Note: Descriptions are shown in the official language in which they were submitted.
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GENE THERAPY OF E~ I Hl~LIAL CELLS WITH ANTI-APOPl'OTIC PROTEINS FOR TRANS-
PLANTAT~ON AND TNFLAMMA1~3RY CONDmONS
Field of the invention
The invention relates to the field of an~i-apoptotic gene therapy for transplantation
and inflammatory conditions. It provides improvements in the field of gene therapy and
tissue and organ transplantation. ~n its broad aspect, it relates to methods of treating
cellular activation processes. ~n parti~ular, i~ is concerned with genetic modification of
endothelial cells to render them less susceptible to an inflammatory, immunological, or
other activatin~ stimulus.
The invention is specifically directed to genetic modification of a cell. in particular
an endothelial celh to render it capable of expressing a polypeptide capable of inhibiting
cellular apopto~is. and to recombinant vectors therefor. Examples of polypeptides capable
of inhibiting apoptosis in mammalian cells include polypeptides having activity of a
mammalian A''O protein, as well as. more generally, polypeptides having anti-apoptotic
activity, in particular cenain proteins of the BCL family.
The invention also concerns the resul~ant ~enetically modified cells, or tissues or
orcans comprising these cells; and non-human lransgenic or somatic ,~,co...binant animals
so modified.
The invention is most particularly directed to transplantation of genetically
modified cells. or grahable tissues or organs comprising such cells, into a m~mm~lian
recipient. The mammalian recipient may be allogeneic or xenogeneic as to the cells.
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Back~ro~md of the invention
The well-characterized problem of "hyperacute njecli~nl~ accompanying
transplantation of organs between discordant species, involving an jmQlçr~ e
immunological response of recipient antibodies and c~n.plc.,lcnI system against the
transplanted organ, has been addressed by various means, including the use of immune
suppressants, as well as donor organs that express factors which inhibit the col~,pie.~.en
system of the recipient (Dalmasso, A.P., Immunopharmacolo~y ~4 (2) [1992] 149-160).
However, a further condition associated with grafted tissue or organs, and w~th
cells subjected to inllammatory processes in general, is the process known as "activation".
In particular. endothelial cell "activation" refers to a continuum of chan~es characterizin~
endothelial cells which are subjected to a stimulus such as a cytotoxic cytokinele.g., tumor necrosis factor (TNF)l, an inflammatory or infectious condition, r~pelr~lsion
injury. atherosclerosis, vasculitis or graft rejection. The endothelium (also referred to as
the "-ascular endothelium") consists of a layer of cells that line the cavities of the heart
and of the blood and Iymph vessels. The initial celluiar response of such cells to an
ac~iv;lting stimulus (often refelTed to as "Type I" activation) typically involves ch~n~Ps in
the cell pheno~vpe, such as retraction of cells from one another, hemorrha~e and edema,
and tra~ micra~ion of leul;ocytes across the endothelium. A stiil further phase of cellular
~c~ tion ("T~pe 11" activa~ion3~ involves transcriptional up-re~ulation of various genes
encodin~ interleu~;ins, adhesion molecules, and procoagulant, prothrombotic col,~ponents
of the coagulation system. For example, E-selectin is a tissue specific molecule which is
expressed exclusively by endothelial cells (EC) upon activation, and therefore is a
~enerall- :~ccepted indicator of Type 11 EC activation (Pober, J.S. and Cotran, R.S.,
Tr~nspl~ntation ,~ 1 19901 537-544).
A reco~nized phenomenon associated with continuous overexpression of such
activlltion proteins, at the expense of normal cell functioning, is the tendency of the cell
to undergo a process of active cellular suicide known as "apoptosis" (G. T. Williarns and
C. A. Smith. Cell 74 l19931 777-779; D.L. Vaux et al., Cell 76 [1994] 777-779).
Apoptosis can be considered as preprogrammed cell death seen in the process of
development. differentiation. or turnover of tissues (Wyllie, A. H. et al., Int. Rev. Cytol.
68 [ 1 98ol ~51-306). Cell death by apoptosis occurs when a cell activates an internally
-
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enro~ d suicide program as a result of either extrinsic or intrinsic signals. Morpho-
logieall~. apoptosis is eharaeterized by loss of eontaet with neighboring eells.concentration of cytoplasm, endonuclease aetivity-~csoei~e~l chromatin
condensation and pyknosis, and seg.l.~ ;on of the n~-elellc, among others.
Disap~ea,dnce of microvilli from the eell surface and vesiele formation on the cell surfaee
(membrane blebbing) are also observed. The rem~inin~ fr~m~nts of apoptotic ~ody cells
are ultimately phagocytosed by neighboring eells (Duvall, E. and Wyllie, A. H.~
Immunolo~y Today 7(4) [1986] 115-119; Trauth, B.C. et al., Science ;~ [1989]
301-30~). Apoptotie eell death is of func~r..~ importanee in infl~mm~tion,
em~rvogenesis and Iymphocvte selection. Avoidance of eell activation and apoptotic cell
death accompanyin~ inflammation in general, and particularly in connection with or~an
transplantation. has become a maior goal for workers in the art. Graft injury and loss
occurring in connection with graft preservation techniques, as well as accc,~ anying ~sraft
rejection. exempiify the vulnerabi}ity of enltoth~lial cells to sueh ~,~,cesses.An identified transcription factor for many of the genes s~sce~ible to
transcriptional up-regulation in response to an activation stimulus such as TNFa, is
"Nuclear Factor KB"~ i.e. NF-lcB (M. Grilli et al., International Review of Cytology 143
1993l 1-61). NF-~cB exists as a preformed transeription faetor in ~he eytoplasm of eells,
which is inactivated by its association with a protein inhibitor of the llcB family. On
exposure to cellular activating stimuli such as lipopolysaceharide ~LPS), TNF, or oxygen
radicals. lhe llcB protein is rapidly phosphorylated and then degraded, thereby liberating
the preformed NF-~cB and allowin~ its transmi~ration tO the rlllcleuc- In the nucleus, the
binding of NF-~cB to certain NF-lcB binding sites (also referred to as "IcB el~-..e~ ") in
promoter regions of the nuclear DNA initiates transcription of genes direetly or indireetly
under the control of said promoters. Genes subjeet to up-regulation by NF-~B upon
stimulatiorl of the cell with TNF, include E-seieetin~ IL-8, and tissue factor, among others
(F.~. Bach et al., ImmunoloFical Reviews 141 ~1994] 1-30; T. Collins, l ~h. lnvest. 68
19931 499-508; M.A. Read et al., J. F.xp. Med. 179 11944] 503-512).
For example, the A20 gene is found to be inducible by TNF or other cellular
activating factors (A.W.Opipari et al.~ J. Biol. Chem. ~ ~1990] 14705-14708;
C.D.Laherty e~ al., I.Biol.Chem. ~~~11993] 5032-5039). There is evidenee that A20
belongs to a sub-set of TNF-inducible genes which assist in ultimately
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conferring recist~nce to TNF-induced apoptosis (M. Tewari et al., J. Immunol. 154 [t995]
1699-1706; A.W. Opipari et al., J. Biol. Chem. ~ [1992] 12424-12427; A.W. Opipari
et al., J. Riol. Chem. ~ ~1990] 14705-14708; Dixit et al. [1989], ~). A. Krikos and
co-workers ~J. Rjol. Chem. ~ llg92] 17971-17976) d~ o~ ted that induction of the
A20 gene by TNFa is also mediated by NF-~B binding sites in the A20 promoter
(see also C.D. Laherty et al., J. Biol. Chem; ~ 119931 5032-5039).
Besides the A20 protein, certain proteins of the BCL (also referred to as BCL-2)famiiy of proteins also exert an anti-apoptotic effect. Such ~.ut~,;ns include BCL-~,
BCL-XL, MCL-I, and Al. However, the precise m~rh~nicm~ by which the A'~0 proteinor BCL proteins excrt an anti-apoptotic effect have not been comple{ely elucidated.
Summar- of the invention
An important means of s.~pp.~Ssillg NF-lcB-m~Aia~ed activation of a cell has nowbeen found. Unexpectedly, it was found that NF-lcB re~ulation of gene transcription is
related to expression of an apoylosis inhibiting (i.e. "anti-apoptotic") protein. Morc
particularly~ it has been found that such a protein can exert a ne~ative fee~lback control
on NF-~cB-mediated gene transcription, namely, the anti-apoptotic protein functions as an
inhibitor of the NF-lcB ~ranscription factor. This observed negative fee~lb~rk effect may
perhaps in certain cases be exerted via an anti-oxidative m~ch~nicm that directly or
indirectly protects the NF-KB-IlcB complex from dissocialing, appa,~nlly by acting
upsl.ea,.l of llcB de~radation. Such inhibitoly function may norrnally assist in p,~v~.~ling
apoptotic cell death. However, under conditions of severe cellular challenge, such as
occurring in connection with transplantation, and particularly xenotransplantation,
expression of the anti-apoptotic protein in a cell may be at insufficient levels, or delayed
relative to the rapid activation of NF-lcB in the cell, so that inhibition of NF-1cB is
rendered ineffective ~o prevent cellular activation and apoptosis.
This finding has now been used to devise a method to treat en~oth~ l or other
cells susceptible to an inflammatory or other activating stimulus, and in particular to trcat
cells. tissues or organs which are subject to transplantation rejection. The method and
other aspects of the invention may be used to treat infl~rnrr ~tion or disease states
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associated with inflammation. e.g;, septic shock, chronic rejection, xeno~raft rejection,
- ~ atherosclerosis (restenosis), vasculitis~ cardiac failure, or auLoi.. ~ r ~ e~ces.
The invention relies on ~ene therapy t~shni~lu~$, utili~in~ an anti-apoptotic ~ene
and its expressed product to inhibit NF-1cB activation in m~mm~ n cells susceptible to
an activating stimulus.
Accordingly, in a first aspect the invention provides a m~m~ n cell ~in
particular. an endothelial cell) which is genetic~11y mo-lif1ed to express an anti-apoptotic
protein which is capable of subst~nti~lly inhibiting NF-lcB activation in the ~,~sence of a
cellular activatin~ stimulus. An example of a "cellular activating stimulus" is tumor
necrosis factor. TNF (i.e. TNFo~).
B~ "NF-~cB activation" is meant NF~ rre~ ted up-regulation of genes which
are directly or indirect]y under the control of an NF-1cB binding site, such as, e.g.,
E-~electin in endothelial cells. In functional terms, NF-lcB activation con~lit.lles the
bindin~ of NF-lcB to )~B regulato~y sequences in the DNA of a cell in a manner sufficient
t~hc~her alonc or in combination with other factors) to initiate transcription of a gene in
o~erati~e a~ ciation ~ith said se~ucnces.
B~ F-~;B inhibition" is meant that NF-lcB binding to NF-KB binding sites in the
nucle;~r D~;A i~ prevented. NF-KB is considered "substantially inhibited" when, for
c.~;lmr)lc. tran~cription of the E-selectin ~ene by an endothelial cell genetic~lly modified
;Iccordin~ tc thc inven~ion and stimulated with TNFo~ is reduced by 60% or greater, and
prcferabl- 8()~,~ or gre~ter, ;md even 90% or ~reater, e.g., 95% and even 99% or grcater,
rclati~e to an unmodifed cell (i.e. a cell not subject to genetic manipulation acco~ing to
the in~ention~ which is al~o stimulated by TNFa.
The in~ention in it~ bro;~der aspects also concerns a method of genetically
modif!in~ m~mmali;~n (e.~.. endothelial) cells to render them less susceptible to an
innammator~ or other immunolo~ical activation stimulus by inserting in these cells, or
pro~enitors thereof. Dl~'A encodin~ an anti-apoptotic protein capab}e of inhibiting NF~
and expressin~ the proteim whereby NF-~B in the cell is subst~n~ y inhibited in ehe
presence of a cellular activatin~ stimulus.
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It was found that inhibition of NF-lcB-initi~t~3 transcription by the anti-apoptotic
proteim such as, e.g., an A'~0 protein, in a ~enetic~ly modified cell is unexpectedly
potent, even at moderate le~els of transfection in vitro with the co..~s~onding A20 gene
(e.g., 0.5 l~g plasmid DNA per approximately 5 x 105 cells), leadin~ to effective
suppression of induction of cytol;ine-inducible genes such as tissue factor, E-selectin and
l~a, all of which are associated with infl~mm~tion.
It will be apparent tha~ such a therapy will be useful in general to treat patients
aMicted with conditions which may benefit from inhibition of NF-lcB activation, such as
inflammation. Such a therapy will also be useful to moderate complications occurring in
connection with organ transplantation, especially where the ~raft recipient is human, and
most particuiarly where the ~rafl is xeno~eneic as to the recipient.
Thus in a further aspect~ the invention comprises a method of transplanting donor
endothelial or other mammalian cells ~e.~., bone marrow stem cells as precursors of
monocytcs. NK cells, or Iymphocvtes: or islet cells). or graftable tissues or organs
comprisinE such cclls. ~o a mammali~n recipient in whose blood or plasma these cells,
ti~ uc~ or org;ln~ ;Irc subjcct to activ;l~ion. which comprises:
gcnclh:;llly modifying~ the donor ccllx. or pro~enitor cells thereof, by inserting therein
D~'A cllcodinc an anti-apoptotic prolein capable of inhibiting NF-lcB, and
~t ) tr;lnspl;~n~ g lhc rcsult;lnl modified donor cells. or tissues or organs comprising these
cclh~. into thc rccipien~. and exprcssin~ in the cells the anti-apGplotic protein, whereby
~F-KB ;Icti~;ltion in the cells is subst;lnti~lly inhibited in the ,olcsence of a cellular
acti~atine ~timulus.
Thc ' modified donor cclls" of step (b) will be understood to refer to cells whiçh
thcm.cclve!i are subjccted ~o ~ cnc~ic modification in s~ep (a), as well as to progeny thereof.
According to a funhcr aspect of the invention~ there are provided donor
endothelial cclls. and tis~iucs ~nd organs comprisin~ such cells, wherein the cells are
gcrlctically modificd to rc,~ul;lbly or constitutively express an anti-apoptotic protein in a
~raf~ recipicnt. ~hercby ~F-lcB is substantially inhibited, for transplantation into a
recipient specie~;. The graft recipient may be allogeneic or xenogeneic as to the donor
cells. tissues or or~ans. In its additional aspects, the invention provides a non-human
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transgenic or somatic recombinant rn~mm~l comprisin~ DNA encoding an anti-apoptotic
protein of a different species; and a method of p~,ya~ g such non-human trans enic or
somatic recombinant mammal. Also within the scope of the invention are vectors for
genetically modifyin6 cells by insertion of anti-apoptotic protein-e~o-~ing
polynucleotides, such as for example retroviral vectors, and especially, adenoviral vectors.
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nescriDtion of the drawinps
Fi~ure 1: Analysis of antibody amnity purified protein extracted from: BAEC
transfected with A20 vector ("A'20"), BAEC transfected with empty pAC vector ("PAC"),
or non-transfected BAEC ("NT") following stim~ tion with TNFa. Also analyzed forcomparison is HUVEC which is either non-stirn~ t~d ("NS") or st~ ted with TNFa
("TNF").
FiYure 2: Luciferase levels in relative light units (RLU) in BAEC co-transfected with
A20 andlor pAC vector ("pAC ) u)gether with the porcine E-selectin promoter region
cloned into a luciferase expressing vector ("porcine E-selectin Reporter"); BAEC are
either non-stimulated ("NS" or "control") or s~imulat~c~ with TNFa ("TNF") or
lipopolysaccharide ("LPS").
Fi~ures 3A-3C: Luciferase levels in BAEC co-tr~ncfect~d with either A20 or pAC
and one of the following promoters cloned into a luciferase vector: (a) human ~L-8
promoter ("IL-8 Reponer") (FIG. 3A); ~b) porcine I cBa promoter ("IlcEk~ Reporter")
FIG. 3B): and (c) porcine tissue faceor (TF) ,~,(""ote.- ~"Tissue Factor Reporter")
(FIG. 3C); and then stimulated with TN3-a or LPS or maintained as a control.
FiQurc 4: Luciferase levels in BAI~C co-transfected with either A20 or pAC and lcB
element~i derived from the porcine E-selectin prull-otcr clon~d into a luciferase vector
("NFIcB Reponer"). and then stimulated with TNFa or LPS or rn~int~in~d as a control.
Fi~ure ~A: Luciferase tevels in BAEC co-transfected with either A20 or pAC and
an RSV-LTR driven 3uciferase vector ("RSV-LUC Reporter").
Fi~ure ~B: '~C-laheled chloramphenicol leYels in counts per minute (CPM), in
BAEC co-transfected ~ ith A20 andlor pAC and an HIV LTR-driven CAT vector
("HIV-CAT Reporter"). Cells are stimulated with the viral c-Tat protein ~"C-Tat") or
maintained as a control.
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_~.
Fi~ures 6A. 6B. 6C: ~uciferase levels in BAEC co-transfected with pAC and eitherBc1-2 or Bcl-X,, ~ogether with either the E-selectin ICp~ Gl (FIG. 6a), the IlcBa l~pollcr
(FIG. 6B), or the NF lcB reporter tFIG. 6C) cloned into a }ueir~lase vector. and then
5tim~ ed with TNF or LPS or ~n~int~inerl as a non-stim~ d control.
Fi~ure 7: Lucifersse levels in BAEC co-transfected with pAC, full length A20, ortruncated A2û clones #3 ["tA20(3)"] or #7 ["tA20(7)"1, together with the E-selectin
reporter cloned into a luciferase vector, and then stim~ ed with TNF or LPS or
rn~in~ined as a non-stim~ ed ("NT") control.
Fi~ure 8: EMSA of nuclear extracts from TNF-sti... Iated ( I ) or non-st~ ted
(-) PAEC infected with adenoviral Bc1-2 ("rAd.Bcl-2") or, as a control, ,B-gal
("rAd.~-Gal"), using a lcB binding oli~onucleotide derived from the human
irnmuno~lobulin (Ig) lc promoter and, for co,l,pa.ison, a cold wild-type NFlcB-speci~lc
probe ("sp-comp."~ and a non-specific co...~ or ("nsp. comp.")(AP-I).
Fi~ure 9: Western blot of rAd.Bcl-2- (or, âS a control, rAd.,B-gal-) infected PAEC
taken prior to ("0"), or ten minutes ("lû' ") or one hundred-twenty minutes ("120' ")
following s~imulation with TNF~ with lK,Ba as shown.
FiQure 10: El~iISA of nuclear extracts from rAd.Bcl-2- (or~ as a control, rAd.,B-gal-)
infected PAEC prior to ("-") or two hours following (~) TNF stimul~ion, using the
transcription factor cAMP responsive cle,..ent ("CRE") as a probe and, for cGI~pâ~ison, a
cold wild-type CRE-specific probe ("sp-comp.") and a non-specific competitor
("nsp. comp.").
Fi~ure 11: Luciferase levels in BAEC co-transfected with either Al or pAC and a
luciferase vector comprisin~ 0.7~1g of either the (A) E-selec~in or (B) NFlcB reporter.
Cells are stimulated with TNF or LPS or non-stimulated (control).
Fi~ure 12: Nothern blot TNF stimulated ~) or nor s~ lfl~d ( ) HUVEC infected
with adenoviral llcBo~ ("rAd.IlcB"a) or A20 ("rAd.A2t)") or, as a controh rAd.~-gal .
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-10-
Pefi~itiorc
"Graft," "transplant" or "implant" are used interch~ng~a~ly to refer to biological material
derived from a donor for transplantation into a l~,cil,i t, and IO the act of placing
such biological material in the l~ci~ient.
"Host or "recipient" refers to the body of the patient in whom donor biologicai material is
grafted.
"Allo~eneic" refers to the donor and l-,cipie~lL being of the same species (also "allograft").
As a subset thereof, "syngeneic" refers to the condition wherein donor and
recipient are genetically identical. "Autologous" refers to donor and recipient
bein~ the same individua}. "Xenogeneic" (and "xenograft"3 refer to the conditionwhere the graft donor and lccipient are of different Cpecies
"A20" refers to a natural mamm~ A20 gene (inrlu~ling the cDNA thereof) or protein,
including derivatives thereof having variations in DNA (or amino acid) sequence
(such as silent mutations or deletions of up to S amino acids) which do not
prejudice the capability of the natural protein to block NF-lcB activation. The A20
gene ~protein) may, for example. be porcine, bovine or human, or may be of a
primate other than human, depending on the nature of the cells to be modified and
the intended recipient species for transplantation.
"A polypeptide having activity of an A20 protein" or "A20 active protein" refers to a
protein which is able to block or suppress NF-1cB activation, and which is at least
705'c, preferably at least 80~o, and more preferably at least 90% (most preferably
at Ic~st 95~7O) homolo~ous to the protein seq~nce of a natural n~mm~iio~l
(e.g., human) A2~ protein ~for example, SEQ. ID. NO. 1 hereof). ln a plefc,l~d
em~odiment, the A'~0 protein of the invention is human and has the amino acid
sequence corresponding to SEQ. ID. NO. 1 herein (as discloscd in A.J.Opipari
et al. 119901, supra). In a further aspect, the A20 gene of the invention is at least
70S~, and more preferably at least 80%, or at least 90% (e.g., at least 95%3
homolo~ous ~o, or corresponds to, SEQ. ID. NO. 2 herein.
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"Bc1-2" refers to a natural m~mrn~ n Bc1-2 gene (including the cDNA thereof) or
- - protein (denoted by capital letters), inclutlin~ derivatives thereof having variations -
in DNA {or amino acid) sequencc (such as silent mutations or deletions of up tO
5 amino acids) which do not pr~judice the capability of the natural protein to
block NF-lcB activation. The Bc1-2 gene (protein) may, for example, be porcine.
bovine or human, or may be of a primate other than human, ~iepen-~in~ on the
nature of the cells to be modified and the int~.nrle~1 icci~icnl species for
transplantation .
"A polypeptide having activity of BCL-2 protein" or "BCL-2 active protein" refers to a
protein which is able to block or su~ ss ~F-lcB activation, and which is at lcast
70'k~ preferably at least 80$~ and more preferably at least 90% (most preferablyat least 95~c) homologous to the protein se~uence of a natural ~ mm~ n
(e.~.. human) BCL-~ (for example, SEQ. ID. NO. 3 hereof). In a ,~lcfc.,cd
embodiment of the invention, the BCL-2 polypeptide of the invention is human
and has the amino acid sequence co.,~s~onding to SEQ. ID. NO. 3 (as disclosed
~ Tsujimoto, Y. and Croce, C.M., PNAS ~ 11986] 5214-S218, and in
wo s~/on6~
Similarl~. "Bci-.~," refers to a natural mammalian BCI XL 8ene (including the cONA
hcrcot ~ or protein ldenoted by capital letters), inclur~ing derivatives thereof having
~;lri;~lions in DNA (or amino acid) sequence (such as silent mutations or deletions
of up to 5 amino acids) which do not prejudice the capability of the natural
protcin ~o blocl; NF-KB activation. The BCI-XL gene (protein) may, for example,
be porcine, bovine or human, or may be of a primate other than human, clepen~lin~
on the nature of the cells ~o be modified and the intencled recipient species for
lran~plantation .
"A pol~pep1idc havin~ activity of BCL-XL protein" or "BCL-XL active protein" rcfers to a
protein ~ hich is able to blocl; or suppress NF-lcB activation, and which is at least
70~k, preferabl~ at least 80~, and more preferably at least 90% (most p.~f~.~blyat leasl 9~k) homolo~ous to the protein seq~er~e of a natural mammalian
~e.~.. human) BCL-XI protein (for example, SEQ. In. NO. 4 hereof). In a
preferred embodiment of the invention, the BCL-XL polypeptidc of the invention is
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- I 2-
human and has the amino acid sequence co.l~sponding to SEQ. ID. NO. 4 (as
also disclosed in WO 95/0064~).
"Al" refers to a natural mammalian Al gene (including the cDNA thereof) or protein,
including derivatives thereof having va~iations in DNA (or amino acid) seq~len~e(such as silent mutations or deletions of up to 5 amino acids) which do not
prejudice the capability of the natural protein to block NF-1cB activation. The Al
gene (protein~ employed in t~e invention may, for example, be po~cine, bovine orhuman. or may be of ~ primate other than human~ depending on the nature of the
cells to be modified and the intended recipient species for transplantation.
"A polypeptide havinc activity of Al protein" or "Al-active protein" refers to a protein
which is able to bloc~; or suppress NF-lcB activation, and which is at least 70%,
preferably at ieast 80~c, and more preferably at least 90% (most preferably at least
95S~) homolo~ous to the prolein sequence of a natural rn~mm~ n (e.~., human)
Al (for e~ample, SEQ. ~D. NO. ~ hereof). In a preferred embodiment of the
invcntion. the Al polypeptide of the invention is hurnan and has the amino acid
scqucncc correspondin~ to SEQ. ID. NO. 5 (as fliselosed in A. Karsan et al.,
Bloo-l. 87. ~;o. 8 IApril 15. 19961 3089-3096).
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Detailed descriDtion
The human A 70 gene was ori~inally cloned as an imn-e~i~t~ early response ~ene
which is rapidly but transientlv expressed following TNF t~ l of human umbilical
vein çndothelial çells (HUVEC) (Opipari et al. [t9901, ~). It is now known that a
protein havin~ A20 activity can also be in-luced by other stimuli such as IL-I in ~UVEC
(Dixit et al. ~19891, supra); CD4~) cross-linking in B cells (Tewari et al. ~199~], supra); or
phorbol l'-rnyristate 13-acetate (PMA) or HTLV-I Tax protein in Jurkat T cells
(Laherty et al. 11993~, ~upra~. An A20 protein is also constitutively present in mature
resting T cells.
A cDNA se~uence of the human A~0 gene obtained from HUVEC, and the
derl~ced amino acid sequence, are pùblished by Opipari et al. [1990], suvra, as indica.ted
hereinabove. TN~-induction of A20 has bcen indicated to be rrl~Ai~r~i throu~h NF-lcB
bindin~ sites in the A't0 promoter, ex~ending from -45 to -54 (5'-GGAAATCCCC-3') and
from -57 to -66 (~'- GGAAAGTCCC-3') of the gene. At the protein level, the de~luced
sequence of 790 amino acids (SEQ. ID. NO. I) contains within its carboxyl terminal half
7 Cys./C!~s~ zinc fin~er repeats: six with the confi~uration Cys-X.l-Cys-X~-Cys-X2-Cys
and one with lhe confi~uration Cys-X.-Cys-XI,-Cys-X.-Cys, wherein X is any amino acid
and the sub.~icripts represent numbers of amino acids between each of the indicated
cvsteines. A no~el fin~er loop domain composed of 11 amino acid residues has also been
identified (Kril;os cl ~1. 1199'1~ supra).
In onc embodimcnl of thi~ invcntion, the "protein having A20 activity" comprisesamino acid residues 386-790 of SEQ, ID. NO, 1, comprising the zinc fin~er region of the
native protein sequrnce (i.e. havin~ 7 zinc bindin~ domains), or a re~ion at least 80%
homologous to said residue~;. Another suit;~ble ~runcated from of the native human
protein consists essentially of residues 373-790 of SEQ. ID. NO. 1 hereof. Otherdeletion mutant.c found to be c~pable of inhibitin~ N~lcB comprise the N-terrninus and
zinc-bindin~ dom~ins of the polypeptide. e.~., amino acids 1-538 of SEQ. ID. NO. I.
It has been found that the A'70 protein acts with specificity to inhibit NFKB. For
cxample. e~pression of ~unB, another TNF or LPS-inducible protein, is not found to be
inhibited by A~0 expression under conditions in which NF~cB is so inhibited.
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The bc1-2 gene was ori~inally cloned from the breakpoint of a t(l4:18)
- - trans]ocation present in many human B cell ly~ c,lllas. In vitro, BCL-2 protein has
been shown to prevent apoptotic cell death selectivel~1 in ccltain cell lines, suggestinC the
existence of multiple independent intracellular rn~oh~ni~rr c of apoplosis, some of
which can be prevented by BCL-2 and others of which are a~pa.c~ltly unaffected by the
~ene ~WO 95/00642). ~ative proteins of the BCL (i.e. BCL-2) family are characterized
by three conserved regions, refelTed to as BCL-2 homology regions 1, 2 and 3
(abbreviated as BH-I, BH-2 and BH-3), that are required for regulation of apoptosis and
protein-protein interaction. Proteins of the BCL family include anti-apoptotic polypeptidcs
such as BCL-2, ~CL-XL (the long form of a splice variant of BCL-X), MCL-l and
BAG-l .
Another member of the BCL family comprises the Al protein. Human Al has
been found to comprise the BHI and BH~7 regions characteristic of the BCL fami~y(A. Karsan et àl., BLood g7, No.8 lApril lS, 1996] 3089-3096; A. Karsan et al.,
J. Biol. Chem. _71 (44) lNovember 1, 19961 27201-27204). Suitable anti-apo~loticpolypeptide~ for use in the invention rnay comprise or consist esserlti~lly of regions BHI
and BH2 of nati-~e ~e.~., human3 Al protein, or an amino acid sequence which in the
a~cre_ate i~ at least 80ck~ preferably at least 90%, and more preferably at least 95%,
homolo~ou.~ to the aC~recate of the BHI and BH2 regions of the native Al protein.
In ~eneral. suitable deletion mutants of the BCL family may comprise, for
example, al lea~t one of the BHI, BH'~. BH3 and BH4 regions of thc native protein, for
example, for each protein. one or more of the following peptide sequences (a.a. = arnino
acid position no.):
BCL-2: about a.a. 10 to about a.a. 30; about a.a. 93 to about a.a. 107; about a.a. 135 to
about a.a.l55; about a.a. 187 to about a.a. 202, of SEQ, ID. NO. 3;
BCL-X,: about a.a. 5 to about a.a. 24; about a.a. 86 to about a.a. 100; about a.a. 129 to
about a.a.l48: about a.a. 180 to about a.a. 195, of SEQ. ID. N0. 4;
Al: about a.a. 27 to about a.a. 45; about a.a. 66 to about a.a. 99; about a.a. 133 to about
a.a. 145, of SEQ. ID. NO. 5.
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Still other BCL family apoptosis-regul~tin~ polypeptides useful in the invention- - may comprise CDN-I and CDN-2 (W0 95115084); MCL-I (Yang et al.,
- J. Cell. Phys. 16611996] 523-S36, psrtieularly a polypeptide eomprising one or more of
amino acid residues 6-25, 209-223, 2~2-272, and 304-319 thereof; and BAG-I (or homo-
or heterodimers thereof with BCL-2 or other BCL family In~mbers) (Takayama et al.,
Cell. 80 [1995~ 279-284).
These anti-apoptotic polypeptides may exist in vivo in the forrn of homodimers or
heterodimers with another anti-apoptotic polypeptide of the BCL family. Sueh anti-
apoptotic polypeptides may also be found in heterodimer eo~.binations with antagonist
polypeptides of the BCL family sueh as BCL-Xs ~the alternatively spliced short forrn of
BCL-X), BAX and BAD.
The present invention also comprises a method of treating the dysfunctional or
activation response of a cell to an inflammatory or other aetivation stimulus, comprising
modifying s~id cell by insertin~ therein DNA encQt3ing an anti-apoptotic protein, in
operative association wilh a suitable promoter, and expressing said anti-apoptotic protein
at effecti~e level~ whereby NF-KB activation in said cell is s~lbst~n~ ly inhibited.
In a panicular aspect~ the invention comprises a method of treating the
dysfunction~l or activation response of a cell to an inflammatory or other activation
stimulus. comprisin~ modifying the cell by inserting therein DNA encoding a polypeptide
having anti-apoptotic activity of an A~0 protein in operative association with a suitable
promoter, and expressin~ the polypeptide at effective levels whereby aetivation in the eell
is substantially inhibited.
It further comprises a method of inhibiting eellular aetivation in a ll,&..l,..alian
subject susceptible to an inflammatory or immunologieal stimulus whieh eomprisesgenetically modifying endothelial cells of the subject, by insertion of DNA eneorling an
~ anti-apoptotic protein capable of inhibiting NF-lcB and expressing that protein, whereby
NF-lcB is substantially inhibiied in the cells in the presenee of a cellular aetivating
stimulus .
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In a further aspect, it comprises a method of treating the activation ,~s~onse of a
- ~ cell to an infl~mm~tory or other stimulus, comprising rnodifying that cell by inserting
therein DNA encoding a polypeptide having anti-apopto~ic activity of a BCL protein
(such as BCL-2 and BCL-XL proteins), a hom~ ner of such a polypeptide, or a
heterodimer of such a polypeptide with another anti-apoptotic protein of the BCL family,
and e~pl._;,sing the polypeptide or dimer at effective levels whereby activation in the cell
is sukst~n-i~lly inhibited.
The invention also includes the cells so modified, and corresponding tissues or
organs comprising such cells.
The protein-encoding region and/or the promoter region of the inserted DNA may
be heterolo~ous. i.e. non-native tO the cell. Altematively, one or both of the protein
cncodin~ re~ions and the promoter region may be native to the cell, provided that the
promoter is other than tl~e promoter which normally controls anti-apoptotic (e.g., A20)
expression in the cell. The protein coding sequence may be under the control of an
-appropriate si~nal sequence, e.g, a nucleus specific signal se~ nce.
Preferably the protein encoding region is under the control of a constitutive orre~ulabie promoter. By ''consliL~Ilive'' is meant substantially continuous transcription of
the ~ene and expression of the protein over the life of the cell. By "regulable" is meant
that transcription of the gene and expression of the protein is related to the presence, or
,~bsence, of a ~iven substance. An embodiment of "regulable" expression comprises
"inducible" expression. i.e. whereby transcription (and thus protein expression) occurs on
demand in rcsponse to a stimulus. The stimulus may comprise endothc~ cell activating
stimuli or a predetermined external stimulus. The endothelial cell activating stimuli may
be any of the stimuli which give rise to changes in the encloth~lium of donor tissue or
organs which stimulate coagulation. The predetermined external stimulus may be a drug,
cyto~;ine or other aBent.
An advantage of employing an inducible promoter for transplantation purposes is
tha~ the desired high level expression of the (e.g., A20) active protein can be obtained on
demand in response to a predetermined stimulus, such as e.g., the pl~sence of tetracycline
in the cellular environrnent. An example of a tetracycline-inducible promoter which is
suitable for use in the invention is disclosed in P.A. Furth et al., ~ ~ [1994
930'~ 9306. Alternatively, an examplc of a regulable promoter system in which
-
CA 02245503 1998-08-13
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- -17-
transcription is initiated by the withdrawal of tetracycline is described in M. Gossen and
- ~ H. Bujard, PNAS 89 ~199~ 5547~5~
Preferably, expression of the (e.g, A20) active protein is induced in l~sponse to a
predetermined external stimulus, and the stim~ c is applied be~l..n;..g imn ~ tely prior
to subjecting the cells to an activating stimulus, so tha~ e~..,ssion is aiready at effective
levels to block NF-lcB activation. For example, cells of a donor ~ (e.g., porcine~
may be genetically modified according to the invention ~y insertion of an anti-apoptotic
gene (e.g., porcine or human) under the control of a plulll~t~r which is inducible by a
druc such as tetracycline. The animal, whether somatic .~eo~ inant or transgenic, may
be r~ised up to the desired level of maturity under tetracycline-free conditions, until such
time as the cell~. or tissue or or~ans comprising the cells, are to be surgically removed for
ransplan~ation purposes. In such case, prior to surgical removal of the or~an, the donor
animal ma~ be administered tetracycline in order to begin inducing high levels of
expression of the anti-apoptotic ~e.g., A20) protein. The organ can then be transplanted
into a recipient (e.~., human), and tetracycline may continue to be administered to the
rccipienl for ~ sufficient time to maintain the protein at the desired levels in the
trallsplalltc~ ccll~ lo inhibit ~-KB activation. Alternatively, after being surgically
rcm~1~ctl from ~hc donor. the or~an can be maintained ex vivo in a tetracycline-cont~ining
medium un~ uch time a~ grafling into a recipient is appropriate.
In another embodiment, expression may be provided to occur as a result of
~ithholding tclracvcline from the cellular environment. Thus, cells of a donor animal
m~y be gcncticallv modified accordin~ to the invention by insertion of a gene encoding
an anti-apoptotic (e.~.~ A'0) protein under the control of a promoter which is blocked by
tctracyclinc. and which is induced in the ahsence of tetracycline. In such case, the animal
may be raised up to the desired level of maturity while being ~Anlini~t~red tetracycline,
until such timc as the cells, tissues of organs of the animal are to be harvested. Prior to
surgic;ll rcmoval, the donor anirnal may be deprived of tetracyclinc in order to begin
inducin~ expression of the protein, and the paticnt in whom the cells, tissue or organs are
transplanted may thereafter also be maintained tetracycline-free for a sufficient time to
maintain appropriate le-els of expression.
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Prefera~oly, the inserted DNA sequçrlees are incG,y~nated into the c~enome of the
cell. Altematively, the inserted sc.luences may be m~ ined in the cell
extrachromosomally. either stably or for a limited period.
The modification of endothelial or other l-.a."lllalian cells according to the
invention may be carried OUl in vivo or ex vivo.
Thus the invention also comprises a method for inhibiting the dysfunc~ional or
activation response of endotheiial cells to an infl~mn~tory or other activation stim-lhl~ in
vivo in a patient in need of such therapy, comprising modifying such cells of the patient
by insenin~ in the cells DNA encodino an anti-apoptotic protein in operative ~csoc;~tion
with a constitutive or inducible promoter and expressing the protein at effective levels
wherebv ~F-KB activation is substantially inhibited. For example, the blood vessels of
an or~an (e.~., a i;idnev) can be temporarily clamped off from the blood circulation of the
patient and the vessels perfused with a solution comprising a trancmi~cible vector
construc~ con~aining the anti-apoptotic (e.~.. A20) ~ene, for a time suf~leient for at least
omc ccll~ of thc or~n ~o be eeneticall~ modified by insertion therein of the vector
cons~ruc~: an~l 011 removal of ~he clamps. blood flow can then be restored to the organ and
it~ norm;ll func~ ninr resumed.
In anothcr a~pcct. cc13 popula~ions can be removed from the patient or a donor
animal. ~cnelic~ modified ex vi~o b~ insenion of vector DNA, and then re-implanted
into thc p;l~ienl or transpiantcd into another recipient. For example, an organ can be
remo-~cd from a palienS or donor. subiected ex vivo to the perfusion step described above,
and thc or~an c;~n be re-~ratled into the pa~ient or implanted into a different l.,cipicnt of
~hc s;lmc or differcnt ~pecie~.
For ~cne deli~er~, rclroviral ~~ectors, and in particular replication-defective
retrovir;ll vcclor~ lacl;ine onc or more of the ~ag, pol, and env sequences required for
retroviral replic;l~ion. are well-known in the art and may be used to transform enciothelial
or other mamm;~ n ccll~. PA3 17 or other producer cell lines producing helper-free viral
vec~or!; are ~cii-dcscribed in the literature (A.D.Miller and C.Buttimore,
Mol..Cell Bic)lo~v 6 11986l '895-~90~). A representative retroviral construct comprises at
lea~t one v ir~l lon~ terminal repeat and promoter sequenres ups~.eal.l of the
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nucleotide se~uence of the llleldpeutic s~lbst~nre and at least one viral long tenninal
repea~ and polyadenylation signal dow~ al.~ of the nucleoticlG sequence.
Vectors derived from adenoviruses, i.e. viruses causing upper respiratory tract
disease and also presen~ in latent infections in primates, are also known in the art. The
ability of adcnoviruses to attach to cells al low alllbic.lt tc.l~ tUI~,S is an advantage in
the transplant settin~ which can facilitate gene transfer during cold preservation of tissue
or organs. Adenoviral-mediated 8ene transfer into vessels or organs by means of
tran~du~ion perfusion as described hereinabove is also a means of genetically modifying
cells in vivo or ex vivo.
AJternative me~ns of tar~eted gene delivery comprisc DNA-protein conjugates,
liposomes, etc.
In ye~ another embodiment. the invention comprises a method for suppressing the
activation response of donor cells, or tissue or organs comprising such cells, upon
transplantation into a rnammalian recipient in whom the cells are sl~ceptible to activation,
which compri~es:
(a) modifyinn the donor cells by introducing therein DNA encodin~ an anti-apoptotic
protein: and
(b) tran~iplantin,~ lhe re~iultanl donor cell~i~ or tissue or organs comprising such cells, into
the recipicnt and expressin_ the protein. whereby NF-KB activation of the cells is
substan~ lly inhibi~ed.
Thc donor ~ipecic~ may be any mammali;m species which is the same or different
from the recipienl species, and which i!i abte to provide the applop~-ate cells, tissue or
organs for transpl~ntation in~o the recipient species.
The donor may be of a species which is allogeneic or xenogeneic to that of the
recipient. The recipient is a mammal, e., a primate, and is preferably human. For
human recipien~s, i~ is envi~;lged that human (i.e. allogeneic) as well as pig (i.e.
xenogeneic) donors witl be suitable, but any other mammalian species (e.g., bovine or
non-human primate) may al~o be suitable as donor.
For example, porcine aortic endothelial cells (PAEC), or the progenitor cells
thereof, can be genetically modified to express porcine or human anti-apoptotic, e.g. A2()
protein at effective levels, for grafting into a human l~cipie.lt. Heterologous DNA
-
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cn~odin~ the A20 or other anti-apoptotic protein can be inserted into the animal or an
ancestor of the animal at the single-cell or early morula stage. The preferred stage is the
single-cell stage, although the process may ~e carried out between the two and eight cell
sta~es. A trans~enic non-human animal can be thereby obt~ Gd which will pass theheterologous DNA on to offspring. In another aspcct genes can be inserted into
somaticlbody cells of the donor animal to provide a somatic recombinant animal, from
whom the DNA construct is not capable of being passed on to offspring (see, e.g.,
Miller, A.D. and Rosman, G.J.. 13iotechni~ues 7 [1989] 980-990).
Appropriate well-known ml-thods of inserting foreign cells or DNA into animal
tissue include micro-injection, embryonic stem cell manipulation, electroporation, cell
gun. transduction. transfection. retroviral infection, adenoviruses, etc. ln one embodil"~nt,
the ~ene is inserted in a particular locus, e.g., the thrombomo~i-llin locus. Subsequently,
the construct is introduced into embryonic stem cells, and the resul~ing progeny express
the construct in a tissue speci~lc manner, paralleling the expression of thrombomodulin,
-i.e. in the vascular endothelium.
Methods of preparin~ transgenic pigs are disclosed in e.g. Pinckert et al., ~ j~,
No. 1 119941 10-15.
Gene~ically modified endothelial cells may be a~minic~red by intravenous or
intra-anerial injection under defined conditions. Tissues or organs comprised thereof
may ;~lso be rcmoved from a donor and grafted into a recipient by well-known surgical
procedures. Prior to implantation, the treated endothelial cells, tissue or organ may be
screened for ~enetically modified cells containin~ and expressing thc construct. For this
purpose~ the vector construct càn also be provided with a second nucleo~ide sequ~r~e
encodin~ an expression product that confers resistance to a select~ble marker subst~nce
~uitable selection markers for screenin~ include the neo ~ene, conferring resist~nre to
neomycin or the neomycin analog, G418.
Althou~h any mammalian cell can be targeted for insertion of the anti-apoptotic
gene. such as monocytes. NK cells, Iymphocytes, or islet cells, the preferred cells for
manipula~ion are endothelial cells. The recipient specics will primarily be human, but
other mammals~ such as non-human primates, may be suitablc recipients.
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In an altemative embodirnent of the invention, thc anti-apGptutic polypeptide, in a
pharm~reuttc~lly acceptable carrier, may be applied directly to cells, tissues or organs in
vivo.
It will be appreciated that the modified donor cells and tissues and organs defined
above have a supple~llenl~ fi~nction in the prevention of xenotransplant ~jec~ion since
complement-me~liatecl events also participate in hyperacute rejection of such transplants
(A.P. Dalmasso et al., Transplantation 52 [1991] 530-533). Therefore, the genetic
material of the cells of the donor organ is typically also altered such that activation of the
complement pathway in the recipient is prevented. This may be done by providing
trans~enic anirnals that express the complement inhibitory factors of the recipient species.
The endothelial cells of a donor organ obtained from such an animal can be modified by
gene therapy techniques to provide the endothelial cells defined above. Alternatively a
vector containin~ DNA encoding a protein having anti-apoptolic (e.g., A20) acti~ity can
be introduced into the trans~enic animal at the single cell or early morula stage. In this
way. the re~;uitin~ transgenic animal will express the complcment inhibitory factors and
will have endothelial cells as defined above.
Thu~ in a further aspect the invention also provides endothelial cells, tissue, donor
or~ans and non-human trans~enic or somatic recombinant anirnals as defined above which
express one or more human complement inhibitory factors.
CA 02245503 1998-08-13
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The following Examples are int~n~led to be illustrative only and not limitativc of
the invention. Cultured BAE~C are transfected with r~po"~ constructs co~cictirlg of
promoters of genes known to be upregulated upon ~C activation, i.e. E-s~kcl;n~ l~oc,
IL-8 and tissue factor.
F.XAMP~.F.S
Materials and r~tho-lc:
The following vectors are utilized in the ~xamples:
"pAC": 8.8 kB plasmid vector con~inin~ a CMV promoter, a pUCI9 polylinker
site, ~nd an SV40 splice/polyA sile (~.Herz and R.D.Gerard, PNAS ~Q [19931 '812-2816).
A20 expression plasmid ("A20" in Figures): human A20 cDNA (Opipari et al.
119901, ~upra) (SEQ. ID. NO. 23, subcloned into the pAC expression vector at the XBal
restriction site.
Bcl-~ and Bcl-x, expression plasrnids: murinc bc1-2 and bcl-xL genes (W. Fang
et al.. J. Imrnunol. ISS [199S} 66-7S). The 830 bp full-length bc1-2 cDNA was
flag-ta~~ed ~nd cloned in the PAW neo-3 expression vector into a Clal/Xbal expression
vector. The 700 bp full-length BCI XL cDNA was also
fla~-tagged and cloned into a Clal/BamHI sites of the PAW neo-3 expression vector
(PAW neo-3 is a 71;b expression plasmid containing ampicillin and neomycin resist~nce
sites and a SFFV-LTR promoter before ~he polylinker cloning site) (SFFV = spleen focus
forming virus).
Porcine E-selectin reporter: bp -1286 to +484 of the porcine E-selectin plUIIIOt~,~
cloned into the pMAMneo-luc plasmid vector by replacing the mm TV promoter
(Clontech, Pnlo Alto, CA) (this includes the first complete intron and exon, as wcll as the
beginning of the 2nd exon up to the ATG site).
Porcine NF-lcB reporter: 4 copies of NF-lcB binding sites derived from the
porcine E-selectin promoter inserted upstream of a TK minitn~1 p,u..-ot~. driving the full
length luciferase gene in a pT3/T7-luc vector (Clontech).
CA 02245503 1998-08-13
WO 97130083 PCT/EP97/0067ti
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The vector backbone is a Bluescript KS+ plasmid (Slr~t~n~, La Jolla CA, USA).
Human IL-8 reporter: human IL-8 (hIL-8) ~ ot~r cloned into p-UB~ luc.
Porcine TF reporter: 1000 to +34 fragment of the porcine TF promoter cloned
into p-UBT luc, a luciferase reporter gene vector (R. de Martin et al., ~ 1'~4 ~19931
137-138), according to the method of T. Moll et al, J. Biol. Chem. ~Q ll995] 3849-3857.
Porcine IlcBa (also referred to as "ECI-6") I~;pGllel. 600 bp fragment of the
porcine ECI-6/IkBa promoter ligated into p-UBT-luc, with the creation of an additional
Hind III siee, as described by R. de Martin et al., Fl~BO J. 12 ~1993J 2773-2779HIV-CAT reporter: -117 bp to the TATA box start of the ~IIV-wt LTR, cloned
upstream of the CAT gene (CAT3N polylinker), pl~,~alcd as described by
K. Zimmerm~nn et al., Virolo~.y 18~ [1991] 874-878.
RSV ~-gal reporter: E. coli ,B-~al gene inserted into the pRc/RSV vector
(ln~itro~en~ San Diego, CA, USA) at the Not I site.
RSV-LUC reporter: full-length luciferase gene cloned into the pRctRSV vector.
A ~ a ~ ~:
Ccll cx1r;1cts ;lre ~ssa,ved for luciferase (or CAT) and g~la~tosidase levels.
al 1 ncit~er;l~e lcvcls (E-selectin~ NF~lcB~ IL-8. TF and IlcBa ~ECI-6~ promoters):
I() ~" Or cellular extract are added to 90 ~1 of a solution con~ ing 24 mM
~Iycyl~lycinc (pH 7.8). ~ mM ATP (pH 7.5) and 10 mM MgSOs. Samples are read on a:~licrolum:ll LB 96P luminome~er (EG+G Berthold) using an injection mix consisting of
~ 1 m.'~1 ~Iyc~l~lycine and 0.1 mM luciferin (Boehrin~er, Mannheim, Germany3.
Lucifcr;l~c activit~ is normalized for ~-gal:-r~osicl~c~ using the following formula:
~lucifera~e ac1i~ily/~-gal activity) x 1000. Luciferase activity is also co,.~,ted for protein
by dividin~ ~hc luciferase ;lCtivity by protein concentr~tion. Norrnalized luciferase
3C~iVily iS gi-en in rela1ive li~hl units (RLU).
CAT levels (HIV LTR activity):
A Promega kit ~Prome~a~ Madison, WI, USA~ is used to incubate cells in
''C-labeled chloramphenicol ;md n-butyryl coenzyme A - cont~ining medium (the CAT
protein transfers the n-but-ryl moiety of the coenzyme to chlo,~J-.rhenicQI). Cells are
extracted inlo xylene. which is mi~ed with scintillation liquid and counted in ascintillation counter (1900 TR. Packard, Downes Grovc, IL, USA). Counts per minute
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(CPM) are nortnalized for ~B-g~ tosidase using the following formula:
(cpm~ a1 activity) x 1000. Si~nificance is determined by Student's t-lest.
c) ~-~alactosidase levels:
The RSV ~-gal reporter serves as a control for transfection err.ciency. The
Tropix, Inc. Galacto-Light protocol (Tropix ~nc., Bedford, MA, USA) is employed to
measure ~-galactosidase levels.
F.xample 1: Transfected ~AFc ex~ress humal~ A20 ~rot~in
Bovine aortic endothelial cells (BAEC) are isolated and cultured in 10 cm platesin Dulbecco's Modified Ea~le Medium (DMEM), supple,nented with L-glutamine
(2 mM), penicillin G (100 units/ml), and fetal calf serum (FCS) (10%). Cells aremaintained ~t 37"C in a humidified inrub~tor with a 159a CO2 atmosphere. When the cells
re;lch 70~k confluency. one ~roup (i.e. approximately I x IQ6 cells) is transfected with 0.5
of the A~Q vector ("A20"); a second ~roup is transfected with 0.5 ~lg of the pAC~cctor ("PAC"): ~nd ;I third ~roup is maintained as a non-transfected ("NT") control. All
tran!ifcclion~ ;Irc done with 16 1l~ lipofectamine. Non-transfected, non-stimulated
Hl'VEC ("!~S") or non-transfected. TNFa-stimulated HUVEC ("TNF") also serves as
control~.
Ccll~ arc ua~ihcd twice ~ ith cysteine and methionine-frce medium (ICN. Lisle, IL,
~ISAI. ;-n~ thcn pl;3ccd in thc s~n1e n-cdium supplemented with 100 I~Ci/ml Tran 35S
lahcllcd cy!i1cinc and methionine (lcN). After four hours, cells are harvested. Tmml~no-
precipila1ion ~ ith polyclon~l rabbit anti-human A20 polyclonal serum on a
polyacrylamidc SDS ~el. a!i shown in FIG. 1, reveals the ~ enee of a 35S-labelled 80 kD
A'~0 protein in thc "A~0" exsract~ but not the "PAC", "NT" or "NS" extracts. This
protcin is comparablc to that scen in the TNF-stimulated HUVEC extrac~ ("TNF").
CA 02245503 1998-08-13
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5_
F.xam~ies 2-4: Gel~ral Procedure
Approximately 3 x 105 ~AEC are plated per well in 6-well plates in ~ ml DMEM
as supplemented and under the conditions described in Example 1. Whcn the cells reach
50~-70% confluency, a total of 1.6 ~g of DNA (comprising test plasmids, Ic?o.t~lconstructs and the ~-gal reporter) and 8 ~lg of li~,~fcc.l~mine are used to transfect the cells
in each well. After incubation of the cells for 5 hours, FCS is added to the cells to make
a final concentration of 10%. After inru~ation for 48 hours, the cells are stimu}ated by
adding tO triplicate wells 100 U/ml of TNFa or 100 ng/ml of lipopolysaccharide (LPS)
(Sigma E.Coli OB~S). Non-stimulated cells serve as control ~"NS" or "control"). Seven
hours after stimula~ion, ~he cells are harvested (in the following Examples all volume or
weight amounts are on a per well basis; the expression "cell population" or "~roup of
cells" refers to the cell population of a sin~le well plate, i.e. estimated to be
approximately 5 x 10~ cells; in the bar graphs, the bars represent the mean of triplicate
values; standard error is represented by a bracket).
F.xample ~: F,selectin reDorter (A~O expression in BAFc inhibits E-selectin
induc~ion in a dose-dependent manner)
BAEC (bo~ine aonic endothelial cells) are cotransfected with 0.7 ,ug of the
porcine E-selectin reponer construct. to~ether with the A~O expression plasmid or the
pAC control plasmid or both. The header portion of FIG. 2 indic;~tes the amount of A20
plasmid pro-~ided to each cell population~ as follows:
lanes 1, 5~ 9: 0 ,ug A~O;
lanes ~. 6, 10: 0.1~5 llg A~O;
lanes 3,7,1 1: 0.5 ~g A'O;
lanes 4.8.1': 0.7 ,ug A'O.
pAC is titralcd wi~h thc A~O plasmid where necessary to bring thc total
concentration of A~O und pAC vector to 0.7 llg per well.
FlG. ~ is a bar graph representing the results of a luciferase assay of each group
of cells. Induction of ~hc luciferase ~ene under the control of the E-selectin promoter is
correlatable to the ~mount in relative light units (RLU) ~etPcte~ in the assay. FIG. 2
demonstrates that stimulation of the cells with TNF or LPS results in substantial increases
in activity of the E-selectin reporter in the untreated control (lane l); or in the stim~ ted
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cells co-transfected with only the pAC control (lanes 5 and 9), where there are 8 and
14-fold incrcases in E-selectin activity. Stim~ ted cells l~a~srccled with the A20
construct show sig-,iricant inhibition of induction of thc E-selectin reporter (lanes 5 v. 8,
9v. 12).
It is also apparent that A'~0 expression inhibits E-selectin induction in a
dose-dependent manner: when 0.125 llg of A20 are used, the inh~bition reaches 53% for
TNF-stimulated cells and 78% for LPS-stim~ t~cl cells (lane 5 v. 6, 9 v. lO~. Virtually
comple~e inhibition is achieved when the amount of A20 used is 0.5 llg and hi~her. as
co"lpal~,d tO the basal levels detected in the non-stirn~ ted BAEC transfected with the
empty vector (lane I v. Ianes 7, 8. 11 and 12). In addition, A20 expression de~l.,ases the
basal. unstimulated activity of the E-selectin l~,po.ler by 2-~old when used at 0.5 llg and
hi~her.
Since maximal inhibition is obtained by t-~n~r.,c,~ g with 0.5 to 0.7 ,ug A20
vector, the concentration of A20 plasmid used to tl~ns~l groups of cells in Examples 3,
and S is selected to be 0.5 ~g.
F.xample 3~ 8.11cB(x (FCJ-fi~ ard TF re~orter cor~ructC
BAEC are cotransfected as described in the General Procedure above with 0.5 ~g
of either the A20 expression plasmid or the pAC control plasmid, and 0.7 tlg of one of
the abo~e-indic;lted reponer constructs, which are up-regulated during EC activation.
FIGS. 3A-3C are bar graphs representin~ ~he results of a luciferase assay for each
reponer tr~nsfection (in FIGS. 3A-3C, as well as FIG.4 and FIG. SA, the presence ("+")
or ~bsence ("-") of A20 or pAC is indic~t~d in the header):
a) 11-8 reporter: When the IL-8 reporter is cotransfected with empty pAC vector,luciferase activity increases ~.5 and '.7-fold after stimulation with TNFa and LPS,
respectively (FIG. 3A, lanes l ~u 3 and ~). However, when the IL-8 reporter is
cotransfected with the A~Q expression plasmid, luciferase levels after TNFa or LPS
stimulation ~re reduced to below that seen with non-stim~ ted pAC-transfected cells
(60~c below the luciferase ~ctivity of unstimulated cells, lane l v. 4 and 6). Further-
more, A20 overexpression decreases the basal luciferase activity of the IL-8 .~;po.t._r by
3-fold tFlG. 3A, lane l v. ~).
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b) TkR(x reporter: The results of the co-transfections pe,t~ ed using the porcine IKBa
(ECI-6) reporter construct are similar to those seen with I~-8. Induction with TNFa and
LPS reaches 1.6 and 3.6-fold, respectively. Inhibition is virtually complete when A20 is
cotransfected with the IkBa reporter. TNFa- or LPS- in-h-ce~ luciferase activities are
also lower than the basal levels noted with the empty vector ~FIG. 3B,
lane 1 v. Ianes 4 and 6). Co-transfection with A20 is found to decrease by 5-fold the
basal level of ECI-6 luciferase activity (FIG. 3B, lanes I v. 2).
c) Tissue factor reporter: In a comparable manner, A20 expression inhibits the 3.5 and
4.5-fold induction of TF reporter activity after TNFa and LPS st~ tion~ respectively
(FIG. 3C, lanes 3, 4, 5, 6). However, a decrease in basal TF reporter activity with A20
co-expression is not observed (FIG. 3C, lane I v. 2~.
Ex~mple 4: NF-lcR re~orter
BAEC are cotransfected according to the General Procedure with 0.5 llg of eitherthe A20 expression plasmid or the pAC control plasmid and 0.7 ~lg of the NF-~B reporter
construct. snd the results are shown in the bar graph comprising FIG. 4. Resultsdemonstrate that A~0 expression abrogates the 12 and 28-fold induction of reporter
activity in response to TNFa and LPS. respectively (FIG. 4, lanes 3 v. 4, 5 v. 6). There
is no apparent significant difference between the basal levels of luciferase activity
between A20 and pAC transfected cells (FIG. 4, lane I v. 2).
All the reporters listed above are known to be highly dependent on NF-lcB.
Activation of these repor~ers by either LPS or TNFo~ is found to be inhibited byexpression of A20, demonstrating that the inhibitory effect of A20 on EC activation
relates, at least in part and perhaps totaliy, to inhibition of NF-lcB.
F.xam~le 5: RS~ .UC and HIV-CAT reporters
To test non-specific or to~cic effects of A'~0 on the transcriptional machinery, cells
are transfected accordin~ to the General Plocedul~ with a constitutive, non-inducible
reporter~ RSV-LUC, which is independent of NF-KB. Also tested is the E~IV-CAT
reporter, which is induced by the viral c-Tat protein through Spl rather than NF-K:B
bindin~ (Zimmermann et al. ~1991], ~). Cells are transfected with 0.5 ,ug of either
A20 or pAC ~RSV-LUC reporter) (as shown in the header of FIG. ~A), or A20 titrated
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with pAC to make up a total of 0.5 ~lg (HIV-CAT reporter) (as shown in the header of
FIG. 5B). For the RSV-LUC reporter, cell groups are either non-stimulated ("Control")
or TNF- or LPS-stimulated. For the HIV-CAT reporter, cells are either unstimulated
("Control") or stimulated with 0.2 ~g of the c-Tat protein. It is found that basal
luciferase activities of the RSV-LUC reporter are comparable to that seen in the A 0 and
pAC transfected BAEC.
FIGS. 5A-5B are bar craphs representing the results of a iuciferase assay. It isapparent that no significant induction is achieved upon TNF or LPS stimulation in either
the pAC- or the A20-expressing cells; luciferase values remain comparable among the ''
~roups (FIG. ~A). With reoard to HIV-CAT, the results demonstrate that A20 expression
affects neither the basal levels nor the 10 to 15-fold induction of the reporter observed
upon stimulation with c-Tat (FIG. ~B, lane 1 v. Ianes 2, 3, 4 and lane 1 v. Ianes 6. 7, 8).
The above demonstrates that expression of A20 prevents gene induction associatedwith endotheliai cell activation. Reporter inhibition is seen when either TNF or LPS is
used to s~imulate the EC, pointing to the broad inhibitory effect of A20 on geneinduction. The similar effect on LPS- and TNF-induced signaling also excludes any
specific association of the action of A20 with the TNF response per se. The basal
expression of the E-selectin, IL-8 and IlcBa reporters is also significantly decreased in
cells expressin.~ A20. Inhibition is found to be dose-dependent.
Expression of A'70 has no apparent effect on either the constitutive activity of the
RSV-LUC reponer or the c-Tat stimulation of the HIV-CAT reporter, which also
demonstrates a lac~; of effect of A20 on Spl, which illustrates the specificity of A20 in
blocking NF-KB activation.
Therefore in addition to its ability to protect cells from apoptosis, expression of
A20 inhibits NF-lcB activation, and thereby inhibits gene induction. This function places
A20 in the category of genes that are dependent on NF-~cB for their induction, but that
subsequently inhibit NF-KB and thus, endothelial cell activation. Such genes presumably
function in necative regulatory loops to regulate the extent and duration of endothelial
cell activation.
While not intending to be bound thereby, it is proposed that an alternative
mechanism exists by which A20 functions as an antioxidant. The full-length human A20
cDNA encodes 7 Cys2/Cys2 repeats, which characterizes it as a Zn finger protein with a
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potentially high Zn binding capacity ~Opipari et al. I1990~, ~). Zn can act as an
antioxidant by two mech~nisms: the protection of sulfhydlyl groups against oxidation and
the inhibition of the production of reactive oxygens by transition metals, mainly iron and
copper. There is evidence that antioxidants such as PDTC can prevent gene induction
associated with EC activation, by inhibition of N~ .B. Cunnin~h~m,
Riochem.E~iophvs.Res.Commun. 215 [1995] 212-218) and also to prevent TNF-me(~i~t~d
apoptosis (T.M. Buttke and P.A. Sandstrom, Immunol. Tod~y 1511994] 7-10). These
findings correlate with the fact that signaling via the TNF receptor results in a rapid rise
in the levels of intracellular reactive oxygen interme~ tes that cause apoptosis via
oxidative damage (Buttke and Sandstrom [1994], ~).
F.xample 6: Adenoviral-mediated transfer of A20 to ~orcinP aortic endothelial cells
A recombinant A''0 adenovirus (rAd.A20) is constructed by homolo~ous
recombination between a transfer vector containing the human A20 cDNA,
pAC.CMV.NLS-A~0, and pJM17, a plasmid-borne form of the adenovirus 5 genome.
Thc encoded A20 protein is unmodified. Homologous ,~,co."bination is perforrned in
'9~ cclls. Clcnal viruses are obtained by limiting dilution cloning in 96-well plates, and
zed h! ~onhern blotting for the presence of A20 mRNA. After identification of a
C recomt~in;lnt A20 adenovirus, amplification is performed in 293 cells. Cesium
chloride purified adent ~irus i~ used to infect porcine aortic endothelial cells (PAEC~ at a
multiplicity of infection (MOI) of 500 to '500/cell. A20 infection is checl~Pd by
;~orthern blol un;llysis of inîected cells. 48 hours after infection, cells are Srimnl~t~l with
l00 U/ml of TNF or l00 ng/ml of LPS. mRNA is extracted 2-6 hours following
EC !itimulation. Nonhern blot analysis shows that A20 adenovirus-infected cells abrogate
by 60-90~k the TNF- and LPS-medi~ted induction of E-selec~in~ IL-g, and IlcBa. The
percenta~e of inhibition is directly correlated to mRNA levels of A2Q (let~cted in infected
cells. In accordance ~ith l~'onhern blot analysis, A20 expression in PAEC inhibits by up
~ ~o 9Q~k the surface expression of E-selectin as assessed by ELISA. Mock-infected cells
as well as PAEC infected ~ith a ~-~alactosidase rAD are used as controls. These results
further demonslrate that expression of A20 inhibits EC activation.
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Fxar-Dle 7: Co-trancfer of BAFC with Bc1-2 and RCI-~L e~.~s;.ion E~lasmids alon~- - with revorter co ~ . I c
Approximately 3 x 10~ bovine aortic cndotl.elial cells obtained from culture in
10 cm plates as described in Example 1, are plated per well in a 6-well plate in 2 ml of
DMEM as supplemented and under the conditions described in Fy~rnrl~ 1. When the
cells reach 50~o-70~ confiuency, a total of 1.5-1.6 ~gtwell of DNA ~test plasmids and
reporter constructs) is added to 8 mg of lipofectamine per weil and incnb~te~l at room
temperature for 30 minutes before being added to the cells in triplicate. In allexperiments, BAEC are co-transfected with 0.5 llg of Bc1-2, BCI-XL or pAC, and 0.7 ,ug
of the E-selectin, ECl-6 (IKBa) or NF-lcB - luciferase (luc~ reporters, as well as 0.3 ~g of
the ~-galactosidase (b-~l) reporter. After S hours incalb~tion~ FCS is added to the
medium to achieve a final concentration of 10~. 48 hours thereafter the cells are
s~imulated with either human recombinant TNF (lOOU/ml) or LPS (lOOnglml), and are
harvested 7 h after stimulation.
Thc effcct of BCL-') and BCL-X~ expression upon EC activation is first studied
using an cndolhclial cell-specific mari;er, E-seiectin. BAEC (3x105to 5xl05 cells) are co-
transfcctcd ~ ith thc porcinc E-selectin reporter construct ~0.7 ~lg) as well as the bc1-2, the
bcl-xl c.~prc!~ion plasmids ~0.~ ~g) or the pAC control (0.5 llg) plasmid in conjunction
~ith thc RS~ tal plasmid (O.~
Thc rc!iults. dcpicted in FIG. fiA. sho~ that BCL-2 and BCL-XL overexpression
Ic;l~s to a siënil~ic;ln~ dccrcasc in the luciferase activity of the E-selectin reporter after
hoth T~F and LPS stimulation. In the pAC control, induction with either TNF or LPS
Icads to a 35- and 50-fold increase in the activity of the E-selectin reporter, ~cspeclively.
BCL-X, exprcssion inhibits TNF- and LPS-induced luciferase activity very significantly,
~his inhibition rcachin, rcspcctivcly 95~k and 90~ of the control following TNF and LPS
stimulation (ialle!i ~ and 7 v. 5. 8). Inhibition is seen to be complete when BCL-2 is
expressc~l in thc ccil.s. No induclion of Ihe E-selectin reporter is seen upon TNF and LPS
stimulation (lancs ~ and 7 v. Ianes 6 and 9). The basal level of luciferase activity of the
E-selec~in rcponcr is not ;lffccted by BGL-~ or BCL-XL expression.
The results of the co-transfections performed using the porcine IlcBa (ECI-6~
reporter cons~ruct (FIG. 6B) are similar to those seen with E-selectin~ Induction with
TNF and LPS reaches ~.5 fold (lanes I v. 4 and 6). BCL-XL and 13CL-2 expression
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cornpletely abolishes TNF- and LPS-in~nced luciferase activity following TNF and LPS
- - stim-ll~tion (lanes 4 and 7 v. ~, 6 and 7, 8). The basal level of luciferase activity of the
I~Ba reporter is not affected by BCL-2 or BCL-XL expression.
BAEC are co-transfected with an NF-~ o,~r construct that is sotely
~pendent upon NF-lc33, and either bcl-xL, bc1-2 or the empty vector, pAC (FIG. 6C).
BCL-XL expression si~nificantly decreases the 10- and 26-fold induction of reporter
activity in response to TNF and LPS, ~~spcctively (lanes 4 and 7 v. 5 and 8). This
inhibition reaches 50Yo and 70qO, respectively. In contrast with BCL-XL, ~CL-2
expression totally abro~ates the TNF and LPS inducibility o~ the NF-~B reporter (lanes 4
and 7 v. 6 and 9). There appears to be no signi~lcant difference in the basal levels of
luciferase activity between 13CL-XL, BCL-2 and pAC ~lanes 1 v. 2 and 3).
Therefore the demonstrated EC inhibitioll is shown to be related to inhibition of
the transcrip~ional faclor NF~
F,xample 8: A20 mutants
A tmncation of the A'~0 ~sene from bp 118~ to 2450 and spanning the 7 Zn
bindin, domains of the molecule is obtained by digestion of the '~.4 kB cDNA with NcoI.
Thi~ fra~ment i~ exprer.~ed a~ a polypeptide of 417 amino acid residues (residues 373 to
790 of SEQ. ID. NO. I ). The truncated A'0 ~ene is cloned into pBac 4 (Promega) and
then subcloned into the pAC expression vector to be used in co-transfection experiments
in BAEC. In these experiments, ~ x 10~ BAEC are plated per well in a 6-well plate with
'' ml of medium as described above. Cells are transfected once they reach 50-70%confluence. 1.5-1.6 ~~/well of DNA (test plasmids and reporter constructs) are added to
4 units of lipofectamine per well and incubaled at room temperature for 30 minutes
before being added to the celhi in triplicate. In this experiment, 0.31lg of the 13-gal
reporter ;s used, with ~.~ ,ug of: A~0, or truncated A'~0 (tA2()), or the control plasmid
pAC, and 0.7 ~g of the E-selecliri-luc reporter. 48 hours after transfection, cells are
~ challenged with either 100 Ulml of TNF or 100 nglml of LPS. Cell extracts are prepared
7 hours after stimulation and assayed for ~-galactosidase and luciferase expression, as
above. Two clones expressing the truncated form of the A20 are tested: clone #3
and #7.
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FIG. 7 shows that expression of the truncated forrn of A20, i.e. consisting
ess~nti~lly of the 7 Zn binding domains of the molecule, inhibits as efficiently as A20
the induction of the E-selectin reporter upon stimlllPtion by TNF or LPS.
F.xample 9: Re~ulable ~ene ex~ress~or~ in trans~enic mice
a) Inducible tetracycline expression system:
A system for temporal regulation of anti-apoptotic gene expression is highly
desirable to inhibit NF-lcB activation on a controllable basis.
An inducible expression system can be employed to regulate anti-apoptotic gene
expression in vivo, in particular the binary plasmid system described by Gossen and
Bujard, PNAS 1199'], ~.e~. which is inducible by the withdrawal of tetracycline; or the
tetracycline-dependent system disclosed by Purth et al., PNf~S [1994), supra. For
example the Gossen and Bujard system employs a ~Irst plasmid cont~ining a bacterial,
tetracycline-sensitive DNA binding protein fused to the HSV-VP16 transcriptionalactivation dornain (t~A) expressed from a cor.sli~ulive CMV promoter. A second plasmid
contains 7 copies of the binding site for tTA, downstream of which the anti-apoptotic
gene is cloned into the vector. When both plasmids are present in a cell, the tTA protein
dri~e.s high le~el transcription of the anti-apoptotic ~ene of the invention. In the presence
of tetrac-cline there is no expression of the anti-apoptotic transgene. In the absence of
tetracycline. there is high level expression of the anti-apoptotic gene (in the Furth et al.
system. the presence of tetracycline promotes expression of the anti-apoptotic gene,
whereas in the absence of tetracycline there is no expression of the anti-apoptotic
transgene ).
b) Trans~enic mice:
For the generation of transgenic mice the anti-apoptotic gene is cloned into a
suitable vector~ for example~ as described by Gossen and Bu~ard, PNAS [1992] ~.
Two sep~rate founder strains are generated for tTA and the anti-apoptotic gene.
Transgenic mice of each strain are rendered homozygous by crossing heterozygous
animals. Hornozygous animals of each strain are bred as lines. Crossing tTA/tTA mice
with~ e.g.. bcl-'/bcl-' mice results in double trans~enic mice carrying both tTA and Bc1-2
transgenes. These crossings are carried out under cover of tetracycline to prevent anti-
apoptotic transgene expression during embryogenesis. Mice carlying the tTA and anti-
CA 02245503 1998-08-13
WO 9713QO83 PCT/EP97/00676
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apoptotic transgene, respectively, are identified by Southern blotting to prevent expression
of the anti-apoptotic gene during embryo&c,.~esis.
Mice that express the anti-apoptotic gene in EC can be used as donors for
xenotransplantation (heart and/or kidney) into rats for modelling yul~oses.
F~xAmple 10: Generation of trnnc~enic PU~
A transgenic pig explessil~g a human anti-apoptotic gene (e.g., A20, bcl-'~, bcl-x~,
Al ) is ~cpa~ed by techniques disclosed in Pinckert et al. [1994~, ~.
F.xample 11: Adenoviral-mediated BC~.-2 exvression ~ bits NF-lcB activation
Nuclear extracts are prepared from rAd.Bcl-2 or rAd.,B-gal-infected PAEC before,and two hours following, treatment with TNF (lOOU/ml). NF-1cB ac~ivation and binding
to a lcB binding o}i~onucleotide derived from the human Immunoglobulin (Ig~ lC promoter
is evaluaeed by electrophoretic mobility shift assay ~EMSA) (FIG. 8~.
Nuclear extracts from PAEC expressing BCL-2 reveal little constitutive, and no
inducible. binding of NF-KB, whereas rAd.~-gal - infected cells demonstrate strong
induction of NF-lcB bindin~, activity following TNF stimulation. Specificity of DNA
binding i~i confirmed by the use of exces~ cold wild-type (specific competitor) or a non-
specific competitor (AP-l ~ probe as controls (lanes 3 and 4).
.xample 1~: BCI~-2 ex~ression in PAFC inhibits IlcRa degradation followin~ TNF
treatment
Cytoplasmic extracts are p~cpa~d prior to, as well as ten minutes or two hours
following, TNF treatment of rAd.Bcl-2 - or rAd.~-gal - infected PAEC. Protein
concentration of the cytoplasmic extracts is qn~ntit~ed by the Bradford method.
IlcBa expression i~ evaluated by Western blot. IlcBa is de~ected using anti-MAD-3 rabbit
polyclon~l IgG anti-serum (Santa-Cruz Biotechnology, Santa Cruz, CA, USA) and
peroxidase-conjugated goat anti-rabbit seconda~y antibody followed by enh~nced
chemiluminescence (ECL) detection (Amersham Corp.).
-
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Results show that BCL-2 expression in PAEC inhibits the usual IlcBa degradation
that occurs lO minutes following TNF sSim~ tion. Results shown are ~cp~tsentative of
3 independent experiments (FIG. 9).
~xamp~e 13: RC~,-2 ex~ression in the F.C does not affect bindir~p of the
tranc-~ri~tion factor, cA~P repor~c;ve e~ ent ~C12F)
To deterrnine whether BCL-2 expression affects nuclear binding to a CRE probe,
nuclear extracts are p,~par.,d from rAd.Bcl-2- or rAd.,~-gal - infected PAEC before, and
two hour~ following, treatment with TNF (lOOU/ml~ and assayed by EMSA
(electrophoretic mobility shift assay) for their binding activity of a radio-labeled
CRE oligonucleotide. No difference is observed between the Bc1-2- and the ~-gal -
infected cells (F~G. ~0).
.xample 14: Function of the Bcl ~ene A1 in er~othelial cells
a) Al expression in EC inhibits TN~- and LPS-induced activation through inhibition of
~F-~B:
HUVEC, when stimulated with TNF, express Al. The maximum induction at the
mRNA le~el occurs at approxirnately three hours following TNF stimulation. Expression
of Al in the EC inhibits activation following TNF and LPS treatment; this inhibitory
effect relates eo inhibition of NF-lcB activation. BAEC are co-transfected with an
expression pl~smid encoding for Al and reporter constructs comprising the promoter
region of E-selec~in linked to the luciferase gene and a r~;~o.l~, solely depen~l~nt upon
NF-KB for its induction (FIG. 11).
b) FYpressjon of Al is dependent on l~lF-~cB:
To ev~luate whether functional NF-KB activity is needed for the induction of Al,it is investigated whether Al continues to be inducible following TNF stimulation in
HUVEC even in the presence of an overexpressed inhibitor of NF-lcB ~i.e. IlcBa or
A20). ~UVEC are infected with the rAd.llcBa, rAd.A20 or the control rAd.,B-gal at an
MOl of lOO. No~hern blot reveals high levels of IlcBa and of A20 mRNA in thc cells.
Forty-eight hours following infection, EC are stimulated with IOOU of TNF for
three hours. RNA is extracted. ~xpression of Al is analyzed by Northern blot analysis.
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Results demonstrate that expression of IlcBa or of A20 inhibits the induction of- - Al 1nessell~er RNA as seen in the control rAd.~-gal-infected cells. Similarly, induction
~ of IlcBa (another NF-B dependent gene) is inhibited in the A20-expressing cells as
col,lpar~,d to controls, further confirrning the ability of A20 to block up-regulation of
NF-lcB dependent genes (FIG. 12).
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SEQUENCE LISTING
(1) GF:NFRA~ IN~ORMATION:
(i) APPLICANT:
(A) NAM~: Novartis AG
(B) STREET: Schwarzwaldallee 215
(C) CITY: Basle
(E) COUNTRY: Switzerland
(~) POSTAL CODE (ZIP): CH~058
(G) TELEPHONF: 61-324 5269
(H) TELEFAX: 61-3~ 7366
(ii) TITLE OF INVENTION: ANTI-APOPTOTIC GENE THERAPY FOR
TRANSPLANTATION AND INFLAMMATORY
CONDITIONS
(iii) NUMBER OF SEQUENCES: 5
~i~ ) COMPUTER READABLE FORM:
A ) .~1EDIUM TYPE: Floppy dis~;
(B~ CO~1PUTER: IBM PC co~"p~tible
C~ OPERATING SYSTEM: PC-DOS/MS-DOS
(Dl SOFTWARE: P;ltentln Rele~e #1.0, Version #1.25 (EPO)
(~ ) CURRENT APPLICATION DATA:
APPLICATIO,N l~'UMBER: WO PCT/EP97/....
(~i) PRIOR APPLICATION DATA:
(A) APPLICATION NUMBER: US 08/601515
(B) FILING DATE: 14-FEB-1996
(~i) PRIOR APPLICATIOI~' DATA:
(A) APPLICATION NUMBER: US 08/634995
(B) FILING DATE: 19-APR-1996
CA 02245503 l998-08-l3
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(2) INFORMATION FOR SF.Q 10 NO. 1:
(i) S3~QUENCE CHARACTERISTIC:S:
(A) LENGTH: 790 amino acids
(B)'rYPE: amino acid
(C) STRANDEDNESS: single
(D3 TOPOLOGY: linear
(ii) MOLE~CULE TYPE: protein
(iii) HYPOTHET~CAL: NO
(iii) ANTI-SENSE: NO
(v) FRAGMENT TYPE: internal
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 1:
~et Ala Glu Gln Val Leu Pro Gln Ala Leu Tyr Leu Ser Asn ~let Ar~
Lys Ala Val Lys Ile Arg Glu Arg Thr Pro Glu Asp Ile Phe Lys Pro
Thr Asn Gly Ile Ile His His Phe Lys Thr Met His Ar~ Tyr Thr Leu
~lu Me~ Phe Arg Thr Cys Gln Phe Cys Pro Gln Phe Arg Glu Ile Ile
His Lys Ala Leu Ile Asp Ary Asn Ile Gln Ala Thr Leu Glu Ser Gln
~ys Lys Leu Asn Trp Cys Arg Glu Val Arg Lys Leu Val Ala Leu Lys
~hr Asn Gly Asp Gly Asn Cys Leu Met His Ala Thr Ser Gln Tyr Met
100 105 llO
~rp Gly Val Gln Asp Thr Asp Leu Val Leu Arg Lys Ala Leu Phe Ser
115 120 125
~hr Leu Lys Glu Thr Asp Thr Arg Asn Phe Lys Phe Arg Trp Gln Leu
130 135 140
Glu Ser Leu Lys Ser Gln Glu Phe Val Glu Thr Gly Leu Cys Tyr Asp
145 150 155 160
~hr Arg Asn Trp Asn Asp Glu Trp Asp Asn Leu Ile Lys Met Ala Ser
165 170 175
~hr Asp Thr Pro Met Ala Arg Ser Gly Leu Gln Tyr Asn Ser Leu Glu
180 185 190
~lu Ile His Ile Phe Val Leu Cys Asn Ile Leu Arg Arg Pro Ile Ile
195 200 205
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Val Ile Ser Asp Lys Met Leu Arg Ser Leu Glu Ser Gly Ser Asn Phe
21~ 215 220
Ala Pro Leu Lys Val Gly Gly Ile Tyr Leu Pro Leu His Trp Pro Ala
225 23Q 235 240
~ln Glu Cys Tyr Arg Tyr Pro Ile Val Leu Gly Tyr Asp Ser His His
24~ 250 255
~he Val Pro Leu Val Thr Leu Lys Asp Ser Gly Pro Glu I le Arg Ala
260 265 270
Val Pro Leu Val Asn Arg Asp Arg Gly Arg Phe Glu Asp Leu Lys Val
275 280 285
His Phe Leu Thr Asp Pro Glu Asn Glu Met Lys Glu Lys Leu Leu Lys
290 295 300
Glu Tyr Leu Met Val Ile Glu Ile Pro Val Gln &ly Trp Asp His Gly
305 310 315 320
~hr Thr His Leu Ile Asn Ala Ala Lys Leu Asp Glu Ala Asn Leu Pro
325 330 335
~ys Glu Ile Asn Leu Val Asp Asp Tyr Phe Glu Leu Val Gln His Glu
340 345 350
Tyr Lys Lys Trp Gln Glu Asn Ser Glu Gln Gly Arg Arg Glu Gly HiS
355 360 365
Ala Gln Asn Pro Met Glu Pro Ser Val Pro Gln ~eu Ser Leu Met Asp
370 375 380
Val Lys Cys Glu Thr Pro Asn Cys Pro Phe Phe Met Ser Val Asn Thr
385 390 395 400
~ln Pro Leu Cys His Glu Cys Ser Glu Arg Arg Gln Lys Asn Gln Asn
405 410 415
~.ys Leu Pro Lys Leu Asn Ser Lys Pro Gly Pro Glu Gly Leu Pro Gly
420 425 430
Met Ala Leu Gly Ala Ser Arg Gly Glu Ala Tyr Glu Pro Leu Ala Trp
435 440 445
Asn Pro Glu Glu Ser Thr Gly Gly Pro His Ser Ala Pro Pro Thr Ala
450 455 460
Pro Ser Pro Phe Leu Phe Ser Glu Thr Thr Ala Met Lys Cys Arg Ser
465 470 475 480
~ro Gly Cys Pro Phe Thr Leu Asn Val Gln His Asn Gly Phe Cys Glu
485 490 49~
~rg Cys His Asn Ala Arg Gln Leu His Ala Ser His Ala Pro Asp His
500 505 510
~hr Arg His Leu Asp Pro Gly Lys Cys Gln Ala Cys Leu Gln Asp Val
515 520 525
CA 02245503 1998-08-13
W097/~W83 PCT~P97/00676
-39-
Thr Arç~ Thr Phe Asn Gly Ile Cys Ser Thr Cys Phe Lys Arg Thr Thr
530 535 540
Ala Glu Ala Ser Ser Ser Leu Ser Thr Ser Leu Pro Pro Ser Cys Hi!;
545 55~ 555 560
~ln Arg Ser Lys Ser Asp Pro Ser Arg Leu Val Arg Ser Pro Ser Pro
565 570 575
~is Ser Cys His Arg Ala Gly Asn Asp Ala Pro Ala Gly CyS Leu Ser
580 585 590
Gln Ala Ala Arg Thr Pro Gly Asp Arg Thr Gly Thr Ser Lys Cys Arg
595 600 605
Lys Ala Gly Cys Val Tyr Phe Gly Thr Pro Glu Asn Lys Gly Phe Cys
610 615 620
Thr Leu Cys Phe Ile Glu Tyr Arg Glu Asrl Lys His Phe Ala Ala Ala
625 630 635 640
~er Gly Lys Val Ser Pro Thr Ala Ser Arg Phe Gln Asn Thr Ile Pro
6g5 650 655
~ys Leu Gly Arg Glu Cys Gly Thr Leu Gly Ser Thr Met Phe Glu Gly
660 665 670
Tyr Cys Gln Lys Cys Phe Ile Glu Ala Gln Asn Gln Arg Phe His Glu
675 680 685
Ala Lys Arg Thr G}u Glu Gln Leu Arg Ser Ser Gln Arg Arg Asp Val
690 695 700
Pro Arg Thr Thr Gln Ser Thr Ser Arg Pro Lys Cys Ala Arg Ala Ser
705 710 715 720
~ys Lys Asn I le Leu Ala Cys Arg Ser Glu Glu Leu Cys Met Glu Cys
725 730 735
~ln His Pro Asn Gln Arg Met Gly Pro Gly Ala His Arg Gly Glu Pro
740 745 750
Ala Pro Glu Asp Pro Pro Lys Gln Arg Cys Arg Ala Pro Ala Cys Asp
755 760 765
His Phe Gly Asn Ala Lys Cys Asn Gly Tyr Cys Asn Glu Cys Phe Gln
770 775 780
Phe Lys Gln Met Tyr Gly
785 790
CA 02245503 1998-08-13
WO g7/30083 ~ 97/00676
-40--
O O O O O O O O O
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t~--~ o ~ 3
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CA 02245503 l998-08-l3
WO 97/30083 PCT/EP97/00676
--41--
O O O O O O O O O O O O O O O O O O O
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r ~ ~ o o ~1 ~ r~ f~ ~ ~ m In ~ ~D
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t, _~ r ¢ t ~, ~C~ 'r ~ r ¢
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r3 ~ ~ r ~ ~ ¢ E~ r~ ~ r
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V ~ ~ U r¢ Jt~ ~U E- r5 ~ J ~¢ V ~ ;,~ rr r~
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Et ~ J V ~ r ,~t U ~¢ ~ V ~ V J I
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CA 02245503 1998-08-13
WO 97/30083 PCT/EP97J00676
--42--
OOOOOOOOOOOOOOr~OOOO
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V ~ V V ~ J l¢ ~ r
~¢ ) ~~ ~ I¢ J C~ V l¢ V '~~
J _) ~ J U J l¢ ~ r U ~ C~ ' J ~ ~ ~ V
J ~J ~) J ~ U C;~ ¢ ~ ct
J ¢ r ~ ) U ~
V O ~ U I t ) ~ ~ ~ I¢ ~¢ V J ;J C~ V
J '~) '~l ~¢ ~ V l¢ J V E~ ~ V '~ V '~ V
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J ) ~ ,r 7 ~¢ CJ ~ ~) J ~ ) I¢ ~)
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VE-~ V ~ ~¢ ~¢ O lct ~J J ) )Et t ~ t t.. ~_
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Vf )' ~ ~ t ~ ~ t r~ t ' ~j t -r~ ~ t _l ~ l~t
V -I t ~ J V ) C~ r~ ~) V ~ t ~ V ~¢ (J ~5
I¢~.)~ ~C~l C~) E-l J~6 ¢ f:~ t E- ~ t c,r t~
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r~ a' J r5 J ;~ tc~ J t cr, _l
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CA 02245503 1998-08-13
WO 97/30083 1 ~ 1 57/00676
-43-
,, ~
O O O O O o o O o o o o o O o o O o o
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3 o V V V r_ 1¢ E~ V ~ ,¢ ;.) V ~ E~
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r3 ~ ~3 ~3 ~ ~ rr ~ ~ ~ ~6 '~ E~
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r~ r~ ~¢ r ~¢ ~ J ~ rJ ~ r~ r~
CA 02245503 1998-08-13
W~ g71~0083 P~ , //00676
--44--
-
O O O O O O O O
O o ~ ~ ~ r~ ~ ~P
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CA 02245503 l998-08-l3
.
W097/~0083 l~l/hl97/00676
- 45-
(2) INF0121~tIATlON FOR S~O ID NO. 3:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 205 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(iii) HYPOTHETICAL: NO
(iii) ANTI-SENSE: NO
(v) FRAGMENT TYPE: internal
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 3:
Met Ala His Ala Gly Arg Thr Gly Tyr Asp Asn Ar~ Glu Ile Val Met
1 5 10 15
Lys Tyr Ile His Tyr Lys Leu Ser Gln Arg Gly Tyr Glu Trp Asp Ala
Gly Asp Val Gly Ala Ala Pro Pro Gly Ala Ala Pro Ala Pro Gly Ile
Phe Ser Ser Gln Pro Gly His Thr Pro His Pro Ala Ala Ser Arg Asp
5t:~ 55 60
Pro Val Ala Arg Thr Ser Pro Leu Gln Thr Pro Ala Ala Pro Gly Ala
Ala Ala Gly Pro Ala Leu Ser Pro Val Pro Pro Val Val His Leu Ala
Leu Arg Gln Ala Gly Asp Asp Phe Ser Arg Arg Tyr Arg Gly Asp Phe
100 105 110
Ala Glu Met Ser Ser Gln Leu His Leu Thr Pro Phe Thr Ala Arg Gly
115 120 125
Arg Phe Ala Thr Val Val Glu Glu Leu Phe Arg Asp Gly Val Asn Trp
130 135 140
Gly Arg Ile Val Ala Phe Phe Glu Phe Gly Gly Val Met Cys Val Glu
145 150 155 160
Ser Val Asn Arg Glu Met Ser Pro Leu Val Asp Asn Ile Ala Leu Trp
165 170 175
Met Thr Glu Tyr Leu Asn Arg His Leu His Thr Trp Ile Gln Asp Asn
180 185 190
~ Gly Gly Trp Val Gly Ala Ser Gly Asp Val Ser Leu Gly
195 200 205
CA 02245503 l998-08-l3
W097/30083 PCT~Pg7/00676
46-
~2) INFORMATION FOR S~O ID NO. 4:
.
(i) SEQUENCE CHARACTERISTICS:
~A) LENGTH: 233 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
~iii) HYPOTHETICAL: NO
(iii) ANTI-SENSE: NO
(v) FRAGMENT TYPE: intemal
(xi) SEQUE~NCE DESCRIPTION: SEQ ID NO: 4:
Met Ser Gln Ser Asn Arg Glu Leu Val Val Asp Phe Leu Ser Tyr Lys
1 5 10 15
Leu Ser Gln Lys Gly Tyr Ser Trp Ser Gln Phe Ser Asp Val Glu Glu
Asn Arg Thr Glu Ala Pro Glu Gly Thr Glu Ser Glu Met Glu Thr Pro
Ser Ala Ile Asn Gly Asn Pro Ser Trp His Leu Ala Asp Ser Pro Ala
Val Asn Gly Ala Thr Gly His Ser Ser Ser Leu Asp Ala Arg Glu Val
Ile Pro Met Ala Ala Val Lys Gln Ala Leu Arg Glu Ala G}y Asp Glu
Phe Glu Leu Arg Tyr Arg Arg Ala Phe Ser Asp Leu Thr Ser Gln Leu
lO0 105 110
His Ile Thr Pro Gly Thr Ala Tyr Gln Ser Phe Glu Gln Val Val Asn
115 120 125
Glu Leu Phe Arg Asp Gly Val Asn Trp Gly Arg Ile Val Ala Phe Phe
130 135 140
Ser Phe Gly Gly Ala Leu Cys Val Glu Ser Val Asp Lys Glu Met Gln
145 150 155 160
Val Leu Val Ser Arg Ile Ala Ala Trp Met Ala Thr Tyr Leu Asn Asp
165 170 175
His Leu Glu Pro Trp Ile Gln Glu Asn Gly Gly Trp A~p Thr Phe Val
180 185 190
Glu Leu Tyr Gly Asn Asn Ala Ala Ala Glu Ser Arg Lys Gly Gln Glu
195 200 205
Arg Phe Asn Arg Trp Phe Leu Thr Gly Met Thr Val Ala Gly Val Val
210 215 220
Leu Leu Gly Ser Leu Phe Ser Arg Lys
225 230
CA 02245503 1998-08-13
WO 97/30083 1 ~ 97/00676
~7-
~2) INFO~MATlON FOI~ SF.O ~D NO. ~:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 175 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(iii) HYPO l llk-l lCAL: NO
(iii) ANTI-SENSE: NO
(v) FRAGMFNT TYPE: internal
(xi) SEQUENCE DESCRIPTION: SFQ ID NO: 5:
Met Thr Asp Cys Glu Phe Gly Tyr Ile Tyr Arg Leu Ala Gln Asp Tyr
Leu Gln Cys Val Leu Gln Ile Pro Gln Pro Gly Ser Gly Pro Ser Lys
Thr Ser Arg Val Leu Gln Asn Val Ala Phe Ser Val Gln Lys Glu V~ 1
Glu Lys Asn Leu Lys Ser Cys Leu Asp Asn Val Asn Val Val Ser Val
Asp Thr Ala Arg Thr Leu Phe Asn Gln Val Met Glu Lys Glu Phe Glu
~s~, Gl;~ Ile Ile Asn Trp Gly Arg Ile Val Thr Ile Phe Ala Phe Glu
Gl,~ Ile Leu Ile Lys Lys Leu Leu Arg G}n Gln Ile Ala Pro Asp Val
100 105 110
Asp Thr Tyr Lys Glu Ile Ser Tyr Phe Val Ala Glu Phe Ile Met Asn
115 120 125
Asn Thr Gly Glu Trp Ile Arg Gln Asn Gly Gly Trp Glu Asn Gly Phe
130 135 140
'v~l Lys Lys Phe Glu Pro Lys Ser Gly Trp Met Thr Phe Leu Glu Val
145 150 155 160
Thr Gly Lys Ile Cys Glu Met Leu Ser Leu Leu Lys Gln Tyr Cys
165 170 175
