1002.5-1003.5+1130.5-1131.5),
respectively. MS analysis was done according to the methods of
Lawrence et al. J Biol Chem. 2008 Nov. 28;283(48):33674-84. Insets
are the corresponding MS spectra for the non-reducing end
structures detected. By combining the known degradative defect with
the mass spectrometry data, the actual structures of the biomarkers
can be deduced or reduced to a few likely structures. The
sensitivity of the method to detect and correctly identify MPS
disease from small cell culture samples indicates that this method
has the sensitivity to work on clinically relevant samples such as
blood or urine.
[0311] Example 6: The non-reducing end biomarker method was used to
measure the accumulation of GAGs in the MPS IIIB mouse model.
Previous reports have had difficulty measuring GAG accumulation in
the brain, despite microscopic validation of lysosomal inclusions.
This has been especially puzzling because the primary phenotype in
MPS IIIB patients are neurological symptoms. We used this
challenging case to determine if this biomarker method can detect
GAG accumulation in difficult tissues. 20 mg (wet weight) tissue
samples were homogenized and total cellular GAGs were purified
using the method described in the specifications. The purified GAGs
were them treated with heparin lyases to liberate the unique
non-reducing ends from the heterogeneous collection of fragments.
The presence of the MPS IIIB biomarkers {(dp3-1Ac,2S),
(dp3(1Ac,3S), and (dp3(1Ac,4S)} was analyzed by LC-MS. These
studies verify that the GAG accumulation is occurring in the
brain.
[0312] Example 7--Diagnosing Disease: The following data is
generated by measuring the abundance of a saturated disaccharide
liberated from GAGs by heparin lyases that accumulate in MPS I
samples. Using the described method, the analyzed saturated
disaccharide elutes at 3.07 minutes.
[0313] Example 7A: In these studies, 9 .mu.L frozen urine samples
from MPS I affected dogs and carrier dogs (unaffected) were
analyzed for the presence of saturated disaccharide liberated from
the non-reducing end of the disease causing glycans. Picomoles of
saturated disaccharide per milliliter of serum are shown in FIG.
35. FIG. 35 illustrates that the assay is useful for detecting the
presence of MPS disease in urine samples.
[0314] Example 7B: In these studies 30 .mu.L frozen serum samples
from MPS I affected dogs and carrier dogs (unaffected) were
analyzed for the presence of saturated disaccharide liberated from
the non-reducing end of the disease causing glycans. Picomoles of
saturated disaccharide per milliliter of serum are shown in FIG.
36. FIG. 36 illustrates that this assay is useful for identifying
the presence of MPS (e.g., MPS I) disease in serum. By using the
unique markers appropriate for other MPS classes, the same is
accomplished for other MPS classes.
[0315] Example 7C: In these studies 30 .mu.L frozen CSF samples
from MPS I affected dogs and carrier dogs (unaffected) were
analyzed for the presence of saturated disaccharide liberated from
the non-reducing end of the disease causing glycans. Picomoles of
saturated disaccharide per milliliter of CSF are shown in FIG. 37.
FIG. 37 illustrates that this method is useful for detecting
disease in CSF from MPS patients.
[0316] Example 7D: To determine if the GAG accumulation assay
described here is able to detect MPS I disease in tissue samples,
we analyzed the GAG accumulation in 2.5 mg samples of cortex taken
from unaffected and affected MPS I dogs. Samples were analyzed for
the presence of saturated disaccharide liberated from the
non-reducing end of the disease causing glycans. Picomoles of
saturated disaccharide per milligram of cortex are shown in FIG.
38. FIG. 38 illustrates this method is useful for detecting MPS
disease in tissue samples from patients.
[0317] Example --Diagnosing Severity of Disease: The following data
is generated by measuring the abundance of a saturated disaccharide
liberated from GAGs by heparin lyases that accumulate in MPS I
samples. Using the described method, this analyzed saturated
disaccharide elutes at 3.07 minutes.
[0318] In these studies 30 .mu.L frozen serum samples from MPS I
affected dogs were analyzed to measure the level of GAG
accumulation in each dog. Because the MPS I dog model is a
naturally occurring and genetically heterogeneous model, there is
significant variation in disease severity between individual dogs.
These differences in disease severity are revealed by variations in
the level of GAG accumulation between the individual dogs using the
methods described here. Samples were analyzed for the presence of
saturated disaccharide liberated from the non-reducing end of the
disease causing glycans. Picomoles of saturated disaccharide per
milliliter of serum are shown in FIG. 39. FIG. 39 illustrates that
this method can be used to detect the response to therapy in serum
from MPS patients.
[0319] Example 9--Monitoring Response to Therapy: The following
data is generated by measuring the abundance of a saturated
disaccharide liberated from GAGs by heparin lyases that accumulate
in MPS I samples. Using the described method, this analyzed
saturated disaccharide elutes at 3.07 minutes.
[0320] Example 9A: In these studies 30 .mu.L frozen CSF samples
from MPS I affected dogs before treatment (untreated) and after
monthly intrathecal (IT) treatment with laronidase (treated).
Samples were analyzed for the presence of saturated disaccharide
liberated from the non-reducing end of the disease causing glycans.
Picomoles of saturated disaccharide per milliliter of CSF are shown
in FIG. 40. FIG. 40 illustrates that this method is useful for
detecting the response to therapy in CSF from MPS patients.
[0321] Example 9B: To determine if the GAG accumulation assay
described here is able to measure different levels of response to
treatments, a group of MPS I dogs who were treated with IT
laronidase were analyzed with respect to their level of therapy
directed antibodies. "Tolerant" dogs are dogs that do not mount a
significant immune reaction to the laronidase treatment while "not
tolerant" dogs mount a significant antibody response. This antibody
response is expected to reduce the efficacy of the enzyme
replacement therapy, laronidase. To determine if the assay can
detect this differential response, 30 .mu.L frozen CSF samples from
MPS I affected dogs before treatment (untreated) and after monthly
intrathecal (IT) treatment with 0.58 mg/kg laronidase (treated).
Samples were analyzed for the presence of saturated disaccharide
liberated from the non-reducing end of the disease causing glycans
and summarized with respect to their tolerance class. The response
to therapy is shown for three different dogs affected with MPS I.
Picomoles of saturated disaccharide per milliliter of CSF are shown
in FIG. 41. FIG. 41 illustrates that this method is useful for
detecting the differential response to therapy in CSF from MPS
patients. In some embodiments, this process is used to guide the
treatment for individual patients.
[0322] Example 9C: To determine if the GAG accumulation assay
described here is able to detect a response to therapy in MPS I
disease in tissue samples treated with IT laronidase, we analyzed
the GAG accumulation in 2.5 mg samples of cortex taken from
unaffected and affected MPS I dogs. Differential response to
therapy is seen based on the tolerance status of the specific dogs.
Samples were analyzed for the presence of saturated disaccharide
liberated from the non-reducing end of the disease causing glycans
and summarized with respect to their tolerance class. The response
to therapy is shown for three different dogs affected with MPS I.
Picomoles of saturated disaccharide per milliliter of CSF are shown
in FIG. 42. FIG. 42 illustrates that this method is useful for
detecting response to therapy in tissue samples from patients.
[0323] Example 9D: In these studies 30 .mu.L frozen serum samples
from MPS I affected dogs before treatment (sample #1) and monthly
samples through weekly IV treatment with laronidase (samples #2, 3,
4 etc.). Serum samples were taken immediately prior to the next IV
infusion. Samples were analyzed for the presence of unsaturated
disaccharides liberated from the non-reducing end of the disease
causing glycans. The response to therapy is shown for three
different dogs affected with MPS I. Picomoles of these structures
per milliliter of serum are shown in FIG. 43A, 43B, and 43C. FIG.
43A, 43B, and 43C illustrate that this method is useful for
detecting the response to therapy in serum from MPS patients.
[0324] Example 10--Diagnosing Carriers: To determine if the GAG
accumulation assay described here is able to detect carriers of the
genetic cause of MPS, we analyzed wild-type dogs and dogs that are
heterozygous for the MPS I causing mutation. Carriers are presumed
to lack any clinical manifestation of MPS I; however, it is
possible that the partial deficiency in a lysosomal enzyme will
lead to low levels of GAG accumulation that can be detected with
this assay. Samples were analyzed for the presence of unsaturated
saturated disaccharide liberated from the non-reducing end of the
disease causing glycans and summarized with respect to their
tolerance class. Picomoles of saturated disaccharide per milligram
of tissue are shown in FIG. 44. FIG. 44 illustrates that the method
described herein is useful for detecting carriers of the genetic
cause of MPS.
* * * * *