U.S. patent application number 11/041164 was filed with the patent office on 2005-10-20 for antisense oligomers and methods for inducing immune tolerance and immunosuppression.
Invention is credited to Iversen, Patrick L., Mourich, Dan V..
Application Number | 20050234002 11/041164 |
Document ID | / |
Family ID | 34826000 |
Filed Date | 2005-10-20 |
United States Patent
Application |
20050234002 |
Kind Code |
A1 |
Mourich, Dan V. ; et
al. |
October 20, 2005 |
Antisense oligomers and methods for inducing immune tolerance and
immunosuppression
Abstract
A method and composition for inducing human dendritic cells to a
condition of reduced capacity for antigen-specific activation of T
cells, and, in mature dendritic cells, increased production of
extracellular IL-10 is disclosed. A population of dendritic cells
is exposed to a substantially uncharged antisense compound
containing 12-40 subunits and a base sequence effective to
hybridize to an expression-sensitive region of a preprocessed or
processed human CD86 transcript identified, in its processed form,
by SEQ ID NO:33, to form a duplex structure between said compound
and transcript having a Tm of at least 45.degree. C. Formation of
the duplex blocks expression of full-length CD86 in said cells,
which in turn leads to reduced capacity for antigen-specific
activation of T cells, and, in mature dendritic cells, increased
production of extracellular IL-10.
Inventors: |
Mourich, Dan V.; (Albany,
OR) ; Iversen, Patrick L.; (Corvallis, OR) |
Correspondence
Address: |
PERKINS COIE LLP
P.O. BOX 2168
MENLO PARK
CA
94026
US
|
Family ID: |
34826000 |
Appl. No.: |
11/041164 |
Filed: |
January 21, 2005 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
60538655 |
Jan 23, 2004 |
|
|
|
Current U.S.
Class: |
514/44A ; 514/81;
514/85 |
Current CPC
Class: |
C12N 2310/3513 20130101;
C12N 2310/3233 20130101; A61K 31/675 20130101; C12N 15/1138
20130101; C12N 2310/11 20130101 |
Class at
Publication: |
514/044 ;
514/081; 514/085 |
International
Class: |
A61K 048/00; A61K
031/675 |
Claims
It is claimed:
1. A method of inducing human dendritic cells to a condition of
reduced capacity for antigen-specific activation of T cells, and,
in mature dendritic cells, increased production of extracellular
IL-10, comprising (a) exposing a population of human dendritic
cells to a substantially uncharged antisense compound containing
12-40 subunits and a base sequence effective to hybridize to an
expression-sensitive region of a preprocessed or processed human
CD86 transcript identified, in its processed form, by SEQ ID NO:33,
to form a heteroduplex structure between said compound and
transcript having a Tm of at least 45.degree. C., (b) by said
forming, blocking expression of full-length CD86 in said cells, and
(c) by said blocking, producing inhibition of the expression of
full-length CD86 on the surface of dendritic cells, and enhanced
expression of extracellular IL-10 by mature dendritic cells.
2. The method of claim 1, wherein the antisense compound to which
the dendritic cells are exposed is composed of
phosphorus-containing intersubunit linkages joining a morpholino
nitrogen of one subunit to a 5' exocyclic carbon of an adjacent
subunit.
3. The method of claim 2, wherein the morpholino subunits in the
compound to which the dendritic cells are exposed are joined by
phosphorodiamidate linkages, in accordance with the structure:
2where Y.sub.1.dbd.O, Z.dbd.O, Pj is a purine or pyrimidine
base-pairing moiety effective to bind, by base-specific hydrogen
bonding, to a base in a polynucleotide, and X is alkyl, alkoxy,
thioalkoxy, amino or alkyl amino, and said heteroduplex structure
formed in step (a) has a Tm of at least 50.degree. C.
4. The method of claim 3, wherein X.dbd.NR.sub.2, where each R is
independently hydrogen or methyl in the compound to which the T
cells are exposed.
5. The method of claim 1, wherein the dendritic cells which are
exposed to said compound include mature dendritic cells, and said
blocking step (b) is effective to enhance expression of
extracellular IL-10 by the dendritic cells.
6. The method of claim 1, wherein said compound is covalently
linked, at one compound end, to an arginine-rich peptide effective
to enhance uptake of said compound into the dendritic cells.
7. The method of claim 6, wherein said arginine-rich peptide has a
sequence selected from the group consisting of SEQ ID NOS: 1 and
2.
8. The method of claim 6, wherein said dendritic cells which are
exposed to said compound include a mixture of immature and mature
dendritic cells, said arginine-rich peptide is an rTAT peptide
having the sequence identified by SEQ ID NO: 1, and the rTAT
peptide is effective to achieve a greater level of intracellular
uptake of the antisense compound into the mature dendritic cells
than is achieved (i) in the immature dendritic cells, or (ii) by
exposing the mature dendritic cells to the antisense compound in
the absence of the rTAT polypeptide.
9. The method of claim 1, wherein said antisense compound is
effective to hybridize to a target region adjacent the start site
of the processed human CD86 transcript, the compound has a base
sequence that is complementary to a target region containing at
least 12 contiguous bases in a processed human CD86 transcript
identified by SEQ ID NO:9, and said blocking step (b) is effective
to block translation of said processed transcript.
10. The method of claim 9, wherein the antisense compound includes
a base sequence selected from the group consisting of: SEQ ID
NOS:21-23 and 32.
11. The method of claim 1, wherein the antisense compound is
effective to hybridize to a splice site of preprocessed human CD68,
and has a base sequence that is complementary to at least 12
contiguous bases of a splice site in a preprocessed human CD86
transcript, and said blocking step (b) is effective to block
processing of a preprocessed CD86 transcript to produce a
full-length, processed CD86 transcript.
12. The method of claim 11, wherein the splice site in the
preprocessed CD86 transcript has a sequence selected from the group
consisting of: SEQ ID NOS:10-14.
13. The method of claim 12, wherein the antisense compound includes
a base sequence selected from the group consisting of: SEQ ID
NOS:24-28.
14. The method of claim 1, for use in inhibiting transplantation
rejection in a human subject receiving an allograft tissue or
organ, wherein said exposing includes administering the antisense
compound to the subject, in an amount effective to inhibit the rate
and extent of rejection of the transplant.
15. The method of claim 14, wherein said administering is carried
out both prior to and following the allograft tissue or organ
transplantation in the subject.
16. The method of claim 14, wherein said administering is carried
out for a selected period of 1-3 weeks.
17. The method of claim 16, which further includes further
administering the antisense compound to the subject, as needed, to
control the extent of transplantation rejection in the subject.
18. The method of claim 1, for use in treating an autoimmune
condition in a human subject, wherein said exposing includes
administering the antisense compound to the subject, in an amount
effective to reduce the severity of the autoimmune condition.
19. The method of claim 18, wherein said administering is carried
out over an extended period of time, as needed, to control the
severity of the autoimmune condition in the subject.
20. A method of inducing mature human dendritic cells to a
condition of increased production of extracellular IL-10,
comprising (a) exposing the population of cells containing mature
dendritic cells to a substantially uncharged antisense compound
containing 12-40 subunits and a base sequence effective to
hybridize to an expression-sensitive region of a preprocessed or
processed human CD-86 transcript identified, in its processed form,
by SEQ ID NO:33, to form a heteroduplex structure between said
compound and transcript having a Tm of at least 45.degree. C., (b)
by said forming, blocking expression of full-length CD86 in said
cells, and (c) by said blocking, enhancing expression of
extracellular IL-10 by the mature dendritic cells.
21. A composition for use in inducing dendritic cells to a
condition of reduced capacity for antigen-specific activation of T
cells, and, in mature dendritic cells, increased production of
extracellular IL-10, said conjugate comprising, a substantially
uncharged antisense compound containing 12-40 subunits and a base
sequence effective to hybridize to an expression-sensitive region
of a preprocessed or processed human CD-86 transcript identified,
in its processed form, by SEQ ID NO:33, to form a heteroduplex
structure between said compound and transcript having a Tm of at
least 45.degree. C.
22. The composition of claim 21, wherein the antisense compound is
composed of phosphorus-containing intersubunit linkages joining a
morpholino nitrogen of one subunit to a 5' exocyclic carbon of an
adjacent subunit.
23. The composition of claim 22, wherein the morpholino subunits in
the compound to which the dendritic cells are exposed are joined by
phosphorodiamidate linkages, in accordance with the structure:
3where Y.sub.1.dbd.O, Z.dbd.O, Pj is a purine or pyrimidine
base-pairing moiety effective to bind, by base-specific hydrogen
bonding, to a base in a polynucleotide, and X is alkyl, alkoxy,
thioalkoxy, amino or alkyl amino, and said heteroduplex structure
has a Tm of at least 50.degree. C.
24. The composition of claim 27, wherein X.dbd.NR.sub.2, where each
R is independently hydrogen or methyl in the compound to which the
T cells are exposed.
25. The composition of claim 21, which further includes an
arginine-rich peptide covalently linked to one end of said
antisense compound, and said peptide is effective to promote uptake
of the composition into dendritic cells.
26. The composition of claim 25, wherein said arginine-rich peptide
is an rTAT peptide having the sequence identified as SEQ ID NO: 1,
and said peptide is effective to achieve a greater level of
intracellular uptake of the antisense compound into the mature
dendritic cells than is achieved (i) in the immature dendritic
cells, or (ii) by exposing the mature dendritic cells to the
antisense compound in the absence of the rTAT polypeptide.
Description
[0001] This application claims priority to U.S. Provisional
Application No. 60,538,655, filed Jan. 23, 2004, which is
incorporated herein by reference in its entirety.
FIELD OF THE INVENTION
[0002] The present invention relates to compounds and methods of
inducing immunological tolerance using a peptide-antisense
conjugate to selectively limit costimulation of nave T-cells by
mature dendritic cells and formation of a cytokine microenvironment
that augments tolerized T-cells.
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BACKGROUND OF THE INVENTION
[0032] Transplantation of allogeneic donor cells, tissues or organs
(transplantation between genetically different individuals of the
same species) is used to treat a variety of conditions (typically
tissue, or organ-failure conditions) and is often the sole or
highly preferred therapeutic option. The list of successfully
transplanted cells, tissues and organs includes kidney, heart,
lung, liver, corneas, pancreas, marrow, skin, and bones. However,
allogeneic transplantation involves significant risks and
drawbacks, including graft rejection, complications from
immunosuppressive therapy and graft-versus host disease which are
frequently highly debilitating or lethal.
[0033] Rejection of allografts is presently understood to be
initiated by the recognition of allogeneic (i.e. donor) major
histocompatibility complex (MHC) molecules by recipient
T-lymphocytes, leading to upregulated cellular and humoral immunity
through activation of T cells. The MHC antigens are typically
presented to the recipient T-lymphocytes by antigen presenting
cells, such as macrophages and dendritic cells. Although
immunosuppressive drugs such as cyclosporine may be used in an
attempt to modulate rejection, these immunosuppressive agents have
severe side effects and often fail to prevent continued rejection
episodes.
[0034] Dendritric cells (DCs) are a family of professional antigen
presenting cells (APCs) that are present in virtually all tissues
of the body. The ability of dendritic cells to capture foreign
antigens, migrate to lymphoid tissues and redistribute antigen-MHC
to the cell surface along with appropriate costimulatory signals
are well known T-cell priming functions for these APCs. In addition
to these immunostimulatory properties, dendritic cells are also
known to play a role in down-regulating immune responses. Certain
subpopulations of dendritic cells, acting as professional APCs,
also maintain and regulate T-cell tolerance in the periphery. There
is thus a need for therapeutic methods and compositions capable of
inducing immunological tolerance with lower toxicity and improved
efficacy.
SUMMARY OF THE INVENTION
[0035] The invention includes, in one aspect, a method of inducing
human dendritic cells to a condition of reduced capacity for
antigen-specific activation of T cells, and, in mature dendritic
cells, increased production of extracellular IL-10. The method
includes exposing a population of human dendritic cells to a
substantially uncharged antisense compound containing 12-40
subunits and a base sequence effective to hybridize to an
expression-sensitive region of a preprocessed or processed human
CD-86 transcript identified, in its processed form, by SEQ ID
NO:33, to form, between the compound and transcript, a heteroduplex
structure having a Tm of at least 45.degree. C. The heteroduplex
formation blocks expression of full-length CD86 in the cells, which
in turn, produces inhibition of the expression of full-length CD86
on the surface of dendritic cells, and produces enhanced expression
of extracellular IL-10 by mature dendritic cells.
[0036] In a preferred embodiment, the antisense compound to which
the dendritic cells are exposed is composed of
phosphorus-containing intersubunit linkages joining a morpholino
nitrogen of one subunit to a 5' exocyclic carbon of an adjacent
subunit. In an exemplary compound, the morpholino subunits in the
compound are joined by phosphorodiamidate linkages, in accordance
with the structure: 1
[0037] where Y.sub.1.dbd.O, Z.dbd.O, Pj is a purine or pyrimidine
base-pairing moiety effective to bind, by base-specific hydrogen
bonding, to a base in a polynucleotide, and X is alkyl, alkoxy,
thioalkoxy, amino or alkyl amino, and the heteroduplex structure
formed has a Tm of at least 50.degree. C. For example, X=NR.sub.2,
where each R is independently hydrogen or methyl in the compound to
which the dendritic cells are exposed.
[0038] The compound may be covalently linked, at one compound end,
to an arginine-rich peptide effective to enhance uptake of the
compound into the dendritic cells. Exemplary arginine-rich peptides
are those having the sequences SEQ ID NOS: 1 or 2. Where the
dendritic cells include a mixture of immature and mature dendritic
cells, the arginine-rich peptide may be an rTAT peptide having the
sequence identified by SEQ ID NO: 1. This peptide is effective to
achieve a greater level of intracellular uptake of the antisense
compound into the mature dendritic cells than is achieved (i) in
the immature dendritic cells, or (ii) by exposing the mature
dendritic cells to the antisense compound in the absence of the
rTAT polypeptide.
[0039] More generally, the rTAT peptide may be coupled to any
antisense or other therapeutic compound to achieve selective uptake
of the compound into mature dendritic cells, relative to uptake in
immature cells.
[0040] Where the antisense compound is effective to hybridize to an
expression-sensitive target region adjacent the start site of the
processed human CD86 transcript, the compound may have a base
sequence that is complementary to a target region containing at
least 12 contiguous bases in a processed human CD86 transcript
identified by SEQ ID NO:9, where the compound is effective to block
translation of the processed transcript. The antisense compound may
have, for example, one of the base sequence identified by SEQ ID
NOS:21-23 and 32.
[0041] Where the antisense compound is effective to hybridize to a
splice site of preprocessed human CD86, the compound may have a
base sequence that is complementary to at least 12 contiguous bases
of a splice site in a preprocessed human CD86 transcript, where the
compound is effective to block processing of a preprocessed CD86
transcript to produce a full-length, processed CD 86 transcript.
The splice site in the preprocessed CD86 transcript may have one of
the sequences identified by SEQ ID NOS:10-14. The antisense
compound may have, for example, one of the base sequences
identified by SEQ ID NOS:24-28.
[0042] For use in inhibiting transplantation rejection in a human
subject receiving an allograft tissue or organ, the compound is
administered to the subject in an amount effective to inhibit the
rate and extent of rejection of the transplant. The compound may be
administered both prior to and following the allograft tissue or
organ transplantation in the subject, and compound administration
may be carried out for a selected period of 1-3 weeks. The compound
may be further administered to the subject, as needed, to control
the extent of transplantation rejection in the subject.
[0043] For use in treating an autoimmune condition in a human
subject, the compound may be administered to the subject, in an
amount effective to reduce the severity of the autoimmune
condition. The compound may be administered over an extended period
of time, as needed, to control the severity of the autoimmune
condition in the subject.
[0044] In another aspect, the invention provides a composition for
use in inducing dendritic cells to a condition of reduced capacity
for antigen-specific activation of T cells, and, in mature
dendritic cells, increased production of extracellular IL-10. The
compound comprises a substantially uncharged antisense compound
containing 12-40 subunits and a base sequence effective to
hybridize to an expression-sensitive region of a preprocessed or
processed human CD-86 transcript identified, in its processed form,
by SEQ ID NO:33, to form a heteroduplex structure between said
compound and transcript having a Tm of at least 45.degree. C.
Exemplary features of the compound are as described above.
[0045] These and other objects and features of the invention will
become more fully apparent when the following detailed description
of the invention is read in conjunction with the accompanying
drawings.
BRIEF DESCRIPTION OF THE FIGURES
[0046] FIG. 1A-D show several preferred morpholino-type subunits
having 5-atom (A), six-atom (B) and seven-atom (C-D) linking groups
suitable for forming polymers.
[0047] FIGS. 2A-D show the repeating subunit segment of exemplary
morpholino oligonucleotides, designated A through D, constructed
using subunits A-D, respectively, of FIG. 1.
[0048] FIGS. 3A-3G show examples of uncharged linkage types in
oligonucleotide analogs.
[0049] FIG. 4 shows the chemical structures of a phosphorodiamidate
morpholino oligomer conjugates and the sequences of peptide
conjugates used in this invention. Fluorescein can be linked to the
3' end of the peptide-PMO conjugate to allow imaging and/or
detection of PMO uptake in intact cells.
[0050] FIG. 5 shows a fluorescence activated cell sorting (FACS)
analysis of the uptake of fluorescein-labeled peptide-PMO
conjugates into dendritic cells subjected to lipopolysaccharide
(LPS) activation.
[0051] FIG. 6 shows antisense PMO to CD86 inhibits expression of
both CD86 and CD80 in dendritic cells.
[0052] FIG. 7 demonstrates that blocking CD86 interactions does not
lead to IL-10 induction in dendritic cells.
[0053] FIGS. 8A and 8B shows that antisense PMO targeting of splice
donor or acceptor sites alters CD86 mRNA.
[0054] FIG. 9 demonstrates that antisense PMO targeted to the CD86
start codon or Exon 10 of the CD86 gene alters the morphology of
the lipopolysaccharide-treated dendritic cells.
DETAILED DESCRIPTION OF THE INVENTION
[0055] I. Definitions
[0056] The terms below, as used herein, have the following
meanings, unless indicated otherwise.
[0057] The terms "CD80" and "CD86" refer to the costimulatory
protein molecules that are expressed on the surface of mature,
antigen presenting dendritic cells. T cell activation is dependent
upon signals delivered through the antigen-specific T cell receptor
and accessory costimulatory receptors on the T cell. The CD28
costimulatory receptor is constitutively expressed on T cells.
Engagement of CD28 on nave T cells by either CD80 or CD86 ligands
on antigen presenting cells (e.g. mature dendritic cells) provides
a potent costimulatory signal to T cells activated through their T
cell receptor. The CD80 and CD86 costimulatory molecules are also
known as B7-1 and B7-2, respectively. The term "B7 molecules" refer
collectively to the CD80 and CD86 molecules.
[0058] The term "antigen-activated T cells" refers to T cells that
become activated after the T cell receptor (TCR) complex and a
co-stimulatory receptor (e.g. CD28 on nave CD4 and CD8 T cells) are
engaged to the extent that a signal transduction cascade is
initiated. Antigen is bound by the TCR in the form of a foreign
peptide in the context of a self MHC molecule, either Class I or
Class II, in the case of CD4 and CD8 T cells respectively,
conferring the antigen specificity of the T cell. Upon activation,
T cells will proliferate and then secrete cytokines or carry out
cytolysis on cells expressing the foreign peptide with self MHC.
Cytokines are growth factors for other T cells or signals for B
cells to produce antibody.
[0059] The term "antigen-activated B cells" refer to either of two
different types of B cell activation, T cell dependent and T cell
independent. T cell independent antigens contain repetitive
identical epitopes and are capable of clustering membrane bound
antibody on the surface of the B cell which can result in
delivering activation signals. T cell dependent activation is in
response to protein antigens where the B cell acts as a
professional antigen presenting cell. Surface antibody bound to
antigen is internalized by the B cell, the antigen processed and
presented as peptides on the B cell surface bound to MHC II
molecules. Responding T cells recognize the peptide as foreign in
the context of self MHC and respond by secreting cytokines and
expression of CD40L. Together these provide a co-stimulatory signal
to the B cell. In either case of B cell activation the cell will
proliferate and differentiate into plasma B cells capable of
secreting antibodies against the antigen.
[0060] The terms "activated dendritic cells" and "mature dendritic
cells" (DCs) refer to professional antigen-presenting cells (APCs)
capable of expressing both MHC class I and II and co-stimulatory
molecules including CD80 (B7-1) and CD86 (B7-2). Two different DC
phenotypes are exhibited depending on maturation state and location
in the body. Immature DCs reside in all tissues and organs as
active phagocytic cells. Mature DCs traffic to secondary lymphoid
organs (e.g. lymph node and spleen) and present peptides derived
from processed protein antigens to T cells in the context of MHC
molecules. Mature DCs also provide the necessary co-stimulatory
signals to T cells by expressing the appropriate surface ligand
(e.g. CD80 and CD86 on DCs bind to CD28 on T cells).
[0061] The terms "antisense oligonucleotides," "antisense
oligomer," and "antisense compound" are used interchangeably and
refer to a compound having a sequence of nucleotide bases and a
subunit-to-subunit backbone that allows the antisense oligomer to
hybridize to a target sequence in an RNA by Watson-Crick base
pairing, to form an RNA:oligomer heterduplex within the target
sequence. The antisense oligonucleotide includes a sequence of
purine and pyrimidine heterocyclic bases, supported by a backbone,
which are effective to hydrogen-bond to corresponding, contiguous
bases in a target nucleic acid sequence. The backbone is composed
of subunit backbone moieties supporting the purine and pyrimidine
heterocyclic bases at positions that allow such hydrogen bonding.
These backbone moieties are cyclic moieties of 5 to 7 atoms in
length, linked together by phosphorous-containing linkages one to
three atoms long.
[0062] A "morpholino" oligonucleotide refers to a polymeric
molecule having a backbone which supports bases capable of hydrogen
bonding to typical polynucleotides, wherein the polymer lacks a
pentose sugar backbone moiety, and more specifically a ribose
backbone linked by phosphodiester bonds which is typical of
nucleotides and nucleosides, but instead contains a ring nitrogen
with coupling through the ring nitrogen. A preferred "morpholino"
oligonucleotide is composed of morpholino subunit structures of the
form shown in FIG. 1A-1D, where (i) the structures are linked
together by phosphorous-containing linkages, one to three atoms
long, joining the morpholino nitrogen of one subunit to the 5'
exocyclic carbon of an adjacent subunit, and (ii) Pi and Pj are
purine or pyrimidine base-pairing moieties effective to bind, by
base-specific hydrogen bonding, to a base in a polynucleotide.
Exemplary structures for antisense oligonucleotides for use in the
invention include the morpholino subunit types shown in FIGS.
1A-1D, with the uncharged, phosphorous-containing linkages shown in
FIGS. 2A-2D, and more generally, the uncharged linkages 3A-3G.
[0063] As used herein, an oligonucleotide or antisense oligomer
"specifically hybridizes" to a target polynucleotide if the
oligomer hybridizes to the target under physiological conditions,
with a thermal melting point (Tm) substantially greater than
37.degree. C., preferably at least 45.degree. C., and typically
50.degree. C.-80.degree. C. or higher. Such hybridization
preferably corresponds to stringent hybridization conditions,
selected to be about 10.degree. C., and preferably about 50.degree.
C. lower than the Tm for the specific sequence at a defined ionic
strength and pH. At a given ionic strength and pH, the Tm is the
temperature at which 50% of a target sequence hybridizes to a
complementary polynucleotide.
[0064] Polynucleotides are described as "complementary" to one
another when hybridization occurs in an antiparallel configuration
between two single-stranded polynucleotides. A double-stranded
polynucleotide can be "complementary" to another polynucleotide, if
hybridization can occur between one of the strands of the first
polynucleotide and the second. Complementarity (the degree that one
polynucleotide is complementary with another) is quantifiable in
terms of the proportion of bases in opposing strands that are
expected to form hydrogen bonds with each other, according to
generally accepted base-pairing rules. An antisense compound may be
complementary to a target region of a target transcript even if the
two bases sequences are not 100% complementary, as long as the
heteroduplex structure formed between the compound and transcript
has the desired Tm stability.
[0065] As used herein the term "analog" with reference to an
oligomer means a substance possessing both structural and chemical
properties similar to those of the reference oligomer.
[0066] As used herein, a first sequence is an "antisense sequence"
or "targeting sequence" with respect to a second sequence or
"target sequence" if a polynucleotide whose sequence is the first
sequence specifically binds to, or specifically hybridizes with,
the second polynucleotide sequence under physiological
conditions.
[0067] As used herein, "effective amount" relative to an antisense
oligomer refers to the amount of antisense oligomer administered to
a subject, either as a single dose or as part of a series of doses,
that is effective to inhibit expression of a selected target
nucleic acid sequence.
[0068] As used herein, an "expression-sensitive region" of a
processed or preprocessed mRNA transcript refers to either (i) a
region including or adjacent the AUG start site of a processed
transcript, where formation of an antisense-transcript heteroduplex
is effective to inhibit translation of the transcript or (ii) a
region including or adjacent a donor or acceptor splice site
junction in a preprocessed transcript, where formation of an
antisense-transcript heteroduplex is effective to inhibit formation
of a full-length processed transcript, either because one or more
exons that would normally be included in the transcript have been
deleted or because the transcript has been truncated at the target
splice site.
[0069] "Dendritic cells" are specialized antigen presenting cells
(APCS) with potent capacity to initiate and direct the
antigen-specific responses of nave T cells. This heterogeneous
population of cells reside in blood and all tissues as two
phenotypically and functionally distinct forms. "Immature DCs" are
highly phagocytic, proficient for antigen processing and
characterized by low-level expression of major histocompatibility
complex (MHC) class II and T cell costimulatory ligands of the B7
family, CD80 and CD86. Maturation can be triggered by various
stimuli derived from either host or pathogen. In response to such
stimuli, "mature DCs" cease phagocytic activity and significantly
increase surface expression of MHC II, CD80 and CD86. Consequently
mature DCs are capable of providing sufficient ligand to trigger T
cell activation through the T cell and costimulatory receptors.
[0070] Abbreviations:
[0071] PMO=phosphorodiamidate morpholino oligomer
[0072] AICD=activation induced cell death
[0073] MHC=major histocompatibility
[0074] TCR=T cell receptor
[0075] DC=dendritic cell
[0076] APC=antigen presenting cell
[0077] II. The Role of Antigen Presenting Cells in Transplantation
and Autoimmune Disorders
[0078] There is evidence from the two signal model for T cell
activation that costimulation by the engagement of dendritic-cell
CD86 molecules with CD28 on T cells is necessary for the complete
induction of T cell responses. The process of antigen presentation
whereby MHC plus peptide antigen are presented by the dendritic
cell to the T cell receptor (TCR) is termed "signal 1". In
circumstances where there is either insufficient or an absence of
costimulation (termed "signal 2") during the process of antigen
presentation, antigen specific tolerance can occur.
[0079] Tolerance produced by a loss in signal 2 can be a result of
clonal deletion (T cell death in the population of cells
recognizing signal 1), anergy (non-responsiveness in the
antigen-specific population on subsequent encounters) or through
the generation of a regulatory population of antigen-specific T
cells. Regulatory T cells can be induced through the presentation
of antigen by immature dendritic cells which express reduced levels
of CD86 compared to mature dendritic cells. Regulatory T cells
provide a form of functional tolerance whereby they produce
inhibitory cytokines (IL-4, IL-10, and TGF-beta) when encountering
antigen and thus inhibit the organism from responding to the
antigen (i.e., immuno-suppression). It has been shown that the
process of generating T regulatory cells can be further facilitated
by providing a third signal ("signal 3") that is the type of
cytokines produced by the antigen presenting cell while providing
signal 1 and 2.
[0080] IL-10, previously termed "cytokine synthesis inhibiting
factor" due to its ability to inhibit cytokine production of most
immune cell types, can provide a signal 3 to T cells. During the
early stages of T cell activation, upon responding to antigen, the
cytokines produced by the dendritic cell presenting antigen will
promote the resulting phenotype of the responding T cell. IL-12 and
IL-4 produced by dendritic cells promote T cell responses of the
Th1 and Th2 phenotypes, respectively. The T cell types in turn
direct the production of cytotoxic T cells or antibodies,
respectively, which are effector cells and molecules capable of
rejecting transplants or producing autoimmune disease. Th3 T cells
exhibit a regulatory phenotype that directly inhibits the
development of any future T cell responses from becoming either a
Th1 or Th2 cell capable of responding to the antigen recognized by
the Th3 cell or the newly responding T cells. Dendritic cells
producing IL-10 can thus induce a tolergenic response by diverting
the development of T cell responses from Th1 and Th2 to a Th3 T
cell type.
[0081] The present invention provides a means to precisely and
specifically alter the manner in which dendritic cells elicit
antigen-specific immune responses from T cells. In particular a
diminution in the level of CD86 protein is achieved by antisense
inhibition targeted to dendritic cells. Experiments conducted in
support of the invention demonstrated that maturing DCs produce
increased amounts of IL-10 as a result of diminished CD86
expression. Moreover, it was determined that the cytoplasmic region
encoded by exon 10 is functionally linked to the regulation of this
cytokine.
[0082] III. Antisense Compound for Targeting Activated Immune
Cells
[0083] A. Antisense Compound
[0084] Antisense oligomers for use in practicing the invention
preferably have the properties: (1) a backbone that is
substantially uncharged, (2) the ability to hybridize with the
complementary sequence of a target RNA with high affinity, that is
a Tm substantially greater than 37.degree. C., preferably at least
45.degree. C., and typically greater than 50.degree. C., e.g.,
60.degree. C.-80.degree. C. or higher, (3) a subunit length of at
least 8 bases, generally about 8-40 bases, preferably 12-25 bases,
and (4) nuclease resistance. In addition, the antisense compound
may have the capability for active or facilitated transport as
evidenced by (i) competitive binding with a phosphorothioate
antisense oligomer, and/or (ii) the ability to transport a
detectable reporter into target cells.
[0085] Candidate antisense oligomers may be evaluated, according to
well known methods, for acute and chronic cellular toxicity, such
as the effect on protein and DNA synthesis as measured via
incorporation of 3H-leucine and 3H-thymidine, respectively. In
addition, various control oligonucleotides, e.g., control
oligonucleotides such as sense, nonsense or scrambled antisense
sequences, or sequences containing mismatched bases, in order to
confirm the specificity of binding of candidate antisense
oligomers. The outcome of such tests is important in discerning
specific effects of antisense inhibition of gene expression from
indiscriminate suppression. Accordingly, sequences may be modified
as needed to limit non-specific binding of antisense oligomers to
non-target nucleic acid sequences.
[0086] Heteroduplex formation. The effectiveness of a given
antisense oligomer molecule in forming a heteroduplex with the
target mRNA may be determined by screening methods known in the
art. For example, the oligomer is incubated in a cell culture
containing an mRNA preferentially expressed in activated
lymphocytes, and the effect on the target mRNA is evaluated by
monitoring the presence or absence of (1) heteroduplex formation
with the target sequence and non-target sequences using procedures
known to those of skill in the art, (2) the amount of the target
mRNA expressed by activated lymphocytes, as determined by standard
techniques such as RT-PCR or Northern blot, (3) the amount of
protein transcribed from the target mRNA, as determined by standard
techniques such as ELISA or Western blotting. (See, for example,
(Pari, Field et al. 1995; Anderson, Fox et al. 1996). For the
purposes of the invention, a preferred test for the effectiveness
of the CD86 antisense oligomer is by measuring the induction of
IL-10 expression and loss of CD86 expression in mature dendritic
cells treated with a CD86 PMO antisense compound.
[0087] Uptake into cells. A second test measures cell transport, by
examining the ability of the test compound to transport a labeled
reporter, e.g., a fluorescence reporter, into cells. The cells are
incubated in the presence of labeled test compound, added at a
final concentration between about 10-300 nM. After incubation for
30-120 minutes, the cells are examined, e.g., by microscopy or FACS
analysis, for intracellular label. The presence of significant
intracellular label is evidence that the test compound is
transported by facilitated or active transport.
[0088] In one embodiment of the invention, uptake into cells is
enhanced by administering the antisense compound in combination
with an arginine-rich peptide linked to the 5' or 3' end of the
antisense oligomer. The peptide is typically 8-16 amino acids and
consists of a mixture of arginine, and other amino acids including
phenylalanine and cysteine, as discussed further below.
[0089] RNAse resistance. Two general mechanisms have been proposed
to account for inhibition of expression by antisense
oligonucleotides (Agrawal, Mayrand et al. 1990; Bonham, Brown et
al. 1995; Boudvillain, Guerin et al. 1997). In the first, a
heteroduplex formed between the oligonucleotide and the viral RNA
acts as a substrate for RNaseH, leading to cleavage of the RNA.
Oligonucleotides belonging, or proposed to belong, to this class
include phosphorothioates, phosphotriesters, and phosphodiesters
(unmodified "natural" oligonucleotides). Such compounds expose the
RNA in an oligomer:RNA duplex structure to hydrolysis by RNaseH,
and therefore loss of function.
[0090] A second class of oligonucleotide analogs, termed "steric
blockers" or, alternatively, "RNaseH inactive" or "RNaseH
resistant", have not been observed to act as a substrate for
RNaseH, and act by sterically blocking target RNA nucleocytoplasmic
transport, splicing, translation, or replication. This class
includes methylphosphonates (Toulme, Tinevez et al. 1996),
morpholino oligonucleotides, peptide nucleic acids (PNA's), certain
2'-O-allyl or 2'-O-alkyl modified oligonucleotides (Bonham, Brown
et al. 1995), and N3'.fwdarw.P5' phosphoramidates (Ding, Grayaznov
et al. 1996; Gee, Robbins et al. 1998).
[0091] A test oligomer can be assayed for its RNaseH resistance by
forming an RNA:oligomer duplex with the test compound, then
incubating the duplex with RNaseH under a standard assay
conditions, as described (Stein, Foster et al. 1997). After
exposure to RNaseH, the presence or absence of intact duplex can be
monitored by gel electrophoresis or mass spectrometry.
[0092] In vivo uptake. In accordance with another aspect of the
invention, there is provided a simple, rapid test for confirming
that a given antisense oligomer type provides the required
characteristics noted above, namely, high Tm, ability to be
actively taken up by the host cells, and substantial resistance to
RNaseH. This method is based on the discovery that a properly
designed antisense compound will form a stable heteroduplex with
the complementary portion of the RNA target when administered to a
mammalian subject, and the heteroduplex subsequently appears in the
urine (or other body fluid). Details of this method are also given
in co-owned U.S. Pat. No. 6,365,351 for "Non-Invasive Method for
Detecting Target RNA," the disclosure of which is incorporated
herein by reference.
[0093] Briefly, a test oligomer containing a backbone to be
evaluated, having a base sequence targeted against a known RNA, is
injected into a mammalian subject. The antisense oligomer may be
directed against any intracellular RNA, including RNA encoded by a
host gene. Several hours (typically 8-72) after administration, the
urine is assayed for the presence of the antisense-RNA
heteroduplex. If heteroduplex is detected, the backbone is suitable
for use in the antisense oligomers of the present invention.
[0094] The test oligomer may be labeled, e.g. by a fluorescent or a
radioactive tag, to facilitate subsequent analyses, if it is
appropriate for the mammalian subject. The assay can be in any
suitable solid-phase or fluid format. Generally, a solid-phase
assay involves first binding the heteroduplex analyte to a
solid-phase support, e.g., particles or a polymer or test-strip
substrate, and detecting the presence/amount of heteroduplex bound.
In a fluid-phase assay, the analyte sample is typically pretreated
to remove interfering sample components. If the oligomer is
labeled, the presence of the heteroduplex is confirmed by detecting
the label tags. For non-labeled compounds, the heteroduplex may be
detected by immunoassay if in solid phase format or by mass
spectroscopy or other known methods if in solution or suspension
format.
[0095] Structural features. As detailed above, the antisense
oligomer has a base sequence directed to a targeted portion of a
cellular gene, preferably the region at or adjacent the start codon
or a processed transcript or a region at or adjacent a splice site
junction of the CD86 mRNA or preprocessed transcript. In addition,
the oligomer is able to effectively inhibit expression of the
targeted gene when administered to a host cell, e.g. in a mammalian
subject. This requirement is met when the oligomer compound (a) has
the ability to be taken up by dendritic cells and (b) once taken
up, form a duplex with the target RNA with a Tm greater than about
45.degree. C., preferably greater than 50.degree. C.
[0096] The ability to be taken up selectively by activated immune
cells requires, in part, that the oligomer backbone be
substantially uncharged. The ability of the oligomer to form a
stable duplex with the target RNA will depend on the oligomer
backbone, the length and degree of complementarity of the antisense
oligomer with respect to the target, the ratio of G:C to A:T base
matches, and the positions of any mismatched bases. The ability of
the antisense oligomer to resist cellular nucleases promotes
survival and ultimate delivery of the agent to the cell
cytoplasm.
[0097] Antisense oligonucleotides of 15-20 bases are generally long
enough to have one complementary sequence in the mammalian genome.
In addition, antisense compounds having a length of at least 12,
typically at least 15 nucleotides in length hybridize well with
their target mRNA. Due to their hydrophobicity, antisense
oligonucleotides tend to interact well with phospholipid membranes,
and it has been suggested that following the interaction with the
cellular plasma membrane, oligonucleotides are actively transported
into living cells (Loke, Stein et al. 1989; Yakubov, Deeva et al.
1989; Anderson, Xiong et al. 1999).
[0098] Morpholino oligonucleotides, particularly phosphoramidate-
or phosphorodiamidate-linked morpholino oligonucleotides have been
shown to have high binding affinities for complementary or
near-complementary nucleic acids. Morpholino oligomers also exhibit
little or no non-specific antisense activity, afford good water
solubility, are immune to nucleases, and are designed to have low
production costs (Summerton and Weller 1997).
[0099] Morpholino oligonucleotides (including antisense oligomers)
are detailed, for example, in co-owned U.S. Pat. Nos. 5,698,685,
5,217,866, 5,142,047, 5,034,506, 5,166,315, 5,185, 444, 5,521,063,
and 5,506,337, all of which are expressly incorporated by reference
herein In one preferred approach, antisense oligomers for use in
practicing the invention are composed of morpholino subunits of the
form shown in the above cited patents, where (i) the morpholino
groups are linked together by uncharged linkages, one to three
atoms long, joining the morpholino nitrogen of one subunit to the
5' exocyclic carbon of an adjacent subunit, and (ii) the base
attached to the morpholino group is a purine or pyrimidine
base-pairing moiety effective to bind, by base-specific hydrogen
bonding, to a base in a polynucleotide. The purine or pyrimidine
base-pairing moiety is typically adenine, cytosine, guanine, uracil
or thymine. Preparation of such oligomers is described in detail in
U.S. Pat. No. 5,185,444 (Summerton et al., 1993), which is hereby
incorporated by reference in its entirety. As shown in this
reference, several types of nonionic linkages may be used to
construct a morpholino backbone.
[0100] Exemplary subunit structures for antisense oligonucleotides
of the invention include the morpholino subunit types shown in
FIGS. 1A-D, each linked by an uncharged, phosphorous-containing
subunit linkage, as shown in FIGS. 2A-2D, respectively. In these
figures, the X moiety pendant from the phosphorous may be any of
the following: fluorine; an alkyl or substituted alkyl; an alkoxy
or substituted alkoxy; a thioalkoxy or substituted thioalkoxy; or,
an unsubstituted, monosubstituted, or disubstituted nitrogen,
including cyclic structures. Alkyl, alkoxy and thioalkoxy
preferably include 1-6 carbon atoms, and more preferably 1-4 carbon
atoms. Monosubstituted or disubstituted nitrogen preferably refers
to lower alkyl substitution, and the cyclic structures are
preferably 5- to 7-membered nitrogen heterocycles optionally
containing 1-2 additional heteroatoms selected from oxygen,
nitrogen, and sulfur. Z is sulfur or oxygen, and is preferably
oxygen.
[0101] FIG. 1A shows a phosphorous-containing linkage which forms
the five atom repeating-unit backbone shown in FIG. 2A, where the
morpholino rings are linked by a 1-atom phosphoamide linkage.
Subunit B in FIG. 1B is designed for 6-atom repeating-unit
backbones, as shown in FIG. 2B. In FIG. 1B, the atom Y linking the
5' morpholino carbon to the phosphorous group may be sulfur,
nitrogen, carbon or, preferably, oxygen. The X moiety pendant from
the phosphorous may be any of the following: fluorine; an alkyl or
substituted alkyl; an alkoxy or substituted alkoxy; a thioalkoxy or
substituted thioalkoxy; or, an unsubstituted, monosubstituted, or
disubstituted nitrogen, including cyclic structures. Z is sulfur or
oxygen, and is preferably oxygen. Particularly preferred morpholino
oligonucleotides include those composed of morpholino subunit
structures of the form shown in FIG. 2B, where X is an amine or
alkyl amine of the form X.dbd.NR.sub.2, where R is independently H
or CH.sub.3, that is where X.dbd.NH.sub.2, X.dbd.NHCH.sub.3 or
X.dbd.N(CH.sub.3).sub.2- , Y.dbd.O, and Z.dbd.O.
[0102] Subunits C-D in FIGS. 1C-D are designed for 7-atom
unit-length backbones as shown for structures in FIGS. 2C and D. In
Structure C, the X moiety is as in Structure B, and the moiety Y
may be methylene, sulfur, or preferably oxygen. In Structure D, the
X and Y moieties are as in Structure B. In all subunits depicted in
FIGS. 1 and 2, each Pi and Pj is a purine or pyrimidine
base-pairing moiety effective to bind, by base-specific hydrogen
bonding, to a base in a polynucleotide, and is preferably selected
from adenine, cytosine, guanine and uracil.
[0103] As noted above, the substantially uncharged oligomer may
advantageously include a limited number of charged linkages, e.g.
up to about 1 per every 5 uncharged linkages. In the case of the
morpholino oligomers, such a charged linkage may be a linkage as
represented by any of FIGS. 2A-D, preferably FIG. 2B, where X is
oxide (--O--) or sulfide (--S--).
[0104] More generally, the morpholino oligomers with uncharged
backbones are shown in FIGS. 3A-3G. Especially preferred is a
substantially uncharged morpholino oligomer such as illustrated by
the phosphorodiamidate morpholino oligomer (PMO) shown in FIG. 3G.
It will be appreciated that a substantially uncharged backbone may
include one or more, e.g., up to 10-20% of charged intersubunit
linkages, typically negatively charged phosphorous linkages.
[0105] Antisense sequence. In the methods of the invention, the
antisense oligomer is designed to hybridize to a region of the
target nucleic acid sequence, under physiological conditions with a
Tm substantially greater than 37.degree. C., e.g., at least
45.degree. C. and preferably 60.degree. C.-80.degree. C., wherein
the target nucleic acid sequence is preferentially expressed in
activated lymphocytes. The oligomer is designed to have
high-binding affinity to the target nucleic acid sequence and may
be 100% complementary thereto, or may include mismatches, e.g., to
accommodate allelic variants, as long as the heteroduplex formed
between the oligomer and the target nucleic acid sequence is
sufficiently stable to withstand the action of cellular nucleases
and other modes of degradation during its transit from cell to body
fluid. Mismatches, if present, are less destabilizing toward the
end regions of the hybrid duplex than in the middle. The number of
mismatches allowed will depend on the length of the oligomer, the
percentage of G:C base pair in the duplex and the position of the
mismatch(es) in the duplex, according to well understood principles
of duplex stability.
[0106] Although such an antisense oligomer is not necessarily 100%
complementary to a nucleic acid sequence that is preferentially
expressed in mature dendritic cells, it is effective to stably and
specifically bind to the target sequence such that expression of
the target sequence is modulated. The appropriate length of the
oligomer to allow stable, effective binding combined with good
specificity is about 8-40 nucleotide base units, and preferably
about 12-25 nucleotides. Oligomer bases that allow degenerate base
pairing with target bases are also contemplated, assuming base-pair
specificity with the target is maintained. mRNA transcribed from
the relevant region of a gene associated with CD86 expression is
generally targeted by the antisense oligonucleotides for use in
practicing the invention, however, in some cases double-stranded
DNA may be targeted using a non-ionic probe designed for
sequence-specific binding to major-groove sites in duplex DNA. Such
probe types are described in U.S. Pat. No. 5,166,315 (Summerton et
al., 1992), which is hereby incorporated by reference, and are
generally referred to herein as antisense oligomers, referring to
their ability to block expression of target genes.
[0107] The antisense compound is targeted against an
expression-sensitive region of a processed or preprocessed CD
transcript, that is, a region which, when bound to the antisense
compound, is effective to inhibit the expression of full-length
CD86 in dendritic cells. In one general embodiment, the
expression-sensitive region is one that includes or is adjacent the
AUG start site of a processed transcript, where formation of an
antisense-transcript heteroduplex is effective to inhibit
translation of the transcript. Here the antisense compound has a
base sequence that is complementary to a target region containing
at least 12 contiguous bases in a processed human CD86 transcript,
in the target region from about -20 to +30 bases with respect to
the A nucleotide of the AUG start site at position 1, and which
includes at least 6 contiguous bases of the sequence identified by
SEQ ID NO: 9. Exemplary antisense sequences include those
identified as SEQ ID NOS: 21-23, and 32.
[0108] In a more specific embodiment, the antisense compounds are
designed to span or cover the three bases +12 to +14 bases (where
the A nucleotide of the AUG start site represents +1). In this
embodiment, the antisense compound may hybridize to a region
spanning these bases, e.g., where the three bases are in the middle
of the target region, or may hybridize to a region predominantly
upstream of and including these bases, e.g., the target bases
extending from -2 to +19 (SEQ ID NO: 23 below), or may hybridize to
a region predominantly downstream of and including these bases,
e.g., the target bases extending from +9 to +30 (SEQ ID NO: 32
below).
[0109] In another general embodiment, the expression-sensitive
region is a splice-site target region that may include (i) an
intron region adjacent, e.g., within 5 bases of, a splice-site
donor or acceptor junction, (ii) a region spanning a donor or
acceptor splice-site junction, or (iii) the exon region adjacent,
e.g., within 5 bases of, a splice-site donor or acceptor junction.
The target region preferably contains at least 12 contiguous bases
in a preprocessed human CD86 transcript, and includes, in exemplary
embodiment, at least 6 contiguous bases of one of the sequences
identified by SEQ ID NOS: 10-14. Exemplary antisense sequences
include those identified as SEQ ID NOS: 24-28.
[0110] However, in some cases, other regions of the CD86 mRNA (SEQ
ID NO: 29) may be targeted, including one or more of, an initiator
or promoter site, a 3'-untranslated region, and a 5'-untranslated
region. Both spliced and unspliced, preprocessed RNA may serve as
the template for design of antisense oligomers for use in the
methods of the invention.
[0111] When the antisense compound is complementary to a specific
region of a target gene (such as the region adjacent the AUG start
codon of the CD86 gene) the method can be used to monitor the
binding of the oligomer to the CD86 RNA.
[0112] The antisense compounds for use in practicing the invention
can be synthesized by stepwise solid-phase synthesis, employing
methods detailed in the references cited above. The sequence of
subunit additions will be determined by the selected base sequence.
In some cases, it may be desirable to add additional chemical
moieties to the oligomer compounds, e.g. to enhance the
pharmacokinetics of the compound or to facilitate capture or
detection of the compound. Such a moiety may be covalently
attached, typically to the 5'- or 3'-end of the oligomer, according
to standard synthesis methods. For example, addition of a
polyethyleneglycol moiety or other hydrophilic polymer, e.g., one
having 10-100 polymer subunits, may be useful in enhancing
solubility. One or more charged groups, e.g., anionic charged
groups such as an organic acid, may enhance cell uptake. A reporter
moiety, such as fluorescein or a radiolabeled group, may be
attached for purposes of detection.
[0113] Alternatively, the reporter label attached to the oligomer
may be a ligand, such as an antigen or biotin, capable of binding a
labeled antibody or streptavidin. In selecting a moiety for
attachment or modification of an oligomer antisense, it is
generally of course desirable to select chemical compounds of
groups that are biocompatible and likely to be tolerated by cells
in vitro or in vivo without undesirable side effects.
[0114] B. Arginine-Rich Polypeptide Moiety
[0115] The use of arginine-rich peptide sequences conjugated to
uncharged antisense compounds, e.g., PMO, has been shown to enhance
cellular uptake in a variety of cells (Wender, Mitchell et al.
2000; Moulton, Hase et al. 2003; Moulton and Moulton 2003)
(Iversen, Moulton et al. U.S. Patent Application No. 60/466,703,
now U.S. publication No. 2004/0265879 A1, published Dec. 30, 2004,
all of which are incorporated herein by reference.
[0116] In one embodiment of the invention, the antisense compound
is covalently linked at its 3' or 5' end to an arginine
rich-peptide effective to enhance uptake of the compound into
dendritic cells relative to uptake in the absence of the peptide.
The arginine-rich peptide is detailed in the above references to
Moulton et al., and described in U.S. patent application.
Preferably, the peptide is composed of d-amino acids, 1-amino
acids, non-natural amino acids or a combination thereof. Exemplary
arginine-rich peptides include those identified by SEQ ID NOS: 1-3,
of which those identified as SEQ ID NOS: 1 and 2 are preferred.
[0117] The transport peptide may significantly enhance cell entry
of attached uncharged oligomer compounds, relative to uptake of the
compound in the absence of the attached transport moiety, and
relative to uptake by an attached transport moiety lacking the
hydrophobic subunits Y. Such enhanced uptake is preferably
evidenced by at least a two-fold increase, and preferably a
four-fold increase, in the uptake of the compound into mammalian
cells relative to uptake of the agent by an attached transport
moiety lacking the hydrophobic subunits Y. Uptake is preferably
enhanced at least twenty fold, and more preferably forty fold,
relative to the unconjugated compound.
[0118] A further benefit of the transport moiety is its expected
ability to stabilize a duplex between an antisense oligomer and its
target nucleic acid sequence, presumably by virtue of electrostatic
interaction between the positively charged transport moiety and the
negatively charged nucleic acid. The number of charged subunits in
the transporter is less than 14, as noted above, and preferably
between 8 and 11, since too high a number of charged subunits may
lead to a reduction in sequence specificity.
[0119] The transport moiety may also lower the effective
concentration of an antisense oligomer to achieve antisense
activity as measured in both tissue culture and cell-free systems.
Cell-free translation systems provide an independent means to
assess the enhanced effect of the transport moiety on the antisense
oligomer's ability to bind to its target and, through steric
blocking, inhibit translation of downstream sequences.
[0120] C. rTAT (P002) Peptide
[0121] In studies conducted in support of the present invention,
several different "arginine-rich" peptide sequences were conjugated
to fluorescent tagged PMO (PMO-fl) and examined to determine the
effect of peptide sequence on uptake into lymphocytes Enhanced
uptake was observed for all arginine-rich peptide-PMO conjugates
tested compared to unconjugated PMO. The P003 and P005
arginine-rich peptides (SEQ ID NOS: 2 and 3, respectively) provide
enhanced uptake into lymphocytes regardless of the cell activation
state. However, among the arginine-rich peptides examined, the rTAT
(P002) peptide [NH.sub.2--RRRQRRKKRC--COOH] (SEQ ID NO: 1) PMO
conjugate exhibited differential uptake into dendritic cells
dependent on cell activation status. PMO uptake was greatly
increased in mature dendritic cells as shown below as well as
activated B cells and CD4 and CD8 T cells when compared to nave
lymphocytes (Mourich, Moulton et al. U.S. Patent Application No.
60/505,418). Furthermore, experiments in support of the invention
demonstrate that the arginine-rich peptide-antisense compounds
conjugates alone do not affect the maturation state of dendritic
cells. This was shown by treating dendritic cells with
arginine-rich peptide-PMO conjugates in the absence of a maturation
stimulus and observing no activation of the dendritic cell
population.
[0122] The rTAT peptide can be synthesized by a variety of known
methods, including solid-phase synthesis. The amino acid subunits
used in construction of the polypeptide may be either I- or d-amino
acids, preferably all I-amino acids or all d-amino acids. Minor (or
neutral) amino acid substitutions are allowed, as long as these do
not substantially degrade the ability of the polypeptide to enhance
uptake of antisense compounds selectively into activated T cells.
One skilled in the art can readily determine the effect of amino
acid substitutions by construction of a series of substituted rTAT
polypeptides, e.g., with a given amino acid substitution separately
at each of the positions along the rTAT chain. Using the above test
for uptake of fluoresceinated PMO-polypeptide conjugate, one can
then determine which substitutions are neutral and which
significantly degrade the transporter activity of the peptide.
Rules for neutral amino acid substitutions, based on common charge
and hydrophobicity similarities among distinct classes of amino
acids are well known and applicable here. In addition, it will be
recognized that the C-terminal cysteine of SEQ ID NO: 1 is added
for purposes of coupling to the antisense compound, and may be
replaced/deleted when another terminal amino acid or linker is used
for coupling.
[0123] The rTAT polypeptide can be linked to the compound to be
delivered by a variety of methods available to one of skill in the
art. The linkage point can be at various locations along the
transporter. In selected embodiments, it is at a terminus of the
transporter, e.g., the C-terminal or N-terminal amino acid. In one
exemplary approach, the polypeptide has, as its C-terminal residue,
a single cysteine residue whose side chain thiol is used for
linking. Multiple transporters can be attached to a single compound
if desired.
[0124] When the compound is a PMO, the transporter can be attached
at the 5' end of the PMO, e.g. via the 5'-hydroxyl group, or via an
amine capping moiety, as described (Moulton and Moulton 2003)
(Iversen, Moulton et al. U.S. Patent Application No. 60/466,703).
Alternatively, the transporter may be attached at the 3' end, e.g.
via a morpholino ring nitrogen, as described (Moulton and Moulton
2003) (Iversen, Moulton et al. U.S. Patent Application No.
60/466,703), either at a terminus or an internal linkage. The
linker may also comprise a direct bond between the carboxy terminus
of a transporter peptide and an amine or hydroxy group of the PMO,
formed by condensation promoted by, for example carbodiimide.
[0125] Linkers can be selected from those which are non-cleavable
under normal conditions of use, e.g., containing a thioether or
carbamate bond. In some embodiments, it may be desirable to include
a linkage between the transporter moiety and compound which is
cleavable in vivo. Bonds which are cleavable in vivo are known in
the art and include, for example, carboxylic acid esters, which are
hydrolyzed enzymatically, and disulfides, which are cleaved in the
presence of glutathione. It may also be feasible to cleave a
photolytically cleavable linkage, such as an ortho-nitrophenyl
ether, in vivo by application of radiation of the appropriate
wavelength.
[0126] For example, the preparation of a conjugate having a
disulfide linker, using the reagent N-succinimidyl
3-(2-pyridyldithio) propionate (SPDP) or succinimidyloxycarbonyl
.alpha.-methyl-.alpha.-(2-pyridyldithio- ) toluene (SMPT), is
described (Moulton and Moulton 2003) (Iversen, Moulton et al. U.S.
Patent Application No. 60/466,703). Exemplary heterobifunctional
linking agents which further contain a cleavable disulfide group
include N-hydroxysuccinimidyl 3-[(4-azidophenyl)dithio] propionate
and others.
[0127] IV. Selective Uptake of rTAT-Antisense Oligomers into
Activated Dendritic Cells
[0128] The present invention provides a method and composition for
delivering therapeutic compounds, e.g., uncharged antisense
compounds, specifically to activated immune cells, e.g.,
antigen-activated T cells, B cells, and mature dendritic cells.
[0129] The ability of the rTat (SEQ ID NO:1, P002) peptide to
enhance uptake of a fluoresceinated PMO antisense compound
selectively into activated mouse dendritic cells is demonstrated in
the study described in Example 1, and with the results shown in
FIG. 5. In this study, cultured mouse dendritic cells were
incubated with fluorescein-labeled P002-PMO conjugate and subjected
to lymphocyte activating substances, as described in Example 1.
Dendritic cells were stained with antibody to determine the extent
of uptake by FACS analysis of the cells. The results show
relatively low uptake of the antisense PMO into non-activated
dendritic cells. Activation by lipopolysaccharide (LPS) caused
significantly increased uptake of the antisense oligomer into
dendritic cells.
[0130] The property of activation-dependent uptake of
peptide-antisense conjugate is not observed with other
arginine-rich peptides, which are known to enhance drug transport
into cells. This is also demonstrated by the study described in
Example 1, and with the results shown in FIG. 5. As seen in these
figures, P003-PMO conjugate (corresponding to the arginine-rich
peptide of SEQ ID NO: 2) is readily taken up by immature dendritic
cells. PMO alone is relatively poorly taken up by immature
dendritic cells, and P002-PMO shows enhanced uptake into LPS
treated dendritic cells.
[0131] In one aspect of the invention, therefore, the P002 peptide
may be conjugated to a substantially uncharged antisense compound,
to enhance its uptake selectively into antigen-activated, mature
dendritic cells, including antigen-activated, mature human
dendritic cells.
[0132] V. Treating Transplantation Rejection and Autoimmune
Disorder
[0133] By manipulating the immune system's normal mechanism for the
generation of immune tolerance to self antigens, the present
invention provides a method and composition that alters the
function and activity of mature dendritic cells in a way that is
advantageous in the treatment of transplantation rejection or
autoimmune disorders, such as multiple sclerosis, lupis, myathenia
gravis, inflammatory bowel disease and rheumatoid arthritis.
[0134] By employing an antisense compound against CD86 (e.g., SEQ
ID NOS: 21-28 and 32), the present invention provides a means to
precisely and specifically block T cell activation to an antigen
presented by a mature dendritic cell. This allows the generation of
a tolerized T cell and dendritic cell population responding to
transplanted tissue, when chronically activated as in an autoimmune
condition, or by an immunogenic therapeutic protein. Where the
antisense compound is linked to an rTAT peptide, the conjugate
preferentially targets activated dendritic cells, thus allowing the
therapy to be made highly specific for mature dendritic cells.
[0135] The generation of tolerized, anergic T-cells using the
compounds and methods of the invention also provides a long-lasting
tolerance that has a variety of therapeutic advantages.
[0136] A. CD86 Antisense Oligomers, T Cell Costimulation and IL10
Production by Mature Dendritic Cells.
[0137] Dendritic cells (DCs) reside and traffic through most
tissues of the body in an immature state. Upon encountering an
inflammatory stimulus changes in DC phenotype rapidly ensue.
Hallmarks of this phenotypic shift termed "maturation" include the
loss of phagocytic function, increased surface expression of MHC
class I, II, adhesion molecules, distinct chemokine receptors and
costimulatory molecules such as B7-1 (CD80) and B7-2 (CD86).
Together these provide mature DCs the ability to traffic to
lymphoid tissue and the capacity to be potent antigen presenting
cells (APCs) to nave T cells. The cascade of signaling events that
follow when CD28 on the responding T cell is engaged by CD86 are
well established. However, little is known about the reciprocity of
events occurring in APCs due to the expression or engagement of
CD80 and CD86 molecules. Studies to determine if antisense
molecules could enter DCs and be used to inhibit the expression of
CD86 molecules led unexpectedly to the observations described in
the present invention that APCs, specifically dendritic cells,
undergo important alterations when B7 molecules are engaged. These
observations include a link between the expression of CD86 and the
regulation of IL-10 expression in bone marrow-derived mature
DCs.
[0138] Exemplary target sequences for the CD86 (B7-2) gene are
listed in Table 1 below. The murine CD86 sequences are noted with
"mu" and are derived from Genbank Accession No. AF065898. The human
CD86 AUG target and targeting sequences are noted with "hu" and
derived from Genbank Accession No. NM006889. The human Exon 6, 7,
and 8 splice donor (sd) and splice acceptor (sa) target and
targeting sequences are derived from Genbank Accession Nos. U17720,
U17721 and U17722, respectively.
1TABLE 1 Exemplary CD86 Target Sequences Oligomer SED ID Target
Sequence (5' to 3') Sp. Nct. Range NO. CD86 AUG
cggaagcacccacgatggaccccag mu 19-43 4 Exon 7sa gctgtttccgtggagacgc
mu 99-117 5 Exon 9sd gccgaatcagcttagcagg mu 833-851 6 Exon 10sa
gcccagcaacacagcctct mu 851-869 7 Exon 11sa gaaaccaaatgcagagtg mu
944-961 8 CD86 AUG catttgtgacagcactatgggactgagtaacattct hu 132-177
9 ctttgtgatg CD86Ex6sa agcttgaggaccctcagcctc hu 170-190 10
CD86Ex6sd gcctcgcaactcttataaatgtg hu 291-313 11 CD86Ex7sa
gaaccaacacaatggagaggga hu 274-295 12 CD86Ex7sd
gagtgaacagaccaagaaaag hu 298-319 13 CD86Ex8sa
agaaaaaatccatatacctgaa hu 223-244 14
[0139]
2TABLE 2 Exemplary CD86 Targeting Sequences Oligomer SEQ ID Target
Sequence (5' to 3') Sp. NO. B7-2 AUG1 CTGGGGTCCATCGTGGGTGC mu 15
B7-2 AUG2 GGGGTCCATCGTGGGTGCTTCCG mu 16 Exon 7sa
GCGTCTCCACGGAAACAGC mu 17 Exon 9sd CCTGCTAAGCTGATTCGGC mu 18 Exon
10sa AGAGGCTGTGTTGCTGGGC mu 19 Exon 11sa CACTCTGCATTTGGTTTC mu 20
CD86 AUG1 GTTACTCAGTCCCATAGTGCTG hu 21 CD86 AUG2
CCATAGTGCTGTCACAAATG hu 22 CD86 AUG3 GAATGTTACTCAGTCCCATAG hu 23
CD86Ex6sa GAGGCTGAGGGTCCTCAAGCT hu 24 CD86Ex6sd
CACATTTATAAGAGTTGCGAGGC hu 25 CD86Ex7sa TCCCTCTCCATTGTGTTGGTTC hu
26 CD86Ex7sd CTTTTCTTGGTCTGTTCACTC hu 27 CD86Ex8sa
TTCAGGTATATGGATTTTTTCT hu 28 CD86 AUG4 CATCACAAAGAGAATGTTACTC hu
32
[0140] B. Treatment Methods
[0141] In one aspect, the invention is directed to methods of
inducing immunological tolerance in vivo in a patient, by
administering to the patient a therapeutically effective amount of
a peptide-conjugated CD86 PMO pharmaceutical composition, as
described herein, e.g., a pharmaceutical composition comprising an
antisense oligomer to CD86.
[0142] The antisense oligomers of the invention can be effective in
the treatment of patients by modulating the immunological response
to allogeneic transplantation or elimination of chronically
activated T cells in the case of autoimmune diseases.
[0143] In one embodiment, a subject is in need of tolerized
dendritic cells and T cells when responding to an allogeneic
transplantation. In this embodiment, a CD86 antisense compound is
administered to the subject in a manner effective to result in
blocking the formation of activated T cells. Typically, the patient
is treated with the conjugate shortly before, e.g., a few days
before, receiving the transplant, then treated periodically, e.g.,
once every 14 days, until immunological tolerance is established.
Immunological tolerance can be monitored during treatment by
testing patient T cells for reactivity with donor MHC antigens in a
standard in vitro test, as detailed below.
[0144] For the treatment of an autoimmune disorder, such as
multiple sclerosis, lupis, myathenia gravis, inflammatory bowel
disease and rheumatoid arthritis, the patient is given an initial
single dose of the CD86 antisense conjugate, then additional doses
on a periodic basis, e.g., every 3-14 days, until improvement in
the disorder is observed. As above, development of immunological
tolerance can be monitored during treatment by testing T cells from
a blood sample for their ability to react with a selected, relevant
antigen in vitro.
[0145] It will be understood that in vivo administration of such a
CD86 antisense compound is dependent upon, (1) the duration, dose
and frequency of antisense administration, and (2) the general
condition of the subject. A suitable dose can be approximated from
animal model studies and extrapolated to patient weight.
[0146] Typically, one or more doses of CD86 antisense oligomer are
administered, generally at regular intervals for a period of about
one to two weeks. Preferred doses for oral administration are from
about 1 mg oligomer/patient to about 25 mg oligomer/patient (based
on an adult weight of 70 kg). In some cases, doses of greater than
25 mg oligomer/patient may be necessary. For IV administration, the
preferred doses are from about 1.0 mg oligomer/patient to about 100
mg oligomer/patient, preferably 5-50 mg oligomer/patient, (based on
an adult weight of 70 kg). The antisense agent is generally
administered in an amount sufficient to result in a peak blood
concentration of at least 200-400 nM antisense oligomer.
[0147] In general, the method comprises administering to a subject,
in a suitable pharmaceutical carrier, an amount of a CD86 antisense
oligomer, e.g., morpholino oligomer, effective to inhibit
expression of CD86 and increase expression of IL-10 in dendritic
cells.
[0148] Effective delivery of an antisense oligomer to the target
nucleic acid is an important aspect of the methods described
herein. In accordance with the invention, such routes of antisense
oligomer delivery include, but are not limited to, inhalation;
transdermal delivery; various systemic routes, including oral and
parenteral routes, e.g., intravenous, subcutaneous,
intraperitoneal, or intramuscular delivery.
[0149] It is appreciated that any methods which are effective to
deliver a CD86 PMO to the cells of an allogeneic transplant or to
introduce the agent into the bloodstream are also contemplated.
[0150] In preferred applications of the method, the subject is a
human subject and the methods of the invention are applicable to
treatment of any condition wherein promoting immunological
tolerance would be effective to result in an improved therapeutic
outcome for the subject under treatment.
[0151] It will be understood that an effective in vivo treatment
regimen using a CD86 antisense compound in the methods of the
invention will vary according to the frequency and route of
administration as well as the condition of the subject under
treatment. Accordingly, such in vivo therapy will generally require
monitoring by tests appropriate to the condition being treated and
a corresponding adjustment in the dose or treatment regimen in
order to achieve an optimal therapeutic outcome.
[0152] C. Ex Vivo Treatment of Human Dendritic Cells
[0153] In another preferred application of the method, autologous
dendritic cells isolated from a human subject can be treated ex
vivo with the CD86 antisense compound in the presence of a
selected, relevant antigen. Studies in several systems have
demonstrated that when dendritic cells are pulsed with antigens ex
vivo, and these cells are subsequently readministered to the human
subject from whom they were isolated, specific immunity can be
established (Lu, Arraes et al. 2004; Mohamadzadeh and Luftig 2004).
A similar strategy can be used to establish, ex vivo, a tolerogenic
population of dendritic cells using the methods and compositions of
the present invention. Dendritic cells are isolated from the
peripheral blood of a human subject using methods well-known to
those skilled in the art. Growth and treatment of the dendritic
cells with the relevant antigen and antisense CD86 antisense will
induce the formation of dendritic cells that, upon readministration
to the subject, will condition the dendritic cells to induce a
T-cell response that suppresses the antigen-specific immunity. This
application of the method is particularly useful in treating an
autoimmune disorder where the immune system is reacting
inappropriately to specific antigens and these antigens can be used
to condition the dendritic cells. An example is the immune-mediated
destruction of myelin in multiple sclerosis (MS). Myelin basic
protein (MBP) and proteolipid protein (PLP) are host proteins which
are thought to be the key antigens in the etiology of this
autoimmune disease (Shevac 2002).
[0154] D. Administration of Anti-CD86 Antisense Oligomers
[0155] Transdermal delivery of an antisense oligomer may be
accomplished by use of a pharmaceutically acceptable carrier. One
example of morpholino oligomer delivery is described in PCT patent
application WO 97/40854, incorporated herein by reference.
[0156] In one preferred embodiment, the oligomer is an anti-CD86
morpholino oligomer, contained in a pharmaceutically acceptable
carrier, and delivered orally. In a further aspect of this
embodiment, the antisense oligomer is administered at regular
intervals for a short time period, e.g., daily for two weeks or
less. However, in some cases the antisense oligomer is administered
intermittently over a longer period of time.
[0157] It follows that a morpholino antisense oligonucleotide
composition may be administered in any convenient vehicle, which is
physiologically acceptable. Such an oligonucleotide composition may
include any of a variety of standard pharmaceutically accepted
carriers employed by those of ordinary skill in the art. Examples
of such pharmaceutical carriers include, but are not limited to,
saline, phosphate buffered saline (PBS), water, aqueous ethanol,
emulsions such as oil/water emulsions, triglyceride emulsions,
wetting agents, tablets and capsules. It will be understood that
the choice of suitable physiologically acceptable carrier will vary
dependent upon the chosen mode of administration.
[0158] In some instances liposomes may be employed to facilitate
uptake of an antisense oligonucleotide into cells. (See, e.g.,
Williams, 1996; Lappalainen, et al., 1994; Uhlmann, et al., 1990;
Gregoriadis, 1979.) Hydrogels may also be used as vehicles for
antisense oligomer administration, for example, as described in WO
93/01286. Alternatively, an oligonucleotide may be administered in
microspheres or microparticles. (See, e.g., Wu et al., 1987).
[0159] Sustained release compositions are also contemplated within
the scope of this application. These may include semipermeable
polymeric matrices in the form of shaped articles such as films or
microcapsules.
[0160] E. Monitoring Treatment
[0161] The efficacy of a given therapeutic regimen involving the
methods described herein, may be monitored, e.g., by conventional
FACS assays for the phenotype of cells in the circulation of the
subject under treatment or cells in culture. Such analysis is
useful to monitor changes in the numbers of cells of various
lineages, in particular, activated T and B cells in response to an
allogeneic transplant.
[0162] Phenotypic analysis is generally carried out using
monoclonal antibodies specific to the cell type being analyzed. The
use of monoclonal antibodies in such phenotypic analyses is
routinely employed by those of skill in the art for cellular
analyses and monoclonal antibodies specific to particular cell
types are commercially available.
[0163] The CD86 PMO treatment regimen may be adjusted (dose,
frequency, route, etc.), as indicated, based on the results of the
phenotypic and biological assays described above.
[0164] From the foregoing, it will be appreciated how various
objects and features of the invention are met. The specific
blockade of dendritic cell activation of T cells capable of
rejecting transplanted tissues is an important therapy for numerous
human diseases where immunological tolerance is beneficial. The
present invention provides a method of specifically blocking the
activation of these cells through the use of antisense oligomers
designed to inhibit CD86 expression, or specific portions of CD86,
during the stage of antigen-specific activation and the generation
of anergic, tolerized T cells. Antisense CD86 mediated suppression
of either chronically activated T cells (i.e. autoimmunity) or nave
T cell responding to alloantigens (transplantation) provides a
potent and specific therapeutic effect.
[0165] Additionally, this treatment method is long lived because
the immune system is unable to replenish antigen-specific T cell
clones once the precursor population is removed from the T cell
repertoire. In addition, by specifically targeting the antisense
CD86 oligomer to mature dendritic cells, immature dendritic cells
would be unaffected, allowing for the patient to respond normally
to foreign antigens as soon as the therapy is withdrawn. Moreover,
the immune status of the patient prior to the antisense CD86
therapy (e.g. immunity provided by previous vaccinations or
infections) would remain intact.
[0166] The following examples illustrate but are not intended in
any way to limit the invention.
[0167] Materials and Methods
[0168] A. Antisense Oligomers and Peptide Conjugates
[0169] PMO synthesis, peptide conjugation and purification were
preformed at AVI BioPharma Inc. (Corvallis, Oreg.) as previously
described (Summerton and Weller 1997). Oligomer sequences were
designed to either block translation by binding to bases
surrounding the AUG start site (CAT) or alter RNA splicing by
blocking splice donor or splice acceptor sites, sa or sd,
respectively. An oligomer with the same base composition of the
B7-2 (AUG) in a scrambled order was synthesized to serve as a
control for oligomer treatment. B7-1 and B7-2 antisense oligomer
sequences were designed using Genbank sequences accession numbers
X60958 and U39459-66, respectively, The PMO sequences and
designations are as follows; B7-1 (AUG) 5'-GCA AGC CAT AGC TTC AGA
TGC-3' (SEQ ID NO:29), B7-2(AUG) 5'-CT GGG GTC CAT CGT GGG
TGC-3'(SEQ ID NO: 15), EXON 7sa 5'-GCG TCT CCA CGG AAA CAG C-3'
(SEQ ID NO:17), EXON 9sd 5'-CCT GCT MG CTG ATT CGG C-3' (SEQ ID
NO:18), EXON 10sa 5'-AGA GGC TGT GTT GCT GGG C-3' (SEQ ID NO:19),
EXON 11 sa 5'-CAC TCT GCA TTT GGT TTC-3' (SEQ ID NO:20), SCRAMBLE
5'-CGT GGT GCA CTG CGT GTG GC-3' (SEQ ID NO:30), 705-FL 5'-CCT CTT
ACC TCA GTT ACA-FL-3' (SEQ ID NO:31). The 3' fluorescein conjugated
oligomer 705-FL targets an irrelevant gene (human b-globin intron
2) and was used to analyze the intracellular delivery properties of
different peptides into cultured DCs. Three different arginine rich
peptide sequences were conjugated separately to the PMOs used in
this study; P002=N-RRRQRRKKRGYC-CONH.sub.2 (SEQ ID NO:1),
P003=N-RRRRRRRRRFFC-CONH.sub.2(SEQ ID NO:2) and
P005=N-RRRQRRKKRGYFFC-CON- H.sub.2 (SEQ ID NO:3).
[0170] B. Generation and Culturing of Bone Marrow Derived DCs
[0171] Murine DCs were generated by culturing marrow flushed from
the femur and tibia of female BALB/c mice obtained from Jackson
Laboratory aged 6-12 weeks. The marrow was minced through a 70
micron nylon cell strainer (BD Falcon) and washed twice after
centrifugation in DMEM+1% fetal bovine serum (FBS) and penicillin
streptomycin and glutamine (PSG). Cell suspension were made in
culture medium (RPMI+10% FBS, PSG and 5.times.10.sup.-5 M
2-mecapotoethanol) supplemented with recombinant mouse GM-CSF
(eBioscience) [25 ng/ml] and seeded onto a 100 mm bacteriological
Petri dish (Falcon) at 2.times.10.sup.5 cells/ml. After 3 days an
additional 5 ml of fresh medium was added containing GM-CSF. The
culture supernatant was removed on the 6.sup.th day and centrifuged
to recover any dislodged cells. The cell pellet was suspended in 10
ml fresh media with GM-CSF, placed back on to the original 100 mm
dish and cultured for an additional 2-4 days prior to treatment
with PMO.
[0172] Non-adherent cells were harvested by gentle pipetting of the
medium which was then transferred to a tube for centrifugation at
room temperature. The cell pellet was washed twice and then
enumerated. The wells of a 12 well plate were seeded with 1.5 ml
[5.times.10.sup.5 cells/ml] in fresh culture medium containing
GM-CSF. PMO working stock [1 mM] in sterile water was added
directly to the wells to obtain a final concentration ranging from
2-20 .mu.M 2-4 hours prior to inducing maturation. Control culture
wells were treated with the equivalent amount of sterile water.
Maturation was induced by the addition of lipopolysaccharide (LPS)
E. coli 026:B6 (Sigma) [1 .mu.g/ml] or anti-CD40 (eBioscience) at
[5 .mu.g/ml] for 16 hours following PMO treatment. Culture
conditions to block binding of cell associated ligand to B7
molecules was carried out by addition of [5 .mu.g/ml] recombinant
chimeric CTLA-4 FC non-cytolytic molecule (Chimerigen Allston,
Mass.). Cultures used for analysis of IL-4 and IL-12 cytokine
production were treated with 1 .mu.l of the protein transport
inhibitor brefeldin A GolgiPlug (BD Pharmingen) for the last 4 hrs
of incubation.
[0173] C. Flow Cytometric Analysis
[0174] Cells were removed from culture wells by scrapping with a 25
cm cell scraper (Sarstedt) and rinsing with 1 ml cold FACS buffer
[PBS+2% FBS+0.2% sodium azide]. The cells were washed twice in cold
FACS buffer after centrifugation and suspended in 50 ml FACS buffer
containing 1 .mu.g anti-mouse CD16/CD32 FC blocking antibody
(eBioscience) for 15 min on ice. The FC blocked samples were
centrifuged and suspended in 50 ml antibody staining reagent for 30
min on ice. The surface staining reagents used were; CD111c-APC (BD
Bioscience) and CD86-PE, CD86-FITC and CD80-PE (eBioscience)
diluted in 50 ml cold FACS buffer. Stains were combined in the 50
ml when dual surface staining was needed for analysis. Cells were
washed thrice by centrifugation in FACS buffer prior to analysis or
use in additional staining procedures.
[0175] Intracellular cytokine staining was performed immediately
following surface staining and washes. The cells were fixed and
permeabilized in 100 .mu.l Cytofix/Cytoperm (Pharmingen) buffer for
20 min on ice. Cell pellets were suspended in 50 .mu.l of 1.times.
PermNVash buffer (Pharmingen) containing either IL-10-FITC or
IL-12-APC and IL-4-FITC (Pharmingen) and incubated for 30 min on
ice. The cells were washed thrice and suspended in 300 ml FACS
buffer prior collection of flow cytometric data on a FACS caliber
cytometer (Becton Dickinson). Cytometric data was analyzed using
FCS Express Software (Denovo Software).
[0176] D. RT-PCR
[0177] After treatment with PMO conjugates for 4 hours and then for
16 hours with LPS [1.0 .mu.g/ml] total cellular RNA was isolated
from the cultured cells using RNAeasy Mini kit (Qiagen) according
to manufacture's instructions. The isolated RNA was treated with
RNAse free DNAse I (2 U) for 30 min at 37.degree. C. to eliminate
contaminating genomic DNA followed by heating to 70.degree. C. for
20 min to inactivation the enzyme. This material was used as
template for single-tube reverse transcription and polymerase chain
reaction using SuperScript One-Step RT-PCR with Platinum Taq enzyme
(Invitrogen). Primers spanning 870 bp of the B7-2 mRNA, forward
primer 5'-GGCAATCCTTATCTTTGTGACAGTC-3' and reverse primer:
5'-TTTGCTGMGCMTTTGGGG-3' were used to examine splice altering
activity of the PMOs. Primers to detect mouse IL-10 mRNA forward
primer 5'-GATCCAGGGATCTTAGCTMCGG-3' and reverse primer
5'-TTCTCTTCCCMGACCCATGAGT- -3' spanning 406 bp were derived from
the Genbank sequence accession number NM.sub.--010548 bases
675-1081.
[0178] The resulting amplicons were fractionated on an EtBr stained
3.0% agarose gel to determine size. Altered splicing patterns and
continuity of the open reading frames were confirmed by sequencing
after insertion into plasmid vector using the TOPO TA cloning kit
according to manufacture's instructions (Invitrogen). At least
three clones harboring the different amplicons were examined.
Identity and sequence alignments were performed by BLAST search
analysis.
EXAMPLE 1
Arginine-Rich Peptides Enhance Uptake of Oligomers into Mature
Dendritic Cells
[0179] Delivery of antisense molecules without substantial
manipulation of the cellular membrane has been an impediment to
targeting gene expression in DCs and other immune cell types. These
procedures often result in extensive damage to the membrane
allowing for only short lived experiments to be conducted. Numerous
arginine-rich peptides were examined as to their ability to deliver
oligomers to various cell types with no manipulation beyond direct
addition to cells cultured under normal conditions. The PMO
chemical structure and peptides used in this study are shown in
FIG. 4. PMO synthesis and conjugation of peptides and or
fluorescein were carried out at AVI BioPharma as previously
described (Summerton and Weller 1997; Moulton, Hase et al. 2003;
Moulton, Nelson et al. 2004). The arginine-rich peptides shown in
FIG. 4 are among several that have been shown to enhance cellular
uptake. Fluorescein can be linked to the 3' end of the peptide-PMO
conjugate to allow imaging and or detection of PMO uptake in intact
cells.
[0180] Using a fluorescein linked PMO, FIG. 5 shows that bone
marrow derived DCs readily take up PMO conjugates of the P003
peptide (FIG. 5, SEQ ID NO:2). In the experiment represented in
this figure an irrelevant control PMO (705,
5'-CCTCTTACCTCAGTTACA-3') was used to measure uptake of
unconjugated and peptide-conjugated PMOs into DCs. Peptides with
similar amino acid content but varied sequence such as P005 (SEQ ID
NO:3) perform similarly. Surprisingly, when PMO conjugates of P002
peptide (SEQ ID NO:1) were tested it was observed that uptake into
DCs was enhanced after stimulation with lipopolysaccharide (LPS)
compared to untreated or immature DCs (FIG. 5).
[0181] The data presented in FIG. 5 were obtained using murine DCs
obtained from murine bone marrow cells cultured for 8 days in
RPMI+10% FBS supplemented with granulocyte macrophage colony
stimulating factor (GM-CSF) (25 ng/ml) and treated in duplicate
wells with either naked or peptide conjugated PMO 705 [5 mM] linked
to fluorescein. One well for each oligomer treatment received LPS
[1 mg/ml]. The cells were cultured for 16 hrs and then harvested,
washed 3 times with PBS, stained with CD11 c-APC and analyzed by
flow cytometry. The histogram indicates the level of fluorescein in
the CD11c positive (i.e. mature DC) cell population.
EXAMPLE 2
Antisense Inhibition of CD86 Expression Also Alters CD80
Expression
[0182] To determine if the enhanced uptake of the PMO into DCs
provided by the peptide conjugates would translate into functional
antisense activity we chose to synthesize oligomers targeting the
translational start site of CD86 (B7-2 AUG1, SEQ ID NO:15) and a
sequence scrambled control oligomer (5'-CGTGGTGCACTGCGTGTGGC-3'). A
considerable reduction in the level of CD86 (B7-2) was observed in
cultured DCs after treatment with the sequence-specific oligomer
and LPS compared to controls (FIG. 6, top histogram). However, when
a measure of the level of CD80 (B7-1) was made under the same
conditions it was observed that a significant reduction was
produced in the cultures treated with antisense to CD86 compared to
controls (FIG. 6, bottom histogram). This was unexpected since the
CD86 sequence shares little homology with that of CD80 and
considerably low levels of homology around the translational start
site. Nearly identical results were observed with regards to a
reduction in CD80 when an oligomer targeting a different sequence
(B7-2 AUG2, SEQ ID NO:16) surrounding the CD86 translational start
site was used (data not shown).
[0183] The data in FIG. 6 were generated using bone marrow derived
DCs treated in duplicate with either P002 peptide conjugated PMO
[20 mM] antisense to CD86 (SEQ ID NO: 15), scrambled PMO sequence
(5'-CGTGGTGCACTGCGTGTGGC-3', SEQ ID NO:30) or media alone for 4
hours. LPS [1.0 mg/ml] was then added to all cultures for 16 hours.
The cells were washed, stained with CD11c-APC antibody (i.e.
specific for mature DCs) and either anti-mouse CD80-PE or CD86-PE
antibodies and analyzed by flow cytometry.
EXAMPLE 3
Increased DC IL-10 Production is Linked to Diminished CD86
Expression and Not Ligand Interaction
[0184] In light of the results seen in Example 2 we examined the
cytokine production profile of the CD86 antisense treated DCs. IL-4
and IL-12 production was not significantly altered in the DCs
receiving the B7-2 AUG1 CD86 antisense oligomer (SEQ ID NO:15)
compared to controls (data not shown). However, IL-10 production
was evident when DCs were treated with a maturation stimulus such
as LPS in conjunction with the CD86 antisense oligomer-P002
conjugate (SEQ ID NO:15) and not the P002 peptide alone or a
scrambled control sequence conjugated to P002 as shown in Table 3
below. The same result was seen when other delivery peptides were
used with SEQ ID NO:15 or alternate sequences targeting the
translational start site (SEQ ID NO:16). Furthermore, IL-10
staining was detectable when the DCs were not permeabilized
indicating that IL-10 was being secreted from the cells where it
could exert an autocrine or paracrine effect (data not shown).
3TABLE 3 Inhibition of CD86 Induces IL-10 Production in LPS-treated
Dendritic Cells % DCs % DCs Treatment CD86 Positive IL10 Positive
Control (No LPS) 6.92 0.10 P002 Peptide (No LPS) 6.47 0.10
Scramble-P002 + LPS 13.4 0.75 B7-2 AUG1(SEQ ID NO: 15)- 2.26 21.1
P002(SEQ ID NO: 1) + LPS
[0185] The data presented in Table 3 was generated using murine
bone marrow derived DCs treated in duplicate with either P002
peptide (SEQ ID NO:1) alone, P002 conjugated to PMO antisense CD86
(SEQ ID NO:15) or scrambled PMO sequence
(5'-CGTGGTGCACTGCGTGTGGC-3', SEQ ID NO:30) at 20 mM or media alone
for 4 hours. LPS [1.0 mg/ml] was then added to the appropriate
cultures for 16 hours. The cell were washed, FC blocked, then
stained with antibody specific for mature DCs (CD11c-APC) and
antibody specific for CD86 (CD86-PE). Intracellular staining with
anti-IL-10-FITC antibody was carried out after fixation and
permeabilization of the cells. The numbers indicate the percentage
of CD11c positive cells (mature DCs) staining positive for IL-10
and CD86 respectively.
[0186] We also examined whether the regulation of IL-10 in the
mature DCs was due to the loss of some interaction with a yet
unknown ligand that is present in bone marrow derived DCs cultures
or whether it might be due to the loss of control through the
absences of CD86 (B7-2). A recombinant form of the CTLA-4 molecule,
a receptor on T cells for CD86, was used to block interactions of
the CD86 molecule in cultured DCs. DCs receiving this treatment
were compared to CD86 antisense-treated DCs as to the levels of
IL-10 produced under two different conditions of DCs maturation.
DCs received a maturation stimulus from either LPS or anti-CD40 and
were then stained for intracellular IL-10. Under either maturation
condition IL-10 was only significantly produced in the
P002-conjugated CD86 antisense (SEQ ID NO: 15) treated cells
compared to control untreated cells (FIG. 7). This suggests that
the regulation of IL-10 production in maturing DCs is linked to
levels of CD86 expression and not those of CD86 interactions with a
ligand.
[0187] The data in FIG. 7, which shows blocking CD86 interactions
does not lead to IL-10 expression in bone marrow derived DCs, were
generated by treatment of murine bone marrow DCs in duplicate with
either media alone, antisense CD86-P003 conjugate [2 mM] or CTLA-4
Ig [5.0 mg/ml] for 4 hours. One of the duplicate cultures was
treated with LPS [1.0 mg/ml] and the other with anti-CD40 [5.0
mg/ml] for 16 hours to induce maturation. The cells were washed, FC
blocked, surface stained for CD11c-APC, fixed and permeabilized for
intracellular staining with anti-IL-10 FITC. Samples were analyzed
by flow cytometry gating on CD11c positive cells.
EXAMPLE 4
Regulation of IL-10 Production in Mature DCs Controlled Through
CD86 Exon 10
[0188] To further examine the question regarding the role of CD86
in the regulation of IL-10 production in maturing DCs we determined
what components of the CD86 polypeptide might be responsible. The
approach taken was to systematically alter the CD86 protein by
limiting expression of either intracellular or extracellular
polypeptide domains. This was done by using antisense oligomers
with sequences targeting splice sites within the unprocessed
message thereby forcing alterations in the mature message to
exclude particular exons (Target and targeting sequences are shown
in Table 1 and Table 2, respectively).
[0189] Employing RT-PCR on total RNA isolated from murine DCs
treated with the different oligomers shows that the predicted
alterations to the CD86 mRNA could be achieved (FIGS. 8A and 8B).
Some cloned sequences exhibited an alternative splice acceptor site
in Exon 8 which has been shown to be used in normal APCs
(Borriello, Oliveros et al. 1995).
[0190] The data in FIGS. 8A and 8B demonstrate that antisense
targeting of splice donor or splice acceptor sites alters CD86
mRNA. The data were generated using bone marrow derived DCs treated
with [10 mM] of either P002-CD86 (SEQ ID NO:15) antisense PMO or
scramble PMO peptide conjugates or P005 PMO peptide conjugates
targeting splice junction sites for exons 7, 9, 10 and 11 (SEQ ID
NOS: 17, 18, 19 and 20, respectively) for 4 hours and then treated
for 16 hours with LPS [1.0 mg/ml]. Total RNA was isolated from each
culture, treated with DNAse free RNAse and used as template
material for single-tube reverse transcription and polymerase chain
reaction using primers spanning an 870 base pair region on the CD86
mRNA. As shown in FIG. 8A, the reaction material was fractionated
on an EtBr stained 3.0% agarose gel to determine the size of the
resulting amplicons. FIG. 8B shows a schematic of the altered
splicing patterns that were observed. The continuity of the open
reading frames were confirmed by sequencing various clones of the
amplicons after insertion into plasmid DNA vector. A schematic
representation of some of the cloned sequences is shown in FIG. 8B
by lines aligned with the wild type CD86 map. The intervening black
lines depict regions where splicing was altered. The relative
positions of the PMOs and the PCR primers are also shown.
EXAMPLE 5
Inhibition of CD86 or CD86 Exon 10 Expression Alters Morphology of
LPS-Treated DCs
[0191] In addition to molecular evidence of the effect of the
splice altering oligomers as described in Example 4, phenotypic
changes in the treated DCs were also observed. Specifically, DCs
treated with the Exon 10 antisense oligomer (SEQ ID NO: 19)
exhibited the greatest production of IL-10 and maintained an
immature phenotype when exposed to LPS (Table 4, below and FIG. 9,
respectively). Table 4, shows that antisense PMO designed to block
Exon 10 expression induces IL-10 in mature dendritic cells. FIG. 9
shows that inhibition of CD86 or Exon 10 expression alters the
morphology of LPS treated DCs. In this experiment, bone marrow
derived DCs were treated as previously described in Example 4.
Prior to staining for flow cytometry the cultured DCs were imaged
by light microscopy. The forward and side light scattering
properties of each culture are shown to the right of each
image.
4TABLE 4 Exon 10 of the CD86 Gene Regulates IL-10 production in
Mature Dendritic Cells % DCs % DCs Treatment CD86 Positive IL10
Positive Control 10.40 0.30 Control (+ LPS) 33.81 0.07 B7-2 AUG
(SEQ ID NO: 15) + LPS 17.93 2.06 EXON 9sd (SEQ ID NO: 18) + LPS
26.62 1.48 EXON 10sa (SEQ ID NO: 19) + LPS 13.36 4.04 EXON 11sa
(SEQ ID NO: 20) + LPS 5.06 0.65
[0192]
Sequence CWU 1
1
33 1 12 PRT Artificial Sequence The reverse of a TAT sequence from
the Human immunodeficiency virus type 1 1 Arg Arg Arg Gln Arg Arg
Lys Lys Arg Gly Tyr Cys 1 5 10 2 12 PRT Artificial Sequence
Synthetic arginine-rich peptide 2 Arg Arg Arg Arg Arg Arg Arg Arg
Arg Phe Phe Cys 1 5 10 3 12 PRT Artificial Sequence Synthetic
arginine-rich peptide 3 Arg Arg Arg Arg Arg Phe Phe Arg Arg Arg Arg
Cys 1 5 10 4 25 DNA Mus musculus 4 cggaagcacc cacgatggac cccag 25 5
19 DNA Mus musculus 5 gctgtttccg tggagacgc 19 6 19 DNA Mus musculus
6 gccgaatcag cttagcagg 19 7 19 DNA Mus musculus 7 gcccagcaac
acagcctct 19 8 18 DNA Mus musculus 8 gaaaccaaat gcagagtg 18 9 46
DNA Homo sapiens 9 catttgtgac agcactatgg gactgagtaa cattctcttt
gtgatg 46 10 21 DNA Homo sapiens 10 agcttgagga ccctcagcct c 21 11
23 DNA Homo sapiens 11 gcctcgcaac tcttataaat gtg 23 12 22 DNA Homo
sapiens 12 gaaccaacac aatggagagg ga 22 13 21 DNA Homo sapiens 13
gagtgaacag accaagaaaa g 21 14 22 DNA Homo sapiens 14 agaaaaaatc
catatacctg aa 22 15 20 DNA Artificial Sequence Antisense oligomer
sequence directed against SEQ ID NO4 15 ctggggtcca tcgtgggtgc 20 16
23 DNA Artificial Sequence Antisense oligomer sequence directed
against SEQ ID NO4 16 ggggtccatc gtgggtgctt ccg 23 17 19 DNA
Artificial Sequence Antisense oligomer sequence directed against
SEQ ID NO5 17 gcgtctccac ggaaacagc 19 18 19 DNA Artificial Sequence
Antisense oligomer sequence directed against SEQ ID NO6 18
cctgctaagc tgattcggc 19 19 19 DNA Artificial Sequence Antisense
oligomer sequence directed against SEQ ID NO7 19 agaggctgtg
ttgctgggc 19 20 18 DNA Artificial Sequence Antisense oligomer
sequence directed against SEQ ID NO8 20 cactctgcat ttggtttc 18 21
22 DNA Artificial Sequence Antisense oligomer sequence directed
against SEQ ID NO9 21 gttactcagt cccatagtgc tg 22 22 20 DNA
Artificial Sequence Antisense oligomer sequence directed against
SEQ ID NO9 22 ccatagtgct gtcacaaatg 20 23 21 DNA Artificial
Sequence Antisense oligomer sequence directed against SEQ ID NO9 23
gaatgttact cagtcccata g 21 24 21 DNA Artificial Sequence Antisense
oligomer sequence directed against SEQ ID NO10 24 gaggctgagg
gtcctcaagc t 21 25 23 DNA Artificial Sequence Antisense oligomer
sequence directed against SEQ ID NO11 25 cacatttata agagttgcga ggc
23 26 22 DNA Artificial Sequence Antisense oligomer sequence
directed against SEQ ID NO12 26 tccctctcca ttgtgttggt tc 22 27 21
DNA Artificial Sequence Antisense oligomer sequence directed
against SEQ ID NO13 27 cttttcttgg tctgttcact c 21 28 22 DNA
Artificial Sequence Antisense oligomer sequence directed against
SEQ ID NO14 28 ttcaggtata tggatttttt ct 22 29 21 DNA Artificial
Sequence Antisense oligomer sequence directed against the start
site of mouse B7-1 transcript 29 gcaagccata gcttcagatg c 21 30 20
DNA Artificial Sequence A scrambled antisense oligomer sequence 30
cgtggtgcac tgcgtgtggc 20 31 18 DNA Artificial Sequence Antisense
oligomer sequence conjugated to fluorescein at the 3' end and
directed against human b-globin intron 2 31 cctcttacct cagttaca 18
32 22 DNA Artificial Sequence Antisense oligomer sequence directed
against the start site of human CD86 transcript 32 catcacaaag
agaatgttac tc 22 33 2717 DNA Homo sapiens 33 aggagcctta ggaggtacgg
ggagctcgca aatactcctt ttggtttatt cttaccacct 60 tgcttctgtg
ttccttggga atgctgctgt gcttatgcat ctggtctctt tttggagcta 120
cagtggacag gcatttgtga cagcactatg ggactgagta acattctctt tgtgatggcc
180 ttcctgctct ctggtgctgc tcctctgaag attcaagctt atttcaatga
gactgcagac 240 ctgccatgcc aatttgcaaa ctctcaaaac caaagcctga
gtgagctagt agtattttgg 300 caggaccagg aaaacttggt tctgaatgag
gtatacttag gcaaagagaa atttgacagt 360 gttcattcca agtatatggg
ccgcacaagt tttgattcgg acagttggac cctgagactt 420 cacaatcttc
agatcaagga caagggcttg tatcaatgta tcatccatca caaaaagccc 480
acaggaatga ttcgcatcca ccagatgaat tctgaactgt cagtgcttgc taacttcagt
540 caacctgaaa tagtaccaat ttctaatata acagaaaatg tgtacataaa
tttgacctgc 600 tcatctatac acggttaccc agaacctaag aagatgagtg
ttttgctaag aaccaagaat 660 tcaactatcg agtatgatgg tattatgcag
aaatctcaag ataatgtcac agaactgtac 720 gacgtttcca tcagcttgtc
tgtttcattc cctgatgtta cgagcaatat gaccatcttc 780 tgtattctgg
aaactgacaa gacgcggctt ttatcttcac ctttctctat agagcttgag 840
gaccctcagc ctcccccaga ccacattcct tggattacag ctgtacttcc aacagttatt
900 atatgtgtga tggttttctg tctaattcta tggaaatgga agaagaagaa
gcggcctcgc 960 aactcttata aatgtggaac caacacaatg gagagggaag
agagtgaaca gaccaagaaa 1020 agagaaaaaa tccatatacc tgaaagatct
gatgaagccc agcgtgtttt taaaagttcg 1080 aagacatctt catgcgacaa
aagtgataca tgtttttaat taaagagtaa agcccataca 1140 agtattcatt
ttttctaccc tttcctttgt aagttcctgg gcaacctttt tgatttcttc 1200
cagaaggcaa aaagacatta ccatgagtaa taagggggct ccaggactcc ctctaagtgg
1260 aatagcctcc ctgtaactcc agctctgctc cgtatgccaa gaggagactt
taattctctt 1320 actgcttctt ttcacttcag agcacactta tgggccaagc
ccagcttaat ggctcatgac 1380 ctggaaataa aatttaggac caatacctcc
tccagatcag attcttctct taatttcata 1440 gattgtgttt ttttttaaat
agacctctca atttctggaa aactgccttt tatctgccca 1500 gaattctaag
ctggtgcccc actgaatctt gtgtacctgt gactaaacaa ctacctcctc 1560
agtctgggtg ggacttatgt atttatgacc ttatagtgtt aatatcttga aacatagaga
1620 tctatgtact gtaatagtgt gattactatg ctctagagaa aagtctaccc
ctgctaagga 1680 gttctcatcc ctctgtcagg gtcagtaagg aaaacggtgg
cctagggtac aggcaacaat 1740 gagcagacca acctaaattt ggggaaatta
ggagaggcag agatagaacc tggagccact 1800 tctatctggg ctgttgctaa
tattgaggag gcttgcccca cccaacaagc catagtggag 1860 agaactgaat
aaacaggaaa atgccagagc ttgtgaaccc tgtttctctt gaagaactga 1920
ctagtgagat ggcctgggga agctgtgaaa gaaccaaaag agatcacaat actcaaaaga
1980 gagagagaga gaaaaaagag agatcttgat ccacagaaat acatgaaatg
tctggtctgt 2040 ccaccccatc aacaagtctt gaaacaagca acagatggat
agtctgtcca aatggacata 2100 agacagacag cagtttccct ggtggtcagg
gaggggtttt ggtgataccc aagttattgg 2160 gatgtcatct tcctggaagc
agagctgggg agggagagcc atcaccttga taatgggatg 2220 aatggaagga
ggcttaggac tttccactcc tggctgagag aggaagagct gcaacggaat 2280
taggaagacc aagacacaga tcacccgggg cttacttagc ctacagatgt cctacgggaa
2340 cgtgggctgg cccagcatag ggctagcaaa tttgagttgg atgattgttt
ttgctcaagg 2400 caaccagagg aaacttgcat acagagacag atatactggg
agaaatgact ttgaaaacct 2460 ggctctaagg tgggatcact aagggatggg
gcagtctctg cccaaacata aagagaactc 2520 tggggagcct gagccacaaa
aatgttcctt tattttatgt aaaccctcaa gggttataga 2580 ctgccatgct
agacaagctt gtccatgtaa tattcccatg tttttaccct gcccctgcct 2640
tgattagact cctagcacct ggctagtttc taacatgttt tgtgcagcac agtttttaat
2700 aaatgcttgt tacattc 2717
* * * * *