Note: Descriptions are shown in the official language in which they were submitted.
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GENE THERAPY WITH MODIFIED p65 PROTEINS
Field of the invent;on
The invention relates to the fields of gene therapy and tissue and organ
transplantation. It concerns genetic modification of endothelial cells, or other m~mm~ n
cells such as hematopoietic cells, to render them less susceptible to an infl~mmzltory
stimulus In particular, it is addressed to genetic modification of endothelial or other
m~3mm~ n cells to render them capable of exl~lGssillg a protein which specifically inhibits
NFKB, whereby NF~ transactivation of infl~mm~tQry and other proteins is suppressed or
inhibited under cell activating conditions.
It is also concerned with transplantation of genetically modified cells, or tissue or
organs comprising such cells, capable of expressing the inhibiting protein; it most
particularly is directed to methods of transplanting modified xenogeneic or allogeneic cells,
tissue or organs; recombinant genes, proteins and vectors for accomplishing same; and the
cells, tissue or organs, as well as non-human transgenic or somatic recombinant ~nim~l~, So
modified.
Ra~k~round of tbe Invention
This invention relates to methods of suppressing mz~mm~ n (e.g. endothelial) cell
activation, and in particular relates to allo- or xenotransplantation of endothelial cells, and
tissues and organs containing them.
A major problem in the successful transplantation of organs between discordant
species is hyperacute rejection of the organ, the main initiators of which are the antibodies
and complement system of the organ recipient. In this respect, one approach to ~tt~ining
prolonged graft life has been to prepare donor organs which express complement regulatory
factors of the recipient.
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However, the cells of the donor organ themselves, i.e. the endothelial cells, also give
rise to rejection by stimnl~ting coagulation in the recipient, and by undergoing physical and
metabolic changes in a process known as "activation". In m~mm~lc, the endothelium (also
known as the "vascular endothelium") consists of a layer of cells that line the cavities of the
heart and of the blood and Iymph vessels. Endothelial cells (EC) normally m~int~in
vascular integrity and blood flow.
The process of activation of donor endothelial cells by graft recipient platelet- and
leukocyte-me~ r~l release of activating agents (e.g. cytokines such as IL-loc), has been
described in the literature [Bach et al., Immunological Reviews 141 (1994) 5-30].
In particular, when "activated" by infl~mm~tory stimuli such as a bacterial endotoxin
comprising a lipopolysaccharide (LPS) or infl~mm~tory cytokines (IL-I, TNFo~), the EC
tend to retract from one another, resulting in leakage of blood cells and plasma proteins (i.e.
hemorrhage and edema), and loss of heparin sulfate and thrombomodulin, among other
proteins, from the EC surface, in turn leading to coagulation and platelet aggregation. The
next stage is characterized by induction in the EC of a number of genes and their products,
including those coding for adhesion molecules that promote host leukocyte adhesion and
extravasation, tissue factor that enhances the pro-coagulant phenotype of the surrounding
cellular environment, cytokines and monokines that also contribute to the attraction and
activation of leukocytes, interleukins, and other procoagulant, prothrombotic components of
the coagulation system. Graft injury and loss seen in allograft and xenograft rejection, as
well as graft preservation-induced endothelial damage, exemplify the vulnerability of
endothelial cells, tissue and organs in the activated condition.
Considerable effort by workers in the art has been directed toward elucidation of
agents which can control endothelial cell activation as well as activation of m~mm~ n cells
in general. However, there continues to exist a critical need for methods of preventing or
minimi7ing the physiological processes associated with endothelial cell activation. In
particular, there is a need to prolong graft organ survival, while minimi7ing toxicity and
other adverse effects often seen with available activation- or inflammzltion-suppressing
agents.
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An identified transcription factor that induces transcription of a wide variety of
genes associated with endothelial cell activation (e.g. immunomodulatory cytokines, cell
surface receptors, procoagulant factors), by binding to specific decameric sequence motifs in
enhancer and promoter elements of said genes (referred to as "~B" sequences), is "Nuclear
Factor KB" (NFlcB).
In its active form NFKB constitutes a dimer comprised of members of the Rel/NFKBfamily of proteins: RelA (p65), Rel (= c-Rel), RelB, NFKBI (pS0) and NF~B2 (p52).
Almost all combinations of these subunits into homodimers and heterodimers have been
isolated, and are reported to have different affinities toward KB sequence motifs. For
example, p50 and pS2 strongly bind such sequence motifs and, when dimerized, are found
to contribute mainly to DNA binding rather than transactivation. On the other hand, p65
(RelA), Rel and RelB carry transcription activation domains of varying strengths, with less
participation in binding. In particular, RelA (alternatively known in the art as "p65"), while
having significantly weaker DNA binding activity than pS0, has been found to be a potent
transcriptional activator.
The Rel/NFKB proteins are characterized in having a highly homologous sequence of
approximately 300 amino acids referred to as the "Rel Homology Domain" (RHD). This
homology also occurs across species; for example, murine and human RelA (p65) have been
found to be strongly homologous. Biochemical analysis has shown that the predominant
form of NFlcB in m~mmzlls (e.g. humans) is a heterodimer comprising pS0 and p65
subunits. The NFlcB heterodimer is constitutively expressed in the cytoplasm of cells. In
unstimul~t~ d cells, NFKB is sequestered in an inactive form in the cytoplasm by binding to
an inhibitor, namely, IKBoc, via the RelA (p65) subunit. In this trimeric form, the IKBo~
appears to mask the nuclear localization signal (NLS) within the Rel homology domain of
the p65 and pS0 subunits of NFKB. However, upon stimulation of cells with specific agents
such as IL-I, TNFa, or lipopolysaccharide (LPS), the IlcBoc protein is rapidly
phosphorylated and proteolytically degraded, liberating the NFKB dimer and thereby
nnm~sking the NLS, and facilitating the rapid translocation of NFKB to the nucleus. In
essence, NFKB functions as a transducer of cytoplasmic signals to the nucleus by a
translocation mech~ni~m- Once in the nucleus, NFKB binds to available lcB sites in control
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elements of the nuclear DNA, and induces transcription of the underlying gene. Inasmuch
as many of the genes associated with endothelial cell activation, such as infl~mm~tory
genes, contain at least one KB site, the activation of NFKB ultimately leads to
transactivation of genes involved in the infl~mm~tory process. By "transactivation" is
meant the regulation of gene activation and transcription by a cellular factor (e.g. NF~cB)
acting in trans (i.e. without being covalently bound to the gene), by binding to or otherwise
influencing the control elements (e.g. promoters, enhancers) which regulate the gene in cis
(i.e. by covalent bonding to the coding part of the gene) [see e.g. Baeuerle and Henkel,
Ann. Rev. Immunol. 12 (1994) 141-179; Fujita et al., Genes & Dev. 6 (1992) 775-787;
Urban et al., Fl\~BO J. 10 (1991) 1817-1825].
One approach to effect inhibition of induction of infl~mm~tory genes under
activating conditions has been to stabilize the NFKB-I~Ba trimer by protecting IlcBa
against phosphorylation and proteases, and thus prevent migration of NFKB to the nucleus.
However, anti-oxidants as well as proteasome inhibitors suffer from non-specificity. At
present there is a lack of available inhibitors of NFlcB that are both specific and effective.
It has remained an objective in the art to identify means of specifically preventing or
suppressing induction of inflammatory genes, especially under endothelial cell activating
conditions.
Summ;~rv of the invention
It has now been found that certain modified proteins can function as potent
inhibitors of NFKB under cellular activating conditions. Such proteins in general have the
properties of:
(a) binding to lcB sequences in control elements (e.g. promoters, enhancers) of cellular
DNA;
(b) dimerizing with another protein containing a Rel homology domain; and
(c) being substantially incapable of transactivating a gene normally subject to
transactivation by NFlcB.
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Such proteins are typically heterologous (i.e. not native) to the cell, and may be
under the control of one or more promoters and/or enhancers which are also heterologous to
the cell.
In particular, it has now been found that the RelA (p65) protein, when modified to
disable its transactivation domain, can function as a potent inhibitor of NF~ induced
transcription of infl~mmatory genes, and that such modified RelA (p65) protein can
competitively bind to DNA sequences bearing a lcB motif, as against the predominant form
of NFlcB comprising a heterodimer of RelA (p65) and p50. Specifically, it has been
observed that reporter gene constructs known to be transcriptionally activated by
endogenous RelA (p65) are rendered resistant to such activation by co-expression of the
above modified RelA (p65) protein.
Suppression of activation by the above modified protein is highly specific. For
example, transcriptional induction of the HIV-LTR by HTLV-1 Tax (which activates NF~B)
is found to be fully inhibited, while NF~ independent HIV Tat-induced transcription from
the HIV-LTR is not so inhibited. Transcriptional inhibition by the altered protein of the
invention is unexpectedly potent, even at low concentrations of the protein, leading to
effective suppression of induction of cytokine-inducible genes such as tissue factor,
E-selectin, IL-8, IL-6 and IlcBa, many of which are associated with inflamm~tion. In
studies in which the endogenous, wild-type RelA (p65) and the altered protein of the
invention are provided on an essentially equimolar basis to cultured endothelial cells,
inhibition has been found in many instances to be 50% or greater, even 75% or greater,
90% or greater, and even 95% or greater, or 100%.
As such, the modified RelA (p65) protein of the invention functions as a "dominant
negative derivative," or alternatively, a "transdominant inhibitor" of the endogenous gene.
The terms "dominant negative" or "transdominant inhibitor" are intended to refer to the
capability of ~.Uppl~;s~ g a normal function of an endogenous protein.
The invention concerns modified human p65 proteins transdominantly inhibiting the
wild-type p65 protein, preferably having the transcription activation domain of the wild-type
protein substantially dysfunctional or deleted, and corresponding DNA coding therefor,
optionally with ancillary sequences for e.g. quantification or recognition, such as a portion
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of human c-myc, and also concerns the use of such modified proteins and DNA in, i.a., the
preparation of a corresponding medicament.
Specifically, the invention concerns proteins, and corresponding DNA coding
therefor, comprising or coding for, essentially, amino acids 1-320 or 2-320 of the wild-type
human p65 amino acid sequence, optionally with ancillary sequences for e.g. quantification
or recognition, such as a portion of human c-myc. They have e.g. the amino acid and
nucleotide sequence disclosed in Seq. Id. No. I and No. 2, or a sequence obtained by
e.g. adding or replacing one to several amino acid residues in Seq. Id. No. 2.
The invention also concerns vector constructs for achieving delivery of modifiedproteins as defined above, or of corresponding DNA coding therefor, to appropriate
recipient cells, tissue or organs, such as vectors for delivering cDNA encoding a modifled
RelA (p65) under the control of a regulable (e.g. inducible) promoter into an endothelial or
other m~mm~ n cell; they are preferably comprising a regulable element such as atetracycline-inducible promoter.
The gene (protein) of the invention may be prepared by well-known recombinant
techniques, e.g. by effecting additions, substitutions or deletions in the nucleotide (amino
acid) sequence of the transactivation domain of the RelA gene (protein), so as to render the
transactivation domain substantially dysfunctional. By "substantially dysfunctional" is
meant that transactivation of at least one infl~mm~tory protein (e.g. E-selectin) is reduced
by at least 50%, and preferably by at least 75%, and more preferably by at least 90%, and
even 100%, relative to transactivation by an equimolar amount of the wild-type protein.
Preferably, the modified gene (protein) of the invention is a deletion mutant (i.e.
truncation) of the naturally occurring gene (protein), whereby at least a portion of the
transactivation domain of the wild-type gene (protein) has been excised. Preferably, the
altered gene (protein) of the invention consists essentially of the Rel homology domain of
the naturally occurring RelA (p65) protein.
While certain mutants derived from RelA (p65) have been prepared by workers in
the art [see Beg et al., Genes & Dev. 6 (1992) 1899-1913; Sun et al., ~cience 259 (1993)
1912-1915], there has been no apparent demonstration of utilization of mut~nt~ as a
transdominant negative competitor of NF~B in activated endothelial cells.
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It has also been found that transcription and expression of the truncated RelA-
derivative can be achieved in a regulable manner, in particular, using a tetracycline-
dependent expression system. For example, tetracycline-induced expression of p65RHD in
stably transfected endothelial cells has been found to inhibit LPS-mediated induction of
endogenous genes such as E-selectin, P-selectin and IKBa.
Therefore, in one aspect, the invention relies on gene therapy techniques, utili7.ing in
its more particular aspects a recombinant gene encoding a mutant RelA (p65) derivative, to
suppress or block NF~ induced activation of m~mm~ n (e.g. endothelial) cells
susceptible to an infl~rnmzttory or immunological stimulus.
Inhibition of NFKB can reduce smooth muscle cell proliferation in addition to
inhibiting endothelial cell activation ~Autieri et al., Biochem. & Biophys. Res. Comm. 213
(1995) 827-836], thereby yielding reduced atherosclerosis and increased graft survival.
Accordingly, the invention in its broader aspects concerns a method of genetically
modifying m~mm~ n (e.g. endothelial) cells to render them less susceptible to aninflammatory or immunological stimulus by conferring on the cells the capability of
expressing a transdominant inhibitor of endogenous NFKB, whereby NFKB-transactivation
of genes is suppressed under activating conditions, as well as the use of such modified cells
in the preparation of a medicament for suppressing NFKB-transactivation of genes under
activating conditions.
The invention also comprises a method of controlling cellular (e.g. endothelial cell)
activation in a m~mm~lian patient, comprising genetically modifying cells of the patient by
inserting therein DNA encoding a transdominant inhibitor of endogenous NFKB, andexpressing functional inhibitor in the nucleus of the cell, whereby NFKB-transactivation of
genes is ~ "Gssed, as well as the use of such modified cells in the preparation of a
medicament for controlling cellular (e.g. endothelial cell) activation in a m~mm~ n patient.
By "endothelial cell activation" or m~mm~ n cellular activation is meant transcriptional
upregulation and synthesis of infl~mm~tory proteins, adhesion molecules and coagulants
(also known as "type II activation"). A generally accepted indicator of type II EC
activation is an elevated level of E-selectin transcription. Preferably the cells or tissue are
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modified in vivo~ i.e. by insertion of a vector comprising the cDNA in the cells while they
remain in the body of the patient. Alternatively, the cells or tissue may be extracted from
the subject, genetically modified ex vivo by insertion of DNA, and then grafted into the
subject. The subject is a vertebrate, in particular a m~mm~l, such as of the porcine or
bovine species, but may also be a primate, and in particular, a human.
It will be apparent that such a therapy will be useful to alleviate infl~mm~toryconditions in a patient (i.e., by syngeneic therapy), and also to moderate complications
occurring in connection with organ transplantation, especially where the graft recipient is
human.
Thus in a further aspect, the invention comprises a method of transplanting donor
allogeneic or xenogeneic m~mm~ n (e.g. endothelial) cells, or tissue or organs to a
m~mms~lian recipient in whom such cells, tissue or organs are subject to infl~mm~tory or
immune activation, which comprises:
(a) genetically modifying the donor cells or progenitor cells thereof or tissue or organ by
inserting therein DNA encoding a transdominant inhibitor of the RelA (p65) protein;
(b) implanting the resultant modified donor cells, tissue or organs into the recipient; and
(c) expressing in the resultant modified cells, tissue or organ, functionally active
transdominant inhibitor, whereby transcriptional activation of infl~mm~tory genes
therein is suppressed.
Activation occurs as a result of cont~cting of the host blood or plasma with thedonor cells, tissue or organs.
Steps (a) and (b) may be carried out in either order; that is, the donor allogeneic or
xenogeneic cells, tissue or organs may be modified or genetically engineered (e.g. by
transfection, transduction, transformation or the like) prior to, or alternatively after,
implantation into the recipient.
For example, endothelial cells of a pig may be genetically modified by insertion of
DNA encoding a heterologous protein comprising a transdominant inhibitor of the RelA
protein of said cell, under the control of a promoter. The modified cells or tissue or organ
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g
may then be grafted into a human recipient. Once transplanted, the donor porcine cells or
tissue or organs express functional heterologous protein, preferably on a regulable basis.
The heterologous protein inserted into such pig cells may be a mutant of a m~mm~ n
Rel/NFKB protein, e.g. RelA (p65); given the homology between proteins of the Rel/NFlcB
family, the mutant human protein is able to exert transdominant inhibition of the
corresponding wild-type porcine protein in the porcine cell.
In one embodiment, a heterologous gene, e.g. a modified human RelA (p65) gene ina suitable vector will be used to modify porcine donor cells or organs in vivo, to render
them transgenic or somatic recombinants, for transplantation purposes.
Somatic recombinant or transgenic donor animals can be obtained by well-known
techniques. The somatic cells of the animal can be a~plup,iately modified in vivo to
provide a somatic recombinant. Alternatively, fertilized oocytes of non-human m~mm~l.c
can also be modiiled by well-known procedures so as to produce a true transgenicexpressing in its cells the desired protein. Cells, tissues and organs which can express the
desired functional protein once transplanted into a recipient (e.g. human) can then be
recruited from the donor animal for transplantation .
Donor cells or tissue can also be genetically modified ex vivo. whereby cells, tissues
or organs extracted from the donor (e.g. pig) and m~int~ine(l in culture are genetically
modified as described above, and then transplanted to a recipient (e.g. a human), where the
graft can then express the desired functional protein. It is preferred that the genetic
modification be done in vivo.
A regulable expression system is disclosed hereunder, particularly for use in
preparing transgenic animals expressing the protein of the invention. By "regulable" is
meant that protein expression, whether increased or decreased, is dependent on the presence,
or addition of, a given substance. An embodiment of "regulable" expression comprises
"inducible" expression, i.e. whereby gene expression is increased by addition of a stimulus.
According to a further aspect of the invention, there are provided graftable
m:~mm~ n (e.g. endothelial) cells, tissue or organs comprising DNA encoding a
transdominant inhibitor of the endogenous RelA (p65) protein, from a donor species, the
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cells, tissue or organ being modified to regulably or constitutively express a transdominant
negative inhibitor of a cellular RelA (p65) protein when transplanted into a graft recipient
of the same or different species as the donor.
The invention also includes m~mm,lli"n (e.g. human) cells transformed by a vector
comprised a modified RelA (p65) gene which is capable of transdominantly inhibiting the
wild-type RelA (p65) protein. Examples of such cells include hematopoietic cells such as
Iymphocytes or stem cells.
In its additional aspects, the invention provides a non-human transgenic m, mm~lcomprising DNA encoding a transdominant inhibitor of a RelA (p65) protein, namely
having cells (e.g. endothelial) or tissue or organs comprising these cells, accordingly
modified; and a method of preparing such non-human transgenic m~mm~l.
.
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Description of Sequence Identi~lers
Se~. Id. No. 1: Nucleotide sequence of the vector construct prepared in Example 1,
comprising the deletion mutant p65RHD at nucleotides 40-996 (i.e. corresponding to
positions 83-1039 of the sequence disclosed by Rubin et al., infra; GenBank accession
number M62399) with the codon for Met at position I and the c-myc sequence at
nucleotides 4-33 (nucleotides 34-39 being derived from the cloning site.)
Se~. Id. No. 2: Amino acid sequence encoded by the vector construct prepared in
Example 1, comprising the deletion mutant p65RHD at residues 1~332 (corresponding to
residues 2-320 of the sequence disclosed by Rubin et al., infra) with Met at position I and
the c-myc sequence at residues 2-11 (residues 12 and 13 being derived from the cloning
site).
~planation of the Figures
Fi~ure 1: Schematic drawing of human RelA (p65) wildtype ("RelA WT") and p65 RHD("p65RHD") constructs ["NH2" and "COOH" refer to the amino and carboxy terminus,respectively; "RHD" refers to the Rel homology domain; "NLS (KRKR)" refers to the
nuclear locali7~tion signal (bracketed)]. The brackets below the RelA WT diagram indicate
the extent of the dimerization domain ("Dimer"), the IlcBa interaction domain ("IlcB") and
the DNA binding domain ("DNA binding"). The numbers refer to the amino acid residues
of wild-type human p65, starting with 1 at the amino terminus.
Fi~ure 2: Repression by the p65 RHD construct of RelA ("p65 WT")-mediated activation
of ECI-6 reporter in BAEC. Different amounts of pRC.CMV/p65RHD ("p65 RHD") were
cotransfected with pCMV4T~p65 (p65 WT).
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Fi~ure 3: Repression by p65 RHD construct of LPS-induced activation of Tissue
Factor (A), ECI-6 (B) and ELAM-I (C) reporter in BAEC. Different amounts of
pRC.CMV/p65 RHD (p65 RHD) were cotransfected.
Fi~ure 4: Tetracycline-regulated expression of p65RHD from pUHD10-3/RHD. Repression
by p65 RHD construct of RelA-mediated activation of ECI-6 reporter in BAEC in the
absence of tetracycline.
Fi~ure ~: Doxycycline-regulated expression of p65RHD in stably transfected BAEC.BAEC were transfected with pUHD172-lneo and pUHD10-3/RHD and selected on G418.
Expression of p65RHD was induced with 2 ,ug/ml doxycycline and whole cell extracts were
prepared at time 0, 8, 16, 24, 48 and 72 hours, separated by SDS-PAGE, blotted on a PVDF
membrane and probed with a polyclonal antibody directed against a N-terminal epitope of
RelA. Bands were revealed using a HRP-conjugated anti-rabbit-IgG antibody and anenhanced chemiluminescent system. Repression of endogenous E-selectin, P-selectin and
IlcBoc induction by TNF:
Top: Northern blot analysis for the indicated genes. Bottom: Western blot analysis
decorated with anti-p65 (N-te~ninus-specific) antibody.
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Definitions
"Graft," "transplant" or "implant" are used interchangeably to refer to biological material
derived from a donor for transplantation into a recipient, and to the act of placing such
biological material in the recipient.
"Host or "recipient" refers to the body of the patient in whom donor biological material is
grafted.
"Allogeneic" refers to the donor and recipient being of the same species (as well as
"allograft"). As a subset thereof, "syngeneic" refers to the condition wherein donor and
recipient are genetically identical. "Autologous" refers to donor and recipient being the
same individual. "Xenogeneic" (and "xenograft") refer to the donor and recipient being of
different species.
Human RelA (p65) protein (gene) refers to the protein (gene) having the amino acid
(cDNA) sequence disclosed by Ruben et al., Science 251 (1991) 1490-1493.
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Detailed Description
NFKB/Rel proteins share a highly homologous sequence of approximately 300 amino
acids referred to as the "Rel homology domain" (RHD). The RHD contains
sequences necessary for DNA binding, nuclear localization, dimerization, and IlcB
binding, but lacks the transcriptional activation function that is found in domains C-terminal
to the RHD in RelA, Rel, and RelB.
A cDNA sequence and ~ ce~ amino acid sequence of human RelA(p65) obtained
from Jurkat T cells was identified by Ruben et al., supra. The amino acid sequence of
551 residues (including the termination codon and the putative initiation codon) encodes a
protein of 60.2 kd. The Rel homology domain has been localized to amino acids 1-300, or
alternatively 1-320 (if the complete IKB recognition site is included).
The modified recombinant protein of the invention preferably comprises a trans-
acting dominant negative derivative of a Rel protein. It comprises at least a portion of the
Rel homology domain (RHD), such that it is capable of a) binding to a gene regulatory
region having affinity for one or more members of the Rel family of proteins; and
b) forming a homo- or hetero-dimer with a second Rel protein. It also has a substantially
dysfunctional or deleted transcriptional activation domain, such that it is substantially
incapable of inducing transcription of genes (e.g. P-selectin, tissue factor or ELAM-1)
which are normally inducible by one or more of the Rel family proteins. It can be derived
recombinantly by rendering the endogenous protein subst~nti~lly unable to transactivate a
gene under the control of a lcB-cont~ining control domain. Preferably, it is a C-terminal
deletion mutant of the p65 protein, whereby at least a portion of the transcriptional
activation domain of the naturally occurring p65 protein has been excised. Alternatively,
the protein (gene) may comprise one or more of the amino acids (nucleotides) normally
constituting the transactivation domain of the wild-type protein (gene), so long as the
transactivation function is rendered dysfunctional, e.g. by one or more mutations or
substitutions of amino acids (nucleotides) or by deletions which are other than truncations.
The resultant mutant protein functions to suppress or block induction of genes whose
expression is dependent on the transcription factor, NFlcB.
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In a preferred embodiment, the protein, and the DNA encoding it, comprise the Rel
homology domain of the p65 (i.e. RelA) protein, but is essentially free of a transcription
activation domain. The transcriptional activation domain of p65 comprises at least two
distinct segments in the C-terminal third. The first consists of a short but strongly
transactivating sequence in the very C-terminus, characterized by a putative o~-helix in the
last 20-24 amino acids. An additional transactivating sequence encompasses amino acids
between position 441 and 518 of the naturally occurring protein [Schmitz and Baeuerle,
EMBO J. 10 (1991) 3805-3817]. It has further been indicated that amino acid residues
between 415 and 550 (containing a leucine zipper-like motif in residues 435-459) constitute
a transactivation domain.
Any or all of the foregoing amino acid sequences may be excised from the
endogenous protein sequence to render a deletion mutant having impaired transactivation
function. In one embodiment, such a mutant protein lacks the carboxyl 250 amino acids of
native human RelA (p65). In other embodiments, the mutant derivative protein maycomprise residues 1-400, or 1-350, or 1-320 of the native human RelA protein.
Alternately, the amino acid sequence of the modified RelA (p65) protein is
substantially dysfunctional with respect to transactivation, and is at least 70%, preferably at
least 80%, and more preferably at least 90% (and even more preferably at least 95%)
homologous to the Rel homology domain of the native p65 protein. Amino acid
residues 222-231 contribute to the formation of homodimers and heterodimers with p50
[Ruben et al., Molecular and Cellular Biolo~y 12 (1992) ~111-454], and are therefore
preferably conserved in the altered protein. In general, a transdominantly acting protein of
the invention can be prepared by truncating from the wild-type p65 sequence the region
which is carboxy-terminal to the NLS.
Preferably, the modified protein consists essentially of the Rel homology domain,
e.g. consists essentially of residues 1-320 or 2-320 (where Met is at position 1) of the native
human protein, either of which is referred to herein as "p65RHD".
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The K:B motifs to which the RelA (p65) binding domain specifically binds, vary
somewhat from gene to gene. An example of a generic KB DNA sequence comprises the
following: 5'-GGGPuNNPyPyCC-3', where Pu is a purine nucleotide (i.e. adenine orguanine); Py is a pyrimidine base (i.e. thymine or cytosine); and N is any of adenine,
cytosine, thymine or guanine.
According to the invention, a m~mm~ n (preferably an endothelial) cell is
transformed by a vector comprising the modified RelA (p65) gene. The gene is taken up
and becomes resident in the nucleus of the cell, where the protein is expressed.Once expressed, the protein is likely to form a homodimer with another altered RelA
(p65) protein through interaction of the respective Rel homology domains. It is believed
that under cellular activating conditions, when NF~B is freed from its association with
IKBa, and translocates to the nucleus, the altered RelA (p65) protein, or homodimer thereof,
has saturated the KB sites to render them incapable of effectively binding the NFKB
heterodimer necessary for transactivation. The competitive binding activity, unexpectedly,
suppresses to a high degree the transactivation function of NFlcB even at low relative
concentration levels of the altered protein.
Therefore the present invention broadly contemplates a method of rendering
endothelial cells or other m~mm~lian cells, or tissue or organs less susceptible to activation
or dysfunction in response to an immune or infl~mm~tory challenge by modifying the cells
or tissue or organs by inserting therein DNA encoding a transdominant inhibitor of NFKB in
operative association with a suitable promoter, and expressing functional transdominant
inhibitor protein by the modified cells at effective levels under normal cell activating
conditions.
The above modification of cells includes introduction of heterologous protein (or
DNA) having the indicated activity. The protein encoding sequence is operably linked with
a promoter sequence, which is typically also heterologous to the cell. The promoter may be
constitutive or regulable (e.g. operate in an appropriate inducible manner).
In one embodiment the modified cells of the invention express the protein
constitutively, i.e. continuously. Thus the DNA coding sequence is operably linked to a
promoter sequence expressing the protein constitutively in said cell.
_
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Alternatively, the modified cells express the protein on a regulable (e.g. inducible)
basis, i.e. the protein coding sequence is operably linked to an inducible promoter, such that
the protein can be expressed immediately before or following cell activation, or on demand
in response to a predetermined external stimulus.
Examples of tetracyline-re~ulable systems particularly suitable for preparation of
transgenic ~nim~l~, have been disclosed by Furth et al., PNAS 91 (1994) 9302-9306
(tetracycline-repressible) and Gossen et al., Science 268 (1995) 1766-1769 (tetracycline-
inducible) (see also Gossen and Bujard, USP 5'464'758) and have been found effective to
regulate expression of the transdominant-acting protein of the invention. In particular, an
inducible tetracycline system comprises:
(a) a first expression plasmid which expresses constitutively (either ubiquitously or tissue-
specifically) a fusion protein between the bacterial tetracycline repressor (which in its
native form is inhibited by Tet and in its mutant form is dependent on Tet), and a
eukaryotic transcription activation domain (e.g. VP16), called tTA; and
(b) a second expression plasmid which contains multiple binding sites (TetO) for the
bacterial tetracycline repressor followed by a minim~l promoter (inactive by itself) and
the gene to be expressed in a regulated manner (e.g. p65RHD).
Depending on which form of the tetracycline repressor (repressible or inducible) is
used, expression of the gene of interest can either be turned off or turned on by
tetracycline. Preferably, expression of the heterologous p65RHD gene from the cells of a
subject (e.g., a transgenic mouse) is rendered inducible by ~iminictration of tetracycline, or
an analog such as doxycycline, to the subject.
Thus the invention comprises a method for inhibiting the dysfunctional or activation
response of vascular endothelial cells, or tissue or organs to an infl~mm~tory or immune
stimulus in a patient in need of such therapy, comprising modification of these cells, or
tissue or organs in the patient as described above.
In a further embodiment, the invention comprises a method for inhibiting graft
transplant rejection in a patient, which comprises:
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(a) modifying donor m~mm~ n (e.g. endothelial) cells, or tissue or organs comprising
these cells, in vivo or in vitro by introducing therein DNA encoding p65RHD under the
control of a suitable promoter;
(b) grafting these donor cells, tissue or organs into the patient; and
(c) expressing in these donor cells, tissue or organs, in the presence of cellular activating
factors, functional p65 RHD protein.
The donor species may be any suitable species which is the same or different from
the recipient species and which is able to provide the app.~ iate endothelial cells, tissue or
organ for transplantation or grafting. In one embodiment, recombinant human protein is
expressed from cells of a non-human m~mm~ n species, which cells have been placed or
grafted into a human recipient. For human recipients, it is envisaged that pig donors will be
suitable, but any other m~mm~ n species (e.g. bovine or primate) may also be suitable.
For example, porcine aortic endothelial cells (PAEC), or the progenitor cells thereof,
can be obtained from porcine subjects, genetically modified, and either re-implanted into the
autologous donor or an allogeneic recipient, or grafted into another subject of a different
species (e.g. human).
The donor cells, tissue or organs may be transgenic or somatic recombinants in the
sense that they contain and express DNA encoding a modified RelA protein. The modified
RelA protein may be a derivative of the wild-type protein which is endogenous to the donor
cell or donor species, or may be a derivative of a wild-type protein which is native to the
species of a graft recipient in whom they are implanted, as is readily ascertainable by one of
skill in the art. Such cells, tissue or organ may continue to express the desired protein
indefinitely for the life of the cell. Modification of m~mm~ n, e.g. endothelial cells
according to the invention can be by any of various means known to the art. In vivo direct
injection of cells or tissue with DNA can be carried out, for example. Appropriate methods
of inserting foreign cells or DNA into animal tissue include microinjection, emblyonic stem
(ES) cell manipulation, electroporation, cell gun, transfection-k, transduction, retroviral
infection, etc. Genes can be inserted into germ cells (e.g. fertilized ova) to produce
transgenic non-human slnim~l~ bearing the gene, which is then passed on to offspring.
Genes can also be inserted into somatic/body cells to provide somatic recombinants, from
whom the gene is not passed on to offspring.
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In one embodiment, gene transcription is subject to an inducible promoter, so that
expression of the recombinant protein can be delayed for a suitable period of time prior to
grafting. In another embodiment. the gene is inserted into a particular locus, e.g. the
thrombomodulin or P-selectin locus. Subsequently, the construct is introduced into
embryonic stem (ES) cells, and the resulting progeny express the construct in their vascular
endothelium.
For gene delivery, retroviral vectors, and in particular replication-defective retroviral
vectors lacking one or more of the gag, pol, and env sequences required for retroviral
replication, are well-known to the art and may be used to transform endothelial cells.
PA 317 or other producer cell lines producing helper-free viral vectors are described in the
literature. A representative retroviral construction comprises at least one viral long terminal
repeat and promoter sequences upstream of the nucleotide sequence of the therapeutic
substance and at least one viral long terminal repeat and polyadenylation signal downstream
of the therapeutic sequence. Vectors derived from adenoviruses, i.e. viruses causing upper
respiratory disease and also present in latent infections in primates, are also known in the
art and may be used as appropriate. The ability of adenoviruses to attach to cells at low
ambient temperatures is an advantage in the transplant setting which can facilitate gene
transfer during cold preservation. Alternative means of targeted gene delivery comprise
DNA-protein conjugates, liposomes, etc.
Cells or cell populations can be treated in accordance with the present invention
in vivo or in vitro. For example, for purposes of in vivo treatments, p65RHD vectors can
be inserted by direct infection of cells, tissues or organs in situ. For example, the vessels of
an organ such as a kidney can be temporarily clamped off from the blood circulation, and
the blood vessels perfused with a solution comprising a tr~n.cmicsihle vector construct
cont~ining the modified RelA gene for a time sufficient for the gene to be inserted into cells
of the organ; and on removal of the clamps, blood flow can then be restored to the organ
and its normal functioning resumed.
In another embodiment, cell modification can be carried out ex vivo. Cell
populations can be removed from the donor or patient, genetically modified by insertion of
vector DNA, and then implanted into the patient or a syngeneic or allogeneic recipient. For
example, an organ can be removed from a donor, subjected ex vivo to the perfusion step
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described above, and the organ can be re-grafted into the donor or implanted into a different
recipient of the same or different species.
Genetically modified endothelial cells may be ~minictered by intravenous or intra-
arterial injection under conventionally defined conditions. Tissue or or~ans comprised
thereof may also be removed from a donor and grafted into a recipient by well-known
surgical procedures. Prior to implantation, the treated endothelial cells or tissue or organs
may be screened for genetically modified cells containing and expressing the construct. For
this purpose, the vector construct can also be provided with a second nucleotide sequence
encoding an expression product that confers resistance to a selectable marker substance.
Suitable selectable markers for screening include the neo gene, conferring resistance to
neomycin, or the neomycin analog G418.
Although any m~mm~ n cell can be targed for insertion of the modified RelA (p65)DNA of the invention, including hematopoietic or stem cells amenable to somatic gene
transfer (e.g. Iymphocytes), endothelial cells are preferred cells for manipulation.
The recipient species will primarily be human, but not exclusively. Other m~mm~lc,
such as non-human primates, may be suitable recipients.
The procedures and techniques to be used in employing the present invention are
known in the art. Insofar as their preparation is not particularly described herein, the
compounds, reagents, vectors, cell-lines, etc. to be used for carrying out the invention are
known and readily available or may be obtained in conventional manner from known and
readily available materials, or equivalent materials may be prepared in conventional manner
from known and readily available materials.
The following Examples are illustrative only and not limitative of the invention.
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li'~ample 1
a) Subclonin~ of p65RHD into expression plasmid:
From plasmid pCMV4T~p65, containing the full length cDNA of human p65 (RelA)
[Ruben et al., Science 251 (1991) 1490-1493; GenBank Accession No M62399] the
p65RHD gene was derived by a PCR-based approach, using as the 5'-sequence,
5'-TAT TGG ATC CTG ACG AAC TGT TCC CCC TCA TC-3', and as the 3'-sequence,
5'-TAC GTG TCG ACT ATT ATC CGC TGA AAG GAC TCT TCT TC-3'. The
conditions under which PCR was carried out are 5 min. at 95~C; and 1 min. 94~C, 1 min.
55~C, 1 min. 72~C for 35 cycles.
The obtained PCR fragment was digested with Bam HI and XbaI to generate
5' overhangs, and cloned along with an oligomer coding for 10 amino acids of the human
c-myc gene and having Hind III-compatible overhangs on the 5' end and BamHI-compatible
overhangs on the 3' end, into HindIII/XbaI cut pRC.CMV (Invitrogen, San Diego,
California, USA).
The resulting construct, pRC.CMV/p65RHD (abbreviated as "p65RHD"), codes for
amino acids 2-320 of human p65 (RelA) preceded by a 13 residue sequence cont~ining 10
residues from the human c-myc gene (used as a recognition sequence for the ATCC
monoclonal antibody CRL 1729 [Evan et al., Molecular ~ Cellular Biolo~y 5 (1985)3610-3616]. Thus, p65RHD essentially comprises the "Rel homology domain" that allows
specific subunit interactions as well as DNA binding.
Seq. Id. No. 1 and No. 2 comprise, respectively, the nucleotide and amino acid
sequence of p65RHD. Fig. 1 comprises a schematic drawing of p65RHD.
Another construct comprising the wild-type human RelA (p65) gene was prepared byanalogous methods and is referred to as pRC.CMV4T~p65 (abbreviated as "p65WT").
b) Reporter constructs:
The porcine ECI-6 (also referred to as IKBa) reporter gene is described by de Martin
et al., EMBO J. 12 (1993) 2773-2779. The porcine tissue factor (TF) reporter is described
by Moll et al., J. Biol. Chem. 270 (1995) 3849-3857.
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In the porcine ~LAM-I (i.e. E-selectin) reporter gene construct [Brostjan et al.,
Transpl.Proc. 28 (1996) 649-651], 3' to the start ATG site, a 3 bp insertion was made,
creating an additional NdeI site. The promoter was cloned into the pMAMneo-luc plasmid
vector (Clontech).
Primary BAEC grown in DMEM supplemented with 10% fetal calf serum (FCS),
penicillin G (50 U/ml) and streptomycin (50 ,ug/ml) were seeded at 3 x 105 cells/30 mm
well, and transfected 18-24 hours later with 700 ng of reporter plasmid in lipofectamine in
DMEM without FCS according to manufacturer's protocol (GIBCO-BRL) for 5 hours.
After addition of FCS to a final concentration of 10%, the cells were allowed to recover for
48 hours. The cells were then stimnl~te~l with 200 ng/ml lipopolysaccharide (LPS) (Sigma,
St. Louis, Missouri, USA) for 7 hours, and extracts were made by freeze/thawing.In one set of experiments, ECI-6 reporter plasmid was used, and the cells were
co-transfected with pRC.CMV/p65RHD (p65RH~) and pCMV4T~p65 ~p65WT), in the
respective nanogram amounts indicated in Fig. 3. The ECI-6 (IKBa) reporter was chosen
because it is usually highly induced by p65 (RelA) and is therefore the most stringent
measure for inhibitory activity by p65 RHD.
In another set of experiments, TF, ECI-6 and ELAM-I reporters were each studied,and the cells were co-transfected with the nanogram amounts of p65RHD indicated in
Fig. 4A-C.
All transfections were done in triplicates and repeated at least three times using two
or more different plasmid preparations. Luciferase (Boehringer) and ~-galactosidase
(Tropix, Bedford, Massachusetts) activities were measured according to the manufacturer's
directions using a 96-well luminescence reader (Bertold). Luciferase readings were
normalized to ~-galactosidase values or protein concentration to account for differences in
transfection efficiency.
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c) ~esults:
Induction of ECI-6 reporter activity by expression of RelA is found to be
well-inhibited by the p65RHD construct, which lacks the C-terminal transactivation domain
and contains a 10 amino acid epitope of the human c-Myc protein at the N-terminus. At
equal nanogram amounts of p65WT and p65RHD, there is found to be almost completeinhibition of the ECI-6 reporter. This indicates that sufflcient p65RHD enters the nucleus
without induction of the cells by LPS. Even in the case of the ECI-6 (IKBa) reporter,
which is induced more than 100-fold, virtual total inhibition is effected by expressing a
1:1 weight (molar) ratio of p65RHD to RelA (amounts of RelA and p65RHD were
confirmed by Western blot analysis.)
Reporter activity induced by LPS (Fig. 3A-C) is also shown to be inhibited in a
dose-dependent manner by p65RHD, reaching maximal levels of 100% at a 1:1 ratio of
reporter plasmid to p65 RHD plasmid, with the exception of E-selectin, which is inhibited
to the extent of about 80%.
Figs. 2 and 3 clearly show that the transcriptional induction of these genes is
dependent on NFlcB. Furtherrnore, the data demonstrate the strong inhibition of NFKB
activity by p65RHD. In some cases (e.g. tissue factor), uninduced, basal reporter activity is
reproducibly reduced by p65RHD expression. The basal transcriptional activity of these
reporters may be due to low level NFlcB activity in llnin(~llce.-l cells that may be a result of
the transfection procedure.
In separate experiments, the transcriptional activity of a promoter lacking NFlcB
sites (RSV-,BGal) was unaffected by p65RHD expression. Additionally, NFlcB-independent,
HIV Tat-merli~t~11 activation of the HIV-LTR was also found to be free from inhibition by
p65RHD, whereas NFlcB-dependent, RelA- or HTLV-I Tax-induced HIV-LTR activation
were completely inhibited.
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Ex~nlple 2:
a) Inducible tetracv~line expression syst~m:
Important for many uses of a genetic inhibitor of NFlcB, such as p65RHD, is
the ability to express the inhibitor only as needed. Homozygous null m~lt~ntc of RelA are
indicated to be lethal in utero [Beg et al., Nature 376 (1995) 167-170]. p65RHD
expression, through a different mech~nicm, also operates to suppress transcription of NF
controlled genes. A system for temporal regulation of p65RHD expression is therefore
highly desirable, since a transgenic animal expressing p65RHD in a regulated fashion can
effectively represent a conditional RelA null mutant. Likewise, in therapeutic use, it may be
desired to inhibit EC activation only on an intermittent basis.
An inducible expression system was employed to regulate p65RHD expression
in vivo~ in particular the binary plasmid system described by Furth et al. IPNAS USA 9]
(1994) 9302-9306]. In this system p65RHD expression is driven by the tetracycline-
sensitive transcriptional activator (tTA) and its expression is repressed by low levels of
tetracycline. In particular, the system employs a first plasmid cont~ining a bacterial,
tetracycline-sensitive DNA binding protein fused to the HSV-VP16 transcriptional activation
domain (tTA) expressed from a constitutive CMV promoter. A second plasmid contains
7 copies of the binding site for tTA, downstream of which the p65RHD gene is cloned into
the vector.
When both plasmids are present in a cell, the tTA protein drives high level
transcription of p65RHD. In the presence of tetracycline there is no expression of p65RHD
and no inhibition of E-selectin reporter activity. In the absence of tetracycline, there is
strong expression of p65RHD and maximal inhibition of the IKBoc reporter activity.
b) Tranc~eni~ mjce:
For the generation of transgenic mice the HindIII/XbaI-cut p65RHD was cloned into
EcoRIlXbaI-cut pUHD10-3 (by filling-in the HindIII and EcoRI sites, respectively, with the
Klenow fragment of E. coli polymerase I). The resulting plasmid was named
pUHD 10-3/RHD.
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Two separate founder strains were generated for tTA and p65 RHD. Crossing
tTA mice with p65RHD mice results in double transgenic mice carrying both transgenes.
These crossings were carried out under cover of tetracycline to prevent p65 RHD expression
during embryogenesis. Mice carrying the tTA and p65RHD transgene, respectively, were
identified by Southern blotting.
As shown in Fig. 4, RelA-me~ t~-l induction of the IlcBoc reporter is completelyinhibited by p65 RHD expressed in the absence of tetracycline, whereas no inhibition is
observed in the presence of tetracycline which represses expression of p65RHD.
To determine the subcellular localization of p65RHD in endothelial cells stably
transfected with the p65 RHD expression plasmid under tetracycline regulation, a c-Myc
specific monoclonal antibody is used. The results shown in Fig. 5, demonstrate that the p65
RHD protein is localized predominantly in the nucleus.
Mice that express p65 RHD in EC can be used as donors for xenotransplantation
(heart and/or kidney) into rats for modelling purposes.
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F.xarrlple 3: Il hibition of inducible endo~enous endothelial cell ~ene expression by
re~ulated expression of p65R~l)
To analyze the effect of NFKB inhibition on endogenous gene expression, bovine
aortic endothelial cells were generated which express p65 RHD in a doxycycline-inducible
manner. Doxycycline is an analog of tetracycline, which also may be used to induce
expression in the system described by Gossen et al., Science 268 (1995) 1766-1769.
Transfections were done as described above using third passage BAEC. The molar
ratio of plasmid pUHD172-lneo and pUHD10-3/RHD used in the transfection was 1:4.24 hours after transfection, cells from three 30 mm wells were trypsinized, pooled, seeded
into 48-well plates, and stable transfectants were selected using 600 ,ugtml Geneticin (Life
Technologies, Grand Island, NY, USA) for 14 days. Only wells with one colony were used
for further experiments. To determine expressing clones, cells were incubated with medium
containing 2 ,ug/ml doxycycline (Sigma, St. Louis, MO, USA) for 24 hours and stained for
p65RHD as described below.
For Northern and Western blot analysis stably transfected BAEC were incubated
with 2 llg/ml doxycycline for 24 hours prior to addition of LPS (200 ng/ml) for
additionally 2 hours. Total RNA and protein were extracted using TRIzol (Life
Technologies, Grand Island, NY, USA) according to the manufacturer's instructions. 20 ,ug
of RNA were separated on an agarose gel cont~ining formaldehyde, transferred to a
Hybond-N nylon membrane (Amersham Life Science Inc., Arlington Heights, IL, USA) and
analyzed by hybridization to radiolabeled cDNA probes of porcine IKBa, bovine E-selectin
and bovine P-selectin. All membranes were probed for glyceraldehyde-3-phosphate
dehydrogenase (GAPDH) mRNA to correct for unequal loading and all qu~ntit~ted IlcB(x,
E-selectin and P-selectin transcript levels were adjusted accordingly.
For Western blot analysis equal amounts of protein were boiled in SDS sample
buffer and electrophoresis under denaturing conditions was carried out on a 10%
polyacrylamide gel. After transfer to a PVDF (polyvinyldifluoridine) membrane
(Immobilon P, Millipore, Bedford, MA, USA) by electroblotting and probing with apolyclonal antibody directed against the N-terminal region of human RelA (Santa Cruz
Biotechnology Inc., Santa Cruz, CA, USA) bands were vi~u~ l using horseradish
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-27-
peroxidase conjugated donkey anti-rabbit IgG (Pierce, Rockford, IL, USA) and theEnhanced ChemiT.Ilminescence assay (Amersham Life Science Inc., Arlington Heights, IL,
USA) according to the manufacturer's inslructions.
Results are shown in Fig. 5: the upper panel (A) shows a Northern blot analysis of
expression of E-selectin, P-selectin and IlcBcx. In the absence of doxycycline these genes
are well inducible by LPS, whereas their induction is strongly inhibited by doxycycline-
induced expression of p65RHD. As a control, GAPDH expression was also analyzed and is
not changed by either LPS or Dox treatment. The lower panel (B) shows the Western blot
decorated with a N-terrninal RelA-specific antibody that detects both endogenous RelA
(upper band) and p65RHD. There is some ~le.t.o.ct~ble p65RHD expression in the absence of
doxycycline which is greatly inhanced upon doxycycline treatment. The low level of
p65RHD expression does not influence gene expression in these cells (lower panel).
This experiment shows that, in addition to reporter constructs, endogenous genes are
also inhibited by p65RHD.
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SEQUENCE LISTING
(1) GENERAL INFORMATION:
(i) APPLICANT:
(A) NAME: Sandoz Ltd.
(B) STREET: Lichtstrasse 35
(C) CITY: Basle
(E) COUNTRY: Switzerland
(F) POSTAL CODE (ZIP): CH~002
(G) TELEPHONE: 61-324 5269
(H) TELEFAX: 61-322 7532
(ii) TITLE OF INVENTION: GENE THERAPY VVITH MODl~ D p65
PROTEINS
(iii) NUMBER OF SEQUENCES: 2
(iv) COMPUTER READABLE FORM:
(A) MEDIUM TYPE: Floppy disk
(B) COMPUTER: IBM PC compatible
(C) OPERATING SYSTEM: PC-DOS/MS-DOS
(D) SOFTWARE: PatentIn Release #1.0, Version #1.25
(EPO)
(v) CURRENT APPLICATION DATA:
APPLICATION NUMBER-~ WO PCT/EP96/
(vi) PRIOR APPLICATION DATA:
(A) APPLICATION NUMBER: US 60/004339
(B) FILING DATE: 26-SEP-1995
(2) INFORMATION FOR Seq. Id No. 1:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 999 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(iii) HYPOTHETICAL: NO
(iii) ANTI-SENSE: NO
(v) FRAGMENT TYPE: internal
(ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 1..999
CA 0223303l l998-03-25
W O 97/12040 PCT/EP96/04216
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CA 02233031 1998-03-25
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V ~Q ~ V ~ V tn E~ h ~ V ~I V r~ V ~1
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V ~ V h ~ V h E~ ,i V >1E~ ~ ~ U ~ f~
V V U ~ I V ~ V ~ V ~ V E~ V ~C
CA 02233031 1998-03-25
W O 97/12040 PCT~EP96/04216
~ ~ o a~
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CA 02233031 1998-03-2~
W O 97/12040 PCTrEP96/04216
-32-
(2)INFO R M ATION FO R Seq.Id. No.2:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 332 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 2:
~et Glu Gln Lys Leu Ile Ser Glu Glu Asp Leu Asp Pro Asp Glu Leu
15~he Pro Leu Ile Phe Pro Ala Glu Pro Ala Gln Ala Ser Gly Pro Tyr
30~al Glu Ile Ile Glu Gln Pro Lys Gln Arg Gly Met Arg Phe Arg Tyr
Lys Cys Glu Gly Arg Ser Ala Gly Ser Ile Pro Gly Glu Arg Ser Thr
Asp Thr Thr Lys Thr His Pro Thr Ile Lys Ile Asn Gly Tyr Thr Gly
80~ro Gly Thr Val Arg Ile Ser Leu Val Thr Lys Asp Pro Pro His Arg
95~ro His Pro His Glu Leu Val Gly Lys Asp Cys Arg Asp Gly Phe Tyr
100 105 110
Glu Ala Glu Leu Cys Pro Asp Arg Cys Ile His Ser Phe Gln Asn Leu
115 120 125
Gly Ile Gln Cys Val Lys Lys Arg Asp Leu Glu Gln Ala Ile Ser Gln
130 135 140
Arg Ile Gln Thr Asn Asn Asn Pro Phe G1I1 Val Pro Ile Glu Glu Gln
145 150 155 160~rg Gly Asp Tyr Asp Leu Asn Ala Val Arg Leu Cys Phe Gln Val Thr
165 170 175~al Arg Asp Pro Ser Gly Arg Pro Leu Arg Leu Pro Pro Val Leu Pro
180 185 190
His Pro Ile Phe Asp Asn Arg Ala Pro Asn Thr Ala Glu Leu Lys Ile
195 200 205
Cys Arg Val Asn Arg Asn Ser Gly Ser Cys Leu Gly Gly Asp Glu Ile
210 215 220
Phe Leu Leu Cys Asp Lys Val Gln Lys Glu Asp Ile Glu Val Tyr Phe
225 230 235 240~hr Gly Pro Gly Trp Glu Ala Arg Gly Ser Phe Ser Gln Ala Asp Val
245 250 255~is Arg Gln Val Ala Ile Val Phe Arg Thr Pro Pro Tyr Ala Asp Pro
260 265 270
Ser Leu Gln Ala Pro Val Arg Val Ser Met Gln Leu Arg Arg Pro Ser
275 280 285
Asp Arg Glu Leu Ser Glu Pro Met Glu Phe Gln Tyr Leu Pro Asp Thr
290 295 300
Asp Asp Arg His Arg Ile Glu Glu Lys Arg Lys Arg Thr Tyr Glu Thr
305 310 315 320
Phe Lys Ser Ile Met Lys Lys Ser Pro Phe Ser Gly
325 330
