U.S. patent application number 14/192696 was filed with the patent office on 2014-07-24 for methods and compositions for determining a graft tolerant phenotype in a subject.
The applicant listed for this patent is The Board of Trustees of the Leland Stanford Junior University, Inserm. Invention is credited to Sophie Brouard, Elaine Mansfield, Minne M. Sarwal, Jean-Paul Soulilou.
Application Number | 20140206566 14/192696 |
Document ID | / |
Family ID | 34811352 |
Filed Date | 2014-07-24 |
United States Patent
Application |
20140206566 |
Kind Code |
A1 |
Sarwal; Minne M. ; et
al. |
July 24, 2014 |
Methods and Compositions for Determining a Graft Tolerant Phenotype
in a Subject
Abstract
Methods are provided for determining whether a subject has a
graft tolerant phenotype. In practicing the subject methods, the
expression of at least one gene in a sample from the subject, e.g.,
a blood sample, is assayed to obtain an expression evaluation for
the at least one gene. The obtained expression evaluation is then
employed to determine whether the subject has a graft tolerant
phenotype. Also provides are compositions, systems and kits that
find use in practicing the subject methods. The methods and
compositions find use in a variety of applications, including the
determination of an immunosuppressive therapy regimen.
Inventors: |
Sarwal; Minne M.; (Portola
Valley, CA) ; Mansfield; Elaine; (Sunnyvale, CA)
; Brouard; Sophie; (Nantes, FR) ; Soulilou;
Jean-Paul; (Nantes, FR) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Inserm
The Board of Trustees of the Leland Stanford Junior
University |
Paris
Palo Alto |
CA |
FR
US |
|
|
Family ID: |
34811352 |
Appl. No.: |
14/192696 |
Filed: |
February 27, 2014 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10585610 |
May 31, 2007 |
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PCT/US05/04799 |
Jan 20, 2005 |
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14192696 |
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60538439 |
Jan 21, 2004 |
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60571471 |
May 14, 2004 |
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Current U.S.
Class: |
506/9 ; 435/6.11;
435/6.12; 435/7.92; 506/16 |
Current CPC
Class: |
C12Q 1/6883 20130101;
C12Q 2600/158 20130101; A61P 37/06 20180101; C12Q 2600/106
20130101 |
Class at
Publication: |
506/9 ; 506/16;
435/7.92; 435/6.12; 435/6.11 |
International
Class: |
C12Q 1/68 20060101
C12Q001/68 |
Claims
1. A method of determining whether a subject has a graft tolerant
phenotype, said method comprising: evaluating the expression of at
least one gene, wherein said at least one gene comprises UHRF1, in
a sample from said subject to determine whether said subject has a
graft tolerant phenotype.
2. The method according to claim 1, wherein said expression of at
least one gene is evaluated by assaying said sample for a nucleic
acid transcript of said gene.
3. The method according to claim 1, wherein said expression of at
least one gene is evaluated by assaying said sample for an
expression product of said gene.
4. The method according to claim 1, wherein said sample is a blood
sample.
5. The method according to claim 4, wherein said blood sample is a
peripheral blood sample.
6. The method according to claim 1, wherein the expression of said
at least one gene, including UHRF1, from Tables 1, 2, 3 and/or 4 is
evaluated.
7. The method according to claim 1, wherein the method comprises:
(a) obtaining an expression profile for said sample from said
subject; and (b) employing said obtained expression profile to
determine whether said subject has a graft tolerant phenotype.
8. The method according to claim 7, wherein said expression profile
is compared to a reference expression profile in said employing
step (b).
9. The method according to claim 7, wherein said expression profile
is determined using a microarray.
10. A method of managing immunosuppressive therapy in a subject
having a graft, said method comprising: (a) evaluating whether said
subject has a graft tolerant phenotype according to claim 1; and
(b) determining a future immunosuppressive therapy protocol based
on said evaluating step (a) to manage immunosuppressive therapy in
said subject.
11. A system for determining whether a subject has a graft tolerant
phenotype comprising: (a) a gene expression evaluation element for
evaluating the expression of at least one gene, wherein said at
least one gene comprises UHRF1, in a sample from said subject to
obtain a gene expression result; and (b) a phenotype determination
element for employing said gene expression result to determine
whether said subject has a graft tolerant phenotype.
12. A reference expression profile for a phenotype that is one of:
(a) graft tolerant; or (b) graft intolerant; wherein said
expression profile is recorded on a computer readable medium.
13. A collection of gene specific primers for evaluating gene
expression, said collection comprising: primers specific for at
least two of the genes, including UHRF1, of Tables 1, 2, 3 and/or
4.
14. An array of probe nucleic acids immobilized on a solid support,
said array comprising: a plurality of probe nucleic acid
compositions, wherein each probe nucleic acid composition is
specific for a gene whose expression profile is indicative of a
graft tolerance, wherein at least two of said probe nucleic acid
compositions correspond to genes listed in Tables 1, 2, 3 and/or
4.
15. A kit comprising: (a) an array according to claim 14; and (b) a
collection of gene specific primers according to claim 13.
16. The method according to claim 7, wherein said expression
profile comprises expression data for UHRF1.
17. The method of claim 7, wherein said expression profile is
determined using quantitative PCR.
18. The method of claim 1, wherein at least five genes is
evaluated.
19. The system of claim 11, wherein at least five genes is
evaluated.
20. A kit for determining whether a subject has a graft tolerant
phenotype, wherein the kit comprises: (a) gene expression
evaluation element for evaluating the expression of at least one
gene, wherein said at least one gene comprises UHRF1, in a sample
from said subject to obtain a gene expression result; and (b) a
phenotype determination element for employing said gene expression
result to determine whether said subject has a graft tolerant
phenotype.
21. The kit of claim 20, wherein at least five genes is evaluated.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] Pursuant to 35 U.S.C. .sctn.119 (e), this application claims
priority to the filing date of the United States Provisional Patent
Application Ser. No. 60/571,471 filed May 14, 2004 and to the
filing date of U.S. Provisional Patent Application Ser. No.
60/538,439 filed on Jan. 21, 2004; the disclosures of which are
herein incorporated by reference.
INTRODUCTION
Background of the Invention
[0002] Transplantation of a graft organ or tissue from a donor to a
host patient is a feature of certain medical procedures and
treatment protocols. Despite efforts to avoid graft rejection
through host-donor tissue type matching, in transplantation
procedures where a donor organ is introduced into a host,
immunosuppressive therapy is generally required to the maintain
viability of the donor organ in the host.
[0003] A variety of immunosuppressive agents have been employed in
transplantation procedures, including azathioprine, methotrexate,
cyclophosphamide, FK-506, rapamycin and corticosteroids. Agents
finding increased use in immunosuppressive therapy due to their
preferential effect on T-cell mediated reactions are the
cyclosporins.
[0004] Following transplantation, administration of the
immunosuppressive agent must be continued indefinitely since the
benefits of immunosuppressive therapy are reversible and graft
rejection may occur once administration of the immunosuppressive
agent is discontinued. While use of immunosuppressive agents, such
as Cyclosporin A, has been reported to prolong the survival of
allogeneic transplants involving skin, heart, kidney, pancreas,
bone marrow, small intestine and lung, use of such agents is not
without undesirable side effects. Examples of undesirable side
effects include increased risk of development of neoplastic disease
conditions, e.g., skin cancer, lymphoma, etc.
[0005] While most recipients who discontinue their
immunosuppressive treatment following a graft go on to suffer
rejection, not all subjects suffer graft rejection. In a few cases,
individuals tolerate their graft without immunosuppression,
suggesting that immune non-responsiveness can be achieved in
clinical practice. The mechanisms of this process are not well
understood, but may involve a combination of clonal deletion,
clonal anergy and the generation of active regulatory T cells.
[0006] Because of the undesirable sides effects and risks of long
term immunosuppressive therapy, it would be desirable to be able
identify those individuals who are tolerant to their graft, i.e.,
graft tolerant, so that immunosuppression could be reduced or even
discontinued in those individuals. Of particular interest would be
the development of a way to identify graft tolerant individuals
without first discontinuing immunosuppressive therapy, thereby
avoiding the risk of graft rejection and damage to the graft
associated therewith. The present invention meets this need.
Relevant Literature
[0007] Publications of interest include: United States Patent
Application No. 2003/0104371.
SUMMARY OF THE INVENTION
[0008] Methods are provided for determining whether a subject has a
graft tolerant phenotype. In practicing the subject methods, the
expression of at least one gene in a sample from the subject, e.g.,
a blood sample, is assayed to obtain an expression evaluation for
the at least one gene. The obtained expression evaluation is then
employed to determine whether the subject has a graft tolerant
phenotype. Also provided are compositions, systems and kits that
find use in practicing the subject methods. The subject methods and
compositions find use in a variety of applications, including the
determination of an immunosuppressive therapy regimen.
[0009] The subject invention provides methods of determining
whether a subject has a graft tolerant phenotype, where the methods
include: evaluating expression of at least one gene in a sample
from the subject to determine whether the subject has a graft
tolerant phenotype. In certain embodiments, the expression of at
least one gene is evaluated by assaying the sample for a nucleic
acid transcript of the gene. In certain embodiments, the expression
of at least one gene is evaluated by assaying the sample for an
expression product of the gene. In certain embodiments, the sample
is a blood sample. In certain embodiments, the blood sample is a
peripheral blood sample. In certain embodiments, the at least one
gene is a gene listed on Tables 1, 2, 3 and/or 4. In certain
embodiments, expression of a least 5 genes from Tables 1, 2, 3
and/or 4 is evaluated. In certain embodiments, expression of at
least 50 genes from Tables 1, 2, 3 and/or 4 is evaluated.
[0010] Also provided are methods of determining whether a subject
has a graft tolerant phenotype by: (a) obtaining an expression
profile for a sample from the subject; and (b) employing the
obtained expression profile to determine whether the subject has a
graft tolerant phenotype. In certain embodiments, the expression
profile is compared to a reference expression profile in the
employing step (b). In certain embodiments, the reference
expression profile is a graft tolerant phenotype expression
profile. In certain embodiments, the reference expression profile
is a graft intolerant phenotype expression profile. In certain
embodiments, the expression profile comprises expression
measurements for at least 5 different genes. In certain
embodiments, the at least 5 different genes are listed in Tables 1,
2, 3 and/or 4. In certain embodiments, the expression profile is
determined using a microarray. In certain embodiments, the
microarray is a genomic array. In certain embodiments, the
microarray is a proteomic array.
[0011] Also provided is a method of managing immunosuppressive
therapy in a subject having a graft, where the method includes: (a)
evaluating whether the subject has an graft tolerant phenotype; and
(b) determining a future immunosuppressive therapy protocol based
on the evaluation of step (a) to manage immunosuppressive therapy
in said subject. In certain embodiments, the evaluating step (a) is
by a method as described above. In certain embodiments, the method
comprises at least reducing immunosuppression in the subject if
said subject is found to have a graft tolerant phenotype. In
certain embodiments, the method comprises discontinuing
immunosuppression in the subject if the subject is found to have a
graft tolerant phenotype.
[0012] Also provided are systems for determining whether a subject
has an graft tolerant phenotype, where the systems include: (a) a
gene expression evaluation element for evaluating expression of at
least one gene in a sample to obtain a gene expression result; and
(b) a phenotype determination element for employing the gene
expression result to determine whether a subject has a graft
tolerant phenotype. In certain embodiments, the gene expression
evaluation element comprises at least one reagent for assaying a
sample for a nucleic acid transcript of said gene. In certain
embodiments, the gene expression evaluation element comprises at
least one reagent for assaying a sample for an expression product
of said gene. In certain embodiments, the gene expression
evaluation element comprises an array. In certain embodiments, the
at least one gene is a gene listed in Tables 1, 2, 3 and/or 4. In
certain embodiments, the phenotype determination element comprises
a reference expression value for the at least one gene. In certain
embodiments, the phenotype determination element comprises a
reference expression profile that includes a reference expression
value for at least one additional gene. In certain embodiments, the
reference expression profile is a graft tolerant phenotype
expression profile. In certain embodiments, the reference
expression profile is a graft intolerant phenotype expression
profile.
[0013] Also provided are kits for determining whether a subject has
a graft tolerant phenotype, where the kits include: (a) a gene
expression evaluation element for evaluating expression of at least
one gene in a sample to obtain a gene expression result; (b) a
phenotype determination element for employing the gene expression
result to determine whether a subject has a graft tolerant
phenotype; and (c) instructions for using said gene expression
evaluation and phenotype determination elements in a method, e.g.,
as described above. In certain embodiments, the gene expression
evaluation element comprises at least one reagent for assaying a
sample for a nucleic acid transcript of said gene. In certain
embodiments, the gene expression evaluation element comprises at
least one reagent for assaying a sample for an expression product
of said gene. In certain embodiments, the gene expression
evaluation element comprises an array. In certain embodiments, the
at least one gene is a gene listed in Tables 1, 2, 3 and/or 4. In
certain embodiments, the phenotype determination element comprises
a reference expression value for the at least one gene. In certain
embodiments, the phenotype determination element comprises a
reference expression profile that includes a reference expression
value for at least one additional gene. In certain embodiments, the
reference expression profile is a graft tolerant phenotype
expression profile. In certain embodiments, the reference
expression profile is a graft intolerant phenotype expression
profile. Also provided are reference expression profiles for a
phenotype that is one of: (a) graft tolerant; or (b) graft
intolerant; wherein the expression profile is recorded on a
computer readable medium. In certain embodiments, the expression
profile includes at least one of the genes from Tables 1, 2, 3
and/or 4. In certain embodiments, the expression profile is a
profile for a phenotype that is allograft tolerant. In certain
embodiments, the expression profile is a profile for a phenotype
that is graft intolerant.
[0014] Also provided by the invention is a collection of reagents
for evaluating gene expression, where the collection includes:
reagents specific for at least two of the genes of Tables 1, 2, 3
and/or 4. In certain embodiments, the reagents are gene specific
primers. In certain embodiments, the collection comprises at least
10 gene specific primers.
[0015] Also provided are arrays of probe nucleic acids immobilized
on a solid support, where the arrays include: a plurality of probe
nucleic acid compositions, wherein each probe nucleic acid
composition is specific for a gene whose expression profile is
indicative of a graft tolerance, wherein at least two of the probe
nucleic acid compositions correspond to genes listed in Tables 1,
2, 3 and/or 4.
[0016] Also provided are kits that include at least one of: (a) an
array according as described above; and (b) a collection of gene
specific primers as described above, where in certain embodiments
the kits include both of these components.
BRIEF DESCRIPTION OF THE FIGURES
[0017] FIG. 1 provides Table 1, which lists 200 representative
genes whose expression differs in OT patients relative to those
with chronic graft injury.
[0018] FIG. 2 provides Table 2, which is an analysis of 591 genes
differentially expressed in PBL samples associated with tolerance
in renal transplantation.
[0019] FIG. 3 provides Table 3, which shows the gene expression
differences between OT and CR samples by KEGG families.
DESCRIPTION OF THE SPECIFIC EMBODIMENTS
[0020] Methods are provided for determining whether a subject has a
graft tolerant phenotype. In practicing the subject methods, the
expression of at least one gene in a sample from the subject, e.g.,
a blood sample, is assayed to obtain an expression evaluation for
the at least one gene. The obtained expression evaluation is then
employed to determine whether the subject has a graft tolerant
phenotype. Also provided are compositions, systems and kits that
find use in practicing the subject methods. The methods and
compositions find use in a variety of applications, including the
determination of an immunosuppressive therapy regimen.
[0021] Before the present invention is further described, it is to
be understood that this invention is not limited to particular
embodiments described, as such may, of course, vary. It is also to
be understood that the terminology used herein is for the purpose
of describing particular embodiments only, and is not intended to
be limiting, since the scope of the present invention will be
limited only by the appended claims.
[0022] Where a range of values is provided, it is understood that
each intervening value, to the tenth of the unit of the lower limit
unless the context clearly dictates otherwise, between the upper
and lower limit of that range and any other stated or intervening
value in that stated range, is encompassed within the invention.
The upper and lower limits of these smaller ranges may
independently be included in the smaller ranges and are also
encompassed within the invention, subject to any specifically
excluded limit in the stated range. Where the stated range includes
one or both of the limits, ranges excluding either or both of those
included limits are also included in the invention.
[0023] Methods recited herein may be carried out in any order of
the recited events which is logically possible, as well as the
recited order of events.
[0024] Unless defined otherwise, all technical and scientific terms
used herein have the same meaning as commonly understood by one of
ordinary skill in the art to which this invention belongs. Although
any methods and materials similar or equivalent to those described
herein can also be used in the practice or testing of the present
invention, the preferred methods and materials are now
described.
[0025] All publications mentioned herein are incorporated herein by
reference to disclose and describe the methods and/or materials in
connection with which the publications are cited.
[0026] It must be noted that as used herein and in the appended
claims, the singular forms "a", "an", and "the" include plural
referents unless the context clearly dictates otherwise. It is
further noted that the claims may be drafted to exclude any
optional element. As such, this statement is intended to serve as
antecedent basis for use of such exclusive terminology as "solely,"
"only" and the like in connection with the recitation of claim
elements, or use of a "negative" limitation.
[0027] The publications discussed herein are provided solely for
their disclosure prior to the filing date of the present
application. Nothing herein is to be construed as an admission that
the present invention is not entitled to antedate such publication
by virtue of prior invention. Further, the dates of publication
provided may be different from the actual publication dates which
may need to be independently confirmed.
[0028] As summarized above, the subject invention is directed to
methods of determining whether a subject has a graft tolerant
phenotype, as well as reagents and kits for use in practicing the
subject methods. In further describing the invention, the subject
methods are described first, followed by a review of the reagents
and kits for use in practicing the subject methods.
Methods of Determining Whether a Subject has a Graft Tolerant
Phenotype
[0029] The subject invention provides methods of determining
whether a patient or subject has a graft tolerant phenotype. By
graft tolerant phenotype is meant that the subject does not reject
a graft organ, tissue or cell(s) that has been introduced into/onto
the subject. In other words, the subject tolerates or maintains the
organ, tissue or cell(s) that has been transplanted to it. As in
known in the transplantation field, the graft organ, tissue or
cell(s) may be allogeneic or xenogeneic, such that the grafts may
be allografts or xenografts. A feature of the graft tolerant
phenotype detected or identified by the subject methods is that it
is a phenotype which occurs without immunosuppressive therapy,
i.e., it is present in a host that is not undergoing
immunosuppressive therapy such that immunosuppressive agents are
not being administered to the host.
[0030] In practicing the subject methods, a subject or patient
sample, e.g., cells or collections thereof, e.g., tissues, is
assayed to determine whether the host from which the assayed sample
was obtained is graft tolerant, i.e., has a graft tolerant
phenotype. Accordingly, the first step of the subject methods is to
obtain a suitable sample from the subject or patient of interest,
i.e., a patient on immunosuppressive therapy and having at least
one graft, e.g., allograft. The sample is derived from any initial
suitable source, where sample sources of interest include, but are
not limited to, many different physiological sources, e.g., CSF,
urine, saliva, tears, tissue derived samples, e.g., homogenates,
and blood or derivatives thereof.
[0031] In certain embodiments, a suitable initial source for the
patient sample is blood. As such, the sample employed in the
subject assays of these embodiments is generally a blood-derived
sample. The blood derived sample may be derived from whole blood or
a fraction thereof, e.g., serum, plasma, etc., where in many
embodiments the sample is derived from blood cells harvested from
whole blood. Of particular interest as a sample source are
peripheral blood lymphocytes (PBL). Any convenient protocol for
obtaining such samples may be employed, where suitable protocols
are well known in the art.
[0032] In practicing the subject methods, the sample is assayed to
obtain an expression evaluation, e.g., expression profile, for one
or more genes, where the term expression profile is used broadly to
include a genomic expression profile, e.g., an expression profile
of nucleic acid transcripts, e.g., mRNAs, of the one or more genes
of interest, or a proteomic expression profile, e.g., an expression
profile of one or more different proteins, where the
proteins/polypeptides are expression products of the one or more
genes of interest. As such, in certain embodiments the expression
of only one gene is evaluated. In yet other embodiments, the
expression of two or more, e.g., about 5 or more, about 10 or more,
about 15 or more, about 25 or more, about 50 or more, about 100 or
more, about 200 or more, etc., genes is evaluated. Accordingly, in
the subject methods, the expression of at least one gene in a
sample is evaluated. In certain embodiments, the evaluation that is
made may be viewed as an evaluation of the transcriptosome, as that
term is employed in the art. See e.g., Gomes et al., Blood (2001
Jul 1) 98(1):93-9.
[0033] In generating the expression profile, in many embodiments a
sample is assayed to generate an expression profile that includes
expression data for at least one gene/protein, usually a plurality
of genes/proteins, where by plurality is meant at least two
different genes/proteins, and often at least about 5, typically at
least about 10 and more usually at least about 20 different
genes/proteins or more, such as 50 or more, 100 or more, etc.
[0034] In the broadest sense, the expression evaluation may be
qualitative or quantitative. As such, where detection is
qualitative, the methods provide a reading or evaluation, e.g.,
assessment, of whether or not the target analyte, e.g., nucleic
acid or expression product, is present in the sample being assayed.
In yet other embodiments, the methods provide a quantitative
detection of whether the target analyte is present in the sample
being assayed, i.e., an evaluation or assessment of the actual
amount or relative abundance of the target analyte, e.g., nucleic
acid in the sample being assayed. In such embodiments, the
quantitative detection may be absolute or, if the method is a
method of detecting two or more different analytes, e.g., target
nucleic acids, in a sample, relative. As such, the term
"quantifying" when used in the context of quantifying a target
analyte, e.g., nucleic acid(s), in a sample can refer to absolute
or to relative quantification. Absolute quantification may be
accomplished by inclusion of known concentration(s) of one or more
control analytes and referencing the detected level of the target
analyte with the known control analytes (e.g., through generation
of a standard curve). Alternatively, relative quantification can be
accomplished by comparison of detected levels or amounts between
two or more different target analytes to provide a relative
quantification of each of the two or more different analytes, e.g.,
relative to each other.
[0035] Genes/proteins of interest are genes/proteins that are
differentially expressed or present at different levels in graft
tolerant and graft intolerant individuals. Representative
genes/proteins of interest in certain embodiments include, but are
not limited to, the genes/proteins provided in Table 1. (Note that
for Table 1, the exact sequence of the clone identified in the
table can be determined through the NCBI Entrez nucleotide database
at located at the website produced by placing "http://www." before:
"ncbi.nlm.nih.gov/entrez/query.fcgi?CMD=search&db=nucleotide";
the sequence for a specific clone is then obtained by entering the
clone ID in quotes as the search term).
[0036] In certain embodiments, at least one of the genes/proteins
in the prepared expression profile is from Tables 1, 2, 3 and/or 4,
where the expression profile may include expression data for 5, 10,
20, 50, 75 or more of, including all of, the genes/proteins listed
in Tables 1, 2, 3 and/or 4. The number of different genes/proteins
whose expression and/or quantity data, i.e., presence or absence of
expression, as well as expression/quantity level, that are included
in the expression profile that is generated may vary, but may be at
least 2, and in many embodiments ranges from 2 to about 100 or
more, sometimes from 3 to about 75 or more, including from about 4
to about 70 or more.
[0037] In certain embodiments, the expression profile obtained is a
genomic or nucleic acid expression profile, where the amount or
level of one or more nucleic acids in the sample is determined,
e.g., the nucleic acid transcript of the gene of interest. In these
embodiments, the sample that is assayed to generate the expression
profile employed in the diagnostic methods is one that is a nucleic
acid sample. The nucleic acid sample includes a plurality or
population of distinct nucleic acids that includes the expression
information of the phenotype determinative genes of interest of the
cell or tissue being diagnosed. The nucleic acid may include RNA or
DNA nucleic acids, e.g., mRNA, cRNA, cDNA etc., so long as the
sample retains the expression information of the host cell or
tissue from which it is obtained. The sample may be prepared in a
number of different ways, as is known in the art, e.g., by mRNA
isolation from a cell, where the isolated mRNA is used as is,
amplified, employed to prepare cDNA, cRNA, etc., as is known in the
differential expression art. The sample is typically prepared from
a cell or tissue harvested from a subject to be diagnosed, e.g.,
via biopsy of tissue, using standard protocols, where cell types or
tissues from which such nucleic acids may be generated include any
tissue in which the expression pattern of the to be determined
phenotype exists, including, but not limited to, peripheral blood
lymphocyte cells, etc, as reviewed above.
[0038] The expression profile may be generated from the initial
nucleic acid sample using any convenient protocol. While a variety
of different manners of generating expression profiles are known,
such as those employed in the field of differential gene expression
analysis, one representative and convenient type of protocol for
generating expression profiles is array-based gene expression
profile generation protocols. Such applications are hybridization
assays in which a nucleic acid that displays "probe" nucleic acids
for each of the genes to be assayed/profiled in the profile to be
generated is employed. In these assays, a sample of target nucleic
acids is first prepared from the initial nucleic acid sample being
assayed, where preparation may include labeling of the target
nucleic acids with a label, e.g., a member of signal producing
system. Following target nucleic acid sample preparation, the
sample is contacted with the array under hybridization conditions,
whereby complexes are formed between target nucleic acids that are
complementary to probe sequences attached to the array surface. The
presence of hybridized complexes is then detected, either
qualitatively or quantitatively. Specific hybridization technology
which may be practiced to generate the expression profiles employed
in the subject methods includes the technology described in U.S.
Pat. Nos.: 5,143,854; 5,288,644; 5,324,633; 5,432,049; 5,470,710;
5,492,806; 5,503,980; 5,510,270; 5,525,464; 5,547,839; 5,580,732;
5,661,028; 5,800,992; the disclosures of which are herein
incorporated by reference; as well as WO 95/21265; WO 96/31622; WO
97/10365; WO 97/27317; EP 373 203; and EP 785 280. In these
methods, an array of "probe" nucleic acids that includes a probe
for each of the phenotype determinative genes whose expression is
being assayed is contacted with target nucleic acids as described
above. Contact is carried out under hybridization conditions, e.g.,
stringent hybridization conditions, and unbound nucleic acid is
then removed.
[0039] The term "stringent assay conditions" as used herein refers
to conditions that are compatible to produce binding pairs of
nucleic acids, e.g., surface bound and solution phase nucleic
acids, of sufficient complementarity to provide for the desired
level of specificity in the assay while being less compatible to
the formation of binding pairs between binding members of
insufficient complementarity to provide for the desired
specificity. Stringent assay conditions are the summation or
combination (totality) of both hybridization and wash
conditions.
[0040] The term "stringent assay conditions" as used herein refers
to conditions that are compatible to produce binding pairs of
nucleic acids, e.g., surface bound and solution phase nucleic
acids, of sufficient complementarity to provide for the desired
level of specificity in the assay while being less compatible to
the formation of binding pairs between binding members of
insufficient complementarity to provide for the desired
specificity. Stringent assay conditions are the summation or
combination (totality) of both hybridization and wash
conditions.
[0041] "Stringent hybridization conditions" and "stringent
hybridization wash conditions" in the context of nucleic acid
hybridization (e.g., as in array, Southern or Northern
hybridizations) are sequence dependent, and are different under
different experimental parameters. Stringent hybridization
conditions that can be used to identify nucleic acids within the
scope of the invention can include, e.g., hybridization in a buffer
comprising 50% formamide, 5.times.SSC, and 1% SDS at 42.degree. C.,
or hybridization in a buffer comprising 5.times.SSC and 1% SDS at
65.degree. C., both with a wash of 0.2.times.SSC and 0.1% SDS at
65.degree. C. Exemplary stringent hybridization conditions can also
include a hybridization in a buffer of 40% formamide, 1 M NaCl, and
1% SDS at 37.degree. C., and a wash in 1.times.SSC at 45.degree. C.
Alternatively, hybridization to filter-bound DNA in 0.5 M
NaHPO.sub.4, 7% sodium dodecyl sulfate (SDS), 1 mM EDTA at
65.degree. C., and washing in 0.1.times.SSC/0.1% SDS at 68.degree.
C. can be employed. Yet additional stringent hybridization
conditions include hybridization at 60.degree. C. or higher and
3.times.SSC (450 mM sodium chloride/45 mM sodium citrate) or
incubation at 42.degree. C. in a solution containing 30% formamide,
1M NaCl, 0.5% sodium sarcosine, 50 mM MES, pH 6.5. Those of
ordinary skill will readily recognize that alternative but
comparable hybridization and wash conditions can be utilized to
provide conditions of similar stringency. In certain embodiments,
the stringency of the wash conditions that set forth the conditions
which determine whether a nucleic acid is specifically hybridized
to a surface bound nucleic acid. Wash conditions used to identify
nucleic acids may include, e.g.: a salt concentration of about 0.02
molar at pH 7 and a temperature of at least about 50.degree. C. or
about 55.degree. C. to about 60.degree. C.; or, a salt
concentration of about 0.15 M NaCl at 72.degree. C. for about 15
minutes; or, a salt concentration of about 0.2.times.SSC at a
temperature of at least about 50.degree. C. or about 55.degree. C.
to about 60.degree. C. for about 15 to about 20 minutes; or, the
hybridization complex is washed twice with a solution with a salt
concentration of about 2.times.SSC containing 0.1% SDS at room
temperature for 15 minutes and then washed twice by 0.1.times.SSC
containing 0.1% SDS at 68.degree. C. for 15 minutes; or, equivalent
conditions. Stringent conditions for washing can also be, e.g.,
0.2.times.SSC/0.1% SDS at 42.degree. C.
[0042] A specific example of stringent assay conditions is rotating
hybridization at 65.degree. C. in a salt based hybridization buffer
with a total monovalent cation concentration of 1.5 M (e.g., as
described in U.S. patent application Ser. No. 09/655,482 filed on
Sep. 5, 2000, the disclosure of which is herein incorporated by
reference) followed by washes of 0.5.times.SSC and 0.1.times.SSC at
room temperature.
[0043] Stringent assay conditions are hybridization conditions that
are at least as stringent as the above representative conditions,
where a given set of conditions are considered to be at least as
stringent if substantially no additional binding complexes that
lack sufficient complementarity to provide for the desired
specificity are produced in the given set of conditions as compared
to the above specific conditions, where by "substantially no more"
is meant less than about 5-fold more, typically less than about
3-fold more. Other stringent hybridization conditions are known in
the art and may also be employed, as appropriate.
[0044] The resultant pattern of hybridized nucleic acid provides
information regarding expression for each of the genes that have
been probed, where the expression information is in terms of
whether or not the gene is expressed and, typically, at what level,
where the expression data, i.e., expression profile (e.g., in the
form of a transcriptosome), may be both qualitative and
quantitative.
[0045] Alternatively, non-array based methods for quantitating the
levels of one or more nucleic acids in a sample may be employed,
including quantitative PCR, and the like.
[0046] Where the expression profile is a protein expression
profile, any convenient protein quantitation protocol may be
employed, where the levels of one or more proteins in the assayed
sample are determined. Representative methods include, but are not
limited to: proteomic arrays, flow cytometry, standard immunoassays
(e.g., ELISA assays), etc.
[0047] Following obtainment of the expression profile from the
sample being assayed, the expression profile is compared with a
reference or control profile to determine the particular graft
tolerant/intolerant phenotype of the cell or tissue, and therefore
host, from which the sample was obtained/derived. The terms
"reference" and "control" as used herein mean a standardized
pattern of gene expression or levels of expression of certain genes
to be used to interpret the expression signature of a given patient
and assign a graft tolerant/intolerant phenotype thereto. The
reference or control profile may be a profile that is obtained from
a cell/tissue known to have the desired phenotype, e.g., tolerant
phenotype, and therefore may be a positive reference or control
profile. In addition, the reference/control profile may be from a
cell/tissue known to not have the desired phenotype, e.g., an
intolerant phenotype, and therefore be a negative reference/control
profile.
[0048] In certain embodiments, the obtained expression profile is
compared to a single reference/control profile to obtain
information regarding the phenotype of the cell/tissue being
assayed. In yet other embodiments, the obtained expression profile
is compared to two or more different reference/control profiles to
obtain more in depth information regarding the phenotype of the
assayed cell/tissue. For example, the obtained expression profile
may be compared to a positive and negative reference profile to
obtain confirmed information regarding whether the cell/tissue has
the phenotype of interest.
[0049] The comparison of the obtained expression profile and the
one or more reference/control profiles may be performed using any
convenient methodology, where a variety of methodologies are known
to those of skill in the array art, e.g., by comparing digital
images of the expression profiles, by comparing databases of
expression data, etc. Patents describing ways of comparing
expression profiles include, but are not limited to, U.S. Pat. Nos.
6,308,170 and 6,228,575, the disclosures of which are herein
incorporated by reference. Methods of comparing expression profiles
are also described above.
[0050] The comparison step results in information regarding how
similar or dissimilar the obtained expression profile is to the
control/reference profile(s), which similarity/dissimilarity
information is employed to determine the phenotype of the
cell/tissue being assayed. For example, similarity with a positive
control indicates that the assayed cell/tissue has a tolerant
phenotype. Likewise, similarity with a negative control indicates
that the assayed cell/tissue has an intolerant phenotype.
[0051] Depending on the type and nature of the reference/control
profile(s) to which the obtained expression profile is compared,
the above comparison step yields a variety of different types of
information regarding the cell/tissue that is assayed. As such, the
above comparison step can yield a positive/negative determination
of a tolerant phenotype of an assayed cell/tissue. In many
embodiments, the above-obtained information about the cell/tissue
being assayed is employed to diagnose a host, subject or patient
with respect to that host's graft tolerance, as described
above.
[0052] The subject methods further find use in pharmacogenomic
applications. In these applications, a subject/host/patient is
first diagnosed for the presence of absence of the graft tolerant
phenotype using a protocol such as the diagnostic protocol
described in the preceding section. The subject is then treated
using a protocol whose suitability is determined using the results
of the diagnosis step. More specifically, where the identified
phenotype is tolerant, a protocol that may include a reduced level
of immunosuppression (i.e., immunosuppression at a level less than
that which is indicated for patients not known to be graft
tolerant), or no immunosuppression, may be employed to manage/treat
the subject. Alternatively, where a patient is identified as having
an intolerant phenotype, full immunosuppressive protocols are then
employed/continued.
[0053] In many embodiments, a host is screened for the presence of
a graft tolerant phenotype following receipt of a graft or
transplant. The host may be screened once or serially following
transplant receipt, e.g., weekly, monthly, bimonthly, half-yearly,
yearly, etc., as long as the host is on immunosuppressive therapy.
In certain embodiments, monitoring of the host expression profile
even after immunosuppressive therapy has been reduced or
discontinued is conducted to determine whether the host has
maintained the tolerogenic expression profile and may continue for
the lifetime of the host.
Databases of Expression Profiles of Phenotype Determinative
Genes
[0054] Also provided are databases of expression profiles of graft
tolerant phenotype determinative genes. Such databases will
typically comprise expression profiles of various cells/tissues
having graft tolerant phenotypes, negative expression profiles,
etc., where such profiles are further described below.
[0055] The expression profiles and databases thereof may be
provided in a variety of media to facilitate their use. "Media"
refers to a manufacture that contains the expression profile
information of the present invention. The databases of the present
invention can be recorded on computer readable media, e.g. any
medium that can be read and accessed directly by a computer. Such
media include, but are not limited to: magnetic storage media, such
as floppy discs, hard disc storage medium, and magnetic tape;
optical storage media such as CD-ROM; electrical storage media such
as RAM and ROM; and hybrids of these categories such as
magnetic/optical storage media. One of skill in the art can readily
appreciate how any of the presently known computer readable mediums
can be used to create a manufacture comprising a recording of the
present database information. "Recorded" refers to a process for
storing information on computer readable medium, using any such
methods as known in the art. Any convenient data storage structure
may be chosen, based on the means used to access the stored
information. A variety of data processor programs and formats can
be used for storage, e.g. word processing text file, database
format, etc.
[0056] As used herein, "a computer-based system" refers to the
hardware means, software means, and data storage means used to
analyze the information of the present invention. The minimum
hardware of the computer-based systems of the present invention
comprises a central processing unit (CPU), input means, output
means, and data storage means. A skilled artisan can readily
appreciate that any one of the currently available computer-based
system are suitable for use in the present invention. The data
storage means may comprise any manufacture comprising a recording
of the present information as described above, or a memory access
means that can access such a manufacture.
[0057] A variety of structural formats for the input and output
means can be used to input and output the information in the
computer-based systems of the present invention. One format for an
output means ranks expression profiles possessing varying degrees
of similarity to a reference expression profile. Such presentation
provides a skilled artisan with a ranking of similarities and
identifies the degree of similarity contained in the test
expression profile.
Reagents and Kits
[0058] Also provided are reagents and kits thereof for practicing
one or more of the above-described methods. The subject reagents
and kits thereof may vary greatly. Reagents of interest include
reagents specifically designed for use in production of the
above-described expression profiles of phenotype determinative
genes, i.e., a gene expression evaluation element made up of one or
more reagents.
[0059] One type of such reagent is an array of probe nucleic acids
in which the phenotype determinative genes of interest are
represented. A variety of different array formats are known in the
art, with a wide variety of different probe structures, substrate
compositions and attachment technologies. Representative array
structures of interest include those described in U.S. Pat. Nos.:
5,143,854; 5,288,644; 5,324,633; 5,432,049; 5,470,710; 5,492,806;
5,503,980; 5,510,270; 5,525,464; 5,547,839; 5,580,732; 5,661,028;
5,800,992; the disclosures of which are herein incorporated by
reference; as well as WO 95/21265; WO 96/31622; WO 97/10365; WO
97/27317; EP 373 203; and EP 785 280.
[0060] In many embodiments, the arrays include probes for at least
1 of the genes listed in Tables 1, 2, 3 and/or 4. In certain
embodiments, the number of genes that are from Tables 1, 2, 3
and/or 4 that is represented on the array is at least 5, at least
10, at least 25, at least 50, at least 75 or more, including all of
the genes listed in Tables 1, 2, 3 and/or 4. The subject arrays may
include only those genes that are listed in Tables 1, 2, 3 and/or
4, or they may include additional genes that are not listed in
Tables 1, 2, 3 and/or 4. Where the subject arrays include probes
for such additional genes, in certain embodiments the number % of
additional genes that are represented does not exceed about 50%,
usually does not exceed about 25%. In many embodiments where
additional "non-Table 1" genes are included, a great majority of
genes in the collection are phenotype determinative genes, where by
great majority is meant at least about 75%, usually at least about
80% and sometimes at least about 85, 90, 95% or higher, including
embodiments where 100% of the genes in the collection are phenotype
determinative genes.
[0061] Another type of reagent that is specifically tailored for
generating expression profiles of phenotype determinative genes is
a collection of gene specific primers that is designed to
selectively amplify such genes. Gene specific primers and methods
for using the same are described in U.S. Pat. No. 5,994,076, the
disclosure of which is herein incorporated by reference. Of
particular interest are collections of gene specific primers that
have primers for at least 1 of the genes listed in one Tables 1, 2,
3 and/or 4, often a plurality of these genes, e.g., at least 2, 5,
10, 15 or more. In certain embodiments, the number of genes that
are from Tables 1, 2, 3 and/or 4 that have primers in the
collection is at least 5, at least 10, at least 25, at least 50, at
least 75 or more, including all of the genes listed in Tables 1, 2,
3 and/or 4. The subject gene specific primer collections may
include only those genes that are listed in Tables 1, 2, 3 and/or
4, or they may include primers for additional genes that are not
listed in Tables 1, 2, 3 and/or 4. Where the subject gene specific
primer collections include primers for such additional genes, in
certain embodiments the number % of additional genes that are
represented does not exceed about 50%, usually does not exceed
about 25%. In many embodiments where additional "non-Table 1",
"non-Table 2", "non-Table 3" or "non-Table 4" genes are included, a
great majority of genes in the collection are phenotype
determinative genes, where by great majority is meant at least
about 75%, usually at least about 80% and sometimes at least about
85, 90, 95% or higher, including embodiments where 100% of the
genes in the collection are phenotype determinative genes.
[0062] The kits of the subject invention may include the
above-described arrays and/or gene specific primer collections. The
kits may further include one or more additional reagents employed
in the various methods, such as primers for generating target
nucleic acids, dNTPs and/or rNTPs, which may be either premixed or
separate, one or more uniquely labeled dNTPs and/or rNTPs, such as
biotinylated or Cy3 or Cy5 tagged dNTPs, gold or silver particles
with different scattering spectra, or other post synthesis labeling
reagent, such as chemically active derivatives of fluorescent dyes,
enzymes, such as reverse transcriptases, DNA polymerases, RNA
polymerases, and the like, various buffer mediums, e.g.
hybridization and washing buffers, prefabricated probe arrays,
labeled probe purification reagents and components, like spin
columns, etc., signal generation and detection reagents, e.g.
streptavidin-alkaline phosphatase conjugate, chemifluorescent or
chemiluminescent substrate, and the like.
[0063] The subject kits may also include a phenotype determination
element, which element is, in many embodiments, a reference or
control expression profile that can be employed, e.g., by a
suitable computing means, to make a phenotype determination based
on an "input" expression profile, e.g., that has been determined
with the above described gene expression evaluation element.
Representative phenotype determination elements include databases
of expression profiles, e.g., reference or control profiles, as
described above.
[0064] In addition to the above components, the subject kits will
further include instructions for practicing the subject methods.
These instructions may be present in the subject kits in a variety
of forms, one or more of which may be present in the kit. One form
in which these instructions may be present is as printed
information on a suitable medium or substrate, e.g., a piece or
pieces of paper on which the information is printed, in the
packaging of the kit, in a package insert, etc. Yet another means
would be a computer readable medium, e.g., diskette, CD, etc., on
which the information has been recorded. Yet another means that may
be present is a website address which may be used via the internet
to access the information at a removed site. Any convenient means
may be present in the kits.
Systems
[0065] Also provided are systems for practicing one or more of the
above-described methods. The subject systems may vary greatly, but
typically include at least a gene expression evaluation element,
e.g., one or more reagents, and a phenotype determination
element.
[0066] Reagents of interest include reagents specifically designed
for use in production of the above-described expression profiles of
phenotype determinative genes, i.e., a gene expression evaluation
element made up of one or more reagents. One type of such reagent
is an array of probe nucleic acids in which the phenotype
determinative genes of interest are represented. A variety of
different array formats are known in the art, with a wide variety
of different probe structures, substrate compositions and
attachment technologies. Representative array structures of
interest include those described in U.S. Pat. Nos.: 5,143,854;
5,288,644; 5,324,633; 5,432,049; 5,470,710; 5,492,806; 5,503,980;
5,510,270; 5,525,464; 5,547,839; 5,580,732; 5,661,028; 5,800,992;
the disclosures of which are herein incorporated by reference; as
well as WO 95/21265; WO 96/31622; WO 97/10365; WO 97/27317; EP 373
203; and EP 785 280.
[0067] In many embodiments, the arrays include probes for at least
1 of the genes listed in Tables 1, 2, 3 and/or 4. In certain
embodiments, the number of genes that are from Tables 1, 2, 3
and/or 4 that is represented on the array is at least 5, at least
10, at least 25, at least 50, at least 75 or more, including all of
the genes listed in Tables 1, 2, 3 and/or 4. The subject arrays may
include only those genes that are listed in Tables 1, 2, 3 and/or
4, or they may include additional genes that are not listed in
Tables 1, 2, 3 and/or 4. Where the subject arrays include probes
for such additional genes, in certain embodiments the number % of
additional genes that are represented does not exceed about 50%,
usually does not exceed about 25%. In many embodiments where
additional "non-Table 1", "non-Table 2", "non-Table 3" or
"non-Table 4" genes are included, a great majority of genes in the
collection are phenotype determinative genes, where by great
majority is meant at least about 75%, usually at least about 80%
and sometimes at least about 85, 90, 95% or higher, including
embodiments where 100% of the genes in the collection are phenotype
determinative genes.
[0068] Another type of reagent that is specifically tailored for
generating expression profiles of phenotype determinative genes is
a collection of gene specific primers that is designed to
selectively amplify such genes. Gene specific primers and methods
for using the same are described in U.S. Pat. No. 5,994,076, the
disclosure of which is herein incorporated by reference. Of
particular interest are collections of gene specific primers that
have primers for at least 1 of the genes listed in one Tables 1, 2,
3 and/or 4, often a plurality of these genes, e.g., at least 2, 5,
10, 15 or more. In certain embodiments, the number of genes that
are from Tables 1, 2, 3 and/or 4 that have primers in the
collection is at least 5, at least 10, at least 25, at least 50, at
least 75 or more, including all of the genes listed in Tables 1, 2,
3 and/or 4. The subject gene specific primer collections may
include only those genes that are listed in Tables 1, 2, 3 and/or
4, or they may include primers for additional genes that are not
listed in Tables 1, 2, 3 and/or 4. Where the subject gene specific
primer collections include primers for such additional genes, in
certain embodiments the number % of additional genes that are
represented does not exceed about 50%, usually does not exceed
about 25%. In many embodiments where additional "non-Table 1",
"non-Table 2", "non-Table 3" or "non-Table 4" genes are included, a
great majority of genes in the collection are phenotype
determinative genes, where by great majority is meant at least
about 75%, usually at least about 80% and sometimes at least about
85, 90, 95% or higher, including embodiments where 100% of the
genes in the collection are phenotype determinative genes.
[0069] The systems of the subject invention may include the
above-described arrays and/or gene specific primer collections. The
systems may further include one or more additional reagents
employed in the various methods, such as primers for generating
target nucleic acids, dNTPs and/or rNTPs, which may be either
premixed or separate, one or more uniquely labeled dNTPs and/or
rNTPs, such as biotinylated or Cy3 or Cy5 tagged dNTPs, gold or
silver particles with different scattering spectra, or other post
synthesis labeling reagent, such as chemically active derivatives
of fluorescent dyes, enzymes, such as reverse transcriptases, DNA
polymerases, RNA polymerases, and the like, various buffer mediums,
e.g. hybridization and washing buffers, prefabricated probe arrays,
labeled probe purification reagents and components, like spin
columns, etc., signal generation and detection reagents, e.g.
streptavidin-alkaline phosphatase conjugate, chemifluorescent or
chemiluminescent substrate, and the like.
[0070] The systems may also include a phenotype determination
element, which element is, in many embodiments, a reference or
control expression profile that can be employed, e.g., by a
suitable computing means, to make a phenotype determination based
on an "input" expression profile, e.g., that has been determined
with the above described gene expression evaluation element.
Representative phenotype determination elements include databases
of expression profiles, e.g., reference or control profiles, as
described above.
[0071] The following examples are offered by way of illustration
and not by way of limitation.
Experimental
I. Methods and Materials
Patient Selection
[0072] The protocol was accepted by the University Hospital Ethical
Committee and the Committee for the Protection of Patients from
Biological Risks. All patients and normal individuals were informed
of the protocol and signed an informed consent. IRB approval was
also obtained to perform DNA microarrays on all patient samples at
Stanford University. The 42 adult participants in this study were
classified into four groups for comparison: operationally tolerant
(OT), chronic rejection (CR), stable, or normal.
[0073] Operationally tolerant patients were kidney graft recipients
with stable graft function who had not taken any immunosuppressive
drugs for at least three years (N=5) with a mean drug free time
period was 8.+-.3.3 years [range 3-12]. Eleven additional patients
who received low doses (.ltoreq.10 mg/day) of Prednisone.RTM. or
Cortancyl.RTM. as the only immunosuppressive drug for at least 3
years before analysis were also classified Op-Tol (or OT). The mean
time period of immunosuppression was 9.8.+-.4.4 years [range 4-15].
The renal function of drug-free tolerant patients and minimally
immunosuppressed patients remained stable and blood creatinemia was
not significantly different before and after immunosuppression
interruption or modification.
[0074] Patients with chronic rejection, patients with stable
function under immunosuppressive maintenance regimen and normal
healthy individuals are used as control groups. The chronic
rejection group included patients with histological chronic
rejection lesions (CR group) associated or not to an allograft
glomerulopathy and with a degradation of their renal function.
Inclusion was evidenced by clinical indices indicative of end-stage
renal disease requiring hemodialysis (N=3) or serum creatine and
proteinuria >1.5 g/day and creatinemia >60 .mu.mol and by
evidence of rejection on kidney biopsies. The stable function group
included patients with stable graft function and under a bitherapy
immunosuppressive regimen including a calcineurin inhibitor
(StaCNI+) or not (StaCNI-). The inclusion of patients with stable
function under standard IS required all following criteria: a)
Patients over 18 years old; b) received kidney transplant between 5
to 8 years; c) patients without acute rejection history; d)
patients with good immunosuppressive treatment compliance (as
judged by medication intake and medical survey); e) patients under
standard dual immunosuppressive maintenance treatment including:
CsA or FK506 and mycophenolate mofetil, azathioprine or
corticosteroids; f) stable kidney function (i.e. .+-.25% of the
mean value of the year that preceded the test) and with a Nankivell
calculated clearance (*please provide reference) above 40 ml/min
after at least five year of transplantation and at one and three
months after the inclusion in the study; g) proteinuria <1.5 g
/day and creatinemia <160 umol after at least five year of
transplantation and at one and three months after the inclusion in
the study; h) blood trough-levels within the International standard
range (75-250 ng/ml) for Neoral and 5-10 ng/ml for Prograf). Normal
individuals included healthy individuals were 25 to 60 years old
(N=3) with a normal blood formula and no known infectious pathology
for at least 6 months prior to the study.
RNA Extraction from Whole Samples
[0075] Blood, harvested in EDTA tubes, was obtained from a
peripheral vein or arterio-venous fistula. Ionogramm, WBC, formula
and CNI blood levels (RIA) were performed on each sample.
Peripheral blood leukocytes (PBL) were separated on a Ficoll layer
(Eurobio, Les Ulis, France) and frozen in Trizol reagent
(Invitrogen, Life Technologies, San Diego, Calif.) for RNA
extraction.
Microarray Hybridization and Data Analysis
[0076] The cDNA microarrays were processed using protocols as
previously described by our laboratory using 2 .mu.g RNA in each
channel (Sarwal, et al., N. Engl. J. Med. (2003) 349: 125-138). A
"common reference" RNA pool (Perou, et al., Nature (2000)
406:747-752) was used as an internal standard with each
hybridization. Sample or reference RNA was subjected to two
successive rounds of amplification before hybridization to the cDNA
microarrays using a protocol essentially as described by Wang et
al. (Wang, et al., Nat. Biotechn. (2000) 18:457-459). Hybridized
microarrays were scanned using GenePix 4000 (Axon Instruments,
Union City, Calif.) and fluorescent images were analyzed with the
GenePix Pro software package. Each microarray contains
approximately 30,000 cDNAs representing over 12,400 unique
genes.
[0077] Array data for the 42 samples were stored in the Stanford
Microarray database (Sherlock, et al., Nuc. Acids Res.
(2001)29:152-155) and gene lists filtered at retrieval. The
hybridized raw data of 30,000 cDNA spots was manually examined for
single spots or blemishes (Alizadeh et al., Cold Spring Harbor
Symp. Quant. Biol. (1999) 64:71-78). These spots or belmishes were
flagged and removed, generating a data file with 22,090 clones
under low stringency (70% representative data and signal/noise
ratio of expression measurements >1.5). Further high-stringency
data filtering, choosing only clones with 90% representative data
and a 4-fold expression level difference at >2 arrays in the
study set generated a file of 2,083 cDNA clones representing 1,846
unique genes. This higher-stringency gene set was further analyzed
for potential biomarkers in this study and in a study of the T-cell
expression profiles from the same patient groups. Cluster (V3.0)
generated hierarchical clusters of the samples measuring similarity
of expression of the genes and similarity across arrays which are
visualized with the TreeView program (Eisen et al., Proc. Nat'l
Acad. Sci USA (1998) 95:14863-8); (See also the website found by
positioning "http://www." in front of "microarrays.org/software."
Significance of gene expression differences among the groups of
samples was performed using
[0078] Statistical Analysis of Microarray (SAM) (Tusher et al.,
Proc. Nat'l Acad. Sci USA (2001) 98:5116-21) using a false
discovery rate threshold of <5%.
Taqman-Based Quantitative RT-PCR
[0079] One hundred nanograms of amplified RNA were subjected to
real-time PCR analysis to assess quantization of marker genes
identified in the microarray screen. Assay-on-demand primer sets
and Taqman EZ RT-PCR core reagents (Applied Biosystems, Foster
City, Calif.) were used according to manufacturer's directions in
20 .mu.L reaction volumes using the 7900HT Sequence Detection
System. Transcript levels were calculated according to the
2-.DELTA..DELTA.Ct method of the manufacturer (1997). The labeled
TaqMan.RTM. probes (Assays-on-demand, Applied Biosystems) used for
these assays include:
TABLE-US-00001 .beta.-Actin: (SEQ ID NO: 01)
5'FAM-TCGCCTTTGCCGATCCGCCGCCCGT-NFQ3'; TK1: (SEQ ID NO: 02)
5'FAM-ACACATGACCGGAACACCATGGAGG-NFQ3'; C1S: (SEQ ID NO: 03)
5'FAM-TTGCCACAGACATAAATGAATGCAC-NFQ3'; EV12A: (SEQ ID NO: 04)
5'FAM-AAGCATTTTGTAAGATTGCCAAGTA-NFQ3'; MMP2: (SEQ ID NO: 05)
5'FAM-GCAGGGCGGCGGTCACAGCTACTTC-NFQ3'; MAPK9: (SEQ ID NO: 06)
5'FAM-GATTGTTTGTGCTGCATTTGATACA-NFQ3'; TCEB3: (SEQ ID NO: 07)
5'FAM-AGACATTCTTGCGGAGACTGGGGTT-NFQ3'; KIFC3: (SEQ ID NO: 08)
5'FAM-CAAGGCCGAGATAGGCCAGGCCATC-NFQ3'; PRAME: (SEQ ID NO: 09)
5'FAM-CGTTTGTGGGGTTCCATTCAGAGCC-NFQ3'; DLK1: (SEQ ID NO: 10)
5'FAM-GATCAACGGCTCCCCCTGCCAGCAC-NFQ3'; CYR61: (SEQ ID NO: 11)
5'FAM-CTGCAGAGCTCAGTCAGAGGGCAGA-NFQ3'; HPRT: (SEQ ID NO: 12)
5'FAM-GGTCAAGGTCGCAAGCTTGCTGGTG-NFQ3'; GAPDH: (SEQ ID NO: 13)
5'FAM-GGGCGCCTGGTCACCAGGGCTGCTT-NFQ3'.
II. Results
[0080] We compared gene expression profiles from RNA extracted
using unfractionated peripheral blood samples from 16 operationally
tolerant (OT) patients (4 without any treatment, 12 with low dose
steroid monotherapy) with that from 11 patients with signs of
chronic rejection (CR), 12 transplant patients with stable function
(S), and from three normal controls (N; healthy un-grafted
individuals). Unsupervised analysis revealed that the majority of
the CR and OT samples segregate into separate groups. In addition,
all of the normal samples and most of the stable samples were
observed to cluster with the CR patient groups. A recent reference
study of whole gene expression profiling using the same cDNA array
platform employed in this study, Whitney et al. (Whitney et al.,
Proc. Nat'l Acad. Sci. USA (2003) 100:1896-901) made at least two
key discoveries that assisted in the interpretation of our
expression results. First, blood-based gene expression profiles
from the same individual were quite stable over time; second, genes
from a common cell origin in PBL samples tend to tightly cluster as
evidenced by correlation analysis between cell counts and
expression measurements.
[0081] Separation of the sample groups was primarily driven by
three gene clusters identified as A, B and C with a large
percentage of the genes expressed at higher levels in the blood
from OT patients than others. By identifying cell signaling genes
tightly clustered to ones of limited tissue expression, one can
then infer the putative cell origin, and immune activation status
of the primary clusters. In addition, genes might be highly
differentially expressed between the two phenotypes of interest, OT
and CR in this study.
[0082] Because the OT and CR patients largely group into separate
branches in the unsupervised expression analysis, close examination
of the observed gene clusters provided insight into potential
functional roles for the major genes involved in the tolerance
signature. Both pro- and anti-apoptotic genes were identified as
highly differentially expressed between OT and CR patients.
Expression patterns of the anti-apoptotic genes TOSO, CASP7 and
FIAM2 in Cluster A parallel both T- and B-cell specific genes
including the T-cell receptor (TCR), HLA class II genes, several
immunoglobulin genes (IGL, IG1 L1, IGJ3, IGHM). This cluster also
contained a number of key cytokines that play a role in the immune
signaling: (CCL20, CCL19, CCL8 and CCR7). This cluster also
contained the T-cell activation antigen CD27, which serves as a
regulator of B-cell activation is the receptor for the
pro-apoptotic CD27-binding protein (SIVA). In contrast, the
pro-apoptosis genes RGN and RIS1 co-clustered with platelet and
T-suppressor cell specific genes CD9 and GFBB2 respectively. It is
this cluster, Cluster C, which contained a number of immune
response genes (C1 s, IER3, and CD59) and several cell cycle
regulated genes (TK1, CCNA2, CDC20, CCND2, CCNA2, CCND1, MCM2 and
CDC6) and GAGE4, an antigen of unknown function that is recognized
by cytotoxic T-cells. IER3 (intermediate early response 3)
functions to protect cells from Fas- or tumor necrosis factor
(TNF.alpha.)-induced apoptosis. CD59, also known as protectin
(MIC1), functions to restrict lysis of erythrocytes and leukocytes
by homologous complement and is involved in signal transduction for
T-cell activation. CD59 interacts with T-cell surface antigen CD2,
a gene also highly differentially expressed in this dataset and
found to co-cluster in Cluster B with NK4, CD5, CD6, CD81, CD8A,
granulysin, and granzyme C and B. Additional NK-specific genes
segregated with this gene group include NKG7 and ICAM3. Thus, both
cytotoxic T-cell antigens and NK-cell specific genes are expressed
in parallel and characterized this cluster. The NK-cell specific
cluster also had elevated expression of IL-2 regulated genes
including CTSW. Together, the expression data shows that at least 3
populations of circulating leukocytes are involved in the tolerance
signature identified: increased expression of T- and B-cell genes
and anti-apoptotic genes serving a protective role (Cluster A);
T-cells expression NK antigens with elevated expression of NK4,
NKG7 and cytotoxic signaling chemokines (Cluster B); and a rapidly
proliferating platelet or suppressor T-cell population modulated by
the pro-apoptotic genes RGN and RIS1 (Cluster C).
Supervised Statistical Analysis of the Expression Profiles
[0083] Of the 2,083 highly differentially expressed genes in this
study, 550 were found to show significant differences in expression
between the OT and CR patient groups using the SAM statistical
analysis tool (Tusher, et al. supra) (q<5%). In contrast, no
significant differences were observed within the 4 tolerant
patients comparing those without any immunosuppressive drug vs. 12
tolerant patients under minimal immunosuppression (steroid
monotherapy) (minimum false discovery rate >16%). This
observation showed that the expression patterns were sufficiently
similar to group all the tolerant patients together in class
prediction analysis and collectively consider them "operationally
tolerant". Far more up-regulated genes are evident in the genes
highly correlated with the OT phenotype: 523 genes were
up-regulated and only 27 down-regulated and these genes clustered
the patient groups in a similar manner as the unsupervised
analysis. This observation showed that the state of operational
tolerance in these patients is not a passive process but is
actively regulated. Further, the expression profiles showed that
the state of operational tolerance involves active transcription
across several hundred genes in concert in peripheral blood cells.
This mechanism is consistent with the observation that a much
larger number of differentially expressed genes were initially
identified using the SAM analysis tool when 22,897 clones were
retrieved from the SMD database with stringency filtering (70%
representative data and signal/noise ratio >1.5). From this
larger gene set, a total of 3,125 cDNA clones representing
.about.1,820 genes were identified in the full gene list with SAM
significance score <5% (data not shown).
[0084] To identify genes whose expression differed in OT and CR
patients from healthy control individuals, each gene was zero
transformed to the average expression level in the 3 normal PBL
samples and supervised analysis with SAM repeated. In this
normalized dataset, 591 significant genes were identified, all of
which are at higher levels in the OT samples than CR (clustered
data not shown).
[0085] The differentially expressed genes that segregate the OT and
CR patient groups include several interesting candidate genes
involved in immune signaling cascades including specific
transcription factors, cell adhesion molecules, cell cycle-specific
genes, inhibitors of inflammation/alloresponse, growth factors, and
genes with important roles in vascular biology. This subset is
provided in Table 2. Each of these gene families are represented by
multiple group members in the identified group of 591 significantly
differentially expressed cDNAs as are B- and T-cell specific genes,
apoptotic gene family members, protein kinases, several cell
matrix/structural proteins as well as other functional categories.
Expression fold differences between OT and CR patients of
representative members of these gene families are summarized in
Table 3. For the remainder of the study, we analyzed both the
normalized data (zero transformed) and clustered data to identify
putative biomarkers predictive of tolerance.
Class Prediction Analysis
[0086] PAM or Predictive Analysis of Microarray data (Tibshirani et
al., Proc. Nat'l Acad. Sci. USA (2002) 99:6567-72) was used to
identify those genes with highest predictive value in identifying a
tolerogenic state in the blood samples. This program identifies a
minimum gene set characteristic of user defined sample groups (the
learning set) and then scores both known and unknown samples based
on similarity to identified expression profile differences.
Normalized expression ratios for OT and CR groups were used as a
learning set and classification scores for the 12 stable patients
determined by PAM analysis using threshold log-ratio difference of
>1.5 (3-fold expression difference between OT and CR (Table 3).
Although there was substantial overlap in the identified genes, the
30 top ranked genes from PAM class prediction analysis using
zero-transformed data (and raw expression values) differ in how
they cluster the samples. The latter dataset segregate OT and CR
samples into separate branches that are in agreement with computed
phenotype goodness-of-fit scores reported by the PAM class
prediction program.
[0087] With the objective of further refining the gene set into a
manageable number of genes for PCR-based analysis, the array data
was analyzed by logistic regression. Class prediction using
logistic regression modeling requires that markers independently
correlate with the test phenotype which initially proved
challenging with the array data due to the extremely tight
clustering of many of the highest ranked genes. In all analysis
performed, the expression of thymidine kinase 1 (TK1) showed the
greatest difference comparing OT vs. CR (T-test p<0.00001) and
highest expression in the tolerant patients. More importantly, the
expression of several hundred genes positively correlated with TK1.
For example, expression of 102 genes correlated with TK1 with
R2>0.7 and 398 genes with R2>0.5 and, at least on the basis
of the array data, these tightly correlating genes consistently had
the highest significance scores. We therefore selected four
candidate genes from the PAM class prediction set, two of which
positively correlate with the tolerant phenotype (TK1 and C1s) and
two of which negatively correlated (EV12A and MAPK9).
Classification based on array data for these four genes give 95.8%
concordant classification and 4.2% discordant classification within
the test groups (Table 3). Remarkably, array-based expression
ratios of TK1 alone yield 94.5% concordant classification and 4.2%
discordant classification (see the web supplement for full output
on the logistic regression and expression ratios for the test
groups). In addition, the expression of TK1 also correlates with
prediction scores generated by both PAM and logistic regression;
R2>0.85 for logistic regression scores and R2>0.80 for PAM
scores. Only 2 of the 39 samples classify discordantly between the
two methods (5% discordance): Chronic patient #88 that
PAM/microarray data profiles as consistent with the tolerant
phenotype and Stable patient #89 which classifies as CR by PAM and
OT by logistic regression. The overall expression profile of Stable
patient #89 is sufficiently distinct from the tolerant patients
that it failed to co-cluster with this group in all three sets of
genes analyzed.
Confirmation of Gene Signature with Taqman PCR
[0088] Real-time quantitative RT-PCR was used to confirm expression
difference between OT and CR patient groups using .beta.-actin as a
reference gene. RNA was available for this analysis from 8 of the
OT patients, 11 CR patients and all 12 stable post-transplant
patients and residual amplified cDNA prepared for the array
expression study was used as the template source. In the small
sample group analyzed, the expression of EV12A was the only gene
tested to reach statistical significance. However, a significant
correlation between array and PCR results were seen for EV12A, TK1
and C1s and average expression of MAPK9 was higher in the CR
patients than OT, also in agreement with the array-based data. To
determine whether all four markers combined would provide greater
statistical power, logistic regression modeling was used to analyze
the PCR data. The overall best the differentiation between OT and
CR groups is obtained a cut-off of 0.02 using a curve described by
the following algorithm (p<0.00005):
1/(1+exp -[10.2373+17.2638(MPK9)+2.7098 (EV12A)-4.8491
(C1S)-10.1332(TK1)])
where gene names are the fold-difference measurements at each
locus. The logistic regression model from these data had correct
prediction of phenotype of 72.2% (sensitivity=75%,
specificity=66.7%).
[0089] TK1 expression by quantitative RT-PCR was still the most
informative of the genes in this analysis, although more
inter-individual variation was observed in the Taqman results and
hence this marker alone failed to reach the predictive power of the
array data. However, we did find that the combined expression
profiles of TK1, MAPK9, EV12A, and C1s, when analyzed by logistic
regression, were in complete agreement with PAM prediction scoring
of the OT and CR patients tested. One of the patients originally
classified as tolerant at the time of the study (Patient #T/C04)
was found to have evidence of chronic injury as evidenced by the
detection of proteinuria. By including the data from this patient
in the CR group of the learning set and repeating the analysis only
using expression measurements on the TK1 locus, cutoffs at 0.10 and
at 0.59 the logistic regression model have correct prediction
probability of 83.3% (sensitivity 100%, specificity 50%) and 83.3%
(sensitivity 91.7% and specificity 66.7%), respectively. Thus, a
cutoff of 0.59 would maximize both the sensitivity and specificity
in the classification by TK1 alone by logistic regression and this
model also gave better fit than the 4-gene profile. However,
agreement among the stable patients between the two test measures
(PAM class prediction of global gene expression profiles vs.
logistic regression analysis of RT-PCR data) was not as conclusive
in that we found consistent classification between the two test
methods in 9 of 12 stable patients (75%). Higher concordance within
the OT and CR patients (100%) might be expected because these are
the learning set in the analyses and the stable patients the test
set (75% concordance). Together these data indicated that the
blood-based expression signature of the tolerogenic state is subtle
and a larger panel of genes, such as the 30 gene sets identified by
PAM class prediction, may be beneficial in certain embodiments for
accurate identification of the tolerance in a diagnostic
setting.
[0090] Additional genes of interest are found in Table 4.
TABLE-US-00002 TABLE 4 Accession Gene Description NM_001003927
EVI2A ecotropic viral integration site 2A NM_005601 NKG7 NATURAL
KILLER CELL GROUP 7 SEQUENCE M38690 CD9 LEUKOCYTE ANTIGEN MIC3
U25931 CD27 TUMOR NECROSIS FACTOR RECEPTOR SUPERFAMILY NM_014009
FOXP3 forkhead box P3 NM_000953 DP1 prostaglandin D2 receptor
III. Discussion and Conclusions
[0091] Identification of blood-based biomarkers with predictive
power in directing clinical outcomes is an active area of clinical
research. We used global gene expression monitoring to test whether
blood-based markers diagnostic of spontaneously achieved tolerance
could be identified in a cohort of long-term renal transplant
patients. By normalizing to the average expression levels in normal
control blood samples, several functional groups of genes were
identified to be at significantly higher levels in the tolerant
patients, where the identified genes included genes characteristic
of rapid cell proliferation, anti-apoptotic activity and
senescence, as well as genes involved in immune signaling cascades
including specific transcription factors, cell adhesion molecules.
Overall the data shows that the spontaneously achieved state of
immune tolerance is an active regulatory process as evidenced by
the large numbers of up-regulated genes relative to normal blood
and by the highly synchronized expression signature that is
observed. Further, this signature was observed in the blood from
.about.25% of long-term stable graft recipients showing that
expression monitoring could become an invaluable method in
realizing the goal of predictably reducing long-term
immunosuppressive therapy when merited. The gene expression
signatures were sufficiently strong that two independent
classification methods, logistic regression and PAM analysis, gave
similar predictive power (.about.90% overall) with differences in
predicted phenotype for only 3 of 39 participants in this pilot
study. Hierarchal clustering of the top 30 genes scored by PAM
separated all patients classified tolerant and from those with
chronic renal injury or graft rejection with 100% concordance to
the logistic regression scoring. Identification of a large number
of differentially expressed genes reported in this study of
spontaneously tolerant adult renal transplant patients provides
several biomarkers for spontaneously achieved immune tolerance. The
gene expression study reported here shows that expression
differences characteristic of spontaneously achieved tolerance can
be detected in whole blood lysates obviating the need for more
invasive methods of sampling.
[0092] It is evident that subject invention provides a convenient
and effective way of determining whether a subject has a graft
tolerant phenotype, without first removing the subject from
immunosuppressive therapy. As such, the subject invention provides
a number of distinct benefits, including the ability to easily
identify subjects undergoing immunosuppressive therapy that have a
graft tolerant phenotype, and therefore may be removed from
immunosuppressive therapy, so that these individuals can avoid the
adverse conditions, as well as costs, associated with such therapy.
As such, the subject invention represents a significant
contribution to the art.
[0093] All publications and patent applications cited in this
specification are herein incorporated by reference as if each
individual publication or patent application were specifically and
individually indicated to be incorporated by reference. The
citation of any publication is for its disclosure prior to the
filing date and should not be construed as an admission that the
present invention is not entitled to antedate such publication by
virtue of prior invention.
[0094] Although the foregoing invention has been described in some
detail by way of illustration and example for purposes of clarity
of understanding, it is readily apparent to those of ordinary skill
in the art in light of the teachings of this invention that certain
changes and modifications may be made thereto without departing
from the spirit or scope of the appended claims.
Sequence CWU 1
1
13125DNAhuman 1tcgcctttgc cgatccgccg cccgt 25225DNAhuman
2acacatgacc ggaacaccat ggagg 25325DNAhuman 3ttgccacaga cataaatgaa
tgcac 25425DNAhuman 4aagcattttg taagattgcc aagta 25525DNAhuman
5gcagggcggc ggtcacagct acttc 25625DNAhuman 6gattgtttgt gctgcatttg
ataca 25725DNAhuman 7agacattctt gcggagactg gggtt 25825DNAhuman
8caaggccgag ataggccagg ccatc 25925DNAhuman 9cgtttgtggg gttccattca
gagcc 251025DNAhuman 10gatcaacggc tccccctgcc agcac 251125DNAhuman
11ctgcagagct cagtcagagg gcaga 251225DNAhuman 12ggtcaaggtc
gcaagcttgc tggtg 251325DNAhuman 13gggcgcctgg tcaccagggc tgctt
25
* * * * *
References