Note: Descriptions are shown in the official language in which they were submitted.
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SOLUBLE TNF RECEPTORS AND THEIR USE IN TREATMENT OF DISEASE
[0001] This application claims the benefit of PCT application Ser. No.
PCT/US2006/04365 1, filed November 10, 2006 and U.S. Provisional application
Ser. No.
60/862,350, filed October 20, 2006, both of which are incorporated by
reference herein in
their entirety.
FIELD OF THE INVENTION
[0002] The present invention relates to tumor necrosis factor (TNF)
antagonists and
corresponding nucleic acids derived from TNF receptors and their use in the
treatment of
inflammatory diseases. These proteins are soluble secreted decoy receptors
that bind to TNF-
a and prevent TNF-a from signaling to cells.
BACKGROUND OF THE INVENTION
[0003] TNF-a is a pro-inflammatory cytokine that exists as a membrane-bound
homotrimer and is released as a homotrimer into the circulation by the
protease TNF-a
converting enzyme (TACE). TNF-a is introduced into the circulation as a
mediator of the
inflammatory response to injury and infection. TNF-a activity is implicated in
the
progression of inflammatory diseases such as rheumatoid arthritis, Crohn's
disease, ulcerative
colitis, psoriasis and psoriatic arthritis (Palladino, M.A., et al., 2003,
Nat. Rev. Drug Discov.
2:736-46). Acute exposure to high TNF-a levels, as experienced during a
massive infection,
results in sepsis. Its symptoms include shock, hypoxia, multiple organ
failure, and death.
Chronic low-level release of TNF-a is associated with malignancies and leads
to cachexia, a
disease characterized by weight loss, dehydration and fat loss.
[0004] TNF-a activity is mediated primarily through two receptors coded by two
different genes, TNF-a receptor type I (hereafter "TNFR1", exemplified by
GenBank
accession number X55313 for human TNFRl) and TNF-a receptor type II (hereafter
"TNFR2", exemplified by GenBank accession number NM 001066 for human TNFR2).
TNFR1 is a membrane-bound protein with a molecular weight of approximately 55
kilodaltons (kDal), while TNFR2 is a membrane-bound protein with a molecular
weight of
approximately 75 kDal. TNFRl and TNFR2 belong to a family of receptors known
as the
TNF receptor (TNFR) superfamily. The TNFR superfamily is a group of type I
transmembrane proteins, with a carboxy-terminal intracellular domain and an
amino-terminal
extracellular domain characterized by a common cysteine rich domain (CRD).
TNFR1 and
TNFR2 have a unique domain in common, called the pre-ligand-binding assembly
domain
(PLAD) that is required for assembly of multiple receptor subunits and
subsequent binding to
TNF-a.
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[0005] TNFR1 and TNFR2 also share a common gene structure, in which the coding
sequence of each extends over 10 exons separated by 9 introns (Fuchs, et al.,
1992, Genoinics
13:219; Santee, et al., 1996, J. Biol. Chem. 35:21151). Most of the
transmembrane domain
sequence is encoded by the seventh exon ("exon 7") (See FIG. 1).
[0006] Experiments in knockout mice lacking both TNFRI and TNFR2 demonstrated
that the injury-induced immune response to brain injury was suppressed,
suggesting that
drugs that target the TNF signaling pathways may be beneficial in treating
stroke or traumatic
brain injury (Bruce, et al., 1996, Nat. Med. 2:788). TNFR2 knockout mice, but
not TNFR1
knockout mice, were resistant to experimentally-induced cerebral malaria
(Lucas, R., et al.,
1997, Eur. J. Immunol. 27:1719); whereas TNFRI knockout mice were resistant to
autoimmune encephalomyelitis (Suvannavejh, G.C., et al., 2000, Cell. Immunol.,
205:24).
These knockout mice are models for human cerebral malaria and multiple
sclerosis,
respectively.
[00071 TNFR2 is present at high density on T cells of patients with
interstitial lung
disease, suggesting a role for TNFR2 in the immune responses that lead to
alveolitis
(Agostini, C., et al., 1996, Am. J. Respir. Crit. Care Med., 153:1359). TNFR2
is also
implicated in human disorders of lipid metabolism. TNFR2 polymorphism is
associated with
obesity and insulin resistance (Fernandez-Real, et al., 2000, Diabetes Care,
23:831), familial
combined hyperlipidemia (Geurts, et al., 2000, Hum. Mol. Genet. 9:2067),
hypertension and
hypercholesterolemia (Glenn, et al., 2000, Hum. Mol. Genet., 9:1943). In
addition, TNFR2
polymorphism is associated with susceptibility to human narcolepsy (Hohjoh,
H., et al., 2000,
Tissue Antigens, 56:446) and to systemic lupus erythematosus (Komata, T., et
al., 1999,
Tissue Antigens, 53:527).
[0008] To simplify further analysis and comparison, the human TNFR2 461 amino
acid
sequence provided in SEQ ID No: 4, GenBank accession number NP_001057, is used
as a
reference unless stated otherwise (FIG. 1). Amino acid 1 is the first amino
acid of the full
length protein human TNFR2, which includes the signal sequence. Amino acid 23
located in
exon 1 is the first amino acid of the mature protein, which is the protein
after cleavage of the
signal sequence. The transmembrane region spans amino acids 258-287. The exon
6/7
junction is located within the codon that encodes residue 263, while the exon
7/8 junction is
located within the codon that encodes residue 289.
[0009] Physiological, soluble fragments of both TNFR1 and TNFR2 have been
identified.
For example, soluble extracellular domains of these receptors are shed to some
extent from
the cell membrane by the action of metalloproteases (Palladino, M.A., et al.,
2003, Nat. Rev.
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Drug Discov. 2:736-46). Additionally, the pre-mRNA of TNFR2 undergoes
alternative
splicing, creating either a full length, active membrane-bound reccptor, or a
sccreted receptor
that lacks exons 7 and 8 (Lainez et al., 2004, Int. Immunol., 16:169)
("Lainez"). The secreted
protein binds TNF-a but does not elicit a physiological response, hence
reducing overall
TNF-a activity. Although an endogenous, secreted splice variant of TNFR1 has
not yet been
identified, the similar genomic structure of the two receptors suggests that a
TNFR1 splice
variant can be produced.
[0010] The cDNA for the splice variant identified by Lainez contains the 113
bp deletion
of exons 7 and 8. This deletion gives rise to a stop codon 17 bp after the end
of exon 6.
Consequently, the protein has the sequence encoded by the first six exons of
the TNFR2 gene
(residues 1-262) followed by a 6 amino acid tail of Ala-Ser-Leu-Ala-Cys-Arg.
[0011] Additional soluble fragments of recombinantly-engineered TNF receptors
are
known. In particular, truncated forms of TNFRI or TNFR2 have been produced
which have
(1) all or part of the extracellular domain or (2) a TNFR extracellular domain
fused to another
protein.
[0012] Smith discloses truncated human TNFR2s, including a protein with
residues 23-
257, which terminates immediately before the transmembrane region, and a
protein with
residues 23-185 (U.S. Pat. No. 5,945,397). Both TNFR2 fragments are soluble
and capable
of binding TNF-a.
100131 Craig discloses that an extracellular domain of human TNFR2 with
residues 23-
257 fused to the Fe region of human IgGi (TNFR:Fc) is a TNF-a antagonist
capable of
reducing inflammation in rat and mice arthritis models (U.S. Pat. No.
5,605,690). TNFR:Fc
is an FDA-approved treatment for certain forms of arthritis, ankylosing
spondylitis, and
psoriasis and is sold under the name etanercept (Enbrel ).
[0014] Moosmayer demonstrated that soluble human TNFR2 proteins containing the
entire intracellular domain are more active TNF antagonists than the
extracellular domain
alone (Moosmayer et al., 1996, J. Interferon Cytokine Res., 16:471). In those
experiments,
Moosmayer compared the activities of solubilized full length TNFR2 (1-461),
with TNFR2
lacking all but the three C-terminal amino acids of the transmembrane region
(ATM) (1-258
joined to 283-461), TNFR extracellular domain (1-258), and TNFR:Fc. The
inhibition of
TNF-mediated cytotoxicity by the ATM protein and solubiIized full length TNFR2
are
comparable. However, their activities are approximately 60-fold higher than
the TNFR2
extracellular domain alone, but approximately seven-fold less than TNFR:Fc.
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[0015] Since excess TNF-a activity is associated with disease pathogenesis,
particularly
for inflammatory conditions, there is a need for TNF-a antagonists and methods
for their use
in the treatment of inflammatory diseases. Concerns have been raised regarding
the side
effects of currently approved protein-based TNF-a antagonists, including
TNFR:Fc; these
concerns include exacerbation of latent tuberculosis, worsening of congestive
heart failure,
and increased risk of lymphoma (Palladino, M.A., et al., 2003, Nat. Rev. Drug
Discov. 2:736-
46). Furthermore, there are patients who do not respond to currently approved
TNF-a
antagonists. Therefore, there is a continuing need to identify new TNF-a
antagonists.
[0016] To that end, Sazani et a1. have shown, inter alia, that by using splice
switching
oligonucleotides (SSOs) it is possible to generate alternatively spliced mRNA
coding for
variant TNFR1 or TNFR2 proteins using the naturally-occurring exon and intron
structure
(U.S. Appl. Ser. No. 11/595,485). In particular, the SSOs lead the cell to
produce mRNAs
that encode novel TNFR proteins that lack only exon 7, which encodes most of
the
transmembrane region of these proteins. Further characterization of the TNFR2
protein
lacking only exon 7 surprisingly showed that it is a particularly stable,
soluble decoy receptor
that binds to and inactivates extracellular TNF-a. This protein unexpectedly
has anti-TNF-a
activity that is at least equivalent to TNFR:Fc.
SUMMARY OF THE INVENTION
[0017] One embodiment of the invention is a protein, either full length or
mature, which
can bind TNF, is encoded by a cDNA derived from a mammalian TNFR gene, and in
the
cDNA exon 6 is followed directly by exon 8 and as a result lacks exon 7 ("TNFR
07"). In
another embodiment, the invention is a pharmaceutical composition comprising a
TNFR A7.
In a further embodiment, the invention is a method of treating an inflammatory
disease or
condition by administering a pharmaceutical composition comprising a TNFR 07.
[0018] In yet another embodiment, the invention is a nucleic acid that encodes
a TNFR
07. In a further embodiment, the invention is a pharmaceutical composition
comprising a
nucleic acid that encodes a TNFR A7.
[0019J In another embodiment, the invention is an expression vector comprising
a nucleic
acid that encodes a TNFR A7. In a further embodiment, the invention is a
method of
increasing the level of a soluble TNFR in the serum of a mammal by
transforming cells of the
mammal with an expression vector comprising a nucleic acid that encodes a TNFR
07.
[0020] In another embodiment, the invention is a cell transformed with an
expression
vector comprising a nucleic acid that encodes a TNFR 07. In a further
embodiment, the
invention is a method of producing a TNFR 07 by culturing, under conditions
suitable to
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express the TNFR 07, a cell transformed with an expression vector comprising a
nucleic acid
that encodes a TNFR 07. In yet another embodiment, the invention is a method
of treating an
inflammatory disease or condition by administering an expression vector
comprising a
nucleic acid that encodes a TNFR A7.
[0021] In yet another embodiment, splice-switching oligomers (SSOs) are
disclosed that
alter the splicing of a mammalian TNFR2 pre-mRNA to produce a mammalian TNFR2
protein, which can bind TNF and where exon 6 is followed directly by exon 8
and as a result
lacks exon 7( `TNFR2 A7 '). One embodiment of the invention is a method of
treating an
inflammatory disease or condition by administering SSOs to a patient or a live
subject. The
SSOs that are administered alter the splicing of a mammalian TNFR2 pre-mRNA to
produce
a TNFR2 A7. In another embodiment, the invention is a method of producing a
TNFR2,&7
in a cell by administering SSOs to the cell.
[0022] The foregoing and other objects and aspects of the present invcntion
are discussed
in detail in the drawings herein and the specification set forth below.
BRIEF DESCRIPTION OF THE DRAWINGS
[0023] FIG. 1 schematically depicts the human TNFR2 structure. Relevant exons
and
introns are represented by boxes and lines, respectively. The signal sequence
and the
transmembrane region are shaded. Residues that form the boundaries of the
signal sequence,
the transmembrane region, and the final residue are indicated below the
diagram. Exon
boundaries are indicated above the diagram; if the 3' end of an exon and the
5' end of the
following exon have the same residue number, then the splice junction is
located within the
codon encoding that residue.
[0024] FIG. 2A graphically illustrates the amount of soluble TNFR2 from SSO
treated
primary human hepatocytes. The indicated SSO was transfected into primary
human
hepatocytes at 50 nM. After -48 hrs, the extracellular media was analyzed by
enzyme linked
immunosorbant assay (ELISA) for soluble TNFR2 using the Quantikine Human sTNF
RII
ELISA kit from R&D Systems (Minneapolis, MN). Error bars represent the
standard
deviation for 3 independent experiments.
[0025J FIG. 2B: Total RNA was analyzed for TNFR2 splice switching by RT-PCR
using
primers specific for human TNFR2. SSOs targeted to exon seven led to shifting
from full
length TNFR2 mRNA (FL) to TNFR22 07 mRNA (A7). SSO 3083 is a control SSO with
no
TNFR2 splice switching ability.
[0026] FIG. 3 shows the splicing products of L929 cells treated with SSO 10-
mers
targeted to mouse TNFR2 exon 7. L929 cells were transfected with the indicated
SSO
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concentration (50 or 100nM), and evaluated for splice switching of TNFR2 by RT-
PCR 24
hrs later. PCR primers were used to amplify froni Exon 5 to Exon 9, so that
"Full Length"
(FL) TNFR2 is represented by a 486 bp band. Transcripts lacking exon 7 (A7) is
represented
by a 408 bp band.
[0027] FIGs. 4A and 4I3 show the splicing products of mice treated with SSO 10-
mers
targeted to mouse TNFR2 exon 7. The indicated SSOs were resuspended in saline,
and
injected i.p. into mice at 25mg/kg/day for 5 days. Mice were prebled before
SSO injection,
and 10 days after the final SSO injection and sacrificed. At the time of
sacrifice, total RNA
from livers was analyzed for TNFR2 splice switching by RT-PCR. FL - full
length TNFR2;
07 - TNFR2 A7 (FIG. 4A). The concentration of TNFR2 d7 in the serum taken
before (Pre)
and after (Post) SSO injection was determined by ELISA using the Quantikines
Mouse
sTNF RII ELISA kit from R&D Systems (Minneapolis, MN) (FIG. 4B). Error bars
represent
the standard error from 3 independent readings of the same sample.
[0028] FIG. 5 depicts the splice switching ability of SSOs of different
lengths. Primary
human hepatocytes were transfected with the indicated SSO and TNFR2 expression
analyzed
by RT-PCR (top panel) and ELISA (bottom panel) as in Figure 2. Error bars
represent the
standard deviation from 2 independent experiments.
[00291 FIGs. 6A and 6B illustrate TNFR2 A7 mRNA induction in the livers of SSO
treated mice. FIG. 6A: Total RNA from the livers of SSO 3274 treated mice were
subjected
to RT-PCR, and the products visualized on a 1.5% agarose gel. The sequence of
the exon 6-
exon 8 junction is shown in FIG. 6B.
[0030] FIGs. 7A and 7B illustrate TNFR2 07 mRNA induction in SSO treated
primary
human hepatocytes. FIG. 7A: Total RNA from SSO 3379 treated cells were
subjected to RT-
PCR, and the products visualized on a 1.5% agarose gel. The sequence of the
exon 6- exon
8 junction is shown in FIG. 7B.
[0031] FIGs. SA and 8B illustrate the dose dependence of TNFR2 pre-mRNA
splicing
shifting by SSO 3378, 3379 and 3384. Primary human hepatocytes were
transfected with 1-
150nM of the indicated SSO. After -48 hrs, the cells were harvested for total
RNA, and the
extracellular media was collected. FIG. 8A: Total RNA was analyzed for TNFR2
splice
switching by RT-PCR using primers specific for human TNFR2. For each SSO,
amount of
splice switching is plotted as a function of SSO concentration. FIG. SB: The
concentration of
soluble TNFR2 in the extracellular media was determined by ELISA and plotted
as a function
of SSO. Error bars represent the standard deviation for at least 2 independent
experiments.
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100321 FIG. 9 graphically illustrates detection of secreted TNFR2 splice
variants from
L929 cells. Cells were transfected with the indicated SSOs. After 72 hrs, the
extracellular
media was removed and analyzed by ELISA. The data are expressed as pg soluble
TNFR2
per mL.
[0033] FIG. 10 shows the splicing products for intraperitoneal (i.p.)
injection of SSO
3274 (top) and 3305 (bottom) in mice. SSO 3274 was injected i.p. at 25
mg/kg/day for either
4 days (4/1 and 4/10) or 10 days (10/1). Mice were sacrificed either 1 day
(4/1 and 10/1) or
days (4/10) after the last injection and total RNA from liver was analyzed by
RT-PCR for
TNFR2 splice switching as described in Figure 3. SSO 3305 was injected at the
indicated
dose per day for 4 days. Mice were sacrificed the next day and the livers
analyzed as with
3274 treated animals.
[00341 FIG. 1 lA graphically illustrates the amount of soluble TNFR2 in mouse
serum 10
days after SSO treatment. Mice were injected i.p. with the indicated SSO or
saline (n=5 per
group) at 25 mg/kg/day for 10 days. Serum was collected 4 days before
injections began and
on the indicated days after the last injection. Sera was analyzed by ELISA as
described in
Figure 2. At day 10, mice were sacrificed and livers were analyzed for TNFR2
splice
switching by RT-PCR (FIG. 11B) as described in Figure 10.
[0035] FIG. 12A graphically illustrates the amount of soluble TNFR2 in mouse
serum 27
days after SSO treatment. Mice were treated as described in Figure 11, except
that serum
samples were collected until day 27 after the last injection. SSOs 3083 and
3272 are control
SSOs with no TNFR2 splice switching ability. At day 27, mice were sacrificed
and livers
were analyzed for TNFR2 splice switching by RT-PCR (FIG. 12B) as described in
Figure 11.
[0036] FIGs. 13A and 13B graphically depict the anti-TNF-a activity in a cell-
based
assay using serum from SSO treated mice, where serum samples were collected 5
days (FIG.
6A) and 27 days (FIG. 6B) after SSO treatment. L929 cells were treated with
either 0.1
ng/mL TNF-a, or TNF-a plus 10% serum from mice treated with the indicated SSO.
Cell
viability was measured 24 hrs later and normalized to untreated cells.
[0037] FIG. 14 graphically compares the anti-TNF-a activity of serum from the
indicated
SSO oligonucleotide-treated mice to recombinant soluble TNFR2 (rsTNFR2)
extracellular
domain from Sigma and to Enbrel using the cell survival assay described in
Figure 13.
[0038] FIGs. 15A and 15B compare the stability of muTNFR2 7 protein (FIG.
15A) and
mRNA (FIG. 15B). Mice were injected at 25 mg/kg/day daily with either SSO,
3272, SSO
3274 or SSO 3305 (n = 5). Mice were bled on the indicated day after the last
injection and
the serum TNFR2 concentration was measured. Total RNA from mice sacrificed on
the
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indicated day after the last injection of SSO was subjected to RT-PCR as
described in Figure
10.
[0039] FIG. 16 plots TNFR2 A7 protein (dashed line) and mRNA (solid line)
levels over
time, as a percentage of the amount of protein or mRNA, respectively, 10 days
after the last
injection.
[0040] FIG. 17 graphically illustrates the dose dependant anti-T'rdF-a
activity of TNFR2
07 expressed in HeLa cells after transfection with TNFR2 A7 mammalian
expression
plasmids. HeLa cells were transfected with the indicated mouse or human TNFR2
A7
plasmid and extracellular media was collected after 48 hrs. The TNFR2 A7
concentration in
the media was determined by ELISA and serial dilutions were prepared. These
dilutions
were assayed for anti-TNF-a activity by the L929 cytoxicity assay as in FIG,
14.
[0041] FIG. 18 shows expressed mouse (A) and human (B) TNFR2 07 protein
isolated
by polyacrylamide gel electrophoresis (PAGE). HeLa cells were transfected with
the
indicated plasmid. After -48 hrs, the extracellular media was collected and
concentrated, and
cells were collected in RIPA lysis buffer. The proteins in the samples were
separated by
PAGE and a western blot was performed using a C-terminal TNFR2 primary
antibody
(Abeam) that recognizes both the human and mouse TNFR2 07 proteins. Media,
extracellular media samples from HeLa cells transfected with the indicated
plasmid; Lysate,
cell lysate from Hela cells transfected with the indicated plasmid. CM,
control media from
untransfected HeLa cells; CL, control cell lysates from untransfected HeLa
cells. +,
molecular weight markers (kDal).
[0042] FIG. 19 shows purified His-tagged human and mouse TNFR2 07.
Unconcentrated
extracellular media containing the indicated TNFR2 A7 protein was prepared as
in Figure 18.
Approximately 32 mL of the media was applied to a 1 mL HisPur cobalt spin
column
(Pierce), and bound proteins were eluted in 1 mL buffer containing 150 mM
imidazole.
Samples of each were analyzed by PAGE and western blot was performed as in
Figure 18.
The multiple bands in lanes 1144-4 and 1319-1 represent variably glycosylated
forms of
TNFR2,&7.
DETAILED DESCRIPTION OF THE INVENTION
[0043] Definitions:
[0044] As used herein, the terms "tumor necrosis factor receptor", "TNF
receptor", and
"TNFR" refer to proteins having amino acid sequences of or which are
substantially similar
to native mammalian TNF receptor sequences, and which are capable of binding
TNF
molecules. In this context, a "native" receptor or gene for such a receptor,
means a receptor
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or gene that occurs in nature, as well as the naturally-occurring allelic
variations of such
receptors and genes.
[0045] The term "mature" as used in connection with a TNFR means a protein
expressed
in a form lacking a leader or signal sequence as may be present in full-length
transcripts of a
native gene.
[0046] The nomenclature for TNFR proteins as used herein follows the
convention of
naming the protein (e.g., TNFR2) preceded by a species designation, e.g., hu
(for human) or
mu (for murine), followed by a A (to designate a deletion) and the number of
the exon(s)
deleted. For example, huTNFR2 A7 refers to human TNFR2 lacking exon 7. In the
absence
of any species designation, TNFR refers generically to mammalian TNFR.
[0047] The term "secreted" means that the protein is soluble, i.e., that it is
not bound to
the cell membrane. In this context, a form will be soluble if using
conventional assays known
to one of skill in the art most of this form can be detected in fractions that
are not associated
with the membrane, e.g., in cellular supernatants or serum.
[0048] The term "stable" means that the secreted TNFR form is detectable using
conventional assays by one of skill in the art, such as, western blots, ELISA
assays in
harvested cells, cellular supernatants, or serum.
[0049] As used herein, the terms "tumor necrosis factor" and "TNF" refer to
the
naturally-occuring protein ligands that bind to TNF receptors. TNF includes,
but is not
limited to, TNF-a and TNF- j3.
[00501 As used herein, the term "an inflammatory disease or condition" refers
to a
disease, disorder, or other medical condition that at least in part results
from or is aggravated
by the binding of TNF to its receptor. Such diseases or conditions include,
but are not limited
to, those associated with increased levels of TNF, increased levels of TNF
receptor, or
increased sensitization or deregulation of the corresponding signaling
pathway. The term
also encompasses diseases and conditions for which known TNF antagonists have
been
shown useful. Examples of inflammatory diseases or conditions include, but are
not limited
to, rheumatoid arthritis, juvenile rheumatoid arthritis, psoriasis, psoriatic
arthritis, ankylosing
spondylitis, inflammatory bowel disease (including Crohn's disease and
ulcerative colitis),
hepatitis, sepsis, alcoholic liver disease, and non-alcoholic steatosis.
[0051] As used herein, the term "hepatitis" refers to a gastroenterological
disease,
condition, or disorder that is characterized, at least in part, by
inflammation of the liver.
Examples of hepatitis include, but are not limited to, hepatitis associated
with hepatitis A
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virus, hepatitis B virus, hepatitis C virus, or liver inflammation associated
with
ischemi a/reperfusion.
[0052] As used herein, the term "TNF antagonist" means that the protein is
capable of
measurable inhibition of TNF-mediated cytotoxicity using standard assays as
are well known
in the art. (See, e.g., Example 1 below, L929 cytotoxicity assay).
[0053] The term "binds TNF" means that the protein can bind detectable levels
of TNF,
preferably TNF-a, as measured by standard binding assays as are well known in
the art (See,
e.g., U.S. Pat. No. 5,945,397 to Smith, cols. 16-17). Preferably, receptors of
the present
invention are capable of binding greater than 0.1 nmoles TNF-a/nmole receptor,
and more
preferably, greater than 0.5 nmoles TNF-a/nmole receptor using standard
binding assays.
[0054] As used herein, the term "regulatory element" refers to a nucleotide
sequence
involved in an interaction of molecules that contributes to the functional
regulation of a
nucleic acid, including but not limited to, replication, duplication,
transcription, splicing,
translation, or degradation of the nucleic acid. The regulation may be
enhancing or inhibitory
in nature. Regulatory elements known in the art include, for example,
transcriptional
regulatory sequences such as promoters and enhancers. A promoter is a DNA
region that is
capable under certain conditions of aiding the initiation of transcription of
a coding region
usually located downstream (in the 3' direction) from the promoter.
[0055] As used herein, the term "operably linked" refers to a juxtaposition of
genetic
elements, wherein the elements are in a relationship permitting them to
operate in the
expected manner. For example, a promoter is operably linked to a coding region
if the
promoter helps initiate transcription of the coding sequence. As long as this
functional
relationship is maintained, there can be intervening residues between the
promoter and the
coding region.
100561 As used herein, the terms "transformation" or "transfection" refer to
the insertion
of an exogenous nucleic acid into a cell, irrespective of the method used for
the insertion, for
example, lipofection, transduction, infection or electroporation. The
exogenous nucleic acid
can be maintained as a non-integrated vector, for example, a plasmid, or
altematively, can be
integrated into the cell's genome.
[0057] As used herein, the term "vector" refers to a nucleic acid molecule
capable of
transporting another nucleic acid to which it has been linked. One type of
vector is a
"plasmid", which refers to a circular double stranded DNA loop into which
additional DNA
segments can be ligated. Another type of vector is a viral vector, wherein
additional DNA
segments can be ligated into the viral genome. Certain vectors are capable of
autonomous
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replication in a host cell into which they are introduced (e.g., bacterial
vectors having a
bacterial origin of replication and episomal mammalian vectors). Other vectors
(e.g., non-
episomal mammalian vectors) are integrated into the genome of a host cell upon
introduction
into the host cell, and thereby are replicated along with the host genome.
Moreover, certain
vectors, expression vectors, are capable of directing the expression of genes
to which they are
operably linked. In general, expression vectors of utility in recombinant DNA
techniques are
often in the form of plasmids. or viral vectors (e.g., replication defective
retroviruses,
adenoviruses and adeno-associated viruses).
[0058] As used herein, the term "isolated protein" refers to a protein or
polypeptide that
is not naturally-occurring and/or is separated from one or more components
that are naturally
associated with it.
[0059] As used herein, the term "isolated nucleic acid" refers to a nucleic
acid that is not
naturally-occurring and/or is in the form of a separate fragment or as a
component of a larger
construct, which has been derived from a nucleic acid isolated at least once
in substantially
pure form, i.e., free of contaminating endogenous materials, and in a quantity
or
concentration enabling identification and manipulation by standard biochemical
methods, for
example, using a cloning vector.
[0060] As used herein the term "purified protein" refers to a protein that is
present in the
substantial absence of other protein. However, such purified proteins can
contain other
proteins added as stabilizers, carriers, excipients, or co-therapeutics. The
term "purified" as
used herein preferably means at least 80% by dry weight, more preferably in
the range of 95-
99% by weight, and most preferably at least 99.8% by weight, of protein
present, excluding
proteins added as stabilizers, carriers, excipients, or co-therapeutics.
[0061] As used herein, the term "altering the splicing of a pre-mRNA" refers
to altering
the splicing of a cellular pre-mRNA target resulting in an altered ratio of
splice products.
Such an alteration of splicing can be detected by a variety of techniques well
known to one of
skill in the art. For example, RT-PCR on total cellular RNA can be used to
detect the ratio of
splice products in the presence and the absence of an SSO.
[0062] As used herein, the term "complementary" is used to indicate a
sufficient degree
of complementarity or precise pairing such that stable and specific binding
occurs between an
oligonucleotide and a DNA or RNA containing the target sequence. It is
understood in the
art that the sequence of an oligonucleotide need not be 100% complementary to
that of its
target. For example, for an SSO there is a sufficient degree of
complementarity when, under
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conditions which permit splicing, binding to the target will occur and non-
specific binding
will be avoided.
[0063] Proteins:
[0064] One embodiment of the present invention is a protein, either full
length or mature,
which is encoded by a cDNA derived from a mammalian TNFR gene, and in the cDNA
exon
6 is followed directly by exon 8 and as a result lacks exon 7. Furthermore the
protein can
bind TNF, preferably TNF-a, and can act as a TNF, preferably TNF-a,
antagonist.
Preferably, TNFR of the present invention is capable of inhibition of TNF-
mediated
cytotoxicity to a greater extent than the soluble extracellular domain alone,
and more
preferably, to an extent comparable to or greater than TNFR:Fc. Mammalian TNFR
according to the present disclosure includes, but is not limited to, human,
primate, murine,
canine, feline, bovine, ovine, equine, and porcine TNFR. Furthermore,
mammalian TNFR
according to the present disclosure includes, but is not limited to, a protein
sequence that
results from one or more single nucleotide polymorphisms, such as for example
those
disclosed in EP Pat. Appl. 1,172,444, as long as the protein retains a
comparable biological
activity to the reference sequence with which it is being compared.
[0065] In one embodiment, the mammalian TNFR is a mammalian TNFR1, preferably
a
human TNFRI. For human TNFRI two non-limiting examples of this embodiment are
given
by huTNFRl A7 which includes the signal sequence as shown in SEQ ID No: 6 and
mature
huTNFRl A7 (amino acids 30-417 of SEQ ID No: 6) which lacks the signal
sequence. The
sequences of these huTNFRl a7 proteins are either amino acids 1-208 of wild
type human
TNFRI (SEQ ID No: 2) which includes the signal sequence or 30-208 of wild type
human
TNFRI for mature huTNFRl A7 which lacks the signal sequence, and in either
case is
followed immediately by amino acids 247-455 of wild type human TNFRI.
[0066] In another preferred embodiment, the mammalian TNFR is a mammalian
TNFR2,
most preferably a human TNFR2. For human TNFR2 two non-limiting examples of
this
embodiment are given by huTNFR2 07 which includes the signal sequence as shown
in SEQ
ID No: 10 or mature huTNFR2 A7 (amino acids 23-435 of SEQ ID No: 10) which
lacks the
signal sequence. The sequences of these huTNFR2 A7 proteins are either amino
acids 1-262
of wild type human TNFR2 (SEQ ID No: 4) which includes the signal sequence or
23-262 of
wild type human TNFR2 for mature huTNFR2 A7 which lacks the signal sequence,
followed
in either case by the amino acid glutamate, because of the creation of a
unique codon at the
exon 6-8 junction, which is followed by amino acids 290-461 of wild type human
TNFR2.
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[0067] The proteins of the present invention also include those proteins that
are
chemically modified. Chemical modification of a protein refers to a protein
where at least
one of its amino acid residues is modified by either natural processes, such
as processing or
other post-translational modifications, or by chemical modification techniques
known in the
art. Such modifications include, but are not limited to, acetylation,
acylation, amidation,
ADP-ribosylation, glycosylation, methylation, pegylation, prenylation,
phosphorylation, or
cholesterol conjugation.
[0068] Nucleic Acids:
[0069] One embodiment of the present invention is a nucleic acid that encodes
a protein,
either full length or mature, which is encoded by a eDNA derived from a
mammalian TNFR
gene, and in the cDNA exon 6 is followed directly by exon 8 and as a result
lacks exon 7.
[00701 Such sequences are preferably provided in the form of an open reading
frame
uninterrupted by internal nontranslated sequences, or introns, which are
typically present in
eukaryotic genes. Genomic DNA containing the relevant sequences can also be
used. In one
embodiment, the nucleic acid is either an mRNA or a eDNA. In another
embodiment, it is
genomic DNA.
[0071] In one embodiment, the mammalian TNFR is a mammalian TNFR1. For this
embodiment, the mammalian TNFR1 is preferably a human TNFR1. For human TNFRI,
two non-limiting examples of this embodiment are nucleic acids which encode
the huTNFRI
A7 which includes the signal sequence as shown in SEQ ID No: 6 and mature
huTNFR1 07
(amino acids 30-417 of SEQ ID No: 6) which lacks the signal sequence.
Preferably, the
sequences of these huTNFR1 07 nucleic acids are nucleotides 1-1251 of SEQ ID
No: 5,
which includes the signal sequence and nucleotides 88-1251 of SEQ ID No: 5
which lacks
the signal sequence. The sequences of these huTNFRl A7 nucleic acids are
either
nucleotides 1-625 of wild type human TNFRl (SEQ ID No: 1) which includes the
signal
sequence or 88-625 of wild type human TNFRI for mature huTNFR2 A7 which lacks
the
signal sequence, and in either case is followed immediately by amino acids 740-
1368 of wild
type human TNFRI.
[0072] In another preferred embodiment, the mammalian TNFR is a mammalian
TNFR2,
most preferably a human TNFR2. For human TNFR2, two non-limiting examples of
this
embodiment are nucleic acids which encode the huTNFR2 A7 which includes the
signal
sequence as shown in SEQ ID No: 10 or mature huTNFR2 A7 (amino acids 23-435 of
SEQ
ID No: 10) which lacks the signal sequence. Preferably, the sequences of these
huTNFR2 07
nucleic acids are nucleotides 1-1305 of SEQ ID No: 9 which includes the signal
sequence and
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nucleotides 67-1305 of SEQ ID No: 9 which lacks the signal sequence. The
sequences of
these huTNFR2 07 nucleic acids are either nucleotides 1-787 of wild type human
TNFR2
(SEQ ID No: 3) which includes the signal sequence or 67-787 of wild type human
TNFR2 for
mature huTNFR2 A7 which lacks the signal sequence, and in either case is
followed
immediately by amino acids 866-1386 of wild type human TNFR2.
[0073] The bases of the nucleic acids of the present invention can be the
conventional
bases cytosine, guanine, adenine and uracil or thymidine. Alternatively,
modified bases can
be used. Other suitable bases include, but are not limited to, 5-
methylcytosine (MeC),
isocytosine, pseudoisocytosine, 5-bromouracil, 5-propynyluracil, 5-propyny-6,
5-
methylthiazoleuracil, 6-aminopurine, 2-aminopurine, inosine, 2,6-
diaminopurine, 7-propyne-
7-deazaadenine, 7-propyne-7-deazaguanine, 2-chloro-6-aminopurine and 9-
(aminoethoxy)phenoxazine.
10074] Suitable nucleic acids of the present invention include numerous
alternative
chemistries. For example, suitable nucleic acids of the present invention
include, but are not
limited to, those wherein at least one of the internucleotide bridging
phosphate residues is a
modified phosphate, such as phosphorothioate, methyl phosphonate, methyl
phosphonothioate, phosphoromorpholidate, phosphoropiperazidate, and
phosphoroamidate.
In another non-limiting example, suitable nucleic acids of the present
invention include those
wherein at least one of the nucleotides contain a 2' lower alkyl moiety (e.g.,
CI -C4, linear or
branched, saturated or unsaturated alkyl, such as methyl, ethyl, ethenyl,
propyl, 1-propenyl,
2-propenyl, and isopropyl).
[0075] Nucleic acids of the present invention also include, but are not
limited to, those
wherein at least one, of the nucleotides is a nucleic acid analogue. Examples
of such
analogues include, but are not limited to, hexitol (HNA) nucleotides, 2'O-4'C-
linked bicyclic
ribofuranosyl (LNA) nucleotides, peptide nucleic acid (PNA) analogues, N3'--
>P5'
phosphoramidate analogues, phosphorodiamidate morpholino nucleotide analogues,
and
combinations thereof.
[0076] Nucleic acids of the present invention include, but are not limited to,
modifications of the nucleic acids involving chemically linking to the nucleic
acids one or
more moieties or conjugates. Such moieties include, but are not limited to,
lipid moieties
such as a cholesterol moiety, cholic acid, a thioether, e.g. hexyl-S-
tritylthiol, a
thiocholesterol, an aliphatic chain, e.g., dodecandiol or undecyl residues, a
phospholipids,
e.g., di-hexadecyl-rac-glycerol or triethylammonium 1,2-di-O-hexadecyl-rac-
glycero-3-H-
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phosphonate, a polyamine or a polyethylene glycol chain, an adamantane acetic
acid, a
palmityl moiety, an octadecylamine or hexylamino-carbonyl-oxycholesterol
moiety.
[0077] Pharmaceutical Compositions and Preparations:
[0078] Other embodiments of the invention are pharmaceutical compositions
comprising
the foregoing proteins and nucleic acids.
[0079] The nucleic acids and proteins of the present invention may be admixed,
encapsulated, conjugated, or otherwise associated with other molecules,
molecule structures,
or mixtures of compounds, as for example liposomes, receptor targeted
molecules, oral,
rectal, topical or other formulations, for assisting in uptake, distribution,
and/or absorption.
[0080] Formulations of the present invention comprise nucleic acids and
proteins in a
physiologically or pharmaceutically acceptable carrier, such as an aqueous
carrier. Thus
formulations for use in the present invention include, but are not limited to,
those suitable for
parenteral administration including intra-articular, intraperitoneal,
intravenous, intraarterial,
subcutaneous, or intramuscular injection or infusion, as well as those
suitable for topical,
ophthalmic, vaginal, oral, rectal or pulmonary administration (including
inhalation or
insufflation of powders or aerosols, including by nebulizer, intratracheal,
and intranasal
delivery). The formulations may conveniently be presented in unit dosage form
and may be
prepared by any of the methods well known in the art. The most suitable route
of
administration in any given case may depend upon the subject, the nature and
severity of the
condition being treated, and the particular active compound which is being
used.
[00811 Pharmaceutical compositions of the present invention include, but are
not limited
to, physiologically and pharmaceutically acceptable salts, i.e., salts that
retain the desired
biological activity of the parent compound and do not impart undesired
toxicological
properties. Examples of such salts are (a) salts formed with cations such as
sodium,
potassium, NH4+, magnesium, calcium, polyamines such as spermine and
spermidine, etc.;
(b) acid addition salts formed with inorganic acids, for example, hydrochloric
acid,
hydrobromic acid, sulfuric acid, phosphoric acid, nitric acid and the like;
and (c) salts formed
with organic acids such as, for example, acetic acid, oxalic acid, tartaric
acid, succinic acid,
maleic acid, fumaric acid, gluconic acid, citric acid, malic acid, ascorbic
acid, benzoic acid,
tannic acid, palmitic acid, alginic acid, polyglutamic acid,
napthalenesulfonic acid,
methanesulfonic acid, p-toluenesulfonic acid, napthalenedisulfonic acid,
polygalacturonic
acid, and the like.
[0082] The present invention provides for the use of proteins and nucleic
acids as set
forth above for the preparation of a medicament for treating a patient
afflicted with an
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inflammatory disorder involving excessive activity of TNF, as discussed below.
In the
manufacture of a medicament according to the invention, the nucleic acids and
proteins of the
present invention are typically admixed with, inter alia, an acceptable
carrier. The carrier
must, of course, be acceptable in the sense of being compatible with other
ingredients in the
formulation and must not be deleterious to the patient. The carrier may be a
solid or liquid.
Nucleic acids and proteins of the present invention are incorporated in
formulations, which
may be prepared by any of the well known techniques of pharmacy consisting
essentially of
admixing the components, optionally including one or more accessory
therapeutic
ingredients.
[0083] Formulations of the present invention may comprise sterile aqueous and
non-
aqueous injection solutions of the active compounds, which preparations are
preferably
isotonic with the blood of the intended recipient and essentially pyrogen
free. These
preparations may contain anti-oxidants, buffers, bacteriostats, and solutes
which render the
formulation isotonic with the blood of the intended recipient. Aqueous and non-
aqueous
sterile suspensions can include, but are not limited to, suspending agents and
thickening
agents. The formulations may be presented in unit dose or multi-dose
containers, for
example, sealed ampoules and vials, and may be stored in freeze-dried
(lyophilized)
condition requiring only the addition of the sterile liquid carrier, for
example, saline or water-
for-injection immediately prior to use.
[0084) In the formulation the nucleic acids and proteins of the present
invention may be
contained within a particle or vesicle, such as a liposome or microcrystal,
which may be
suitable for parenteral administration. The particles may be of any suitable
structure, such as
unilamellar or plurilameller, so long as the nucleic acids and proteins of the
present invention
are contained therein. Positively charged lipids such as N-[1-(2,3-
dioleoyloxy)propyl]-
N,N,N-trimethyl-ammoniummethylsulfate, or "DOTAP," are particularly preferred
for such
particles and vesicles. The preparation of such lipid particles is well known
(See references
in U.S. Pat. No. 5,976,879 col. 6).
[0085] Expression Vectors and Host Cells:
[0086] The present invention provides expression vectors to amplify or express
DNA
encoding mammalian TNFR of the current invention. The present invention also
provides
host cells transformed with the foregoing expression vectors. Expression
vectors are
replicable DNA constructs which have synthetic or eDNA-derived DNA fragments
encoding
mammalian TNFR or bioequivalent analogues operably linked to suitable
transcriptional or
translational regulatory elements derived from mammalian, microbial, viral, or
insect genes.
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A transcriptional unit generally comprises an assembly of (a) a genetic
element or elements
having a regulatory role in gene expression, such as, transcriptional
promoters or enhancers,
(b) a structural or coding sequence which is transcribed into mRNA and
translated into
protein, and (c) appropriate transcription and translation initiation and
termination sequences.
Such regulatory elements can include an operator sequence to control
transcription, and a
sequence encoding suitable mRNA ribosomal binding sites. The ability to
replicate in a host,
usually conferred by an origin of replication, and a selection gene to
facilitate recognition of
transformants, can additionally be incorporated.
[0087] DNA regions are operably linked when they are functionally related to
each other.
For example, DNA for a signal peptide (secretory leader) is operably linked to
DNA for a
polypeptide if it is expressed as a precursor which participates in the
secretion of the
polypeptide; a promoter is operably linked to a coding sequence if it controls
the transcription
of the sequence; or a ribosome binding site is operably linked to a coding
sequence if it is
positioned so as to permit translation. Generally, operably linked means
contiguous and, in
the case of secretory leaders, contiguous and in reading frame. Structural
elements intended
for use in yeast expression systems preferably include a leader sequence
enabling
extracellular secretion of translated protein by a host cell. Alternatively,
where recombinant
protein is expressed without a leader or transport sequence, it may include an
N-terminal
methionine residue. This residue may optionally be subsequently cleaved from
the expressed
protein to provide a final product.
100881 Mammalian TNFR DNA is expressed or amplified in a recombinant
expression
system comprising a substantially homogeneous monoculture of suitable host
microorganisms, for example, bacteria such as E. coli or yeast such as S.
cerevisiae, which
have stably integrated (by transformation or transfection) a recombinant
transcriptional unit
into chromosomal DNA or carry the recombinant transcriptional unit as a
component of a
resident plasmid. Recombinant expression systems as defined herein will
express
heterologous protein either constitutively or upon induction of the regulatory
elements linked
to the DNA sequence or synthetic gene to be expressed.
[0089] Transformed host cells are cells which have been transformed or
transfected with
mammalian TNFR vectors constructed using recombinant DNA techniques.
Transformed
host cells ordinarily express TNFR, but host cells transformed for purposes of
cloning or
amplifying TNFR DNA do not need to express TNFR. Suitable host cells for
expression of
mammalian TNFR include prokaryotes, yeast, fungi, or higher eukaryotic cells.
Prokaryotes
include gram negative or gram positive organisms, for example E. coli or
bacilli. Higher
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eukaryotic cells include, but are not limited to, established insect and
mammalian cell lines.
Cell-free translation systems can also be employed to produce mammalian TNFR
using
RNAs derived from the DNA constructs of the present invention. Appropriate
cloning and
expression vectors for use with bacterial, fungal, yeast, and mammalian
cellular hosts are
well known in the art.
[0090] Prokaryotic expression hosts may be used for expression of TNFR that do
not
require extensive proteolytic and disulfide processing. Prokaryotic expression
vectors
generally comprise one or more phenotypic selectable markers, for example a
gene encoding
proteins conferring antibiotic resistance or supplying an autotrophic
requirement, and an
origin of replication recognized by the host to ensure amplification within
the host. Suitable
prokaryotic hosts for transformation include E. coli, Bacillus subtilis,
Salmonella
typhimurium, and various species within the genera Pseudomonas, Streptomyces,
and
Staphyolococcus, although others can also be employed as a matter of choice.
[0091] Useful expression vectors for bacterial use can comprise a selectable
marker and
bacterial origin of replication derived from commercially available plasmids
comprising
genetic elements of the well known cloning vector pBR322 (ATCC 37017). These
pBR322
"backbone" sections are combined with an appropriate promoter and the
structural sequence
to be expressed. pBR322 contains genes for ampicillin and tetracycline
resistance and thus
provides simple means for identifying transformed cells. Such commercial
vectors include,
for example, the series of Novagen pET vectors (ENID Biosciences, Inc.,
Madison, Wis.).
[0092] Promoters commonly used in recombinant microbial expression vectors
include
the lactose promoter system, and the X PL promoter, the T7 promoter, and the
T7 lac
promoter. A particularly useful bacterial expression system, Novagen pET
system (EMD
Biosciences, Inc., Madison, Wis.) employs a T7 or T7 lac promoter and E. coli
strain, such as
BL21(DE3) which contain a chromosomal copy of the T7 RNA polymerase gene.
[00931 TNFR proteins can also be expressed in yeast and fungal hosts,
preferably from
the genus Saccharomyces, such as S. cerevisiae. Yeast of other genera, such as
Pichia or
Kluyveromyces can also be employed. Yeast vectors will generally contain an
origin of
replication from the 2 yeast plasmid or an autonomously replicating sequence
(ARS),
promoter, DNA encoding TNFR, sequences for polyadenylation and transcription
termination
and a selection gene. Preferably, yeast vectors will include an origin of
replication and
selectable marker permitting transformation of both yeast and E. coli, e.g.,
the ampicillin
resistance gene of E. coli and S. cerevisiae TRPI or URA3 gene, which provides
a selection
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marker for a mutant strain of yeast lacking the ability to grow in tryptophan
or uracil,
respectively, and a promoter derived from a highly expressed yeast gene to
induce
transcription of a structural sequence downstream. The presence of the TRP1 or
URA3
lesion in the yeast host cell genome then provides an effective environment
for detecting
transformation by growth in the absence of tryptophan or uracil, respectively.
[0094] Suitable promoter sequences in yeast vectors include the promoters for
metallothionein, 3-phosphoglycerate kinase or other glycolytic enzymes , such
as enolase,
glyceraldehyde-3-phosphate dehydrogenase, hexokinase, pyruvate decarboxylase,
phosphofructokinase, glucose-6-phosphate isomerase, 3-phosphoglycerate mutase,
pyruvate
kinase, triosephosphate isomerase, phosphoglucose isomerase, and glucokinase.
Suitable
vectors and promoters for use in yeast expression are well known in the art.
[0095] Preferred yeast vectors can be assembled using DNA sequences from pUC
18 for
selection and replication in E. coli (Amp` gene and origin of replication) and
yeast DNA
sequences including a glucose-repressible ADH2 promoter and a-factor secretion
leader. The
yeast a-factor leader, which directs secretion of heterologous proteins, can
be inserted
between the promoter and the structural gene to be expressed. The leader
sequence can be
modified to contain, near its 3' end, one or more useful restriction sites to
facilitate fusion of
the leader sequence to foreign genes. Suitable yeast transformation protocols
are known to
those of skill in the art.
[0096] Host strains transformed by vectors comprising the ADH2 promoter may be
grown for expression in a rich medium consisting of 1% yeast extract, 2%
peptone, and 1%
or 4% glucose supplemented with 80 g/rnl adenine and 80 g/ml uracil.
Derepression of the
ADH2 promoter occurs upon exhaustion of medium glucose. Crude yeast
supernatants are
harvested by filtration and held at 4 C. prior to further purification.
[0097] Various mammalian or insect cell culture systems are also
advantageously
employed to express TNFR protein. Expression of recombinant proteins in
mammalian cells
is particularly preferred because such proteins are generally correctly
folded, appropriately
modified and completely functional. Examples of suitable mammalian host cell
lines include
the COS-7 lines of monkey kidney cells, and other cell lines capable of
expressing an
appropriate vector including, for example, L cells, such as L929, C127, 3T3,
Chinese hamster
ovary (CHO), HeLa and BHK cell lines. Mammalian expression vectors can
comprise
nontranscribed elements such as an origin of replication, a suitable promoter,
for example, the
CMVie promoter, the chicken beta-actin promoter, or the composite hEF1-HTLV
promoter,
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and enhancer linked to the gene to be expressed, and other 5' or 3' flanking
nontranscribed
sequences, and 5' or 3' nontranslated sequences, such as necessary ribosome
binding sites, a
polyadenylation site, splice donor and acceptor sites, and transcriptional
termination
sequences. Baculovirus systems for production of heterologous proteins in
insect cells are
known to those of skill in the art.
[0098] The transcriptional and translational control sequences in expression
vectors to be
used in transforming vertebrate cells can be provided by viral sources. For
example,
commonly used promoters and enhancers are derived from Polyoma, Adenovirus 2,
Simian
Virus 40 (SV40), human cytomegalovirus, such as the CMVie promoter, HTLV, such
as the
composite hEF1-HTLV promoter. DNA sequences derived from the SV40 viral
genome, for
example, SV40 origin, early and late promoter, enhancer, splice, and
polyadenylation sites
can be used to provide the other genetic elements required for expression of a
heterologous
DNA sequence.
[0099] Further, mammalian genomic TNFR promoter, such as control and/or signal
sequences can be utilized, provided such control sequences are compatible with
the host cell
chosen.
[0100] In preferred aspects of the present invention, recombinant expression
vectors
comprising TNFR cDNAs are stably integrated into a host cell's DNA.
[0101] Accordingly one embodiment of the invention is a method of treating an
inflammatory disease or condition by administering a stable, secreted, ligand-
binding form of
a TNF receptor, thereby decreasing the activity of TNF for the receptor. In
another
embodiment, the invention is a method of treating an inflammatory disease or
condition by
administering an oligonucleotide that encodes a stable, secreted, ligand-
binding form of a
TNF receptor, thereby decreasing the activity of TNF for the receptor. In
another
embodiment, the invention is a method of producing a stable, secreted, ligand-
binding form
of a TNF receptor.
[0102] The following aspects of the present invention discussed below apply to
the
foregoing embodiments.
[0103] The methods, nucleic acids, proteins, and formulations of the present
invention are
also useful as in vitro or in vivo tools.
[0104] Embodiments of the invention can be used to treat any condition in
which the
medical practitioner intends to limit the effect of TNF or a signalling
pathway activated by it.
In particular, the invention can be used to treat an inflammatory disease. In
one embodiment,
the condition is an inflammatory systemic disease, e.g., rheumatoid arthritis
or psoriatic
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arthritis. In another embodiment, the disease is an inflammatory liver
disease. Examples of
inflammatory liver diseases include, but are not limited to, hepatitis
associated with the
hepatitis A, B, or C viruses, alcoholic liver disease, and non-alcoholic
steatosis. In yet
another embodiment, the inflammatory disease is a skin condition such as
psoriasis.
[0105] The uses of the present invention include, but are not limited to,
treatment of
diseases for which known TNF antagonists have been shown useful. Three
specific TNF
antagonists are currently FDA-approved. The drugs are etanercept (Enbrel ),
infliximab
(Remicade ) and adalimumab (Humira ). One or more of these drugs is approved
for the
treatment of rheumatoid arthritis, juvenile rheumatoid arthritis, psoriasis,
psoriatic arthritis,
ankylosing spondylitis, and inflammatory bowel disease (Crohn's disease or
ulcerative
colitis).
[0106] Protein Expression and Purification:
[0107] When mammalian or insect cells are used, properly expressed TNFR
protein will
be secreted into the extracellular media. The protein is recovered from the
media, and is
concentrated and is purified using standard biochemical techniques. After
expression in
mammalian cells by lentiviral or AAV transduction, plasmid transfection, or
any similar
pr,ocedure, or in insect cells after baculoviral transduction, the
extracellular media of these
cells is concentrated using concentration filters with an appropriate
molecular weight cutoff,
such as Amicon filtration units. To avoid loss of TNFR protein, the filter
should allow
proteins to flow through that are at or below 50 kDal.
[0108] When TNFR protein is expressed in bacterial culture it can be purified
by standard
biochemical techniques. Bacteria are lysed, and the cellular extract
containing the TNFR is
desalted and is concentrated.
[0109] In either case, the TNFR protein is preferably purified by affinity
chromatography. The use of column chromatography with an affinity matrix
comprising
TNF-a is preferred. Alternatively, an affinity purification tag can be added
to either the N- or
the C-terminus of the TNFR protein. For example, a polyhistidine-tag (His-
tag), which is an
amino acid motif with at least six histidines, can be used for this purpose
(Hengen, P., 1995,
Trends Biochem. Sci. 20:285-86). The addition of a His-tag can be achieved by
the in-frame
addition of a nucleotide sequence encoding the His-tag directly to either the
5' or 3' end of
the TNFR open reading frame in an expression vector. One such nucleotide
sequence for the
addition of a C-terminal His-tag is given in SEQ ID No: 126. When a His-tag is
incorporated
into the protein, a nickel or cobalt affinity column is employed to purify the
tagged TNFR,
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and the His-tag can optionally then be cleaved. Other suitable affinity
purification tags and
methods of purification of proteins with those tags are well known in the art.
101101 Alternatively, a non-affinity based purification scheme can be used,
involving
fractionation of the TNFR extracts on a series of columns that separate the
protein based on
size (size exclusion chromatography), charge (anion and cation exchange
chromatography)
and hydrophobicity (reverse phase chromatography). High performance liquid
chromatography can be used to facilitate these steps.
[01111 Other methods for the expression and purification of TNFR proteins are
well
known (See, e.g., U.S. Pat. No. 5,605,690 to Jacobs).
[0112] Use of proteins for the treatment of inflammatory diseases:
[01131 For therapeutic use, purified TNFR proteins of the present invention
are
administered to a patient, preferably a human, for treating TNF-dependent
inflammatory
diseases, such as arthritis. In the treatment of humans, the use of huTNFRs is
preferred. The
TNFR proteins of the present invention can be administered by bolus injection,
continuous
infusion, sustained release from implants, or other suitable techniques.
Typically, TNFR
therapeutic proteins will be administered in the form of a composition
comprising purified
protein in conjunction with physiologically acceptable carriers, excipients or
diluents. Such
carriers will be nontoxic to recipients at the dosages and concentrations
employed.
Ordinarily, the preparation of such compositions entails combining the TNFR
with buffers,
antioxidants such as ascorbic acid, polypeptides, proteins, amino acids,
carbohydrates
including glucose, sucrose or dextrins, chelating agents such as EDTA,
glutathione and other
stabilizers and excipients. Neutral buffered saline or saline mixed with
conspecific serum
albumin are exemplary appropriate diluents. Preferably, product is formulated
as a
lyophilizate using appropriate excipient solutions, for example, sucrose, as
diluents.
Preservatives, such as benzyl alcohol may also be added. The amount and
frequency of
administration will depend of course, on such factors as the nature and the
severity of the
indication being treated, the desired response, the condition of the patient
and so forth.
[0114] TNFR proteins of the present invention are administered systemically in
therapeutically effective amounts preferably ranging from about 0.1 mg/kg/week
to about 100
mg/kg/week. In preferred embodiments, TNFR is administered in amounts ranging
from
about 0.5 mg/kg/week to about 50 mg/kg/week. For local administration, dosages
preferably
range from about 0.01 mg/kg to about 1.0 mg/kg per injection.
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[0115] Use of expression vectors to increase the levels of a TNF antagonist in
a mammal:
[0116] The present invention provides a process of increasing the levels of a
TNF
antagonist in a mammal. The process includes the step of transforming cells of
the mammal
with an expression vector described herein, which drives expression of a TNFR
as described
herein.
[0117] The process is particularly useful in large mammals such as domestic
pets, those
used for food production, and primates. Exemplary large mammals are dogs,
cats, horses
cows, sheep, deer, and pigs. Exemplary primates are monkeys, apes, and humans.
[0118] The mammalian cells can be transformed either in vivo or ex vivo. When
transformed in vivo, the expression vector are administered directly to the
mammal, such as
by injection. Means for transforming cells in vivo are well known in the art.
When
transformed ex vivo, cells are removed from the mammal, transformed ex vivo,
and the
transformed cells are reimplanted into the mammal.
[0119] Splice-switching; oligomers (SSOs):
[0120] In another aspect, the present invention employs splice switching
oligonucleotides
or splice switching oligomers (SSOs) to control the alternative splicing of
TNFR2 so that the
amount of a soluble, ligand-binding form that lacks exon 7 is increased and
the amount of the
integral membrane form is decreased. The methods and compositions of the
present
invention can be used in the treatment of diseases associated with excessive
TNF activity.
[0121] Accordingly, one embodiment of the invention is a method of treating an
inflammatory disease or condition by administering SSOs to a patient. The SSOs
that are
administered alter the splicing of a pre-mRNA to produce a mammalian TNFR2
protein that
lacks exon 7. In another embodiment, the invention is a method of producing a
mammalian
TNFR2 protein that lacks exon 7 in a cell by administering SSOs to the cell.
[0122] The length of the SSO (i.e. the number of monomers in the oligomer) is
similar to
an antisense oligonucleotide (ASON), typically between about 8 and 30
nucleotides. In
preferred embodiments, the SSO will be between about 10 to 16 nucleotides. The
invention
can be practiced with SSOs of several chemistries that hybridize to RNA, but
that do not
activate the destruction of the RNA by RNase H, as do conventional antisense
2'-deoxy
oligonucleotides. The invention can be practiced using 2'O modified nucleic
acid oligomers,
such as where the 2'O is replaced with -O-CH3, -O-CH2-CH2-O-CH3a -O-CH2-CHZ-
CH2-
NHZ, -O-CH2-CH2-CH2-OH or -F, where 2'O-methyl or 2'O-methyloxyethyl is
preferred.
The nucleobases do not need to be linked to sugars; so-called peptide nucleic
acid oligomers
or morpholine-based oligomers can be used. A comparison of these different
linking
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chemistries is found in Sazani, P. et al., 2001, Nueleic Acids Res. 29:3695.
The term splice-
switching oligonucleotide is intended to cover the above forms. Those skilled
in the art will
appreciate the relationship between antisense oligonucleotide gapmers and
SSOs. Gapmers
are ASON that contain an RNase H activating region (typically a 2'-
deoxyribonucleoside
phosphorothioate) which is flanked by non-activating nuclease resistant
oligomers. In
general, any chemistry suitable for the flanking sequences in a gapmer ASON
can be used in
an SSO.
[0123J The SSOs of this invention may be made through the well-known technique
of
solid phase synthesis. Any other means for such synthesis known in the art may
additionally
or alternatively be used. It is well known to use similar techniques to
prepare
oligonucleotides such as the phosphorothioates and alkylated derivatives.
[0124] The bases of the SSO may be the conventional cytosine, guanine, adenine
and
uracil or thymidine. Alternatively, modified bases can be used. Of particular
interest are
modified bases that increase binding affinity. One non-limiting example of
preferred
modified bases are the so-called G-clamp or 9-(aminoethoxy)phenoxazine
nucleotides,
cytosine analogues that form 4 hydrogen bonds with guanosine. (Flanagan, W.M.,
et al.,
1999, Proc. Natl. Acad. Sci. 96:3513; Holmes, S.C., 2003, Nucleic Acids Res.
31:2759).
Specific examples of other bases include, but are not limited to, 5-
methylcytosine (MeC),
isocytosine, pseudoisocytosine, 5-bromouracil, 5-propynyluracil, 5-propyny-6,
5-
methylthiazoleuracil, 6-aminopurine, 2-aminopurine, inosine, 2,6-
diaminopurine, 7-propyne-
7-deazaadenine, 7-propyne-7-deazaguanine and 2-chloro-6-aminopurine.
[0125] A particularly preferred chemistry is provided by locked nucleic acids
(LNA)
(Koshkin, A.A., et al., 1998, Tetrahedron 54:3607; Obika, S., et al., 1998,
Tetrahedron Lett.
39:5401). As used herein, the terms "LNA unit", "LNA monomer", "LNA residue",
"locked
nucleic acid unit", "locked nucleic acid monomer" or "locked nucleic acid
residue", refer to a
bicyclic nucleoside analogue. LNA units and methods of their synthesis are
described in
inter alia WO 99/14226, WO 00/56746, WO 00/56748, WO 01/25248, WO 02/28875, WO
03/006475 and WO 03/095467. The LNA unit may also be defined with respect to
its
chemical formula. Thus, an "LNA unit", as used herein, has the chemical
structure shown in
Formula I below:
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Formula I
~
0 Y---)( B
Y__
or
IA 1B
[0126] wherein,
[0127] X is selected from the group consisting of 0, S and NRH, where R is H
or C1-C4-
alkyl;
[0128] Y is (-CH2),, where r is an integer of 1-4; and
[0129] B is a base of natural or non-natural origin as described above.
[0130] In a preferred embodiment, r is 1 or 2, and in a more preferred
embodiment r is 1.
[0131] When LNA nucleotides are employed in an SSO it is preferred that non-
LNA
nucleotides also be present. LNA nucleotides have such high affinities of
hybridization that
there can be significant non-specific binding, which may reduce the effective
concentration
of the free-SSO. When LNA nucleotides are used they may be altemated
conveniently with
2'-deoxynucleotides. The pattern of alternation is not critical. Alternating
nucleotides,
alternating dinucleotides or mixed patterns, e.g., LDLDLD or LLDLLD or LDDLDD
can be
used. For example in one embodiment, contains a sequence of nucleotides
selected from the
group consisting of: LdLddLLddLdLdLL, LdLdLLLddLLLdLL, LMLMMLLMMLMLMLL,
LMLMLLLMMLLLMLL, LFLFFLLFFLFLFLL, LFLFLLLFFLLLFLL, LddLddLddL,
dLddLddLdd, ddLddLddLd, LMMLMMLMML, MLMMLMMLMM, MMLMMLMMLM,
LFFLFFLFFL, FLFFLFFLFF, FFLFFLFFLF, dLdLdLdLdL, LdLdLdLdL,
MLMLMLMLML, LMLMLMLML, FLFLFLFLFL, LFLFLFLFL, where L is a LNA unit, d
is a DNA unit, M is 2'MOE, F is 2'Fluoro.
[0132] When 2'-deoxynucleotides or 2'-deoxynucleoside phosphorothioates are
mixed
with LNA nucleotides it is important to avoid RNase H activation. It is
expected that
between about one third and two thirds of the LNA nucleotides of an SSO will
be suitable.
When affinity-enhancing modifications are used, including but not limited to
LNA or G-
clamp nucleotides, the skilled person recognizes it can be necessary to
increase the proportion
of such affinity-enhancing modifications.
[0133] Numerous alternative chemistries which do not activate RNase H are
available.
For example, suitable SSOs can be oligonucleotides wherein at least one of the
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internucleotide bridging phosphate residues is a modified phosphate, such as
methyl
phosphonate, methyl phosphonothioate, phosphoromorpholidate,
phosphoropiperazidate, and
phosphoroamidate. For example, every other one of the intemucleotide bridging
phosphate
residues may be modified as described. In another non-limiting example, such
SSO are
oligonucleotides wherein at least one of the nucleotides contains a 2' lower
alkyl moiety
(e.g., Ci-C4, linear or branched, saturated or unsaturated alkyl, such as
methyl, ethyl, ethenyl,
propyl, 1-propenyl, 2-propenyl, and isopropyl). For example, every other one
of the
nucleotides may be modified as described. (See references in U.S. Pat.
5,976,879 col. 4). For
in vivo use, phosphorothioate linkages are preferred.
[01341 The length of the SSO will be from about 8 to about 30 bases in length.
Those
skilled in the art appreciate that when affinity-increasing chemical
modifications are used, the
SSO can be shorter and still retain specificity. Those skilled in the art will
further appreciate
that an upper limit on the size of the SSO is imposed by the need to maintain
specific
recognition of the target sequence, and to avoid secondary-structure forming
self
hybridization of the SSO and by the limitations of gaining cell entry. These
limitations imply
that an SSO of increasing length (above and beyond a certain length which will
depend on the
affinity of the SSO) will be more frequently found to be less specific,
inactive or poorly
active.
[01351 SSOs of the invention include, but are not limited to, modifications of
the SSO
involving chemically linking to the SSO one or more moieties or conjugates
which enhance
the activity, cellular distribution or cellular uptake of the SSO. Such
moieties include, but are
not limited to, lipid moieties such as a cholesterol moiety, cholic acid, a
thioether, e.g. hexyl-
S-tritylthiol, a thiocholesterol, an aliphatic chain, e.g., dodecandiol or
undecyl residues, a
phospholipids, e.g., di-hexadecyl-rac-glycerol or triethylammonium 1,2-di-O-
hexadecyl-rac-
glycero-3-H-phosphonate, a polyamine or a polyethylene glycol chain, an
adamantane acetic
acid, a palmityl moiety, an octadecylamine or hexylamino-carbonyl-
oxycholesterol moiety.
[0136] It is not necessary for all positions in a given SSO to be uniformly
modified, and
in fact more than one of the aforementioned modifications may be incorporated
in a single
compound or even at a single nucleoside within an SSO.
[01371 The SSOs may be admixed, encapsulated, conjugated, or otherwise
associated
with other molecules, molecule structures, or mixtures of compounds, as for
example
liposomes, receptor targeted molecules, oral, rectal, topical or other
formulation, for assisting
in uptake, distribution, and/or absorption.
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t01381 Those skilled in the art appreciate that cellular differentiation
includes, but is not
limited to, differentiation of the spliceosome. Accordingly, the activity of
any particular SSO
can depend upon the cell type into which they are introduced. For example,
SSOs which are
effective in one cell type may be ineffective in another cell type.
101391 The methods, oligonucleotides, and formulations of the present
invention are also
useful as in vitro or in vivo tools to examine splicing in human or animal
genes. Such
methods can be carried out by the procedures described herein, or
modifications thereof
which will be apparent to skilled persons.
[0140] The SSOs disclosed herein can be used to treat any condition in which
the medical
practitioner intends to limit the effect of TNF or the signalling pathway
activated by TNF. In
particular, the invention can be used to treat an inflammatory disease. In one
embodiment,
the condition is an inflammatory systemic disease, e.g., rheumatoid arthritis
or psoriatic
arthritis. In another embodiment, the disease is an inflammatory liver
disease. Examples of
inflammatory liver diseases include, but are not limited to, hepatitis
associated with the
hepatitis A, B, or C viruses, alcoholic liver disease, and non-alcoholic
steatosis. In yet
another embodiment, the inflammatory disease is a skin condition such as
psoriasis.
[0141] The uses of the present invention include, but are not limited to,
treatment of
diseases for which known TNF antagonists have been shown useful. Three
specific TNF
antagonists are currently FDA-approved. The drugs are etanercept (Enbrel ),
infliximab
(Remicade(D) and adalimumab (Humira(&). One or more of these drugs is approved
for the
treatment of rheumatoid arthritis, juvenile rheumatoid arthritis, psoriasis,
psoriatic arthritis,
ankylosing spondylitis, and inflammatory bowel disease (Crohn's disease or
ulcerative
colitis).
/[0142] The administration of the SSO to subjects can be accomplished using
procedures
developed for ASON. ASON have been successfully administered to experimental
animals
and human subjects by intravenous administration in saline in doses as high as
6 mg/kg three
times a week (Yacysyhn, B.R., et al., 2002, Gut 51:30 (anti-ICAM-1 ASON for
treatment of
Crohn's disease); Stevenson, J., et al., 1999, J. Clinical Oncology 17:2227
(anti-RAF-1
ASON targeted to PBMC)). The pharmacokinetics of 2`O-MOE phosphorothioate
ASON,
directed towards TNF-a has been reported (Geary, R.S., et al., 2003, Drug
Metabolism and
Disposition 31:1419). The systemic efficacy of mixed LNAIDNA molecules has
also been
reported (Fluiter, K., et al., 2003, Nucleic Acids Res. 31:953).
[0143] The systemic activity of SSO in a mouse model system was investigated
using
2'O-MOE phosphorothioates and PNA chemistries. - Significant activity was
observed in all
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tissues investigated except brain, stomach and dermis (Sazani, P., et al.,
2002, Nature
Biotechnology 20, 1228).
[0144] In general any method of administration that is useful in conventional
antisense
treatments can be used to administer the SSO of the invention. For testing of
the SSO in
cultured cells, any of the techniques that have been developed to test ASON or
SSO may be
used.
[0145] Formulations of the present invention comprise SSOs in a
physiologically or
pharmaceutically acceptable carrier, such as an aqueous carrier. Thus
formulations for use in
the present invention include, but are not limited to, those suitable for
parenteral
administration including intraperitoneal, intraarticular, intravenous,
intraarterial,
subcutaneous, or intramuscular injection or infusion, as well as those
suitable for topical,
ophthalmic, vaginal, oral, rectal or pulmonary (including inhalation or
insufflation of
powders or aerosols, including by nebulizer, intratracheal, intranasal
delivery) administration.
The formulations may conveniently be presented in unit dosage form and may be
prepared by
any of the methods well known in the art. The most suitable route of
administration in any
given case may depend upon the subject, the nature and severity of the
condition being
treated, and the particular active compound which is being used.
[0146] Pharmaceutical compositions of the present invention include, but are
not limited
to, physiologically and pharmaceutically acceptable salts ,i.e, salts that
retain the desired
biological activity of the parent compound and do not impart undesired
toxicological
properties. Examples of such salts are (a) salts formed with cations such as
sodium,
potassium, NH4}, magnesium, calcium, polyamines such as spermine and
spermidine, etc.;
(b) acid addition salts formed with inorganic acids, for example, hydrochloric
acid,
hydrobromic acid, sulfuric acid, phosphoric acid, nitric acid and the like;
and (c) salts formed
with organic acids such as, for example, acetic acid, oxalic acid, tartaric
acid, succinic acid,
maleic acid, fumaric acid, gluconic acid, citric acid, malic acid, ascorbic
acid, benzoic acid,
tannic acid, palmitic acid, alginic acid, polyglutamic acid,
napthalenesulfonic acid,
methanesulfonic acid, p-toluenesulfonic acid, napthalenedisulfonic acid,
polygalacturonic
acid, and the like.
[0147] The present invention provides for the use of SSOs having the
characteristics set
forth above for the preparation of a medicament for increasing the ratio of a
mammalian
TNFR2 protein that lacks exon 7 to its corresponding membrane bound form, in a
patient
afflicted with an inflammatory disorder involving TNF-a, as discussed above.
In the
manufacture of a medicament according to the invention, the SSOs are typically
admixed
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with, inter alia, an acceptable carrier. The carrier must, of course, be
acceptable in the sense
of being compatible with any other ingredients in the formulation and must not
be deleterious
to the patient. The carrier may be a solid or liquid. SSOs are incorporated in
the
formulations of the invention, which may be prepared by any of the well known
techniques of
pharmacy consisting essentially of admixing the components, optionally
including one or
more accessory therapeutic ingredients.
101481 Formulations of the present invention may comprise sterile aqueous and
non-
aqueous injection solutions of the active compounds, which preparations are
preferably
isotonic with the blood of the intended recipient and essentially pyrogen
free. These
preparations may contain anti-oxidants, buffers, bacteriostats, and solutes
which render the
formulation isotonic with the blood of the intended recipient. Aqueous and non-
aqueous
sterile suspensions can include, but are not limited to, suspending agents and
thickening
agents. The formulations may be presented in unit dose or multi-dose
containers, for
example, sealed ampoules and vials, and may be stored in freeze-dried
(lyophilized)
condition requiring only the addition of the sterile liquid carrier, for
example, saline or water-
for-injection immediately prior to use.
[0149] In the formulation the SSOs may be contained within a particle or
vesicle, such as
a liposome, or microcrystal, which may 'be suitable for parenteral
administration. The
particles may be of any suitable structure, such as unilamellar or
plurilameller, so long as the
SSOs are contained therein. Positively charged lipids such as N-[1-(2,3-
dioleoyloxy)propyl]-
N,N,N-trimethyl-ammoniummethylsulfate, or "DOTAP," are particularly preferred
for such
particles and vesicles. The preparation of such lipid particles is well known.
[See references
in U.S. Pat. 5,976,879 col. 6]
[0150] The SSO can be targeted to any element or combination of elements that
regulate
splicing, including the 3'splice site, the 5' splice site, the branch point,
the polypyrimidine
tract, exonic splicing ehancers, exonic splicing silencers, intronic splicing
enhancers, and
intronic splicing silencers.
[0151] Those skilled in the art can appreciate that the invention as directed
toward human
TNFR2 can be practiced using SSO having a sequence that is complementary to at
least 8, to
at least 9, to at least 10, to at least 11, to at least 12, to at least 13, to
at least 14, to at least 15,
preferably between 10 and 16 nucleotides of the portions of the TNFR2 gene
comprising
exons 7 and its adjacent introns. SEQ ID No: 13 contains the sequence of exon
7 of TNFR2
and 50 adjacent nucleotides of the flanking introns. For example, SSO targeted
to human
TNFR2 can have a sequence selected from the sequences listed in Table 1. When
affinity-
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enhancing modifications are used, including but not limited to LNA or G-clamp
nucleotides,
the skilled person recognizes the length of the SSO can be correspondingly
reduced. The
pattern of alternation of LNA and conventional nucleotides is not.important.
Table 1: SSOs Tar eted to Human TNFR2
SEQ ID. Name Sequence 5' to 3'
14 3378 CCA CAA TCA GTC CTA G
15 SK101 A CAA TCA GTC CTA G
16 SK102 AA TCA GTC CTA G
17 SK103 TCA GTC CTA G
18 SK104 CCA CAA TCA GTC CT
19 SK105 CCA CAA TCA GTC
20 SK106 CCA CAA TCA G
21 SK107 CA CAA TCA GTC CTA
22 SK108 CA CAA TCA GTC C
23 SK109 A CAA TCA GTC CT
24 SK110 CAA TCA GTC CTA
25 SK111 CA CAA TCA GT
26 SK112 A CAA TCA GTC
27 SK113 CAA TCA GTC C
28 SK114 AA TCA GTC CT
29 SK115 A TCA GTC CTA
30 3379 CAG TCC TAG AAA GAA A
31 SK117 G TCC TAG AAA GAA A
32 SK118 CC TAG AAA GAA A
33 SK119 TAG AAA GAA A
34 SK120 CAG TCC TAG AAA GA
35 SK121 CAG TCC TAG AAA
36 SK122 CAG TCC TAG A
37 SK123 AG TCC TAG AAA GAA
38 SK124 AG TCC TAG AAA G
39 SK125 G TCC TAG AAA GA
40 SK126 TCC TAG AAA GAA
41 SK127 AG TCC TAG AA
42 SK128 G TCC TAG AAA
43 SK129 TCC TAG AAA G
44 SK130 CC TAG AAA GA
45 SK131 C TAG AAA GAA
46 3384 ACT TTT CAC CTG GGT C
47 SK133 T TTT CAC CTG GGT C
48 SK134 TT CAC CTG GGT C
49 SK135 CAC CTG GGT C
50 SK136 ACT TTT CAC CTG GG
51 SK137 ACT TTT CAC CTG
52 SK138 ACT TTT CAC C
53 SK139 CT TTT CAC CTG GGT
54 SK140 CT TTT CAC CTG G
55 SK141 T TTT CAC CTG GG
56 SK142 TTT CAC CTG GGT
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57 SK143 CT TTT CAC CT
58 SK144 T TTT CAC CTG
59 SK145 TTT CAC CTG G
60 SK146 TT CAC CTG GG
61 SK147 T CAC CTG GGT
[0152] Those skilled in the art will also recognize that the selection of SSO
sequences
must be made with care to avoid a self-complementary SSO, which may lead to
the formation
of partial "hairpin" duplex structures. In addition, high GC content should be
avoided to
minimize the possibility of non-specific base pairing. Furthermore, SSOs
matching off-target
genes, as revealed for example by BLAST, should also be avoided.
[0153] In some situations, it may be preferred to select an SSO sequence that
can target a
human and at least one other species. These SSOs can be used to test and to
optimize the
invention in said other species before being used in humans, thereby being
useful for
regulatory approval and drug development purposes. For example, SSOs with
sequences
selected from SEQ ID Nos: 14, 30, 46, 70 and 71 which target human TNFR2 are
also 100%
complementary to the corresponding Macaca Mullata sequences. As a result these
sequences
can be used to test treatments in monkeys, before being used in humans.
[0154] It will be appreciated by those skilled in the art that various
omissions, additions
and modifications may be made to the invention described above without
departing from the
scope of the invention, and all such modifications and changes are intended to
fall within the
scope of the invention, as defined by the appended claims. All sequence
citations, references,
patents, patent applications or other documents cited referred to herein are
incorporated by
reference.
Example 1
Materials and Methods
[0155] Oligonucteotides. Table 3 lists chimeric locked nucleic acid (LNA) SSOs
with
alternating 2'deoxy- and 2'O-4'-(methylene)-bicyclic-ribonucleoside
phosphorothioates and
having sequences as described in U.S. Appi. No. 11/595,485. These were
synthesized by
Santaris Pharma, Denmark. For each SSO, the 5'-terminal nucleoside was a 2'O-
4'-
rnethylene-ribonucleoside and the 3'-terminal nucleoside was a 2'deoxy-
ribonucleoside.
Table 4 shows the sequences of chimeric LNA SSOs with alternating 2'-O-methyl-
ribonucleoside-phosphorothioates (2'-OMe) and 2'O-4'-(methylene)-bicyclic-
ribonucleoside
phosphorothioates. These were synthesized by Santaris Pharma, Denmark. The LNA
is
shown in capital letters and the 2'-OME is shown in lower case letters.
[0156] Cell culture and transfections. L929 cells were maintained in minimal
essential
media supplemented with 10% fetal bovine serum and antibiotic (37 C, 5% C02).
For
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transfection, L929 cells were seeded in 24-well plates at 105 cells per well
and transfected 24
hrs later. Oligonucleotides were complexed, at the indicated concentrations,
with 2 L of
LipofectamineTM 2000 transfection reagent (Invitrogen) as per the
manufacturer's directions.
The nucleotide/lipid complexes were then applied to the cells and incubated
for 24 hrs. The
media was then aspirated and cells harvested with TRI-ReagentTM (MRC,
Cincinnati, OH).
[0157] RT-PCR. Total RNA was isolated with TRI-Reagent (MRC, Cincinnati, OH)
and
TNFRI or TNFR2 mRNA was amplified by GeneAmp RT-PCR using rTth polyinerase
(Applied Biosystems) following supplier directions. Approximately 200 ng of
RNA was
used per reaction. Primers used in the examples described herein are included
in Table 2.
Cycles of PCR proceeded: 95 C, 60 see; 56 C, 30 sec; 72 C, 60 sec for 22-30
cycles total.
[0158] In some instances a Cy5-labeled dCTP (GE Healthcare) was included in
the PCR
step for visualization (0.1 L per 50 L PCR reaction). The PCR products were
separated on
a 10% non-denaturing polyacrylamide gel, and Cy5-labeled bands were visualized
with a
TyphoonTM 9400 Scanner (GE Healthcare). Scans were quantified with
ImageQuantTM (GE
Healthcare) software. Alternatively, in the absence of the inclusion of Cy5 -
labeled dCTP, the
PCR products were separated on a 1.5% agarose gel containing trace amounts of
ethidium
bromide for visualization.
[0159] PCR. PCR was performed with Platinum Taq DNA Polymerase (Invitrogen)
according to the manufacturer's directions. For each 50 L reaction,
approximately 30 pmol
of both forward and reverse primers were used. Primers used in the examples
described
herein are included in Table 2. The thermocycling reaction proceeded, unless
otherwise
stated, as follows: 94 C, 3 minutes; then 30-40 cycles of 94 C, 30 sec; 55 C,
30 sec; and
72 C, 105 sec; followed by 72 C, 3 minutes. The PCR products were analyzed on
1.5%
agarose gels and visualized with ethidium bromide.
Table 2: RT-PCR and PCR Primers
SEQ Name Sequence 5' to 3'
ID.
Human TNFR2
74 TR001 ACT GGG CTT CAT CCC AGC ATC
75 TR002 CAC CAT GGC GCC CGT CGC CGT CTG G
76 TR003CGA CTT CGC TCT TCC AGT TGA GAA GCC CTT GTG CCT GCA G
77 TR004 TTA ACT GGG CTT CAT CCC AGC ATC
78 TR005CTG CAG GCA CAA GGG CTT CTC AAC TGG AAG AGC GAA GTC G
79 TR026TTA ACT GGG CTT CAT CCC AGC
80 TR027CGA TAG AAT TCA TGG CGC CCG TCG CCG TCT GG
81 TR028CCT AAC TCG AGT TAA CTG GGC TTC ATC CCA GC
82 TR029GAC TGA GCG GCC GCC ACC ATG GCG CCC GTC GCC GTC TGG
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83 TR030 CTA AGC GCG GCC GCT TAA CTG GGC TTC ATC CCA GCA TC
84 TR047 CGT TCT CCA ACA CGA CTT CA
85 TR048 CTT ATC GGC AGG CAA GTG AGG
86 TR049 ACT GAA ACA TCA GAC GTG GTG TGC
87 TR050 CCT TAT CGG CAG GCA AGT GAG
Human TNFR1
88 TR006 CCT CAT CTG AGA AGA CTG GGC G
89 TR007 GCC ACC ATG GGC CTC TCC ACC GTG C
90 TR008GGG CAC TGA GGA CTC AGT TTG TGG GAA ATC GAC ACC TG
91 TR009 CAG GTG TCG ATT TCC CAC AAA CTG AGT CCT CAG TGC CC
92 TR010CAC CAT GGG CCT CTC CAC CGT GC
93 TRO11 TCT GAG AAG ACT GGG CG
94 TR03ICGA TAG GAT CCA TGG GCC TCT CCA CCG TGC
95 TR032 CCT AAC TCG AGT CAT CTG AGA AGA CTG GGC G
96 TR033GAC TGA GCG GCC GCC ACC ATG GGC CTC TCC ACC GTG C
97 TR034 CTA AGC GCG GCC GCT CAT CTG AGA AGA CTG GGC G
Mouse TNFR2
98 TR012 GGT CAG GCC ACT TTG ACT GC
99 TR013CAC CGC TGC CCC TAT GGC G
100 TR014 CAC CGC TGC CAC TAT GGC G
101 TR015 GGT CAG GCC ACT TTG ACT GCA ATC
102 TR016 GCC ACC ATG GCG CCC GCC GCC 'CTC TGG
103 TR017GGC ATC TCT CTT CCA ATT GAG AAG CCC TCC TGC CTA CAA AG
104 TR018CTT TGT AGG CAG GAG GGC TTC TCA ATT GGA AGA GAG ATG CC
105 TR019 GGC CAC TTT GAC TGC AAT CTG
106 TR035CAC CAT GGC GCC CGC CGC CCT CTG G
107 TR036TCA GGC CAC TTT GAC TGC AAT C
108 TR037 CGA TAG AAT TCA TGG CGC CCG CCG CCC TCT GG
109 TR038CCT AAC TCG AGT CAG GCC ACT TTG ACT GCA ATC
110 TR039 GAC TGA GCG GCC GCC ACC ATG GCG CCC GCC GCC CTC TGG
111 TR040 CTA AGC GCG GCC GCT CAG GCC ACT TTG ACT GCA ATC
112 TR045 GAG CCC CAA ATG GAA ATG TGC
113 TR046 GCT CAA GGC CTA CTG CAT CC
Mouse TNFR1
114 TR020 GGT TAT CGC GGG AGG CGG GTC G
115 TR021 GCC ACC ATG GGT CTC CCC ACC GTG CC
116 TR022 CAC AAA CCC CCA GGA CTC AGT TTG TAG GGA TCC CGT GCC T
117 TR023AGG CAC GGG ATC CCT ACA AAC TGA GTC CTG GGG GTT TGT G
118 TR024 CAC CAT GGG TCT CCC CAC CGT GCC
119 TR025 TCG CGG GAG GCG GGT CGT GG
120 TR041CGA TAG TCG ACA TGG GTC TCC CCA CCG TGC C
121 TR042 CCT AAG AAT TCT TAT CGC GGG AGG CGG GTC G
122 TR043GAC TGA GCG GCC GCC ACC ATG GGT CTC CCC ACC GTG CC
123 TR044 CTA AGC GCG GCC GCT TAT CGC GGG AGG CGG GTC G
[0160] Human hepatocyte cultures. Human hepatocytes were obtained in
suspension
either from ADMET technologies, or from The UNC Cellular Metabolism and
Transport
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Core at UNC-Chapel Hill. Cells were washed and suspended in RPMI 1640
supplemented
with 10% FBS, I g/mL human insulin, and 13 nM Dexamethasone. Hepatocytes were
plated in 6-well plates at 0.5 x 106 cells per plate in 3 mL media. After 1-
1.5 hrs, non-
adherent cells were removed, and the media was replaced with RPMI 1640 without
FBS,
supplemented with 1 g/mL human insulin, and 130 nM Dexamethasone.
[0161] For delivery of SSOs to hepatocytes in 6-well plates, 10 L of a 5 M
SSO stock
was diluted into 100 L of OPTI-MEMTM, and 4 L of LipofectamineTM 2000 was
diluted
into 100 L of OPTI-MEMTM. The 200 L complex solution was then applied to the
cells in
the 6-well plate containing 2800 L of media, for a total of 3000 L. The
final SSO
concentration was 17 nM. After 24 hrs, cells were harvested in TRI-ReagentTM.
Total RNA
was isolated per the manufacturer's directions. Approximately 200 ng of total
RNA was
subjected to reverse transcription-PCR (RT-PCR).
[0162] ELISA. To determine the levels of soluble TNFR2 in cell culture media
or sera,
the Quantikine Mouse sTNF RII ELISA kit from R&D Systems (Minneapolis, MN) or
Quantikine Human sTNF RII ELISA kit from R&D Systems (Minneapolis, MN) were
used. The antibodies used for detection also detect the protease cleavage
forms of the
receptor. ELISA plates were read using a microplate reader set at 450 nm, with
wavelength
correction set at 570 nm.
[0163] For mouse in vivo studies, blood from the animals was clotted for 1
hour at 37 C
and centrifuged for 10 min at 14,000 rpm (Jouan BRA4i centrifuge) at 4 C. Sera
was
collected and assayed according to the manufacturer's guide, using 50 L of
mouse sera
diluted 1:10.
[0164] L929 cytotoxicity assay. L929 cells plated in 96-well plates at 104
cells per well
were treated with 0.1 ng/mL TNF-a and 1 gg/mL actinomycin D in the presence of
10%
serum from mice treated with the indicated oligonucleotide in 100 L total of
complete MEM
media (containing 10% regular FBS) and allowed to grow for -24 hrs at 37 C.
Control lanes
were plated in 10% serum from untreated mice. Cell viability was measured 24
hrs later by
adding 20 L CeIlTiter 96 AQ1eous One Solution Reagent (Promega) and
measuring
absorbance at 490 nm with a microplate reader. Cell viability was normalized
to untreated
cells.
[0165] Western blots. Twenty pL of media or 20 g of lysate were loaded in
each well of
a 4-12% NuPAGE polyacrylamide gel (Invitrogen). The gel was run 40 min at
20OV. The
protein was transferred, for 1 hr at 30V, to an InvitrolonTM PVDF membrane
(Invitrogen),
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which was then blocked with StartingBlock Blocking Buffer (Pierce) for 1 hr
at room
temperature. The membrane was incubated for 3 hrs at room temperature with a
rabbit
polyclonal antibody that recognizes the C-terminus of human and mouse TNFR2
(Abcam),
Following three washes in PBS-T buffer (1 xPBS, 0.1% Tween-20), the membrane
was
incubated for one hour at room temperature with secondary goat anti-rabbit
antibody
(Abeam) and again washed three times with PBS-T buffer. The protein was then
detected
with ECL PlusTM (GE Healthcare), according to the manufacturer's
recommendations and
then photographed.
Example 2
SSO Splice Switching Activity with TNFR mRNA
[0166] Table 3 shows the splice switching activities of SSOs having sequences
as
described in U.S. Appl. No. 11/595,485 and targeted to mouse and human TNFRs.
Of SSOs
targeted to mouse TNFR2 exon 7, at least 8 generated some muTNFR2 A7 mRNA. In
particular, SSO 3312, 3274 and 3305 induced at least 50% skipping of exon 7;
SSO 3305
treatment resulted in almost complete skipping. Of SSOs transfected into
primary human
hepatocytes, and targeted to human TNFR2 exon 7, at least 7 SSOs generated
some
huTNFR2 d7 mRNA. In particular, SSOs 3378, 3379, 3384 and 3459 induced at
least 75%
skipping of exon 7 (FIG. 2B), and significant induction of huTNFR2 A7 into the
extracellular
media (FIG. 2A).
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Table 3: SSO Splice Switching Activity
SEQ ID. Name Activi
Mouse TNFR2
3272 -
3304 -
3305 +
3306 +
3307 +
3308 +
3309 +
3310 -
3311 +
62 3274 +
3312 +
3273 -
Mouse TNFR7.
3333 +
Huanan TNFR2
14 3378 +
30 3379 +
3380 -
70 3381 +
71 3382 +
3383 -
46 3384 +
72 3459 +
3460 -
73 3461 +
Control
3083 -
[0167J Table 4 contains the sequences of 10 nucleotide chimeric SSOs with
alternating
2'-O-methyl-ribonucleoside-phosphorothioates (2'-OMe) and 2'0-4'-(methylene)-
bicyclic-
ribonucleoside phosphorothioates. These SSOs are targeted to exon 7 of mouse
TNFR2.
Table 4: LNA/2'-OMe-ribonucleoside hos horothioate chimeric mouse targeted SSO
SEQ ID. Name Sequence 5' to 3' *
62 3274 AgAgCaGaAcCtTaCt
63 3837 gAaCcTuAcT
64 3838 aGaGcAgAaC
65 3839 gAgCaGaAcC
66 3840 aGcAgAaCcT
67 3841 gCaGaAcCuT
68 3842 cAgAaCcTuA
69 3843 aGaAcCuTaC
*Capital letters are 2'0-4'-(methylene)-bicyclic-
ribonucleosides; lowercase letters are 2'-OMe
[01681 To analyze the in vitro splice-switching activity of the SSOs listed in
Table 4,
L929 cells were cultured and seeded as described in Example 1. For delivery of
each of the
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SSOs in Table 4 to the L929 cells, SSOs were diluted into 50 L of OPTI-MEMTM,
and then
50 L LipofectamineTM 2000 mix (1 part LipofectamineTM 2000 to 25 parts OPTI-
MEMTM)
was added and incubated for 20 minutes. Then 400 L of serum free media was
added to the
SSOs and applied to the cells in the 24-well plates. The final SSO
concentration was either
50 or 100 nM. After 24 hrs, cells were harvested in 800 L TRI-ReagentTM.
Total RNA was
isolated per the manufacturer's directions and analyzed by RT-PCR (FIG. 3)
using the
forward primer TR045 (SEQ ID No: 112) and the reverse primer TR046 (SEQ ID No:
113).
[0169] To analyze the in vivo splice-switching activity of the SSOs listed in
Table 4, mice
were injected with the SSOs listed in Table 4 intraperitoneal (i.p.) at 25
mg/kg/day for 5 days.
Mice were bled before injection and again 1, 5 and 10 days after the last
injection. The
concentration of soluble TNFR2 A7 in the sera taken before the first injection
and 10 days
after the last injection were measured by ELISA (FIG. 4B). The mice were
sacrificed on day
and total RNA from 5-10 mg of the liver was analyzed by RT-PCR (FIG. 4A) using
the
forward primer TR045 (SEQ ID No: 112) and the reverse primer TR046 (SEQ ID No:
113).
[0170] Of the 10 nucleotide SSOs subsequences of SSO 3274 tested in vitro, all
of them
generated at least some muTNFR2 07 mRNA (FIG. 3). In particular, SSO 3839,
3840 and
3841 displayed greater splice-switching activity than the longer 16 nucleotide
SSO 3274 from
which they are derived. The three 10 nucleotide SSOs, 3839, 3840, 3841, that
demonstrated
the greatest activity in vitro also were able to generate significant amounts
of muTNFR2 A7
mRNA (FIG. 4A) and soluble muTNFR2 d7 protein (FIG. 4B) in mice in vivo.
[0171] To assess the effect of SSO length on splice switching activity in
human TNFR2,
cells were treated with SSOs of different lengths. Primary human hepatocytes
were
transfected with the indicated SSOs selected from Table 1. These SSOs were
synthesized by
Santaris Pharma, Denmark with alternating 2'deoxy- and 2'O-4'-(methylene)-
bicyclic-
ribonucleoside phosphorothioates. For each SSO, the 5'-terminal nucleoside was
a 2'O-4'-
methylene-ribonucleoside and the 3'-terminal nucleoside was a 2'deoxy-
ribonucleoside.These SSOs were either 10-, 12-, 14- or 16-mers. The
concentration of
soluble TNFR2 A7 was measured by ELISA (FIG. 5, top panel). Total RNA was
analyzed by
RT-PCR for splice switching activity (FIG. 5, bottom panel).
Example 3
Analysis of the Splice Junction of SSO-induced TNFR2 Splice Variants
[0172] To cbnfirm that the SSO splice switching, both in mice and in human
cells, leads
to the expected TNFR2 07 mRNA, SSO-induced TNFR2 A7 mRNA was analyzed by RT-
PCR and was sequenced.
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[0173] Mice. Mice were injected with SSO 3274 intraperitoneal (i.p.) at 25
mg/kg/day
for 10 days. The mice were then sacrificed and total RNA from the liver was
analyzed by
RT-PCR using the forward primer TR045 (SEQ ID No: 112) and the reverse primer
TR046
(SEQ ID No: 113). The products were analyzed on a 1.5% agarose gel (FIG. 6A)
and the
product for the TNFR2 A7 was isolated using standard molecular biology
techniques. The
isolated TNFR2 A7 product was amplified by PCR using the same primers and then
sequenced (FIG. 6B). The sequence data contained the sequence
CTCTCTTCCAATTGAGAAGCCCTCCTGC (nucleotides 777-804 of SEQ ID No: 11),
which confirms that the SSO-induced TNFR2 A7 mRNA lacks exon 7 and that exon 6
is
joined directly to exon 8.
[0174] Human hepatocytes. Primary human hepatocytes were transfected with SSO
3379
as described in Example 1. Total RNA was isolated 48 hrs after transfection.
The RNA was
converted to cDNA with the SuperscriptTM II Reverse Transcriptase (Invitrogen)
using
random hexamer primers according to the manufacturer's directions. PCR was
performed on
the cDNA using the forward primer TR049 (SEQ ID No: 86) and the reverse primer
TR050
(SEQ ID No: 87). The products were analyzed on a 1.5% agarose gel (FIG. 7A).
The band
corresponding to TNFR2 07 was isolated using standard molecular biology
techniques and
then sequenced (FIG. 7B). The sequence data contained the sequence
CGCTCTTCCAGTTGAGAAGCCCTTGTGC (nucleotides 774-801 of SEQ ID No: 9),
which confirms that the SSO-induced TNFR2 07 mRNA lacks exon 7 and that exon 6
is
joined directly to exon 8.
Example 4
SSO Dose-Dependent Production of TNFR2 /S7 Protein in Primary Human
Hepatocytes
[0175] The dose response of splice-switching activity of SSOs in primary human
hepatocytes was tested. Human hepatocytes were obtained in suspension from
ADMET
technologies. Cells were washed three times and suspended in seeding media
(RPMI 1640
supplemented with L-Glut, with 10% FBS, penicillin, streptomycin, and 12 nM
Dexamethasone). Hepatocytes were evaluated for viability and plated in 24-
well, collagen-
coated plates at 1.0 x 105 cells per well. Typically, cell viability was 85-
93%. After
approximately 24 hrs, the media was replaced with maintenance media (seeding
media
without FBS).
[0176] For delivery of each of the SSOs to the hepatocytes, SSOs were diluted
into 50 L
of OPTI-MEMTM, and then 50 L LipofectamineTM 2000 mix (1 part LipofectamineTM
2000
to 25 parts OPTI-MEMTM) was added and incubated for 20 minutes. The SSOs were
then
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applied to the cells in the 24-well plates. The final SSO concentration ranged
from 1 to 150
nM. After 48 hrs, cells were harvested in 800 L TRI-ReagentTM.
[0177] Total RNA from the cells was analyzed by RT-PCR using the forward
primer
TR047 (SEQ ID No: 84) and the reverse primer TR048 (SEQ ID No: 85) (FIG. 8A).
The
concentration of soluble TNFR2 A7 in the serum was measured by ELISA (FIG.
SB). Both
huTNFR2 07 mRNA (FIG. 8A) and secreted huTNFR2 07 protein (FIG. 8B) displayed
dose
dependent increases.
Example 5
Secretion of TNFR2 Splice Variants from Murine Cells
[0178] The ability of SSOs to induce soluble TNFR2 protein production and
secretion
into the extracellular media was tested. L929 cells were treated with SSOs as
described in
Example 1, and extracellular media samples were collected -48 hrs after
transfection. The
concentration of soluble TNFR2 in the samples was measured by ELISA (FIG. 9).
SSOs that
best induced shifts in RNA splicing, also secreted the most protein into the
extracellular
media. In particular, SSOs 3305, 3312, and 3274 increased soluble TNFR2 at
least 3.5-fold
over background. Consequently, induction of the splice variant mRNA correlated
with
production and secretion of the soluble TNFR2.
Example 6
In Vivo Injection of SSOs Generated muTNFR2 A7 mRNA in Mice
[01791 SSO 3305 in saline was injected intraperitoneal (i.p.) daily for 4 days
into mice at
doses from 3 mg/kg to 25 mg/kg. The mice were sacrificed on day 5 and total
RNA from the
liver was analyzed by RT-PCR. The data show splice switching efficacy similar
to that found
in cell culture. At the maximum dose of 25 mg/kg, SSO 3305 treatment induced
almost full
conversion to A7 mRNA (FIG. 10, bottom panel).
[0180] A similar experiment with SSO 3274 induced about 20% conversion to A7
mRNA. To optimize SSO 3274 induction of A7 mRNA, both the dose regimen and the
time
from the last injection to the sacrifice of the animal were varied. SSO 3274
was injected
(i.p.) into mice daily for 4 days. SSO treatment induced about 30% conversion
to A7 mRNA
in mice analyzed on day 15, whereas a 20% shift was observed in mice analyzed
on day five
(FIG. 10, top panel). Furthermore, mice given injections for 10 days, and
sacrificed on day
11 showed a 50% induction of A7 mRNA (FIG. 10, top). These in vivo data
suggest that
TNFR2 SSOs can produce muTNFR2 A7 mRNA for at least 10 days after
administration.
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Example 7
Circulatory TNFR2 A7
[0181] Mice were injected with SSO 3274, 3305, or the control 3083
intraperitoneal (i.p.)
at 25 mg/kg/day for 10 days. Mice were bled before injection and again 1, 5
and 10 days
after the last injection. The concentration of soluble TNFR2 07 in the serum
was measured.
SSO treatment induced soluble TNFR2 07 protein levels over background for at
least 10 days
(FIG. 11).
[0182] To test the effects at longer time points, the experiment was repeated,
except that
serum samples were collected until day 27 after the last injection.. The
results show only a
slight decrease in soluble TNFR2 A7 levels 27 days after the last SSO
injection (FIG. 12).
Example 8
Anti-TNF-a Activity in Mice Serum
101831 The anti-TNF-a activity of serum from SSO 3274 treated mice was tested
in an
L929 cytotoxicity assay. In this assay, serum is assessed for its ability to
protect cultured
L929 cells from the cytotoxic effects of a fixed concentration of TNF-a as
described in
Example 1. Serum from mice treated with SSO 3274 but not control SSOs (3083 or
3272)
increased viability of the L929 cells exposed to 0.1 ng/mL TNF-a (FIG. 13).
Hence, the SSO
3274 serum contained TNF-a antagonist sufficient to bind and to inactivate TNF-
a, and
thereby protect the cells from the cytotoxic effects of TNF-a. This anti-TNF-a
activity was
present in the serum of animals 5 and 27 days after the last injection of SSO
3274.
Example 9
Comparison of SSO Generated TNFR2 A7 to other anti-TNF-a antagonists
[0184] L929 cells were seeded as in Example 8. Samples were prepared
containing 90
L of serum-free MEM, 0.1 ng/ml TNF-a and 1 g/ml of actinomycin D, with either
(i)
recombinant soluble protein (0.01-3 pg/mL)) from Sigmag having the 236 amino
acid
residue extracellular domain of mouse TNFR2, (ii) serum from SSO 3274 or SSO
3305
treated mice (1.25-10%, diluted in serum from untreated mice; the
concentration of TNFR2
A7 was determined by ELISA) or (iii) Enbrel (0.45-150 pg/ml) to a final
volume of 100 l
with a final mouse serum concentration of 10%. The samples were incubated at
room
temperature for 30 minutes. Subsequently, the samples were applied to the
plated cells and
incubated for -24 hrs at 37 C in a 5% CO2 humidified atmosphere. Cell
viability was
measured by adding 20 L Ce1lTiter 96 AQõ,oUS One Solution Reagent (Promega)
and
measuring absorbance at 490 nm with a microplate reader. Cell viability was
normalized to
untreated cells and plotted as a function of TNF antagonist concentration
(FIG. 14).
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Example 10
Stability of TNFR2 07 mRNA and protein
[0185] Mice were treated with either SSO 3274 or 3272 (control) (n=5) by i.p.
injection
at a dose of 25 mg/kg/day daily for five days . Mice were bled before
injection and again 5,
15, 22, 27, and 35 days after the last injection. The concentration of soluble
TNFR2 A7 in
the serum was measured (FIG. 15A). Splice shifting of TNFR2 in the liver was
also
determined at the time of sacrifice by RT-PCR of total RNA from the liver
(FIG. 15B).
Combined with data from Example 7, a time course of TNFR2 mRNA levels after
SSO
treatment was constructed, and compared with the time course of TNFR2,A7
protein in serum
(FIG. 16). The data show that TNFR2 A7 mRNA in vivo decays at a rate
approximately 4
times faster than that of TNFR2 07 protein in serum. On day 35, TNFR2 07 mRNA
was
only detectable in trace amounts, whereas TNFR2 A7 protein had only decreased
by 20%
from its peak concentration.
Example 11
Generation of Human TNFR2 A7 eDNA
[01861 A plasmid containing the full length human TNFR2 cDNA was obtained
commercially from OriGene (Cat. No: TC 119459, NM 00 1066.2). The cDNA was
obtained
by performing PCR on the plasmid using reverse primer TR001 (SEQ ID No: 74)
and
forward primer TR002 (SEQ ID No: 75). The PCR product was isolated and was
purified
using standard molecular biology techniques, and contains the 1383 bp TNFR2
open reading
frame without a stop codon.
[0187] Alternatively, full length human TNFR2 cDNA is obtained by performing
RT-
PCR on total RNA from human mononuclear cells using the TR001 reverse primer
and the
TR002 forward primer. The PCR product is isolated and is purified using
standard molecular
biology techniques. `
[0188) To generate human TNFR2 A7 cDNA, two separate PCR reactions were
performed on the full length human TNFR2 cDNA, thereby creating overlapping
segments of
the TNFR2 07 eDNA. In one reaction, PCR was perforned on full length TNFR2
cDNA
using the forward primer TR003 (SEQ ID No: 76) and the reverse primer TR004
(SEQ ID
No: 77). In the other reaction, PCR was performed on full length TNFR2 eDNA
using the
reverse primer TR005 (SEQ ID No: 78) and the TR002 forward primer. Finally,
the 2
overlapping segments were combined, and PCR was performed using the TR002
forward
primer and the TR004 reverse primer. The PCR product was isolated and was
purified using
standard molecular biology techniques, and was expected to contain the 1308 bp
TNFR2 A7
open reading frame with a stop codon (SEQ ID No: 9).
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[0189] Similarly, by using the TR001 reverse primer instead of the TR004
reverse primer
in these PCR reactions the 1305 bp human TNFR2 A7 open reading frame without a
stop
codon was generated. This allows for the addition of in-frame C-terminal
affinity
purification tags, such as His-tag, when the final PCR product is inserted
into an appropriate
vector.
Example 12
Generation of Human TNFR1 07 cDNA
[0190] A plasmid containing the full length human TNFR2 cDNA is obtained
commercially from OriGene (Cat. No: TC 127913, NM 001065.2). The cDNA is
obtained by
performing PCR on the plasmid using the TR006 reverse primer (SEQ ID No: 88)
and the
TR007 forward primer (SEQ ID No: 89). The full length human TNFR1 eDNA PCR
product
is isolated and is purified using standard molecular biology techniques.
[0191] Alternatively, full length human TNFR1 cDNA is obtained by performing
RT-
PCR on total RNA from human mononuclear cells using the TR006 reverse primer
and the
TR007 forward primer. The full length human TNFRI cDNA PCR product is isolated
and is
purified using standard molecular biology techniques.
[0192] To generate human TNFR1 d7 cDNA, two separate PCR reactions are
performed
on the full length human TNFR 1 cDNA, thereby creating overlapping segments of
the
TNFRI A7 cDNA. In one reaction, PCR is performed on full length TNFR1 cDNA
using the
TR008 forward primer (SEQ ID No: 90) and the TR006 reverse primer. In the
other reaction,
PCR is performed on full length TNFR1 cDNA using the TR009 reverse primer (SEQ
ID No:
91) and the TR010 forward primer (SEQ ID No: 92). Finally, the 2 overlapping
segments are
combined, and PCR is performed using the TR010 forward primer and the TR006
reverse
primer. The PCR product is isolated and is purified using standard molecular
biology
techniques, and contains the 1254 bp human TNFR1 A7 open reading frame with a
stop
codon (SEQ ID No: 5).
[0193) Alternatively, by using the TR011 reverse primer (SEQ ID No: 93)
instead of the
TR006 reverse primer in these PCR reactions the 1251 bp human T1VFR1 A7 open
reading
frame without a stop codon is generated. This allows for the addition of in-
frame C-terminal
affinity purification tags, such as His-tag, when the final PCR product is
inserted into an
appropriate vector.
Example 13
Generation of Murine TNFR2 A7 cDNA
[0194] To generate full length murine TNFR2 cDNA, PCR was performed on the
commercially available FirstChoiceTM PCR-Ready Mouse Liver cDNA (Ambion, Cat.
No:
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AM3300) using the TRO12 reverse primer (SEQ ID No: 98) and the TRO13 forward
primer
(SEQ ID No: 99). The full length murine TNFR2 cDNA PCR product is isolated and
is
purified using standard molecular biology techniques. Then by performing PCR
on the
resulting product using the TR014 forward primer (SEQ ID No: 100) and the
TR012 reverse
primer the proper Kozak sequence was introduced.
[0195] Alternatively, full length murine TNFR2 cDNA is obtained by performing
RT-
PCR on total RNA from mouse mononuclear cells or mouse hepatocytes using the
TR015
reverse primer (SEQ ID No: 101) and the TR016 forward primer (SEQ ID No: 102).
The full
length murine TNFR2 cDNA PCR product is isolated and is purified using
standard
molecular biology techniques.
[0196] To generate murine TNFR2 A7 eDNA, two separate PCR reactions were
performed on the full length murine TNFR2 cDNA, thereby creating overlapping
segments of
the TNFR2 A7 cDNA. In one reaction, PCR was performed on full length TNFR2
cDNA
using the TR017 forward primer (SEQ ID No: 103) and the TR015 reverse primer.
In the
other reaction, PCR was performed on full length TNFR2 cDNA using the TROI
S'reverse
primer (SEQ ID No: 104) and the TR016 forward primer. Finally, the 2
overlapping
segments were combined, and PCR was performed using the TR016 forward primer
and the
TRO15 reverse primer. The PCR product was isolated and was purified using
standard
molecular biology techniques, and was expected to contain the 1348 bp murine
TNFR2 A7
open reading frame with a stop codon (SEQ ID No: 11).
[0197] Alternatively, by using the TR019 reverse primer (SEQ ID No: 105)
instead of the
TR015 reverse primer in these PCR reactions the 1345 bp murine TNFR2 07 open
reading
frame without a stop codon was generated. This allows for the addition of in-
frame C-
terminal affinity purification tags, such as His-tag, when the final PCR
product is inserted
into an appropriate vector.
Example 14
Generation of Murine TNFR1 A7 cDNA
[0198) To generate full length murine TNFRI cDNA, PCR is performed on the
commercially available FirstChoiceTM PCR-Ready Mouse Liver cDNA (Ambion, Cat.
No:
AM3300) using the TR020 reverse primer (SEQ ID No: 114) and the TR021 forward
primer
(SEQ ID No: 115). The full length murine TNFRI cDNA PCR product is isolated
and is
purified using standard molecular biology techniques.
[0199] Alternatively, full length murine TNFR1 cDNA is obtained by performing
RT-
PCR on total RNA from mouse mononuclear cells using the TR020 reverse primer
and the
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TR021 forward primer. The full length murine TNFR1 cDNA PCR product is
isolated and is
purified using standard molecular biology techniques.
102001 To generate murine TNFRl A7 cDNA, two separate PCR reactions are
performed
on the full length human TNFR1 cDNA, thereby creating overlapping segments of
the
TNFR1 A7 cDNA. In one reaction, PCR is performed on full length TNFRI cDNA
using the
TR022 forward primer (SEQ ID No: 116) and the TR020 reverse primer. In the
other
reaction, PCR is performed on fu111ength TNFR1 cDNA using the TR023 reverse
primer
(SEQ ID No: 117) and the TR024 forward primer (SEQ ID No: 118). Finally,'the 2
overlapping segments are combined, and PCR is performed using TR024 forward
primer and
the TR020 reverse primer. The 1259 bp PCR product is isolated and is purified
using
standard molecular biology techniques, and contains the 1251 bp murine TNFR1
07 open
reading frame with a stop codon (SEQ ID No: 7).
[0201] Alternatively, by using the TR025 reverse primer (SEQ ID No: 119)
instead of the
TR020 reverse primer in these PCR reactions the 1248 bp murine TNFR1 A7 open
reading
frame without a stop codon is generated. This allows for the addition of in-
frame C-terminal
affinity purification tags, such as His-tag, when the final PCR product is
inserted into an
appropriate vector.
Example 15
Construction of Vectors for the Expression of Human TNFR2 A7 in Mammalian
Cells
[0202] For expression of the human TNFR2 A7 protein in mammalian cells, a
human
TNFR2 A7 eDNA PCR product from Example 11 was incorporated into an appropriate
mammalian expression vector. The TNFR2 A7 eDNA PCR product from Example 11,
both
with and without a stop codon, and the pcDNATM3.1D/V5-His TOPO expression
vector
(Invitrogen) were blunt-end ligated and isolated according to the
manufacturer's directions.
Plasmids containing inserts encoding human TNFR2 A7 were transformed into
OneShot
Top10 competent cells (Invitrogen), according to the supplier's directions.
Fifty L of the
transformation mix were plated on LB media with 100 g/mL of ampicillin and
incubated
overnight at 37 C. Single colonies were used to inoculate 5 mL cultures of LB
media with
100 g/mL ampicillin and incubated overnight at 37 C. The cultures were then
used to
inoculate 200 mL of LB media with 100 gg/mL of ampicillin and grown oveinight
at 37 C.
The plasmids were isolated using GenEluteTM Plasmid Maxiprep kit (Sigma)
according to
manufacturer's directions. Purification efficiency ranged from 0.5 to 1.5 mg
of plasmid per
preparation.
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[0203] Three human TNFR2 07 clones (1319-1, 1138-5 and 1230-1) were generated
and
sequenced. Clone 1319-1 contains the human TNFR2 A7 open reading frame without
a stop
codon followed directly by an in-frame His-tag from the plasmid; while clones
1138-5 and
1230-1 contain the TNFR2 A7 open reading frame followed immediately by a stop
codon.
The sequence of the His-tag from the plasmid is given in SEQ ID No: 126. The
sequences of
the TNFR2 A7 open reading frames of clones 1230-1 and 1319-1 were identical to
SEQ ID
No: 9 with and without the stop codon, respectively. However relative to SEQ
ID No: 9, the
sequence (SEQ ID No: 125) of the TNFR2 A7 open reading frames of clone 1138-5
differed
by a single nucleotide at position 1055 in exon 10, with an A in the former
and a G in the
later. This single nucleotide change causes the amino acid 352 to change from
a glutamine to
an arginine.
Example 16
Expression of Human TNFR2 A7 in E. colf
[0204] For expression of the human TNFR2 A7 protein in bacteria, a human TNFR2
d7
cDNA from Example 11 is incorporated into an appropriate expression vector,
such as a pET
Directional TOPOO expression vector (Invitrogen). PCR is performed on the PCR
fragment
from Example 11 using forward (TR002) (SEQ ID No: 75) and reverse (TR026) (SEQ
ID
No: 79) primers to incorporate a homologous recombination site for the vector.
The resulting
PCR fragment is incubated with the pET101 /D-TOPO vector (Invitrogen)
according to the
manufacturer's directions, to create the human TNFR2 07 bacterial expression
vector. The
resulting vector is transformed into the E. coli strain BL21(DE3). The human
TNFR2 A7 is
then expressed from the bacterial cells according to the manufacturer's
instructions.
Example 17
Expression of Human TNFR2 07 in insect cells
[0205] For expression of the human TNFR2 A7 protein in insect cells, a human
TNFR2
07 cDNA from Example 11 is incorporated into a baculoviral vector. PCR is
performed on a
human TNFR2 A7 cDNA from Example 11 using forward (TR027) (SEQ ID No: 80) and
reverse (TR028) (SEQ ID No: 81) primers. The resulting PCR product is digested
with the
restriction enzymes EcoRI and Xhol. The digested PCR product is ligated with a
EcoRI and
Xhol digested pENTRTM Vector (Invitrogen), such as any one of the pENTRTMIA,
pENTRTM2B, pENTRTM3C, pENTRTM4, or pENTRTM 11 Vectors, to yield an entry
vector.
The product is then isolated, amplified, and purified using standard molecular
biology
techniques.
[0206] A baculoviral vector containing the human TNFR2 A7 cDNA is generated by
homologous recombination of the entry vector with BaculoDirectTM Linear DNA
(Invitrogen)
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using LR ClonaseTM (Invitrogen) according to the manufacturer's directions.
The reaction
mixture is then used to infect Sf9 cells to generate recombinant baculovirus.
After harvesting
the recombinant baculovirus, expression of human TNFR2 07 is confirmed.
Amplification
of the recombinant baculovirus yields a high-titer viral stock. The high-titer
viral stock is
used to infect Sf9 cells, thereby expressing human TNFR2 07 protein.
Example 18
Generation of Adeno-Associated viral vectors for the expression of Human TNFR2
A7
[0207] For in vitro or in vivo delivery to mammalian cells of the human TNFR2
A7 gene
for expression in those mammalian cells, a recombinant adeno-associated virus
(rAAV)
vector is generated using a three plasmid transfection system as described in
Grieger, J., et
al., 2006, Nature Protocols 1:1412. PCR is performed on a purified human TNFR2
A7 PCR
product of Example 11, using forward (TR029) (SEQ ID No: 82) and reverse
(TR030) (SEQ
ID No: 83) primers to introduce unique flanking Notl restriction sites. The
resulting PCR
product is digested with the Notl restriction enzyme, and isolated by standard
molecular
biology techniques. The Notl-digested fragment is then ligated to Notl-
digested pTR-UF2
(University of North Carolina (UNC) Vector Core Facility), to create a plasmid
that contains
the human TNFR2 07 open reading frame, operably linked to the CMVie promoter,
flanked
by inverted terminal repeats. The resulting plasmid is then transfected with
the plasmids
pXX680 and pHelper (UNC Vector Core Facility) into HEK-293 cells, as described
in
Grieger, J., et al., to produce rAAV particles containing the human TNFR2 07
gene where
expression is driven by the strong constitutive CMVie promoter: The virus
particles are
harvested and purified, as described in Grieger, J., et al., to provide an
rAAV stock suitable
for transducing mammalian cells.
Example 19
Expression of Human TNFR1 A7 in E. coli
[0208] For expression of the human TNFR1 A7 protein in bacteria, the cDNA from
Example 12 is incorporated into an appropriate expression vector, such as a
pET Directional
TOPO expression vector (Invitrogen). PCR is performed on the cDNA from
Example 12
using forward (TRO10) (SEQ ID No: 92) and reverse (TR006) (SEQ ID No: 88)
primers to
incorporate a homologous recombination site for the vector. The resulting PCR
fragment is
incubated with the pET101/D-TOPO vector (Invitrogen) according to the
manufacturer's
directions, to create the human TNFRI d7 bacterial expression vector. The
resulting vector is
transformed into the E. colf strain BL21(DE3). The human TNFR1 07 is then
expressed
from the bacterial cells according to the manufacturer's instructions.
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Example 20
Expression of Human TNFRl 07 in mammalian cells
[02091 For expression of the human TNFRI 07 protein in mammalian cells, a
human
TNFR1 07 cDNA PCR product from Example 12 is incorporated into an appropriate
mammalian expression vector. human TNFRl A7 cDNA PCR product from Example 12
and
the pcDNAT"'3.1D1V5-His TOPO expression vector (Invitrogen) are blunt-end
ligated
according to the manufacturer's directions. The product is then isolated,
amplified, and
purified using standard molecular biology techniques to yield the mammalian
expression
vector. The vector is then transfected into a mammalian cell, where expression
of the human
TNFR1 A7 protein is driven by the strong constitutive CMVie promoter.
Example 21
Expression of Human TNFRl 07 in insect cells
102101 For expression of the human TNFR1 A7 protein in insect cells, the cDNA
from
Example 12 is incorporated into a baculoviral vector. PCR is performed on the
eDNA from
Example 12 using forward (TR031) (SEQ ID No: 94) and reverse (TR032) (SEQ ID
No: 95)
primers. The resulting PCR product is digested with the restriction enzymes
EcoRI and
Xhol. The digested PCR product is ligated with a EcoRI and XhoI digested
pENTRTM
Vector (Invitrogen), such as any one of the pENTRTMIA, pENTRTM2B, pENTRTM3C,
pENTRTM4, or pENTRTMI l Vectors, to yield an entry vector. The product is then
isolated,
amplified, and purified using standard molecular biology techniques.
[0211] A baculoviral vector containing the human TNFR1 A7 cDNA is generated by
homologous recombination of the entry vector with BaculoDirectTM Linear DNA
(Invitrogen)
using LR ClonaseTM (Invitrogen) according to the manufacturer's directions.
The reaction
mixture is then used to infect Sf9 cells to generate recombinant baculovirus.
After harvesting
the recombinant baculovirus, expression of human TNFRI 07 is confirmed.
Amplification
of the recombinant baculovirus yields a high-titer viral stock. The high-titer
viral stock is
used to infect Sf9 cells, thereby expressing human TNFRl 07 protein.
Example 22
Generation of Adeno-Associated viral vectors for the expression of Human TNFR1
A7
[0212] For in vitro or in vivo delivery to mammalian cells of the human TNFRl
t17 gene
for expression in those mammalian cells, a recombinant adeno-associated virus
(rAAV)
vector is generated using a three plasmid transfection system as described in
Grieger, J., et
al., 2006, Nature Protocols 1:1412. PCR is performed on the purified human
TNFR1 A7
PCR product of Example 12, using forward (TR033) (SEQ ID No: 96) and reverse
(TR034)
(SEQ ID No: 97) primers to introduce unique flanking Notl restriction sites.
The resulting
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PCR product is digested with the Notl restriction enzyme, and isolated by
standard molecular
biology techniques. The Notl-digested fragment is then ligated to Notl-
digested pTR-UF2
(University of North Carolina (iTNC) Vector Core Facility), to create a
plasmid that contains
the human TNFR1 A7 open reading frame, operably linked to the CMVie promoter,
flanked
by inverted terminal repeats. The resulting plasmid is then transfected with
the plasmids
pXX680 and pHelper (UNC Vector Core Facility) into HEK-293 cells, as described
in
Grieger, J., et al., to produce rAAV particles containing the human TNFR1 07
gene where
expression is driven by the strong constitutive CMVie promoter. The virus
particles are
harvested and purified, as described in Grieger, J., et al., to provide an
rAAV stock suitable
for transducing mammalian cells. _
Example 23
Construction of Vectors for the Expression of Mouse TNFR2 07 in mammalian
cells
[0213] For expression of the murine TNFR2 A7 protein in mammalian cells, a
murine
TNFR2 A7 cDNA PCR product from Example 13 was incorporated into an appropriate
mammalian expression vector. The TNFR2 A.7 cDNA PCR product from Example 13,
both
with and without a stop codon, and the pcDNATM3.1D/V5-His TOPO expression
vector
(Invitrogen) was blunt-end ligated and isolated according to the
manufacturer's directions.
Plasmids containing inserts encoding murine A7 TNFR2 were transformed into
OneShot
ToplO competent cells (Invitrogen), according to the supplier's directions.
Fifty L of the
transformation mix were plated on LB media with 100 g/mL of ampicillin and
incubated
overnight at 37 C. Single colonies were used to inoculate 5 mL cultures of LB
media with
100 g/mL ampicillin and incubated overnight at 37 C. The cultures were then
used to
inoculate 200 mL of LB media with 100 g/mL of ampicillin and grown overnight
at 37 C.
The plasmids were isolated using GenEluteTM Plasmid Maxiprep kit (Sigma)
according to
manufacturer's directions. Purification efficiency ranged from 0.5 to 1.5 mg
of plasmid per
preparation.
[0214] Two murine TNFR2 d7 clones (1144-4 and 1145-3) were generated and
sequenced. Clone 1144-4 contains the murine TNFR2 A7 open reading frame
without a stop
codon followed directly by an in-frame His-tag from the plasmid; while clone
1145-3
contains the TNFR2 A7 open reading frame followed immediately by a stop codon.
The
sequence of the His-tag from the plasmid is given in SEQ ID No: 126. Relative
to SEQ ID
No: 11, the sequence (SEQ ID No: 124) of the TNFR2 A7 open reading frames of
the two
clones, 1144-4 and 1145-3, differed by a single nucleotide at eleven
positions. As a result of
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these single nucleotide changes there are four amino acid differences relative
to SEQ ID No:
12.
Example 24
Expression of Murine TNFR2 07 in E. coli
[0215] For expression of the mouse TNFR2 A7 protein in bacteria, a murine
TNFR2 A7
cDNA from Example 13 is incorporated into an appropriate expression vector,
such as a pET
Directional TOPO expression vector (Invitrogen). PCR is performed on the PCR
fragment
from Example 13 using forward (TR035) (SEQ ID No: 106) and reverse (TR036)
(SEQ ID
No: 107) primers to incorporate a homologous recombination site for the
vector. The
resulting PCR fragment is incubated with the pET101/D-TOPO vector
(Invitrogen)
according to the manufacturer's directions, to create the murine TNFR2 A7
bacterial
expression vector. The resulting vector is transformed into the E. coli strain
BL21(DE3).
The murine TNFR2 A7 is then expressed from the bacterial cells according to
the
manufacturer's instructions.
Example 25
Expression of Mouse TNFR2 07 in insect cells
[0216] For expression of the murine TNFR2 07 protein in insect cells, the cDNA
from
Example 13 is incorporated into a baculoviral vector. PCR is performed on the
cDNA from
Example 13 using forward (TR037) (SEQ ID No: 108) and reverse (TR038) (SEQ ID
No:
109) primers. The resulting PCR product is digested with the restriction
enzymes EcoRI and
Xhol. The digested PCR product is ligated with a EcoRI and Xhol digested
pENTRTM
Vector (Invitrogen), such as any one of the pENTRTM1A, pENTRTM2B, pENTRTM3C,
pENTRTM4, or pENTRTM l 1 Vectors, to yield an entry vector. The product is
then isolated,
amplified, and purified using standard molecular biology techniques.
[0217] A baculoviral vector containing the murine TNFR2 A7 cDNA is generated
by
homologous recombination of the entry vector with BaculoDirectTM Linear DNA
(Invitrogen)
using LR ClonaseTM (Invitrogen) according to the manufacturer's directions.
The reaction
mixture is then used to infect Sf9 cells to generate recombinant baculovirus.
After harvesting
the recombinant baculovirus, expression of murine TNFR2 A7 is confirmed.
Amplification
of the recombinant baculovirus yields a high-titer viral stock. The high-titer
viral stock is
used to infect Sf9 cells, thereby expressing murine TNFR2 A7 protein.
Example 26
Generation of Adeno-Associated viral vectors for the expression of Murine
TNFR2 d7
[0218] For in vitro or in vivo delivery to mammalian cells of the murine TNFR2
07 gene
for expression in those mammalian cells, a recombinant adeno-associated virus
(rAAV)
vector is generated using a three plasmid transfection system as described in
Grieger, J., et
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al., 2006, Nature Protocols 1:1412. PCR is performed on the purified murine
TNFR2 A7
PCR product of Example 13, using forward (TR039)(SEQ ID No: 110) and reverse
(TR040)(SEQ ID No: 111) primers to introduce unique flanking NotI restriction
sites. The
resulting PCR product is digested with the Notl restriction enzyme, and
isolated by standard
molecular biology techniques. The Notl-digested fragment is then ligated to
Notl-digested
pTR-UF2 (University of North Carolina (UNC) Vector Core Facility), to create a
plasmid
that contains the murine TNFR2 A7 open reading frame, operably linked to the
CMVie
promoter, flanked by inverted terminal repeats. The resulting plasmid is then
transfected
with the plasmids pXX680 and pHelper (UNC Vector Core Facility) into HEK-293
cells, as
described in Grieger, J., et al., to produce rAAV particles containing the
murine TNFR2 A7
gene where expression is driven by the strong constitutive CMVie promoter. The
virus
particles are harvested and purified, as described in Grieger, J., et al., to
provide an rAAV
stock suitable for transducing mammalian cells.
Example 27
Expression of Murine TNFR1 07 in E. coli
[0219] For expression of the mouse TNFR1 A7 protein in bacteria, the cDNA from
Example 14 is incorporated into an appropriate expression vector, such as a
pET Directional
TOPO expression vector (Invitrogen). PCR is performed on the cDNA from
Example 14
using forward (TR024)(SEQ ID No: 118) and reverse (TR020)(SEQ ID No: 114)
primers to
incorporate a homologous recombination site for the vector. The resulting PCR
fragment is
incubated with the pET101/D-TOPO vector (Invitrogen) according to the
manufacturer's
directions, to create the murine TNFR1 A7 bacterial expression vector. The
resulting vector is
transformed into the E. colr strain BL21(DE3). The murine TNFRI A7 is then
expressed
from the bacterial cells according to the manufacturer's instructions.
Example 28
Expression of Mouse TNFR1 A7 in mammalian cells
[0220] For expression of the murine TNFR1 A7 protein in mammalian cells, a
murine
TNFRl A7 cDNA PCR product from Example 14 is incorporated into an appropriate
mammalian expression vector. The murine TNFR1 A7 cDNA PCR product from Example
14 and the pcDNATM3.1D/V5-His TOPO expression vector (Invitrogen) are blunt-
end
ligated according to the manufacturer's directions. The product is then
isolated, amplified,
and purified using standard molecular biology techniques to yield the
mammalian expression
vector. The vector is then transfected into a mammalian cell, where expression
of the murine
TNFRI 07 protein is driven by the strong constitutive CMVie promoter.
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Example 29
Expression of Mouse TNFR1 07 in insect cells
[0221] For expression of the murine TNFR1 A7 protein in insect cells, the cDNA
from
Example 14 is incorporated into a baculoviral vector. PCR is performed on the
cDNA from
Example 14 using forward (TR041)(SEQ ID No: 120) and reverse (TR042) (SEQ ID
No:
121) primers. The resulting PCR product is digested with the restriction
enzymes EcoRI and
Xhol. The digested PCR product is ligated with a EcoRI and XhoI digested
pENTRTM
Vector (Invitrogen), such as any one of the pENTRTMIA, pENTRTM2B, pENTRTM3C,
pENTRTM4, or pENTRTMl 1 Vectors, to yield an entry vector. The product is then
isolated,
amplified, and purified using standard molecular biology techniques.
[0222] A baculoviral vector containing the murine TNFR1 07 cDNA is generated
by
homologous recombination of the entry vector with BaculoDirectTM Linear DNA
(Invitrogen)
using LR ClonaseTM (Invitrogen) according to the manufacturer's directions.
The reaction
mixture is then used to infect Sf9 cells to generate recombinant baculovirus.
After harvesting
the recombinant baculovirus, expression of murine TNFRI A7 is confirmed.
Amplification
of the recombinant baculovirus yields a high-titer viral stock. The high-titer
viral stock is
used to infect Sf9 cells, thereby expressing murine TNFR1 07 protein.
Example 30
Generation of Adeno-Associated viral vectors for the expression of Murine
TNFR1 A7
[02231 For in vitro or in vivo delivery to mammalian cells of the murine TNFR1
A7 gene
for expression in those mammalian cells, a recombinant adeno-associated virus
(rAAV)
vector is generated using a three plasmid transfection system as described in
Grieger, J., et
al., 2006, Nature Protocols 1:1412. PCR is performed on the purified murine
TNFR1 A7
PCR product of Example 13, using forward (TR043)(SEQ ID No: 122) and reverse
(TR044)(SEQ ID No: 123) primers to introduce unique flanking Notl restriction
sites. The
resulting PCR product is digested with the NotI restriction enzyme, and
isolated by standard
molecular biology techniques. The NotI-digested fragment is then ligated to
NotI-digested
pTR-UF2 (University of North Carolina (UNC) Vector Core Facility), to create a
plasmid
that contains the murine TNFRI 07 open reading frame, operably linked to the
CMVie
promoter, flanked by inverted terminal repeats. The resulting plasmid is then
transfected
with the plasmids pXX680 and pHelper (UNC Vector Core Facility) into HEK-293
cells, as
described in Grieger, J., et al., to produce rAAV particles containing the
murine TNFR1 A7
gene where expression is driven by the strong constitutive CMVie promoter. The
virus
particles are harvested and purified, as described in Grieger, J., et al., to
provide an rAAV
stock suitable for transducing mammalian cells.
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Example 31
Generation of Lentiviral vectors for the expression of TNFR A7
[0224] For in vitro or in vivo delivery to mammalian cells of a TNFR A7 gene
for
expression in those mammalian cells, a replication-incompetent lentivirus
vector is generated.
A PCR product from Example 16, Example 19, Example 24 or Example 27 and the
pLenti6/V5-D-TOPOV vector (Invitrogen) are blunt-end ligated according to the
manufacturer's directions. The resulting plasmid is transformed into E. coli,
amplified, and
purified using standard molecular biology techniques. This plasmid is
transfected into 293FT
cells (Invitrogen) according to the manufacturer's directions to produce
lentivirus particles
containing the TNFR 07 gene where expression is driven by the strong
constitutive CMVie
promoter. The virus particles are harvested and purified, as described in
Tiscornia, G., et al.,
2006, Nature Protocols 1:241, to provide a tentiviral stock suitable for
transducing
mammalian cells.
Example 32
Expression of TNFR2 A7 in Mammalian Cells
[0225] The plasmids generated in Example 15 and Example 23 were used to
express
active protein in mammalian HeLa cells, and the resulting proteins were tested
for anti-TNF-
a activity. HeLa cells were seeded in at 1.0 x 105 cells per well in 24-well
plates in SMEM
media containing L-glutamine, gentamicin, kanamycin, 5% FBS and 5% HS. Cells
were
grown overnight at 37 C in a 5% CO2 humidifed atmosphere. Approximately 250 ng
of
plasmid DNA was added to 50 L of OPTI-MEMTM, and then 50 L LipofectamineTM
2000
mix (I part LipofectamineTM 2000 to 25 parts OPTI-MEMTM) was added and
incubated for
20 minutes. Then 400 L of serum free media was added and then applied to the
cells in the
24-well plates. After incubation for -48 hrs at 37 C in a 5% CO2 humidified
atmosphere, the
media was collected and the cells were harvested in 800 L TRI-ReagentTM.
Total RNA was
isolated from the cells per the manufacturer's directions and analyzed by RT-
PCR using the
forward primer TR047 (SEQ ID No: 84) and the reverse primer TR048 (SEQ ID No:
85) for
human TNFR2 07, or the forward primer TR045 (SEQ ID No: 112) and the reverse
primer
TR046 (SEQ ID No: 113) for mouse TNFR2 A7. The concentration of soluble TNFR2
in the
media was measured by ELISA.
[0226] The anti-TNF-a activity of the above media was tested in an L929
cytotoxicity
assay. L929 cells were plated in 96-well plates at 2 x 104 cells per well in
MEM media
containing 10% regular FBS, penicillin and streptomycin and grown overnight at
37 C in a
5% CO2humidified atmosphere. The media samples were diluted 1, 2, 4, 8 and 16
fold with
media from non-transfected HeLa cells. Ninety L of each of these samples was
added to 10
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L of serum-free media, containing 1.0 ng/ml TNF-a and 1 g/ml of actinomycin
D. The
media from the cells were removed and replaced with these 100 L samples. The
cells were
then grown overnight at 37 C in a 5% CO2 humidified atmosphere. Twenty L
CellTiter
96 AQ1eoõ5 One Solution Reagent (Promega) was then added to each well. Cell
viability
was measured 4 hrs later by measuring absorbance at 490 nm with a microplate
reader. Cell
viability was normalized to untreated cells nd plotted as a function of TNF
antagonist
concentration (FIG. 17).
[0227] The data from this example and from Example 9 were analyzed using the
GraphPad Prism software to determine the EC50 value for each antagonist. For
each
antagonist from these examples a sigmoidal dose-response curve was fit by non-
linear
regression with the maximum and minimum responses held fixed to 100% and 0%,
respectively. The EC50 values shown in Table 5 correspond to a 95% confidence
level, and
each curve had an r2 value ranging from 0.7 to 0.9.
Table 5: Activi of TNF-a anta onists
TNF-a Antagonist ECso
n/nzL
Etanercept 1.1 0.5
ecombinant soluble TNFR2 (rsTNFR2) 698 180
SSO 3305 treated mice serum (mouse TNFR2 A7) 0.6 0.2
SSO 3274 treated mice serum (mouse TNFR2 A7) 0.8 0.3
xtracellular media from 1144-4 transfected HeLa cells (mouse TNFR2 A7) 2.4
:4:1.4
xtracellular media from 1145-3 transfected HeLa cells (mouse TNFR2 A7) 2.4 J=
0.8
xtracellular media from 1230-1 transfected HeLa cells (human TNFR2 A7) 1.4
1.1
xtracellular media from 1319-1 transfected HeLa cells (human TNFR2 A7) 1.7
1.0
xtracellular media from 1138-5 transfected HeLa cells (human TNFR2 07) 1.8 zL
1.1
Example 33
Expression and Purification of TNFR2 07 in Mammalian Cells
[0228] The plasmids generated in Example 15 and Example 23 were used to
express and
purify TNFR2 A7 from mammalian HeLa cells. HeLa cells were plated in 6-well
plates at 5
x 105 cells per well, and grown overnight at 37 C, 5% C02, in humidified
atmosphere. Each
well was then transfected with 1.5 g of plasmid DNA using either 1144-4
(mouse TNFR2
A7 with His-tag), 1145-1 (mouse TNFR2 07, no His-tag), 1230-1 (human TNFR2 A7,
no
His-tag) or 1319-1 (human TNFR2 A7 with His-tag) plasmids. Media was collected
-48 hrs
after transfection and concentrated approximately 40-fold using Amicon MWCO
30,000
filters. The cells were lysed in 120 L of RIPA lysis buffer (Invitrogen) with
protease
inhibitors (Sigma-aldrich) for 5 minutes on ice. Protein concentration was
determined by the
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CA 02666981 2009-04-20
WO 2008/051306 PCT/US2007/010556
Bradford assay. Proteins were isolated from aliquots of the cell lysates and
the extracellular
media and analyzed by western blot for TNFR2 as described in Example 1(FIG.
18).
102291 Human and mouse TNFR2 tA7 with a His-tag (clones 1319-1 and 1144-4,
respectively) were purified from the above media by affinity chromatography.
HisPurTM
cobalt spin columns (Pierce) were used to purify mouse and human TNFR2 A7
containing a
His-tag from the above media. Approximately 32 mL of media were applied to a I
mL
HisPurTM column equilibrated with 50 mM sodium phosphate, 300 mM sodium
chloride, 10
mM imidazole buffer (pH 7.4) as recommended by the manufacturer. The column
was then
washed with two column volumes of the same buffer and protein was eluted with
1 mL of 50
mM sodium phosphate, 300 mM sodium chloride, 150 mM imidazole buffer (pH 7.4).
Five
L of each etuate were analyzed by Western blot as described above (FIG. 19).
TNFR2 A7
appears in the eluate and the multiple bands represent variably glycosylated
forms of TNFR2
7. As negative controls, the TNFR2 A7 proteins expressed from plasmids 1230-1
or 1145-1
which do not contain a His-tag where subjected to the above purification
procedure. These
proteins do not bind the affinity column and do not appear in the eluate (FIG.
19).
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