Note: Descriptions are shown in the official language in which they were submitted.
wo 96~fJ6933 ~ 1 ~ 7 '1 ~ ~
GENETHERAPY FOR TRANSPLANTATION
AND INFLAMMATORY OR THROMBOTIC CONDITIONS
Field of the '
The invention provides i...~.u.. in the field of gene therapy for i .'
and ~ y and thrombotic conditions. In particular, it is concemed with genetic
...... I:'i. ~d.," of endothelial cells to render them less susceptible to an r~ y or
~ ' O ' stimulus. It is aiso concemed with i . ' of genetically modified
tissues or organs and most particularly is directed to a method of, , ' _ genetically
modifed endothelial cells or tissue or organs into a ' recipient of the same or
different species, . .1.' --: vectors for ~f~..,.,l.lNI,: ,g same, and the cells, tissue or
organs, as well as non-human transgenic animais, so modified.
' 'of ' i
The ' ' ' consists of the layer of cells which form the lining of blood vesseis,e.g. arteries, veins and capillaries. While the . k ~ ' (also referred to as "vascular
~ 1. i; ") was once thought to serve merely as a passive conduit for the blood and
blood serum, it is now recognized that the condition of the endotheliai tissue is critical for
~ _ the norrnai anti-s 1,, ' y state of circuiating blood. In particular, the
tl ' ' ' (l~i) protein which is ~ by endothelial cells and is distributed
as a surface oly~, throughout the ~ ' ' ' of arteries, veins and capillaries,
constitutes a ' '. ' anti-coagulant molecule which is pivotal in the
well-: ,.. ~ . ;.. .1 Protein C ~ ;",, pathway. In this pathway, Ih~
functions as a binding receptor of circulating thrombin, removing thrombin from circulation
and thereby precluding it from, among other functions, clotting fibrinogen or activating
platelets. Most ;...~,. '3" once bound by ' . 1 ' ' . the thrombin binds up
circulating, inactive Protein C, which is thus converted to the activated fomm. The action of
activated Protein C on still other co-factors of the coagulant pathway, i.e Protein S,
ultimately assists in an anti-. ~ ' y steady-state.
SUBSTITUTE SHEET ~RULE 26)
Wo 961~6933 ~ i 9 l ~ 2 ~
--2-
In response to injury, h~ or ;~ challenge, such as occurring in
connection with allogeneic or xenogeneic . ' of tissues or organs, as well as
other '' ~ conditions such as various; diseases or sepsis, the
. ' ' ' is changed to a iyaf~ Liunai or "activated" pro~ ' y state. An aspect
of this conversion occurring in the presence of cytokines such as tumor necrosis factor
f~lNF or TNF~), is the ioss or ~ L";~" of Liu~ ' ' ' from the endothelial cell
surface. This t~duction in li -- .1,~,.,..~1..1;.. has been ascribed to iu...-lc~ulalion at the
' level [Yu et al., J. Biol. Chem. 2667 (1992) 23231-23247].
Thus, ~ y or ~, ~ challenge, such as: tmnCp~ on of
tissues or organs, presents a significant threat of ~ ,lvv,la~,.,l~ thrombosis, i.e. u~ g;. ~i
clotting of the blood in the Illi~,lVV~.U~ UI~, of the ' ' ' where i' ' ' ' is
ordinarily most active, and
For the above reasons, in connection with i . ' ' ' synthetic materials such as
prosthetic arteriai grafts, Ito and co-woricer, for example, have used Lhlu~ in protein
as an adherent surface coating (USP 5'126'140). However, this approach has limited
usefulness where live grafts are concemed.
The problem of endothelial celi dJ ~ or "activation" in the organ transplantsetting has been addressed largely by efforts to disable the host ' ~g ' response,
such as with cytotoxic drugs, anti . .- ~ ~,ulLi~,v t~,.v;da, non-speciffc
., such as ~"~,luafJ.,lh~ A, and the like.
Thete exists a criticai need for a method to aileviate LiL ~ o~ y ~uu~ ulyhlg
activation of endotheliai ceils, and in particular, to prolong organ transplant survival, while
minimizing toxicity and other adverse effects associated with iarge do~ses of
SUBSTITUTE SHEET (RULE 26~
wo96106933 2~ 9 jT4~ r~"L~
-3-
of the invention
The invention utilizes gene therapy techniques to alleviate the 1l~ v~ y (i.e.
the tendency to promote blood clotting) of vascular endothelial cells susceptible to an
' '' y or ~ ' stimulus.
Endothelial cells have been modified by rendering them capable of expressing
functional Ihl~ , on a regulable or u~.. DLiluiiv., basis, whereby ~hllJ 1.. ~i.. l;"
expression is maintained even in the presence of endothelial cell activating factors. By
"functional" is meant that the expressed 11ll'. ,I.v,...,~l~ l; protein of said endothelial cell can
bind thrombin in the cellular .,,,vi., (for example, in the presence of blood of a graft
recipient), and that the IIL"~ "I;" thrombin complex thus formed can catalyze the
activation of zymogen (i.e. inactive) protein C to provide activated protein C. By
"regulable" is meant that protein expression, whether increased or decreased. is dependent
on the presence of, or addition of. a given substance. The temm "regulable expression"
includes "inducible expression", whereby gene expression is increased by addition of a
stimulus. By "conslitutive" is meant that protein expression is essentially; ~.1.. 1.. ,. of
endothelial cell activation. Thus, ~ , expression of ~ l; by an
endothelial cell is ' ' "!~ free of down-regulation by TNFa or other activating factors.
Likewise, a constitutive promoter of tll. ~ l; is capable of directing ~ of
the gene even in the presence of serum .Iu.... , ' y factors such as TNF.
Accordingly, the invention concems a method of genetically modifying .. --.,
preferably endothelial cells to render them less susceptible to an r~ y or
' O ' stimulus, preferably by inserting into these cells, or the progenitors of these
cells, DNA encoding functional lI.. ~.. I... I~.'i protein in operative association with a
suitable promoter. whereby ~LIv~u~ is expressed under endothelial cellular
activating conditions.
The invention a}so comprises a method of inhibiting thrombosis in a
subject susceptible to an i r~ y or immune stimulus which comprises inserting into
cells, tissue or an organ of the subject, DNA encoding Ih V ~ '-l in operative
association with a promoter, whereby functional tlll. ' ' " is expressed from these
modified cells in the presence of cellular activating factors.
SUESTITUTE SHEET (RULE 26~
W0 96/06933 r~ L~ 79
~ 1 ~ 7 i~
Preferably the cells or tissue are modifed in vivo, i.e. whiie remaining in the body of
the patient. Altematively, cells or tissue may be extracted from the subject and transferred
to in vitro culture, where tbey are genotically modifed i~y insertion of DNA as described
herein. and then grafted into the subject or a different recipient. The subject is a vertebrate,
in particular a mammal, such as a pig, but may also be a primate, and in particuiar, a
human. Such a tnerapy will be useful to relieve i " ~ conditions or immune
conditions, such as - S~, diseases, in a patient, especially where the patient is a
human.
In a further aspect the invention comprises a method of u ~ ; g donor allogeneicor xenogeneic endothelial cells, or tissue or organs containing endothelial cells to a
mammaiian recipient in whom such cells, tissue or organ are subject to i '' y or
immune activuion, which compnses:
(a) genetically modifying these donor cells or progenitor cells thereof or tissue or organ by
inserting therein DNA encoding tl , 1, ,.1 11 under the control of a promoter
(b) implanting the resultant modified donor cells, tissue or organ into the recipient; and
(c) allowing expression ftom the resultant modifed cells, tissue or organ of r,..
active ~LI. ~ ' '
Steps (a) and (b~ may be carried out in either order; that is, the donor allogeneic or
xenogeneic cells, tissue or organ may be moclified or genetically enginoered ~e.g., by
r~ncF~-inn ' : or the liice) prior to, or '~.~ .ly after,
;.... into the recipient.
The promoter may be eitner cul~ ti~, or reguiable, e.g. inducible.
For example, endothelial cells or tissue recwited from a pig may be geneticaily
modified ~yQ by insertion of DNA encoding human 1l - ..1... - ..1~.1~ - under the control
of a suitable promoter, and the modified cells or tissue, having been rendered "transgenic"
thereby if ,...,.I:r;. ~i,... has occurred in the germ line or alL,....ui~,ly, somatic
if ...n~ ;.... has occurred in the somatic cells, are then grafted into the recipient, e.g. a
human. Once, , ' 1, the modified cells or tissue will express functional human
i' ~ ' ' at the graft site, even in the presence of othcrwise du.. " ' y factorssuch as TNF. Genetic "~ of the donor mammal can be carried out at an
embryonic stage, by well-known tcchniques, so as to produce a transgenic or somatic
animai expressing the desired recipient protein. The donor, e.g. pig endothelial
SUBSTITUTE SHEET lRULE 26)
W0 96/06933 2 1 9 ~ ~ 2 1 r ~ 0~79
cells or tissue can also be genetically modified by ex vivo means, whereby cells. tissues or
organs extracted from the donor and maintained in culLure are genetically modified as
described above, and then i , ' ' to the recipient. where the graft can then express the
desircd functional recipient il.l.J..~ substantially free of down-regulation in the
presence of endothelial cell activating factors.
It is preferable that the genetic "... I:r. ~...,. be done in vivo rather than in vitro (i.e.
x vivo). The preferred in vivo method is by raising up of a transgenic mammal, e.g. a pig,
to be used as a source of donor cells, tissues and organs expressing the desired functional
ptotein.
Particularly suitable promoters and vector constructs have been identifled with which
to modify endothelial cells or tissues, and in particular porcine endothelial cells [e.g. porcine
aottic endothelial cells (PAEC) or porcine .. uv ' endothelial cells in general], so as
to render ihem cul~ ilLd~. or regulable expressers of functiona Ml.. ,.,.l.. l.. l;
In this regard, in one c ~I,o~ , a construct is described hereafter which comprises
the IL., L ' ' DNA in operative association with the Herpes simplex thymidine
kinase (tk) promoter.
A retroviral construct is further utilized which comprises-
(a) a 5'-long terminai repeat (LTR) of a retrovirus such as Moloney murine leukemia virus
(Mo-Mu-LV));
(b) a retroviral packaging signal such as ~, du.. from the LTR;
(c) DNA encoding L;..~ L ' " in operative association with the llerpes simplex
thymidine kinase promoter du.. from the LTR; and
(d) a 3'-long terminal repeat.
The vector may also comprise at least a portion of the retroviral gag coding region
(e.g. about 400 nucleotides thereofl, generally du.. from the packaging signal
sequence, i.e. normally ~. The 3'-LTR also preferably includes a pol~lu..,~l~iu.. sequence.
Preferably, a selectable marker such as the neo gene, conferring resistance to neomycin or
an analog thereof, may be placed under the control of the S'-LTR, generally du... of
the packaging signal sequence. An example of such a plasmid is pNTK-2.TM.CWI,
illustrated in FIG. 3.
SUBSTITUTE SHEET (RULE 26)
wo g~/06933 2 1~ 7 ~ ~ 7 -6- r~ 79
Adenoviral constructs may also be utilized, and are prepared e.g. as descnbed
hereunder. In particular, perfusion of diseased vessels with an adenovirai construct
encoding TM can be effected to introduce the TM into the ~ in the diseased
vessels.
According to a further aspect of the invention, there is provided graftable mammalian
endotheliai cells, tissue or organs comprising DNA encoding Ll.~ -o~ b~ under the
control of a promoter, especiaily graftable endotheliai cells, tissue or organs of a donor
species, the cells, tissue or organs being modified to express tl..u,.li,. ' ' of
a graft recipien~ species which is the same or a different species as the donor. Expression
may be constitutive or regulable.
A further aspect of the invention provides for a non-human transgenic mammal
comprising DNA encoding IL,~ ;.. of a different species, particularly haYing
. ' ' ' endotheliai cells or tissue modified as described above, and a method ofpreparing a non-human transgenic mammal comprising DNA encoding tl.., ' ' ' of adiffexnt species. An example of such a mamrnal is a transgenic mouse expressing human
TM from its endotheliai (sr other) cells. A further example is a transgenic pig expressing
human TM from its endotheliai (or other) cells.
In an illustrative . ~ -- - ~.1 the invcntion comprises a method of 1~ g into
a human, an organ comprising porcine endotheliai cells genetically modi~led to express
functional human i' ' ' "
SUBSTITUTE SHEET (RULE 26)
w096l06933 219 ~ 4 27 r~ /Y
-7-
~ ' ofthe~' '
FIG.l Restriction map of plasmid pNTK-2.
Fl&. 2a Schematic view of plasmid pUC19.TM15 encoding human TM.
FIG. 2b Schematic view of the insert of plasmid pUC19.TM.CWI
(in FIGS. 2a and 2b. the bold lines indicate plasmid backbone sequences).
FIG.3 Schematic view of the expression vector pNTK-2.TM.CWI
FIG.4JI FACscan analysis of the NTK.TM.CWI producer cell pool.
FIG.4b Bar graph depicting relative expression levels of TM clones (arbitrary units) from
limiting dilution of the NTK-2.TM.CWI producer pool.
FIG.5 Northern analysis of NTK.TM.CWI transduced cells.
FIG.6 Nuclease Sl analysis of ~Ll~ expression.
FIG.7 Illustrated principle of the ~.Ll~,llloO_.l;" assay for activated Protein C generation.
FIG.8 Rate of S-2366 clesvage at 37~C:
dotted squares: ~ ' ' PAEC
lozenges: NTK-2.TM.CWI trsnsduced and selected PAEC
dark squares: EAhy926 cell line.
FIG.9 Bar graph illustrating activity of tl..~ ~1 ' ' as reflected by maximal rate of
activated Protein C (APC) production, following treatment of cells with TNF.
FIG.10 TM.CWI comprising the native human 1~ cDNA sequence
(SEQ ID No.l) and NTK-2.TM.CWI restriction analysis.
SUBSTITUTE SHEET (RULE 26)
W096~06933 2 19 7~ ? ~ ;r.~ 79
--8--
r-~ .
"Graft", "transplant" or "implant" are used ' _ "~ to refer to biological
material derived from a donor for: , ' into a recipient, and to thc act of
placing such biolog;cal material in the recipient.
"Host" or rrecipient" refers to the body of the patient in whom donor biological material is grafted.
"Transgenic" refers to animals bearing foreign genes, ;,~ r ~. ~Iy of whether the gene is
in the ger n line or in the somatic cells.
"Allogeneic" (and "allograft") refer to the donor and recipient being of the same
species. As a subset thereof, "syngeneic" refers to the condition wherein donor and
recipient are geneticnlly identical. "Autologous" refers to donor and recipicnt being
the sarne individual. "Xenogeneic" ~and "xenograftn) refer to the condition where the
graft donor and recipient are of diffetent species
"Mo-Mu-LTR": Moloney Murine Leukemia Virus long term;nal repeat DNA sequence.
"polyA": DNA sequence encoding a l~ul,y~.~.~l~iu~ signal.
"neo": the gene encoding neomycin 1 . , which confers resistance to
neomycin or neomycin analogs such as G418, which are toxic m most
eukaryotic and other host cells.
"promoter" a DNA sequence that directs i , of DNA into RNA.
"tk" Herpes Simplex thymidine kinase.
"PAEC" porcine aortic endothelial cells.
NHUVEC" human umbilical vein endothelial cells.~EAhy926 HUVEC": : .u, ~ human umbilical endothelial cell line expressing
human TM~TNF': tumor neCrQsis factor.~Tl.... '., ' " " ~TM) refers to the natural lL. L ~ ' " gene (including
the cDNA thereof~ or protein, and include derivatives thereof having
variations in DNA (or amino acid) sequence (such as silent mutations or
deletions) which do not prejudice the ~ ' '.y of natural ~.- ' ' '
(USP 5'273'962 and USP 5'256'770~.
SUBSTITUTE SHEET (RULE 26)
W096~06933 2~17~ r~ . cr~l79
g
of the jnventjon
The gene encoding native li"~ has been isolated and sequenccd from
several species, both in its generic fomn and as cDNA. The complete cDNA sequence for
human ~ u ' '', derived from human umbilicai vein endothelial cells, comprising an
open reading frame of 1'725 base pairs~ encoding a 60.3 kDa (M, = 60'328) protein of
575 amino acids, is disciosed by Majerus, USP 4'912'207, and is aiso depicted for
nucleotide positions 170 to 1'894 in FIG. 10 (SEQ ID No.1) hereof.
The invention broadly comprises rendering endothelial cells or tissue or organs less
susceptible to activation or i~ ' in response to an immune challenge, by
,.. nr. 1;l.. of these endothelial cells, tissue or organs by insertion therein of DNA
encoding Li"....l~ ;.. in operative association with an appropriate promoter and
expressing functional Lhlu.~ ' " from these modified cells at effective leveis.
The promoter may be ' 'vc or regulable, e.g. operate in a~ , 'y inducible
manner. In one ~ the endothelial cells of the invention can express the TM
protein uu~ iluL~.,ly, i.e m '~, thus the TM coding sequence is operably linked to
a promoter sequence expressing the protein ~.ly in said cell. Altematively, the
endothelial cells can express L..~ on a regulable, e.g. inducible basis, i.e. the
TM coding sequence is operably linked to an inducibie promoter, such that the protein can
be expressed ' '~ before or following endothelial cell activation, or on demand in
response to a I ' ' extemal stimulus. In a further ~ -~u~ the TM cDNA can
be operably linked to an endothelial cell specifc promoter. All the above promotcrs are
preferably other than the promoter which is native to the TM protein encoding DNA
sequence.
Thus the invention in one aspect addresses the i,~fb.~,Lullal or activation response of
vascular endotheliai cells or tissue to an ' ~ y or immune stimulus in a patient
following such ,,..~.I;r.. -~;.... It pemlits inhibition of thrombosis in a patient in need of such
therapy following genetic .. I.r.. ~;.. " of donor endothelial cells or tissue in vivo or in vitro
by inserting therein DNA encoding i' ' ' ' protein under the control of an
'l~p ~r ' , e.g. regulable or ~,u..~liluLi~, promoter, grafting of these donor cells or tissue
into a recipient, and thereby ailowing expression of functionai i' 1 -- - 1 1; from these
donor cells or tissue.
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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 appropriate endothelial cells. tissue or
organs for n ,~ or grafting. In a preferred ....l.o~ ,I human TM proiein isexpressed from cclls of a different m~ 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 ~ nn~ species, e.g. bovine, may be suitable. For example,
porcine endothelial cells, or the progcnitor cells thereof, can be recruited from porcine
subjects, genetically modified, and implanted into either the autologous donor or into
another ' e.g. human subject.
The donor cells or tissue may bc transgenic in the sense that they contain and express
DNA encoding the ~L~ I;.. protein of a graft recipient species or of a different
species in whom they are implanted. Such transgenic cells or tissue may continue to
express ll.l.. l.. . h ~ indefiniteiy for the life of the cell. tissue or organ.
of endothelial cells according to the invention can be by any of various
means known to the sicilled worker. In vivo direct injection of cells or tissue with DNA
can be carried out, for example. Methods of producing transgenic animals are becoming
more ~;~;.~,l.~l, appropriatc methods of inserting foreign cells or DNA into animai tissue
include ; embryonic stem OE~ cell , ~ ~ n~ ,.li...l cell gun,
il r I; ~ retrovirai infection. etc.
Retroviral vectors, and in patticular, rcplication-defective retroviral vectors lacking one
or more of the gag, pol, and env sequences required for retroviral replication, ~re
well-known to the art and may be nscd to transfomm endothelial cells. PA317 or other
producer cell iines producing helper-free viral vectors arc .. " ' ' ~ in the literature. e.g.
in Miller and Buttimore, ~ ll Binl (1986), 2895-2902; USP 5'219'740;
USP 4'861'719; USP 5'124'263; and USP 4'650'764.
A ~ -~.llaLi.. retroviral construct suitable for purpores of the invention comprises
at least one viral long terminal repeat and promoter sequence upstream of the nucleotide
sequence of the therapeutic substance, and at least one viral long terrninal repeat and
!)ul~ld~,,,yl,lLiOIl signal do....s~ l of the therapeutic sequence. It can be, for example, the
Moloney Murine Leukemia Virus (Mo-Mu-LV)-derived N2 pro-viral vector in which the
gene of interest is flanked by a 5'- and a 3'- long terminal ropeats (LTR's~ and a packaging
SUBSTITUTE SHEET ~RULE 26)
w09610~933 2 1 ~ 7
-Il-
signal sequence l:hJ....sti.~.,.. of the 5'-LTR. N2 also contains ~ '.y 400 base pairs
of the gag coding region of Mo-Mu-MLV. Additionally, a large ponion of the
Mo-Mu-MLV coding sequence has been deleted in N2 and replaced with the bacterialnev,,.~, rosistance 8ene (neo) [Eglitis et al., Science 230 (1985) 1395-13981.
Porcine endothelial cells, such as porcine aonic endothelial cells, have been difficult
to manipulate by typical genetic ~..~;;..~.;,.~, techniques. Various vector constructS for
cu~it;~ . expression of Ih,.. ~ in ' cells are given below in Table 1.
It has , ' ~'~, been found that a particular retroviral construct provides superior viral
titre as well as protein expression of the transduced tl..u ~ The exemplifed
pNTK-Z vector has been found superior to e.g. the other retro-iral contructs indicated in
Table I below for deiivering high levels of viral titre and Llllu ~ ; expression:
Table 1:
Vectorneo resistnnce gene driven by: cDNA
driven by
LXSN, SV 40 5' LTR ~.u...vt~
pBC140 5LTR CMV
~.u................................................ .................. ..ut.,,/, '
pCMVLTRtkneo5 Thymidine 5' LTR
kinase promoter
pNTK-2 5' LTR Thymidine
icinase promoter
CMV = ~ tVll..~5~1iU~;lh..
LXSN = Long terminal repeat - ~ ~insert) - SV40 promoter - ~leomycin, , -
gene
SV = simian virus
As a rcsult of an apparent synergy between the promoter-containing 5'-long terminai
repeat sequence of the present pNTK-2 vector and its Herpes simplex tk promoter sequence,
attractive 11.. ~ .h.,.. i.. l; expression levels are obtainable from ~ 1 endotheliai
cells.
Vectors derived from rdl.l~...VV;lh,~.D, i.e. viruses causing upper respiratory tract disease
and also present in latent infections in primates, are aiso generally known and may be used.
The ability of ~L..~ to attach to cells at low ambient i , is an advantage in
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the transplrnt setting which can facilitate gene transfer during cold tnca~ iull. Adenoviral
vector constructs can be prepared in various ways to effect expression of ~L~
protein. In such vectors, the TM gene may be under the control of onc or more different
promoters. One such promoter is the known CMV p-ul--utl,~
An adenoviral vector for the delivery of TM into endotheliai ceils may e.g. be
constructed as follows:
(a) A TM transfer vector can comprisc the TM gene (e.g. construct TM.CWI ) du~ ou~
and under control of the CMV ~IU.~IUtU~ ' with polyA sequences du.. ~oUc. llll of
the gene. Flanking this construct are sdenovirai sequences necessary to promote
with a suitable vector. For example, such sequences may comprise
about 400 bp of the adenoviral sequence upstream and about 3 kb of the adenoviral
sequence du..l.o.l~ln which is numbered ~,~ul"v ~y 3û00-6000 relative to the
upstream 400 bp sequence.
(b) An adenoviral vector construct comprising the aderioviral genome with an inseri in the
El coding region, whereby the insert prevents the plasmid from giving rise to
(c) 293 cells are a continuous line deriving from human fetal kidney, containing and
expressing proteins of the adenoviral El region (i.e. in trans) necessary for replication
and related functions in an adenovoral vector which iacks this region. For example,
293 cells can contain and express the adenovirai Ela and Elb proteins needed forreplication-related functions.
The ~ ,lh ~ ll of YeCtOr (a) uid vector (b~ by cGtransfecsiûn into 293 cells results
in formation of adenoviral particies cont~ining tbe DNA encoding TM, these particles being
preferably lacking in one or more sequences of the adenoviral region, and thus being
preferably neplication defective. Co-n - r_, 1;.." is done while the cells are overlayed with
medium containing I % agarose so that the pure ' virus or virai vectors are
obtained directly as visible Cl~i~ ' . in the monolayer upon Iysing of virus
producing cells. The ' virus may be ~ ~ and expanded physically by,
for example, PCR and Southern blot and by expression using e.g. huTM monoclonal
antibody prepared by known procedures. The resulting replication-defective ~ .u
virus as obtained, for example, by freeze-thaw Iysis of infected 293 cells, is particulariy
effccdve for constitutive production of TM protein, in particular in endothelial cells.
SUBSTITUTE SHET ~RULE 25~
wo s6l06933 2~ r~ 7g
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An adenoviral vector contruct for use in the invention comprises an expression
cassette comprising the Li..~ DNA in operative association with the cmv
promoter, and a poly A sequence iu.. ' therefrom. The expression cassette is
introduced into the adenovirus genome by ~u ~ of two plasmids in human
embryonic kidney cells (293 cell line) which prove the presence of El genes in trans and
thus pemmit replication of an adenovirus vector which lacks the El region.
An altemative approach to targeted gene delivery is through the fommation of a
DNA-protein complex. This type of gene transfer substrate is constructed by conjngaling
the ~ I '; gene to a poly~ JLi ic ligand for an acceptor on an endothelial cell.
Cells or cell r-r ' " can be treated in accordance with the present invention
in vivo or in vitro. For example, for purposes of in vivo treatments, LI..~J ~
vectors of the present invention can be inserted by direct infection of cells, tissues or organs
in situ. Thus the vessels of an organ such as a kidney can be t~,...!,ul~iiy clamped off
in Yivo from the blood circulation and the blood vessels perfused with a soiution comprising
a i " ' vector construct containing the subject Lhu ~1~ ' ' gene, for a time
suffficient for the gene to be inserted into cells of the organ; and, on removal of ciamps,
blood fow can then be restored to the organ. In anotber ~- ~.o~ . cellular ~ ..1;l;. ~,i....
can be carried out ex vivo in cultured cells. Cell ~ can be removed from the
donor or patient, geneically modifed by insertion of vector DNA, and then implanted into
the patient or another recipient. Additionaily, an organ can be removed from a donor,
subjected ex vivo to tbe perfusion step described above, and the organ can be re-grafted into
the donor or implanted in a different recipient who is of the same or a different species.
Geneticaily modified endotheliai cells may be introduced e.g. in reseeded denuded
vessels or prostheses at the site of a transplant. Tissue or organs comprised thereof may
also be removed from a donor and grafted into a recipient by well-known surgicalprocedures. Prior to ' . ' the treated endotheliai cells or tissue 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
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~O961~6933 2 ~ 2~ r ~ ~?g
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selectable markers for screening include the neo gene, conferring resistance to neomycin, or
the neomycin anaiog G418.
Although any ' cells can be targeted for insertion of the Liu.. - Iu,..,,,,i,,l;..
gene, endothelial cells are the preferred cells For I , ' The recipient species will be
pnmarily but not exclusively human. Other mammais, such as primates, may be suitable
recipients.
An r~ _y stimuius which leads to activation of endothelial cells by serum
factors such as TNF occurs in a wide variety of ~ I.nl..j3~ conditions, including injuries,
bums, septic shock,: ' disease, allogeneic or ~ vO~ ;n,~
surgery, etc. Further diseases include those where there is an increase in propensity for
thrombus formation (e.g. 2llh~u:>~h~u~ and thrombotic conditions such as ischemic heart
disease, i~lh..U~,L,~v~;5, multiple sclerosis, intracranial tumors, Ll--v -~ -I-olicm and
i.,~tJ.Ili~,~...;,l, thror~nphl~hi~ic ~ Iul,uLluul.,l,v~is, cerebral thrombosis, coronary thrombosis
and retinai thrombosis), as well as those following parturition or surgicai operationC such as
coronary arLery bypass surgery, angioplasty and prosthetic heart valve b~
Insofar as the preparation of the reagents, starting materiais or cell lines mentiuned is
not speciflcally described herein, they are known and available or they, or equivaients
thereof, may be prepared from known available producrs according to known procedures.
The following Exampie is by way of illustration only and is not intended to be
limihtive in any respect of the invention herein described and claimed. All
are in degrees Centigrade.
SUBSTITUTE SHEET (RULE 26)
wo 96/06g33 2 ~ ~ 7 ~ 2 7 PCI'IEP95~03379
~ -15-
k~
Human lhl ....I....l ~ '; cDNA is obtained from the American Type Culture
Collection (Rockville, MD, USA~ cloned in the EcoRI site of plasmid pUC19 to form
pUC19TM15 (ATCC Accession No. 61348).
Plasmid pNTK-2 is derived from pXTl [Boulter et ai., Nucleic Ari~c Research 15
(17) (1987) 7194, and further described in Fig. 1.
The PA317 cell line (ATCC CRL 9078) contains an ,' ~,uL.;c, replication-defective
retroviral construct lacking in the viral packaging signal but encoding the gag, pol, and env
protein packaging genes.
Pig aortic endothelial cells (PAEC) are purified from aorta obtained e.g. from the
Deaconess Hospital Animal T D~ riPc (Boston, MA, USA~. Passage 4 human umbilicalvein endothelial cells (IIUVECl originate from Children's Hospital (Boston, MA, USA).
The PA317 cells and PAEC are maintained on DMEM media (Gibco) with
10% heat-inactivated fetal calf serum, penicillin and S~ VlllJ.'
l~h~
a. Construction of pNTK-2.TM.CWI
Digestion of plasmid pUC19.TM15 with BstXI and Hindm produces a truncated
cDNA secuence from which the 3': - ' ' region, including IJVIyUCll,llyldlivll sites of
the TK gene and a small segment of the adjacent pUC polylinker, are deleted. The excised
segment is replaced by a synthetic linker of 29 nucleotides plus overhangs encoding
Sail, EcoR1 and BamHI sites, and is ~. h ~ y depicted in FIG. 2b as
pUC.TM.CW 1.
The truncated sequence TM.CWI is cut out Or pUC.TM.CWI at the Sal-l site and
cloned into pNTK-2 as a Sall fragment fused to the Xhol site in pNTK-2 to give
pNTK-2.TM.CWI, crh~-mD-ir~liy depicted in FIG. 3.
b. TrPnc~ rrinn of producer rPllc
Plasmid pNTK-2.TM.CWI is linearized in the plasmid backbone with Scal, and the
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Iinearized plasmid pNTK-2.TM.CWI is transduced by ~ u~y~ into PA 317 cell
culture.
c. Cnntrol i~r~ urrrs
A plasmid is prepared by the methods described under a. above~ except that it islacking the ~"., ~ gene. This plasmid is also transduced by elc.,LIuy~ ion into
PA317 cells to produce "empty" provirus, i.e. viral titres containing the neo gene but not the
b. ' '' pne.
d, Selection and analysis of producer cl ,
Neomycin selection of the transfected PA317 cells is carried out for at least 14 days
in at Icast 600 ilglml G4]8 ~Gibco Grand Island NY Cat. No. 11811-031). The resulting
NTK-2.TM.CWI producer pool is sub-cloned by limiting dilution to yield 29 sub-clones.
S, from the sub-clones is harvested in the late log, early stationary phase and spun
at 2000 g for 10 minutes to remove producer cells and debris, then purified in a single cycle
of rapid freeze-thawing in liquid nitrogen.
e. FA~' 1 ' '
FACscan anaiysis of the NTK-2.TM.CWI producer pool is carried out using
anthlu, ~ ~ " ' ' antibody llBI and FITC conjugated secondary reagents
according to the procedure described by Hatiow and Lane, Antihntlil c A L '
Manual, Cold Spring Harbor Laboratory Publication. Ch. 10 ~1988). In FIG. 4~, the
unfilled area shows producer cells incubated with ihe secoùdary reagent alone. The filled
area (secondary reagent and 11 Bl ) c~ to the level of r' .. i~ per cell.
increasing left to right. The NTK-2.TM.CWI producer pool produces a medium titre virus
~1-2 x 104 infectious unitslml). Ayyl~ 80% of the NTK-2.TM.CWI clones are
found to be TM expressing. ]0-1~% of the .~ubclones produce vinis which confers G4i8
resistance but not NTK-2.TM.CWI expression (FIG. 4bl.
fi J r ' ' of 3T3 rPilc
3T3 fibrobiasts are infected with purified virai supernatant and selected with G418 14
days thereafter. Of the 29 sub-clones, 22 produce viral titres on the 3T3 fibroblasts. By
romp-i~rn. otber retrovira] constructs containing NTK-2.TM.CWI under the control of
SUBSTITUTE SHEET (RULE 263
wo g6fOfig33 ,'~ ~ ~ 7 4 ~ ~ r~ 79
stronger constitutive promoters, such as ~ described in Table 1. produce low viral
tiv~cs. Selection of a single clone, A5, results in an "~ t~ doubling of the
frequency as judged by anti-TM i.,..., -~ v ~ y, from 30-40~o to
60 80%, I~D~ ~IY~
g. J ~ of p~F.~
PAEC in the log phase of growth are seeded at 0.5 - 2 x 109 cells per
30 mm-diameter petri dish and incubated with the purifled A5 supematant in the presence
of S ,ug/ml polybrene surfactant over 24 hours.
h. P.AFC ~ ' with h~t~ri~ nl1ntnYin Pntl h~m~ TNF
Cnnfi-m~rinn that the transduced PAEC retains , ' ' ~y to activation is obtainedby carrying out an ELISA with anti-human TM .".,, 1,,,~ antibody IA4 on PAEC clones
treated with 100 ng/ml of bacterial cell wall l;~ vlyD~uL~lide (LPS) or TNF, known cell
activators. The cells respond to stimulation by up-regulating E-selectin, which is known to
be up-regulated during cellular activation. Stimll~tinn results in some reduction of
J~I ,I; activity, as also measurcd by ELISA with IA4, which appears largely
attributable to ~r ' ' r ' (i.e. the oxidation tendency of m~ thinni~o of
the protein) rather than reduced expression. TM expression and APC production inresponse to stimulus of various cell lines with TNF is shown in FIG. 9 (error bars
+ I standard deviation). The activity of the ~..1,,,. , TM protein expressed by HUVEC
is essentially fully .Iv...... , ' ' by TNF-~, whereas human TM expressed by ~ransduced
PAEC is resistant to .,~Iv~;.. ., - ' ' down .~ liv
j, I ' ' ~mrly
The n - ~ --- efficiency of - T " lly growing PAEC is detemmined 24 hours
after infection by indirect ' , ' staining with antibody IA4. The transducedPAEC are washed with phosphate buffercd saline (PBS), then fixed for 10 minutes with
0.05 % ~jlu~ lJ-,h,~Jc at room t....1. m~ - After blocking in 5 ~o goat serum in PBS, the
cclls are incubated for I hour in 2 % goat serum in PBS at room I ..~ l with
anti-human ih ~ -v- ...n~ ' IA4 diluted 1:350;l .r' ' goat anti-mouse
antibody (Pierce 31802. diluted 1:2000) and DLI~ '' peroxidase (Pierce, diluted I
:2000). The presence of antigen was detected using aminoethyl-carbazole. Good
SUBSTITUTE SHEET (RULE 26)
wo s6l06~33 ~ 1 9 7 ~ C ~Y
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fre4uencies are obtained with a single round of infection using supernatant
hatvested from the A5 clone. In . ' ' PAEC~ . message is
negligable and no protein is detected.
j. Nonh..T~, ~
Total RNA is then isolated from the transduced, pu~ s~,lcctcd PAEC by the
isulLu~ ~ / acid phenol method. FIG. S shows the Notthem blot of RNA
(16 h exposure) from PAEC culture cells probed with the EcoRI fragment of
pUC.TM.CWI; ' ' PAEC; EAhy926; NIH 3T3; and NTK-2.TM.CWI transduced.
positive selected NIH3T3~ The lancs contain 10 ,ug RNA. The i..;~ ' size of
tl,.. L ~ ' mRNA is 3.7 kilobases.
Two major RNA species I f~ (I) a 3-35 kb sequence, uu~c~pundi~lg to the
tk driven transcript; and (2~ an ~~ ly 5.5 kb sequence l~yLd~li~LIc with a Th,I
probe and a probe for G418 resistance, evidencing , ' ~ ld~u~;L, starting with
the LTR driven n-..~. .;~.;;~... and continuing from the neo gene through the tk and TM
sequence, ~UIII ~ to a 5~-LTR to 3~TR transcript.
k. ~31tl~1..,.cf. Sl ~nlllySic
Steady state levels of human T~ u.lh.g mRNA are determined by Sl nuclease
protection analysis. TM15 cloned in plasmid pECS M13+ at the EcoRI site is digested with
Pvull and Xmal to produce a 530 bp probe for Sl nuclea_e protection. The protected
fragments seen in the transduced cells correspond to the 260 bp fragment that would be
expected from the EcoRI site to ~he Xmai site in TM15. The wild type human
' . I ' ' yields a protected fragment 23 bp shorter. S I nuclease anaiysis (3 days
exposure) of RNA from transduced cells .L .. -:.m~ - significant steady-state expression of
human T~l e...,o.li..g mRNA (FIG. 6).
n~ivci~ of TM expression.
A compatison is made between the level of expression of TM in transduced PAEC ascompared with the high-expressing EAhy926 cell line, and with human umbilical cord vein
endothelial cells (HUVEC), which expresses human TM ~.u-~liluL~,ly.
The comparison is made by first estimating the absolute number of LL..,...I,..----,.I n;,.
molecules per cell on the iEAhy9~o cell line since all cells should express the molecule,
SUBSTITUTE SHEET (RULE 26j
wo 96106933 ~ 1 ~ 7 ~ ~ ~ P~ ~ Y
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using Scatchard analysis with "~-~ nl ~ i IA4. The levels of expression of the PAEC and
EAhy926 ate of similar magnitude. In a Cu~ ~dli~, ELISA. the EAhy926 cells express an
average of 2.5 x 105 molecules per cell. and the G418-selected NTR-2.TM.CWI -
transduced cells at least 1.5x 105 mûlecules per cell.
~ m. Biolo~ir~l ~r~ivity
r ~ ~.y of expressed human TM is quantified by co-factor assay on confluent
human TM-expressing PAEC, empty vector-transduced PAEC, HUVEC and EAhy926 (FIG.
7). The cells are grown to confluence in 24-well plates. The culture medium is removed
and wells washed with PBS. The cell cultures are incubated with 170 ,ul of the following
substrate co-factor solution:
0.8 ,ug human Protein C (purified from pooled plasma (Amencan Red Cross)
0.1; ~ units of bovine thrombin in Ix cofactor buffer (comprising 50 mM
tris-HCI, pH 8.0; 100 mM NaCI; 10 mM CaCI2; 0.1% BSA),
for 60 minutes at 37~ in a CO2 incubator as described in Owen, W. and Esmon, C., J. Biol.
Chem. 2~ (1982) 859 and Freyssinet et al., Binrh~m. J. ~ (1986) 151. To quench the
reaction, 3 units of leech hirudin (a thrombin binder) is added to e~h culture. Activated
Protein C generated during thc above incubation is assayed kinetically using thesubstrate, S-2366 by combining 159 ~1 of quenched reaction mixture and 50 ,ul
of substrate and reading the OD at 405 nm in a microplate reader. Activity of the
i' . l ' ' is expressed as the maximum rate of cleavage of the chromogen in mOD
units/min. The ~1.. - ..i... . A P... expressed by the PAEC is found able to catalyze thrombin
mediated action of human protein C, using both human and bovine thrombin. By
no significant 1' . ..1. ~ ' activity is detected on the ' ' PAEC
(FIG. 8), and less activity is seen with the EAhy926 cell line.
The activity of the tlll~ ' is .~ to that seen with HUVEC.
n. ('Inttir~p ctl~A;.c
A microwell i~i~ clotting experiment can !' that transduced PAEC
causes an extension of clotting times when activated with human plasma followed by
freeze-thaw disruption of the cells.
SUBSTITUTE SHEET (RULE 26)
W096106933 21~ r~l,~,s ~Y
-ZO-
S~
(I) GENERAL INFORMATION:
(i) APPLICANT:
(A) NAME: Sandoz Ltd. .
(B) STREET: 1. ~ ~ 35
(C) CITY: Basle
(E) COUNTRY: Switzerland
(F) POSTAL CODE (ZIP~: CH 4002
(G) TELEPHONE: 61-324 ~269
(H~ TELEFAX: 61-322 7532
(A) NAME: New England Deaconess Hospital ~ n
(B) STREET: 185 Pilgrim Road
(C) CITY: Boston
(D) STATE: MA
(E) COUNTRY: U.S.A.
(F) POSTAL CODE (ZIP): 02215
(A) NAME: BACH, Fritz H.
(B) STREEI: B. Blossom Lane
(C) CITY: ~' ' - By-The-Sea. Boston
iD) STATE: MA
(E) COUNTRY: U.S A.
(F) POSTAL CODE (ZIP): 01966
(A) NAME: Wl~IGHTON, Chdstopher
(B) STREET: 17, Stearns Road, Unit 5
(C) CITY: Brookline
(D) STATE: MA
(E~ COONTRY: U.S.A.
(F) PosTAL CODE (ZIP): 0214~
(ii) TITLE OF INYENlION: GENE THERAPY FOR TRANSPLANTATION AND
INFLAMMATORY OR IHROMBOTIC CONDlTlONS
(iii) NUMBER OF SEOUENCES: I
(iv) COMPUTER READABLE FORM:
(A) MEDIUM TYPE: Floppy disk
(B) COMPUTER: IBM PC compatible
(C) OPERATING SYSTEM: PC-DOS/MS-DOS
(D) SOFTWARE: Patentln Relesse #1.0, Version #1.25 (EPO)
(v) CURRENT APPLICATION DATA:
APPLICATION NUMBER: WO PCTIEP 95/.....
(vi) PRIOR APPLICATION DATA:
(A) APPLICATION NUMBER: US 081296'945
(B) F~ING DATE: 26-AUG-1994
wo 96/.,6933
-21- ~ 7 ~ 79
(2) INFORMATION FOR SEQ ID NO: 1:
(i) SEQUENCE CHARACTERISTICS:
~A) LENGTH: 1946 bsse pairs
(B) TYPE: nucleic . cid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(iii) ~IYI~ul~kllCAL: NO
(iii) ANTI-SENSE: NO
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Homo sapiens
(x) PUBLICATION INFORMATION:
(H) DOCUMENT NUMBER: US 4'912'207 P
(I) FILING DATE: 06-MAY-1987
(J) PUBLICATION DATE: 27~ 1990
SEQUENCE DESCRII~ION: SEQ ID NO: 1:
GAATTCTAGA GTCGACTGCT ACCGGCAGCG CGCAGCGGCA AGAAGTGTCT GGGCTGGGAC 60
GGACAGGAGA GGCTGTCGCC ATCGGCGTCC TGTGCCCCTC TGCTCCGGCA CGGCCCTGTC 120
GCAG~GCCCG CGCTTTCCCC GGCGCCTGCA CGCGGCGCGC C~GGGTAACA TGCTTGaGG~ 180
CCTGGTCCTT GGCGCGCTGG CCCTGGCCGG CCTGGGGTTC CCCGCACCCG CAGAGCCGCA 240 5-.
GCCGGGTGGC AGCCAGTGCG TCGAGCACGA CTGCTTCGCG CTCTACCCGG GCCCCGCGAC 300
CTTCCTCA~T GCCAGTCAGA TCTGCGACGG ACTGCGGG~C CACCTAATC-A CAGTGCGCTC 360
CTCGGTGGCT GCCGATGTCA TTTCCTTGCT ACTGAACGGC GACGGCGGCG TTGGCCGCCG 420
GCGCCTCTGG ATCGGCCTGC AGCTGCCACC CGGCTGCGGC GACCCCAAGC GCCTCGGGCC 480
CCTGCGCGGC TTCCAGTGGG TTACGGGAGA CAACAACACC AGCTATAGCA GGTGGGCACG 540
GCTCGACCTC AATGGGGCTC CCCTCTGCGG'CCCGTTGTGC GTCGC~ r CCGCTGCTGA 600
GGCCACTGTG CCCAGCGAGC CGATCTGGGA GGAGCAGCAG TGCGAAGTGA AGGCCGATGG 660
~ll'C'~ GC GAGTTCCACT TCCCAGCCAC CTGCAGGCCA CTGGCTGTGG AGCCCGGCGC 720
CGCGGCTGCC GCCGTCTCGA TCACCTACGG CACCCCGTTC GCGGCCCGCG GAGCGGACTT 780
WO gG106913 ~ C 11~ /Y
--23-
oooooooooooo
O O O ~ C~ ~ O ~ ~ O
~r o ~D O O ~1~ ~ r~
C ~ s ~ C ~- ~ C
C ~ ' f ~ ~ C
f~ -~ J - _ r~ r , , -r r
fr I f r ' , ~ ~ ~ r
S -~ - _
- J
~ I J J ~ -
r "~ ~ r r
r _l
J
,~ t , 1~ r C ~ ''
;~ S -I 1~: C - - ~ ~
J -- J ~, ~ r
J ~ r ~ ~ C' _ _. ~ _ C
C ~ J
r,~ r ~ r
_ _ s
C _ _ _
~' 5 ~ ~ , c)
,, , , ~_ _ ., _ . _ J
? - - -, - - f~
, . ~
- - ~ r ~ s ~ J
J
C fl :~ ~ 3
¢ , ,~ .
-- s
f I . . ,.~ ~ .
~ ~ ' C~
u~ 3 ~ , ~ c~ . ,
' ' u~ u~ , :
u ,' ~u
~. ~ U ~ ~ ~ U
fl - E~ ~ ~ C
J ~ _ V J C .~ r}
CTTGACTCCT CCGGCCGTGG GGCTC¢TGCA TTCGGGCTTG CTCATAGGCA TCTCCATCGC 1740
GAGCCTGTGC CTGGTGGTGG CG~lllGGC G~TC~l~lGC CACCTGCGCA AGAAGCAGGG 1800
CGCCGCCAGG GCCAAGATGG AGTACAAGTG CGCGGCCCCT TCCAAGGAGG TAGTGCTGCA 1860
GCA~T~G~G ACCGAGCGGA CGCCGCAGAG ACTCTGAGCG~GCCTCCGTCC AGGAGCGTCG 1920
~CGACGAATT CGGATCCGAC AAGCTT 1946
.
